regarding the mechanisms of ozone toxicity are adequately
Research Council's publication, “Ozone and Other Phoro~
ce it to say that the oxidant property of ozone

£ an oxygen singlet when ozone breaks down. This
electron orbit and, thus, acts as an avid electron
ree radicals are produced that themselves act as
radical chain reaction. Carbon to carbon
are easily ox: ed by ozone. Because such
D cell membranes, such oxidations are believed «=
echanisms of cellular injury by ozone. Ionizing radia~
mechanisa; ities of ozone and ionizing

E and C are free radical scavengers, leading to the idea that these
ar compounds may prote against ozone toxicity. Goldstein et al. (16)
claimed that p-aminobenzoic acid (PABA), p-aminchippuric acid, and anthranillic
rotected against czone toxicity in vitro and in vivo. It was reported

ppav ozone. O, rats injected with PABA survived almost twice as long as
untreateé rats when exposed te 15 ppmv ozone. Because these acids are nontoxic
the authors suggested that they may have use in pro~
rom ozone.

heorized that ozone toxicity represented
seen in vitamin E deficiency. These
0 ppmv ezone and determined the time for
population. Vitamin E supplement in

and coworkers (2

200 zg of v in E prevent

body inclusions in human BBC's in vitre. yi oleate ozonide has been proposed

an intermediary in ozone toxicity. Heinz bodies are believed te be polymers
+ emoglobin) attached to the inner surface of the RBC

int out that release of histamine, serotonin, brady—
S have been suggested as possible mediators of ozone-
ctions in the pulmonary airways. Rats were exposed for
Exposure began 60 to 90 min after treatment with aspirin,
cortisone, or heparin. Control rats were treated with equiva—
er. At the end of the exposure period the rats

ozone~induced pulmonary edema. Indomethacin and heparin produced significant
reductions in pulmonary edema in ozone-treated rats. The authors interpret their
findings to mean that one or more of the prostaglandins are involved in ozone
toxicity and that some anti-inflammatory drugs ameliorate the effects of czone.



The number of mac-ophages in random microscopic fields along the terminal air-
ways were related to ozone level. Dogs that were exposed to 1.0 ppmv for 16 h
(16 ppmv-h) showed less than half the response (5 cells/field) shown by dogs
exposed to 2.0 ppmv (13 cells/field) for $ h (16 ppmv-h). Likewise, dogs
exposed to 1.0 ppmv for 24 h (24 ppmv-h) showed about half the response of
dogs exposed to 3.0 ppmv for 8h (24 ch). This work shows that equal doses
(CxT) did aot produce equal responses; ozone level determined the extent of
damage.

Horvath and his associates (21} exposed healthy young men to various
levels of ozone in an atmospheric chamber. The subjects underwent various
levels of exercise while being exposed. The level of ventilation produced
by exercise was not as important as was ozone concentration in predicting the
degree of pulmonary function alteration. The effective dose was calculated as
the product of ozone concentration, ventilation volume and time of exposure.
With the exception of respiratory frequency, the majority of the variance was
explained by ozone concentration rather than by ventilation during the exposure.

Silverman and others (36) exposed 28 young healthy adults (20 men, 8 women)
to 0.37, 0.50, and 0.75 ppmv ozone for 2 h together with exercise calculated to
increase pulmonary ventilation 2.5 tines. Effective dose was calculated as the
Product of concentration, duration of exposure and ventilation. They found that
exposure to a high concentration of ozone for a short period had more effect
than did a lower concentration for a longer period, thus implying that there is
not only a threshold effective dose, but also a threshold effective concentration.

Coffin, Gardner, Sidorenko, and Pinigin (/,14) exposed white mice and rats
to benzene, SOQ and NO2 and demonstrated for NO2Q exposure only that brief peak
levels were more toxic than was continuous exposure to lower levels, thus indi-
cating that the prime importance of concentration of an irritant gas is a phenom—
enon not confined to ozone.

Intuitively, it can be seen that concentration of ozone should be more
important in causing damage than would duration of exposure. In order to reach
the cells lining the respiratory passages, ozone must diffuse through the sur-
face mucus. The diffusion gradient will determine how rapid and how deep penetra-
tion will be; thus, the mass transfer of ozone into respiratory tissue will depend
on the concentration at the mucous surface. An equation that defines the rate of
movement of a gas from the lumen of the respiratory tract to the tissue is as
follows (24):

Gm = -KgA (Cy-C2)

dt

Kg is the mass transfer coefficient in centimeters per second, A is exposed
airway surface, C; is the ozone concentration in the airway lumen and Co is the
concentration cf ozone in the airway wall. The expression (C,- Co) is the con-
centration gradient. Because ozone is rapidly destroyed in tissue, a steady—
state diffusion profile will quickly develop and penetration of the gas into the
airway wall will depend primarily on C,. Implicit in this line of reasoning is
the idea that ozone is evenly distributed and held et a constant concentration
in the air of the bronchiole, alveolar duct or atvevlus-—-probably by a valid

{lt



14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

Gardner, D. E., D. L. Coffin, M. A. Pinigin, and G. I. Sidorenko: Role
of Time as a Factor in the Toxicity of Chemical Compounds in Intermittent
and Continucus Exposures. Part I. Effects of Continuous Exposure, J.
TOX. ENVIRON. HEALTH, 3:811-820, 1977.

Giri, S. N., J. Benson, D. M. Siegel, S. A. Rice, and M. Schiedt:
Effects of Pretreatment with Anti-Inflammatory Drugs on Ozone~Induced
Leng Damage in Rats, PROC. SOC. EXP. BIOL. MED., 150:810-814, 1975.

Goldstein, B., M. R. Levine, R. Cuzzi-Spada, R. Cardenos, R. D. Buckley,
and 0. J. Balchum: p-Aminobenzoic Acid as a Protective Agent in Ozone
Toxicity, ARCH. ENVIRON. HEALTH, 30:234-236, 1972.

