V-/EPA
                                   United States
                                   Environmental Protection
                                   Agency
                                   Health Effects Research
                                   Laborarory
                                   Research Triangle Park NC 27711
                                   Research and Development
                                   EPA-600/S1-81-021  May 1981
Project  Summary
                                   Tracheal  Organ  Culture  as  Air
                                   Pollution  Damage  Indicator
                                   Leonard J. Schiff
                                    This report presents the results of a
                                   study conducted to determine the
                                   effects of various energy-related ef-
                                   fluents on respiratory tract epithelial
                                   tissue. Measurement of mucociliary
                                   activity and characterization of the
                                   morphological alterations induced by
                                   such effluents was carried out  in
                                   hamster trachea) organ culture. Dif-
                                   ferent combinations of in vivo and in
                                   vitro exposure and/or maintenance
                                   were used to determine the relation-
                                   ship between in vivo exposure danger
                                   and adverse effects observed in organ
                                   tissue.
                                    The pollutants assayed included
                                   particulate effluents from mobile and
                                   stationary sources of both conven-
                                   tional and advanced energy processes.
                                   Included were fly ash (from coal-fired
                                   and oil-fired sources), cigarette smoke
                                   condensate, and diesel fuel exhaust
                                   extract, with benzo(a)pyrene serving
                                   as a positive control.
                                    Both acute (72-hr) and  long-term
                                   (14-day) studies permitted assessment
                                   of tissue specific effects. In addition to
                                   acute and long-term toxicity studies,
                                   testing was conducted to determine
                                   the effects of selected particulate
                                   effluents on the pathogenesis of viral
                                   infections.
                                    This Project  Summary was devel-
                                   oped by EPA's Health Effects Research
                                   Laboratory, Research Triangle Park,
                                   NC, to, announce key findings of the
                                   research project that is fully docu-
                                   mented in a separate report of the
                                   same title (see Project Report ordering
                                   information at back).
                                   Introduction
                                     The Health Effects Research Labora-
                                   tory of the U.S. Environmental Protection
                                   Agency is presently involved in research
                                   to evaluate the health effects of human
                                   exposure to airborne environmental
                                   pollutants. Past research has established
                                   a link between exposure to air pollution
                                   and an increased frequency and severity
                                   of acute respiratory disease. Under
                                   certain exposure conditions, environ-
                                   mental pollutants may impair the func-
                                   tion of important defense mechanisms
                                   of the respiratory tract epithelial tissue.
                                   Pathologic consequences could include
                                   shifts in normal cell populations, struc-
                                   tural abnormalities of cells and cell
                                   organelles, and changes in normal cell
                                   repair processes. These adverse effects
                                   could  render the tracheobronchial
                                   epithelium more susceptible to respira-
                                   tory infections, as well as more vulner-
                                   able to non-infectious pollutant agents.
                                     Many of the most dangerous particu-
                                   late emissions commonly found in the
                                   ambient air are produced by conven-
                                   tional and advanced energy processes,
                                   such as the combustion of coal and oil.
                                   Thus, examination of the health effects
                                   resulting from fugitive emissions from
                                   these  processes is becoming increas-
                                   ingly important.

                                   Summary
                                     In support of EPA's research program
                                   in this area, IIT Research Institute con-
                                   ducted a study to obtain data on hazard-
                                   ous energy-related pollutants suspected
                                   to interfere with normal respiratory
                                   function. Although examination of the

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health effects of potential environmental
pollutants has traditionally been based
on chronic rodent bioassays, these bio-
assays are complex and require control
of many variables over  several years'
time. Thus, NT proposed to utilize in vitro
methods  in hamster trachea! organ
culture to provide a more rapid, sensitive,
and relevant test system for screening a
variety of hazardous pollutants. In order
to test  the  suitability of the in vitro
method as a rapid  screening model,
both  in vivo and in  vitro  studies of
pollutant exposure effects on tracheal
mucociliary transport parameters and
cytopathology were conducted  and the
results compared.
