DEPARTMENT OF HEALTH AND HUMAN SERVICES 

Centers for Disease Control and Prevention 

National Institute for Occupational Safety and Health

Criteria for a Recommended Standard

Occupational Exposure to Diacetyl 

and 2,3-Pentanedione
On the cover: The chemical structure of diacetyl
Criteria for a Recommended Standard

Occupational Exposure to 

Diacetyl and 2,3-Pentanedione

DEPARTMENT OF HEALTH AND HUMAN SERVICES

Centers for Disease Control and Prevention

National Institute for Occupational Safety and Health

McKernan LT, Niemeier RT, Kreiss K, Hubbs A, Park R, 

Dankovic D, Dunn KH, Parker J, Fedan K, Streicher R, Fedan 

J, Garcia A, Whittaker C, Gilbert S, Nourian F, Galloway E, 

Smith R, Lentz TJ, Hirst D, Topmiller J, Curwin B
ii Occupational Exposure to Diacetyl and 2,3-Pentanedione

This document is in the public domain and may be freely copied or reprinted.

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Suggested Citation

NIOSH [2016]. Criteria for a recommended standard: occupational exposure to diacetyl and 

2,3-pentanedione. By McKernan LT, Niemeier RT, Kreiss K, Hubbs A, Park R, Dankovic D, Dunn 

KH, Parker J, Fedan K, Streicher R, Fedan J, Garcia A, Whittaker C, Gilbert S, Nourian F, Galloway E, 

Smith R, Lentz TJ, Hirst D, Topmiller J, Curwin B. Cincinnati, OH: U.S. Department of Health and 

Human Services, Centers for Disease Control and Prevention, National Institute for Occupational 

Safety and Health, DHHS (NIOSH) Publication No. 2016-111.

DHHS (NIOSH) Publication No. 2016-111

October 2016
Occupational Exposure to Diacetyl and 2,3-Pentanedione iii

Foreword

When the U.S. Congress passed the Occupational Safety and Health Act of 1970 (Public Law 91-596), 

it established the National Institute for Occupational Safety and Health. Through the Act, Congress 

charged NIOSH with recommending occupational safety and health standards and describing exposure levels that are safe for various periods of employment, including but not limited to the exposures at which no employee will suffer diminished health, functional capacity, or life expectancy 

because of his or her work experience.

Criteria documents contain a critical review of the scientific and technical information about the 

prevalence of hazards, the existence of safety and health risks, and the adequacy of control methods. 

By means of criteria documents, NIOSH communicates these recommended standards to regulatory agencies, including the Occupational Safety and Health Administration, health professionals in 

academic institutions, industry, organized labor, public interest groups, and others in the occupational safety and health community.

This criteria document is derived from the NIOSH evaluation of critical health effects studies of 

occupational exposure to diacetyl and 2,3-pentanedione. It provides recommendations for controlling workplace exposures including recommended exposure limits derived by using current quantitative risk assessment methodology on human and animal health effects data. 

Using cross-sectional pulmonary function data from diacetyl-exposed employees, NIOSH conducted assessments to determine the exposure-response relationship and to identify risk of pulmonary function decrease at various levels of diacetyl exposure. NIOSH found that a relationship exists 

between diacetyl exposures and lower pulmonary function. Utilizing this analysis, NIOSH recommends keeping exposure to diacetyl below a concentration of 5 parts per billion as a time-weighted 

average during a 40-hour work week. To further protect against effects of short-term exposures, 

NIOSH recommends a short-term exposure limit for diacetyl of 25 parts per billion for a 15-minute 

time period.

In many operations, 2,3-pentanedione is being used to substitute for diacetyl. Published toxicological studies indicate that 2,3-pentanedione exposure can cause damage similar to that caused by 

diacetyl in laboratory studies. Therefore, NIOSH recommends keeping occupational exposure to 

2,3-pentanedione below a level comparable to the level recommended for diacetyl. However, the 

recommended sampling and analytical method can only reliably quantify it to 9.3 parts per billion 

in an 8-hour sample. NIOSH also recommends a short-term exposure limit for 2,3-pentanedione of 

31 parts per billion during a 15-minute period.

Engineering and work practices are available to control diacetyl and 2,3-pentanedione exposures 

below the recommended exposure limits. A hierarchy of controls including elimination, substitution, engineering controls, administrative controls, and the use of personal protective equipment 

should be followed to control workplace exposures.
Foreword

iv Occupational Exposure to Diacetyl and 2,3-Pentanedione

NIOSH urges employers to disseminate this information to employees and customers. NIOSH also 

requests that professional and trade associations and labor organizations inform their members 

about the hazards of occupational exposure to these flavoring compounds.

NIOSH appreciates the time and effort of the expert peer, stakeholder, and public reviewers whose 

comments and input strengthened this document.