Hackney, J. D., W. S. Linn, R. D. Buckley, E. E. Pedersen, S. Karuza,

D. C. Law, and D. A. Fischer: Physiological Effects of Air Pollutants

in Humans Subjected to Secondary Stress, Air Resources Board, Resources
Agency, 1709 ilth St., Sacramento, CA. Report No. ARB-R-2-372-74-25, 1974.

Hackney, J. D., W. S. Linn, S. K. Karuza, R- D. Buckley, D. C. Law,

D. V. Bates, M. Hazucha, L. D. Pengelly, and F. Silverman: Effects of
Qzone Exposure in Canadians and Southern Californians, ARCH. ENVIRON.
HEALTH, 32:110-116, 1977.

Hackney, J. D., W. S. Linn, J. G. Mohler, and C. Collier: Adaptation to
Short-Term Respiratory Effects of Ozone in Men Exposed Repeatedly, J.
APPL. PHYSIOL.: RESP. ENVIRON. EXERCISE PHYSIOL., 43:82-85, 1977.

Higgins, E. A., M. T. Lategola, J. M. McKenzie, C. E. Melton, and

J. A. Vaughan: Effects of Ozone on Exercising and Sedentary Adult Men
and Women Representative of the Flight Attendant Populaticn. FAA Office
of Aviation Medicine Report No. FAA~AM-79-20, July 1979.

Hervath, Steven M., Jeffrey A. Gliner, Lawrence J. Folinsbee, and
John F. Bedi: Physiological Effects of Exposure to Different Concen-
trations of Ozone. Report to the California Air Resources Board and
the National Institutes of Health (Grant No. ESO 1143), 1978.

Linn, W. S., R. D. Buckley, C. E. Spier, R. D. Blessey, M. P. Jones,
D. A. Fischer, and J. D. Hackney: Health Effects of Ozone Exposure in
Asthmatics, AM. REV. RESP. DIS., 117:835-843, 1978.

MacEwen, J. D. and R. P. Geckler: Comparative Studies of 90-Day Continuous
Exposure to 03, NOQ and CCl, at Reduced and Ambient Pressures. AMRL-TR-
67-68. 1963.

McJilton, C., J. Thieike, and R. Frank: Ozone Uptake Model for the
Respiratory System. Paper presented at the American Industrial Hygiene
Association Conference, San Francisco, CA, May 14-19, 1972.

McNerney, J. M., and J. 0. MacEwen: Comparative Toxicity Studies at

Reduced and Ambient Pressures. I. Acute Response. AM. IND. HYG. ASSOC.
3., 1965.

13


exposure. Its lungs were hemorrhagic and edematous. Other animals that died
were described as showing signs consistent with ozone poisoning. Monkeys were
entirely unaffected by the experiment. In anether series of studies involving
acute exposure to ozone for 2 weeks, these workers showed that 800 ug/m3 (00.40
ppmv) in air at ambient pressure of 700 mm Hg caused the deaths of 5 out of 5
dogs, 2 out of 4 monkeys, 50 out of 50 rats, 34 out of 40 mice, and 8 out of 8
guinea pigs. Again, the authors did not provide the distribution of deaths
over the 2-week period.

Freeman et al. (12) exposed dogs to various levels of ozone for 18 months.
Dogs were exposed te 1.0 ppmv for 8, 16, or 24 n/d and others to 2.0 or 3.9
ppnv for 8 n/d- The animals' lungs were examined by light and electron
microscopy. The response was dependent on the concentration of ozone; the
earliest response (1.0 ppmv for 8 h/d) consisted of macrophages in and around
the bronchiolar-ductal region and in adjacent alveoli. Macrophages increased
with higher concentraticns of ozone and fibrosis appeared. At higher concentra—
tions, peribronckiolar collars of fibroblasts were apparent, reducing the caliber
ef the small airways.

Bartlett et al. (2) exposed 3- to 4-week-old rats to 0.20 ppmv ozone for
i month. Ozone did not apparently affect health or lung growth in exposed rats.
The ozone-exposed rats were morphometrically indistinguishable from the air-
exposed controls. Lung volume, however, was 16 percent greater in the exposed
group than in the control group. This overdistension was reflected in increased
chord length end in alveolar surface area. The authors speculate that ozone
adversely affected characteristics of lung collagen.

Penha and Werthamer (32) exposed tumor-resistant mice to 2.5 ppmv ozone for
120 d. The exposure regimen consisted of 2 h exposures of mice in groups of 10
to ozone-containing air or to air only each day of the study. Histological study
of the lungs showed proliferative changes in the tracheal epithelium of ozone-
treated mice around the 45th day of exposure. Celluiar proliferation in the
bronchioles formed papillary projections around the 80th day, becoming more pro-
nounced by the 120th day. Ozone-exposed mice were alsc more susceptible to myco-
plasma infection than were controls. In 120 days postexposure, most lung
pathology reversed. The authors concluded that ozone should be added to the
list of pulmonary carcinogenic compounds.

The respiratory system of monkeys most nearly resembles that of man;
therefore, studies on primates are of special value. Unfortunately, because
of acquisition cests and difficulty and expense of housing, few institutions
are equipped to carry out studies on primates. Dungworth et al. (10) studied
effects of ozone levels representative of those found in the California South
Coast Basin on rhesus and bonnet monkeys. Monkeys were exposed to concentrations
of ozone ranging from 0.20 to 0.80 ppmv, 8 h/d for 7 d. Companicn controls con-
sisted of identically treated monkeys, except that they were exposed to air only.
At the end of the exposure period, ali animais were killed, their lungs removed
and examined histolugically. The authors state, "The most obvious and consist-
ent pulmonary iesion in all exposed nonkeys occurred in respiratery bronchioles
The extent and severity of damage, but not its nature, varied wi he level of

wu


The number of macrophages in randor icroscopic fields along the terminal air-
ways were related to ozone level. ..gs that were exposed to 1.0 ppmv for 16 h
(16 ppmv-h) showed less than half the response (5 cells/field) shown by dogs
exposed to 2.0 ppmv (13 cells/field) for 8 h (16 ppmv-h). Likewise, dogs
exposed to 1.0 ppmv for 24 h (24 ppmv-h) showed about half the response of
dogs exposed to 3.0 ppmv for 8 h (24 ppmv-h). This work shows that equal doses
(CxT) did not produce equal responses; ozone level determined the extent of
damage.

