United States Office of Potiufcn fePA-74
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EPA-748-R-001
WORKSHOP REPORT ON
CHRONIC INHALATION TOXICITY
AND CARCINOGENICnY 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 Carcinogenicity and Biomarkers of
Toxicologic Effects 43
Mechanisms of Toxichy 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 EL Special thanks are extended to Dr. Gunter 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
lexicological 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 Me*hods
• The term "respirable fiber" should ay DC used with a species modifier, i.e.,
"human respirable" or "rat respirab.
• Rodent inhalation studies should use an aerosol which is enriched with rodent
respirable fibers and fibers with lengths of >20 um 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 hmg 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 bronchiolization, 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 Toricitv and Carcinogenicitv and Biomarkers of Toiicologic
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; (rv) species comparisons of fiber-
induced pulmonary effects in vivo and in vitro; (v) use of pleural 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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Intratracheal instillation was considered to be an acceptable alternative to
inhalation exposure for short-term screening studies. Intratracheal instillation
was not recommended for assessing the carcinogenic potential in long-term
studies.
The intracavhary 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 DDT). Appendix IV is
the "Background Information as Basis for Workshop Discussions," prepared by Dr.
Gunter 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 hs 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 toxichy and carcinogenichy 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 following 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) Mechgni$ms of Toxichy 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 IK).
The consultants were asked to keep in mind that needs for information from
toxicologjcal testing differ among countries. In the U.S., risks must be quantitatively
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assessed hi 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 0ength: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 fim. "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
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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-^im 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. jim;
• fibers with lengths of at least 20 urn or fibers with high aspect ratios.
The fraction of long fibers (>20 urn) should be specified; 10% to 20% would seem
appropriate, but not enough information is available on which to base a specified
percentage.
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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 maximise 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 h 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 um 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 view, 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 um and a geometric mean length of longer than 15 um 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 charged fibers is greater, both in the
environment and in the lung. It was agreed that charge of fibers in exposure aerosols
must be reduced, to reduce erratic effects, such 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
paniculate 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.
Interlaboratory 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, rt 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
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(and particle) size, using measurements of average longest length and average
narrowest dimension. It was agreed that regardless of how the exposure aerosol is
produced, h 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 inter-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 by 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. Gxinter 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
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mesothelioma 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 to. 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.
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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 Osborne-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 in 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
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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.
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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, h 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 in 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 King 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 h 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
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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.
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Steady-state King burden is a function of clearance rate, minute volume per
unit hmg mass, deposition rate, particle solubility, and air concentration. It was
suggested that the retention half-time be determined in a preliminary subchronic study
and used in 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 in significant toxitity 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 MTD 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 in
lavage fluid). Data on cell proliferation, histopatKology, and increased lung weight
should also be considered. Impaired clearance could be measured either as a
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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.
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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 officers?
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.
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Discussion: Sacrifice at 3 months is needed for comparison with the results of
i
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
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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 carcinogenicity data for asbestos. 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, amosfte 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 collectec a positive control would be superfluous
and an unnecessary expense.
Among those fevering 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 usec with a particular species or strain; if a
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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 tact
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
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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 negati /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 n error is
controlled at 0.2.
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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. Bronchoalveolar 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
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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 sinus 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
Pathologists.
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.
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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.
Bronchoalveolar 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)
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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.
Quantitation 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.
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The 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
Toxicologic Pathologists and other such organizations, which spell out the conditions
under which blinding 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 Disposition/Dosimetry 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
b=f_5*x———x Dep x AirConc.
In* Lung Mass
where f is the fraction of time exposed per week, TV£ relates to a clearance
term, hi denotes the natural logarithm, MV is the minute volume, and Dep is
the deposition fraction.
T/2 for the alveolar region most likely is not a single term for fibers; rather,
there is an alveolar *TH for fast clearance and an alveolar *TK for slower
clearance.
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• For rat-respirability, the fiber must have an aerodynamic diameter of less than 3
urn (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, but not answered: At what dissolution
rate does chemical toxicity, rather than "particle/fiber11 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.
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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.
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It was suggested that three types of studies actually are needed: a 3-month
study to establish toxichy, a 24-month to measure chronic effects, and a S-day
exposure with long-term follow-up, which would be important in looking at fiber
durability in vivo. The S-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 MTD 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.
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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 not 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 pleural data in
quantitative risk assessment and the cost of repeating studies, investigators should be
encouraged to collect pleural burden samples and keep them available for future
analysis.
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Discussion (Lung Burden Analysis): Lung burden analysis should be
required, despite the fact that it requires ext-a 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 tung 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 hing burden analysis.
With respect to mesotheliomas, a major issue is whether fibers reach the
pleura. Collection of pleura! burden samples Of 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.
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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, in
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 alter 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 imetry information could be obtained as part
of the chronic study to help in characterizing the human hazard and
exposure-dose-response assessment?
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 bioassay or
development of new molecular or biochemical techniques.
Discussion: For eventual validation of mathematical models, in vivo
determination of detailed or localized deposition patterns would be valuable. However,
such measurements are not needed for the purpose of chronic toxicity and
carcinogenicrty testing.
Question 2: With regard to the selection of the human dosimeter, is milligrams
per kilogram body weight adjusted for differences in metabolic rate appropriate
for fibers? For diesd 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: The consultants do not endorse use of
milligrams per kilogram body weight as a viable human dosimeter, even after
adjustment for species differences in metabolic rates. Various dose metrics can be
computed, but none of them affect the design of subchronic or chronic studies!
42
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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 Toiicitv 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, H 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 carcinogenichy 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, pleural 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 NIH
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 Toiicologic 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
in types of studies — as defined in the CUT 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 TH (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 HI 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 D 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 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 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 (eg., inflammation, cell proliferation, fibrosis) in 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 materials for longer-term studies. Short-term
screening studies should include (but not be limited to) analysis of BAL fluid for
markers of cell injury and inflammation histopathology, 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 toxichy). 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 In 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 offered to EPA at this time regarding the use
of specific tests. Problems with existing in ro cell response data include the lack of
adequate characterization of test fibers. Controlled in vitro cellular assays are
considered very important for delineating mechanisms 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 HI 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 toxtcfty 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. 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 Lp/ 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 bohis doses of fibers much over 1 mg
may introduce artifactual effects, such as clogging of terminal bronchioles, it was
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
i
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 mesothelhim 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
1-1
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WORKSHOP ON CHRONIC INHALATION OXICITY AND CARCINOGENICITY
TESTING OF RESPIRABLE i IBROUS PARTICLES
WORKSHOP AGENDA
Workshop Chair, Dr. Gunter 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
920.AM Discussion of Issues on: "Exposure" :
