Annex D. Controlled Human 

Exposure Studies 

Table D-1. Controlled human exposure studies. 

Study Subjects Exposure Findings 

Adir et al. (1999, 

001026) 

15 healthy nonsmokers 

Gender: M 

Age: 22-34 yr 

Inhaled Concentration: Not provided 

Exposure Duration: 3 min 45 s 

COHb Concentration: 4-6% 

COHb Analysis: CO-oximeter (IL-282) 

Exposures to CO and room air were separated 

by 1 mo, with the order of exposure randomly 

assigned. 

Exposure to CO resulted in a decrease in postexposure 

exercise duration (Bruce protocol) relative to clean air 

exposure in 13 out of 15 subjects (p = 0.0012). Statistically 

significant decreases in METs were also reported following CO 

exposure (p = 0.0001). No CO-induced changes in HR, BP, 

ECG parameters, or myocardial perfusion were observed. 

Bathoorn et al. (2007, 

193963) 

19 former smokers with 

COPD 

Gender: 18 M/1 F 

Age: 66-70 yr 

Inhaled Concentration: 100 ppm (9 subjects) or 

125 ppm (10 subjects) 

Exposure Duration: 2 h on each of 4 

consecutive days 

COHb Concentration: 2.7% (following 4th day 

exposure) 

COHb Analysis: Not provided 

Exposures to CO and room air conducted were 

separated by at least 1 wk, using a randomized 

crossover design. 

Following the 4th day of exposure, CO inhalation reduced 

sputum eosinophils relative to room air and also increased the 

provocative concentration of methacholine required to cause a 

20% reduction in FEV1. Neither of these effects were shown to 

reach statistical significance. No changes in sputum 

neutrophils, white blood cell counts or serum C-reactive 

protein (CRP) were observed. Although this study appears to 

demonstrate some evidence of an anti-inflammatory effect of 

CO among subjects with COPD, it must be noted that 2 of 

these patients experienced exacerbations of COPD during or 

following CO exposure, with 1 patient requiring hospitalization 

2 mo after exposure (initial symptoms first experienced 1 wk 

postexposure). 

Hanada et al. (2003, 

193915) 

20 healthy adults 

Gender: M 

Age: 26 ± 1 yr 

Inhaled Concentration: Not provided 

Exposure Duration: 20 min 

COHb Concentration: 20-24% 

COHb Analysis: CO-oximeter (OSM-3) 

15 subjects exposed for 20 min (10 min rest, 

5 min handgrip exercise, 2 min postexercise 

ischemia, 3 min recovery) under the following 4 

conditions: (1) normoxia (inspiratory O2 fraction 

21.4%); (2) hypoxia (inspiratory O2 fraction 

10.3%); (3) CO + normoxia; and (4) CO + 

hyperoxia (inspiratory O2 fraction 95.9%). Trials 

involving exposure to CO were conducted last in 

this sequence. Each of the 4 conditions was 

separated from the next by 20 min of rest. 

5 subjects served as controls (4 consecutive 20 

min periods of normoxia). 

Blood oxygenation, BP, HR and respiratory rate were 

measured during exposure. Muscle sympathetic nerve activity 

(MSNA) and leg hemodynamics were evaluated in two subsets 

of the study group (n = 8 and 7, respectively). Arterial oxygen 

saturation (pulse oximetry) was significantly lower, and resting 

HR and ventilation significantly higher during the period of 

hypoxia compared to the other periods; none of these 

measures were affected by exposure to CO. MSNA was 

shown to increase during hypoxia and CO exposure relative to 

normoxia. Neither hypoxia nor CO was found to affect leg 

blood flow or vasoconstriction. 

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

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January 2010 D-1 
Study Subjects Exposure Findings 

Kizakevich et al. (2000, 

052691) 

16 healthy nonsmokers 

Gender: M 

Age: 18-29 yr 

Inhaled Concentration: Initial short term 

(4-6 min) exposure to 1,000 or 3,000 pap, 

followed by exposures to 27, 55, 83, or 100 ppm 

to maintain COHb concentration. 

Exposure Duration: 4-6 min at 1,000 or 

3,000 pap, followed by 20 min at 27, 55, 83, or 

100 ppm. 

Target COHb Concentrations: 5, 10, 15, and 

20% 

COHb Analysis: CO-oximeter (IL-282) 

Subjects exposed on 4 separate days to 

increasing CO concentrations during either 

upper-body exercise (hand-crank) or lower-body 

exercise (treadmill). Targeted COHb 

concentrations were initially attained using shortterm (4-6 min) exposures to CO at 

concentrations of 1,000 or 3,000 ppm. Chamber 

exposures were then conducted at CO 

concentrations required to maintain COHb levels 

of <2% (room air), 5% (27 ppm), 10% (55 ppm), 

15% (83 ppm), and 20% (100 ppm). 