Horvath and his associates (21) exposed healthy young men to various
levels of ozone in an atmospheric chamber. The subjects underwent various
leveis of exercise while being exposed. The level of ventilation produced
by exercise was not as important as was ozone concentration in predicting the
degree of pulmonary function alteration. The effective dose was calculated as
the product of ozone concentration, ventilation volume and time of exposure.
With the exception of respiratory frequency, the majority of the variance was
explained by ozone concentration rather than by ventilation during the exposure.

Silverman and others (36) exposed 28 young healthy adults (20 men, 8 women)
to 0.37, 0.50, and 0.75 ppmv ozone for 2 h together with exercise calculated to
increase pulmonary ventilation 2.5 times. Effective dose was calculated as the
product of concentration, duration of exposure and ventilation. They found that
exposure to a high concentration of ozone for a short period had more effect
than did a lower concentration for a longer period, thus implying that there is
not only a threshold effective dose, but also a threshold effective concentration-

Coffin, Gardner, Sidorenko, and Pinigin (7,14) exposed white mice and rats
to benzene, SQg and NOg and demonstrated for NG2 exposure only that brief peak
levels were more toxic than was continuous exposure to lower levels, thus indi-
cating thet the prime importance of concentration of an irrirant gas is a phenom—
enon not confined to ozone.

intuitively, it can be seen that concentration of ozone should be more
important in causing damage than would duration of exposure. In order to reach
the cells lining the respiratory passages, ozone must diffuse through the sur-
face mucus. The diffusion gradient wiil determine how rapid and how deep penetra-
tion will be; thus, the mass transfer of ozone into respiratory tissue will depend
on the concentration at the mucous surface. An equation that defines the rate of
movement of a gas from the lumen of the respiratory tract to the tissue is as
follows (24):

dm = -KeA (C)-Cp)
at

Kg is the mass transfer coefficient in centimeters per second, A is exposed
airway surface, C; is the ozone concentration in the airway lumen and Cp is the
concentration of ozone in the airway wall. The expression (C,- Cy) is the con~
centration gradient. Because ozone is rapidly destroyed in tissue, a steady—
state diffusion vrofile will quickly develop and penetration of the gas into the
airway wall will depend primarily on C). Implicit in this line of reasoning is
the idea that ozone is evenly distributed and held at a constant concentration
in the air of the bronchiole, alveolar duct or alveolus--probably by a valid


EFFECTS OF LONG-TERM EXPOSURE TO
LOW LEVELS OF OZONE: A REVIEW

The literature regarding the effects of ozone on living systems is mountainous.
Most of this literature relates to the effects of urban/industrial air pollution
(mixed gases plus particulates) on humans, animals, and vegetation. Much of the
literature is concerned with industrial uses of ozone. Ali of this literature is,
some extent, pertinent to the subject of this review, but for obvious reasons
it cannot all be cited. To de so would be to defeat the purpose of a review,
which is to collect and clarify information about one segment of a larger question.
This review, then, will be concerned with information related co long-term expo-
sure of humans anc animals to low levels of ozone.

Having set these limits, it then becomes incumbent on the reviewer to define
“long term" and "low level." The definitions will be based on exposures that
would Je expected to occur in airline operations. Such exposures would be inter-
mittent rather than continucus and the levels of ozone encountered in flight would
ordinarily not be greater than about 2,000 :g/m? (~L.0 part per million by volume -
ppmv at sea level) (33). The duratioa that qualifies for "long term will be con-
sidered to be any exposure, continuous or intermittent, that goes on for days
rather than hours. ‘Net all the experiments cited in this review are directed at
effects of low levels of ozone or effects of long-term exposure; however, there
are aspects of each of these experiments that bear on the question of effeccs of
chronic exposure to lew levels of ozone.

A "dese" of ozone is defined as the product of concentration (C) of the gas
and the duration of exposure (T). This concept has led to the formlation of the
"Time-~Weighted Average" (TWA) as an index of the severity of exposure. The TWA
is calculated according to the following equation (1):

twa = 2C5Tp
it

Where: Cz; = Concentration of the *th sample
Ty Sampling time of the 1ch sample
i = Any one sample during the time to which the

TWA applies.

The concept on which this formula is based assumes that sampling periods
(e.g., 10 min) rather than continuous measurement will be used. The sum of the
CxT values thus obtained is divided by the total sampling time to give the TWA.
When the TWA is used in conjunction with a peak limit, harmful exposures should
be prevented. The TWA alone only fells, at the end of exposure, what the average
exposure was.

It is clear from the literature that concentration is more important than
duration of exposure in causing ozone damage. Freeman et al. (12) exposed dogs
te texic levels cf azone foc various yeriods of time after which the dogs were
sacrificed and their lungs and respiratory passages examined histologically.



26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

Menzel, D. B., R. J. Slaughter, A. M. Bryant, and H. 0. Jauregui: Prevention
of Ozonide-Induced Heinz Bodies in Human Erythrocytes by Vitamin E, ARCH.
ENVIRON. HEALTH, 30:234-236, 1975.

Miller, F., J. W. Illings, and D. E. Gardner: Effect of Urban Ozone Levels

on Laboratory-Induced Respiratory Infections. Toxicology Letters, 2:163-169,
1978.

