### Draft

– Do Not Cite or Quote 7

- 1 June 2012 

## 7 INTEGRATED HEALTH EF FECTS OF LONG

- TERM OZONE
EXPOSURE

7.1 Introduction

This chapter reviews, summarizes, and integrates the evidence on relationships between 

### 1

health effects and long

- term exposures to O 3 . Both epidemiologic and toxicological 

### 2

studies provide a b asis for examining long

- term O 3 exposure health effects for respiratory 

### 3

effects, cardiovascular effects, reproductive and developmental effects, central nervous 

### 4

system effects, cancer outcomes, and mortality. Long

- term exposure has been defined as 

### 5

a durati on of approximately 30 days (1 month) or longer 1 . However, in order to 

### 6

characterize the weight of evidence for the effects of O 3 on reproductive and 

### 7

developmental effects in a consistent, cohesive and integrated manner, results from both 

### 8

short

- term and lon g

- term expos ure periods are included in that section

, and are identified 

### 9

accordingly in the text and tables

. 

### 10

Conclusions from the 2006 O 3 AQCD ( U.S. EPA, 2006b ) are summarized briefly at the 

### 11

beginning of each section, and the evaluation of evidence from recent studies builds upon 

### 12

what was available during the previo us review. For each health outcome (e.g., respiratory 

### 13

disease, lung function), results are summarized for studies from the specific scientific 

### 14

discipline, i.e., epidemiologic and toxicological studies. The major sections 

### 15

(i.e., respiratory, cardiovascular, mortality, reproductive/developmental, cancer) conclude 

### 16

with summaries of the evidence for the various health outcomes within that category and 

### 17

integration of the findings that lead to conclusions regarding causality based upon the 

### 18

framework described in the Preamble to this ISA. Determination of causality is made for 

### 19

the overall health effect category, such as respiratory effects, with coherence and 

### 20

plausibility being based on evidence from across disciplines and also across the suite of 

### 21

related health ou tcomes, including cause

- specific mortality. 

### 22

As mentioned in Chapter 2 (Section 2.3 ), epidemiologic studies generally present O 3 - 

### 23

related effect estimates for mortality and morbidity health outcomes based on an 

### 24

incremental change in exposure. Studies traditionally present the relative risk per an 

### 25

incremental change equal to the interquartile range in O 3 concentrations or some other 

### 26

arbitrary value (e.g., 10 ppb). Additionally, various exposure metrics are used in O 3 

### 27

epidemiologic studies, with the three most common being the m aximum 1

- h average 

### 28

within a 24

- h period (1

- h max), the maximum 8

- h average within a 24

- h period 

### 29

(8

- h max), and 24

- h average (24

- h avg). For the purpose of presenting results from 

### 30

1 Unless otherwise specified, the term “chronic” generally refers to an annual exposure duration for epidemiology studies and a duration of greater than 10% of the lifespan of the animal in toxicological studies.

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- 2 June 2012 studies that use different exposure metrics, EPA consistently applies the sam e O 3 

### 1

increments to facilitate comparisons between the results of various studies that may 

### 2

present results for different incremental changes. Differences due to the use of varying 

### 3

exposure metrics (e.g., 1

- h max, 24

- h avg) become less apparent when averaged across 

### 4

longer exposure periods, because levels are typically lower and less variable. As such, 

### 5

throughout this chapter an increment of 10 ppb was consistently applied across studies, 

### 6

regardless of exposure metric, to facilitate comparisons between the res ults from these 

### 7

studies. 

### 8



## 7.2 Respiratory Effects

Studies reviewed in the 2006 O 3 AQCD examined evidence for relationships between 

### 9

long

- term O 3 exposure (several months to yearly) and effects on respiratory health 

### 10

outcomes including declines in lung function, i ncreases in inflammation, and 

### 11

development of asthma in children and adults. Animal toxicology data provided a clearer 

### 12

picture indicating that long

- term O 3 exposure may have lasting effects. Chronic exposure 

### 13

studies in animals have reported biochemical and morphological changes suggestive of 

### 14

irreversible long

- term O 3 impacts on the lung. In contrast to supportive evidence from 

### 15

chronic animal studies, the epidemiologic studies on longer

- term (annual) lung function 

### 16

declines, inflammation, and new asthma develo pment remained inconclusive. 

### 17

Several studies reviewed in the 2006 O 3 AQCD ( Horak et al., 2002 ; Frischer et al., 1999 ) 

### 18

collectively indicated that O 3 exposure averaged over several summer months was 

### 19

associated with smaller increases in lung function growth in children. For longer 

### 20

averaging periods (annual), the definitive analysis in the Children’s Healt h Study (CHS) 

### 21

reported by Gauderman et al. (2004 ) provided little evidence that such long

- term 

### 22

exposure to ambient O 3 was associated with significa nt deficits in the growth rate of lung 

### 23

function in children in contrast to the effects observed with other pollutants such as acid 

### 24

vapor, NO 2 , and PM 2.5 . Limited epidemiologic research examined the relationship 

### 25

between long

- term O 3 exposures and inflammation. Consistent with evidence of 

### 26

inflammation and allergic responses reported in experim ental studies, an association 

### 27

between 30

- day average O 3 and increased eosinophil levels was observed in an Austrian 

### 28

study ( Frischer et al., 2001 ) . The cross

- s ectional studies available for the 2006 O 3 AQCD 

### 29

detected no associations between long

- term O 3 exposures and asthma prevalence, asthma



### 30

related symptoms or allergy to common aeroallergens in children after controlling for 

### 31

covariates. However, longitudinal st udies provided evidence that long

- term O 3 exposure 

### 32

influences the risk of asthma development in children ( McConnell et al., 2002 ) and adults 

### 33

( McDonnell et al., 1999a ; Greer et al., 1993 )

. 

### 34

### Draft

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- 3 June 2012 New evidence presented below reports interactions between genetic v ariants and long



### 1

term O 3 exposure in effects on new onset asthma in U.S. cohorts in multi

- community 

### 2

studies where protection by specific oxidant gene variants was restricted to children 

### 3

living in low O 3 communities. Related studies report coherent relation ships between 

### 4

respiratory symptoms among asthmatics and long

- term O 3 exposure. Short

- term exposure 

### 5

to O 3 is associated with increases in respiratory symptoms and asthma medication use in 

### 6

children with asthma (Section 6.2.4.1 ) and asthma hospitalizations in children 

### 7

(Section 6.2.7.2 ). A new line of evidence reports a positive concentration

- response 

### 8

relationship between first asthma hospitalization and long

- term O 3 exposure. Results 

### 9

from recent studies examining pulmonary function, inflammation, and allergic re sponses 

### 10

are also presented. 

### 11

7.2.1 Asthma

7.2.1.1 New Onset Asthma

Asthma is a heterogeneous disease with a high degree of temporal variability. Its 

### 12

progression and symptoms can vary within an individual’s experience over time. The 

### 13

course of asthma may vary markedly bet ween young children, older children and 

### 14

adolescents, and adults. This variation is probably more dependent on age than on 

### 15

symptoms ( NHL BI, 2007 ) . Longitudinal cohort studies have examined associations 

### 16

between long

- term O 3 exposures and the onset of asthma in adults and children 

### 17

( McConnell et al., 2002 ; McDonnell et al., 1999a ; Greer et al., 1993 ) , with results 

### 18

indicating a direct effect of long

- term O 3 exposure on asthma risk in adults and effect 

### 19

m odification by O 3 in children. 

### 20

Associations between long

- term O 3 exposure and new cases of asthma were reported in a 

### 21

cohort of nonsmoking adults in California ( McDonnell et al., 1999a ; Greer et al., 1993 )

. 

### 22

The Adventist Health and Smog (AHSMOG) study cohort of 3,914 (age 27 to 87 years, 

### 23

36% male) was drawn from nonsmoking, non

- Hispanic white California Seventh Day 

### 24

Adventists, who wer e surveyed in 1977, 1987, and 1992. To be eligible, subjects had to 

### 25

have lived 10 or more years within 5 miles of their current residence in 1977. Residences 

### 26

from 1977 onward were followed and linked in time and space to interpolate 

### 27

concentrations of O 3 , P M 10 , SO 4 2

- , SO 2 , and NO 2 . New asthma cases were defined as self



### 28

reported doctor

- diagnosed asthma at either the 1987 or 1992 follow

- up questionnaire 

### 29

among those who had not reported having asthma upon enrollment in 1977. During the 

### 30

10

- year follow

- up (1977 t o 1987), the incidence of new asthma was 2.1% for males and 

### 31

2.2% for females ( Greer et al., 1993 ) . Ozone concentration data were not provided. A 

### 32

### Draft

– Do Not Cite or Quote 7

- 4 June 2012 relative risk of 3.12 (95% CI: 1.16, 5.85) per 10

- ppb increase in annual mean O 3 

### 1

(exposure metric not stated) was observed in males, compared to a relative risk of 0.94 

### 2

(95% CI: 0.65, 1.34) in females. 

### 3

In the 15

- year follow

- up study (1977

- 1992), 3.2% of the eligible males and a slightly 

### 4

greater 4.3% o f the eligible females developed adult asthma ( McDonnell et al., 1999a )

. 

### 5

The mean 20

- year average (1973

- 1992) for 8

- h avg O 3 (9 a.m. to 5 p.m.) was 46.5 ppb 

### 6

(SD 15.3). For males, the relative risk of developing asthma was 1.31 (95% CI: 1.01, 

### 7

1.71) per 10

- ppb increase in 8

- h avg O 3 . O nce again, there was no evidence of a positive 

### 8

association between O 3 and new

- onset asthma in females (relative risk of 0.94 [95% CI: 

### 9

0.87, 1.02]). The lack of an association does not necessarily indicate no effect of O 3 on 

### 10

the development of asthma among females. For example, differences between females 

### 11

and males in time

- activity patterns may influence relative exposures to ambient O 3

. 

### 12

During summer 1992, the mean (SD) hours per week spent outdoors for male and female 

### 13

asthma cases were 13.8 (10.6) and 11.4 (10.9), respectively, indicating potential greater 

### 14

misclassification of exposure in females. None of the other pollutants (PM 10 , SO 4 2

- , SO 2 , 

### 15

and NO 2 ) were associated with development of asthma in either males or females. 

### 16

Adjusting for copollutants did not diminish the association between O 3 and asthma 

### 17

incidence for males. In no case was the O 3 coefficient reduced by more than 10% in the 

### 18

two

- pollutant models compared to the model containing O 3 alone. The consistency of the 

### 19

results in the two studies with di fferent follow

- up times, as well as the independent and 

### 20

robust association between annual mean O 3 concentrations and asthma incidence, provide 

### 21

supportive evidence that long

- term O 3 exposure may be associated with the development 

### 22

of asthma in adult males. H owever, because the AHSMOG cohort was drawn from a 

### 23

narrow subject definition, the representativeness of this cohort to the general U.S. 

### 24

population may be limited. 

### 25

In children, the relationship between long

- term O 3 exposure and new onset asthma has 

### 26

been ext ensively investigated in the CHS. In this cohort, evidence provides stronger 

### 27

support for long

- term O 3 exposure modifying the risk of new onset asthma associated 

### 28

with other potential risk factors than having a main effect on new onset asthma. Initiated 

### 29

in t he early 1990s, the CHS was originally designed to examine whether long

- term 

### 30

exposure to ambient pollutants was related to chronic respiratory outcomes in children in 

### 31

12 communities in southern California ( Peters et al., 1999b ; Peters et al., 1999a ) . New



### 32

onset asthma was classified as having no prior history of asthma at study entry with 

### 33

subsequent report of physician

- diagnosed asthma at follow

- up with the date of onset 

### 34

assigned to be the midpoint of the interval between the interview date when asthma 

### 35

diagnosis was first reported and the previous interview date. In a cohort recruited during 

### 36

2002

- 2003 and followed for three years beginning in kindergarten or first grade, 

### 37

McConnell et al. (2010 ) reported a hazard ratio for new onset asthma of 0.76 (95% CI: 

### 38

### Draft

– Do Not Cite or Quote 7

- 5 June 2012 0.38, 1.54) comparing the commu nities with the highest (59.8 ppb) and lowest (29.5 ppb) 

### 1

annual average of 8

- h avg (10 a.m.

- 6 p.m.) O 3 . With adjustment for school and residential 

### 2

modeled non

- freeway traffic

- related exposure, the estimated HR for O 3 was 1.01 

### 3

(95% CI: 0.49, 2.11). 

### 4

Similarl y in a cohort recruited in 1993, asthma risk was not higher for residents of the six 

### 5

high

- O 3 communities versus residents of the six low

- O 3 communities ( McConnell et al., 

### 6

2002 ) . In this study, 3,535 initially nonasthmatic children (ages 9 to 16 years at 

### 7

enrollment) were followed for up to 5 years, during which 265 cases of new

- onset asthma 

### 8

were identified. Communities were stratified by 4

- year average 1

- h max O 3 levels, with 

### 9

six high

- O 3 communities (mean 75.4 ppb [SD 6.8]) and six low

- O 3 communities (mean 

### 10

50.1 ppb [SD 11.0]). Within the high

- O 3 communities, asthma risk was 3.3 (95% CI: 1.9, 

### 11

5.8) times greater for children who played three or more sports as compared with children 

### 12

who played no sports. None of the children who lived in high

- O 3 communities and played 

### 13

three or more sports had a fami ly history of asthma. In models with individual sports 

### 14

entered as dummy variables, only tennis was significa ntly associated with asthma and 

### 15

only in the high O 3 communities. This association was absent in the low

- O 3 communities 

### 16

(relative risk of 0.8 [95% CI : 0.4, 1.6]). The overall observed pattern of effects of sports 

### 17

participation on asthma risk was robust to adjustment for SES, history of allergy, family 

### 18

history of asthma, insurance, maternal smoking, and BMI. 

### 19

Analyses aimed at distinguishing the effects of O 3 from effects of other pollutants 

### 20

indicated that in communities with high O 3 and low levels of other pollutants there was a 

### 21

4.2

- fold (95% CI: 1.6, 10.7) increased risk of asthma in children playing three or more 

### 22

sports, compared to children who played no sports. The relative risk in children playing 

### 23

three or more sports was slightly lower (3.3 [95% CI: 1.6, 6.9]) in communities with a 

### 24

combination of high levels of O 3 and other pollutants. Ozone concentrations were not 

### 25

strongly correlated with PM 10 , PM 2 .5 , NO 2 , or inorganic acid vapors, and no associations 

### 26

with asthma were found for these other pollutants. These results provide additional 

### 27

support that the effects of physical activity on asthma are modified by long

- term 

### 28

O 3 exposure. Overall, the results f rom McConnell et al. (2002 ) suggest that playing spo rts 

### 29

may indicate greater outdoor activity when O 3 levels are higher and an increased 

### 30

ventilation rate, which may lead to increased O 3 exposure. It should be noted, however, 

### 31

that these findings were based on a small number of new asthma cases (n = 29 among 

### 32

children who played three or more sports) and were not based on a priori hypotheses. 

### 33

Recent studies from the CHS provide evidence for gene

- environment interactions in 

### 34

effects on new

- onset asthma by indicating that the lower risks associated with specific 

### 35

genetic variants are found in children who live in lower O 3 communities ( Islam et al., 

### 36

2009 ; Islam et al., 2008 ; Oryszczyn et al., 2007 ; Lee et al., 2004b ; Gilliland et al., 2002 )

. 

### 37

### Draft

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- 6 June 2012 Risk for new

- onset asthma is related in part to genetic susceptibility, behavioral factors 

### 1

and environmental exposure ( Gilliland et al., 1999 ) . Gene

- environment interactions in 

### 2

asthma have been well discussed in the literature ( von Mutius, 2009 ; Holgate et al., 2007 ; 

### 3

Martinez, 2007a

, b ; Rahman et al., 2006 ; Hoffjan et al., 2005 ; Kleeberger and Peden, 

### 4

2005 ; Ober, 2005 ) . Complex chronic diseases, such as asthma, are partially the result of a 

### 5

sequence of biochemical reactions involving exposures to various environmental agents 

### 6

metabolized by a number of different genes ( Conti et al., 2003 ) . Oxidative stress has been 

### 7

proposed to underlie these mechanistic hypotheses ( Gilliland et al., 1999 ) . Genetic 

### 8

variants may impact disease risk directly or modif y disease risk by affecting internal dose 

### 9

of pollutants and other environmental agents and/or their reaction products or by altering 

### 10

cellular and molecular modes of action. Understanding the relation between genetic 

### 11

polymorphisms and environmental exposure can help identify high

- risk subgroups in the 

### 12

population and provide better insight into pathway mechanisms for these complex 

### 13

diseases. 

### 14

CHS analyses have found that asthma risk is related to interactions between O 3 and 

### 15

variants in genes for enzymes such a s heme

- oxygenase (HO

- 1), arginases (ARG1 and 2), 

### 16

and glutathione S transferase P1 (GSTP1) ( Himes et al., 2009 ; Islam et al., 2008 ; Li et al., 

### 17

2008 ; Hanene et al., 2007 ; Ercan et al., 2006 ; Li et al., 2006a ; Tamer et al., 2004 ; 

### 18

Gilliland et al., 2002 ) . Biological plausibility for these findings is provided by evidence 

### 19

that these enzymes have antioxidant and/or anti

- inflammatory activity and participate in 

### 20

well recognized modes of action in asthma pathogenesis. Further, several lines of 

### 21

evidence demonstrate that secondary oxidation products of O 3 initiate the key modes of 

### 22

action th at mediate downstream health effects (Section 5.3.2 ). For example, HO

- 1 has 

### 23

been found to respond rapidly to oxidants, have anti

- inflammatory and a nti

- oxidant 

### 24

effects ( Exner et al., 2004 ) , relax airw ay smooth muscle, and be induced in airways 

### 25

during asthma ( Carter et al., 2004 ) . The GSTP1 Val/Val genotype has been associated 

### 26

with increased risk of havi ng atopic asthma ( Tamer et al., 2004 ) . Gene

- environment 

### 27

interactions are discussed in greater detail in Section 5.4.2.1

. 

### 28

Islam et al. (2008 ) found that functional polymorphisms of the heme oxygenase

- 1 gene 

### 29

(HMOX

- 1, [(GT)n repeat]) influenced the risk of new

- onset asthma, depending on 

### 30

ethnicity and long

- term community O 3 concentrations. Ozone

- gene interactions were not 

### 31

found for variants in other antioxidant genes: catalase (CAT [

- 262C >T

- 844C >T0]) or 

### 32

and manganese superoxide dismu tase (MNSOD, [Ala

- 9Val]). Analyses were restricted to 

### 33

children of Hispanic (n = 576) or non

- Hispanic white ethnicity (n = 1,125) and were 

### 34

conducted with long

- term pollutant levels averaged from 1994 to 2003. The effect of 

### 35

ambient air pollution on the relat ionship between genetic polymorphism and new

- onset 

### 36

asthma was assessed using Cox proportional hazard regression models where the 

### 37

community specific average air pollution levels were fitted as a continuous variable 

### 38

### Draft

– Do Not Cite or Quote 7

- 7 June 2012 together with the appropriate interaction terms for genes and air pollutants and a random 

### 1

effect of community ( Berhane et al., 2004 )

. 

### 2

Over the follow

- up period, 160 new cases of asthma were diagnosed ( Islam et al., 2008 )

. 

### 3

For HMOX

- 1, the interaction (p = 0.003) indicated a greater protective ef fect of the 

### 4

S

- allele (short, <23 (GT)n repeats) compared to the L

- allele (long, >23 repeats) among 

### 5

non

- Hispanic white children who lived in the low O 3 community (nonparallelism 

### 6

presented in Figure 7

- 1 ). Among children residing in low

- O 3 communities, the hazard 

### 7

ratio (HR) of new onset asthma associated with the S

- allele was 0.44 (95% CI: 0.23, 

### 8

0.83) compared to non

- Hispanic white children who lived in lo w O 3 communities and had 

### 9

no S

- alleles. Biological plausibility for these results is provided by evidence that the 

### 10

S

- allele variant of HMOX

- 1 is more readily induced than those with more numerous 

### 11

repeats. The S

- allele was found to have a less protective eff ect in non

- Hispanic white 

### 12

children who resided in high O 3 communities (HR = 0.88; [95% CI: 0.33, 2.34] compared 

### 13

to non

- Hispanic white children in low O 3 communities with no S

- allele). Because 

### 14

HMOX

- 1 variants were not associated with asthma risk in Hispanic children, effect 

### 15

modification by O 3 was not investigated. No significa nt interactions were observed 

### 16

between PM 10 or other pollutants and the HMOX

- 1 gene ; quantitative results were not 

### 17

presented. Average O 3 levels showed low correlation with the other moni tored pollutants. 

### 18

The authors did not consider the lack of adjustment for multiple testing to be a concern in 

### 19

this analysis because the selection of the genes was based on a priori hypotheses defined 

### 20

by a well

- studied biological pathway, in which oxidative stress serves as the link among 

### 21

O 3 exposure, enzyme activity, and asthma. 

### 22

Collectively, results from Islam et al. (2008 ) indicate that a variant in HMOX

- 1 that 

### 23

produces a more readily inducible enzyme is associated with lower risk of new

- onset 

### 24

asthma in children who live in low O 3 communities. Results were not presented for the 

### 25

main effects relating new

- onset asthma to O 3 e xposure. However, they do indicate that 

### 26

that in environments of low ambient O 3 , enzymes with greater antioxidative activity may 

### 27

have the capacity to counter any temporary imbalance in an oxidant

- antioxidant 

### 28

relationship. However, in the presence of high ba ckground O 3 , the protective effect may 

### 29

be attenuated because with higher exposure to oxidants, the antioxidant genes may be at 

### 30

their maximal level of inducibility, and variation in promoters no longer affects levels of 

### 31

expression. Supporting evidence is pr ovided by Schroer et al. (2009 ) , who found that 

### 32

infants with multiple environmental exposures were at increased risk of wheeze 

### 33

regardless of variant in GSTP1, which encodes a gene with antioxidant activity. 

### 34

### Draft

– Do Not Cite or Quote 7

- 8 June 2012 Note: An interaction p

- value of 0.003 was obtained from the hierarchical two stage Cox proportional hazard model fitting the community specific O 3 and controlling for random effect of the communities. The interaction indicates there is a greater protective effect of having a heme

- oxygenase S

- allele compared to having the L

- allele among children living in communities with lower long

term ambient ozon e concentrations. The HRs are off

- set as opposed to overlapping in the figure to allow clearer presentation of the
results.
Source: Developed by EPA with data from Islam et al. (2008 ) (data used with permission of American Thoracic Society).



### Figure 7

- 1 Interaction of heme

- oxygenase genetic variants and O 3 

### level on the Hazard Ratio (HR) of new

- onset asthma in the 12 Children’s Health Study communities.

Expanding on the results of McConnell et al. (2002 )

, Islam et al. (2009 ) provided 

### 1

evidence that variants in GSTM1 and GSTP1 may influence associations between 

### 2

outdoor exercise and new onset asthma. A primary conclusion that the authors ( Islam et 

### 3

al., 2009 ) reported was that the GSTP1 Ile/Ile and GSTM1 null genotypes increased risk 

### 4

of new onset asthma during adolescence. The highest risk was found for participation in 

### 5

three or more team sports (compared to no sports) among children with GSTP1 Ile/Ile 

### 6

genotype living in high

- O 3 communities (HR: 6.15, [95% CI: 2.2, 7.4]). No three

- way 

### 7

interaction was found for GSTM1. These results demonstrate the potenti al importance of 

### 8

a combination of genetic variability, O 3 exposure, and outdoor activity on asthma risk. It 

### 9

is important to note that while some studies have found a modifying role of air pollution 

### 10 Interaction of Gene presence and Ozone Level on the Hazard Ratio of New Onset Asthma (P-value of 0.003)

0.94 0.88 1

0.44

0 0.5

1 1.5

2 2.5

3

Community Mean Ozone LevelHR

Children with no S-Allele

Children with S-Allele

Low (38.4 ppb) High (55.2 ppb) (2.43) (2.34)

(0.36) (0.33) (0.83)

(0.28)Reference

(Confidence limits based on comparison with reference group)

### Draft

– Do Not Cite or Quote 7

- 9 June 2012 on the association between GSTP1 Ile/Ile and asthma in chi ldren ( Lee et al., 2004b )

, 

### 1

others have found that the GSTP1 Val/Val variant to be associated with greater asthma 

### 2

prevalence and increase the risk of O 3 - associated respi ratory morbidity (see discussion in 

### 3

Section 6.2.4.1 ). 

### 4

The CHS also provided evidence of interactions between O 3 exposure and variants in 

### 5

genes for arginase ( Salam et al., 2009 ) . Arginase catalyzes the conversion of L

- arginine. 

### 6

Because L

- arginine is a precursor of NO, higher arginase activity can limit production of 

### 7

NO and subsequent nitrosative stress. Epidemiologic evidence of associati ons of arginase 

### 8

variants with asthma are limited ( Li et al., 2006a ) ; however, asthmatic subjects have been 

### 9

found to have higher arginase activity than non

- asthmatic subjects ( Morris et al., 2004 )

. 

### 10

The modifying effect of O 3 and atopy on the association between ARG1 and ARG2 

### 11

haplotypes and asthma were evaluated using likelihood ratio tests with appropriate 

### 12

interaction terms. Having more copies of the ARG1h4 haplotype (compared to having 

### 13

zero copies) was associated with lower odds of asthma, particularly among children with 

### 14

atopic asthma living in high O 3 communities (OR: 0.12; [95% CI: 0.04, 0.43]). Having 

### 15

more copies of the ARG2h3 haplotype (compared to having zero copies) was associated 

### 16

with increased risk of childhood

- onset asthma among children in both low and high O 3 

### 17

communities. The implications of findings are somewhat limited because the functional 

### 18

r elevance of the ARG1 and ARG2 variants is not clear. 

### 19

7.2.1.2 Prevalence of Asthma and Asthma Symptoms

Some cross

- sectional studies reviewed in the 2006 O 3 AQCD observed positive 

### 20

relationships between chronic exposure to O 3 and prevalence of asthma and asthmatic 

### 21

sy mptoms in school children ( Ramadour et al., 2000 ; Wang et al., 1999 ) while others 

### 22

( Kuo et al., 2002 ; Charpin et al., 1999 ) did not. Recent studies provide additional 

### 23

evidence. 

### 24

In a cross

- sectional nationwide study of 32,672 Taiwanese school children, Hwang et al. 

### 25

(2005 ) assessed the effects of air pollutants on the risk of asthma. The study population 

### 26

was recruited from element ary and middle schools within 1 km of air monitoring stations. 

### 27

The risk of asthma was related to O 3 in the one

- pollutant model. The addition of other 

### 28

pollutants (NO X , CO 2 , SO 2 , and PM 10 ), in two

- pollutant and three

- pollutant models, 

### 29

increased the O 3 risk e stimates. The prevalence of childhood asthma was assessed in 

### 30

Portugal by contrasting the risk of asthma between a high O 3 rural area and an area with 

### 31

low O 3 levels ( Sousa et al., 2011 ; Sousa et al., 2009 ; Sousa et al., 2008 ) . The locations 

### 32

were selected to provide a difference in O 3 levels without the confounding effects of 

### 33

other pollutants. Both evaluation for asthma symptoms and FEV 1 suggested that O 3 

### 34

### Draft

– Do Not Cite or Quote 7

- 10 June 2012 increased asthma prevalence. Clark et al. (2010 ) investigated the effect of exposure to 

### 1

ambient air pollution in utero and during the first year of life on risk of subsequent 

### 2

incidence asthma diagnosis up to 3

- 4 years of age in a population

- based nested case



### 3

control stud y for all children born in southwestern British Columbia in 1999 and 2000 

### 4

(n = 37,401; including 3,482 [9.3%] with asthma). Air pollution exposure for each 

### 5

subject was estimated based on their residential address history using regulatory 

### 6

monitoring data, l and use regression modeling, and proximity to stationary pollutant 

### 7

sources. Daily values from the three closest monitors within 50 km were used to calculate 

### 8

exposures. Traffic

- related pollutants were associated with the highest risk. Ozone was 

### 9

inversely co rrelated with the primary traffic

- related pollutants (r =

- 0.7 to

- 0.9). The low 

### 10

reliability of asthma diagnosis in infants makes this study difficult to interpret ( Martinez 

### 11

et al., 19 95 ) . In a cross

- sectional analysis, Akinbami e t al. (2010 ) examined the 

### 12

association between chronic exposure to outdoor pollutants (12

- month avg levels by 

### 13

county) and asthma outcomes in a national sample of children ages 3

- 17 years living in 

### 14

U.S. metropolitan areas (National Health Interview Survey , N = 34,073). A 5

- ppb 

### 15

increase in estimated 8

- h max O 3 concentration (annual average) yielded a positive 

### 16

association for both currently having asthma and for having at least 1 asthma attack in the 

### 17

previous year, while the adjusted odds ratios for other po llutants were not statistically 

### 18

significa nt. Models in which pollutant value ranges were divided into quartiles produced 

### 19

comparable results. Multi

- pollutant models (SO 2 and PM) produced similar results. The 

### 20

median value for 12

- month avg O 3 levels was 39.5 ppb and the IQR was 35.9

- 43.7 ppb. 

### 21

The adjusted odds for current asthma for the highest quartile (49.9

- 59.5 ppb) of estimated 

### 22

O 3 exposure was 1.56 (95% CI: 1.15, 2.10) with a positive concentration

- response 

### 23

relationship apparent from the lowest quartile to the highest. Thus, this cross

- sectional 

### 24

analysis and Hwang et al. (2005 ) provid es further evidence relating O 3 exposure and the 

### 25

risk of asthma. 

### 26

Relationships between long

- term exposure and respiratory symptoms in asthmatic 

### 27

children also were examined in the CHS. McConnell et al. (1999 ) examined the 

### 28

association between O 3 levels and the prevalence of chronic lower respiratory tract 

### 29

symptoms in 3,676 cohort children with asthma. In this cross

- sectional study, bronchitis, 

### 30

phlegm, and cough were not associated with annual mean 1

- h max O 3 concentrations in 

### 31

children with asthma or wheeze. All other pollutants examined (PM 10 , PM 2.5 , NO 2 , and 

### 32

gaseous acid) were associated with an increase in phlegm but not cough. The mean 

### 33

annual average 1

- h max O 3 concentration was 65.6 ppb (range 35.5 to 97.5) across the 

### 34

12 communities. In anothe r CHS analysis, McConnell et al. (2003 ) evaluated 

### 35

relationships between air pollutants and bronchitic symptoms among 475 children with 

### 36

asthma. The mean 4

- year average 8

- h avg O 3 (10 a.m.

- 6 p.m.) concentration was 

### 37

47.2 ppb (range 28.3 to 65.8) across the 12 communities. For a 10

- ppb increase in 

### 38

8

- h avg O 3 averaged over 4 years, the between

- community odds ratio was 0.90 (95% CI: 

### 39

### Draft

– Do Not Cite or Quote 7

- 11 June 2012 0.82, 1.00) whereas the within

- community (i.e., difference between one

- and four

- year 

### 1

ave rage) odds ratio was larger, i.e., 1.79 (95% CI: 1.00, 3.21). The authors commented 

### 2

that if the larger within

- community effect estimates were correct, then other cross



### 3

sectional (between

- community) studies might have underestimated the true effect of air 

### 4

p ollution on bronchitic symptoms in children. These differences might be attributable to 

### 5

confounding by poorly measured or unmeasured risk factors that vary between 

### 6

communities. Within community effects may more accurately represent risk associated 

### 7

with pol lutant exposure because the analyses characterize health effects associated with 

### 8

changing pollutant concentrations within a community, thereby minimizing potential 

### 9

confounding by factors that are constant over time within a community. PM 2.5 , NO 2 , and 

### 10

organ ic carbon also were associated with bronchitic symptoms. In two

- pollutant models, 

### 11

the within

- community effect estimates for O 3 were markedly reduced and no longer 

### 12

statistically significa nt in some cases. 

### 13

CHS also examined interactions between TNF

- α 308 ge notype and long

- term O 3 

### 14

exposure in the occurrence of bronchitic symptoms among children with asthma ( Lee et 

### 15

al., 2009b ) . Increased airway levels of t he cytokine TNF

- α has been related to 

### 16

inflammation, and the GG genotype has been linked to lower expression of TNF

- α

. 

### 17

Asthmatic children with the GG genotype had a lower prevalence of bronchitic symptoms 

### 18

compared with children carrying at least one A

- allel e (e.g., GA or AA genotype). Low



### 19

versus high

- O 3 strata were defined as less than or greater than 50

- ppb O 3 avg. Asthmatic 

### 20

children with TNF

- 308 GG genotype had a significa ntly reduced risk of bronchitic 

### 21

symptoms with low

- O 3 exposure (OR: 0.53 [95% CI: 0.3 1, 0.91]). The risk was not 

### 22

reduced in children living in high

- O 3 communities (OR: 1.42 [95% CI: 0.75, 2.70]). The 

### 23

difference in genotypic effects between low

- and high

- O 3 environments was statistically 

### 24

significa nt among asthmatics (P for interaction = 0.0 1), but not significa nt among non



### 25

asthmatic children. Figure 7

- 2 presents adjusted O 3 community

- specific regression 

### 26

coefficients plotted against ambient O 3 concentration, using weights proportional to the 

### 27

inverse variance. Investigators further reported no substantial differences in the effect of 

### 28

the GG genotype on bronchitic symptoms by long

- term exposure to PM 10 , PM 2.5 , NO 2 , 

### 29

acid vapor, or second

- hand smoke. 

### 30

### Draft

– Do Not Cite or Quote 7

- 12 June 2012 Note: Using indicator variables for each category of genotype and O 3 exposure, investigators ca lculated effect estimates for TNF

- α GG genotype on the occurrence of bronchitic symptoms among children with asthma.
Source: Reprinted with permission of John Wiley & Sons, ( Lee et al., 2009b )

.



### Figure 7

- 2 Ozone modifies the effect of TNF GG genotype on bronchitic symptoms among children with asthma in the CHS.

Another CHS analyses reported interrelationships between variants in CAT and 

### 1

myeloperoxidase (MPO) genes, ambient pollutants, and respiratory

- related school 

### 2

absences for 1,136 Hispanic and non

- Hispanic white cohort children ( Wenten et al., 

### 3

2009 ) . A related study ( Gilliland et al., 2001 ) , found increased O 3 exposure t o be related 

### 4

greater school absenteeism due to respiratory illness but did not consider genetic variants. 

### 5

Wenten et al. (2009 ) hypothesized that variation in the level or function of ant ioxidant 

### 6

enzymes would modulate respiratory illness risk, especially under high levels of 

### 7

oxidative stress expected from high ambient O 3 exposure. The joint effect of variants in 

### 8

these two genes (genetic epistasis) on respiratory illness was examined becau se the 

### 9

enzyme products operate on the same substrate within the same biological pathway. Risk 

### 10

of respiratory

- related school absences was elevated for children with CAT GG plus MPO 

### 11

GA or AA genotypes (RR: 1.35 [95% CI: 1.03, 1.77] compared to GG for both ge nes) 

### 12

and reduced for children with CAT GA or AA plus MPO GA or AA (RR: 0.81 [95% CI: 

### 13

0.55, 1.19] compared to GG for both genes). Both CAT GG and MPO GA or AA 

### 14

genotypes produce a lower activity enzyme. In analyses that stratified communities into 

### 15

high and l ow O 3 exposure groups by median levels (46.9 ppb), the protective effect of 

### 16

CAT GA or AA plus MPO GA or AA genotype was largely limited to children living in 

### 17

### Draft

– Do Not Cite or Quote 7

- 13 June 2012 communities with high ambient O 3 levels (RR: 0.42 [95% CI: 0.20, 0.89]). The 

### 1

association of respir atory

- illness absences with functional variants in CAT and MPO that 

### 2

differ by air pollution levels illustrates the need to consider genetic epistasis in assessing 

### 3

gene

- environment interactions. 

### 4

Collective evidence from CHS provides an important demonstrati on of gene

- environment 

### 5

interactions. In the complex gene

- environment setting a modifying effect might not be 

### 6

reflected in an exposure main effect. The simultaneous occurrence of main effect and 

### 7

interaction effect can occur. The study of gene

- environment in teractions helps to dissect 

### 8

disease mechanisms in humans by using information on susceptibility genes to focus on 

### 9

the biological pathways that are most relevant to that disease ( Hunter, 2005 )

. 

