Lovelace Respiratory Research Institute

www.LRRI.org

2010Short-2

EPIDEMIOLOGY FINDS ASSOCIATIONS BETWEEN PM AND NUMEROUS HEALTH EFFECTS

Mortality (non-accidental, non-homicide deaths)

Daily mortality rates (time series studies)

Shortened life span (cohort studies)

Morbidity (illness)

Respiratory

Symptoms

Reduced lung function

Lung inflammation

Exacerbation of asthma

Incidence of infections

Allergic sensitization

Reduced rate of lung growth

Medication use, physician visits and hospital admissions

Cardiovascular

ECG abnormalities

Implanted cardioverter events

Heart attacks

Athersclerosis

The list is growing !

Other

Premature birth

Low birth weight

Altered maternal hormones

Developmental effects:

Reduced thymus size

Reproductive system

CNS Inflammation

Cognitive defects

Metabolic Disorders

2010Short-3

Tools for Evaluation of Toxicity

2010Short-4

SOURCE APPORTIONMENT OF IMPACT OF PM10 ON RESPIRATORY AND

CARDIOVASCULAR HOSPITAL ADMISSIONS Janssen et al., Environ. Health Perspect. 110: 43, 2002

Time series study of PM10 vs. hospital admissions in 14 U.S. cities

1985 –1994, adjusted for weather, day, season, and air conditioning

Proportional source contributions from Emission Inventory EstimatesVehicle and oil combustion-derived PM had the greatest impact

% change in regression coefficient for interquartile change in PM10 (*p<0.05)

PM10 Source CVD COPD Pneumonia

Highway vehicles +58* +48 +61

Highway diesels +56* +47 +56

Oil combustion +38* +2 +27

Wood burning +3 +1 +2

Coal combustion +1 +0 +2

Metal processing +29* +3 +12

Fugitive dust 49* 11 31

*P < .05

2010Short-5

EFFECTS OF PROXIMITY TO TRAFFIC ON LUNG FUNCTION OF CHILDREN

Brunekreef et al., Epidemiology 8:298, 1997

Lung function of 877 9 yr olds and air pollution at schools in 6 locations

Measured black smoke (proxy for diesel), PM10, and NO2

Traffic counts (cars & trucks), distance from motorway, & wind direction

2010Short-6

IF MOBILE SOURCE COMBUSTION IS IMPLICATED, WHAT PART OF EMISSIONS ARE MOST IMPORTANT?

2010Short-7

C57Bl6N Male Mice Exposed by Inhalation 6h/day 7 daysExposed to whole exhaust with PM range from 30-3000 µg/m3

Exhaust generated from Yanmar Genset: No 2 fuel, steady stateComposition of exhaust varied by change in engine operation

Group 1: Implanted with telemeters to monitor heart rate/varianceGroup 2: Challenged with respiratory syncytial virus at end of exposure

Evaluated 4 days later for viral persistance/pathology

2010Short-8

ENGINE OPERATION CONDITIONS HAD MARKED IMPACT ON COMPOSITION

2010Short-9

HIGH BLACK CARBON CONTENT/LOW ORGANICS LED TO INCREASED SUSCEPTIBILITY TO INFECTION

2010Short-10

HIGH ORGANIC (VOLATILE AND NON) HAD LARGEST IMPACT ON CARDIOVASCULARPHYSIOLOGY

2010Short-11

THE BEHAVIOR OF ULTRAFINES AND NANOPARTICLES WILL DEPEND ON THEIR COMPOSITION, AS WELL AS THEIR SIZE

Solubility determines whether mass or number/surface area is the most important dose metric

2010Short-12

BOTTOM LINES1. Epidemiology associations have been found with environmental

Pollution and many health outcomes

2. Mobile-Source PM appears to be especially important

3. Careful consideration of the composition and type of exhaust

Needs to be considered when interpreting results

Modern diesel is much different than old diesel

2010Short-13

1. Collected PM and vapor-phase SVOCs during urban driving cycle

2. Analyzed composition in detail

3. Tested the re-combined fractions by instillation into rat lungs

IDENTIFYING TOXIC COMPONENTS OF ENGINE EMISSION

SAMPLES

[Seagrave et al. Toxicol. Sci. 70: 212-226, 2002]