Miller, Frederick J., Daniel B. Menzel, and David L. Coffin: Similarity

Between Man and Laboratcry Animals in Regional Deposition of Ozone, ENVIRON.
RES., 17:84-101, 1978.

Mittler, Sidney, Delores Hedrick, Maurice King, and Allan Gaynor: Toxicity
ef Ozone. I. Acute Toxicity. IND. MED. SURG., 25:301-306, 1956.

Mictler, Sidney, Maurice King, and Betty Burkhardt: Toxicity of Ozone. III.
Chronic Toxicity. AMA ARCH. IND. HEALTH, 15:191-197, 1957.

National Research Council, Assembly of Life Sciences, Division #f Medical
Sciences. Ozone and Other Photochemical Cxidants. Committee on Medical
and Biologic Effects of Environmental Pollutants, National Academy of
Sciences, Washington, D.C., 1977.

Penha, P. D. and S. Werthamer: Pulmonary Lesions Induced by Long-Term
Exposure to Ozone, ARCH. ENVIRON. HEALTH, 29:262-289, 1974.

Perkins, Porzer J., J. D. Holdeman, and G. D. Nastrom: Simultaneous Cabin
and Ambient Ozone Measurements on Two Boeing 747 Airplanes, Vol. 1. Revort
No. NASA TM-79166; FAA-EE-79-05, 1979.

Roehm, G. N., J. G. Hadley, and D. B. Menzel: The Influence of Vitamin E on
Lung Fatty Acids of Rat. Exposed to Ozone, ARCH. ENVIRON. HEALTH, 24:237-242,
1972.

Schwartz, W., DB. L. Dungworth, M. G. Mustafa, B. K. Tarkington, and W. S.
Tyler: Pulmonary Responses of Rats to Ambient Levels of Ozone, LAB. TNVEST.,
34:565-578, 1976.

Silverman, F., L. J. Folinsbee, J. Barnard, and R. J. Shephard: Pulmonary
Function Changes in Ozone - Interaction of Concentration and Ventilation,

J. APPL. THYSIOL., 41:859-864, 1976.

Stokinger, H. E., W. D. Wagner, and 0. J. Dobrogorski: Ozone Toxicity Studies:
ILL. Chronic Effects. AMA ARCH. IND. HEALTH, 16:514-522, 1957.

Stokinger, H. E.: Ozone Toxicology, ARCH. ENVIRON. HEALTH, 10:719-731, 1965.

Watanabe, S., R. Frank, and ©. Yokoyama: Acute Effects of Ozone on Lungs of
Cats. I. Functional. AM. REV. RESP. DIS.: 108:1141-1i51, 1973.

14


In a later study, Mittler et al. (30) exposed young rats to "low" concen-
trations of ozone (0.6-2.4 ppmv) 5 d/wk for 4 wk and compared their mean weight
gain with that of control rats. Weight gain was significantly inbibited in the
ozone-exposed animals by doses ranging from 0.6 ppmv for 10 d to 2.4 ppmv for
3d. Exposure ef mice to 2.4 ppmv continuously for 10 d caused the deaths of
21 of the 102 animals tested. All effects seen in rats and mice were confined
to their lungs. Hemorrhage and pulmonary edema were common autopsy findings.

Stokinger et al. (37) exposed hamsters, rats, guinea pigs, and dogs to
ozone levels that, at that time, were considered to be low (0.5-2.3 ppmv; av.,
1.9 ppmv) for 6 t/d, 5 d/wk over a total period of 433 d. Rats and guinea pigs
showed the highest mortality rate (33 and 30 percent, respectively) associated
with ozone exposure. Ozone~exposed hamsters showed a lower mortality rate than
did contrels (11 percent as opposed to 50 percent) and no dogs, neither controls
nor ozone-exposed, died. Mean weight gain was less in ozone-exposed rats than
in controls, but the other species showed no significant effects. Examination
of the dogs’ eyes showed no effect of ozone after } year, either ophthalmolog—
ically while they were alive or histologically after sacrifice. Arterial blood
oxygen saturation in guinea pigs was affected secondary to hypoxia brought on
by pneumonia, pneumonitis or bronchiolitis.

There were no effects of ozone on arterial blood oxygen saturation in dogs.
Lung pathology was qualitatively similar in all species examined except dogs,
with guinea pigs showing the greatest damage. Terminal bronchiolar epithelium
was hyperplastic, causing stenosis of tubes. Sloughing of the epithelium and
leucocytic invasion were also common findings. Fibrosis extended from the
bronchioles into the alveolar ducts. Emphysema was evident. Dogs showed only
irritation of the trachea and large bronchi. Stokinger felt that the observed
species differences could be accounted for by nasal scrubbing being better in
some species than in others and by length of the respiratory tract. Unfortunately,
these authors did not relate mortality rate to duration of exposure. Such data
would be useful in assessing long-term effects. As it stands, it is not known
whether or not most of the deaths occurred early in the experiment, thus qual-
ifying as acute rather than chronic effects.

Later, in a review of ozone toxicology (38), Stokinger summarized the effects
of chronic exposure to damaging levels of ozope as being (i) premature aging,
evidenced in rabbits by premature calcification of the sternocostal cartilage,
depletion of body fat, coarseness of the coat, dull cornea and sagging conjunctivae;
(ii) lung-tumorigenesis (adenoma) in a lung tumor-susceptible strain of mice; and
(iii) chronic pulmonary effects in guinea pigs, hamsters, rats, and mice. These
effects consisted of emphysematous changes, chronic bronchitis, bronchiolitis, and
fibrotic changes.