  The primary pollutant used  in the
study was coal-fired fly ash, obtained
from  a coal-fueled power plant using
Eastern Kentucky coal. Additional par-
ticulate pollutants tested in tracheal
organ culture were  oil-fired  fly  ash,
diesel fuel exhaust extract, and cigarette
smoke condensate. These air pollutants
contain a variety of chemical carcino-
gens, the most commonly recognized of
which is the well known carcinogen
benzo(a)pyrene. In order to establish the
relative potency  of  the various  test
substances, the effect of benzo(a)pyrene
on tracheal organ culture was compared
to the effects observed for the other
pollutants. Thus, benzo(a)pyrene served
as a positive control for the study.
  Cytotoxicity  resulting from exposure
to test compounds was expressed as
alterations in ciliary  activity and cell
morphology. Both the short-term and
long-term toxic effects of the pollutants
on these factors were  investigated. The
primary indicator of pollution damage
used was cilia beating frequency.
Morphological changes  were  detected
using light microscopy  and scanning
electron microscopy.
  Suckling  and adult golden Syrian
hamsters were employed in the in vivo
studies. In  addition, whole tracheal
explants from donor  Fischer 344 rats
and Alexander B10 FiD hamsters were
inserted with fly ash pellets and trans-
planted through subcutaneous grafting
to recipient rats and  hamsters. For in
vitro studies, tracheal  ring explants and
whole tracheal explants  were prepared
and maintained in culture. Tracheal
organ culture  techniques were devel-
oped to maintain differentiated respira-
tory tract  epithelial tissue.
  Cilia beating frequency was measured
for each tracheal explant at four separate
areas on the lumen side, and the average
recorded as beats per minute. The cilia
beating frequency of whole  tracheas
was observed by focusing on the mucosa
through  the explant. After  an  initial
incubation period, the baseline cilia
beating frequency for each explant was
determined.
  In vivo inhalation exposure to respi-
rable-size coal-fired fly ash was accom-
plished using  suckling and  adult
hamsters. The hamsters were exposed
to fly ash in a 432-liter capacity plastic
chamber at a mass concentration of 2
mg/m3 for 3 h/day, 5 days/week for 2
weeks. The chamber was maintained at
25-29°C and 30-55% RH. Mass con-
centration of the particle aerosol in the
exposure chamber was monitored using
an aerosol, smoke, and dust photometer.
In addition, periodic  determination of
the aerosol concentration  was also
made  using  a condensation nuclei
counter.  The desired mass concentra-
tion for the trials was 2000 fjg/m3 of the
fly ash particles; the  actual concentra-
tion was 1987 /jg/m3.
  In vivo exposure of hamsters to coal-
fired fly ash resulted in a reduction of
cilia beating frequency and cytological
alterations  in the tracheal mucociliary
epithelium compared to ambient air
controls. The tracheal epithelium of
animals experiencing from five to ten 3-
h  exposures  showed large   areas of
based cell hyperplasia and stratification.
The recovery time from damage to the
ciliated epithelium was directly propor-
tional to the number of 3-h exposures.
  Whole tracheas from adult B10 FiD
Alexander hamsters were grafted sub-
cutaneously (heterotopic  tracheal
transplants). Epithelial changes after 2
months in untreated tracheas and
tracheas with beeswax (cholesterol
[1.9]  pellets only) were  essentially
normal, resembling that of the  host's
own trachea. Pellets containing coal-
fired fly ash induced mild focal  hyper-
plasia and occasional areas of squamous
metaplasia. In addition, presarcomatous
lesions of connective and cartilagenous
tissue was observed.
  For in vitro inhalation exposure
studies, fly  ash (coal-fired and oil-fired)
was suspended in medium at 5, 10, 50,
100, and 500 x/g/ml. Tracheal ring ex-
plants were exposed to the fly ash for 1
or 3 h/day, 5 days/week for 2 weeks.
Culture dishes were  placed  in a con-
trolled atmosphere chamber and main-
tained in 5% COz and balanced air. The
chamber was then placed on a platform
which rocked at 10cycles/min, causing
the medium containing the fly ash agents
to flow continuously over the epithelial
surface.