John Howard, MD

Director, National Institute for 

 Occupational Safety and Health

Centers for Disease Control and Prevention
Occupational Exposure to Diacetyl and 2,3-Pentanedione v

Executive Summary

Diacetyl and its substitute, 2,3-pentanedione, are widely used flavoring compounds. There have been 

extensive reports of serious respiratory disease and decreased lung function in employees exposed to 

diacetyl. The NIOSH objective in establishing recommended exposure limits (RELs) for diacetyl and 

2,3-pentanedione is to reduce the risk of respiratory impairment (decreased lung function) and the 

severe irreversible lung disease obliterative bronchiolitis associated with occupational exposure to these 

compounds. In this disease the smallest airways in the lungs, the bronchioles, become scarred and constricted, blocking the movement of air. In addition, maintaining exposures below the RELs will help 

prevent other adverse health effects including but not limited to irritation of the skin, eyes, and respiratory tract in exposed employees. The recommendation to limit exposure to diacetyl and 2,3-pentanedione is based upon data from human and animal studies and the quantitative risk assessment; however, 

additional considerations include sampling and analytical feasibility and the achievability of engineering controls.

Diacetyl is used extensively in the food flavoring and production industries, and occupational exposure to this substance has been associated with severe respiratory impairment and the disease obliterative bronchiolitis. 2,3-Pentanedione, which has been used as a substitute for diacetyl, is also of concern 

because of structural similarities with diacetyl and because animal studies show similar toxicity for the 

respiratory tract [Hubbs et al. 2012; Morgan et al. 2012; Morgan et al. 2016].

The first observation of obliterative bronchiolitis in a food production employee may have occurred in 

1985 in a facility where diacetyl was listed among ingredients used in making flavorings for the baking 

industry [NIOSH 1985]. The link between exposure to diacetyl and lower pulmonary function was 

confirmed in the early 2000s, and research further showed that diacetyl exposure leads to a decrease in 

pulmonary function [Kreiss et al. 2002]. Occupational exposures to diacetyl have been assessed in a variety of food production and flavoring facilities [Kanwal et al. 2006; Martyny et al. 2008; NIOSH 2003a, b, 

2004a, b, 2006, 2007, 2008a, b, 2009, 2011]. 

Another compound, acetoin, was present along with diacetyl in many of the workplaces where obliterative bronchiolitis occurred in employees who made or used diacetyl [Kullman et al. 2005; van Rooy et 

al. 2007]. However, current data indicate that acetoin is considerably less hazardous than diacetyl and it 

does not have the reactive α-dicarbonyl group, which has been implicated in the toxicity of diacetyl and 

2,3-pentanedione [Hubbs et al. 2016; National Toxicology Program 2015; Zaccone et al. 2013].

Mean diacetyl air concentrations measured at the first microwave popcorn facility where obliterative 

bronchiolitis was reported were highest in the mixing room (57.2 parts per million [ppm]), followed 

by the packaging area (2.8 ppm) [Kanwal et al. 2011]. Mean personal diacetyl air concentrations at five 

other microwave popcorn plants were lower: 0.023 to 1.16 ppm in the mixing room and 0.35 to 1.33 ppm 

in the packaging rooms/areas [Kanwal et al. 2006]. Mean full-shift diacetyl air concentrations measured 
Executive Summary

vi Occupational Exposure to Diacetyl and 2,3-Pentanedione

at flavor manufacturing facilities ranged from 0.07 ppm to 2.73 ppm [Kanwal et al. 2006; Martyny et al. 

2008; NIOSH 2003a, b, 2004a, b, 2006, 2007, 2008a, b, 2009, 2011]. 

In addition to cases consistent with obliterative bronchiolitis in flavoring manufacturing, diacetyl manufacturing, and microwave popcorn production, case reports have surfaced in other industries in which 

flavorings are introduced. In cookie manufacturing with artificial butter flavoring in Brazil, four cases 

of bronchiolitis were described in young men, aged 24 to 27 years, who had worked between 1 and 3 

years handling flavorings in preparation of cookie dough [Cavalcanti et al. 2012]. In a coffee production 

plant, two cases have biopsy confirmation of obliterative bronchiolitis among employees with artificial 

flavorings exposure in the production of roasted coffee beans and ground coffee [CDC 2013]. In 2012, 

NIOSH conducted a health hazard evaluation (HHE) involving 75 current employees (88% participation) [Bailey et al. 2015]. Excluding the five sentinel former employees (all never-smokers under age 42), 

standardized morbidity ratios were elevated 1.6-fold for shortness of breath and 2.7-fold for obstructive 

spirometric abnormalities.

Investigations of severe lung disease consistent with obliterative bronchiolitis among diacetylexposed employees presented in Chapter 3 have provided substantial evidence of a causal relationship between diacetyl exposure and development of this disease. These findings in conjunction 

with laboratory experiments providing biological plausibility, meet the standard criteria used to 

determine causation: that an exposure is the likely cause of specific health effects [Gordis 1996; 

Hill 1965]. 

NIOSH has reviewed the literature on diacetyl toxicology and exposures in the workplace and subsequently conducted a quantitative risk assessment. The quantitative risk assessment used to derive 

the REL was based solely on human (employee) data, but the results were informed and corroborated 

by animal risk assessments. On the basis of a quantitative risk assessment of data collected in a series 

of NIOSH health hazard investigations (full description in Chapter 5), NIOSH has concluded that 

employee exposure to diacetyl is associated with a reduction in lung function. Specifically, a statistically 

significant exposure-associated reduction in the forced expiratory volume in one second/forced vital 

capacity (FEV1/FVC) ratio and percent predicted FEV1 (ppFEV1)and an exposure-associated estimated 

incidence of symptomatic obstructive lung disease were observed. NIOSH quantified these exposureresponse relationships and determined the exposure levels that correspond to a variety of risks (Chapter 

5, Table 5.35). Lifetime risks in the range of 1:1,000 corresponded to working lifetime diacetyl exposure 

of approximately 5 parts per billion (ppb). Once the risks were characterized, NIOSH examined the 

analytical methods (OSHA Methods 1012 and 1016) and available engineering controls and determined 

that they supported establishing a REL at that level. 