[Zielinska et al., J. Air Waste Man. Assoc. 54: 1138-1150, 2004]

2010Short-14

SAMPLES HAD A 5-FOLD RANGE OF POTENCY

Lung Inflammation

(5 parameters)

2010Short-15

Laboratory Results Also Suggest SOA NOT More Toxic than Primary Sources

Collected Ambient Samples

Explored as Described for Motor Vehicle Study

Source ApportionmentToxicity

2010Short-16

-0.40

-0.30

-0.20

-0.10

0.00

0.10

0.20

0.30

0.40

-0.20 -0.10 0.00 0.10 0.20 0.30

Seco

nd C

ompo

nent

First Component

Loadings plot - 2 components

NH4

NitrateSulfate

EC

OC

As

Cu

Earth Me

Mn

Pb

SeZn

aromatic

branch a

carboxyl

Choleste

dioic ac

Hop and

Levogluc

n-alkane

Nonanal

PAH < 25PAH>252

resin ac

ALP

Cells

LDHlung:BW

LYMPH

MACSPMN

Protein

PCA/PLS Reveals Composition:Toxicity Associations

SOA “Indicators”

Methods reported in McDonald et al.,2004

(diacids)

2010Short-17

“The Working Group found that diesel exhaust is a cause of lung cancer (sufficient evidence) and also noted a positive association (limited evidence) with an increased risk of bladder cancer (Group 1).

The Working Group concluded that gasoline exhaust was possibly carcinogenic to humans (Group 2B), a finding unchanged from the previous evaluation in 1989.”

IARC press release, 2012

IARC: Diesel Exhaust Carcinogenic

2010Short-18

CANCER HAZARD

Mutagenicity

Interest heightened in 1970s with application of Ames testReverse mutations in Salmonella bacteria

Many labs studied many variables through 1980s (huge literature)

Biodirected fractionation pointed toward nitro-aromatic compounds

Carcinogenicity

Several large-scale gasoline and diesel rodent inhalation studiesU.S., Germany, Switzerland, Japan

Results generally consistent across studies

Extreme exposures increased lung tumors in rats

No increase in mice or Syrian hamsters

Gasoline studies did not cause cancer in any species

Led to recognition of “particle overload” phenomenon

Kotin et al., Indust. Health 11:113, 1955 (USC)Solvent extracts of diesel and gasoline emissions caused tumors in mouse skin

painting assay

Related to source, operating condition, and aromatic hydrocarbons

2010Short-19

ORIGINAL LOVELACE DIESEL STUDY (early 1980s)Mauderly et al. Fundam. Appl. Toxicol. 9:208-221, 1987

Male & female F344 rats, n=221-230 examined for tumors/group

Exposed 7 hr/day, 5 days/wk for up to 30 mo1980 5.7L GM engines on FTP, burning certification fuel 0, 350, 3470, or 7080 µg total chamber PM/m3 (control 10

µg/m3 )

High level = 30 ppm CO, 11 ppm NOx, 13 ppm HC

Results:No effect on clinical signs, body weight, or survivalProgressive accumulation of soot in lung at mid & high levelsProgressive inflammatory and fibrotic lung disease at mid &

high levelsLung tumor incidences (benign + malignant)

C = 0.9%, L = 1.3%, M = 3.6%, H = 12.8%

Low level (340 µg exhaust PM/m3): No progressive lung disease or increase in lung tumors

No significant effects on hematology, serum chemistry, bronchoalveolar lavage, pulmonary function, lymphocyte function

in bronchial lymph odes, or histopathology (except for PM in some macrophages)

2010Short-20

ORIGINAL LRRI CARCINOGENICITY STUDY

Mauderly et al. Fundam. Appl. Toxicol. 9:208-221, 1987

Rats exposed 7 hr/day, 5 days/wk x 30 mo to whole diesel exhaust

10

0

0

20

100 200 300

Some estimated risks for humans exposed at much lower levels by linear extrapolation of these data

No significant carcinogenicity in CD-1 mice exposed simultaneously

2010Short-21

THE LACK OF CARCINOGENICITY FROM LOW EXPOSURES WAS TOO READILY IGNORED

Mauderly, in Environmental Toxicants , Lippmann Ed., Wiley, 2000

Increased tumors resulted only from exposures that caused preceding, chronic-active inflammation and progressive epithelial hyperplasia-metaplasia