### 10

The French Epidemiology study on Genetics and Environment of Asthma (EGEA) 

### 11

investigated the relationship between ambient air pollution and asthma severity in a 

### 12

cohort in five French cities (Paris, Lyon, Marseille, Montpellier, and Grenoble) ( Rage et 

### 13

al., 2009b ) . In this cross

- sectional study, asthma severity over the past 12 months was 

### 14

assessed among 328 adult asthmatics using two methods: (1) a four

- class severity score 

### 15

that integrated clinical events and type of treatment; and (2) a five

- level asthma score 

### 16

based only on symptoms. Two measures of exposure were also assess ed: (1) [first 

### 17

method]) closest monitor data from 1991 to 1995 where a total of 93% of the subjects 

### 18

lived within 10 km of a monitor, but where 70% of the O 3 concentrations were 

### 19

back

- extrapolated values; and (2) [second method]) a validated spatial model th at used 

### 20

geostatistical interpolations and then assigned air pollutants to the geocoded residential 

### 21

addresses of all participants and individually assigned exposure to ambient air pollution 

### 22

estimates. Higher asthma severity scores were significa ntly related to both the 8

- h avg O 3 

### 23

during April

- September and the number of days with 8

- h O 3 averages above 55 ppb. Both 

### 24

exposure assessment methods and severity score methods resulted in very similar 

### 25

findings. Effect estimates of O 3 were similar in three

- pollutant m odels. No PM data were 

### 26

available. Since these estimates were not sensitive to the inclusion of ambient NO 2 in the 

### 27

three

- pollutant models, the authors viewed the findings not to be explained by particles 

### 28

which usually have substantial correlations between P M and NO 2 . Effect estimates for O 3 

### 29

in three

- pollutant models including O 3 , SO 2 , and NO 2 yielded OR for O 3 - days of 2.74 

### 30

(95% CI: 1.68, 4.48) per IQR days of 10

- 28 (+18) ppb. The effect estimates for SO 2 and 

### 31

NO 2 in the three

- pollutant model were 1.33 (95% CI : 0.85, 2.11) and 0.94 (95% CI: 0.68, 

### 32

1.29) respectively. Taking into account duration of residence did not change the result. 

### 33

This study suggests that a higher asthma severity score is related to long

- term O 3 

### 34

exposure. 

### 35

An EGEA follow

- up study ( Jacquemin et al., In Press ) , examines the relationship 

### 36

bet ween asthma and O 3 , NO 2 , and PM 10 . New aspects considered include: (1) 

### 37

### Draft

– Do Not Cite or Quote 7

- 14 June 2012 examination of three domains of asthma control (symptoms, exacerbations, and lung 

### 1

function); (2) levels of asthma control (controlled, partially controlled, and uncontrolled 

### 2

asthma); an d (3) PM 10 and multi

- pollutant analysis. In this cross

- sectional analysis, 

### 3

EGEA2 studied 481 adult subjects with current asthma from 2003 to 2007. The IQRs 

### 4

were 11 (41

- 52) μg/m 3 for annual O 3 and 13 (25

- 38) μg/m 3 for summer (April



### 5

September) O 3 . The associ ation between asthma control and air pollutants was expressed 

### 6

by ORs (reported for one IQR of the pollutant), derived from multinomial logistic 

### 7

regression. For each factor, the simultaneous assessment of the risk for uncontrolled 

### 8

asthma and for partly cont rolled asthma was compared with controlled asthma using a 

### 9

composite of the three domains. In crude and adjusted models, O 3 - sum and PM 10 were 

### 10

positively associated with partly controlled and uncontrolled asthma, with a clear gradient 

### 11

from controlled, partly controlled (OR = 1.53, 95% CI: 1.01, 2.33) and uncontrolled 

### 12

(OR = 2.14, 95% CI: 1.34, 3.43) (from the multinomial logistic regression)

. 

### 13

Separately, they used a composite asthma control classification that used the ordinal 

### 14

logistic regression for risk comparing controlled to partly controlled asthma and 

### 15

comparing partly controlled to uncontrolled asthma. For these two pollutants, the ORs 

### 16

assessed using the ordinal logistic regression were significa nt (ORs were 1.69 (95% CI: 

### 17

1.22, 2.34) and 1.35 (95% CI: 1.13, 1.64) for O 3 - sum and PM 10 , respectively). For two 

### 18

pollutant models using the ordinal logistic regression, the adjusted ORs for O 3 - s um and 

### 19

PM 10 included simultaneously in a unique model were 1.50 (95% CI: 1.07, 2.11) for O 3 - 

### 20

sum and 1.28 (95% CI: 1.06, 1.55) for PM 10 , respectively. This result suggests that the 

### 21

effects of both pollutants are independent. 

### 22

The analysis of the association s between air pollution for all asthma subjects and each 

### 23

one of the three asthma control domains showed the following: (1) for lung function 

### 24

defined dichotomously as percent predicted FEV 1 value <or >=80 (OR = 1.35, 95% CI: 

### 25

0.80, 2.28 for adjusted O 3 - sum); (2) for symptoms defined as asthma attacks or dyspnea 

### 26

or woken by asthma attack or shortness of breath in the past three months (OR = 1.59, 

### 27

95% CI: 1.10, 2.30 for adjusted O 3 - sum); and for exacerbations defined at least one 

### 28

hospitalizations or ER visits i n the last year or oral corticosteroids in the past three 

### 29

months (OR = 1.58, 95% CI: 0.97, 2.59 for adjusted O 3 - sum). Since the estimates for 

### 30

both pollutants were more stable and significa nt when using the integrated measure of 

### 31

asthma control, this indicat es that the results are not driven by one domain. These results 

### 32

support an effect of long

- term exposure to O 3 on asthma control in adulthood in subjects 

### 33

with pre

- existing asthma. 

### 34

Goss et al. (2004 ) investigated the effect of O 3 on pulmonary exacerbations and lung 

### 35

function in individuals over the age of 6 years with cystic fibrosis (n = 11,484). The study 

### 36

included patients enrolled in the Cystic Fibrosis Foundation National Patient Registry, 

### 37

### Draft

– Do Not Cite or Quote 7

- 15 June 2012 which contains demographic and clinical data collected annually at accredited centers for 

### 1

cystic fibrosis. For 1999 through 200 0, the annual mean O 3 concentration, calculated 

### 2

from 1

- h averages from 616 monitors in the U.S. EPA Aerometric Information Retrieval 

### 3

System (AIRS), was 51.0 ppb (SD 7.3). Exposure was assessed by linking air pollution 

### 4

values from AIRS with the patient’s ho me ZIP code. No clear association was found 

### 5

between annual mean O 3 and lung function parameters. However, a 10 ppb increase in 

### 6

annual mean O 3 was associated with a 10% (95% CI: 3, 17) increase in the odds of two or 

### 7

more pulmonary exacerbations. Significa nt excess odds of pulmonary exacerbations also 

### 8

were observed with increased annual mean PM 10 and PM 2.5 concentrations. The O 3 effect 

### 9

was robust to adjustment for PM 10 and PM 2.5 , 8% (95% CI: 1, 15) increase in odds of two 

### 10

or more pulmonary exacerbations per 1 0 ppb increase in annual mean O 3

. 

### 11

7.2.2 Asthma Hospital Admissions and ED Visits

The studies on O 3 - related hospital discharges and emergency department (ED) visits for 

### 12

asthma and respiratory disease that were available in the 2006 O 3 AQCD mainly looked 

### 13

at the da ily time metric. Collectively the short

- term O 3 studies presented earlier in 

### 14

Section 6.2.7.5 indicate that there is evidence for increases in both hospital admissions 

### 15

and ED visits related to both all respiratory outcomes and asthma with stronger 

### 16

associations in the warm months. New studies evaluated long

- term O 3 exposure metrics, 

### 17

providing a new line of evidence that suggests a positive exposure

- response relationship 

### 18

between first asthma hospital admission and long

- term O 3 exposure. 

### 19

An ecologic study ( Moore et al., 2008 ) evaluated time trends in associations between 

### 20

declining warm

- season O 3 concentrations and hospitalization for asthma in children in 

### 21

California’s South Coast Air Basin who ranged in age from birth to 19 years. Quarterly 

### 22

average concentrations from 195 spatial grids, 10×10 km, were used. Ozone was the only 

### 23

pollutant associated with increased hospital admissions over the study period. A linear 

### 24

relation was observed for asthma hospital discharges ( Moore et al., 2008 ) . A matched 

### 25

case

- control study ( Karr et al., 2007 ) was conducted of infant bronchiolitis (ICD 9, code 

### 26

466.1) hospitalization and two measures of long

- term pollutant exposure (the month prior 

### 27

to hospitalizatio n and the lifetime average) for O 3 in the South Coast Air Basin of 

### 28

southern California among 18,595 infants born between 1995 and 2000. Ozone was 

### 29

associated with reduced risk in the single

- pollutant model, but this relation did not persist 

### 30

in multi

- polluta nt models (CO, NO 2 , and PM 2.5 ). 

### 31

In a cross

- sectional study, Meng et al. (2010 ) examined associations between air 

### 32

pollution and asthma morbidity in the San Joaquin Valley in California by using the 2001 

### 33

California Health Interview Survey data from subjects ages 1 to 65+ who reported 

### 34

### Draft

– Do Not Cite or Quote 7

- 1 6 June 2012 physician

- diagnosed asthma (n = 1,502). Subjects were assigned annual average 

### 1

concentrations for O 3 based on residential ZIP code and the closet air monitor ing station 

### 2

within 8 km but did not have data on duration of residence. Multi

- pollutant models for O 3 

### 3

and PM did not differ substantially from single

- pollutant estimates, indicating that 

### 4

pollutant multi

- collinearity is not a problem in these analyses. The authors reported 

### 5

increased asthma

- related ED visits or hospitalizations for O 3 (OR = 1.49; [95% CI: 1.05, 

### 6

2.11] per 10 ppb) for all ages. Positive associations were obtained for symptoms, but 

### 7

95% confidence intervals included null values. Associations for symptoms for adults 

### 8

(ages 18 +) were observed (OR = 1.40; [95% CI: 1.02, 1.91] per 10 ppb). 

### 9

Associations between air pollution and poorly controlled asthma among adults in 

### 10

Los Angeles and San Diego Counties were investigated using the California Health 

### 11

In terview Survey data collected between November 2000 and September 2001 ( Meng et 

### 12

al., 2007 ) . Each respondent was assigned an annual average concentration measured at 

### 13

the nearest station within 5 miles of the residential cross

- street intersection. Poorly 

### 14

controlled asthma was defined as having daily or weekly asthma symptoms or at least one 

### 15

ED visit or hospitalization because of asthma during the past 12 mon ths. This cross



### 16

sectional study reports an OR of 3.34 (95% CI: 1.01, 11.09) for poorly controlled asthma 

### 17

when comparing those 65 years of age and older above the 90th percentile (28.7 ppb) 

### 18

level to those below that level. Co

- pollutant (PM) analysis produce d similar results. 

### 19

Evidence associating long

- term O 3 exposure to first asthma hospital admission in a 

### 20

concentration

- response relationship is provided in a retrospective cohort study ( Lin et al., 

### 21

2008b ) . This study investigated the association between chronic exposure to O 3 and 

### 22

childhood asthma admissions (defined as a principal dia gnosis of ICD9, code 493) by 

### 23

following a birth cohort of 1,204,396 eligible births born in New York State during 

### 24

1995

- 1999 to first asthma admission or until 31 December 2000. There were 10,429 

### 25

(0.87%) children admitted to the hospital for asthma between 1 and 6 years of age. The 

### 26

asthma hospitalization rate in New York State in 1993 was 2.87 per 1,000 ( Lin et al., 

### 27

1999 ) . Three annual indicators (all 8

- h max from 10:00 a.m. to 6:00 p.m.) were used to 

### 28

define chronic O 3 exposure: (1) mean concentration during the follow

- up period 

### 29

(41.06 ppb); (2) mean concentration during the O 3 season (50.62 ppb); and (3) proportion 

### 30

of follow

- up days with O 3 levels >70 ppb. In this study the authors aimed to predict the 

### 31

risk of having asthma admissions in a birth cohort, but the time to the first admission in 

### 32

chi ldren that is usually analyzed in survival models was not their primary interest. The 

### 33

effects of co

- pollutants were assessed and controlled for using the Air Quality Index 

### 34

(AQI). Interaction terms were used to assess potential effect modifications. A posit ive 

### 35

association between chronic exposure to O 3 and childhood asthma hospital admissions 

### 36

was observed indicating that children exposed to high O 3 levels over time are more likely 

### 37

to develop asthma severe enough to be admitted to the hospital. The various fa ctors were 

### 38

### Draft

– Do Not Cite or Quote 7

- 17 June 2012 examined and differences were found for younger children (1

- 2 years), poor 

### 1

neighborhoods, Medicaid/self

- paid births, geographic region and others. As shown in 

### 2

Figure 7

- 3 , positive concentration

- response relationships were observed. Asthma 

### 3

admissions were significa ntly associated with increased O 3 levels for all chronic exposure 

### 4

indicators (ORs, 1.16

- 1.68). When estimating the O 3 effect using the exceedance 

### 5

proportion, an increase was observed (OR = 1.68; [95% CI: 1.64, 1.73]) in hospital 

### 6

admissions with an IQR (2.51%) increase in O 3 . A proportional hazards model for the 

### 7

New York City data was run as a sensitivity analysis and it yielded simila r results 

### 8

between asthma admissions and chronic exposure to O 3 (Cox model: HR = 1.14, 

### 9

[95% CI: 1.124, 1.155] is similar to logistic model results: OR = 1.16 [95% CI: 1.15, 

### 10

1.17]) ( Lin, 2010 ) . Thus, this study provides evidence associating long

- term O 3 exposure 

### 11

to first asthma hospital admission in a concentration

- response relationship. 

### 12

In considering relationships between long

- term pollutant exposure and chronic disease 

### 13

health endpoints, Künzli (2012 ) offers two hypotheses relevant to research on air 

### 14

pollution and chronic disease where chronic pathologies are found with acute expressions 

### 15

of the chronic disease: “H1: Exposure provides a basis for the development of the 

### 16

underlying chronic pathology, whic h increases the pool of people with chronic conditions 

### 17

prone to exacerbations; H2: Exposure triggers an acute event (or a state of frailty that 

### 18

results in an event with a delay of a few days or weeks) among those with the disease.” 

### 19

Künzli (2012 ) states if associations of pollutio n with events are much larger in the long



### 20

term studies, it provides some indirect evidence in support of H1. If air pollution 

### 21

increases the pool of subjects with the chronic pathology (H1), more acute events are 

### 22

expected to be seen for higher exposures sin ce events due to various causes are part of the 

### 23

chronic disease pathway. 

### 24

Künzli (2012 ) makes such a comparison noting larger associations with long

- term NO 2 

### 25

exposures for adult asthma hospital admissions ( Andersen et al., 2012 ) as compared to 

### 26

short

- term NO 2 exposures for asthma hospital admissions ( Peel et al., 2005 ) . In a further 

### 27

example, Pope (2007 ) makes similar conclusions comparing long

- term PM mortality 

### 28

study results to short

- term PM mortality studies. The results of Lin et al. (2008b ) for first 

### 29

asthma hospital admission, presented below, show effect estimates that are larger than 

### 30

those reported in a study of asthma hospital admis sions in New York State by Silverman 

### 31

a nd Ito (2010 ) , discussed in Chapter 6 (both studies are for young children). This 

### 32

provides some support for the hypothesis that O 3 exposure may not only have triggered 

### 33

the events but also increased the pool of asthmatics. 

### 34

### Draft

– Do Not Cite or Quote 7

- 18 June 2012 Note : Adjusted for child’s sex, age, birth weight, and gestational age; maternal race, ethnicity, age, education, insurance, and smoking status during pregnancy; and regional poverty level and temperature. ORs by low, medium, and high exposure are shown
for Ne w York City (NYC: low [37.3 ppb], medium [37.3

- 38.11 ppb], high [38.11+ ppb] and other New York State regions (Other NYS regions: low [42.58 ppb], medium [42.58

- 45.06 ppb], high [45.06+ ppb]) for first asthma hospital admission.

Source: Lin (2010 ); Lin et al. (2008b )



### Figure 7

- 3 Ozone

- asthma concentration

- response relationship using the mean
concentration du ring the entire follow

- up period for first asthma hospital admission.

7.2.3 Pulmonary Structure and Function

7.2.3.1 Pulmonary Structure and Function: Evidence from Epidemiology Studies

The definitive 8

- year follow

- up analysis of the first cohort of the CHS, which is 

### 1

di scussed in Section 7.2 ( Gauderman et al., 2004 ) , provided little evidence that long

- term 

### 2

exposure to ambient O 3 was associated with significa nt deficits in the growth rate of lung 

### 3

function in children. A later CH S study ( Islam et al., 2007 ) examined relat ionships 

### 4

between air pollution, lung function, and new onset asthma and reported no substantial 

### 5

differences in the effect of O 3 on lung function. Ozone concentrations from the least to 

### 6

most polluted communities (mean annual average of 8

- h avg O 3 ) ranged fr om 30 to 

### 7

65 ppb, as compared to the ranges observed for the other pollutants, which had 4

- fold

- to 

### 8

8

- fold differences in concentrations. In a more recent CHS study, Breton et al. (2011 ) 

### 9

hypothesized that genetic variation in genes on the glutathione metabolic pathway may 

### 10

### Draft

– Do Not Cite or Quote 7

- 19 June 2012 influence the association between ambient air pollutant exposures and lung function 

### 1

growth in chil dren. They investigated whether genetic variation in glutathione genes 

### 2

GSS, GSR, GCLC, and GCLM was associated with lung function growth in healthy 

### 3

children using data collected on 2,106 children over an 8

- year time

- period as part of the 

### 4

Children’s Health Study. Breton et al. (2011 ) found that variation in the GSS locus was 

### 5

associated with differences in risk of child ren for lung function growth deficits associated 

### 6

with NO 2 , PM 10 , PM 2.5 , elemental carbon, organic carbon, and O 3 . The negative effects of 

### 7

air pollutants were largely observed within participants who had a particular GSS 

### 8

haplotype. The effects ranged from

- 124.2 to

- 149.1 mL for FEV 1 , - 92.9 to

- 126.7 mL for 

### 9

FVC and

- 193.9 to

- 277.9 mL/sec for MMEF for all pollutants except O 3 , for which some 

### 10

positive associations were reported: 25.9 mL for FEV 1 ; 0.1 mL for FVC, and 

### 11

166.5 mL/sec for MMEF. Ozone was associated with larger decreases in lung function in 

### 12

children without this haplotype, when compared to the other pollutants with values of 

### 13

- 76.6 mL for FEV 1 , - 17.2 mL for FVC, and

- 200.3 mL/sec for MME F, but only the 

### 14

association with MMEF was statistically significa nt. 

### 15

As discussed in the 2006 O 3 AQCD, a study of freshman students at the University of 

### 16

California, Berkeley reported that lifetime exposure to O 3 was associated with decreased 

### 17

measures of sm all airways (<2 mm) function (FEF 75 and FEF 25

- 75 ) ( Tager et al., 2005 )

. 

### 18

There was an interaction with the FEF 25

- 75 /FVC r atio, a measure of intrinsic airway size. 

### 19

Subjects with a large ratio (indicating an increased airway size relative to their lung 

### 20

volume) were less likely to have decreases in FEF 75 and FEF 25

- 75 for a given estimated 

### 21

lifetime exposure to O 3

. Kinney and Lippmann (2000 ) examined 72 nonsmoking adults 

### 22

(mean age 20 years) from the second

- year class of students at the U.S. Military Academy 

### 23

in West Point, NY, and reported results that appear to be consistent with a decline in lung 

### 24

function tha t may in part be due to O 3 exposures over a period of several summer months. 

### 25

Ihorst et al. (2004 ) examined 2,153 children with a median ag e of 7.6 years and reported 

### 26

pulmonary function results which indicated that significa ntly lower FVC and FEV 1 

### 27

increases were associated with higher O 3 exposures over the medium

- term of several 

### 28

summer months, but not over several months in the winter. Semi

- a nnual mean O 3 

### 29

concentrations ranged from 22 to 54 ppb during the summer months and 4 to 36 ppb 

### 30

during the winter months. Further, over the longer

- term 3.5

- year period Ihorst et al. 

### 31

(2004 ) found that higher mean summer months O 3 levels were not associated with growth 

### 32

rates in lung function and for FVC and FEV 1 , in contrast to the significa nt medium

- term 

### 33

effects. Frischer et al. (1999 ) found that higher O 3 over one summer season, one winter 

### 34

season, and greater increases from one summer to the next over a three

- year period were 

### 35

associated with smaller increases in lung function growth, indicating both medium and 

### 36

longer

- term effects. 

### 37

### Draft

– Do Not Cite or Quote 7

- 20 June 2012 ( Mortimer et al., 2008a

, b ) examined the association of prenatal and lifetime exposures to 

### 1

air pollutants with pulmonary function and allergen se nsitization in a subset of asthmatic 

### 2

children (ages 6

- 11) included in the Fresno Asthmatic Children’s Environment Study 

### 3

(FACES). Monthly means of pollutant levels for the years 1989

- 2000 were created and 

### 4

averaged separately across several important develop mental time

- periods, including: the 

### 5

entire pregnancy, each trimester, the first 3 years of life, the first 6 years of life, and the 

### 6

entire lifetime. In the first analysis ( Mortimer et al., 2008a ) , negative effects on 

### 7

pulmonary function were found for exposure to PM 10 , NO 2 , and CO during key neonatal 

### 8

and early life developmental peri ods. The authors did not find a negative effect of 

### 9

exposure to O 3 within this cohort. In the second analysis ( Mortimer et al., 2008b )

, 

### 10

sensitization to at least one allergen was associated, in general, with higher levels of CO 

### 11

and PM 10 during the entire pregnancy and second trimester, and higher PM 10 during the 

### 12

first 2 yea rs of life. Lower exposure to O 3 during the entire pregnancy or second trimester 

### 13

was associated with an increased risk of allergen sensitization. Although the pollutant 

### 14

metrics across time periods were correlated, the strongest associations with the outcom es 

### 15

were observed for prenatal exposures. Though it may be difficult to disentangle the effect 

### 16

of prenatal and postnatal exposures, the models from this group of studies suggest that 

### 17

each time period of exposure may contribute independently to different dim ensions of 

### 18

school

- aged children’s pulmonary function. For 4 of the 8 pulmonary

- function measures 

### 19

(FVC, FEV 1 , PEF, FEF 25

- 75 ), prenatal exposures were more influential on pulmonary 

### 20

function than early

- lifetime metrics, while, in contrast, the ratio of measur es (FEV 1 /FVC 

### 21

and FEF 25

- 75 /FVC) were most influenced by postnatal exposures. When lifetime metrics 

### 22

were considered alone, or in combination with the prenatal metrics, the lifetime measures 

### 23

were not associated with any of the outcomes. This suggests that the timing of the O 3 

### 24

exposure may be more important than the overall dose, and prenatal exposures are not 

### 25

just markers for lifetime or current exposures. 

### 26

Latzin et al. (2009 ) examined whether prenatal exposure to air pollution was associated 

### 27

with lung function changes in the newborn. Tidal breathing, lung volume, ventilati on 

### 28

inhomogeneity and eNO were measured in 241 unsedated, sleeping neonates 

### 29

(age = 5 weeks). Consistent with the previous studies, no association was found for 

### 30

prenatal exposure to O 3 and lung function. 

### 31

In a cross

- sectional study of adults, Qian et al. (2005 ) examined the association of long



### 32

term exposure to O 3 and PM 10 with pulmonary function from data of 10,240 middle

- age d 

### 33

subjects who participated in the Atherosclerosis Risk in Communities (ARIC) study in 

### 34

four U.S. communities. A surrogate for long

- term O 3 exposure from daily data was 

### 35

determined at the individual level. Ozone was significa ntly and negatively associated 

### 36

wi th measures of pulmonary function. 

### 37

### Draft

– Do Not Cite or Quote 7

- 21 June 2012 To determine the extent to which long

- term exposure to outdoor air pollution accelerates 

### 1

adult decline in lung function, Forbes et al. (2009b ) studied the association between 

### 2

chronic exposure to outdoor air pollution and lung function in approximately 42,000 

### 3

adults aged 16 and older who were representatively sampled cross

- sectionally from 

### 4

participants in the Health Survey for England (1995, 1996, 1997, and 2001). FEV 1 was 

### 5

not associated with O 3 concentrations. In contrast to the results for PM 10 , NO 2 , and SO 2; 

### 6

combining the results of all the survey years showed that a 5

- ppb difference in O 3 was 

### 7

counter

- intuitively associated with a higher FEV 1 by 22 mL. 

### 8

In a prospective cohort study consisting of school

- age, non

- asthmatic children in 

### 9

Mexico City (n = 3,170) wh o were 8 years of age at the beginning of the study, Rojas



### 10

Martinez et al. (2007 ) evaluated the association between long

- term exposure to O 3 , PM 10 

### 11

and NO 2 and lung function growth every 6 months from April 1996 through May 1999. 

### 12

Exposure dat a were provided by 10 air quality monitor stations located within 2 km of 

### 13

each child’s school. Over the study period, 8

- h O 3 concentrations ranged from 60 ppb 

### 14

(SD, ± 25) in the northeast area of Mexico City to 90 ppb (SD, ± 34) in the southwest, 

### 15

with an ov erall mean of 69.8 ppb. In multi

- pollutant models, an IQR increase in mean O 3 

### 16

concentration of 11.3 ppb was associated with an annual deficit in FEV 1 of 12 mL in girls 

### 17

and 4 mL in boys. Single

- pollutant models showed an association between ambient 

### 18

pollutan ts (O 3 , PM 10 , and NO 2 ) and deficits in lung function growth. While the estimates 

### 19

from co

- pollutant models were not substantially different than single pollutant models, 

### 20

independent effects for pollutants could not be estimated accurately because the traffi c



### 21

related pollutants were correlated. To reduce exposure misclassification, 

### 22

microenvironmental and personal exposure assessments were conducted in a randomly 

### 23

selected subsample of 60 children using passive O 3 samplers. Personal O 3 concentrations 

### 24

were corre lated (p <0.05) with the measurements obtained from the fixed

- site air 

### 25

monitoring stations. 

### 26

In the 2006 O 3 AQCD, few studies had investigated the effect of chronic O 3 exposure on 

### 27

pulmonary function. The strongest evidence was for medium

- term effects of ex tended O 3 

### 28

exposures over several summer months on lung function (FEV 1 ) in children, i.e., reduced 

### 29

lung function growth being associated with higher ambient O 3 levels. Longer

- term 

### 30

studies (annual), investigating the association of chronic O 3 exposure on lun g function 

### 31

(FEV 1 ) such as the definitive 8

- year follow

- up analysis of the first cohort ( Gauderman et 

### 32

al., 2004 ) provides little evidence that long

- term exposure to ambient O 3 at current levels 

### 33

is associated with significa nt deficits in the growth rate of lung function in children. 

### 34

Analyses indicated that there was no evidence that either 8

- h avg O 3 (10 a.m. to 6 p.m.) 

### 35

or 24

- h avg O 3 was associated with any measure of lung function growth over a 4

- year 

### 36

(age 10 to 14 years; ( Gauderman et al., 2000 ) ) or 8

- year (age 10 to 18 years; ( Gauderman 

### 37

et al., 2004 ) ) period. However, most of the other pollutants examined (including PM 2.5 , 

### 38

### Draft

– Do Not Cite or Quote 7

- 22 June 2012 NO 2 , a cid vapor, and elemental carbon) were found to be significa ntly associated with 

### 1

reduced growth in lung function. In addition, there was only about a 2

- to 2.5

- fold 

### 2

difference in O 3 concentrations from the least to most polluted communities (mean 

### 3

annual ave rage of 8

- h avg O 3 ranged from 30 to 65 ppb), versus the ranges observed for 

### 4

the other pollutants (which had 4

- to 8

- fold differences in concentrations). 

### 5

Short

- term O 3 exposure studies presented in Section 6.2.1 .2 provide a cumulative body of 

### 6

epidemiologic evidence that strongly supports associations between ambient O 3 exposure 

### 7

and decrements in lung function among children. For new studies of long

- t erm O 3 

### 8

exposure relationship to pulmonary function, one study, where O 3 and other pollutant 

### 9

levels were higher (90 ppb at high end of the range) than those in the CHS, observes a 

### 10

relationship between O 3 concentration and pulmonary function declines in scho ol

- aged 

### 11

children. Two studies of adult cohorts provide mixed results where long

- term exposures 

### 12

were at the high end of the range with levels of 49.5 ppb in one study and 27 ppb IQR in 

### 13

the other. Toxicological studies examining monkeys have provided data f or airway 

### 14

resistance in an asthma model but this is difficult to compare to FEV 1 results. Thus there 

### 15

is little new evidence to build upon the very limited studies of pulmonary function 

### 16

(FEV 1 ) from the 2006 O 3 AQCD. 

### 17

7.2.3.2 Pulmonary Structure and Function: Eviden ce from Toxicological Studies and Nonhuman Primate Asthma
Models

Long

- term studies in animals allow for greater insight into the potential effects of 

### 18

prolonged exposure to O 3 , that may not be easily measured in humans, such as structural 

### 19

changes in the res piratory tract. As reviewed in the 1996 and 2006 O 3 AQCDs and 

### 20

Chapter 5 of this ISA, there are both qualitative and quantitative uncertaintie s in the 

### 21

extrapolation of data generat ed by rodent toxicology studies to the understanding of 

### 22

health effects in humans. Despite these uncertainties, epidemiologic studies observing 

### 23

functional changes in humans can attain biological plausibility, in conju n c tion with long



### 24

term toxicological studies, particularly O 3 - inhalation studies performed in non

- human 

### 25

primates whose respiratory system most closely resembles that of the human. A n 

### 26

important series of studies have used nonhuman primates to examine the effec t of O 3 

### 27

alone or in combination with an inhaled allergen, house dust mite antigen, on 

### 28

morphology and lung function. These animals exhibit the hallmarks of allergic asthma 

### 29

defined for humans, including: a positive skin test for HDMA with elevated levels of IgE 

### 30

in serum and IgE

- positive cells within the tracheobronchial airway walls; impaired 

### 31

airflow which is reversible by treatment with aerosolized albuterol; increased abundance 

### 32

of immune cells, especially eosinophils, in airway exudates and bronchial lavage ; and 

### 33

### Draft

– Do Not Cite or Quote 7

- 23 June 2012 development of nonspecific airway responsiveness ( NHLBI, 2007 )

. Hyde et al. (2006 ) 

### 1

compared asthma models of rodents (mice) and the nonhuman primate model to 

### 2

re sponses in humans and concluded that the unique responses to inhaled allergen shown 

### 3

in the rhesus monkeys make it the most appropriate animal model of human asthma. 

### 4

These studies and others have demonstrated changes in pulmonary function and airway 

### 5

morphol ogy in adult and infant nonhuman primates repeatedly exposed to 

### 6

environmentally relevant concentrations of O 3 ( Joad e t al., 2008 ; Carey et al., 2007 ; 

### 7

Plopper et al., 2007 ; Fanucchi et al., 2006 ; Joad et al., 2006 ; Evans et al., 2004 ; Larson et 

### 8

al., 2004 ; Tran et al., 2004 ; Evans et al., 2003 ; Schelegle et al., 2003 ; Fanucchi et al., 

### 9

2000 ; Hyde et al., 1989 ; Harkema et al., 1987a ; Harkema et al., 1987b ; Fujinaka et al., 

### 10

1985 ) . Many of the obse rvations found in adult monkeys have also been noted in infant 

### 11

rhesus monkeys, although a direct comparison of the degree of effects between adult and 

### 12

infant monkeys has not been reported. The findings of these nonhuman primate studies 

### 13

have also been obser ved in rodent studies discussed at the end of this section and 

### 14

included in Table 7

- 1

. 

### 15

The initial observations in adult nonhuman primates have been expanded in a series of 

### 16

experiments using infant rhesus monkeys repeatedly exposed to 0.5 ppm O 3 starting at 

### 17

1 month of age 1 ( Plopper et al., 2007 ) . The purpose of these studies, designed by Plopp er 

### 18

and colleagues, was to determine if a cyclic regimen of O 3 inhalation would amplify the 

### 19

allergic responses and structural remodeling associated with allergic sensitization and 

### 20

inhalation in the infant rhesus monkey. In terms of pulmonary function change s, after 

### 21

several episodic exposures of infant monkeys to O 3 , they observed a significa nt increase 

### 22

in the baseline airway resistance, which was accompanied by a small increase in airway 

### 23

responsiveness to inhaled histamine ( Schelegle et al., 2003 ) , although neither 

### 24

measurement was statistically different from filtered air control values. Exposure of 

### 25

animals to inhaled house dust mite antigen alone also produced small but not statistically 

### 26

significa nt change s in baseline airway resistance and airway responsiveness, whereas the 

### 27

combined exposure to both (O 3 + antigen) produced statistically significa nt and greater 

### 28

than additive changes in both functional measurements. This nonhuman primate evidence 

### 29

of an O 3 - in duced change in airway resistance and responsiveness supports the biologic 

### 30

plausibility of long

- term exposure to O 3 contributing to the effects of asthma in children. 

### 31

To understand which conducting airways and inflammatory mechanisms are involved in 

### 32

O 3 - ind uced airway hyperresponsiveness in the infant rhesus monkey, a follow

- up study 

### 33

examined airway responsiveness ex vivo in lung slices ( Joad et al., 2006 ) . Using video 

### 34

microscopy to morphometrically evaluate the response of bronchi and respiratory 

### 35

1 Schelegle et al. (2003 ) used a two

- by

- two block design . Twenty

- four infant rhesus monkeys (30 days old) were exposed to 11 episodes (total of 6

- months exposure period) of filtered air (FA), house dust mite allergen (HDMA), O 3 (5 days each followed by 9 days of FA). Ozone was delivered for 8h/day at 0.5 ppm. Twelve of the monkeys (HDMA

, and H DMA + O 3 groups) were sensitized to house dust mite allergen (HDMA, confirmed by skin testing). To evaluate the potential for recovery, th e 5 months of exposure were followed by another 6 months in FA until the monkeys were reevaluated at 12 months of age.

### Draft

– Do Not Cite or Quote 7

- 24 June 2012 bronchioles to methacholine, (a bronchoconstricting agent commonly used to evaluate 

### 1

airway responsiveness in asthmatics), the investigators observed differential effects for 

### 2

the two airway sizes. While episodic exposure to O 3 alone (0.5 ppm) had little effect on 

### 3

ex vivo airway responsiveness in bronchi and respiratory bronchioles, exposure to dust 

### 4

mite antigen alone produced airway hyperrespon siveness in the large bronchi, whereas 

### 5

O 3 + antigen produced significa nt increases in airway hyperresponsiveness only in the 

### 6

respiratory bronchioles. These results suggest that ozone’s effect on airway 

### 7

responsiveness occurs predominantly in the smaller bro nchioles, where dosimetric 

### 8

models indicate the dose would be higher. 

### 9

The functional changes in the conducting airways of infant rhesus monkeys exposed to 

### 10

either O 3 alone or O 3 + antigen were accompanied by a number of cellular and 

### 11

morphological changes, in cluding a significa nt 4

- fold increase in eosinophils, (a cell type 

### 12

important in allergic asthma), in the bronchoalveolar lavage of infant monkeys exposed 

### 13

to O 3 alone. Thus, these studies demonstrate both functional and cellular changes in the 

### 14

lung of infant monkeys after cyclic exposure to 0.5 ppm O 3 . This concentration, provides 

### 15

relevant information to understanding the potentially damaging effects of ambient O 3 

### 16

exposure on the respiratory tract of humans. No concentration

- response data, however, 

### 17

are available from these nonhuman primate studies. 

### 18

In addition to these functional and cellular changes, significa nt structural changes in the 

### 19

respiratory tract have be en observed in infant rhesus monkeys exposed to O 3 . During 

### 20

normal respiratory tract development, conducting airways increase in diameter and length 

### 21

in the infant rhesus monkey. Exposure to O 3 alone (5 days of 0.5 ppm O 3 at 8 h/day, 

### 22

followed by 9 days of fi ltered air exposures for 11 cycles), however, markedly affected 

### 23

the growth pattern of distal conducting airways ( Fanucchi et al., 2006 ) . Whereas the first 

### 24

alveolar outpocketing occurred at airway generation 13 or 14 in filtered air

- control infant 

### 25

monkeys, the most proximal alveolariz ed airways occurred at an average of 10 airway 

### 26

generations in O 3 - exposed monkeys. Similarly, the diameter and length of the terminal 

### 27

and respiratory bronchioles were significa ntly decreased in O 3 - exposed monkeys. 

### 28

Importantly, the O 3 - induced structural path way changes persisted after recovery in 

### 29

filtered air for 6 months after cessation of the O 3 exposures. These structural effects were 

### 30

accompanied by significa nt increases in mucus goblet cell mass, alterations in smooth 

### 31

muscle orientation in the respiratory bronchioles, epithelial nerve fiber distribution, and 

### 32

basement membrane zone morphometry. These latter effec ts are noteworthy because of 

### 33

their potential contribution to airway obstruction and airway hyperresponsiveness which 

### 34

are central features of asthma

. 

