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## 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 basis 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 durat ion of approximately 30 days (1 month) or longer. 

### 6

Conclusions from the 2006 O 3 AQCD are summarized briefly at the beginning of each 

### 7 section, and the evaluation of evidence from recent studies builds upon what was 

### 8 available during the previous review. For each health outcome (e.g., respiratory disease, 

### 9 lung function), results are summarized for studies from the specific scientific discipline, 

### 10 i.e., epidemiologic and toxicological studies. The major sections (i.e. respiratory, 

### 11 cardiovascular, mortality, repr oductive/developmental, cancer) conclude with summaries 

### 12 of the evidence for the various health outcomes within that category and integration of 

### 13 the findings that lead to conclusions regarding causality based upon the framework 

### 14 described in Chapter 1. Deter mination of causality is made for the overall health effect 

### 15 category, such as respiratory effects, with coherence and plausibility being based on 

### 16 evidence from across disciplines and also across the suite of related health outcomes, 

### 17 including cause

- specifi c mortality. 

### 18



## 7.2 Respiratory Effects

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

### 19 long

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

### 20 outcomes including declines in lung function, increases i n inflammation, and 

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

### 22 picture indicating that long

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

### 23 studies in animals have reported biochemical and morphologi cal changes suggestive of 

### 24 irreversible long

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

### 25 chronic animal studies, the epidemiologic studies on longer

- term (annual) lung function 

### 26 declines, inflammation, and new asthma development rema ined inconclusive. 

### 27Draft

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- 2 September 2011 Several studies ( Horak et al., 2002 ; Frischer et al., 1999 ) collective ly indicated that O 3 

### 1 exposure over several summer months was associated with smaller increases in lung 

### 2 function growth in children. For longer time periods (annual), the definitive analysis in 

### 3 the Child Health Study (CHS) reported by Gauderman et al. ( 2004 ) provided little 

### 4 evidence that such long

- term exposure to ambient O 3 was associated with significant 

### 5 deficits in the growth rate of lung function in children in contrast to the effects observed 

### 6 with other pollutants such as acid vapor, NO 2 , and PM 2.5 . Asthmatic children with 

### 7 GSTM1 null genotype were found to be more susceptible to the impact of O 3 exposure 

### 8 (over a 12 week study period) on small airways function in Mexico ( Romieu et al., 

### 9
2004a ) . Limited epidemiologic research examined the relationship between long

- term O 3 

### 10 exposures and inflammation. Evidence of inflammation and allergic responses consistent 

### 11
with known effects of O 3 exposure (30 day mean) s uch as increased eosinophil levels 

### 12 were observed in an Austrian study ( Frischer et al., 2001 ) . The cross

- sectional surveys 

### 13
available for the 2006 O 3 AQCD detected no associations between long

- term O 3 

### 14 exposures and asthma prevalence, asthma

- related symptoms or allergy to common 

### 15 aeroallergens in children after controlling for covariates. 

### 16

New evidence presented below reports consistent associations betwe en long

- term O 3 

### 17 exposure and new

- onset asthma related to genotype in U.S. cohorts in multi

- community 

### 18 studies. Related studies report coherent relationships between respiratory symptoms 

### 19 among asthmatics and long

- term O 3 exposure. Short

- term exposure to O 3 i s associated 

### 20 with increases in respiratory symptoms and asthma medication use, as summarized in 

### 21
Section 6.2.4.2. A new line of evidence reports a positive exposure response relationship 

### 22 between first asthma hospitalization and long

- term O 3 exposure. Result s from recent 

### 23 studies examining pulmonary function, inflammation, and allergic responses are also 

### 24 presented. 

### 25

7.2.1 New Onset Asthma

Risk for new

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

### 26 and environmental exposure ( Gilliland et al., 1999 ) . Complex chronic diseases, such as 

### 27 asthma, are partially the result of a sequence of biochemical reactions involving 

### 28 exposures to various environmental agents metabolized by a number of different genes 

### 29 ( Conti et al., 2003 ) . Understanding the relation between genetic polymorphisms and 

### 30 environmental exposure can help identify high

- risk subgroups in the population and 

### 31 provide better insight into pathwa y mechanisms for these complex diseases. Oxidative 

### 32 stress likely underlies these mechanistic hypotheses ( Gilliland et al., 1999 ) . Susceptibility 

### 33 genes act through modification of disease risk asso ciated with environmental factors. 

### 34 Epidemiologic investigation of hypotheses of possible mechanisms involving the gene

- 

### 35Draft

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- 3 September 2011 environmental (GxE) interaction involves statistical analysis of these interactions for the 

### 1
risk of new

- onset asthma in children being in fluenced by exposure to air pollution 

### 2 ( Gauderman, 2002

, 2001 ; Gilliland et al., 1999 )

. 

### 3

Evidence for the potential importance of genetic susceptibility and behavioral factors on 

### 4 new onset asthma are provided by several recent studies ( Himes et al., 2009 ; Islam et al., 

### 5
2008 ; Li et al., 2008 ; Hanene et al., 2007 ; Ercan et al., 2006 ; Li et al., 2006a ; Tamer et 

### 6 al., 2004 ; Gilliland et al., 2002 )

. Evidence for a gene

- pollution interaction in the 

### 7 pathogenesis of asthma are supported by recent study findings ( Islam et al., 2009 ; Islam 

### 8 et al., 2008 ; Oryszczyn et al., 2007 ; Lee et al., 2004b ; Gilliland et al., 2002 )

. 

### 9

Evidence for associations between long

- term exposure to O 3 and new

- onset asthma is 

### 10 provided by new studies from the CHS. Initiated in the early 1990 ’ s, the CHS was 

### 11 originally designed to examine whether long

- term exposure to ambient pollutants was 

### 12 related to chronic respiratory ou tcomes in children in 12 communities in southern 

### 13
California ( Peters et al., 1999a ; Peters et al., 1999b ) . About 10 years later, the CHS 

### 14 inaugurated a series of genetic studies ( Gilliland et al., 1999 ) nested within the CHS 

### 15 cohort by obtaining biological samples from the study subjects (buccal cells). These new 

### 16 studies examined the relationship between health outcomes, genetic susceptibility, 

### 17
behavioral fact ors and environmental exposure. 

### 18

First, the hypothesis that the functional polymorphisms of HMOX

- 1 [(GT)n repeat], CAT 

### 19 (

- 262C > T

- 844C > T0, and MNSOD (Ala

- 9Val) are associated with new

- onset asthma 

### 20 was evaluated, and then whether the effects of these vari ants varied by exposure to O 3 

### 21 ( Islam et al., 2008 )

. HMOX1 [heme oxygenase (decycling) 1] is a human gene that 

### 22 encodes for the enzyme heme oxygenase. Heme ox ygenase 1 (HO

- 1) is an enzyme that 

### 23 catalyzes the metabolism of heme. The heme iron serves as a source or sink of electrons 

### 24 during electron transfer or redox chemistry, so the presence of the HMOX1 gene, and 

### 25 therefore the generation of heme oxygenase, prote cts against oxidative stress in the body. 

### 26 The authors observed that functional promoter variants in CAT and HMOX

- 1 showed 

### 27 ethnicity

- specific associations with new

- onset asthma and that oxidant gene protection 

### 28 was restricted to children living in low

- O 3 com munities. 

### 29

The subjects were drawn from the CHS cohort. Children with a history of asthma or 

### 30 wheeze were excluded from this analysis. Analyses were restricted to children of 

### 31 Hispanic (n = 576) or non

- Hispanic white ethnicity (n = 1,125). New

- onset asthma w as 

### 32 classified as having no prior history of asthma at study entry with subsequent report of 

### 33 physician

- diagnosed asthma at follow

- up with the date of onset assigned to be the 

### 34 midpoint of the interval between the interview date when asthma diagnosis was firs t 

### 35 reported and the previous interview date. As a sensitivity analysis, the asthma definition 

### 36
was restricted to those new

- onset asthma cases who also used an inhaler (n = 121). They 

### 37Draft

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- 4 September 2011 calculated long

- term mean pollutant levels (1994

– 2003) to assign exposure to children 

### 1 in each community for use in the statistical analysis. The effect of ambient air pollution 

### 2 on the relationship between genetic polymorphism and new

- onset asthma was assessed 

### 3 using models where the community specific average air pollution level s were fitted as a 

### 4 continuous variable together with the appropriate interaction terms for genes and air 

### 5 pollutants ( Berhane et al., 2004 ) . Cox proportional hazard regression models were fitted 

### 6 to the data. A stratified analysis for the two independent fourth

- grade cohorts of the study 

### 7

po pulation recruited in 1993 and 1996 was conducted to assess whether the results could 

### 8 be replicated in independent groups of children. 

### 9

Over the follow

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

. 

### 10
The evidence indicated that the effect of variation in the HMOX

- 1 gene on risk of new



### 11 onset asthma differed by ambient O 3 level. An interaction P value was reported of 0.003 

### 12
from the hier archical two stage Cox proportional hazard model fitting the community



### 13 specific O 3 and PM 10 levels (continuous) and controlling for random effect of the 

### 14 communities. Average O 3 levels showed low correlation with the other monitored 

### 15 pollutants. The interact ion indicated a greater effect (association) of community O 3 level 

### 16 among children with the gene than with children without the gene. Alleles with 23 or 

### 17 fewer (GT)n repeats are categorized as short (S). The S

- allele variant of this protective 

### 18 enzyme is more readily induced than those with more numerous repeats. The largest 

### 19 protective effect of the (GT)n repeat polymorphism of HMOX

- 1 was observed for 

### 20
children who were S

- allele carriers and resided in low

- O 3 communities with Hazard 

### 21 Ratio (HR) of 0.44 (95% CI: 0.23, 0.83). The ratio of HR of S

- allele carriers who resided 

### 22 in high O 3 communities (HR=0.88; [95% CI: 0.33, 2.34]) was twofold greater than in 

### 23
those who resided in the low

- O 3 communities (HR=0.44). The non

- parallelism of the two 

### 24
lines in An interaction p

- value of 0.003 was obtained from the hierarchical two stage Cox 

### 25 proportional hazard model fitting the community specific O 3 and controlling for random 

### 26 effect of the communities. The interaction indicates there is a gr eater effect (association) 

### 27 of community O 3 level on children with the gene than with children without the gene. 

### 28
The HRs are off

- set as opposed to overlapping in the figure to allow clearer presentation 

### 29
of the results. 

### 30

Figure 7

- 1 illustrates the interaction: Children with the S

- allele have protection against 

### 31 the onset of asthma; however, in high

- O 3 communities, this protection is attenuated. The 

### 32 results from sensitivity analyses on the two fourth

- grade cohorts, and the inhaler 

### 33 definition for asthma were both consistent with the main results. An analysis related to 

### 34
children ’ s participation in sports or time spent outdoors produced the same outcome. No 

### 35 significant interactions were observed be tween PM 10 or other pollutants and the HMOX

- 

### 36 1 gene ; quantitative results were not presented. A potential concern for not adjusting for 

### 37 multiple testing was considered by the authors as not a factor in this analysis because the 

### 38Draft

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- 5 September 2011 selection of the genes was based on a priori hypotheses defined by a well

- studied 

### 1 biological pathway. Thus in this cohort in southern California, Islam et al. ( 2008 ) related 

### 2

new

- onset asthma to O 3 exposure in genetically susceptible children. 

### 3

Source: Developed by EPA with data from Islam et al. ( 2008 ) (us ed by permission of American Thoracic Society ). 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 interacti on indicates there is a greater effect (association) of community O 3 level on children with the gene than with children without the gene. The HRs are off

- set as opposed to overlapping in the figure to allow clearer presentation of the results.



### Figure 7

- 1 Interaction of gene presence and O 3 

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

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

.

Related to the findings in Islam et al. ( 2008 ) discussed above, Islam et al. ( 2009 ) 

### 4 examined whether GSTP1, GSTM1, exercise and O 3 exposure have interrelated effects 

### 5 on the pathogenesis of asthma. A modifying role of air pollution on the association 

### 6
between Ile105Val and asthma in a cohort of children had been observed ( Lee et al., 

### 7
2004b ) , but the study did not examine O 3 specifically or consider exercise. A primary 

### 8
conclusion that the authors ( Islam et al., 2009 ) reported was that the common functional 

### 9 variants of GSTP1 and GSTM1 null genotypes modulate the risk of new onset asthma 

### 10
dur ing adolescence. Children who had the GSTM1 null genotype were at 1.6

- fold (95% 

### 11 CI: 1.2, 2.2) increased risk of developing new onset asthma compared with those without 

### 12 the null genotype. Further, the CHS investigators examined the complex interrelationship 

### 13 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

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- 6 September 2011 of antioxidant defenses with asthma risk with increasing doses of O 3 , resulting from 

### 1 increasing ventilation associated with vigorous exercise characterized by the number of 

### 2 team sports played. In an earlier analysis, McConnell et al. ( 2002 ) had repo rted that the 

### 3 risk of new onset asthma was associated with outdoor exercise, especially in high O 3 

### 4 communities but did not consider genetic variants. In this new study, Islam et al. ( 2009 ) 

### 5 find a six fold increased risk of asthma (HR=6.15, [95% CI: 2.2, 7.4]) for children who 

### 6 were homozygous for Ile105, participated in three or more team sports and lived i n 

### 7 high

- O 3 communities, demonstrating the potential importance of a combination of 

### 8 genetic variability, O 3 exposure and behavior on asthma risk. 

### 9

Epidemiologic evidence of associations of arginase variants with asthma are limited ( Li 

### 10

et al., 2006a ) . Asthmatic subjects have higher arginase activity than non

- asthmatic 

### 11 subjects ( Morris et al., 2004 ) . NO is a me diator of nitrosative stress synthesized from L



### 12 arginine by nitric oxide synthases. In the CHS, Salam et al. ( 2009 ) examined whether 

### 13 arginase variants (ARG1 and ARG2 genes) were associated with asthma and whether 

### 14 atopy and exposures to smoking and air pollution influence the a ssociations. The 

### 15 modifying effect of O 3 and atopy on the association between haplotypes and asthma were 

### 16 evaluated using likelihood ratio tests with appropriate interaction terms. They found that 

### 17 both ARG1 and ARG2 genetic loci were associated with childhoo d

- onset asthma. The 

### 18 effect of the ARG1 haplotype varied by the child ’ s history of atopy and ambient O 3 . 

### 19 Among atopic children living in high O 3 communities, those carrying the ARG1 

### 20 haplotype had reduced asthma risk (Odds Ratio [OR] per ARG1h4 haplotype cop y = 

### 21 0.12; [95% CI: 0.04, 0.43]; P heterogeneity across atopy/O 3 categories = 0.008). 

### 22

Further, the CHS presents results examining the relationship of new onset asthma with 

### 23
traffic

- related pollution near homes and schools ( McConnell et al., 2010 ) . Asthma risk 

### 24
increased with modeled traffic

- related pollution exposure from roadways near homes and 

### 25
near schools. T he HR was 0.76 (95% CI: 0.38, 1.54) across the range of ambient O 3 

### 26 exposure in the communities. With adjustment for school and residential non

- freeway 

### 27
traffic

- related exposure, the estimated HR for O 3 was 1.01 (95% CI: 0.49, 2.11). Gene 

### 28
variants were not e valuated in this study. 

### 29

Some cross

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

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

### 31 symptoms in school children ( Ramadour et al., 2000 ; Wang et al., 1999 ) whil e others 

### 32 ( Kuo et al., 2002 ; Charpin et al., 1999 ) did not. Recent studies provide ad ditional 

### 33
evidence. 

### 34

In a cross

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

### 35 ( 2005 ) assessed the effects of air pollutants on the risk of asthma. The study population 

### 36 was recruited from elementary and middle schools within 1 km of air monitoring stations. 

### 37Draft

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- 7 September 2011 The risk of asthma was related to O 3 in the one

- pollutant mode l. The addition of other 

### 1 pollutants (NO x , CO 2 , SO 2 , and PM 10 ), in two

- pollutant and three

- pollutant models, 

### 2
increased the O 3 risk estimates. The prevalence of childhood asthma was assessed in 

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

### 4
low O 3 levels ( Sousa et al., 2011 ; Sousa et al., 2009 ; Sousa et al., 2008 ) . The locations 

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

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

### 7 increased asthma prevalence. Clark et al. ( 2010 ) investigated the effect of exposure to 

### 8 ambient air pollu tion in utero and during the first year of life on risk of subsequent 

### 9 incidence asthma diagnosis up to 3

- 4 years of age in a population

- based nested case



### 10 control study for all children born in southwestern British Columbia in 1999 and 2000 

### 11 (n=37,401; inclu ding 3,482 [9.3%] with asthma). Air pollution exposure for each subject 

### 12 was estimated based on their residential address history using regulatory monitoring data, 

### 13 land use regression modeling, and proximity to stationary pollutant sources. Daily values 

### 14
fro m the three closest monitors within 50 km were used to calculate exposures. Traffic



### 15 related pollutants were associated with the highest risk. Ozone was inversely correlated 

### 16 with the primary traffic

- related pollutants (r =

- 0.7 to

- 0.9). The low reliability of asthma 

### 17 diagnosis in infants makes this study difficult to interpret ( Martinez et al., 1995 ) . In a 

### 18

cross

- sectional analysis, Akinbami et al. ( 2010 ) examined the association between 

### 19 chronic exposure to outdoor pollutants (12

- month avg le vels by county) and asthma 

### 20 outcomes in a national sample of children ages 3

- 17 years living in U.S. metropolitan 

### 21 areas (National Health Interview Survey, N = 34,073). A 5

- ppb increase in estimated 8

- h 

### 22
max O 3 concentration (annual average) yielded a positiv e association for both currently 

### 23 having asthma and for having at least 1 asthma attack in the previous year; while the 

### 24 adjusted odds ratios for other pollutants were not statistically significant. Models in 

### 25 which pollutant value ranges were divided into qu artiles produced comparable results. 

### 26
Multi

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

### 27

12

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

- 43.7 ppb. The adjusted odds 

### 28 for current asthma for the highest quartile (49.9

- 5 9.5 ppb) of estimated O 3 exposure was 

### 29 1.56 (95% CI: 1.15, 2.10) with a positive dose

- response relationship apparent from the 

### 30 lowest quartile to the highest. Thus, this cross

- sectional analysis and Hwang et al. ( 2005 ) 

### 31 provides further evidence relating O 3 exposure and the risk of asthma. 

### 32

The occurrence of bronchitic symptoms among children with as thma was investigated in 

### 33 the CHS examining the role of gene

- environment interactions and long

- term O 3 

### 34 exposure. Lee et al. ( 2009b ) studied associatio ns of TNF

- 308 genotype with bronchitis 

### 35 symptoms among asthmatic children and investigated whether associations vary with 

### 36
ambient O 3 exposure since increased airway TNF may be related to inflammation. 

### 37 Asthmatic children with the GG genotype had a lower prev alence of bronchitic symptoms 

### 38 compared with children carrying at least one A

- allele (e.g., GA or AA). Low

- versus high

- 

### 39Draft

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- 8 September 2011 O 3 strata were defined as less than or greater than 50

- ppb O 3 avg. Asthmatic children 

### 1
with TNF

- 308 GG genotype had a significantly reduce d risk of bronchitic symptoms with 

### 2
low

- O 3 exposure (OR=0.53; [95% CI: 0.31, 0.91]). The risk was not reduced in children 

### 3 living in high

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

### 4 genotypic effects between low

- and high

- O 3 environments was statistically significant 

### 5 among asthmatics (P for interaction = 0.01), but insignificant among non

- asthmatic 

### 6 children. Using indicator variables for each category based on genotype and O 3 exposure, 

### 7
Lee et al. ( 2009b ) calculated the effect of TNF

- 308 GG genotype on the occurrence of 

### 8 bronchitic symptoms among children with asthma. Figure 7

- 2 presents adjusted O 3 

### 9 community

- specific beta

- coefficients plotted against ambient O 3 concentration, using 

### 10 weights proportional to the inverse variance. They further report that they found no 

### 11
subs tantial differences in the effect of the GG genotype in asthmatic children in relation 

### 12 to exposure to PM 10 , PM 2.5 , NO 2 , acid vapor or second

- hand smoke exposure. These 

### 13 results suggest a role of gene

- environment interactions such as long

- term O 3 exposure on 

### 14 the occurrence of bronchitic symptoms among children with asthma. 

### 15

Source: Reprinted with permission of John Wiley & Sons ( Lee et al., 2009b )

.


### Figure 7

- 2 Ozone modifies the effect of TNF G

- 308A genotype on bronchitic symptoms among children with asthma in the CHS

. Using indicator variables for each category based on genotype and O 3 

### exposure, betas were calculated of TNF

- 308 GG genotype on the occurrence of bronchitic symptoms among children with asthma.Draft

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- 9 September 2011 The French Epidemiology study on Genetics and Environment of Asthma (EGEA) 

### 1 investigated the relationship between ambient air pollu tion and asthma severity in a 

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

### 3 al., 2009a ) . In this cross

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

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

- class severity score 

### 5 that integrated clinical eve nts and type of treatment; and (2) a five

- level asthma score 

### 6 based only on symptoms. Two measures of exposure were also assessed: (1 [first 

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

### 8
lived within 10 km of a monitor , but where 70% of the O 3 concentrations were back



### 9 extrapolated values; and (2 [second method]) a validated spatial model that used 

### 10 geostatistical interpolations and then assigned air pollutants to the geocoded residential 

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

### 12 estimates. Higher asthma severity scores were significantly related to both the 8

- h avg O 3 

### 13 during April

- September and the number of days with 8

- h O 3 avgs above 55 ppb. Both 

### 14 exposure assessment method s and severity score methods resulted in very similar 

### 15 findings. Effect estimates of O 3 were similar in three

- pollutant models. No PM data were 

### 16
available. Since these estimates were not sensitive to the inclusion of ambient NO 2 in the 

### 17
three

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

### 18 which usually have substantial correlations between PM and NO 2 . Effect estimates for 

### 19
O 3 in three

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

### 20 2.74 (95% CI: 1.68, 4. 48) per IQR days of 10

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

### 21
and NO 2 in the three

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

### 22 0.68, 1.29) respectively. Taking into account duration of residence did not change the 

### 23
result. This stud y suggests that a higher asthma severity score is related to long

- term O 3 

### 24

exposure. 

### 25

An EGEA follow

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

### 26
between asthma and O 3 , NO 2 , and PM 10 . New aspects considered include: 1) 

### 27 examination of three domains of asthma control (symptoms, exacerbatio ns, and lung 

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

### 29 asthma); and 3) PM 10 and multi

- pollutant analysis. In this cross

- sectional analysis, 

### 30 EGEA2 studied 481 adult subjects with current asthma from 2003 to 2 007. The IQRs 

### 31 were 11 (41

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

- 38) μg/m 3 for summer (April



### 32 September) O 3 . The association between asthma control and air pollutants was expressed 

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

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

### 35 asthma and for partly controlled asthma was compared with controlled asthma using a 

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

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

### 38Draft

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- 10 September 2011 from controlled, partly controlled (OR=1.53, 95%CI: 1.01, 2.33) and uncontrolled 

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

### 2

Se parately, they used a composite asthma control classification that used the ordinal 

### 3 logistic regression for risk comparing controlled to partly controlled asthma and 

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

### 5

asse ssed using the ordinal logistic regression were significant (ORs were 1.69 (95% CI: 

### 6 1.22, 2.34) and 1.35 (95% CI: 1.13, 1.64) for O 3 - sum and PM 10 , respectively). For two 

### 7 pollutant models using the ordinal logistic regression, the adjusted ORs for O 3 - sum an d 

### 8

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

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

### 10 effects of both pollutants are independent. 

### 11

The analysis of the associations bet ween air pollution for all asthma subjects and each 

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

### 13 defined dichotomously as % predicted FEV 1 value < or > =80 (OR=1.35, 95%CI: 0.80, 

### 14 2.28 for adjusted O 3 - sum); 2) for sympt oms defined as asthma attacks or dyspnoea or 

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

### 16 95%CI: 1.10, 2.30 for adjusted O 3 - sum); and for exacerbations defined at least one 

### 17 hospitalizations or ER visits in the last year or oral corticosteroids in the past three 

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

### 19 pollutants were more stable and significant when using the integrated measure of asthma 

### 20 control, this indicates that the results are not driven by one domain. These results support 

### 21 an effect of long

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

### 22

pre

- existing asthma. 

### 23

The interrelationships between variants in catalase (CAT) and myeloperoxidase (MPO) 

### 24 genes, ambi ent pollutants, and acute respiratory illness were investigated in a national 

### 25
U.S. cohort ( Wenten et al., 2009 ) . Health information, air pollution, and incident 

### 26 respiratory

- related school absences were a scertained in January

- June 1996 for 1,136 

### 27 Hispanic and non

- Hispanic white U.S. elementary schoolchildren as part of the 

### 28 prospective Air Pollution and Absence Study, a population based cohort study conducted 

### 29 as part of the CHS. A related earlier study ( Gilliland et al., 2001 ) , which was discussed in 

### 30
the 2006 O 3 AQCD, examined the effects of ambient air pollution on school absenteeism 

### 31 due to respiratory illness without a genetic aspect to the study. In a new study Wenten et 

### 32
al. ( 2009 ) hypothesized that variation in the level or function of these enzymes would 

### 33
modulate res piratory illness risk, especially under high levels of oxidative stress. The 

### 34 joint effect of these two genes on respiratory illness was examined. Risk of respiratory



### 35 related school absences was elevated for children with the CAT (G/G) and MPO (G/A or 

### 36 A/A) genes (relative risk = 1.35, [95% CI: 1.03, 1.77]; P

- interaction = 0.005). To assess 

### 37Draft

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- 11 September 2011 effects of long

- term average levels of O 3 on acute effects, communities were divided into 

### 1 high and low exposure groups by median levels (46.9 ppb O 3 ). The epistatic effect of 

### 2 CAT and MPO variants was evident in communities exhibiting high ambient O 3 levels 

### 3 (P

- interaction = 0.03). The association of respiratory

- illness absences with functional 

### 4 variants in CAT and MPO that differ by air pollution levels illustrates the need t o 

### 5 consider genetic epistasis in assessing gene

- environment interactions. In high O 3 

### 6 communities, CAT/MPO genotypes that resulted in decreased oxidative stress were 

### 7 associated with a decreased risk of respiratory related school absences compared with the 

### 8
CA T/MPO wild

- type genotype (Relative Risk [RR] = 0.42, [95% CI: 0.20, 0.89]). 

### 9

7.2.2 Asthma Hospital Admissions and ED Visits

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

### 10 asthma and respiratory disease that were available i n the 2006 O 3 AQCD mainly looked 

### 11 at the daily time metric. Collectively the short

- term O 3 studies presented earlier in 

### 12
Section 6.2.7.5 indicate that there is evidence for increases in both hospital admissions 

### 13
and ED visits related to both all respiratory o utcomes and asthma with stronger 

### 14 associations in the warm months. New studies evaluated long

- term O 3 exposure metrics 

### 15 providing a new line of evidence that suggests a positive exposure

- response relationship 

### 16 between first asthma hospital admission and long

- term O 3 exposure. 