It should be noted that diacetyl and 2,3-pentanedione are found in cigarette smoke [Fujioka and 

Shibamoto 2006; Pierce et al. 2014; Polzin et al. 2007] and some flavored e-cigarettes [Allen et al. 2016; 

Farsalinos et al. 2015]. As extensively discussed in Chapter 3, increased prevalence of airway obstruction 

and decreased FEV1 can be identified in smokers who are exposed to diacetyl in comparison to prevalence in smokers in the U.S. population. Most importantly, because diacetyl causes obstructive lung 

disease and because smoking causes obstructive lung disease, the presence of diacetyl in cigarette smoke 

in no way diminishes the need to control diacetyl exposures in employees. 

NIOSH concludes that the toxicological responses to diacetyl observed in animal studies support the 

conclusions of the epidemiologically-based risk assessment for diacetyl. Further, the animal-based 
Executive Summary

Occupational Exposure to Diacetyl and 2,3-Pentanedione vii

risk assessment presented in Chapter 6 corroborates the epidemiologic assessment by demonstrating 

a causal link between diacetyl exposure and respiratory health effects and by showing a clear doseresponse relationship in exposed animals as was observed in employees exposed to diacetyl in the epidemiologic assessment.

On this basis, NIOSH recommends a REL of 5 ppb for diacetyl as a time-weighted average (TWA) for up 

to 8 hours/day during a 40-hour workweek. NIOSH has determined that employees exposed to diacetyl 

at this level for 8 hours a day, 40 hours a week for a 45-year working lifetime should have no more than 

a 1/1,000 excess risk of lung function falling below the lower limit of normal due to diacetyl exposure. 

To ensure that employee exposures are routinely below the REL for diacetyl, NIOSH also recommends 

using an action level (AL) of 2.6 ppb with the exposure monitoring program to ensure that all control 

efforts (engineering controls, medical surveillance, and work practices) are in place and working properly. When exposures exceed the AL, employers should take corrective action (determine the source of 

exposure, identify methods for controlling exposure) to ensure that exposures are maintained below the 

REL. NIOSH has concluded that the use of an AL in conjunction with periodic monitoring of employee 

exposures (described in Chapter 10) is helpful to protect employees.

NIOSH is also recommending a short-term exposure limit (STEL) for diacetyl of 25 ppb for a 15-minute 

time period. The establishment of a STEL is based on the concern that peak exposures may have greater 

toxicity than the same total dose spread out over a longer period of time. 

2,3-Pentanedione is used in many operations; it is structurally similar to diacetyl because it is a 5-carbon 

alpha diketone, whereas diacetyl is a 4-carbon alpha diketone. Published toxicology studies indicate that 

2,3-pentanedione exposure can cause damage to the lining of airways similar to that caused by diacetyl 

in laboratory studies [Hubbs et al. 2012; Morgan et al. 2012; Morgan et al. 2016]. Therefore, NIOSH 

recommends controlling occupational exposure to 2,3-pentanedione to a level comparable to that recommended for diacetyl. However, analytical limitations allow 2,3-pentanedione to be reliably measured 

only above 9.3 ppb. This recommended exposure limit is slightly higher than the recommended exposure limit for diacetyl. NIOSH recommends keeping exposure to 2,3-pentanedione below 9.3 ppb in an 

8-hour average during a 40-hour work week. NIOSH has estimated that employees exposed to 2,3-pentanedione at this concentration should have a similar risk of decreased pulmonary function as employees exposed to diacetyl. NIOSH also recommends a short-term exposure limit for 2,3-pentanedione of 

31 ppb during a 15-minute period.

Research into various flavoring industries has led to the development of engineering controls that may 

help reduce employee exposure to diacetyl, 2,3-pentanedione, and other chemicals. Chapter 8 describes 

engineering controls for the industries where diacetyl is handled or used within products. Table 8.2 in 

Chapter 8 provides a summary of NIOSH-evaluated engineering control efficiencies for the mixing of 

food flavorings. NIOSH acknowledges that the frequent use of personal protective equipment (PPE), 

including respirators, may be required for some employees who handle diacetyl, 2,3-pentanedione, 

diacetyl-containing flavorings or flavored products. The frequent use of PPE may be required during job 

tasks for which (1) routinely high airborne concentrations of diacetyl or 2,3-pentanedione (e.g., pouring, 

mixing, packaging) exist, (2) the airborne concentration of diacetyl or 2,3-pentanedione is unknown or 

unpredictable, and (3) job tasks are associated with highly variable airborne concentrations because of 

environmental conditions or the manner in which the job task is performed. In all work environments 
Executive Summary

viii Occupational Exposure to Diacetyl and 2,3-Pentanedione

where diacetyl, 2,3-pentanedione, diacetyl-containing flavorings, or flavored products are found control 

of exposure through engineering controls should be the highest priority.