  At various intervals, alterations in
ciliary activity and cytology were docu-
mented by light microscopy and scanning
electron microscopy.  Tracheal cultures
exposed for 1 or 3  h/day showed a
decrease in ciliary activity that paralleled
cytopathological changes following
seven exposures to 50 fjg oil-fired fly
ash/ml or higher. Morphological altera-
tions consisted of diffuse basal cell
hyperplasia  and stratified metaplastic
epithelium.  Whole suckling hamster
tracheas in organ culture exposed for 1
or 3 h/day to 10 and 50//g coal-fired fly
ash/ml produced cornifying epidermoid
metaplasia after  seven exposures. The
most characteristic finding of surface
cells was a number of broad metaplastic
areas with keratin for formation.
  Oil-fired fly ash produced a .more
pronounced  effect than coal-fired fly
ash. Complete cessation of ciliary activity,
occurred after seven exposures to 50 fjg
oil-fired fly ash/ml. The epithelium of
cultures exposed to 10/ug/ml or greater
of oil-fired fly ash showed pathological
alterations proportional to the concen-
tration of fly ash and  the period of time
exposed. Histological sections of treated
explants showed distinct cytopathologic
changes which preceded ciliostasis.
  In vitro exposures of hamster tracheal
epithelium to cigarette smoke condensate
(CSC) and diesel fuel exhaust extract
(DFEE) were performed to elucidate
functional and morphological changes.
Early  expression of the  cytotoxicity of
both materials was indicated by altered
ciliary activity and cytopathology. Con-
trols exposed to  DMSO (DFEE solvent)
and  acetone (CSC solvent)  showed
changes in the mucosa at a concentra-
tion of 0.25% and 0.2% nine days after
initial exposure. At 50 and 100 /jg ciga-
rette smoke condensate or  diesel fuel
exhaust extract, histopathological alter-
ations included  stratified metaplastic
epithelium (33%) and epidermoid
metaplasia (20-25%). At these concen-
trations, ciliary activity and cytopathology
were not altered. Ring explants exposed
to 1  and 3 fjg benzo(a)pyrene/ml, the
prototype  carcinogenic polycyclic
aromatic hydrocarbon  and a positive
control for the organ culture bioassay,
showed epithelial changes progressing
to stratified metaplasia. These changes
were reflected as an alteration in
cytopathology and a decrease in beating
frequency.
                                                                                                                     I

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  In separate experiments, in vivo and
n vitro tests were used to determine the
nteractions of fly ash and  influenza
rirus (e.g., what effect the virus had on
he expression of fly ash damage).
  In vivo experiments to study the inter-
iction of fly ash and influenza A/PR-8
'irus were conducted using adult male
 olden Syrian hamsters.  In the first
sxperiment, influenza virus replication
vas measured in tracheal epithelium
ifter aerosol infection. Virus aerosol
nfection of hamsters was immediately
ollowed by exposure to fly ash  in the
iecond experiment. The third experi-
nent  was conducted similarly to the
iecond,  except that fly ash  exposure
vas delayed until one  day after virus
nfection.  In  the last experiment,
lamsters were exposed to fly ash prior
o infection by the virus.
  Results from the four in vivo experi-
nents indicated that hamsters exposed
o a pollutant during an on-going infec-
ion (i.e., infected by aerosolized in-
luenza virus and 24 h later exposed to 1
ng fly ash/m3 for 3 h/day, 5 days/week
or 2 weeks) produced greater cytotoxic
sffects than hamsters exposed to fly ash
  or 2 weeks  prior to virus  infection.
"oxicity was  mainly expressed as
lytopathological and  histopathological
 'terations along the luminal  surface.
  Tracheal cultures from hamsters in-
ected with  influenza virus aerosol and
hen exposed in vitro  to  10 or  50 ug
:oal-fired fly ash/ml showed morpho-
ogical alterations  that were similar to
hose  observed in  the in  vivo experi-
nents. In comparison loin vitro exposure
o fly ash alone, histological changes in
he virus-fly ash treatment group were
ess severe. Initial changes were char-
icterized by focal loss of cilia and
 toughing of superficial epithelium.