### 17

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

### 18 declining warm

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

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

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

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

### 22

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

### 23

case

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

### 24 466.1) hospitalization and two measures of long

- term pollutant exposure (the month prior 

### 25 to hospitalization and the lifetime average) for O 3 in the South Coast Air Basin of 

### 26 southern California among 18,5 95 infants born between 1995 and 2000. Ozone was 

### 27 associated with reduced risk in the single

- pollutant model, but this relation did not persist 

### 28
in multi

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

### 29

In a cross

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

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

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

### 32 physician

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

### 33
concentrations for O 3 based on residential ZIP code and the closet air monitoring station 

### 34Draft

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- 12 September 2011 within 8 km but did not have data on duration of residence. Multi

- pollutant models for O 3 

### 1
and PM did not differ substantially from single

- pollutant estimates, indicating that 

### 2 pollutant multi

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

### 3
increased asthma

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

### 4 2.11] pe r 10 ppb) for all ages. Positive associations were obtained for symptoms but 95% 

### 5 CIs included null values. Associations for symptoms for adults (ages 18 +) were observed 

### 6 (OR=1.40; [95% CI: 1.02, 1.91] per 10 ppb). 

### 7

Associations between air pollution and poo rly controlled asthma among adults in 

### 8

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

### 9 Interview Survey data collected between November 2000 and September 2001 ( Meng et 

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

### 11
the nearest station within 5 miles of the residential cross

- street intersection. Poorly 

### 12
controlled asthma was defined as having daily or weekly asthma symptoms or at least one 

### 13 ED visit or hospitalization because of asthma during the past 12 months. This cross



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

### 15 when comparing those 65 year s of age and older above the 90th percentile (28.7 ppb) 

### 16
level to those below that level. Multi

- pollutant (PM) analysis produced similar results. 

### 17

Evidence associating long

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

### 18
concentration

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

### 19
2008b ) . This study investigated the association between chronic exposure to O 3 and 

### 20 childhood asthma admissions (defined as a principal diagnosis of ICD9, code 493) by 

### 21 following a birth cohort of 1,204,396 eligible births born in New York State during 1995

- 

### 22
1999 to firs t asthma admission or until 31 December 2000. There were 10,429 (0.87%) 

### 23 children admitted to the hospital for asthma between 1 and 6 years of age. The asthma 

### 24 hospitalization rate in New York State in 1993 was 2.87 per 1,000 ( Lin et al., 1999 )

. 

### 25 Three annual indicators (all 8

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

### 26
chronic O 3 exposure: (1) mean concentration during the follow

- up period (41.06 ppb); (2) 

### 27 mean concentration during the O 3 season (50.62 ppb); and (3) proportion of follow

- up 

### 28 days with O 3 levels >70 ppb. In this study t he authors aimed to predict the risk of having 

### 29 asthma admissions in a birth cohort, but the time to the first admission in children that is 

### 30 usually analyzed in survival models was not their primary interest. The effects of 

### 31

co

- pollutants were assessed and c ontrolled for using the Air Quality Index (AQI). 

### 32 Interaction terms were used to assess potential effect modifications. A positive 

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

### 34 was observed indicating that children expo sed to high O 3 levels over time are more likely 

### 35 to develop asthma severe enough to be admitted to the hospital. The various factors were 

### 36 examined and differences were found for younger children (1

- 2 years), poor 

### 37 neighborhoods, Medicaid/self

- paid births, ge ographic region and others. As shown in 

### 38Draft

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- 13 September 2011 Adjusted for child ’ s sex, age, birth weight, and gestational age; maternal race, ethnicity, age, education, 

### 1

insurance, and smoking status during pregnancy; and regional poverty l evel and temperature. ORs by low, 

### 2 medium, and high exposure are shown for New York City (NYC: low [37.3 ppb], 

### 3 medium [37.3

- 38.11] ppb, high [38.11 + ppb]) and other New York State regions (Other 

### 4 NYS regions: low [42.58 ppb], medium [42.58

- 45.06 ppb], hig h [45.06+ ppb]) for first 

### 5 asthma hospital admission. 

### 6

Figure 7

- 3 , positive concentration

- response relationships were observed. Asthma 

### 7 admissions were significantly associated with increased O 3 levels for all chronic 

### 8 exposure indicators (ORs, 1.16

- 1.68). Wh en estimating the O 3 effect using the 

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

### 10 hospital admissions with an IQR (2.51%) increase in O 3 . A proportional hazards model 

### 11
for the New York City data was run as a sensitivit y analysis and it yielded similar results 

### 12 between asthma admissions and chronic exposure to O 3 (Cox model: HR = 1.14, [95% 

### 13 CI: 1.124, 1.155] is similar to logistic model results: OR = 1.16 (95% CI: 1.15, 1.17) 

### 14 ( Lin, 2010 ) . Thus, this study provides evidence associating long

- term O 3 exposure to f irst 

### 15 asthma hospital admission in a concentration

- response relationship. 

### 16

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 lev el and temperature. ORs by low, medium, and high exposure are shown for New 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.



### Figure 7

- 3 Ozone

- asthma concentration

- response relationship using the mean concentration during the entire follow

- up period

.Draft

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- 14 September 2011 

### 7.2.3 Pulmonary Structure and Functio n

The definitive 8

- year follow

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

### 1
discussed in Section 7.2 ( Gauderman et al., 2004 ) , provided little evidence that long

- term 

### 2 exposure to ambient O 3 was associated with significant deficits in the growth rate of lung 

### 3
function in children. A later CHS study ( Islam et al., 2007 ) examined re lationships 

### 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 from 30 to 

### 7
65 ppb, as compared to the ranges observed for the other pollutants, which had four

- to 

### 8 eightfold 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 influence the association between ambient air pollutant exposures and lung function 

### 11 growth in children. They i nvestigated whether genetic variation in glutathione genes 

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

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

- year time

- period as part of the 

### 14
Children ’ s Health Study. Breto n et al. ( 2011 ) found that variation in the GSS locus was 

### 15 associated with differences in susceptibility of children for lung funct ion growth deficits 

### 16
associated with NO 2 , PM 10 , PM 2.5 , elemental carbon, organic carbon, and O 3 . The 

### 17 negative effects of air pollutants were largely observed within participants who had a 

### 18 particular GSS haplotype. The effects ranged from

- 124.2 to

- 149.1 mL for FEV 1 , - 92.9 

### 19

to

- 126.7 mL for FVC and

- 193.9 to

- 277.9 mL/s for MMEF for all pollutants except O 3 , 

### 20 for which some positive associations were reported: 25.9 mL for FEV 1 ; 0.1 mL for FVC, 

### 21
and 166.5 mL/s for MMEF. Ozone was associated with larger decreases in lung function 

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

- 

### 23

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

- 200.3 mL/s for MMEF, but only the 

### 24 association with MMEF was statistically significant. 

### 25

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

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

### 27 measures of small airways (<2 mm) function (FEF 75 and FEF 25

- 75 ) ( Tager et al., 2005 )

. 

### 28
There was an interaction with the FEF 25

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

### 29 Subjects with a large ratio were less likely t o have decreases in FEF 75 and FEF 25

- 75 for a 

### 30 given estimated lifetime exposure to O 3 . Kinney and Lippmann ( 2000 ) examined 72 

### 31 nonsmoking adults (mean age 20 years) from the second

- year class of students at the U.S. 

### 32 Military Academy in West Point, NY , and reported results that appear to be consistent 

### 33 with a decline in lung function that may in part be due to O 3 exposures over a period of 

### 34
several summer months. Ilhorst et al. ( 2004 ) examined 2,153 children with a median age 

### 35
of 7.6 years and reported pulmonary function results which indicated that significantly 

### 36
lower FVC and FEV 1 increases were associated with higher O 3 exposures over the 

### 37Draft

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- 15 September 2011 medium

- te rm of several summer months, but not over several months in the winter. 

### 1
Semi

- annual mean O 3 concentrations ranged from 22 to 54 ppb during the summer 

### 2
months and 4 to 36 ppb during the winter months. However, over the longer

- term 

### 3
3.5

- year period Ilhorst et al. ( 2004 ) found no associations between increases in lung 

### 4
function and mean summer months O 3 levels for FVC and FEV 1 , in contrast to the 

### 5 significan t medium

- term effects. Frischer et al. ( 1999 ) showed results similar to the 

### 6
Ilhorst et al. ( 2004 ) study. 

### 7

Mortimer et al. ( 2008a

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

### 8 air pollutants with pulmonary function and allergen sensitization in a subset of asthmatic 

### 9 children (ages 6

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

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

- 2000 were created and 

### 11 averaged separately across several important developmental time

- periods, including: the 

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

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

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

### 15 and early life developmental periods. The authors did not find a negative effect of 

### 16 exposure to O 3 within thi s cohort. In the second analysis ( Mortimer et al., 2008b )

, 

### 17 sensitization to at least one allergen was ass ociated, in general, with higher levels of CO 

### 18
and PM 10 during the entire pregnancy and second trimester, and higher PM 10 during the 

### 19 first 2 years of life. Lower exposure to O 3 during the entire pregnancy or second trimester 

### 20
was associated with an increased risk of allergen sensitization. Although the pollutant 

### 21 metrics across time periods were correlated, the strongest associations with the outcomes 

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

### 23 of prenatal and pos tnatal exposures, the models from this group of studies suggest that 

### 24 each time period of exposure may contribute independently to different dimensions of 

### 25
school

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

- function measures 

### 26 (FVC, FEV 1 , PEF, FEF 25

- 75 ), prenatal exposures were more influential on pulmonary 

### 27 function than early

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

### 28
and FEF 25

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

### 29
were considered alone, or in combination with the prenatal metrics, the lifetime measures 

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

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

### 32 just marke rs for lifetime or current exposures. 

### 33

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

### 34 with lung function changes in the newborn. Tidal breathing, lung volume, ventilation 

### 35 inhomogeneity and eNO were measured in 241 u nsedated, sleeping neonates (age = 

### 36
5 weeks). Consistent with the previous studies, no association was found for prenatal 

### 37 exposure to O 3 and lung function. 

### 38Draft

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- 16 September 2011 In a cross

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



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

- aged 

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

### 3 four U.S. communities. A surrogate for long

- term O 3 exposure from daily data was 

### 4 determined at the individual level. Ozone was significantly and negatively associated 

### 5 with measures of pulmonary function. 

### 6

To determine th e extent to which long

- term exposure to outdoor air pollution accelerates 

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

### 8 chronic exposure to outdoor air pollution and lung function in approxim ately 42,000 

### 9 adults aged 16 and older who were representatively sampled cross

- sectionally from 

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

### 11
not associated with O 3 concentrations. In contrast to the results for PM 10 , NO 2 , and SO 2; 

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

- ppb difference in O 3 was 

### 13

counter

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

### 14

In a prospective cohort study consisting of school

- age, non

- asthmatic children in 

### 15
Mexico City ( n = 3,170) who were 8 years of age at the beginning of the study, Rojas



### 16
Martinez et al. ( 2007 ) evaluated the association between long

- term exposure to O 3 , PM 10 

### 17
and NO 2 and lung function gr owth every 6 months from April 1996 through May 1999. 

### 18 Exposure data were provided by 10 air quality monitor stations located within 2 km of 

### 19
each child ’ s school. Over the study period, 8

- h O 3 concentrations ranged from 60 ppb 

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

### 21
an overall mean of 69.8 ppb. In multi

- pollutant models, an IQR increase in mean O 3 

### 22
concentration of 11.3 ppb was associated with an annual deficit in FEV 1 of 12 mL in 

### 23 girls and 4 mL in boys. Single

- pollutant models showed an association between ambient 

### 24 pollutants (O 3 , PM 10 and NO 2 ) and deficits in lung function growth. While the estimates 

### 25
from copollutant models were not substantially different than single pollutant models, 

### 26 independent effects for p ollutants could not be estimated accurately because the traffic



### 27 related pollutants were correlated. To reduce exposure misclassification, 

### 28 microenvironmental and personal exposure assessments were conducted in a randomly 

### 29 selected subsample of 60 children us ing passive O 3 samplers. Personal O 3 concentrations 

### 30 were correlated (p < 0.05) with the measurements obtained from the fixed

- site air 

### 31 monitoring stations. 

### 32

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

### 33 pulmonary fun ction. The strongest evidence was for medium

- term effects of extended O 3 

### 34 exposures over several summer months on lung function in children, i.e., reduced lung 

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

- term studies, 

### 36 investigating the association of chronic O 3 exposure on lung function such as the CHS, 

### 37Draft

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- 17 September 2011 were inconclusive. Short

- term O 3 exposure studies presented in Section 6.2.1.2 provide a 

### 1 cumulative body of epidemiologic evidence that str ongly supports associations between 

### 2
ambient O 3 exposure and decrements in lung function among children. For new studies 

### 3 of long

- term O 3 exposure relationship to pulmonary function, one study, where O 3 and 

### 4 other pollutant levels were higher (90 ppb at high end of the range) than those in the 

### 5 CHS, observes a relationship between O 3 concentration and pulmonary function declines 

### 6
in school

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



### 7 term exposures were at the high end of the rang e with levels of 49.5 ppb in one study and 

### 8 27 ppb IQR in the other. Thus there is little new evidence to build upon the very limited 

### 9
studies from the 2006 O 3 AQCD. 

### 10

7.2.3.1 Pulmonary Structure and Function: Evidence from Toxicological Studies

As reviewed in the 199 6 and 2006 O 3 AQCDs, there are both qualitative and quantitative 

### 11 uncertainties in the extrapolation of data generated by rodent toxicology studies to the 

### 12 understanding of health effects observed in humans, as documented by epidemiologic and 

### 13 controlled expo sure studies. Chief among these data extrapolation issues are the 

### 14 differences between rodent and human respiratory physiology, cellular makeup, 

### 15 dosimetry, and morphometry (see Chapter 5). However, important evidence is available 

### 16
from O 3 - inhalation studies performed in nonhuman primates whose respiratory system 

### 17 most closely resembles that of the human. A long series of studies have used nonhuman 

### 18 primates to examine the effect of O 3 alone or in combination with an inhaled allergen, 

### 19 house dust mite antigen, on morphology and lung function. These studies, by Plopper and 

### 20 colleagues, have demonstrated changes in pulmonary function and airway morphology in 

### 21 adult and infant nonhuman primates repeatedly exposed to environmentally relevant 

### 22
concentrations of O 3 ( Joad et al., 2008 ; Carey et al., 2007 ; Plopper et al., 2007 ; Fanucchi 

### 23 et al., 2006 ; Joad et al., 2006 ; Evans et al., 2004 ; Larson et al., 2004 ; Tran et al., 2004 ; 

### 24 Evans et al., 2003b ; Schelegle et al., 2003 ; Fanucchi et al., 2000 ; Hyde et al., 1989 ; 

### 25 Harkema et al., 1987a ; Harkema et al., 1987b ; Fujinaka et al., 1985 ) . The findings of 

### 26 these nonhuman primate studies have also been observed in rodent studies discussed at 

### 27
the end of this section and included in Table 7

- 1

. 

### 28

Since publication of the 1996 and 2006 O 3 AQCDs, the initial observations in adult 

### 29 nonhuman primates have been expanded in a series of experiments using infant rhesus 

### 30 monkeys repeatedly expo sed to 0.5 ppm O 3 starting at 1 month of age ( Plopper et al., 

### 31
2007 ) . Many of the observations found in adult monkeys have also been noted in infant 

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

### 33 infant monkeys has not been reported. In terms of pulmonary function changes, after 

### 34Draft

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- 18 September 2011 several episodic exposures of infant monkeys to O 3 (each cycle: 5 days of 0.5 ppm O 3 at 

### 1 8 h/day, followed by 9 days of filtered air exposure), they observed more than a doubling 

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

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

### 4

measur ement was statistically different from filtered air control values. Exposure of 

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

### 6 significant changes in baseline airway resistance and airway responsiveness, whereas the 

### 7 combined exposure to both (O 3 + antigen) produced statistically significant and greater 

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

### 9
of an O 3 - induced change in airway responsiveness supports the biologic plausibility of 

### 10 long

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

### 11 which conducting airways and inflammatory mechanisms are involved in O 3 - induced 

### 12 airway hyperresponsiveness in the infant rhesus monkey, a follow

- up st udy examined 

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

### 14 to morphometrically evaluate the response of bronchi and respiratory bronchioles to 

### 15 methacholine, (a bronchoconstricting agent commonly used to evaluate airway 

### 16 responsiveness in asthmatics), the investigators observed differential effects for the two 

### 17 airway sizes. While episodic expo sure to O 3 alone (0.5 ppm) had little effect on ex vivo 

### 18 airway responsiveness in bronchi and respiratory bronchioles, exposure to dust mite 

### 19 antigen alone produced airway hyperresponsiveness in the large bronchi, whereas O 3 + 

### 20 antigen produced significant in creases in airway hyperresponsiveness only in the 

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

### 22 responsiveness occurs predominantly in the smaller bronchioles, where dosimetric 

### 23 models indicate the dose would be higher. 

### 24

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

### 25
either O 3 alone or O 3 + antigen were accompanied by a number of cellular and 

### 26 morphological changes, including a significant fourfold increase in eosinophils, (a cell 

### 27 type import ant in allergic asthma), in the bronchoalveolar lavage of infant monkeys 

### 28 exposed to O 3 alone. Thus, these studies demonstrate both functional and cellular 

### 29 changes in the lung of infant monkeys after cyclic exposure to 0.5 ppm O 3 . This 

### 30 concentration, while higher than those used in controlled human exposure studies, 

### 31 provides relevant information to understanding the adverse effects of ambient O 3 

### 32 exposure on the respiratory tract of humans. No concentration

- response data, however, 

### 33
are available from these non human primate studies. 

### 34

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

### 35 respiratory tract have been observed in infant rhesus monkeys exposed to O 3 . During 

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

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

### 38Draft

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- 19 September 2011 followed by 9 days of filtered air exposures for 11 cycles), however, markedly affected 

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

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

- control infant 

### 3 monkeys, the most proximal alveolarized airways occurred at an average of 10 airway 

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

### 5 and respiratory bronchioles were significantly decreased in O 3 - exposed monkeys. 

### 6 Importantly, the O 3 - induced structural pathway changes persisted after recovery in 

### 7
filtered air for 6 months after cessation of the O 3 exposures. These structural effects were 

### 8 accompanied by significant increases in mucus goblet cell mass, alterations in smooth 

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

### 10 basement membrane zone morphometry. These latter effects are sig nificant because of 

### 11 their potential contribution to airway obstruction and airway hyperresponsiveness which 

### 12
are central features of asthma. 

### 13

As noted above, a significant increase in airway responsiveness to inhaled histamine 

### 14
occurred in infant rhesus monke ys exposed to O 3 + house dust mite antigen, but not to O 3 

### 15
alone ( Schelegle et al., 2003 ) . To study the underlying mechanisms of this airway 

### 16 hyperresponsiveness, these investigators evaluated the effect of exposure to O 3 alone and 

### 17 in combination with (+) antigen on non

- specific airway responsiveness to methacholine 

### 18 at different airway generations. After exposure to filtered air, O 3 , antigen, or O 3 + 

### 19 antigen, the bronchi and respiratory bronchioles of 6

- month

- old monkeys were 

### 20 challenged ex vivo with methacholine. Exposure to O 3 alone had no significant effect on 

### 21 airway responsiveness to methacholine in either airway, whereas O 3 + antigen produced 

### 22 a significant increase in airway responsiveness in the respiratory bronchiole s but not the 

### 23 larger bronchi. 

### 24

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

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

### 26 was also examined in infant rhesus monkeys. Mast cells, which contribute to asthma via 

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

### 28
alone had little effect on mast cell num bers and the response of animals exposed to O 3 + 

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

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

### 31 allergic asthma ( Van Winkle et al., 2010 ) . Increases in CD4+ and CD8+ lymphocytes 

### 32
were observed at 6 months of age in the blood and bronchoalveolar lavage fluid of infant 

### 33 rhesus monkeys exposed to O 3 + antigen but not in monkeys exposed to either agent 

### 34
alone ( Mil ler et al., 2009 ) . Activated lymphocytes (i.e., CD25+ cells) were 

### 35 morphometrically evaluated in the airway mucosa and significantly increased in infant 

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

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

### 38Draft

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- 20 September 2011 Ozone had only a small effect on these sets of immune cells and did not produce a strong 

### 1 interaction with an inhaled allergen in this nonhuman primate model. 

### 2

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

### 3 epithelial cells are thought to play a contributing role to airway hyperresponsiveness. As 

### 4
noted in the 2006 O 3 AQCD, exposure of infant rhesus monkeys altered the normal 

### 5 development of neural innervat ion in the epithelium of the conducting airways ( Larson et 

### 6 al., 2004 ) . Whereas, a significant reduction in airway innervation occurred after exposure 

### 7
to O 3 alone, a significantly greater reduction o ccurred in monkeys exposed to O 3 + 

### 8 antigen. This reduction in overall airway innervation was accompanied, however, by an 

### 9 increase in the abundance of protein gene product 9.5, a nonspecific neural marker. 

### 10 Significant increases in protein gene product 9.5 w ere still observed in O 3 alone

- and O 3 

### 11 + antigen

- exposed infant monkeys after a 6

- month recovery protocol ( Kajekar et al., 

### 12
2007 ) . Thus, in addition to structural, immune, and inflammatory effect s, exposure to O 3 

### 13 produces alterations in airway innervation which may contribute to O 3 - induced 

### 14
exacerbation of asthma. 

### 15

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

### 16 exposure can increase collagen synthesis and deposition, inducing fibrotic

- like changes in 

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

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

### 19 cross links that appear to be irreversible ( Reiser et al., 1987 )

. Generally changes in 

### 20 collagen content have been observed in rats exposed to 0.5 ppm O 3 or higher, although 

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

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

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

### 24
demonstrated that O 3 - induced collagen deposition in mice is dependent on the activity of 

### 25
TGF

- β ( Katre et al., 2011 ) . Sex differences have been observed with respec t to increased 

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

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

. Few other 

### 28 long

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

### 29 evaluated males. It is unclear what the long

- term effects of these structural changes may 

### 30 be. A number of studies indicate that structural changes in the respir atory system are 

### 31 persistent or irreversible. For example, O 3 - induced hyperplasia was still evident in the 

### 32 nasal epithelia of rats 13 weeks after recovery from 0.5 ppm O 3 exposure ( Harkema et 

### 33 al., 1999 ) . In a study of episodic exposure to 0.25 ppm O 3 , Chang et al. ( 1992 ) observed 

### 34 no reversal of basement membrane thickening in rat lungs up to 17 weeks post

- exposure. 

### 35 Episodic exposure (0.25 ppm O 3 , every other month) of monkeys induced equivalent 

### 36 morphological changes compared to continuously exposed animals, even though they 

### 37Draft

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- 21 September 2011 were exposed for half the time and evaluation occurred a month after exposure ceased as 

### 1 opposed to immediately ( Tyler et al., 1988 )

. 

### 2Draft

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- 22 September 2011 

### Table 7

- 1 Respirat ory effects in nonhuman primates and rodents resulting from long

- term O 3 

### exposure

Study Model O 3 (ppm) Exposure Duration Effects

Catalano et al. ( 1995a ; 1995 b ) ; Chang et al. ( 1995 ) ; Harkema et al. ( 1997a ; 1997b ; 1994 ) ; Last et al. ( 1994 ) ; Pinkerton et al. ( 1995 ) ; Plopper et al. ( 1994 ) ; Stockstill et al. ( 1995 ) ; Pinkerton et al. ( 1998 ) 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 in rats exposed to 0.12 ppm O 3 . Other effects (e.g., mucous cell metaplasia in the nose and mild fibrotic response in the parenchym a, increased collagen in CAR of females) observed at 0.5 to 1.0 ppm. Some morphometric changes such as epithelial thickening and bronchiolarization occurred after 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 Continuous: 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 epithelia l 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 fiber s. 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 development not significantly affected. Increased Type 1 and 2 epithelial cells and AM in CAR alveoli, thickene d Type 1 cells with smaller volume and less surface coverage as assessed by TEM (adults and juveniles). In adults, smaller but statistically significant similar changes at 0.12 ppm, suggesting linear concentration

- response relationship. No statistically si gnificant age

- related effects observed. Tyler et al. ( 1988 ) Monkey; male, M acaca fascicularis, 7 mo old 0.25 8 h/day, 7 days/week, Daily for 18 mo or episodically every other mo for 18 mos Episodic group evaluated 1 mo post exposure 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 and 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 week s old 0.25
0.5 8 h/day, 7 days/week for 13 weeks Mucous cell hyperplasia in nasal epithelium after exposure 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 exp osure with increased collagen and bronchiolization still present after a recovery period.
Katre et al. ( 2011 ) Mice; male, C57BL/6, 6

- 8 week sold 0.5 8 h/day, [5 days/week O3, and 2 days filtered 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

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- 23 September 2011 Study Model O 3 (ppm) Exposure Duration Effects Schelegle et al. ( 2003 ) ; Monkey; Rhesus, 30 days old* 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.
Larson et al. ( 2004 ) Monkey;Macaca mul atta, 30 days old* 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 o f neural innervation in epithelial compartment of developing lungs. Plopper et al. ( 2007 ) Monkey; Rhesus, 30 days old* 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 fo llowed 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.