NIOSH recommends that employers develop and implement comprehensive occupational safety and 

health programs to protect employees with potential exposure to diacetyl, 2,3-pentanedione, and other 

potentially hazardous flavoring compounds. This program should include periodic exposure and medical evaluation and monitoring exposure controls and appropriate employee training on potential health 

effects, respiratory protection, and use of controls. Employers should (1) determine employee exposure 

to diacetyl, 2,3-pentanedione, and other flavoring compounds used in the workplace; (2) evaluate the 

effectiveness of work practice and engineering controls; and (3) facilitate the selection of appropriate 

personal protective equipment. Because diacetyl and 2,3-pentanedione are found in cigarette smoke 

[Fujioka and Shibamoto 2006; Pierce et al. 2014; Polzin et al. 2007] and e-cigarettes, NIOSH also recommends that all employers make tobacco cessation programs available to employees and have workplaces 

that are free of tobacco smoking and vaping with flavored nicotine delivery systems [NIOSH 2015]. 

All permanent, temporary, and contract employees who work in or enter areas where diacetyl, 2,3-pentanedione, or similar flavoring compounds or products that contain these compounds are used or produced should be included in the medical monitoring program. Employees who work in or enter these 

areas for a total of 40 or more hours per year should be included in the medical monitoring program. 

Because of the potentially rapid progression and grave consequences of flavoring-related lung disease, it 

is important that the medical monitoring program director be able to quickly evaluate clinical data and 

make medical judgments about appropriate diagnostic and therapeutic measures, including medical 

removal. For this reason, the medical monitoring program director should be a licensed physician with 

training and experience in identifying and preventing occupational lung disease. The medical program 

that includes the following:

■ good quality spirometry testing for pulmonary function

■ medical evaluation for employees found with abnormal spirometry

■ removal from exposure pending medical evaluation

■ analysis of group medical surveillance and longitudinal spirometry data to assess workrelated risk factors on the basis of job, task, area, and other exposure indices

The purpose of this epidemiologic surveillance is to assist monitoring physicians in assessing the likelihood of work-related causes of abnormalities and to prioritize interventions, if needed. Identifying 

excessive declines in spirometry, even if absolute spirometric values remain within the normal range, 

offers the best opportunity to intervene before progression to symptomatic impairment and to prevent 

the development of clinically significant occupational lung disease. The rapid onset and progression of 

diacetyl-related lung disease requires more frequent medical monitoring evaluations be done than with 

slowly progressive occupational lung diseases, such as silicosis and coal employees pneumoconiosis. 

While the focus of this document is on diacetyl and 2,3-pentanedione, NIOSH has concern regarding 

other volatile and reactive flavorings potentially capable of producing similar toxic effects. Therefore, 

NIOSH recommends that such exposures be carefully considered and controlled in consultation with 

workplace safety professionals and the recommendations contained within this criteria document.
Occupational Exposure to Diacetyl and 2,3-Pentanedione ix

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Occupational Exposure to Diacetyl and 2,3-Pentanedione xiii

Contents

Foreword . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . iii

Executive Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .v

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix

Abbreviations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xix

Glossary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xxv

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .xxvii

1 Introduction 1

1.1 Purpose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.2 Scope . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1

1.3 Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

1.4 Chemical and Physical Properties . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

1.4.1 How Diacetyl and 2,3-Pentanedione are Prepared . . . . . . . . . . . . . . . . . . . 4

1.5 Production Uses and Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

1.6 Potential for Exposures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

2 Assessing Occupational Exposure in Employees 15

2.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.2 Time-integrated Air Sampling and Analytical Methods for Diacetyl 

and 2,3-Pentanedione Vapor . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.2.1 OSHA Methods 1012 and 1013 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 15

2.2.2 OSHA Method 1016 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.2.3 OSHA Method PV2118 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

2.2.4 NIOSH Method 2557 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

2.2.5 Other Air Sampling Method(s) in Development . . . . . . . . . . . . . . . . . . . . 17

2.2.6 NIOSH Method 2549 – Qualitative Determination of 

Volatile Organic Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.3 Sampling for Diacetyl and 2,3-Pentanedione in Airborne 

Dust and in Bulk Materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.3.1 Size-Selective Air Sampling for Dust . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.3.2 Sampling for Diacetyl and 2,3-Pentanedione in Airborne Dust . . . . . . . . . . . . 19

2.3.3 Bulk Liquids and Solids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

2.4 Real-time Techniques for Diacetyl and Other Flavoring Compounds . . . . . . . . . . . 20

2.4.1 Photoionization Detectors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.4.2 Infrared Analyzers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

