United States        Office of Pollution
        Environmental Protection    Prevention and Toxict,
        Agency          Washington, DC 20460
&EPA   Workshop Report Or«
        Chronic Inhalation
        Toxicity And
        Carcinogenicity Testing
        Of Respirable Fibrous
        Particles
    Recycled/Recyclable • Printed with Vegetable Oil Based Inks on 100% Recycled Paper (50% Posteonsumer)

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                                      EPA-748-R-001
           WORKSHOP REPORT ON
      CHRONIC INHALATION TOXICTTY
     AND CARCBVOGENICITY TESTING OF
      RESPIRABLE FIBROUS PARTICLES
                May 8-10,1995

              Omni Europa Hotel
           Chapel Hill, North Carolina
               Sponsored by the
      U.S. Environmental Protection Agency

            in Collaboration with the

 National Institute of Environmental Health Sciences
National Institute for Occupational Safety and Health
   Occupational Safety and Health Administration
                Assembled by
       Research Evaluation Associates, Inc.

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                             DISCLAIMER

The proceedings of this workshop were prepared by Research and Evaluation
Associates, Inc., for the EPA's Office of Pollution Prevention and Toxics, under
Contract Number 68-D1-0136, Work Assignment 402.

The opinions, findings, conclusions, or recommendations presented in  these
proceedings are those of the Consultant Panel and do not necessarily reflect the
views of the  Office of Pollution Prevention and Toxics, U.S. Environmental
Protection Agency.

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                           Table of Contents

                                                                     Page
Acknowledgements	    iii

Workshop Summary	1

Workshop Goals  	5

Session 1: Inhalation Exposures: Materials and Methods                      8
       Definition of Fibers  	8
       Selection Criteria for Suitable Test Materials  	11
       Characterization of Test Fibers	 14
       Exposure Conditions and Methods	17

Session 2: Study Design	18
       Animal Species/Strain/Sex Selection  	18
       Selection of Exposure Concentrations 	23
       Exposure Regimen and Observation Period 	25
       Numbers of Animals and Interim Sacrifices 	26
       Use of Positive Control  	27
       Criteria for a Negative Inhalation Test	30

Session 3: Histopathologic Evaluation	31

Session 4: Fiber Disposition and Dosimetry and
    Interspecies Considerations	35
       Pre-chronic Studies  	36
       Lung Burden Analysis	! . . 38
       Chronic Study	  41
       Dosimetry and Interspecies Considerations	42

Session 5: Mechanisms of Toxicity and Carcinogeniciry and Biomarkers     of
Toxicologic Effects	43
       Mechanisms of Toxicity and Carcinogenicity	43
       Biomarkers of Toxicologic Effects	46

Session 6: Screening Battery  	46

APPENDIX I - Workshop Agenda

APPENDIX n - List of Workshop Participants/Consultant Panel

APPENDIX m - Issue Paper

APPENDIX IV- Background Information as Basis for Workshop Discussions

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                            Acknowledgements

The Office of Pollution Prevention and Toxics gratefully acknowledges the time, effort,
and input provided by the Interagency Steering Committee, Consultant Panel, and the
invited participants during this intensive 3-day workshop. The workshop participants are
identified in Appendix n. Special thanks are extended to Dr. Giinter Oberdorster for the
work he performed as chair of the workshop, and for the preparation of the "Background
Information as Basis for Workshop Discussion." A number of consultants (including Dr.
Susan Dakin)  are also thanked for serving as rapporteurs and/or session chairs (see
Appendix n).
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                           Workshop Summary


       EPA, in conjunction with NIEHS, NIOSH, and OSHA, convened a workshop

in May 1995, on "Chronic Inhalation Toxicity and Carcinogenicity Testing of

Respirable Fibrous Particles." A panel of consultants discussed methodologies for

toxicological screening and chronic exposure testing of fibers with the goal to advise

EPA on the development of testing guidelines. EPA sought guidance from the

scientific community on the following issues:

—     the optimal design and conduct of studies of the health effects of chronic
       inhalation exposure of animals to respirable fibers;

—     identification of preliminary studies needed to guide design of a chronic
       exposure study;

—     evaluation of which mechanistic studies would provide information useful in
       design and interpretation of inhalation exposure studies and in extrapolation of
       study results to potential effects in exposed humans;

—     identification and evaluation of screening tests that could be used to develop a
       minimum data set for (a) making decisions about the potential health hazards
       of fibers and (b) prioritizing the need for further testing in a chronic inhalation
       study.

       These issues were addressed in six sessions during the workshop. Although the

experts believed that inhalation exposure is the most appropriate route of

administration for testing fibers, they recommended that a multi-dose chronic

inhalation study of asbestos fibers be conducted in rodents to validate the sensitivity

and specificity of chronic inhalation studies in predicting the human health effects of

fibrous particles. Other major conclusions and recommendations are summarized as

follows:

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Inhalation Exposures; Materials and Methods

•      The term "respirable fiber" should alway  oe used with a species modifier, i.e.,
       "human respirable" or "rat respirabl

•      Rodent inhalation studies should use an aerosol which is enriched with rodent
       respirable fibers and fibers with lengths of >20 \im or fibers with high aspect
       ratios.

•      The complete bivariate length and diameter distribution -including fibrous and
       non-fibrous particles - should be determined in the aerosol and in the lung.

Study Design

•      The rat is the preferred animal for fiber inhalation studies; however,
       investigators and regulatory agencies are encouraged to investigate health
       effects of fibers in a second species, particularly the hamster.

•      Criteria for selecting a suitable rat strain for fiber testing should include: (i) a
       low background rate of neoplasia; (ii) a low background rate of pulmonary
       disease; (iii) longevity; (iv) a history of laboratory use.

•      Use of both sexes for fiber testing was not thought to be mandatory.

•      Either nose-only or whole-body exposure can be used.

•      A subchronic study should be conducted with the goals to establish lung
       burdens and potential target sites to aid in the design of the chronic study and
       to evaluate toxicity with respect to a variety of important biological endpoints.

•      The 90-day subchronic study should include a follow-up period to  assess
       reversibility of effects.

•      A practical upper limit concentration (Maximum Aerosol Concentration
       [MAC]) was not recommended. Instead, the MAC should be based on
       achieving the Maximum Tolerated Dose (MTD) in the lung. The MTD is
       defined by alterations in alveolar macrophage-mediated particle clearance rate,
       normalized fiber lung burden, cell proliferation, histopathology, quantitated
       inflammation, and lung weight.

•      The chronic study should be performed with 3 exposure levels with the highest
       showing  significant effects indicative of the MTD and 2 appropriately spaced
       lower exposure concentrations.

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•      The chronic study in rats should be a lifetime study, with exposures terminated
       at 24 months and the study terminated when survival of the control group
       reaches 20%.

•      Interim sacrifices should be made in the chronic study at 3, 6, 12, 18, and 24
       months to evaluate specific parameters of importance for toxicity assessment.

•      A positive control need not be included in every study. The result of a chronic
       inhalation study with fibers is acceptable as negative if the study was properly
       designed and conducted and if the health effects of concern are not
       significantly more frequent in the exposure groups than in the control groups.

Histopathologic Evaluation

•      Testing guidelines should specify procedures for histopathological grading of
       lung lesions to replace the Wagner scoring system. Neoplastic endpoints
       should include epithelial hyperplasia, alveolar bronchioUzation, metaplasia,
       adenomas, mesotheliomas, carcinomas, and keratin cysts.

•      To the fullest extent possible, tissues from the chronic study should be
       preserved in such a way that other measurements or analyses can be conducted
       later.

Fiber Disposition and Dosimetrv and Interspecies  Considerations

•      Lung burden analysis should be performed at 3, 6, 12, 18, and 24 months of
       exposure not only to assist in establishing the chronic exposure levels, but also
       to quantify aspects of risk assessment related to dosimetric adjustment before
       extrapolation.

•      Impairment of clearance should be assessed via challenge with a labelled
       particle.

Mechanisms of Toxicitv and Carcinogenicitv and Biomarkers of Toxicoloeic
Effects

       No mechanistic studies are recommended, but priority should be given to
       research with respect to (i) development of short-term in vivo assays; (ii)
       investigations of importance of oncogenes and tumor suppressor genes; (iii)
       development of transgenic animal models; (iv) species comparisons of fiber-
       induced pulmonary effects in vivo and in vitro, (v) use of pleura! lavage to
       evaluate predictive markers of response.

       A tiered testing approach (in vitro studies on durability and in vivo short-term
       studies on toxicity and biopersistence) will provide useful information to
       prioritize materials for further testing.

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Intratraeheal instillation was considered to be an acceptable alternative to
inhalation exposure for short-term screening studies. Intratraeheal instillation
was not recommended for assessing the carcinogenic potential in long-term
studies.

The intracavitary test was viewed as being primarily useful in conjunction with
inhalation exposure studies and as a research tool. Additional information is
required, including the dose and dimensions of fibers reaching the mesothelium
after inhalation exposure.

Intraperitoneal injection studies can provide information on the interaction of
fibers with mesothelial cells. Dose levels should be selected so  that an MTD is
achieved but not exceed several-fold.

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                             Workshop Goals







       The Workshop on Chronic Inhalation Toxicity and Carcinogenicity Testing of




Respirable Fibrous Particles was held on May 8 through 10, 1995, at the Omni Europa




Hotel in Chapel Hill, NC. A workshop agenda and list of participants are included in




Appendices I and n, respectively.  This report summarizes the discussion and




recommendations of the panel of consultants convened by the U.S. Environmental




Protection Agency (EPA) and the other sponsoring agencies to address the specific




questions raised in the "Issue Paper for Workshop Discussion" prepared by the




Inter-Agency Steering Committee on Fiber Testing (see Appendix HI). Appendix IV is




the "Background Information as Basis for Workshop Discussions," prepared by Dr.




Giinter Oberdorster. Following a summary of the workshop goals, the report is




organized in six sections corresponding to the six workshop sessions.




       The workshop goals were  outlined by Dr. Charles Auer, Director of EPA's




Chemical Control Division, and Dr. Vanessa Vu, Chair of the Inter-Agency Steering




Committee on Fiber Testing.




       An important task for the U.S. EPA and the other agencies sponsoring this




workshop is to identify health risks posed by toxic substances and to reduce these




risks. Natural and synthetic fibers  are one group of substances of potential concern.




Although many of these fibers have wide industrial and commercial applications,




limited information is available about their toxic properties or about public or




occupational exposure to them. EPA has established the category of "respirable fibers"




as priority substances on its Master Testing List for health effects and exposure

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testing, to evaluate the health risks associated with respirable fibers and to determine

whether risk reduction measures are nee  d.

      . The health endpoints of concern for respirable fibers are development of

respiratory diseases, including cancer, as a result of chronic inhalation exposure. In

humans, inhalation of asbestos and erionite fibers has been associated with the

development of nonmalignant and malignant diseases, primarily of the lung, pleura,

and peritoneum. Although the mechanisms by which fibers induce disease are not well

understood, they are believed to depend on the physical properties of the fibers and on

their respirability (i.e., their ability to enter the respiratory tract and penetrate into the

alveolar region of the lung).

       EPA recognizes that the current health effects testing guidelines for chronic

inhalation toxicity and carcinogenicity studies are not  specific enough for testing of

fibers. EPA needs to develop standardized guidelines for health effects testing of

fibrous substances to be used in future rulemaking, negotiated enforceable consent

agreements or voluntary testing programs (such as product stewardship programs) and

to obtain the toxicologic information needed for risk assessment. However, no

standardized protocols for toxicologic screening or chronic exposure testing of fibers

have been generally agreed upon. Thus, EPA's goal for this workshop is to obtain

guidance from the scientific community on the folio whig issues:

(1)    Issues in the optimal design and conduct of studies of the health effects of
       chronic inhalation exposure of animals to respirable fibers.

(2)    Identification of preliminary studies needed to guide design of a chronic
       exposure study.

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(3)    Evaluation of which mechanistic studies would provide information useful in
       design and interpretation of exposure studies and in extrapolation of study
       results to potential effects in exposed humans.

(4)    Identification and evaluation of screening tests that could be used to develop a
       minimum data set for (a) making decisions about the potential health hazards
       of fibers and (b) prioritizing the need for further  testing in a chronic inhalation
       study.

       These issues were grouped  into six sessions for discussion during the

workshop,  corresponding to numbered sections in the Issue Paper for Workshop

Discussion:

(1)    Inhalation Exposures:  Materials and Methods (Issue Paper section 3.1)

(2)    Study Design (Issue Paper section 3.2)

(3)    Histopathologic Evaluation (Issue Paper section 3.7)

(4)    Fiber Disposition (Issue Paper section 3.3) and Dosimetry and Interspecies
       Considerations (Issue  Paper section 3.4)

(5)    Mechanisms of Toxicity and Carcinogenicity (Issue Paper section 3.5) and
       Biomarkers of Toxicologic  Effects (Issue Paper  section 3.6)

(6)    Screening Battery (Issue Paper section 3.8)

The consultants were asked to review a background document which deals in the

major issues related to lexicological testing of fibrous substances (Oberdorster, 1995,

Appendix  IV) and to address the specific questions listed under each issue in the Issue

Paper (Appendix ffl).

       The consultants were  asked to keep in mind that needs for information from

toxicological testing differ among countries. In the U.S., risks must be quantitatively
   i
assessed in order for control action to be taken. Therefore, studies of the effects of

chronic inhalation exposure to fibers are necessary; studies of exposure via instillation

or implantation are not sufficient for regulatory purposes. The protocols EPA adopts

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' for lexicological testing of fibers must be scientifically sound and must span the range

of information needs, from screening to testing of effects by chronic inhalation

exposure.

       In the summaries below, the consultants' comments in each session are

organized according to the lists of questions in the Issue Paper. Under each question

or group of questions, the panel's conclusions and recommendations, as summarized

by the rapporteur for the session, are stated first, followed by a summary of the

discussion leading to those conclusions and recommendations. (In cases where the

rapporteur's conclusions and recommendations fully summarize the discussion, a

separate "discussion" section is not included.)


       Session 1: Inhalation Exposures: Materials and Methods

       The Exposure session was chaired by Dr. James Vincent, and Dr. David

Bernstein served as rapporteur. Because Dr. Vincent was delayed, Workshop Chair

Dr. Gunter Oberdorster opened the session and served as session chair until Dr.

Vincent arrived.


Definition of Fibers

       Question 1: Is an aspect ratio of equal to or greater than 3:1 an acceptable
       definition of a fiber? If not, how should the definition be modified to
       encompass the varying range of sizes and shapes of naturally occurring and
       synthetic fibrous substances?

       Conclusions and Recommendations: A fiber is defined as a  particle having

an aspect ratio of at least 3:1 (length:diameter) and being structurally continuous.

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       Discussion: It was noted that particle sizing (based on average maximum

length and average minimum diameter) results in a bimodal bivariate distribution, with

the dip between the modes falling at an aspect ratio of 3:1 to 4:1. Thus, 3:1 is a good

aspect ratio to use to distinguish between fibers and non-fibrous particles. It was

concluded that, because of present uncertainty about the importance of fiber length in

health effects, no lower length limit for defining fibers should be specified.

       It was acknowledged that fiber shape is important. However, it was agreed that

shape (e.g., whether the fiber has parallel or non-parallel sides) should not be used to

define whether a particle is a fiber. It also was agreed that the propensity of particles

to split after being deposited in the lung should not be considered in the definition of

"fiber." Biological activity should not be built into the definition; rather, fiber shape

and behavior in the lung should be taken into account in considering respirability and

biological effects of fibers.

       Nonetheless, it was agreed that a fiber must be structurally continuous (a solid

object); for example, a string of spherical particles does not constitute a fiber.


       Question 2: What would be appropriate definitions of human "thoracic" fibers
       and "respirable" fibers?

       Question 3: What would be a suitable  definition of rat "respirable" fibers?

       Conclusions and Recommendations: Respirability should be defined on the

basis of experimental data, rather than calculated data. The term "respirable fiber"

should always be used with a species modifier, such as "human-respirable" or

"rat-respirable." "Respirable" means that the particle in question can penetrate to the

alveolar region upon inhalation. A "rat-respirable fiber" is defined as a fiber having an

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aerodynamic diameter of less than 3 um. "Thoracic" fibers were defined by the




participants only in general terms as those fibers penetrating to the conducting airways




upon inhalation.




       Discussion: It was noted that because the rat is a compulsory nose-breather,




the size range of fibers that reach the human thoracic and alveolar regions are a subset




of the size range of fibers that can reach the human thoracic and alveolar regions.




Furthermore, fibers must reach the rat's alveolar region to have a biological effect. It




was noted that rats and humans also differ in inhalability of a given fiber (i.e., its




probability of being inspired). Interception and impaction of fibers in the nose and in




the upper respiratory tract principally controls the shape of the distribution of fibers




that reach the deep lung.




       A consequence of species differences in deposition is that fibers with potential




human health consequences may be too large (in particular, too thick) to be respired




by the rat. For instance,  many fibers in the workplace environment are not inhalable




and respirable by the rat. The problem is how to use animals in an inhalation assay to




test the effects of fibers of concern for human health and extrapolate the results to




human health effects when these fibers are not respirable by the test animal.




       It was agreed that separate definitions are needed for "rat-respirable fibers,"




"human-respirable fibers," and "human thoracic fibers." Rat-respirability of a fiber




must be defined in order to design inhalation exposure studies. Respirability of a fiber




depends on the species and on whether the fiber penetrates to the alveoli or small




bronchioles. For many types of fibers, what reaches the lung has been well




characterized. In the rat, fibers of 0.8- to 0.9-um mass median aerodynamic diameter
                                       10

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(MMAD) get into the lung. Much larger fibers also have a certain probability of

getting into the lung. The deposition efficiency for fibers of 3-um aerodynamic

diameter is ~6%. An upper limit of 3-um aerodynamic diameter will be effective in

capturing rat-respirable particles. However, the actual dose and size distribution

(bivariate) deposited in the lung in a study must be characterized.


Selection Criteria for Suitable Test Materials

       Question 1: For a given fiber type, should inhalation studies be performed
       using samples with the greatest potential for pathogenic effects (e.g., long, thin
       fibers)?

       Question 2: Should fiber samples for testing be prepared so that they are
       rodent-respirable, or should they represent a human-respirable sample (or
       fibers conforming to a human thoracic particle definition)?

       Question 3: Should the test fibers reflect what are actually present at the
       workplace and/or non-occupational environments?

       Question 4: In the case of new fibers, how should the test materials be
       selected?

       Conclusions and Recommendations: Rodent inhalation exposure studies

should use an exposure aerosol that is, as far as is technically feasible, enriched with

the following fiber size fractions:

       •  rat-respirable fibers: aspect ratio of at least 3:1 and aerodynamic diameter
           less than 3. jam;

       •  fibers with lengths of at least 20 \im or fibers with high aspect ratios.

The fraction of long fibers (>20 \im) should be specified; 10% to 20% would seem

appropriate, but not enough information is available on which to base a specified

percentage.
                                       11

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       The aerosolized fibers should be discharged to Boltzmann equilibrium before




being delivered to the test species.




       It was suggested that to maximize sensitivity of animal inhalation exposure




studies to health effects of fibers, the test material should consist of rat-respirable




fibers and should be enriched with the most potent fraction (i.e., long, thin fibers). If




the study results are negative and it can be shown that fiber mass loading and fiber size




distillation in the lung are not sufficient, then the fiber would be considered to not have




been adequately tested.




       Discussion: It was noted that disease processes appear to depend on the




presence of long, thin fibers (i.e., <1 \im in diameter and 20 urn or longer). Therefore,




a single figure such as the geometric mean should never be the only selection factor;




the number of fibers (especially long ones) per unit air must be considered.




       However, different classes of fibers may be related to different health




endpoints, and new fibers will not necessarily act in the same way as asbestos. The




important point is to ensure that  all size fractions that could have a health effect are




tested, particularly a sufficient concentration of long fibers. For design of an adequate




study, test-material selection criteria are needed to ensure an appropriate fiber size




distribution.




       Dr. Paul Baron described a dielectrophoresis technique that has been




developed for classifying fibers by length, which works for any conducting fiber.




       Two basic approaches to test-material selection were debated at length: (1)




providing an exposure representative of the workplace atmosphere or (2) sampling the
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workplace atmosphere and selecting the rat-respirable portion to use as the exposure




atmosphere.




       According to the first yiew, in providing data for risk assessment, it is hard to




isolate the question of test-material selection from other considerations. For example,




fibers respirable by humans may not be rat-respirable, and fibers present in the




workplace may be preferentially respirable by humans. Test-material selection must




consider these factors. Perhaps a different testing strategy is needed to determine the




effects of fibers important to human health but not respirable by rats.




       According to the second view, the minimum requirement for the test material is




that the fibers be rat-respirable; this could cause a dilemma in that longer (with larger




aerodynamic diameter) human-respirable fibers may not be tested, although they are




potentially more dangerous. The question is whether a fiber length distribution should




also be specified. Because of the need to evaluate new, as well as existing, fibers, it is




not sufficient to specify that exposure should be "representative of the workplace




atmosphere"; a more general guideline is needed. It was suggested that prescribing




specified percentage or air concentration of long fibers in the aerosol would allow




comparability among studies.




       Practical considerations in specifying for the proportion of long fibers were




noted. For example, the ICRP (International Cooperative Research Programme on the




Assessment of MMF's Toxicity) suggestion of a geometric mean fiber diameter close




to 0.8 \im and a geometric mean length of longer than 15 urn may be too specific. It




was suggested that a specification based on aspect ratio would be more appropriate,




and that practical considerations in providing exposures be considered.
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       It was noted that deposition of char ad fibers is greater, both in the

environment and in the lung. It was agreec    t charge of fibers in exposure aerosols

must be reduced, to reduce erratic effects,  .ch as increases in nasal deposition relative

to pulmonary deposition. For standardization of inhalation exposure studies, the

charge should be reduced to Boltzmann equilibrium.


Characterization of Test Fibers

       Question 1: At a minimum, what aspects of the test  samples need to be
       characterized (e.g., fiber morphology, dimension, size distribution,
       aerodynamic diameter, chemistry, density, solubility, surface characteristics,
       the ability of a fiber to split longitudinally or cross-sectionally)?

       Question 2: Should the presence of chemical and/or mineralogical impurities
       and trace metals  also be characterized, since they may also contribute to
       toxicity?

       Question 3: Should efforts be made to assess the contribution of non-fibrous
       particulate materials, since they may be substantial and could add significantly
       to total lung burden in terms of mass?

       Question 4: Are there any specific analytical methods that should be required
       to be used to characterize certain chemical and physical properties of bulk
       materials or individual fibers present in the aerosol and lung tissues?

       Question 5: Are the available methods for the measurement of fiber size
       distributions considered adequate? Are there any new and improved methods
       that can be used  for measuring fiber size distributions?

       Conclusions and Recommendations: The complete bivariate length and

diameter distribution should be determined in the aerosol and in the lung via electron

microscopy. This bivariate distribution should include the non-fibrous particles present

in the aerosol. For non-fibrous particles, "length" and "diameter"  can be determined as

the "longest length" and "narrowest dimension." The aerodynamic size distribution

should be determined; a cascade impactor can be used for this purpose. WHO
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counting rules and sizing rules should be used and results should be evaluated




statistically to assure sufficient sensitivity.




       Intel-laboratory validation should be provided for all counting and sizing




methods. In addition, it is recommended that sizing techniques be used that permit




returning to the same fields and fibers in the event it is necessary to confirm counting.




       Routine monitoring to control the day-to-day aerosol concentration can be




performed using phase-contrast optical microscopy (PCOM) and gravimetric




techniques.




       Discussion: It was noted that, especially in the older literature, fibers have




been described in terms of geometric and aerodynamic mean dimensions, which is not




sufficient. It is important to know the bivariate length and diameter distributions of




fibers.




       Given that the presence of non-fibrous particles in the exposure atmosphere




may condition the lung's response to fibers, it was debated whether the ratio of




particles to fibers should be the same as in the workplace atmosphere, and whether




other substances present in the workplace atmosphere (such as binders) should be




included in the exposure aerosol. It was suggested that trying to reproduce the




workplace atmosphere was too narrow a focus for testing (especially since workplace




atmospheres are affected by machine-specific processes), and that this approach would




confound testing for health effects specific to fibers. Questions of synergistic effects of




fibers and other substances  present in the workplace atmosphere are a separate issue.




       Nonetheless, some fiber-related  particles will inevitably be present in the




exposure aerosol; these should be accounted for in the bivariate distribution of fiber
                                       15

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(and particle) size, using measurements of average longest length and average




narrowest dimension. It was agreed that reg'ydless of how the exposure aerosol is




produced, it must be characterized in terms of fiber and particle size distribution and




also chemical composition. However, particle-by-particle elemental analysis is not




necessary. The aerodynamic size distribution should also be determined; cascade




impactors are appropriate for this purpose.




       For reporting of results, fiber size distributions should be determined by




scanning electron microscopy (SEM) or transmission electron microscopy (TEM), not




PCOM. However, PCOM is acceptable for routine monitoring of exposure




atmospheres. Two dimensions must be measured for every fiber or particle observed.




Semi-automated image-analysis systems can be used to speed the process.




       With regard to the counting rule, it was suggested that a 100-fiber stopping




rule is not sufficient, and that at least 300 fibers should be measured, according to the




WHO counting and sizing rules. It was also suggested that a statistician should be




involved in this determination. The numbers of fibers of a particular length should be




used as the criterion; the number of fibers of a particular aspect ratio is not sufficient




(e.g., fibers with a high aspect ratio could be short and thin).




       The need for quality assurance (QA) in measurement of fiber size distributions




to ensure niter-laboratory consistency was stressed. This might be accomplished




through a round-robin exchange. For QA purposes, the methods  should permit




returning to the fibers that were described, in case of disagreement.
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Exposure Conditions and Methods

       Question 1: Should there be a requirement for the use of any particular
       methods for generating fibrous aerosols?

       Conclusions and Recommendations: The guidelines should not specify a

particular aerosol generation system, but should require that the exposure system be

validated by the investigator (e.g., with respect to airborne fiber size distribution and

the target dose in the lung). It should be demonstrated that the generation system does

not contaminate the fibers.

       Question 2: Are both methods of exposure (whole-body and nose-only
       exposures) acceptable? Is there a preferred method that should be
       recommended?

       Question 3: How often should exposure atmosphere be monitored with regard
       to fiber number and mass concentration, size distribution, and chemical
       analysis?

       Conclusions and Recommendations: Either nose-only or whole-body

exposure can be used. The target exposure concentrations should be measured by

electron microscopy (fiber number and bivariate size distribution) to confirm the dose

delivered to the animals.

       Discussion: It was noted that although whole-body exposure results in

secondary exposure of the animal, this effect is probably small relative to

inter-individual variability in lung burden resulting from the primary exposure. After

animals are removed from a whole-body exposure, they are surrounded oy a cloud of

fibers; the different exposure groups should be kept isolated after the exposure ends.

       An important consideration is that nose-only exposure can be very stressful to

the animal. However, because quantities of test materials often are limited, nose-only

exposure may be the only practical method. Although there are trade-offs between the

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advantages and disadvantages of whole-body and nose-only exposure, the ultimate

determinant of adequate exposure is the lung burden. It was agreed that either type of

exposure is acceptable.

                         Session 2: Study Design

       The Study Design session was chaired by Dr. Gunter Oberdorster, and Dr. Neil

Johnson served as rapporteur.

Animal Species/Strain/Sex Selection

       Question 1: Is it necessary to test fibers of unknown activity in a second
       animal species? If yes, what would be an appropriate second species (e.g.,
       Syrian Golden hamster, Chinese hamster)? If not, what are the scientific
       reasons? Are there any circumstances that warrant testing in a second species?

       Conclusions and Recommendations: Given the present limited knowledge of

the effects of fibers in species other than the rat, testing of fibers in a second animal

species is not strongly recommended; however, investigators should be encouraged to

investigate health effects of fibers in another species, particularly the hamster. In the

future, transgenic animals may prove useful for testing.

       Discussion: A general principle in testing is to use the species that is the best

surrogate for humans for each health endpoint of concern. For a species to be useful in

testing fibers of unknown effects, a fiber known to cause a particular health effect in

humans should be shown to produce the same effect in the animal model.

       In hamsters, several known human carcinogens (e.g., chrysotile asbestos) do

not produce lung cancer or mesothelioma; however, in one study, refractory ceramic

fibers (RCF) did produce mesothelioma. In mice, inhalation exposure to fibers has

never been shown to produce lung cancer. In one study, chrysotile did not produce
                                      18

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mesothdioma in mice, although it did in rats. The issue is complicated by strain




differences. For example, in particle inhalation exposure studies with mice, there are




high- and low-responding strains with respect tq inflammation; the high-responding




strains also have high rates of spontaneous tumor formation. Insufficient data are




available for the hamster to decide whether it is an appropriate second species. It was




noted that high inflammatory responses in rats, which complicate data interpretation,




can be avoided by not using excessively high doses (i.e., avoiding the equivalent of




"overload" for non-fibrous particles).




       Deposition and clearance rates of particles vary among rodent species; with




respect to clearance, the guinea pig resembles humans more closely than other rodent




species, including the rat However, because guinea pigs are larger than rats and live




twice as long, their use more than doubles the cost of a chronic inhalation exposure




study.




       The consultants were reminded that EPA uses testing not only for qualitative




determination of carcinogenic potential, but also for quantitative risk assessment, and




has historically required the use of two species.




       There was general agreement that at present only the rat model has a sufficient




database to be recommended for inhalation exposure studies with fibers, but that the




agencies should be encouraged to look at the hamster as a possible second species. At




present, not enough is known to recommend that only one species (i.e., the rat) is




needed; however, not enough is known to recommend an appropriate second species,




either. It was suggested that for the future, the use of transgenic animals should be




explored.
                                       19

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       Question 2: Do different animal strains respond with different sensitivities to
       fibrous particles? If yes, what is the most appropriate strain of rodent species
       to be used?

       Conclusions and Recommendations: Although it was acknowledged that rat

strains clearly differ in suitability for inhalation exposure studies with fibers, the panel

could not agree on whether to recommend a particular strain to use (or to avoid), but

proposed a set of criteria for choosing a strain. It was agreed that criteria for a suitable

strain include (1) a low background rate of neoplasia, (2) a low background rate of

pulmonary disease, (3).longevity, and (4) a history of laboratory use.

       Discussion: Some members suggested that the Fischer-344 rat is not a good

model for inhalation exposure studies, because tumors appear late, and at two years, at

least 80% of the animals have leukemia. The main argument for using the Fischer-344

rat is the large body of data that exist for the strain. It also was suggested that the

Fischer-344 rat would be acceptable for detecting lung tumors. The Sprague-Dawley

rat has the disadvantages of being relatively large and prone to developing mammary

tumors. The usefulness of the Osborae-Mendel rat is limited by the lack of baseline

data for this strain.

       The Wistar rat was suggested to be a more appropriate choice because the

background tumor rate at 30 months is lower and leukemia is rare, increasing

confidence that the effects seen hi the oldest rats are treatment-related. However,

fewer data exist for this strain, and in one study with Wistar rats, crocidolite did not

produce lung tumors (possibly because the dose of long fibers was too low). Also,

different stocks of Wistar rats appear to differ. For example, good results were

obtained with Hanover-derived Wistar rats, which were long-lived and had low
                                      20

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background disease rates, and for which much control background data was available.

Results with a Wistar stock in the Netherlands were poorer.

       It was noted that choice of strain is a statistical issue, in that with a high

background tumor rate, more animals are needed in order to detect an exposure effect.

Specification of one strain would provide a "level playing field" for detection of

effects. However, because characteristics such as background disease incidence vary

even within strains, it was suggested that rather than specifying a strain, the testing

guidelines give the specifications for a suitable stock, but leave the choice to the

investigator. These specifications should include longevity, a low rate of neoplasia,

minimum confounding pulmonary pathology, and availability of good baseline data.

       Question 3: Should both sexes of the animal be used? If not, which sex is more
       suitable, and why?

       Conclusions and Recommendations: It was agreed that whether to use  one

or both sexes should be left to the investigator, and that if one sex is used, the choice

should depend on factors related to the strain, test  material, and endpoint studied.

However, if cost was not a factor, testing in both, sexes should be encouraged because

data on sex differences in response to inhalation exposure to fibers are limited.

       Discussion: Information on sex differences in response to fiber inhalation

exposure is limited. Which sex is most sensitive to  inhalation exposure effects of fibers

appears to depend on the strain or stock, test material, and endpoint being studied.

Also, it seems that sensitivity differences appear to be relatively small and unlikely to

affect the outcome of a lifetime study. The sexes may also differ in longevity,

spontaneous incidence of disease, and the age'at which treatment-related effects are

first seen.

                                       21

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       It was generally agreed that if cost were not a consideration, it would be best

to include both sexes. Using both sexes need not double the cost of a study, because

the main test and the test of a sex-specific effect can have different statistical power. If

the sexes do not differ in their response, it may be appropriate to combine both males

and females for statistical purposes. However, it is not clear whether the benefit of

including both sexes will necessarily justify the increased cost. The participants

concluded that presently there is no evidence of a sex difference hi response to inhaled

fibers, in contrast to non-fibrous particles where females may be more sensitive. Thus

a single sex is adequate.


Selection of Exposure Concentrations

       Question 1: What criteria can be used to determine the maximum aerosol
       concentration (MAC) in inhalation studies of fibrous particulates and to judge
       whether a MAC has been reached or exceeded?

       Question 2: Is it necessary to include a satellite group exposed to a fiber
       concentration exceeding the MAC for.the evaluation of lung pathology other
       than neoplasia?

       Question 3: What preliminary studies would be useful and important for
       setting appropriate exposure  concentrations (e.g., 90-day and/or shorter-term
       inhalation studies, in vitro solubility, in vivo biopersistence studies)?

       Question 4: The NTP generally employs an upper limit exposure concentration
       of 100 mg/m3 for relatively insoluble non-fibrous particles of low toxicity. In
       view of potential particle "overload," should a practical upper limit
       concentration also be set for  fibrous particles?

       Conclusions and Recommendations: A practical upper limit concentration

was not endorsed since it would depend on fiber type, and no one number could be

determined that applies to all fibers.  The MAC should be based on the total number of

inhaled particles (fibers and non-fibrous particles combined). The MAC should be set
                                       22

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based on the following functional parameters determined in a 90-day subchronic




inhalation study: altered alveolar macrophage medicated particle clearance rate, fiber




lung burden normalized to exposure concentration, cell proliferation, histopathology,




inflammation (quantitatively determined as percentage increase in polymorphonuclear




leukocytes [PMNs] in lung lavage samples) and lung weight. It was suggested that an




appropriate lung burden of critical fibers (long and thin) should be achieved, but no




number was suggested. These parameters should be considered together, rather than




individually, in an attempt to define a maximum tolerated dose (MTD) for the chronic




study. The MTD is the lung dose achieved with the MAC. For the chronic study,.three




exposure levels should be used; the high exposure concentration and resulting lung




dose should show significant effects in the above parameters (MTD), and the lower




doses should be appropriately spaced and be selected based on results from the 90-day




study and from previous studies with the particular fiber. Ancillary studies should be




conducted to determine in vitro solubility and in vivo biopersistence.




       Discussion: Several approaches to determining the MAC were suggested: (1)




starting with the maximum lung burden that one would want to test, (2) starting with




the expected occupational or user exposure to be, then multiplying by an appropriate




factor (e.g., 100 or 1,000), or (3) starting with an upper limit beyond which exposure




testing would be nonsense, because, for example, some fibers show no toxicity at




levels as high as 1,000 fibers/ml. It was noted that the upper limit for the exposure




concentration would depend on fiber type, but that no one number could be




determined that applies to all fibers.
                                      23

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       Steady-state lung burden is a function of clearance rate, minute volume per




unit lung mass, deposition rate, particle solubility, and air concentration. It was




suggested that the retention half-time be determined hi a preliminary subchronic study




and used hi designing the chronic study. Biopersistence, durability, and solubility of




the fiber also are important in determining exposure concentrations.




       It was suggested that the highest exposure concentration should be a level that




would result hi significant toxicity as determined by the parameters listed above, and




that the other concentrations should be below this level. An absolute ceiling of 1,000




fibers/cm3 was discussed but was not endorsed by the group. It was noted that the




clearance rate is generally lower for fibers than for spherical particles,  and that a MAC




will be based on the actual exposure atmosphere, including both fibers and non-fibrous




particles (i.e., total inhaled objects).




       It was noted that the concept of MID is increasingly focusing on the target




organ, rather than systemic toxicity. For minimally toxic substances, the requirement is




to demonstrate that the target organ has been adequately dosed; the highest exposure




level used will affect particle clearance and produce an inflammatory response.




       It was suggested that generally the exposure concentrations should be derived




from data on particle clearance impairment and inflammatory responses observed in a




90-day study, with the MAC set at a level corresponding to the MTD at which




clearance is impaired and an inflammatory response is observed (e.g.,  -20% PMN hi




lavage fluid). Data on cell proliferation, histopathology, and increased lung weight




should also be considered. Impaired clearance could be measured either as a
                                       24

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disproportionately increased load of fibers in the lung or as impaired clearance of

separately administered test particles. However, present data probably are not

sufficient to set levels for these parameters; for each study, all the parameters in the

90-day study should be considered in setting reasonable dose levels for the chronic

study, and these should be presented to the agency before the chronic study is

conducted.


Exposure Regimen and Observation Period

       Question 1: Is the exposure regimen as specified in EPA's guidelines
       appropriate for the testing of fibers?

       Question 2: Is it necessary to recommend when final sacrifice be carried out?
       If so, when would it be?

       Conclusions and Recommendations: The chronic inhalation exposure study

with fibers should be a lifetime study, with exposure terminated at 24 months in rats

and the study terminated when survival of the  control group reaches 20%. Due to the

shorter lifespan of hamsters, their exposure duration could be shorter.

       Discussion: Because tumors resulting  from inhalation exposure to fibers tend

to be late-developing, mostly appearing after 18 to 24 months, the chronic inhalation

exposure study should be a lifetime study, with exposure terminated at 24 months.

However, waiting until every animal has died probably will not increase sensitivity

appreciably and would raise costs. Therefore,  a practical cutoff point should be

specified, such as termination of the study at 20% survival in the control group. In the

Wistar rat, 20% survival normally is reached at about 30 months.
                                      25

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       Objections were raised to "approximately 20% survival... in one of the test

exposure groups" as a criterion for termination of the study, because the highest

exposure group could reach that survival level while most of the animals in the other

groups were still alive. Statistical power is lost if termination is based on survival of

exposure groups (even the low-dose group). Thus, termination should be based on

20% survival of the control group.

       It was noted that several fibers that have been shown to be carcinogenic after a

24-month exposure also were carcinogenic after a 12-month exposure and lifetime

observation. However, to provide confidence in negative results, a 24-month exposure

should be required.  The use of a 12-month only exposure plus observation period

needs further validation.


Numbers of Animals and Interim Sacrifices

       Question: Should interim sacrifices be recommended for the testing of fibers?
       If yes, what would be an appropriate interim sacrifice schedule and design
       (e.g., number of animals per group, duration of exposure and recovery period)?

       Conclusions and Recommendations: Interim sacrifices are essential and

should be made at 3, 6, 12, 18, and 24 months in rats. The endpoints evaluated at

these times should be the same as in the subchronic study. Since hamsters do not live

as long as rats, a study of 24 months exposure may not be possible in this species.

Lung clearance of particles in live animals also should be measured at set intervals.

Investigators should be encouraged also to follow recovery in animals exposed for

shorter periods and sacrificed at the same intervals! The number of animals will depend

on the specific study design.
                                     26

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       Discussion: Sacrifice at 3 months is needed for comparison with the results of
               t
the subchronic exposure'study, and interim sacrifices at other timepoints are needed to

follow the development of lesions. Interim sacrifices also are critical for determining

the lung burden at different stages of exposure. For extrapolation, it was suggested

that interim sacrifices be evenly spaced on a logarithmic scale at 3, 6, 12, and 18

months (which is the breaking point).

       All endpoints used in the subchronic study should be measured in the interim

sacrifices, and clearance in live animals should be measured at 9 and 18 months. To

study recovery, it was suggested that shorter exposure times (e.g., 9 and 18  months)

.be built into the chronic study, with clearance tests and sacrifices  at the same intervals

as for the animals exposed for 24 months.


Use of Positive Control

       Question 1: Should a positive control be included in the chronic inhalation
       study with fibers? If not, why?

       Question 2: If yes, what might be appropriate criteria for  selecting as a
       positive control asbestos fibers with fiber size distribution similar to the test
       material?

       Question 3: How many exposure concentrations of a positive control should
       be conducted? If only one exposure concentration is used, should it be
       comparable to the highest exposure concentration of the test material in terms
       of fiber concentration or fiber lung burden? Or should it be at an exposure
       concentration expected to induce tumor effects?

       Conclusions and Recommendations: A positive control need not be included

in every study, but each new test system (including use of a different animal  species or

strain) should be validated with a positive control material. In addition, it was

generally agreed that a chronic multidose asbestos inhalation study in rats is critically
                                       27

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needed to validate and calibrate the chronic rat inhalation assay (a) for evaluation.of




the toxic and carcinogenic potential and potency of other fibers and (b) for comparison




with known human carcinogenichy data for .sbestos. It was strongly recommended by




the panelists that priority should be given to conduct such a multidose asbestos




inhalation study. The exposure levels should be based on the outcome of a subchronic




90-day inhalation study using the same criteria for deriving the MAC and MTD as is




used for the testing of other fibers. The most appropriate asbestos type for this




material would be crocidolite, because good human epidemiology data are available.




However, amosite may also be an appropriate positive control.




       Discussion: The purpose of a positive control is not to compare the effects of




the test fiber with those of a known human carcinogen, but to  confirm that the test




system is capable of detecting the effects of concern. However, the consultants




debated at length the issues of (1) whether a positive control is needed at all, (2)




whether a positive control needs to be included in every study, and (3) whether a




positive control should be used to determine the  sensitivity of the test system.




       Concerning the need for any positive control, it was argued that a test system




must be demonstrated to be capable of detecting the health effects of interest. On the




other hand, it was argued that given the size and design of chronic inhalation studies




and the kinds and amounts of data collected, a positive control would be superfluous




and an unnecessary expense.




       Among those favoring the use of positive controls, one view was that a




positive control (a fiber known to be a human carcinogen) should be.required to




validate a test system the first time it is used with a particular species or strain; if a
                                      28

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laboratory has previously produced positive results with a particular test system, a




positive control would not be required. Another view was that a positive control's




purpose is to control for variability in the test system over time and to validate each




experiment; thus, a positive control (using a standardized positive control substance)




must be included in every study to test whether the system is capable of detecting




effects under the specific conditions of the study.




       The question was raised of how many positive control dose levels should be




used. If the purpose is simply to validate the system, one dose level known to produce




effects is sufficient; this could be determined in a short-term study. If the purpose is to




test the sensitivity of the system, a multi-dose positive control is needed. Concern was




expressed by some as to whether the rat inhalation exposure system is sensitive




enough; others considered the system to be, if anything, over-sensitive. The




consultants were divided over the issue of whether a positive control is needed to test




the sensitivity of the test system.