Moderate epithelial vacuolation was
 bserved. Only occasional  areas of
tratified metaplastic epithelium ap-
 eared,  indicating the virus retards
netaplastic changes. These changes
/ere the same whether exposed im-
lediately after infection or 24 h post-
nfection. Maximum infectious virus
iters in fly ash treatment groups were
eached earlier than in the virus infected
ontrol explants.

 onclusions
 The  results of this study indicate that
ilia beating frequency and cytopathology
 lone  are  not  adequate as  pollution
amage  indicators. To detect the full
xtent of cytological damage, these
indicators should be used in conjunction
with light microscopy and scanning
electron microscopy examinations.
  Toxicity testing results showed that
hamster tracheal organ cultures can be
used to study acute effects of fly ash on
respiratory ciliated epithelium, and that
the toxic effects produced by the two fly
ash  species are the result of different
mechanisms of inducing injury. The
toxicity of oil-fired fly ash was found
directly proportional to the concentration
and duration of exposure. Morphological
observations indicated that cytotoxic
concentrations of oil-fired  fly ash were
below the level that produced ciliostasis.
In addition, the  transformation of
tracheobronchial epithelium to epider-
moid metaplasia by coal-fired fly ash
was shown to be similar in response to
the effects of carcinogens, vitamin A
deficiency, and nutritional influences//?
vitro. Results also indicated a correlation
between  fly  ash-induced  changes in
tracheobronchial epithelium of hamsters
exposed in vivo (2 mg/m3) and effects
observed in hamster tracheal epithelium
exposed in organ culture (5-10 A/g/ml).
  The organ culture assay permitted
toxicity ranking of the pollutants tested.
In order of decreasing toxicity, they
were: oil-fired fly ash, cigarette smoke
condensate, coal-fired fly ash, and
diesel fuel exhaust extract.
  Studies of the effect of  influenza
A/PR-8 virus on the expression of  fly
ash damage demonstrated that tracheal
explants infected 24 h prior to coal-fired
fly ash exposure produce a significantly
greater response than explants exposed
immediately after infection. These find-
ings may have bearing on the hypothesis
that influenza may stimulate the trans-
formation  of tracheobronchial epithelium
to epidermoid metaplasia by (1) altering
the permeability of target cells, so that
the penetration of the fly ash is facili-
tated, (2) increasing the mitotic activity
of the cells providing a favorable condi-
tion for an attack by the aromatic hydro-
carbons and trace elements that consti-
tute the soluble fraction of the fly ash, or
(3) interfering with the detoxification
mechanisms of the target cells, leading
to an  increased persistance  of the
aromatic hydrocarbons.
  Environmental exposure to airborne
particulates has been linked to chronic
respiratory disease, such as chronic
bronchitis. Upon inhalation,  pollutants
are deposited to a large extent in the
trachea where they are removed by
mucociliary mechanisms, or phagocy-
 tized. The effects of the various pol-
 lutants on cells of the tracheobronchial
 epithelium were in many instances
 complex. Each of the real-world pol-
 lutants assayed produced a unique re-
 sponse, probably related to chemical
 composition and size of the particles. It
 is recognized that smaller particles are
 phagocytized by mucosal cells while the
 larger particles provoke  hyperplastic
 and  metaplastic  changes. One might
 speculate that exposure to the particulate
 pollutants affects the structure and
 functional continuity of the epithelium,
 thereby contributing to chronic bronchitis.

 Recommendations
   Organ cultures of hamster trachea
 consisting in part of mucociliary epithe-
 lium  have permitted study of the effects
 of toxic agents on this tissue. The ring
 and whole tracheal organ culture prepa-
 rations were found to be well suited to
 evaluate a number  of parameters on
 viable explants, as well as fixed tissue.