Chair, Dr. James Vincent
1030 AM Coffee Break
11.00 AM Discussion of Issues on: 'Exposure" (cont.)
12.00 PM Summary of Discussions
Rapporteur, Dr. David Bernstein •
1230 PM Lunch
130 PM Discussion of Issues on "Study Design"
Chair, Dr. Gunter Oberdorster
330 PM Coffee Break
4.00 PM Discussion of Issues on: "Study Design" (cont)
5.00 PM Summary of Discussions
Rapporteur, Dr. NeUJohnson
530 PM Adjourn
1-2
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Tuesday May 9,1995
830 AM Discussion of Issues on: "Histopathology"
Chair, Dr. Paul Nettesheim
1030 AM Summary of Discussions
Rapporteur, Dr. John Davis
11.00 AM Coffee Break
1130 AM Discussion of Issues on: 'Disposition/Dosimetry"
Chair, Dr. Otto Raabe
1230 PM Lunch
130 PM Discussion of Issues on: 'Disposition/Dosimetry'
(cont)
230 PM Summary of Discussions
Rapporteur, Dr. Fred Miller
3.00 PM Coffee Break
3.15 PM Discussion of Issues on: "Mechanisms/Biomarkers of Effects'"
Chair, Dr. Agnes Kane
5.15 PM Summary of Discussions
Rapporteur, Dr. David Warheit
5.40 PM Adjourn
1-3
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Wednesday
May 10,1995
830AM
10.00AM
1030AM
12.00PM
1230PM
1.00PM
Discussion of Issues on: "Screening Battery"
Chair, Dr. Kevin Driscott
Coffee Break
Discussion of Issues on: "Screening Battery" (cont)
Summary of DlSCUSSionS
Rapporteur, Dr. Ernest McConnett
Workshop Chair's Summary
Dr. Gunter Oberdorster
Adjourn
1-4
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APPENDIX H
List of Workshop Participants/Consultant Panel
H-l
•»
\
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WORKSHOP ON CHRONIC INHALATION TOXICITY AND CARCINOGENICITY
TESTING OF RESPiRABLE RBROUS 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. Carl Barrett*
National Institute of Environmental
Hearth Sciences
Telephone: (919) 541-3464
FAX: (919) 541-7784
Dr. David Dankovte
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 TOXICfTY AND CARCINOGENICITY
TESTING OF RESPIRABLE RBROUS PARTICLES
Consultants
Dr. GOnter Oberd6rster [Chair]
Department of Environmental Medidne
University of Rochester
575 EJmwood 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 Drlscoll
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
Bfomedteal 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
H-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
Dr. Paul Nettesheim
National Institute of Environmental Health
Sciences
111 T.W. Alexander Drive
Building 101, MD-D201
Research Triangle Park, NO 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, Bkton 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
H-4
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WORKSHOP ON CHRONIC INHALATION TOXICfTY AND CARCINOGENICITY
TESTING OF RESPIRABLE RBROUS 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
US. EPA-MD 77
Research Triangle Park, NC 27711
Telephone: (919) 541-2623
FAX: (919) 541-0239
Dr. Jean Blgnon
INSERM139 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-A(>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
Shetoyvflle, 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, QA
Telephone: (404) 944-4740
FAX: (404) 944-4745
Dr. Jeffrey Everftt
cirr
Research Triangle Park, NC
Telephone: (919) 558-1268
FAX: (919) 558-1300
II-5
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USTOF 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, FL 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 Melnlck
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. II60606
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) 455-5406
H-6
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LIST OF PARTICIPANTS
(Continued)
Dr. Nagui RlzkaJlah
University of North Carolina
212 Rnley 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, Mettlers 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 ffl
Issue Paper
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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
US. 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
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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.
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AUTHORS
This document was prepared by an Inter-Agency 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
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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
carcinogenicity 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 (NffiHS), 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:
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(1) issues dealing with the design and conduct of chronic inhalation studies of fibers;
(2) what preliminary studies would be usef 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
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 oncogenicity, 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 hi 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; Mcdellan et aL, 1992; ISRTP, 1994). A tiered-approach for evaluating the
toxicity and cartinogeniciry of new fibers or untested fibers has also been recommended
by workshop participants sponsored by the Chemical Industry Institute of Toxicology
(CUT) as a guideline for research purposes (McClellan et al., 1992).
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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
"Draft Protocol for Inhalation Oncogenicity 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?
3.12. 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 ifonfy 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 (Le., generally
long and thin fibers)" (Mcdellan et al., 1992). The ICRFs draft protocol even specifies
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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 tha 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 paniculate
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
ICRFs draft protocol specifies methods using SEM (scanning electron microscopy); on
the other hand, others have recommended either method (e.g. CUT 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 ICRFs draft protocol
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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 CUT 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 verify the integrity of the aerosolized fibers and that constant fiber
concentrations are maintained throughout the exposure period.
Certain study protocols (e.g. the ICRFs 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
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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 lexicological studies, then* 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 onfy 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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322. 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 participates.
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 7-10, 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 an 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 Regbnen 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
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protocols (e.g. ICRFs) 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)?
3.2.5. 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 CUT 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 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 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 stuffy.
Should a subchronic 90-day study be recommended prior to conducting the
chronic study? Or, should it be made optional?
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What are the primary goals of the subchronic study?
Are there any specific methods that should be recommended to measure the
effect if fibers on lung clearance (e.g. use of radiolabeled particles) ?
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. Dosimetiy 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, Le., 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 CUT
workshop which include: product cycle evaluation and physical and chemical
characterization (Tier I); in vitro solubility and durability, in vitro cell toxicity (Tier n);
short-term inhalation studies- ideally for 3 months (Tier HI); and chronic studies (Tier
IV). No recommendations were made with regard to the specific tests to be included in
tiern.
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 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 (Lp. 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 time 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)
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Table 1. Histologic grading of pulaonary changes related to eHe inhalarloa of fibre*
itenal
1 Mo lesion observed.
IU14
Cellular change
flbroaia
of the tenrifial bronefaiojcs
A f«v •aeropnagea la Che
aod «lv*oli
Frescncc of cuboi4al «*ieh*lioa llnian the proxijul alvoolt
(bronchioltzaclon). Ho collagoa b«c roeleulia fibnta ««y
be presvac IB intcradciui ac the Jwaceloa of rho uxviaal
bronchiole «nd «l«*olu«. Iiadoal mvcTophmt/em arc a»r«
«ooapictou» and •oooouclear calls wty bo found in the
4 Hinlaal collagen d*pa*itl0« at lerel of ecraritaal bronchiole
aod alveola*. Increased broaeMollsacloa with associated
wuculd debris suggesting glandular pattern.
> Intetlobular liaklag of lesion deaerlbed in grade * and
iccreaaed severity of fibre*!*.
ie apparoac.
Hiniawl
Hi Id
iecreaaed severity of
noderace « Early coaavUdatlee,. Vanncbywa doer
7 Harked fibrosi* aad coavalidaeion.
. 8 Complete obstruct Ion of ooec airway*.
Mon-fibrocie lesions.
McConneil* K£^ WagB«r. J.C. SUdmore. J.W.
d gLaM mieroflbre (JM
100). In: Biological
of Maa-made Mineral Fibre*. WorW Hodth Orpuuxatton, l»M.pp.
ffl A- 1
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Table 2. Claeeification ard criteria u»ed for (he diagnosis of
proliferative leeiove induced by exposure Co inhaled fibres
Broaenoalvoolar hyoerolaaia (BAfr)
1. ftatcncion of alveolar architecture
2. Air •»«€•• «r« lined by cuboidcl cells
3. Lack of cellular aad uuclcar atypla
4. Hay or aay ooc to oigalficaaC amount! of ftbresis
Adeneaa
1. Loaa of •ro-«iiaei-| arcblCcctun
2. Papillary tarnation into air tpaca*
3. Mueloar unifomicy
«. Hay or «ay not b« Mtaplaaia to other cell type*
3. Coeetreeaion but ao inwaeioo'of adjacent ttaauej
Adanocarcineoja
1. Eirpaaoion, coefroaaioo, aad iavaaion «f edjaceoc Citeues
2. Acypia (nuclear and/or cellular)
). Pleeeeirphifli , ' -
4. Hetaacaae*
S. Hetaplaoie e^^chor eoll tyfee ia often preaenc
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APPENDIX IV
Background Information as Basis for workshop Discussions
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DEVELOPING TEST GUIDELINES
FOR
RESPIRABLE FIBROUS PARTICLES:
Background Information as Basis for Workshop Discussions
Gunter Oberddrster, D.V.M., Ph.D.
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Disclaimer
This report was prepared by Dr. G&nter Oberddrster 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.
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TABLE OF CONTENTS
Page *
1. Introduction 1
1.1 Need for Testing Guidelines ;.. 1
1.2 Purpose of Background Paper 2
1.3. Overview of Concepts and Principles of ToxJcological Testing 2
1.4 Scientific controversies sinrotinding fiber testing and cancer classification... 5
1.4.1 Respirabilitv of fibrous and non-fibrous particles 5
1.4.2 The Maximum Tolerated Dose 7
1.4.3 Biopersistence vs. biodurability and chronic toxicitv 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 foj fiber testing from CUT Workshop 20
2.3 Test Protocols from International Cooperative Research Programme 22
2.3J Chronic inhalqjfton study for caicinogenicity testing 23
2.3.2 Intracavitarv Testing 24
2.3.3 Biopersistence Studies 26
2.3.4 In vitro studies 27
2.3.4.1 Acellular tests 27
2.3.4.2 Cellular tests 28
3. Evaluation of Available Teat Methods. 29
3.1 In vitro cellular asgayft 4 29
3.2. In vitro and in vivo durability studies 29
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Table of Contents Continued: Page #
3.3 Intracavjtajy Studies, • 30
3.4 Chroniq inhalation studies - mqjor issues for 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 Histopathology 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
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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 et al., 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 mere is an urgent need that specific testing
guidelines be developed. A 1991 workshop (McClellan ctal., 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
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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 die 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 die 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 men 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 Toxicolopcal 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
die 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 the 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! mesotheliomas (Glass et al., 1992; Mast et a/., 1992).