       It was agreed that a positive control fiber need not resemble the test fiber, it




should be the most potent fiber known to produce the effect of interest. Large




numbers of positive control animals would not be necessary, and the positive control




could be run concurrently with the chronic study.




       One reason for concern about whether a positive control is needed is the fact




that some studies have failed to detect positive effects of asbestos. It was suggested




that an effort be made to understand why these studies gave negative results (e.g., the




fibers used were too short, or the dose to the lung was not confirmed). Once it is




understood what factors account for negative results with substances known to have
                                       29

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human health effects, these factors can be accounted for in study design specifications,

and a positive control should not be required. It was suggested that the "overload"

effects being looked for in the subchronic study are, in effect, a positive control, in

terms of sufficient dosing.

       The consultants were reminded that EPA generally does not require positive

controls, but the main reason for concern is that some studies with known

carcinogenic fibers have produced negat /e results. Therefore, the main issue is

whether a laboratory should be required to validate its test system. A separate issue is

the level of confidence with which a negative result can be accepted in the absence of a

positive control, even with a "validated" system.


Criteria for a Negative Inhalation Test

       Question: What might be suitable criteria for the acceptance of an inhalation
       study with fibers as negative (no tumors, achievement of a MAC, appropriately
       spaced lower concentrations, adequate animal survival, use of an appropriate
       positive control)?

       Conclusions and  Recommendations: For acceptance of the results of a

chronic inhalation exposure study with fibers as negative, the study must have been

designed and conducted according to the criteria outlined above,  the health effects of

concern must not be significantly more frequent in the exposure groups than in the

control group. In order to detect a positive effect, the power of the study should be

such that a as the type I error is controlled at 0.05 and P as the type II error is

controlled at  0.2.
                                       30

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       Discussion: It was suggested that a negative study is one in which the highest

background disease rate is used and both type I and type n error are controlled. Three

dose levels must be used and the high dose meets the criteria for MTD.


                 Session 3: Histopathologic Evaluation

       The Histopathology session was chaired by Dr. Paul Nettesheim, and Dr. John

Davis served as rapporteur. In this session, in addition to the following questions

raised in the Issue Paper, the panel discussed what needs to be examined and measured

in the histopathological evaluation.

       Question 1: Is it necessary to utilize a standardized scoring system for the  •
       evaluation of cellular changes and fibrosis in the lung?

       Question 2: The ICRP has recommended that the Wagner scoring system be
       revised to take into account the limitation [that it does not consider the mass of
       the lung tissue involved]. What specific modifications must be made before it
       can be adopted for inclusion in the test guidelines?

       Conclusions and Recommendations: The 90-day subchronic study should

include a follow-up period to assess reversibility of effects at several time points after

the end of exposure. Brpnchoalveolar lavage (BAL) is recommended for evaluation of

inflammatory response (e.g., protein content, enzymes, presence of inflammatory

cells); however, because of the expense involved (i.e., more animals are needed),

consensus was not reached on whether it should be required. Lung weight also should

be measured. Early fibrosis should be assessed through histological examination.

Cellular proliferation should be measured by the bromodeoxyuridine technique.

Although the primary purpose of the subchronic study recommended here is

range-finding for the chronic study, the panelists agreed that further development of
                                      31

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the subchronic study and collection of specific data may improve its predictive ability




enough that it could eventually replace the chronic study.




       In the chronic study, use of the Wagner scoring system to evaluate progression




of fibrosis has the disadvantage of being purely qualitative and inconsistently applied.




To  promote more quantitative evaluation, the testing guidelines should specify set




procedures for grading of lesions and for lung preparation. Further research is needed




before other quantitative histopathological methods can be recommended for




large-scale testing. However, a promising approach could be quantitation of collagen




deposits using sirius red and evaluation with polarized light.




       Neoplastic endpoints recorded should include epithelial hyperplasia,  alveolar




bronchiolization, metaplasia, adenomas, mesotheliomas, and carcinomas. Keratin cysts




should be identified as such, to permit subsequent evaluation, and it must be stated




whether or not a cyst presents evidence of invasion or dysplasia. A dissecting




microscope should be used to examine for mesotheliomas. In distinguishing  between




hyperplasia and mesothelioma,  standard diagnostic criteria should be applied to




identified lesions. Established published guidelines on the use of blinding in




histopathology should be followed, e.g., those published by the Society of American




Pathologjsts.




       Discussion: In the subchronic study, more emphasis should be placed on the




potential reversibility of non- or pre-neoplastic lesions and on how to define




pre-neoplastic lesions. One- and three-month recovery groups should be used after the




three-month exposure.
                                       32

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       Histopathology is critical for information on the location, distribution, and


nature of lesions, and new staining procedures are allowing more histopathological


effects to be seen. Histopathological evaluation should incorporate both qualitative


description of lesions and rigorous quantitation.


It was suggested that cellular proliferation be measured by the bromodeoxyuridine


technique.


       Brohchoalveolar lavage was recommended for quantitation of inflammation.


Concern about the cost of BAL vs. histopathology for assessing inflammation was
    /

raised, as the same lungs typically are not used for BAL and histopathology. It was


noted that the purpose of the subchronic study is to provide dose-ranging for the


chronic study, and that running the chronic study at inappropriate dose levels would be


even more costly. It was suggested that to conserve animals, the left lobe could be


used for histopathology and the right lobe for BAL. The question was raised of


whether BAL is really representative of the state of the lung. It was suggested that to


adequately represent the state of the lung, BAL must be done at several time points.


Although the value of BAL for quantitation of inflammation was generally agreed on,


the consultants disagreed as to whether it should be made a requirement, especially


given that it has not previously been used for this purpose.


       For the chronic study, the nature and extent of non-neoplastic lesions (fibrosis)


should be reported in more quantitative terms than "minimal," "mild," or "moderate."


However, it was acknowledged that evaluation of pathological changes can be at best


semi-quantitative. Development of a more quantitative scale based on the Wagner


system for scoring of fibrosis (e.g., based on counting and measurement of lesions)
                                      33

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was recommended; however, such an evaluation would be slow and expensive and




thus might be more appropriate for research purposes than for inclusion in testing




guidelines.




       Quantisation of incidence of non-neoplastic effects provides additional




information on degree of response, a refinement that would be useful in setting of




exposure levels and in risk assessment. If tissues are fixed and saved at the time of the




study, they can be returned to later for quantitative analysis, as warranted by other




study results. The testing guidelines should specify methodology for fixing lungs and




standard operating procedures based on GLP guidelines.




       The feasibility of other biochemical or staining techniques for quantitative




histopathology was discussed, and it was generally agreed that such methods are not




yet applicable to large-scale testing; further research is needed on quantitative




histopathology. The Sinus red method looks promising for quantitation of collagen




deposition.




       For evaluation of neoplastic effects, quantitative data are needed. However,




fibrosis often obscures diagnosis of epithelial hyperplasia. It was agreed that




measurement of tumor size is impractical. Cystic keratinizing lesions should be




recorded as such, not simply as metaplasia or adenoma. In detecting mesotheliomas,




sampling is important; a dissecting microscope may be needed to examine the covering




of the lung. A complete necropsy should be done, including careful examination of the




pleura! cavity. It was noted that in the rat, early mesotheliomas, easily missed without




careful examination, may progress to become life-threatening tumors.
                                       34

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       Tlie issue of whether slides should be read in blinded fashion was discussed. It

was suggested that EPA follow the established guidelines of the Society of

ToxicolQgic Pathologists and other such organizations, which spell out the conditions

under which bonding is called for. It was suggested that whether to examine organs

beyond the respiratory tract should be decided on a case-by-case basis, depending on

such factors as fiber solubility. In all studies, tissues should be saved.


            Session 4: Fiber Disposition and Dosimetry and
                       Interspecies Considerations

       The Disposhion/Doshnetry session was chaired by Dr. Otto Raabe, and Dr.

Fred Miller served as rapporteur.

       The session began with presentations of background information on fiber

disposition by Dr. Raabe and Dr. Yu. The following main points were made in the

presentations and ensuing discussion:

•     The air concentration is not the dose; the dose is the concentration of material
       in the lung (as defined by a given dose metric, such as the number of long
       fibers per unit surface area or gram of tissue).

•     The concentration in tissue is reflective of a dose rate. A steady-state lung
       burden, b, can be approximated as

                              xDepxAirOonc.
                In5   LtmgMass
       where f is the fraction of time exposed per week, TV4 relates to a clearance
       term, m denotes the natural logarithm, MV is the minute volume, and Dep is
       the deposition fraction.

       TV4 for the alveolar region most fikeh/ is not a single term for fibers; rather,
       there is an alveolar *TH for fast clearance and an alveolar ®TH for slower
       clearance.
                                      35

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•      For rat-respirability, the fiber must h -;ve an aerodynamic diameter of less than 3
       jim (an upper limit, not the MMAD/ Fibers of aerodynamic diameter greater
       than 3 could pose a risk to humans, but they do not enter the alveolar region in
       the rat.

•      Dissolution rate and breakage affect the clearance of fibers; dissolution of short
       fibers is more influenced by pH, because they can be phagocytized and are
       exposed to the acidic milieu in the phagolysosome.

       The following questions were raised, out not answered: At what dissolution

rate does chemical toxicity, rather than "particle/fiber" toxicity, become the issue? Or

analogously, is there a residence time beyond which concern about fibrosis or

carcinoma increases?

Pre-chronic Studies

       Question 1: Should a subchronic 90-day study be recommended prior to
       conducting the chronic study? Or should it be made optional?

       Conclusions and Recommendations: A subchronic study should be

conducted unless sufficient data are available from other studies to allow the proper

setting of chronic exposure concentrations. In any case, the burden of proof is on the

investigators.

       Question 2: What are the primary goals of the
       subchronic study?

       Conclusions and Recommendations: The primary goals of the subchronic

study are (1) to establish lung burdens and potential target sites to aid in design of the

chronic study and (2) to evaluate toxicity for a variety of important biological

endpoints. Other studies would be complementary, such as replica cast studies to

identify hot-spot locations of deposition; however, these studies are ancillary and

should not be required.
                                      36

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       Impairment of clearance should be assessed via challenge with a tagged

particle. Clearance should be assessed after the 90-day exposure period. The clearance

of the labelled particles should be measured over a period of a few months.

       Question 3: What specific data related to fiber disposition should be obtained '
       in the subchronic study?

       Conclusions and Recommendations: Data should be obtained on lung

burdens (a) to assist in establishing the chronic exposure levels or aerosol generation

changes needed to get more fibers deep into the lung and (b) to quantify aspects of

risk assessment related to .dosimetric adjustments before extrapolation. Data also

should be obtained on fiber deposition in the nasal cavity and the fiber burden in the

thoracic lymph nodes, and  collection of pleura! tissues is encouraged.

       Question 4: Are there any specific methods that should be recommended to
       measure the effects of fibers on lung clearance (e.g.,  use of radiolabeled
       particles)?

       Conclusions and Recommendations: Although no  specific method is

recommended, the method chosen should be validated. Also, it is important to

distinguish between fiber clearance and clearance of the test  particle used in the

challenge.

       Discussion (Pre-chronic Studies): The goal of the subchronic study is to gain

an idea of fiber burdens and potential target sites, for consideration in design of the

chronic study. It was suggested that a subchronic study should evaluate clearance and

factors affecting clearance, including the effect of fiber shape on macrophage function

and the effects of fiber length and diameter on breakage or dissolution. Animals should

be studied during a recovery period of up to 6 months.
                                      37

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       It was suggested that three types of studies actually are needed: a 3-month

study to establish toxicity, a 24-month to m  sure chronic effects, and a 5-day

exposure with long-term follow-up, which would be important in looking at fiber

durability in vivo. The 5-day exposure group could be a subgroup in the subchronic

study.

       Increased attention to deposition in the nasal cavity was recommended, for

information on the effects of changed aerosol composition on deposition in the deep

lung and for use in quantitative risk estimates. More detailed information on the lymph

nodes also would be desirable. Studies of aerosol deposition in replica casts or models

of the lungs and the nasal cavity of laboratory animals would be desirable to

complement the subchronic  study. A major difficulty in modeling is the lack of

information on deposition in the nasal-pharyngeal region.

       An exception to the  requirement for a preliminary subchronic study could be

made in cases where enough data already exists to allow determination of dose levels

for the chronic study. If a subchronic study is not done, attainment of a MID must be

assessed at three months; if a MTD has not been attained, additional higher dose

groups should be added to the study to assure that the MTD was reached.

Lung Burden Analysis

       Question 1: Should lung burden analysis be included in the subchronic and
       chronic studies?

       Conclusions and  Recommendations: Lung burden analysis should be

included in the subchronic and chronic studies even if extra animals need to be added

to the study.
                                      38

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       Question 2: If yes, should the procedure as recommended by the ICRP be
       adopted? As discussed on page 36 of the background document, there are data
       to indicate that fiber burden data based only on the accessory lung lobe may
       not be representative of the whole lung because of nonuniform pattern of
       deposition. In view of these findings, what changes should be recommended?

       Conclusions and Recommendations: The panel does hot endorse adoption of

the procedure recommended by the ICRP. For fiber burden analysis, one of the two

lungs (left or right) should be used, rather than only the accessory lobe. It may be

possible to determine a correction factor during subchronic studies that would allow

the use of only one lobe in the chronic studies. However, disease development could

change deposition patterns and invalidate a correction factor. Five to six animals per

exposure group should.be studied at each time point.

       Question 3: Should any specific methods for lung ashing be recommended?

       Conclusions and Recommendations: Rather than "lung ashing," the proper

term is "lung digestion," because the guidelines will apply to other types of fibers in

addition to man-made vitreous fibers. No specific lung digestion method is

recommended. The investigator must show that the fibers are not affected by the

method used to harvest them from the lung tissue.

       Question 4: How often should lung burden analysis be performed (at interim
       and final sacrifice time points?)?

       Conclusions and Recommendations: Lung burden analyses should be

required after 3, 6, 12, 18, and 24 months of exposure. Pleura! burden analysis is not

recommended at this time. However, in view of the potential use of the pleura! data in

quantitative risk assessment and the cost  of repeating studies, investigators should be

encouraged to collect pleura! burden samples and keep them available for future

analysis.

                                     39

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       Discussion (Lung Burden Analysis): Lung burden analysis should be




required, despite the fact that it requires extra animals (because the lung is destroyed).




To reduce the numbers of animals required, one side of the lung could be used for lung




burden analysis and the other for pathology. Although this would entail assumptions




about disposition patterns, use of the whole lung for lung burden analysis would be a




luxury. Lung burden and fiber size distribution should be reported as number of fibers




per gram of dry lung tissue. The burden should be extrapolated to the whole lung.




       The method for lung burden analysis should be left open, but it must be




validated. At least half the lung should be used, and at least five or six animals per




exposure group. This sample size is sufficient to estimate the mean, though not the




population variability.  The term "digestion" should be used instead of "ashing." The




method for lung digestion should be left open, but, as lung digestion can damage




fibers, the method must be validated. Lung burden analysis should be performed at 3,




6, 12, 18, and 24 months. These time points are needed to avoid reliance on the high




dose for extrapolation, because the breaking point is at 18 months. A research need is




to validate  the use of the accessory lobe for lung burden analysis.




       With respect to mesotheliomas, a major issue is whether fibers reach the




pleura. Collection of pleura! burden samples (if only to be preserved for future




analysis) would be highly desirable, but should not be a requirement because of




considerable cost and  difficulty in assessing pleura! tissues.
                                       40

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Chronic Study

           Question: What additional information on fiber disposition should be
           obtained in the chronic inhalation study?

       Conclusions and Recommendations: At an early and late timepoint (e.g., 9

and 18 months of exposure) it is desirable that animals are tested for impaired

clearance of a pulse of a small labeled spherical particle (i.e., one whose deposition

would be primarily in the alveolar region).

       It would be desirable, though not required, to  obtain data on translocation, hi

order to build a more detailed compartmental model for fiber disposition. This would

.involve obtaining data on free fibers versus those associated with macrophages,

alveolar interstitial burden, and so forth. An optional item would be to remeasure the

physical density of fibers recovered from animals after various lengths of exposure.

These measurements could allow the extent of dissolution and leaching of the fibers to

be inferred.

       Discussion (Chronic Study): Measuring the density of recovered fibers

including their chemical composition would be desirable, for establishment of a

dosimetry model, but the measurements are expensive and not important for risk

assessment. Also desirable, but not required, would be data on translocation of

different types of fibers, to provide information on compartmentalization of deposited

fibers. Compartmentalization is important for estimating human dose levels.
                                      41

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Dosimetrv and Intersoecies Considerations

       Question 1: Which additional dc 
-------
     Session 5: Mechanisms of Toxicity and Carcinogenicity and
                   Biomarkers of Toxicologic Effects

       The Mechanisms/Biomarkers of Effects session was chaired by Dr. Agnes

Kane, and Dr. David Warheit served as rapporteur.

Mechanisms of Toxicitv and Carcinogenicitv

       Question: Should any mechanistic studies be recommended? Which and when
       (prior to the chronic study, in parallel with the chronic study, and/or
       subsequent to the chronic study?)?

       Conclusions and Recommendations: No mechanistic studies are

recommended at this time. However, investigators should be encouraged to obtain

mechanistic information as far as possible during the course of subchronic or chronic

inhalation studies. A high research priority should be to determine whether fiber

carcinogenesis is a direct effect or an indirect effect related to inflammation. It was

suggested that the most promising approach for obtaining mechanistic information is

to isolate target cells after in vivo exposure for use in subsequent in vitro studies.

       Other priorities for research include (1) development of short-term in vivo

assays With ex vivo/in vitro investigations in appropriate target cell populations, (2)

investigation of oncogenes and tumor suppressor genes in human and rodent tumors,

(3) development of transgenic animal models, (4) species comparisons of fiber-induced

pulmonary effects in vivo and in vitro, and (5) use of pleura! lavage to evaluate

predictive markers of response.

       Discussion: As background, Dr. Kane summarized the current hypotheses

concerning mechanisms of fiber effects and the types of mechanistic studies that have

been conducted. She noted the. importance of determining the relevance of study
                                     43

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findings to humans and the potential for mechanistic study methods to be developed as




screening assays.




       With respect to short-term genotoxicity assays, it was noted that most positive




studies with fibers have been obtained by assessing responses to DNA damage (e.g.,




assays for unscheduled DNA synthesis). Results with point mutations have generally




been negative or borderline. Recent studies in one laboratory detected multilocus




deletions induced by asbestos. Assays for aneuploidy have shown some sensitivity to




fiber length. It is not yet clear whether these in vitro genotoxicity assays with fibers are




relevant to in vivo animal exposures or to human health, or whether they can be




developed for screening of fibers. The need for a good negative control fiber was




noted. Any assay developed for screening would have to be standardized. Given that




tumor formation is a chronic effect, it is important to note that short-term assays do




not address questions of fiber durability. For dosimetry, realistic in vivo doses to cells




need to be determined. The appropriateness of in vitro assays may also depend on the




cell type.




       The question was raised of how to separate genotoxic effects due to the




fibrous nature of a substance from effects due to its chemical composition. Hypotheses




concerning physical versus chemical mechanisms of fiber carcinogenicity have  not been




adequately tested. Furthermore, mechanisms may differ between different types of




fibers, and indirect mechanisms may be important. Understanding of mechanism will




influence the type of risk assessment model to be applied  in evaluating fiber effects.




Studies characterizing mutation in vivo (e.g., aneuploidy in rat  and human mesothelial




and bronchial epithelial cells) are needed to explore potential mechanisms. Mechanistic
                                       44

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studies are complicated by species differences in different target cells as well as




variability between cell lines derived from different human donors.




       The possibility of using short-term animal studies for screening was raised.




Because pleura! disease is specific to fibers, pleura! lavage might be a promising




approach. Short-term inhalation bioassays in the rat and hamster, looking at such




factors as biopersistence, cellular proliferation, and inflammatory response, might be




encouraged as a preliminary step towards development of in vitro assays. Research is




needed to correlate preneoplastic and neoplastic markers with tumor development.




Important research goals are to understand whether rats and humans have a common




pathway to tumor development (e.g., by looking at major oncogenes or tumor




suppressor genes) and to understand the possible role of the inflammatory response in




tumor formation. Transgenic mice or rats could be used to look at expression of




specific genes or to evaluate direct versus indirect mechanisms.




       It was suggested that the government issue a Request for Proposals for




research on mechanisms of fiber carcinogenichy. It was noted that NIEHS and NTH




are currently funding grants for research on mechanisms of toxicity of non-fibrous




particles and fibers.




       Earlier recommendations that tissues from chronic studies be saved and




preserved for future analysis were reconfirmed; in particular, tissues could be frozen




for later molecular studies. If retrospective studies are deemed important, priorities,




standards, and requirements for necropsy, fixing, and archiving need to be specified.
                                      45

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Biomarkers of Toxicologic Effects

       Question 1: Which specific biomarkers of toxicity and carcinogenicity should
       be measured in the subchronic study (e.g., BALF analysis, cytotoxicity, cell
       proliferation)?

       Question 2: Should BALF analysis be made mandatory for the chronic study?

       Question 3: If yes, should the procedure as specified in the ICRP's protocol be
       adopted? Are there any modifications that should be considered?

       Conclusions and Recommendations: The subchronic study should include

analysis of BAL fluid and measurement of cell proliferation. BAL fluid analysis should

be required in the chronic study. Some modification of the ICRP protocol should be

adopted.


                      Session 6: Screening Battery

       The Screening Battery session was chaired by Dr. Kevin Driscoll, and Dr.

Ernest McConnell served as rapporteur.

       Question 1: Recognizing that no single screening study can accurately predict
       the in vivo responses from long-term exposure to fibers, can Tier n and Tier
       HI types of studies — as defined in the CITT workshop proceedings — be
       used to screen and set priorities with regard to confirmatory testing in a
       chronic study to obtain more definitive information for risk assessment
       purposes? If not, why not?

       Conclusions and Recommendations: Appropriately designed Tier n (in

vitro) and Tier HI (short-term in vivo) studies can provide useful information to assess

the relative potential of fibrous materials to cause toxicity in the lung and associated

tissues. Along with other information (e.g., from Tier I assessments), data from a

battery of Tier n and IE studies can provide key information to prioritize materials for

further chronic testing. At present, no single assay or battery of short-term assays can
                                     46

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predict the outcome of a chronic inhalation bioassay with respect to carcinogenic

effects.

       Question 2: If yes, what specific tests or combinations of tests can be utilized
       in this screening battery?

       Conclusions and Recommendations: Tier n In Vitro Tests.

Solubility/durability can influence the lung's response to long-term inhalation of fibers.

In most instances, rates of in vitro and in vivo solubility correlate well, although the

absolute rates may differ. Therefore, in vitro assays providing information on fiber

•solubility/durability can provide useful information for prioritizing groups of fibers for

further testing.

       It was suggested that in vitro solubility alone could not be used to rule out

further testing,  and that fibers should be evaluated man in vivo test system for toxicity

and in vivo dissolution.

       Although other characteristics of fibers that can be examined in acellular in

vitro tests were discussed, no general agreement was reached on their value as routine

tests for assessing the potential of fibers to cause toxicity.

       In vitro cell or tissue culture assays can potentially provide useful information

on fiber toxicity; however, these systems are not yet well enough validated or

understood to be recommended for routine use in screening fibers toxicity.

       Tier n (Short-Term In Vivo Studies). There is no standardized protocol for

short-term respiratory tract  exposures to fibers (i.e., less than 3-month exposure)

followed by characterization of the lung response periodically over several weeks.

Nonetheless, this type of study is a useful tool for assessing the relative ability of fibers

to produce nonneoplastic effects (e.g., inflammation, cell proliferation, fibrosis) hi the

                                        47

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lung. Thus, information from short-term in vivo studies, combined with data from Tier




I studies^ would be useful for prioritizing me nals for longer-term studies. Short-term




screening studies should include (but not be united to) analysis of BAL fluid for




markers of cell injury and inflammation, bistopathology, and assessment of lung fiber




dose. Study design should include assessments of dose-response relationships and,




when possible, comparisons to physically and chemically similar "control" fibers for




which chronic lung effects already have been evaluated. Exposure concentrations




should include at least one level that elicits significant lung effects, to provide a basis




for comparison of both the nature and persistence of the response to the fibers. In




addition, it would be useful to assess fiber biopersistence following a short-term




exposure (e.g., a five-day exposure, followed by monitoring for several weeks).




       Discussion: Solubility/durability of fibers was recognized as an important




characteristic influencing the behavior of fibers in the lung. The relative in vitro




solubility of fibers has been related to their relative in vivo dissolution rates (although




actual in vivo dissolution rates cannot be accurately predicted from in vitro rates).




Highly soluble fibers are likely to be less toxic than less-soluble ones (however,




solubility per se is not considered the only determinant of toxicity). Evaluation of fiber




solubility and durability characteristics in vitro provides information useful for




prioritizing fibers for further testing.




       Although fairly well defined in vitro tests for fiber solubility/durability are




being used by several laboratories, no standardized protocol has been agreed upon.




For standardization of approaches, control fibers with known solubilities/durabilities




are needed;  one or more standard fibers should be established and made available from
                                       48

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a repository. It was noted that changing the flow rate in solubility tests changes the




extremes of the scale, but not the ranking of fiber solubility. Also, for each




experimental system, a flow rate exists above which the results for solubility are




independent of the test system. The test protocol in the background document, "Draft




Protocol for/w Vitro Acellular Tests - Durability," was characterized as preliminary; it




represents a guideline for issues that should be addressed in development of a testing




method. It was noted that some companies are currently developing an in vitro test of




fiber breakage rates using SEM. The question was raised whether fiber breakage




should be tested in a cellular system; it was noted that acellular systems may be better




developed.




       For screening, a key question is whether, and over what time frame, fibers




dissolve in the lung. Knowing how dissolution occurs was considered by some




panelists to be useful but not essential. It was suggested that a threshold dissolution




rate could be established beyond which the dissolved fiber should be considered as a




chemical rather than as a fiber in development of a safety testing program. It was




noted that both biopersistence and bioactivity (i.e., toxicity) need to be taken into




consideration when materials are prioritized for further testing.




       It also was suggested that acellular in vitro systems should be used to evaluate




fibers for surface reactivity. The ability of fibers to generate or adsorb free radicals can




be assessed in vitro; this property of some fibers can influence their toxicity and is




worth evaluating.




       Tier n tests should also include in vitro cellular response assays. However,




although such assays can provide useful information, methods have not been
                                       49

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standardized, and little guidance can be offr  ?d to EPA at this time regarding the use




of specific tests. Problems with existing in    o cell response data include the lack of




adequate characterization of test fibers. Controlled in vitro cellular assays are




considered very important for delineating i  -hanisms of fiber toxicity and




carcinogenicity. However, in vitro cellular assays for toxicity screening need to be




based on an understanding of mechanisms operating in vivo. Development of




mechanistically based cellular response assays is an important research need.




       Tier TTT short-term in vivo exposure studies would provide useful information




for a preliminary assessment of the relative potential of fibers to produce




non-neoplastic effects such as inflammation, cell proliferation, and fibrosis in the lung.




Data from short-term studies could be used along with other information to prioritize




materials for further testing. To the extent that non-neoplastic effects contribute to




carcinogenic effects, short-term assays may provide information on potential




carcinogenicity; however, more information is needed  before these types of




assessments can be made. A typical short-term study is envisioned to entail a short-




term inhalation or intratracheal instillation exposure followed by periodic assessment




of lung response over six to eight weeks. Short-term toxicity studies for screening and




rank-ordering materials would include an assessment of dose response (with at least




three dose levels), using at least one exposure concentration that  results in lung




responses. The inclusion of an effect level was considered critical, to allow comparison




between fibers of both the nature and the persistence of lung effects. Endpoints




examined in short-term screening studies should include analysis of BAL fluid for




indicators of lung injury and inflammation, histopathology, and an assessment  of fiber
                                       50

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dose. Other endpoints that provide insight into mechanisms such as assessments of cell

proliferation and fiber biopersistence should be considered. A five-day inhalation

exposure, followed by monitoring for several months, was suggested for studying

biopersistence. To the extent possible, studies should include control fibers that are

physically and chemically similar to the test fibers and for which chronic lung effects

have already been characterized. In design of such short-term studies, it should be kept

in mind that the objectives are to screen for toxicity, to assess relative effects, and to

prioritize materials for long-term testing. These studies should be time and cost

effective.

       Question 3: Given that in vivo studies using non-inhalation methods of
       exposure (e.g., intraperitoneal injection, intratracheal instillation studies) have
       been proven useful in identifying the potential health hazard to humans, should
       they be considered acceptable as an alternative screening test or an adjunct to
       short-term inhalation studies in a screening battery?

       Conclusions and Recommendations: Intratracheal Instillation.

Intratracheal instillation was considered by a majority of the panel members to be an

acceptable alternative to inhalation exposure for short-term screening studies to assess

the relative biopersistence and relative non-neoplastic toxicity of fibers  in the lung

provided low doses are used. Intratracheal instillation allows a known amount of test

material to be administered to the lung in  a manner not requiring the resources (i.e.,

exposure facility and level of research funding) needed for inhalation exposure.

Moreover, human-respirable fibers not respirable by the rat may be evaluated via

intratracheal instillation, although care should be taken to avoid higher doses of longer

fibers which may result in clumping. However, the intratracheal instillation delivers

materials at a much higher dose rate than does inhalation, and care must be taken to
                                       51

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ensure that the responses observed after intratracheal instillation are comparable to




what would be expected after inhalation. Fiber doses should be low enough to




minimize problems of fiber clumping and overwhelming of lung defense mechanisms.




It would be useful to include a "control" fiber for which the lung tissue response after




inhalation has already been characterized to demonstrate that intratracheal instillation




produces a response similar to that expected after inhalation. The majority thought




that intratracheal instillation should not be recommended for assessing the




carcinogenic potency in long term studies.




       Intraperitoneal Injection. Intraperitoneal injection studies can provide




information on the interaction of fibers with mesothelial cells. However, for screening




or rank ordering the potential toxicity of fibers hi the lung based on intraperitoneal




injection studies, the behavior in the lung (e.g., clearance, translocation) of the fibers




being evaluated must be taken into account. The dose levels for intraperitoneal




injection studies should be selected so  that a MTD is achieved, but not exceeded




several-fold. What constitutes a MTD  for i.p. study remains to be defined. There was




little discussion on this subject.




       Discussion: A majority of the panel supported the use  of intratracheal




instillation, in addition to inhalation, as a method of exposure in short-term screening




studies for rank-ordering fibers with respect to their non-neoplastic effects in the lung.




Intratracheal instillation provides a time- and cost-effective exposure method for




screening studies. Because administration of bolus doses of fibers much over 1 mg




may introduce artifactual effects, such as clogging of terminal bronchioles, it was




emphasized that low doses should be used. Intratracheal instillation may be especially







                                       52

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useful in screening non-rat-respirable fibers that are respirable by humans. Control




fibers that have been previously examined in inhalation studies should be included.




Similarities in the lung response to the control fiber after intratracheal instillation and




after inhalation would provide added assurance that intratracheal instillation did not




result in artifactual effects.




       It was suggested that intratracheal instillation could also be used to assess




biodurability, if clearance rates following intratracheal instillation and inhalation




exposure are shown to be similar. This method of exposure can assure equal lung




burdens. However, sizing of fibers would be important. Reservations were expressed




about combining durability and toxicity endpoints in the same screening-level study. It




was suggested that if further validated and standardized, intratracheal instillation




studies might eventually replace short-term inhalation exposure studies, although there




was no consensus among the experts on this suggestion. However, additional work is




needed to determine whether clearance half-times are similar. It was noted that for




some types of fibers, intratracheal instillation produces a different pattern of tumor




formation. In this respect, although intratracheal instillation may be useful for short-




term screening studies, most of the panelists were of the opinion that it may not be




appropriate for assessing carcinogenic effects in long-term studies. However, some




panelists thought it to be useful for evaluating the carcinogenic potential of a fiber.




       It was suggested that exposure by intraperitoneal injection also be considered




for use in short-term screening tests. Response would be characterized using the same




endpoints as in short-term inhalation or intratracheal instillation screening studies.




Intraperitoneal injection studies can provide information on the interaction of fibers
                                        53

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with mesothelial cells. Additional information — e.g., fiber translocation from alveolar

to pleura! sites — is needed to validate intraperitoneal injection tests for routine use in

screening fibers.

       Question 4: Appendix D of the background document is the ICRP's draft
       protocol for intracavitary testing (i.p. study). Would a positive finding using
       this protocol constitute a potential hazard to humans, or would a positive.
       finding need to be followed by a chronic inhalation study to confirm the
       hazard?

       Question 5: As discussed on page 42 of the background document, questions
       have been raised as to the appropriateness of using a large total dose of 250
       mg in the i.p. test. The issue of the MTD for i.p. studies requires further
       discussion (if time permits).

       Conclusions and Recommendations: The intracavitary test is primarily useful

in conjunction with inhalation exposure studies and as a research tool. Doses  currently

used are extremely high, and dose-response information needs to be developed,

specifically for very low dose levels.  Interpretation of the intracavitary test with

respect to assessment  of potential health hazards requires additional information —

specifically, whether the fibers tested by the intracavitary method would ever  reach

mesothelial tissue after inhalation exposure and, if so, how the dose and the

dimensions of fibers reaching the mesothelium after inhalation compare to the material

tested by the intracavitary method. The results from intracavitary testing may be useful

for prioritizing materials for chronic testing.

       Discussion: This system has not been validated for quantifying penetration of

fibers to the pleura! cavity and their survival there.  This test may identify a hazard for

mesothelioma, but it cannot eliminate the need for inhalation exposure testing for lung

cancer. Moreover, fiber properties responsible for carcinogenic effects may be

evaluated by this technique. If the intraperitoneal injection test shows that a fiber has

                                       54

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carcinogenic potential in the mesothelium, the more-expensive inhalation exposure




testing can demonstrate whether the fiber actually reaches the mesothelium. However,




more information is needed, linked to dosimetry, to determine how useful the test may




be for hazard identification (the dose levels currently used are too high for the results




to be meaningful for this purpose, especially as the dose is delivered instantaneously,




rather than over a lifetime. A small minority of the panelists thought that does used in




the i.p. test are appropriate since they adequately identified carcinogenic fibers in the




past). Future testing should be done at much lower dose levels and should establish




dose-response effects. Criteria should be established for a MTD via IP administration.
                                       55

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 APPENDIX I
Workshop Agenda
    I- 1

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  WORKSHOP ON CHRONIC INHALATION  OXICITY AND CARCINOGENICITY
               TESTING OF RESPIRABLE i- IBROUS PARTICLES

                            WORKSHOP AGENDA

                     Workshop Chair, Dr. Giinter Oberdorster


Monday                 May 8,1995


 8.00 AM                Registration

 9.00 AM                Opening Remarks
                        Charles Auer, Director, Chemical Control Division, USEPA

 9.10 AM                Overview of Issues
                        Dr. Vanessa Vu, USEPA

 9.20.AM                Discussion of Issues on: "Exposure"
                        Chair, Dr. James Vincent

10.30 AM                Coffee Break

11.00 AM                Discussion of Issues on: "Exposure" (cont.)

12.00 PM                Summary of Discussions
                        Rapporteur, Dr. David Bernstein


12.30 PM                Lunch


 1.30 PM                 Discussion of Issues on  "Study Design"
                        Chair, Dr. Giinter Oberdorster

3.30 PM                 Coffee Break

4.00 PM                 Discussion of Issues on: "Study Design" (cont.)

5.00 PM                 Summary of Discussions
                        Rapporteur, Dr. Neil Johnson

5.30 PM                 Adjourn
                                   1-2

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Tuesday
 May 9,1995
 830AM


10.30 AM


11.00AM

1130AM
 Discussion of Issues on: "Histopathology"
 Chair, Dr. Paul Nettesheim

 Summary of Discussions
 Rapporteur, Dr. John Davis

 Coffee Break

 Discussion of Issues on: "Disposition/Dosimetiy*
 Chair, Dr. Otto Raabe
1230 PM
Lunch
 130PM


 230PM


 3.00PM

 3.15PM
Discussion of Issues on:  "Disposition/Dosimetry"
(conk)

Summary of Discussions
Rapporteur, Dr. Fred Miller

Coffee Break

Discussion of Issues on:  "Mechanisms/Biomarkers of Effects'
Chair, Dr. Agnes Kane
5.15PM
Summary of Discussions
Rapporteur, Dr. David Warheit
5.40 PM
Adjourn
                                       1-3

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Wednesday               May 10,1995
 8.30 AM                Discussion of Issues on: "Screening Battery"
                         Chair, Dr. Kevin Driscoll

10.00 AM                Coffee Break

10.30 AM                Discussion of Issues on: "Screening Battery" (eont.)

12.00 PM                Summary of Discussions
                         Rapporteur, Dr. Ernest McConnell

12.30 PM                Workshop Chair's Summary
                         Dr. Gunter Oberdorster

 1.00 PM                 Adjourn
                                      1-4

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            APPENDIX H





List of Workshop Participants/Consultant Panel
               n-1

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      WORKSHOP ON CHRONIC INHALATION TOXICITY AND CARCINOGENICITY
                  TESTING OF RESPIRABLE FIBROUS PARTICLES

                       INTERAGENCY STEERING COMMITTEE
Dr. Vanessa Vu [Chair]
U.S. Environmental Protection Agency
Office of Pollution Prevention
 and Toxics
Telephone:   (202) 260-1243
FAX:        (202) 260-1283

Dr. Paul Baron
National Institute for Occupational
 Safety and Health
Telephone:   (513) 841-4278
FAX:        (513) 841-4500

Dr. Cart Barrett*
National Institute of Environmental
 Health Sciences
Telephone:   (919) 541-3464
FAX:        (919) 541-7784

Dr. David Dankovic
National Institute for Occupational
 Safety and Health
Telephone:   (513) 533-8329
FAX:         (513) 533-8588

Dr. David Lai
U.S. Environmental Protection Agency
Office of Pollution Prevention
 and Toxics
Telephone:   (202) 260-6222
FAX:         (202) 260-1279

Dr. Ted Martonen
U.S. Environmental Protection Agency
Office of Research and Development
Health Effects Research Laboratory
Telephone:   (919) 541 -7875
FAX:        (919) 541-4284

Dr. William Pepelko
U.S. Environmental Protection Agency
Office of Research and Development
Telephone:   (202) 260-5904
 FAX:        (202) 260-3803
Dr. Joseph Roycroft
National Institute of Environmental Health
 Sciences
Telephone:   (919) 541-3627
FAX:        (919) 541-4714

Dr. Loretta Schuman
U.S. Department of Labor
Occupational Safety and Health
Administration
Telephone:   (202) 219-7111
FAX:        (202) 219-7125
       Did not attend workshop.
                                       n-2

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        WORKSHOP ON CHRONIC INHALATION TOXICITY AND CARCINOGENICITY
                    TESTING OF RESPIRABLE RBROUS PARTICLES
                                    Consultants
 Dr. Gunter Oberdorster [Chair]
 Department of Environmental Medicine
 University of Rochester
 575 Elmwood Avenue
 Rochester,  NY 14642
 Telephone:   (716) 275-3804
 FAX:         (716) 256-2631

 Dr. David Bernstein
 40 ch. de la Petite-Boissiere
 Geneva, Switzerland CH 1280
 Telephone:   011 -41 -22-735-0043
 FAX:         011-41-22-735-1463

 Dr. John M. G. Davis
 Institute of Occupational Medicine, LTD.
 Department of Pathology
 8 Roxburgh Place
 Edinburgh, Scotland EH 89 SU
Telephone:    011 -44-31 -667-5131
 FAX-         011 -44-31 -667-0136

 Dr. Kevin Driscoll
 Procter and Gamble Co.
One Procter and Gamble Plaza
Cincinnati, OH 45202
Telephone:    (513) 627-2360
 FAX:         (513) 627-0400

 Dr. David Groth
 602 Main Street
 Cincinnati, OH 45202
 Telephone:   (513) 579-1361
 FAX:         (513) 579-1476

 Dr. Thomas W. Hesterberg
 Health, Safety and Environmental Department
 Schuller International, Inc.
 10100 West Ute Avenue
 Littleton, CO  80127
 P.O. Box 5108
 Denver, CO 80217-5108
 Telephone:   (303) 978-3119
 FAX:        (303) 978-2358
 Dr. David Johnson
 University of Oklahoma
 801 Northeast 13th, Room 413
 Oklahoma City, OK 73190
 Telephone:   (405) 271-2070
 FAX:        (405)271-1971

 Dr. Neil F. Johnson
 Inhalation Toxicology Research Institute
 Bldg. 9217, Area Y, KAFB East (87115)
 P.O. Box 5890
 Albuquerque, NM 87185
 Telephone:   (505) 845-1189

 Dr. Agnes Kane
 Brown University
 BFomedical Center, Box 6R
 Providence, Rl 02912
Telephone-   (401) 863-1110
 FAX:        (401) 863-2044

Dr. Ernest E. McConnell
3028 Ethan Lane
Raleigh, NC 27612
Telephone:   (919) 848-1576
 FAX:        Same

 Dr. Fred Miller
 Chemical  Industry Institute of Toxicology
 6 Davis Drive
 Research  Triangle Park, NC  27709
 Telephone:   (919)558-1268
 FAX:        (919) 558-1300

 Dr. Owen Moss
 Chemical  Industry Institute of Toxicology
 6 Davis Drive
 Research Triangle Park, NC 27709
 Telephone:    (919) 558-1268
 FAX:         (919)558-1300
                                       II-3

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                                     Consultants
                                      (Continued)
 Dr. Hartwig Muhle
 Fraunhoter-lnstitut for Toxikologie und
 Aerosoitor
 Nikolai-Fuchs-Str. 1
 30625 Hannover, Germany
 Telephone:   011 -49-511 -535-451
 FAX:        011-49-511-525-155

 Or. Paul Nettesheim
 National Institute of Environmental Health
 Sciences
 111 T.W. Alexander Drive
 Building 101, MD-D201
 Research Triangle Park, NC 27709
 Telephone:   (919) 541-3540
 FAX:        (919) 541-4133

 Dr. Friedrich Pott
 Medical Institute
 Dusseldorf, Germany 40225
 Telephone:   011 -49-211 -338-9304
 FAX:         011-49-211-319-0910

 Dr. Otto Raabe
 University of California
 Institute of Toxicology and Environmental
 Health
 Davis, California 95616
Telephone:   (916)752-7754
 FAX:         (916) 752-5300

 Dr. James H. Vincent
 University of Minnesota
 Division of Environmental and Occupational
 Health
 School of Public Health
 Box 807 Mayo, 420 Delaware Street, S.E.
 Minneapolis, MN  55455
 Telephone:   (612) 624-2967
 FAX:        (612) 626-0650
Dr. David Warheit
Stein Haskell Research Center
Dupont Haskell Laboratory
P. O. Box 50, EJkton Rd
Newark, DE 19714
Telephone:   (302) 366-5322
FAX:       (302)366-5207