### 35

Because many cellular and biochemical factors are known to contribute to allergic 

### 36

asthma, the effect of exposure to O 3 alone or O 3 + antigen on immune system parameters 

### 37

### Draft

– Do Not Cite or Quote 7

- 25 June 2012 was also examined in infant rhesus monkeys. Mast cells, which contribute to asthma via 

### 1

the release of potent proteases, were elevated in animals exposed to antigen alone but O 3 

### 2

alone had little effect on mast cell numbers and the response of animals exposed to O 3 + 

### 3

antigen was not different from that of animals exposed to antigen alone; thus suggesting 

### 4

that mast cells played little role in the interaction between O 3 and antigen in this model of 

### 5

allergic asthma ( VanWinkle et al., 2010 ) . Increases in CD4+ and CD8+ lymphocytes 

### 6

were observed at 6 months of age in the blood and bronchoalveolar lavage fluid of infant 

### 7

rhesus monkeys exposed to O 3 + antigen but not in monkeys exposed to eit her agent 

### 8

alone ( Miller et al., 2009 ) . Activated lymphocytes (i.e., CD25+ cells) were 

### 9

morphometrically evaluated in the airway mucosa and signi fica ntly increased in infant 

### 10

monkeys exposed to antigen alone or O 3 + antigen. Although O 3 alone had no effect on 

### 11

CD25+ cells, it did alter the anatomic distribution of CD25+ cells within the airways. 

### 12

Ozone had only a small effect on these sets of immune cells and did not produce a strong 

### 13

interaction with an inhaled allergen in this nonhuman pr imate model. 

### 14

In addition to alterations in the immune system, nervous system interactions with 

### 15

epithelial cells are thought to play a contributing role to airway hyperresponsiveness. A 

### 16

critical aspect of postnatal lung development is the laying of nerve ax ons with specific 

### 17

connections serving to maintain lung homeostasis. Aberrant innervation patterns may 

### 18

underlie allergic airways disease pathology and long

- term decrements in airway function. 

### 19

As noted in the 2006 O 3 AQCD, exposure of infant rhesus monkeys a ltered the normal 

### 20

development of neural innervation in the epithelium of the conducting airways ( Larson et 

### 21

al., 2004 )

. Significa nt mean reductions in nerve fiber density were observed in the 

### 22

midlevel airways of animals exposed to O 3 alone (49% reduction), and O 3 + antigen (55% 

### 23

reduction). Moreover, the morphology of nerve bundles was altered. The persistence of 

### 24

these effects was examined after a 6

- month recovery period, and although nerve 

### 25

distribution remained atypical, there was a dramatic increase in airway nerve density 

### 26

(hype rinnervation) ( Kajekar et al., 2007 ) . Thus, in addition to structural, immune, and 

### 27

inflammatory effects, exposure to O 3 produces alterations in airway innervation which 

### 28

may c ontribute to O 3 - induced exacerbation of asthma. Evaluation of the pathobiology of 

### 29

airway remodeling in growing lungs of neonates using an animal model where exposure 

### 30

to allergen generates reactive airway disease with all the hallmarks of asthma in humans 

### 31

i llustrates that exposure to O 3 and allergen early in life produces a large number of 

### 32

disruptions of fundamental growth and differentiation processes. 

### 33

A number of studies in both nonhuman primates and rodents demonstrate that O 3 

### 34

exposure can increase colla gen synthesis and deposition, inducing fibrotic

- like changes in 

### 35

the lung ( Last et al., 1994 ; Chang et al., 1992 ; Moffatt et al., 1987 ; Reiser et al., 1987 ; 

### 36

Last et al., 1984 ) . Increased collagen content is often associated with elevated abnormal 

### 37

cross links that appear to be irreversible ( Reiser et al., 1987 ) . Generally changes in 

### 38

### Draft

– Do Not Cite or Quote 7

- 26 June 2012 collagen content have been observed in rats exposed to 0.5 ppm O 3 or higher, althou gh 

### 1

extracellular matrix thickening has been observed in the lungs of rats exposed to an urban 

### 2

pattern of O 3 with daily peaks of 0.25 ppm for 38 weeks ( Chang et al., 1992 ; Chang et al., 

### 3

1991 ) . A more recent study using an urban pattern of exposure to 0.5 ppm O 3 

### 4

demonstrated that O 3 - induced collagen deposition in mice is dependent on the activity of 

### 5

TGF

- β ( Katre et al., 2011 ) . Sex differences have been observed with respect to incr eased 

### 6

centriacinar collagen deposition and crosslinking, which was observed in female but not 

### 7

male rats exposed to 0.5 and 1.0 ppm O 3 for 20 months ( Last et al., 1994 ) . Few other 

### 8

long

- term exposure morphological studies have presented sex differences and most only 

### 9

evaluated males. 

### 10

As described in the 1996 and 2006 O 3 AQCDs, perhaps the largest chronic O 3 study was 

### 11

a n NIEHS

- NTP/HEI funded rodent study conducted by multiple investigators studying a 

### 12

number of different respiratory tract endpoints ( Catalano et al., 1995b ) . Rats were 

### 13

exposed to 0.12, 0.5, or 1.0 ppm O 3 for 6 h/day and 5 d/week for 20 months. The most 

### 14

prominent changes were observed in the nasal cavity where a large fraction of O 3 is 

### 15

absorbed. Alterations in nasal function (increase d mucous flow) and structure (goblet cell 

### 16

metaplasia) were observed at 0.5 and 1.0 ppm but not 0.12 ppm O 3 . In the lung, the 

### 17

centriacinar region (CAR) was the anatomical site most affected by O 3 . The epithelial cell 

### 18

lining was changed to resemble that seen in respiratory bronchioles and the interstitial 

### 19

volume was increased. Biochemical analyses demonstrated increased collagen and 

### 20

glycoaminoglycans, an observation that supported the structural changes. As in the nose, 

### 21

these changes were observed only at the two highest exposure concentrations. 

### 22

Importantly, despite these morphologic and biochemical changes after 20 months of 

### 23

exposure, detailed pulmonary function testing revealed little to no measurable change in 

### 24

function. Thus, minor respiratory tract changes were observed after chronic exposure to 

### 25

O 3 up to 1.0 ppm in the F344 rat model. 

### 26

It is un clear what the long

- term impact of O 3 - induced structural changes may be. 

### 27

Simulated seasonal (episodic) exposure studies suggest that such exposures might have 

### 28

cumulat ive impacts, and a number of studies indicate that structural changes in the 

### 29

respiratory system are persistent or irreversible. For example, O 3 - induced hyperplasia 

### 30

was still evident in the nasal epithelia of rats 13 weeks after recovery from 0.5 ppm O 3 

### 31

exp osure ( Harkema et al., 1999 ) . In a study of epis odic exposure to 0.25 ppm O 3 , Chang 

### 32

et al. (1992 ) observed no r eversal of basement membrane thickening in rat lungs up to 17 

### 33

weeks post

- exposure. Thickening of the sub

- basement membrane is one of the persistent 

### 34

structural features observed in human asthmatics ( NHLBI, 2007 ) . Episodic exposure 

### 35

(0.25 ppm O 3 , every other month) of young monkeys induced equivalent morphological 

### 36

changes compared to continuously exposed animals, even though they were exp osed for 

### 37

half the time and evaluation occurred a month after exposure ceased as opposed to 

### 38

### Draft

– Do Not Cite or Quote 7

- 27 June 2012 immediately ( Tyler et al., 1988 ) . Notably, episodic O 3 exposure increased total lung 

### 1

collagen content, chest wall compliance, and inspiratory capacity, suggesting a delay in 

### 2

lung maturation in episodically

- exposed animals. These ch anges were in contrast to the 

### 3

continuously exposed group, which did not differ from the air exposed group in these 

### 4

particular parameters but did exhibit greater bronchiolitis than the episodically exposed 

### 5

animals. In a study by Harkema and colleagues ( Harkema et al., 1993

, 1987b ) , monkeys 

### 6

(both males and females) were acutely exposed for 8 h/day to 0.15 ppm O 3 (6 days) or 

### 7

chronically to 0.15 ppm or 0.3 ppm O 3 (90 days). For most endpoints in the nasal cavity, 

### 8

the observed morphologic changes and inflammation were greate r in the monkeys 

### 9

exposed for 6 days compared to 90 days, whereas in the respiratory bronchioles of the 

### 10

same animals, there were no significa nt time or concentration dependent differences 

### 11

(increased epithelial thickness and proportion of cuboidal cells) bet ween the 6 and 90 day 

### 12

exposure groups. 

### 13

Stokinger (1962 ) reported that chronic bronchitis, bronchiolitis, and emphysematous and 

### 14

fibrotic changes develop in the lung tissues of mice, rats, hamsters, and guinea pigs 

### 15

exposed 6 h/day, 5 days/week for 14.5 months to a concentration slightly above 1 ppm 

### 16

O 3 . Rats continuously exposed for 3 to 5 months to 0.8 ppm O 3 develop a disease that 

### 17

resembles emphysema, and they finally die of respiratory failure ( Stephens et al., 1976 )

. 

### 18

Ozone results in a greater response of fibroblasts in the lesion, thickening of the alveolar 

### 19

septae, and an increase in number of alveolar macrophages in the proximal alv eoli. 

### 20

### Draft

– Do Not Cite or Quote 7

- 28 June 2012 Table 7

- 1 Respiratory effects in nonhuman primates and rodents resulting from long

- term ozone exposure

Study Model O 3 (ppm) Exposure Duration Effects

Pinkerton et al. (1998 ); Harkema et al. (1997a ); Harkema et al. (1997b ); Catalano et al. (1995b ); Catalano et al. (1995a ); ( Chang et al., 1995 ); Pinkerton et al. (1995 );
Stockst ill et al. (1995 ); Harkema et al. (1994 ); Last et al. (1994 ); Plopper et al. (1994 ) Rat, male and female, Fischer F344,

6

- 8 weeks old 0.12

0.5

1.0 6 h/day, 5 days/week for
20 months Effects similar to (or a model of) early fibrotic human disease were greater in the periacinar region than in terminal bronchioles. Thickened alveolar septa observed at 0.12 ppm O 3 . Other effects (e.g., mucous cell metaplasia in the nose, mild fibrotic response in the parenchyma, and increased collagen in CAR of females) observed

at 0.5 to 1.0 ppm. Some morphometric changes (epithelial thickening and bronchiolarization)
occurred aft er 2 or 3 months of exposure to

1.0 ppm.
Herbert et al. (1996 ) Mice, male and female, B6C3F1, 6

- 7
weeks old, 0.12

0.50

1.0 6 h/day, 5 days/week for
24 and 30 months Similar to the response of rats in the same study (see rat above). Effects were seen in the nose and centriacinar region of the lung at 0.5 and 1.0 ppm.

Chang et al. (1991 ) Rat, male, F344,

6 weeks old Continuous: 0.12

or 0.25
Episodic/urban: baseline 0.06; peak 0.25 Continuou s: 12 h/day for

6 weeks
Simulated urban pattern; slow rise to peak 9 h/day, 5 days/week,

13 weeks Increased Type 1 and 2 epithelial volume assessed by TEM. Linear relationship observed between increases in Type 1 epithelial cell volume
and concentration x time product. Degree of injury not related to pattern of exposure (continuous or episodic).

Chang et al. (1992 ) Rat, male, F344,
6 weeks old baseline 0.06; peak 0.25 Slow rise to peak

9 h/day, 5 days/week,

13 and 78 weeks

Recovery in filtered
air for 6 or 17 weeks Progressive epithelial hyperplasia, fibroblast proliferation, and interstitial matrix accumulation observed using TEM. Interstitial matrix thickening due to deposition of basement membrane and collagen fibers. Partial recovery of interstitial matrix during follow

- up periods in air; but no
resolution of basement membrane thickening.

Barry et al. (1985 ); ( 1983 ) Rat, male, 1 day old
or 6 weeks old 0.12 (adults only)

0.25 12 h/day for 6 weeks Lung and alveolar dev elopment not significantly affected. Increased Type 1 and 2 epithelial cells and AM in CAR alveoli, thickened Type 1 cells with smaller volume and less surface coverage as assessed by TEM (adults and juveniles). In adults, smaller but statistically signifi cant similar changes
at 0.12 ppm, suggesting linear concentration

response relationship. No statistically significant

age

- related effects observed.

Tyler et al. (1988 ) Monkey; male,
Macaca fascicularis

, 7 mo old 0.25 8 h/day, 7 days/week, Daily for 18 mo or episodically every other
month for 18 mo

Episodic group
evaluated 1 mo postexposure Increased collagen content, chest wall compliance, and inspiratory capacity in episodic group only. Respiratory bronchiolitis in both groups. Episodically exposed group incurred greater alterations in physiology a nd biochemistry and equivalent changes in morphometry even though exposed for half the time as the daily

exposure group.
Harkema et al. (1999 ) Rat, male, Fischer F344/N HSD, 10

- 14
weeks old 0.25

0.5 8 h/day, 7 days/week for
13 weeks Mucous cell hyperplasia in nasal epithelium after

expos ure to 0.25 and 0.5 ppm O 3 ; still evident after 13 weeks recovery from 0.5 ppm O 3 exposure.
Van Bree et al. (2002 ) Rat, male, Wistar, 7 weeks old,

n = 5/group 0.4 23.5 h/day for 1, 3, 7, 28,or 56 days Acute inflammatory response in BALF reached a maximum at day 1 and resolved within 6 days during exposure. Centriacinar region inflammatory responses throughout O 3 exposure with increased collagen and bronchiolization still present after a recovery period.

Katre et al. (2011 ) Mice; male, C57BL/6,

6

- 8 weeks old 0.5 8 h/day, [5 days/week
O 3 , and 2 days f iltered air] for 5 or 10 cycles Sustained elevation in TGF

- β and PAI

- 1 in lung (5 or 10 cycles); elevated α

- SMA and increased collagen deposition in airway walls (after 10 cycles). Collagen increase shown to depend on
TGF

- β.

### Draft

– Do Not Cite or Quote 7

- 29 June 2012 Study Model O 3 (ppm) Exposure Duration Effects

Schelegle et al. (2003 ) ; Monkey; Rhesus,
30 days old a 0.5 8 h/day for 5 days, every 5 days for a total of 11 episodes Goblet cell metaplasia, increased AHR, and increased markers of allergic asthma (e.g., eosinophilia) were observed, suggesting that episodic exposure to O 3 alters postnatal morphogenesis and epithelial differentiation and enhances the allergic effects of house dust mite allergen in the lungs of infant primates.

Harkema et al. (1993

, 1987b ) Monkey; Macac a radiata , M, F

2

- 6 years old 0.15

0.3 8 h/day for 90 days Significant increase in epithelial thickness in respiratory bronchioles which was accompanied by increase in cuboidal cells; nasal lesions consisted of ciliated cell necrosis and secretory
cell hyperplasia; no concentration response
effects

Larson et al. (2004 ) Monkey; Macaca
mulatta , 30 days old a 0.5 11 episodes of 5 days each, 8 h/day followed by 9 days of recovery O 3 or O 3 + house dust mite antigen caused changes in density and number of airway epithelial nerves in small conducting airways. Suggests episodic O 3 alters pattern of neural innervation in epithelial compartment of developing lungs.

Plopper et al. (2007 ) Monkey; Rhesus,
30 days old a 0.5 5 months of episodic exposure; 5 days O 3 followed by 9 days of filtered air, 8h/day. Non

- significant increases airway resistance and airway responsiveness with O 3 or inhaled allergen alone. Allergen + O 3 produced additive changes in
both measures.

Fanucchi et al. (2006 ) Monkey; male Rhesus,30 days old 0.5 5 months of episodic exposure; 5 days O 3 followed by 9 days of filtered air, 8h/day. Cellular changes and significant structural changes in the distal respiratory tract in infant rhesus monkeys exposed to O 3 postnatally.

Reiser et al. (1987 ) Monkey; male and female Cynomolgus

6

- 7 mo old 0.61 8 h/day for 1 year Increased lung collagen content associated with
elevated abnormal cross links that were irreversibly deposited.

a sex not reported

Collectively, evidence from animal studies strongly suggests that chronic O 3 exposure is 

### 1

capable of damaging the distal airways and proximal alveoli, resulting in lung tissue 

### 2

remodeling and leading to apparent irreversible changes. Potentially, persistent 

### 3

inflammation and interstitial remodeling play an important role in the progr ession and 

### 4

development of chronic lung disease. Further discussion of the modes of action that lead 

### 5

to O 3 - induced morphological changes can be found in Section 5.3.7 . The findings 

### 6

reported in chronic animal studies offer insight into potential biological mechanisms for 

### 7

the suggested association between seasonal O 3 exposure and reduced lung function 

### 8

development in children as observed in epidemiologic studies (see Section 7.2.3 ). 

### 9

Discussion of mechanisms invo lved in lifestage susceptibility and developmental effects 

### 10

can be found in Section 5.4.2.4

. 

### 11

7.2.4 Pulmonary Inflammation, Injury, and Oxidative Stress

The 2006 O 3 AQCD stated that the extensive human clinical and animal toxicological 

### 12

evidence, together with the limited epidemiologic evidence available, suggests a causal 

### 13

role for O 3 in inflammatory responses in the airways. Short

- term exposure epidemiologic 

### 14

### Draft

– Do Not Cite or Quote 7

- 30 June 2012 studies discussed earlier in Section 6.2.3.2 show consistent associations of O 3 exposure 

### 1

and increased airway inflammation and oxidative stress. Fur ther discussion of the 

### 2

mechanisms underlying inflammation and oxidative stress responses can be found in 

### 3

Section 5.3.3 . Though the majority of recen t studies focus on short

- term exposures, 

### 4

several epidemiologic and toxicology studies of long

- term exposure add to observations 

### 5

of O 3 - induced inflammation and injury. 

### 6

Inflammatory markers and peak expiratory pulmonary function were examined in 37 

### 7

allergic children with physician

- diagnosed mild persistent asthma in a highly polluted 

### 8

urban area in Italy and then again 7 days after relocation to a rural location with 

### 9

significa ntly lower pollutant levels ( Renzetti et al., 2009 ) . The authors obser ved a 4

- fold 

### 10

decrease in nasal eosinophils and a statistically significa nt decrease in fractional exhaled 

### 11

nitric oxide along with an improvement in lower airway function. Several pollutants were 

### 12

examined, including PM 10 , NO 2 , and O 3 , though pollutant

- specific results were not 

### 13

presented. These results are consistent with studies showing that traffic

- related exposures 

### 14

are associated with increased airway inflammation and reduced lung function in children 

### 15

with asthma and contribute to t he notion that this negative influence may be rapidly 

### 16

reversible. Exhaled NO (eNO) has been shown to be a useful biomarker for airway 

### 17

inflammation in large population

- based studies ( Linn et al., 2009 ) . Thus, while the time 

### 18

scale of 7 days between examinations for eNO needs to be evaluated for appropriateness, 

### 19

the results suggest that inflammatory responses are reduced when O 3 levels are decreased. 

### 20

Chest radiographs (CXR) of 249 children in Mexico City who were chronically exposed 

### 21

to O 3 and PM 2.5 were analyzed by Calderón

- Garcidueñas et al. (2006 ) . They reported an 

### 22

association between chronic exposures to O 3 and other pollutants and a significa nt 

### 23

increase in abnormal CXR’s and lung CTs suggestive of a bronchiolar, peribronchiolar, 

### 24

and/or alveolar duct inflammatory process, in clinically healthy children with no risk 

### 25

factors for lung disease. These CXR and CT resul ts should be viewed with caution 

### 26

because it is difficult to attribute effects to O 3 exposure. 

### 27

In a cross

- sectional study, Wood et al. (2009 ) examined the association of outdoor air 

### 28

pollution with respiratory phenotype (PiZZ type ) in alpha 1

- antitrypsin deficiency 

### 29

(α

- ATD) from the U.K. α

- ATD registry. This deficiency leads to exacerbated responses 

### 30

to inflammatory stimuli. In total, 304 PiZZ subjects underwent full lung function testing 

### 31

and quantitative high

- resolution computed tom ography to identify the presence and 

### 32

severity of COPD

– emphysema. Mean annual air pollution data for 2006 was matched to 

### 33

the location of patients’ houses and used in regression models to identify phenotypic 

### 34

associations with pollution controlling for cova riates. Relative trends in O 3 levels were 

### 35

assessed to validate use of a single year’s data to indicate long

- term exposure and 

### 36

validation; data showed good correlations between modeled and measured data ( Stedman 

### 37

### Draft

– Do Not Cite or Quote 7

- 31 June 2012 and Kent, 2008 ) . Regression models showed that estimated higher exposure to O 3 

### 1

exposure was associated with worse gas transfer and more severe emphysema, albeit 

### 2

acc ounting for only a small proportion of the lung function variability. This suggests that 

### 3

a gene

- specific group demonstrates a long

- term O 3 exposure effect. 

### 4

The similarities of nonhuman primates to humans make them attractive models in which 

### 5

to study the ef fects of O 3 on the respiratory tract. The nasal mucous membranes, which 

### 6

protect the more distal regions of the respiratory tract, are susceptible to injury from O 3

. 

### 7

Carey et al. (2007 ) conducted a study of O 3 exposure in infant rhesus macaques, whose 

### 8

nasal airways closely resemble that of humans. Monkeys were exposed either acutely for 

### 9

5 days (8 h/day) to 0.5 ppm O 3 , or episodically for several biweekly cycles alternating 

### 10

5 days of 0.5 ppm O 3 with 9 days of fi ltered air (0 ppm O 3 ), designed to mimic human 

### 11

exposure (70 days total). All monkeys acutely exposed to O 3 had moderate to marked 

### 12

necrotizing rhinitis, with focal regions of epitheliar exfoliation, numerous infiltrating 

### 13

neutrophils, and some eosinophils. T he distribution, character, and severity of lesions in 

### 14

episodically exposed monkeys were similar to that of acutely exposed animals. Neither 

### 15

group exhibited the mucous cell metaplasia proximal to the lesions, observed in adult 

### 16

monkeys exposed continuously to 0.3 ppm O 3 in another study ( Harkema et al., 1987a )

. 

### 17

Adult monkeys also exhibit attenuation of inflammatory responses with continued daily 

### 18

exposure ( Harkema et al., 1987a ) , but inflammation did not resolve over time in young 

### 19

episodically exposed monkeys ( Carey et al., 2011 ) . Inflammation in conducting airways 

### 20

has also be en observed in rats chronically exposed to O 3 . Using an agar

- based technique 

### 21

to fill the alveoli so that only the rat bronchi are lavaged, a 90

- day exposure of rats to 

### 22

0.8 ppm O 3 (8 h/day) elicited significa ntly elevated pro

- inflammatory eicosanoids PGE 2 

### 23

a nd 12

- HETE in the conducting airway compared to filtered air

- exposed rats ( Schmelzer 

### 24

et al., 2006 )

. 

### 25

Persistent inflammation and injur y leading to interstitial remodeling may play an 

### 26

important role in the progression and development of chronic lung disease. Chronic 

### 27

airway inflammation is an important component of both asthma and COPD. The 

### 28

epidemiological evidence supporting an associatio n between long

- term exposure to O 3 

### 29

and inflammation or injury is limited. However, animal studies clearly demonstrate O 3 - 

### 30

induced inflammation and injury, which may or may not attenuate with chronic exposure 

### 31

depending on the model. Further discussion of how O 3 initiates inflammation can be 

### 32

found in Section 5.3.3

. 

### 33

### Draft

– Do Not Cite or Quote 7

- 32 June 2012 7.2.5 Allergic Responses

The association of air pollutants with childhood respiratory allergies was examined in the 

### 1

U.S. using the 1999

- 2005 National Health Interview Survey of approximately 70,000 

### 2

children, and ambient air pollution data from the U.S. EPA, with monitors within 20 

### 3

miles of each child’s residential block ( Parker et al., 2009 ) . The authors examined the 

### 4

associations between the reporting of respiratory allergy or hay fever and medium

- term 

### 5

exposure to O 3 over several summer months, controlling for demographic and geographic 

### 6

factors. Increased respiratory allergy/hay fever was associated with increased O 3 levels 

### 7

(adjusted OR per 10 ppb = 1.20; [95% CI: 1.15, 1.26]). These associa tions persisted after 

### 8

stratification by urban

- rural status, inclusion of multiple pollutants (O 3 , SO 2 , NO 2 , PM), 

### 9

and definition of exposure by differing exposure radii; smaller samples within 5 miles of 

### 10

monitors were remarkably similar to the primary resul ts. No associations between the 

### 11

other pollutants and the reporting of respiratory allergy/hay fever were apparent. 

### 12

Ramadour et al. (2000 ) reported no relationship between O 3 levels and rhinitis symptoms 

### 13

and hay fever. Hwang et al. (2006 ) report the prevalence of allergic rhinitis (adjusted OR 

### 14

per 10 ppb = 1.05; [95% CI: 0.98, 1.12]) in a large cross

- sectional study in Taiwan. In a 

### 15

large cross

- sectional study in France, Penard

- Morand et al. (2005 ) reported a positive 

### 16

relationship between lifetime allergic rhinitis and O 3 exposure in a two

- pollutant model 

### 17

with NO 2 . These studies related positive outcomes of allerg ic response and O 3 exposure 

### 18

but with variable strength for the effect estimates. A toxicological study reported that 

### 19

five weeks of continuous exposure to 0.4 ppm O 3 (but not 0.1 or 0.2 ppm O 3 ) augmented 

### 20

sneezing and nasal secretions in a guinea pig model o f nasal allergy ( Iijima and 

### 21

Kobayashi, 2004 ) . Nasal eosinophils, which participate in allergic disease and 

### 22

inflammation, and allergic antibody levels in serum were also elevated by exposure to 

### 23

concent rations as low as 0.2 ppm ( Iijima and Kobayashi, 2004 )

. 

### 24

Nasal eosinophils were observed to decrease by 4

- fold in 37 atopic, mildly asthmatic 

### 25

children 7 days after relocation from a highly polluted ur ban area in Italy to a rural 

### 26

location with significa ntly lower pollutant levels ( Renzetti et al., 2009 ) . Inflammatory 

### 27

and allergic effects of O 3 exposure (30 day mean) such as increased eosinophil levels 

### 28

were observed in children in an Austr ian study ( Frischer et al., 2001 ) . Episodic exposure 

### 29

of infant rhesus monkeys to 0.5 ppm O 3 for 5 months appears to significa ntly increase the 

### 30

number and prop ortion of eosinophils in the blood and airways (lavage) [protocol 

### 31

described above in Section 7.2.3.1 for Fanucchi et al. (2006 ) ] ( Maniar

- Hew et al., 2011 )

. 

### 32

These changes were not evident at 1 year of age (6 months after O 3 exposure ceased). 

### 33

Increased eosinophils levels have also been observed after acute or prolonged exposures 

### 34

to O 3 in adult bonnet and rhesus monkeys ( Hyde et al., 1992 ; Eustis et al., 1981 )

. 

### 35

### Draft

– Do Not Cite or Quote 7

- 33 June 2012 Total IgE levels were related to air pollution levels in 369 adult asthmatics in five French 

### 1

centers using generalized estimated equations (GEE) as part of the EGEA study described 

### 2

earlier ( Rage et al., 2009a ) . Geostatistical models were performed on 4×4 km grids to 

### 3

assess individual outdoor air pollution exposure that was assigned to subject’s home 

### 4

address. Ozone concentrations were positively related to total IgE levels and an increase 

### 5

of 5 ppb of O 3 resulted in an increase of 20.4% (95% CI: 3.0, 40.7) in total IgE levels. 

### 6

Nearly 75% of the subjects were atopic. In two

- pollutant models including O 3 and NO 2, the 

### 7

O 3 effect estimate was decreased by 25% while the NO 2 effect estimate was decreased by 

### 8

57%. Associations were not sensitive to adjustment for covariates or the season of IgE 

### 9

measurements. These cro ss

- sectional results suggest that exposure to O 3 may increase 

### 10

total IgE in adult asthmatics. 

### 11

Although very few toxicological studies of long

- term exposure examining allergy are 

### 12

available, short

- term exposure studies in rodents and nonhuman primates demons trate 

### 13

allergic skewing of immune responses and enhanced IgE production. Due to the 

### 14

persistent nature of these responses, the short

- term toxicological evidence lends 

### 15

biological plausibility to the limited epidemiologic findings of an association between 

### 16

lon g

- term O 3 exposure and allergic outcomes. 

### 17

7.2.6 Host Defense

Short

- term exposures to O 3 have been shown to cause decreases in host defenses against 

### 18

infectious lung disease in animal models. Acute O 3 - induced suppression of alveolar 

### 19

phagocytosis and immune functio ns observed in animals appears to be transient and 

### 20

attenuated with continuous or repeated exposures, although chronic exposure (weeks, 

### 21

months) has been shown to slow alveolar clearance. In an important study investigating 

### 22

the effects of longer term O 3 expo sure on alveolobronchiolar clearance, rats were exposed 

### 23

to an urban pattern of O 3 (continuous 0.06 ppm, 7 days/week with a slow rise to a peak of 

### 24

0.25 ppm and subsequent decrease to 0.06 ppm over a 9 h period for 5 days/week) for 

### 25

6 weeks and were exposed 3 days later to chrysotile asbestos, which can cause pulmonary 

### 26

fibrosis and neoplasia ( Pinkerton et al., 1989 ) . After 30 days, the lungs of the O 3 - exposed 

### 27

animals had twice the number and mass of asbestos fibers as the air

- exposed rats. 

### 28

However, chronic exposures of 0.1 ppm do not cause greater effects on infectivity than 

### 29

short exposures, due to defense parameters becoming reestablished with prolonged 

### 30

exposures. No detrimental effects were seen with a 120

- day exposure to 0.5 ppm O 3 on 

### 31

acute lung injury from influenza virus administered immediately before O 3 exposure 

### 32

started. Howe ver, O 3 was shown to increase the severity of postinfluenzal alveolitis and 

### 33

lung parenchymal changes ( Jakab and Bassett, 1990 ) . A recent study by Maniar

- Hew et 

### 34

al. (2011 ) demonstrated that the immune system of infant rhesus monkeys episodically 

### 35

### Draft

– Do Not Cite or Quote 7

- 34 June 2012 exposed to 0.5 ppm O 3 for 5 months 1 appeared to be alter ed in ways that could diminish 

### 1

host defenses. Reduced numbers of circulating leukocytes were observed, particularly 

### 2

polymorphonuclear leukocytes (PMNs) and lymphocytes, which were decreased in the 

### 3

blood and airways (bronchoalveolar lavage). These changes d id not persist at 1 year of 

### 4

age (6 months postexposure); rather, increased numbers of monocytes were observed at 

### 5

that time point. Challenge with LPS, a bacterial ligand that activates monocytes and other 

### 6

innate immune cells, elicited lower responses in O 3 - exposed animals even though the 

### 7

relevant reactive cell population was increased. This was observed in both an in vivo 

### 8

inhalation challenge and an ex vivo challenge of peripheral blood mononuclear cells. 

### 9

Thus a decreased ability to respond to pathogenic sig nals was observed six months after 

### 10

O 3 exposure ceased, in both the lungs and periphery. 

### 11

7.2.7 Respiratory Mortality

A limited number of epidemiologic studies have assessed the relationship between long



### 12

term exposure to O 3 and mortality. The 2006 O 3 AQCD conclude d that an insufficient 

### 13

amount of evidence existed “to suggest a causal relationship between chronic O 3 

### 14

exposure and increased risk for mortality in humans” ( U.S. EPA, 2006b ) . Though total 

### 15

and cardio

- pulmonary mortality were considered in these studies, respiratory mortality 

### 16

was not specifically considered. In the mos t recent follow

- up analysis of the ACS cohort 

### 17

( Jerrett et al., 2009 ) , cardiopulmonary deaths were subdivided into respiratory and 

### 18

cardiovascular, separately, as opposed to combined in the Pope et al. (2002 ) work. A 

### 19

10

- ppb increment in exposure to O 3 elevated the risk of death from respiratory causes and 

### 20

this effect was robust to the inclus ion of PM 2.5 . The association between increased O 3 

### 21

concentrations and increased risk of death from respiratory causes was insensitive to the 

### 22

use of a random

- effects survival model allowing for spatial clustering within the 

### 23

metropolitan area and state of re sidence, and to adjustment for several ecologic variables 

### 24

considered individually. Additionally, a recent study ( Zanobetti and Schwartz, 2011 ) 

### 25

observed an association between long

- term exposure to O 3 and elevated risk of mortality 

### 26

among Medicare enrollees that had previously experienced an emergency hospital 

### 27

admission due to COPD. 

### 28

7.2.8 Summary and Causal Determination

The epidemiologic studies reviewed in the 2006 O 3 AQCD detected no associations 

### 29

between l ong

- term (annual) O 3 exposures and asthma

- related symptoms, asthma 

### 30

1 Exposure protocol is described above in Section 7.2.3.2 for Fanucchi et al. (2006 )

.

### Draft

– Do Not Cite or Quote 7

- 35 June 2012 prevalence, or allergy to common aeroallergens among children after controlling for 

### 1

covariates. Little evidence was available to relate long

- term exposure to ambient O 3 

### 2

concentrations with deficits in the growth rate of lung function in children. Additionally, 

### 3

limited evidence was available evaluating the relationship between long

- term O 3 

### 4

concentrations and pulmonary inflammation and other endpoints. From toxicological 

### 5

studies, it appeared t hat O 3 - induced inflammation tapered off during long

- term 

### 6

exposures, but that hyperplastic and fibrotic changes remained elevated and in some 

### 7

cases even worsened after a postexposure period in clean air. Episodic exposures were 

### 8

also known to cause more seve re pulmonary morphologic changes than continuous 

### 9

exposure ( U.S. E PA, 2006b )

. 

### 10

The recent epidemiologic evidence base consists of studies using a variety of designs and 

### 11

analysis methods evaluating the relationship between long

- term exposure to ambient O 3 

### 12

concentrations and measures of respiratory health effects and mor tality conducted by 

### 13

different research groups in different locations. See Table 7

- 2 for O 3 concentrations 

### 14

associated with selected studies. Table 7

- 2 is organized by longitudinal and cross



### 15

sectional studies both presented alphabetically. The positive results from various designs 

### 16

and locations support a relationship between long

- term exposure to ambient O 3 

### 17

concentrations and respiratory health effects and mortality. 

### 18

Earlier studies reported associations of new

- onset asthma and O 3 in an adult cohort in 

### 19

California ( McDonnell et al., 1999a ; Greer et al., 1993 ) but only in males. In the CHS 

### 20

cohort of children in 12 Southern Cal ifornia communities, long

- term exposure to O 3 

### 21

concentrations was not associated with increased risk of developing asthma ( McConnell 

### 22

et al., 2010 ) ; however, greater outdoor exercise was associated with development of 

### 23

asthma in children living in communities with higher ambient O 3 concentrations 

### 24

( McConnell et al., 2002 ) . Recent CHS studies examined interactions among genetic 

### 25

variants, long

- term O 3 exposure, and new onset asthma in children. These prospective 

### 26

cohort studies are methodologically rigorous epidemiology studies, and evidence 

### 27

indicates gene

- O 3 interactions. These studies have provided data supporting decreased 

### 28

risk of certain different genetic vari ants on new onset asthma (e.g., HMOX

- 1, ARG) that 

### 29

is limited to children either in low ( Islam et al., 2008 ) or high ( Salam et al., 2009 ) O 3 

### 30

communities. Gene

- environment intera ction also was demonstrated with findings that 

### 31

greater outdoor exercise increased risk of asthma in GSTP1 Ile/Ile children living in high 

### 32

O 3 communities ( Islam et al., 2009 ) . Biological plausibility for these these gene

- O 3 

### 33

environment interactions is provided by evidence that these enzymes have antioxidant 

### 34

and/or anti

- inflammatory activity and participate in well recognized modes of action in 

### 35

asthma pathogenesis. As O 3 is a source of oxidants in the airways, oxidative stress serves 

### 36

as the link among O 3 exposure, enzyme activity, and asthma. 

### 37

### Draft

– Do Not Cite or Quote 7

- 36 June 2012 Table 7

- 2 Summary of selected key new studies examining annual ozone exposure and respiratory health effects

Study; Health Effect;
Location Annual Mean O 3 Concentration (ppb) O 3 Range (ppb)
Percentiles

Longitudinal

Islam et al. (2008 ) ; New

- onset asthma;
CHS 55.2 high vs. 38.4 low communities 10:00 a.m. to 6:00 p.m. average See left

Islam et al. (2009 ) ; New

- onset asthma;
CHS 55.2 high vs. 38.4 low communities 10:00 a.m. to 6:00 p.m. See left

Lin et al. (2008b ) ; First asthma hospital admission;
New York State

- 10 regions Range of mean O 3 concentra tions over the
10 New York Regions 37.51 to 47.78

8

- h max 10:00 a.m. to 6:00 p.m. See left

Salam et al. (2009 ) ; Childhood onset asthma;
CHS O 3 greater than or less than 50 ppb See left

Cross

- sectional

Akinbami et al. (2010 ) ; Current asthma
United States 12 month median 39.8

8hr max IQR

35.9 to 43.7

Hwang et al. (2005 ) ; Prevalence of asthma;
Taiwan Mean 23.14 Range

18.65 to 31.17

Jacquemin et al. ( In Press ) ; Asthma control in adults;
Five French cities Median 46.9 ppb;

8

- h average 25th

- 75th

41

- 52

. Lee et al. (2009b ) ; Bronchi tic symptoms in asthmatic children;
CHS Above and below 50 ppb See left

Meng et al. (2010 ) ; Asthma ED visits or hospitalizations; San Joaquin Valley, CA Median 30.3 ppb

Yearly b ased on hourly 25

- 75% range
27.1 to 34.0

Moore et al. (2008 ) ; Asthma hospital admissions;
South Coast Basin Median 87.8 ppb
Quarterly 1hr daily max Range
28.6 to 199.9

Rage et al. (2009a ) ; Asthma severity;
Five French ci ties Mean 30 ppb

8

- h average 25th

- 75th

21

- 36

Wenten et al. (2009 ) ; Respiratory school absence,
U.S. Median 46.9 ppb;

10a.m.