This raises the question whether in future chronic inhalation studies both species should be
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investigated, an issue which will be discussed at the workshop. Likewise, should a result from a
ch nic animal inhalation study indicating a non-linear tumor response be interpreted to also
indicate non-linearity in humans? And, should results derived from Lp. administration of fibers be
used for hazard identification and risk chanjctp"7?^"^
The experimental lexicologist is faced with these and many other questions related to
toxicological testing principles. A most important issue is dose selection in a chronic
carcinogenitity 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 era/on 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 histopathological changes to be evaluated
as wen as determining toxicokinetics and metabolic parameters. (IARC, 1980; Occupational Safety
and Health Administration, 1980; OECD, 1981; US EPA, 1982; NIP, 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, in 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 NC1/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 MID and subsequent lower doses at one-half and one-quarter of
the MTD (Baseman, 1985).
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Existing guidelines, however, are rather vague with respect to defining the MID, 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 era/., 1977;
1988) as well as mathematical deposition models (Yeh and Schum, 1980) also permit a very good
description about deposition efficiencies of inhaled spherical particles in this species. For example,
panicles larger than -5 |im are not respirable for rats, whereas they are well respirable for humans
which shows that 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 on>-
•IV-5
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nasal brtathers. 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 particulate 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 et al.,
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
Timbrell's work (1965) suggested that the maTimnm diameter of respirable asbestos fibers
is 3.5 urn; his more recent studies (Timbrell, 1982) showed asbestos fiber diameters of up to 4.1
HTD 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 mis figure can be used for converting data in Fig. 1 to geometric diameters.
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The differences in fiber rcspirability 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 urn thick and 28 \un long fiber (specific density = 2.7) has an aerodynamic
diameter of 3 um (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.
1.4J2 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 marimal aerosol exposure concentration in chronic particle inhalation
studies was discussed (Lewis ft a/., 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 tun. Similar to the respirability for fibers, human respirability
for non-fibrous spherical particles is also different from mat 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; if q/as «1y» mXVTprf^fd tM ft*f retained test material in th<» 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 TiO^ the term particle overload being reached at high exposure
concentrations may not be appropriate for fibers (Oberddrster ctol., 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
»
intrinsically higher cytotoxic 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 mat 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 etal. (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. biodurability and chronic toxicity:
•A generally accepted concept in fiber toxicology is that the biopersistence of
i
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 toxicological 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 a d. (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 ctal., 1992) and
non-human primates (Kendall, 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
toxicological 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 intratracheal
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-determined 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 et a/., 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 (Oberdorster Gal., 1992; Muhle et a/., 1994). Thus, recognizing the limitations
of the Lt 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.
1.4.43 Intracavitary injections.
The disadvantage of non-physiological administration associated with the
intratracheal instillation method is more obvious with routes of administration which circumvent
altogether the physiological route of entry into the respiratory tract via conducting airways, i.e.,
injections into the pleura! 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 era/., 1977; Pott, 1987). Proponents of this method view it as an excellent and easy to
perform bioassay to determine bom 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 mat 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 bom, 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 thflt 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, mat 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, mere is good evidence mat 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
era/., 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
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delivered dose has to be considered carefully. OveraL .ntracavitary 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 cartinogenicity, in vitro studies have been performed using different types of lung
cells, e.g., tracheal or bronchial epithelial cells (Mossman et a/., 1986), mesothelial cells
(Kuwahara era/., 1994), alveolarmacrophages (Donaldson era/., 1992). Such studies are very
valuable for uncovering basic mechanisms of fiber carcinogcnesis 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 ei a/., 1984; 1985; Marafante et a/., 1987; Krcyling etol.,
1990; Krcyling, 1997) and it appears that dissolution rates inside AM phagolysosomes arc 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 dmiiiataH 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 ol., 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
(biodurabiUty, 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 standard! ration 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 mat epidemiologk 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 mat 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
intraperitoneaU intratracheal or subcutaneous injection. The draft guidelines define the highest
dose to be used as one mat 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 mat 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 flnjrnpls 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 die 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
die 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 die draft guidelines on data other than tumor data
per se since they thought diose data to be critical which consider the mechanisms of carcinogenicity
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 mammalian species, and rats and mice 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 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 (SO 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 die 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 gnfrnat*
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 man 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 IS air changes per hour, insuring an adequate oxygen content of
19% and an evenly distributed exposure atmosphere. Crowding of the animals should be
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 the 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 timcpoint 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
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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 mat 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 Cl^r Workshop:
Participants at a 1991 workshop specifically discussed approaches to evaluating the
toxicity and carcinogenicity of man-made fibers (McClellan et a/., 1992). Although primary
conclusions from mis 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 tiercd-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 die 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
i
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 Jim for aerodynamic diameter, it appears that human respirability is
addressed here (see Section 1.4.1).
The use of the rat as die 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 rumors 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
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,
toxkity 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 in 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
i
fiber testing. The detailed draft protocols of this workshop are attached as Appendices C-E.
2.3.1 Chronic inhalation study for carrinogenicity 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 bv 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
are to be used with the hihest bein 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 k^fd. 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 1 Vmin. For evaluation
of fiber retention kinetics and additional toxicological parameters the use of bronchoalveolar livtfe
parameters is recommended. All animals are to be necropsied 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 \Jan long, £ 3 ^m 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 mat 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 die number should be 50 rats/dose and sex + 50 control
animals which are injected with saline.
It is also cmpbasiTcd 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 5Q
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 mp 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 Poet
(personal communication) die dust should be suspended in Nad solution widi ultrasonic treatment
and kept in suspension by a magnetic mixer while die injections are being performed. Injection is
carried out under CO2 anesdiesia on die left side into die lower part of die abdominal cavity is a
rule. The cannula should have a diameter of 2 mm. It may be necessary to use higher volumes
tiian 2 mL for suspending 50 mg sufficiendy. In addition to die tissues cited above, die uterus
should pl^Q be excised in female «nimqi$ for histological examination.
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2.3.3 Biopersistence Studies:
The determination of die 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 long. Biopersistence includes a number of mechanisms which all
contribute to the retention of a fiber in die 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
mg/m3 of rat respirable fibers should be used. If fibers are very soluble (as determined by in vitro
dissolution rates of greater man 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 die pleura after appropriate recovery of pleura! tissue (Bennudez, 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
electxommcroscopy 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^CXj. 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.
tHssolution 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 era/., 1994).
Toxicity of fibers to specific cells in vitro has also been performed using alveolar
macrophages or macrophage-derived cell lines or pleura! mesothelial cells. Endpoints to be
determined include release of LDH as a simple toxicity assay or dye exclusion assays which could
become grpiufonfliy^ 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 smnHpnKM* these more complex tests which are mainly
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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
amphibole 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 standardirr 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 halfrimr, 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 hfliftimg 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 virro 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
firy 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
N
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,
t
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 die rat?
t
It appears that results of intraperitoneal injections of fibers can be very useful for
establishing the relative toxicity (ranking) among different fiber types. Thus, mis 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 mat 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 Characterizati n of test sc iles - size, chemical comosition, surface.
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 GET Workshop (McQellan 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 v s. human resoirability;
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 CHT 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 AniiltnaJ selection:
The rat is die 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 body weight
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 mis 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 ^xpo/sure conditions and methods:
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 halftone 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 Histopathology:
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 CUT 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 etal., 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 minimpm of 3 animals for each 6-month
•
sacrifice timcpoint 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 AflalytJCalmcthpds 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 king 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 fim 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
i
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 JQ 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 P-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 die 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.«., 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
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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,
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 rcspirable 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, ip. 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 et al., 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
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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.g., 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
genera] 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
MID 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 MID. 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 al.t 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 MID 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 antioxidint
capacities.