* 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 m echanisms 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 involved 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 AQC D 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
studies disc ussed earlier in Section 6.2.3.2 show consistent associations of O 3 exposure 

### 15 and increased airway inflammation and oxidative stress. Further discussion of the 

### 16 mechanisms underlying inflammation and oxidative stress responses can be found in 

### 17
Section 5.3.3. Though the majority of recent studies focus on short

- term exposures, 

### 18Draft

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- 24 September 2011 several epidemiologic and toxicology studies of long

- term exposure add to observations 

### 1
of O 3 - induced inflammation and injury. 

### 2

Inflammatory markers and peak expiratory pulmonary function w ere examined in 37 

### 3 allergic children with physician

- diagnosed mild persistent asthma in a highly polluted 

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

### 5 significantly lower pollutant levels ( Renzetti et al., 2009 ) . The authors observed a 

### 6 fourfold decrease in nasal eosinophils and a statistically significant decrease in fractional 

### 7 exhaled nitric oxide along with an improvement in lower airway function. Several 

### 8 pollutants were examined, including PM 10 , NO 2 , and O 3 , though pollutant

- specific 

### 9 results were not presented. These results are consistent with studies showing that traffic



### 10 related exposures are associated with increased airway inflammation and reduced lung 

### 11
function in children with asthma and contribut e to the notion that this negative influence 

### 12 may be rapidly reversible. Exhaled NO (eNO) has been shown to be a useful biomarker 

### 13 for airway inflammation in large population

- based studies ( Linn et al., 2009 ) . Thus, while 

### 14
the time sc ale of 7 days between examinations for eNO needs to be evaluated for 

### 15 appropriateness, the results suggest that inflammatory responses are reduced when O 3 

### 16
levels are decreased. 

### 17

Chest radiographs (CXR) of 249 children in Mexico City who were chronically expo sed 

### 18
to O 3 and PM 2.5 were analyzed by Calderón

- Garcidueñas et al. ( 2006 ) . They reported an 

### 19 association between chronic exposures to O 3 and other pollutants and a significant 

### 20
increase in abnormal CXR ’ s and lung CTs suggestive of a bronchiolar, peribronchiolar, 

### 21 and/or alveolar duct inflammatory process, in clinically healthy children with no risk 

### 22 factors for lung disease. These CXR and CT results should be viewed w ith caution 

### 23
because it is difficult to attribute effects to O 3 exposure. 

### 24

In a cross

- sectional study, Wood et al. ( 2009 ) examined the association of outdoor air 

### 25 pollution with respiratory phenotype (PiZZ type) in alpha 1

- Antitrypsin defic iency (α



### 26 ATD) from the U.K. α

- ATD registry. In total, 304 PiZZ subjects underwent full lung 

### 27 function testing and quantitative high

- resolution computed tomography to identify the 

### 28 presence and severity of COPD

– emphysema. Mean annual air pollution data for 2006 

### 29 was matched to the location of patients ’ houses and used in regression models to identify 

### 30 phenotypic associations with pollution controlling for covariates. Relative trends in O 3 

### 31 levels were assessed to validate use of a single year ’ s data to indicate long

- term exposure 

### 32 and validation; data showed good correlations between modeled and measured data 

### 33 ( Stedman and Kent, 2008 ) . Regression models showed that estimated higher exposure to 

### 34
O 3 exposure was associated with worse gas transfer and more severe emphysema, albeit 

### 35

acc ounting for only a small proportion of the lung function variability. This suggests that 

### 36

a gene

- specific group demonstrates a long

- term O 3 exposure effect. 

### 37Draft

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- 25 September 2011 The similarities of nonhuman primates to humans make them attractive models in which 

### 1 to study the ef fects of O 3 on the respiratory tract. The nasal mucous membranes, which 

### 2 protect the more distal regions of the respiratory tract, are susceptible to injury from O 3 . 

### 3 Carey et al. ( 2007 ) conducted a study of O 3 exposure in infant rhesus macaques, whose 

### 4 nasal airways closely resemble that of humans. Monkeys were expos ed either acutely for 

### 5
5 days (8 h/day) to 0.5 ppm O 3 , or episodically for several biweekly cycles alternating 

### 6
5 days of 0.5 ppm O 3 with 9 days of filtered air (0 ppm O 3 ), designed to mimic human 

### 7 exposure (70 days total). All monkeys acutely exposed to O 3 h ad moderate to marked 

### 8 necrotizing rhinitis, with focal regions of epitheliar exfoliation, numerous infiltrating 

### 9 neutrophils, and some eosinophils. The distribution, character, and severity of lesions in 

### 10 episodically exposed monkeys were similar to that of acutely exposed animals. Neither 

### 11 group exhibited mucous cell metaplasia proximal to the lesions, a protective adaptation 

### 12 observed in adult monkeys exposed continuously to 0.3 ppm O 3 in another study 

### 13 ( Harkema et al., 1987a ) . Adult monkeys also exhibit attenuation of inflammatory 

### 14 responses with continued daily exposure ( Harkema et al., 1987a ) , but inflamma tion did 

### 15 not resolve over time in young episodically exposed monkeys ( Carey et al., 2011 )

. 

### 16 Inflammation in conducting airways has also been observed in rats chronically exposed to 

### 17
O 3 . Using an agar

- based technique to fill the alveo li so that only the rat bronchi are 

### 18 lavaged, a 90

- day exposure of rats to 0.8 ppm O 3 (8 h/day) elicited significantly elevated 

### 19

pro

- inflammatory eicosanoids PGE 2 and 12

- HETE in the conducting airway compared to 

### 20
filtered air

- exposed rats ( Schmelzer et al., 2006 )

. 

### 21

7.2.5 Allergic Responses

The association of air pollutants with childhood respiratory allergies was examined in the 

### 22 U.S. using the 19 99

- 2005 National Health Interview Survey of approximately 70,000 

### 23 children, and ambient air pollution data from the U.S. EPA, with monitors within 20 

### 24
miles of each child ’ s residential block ( Parker et al., 2009 ) . The authors examined the 

### 25 associations between the reporting of respiratory allergy or hay fever and medium

- term 

### 26 exposure to O 3 over several summer month s, controlling for demographic and geographic 

### 27 factors. Increased respiratory allergy/hay fever was associated with increased O 3 levels 

### 28 (adjusted OR per 10 ppb = 1.20; [95% CI: 1.15, 1.26]). These associations persisted after 

### 29 stratification by urban

- rural s tatus, inclusion of multiple pollutants (O 3 , SO 2 , NO 2 , PM), 

### 30 and definition of exposure by differing exposure radii; smaller samples within 5 miles of 

### 31 monitors were remarkably similar to the primary results. No associations between the 

### 32 other pollutants and the reporting of respiratory allergy/hay fever were apparent. 

### 33
Ramadour et al. ( 2000 ) reported no relationship between O 3 levels and rhinitis symptoms 

### 34 and hay fever. Hwang et al. ( 2006 ) report the prevalence of allergic rhinitis (adjusted OR 

### 35Draft

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- 26 September 2011 per 10 ppb = 1.05; [95% CI: 0.98, 1.12]) in a large cross

- sectional study in Taiwan . In a 

### 1 large cross

- sectional study in France, Penard

- Morand et al. ( 2005 ) reported a positive 

### 2 relationship be tween lifetime allergic rhinitis and O 3 exposure in a two

- pollutant model 

### 3
with NO 2 . These studies related positive outcomes of allergic response and O 3 exposure 

### 4 but with variable strength for the effect estimates. A toxicological study reported that 

### 5
five w eeks of continuous exposure to 0.4 ppm O 3 (but not 0.1 or 0.2 ppm O 3 ) augmented 

### 6 sneezing and nasal secretions in a guinea pig model of nasal allergy ( Iijima and 

### 7 Kobayashi, 2004 ) . Nasal eosinophils, wh ich participate in allergic disease and 

### 8 inflammation, and allergic antibody levels in serum were also elevated by exposure to 

### 9
concentrations as low as 0.2 ppm ( Iijima and Kobayashi, 2004 )

. 

### 10

Nasal eosi nophils were observed to decrease by fourfold in 37 atopic, mildly asthmatic 

### 11
children 7 days after relocation from a highly polluted urban area in Italy to a rural 

### 12 location with significantly lower pollutant levels ( Renzetti et al., 2009 ) . Inflammatory 

### 13 and allergic effects of O 3 exposure (30 day mean) such as increased eosinophil levels 

### 14 were observed in children in an Austrian study ( Frisch er et al., 2001 ) . Episodic exposure 

### 15 of infant rhesus monkeys to 0.5 ppm O 3 for 5 months appears to significantly increase the 

### 16 number and proportion of eosinophils in the blood and airways (lavage) [protocol 

### 17
described above in 7.2.3.1 for Fanucchi et al. ( 2006 ) ] ( Maniar

- Hew et al., 2011 ) . These 

### 18 changes were not evident at 1 year of age (6 months after O 3 exposure ceased). Increased 

### 19 eosinophils levels have also been observed after acute or prolonged exposures to O 3 in 

### 20 adult bonnet and rhesus monkeys ( Hyde et al., 1992 ; Eustis et al., 1981 )

. 

### 21

Total IgE levels were related to air pollution levels in 369 adult asthmatics in five French 

### 22 centers using generalized estimated equ ations (GEE) as part of the EGEA study described 

### 23
earlier ( Rage et al., 2009b ) . Geostatistical models were performed on 4×4 km grids to 

### 24 assess individual outdoor air pollution exposure that was assigned to subject ’ s home 

### 25
address. Ozone concentra tions were positively related to total IgE levels and an increase 

### 26
of 5 ppb of O 3 resulted in an increase of 20.4% (95% CI: 3.0, 40.7) in total IgE levels. 

### 27 Nearly 75% of the subjects were atopic. In two

- pollutant models including O 3 and NO 2, 

### 28

the O 3 effect estimate was decreased by 25% while the NO 2 effect estimate was decreased 

### 29 by 57%. Associations were not sensitive to adjustment for covariates or the season of IgE 

### 30
measurements. These cross

- sectional results suggest that exposure to O 3 may increase 

### 31
total I gE in adult asthmatics. 

### 32

Although very few toxicological studies of long

- term exposure examining allergy are 

### 33 available, short

- term exposure studies in rodents and nonhuman primates demonstrate 

### 34 allergic skewing of immune responses and enhanced IgE productio n. Due to the 

### 35 persistent nature of these responses, the short

- term toxicological evidence lends 

### 36Draft

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- 27 September 2011 biological plausibility to the limited epidemiologic findings of an association between 

### 1 long

- term O 3 exposure and allergic outcomes. 

### 2

7.2.6 Host Defense

Short

- term exp osures to O 3 have been shown to cause decreases in host defenses against 

### 3 infectious lung disease in animal models. However, acute O 3 - induced suppression of 

### 4 alveolar phagocytosis and immune functions observed in animals appears to be transient 

### 5
and attenuate d with continuous or repeated exposures. Chronic exposures (weeks, 

### 6 months) of 0.1 ppm do not cause greater effects on infectivity than short exposures, due 

### 7 to defense parameters becoming reestablished with prolonged exposures, although 

### 8 chronic exposure has been shown to slow alveolar clearance. No detrimental effects were 

### 9
seen with a 120

- day exposure to 0.5 ppm O 3 on acute lung injury from influenza virus 

### 10 administered immediately before O 3 exposure started. However, O 3 was shown to 

### 11 increase the severity of postinfluenzal alveolitis and lung parenchymal changes ( Jakab 

### 12 and Bassett, 1990 ) . Little new evidence has become available to address the effects of 

### 13 long

- term exposure on host defense mechani sms. However, a recent study by Maniar



### 14
Hew et al. ( 2011 ) demonstrated that the immune system of infant rhesus monkeys 

### 15 episodically exposed to 0.5 ppm O 3 for 5 months 1 appeared to be altered in ways that 

### 16
could diminish host defenses. Reduced numbers of circulating leukocytes were observed, 

### 17 particularly polymorphonuclear leukocytes (PMNs) and lymphocytes, which were 

### 18
decreased in the bl ood and airways (bronchoalveolar lavage). These changes did not 

### 19 persist at 1 year of age (6 months postexposure); rather, increased numbers of monocytes 

### 20 were observed at that time point. Challenge with LPS, a bacterial ligand that activates 

### 21 monocytes and o ther innate immune cells, elicited lower responses in O 3 - exposed 

### 22 animals even though the relevant reactive cell population was increased. This was 

### 23 observed in both an in vivo inhalation challenge and an ex vivo challenge of peripheral 

### 24
blood mononuclear cel ls. Thus a decreased ability to respond to pathogenic signals was 

### 25
observed six months after O 3 exposure ceased, in both the lungs and periphery. 

### 26

7.2.7 Respiratory Mortality

A limited number of epidemiologic studies have assessed the relationship between long



### 27

ter m exposure to O 3 and mortality. The 2006 O 3 AQCD concluded that an insufficient 

### 28
amount of evidence existed “ to suggest a causal relationship between chronic O 3 

### 29 exposure and increased risk for mortality in humans ” ( U.S. EPA, 2006b ) . Thoug h total 

### 30

1 Exposure protocol is described above in Section 7.2.3.1 for Fanucchi et al. ( 2006 )Draft

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- 28 September 2011 and cardio

- pulmonary mortality were considered in these studies, respiratory mortality 

### 1 was not specifically considered. In the most recent follow

- up analysis of the ACS cohort 

### 2 ( Jerrett et al., 2009 ) , cardiopulmonary deaths were subdivided into respiratory and 

### 3 cardiovascular, separately, as opposed to combined in the Pope et al. ( 2002 ) work. A 10

- 

### 4 ppb increment in exposure to O 3 elevated the risk of death from respiratory causes and 

### 5
this effect was robust to the inclusion of PM 2.5 . The association between increased O 3 

### 6
concentrations and inc reased risk of death from respiratory causes was insensitive to the 

### 7
use of a random

- effects survival model allowing for spatial clustering within the 

### 8 metropolitan area and state of residence, and to adjustment for several ecologic variables 

### 9
considered indi vidually. Additionally, a recent study ( Za nobetti and Schwartz, In Press ) 

### 10 observed an association between long

- term exposure to O 3 and elevated risk of mortality 

### 11 among Medicare enrollees that had previously experienced an emergency hospital 

### 12
admission due to COPD. 

### 13

7.2.8 Summary and Causal Determinatio n

The epidemiologic studies reviewed in the 2006 O 3 AQCD detected no associations 

### 14 between long

- term (annual) O 3 exposures and asthma

- related symptoms, asthma 

### 15 prevalence, or allergy to common aeroallergens among children after controlling for 

### 16
covariates. Li ttle evidence was available to relate long

- term exposure to current ambient 

### 17
O 3 concentrations to deficits in the growth rate of lung function in children. Additionally, 

### 18 limited evidence was available evaluating the relationship between long

- term O 3 levels 

### 19 and pulmonary inflammation and other endpoints. From toxicological studies, it appeared 

### 20
that O 3 - induced inflammation tapered off during long

- term exposures, but that 

### 21 hyperplastic and fibrotic changes remained elevated and in some cases even worsened 

### 22
after a postexposure period in clean air. Episodic exposures were also known to cause 

### 23 more severe pulmonary morphologic changes than continuous exposure ( U.S. EPA, 

### 24
2006b )

. 

### 25

The new epidemiologic evidence base consists of studies using a variety of designs and 

### 26 analysis methods evaluating the relationship between long

- term annual measures of 

### 27 exposure to ambient O 3 and measures of respiratory morbidity conducted by different 

### 28 research groups in different locations. See Table 7

- 2 for O 3 concentrations associated 

### 29 with selected studies. The positive results from various designs and locations support an 

### 30 association between long

- term O 3 concentrations and respiratory morbidity. 

### 31

New studies examined the relationship between long

- term O 3 exposure and new onset 

### 32 asthma in children. Studies have provided evidence for a relationship between different 

### 33 genetic variants (HMOX, GST, ARG) that, in combination wit h O 3 exposure, are related 

### 34Draft

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- 29 September 2011 to new onset asthma ( Islam et al., 2009 ; Salam et al., 2009 ; Islam et al., 2008 ) . These 

### 1 studies involve two separate co horts in 12 California communities of the CHS. These 

### 2 prospective cohort studies represent strong evidence because they are methodologically 

### 3 rigorous epidemiology studies. The stratified analysis for the two independent fourth



### 4 grade cohorts of the study pop ulation recruited in 1993 and 1996 yielded consistent 

### 5 results and provided replication in independent groups of children. 

### 6

Table 7

- 2 Summary of selected key new studies examining annual ozone exposure and resp iratory health effects

Study; Health Effect; Location Annual Mean O 3 Concentration (ppb) O 3 Range (ppb)
Percentiles

Akinbami et al. ( 2010 ) ; current asthma United States 12 month median 39.8 IQR 35.9 to 43.7 Hwang et al. ( 2005 ) ; prevalence of asthma
Taiwan Mean 23.14 Range 18.65 to 31.17

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. 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

Salam et al. ( 2009 ) ; childhood onset asthma; CHS O 3 greater than or less than 50 ppb See left
Lin et al. ( 2008b ) ; first asthma hospital admission; New York State

- 10 regions Range of mean O3 concentrations over the 10 New Y ork Regions 37.51 to 47.78 See left

Moore et al. ( 2008 ) ; asthma hospital admissions; South Coast Basin Median 87.8 ppb Range 28.6 to 199.9 Meng et al. ( 2010 ) ; asthma ED visits or hospitalizations; San Joaquin Valley, CA Median 30.3 ppb 25

- 75% range
27.1 to 34.0

Lee et al. ( 2009b ) ; bronchitic symptoms in asthmatic children; CHS Above and below 50 ppb See left

Rage et al. ( 2009b ) ; asthma severity; five French cities Mean 30 ppb 25th

- 75th 21

- 36 Jacquemin et al. ( In Press ) ; asthma control in adults; five French cities Median 46.9 ppb; 25th

- 75th 41

- 52
Wenten et al. ( 2009 ) ; respiratory school absence, U.S. Median 46.9 ppb; 10:00 a.m.

– 6:00 p.m. Min

- Max 27.6

- 65.3

Studies using a cross

- sectional design provide support for a relationship between long



### 7
term O 3 exposure and health effects in asthmatics. A long

- term O 3 exposure study relates 

### 8 bronchitic symptoms to TNF

- 308 genotype asthmatic children with ambient O 3 exposure 

### 9
in the CHS ( Lee et al., 2009b ) . A study in five French cities reports effects on asthma 

### 10 severity related to long

- term O 3 exposure ( Rage et al., 2009a ) . A follow

- up study of this 

### 11
cohort ( Jacquemin et al., In Press ) supports an effect of long

- term O 3 - sum exposure on 

### 12 asthma control in adulthood in subjects with pre

- existing asthma. Akinbami et al. ( 2010 ) 

### 13 and Hwang et al. ( 2005 ) provides further evidence relating O 3 exposures and the risk of 

### 14
asthma. For the respiratory health of a cohort based on the general U.S. population, risk 

### 15Draft

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- 30 September 2011 of respiratory

- related school absences was elevated for children with the CAT and MPO 

### 1 variant genes related to communities with high ambient O 3 levels ( Wenten et al., 2009 )

. 

### 2

Chronic O 3 exposure was related to first childhood asthma hospital admissions in a 

### 3 positive concentration

- response relationship in a New York State birth cohort ( Lin et al., 

### 4
2008b ) . A separate hospitalization cross

- sectional study in San Joaquin Valley in 

### 5 California reports similar findings ( Meng et al., 2010 ) . Another study relates asthma 

### 6 hospital admissions to quarterly average O 3 in the South Coast Air Basin of California 

### 7 ( Moore et al., 2008 )

. 

### 8

Information from toxi cological studies indicates that long term exposure to O 3 during 

### 9 gestation or development can result in irreversible morphological changes in the lung, 

### 10 which in turn can influence pulmonary function. Studies by Plopper and colleagues have 

### 11
demonstrated chan ges in pulmonary function and airway morphology in adult and infant 

### 12 nonhuman primates repeatedly exposed to environmentally relevant concentrations of O 3 

### 13 ( Fanucchi et al., 2006 ; Joad et al., 2006 ; Sch elegle et al., 2003 ; Harkema et al., 1987b )

. 

### 14 This nonhuman primate evidence of an O 3 - induced change in airway responsiveness 

### 15 supports the biolog ic plausibility of long term exposure to O 3 contributing to the adverse 

### 16 effects of asthma in children. Results from epidemiologic studies examining long

- term 

### 17
O 3 exposure and pulmonary function effects are inconclusive with some new studies 

### 18 relating effects at higher exposure levels. The results from the CHS described in the 2006 

### 19
O 3 AQCD remain the definitive line of evidence. Other cross

- sectional studies provide 

### 20
mixed results. 

### 21Draft

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- 31 September 2011 

### 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

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)

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

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)

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

Rage et al. ( 2009a )
Asthma severi ty in adults IQR 28.5

- 33.9 ppb 2.53 (1.69, 3.79) NA No PM data Three pollutant (O 3 , NO 2 , SO 2 ) 2.74 (1.68, 4.48) 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) PM10
1.29 (0.99, 1.69) Did not differ NA

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)

Rojas

- Martinez et al. ( 2007 )
FEV 1 (mL) Deficit
Girls 11.3 ppb IQR - 24 (

- 30,

- 19) PM 10 IQR 36.4 ug/m3

- 29(

- 36,

- 21) - 17 (

- 23,

- 12) - 24 (

- 31,

- 16)

Parker et al. ( 2009 ) Respiratory allergy 10 ppb 1.24 (1.15, 1.34) 1.23 (1.04, 1.46) Multi

- pollutant 1.18 (1.09, 1.27) 1.29 (1.07, 1.56)

The highest quartile is shown for all results.
NA = not available

Several studies (see Table 7

- 3 ) provide results from studies that adjusted for potential 

### 1 confounders, presenting results for both O 3 and PM (single and multipollutant models) as 

### 2 well as other pollutants where PM effects we re not provided. As shown in the table, O 3 

### 3 associations are generally robust to adjustment for potential confounding by PM. 

### 4

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

### 5 toxicological evidence, together with the limited epidemiolo gic evidence available, 

### 6Draft

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- 32 September 2011 suggests a causal role for short

- term O 3 exposure in inflammatory responses in the 

### 1 airways. Though the majority of recent studies focus on short

- term exposures, several 

### 2 epidemiologic and toxicological studies of long

- term exposure a dd to observations of O 3 - 

### 3 induced inflammation and injury. Toxicological studies in rodents and nonhuman 

### 4 primates indicate that chronic O 3 exposure causes structural changes in the respiratory 

### 5 tract, and simulated seasonal exposure studies suggest that such exposures might have 

### 6 cumulative impacts. The strongest epidemiologic evidence for a relationship between 

### 7 long

- term O 3 exposure and respiratory morbidity is provided by new studies that 

### 8 demonstrate associations between long

- term measures of O 3 exposure and new

- onset 

### 9 asthma in children and increased respiratory symptom effects in asthmatics. While there 

### 10 are currently a limited number of studies in this data base, the U.S. multi

- community 

### 11 prospective cohort studies are methodologically rigorous epidemiologic studies. Asthma 

### 12 risk is related to complex relationships between genetic variability, environmental O 3 

### 13 exposure, and behavior. The genes, evaluated in these studies, are both key candidates in 

### 14 the oxidative stress pathway and have been shown to play an imp ortant role in asthma 

### 15 development. Reduced risk for asthma development is reported in some studies in 

### 16 children living in low

- O 3 communities. Mean O 3 concentrations in the studies (10:00 

### 17 a.m. to 6:00 p.m.) ranged from 28.6 to 45.5 ppb in low O 3 communities 

### 18 (mean = 38.4 ppb) and from 46.5 to 64.9 ppb in high O 3 communities (mean = 55.2 ppb). 

### 19
These CHS multi

- community studies form a foundation for the evidence base in which 

### 20 findings for several genes indicate the breath of the evidence across different gene 

### 21

v ariants. The several other studies with different designs, analysis, locations and 

### 22 researchers provide a cumulative collective body of evidence informing these 

### 23 relationships. The other studies in the new data base provide coherent evidence for long



### 24
term O 3 exposure and respiratory morbidity effects such as first asthma hospitalization 

### 25 and respiratory symptoms in asthmatics. Studies considering other pollutants provide data 

### 26 suggesting that the effects related to O 3 are independent from potential effects of t he 

### 27 other pollutants. Some studies provide evidence for a positive concentration

- response 

### 28 relationship. Short

- term studies provide supportive evidence with increases in respiratory 

### 29 symptoms and asthma medication use, hospital admissions and ED visits for al l 

### 30 respiratory outcomes and asthma, and decrements in lung function in children. The above 

### 31 discussion of the recent epidemiologic and toxicological data base provides a compelling 

### 32 case to support the hypothesis that a relationship exists between long

- term e xposure to 

### 33
ambient O 3 and measures of respiratory morbidity. The 2006 O 3 AQCD concluded the 

### 34 evidence was suggestive but inconclusive at that time. The new epidemiological data 

### 35 base, combined with toxicological studies in rodents and nonhuman primates, 

### 36

prov ides biologically plausible evidence that there is likely to be causal 

### 37 relationship between long

- term exposure to O 3 and respiratory morbidity. 

### 38Draft

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- 33 September 2011 

## 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 

### 1
considered in the 2006 O 3 AQCD. However, recent studies have assessed the chronic 

### 2
effects of O 3 exposure on cardiovascular morbidity ( Chuang et al., 2011 ; Forbes et al., 

### 3
2009a ; Chen et al., 2007 ) . The association between O 3 exposure and markers of lipid 

### 4 peroxidation and anti oxidant capacity was examined among 120 nonsmoking healthy 

### 5 college students, aged 18

- 22 years, from the University of California, Berkeley (Feb

- Jun 

### 6 2002) ( Chen et al., 2007 ) . By design, students were chosen from geographic areas so they 

### 7 had experienced different lev els of O 3 over their lifetimes and during recent summer 

### 8 vacation in either greater Los Angeles (LA) or the San Francisco Bay Area (SF). A 

### 9 marker of lipid peroxidation, 8

- isoprostane (8

- iso

- PGF) in plasma, was assessed. This 

### 10 marker is formed continuously un der normal physiological conditions but has been found 

### 11 at elevated concentrations in response to environmental exposures. A marker of overall 

### 12 antioxidant capacity, ferric reducing ability of plasma (FRAP), was also measured. The 

### 13
lifetime O 3 exposure estima tes (estimated monthly average) did not show much overlap 

### 14 between the two geographic areas [median (range): LA, 42.9 ppb (28.5

- 65.3); SF, 26.9 

### 15 ppb (17.6

- 33.5)]. Estimated lifetime O 3 exposure was related to 8

- iso

- PGF [β = 0.025 

### 16 (pg/mL)/8

- h ppb O 3 , p = 0.00 07]. For the 17

- ppb cumulative lifetime O 3 exposure 

### 17 difference between LA and SF participants, there was a 17.41

- pg/mL (95% CI: 15.43, 

### 18 19.39) increase in 8

- iso

- PGF. No evidence of association was observed between lifetime 

### 19
O 3 exposure and FRAP [β =

- 2.21 (p g/mL)/8

- h ppb O 3 , p = 0.45]. The authors note that 

### 20
O 3 was highly correlated with PM 10

- 2.5 and NO 2 in this study population; however, their 

### 21
inclusion in the O 3 models did not substantially modify the magnitude of the associations 

### 22
with O 3 . Because the lifeti me exposure results were supported by shorter

- term exposure 

### 23 results from analyses considering O 3 concentrations up to 30 days prior to sampling, the 

### 24 authors conclude that persistent exposure to O 3 can lead to sustained oxidative stress and 

### 25 increased lipid peroxidation. However, because there was not much overlap in lifetime 

### 26
O 3 exposure estimates between LA and SF, it is possible that the risk estimates involving 

### 27
the lifetime O 3 exposures could be confounded by unmeasured factors related to other 

### 28
differences between the two cities. 

### 29

Forbes et al. ( 2009a ) used the annual average exposures to assess the relationship 

### 30 between chronic ambient air pollution and levels of fibrinogen and C

- reactive protein 

### 31Draft

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- 34 September 2011 (CRP) in a cross

- sectional study conducted in England. Data were co llected from the 

### 1 Health Survey of England for 1994, 1998, and 2003. The sampling strategy was designed 

### 2 to obtain a representative sample of the English population; however, due to small group 

### 3 sizes, only data from white ethnic groups were analyzed. For ana lyses, the annual 

### 4
concentrations of O 3 were averaged for the year of data collection and the previous year 

### 5 with the exception of 1994 (because pollutant data were not available for 1993). Median 

### 6
O 3 concentrations were 26.7 ppb, 25.4 ppb, and 28 ppb for 1994, 1998, and 2003, 

### 7 respectively. Year specific adjusted effect estimates were created and combined in a 

### 8

meta

- analysis. No evidence of association was observed for O 3 and levels of fibrinogen 

### 9
or CRP (e.g., the combined estimates for the percent change in fibrinogen and CRP for a 

### 10
10 ppb increase in O 3 were

- 0.28 [95% CI:

- 2.43, 1.92] and

- 3.05 [95% CI:

- 16.10, 

### 11 12.02], respectively). 

### 12

A study was performed in Taiwan to examine the association between l ong

- term O 3 

### 13 concentrations and blood pressure and blood markers using the Social Environment and 

### 14 Biomarkers of Aging Study (SEBAS) ( Chuang et al., 2011 ) . Individuals included in the 

### 15 study were 54 years of age and older. The mean annual O 3 concentration during the study 

### 16 period wa s 22.95 ppb (SD 6.76 ppb). Positive associations were observed between O 3 

### 17 concentrations and both systolic and diastolic blood pressure [changes in systolic and 

### 18 diastolic blood pressure were 21.51mmHg (95% CI: 16.90, 26.13) and 20.56 mmHg 

### 19 (95% CI: 18.14, 2 2.97) per 8.95 ppb increase in O 3 , respectively). Increased O 3 

### 20 concentrations were also associated with increased levels of total cholesterol, fasting 

### 21 glucose, hemoglobin A1c, and neutrophils. No associations were observed between O 3 

### 22
concentrations and tri glyceride and IL

- 6 levels. The observed associations were reduced 

### 23 when other pollutants were added to the models. Further research will be important for 

### 24 understanding the effects, if any, of chronic O 3 exposure on cardiovascular morbidity 

### 25
risk. 

### 26

7.3.1.2 Cardiovascu lar Toxicology

Three new studies have investigated the cardiovascular effects of long

- term exposure to 

### 27
O 3 in animal models (See Table 7

- 4 for study det ails). In addition to the short

- term 

### 28 exposure effects described in Section 6.3.3, a recent study found that O 3 exposure in 

### 29 genetically hyperlipidemic mice enhanced aortic atherosclerotic lesion area compared to 

### 30 air exposed controls ( Chuang et al., 2009 ) . Chuang et al. ( 2009 ) not only provided 

### 31 evidence for increased atherogenesis in susceptible mice, but also reported an elevated 

### 32

vascu lar inflammatory and redox state in wild

- type mice and infant primates 

### 33 (Section 6.3.3.2). This study is compelling in that it identifies biochemical and cellular 

### 34 events responsible for transducing the airway epithelial reactions of O 3 into 

### 35Draft

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- 35 September 2011 proinflammatory responses that are apparent in the extrapulmonary vasculature ( Cole and 

### 1 Freeman, 2009 )

. 