– 6 p.m. average Min

- Max
27.6

- 65.3

Studies usi ng a cross

- sectional design provide support for a relationship between long



### 1

term O 3 exposure and health effects in asthmatics. A long

- term O 3 exposure study relates 

### 2

bronchitic symptoms to TNF

- 308 genotype asthmatic children with ambient O 3 exposure 

### 3

in the CHS ( Lee et al., 2009b ) . A study in five French cities reports effects on asthma 

### 4

severity related to long

- term O 3 exposure ( Rage et al., 2009b ) . A follow

- up study of this 

### 5

### Draft

– Do Not Cite or Quote 7

- 37 June 2012 cohort ( Jacquemin et al., In Press ) supports an effect of cumulative long

- term O 3 

### 1

exposure o n asthma control in adulthood in subjects with pre

- existing asthma. Akinbami 

### 2

et al. (2010 ) and Hwang et al. (2005 ) provide fur ther evidence relating O 3 exposures and 

### 3

the risk of asthma. For the respiratory health of a cohort based on the general U.S. 

### 4

population, risk of respiratory

- related school absences was elevated for children with the 

### 5

CAT and MPO variant genes related to com munities with high ambient O 3 levels 

### 6

( Wenten et al., 2009 )

. 

### 7

Long

- term O 3 exposure was related to first childhood asthma hospital admissions in a 

### 8

positive concentration

- response relationship in a New York State birth cohort ( Lin et al., 

### 9

2008b ) . A separate hospitalization cross

- sectional study in San Joaquin Valley, California 

### 10

reports similar findings ( Meng et al., 2010 ) . Another study relates asthma hospital 

### 11

admissions to quarterly average O 3 in the South Coast Air Basi n of California ( Moore et 

### 12

al., 2008 )

. 

### 13

Information from toxicological studies indicates that long term exposure to O 3 during 

### 14

gestation or development can result in irrever sible morphological changes in the lung, 

### 15

which in turn can influence the function of the respiratory tract . Studies by Plopper and 

### 16

colleagues using an allergic asthma model have demonstrated changes in pulmonary 

### 17

function and airway morphology in adult and infant nonhuman primates repeatedly 

### 18

exposed to environmentally relevant concentrations of O 3 ( Fanucc hi et al., 2006 ; Joad et 

### 19

al., 2006 ; Schelegle et al., 2003 ; Harkema et al., 1987b ) . This nonhuman primate 

### 20

evidence of an O 3 - induced change in airway responsiveness supports the biologic 

### 21

plausibility of long term exposure to O 3 contributing to effects of asthma in children. 

### 22

Results from epidemiologic studies examining long

- term O 3 exposure and pulmonary 

### 23

function effects are inconclusive with some new studies relating effects at higher 

### 24

exposure levels. The definitive 8

- year foll ow

- up analysis of the first cohort of the CHS, 

### 25

which is discussed in Section 7.2 ( Gauderman et al., 2004 ) , provided little evidence that 

### 26

long

- term exposure to ambient O 3 was associated with significa nt deficits in the growth 

### 27

rate of lung function in children. Other cross

- sectional studies provide mixed results. 

### 28

Several studies (see Table 7

- 3 ) provide results adjusted for potential confounders, 

### 29

presenting results for both O 3 and PM (single and multipollutant models) as well as other 

### 30

pollutants where PM effects were not provided. As shown in the t able, O 3 associations 

### 31

are generally robust to adjustment for potential confounding by PM. 

### 32

### Draft

– Do Not Cite or Quote 7

- 38 June 2012 Table 7

- 3 Studies providing evidence concerning potential confounding by PM for available endpoints.

Study Endpoint Exposure Single
Pollutant O 3 Single Pollutant

PM O 3 with PM PM with O 3

Asthma Related Health Effect Endpoint

Akinbami et al. (2010 )

Asthma prevalence in
children IQR

35.9

- 43.7 ppb 1.56 (1.15, 2.10) PM 2.5 1.43 (0.98, 2.10) Adjusted for
SO 2 ,PM 2.5 ,PM 10 1.86 (1.02

- 3.40)

Adjusted for
PM 2.5 , PM 10 1.36 (0.91

- 2.02) PM 2.5 1.24 (0.70

- 2.21)

PM 2.5 1.26
0.80

- 1.98)

Hwang et al. (2005 )

Asthma risk in children 10 ppb O 3 1.138 (1.001, 1.293 0.934 (0.909, 0.960) PM 10 1.253 (1.089, 1.442) 0.925 (0.899, 0.952)

Jacquemin et al. (In
Press )

Asthma control in adults IQR 25

- 38 ppb O 3

summer 1.69 (1.22, 2.34) 1.33 (1.06, 1.67) PM 10 1.50 (1.07, 2.11) 1.28 (1.06, 1.55)

Lee et al. (2009b )

Bronchitic symptoms
asthmatics High O 3 >50 ppb 1.42 (0.75, 2.70) NA No substantial
differences

PM 10 , PM 2.5 NA

Lin et al. (2008b )

Asthma admissions in
children IQR 2.5% 1.16 (1.15, 1.17) NA Air Quality Index

1.24 (1.23, 1.25) NA

Meng et al. (2007 )

Asthma control 1 ppm 1.70 (0.91, 3.18) PM 10 2.06 (1.17, 3.61)

women Did not differ NA

Meng et al. (2010 )

Asthma ED visits, Hospitalization 10 ppb 1.49 (1.05, 2.11) PM 10 1.29 (0.99, 1.69) Did not differ NA

Rage et al. (2009b )

Asthma severity in adults IQR

28.5

- 33.9 ppb 2.53 (1.69, 3.79) NA No PM data

Thr ee pollutant (O 3 , NO 2 , SO 2 )

2.74 (1.68, 4.48) NA

Other Respiratory Health Effect Endpoints

Karr et al. (2007 )

Bronchiolitis

Hospitalization 10 ppb 0.92 (0.88, 0.96) 1.09 (1.04, 1.14) PM 2.5 1.02 (0.94, 1.10) 1.09 (1.03, 1.15)

Parker et al. (2009 )

Respiratory allergy 10 ppb 1.24 (1.15, 1.34) 1.23 (1.04, 1.46) Multi

- pollu tant
1.18 (1.09, 1.27) 1.29 ( 1.07, 1.56)

Rojas

- Martinez et al. (2007 )

FEV 1 (mL) Deficit

Girls 11.3 ppb IQR

- 24 (

- 30,

- 19) PM 10 IQR 36.4 ug/m 3

- 29(

- 36,

- 21) - 17 (

- 23,

- 12) - 24 (

- 31,

- 16)

The highest quartile is shown for all results

NA = not available

### Draft

– Do Not Cite or Quote 7

- 39 June 2012 There is limited evidence for an association between long

- term exposure to ambient O 3 

### 1

concentrations and respiratory mortality ( Jerrett et al., 2009 ) and this effect was robust to 

### 2

the inclusion of PM 2.5 . The association between increased O 3 c oncentrations and 

### 3

increased risk of death from respiratory causes was insensitive to a number of different 

### 4

model specifications. Additionally, there is evidence that long

- term exposure to O 3 is 

### 5

associated with mortality among individuals that had previousl y experienced an 

### 6

emergency hospital admission due to COPD ( Zanobetti and Schwartz, 2011 )

. 

### 7

Taken together, the recent epidemiologic studies of respiratory health effects (including 

### 8

respiratory symptoms, new

- onset asthma and respiratory mortality) combined with 

### 9

toxicological studies in rodents and nonhuman primates, provide biologically plausible 

### 10

evidence that there is likely to be a causal relationship between long

- term exposure 

### 11

to O 3 and respiratory effects. The strongest epidemiologic evidence f or a relationship 

### 12

between long

- term O 3 exposure and respiratory effects is provided by studies that 

### 13

demonstrate interactions between exercise or different genetic variants and long

- term 

### 14

measures of O 3 exposure on new

- onset asthma in children; and increased respiratory 

### 15

symptom effects in asthmatics. Additional studies of respiratory health effects and a 

### 16

study of respiratory mortality provide a collective body of evidence supporting these 

### 17

relationships. Studies considering other pollutants provide data sugges ting that the effects 

### 18

related to O 3 are independent from potential effects of the other pollutants. Some studies 

### 19

provide evidence for a positive concentration

- response relationship. Short

- term studies 

### 20

provide supportive evidence with increases in respirato ry symptoms and asthma 

### 21

medication use, hospital admissions and ED visits for all respiratory outcomes and 

### 22

asthma, and decrements in lung function in children. The recent epidemiologic and 

### 23

toxicological data base provides a compelling case to support the hy pothesis that a 

### 24

relationship exists between long

- term exposure to ambient O 3 and measures of 

### 25

respiratory health effects. 

### 26



## 7.3 Cardiovascular Effects

7.3.1 Cardiovascular Disease

7.3.1.1 Cardiovascular Epidemiology

Long

- term exposure to O 3 and its effects on cardiovascular morbidity were not 

### 27

considered in the 2006 O 3 AQCD. However, recent studies have assessed the chronic 

### 28

effects of O 3 concentration on cardiovascular morbidity ( Chuang et al., 2011 ; Forbes et 

### 29

### Draft

– Do Not Cite or Quote 7

- 40 June 2012 al., 200 9a ; Chen et al., 2007a ) . The association between O 3 concentration and markers of 

### 1

lipid peroxidation and antioxidant capacity was examined among 120 nonsmoking 

### 2

healthy college students, aged 18

- 22 years, from the University of California, Berkeley 

### 3

(Feb ruary

— Jun e 2002) ( Chen et al., 2007a ) . By design, students were chosen from 

### 4

geographic areas so they had experienced different concentrations of O 3 over their 

### 5

lifetimes and during recent summer vacation in either grea ter Los Angeles (LA) or the 

### 6

San Francisco Bay Area (SF). A marker of lipid peroxidation, 8

- isoprostane (8

- iso

- PGF) 

### 7

in plasma, was assessed. This marker is formed continuously under normal physiological 

### 8

conditions but has been found at elevated concentratio ns in response to environmental 

### 9

exposures. A marker of overall antioxidant capacity, ferric reducing ability of plasma 

### 10

(FRAP), was also measured. The lifetime average O 3 concentration estimates (from 

### 11

estimated monthly averages) did not show much overlap be tween the two geographic 

### 12

areas [median (range): LA, 42.9 ppb (28.5

- 65.3); SF, 26.9 ppb (17.6

- 33.5)]. Estimated 

### 13

lifetime average O 3 concentration was related to 8

- iso

- PGF [β = 0.025 (pg/mL)/8

- h ppb 

### 14

O 3 , p = 0.0007]. For the 17

- ppb lifetime O 3 concentration d ifference between LA and SF 

### 15

participants, there was a 17.41

- pg/mL (95% CI: 15.43, 19.39) increase in 8

- iso

- PGF. No 

### 16

evidence of association was observed between lifetime O 3 concentration and FRAP 

### 17

[β =

- 2.21 (pg/mL)/8

- h ppb O 3 , p = 0.45]. The authors note th at O 3 was highly correlated 

### 18

with PM 10

- 2.5 and NO 2 in this study population; however, their inclusion in the O 3 models 

### 19

did not substantially modify the magnitude of the associations with O 3 . Because the 

### 20

average lifetime concentration results were supported by shorter

- term exposure period 

### 21

results from analyses considering O 3 concentrations up to 30 days prior to sampling, the 

### 22

authors conclude that persistent exposure to O 3 can lead to sustained oxidative stress and 

### 23

increased lipid peroxidation. However, becau se there was not much overlap in average 

### 24

lifetime O 3 concentration estimates between LA and SF, it is possible that the risk 

### 25

estimates involving the lifetime O 3 exposures could be confounded by unmeasured 

### 26

factors related to other differences between the tw o cities. 

### 27

Forbes et al. (2009a ) used the annual average exposures to assess the relationship 

### 28

between chronic ambient air pollution and levels of fibrinogen and C

- reactive protein 

### 29

(CRP) in a cross

- sectional study conducted in England. Data were c ollected from the 

### 30

Health Survey of England for 1994, 1998, and 2003. The sampling strategy was designed 

### 31

to obtain a representative sample of the English population; however, due to small group 

### 32

sizes, only data from white ethnic groups were analyzed. For an alyses, the annual 

### 33

concentrations of O 3 were averaged for the year of data collection and the previous year 

### 34

with the exception of 1994 (because pollutant data were not available for 1993). Median 

### 35

O 3 concentrations were 26.7 ppb, 25.4 ppb, and 28 ppb for 19 94, 1998, and 2003, 

### 36

respectively. Year specific adjusted effect estimates were created and combined in a 

### 37

meta

- analysis. No evidence of association was observed for O 3 and levels of fibrinogen 

### 38

or CRP (e.g., the combined estimates for the percent change in f ibrinogen and CRP for a 

### 39

### Draft

– Do Not Cite or Quote 7

- 41 June 2012 10 ppb increase in O 3 were

- 0.28 [95% CI:

- 2.43, 1.92] and

- 3.05 [95% CI:

- 16.10, 

### 1

12.02], respectively). 

### 2

A study was performed in Taiwan to examine the association between long

- term O 3 

### 3

concentrations and blood pressure and blood ma rkers using the Social Environment and 

### 4

Biomarkers of Aging Study (SEBAS) ( Chuang et al., 2011 ) . Individuals included in the 

### 5

study were 54 years of age and older. The mean annual O 3 concentration during the study 

### 6

period was 22.95 ppb (SD 6.76 ppb). Positive associations were obs erved between O 3 

### 7

concentrations and both systolic and diastolic blood pressure [changes in systolic and 

### 8

diastolic blood pressure were 21.51mmHg (95% CI: 16.90, 26.13) and 20.56 mmHg 

### 9

(95% CI: 18.14, 22.97) per 8.95 ppb increase in O 3 , respectively). Increas ed O 3 

### 10

concentrations were also associated with increased levels of total cholesterol, fasting 

### 11

glucose, hemoglobin A1c, and neutrophils. No associations were observed between O 3 

### 12

concentrations and triglyceride and IL

- 6 levels. The observed associations were reduced 

### 13

when other pollutants were added to the models. Further research will be important for 

### 14

understanding the effects, if any, of chronic O 3 exposure on cardiovascular morbidity 

### 15

risk. 

### 16

7.3.1.2 Cardiovascular Toxicology

Three new studies have investigated the ca rdiovascular effects of long

- term exposure to 

### 17

O 3 in animal models (See Table 7

- 3 for study details). In addition to the short

- term 

### 18

exposure effects de scribed in Section 6.3.3 , a recent study found that O 3 exposure in 

### 19

genetically hyperlipidemic mice enhanced aortic atherosclerotic lesion area compa red to 

### 20

air exposed controls ( Chuang et al., 2009 )

. Chuang et al. (2009 ) not only provided 

### 21

evidence for increased atherogenesis in susceptible mice, but also reported an elevated 

### 22

vascular inflammatory and redox state in wild

- type mice and infant primates 

### 23

(Section 6.3.3 ). This study is compelling in that it identifies biochemical and cellular 

### 24

events responsible for transducing the airway epithelial reactions of O 3 into 

### 25

proinflammatory responses that are apparent in the extrapulmonary vasculature ( Cole and 

### 26

Freeman, 2009 )

. 

### 27

Another recent study provides further evidence for increased vascular in flammation and 

### 28

oxidation and long term effects in the extrapulmonary space. Rats episodically exposed to 

### 29

O 3 for 16 weeks presented marked increases in gene expression of biomarkers of 

### 30

oxidative stress, thrombosis, vasoconstriction, and proteolysis ( Kodavanti et al., 2011 )

. 

### 31

Ozone exposure upregulated aortic mRNA expression of heme oxygenase

- 1 (HO

- 1), 

### 32

tissue plasminogen activator (tPA), plasminogen activator inhibitor

- 1 (PAI

- 1), von 

### 33

Willebrand factor (vWf), thrombomodulin, endothelial nitric oxide synthase (eNOS), 

### 34

### Draft

– Do Not Cite or Quote 7

- 42 June 2012 endothelin

- 1 (ET

- 1), matrix metalloprotease

- 2 (MMP

- 2), matrix metalloprotease

- 3 

### 1

(MMP

- 3), and tissue inhibitor of matri x metalloprotease

- 2 (TIMP

- 2). In addition, O 3 

### 2

exposure depleted some cardiac mitochondrial phospholipid fatty acids (C16:0 and 

### 3

C18:1), which may be the result of oxidative modifications. The authors speculate that 

### 4

oxidatively modified lipids and proteins p roduced in the lung and heart promote vascular 

### 5

pathology through activation of lectin

- like oxidized

- low density lipoprotein receptor

- 1 

### 6

(LOX

- 1). Activated LOX

- 1 induces expression of a number of the biomarkers induced by 

### 7

O 3 exposure and is considered pro

- at herogenic. Both LOX

- 1 mRNA and protein were 

### 8

increased in mouse aorta after O 3 exposure. This study provides a possible pathway and 

### 9

further support to the observed O 3 induced atherosclerosis. 

### 10

Vascular occlusion resulting from atherosclerosis can block blood flow through vessels 

### 11

causing ischemia. The restoration of blood flow or reperfusion can cause injury to the 

### 12

tissue from subsequent inflammation and oxidative damage. Ozone exposure enhanced 

### 13

the sensitivity to myocardial ischemia

- reperfusion (I/R) injury i n rats while increasing 

### 14

oxidative stress levels and pro

- inflammatory mediators and decreasing production of 

### 15

anti

- inflammatory proteins ( Perepu et al., 2010 ) . Both long

- and short

- term O 3 exposure 

### 16

decreased the left ventricular developed pressure, rate of change of pressure 

### 17

development, and rate of change of pressure decay and incr eased left ventricular end 

### 18

diastolic pressure in isolated perfused hearts (Section 6.3.3 for short

- term exposure 

### 19

discussion). In this ex vivo heart model, O 3 induced oxidative stress by decreasing SOD 

### 20

enzyme activity and increasing malondialdehyde levels. Ozone also elicited a 

### 21

proinflammatory state evident by an increase in TNF

- α and a decrease in the 

### 22

anti

- inflammatory cytokine IL

- 10. The authors conc lude that O 3 exposure will result in a 

### 23

greater I/R injury. 

### 24

Overall, the few animal studies that have been conducted suggest that long

- term O 3 

### 25

exposure may result in cardiovascular effects. These studies demonstrate O 3 - induced 

### 26

atherosclerosis and injury. In addition, evidence is presented for a potential mechanism 

### 27

for the development of vascular pathology that involves increased oxidative stress and 

### 28

proinflammatory mediators, activation of LOX

- 1 by O 3 oxidized lipids and proteins, and 

### 29

upregulation of genes r esponsible for proteolysis, thrombosis, and vasoconstriction. 

### 30

Further discussion of the mechanisms that may lead to cardiovascular effects from O 3 

### 31

exposure can be found in Section 5.3.8

. 

### 32

### Draft

– Do Not Cite or Quote 7

- 43 June 2012 Table 7

- 3 Characterization of Study Details for Section 7.3.1.2.

Study Model O 3 (ppm) Exposure Duration Effects

Chuang et al. (2009 ) Mice; ApoE - /

- ; M;

6 weeks 0.5 8 wks, 5 days/week,

8 h/day Enhanced aortic atherosclerotic lesion area compared to air controls.

Kodavanti et al. (2011 ) Rat; Wistar; M;

10

- 12 weeks 0.4 16 wks, 1 day/week,

5 h/day Increased vascular inflammation and oxidative stress, possibly through
activation of LOX

- 1 signaling.

Perepu et al. (2010 ) Rat; Sprague

- Dawley; Weight: 50

- 75 g 0.8 56 days, 8 h/day Enhanced the sensitivity to myocardial I/R injury while increasing oxidative stress and pro

- inflammatory mediators and decreasing production of
anti

- inflammatory proteins.

No previous studies investigated cardiovascular effects from long

- term exposure to O 3

. For details, see Section 7.3.1.2



### 7.3.2 Cardiovascular Mortality

A limited number of epidemiologic studies have assessed the relationship between long



### 1

term exposure to O 3 and mortality. The 2006 O 3 AQCD concluded that an insuffic ient 

### 2

amount of evidence existed “to suggest a causal relationship between chronic O 3 

### 3

exposure and increased risk for mortality in humans” ( U.S. EPA, 2006b ) . Though total 

### 4

and cardio

- pulmonary mortality were considered in these studies, cardiovascular 

### 5

mortality was not specifically considered. In the most recent follow

- up analysis of the 

### 6

ACS cohort ( Jerrett et al., 2009 ) , cardiopulmonary deaths were subdivided into 

### 7

respiratory and cardiovascular, separately, as opposed to combined in the Pope et al. 

### 8

(2002 ) work. A 10

- ppb increment in exposure to O 3 elevated the risk of death from the 

### 9

cardiopulmonary, cardiovascular, and ischemic heart disease. Inclusion of PM 2.5 as a 

### 10

copollutant attenuated the association with exposure to O 3 for all of the cardiovascular 

### 11

endpoints to become null. Additionally, a recent study ( Zanobetti and Schwartz, 2011 ) 

### 12

observed an association between long

- term exposure to O 3 and elevated risk of mortality 

### 13

among Medicare enrollees that had previously experienced an emergency hospital 

### 14

admission due to congestive heart failure (CHF) or myocardial infarction (MI). 

### 15

7.3.3 Summary and Causal Determination

Previous AQCDs did not address t he cardiovascular effects of long

- term O 3 exposure due 

### 16

to limited data availability. The evidence remains limited; however the emerging data is 

### 17

supportive of a role for O 3 in chronic cardiovascular diseases. Few epidemiologic studies 

### 18

have investigated cardiovascular morbidity after long

- term O 3 exposure, and the majority 

### 19

only assessed cardiovascular disease related biomarkers. The studies used annual or 

### 20

### Draft

– Do Not Cite or Quote 7

- 44 June 2012 multi

- year averages of air monitoring data for exposure assessment. As described in 

### 1

Section 4.6 , this exposure assignment method is typical of long

- term epidemiologic 

### 2

studies, and analyses suggest that annual average concentrations are representative of 

### 3

exposure metrics accounting for residential mobility. A study on O 3 and cardiovascular 

### 4

mortality reported no association after adjustment for PM 2.5 levels. Further epide miologic 

### 5

studies on cardiovascular morbidity and mortality after long

- term exposure have not been 

### 6

published. 

### 7

Toxicological evidence on long

- term O 3 exposure is also limited but three strong 

### 8

toxicological studies have been published since the previous AQCD. These studies 

### 9

provide evidence for O 3 enhanced atherosclerosis and I/R injury, corresponding with 

### 10

development of a systemic oxidative, proinflamma tory environment. Further discussion 

### 11

of the mechanisms that may lead to cardiovascular effects can be found in Section 5.3.8

. 

### 12

Although questions exi st for how O 3 inhalation causes systemic effects, a recent study 

### 13

proposes a mechanism for development of vascular pathology that involves activation of 

### 14

LOX

- 1 by O 3 oxidized lipids and proteins. This activation may also be responsible for O 3 

### 15

induced changes in genes involved in proteolysis, thrombosis, and vasoconstriction. 

### 16

Taking into consideration the findings of toxicological studies, and the emerging 

### 17

evidence from epidemiologic studies, the generally limited body of evidence is 

### 18

suggestive of a causal rel ationship between long

- term exposures to O 3 and 

### 19

cardiovascular effects. 

### 20



## 7.4 Reproductive and Developmental Effects

Although the body of literature characterizing the health effects associated with exposure 

### 21

to O 3 is large and continues to grow, the research f ocusing on adverse birth outcomes is 

### 22

relatively small. Among these studies, various measures of birth weight and fetal growth, 

### 23

such as low birth weight (LBW), small for gestational age (SGA), and intrauterine 

### 24

growth restriction (IUGR), and preterm birth (< 37

- week gestation; [PTB]) have received 

### 25

more attention in air pollution research, while congenital malformations are less studied. 

### 26

There are also recent studies on reproductive and developmental effects and infant 

### 27

mortality. 

### 28

A major issue in studying envir onmental exposures and reproductive and developmental 

### 29

effects (including infant mortality) is selecting the relevant exposure period, since the 

### 30

biological mechanisms leading to these outcomes and the critical periods of exposure are 

### 31

poorly understood. To a ccount for this, many epidemiologic studies evaluate multiple 

### 32

exposure periods, including long

- term (months to years) exposure periods, such as entire 

### 33

pregnancy, individual trimesters or months of pregnancy, and short

- term (days to weeks) 

### 34

### Draft

– Do Not Cite or Quote 7

- 45 June 2012 exposure periods such as the days and weeks immediately preceding birth. Due to the 

### 1

length of gestation in rodents (18

- 24 days, on average), animal toxicological studies 

### 2

investigating the effects of O 3 generally utilize short

- term exposure periods. Thus, an 

### 3

epidemiologic s tudy that uses the entire pregnancy as the exposure period is considered 

### 4

to have a long

- term exposure period (about 40 weeks, on average), while a toxicological 

### 5

study conducted with rats that also uses the entire pregnancy as the exposure period is 

### 6

conside red to have a short

- term exposure period (about 18

- 24 days, on average). In order 

### 7

to characterize the weight of evidence for the effects of O 3 on reproductive and 

### 8

developmental effects in a consistent, cohesive and integrated manner, results from both 

### 9

shor t

- term and long

- term exposure periods are included in this section and are identified 

### 10

accordingly in the text and tables throughout this section. 

### 11

Due to the poorly understood biological mechanisms and uncertainty regarding relevant 

### 12

exposure studies, all o f the studies of reproductive and developmental outcomes, 

### 13

including infant mortality, are evaluated in this section. Infant development processes, 

### 14

much like fetal development processes, may be particularly sensitive to O 3 - induced 

### 15

health effects. Exposures proximate to the death may be most relevant if exposure causes 

### 16

an acute effect. However, exposure occurring in early life might affect critical growth and 

### 17

development, with results observable later in the first year of life, or cumulative exposure 

### 18

during t he first year of life may be the most important determinant. In dealing with the 

### 19

uncertainties surrounding these issues, studies have considered several exposure metrics 

### 20

based on different periods of exposure, including both short

- and long

- term exposure 

### 21

p eriods. In the toxicological literature,a challenge in interpreting data from studies that 

### 22

use very young murine pups, is that pups can have differential exposure to O 3 doses, 

### 23

versus their respective dams, because of the physiology and behavior associated with the 

### 24

early postnatal period. Namely, young pups tend to nuzzle close to their mothers and are 

### 25

often housed in cages with litter used in nest formation. Both the dam's fur and the 

### 26

bedding can absorb and react with O 3 , decreasing the dose that a young an imal might 

### 27

receive. The reproductive and developmental studies are characterized in this chapter, as 

### 28

they contribute to the weight of evidence for an effect of O 3 on reproductive and 

### 29

developmental effects. 

### 30

Infants and fetal development processes may be particularly at

- risk for O 3 - induced health 

### 31

effects, and although the physical mechanisms are not fully understood, several 

### 32

hypotheses have been proposed; these include: oxidative stress, systemic inflammation, 

### 33

vascular dysfunction and impaired immune funct ion (Section 5.3 ). Study of these 

### 34

outcomes can be difficult given the need for detailed exposure data and potential 

### 35

residential movement of mothers during pregnancy. Air pollution epidemiologic studies 

### 36

reviewed in the 2006 O 3 AQCD examined impacts on birth

- related endpoints, including 

### 37

intrauterine, perinatal, postneonatal, and infant deaths; premature births; intrauterine 

### 38

### Draft

– Do Not Cite or Quote 7

- 46 June 2012 growth retardation; very low birth weight (weight <1,500 grams) and low birth weight 

### 1

(weight <2,500 grams); and birth defects. However, in the limited number of studies that 

### 2

investigated O 3 , no associations were found between O 3 and birth outcomes, with the 

### 3

possible exception of birth defects. 

### 4

Several recent articles have reviewed methodological issues relating to the study of 

### 5

outdoor air pollution and adverse birth outcomes ( Chen et al., 2010a ; Woodruff et al., 

### 6

2009 ; Ritz and Wilhelm, 2008 ; Slama et al., 2008 ) . Some of the key challenges to 

### 7

interpretation of these study results include the difficulty in assessing exposu re as most 

### 8

studies use existing monitoring networks to estimate individual exposure to ambient air 

### 9

pollution; the inability to control for potential confounders such as other risk factors that 

### 10

affect birth outcomes (e.g., smoking); evaluating the exposure window (e.g., trimester) of 

### 11

importance; and limited evidence on the physiological mechanism of these effects ( Ritz 

### 12

and Wilhelm, 2008 ; Slama et al., 2008 )

. 

### 13

Ove rall, the evidence for an association between exposure to ambient O 3 and 

### 14

reproductive and developmental outcomes is growing, yet remains relatively small. 

### 15

Recently, an international collaboration was formed to better understand the relationships 

### 16

between ai r pollution and adverse birth outcomes and to examine some of these 

### 17

methodological issues through standardized parallel analyses in datasets from different 

### 18

countries ( Woodruff et al., 2010 ) . Initial results from this collaboration have examined 

### 19

PM and birth weight ( Parker et al., 2011 ) ; work on O 3 has not yet been performed. 

### 20

Altho ugh early animal studies ( Kavlock et al., 1980 ) found that exposure to O 3 in the late 

### 21

gestation of pregnancy in rats led to some abnormal reproductive performances for 

### 22

neonates, to da te human studies have reported inconsistent results for the association of 

### 23

ambient O 3 concentrations and birth outcomes. 

### 24

7.4.1 Effects on Sperm

A limited amount of research has been conducted to examine the association between air 

### 25

pollution and male reproductiv e outcomes, specifically semen quality. To date, the 

### 26

epidemiologic studies have considered various exposure durations before semen 

### 27

collection that encompass either the entire period of spermatogenesis (i.e., 90 days) or 

### 28

key periods of sperm development tha t correspond to epididymal storage, development of 

### 29

sperm motility, and spermatogenesis. In an analysis conducted as part of the Teplice 

### 30

Program, 18

- year

- old men residing in the heavily polluted district of Teplice in the Czech 

### 31

Republic were found to be at greater risk of having abnormalities in sperm morphology 

### 32

and chromatin integrity than men of similar age residing in Prachatice, a less polluted 

### 33

district ( Selevan et al., 2000 ; Sram et al., 1999 ) . A follow

- up longitudinal study 

### 34

### Draft

– Do Not Cite or Quote 7

- 47 June 2012 conducted on a subset of the same men from Teplice revealed associations between total 

### 1

episodic air pollution and abnormalities in sperm chromatin ( Rubes et al., 2005 ) . A 

### 2

limitation of these studies is that they did not identify specific pollutants or their 

### 3

concentrations. 

### 4

More recent epidemiologic studies conducted in the U.S. have also reported associations 

### 5

be tween ambient air pollution and sperm quality for individual air pollutants, including 

### 6

O 3 and PM 2.5 . In a repeated measures study in Los Angeles, CA, Sokol et al. (2006 ) 

### 7

reported a reduction in average sperm concentration during three exposure windows 

### 8

(short

- term exposures of 0

- 9, 10

- 14, and 70

- 90 days before semen collection, as well as 

### 9

long

- term exposures of 0

- 90 days before semen collection) associated with high ambient 

### 10

levels of O 3 in healthy sperm donors. This effect persisted under a joint additive model 

### 11

for O 3 , CO, NO 2 and PM 10 . Th e authors did not detect a reduction in sperm count. Hansen 

### 12

et al. (2010 ) investigated the effect of exposure to O 3 and PM 2.5 (using the same exposure 

### 13

windows used by Sokol et al. (2006 ) on sperm quality in three southeastern counties 

### 14

(Wake County, NC; Shelby County, TN; Gal veston County, TX). Outcomes included 

### 15

sperm concentration and count, morphology, DNA integrity and chromatin maturity. 

### 16

Overall, the authors found both protective and adverse effects, although some results 

### 17

suggested adverse effects on sperm concentration, c ount and morphology. 

### 18

The biological mechanisms linking ambient air pollution to decreased sperm quality have 

### 19

yet to be determined, though O 3 - induced oxidative stress, inflammatory reactions, and the 

### 20

induction of the formation of circulating toxic species have been suggested as possible 

### 21

mechanisms (see Section 5.3.8 ). Decremental effects on testicular morphology have been 

### 22

demonstrated in a toxicological study with histological evidence of O 3 - induced depletion 

### 23

of germ cells in testicular tissue and decreased seminiferous tubule epithelial layer. 

### 24

Jedlinska

- Krakowska et al. (2006 ) demonstrated histopathological evidence of impaired 

### 25

spermatogenesis (round spermatids/ spermatocytes, giant spermatid cells, and focal 

### 26

epithelial desquam ation with denudation to the basement membrane). The exposure 

### 27

protocol used five

- month

- old adult rats exposed to O 3 as adults (long

- term exposure, 

### 28

0.5 ppm, 5 h/day for 50 days). This degeneration could be rescued by vitamin E 

### 29

administration, indicating an antioxidant effect. Vitamin C administration had no effect at 

### 30

low doses of ascorbic acid and exacerbated the O 3 - dependent damage at high doses, as 

### 31

would be expected as vitamin C can be a radical generator instead of an antioxidant at 

### 32

higher doses. In summa ry, this study provided toxicological evidence of impaired 

### 33

spermatogenesis with O 3 exposure that was rescued with certain antioxidant 

### 34

supplementation. 

### 35

Overall, there is limited epidemiologic evidence for an association with O 3 concentration 

### 36

and decreased sperm concentration. A recent toxicological study provides limited 

### 37

### Draft

– Do Not Cite or Quote 7

- 48 June 2012 evidence for a possible biological mechanism (histopathology showing impaired 

### 1

spermatogenesis) for such an association. 

### 2

7.4.2 Effects on Reproduction

Evidence suggests that exposure to air pollut ants during pregnancy may be associated 

### 3

with adverse birth outcomes, which has been attributed to the increased sensitivity of the 

### 4

fetus due to physiologic immaturity. Gametes (i.e., ova and sperm) may be even more at



### 5

risk, especially outside of the human body, as occurs with assisted reproduction. Smokers 

### 6

require twice the number of in vitro fertilization (IVF) attempts to conceive as non



### 7

smokers ( Feichtinger et al., 1997 ) , suggesting that a preconception exposure can be 

### 8

harmful to pregnancy. A recen t study used an established national

- scale, log

- normal 

### 9

kriging method to spatially estimate daily mean concentrations of criteria pollutants at 

### 10

addresses of women undergoing their first IVF cycle and at their IVF labs from 2000 to 

### 11

2007 in the northeastern U.S. ( Legro et al., 2010 ) . Increasing O 3 concentration at the 

### 12

patient’s address during ovulation ind uction (short

- term exposure, ~12 days) was 

### 13

significa ntly associated with an increased chance of live birth (OR = 1.13, [95% CI: 1.05, 

### 14

1.22] per 10 ppb increase), but with decreased odds of live birth when exposed from 

### 15

embryo transfer to live birth (long

- te rm exposure, ~200 days) (OR = 0.79, [95% CI: 0.69, 

### 16

0.90] per 10 ppb increase). After controlling for NO 2 in a copollutant model, however, O 3 

### 17

was no longer significa ntly associated with IVF failure. The results of this study suggest 

### 18

that short

- term exposure to O 3 during ovulation was beneficial (perhaps due to early 

### 19

conditioning to O 3 ), whereas long

- term exposure to O 3 (e.g., during gestation) was 

### 20

detrimental, and reduced the likelihood of a live birth. 

### 21

In most toxicological studies, reproductive success app ears to be unaffected by O 3 

### 22

exposure. Nonetheless, one study has reported that 25% of the BALB/c mouse dams in 

### 23

the highest O 3 exposure group (1.2 ppm, short

- term exposure GD9

- 18) did not complete 

### 24

a successful pregnancy, a significa nt reduction ( Sharkhuu et al., 2 011 ) . Ozone 

### 25

administration (continuous 0.4, 0.8 or 1.2 ppm O 3 ) to CD

- 1 mouse dams during the 

### 26

majority of pregnancy (short

- term exposure, PD7

- 17, which excludes the 

### 27

pre

- implantation period), led to no adverse effects on reproductive success (proportion of 

### 28

successful pregnancies, litter size, sex ratio, frequency of still birth, or neonatal mortality) 

### 29

( Bignami et al., 1994 ) . There was a nearly statistically significa nt increase in pregna ncy 

### 30

duration (0.8 and 1.2 ppm O 3 ). Initially, dam body weight (0.8 and 1.2 ppm O 3 ), water 

### 31

consumption (0.4, 0.8 and 1.2 ppm O 3 ) and food consumption (0.4, 0.8 and 1.2 ppm O 3 ) 

### 32

during pregnancy were decreased with O 3 exposure but these deficits dissipated a week or 

### 33

two after the initial exposure ( Bignami et al., 1994 ) . The anorexigenic effect of O 3 

### 34

exposure on the pregnant dam appears to dissipate with time; the dams seem to adapt to 

### 35

### Draft

– Do Not Cite or Quote 7

- 49 June 2012 the O 3 exposure. In males, data exist showing morphological evidence of altered 

### 1

spermatogen esis in O 3 exposed animals ( Jedlinska

- Krakowska et al., 2006 ) . Some 

### 2

evidence suggests that O 3 may affec t reproductive success when combined with other 

### 3

chemicals. Kavlock et al. (1979 ) showed that O 3 acted synergistically with sodium 

### 4

salicylate to increase the rate of pup resorptions after midgestational exposure (1.0 ppm 

### 5

O 3 , short

- term exposure, GD9

- GD12). At low concentrations of O 3 exposure, 

### 6

toxicological studies show r eproductive effects to include a transient anorexigenic effect 

### 7

of O 3 on gestational weight gain, and a synergistic effect of O 3 on salicylate

- induced pup 

### 8

resorptions; other fecundity, pregnancy

- and gestation

- related outcomes appear 

### 9

unaffected by O 3 exposu re. 