2.4.3 Photoacoustic Spectroscopy (Infrared Absorbance) Techniques . . . . . . . . . . . 22

2.5 Industrial Hygiene Surveys and Exposure Assessments . . . . . . . . . . . . . . . . . . . 22
Contents

xiv Occupational Exposure to Diacetyl and 2,3-Pentanedione

2.5.1 NIOSH Microwave Popcorn Production Exposure Assessments . . . . . . . . . . . 23

2.5.2 Other Microwave Popcorn Production Exposure Assessments . . . . . . . . . . . . 23

2.5.3 NIOSH Flavoring Manufacturing Exposure Assessments . . . . . . . . . . . . . . . 23

2.5.4 Other Flavoring Manufacturing Exposure Assessments . . . . . . . . . . . . . . . . 28

2.5.5 NIOSH Flavored Food Production Exposure Assessments . . . . . . . . . . . . . . 29

2.5.6 OSHA Site Visits Related to Diacetyl and Flavorings that Contain Diacetyl . . . . . 31

2.5.7 Other Exposure Assessments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 33

3 Effects of Exposure in Employees 37

3.1 Obstructive Lung Disease Consistent with Obliterative Bronchiolitis . . . . . . . . . . . 37

3.1.1 Bronchiolar Disease and Terminology . . . . . . . . . . . . . . . . . . . . . . . . . . 43

3.1.2 Evidence from Field Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45

3.2 Restrictive Spirometry in Flavoring-exposed Workforces . . . . . . . . . . . . . . . . . . 59

3.2.1 Index Plant Findings Regarding Restriction . . . . . . . . . . . . . . . . . . . . . . . 60

3.2.2 NIOSH Findings of Restrictive Spirometry at Other Microwave Popcorn Plants . . 62

3.2.3 NIOSH Findings of Restrictive Spirometry at Flavoring Manufacturing Plants . . . 62

3.3 Rapid Lung Function Decline . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

3.4 Asthma . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

3.5 Mucous Membrane Irritation (Eye, Upper Respiratory) . . . . . . . . . . . . . . . . . . . 70

3.6 Dermatologic Effects . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

3.7 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75

4 Toxicology of Diacetyl and 2,3-Pentanedione 79

4.1 Chemistry and Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

4.1.1 Diacetyl and 2,3-Pentanedione in Food . . . . . . . . . . . . . . . . . . . . . . . . . 79

4.1.2 Metabolism in Mammalian Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

4.2 In Vivo and in Vitro Toxicology Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

4.2.1 In Vivo Toxicology of Orally Administered Diacetyl . . . . . . . . . . . . . . . . . . 87

4.2.2 Effects of Topically Applied Diacetyl and 2,3-Pentanedione in Vivo . . . . . . . . . 87

4.2.3 Toxicology of Inhaled Diacetyl in Vivo . . . . . . . . . . . . . . . . . . . . . . . . . 88

4.2.4 In Vitro Toxicology of Diacetyl and 2,3-Pentanedione . . . . . . . . . . . . . . . . . 90

4.2.5 Toxicology of Inhaled Diacetyl Substitutes . . . . . . . . . . . . . . . . . . . . . . . 92

4.2.6 Diacetyl and 2,3-Pentanedione in Cigarette Smoke . . . . . . . . . . . . . . . . . . 93

4.2.7 Relevance of Diacetyl Animal Studies to Humans . . . . . . . . . . . . . . . . . . . 95

4.3 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 99

5 Quantitative Risk Assessment Based on Employee Data 105

5.1 Methods: Study Population, Exposure Assessment, and Outcomes . . . . . . . . . . . . . 106

5.1.1 Study Population . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 106

5.1.2 Environmental Assessment and Exposure Estimation . . . . . . . . . . . . . . . . . 107

5.1.3 Work History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
Contents

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5.1.4 Outcomes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

5.2 Methods: Analysis of Exposure Response . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

5.2.1 Exposure Metrics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 110

5.2.2 Models of Percent Predicted FEV1 and FEV1/FVC . . . . . . . . . . . . . . . . . . . 111

5.2.3 Models of the Incidence of Pulmonary Obstruction . . . . . . . . . . . . . . . . . . 111

5.3 Results: Exposure Response . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 112

5.3.1 Cross-Sectional Pulmonary Function Changes . . . . . . . . . . . . . . . . . . . . . 112

5.3.2 Longitudinal Analyses of ppFEV1 at Company G . . . . . . . . . . . . . . . . . . . . 118

5.3.3 Incidence of Pulmonary Impairment at Company G . . . . . . . . . . . . . . . . . . 118

5.3.4 Evidence of Variable Susceptibility to Diacetyl Effects . . . . . . . . . . . . . . . . . 118

5.3.5 Interpretation of Modeling Results . . . . . . . . . . . . . . . . . . . . . . . . . . . 125

5.4 Human Data-based Assessment of Risks . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

5.4.1 Benchmark Dose . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 129

5.4.2 Excess Lifetime Risk for Pulmonary Impairment . . . . . . . . . . . . . . . . . . . 131

5.5 Sensitivity Analyses and Alternate Hypotheses . . . . . . . . . . . . . . . . . . . . . . . . 135

5.6 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 137

5.7 Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 138

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141

6 Quantitative Risk Assessment Based on Animal Data . . . . . . . . . . . . . . 145

6.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

6.1.1 Diacetyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

6.1.2 2,3-Pentanedione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145

6.2 Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

6.2.1 Data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 146

6.2.2 Analytical approach . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 148

6.3 Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

6.3.1 Diacetyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158

6.3.2 2,3-Pentanedione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

6.4 Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

6.4.1 Diacetyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 164

6.4.2 2,3-Pentanedione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 169

6.5 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 170

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171

7 Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione 173

7.1 Health Effect Studies of Employees Exposed to Diacetyl . . . . . . . . . . . . . . . . . . . 173

7.2 Toxicological Studies of Diacetyl . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 173

7.3 Quantitative Risk Assessment for Deriving the Recommended Exposure Limit . . . . . 174

7.4 Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

7.5 Recommended Exposure Limits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175

7.5.1 Recommended Exposure Limit for Diacetyl . . . . . . . . . . . . . . . . . . . . . . 175

7.5.2 Recommended Exposure Limit for 2,3-Pentanedione . . . . . . . . . . . . . . . . . 176

7.6 Rationale for the Recommended Exposure Limit . . . . . . . . . . . . . . . . . . . . . . . 177
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xvi Occupational Exposure to Diacetyl and 2,3-Pentanedione