### 2

Another recent study provides further evidence for increased vascular inflammation and 

### 3 oxidation and long term effects in the extrapulmonary space. Rats episodically exposed to 

### 4
O 3 for 16 weeks presented marked increases in gene expression of biomarkers of 

### 5 oxidative stress, thrombosis, vasoconstriction, and proteolysis ( Kodavanti et al., 2011 )

. 

### 6 Ozone exposure upregulated aortic mRNA expression of heme oxygenase

- 1 (HO

- 1), 

### 7 tissue plasminogen activator (tPA), plasminogen activator inhibitor

- 1 (PAI

- 1), von 

### 8 Willebrand factor (vWf), thrombomodulin, endothelial nitric oxide synthase (eNOS), 

### 9
endothelin

- 1 (ET

- 1), matrix metalloprotease

- 2 (MMP

- 2), matrix metalloprotease

- 3 

### 10 (MMP

- 3), and tissue inhibitor of matrix metalloprotease

- 2 (TIMP

- 2). In addition, O 3 

### 11 exposure depleted some cardiac mitochondrial phospholipid fat ty acids (C16:0 and 

### 12 C18:1), which may be the result of oxidative modifications. The authors speculate that 

### 13 oxidatively modified lipids and proteins produced in the lung and heart promote vascular 

### 14 pathology through activation of lectin

- like oxidized

- low den sity lipoprotein receptor

- 1 

### 15 (LOX

- 1). Activated LOX

- 1 induces expression of a number of the biomarkers induced by 

### 16
O 3 exposure and is considered pro

- atherogenic. Both LOX

- 1 mRNA and protein were 

### 17
increased in mouse aorta after O 3 exposure. This study provides a possible pathway and 

### 18 further support to the observed O 3 induced atherosclerosis. 

### 19

Vascular occlusion resulting from atherosclerosis can block blood flow through vessels 

### 20 causing ischemia. The restoration of blood flow or reperfusion can cause injury to th e 

### 21 tissue from subsequent inflammation and oxidative damage. Ozone exposure enhanced 

### 22 the sensitivity to myocardial ischemia

- reperfusion (I/R) injury in rats while increasing 

### 23 oxidative stress levels and pro

- inflammatory mediators and decreasing production of anti



### 24 inflammatory proteins ( Perepu et al., 2010 ) . Both long

- and short

- term O 3 exposure 

### 25 decreased the left ventricular developed pressure, rate of change of pressure 

### 26 development, and rate of change of pressure decay and increased left ventricular end 

### 27 diastolic pressure in isolated perfused hearts (Section 6.3.3.2 for short

- term e xposure 

### 28 discussion). In this ex vivo heart model, O 3 induced oxidative stress by decreasing SOD 

### 29 enzyme activity and increasing malondialdehyde levels. Ozone also elicited a 

### 30 proinflammatory state evident by an increase in TNF

- α and a decrease in the anti



### 31
in flammatory cytokine IL

- 10. The authors conclude that O 3 exposure will result in a 

### 32 greater I/R injury. 

### 33Draft

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- 36 September 2011 

### Table 7

- 4 Characterization of study details for Section 7.3.1.2

Study Model O 3 (ppm) Exposure Duration Effe cts

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 a l. ( 2011 ) Rat; Wistar; M; 10

- 12 weeks 0.4 16 wks, 1 day/week, 5 h/day Increased vascular inflammation and oxidative stress, po ssibly 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
.



### 7.3.2 Cardiac 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 insufficient 

### 2

a mount 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, In Press ) 

### 12
observed an associatio n 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 the 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 epidemiolog ic studies 

### 18 have investigated cardiovascular morbidity after long

- term O 3 exposure, and the majority 

### 19 only assessed cardiovascular disease related biomarkers. A study on O 3 and 

### 20 cardiovascular mortality reported no association after adjustment for PM 2.5 level s. 

### 21Draft

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- 37 September 2011 Further epidemiologic studies on cardiovascular morbidity and mortality after long

- term 

### 1 exposure have not been published. 

### 2

Toxicological evidence on long

- term O 3 exposure is also limited but three strong 

### 3 toxicological studies have been published since t he previous AQCD. These studies 

### 4 provide evidence for O 3 enhanced atherosclerosis and I/R injury, corresponding with 

### 5 development of a systemic oxidative, proinflammatory environment. Further discussion 

### 6 of the mechanisms that may lead to cardiovascular effec ts can be found in Section 5.3.8. 

### 7 Although questions exist for how O 3 inhalation causes systemic effects, a recent study 

### 8 proposes a mechanism for development of vascular pathology that involves activation of 

### 9
LOX

- 1 by O 3 oxidized lipids and proteins. This a ctivation may also be responsible for O 3 

### 10 induced changes in genes involved in proteolysis, thrombosis, and vasoconstriction. 

### 11 Taking into consideration the findings of toxicological studies, and the emerging 

### 12 evidence from epidemiologic studies, the generall y limited body of evidence is 

### 13 suggestive of a causal relationship between long

- term exposures to O 3 and 

### 14
cardiovascular effects. 

### 15



## 7.4 Reproductive and Developmental Effects

Although the body of literature is growing, the research focusing on adverse birth 

### 16 outcomes is small. Among these studies, various measures of birth weight and fetal 

### 17 growth, such as low birth weight (LBW), small for gestational age (SGA), and 

### 18 intrauterine growth restriction (IUGR), and preterm birth (<37

- week gestation; [PTB]) 

### 19
have recei ved more attention in air pollution research, while congenital malformations 

### 20 are less studied. There are also new studies on reproductive and developmental effects. 

### 21

Infants and fetal development processes may be particularly susceptible to O 3 - induced 

### 22
healt h effects, and although the physical mechanisms are not fully understood, several 

### 23 hypotheses have been proposed; these include: oxidative stress, systemic inflammation, 

### 24 vascular dysfunction and impaired immune function (Section 5.3). Study of these 

### 25

outcome s can be difficult given the need for detailed exposure data and potential 

### 26 residential movement of mothers during pregnancy. Air pollution epidemiologic studies 

### 27
reviewed in the 2006 O 3 AQCD examined impacts on birth

- related endpoints, including 

### 28
intrauterin e, perinatal, postneonatal, and infant deaths; premature births; intrauterine 

### 29 growth retardation; very low birth weight (weight <1,500 grams) and low birth weight 

### 30 (weight <2,500 grams); and birth defects. However, in the limited number of studies that 

### 31
inve stigated O 3 , no associations were found between O 3 and birth outcomes, with the 

### 32 possible exception of birth defects. 

### 33Draft

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- 38 September 2011 Several recent articles have reviewed methodological issues relating to the study of 

### 1 outdoor air pollution and adverse birth outcomes ( Chen et al., 2010a ; Woodruff et al., 

### 2
2009 ; Ritz and Wilhelm, 2008 ; Slama et al ., 2008 ) . Some of the key challenges to 

### 3 interpretation of these study results include the difficulty in assessing exposure as most 

### 4 studies use existing monitoring networks to estimate indiv idual exposure to ambient air 

### 5 pollution; the inability to control for potential confounders such as other risk factors that 

### 6 affect birth outcomes (e.g., smoking); evaluating the exposure window (e.g., trimester) of 

### 7 importance; and limited evidence on the p hysiological mechanism of these effects ( Ritz 

### 8 and Wilhelm, 2008 ; Slama et al., 2008 ) . Recently, an international collaboration was 

### 9 formed to better understand the relationships between air pollution and adverse birth 

### 10 outcomes and to examine some of these methodological issues through standardized 

### 11 parallel analyses in datasets from differe nt countries ( Woodruff et al., 2010 )

. Initial 

### 12 results from this collaboration have examined PM and birth weight ( Parker et al ., 2011 ) ; 

### 13
work on O 3 has not yet been performed. Although early animal studies ( Kavlock et al., 

### 14
1980 ) found that exposure to O 3 in the late gestation of pregnancy in rats led to some 

### 15 abnormal reproductive performances for neonates, to date human studies have reported 

### 16
inconsistent results for the association of ambient O 3 on birth outcomes. 

### 17

7.4.1 Effects on Sperm

A limited amount of resear ch has been conducted to examine the association between air 

### 18 pollution and male reproductive outcomes, specifically semen quality. To date, the 

### 19 epidemiologic studies have considered various exposure durations before semen 

### 20 collection that encompass either t he entire period of spermatogenesis (i.e., 90 days) or 

### 21 key periods of sperm development that correspond to epididymal storage, development of 

### 22 sperm motility, and spermatogenesis. In an analysis conducted as part of the Teplice 

### 23 Program, 18

- year

- old men resi ding in the heavily polluted district of Teplice in the Czech 

### 24 Republic were found to be at greater risk of having abnormalities in sperm morphology 

### 25 and chromatin integrity than men of similar age residing in Prachatice, a less polluted 

### 26
district ( Selevan et al., 2000 ; Sram et al., 1999 ) . A follow

- up longitudinal study 

### 27 conducted on a subset of the same men from Teplice revealed associations between total 

### 28 episodic air pollution and abnormalities in spe rm chromatin ( Rubes et al., 2005 ) . A 

### 29 limitation of these studies is that they did not identify specific pollutants and their 

### 30
concentrations. 

### 31

More recent epidemiologic studies conducted in the U.S. have also reported associations 

### 32 between ambient air pollution and sperm quality for individual air pollutants, including 

### 33
O 3 and PM 2.5 . In a repeated measures study in Los Angeles, CA, Sokol et al. ( 2006 ) 

### 34 reported a reduction in average sperm concentration during three exposure windows (0

- 9, 

### 35Draft

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- 39 September 2011 10

- 14, and 70

- 90 days before semen collection) associated with high ambient levels of 

### 1
O 3 in healthy sperm donors. This effect persisted under a joint additive model for O 3 , 

### 2 CO, NO 2 and PM 10 . The authors did not detect a reduction in sperm count. Hansen et al. 

### 3 ( 2010 ) investigated the effect of exposure to O 3 and PM 2.5 (using the same exposure 

### 4 windows used by Sokol et al. ( 2006 ) on sperm quality in three southeastern counties 

### 5 (Wake County, NC; Shelby County, TN; Galveston County, TX). Outcomes included 

### 6 sperm concentration and count, morphology, DNA integri ty and chromatin maturity. 

### 7 Overall, the authors found both protective and adverse effects, although some results 

### 8 suggested adverse effects on sperm concentration, count and morphology. 

### 9

The biological mechanisms linking ambient air pollution to decreased s perm quality have 

### 10 yet to be determined, though O 3 - induced oxidative stress, inflammatory reactions, and 

### 11 the induction of the formation of circulating toxic species have been suggested as 

### 12 possible mechanisms (see Section 5. 3.8). Decremental effects on test icular morphology 

### 13 have been demonstrated in toxicological studies with histological evidence of O 3 - induced 

### 14 depletion of germ cells in testicular tissue and decreased seminiferous tubule epithelial 

### 15 layer. Jedlinska

- Krakowska et al. ( 2006 ) demonstrated histopathological evidence of 

### 16 impaired spermatogenesis (round spermatids/ spermatocytes, giant spermatid cells, and 

### 17
foca l epithelial desquamation with denudation to the basement membrane). The exposure 

### 18 protocol used five month old adult rats exposed to O 3 as adults (0.5 ppm, 5 h/day for 

### 19
50 days). This degeneration could be rescued by vitamin E administration, indicating an 

### 20
antioxidant effect. Vitamin C administration had no effect at low doses of ascorbic acid 

### 21
and exacerbated the O 3 - dependent damage at high doses, as would be expected as 

### 22 vitamin C can be a radical generator instead of an antioxidant at higher doses. In 

### 23

summa ry, this study provided toxicological evidence of impaired spermatogenesis with 

### 24
O 3 exposure that was rescued with certain antioxidant supplementation. 

### 25

Overall, there is limited epidemiologic evidence for an association with O 3 concentration 

### 26
and decreased sperm concentration. A recent toxicological study provides limited 

### 27 evidence for a possible biological mechanism (histopathology showing impaired 

### 28 spermatogenesis) for such an association. 

### 29

7.4.2 Effects on Reproduction

Evidence suggests that exposure to air pollut ants during pregnancy is associated with 

### 30 adverse birth outcomes, which has been attributed to the increased susceptibility of the 

### 31 fetus due to physiologic immaturity. Gametes (i.e., ova and sperm) may be even more 

### 32 susceptible, especially outside of the hum an body, as occurs with assisted reproduction. 

### 33 Smokers require twice the number of in vitro fertilization (IVF) attempts to conceive as 

### 34Draft

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- 40 September 2011 non

- smokers ( Feichtinger et al., 1997 ) , suggesting that a preconception exposure can be 

### 1 harmful to pregnancy. A recent study used an established national

- scale, log

- normal 

### 2 kriging method to spatially estimate daily mean concentrations of criteria pollutants at 

### 3 addresses of women undergoing the ir first IVF cycle and at their IVF labs from 2000 to 

### 4
2007 in the northeastern U.S. ( Legro et al., 2010 )

. Increasing O 3 concentration at the 

### 5 patient ’ s address was significantly associated with an increased chance of live birth 

### 6 during ovulation induction (OR=1.13, [95% CI: 1.05, 1.22] per 10 ppb increase), but with 

### 7 decreased odds of live birth when exposed from embryo transfer to live birth (OR=0.79, 

### 8 [95% CI: 0.69, 0.90] per 10 ppb increase). After controlling for NO 2 in a copollutant 

### 9 model, however, O 3 was no longer significantly associated with IVF failure. The results 

### 10 of this study suggest that exposure to O 3 during ovulation was beneficial (perhaps due to 

### 11 early conditioning to O 3 ), whereas later exposure to O 3 (e.g., during gestation) was 

### 12 detrimental, and reduced the likelihood of a live birth. 

### 13

In most toxicological studies, reproductive success appears to be unaffected by O 3 

### 14 exposure. Nonetheless, one study has reported that 25% of the BALB/c mouse dams in 

### 15 the highest O 3 exposure group (1.2 ppm, GD9

- 18) did not complete a successful 

### 16 pregnancy, a significant reduction ( Sharkhuu et al., 2011 )

. Ozone administration 

### 17 (co ntinuous 0.4, 0.8 or 1.2 ppm O 3 ) to CD

- 1 mouse dams during the majority of 

### 18 pregnancy (PD7

- 17, which excludes the pre

- implantation period), led to no adverse 

### 19 effects on reproductive success (proportion of successful pregnancies, litter size, sex 

### 20 ratio, freq uency of still birth, or neonatal mortality) ( Bignami et al., 1994 ) . There was a 

### 21 nearly statistically significant increase in pregnancy duration (0.8 and 1.2 ppm O 3 ). 

### 22 Initially, dam body weight (0.8 and 1.2 ppm), water consumption (0.4, 0.8 and 1.2 ppm 

### 23
O 3 ) and food co nsumption (0.4, 0.8 and 1.2 ppm) during pregnancy were decreased with 

### 24
O 3 exposure but these deficits dissipated a week or two after the initial exposure 

### 25 ( Bignami et al., 1994 ) . The anorexigenic effect of O 3 exposure on the pregnant dam 

### 26 appears to dissipate with time; the dams seem to adapt to the O 3 exposure. In males, data 

### 27 exist showing morphological evidence of altered spermatogenesis in O 3 exposed animals 

### 28 ( Jedlinska

- Krakowska et al., 2006 ) . Some evidence suggests that O 3 may affect 

### 29 reproductive success when combined with other chemicals. Kavlock et al. ( 1979 ) showed 

### 30
that O 3 acted synergistically with sodium salicylate to increase the rate of pup resorptions 

### 31 after midgestational exposure (1.0 ppm O 3 , GD9

- 12). At low doses of O 3 exposure, 

### 32 toxicological studies show reproductive effects to include a transient anorexigenic effect 

### 33
of O 3 on gestational weight gain, and a synergistic effect of O 3 on salicylate

- induced pup 

### 34 resorptions; other fecundity , pregnancy and gestation related outcomes appear unaffected 

### 35 by O 3 exposure. 

### 36Draft

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- 41 September 2011 Collectively, there is very little epidemiologic evidence for the effect of O 3 on 

### 1 reproductive success, and the reproductive success in rats appears to be unaffected in 

### 2 toxicolog ical studies of O 3 exposure. 

### 3

7.4.3 Birth Weight

With birth weight routinely collected in vital statistics and being a powerful predictor of 

### 4 infant mortality, it is the most studied outcome within air pollution

- birth outcome 

### 5 research. Air pollution researchers h ave analyzed birth weight as a continuous variable 

### 6 and/or as a dichotomized variable in the form of LBW (<2,500 g [5 lbs, 8 oz]). 

### 7

Birth weight is primarily determined by gestational age and intrauterine growth, but also 

### 8 depends on maternal, placental and f etal factors as well as on environmental influences. 

### 9 In both developed and developing countries, LBW is the most important predictor for 

### 10 neonatal mortality and is a significant determinant of postneonatal mortality and 

### 11 morbidity. Studies report that infant s who are smallest at birth have a higher incidence of 

### 12 diseases and disabilities, which continue into adulthood ( Hack and Fanaroff, 1999 )

. 

### 13

The strongest evidence for an effect of O 3 on birth weight comes from the Children ’ s 

### 14 Health Study conducted in southern California. In this study, Salam et al. ( 2005 ) report 

### 15 that maternal exposure to 24

- h avg O 3 concentrations averaged over the entire pregnancy 

### 16 was associated with reduced birth weight (39.3 g decrease [95% CI:

- 55.8,

- 22.8] in birth 

### 17 weight per 10 ppb and 8

- h avg ( 19.2

- g decrease [95% CI:

- 27.7,

- 10.7] in birth weight per 

### 18
10 ppb). This effect was stronger for concentrations averaged over the second and third 

### 19
trimesters. PM 10 , NO 2 and CO concentrations averaged over the entire pregnancy were 

### 20 not statistically signifi cantly associated with birth weight, although CO concentrations in 

### 21
the first trimester and PM 10 concentrations in the third trimester were associated with a 

### 22 decrease in birth weight. Additionally, the authors observed a concentration

- response 

### 23 relationship of birth weight with 24

- h avg O 3 concentrations averaged over the entire 

### 24 pregnancy that was clearest above the 30

- ppb level (see Figure 7

- 4). Relative to the 

### 25
lowest decile of 24

- h avg O 3 , estimates for the next 5 lowest deciles were approximately 

### 26

- 40 g to

- 50 g, with no clear trend and with 95% confidence bounds that included zero. 

### 27 The highest four deciles of O 3 exposure showed an approximately linear decrease in birth 

### 28 weight, and all four 95% CIs excluded zero, and ranged from mean decreases of 

### 29
74 grams to decreases of 148 grams. 

### 30Draft

– Do Not Cite or Quote 7

- 42 September 2011 Source: Salam et al. ( 2005 ) Deficits are plotted against the decile

- g roup

- 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.



### Figure 7

- 4 Birthweight defic it by decile of 24

- h avg O 3 

### concentration averaged over the entire pregnancy compared with the decile group with the lowest O 3 

### exposure.

Several additional studies conducted in the U.S. and Canada also investigated the 

### 1
association between ambient O 3 conce ntrations and birth weight and report some weak 

### 2
evidence for an association. Morello

- Frosch et al. ( 2010 ) estimated ambient O 3 

### 3 concentrations throughout pregna ncy and for each trimester in the neighborhoods of 

### 4 women who delivered term singleton births between 1996 and 2006 in California. A 10

- 

### 5 ppb increase in O 3 averaged across the entire pregnancy was associated with a 5.7

- g 

### 6 decrease (95% CI:

- 6.6,

- 4.9) in birt h weight when exposures were calculated using 

### 7
monitors within 10 km of the maternal address at date of birth. When the distance from 

### 8 the monitor was restricted to 3 km, the decrease in birth weight associated with a 10

- ppb 

### 9
increase in O 3 increased to 8.9 g (95% CI:

- 10.6,

- 7.1). These results persisted in 

### 10 copollutant models and in models that stratified by trimester of exposure, SES, and race. 

### 11
Darrow et al. ( 2011a ) did not observe an association with birth weight and O 3 

### 12 concentrations during two exposu re periods of interest (i.e., the first month and last 

### 13 trimester), but did find an association with reduced birth weight when examining the 

### 14 cumulative air pollution concentration during the entire pregnancy period. Additionally, 

### 15 they observed effect modifi cation by race and ethnicity, such that associations between 

### 16 birth weight and third

- trimester O 3 concentrations were significantly stronger in 

### 17Draft

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- 43 September 2011 Hispanics and non

- Hispanic African Americans than in non

- Hispanic whites. Chen et al. 

### 1 ( 2002 ) used 8

- h avg O 3 concentrations to create exposure variables based on average 

### 2 maternal exposure for each trimester. Ozone was not found to be related to birth weight 

### 3 in single

- pollutant models, though the O 3 effect during the third trimester was borderline 

### 4 statistically significant in a copollutant model with PM 10 . 

### 5

Several studies found no association between ambient O 3 concentrations and birth 

### 6 weight. Wilhelm and Ritz ( 2005 ) extended previous analyses of term LBW ( Ritz et al., 

### 7
2000 ; Ritz and Yu , 1999 ) to include the period 1994

- 2000. The authors examined varying 

### 8 residential distances from monitoring stations to see if the distance affected risk 

### 9 estimation, exploring the possibility that effect attenuation may result from local pollutant 

### 10 heterogeneity i nadequately captured by ambient monitors. As in their previous studies, 

### 11
the authors observed associations between elevated concentrations of CO and PM 10 both 

### 12 early and late in pregnancy and risk of term LBW. After adjusting for CO and/or PM 10 

### 13
the authors d id not observe associations between O 3 and term LBW in any of their 

### 14
models. Brauer et al. ( 2008 ) evaluated the impacts of air pollution (CO, NO 2 , NO, O 3 , 

### 15
SO 2 , PM 2.5 , PM 10 ) on birth weight for the period 1999

- 2002 using spatiotemporal 

### 16 residential exposure metrics by month of pregnancy in Vancouver, BC. Quantitative 

### 17 results were not presented for the association between O 3 and LBW, though the authors 

### 18
observed associations that were la rgely protective. Dugandzic et al. ( 2006 ) examined the 

### 19 association between LBW and ambient levels of air pollutants by trim ester of exposure 

### 20 among a cohort of term singleton births from 1988

- 2000. Though there was some 

### 21
indication of an association with SO 2 and PM 10 , there were no effects for O 3 . 

### 22

Similarly, studies conducted in Australia, Latin America, and Asia report limited 

### 23
evidence for an association between ambient O 3 and measures of birth weight. In Sydney, 

### 24 Australia, Mannes et al. ( 2005 ) found that O 3 concentrations in the second trimester of 

### 25

p regnancy had small adverse effects on birth weight (7.5

- g decrease; [95 % CI:

- 13.8, 

### 26 1.2] per 10 ppb), although this effect disappeared when the analysis was limited to births 

### 27 with a maternal address within 5 km of a monitoring station (87.7

- g increase; [9 5% CI: 

### 28 10.5, 164.9] per 10 ppb). Hansen et al. ( 2007 ) reported that trimester and monthly 

### 29 specific exposures to all pollutants were not statistically significantly associated with a 

### 30 reduction in birth weight in Brisbane, Australia. In Sao Paulo, Brazil, Gouveia et al. 

### 31 ( 2004 ) found that O 3 exhibited a small inverse relation with birth weight over the third 

### 32 trimester (6.0

- g decrease; [95% CI:

- 30.8, 18.8] per 10 ppb). Lin et al. ( 2004b ) reported a 

### 33 positive, though not statistical ly significant, exposure

- response relationship for O 3 during 

### 34 the entire pregnancy in a Taiwanese study. In a study performed in Korea, Ha et al. 

### 35 ( 2001 ) reported no O 3 effect during the first trimester of pregnancy, but they found that 

### 36 during the third trimester of pregnancy O 3 was associated wi th LBW (RR=1.05 [95% CI: 

### 37 1.02, 1.08] per 10 ppb). 

### 38Draft

– Do Not Cite or Quote 7

- 44 September 2011 

### Table 7

- 5 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 ) Californi a, 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. ( 2011a ) Atlanta, GA (N=406,627) 8

- h max: 44.8 Population

- weighted s patial average Entire pregnancy:

- 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, US (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 ) Van couver, 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

- 1.2 4) d Mannes et al. ( 2005 ) Sydney, Australia (n=138,056) 1

- h max: 31.6 City

- wide 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 City

- wide 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 City

- wide 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 pregnanc y: 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 City

- wide 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

- 5 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 

### 4Draft

– Do Not Cite or Quote 7

- 45 September 2011 results of the Children ’ s Health Study. Additional studies conducted in the U.S., Canada, 

### 1 Australia, Latin America, an d Asia provide limited and inconsistent evidence to support 

### 2 the effect reported in the Children ’ s Health Study. The toxicological literature on the 

### 3
effect of O 3 on birth weight is sparse. In some studies, the reporting of birth weight may 

### 4
be avoided becaus e birth weight can be confounded by decreased litter size resulting 

### 5 from an increased rate of pup resorption (aborted pups) in O 3 exposed dams. In one 

### 6 toxicological study by Haro and Paz ( 1993 ) , no dif ferences in litter size were observed 

### 7 and decreased birth weight in pups from dams who were exposed to 1ppm O 3 during 

### 8 pregnancy was reported. A second animal toxicology study recapitulated these finding 

### 9 with pregnant BALB/c mice that exposed to O 3 (1.2 ppm , GD9

- 18) producing pups with 

### 10 significantly decreased birth weights ( Sharkhuu et al., 2011 )

. 