### 10

Collectively, there is very little epidemiologic evidence for the effect of short

- or long



### 11

term exposure to O 3 on reproductive success, and the reproductive success in rats appears 

### 12

to be unaffected in toxicological studies of short

- term exposure to O 3

. 

### 13

7.4.3 Birth Weight

With birth weight routinely collected in vital statistics and being a powerful predictor of 

### 14

infant mortality, it is the most studied outcome within air pollution

- birth outcome 

### 15

research. Air pollution researchers have analyzed birth weight a s a continuous variable 

### 16

and/or as a dichotomized variable in the form of LBW (<2,500 g [5 lbs, 8 oz]). 

### 17

Birth weight is primarily determined by gestational age and intrauterine growth, but also 

### 18

depends on maternal, placental and fetal factors as well as on environmental influences. 

### 19

In both developed and developing countries, LBW is the most important predictor for 

### 20

neonatal mortality and is a significa nt determinant of postneonatal mortality and 

### 21

morbidity. Studies report that infants who are smallest at birth have a higher incidence of 

### 22

diseases and disabilities, which continue into adulthood ( Hack and Fanaroff, 1999 )

. 

### 23

The strongest evidence for an effect of O 3 on birth weight comes from the Children’s 

### 24

Health Study conducted in southern California. In this study, Salam et al. (2005 ) report 

### 25

that maternal exposure to 24

- h avg O 3 concentrations averaged over the entire pregnancy 

### 26

was associated with reduced birth weight (39.3 g decrease [95% CI:

- 55.8,

- 22.8] in birth 

### 27

weight per 10 ppb and 8

- h avg (19.2

- g decrease [95% CI:

- 27.7,

- 10.7] in birth weight per 

### 28

10 ppb). This effect was stronger for concentrations averaged over the second and third 

### 29

trimesters. PM 10 , NO 2 and CO concentrations averaged over the entire pregnancy were 

### 30

not statistically significa ntl y associated with birth weight, although CO concentrations in 

### 31

the first trimester and PM 10 concentrations in the third trimester were associated with a 

### 32

decrease in birth weight. Additionally, the authors observed a concentration

- response 

### 33

relationship of bi rth weight with 24

- h avg O 3 concentrations averaged over the entire 

### 34

### Draft

– Do Not Cite or Quote 7

- 50 June 2012 pregnancy that was clearest above the 30

- ppb level (see Figure 7

- 4 ). Relative to t he 

### 1

lowest decile of 24

- h avg O 3 , estimates for the next 5 lowest deciles were approximately 

### 2

- 40 g to

- 50 g, with no clear trend and with 95% confidence bounds that included zero. 

### 3

The highest four deciles of O 3 exposure showed an approximately linear decrea se in birth 

### 4

weight, and all four 95% CIs excluded zero, and ranged from mean decreases of 

### 5

74 grams to decreases of 148 grams. 

### 6

Note: Deficits are plotted against the decile

- group

- specific median O 3 exposure. Error bars represent 95% CIs. Indicator variables
for each decile of O 3 exposure (except the least

- exposed group) were included in a mixed model.

Source: Salam et al. (2005 ) .’



### Figure 7

- 4 Birthweight deficit by decile of 24

- h avg ozone concentration averaged over the entire pregnancy compared with the decile group with the lowest ozone exposure.

Several additional studies conducted in the U.S. and Canada also investigated the 

### 7

association between ambient O 3 concentrations and birth weight and report some weak 

### 8

evidence for an association. Morello

- Frosch et al. (2010 ) estimated ambient O 3 

### 9

concentrations throughout pregnancy and for each trimester in the neighborhoods of 

### 10

women who delivered term singleton births between 1996 and 2006 in California. A 

### 11

10

- ppb increase in the O 3 concentration averaged across the entire pregnancy was 

### 12

associated with a 5.7

- g decrease (95% CI:

- 6.6,

- 4.9) in birth weight when exposures 

### 13

were calculated using monitors within 10 km of the maternal address at date of birth. 

### 14

### Draft

– Do Not Cite or Quote 7

- 51 June 2012 When the di stance from the monitor was restricted to 3 km, the decrease in birth weight 

### 1

associated with a 10

- ppb increase in O 3 concentration was 8.9 g (95% CI:

- 10.6,

- 7.1). 

### 2

These results persisted in copollutant models and in models that stratified by trimester of 

### 3

exposure, SES, and race. Darrow et al. (2011b ) did not observe an association with birth 

### 4

weight and O 3 concentrations during two exposure periods of interest (i.e., the first month 

### 5

and last trimester), but did find an association with reduced birth weight w hen examining 

### 6

the cumulative air pollution concentration during the entire pregnancy period. 

### 7

Additionally, they observed effect modification by race and ethnicity, such that 

### 8

associations between birth weight and third

- trimester O 3 concentrations were 

### 9

signi fica ntly stronger in Hispanics and non

- Hispanic African Americans than in non



### 10

Hispanic whites. Chen et al. (2002 ) used 8

- h avg O 3 concentrations to create exposure 

### 11

variables based on average maternal exposure for each trimester. Ozone was not found to 

### 12

be related to birth weight in single

- poll utant models, though the O 3 effect during the third 

### 13

trimester was borderline statistically significa nt in a copollutant model with PM 10

. 

### 14

Several studies found no association between ambient O 3 concentrations and birth 

### 15

weight. Wilhelm and Ritz (2005 ) extended previous analyses of term LBW ( Ritz et al., 

### 16

2000 ; Ritz and Yu, 1999 ) to include the period 1994

- 2000. The authors examined varying 

### 17

residential distances from monitoring stations to see if the distance affected risk 

### 18

estimation, exploring the possibility that effect attenuation may result from local pollutant 

### 19

heterogeneity inadequately captured by ambi ent monitors. As in their previous studies, 

### 20

the authors observed associations between elevated concentrations of CO and PM 10 both 

### 21

early and late in pregnancy and risk of term LBW. After adjusting for CO and/or PM 10 

### 22

the authors did not observe associations between O 3 and term LBW in any of their 

### 23

models. Brauer et al. (2008 ) evaluated the impacts of air pollution (CO, NO 2 , NO, O 3 , 

### 24

SO 2 , PM 2.5 , PM 10 ) on birth weight for the period 1999

- 2002 using spatiotemporal 

### 25

residential exposure metrics by month of pregnancy in Va ncouver, BC. Quantitative 

### 26

results were not presented for the association between O 3 and LBW, though the authors 

### 27

observed associations that were largely protective. Dugandzic et al. (2006 ) examined the 

### 28

association between LBW and ambient levels of air pollutants by trimester of exposure 

### 29

among a cohort of term singleton births from 1988

- 2000. Though there w as some 

### 30

indication of an association with SO 2 and PM 10 , there were no effects for O 3

. 

### 31

Similarly, studies conducted in Australia, Latin America, and Asia report limited 

### 32

evidence for an association between ambient O 3 and measures of birth weight. In Sydney, 

### 33

Australia, Mannes et al. (2005 ) found that O 3 concentrations in the second trimester of 

### 34

pregnancy had small adverse effects on birth weight (7.5

- g decrease; [95% CI:

- 13.8, 1.2] 

### 35

per 10 ppb), although this effect disappeared when the analysis was limi ted to births with 

### 36

a maternal address within 5 km of a monitoring station (87.7

- g increase; [95% CI: 10.5, 

### 37

164.9] per 10 ppb). Hansen et al. (2007 ) reported that trimester and monthly specific 

### 38

### Draft

– Do Not Cite or Quote 7

- 52 June 2012 exposures to all pollutants were not statistically significa ntly associated with a reduction 

### 1

in birth weight in Brisbane, Australia. In Sao Paul o, Brazil, Gouveia et al. (2004 ) found 

### 2

that O 3 exhibited a small inverse relation with birth weight over the third trimester (6.0

- g 

### 3

decrease; [95% CI:

- 30.8, 18.8] per 10 ppb). Lin et al. (2004b ) reported a positive, though 

### 4

not statistically significa nt, exposure

- response relationship for O 3 during the entire 

### 5

pregnancy in a Taiwanese study. In a study performed in Korea, Ha et al. (2001 ) reported 

### 6

no O 3 effect during the first trimester of pregnancy, but they found that during the third 

### 7

trimester of pregnancy O 3 was associ ated with LBW (RR = 1.05 [95% CI: 1.02, 1.08] per 

### 8

10 ppb). 

### 9

Table 7

- 4 Brief Summary of Epidemiologic Studies of Birth Weight.

Study Location
Sample Size Mean O 3 (ppb) Exposure assessment Effect Estimate a (95% CI)

Salam et al. (2005 ) California, U.S.

(n = 3,901) 24

- h avg:

27.3

8 h:

50.6 ZIP code level Entire pregnancy:

- 39.3 g (

- 55.8,

- 22.8)

T1:

- 6.1 g (

- 16.8, 4.8)

T2:

- 20.0 g (

- 31.7,

- 8.4)

T3:

- 20.7 g (

- 32.1,

- 9.3)

Morello

- Frosch et al. (2010 ) California, U.S. (n = 3,545,177) 24

- h avg:

23.5 Nearest Monitor (within 10, 5, 3 km) Entire pregnancy:

- 5.7 g (

- 6.6,

- 4.9)

T1:

- 2.1 g (

- 2.9,

- 1.4)

T2:

- 2.3 g (

- 3.1,

- 1.5)

T3:

- 1.3 g (

- 2.1,

- 0.6)

Darrow et al. (2011b ) Atlanta, GA

(N=406,627) 8

- h max:

44.8 Population

- weighted spatial average Entire preg nancy:

- 12.3 g (

- 17.8,

- 6.8)

First 28 days

- 0.5 g (

- 3.0, 2.1)

T3:

- 0.9g (

- 4.5, 2.8)

Chen et al. (2002 ) Northern Nevada,
U.S.

(n = 36,305) 8

- h:

27.2 County level Entire pregnancy:
20.9 g (6.3, 35.5)
T1: 23.4 g (

- 35.6 , 82.4)

T2:

- 19.4 g (

- 77.0, 38.2)
T3: 7.7 g (

- 50.9, 66.3)

Wilhelm and Ritz (2005 ) Los Angeles County,
CA

(n = 136,134) 1

- h:

21.1

- 22.2 Varying distances from
monitor T1: NR

T3: NR

6 weeks before birth: NR

Brauer et al. (2008 ) Vancouver, BC,
Canada

(n = 70,249) 24

- h avg:

14 Nearest Monitor (within 10 km)

Inverse Distance Weighting (IDW) Entire pregnancy: NR
First 30 days of pregnancy: NR

Last 30 days of pregnancy: NR

T1: NR

T3: NR

Dugandzic et al. (2006 ) Nova Scotia, Canada

(n = 74,284) 24

- h avg:

21 Nearest Monitor (within 25 km) T1: 0.97 (0.81, 1.18) d

T2: 1.06 (0.87, 1.27) d

T3: 1.01 (0.83

- 1.24) d

### Draft

– Do Not Cite or Quote 7

- 53 June 2012 Study Location
Sample Size Mean O 3 (ppb) Exposure assessment Effect Estimate a (95% CI)

Mannes et al. (2005 ) Sydney, Australia

(n = 138,056) 1

- h max:

31.6 Citywide avg and
<5 km from monitor T1:

- 0.9 g (

- 6.6, 4.8)

T2:

- 7.5 g (

- 13.8, 1.2)

T3:

- 4.5 g (

- 10.8, 1.8)

Last 30 days:

- 1.1 g (

- 5.6, 3.4)

Hansen et al. (2007 ) Brisbane, Australia
(n = 26,617) 8 h max:

26.7 Citywide avg T1: 2.8 g (

- 10.5, 16.0)
T2: 4.4 g (

- 11.4, 20.1)
T3: 11.3 g (

- 4.4, 27.1)

Gouveia et al. (2004 ) Sao Paulo, Brazil
(n = 179,460) 1

- h max:

31.5 Citywide avg T1:

- 3.2 g (

- 25.6, 19)

T2:

- 0.2 g (

- 23.8, 23.4)

T3:

- 6.0 g (

- 30.8,

- 18.8)

Lin et al. (2004b ) Kaohsiung and Taipei, Taiwan
(n = 92,288) 24

- h avg: 15.86

- 47.78 Nearest monitor (within 3 km) Entire pregnancy: 1.13 (0.92, 1.38) c

T1: 1.02 (0.85, 1.22) c

T2: 0.93 (0.78, 1.12) c

T3: 1.05 (0.87, 1.26) c

Ha et al. (2001 ) Seoul , Korea
(n = 276,763) 8

- h avg: 22.4

- 23.3 b Citywide avg T1: 0.87 (0.81, 0.94) c

T3: 1.05 (1.02, 1.08) c

a Change in birthweight per 10 ppb change in O 3

b Median

c Odds ratios of LBW; Highest quartile of exposure compared to lowest quartile of exposure

d Relative risk of LBW per 10 ppb change in O 3
T1 = First Trimester, T2 = Second Trimester, T3 = Third Trimester

NR: No quantitative results reported

Table 7

- 4 provides a brief overview of the epidemiologic studies of birth weight. In 

### 1

summary, only the Children’s Health Study conducted in southern California ( Salam et 

### 2

al., 2005 ) provides strong evidence for an effect of ambient O 3 on birth weight. The study 

### 3

by Morello

- Frosch et al. (2010 ) , also conducted in California, provides support for the 

### 4

results of the Children’s Health Study. Additional studies, conducted in the U.S., Canada, 

### 5

Australia, Latin America, and Asia, provide limited and inconsistent evidence to support 

### 6

th e effect reported in the Children’s Health Study. The toxicological literature on the 

### 7

effect of O 3 on birth weight is sparse. In some studies, the reporting of birth weight may 

### 8

be avoided because birth weight can be confounded by decreased litter size resu lting 

### 9

from an increased rate of pup resorption (aborted pups) in O 3 exposed dams. In one 

### 10

toxicological study by Haro and Paz (1993 ) , no differences in litter size were observed 

### 11

and decreased b irth weight in pups from dams who were exposed to 1ppm O 3 during 

### 12

pregnancy (short

- term exposure, ~22 days) was reported. A second animal toxicology 

### 13

study recapitulated these finding with pregnant BALB/c mice that exposed to O 3 

### 14

(1.2 ppm, short

- term exposure , GD9

- 18) producing pups with significa ntly decreased 

### 15

birth weights ( Sharkhuu et al., 2011 )

. 

### 16

### Draft

– Do Not Cite or Quote 7

- 54 June 2012 7.4.4 Preterm Birth

Preterm birth (PTB) is a syndrome ( Romero et al., 2006 ) that is char acterized by multiple 

### 1

etiologies. It is therefore unusual to be able to identify an exact cause for each PTB. In 

### 2

addition, PTB is not an adverse outcome in itself, but an important determinant of health 

### 3

status (i.e., neonatal morbidity and mortality). Alth ough some overlap exists for common 

### 4

risk factors, different etiologic entities related to distinct risk factor profiles and leading 

### 5

to different neonatal and postneonatal complications are attributed to PTB and measures 

### 6

of fetal growth. Although both restr icted fetal growth and PTB can result in LBW, 

### 7

prematurity does not have to result in LBW or growth restricted babies. 

### 8

A major issue in studying environmental exposures and PTB is selecting the relevant 

### 9

exposure period, since the biological mechanisms lead ing to PTB and the critical periods 

### 10

of vulnerability are poorly understood ( Bobak, 2000 ) . Short

- term exposures proximate to 

### 11

the birth may be most relevant if exposure causes an acute effect. However, exposure 

### 12

occurring in early gestation might affect placentation, with results observable later in 

### 13

pregnancy, or cumulative exposure duri ng pregnancy may be the most important 

### 14

determinant. The studies reviewed have dealt with this issue in different ways. Many 

### 15

have considered several exposure metrics based on different periods of exposure. Often 

### 16

the time periods used are the first month (or first trimester) of pregnancy and the 

### 17

last month (or 6 weeks) prior to delivery. Using a time interval prior to delivery 

### 18

introduces an additional problem since cases and controls are not in the same stage of 

### 19

development when they are compared. For example , a preterm infant delivered at 

### 20

36 weeks is a 32

- week fetus 4 weeks prior to birth, while an infant born at term 

### 21

(40 weeks) is a 36

- week fetus 4 weeks prior to birth. 

### 22

Recently, investigators have examined the association of PTB with both short

- term 

### 23

(i.e., hours, days, or weeks) and long

- term (i.e., months or years) exposure periods. Time



### 24

series studies have been used to examine the association between air pollution 

### 25

concentrations during the days immediately preceding birth. An advantage of these time



### 26

serie s studies is that this approach can remove the influence of covariates that vary across 

### 27

individuals over a short period of time. Retrospective cohort and case

- control studies 

### 28

have been used to examine long

- term exposure periods, often averaging air polluti on 

### 29

concentrations over months or trimesters of pregnancy. 

### 30

Studies of PTB fail to show consistency in pollutants and periods during pregnancy when 

### 31

an effect occurs. For example, while some studies find the strongest effects associated 

### 32

with exposures early i n pregnancy, others report effects when the exposure is limited to 

### 33

the second or third trimester. However, the effect of air pollutant exposure during 

### 34

pregnancy on PTB has a biological basis. There is an expanding list of possible 

### 35

### Draft

– Do Not Cite or Quote 7

- 55 June 2012 mechanisms that may expla in the association between O 3 exposure and PTB (see 

### 1

Section 5.4.2.4 ). 

### 2

Many studies of PTB compare exposure in quartiles, using the lowest quartile as the 

### 3

reference (or control) group. No studies use a truly unexposed control group. If exposure 

### 4

in the lowest quartile confers risk, than it may be difficult to demonstrate additional risk 

### 5

associated with a higher quartile. Thus negative studies must be i nterpreted with caution. 

### 6

Preterm birth occurs both naturally (idiopathic PTB), and as a result of medical 

### 7

intervention (iatrogenic PTB). Ritz et al. (2007 ); ( 2000 ) e xcluded all births by Cesarean 

### 8

section to limit their studies to idiopathic PTB. No other studies attempted to distinguish 

### 9

the type of PTB, although air pollution exposure maybe associated with only one type. 

### 10

This is a source of potential effect misclassif ication. 

### 11

Generally, studies of air pollution and birth outcomes conducted in North America and 

### 12

the United Kingdom have not identified an association between PTB and maternal 

### 13

exposure to O 3 . Most recently, Darrow et al. (2009 ) used vital record data to construct a 

### 14

retrospective cohort of 476,489 births occurring between 1994 and 2004 in 5 central 

### 15

counties of metropolitan Atlanta. Using a time

- series approach, the authors examined 

### 16

aggregated daily counts of PTB in relation to ambient levels of CO, NO 2 , SO 2 , O 3 , PM 10 , 

### 17

PM 2.5 and speciated PM measurements. This study investigated 3 gestational windows of 

### 18

short

- and long

- term exposure: the final week of gestation (short

- term exposure), and the 

### 19

first month of gestation and the final 6 weeks of gestation (long

- term exposur e). The 

### 20

authors did not observe associations of PTB with O 3 concentrations for any of the 

### 21

exposure periods. 

### 22

A number of U.S. studies were conducted in southern California, and report somewhat 

### 23

inconsistent results. Ritz et al. (2000 ) evaluated the effect of air pollution (CO, NO 2 , O 3 , 

### 24

PM 10 ) exposure during pregn ancy on the occurrence of PTB in a cohort of 97,518 

### 25

neonates born in southern California between 1989 and 1993. The authors use both short



### 26

and long

- term exposure windows, averaging pollutant measures taken at the closest air



### 27

monitoring station over distin ct periods, such as 1, 2, 4, 6, 8, 12, and 26 weeks before 

### 28

birth and the whole pregnancy period. Additionally, they calculated average exposures 

### 29

for the first and second months of pregnancy. The authors found no consistent effects 

### 30

associated with O 3 concen tration over any of the pregnancy periods in single or 

### 31

multipollutant models. Wilhelm and Ritz (2005 ) extended previous analyses of PTB ( Ritz 

### 32

et al., 2000 ; Ritz and Yu, 1999 ) in California to include 1994

- 2000. The authors 

### 33

examined varying residential distances from monitoring stations to see if the distance 

### 34

affected risk estimation, because effect attenuation may result from local pollutant 

### 35

heterogeneity inadequately captured by ambient monitors. The authors analyzed the 

### 36

association between long

- term O 3 exposure during varying periods of pregnancy and 

### 37

### Draft

– Do Not Cite or Quote 7

- 56 June 2012 PTB, finding a positive association betwe en O 3 levels in both the first trimester of 

### 1

pregnancy (RR = 1.23 [95% CI: 1.06, 1.42] per 10 ppb increase in 24

- h avg O 3 ) and the 

### 2

first month of pregnancy (results for first trimester exposure were similar, but slightly 

### 3

smaller, quantitative results not pr esented) in models containing all pollutants. No 

### 4

association was observed between O 3 in the 6 weeks before birth and preterm delivery. 

### 5

Finally, Ritz et al. (2007 ) conducted a case

- control survey nested within a birth cohort 

### 6

and assessed the extent to which residual confounding and exposure misclassification 

### 7

impacted air pollution effect estimates. The authors calculated mean long

- term exposure 

### 8

levels for three gestational periods: the entire pregn ancy, the first trimester, and the last 

### 9

6 weeks before delivery. Though positive associations were observed for CO and PM 2.5 , 

### 10

no consistent patterns of increase in the odds of PTB for O 3 or NO 2 were observed. 

### 11

A study conducted in Canada evaluated the impa cts of air pollution (including CO, NO 2 , 

### 12

NO, O 3 , SO 2 , PM 2.5 , and PM 10 ) on PTBs (1999

- 2002) using spatiotemporal residential 

### 13

exposure metrics by month of pregnancy (long

- term exposure) in Vancouver, BC ( Brauer 

### 14

et al., 2008 ) . The authors did not observe consistent associations with any of the 

### 15

pregnancy average exposure metrics except for PM 2.5 for PTB. The O 3 associations were 

### 16

largely protective, and no quantitative results were presented for O 3 . Additionally, Lee et 

### 17

al. (2008c ) used time

- series techniques to investigate the associations of short

- term 

### 18

exposure to O 3 and PTB in London, England. In addition to exposure on the day of birth, 

### 19

cumulative exposure up to 1 week before birth was investigated. The risk of PTB did not 

### 20

incre ase with exposure to the levels of ambient air pollution experienced by this 

### 21

population. 

### 22

Conversely, studies conducted in Australia and China provide evidence for an association 

### 23

between ambient O 3 and PTB. Hansen et al. (2006 ) reported that long

- term exposure to 

### 24

O 3 during the first trimester was associated with an increased risk of PTB (OR = 1.38, 

### 25

[95% CI: 1.14, 1.69] per 10 ppb increase). Although the test for trend was significa nt due 

### 26

to the strong effect in the highest quartile, there was not an obvious exposure

- response 

### 27

pattern across the quartiles of O 3 during the first trimester. The effect estimate was 

### 28

diminished and lost statistical significa nce when PM 10 was included in the model 

### 29

(OR = 1.23, [95% CI: 0.97, 1.59] per 10 ppb increase). Maternal exposure to O 3 during 

### 30

the 90 days prior to birth showed a weak, positive association with PTB (OR = 1.09, 

### 31

[95% CI: 0.85, 1.39] per 10 ppb increase). Jalaludin et al. (2007 ) found that O 3 levels in 

### 32

the month and three months preceding birth had a statistically significa nt association wi th 

### 33

PTB. Ozone levels in the first trimester of pregnancy were associated with increased risks 

### 34

for PTBs (OR = 1.15 [95% CI: 1.05, 1.24] per 10 ppb increase in 1

- h max O 3 

### 35

concentration), and remained a significa nt predictor of PTB in copollutant models (ORs 

### 36

between 1.07 and 1.10). Jiang et al. (2007 ) examined the effect of short

- and long

- term 

### 37

exposure to air pollution on PTB, includin g risk in relation to levels of pollutants for a 

### 38

### Draft

– Do Not Cite or Quote 7

- 57 June 2012 single day exposure window with lags from 0 to 6 days before birth. An increase of 

### 1

10 ppb of the 8

- week avg of O 3 corresponded to 9.47% (95% CI: 0.70, 18.7%) increase in 

### 2

PTBs. Increases in PTB were also obse rved for PM 10 , SO 2 , and NO 2 . The authors did not 

### 3

observe any significa nt effect of short

- term exposure to outdoor air pollution on PTB 

### 4

among the 1

- day time windows examined in the week before birth. 

### 5

Little data is available from toxicological studies; a s tudy reported a nearly statistically 

### 6

significa nt increase in pregnancy duration (short

- term exposure) in mice when exposed to 

### 7

0.8 or 1.2 ppm O 3 . This phenomenon was most likely due to the anorexigenic effect of 

### 8

relatively high O 3 concentrations ( Bignami et al., 1994 )

. 

### 9

Table 7

- 5 provides a brief overview of the epidemiologic studies of PTB. In summary, 

### 10

the evidence is consistent when examining short

- term exposure to O 3 during late 

### 11

pregnancy and reports no association with PTB. However when long

- term exposure to O 3 

### 12

early in pregnancy is examined the results are inconsistent. Generally, studies conducted 

### 13

in the U.S., Canada, and England find no association with O 3 and PTB, while studies 

### 14

conducted in Australia and China report an O 3 effect on PTB. 

### 15

### Draft

– Do Not Cite or Quote 7

- 58 June 2012 Table 7

- 5 Brief summary of epidemiologic studies of PTB

Study Location
Sample Size Mean O 3 (ppb) Exposure

assessment Effect Estimate a (95% CI)

Darrow et al. (2009 ) Atlanta, GA

(n = 476,489) 8

- h max: 44.1 Population

- weighted spatial averages

Nearest Monitor (within 4 miles) First month: 0.98 (0.97, 1.00)

Last week: 0.99 (0.98, 1.00)

Last 6 weeks: 1.00 (0.98, 1.02)

Ritz et al. (2000 ) California, U.S.

(n = 97,158) 8 h: 36.9 <2 mi of monitor First month: NR

Last 6 weeks: NR

Wilhelm and Ritz (2005 ) Los Angeles, CA

(n = 106,483) 1 h: 21.1

- 22.2 Varyi ng distances to
monitor First month: 1.23 (1.06, 1.42)

T1: NR
T2: 1.38 (1.14, 1.66)

Last 6 weeks: NR

Ritz et al. (2007 ) Los Angeles, CA

(n = 58,316) 24

- h avg: 22.5 Nearest monitor to
ZIP code Entire pregnancy: NR
T1: 0.93 (0.82, 1.06)

Last 6 weeks: NR

Brauer et al. (2008 ) Vancouver, BC,
Canada

(n = 70,249) 24

- h avg:

14 Nearest Monitor (within 10 km)

Inverse Distance Weighting (IDW) Entire pregnancy: NR
First 30 days of pregnancy: NR
Last 30 days of pregnancy: NR

T1: NR

T3: NR

Lee et al. (2008c ) London, UK 24

- h avg: NR 1 monitor Lag 0: 1.00 (1.00, 1.01)

Hansen et al. (2006 ) Brisbane,
Australia
(n = 28,200) 8

- h max:

26.7 Citywide avg T1: 1.39 (1.15, 1.70)

T3: 1.09 (0.88, 1.39)

Jalaludin et al. (2007 ) Sydney,
Australia
(n = 123,840) 1

- h max:

30.9 Citywide avg and <5
km from monitor First month: 1.04 (0.95, 1.13)

T1: 1.15 (1.05, 1.24)

T3: 0.98 (0.89, 1.07)

Last month: 0.98 (0.88, 1.06)

Jiang et al. (2007 ) Shanghai, China

(n = 3,346 preterm births) 8

- h avg:

32.7 Citywide avg 4 wks before birth: 1.06 (1.00, 1.12)

6 wks before birth: 1.06 (0.99, 1.13)

8 wks before birt h: 1.09 (1.01, 1.19)
L0: NR (results presented in figure)
L1: NR (results presented in figure)
L2: NR (results presented in figure)
L3: NR (results presented in figure)
L4: NR (results presented in figure)
L5: NR (results presented in figure)

L6: NR (results presented in figure)

a Relative risk of PTB per 10 ppb change in O 3

. T1 = First Trimester, T2 = Second Trimester, T3 = Third Trimes t er.

L0 = Lag 0, L1= Lag 1, L2 = Lag 2, L3 = Lag 3, L4 = Lag 4, L5 = Lag 5, L6 = Lag 6.

NR: No quantitative results reported.

### Draft

– Do Not Cite or Quote 7

- 59 June 2012 7.4.5 Fetal Growth

Low birth weight has often been used as an outcome measure because it is easily 

### 1

available and accurately recorded on birth certificates. However, LBW may result from 

### 2

either short gestation, or inadequate growth in utero. Most of t he studies investigating air 

### 3

pollution exposure and LBW limited their analyses to term infants to focus on inadequate 

### 4

growth. A number of studies were identified that specifically addressed growth restriction 

### 5

in utero by identifying infants who failed to m eet specific growth standards. Usually 

### 6

these infants had birth weight less than the 10th percentile for gestational age, using an 

### 7

external standard. Many of these studies have been previously discussed, since they also 

### 8

examined other reproductive outcomes (i.e., LBW or PTB). 

### 9

Fetal growth is influenced by maternal, placental, and fetal factors. The biological 

### 10

mechanisms by which air pollutants may influence the developing fetus remain largely 

### 11

unknown. Several mechanisms have been proposed, and are the same a s those 

### 12

hypothesized for birth weight (see Section 5.4.2.4 ). Additionally, in animal toxicology 

### 13

studies, O 3 causes transient anorexia in exposed pregnant dams. This may be one of many 

### 14

possible contributors to O 3 - dependent decreased fetal growth. 

### 15

A limitation of environmental studies that use birth weight as a proxy measure of fetal 

### 16

growth is that patterns of fe tal growth during pregnancy cannot be assessed. This is 

### 17

particularly important when investigating pollutant exposures during early pregnancy as 

### 18

birth weight is recorded many months after the exposure period. The insult of air 

### 19

pollution may have a transient effect on fetal growth, where growth is hindered at one 

### 20

point in time but catches up at a later point. For example, maternal smoking during 

### 21

pregnancy can alter the growth rate of individual body segments of the fetus at variable 

### 22

developmental stages, as t he fetus experiences selective growth restriction and 

### 23

augmentation ( Lampl and Jeanty, 2003 )

. 

### 24

The t erms small

- for

- gestational

- age (SGA), which is defined as a birth weight <10th 

### 25

percentile for gestational age (and often sex and/or race), and intrauterine growth 

### 26

retardation (IUGR) are often used interchangeably. However, this definition of SGA does 

### 27

have limitations. For example, using it for IUGR may overestimate the percentage of 

### 28

“growth

- restricted” neonates as it is unlikely that 10% of neonates have growth 

### 29

restriction ( Wollmann, 1998 ) . On the other hand, when the 10th percentile is based on the 

### 30

distribution of live births at a population level, the percentage of SGA among PTB is 

### 31

most likely underestimated ( Hutcheon and Platt, 2008 ) . Nevertheless, SGA represents a 

### 32

statistical description of a small neonate, whereas the term IUGR is reserved for those 

### 33

with clinical evidence of abnormal growth. Thus all IUGR neonates will be SGA, but not 

### 34

all SGA neonates with be IUGR ( Wollmann, 1998 ) . In the following section the terms 

### 35

SGA and IUGR are r eferred to as each cited study used the terms. 

### 36

### Draft

– Do Not Cite or Quote 7

- 60 June 2012 Over the past decade a number of studies examined various metrics of fetal growth 

### 1

restriction. Salam et al. (2005 ) assessed the effect of increas ing O 3 concentrations on 

### 2

IUGR in a population of infants born in California from 1975

- 1987 as part of the 

### 3

Children’s Health Study. The authors reported that maternal O 3 exposures averaged over 

### 4

the entire pregnancy and during the third trimester were associ ated with increased risk of 

### 5

IUGR. A 10

- ppb difference in 24

- h maternal O 3 exposure during the third trimester 

### 6

increased the risk of IUGR by 11% (95% CI: 0, 20%). Brauer et al. (2008 ) evaluated the 

### 7

impacts of air pollution (CO, NO 2 , NO, O 3 , SO 2 , PM 2.5 , PM 10 ) on S GA (1999

- 2002) using 

### 8

spatiotemporal residential exposure metrics by month of pregnancy in Vancouver, BC. 

### 9

The O 3 associations were largely protective (OR = 0.87, [95% CI: 0.81, 0.93] for a 

### 10

10 ppb increase in inverse distance weighted SGA), and no additiona l quantitative results 

### 11

were presented for O 3

. Liu et al. (2007b ) examined the association between IUGR among 

### 12

singleton term live births and SO 2 , NO 2 , CO, O 3 , and PM 2.5 in 3 Canadian cities for the 

### 13

period 1985

- 2000. No increase in the risk of IUGR in relation to exposure to O 3 averaged 

### 14

over each month and trimester of pregnancy was noted. 

### 15

Three studies conducted in Australia provide evidence for an as sociation between 

### 16

ambient O 3 and fetal growth restriction. Hansen et al. (2007 ) e xamined SGA among 

### 17

singleton, full

- term births in Brisbane, Australia in relation to ambient air pollution (bsp, 

### 18

PM 10 , NO 2 , O 3 ) during pregnancy. They also examined head circumference and crown



### 19

heel length in a subsample of term neonates. Trimester specific exposures to all pollutants 

### 20

were not statistically significa ntly associated with a reduction in head circumference or 

### 21

an increased risk of SGA. When monthly

- specific exposures were examined, the authors 

### 22

observed an increased risk of SGA associated with ex posure to O 3 during month 4 

### 23

(OR = 1.11 [95% CI: 1.00, 1.24] per 10 ppb increase). In a subsequent study, Hansen et 

### 24

al. (2008 ) examined the possible associations between fetal ultrasonic measurements and 

### 25

ambient air pollution (PM 10 , O 3 , NO 2 , SO 2 ) during early pregnancy. This study had two 

### 26

strengt hs: (1) fetal growth was assessed during pregnancy as opposed to at birth; and (2) 

### 27

there was little delay between exposures and fetal growth measurements, which reduces 

### 28

potential confounding and uses exposures that are concurrent with the observed growth 

### 29

p attern of the fetus. Fetal ultrasound biometric measurements were recorded for biparietal 

### 30

diameter (BPD), femur length, abdominal circumference, and head circumference. To 

### 31

further improve exposure assessment, the authors restricted the samples to include o nly 

### 32

scans from women for whom the centroid of their postcode was within 14 km of an air 

### 33

pollution monitoring site. Ozone during days 31

- 60 was associated with decreases in all 

### 34

of the fetal growth measurements, and a 1.78 mm reduction in abdomen circumferen ce 

### 35

per 10 ppb increase in O 3 concentration, though this effect did not persist in copollutant 

### 36

models. The change in ultrasound measurements associated with O 3 during days 31

- 60 of 

### 37

gestation indicated that increasing O 3 concentration decreased the magnitude of 

### 38

ultrasound measurements for women living within 2 km of the monitoring site. The 

### 39

### Draft

– Do Not Cite or Quote 7

- 61 June 2012 relationship decreased toward the null as the distance from the monitoring sites increased. 

### 1

When assessing effect modification due to SES, there was some evidence of effec t 

### 2

modification for most of the associations, with the effects of air pollution stronger in the 

### 3

highest SES quartile. In the third study, Mannes et al. (2005 ) estimated the effects of 

### 4

pollutant (PM 10 , PM 2.5 , NO 2 , CO and O 3 ) exposure in the first, seco nd and third trimesters 

### 5

of pregnancy and risk of SGA in Sydney, Australia. Citywide average air pollutant 

### 6

concentrations in the last month, third trimester, and first trimester of pregnancy had no 

### 7

effect on SGA. Concentrations of O 3 in the second trimester of pregnancy had small but 

### 8

adverse effects on SGA (OR = 1.10 [95% CI: 1.00, 1.14] per 10 ppb increment). This 

### 9

effect disappeared when the analysis was limited to births with a maternal address within 

### 10

5 km of a monitoring station (OR = 1.00 [95% CI: 0.60, 1.79] per 10 ppb increment). 