 These cultures were maintained for
 weeks and monitored while  being ex-
 posed for a specific period under com-
 pletely defined conditions. The ability to
 prepare a large number of tracheal
 cultures permitted multiple replicate
 exposures at each experimental point
 and a wide range of concentrations. Our
 results demonstrated that  changes in
 any single parameter were insufficient
 indications of the toxic effects of expo-
 sure to a test substance, and we there-
 fore  recommend that cilia beating
 frequency data be correlated with
 morphological and biochemical param-
 eters.
  The increasing number of pollutants
 being emitted into the environment and
 the backlog of  potentially hazardous
 chemicals used in industry make a rapid,
 reproducible, and relatively inexpensive
 test system for evaluating toxicity a
 pressing need. Hamster tracheal organ
 cultures provide the  necessary criteria
 to facilitate identification of these toxic
 agents. Additional investigations are
 therefore needed to  determine the re-
 sponse of mammalian respiratory tract
 tissue to a variety of complex  mixtures.
  The studies should be expanded using
 organ cultures  of tracheal epithelium
 and heterotopic tracheal grafts as a
bioassay for respiratory tract carcino-
 genicity to evaluate environmental pol-
 lutants as potential carcinogens. The
criteria for assessment  would include
histologic and cytologic  changes, DNA
synthesis, and tumor  production. In this
                                                                                     1 US GOVERNMENT PRINTING OFFICE 1981757-01Z/7095

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    way, the questions concerning the
    interrelationships between differenti-
    ation, cell  population kinetics,  and
    various stages in carcinogenesis could
    possibly be clarified.
           Leonard J Schiff is with Life Sciences Research Division, in'Research Institute,
             Chicago, IL 60616.
           Judith A. Graham is the EPA Project Officer (see below).
           The complete report, entitled "Trachael Organ Culture as Air Pollution Damage
             Indicator," (Order No. PB 81-168 999; Cost: $9.50, subject to change! will be
             available only from.
                   National Technical Information Service
                   5285 Port Royal Road
                   Springfield, VA 22161
                   Telephone. 703-487-4650
           The EPA  Project Officer can be contacted at'
                   Health Effects Research Laboratory
                   U.S. Environmental Protection Agency
                   Research Triangle Park, NC 27711
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
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x°/EPA
                             United States
                             Environmental Protection
                             Agency
                             Health Effects Research
                             Laboratory
                             Research Triangle Park NC 27711
                             Research and Development
                             EPA-600/S1-81-022  May 1981
Project  Summary
                             Effects  of  Etiologically Defined
                             Respiratory Infections on Lung
                             Function  and  Its  Growth in an
                             Area  of Low Air  Pollution  -
                             Spirometry in  Young  Children
                             When  Illness-Free
                             Albert M. Collier, Gerald L. Strope, Ronald W. Helms, Lisa Morrisey Lavange,
                             Wallace A. Clyde, Jr., Russel Pimmel, Floyd W.  Denny, Frederick W.
                             Henderson, and Gerald W. Fernald
                               This longitudinal study was per-
                             formed in a group of 3-12 year old
                             children to document normal lung
                             growth patterns as measured by spi-
                             rometry. By clinical and laboratory
                             parameters, these children were free
                             of illness at the time of study and had
                             been for the preceding 21 days. Spiro-
                             metry was performed prospectively
                             over a period of six years in 69 children
                             (27 black females, 23 black males, 10
                             white females, and 9 white males)
                             from a day care center. Eight-hundred
                             fifteen spirometric tests were made on
                             these children. Linear regres-
                             sion analyis by a method of weighted
                             least squares was found to adequately
                             describe the data over a height range
                             of 100-150 cm and was performed on
                             six spirometric parameters: forced
                             vital capacity (FVC), forced expiratory
                             volume in one second (FEV,), peak
                             expiratory flow (PEF), forced expira-
                             tory flow during the middle half of the
                             FVC (FEF25-7s%), and maximum expira-
                             ory flows after 50% and 75% of the
                             FVC have been exhaled (Vmax5o% and
                             Vmax75%, respectively). Ninety-five
                             percent confidence limits for the
                             regression lines and 95% prediction
                             intervals for individual observations
                             were also computed. There were sig-
                             nificant differences between the re-
                             gression lines (considering slope and
                             intercept) for all six parameters when
                             black females were compared to black
                             males, white females to white males,
                             black females to white females and
                             black males to white males, except for
                             Vmax?5% for the comparison of black
                             females to black males and white
                             females to white males. These slopes
                             and intercepts were similar to those
                             reported  by others for children of
                             similar age and height. The 95% confi-
                             dence limits and 95% prediction inter-
                             vals were proportional to height and
                             were similar to estimates of parameter
                             variability reported by others. This
                             study demonstrates that spirometry
                             can be performed reliably at an early
                             age in a day care center population,
                             that there are significant racial and
                             sexual differences in spirometric vol-
                             ume and flow parameters and that the
                             variability of these measurements is

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proportional to height rather than
being a constant.