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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, /.«., affecting lung
clearance mechanisms? Another possibility is to consider human exposure scenarios to be
expected and men 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 Lp. injection studies requires some further consideration. The
peritoneal cavity consists of a large surface area of-600 cm2 in the rat (Rubin etal., 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 man 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 paniculate 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 «npha«TftH 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
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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 ampiubole
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 era/. (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, (histopathology) 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
-------
It should also be considered whether a tiered testing approach can be used, starting with
acc.lhilar 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 CUT Workshop
CIable2).
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 a d. (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. Lp. injection vs. intratracheal instillation for oncogenicity testing
rat vs. human respirabiliry
F-344 rat vs. other rat strain, sex and animal numbers
nose-only exposure and stress vs. whole-body exposure
definition of NTTD or MFTD
— for inhalation
- for Lp. injection
dosimetry for ip. injection (surface area dose)
hamster as second species for evaluation of mesothelioma
histppamology scoring system - e.g., Wagner Scale vs. other
positive control group - appropriate amphibole sample
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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6. Literature Cited:
ACGIH (1994). American Conference of Governmental Industrial Hygienists: 1993-1994.
Threshold Limit Values for chemical substances and physical agents and biological exposure
indices. ACGIH, Cincinnati, OH.
Bermudez, E. (1994). Recovery of particles from the pleura! cavity using agarose casts: A novel
method for the determination of fiber dose to the rat pleura. Inhalation Toxicology 6: 115-
124.
Coin, P.G., Roggli, V.L., Brody, A.R. (1992). Deposition, clearance, and translocation of
chrysotile asbestos from peripheral and central regions of the rat lung. Environmental
Research 5*: 97-116.
Donaldson, K., Li, XY, Dogra, S., Miller, B.G. and Brown, G.M (1992) Asbestos-stimulated
tumour necrosis factor release from alveolar macrophages depends on fibre length and
opsonization. Pathology168: 243-248.
EPA (1982). Environmental Protection Agency Health Effects Test Guidelines. Washington,
D.C.: Office of Pesticides and Toxic Substances.
EPA (1986a). Hie Risk Assessment Guidelines of 1986. EPA/600/8-87/045, August, 1987.
EPA (1986b). Position paper on Maximum Tolerated Dose (MTD) in oncogenicity studies.
Chemical Regulation Reporter, May 9,1986.
EPA (1992). Code of Federal Regulations, Part 798.3300/3320. Health Effects Test Guidelines,
Subpart D • Chronic Exposure, pgs. 793-805.
EPA (1994). EPA Draft Revisions to Guidelines for Carcinogen Risk Assessment, Review draft,
7/94. The Bureau of National Affairs, Inc., Washington, DC
Glass, LJL, Mast, R.W., Hesterberg, TJL, Anderson, r., McConnell, E.E. and Bernstein, D.M.
(1992). Inhalation oncogenicity .study of refractory ceramic fiber (RCF) in rats - Final
results. The Toxicologist 12(1): 377.
Haseman, J.K. .(1985). Issues in carcinogenicity testing: Dose selection. Fundamental and
Applied Toxicology S: 66-78.
IARC(1980). International Agency for Research on Cancer. Long-term and short-term screening
assays for carcinogens: A critical appraisal. IARC Monograph Evaluating Carcinogenic Risk
of Chemicals to Man. SuppL 2, pp 21-83.
IV-45
-------
IARC (1993). IARC Monographs on the Evaluation of Carcinogenic Risks to Humans. \bl. 58.
Beryllium, cadmiutfl. ryypt^gv and exposures in the gla^s rflgpufacturing industry. 444 DCS,
IARC, France.
Infante, P.P., Schuman, LJX, Dement, J. and Huff, J. (1994). Commentary - Fibrous Glass
and Cancer. Am. J. Industrial Med. 26: 559-584.
ILSI (1984). International Life Sciences Institute. The selection of doses in chronic
toxicity/carcinogenicity studies. In Current Issues in Toxicology. (H.G Grice, Ed.), pp. 9-49.
Springer Verlag, New York.
Klingholz, R. and Steinkopf, B.(1982). Das verhalten von Kflnstlichen Mineralfascrn in einer
physiologischenModellflussigkeitundinWasser. Staub Reinhalt. Luft 42: 69-76.
Kreyling, W.G., Godleski, J.J., Kariya, ST., Rose, R.M., Brain, JJX (1990). In vitro
dissolution of uniform cobalt oxide particles by human and canine alveolar macrophages.
Am. J. Resplr. Cell Mol. Biol 2: 413-422.
Kreyling, W.G. (1992). Intracellular particle dissolution in alveolar macrophages.
Environmental Health Perspectives 9 7: 121-126.
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,
Hbronectin. Am. /. Respir. Cell Mol. Biol. 10: 167-176.
Law, BJX, 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, KJ*., Trochimowicz, HJ. Reinhardt, CJF. (1985). Pulmonary response of rats exposed to
titanium dioxide (TKXj) by inhalation for two years. Tox.andAppl.Pharma.T9: 179-192.
Leineweber, JJP.(1984). The solubility of fibres in vitro and in vivo. In; Biolopcal Effects of
Man-Marie Mineral Fibres. \bl 2,87-101. WHO/IARC Meeting, Copenhagen.
Lewis, T.R., Morrow, P.E., Mcdcllan, R.O., Raabe, O.B., Kennedy, G.R., Schartz B.A..
Goche, TJ., RoycrotX JJL, and Chabra, R.S. (1989). Establishing aerosol exposure
concentrations for inhalation tenacity studies. Tax. Appl. Pharmacol., 9 9: 377-383.
Landborg, M., Camner, P., l-inH, B.(1984 ). Ability of rabbit alveolar macrophages to dissolve
»
metals. Exper.LungRes.il 11-22.
IV-46
-------
Lundborg, M, FH^nd, A., Lind, B, Camncr, 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, TJi., 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.
McClellan, R.O., Miller, J.F., Hesterberg, T.W., Warheit, D.B., Bunn, W.B., Kane, A.G.,
Lippmann, M, Mast, R.W., McConnell, E£. and Reinhardt, C F. (1992). Approaches to
evaluating the toricity and carcinogcnicity of man-made fibers: Summary of a workshop held
November 11*13, 1991, Durham, NC. Regulatory Toxicology and Pharmacology 16: 321-
364.
Morgan, A. and Holmes, A. (1986). Solubility of asbestos and man-made mineral fibers in vitro
toad in vivo: Its significance in lung disease. Environmental Research 3 9: 475-484.
Morgan, A., Holmes, A., Gold, C. (1971). Studies of the solubility of constituents of chrysotile
asbestos in vivo using radioactive tracer techniques. Environ. Res. 4: 558-570.
Morrow, PJ2. (1988). Possible mechanisms to explain dust overloading of the lungs. Fundam.
Appl. Toxicol. 10, 369-384.
Mossman, B., Marsh, J., Shatos, M (1986). Alteration of superoxide dismutase activity in
trachea! epithelial cells by asbestos and inhibition of cytotoxicity by antioxidants. Lab. Invest.
54: 204-212.
Muhle, H., Bellmann, B., Creutzenberg, 0.. Fuhst, R., Koch, W., Mohr, U., Takenaka, S.,
Morrow, P.E., Kilpper, R., MacKenzie, J. and Mermelstein, R. (1990). Subchronic
inhalation study of toner in rats. InhalationToxicologyl: 341-360.
IV-47
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Muhle, H., Bellmann, B. and Pott, F. (1994). Comparative investigations of the biodurability of
mineral fibers in the rat lung. Environ. Health Perspect. 102 (Suppl 5): 163-168.
k
NRC(1983). National Research Council Commission on Life Sciences. Risk assessment in the
Federal Government: Managing the process^ Washington, DC, National Academy Press.
NRC Report "Science and Judgement in Risk Assessment? (1994).
NTP (1984). National Toxicology Program. Report of the NTP Ad Hoc Panel on Chemical
Carcinogenesis Testing and Evaluation. Research Triangle Park, NC: National Toxicology
Program.