### 11

Very little information from toxicological studies is available to address effects on fetal 

### 12

growth. However, there is evidence to suggest that prenatal (short

- term) exposure to O 3 

### 13

can affect postnatal growth. A few studies reported that mice or rats exposed 

### 14

developmentally (gestationally ± lactationally) to O 3 had deficits in body weight gain in 

### 15

the postpartum period ( Bignami et al., 1994 ; Haro and Paz, 1993 ; Kavlock et al., 1980 )

. 

### 16

Table 7

- 6 provides a brief overview of the epidemiologic studies of fetal growth 

### 17

restriction. In summary, the evidence is inconsistent when examining exposure to O 3 and 

### 18

fetal growth restriction. Similar to PTB, studies conducted in Australia have reported an 

### 19

effect of O 3 on fetal growth, whereas studies conducted in other areas generally have not 

### 20

found such an effect. This m ay be due to the restriction of births to those within 2

- 14 km 

### 21

of a monitoring station, as was done in the Australian studies. 

### 22

### Draft

– Do Not Cite or Quote 7

- 62 June 2012 Table 7

- 6 Brief summary of epidemiologic studies of fetal g rowth.

Study Location
(Sample Size) Mean O 3 (ppb) Exposure

assessment Effect Estimate a (95% CI)

Salam et al. (2005 ) California, U.S.

(n = 3901) 24

- h avg:

27.3

8 h:

50.6 ZIP code level Entire pregnancy: 1.16 (1.00, 1.32)

T1:1.00 (0.94, 1.11)

T2: 1.06 (1.00, 1.12)

T3: 1.11 (1.00, 1.17)

Brauer et al. (2008 ) Vancouver, BC, Canada

(n = 70,249) 24

- h avg:

14 Nearest Monitor (within 10 km)

Inverse Distance Weighting (IDW) Entire pregnancy: NR
First 30 days of pregnancy: NR

Last 30 days of pregnancy: NR

T1: NR

T3: NR

Liu et al. (2007b ) Calgary, Edmonton, and Montreal, Canada

(n = 16,430) 24

- h avg:

16.5

1

- h max:

3 1.2 Census Subdivision

avg Entire pregnancy: NR (results presented in figure)
T1: NR (results presented in figure)
T2: NR (results presented in figure)
T3: NR (results presented in figure)

Hansen et al. (2007 ) Brisbane, Australia
(n = 26,617) 8

- h max:

26.7 Citywide avg T1: 1.01 (0.89, 1.15)

T2: 1.00 (0.86, 1.17)

T3: 0.83 (0.71, 0.97)

Hansen et al. (2008 ) Brisbane, Au stralia
(n = 15,623) 8

- h avg:

24.8 Within 2 km of monitor M1:

- 0.32 (

- 1.56, 0.91) b

M2:

- 0.58 (

- 1.97, 0.80) b

M3: 0.26 (

- 1.07, 1.59) b

M4: 0.11 (

- 0.98, 1.21) b

Mannes et al. (2005 ) Sydney, Australia

(n = 138,056) 1

- h max:

31.6 Citywide avg and
<5 km from monitor T1: 0.90 (0.48, 1.34)

T2: 1.00 (0.60, 1.79)

T3: 1.10 (0.66, 1.97)
Last 30 days of pregnancy: 1.10 (0.74, 1.79)

a Relative risk of fetal growth restriction per 10 ppb change in O 3 , unless otherwise noted.

b Mean change in fetal ultrasonic measure of head circumference recorded between 13 and 26 weeks gestation for a 10

- ppb increase in maternal exposure to O 3 during early pregnancy

T1 = First Trimester, T2 = Second Trimester, T3 = Third Trimester

M1 = Month 1, M2 = Month 2, M3 = Month 3, M4 = Month 4

NR: No quantitative results reported



### 7.4.6 Postnatal Growth

Postnatal weight and height are routinely measured in children as indicators of growth 

### 1

and somatic changes. Toxicological studies often follow these endpoints to ascertain if a 

### 2

known exposure has an effect in the postnatal window, an effect which can be permanent. 

### 3

Time

- pregnant BALB/c mice were exposed to O 3 (0, 0.4, 0.8, or 1.2 ppm) GD9

- 18 

### 4

(short

- term exposure) with parturition at GD20

- 21 ( Sharkhuu et al., 2011 ) . As the 

### 5

offspring aged, postnatal litter body weight continued to be significa ntly decreased in the 

### 6

highest concentration (1.2 ppm) O 3 group at PND3 and PND7. When the pups were 

### 7

### Draft

– Do Not Cite or Quote 7

- 63 June 2012 weighed separately by sex at PND42, the males with the highest concentration of O 3 

### 1

exposure (1.2 ppm, GD9

- 18) had significa nt decrements in body weight ( Sharkhuu et al., 

### 2

2011 )

. 

### 3

Significa nt decrements in body weight at 4 weeks of age were reported in C57Bl/6 mice 

### 4

that were exposed to postnatal O 3 (short

- term exposure, PND2

- 28 exposure, 1 ppm O 3 , 

### 5

3 hours/day, 3 days/week) ( Auten et al., 2012 ) . Animals with co

- exposure to in utero DE 

### 6

(short

- term exposure, dam GD9

- GD17; inhalation 0.5 or 2.0 mg/m 3 O 3 ; 4 h/day via 

### 7

inhalation; or oropharyngeal aspiration DEPs, 2×/week) + postnatal O 3 (aforementioned 

### 8

short

- term exposure) also had significa ntly reduced body weight. 

### 9

7.4.7 Birth Defects

Despite the growing body of literature e valuating the association between ambient air 

### 10

pollution and various adverse birth outcomes, relatively few studies have investigated the 

### 11

effect of temporal variations in ambient air pollution on birth defects. Heart defects and 

### 12

oral clefts have been the fo cus of the majority of these recent studies, given the higher 

### 13

prevalence than other birth defects and associated mortality. Mechanistically, air 

### 14

pollutants could be involved in the etiology of birth defects via a number of key events 

### 15

(see Section 5.4.2.4 ). 

### 16

Several studies have been conducted examining the relationship between O 3 exposure 

### 17

during pregnancy and birth defects and reported a positive association with cardiac 

### 18

defects. The earliest of these studies was conducted in southern California ( Ritz et al., 

### 19

2002 ) . This study evaluated the effect of air pollution on the occurrence of cardiac birth 

### 20

defects in neonates and fetuses delivered in southern California in 1987

- 1993. Maternal 

### 21

exposure estimates were based on data from the fixed site closest to the mother’s ZIP 

### 22

code area. When using a case

- control design where cases were matched to 10 randomly 

### 23

selected controls, results showed increased risks for aortic artery and valve defects 

### 24

(OR = 1.56 [95% CI: 1.16, 2.09] per 10 ppb O 3 ), pulmonary artery and valve anomalies 

### 25

(OR = 1.34 [95% CI: 0.9 6, 1.87] per 10 ppb O 3 ), and conotruncal defects (OR = 1.36 

### 26

[95% CI: 0.91, 2.03] per 10 ppb O 3 ) in a dose

- response manner with second

- month O 3 

### 27

exposure. A study conducted in Texas ( Gilboa et al., 2005 ) looked at a similar period of 

### 28

exposure but reported no association with most of the birth defects studied (O 3 

### 29

concentration was studied using quartiles with the lowest representing <18 ppb and the 

### 30

highest representing ≥ 31 ppb). The authors found slightly elevate d odds ratios for 

### 31

pulmonary artery and valve defects. They also detected an inverse association between O 3 

### 32

exposure and isolated ventricular septal defects. Overall, this study provided some weak 

### 33

evidence that air pollution increases the risk of cardiac de fects. Hansen et al. (2009 ) 

### 34

### Draft

– Do Not Cite or Quote 7

- 64 June 2012 investigated the possible association between ambient air pollution concentrations 

### 1

averaged over weeks 3

- 8 of pregnancy and the risk of cardiac defects. When analy zing all 

### 2

births with exposure estimates for O 3 from the nearest monitor there was no indication for 

### 3

an association with cardiac defects. There was also no adverse association when 

### 4

restricting the analyses to only include births where the mother resided wit hin 12 km of a 

### 5

monitoring station. However, among births within 6 km of a monitor, a 10 ppb increase 

### 6

in O 3 was associated with an increased risk of pulmonary artery and valve defects 

### 7

(OR = 8.76 [95% CI: 1.80, 56.55]). As indicated by the very wide credible intervals, 

### 8

there were very few cases in the sensitivity analyses for births within 6 km of a monitor, 

### 9

and this effect could be a result of type I errors. Dadvand et al. (2011 ) investigated the 

### 10

association between maternal exposure to ambient air pollution concentrations averaged 

### 11

over weeks 3

- 8 of pregnancy and the occurrence of cardiac birth defects in England. 

### 12

Similar to Hansen et al. (2009 ) , they found no associations with maternal exposure to O 3 

### 13

except for when the analysis was limited to those subjects residing within a 16 km 

### 14

distance of a moni toring station (OR for malformations of pulmonary and tricuspid 

### 15

valves=1.64 [95% CI: 1.04, 2.60] per 10 ppb increase in O 3 ). 

### 16

Despite the association between O 3 and cardiac defects observed in the above studies, a 

### 17

recent study did not observe an increased r isk of cardiac birth defects associated with 

### 18

ambient O 3 concentrations. The study, conducted in Atlanta, GA, examined O 3 exposure 

### 19

during weeks 3

- 7 of of pregnancy and reported no association with risk of cardiovascular 

### 20

malformations ( Strickland et al., 2009 )

. 

### 21

Several of these studies have also examined the relationship between O 3 exposure during 

### 22

pregnancy and oral cleft defects. The study by Ritz et al. (2002 ) evaluated the effect of air 

### 23

pollution on the occurrence of orofacial birth defects and did not observe strong 

### 24

associat ions between ambient O 3 concentration and orofacial defects. They did report an 

### 25

OR of 1.13 (95% CI: 0.90, 1.40) per 10 ppb during the second trimester for cleft lip with 

### 26

or without cleft palate. Similarly, Gilboa et al. (2005 ) reported an OR of 1.09 (95% CI: 

### 27

0.70, 1.69) for oral cleft defects when the fourth quartile was contrasted with the first 

### 28

quartile of exposure during 3

- 8 weeks of pregnancy. Hansen et al. (2009 ) reported no 

### 29

indication for an association with cleft defects and air pollution concentrations averaged 

### 30

over weeks 3

- 8 of pregnancy. Hwang and Jaakkola (2008 ) conducted a population

- based 

### 31

case

- control study to investigate exposure to ambient air pollution and the risk of cleft lip 

### 32

with or without cleft palate in Taiwan. The risk of clef t lip with or without cleft palate 

### 33

was increased in relation to O 3 levels in the first gestational month (OR = 1.17 [95% CI: 

### 34

1.01, 1.36] per 10 ppb) and second gestational month (OR = 1.22 [95% CI: 1.03, 1.46] 

### 35

per 10 ppb), but was not related to any of the other pollutants. In three

- pollutant models, 

### 36

the effect estimates for O 3 exposure were stable for the four different combinations of 

### 37

pollutants and were all statistically significa nt. Marshall et al. (2010 ) compared estimated 

### 38

### Draft

– Do Not Cite or Quote 7

- 65 June 2012 exposure to ambient pollutants during early pregnancy (6 week period from 5 to 10 

### 1

weeks into the gestational period) among mothers of chil dren with oral cleft defects to 

### 2

that among mothers of controls. The authors observed no consistent elevated associations 

### 3

between any of the air pollutants examined and cleft malformations, though there was a 

### 4

weak association between cases of cleft palate o nly and increasing O 3 concentrations. 

### 5

This association increased when cases and controls were limited to those with residences 

### 6

within 10 km of the closest O 3 monitor (OR = 2.2 [95% CI: 1.0, 4.9], comparing highest 

### 7

quartile [>33 ppb] to lowest quartile [<15 ppb]). 

### 8

A limited number of toxicological studies have examined birth defects in animals 

### 9

exposed gestationally to O 3

. Kavlock et al. (1979 ) exposed pregnant rats to O 3 for precise 

### 10

periods during organogenesis. No significa nt teratogenic effects were found in rats 

### 11

exposed 8 h/day to concentrations of O 3 varying from 0.44 to 1.97 ppm during early 

### 12

(days 6

- 9), mid (days 9

- 12), or late (days 17 to 20) gestation, or the entire period of 

### 13

organogenesis (days 6

- 15) (short

- term exposures). Earlier research found eyelid 

### 14

malformation following gestational and postnatal exposure to 0 .2 ppm O 3 ( Veninga, 

### 15

1967 )

. 

### 16

Table 7

- 7 provides a brief overview of the epidemiologic studies of birth defects. These 

### 17

studies have focused on cardiac and oral cleft defects, and the results from these studies 

### 18

are not entirely consistent. This inconsistency could be due to the absence of tr ue 

### 19

associations between O 3 and risks of cardiovascular malformations and oral cleft defects; 

### 20

it could also be due to differences in populations, pollution levels, outcome definitions, or 

### 21

analytical approaches. The lack of consistency of associations betwee n O 3 and 

### 22

cardiovascular malformations or oral cleft defects might be due to issues relating to 

### 23

statistical power or measurement error. A recent meta

- analysis of air pollution and 

### 24

congenital anomalies concluded that there was no statistically significa nt in crease in risk 

### 25

of congenital anomalies and O 3 ( Vrijheid et al., 2011 ) . These authors note that 

### 26

heterogeneity in the results of these studies may be due to inherent differences in study 

### 27

location, study de sign, and/or analytic methods, and comment that these studies have not 

### 28

employed some recent advances in exposure assessment used in other areas of air 

### 29

pollution research that may help refine or reduce this heterogeneity. 

### 30

### Draft

– Do Not Cite or Quote 7

- 66 June 2012 Table 7

- 7 Brief summary of epidemiologic studies of birth defects

Study Outcomes
Examined Location
(Sample Size) Mean O 3 (ppb) Exposure
Assessment Exposure
Window

Ritz et al. (2002 ) Car diac and Cleft
Defects Southern California

(n = 3,549 cases; 10,649 controls) 24

- h avg:

NR Nearest Monitor (within 10 mi) Month 1,2,3

Trimester 2,3

3

- mo period prior to conception

Gilboa et al. (2 005 ) Cardiac and Cleft
Defects 7 Counties in TX

(n = 5,338 cases; 4,580 controls) 24

- h avg:

NR Nearest Monitor Weeks 3

- 8 of gestation

Hwang and Jaakkola (2008 ) Oral Cleft Defects Taiwan
(n = 653 cases; 6,530 controls) 24

- h avg:

27.31 Inverse Distance Weighting (IDW) Months 1,2,3

Strickland et al. (2009 ) Cardiac Defects Atlanta, GA

(n = 3,338 cases) 8

- h max:

39.8

- 43.3 Weighted citywide

avg Weeks 3

- 7 of gestation

Hansen et al. (2009 ) Cardiac and Cleft
Defects Brisbane, Australia
(n = 150,308 births) 8

- h max:

25.8 Nearest Monitor Weeks 3

- 8 of gestation

Marshall et al. (2010 ) Oral Cleft Defects New Jersey
(n = 717 cases; 12,925 controls) 24

- h avg:

25 Nearest Monitor (within 40 km) Weeks 5

- 10 of gestation

Dadvand et al. (2011 ) Cardiac Defects Northeast England
(n = 2,140 cases; 14,256 controls) 24

- h avg:

18.8 N earest Monitor Weeks 3

- 8 of gestation`1



### 7.4.8 Developmental Respiratory Effects

The issue of prenatal exposure has assumed increasing importance since ambient air 

### 1

pollution exposures of pregnant women have been shown to lead to adverse pregnancy 

### 2

outcomes, as well as to respiratory morbidity and mortality in the first year of life. 

### 3

Growth and development of the respiratory system take place mainly during the prenatal 

### 4

and early postnatal periods. This early developmental phase is thought to be very 

### 5

important in determining long

- term lung growth. Studies have recently examined this 

### 6

emerging issue. Several studies were included in Section 7.2.1 and Section 7.2.3 , and are 

### 7

included here because they reported both prenatal and post

- natal exposure periods. 

### 8

Mortimer et al. (2008a ); ( 2008b ) examined the association of prenatal and lifetime 

### 9

exposures to air pollutants with pulmonary function and allergen sensitization in a su bset 

### 10

of asthmatic children (ages 6

- 11) included in the Fresno Asthmatic Children’s 

### 11

Environment Study (FACES). Monthly means of pollutant levels for the years 1989

- 2000 

### 12

were created and averaged separately across several important developmental time



### 13

periods , including the entire pregnancy, each trimester, the first 3 years of life, the first 

### 14

6 years of life, and the entire lifetime. The 8

- h avg O 3 concentrations were approximately 

### 15

### Draft

– Do Not Cite or Quote 7

- 67 June 2012 50 ppb for each of the exposure metrics (estimated from figure). In the first analysis 

### 1

( Mortimer et al., 2008a ) , negative effects on pulmonary function were found for exposure 

### 2

to PM 10 , NO 2 , and CO during key neonatal and early life developmental periods. The 

### 3

authors did not find a negative effect of exposure to O 3 among this cohort. In the second 

### 4

analysis ( Mortimer et al., 2008b ) , sensitization to at least one allergen was associated, in 

### 5

general, with higher levels of CO and PM 10 during the entire pregnancy and second 

### 6

trimester and higher PM 10 during the first 2 years of life. Lower exposure to O 3 during the 

### 7

entire pregnancy or second trimester was associated with an increased risk of allergen 

### 8

sensitization. Although the pollutant metrics across time periods are correlated, the 

### 9

strongest associations with the outcomes were observed for prenatal exposures. Though it 

### 10

may be difficult to disentangle the effect of prenatal and postnatal exposures, the models 

### 11

from this group of studies suggest that each time period of exposure may contribute 

### 12

independently to different dimensions of school

- aged children’s pulmonary function. For 

### 13

4 of the 8 pulmonary

- function measures (FVC, FEV 1 , PEF, FEF 25

- 75 ), prenatal exposures 

### 14

were more influential on pulmonary function than early

- lifetime metrics, while, in 

### 15

contrast, the ratio of measures (FEV 1 /FVC and FEF 25

- 75 /FVC) were most influenced by 

### 16

postnatal exposures. When lifetime metrics were considered alone, or in combination 

### 17

with the prenatal metrics, the lifetime measures were not associated with any of the 

### 18

outcomes, suggesting the timing of the exposure may be more important than the overall 

### 19

dose and prenatal exposures are not just markers for lifetime or current exposures. 

### 20

Clark et al. (2010 ) investigated the effect of exposure to ambient air pollution in utero 

### 21

and during the first year of life on risk of subsequent asthma diagnosis (incident asthma 

### 22

diagnosis up to age 3

- 4) in a population

- based nested case

- control study. Air pollution 

### 23

exposure for each subject based on their residential address history was estimated using 

### 24

regulatory monitoring data, land use regression modeling, and proximity to stationary 

### 25

pollution sources. An average exposure was calculated for t he duration of pregnancy 

### 26

(~15 ppb) and the first year of life (~14 ppb). In contrast to the Mortimer et al. (2008a ); 

### 27

( 2008b ) studies, the effect estimates for first

- year exposure were generally larger than for 

### 28

in utero exposures. However, similar to the Mortimer et al. studies, the observed 

### 29

associations with O 3 were largely protective. Because of the relatively high correl ation 

### 30

between in utero and first

- year exposures for many pollutants, it was difficult to discern 

### 31

the relative importance of the individual exposure periods. 

### 32

Latzin et al. (2009 ) examined whether prenatal exposure to air pollution was associated 

### 33

with lung function changes in the newborn. Tidal breathing, lung volume, ve ntilation 

### 34

inhomogeneity and eNO were measured in 241 unsedated, sleeping neonates (age= 

### 35

5 weeks). The median of the 24

- h avg O 3 concentrations averaged across the post

- natal 

### 36

period was ~44 ppb. Consistent with the previous studies, no association was found for 

### 37

prenatal exposure to O 3 and lung function. 

### 38

### Draft

– Do Not Cite or Quote 7

- 68 June 2012 The new toxicological literature since the 2006 O 3 AQCD, covering respiratory changes 

### 1

related to developmental O 3 exposure, reports ultrastructural changes in bronchiole 

### 2

development, alterations in placental and pup cytokines, and increased pup airway hyper



### 3

reactivity. These s tudies are detailed below. 

### 4

Fetal rat lung bronchiole development is triphasic, comprised of the glandular phase 

### 5

(measured at GD18), the canalicular phase (GD20), and the saccular phase ( GD21). The 

### 6

ultrastructural lung development in fetuses of pregnant rats exposed to 1

- ppm O 3 (12 

### 7

h/day, out to either GD18, GD20 or GD21) was examined by electron microscopy during 

### 8

these three phases. In the glandular phase, bronchiolar columnar epithelial cells in fetuses 

### 9

of dams exposed to O 3 had cytoplasmic damage and swollen mitochondria. Bro nchial 

### 10

epithelium at the canalicular phase in O 3 exposed pups had delayed maturation in 

### 11

differentiation, i.e., glycogen abundance in secretory cells had not diminished as it should 

### 12

with this phase of development. Congruent with this finding, delayed matura tion of 

### 13

tracheal epithelium following early neonatal O 3 exposure (1 ppm, 4

- 5 h/day for first week 

### 14

of life) in lambs has been previously reported ( Mariassy et al., 1990 ; Mariassy et al., 

### 15

1989 ) . Also at the canalicular phase, atypical cells were seen in the bronchiolar lumen of 

### 16

O 3 - exposed rat fetuses. Finally, in the saccular phase, mitochondrial degradation was 

### 17

present in the non

- ciliated bronchiolar cells o f rats exposed in utero to O 3 . In conclusion, 

### 18

O 3 exposure of pregnant rats produced ultra

- structural damage to near

- term fetal 

### 19

bronchiolar epithelium ( López et al., 2008 )

. 

### 20

Exposure of laboratory animals to multiple airborne pollutants can differentially aff ect 

### 21

pup physiology. One study showed that exposure of C57BL/6 mouse dams to 0.48 mg 

### 22

PM intratracheally twice weekly for 3 weeks during pregnancy augmented O 3 - induced 

### 23

airway hyper

- reactivity in juvenile offspring. Maternal PM exposure also significa ntly 

### 24

inc reased placental cytokines above vehicle

- instilled controls. Pup postnatal O 3 exposure 

### 25

(1 ppm 3 h/day, every other day, thrice weekly for 4 weeks) induced significa ntly 

### 26

increased cytokine levels (IL

- 1β, TNF

- α, KC, and IL

- 6) in whole lung versus postnatal 

### 27

a ir exposed groups; this was further exacerbated with gestational PM exposure ( Auten et 

### 28

al., 2009 ) . In fu rther studies by the same laboratory, O 3 - induced AHR was studied in 

### 29

rodent offspring after dam gestational exposure to inhaled diesel exhaust ( Auten et al., 

### 30

2012 ) . Pregnant C57Bl/6 mice were exposed to diesel exhaust GD9

- 17 (0.5 or 2.0 mg/m 3 

### 31

O 3 , 4h/day) via inhalation or in a separate set of animals via oropharyngeal aspiration of 

### 32

freshly generated DEPs (2×/week). Postnatally, the offspring were exposed to O 3 starting 

### 33

at PND2 (1 ppm O 3 , 3 hours/day, 3 days/week for 4 weeks). Juvenile mice were then 

### 34

subjected to measurements of pulmonary mechanisms (at 4 weeks of age and then at 8 

### 35

weeks of age). Increased inflammation of the placenta and lungs of DE exposed fetuses 

### 36

was reported at GD18. In animals with postnatal O 3 exposure alone, elevated 

### 37

inflammation was seen with significa nt increased levels of BAL cytokines; these O 3 - 

### 38

### Draft

– Do Not Cite or Quote 7

- 69 June 2012 related elevated levels were significa ntly exacerbated with pren atal DE exposure 

### 1

(DE+O 3 ). At PND28, DE+O 3 exposed offspring had significa nt impairment of alveolar 

### 2

development as measured with secondary alveolar crest development, a finding that was 

### 3

absent in all other exposure groups (O 3 alone, DE alone). Postnatal O 3 exposure induced 

### 4

AHR in methacholine challenged animals at 4 weeks of age and was exacerbated with the 

### 5

higher dose of DE exposure (DE+O 3 ). At 8 weeks of age, O 3 exposed pups had persistent 

### 6

AHR (+/

- DE) that was significa ntly augmented in DE+O 3 pups. In summ ary, gestational 

### 7

DE exposure induced an inflammatory response which, when combined with postnatal O 3 

### 8

exposure impaired alveolar development, and caused an exacerbated and longer

- lasting 

### 9

O 3 - induced AHR in offspring. 

### 10

A series of experiments using infant rhes us monkeys repeatedly exposed to 0.5 ppm O 3 

### 11

starting at one

- month of age have examined the effect of O 3 alone or in combination with 

### 12

an inhaled allergen on morphology and lung function ( Plopper et al ., 2007 ) . Exposure to 

### 13

O 3 alone or allergen alone produced small but not statistically significa nt changes in 

### 14

baseline airway resistance and airway responsiveness, but the combined exposure to both 

### 15

O 3 + antigen produced statistically significa nt and grea ter than additive changes in both 

### 16

functional measurements. Additionally, cellular changes and significa nt structural 

### 17

changes in the respiratory tract have been observed in infant rhesus monkeys exposed to 

### 18

O 3 ( Fanucchi et al., 2006 ) . A more detailed description of these studies can be found in 

### 19

Section 7.2.3 (Pulmonary Structure and Function), with mechanistic information found in 

### 20

Section 5.4.2.4

. 

### 21

Lung immunological response in O 3 exposed pups was followed by analyzing BAL and 

### 22

lung tissue. Sprague Dawley (SD) pups were exposed to a single 3h exposure of air or O 3 

### 23

(0.6 ppm) o n PND 13 ( Han et al., 2011 ) . Bronchoalveolar lavage (BAL) was performed 

### 24

10 hours after the end of O 3 exposure. BALF polymorphonuclear leukocytes (PMNs) and 

### 25

total BALF protein were significa ntly elevated in O 3 exposed pups. Lung tissue from O 3 

### 26

exposed pups had significa nt elevations of manganese superoxide dismutase (SOD) 

### 27

protein and significa nt decrements of extra

- cellular SOD protein. 

### 28

Various immunological outcomes were followed in offspring after their pregnant dams 

### 29

(BALB/c mice) were exposed gestationally to O 3 (0, 0.4, 0.8, or 1.2 ppm, GD9

- 18) 

### 30

( Sharkhuu et al., 2011 ) . Delayed type hypersensitivity (DTH) was initiat ed with initial 

### 31

BSA injection at 6 weeks of age and then challenge 7 days later. The normal edematous 

### 32

response of the exposed footpad (thickness after BSA injection) was recorded as an 

### 33

indicator of DTH. In female offspring, normal footpad swelling with BSA injection that 

### 34

was seen in air exposed animals was significa ntly attenuated with O 3 exposure (0.8 and 

### 35

1.2 ppm O 3 ), implying immune suppression of O 3 exposure specifically in DTH. Humoral 

### 36

immunity was measured with the sheep red blood cell (SRBC) response. Animals 

### 37

### Draft

– Do Not Cite or Quote 7

- 70 June 2012 received primary immunization with SRBC and then blood was drawn for SRBC IgM 

### 1

measurement. A SRBC booster was given 2 weeks later with blood collected 5 days after 

### 2

booster for IgG measurement. Maternal O 3 exposure had no effect on humoral immunity 

### 3

in the offspring as measured by IgG and IgM titers after SRBC primary and booster 

### 4

immunizations ( Sharkhuu et al., 2011 )

. 

### 5

Toxicity assessment and allerg en sensitization was also assessed in these O 3 exposed 

### 6

offspring. At PND42, animals were euthanized for analysis of immune and inflammatory 

### 7

markers (immune proteins, inflammatory cells, T

- cell populations in the spleen). A subset 

### 8

of the animals was intra

- n asally instilled or sensitized with ovalbumin on either PND2 

### 9

and 3 or PND42 and 43. All animals were challenged with OVA on PND54, 55, and 56. 

### 10

One day after final OVA challenge, lung function, lung inflammation and immune 

### 11

response were determined. Offsprin g of O 3 exposed dams that were initially sensitized at 

### 12

PDN3 (early) or PND42 (late) were tested to determine the level of allergic sensitization 

### 13

or asthma

- like inflammation after OVA challenge. Female offspring sensitized early in 

### 14

life developed significa n t eosinophilia (1.2 ppm O 3 ) and elevated serum OVA

- specific 

### 15

IgE (1.2 ppm O 3 ), which is a marker of airway allergic inflammation. The females that 

### 16

were sensitized early also had significa nt decrements in BALF total cells, macrophages, 

### 17

and lymphocytes (1.2 p pm O 3 ). Offspring that were sensitized later (PND42) in life did 

### 18

not develop the aforementioned changes in BALF, but these animals did develop modest, 

### 19

albeit significa nt neutropenia (0.8 and 1.2 ppm O 3 ) ( Sharkhuu et al., 2011 )

. 

### 20

BALF cytology in non

- sensitized animals was followed. BALF of offspring born to dams 

### 21

exposed to O 3 was relatively unaffected (cytokines, inflammatory cell numbers/types) as 

### 22

were splenic T

- cell subpopulations. LDH was significa ntly elevated in BALF of females 

### 23

whose mothers were exposed to 1.2 ppm during pregnancy ( Sharkhuu et al., 2011 ) . In 

### 24

summary, the females born to mothers exposed to O 3 developed modest 

### 25

immunocompromise. Males were unaffected ( Sharkhuu et al., 2011 )

. 

### 26

Overall, animal toxicological studies have reported ultrastructural changes in bronchiole 

### 27

development, alterations in placental and pup cytokines, and increased pup airway hyper



### 28

reactivity related to exposure to O 3 during the developmental period. Epidemiologic 

### 29

studies have found no association between prenatal exposure to O 3 and growth and 

### 30

development of the respiratory system. Fetal origins of disease have received a lot of 

### 31

attention recently, thus additional research to further explore the inconsistencies between 

### 32

these two lines of evidence is warranted. 

### 33

### Draft

– Do Not Cite or Quote 7

- 71 June 2012 7.4.9 Developmental Central Ne rvous System Effects

The following sections describe the results of toxicological studies of O 3 and 

### 1

developmental central nervous system effects. No epidemiologic studies of this 

### 2

association have been published. 

### 3

7.4.9.1 Laterality

Two reports of laterality changes in mice developmentally exposed to O 3 have been 

### 4

reported in the literature. Mice developmentally exposed to 0.6 ppm O 3 (6 days before 

### 5

breeding to weaning at PND21) showed a turning preference (left turns) distinct from air 

### 6

exposed controls (clockwise turns) ( Dell'Omo et al., 1995 ) ; in previous studies this 

### 7

behavior in mice has been found to correlate with specific structural asymmetries of the 

### 8

hippocampal mossy fi ber projections ( Schöpke et al., 1991 ) . The 2006 O 3 AQCD 

### 9

evidence for the effect of O 3 on laterality or handedness demonstrated that rats exposed to 

### 10

O 3 during fetal and neonatal life showed limited, gender

- specific changes in handedness 

### 11

after exposure to the intermediate concentration of O 3 (only seen in female mice exposed 

### 12

to 0.6 ppm O 3 , and not in males at 0.6 ppm or in either sex of 0.3 or 0.9 ppm O 3 with 

### 13

exposure from 6 days before breeding to PND26) ( Petruzzi et al., 1999 )

. 

### 14

7.4.9.2 Brain Morphology and Neurochemical Changes

The nucleus tractus solit arius (NTS), a medullary area of respiratory control, of adult 

### 15

animals exposed prenatally to 0.5 ppm O 3 (12h/day, ED5

- eD20) had significa ntly less 

### 16

tyrosine hydroxylase staining versus control ( Boussouar et al., 2009 ) . Tyrosine 

### 17

hydroxylase is the rate

- limiting enzyme for dopamine synthesis and serves as a precursor 

### 18

for catecholamine synthesis; thus, decreased staining is used as a marker of dopaminergic 

### 19

or catecholaminergic cell or activity loss in these regions and thus functions in neuronal 

### 20

plasticity. After physical restraint stress, control animals respond at the histological level 

### 21

with Fos activation, a marker of neuronal activity, and tyrosine hydroxylase activation in 

### 22

the NTS , a response which is absent or attenuated in adult animals exposed prenatally to 

### 23

0.5 ppm O 3 ( Boussouar et al., 2009 ) when compared to control air exposed animals who 

### 24

also were r estrained. The O 3 - exposed offspring in this study were cross

- fostered to 

### 25

control air exposed dams to avoid O 3 - dependent dam related neonatal effects on offspring 

### 26

outcomes (i.e., dam behavioral or lactational contributions to pup outcomes) ( Boussouar 

### 27

et al., 2009 )

. 

### 28

### Draft

– Do Not Cite or Quote 7

- 72 June 2012 Developmental exposure to 0.3 or 0.6 ppm O 3 prior to mating pair formation through 

### 1

GD17 induced significa nt increased levels of BDNF in the striatum of adult (PND140) 

### 2

O 3 exposed offspring as compared to control air exposed animals; these O 3 - exposed 

### 3

animals also had significa ntly decreased level of NGF in the hippocampus versus control 

### 4

( Santucci et al., 2006 )

. 

### 5

Changes in the pup cerebellum with prenatal 1 ppm O 3 exposure inc lude altered 

### 6

morphology ( Romero

- Velazquez et al., 2002 ; Rivas

- Manzano and Paz, 1999 ) , decreased 

### 7

total area ( Romero

- Velazquez et al., 2002 ) , decreased number of Purkinje cells ( Romero



### 8

Velazquez et al., 2002 ) , and altered monoamine neurotransmitter content with the 

### 9

catecholamine system affected and the indoleamine system unaffected by O 3 ( Gonzalez



### 10

Pina et al., 2008 )

. 

### 11

7.4.9.3 Neurobehavioral Outcomes

O 3 administration to dams during pregnancy with or without early neonatal exposure has 

### 12

been shown to contribute to multiple neurobehavioral outcomes in offspring that are 

### 13

described in further detail below. 

### 14

O 3 administration (0.4, 0.8 or 1.2 ppm O 3 ) during th e majority of pregnancy (PD7

- 17) of 

### 15

CD

- 1 mice did not affect pup behavioral outcomes including early behavioral ultrasonic 

### 16

vocalizations and more permanent later measurements (PND60 or 61) including pup 

### 17

activity, habituation and exploration and d

- amphetami ne

- induced hyperactivity ( Bignami 

### 18

et al., 1994 ) ; these pups were all cross

- fostered or reared on non

- O 3 exposed dams. 

### 19

Testing for aggressive behavior in mice continuously exposed to O 3 (0.3 or 0.6 ppm from 

### 20

30 days prior to mating to GD17) revealed that mice had signifi ca ntly increased 

### 21

defensive/ submissive behavior (increased freezing posturing on the first day only of a 

### 22

multiple

- day exam) versus air exposed controls ( Santucci et al., 2006 ) . Similarly, 

### 23

continuous exposure of adult animals to O 3 induced significa nt increases in fear behavior 

### 24

and decreased aggression as measured by significa ntly decreased freezing behavior 

### 25

( Petruzzi et al., 1995 )

. 

### 26

Developmentally exposed animals also had significa ntly decreased amount of time spe nt 

### 27

nose sniffing other mice ( Santucci et al., 2006 ) ; this social behavior deficit, decreased 

### 28

sniffing time, was not found in an earlier study with similar exposures ( Petruzzi et al., 

### 29

1995 ) , but sniffing of specifi c body areas was measured in Santucci et al. (2006 ) and total 

### 30

number of sniffs of the entire body was measured in Petruzzi et al. (1995 ) . The two 

### 31

toxicology studies exploring social behavior (sniffing) employ different study designs 

### 32

and find opposite effects in animals exposed to O 3. 

### 33

### Draft

– Do Not Cite or Quote 7

- 73 June 2012 7.4.9.4 Sleep Aberrations after Developmental Ozone Exposure

The effect of gestational O 3 exposure (1 ppm O 3 daily for 12h/day, during dark period for 

### 1

the entire pregnancy ) on sleep patterns in rat offspring was followed using 24 h 

### 2

polysomnographic recordings at 30, 60 and 90 days of age ( Haro and Paz, 1993 )

. 

### 3

Ozone

- exposed pups manifested with inverted sleep

- wake p atterns or circadian rhythm 

### 4

phase

- shift. Rat vigilance was characterized in wakefulness, slow wave sleep (SWS), and 

### 5

paradoxical sleep (PS) using previously characterized criteria. The O 3 exposed offspring 

### 6

spent longer time in the wakefulness state during t he light period, more time in SWS 

### 7

during the period of darkness, and showed significa nt decrements in PS. Chronic O 3 

### 8

inhalation significa ntly decreased the duration of PS during both the light and dark 

### 9

periods ( Haro and Paz, 1993 ) . These effects were consistent at all time periods measured 

### 10

(30, 60 and 90 days of age). These sleep effects reported after developmental exposures 

### 11

expand upon the existing literature on sleep aberrations in adult animals exposed t o O 3 

### 12

[rodents: ( Paz and Huitron

- Resendiz, 1996 ; Arito et al., 1992 ) ; and cats: ( Paz and Bazan



### 13

Perkins, 1992 ) ]. A role for inhibition of cyclooxygenase

- 2 and the interleukins and 

### 14

prostaglandins in the O 3 - dependent sleep changes potentially exists with ev idence from a 

### 15

publication on indomethacin pretreatment attenuating O 3 - induced sleep aberrations in 

### 16

adult male animals ( Rubio and Paz, 2003 )

. 