Dr. C.P. Yu
Department of Aerospace
State University of New York
Buffalo, NY
Telephone:   (716)  645-2593
FAX:        (716)  688-3875
                                         n-4

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       WORKSHOP ON CHRONIC INHALATION TOXICITY AND CARCINOGENICITY
                   TESTING OF RESPIRABLE FIBROUS PARTICLES

                              LIST OF PARTICIPANTS
 Dr. Charles Axten
 44 Cana Center Plaza, #310
 Alexandria, VA 22314
 Telephone:   (703) 684-0084
 FAX:        (703) 684-0427

 Mr. Michael E. Beard
 U.S. EPA-MD 77
 Research Triangle Park, NC 27711
 Telephone:   (919) 541 -2623
 FAX:        (919) 541-0239

 Dr. Jean BIgnon
 INSERM 139 France
 CHV - Henri Mondor
 Creteil 94010 CEDEX
 FAX:        011-33-149-81-3533

 Dr. Gary Boorman
 NIEHS
 111 T.W. Alexander Drive
 Research Triangle Park, NC 27709
 Telephone:   (919) 541 -5716
 FAX:        (919) 541-4714

 J. Robert Buchanan
 NIEHS MD-AO-02
 111 T.W. Alexander Drive
 P.O. Box 12233
 Research Triangle Park, NC 27709

 Dr. John Bucher
 NIEHS
 111 T.W. Alexander Drive
 Research Triangle Park, NC 27709
 Telephone:   (919) 541-4532
 FAX:        (919) 541-0295

 Dr. Michael Butler
 H.R.C.
 51 Monroe Street, Suite 1402
. Rockville, MD 20850
 Telephone:   (301) 762-8823
 -FAX:        (301) 762-1588
 Dr. James Cason
 1625 Buffalo Avenue
 Niagara Falls, NY 14302
 Telephone:   (716) 278-2062
 FAX:       (716) 278-2006

 Dr. Jerry Chase
 10100 W. Ute Avenue
 Littleton, CO 80217
 Telephone:   (303) 978-3119
 FAX:       (303) 978-2358

 Dr. David Chan
 Texas Instruments, Inc.
 P.O. Box 655012, MS 81
 Dallas, TX 75265
 Telephone:   (214)995-7204
 FAX:        (214) 995-7004

 Dr. Richard Cunningham
 240 Elizabeth Court
 Shelbyville, IN 46143
 Telephone:    (317) 398-3675
 FAX:        (317) 398-4434, X8801

 Dr. Raymond David
•Eastman Kodak Company
 Rochester, NY 14652-6272
 Telephone:   (716) 588-4763
 FAX:       (716) 722-7561

 Dr. Jim Dunn
 Amoco Corporation
 Atlanta, GA
 Telephone:   (404) 944-4740
 FAX:       (404) 944-4745

 Dr. Jeffrey Everitt
 CUT
 Research Triangle Park, NC
 Telephone:   (919) 558-1268
 FAX:       (919) 558-1300
                                  II-5

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                              LIST OF PARTICIPANTS
                                   (Continued)
Marian Frolley
AYSA
P.O. Box 99
Gastonia, NC 28053

Dr. Tom Goehl
NIEHS
111 T.W. Alexander Drive
Research Triangle Park, NC 27709
Telephone:   (919) 541-7961
FAX:        (919) 541-0273

Dr. John Hadley
Owens Coming
2790 Columbus Road. R 116
Evansville, OH 43023
Telephone:   (614) 321-7228
FAX:        (614) 321-7529

Dr. Ron Howell
NC DEHNR - Asbestos Board
P.O. Box 27687
Raleigh, NC 27511
Telephone:   (919)733-0502
FAX:        (919) 733-8493

Dr. William Jameson
NIEHS
111 T.W. Alexander Drive
Research Triangle Park, NC 27709
Telephone:   (919) 541-4096
FAX:        (919) 541-2242

Dr. William P. Kelly
Carbom DVM
P.O. Box 808
Niagara Falls, NY 14302
Telephone:   (716) 278-2187
FAX:        (716) 278-2319

Dr. Alan Koenig
Center for Applied Engineering
10301 9th Street, N.
St. Petersburg, F1_ 33716
Telephone:   (813) 578-5340
FAX:         (813) 578-4280
 Dr. Kl Poong Lee
 Atsuka Chemical Co.
 747 Third Avenue, 26th Floor
 New York, NY 10017
 Telephone:   (302) 454-7777
 FAX:        (212) 826-5094

 Dr. Ron Melnick
 NIEHS
 111 T.W. Alexander Drive
 Research Triangle Park, NC 27709
 Telephone:   (919) 541-4142
 FAX:        (919)541-7666

 Dr. Dan Morgan
 NIEHS
 111 T.W. Alexander Dr.
 Research Triangle Park, NC 27709
Telephone:   (919) 541-2264
 FAX:        (919) 541-0356

Dr. Betty Muchak
PB Associates
714 Ninth Street, Suite G-3
Durham, NC 27709
Telephone:  (919) 286-7193
FAX:       (919) 286-7369

Dr. Rod Musselman
USG
 124 S. Franklin
Chicago, IL 60606
Telephone:   (312) 606-5854
 FAX:        (312) 606-3906

 Dr. Roger Reinhold
 Allied Signal Inc.
 101 Columbia Road
 Morristown, NJ 07962-1139
 Telephone:   (201) 455-2590
 FAX:        (201) 455-5408
                               II-6

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                             UST OF PARTICIPANTS
                                   (Continued)
Dr. Nagui Rizkallah
University of North Carolina
212 Fmley Golf Course Road
Chapel Hill,  NC 27514
Telephone:   (919) 962-5720
FAX:        (919)962-0227

Dr. Philip Robinson
US EPA-CMD/Oppts (7404)
401 M. Street, SE
Washington, DC 20460
Telephone:   (202) 260-0001
FAX:        (202) 260-3910

Dr. Christiana Rydman
Parkex Insulation
54186 Skevde
Sweden
Telephone:   011 -46-500-469-296
FAX:        011 -46-500-469-297

Dr. Klaus Sachsse
RK
Switzerland
Telephone:   41-61-901-9044
FAX:        41-61-901-9046

Dr. Jacqueline Smith
EXXON Biomedical Sciences, Inc.
CN 2350, Mettiers Road
East Millston, NJ 08875-2350
Telephone:   (908) 873-6261
FAX:        (908) 873-6009

Mr. Stan  Stasiewicz
NIEHS
111 T.W.  Alexander Drive
P.O. Box  12233
Research Triangle Park, NC 27705
Telephone:   (919) 541-7638
FAX:        (919) 541-4714
Dr. William C. Thomas
Hoechst Celanese
P.O. Box 2500
Somerville, NJ 08801
Telephone:   (908) 231-4485
FAX:        (908) 231-4554

Dr. Tony Wells
Owens Coming Canada
4100 Yonge Street
Toronto, ONT, M2P 2B6
Telephone:   (416)484-6760
FAX:        (416) 484-6761

Dr. Michael Werley
Battelle Columbus Operations
505 King Avenue
Columbus, OH
Telephone:   (614) 424-5973
FAX:        (614) 424-3268
                                       H-7

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APPENDIX





  Issue Paper
    m-i

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           ISSUE PAPER FOR WORKSHOP DISCUSSION






CHRONIC INHALATION TOXICITY AND CARCINOGENICITY TESTING




               OF RESPIRABLE FIBROUS PARTICLES
                         May 8-10, 1995



                    Chapel Hill, North Carolina
                    Workshop Sponsored by the




                 U.S. Environmental Protection Agency




                     in Collaboration with the




          National Institute of Environmental Health Sciences




          National Institute for Occupational Safety and Health




            Occupational Safety and Health Administration






                              m-ii

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                                 DISCLAIMER
      This document is a working paper for workshop discussion purposes only and does
not constitute the policy of the U.S. Environmental Protection Agency and its
collaborating Agencies.  Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
                                     ffl-iii

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                                   AUTHORS
      This document was prepared by an Inter vgency Steering Committee on fiber
testing.  The Committee membership consisted of the following individuals:
Paul Baron, NIOSH, Cincinnati, Ohio


Carl Barrett, NIEHS, Research Triangle Park, North Carolina


David Dankovic, NIOSH, Cincinnati, Ohio


David Lai,  USEPA, Office of Pollution Prevention and Toxics, Washington, D.C.
Ted Martonen, USEPA, Office of Research and Development, Research Triangle Park,
             North Carolina
William Pepelko, USEPA, Office of Research and Development, Washington, D.C.


Joseph Roycroft, NIEHS, Research Triangle Park, North Carolina


Loretta Schuman, OSHA, Washington, D.C.


Vanessa Vu (Chair), USEPA, Office of Pollution Prevention and Toxics, Washington, D.C.
                                      Hl-iv

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 1.     BACKGROUND

        An important task for environmental protection is to identify, and subsequently
 to prevent, eliminate, or mitigate the risks to human health and the environment posed
 by toxic substances.  Natural and synthetic fibers are one group of substances that have
 been identified to be of potential concern.  Many of these fibers have wide industrial and
 commercial applications, but there is limited, inconclusive,  or virtually no information
 about their health effects and/or exposure to workers, consumers, and the general public.
 As a result, the U.S. Environmental Protection Agency (EPA) has added a "respirable
 fibers" category as priority substances for health effects and exposure testing to obtain.
 the necessary data to evaluate the extent and magnitude of health risks to the exposed
 individuals and populations. This would then allow the Agency to determine whether or
 not there is a basis for any risk reduction measures.

       The health endpoints of potential concern for respirable fibers are the potential
 development of respiratory diseases including cancer from chronic inhalation exposure.
 In humans, the inhalation of asbestos and erionite fibers has been associated with the
 development of non-malignant and malignant diseases, primarily of the lung, pleura, and
 peritoneum.  The mechanisms by which these fibers induce diseases in humans are not
 clearly understood.  It is generally believed, however, that the potential toxicity and
 carciriogenicity of a given fiber type appear to be dependent upon the respirability of the
 particle, i.e., the ability of the fiber to enter the respiratory tract and penetrate into the
 alveolar region of the lung, and on the nature of the fiber.

       EPA recognizes that the current health effects test guidelines for chronic
 inhalation toxicity and/or carcinogenicity are not specific enough for the testing of
 fibrous substances.  Thus, there is a need for EPA to develop standardized health effects
 test guidelines for fibrous substances that can be" used by EPA in future rulemaking,
 negotiated enforceable consent agreement, or voluntary action to obtain the  necessary
 toxicologic information for  risk assessment. However, at present, there is no general
 agreement upon test protocols for chronic inhalation toxicity and carcinogenicity testing
 of fibers for regulatory purposes. It is, therefore, important for the Agency to obtain
 input from the scientific community on a number of issues related to fiber testing prior
 to the development of proposed standardized study protocol(s)  for respirable fibers.

 2.     GOAL OF WORKSHOP

       EPA, in collaboration with the National Institute of Environmental Health
 Sciences (NIEHS), the National Institute for Occupational Safety and Health (NIOSH),
 and the Occupational Safety and Health Administration (OSHA) through an interagency
working group has identified a number of scientific issues related to fiber testing that
requires further evaluation  by expert scientists.  The goal of the workshop is to obtain
scientific evaluations and recommendations from outside expert scientists on:
                                       m-i

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(1)    issues dealing with the design and conduct of chronic inhalation studies of fibers;

(2)    what preliminary studies would be useful guides in designing the chronic study;

(3)    what mechanistic studies would be important adjuncts to the chronic study to
       enable better interpretation of study results and extrapolation of potential effects
       in exposed humans; and

(4)    which or which combination of the available screening studies constitute a
       minimum data set which can be used to make decisions about the potential health
       hazard of the fiber in question, and  prioritize the need for further testing in a
       chronic inhalation study.

       The invited experts are being asked to review the background document
(Oberdorster, 1995) which provides an overview of major issues related to toxicologic
testing of respirable fibrous particles.  The  background document only serves as a basis
for further workshop discussions and does not represent EPA's policy.  The following
sections provide specific issues to be discussed at the workshop.  These issues are
grouped into several topics for discussion purposes according to the workshop agenda.


3.     ISSUES FOR DISCUSSION AND EVALUATION

       EPA's health  effects test guidelines for oncogeniciry, and combined chronic
toxicity and oncogenicity are widely accepted by the scientific and regulatory
communities for the  testing of chemical substances (Appendix A of the background
document).  EPA's guidelines are essentially similar to those of the Organization for
Economics Cooperation and Development  (OECD) and the National Toxicology
Program (NTP). It is recognized, however, that these guidelines need to be modified to
take into account testing issues which are unique to fibrous particles,

       Numerous test systems and/or protocols have been developed and utilized by. the
scientific community for evaluating the fibrogenic and carcinogenic potential of fibrous
particles. As discussed in the background document (Oberdorster, 1995), there has been
considerable debate about the scientific validity and utility of available test methods.
This subject along with research needs for better understanding of the mechanisms of
fiber-induced disease have been the topics  of discussion at several scientific conferences,
workshops, and expert meetings, sponsored by various organizations (e.g.  Dement, 1990;
WHO, 1992; McCleUan et al.,  1992; ISRTP, 1994).  A tiered-approach  for evaluating the
toxicity and carcinogenicity of new fibers or untested fibers has  also been recommended
by workshop participants sponsored by the  Chemical Industry Institute  of Toxicology
(GET) as a guideline for research purposes (McClellan et al., 1992).
                                       m-2

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       Other organizations such as the International Cooperative Research Programme
 (ICRP) has recently developed a draft protocol for the assessment of synthetic fiber's
 toxicity, as a part of a tiered-approach testing program. This protocol, known as the
 'TJraft Protocol for Inhalation Oncogeniciry Study with Fibers" is appended in Appendix
 C of the background document. This protocol gives more specific details on the design
 and conduct of the chronic study.  This draft protocol, however, does not provide specific
 guidance on the selection of exposure concentrations. There are also some  differences
 between this particular study protocol and the EPA's test protocols with  regard to certain
 standard requirements of the design of the study (e.g. species, strain, gender, exposure
 method). Thus, there is a need for examining and articulating the scientific bases for any
 recommended changes for the testing of fibrous particles.

 3.1.   Inhalation Exposures: Materials and Methods

 3.1.1.  Definition of Fibers

       Fibers are generally defined as elongated particles with a length-to-diameter ratio
 (i.e., aspect ratio) equal to or greater than 3 to 1. This definition is presumed to  include
 particles with varying shapes such as rod.-like, curly, or acicular  (needle-like) shapes, and
 having different structural units commonly referred as to fibers, fibrils, or whiskers.

       Is this an acceptable definition of a fiber? If not,  how should it be modified to
       encompass the varying range of sizes and shapes of naturally occurring and
       synthetic fibrous substances?

       What would be an appropriate definition of a human "respirable"  fiber?

 3.1.2.  Selection Criteria for Suitable Test Materials

       As discussed on pages 5-7 of the background document, there are considerable
 differences in fiber respirability between humans and laboratory rodents. This
 observation raises several questions with respect to  the choices of fiber samples to be
 tested, recognizing the inherent limitations of using rodent species as  surrogates of
 humans in inhalation studies, and  the need for optimizing the study conditions while still
 being able to obtain pertinent toxicologic information for extrapolations to humans.

       For example, participants of a CUT workshop have recommended that "Samples
 used for fiber exposures in experimental studies should be representative of the respirable
fraction found in industrial or other environments.  Therefore, even if.onfy small percentages
 of respirable-sized fiber samples are identified in the workplace, experimental studies should
 be carried out using samples with the greatest potential for pathogenic effects (i.e., generally
 long and thin fibers)" (McClellan et al, 1992).  The  ICRPs draft protocol even specifies
                                        ffl-3

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that "The bulk fiber used for aerosol generation should be prepared or pre-selected to have
a nominal geometric mean fiber diameter close to 0.8 micron and a geometric mean length
of longer than 15 microns".

      For a given fiber type, should inhalation studies be performed using samples with
      the greatest potential for pathogenic effects (e.g. long, thin fibers)?

•     Should fiber samples for testing be prepared so that they are rodent-respirable, or
      should they represent a human respirable sample?

      Should the test fibers reflect what are actually present at the workplace and/or
      non-occupational environments?

      In the case of new fibers, how should the test materials be selected?

3.13. Characterization of Test Fibers

      There is considerable evidence to suggest the importance of fiber characteristics
in relation to disease outcomes. Thus, it is desirable to obtain data on a number of the
physical and chemical properties of the particles. These data will also enable the
investigator to make some preliminary estimates of the lung burden of the material at a
given exposure concentration, the behavior of the particle in the lung, and to some
extent, its expected toxicity.

      At a minimum, what aspects of the test samples need to be characterized (e.g.
      fiber morphology, dimension, size distribution, aerodynamic diameter, chemistry,
      density, solubility, surface characteristics)?

      Should the presence of chemical and/or mineralogical impurities, and trace
      metals also be characterized, since they may also contribute  to toxicity?

      Should efforts be made to assess the contribution of non-fibrous particulate
      materials since they may be substantial and could add significantly to total lung
      burden in terms of mass?

      For fiber number calculations, CUT workshop participants and the ICRP
recommended the use of NIOSH 7400 PCOM (phase contrast optical microscopy)
technique. For determining bivariate distributions of fiber length and diameter, the
ICRP's draft protocol specifies methods using SEM (scanning electron microscopy); on
the other hand, others have recommended  either method (e.g. CHT workshop).  With
regard to fiber counting and sizing rules, there are two available methods- NIOSH and
WHO rules. A modification of WHO/EURO counting and sizing rules are specified  in
the ICRP's draft protocol.
                                       m-4

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      There is also an issue of what would be a suitable method to measure the mass
median aerodynamic diameter (MMAD)- the use of cascade impactors versus spiral duct
centrifugation.  The former method has been recommended at the CHT workshop.  No
specific guidance is given on this measurement in other study protocols (e.g. the ICRP's
protocol).

•     Are there any specific analytical methods that should be required to be used  to
      characterize certain chemical and physical properties of bulk materials or
      individual fibers present in the aerosol and lung tissues?

3.1.4. Exposure Conditions and Methods

      As discussed on page 11 and pages 33-34 of the background document, there are
advantages and disadvantages associated with either method of exposure- whole body
exposure and nose-only exposure. Both methods are considered acceptable by EPA and
other regulatory authorities as appropriate methods for inhalation testing of chemical
substances.  Regardless of the method used, fiber samples need to be aerosolized in such
a way that they are evenly distributed in the chamber atmosphere and that there is
adequate sampling to verity the integrity of the  aerosolized fibers and that constant fiber
concentrations are maintained throughout the exposure period.

      Certain study protocols (e.g. the ICRP's draft protocol) specifies the preference
for the use of nose-only exposure method, the use of a piston brush feed aerosol
generator, and the frequency of exposure atmosphere monitoring (daily for mass
concentration, weekly for fiber concentration and bivariate size distribution, every three
months  for chemical analysis). No reasons  are given concerning these specific
recommendations.

      Should there be a requirement for the use of any  particular methods for
      generating fiber aerosol?

      Are both methods  of exposure (whole body and nose-only exposures) acceptable?
      Is there a preferred method that should be recommended?

      How often should exposure atmosphere be monitored with regard, to fiber number
      and mass concentration, size distribution, and chemical analysis?

32.     Study Design

3.2.1. Animal Species/Strain/Sex Selection

      EPA's test guidelines for oncogenicity require  that a compound of unknown
activity shall be tested on two mammalian species via oral, inhalation, or dermal route of
exposure.  Rats and mice of both sexes are  the species of choice without specifying more

                                      m-5

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precisely any specific strains, except that commonly used laboratory strains shall be
employed.  Justification when selecting other species has to be provided. Rats and mice
are the species of choice mainly because of their relatively short life spans, their
widespread use in toxicological studies, their susceptibility to tumor induction, and the
limited cost of their maintenance.  On the other hand, for combined chronic toxicity and
oncogenicity study, the rat is the species of choice.

       Inhalation studies with asbestos fibers in rats have demonstrated to be an
appropriate experimental model for the identification of asbestos-induced human
diseases, primarily fibrosis and cancer of the lung. The low mesothelioma rate  induced
in rats via inhalation compared with the rate of crocidolite-induced mesotheliomas in
humans indicate that the rat inhalation model may not be adequately sensitive to identify
the potential ability of fibers of unknown activity to induce mesothelioma in humans,
unless the fiber in question is expected to be a potent mesothelioma inducer such as
erionite fiber. Since induction of mesothelioma is also a health endpoint of concern,
testing in a second rodent species may be necessary to ensure that all potential  health
effects would be properly identified.

       On the other hand, questions have been raised about the validity and utility of
using either the mouse or the hamster as the second species for carcinogenicity testing of
fibers. The concern is that there have been fewer studies using mice and hamsters with
asbestos fibers and results obtained to date seem to indicate that they may not be
suitable animal models for predicting asbestos fiber-induced diseases. The mouse
generally does not respond to tumor induction by asbestos fibers via inhalation. In the
case of the hamster, this  species appears to be more sensitive than the rat with respect to
fiber-induced mesothelioma, but less sensitive to the induction of lung tumors and
fibrosis than the rat.

       There has been considerable debate about the need for a second species testing.
For example, at the CUT workshop, the rat has been recommended as the only species
to be tested in inhalation studies.  It is stated that 'While it may be desirable to study two
species, the participants agreed that only the rat is necessary, in view of the extraordinary
expense of conducting inhalation studies" (McClellan et aL, 1992). The ICRP's draft
protocol also specifies the use of rat and even specifies the use of male Fischer 344 ra.ts
only.  No specific reasons are provided.

      Is it necessary to test fibers of unknown activity in a second animal species? If yes,
      what would be an appropriate second species? If not, what are the scientific
      reasons?  Are there any circumstances that warrant testing in a second species?

      Do different animal strains respond with different sensitivities to fibrous  particles?
      If yes, what is the  most appropriate strain of rodent species to be used?

      Should both sexes of the animal be used? If not, which sex is more suitable, and
      why?
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             Selection of Exposure Concentrations

       EPA's test guidelines for oncogenicity require at least three exposure
concentrations and a sham-exposed (filtered air only) control group. The highest
concentration level should elicit signs of minimal toxicity without substantially altering
the normal life span other than tumor formation.  The lowest exposure level should not
induce any indications of toxicity and the intermediate concentration(s) should be
established in a mid-range between high and low levels.  There is no specific guidance
on how to select aerosol concentrations for inhalation studies  of particulates.

       For combined chronic toxicity and oncogenicity studies, EPA's guidelines require
the use of a high concentration treated and control satellite group designed to assess the
evaluation of pathology other than neoplasia. The highest concentration for satellite
animals should be chosen so  as to produce frank toxicity, but not excessive lethality.

       As discussed on pages 7-iO, 34-35, and 40-42 of the background document, several
proposed criteria (e.g. effect on lung clearance and pulmonary function, chronic
inflammatory responses, cell proliferation, histopathological changes) may be used to
define the highest fiber concentrations to be tested in a chronic study, also known as the
maximum aerosol concentration or MAC.

•      What criteria can be used to determine the MAC in inhalation studies of fibrous
       particulates and to judge whether a MAC has been reached or exceeded?

       Is it necessary to include a satellite group exposed to a fiber concentration
       exceeding the MAC for the evaluation of lung pathology other than neoplasia?

       What preliminary studies would be useful and important for setting appropriate
       dose levels (e.g. 90-day and/or shorter-term inhalation  studies, in vitro solubility,
       in vivo  biopersistence studies)?

       The NTP generally employs ah upper limit exposure concentration of 100 mg/m3
       for relatively insoluble particles of low toxicity.  In view of potential particle
       "overload", should a practical upper limit concentration also be set for fibrous
       particles?

323.  Exposure Regimen and Observations Period

       EPA's test guidelines require that the animals are exposed to the test substance
for 6 hours per day, 5 day per week over a period of at least 24 months for rats, and 18
months for mice and hamsters.  Termination of the study should be at 24 months and
not longer than 30 months for rats, and at 18 months and not longer than 24 months for
mice and hamsters. However, termination of the study is acceptable when the number of
survivors of the lower exposure  groups or of control reaches 25 percent.  Other study

                                       m-7

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protocols (e.g. ICRP's) specify that all remaining animals will be sacrificed when
approximately 20 percent survival is reached in one of the test exposure groups.

       Experience with asbestos fibers indicates that fiber-induced lung tumors or
mesothelioma in rats occur at relatively advanced age.  Thus, it  would be desirable to
allow the animals to live out their life span after the two-year exposure is completed.
On the other hand, there are disadvantages of a lifetime study.  These include the high
mortality rate in rats over 2 years of age, and the high incidence of age-related
spontaneous non-neoplastic  and neoplastic lesions which would make interpretations of
study findings difficult.

       Is the exposure regimen as specified in EPA's guidelines  appropriate for the
       testing of fibers?

       Is it necessary to recommend when final sacrifice be carried out?  If so, what
       would it be?

32.4.  Numbers of Animals and Interim Sacrifices

       EPA's test guidelines require at least 100 animals (50 males and 50 females) be
used for each exposed and negative control groups.  Satellite exposed and control groups
consisting of 20 males and 20 females are to be used in the combined chronic toxicity
and oncogenicity study.  Additional animals are used if interim sacrifices are planned.
However, this is optional.

       Should interim sacrifices be recommended for the testing of fibers? If yes, what
       would be an appropriate interim sacrifice schedule and design (e.g. number of
       animals per group, duration of exposure and recovery period)?

32.5.  Use of Positive Control

       The use of positive control is not required in EPA's test guidelines for chronic
toxicity and oncogenicity testing. However, in view of the complexity of conducting an
inhalation study with fibrous particles, it may be useful to consider including a group of
positive control to validate the reliability  of the testing system.  Asbestos fibers are most
often used as a positive control, but exposure-dose-response relationships have not yet
been established for any types of asbestos fibers.  Moreover,  standardized UICC
reference materials (e.g.  UICC crocidolite) have been considered not suitable because of
their short fiber length.  It should be pointed out that the ICRP and the  CIIT workshop
participants did not make any specific recommendations with regard to the use of
positive control in the chronic inhalation  study.

       Should a positive control be included in the chronic inhalation study with fibers?
       If not, why?

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      If yes, what might be appropriate criteria for selecting a positive control- asbestos
      fibers with fiber size distribution similar to the test material?

      How many exposure levels of a positive control be conducted? If only one
      exposure level is used, should it be comparable to the highest exposure level of
      the test material in terms of fiber concentration or fiber lung burden?  Or, should
      it be at an exposure level expected to induce tumor effects?

3.2.6. Criteria far a Negative Inhalation Test
                                                                         i
      EPA generally considers an oncogenicity study to be negative if there is an
absence of tumor effects in an adequately sensitive and well-conducted study.  The key
issue which needs to be defined is what constitutes "an adequately sensitive study" for the
testing of fibrous particles.

      What might be suitable criteria for the acceptance of an inhalation study with
      fibers as negative? (no tumors, achievement of a MAC, appropriately spaced low
      doses, adequate animal survival, use  of an appropriate positive control?)

33.   Fiber Disposition

      The ability of fibers to induce disease seems to be dependent on their site of
deposition and their ability to biopersist in the lung. Thus, it would be desirable to fully
characterize the deposition, translocation, and clearance of the test  fibers as well as their
lung retention.

33.1. Pre-chronic Studies

      It has been suggested that for the studying of particles, the primary goals of a
subchronic study should include: (a) an evaluation of patterns of particle deposition
(including hot spot of deposition), translocation, and clearance, and determination of the
lung burden at which impaired clearance occurs; and (b) an evaluation of toxicity and
mechanisms of pulmonary toxicity.

      EPA generally recommends a subchronic 90-day study to help establishing suitable
study conditions for the chronic study, especially for setting appropriate exposure levels.
The subchronic study would also provide important toxicologic information to be used in
conjunction with results of other mechanistic studies to help the interpretation of the
chronic  study findings.  The NTP recommends conducting both a 14-day and a 90-day
study before performing the chronic study,

      Should a subchronic 90-day study be recommended prior to conducting the
      chronic study? Or, should it be made optional?
                                       m-9

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       What are the primary goals of the subchroriic study?

       Are there any specific methods that should be recommended to measure the
       effects of fibers on lung clearance (e.g. use of radiolabeled particles) ?

3.3.2   Lung Burden Analysis

       Lung burden analysis is not a requirement in EPA's study protocol for chronic
toxicity and oncogenicity testing.  However, lung burden data would provide useful data
on biopersistence of the test fibers and serve as a better measure of internal dose.

       Should lung burden analysis be included in the subchronic and chronic studies?

       If yes,  should the procedure as recommended by the ICRP be adopted?  As
       discussed on page 36 of the background document, there are data to indicate that
       fiber burden data based only on the accessory lung lobe may not be representative
       of the  whole lung because of potential hot spot of deposition.  In view of these
       findings, what changes should be recommended?

       Should any specific methods for lung ashing be recommended?

       How often should lung burden analysis be performed (at interim and final
       sacrifice time points)?

3.4.    Dosimetry and Interspecies Considerations

       The size distribution of fibers that deposit in the lungs of rodents may be different
from those in the lungs of humans because of anatomical and physiological differences.
Deposition and clearance mathematical models have been  developed  to relate the fiber
lung burden to biological effects which may serve as useful exposure-dose-response
models for human risk assessment.

       Which additional dosimetry information could be obtained as a part of the
       chronic study to help in characterizing the human hazard and exposure-dose-
       response assessment?

3.5.    Histopathologic Evaluation

       It  has been suggested at the CUT workshop to use the Wagner scoring system
(Appendix A) for the evaluation of pulmonary fibrosis to enable direct comparison of
effects induced by different types of fibers. The Wagner scoring system has also been
utilized in recent studies on synthetic mineral fibers conducted by RCC Laboratory.  This
system, however, does not consider the mass of the lung tissue  involved.  Other methods
                                      m-io

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which have been used by other investigators include a morphometric approach to
determine the percentage of lung tissue involved in fibrotic lesions, and in another
method, total lung collagen is measured as an indicator of lung fibrosis.

      Is it necessary to utilize a standardized scoring system for the evaluation of
      cellular changes and fibrosis in the lung?

      The ICRP has recommended that the Wagner scoring system be revised to take
      into account the limitation as discussed above.  What specific modifications need
      to be made before it can be adopted for the inclusion in the test guidelines?

3.6.   Biomarkers of Toxicological Effects

      Recent studies in rats have demonstrated the value of bronchoalveolar lavage
fluid  (BALF) analysis in subchronic studies in predicting the chronic effects of exposure
to a variety of particles.  This will enable the investigator to better select the dose level
for the  chronic studies as well as to help understand the biochemical and cellular
sequence  of events of particle-induced toxicity and carcinogenicity.

      Which specific biomarkers of toxicity and carcinogenicity should be measured in
      the subchronic study (e.g. BALF analysis, cytotoxicity, cell proliferation)?

      Should BALF analysis be made mandatory for chronic study?

      If yes, should the procedure as specified in the ICRP's protocol be adopted?  Are
      there any modifications that should be considered?

3.7.   Mechanistic Studies

      In assessing the potential toxicologic and carcinogenic effects of respirable fibers
in humans, it is desirable to consider the differences of species responses and the
understanding of the mechanisms of fiber-induced toxicity and carcinogenicity. This may
allow an improved basis for extrapolating observed effects in the test species exposed to
high concentrations to humans exposed to relatively lower levels generally found in the
workplace and the general environment.

      A number of cell culture systems have been developed to assess the cytoxicity, cell
proliferation, genetic alterations, to better elucidate the mechanisms of fiber-induced
pathogenesis, i.e., chronic pulmonary inflammation, fibrogenesis, and oncogenesis (as
reviewed in the background document and report by McClellan et al., 1992).

      Should additional mechanistic studies be recommended? Which and when? (prior
      to the chronic study, in parallel with the chronic study,  and/or subsequent to the
      chronic study?)

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3.8.    Screening Battery

       It is generally agreed that the only way to be certain that a fiber would not
produce long-term effects including tumorigenesis is to conduct a chronic study in rodent
species.  On the other hand, it is recognized that there is considerable economic burden
involved in the testing all fibrous materials available in commerce. Thus, from a
practical perspective, it may be desirable to develop a testing approach that may be
useful for screening large number of fibrous substances and identifying and prioritizing
those that are of most concern.

       A screening approach for research purposes was recommended at the GIFT
workshop which include: product cycle evaluation and physical and chemical
characterization (Tier I); in vitro solubility and durability, in vitro  cell toxicity (Tier II);
short-term inhalation studies- ideally for 3 months (Tier ffl); and chronic studies (Tier
IV).  No recommendations were made with regard to the specific tests to be included in
tier H.

       Recognizing that no single  screening study can accurately predict the in vivo
       responses from long-term exposure to fibers, can Tier n and Tier  III types of
       studies be used to screen and set priority with regard to confirmatory testing in a
       chronic study to obtain more definitive information for risk assessment purposes?
       If not, why?

       If yes, what specific tests or combinations of tests  can be utilized in this screening
       battery?

       Given that in vivo studies using non-inhalation method of exposure (e.g.
       intraperitoneal injection, intratracheal instillation studies) have been proven useful
       in identifying the potential  health hazard to humans, should they be considered
       acceptable as an alternative screening test or an adjunct to  short-term inhalation
       studies in a screening battery?

       Appendix D of the background document is the ICRP's draft protocol for
       intracavitary testing (i.p. study).  Would a positive finding using this protocol
       constitute a potential hazard to humans, or would a positive finding need to be
       followed by a chronic, inhalation study to confirm  the hazard?

      As discussed on page 42 of the background document, questions have been raised
as to the appropriateness of using a large total dose of 250 mg in the i.p. test. The issue
of the MTD for i.p. studies require further discussion (if tune permits).
                                       m-12

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4.    REFERENCE

Dement JM (1990). Overview: Workshop on fiber toxicology research needs.
Environmental Health Perspectives 88:261-268.

ISTRP (1994). International Society of Regulatory Toxicology and Pharmacology.
Proceedings Symposium on Synthetic Vitreous Fibers: Scientific and Public Policy Issues.
Regulatory Toxicology and Pharmacology 20: S1-S222.

McClellan RO,  Miller FJ, Hesterberg TH, Warheit DB. et al. (1992). Approaches to
evaluating the toxicity and  carcinogenicity of man-made fibers: Summary of a workshop
held November 11-13, 1991, Durham, NC. Regulatory Toxicology and Pharmacology 16:
321-364.

Oberdorster G (1995). Developing test guidelines for respirable fibrous particles:
Background information as basis for workshop discussions. Prepared for the U.S.
Environmental Protection-Agency under Purchase Order Number 4W 5147 NASA.

WHO (1992). World Health Organization. Validity of methods for assessing the
carcinogenicity of man-made fibres. Executive Summary of a WHO Consultation. World
Health Organization Regional Office for Europe.

5.    APPENDIX A

      Wagner's Histologic Grading of Pulmonary Changes Related to the Inhalation of
      Fibers (see attachment)
                                     m-13

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   Table 1.  Hiccologic grading of p«tl*onary change* related to the inhalation of fibre*
                                        Me  Leatoo observed.
   Cal lular
                         Kiniaetl


                         lUld
A  few •acrephage* la the 1
•ad alveoli
                                            of the terminal bronchioles
Frescncc oC cuboid*! epleh«litw linioit th« proxiiul «lv«oti
(bronehioLtzacloR).  Ho collagen buc  reeieulia  fibre* ^y
be present la iaterscieto*. «c Che Juaeeloa of the  ceniaal
bronchial* 4Ad *lvealu>.  UaUa*l ii*cropb«gc* are  ear*
•coaapicuBUs and eotweuelear cell* way be  Couad  in  the
lacervtlciiai.
   Fibre*!*
Mild


Itooerate

Severe
                                   6

                                   7

                                   8
Hinlfeal eollagea dopoaicioa at level et terminal bronchiole
aod alveola*.  lacnaaed bronchtolixaclon with asaoclated
•ucaid debri* *ttgae*tia« glandular pattern.

Inteclobular linking of le*ion 4e*crlbwl in zrade « and
iacreaaed *everltv of fibre*!*.

Early eooaeUdatioa.  Parcncliymal decree** 1* eppareac.

Harked fibroai* aad cancalidatien.

Covplece obstruct ion of «o*c ainmv*.
         Non-fibroclc  lesion*.
              *    ,       r. J.C. Skidmons, J.W. and Moore. JJ^ A oompejmrhm •tudy of tb«
flbrog^iie ejid cnvinoeente efleete of in(X (^Ba^
100).ln: Binlnrical EfTer*- "f ^^^^eMineral Fibre*. WoridHe.lthOrgmnit.tion, 19M.pp.
                                                m A- i

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Table 2.  Classification  and criteria uccd for she diagnosis of
          proliferative lesions  induced by exposure co Inhaled fibre
       Bronehoalveolar hyperpii sta (BAft)
          1.  Recent ion of alveolar architecture
          2.  Air space* are lined by cuboldel.ccllf
          3.  Lack of cellular and nuclear atypia
          4.  Kay or may not b« aifnif leant aaounti  of  flbroslc

      Adenoma
          1.  Lose of ;pre-exiatl-| architecture
          2.  Papillary foneeion Into air space*
          3.  Nuclear uniformity
          t>.  Hay or nay  not  be netaplaeia to other cell typei
          5.  Co«preeeion but no ihvacioo  of adjacent tteiuu

     Adenocercino«a
          1.  Expansion,  coee>ree»ioa and invasion pf adjacent tiaeucs
          2.  Acypie (naclear end/or cellular)
          3.   Pleottorphifo
          it.   Hetaetases
          3.   Metaplasia  e^%eh«r  ««11  type* is of cm present
                              III A - 2

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           APPENDIX IV





Background Information as Basis for workshop Discussions
               IV-i

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          DEVELOPING TEST GUIDELINES
                      FOR
         RESPIRABLE FIBROUS PARTICLES:
Background Information as Basis for Workshop Discussions
          Gunter Oberdorster, D.V.M., Ph.D.
                      IV-ii

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                                  Disclaimer

       This report was prepared by Dr. GUnter Oberdorster for the U. S. Environmental
Protection Agency under procurement number 4W 5147 NASA. It is made available for scientific
discussions and evaluation and does not represent the official position or policies of the US.
Environmental Protection Agency.
                                     IV-iii

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                               TABLE OF CONTENTS
                                                                               Page #
1.     Tntroduction	  1
       1.1   Need for Testing Guidelines	  1
       1.2   Purpose of Background Paper	-.	 2
       1.3.   Overview of Concepts and Principles of Toxicological Testing	 2
       1.4   Scientific controversies surrounding fiber testing and cancer classification... 5
              1.4.1  Respirability of fibrous and non-fibrous particles	 5
              1.4.2  The Maximum Tolerated Dose	 7
              1.4.3" Biopersistence vs. biodurability and chronic toxicity	 10
              1.4.4  Methods of dosing	 11
                     1.4.4.1  Inhalation	  11
                     1.4.4.2  Intratracheal  instillation	 11
                     1.4.4.3  Intracavitary  injections  	 12
                     1.4.4.4 In vitro assays  	 14
       1.5   Risk Assessment Guidelines	  15
2.     Review of available test protocols	  17
       2.1.   EPA health effects test-guidelines	  17
       2.2   Recommendations for fiber testing from GIFT Workshop	  20
       2.3   Test Protocols from International Cooperative Research Programme	 22
             2.3.1 Chronic inhalation study for carcinogenicity testing	,.  23
             2.3.2  Intracavitary Testing	  24
             2.3.3  Biopersistence Studies	  26
             2.3.4  Invitrostudies...	  27
                    2.3.4.1   Acellular  tests  	*.	  27
                    2.3.4.2   Cellular  tests 	  28
3.     Evaluation of Available Test Methods.   	„	  29
       3.1   In vitro cellular assays	  29
       3.2.   In vitro and in vivo durability studies..;	  29  .
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Table of Contents Continued:                                                    Page #

       3.3   Intracavitary studies	 30

       3.4   Chronic^ inhalation studies - major issues fen* discussion:  	 32

             3.4.1  Characterization of test samples - size, chemical composition.
                    surface properties	 32

             3.4.2  Rat vs. human respirability	 32.

             3.4.3  Animal selection	  33

             3.4.4  Exposure conditions and methods	  34

             3.4.5  Exposure concentration - MTD prediction	  34

             3.4.6  Positive control group	  35

             3.4.7  Gross Examinations	  36

             3.4.8  Histopathologv	  36

             3.4.9  Lung burden analysis	  36

             3.4.10 Analytical methods for fiber measurements  	  37

             3.4.11 Fiber counting	,-	  37

             3.4.12 Clinical observations	  38

             3.4.13 Bronchoalveolar lavage	  38

4.     Possible Criteria  for  Accenting a  Negative Inhalation Test	  38

5.     Summary of Issues for Workshop  Discussion	...  39

6.     Literature Cited	  45

Tables and Figures

Appendices
       Appendix A: EPA Health Effects Test Guidelines
       Appendix B: Summary of CUT Workshop Conclusions
       Appendix C: Draft protocol for inhalation oncogenicity study with fibers
             Addendum to oncogencity protocol
       Appendix D: Draft protocol for intracavitary testing
       Appendix E: Draft protocol for inhalation biopersistence study with fibers
       Appendix F:  Intratracheal instillation for biopersistence evaluation
     ,  Appendix G: Draft protocol for in vitro acellular tests - durability
                                         W-v

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                        DEVELOPING TEST GUIDELINES
                                        FOR
                      RESPIRABLE FIBROUS PARTICLES:
           Background Information as Basis for Workshop  Discussions

1.     Introduction
       1.1   Need for testing guidelines:
       There is increasing concern about the potential health risks of respirable fibers, both at the
workplace and in ambient air. Specifically, controversy has developed with respect to classification
of man-made vitreous fibers (MMVF). Table 1 shows the main types of these fibers according to
WHO (1988).  The recent listing of respirable glass wool in the National Toxicology Program's
(NTP) Seventh Annual Report on Carcinogens raised considerable concern among the glass fiber
industry and generated intensive debates among scientists from regulatory agencies and industry
about the adequacy of this listing (Infante el a/., 1994).  Arguments  centered around  the
interpretation, usefulness and appropriateness of specific animal tests including: Use of a relevant
test model; meeting adequate study design criteria for carcinogen identification, not achieving the
Maximum Tolerated Dose (MTD) or, on the other hand, exceeding the MTD; use of the appropriate
animal species, etc.; in short: It was criticized that study protocols have not met scientific criteria
and were unsound.
       At present, there are no generally agreed upon protocols for carcinogenicity testing of
fibers,  which appears to be the main  stumbling block to accepting laboratory test data for
classification of man-made fibers, existing ones as well as new ones to be developed. This lack of
accepted study protocols is a major deficiency causing .great uncertainties  when it comes to
regulating potentially hazardous fibrous particles, and there is an urgent need that specific testing
guidelines be developed. A 1991 workshop (McClellan etal., 1992) addressed approaches to
evaluating the toxicity and carcinogenicity of man-made fibers. Participants recommended general
approaches for fiber testing without going into specific details about testing, protocols or definition
of an MTD.  Participants of another workshop held in Paris in October, 1994  were charged with
                                        IV-1