  This final report was submitted in
fulfillment of Grant #R-804577 by
the University of North Carolina under
the sponsorship of the U.S. Environ-
mental Protection Agency. This report
covers the period from  August 1,
1976  to  April  30, 1980 and  was
completed as of April 30,  1980.
  This Project Summary  was devel-
oped by EPA's Health Effects Research
Laboratory, Research Triangle Park.
NC, to announce key findings of the
research project that is fully docu-
mented in a separate report of the
same title (see Project Report ordering
information at back).

Introduction
  The growth of the lung in children is a
dynamic  process characterized by an
increase in alveolar, number followed by
an increase in size of airways and air
spaces in  the lung. Other researchers
have shown that the  total number of
airways is the same  in children  and
adults. The total complement of bronchi
al generations is present by the end of
the sixteenth week of gestation and the
spaces which will contain air  begin to
change to an alveolar pattern during the
latter part of gestation During the  first
weeks of life, typical alveoli appear and
lung growth for the first four years of life
consists predominantly of an  increase
in alveolar number At this point, increase
in lung size changes to a pattern of
growth consisting of an increase in size
of airways and alveoli The physiologic
importance of this growth  pattern has
been demonstrated by other researchers
who found that the conductance of
peripheral airways (beyond the  fifteenth
bronchial  generation) increases dra-
matically  at about five years of age,
while no change occurs in the central
airways.
  Most reference data for pulmonary
function  tests in children have been
generated on newborns and on children
over five or six years of age. Pulmonary
function tests from infancy to five years
of age have not been well documented
and it is not known what effect the
relative lower conductance of the pe-
ripheral airways might have on measure-
ments of lung function. It is  also not
known what effects acute respiratory
illnesses might have on measurements
of pulmonary function and  if these
effects are more easily detected because
conductance of the peripheral airways
is lower. Previous research has shown
that the peripheral fraction of total
respiratory resistance measured by the
forced random noise technique in a
group of children, whose average age
was 46  months, to be significantly
greater than  in a group of children
whose average age was 61  months.
(Conductance of the peripheral airways
was less in younger children than in the
older children.) Other researchers have
demonstrated a  greater  number of
significantly decreased spirometric
parameters during acute upper respira-
tory illness in  a group of children less
than 84 months of age, ascomparedtoa
group of children greater than 84 months
of age. These  data suggest that there
are differences in lung function  during
early life attributable to developmental
phenomena m the airways and that
these differences  may be important in
the manifestation of disease.
  Additionally, data of pulmonary func-
tion in children have been collected in a
cross-sectional fashion. No data exist
which have been systematically gener-
ated in a  longitudinal fashion.  These
data are important to determine  if lung
function development in children pro-
gresses in a predictable fashion much
the same as height and weight develop-
ment, or if the growth of the lung might
progress in another as yet undetermined
pattern.
  This report outlines the analysis of the
spirometry data collected over a six-year
period at the Frank Porter Graham Child
Development Center. Only  information
gathered  on the children  when they
were  well has been included in this
analysis.  The data have been analyzed
in a cross-sectional fashion so that later
analyses can be performedtodetermme
what the relationships of each individual
child's growth  pattern is to the overall
growth pattern. Because of the known
effects of race and sex on lung function,
the children have been separated into
four groups for these analyses: black
females and males and white females
and males.