### 17

7.4.10 Early Life Mortality

Infants may be particularly at risk for the effects of air pollution. Within the first year of 

### 18

life, infants develop rapidly; therefore their sensitivity may change within weeks or 

### 19

months. During the neonatal and post

- neonatal periods, the developing lung is highly 

### 20

sensitive to environmental toxicants. The lung is not well developed at birth , with 80% of 

### 21

alveoli being formed postnatally. An important question regarding the association 

### 22

between O 3 and infant mortality is the critical window of exposure during development 

### 23

for which infants are at risk. Several age intervals have been explored: n eonatal 

### 24

(<1 month); postneonatal (1 month to 1 year); and an overall interval for infants that 

### 25

includes both the neonatal and postneonatal periods (<1 year). Within these various age 

### 26

categories, multiple causes of deaths have been investigated, particularl y total deaths and 

### 27

respiratory

- related deaths. The studies reflect a variety of study designs, exposure 

### 28

periods, regions, and adjustment for confounders. As discussed below, a handful of 

### 29

studies have examined the effect of ambient air pollution on neonatal and postneonatal 

### 30

mortality, with the former the least studied. These studies varied somewhat with regard to 

### 31

the outcomes and exposure periods examined and study designs employed. 

### 32

### Draft

– Do Not Cite or Quote 7

- 74 June 2012 7.4.10.1 Stillbirth

Pereira et al. (1998 ) investigated the association among daily counts of intrauterine 

### 1

mortality (over 28 weeks of g estation) and air pollutant concentrations in Sao Paulo, 

### 2

Brazil from 1991 through 1992. The association was strong for NO 2 , but lesser for SO 2 

### 3

and CO. These associations exhibited a short lag time, less than 5 days. No significa nt 

### 4

association was detected between short

- term O 3 exposure and intrauterine mortality. 

### 5

7.4.10.2 Infant Mortality, Less than 1 Year

Ritz et al. (2006 ) linked birth and death certificat es for infants who died between 1989 

### 6

and 2000 to evaluate the influence of outdoor air pollution on infant death in the South 

### 7

Coast Air Basin of California. The authors examined short

- and long

- term exposure 

### 8

periods 2 weeks, 1 month, 2 months, and 6 months before each case subject’s death and 

### 9

reported no association between ambient levels of O 3 and infant mortality. Similarly, 

### 10

Diaz et al. (2004 ) analyzed the effects of extreme temperatures and short

- term exposure 

### 11

to air pollutants on daily mortality in children less than 1 year of age in Madrid, Spain, 

### 12

from 1986 to 1997 and observed no statistically significa nt association between mortality 

### 13

a nd O 3 concentrations. Hajat et al. (2007 ) analyzed time

- series data of daily infant 

### 14

mortality counts in 10 major cities in the UK to quantify any associations with short

- term 

### 15

changes in air pollution . When the results from the 10 cities were combined there was no 

### 16

relationship between O 3 and infant mortality, even after restricting the analysis to just the 

### 17

summer months. 

### 18

Conversely, a time

- series study of infant mortality conducted in the southwestern part of 

### 19

Mexico City in the years 1993

- 1995 found that infant mortality was associated with 

### 20

short

- term exposure to NO 2 and O 3 3

- 5 days before death, but not as consistently as with 

### 21

PM. A 10

- ppb increase in 24

- h avg O 3 was associated with a 2.78% increase ( 95% CI: 

### 22

0.29, 5.26%) in infant mortality (lag 3) ( Loomis et al., 1999 ) . This increase was 

### 23

attenuated, although still positive when evaluated in a two

- pollutant model with PM 2.5

. 

### 24

One

- hour max concentrations of O 3 exceeded prevailing Mexican and international 

### 25

standards nearly every day. 

### 26

7.4.10.3 Neonatal Mortality, Less than 1 Month

Several studies have evaluated ambient O 3 concentrations and neonatal mortality and 

### 27

observed no association. Ritz et al. (2006 ) linked birth and death cert ificates for infants 

### 28

who died between 1989 and 2000 to evaluate the influence of outdoor air pollution on 

### 29

### Draft

– Do Not Cite or Quote 7

- 75 June 2012 infant death in the South Coast Air Basin of California. The authors examined short

- and 

### 1

long

- term exposure periods 2 weeks, 1 month, 2 months, and 6 months before each case 

### 2

subject’s death and reported no association between ambient levels of O 3 and neonatal 

### 3

mortality. Hajat et al. (2007 ) analyzed time

- series data of daily infant mortality counts in 

### 4

10 major cities in the UK to quantify any associations with short

- term changes in air 

### 5

pollution. When the results from the 10 cities were combined there was no relationship 

### 6

between O 3 and neonatal mortality, even after restricting the analysis to just the summer 

### 7

months. Lin et al. (2004a ) assessed the impact of short

- term changes in air pollutants on 

### 8

the number of daily neonatal deaths in Sao Paulo, Brazil. The authors observed no 

### 9

association between ambient levels of O 3 and neonatal mortality. 

### 10

7.4.10.4 Postneonatal Mortality, 1 Month to 1 Year

A number of studies focused on the postneonatal period when exami ning the effects of O 3 

### 11

on infant mortality. Ritz et al. (2006 ) linked birth and death certificates for infants who 

### 12

died between 1989 and 2000 to ev aluate the influence of outdoor air pollution on infant 

### 13

death in the South Coast Air Basin of California. The authors examined short

- and long



### 14

term exposure periods 2 weeks, 1 month, 2 months, and 6 months before each case 

### 15

subject’s death and reported no a ssociation between ambient levels of O 3 and 

### 16

postneonatal mortality. Woodru ff et al. (2008 ) evaluated the county

- level relationship 

### 17

between cause

- specific postneonatal infant mortality and long

- term early

- life exposure 

### 18

(first 2 months of life) to air pollutants across the U.S. Similarly, they found no 

### 19

association between O 3 ex posure and deaths from respiratory causes. In the U.K., Hajat et 

### 20

al. (2007 ) analyzed time

- series data of daily infant mortality counts in 10 major cities to 

### 21

quantify any associations with short

- term changes in air pollution. When the results from 

### 22

the 10 cities were combined there was no relationship between O 3 and postneonatal 

### 23

mortality, even after restricting the analysis to just the summer months. In Ciudad Juarez, 

### 24

Mexico, Romieu et al. (2004a ) examined the daily number of deaths between 1997 and 

### 25

2001, estimating the modifying effect of SES on the risk of postneonatal mortality. 

### 26

Ambient O 3 concentrations were not related to infant mortality overall, or in any of the 

### 27

SES groups. In a follow

- up s tudy, Carbajal

- Arroyo et al. (2011 ) evaluated the 

### 28

relationship of 1

- h daily max O 3 levels with postneonatal infant mortality in the 

### 29

Mexico City Metropolitan Area between 1997 and 2005. Generally, short

- ter m exposure 

### 30

to O 3 was not significa ntly related to infant mortality. However, upon estimating the 

### 31

modifying effect of SES on the risk of postneonatal mortality, the authors found that O 3 

### 32

was statistically significa ntly related to respiratory mortality among those with low SES. 

### 33

In a separate analysis, the effect of PM 10 was evaluated with O 3 level quartiles. PM 10 

### 34

alone was related to a significa nt increase in all

- cause mortality. The magnitude of this 

### 35

### Draft

– Do Not Cite or Quote 7

- 76 June 2012 effect remained the same when only the days when O 3 was in the lowest quartile were 

### 1

included in the analyses. However, when only the days when O 3 was in the highest 

### 2

quartile were included in the analyses, the magnitude of the PM 10 effect increased 

### 3

dramatically (OR = 1.06 [95% CI: 0.909, 1.241] for PM 10 on days wi th O 3 in lowest 

### 4

quartile; OR = 1.26 [95% CI: 1.08, 1.47] for PM 10 on days with O 3 in the highest quartile. 

### 5

These results suggest that while O 3 alone may not have an effect on infant mortality, it 

### 6

may serve to potentiate the observed effect of PM 10 on infan t mortality. 

### 7

Tsai et al. (2006 ) used a case

- crossover analysis to examine the relationship between 

### 8

short

- term exposure to air pollution and postneonatal mortality in Kaohsiung, Taiwan 

### 9

during the period 1994

- 2000. The risk of postneonatal deaths was 1.023 (95% CI: 0.564, 

### 10

1.858) per 10

- ppb increase in 24

- h avg O 3 . The confidence interval for this effect 

### 11

estimate is very wide, likely due to the small number of infants that died each day, 

### 12

making it difficult to interpret this result. Several other studies conducted in Asia d id not 

### 13

find any association between O 3 concentrations and infant mortality in the postneonatal 

### 14

period. Ha et al. (2003 ) conducted a daily time

- series study in Seoul, Korea to evaluate 

### 15

the effect of short

- term changes in ambient 8

- h O 3 concentrations on postneonatal 

### 16

mortality. Son et al. (2008 ) examined the relationship between air pollution and 

### 17

postneonatal mortality from all causes among firstborn infants in Seoul, Korea during 

### 18

1999

- 2003. Yang et al. (2006 ) used a case

- crossover analysis to examine the relationship 

### 19

between air pollution exposure and postneonatal mortality in Taipei, Taiwan for the 

### 20

period 1994

- 2000. The authors observed no associations between ambient levels of O 3 

### 21

and postneonatal mortality. 

### 22

7.4.10.5 Sudden Infant Death Syndrome

The strongest evidence for an association between ambient O 3 concentrations and SIDS 

### 23

comes from a study that evaluated the county

- level relationship bet ween SIDS and long



### 24

term early

- life exposure (first 2 months of life) to air pollutants across the U.S. 

### 25

( Woodruff et al., 2008 ) . The authors observed a 1.20 (95% CI: 1.09, 1.32) odds ratio for a 

### 26

10

- ppb increase in O 3 and deaths from SIDS. There was a monotonic increase in odds of 

### 27

SIDS for each quartile of O 3 exposure compared with the lowes t quartile (highest quartile 

### 28

OR = 1.51; [95% CI: 1.17, 1.96]). In a multi

- pollutant model including PM 10 or PM 2.5 , 

### 29

CO and SO 2 , the OR for SIDS and O 3 was not substantially lower than that found in the 

### 30

single

- pollutant model. When examined by season, the re lationship between SIDS deaths 

### 31

and O 3 was generally consistent across seasons with a slight increase for those babies 

### 32

born in the summer. When stratified by birth weight, the OR for LBW babies was 1.27 

### 33

(95% CI: 0.95, 1.69) per 10

- ppb increase in O 3 and the OR for normal weight babies was 

### 34

1.16 (95% CI: 1.01, 1.32) per 10

- ppb increase in O 3

. 

### 35

### Draft

– Do Not Cite or Quote 7

- 77 June 2012 Conversely, two additional studies reported no association between ambient levels of O 3 

### 1

and SIDS. Ritz et al. (2006 ) linked birth and death certificates for infants who died 

### 2

between 1989 and 2000 to evaluate the influence of outdoor air pollution on infant death 

### 3

in the South Coast Air Basin of California. The autho rs examined short

- and long

- term 

### 4

exposure periods 2 weeks, 1 month, 2 months, and 6 months before each case subject’s 

### 5

death and reported no association between ambient levels of O 3 and SIDS. Dales et al. 

### 6

(2004 ) used time

- series analyses to compare the daily mortality rates for SIDS and short



### 7

term air pollution concentrations in 12 Canadian cities during the period of 1984

- 1999. 

### 8

Increased daily rates of SIDS were associated with previous day increases in the levels of 

### 9

SO 2 , NO 2 , and CO, but not O 3 or PM 2.5

. 

### 10

Table 7

- 8 provides a brief overview of the epidemiologic studies of infant mortality. 

### 11

These studies have focused on short

- term exposure windows (e.g., 1

- 3 days) and long



### 12

term exposure windows (e.g., up to 6 months). Collectively, they provide no evidence for 

### 13

Table 7

- 8 Brief summary of infant mortality studies.

Study Location Mean O 3 (ppb) Exposure
Assessment Effect Estimate a (95% CI):

Pereira et al. (1998 ) Sao Paulo, Brazil 1

- h max: 33.8 Citywide avg L0

- 2: 1.00 (0.99, 1.01)

Diaz et al. (2004 ) Madrid, Spain 24

- h avg: 11.4 Citywide avg NR

Loomis et al. (1999 ) Mexico City,
Mexico 24

- h avg: 44.1

1

- h max: 163.5 1 monitor L0: 0.99 (0. 97, 1.02)

L1: 0.99 (0.96, 1.01)

L2: 1.00 (0.98, 1.03)

L3: 1.03 (1.00, 1.05)

L4: 1.01 (0.98, 1.03)

L5: 1.02 (0.99, 1.04)

L0

- 2: 1.02 (0.99, 1.05)

Ritz et al. (2006 ) Southern
California 24

- h avg: 21.9

- 22.1 Nearest Monitor 2 weeks before death: 1.03 (0.93, 1.14)

1 mo before death: NR

2 mo before death: 0.93 (0.89, 0.97)

6 mo before death: NR

Hajat e t al. (2007 ) 10 Cities in the
UK 24

- h avg: 20.5

- 42.6 Citywide avg L0

- 2: 1.00 (0.96, 1.06)

Lin et al. (2004a ) Sao Paulo, Brazil 24

- h avg: 38.06 Citywide avg L0: 1.00 (0.99, 1.01)

Ha et al. (2003 ) Seoul, South
Korea 8

- h avg: 21.2 Citywide avg L0: 0.93 (0.90, 0.96)

Romieu et al. (2004a ) Ciudad Juarez,
Mexico 8

- h avg: 43.43

- 55.12 Citywide avg L1: 0.96 (0.90, 1.03)

L2: 0.97 (0.91, 1.04)

L0

- 1 cum: 0.96 (0.89, 1.04)

L0

- 2 cum: 0.94 (0.87, 1.02)

### Draft

– Do Not Cite or Quote 7

- 78 June 2012 Study Location Mean O 3 (ppb) Exposure
Assessment Effect Estimate a (95% CI):

Carbajal

- Arroyo et al. (2011 ) Mexico City,
Mexico 1

- h max: 103.0 Citywide avg L0: 1.00 (0.99, 1.00)

L1: 0.99 (0.99, 0.99)

L2: 0.99 (0.99, 1.00)

L0

- 2: 0.99 (0.99, 1.00)

Son et al. (2008 ) Seoul, South
Korea 8

- ha avg:
25.61 Citywide avg L(NR): 0.984 (0.976, 0.992) b

Tsai et al. (2006 ) Kaohsiung,
Taiwan 24

- h avg: 23.60 Citywide avg L0

- 2 cum: 1.02 (0.56, 1.86)

Woodruff et al. (2008 ) Nationwide, U.S. 24

- h avg: 26.6 County wide avg First 2 mo of life: 1.04 (0.98, 1.10)

Yang et al. (2006 ) Taipei, Taiwan 24

- h avg: 18.14 Citywide avg L0

- 2 cum:1.00 (0.62, 1.61)

Dales et al. (2004 ) 12 Canadian
cities 24

- h: 31.77 Citywide avg L0: NR

L1: NR

L2: NR

L3: NR

L 4: NR

L5: NR
Multiday lags of 2

- 6 days: NR

a Relative risk of infant mortality per 10 ppb change in O 3

b No increment provided

L0 = Lag 0, L1= Lag 1, L2 = Lag 2, L3 = Lag 3, L4 = Lag 4, L5 = Lag 5, L6 = Lag 6

NR: No quantitative results reported



### Table 7

- 9 Summary of key reproductive and developmental toxicological

s tudies.

Study Model O 3 (ppm) Exposure Duration Effects

Sharkhuu et al. (2011 ) Pregnant mice; BALB/c; F;
GD9

- 18; effects in offspring 0.4, 0.8, or

1.2 Continuously for 10 consecutive days Dams: Decreased number of dams reaching parturition. Offspring: ( 1 )

- Decreased birth weights. ( 2 )

- Decreased rate of postnatal growth (body weight). ( 3 )

- impaired delayed type hypersensitivity. ( 4 )

- No effec t on humoral immunity. ( 5 )

- Significa ntly affected allergic airway inflammation markers (eosinophilia, IgE) in female offspring sensitized early in life.

6

- BALF LDH significa ntly elevated in female offspring.

Bignami et al. (1994 ) Pregnant CD

- 1 dams (PD7

- 1 7) 0.4, 0.8
or 1.2 Continuous Reproductive success was not affected by O 3 exposure (PD7

- 17, proportion of successful pregnancies, litter size, ex ratio, frequency of still birth, or neonatal mortality). Ozone acted as a transient anorexigen in pregnant dams.

Haro and Paz (1993 ) Rat dams, Exposure over the entirety of

pregnancy; 1.0 12h/day during dark cycle Decreased birth weight and postnatal body weight of offspring
out to PND 90. Ozone

- exposed pups manifested with inverted sleep

- wake patterns or circadian rhythm phase

- shift.

López et al. (2008 ) Rats; Pregnant dams; GD1

- GD18, GD20,
or GD21. 1.0 (12 h/day, out to either GD18, GD20 or GD21) O 3 induced delayed maturation of near term rodent bronchioles, with ultra

- structural damage to bronchiolar epithel ium.

### Draft

– Do Not Cite or Quote 7

- 79 June 2012 Study Model O 3 (ppm) Exposure Duration Effects

Auten et al. (2009 ) C57BL/6

mouse pups 1.0 3 h/day, every ot her day, thrice weekly for 4
weeks Postnatal O 3 exposure significa ntly increased lung inflammatory cytokine levels; this was further exacerbated with gestational PM exposure.

Plopper e t al. (2007 ) Infant rhesus monkeys 0.5 Postnatal, PND30

- 6month of age, 5 months of cyclic exposure, 5 days O 3 followed by 9 days of filtered air, 8h/day. Non

- significa nt increases airway resistance and airway responsiveness with O 3 or inhaled allergen a lone. Allergen +
O 3 produced additive changes in both measures.

Fanucchi et al. (2006 ) Infant male
Rhesus monkeys, post

natal exposure 0.5 5 months of episodic exposure, age 1 month

age 6 months, 5 days
O 3 followed by 9 days
of filtered air, 8h/day. Cellular changes and significa nt structural changes in the distal respiratory tract in infant rhesus monkeys exposed to O 3 postnatally.

Dell'Omo et al. (1995 ) CD

- 1 Mouse dams and pups 0.6 6 days before breeding to weaning at PND21 Laterality changes in offspring: Ozone exposed pups showed a turning preference (left turns) distinct from air exposed controls (clockwise turns) as adults.

Santucci et al. (2006 ) CD

- 1 Mouse
dams 0.3 or
0.6 Dam exposure prior to mating through GD17. Developmental O 3 caused increased defensive/submissive behavior in offspring. O 3 exposed offspring also had significa nt elevations of striatal BDNF and hippocampal NGF v. air exposed controls.

Han et al. (2011 ) Rat; Sprague Dawley, M & F;
PND13 0.6 3 h, BALF examined
10h after O 3 exposure BALF polymorphonuclear leukocytes and total BALF protein

were significa ntly elevated in O 3 exp osed pups. Lung tissue
from O 3 exposed pups had significa nt elevations of manganese superoxide dismutase (SOD) protein and significa nt decrements of extra

- cellular SOD protein.

Campos

Bedolla et al. (2002 ) Pregnant Rats; Sprague Dawley (GD5, GD10, or GD18) 3.0 1 h on one day of gestation, uteri
collected 16

- 18 h later Ozone inhalation modifies the contractile response of the pregnant uterus. The O 3 exposed pregnant uteri had significa nt increases in the maximum response to acetyl choline stimulation at GD5 and 10; they also had a significa nt increase in maximal response to oxytocin at GD 5.

Kavlock et al. (1980 ) CD

- 1 mice; (pregnancy day

7

- 17) 0.4, 0.8
and 1.2 Continuous, pregnancy day 7

- 17 O 3 induced d ecrements in postnatal body weight gain. When
O 3 was co

- administered with sodium salicylate, O 3 synergistically increased the rate of pup resorption (1.0 ppm GD9

- 12).

Jedlinska

Krakowska et al. (2006 ) 5 month old
male Wistar
Hannover rats 3.0 0.5 ppm, 5h/day for

50 days Histopathological evidence of impaired spermatogenesis (round spermatids / 21 spermatocytes, giant spermatid cells, and focal epithelial desquamation with denudation to the 22 basement membrane). Vitamin E exposure concomitant with
O 3 protected against pathological changes but Vitamin C did

not.



### 7.4.11 Summary and Causal Determination

The 2006 O 3 AQCD concluded that the limited number of studies that investigated O 3 

### 1

demonstrated no associations between O 3 and birth outcomes, with the possible exception 

### 2

of birth defects. The current review included an expanded body of evide nce on the 

### 3

associations between O 3 and reproductive and developmental effects. Recent 

### 4

epidemiologic and toxicological studies provide evidence for an effect of prenatal 

### 5

exposure to O 3 on pulmonary structure and function, including lung function changes in 

### 6

the newborn, incident asthma, ultrastructural changes in bronchiole development, 

### 7

### Draft

– Do Not Cite or Quote 7

- 8 0 June 2012 alterations in placental and pup cytokines, and increased pup airway hyper

- reactivity. 

### 1

Also, there is limited toxicological evidence for an effect of prenatal and early life 

### 2

e xposure on central nervous system effects, including laterality, brain morphology, 

### 3

neurobehavioral abnormalities, and sleep aberration. Recent epidemiologic studies have 

### 4

begun to explore the effects of O 3 on sperm quality, and provide limited evidence for 

### 5

decrements in sperm concentration, while there is limited toxicological evidence for 

### 6

testicular degeneration associated with O 3

. 

### 7

While the collective evidence for many of the birth outcomes examined is g enerally 

### 8

inconsistent (including birth defects), there are several well

- designed, well

- conducted 

### 9

studies that indicate an association between O 3 and adverse outcomes. For example, as 

### 10

part of the southern California Children’s Health Study, Salam et al. (2005 ) observed a 

### 11

concentration

- response relationship of decreasing birth weight with increasing O 3 

### 12

concentrations averaged over the entire pregnancy that was clearest above the 30

- ppb 

### 13

level (see Figure 7

- 4 ). Similarly, Hansen et al. (2008 ) utilized fetal ultrasonic 

### 14

measurements and found a change in ultrasound measurements associated with O 3 during 

### 15

days 31

- 60 of gestation indicated that increasing O 3 concentration decreased an 

### 16

ultrasound measurement for women living within 2 km of the monitoring site. 

### 17

The weight of evidence does not indicate that prenatal or early life O 3 concentrations are 

### 18

associated with infant mortality. Collectiv ely, there is limited though positive 

### 19

toxicological evidence for O 3 - induced developmental effects, including effects on 

### 20

pulmonary structure and function and central nervous system effects. Limited 

### 21

epidemiologic evidence for an effect on prenatal O 3 exposur e on respiratory development 

### 22

provides coherence with the effects observed in toxicological studies. There is also 

### 23

limited epidemiologic evidence for an association with O 3 concentration and decreased 

### 24

sperm concentration. A recent toxicological study provid es limited evidence for a 

### 25

possible biological mechanism (histopathology showing impaired spermatogenesis) for 

### 26

such an association. Additionally, though the evidence for an association between O 3 

### 27

concentrations and adverse birth outcomes is generally incons istent, there are several 

### 28

influential studies that indicate an association with reduced birth weight and restricted 

### 29

fetal growth. 

### 30

Some of the key challenges to interpretation of these study results include the difficulty in 

### 31

assessing exposure as most stud ies use existing monitoring networks to estimate 

### 32

individual exposure to ambient air pollution (see Section 4.6 ); the inability to control for 

### 33

potent ial confounders such as other risk factors that affect birth outcomes (e.g., smoking); 

### 34

evaluating the exposure window (e.g., trimester) of importance; integrating the results 

### 35

from both short

- and long

- term exposure periods; integrating the results across a variety 

### 36

### Draft

– Do Not Cite or Quote 7

- 81 June 2012 of reproductive and developmental outcomes; and limited evidence on the physiological 

### 1

mechanism of these effects. 

### 2

Taking into consideration the positive evidence for developmental and reproductive 

### 3

outcomes from toxicological and epidemiological st udies, and the few influential birth 

### 4

outcome studies, the evidence is suggestive of a causal relationship between 

### 5

exposures to O 3 and reproductive and developmental effects

. 

### 6



## 7.5 Central Nervous System Effects

7.5.1 Effects on the Brain and Behavior

The 2006 O 3 AQCD included toxicological evidence that acute exposures to O 3 are 

### 7

associated with alterations in neurotransmitters, motor activity, short and long term 

### 8

memory, and sleep patterns. Additionally, histological signs of neurodegeneration have 

### 9

been observed. Reports of headache, dizziness, and irritation of the nose with O 3 

### 10

exposure are common complaints in humans, and some behavioral changes in animals 

### 11

may be related to these symptoms rather than indicative of neurotoxicity. Research in the 

### 12

area of O 3 - induce d neurotoxicity has notably increased over the past few years, and 

### 13

recent studies examining the effects of long

- term exposure have demonstrated 

### 14

progressive damage in various regions of the brains of rodents in conjunction with altered 

### 15

behavior. Evidence fr om epidemiologic studies has been more limited. A recently 

### 16

published epidemiologic study examined the association between O 3 concentration and 

### 17

neurobehavioral effects. Chen and S chwartz (2009 ) utilized data from the NHANES III 

### 18

cohort to study the relationship between O 3 concentrations (mean annual O 3 

### 19

concentration 26.5 ppb) and neurobehavioral effects among adults aged 20

- 59 years. 

### 20

Annual O 3 concentration was determined using i nverse distance weighting for county of 

### 21

residence and adjacent counties (for more information on inverse distance weighting and 

### 22

other methods for exposure assessment, see Section 4.5.1 and 4.6 ). The authors observed 

### 23

an association between annual O 3 concentration and tests measuring coding abilit y 

### 24

(symbol

- digit substitution test) and attention/short

- term memory (serial

- digit learning 

### 25

test). Each 10

- ppb increase in annual O 3 concentration corresponded to an aging

- related 

### 26

cognitive performance decline of 3.5 yr for coding ability and 5.3 years for 

### 27

a ttention/short

- term memory. These associations persisted in both crude and adjusted 

### 28

models. There was no association between O 3 concentration and reaction time tests. The 

### 29

authors concluded that overall, there is an association between long

- term O 3 

### 30

concentr ation and reduced performance on neurobehavioral tests. 

### 31

### Draft

– Do Not Cite or Quote 7

- 82 June 2012 A number of recent toxicological studies demonstrate various perturbations in neurologic 

### 1

function or histology with long

- term exposure to O 3 , including changes similar to those 

### 2

observed in neurodegene rative disorders such as Parkinson’s and Alzheimer’s disease 

### 3

pathologies in relevant regions of the brain ( Table 7

- 10 ). The central nervous system is 

### 4

very sensitive to oxidative stress, due in part to its high content of polyunsaturated fatty 

### 5

acids, high rate of oxygen consumption, and low antioxidant enzyme capacity. Oxidative 

### 6

stress has been identified as one of the pa thophysiological mechanisms underlying 

### 7

neurodegenerative disease ( Simonian an d Coyle, 1996 ) , and it is believed to play a role in 

### 8

altering hippocampal function, which causes cognitive deficits with aging ( V anguilder 

### 9

and Freeman, 2011 ) . A particularly common finding in studies of O 3 - exposed rats is lipid 

### 10

peroxidation in the brain, especially in the hippocampus, which is important for higher 

### 11

cognitive function including contextual memory acquisition. Perfor mance in passive 

### 12

avoidance learning tests is impaired when the hippocampus is injured. For example, in a 

### 13

subchronic study, exposure of rats to 0.25 ppm O 3 (4 h/day) for 15

- 90 days caused a 

### 14

complex array of responses, including a time

- dependent increase in lipid peroxidation 

### 15

products and immunohistochemical changes in the hippocampus that were correlated 

### 16

with decrements in passive avoidance behavioral tests ( Rivas

- Arancibia et al., 2010 )

. 

### 17

Changes included increased numbers of ac tivated microglia, a sign of inflammation, and 

### 18

progressive neurodegeneration. Notably, continued exposure tends to bring about 

### 19

progressive, cumulative damage, as shown by this study ( Rivas

- Arancibia et al., 2010 ) 

### 20

and others ( Santiago

- López et al., 2010 ; Guevara

- Guzmán et al., 2009 ; Angoa

- Pérez et 

### 21

al., 2006 ) . The effects of O 3 on passive avoidance test performance were particularly 

### 22

evident at 90 days for both short

- and long

- term memory. The greatest extent of cell loss 

### 23

was also observed at this time point, whereas lipid peroxidation did not increase much 

### 24

beyond 60 days of expo sure. 

### 25

The substantia nigra is another region of the brain affected by O 3 , and seems particularly 

### 26

sensitive to oxidative stress because the metabolism of dopamine, central to its function, 

### 27

is an oxidative process perturbed by redox imbalance. Oxidative stre ss has been 

### 28

implicated in the premature death of substantia nigra dopamine neurons in Parkinson’s 

### 29

disease. Progressive damage has been found in the substantia nigra of male rats after 15, 

### 30

30, and 60 days of exposure to 0.25 ppm O 3 for 4 h/day. Santiago

- López et al. (2010 ) 

### 31

observed a reduction dopaminergic neurons within the substantia nigra over time, with a 

### 32

complete loss of normal morphology in the remaining cells and virtually no dopamine 

### 33

immunoreactivity at 60 days. This was accompanied by an increase in p53 levels and 

### 34

nuclear translocation, a process associated with programmed cell death. Similarly, 

### 35

Angoa

- Pérez et al. (2006 ) have shown progressive lipoperoxidation in the substantia 

### 36

nigra and a decrease in nigral neurons in ovariectomized female rats exposed to 0.25 ppm 

### 37

O 3 , 4h/day, for 7

- 60 days. Lipid peroxidation effectively doubled between the 30 and 

### 38

60 day time points. Total nigral cell number was also diminished to the greatest extent at 

### 39

### Draft

– Do Not Cite or Quote 7

- 83 June 2012 60 days, and cell loss was p articularly evident in the tyrosine hydroxylase positive cell 

### 1

population (90%), indicating a selective loss of dopamine neurons or a loss of dopamine 

### 2

pathway functionality. 

### 3

The olfactory bulb also undergoes oxidative damage in O 3 - exposed animals, in some 

### 4

c ases altering olfactory

- dependent behavior. Lipid peroxidation was observed in the 

### 5

olfactory bulbs of ovariectomized female rats exposed to 0.25 ppm O 3 (4 h/day) for 30 or 

### 6

60 days ( Guevara

- Guzmán et al., 2009 ) . O 3 also induced decrements in a selective 

### 7

olf actory recognition memory test, which were significa ntly greater at 60 days compared 

### 8

to 30 days, and the authors note that early deficits in odor perception and memory are 

### 9

components of human neurodegenerative diseases. The decrements in olfactory memory 

### 10

d id not appear to be due to damaged olfactory perception based on other tests early on, 

### 11

but by 60 days deficits in olfactory perception had emerged. 

### 12

Memory deficits and associated morphological changes can be attenuated by 

### 13

administration of α

- tocopherol ( Guerrero et al., 1999 ) , taurine ( Rivas

- Arancibia et al., 

### 14

2000 ) , and estradiol ( Guevara

- Guzmán et al., 2009 ; Angoa

- Pérez et al., 2006 ) , all of 

### 15

which have antioxidant properties. In the study by Angoa

- Pérez et al. (2006 ) described 

### 16

above, estradiol seemed particularly effective at protecting against lipid peroxidation and 

### 17

nigral cell loss at 60 days compared to sh orter exposure durations. The same was true for 

### 18

amelioration of decrements in olfactory recognition memory ( Guevara

- Guzmán et al., 

### 19

2009 ) , although protection against lipid peroxidation was similar for the 30 and 60 day 

### 20

exposures. 

### 21

CNS effects have also been demonstrated in adult mice whose only exposure to O 3 

### 22

occurred while in utero, a period particularly critical for brain development. Santucci et 

### 23

al. (2006 ) investigated behavioral effects and gene expression after in utero exposure of 

### 24

mice to 0.3 or 0.6 ppm O 3 . Exposure began 30 days prior to mating and continued 

### 25

throughout gestation. Testing of adult animals demonstrated increased 

### 26

defensive/submissive behavior and reduced social investigation were observed in both the 

### 27

0.3 and 0.6 ppm O 3 groups. C hanges in gene expression of brain

- derived neurotrophic 

### 28

factor (BDNF, increased in striatum) and nerve growth factor (NGF, decreased in 

### 29

hippocampus) accompanied these behavioral changes. BDNF and NGF are involved in 

### 30

neuronal organization and the growth, ma intenance, and survival of neurons during early 

### 31

development and in adulthood. This study and two others using short

- term exposures 

### 32

demonstrate that CNS effects can occur as a result of in utero exposure to O 3 , and 

### 33

although the mode of action of these effec ts is not known, it has been suggested that 

### 34

circulating lipid peroxidation products may play a role ( Boussouar et al., 2009 )

. 

### 35

Importantly, these CNS effects occurred in rodent mo dels after in utero only exposure to 

### 36

(semi

- ) relevant concentrations of O 3

. 

### 37

### Draft

– Do Not Cite or Quote 7

- 84 June 2012 Table 7

- 10 Central nervous system effects of l ong
- term ozone exposure in r ats.

Study Model O 3 (ppm) Exposure Duration Effects

Angoa

- Pérez et al. (2006 ) Rat; Wistar; F; Weight: 300 g; Ovariectomized 0.25 7 to 60 days, 4 h/day,

5 days/week Long

- term estradiol treatment protected against O 3 - induced oxidative damage to nigral dopamine neurons, lipid peroxidation, and loss of tyrosine hydrolase

- immunopositive cells.

Guevara

Guzmán et al. (2009 ) Rat; Wistar; F; Weight: 264 g; Ovariectomized 0.25 30 and 60 days, 4h/day Long

- term estradiol treatment protected against O 3 - induced oxidative stress and
decr eases in α and β estrogen receptors and dopamine β

- hydroxlyase in olfactory bulb, and deficits in olfactory social recognition memory and chocolate recognition.

Rivas

- Arancibia et al. (2010 ) Rat; Wistar; M; Weig ht: 250

- 300 g 0.25 15 to 90 days, 4h/day Ozone produced significa nt increases in lipid peroxidation in the hippocampus, and altered the number of p53 positive immunoreactive cells, activated and phagocytic microglia, GFAP immunoreactive cells, double corti ne cells,
and short

- and long

- term memory

retention latency

Santiago

- López et al. (2010 ) Rat; Wistar; M; Weight: 250

- 300 g 0.25 15, 30, and 60 days,

4 h/day Progressive loss of dopamine reactivity in the substantia nigra, along with morphological changes. Increased p53
levels and nuclear translocation.

Santucci et al. (2006 ) Mice; CD

- 1; M;

18 weeks old 0.3; 0.6 Females continuously exposed from 30 days prior to breeding until
GD17 Upon behavioral challenge with another
mal e, there was a significa nt increase in defensive and freezing postures and decrease in the frequency of nose

sniffing. These behavioral changes were accompanied by a significa nt increase in
BDNF in the striatum and a decrease of NGF in the hippocampus.



### 7.5.2 Summary and Causal Determination

The 2006 O 3 AQCD included toxicological evidence that acute exposures to O 3 are 

### 1

associated with alterations in neurotransmitters, motor activity, short and long term 

### 2

memory, and sleep patterns. Additionally, histological s igns of neurodegeneration have 

### 3

been observed. However, evidence regarding chronic exposure and neurobehavioral 

### 4

effects was not available. Recent research in the area of O 3 - induced neurotoxicity has 

### 5

included several long

- term exposure studies. Notably, the first epidemiologic study to 

### 6

examine the relationship between O 3 exposure and neurobehavioral effects observed an 

### 7

association between annual O 3 levels and an aging

- related cognitive performance decline 

### 8

in tests measuring coding ability and attention/short

- term memory. This observation is 

### 9

supported by studies in rodents which demonstrate progressive oxidative stress and 

### 10

damage in the brain and associated decrements in behavioral tests, including those 

### 11

measuring memory, after subchronic exposure to 0.25 ppm O 3 . Additionally, 

### 12

### Draft

– Do Not Cite or Quote 7

- 85 June 2012 neurobehavioral changes are evident in animals whose only exposure to O 3 occurred in 

### 1

utero. Collectively, the limited epidemiologic and toxicological evidence is coherent and 

### 2

suggestive of a causal relationship between O 3 exposure and CNS effects. 