At all levels of upper- and lower-body exercise, exposures to 

CO resulted in increases in HR, cardiac output, and cardiac 

contractility relative to clean-air exposures. Increases in HR 

reached statistical significance at COHb concentrations ≥ 5%, 

and increases in both cardiac output and cardiac contractility 

reached statistical significance at COHb concentrations 

≥ 10%. CO exposure during exercise was not observed to 

cause ventricular arrhythmias or affect ECG wave shape (no 

evidence of ST-segment depression) at COHb concentrations 

≤ 20%. 

Mayr et al. (2005, 

193984) 

13 healthy nonsmokers 

Gender: M 

Age: 18-38 yr 

Inhaled Concentration: 500 ppm 

Exposure Duration: 1 h 

COHb Concentration: 7% 

COHb Analysis: CO-oximeter (AVL 912) 

Subjects exposed to both CO and clean air with 

exposures separated by a 6-wk period. 

Immediately following exposure, subjects were 

administered an intravenous bolus dose 

(2 ng/kg) of lipopolysaccharide (LPS). 

Infusion of LPS significantly increased plasma concentrations 

of TNF-α, CRP, IL-6, and IL-8, with no difference in the 

inflammatory response between clean-air and CO exposures. 

Morse et al. (2008, 

097980) 

12 healthy nonsmokers 

Gender: M 

Age: 25 ± 2.9 yr 

Inhaled Concentration: 3,000 ppm 

Exposure Duration: 3-8 min 

COHb Concentration: 6.2% 

COHb Analysis: Electrochemical sensor 

(Smokerlyzer) measuring CO in exhaled breath 

Exposures conducted on2 separate occasions to 

room air (6 min) and CO. Subjects were exposed 

to CO until COHb reached 6% (3- to 8-min 

exposures). 

Leg strength and muscle fatigue were evaluated immediately 

following exposure. CO exposure did not affect muscle 

strength (maximal voluntary isometric contraction) but did 

cause a statistically significant increase in muscle fatigue 

(p < 0.05). 

Ren et al. (2001, 

193850) 

12 healthy adults 

(10 nonsmokers and 1 

smoker) 

Gender: 9 M/3 F 

Age: 20-32 yr 

Inhaled Concentration: 0.4% (4,000 ppm) 

Exposure Duration: 10-30 min at 0.4% followed 

by ~ 8-h with periodic exposure to maintain 

COHb concentration 

COHb Concentration: 10% 

COHb Analysis: Not provided 

Each subject underwent 4 different 8-h 

experimental protocols: (1) isocapnic hypoxia 

(end-tidal PO2 held at 55 mmHg); (2) withdrawal 

of 500 mL of venous blood at the start of an 8-h 

period; (3) CO exposure at a concentration 

required to maintain a COHb level of 10%; and 

(4) a control exposure where subjects breathed 

room air with no intervention. 

A statistically significant increase in ventilation was observed 

following hypoxia, but no such increase was found following 

any of the other 3 protocols, including exposure to CO. One 

subject felt faint during the blood withdrawal protocol and did 

not complete the study. 

January 2010 D-2 
Study Subjects Exposure Findings 

Resch et al. (2005, 

193853) 

15 healthy nonsmokers 

Gender: M 

Age: 27 ± 4 yr 

Inhaled Concentration: 500 ppm 

Exposure Duration: 1 h 

COHb Concentration: ~ 10% 

COHb Analysis: CO-oximeter (AVL 912) 

Exposures to CO and synthetic air control were 

separated by a period of at least 1 wk. 

COHb levels averaged 5.6% after 30 min and 9.4% after 60 

min of exposure. Statistically significant increases in retinal 

blood flow, retinal vessel diameter, and choroidal blood flow 

were observed with CO exposure relative to synthetic air at 

both time points. Exposure to CO did not affect oxygen 

saturation of arterial blood. 

Vesely et al. (2004, 

194000) 

10 healthy nonsmokers 

Gender: M 

Age: 22-52 yr 

Inhaled Concentration: 1,200 ppm 

Exposure Duration: 30-45 min 

COHb Concentration: 10% 

COHb Analysis: CO-oximeter (OSM-3) 

Prior to and following exposure, subjects 

performed hypoxic and hyperoxic rebreathing 

tests. Four subjects were exposed to hypoxic 

conditions first, while 6 subjects were exposed to 

hyperoxic conditions first, both prior to and 

following CO exposure. 