Conclusions
  Over the six year study period, 69
children were followed for an average of
3.5 years each  and had an average of
3.14 measurements made per year. The
children, in general, were 90 to 155cm.
tall and spanned an age range of 21/2 to
12  years. Only data from the children
whose heights were between 100 and
150 cm., however, were included in the
regression analyses
  The regression coefficients and stan-
dard errors of the six spirometric param-
eters computed by weighted least squares
analysis for  the  four race/sex groups
are shown in Table 1. The coefficients
for the 95% confidence and 95% predic-
tion limits are also listed. Plots of each
discrete data point along with the
regression lines and 95% confidence
and prediction intervals for each param-
eter in each of the four race/sex groups
reveal an excellent fit of the data to the
regression lines. Comparisons of plots
of the regressions for the four race/sex
groups in general reveal that, for any
given  height, white children have larger
lung function parameters than black
children. The p-values for the tests of no
difference comparing the regression
lines (including slope and intercept)
between the important race/sex group-
ings are shown  in Table 2. All  of these
comparisons are significantly different
except for Vmax7s% for black  females
compared to black males and for white
females compared to white males. The
variability para meters (9 5% prediction
limits) as computed in these analyses
are similar to variability as computed by
more classical methods (e.g.,  Mean ±
2SD)  This suggests that making mea-
surements in a longitudinal  fashion
does not reduce the expected variability,
as demonstrated below.
1.  The feasibility of obtaining reproduc-
   ible spirometric tests of lung function
   in small children as young  as three
   years of age
2.  Significant racial and sexual differ-
   ences in most spirometric parameters
   of lung function  in young children.
3.  That  regression lines of spirometric
   parameters of lung function can  be
   adequately described in young chil-
   dren  by a linear regression  over the
   height range  of 100-1 50 cm.
4.  That  parameters of variability are
   proportional  to height rather than
   being a constant over the region of
   interest.
Recommendations
  To ascertain the effects of acquired
inslut on  the lung such as from  air
pollution on infectious agents, additional
reference data from populations studied
in an area with  minimal  air pollution,
and from  populations whose illness
patterns have been closely documented,
are needed. Until the effects of lower

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 Table 1.     Weighted Regression Coefficients* for Spirometric Parameters
                                   Black Females
                                                                  Black Males
Parameter6
FVC, '
FEV,. '
PEF. '/sec
FEFZ5-7s%, '/sec
VmaxsoVo. '/sec
Vmax75%, '/sec
be
-1.966+0.084
-1.533+0.076
-3.112±0.290
-0.493±0.211
-0.820+0.245
0.243+0.177
mf
0.0267±0.0008
0.0222 ±0.000 7
0.0531 ±0.0026
0.0195±0.0019
0.0245±0.0022
0.0073+0.0016
Kh
(xlO3)
0.415
0.378
1.477
1.041
1.213
0.865
Lc
Ix102)
0.371
0.334
1.281
0.931
1.084
0.781
b
-2.601 ±0.1 00
-2. 191 ±0.090
-4.913±0.343
-1.650±0.246
-1. 888 ±0.291
-0.297 ±0.209
m
0.0324±0.0009
0.0281+0.0008
0.0685±0.0031
0.0292+0.0022
0.0330±0.0026
0.01 1 9±O.O01 9
K
(x103)
0.485
0.437
1.692
1.231
1.478
1.032
L
(x102l
0.372
0.335
1.284
0.933
1.087
0.783
                                   White Females
                                                                  White Males
   Parameter
                                        m
                                   K
                                 (x103)
  L
(x102)
                                                                                         m
                          K      L
                        (x102)   fx103j
 FVC, '
 FEVi. '
 PEF, '/sec
 FEF25-75%, '/sec
 VmaxsoVo, '/sec
 Vmax75a/o, '/sec
-2.969+0.165   0.0365+0.0013   0.708   0.374  -2.442±0.152
-2.469+0.148   0.0313±0.0012   0.639   0.336  -1.676±0.136
-3.846±0.569   0.0611 ±0.0046   2.393   1.288  -1.378+0.523
-1.644+0.414   0.0316+0.0033   1.739   0.936  -0.077±0.380
-2.111+0.482   0.0380±0.0039   2.026   1.090  -0.287±0.443
-0.256+0.347   0.0130+0.0028   1.485   0.786   0.466+0.319
                        0.0331±0.0013   0.663   0.374
                        0.0256+0.0011   0.581   0.336
                        0.0402±0.0043   2.227   1.288
                        0.0174±0.0032   1.618   0.937
                        0.0209 ±0.0037   1.885   1.091
                        0.0067+0.0026   1.390   0.786
"Regression equations have the form y=mx + b, where y represents the parameters; m, the slope with units equal to those of the
 parameter divided by cm; b, the intercept with units identical to the parameter; and x. the height in cm.