7.7 Controlling Diacetyl and 2,3-Pentanedione Exposures in the Workplace . . . . . . . . . 177

7.8 Hazards Associated with Diacetyl Substitutes . . . . . . . . . . . . . . . . . . . . . . . . . 178

7.9 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 179

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 181

8 Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione 183

8.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183

8.2 Engineering Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

8.2.1 General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 184

8.2.2 Primary Production Processes and Controls . . . . . . . . . . . . . . . . . . . . . . 185

8.2.3 Summary of Capture Efficiencies of Control Approaches . . . . . . . . . . . . . . . 191

8.3 Administrative Controls . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

8.3.1 Good Housekeeping Practices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

8.3.2 Closed Transfers, Containers, and Processes . . . . . . . . . . . . . . . . . . . . . . 193

8.3.3 Hygiene Procedures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 194

8.3.4 Reduced Process Temperatures for Priority Flavoring Compounds . . . . . . . . . 194

8.3.5 Cleaning Practices for Equipment and Tools . . . . . . . . . . . . . . . . . . . . . . 194

8.3.6 Limit Access to Priority Flavoring Compounds . . . . . . . . . . . . . . . . . . . . 195

8.3.7 Informing Employees about the Hazard . . . . . . . . . . . . . . . . . . . . . . . . . 195

8.3.7.3 Classifying mixtures containing diacetyl and 2,3-pentanedione . . . . . . . . . . 200

8.3.7.4 Labeling and posting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202

8.3.7.5 Training . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 203

8.4 Respiratory Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204

8.5 Dermal, Eye, and Face Protection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 221

9 Medical Monitoring and Surveillance of Exposed Employees . . . . . . . . . . 225

Medical Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

Medical Surveillance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225

9.1 Medical Monitoring Program Director . . . . . . . . . . . . . . . . . . . . . . . . . . . . 226

9.2 Employees to Include in the Medical Monitoring Program . . . . . . . . . . . . . . . . . 226

9.3 Medical Monitoring Program Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

9.3.1 Questionnaire . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 227

9.3.2 Spirometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 228

9.4 Frequency of Medical Monitoring Evaluations . . . . . . . . . . . . . . . . . . . . . . . . 231

9.5 Reporting Medical Monitoring Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . 232

9.6 Early Identification of Affected Employees . . . . . . . . . . . . . . . . . . . . . . . . . . 233

9.7 Continuity of Medical Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 234

9.8 Tests Used in Medical Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

9.8.1 Spirometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

9.8.2 Other Pulmonary Function Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

9.8.3 High-resolution Computerized Tomography . . . . . . . . . . . . . . . . . . . . . . 235

9.8.4 Lung Biopsy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 235

9.8.5 Determining Diagnosis Responsible for Lung Disease . . . . . . . . . . . . . . . . . 236
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9.9 Response to Identification of Work-related Lung Disease . . . . . . . . . . . . . . . . . . 238

9.10 Medical Surveillance Analyses . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 238

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 241

10 Exposure Monitoring in Occupational Safety and Health Programs . . . . . . 245

10.1 Exposure Monitoring Program Goals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

10.2 Exposure Monitoring Program Elements . . . . . . . . . . . . . . . . . . . . . . . . . . . 245

10.2.1 Objectives of Sampling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 246

10.2.2 What to Sample (Specific Agents and Physical States) . . . . . . . . . . . . . . . . 246

10.2.3 Whom and Where to Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 247

10.2.4 How to Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 248

10.2.5 When to Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249

10.2.6 How Long to Sample . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

10.2.7 How Many Samples to Collect . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

10.2.8 Sample Handling, Storage, and Shipment . . . . . . . . . . . . . . . . . . . . . . . 250

10.3 Outcomes of Exposure Monitoring . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

10.3.1 Interpretation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250

10.3.2 Notification of Employees . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 252

11 Research Needs 253

12 Appendices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Appendix A . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

OSHA PV2118 (Diacetyl) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257

Appendix B . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

OSHA 1012 (Acetoin and Diacetyl) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 267

Appendix C . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Acetoin Diacetyl 1013 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 303

Appendix D . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

2, 3-Pentanedione . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 329

Appendix E . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 347

Volatile Organic Compounds (Screening) 2549 . . . . . . . . . . . . . . . . . . . . . . . . 347

Appendix F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Correcting Diacetyl Concentrations from Air Samples 

Collected with NIOSH Method 2557 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 357

Appendix G . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

JEM Tables for Four Plants . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 371

Appendix H . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 377

Development of a Job Exposure Matrix for Company G . . . . . . . . . . . . . . . . . . . 377

Appendix I . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389

Typical Protocol for Collecting Air Samples for Diacetyl and 2, 3-Pentanedione . . . . . 389
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Occupational Exposure to Diacetyl and 2,3-Pentanedione xix