Problems of exposure of spacecraft crews to on-board air poliutants prompted
an investigation into the effect of reduced ambient gas pressure on ozone toxicity
(23,25). These workers exposed dogs, rats, mice, guinea pigs, and rhesus monkeys
for 90 d to 190 xg /m> (0.10 ppmv) ozone mixed with either air or 100 percent
oxygen at ambient and reduced pressures. Reduced pressure accounted for a lower
mortality rate in ozone-exposed animals. However, as with Stckinger's chronic-
exposure experiments, these workers did not state when in the 90-d period the
animals died. One dog was sacrificed when it was believed to be dying of ozone

-


FAA-AM-70-16

EFFECTS OF LONG-TERM EXPOSURE TO LOW LEVELS OF OZONE: A REVIEW

C. E. Melton ORI GINAL

Civil Aerome+ical Institute

Federal Aviation Administration ip [aD
Oxiahoma City, Okiahoma 1: i

September 1980

Document is available to the public through the
National Technicai Informaticn Service
Springrield, Virginia 22161

Prepared for
@.S. DEPARIMENT OF TRANSPORTATION
Federai Aviation Ad stration
Office of Aviation Medicine
Washington, D.C. 20591



were no clinical changes attributable to ozone ner were there any significant
changes in forced wital capacity (FYC) or in ferced expiratory volume (FEV).
Group B experienced no subjective symptoms and had 0.80 URI/person during the
experimental period. FVC trended downward, but without statistical significance.
FEV was significantly depressed. Ali parameters returned to normal in 6 weeks.

The experinents of Hackney et al. (17) do not qualify as "chroricTM exposure

but do vield valuable formation regarding dose-response relationships. A
group of normal adult men were exposed for 2 h on each of 2 successive days to
0.37 or 0.50 ppmv ozone. Significant effects were found on forced expiratory
parameters by 0.50 ppmv. There were no significant effects of exposure te 0.37
ppmv. The interesting comment wes made, but net elaborated upon, that "The
group changes in physiological measurements did not achieve statistical signi
cance until the sccond exposure day in many cases, and in most cases the decre—
ments in function were markedly worse on the second day.” Hackney offers the
opinion that the “zero effect v
induced alterations in blood parameters (several sulfhydrvl enzymes and red bicod
cell (RBC) fragility) teok place after 2.75 h of exposure to 0.59 ppav ozone.

Ci

ianey and coworkers (4) exposed healthy voumg nonsmoking men to 0.40 ppav
ozone (784 ug/m?) for 4 8 on each of 4 successive days with and without exercise.
Nine blood biochemical parameters were measured including serum glutamic-pyruvic
acid transaminase (5 ). alkaline phosphatase, lactic acid dehydrogenase, RBC
cholinesterase, RBC glucose-6-phosphate dehydrogenase (C6PDH), RBC and serum

hai reductase, serum vitamin E and several serum proteins. effects of
or in combination with exercise were found on most of the parameters.
eects were found on serum vitamin E, GSPDE and complement C3 by 0.4

m with exercise.

exposure were inconsistent. For RBC G6PDH the ozone by
ificant ndicatedé that heavy exercise was

t exercise in causing G6PDE levels to vary. Comple-

e by exercise ct; for the heavy exercise

with time.

and Horvath (1 ssed the question of cumulative
They used three groups of
ci exposed te a di 2 hyd for 3d. Group 1

)} was exposed to 6.20 >: Group 2 2 (10 subjects) to 0.35 ppmv and
ubjects) to 0.50 ppmv. Additicnaily, two subjects were exposed 2
/& on 4 consecutive d@ to 0.50 ppmv czone. un the davs preceding and followiag
ozone exposure, subjects underwens an ideatical protocol while being exposed to
In this closely controlled experiment these workers showed

i al fect of ozone on all forced expiratory parameters
followed by a decreased effect on the third day of
cunulative effect and adaptation s also apparent
ese effects were arent only
ect either sub-
thresheld level of

the second
exposure.

in the two
x 0.35

Areshold” lies between 0.25 and 0.50 ppmv. Oxidanz-—


ozone exposure. There was a progressive decrease in amount of damage from proximal
to distal erders of bronchicles and the farthest point reached depended on dosage.
With 0.2 ppmv, the lesion was largely limited te the proximal generation of respir—
afory bronchioles whereas with 0.8 ppmv the damage extended to invoive, minimally,
pruximal porticns of alveolar ducts." The authors also state thet the threshold
for detectable piochemical and morphological effect of the same chronic regimen in
rats was 0.1 ppmv. They conclude, because the threshold leveis in bonnet monkeys
was less than 5.2 ppnv, t similar effects would be produced in sensitive people
by a level of ozone in the neighborhood of 0.2 In rats exposed to Q.2 oprv
ozone for 30 d, the effect reached a peak by the 4th day and leveled off

Fukase et al. (13) exposed mice fer 3
ezone and examined their lungs for reduced
anc control mice were exercised in 2 motor-
percent

to 9.2, 0.5, or 1.0 ppav
(GSH) levels. Experimental
Body weight, as
decreased in the ozone-e i group as a function of ozone
and GSE level increased with dosage. Exercise increased

of Streptucoceus pyrogenes
re (9.08-1.0 ppm).
sas enhanced and such

h

controis
tality at zero time
expesure to ozone,
stexposure.

be

(i) Common Laboratory smali

Ss and The reason
cf cilées r to differences
in ne TesD ferent resz
at (ii) Ozone damage is confined te
pu. effects were described at dosages below
L affects susceptibi te lung infection and the
e xposure. (iv) Exe exacerbates the effects
Fo of éata relating level and duration of

19)

ae)

yt

EU
we.
im oups of men 3 ¢
rod: these enco
the:

6



assump..ou. Accordingly, ao matter how long a small mass of ovone acts, beyond
some undefined minimum time probabiy measurable in seconds or minutes, the
response or damage would not be increased because depth of penetration into the
tissue would not be increased. Stated another way, effect is primarily dependent
on the partial pressure of czone in the airway gas.