### 11

7.4.4 Preterm Birth

Preterm birth (PTB) is a syndrome ( Romero et al., 2006 ) that is characterize d by multiple 

### 12 etiologies. It is therefore unusual to be able to identify an exact cause for each PTB. In 

### 13 addition, PTB is not an adverse outcome in itself, but an important determinant of health 

### 14 status (i.e., neonatal morbidity and mortality). Although som e overlap exists for common 

### 15 risk factors, different etiologic entities related to distinct risk factor profiles and leading 

### 16 to different neonatal and postneonatal complications are attributed to PTB and measures 

### 17 of fetal growth. Although both restricted fe tal growth and PTB can result in LBW, 

### 18 prematurity does not have to result in LBW or growth restricted babies. 

### 19

A major issue in studying environmental exposures and PTB is selecting the relevant 

### 20 exposure period, since the biological mechanisms leading to P TB and the critical periods 

### 21 of vulnerability are poorly understood ( Bobak, 2000 ) . Exposures proximate to the birth 

### 22 may be most relevant if exposure causes an acute effect. However, exposure occurring in 

### 23 early gestation might affect placentation, with results observable later in pregnancy, or 

### 24 cumulative exposure during pregnancy may be the most im portant determinant. The 

### 25 studies reviewed have dealt with this issue in different ways. Many have considered 

### 26 several exposure metrics based on different periods of exposure. Often the time periods 

### 27
used are the first month (or first trimester) of pregnancy and the last month (or 6 weeks) 

### 28 prior to delivery. Using a time interval prior to delivery introduces an additional problem 

### 29 since cases and controls are not in the same stage of development when they are 

### 30 compared. For example, a preterm infant delivered at 36 weeks is a 32

- week fetus 

### 31

4 weeks prior to birth, while an infant born at term (40 weeks) is a 36

- week fetus 4 weeks 

### 32 prior to birth. 

### 33Draft

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- 46 September 2011 Recently, investigators have examined the association of PTB with both short

- term (i.e., 

### 1 hours, days, or weeks) and lon g

- term (i.e., months or years) exposure periods. Time



### 2 series studies have been used to examine the association between air pollution 

### 3 concentrations during the days immediately preceding birth. An advantage of these time



### 4 series studies is that this approach can remove the influence of covariates that vary across 

### 5 individuals over a short period of time. Retrospective cohort and case

- control studies 

### 6 have been used to examine long

- term exposure periods, often averaging air pollution 

### 7
concentrations over months o r trimesters of pregnancy. 

### 8

Reported studies fail to show consistency in pollutants and periods during pregnancy 

### 9 when an effect occurs. For example, while some studies find the strongest effects 

### 10 associated with exposures early in pregnancy, others report ef fects when the exposure is 

### 11 limited to the second or third trimester. However, the effect of air pollutant exposure 

### 12 during pregnancy on PTB has a biological basis. There is an expanding list of possible 

### 13 mechanisms that may explain the association between O 3 exposure and PTB (see 

### 14
Section 5. 4.2.4). 

### 15

Many studies of PTB compare exposure in quartiles, using the lowest quartile as the 

### 16 reference (or control) group. No studies use a truly unexposed control group. If exposure 

### 17 in the lowest quartile confers risk, tha n it may be difficult to demonstrate additional risk 

### 18 associated with a higher quartile. Thus negative studies must be interpreted with caution. 

### 19

Preterm birth occurs both naturally ( idiopathic preterm ), and as a result of medical 

### 20 intervention ( iatrogenic pr eterm ). Ritz et al. ( 2007 ; 2000 ) excluded all births by Cesarean 

### 21 section to limit their studies to idiopathic preterm. No other studies attempted to 

### 22 distinguish the type of PTB, although air pollution exposure maybe associated with only 

### 23 one type. This is a sour ce of potential effect misclassification. 

### 24

Generally, studies of air pollution

- birth outcome conducted in North America and the 

### 25 United Kingdom have not identified an association between PTB and maternal exposure 

### 26
to O 3 . Most recently, Darrow et al. ( 2009 ) used vital record data to construct a 

### 27 retrospective cohort of 476,489 births occurring between 1994 and 2004 in 5 central 

### 28 counties of metropolitan Atlanta. Using a time

- series approach, the authors examined 

### 29 aggregated daily counts of PTB in relation to ambient levels of CO, NO 2 , SO 2 , O 3 , PM 10 , 

### 30

PM 2.5 and speciated PM measurements. This study investigated 3 gestational windows of 

### 31 exposure: the first month of gestation, the final week of gestati on, and the final 6 weeks 

### 32 of gestation. The authors did not observe associations of PTB with O 3 . 

### 33

A number of U.S. studies were conducted in southern California, and report somewhat 

### 34
inconsistent results. Ritz et al. ( 2000 ) evaluated the effect of air pollution (CO, NO 2 , O 3 , 

### 35

PM 10 ) exposure during pregnancy on the occurrence of PTB in a cohort of 97,518 

### 36Draft

– Do Not Cite or Quote 7

- 47 September 2011 neonates born in southern California between 1989 and 1993. The authors use both short



### 1 and long

- term exposure windows, averaging pollutant measures taken at the closest air



### 2
monitor ing station over distinct periods, such as 1, 2, 4, 6, 8, 12, and 26 weeks before 

### 3 birth and the whole pregnancy period. Additionally, they calculated average exposures 

### 4
for the first and second months of pregnancy. The authors found no consistent effects fo r 

### 5
O 3 over any of the pregnancy periods in single or multi

- pollutant models. Wilhelm and 

### 6
Ritz ( 2005 ) extended previous analyses of PTB ( Ritz et al., 2000 ; Ritz and Yu, 1999 ) in 

### 7
California to include 1994

- 2000. The authors examined varying residential distances 

### 8 from monitoring stations to see if the dist ance affected risk estimation, because effect 

### 9 attenuation may result from local pollutant heterogeneity inadequately captured by 

### 10 ambient monitors. The authors analyzed the association between O 3 exposure during 

### 11 varying periods of pregnancy and PTB, finding a positive association between O 3 levels 

### 12 in both the first trimester of pregnancy (RR=1.23 [95% CI: 1.06, 1.42] per 10 ppb 

### 13
increase in 24

- h avg O 3 ) and the first month of pregnancy (results for first trimester 

### 14 exposure were similar, but slightly smaller, quantitative results not presented) in models 

### 15 containing all pollutants. No association was observed between O 3 in the 6 weeks before 

### 16 birth and preterm delivery. Finally, Ritz et al. ( 2007 ) conducted a case

- co ntrol survey 

### 17 nested within a birth cohort and assessed the extent to which residual confounding and 

### 18 exposure misclassification impacted air pollution effect estimates. The authors calculated 

### 19 mean exposure levels for three gestational periods: the entire pr egnancy, the first 

### 20 trimester, and the last 6 weeks before delivery. Though positive associations were 

### 21
observed for CO and PM 2.5 , no consistent patterns of increase in the odds of PTB for O 3 

### 22
or NO 2 were observed. 

### 23

One study conducted in Canada evaluated the impacts of air pollution (including CO, 

### 24
NO 2 , NO, O 3 , SO 2 , PM 2.5 , and PM 10 ) on PTBs (1999

- 2002) using spatiotemporal 

### 25 residential exposure metrics by month of pregnancy in Vancouver, BC ( Brauer et al., 

### 26
2008 ) . The authors did not observe consistent associations with any of the pregnancy 

### 27 average exposure metrics except for PM 2.5 for PTB. The O 3 associations were largely 

### 28 protective, and no quantitative results were presented for O 3 . Additionally, Lee et al. 

### 29 ( 2008c ) used time

- series techniques to investigate the short

- term associations of O 3 and 

### 30 PTB in London, England. In addition to exposure on the day of birth, cumulative 

### 31 exposure up to 1 week before birth was investigated . The risk of PTB did not increase 

### 32 with exposure to the levels of ambient air pollution experienced by this population. 

### 33

Conversely, studies conducted in Australia and China provide evidence for an association 

### 34
between ambient O 3 and PTB. Hansen et al. ( 2006 ) reported that exposure to O 3 during 

### 35 the first trimester was associated with an increased risk of PTB (OR=1.38, [95% CI: 

### 36 1.14, 1.69] per 10 ppb increase). Although t he test for trend was significant due to the 

### 37 strong effect in the highest quartile, there was not an obvious exposure

- response pattern 

### 38Draft

– Do Not Cite or Quote 7

- 48 September 2011 across the quartiles of O 3 during the first trimester. The effect estimate was diminished 

### 1 and lost statistical significan ce when PM 10 was included in the model (OR=1.23, [95% 

### 2 CI: 0.97, 1.59] per 10 ppb increase). Maternal exposure to O 3 during the 90 days prior to 

### 3 birth showed a weak, positive association with PTB (OR=1.09, [95% CI: 0.85, 1.39] per 

### 4
10 ppb increase). Jalaludi n et al. ( 2007 ) found that O 3 levels in the month and 

### 5
three months preceding birth had a statistically significant association with PTB. Ozone 

### 6 levels in the first trimester of pregnancy were associated with increased risks for PTBs 

### 7 (OR=1.15 [9 5% CI: 1.05, 1.24] per 10 ppb increase in 1

- h max O 3 concentration), and 

### 8 remained a significant predictor of PTB in copollutant models (ORs between 1.07 and 

### 9 1.10). ORs increased for first month of pregnancy when restricted to within 5 km of a 

### 10 monitoring st ation (OR=1.60, [95% CI: 1.27, 2.03]), but did not show a cumulative effect 

### 11
for first 3 months of pregnancy (OR=0.81, [95% CI: 0.67, 0.98]). Jiang et al. ( 2007 ) 

### 12 examined the acute effect of air pollution on PTB, including risk in relation to levels of 

### 13 pollutants for a single day exposure window with lags from 0 to 6 days before birth. An 

### 14
increase of 10 ppb of the 8

- week avg of O 3 corresponded to 9.47 % (95% CI: 0.70, 

### 15 18.7%) increase in PTBs. Increases in PTB were also observed for PM 10 , SO 2 , and NO 2 . 

### 16 The authors did not observe any significant acute effect of outdoor air pollution on PTB 

### 17 among the 1

- day acute time windows examined in the week bef ore birth. 

### 18

Little data is available from toxicological studies; one study reported a nearly statistically 

### 19 significant increase in pregnancy duration in mice when exposed to 0.8 or 1.2 ppm O 3 . 

### 20 This phenomenon was most likely due to the anorexigenic effect of relatively high O 3 

### 21
concentrations ( Bignami et al., 1994 )

. 

### 22Draft

– Do Not Cite or Quote 7

- 49 September 2011 

### Table 7

- 6 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, US (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 Varying 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 City

- wide 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 City

- wide avg and <5 km from monitor First month: 1.604 (1.268, 2.030) b T1: 0.807 (0.668, 0.976) b T3: 1.011 (0.910, 1.124) b Last month: 0.984 (0.906, 1.069) b Jiang et al. ( 2007 ) Shanghai, China (n=3,346 preterm births) 8

- h avg:
32.7 City

- wide avg 4 wks before birth: 1.06 (1.00, 1.12) 6 wks before birth: 1.06 (0.99, 1.13) 8 wks before birth: 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 O3.

b Relative risk of PTB per 1 ppb change in O3. T1 = First Trimester, T2 = Second Trimester, T3 = Third Trimester 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

- 6 provi des a brief overview of the epidemiologic studies of PTB. In summary, 

### 1 the evidence is consistent when examining shorter

- term, late

- pregnancy exposure to O 3 

### 2 and reports no association with PTB. However when long

- term exposure to O 3 early in 

### 3 pregnancy is exa mined the results are inconsistent. Studies conducted in the U.S., 

### 4Draft

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- 50 September 2011 Canada, and England find no association with O 3 and PTB, while studies conducted in 

### 1 Australia and China report an O 3 effect on PTB. 

### 2

7.4.5 Fetal Growth

Low birth weight has often been used as an outcome measure because it is easily 

### 3 available and accurately recorded on birth certificates. However, LBW may result from 

### 4 either short gestation, or inadequate growth in utero. Most of the studies investigating air 

### 5 pollution exposure and LBW limited thei r analyses to term infants to focus on inadequate 

### 6 growth. A number of studies were identified that specifically addressed growth restriction 

### 7 in utero by identifying infants who failed to meet specific growth standards. Usually 

### 8 these infants had birth weigh t less than the 10th percentile for gestational age, using an 

### 9 external standard. Many of these studies have been previously discussed, since they also 

### 10 examined other reproductive outcomes (i.e., LBW or PTB). 

### 11

Fetal growth is influenced by maternal, placenta l, and fetal factors. The biological 

### 12 mechanisms by which air pollutants may influence the developing fetus remain largely 

### 13 unknown. Several mechanisms have been proposed, and are the same as those 

### 14 hypothesized for birth weight (see Section 5. 4.2.4). Additi onally, in animal toxicology 

### 15 studies, O 3 causes transient anorexia in exposed pregnant dams. This may be one of 

### 16 many possible contributors to O 3 - dependent decreased fetal growth. 

### 17

A limitation of environmental studies that use birth weight as a proxy measur e of fetal 

### 18 growth is that patterns of fetal growth during pregnancy cannot be assessed. This is 

### 19 particularly important when investigating pollutant exposures during early pregnancy as 

### 20 birth weight is recorded many months after the exposure period. The insu lt of air 

### 21 pollution may have a transient effect on fetal growth, where growth is hindered at one 

### 22 point in time but catches up at a later point. For example, maternal smoking during 

### 23 pregnancy can alter the growth rate of individual body segments of the fetu s at variable 

### 24 developmental stages, as the fetus experiences selective growth restriction and 

### 25 augmentation ( Lampl and Jeanty, 2003 )

. 

### 26

The terms small

- for

- gestational

- age (SGA), which is defined as a birth weight <10th 

### 27 percentile for gestational age (and often sex and/or race), and intrauterine growth 

### 28 retardation (IUGR) are often used interchangeably. Ho wever, this definition of SGA does 

### 29 have limitations. For example, using it for IUGR may overestimate the percentage of 

### 30

“ growth

- restricted ” neonates as it is unlikely that 10% of neonates have growth 

### 31
restriction ( Wollmann, 1998 ) . On the other hand, when the 10th percentile is based on the 

### 32 distribution of live births at a population level, the percentage of SGA among PTB is 

### 33 most likely underestimated ( Hutcheon and Platt, 2008 ) . Nevertheless, SGA represents a 

### 34Draft

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- 51 September 2011 statistical description of a small neonate, whereas the term IUGR is rese rved for those 

### 1 with clinical evidence of abnormal growth. Thus all IUGR neonates will be SGA, but not 

### 2
all SGA neonates with be IUGR ( Wollmann, 1998 ) . In the following section the terms 

### 3 SGA and IUGR are referred to as each cited study used the terms. 

### 4

Over the past decade a number of studies examined various metrics of fetal growth 

### 5
restriction. Salam et al. ( 2005 ) assessed the effect of increasing O 3 concentrations on 

### 6 IUGR in a population of infants born in California from 1975

- 1987 as part of the 

### 7
Children ’ s Health Study. T he authors reported that maternal O 3 exposures averaged over 

### 8 the entire pregnancy and during the third trimester were associated with increased risk of 

### 9
IUGR. A 10

- ppb difference in 24

- h maternal O 3 exposure during the third trimester 

### 10
increased the risk of IUGR by 11% (95% CI: 0, 20%). Brauer et al. ( 2008 ) evaluated the 

### 11 impacts of air pollution (CO, NO 2 , NO, O 3 , SO 2 , PM 2.5 , PM 10 ) on SGA (1999

- 2002) 

### 12 using spatiotemporal residential exposure metrics by month of pregnancy in Vancouver, 

### 13
BC. The O 3 associations were largely protective (OR= 0.87, [95% CI: 0.81, 0.93] for a 

### 14
10 ppb increase in inverse distance weighted SGA), and no additional quantitative results 

### 15 were presented for O 3 . Liu et al. ( 2007b ) examined the association between IUGR among 

### 16 singleton term live births and SO 2 , NO 2 , CO, O 3 , and PM 2.5 in 3 Canadian cities for the 

### 17 period 1985

- 2000. No increase in the risk of IUGR in relation to exposure to O 3 averaged 

### 18 over each month and trimester of pregnancy was noted. 

### 19

Three studies conducted in Australia p rovide evidence for an association between 

### 20
ambient O 3 and fetal growth restriction. Hansen et al. ( 2007 ) examined SGA among 

### 21 singleton, full

- term births in Brisbane, Australia in relation to ambient air pollution (bsp, 

### 22

PM 10 , NO 2 , O 3 ) during pregnancy. They also examined head ci rcumference and crown



### 23 heel length in a subsample of term neonates. Trimester specific exposures to all pollutants 

### 24 were not statistically significantly associated with a reduction in head circumference or 

### 25 an increased risk of SGA. When monthly

- specific expo sures were examined, the authors 

### 26 observed an increased risk of SGA associated with exposure to O 3 during month 4 

### 27 (OR=1.11 [95% CI: 1.00, 1.24] per 10 ppb increase). In a subsequent study, Hansen et al. 

### 28 ( 2008 ) exami ned the possible associations between fetal ultrasonic measurements and 

### 29 ambient air pollution (PM 10 , O 3 , NO 2 , SO 2 ) during early pregnancy. This study had two 

### 30 strengths: (1) fetal growth was assessed during pregnancy as opposed to at birth; and (2) 

### 31
there wa s little delay between exposures and fetal growth measurements, which reduces 

### 32 potential confounding and uses exposures that are concurrent with the observed growth 

### 33 pattern of the fetus. Fetal ultrasound biometric measurements were recorded for biparietal 

### 34
d iameter (BPD), femur length, abdominal circumference, and head circumference. To 

### 35 further improve exposure assessment, the authors restricted the samples to include only 

### 36 scans from women for whom the centroid of their postcode was within 14 km of an air 

### 37 pol lution monitoring site. Ozone during days 31

- 60 was associated with decreases in all 

### 38Draft

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- 52 September 2011 of the fetal growth measurements, and a 1.78 mm reduction in abdomen circumference 

### 1 per 10 ppb increase in O 3 concentration, though this effect did not persist in copolluta nt 

### 2 models. The change in ultrasound measurements associated with O 3 during days 31

- 60 of 

### 3 gestation indicated that increasing O 3 concentration decreased the magnitude of 

### 4 ultrasound measurements for women living within 2 km of the monitoring site. The 

### 5
relati onship decreased toward the null as the distance from the monitoring sites increased. 

### 6 When assessing effect modification due to SES, there was some evidence of effect 

### 7 modification for most of the associations, with the effects of air pollution stronger in the 

### 8 highest SES quartile. In the third study, Mannes et al. ( 2005 ) estimated the effects of 

### 9 pollutant (PM 10 , PM 2.5 , NO 2 , CO and O 3 ) exposure in the first, second and third 

### 10 trimesters of pregnancy and risk of SGA in Sydney, Australia. Citywide average air 

### 11 pollutant concentrations in the last month, third trimester, and first trimester of pregnancy 

### 12
had no effect on SGA. Concentrations of O 3 in the second trimester of pregnancy had 

### 13 small but adverse effects on SGA (OR=1.10 [95% CI: 1.00, 1.14] per 10 ppb increment). 

### 14 This effect disappeared when the analysis was limited to births with a maternal address 

### 15 within 5 km of a monitoring station (OR=1.00 [95% CI: 0.60, 1.79] per 10 ppb 

### 16 increment). 

### 17

Very little information from toxicological studies is available to address effects on fetal 

### 18 growth. However, there is evidence to suggest that prenatal exposure to O 3 can affect 

### 19 postnatal growth. A few studies reported that mice or rats exposed developmentally 

### 20 (gestationally ± lactationally) to O 3 had deficits in body weight gain in the postpartum 

### 21 period ( Bignami et al., 1994 ; Haro and Paz, 1993 ; Kavlock et al., 1980 )

. 

### 22

Table 7

- 7 provides a brief overview of the epidemiologic studies of fetal growth 

### 23 restriction. In summary, the evidence is inconsistent when exa mining exposure to O 3 and 

### 24 fetal growth restriction. Similar to PTB, studies conducted in Australia have reported an 

### 25
effect of O 3 on fetal growth, whereas studies conducted in other areas have not found 

### 26 such an effect. This may be due to the restriction of births to those within 2

- 14 km of a 

### 27 monitoring station, as was done in the Australian studies. 

### 28Draft

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### Table 7

- 7 Brief summary of epidemiologic studies of fetal growth

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:
2 7.3
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:
31.2 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 City

- wide 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, Australia (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 City

- wide 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
. b Mean change in fetal ultrasonic measure of head circumference recorded between 13 and 26 weeks gestation for a 10

- ppb increase in maternal exposur e 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

Time

- pregnant BALB/c mice were exposed to O 3 (0, 0.4, 0.8, or 1.2 ppm) GD9

- 18 with 

### 1 parturition at GD20

- 21 ( Sharkhuu et al., 2011 )

. As the offspring aged, postnatal litter 

### 2 body weight continued to be significantly decreased in the highest dose (1.2 ppm) O 3 

### 3 group at PND3 and PND7. When the pups were weighed separately by sex at PND42, the 

### 4 males with the highest dose of O 3 exposure (1.2 ppm, GD9

- 18) had significant 

### 5 decrements in body weight ( Sharkhuu et al., 2011 )

. 

### 6Draft

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- 54 September 2011 

### 7.4.7 Birth Defects

Despite the growing body of literature evaluating the assoc iation between ambient air 

### 1 pollution and various adverse birth outcomes, relatively few studies have investigated the 

### 2 effect of temporal variations in ambient air pollution on birth defects. Heart defects and 

### 3 oral clefts have been the focus of the majority of these recent studies, given the higher 

### 4 prevalence than other birth defects and associated mortality. Mechanistically, air 

### 5 pollutants could be involved in the etiology of birth defects via a number of key events 

### 6 (see Section 5. 4.2.4). 

### 7

Several studies have been conducted examining the relationship between O 3 exposure 

### 8 during pregnancy and birth defects and reported a positive association with cardiac 

### 9
defects. The earliest of these studies was conducted in southern California ( Ritz et al., 

### 10
2002 ) . This study evaluated the effect of air pollution on the occurrence of cardiac birth 

### 11
defects in neonates and fetuses delivered in southern California in 1987

- 1993. Maternal 

### 12 exposure estimates were based on data from the fixed site closest to the mother ’ s ZIP 

### 13 code area. When using a case

- control design where cases were matched to 10 randomly 

### 14 selected controls, results showed increased risks for aortic artery and valve defects 

### 15 (OR=1.56 [95% CI: 1.16, 2.09] per 10 ppb O 3 ), pulmonary artery and va lve anomalies 

### 16 (OR=1.34 [95% CI: 0.96, 1.87] per 10 ppb O 3 ), and conotruncal defects (OR=1.36 [95% 

### 17 CI: 0.91, 2.03] per 10 ppb O 3 ) in a dose

- response manner with second

- month O 3 

### 18

exposure. A study conducted in Texas ( Gilboa et al., 2005 ) looked at a similar period of 

### 19 exposure but reported no association with most of the birth defects studied (O 3 

### 20
concentration was studied u sing quartiles with the lowest representing <18 ppb and the 

### 21 highest representing ≥ 31 ppb). The authors found slightly elevated odds ratios for 

### 22 pulmonary artery and valve defects. They also detected an inverse association between 

### 23
O 3 exposure and isolated v entricular septal defects. Overall, this study provided some 

### 24 weak evidence that air pollution increases the risk of cardiac defects. Hansen et al. ( 2009 ) 

### 25 investigated the possible association between ambient air pollution and the risk of cardiac 

### 26
defects. When analyzing all births with exposure estimates for O 3 from the nearest 

### 27
monitor there was no indication for an association with cardiac defects. There was also 

### 28 no adverse association when restricting the analyses to only include births where the 

### 29 mother resided within 12 km of a monitoring station. However, among births within 6 km 

### 30 of a monitor, a 10 ppb increase in O 3 was associated with an increased risk of pulmonary 

### 31 artery and valve defects (OR=8.76 [95% CI: 1.80, 56.55] ). As indicated by the very wide 

### 32 credible intervals, there were very few cases in the sensitivity analyses for births within 6 

### 33 km of a monitor, and this effect could be a result of type I errors. Dadvand et al. ( 2011 ) 

### 34 investigated the association between maternal exposure to ambient air pollution and the 

### 35 occurrence of cardiac birth defects in England. Similar to Hansen et al. ( 2009 ) , they 

### 36 found no associations with maternal exposure to O 3 except for when the analysis was 

### 37Draft

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- 55 September 2011 limited to those subjects residing within a 16 km distance of a monitoring station (OR for 

### 1 malformations of pulmonary and tr icuspid valves=1.64 [95% CI: 1.04, 2.60] per 10 ppb 

### 2
increase in O 3 ). 

### 3

Despite the association between O 3 and cardiac defects observed in the above studies, a 

### 4 recent study did not observe an increased risk of cardiac birth defects associated with 

### 5
ambient O 3 concentrations. The study, conducted in Atlanta, GA, examined O 3 exposure 

### 6 during the third through seventh week of pregnancy and reported no association with risk 

### 7 of cardiovascular malformations (mean long

- term average of 8

- h O 3 concentrations 

### 8 excluding No vember through February ranged by 5

- year groups from 39.8 to 43.3 ppb) 

### 9 ( Strickland et al., 2009 )

. 