Oberddrster, O., Boose, Ch., Pott, F., Pfeiffer, U. (1980). In vitro dissolution rates of trace
elements from mineral fibers in simulated lung fluid. In: The in vivo effects of mineral dusts.
Eds., Brown, Gormley, Chemberlin, Davis, Academic Press, pp. 183-189.
Oberdorster, G. (1994). Macrophage-associated responses to chrysotile. Annals Occupational
Hygiene 3&Q*o. 4): 601-615.
Oberddrster, G., Ferin, J., Morrow, PJL (1992). Volumetric loading of alveolar macrophages
(AM): A possible basis for diminished AM-mediated particle clearance. Exp. Lung Res. 18:
87-104.
OSHA (1980). Occupational Safety and Health Administration. Identification, classification and
regulation of potential occupational carcinogens. Fed. Regist. 4 5: (IS) 5001-5296.
OSTP (1985). Office of Science and Technology Policy. Chemical carcinogens: A review of the
science and its associated principles. Fed. Reg., II: 10371-10442.
OECD: (1981) Organization for Economic Co-operation and Development Guidelines for Testing
Chemicals. Paris, France.
Phalen, RJ7. (1984). Inhalation Studies: Foundations and Techniques. CRC Press, Inc., FL.
277 pgs.
Pott, F., Ziem, U., Reiffer, F-j., Huth, F., Ernst, H. and Mohr, U. (1987). Carcinogenicity
studies on fibres, metal compounds and some other dusts in rats. Exp.Pathol.32: 129-152.
Raabc, O.G., Yen, H.C, Newton, G J., Phalen, RJ., Velasquez, D J. (1977). Deposition of
inhaled monodisperse aerosols in small rodents. In Inhaled Particles, IV, Pan /,
(W.H.Walton and B. McGovern, Eds.) pp. 3-21. Pergamon Press.
IV-48
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Raabe, O. G., Al-Bayati, M.A., Teague, S.V. and Rasolt, A. (1988). Regional deposition of
inhaled monodisperse coarse and fine aerosol particles in small laboratory animals. Ann.
occup. Hyg. 32 (SuppL 1): 53-63 (Inhaled Particles VD.
Rendall, R.E.G. (1988). The retention and clearance of inhaled glass fibre and different varieties
of asbestos by the lung. Master of Science Thesis (dissertation), University of Witwatersrand,
Johannesburg, pg. 281-325.
Rubin, J., Clawson, M., Planch, A., Jones, Q. (1988). Measurements of peritoneal surface area
in man and rat Am. J. Med. Sci. 295(5): 453-458.
Sontag, J.M., Page, NJ*. and Saffiotti, U. (1976): Guidelines for Carcinogen Bioassay in Small
Rodents. DHHS Publication (NIH) 76-801, Washington, D.C.
Stanton, M. E, Layard, M., Tegeris, A., Miller, E., May, M, Kent, E. (1977). Carcinogenicity
of fibrous glass: Pleura! response in the rat in relation to fiber dimension. J. National Cancer
7/wr.58(3): 587-603.
TRGS (1994). Technische Regeln fiir Gefahrstoffe, 905, Bundesarbeitsblatt 6, p. 75, Ministry for
Labor, Germany.
Timbrell, V. (1965). The inhalation of fibrous dusts. Ann. N.Y. Acad. Sci. 132: 255-273.
Umbrell, V. (1982). Deposition and retention of fibres in the human lung. Ann. occup. Hyg. 2 6
(Nos. 1-4): 347-369 flnhaled Particles V).
Yen, H-C, and Schum, M. (1980). Theoretical evaluation of aerosol deposition in anatomical
models of mammalian long airways. Bull Math Biol 4 2:1-15.
Wagner, J. C, Berry, G., Skidmore, J.W., Timbrell, V. (1974). The effects of the inhalation of
asbestos in rats. Brit. J. Can. 2 9: 252-269.
, C.P., Zhang, L., OberdOrster, G., Mast, R.W., Glass, L.R. and Utell, MJ. (1994).
Clearance of refractory ceramic fibers (RCF) from the rat lung: Development of a model.
Environmental Research 65: 243-253.
IV-49
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TABLE 1
MAN MADE VITREOUS FIBERS (MMVF1
(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 (RCF)
- kaolin clay-based
- high purity
• others
Special Purpose Fibers
• glass microfibers
Composition
(6-15 fun diameter)
borosilicate, calcium-aluminum silicate glass
(2-9 tun diameter)
borosilicate, calcium-aluminum silicate glass
natural igneous rock with high Ca, Mg .
melted slag (iron, steel, copper) calcium-aluminum silicate
(1.2 - 3 urn diameter)
blends of alumina and silica, with other refractory oxides
(0.1 • 3 iun diameter)
borosilicate, calcium-aluminum silicate glass
General properties: Amorphous; do not break longitudinally, break transversely; melting > 1000°C; chemically non-reactive
IV-50
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Table 2: Ticred-approach to evaluating new fibers for characterizing toxicity and carcinogenicity.
(Mcdellancra/., 1992)
Product cycle evaluation
and
Physical and chemical characterization
In vitro solubility In vitro cell
and durability toxicity
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
-------
0.35
0.3
c 0.25
O
S
o
IQ.IS
o
Q.
O
Q 0.1
0.05
Human (mouth-breathing, at rest)
FIGURE 1
2345
Aerodynamic Diameter (Mm)
6
0.35
0.3
c 0.25
JO
2 0.2
O
0.15
O
Q.
0.1
0.05
Rat
12 3 4 5
Aerodynamic Diameter (A*/n)
IV -52
FIGURE 2
-------
FATE OF FIBERS AFTER DEPOSITION IN RESPIRA TORY TRACT
Physical/Mechanical Processes
(translocation; splitting; breaking)
Chemical Processes
(biodurability; dissolution; leaching)
(in vivo vs. in vitro)
BIOPERSISTENCE
Effects
IV-53
FIGURE- 4
-------
10
8
k.
0)
0
.£2 6
o
.o
05
T3
2
0)
0
^=20
0
2 3
Fiber Diameter
IV-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.449-461, July 1, 1994)
IVA-1
-------
Environmental Protection Agency
(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) Reference*. For additional back-
ground information on this test guide-
line the following references should be
consulted:
(1) Benitz. KJ. "Measurement of
Chronic Toxicity." Methods of Toxi-
cology- Ed. G.E. Paget. (Oxford:
Blackwell Scientific Publications. 1970)
pp. 82-131.
(2) D'Aguanno. W. "Drug Safety
Evaluation—Pre-Clinlcal Consider-
ations." Induttrial Pharmacology:
Neurolepttcs. Vol. I. Ed. 3. Fielding and
H. Lai. (Mt. Klaoo: Futura Publishing
CO. 1974) pp. 317-332.
(3) Fltzhugh, O.O. Third Printing:
1976. "Chronic Oral Toxicity." Ap-
praisal of the Safety of Chemicals tn
Foods, Drugs and Cosmetics. The Asso-
ciation of Food and Drug Officials of
the United States (1956. 3rd Printing
1975) pp. 36-45.
(4) Ooldenthal. EJ.. D'Aguanno. W.
"Evaluation of Drags." Appraisal of the
Safety of Chemicals in Foods, Drugs, and
Cosmetics. The Association of Food and
Drug Officials of the United States
(1956. 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. (Rockvllle: National
Center for Toxicologlcal Research,
1972).
(7) Page. N.P. "Chronic Toxicity and
Carcinogen!city Guidelines." Journal of
Environmental Pathology and Toxicology,
1:161-182 (1977).
§798.3300
(8) Schwartz. E. "Toxicology of
Neuroleptlc Agents," Industrial Phar-
macology: Neuroleptics Ed. S. Fielding
and H. Lai. (Mt. Klsco. 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. 1976).
(11) World Health Organization.
"Part L 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-Cllnlcal Testing of
Drug Safety." WHO Technical Report
Series No. 341. (Geneva: World Health
Organization. 1966).
[SO PR 38387. Sept. 27. 1985. •» amended at M
FR 21064. May 16.1969]
179&3300 Oneogeniclty.