This principle was again demonstrated by Watanabe, Frank, and Yokoyama (39)
on anesthetized, paralyzed, mechanicaily ventilated cats. Ozone was adminis-
tered through a tracheal cannula in concentrations of 0.26, 0.50, and 1.0 ppm.
The duration of exposure to ozone ranged from 2.6 to 6.5 h. A fourfold increase

in concentration of ozone produced a fivefold increase in luag resistance at lh
and 4 h.

The operational meaning of the predominant importance of ozone concentration
over duration of exposure is that exposure te an ozone concentration that is
acutely innocuous is also innocuous over the long term. As far as pulmonary
effects and subjective symptoms in healthy humans are concerned, concentrations
at or below 0.25 ppmv appear to lie below the threshold for such effects. In
experiments on human subjects in this laboratory, 0.25 ppmv for 4 h with exercise

had no effect; whereas, 0.3 ppmv for 3 h with exercise had effects on all sub-
jects (26).

An extensive literature review on ozone effects was prepared by the American
Society for Testing and Materials (ASTM) (1). The appendix to this document
cites instances of human exposure and shows only one case of a biological effect
of ozone, other than odor, below 0.30 ppmv. These observations emphasize that
below ozone concentrations of about 0.30 ppmv, the concentration gradient from
airway lumen te tissue is so shallow that the probability of ozone traversing
the mucus covering the respiratory epithelium before it is destroyed is prac-
tically nonexistent. This concept has been mathematically modeled by Miller,
Menzel, and Coffin (28).

Animal Studies: Animals vary widely in their susceptibility to ozone, thus
greatly complicating the choice of surrogates for humans. Such surrogates are
important because studies on humans must be limited to exposures that are not
damaging and evaluations must be noninvasive. With animals, exposure regimens
can b> widely variable and organ and tissue studies can be carried out on all
subjects.

Mittler et al. (29,30) have shown that LD-50's for different animals vary
greatly. They exposed mice, rats, guinea pigs, rabbits, and cats to levels of
ozone ranging from about 12.0 to 60.0 ppmv. The 3-h LD-5%'s were 21.0 ppmv for
mice, 21.8 for rats, 51.7 for guinea pigs, 36.6 for rabbits, and 34.5 for cats.
The differences in susceptibility could not be accounted fer on the basis of
body weight or metabolic rate.

Birds exhibit relatively great resistance to ozone. Clamann (6) cites work
showing that young turkeys were not killed by 417.0 ppmv ozone for 3 hs; pigeons
withstood 67.3 ppmv for 3 h; some parakeets died in 25.0 ppmv ozone while others
did not; 54.0 ppmv ozone for 3 h was not lethal for canaries.


EFFECTS OF LONG-TERM EXPOSURE TO
LOW LEVELS OF OZONE: A REVIEW

The literature regarding the effects of ozone on living systems is mountainous.
Most of this literature reiaces to the effects of urban/industrial air pcllution
(mixed gases plus particulates) on humans, animals, and vegetation. Much of the
literature is concerned with industrial uses of ozone. Ali of this literature is,
to Some extent, pertinent to the subject of this review, but for obvious reasons
it cannot all be cited. Ta do so would be to defeat the purpose of a review,
which is to collect and clarify information about one segment of a larger question.
This review, then, will be concerned with information related to long-term expo-
sure of humans and animals to low levels of ozone.

Having set these limits, it then becomes incumbent on the reviewer to define
“long term" and “low level." The definitions will be based on exposures that
would be expected to cccur in airline operations. Suck exposures would be inter-
mittent rather than continuous and the levels of ozone encountered in flight would
ordinarily not be greater than about 2,000 ug/m” (71.0 part per million by volume -
ppmv at sea level) (33). The duration that qualifies for "long term" will be con-
sidered to be any exposure, continuous or intermittent, that goes on for days
rather than hours. Not all the experiments cited in this review are directed at
effects of low leveis of ozone or effects of long-term exposure; however, there
are aspects of each of these experiments that bear on the ques m of effects of
chronic exposure to low levels of ozone.

A "dose" of ozone is defined as the product of concentration (C) of the gas
and the duration of exposure (T). This concept has led to the formulation of the
"Time-Weighted Average" (TWA) as an index of the severity of exposure. The TWA
is calculated according to the following equation (1):

twa = 2C5Ty

= Concentration of the ith sample
= Sampling time of the ith sample
Any one sample during the time to which the TWA applies.

Where:

oO
bee
u

The concept on which this formula is based assumes that sampling periods
(e.g., 10 min) rather than continuous measurement will be used. The sum of the
CxT values thus obtained is divided by the total sampling time to give the TWA.
When the TWA is used in conjurction with a peak limic, harmful exposures should
be prevented. The TVA alone only telis, at the end of exposure, what the average
exposure was.

It is clear from the literature that concentration is more important than
duration of exposure in causing ozone damage. Freeman et al. (12) exposed dogs
to toxic levels of ozone for various pericds of time after which the dogs were
sacrificed and their lungs and respiratory passages examined histologically.

oe

H


Evolution of ethane and pentane when unsaturated fatty acids undergo peroxi-
dation is a fairly common reaction. It takes place, for example, when fats
become rancid and it also occurs during the peroxidation of lipids by ozone in
vivo. Dumelin, Dillard, and Tappel (9) measured ethane and pentane in expired
air of rats on vitamin E-deficient diets and on vitamin E-enriched diets after
the rats were exposed to 1.0 ppmv ozone for 1h. Vitamin E supplementation was
associated with significantly decreased amounts of ethane and pentane in the rats'
expired air, indicating protection against lipid peroxidation by ozone.