bNmety-five percent confidence limits (for the regression line) for any height between 100 and 150 cm can be computed using the
 form (mx + b) ± Kx, where K represents the 95% confidence coefficient.
c Ninety-five percent prediction limits (for an individual observation) for any height between 100 and 150 cm can be computed using
 the form (mx + b) ± Lx, where L represents the 95% prediction coefficient.
^Parameters are FVC (forced vital capacity), FEV\ (forced expiratory volume in one second), PEF (peak expiratory flow), FEF25-750/o
 (forced expiratory flow during the middle half of the FVC), VmaxSo%, (maximum expiratory flow after 50% of the FVC has been
 exhaled), and Vmax7S% (maximum expiratory flow after 75% of the FVC has been exhaled).
eb = intercept + S.E.
'm = slope + S.E.
respiratory illnesses during infancy on
lung growth and development are better
understood, populations whose entire
life history of  respiratory illness is
known should be studied. Since lower
respiratory illnesses during infancy may
be related to altered lung function later
in life, these illnesses need to be docu-
mented  Documentation should not only
include determination of the etiology of
the illness and  other clinical data but
should also include attempts to charac-
terize  changes m lung function during
these illnesses, as well as when illness
free. This will necessitate the continued
effort in developing tests of lung function
which can be utilized in very young
children. Also, reference data for tests
of pulmonary function are needed which
have been collected in a longitudinal
fashion  in illness-free, non-smoking
individuals 13-25 years of age who are
living in an  area relatively free of
atmospheric pollution.
                     Table 2.     P-Values for Tests of No Differences Between the Indicated
                                Regression Lines
                      Comparisons*
FVC    FEVi
PEF     FEF25-75%  Vmaxso%
Vmax75%
BF vs BM"
WF vs WM
BF vs WF
BM vs WM
Oc
0
0
0
O
0
0
0
 US GOVERNMENT PRINTING OFFICE 1981-757-012/7099

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     Albert M.  Collier, Gerald L. Strope, Ronald W. Helms, Lisa Morrisey Lavange,
       Wallace A. Clyde, Jr.. Russel Pimmel, Floyd W. Denny, Frederick W. Hender-
       son, and Gerald W. Fernald are with the University of North Carolina School of
       Medicine, Chapel Hill, NC 27514.
     Ralph W.  Stacy is the EPA Project Officer (see below/.
     The  complete report, entitled  "Effects of Etiologically Defined Respiratory
       Infections on Lung Function and Its Growth in an Area of Low Air Pollution -
       Spirometry in Young Children When Illness-Free," (Order No, PB 81-173 338;
       Cost:  $8.00, subject to change)  will be available only  from:
             National Technical Information Service
             5285 Port Royal Road
             Springfield,  VA  22161
             Telephone: 703-487-4650
     The EPA Project Officer can be contacted at:
             Health Effects Research Laboratory
             U.S. Environmental Protection Agency
             Research  Triangle Park, NC 27711
United States
Environmental Protection
Agency
                               Center for Environmental Research
                               Information
                               Cincinnati OH 45268
Postage and
Fees Paid
Environmental
Protection
Agency
EPA 335
Official Business
Penalty for Private Use $300
230
              DKAPBUR*
           AGO  LI,
                        srkcKr

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