(a) Purpose. The objective of a long-
term oncogenicity study Is to observe
test animals for a major portion of
their life span for the development of
neoplastic lesions during or after expo-
sure to various doses of a test sub-
stance by an appropriate route of ad-
ministration.
(b) Test procedures—(1) Animal selec-
tton—(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 aelection.
(11) Age. (A) Doslnc of rodents shall
begin as soon as possible After weaning.
ideally before the animal* are 6 weeks
old. but in no case more 'ban 8 weeks
old.
(B) At commencement of the study.
the weight variation of animals used
IVA-2
-------
§798.3300
40 CFR Ch. I (7-1-94 Edttton)
shall not exceed ±20 percent of the
mean weight for each sex.
(C) Studies using prenatal or
neonatal
-------
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 ant1 ensure that the
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 IS 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 irrtnimti* 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
development. The day of onset, loca-.
tton, 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 BP
-------
§798.3300
40 CFR Ch. I (7-1-94 Edition)
(11) During each exposure period the
actual concentrations of the test sub-
stance shall 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 examinatioiu. 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 animals in the highest doe-
age group and the controls. If these
data, or data from the pathological ex-
amination indicate -a need, then the 12-
and 18-month blood smears from other
dose levels shall also be examined. Dif-
ferential blood counts shall be per-
formed for the next lower gronp(8) if
there is a major discrepancy between
the highest group and the controls. If
clinical observations suggest a deterio-
ration in health of the animal* daring.
the study, a differential blood count of
the affected ajUmaJn shall be per-
formed.
(10) Gnu necropsy. (1) A complete
gross examination shall be performed
on all animals, including those which
died during the experiment or were
killed in moribund conditions.
(11) The following organs and tissues
or representative samples thereof, shall
be preserved in a suitable medium for
possible future hlstopathologlcal exam-
ination: All gross lesions and tumors of
all animals shall be preserved; brain-
Including sections of medulla/pons,
cerebellar cortex and cerebral cortex;
pituitary; thyroid/parathyroid; thy-
mus; lungs; trachea; heart; spinal cord
at three levels—cervical, midthoraclc
and lumbar; sternum and/or femur with
bone marrow; salivary glands; liver;
spleen; kidneys; adrenals; esophagus;
stomach; duodenum; jejunum; lleum;
cecum; colon; rectum; urinary bladder;
representative lymph nodes; pancreas;
gonads; uterus; accessory genital or-
gans (epldldymls, prostate, and. if
present, seminal vesicles); mammary
gland; skin; musculature; peripheral
nerve; and eyes. In inhalation studies,
the entire respiratory tract shall be
preserved, including nasal cavity, phar-
ynx. larynx and paranasal sinuses. In
dermal studies, skin from sites of skin
pa String shall be examined and pre-
served.
(Ill) Inflation of lungs and urinary
bladder with a fixative Is the optimal
method for preservation of. these tis-
sues. The proper inflation and fixation
of the lungs in Inhalation studies is re-
quired for appropriate and valid
histopathologlcal examination.
(Iv) If other clinical examinations are
carried out. the information obtained
from these- procedures shall be avail-
able before microscopic examination,
since they may provide significant
guidance to the pathologist.
(11) Hlstopathology • (i) The following
hlstopathology shall be performed:
(A) Full hlstopathology on organs
and tissues listed above of all %"1*pal«
in the control and high dose groups and
all ajnimain that died or were killed
during the study.
(B) All gross lesions in all
(C) Target organs in all
(II) If A «fyntfj<»»«fc difference is ob-
served In hyperplastlc. pre-neoplastlc
or neoplastlc lesions between the high-
est dose and control groups, micro-
scopic examination «>">>i be made on
that particular organ or tissue of all
study.
(ill) If excessive early deaths or other
problems occur in the high dose group.
compromising the significance of the
data, the next lower dose level shall be
examined for complete hlstopathology.
(Iv) In case the results of an experi-
ment give evidence of substantial al-
teration of the «mimai«' normal longev-
ity or the induction of effects that
might affect a neoplastlc response, the
next lower dose level shall be examined
rally as described in this section.
IVA-5
-------
Environmental Protection Ager cy
§798.3300
(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 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, 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 oncogenlc toxiclty study
shall be evaluated in conjunction with
the findings of preceding studies and
considered in terms of the toxic effects.
the necropsy and hlstopathologlcal
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
bloavallabllity 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 la lost due to autolysls. can-
nibalism, or management problems;
and survival In each group should be no
less than 60 percent at 18 months for
mice and hamsters and at 34 month*
for rats.
(3) Test report. (1) In addition to the
reporting requirements aa specified
under 40 CFR part 782. subpart J the
following specific Information shall be
reported:
(A) Group animal data. Tabulation of
toxic response data by specie*, strain.
sex and exposure level for
(1) Number of *n
-------
1798.3320
Teratogenldty. M*"*"**"1 of Health and
Welfare. (Canada: Department of
Health and Welfare, 1975).
(2) Food and Drug Administration
Advisory Committee on Protocols for
Safety Evaluation: Panel on Cardno-
genesls. "Report on Cancer Testing In
the Safety of Food Additives and Pes-
tiddes," Toxicology and Applied Phar-
macology. 20:419-438 (1971).
(3) International Union Against Can-
cer. "Carclnogenldty Testing." IUCC
Technical Report Series, Vol. 2., Ed. L
Berenblum. (Geneva: International
Union Against Cancer, 1969).
(4) Leong. B.K.J.. Laskln. S. "Num-
ber and Spedes of Experimental Ani-
mals for Inhalation Gardnogenidty
Studies" Paper presented at Con-
ference on Target Organ Toxidty. Sep-
tember 1976, Cincinnati, Ohio.
(6) National Academy of Sdences.
"Prlndples and Procedures for Evalu-
ating the Toxidty 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 Coundl. National
Academy of Sdences, Washington. DC
(1977).
(6) National Cancer Institute. Report
of the Subtask Group on Carcinogen Test-
ing to the tnteragency Collaborative
Group on Environmental Carctnogenesis.
(Bethesda: United States National Can-
cer Institute. 1976).
(7) National Center for Toxicologlcal
Research. "Appendix B." Report of
Chronic Studies Task Force Committee.
April 13-21 (Rockvllle: National Center
for Toxicologlcal Research. 1972).
(8) Page. N.P. "Chronic Toxidty and
Cardnogenldty Guidelines." Journal of
Environmental Pathology and Toxicology.
1:161-182 (1977).
(9) Page, NJ>. "Concepts of a Bio-
assay Program In Environmental Car-
dnogenesis," Advances in Modern Toxi-
cology Vol. J. Ed. Krayblll and
Mehlman. (Washington, DC: Hemi-
sphere Publishing Corporation, 1977)
pp. 87-171.
(10) Sontag. JJtf.. Page N.P..
Safflotti. U. Guidelines for Carcinogen
Bioassay in Small Rodents. NCI-CS-TR-
L (Bethesda: United States Cancer In-
stitute. Division of Cancer Control and
40 CFR Ot I (7-1-94 Edition)
Prevention. Carcinogenesis 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.
"Prlndples for the Testing and Evalua-
tion of Drugs for Cardnogenldty,"
WHO Technical Report Series No. 42S.
(Geneva: World Health Organization,
1969).
(13) World Health Organization.
"Part L Environmental Health Criteria
6," Principles and Methods for Evaluat-
ing the Toxidty of Chemicals. (Geneva:
World Health Organization, 1978).
[60 PR 30387. Sept. 27. 1986, a* amended at 03
PR 19076. May 30, 1987; M PR 21004. May IB.
1989]
I79&S320 Combined chronic tudcity/
oneofBiiietty.
(a) Purpose. The objective of a com-
bined chronic toxldty/oncogenidty
study Is to determine the effects of a
substance in a mammaJiM species fol-
lowing prolonged and repeated expo-
sure. The application of this guideline
should generate data which Identify
the majority of chronic and oncogenic
effects and determine dose-response re-
lationships. The design and conduct
should allow for the detection of neo-
plastic effects and a determination of
oncogenic potential as well as general
toxidty, Including neurological, phys-
iological, biochemical, and
hematologlcal effects and exposure-re-
lated morphological (pathology) ef-
fects.