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 developing specific testing protocols; however, attendance of expert scientists in the field was
 limited so that a general acceptance of the suggested protocols is not assured and important issues
 such as the MTD were not addressed satisfactorily.
       1.2    Purpose of Background Paper:
              Given the lack of a consensus as to what constitutes an appropriate testing protocol,
 the EPA has decided to convene a workshop with the goal to develop  scientifically sound
 guidelines for evaluating the toxic and carcinogenic potential of fibrous particles. This background
 paper was written in preparation of this workshop. The need for this workshop is also based on
 the recent addition of a "respirable fibers" category to EPA's list of chemical testing priorities
 known as the Master Testing List  (MTL).  It is generally recognized that EPA's current test
 guidelines for chronic inhalation toxicity and carcinogenicity are not considered specific enough for
 the testing of fibrous substances. Thus, there is a need for EPA to develop standard health effects
 test guidelines for respirable fibers that can be used by EPA in future rule-making or negotiated
 consent agreements.
       Prior to the development of such test guidelines, it is essential to critically evaluate currently
 available methodologies for evaluating the potential toxic and carcinogenic effects from inhalation
 exposure to fibrous particles. This will serve as the basis for further discussion and debate by an
 ad hoc expert panel to be convened by EPA in 1995 to obtain scientific input from outside experts.
 These evaluations will then be used in the development of EPA's  test guidelines for respirable
 fibers.  The goals of this paper are to prepare an evaluative report of available test methods and
 study designs for assessing the potential chronic toxic and carcinogenic effects of inhaled fibrous
 particles.  Brief overview of concepts of lexicological testing and of scientific controversies
 surrounding fiber testing and risk assessment will be given first
       1.3.    Overview of Concepts and Principles of Toxicological Testing:
             Toxicological testing is concerned with identifying acute and chronic effects of
agents after  different routes of exposure,  normally performed in experimental animals.
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lexicological testing also includes quantitation of a hazard and uncovering mechanisms of action to
better predict a potential health hazard to humans and to develop exposure standards designed to
protect human health.  In view of available numerous lexicological test methodologies it becomes
oftentimes very difficult to compare results from one test to those of others, even if the tests are
performed only slightly differently. It is, therefore, critical to establish standardized test guidelines
based on accepted sound scientific principles and methods so that results can be used more easily
by the scientific and regulatory communities for purposes of risk assessment
       The National Academy of Sciences (NRC, 1983) developed a risk assessment paradigm
which includes four  steps:  Hazard Identification, Dose-Response Assessment,  Exposure
Assessment and Risk Characterization. Toxicological studies are aimed at these individual steps by
obtaining sufficient information for developing risk management rules and  guidelines, e.g.,
standards for occupational or environmental exposures.  To achieve this  goal, studies in
experimental animals are performed routinely to identify a specific hazard (Hazard Identification)
and to establish dose-response relationships to further characterize the adverse effect (Dose-
Response Assessment). While such studies are mostly based on well developed sound scientific
principles methodologies for the process of risk characterization are most often based on a number
of unproven assumptions when attempts are made to extrapolate from results of animal studies to
humans. This results in considerable uncertainties with regard to quantitative and even qualitative
risk assessment, since different animal species can show very different responses.  However, in
the absence of essential information about the relevancy for humans of a mechanism involved in an
observed adverse response in experimental animals it is prudent from a medical-preventive point-
of-view to use tiie data from the most sensitive animal species (EPA, 1986). For example, results
of a recent chronic inhalation study with refractory ceramic fibers (RCF) in rats and hamsters
showed that rats were most sensitive with respect to inducing lung tumors whereas hamsters
responded with the development of pleura! mesothelipmas (Glass et cd.,  1992; Mast  et al., 1992).
This raises the question whether in future chronic inhalation studies both species should be
                                         IV-3

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investigated, an issue which will be discussed at the workshop. Likewise, should a result from a
cb  nic animal inhalation study indicating a non-linear tumor response be interpreted to also
indicate non-linearity in humans? And, should results derived from i.p. administration of fibers be
used for hazard identification and risk characterization?
       The experimental toxicologist is faced with these and many other questions related to
toxicological testing principles.   A most important issue is  dose selection in a  chronic
carcinogenicity bioassay.  Very high doses administered chronically to experimental animals may
result in premature death; or in organ specific cell proliferative responses which may lead to an
increased tumor response. In either case, an observed tumor response may not reflect  the true
carcinogenic potency of the compound under study, and the concept of MTD was introduced in
1976 by Sontag etal.in an attempt to define the highest dose level in a chronic study that will not
alter the animal's normal longevity from effects other than carcinogenicity. It was suggested to
select the MTD from a subchronic study causing a decrement in weight gain of approximately
10%.
       Similar approaches to the MTD to be used in chronic studies have been recommended by
various agencies which, to a variable degree, emphasize histopathplogical changes to be evaluated
as well as determining toxicokinetics and metabolic parameters. (IARC, 1980; Occupational Safety
and Health Administration, 1980;  OECD, 1981; US EPA,  1982; NTP, 1984; International Life
Sciences Institute, 1984; Office of Science and Technology Policy, 1985). In a policy statement
discussing this issue, EPA (1986b) also included as a parameter indicating the MTD significant
weight changes of the target organ and, hi addition, emphasized collection of general toxicity data
as well as the necessity that the effects cause no shortening of life.  This policy, however, has not
been discussed further by the agency.  The present standard NCI/NTP design for a  chronic
carcinogenicity study requires a life-long exposure at three dose levels in rodents, with exposures
for the highest dose being at the MTD and subsequent lower doses at one-half and one-quarter of
the MTD (Haseman, 1985).
                                            IV-4

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       Existing guidelines, however, are rather vague with respect to defining the MTD, and
specifically do not mention inhalation studies. It is, therefore, a major challenge to both define
parameters which indicate that an MTD is achieved and to reach consensus about its applicability in
chronic inhalation studies.
       1.4   Scientific controversies surrounding fiber testing and cancer classification:
             Inhalation studies pose specific problems due to the uncertainty of the dose
delivered to the target organ.  Thus, responses are initially observed as exposure-response
relationships rather than dose-response relationships. Respiratory tract dosimetry needs to take
into account both deposition of inhaled substances as well as their retention kinetics in the lung
which are different between paniculate material and gases. Particle deposition and retention
throughout the respiratory tract can be very different depending on a number of physico-chemical
properties including particle size, shape and solubility.
             1.4.1 Respirability of fibrous and non-fibrous particles:
                    An important distinction exists between the "inhalability" and "respirability"
of inhaled particles. As defined by ACGIH (1994), inhalable particles are those that are hazardous
when deposited anywhere in the respiratory tract, whereas respirable particles are hazardous when
deposited in the gas exchange region. In addition, a Thoracic Paniculate Mass is defined for those
materials that are hazardous when deposited anywhere  within the lung airways (conducting
airways) and the  gas exchange  region.  For spherical non-fibrous particles respective curves
defining the 3 categories are well described (ACGIH, 1994). Studies in rats (Raabe etal.,  1977;
1988) as well as mathematical deposition models (Yeh and  Sebum, 1980) also permit a very good
description about deposition efficiencies of inhaled spherical particles in this species. For example,
particles larger than -5 pm are not respirable for rats, whereas they are well respirable for humans
which shows mat it is important for respiratory tract dosimetry to recognize these differences when
interpreting and extrapolating results from rats to humans. In addition, of course, it needs to be
considered that rodents are obligatory nose-breathers whereas humans are most often mixed oro-
                                           -IV-5

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nasal breathers.  In the worst case of only oral-breathing significantly more inhaled particles
penetrate to the alveolar region.
       It becomes even more difficult when fibrous particles are concerned. Several factors need
to be considered in terms of adverse effects and respiratory tract dosimetry. One is that humans
develop lung tumors both in the conducting airways and in peripheral regions, with conducting
airway tumors probably being the major portion. In contrast, lung tumors in rats after inhalation of
fibers are induced in the peripheral region.  This indicates that respirable fibers are the most
important ones for the rat yet for humans fibers conforming to the thoracic paniculate fraction may
have to be considered which includes more than the respirable fraction.  Secondly, respirable fibers
for humans and rats can represent very different fractions, which - as mentioned above - becomes
even more obvious when human mouth-breathing is considered. For example, Figs. 1 and 2 show
alveolar deposition efficiencies for fibers of different aerodynamic diameters and different aspect
ratios (P) between rats and mouth-breathing humans (data from work in progress by Yu etal.,
1994). The fiber deposition curves are contrasted with those for spherical particles.  It is quite
clear from these curves that respirability for rats is very different from respirability in humans.
Deposition curves for hamsters are very similar to those for the rat
       TimbrelTs work (1965) suggested that the maximum diameter of respirable asbestos fibers
is 3.5 (im; his more recent studies (Timbrell, 1982) showed asbestos fiber diameters of up to 4.1
|im in human lung tissue which is in agreement  with Fig. 1 showing the results of  deposition
modeling of inhaled fibers in humans.  Figure 1 expresses the  fibers with their aerodynamic
diameter which can be derived from their geometric diameter and their specific density. Since  the
density of asbestos fibers is 2.4 - 3.3 g/cm3 their aerodynamic diameters are greater than their
geometric diameters. Fig. 3 shows the relationship of these two parameters for fibers of different
aspect ratios with a specific density of 2.7 cm3 (i.e., RCF fibers) for random orientation of fibers
so that this figure can be used for converting data in Fig. 1 to geometric diameters.

                                           IV-6

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      The differences in fiber respirability between humans and laboratory rodents raises several
questions with respect to lexicological testing of fibers:  1) Should fiber samples for testing be
prepared so that they are rodent-respirable; should they represent a human respirable sample; or
should they reflect what is actually present at the workplace? 2) If the longer human respirable
fibers are significant for chronic adverse effects, will these be testable by rat inhalation studies?
For example, a 1.4 ^m thick and 28 \im long fiber (specific density = 2.7) has an aerodynamic
diameter of 3 urn (Fig. 3); this fiber is not respirable by the rat, but more than 20% of inhaled
fibers of this size are deposited in the human alveolar region. 3) Can we appropriately test human
respirable fibers by inhalation in laboratory rodents?  Or is testing not necessary since the fraction
of those fibers which are respirable by humans but not by rodents is so small that they can be
neglected? 4) Should fibers conforming to a human Thoracic Particle Definition be considered as
well? And, indirectly also related to the issue of respirability: 5) What should the maximum fiber
concentration be which is to be used in an inhalation assay? There are no simple answers to most
of these questions, they require thoughtful discussions.
             1.4.2  The Maximum Tolerated Dose:
                   With respect to the highest exposure concentration to be used in a chronic
inhalation study some  guidance may  be obtained from recommendations  of an NTP-sponsored
workshop at which the maximal aerosol exposure concentration in chronic particle inhalation
studies was  discussed (Lewis et al., 1989).  The emphasis of this workshop was on highly
insoluble non-fibrous particles of low toxicity, formerly referred to as "nuisance" particles.  At
issue here was the phenomenon of lung particle overload with such particles which has been
observed in a number of chronic inhalation studies in rats and subsequently was linked to the
occurrence of chronic  adverse effects such as fibrosis and lung tumors (Morrow,  1988). The
workshop participants  recommended  that the mass median aerodynamic diameter of the  inhaled
particles should be restricted to <3 urn. Similar to the rcspirability for fibers, human respirability
for non-fibrous spherical particles is also different from that of rodents (see  Figs. 1 and 2) and thus
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larger particles not respirable by rats would be excluded by this recommendation.  However,
dissimilar to the fibers, non-fibrous low toxicity particles of larger-size are not expected to have a
greater adverse effect than smaller ones; it seems to be well established now that the overloaded
lung is mainly characterized by the phagocytized particle load (non-fibrous, low toxicity particles)
of the alveolar macrophages which eventually will affect their normal clearance function (Morrow,
1988).
       The NTP workshop participants also addressed indirectly the question of the Maximum
Tolerated Dose and suggested that testing should not be performed at the highest technologically
feasible concentration, but the highest inhaled concentration should rather lead to  a minimal
interference with  lung defense  mechanisms, i.e., lung clearance.   In addition, two lower
concentrations should not lead to interference with normal clearance and particle accumulation in
the lung. Accordingly, a respective workshop recommendation was that the determination of the
highest chronic exposure concentration should be based either on model predictions of particle
deposition and retention kinetics or on a subchronic (90-day) study performed at several exposure
levels. A potential particle overload should be determined in this subchronic study by the use of
labeled paniculate probes inhaled by the test animals and followed by determining their subsequent
clearance from the lung. Finally, it was also recommended that the retained test material in the lung
should be measured as a function of time which would give very important information to decide
whether particle accumulation in the lung at different exposure concentrations followed linear or
non-linear kinetics. Adherence to these recommendations would allow the identification of any
potential lung particle overload related deviation from the normal accumulation kinetics.
       Since fibrous particles cannot be categorized as particles of low toxicity in contrast - for
example - to non-fibrous TiO2, the term particle overload being reached at high exposure
concentrations may not be appropriate for fibers (Oberd5rster era/.,  1994); fibers do not lead to a
volumetric overloading of alveolar macrophages as is known for spherical particles, but cause
effects on clearance mechanisms at much lower lung burdens (Yu et a/., 1994).  Several
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mechanisms might be responsible for this, one being that longer fibers in general cannot be fully
phagocytized by alveolar macrophages, and another being that certain fibers may have an
k
intrinsically higher cytotbxic potency than others and than TiO2.  Even non-fibrous particles have
vastly.different cytotoxic properties, e.g., crystalline SiO2 vs. TiO2, and the same will be true for
different fiber types.
       Whether an adverse effect on lung clearance function induced by fibers can also be used to
decide that an MTD has been reached or exceeded needs to be discussed. Obviously, for spherical
low toxicity particles, the state of lung overload is equivalent to having reached an MTD. Thus, in
an effort to define the MTD specifically for chronic inhalation studies with low toxicity spherical
particles Muhle et al. (1990) introduced the term Maximum Functionally Tolerated Dose (MFTD)
for such situations.  They arbitrarily defined this dose as a lung burden causing a 2- to 4-fold
prolongation of particle clearance based on their results with a number of particle inhalation studies
in rats.
       While a parameter defining overload specifically for inhalation studies would be very useful
and while the concept of the MFTD in particular would allow one to decide if a chronic particle
inhalation study had been performed at irrelevantly high exposure concentrations, the concept of
MFTD has not been generally adopted or accepted by inhalation lexicologists or regulatory
agencies.  A consensus on how to deal with this issue in inhalation toxicology is urgently needed,
particularly since the problems associated with the interpretation of particle overload studies
specifically and with high dose particle studies in general are widely acknowledged.  It should be
kept in mind, however, that the MFTD as defined by Muhle etal. (1990) should primarily be
applied to highly  insoluble particles of low intrinsic toxicity; the broader question of whether to
extend the MFTD concept to fibrous particles or to inhaled materials in general requires additional
thorough discussion.

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              1.4.3 Biopersistence vs. biodurabilitv and chronic
                   •A generally accepted concept in fiber toxicology is that the biopersistence of
t
a fiber after lung deposition relates to its chronic toxicity and carcinogenicity.  Biopersistence is the
result of a number of parallel processes which determine the fate of a fiber in the lung (Fig. 4),
These include physical/mechanical and chemical processes, and a fiber of low biopersistence is
assumed to have a lower toxic and carcinogenic potential than a highly biopersistent fiber. A
distinction needs to be made between biopersistence, biodurability and durability. Biodurability
refers to in vivo processes whereas durability refers to results from in vitro studies which may be
very different from biodurability (see further discussion of durability in Section 1.3.4.4, in vitro
assays). A proposal by regulatory agencies in Germany for the initial carcinogenicity classification
of man-made fibers is entirely based on the durability of a fiber (TRGS, 1994) which is estimated
from fiber composition based on specific metal oxides. Although such a proposal may be
premature - the formula needs to be validated before industry moves ahead to design fibers to
satisfy the formula rather than relying on a lexicological test  - it nevertheless emphasizes the
importance which is placed on fiber durability.  This emphasis may be justified in  general,
however, as the example of chrysotile clearly shows, even fibers of very low durability can be as
toxic and carcinogenic as highly durable fibers: Inhalation studies in rats by Wagner etd. (1974)
with chrysotile and amphibole asbestos  showed both to have a very similar fibrogenic  and
carcinogenic potency; yet the same study as well as newer studies in rats  (Coin etal., 1992) and
non-human primates (Rendall, 1988) demonstrated that chrysotile is rapidly eliminated from the
lung, probably due to dissolution processes. Thus, reliance solely on durability  or even
biodurability and biopersistence may not be appropriate for classification of fibers without further
lexicological testing.
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              1.4.4 Methods of dosing:
                   1.4.4.1  Inhalation.
                   A number of different test strategies has been used and advocated for
evaluating the toxic potential of particles. Foremost are inhalation studies in laboratory rodents,
mostly performed in rats.  Inhalation is obviously the most physiological route of exposure and
most relevant to the human situation. However, as pointed out above, breathing modes (nose vs.
mouth) and respirability (dosimetry) can be quite different between rodents and humans. Both
whole body exposures and nose-only exposures  have been developed, each having its role for
specific applications (Phalen, 1984). Generally, whole body exposures require more test material
and will result in the dusting of the whole animal, while nose-only exposures restrict external
contamination of the animal to the head and neck region. This avoids significant contamination of
the laboratory environment in the post-exposure phases which is highly important for paniculate
material of greater toxicity. On the other hand, there is greater stress on the animals during restraint
for hours in the nose-only tubes which might have an influence on toxicity outcome. Principally,
results from rodent inhalation studies are considered appropriate for risk characterization purposes
with the caveat that extrapolation to humans has to be performed cautiously by considering
important species differences.
                   1.4.4.2 Intratracheal instillation.
                   Another mode of administration to the respiratory tract is  via intratrachcal
instillation. Intratracheal instillation of particles to be tested has been performed frequently in rats,
hamsters and mice.  In contrast to inhalation, the particles are delivered within a fraction of a
second at doses which are unevenly distributed through the lung, forming hot spots of deposition
which can give rise to local acute inflammatory responses. Thus, this method is considered non-
physiological and may not be suitable for purposes of risk characterization but could well be used
for comparative  studies establishing dose-response relationships for the purpose of hazard
identification. The advantage obviously is that human respirable particles, specifically fibers, can
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be delivered to the rodent lung at pre-detennined doses. Repeated instillations in weekly intervals
for up to 20 weeks - or even longer - can be performed and animals are kept for the rest of their
lives to evaluate carcinogenic responses (e.g., Pott era/., 1987).  One-time instillations of non-
inflammatory doses have been shown to be useful for evaluating pulmonary retention of non-
fibrous and fibrous particles which seems to be in very good agreement with  results from
inhalation studies (OberdSrster etal., 1992; Muhle el al., 1994). Thus, recognizing the limitations
of the i.L instillation studies they can be very useful for answering specific questions regarding
comparative pulmonary toxicity and retention characteristics of particles but results are not well
suitable for risk characterization.
                   1.4.4.3 Intracavitary injections.
                  The disadvantage of non-physiological administration associated with the
intratracheal instillation method is more obvious with routes of administration which circumvent
         i
altogether the physiological route of entry into the respiratory tract via conducting airways, i.e.,
injections into the pleural cavity and peritoneal cavity. Both are lined with mesothelial cells and,
therefore, a carcinogenic response is characterized by the development of mesotheliomas when
fibrous particles are administered in sufficiently high doses. The method of administration has the
advantage of being less work intensive and time-consuming than the inhalation mode, and repeated
injections over several months (weekly intervals) can be performed. Intracavitary injections have
successfully been used to demonstrate the carcinogenicity of asbestos and man-made fibers
(Stanton etal., 1977; Pott, 1987). Proponents of this method view it as an excellent and easy to
perform bioassay to determine both the carcinogenic potential and carcinogenic potency of a fiber
with the results not only being relevant for the development of mesothelioma but also relevant for
indicating a carcinogenic risk for lung tumors. The fact that the intrapleural and intraperitoneal
injection of different asbestos fibers results in a tumorigenic response which has also been found in
exposed humans either as mesothelioma, lung cancer or both, is cited as convincing evidence for
the relevancy of this mode of application.

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       Indeed, IARC accepts findings of carcinogenicity from this route of administration, and in
its Preamble on the Evaluation of Carcinogenic Risks to Humans (e.g., IARC, 1993) makes the
very general statement"... in the absence of adequate data on humans, it is biologically plausible
and prudent to regard all agents and mixtures  for  which there is sufficient evidence  of
carcinogenicity in experimental animals as if they presented a carcinogenic risk to humans."
Opponents of the intracavitary routes of administration charge that in addition to being highly non-
physiological this method also bypasses all defense mechanisms of the respiratory tract, and leads
to massive local doses with significant inflammatory responses. It would not be relevant for
human exposure and could at best be used for identifying a hazard to mesothelial cells but not to
cells of conducting airways and alveolar region. Moreover, cell types are very different between
the two regions and it cannot be assumed that responses will be the same.
       It is, indeed, quite obvious that inhalation of fibrous particles will lead to a very slow build-
up only in the lung; defenses such as different local clearance mechanisms including alveolar
macrophages, other inflammatory cells, mucociliary escalator and dissolution processes are active
to remove fibers after deposition by inhalation. Transmigration to the pleura from the alveolar
space occurs for a limited number of fibers, and it appears to be different for different types of
fibers. It seems, therefore, that intraperitoneal or intrapleural injections are useful for identifying a
potential of a fiber for inducing mesothelioma but not necessarily for induction of lung cancer.
       The interpretation of tumor responses observed after intracavitary injection has  to  be
correlated with the delivered doses.  For example, there is good evidence that any highly insoluble
low toxicity non-fibrous particle will cause lung tumors in the rat if inhaled chronically at high
enough concentrations, as well demonstrated with TiO2 inhaled over two years at 250 mg/m3 (Lee
etal., 1984). By analogy, any highly insoluble fibrous particle administered to the lungs of a rat in
sufficiently high doses also has the potential to induce lung tumors, and if administered directly to
mesothelial sites in sufficiently high doses may cause, mesothelioma as well Yet, the relevancy for
human extrapolation may be questionable, in each of these  cases the appropriateness of the
                                         IV- 13

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delivered dose has to be considered carefully.  OveraL Jitracavitaiy tests using appropriate doses
can be useful for purposes of hazard identification wr  -espect to mesothelioma but should not be
used for purposes of risk characterization.
                   1.4.4.4 In vitro assays.
                   Of great importance for using results from rodent bioassays for extrapolation
to humans is the knowledge about underlying mechanisms. To uncover mechanisms of action of
fiber-induced carcinogenicity, in vitro studies have been performed using different types of lung
cells, e.g., tracheal or bronchial epithelial cells (Mossman et ol., 1986), mesothelial cells
(Kuwahara etol., 1994), alveolar macrophages (Donaldson etol., 1992).  Such studies are very
valuable for uncovering basic mechanisms of fiber carcinogenesis but they remain to be developed
for use as standard testing protocols of carcinogenicity.
       Other in vitro assays are aimed at the question of particle dissolution. Cultures of alveolar
macrophages from animals and humans have been used successfully to demonstrate dissolution of
different metal oxide particles (Lundborg et ol., 1984; 1985; Marafante et ol., 1987; Kreyling etol.,
1990; Kreyling, 1992) and it appears that dissolution rates inside AM phagolysosomes are similar
between the different species.  Standardization of this in vitro assay for fibrous particles would be
useful for evaluating an important component of the biopersistence of fibers.  Additional tests
related to fiber durability in vitro are acellular systems for measuring leaching of specific elements
from fibers using simulated lung fluid (Gamble's solution) (Morgan and Holmes, 1986; Klingholz
and Steinkopf,  1982; Leineweber,  1984; Law et a/., 1990).  Different methods of chemical
analysis have been used, including  neutron activation of  fiber samples  and subsequent
measurement of specific leached radioisotopes (Morgan etol., 1971; Oberddrster et a/.,  1980).
Static or dynamic flow through systems with or without recirculation have been used. However,
results from different laboratories are difficult to compare because of differing conditions with
respect to temperature, fluid composition, fluid flow, etc. Thus, it is no surprise when dissolution
rates observed in vitro do not correlate with those observed in vivo. Moreover, in vivo dissolution
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is often determined by measuring a decrease in fiber diameter over time after deposition in the lung
which is different from measuring the loss of certain elements (leaching) during in vitro dissolution
(biodurability, see Section 1.4.3). The latter determines a mass  loss by which physical fiber
dimension is not necessarily affected, and the former determines a loss in physical fiber appearance
where additional mass loss through leaching processes is unknown. Differences between in vitro
and in vivo may not only be qualitative but also quantitative, and a standardization of the testing
protocol is needed.
       1.5    Risk Assessment Guidelines:
              EPA has published general guidelines which govern certain aspects of experimental
carcinogenicity testing. However, no specific guidelines for carcinogenicity testing by inhalation,
or even more specifically for fibers, have been issued.  In its 1986 Risk Assessment Guidelines,
EPA addressed policy issues related to carcinogenicity testing and evaluation to promote high
technical quality and agency-wide consistency in the risk assessment process (EPA, 1986a).  The
four steps of risk assessment including hazard identification, dose-response assessment, exposure
assessment and risk characterization were briefly reviewed, and specifically the elements of hazard
identification were discussed in greater detail with respect to the use of animal studies.  It was
recognized that epidemiologic studies are inherently capable of detecting only comparatively large
increases in the relative risk of cancer  It was also stated that because it is possible that human
sensitivity is as high as the most sensitive responding animal species, in the absence of evidence to
the contrary, the biologically acceptable data set from long-term animal studies showing the greatest
sensitivity should generally be given the  greatest emphasis.
       The issue of the Maximum Tolerated Dose was also addressed without giving specific
recommendations as to what constitutes an MTD. Thus, the expectation is that long-term animal
studies at or near the MTD will be used to insure an adequate power for the detection  of
carcinogenic activity. It was further stated that negative long-term animal studies at exposure levels
above the MTD may not be acceptable if animal survival is so impaired that the sensitivity of the
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study is significantly reduced.  Specifically, the OSTP (1985) was cited which states that the
carcinogenic effects of agents may be influenced by non-physiological responses induced in the
model systems (such as extensive organ damage, radical disruption  of hormonal function,
saturation of metabolic pathways, formation of stones in the urinary tract, saturation of DNA repair
with a functional loss of the system).  Testing regimens inducing these responses should be
evaluated for their relevance to the human response to an agent, and evidence from such a study,
whether positive or negative, must be carefully reviewed.  Additionally, it was suggested in EPA's
guidelines that positive studies at levels above the MTD should be carefully reviewed to insure that
the responses are not due to factors which do not operate at exposure levels below the MTD.
Although these are important statements, they are rather vague and leave a lot of room for
individual interpretations.
       In July, 1994, EPA issued draft Revisions to Guidelines for Carcinogen Risk Assessment
which have been reviewed since then (EPA, 1994). These guidelines will eventually replace the
1986 Carcinogen Risk Assessment Guidelines and are basically structured in a similar way
although in much more detail. A discussion of the relevance of high doses being used in animal
studies is more extensive, although still not satisfactory, and missing in the present draft is a
discussion on  routes of exposures other than ingestion, inhalation or dermal  such as
intraperitoneal, intratracheal or subcutaneous injection. The draft guidelines define the highest
dose to be used as one that in an animal lifetime produces some toxic effects without either unduly
affecting mortality from  effects other than cancer or producing significant adverse effects on the
animals' health.  It is deemed mandatory to reach an adequately high dose since otherwise the
sensitivity of the study is reduced; on the other hand it is recognized that excessive general toxiciry
or toxicity in a tumor target tissue raises the question whether tumor effects are specific to the agent
or are non-specific  effects secondary to the toxicity. This poses a problem, as  discussed in the
review draft, since reducing the high dose to  avoid toxicity would reduce the sensitivity of a
protocol that may at best be able to detect an increase of tumor incidence of 10% if no spontaneous

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 background incidence is present  A greater sensitivity for tumor detection would require the use of
 thousands of animals per assay which is obviously not a feasible option.
       The review draft guidelines discuss further that among confounding factors potentially
 identifiable in a cancer bioassay  are findings of significant toxicity manifested by clinical signs,
 hematological or chemistry measures or changes in organ weight, morphology and histopathology.
 These findings may  indicate interference  with the carcinogenic process and could obscure
 interpretation of the results. Of importance is also the statement in the draft guidelines that absence
 of tumor effects in an adequately sensitive  and well conducted study is accepted as a negative
 finding, as are studies with undue effects on mortality or health that show no tumor effects and that
 include lower doses which are appropriately spaced.  It is also pointed out in these draft guidelines
 for carcinogen risk assessment that results from short-term in vitro and in vivo studies are useful in
 the interpretation of epidemiological and animal data with respect to possible modes of action.
       A workshop was  held in September, 1994 to discuss  these proposed new cancer
 assessment guidelines and provide EPA with suggestions and recommendations for inclusion in a
 final document One of the key suggestions was that there is a need to expand the discussion on
 the Maximum Tolerated Dose. It should be recognized that testing at the MTD level may produce
 results irrelevant to humans and may increase sensitivity at the price of specificity. In general, the
 workshop participants supported die emphasis of the draft guidelines on data other than tumor data
per se since they thought those data to be critical which consider the mechanisms of carcinogenitity
 and which bear on the significance of extrapolation of experimental data to humans.
 2.    Review  of available test protocols
       2.1.    EPA health effects test-guidelines:
              EPA's Health Effects Guidelines of 1992 (EPA, 1992) give more specific details on
the conduct of long-term oncogenicity studies in general which are briefly summarized in the
following paragraphs.  The complete text of the guidelines is attached as Appendix A.
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       The section on oncogenicity within these test guidelines require that a compound of
unknown activity shall be tested on two mammal   species, and rats and mice are the species of
choice without specifying more precisely any specie,: strains except that commonly used laboratory
strains shall be employed. Justification when selec Jig other species has to be provided
       Dosing of the rodents shall begin as soon as possible after  weaning, ideally before the
animals are 6 weeks old, but in no case more than 8 weeks. Bodyweight variation of the animals
used should not exceed ±20% of the mean weight for each sex.
       At least 100 animals (50 females and 50 males) shall be used for each dose level and for the
concurrent control. Numbers have to be increased if interim sacrifices are planned so that the
number of animals at the termination of the study should be adequate for a meaningful and valid
statistical evaluation of long-term exposure. For this purpose, survival in all groups should not fall
below 50% at the time of termination.
       Of importance is the use of a concurrent  control group  of untreated or sham-treated
animals. In addition, the use of historical  control data is desirable for  assessing the significance of
changes observed in exposed animals.
       For purposes of risk assessment  at least 3 dose levels shall be used in addition to the
control group.  The high dose level should show signs of minimal toxicity without substantially
altering the normal lifespan, and  the lowest dose should not interfere  with normal growth
development and longevity of the animals or should not cause any  indication of toxicity. The
lowest dose should not be lower than 10% of the highest dose and the selection of the dose levels
should be based on either existing data or preferably on the results of subchronic studies (range-
finding studies).
       With respect to the exposure concentrations a 7-day/week dosing over a period of at least
24 months for rats and 18 months for mice is suggested. However, a  5 day/week basis is also
acceptable based on practical considerations.

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       Specific requirements are given for inhalation studies with respect to the dynamic airflow
changes which should be 12 to 15 air changes per hour, insuring an adequate oxygen content of
19% and an evenly distributed exposure atmosphere.  Crowding of the animals  should be
minimized by individual caging where the total volume of the test animals shall not exceed 5% of
the test chamber volume, or nose-only exposures should be used.  The daily exposure is for a 6 hr.
timeperiod in which feed and water should be withheld. The temperature should be maintained at
22± 2°C with a relative humidity between 40-60% as far as practicable.
       Bodyweights are to be recorded for all animals once per week during the first 13 months,
and thereafter at least once every 4 weeks.
       The chamber airflow rates shall be monitored continuously and recorded at least once every
30 minutes. Likewise, temperature and humidity shall be monitored in the same way.  The
exposure concentration should be held as constant as practicable and be monitored continuously
with recordings at least 3 times during the test period, i.e., at the beginning, at an intermediate time
and at me end of the period. The stability of the aerosol concentration and size distribution should
be established during the development of the generating system, and the exposure analysis shall be
repeated as often as necessary to determine the consistency of the experimental conditions.
       Clinical examinations are specified to be performed at 12,18 months and at sacrifice at
which timepoint a blood smear shall be obtained from all animals, and differential blood count be
evaluated from those animals of the highest dose group and the controls. Additional examination
of the other dose groups are to be performed if indicated by the results of the highest dose group.
It is further stated that a complete gross examination shall be performed on all animals and
essentially all important organs and tissues be preserved for future histopathological examination.
Specifically for inhalation studies it is stated that the entire respiratory tract shall be preserved
including nasal cavity, pharynx, larynx and perinasal sinuses. As the optimal method preservation
of these tissues the inflation of lungs with a fixative is recommended  Full histopathology is to be
performed on these organs and tissues of all animals in the control and high dose groups. In case
                                       IV -  19

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of substantial alteration of the animals' normal longevity or induction of effects which could have
affected a neoplastic response  the  next  lower dose level will also be  examined fully by
histopathology.
       The guidelines also list criteria for a negative test to be accepted which include that no more
than 10% of any group is lost due to autolysis or cannibalism and  that survival in each group
should be no less than 50% at 18 months for mice and hamsters and at 24 months for rats.
       Finally, the guidelines require that a test report be provided on all group animal data, test
conditions and exposure data.
       These test guidelines also contain a section on combined chronic toxicity/oncogenicity
studies which essentially are the same as the ones issued for oncogenicity alone.  The main
difference is that the test animals also include at least 40 rodents (20 females and 20 males) which
should be used for satellite dose groups and a satellite control group for additional toxicity testing.
       In these testing guidelines no mention is made about using a positive control Although the
guidelines are very general they  include also the inhalation mode of administration but do not
specifically mention fibrous particles.
       2.2    Recommendations for fiber testing from CllT Workshop:
              Participants at a 1991 workshop specifically discussed approaches to evaluating the
toxicity and carcinogenicity of man-made  fibers (McClellan et al.,  1992).  Although primary
conclusions from this workshop have been published (Appendix B), total agreement on a specific
approach to evaluating the toxicity and carcinogenicity of man-made fibers was not reached.  As
stated by the authors, the recommended approaches are, instead,  intended  as  guidelines for
research so that the resulting information will be useful for making informed judgements about
potential effects in people from exposure to man-made fibers. Thus, the primary conclusions
reached  at the workshop could serve as  the basis for further discussion for  the setting of
appropriate guidelines for  fiber toxicity and carcinogenicity testing; they should not be used blindly
as a standard approach to testing.
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       With respect to the lexicological evaluation of effects of fibers, a tiered-approach of testing
was suggested  consisting  of (I)  product cycle evaluation and  physical and  chemical
characterization; (II) in vitro  solubility and durability testing as well as in vitro cell toxicity
assessment; (HI) short-term inhalation studies in which fiber deposition and retention and
respiratory tract toxicity are evaluated which is followed by (IV) long-term inhalation studies which
also evaluate fiber deposition and retention as well as respiratory tract toxicity (Table 2).
       A description of the individual tests  in this tiered-approach was given in the  published
report and it was specifically stressed that the short-term in vivo laboratory animal studies should
be used to assess several endpoints. These include evaluation of the number and size dimensions
of the fibers retained in the respiratory  tract  since these  parameters would provide crucial
information of the actually inhaled deposited fibers after inhalation by the experimental animals.
No recommendation was made with respect to sample selection as to whether non-fibrous particles
should be removed or retained.
       In addition to evaluating dosimetric aspects, the short-term in vivo inhalation study would
also provide information about the potential for pulmonary inflammation and fibrosis using specific
biomarkers, for investigating mechanisms of fiber-induced pulmonary injury, for  assessing
pulmonary cell proliferation rates and for  determining biopersistence based on measurement of
fiber retention by serial sacrifice of the exposed animals. The ideal timeperiod for this subchronic
study was given with 3 months of exposure; major emphasis was to be placed on histopathological
evaluations of the respiratory tissues and determining exposure-response relationships.
       With respect to long-term inhalation studies as the final step in this tiered-approach, a two-
year study was recommended with the rat as the most appropriate animal species. Three exposure
levels plus a sham-exposed control group should be used, and the highest level should be set at or
near the MTD. The MTD is defined as the dose that does not decrease the lifespan of the exposed
animals other than by tumor formation, it should produce a minimal degree of alteration in
pulmonary clearance compared to the lower exposure concentrations.  It was not specifically stated
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that the MTD should be derived from respective measurements in the subchronic study and how it
should be done. The lowest exposure level was suggested to be either at a plausible upper-bound
level of human exposure and/or at an exposure concentration expected to produce no observed
effect  Further it was stated that the fiber dimension should be such that they are respirable without
specifying whether respirability for humans or respirability for rodents was meant Based on the
numbers given, i.e., <3.5 |im for aerodynamic diameter, it appears that human respirability is
addressed here (see Section 1.4.1).
       The use of the rat as the preferred animal species was justified by its similarity with humans
in terms of tumorigenic and pulmonary fibrotic responses to fibers as well as the availability of a
comparatively large data base for rats.  A second species was not recommended due to the
extraordinary expenses for conducting inhalation studies.  In addition to  interim sacrifice
timepoints every six months, it was also deemed useful to include a post-exposure recovery group
to investigate whether lesions may be reversible. Lung burden data would again give useful
information about the biopersistence of the tested fibrous particles.  A major emphasis was on a
detailed macroscopic and light microscopic histopathological evaluation of lung tissues including
inflammation, fibrosis, lung tumors and mesotheliomas. Additional specific parameters to be
measured included cell proliferation assays and also analysis of bronchoalveolar lavage fluid
samples as well as pulmonary function studies.
       2.3    Test Protocols from the Workshop of the International Cooperative Research
             Programme:
             As mentioned in the Introduction, a one-day workshop was held in  Paris in
September, 1994, in an attempt to  discuss and develop protocols for testing of fiber durability,
toxicity and carcinogenicity. This workshop was organized by the French University of Paris and
the French National Institute of Research and Security and co-sponsored by the French Ministries
of Health, Labor, Environment and  Industry.  Several working groups  were charged with
developing standard protocols considering inhalation, intratracheal instillation, intraperitoneal
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injection and i'n vitro studies. These draft protocols are briefly summarized here and shall represent

a basis from which further details can be developed which could be incorporated into guidelines for
t
fiber testing. The detailed draft protocols of this workshop are attached as Appendices C-E.

              2.3.1  Chronic inhalation study for carcinogenicity testing:

                    The workshop protocol for this test is given in Appendix C.  This protocol

essentially follows the exposure regimen used in the long-term inhalation study with refractory

ceramic fibers and glass fibers performed at the RCC Laboratory in Switzerland. Main points of

the testing protocol are the use of the Fischer-344 rat, male animals only, which are exposed by

nose-only inhalation exposure to well characterized fiber test atmospheres which should be

respirable by the rat. Exposure durations are for 6 hours/day, 5 days/week for 104 weeks total

with a subsequent non-exposure period lasting until about 20%  survival in one of the exposure

groups is reached.  The fiber aerosol generator has to be validated to show that it does not

significantly grind or contaminate the bulk fibers provided. Three exposure concentrations at a

minimum are  to be used with the highest being at the Maximum Tolerated Dose. No further

specifics are given to define the Maximum Tolerated Dose. The exposure atmosphere is to be

monitored at least once per day in terms of its gravimetric (mg/m3) concentration, at least once per

week in terms of fiber number concentration and also at least once per week to determine the

bivariate size distribution by scanning electronmicroscopy,  and every three months a chemical

analysis of the fiber sample should be undertaken. The age of the animals at delivery is between fii

12 weeks, and there should be 140 animals per group; interim sacrifice timepoints every three

months are scheduled at which times six animals  are to be killed.  The final sacrifice occurs when

about 20% survival is reached in one of the exposure groups. The exposure system consists of a

nose-only exposure in which the airflow to each animal is approximately 11/min. For evaluation

of fiber retention kinetics and additional lexicological parameters the use of bronchoalveolar lavtge

parameters is recommended. All animals  are to be necrppsied and all tissues be examined and

preserved; the lungs will be inflated with  a fixative for fixation and the accessory lobe will be


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moved prior to fixation and frozen at or below -20°C for further processing by low-temperature
ashing to determine lung fiber burdens. Fiber analysis includes number concentration and bivariate
size distribution and chemistry. The counting rules are those provided by the WHO/EURO
Guidelines (i.e., fibers £ 5 |im long, £3\un thick, and with an aspect ratio of 1:3 or greater).
Additional procedures are detailed in the attached Appendix C, including recording of bivariate size
distribution and specific stopping rules for counting.  Appropriate statistical analysis and data
reporting has to be performed as well.
       The workshop participants also discussed how the histopathological evaluation should be
performed and suggested to use a revised cellular/fibrosis scoring system replacing the old Wagner
Scale. Moreover, there was a strong suggestion to also establish a resource program with the aim
to develop shorter-term methodologies for the evaluation of fiber toxicity.  Endpoints to be
considered should be cellular proliferation, induction of fibrosis as predictors for long-term effects
when animals are exposed for only 6 months by inhalation. With respect to quantification of
cellular proliferation specific methodologies using bromodeoxyuridine via osmotic minipumps
were given, or alternatively labelling for PCNA was suggested.  Finally, based on newer data, it
was suggested to also include an assay to evaluate mutation frequency of alveolar epithelial cells,
specifically the HPRT gene mutation assay, in a short-term testing protocol.  This might be
particularly useful to evaluate mutagenic responses in vivo of inhaled fibers. These additional
points are also listed as an addendum to Appendix C.
             2.3.2  Intracavitary Testing:
                    Fibers have been administered in the past by intrapleural and intraperitoneal
injections. However, intraperitoneal injection has become the most widely used method for
evaluating carcinogenicity of fibers and thus the focus will be on this route of administration.
Appendix D lists the draft protocol that was developed at the 1994 workshop in Paris which may
serve  as a basis for developing a more detailed protocol for the guidelines.  The rat is listed the
animal of choice for an Lp. injection study. Wistar rats have been used mostly in the past within
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this test in contrast to the F-344 rat used in inhalation studies.  However, it may be desirable to
perform the long-term inhalation as well as the intraperitoneal injection studies in the same rat
species. Both sexes should be used, and the number should be 50 rats/dose and sex + 50 control
animals which are injected with saline.
       It is also emphasized in the attached protocol for intracavitary testing that a positive control
in addition to the negative control should be used which preferentially should be UICC crocidolite
administered at the standard  3 doses. Suggested doses to be used for each dust sample  are 1 x
109,1 x 108,1 x 107 fibers which should be injected after being suspended in phosphate buffered
saline in an injection volume  of 2 ml.  The maximum dose in terms of mass should not exceed 50.
mg per injection, and when more than 1 injection is required to obtain the full dose then  weekly
intervals of injections should  be performed; however, the maximum overall dose in terms of mass
should not be more than 250 mg of fibers. Since injection by syringe leads to losses of fibers this
should be determined in pre-test studies by appropriate tests. The maintenance of the animals and
duration of studies as well as  handling for necropsy and histology are similar to  that described for
the long-term inhalation study as are the recording requirements.  Any macroscopically visible
tumor has to be confirmed by histological diagnosis; serosal tissues from organs  not showing
tumors macroscopically should be prepared for histology, i.e., diaphragm, liver, spleen, pancreas,
mesentery and omentum phis  gut segments.
       The draft protocol developed by the participants at the Paris workshop does not specify the
technique of intraperitoneal  injection which needs some further  attention. According to Pott
(personal communication) the dust should be suspended in NaCl solution with ultrasonic treatment
and kept in suspension by a magnetic mixer while the injections are being performed. Injection is
carried out under CO2 anesthesia on the left side into the lower part of the abdominal cavity as  a
rule. The cannula should have a diameter of 2 mm. It may be necessary to use higher volumes
than 2 mL for suspending 50 mg sufficiently. In addition to the tissues cited above, the uterus
should also be excised in female animals for histological examination.
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              2.3.3  Biopersistence Studies:
                    The determination of the biopersistence of a fiber is based on the hypothesis
that fibers of low biopersistence are less toxic and carcinogenic than those which have a very high
persistence after inhalation in the lung. Biopersistence includes a number of mechanisms which all
contribute to the retention of a fiber in the lung, i.e., mechanical clearance processes, translocation
processes, and chemical dissolution processes (Fig. 4).  Appendix E shows a respective draft
protocol for biopersistence testing as proposed by the workshop participants in Paris in September,
1994. The preferred method of exposure is by inhalation, and a 5-day inhalation period is thought
to be optimal  However, under specific circumstances (radioactively labeled fibers, availability of
only a small fiber sample) intratracheal instillation could be considered as well if the necessary
precautionary measures are taken (see Appendix F).
       "The animal of choice is the Fischer-344 rat, male animals only. The exposure is for 6
hours/day during the 5 days of exposure. At predetermined intervals after exposure groups of
animals are killed for assessment of fiber lung burdens. Fibers with respect to diameter and length
are prepared as described for the chronic oncogenicity inhalation study, and a concentration of 30
mgAn3 of rat respirable fibers should be used If fibers are very soluble (as determined by in vitro
dissolution rates of greater than 200 ng x cm*2 x h*1) then exposure concentrations can be increased
to 40 mg/m3.  It is important that the 5-day inhalation does not lead to significant inflammatory
responses in the lung which should be evaluated by determining inflammatory parameters in the
lung lavage fluid in animals sacrificed at 1 and 28 days post-exposure (PMN count, lavage protein,
LDH level).  Since  significant inflammation may interfere  with subsequent parameters of
biopersistence the tests should be repeated if this occurs.  For an evaluation of fiber retention
kinetics in different respiratory tract compartments, additional  animals can also be added to
determine fibers recoverable in the B AL fluid, in the lavaged lung, in the thoracic lymph nodes and
also in the pleura after appropriate recovery of pleura! tissue (Bermudez, 1994).