Chow and Kaneko (5) studied the effect of dietary vitamin E on rats exposed
continuously for 7 d to 0.8 ppmv ozone. Control rats were kept on a vitamin E-
deficient diet for 3 or 4 months; experimental rats were fed the same basic diet
but with vitamin E supplementation. Ozone caused a significant increase in activ-
ity of glutathione (GSH) peroxidase, pyruvate kinase and lactic dehydrogenase,
and a decrease in GSH level in RBC's of vitamin E-deficient rats but not in those
that received vitamin E supplementation.

The free radical mechanism of ozone toxicity appears to be emerging as the
most acceptabie theory. Such toxicity is apparently ameliorated by free radical
scavengers, the most common of which is vitamin E.

Conclusicns:

The Naticnal Research Council has sponsored an excellent review of all
aspects of ozone pollution (31) that covers the literature through 1976. The
present review has, therefore, been focused primarily on literature that has
appeared since 1976, though some papers of an earlier date have been included
for perspective and completeness. Not manv papers have appeared in the last 4
years that are directly pertinent to long-term effects of ozone, though some of
them certainly are pertinent to the problem of ozone contamination of airliner
cabins. This literature review does allow certain conclusions to be drawn and
points to areas wheve our knowledge is deficient.

The evidence is clear that there is a threshold level for toxic manifesta-
tions of ozone exposure as well as a threshold dose. Dose is defined as the

product of ozone concentration and duration of exposure. Of these two determinants,

ozone concentration is by far the most important.

Operationally, this may mean that peak concentrations are more important than
the duration of exposure in assessing ozone-induced symptoms. For example, brief
exposures to 1.0 ppmv may be more effective in causing “ozone sickness" than
would be sustained exposure to 0.20 ppmv. Furthermore, the effects of exposures
to 1.0 ppmv would not be reversed by subsequent flight segments when there was
ne ozone present; thus, a time-weighted average may not give an accurate index
of exposure without due consideration being given to peak exposures. Federal
Aviation Regulaticns, Part 121.220, limits peak ozone levels to 0.25 ppmv, sea
level equivalent. From the available literature one would conclude that this
level is innocuous to healthy humans for indefinite exposure. The Literature
does not adequately address the effect such a level would have on people with
various diseases, particularly pulmonary diseases. Asthmatics have been exposed
to 0.20 ppmv ozone for 2 h experimentally (22). This dose of ozone caused no
measurabie changes in pulmonary function of asthmatic subjects, but some blood
biochemical changes were ascribed to ozone. No other experiments on humans with
other pulmonary diseases are known.

10



10.

il.

12.

13.

REFERENCES

American National Standard Institute (ANSI), American Society for Testing and
Materials (ASTM); Standard Practice for Safety and Health Requirements Relat~
ing to Occupational Exposure to Ozone. Standard Practice ANSI/ASTM E591-77
developed under the jurisdiction of ASTM Committee E-34 on Occupational Health
and Safety Aspects of Materials, Physical and Biological Agents.

Bartlett, D., Jz., C. S. Faulkner, H. Cook, and K. Cook: Effect of Chronic
Ozone Exposure on Lung Elasticity in Young Rats, J. APPL. PHYSICL., 37:92-96,
1977.

Bennett, G.: Ozone Contamination of High Altitude Aircraft Cabins, AEROSP.
MED., 33:969-973, 19€2.

Chaney, S., P. BeWitt, W. Blomquist, K. Muller, F. Bruce, and G. Goldstein:
Biochemical Changes in Humans Upon Exposure to Ozone and Exercise, EPA~600/
1-79-026, Health Effects Research Laboratory, U.S. Environmentel Protection
Agency, Chapel Hill, NC 27514, 1979.

Chow, C. K. and J. J. Kaneko: Influence of Dietary Vitamin E on the Red
Cells of Ozone-Exposed Rats, ENVIRON. RES., 19:49-55, 1979.

Clamann, Hans G.: Physical and Medical Aspects of Ozone, from p. 151,
Physics and Medicine of the Atmosphere and Space, Otis E. Benson, Jr., and
Hubertus Strughold (Eds-), John Wiley and Sons, New York, London, 196C.

Coffin, D. L., D. E. Gardner, G. I. Sidorenko, and M. A. Pinigin: Role of
Time aS a Factor in the Toxicity of Chemical Compounds in Intermittent and
Continuous Exposures. Part TI. Effects of Intermittent Exposure, J. TOX.
ENVIRON. HEALTH, 3:821-828, i977.

Douglas, J. S., G. Curry, and S. A. Geffkin: Superoxide Dismutase and Pul-
monary Ozone Toxicity, LIFE SCIENCES, 20:1187-1192, 1979.

Dumelin, E. E., C. J. Dillard, and A. L. Tappel: Effect of Vitamin E and
Ozone on Pentane and Ethane Expired by Rats, ARCH. ENVIRON. HEALTH, 33:
129-135, 1978.

Dungworth, D. L., W. L. Castleman, C. K. Chow, P. W. Meliick, M. G. Mustafa,
B. Tarkington, and W. S. Tyler: Effect of Ambient Levels of Ozone on
MonkeyS, FED. PROC., 34:1670-1674, 1975.

Folinsbee, L., J. F. Bedi, and S. M. Horvath: Respiratory Responses in
Humans Repeatedly Exposed to Low Concentrations of Ozone, AM. REV. RESP.
DIS., 121:431-439, 1980.

Freeman, Gustave, Robert J. Stephens, David L. Coffin, and Jerry Stara:
Changes in Dogs’ Lungs Aiter Long-Term Exposure to ‘zone, ARCH. ENVIRON.
HEALTH, 26:209-216, 1973.

Fukase, O., K. Isomura, and H. Watanabe: Effects of Exercise on Mice
Exposed to Ozone, ARCH. ENVIRON. HEALTH, 33:198-201, 1978.

12


Technicel Report Documentation Page

T. Report No. 2. Government Ac

n No. “T 3. Recipient's Catalog No.