(b) Test procedures (1) Animal selec-
tion—<1) Species and strain. Preliminary
studies providing data on acute.
snbchronlc. and metabolic responses
should have been carried out to permit
an appropriate choice of animals (spe-
des and strain). As discussed In other
guidelines, the mouse and rat have
been most widely used for assessment
of oncogenic potential, while the rat
and dog have been most often studied
for chronic toxidty. The rat Is the spe-
des of choice for combined chronic
toxidty and oncogenidty studies. The
provisions of this guideline are de-
signed primarily for use with the rat as
the test spedes. If other species are
used, the tester should provide jus-
IVA-7
-------
Environmental Protodon Agency
tiflcatlon/reasoning tor their selection.
The strain selected should be suscep-
tible to the oncogenic or toxic effect of
cue 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. HO percent of the
mean weight for each sex.
(C) Studies uidng prenatal or
neonatal f«<»«*i« 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 nnlllparous
and nonpregnant.
(iv) Number*. (A) At least 100 rodents
(SO females and 60 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 dose group(s) and the
satellite control group. The purpose of
the satellite group is to allow for the
evaluation of pathology other than ne-
oplasla.
(B) If interim sacrifices are planned.
the number of •«ni« employed in the test) is desir-
able for sssessing the significance of
changes observed in exposed •J>
-------
§798.3320
40 CFR Ch. I (7-1-94 Edition)
(71) 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 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 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:
(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. me-
dian aerodynamic diameter of particles
with standard deviation from the
mean).
(d) Reference*. For additional back-
ground information on this test guide-
line the following references should be
consulted:
(1) Benltc. K.F. "Measurement of
Chronic Toxiclty." Methods of Toxi-
cology. Bd. GJS. Paget. (Oxford:
Blackwell Scientific Publications, 1970)
pp. 82-131.
(2) D'Aguanno, W. "Drug Safety
Evaluation—Pre-Clinlcal Consider-
ations." "Industrial Pharmacology:
NeurolepOcs, Vol. I Ed. S. Fielding and
H. Lai. (ML Klsco. New York: Futura
Publishing Co.. 1974) pp. 317-332.
(3) Department of Health and Wel-
fare. The Testing of Chemicals for Car-
cinogenicity. Mutagentctty. Teratogeni-
ctty. Minister of Health and Welfare.
(Canada: Department of Health and Wel-
fare. 197S).
(4) Fitzhugh. O.Q. "Chronic Oral Tox-
iclty," 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.
(6) Food and Drug Administration
Advisory Committee on Protocols for
Safety Evaluation: Panel on Carcino-
genesls. "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 tn Poods. 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. "Carcinogenicity Testing," IUCC
Technical Report Series Vol. 2, Ed. L
Berenblum. (Geneva: International
Union Against Cancer. 1969).
(8) Leong. B.K.J., and Laskln. 8.
"Number and Species of Experimental
Anlmaln for Inhalation Carcinogenicity
Studies." Paper presented at Con-
ference on Target Organ Toxiclty. Sep-
tember, 1975. Cincinnati. Ohio.
(9) 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).
(10) National Cancer Institute. Report
of the Subtask Group on Carcinogen Test-
ing to the hiteragency Collaborative
Group on Environmental Cardnogenesis.
(Bethesda: United States National Can-
cer Institute. 1976).
(11) National Center for Toxi-
cologlcal. Report of Chronic Studies Task
Force Research Committee. "Appendix B.
(RockvUle: National Center for Toxi-
cological Research, 1872)).
(12) Page. N.P. "Chronic Toxiclty and
Carcinogenicity 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
-------
Envtronm«ntd Protection Ag»*ncy
individual caging. As a general rule, t
ensure stability of a chamber atmot
phere. the total "volume" of the tet
Animals should not exceed 5 percent <.
the volume of the test chamber. Altei
natively, oro-nasal. head only, or whole
body Individual chamber exposure ma-
be used.
(B) The temperature at which the
test Is performed should be maintained
»t 22 'C (12*). Ideally, the relative hu-
midity should be maintained between
40 to 9 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 coBtrol system should be used.
The rate of air flow 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) Ofcterartton of animals, (i) Each
animal should be handled and its phys-
ical condition appraised at least once
each day.
(11) Additional observations should be
made dally with appropriate actions
taken to mlnlmlm loss Of ^Jn
-------
§798.3320
40 CFR Ch. I (7-1-94 Edition)
should be performed. A differential
blood count should be performed on
samples from anlrr"^* 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
hematologlcal effects were notad In the
subchronlc test, hematologlcal 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 glutamio-pyrn-
vlc transamlaase (now known as serum
alanine azninotransferase), serum glu-
tamlc oxaloacetic transamlnase (now
known as serum aspartate aminotrans-
ferase). ornithine deearboxylase.
gamma glntamyl transpeptidase. blood
urea nitrogen, albumen, oreatinine
phospholdnase. total cholesterol, total
blllrubin and total serum protein meas-
urements. Other determinations which
may be necessary for an adequate toxi-
oologlcal evaluation include analyses
of llplds. hormones, acid/base balance.
methemoglobin and chollnesterase 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
hematologlcal examination above.
should be collected for analysis. The
following determinations should be
made from either individual animals or
on a pooled sample/sex/group for ro-
dents: appearance (volume and specific
gravity), protein, glucose, ketones, bil-
Irnbln. occult blood (semi-quan-
titatlvely) and microscopy of sediment
(semi-quantitatively).
(B) Ophthalmologies! 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 animaia should be exam-
ined.
(10) Grots necropsy. (1) A complete
gross examination should be performed
on all animals, 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-
Ical examination: All gross lesions and
tumors; brain-Including sections of me-
dulla/pons, cerebellar cortex, and cere-
bral cortex; pituitary; thyroid/parathy-
roid; thymns; lungs; trachea; heart;
sternum and/or femur with bone mar-
row; salivary glands: liven spleen; kid-
neys; adrenals; esophagus; stomach;
duodenum; jejunum; Ueum; cecum;
colon; rectum; urinary bladder rep-
resentative lymph nodes; pancreas; go-
nads; uterus; accessory genital organs
(epldldymis, prostate, and. If present,
seminal vesicles); female mammary
gland; aorta; gall bladder (if present);
skin; musculature; peripheral nerve;
spinal cord at three levels— cervical.
midthoradc. 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.
(lv) Inflation of lungs and urinary
bladder with a fixative Is the optimal
method for preservation of these tis-
IVA-11
-------
Environmental Protection Agency
goes. The proper inflation at fixation
of the lungs in inhalation undies is
considered essential for appropriate
histopathologlcal nxamlna-
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
biatopathology should be performed:
(A) Full histopathology on the or
g*ns and tissues, listed above, of all
non-rodents, of all rodents In the con- •
trol and high dose groups and of all 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
>n
-------
§798.3320
40 CFR Ch. I (7-1-94 Edttion)
order to elucidate a chronic toxl-
eologlcal proflle of the teat substance.
If more than one dose level la selected
for satellite dose groups, the doses
should be spaced to produce a grada-
tion of tortc 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 »n««n*i« in the satellite
groups which should be dosed for 12
months.
(6) Observation period. It Is necessary
that the duration of the oncogenidty
test comprise the majority of the nor-
mal life span of the Mi<*na.i« to be used.
It has been suggested that the duration
of the study should be for the entire
lifetime of all *™«*M.I« However, a few
*rHmti« may greatly exceed the aver-
age lifetime and the duration of the
study may be unnecessarily extended
and complicate the conduct and eval-
uation of the study. Bather, a finite pe-
riod covering the majority of the ex-
pected life span of the strain Is pre-
ferred since the probability is high
that, for the great majority of chemi-
cals. Induced tumors will occur within
such an observation period. The follow-
ing guidelines are recommended:
(1) Generally, the termination of the
•tody should be at 18 months for mice
and hamsters and 24 months for rats;
however, for certain strains of ««
-------
gnvlronnwntar Protection Agwtcy
(14) Schwartz, E. 1374. "Toxicology of
Keurolcptlc Agents." Industrial Phar-
macology: Neurolepttcs. Ed. S. Fielding
and H. Lai. (Mt. Kisco. New York:
putura Pabllahing Co. 1974) pp. 203-221.