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       Regular sacrifice timepoints are recommended depending on the in vitro durability of a
given fiber as shown in the table in Appendix E, pg. 3. At a minimum animals should be killed at
2 or 3 days and 4 weeks and 3 months after exposure. With respect to exposure methodology,
fiber aerosol preparation and generation, monitoring and counting and sizing, the same procedures
as presented under Chronic Oncogenicity Study should be used.  For determination of the lung
fiber content lungs should initially be cleaned from adjacent tissues and frozen at or below -20°C
for further storage. Fiber recovery should be performed using low-temperature ashing, and both
fibrous and non-fibrous particles should be analyzed by number concentration, bivariate size
distribution and chemistry.
              2.3.4 In vitro studies:
                  2.3.4.1 Acellular tests.
                  Protocols for assessing in vitro the  durability of a specific fiber can vary
considerably as discussed in the Background section.  Appendix G is the draft protocol developed
at the September, 1994 Paris workshop which could form a starting point for a standardization of
the in vitro durability methodology for fibers. In general, the acellular test systems of solubility
testing consist of short-term and long-term continuous flow experiments. Length and diameter
distribution of the fibers should be determined before the tests are started (bivariate distribution).
Chemical composition  is determined as  well  and fibers are characterized by scanning
electronmicroscopy for testing. From the geometrical characterization a specific surface area of the
sample can be calculated; following this the sample should be placed in a polyethylene cell with a
continuous fluid flow at either pH 4.5  (to simulate  the pH inside a phagolysosome  of
macrophages) or pH 7 (model of extracellular fluid, modified Gamble's Solution).  The test is
performed at  a temperature of 37°C for up to 60 days. The dissolution rates are monitored by
analysis of SiO2 and CaO, and possibly  also Fe2Oj, MgO  and I^Oj.  SiO2 dissolution is an
approximate indicator of the dissolution of the fiber network, whereas CaO is indicating in addition
the leaching at the surface of a fiber (specifically for glass fibers). At the end of the test and after
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washing in deionized water, fibers should be examined by scanning electronmicroscopy, and EDX
analyses of the surface of the fibers be performed for evaluating the thickness of a leached layer.
Dissolution velocities should be calculated from initial SiO2 content and the SiO2 analysis in the
eluate as well as from the initial diameter distribution and are given in nm/day or ng/cm2 x hr.
Standardization of the dissolution period (e.g., 14 days) is necessary to more easily compare
different dissolution rates of different fiber samples. Specifics for standardization of flow rates
need to be given since these will affect significantly fiber dissolution rates.
                   2.3.4.2 Cellular tests.
                   Additional in vitro tests using cell  systems have been described to assess
toxicity of fibers as well as in vitro cellular dissolution rates. For example, alveolar macrophages
from rats or alveolar macrophage cell lines can be used. The dissolution of the fibers is determined
by measuring the amounts of Si, Al and Fe dissolved from fibers after exposure in cell culture.
Like  with the  acellular  test  systems, the additional evaluation of fiber surfaces by
electronmicroscopy could be included after having been in culture  for a standardized period.
Differences between intracellular and acellular dissolution have been reported (Luoto etal., 1994).
      Toxicity of fibers to specific cells in vitro has also been  performed using alveolar
macrophages or macrophage-derived cell lines or pleural mesothelial cells.  Endpoints to be
determined include release of LDH as a simple toxicity assay or dye exclusion assays which could
become standardized tests. More involved endpoints consider evaluation of unscheduled DNA
synthesis and determination of DNA  repair or mutation  frequencies in cells or cell lines after in
vitro fiber exposure.  Other in vitro  tests, both after in vivo and after in vitro exposures to fibers,
have been described which can be very useful for elucidating mechanisms of fiber toxicity and
carcinogenicity. These tests have numerous variations depending on individual questions to be
studied and it may not be advisable to try to standardize these more complex tests which are mainly
aimed at mechanisms.
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3.     Evaluation of Available  Test Methods.
       In general, protocols recommended at the CUT and Paris workshops should form a basis
for further discussions.  These are attached as appendices. However, there are a number of
specific points which need to be considered and which are addressed below.
       3.1    In vitro cellular assays:
              As already mentioned before, a number of tests using either cell lines or freshly-
isolated pulmonary cells are being used.  In vitro cells will  be particularly useful for initial
evaluation of fiber toxicity in comparison to a well-known and well-characterized fiber like an
ampbibole asbestos or also a non-fibrous particle such as TiO2.  Endpoints to be evaluated could
be simply cell viability, phagocytosis by alveolar macrophages, cell proliferation, expression and
induction of specific mediators .including cytokines, growth factors, antioxidants, and other studies
directed at the mechanisms of fiber toxicity and carcinogenicity. Simple cellular in vitro  assays
may give a useful initial assessment about the toxicity of a fiber and more involved assays can be
very useful for studying specific mechanisms at a cellular/molecular level.  A difficulty
encountered is to quantify the dose of administered fibers per cell since both cell adherence as well
as endocytosis/phagocytosis has to be considered
       3.2.   In vitro and in vivo durability studies:
              As discussed  in  Section  1.4.4 biodurability is considered  a  most important
parameter correlated with chronic fiber toxicity. However, as the example of chrysotile shows, a
short biopersistence may not always be equivalent to low toxicity.  An important issue is that
presently results from in vitro and  in vivo durability tests do not appear to correlate well, and it
would be useful to develop relevant in vitro assays which would mimic more closely biodurability.
For example, by changing flow conditions in the in vitro assay as well as fluid composition and
pH it may be possible to establish and standardize better predictive in vitro durability assays.  In
addition, it should also be considered to evaluate fiber breakage as part of the in vitro durability
studies.
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       An important aspect which should not be overlooked is that biodurability has potentially a
much greater importance for fiber retention in humans than in experimental animals. For example,
a dissolution rate of 0.35% per day (equivalent to a t ilftime of 200 days) will have much less of an
impact on overall biopersistence in the rat than in humans. That is because humans have a much
longer pulmonary retention halftime due to mechanical clearance mechanisms (400-700 days for
non-fibrous particles) than rats (-60-70 days) so that fiber dissolution in this example shortens
biopersistence of the fiber in humans much more than it does in rats.
       Even though in vitro dissolution rates may not be the same as those determined in vivo, it
would be useful to find out if the relative ranking of fiber dissolution rates between different fibers
is the same when performed in vitro vs.  in vivo.  Thus, a minimal requirement for an appropriate
in vitro durability assay should be that it gives the same ranking order for different fiber types as is
observed in vivo.
       When interpreting results of in vivo persistence assays, potential interference of pulmonary
inflammation on biopersistence due to high doses should be taken into account Dose levels from
short-term inhalation or intratracheal instillation should be sufficiently low to avoid significant
inflammatory responses.  In vivo assays of fiber persistence can either be expressed on a basis of
retained fiber mass or retained fiber number.  Retention expressed by fiber number should consider
different categories of fiber dimensions; however, during the clearance phase fiber dimensions can
change so that fibers can move from one  category into another, e.g., from a longer to a shorter due
to breakage, or from a thicker to a thinner due to dissolution. High leaching rates can also change
the mass without changing fiber dimensions and consequently influence retention curves based on
mass.  A comprehensive clearance model incorporating mechanical clearance, dissolution and
leaching, breakage and transport to other lung structures would be desirable.
       3.3    Intracavitarv studies;
             As discussed in Section 1.3, there  is considerable controversy regarding the use of
the results of intracavitary testing for purposes of risk assessment Primarily, the dose issue is of
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major concern which needs careful discussion in terms of the dose parameter to be used: Should it


be fiber mass, total fiber numbers (e.g., 1 x 107 -1 x 109), fiber numbers per bodyweight, or fiber


numbers per available surface area of the peritoneal or pleura! cavity? Should additional parameters


be determined, e.g., inflammatory cell influx, cell  proliferative responses of the  serosal lining,
                     i

persistence of such responses, in order to obtain a  more complete understanding of events and


sequences of responses being induced in the peritoneal cavity after injection of fibers?  Other


questions pertain to possible species differences in response, e.g., does the hamster respond more


sensitively after direct injection of fibers into its peritoneal or pleura! cavity than the rat? Or,  is the


apparent greater sensitivity of the hamster with respect to mesothelioma induction seen after


inhalation studies due to a different transport mechanism of the fibers from the alveolar space to the


pleura compared to the rat?
                                           i

       It appears that results  of intraperitoneal injections of fibers can be very useful for


establishing the relative toxicity (ranking) among different fiber types. Thus, this test may be very


valuable for purposes of hazard identification once it has been determined what the appropriate


dosing scheme should be. Doses recommended by  the participants of the Paris Workshop  of 50


mg per injection or 250 mg total are based on a number of 1 x 109 WHO fibers, and these are very


high doses administered to the available surface area in the peritoneal cavity.  A careful assessment


of the MTD needs to be performed for the intracavitary injection. Additional consideration should


be given to recommendations that both sexes of animals should  be tested, the use of positive


(which amphibole asbestos should be used?) and  negative control animals, histopathological


evaluation of all serosal surfaces (organs), and in addition to evaluating tumors also recording of


granulomas and fibrotic reactions.
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       3.4   Chronic inhalation studies - major issues for discussion!
             3.4.1  Characterizatk n of test sn nples - size, chemical composjdon. surface.
                    properties:
                    Since physical and chemical properties of fibers have been correlated with
their long-term toxicity and carcinogenicity, respective parameters need to be carefully evaluated.
Thus, bivariate distributions of fiber diameter and length need to be available for samples to be
tested. Analysis of chemical composition of the fibers, both individual fibers as well as bulk
samples, should be performed. Moreover, the percentage and size of non-fibrous particles should
also be determined since they could be a major portion of the test material Consideration should
also be given to samples which are used as large diameter products which may not be respirable for
humans, or only be respirable for humans to a limited degree; then the question becomes whether
samples of this material should be prepared to make them respirable for rats.
       Other recommendations made at the GOT Workshop (McClellan etal., 1992) included
number calculations of fibers using the NIOSH 7,400 PCOM phase contrast optical microscopy
technique, and measurements of aerodynamic diameters (MMAD and geometric standard deviation)
using cascade impactors. However, cascade impactors may only give an estimate of aerodynamic
diameters since fibers may behave differently in an impactor than in the airways. It should also be
decided whether light microscopy (PCOM) or scanning electronmicroscopy is to.be  used for
determining fiber length and diameter since results will differ. Use of SEM should be preferable.
             3.4.2  Rat vs. human respirabilitv:
                    As discussed in Section 1.4.1 and shown in Figures  1 and 2, there are
significant differences with respect to respirability between rodents and humans.  This is an
important issue deserving further discussion.  Participants at the CUT Workshop recommended to
use human respirable fiber samples, whereas participants at the Paris Fiber Workshop suggested to
use rat respirable fiber samples.  Model calculations predict significant differences in pulmonary
deposition in the rat between these two. It would be useful to verify  experimentally in  specifically
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designed short-term inhalation studies which portion and size distributions of a specific fiber

sample will deposit in a rat lung.

       As also discussed in this background paper, it needs to be evaluated as to whether fiber

sizes which are not respirable by the rat yet predictably deposit in the human lung should be forced

into the rat lung by using instillation in additional tests. Obviously, the question that needs to be

answered first is what fiber fractions will be encountered at the workplace, and subsequent

decisions have to be based on the answer to this question.

              3.4.3 Animal selection:

                    The rat is the preferred animal for testing. Since there is no indication from

inhalation studies with fibers that female and male rats may respond differently, use of one sex

(male) may be appropriate. The numbers should be high enough to allow for sufficient numbers of

animals to be evaluated at different sacrifice timepoints, which means that at least 100 animals per

group and concentration should be used; numbers have to be increased for any additional tests to

be performed. With respect to age at the start of exposure,  it is generally accepted that for

inhalation studies 8-10 week-old animals should be used Using younger animals (i.e., 6 weeks

of age as suggested by EPA's Health Effects Guidelines) might be risky since the animals  -

depending on the exposure method - may be under a greater stress and will not gain bodyweight

appropriately.

       A further question relates to the use of a  second species, i.e., the hamster, since this

species has been shown to react more sensitively with induction of mesotheliomas than rats do.

Presently, it is not known  why this significant species difference exists, and it needs to be

considered whether the hamster should be a mandatory second  test species in future testing

protocols.  Further research into this issue of species differences is necessary.  Additionally,

consideration of intratracheal instillation methods should be considered which may facilitate species

comparison.  Again, doses to be administered by instillation have to be carefully selected with

respect to the MTD.
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             3.4.4  Exposure conditions and
                    Fiber samples need to be aerosolized in such a way that they are evenly
distributed in the chamber atmosphere.  Fiber dispersion has to be adequately determined by
sampling throughout the exposure period as  recommended by the participants of the Paris
Workshop.  To avoid major contamination of external body surfaces, nose-only exposures may be
preferable to whole-body exposures. However, as mentioned above, it needs to be considered that
this poses a greater stress for the animals which is even more important for young animals of
smaller bodyweight, especially for hamsters. This may have an impact on the long-term outcome
and it needs to be stressed in any respective inhalation protocol that an adequate adaptation period
is used and that the animals are of sufficient age before being exposed. For example, 6 weeks of
age may be too early, especially for hamsters, and one might consider to select animals based on a
minimal bodyweight in addition to age.  The duration of the chronic inhalation study in rats is
generally about 2 years, with 6 hr/day of exposure for 5 days/week.  Killing groups of animals
every 6 months is advantageous to determine fiber burden parameters and fiber accumulation as
well as histopathological changes. This also provides further opportunities to perform mechanistic
studies with respect to evaluating predictive endpoints for chronic effects. A subsequent recovery
period should be included until -20% survival in one of the groups has occurred.
             3.4.5  Exposure concentration - MTD prediction:
                    In addition to three fiber-exposed groups, a sham-exposed (filtered air only)
control group is to be included which otherwise is kept the same way as the animals inhaling the
fiber aerosol. A most important question  relates to the MTD which should at best be determined
before the start of the chronic study by performing a 3-month subchronic study.  Two lower dose
levels should be appropriately spaced so that the lowest dose is at a level which does not induce
any observed effects. Changes in the rate  of bodyweight increase are not necessarily indicative of
an MTD in inhalation studies, and other parameters need to be found. Effect on lung clearance
mechanisms could be one such parameter which would define a Maximum Functionally Tolerated
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Dose.  For example, a doubling of normal alveolar retention halftime of test particles could be
used.  Other parameters to be considered could be lung lavage parameters with respect to
inflammatory response (increase of lavage PMN above a certain level, increase in lavage protein,
etc.) which could well be determined in the subchronic study. Additional parameters would be an
increase in cell proliferation, and an acceptable level of proliferation rates for specific cell types
needs to be defined.
       In the context of performing a subchronic study for MTD prediction, it is appropriate to
discuss the feasibility of using short-term inhalation studies (3-6 mos.) for predicting long-term
outcome. Although presently there may not be enough mechanistic information to determine
unequivocally which parameter may be best to use for such tests, this alternative to long-term
exposures should nevertheless be discussed. To perform a two-year inhalation study in rodents,
potentially in two species, to evaluate a new fiber product may not be necessary for each case, and
there is  a need to design short-term  tests.  The challenge  is to find endpoints which  are
scientifically acceptable as predictors for carcinogenicity.  The strategy is to compare such
endpoints from fibers of extreme carcinogenic potencies - highest and lowest - with those of the
fiber under test  Predictive parameters for long-term effects could include  fibrosis, but also
parameters more directly related to tumorigenesis,  i.e., assessing the mutation frequency of
alveolar epithelial cells.  A suggestion by Paris Workshop participants included as Addendum to
Appendix C lists this as well as other potential predictive short-term markers for chronic outcome.
              3.4.6 Positive control group:
                     Several long-term inhalation studies in the past with asbestos (crocidolite)
did not show lung tumor induction in rats. This failure may  be viewed as a limitation of the
inhalation mode of application; however, potential confounders in inhalation studies can be the
method of aerosol dispersion affecting fiber inhalability as well  as respirability, and consequently
lung burdens can be lower than expected. It is, therefore, mandatory to always determine fiber
burdens after and during inhalation studies, and to assure that a study is adequately sensitive a
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positive control group using an amphibole asbestos should be included as well. In this context, it
needs to be pointed out that there is an urgent need to obtain data from a well-performed chronic
inhalation study with asbestos at different exposure levels to which future inhalation studies can be
compared.  This would also  provide an opportunity for relative ranking of the toxicity and
carcinogenicity of different types of man-made vitreous fibers which at present cannot be done
appropriately.
              3.4.7  Gross Examinations:
                    Detailed macroscopic examinations of the respiratory tract surfaces and
other organs should be made on all animals including those which died during the experiment or
were killed because of moribund condition.
              3.4.8  Histopathologv:
                    As suggested in EPA's Health Effects Test Guidelines (EPA,  1992), the
entire respiratory tract should be preserved including nasal cavity, pharynx, larynx and perinasal
sinuses and the lungs including trachea and conducting airways. For preservation of the lungs
inflation with the appropriate fixative is  recommended as stated in EPA's guidelines.
Standardization of the inflation pressure, i.e., 20 cm H2O, should also be considered.  Use of the
Wagner Scale for fibrosis scoring can create difficulties and disagreement among pathologists, and
other scoring systems may  be better suited  as also pointed  out in the CIIT Workshop
recommendations.
              3.4.9  Lung burden analysis:
                    The recommendation from the Paris Workshop was to use fiber burdens of
the accessory lung lobe to determine total lung burden. However, it needs to be kept in mind that
distribution of inhaled paniculate material throughout the different lung lobes in a rat is not uniform
(Raabe  era/., 1977).  These  authors found significant differences between the  lobes, which
additionally differed for different particle sizes. Thus, it is difficult to predict from fiber burden in
the accessory lobe only total lung burdens, and if this is the objective then the whole lung needs to
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be used for ashing and fiber counting. Fiber accumulation kinetics and subsequent clearance
kinetics can be determined by having available data from a minimum of 3 animals for each 6-month
 i
sacrifice timepoint  Standardized operating procedures for ashing of lung lobes need to be
established and validated to assure that fibers are not lost during the preparation of tissues. Fiber
lung burdens should be expressed as fiber numbers per lung (or also per g lung); attempts to
convert number burdens into mass burdens have to take into account that fiber density may be less
than that of the bulk sample due to leaching processes in vivo.  This creates a problem for
determining fiber retention halftimes based  on the retained mass;  specific fiber categories
(dimensions) need to be considered separately.
             3.4.10 Analytical methods for fiber measurements:
                    Fiber dimensions need to be determined in the bulk material to be used for
aerosol generation, in the aerosolized phase and during and after exposure in lung tissues. This
includes determination of bivariate fiber diameter and length, fiber concentration in terms of fiber
numbers per cm3 (in air) or fiber numbers per lung and per g lung tissue.  Efforts should be made
also to assess the contribution of non-fibrous paniculate material since mis may be substantial and
could add significantly to total lung burden in terms of mass.
             3.4.11 Fiber counting:
                    Use of optical microscopy (PCOM) or electronmicroscopy (SEM) needs to
be specified, SEM might be  the preferred method because of its greater sensitivity (see Counting
Rules, Appendix C, Paris Workshop). Fiber counting is generally performed according to WHO
rules, i.e., fiber ^5 Jim in length, <3  ^m in diameter and aspect ratio of >3:1.  The NIOSH fiber
definition has the same length and diameter requirements but an aspect ratio of >5:1. NIOSH fiber
counts are, therefore, less than WHO fiber counts, and both are less than total fiber numbers.  Use
of NIOSH rules would increase the number of "non-fibrous" particles.  In any event,  it is
important to also include counting of non-fibrous paniculate material  since this can add
substantially to lung burdens that could significantly influence chronic outcome as shown in
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previous inhalation studies.  Additional counting rules as well as stopping rules for counting were
recommended by the Paris Workshop participants and are given in Appendix C.
              3.4.12  Clinical observations:
                    Clinical observations during an inhalation study are mostly restricted to
observing weight gain and survival. Since bodyweight development is a sensitive sign of general
toxicity this appears to be  adequate.  EPA's Health Effects Test Guidelines (1992) require in
addition blood samples to be taken from all animals at 12,18 and 24 months during a long-term
oncogenicity study. While this is quite appropriate for agents that may cause effects in blood cell
counts and composition, it would pose an additional stress on the animals in a long-term inhalation
study and may not be necessary. However, participants should discuss this requirement further.
              3.4.13  Bronchoalveolar lavage!
                    Very useful data can be obtained from the analysis of bronchoalveolar
lavages. Specifically for subchronic studies but also for chronic  studies, it should be required to
perform and analyze bronchoalveolar lavages.  Lavages can be done in situ with the lungs
remaining in the thoracic cavity or on the excised lungs, the latter method achieves a more
exhaustive cell yield and may be preferable. For a rat lung a lavage volume of 5 ml is appropriate,
and lavages should be repeated to give a total of 10 samples.  Lavage parameters to be determined
include total cell count, cell differential (PMN, alveolar macrophages, lymphocytes, and others),
cell viability, protein, LDH and (3-glucuronidase as examples of cytoplasmic  and lysosomal
enzymes. Protein and enzymes are best determined in the pooled first two lavages, where their
concentrations are highest. Cytospins of lavaged cells can be saved for later analysis of specific
molecular endpoints for a mechanistic evaluation if necessary. Likewise, lavage fluid supernatant
can also be stored frozen.
4.    Possible Criteria for Accepting a Negative Inhalation Test.
      EPA's draft guidelines for carcinogenicity addressed the issue of acceptable negative tests
by stating that the absence of tumor effects in an adequately sensitive and well-conducted study is

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accepted as a negative finding, and so are also studies with undue effects on mortality or health that
show no tumor effects and that include lower doses which are appropriately spaced.  If this
definition is acceptable the main question becomes what constitutes adequate sensitivity in a study.
Most important here is the issue of the dose, i.e., the upper limit for a high dose.  As was
discussed in this background paper several parameters could be used to define such dose,  e.g.,
impaired clearance, excessive inflammation, cell proliferation, significant fibrotic changes and
perhaps others. Anticipated human exposure levels need to be considered as well  It should also
be demonstrated that a sufficient dose of inhaled fibers reached sensitive sites in the lungs of the
experimental animals. Thus, determination of lung burdens including the adequacy of numbers of
long fibers with high aspect ratios becomes a very important part of chronic inhalation studies.
Furthermore, a positive control should be included demonstrating  the sensitivity of the model
system in order to accept a study as negative.  Obviously, animal survival needs to be adequate.
The following criteria should be discussed for the acceptance of an inhalation study as negative if
no tumors are observed:
       -  MTD is achieved in high exposure group, i.e., high lung burden is present
       -  appropriately spaced lower doses
       -  positive control group
       -  adequate animal survival (at least for lower exposure groups if increased mortality in
          highest group).
Additionally, the number of non-fibrous particles should be considered which may affect fibrosis
and tumorigenicity outcome.
5.     Summary of Issues  for Workshop  Discussion.
       Guidelines for toxicity and carcinogenicity testing should provide a detailed description of
the testing protocol which should be based on accepted sound scientific principles and methods. In
addition, justification and purpose of the testing should be included, and potential pitfalls, caveats
and disadvantages associated with a specific method may also be pointed out The protection from
exposure to unsafe agents, either in the general environment or at the workplace, should be the
prime concern when establishing testing guidelines.  However, detailed mechanistic information is
                                            IV-39

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lacking as a rule, and there are many species specific differences with respect to dosimetry,
metabolism, cell kinetics, defense mechanisms, etc. which affect the lexicological outcome. Thus,
 i
it is important to develop test information that includes such mechanistic data to better extrapolate
results from animal studies to humans.
       When comparing the two most widely used methods of fiber oncogenicity testing, i.e.,
long-term inhalation vs. intraperitoneal injection, it appears that the inhalation route of exposure as
the most physiological one should have an advantage over any other method. However, as pointed
out in Section 1.4.1, an important issue to be considered is the respirability of a given fiber sample
in rats vs. that in humans.  The dilemma is that there is this apparently most relevant mode of
administration yet potentially important fiber sizes with respect to adverse effects in humans may
not be respirable by the experimental animal. Thus, while inhalation in general may be viewed as
the "gold standard" when testing airborne agents the rat cannot necessarily be considered the gold
standard model for human extrapolation as far as certain sizes of airborne fibers are concerned. In
contrast, i.p. injection of a fiber sample can be performed with any fiber size and could, therefore,
potentially also permit evaluation of  non rat-respirable fibers.  However, as discussed in the
Section 1.4.4.3, the peritoneal cavity is  not equivalent to the alveolar space with respect to
structure, cell types, clearance and other defense mechanisms, and it is highly controversial which
doses are appropriate to be administered Lp. (see below). Intratracheal instillation could be viewed
as a potential compromise since human respirable samples can be administered directly into the
lung at defined doses. Indeed, studies in the past have shown that this route of administration will
identify carcinogenic fibers although  only a limited number of studies is available for a more
complete evaluation (Pott era/., 1987).  Obvious disadvantages are the uneven dosing of different
lung regions with potentially significant local responses due to a high fiber burden.  This aspect
needs to be discussed further at the workshop.
       Use of test results from long-term studies, either inhalation or injection, for the individual
steps within the risk assessment paradigm needs to be evaluated. For example, using results from
                                          IV-40

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a chronic inhalation study in rats for human risk characterization may have to be based on results of
a positive control, e.#•., amphibole asbestos. (It should be re-emphasized here that we do not have
results of a well-designed chronic rat inhalation study with multiple doses of asbestos which would
be needed as a basis for comparison). Although testing guidelines to be developed cannot and
should not give a detailed description about the individual steps for the extrapolation process the
general principles could be outlined as part of the objective and justification for establishing them.
On the other hand, it may be argued that.it is sufficient to compare testing results for different
fibers with each other to obtain a comparative relative ranking.
       The necessity to perform an appropriate range-finding study of shorter duration before
starting the chronic study needs some discussion as well. Specifically, this is important for the
characterization of an MTD which has been addressed in the newer EPA draft guidelines (EPA,
1994) and is of great significance for any long-term testing.  Obviously, attempts to characterize an
MTD before chronic testing should not only be mandatory for inhalation studies but also for i.p.
injection  studies and intratracheal instillation studies; appropriate relevant parameters which indicate
that the MTD is reached need to be defined for each mode of exposure.  Bodyweight changes alone
are not necessarily a good indicator of the MTD. For example, in the 2-year rat inhalation study
using TiO2 at the excessively high concentration of 250 mg/m3 no change in bodyweight occurred
(Lee et at., 1985). We need to find and define more appropriate parameters for the MTD.
      The dilemma obviously is how to decide when an observed response seen in a target organ
truly indicates an MTD rather than reflecting a normal host defense response. For example, when
is  an increase in activation and recruitment of alveolar macrophages and PMN or in cell
proliferation - seen as an inflammatory response to a high dose - an indication of the MTD being
exceeded and when is it a "physiological" response to the agent even at lower doses which does
not reflect an MTD? Maybe it is possible to define a level of inflammatory PMN influx into the
lung which  does not overwhelm normal defense mechanisms, e.g., pulmonary antioxidam
capacities.
                                         IV-41

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       Perhaps several criteria need to be met before deciding that an MTD has been reached, e.g.,
a certain level of cell proliferation + inflammatory cell influx + epithelial leakiness + effect on
functional parameter + altered retention kinetics, supported by other parameters related to lung
morphology.  Alternatively or in addition, could a functional parameter for the MTD be defined
such as the one proposed for spherical particles  in terms of the MFTD, i.e., affecting lung
clearance mechanisms?  Another possibility is to consider human exposure scenarios to be
expected and then use a factor 10 or more higher exposure as the maximum for a testing protocol.
It should be emphasized that a very careful evaluation of exposure-dose-response relationship
needs to be performed for defining MTD parameters for any testing protocol, whether inhalation or
injection studies.
       The issue of the MTD for i.p. injection studies requires some further consideration. The
peritoneal cavity consists of a large surface area of -600 cm2 in the rat (Rubin et al., 1988). If the
recommended maximum dose for the i.p. test, i.e., 50 mg per injection and 250 mg total
(Appendix D) would be deposited in the alveolar region of a rat lung (-4400 cm2 surface area) this
would be equivalent to an amount of 360 mg/rat lung per dose, or 1.8 g per rat lung maximally; the
latter is more than the weight of a rat lung. This is certainly highly excessive and would not be
acceptable as lung burden in a testing scheme, and the question is as to how peritoneal surface area
burdens can be adjusted accordingly.  This is of even greater importance considering that the
peritoneal cavity is not designed to be exposed to environmental particulate compounds, i.e., it
lacks respective cellular and other defenses, quite in contrast to the alveolar region of the lung.
       Protocols for fiber testing which have been published or are proposed in draft form (see
Appendices A-F) have emphasized the rat as the appropriate species for carcinogenicity testing of
fibers. However, since it is well known that the hamster responds with greater sensitivity with
respect to induction of mesothelioma after inhalation of fibers the use of this species as a required
test model should be considered as well
                                            IV-42

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       Inclusion of a positive control has not been addressed well in proposed guidelines for fiber
oncogenicity testing, except for the i.p. test (see Appendix D) where it is proposed to use UICC
crocidolite.  However, UICC crocidolite may not be the appropriate fiber sample as a positive
control since it seems to be of rather short fiber length.  Therefore, preparation of an amphibole
asbestos sample should be considered with appropriate length and diameter distribution. Most
importantly, as stated before, exposure-dose-response relationships need to be established at least
once for the positive control
       With respect to an evaluation of biopersistence it should be remembered that biopersistence
may not be the most important parameter correlated with adverse fiber effects, as demonstrable by
the example with chrysotile (see Section 1.4.3). There also appears to be a discrepancy between
results of durability derived from in vivo vs. in vitro dissolution studies because - as pointed out
before - both methods measure different parameters.  Appropriate adjustments of flows and other
conditions for in vitro  durability testing could be made to achieve better agreement between the two
methods. The goal would be to derive dissolution rates from an in vitro assay which are relevant
for in vivo.  Should pulmonary cells be used in the in vitro  assays? Luoto etal. (1994) reported
that marked differences can be found when in vitro studies of fiber dissolution are performed either
in a cell-free system or using alveolar macrophages which ingest the fibers.
       A further issue of general interest is as to whether new test methods of shorter duration  can
be established which are not as costly as long-term inhalation studies.  Having to test any newly
developed fiber in a 2-year inhalation study will become prohibitively expensive and  the
development of respective short-term tests from which long-term effects can be predicted would be
very desirable. Pulmonary parameters to be evaluated in such short-term tests (3-6 months) could
be fibrosis, (bistopathology) clearance effects,  fiber kinetics, evaluation of pleura! dosimetry, and
specific mechanistic studies including measuring mutation frequency in alveolar epithelial cells. Of
importance in this  regard would also be the cellular and biochemical evaluation of lung lavages
which contain very useful information about long-term effects.
                                        IV-43

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       It should also be considered whether a tiered testing approach can be used, starting with

acellular in vitro tests, then continuing with short-term intratracheal instillations before moving on

to inhalation studies, somewhat modified from what has been proposed by the CIIT Workshop

(Table 2).

       Additional topics for discussion include the question about the appropriateness of the

Fischer-344 rat as a standard rat strain. Although this strain is the standard NTP rat, longevity of

the F-344 rat appears to be less than that of Wistar rats.  Given the restrictions on use of animals

for testing, a justification for using only male rats can be made.

       Finally, with respect to fiber dosimetry and fiber counting the adequacy of using the

accessory lobe only for determination of lung burden needs to be considered.  Is this lobe

representative of all other lobes? As shown by Raabe etd. (1977), lobar deposition of particles is

not uniform among the different lobes of the rat lung, and changes also with particle size.

       In summary, for the development of standardized testing protocols for oncogenicity and

biopersistence of fibers (including inhalation, injection, instillation and acellular test systems) the

following issues have been identified to be discussed further at the workshop:

          inhalation vs. i.p. injection vs. intratracheal instillation for oncogenicity testing
          rat vs. human respirability
          F-344 rat vs. other rat strain, sex and animal numbers
          nose-only exposure and stress vs. whole-body exposure
          definition of MTD or MFTD
             —for inhalation
             - for i.p. injection
          dosimetry for Lp. injection (surface area dose)
          hamster as second species for evaluation of mesothelioma
          histppathology scoring system - e.g., Wagner Scale vs. other
          positive control group - appropriate amphibole sample
          acceptance of a negative test outcome
          in vivo vs. in vitro biopersistence
          predictive shorter-term test methods
          tiered testing approach
          representative fiber burden in lung from one lobe
          fiber counting rules
          extrapolation for purposes of risk assessment


Additional issues not discussed specifically here but mentioned in. proposed protocols and/or

guidelines may come up as well

                                      IV-44

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Bermudez, E. (1994). Recovery of particles from the pleura! cavity using agarose casts: A novel
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Coin, P.O., Roggli,  V.L., Brody, A.R. (.1992). Deposition, clearance, and translocation of
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EPA (1994). EPA Draft Revisions to Guidelines  for Carcinogen Risk Assessment, Review draft,
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IARC (1993).  IARC Monographs on the Evaluation of Carcinogenic Risks to Humans.  Vol. 58.
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Infante, P.P., Schuman, LJD., Dement, J. and Huff, J. (1994). Commentary ~ Fibrous Glass
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ELSI (1984).   International Life Sciences Institute.   The selection  of  doses in  chronic
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Klingholz, R. and Steinkopf, B.(1982). Das verhalten von Kiinstlichen Mineralfasera in einer
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Kreyling, W.G., Godleski, J.J.,  Kariya,  S.T., Rose, R.M., Brain,  J.D.  (1990).   In vitro
    dissolution of uniform cobalt oxide particles by human and canine alveolar macrophages.
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Kreyling, W.G.  (1992).  Intracellular particle dissolution  in alveolar macrophages.
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Kuwahara, M, Kuwahara, M, Verma, K., Ando, T., Hemenway,  D.R., Kagan, E. (1994).
    Asbestos exposure stimulates pleura! mesothelial cells to secrete the fibroblast chemoattractant,
    Fibronectin. Am. J. Respir. Cell Mol. Biol. 10:  167-176.
Law, B.D., Bunn, W.B., Hesterberg, T.W. (1990). Solubility of polymeric organic fibers and
    man-made vitreous fibers in Gamble's Solution.  Inhalation Toxicology 2: 321-339.
Lee, K.P., Trochimowicz, HJ. Reinhardt, CJ7. (1985). Pulmonary response of rats exposed to
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Leineweber, JJ*.(1984). The solubility of fibres in vitro and in vivo. ]ni  Biological Effects of
    Man-Made Mineral Fibres. \bl. 2, 87-101. WHO/IARC Meeting, Copenhagen.
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    Goche, T.J., Roycroft, J.H., and Chabra, R.S. (1989).    Establishing aerosol exposure
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Lundborg, M., Camner, P., Lind, B.(1984 ). Ability of rabbit alveolar macrophages  to dissolve
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Lundborg, M., Eklund, A., Lind, B, Camner, P.(1985).  Dissolution of metals by human and
   rabbit alveolar macrophages. Brit. J. Ind. Med. 4 2: 642-645.
Luoto, K.,  Kolopainen,. M., Karppinen, K.,  Perander, M.  and Savolainen, K.  (1994).
   Dissolution of man-made vitreous fibers in rat alveolar macrophage culture and Gamble's
   saline solution:  Influence of different  media and chemical composition of the fibers.
   Environm. Health Perspect. 102(Suppl. 5): 103-107.
Marafante, E., Lundborg,  M., Vahter, M., Camner, P.  (1987).  Dissolution of two arsenic
   compounds by rabbit alveolar macrophages in vitro. Fundamental & Applied Toxicology 8:
   382-388.
Mast, R.W., McConnell, E.E., Glass, L.R., Hesterberg, T.H., Anderson, R. and Bernstein,
   D.M. (1992). Inhalation oncogenicity study of kaolin refractory ceramic fiber (RCF)  in
   hamsters - Final results. The Toxicologist 12(1): 377.
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   Lippmann, M., Mast, R.W., McConnell, EE. and Reinhardt, C F. (1992). Approaches  to
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Raabe, O. G., Al-Bayati, M.A., Teague, S.V. and Rasolt, A. (1988). Regional deposition of
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                                        IV-49

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                                                 TABLE 1
                           MAN MADE VITREOUS FIBERS (MMVFl
                                        (Synthetic Inorganic Fibers)
          Types (WHO, 1988)

      Continuous Glass Filament
        • glass filament (textile glass)

      Insulation Wool
        • glass wool
        • rock wool
        • slag wool

      Refractory Fibers
        • refractory ceramic fibers (RGF)
           - kaolin clay-based
           - high purity
        • others

      Special Purpose Fibers
        • glass microfibers
            Composition

        (6-15 \un  diameter)
borosilicate, calcium-aluminum silicate glass

        (2-9 \im diameter)
borosilicate, calcium-aluminum silicate glass
natural igneous rock with high Ca, Mg
melted slag (iron, steel, copper) calcium-aluminum silicate

        (1.2 - 3 juifi diameter)
blends of alumina and silica, with other refractory oxides
        (0.1  - 3 pm diameter)
borosilicate, calcium-aluminum silicate glass
General properties'.  Amorphous; do not break longitudinally, break transversely; melting > 1000°C; chemically non-reactive
                                                    rv-50

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2: Tiered-approach to evaluating new fibers for characterizing toxicity and carcinogenicity.