### 10

Several of these studies have also examined the relationship between O 3 exposure during 

### 11 pregnancy and oral cleft defects. The study by Ritz et al. ( Ritz et al., 2002 ) evaluated the 

### 12 effect of air pollution on the occurrence of orofacial birth defects and did not observe 

### 13 strong associations between ambient O 3 concentration and orofacial defects. They did 

### 14 report an OR of 1.13 (95% CI: 0.90, 1.40) per 10 ppb during the second trimester for cleft 

### 15 lip with or without cleft palate. Similarly, Gilboa et al. ( Gilboa et al., 2005 ) reported and 

### 16 OR of 1.09 (95% CI: 0.70, 1.69) for oral cleft defects when the fourth quartile was 

### 17 contrasted with the first quartile of exposure during 3

- 8 weeks of pregnancy. Hansen et 

### 18
al. ( 2009 ) reported no indication for an association with cleft defects. Hwang and Jaakola 

### 19 ( 2008 ) conducted a population

- based case

- control study to investigate exposure to 

### 20 ambient air pollution and the risk of cleft lip with or without cleft palate in Taiwan. The 

### 21 risk of cleft lip wi th or without cleft palate was increased in relation to O 3 levels in the 

### 22 first gestational month (OR=1.17 [95% CI: 1.01, 1.36] per 10 ppb) and second gestational 

### 23 month (OR=1.22 [95% CI: 1.03, 1.46] per 10 ppb), but was not related to any of the other 

### 24 pollu tants. In three

- pollutant models, the effect estimates for O 3 exposure were stable for 

### 25 the four different combinations of pollutants and were all statistically significant. 

### 26
Marshall et al. ( 2010 ) compared estimated exposure to ambient pollutants during early 

### 27 pregnancy among mothers of children with oral cleft defects to that among mothers of 

### 28 controls. The authors observed no consistent elevated associations between any of the air 

### 29 pol lutants examined and cleft malformations, though there was a weak association 

### 30 between cases of cleft palate only and increasing O 3 concentrations. This association 

### 31
increased when cases and controls were limited to those with residences within 10 km of 

### 32
the closest O 3 monitor (OR=2.2 [95% CI: 1.0, 4.9], comparing highest quartile [>33 ppb] 

### 33 to lowest quartile [<15 ppb]). 

### 34

A limited number of toxicological studies have examined birth defects in animals 

### 35 exposed gestationally to O 3 . Kavlock et al. ( 1979 ) exposed pregnant rats to O 3 for precise 

### 36 periods during organogenesis. No significant teratogenic effects were found in rats 

### 37Draft

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- 56 September 2011 exposed 8 hr/day to concentrations of O 3 varying from 0.44 to 1.97 ppm during early 

### 1 (days 6

- 9), mid (days 9

- 12), or late (days 17 to 20) gestation, or the entire period of 

### 2 organogenesis (days 6

- 15). Earlier research f ound eyelid malformation following 

### 3 gestational and postnatal exposure to 0.2 ppm O 3 ( Veninga, 1967 )

. 

### 4

Table 7

- 8 provides a brief overview of the epidemiologic studies of birth defects. These 

### 5 studies have focused on cardiac and oral cleft defects, and the results from these studies 

### 6 are not entirely consistent. This inconsistency could be due to the absence of true 

### 7

a ssociations between O 3 and risks of cardiovascular malformations and oral cleft defects; 

### 8 it could also be due to differences in populations, pollution levels, outcome definitions, or 

### 9 analytical approaches. The lack of consistency of associations between O 3 and 

### 10 cardiovascular malformations or oral cleft defects might be due to issues relating to 

### 11 statistical power or measurement error. A recent meta

- analysis of air pollution and 

### 12 congenital anomalies concluded that there was no statistically significant increa se in risk 

### 13 of congenital anomalies and O 3 ( Vrijheid et al., 2011 )

. These authors note that 

### 14 heterogeneity in the results of these studies may be due to inherent differences in study 

### 15 location, study design , and/or analytic methods, and comment that these studies have not 

### 16 employed some recent advances in exposure assessment used in other areas of air 

### 17 pollution research that may help refine or reduce this heterogeneity. 

### 18

Table 7

- 8 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 ) Cardiac 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. ( 2005 ) 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 Jaakola ( 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 City

- wide avg Weeks 3

- 7 of gestation

Hansen et al. ( 2009 ) Cardia c 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 Defe cts Northeast England (n=2,140 cases; 14,256 controls) 24

- h avg:
18.8 Nearest Monitor Weeks 3

- 8 of gestation`1Draft

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- 57 September 2011 

### 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
Gro wth 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 emergi ng issue. Several studies were included in Sections 7.2.1 and 7.2.3, and are 

### 7 included here because they reported both prenatal and post

- natal exposure periods. 

### 8

Mortimer et al. ( 2008a

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

### 9 air pollutants with pulmonary function and allergen sensitization in a subset of asthmatic 

### 10 children (ages 6

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

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

- 2000 were created and 

### 12 averaged separately across several important developmental time

- periods, including the 

### 13 entire pregnancy, each trimester, the first 3 years of life, the first 6 years o f life, and the 

### 14
entire lifetime. The 8

- h avg O 3 concentrations were approximately 50 ppb for each of the 

### 15 exposure metrics (estimated from figure). In the first analysis ( Mortimer et al., 2008a )

, 

### 16 negative effects on pulmonary function were found for exposure to PM 10 , NO 2 , and CO 

### 17 during key neonatal and early life developmental periods. The authors did not find a 

### 18 negative effect of exposure to O 3 among this cohort. In the second analysis ( Mort imer et 

### 19 al., 2008b ) , sensitization to at least one allergen was associated, in general, with higher 

### 20
levels of CO and PM 10 during the entire pregnancy and second trimester and higher PM 10 

### 21 during the first 2 years of life. Lower exposure to O 3 during the entire pregnancy or 

### 22 second trimester was associated with an increased risk of allergen sensitization. Although 

### 23 the pollutant metrics across time periods are correlated, the strongest associations with 

### 24 the outcomes were observed for prenatal exposures. Thou gh it may be difficult to 

### 25 disentangle the effect of prenatal and postnatal exposures, the models from this group of 

### 26 studies suggest that each time period of exposure may contribute independently to 

### 27
different dimensions of school

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

### 28 pulmonary

- function measures (FVC, FEV 1 , PEF, FEF 25

- 75 ), prenatal exposures were 

### 29 more influential on pulmonary function than early

- lifetime metrics, while, in contrast, the 

### 30 ratio of measures (FEV 1 /FVC and FEF 25

- 75 /FVC) were most infl uenced by postnatal 

### 31 exposures. When lifetime metrics were considered alone, or in combination with the 

### 32 prenatal metrics, the lifetime measures were not associated with any of the outcomes, 

### 33 suggesting the timing of the exposure may be more important than th e overall dose and 

### 34 prenatal exposures are not just markers for lifetime or current exposures. 

### 35Draft

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- 58 September 2011 Clark et al. ( 2010 ) investigated the effect of exposure to ambient air pollution in utero 

### 1 and during the first year of life on risk of subsequent asthma diagnosis (incident asthma 

### 2 diagnosis up to age 3

- 4) in a population

- based nested case

- control study. Air pollution 

### 3 exposure for each subject based on their residential address history was estimated using 

### 4 regulatory monitoring data, land use regression modeling, and proximity to stationary 

### 5 pollution sources. An average exposure was calculated for the duration of pregnancy 

### 6 (~15 ppb) and the first year of life (~14 ppb). In contrast to the Mortimer et al. studies 

### 7 ( 2008a

, b ) , the effect estimates for first

- year exposure were generally larger than for in 

### 8 utero exposures. However, similar to the Mortimer et al. studies, the observed 

### 9
associations with O 3 were largely protective. Because of the relatively high correlation 

### 10
between in utero and first

- year exposures for many pollutants, it was difficult to discern 

### 11 the relative importance of the individual exposure periods. 

### 12

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

### 13 with lung function changes in the newborn. Tidal breathing, lung volume, ventilation 

### 14 inhomogeneity and eNO were measured in 241 unsedated, sleeping neonates (age= 

### 15
5 weeks). The median of the 24

- h avg O 3 concentrations averaged across the post

- natal 

### 16 period was ~44 ppb. Consistent with the previous studies, no association was found for 

### 17 prenatal exposure to O 3 and lung function. 

### 18

The new toxicological literature since the 2006 O 3 AQCD, covering respiratory changes 

### 19 related to developmental O 3 exposure, reports ultrastructural changes in bronchiole 

### 20 development, alterations in placental and pup cytokines, and increased pup airway hyper



### 21 reactivity. These studies are detailed below. Older studies are discussed wh ere new 

### 22
information is not available. 

### 23

Fetal rat lung bronchiole development is triphasic, comprised of the glandular phase 

### 24 (measured at GD18), the canalicular phase (GD20), and the saccular phase (GD21). The 

### 25 ultrastructural lung development in fetuses of pregnant rats exposed to 1

- ppm O 3 (12 

### 26 h/day, out to either GD18, GD20 or GD21) was examined by electron microscopy during 

### 27 these three phases. In the glandular phase, bronchiolar columnar epithelial cells in fetuses 

### 28 of dams exposed to O 3 had cytoplasmic dam age and swollen mitochondria. Bronchial 

### 29 epithelium at the canalicular phase in O 3 exposed pups had delayed maturation in 

### 30 differentiation, i.e., glycogen abundance in secretory cells had not diminished as it should 

### 31 with this phase of development. Congruent with this finding, delayed maturation of 

### 32 tracheal epithelium following early neonatal O 3 exposure (1 ppm, 4

- 5 h/day for 

### 33
first week of life) in lambs has been previously reported ( Mariassy et al. , 1990 ; Mariassy 

### 34 et al., 1989 ) . Also at the canalicular phase, atypical cells were seen in the bronchiolar 

### 35
lumen of O 3 exposed rat fetuses. Finally, in the saccular phase, mitochondrial 

### 36 degradation was present in the non

- ciliated bronchiolar cells of rats exposed in utero to 

### 37Draft

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- 59 September 2011 O 3 . In conclusion, O 3 exposure of pregnant rats produced ultra

- structural damage to near



### 1
term feta l bronchiolar epithelium ( López et al., 2008 )

. 

### 2

Exposure of laboratory animals to multiple airborne pollutants can differentially affect 

### 3 pup physiology. One study showed that exposure of C57BL/6 mouse dams to 0.48 mg 

### 4 PM intratracheally twice weekly for 3 weeks during pregnancy augmented O 3 - induced 

### 5 airway hyper

- reactivity in juvenile offspring. Maternal PM exposure also significantly 

### 6 increased placental cytokines above vehicle

- instilled controls. Pup postnatal O 3 exposure 

### 7 (1 ppm 3 h/day, every other day, thrice w eekly for 4 weeks) induced significantly 

### 8 increased cytokine levels (IL

- 1β, TNF

- α, KC, and IL

- 6) in whole lung versus postnatal 

### 9 air exposed groups; this was further exacerbated with gestational PM exposure ( Auten et 

### 10 al., 2009 )

. 

### 11

A series of experiments using infant rhesus monkeys repeatedly exposed to 0.5 ppm O 3 

### 12 starting at one

- month of age have examined the eff ect of O 3 alone or in combination with 

### 13 an inhaled allergen on morphology and lung function ( Plopper et al., 2007 ) . Exposure to 

### 14
O 3 alone or allergen alone produced small but not statistically signi ficant changes in 

### 15 baseline airway resistance and airway responsiveness, but the combined exposure to both 

### 16
O 3 + antigen produced statistically significant and greater than additive changes in both 

### 17 functional measurements. Additionally, cellular changes and significant structural 

### 18 changes in the respiratory tract have been observed in infant rhesus monkeys exposed to 

### 19
O 3 ( Fanucchi et al., 2006 )

. A more detailed description of these studies can be found in 

### 20
Section 7.2.3 (Pulmonary Structure and Function), with mechanistic information found in 

### 21
Section 5.4.2.4. 

### 22

Lung immunological response in O 3 exposed pups was followed by analyzing BAL and 

### 23 lung tissue. Sprague Dawley (SD) pups were exposed to a single 3h exposure of air or O 3 

### 24 (0.6 ppm) on PND 13 ( Han et al., 2011 )

. Bronchoalveolar lavage (BAL) was performed 

### 25
10 hours after the end of O 3 exposure. BALF polymorphonuclear leukocytes (PMNs) and 

### 26 total BALF protein were significantly elevated in O 3 exposed pu ps. Lung tissue from O 3 

### 27 exposed pups had significant elevations of manganese superoxide dismutase (SOD) 

### 28 protein and significant decrements of extra

- cellular SOD protein. 

### 29

Various immunological outcomes were followed in offspring after their pregnant dams 

### 30 (B ALB/c mice) were exposed gestationally to O 3 (0, 0.4, 0.8, or 1.2 ppm, GD9

- 18) 

### 31 ( Sharkhuu et al., 2011 ) . Delayed type hypersensitivity (DTH) was initiated with initial 

### 32 BSA injection at 6 weeks of age and then challenge 7 days later. The normal edematous 

### 33

response o f the exposed footpad (thickness after BSA injection) was recorded as an 

### 34
indicator of DTH. In female offspring, normal footpad swelling with BSA injection that 

### 35 was seen in air exposed animals was significantly attenuated with O 3 exposure (0.8 and 

### 36 1.2 ppm O 3 ), implying immune suppression of O 3 exposure specifically in DTH. 

### 37Draft

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- 60 September 2011 Humoral immunity was measured with the sheep red blood cell (SRBC) response. 

### 1 Animals received primary immunization with SRBC and then blood was drawn for 

### 2 SRBC IgM measurement. A SRBC boost er was given 2 weeks later with blood collected 

### 3 5 days after booster for IgG measurement. Maternal O 3 exposure had no effect on 

### 4 humoral immunity in the offspring as measured by IgG and IgM titers after SRBC 

### 5 primary and booster immunizations ( Sharkhuu et al., 2011 )

. 

### 6

Toxicity assessment and allergen sensitization was also assessed in these O 3 exposed 

### 7 offspring. At PND42, animals were euthanized for analysis of immune and inflammatory 

### 8 markers (immune proteins, inflammatory cells, T cell populations in the spleen). A subset 

### 9
of the animals was intra

- nasally instilled or sensitized with ovalbumin on either PND2 

### 10
and 3 or PND42 and 43. All animals were challenged with OVA on PND54, 55, and 56. 

### 11 One day after final OVA challenge, lung function, lung inflammation and immune 

### 12 response were determined. Offspring of O 3 exposed dams that were initially sensitized at 

### 13 PDN3 (early) or PND42 (late) were tested to determine the level of allergic sensitization 

### 14
or asthma

- like inflammation after OVA challenge. Female offspring sensitized early in 

### 15 life developed significant eosinophilia (1.2 ppm O 3 ) and elevated serum OVA

- specific 

### 16 IgE (1.2 ppm O 3 ), which is a marker of airway allergic inflammation. The females that 

### 17 were sensitized early also had significant decrements in BALF total cells, m acrophages, 

### 18 and lymphocytes (1.2 ppm O 3 ). Offspring that were sensitized later (PND42) in life did 

### 19 not develop the aforementioned changes in BALF, but these animals did develop modest, 

### 20 albeit significant neutropenia (0.8 and 1.2 ppm O 3 ) ( Sharkhuu et al., 2011 )

. 

### 21

BALF cytology in non

- sensitized animals was followed. BALF of offspring born to dams 

### 22 exposed to O 3 was relatively unaffected (cytokines, inflammatory cell numbers/types) as 

### 23 were splenic T cell subpopulations. LDH was significantly elevated in BALF of femal es 

### 24 whose mothers were exposed to 1.2 ppm during pregnancy ( Sharkhuu et al., 2011 )

. In 

### 25 summary, the females born to mothers exposed to O 3 developed modest 

### 26 immunocompromise. Males were unaffected ( Sharkhuu et al., 2011 )

. 

### 27

Overall, animal toxicological studies have reporte d ultrastructural changes in bronchiole 

### 28 development, alterations in placental and pup cytokines, and increased pup airway hyper



### 29 reactivity related to exposure to O 3 during the developmental period. Epidemiologic 

### 30 studies have found no association between pr enatal exposure to O 3 and growth and 

### 31 development of the respiratory system. Fetal origins of disease have received a lot of 

### 32 attention recently, thus additional research to further explore the inconsistencies between 

### 33
these two lines of evidence is warranted

. 

### 34Draft

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### 7.4.9 Developmental Central Nervous 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 rep orts 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

expose d controls (clockwise turns) ( Dell'Omo et a l., 1995 ) ; in previous studies this 

### 7 behavior in mice has been found to correlate with specific structural asymmetries of the 

### 8 hippocampal mossy fiber 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 

### 10
to O 3 during fetal and neonatal life showed limited, gender

- specific changes in 

### 11 handedness after exposu re to the intermediate dose of O 3 (only seen in female mice 

### 12 exposed 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 

### 13 with 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 solitarius (NTS), a medullary area of respiratory control, of adult 

### 15 animals exposed prenatally to 0.5 ppm O 3 (12h/day, ED5

- ED20) had significantly 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 restrained. 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 

### 26 offspring outcomes (i.e., dam behavioral or lactational contributions to pup outcomes) 

### 27 ( Boussouar et al., 2009 )

. 

### 28Draft

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- 62 September 2011 Developmental exposure to 0.3 or 0. 6 ppm O 3 prior to mating pair formation through 

### 1 GD17 induced significant 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 significantly decreased l evel 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 include 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 outco mes 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 the majority of pregnancy (PD7

- 17) of 

### 15
CD

- 1 mice did not affect pup behavioral outcomes including early behavioral ultrasonic 

### 16
vocalizations and mo re permanent later measurements (PND60 or 61) including pup 

### 17 activity, habituation and exploration and d

- amphetamine

- 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 significantly increased 

### 21 defensive/ submissive behavior (increased freezing posturing on the first day only of a 

### 22 multiple

- day exam) versus air expos ed controls ( Santucci et al., 2006 ) . Similar to this and 

### 23 as reported in previous AQCDs, continuous exposure of adult animals to O 3 induced 

### 24 significant increases in fear behavior and decreased aggression as measured by 

### 25 significantly decreased freezing behavior ( Petruzzi et al., 1995 )

. 

### 26

Developmentally exposed animals also had significantly decreased amount of time spent 

### 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 specific 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. 

### 33Draft

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- 63 September 2011 

### 7.4.9.4 Sleep Aberrations a fter Developmental Ozone Exposure

The effect of gestational O 3 exposure (1 ppm O 3 , 12h/day, during dark period) on sleep 

### 1 patterns in rat offspring was followed using 24 h polysomnographic recordings at 30, 60 

### 2
and 90 days of age ( Haro and Paz, 1993 ) . Ozone

- exposed pups manifested with inverted 

### 3 sleep

- wake patterns or circadian rhythm phase

- shift. Rat vigilance was characterized in 

### 4 wakefulness, slow wave sleep (SWS), and paradoxical sleep (PS) using previous ly 

### 5
characterized criteria. The O 3 exposed offspring spent longer time in the wakefulness 

### 6 state during the light period, more time in SWS during the period of darkness, and 

### 7 showed significant decrements in PS. Chronic O 3 inhalation significantly decreased t he 

### 8 duration of PS during both the light and dark periods ( Haro and Paz, 1993 ) . These effects 

### 9 were consistent at all time periods measured (30, 60 and 90 days of age). These sleep 

### 10 effects reported after d evelopmental exposures expand upon the existing literature on 

### 11 sleep aberrations in adult animals exposed to O 3 [rodents: ( Paz and Huitron

- Resendiz, 

### 12
1996 ; Arito et al., 1992 ) ; and cats: ( Paz and Bazan

- Perkins, 1992 ) ]. A role for inhibition 

### 13 of cyclooxygenase

- 2 and the interleukins and prostaglandins in the O 3 - dependent sleep 

### 14 changes potentially exists with evidence from a publication on indomethacin pretreatment 

### 15 attenuating O 3 - induced sleep aberrations in adult male animals ( Rubio and Paz, 2003 )

. 

### 16

7.4.10 Early Life Mortality

Infa nts may be particularly susceptible to the adverse effects of air pollution. Within the 

### 17
first year of life, infants develop rapidly; therefore their susceptibility may change within 

### 18 weeks or months. During the neonatal and post

- neonatal periods, the develo ping lung is 

### 19 highly susceptible to environmental toxicants. The lung is not well developed at birth, 

### 20 with 80% of alveoli being formed postnatally. An important question regarding the 

### 21
association between O 3 and infant mortality is the critical window of exp osure during 

### 22 development for which infants are susceptible. Several age intervals have been explored: 

### 23 neonatal (<1 month); postneonatal (1 month to 1 year); and an overall interval for infants 

### 24 that includes both the neonatal and postneonatal periods (<1 ye ar). Within these various 

### 25 age categories, multiple causes of deaths have been investigated, particularly total deaths 

### 26 and respiratory

- related deaths. The studies reflect a variety of study designs, exposure 

### 27 periods, regions, and adjustment for confounders. As discussed below, a handful of 

### 28 studies have examined the effect of ambient air pollution on neonatal and postneonatal 

### 29 mortality, with the former the least studied. These studies varied somewhat with regard to 

### 30 the outcomes and exposure periods examined a nd study designs employed. 

### 31

A major issue in studying environmental exposures and infant mortality is selecting the 

### 32 relevant exposure period, since the biological mechanisms leading to death and the 

### 33Draft

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- 64 September 2011 critical periods of exposure are poorly understood. Both s hort

- term (days to weeks) and 

### 1 long

- term (months to years) exposure studies are included in this section and are 

### 2 characterized accordingly in the text and tables. All studies of infant mortality are 

### 3 included in the Reproductive and Developmental Effects sec tion, as opposed to the 

### 4
sections devoted to all

- and cause

- specific mortality, because infant development 

### 5 processes, much like fetal development processes, may be particularly susceptible to O 3 - 

### 6
induced health effects. Exposures proximate to the death may b e most relevant if 

### 7 exposure causes an acute effect. However, exposure occurring in early life might affect 

### 8 critical growth and development, with results observable later in the first year of life, or 

### 9 cumulative exposure during the first year of life may be the most important determinant. 

### 10 The studies reviewed below have dealt with this issue in different ways. Many have 

### 11 considered several exposure metrics based on different periods of exposure. 

### 12

7.4.10.1 Stillbirth

Pereira et al. ( 1998 ) investigated the association among daily counts of intrauterine 

### 13 mortality (over 28 weeks of gestation) and air pollutant concentrations in Sao Paulo, 

### 14 Brazil from 1991 through 1992. The association was strong for NO 2 , but lesser for SO 2 

### 15 and CO. These associations exhibited a short lag time, less than 5 days. No significant 

### 16
association was detected between short

- term O 3 exposure and intrauterine mortality. 

### 17

7.4.10.2 Infant Mortality, Less than 1 Year

Ritz et al. ( 2006 ) linked birth and death certi ficates for infants who died between 1989 

### 18 and 2000 to evaluate the influence of outdoor air pollution on infant death in the South 

### 19
Coast Air Basin of California. The authors examined short

- and long

- term exposure 

### 20 periods 2 weeks, 1 month, 2 months, and 6 m onths before each case subject ’ s death and 

### 21 reported no association between ambient levels of O 3 and infant mortality. Similarly, 

### 22
Diaz et al. ( 2004 ) a nalyzed the effects of extreme temperatures and short

- term exposure 

### 23 to air pollutants on daily mortality in children less than 1 year of age in Madrid, Spain, 

### 24 from 1986 to 1997 and observed no statistically significant association between mortality 

### 25
and O 3 concentrations. Hajat et al. ( 2007 ) analyzed time

- series data of daily infant 

### 26 mortality counts in 10 major cities in the UK to quantify any associations with short

- term 

### 27 changes in air pollution. When the results from the 10 cities were combined there was no 

### 28 relationship between O 3 and infant mortality, even after restricting the analysis to just the 

### 29
summer months. 

### 30Draft

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- 65 September 2011 Conversely, a time

- series study of infant mortality conducted in the southwestern part of 

### 1
Mexico Ci ty in the years 1993

- 1995 found that infant mortality was associated with 

### 2
short

- term exposure to NO 2 and O 3 3

- 5 days before death, but not as consistently as with 

### 3
PM. A 10

- ppb increase in 24

- h avg O 3 was associated with a 2.78% increase (95% CI: 

### 4 0.29, 5.26%) in infant mortality (lag 3) ( Loomis et al., 1999 ) . This increase was 

### 5 attenuated, although still positive when evaluated in a two

- pollutant model with PM 2.5 . 