Abbreviations

μg Microgram

μg3 Microgram per meter cubed

 Square root of cumulative exposure

A, B Subscripts denoting experimental animal and target species, respectively

ACGIH American Conference of Governmental Industrial Hygienists

ACOEM American College of Occupational and Environmental Medicine 

AH Absolute humidity

APF Assigned protection factor

AL Action level

Arg5 5th arginine

ATS/ERS American Thoracic Society/European Respiratory Society 

Avg(DA) Average exposure to diacetyl

BFV Butyric acid in butter flavoring vapors

BLS Bureau of Labor Statistics 

BMC Benchmark concentration

BMCL Lower one-sided 95% confidence limit

BMD Benchmark dose

BMDL Lower bound on the benchmark dose

BMI Body mass index

BOOP Bronchiolitis obliterans organizing pneumonia

BOS Bronchiolitis obliterans syndrome

Cal/OSHA California Occupational Safety and Health Administration

CAS Chemical Abstract Service

CD Crossdraft 

CDC Centers for Disease Control and Prevention

CDPH California Department of Public Health

Cdyn Dynamic lung compliance

CFD Computational fluid dynamic

CFD/PBPK Computational fluid dynamic/physiologically-based 

pharmacokinetic model
Abbreviations

xx Occupational Exposure to Diacetyl and 2,3-Pentanedione

cfm Cubic feet per minute

CFR Code of Federal Regulations

CI Confidence interval

cm Centimeter

COP Cryptogenic organizing pneumonitis

COPD Chronic obstructive pulmonary disease

CT Computed tomography

DA Diacetyl

Cum(DA) Cumulative exposure to diacetyl

DCXR Dicarbonyl/L-xylulose reductase

DLCO Diffusing capacity for carbon monoxide

DNPH 2,4-Dinitrophenylhydrazine

DOT Department of Transportation

EPA Environmental Protection Agency

ERS European Respiratory Society

ET Extrathoracic, also subscript denoting the extrathoracic region 

eV Electron volt

EX Exhaust flow rate

FASEB Federation of American Societies for Experimental Biology

FDA Food and Drug Administration

FEMA Flavor and Extract Manufacturers Association

FEV1 Forced expiratory volume in one second

fpm Feet per minute

FTIR Fourier transform infrared gas analyzer

FVC Forced vital capacity

GC-ECD Gas chromatography using an electron capture detector

GC-FID Gas chromatography using a flame ionization detector

GC-MS Gas chromatography-mass spectrometry

GC-NPD Gas chromatography-nitrogen/phosphorus detection

GHS Globally Harmonized System of Classification and Labelling of Chemicals

g/kg Grams per kilogram

HCI Health Canada Initiative

HCS Health communication standard

HDS Helium diffusion sampler
Abbreviations

Occupational Exposure to Diacetyl and 2,3-Pentanedione xxi

HEC Human equivalent concentration

HEPA High-efficiency particulate air

HHE Health hazard evaluation

HPLC-UV High pressure liquid chromatography- ultraviolet

HRCT High resolution computed tomography

HSE Health and Safety Executive

IATA International Air and Transport Association

ICD International Classification of Diseases

ICRP International Commission on Radiological Protection

IOSHA Indiana Occupational Safety and Health Administration

IR Infrared 

ISO International Organization for Standardization

IUPAC International Union of Pure and Applied Chemistry

JEM Job exposure matrix

Km Michaelis constant 

LC50 Lethal concentration for 50% of exposed population

LD50 Lethal dose for 50% of dosed population

LEV Local exhaust ventilation

LLD Longitudinal limit of decline

L/min Liters per minute 

LLofN Lower limit of normal

LOD Limit of detection

LOQ Limit of quantification

LR Likelihood ratio

LRT Likelihood ratio test

mg/mL Milligrams per milliliter

mg/m3 Milligrams per meter cubed

mL Milliliter

mL/min Milliliters per minute

mM Millimoles 

mV Millivolt

NADH Nicotinamide adenine dinucleotide

NADPH Nicotinamide adenine dinucleotide phosphate

NAICS North American Industry Classification System
Abbreviations

xxii Occupational Exposure to Diacetyl and 2,3-Pentanedione

ND Not detected

NHANES III Third National Health and Nutrition Examination Survey

NHANES National Health and Nutrition Examination Survey

NIEHS National Institute for Environmental Health and Safety

NIOSH National Institute for Occupational Safety and Health

NTP National Toxicology Program

OEL Occupational exposure limit

OR Odds ratio

OSHA Occupational Safety and Health Administration

OV-HE Organic vapor-high efficiency particulate

PAPR Powered air-purifying respirator

PBPK Physiologically based pharmacokinetic model

PD 2,3-pentanedione

PEL Permissible exposure limit

PFT Pulmonary function test

PTFE Polytetrafluoroethylene

PID Photoionization detector

POD Point of departure

ppb Parts per billion

PPE Personal protective equipment

ppFEV1 Percent of predicted FEV1

ppm Parts per million

QA Quality assurance

QC Quality control

REL Recommended exposure limit

RfC Reference concentration

RGDR Regional gas dose ratio

RH Relative humidity

RL Basal airway resistance

RQL Reliable quantitation limit

RR Rate ratio

SA Surface area (cm2)