                           (Mcdellaneraf.,  1992)
          I                         Product cycle evaluation
                                             and
                              Physical and chemical characterization

                                   /                    y
         n                  In vitro solubility          In vitro cell
                               and durability            toxicity


        m                       Short-term inhalation studies
                                  —Fiber deposition and retention
                                  —Respiratory tract toxicity


        IV                       Long-term inhalation studies
                                  —Fiber deposition and retention
                                  —Respiratory tract toxicity
                                    IV-51

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   0.35
    0.3
c 0.25
O
O
Q.
O
   0.1
  0.05
             Human (mouth-breathing, at rest)
                1234
                        Aerodynamic Diameter
                                                                 _  ' FIGURE 1
  0.35
   0.3
c 0.25
C

f
O
Q.
0)
   0.2
  o.15
  0.05
   . 0
           Rat
                        234
                        Aerodynamic Diameter
                                                      Sphen
                                                       0=3
                                                       £=10
                                   IV-52
                                                                      FIGURE 2

-------
FATE OF FIBERS AFTER DEPOSITION IN RESPIRATORY TRACT
Physical/Mechanical Processes
   (translocation; splitting; breaking)
   Chemical Processes
(biodurability; dissolution; leaching)
      (in vivo Vs. in vitro)
                         BIOPERSISTENCE
                              Effects
                                IV-53
                                                             FIGURE 4

-------
   10
I
v_
0
    8
.53  6
Q
o
03
T3
O
CD
    0
     0
                                23
                               Fiber Diameter
                                      W-54
                                                                        FIGURE 3

-------
                 APPENDIX A
       EPA's Health Effects Testing Guidelines
Oncogenicity and Combined Chronic Toxicity/Oncogenicity

 (Code of Federal Regulations, Title 40, part 798.3300 -
         798.3320, pp.449^61, July 1, 1994)
                      IV A- 1

-------
Environmental Protection Agency
                            §798.3300
  (J)  Nominal  concentration  (total
amount of test substance fed  into the
inhalation equipment divided by  vol-
ume of air).
  (4)  Actual  concentration  in  test
breathing zone.
  (5)  Particle  size  distribution  (e.g.,
median aerodynamic diameter of  par-
ticles with standard deviation from the
mean).
  (d)  References. For additional back-
ground information  on this test guide-
line the following references should be
consulted:
  (1)  Benitz,  K.F.  "Measurement of
Chronic  Toxicity,"  Methods of Toxi-
cology.   Ed.  GJB.   Paget.   (Oxford:
Blackwell Scientific Publications, 1970)
pp. 82-131.
  (2)  D'Aguanno.  W.  "Drug  Safety
Evaluation—Pre-Clinlcal     Consider-
ations,"     Industrial    Pharmacology:
Neuroleptics. Vol. I. Ed. S. Fielding and
H. Lai.  (Mt. Kisco:  Futura Publishing
Co. 1974) pp. 317-332.
  (3)  Fitzhugh,  O.G. Third Printing:
1975.  "Chronic  Oral  Toxicity,"  Ap-
praisal of the  Safety  of Chemicals in
Foods, Drugs and Cosmetics. The Asso-
ciation  of Food and Drug Officials of
the United States (1959, 3rd Printing
1975) pp. 36-45.
  (4) Goldenthal. E.L. D'Aguanno, W.
"Evaluation of Drugs," Appraisal of the
Safety of Chemicals in Foods, Drugs, and
Cosmetics. The Association of Food and
Drug Officials of the  United States
(1959. 3rd Printing 1975) pp. 60-67.
  (5) National  Academy of Sciences.
"Principles and Procedures for Evalu-
ating the Toxicity of Household Sub-
stances,"  a  report  prepared  by  the
Committee  for  the  Revision  of NAS
Publication 1138, under the auspices of
the  Committee  on  Toxicology,  Na-
tional   Research   Council,   National
Academy  of Sciences, Washington, DC
(1977).
  (6) National Center for Toxicologlcal
Research.  "Appendix  B,"   Report  of
Chronic  Studies  Task Force Committee,
April 13-21, 1972. (Rockville:  National
Center  for  Toxicological  Research,
1972).
  (7) Page, N.P.  "Chronic Toxicity and
Carcinogenicity Guidelines," Journal of
Environmental Pathology and Toxicology,
1:161-182 (1977).
  (8)   Schwartz,   E.  "Toxicology   of
Neuroleptic  Agents," Industrial Phar-
macology: Neuroleptics Ed.  S.  Fielding
and H. Lai. (Mt. Kisco, Futura Publish-
ing Co., 1974) pp. 203-221.
  (9)  United States Pharmaceutical
Manufacturers Association. Guidelines
for the Assessment of Drug and Medical
Device Safety in Animals. (1977).
  (10)  World  Health   Organization.
"Guidelines for Evaluation of Drugs for
Use in Man," WHO Technical Report Se-
ries No. 563. (Geneva: World Health  Or-
ganization, 1975).
  (11)  World  Health   Organization,
"Part I. Environmental Health Criteria
6," Principles and Methods for  Evaluat-
ing the Toxicity  of Chemicals. (Geneva:
World Health Organization. 1978).
  (12)  World  Health   Organization.
"Principles  for Pre-Clinlcal Testing of
Drug Safety." WHO  Technical Report
Series No. 341. (Geneva: World Health
Organization, 1966).
[SO FR 39387, Sept. 27, 1985, as amended at 54
FR 21064. May 16.1989]

S79&3300 Oncogenicity.
  (a) Purpose. The objective of a long-
term oncogenlcity study  is to observe
test animals for a major  portion of
their life span for the development of
neoplastlc lesions during  or after expo-
sure to  various  doses of a test sub-
stance by an appropriate route of  ad-
ministration.
  (b) Test procedures—(I)  Animal selec-
tion—(1) Species and strain. A compound
of unknown activity shall be tested on
two mammalian species. Rats and mice
are the  species  of  choice  because of
their relatively  short life  spans,  the
limited  cost of  their   maintenance.
their  widespread   use   in   pharma-
cological  and  lexicological  studies,
their susceptibility  to  tumor induc-
tion, and the availability of inbred or
sufficiently    characterized   strains.
Commonly  used   laboratory   strains
shall be employed. If other species are
used, the tester shall provide justifica-
tion/reasoning for their selection.
  (11) Age. (A) Dosing of  rodents shall
begin as soon as possible a/ier weaning,
ideally before the animjUm are  6 weeks
old, but in no case more  r ban  8 weeks
old.
  (B) At  commencement  of the study,
the weight  variation  of  Animals used
                                   IV A-2

-------
 §798.3300
          40 CFR Ch. I (7-1-94 Edition)
 shall  not exceed ±20 percent  of  the
 mean weight for each sex.
   (C)   Studies   using  prenatal   or
 neonatal animals may be recommended
 under special conditions.
 ,  (ill) Sex.  (A)  Animals  of each  sex
 shall be used at each dose level.
   (B) The females shall be nulliparous
 and non-pregnant.
   (iv) Numbers. (A) For rodents, at least
 100  animals (50  females and SO  males)
 shall  be used at each dose level and
 concurrent control.
   (B) If interim sacrifices are planned
 the  number shall be Increased  by  the
 number of animaia scheduled to be sac-
 rificed during the course of the study.
   (C) The number of animals at the ter-
 mination  of the  study should be ade-
 quate for a meaningful and valid statis-
 tical evaluation of long term exposure.
 For a valid  interpretation of negative
 results.  It is essential that survival in
 all groups does  not fall  below SO per-
 cent at the time of termination.
   (2) Control groups,  (i) A concurrent
 control  group is required. This group
 shall be an untreated or sham treated
 control group or, if a vehicle is used in
 administering' the test substance, a ve-
 hicle control group. If the toxic prop-
 erties of the vehicle are not known or
 cannot  be made available,  both un-
 treated and vehicle control  groups are
 required.
  (11) In special circumstances such  as
 in Inhalation studies involving aerosols
 or  the   use  of  an  emulslfier   of
 uncharacterized  biological activity  in
 oral  studies,  a  concurrent negative
 control  group shall  be .utilized. The
 negative control group shall be treated
 In the same manner  as all  other test
 animals  except that this control group
 shall not be  exposed to either the test
 substance or any vehicle.
  (Ill)  The use  of historical control
 data (i.e.,  the Incidence of tumors and
 other suspect lesions normally  occur-
 ring under the same laboratory  condi-
 tions and in the same strain of animals
 employed  in the  test) is desirable  for
 assessing  the significance  of changes
 observed in exposed animals.
  (3) Dose levels  and dose selection.  (1)
 For  risk assessment purposes, at least
3 dose levels shall be used, in addition
 to the concurrent control group. Dose
 levels should be  spaced to produce a
 gradation of chronic effects.
  (11) The high dose level should elicit
 signs of minimal  toxlclty without sub-
 stantially  altering  the  normal  life
 span.
  (ill) The lowest dose should not inter-
 fere with normal  growth, development
 and longevity of the  animal; and  it
 should not otherwise cause any indica-
 tion of toxiclty. In general, this should
 not be  lower than ten  percent  of the
 high dose.
  (Iv) The Intermediate dose(s) should
 be  established in  a mid-range  between
 the high and low doses, depending upon
 the toxicoklnetlc properties  of  the
 chemical. If known.
  (v) The selection of these dose levels
 should be based on existing data, pref-
 erably on the  results  of subchronlc
 studies.
  (4) Exposure conditions. The  *"
-------
 Environmental Protection Agency
                            §798.3300
 in the  diet,  exposure shall be continu-
 ous.
   (C) For a  diet mixture,  the highest
 concentration should not exceed 5 per-
 cent.
   (ii) Dermal studies. (A) The animals
 are treated by topical application with
 the test substance, ideally for at least
 6 hours per day.
   (B) Fur should  be clipped  from the
 dorsal area of the trunk of the test ani-
 mals. Care should be taken to avoid ab-
 rading  the skin which could alter its
 permeability.
   (C) The test  substance shall be ap-
 plied uniformly over  a shaved  area
 which is approximately 10 percent of
 the total body surface area. With high-
 ly  toxic  substances, the surface  area
 covered may be  less, but as much of the
 area shall be covered with as thin and
 uniform a film as possible.
  (D) During the exposure  period, the
 test substance  may be held. If  nec-
 essary,  in contact with the skin with a
 porous  gauze dressing and non-irritat-
 ing tape. The test site should be  fur-
 ther covered in a suitable manner to
 retain the gauze dressing and test sub-
 stance  and ensure  that  the  animals
 cannot ingest the test substance.
  (ill) Inhalation studies. (A)  The  ani-
 mals  shall be  tested  with inhalation
 equipment designed to sustain a mini-
 mum  dynamic air flow of 12  to 15 air
 changes per hour,  ensure an  adequate
 oxygen  content of 19  percent and an
 evenly  distributed  exposure  atmos-
 phere. Where a chamber is used, its de-
 sign should minimize crowding of the
 test animals  and .maximize their expo-
 sure to  the test  substance.  This is best
 accomplished by individual caging. To
 ensure stability of a chamber atmos-
 phere, the total "volume" of the  test
 animals shall not  exceed 5 percent of
 the volume of the test chamber. Alter-
 natively,   oro-nasal,  head-only,   or
whole-body individual chamber expo-
sure may be used.
  (B) The  temperature at  which  the
test is performed should be maintained
at 22 °C (±2°). Ideally, the relative  hu-
midity should be maintained between
40 to  60  percent,  but  in  certain  in-
stances  (e.g.  tests of aerosols, use of
water  vehicle) this may not  be prac-
ticable.
   (C) Feed and water shall be withheld
 during each daily 6-hour exposure pe-
 riod.
   (D)  A  dynamic  inhalation system
 with a  suitable  flow  control system
 shall be used. The rate of air flow shall
 be adjusted to ensure that conditions
 throughout  the  equipment are essen-
 tially the same. Maintenance of slight
 negative pressure inside the chamber
 will prevent leakage of the test  sub-
 stance into the surrounding areas.
   (7) .Observations of animals, (i)  Each
 animal shall be observed daily and if
 necessary should  be handled to  ap-
 praise its physical condition.
   (11) Additional observations shall be
 made  dally  with appropriate  actions
 taken to minimize  loss of animals to
 the study (e.g., necropsy or  refrigera-
 tion of those animals found  dead and
 isolation or  sacrifice of weak or mori-
 bund animals).
   (ill)  Clinical signs  and  mortality
 shall be recorded  for all animals. Spe-
 cial attention should be paid to tumor
 develqpment. The day of onset,  loca-.
 tlon, dimensions,  appearance and pro-
 gression of each grossly visible or pal-
 pable tumor shall be recorded.
   (iv) Body weights shall be recorded
 individually  for all animals once  a
 week during the first 13 weeks of the
 test period and at least once every 4
 weeks thereafter unless  signs of clini-
 cal  toxlclty  suggest more  frequent
 weighings  to facilitate  monitoring of
 health status.
  (v) When the test substance  is admin-
 istered in  the feed or drinking water.
 measurements  of  feed  or  water  con-
 sumption,  respectively, shall  be deter-
 mined weekly during the first 13 weeks
 of the study and then at approximately
 monthly intervals unless health status
 or body weight changes  dictate other-
 wise.
  (vi) At the end of the study period all
 survivors are sacrificed. Moribund ani-
 mals shall be removed and sacrificed
 when noticed.
  (8) Physical measurements. For inhala-
 tion studies,  measurements  or mon-
 itoring should be  made of  the follow-
 ing:
  (1) The rate of air flow shall be mon-
itored continuously and recorded at in-
 tervals of at least once every 30 min-
utes.
                                  IV A-4

-------
 §798.3300
          40 CFR Ch. I (7-1-94 Edition)
   (11) During each exposure period the
 actual concentrations of the test sub-
 stance snail be  held as constant as
 practicable,  monitored  continuously
 and recorded at least three times dur-
 ing the test period: at the beginning, at
 an intermediate time and at the end of
 the period.
   (ill) During the development  of the
 generating system, particle size  analy-
 sis shall  be performed to establish the
 stability  of  aerosol concentrations
 with respect to particle size. During ex-
 posure, analyses shall be conducted as
 often  as necessary  to determine the
 consistency of particle  size, distribu-
 tion, and homogeneity of the exposure
 stream.
   (iv) Temperature and humidity shall
 be monitored continuously, but shoud
 be recorded at intervals of at least once
 every 30 minutes.
   (9)   Clinical  examinations.  At  12
 months, 18 months, and  at sacrifice, a
 blood smear shall be obtained from all
             differential  blood  count
 shall  be performed  on blood  smears
 from those «.n
-------
 Environmental Protection Ager ;y

  (v) An attempt shall be mad? to cor-
 relate gross observations  with micro-
 scopic findings.
  (c) Data and reporting—(1) Treatment
 of results. (1) Data shall be summarized
 in tabular form, showing for each test
 group  the number of animals  at  the
 start of the test, the nvmber of ani-
 mals showing lesions, the types of le-
 sions and the percentage  of animals
 displaying each type of lesion.
  (ii) All observed results, quantitative
 and incidental, shall be evaluated  by
 an appropriate statistical method. Any
 generally accepted statistical  method
 may be  used;  the statistical methods
 shall be selected during the design of
 the study.
  (2) Evaluation of study results. (1) The
 findings of an oncogenic toxicity study
 shall be evaluated in conjunction with
 the findings of preceding  studies and
 considered in terms of the toxic effects,
 the  necropsy  and   histopathologlcal
 findings. The evaluation shall  Include
 the relationship between  the  dose, of
 the test substance and the presence, in-
 cidence and  severity of abnormalities
 (including behavioral and  clinical  ab-
 normalities), gross lesions. Identified
 target organs, body weight changes, ef-
 fects on mortality and any other gen-
 eral or specific toxic effects.
  (11) In any study which demonstrates
 an absence of toxic effects, farther In-
 vestigation to establish absorption and
 bioavallablllty of the test substance
 should be considered.
  (Ill) In order for a negative test to be
 acceptable. It shall meet the following
 criteria: no more than  10  percent  of
 any group Is lost due to autolysls, can-
 nibalism,  or  management problems;
 and survival in each group should be no
 less than 50  percent at 18 months  for
 mice and hamsters and  at 24 months
 for rats.
  (3) Test  report. (I) In addition to the
reporting  requirements  as specified
under 40 CFR part 792, subpart J the
following specific Information shall be
reported:
  (A) Group animal data. Tabulation of
 toxic response  data by species, strain,
sex and exposure level for:
  (1) Number of a.nimn.in dying.
  (2) Number of animals showing signs
 of toxicity.
  (3) Number of animaia exposed.
                            §798.3300
                          •
  (B) Individual animal data:  (1) Time of
 death during the study or whether ani-
 mals survived to termination.
  (2) Time  of  observation of  each ab-
 normal sign and its subsequent course.
  (3) Body weight data.
  (4) Feed and water consumption data,
 when collected.
  (5) Results  of ophthalmologies!  ex-
 amination, when performed.
  (6) Hematological tests employed and
 all results.
  (7) Clinical  biochemistry  tests em-
 ployed and all results.
  (8) Necropsy findings.
  (9)  Detailed  description  of   all
 histopathologlcal findings.
  (10) Statistical treatment  of results,
 where appropriate.
  (11) Historical control data.  If taken
 Into account.
  (11) In addition, for inhalation studies
 the following shall be reported:
  (A) Test conditions. (1) Description of
 exposure  apparatus  Including design,
 type, dimensions, source of air. system
 for   generating   partlculates    and
 aerosols,  method of conditioning air.
 treatment of exhaust air and the meth-
 od  of housing the animals  in a test
 chamber.        '     /
  (2)  The  equipment  for measuring
 temperature, humidity, and paniculate
 aerosol concentrations and size shall be
 described.
  (B) Exposure data. These shall be tab-
 ulated and presented with mean values
 and  a  measure  of  variability  (e.g..
 standard deviation) and shall include:
  (7) Airflow rates through the Inhala-
 tion equipment.
  (2) Temperature and humidity of air.
  (J)  Nominal  concentration  (total
 amount of test substance fed into the
 Inhalation equipment divided  by vol-
 ume of air).
  (4)  Actual   concentration   in  test
 breathing zone.
  (5)  Particle  size  distribution  (e.g..
median  aerodynamic diameter of par-
 ticles with standard deviation from the
mean).
  (d) References. For additional  back-
ground information on this test guide-
line  the following references should to
consulted:
  (1)  Department   of   Health   and
Welfare.   The  Testing  of  Chemical*
for   Carcinogenlcity,   Mutagenicity.
                                   IV A-6

-------
 §798.3320
          40 CFR Ch. I (7-1-94 Edition)
 Teratogeniclty. Minister of Health and
 Welfare.   (Canada:   Department   of
 Health and Welfare, 1975).
   (2) Food  and Drag Administration
 Advisory Committee on Protocols  for
 Safety Evaluation: Panel  on Carclno-
 genesis. "Report on Cancer Testing In
 the Safety of Food Additives and Pes-
 ticides,"  Toxicology  and Applied  Phar-
 macology. 20:419-438 (1971).
   (3) International Union Against Can-
 cer.  "Carclnogenlclty Testing,"  IUCC
 Technical Report Series. Vol. 2., Ed. I.
 Berenblum.   (Geneva:   International
 Union Against Cancer, 1969).
   (4) Leong. B.K.J.,  Laskln.  S. "Num-
 ber and Species of Experimental Ani-
 mals  for  Inhalation  Carclnogenlcity
 Studies"   Paper   presented  at  Con-
 ference on Target Organ Toxiclty, Sep-
 tember 1975, Cincinnati, Ohio.
   (5) National  Academy  of Sciences.
 "Principles  and Procedures for Evalu-
 ating the  Toxiclty of Household Sub-
 stances."  A report prepared by the
 Committee  for the  Revision of NAS
 Publication  1138. under the auspices of
 the  Committee on  Toxicology,  Na-
 tional  Research  Council.  National
 Academy of Sciences, Washington. DC
 (1977).
   (6) National Cancer Institute. Report
 of the Subtask Group on Carcinogen Test-
 ing  to the   Interagency  Collaborative
 Group on Environmental Carctnogenesis.
 (Bethesda: United States National Can-
 cer Institute, 1976).
   (7) National Center for Toxicological
 Research.  "Appendix  B,"  Report   of
 Chronic Studies Task Force Committee.
 April 13-21 (RockvlUe: National Center
 for Toxicological Research. 1972).
   (8) Page, N.P. "Chronic Toxiclty and
 Carclnogenicity Guidelines," Journal of
 Environmental Pathology and Toxicology.
 1:161-182 (1977).
  (9) Page,  N.P. "Concepts of a Bio-
 assay Program in  Environmental Car-
 clnogenesia," Advances in Modern Toxi-
 cology  Vol.   3,   Ed.   KrayblU  and
 Mehlman.  (Washington,  DC:  Hemi-
 sphere  Publishing  Corporation,  1977)
 pp. 87-171.
  (10)   Sontag,   J.M..  Page   N.P.,
 Safflotti,  U. Guidelines for Carcinogen
Bioassay in Small Rodents. NCI-CS-TR-
1.  (Bethesda: United States Cancer In-
stitute, Division of Cancer Control and
 Prevention.  Carclnogenesls  Bioassay
 Program, 1976).
   (11) United States  Pharmaceutical
 Manufacturers  Association. Guidelines
 for the Assessment of Drug and Medical
 Device Safety in Animals. (1977).
   (12)  World  Health   Organization.
 "Principles for the Testing and Evalua-
 tion  of Drugs  for  Carclnogenlclty."
 WHO  Technical  Report Series No.  428.
 (Geneva:  World Health  Organization,
 1969).
   (13)  World  Health   Organization.
 "Part L Environmental Health Criteria
 6," Principles and Methods for Evaluat-
 ing the Toxiclty  of Chemicals. (Geneva:
 World Health Organization, 1978).
 [60 FR 39397, Sept. 27, 1965, as amended at 83
 PR 19075. May 20. 1987; 64 FR 21064. May 16.
 1969]

 S7B&3320  Combined chronic tozicity/
    oncogenicity.
   (a)  Purpose. The objective of a com-
 bined  chronic  toxlcity/oncogenlclty
 study is to determine the effects of a
 substance in a mammalian species fol-
 lowing  prolonged and repeated  expo-
 sure.  The application of this guideline
 should  generate data  which identify
 the majority  of chronic and oncogenlo
 effects and determine dose-response re-
 lationships.  The design  and conduct
 should allow  for the detection of neo-
 plastic  effects and a determination of
 oncogenlc potential as well as general
 toxldty. including neurological,  phys-
 iological,      biochemical,       and
 hematologlcal effects and exposure-re-
 lated  morphological  (pathology)  ef-
 fects.
  (b)  Test procedures—<1)  Animal  selec-
 tion—
-------
 Environmental Protection Agency

 tiflcation/reasoning for their selection.
 The strain selected should be suscep-
 tible to the oncogenic or toxic effect of
 the class of substances being tested, if
 Known, and provided it does not have a
 spontaneous background  too high  for
 meaningful   assessment.   Commonly
 used laboratory strains should be em-
 ployed.
  (11) Age. (A) Dosing  of rats  should
 begin as soon as possible after weaning,
 ideally before the rats are 6 weeks -old,
 but in no case more than  8 weeks old.
  (B) At commencement of the study,
 the weight variation of animals used
 should not exceed. i20  percent  of  the
 mean weight for each sex.
  (C)  Studies   using   prenatal   or
 neonatal animals may be recommended
 under special conditions.
  (ill) Sex. (A) Equal numbers of ani-
 mals of each sex should be  used at each
 dose level.
  (B) The females should be nulliparOus
 and nonpregnant.
  (iv) Numbers. (A) At least 100 rodents
 (SO females and  SO males) should  be
 used at each dose  level  and concurrent
 control for those  groups not  Intended
 for early sacrifice. At least 40 rodents
 (20 females and  20 males) should  be
 used for satellite doae groupfc) and the
 satellite  control group.  The purpose of
 the satellite group is to allow for the
 evaluation of pathology other than ne-
 oplasia.
  (B) If interim sacrifices are planned,
 the number of a.nim«.ia should  be  In-
 creased  by the   number  of  animals
 scheduled to be sacrificed during the
 course of the study.
  (C) The number of animals at the ter-
 mination  of each  phase of the study
 should be  adequate for a meaningful
 and valid statistical evaluation of long
 term exposure. For a valid interpreta-
 tion  of negative results, it Is essential
 that survival in  all groups  not fall
 below 50  percent at the time of termi-
 nation.
  (2) Control groups. (1) A concurrent
 control group (50 females and 50 males)
and  a  satellite control group (20  fe-
 males and 20 males) are recommended.
 These groups  should be untreated  or
 sham treated control groups or, If a ve-
 hicle is used in administering the test
 substance,  vehicle control groups.  If
 the toxic properties of the vehicle are
                            §798.3320

 not known or  cannot be made avail-
 able, both untreated and vehicle  con-
 trol groups are recommended. Animals
 in the satellite control group should be
 sacrificed at the same time the sat-
 ellite test group Is terminated.
   (11) In special circumstances  such as
 inhalation studies Involving aerosols or
 the   use   of   an   emulsifler    of
 uncharacterlzed biological activity in
 oral  studies,  a  concurrent negative
 control group should be utilized.  The
 negative control group should be treat-
 ed in the same manner as all other test
 animals, except that this control group
 should not be exposed to the test sub-
 stance or any vehicle.
   (Ill) The  use of  historical  control
 data  (I.e., the Incidence of tumors and
 other   suspect   lesions    normally
 occurlng  under the same  laboratory
 conditions and in the same strain  of
 animals employed in the test) is desir-
 able  for assessing the significance  of
 changes observed In exposed animals.
   (3)  Dose levels and dose selection, (i)
 For risk assessment purposes, at least
 three dose levels should be used, in ad-
 dition to the concurrent control group.
 Dose  levels should be spaced  to produce
 a gradation of effects.
   (11) The highest  dose level  in rodents
 should elicit signs of toxlclty without
 substantially  altering the normal life
 span due to effects other than tumors.
   (Ill) The  lowest  dose  level  should
 produce no evidence of toxlclty. Where
 there is a usable estimation of human
 exposure, the lowest dose level should
.exceed this even though this dose level
 may result In some signs of toxiclty.
  (ly) Ideally, the  intermediate  dose
 level(s) should  produce  minimal  ob-
 servable toxic effects. If more than one
 Intermediate dose is used the dose lev-
 els should be  spaced to produce a gra-
 dation of toxic effects.
  (v)  For  rodents, the Incidence of  fa-
 talities in low  and  intermediate dose
 groups and  in the controls  should be
 low to permit a meaningful evaluation
 of the results.
  (vi) For chronic toxicological  assess-
 ment, a high dose treated satellite and
 a concurrent control  satellite  group
 should be Included in the study  design.
 The highest dose  for satellite animals
 should be chosen so as to produce frank
 toxlcity, but not excessive lethality, in
                                  IV A-8

-------
 §798.3320
          40 CFR Ch. I <7-l-*4 Edition)
  (11) Historical control data. If taken
 Into account.
  (11) In addition, for Inhalation studies
 the following should be reported:
  (A) Test Conditions. (7) Description of
 exposure apparatus  including design,
 type, dimensions, source of air, system
 for  • generating   participates   and
 aerosols, method of conditioning air,
 treatment of exhaust air and the meth-
 od of housing  the animals in a  test
 chamber.
  (2)  The  equipment  for  measuring
 temperature, humidity, and partlculate
 aerosol concentrations and size should
 be described.
  (B) Exposure  data. These  should be
 tabulated and presented with mean val-
 ues and a measure of variability (e.g.
 standard deviation) and should include:
  (1)  Airflow races through the inhala-
 tion equipment.
  (2) Temperature and humidity of air.
  (3)   Nominal   concentration  (total
 amount of  test substance fed  into the
 Inhalation  equipment divided by  vol-
 ume of air).
  (4)  Actual  concentration  in   test
 breathing zone.
  (5) Particle size distribution  (e.g. me-
 dian aerodynamic diameter of particles
 with   standard  deviation  from  the
 mean).
  (d)  References. For additional back-
 ground information on this test guide-
 line the following references should be
 consulted:
  (1)  Benltz, K.F.  "Measurement of
 Chronic  Toxlcity."   Methods  of  Toxi-
 cology.   Ed.  O.E.   Paget.   (Oxford:
 BlackweU Scientific Publications, 1970)
 pp. 82-131.
  (2)  D'Aguanno.  W.  "Drug  Safety
 Evaluation—Pre-Clinical     Consider-
 ations,"    "Industrial   Pharmacology:
 Neuroleptics. Vol. I Ed.  S.  Fielding and
 H. Lai. (Mt. Kisco, New York: Futura
 Publishing Co.. 1974) pp. 317-332.
  (3)  Department of  Health and  Wel-
 fare.  The Testing of Chemicals for Car-
 cinogenicity, Mutagenicity; Teratogeni-
 city.  Minister of  Health  and  Welfare.
 (Canada: Department of Health and Wel-
fare, 1975).
  (4) Fltzhugh, O.G. "Chronic Oral Tox-
 icity," Appraisal of the Safety of Chemi-
 cals in Foods, Drugs and Cosmetics.  The
 Association of Food and Drug  Officials
of the United States (1959, 3rd Printing
1975). pp. 36-45.
  (5)  Food  and  Drug  Administration
Advisory  Committee on  Protocols  for
Safety Evaluation: Panel on Carclno-
genesis. "Report on Cancer Testing in the
Safety of Food Additives and Pesticides,"
Toxicology and  Applied Pharmacology.
20:419-438(1971).
  (6) Ooldenthal, E.I., and D'Aguanno.
W.  "Evaluation  of Drugs," Appraisal of
the Safety of Chemicals in Foods,  Drugs,
and Cosmetics. The Association of Food
and Drug Officials of the United States
(1959, 3rd printing 1975) pp.60-67.
  (7) International Union Against Can-
cer. "Carcinogenlclty Testing," IUCC
Technical  Report Series Vol. 2,  Ed.  L
Berenblum.   (Geneva:    International
Union Against Cancer, 1969).
  (8)  Leong. B.K.J., and Laskin,  8.
"Number  and Species of Experimental
Animals for Inhalation Carcinogenlclty
Studies,"  Paper  presented  at  Con-
ference on Target Organ Toxlcity. Sep-
tember, 1975. Cincinnati, Ohio.
  (9)  National  Academy  of Sciences.
"Principles  and Procedures for Evalu-
ating the Toxlcity of Household Sub-
stances,"  A report prepared  by  the
Committee  for  the  Revision of NAS
Publication 1138, under the auspices of
the  Committee  on  Toxicology, Na-
tional  Research  Council.   National
Academy  of Sciences, Washington. DC
(1977).
  (10) National Cancer Institute. Report
of the Subtask Group on Carcinogen Test-
ing  to  the  Interagency  Collaborative
Group on  Environmental Carcinogenesis.
(Bethesda: United States National Can-
cer Institute, 1976).
  (11)  National   Center  for   Toxi-
cologlcal. Report of Chronic Studies Task
Force Research Committee.  "Appendix B,
(Rockville:  National  Center  for  Toxi-
cological Research, 1972)).
  (12) Page. N.P. "Chronic Toxicity and
Carcinogenlclty   Guidelines,"  Journal
Environmental Pathology and Toxicology.
1:161-182 (1977).
  (13) Page, N.P.  "Concepts  of a Bio-
assay Program in Environmental Car-
cinogenesis," Advances in Modern Toxi-
cology Volume  3,  Ed.  Kraybill and
Mehlman.  (Washington,  D.C.:  Hemi-
sphere Publishing Corp.,  1977) pp. 87-
171.
                                  IVA-9

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 Environmental Protection Agency

 individual caging:. As a general rule, to
 ensure  stability  of a chamber  atmos-
 phere, tae total  "volume"  of the test
 animals should not exceed 5 percent of
 the volume of the test chamber. Alter-
 natively, oro-nasal, head only, or whole
 body individual chamber exposure may
 be used.
  (B) The temperature  at  which  the
 test is performed should be maintained
 at 22 "C (±2*). Ideally, the relative hu-
 midity  ihould.be maintained between
 40 to 6B  percent, but in certain in-
 stances (e.g., tests of aerosols,  use of
 water vehicle) this may not  be prac-
 ticable.
  (C) Feed and water should  be with-
 held during each  dally 6-hour exposure
 period.
  (D) A  dynamic  inhalation  system
 with  a  suitable analytical  concentra-
 tion  control  system  should be used.
 The rate of air now should be adjusted
 to ensure that conditions throughout
 the  equipment   are   essentially  the
 same. Maintenance of slight negative
 pressure inside the chamber will pre-
 vent leakage  of the test substance into
 the surrounding areas.
  (7)  Observation  of animals. (1) Each
 animal ihould be  handled and its phys-
 ical condition appraised at  least once
 each day.
  (11) Additional observations should be
 made daily  with  appropriate actions
 taken to minimize loss of animals to
 the study  (e.g., necropsy or refrigera-
 tion  of those %nl""d« found dead and
 isolation or sacrifice of weak or mori-
 bund animals).
  (ill) Clinical signs  and  mortality
 should be recorded for all animals. Spe-
 cial attention should be paid to  tumor
 development.  The time of onset, loca-
 tion,  dimensions,  appearance and pro-
 gression of each grossly visible or pal-
 pable tomor should be recorded.
  (iv) Body weights should be recorded
 individually  for  all  animals  once  a
week  during the first 13 weeks  of the
 test period and at least once every 4
weeks thereafter,  unless signs  of clini-
cal  toxlcity  suggest  more frequent
weighings  to  facilitate monitoring of
health status.
  (v) When the test substance is admin-
istered in  the feed or drinking  water,
measurements of  feed or water con-
sumption,  respectively, should be de-
                            §798.3320

 tennmed weekly during  the first  13
 weeks of the study and then at approxi-
 mately   monthly  intervals   unless
 health status or body weight changes
 dictate otherwise.
   (vl) At the end of the study period,
 all survivors are  sacrificed.  Moribund
 animals  should be  removed and  sac-
 rificed when noticed.
   (8) Physical measurements. For inhala-
 tion  studies, measurements or mon-
 itoring should be made of the follow-
 ing:
   (1) The rate of airflow should be mon-
 itored continuously, but should be re-
 corded at Intervals of  at least once
 every 30  minutes.
   (11) During each exposure period the
 actual concentrations of the test sub-
 stance should  be  held as constant as
 practicable,  monitored   continuously
 and recorded at least  three times dur-
 ing the  test period: At  the beginning,
 at an Intermediate time  and at the end
 of the period.
   (ill)  During the development of the
 generating system, particle size analy-
 sis should be performed to establish the
 stability of  aerosol  concentrations.
 During exposure,  analyses should be
 conducted as often as  necessary to de-
 termine the consistency of particle size
 distribution  and homogeneity  of  the
 exposure stream.
  (ir)  Temperature  and  humidity
 should be monitored continuously,  but
 should be recorded  at Intervals of at
 least once every 30 minutes.
  (9) Clinical examinations. (1) The  fol-
 lowing examinations should  be  made
 on at least  20 rodents of each sex  per
 dose level:
  (A)  Certain hematology  determina-
 tions (e.g.. hemoglobin content, packed
 cell volume, total red blood cells, total
 white blood  cells, platelets,  or  other
 measures of clotting potential) should
 be performed at  termination  and
 should be performed  at 3 months.  6
 months and at approximately 6-month
 intervals thereafter  (for those groups
 on test for longer than 12 months) on
 blood samples collected from 20 rodents
 per sex of all groups. These collections
should be from the same animals at
each Interval. If clinical observations
suggest a deterioration In health of the
anlmaln during the study, a differential
blood count  of the affected animals
                                IV A-10

-------
 §798.3320
          40 CFR Ch. I (7-1-94 Edition)
 should be  performed.  A  differential
 blood count  should be performed on
 samples from animals in the highest
 dosage group and  the controls.  Dif-
 ferential  blood counts should be  per-
 formed for the next lower group(s) if
 there is a major discrepancy between
 the highest group and the controls. If
 hematological effects were noted in the
 subchronlc test, hematological testing
 should be performed at 3, 6,12.18 and 24
 months for a year study.
  (B) Certain clinical  biochemistry de-
 terminations on blood should be  car-
 ried out at least three times during the
 test period: Just prior to  initiation of
 dosing' (baseline data), near the middle
 and at the end of the test period. Blood
 samples should be  drawn for clinical
 measurements  from at least ten ro-
 dents per sex of all  groups; if possible,
 from the same rodents at each time in-
 terval. Test areas which are considered
 appropriate to  all studies: electrolyte
 balance, carbohydrate metabolism and •
 liver and kidney function. The  selec-
 tion of specific tests will be influenced
 by observations on the mode  of action
 of  the substance and  signs of clinical
 toxlclty.  Suggested  chemical  deter-
 minations: Calcium, phosphorus, chlo-
 ride, sodium,  potassium,  fasting  glu-
 cose (with period of fasting appropriate
 to  the species), serum glutamic-pyru-
 vlc transaminase (now known as serum
 alanine amlnotransferase), serum  glu-
 tamic oxaloacetic transaminase (now
 known as serum aspartate amlnotrans-
 ferase).    ornithine   decarboxylase.
 gamma glutamyl transpeptidase, blood
 area  nitrogen,  albumen,  creatlnlne
 phosphoklnase,  total cholesterol, total
 blllrubin and total serum protein  meas-
 urements. Other determinations which
 may be necessary for an adequate toxl-
 cological  evaluation include  analyses
 of llplds, hormones,  acid/base balance,
 methemoglobin and  cholinesterase ac-
 tivity. Additional clinical biochemis-
 try may be employed  where necessary
 to extend the investigation of observed
 effects.
  (11)  The .following  should   be   per-
 formed on at least 10 rodents of each
sex per dose level:
  (A) Urine samples  from the  same ro-
dents  at   the  same  intervals   as
hematological  examination   above,
should be collected  for analysis.  The
 following  determinations  should  be
 made from either individual animals or
 on a pooled  sample/Bex/group for ro-
 dents: appearance (volume and specific
 gravity), protein, glucose, ketones, bil-
 irubin.  occult   blood   (semi-quan-
 titatlvely) and microscopy of sediment
 (semi-quantltatlvely).
  (B)  Ophthalmological examination,
 using an ophthalmoscope or equivalent
 suitable equipment,  should be  made
 prior to the administration of the test
 substance  and at the  termination of
 the study. If changes in the eyes are
 detected, all animals should be exam-
 ined.
  (10)  Gross  necropsy, (i) A complete
 gross examination should be performed
 on all ttTiiTna.ig, including those which
 died during  the experiment or were
 killed in moribund conditions.     *
  (11)  The  liver,   kidneys,  adrenals,
 brain and  gonads  should  be weighed
 wet. as soon as possible after dissection
 to avoid drying. For these organs, at
 least 10 rodents  per sex  per group
 should be weighed.
  (ill) The following organs and tissues,
 or  representative   samples  thereof.
 should be preserved in a suitable me-
 dium for possible future hlstopatholog-.
 leal examination: All gross lesions and
 tumors; brain-Including sections of me-
 dulla/pons, cerebellar cortex, and cere-
 bral cortex; pituitary; thyroid/parathy-
 roid;  thymus;  lungs;  trachea;  heart;
 sternum and/or femur with bone mar-
 row; salivary glands; liver;  spleen; kid-
 neys;  adrenals: esophagus;  stomach;
 duodenum;  jejunum;  ileum;  cecum;
 colon;  rectum;  urinary bladder;  rep-
 resentative lymph  nodes: pancreas; go-
 nads; uterus; accessory genital organs
Xepididymis, prostate,  and. If present.
seminal  vesicles);  female  mammary
gland;  aorta; gall bladder (if present);
skin;  musculature;  peripheral nerve;
spinal  cord at three  levels—cervical,
mldthoradc,  and lumbar, and eyes. In
inhalation  studies,   the  entire  res-
piratory tract, including nose, phar-
ynx,   larynx   and   paranasal  sinuses
should be examined and preserved. In
dermal studies, skin from sites of skin
painting should be  examined and  pre-
served.
  (iv) Inflation of lungs and urinary
bladder with  a fixative is the optimal
method for preservation of these tis-
                                  IVA-11

-------
 Environmental Protection Agency
                           §798.3320
 sues. The proper Inflation at  fixation
 of the  lungs  In Inhalation studies Is
 considered  essential  for appropriate
 and  valid histopathological examina-
 tion.
  (v) If other clinical examinations are
 carried out, the information  obtained
 from these procedures should  be avail-
 able  before microscopic  examination,
 since  they may provide  significant
 guidance to the pathologist.
  (11) Histopathology. (i) The following
 histopathology should be performed:
  (A) Full histopathology on the or-
 gans and tissues, listed  above,  of all
 non-rodents, of all rodents in the  con- •
 trol  and .high dose groups and  of ail ro-
 dents that died or were  killed  during
 the study.
  (B) All gross lesions In all animals.
  (C) Target organs in all animals.
  (D) Lungs, liver and kidneys of all
 animals. Special attention to examina-
 tion of the  lungs of rodents should be
 made for evidence of  Infection since
 this  provides an assessment of  the
 state of health of the animals.
  (ii) If excessive early deaths or other
 problems  occur in the high dose group
 compromising the significance  of the
 data, the  next dose level should be ex-
 amined for complete histopathology.
  (ill) In case the results of the experi-
 ment give evidence  of  substantial al-
 teration of the animals' normal longev-
 ity or  the  induction  of effects  that
might affect a toxic response,  the  next
 lower dose level  should be examined as
described above.
  (iv) An  attempt should be  made to
correlate  gross observations with mi-
croscopic findings.
  (c) Data and reporting—(1) Treatment
of results, (i) Data .should be summa-
rized in tabular form, showing for each
test  group the number of animals at
the start of the test, the number of ani-
mals showing  lesions, the types of le-
sions and the percentage of  animals
displaying each type of lesion.
  (11) All observed results, quantitative
and incidental, should be evaluated by
an appropriate statistical method.  Any
generally  accepted statistical  methods
may  be used;  the statistical  methods
should be  selected during the design of
the study.
  (2)  Evaluation of study results, (i) The
findings of  a combined  chronic  tox-
 iclty/oncogenlclty  study   should  be
 evaluated in conjunction with the find-
 ings  of preceding studies and  consid-
 ered  In terms of the toxic  effects, the
 necropsy  and  histopathological  find-
 ings. The evaluation will Include the
 relationship between the  dose  of the
 test substance and the presence, inci-
 dence and  severity  of abnormalities
 (including behavioral and clinical ab-
 normalities),  gross  lesions, identified
 target organs, body weight changes, ef-
 fects on mortality and any other gen-
 eral or specific toxic effects.
  (ii) in any study which demonstrates
 an absence  of toxic effects, further in-
 vestigation  to establish absorption and
 bioavailabllty of the  test  substance
 should be considered.
  (ill) In order for a negative test to be
 acceptable.  It should meet  the  follow-
 ing criteria: No more than 10 percent of
 any group Is lost due to autolysls, can-
 nibalism,  or  management  problems;
 and survival In each.group  Is no less
 than  SO $ercent  at 18 months for mice
 and hamsters and at 24 months for rats.
  (3)  Test report, (i) In addition  to the
 reporting  requirements  as specified
 under 40 CFR part 792, subpart J the
 following  specific Information  should
 be reported:
  (A)  Group animal data. Tabulation of
 toxic response data by  species,  strain.
 sex and exposure level for:
  (7) Number of animals dying.
  (2) Number of  animals showing signs
 oftoxicity.
  (J) Number of animals exposed..
  (B)  Individual animal data.  (/) Time of
 death during the study or whether ani-
 mals survived to termination.
  (Z) Time of observation of each ab-
 normal sign and Its subsequent course.
  (J) Body weight data.
  (4) Feed and water consumption data.
 when collected.
  (5)  Results of ophthalmologlcal  ex-
amination, when performed.
  (5) Hematological tests employed and
all results.
  (7)  Clinical  biochemistry  tesu em-
ployed and all results.
  (8) Necropsy findings.
  (9)   Detailed   description  of  all
histopathological findings.
  (10)  Statistical treatment  of  recults
where appropriate.
                                 IV A- 12

-------
 §798.3320
          40 CFR Ch. I (7-1-94 Edition)
 order  to  elucidate  a  chronic  toxl-
 eologlcal profile of the test substance.
 If more than one dose level Is selected
 for  satellite  dose groups,  the doses
 should be spaced to produce a grada-
 tion of toxic effects.
  (4) Exposure  conditions. The animals
 are dosed with the test substance Ideal-
 ly on a 7-day per week basis over a pe-
 riod of at least 24 months for rats, and
 18 months for  mice and hamsters, ex-
 cept for the a.n
-------
 Environmental Protection Agency

  (14) Schwartz, E. 1374. "Toxicology of
 jjeuroleptic  Agents,"  Industrial  Phar-
 macology: NeuToleptics.  Ed.  S.  Fielding
 and H.  Lai. (Mt.  Kisco.  New York:
 Futura Publishing Co.  1974) pp. 203-221.
  (15)  Sontag, J.»t, Page. N;P.. and
 Saffiotti. U.  Guidelines for Carcinogen
 gioassay in Small Rodents. NCI-CS-TR-
 1 (Bethesda: United States Cancer In-
 stitute, Division of Cancer Control and
 prevention,  Carcinogenesis  Bioassay
 program, 1976).
  (16)  United  States  Pharmaceutical
 Manufacturers Association. Guidelines
 for the Assessment of Drug and  Medical
 Device Safety in Animals. (1977).
  (17)   World   Health  Organization.
 "Principles for the Testing and Evalua-
 tion  of Drugs for  Carcinogenicity,"
 WHO  Technical Report Series  No. 426.
 (Geneva:  World  Health Organization,
 1969).
  (18)   World   Health  Organization.
 "Guidelines for Evaluation of Drugs for
 Use in Man." WHO Technical Report Se-
 ries No. S63. (Geneva: World Health Or-
 ganization, 1975).
  (19)   World   Health  Organization.
 "Part I. Environmental Health Criteria
 6," Principles and Methods for Evaluat-
 ing the Toxicity of Chemicals. (Geneva:
 World Health Organization,  1978).
  (20)  World  Health  Organization.
 "Principles for Pre-Clinlcal Testing of
 Drug Safety." WHO Technical Report
 Series No. 341. (Geneva: World  Health
 Organization, 1966).