FAA-AM~80-16

[4 Tike ond Subtitle 5. Report Date
H September 1980

6. Performing Orgenizetion Code

EFFECTS OF LONG-TERM EXPOSURE TO LCW LEVELS Ol OZONE:

A REVIEW
i 8. Performing Organization Repor: No.
1 7. Author's}

C. E. Melton

9. Performing Organ: zation Name ond Address 10. Work Unt No. (TRAIS)

FAA Civil Aeromedical institute
P.O. Box 25082
| Oklahoma City, Oklahoma 73125

2. Contract or Gront No.

13. “Type of Report and Perice Covered

112. Sconsering Agency Nome ord Addvess

j Office iation Medici:
fic of Aviatio Medicine — OAM Report

Federai Aviation Administration

860 Independence Avenue, S.W. 14. Sponsoring Agency Code
Washington, D.C. 20591

15. Supplementary Notes

| Work was done under Task No. ATM“-E~-80-PHY~124.

{ 16. Abstract

{ Available literature regarding long-term effects of czone on animals and humans is
reviewed. Emphasis is placed on reports that have appeared since 1976, tut some
earlier reports are cited for completeness and perspective.

This review shows that ozone concentration is more imrortant than is duration of
exposure ia determining the effectiveness of an ozone exposure (dose). This con-
clusion cails into question the validity of the Time-Weighted Average (TWA) as an
index of severity of ozone exposure. The literature review further reveals that
there is wide variation in susceptibility of different animal species to ozone,
making it difficult te apply results of animal experiments to humans. It further
appears that a dose of ozone that is acucely innocuous is also innocucus over the
long term.

j The effects of a symptom-producing dose of ozone are initially cumulative for the
| first two or three exposures, then an adaptive response may ensue that involves a
plateau of response or even a reversal. These effects are shown by both animals
and humans. The mechanisms are unknown.

Ozone probably causes damage by free radical formation. Free radical scavengers

such as vitamins £ and C may provide protection against ozone damage.

PU7. Key Words ] 18. Orstribution Statement

i {Document is available to the public through
Ozone toxicity }the National Technical Information Service,
Chronic effects of ozone jSpringfield, Virginia 22161

Ozone in aviation |

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for its content or use thereof.


ese observations
Rresholé ozone level F. obably detween
(ii) Once the threshold level is exceeded, impediment to

2i bronchioles ensees quickly—orobably in a matter oF minutes.
cumla’

on: it has been known for many years that the magnitude of the
te ozone is not linear with respect to time, but that a response

eau develops after the first few exposures. This phenomenon has been var-
ously called “adaptation,” "adjustment," “tolerance,”
occurs in animals as well as humans.

" or “resistance.” It

Hackmey et al. (18) have studied the phenomenon of adaptation in four
resicents of Southern California and four residents of Toronto ané Hamilton,
Ontario, Canada, thus providing < comparison suscepti ty in smog--~-rosed
and nonexposed people. Subjects were exposed tc purified air and, on a.other
eccasion, to 0.37 ppmy ozone in an experimental chamber. Exposure lasted 2 h

curing ich they exercised haif the time at a level that produced a doubling of
ventilation. Forced expiratery measaremenis were made every 30 mia during expo-

sure and other measurements were made ee: Sur2 and postexpesure. Two of the
Canadian subjects developed clinical ess during exposure with cough, sud-

Stermmal discomfort and upper 2: y irritation. The other two Canadians showed
ation; one eSe also complained of chest discomfort. Two
iS reported tation; @ other two reported no
rced expiratcry parameters were not Significantiy
EVC, and tot.

Zit

>
howed neo changes. Canadians also showed increased R5C flagili vasa
result of ozone exposure, whereas Califorziams did aot. These experiments indi-
erence in suscept to exposure te a low level of ozone between
ans and nonadapted Canadians.

Five of the subjects showed cumulative effec
expiratory parameters for the
values near control values. Cne sv
changes from control throughevt the experiment.

Bougi Curry, and G
expesing mice to a “low concentration of ozon
exposing them together with controls to a lett
ppv). Ali animals showed effects of the high écse
conditioned controis died wrile only 30 percent of

of ozone (36.9
1

succunt thors hypothesized that induction a!
DY pretreatment with a now al dose of ozone =

the subsequent dose oF ozone, the toxicity of
Superoxide radical, No such relationship was



Animal experiments point to increased susceptibility to streptococcal
infection during ozone exposure. Thus, one might expect to find a higher rate
of illness in people chronicaliy exposed to ozone, but Bennett's experiment (3)
on human subjects actually points to the contrary conclusion--that ozone pro~
tects against upper respiratory infection.

It is difficult, if ust impossible, to draw meaningful conclusicns from
animal experiments about hazards to humans of long-term exposure to ozone.
Laboratory animals differ both quantitatively and qualitatively from humans
in their responses to ozone.

Repeated daily exposure to ozone causes 2 peculiar biphasic response. For
the first day or so there is a so-called cumulative effect, a worsening of the
response. Then a stabilization period, or even a reversal, occurs when the
individual “adapts" to ozone. The mechanism involved in these responses is
completely unknown. It could be that the rate of repair increases, that the
induction of some unidentified enzyme occurs. or that damage merely attains a
steady state with repair. These phenomena show considerable variability and
occur in animals as well as in humans. Apparently, the adaptation is per-
manent and would tend to lessen the hazard of chronic ozone exposure to
aircrewmembers.

The consensus favors the free radical mechanism of ozone toxicity. Investi-
gations into the effectiveness of free radical scavengers in mitigating ozone
toxicity have not been as extensive as one could wish; however, it seems clear
from animal experiments that vitamin E is effective in reducing mortality from
high doses of ozone. It seems to be a safe assumption that vitamin E would like-
wise reduce the toxicity of low doses of ozone and help prevent cumulative
effects. Some of the individual variability in susceptibility to ozone may be
based on dietary content of free radical scavengers.

11