Ventilation rate was observed to significantly increase during 

hypoxic rebreathing relative to hyperoxic rebreathing. 

However, exposure to CO had no effect on ventilation under 

either hypoxic or hyperoxic conditions. The authors concluded 

that exposure to low levels of CO does not significantly affect 

chemoreflex sensitivity of the CO2-induced stimulation of 

ventilation. 

Zevin et al. (2001, 

021120) 

12 healthy smokers 

Gender: M 

Age: 27-47 yr 

Inhaled Concentration: 1,200-1,500 ppm 

Exposure Duration: 10 min each h, 16 h each 

day, over 7 days 

COHb Concentration: 5-6% 

COHb Analysis: CO-oximeter (Ciba Corning 

2500) 

Exposures were conducted over 21 consecutive 

days under3 different protocols, with each 

protocol lasting 7 days. In 1 protocol, subjects 

smoked 20 cigarettes per day, 1 every 45 min. In 

the other 2 protocols, every 45 min (20 times per 

day) subjects breathed either air or CO from a 

1-liter bag once per min for 10 min at a time. 

Subjects completed all 3 protocols, with 6 

subjects exposed sequentially to CO, smoking, 

then air, and the other 6 exposed sequentially to 

air, smoking, then CO. 

COHb levels were similar during smoking and exposure to CO, 

with average concentrations of 6% and 5%, respectively. 

Blood was drawn on day 4 of each exposure and analyzed for 

CRP, plasma platelet factor 4, and white blood cell count. 

Plasma levels of CRP and platelet factor 4 were significantly 

elevated with smoking but not with CO exposure, relative to air 

control. HR and BP were evaluated on day 3 of each protocol. 

Cigarette smoke but not CO was observed to significantly 

increase HR, while no difference in BP was observed between 

any of the 3 exposures. 

January 2010 D-3 
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monoxide on exercise performance and myocardial perfusion in young healthy men. Occup Environ Med, 56: 535-

538. 001026

Bathoorn E; Slebos DJ; Postma DS; Koeter GH; van Oosterhout AJ; van der Toorn M; Boezen HM; Kerstjens HA (2007). 

Anti-inflammatory effects of inhaled carbon monoxide in patients with COPD: A pilot study. Eur Respir J, 30: 

1131-1137. 193963

Hanada A; Sander M; González-Alonso J (2003). Human skeletal muscle sympathetic nerve activity, heart rate and limb 

haemodynamics with reduced blood oxygenation and exercise. J Physiol, 551: 635-647. 193915

Kizakevich PN; McCartney ML; Hazucha MJ; Sleet LH; Jochem WJ; Hackney AC; Bolick K (2000). Noninvasive 

ambulatory assessment of cardiac function in healthy men exposed to carbon monoxide during upper and lower 

body exercise. Eur J Appl Physiol, 83: 7-16. 052691

Mayr FB; Spiel A; Leitner J; Marsik C; Germann P; Ullrich R; Wagner O; Jilma B (2005). Effects of carbon monoxide 

inhalation during experimental endotoxemia in humans. Am J Respir Crit Care Med, 171: 354-360. 193984

Morse CI; Pritchard LJ; Wust RC; Jones DA; Degens H (2008). Carbon monoxide inhalation reduces skeletal muscle 

fatigue resistance. Eur Phys J A, 192: 397-401. 097980

Ren X; Dorrington KL; Robbins PA (2001). Respiratory control in humans after 8 h of lowered arterial PO2, hemodilution, 

or carboxyhemoglobinemia. J Appl Physiol, 90: 1189-1195. 193850

Resch H; Zawinka C; Weigert G; Schmetterer L; Garhofer G (2005). Inhaled carbon monoxide increases retinal and 

choroidal blood flow in healthy humans. Invest Ophthalmol Vis Sci, 46: 4275-4280. 193853

Vesely AE; Somogyi RB; Sasano H; Sasano N; Fisher JA; Duffin J (2004). The effects of carbon monoxide on respiratory 

chemoreflexes in humans. Environ Res, 94: 227-233. 194000

Zevin S; Saunders S; Gourlay SG; Jacob P III; Benowitz NL (2001). Cardiovascular effects of carbon monoxide and 

cigarette smoking. J Am Coll Cardiol, 38: 1633-1638. 021120

 

Note: Hyperlinks to the reference citations throughout this document will take you to the NCEA HERO database (Health and 

Environmental Research Online) at http://epa.gov/hero. HERO is a database of scientific literature used by U.S. EPA in the process of 

developing science assessments such as the Integrated Science Assessments (ISAs) and the Integrated Risk Information System (IRIS). 

January 2010 D-4