 (SO FR 39397. Sept. 27, 19
 FR 21064. May 16.1989]
                                             §798.4100

                  not fall to identify substances with sig-
                  nificant allergenic potential, while at
                  the same  time avoiding false negative
                  results.
                    (b) Definitions.  (1) Skin sensitization
                  (allergic  contact  dermatitis)  is  an
                  inununologically  mediated  cutaneous
                  reaction to a substance. In the human,
                  the responses may be characterized by
                  pruritis,  erythema,  edema,  papules,
                  vesicles, bullae,  or a combination of
                  these.  In  other  species the reactions
                  may differ  and  only  erythema  and
                  edema may be seen.
                    (2) Induction period is a period of at
                  least 1  week following a sensitization
                  exposure during which a hypersensitive
                  state is developed.
                    (3) Induction exposure is an experi-
                  mental exposure  of a subject to a test
                  substance with the intention of induc-
                  ing a hypersensitive state.
                    (4) Challenge exposure is an experi-
                  mental  exposure  of a previously treat-
                  ed subject to a test substance following
                 . an  induction  period,  to  determine
                  whether the subject will react in a
                  hypersensitive manner.
                    (c) Principle of the test method. Follow-
                  ing initial exposure(s)  to  a test  sub-
                  stance,  the  «pi
-------
                                    APPENDIX B
Summary of mT Workshop Conclusions (for full workshop report, see McClellan era/., 1992):
       •   AIT fiber types capable of depositing in the thorax are not alike in their pathogenic
          potential.
       •   Only fiber samples with dimensions similar to those which humans can inhale should
          be tested.
       •   A complete characterization (i.e., dimensions, fiber number, mass, and aerodynamic
          diameter) of the fiber aerosol and retained dose is essential
       •   Appropriate aerosol generation methods must be used for inhalation studies in order to
          preserve fiber lengths.
       •   A tiered-approach to toxicity evaluation is recommended that includes:
             1. In vitro screening for durability, surface properties, cytotoxicity, and similar
                properties, etc.',
             2. Short-term inhalation or other in vivo studies;
             3. That chronic inhalation studies are the "gold  standard" (i.e., provide most
                appropriate data for risk characterization).
       •   The rat is the most appropriate species for inhalation studies.
       •   In chronic inhalation studies, animals should be retained to at least 20% survival after
          2-year exposure.
       •   Serial lung burden analyses are an essential component of inhalation studies and are
          essential for understanding exposure-dose-response relationships.
       •   Studies oriented to understanding mechanisms of toxicity and carcinogenicity are
          important adjuncts to traditional toxicity studies.
       •   Histopathological analyses of tissues of the respiratory tract represent  primary
          endpoints for evaluating effects of inhaled fibers.  Major effects include pulmonary
          fibrosis, lung tumors, and mesotheliomas.  Experimental tissues should be archived for
          future studies; wherever possible, handling and preservation of tissues should be done
          in a way mat maximizes their future use in mechanistic studies.
       •   Potential human exposures throughout  the entire life-cycle of the fiber  must be
          considered and fibrous material for toxicologic studies prepared accordingly.
       .   Intracavity studies are  inappropriate  for  risk characterization but can play a useful
          screening role in assessing fiber toxicity.
                                          IVB- 1

-------
                                     APPENDIX C

            Draft Protocol for Inhalation Oncogenicity Study  with Fibers


                       Prepared by the Inhalation Working Group of the
        International Cooperative Research Programme on the Assessment of MMFs Toxicity
                                     September, 1994
Preamble:
This testing protocol is intended to be used as part of a tiered-approach to the
evaluation of fibers. This approach involves the evaluation of the acellular in
vitro dissolution rate, in vivo biopersistence and in vivo long-term lexicological
effects.
GLP Regulations:

Objective:


Outline of Method-
Animals:
Control:

Fiber Size
   Preparation:
OECD, USEPA 40 CFR Part 792 etc., or their successors as appropriate.

To assess the dose-response relation of the potential pathogenic  and/or
oncogenic effects of chronic inhalation exposure to inhaled fibers in rats.

Laboratory rats are exposed by nose-only inhalation to well characterized fiber
test atmospheres which have been optimized to be largely rat respirable.  The
exposure  duration is  6 hours/day, 5 days/week for 104 weeks, with a
subsequent non-exposure period lasting until ~20% survival in one of the test
fiber groups.  Whole-body exposure may be used with proper validation (see
below 'Exposure').

Rat, Fischer-344, supplied specific pathogen free (SPF),virus antigen-free
(VAF+) and maintained under optimum hygienic condition (OHC), males only.
Upon receipt and at selected intervals throughout the study, a sentinel group of
rats should be analyzed for bacteriological and viral contamination.  Another
strain of laboratory rats may be used providing it is validated in comparison to
the Fischer-344 rat

Filtered air
The bulk fiber used for aerosol generation should be prepared or pre-selected to
have a nominal geometric mean fiber diameter close to 0.8 ^m and a geometric
mean length of longer than 15 Jim.
                                          IVC- 1

-------
Exposure
   Concentrations:   At least 3 exposure concentrations with the highest at the Maximum Tolerated
                    Dose (MTD), (Expressed gravinietrically as mg/m3 of rat respirable aerosol)
                    (Note:  The gravimetric concentration is used here as this is usually the method
                    for daily control of the test atmosphere. All results should also be reported as
                    the number of fibers/cm3).

Number of animals/group: 140 (assigned by randomization)
                    Optional sub-groups:  Additional animals may be included at the interim
                    sacrifices 1) to assess the exposure free recovery and 2) for broncho-alveolar
                    lavage and cytology (see below).
Animal.*?* age at
    delivery:
8-12 weeks
Interim sacrifice time points and number of animals:
                    WEEK           Number of Animals
                      13                     6
                      26                     6
                      52                     6
                      78                     6
                     104                     6
Final sacrifice:
Recovery Animals:
Exposure:
Fiber Aerosol
    Generation:
All remaining animals will be killed when -20% survival is reached in one of
the test fiber exposure groups.

Optional - At the time of each scheduled sacrifice, at least 6 rats per group will
be removed from exposure and maintained without further exposure. These
animals will be sacrificed at 104 weeks.  Analysis will include all endpoints
specified for scheduled interim sacrifices.

Flow-past, nose-only exposure with an air flow to each animal of ~1 liter/min.
is recommended. The testing facility must provide documentation showing the
uniformity at the top, middle and bottom level of the exposure system - this
must be within ±10%.

Whole-body exposure may be used with proper validation. It is recommended
that this validation should be based upon equivalent number and bivariate size
distribution of fibers in the lungs as compared to similar nose-only exposures.
The fibers will be generated using a piston fed brush feed aerosol generator or
other generator which has been validated by the testing facility with data that
shows that the aerosol generator does not significantly grind or contaminate the
bulk fiber provided.
                                               IVC-2

-------
Exposure system
    monitoring daily:  • Airflow rate
                    • Oxygen concentration
                    • Temperature & humidity

Exposure atmosphere
    monitoring:      • Gravimetric (mg/m3) - at least once per day
                    • Fiber Number by phase contrast optical microscopy (PCOM) (f/cm3) -
                       at least once per week
                    • Bivariate size distribution scanning election microscopy (SEM) (\uri) -
                       at least once per week
                    • Chemical analysis, 1 fiber sample taken every 3 months.

Counting and Sizing Rules: The general guidelines provided by the WHO/EURO are recommended
                    with the following additional procedures:
                    •  Sizing of length and diameters  to be performed using  an  SEM at a
                       magnification of at least 2000. All objects which seen at this magnification
                       are to be counted. No lower or upper limit is to be imposed on either length
                       or diameter.
                    •  The bivariate length and diameter are to be recorded individually for each
                       fiber measured.
                    •  When sizing, an object is to be accepted as a fiber if the ratio of length to
                       diameter was at least 3:1. All other objects are considered particles.
                    •  STOPPING RULES:
                       At least enough fields of view are to be counted for evaluation so that a total
                       of 0.15 mm2 of the filter surface is examined.
                       1. NON-FIBROUS PARTICLES:  The recording of particles is to  be stopped
                       when a total of 30 particles is observed.
                       2. FIBERS: The evaluation of fibers is to be stopped when 300 WHO fibers
                       (1 £ 5 |im, d £  3 Jim) (WHO, 1985) or a total of 1000 fibers and non-
                       fibrous particles are  recorded, or 1 mm2 of the filter surface has  been
                       examined, even if a total of 300 countable WHO fibers is not reached.
                       3. LONG FIBERS: If 20 fibers equal or longer than 20 Jim have not been
                       recorded by 2 above, then the evaluation of those fibers ^20 pm  is to
                       continue until 20 fibers with d < 3 Jim and 1 > 20 jam are recorded or a filter
                       surface area of 5 mm2 has been examined whichever comes first (Fibers <
                       20 Jim are not recorded in this step). NOTE:  To facilitate analysis and to
                       aid in comparison with previous data, it is suggested that data in the above 3
                       categories be recorded separately.

Clinical observations:  • Mortality
                    • Clinical signs
                    • Body weights
                    • Disease screening (viral, bacterial & parasites) of sentinel animals
                                              IVC-3

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Necropsy:           All animals will be necropsied.  All tissues (as specified for oncogenicity
                    studies by the OECD guidelines^ will be examined and preserved.
                    In addition: Lungs are to be we- ™hed when removed:
                    - accessory lobe removed prior; fixation, weighed & frozen at or below -20°C
                    -remaining lung to be weight   inflated to validated standard pressure and
                    fixed with formalin or Karnovski 's fixative.
                    - 35 mm slides of the lungs taken before and after inflation.
                    A dissecting microscope could be used to help identify any potential lesions.

Broncho-
    Alveolar Lavage:  At the time of the 12 and 24 month sacrifices, subgroups of 5 rats/group should
                    be subjected to Broncho-Alveolar Lavage (B AL). It is recommended that the
                    lavage fluid should be analyzed for differential cell counts, total protein, lactate
                    dehydrogenase,  p-glucuronidase and N-acetylglucosamidase.  As mentioned
                    above, B AL at additional time points is optional.

Lung Dust Content:   The accessory lobe will  be processed  by low temperature ashing or other
                    method using a validated lung burden recovery system (The method of standard
                    additions should be employed using fibers injected into otherwise unexposed
                    lungs and lung  and solution blanks).  The fibers should be  analyzed for
                    number, bivariate size distribution and chemistry.

Histotechnology:     Left lobe and cranial lobe of right lung - divided longitudinally (with left lobe
                    cut through main stem bronchus if technically possible).

                    Other organs: Nasal cavity/turbinates, trachea, mediastinal lymph nodes, liver,
                    kidneys, heart, spleen and all gross lesions.

Histopathology:      Lungs: Scoring of macrophage, fibrosis, bronchiolization, pleura! thickening
                    Neoplastic Evaluation (tumors)
                    - 35 mm slides of representative lesions
                    OPTIONAL: A validated quantitative method of assessing pneumoconiosis may
                    be used (e.g., David etal., 1978).

Statistics:            - Body  weight and lung lobe weights
                    - Survival analysis
                    - Tumor incidence

Data and reporting: (Taken from EPA Health Effects Test Guidelines, 1992)
       (1) Treatment of results.
          (a) Data will be summarized in tabular form, showing for each test group the number of
          animals at the start of the test, the number of animals showing lesions, the type of lesions
          and die percentage of animals displaying each type of lesion.
          (b) All observed results, quantitative and inHHpnt^i, <;h^i fr» qvai^atgH by an appropriate
          statistical method.  Any generally accepted statistical method may be used; the statistical
          methods shall be selected during the design of the study.

                                           IV C-4

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       (2) Evaluation of study results.
          (a) The findings of an oncogenic toxicity study shall be evaluated in conjunction with the
          findings of preceding studies and considered in terms of the toxic effects, the necropsy and
          histopathological findings. The evaluation shall include the relationship between die dose
          of the test substance and the presence, incidence and severity of abnormalities (including
          behavioral and clinical abnormalities), gross lesions, identified target organs, body weight
          changes, effects on mortality and any other general or specific toxic effects.
          (b) In order for a negative test to be acceptable, it shall meet the following criteria: No
          more than 10% of any group is lost due to autolysis, cannibalism, or management
          problems; and survival in each group should be no less than 50% at 24 months for rats.
       (3) Test report.
          (a)  Group animal data. Tabulation of toxic response data by species, strain, sex and
          exposure level for
                 (a)  Number of animals dying.
                 (b)  Number of pnimaly showing signs of toxicity.
                 (c)  Number of animals exposed
          (b) Individual animal data.
                 (a)  Time of death during the study or whether animals survived to termination.
                 (b)  Time of observation of each abnormal sign and its subsequent course.
                 (c)  Body weight data.
                 (d)  Necropsy findings.
                 (e)  Detailed description of all histopathological findings.
                 (f) Statistical treatment of results, where appropriate.
                 (g)  Historical or positive control data, if taken into account
       (4) Test conditions.
          (a) Description of exposure apparatus including design, type, dimensions, source of air,
          system for generating particulates and aerosols, method of conditioning air, treatment of
          exhaust air and the  method of housing the ?nimqls in a test chamber.
          (b)  The equipment for  measuring temperature, humidity, and paniculate aerosol
          concentrations and size shall be described.
       (5) Exposure data. These shall be tabulated and presented with mean values and a measure of
variability (e.g., standard deviation) and shall include:
          (a)  Airflow rates through the inhalation equipment
          (b) Temperature and humidity of air.
          (c)  Nominal concentration  (total amount of test substance fed into the inhalation equipment
          divided by volume  of air).
          (d) Actual concentration in test breathing zone expressed as mg/m3 and as f/cm3.
          (e)  Fiberdata:
              Complete summary statistics of total fiber count and bivariate size distribution of the
              bulk fiber, fibers in the aerosol and fibers recovered following lung digestion should be
              reported at each sacrifice time point The number of particles should also be reported.
              In addition, the rat respirable aerosol exposure concentration should be reported. The
              fiber concentration in the lung should be given as fibers per whole lung with the lung
              weights provided^eparately.
                                             IV C-5

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                                    ADDENDUM
                         TO  ONCOGENICITY PROTOCOL
1.     Histopathological evaluation: One of the histolqgical endpoints of the oncx>genicity study is
       quantification of the cellular changes and fibrosis in the lung. While the Wagner scale has
       provided some interesting information, a standardized scoring system better adapted to the
       response seen in rodents would be more useful In addition, not only should the types of
       cellular and fibrotic lesions be scored and reported but the extent of the lung involved with
       those lesions as well. This working group strongly supports a joint EURIMA-NAIMA
       program to produce a revised cellulaf/fibrosis scoring system for fibers.

2.     Due to the length of time, number of animals and cost requires for the fiber inhalation
       oncogenicity study, a research program should be established to define a shorter term study
       for the evaluation of fiber toxicity.  This study should examine the cellular response,
       proliferation and the  creation of fibrosis in the lung after 6-12 months of inhalation
       exposure.  These results should be compared to the tumorigenic response observed in
       chronic  fiber inhalation studies.  It is recommended as well that the study design be
       discussed with regulators such as the USEPA as part of the protocol development process.

3.     Quantification of cellular proliferation: A quantification of cellular proliferation  by using
       subcutaneous administration of bromodeoxyuridine (BrDU)  via  osmotic minipumps
       implanted for a period of 3-4 days prior to sacrifice and subsequent quantification of
       labeled cells.  This would require an additional group of animals in an oncogenicity study
       which, however, would provide useful information on cell proliferative responses in the
       alveolar region as well as of the pleura! lining. With respect to cell proliferative responses
       of the pleura! lining, it needs to be investigated whether a short-term pulse delivery of
       BrDU or a 3-4 day osmotic minipump delivery is best suited. Alternatively, evaluation of
       cell proliferation could be performed by evaluating PCNA labeling which would avoid
       administration of BrDU altogether. This approach needs additional comparative research.

4.     Evaluation of mutation frequency: Evaluation of mutation frequency of type n cells of the
       HPRT gene should be investigated as an endpoint for short-term tests as it  provides
       relevant information about the in vivo  mutation potential of inhaled particulate matter,
       fibrous and non-fibrous, and could possibly be used to rank the carcinogenicity potency of
       different fibers.  Respective methodologies are available now involving isolation of type n
       cells after in vivo exposures and apply HPRT mutation assays to type n cell cultures.
                                          rvc-6

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                                       APPENDIX  D
                          Draft Protocol for Intracavitary Testing
              Prepared by the Working Group on Intracavitary Testing of the
  International Cooperative Research Programme  on the Assessment of MMFs Toxicity
                                      September, 1994
Species:

Strain:
Rat

Wistar  Source of supply to be listed in reports although it is accepted that all
        laboratories in the world cannot use the same source of supply.
Animal husbandry:         Animal must be SPF and Barrier Maintained throughout the study.

Sex:            Use of both sexes desirable. Where only one sex is proposed then this should be male.

Age:            8-10 weeks at first injection.

Food & Water   Ad libitum.

Dose:           Tb be calibrated by numbers of fibers. The WHO fiber criteria should be used.
                    Three doses to be used for each dust sample. IxlO9, 1 xlO8, Ix 107  fibers. Dust
                to be injected suspended in PBS. Injection volume 2 ml Maximum dust mass for each
                injection SO mg. When more than one injection is required to obtain the full dose,
                injections should be at weekly intervals.  Maximum overall dose to be 250 mg of dust
                Where more than this mass is required to inject IxlO9 WHO fibers this indicates that
                either fibers are too thick to be important or that too much non-fibrous paniculate
                material is present  The variation between  injected doses, which is inevitable when
                syringe injection is used, should be quantified for each dust by weighing 20 doses that
                have been 'injected* into weighing bottles before drying and weighing. These samples
                will have been suspended in distilled water to avoid the complication of salt crystals in
                dried dust

Fiber sizing:     Fiber sizing should be undertaken by Scanning Electron Microscopy.  The counting and
                sizing protocol should involve counting the first 500 fibers of all sizes found (aspect
                ratio >3-10. This count should be used to estimate the mass required for the proposed
                dose of WHO fibers. In addition the first 100 fibers counted should be measured
                (length and diameter). The number of non-fibrous particles present in the  area covered
                by die first 100 fibers should be recorded. All this data should be included in reports.

Animal numbers: 50 rats  per dose plus 50 controls injected with saline only. Where more than one
                injection is required to obtain one of the doses then the controls should receive the same
                number of saline injections.  Negative control animals should be included with each
                group of tests.

                In addition to negative control data a laboratory should be required to provide positive
                control data.  This need not be for each batch of tests.  One positive control data set
                every two years should suffice.  Positive control dust should be UICC crocidolite
                administered at the standard three doses.

                                             IVD- 1

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Animal
 maintenance:
Duration of
 studies:
Necropsy:


Histology:
Animals should be weighed throughout the study.  Once each week for the first 13
weeks followed by once each month for the rest of the study. Animals will be killed
when they show signs of debilitation. Whether an animal has been killed or found dead
shall be recorded in the autopsy report
A study shall be terminated when the last of the three test groups has fallen to 20%
survival.

Apart from a routine description of any tumors present, the presence of granulomas and
fibrous adhesions should be recorded.

When an obvious tumor is present three blocks of tissue from separate tumor areas to
confirm the diagnosis.

Where no tumor is visible the following blocks of tissue should be taken:
   (A)  Diaphragm/liver
   (B)  Liver/spleen/pancreas
   (Q  Mesentery plus gut segments
   (D)  Qmentum plus gut segments
                                             IVD-2

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                                       APPENDIX E

            Draft Protocol for Inhalation  Biopersistence Study  with Fibers


                        Prepared by the Inhalation Working Group of the
        International Cooperative Research Programme on the Assessment of MMFs Toxicity
                                       September, 1994
Preamble:
GLP Regulations:

Objective:


Outline of Method-
Animals;
Control:
This testing protocol is intended to be used as part of a tiered-approach to the
evaluation of fibers.  This approach involves the evaluation of the acellular in
vitro dissolution rate1, in vivo biopersistence and in vivo long-term lexicological
effects.

This protocol defines many of the key parameters for evaluating biopersistence
and permits inter-fiber and inter-laboratory comparison of results.  It is
recognized, however, that additional approaches may be more suitable for
addressing special questions.

OECD, USEPA 40 CFR PART 792 etc., or their successors as appropriate.

To assess the in vivo pulmonary biopersistence of the inhaled fibrous and non-
fibrous particles in the rat

Following preliminary characterization of the test material, laboratory rats are
exposed by  inhalation for 5 consecutive days to well characterized fiber test
atmospheres which have been optimized to be largely rat respirable. Following
the end of the  exposure period, subgroups of animals are sacrificed at pre-
determined  intervals and the lung burden determined  by suitably validated
extraction and measurement methods.

Rat, Fischcr-344, supplied specific pathogen free (SPF), virus antigen-free
(VAF+) and maintained under optimum hygienic condition(OHC), males only.
Upon receipt and at selected intervals throughout the study, a sentinel group of
rats should be analyzed for bacteriological and viral contamination.  Another
strain of laboratory rats may be used providing it is validated in comparison to
the Fischer-344 rat

Filtered air
    1 Prior to the evaluation of fibers by inhalation for biopersistence, it may be useful depending upon the
characteristics of die fiber under test, to expose a group of animals by intratracheal instillation and examine the broocho-
alveolar lavage fluid in order to evaluate the cytotoxic response. In such an evaluation, a positive and negative control
should be included.
                                          WE- 1

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Fiber Size
   Preparation:      The bulk used for aerosol generation should be prepared or pre-selected to have
                    a nominal geometric mean fiber diameter close to 0.8 Jim and a geometric mean
                    length of at least approximately 15 |im, if technically feasible. This size range
                    corresponds to that found in most industrial hygiene situations.

Concentrations:      For MMVF, a recommended value of 30 mg/m3 of rat respirable aerosol. For
                    very soluble fibers (dissolution rate > 200 ng-cm'2^*1) an exposure group at
                    40 mg/m3 should be included.  (Note: A gravimetric concentration is stated as
                    this is usually the method for daily control of the test atmosphere.  All results
                    should also be reported as the number of fibers/cm3).

Optional evaluation:  At 1 (or 3) and 28 days post-exposure, subgroups of 5 animals may be included
                    for bronchial-alveolar lavage2.  If the fiber illicits an elevated response PMN
                    (>5%; Note: This value should be reevaluated after further experience) in the
                    BAL at either 1 day or 28 days post-exposure, the study should be repeated at a
                    reduced exposure concentration (with an air control).

Exposure duration:   6 hours/day for 5 consecutive days

Number animals/group/sacrifice interval:
                    7: 5 for analysis and 2 optional spares (assigned by randomization)

Optional evaluation:  Additional animals may be added for examination at 1 day and 28 days post-
                    exposure of the following endpoints:
                    - Fibers recovered in the BAL fluid
                    - Fibers recovered from the lavaged lung following digestion
                    - Fibers in the thoracic lymph nodes following digestion
                    - Using the method of Bermudez (1994) or a suitably validated equivalent
                       method, recovering and analyzing the fibers from the pleura! cavity.

Animals' age
   at delivery:       8-12 weeks
   2 Bronchial alveolar lavage: In order to maximize the recovery of cellular contents, the lungs should be excised and
an exhaustive lavage consisting of 10 lavages with 5 ml saline each should be performed under slight massage of the
excised lung.

                                        IVE-2

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Sacrifice time points and number of animals analyzed: Sacrifice time points are adjusted as shown
                    below depending upon fiber in vitro durability (using the standard method
                    agreed upon by the Working Group on In Vitro Dissolution) @);
Sacrifice^



1 hour(4)
Iday
2 or 3 days
14 days
4 weeks
3 months
6 months
12 months
If in virro
dissolution
rate
£50
(3)
(3)
7
(3)
7
(3)
7
7
••• Number of Animals •<
If in vitro
dissolution
rate
>50 - £200
ng- cm"^* h"*
(3)
7
7
(3)
7
7
7
7
»•••••••••••••••••
If in vitro
dissolution
rate
>200
7
7
7
7
7
7
(3)
7
                    (*) It is recommended that all studies include a minimum of 2 or 3 days, 4
                       weeks and 3 months sacrifice time points.
                    ® The background level/limit of detection for the fibers in the treated lungs
                       should be determined based upon the control lungs. At each sacrifice, if
                       an exposure group has a mean fiber concentration below the limit of
                       detection, the remaining animals in that group should be terminated at
                       the next scheduled sacrifice.
                    ® OPTIONAL: These groups may be included with the animals exposed,
                       necropsied,the lungs removed and ashed and then maintained frozen
                       (-20°C) for optional processing.
                    (4) If the 1  hour time point is measured, it is recommended that the early
                       transitions from 1 hour to 1 or more days be considered separately from
                       long-term clearance.

Exposure method:    Flow-past, nose-only exposure with an air flow to each animal of ~1 liter/min.
                    The testing facility must provide documentation showing the uniformity at the
                    top, middle and bottom level of the exposure system - this must be within
                    ±10%. Animals should be adapted to the system for several days prior to
                    exposure.

                    Whole body exposure may be used with proper validation. It is recommended
                    that this validation should be based upon equivalent number and bivariate size
                    distribution of fibers in the lungs as compared to similar nose-only exposures.
                                          IVE-3

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Fiber Aerosol
   Generation:       The fibers will be generated using a piston fed brush feed aerosol generator or
                    other generator which has been validated by the testing facility with data that
                    shows that the aerosol generator does not significantly grind or contaminate the
                    bulk fiber orovided.

Exposure system
   monitoring daily:  •  Airflow rate
                    •  Oxygen concentration
                    •  Temperature & humidity

Exposure atmosphere
   monitoring:       •  Gravimetric (mgAn3) - at least once per day
                    •  Fiber Number by phase contract optical microscopy (PCOM) (f/cm3) - at least
                      once per day. If the fiber has a significant fraction below the PCOM detection
                      limit, then SEM is recommended 3 times per week.
                    •  Bivariate size distribution scanning electron microscopy (SEM) (urn) - at least
                      twice per week.
                    •  Chemical analysis, 1 filter sample taken for possible analysis.

Counting and Sizing
   Rules:           The general guidelines provided by the WHO/EURO are recommended with the
                    following additional procedures:
                    •   Sizing of length and diameters to be performed using an SEM at a
                       magnification of at least 2000. All objects which seen at this magnification
                       are to be counted. No lower or upper limit is to be imposed on either length
                       or diameter.
                    •   The bivariate length and diameter are to be recorded individually for each
                       fiber measured.
                    •   When sizing, an object is to be accepted as a fiber if the ratio of length to
                       diameter was at least 3:1. All other objects are considered particles.
                    •   STOPPING RULES:
                       Enough fields of view are to be counted for evaluation so that at least a total
                       of 0.15 mm2 of the filter surface (for 25 mm dia.) is examined.
                       1.  NON-FIBROUS PARTICLES: The recording of particles is to be stopped
                       when a total of 30 particles is observed.
                       2.  FIBERS: The evaluation of fibers is to be stopped when 300 WHQ fibers
                       (1  > 5 nm, d <  3 ^m) (WHO, 1985) or a total of 1000 fiben and non-
                       fibrous particles are recorded, or 1 mm2 of the filter surface has been
                       examined, even if a total of 300 countable WHO fibers is not reached.
                       3.  LONG FIBERS (OPTIONAL): If 20 fibers equal or longer than 20 tun have
                       not been recorded by 2 above, then the evaluation of those fibers £20 Hm is
                       to continue (at lower magnification, lOOOx) until 20 fibers with d £ 3 |im
                       and 1 £ 20 pirn are recorded or a filter surface area of 5 mm2 has been
                       examined whichever comes first  (Fibers < 20 |im are not recorded in this

                                                      IV E-4

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Clinical observations:
Necropsy:
Lung Dust Content:
Statistics:
Suggestions for
   Reporting:
    step).  NOTE: To facilitate analysis and to aid in comparison with previous
    data, it is suggested that data in the above 3 categories be recorded
    separately.

• Mortality, twice a day
• Clinical signs, weekly
• Body weights, weekly first 13 weeks and every 2 weeks thereafter.

All animals will be necropsied.
Lungs are to be dissected from the cardio-pulmonary vasculature, weighed
and frozen at or below -20°C.
Spare animals, if included, should be considered for histopathological analysis,
broncho-alveolar lavage and subsequent cellular determination and scanning
electron microscopy.

The entire lung  (all lobes) will  be processed by low  temperature ashing or
another method using a validated test system (standard additions with fibers and
blanks).  For fiber types that cannot be so processed other digestion systems
should be developed and validated.  The fibers and non-fibrous particles should
be analyzed for number, bivariate size distribution and chemistry. The counting
and stopping rules are defined  above.   OPTION:  Representative SEM
photomicrographs of the fibers in the lung and following digestion.

Either linear and non-linear regression methods can be  used. The investigator
should assure that the model chosen provides  a good fit to the data (r2 > 0.85).
Data points that are below the limit of detection as determined using standard
statistical procedures should be clearly identified and should not be used in the
regression analysis.   When using non-linear regression analysis, it is
recommended that the loss function be weighted by the inverse of the variance.
Other weightings can be used if shown to be more appropriate.
It is suggested that the following should be reported:
•  Summary statistics of fiber count and bivariate size distribution of the bulk
   fiber, fibers in the aerosol and fibers recovered following lung digestion at
   each sacrifice time point
•  The fiber concentration in the lung should be given as fibers per whole lung
   with the lung weights provided separately.
•  The number and size of non-fibrous particles, if technically feasible.
•  The rat respirable aerosol concentration.
•  The retained fiber volume or mass determined either by calculation based on
   the bivariate size distribution and density3 or on the silicon content in the lung
   as determined by chemical analysis.  If technically possible, the mass of the
   non-fibrous particles should be presented as well.
' It should be noted that the use of the fiber's original density in this calculation may provide erroneous results if the fiber
is forming a leached layer which has a different density than the fiber core.

                                                    IVE-5

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If the 1 hour time point is measured, it is recommended that the early
transitions from 1 hour to 1 or more days be considered separately from
long-term clearance.
Statistical extrapolation of the clearance curves for total lung burden and by
length intervals should  be reported.  Clearance  can be fit  to single
exponential or a more complex model if information on the different
mechanisms which result in clearance is available.
              IV E-6

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                                    APPENDIX F

               Intratracheal instillation for Biopersistence Evaluation
       Inhalation represents the natural route of uptake and leads to a distribution of fibers which
is more uniform than that resulting from intratracheal instillation.  However, under certain
circumstances the use of the intratracheal instillation may be considered. Examples are the use of
radioactively labeled fibers and the availability of a lmrit*Jl amount of sized fibers.

       The instillation procedure can be performed using either single or multiple injections.  The
distribution of the fiber suspensions should be checked to avoid the formation of fiber aggregates
in the application suspension. In addition, laboratories who use this method should validate the
homogeneity of the distribution of fibers in the lung by SEM.  The animals are anesthetized with
Halpthane, the instillation performed via the mouth and the instillation should be performed when
the animal is at FRC The fibers are usually suspended  in 0.9%  Nad. The instilled volume
should not exceed 0.2 to 0.3 ml and the fiber concentration should not exceed 1 mg/ml and
preferably should be less. In addition, to determine if the instilled amount has caused any adverse
reactions in die  lung, additional animals should be similarly exposed and then lavaged 24 hr. later
and an analysis of die BAL performed.

       The total lung burden used should be in the range of 0.1 to 0.5 mg which is similar to that
documented for short-term inhalation studies described above.

       Intratracheal instillation is sometimes considered for die application of larger diameter fibers
in the range of human respirable or die investigation of die effect of high fiber lung burden such as
found following chronic inhalation exposure. However, it is not clear if rnese applications result in
artifacts due to die sudden introduction of fibers with diameters not normally encountered by die rat
or die acute application of an enormous number of fibers. Human respirable fibers are considered
to be tiiose witii geometric mean diameters of -3 |im and less. The lung burden corresponding to
drat found following chronic inhalation exposure hi rats is 1-2 mg.

       Whenever intratracheal instillation is used, die results should be validated by comparison
using some of die fibers with those results obtained by inhalation exposure.
                                         IVF- 1

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                                    APPENDIX  G

              Draft Protocol for In Vitro Acellular Tests - Durability.

                  Prepared by Acellular  Test Working Group  of the
       Cooperative Research Programme on the Assessment of MMF Toxicity
       An in vitro acellular standard test has to fulfill some essential requirements:

       — The test procedure has to consider the chemical environments which can significantly
          affect die dissolution of MMFs:
              -  the chemical dissolution in an extracellular model fluid (pH -7.5)
              -  and the chemical dissolution in an intracellular model fluid (pH -4.5 - 5)

       — The model fluids have to contain all essential components of a physiological fluid,
          which can significantly influence the dissolution.

       — The experimental conditions have to be adjusted to a suitable level considering
              -  the-detection limits for the analysis of relevant dissolved fiber components
              —  the ability to ensure stable conditions
              —  a suitable resolution of the dissolution behavior of the fibers

       — The test has to deliver reliable results within a certain accuracy, independent of the
          laboratory.

Test Equipment

       The test cells* the tubes for the physiological fluid and the collecting bottles consist of
materials stable against SBPFs (Simulated Body/Physiological Model Fluids). Before running a
test, the equipment has to be examined for leakages.  The fiber samples are placed in the cells
between two micropore filters (0.2 - 0.4 Jim) to prevent particles from leaving the cells. The free
volume which can be occupied by the fibers may vary between 2 cm3  and 10 cm3, approximately.
The free cell volume outside the micropore filters should not exceed the volume for the fiber
samples.  The whole system starting  from the supply container of the  model fluid up to the
collecting container has to be kept at 37±1°C during a test run.

Performance of the CFT (Continuous Flow Test)

       The fiber samples have to be stored in a desiccator with a drying agent if no characterization
or dissolution test is performed

       According to the specific surface area of the fibers and the desired F/A ratio (Flow rate/fiber
surface area) the respective amount of dried (110°C to constant weight) fibers is weighed by a
balance with an accuracy of ±1 mg, approximately. After filling the cells with the fibers and SBPF
the closed cells are ultrasonicated for 10 s. After the connection of the  tubes the test will be started.


                                                IVG-1

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At die end of the experiment the remaining fibers are rinsed in deionized water, dried to a constant
weight and inspected by SEM

       Three cells will be used for each set of experimental conditions. For each pH/flow rate
combination one blank cell will be run in parallel, which is performed in the same way as the fiber
containing cells, but without fibers.

       The F/A ratio has to be understood as the flow rate of the solution divided by the initial
surface area of the fiber sample expressed as jim/s.  F has to be kept constant within ±10% of the
nominal value.  The respective F/A is adjusted either by the flow rate of the solution or by the
amount of fibers. Since so-called technical fibers with the respective diameter distribution are
applied, the amount of fiber samples should be more than 50 mg per cells.  If the required flow rate
is too high, slightly lower F/A ratios will be adjusted.  The exact values will be fixed when the
fiber samples are available.

       The composition of the solution for both pHs will be defined in time, according to the
requirements outlined above.

Analytical investigations

       The dissolution is monitored by the  chemical analysis of the eluate in the collecting
container. The analysis will be performed at 1,4,7,14,28 and 42 days after starting the test The
eluate is analyzed for Si, Ca and some other elements suitably analyzed (B, Al,...) by ICP-AES
and AAS, respectively, using defined aliquots of the eluate and standard dilutions.

       The pH of the solution has to be measured before and after the cell once a day, if possible,
and just before the eluate is removed for analysis. Just after removing the collecting container for
the analysis the weight of the eluate has to be determined.

Calculations

       There are no standard rules for computing the dissolution rate or dissolution velocity which
should be done based on the standard characterization (chemical composition and diameter
distribution) and the  analytical data.
                                            IVG-2

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REC.l LATORY r 1996 I
ARTICLE NO 0128
                                    WORKSHOP  REPORT

            Chronic  Inhalation Toxicity  and Carcinogenicity Testing
                             of Respirable  Fibrous Particles1

       VANESSA Vu.* J. CARL BARRETT,t JOSEPH ROYCROFT,! LORETTA SCHUMAN,* DAVID DANKOVIC,§
                   PAUL BARON,§ TED MARTONEN," WILLIAM PEPELKO,|| AND DAVID LAI*'2
 ^Office of Pollution Prevention and Toxics !7403>, US. Environmental Protection Agency, Washington, DC 20460: ^National Institute of
      Environmental Health Sciences, Research Triangle Park, North Carolina; ^.Occupational Safety and Health Administration,
    Washington. DC: ^National Institute for Occupational Safety and Health, Cincinnati, Ohio; ^Office of Research and Development,
      L~ S  Environmental Protection Agency. Research Triangle Park, North Carolina; and \\Office of Research and Development.
                               U.S. Environmental Protection Agency, Washington, DC

                                          Received September 7, 1996
  On May 8-10, 1995, a workshop on chronic inhala-
tion toxicity and carcinogenicity testing of respirable
fibrous particles was held in Chapel Hill, North Caro-
lina. The workshop was sponsored by the Office of Pol-
lution Prevention and Toxics, U.S. Environmental Pro-
tection Agency  (EPA), in collaboration with the Na-
tional  Institute  of Environmental Health  Sciences
(NTEHS),  the National  Institute  for Occupational
Safety and Health (NIOSH), and the Occupational
Safety and Health Administration (OSHA). The goal of
the workshop was  to obtain input from the scientific
community on a number of issues related to fiber test-
ing. Major issues for discussion were: (i) the optimal
design and conduct of studies of the health effects of
chronic inhalation exposure of animals to fibers; (ii)
preliminary studies which would be useful guides in
designing the chronic exposure study; (iii) mechanis-
tic  studies which would be  important adjuncts to the
chronic exposure study to enable better interpretation
of study results  and extrapolation of potential effects
in exposed humans; and (iv) available screening tests
which can be  used  to develop a minimum data set for
(a) making decisions about the potential health hazard
of the  fibers and (b) prioritizing the need for further
testing in a chronic inhalation study. After extensive
discussion and debate of the workshop issues, the gen-
eral consensus of the expert panel is that chronic inha-
lation  studies of fibers in the rat are the most appro-
  1 This Workshop Report has been reviewed and approved by the
Office of Pollution Prevention and Toxics, U.S. Environmental Pro-
tection Agency. Approval does not signify that the contents necessar-
ily reflect the views and policies of the Agency. The contents of this
report also do not necessarily reflect the views and policies of NIEHS,
NIOSH, or OSHA.
  2 To whom correspondence should be addressed.
priate tests for predicting inhalation hazard and risk
of fibers to humans. A number of guidances specific
for the design and conduct of prechronic and chronic
inhalation studies of fibers  in rodents were recom-
mended. For instance, it was  recommended that along
with other information (decrease in body we ght, sys-
temic toxicity, etc.), data should be obtained on lung
burdens and bronchoalveolar lavage fluid at alysis to
assist in establishing the chronic exposure  levels.
Lung burden data are also important for quantifying
aspects of risk assessment related to  dosimetric  ad-
justments before extrapolation. Although mechanistic
studies  are not recommended as part of the standard
chronic inhalation studies, the expert panel stressed
the need for obtaining mechanistic information as far
as possible during the course of subchronic or chronic
inhalation studies. At present, no single assay and bat-
tery of short-term assays can predict the outcome of a
chronic inhalation bioassay  with respect to carcino-
genic effects.  Meanwhile, several short-term in vitro
and in vivo studies that may be useful to assess  the
relative potential of fibrous substances to cause lung
toxicity/carcinogenicity have been identified.  • 1996
Academic Press, Inc.
                  INTRODUCTION

  An important task for environmental protection is
to identify and subsequently to prevent, eliminate, or
mitigate the risks to human health and the environ-
ment posed by toxic substances. Natural and synthetic
fibers are one group of substances that have been iden-
tified to be of potential concern. Many  of these fibers
have wide industrial and commercial applications, and
for some there are limited, inconclusive, or virtually no
0273-2300/96 $18.00
Copyright £ 1996 by Academic Press, Inc.
All rights of reproduction in any form reserved.
                                                   202

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                      CHRONIC INHALATION TOXICITY AND CARCINOGENICITY TESTING
                                              203
information about their health effects and/or exposure
to workers, consumers, and the general public. As a
result,  the U.S. Environmental Protection Agency
(EPA) has added a "respirable fibers" category as prior-
ity substance for health effects and exposure testing to
obtain the necessary data to evaluate the extent and
magnitude of health risks to exposed individuals and
populations. This would then allow the Agency to deter-
mine whether there is a basis  for any risk reduction
measures.
  The health endpoints of concern for respirable fibers
are the potential development of respiratory diseases
including cancer from chronic inhalation exposure. In
humans, the inhalation of asbestos and erionite fibers
has been associated with the development of nonmalig-
nant and malignant diseases, primarily of the lung,
pleura, and  peritoneum. The mechanisms by which
these fibers induce diseases in humans are not clearly
understood. It is generally believed, however, that the
potential toxicity and carcinogenicity of a given fiber
type appear to be dependent upon the respirability and
biopersistence of the particle, i.e., the ability of the fiber
to enter the  respiratory tract and penetrate into  the
alveolar region of the lung, and to be retained in  the
lung.
  EPA recognizes that the current health effects test
guidelines for chronic inhalation toxicity and/or carci-
nogenicity studies are  not sufficiently specific for  the
testing of fibrous substances. Thus, there is a need  for
EPA to develop standardized health effects test guide-
lines for fibrous substances  that can be used by EPA
in future rulemaking,  negotiated enforceable consent
agreement, or voluntary action to obtain the necessary
toxicologic information for risk assessment. However,
at present, there is no general agreement upon test
protocols for chronic inhalation toxicity and carcinoge-
nicity testing of fibers for regulatory purposes. It is,
therefore,  important for the Agency to obtain input
from the scientific community on a number of issues
related to fiber testing prior to the development of stan-
dardized health effects test guidelines for fibrous sub-
stances.
  On May 8-10,  1995, a workshop on chronic inhala-
tion toxicity  and carcinogenicity testing of respirable
fibrous particles was held at Chapel Hill, North Caro-
lina. The workshop was sponsored by the Office of Pol-
lution Prevention and Toxics,  EPA, in collaboration
with the National Institute of Environmental Health
Sciences (NIEHS), the National Institute for Occupa-
tional Safety and Health (NIOSH),  and the Occupa-
tional Safety and Health Administration (OSHA). The
Steering Committee ("authors of the present paper),
which was responsible for the  planning of the  work-
shop, was composed of representatives of each of these
health protection/regulatory government agencies. The
workshop was conducted by Research Evaluation Asso-
ciates, Inc., an EPA contractor who was responsible  for
convening the expert panel which was composed of 19
international expert scientists in inhalation toxicology.
The expert panel (see Appendix) reviewed, evaluated,
and commented on the  scientific  issues of the work-
shop. Dr. Giinter Oberdorster of the University of Roch-
ester was the workshop chair  and a number of other
members of the expert panel served as session chairs
and rapporteurs. More than 60 participants from  the
government, industry, academia, and the general pub-
lic attended this workshop. The workshop agenda,  list
of participants, and the full account of the panel discus-
sions have been published in an EPA report (USEPA,
1996). The present report summarizes the workshop
issues identified in an  issue paper prepared by  the
Steering Committee and the panel's conclusions and
recommendations prepared by the rapporteurs of  the
workshop.