### 6
One

- hour max concentrations of O 3 exceeded prevailing Mexican and international 

### 7 standards nearly every day. 

### 8

7.4.10.3 Neonatal Morta lity, Less than 1 Month

Several studies have evaluated ambient O 3 concentrations and neonatal mortality and 

### 9
observed no association. Ritz et al. ( 2006 ) lin ked birth and death certificates for infants 

### 10 who died between 1989 and 2000 to evaluate the influence of outdoor air pollution on 

### 11
infant death in the South Coast Air Basin of California. The authors examined short

- and 

### 12 long

- term exposure periods 2 weeks, 1 month, 2 months, and 6 months before each case 

### 13 subject ’ s death and reported no association between ambient levels of O 3 and neonatal 

### 14 mortality. Hajat et al. ( 2007 ) analyzed time

- series data of daily infant mort ality counts in 

### 15 10 major cities in the UK to quantify any associations with short

- term changes in air 

### 16 pollution. When the results from the 10 cities were combined there was no relationship 

### 17
between O 3 and neonatal mortality, even after restricting the analy sis to just the summer 

### 18
months. Lin et al. ( 2004a ) assessed the impact of short

- term changes in air pollutants on 

### 19 the number of daily neonatal deaths in Sao Paulo, Brazil. The authors observed no 

### 20
association between ambient levels of O 3 and neonatal mortality. 

### 21

7.4.10.4 Postneonatal Mortality, 1 Month to 1 Year

A number of studies focused on the postneonatal period when examin ing the effects of 

### 22
O 3 on infant mortality. Ritz et al. ( 2006 ) linked birth and death certificates for infants 

### 23
who died between 1989 and 2000 to evaluate th e influence of outdoor air pollution on 

### 24
infant death in the South Coast Air Basin of California. The authors examined short

- and 

### 25 long

- term exposure periods 2 weeks, 1 month, 2 months, and 6 months before each case 

### 26 subject ’ s death and reported no associatio n between ambient levels of O 3 and 

### 27 postneonatal mortality. Woodruff et al. ( 2008 ) evaluated the county

- level relationship 

### 28
between cause

- specific postneonatal infant mortality and long

- term early

- life exposure 

### 29 (first 2 months of life) to air pollutants across the U.S. Similarly, they found no 

### 30
association between O 3 exposure and deaths f rom respiratory causes. In the U.K., Hajat 

### 31
et al. ( 2007 ) analyzed time

- series data of daily infant mortality counts in 10 major cities 

### 32Draft

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- 66 September 2011 to quantify any associations with short

- term changes in air pollution. When the results 

### 1 from the 10 cities were combined there was no relationship between O 3 and postneonatal 

### 2 mortality, even after restricting the analysis to just the summer months. In Ciudad Juarez, 

### 3 Mexico, Romieu et al. ( 2004b ) examined the daily number of deaths bet ween 1997 and 

### 4 2001, estimating the modifying effect of SES on the risk of postneonatal mortality. 

### 5
Ambient O 3 concentrations were not related to infant mortality overall, or in any of the 

### 6 SES groups. In a follow

- up study, Carbajal

- Arroyo ( 2011 ) evaluated the relationship of 

### 7
1

- h daily max O 3 levels with postneonatal infant mortality in the Mexico City 

### 8 Metropolitan Area between 1997 and 2005. Generally, short

- term exposure to O 3 was not 

### 9 significantly related to infant mortality. Howe ver, upon estimating the modifying effect 

### 10 of SES on the risk of postneonatal mortality, the authors found that O 3 was statistically 

### 11 significantly related to respiratory mortality among those with low SES. In a separate 

### 12 analysis, the effect of PM 10 was eval uated with O 3 level quartiles. PM 10 alone was related 

### 13 to a significant increase in all

- cause mortality. The magnitude of this effect remained the 

### 14 same when only the days when O 3 was in the lowest quartile were included in the 

### 15 analyses. However, when only t he days when O 3 was in the highest quartile were 

### 16 included in the analyses, the magnitude of the PM 10 effect increased dramatically 

### 17 (OR=1.06 [95% CI: 0.909, 1.241] for PM 10 on days with O 3 in lowest quartile; OR=1.26 

### 18 [95% CI: 1.08, 1.47] for PM 10 on days wi th O 3 in the highest quartile. These results 

### 19 suggest that while O 3 alone may not have an effect on infant mortality, it may serve to 

### 20 potentiate the observed effect of PM 10 on infant mortality. 

### 21

Tsai et al. ( 2006 ) used a case

- crossover analysis to examine the relationship between 

### 22
short

- term exposure to air pollution and postneonatal mortality in Kaohsiung, Taiwan 

### 23 during the period 1994

- 2000. The risk of postneonatal deaths was 1.023 (95% CI: 0.564, 

### 24 1.858) per 10

- ppb increase in 24

- h avg O 3 . The confidence interval for this effect 

### 25
estima te is very wide, likely due to the small number of infants that died each day, 

### 26 making it difficult to interpret this result. Several other studies conducted in Asia did not 

### 27 find any association between O 3 concentrations and infant mortality in the postneon atal 

### 28 period. Ha et al. ( 2003 ) conducted a daily time

- series study in Seoul, Korea to evaluate 

### 29
the effect of short

- term changes in ambient 8

- h O 3 concentrations on postneonatal 

### 30 mortality. Son et al. ( 2008 ) examined the relationship bet ween air pollution and 

### 31 postneonatal mortality from all causes among firstborn infants in Seoul, Korea during 

### 32
1999

- 2003. Yang et al. ( 2006 ) used a case

- crossover analysis to examine the relationship 

### 33 between air pollution exposure and postneonatal mortality in Taipei, Taiwan for the 

### 34 period 1994

- 2000. The authors observed no associations between ambient levels o f O 3 

### 35 and postneonatal mortality. 

### 36Draft

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- 67 September 2011 

### 7.4.10.5 Sudden Infant Death Syndrome

The strongest evidence for an association between ambient O 3 concentrations and SIDS 

### 1 comes from a study that evaluated the county

- level relationship between SIDS and long



### 2 term early

- life exposu re (first 2 months of life) to air pollutants across the U.S. ( Woodruff 

### 3 et al., 2008 ) . The authors observed a 1.20 (95% CI: 1.09, 1.32) odds ratio for a 10

- ppb 

### 4
increase in O 3 and deaths from SIDS. There was a monotonic increase in odds of SIDS 

### 5
for each quartile of O 3 exposure compared with the lowest quartile (highest quartile 

### 6 OR=1.51; [95% CI: 1.17, 1.96]). In a multi

- pollutant model including PM 10 or PM 2.5 , CO 

### 7
and SO 2 , the OR for SIDS and O 3 was not substantially lower than that found in the 

### 8 sing le

- pollutant model. When examined by season, the relationship between SIDS deaths 

### 9
and O 3 was generally consistent across seasons with a slight increase for those babies 

### 10 born in the summer. When stratified by birth weight, the OR for LBW babies was 1.27 

### 11 (95 % CI: 0.95, 1.69) per 10

- ppb increase in O 3 and the OR for normal weight babies was 

### 12 1.16 (95% CI: 1.01, 1.32) per 10

- ppb increase in O 3 . 

### 13

Conversely, two additional studies reported no association between ambient levels of O 3 

### 14
and SIDS. Ritz et al. ( 2006 ) linked birth and death certificates for infants who died 

### 15 between 1989 and 2000 to evaluate the influence of outdoor air pollution on infant death 

### 16
in the Sou th Coast Air Basin of California. The authors examined short

- and long

- term 

### 17 exposure periods 2 weeks, 1 month, 2 months, and 6 months before each case subject ’ s 

### 18 death and reported no association between ambient levels of O 3 and SIDS. Dales et al. 

### 19 ( 2004 ) used time

- series analyses to compare the daily mortality rates for SIDS and short



### 20 term air pollution con centrations in 12 Canadian cities during the period of 1984

- 1999. 

### 21 Increased daily rates of SIDS were associated with previous day increases in the levels of 

### 22
SO 2 , NO 2 , and CO, but not O 3 or PM 2.5 . 

### 23

Table 7

- 9 provides a brief overview of the epidemiologic studies of infant mortality. 

### 24
These studies have focused on short

- term exposure windows (e.g., 1

- 3 days) and long



### 25 term exposure windows (e.g., up to 6 months). Collectively, they provide no evidence for 

### 26
an association between ambient O 3 concentrations and infant mortality. 

### 27Draft

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- 68 September 2011 

### Table 7

- 9 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 wk 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 et 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. ( 2004b ) 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) 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, US 24

- h avg: 26.6 County wide avg First 2 mo of life: 1.04 (0.98, 1.10) Yang et al. ( 2006 ) T aipei, Taiwan 24

- h avg: 18.14 Citywide avg L0

- 2 cum:1.00 (0.62, 1.61)
Dales et al. ( 2004 ) 12 Canadi an cities 24

- h: 31.77 Citywide avg L0: NR
L1: NR
L2: NR
L3: NR
L4: 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 = La g 5, L6 = Lag 6 NR: No quantitative results reportedDraft

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- 69 September 2011 

### Table 7

- 10 Summary of Key Reproductive and Developmental Toxicological Studies

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

- I mpaired delayed type hypersensitivity.4

- No effect on humoral immunity. 5

- Significantly affected allergic airway inflammation markers (eosinophilia, IgE) in female offspring sensitized early in life. 6

- BALF LDH significantly elevated in female offspring.
B ignami et al. ( 1994 ) Pregnant CD

- 1 dams (PD7

- 17) 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 t ransient 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 m aturation of near term rodent bronchioles, with ultra

- structural damage to bronchiolar epithelium.

Auten et al. ( 2009 ) C57BL/6 mouse pups 1.0 3 h/day, every other day, thrice weekly for 4 weeks Postnatal O 3 exposure significantly increased lung inflammatory cytokine levels; this was further exacerbated with gestational PM exposure. Plopper et 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

- 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 ) 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 significant 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 off spring: 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 significant 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 significantly elevated in O 3 exposed pups. Lung tissue from O 3 exposed pups had significant elevations of manganese superoxide dismutase (SOD) protein and significant decrements of extra

- cellular SOD protein. Campos

Bedolla et al. ( 2002 ) Pregnant Rats; Sprague Dawley (GD5, 10 or 18) 3.0 1h 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 significant increases in the maximum response to acetyl choline stimulation at GD5 and 10; they also had a significant 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 decrements in postnatal body weight ga in. 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 epithelia l desquamation with denudation to the 22 basement membrane). Vitamin E exposure concomitant with O 3 protected against pathological changes but Vitamin C did not.Draft

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- 70 September 2011 

### 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 

### 2 exception of birth defects. The current review included an expanded body of evidence on 

### 3
the associations b etween 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 alterations in placental and pup cytokines, and increased pup airway hyper

- reactivity. 

### 8 Also, there is limited toxicological evidence for an effect of prenatal and early life 

### 9 exposure on central nervou s system effects, including laterality, brain morphology, 

### 10 neurobehavioral abnormalities, and sleep aberration. Recent epidemiologic studies have 

### 11 begun to explore the effects of O 3 on sperm quality, and provide limited evidence for 

### 12 decrements in sperm conce ntration, while there is limited toxicological evidence for 

### 13 testicular degeneration associated with O 3 . 

### 14

While the collective evidence for many of the birth outcomes examined is generally 

### 15 inconsistent (including birth defects), there are several well

- desig ned, well

- conducted 

### 16
studies that indicate an association between O 3 and adverse outcomes. For example, as 

### 17 part of the southern California Children ’ s Health Study, Salam et al. ( 2005 ) observed a 

### 18
concentration

- response relationship of decreasing birth weight with increasing O 3 

### 19 concentrations averaged over the entire pregnancy that was clearest above the 30

- ppb 

### 20
level ( see Figure 7

- 4 ) . Simiarly, Hansen et al. ( 2008 ) utilized fetal ultrasonic 

### 21 measurements and found a change in ultrasound measurements associated with O 3 during 

### 22 days 31

- 60 of gestation indicated that increasin g O 3 concentration decreased an 

### 23 ultrasound measurement for women living within 2 km of the monitoring site. 

### 24

There is no evidence that prenatal or early life O 3 concentrations are associated with 

### 25 infant mortality. Collectively, there is limited though posit ive toxicological evidence for 

### 26
O 3 - induced developmental effects, including effects on pulmonary structure and function 

### 27 and central nervous system effects. Limited epidemiologic evidence for an effect on 

### 28 prenatal O 3 exposure on respiratory development provi des coherence with the effects 

### 29 observed in toxicological studies. There is also limited epidemiologic evidence for an 

### 30
association with O 3 concentration and decreased sperm concentration. A recent 

### 31 toxicological study provides limited evidence for a possible biological mechanism 

### 32 (histopathology showing impaired spermatogenesis) for such an association. 

### 33 Additionally, though the evidence for an association between O 3 concentrations and 

### 34 adverse birth outcomes is generally inconsistent, there are several influent ial studies that 

### 35 indicate an association with reduced birth weight and restricted fetal growth. Taking into 

### 36Draft

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- 71 September 2011 consideration the positive evidence for developmental and reproductive outcomes from 

### 1 toxicological and epidemiological studies, and the few influent ial birth outcome studies, 

### 2
the evidence is suggestive of a causal relationship between long

- term exposures to O 3 

### 3 and reproductive and developmental effects. 

### 4



## 7.5 Central Nervous System Effects

7.5.1 Effects on the Brain and Behavior

The 2006 O 3 AQCD included toxicol ogical evidence that acute exposures to O 3 are 

### 5 associated with alterations in neurotransmitters, motor activity, short and long term 

### 6 memory, and sleep patterns. Additionally, histological signs of neurodegeneration have 

### 7 been observed. Reports of headache, dizziness, and irritation of the nose with O 3 

### 8 exposure are common complaints in humans, and some behavioral changes in animals 

### 9 may be related to these symptoms rather than indicative of neurotoxicity. Research in the 

### 10
area of O 3 - induced neurotoxicity has no tably increased over the past few years, and new 

### 11 studies examining the effects of long

- term exposure have demonstrated progressive 

### 12 damage in various regions of the brains of rodents in conjunction with altered behavior. 

### 13 Evidence from epidemiologic studies has been more limited. A recently published 

### 14 epidemiologic study examined the association between O 3 exposure and neurobehavioral 

### 15
effects. Chen et al. ( 2009 ) utilized data from the NHANES III c ohort to study the 

### 16 relationship between O 3 levels (mean annual O 3 concentration 26.5 ppb) and 

### 17 neurobehavioral effects among adults aged 20

- 59 years. The authors observed an 

### 18 association between annual exposure to O 3 and tests measuring coding ability (symbol



### 19 digit substitution test) and attention/short

- term memory (serial

- digit learning test). Each 

### 20
10

- ppb increase in annual O 3 levels corresponded to an aging

- related cognitive 

### 21 performance decline of 3.5 yr for coding ability and 5.3 years for attention/short

- term 

### 22 memory. These associations persisted in both crude and adjusted models. There was no 

### 23
association between O 3 levels and reaction time tests. The authors concluded that overall, 

### 24
there is an association between l ong

- term O 3 exposure and reduced performance on 

### 25
neurobehavioral tests. 

### 26

A number of new toxicological studies demonstrate various perturbations in neurologic 

### 27 function or histology with long

- term exposure to O 3 , including changes similar to those 

### 28
observed in neurodegenerative disorders such as Parkinson ’ s and Alzheimer ’ s disease 

### 29 pathologies in relevant regions of the brain ( Table 7

- 11 ). The central nervous system is 

### 30 very sensitive to oxidative stress, due in part to its high content of polyunsaturated fatty 

### 31 acids, high rate of oxygen consumption, and low antioxidant enzyme capacity. Oxidative 

### 32Draft

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- 72 September 2011 stress has been identified as one of the pathophysiological mecha nisms underlying 

### 1 neurodegenerative disease ( Simonian and Coyle, 1996 ) , and it is believed to play a role in 

### 2 altering hippocampal function, which causes cognitive deficits with aging ( Vanguilder 

### 3 and Freeman, 2011 ) . A particularly common finding in studies of O 3 - exposed rats is lipid 

### 4 peroxidation in the brain, especially in the hippocampus, which is important for higher 

### 5 cognitive function including contextual memory acquisition. Performance in passive 

### 6
avoid ance learning tests is impaired when the hippocampus is injured. For example, in a 

### 7 subchronic study, exposure of rats to 0.25 ppm O 3 (4 h/ day ) for 15

- 90 days caused a 

### 8 complex array of responses, including a time

- dependent increase in lipid peroxidation 

### 9

pro ducts and immunohistochemical changes in the hippocampus that were correlated 

### 10 with decrements in passive avoidance behavioral tests ( Rivas

- Arancibia et al., 2010 )

. 

### 11 Changes included increased numbers of activated microglia, a s ign of inflammation, and 

### 12 progressive neurodegeneration. Notably, continued exposure tends to bring about 

### 13 progressive, cumulative damage, as shown by this study ( Rivas

- Arancibia et al., 2010 ) 

### 14
and others ( Santiago

- López et al., 2010 ; Guevara

- Guzmán et al., 2009 ; Angoa

- Pérez et 

### 15 al., 2006 ) . The effects of O 3 on passive avoidance test performance were particularly 

### 16 evident at 90 days for both short

- and long

- term memory. The greatest extent of cell loss 

### 17 was also observed at this time point, whereas lipid peroxidation did not increase much 

### 18 beyond 60 days of exposure. 

### 19

The substantia nigra is another region of the brain affected by O 3 , and seems particularly 

### 20 sensitive to oxidative stress because the metabolism of dopamine, central to its functi on, 

### 21 is an oxidative process perturbed by redox imbalance. Oxidative stress has been 

### 22 implicated in the premature death of substantia nigra dopamine neurons in Parkinson ’ s 

### 23 disease. Progressive damage has been found in the substantia nigra of male rats after 15, 

### 24 30, and 60 days of exposure to 0.25 ppm O 3 for 4 h/day. Santiago

- López and colleagues 

### 25 ( 2010 ) observed a reduction dopaminergic neurons within the substantia nigra over time, 

### 26 with a complete loss of normal morphology in the remaining cells and virtually no 

### 27 dopamine immunoreactivity at 60 days. This was accompanied by an increase in p53 

### 28 levels and nuclear translocation, a process associated with programmed cell death. 

### 29 Similarly, Angoa

- Pérez et al. ( 2006 ) have shown progressive lipoperoxidation in the 

### 30
substanti a nigra and a decrease in nigral neurons in ovariectomized female rats exposed 

### 31 to 0.25 ppm O 3 , 4h/day, for 7

- 60 days. Lipid peroxidation effectively doubled between 

### 32 the 30 and 60 day time points. Total nigral cell number was also diminished to the 

### 33
greate st extent at 60 days, and cell loss was particularly evident in the tyrosine 

### 34 hydroxylase positive cell population (90%), indicating a selective loss of dopamine 

### 35 neurons or a loss of dopamine pathway functionality. 

### 36

The olfactory bulb also undergoes oxidativ e damage in O 3 - exposed animals, in some 

### 37 cases altering olfactory

- dependent behavior. Lipid peroxidation was observed in the 

### 38Draft

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- 73 September 2011 olfactory bulbs of ovariectomized female rats exposed to 0.25 ppm O 3 (4 h/day) for 30 or 

### 1
60 days ( Guevara

- Guzmán et al., 2009 ) . O 3 a lso induced decrements in a selective 

### 2 olfactory recognition memory test, which were significantly greater at 60 days compared 

### 3 to 30 days, and the authors note that early deficits in odor perception and memory are 

### 4 components of human neurodegenerative disea ses. The decrements in olfactory memory 

### 5 did not appear to be due to damaged olfactory perception based on other tests early on, 

### 6 but by 60 days deficits in olfactory perception had emerged. 

### 7

Memory deficits and associated morphological changes can be attenu ated by 

### 8
administration of α

- tocopherol ( Guerrero et al., 1999 ) , taurine ( Rivas

- Arancibia et al., 

### 9
2000 ) , and estradiol ( Guevara

- Guzmán et al., 2009 ; Angoa

- Pérez et al., 2006 ) , all of 

### 10 which have antioxidant properties. In the study by Angoa

- Pérez et al. ( 2006 ) described 

### 11 above, estradiol seemed particularly effective at protecting against lipid peroxidation and 

### 12 nigral cell loss at 60 days compared to shorter exposure durations. The same was true for 

### 13 amelioration of decrements in olfactory recognition memory ( Guevara

- Guzmán et al., 

### 14
2009 ) , although protection against lipid peroxidation was similar for the 30 and 60 day 

### 15

exposures. 

### 16

Table 7

- 11 Central nervous system effects of long

- term O 3 

### exposure in rats

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 d ays, 4 h/day, 5 days/wk 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 decreases in α and β estrogen receptors and dopamine β

hydroxlyase in olfactory bulb, and deficits in olfactory social recognition me mory and chocolate recognition.
Rivas

- Arancibia et al. ( 2010 ) Rat; Wistar; M; Weight: 250

- 300 g 0.25 15 to 90 days, 4h/day Ozone produced significant increases in lipid peroxidation in the hippocampus, and altered the number of p53 p ositive immunoreactive cells, activated and phagocytic microglia, GFAP immunoreactive cells, double cortine 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 pri or to breeding until GD17 Upon behavioral challenge with another male, there was a significant increase in defensive and freezing postures and decrease in the frequency of nose

- sniffing. These behavioral changes were accompanied by a significant increase in BDNF in the striatum and a decrease of NGF in the hippocampus.Draft

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- 74 September 2011 CNS effects have also been demonstrated in adult mice whose only exposure to O 3 

### 1
occurred while in utero, a period particularly critical for brain development . Santucci et 

### 2
al. ( 2006 ) investigated behavioral effects and gene expression after in utero exposure of 

### 3
mice to 0.3 or 0.6 ppm O 3 . Exposure began 30 days prior to mating and continued 

### 4 throughout gestation. Testing of adult animals demonstrated increased 

### 5
defensive/submissive beha vior and reduced social investigation were observed in both the 

### 6 0.3 and 0.6 ppm O 3 groups. Changes in gene expression of brain

- derived neurotrophic 

### 7 factor (BDNF, increased in striatum) and nerve growth factor (NGF, decreased in 

### 8 hippocampus) accompanied the se behavioral changes. BDNF and NGF are involved in 

### 9 neuronal organization and the growth, maintenance, and survival of neurons during early 

### 10 development and in adulthood. This study and two others using short

- term exposures 

### 11
demonstrate that CNS effects can occur as a result of in utero exposure to O 3 , and 

### 12 although the mode of action of these effects is not known, it has been suggested that 

### 13 circulating lipid peroxidation products may play a role ( Boussouar et al., 2009 )

. 

### 14 Importantly, these CNS effects occurred in rodent models after in utero only exposure to 

### 15 (semi

- ) relevant concentrations of O 3 . 

### 16

7.5.2 Summary and Causal Determination

The 2006 O 3 AQCD included toxicological evidence that acute exposures to O 3 are 

### 17 associated with alterations in neurotransmitters, motor activity, short and long term 

### 18 memory, and sleep patterns. Additionally, histological signs of neurodegeneration have 

### 19 been observed. However, evidence regarding chronic exposu re and neurobehavioral 

### 20
effects was not available. Recent research in the area of O 3 - induced neurotoxicity has 

### 21 included several long

- term exposure studies. Notably, the first epidemiologic study to 

### 22 examine the relationship between O 3 exposure and neurobehav ioral effects observed an 

### 23
association between annual O 3 levels and an aging

- related cognitive performance decline 

### 24 in tests measuring coding ability and attention/short

- term memory. This observation is 

### 25 supported by studies in rodents which demonstrate progr essive oxidative stress and 

### 26 damage in the brain and associated decrements in behavioral tests, including those 

### 27 measuring memory, after subchronic exposure to 0.25 ppm O 3 . Additionally, 

### 28 neurobehavioral changes are evident in animals whose only exposure to O 3 occurred in 

### 29

utero . Collectively, the limited epidemiologic and toxicological evidence is coherent and 

### 30 suggestive of a causal relationship between O 3 exposure and CNS effects. 

### 31Draft

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- 75 September 2011 

## 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 

### 1 proliferation of cells in the respiratory tract, have suggested that O 3 could act as a 

### 2 carcinogen. However, toxicological studies of tumorigenesis in the rodent lung have 

### 3 yielded mixed an d often confusing results, and the epidemiologic evidence is equally 

### 4
conflicted. The 2006 O 3 AQCD concluded that, “ the weight of evidence from recent 

### 5 animal toxicological studies and a very limited number of epidemiologic studies do not 

### 6 support ambient O 3 as a pulmonary carcinogen ” 2 ( U.S. EPA, 2006b )

. 

### 7

Multiple epidemiologic studies reported in the 2006 O 3 AQCD examined the association 

### 8
between O 3 exposure and cancer. The largest of these studies, by Pope et al. ( 2002 )

, 

### 9 included 500,000 adults from the American Cancer Society ’ s (ACS) Cancer Prevention II 

### 10 study. In this study, no association was observed between O 3 and lung cancer mortality. 

### 11 The Adventist Health Study of Smog (AHSMOG) also examined the association between 

### 12
O 3 and lung cancer mortality ( Abbey et al., 1999 ) . There was a positive association 

### 13
between O 3 levels and lung cancer mortality among men. No association was reported for 

### 14 women. Another study using the AHSMOG cohort assessed the risk of incident lung 

### 15

cancer ( Beeson et al., 1998 ) . Among males, an association with incidence of lung cancer 

### 16 was observed with increasing O 3 concentrations. When stratified by smoking status, the 

### 17 association persisted among never smokers but was null for former smokers. No 

### 18
association was detected for females. The Six Cit ies Study examined various air 

### 19 pollutants and mortality but did not specifically explore the association between O 3 

### 20
concentrations and lung cancer mortality due to low variability in O 3 levels across the 

### 21
cities ( Dockery et al., 1993 ) . An ecologic study performed in Sao Paulo City, Brazil 

### 22
examined the correlations between O 3 levels in four of the city districts and incident 

### 23 cancer of the larynx and lung re ported in 1997 ( Pereira et al., 2005 ) . A correlation 

### 24 between the average number of days O 3 levels exceeded air quality standards from 1981 

### 25 to 1990 and cancer incidence was present for larynx cancer but not for lung cancer. 

### 26

Early toxicological research demonstrated lung adenoma 3 acceleration in mice with daily 

### 27 exposure to 1 ppm over 1 5 months ( Stokinger, 1962 ) . Later work demonstrated a 

### 28 significant increase in lung tumor numbers in one strain of mouse (A/J) but not anot her 

### 29

2 The toxicological evidence is presented in detail in Table 6

- 18 on p. 6

- 116 of the 1996 O 3 AQCD and Table AX5

- 13 on p. AX5

- 43 of the 2006 O 3 AQCD.

3 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.”Draft

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- 76 September 2011 after exposure to 0.3

- 0.8 ppm O 3 ( Last et al., 1987 ; Hassett et al., 1985 ) . The A/J mouse 

### 1 strain is known to have a high incidence of sponta neous adenomas, and further studies 

### 2 using this strain found a statistically significant increase in lung tumor incidence after a 

### 3
9

- month exposure to 0.5 ppm and incidence and multiplicity after a 5 month exposure to 

### 4
0.12 ppm with a 4

- month recovery period ( Witschi et al., 1999 ) . However, these findings 

### 5
were discounted b y the study authors due to the lack of a clear dose response, and results 

### 6 from the Hassett et al. 1985 and Last et al. 1987 studies were retrospectively deemed 

### 7 spurious based on what appeared to be unusually low spontaneous tumor incidences in 

### 8
the control groups ( Witschi, 1991 ) . A study of carcinogenicity of O 3 by the National 

### 9 Toxicology Program ( NTP, 1994 ) reported increased incidences of alveolar/ bronchiolar 

### 10 adenoma or carcinoma (combined) in female B6C3F 1 mice exposed over 2 years to 

### 11
1.0 ppm O 3 , but not 0.12 or .5 ppm. No effect was detected in male mice. For a lifetime 

### 12 exposure to 0.5 or 1.0 ppm O 3 , an increase in the number of female mice with a denomas 

### 13 (but not carcinomas or total neoplasms) was found. The number of total neoplasms was 

### 14 also unaffected in male mice, but there was a marginally increased incidence of 

### 15 carcinoma in males exposed to 0.5 and 1.0 ppm. Thus there was equivocal evidence of 

### 16 carcinogenic activity in male mice and some evidence of carcinogenic activity of O 3 in 

### 17
females. Some semblance of a dose

- response relationship was also evident in this study. 

### 18 Experimental details of the NTP study are available in Table 6

- 19 on p. 6

- 121 of the 1996 

### 19
O 3 AQCD. 

### 20

In Fischer

- 344/N rats (50 of each sex per group), neither a 2

- yea r nor lifetime exposure to 

### 21
O 3 ranging from 0.12 to 1.0 ppm was found to be carcinogenic ( Boorman et al., 1994 )

. 