2010Short-22

RAT LUNG TUMORS IN OLD DIESEL STUDIES WERE PRIMARILY DUE TO LUNG LOADING WITH PM

Nikula et al., Fundam. Appl. Toxicol. 25: 80-94, 1995

Male & Female F344 rats exposed to diesel exhaust (DE) or carbon black (CB)16 hr/day, 5 days/wk at 2 PM concentrations

1988 6.2L GM engine on FTP burning certification fuel

2390 & 6280 µg PM/m3 exhaust PM

10 & 27 ppm CO

9.5 & 27.2 ppm NOx (0.7 & 3.8 ppm NO2)

6.5 & 8.1 ppm HC

Cabot Elftex-122410 & 6500 µg PM/m3 CB

Control

50 µg PM/m3

Lung tumor results (n = 210-213):

DE CB

High level 17.9% 15.2%Low level 6.2% 4.7%Control 1.4%

2010Short-23

THE LUNG “OVERLOADING” CONCEPT EVOLVED

Heinrich et al. Inhal. Toxicol. 7:533-556, 1995

Other poorly-soluble, respirable PM fell on the same exposure-response line

Same exposures were not tumorigenic in normal test strains of mice

Mutagen-free carbon blacks gave same result in two studies

Consensus evolved that results from “overloaded” rats should not be used to estimate risks to humans

Patterns of particle retention in the lung differ between rats and humans

Threshold Nonspecific Not reliable for estimating human risk Species-specific

2010Short-24

Bigchanges!

OLD DIESEL NEW DIESEL

Pre-1990

2010Short-25

NTDE: Less PM and Composition Very Different

TDE NTDE

[Kittelson, 1998] [ACES, 2009]

25

2010Short-26

Pre-Trap Post-Trap

Sulfate/Nitrate

Hydrocarbons (Fuel + Lube derived)

Elemental Carbon + Ash

≥0.1 g/bhp-hr

0.01 g/bhp-hr

Particulate Matter Composition Breakdown

Cummins ISM 2007; Liu et al. , Aerosol Science and Technology, 43/11: 1142-52 2009

-99%Mass

2010Short-27

Combustion and dilution Air Supply

Engine Dynamometer

Engine Cooling heat Exchanger

Primary Dilution Tunnel

Dynamometer Power Supply and Controls

Fuel “Day Tank”

Engine Exhaust Injection Point

Diluted Exhaust Extraction Point

ACES Engine and Primary Dilution System

2010Short-28

28

2010Short-29

CORE BIOSCREENING STUDY DESIGN

Chronic Carcinogenicity Bioassay of Wistar Han Rats:

Expose 288/group 16 hr/day, 5 days/wk for 24-30 months

3 dilutions of whole emissions + clean air controls

166/group committed to carcinogenesis bioassay

122/group allocated for interim evaluations at 1, 3, 12, & 24 months Pulmonary function (3, 12, & 24 mo)Lung lavage, lung tissue & cell proliferation

Hematology & serum chemistry (3, 12, & 24 mo)

Histopathology

2010Short-30

MAIN EXHAUST DUCT

MAIN SUPPLY AIR DUCT

H-2000 EXPOSURE CHAMBER

2ndary CHAMBER DILUTION

Muffler

MAIN CHAMBER DILUTION

Diesel Dilution Tunnel

(Note: Drawing is not to scale)

Sampling Probe flange

CompressedAir

Air-Vac

Primary Dilution Tunnel

Extraction probe flange

Compressed air

Exhaust Extraction and Secondary Dilution Systems

2010Short-31

31

ATMOSPHERE COMPOSITION

Real-time particle mass

Real-time particle number

2010Short-32

Biological Response in Rats

2010Short-33

Histopathology in Rats at 3 Months:

Higher Power View of Previous Slide

Control

High

Thickening of alveolar ductseptae

Macrophage

2010Short-34

3 months

12 months

Enhanced epithelial hyperplasia at 12 months. Only observed at high level, but in both sexes.

Histopathology in Rats at 3 and 12 Months:

2010Short-35

ACES Findings

In a nutshell; we did not observe any exposure related evidence of cancer