### 3



## 7.6 Carcinogenic and Genotoxic Potential of Ozone

7.6.1 Introduction

The radiomimetic and clastogenic qualities of O 3 , combined with its ability to stimulate 

### 4

proliferation of cells in the respiratory tract, have suggested that O 3 could act as a 

### 5

carcinogen. However, toxicological studies of tumorigenesis in the rodent lung have 

### 6

yielded mixed and often confusing results, and the epidemiologic evidence is equally 

### 7

conflicted. The 2006 O 3 AQCD concluded that, “the weight of evidence from recent 

### 8

animal toxicologic al studies and a very limited number of epidemiologic studies do not 

### 9

support ambient O 3 as a pulmonary carcinogen” 1 ( U.S. EPA, 2006b )

. 

### 10

Multiple epidemiologic studies reported in the 2006 O 3 AQCD examined the association 

### 11

between O 3 concentration and cancer. The largest of these studies, by Pope et al. (2002 )

, 

### 12

included 500,000 adults from the American Cancer Society’s (ACS) Cancer Preventio n II 

### 13

study. In this study, no association was observed between O 3 concentration and lung 

### 14

cancer mortality. The Adventist Health Study of Smog (AHSMOG) also examined the 

### 15

association between O 3 concentration and lung cancer mortality ( Abbey et al., 1999 )

. 

### 16

The re was a positive association between O 3 concentrations and lung cancer mortality 

### 17

among men. No association was reported for women. Another study using the AHSMOG 

### 18

cohort assessed the risk of incident lung cancer ( Beeson et al., 1998 ) . Among males, an 

### 19

association with incidence of lung cancer was observed with increasing O 3 

### 20

concentrations. When stratified by smoking status, the association persisted among never 

### 21

smokers but was null for former smokers. No assoc iation was detected for females. The 

### 22

Six Cities Study examined various air pollutants and mortality but did not specifically 

### 23

explore the association between O 3 concentrations and lung cancer mortality due to low 

### 24

variability in O 3 concentrations across the cities ( Dockery et al., 1993 ) . An ecologic study 

### 25

performed in Sao Paulo City, Brazil examined the correlations between O 3 concentrations 

### 26

in four of the city districts and incident cancer of the larynx and lung reported in 1997 

### 27

( Pereira et al., 2005 ) . A correlation between the average number of days O 3 

### 28

concentrations exceeded air quality standards from 1981 to 1990 and cancer incidence 

### 29

was present for larynx cancer but not for lung cancer. 

### 30

1 The toxicological evidence is presented in detail in Table 6

- 18 on page 6

- 116 of the 1996 O 3 AQCD and Table AX5

- 13 on page AX5

- 43 of the 2006 O 3 AQCD.

### Draft

– Do Not Cite or Quote 7

- 86 June 2012 Early toxicological research demonstrated lung adenoma 1 acceleration in mice with daily 

### 1

exposure to 1 ppm over 15 months ( Stokinger, 1962 ) . Later work demonstrated a 

### 2

significa nt increase in lung tumor numbers in one strain of mouse (A/J) but not anot her 

### 3

after exposure to 0.3

- 0.8 ppm O 3 ( Last et al., 1987 ; Hassett et al., 1985 ) . The A/J mouse 

### 4

strain is known to have a high incidence of spontaneous adenomas, and further studies 

### 5

using this strain found a statistically significa nt increase in lung tumor incidence after a 

### 6

9

- month exposure to 0.5 ppm and incidence and multiplicity after a 5 month exposure to 

### 7

0.12 ppm with a 4

- month recovery period ( Witschi et al., 1999 ) . However, these findings 

### 8

were discounted by the study authors due to the lack of a clear concentration

- response, 

### 9

and results from the Hassett et al. 1985 and Last et al. 1987 studies were retrospectively 

### 10

deemed spurious based on what appeared to be unusually low spontaneous tumor 

### 11

incidences in the control gr oups ( Witschi, 1991 ) . A study of carcinogenicity of O 3 by the 

### 12

National Toxicology Program ( NTP, 1994 ) reported increased incidences of 

### 13

alveolar/bronchiolar adenoma or carcinoma (combined) in female B6C3F 1 mice exposed 

### 14

over 2 years to 1.0 ppm O 3 , bu t not 0.12 or .5 ppm. No effect was detected in male mice. 

### 15

For a lifetime exposure to 0.5 or 1.0 ppm O 3 , an increase in the number of female mice 

### 16

with adenomas (but not carcinomas or total neoplasms) was found. The number of total 

### 17

neoplasms was also unaffe cted in male mice, but there was a marginally increased 

### 18

incidence of carcinoma in males exposed to 0.5 and 1.0 ppm. Thus there was equivocal 

### 19

evidence of carcinogenic activity in male mice and some evidence of carcinogenic 

### 20

activity of O 3 in females. Experim ental details of the NTP mouse study are available in 

### 21

Table 6

- 19 on page 6

- 121 ( U.S. EPA, 1996o ) of the 1996 O 3 AQCD. 

### 22

In Fischer

- 344/N rats (50 of each sex per group), neither a 2

- year nor lifetime exposure to 

### 23

O 3 ranging from 0.12 to 1.0 ppm was found to be carcinogenic ( Boorman et al., 1994 ; 

### 24

NTP, 1994 ) . However, a marginally significa nt carcinogenic effect of 0.2 ppm O 3 was 

### 25

reported in a study of male Sprague

- Dawley rats exposed for 6 months (n = 50) 

### 26

( Monchaux et al., 1996 ) . These two studies also examined co

- carcin ogenicity of O 3 with 

### 27

NNK 2 ( Boorman et al., 1994 ) or a relatively high dose of radon ( Monchaux et al., 1996 )

, 

### 28

finding no enhancement of NNK related tumors and a slight non

- significa nt increase in 

### 29

tumor incidence after combined exposure with radon, respectively. Another study 

### 30

exploring co

- carcinogenicity was conducte d in hamsters. Not only was there no 

### 31

enhancement of chemically induced tumors in the peripheral lung or nasal cavity, but 

### 32

results suggested that O 3 could potentially delay or inhibit tumor development ( Witschi et 

### 33

al., 1993 ) . Thus there is no concrete evidence that O 3 can act as a co

- carcinogen. 

### 34

1 NOTE: Although adenomas are benign, over time they may progress to become malignant, at which point they are called
adenocarcinomas

. Adenocarcinoma is the predominant lung cancer subtype in most countries, and is the only lung cancer found in
nonsmokers. From page 8

- 33 of the 1970 O 3 AQCD: “No true lung cancers have been reported, however, from experimental exposures to either O 3 alone or any other combination or ingredient of photochemical oxidants.”

2 4

- (N

- nitrosomethylamino)

- 1

- (3

- pyridyl)

- 1

- butanone

### Draft

– Do Not Cite or Quote 7

- 87 June 2012 Immune surveillance is an important defense against cancer, and it should be noted that 

### 1

natural killer (NK) cells, which destroy tumor cells in the lung, appear to be inhibited by 

### 2

higher concentrations of O 3 and either unaffected or stimulated at lower concentrations 

### 3

(Section 6.2.5.4 , Infection and Adaptive Immunity). This aspect of tumorigenesis adds 

### 4

yet another layer of complexity which may be reflected by conflicting results across 

### 5

studies. 

### 6

The following sections will examine epidemiologic st udies of cancer incidence and 

### 7

mortality and toxicological studies that have been published since the 2006 O 3 AQCD. 

### 8

An epidemiologic study has been published with cancer as the outcome; most 

### 9

epidemiologic studies examine markers of exposu re

. 

### 10

7.6.2 Lung Cancer Incidence and Mortality

A recent re

- analysis of the full ACS CPSII cohort by the Health Effects Institute is the 

### 11

only epidemiologic study that has explored the association between O 3 concentration and 

### 12

cancer mortality since the last O 3 AQCD. Krewski et al. (2009 ) conducted an extended 

### 13

follow

- up of the cohort (1982

- 2000). Mean O 3 concentration [obtained from the 

### 14

A erometric Information Retrieval System (AIRS) for 1980] were 22.91 ppb for the full 

### 15

year and 30.15 ppb for the summer months (April

- September). No association was 

### 16

reported between lung cancer mortality and O 3 concentration (HR = 1.00 [95% CI: 

### 17

0.96

- 1.04] pe r 10 ppb O 3 ). Additionally, no association was observed when the analysis 

### 18

was restricted to the summer months. There was also no association present in a sub



### 19

analysis of the cohort examining the relationship between O 3 concentration and lung 

### 20

cancer mortali ty in the Los Angeles area. 

### 21

Since the 2006 O 3 AQCD, two toxicological studies have examined potential 

### 22

carcinogenicity of O 3 ( Kim and Cho, 2009a

, b ) . Looking across both studies, which used 

### 23

the same mouse strain as the National Toxicology Program study descr ibed above ( NTP, 

### 24

1994 ) , 0.5 ppm O 3 alone or in conjunction with chemical tumor inducers did not enhance 

### 25

lung tumor incidence in males or females. However, a 10% incidence of oviductal 

### 26

carcinoma was observed in mice exposed to 0.5 ppm O 3 for 16 weeks. The impl ications 

### 27

of this observation are unclear, particularly in light of the lack of statistical information 

### 28

reported. Additionally, there is no mention of oviductal carcinoma after 32 weeks of 

### 29

exposure, and no oviductal carcinoma was observed after one year of exposure. The NTP 

### 30

study did not report any increase in tumors at extrapulmonary sites. 

### 31

### Draft

– Do Not Cite or Quote 7

- 88 June 2012 7.6.3 DNA Damage

The potential for genotoxic effects relating to O 3 exposure was predicted from the 

### 1

radiomimetic properties of O 3 . The decomposition of O 3 in water produces OH and HO 2 

### 2

radicals, the same species that are generally considered to be the biologically active 

### 3

products of ionizing radiation. Ozone has been observed to cause degradation of DNA in 

### 4

a number of different models and bacterial strains. The toxic effects of O 3 have been 

### 5

generally assumed to be confined to the tissues directly in contact with the gas, such as 

### 6

the respiratory epithelium. Due to the highly reactive nature of O 3 , little systemic 

### 7

absorption is predicted. Zelac et al. (1971a ); ( 1971b ) , however, reported a significa nt 

### 8

increase in chromosome aberrations in peripheral blood lymphocytes from Chinese 

### 9

hamsters exposed to 0.2 ppm for 5 hours. Other in vivo exposure studies found increase d 

### 10

DNA strand breaks in respiratory cells from guinea pigs ( Ferng et al., 1997 ) and mice 

### 11

( Bornholdt et al., 2002 ) but only with exposure to higher concentrations of O 3 (1 ppm for 

### 12

72 hours and 1 or 2 ppm fo r 90 minutes, respectively). In other studies there were no 

### 13

observations of chromosomal aberrations in germ cells, but mutagenic effects have been 

### 14

seen in offspring of mice exposed to 0.2 ppm during gestation (blepharophimosis or 

### 15

dysplasia of the eyelids). The overall evidence for mutagenic activity from in vitro 

### 16

studies is positive, and in the National Toxicology Program report described above, O 3 

### 17

was found to be mutagenic in Salmonella, with and without S9 metabolic activation. No 

### 18

recent toxicological stu dies of DNA damage have become available since the 2006 O 3 

### 19

AQCD. 

### 20

A number of epidemiologic studies looked at the association between O 3 and DNA and 

### 21

cellular level damages. These changes may be relevant to mechanisms leading to cancers 

### 22

development and serve as early indicators of elevated risk of mutagenicity. 

### 23

Two studies performed in California examined cytogenetic damage in relation to O 3 

### 24

exposures. Huen et al. (2006 ) examined cytogenetic damage among African American 

### 25

children and their mothers in Oakland, CA. Increased O 3 (mean monthly 8

- h O 3 

### 26

concentrations ran ged from about 30 ppb in April to 14 ppb in November) was associated 

### 27

with increased cytogenetic damage (micronuclei frequency among lymphocytes and 

### 28

buccal cells) even after adjustment for household/personal smoking status and distance



### 29

weighted traffic dens ity. Chen et al. (2006a ) recruited college students at the University 

### 30

or California, Berkele y who reported never smoking and compared their levels of 

### 31

cytogenetic damage (micronuclei frequency from buccal cells) in the spring and fall. 

### 32

Cytogenetic damage was greater in the fall, which the authors attributed to the increase in 

### 33

O 3 over the summer. H owever, O 3 levels over 2, 7, 10, 14, or 30 days (concentrations not 

### 34

given) before collection of buccal cells did not correlate with cytogenetic damage. 

### 35

Estimated lifetime O 3 exposure was also not correlated with cytogenetic damage. 

### 36

### Draft

– Do Not Cite or Quote 7

- 89 June 2012 Additionally, the author s exposed a subset of the students (n = 15) to 200 ppb O 3 for 

### 1

4 hours while the students exercised intermittently. Ozone was found to be associated 

### 2

with an increase in cytogenetic damage in degenerated cells but not in normal cells 

### 3

9

- 10 days after exposure . Increased cytogenetic damage was also noted in peripheral 

### 4

blood lymphocytes collected 18 hours after exposure. 

### 5

A study performed in Mexico recruited 55 male workers working indoors (n = 27) or 

### 6

outdoors (n = 28) in Mexico City or Puebla, Mexico in order to study the relationship 

### 7

between O 3 and DNA damage (detected from peripheral blood samples using the Comet 

### 8

assay) ( Tovalin et al., 2006 ) . The median estimated daily O 3 concentrations were 

### 9

estimated to be 28.5 ppb for outdoor workers and 5.1 ppb for indoor workers in 

### 10

Mexico City and 36.1 ppb for outdoor workers and 19.5 ppb for indoor workers in 

### 11

Puebla. Overall, a positive correlation between O 3 levels and DNA d amage was 

### 12

observed. However, when examining the relationship by city and workplace, only DNA 

### 13

damage in outdoor workers in Mexico City remained correlated with O 3 levels. 

### 14

Three studies examining the relationship between O 3 concentration and DNA

- level 

### 15

damag e have been performed in Europe. The largest of these studies was the GenAir 

### 16

case

- control study, which was nested within the European Prospective Investigation into 

### 17

Cancer and Nutrition (EPIC) study, and included individuals recruited between 1993 and 

### 18

1998 from ten European countries. Only non

- smokers (must not have smoked for at least 

### 19

10 years prior to enrollment) were enrolled in the study. The researchers examined DNA 

### 20

adduct levels (DNA bonded to cancer

- causing chemicals) and their relationship with O 3 

### 21

c oncentrations (concentrations not given) ( Peluso et al., 2005 ) . A positive association was 

### 22

seen between DNA adduct levels and O 3 concentrations from 1990

- 1994 but not O 3 

### 23

concentrations from 1995

- 1999. In adjusted analyses with DNA adduct levels 

### 24

dichotomized as high and low (detectable versus non

- detectable), the OR was 1.97 

### 25

(95% CI: 1.08, 3.58) when comparing the upper tertile of O 3 concentration to the lower 

### 26

two tertiles. Two other European studies were conducted in Florence, I taly. The most 

### 27

recent of these enrolled individuals from the EPIC study into a separate study between 

### 28

March and September of 1999 ( Palli et al., 2009 ) . The purpose of the study was to 

### 29

examine oxidative DNA damage (determined by Comet ass ay using blood lymphocytes) 

### 30

in association with varying periods of O 3 exposure. The researchers observed that longer 

### 31

periods of high O 3 concentrations (values not given) were more strongly correlated with 

### 32

oxidative DNA damage than shorter periods of time ( i.e., the rho [p

- value] was 0.26 

### 33

[0.03] for 0

- 10 days and 0.35 [0.002] for 0

- 90 days). This correlation was stronger among 

### 34

men compared to women. The correlations for all time periods had p

- values <0.05 for ex



### 35

and never

- smokers. For current smokers, the c orrelation was only observed among time 

### 36

periods ≤ 25 days. When adjusted for age, gender, smoking history, traffic pollution 

### 37

exposure, period of blood draw, and area of residence, the association between O 3 

### 38

### Draft

– Do Not Cite or Quote 7

- 90 June 2012 concentrations and oxidative DNA damage was posit ive for O 3 concentrations 0

- 60 days, 

### 1

0

- 75 days, and 0

- 90 days prior to blood draw. Positive, statistically significa nt 

### 2

associations were not observed among shorter time periods. The other study performed in 

### 3

Florence recruited healthy volunteers who reporte d being non

- smokers or light smokers 

### 4

( Giovannelli et al., 2006 ) . The estim ated O 3 concentrations during the study ranged from 

### 5

approximately 4

- 40 ppb for 3

- day averages, 5

- 35 ppb for 7

- day averages, and 

### 6

7.5

- 32.5 ppb for 30

- day averages. Ozone concentrations were correlated with DNA 

### 7

strand breaks (measured from blood lymphocytes) over longer exposure periods (p

- value: 

### 8

0.002 at 30 days, p

- value: 0.04 at 7 days; p

- value: 0.17 at 3 days). This association was 

### 9

robust to control for temperature, solar radiation, gender, and age. No association was 

### 10

seen between O 3 concentrations and meas ures of oxidative DNA damage at 3, 7, or 

### 11

30 days. 

### 12

7.6.4 Summary and Causal Determination

The 2006 O 3 AQCD reported that evidence did not support ambient O 3 as a pulmonary 

### 13

carcinogen. Since the 2006 O 3 AQCD, very few epidemiologic and toxicological studies 

### 14

have been published that examine O 3 as a carcinogen, but collectively, study results 

### 15

indicate that O 3 may contribute to DNA damage. O 3 concentrations in most 

### 16

epidemiologic studies were measured using air monitoring data. For more information on 

### 17

long

- term exposu re assessment, see Section 4.6.3.2 Overall, the evidence is inadequate 

### 18

to determine if a causal relationship exists between ambient O 3 exposures and 

### 19

cancer. 

### 20



## 7.7 Mortality

A limited number of epidemiologic studies have assessed the relationship between long



### 21

te rm exposure to O 3 and mortality in adults. The 2006 O 3 AQCD concluded that an 

### 22

insufficient amount of evidence existed “to suggest a causal relationship between chronic 

### 23

O 3 exposure and increased risk for mortality in humans” ( U.S. EPA, 2006b ) . In addition 

### 24

to the infant mortality studies discussed in Section 7.4.10 , additional studies have been 

### 25

conducted among adults since the last review; an ecologic study that finds no association 

### 26

between mortality and O 3 , several re

- analyses of the ACS cohort, one of which 

### 27

specifically points to a relationship between long

- term O 3 exposure and an increased risk 

### 28

of respiratory mortality, and a study of four cohorts of persons with potentially 

### 29

predisposing conditions. Thes e studies supplement the evidence from long

- term cohort 

### 30

studies characterized in previous reviews of O 3, and are summarized here briefly. 

### 31

### Draft

– Do Not Cite or Quote 7

- 91 June 2012 In the Harvard Six Cities Study ( Dockery et al., 1993 ) , adjusted mortality rate ratios were 

### 1

examined in relation to long

- term mean O 3 concentrations in six cities: Topeka, KS; St. 

### 2

Louis, MO; Portage, WI; Harriman, TN; Steubenville, OH; and Watertown, MA. Mean 

### 3

O 3 concentrations from 1977 to 1985 ranged from 19.7 ppb in Watertown to 28.0 ppb in 

### 4

Po rtage. Long

- term mean O 3 concentrations were not found to be associated with 

### 5

mortality in the six cities. However, the authors noted that “The small differences in O 3 

### 6

levels among the (six) cities limited the power of the study to detect associations betwe en 

### 7

mortality and O 3 levels.” In addition, while total and cardio

- pulmonary mortality were 

### 8

considered in this study, respiratory mortality was not specifically considered. 

### 9

In a subsequent large prospective cohort study of approximately 500,000 U.S. adults, 

### 10

Pope et al. (2002 ) examined the effects of long

- term exposure to air pollutants on 

### 11

mor tality (American Cancer Society, Cancer Prevention Study II). All

- cause, 

### 12

cardiopulmonary, lung cancer and other mortality risk estimates for long

- term O 3 

### 13

exposure are shown in Figure 7

- 5 . While consistently positive associations were not 

### 14

observed between O 3 and mortality (effect estimates labeled A in Figure 7

- 5 ) , the 

### 15

mortality risk estimates were larger in magnitude when analyses considered more 

### 16

accurate exposure metrics, increasing when the entire period was considered (effect 

### 17

estimates labeled B in Figure 7

- 5 ) and becoming marginally significa nt when the 

### 18

exposure estimate was restricted to the summer months (July to September; effect 

### 19

estimates labeled C in Figure 7

- 5 ), especially when considering cardiopulmonary deaths. 

### 20

In contrast, consistent positive and significa nt effects of PM 2.5 were observed for both 

### 21

lung cancer and cardio

- pulmonary mor tality. 

### 22

### Draft

– Do Not Cite or Quote 7

- 92 June 2012 Years of Data Collection Number of
Metropolitan Areas Number of Participants (in thousands) 1

- h max O 3 Mean (SD)

A 1980

- 1981 134 559 47.9 (11.0)

B 1982

- 1998 119 525 45.5 (7.3)

C 1982

- 1998 (July

– Sept) 134 557 59.7 (12.8)

Source: Reprinted with permission of American Medical Association Pope et al. (2002 )

.



### Figure 7

- 5 Adjusted ozone

- mortality relative risk estimates (95% CI) by time period of analysis per subject

- weighted mean ozone concentration in the Cancer Prevention Study II by the American Cancer Society.

A study by Abbey et al. (1999 ) examined the ef fects of long

- term air pollution exposure, 

### 1

including O 3 , on all

- cause (n = 1,575), cardiopulmonary (n = 1,029), nonmalignant 

### 2

respiratory (n = 410), and lung cancer (n = 30) mortality in the long

- term prospective 

### 3

Adventist Health Study of Smog (AHSMOG) of 6 ,338 nonsmoking, non

- Hispanic white 

### 4

individuals living in California. A particular strength of this study was the extensive 

### 5

effort devoted to assessing long

- term air pollution exposures, including interpolation to 

### 6

residential and work locations from monito ring sites over time and space. No associations 

### 7

with long

- term O 3 exposure were observed for all cause, cardiopulmonary, and 

### 8

nonmalignant respiratory mortality. In a follow

- up, Chen et al. (2005 ) utilized data from 

### 9

the AHSMOG study and reported no evidence of associations between long

- term O 3 

### 10

exposure (mean O 3 concentration 26.2 ppb) and fatal c oronary heart disease. Thus, no 

### 11

association of chronic O 3 exposure with mortality outcomes has been detected in this 

### 12

study. 

### 13

Lipfert et al. (2003 ); ( 2000 ) reported positive effect s on all

- cause mortality for peak O 3 

### 14

exposures (95th percentile levels) in the U.S. Veterans Cohort study of approximately 

### 15

50,000 middle

- aged men recruited with a diagnosis of hypertension. The actual analysis 

### 16

### Draft

– Do Not Cite or Quote 7

- 93 June 2012 involved smaller subcohorts based on exposure and mortality follow

- up periods. Four 

### 1

separate exposure periods were associated with three mortality follow

- up periods. For 

### 2

concurrent exposure periods, peak O 3 was positively associated with all

- cause mortality, 

### 3

with a 9.4% (95% CI: 0.4, 18.4) excess risk per mean 95th percentile O 3 less estimated 

### 4

background level (not stated). “Peak” refers, in this case, to the 95th percentile of 

### 5

the hourly measurements, averaged by year and county. In a further analysis, Lipfert et al. 

### 6

(2003 ) reported the strongest positive association for concurrent exposure to peak O 3 for 

### 7

the subset of subjects with low diastolic blood pressure during the 1982 to 1988 period. 

### 8

Two more recent studies of this cohort focused specifically on traffic density ( Lipfert et 

### 9

al., 2006a ; 2006b )

. Lipfert et al. (2006b ) concluded that: “Traffic density is seen to be a 

### 10

significa nt and robust predictor of survival in this cohort, more so than ambient air 

### 11

quality, with the possible ex ception of O 3 ,” reporting a significa nt O 3 effect even with 

### 12

traffic density included in the model: RR = 1.080 per 40 ppb peak O 3 (95% CI: 1.019, 

### 13

1.146). However, in Lipfert et al. (2006a ) , which considers only the EPA Speciation 

### 14

Trends Network (STN) sites, O 3 drops to non

- significa nt predictor of total mortality for 

### 15

this cohort. The authors acknowledge that: “Peak O 3 has been important in analyses of 

### 16

this cohort for previous periods, but in the STN data set, this variable has limited range 

### 17

and somewhat lower values and its small coefficient of variation results in a relatively 

### 18

lar ge standard error.” The restriction to subjects near STN sites likely reduced the power 

### 19

of this analysis, though the size of the remaining subjects considered was not reported in 

### 20

this paper. In addition, these various Veterans Cohort studies considered onl y total 

### 21

mortality, and did not consider mortality on a by

- cause basis. 

### 22

An ecological study in Brisbane, Australia used a geospatial approach to analyze the 

### 23

association of long

- term exposure to gaseous air pollution with cardio

- respiratory 

### 24

mortality, in th e period 1996

- 2004 ( Wang et al., 2009c ) . A generalized estimating 

### 25

equations model was employed to investigate the impact of NO 2 , O 3 and SO 2 , but PM 

### 26

was not addressed. The results indicated that long

- term exposure to O 3 was not associated 

### 27

with cardio

- respiratory mortality, but the fact that this study considere d only one city, and 

### 28

that the range of O 3 exposure across that city (23.7

- 35.6 ppb) was low and slight in 

### 29

variation in comparison to the range of other pollutants across the city, limited study 

### 30

power. In addition, confounding factors (e.g., smoking) could not be addressed at the 

### 31

individual level in this ecological study. Respiratory mortality was not evaluated 

### 32

separately. 

### 33

A recent study by Zanobetti and Schwartz examined whether year

- to

- year variations in 

### 34

8

- h mean daily O 3 concentrations for the summer (May

- September) around their city



### 35

specific long

- term trend were associated with year

- to

- year variation s in mortality around 

### 36

its long

- term trend. This association was examined among Medicare participants with 

### 37

potentially predisposing conditions, including COPD, diabetes, CHF, and MI, defined as 

### 38

### Draft

– Do Not Cite or Quote 7

- 94 June 2012 patients discharged alive after an emergency admission for one of these four conditions. 

### 1

The analyses was repeated in 105 cities using available data from 1985 through 2006, and 

### 2

the results were combined using methods previously employed by these authors 

### 3

( Zanobetti et al., 2008 ; Zanobetti and Schwartz, 2007 ) . This study design eliminated 

### 4

potential confounding by factors that vary across city, which is a common concern in 

### 5

m ost air pollution cohort studies, and also avoided both confounding by cross

- sectional 

### 6

factors that vary by city and the short

- term factors that confound daily time

- series studies, 

### 7

but are not present in annual analyses. The average 8

- h mean daily summer O 3 

### 8

concentrations ranged from 15.6 ppb (Honolulu, HI) to 71.4 ppb (Bakersfield, CA) for 

### 9

the 105 cities. The authors observed associations between yearly fluctuations in summer 

### 10

O 3 concentrations and mortality in each of the four cohorts; the hazard ratios (p er 10 ppb 

### 11

increment) were 1.12 (95% CI: 1.06, 1.17) for the CHF cohort, 1.19 (95% CI 1.12, 1.25) 

### 12

for the MI cohort, 1.14 (95% CI: 1.10, 1.21) for the diabetes cohort, and 1.14 (95% CI: 

### 13

1.08, 1.19) for the COPD cohort. A key advantage to this study is that fluctuations from 

### 14

summer to summer in O 3 concentrations around long

- term level and trend in a specific 

### 15

city are unlikely to be correlated with most other predicators of mortality risk; except for 

### 16

temperature, which was controlled for in the regression. Key limitations of the study were 

### 17

the inability to control for PM 2.5 , since it was not reliably measured in these cities until 

### 18

1999, and the inability to separate specific causes of death (e.g., respiratory, 

### 19

cardiovascular), since Medicare does not provide th e underlying cause of death. 

### 20

In the most recent follow

- up analyses of the ACS cohort ( Jerrett et al., 2009 ; Smith et al., 

### 21

2009a ) , the effects of long

- term exposure to O 3 were evaluated alone, as well as in 

### 22

copollutant models with PM 2.5 and components of PM 2.5

. Jerrett et al. (2009 ) utilized the 

### 23

ACS cohort with data from 1977 through 2000 (mean O 3 concentration ranged from 33.3 

### 24

to 104.0 ppb) and subdivided c ardiopulmonary deaths into respiratory and cardiovascular, 

### 25

separately, as opposed to combined into one category, as was done by Pope et al. (2002 )

. 

### 26

Increases in exposure to O 3 were associated with an elevated risk of death from 

### 27

cardiopulmonary, cardiovascular, ischemic heart disease, and respiratory causes. 

### 28

Consistent with study hypotheses, inclusion of PM 2.5 concentrations measured in 

### 29

1999

- 2000 (the earliest years for which it was available) as a copollutant attenuated the 

### 30

association with O 3 for all end points except death from respiratory causes, for which a 

### 31

significa nt association persis ted ( Table 7

- 11 ). The association between increased O 3 

### 32

concentrations and increased risk of death from respiratory causes was insensitive to the 

### 33

use of a random

- effects survival model allowing for spatial clustering within the 

### 34

metropolitan area and state of residence, and adjustment for several ecologic variables 

### 35

considered individually. Subgroup analyses showed that temperature and region of 

### 36

country, bu t not sex, age at enrollment, body

- mass index, education, or PM 2.5 

### 37

concentration, modified the effects of O 3 on the risk of death from respiratory causes 

### 38

(i.e., risks were higher at higher temperature, and in the Southeast, Southwest, and Upper 

### 39

### Draft

– Do Not Cite or Quote 7

- 95 June 2012 Midwest). O zone threshold analyses indicated that the threshold model was not a better 

### 1

fit to the data (p >0.05) than a linear representation of the overall O 3 - mortality 

### 2

association. Overall, this new analysis indicates that long

- term exposure to PM 2.5 

### 3

increases risk of cardiac death, while long

- term exposure to O 3 is specifically associated 

### 4

with an increased risk of respiratory death, and suggests that combining cardiovascular 

### 5

and respiratory causes of mortality into one category for analysis may obscure any effect 

### 6

t hat O 3 may have on respiratory

- related causes of mortality. 

### 7

Table 7

- 11 Relative risk (and 95% CI) of death attributable to a 10

- ppb change
in the ambient ozone concentration.

Cause of Death O 3 (96 MSAs) a O 3 (86 MSAs) a O 3 +PM 2.5 (86 MSAs) a

Any Cause 1.001 (0.996, 1.007) 1.001 (0.996, 1.007) 0.989 (0.981, 0.996)

Cardiopulmonary 1.014 (1.007, 1.022) 1.016 (1.008, 1.024) 0.992 (0.982, 1.003)

Respiratory 1.029 (1.010, 1.048) 1.027 (1.007, 1.046) 1.040 (1.013, 1.067)

Cardiovascular 1.011 (1.003, 1.023) 1.014 (1.005, 1.023) 0.983 (0.971, 0.994)

Ischemic Heart Disease 1.015 (1.003, 1.026) 1.017 (1.006, 1.029) 0.973 (0.958, 0.988)

a Ozone concentrations were measured from April to September during the years from 1977 to 2000, with follow

- up from 1982 to 2000; changes in the concentration of PM 2.5 of 10 μg/m 3 were recorded for members of the cohort in 1999 and 2000.
Source: Reprinted with permission of Massachusetts Medical Society ( Jerrett et al., 2009 )

.

In a similar analysis, Smith et al. (2009a ) used data from 66 Metropolitan Statistical 

### 8

Areas ( MSAs ) in the ACS cohort to exa mine the association of O 3 concentrations during 

### 9

the warm season and all

- cause and cardiopulmonary mortality. Mortality effects were 

### 10

estimated in single pollutant and copollutant models, adjusting for two PM 2.5 constituents, 

### 11

sulfate, and EC. When all

- cause mortality was investigated, there was a 0.8% (95% CI: 

### 12

- 0.31, 1.9) increase associated with a 10 ppb increase in O 3 concentration. This 

### 13

association was diminished when sulfate or EC were included in the model. There was a 

### 14

2.48% (95% CI: 0.74, 4.3) increase in cardiopulmonary mortality associated with a 

### 15

10 ppb increase in O 3 concentration. The cardiopulmonary association was robust to 

### 16

adjustment for sulfate, and diminished, though still positive, after adjustment for EC 

### 17

(1.63% increase; 95% CI:

- 0.41, 3.7). Smith et al. (2009a ) did not specifically separate 

### 18

out cardiovascular and respiratory causes of death from the cardiopu lmonary category, as 

### 19

was done by Jerrett et al. (2009 )

. 

### 20

7.7.1 Summary and Causal Determination

The The 2006 O 3 AQCD concluded that an insufficient amount of evidence existed “to 

### 21

suggest a causal relationship between chronic O 3 exposure and increased risk for 

### 22

### Draft

– Do Not Cite or Quote 7

- 96 June 2012 mortality in humans” ( U.S. EPA, 2006b )

. Several additional studies have been conducted 

### 1

since the last review that evaluate cause

- specific and total mortality. An ecologic study 

### 2

conducted in Australia observed no association between cardiopulmonary mortality and 

### 3

O 3 ( Wang et al., 2009c )

. Two reanalyses of the ACS cohort were conducted; one 

### 4

provides weak evidence for an association with cardiopulmonary mortality ( Smith et al., 

### 5

2009a ) while the other specifically points to a relati onship between long

- term O 3 

### 6

exposure and an increased risk of respiratory mortality ( Jerrett et al., 2009 ) . Most 

### 7

recently, a study of four cohorts of Medicare enrollees with potentially predisposing 

### 8

conditions observed associations between O 3 and total mo rtality among each of the 

### 9

cohorts ( Zanobetti and Schwartz, 2011 )

. 

### 10

When considering the entire body of evidence, there is limited support for an association 

### 11

with long

- term exposure to ambient O 3 and total mortality. There is inconsistent evidence 

### 12

for an association between long

- term exposure to ambient O 3 and cardiopulmonary 

### 13

mortality, with several analyses from the ACS cohort reporting some positive 

### 14

associations ( Smith et al., 2009a ; Pope et al., 2002 ) while other studies reported no 

### 15

association ( Wang et al., 2009c ; Abbey et al., 1999 ; Dockery et al., 1993 ) . The strongest 

### 16

evidence for an association between long

- term exposure to ambient O 3 concentrations 

### 17

and mortality is derived from associations reported in the Jerrett et al. (2009 ) study for 

### 18

respiratory mortality that remained robust after adjusting for PM 2.5 concentrations. 

### 19

Finally, a recent analysis reported associations of ambient O 3 concentrations and total 

### 20

mortality in potentially at

- risk popul ations in the Medicare Cohort ( Zanobetti and 

### 21

Schwartz, 2011 ) , while earlier studies generally report no associations with total 

### 22

mortality ( Lipfert et al., 2006a ; Lipfert et al., 2003 ; Pope et al., 2002 ; Abbey et al., 1999 ; 

### 23

Dockery et al. , 1993 ) . Studies of cardiopulmonary and total mortality provide limited 

### 24

evidence for an association with long

- term exposure to ambient O 3 concentrations. The 

### 25

study by Jerrett et al. (2009 ) observes an association between long

- term exp osure to 

### 26

ambient O 3 concentrations and respiratory mortality remained robust after adjusting for 

### 27

PM 2.5 concentrations. Coherence and biological plausibility for this observation is 

### 28

provided by evidence from epidemiologic, controlled human exposure, and an imal 

### 29

toxicological studies for the effects of short

- and long

- term exposure to O 3 on respiratory 

### 30

effects (See Sections 6.2 and 7.2). Respiratory mortality is a relatively small portion of 

### 31

total mortality [about 7.6% of all deaths in 2010 were due to respir atory causes ( Murphy 

### 32

et al., 2012 ) ], thus it is not surprising tha t the respiratory mortality signal may be difficult 

### 33

to detect in studies of cardiopulmonary or total mortality. Based on the recent evidence 

### 34

for respiratory mortality along with limited evidence for total and cardiopulmonary 

### 35

mortality, the evidence is sug gestive of a causal relationship between long

- term O 3 

### 36

exposures and total mortality. 

### 37

### Draft

– Do Not Cite or Quote 7

- 97 June 2012 

## 7.8 Overall Summary

The evidence reviewed in this chapter describes the recent findings regarding the health 

### 1

effects of long

- term exposure to ambient O 3 concentrations. Table 7

- 12 provides an 

### 2

overview of the causal determinations for each of the health categories evaluated. 

### 3

Table 7

- 12 Summary of causal determinations for long

- term exposures to

ozone.

Health Category Causal Determination

Respiratory Effects Likely to be a causal relationship

Cardiovascular Effects Suggestive of a causal relationship

Reproductive and Developmental Effects Suggestive of a causal relationship

Central Nervous System Effects Suggestive of a causal relationship

Carcinogenicity and Genotoxicity Inadequate to infer a causal relationship

Total Mortality Suggestive of a causal relationship

### Draft

– Do Not Cite or Quote 7

- 98 June 2012 

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