### 22 However, a marginally significant carcinogenic effect of 0.2 ppm O 3 was reported in a 

### 23 study of male Sprague

- Dawley rats exposed for 6 months (n = 50) ( Monchaux et al., 

### 24
1996 ) . These two studies also examined co

- carcinogenicity of O 3 with NNK 4 ( Boorman 

### 25 et al., 1994 ) or a rel atively high dose of radon ( Monchaux et al., 1996 ) , finding no 

### 26 enhancement of NNK related tumors and a slight non

- significant increase in tumor 

### 27 incidence after combined exposure with radon, respectively. Another study exploring co



### 28

carc inogenicity was conducted in hamsters. Not only was there no enhancement of 

### 29 chemically induced tumors in the peripheral lung or nasal cavity, but results suggested 

### 30
that O 3 could potentially delay or inhibit tumor development ( Witschi et al., 1993 ) . Thus 

### 31
there is no concrete evidence that O 3 can act as a co

- carcinogen. 

### 32

Immune surveillance is an important defense against cancer, and it sh ould be noted that 

### 33 natural killer (NK) cells, which destroy tumor cells in the lung, appear to be inhibited by 

### 34 higher doses of O 3 and either unaffected or stimulated at lower doses (Section 6.2.5.4, 

### 35

4 4

- (N

- nitros omethylamino)

- 1

- (3

- pyridyl)

- 1

- butanoneDraft

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- 77 September 2011 Infection and Adaptive Immunity). This aspect of tumorige nesis adds yet another layer of 

### 1 complexity which may be reflected by conflicting results across studies. 

### 2

The following sections will examine epidemiologic studies of cancer incidence and 

### 3 mortality and toxicological studies that have been published since th e 2006 O 3 AQCD. 

### 4 An epidemiologic study has been published with cancer as the outcome; most 

### 5 epidemiologic studies examine markers of exposure or susceptibility. 

### 6

7.6.2 Lung Cancer Incidence and Mortality

A recent re

- analysis of the full ACS CPSII cohort by the H ealth Effects Institute is the 

### 7 only epidemiologic study that has explored the association between O 3 and cancer 

### 8 mortality since the last O 3 AQCD. Krewski et al. ( 2009 ) conducted an extended follow



### 9 up of the cohort (1982

- 2000). Mean O 3 levels [obtained from the Aerometric Information 

### 10 Retrieval System (AIRS) for 1980] were 22.91 ppb for the full year and 30.15 ppb for the 

### 11 summer months (April

- September). No association was reported between lung cancer 

### 12 mortality and O 3 (HR=1.00 [95% CI: 0 .96

- 1.04] per 10 ppb O 3) . Additionally, no 

### 13
association was observed when O 3 was restricted to the summer months. There was also 

### 14 no association present in a sub

- analysis of the cohort examining the relationship between 

### 15
O 3 and lung cancer mortality in the Lo s Angeles area. 

### 16

Since the 2006 O 3 AQCD, two toxicological studies have examined potential 

### 17 carcinogenicity of O 3 ( Kim and Cho, 2009a

, b ) . Looking across both studies, which used 

### 18 the same mouse strain as the National Toxicology Program study described above ( NTP, 

### 19
1994 ) , 0.5 ppm O 3 alone or in conjunction with chemical tumor inducers did not enhance 

### 20 lung tumor incidence in males or females. However, a 10% incidence of oviductal 

### 21 carcinoma was observed in mice exposed to 0.5 ppm O 3 for 16 weeks. The implicatio ns 

### 22 of this observation are unclear, particularly in light of the lack of statistical information 

### 23 reported. Additionally, there is no mention of oviductal carcinoma after 32 weeks of 

### 24 exposure, and no oviductal carcinoma was observed after one year of exposu re. The NTP 

### 25 study did not report any increase in tumors at extrapulmonary sites. 

### 26

7.6.3 DNA Damage

The potential for genotoxic effects relating to O 3 exposure was predicted from the 

### 27 radiomimetic properties of O 3 . The decomposition of O 3 in water produces OH and H O 2 

### 28 radicals, the same species that are generally considered to be the biologically active 

### 29 products of ionizing radiation. Ozone has been observed to cause degradation of DNA in 

### 30
a number of different models and bacterial strains. The toxic effects of O 3 hav e been 

### 31Draft

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- 78 September 2011 generally assumed to be confined to the tissues directly in contact with the gas, such as 

### 1 the respiratory epithelium. Due to the highly reactive nature of O 3 , little systemic 

### 2 absorption is predicted. Zelac et al. ( 1971a

, b ) , however, reported a significant increase in 

### 3 chromosome aberrations in peri pheral blood lymphocytes from Chinese hamsters 

### 4 exposed to 0.2 ppm for 5 hours. Other in vivo exposure studies found increased DNA 

### 5 strand breaks in respiratory cells from guinea pigs ( Ferng et al., 1997 ) and mice 

### 6 ( Bornholdt e t al., 2002 ) but only with exposure to higher doses of O 3 (1 ppm for 72 hours 

### 7
and 1 or 2 ppm for 90 minutes, respectively). In other studies there were no observations 

### 8 of chromosomal aberrations in germ cells, but mutagenic effects have been seen in 

### 9
off spring of mice exposed to 0.2 ppm during gestation (blepharophimosis or dysplasia of 

### 10 the eyelids). The overall evidence for mutagenic activity from in vitro studies is positive, 

### 11 and in the National Toxicology Program report described above, O 3 was found to be 

### 12 mutagenic in Salmonella

, with and without S9 metabolic activation. No new 

### 13 toxicological studies of DNA damage have become available since the 2006 O 3 AQCD. 

### 14

A number of epidemiologic studies looked at the association between O 3 and DNA and 

### 15 cellular level damages. These changes may be relevant to mechanisms leading to cancers 

### 16 development and serve as early indicators of elevated risk of mutagenicity. 

### 17

Two studies performed in California examined cytogenetic damage in relation to O 3 

### 18 exposures. Huen et al. ( 2006 ) examined cytogenetic damage among African American 

### 19 children and their mothers in Oakland, CA. Increased O 3 (mean monthly 8

- h O 3 

### 20 concentrations ranged from about 30 ppb in April to 14 ppb in November) was associated 

### 21 with increased cytogenetic damage (micronuclei frequency among lymphocytes and 

### 22 buccal cells) even after adjustment for household/personal smoking status and distance



### 23 weighted traffic density. Chen et a l. ( 2006a ) recruited college students at the University 

### 24 or California, Berkeley who reported never smok ing and compared their levels of 

### 25 cytogenetic damage (micronuclei frequency from buccal cells) in the spring and fall. 

### 26 Cytogenetic damage was greater in the fall, which the authors attributed to the increase in 

### 27
O 3 over the summer. However, O 3 levels over 2, 7, 10, 14, or 30 days (concentrations not 

### 28 given) before collection of buccal cells did not correlate with cytogenetic damage. 

### 29
Estimated lifetime O 3 exposure was also not correlated with cytogenetic damage. 

### 30 Additionally, the authors exposed a subset of the students (n=15) to 200 ppb O 3 for 

### 31

4 hours while the students exercised intermittently. Ozone was found to be associated 

### 32 with an increase in cytogenetic damage in degenerated cells but not in normal cells 9

- 

### 33
10 days after exposure. Increased cytogenetic damage was also noted in peripher al blood 

### 34 lymphocytes collected 18 hours after exposure. 

### 35

A study performed in Mexico recruited 55 male workers working indoors (n=27) or 

### 36 outdoors (n=28) in Mexico City or Puebla, Mexico in order to study the relationship 

### 37Draft

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- 79 September 2011 between O 3 and DNA damage (detected from peripheral blood samples using the Comet 

### 1 assay) ( Tovalin et al., 2006 ) . The median estimated daily O 3 concentrations were 

### 2
estimated to be 28.5 ppb for ou tdoor workers and 5.1 ppb for indoor workers in 

### 3
Mexico City and 36.1 ppb for outdoor workers and 19.5 ppb for indoor workers in 

### 4 Puebla. Overall, a positive correlation between O 3 levels and DNA damage was 

### 5 observed. However, when examining the relationship by city and workplace, only DNA 

### 6 damage in outdoor workers in Mexico City remained correlated with O 3 levels. 

### 7

Three studies examining the relationship between O 3 and DNA

- level damage have been 

### 8 performed in Europe. The largest of these was the GenAir case

- c ontrol study, which was 

### 9 nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) 

### 10 study, and included individuals recruited between 1993 and 1998 from ten European 

### 11 countries. Only non

- smokers (must not have smoked for at least 1 0 years prior to 

### 12 enrollment) were enrolled in the study. The researchers examined DNA adduct levels 

### 13 (DNA bonded to cancer

- causing chemicals) and their relationship with O 3 concentrations 

### 14 (concentrations not given) ( Peluso M Hainaut et al., 2005 ) . A positive association was 

### 15
seen between DNA adduct levels and O 3 concentrations from 1990

- 1994 but not O 3 

### 16
lev els from 1995

- 1999. In adjusted analyses with DNA adduct levels dichotomized as 

### 17 high and low (detectable versus non

- detectable), the OR was 1.97 (95% CI: 1.08, 3.58) 

### 18 when comparing the upper tertile of O 3 concentration to the lower two tertiles. Two other 

### 19 European studies were conducted in Florence, Italy. The most recent of these enrolled 

### 20 individuals from the EPIC study into a separate study between March and September of 

### 21
1999 ( Palli et al., 2009 ) . The purpose of the study was to examine oxidative DNA 

### 22 damage (determined by Comet assay using blood lymphocytes) in association with 

### 23 varying periods of O 3 exposure. The researchers observed that longer periods of high O 3 

### 24 exposure (concentrat ions not given) were more strongly correlated with oxidative DNA 

### 25 damage than shorter exposures (i.e., the rho [p

- value] was 0.26 [0.03] for 0

- 10 days and 

### 26 0.35 [0.002] for 0

- 90 days). This correlation was stronger among men compared to 

### 27
women. The correlatio ns for all time periods had p

- values <0.05 for ex

- and never



### 28 smokers. For current smokers, the correlation was only observed among time periods ≤ 

### 29
25 days. When adjusted for age, gender, smoking history, traffic pollution exposure, 

### 30 period of blood draw, and area of residence, the association between O 3 levels and 

### 31 oxidative DNA damage was positive for O 3 levels 0

- 60 days, 0

- 75 days, and 0

- 90 days 

### 32 prior to blood draw. Positive, statistically significant associations were not observed 

### 33 among shorter time periods . The other study performed in Florence recruited healthy 

### 34 volunteers who reported being non

- smokers or light smokers ( Giovannelli et al., 2006 )

. 

### 35
The estimated O 3 levels during the study ranged from approximately 4

- 40 ppb for 3

- day 

### 36 avgs, 5

- 35 ppb for 7

- day avgs, and 7.5

- 32.5 ppb for 30

- day avgs. Ozone concentrations 

### 37
were correlat ed with DNA strand breaks (measured from blood lymphocytes) over longer 

### 38 exposure periods (p

- value: 0.002 at 30 days, p

- value: 0.04 at 7 days; p

- value: 0.17 at 

### 39Draft

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- 80 September 2011 3 days). This association was robust to control for temperature, solar radiation, gender, 

### 1 and age . No association was seen between O 3 concentrations and measures of oxidative 

### 2 DNA damage at 3, 7, or 30 days. 

### 3

7.6.4 Summary and Causal Determination

The 2006 O 3 AQCD reported that evidence did not support ambient O 3 as a pulmonary 

### 4 carcinogen. Since the 2006 O 3 AQCD, very few epidemiologic and toxicological studies 

### 5 have been published that examine O 3 as a carcinogen, but collectively, study results 

### 6
indicate that O 3 may contribute to DNA damage. Overall, the evidence is inadequate to 

### 7
determine if a causal relationship exists between ambient O 3 exposures and 

### 8

cancer. 

### 9



## 7.7 Mortality

A limited number of epidemiologic studies have assessed the relationship between long



### 10 term exposure to O 3 and mortality in adults. The 2006 O 3 AQCD concluded that an 

### 11
insufficient amoun t of evidence existed “ to suggest a causal relationship between chronic 

### 12
O 3 exposure and increased risk for mortality in humans ” ( U.S. EPA, 2006b ) . In addition 

### 13 to the infant mortality studies discussed in Section 7.4.9 , additional studies have been 

### 14 conducted among adults since the last review; an ecologic study that finds no association 

### 15 between mortality and O 3 , several reanalyses of the ACS cohort, one of which 

### 16 specifically points to a relationship between long

- term O 3 exposure and an increased risk 

### 17 of respiratory mortality, and a study of four cohorts of persons with potentially 

### 18 predisposing conditions. These studies supplement the evidence from long

- term cohort 

### 19 studies characterized in previous reviews of O 3, and are summarized here briefly. 

### 20

In the Harvard Six Cities Study ( Dockery e t al., 1993 ) , adjusted mortality rate ratios were 

### 21 examined in relation to long

- term mean O 3 concentrations in six cities: Topeka, KS; St. 

### 22 Louis, MO; Portage, WI; Harriman, TN; Steubenville, OH; and Watertown, MA. Mean 

### 23
O 3 concentrations from 1977 to 1985 ranged from 19.7 ppb in Watertown to 28.0 ppb in 

### 24 Portage. Long

- term mean O 3 concentrations were not found to be associated with 

### 25 mortality in the six cities. However, the authors noted that “ The small differences in O 3 

### 26 levels among the (six) cities limited the power of the study to detect associations between 

### 27 mortality and O 3 levels. ” In addition, while total and cardio

- pulmonary mortality were 

### 28 considered in this study, respiratory mortality was not specifically considered. 

### 29

In a subsequent large prospective cohort study of approximately 500,000 U.S. adults, 

### 30 Pope et al. ( 2002 ) examined the effects of long

- term exposure to air pollutants on 

### 31Draft

– Do Not Cite or Quote 7

- 81 September 2011 mortality (American Cancer Society, Cancer Prevention Study II). All

- cause, 

### 1 cardiopulmonary, lung cancer and other mortality risk estimates for long

- term O 3 

### 2 exposure are shown in Figure 7

- 5. While consistently positive associations were not 

### 3
observed between O 3 and mortality (effect estimates labeled A in Figure 7

- 5 ) , the 

### 4 mortality risk est imates were larger in magnitude when analyses considered more 

### 5 accurate exposure metrics, increasing when the entire period was considered (effect 

### 6
estimates labeled B in Figure 7

- 5 ) and becoming marginally significant when the 

### 7 exposure estimate was restricted to the summer months (July to September; effect 

### 8
estimates labeled C in Figure 7

- 5 ), especially when considering cardiopulmonary deaths. 

### 9 In contrast, consistent positive and significant effects of PM 2.5 were observed for both 

### 10 lung cancer and cardio

- pulmonary mortality. 

### 11

Years of Data Collection Nu mber 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 )

.


### Fig ure 7

- 5 Adjusted ozone

- mortality relative risk estimates (95% CI) by time period of analysis per subject

- weighted mean O 3 

### concentration in the Cancer Prevention Study II by the American Cancer Society.

A study by Abbey et al. ( 1999 ) examined the effects of long

- term air pollution exposure, 

### 12 including O 3 , on all

- cause (n = 1,575), cardiopulmonary (n = 1,029), nonmalignant 

### 13 respiratory (n = 410), and lung cancer (n = 30) mortality in the long

- term prospecti ve 

### 14 Adventist Health Study of Smog (AHSMOG) of 6,338 nonsmoking, non

- Hispanic white 

### 15 individuals living in California. A particular strength of this study was the extensive 

### 16Draft

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- 82 September 2011 effort devoted to assessing long

- term air pollution exposures, including interpolatio n to 

### 1 residential and work locations from monitoring sites over time and space. No associations 

### 2 with long

- term O 3 exposure were observed for all cause, cardiopulmonary, and 

### 3 nonmalig nant respiratory mortality. In a follow

- up, Chen et al. ( 2005 ) utilized data from 

### 4 the AHSMOG study and reported no evidence of associations between long

- term O 3 

### 5

expo sure (me an O 3 concentration 26.2 ppb) and fatal coronary heart disease. Thus, no 

### 6
associ ation of chronic O 3 exposure with mortality outcomes has been detected in this 

### 7 study. 

### 8

Lipfert et al. ( 2003

, 2000 ) reported positive effects on all

- cause mortality for peak O 3 

### 9 exposures (95th percentile levels) in the U.S. Veterans Cohort study of approximately 

### 10 50,000 middle

- aged men recruited with a diagnosis of hypertension. The actual analysis 

### 11
involved smaller su bcohorts based on exposure and mortality follow

- up periods. Four 

### 12 separate exposure periods were associated with three mortality follow

- up periods. For 

### 13 concurrent exposure periods, peak O 3 was positively associated with all

- cause mortality, 

### 14 with a 9.4% (95% CI: 0.4, 18.4) excess risk per mean 95th percentile O 3 less estimated 

### 15 background level (not stated). “ Peak ” refers, in this case, to the 95th percentile of 

### 16
the hourly measurements, averaged by year and county. In a further analysis, Lipfert et al. 

### 17 ( 2003 ) reported the strongest positive association for concurrent exposure to peak O 3 for 

### 18 the subset of subjects with low diastolic blood pressure during the 1982 to 1988 period. 

### 19 Two more recent studies of this cohort focused specifically on traffic density ( Lipfert et 

### 20 al., 2006a ; 2006b ) . Lipfert ( 2006b ) concluded that: “ Traffic density is seen to be a 

### 21 significant and robust predictor of survival in this cohort, more so than ambient air 

### 22 quality, with the possible exception of O 3 , ” reporting a significant O 3 effect even with 

### 23 traffic density included in the model: RR=1.080 per 40 ppb peak O 3 (95% CI: 1.019, 

### 24 1.146). However, in Lipfert ( 2006a ) , which considers only the EPA Speciation Trends 

### 25 Network (STN) sites, O 3 drops to non

- significant predictor of total mortality for this 

### 26 cohort. The authors acknowledge that: “ Peak O 3 has been important in analyses of this 

### 27 cohort for previous periods, but in the STN data set, this v ariable has limited range and 

### 28 somewhat lower values and its small coefficient of variation results in a relatively large 

### 29
standard error. ” The restriction to subjects near STN sites likely reduced the power of this 

### 30 analysis, though the size of the remaining subjects considered was not reported in this 

### 31 paper. In addition, these various Veterans Cohort studies considered only total mortality, 

### 32 and did not consider mortality on a by

- cause basis. 

### 33

An ecological study in Brisbane, Australia used a geospatial appro ach to analyze the 

### 34 association of long

- term exposure to gaseous air pollution with cardio

- respiratory 

### 35 mortality, in the period 1996

- 2004 ( Wang et al., 2009c ) . A generalized estimating 

### 36 equations model was employed to investigate the impact of NO 2 , O 3 and SO 2 , but PM 

### 37
was not addressed. The results indicated that long

- term exposure to O 3 was not 

### 38Draft

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- 83 September 2011 associated with cardio

- respiratory mortality, but the fact that this study considered only 

### 1 one city, and that the range of O 3 exposure across that city (23.7

- 35.6 ppb) was low and 

### 2 slight in variation in comparison to the ra nge of other pollutants across the city, limited 

### 3 study power. In addition, confounding factors (e.g., smoking) could not be addressed at 

### 4 the individual level in this ecological study. Respiratory mortality was not evaluated 

### 5 separately. 

### 6

A recent study by Za nobetti and Schwartz ( In Press ) examined whethe r year

- to

- year 

### 7
variations in 8

- h mean daily O 3 concentrations for the summer (May

- September) around 

### 8 their city

- specific long

- term trend were associated with year

- to

- year variations in 

### 9 mortality around its long

- term trend. This association was examined amon g Medicare 

### 10 participants with potentially predisposing conditions, including COPD, diabetes, CHF, 

### 11 and MI, defined as patients discharged alive after an emergency admission for one of 

### 12 these four conditions. The analyses was repeated in 105 cities using avail able data from 

### 13 1985 through 2006, and the results were combined using methods previously employed 

### 14 by these authors ( Zanobetti et al., 2008 ; Zanobetti and Schw artz, 2007 )

. This study 

### 15 design eliminated potential confounding by factors that vary across city, whi ch is a 

### 16 common concern in most air pollution cohort studies, and also avoided both confounding 

### 17 by cross

- sectional factors that vary by city and the short

- term factors that confound daily 

### 18
time

- series studies, but are not present in annual analyses. The aver age 8

- h mean daily 

### 19
summer O 3 concentrations ranged from 15.6 ppb (Honolulu, HI) to 71.4 ppb 

### 20 (Bakersfield, CA) for the 105 cities. The authors observed associations between yearly 

### 21
fluctuations in summer O 3 concentrations and mortality in each of the four co horts; the 

### 22 hazard ratios (per 10 ppb increment) were 1.12 (95% CI: 1.06, 1.17) for the CHF cohort, 

### 23 1.19 (95% CI 1.12, 1.25) for the MI cohort, 1.14 (95% CI: 1.10, 1.21) for the diabetes 

### 24 cohort, and 1.14 (95% CI: 1.08, 1.19) for the COPD cohort. A key advan tage to this study 

### 25
is that fluctu ations from summer to summer in O 3 concentrations around long

- term level 

### 26 and trend in a specific city are unlikely to be correlated with most other predicators of 

### 27 mortality risk, except for temperature, which was controlle d for in the regression. Key 

### 28 limitations of the study were the inability to control for PM 2.5 , since it was not reliably 

### 29 measured in these cities until 1999, and the inability to separate specific causes of death 

### 30 (e.g., respiratory, cardiovascular), since Medicare does not provide the underlying cause 

### 31
of death. 

### 32

In the most recent follow

- up analyses of the ACS cohort ( Jerrett et al., 2009 ; Smith et al., 

### 33
2009a ) , the effects of long

- term exposure to O 3 were evaluated alone, as well as in 

### 34 copollutant models with PM 2.5 and components of PM 2.5 . Jerrett et al. ( 2009 ) utilized the 

### 35
ACS cohort with data from 1977 through 2000 (mean O 3 concentration ranged from 33.3 

### 36
to 104.0 ppb) and subdivided cardiopulmonary deaths into respiratory and cardiovascular, 

### 37 separately, as opposed to combined into one category, as was done by Pope et al. ( 2002 )

. 

### 38Draft

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- 84 September 2011 Increases in exposure to O 3 were associated with an elevated risk of death from 

### 1 cardiopulmonar y, cardiovascular, ischemic heart disease, and respiratory causes. 

### 2
Inclusion of PM 2.5 concentrations measured in 1999

- 2000 as a copollutant attenuated the 

### 3
association with O 3 for all end points except death from respiratory causes, for which a 

### 4 significant association persisted ( Table 7

- 12 ). The association between increased O 3 

### 5 concentrations and increased risk of death from respiratory causes was insensi tive to the 

### 6
use of a random

- effects survival model allowing for spatial clustering within the 

### 7 metropolitan area and state of residence, and adjustment for several ecologic variables 

### 8 considered individually. Subgroup analyses showed that temperature and reg ion of 

### 9 country, but not sex, age at enrollment, body

- mass index, education, or PM 2.5 

### 10 concentration, modified the effects of O 3 on the risk of death from respiratory causes 

### 11 (i.e., risks were higher at higher temperature, and in the Southeast, Southwest, and Upper 

### 12 Midwest). Ozone threshold analyses indicated that the threshold model was not a better 

### 13 fit to the data (p > 0.05) than a linear representation of the overall O 3 - mortality 

### 14 association. Overall, this new analysis indicates that long

- term exposure to PM 2.5 

### 15 increases risk of cardiac death, while long

- term exposure to O 3 is specifically associated 

### 16 with an increased risk of resp iratory death, and suggests that combining cardiovascular 

### 17 and respiratory causes of mortality into one category for analysis may obscure any effect 

### 18
that O 3 may have on respiratory

- related causes of mortality. 

### 19

Table 7

- 12 Relative risk (and 95% CI) of death attributable to a 10

- ppb change in the ambient O 3 

### concentration*

Cause of Death O 3 (96 MSAs) O 3 (86 MSAs) O 3 +PM 2.5 (86 MSAs) 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) * 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 PM2.5 of 10 μg per cu bic meter 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 MSAs in the ACS cohort to 

### 20
examine the association of O 3 concentrations during the warm season and all

- cause and 

### 21 cardiopulmonary mortality. Mortality effects were est imated in single pollutant and 

### 22 copollutant models, adjusting for two PM 2.5 constituents, sulfate and EC. When all

- cause 

### 23 mortality was investigated, there was a 0.8% (95% CI:

- 0.31, 1.9) increase associated 

### 24 with a 10 ppb increase in O 3 concentration. This a ssociation was diminished when sulfate 

### 25
or EC were included in the model. There was a 2.48% (95% CI: 0.74, 4.3) increase in 

### 26 cardiopulmonary mortality associated with a 10 ppb increase in O 3 concentration. The 

### 27Draft

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- 85 September 2011 cardiopulmonary association was robust to adjust ment for sulfate, and diminished, though 

### 1 still positive, after adjustment for EC (1.63% increase; 95% CI:

- 0.41, 3.7). Smith et al. 

### 2 ( 2 009a ) did not specifically separate out cardiovascular and respiratory causes of death 

### 3 from the cardiopulmonary category, as was done by Jerrett et al. ( 2009 )

. 

### 4

7.7.1 Summary and Causal Determination

The 2006 O 3 AQCD concluded that an insufficient amount of evidence existed “ to 

### 5 suggest a causal relationship between chronic O 3 exposure and increased risk for 

### 6 mortality in humans ” ( U.S. EPA, 2006b ) . Several additional studies have been conducted 

### 7 since the last review, including an ecologic study that finds no association between 

### 8 mortality and O 3 ( Wang et a l., 2009c ) , a study of four cohorts of Medicare enrollees with 

### 9 potentially predisposing conditions that observes associations between O 3 and mortality 

### 10 among each of the cohorts ( Zanobetti and Schwartz, In Press ) , and reanalyses of the ACS 

### 11 cohort that provide weak evidence for an association with cardiopulmonary mortality 

### 12 ( Smith et al., 2009a ) and specifically point to a relationship between long

- term O 3 

### 13 exposure and an in creased risk of respiratory mortality ( Jerrett et al., 2009 ) . The findings 

### 14
from the Jerrett et al. ( 2009 ) study are consistent and coherent with the evidence from 

### 15 epidemiologic, controlled human exposure, and animal toxicological studies for the 

### 16
effects of short

- and long

- term exposure to O 3 on respiratory effects. Additionally, the 

### 17
evidence for short

- and long

- term respiratory morbidity provides biological plausibility 

### 18 for mortality due to respiratory disease. Collectively, the evidence is suggestive of a 

### 19 causal relationship between long

- term O 3 exposures and mortality. 

### 20



## 7.8 Overall Summary

The evidence reviewed in this chapter describes the recent findings regarding the health 

### 21 effects of long

- term exposure to ambient O 3 concentrations. Table 7

- 13 provides an 

### 22 overview of the causal determinations for each of the health categories evaluated. 

### 23Draft

– Do Not Cite or Quote 7

- 86 September 2011 

### Table 7

- 13 Summary of causal determinations for l ong - 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
Mortality Suggestive of a causal relationship



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