(15) SonUg. J.NL. Page. N.P.. and
Safflotti, U. Guidelines for Carcinogen
Bioassay in Small Rodents. NCI-CS-TR-
1 (Bethesda: United SUtes Cancer In-
stitute, Division of Cancer Control and
prevention. Carclnogenesls 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 Tasting and Evalua-
tion of Drugs for Carcinogenlcity."
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. 563. (Geneva: World Health Or-
ganization, 1975).
(19) World Health Organization.
"Part I. Environmental Health Criteria
6." Principles and Methods for Evahiat-
mg the Tozidty of Chemicals. (Geneva:
World Health Organization. 1978).
(20) World Health Organization.
"Principles for Pre-CUnlcal Testing of
Drug Safety." WHO Technical Report
Series No. 341. (Geneva: World Health
Organization, 1966).
(SO FR 3WB7. Sept. 77, UK. u amended at M
FR 21004. May 16.IBM]
Subport E—Specific Organ/Tissue
ToxJciy
I7B&4100 Dsnuls«a
(a) Purpose, In the assessment and
•valuation of the toxic characteristics
of a substance, determination of its po-
tential to provoke skin sensitization
reactions Is Important. Information de-
rived from tests for skin sensitization
•erves to identify the possible hazard
to a population repeatedly exposed to a
test substance. While the desirability
of skin sensitization testing Is recog-
nized, there Are some real differences
of opinion about the best method to
use. The test selected should be a reli-
able screening procedure which should
S 7984100
not fail to identify substances with sig-
nificant allergenlc potential, while at
the same time avoiding false negative.
results.
(b) Definitions. (1) Skin sensitization
(allergic contact dermatitis) is an
Immunologlcally mediated cutaneous
reaction to a substance. In the human.
the responses may be characterized by
prurltls. 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.
(o) Principle of the test method. Follow-
ing initial exposnre
-------
APPENDIX B
Summary of CTTT Workshop Conclusions (for full workshop report, see McClellan etal., 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 die 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 that maTitniTpe 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 die 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 Pan 792 etc., or their successors as appropriate.
Ib 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 tree (SPF),virus antigen-free
(VAF+) and maintained under optimum hygienic condition (OHQ, 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 |im and a geometric
mean length of longer than IS nm.
IVC- 1
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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).
Animals* age at
delivery:
8-12 weeks
Interim sacrifice rime points and number of animals:
WEEK Number of AlUITIfllS
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
mat 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
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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 electron microscopy (SEM) (pm) -
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
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 \un, d £ 3 Jim) (WHO, 1985) or a total of 1000 fibers and non-
fibrous panicles 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 inn is to
continue until 20 fibers with d < 3 pm and 1 £ 20 Jim are recorded or a filter
surface area of 5 mm2 has been examined whichever comes first. (Fibers <
20 urn 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
signs
Body weights
Disease screening (viral, bacterial & parasites) of sentinel animals
IVC-3
-------
Necropsy: All animals will be necropsied. All tissues (as specified for oncogenicity
studies by die OEGD guideline- will be examined and preserved.
In addition: Lungs are to be w ned 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 Kamovsk:'s fixative.
- 35 mm slides of die 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, lactatc
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 et a/., 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 i*™*****^ «h^u h» m/piimtM by an appropriate
statistical method. Any generally accepted statistical method may be used; die mrisrical
methods shall be selected during die design of die 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, die 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 animals 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 animals 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 tffb'^ff^ 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) Fiber data:
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 separately.
IV C-5
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ADDENDUM
TO ONCOGENICI Y PROTOCOL
1. Histopathological evaluation: One of the hi tological endpoints of the oncogenicity study is
quantification of the cellular changes and t.orosis 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 cellular/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 brompdeoxyuridine (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 paniculate 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: To 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 50 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 aU 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 the 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 men 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
-------
Animal
maintenance:
Duration of
studies:
Necropsy:
Histology:
Animals should be weighed throu^ t the study. Once each week for the first 13
weeks followed by once each month IOT 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) Livo/splccn/pancreas
(Q Mesentery plus gut segments
(D) Omcntum plus gut segments
IVD-2
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APPENDIX E
Draft Protocol for Inhalation Biopersistence Study with Fibers
Prepared by die 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, Fischer-344, supplied specific pathogen tree (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 an-
Prior to the evaluation of fibers by inhalation for biopersistence, it may be useful depending opoa the
cferistics of the fiber under tfst. to expose a group of aninmiq by intratncheal instillation and <**a»ntt^ the broncho-
alveolar lavage fluid in order to evaluate die cytotoxic response. In such an evaluation, a positive and oepuw control
IVE-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 Jim, 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'^h'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 lavage*. 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/sacrifioe 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 thelavaged 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
JBronchial alveolar lavage: In order to 1*9*™**? the recovery of cellular contents, the lungs should be excised and
an exhaustive lavage conning of 10 lavages with 5 ml saline each should be performed under slight massage of the
extisedkng.
IVE-2
-------
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) W;
Number of Animals ••<
•»•«••••••••»»»«»
Sacrifice® If in vitro If in vitro If in vitro
dissolution dissolution dissolution
rate rate rate
>50-£200 >200
_ 2 , .1
ng* cm"** h"1 ng* an'** n"
1 hour(4)
Iday
2 or 3 days
14 days
4 weeks
3 months
6 months
12 months
(3)
(3)
7
(3)
7
(3)
7
7
(3)
7
7
(3)
7
7
7
7
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°Q 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
mat 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:
Exposure system
monitoring daily:
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.
• Airflow rate
• Oxygen concentration
• Temperature & humidity
Exposure atmosphere
Counting and Sizing
Rules:
• 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.
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
• 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 WHO fibers
(1 £ 5 Jim, d £ 3 urn) (WHO, 1985) or a total of 1000 fibers and non-
fibrous particles are recorded, or 1 mm2 of die 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 urn have
not been recorded by 2 above, then the evaluation of those fibers ^20 iim is
to continue (at lower magnification, lOOOx) until 20 fibers with d £ 3 \ua
and 1 £ 20 \un are recorded or a filter surface area of 5 mm2 has been
examined whichever comes first (Fibers < 20 |im are not recorded in this
IVE-4
-------
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, twice a day
• Clinical signs, weekly
• Body weights, weekly first 13 weeks and every 2 weeks thereafter.
Necropsy:
Lung Dust Content:
Statistics:
Suggestions for
Reporting:
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 mat 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 density1 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 calcdatioa may provide erroneous results if the Tiber
is forming a leached layer which bar a different density than the fiber core.
IVE-5
-------
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.
IVE-6
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APPENDIX F
Intratrachcal 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 die use of
radioactively labeled fibers and the flvailflhility pf a limited arnmint of sized fihery.
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
Halothane, die instillation performed via die mouth and the instillation should be performed when
die 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 die fiber concentration should not exceed 1 mg/ml and
preferably should be less. In addition, to determine if die instilled amount has caused any adverse
reactions in me lung, additional animals should be similarly exposed and then lavaged 24 he later
and an analysis of die BAL performed.
The total lung burden used should be in die 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 Hiany»jfr fibers
in die 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 tiiese applications result in
artifacts due to die sudden introduction of fibers witii diameters not normally encountered by die rat
or die acute application of an enormous number of fibers. Human respirable fibers are considered
to be rnose widi geometric mean diameters of-3 \un and less. The lung burden corresponding to
dial found following chronic inhalation exposure in rats is 1-2 mg.
Whenever intratracheal instillation is used, me results should be validated by comparison
using some of die fibers with tiiose 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
i
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 nricropore filters (0.2 - 0.4 pm) to prevent particles from leaving the cells. The tree
volume which can be occupied by the fibers may vary between 2 cm3 and 10 cm3, approximately.
The tree 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.
Pei fun iiano*. of the U*T (Continuous Flow Tesrt
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 10s. After the connection of the tubes Ac test will be started.
IVG-1
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At the 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 cxpe .ncntal 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 |im/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 man 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.
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.
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