              WORKSHOP OBJECTIVE

  The objective of the workshop was to obtain guidance
from the scientific  community  on  a number of issues
related  to chronic inhalation  toxicity/carcinogenicity
testing of fibrous substances. Major issues involved in-
clude the following:

  (1) the optimal design and conduct of studies of  the
health effects of chronic inhalation exposure of animals
to fibers;
  (2) preliminary  studies  which would  be  useful
guides in designing the chronic exposure study:
  (3) mechanistic  studies which would be important
adjuncts to the chronic exposure study to enable better
interpretation of study results and extrapolation of po-
tential effects in exposed humans; and
  (4) available  screening tests which  can be used to
develop  a minimum data set for (a) making decisions
about the potential hea'lth hazard  of the fibers and (b)
prioritizing the need for further testing in a chronic
inhalation study.

  The expert panel was  asked to review a background
document (Oberdorster,  1995; in Appendix IV. USEPA,
1996) which provides  an overview of major is>ues  re-
lated to toxicologic testing of respirable fibrous parti-
cles and an issue paper  (Appendix III, USEPA. 1996)
which presents the issues  for workshop discussions.

WORKSHOP ISSUES AND PANEL RECOMMENDATIONS

  The workshop issues were grouped into sevi-n major
topics for discussion purposes.

      I. Inhalation Exposures: Materials and Methods

I.I. Definition of Fibers

  Fibers are  generally defined as elongated  particles
with a length-to-diameter ratio (i.e., aspect ratm < equal

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204
XT' ET AL.
to or greater than 3  to 1. This definition is presumed
to include particles with varying shapes such as rod-
like, curly, or acicular (needle-like) shapes, and having
different structural units commonly referred to  as fi-
bers, fibrils, or whiskers.
  Fibers of most concern to humans appear to be con-
fined to the thoracic particulate fraction. For instance,
it has  been shown that humans exposed to  asbestos
fibers develop lung tumors both in the conducting air-
ways and in  the peripheral regions. In contrast, lung
tumors are induced  in the peripheral regions in rats
after inhalation of fibers, indicating that respirable fi-
bers are the most hazardous ones  for rats.  Further-
more, longer human respirable fibers which  are haz-
ardous  to humans may. not be  respirable by the rat.
On the other hand, because of the smaller size of rat
phagocytic cells, shorter fibers may  be carcinogenic in
rats but may be readily cleared in humans.
  Question 1: Is an aspect ratio  of equal to or greater
than 3:1 an acceptable definition of a fiber? If not, how
should  the definition be modified  to encompass the
varying  range of sizes and shapes of naturally oc-
curring and synthetic fibrous substances?
  Question 2: Since  fibers of potential health concern
to humans are those that are deposited in the entire
lung, not just in the gas exchange region, a more correct
term would be thoracic fibers rather than respirable
fibers. What would be appropriate definitions of human
"thoracic" fibers and "respirable" fibers? What would
be a suitable definition of rat "respirable"  fibers?

  Conclusions and recommendations:  A fiber is de-
fined as a particle having an aspect ratio of at least 3:1
ilength:diameter) and being structurally continuous.
  Respirability should be defined on  the basis of experi-
mental data,  rather than calculated data. The  term
"respirable fiber"  should always be used with a species
modifier, such as "human-respirable" or "rat-respira-
ble." "Respirable" means that the particle in  question
can penetrate to  the alveolar region upon inhalation.
A "rat-respirable  fiber" is defined as a fiber having an
aerodynamic diameter of less than 3 ^m- "Thoracic"
fibers were defined by the participants only in general
terms as those fibers penetrating to the conducting air-
ways upon inhalation.

1.2. Selection Criteria for Suitable Test Materials

  As discussed above, there are considerable differ-
ences in fiber inhalability and respirability  between
humans and laboratory  rodents.  This  observation
raises several questions with respect to the choices of
fiber samples to be tested, recognizing the inherent lim-
itations  of using  rodent species as  surrogates of hu-
mans in inhalation studies, and the need for optimizing
the study conditions while still being  able  to obtain
pertinent toxicologic information for extrapolations to
humans.
        Question 1: For a given fiber type, should inhalation
      studies be performed using samples with the greatest
      potential for pathogenic effects (e.g., long, thin fibers)?
        Question 2: Should fiber samples for testing be pre-
      pared so that they are rodent-respirable. or should they
      represent a  human-respirable sample (or  fibers con-
      forming to a human thoracic particle definition)?
        Question 3: Should the test fibers reflect what are
      actually present at  the workplace  and/or nonoccupa-
      tional environments?
        Question 4: In the case of new  fibers, how should the
      test materials be selected?

        Conclusions  and  recommendations.   It  was  sug-
      gested that to maximize sensitivity of animal inhala-
      tion exposure studies for determining health effects of
      fibers, the test material should consist of rat-respirable
      fibers and should be enriched with the most  potent
      human-respirable fraction (i.e., long, thin fibers); there-
      fore, rodent inhalation exposure  studies should use an
      exposure aerosol that is, as far as is technically feasi-
      ble, enriched with the following fiber size fractions: rat-
      respirable fibers with aspect ratio of at least 3:1 and
      aerodynamic diameter less than 3 urn, and human-re-
      spirable fibers with lengths of at least 20 pm or fibers
      with high aspect ratios. The fraction of long fibers (>20
      /mi) should be specified; 10 to 20%  enrichment would
      seem appropriate, but not enough information is avail-
      able on which to base a specified percentage. The aero-
      solized fibers should be discharged to Boltzmann equi-
      librium before being delivered to the test species.
        If the study results are negative and it can be  shown
      that fiber mass  loading and  fiber size distribution in
      the lung are not sufficient, then the fiber would be con-
      sidered to not have been adequately tested.

      7.3.  Characterization of Test Fibers

        There is considerable evidence to suggest the  impor-
      tance of fibej" characteristics in relation to disease out-
      comes. Thus, it is desirable to obtain data on a number
      of the physical and chemical properties of the particles.
      These data will also enable the investigator tn  make
      some preliminary estimates of the lung burden »i the
      material at a given exposure concentration, the behav-
      ior of the particle in the lung, and to some extern, its
      expected toxicity.
        Question 1: At a minimum, what aspects of tin-  test
      samples need to be characterized (e.g., fiber mnrphol-
      ogy, dimension,  size distribution, aerodynamic diame-
      ter, chemistry, density,  solubility, surface char.uu-ns-
      tics, the ability of a fiber to split longitudinally nr t m-s-
      sectionally)?
        Question 2: Are there any specific analytical run hod.s
      that should be required to be used to character!/.  . er-
      tain chemical and physical properties of bulk m.iti nals
      or individual fibers present  in  the aerosol ami  inng
      tissues?

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                      CHRONIC INHALATION TOXICITY AND CARCINOGENICITY TESTING
                                               205
  Question 3: Are the available methods for the mea-
surement of fiber  size distributions considered  ade-
quate? Are there any new and improved methods that
can be used for measuring fiber size distributions?

  Conclusions and  recommendations.  The complete
bivariate length and diameter distribution should  be
determined in the aerosol and in the lung via electron
microscopy. This bivariate distribution should include
the nonfibrous particles present in the aerosol. For non-
fibrous particles, "length" and "diameter" can be deter-
mined as the "longest length" and "narrowest dimen-
sion." The aerodynamic size distribution should be de-
termined; a cascade impactor  can be  used for this
purpose. The World Health Organization (WHO) count-
ing rules and sizing rules should be used and results
should be evaluated statistically to assure sufficient
sensitivity. Interlaboratory validation should be pro-
vided for all counting and sizing methods. In addition,
it is recommended that sizing techniques be used that
permit returning to the same fields and fibers in the
event it is necessary to confirm counting. Routine moni-
toring to control the day-to-day aerosol concentration
can be performed using phase-contrast optical micros-
copy (PCOM) and gravimetric techniques.

1.4. Exposure Conditions and Methods

  There are certain disadvantages associated with  ei-
ther whole-body nose-only exposure: greater stress of
the animals with nose-only method and ingestion from
grooming with whole-body method.  Both methods are
considered acceptable by EPA and other regulatory au-
thorities as appropriate methods for inhalation testing
of chemical substances.  Regardless of the exposure
method used, fiber samples need to be aerosolized in
such  a way that they are evenly distributed  in the
chamber atmosphere and that there is adequate sam-
pling to verify the integrity of the aerosolized fibers
and that constant fiber concentrations are maintained
throughout the  exposure  period.
  Question 1: Should there be  a requirement for the
use of any particular method for  generating fibrous
aerosols?
  Question 2: Are both methods of exposure (whole-
body and nose-only exposures)  acceptable?  Is there a
preferred method that should be recommended?
  Question 3: How often  should exposure atmosphere
be monitored with regard to  fiber number and mass
concentration, size distribution, and chemical analysis?

  Conclusions and recommendations.  The test guide-
lines should not specify a  particular aerosol generation
system, but should  require that the exposure system
be validated by the investigator (e.g., with respect to
airborne fiber size distribution and  the target dose in
the lung). It should be demonstrated that the genera-
tion system does not contaminate the fibers.
  Either nose-only or whole-body exposure can be used.
The  target exposure  concentrations should be mea-
sured regularly during the course of the study by elec-
tron microscopy (fiber number and bivariate size distri-
bution)  to confirm the dose delivered to the animals.
The  frequency of exposure  atmosphere monitoring
should be daily for mass concentration, weekly for fiber
concentration  and bivariate size distribution, every 3
months  for chemical analysis.

      II. Study Design for Chronic Inhalation Studies

ILL  Animal Species/Strain/Sex Selection

  EPA's current  test guidelines for oncogenicity re-
quire that  a compound  of unknown activity shall  be
tested on two mammalian species via oral, inhalation,
or dermal route of exposure. Rats and  mice of both
sexes are the species of choice without specifying more
precisely any  specific strains, except that commonly
used laboratory strains shall be employed. Justification
when selecting other  species  must be provided. Rats
and mice are the species of choice  mainly because of
their relatively short life spans, their widespread use
in toxicological studies,  their susceptibility to tumor
induction, and the limited cost of their maintenance.
However, for combined chronic toxicity and oncogenic-
ity study, the rat is the species of choice.
  Inhalation studies with asbestos  fibers in  rats have
been  demonstrated  to be appropriate experimental
models  for the identification  of asbestos-induced hu-
man diseases, primarily fibrosis and cancer of the lung.
The low mesothelioma rate induced in rats via inhala-
tion, compared with the rate of crocidolite-induced me-
sotheliomas in humans,  indicates that the rat inhala-
tion model may not be adequately sensitive to identify
the potential ability  of fibers of unknown activity  to
induce  mesothelioma in humans.  Exceptions occur
when the fiber in question is expected to be a potent
mesothelioma  inducer such as erionite fiber. Since in-
duction  of mesothelioma is also a health end point of
concern, testing in a second rodent species may he nec-
essary to ensure that all potential health effects would
be properly identified.
  On the other hand, questions have been raised about
the validity and utility  of using either the  mouse  or
the hamster as the second species  for carcinoijemcity
testing of fibers. The concern is that there have been
fewer studies using mice and hamsters with asbestos
fibers and,  although many of these studies have been
considered  limited (e.g.,  using short asbestos fibers,
short duration of exposure), results obtained to date
seem to indicate that they may not  be suitable animal
models for  predicting asbestos fiber-induced diseases.
The mouse generally does not respond to tumor induc-
tion by asbestos fibers via inhalation. In the ca.-r of the
hamster, this species appears to be more sensitm- than
the rat with respect to fiber-induced mesothelioma. but

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                                               \"U ET AL.
less sensitive to the  induction of lung tumors and fi-
brosis than the rat.
  Question 1: Is it necessary to test fibers of unknown
activity in a second animal species? If yes, what would
be an appropriate second  species (e.g., Syrian Golden
hamster, Chinese hamster)? If not, what are the scien-
tific reasons? Are  there any circumstances that  war-
rant testing in a second species?
  Question 2: Do different  animal strains respond with
different sensitivities to fibrous particles? If yes, what
is the most  appropriate strain of rodent species to be
used?
  Question 3: Should both  sexes of the animal be used?
If not, which sex is more suitable, and why?

  Conclusions and recommendations.   Given the pres-
ent limited knowledge of the effects of fibers in species
other than the rat, testing of fibers in a second animal
species is not strongly recommended;  however, investi-
gators  should be encouraged to investigate health ef-
fects of fibers in another species, particularly the ham-
ster. In the future, transgenic animals may prove use-
ful for testing.
  Although  it  was  acknowledged that rat  strains
clearly differ  in suitability for inhalation exposure
studies  with fibers, the  panel could not agree  on
whether  to recommend a particular strain to use (or to
avoid), but  proposed a set of criteria  for choosing a
strain. It was agreed that  criteria for a suitable strain
include (1) a low background  rate of neoplasia, (2) a
low background rate of pulmonary disease,  (3) longev-
ity, and (4) a history of laboratory use.
  It was  agreed that whether to use one or  both sexes
should be left to the investigator, and that if one sex
is used, the  choice should  depend on  factors related to
the strain, test material, and endpoint studied. How-
ever, if cost was not a factor, testing  in both sexes
should be encouraged because data on sex differences
in response to inhalation exposure to fibers are limited.

II.2.  Selection of Exposure Concentrations

  EPA's  current  test guidelines  for oncogenicity  re-
quire at least  three exposure concentrations and a
sham-exposed (filtered air only) control group. The
highest concentration  should  elicit signs of minimal
toxicity without substantially altering the normal life
span other than tumor formation. The lowest exposure
concentration should not induce any indications of tox-
icity and the intermediate concentration(s) should be
established in a mid-range between high and low con-
centrations. There is no specific  guidance  on  how to
select aerosol concentrations for inhalation studies of
particulate matter.
  For combined chronic toxicity and oncogenicity stud-
ies, EPA's guidelines require the use  of a high concen-
tration treated  and control satellite group designed to
evaluate pathology other than neoplasia. The highest
concentration for satellite animals should be chosen to
produce frank toxicity, but not excessive lethality.
  Recently,  a number of criteria (e.g., effect on lung
clearance and pulmonary function, chronic inflamma-
tory responses, cell  proliferation,  histopathological
changes) have been proposed to define the highest fiber
concentrations  to  be  tested in a chronic study, also
known as the maximum aerosol concentration or MAC.
  Question 1: What criteria can be used to determine
the maximum aerosol concentration (MAC) in inhala-
tion studies of fibrous  particulates  and  to judge
whether  a MAC has been reached or exceeded?
  Question 2: What preliminary studies would be useful
and important for setting appropriate exposure concen-
trations (e.g., 90-day and/or shorter-term inhalation stud-
ies, in vitro solubility, in vivo biopersistence studies)?
  Question 3: The National Toxicology Program (NTP)
generally employs  an upper limit exposure concentra-
tion of 100 mg/m3 for relatively insoluble nonfibrous
particles of low toxicity.  In view of potential particle
"overload," should a practical upper limit concentration
also be set for fibrous particles?

  Conclusions and recommendations.  A. practical up-
per limit concentration was not endorsed since it would
depend on fiber type, and no one number could be deter-
mined that applies to all fibers. The MAC should be
based on the total  number of inhaled particles (fibers
and nonfibrous particles  combined). The MAC should
be considered based on a combination of the following
parameters determined in a 90-day subchronic inhala-
tion study: altered  alveolar macrophage-mediated par-
ticle clearance  rate, fiber lung burden normalized to
exposure concentration, cell proliferation, histopathol-
ogy, inflammation  (quantitatively determined as per-
centage  increase  in  polymorphonuclear leukocytes
[PMNs] in lung lavage samples) and lung weight. It
was suggested that an appropriate lung burden of criti-
cal fibers (long and thin) should be  achieved, but  no
number was  suggested. These parameters should  be
considered together, rather than individually, in an  at-
tempt to define a maximum tolerated dose (MTD)  for
the chronic study. The MTD is the lung dose achieved
with the MAC.  For the chronic study, three exposure
concentrations should be  used; the high exposure con-
centration and resulting lung dose should show sig-
nificant effects  in  the above parameters (MTD), and
the lower doses should be appropriately spaced and be
selected based  on  results from  the 90-day study and
from previous studies with the particular fiber. Ancil-
lary studies should be conducted to determine in vitro
solubility and in vivo biopersistence.

II.3. Exposure Regimen and Observation Period
  EPA's  current test guidelines  require that the ani-
mals are exposed to the test substance for 6 hr per day,
5 days per week over a period of at least 24 months  for

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                      CHRONIC INHALATION TOXICITY AND CARCINOGENICITY TESTING
                                                                                                   207
rats, and 18 months for mice and hamsters. Termina-
tion of the study should be at 24 months and not longer
than 30 months for rats, and at 18 months  and not
longer than 24 months  for mice and hamsters. How-
ever, termination of the study is acceptable when the
number of survivors of the lower exposure groups or of
controls reaches 25%.
  Experience with asbestos fibers indicates that fiber-
induced lung tumors or  mesothelioma in rats  occur at
relatively advanced age. Thus, it would be desirable to
allow the animals to live out their life span after the
2-year exposure is completed. On the other hand, there
are disadvantages of a  lifetime study. These include
the high mortality rate in rats over 2 years of age, and
the high incidence pf age-related spontaneous  nonneo-
plastic and neoplastic lesions which would make inter-
pretation of study findings difficult.
  Question 1: Is the exposure regimen as specified in
EPA's guidelines appropriate for the testing of fibers?
  Question 2: Is it necessary to recommend when final
sacrifice be carried out?  If so, when would it be?

  Conclusions and recommendations.  The chronic in-
halation exposure study with fibers should be a lifetime
study with exposure terminated at 24 months in rats
and the study terminated when survival of the control
group reaches 20%. Due to the shorter life span of ham-
sters, their exposure duration could be shorter, based
upon survival/lifetime expectancy.

II.4. Numbers of Animals and Interim Sacrifices

  EPA's current test guidelines require that  at least
100 animals (50 males and 50 females) are to  be used
for each exposed and sham-exposed control groups. Sat-
ellite exposed and control groups consisting of 20 males
and 20 females are to be used in the combined chronic
toxicity and oncogenicity study. Additional animals are
used if interim sacrifices are planned. However, this is
optional.
  Question: Should interim sacrifices be recommended
for the testing of fibers? If yes, what would be an appro-
priate interim sacrifice schedule and design (e.g., num-
ber of animals per group, duration of exposure and
recovery period)?

  Conclusions and recommendations.   Interim sacri-
fices are essential and should be made at 3, 6, 12, 18,
and 24  months  in rats.  The endpoints evaluated at
these  times should be the same as in the subchronic
study. Since hamsters do not live as  long as rats, a
study of 24 months  exposure may not be  possible in
this species. Lung clearance of particles in live  animals
also should be measured at set intervals. Investigators
should be encouraged also to follow recovery in  animals
exposed for shorter periods and sacrificed at the same
intervals. The number of animals will depend on the
specific study design.
II.5. Use of Positive Control
  The use of a positive control is not required in EPA's
current test guidelines for chronic toxicity and oncoge-
nicity testing. However, in view of the complexity of
conducting an inhalation study with fibrous particles,
it may be useful to consider including a group of posi-
tive controls  to validate the reliability of the testing
system. Asbestos fibers are most often used as a posi-
tive control, but exposure-dose-response relationships
have not yet been established for any types of asbestos
fibers. Moreover, standardized UICC reference materi-
als  (e.g., UICC crocidolite) have been considered  not
suitable because of their short  fiber length.
  Question 1: Should a positive control be included in
the chronic inhalation study with fibers? If not, why?
  Question 2: If yes, what might be appropriate criteria
for  selecting as a positive control asbestos fibers with
fiber size distribution similar to the test material?
  Question 3: How many exposure concentrations of a
positive control should be conducted? If only one expo-
sure concentration is used, should it be comparable to
the highest exposure concentration of the test material
in terms of fiber concentration or fiber lung burden?
Or should it be at an exposure  concentration expected
to induce tumor effects?
  Conclusions and recommendations.  A positive con-
trol need not be included in every study, but each new
test system (including use of a different animal species
or strain) should be validated  with a positive control
material.
  In addition, it was generally agreed that  a  chronic
multidose asbestos inhalation study in rats is critically
needed to validate and calibrate the chronic rat inhala-
tion assay (a) for evaluation of the toxic and carcino-
genic potential and potency of  other fibers and (b)  for
comparison with known human  carcinogenicity data
for asbestos. It was strongly recommended by the pan-
elists that; priority should be given to conduct such a
multidose asbestos inhalation study. The exposure con-
centrations should be based on the outcome of a sub-
chronic 90-day inhalation study using the same criteria
for deriving the MAC and MTD as is used for the test-
ing of other fibers.

77.6. Criteria  for a Negative Inhalation Test
  EPA generally considers an oncogenicity study to be
negative if there is an absence of tumor effects in an
adequately sensitive and  well-conducted study. The
key issue which needs to be defined is what constitutes
"an adequately sensitive study" for the testing of fi-
brous particles.
  Question: What might be suitable criteria for the ac-
ceptance of an inhalation study with fibers as negative
(no  tumors,  achievement of a MTD,  appropriately
spaced  lower  concentrations,  adequate  animal sur-
vival, use of an appropriate positive control i'.'

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                                               VU ET AL.
  Conclusions and recommendations.  For acceptance
of the results of a chronic inhalation exposure study
with fibers as negative, the study must have been de-
signed and conducted according to the criteria outlined
previously; the  health effects of concern must not be
sirrnificantly  more frequent in the exposure  groups
than in the control group. In order to detect a positive
effect, the power of the study should be such that a as
the type I error is controlled at 0.05 and as the type II
error is controlled at 0.2.

                III. Prechronic Studies

  EPA generally recommends a subchronic  90-day
study to help establish suitable study conditions for the
chronic study, especially  for setting appropriate expo-
sure concentrations. The subchronic study would also
provide important toxicologic information to be used in
conjunction with results of other mechanistic studies to
help in the interpretation of the chronic study findings.
  It has been suggested that for the testing of particle
toxicity, the primary goals of a subchronic study should
include: (a) an evaluation of patterns of particle deposi-
tion (including  hot spots of deposition), translocation,
and clearance,  and determination of the lung burden
at which impaired clearance occurs; and (b) an evalua-
tion of toxicity and mechanisms of pulmonary toxicity.
  Question 1: Should a subchronic 90-day study be rec-
ommended prior to conducting the chronic study? Or
should it be made optional?
  Question 2: What are the primary goals of the sub-
chronic study?
  Question 3: What specific data related to fiber dispo-
sition should be obtained in the subchronic study?
  Question 4:  Are there any specific methods that
should be recommended to measure the effects of fibers
on lung clearance (e.g., use of radiolabeled particles)?

  Conclusions  and recommendations.  A subchronic
study should be conducted unless sufficient data are
available  from  other studies to  allow for the proper
design of the chronic study. The primary goals of the
subchronic study are (1) to establish lung burdens and
potential  target sites to  aid in design of the chronic
study and (2) to evaluate toxicity for a variety of im-
portant biological endpoints.
  Lung burden analysis and bronchoalveolar lavage
fluid (BALF) are recommended in prechronic inhala-
tion studies of fibers (as well as in chronic studies, see
sections  below).  Early  fibrosis   should be assessed
through histological examination. Other studies would
be complementary, such as replica cast studies to iden-
tify  hot-spot locations of deposition; however, these
studies are ancillary and should not be required.
  Impairment of clearance should be assessed via chal-
lenge with a tagged particle. Clearance should be as-
sessed after the 90-day exposure period. The clearance
of the labeled particles should be measured over a pe-
riod of a few months. Although no specific method is
recommended, the method chosen should be validated.
Also, it is important to distinguish between fiber clear-
ance and clearance of the test particle used in the chal-
lenge.

           IV. Fiber Disposition and Dosimetry
IV. 1. Lung Burden Analysis
  Lung burden analysis is not a requirement in EPA's
current study protocol for chronic toxicity and oncoge-
nicity testing of chemicals. However, lung burden data
would provide useful data on biopersistence of the test
fibers and serve as a better measure of internal dose.
  Question 1: Should lung burden analysis be included
in the subchronic and chronic studies?
  Question 2: If yes,  should the procedure as recom-
mended by the International Cooperative Research
Programme (ICRP) be adopted? There are data to indi-
cate that fiber burden data based only on the accessory
lung lobe (as recommended  by the ICRP)  may not be
representative of the whole lung because of nonuniform
pattern of deposition. In view of these  findings, what
changes should be recommended?
  Question 3: Should any specific methods for lung ash-
ing be recommended?
  Question 4: How often should lung burden analysis
be  performed  (at interim  and  final  sacrifice  time
points)?
  Conclusions and recommendations.   Lung burden
analysis  should be included in  the subchronic  and
chronic studies  even if extra animals need  to be added
to the study. Data should be obtained on lung burdens
(a) to assist in establishing the chronic exposure levels
or aerosol generation changes needed to  get more fibers
deep into the lung and (b) to quantify  aspects of risk
assessment related to dosimetric adjustments before
extrapolation. Data also should be obtained on  fiber
deposition in the  nasal cavity and the fiber burden in
the thoracic lymph nodes, and collection of pleural tis-
sues is encouraged.
  For fiber burden analysis, one of the  two lungs (left
or right) should be used, rather than only the accessory
lobe (as recommended by the ICRP). It may be possible
to determine a correction  factor during subchronic
studies that would allow the use of only one lobe in the
chronic studies. However, disease development could
change deposition patterns and invalidate  a correction
factor. Five to six animals per exposure group should
be studied  at each time  point.
  Rather than "lung ashing," the proper term is "lung
digestion,"  because the  guidelines will  apply to other
types of fibers in addition to  man-made  vitreous fibers.
No specific  lung digestion method is recommended. The
investigator must show that the fibers are  not affected
by the method used  to harvest them  from the  lung
tissue.

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                      CHRONIC INHALATION TOXICITY AND CARCIXOGEXICITY TESTING
                                               209
  Lung burden  analyses should be required after  3,
6, 12, 18, and 24 months of exposure. Pleural burden
analysis is not recommended at this time. However,  in
view of the potential use of the pleural data in quantita-
tive risk assessment and the cost of repeating studies,
investigators should be encouraged to collect pleural
burden samples and keep  them available for future
analysis.

IV.2.  Biomarkers of Toxicologic Effects

  Recent studies in rats have demonstrated the value
of BALF analysis in subchronic studies in predicting
the chronic effects of exposure to a variety of particles.
This will enable the investigator to better select the
exposure concentrations for the chronic studies as well
as to help in understanding the biochemical and cellu-
lar sequence of events of particle-induced toxicity and
carcinogenicity.
  Question 1: Which specific biomarkers of toxicity and
carcinogenicity should be measured in the subchronic
study (e.g., BALF analysis, cytotoxicity, cell prolifera-
tion;?
  Question 2: Should BALF analysis be made manda-
tory for the chronic study?
  Question 3: If yes, should the procedure as specified
in the ICRP's protocol be adopted? Are there any modi-
fications that should be considered?

  Conclusions and recommendations.  The subchronic
study should include analysis of BALF for evaluation of
inflammatory response (e.g., protein content, enzymes,
presence of inflammatory cells)  and measurement  of
cell proliferation. BALF analysis should be required  in
the chronic study.

FV.3.  Dosimetry and Interspecies Considerations

  The size distribution of fibers that deposit in the
lungs of rodents may be different from those in the
lungs of humans because of anatomical and physiologi-
cal differences (e.g., lung surface area, respiratory ex-
change rates, clearance  rates, dissolution rates). Depo-
sition and clearance mathematical models  have been
developed to relate the  fiber lung burden to biological
effects which may serve as  useful exposure-dose-re-
sponse models for human risk assessment.
  Question 1: Which additional dosimetry information
could be obtained as part of the chronic study to help  in
characterizing the human hazard and exposure-dose-
response assessment?
  Question 2: With regard to the selection of the human
dosimeter, is milligrams per kilogram body weight ad-
justed for differences in metabolic rate appropriate for
fibers? For diesel exhaust, EPA utilizes milligrams per
unit alveolar surface area as the dosimeter without an
adjustment for metabolic rate. Is this reasonable for
fibers, or are their mechanisms sufficiently different?
  Conclusions and  recommendations.  Quantitative
information on the fiber burden at airway bifurcations
or other localized sites would be useful. To the fullest
extent possible,  tissues should be preserved in  such
a way that other measurements or  analyses can be
conducted later, depending on the outcome of the bioas-
say or development of new molecular or biochemical
techniques.
  The expert panel did not endorse use of milligrams
per kilogram body weight as a viable human dosimeter,
even after adjustment for species differences in meta-
bolic rates. Various dose metrics can be computed, but
none of them  affect the design of subchronic or chronic
studies.

                V. Mechanistic Studies

  The mechanisms by which fibers induce fibrosis and
cancer are not known, but are thought to be mediated
via reactive oxygen species and growth factor  path-
ways. Fibrous particles  may also cause  mutagenic
events through induction of DNA strand breaks, clasto-
genic effects, deletions, and interference with the spin-
dle apparatus of dividing cells. Mechanistic studies are
generally not required as part of EPA's test guidelines.
However,  in  assessing the potential chronic toxicity
and carcinogenic effects of respirable fibers in humans,
it is desirable to  consider the  differences of species re-
sponses and the  understanding of the mechanisms of
fiber-induced toxicity and carcinogenicity. This  may
allow an improved basis for extrapolating observed ef-
fects in the test species exposed to high concentrations
to humans exposed to relatively lower concentrations
generally found in the workplace and the general envi-
ronment.
  In vivo and in vitro mechanistic studies have been
developed and proven useful and valuable in elucidat-
ing the mechanisms of fiber-induced pathogenesis, i.e.,
chronic pulmonary inflammation, fibrogenesis, and on-
cogenesis'. Evaluative endpoints may include cytotoxic-
ity, phagocytosis, cell proliferation, expression and in-
duction of specific mediators (e.g., cytokines, growth
factors, antioxidants), unscheduled DNA synthesis, de-
termination of DNA repair, or mutational frequencies
of target cells (e.g., HRPT mutation assays of type II
cells).
  Question: Should any mechanistic studies be recom-
mended? Which and when (prior to the chronic study,
in parallel with the chronic study, and/or subsequent
to the chronic study)?

  Conclusions and recommendations.   No mechanistic
studies are recommended at this time. However, inves-
tigators should be encouraged to obtain  mechanistic
information as far as  possible during the course of sub-
chronic or chronic inhalation  studies. A high research
priority should be to  determine whether fiber carcino-
genesis is a direct effect or an indirect effect related to

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2K3
ET AL.
inflammation. It was suggested that the most promis-
ing approach for obtaining mechanistic information is
to isolate target cells after in vivo exposure for use in
subsequent  in  vitro studies. Other priorities for re-
search include <1) development of short-term in vivo
a" 3ays with ex vivo/in vitro investigations in appro-
priate target cell populations. (2) investigation of onco-
genes and tumor suppressor genes  in human and ro-
dent tumors,  (3) development of transgenic animal
models. (4) species comparisons of fiber-induced pulmo-
nary effects  in vivo  and in vitro, and (5) use of pleural
lavage to evaluate predictive markers of response.

             VI. Histopathologic Evaluation

   In this session, in addition to the following questions
raised in  the  issue paper, the panel  discussed what
needs to be examined and measured in the histopatho-
logical evaluation.
   The use of the Wagner scoring system has been sug-
gested at the Chemical Industry Institute of Toxicology
(CUT) workshop (McClellanet al, 1992) for the evalua-
tion of pulmonary fibrosis to enable direct comparison
of effects induced  by  different  types of fibers. The
Wagner scoring system has also been utilized in recent
studies on synthetic mineral fibers. This system, how-
ever, does not consider the mass of the lung tissue in-
volved. One  method that has been used by other inves-
tigators includes  a  morphometric approach to deter-
mine the percentage of lung tissue involved in fibrotic
lesions. In another method, total lung  collagen is mea-
sured as an  indicator of lung fibrosis.
   Question  1: Is it necessary to utilize a standardized
scoring system for the evaluation of cellular changes
and fibrosis  in the lung?
   Question 2: The International Cooperative Research
Programme  (cited in USEPA, 1996) has recommended
that the Wagner scoring system be revised to take into
account the limitation [that it does not  consider the
mass of the lung tissue involved]. What specific modifi-
cations must be made before it can be adopted for inclu-
sion in the test guidelines?

   Conclusions and  recommendations.   In the chronic
study, use of the Wagner scoring system to evaluate
progression  of fibrosis has the disadvantage of being
purely qualitative and inconsistently  applied. To pro-
mote more quantitative evaluation, the testing guide-
lines should specify set procedures for grading of le-
sions and for lung preparation. Further research  is
needed before other  quantitative histopathological
methods can be recommended for large-scale testing.
However, a  promising  approach could be quantitation
of collagen  deposits using sirius red and evaluation
with  polarized light.  Neoplastic endpoints recorded
should include epithelial hyperplasia, alveolar bronchi-
olization,  metaplasia,  adenomas,  mesotheliomas, and
carcinomas. Keratin cysts should be identified as such,
  to permit subsequent evaluation, and it must be stated
  whether or not a cyst presents evidence of invasion or
  dysplasia. A dissecting microscope should be  used to
  examine for mesotheliomas. In distinguishing between
  hyperplasia and mesothelioma, standard diagnostic
  criteria should be applied to identified lesions. Estab-
  lished published guidelines on the use of blinding in
  histopathology should be followed, e.g., those published
  by the Society of American Pathologists.

                   VII. Screening Battery

    Numerous test systems and/or protocols have been
  developed and utilized by the scientific community for
  evaluating the fibrogenic and carcinogenic potential of
  fibrous particles. However, there has been considerable
  debate about the scientific validity and utility of avail-
  able test methods.  This subject along  with research
  needs for better understanding of the  mechanisms of
  fiber-induced disease have been the topics of discussion
  at several scientific conferences, workshops, and expert
  meetings, sponsored by various organizations (e.g., De-
  ment, 1990; WHO, 1992; McClellanetal, 1992; ISRTP,
  1994). A tiered approach for evaluating the toxicity and
  carcinogenicity of new fibers or untested fibers has also
  been  recommended at a workshop sponsored by the
  CUT  as a guideline for research purposes (McClellan
  et al, 1992).
    Other organizations  such as the  ICRP have recently
  developed a draft protocol for the  assessment of syn-
  thetic  fiber's toxicity,  as a part of a  tiered-approach
  testing program (see USEPA, 1996).
    Question  1: Recognizing that no single  screening
  study can accurately predict the in  vivo responses from
  long-term exposure to fibers, after evaluating the phys-
  ical and chemical properties of the fibers (Tier I), can
  Tier II and Tier III types'of studies as  defined in the
  CITT" workshop proceedings (McClellan et al, 1992) be
  used to screen and set priorities with regard to confir-
  matory testing in a chronic study to obtain more defin-
  itive information for risk assessment purposes? If not,
  why not?
    Question 2: If yes, what specific tests or combinations
  of tests can be utilized in this screening battery?
    Question 3: Given that in vivo studies using noninha-
  lation methods of exposure (e.g., intraperitoneal injec-
  tion,  intratracheal  instillation  studies)  have  been
  proven useful in identifying the potential health haz-
  ard to humans, should they be considered acceptable
  as an alternative screening test or  an adjunct to short-
  term  inhalation studies in a screening battery?
    Question 4: Intracavitary testing (ip study) of fibers
  has been proposed by the ICRP (Oberdorster,  1995;
  see USEPA, 1996). Would a positive finding using this
  protocol constitute  a potential hazard to  humans,  or
  would a positive finding need to be followed by a chronic
  inhalation study to confirm the hazard?

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                       CHRONIC INHALATION' TOXICITY AND CARCINOGENICITY TESTING
                                               211
  Conclusions and recommendations.   Appropriately
designed Tier II (in vitro) and Tier III (short-term in
vivo) studies can provide useful  information to assess
the relative potential of fibrous materials to cause tox-
icity in the lung and  associated tissues. Along with
other information, data from a battery  of Tier II and
III  studies can provide key information to prioritize
materials for further chronic testing. At present, no
single assay or battery of short-term assays can predict
the outcome of a chronic inhalation bioassay with re-
spect to carcinogenic effects.

  Tier II in vitro tests.  Solubility/durability can in-
fluence the lung's response to long-term inhalation
of fibers. In most instances, rates of in vitro and in
vivo solubility correlate well, although the absolute
rates may differ. Therefore, in vitro assays providing
information on fiber solubility/durability can provide
useful information for prioritizing groups of fibers for
further testing. It was suggested that in vitro solubil-
ity alone could not be used to rule out further testing,
and that fibers should be evaluated in an in vivo test
system for toxicity and in vivo dissolution. Although
other characteristics of fibers that can be examined
in acellular in vitro tests were discussed, no general
agreement  was reached  on their value as routine
tests  for assessing  the potential of fibers to cause
toxicity. In vitro cell or tissue culture assays can po-
tentially provide useful information on fiber toxicity;
however, these systems are not yet well enough vali-
dated or understood to be recommended for routine
use in screening fibers toxicity.

  Tier III (short-term  in  vivo studies).   There is no
standardized protocol for short-term respiratory tract
exposures to fibers 'i.e., less than 3-month exposure)
followed by characterization of the lung response peri-
odically over several weeks. Nonetheless, this type of
study is a useful tool for assessing the relative  ability
of fibers to produce nonneoplastic effects (e.g., inflam-
mation, cell proliferation, fibrosis) in the lung. Thus,
information from short-term in vivo studies, combined
with data from Tier I studies (physicochemical proper-
ties),  would  be  useful for prioritizing  materials  for
longer-term  studies. Short-term  screening  studies
should include (but not be limited to) analysis of BALF
for markers of cell injury and inflammation, histopa-
thology, and assessment of lung fiber dose. Study de-
sign should include assessments of dose-response rela-
tionships and, when possible, comparisons to  physi-
cally and chemically similar "control" fibers for which
chronic lung effects already have been evaluated. Expo-
sure concentrations  should include at least one level
that elicits significant lung effects, to provide a basis
for comparison of both the nature and persistence of the
response to the fibers. In addition, it would be useful to
assess fiber biopersistence following a short-term expo-
sure (e.g., a 5-day exposure, followed by monitoring for
several weeks).

  Intratracheal  instillation.   Intratracheal  instilla-
tion was considered by a majority of the panel members
to be an acceptable alternative to inhalation exposure
for  short-term screening studies  to assess the relative
biopersistence and relative  nonneoplastic toxicity of
fibers in  the  lung provided  that low  doses are used.
Intratracheal instillation allows a known amount of
test material to be administered  to the lung in a man-
ner not requiring the resources (i.e., exposure facility
and level  of research funding) needed for inhalation
exposure. Moreover, human-respirable fibers not respi-
rable  by  the  rat may be evaluated via intratracheal
instillation, although care should be  taken to  avoid
higher doses of longer fibers which may result in clump-
ing. However, the intratracheal instillation  delivers
materials at a much higher dose rate than does inhala-
tion, and care must be taken to ensure that the re-
sponses observed after intratracheal  instillation are
comparable to what would be expected after inhalation.
Fiber doses should be low enough  to minimize problems
of fiber clumping and  overwhelming  of lung defense
mechanisms.  It would be useful  to include a "control"
fiber for which the lung tissue response after inhalation
has already been characterized to demonstrate that in-
tratracheal instillation produces  a response similar to
that expected after inhalation. The majority thought
that intratracheal instillation should not be recom-
mended for assessing the carcinogenic  potency in long-
term studies.

  Intraperitoneal injection.   Intraperitoneal injection
studies can provide information  on the interaction of
fibers with mesothelial cells. However,  for screening or
rank ordering the potential toxicity of fibers in the lung
based on intraperitoneal injection studies, the behavior
in the lung (e.g., clearance, translocation) of the fibers
being evaluated must be taken into account. What con-
stitutes a MTD for  ip study remains to be  defined.
There was little discussion on this subject.

                  CONCLUSIONS

  EPA's health effects test guidelines  for oncogenicity
and combined chronic toxicity and oncogenicity are
widely accepted by the scientific and  regulatory  com-
munities for the testing of chemical substances (EPA,
1992).  EPA's  guidelines are similar to  those of the Or-
ganization for Economics Cooperation and  Develop-
ment (OECD) and the National Toxicology Program
(NTP). It is recognized, however,  that these guidelines
need to be modified to take into account testing issues
which are unique to fibrous particles.
  Numerous  test  systems  and/or   protocols   (e.g.,
McClellan, 1992, WHO, 1992) have been developed and
utilized by the scientific community for evaluating the

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212
\T ET AL.
fibrogemc and carcinogenic potential of fibrous parti-
cles.  However,  there has  been  considerable debate
about the scientific validity and utility of available test
methods. There are also some  differences  between
these study protocols and the EPA's test protocols with
regard to certain standard requirements of the design
of the study  (e.g.,  species,  strain,  gender,  exposure
method). Thus, there is a need for examining and artic-
ulating  the scientific  bases  for any  recommended
changes for the testing of fibrous particles.
  EPA, in collaboration with the National Institute of
Environmental Health Sciences (NIEHS), the National
Institute for Occupational Safety and Health (NIOSH),
and the Occupational Safety and Health Administra-
tion (OSHA) through an interagency working group,
has identified a number of scientific issues related to
fiber testing that requires further evaluation by expert
scientists.
  In this workshop, the expert panel concluded that at
present, no single assay or battery of short-term assays
can predict the outcome of a  chronic inhalation  bioas-
say with respect to carcinogenic effects;  a number of
guidances specific  for the design and conduct of pre-
chronic and chronic inhalation studies of fibers in labo-
ratory rodents were recommended.  Overall, the objec-
tive of the workshop has been accomplished. The panel
recommendations shall be considered for incorporation
into a proposed health effects test guidelines being de-
veloped by EPA.

                     APPENDIX

Expert Panel

  Giinter Oberdorster (Chair), University of Rochester,
Rochester, New York
  David Berstein, Consultant, Switzerland
  John  Davis,  Institute of  Occupational  Medicine,
United Kingdom
  Kevin  Driscoll,  Procter  and  Gamble  Co., United
States
  David Groth, Consultant, United  States
        Thomas  Hesterberg,  Schuller  International  Inc.,
      United States
        David Johnson, University of Oklahoma,  Norman,
      Oklahoma
        Neil Johnson, Inhalation Toxicology Research Insti-
      tute, United States
        Agnes Kane, Brown  University, Providence, Rhode
      Island
        Ernest McConnell, Consultant,  United States
        Fred Miller, Chemical Industry Institute of Toxicol-
      ogy, United States
        Owen Moss, Chemical Industry Institute of Toxicol-
      ogy, United States
        Hartwig  Muhle, Fraunhoter-Instit fur Toxikologie
      und Aerosoitor, Germany
        Paul Nettesheim, National Institute of Environmen-
      tal Health Sciences,  United States
        Friedrich Pott, Medical Institute, Germany
        Otto Raabe, University of California, Davis, California
        James Vincent, University of Minnesota, Minnesota
        David Warheit, DuPont  Haskell Research Labora-
      tory, United States
        C. P. Yu, State University of New York, New York

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