SAR Supplied-air respirator

SCBA Self-contained breathing apparatus
Abbreviations

Occupational Exposure to Diacetyl and 2,3-Pentanedione xxiii

SD Standard deviation

SDS Safety data sheet

SMR Standardized mortality ratio

SPIROLA Spirometry Longitudinal Data Analysis

SPME Solid-phase microextraction

STEL Short-term exposure limit

TB Tracheobronchial region

TD-GC-MS Thermal desorption-gas chromatography-mass spectrometry

TERA Toxicology Excellence for Risk Assessment 

TLC Total lung capacity 

TWA Time weighted average

UF Uncertainty factor

VE Minute volume (mL/min = cm3/min)

VOC Volatile organic compound
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Occupational Exposure to Diacetyl and 2,3-Pentanedione xxv

Glossary

2,3-Pentanedione: A diketone (Chemical Abstracts Service No. 600-14-6) used as a synthetic flavoring 

agent and aroma carrier. It has a buttery taste and smell. It may be used as either a solid (powder) or as 

a liquid. It is structurally very similar to diacetyl. 

Acetoin: A hydroxy ketone (Chemical Abstracts Service No. 513-86-0) found in butter flavoring. 

Acetoin may be converted to diacetyl through oxidation.

American Conference of Governmental Industrial Hygienists threshold limit value: Voluntary exposure guidelines recommended by ACGIH, a professional organization, for use by industrial hygienists 

and others trained in this discipline to assist in the control of health hazards.

Asthma: A chronic inflammatory airway disease that causes episodic wheezing, shortness of breath, 

chest tightness, and coughing.

Bronchiolitis obliterans: Disease processes that show some degree of inflammation, narrowing, or 

obliteration of small airways (bronchioles) in the lung. Historically, classified into 2 groups: proliferative bronchiolitis obliterans and constrictive (obliterative) bronchiolitis obliterans which is the type that 

has been associated with flavorings exposure.

Constrictive (obliterative) bronchiolitis: A potentially fatal, irreversible lung disease with symptoms 

of dry cough and shortness of breath. On biopsy, the bronchioles are compressed and narrowed by 

either fibrosis or inflammation. Constrictive (obliterative) bronchiolitis often is characterized by fixed 

airway obstruction, but pathologic cases sometimes have normal or restrictive spirometry.

Diacetyl: An alpha-diketone (Chemical Abstracts Service No. 431-03-8) used as a synthetic flavoring 

agent and aroma carrier. It has a buttery taste and smell. It may be used as either a solid (powder) or as 

a liquid.

Emphysema: An irreversible progressive disease of the lungs that destroys the alveolar tissues of 

the lungs.

Encapsulated powder: Ingredients such as diacetyl or other flavor enclosed within a material to 

decrease volatility and allow a subsequent release or flavor burst. 

Fibrosis: A condition in which lung tissue is replaced over time with scar tissue. This process restricts 

the lungs and reduces total lung capacity.

Fixed airways obstruction: A respiratory problem marked by reduced airflow out of the lungs that, 

unlike asthma, is not reversible with a bronchodilator medication.

Gas chromatography/mass spectrometry: A method of analyzing mixtures of chemicals qualitatively 

and quantitatively.

Mid-expiratory flow rate: The maximum rate of airflow measured between exhaled volumes of 25% 

and 75% of the forced vital capacity as measured during a forced exhalation.

Obliterative bronchiolitis: See constrictive bronchiolitis.

Occupational exposure limit: Levels of exposure that most employees may be exposed to for up to 10 

hours per day, 40 hours per week, for a working lifetime, without experiencing adverse health effects.
Glossary

xxvi Occupational Exposure to Diacetyl and 2,3-Pentanedione

N-95 filtering facepiece respirator: A term that describes the class of respirators that use N95 filters 

to remove particles from the air that is breathed through them. An N95 filter removes at least 95% 

of airborne particles during “worst case” testing using a “most-penetrating” sized particle during 

NIOSH testing.

NIOSH recommended exposure limit: An 8- or 10-hour time-weighted average, ceiling, or short 

term exposure concentration recommended by NIOSH that is based on an evaluation of the health 

effects data. 

OSHA permissible exposure limit: Regulatory limits indicating an 8-hour time weighted average 

exposure unless otherwise noted; a (c) designation denotes a ceiling limit. 

Organic vapor cartridge: Devices used in respirators to remove organic vapors from the air.

Priority flavoring (or priority chemical, substance, etc.): Generally used in reference to flavoring compounds on the Flavor and Extract Manufacturers Association priority list [FEMA 2004].

Personal protective equipment: Respirators, work gloves, work boots, and other equipment that 

reduce or eliminate employee exposure to hazards.

Prevalence: The number of cases of a disease or condition present in a particular population at a 

given time.

Respiratory rate: The number of breaths taken within a certain amount of time, commonly measured 

in breaths per minute.

Safety data sheet: A listing of a hazardous chemical’s health and physical hazards, exposure limits, and 

precautions (formerly known as material safety data sheet).

Silicosis: a respiratory disease caused by inhaling silica dust.

Slurry: a mixture of liquid and powder ingredients.

Spirometer/Spirometry: An instrument and method for performing a pulmonary function test that 

measures the volume or flow of air that can be inhaled or exhaled to assess lung function.

Starter distillate: A steam distillate of the culture of bacteria grown on a medium consisting of skim 

milk usually fortified with about 0.1% citric acid. It contains mostly water, and the remainder is a 

mixture of butter-like flavor compounds. The major flavoring ingredient is diacetyl, but starter distillate also contains minor amounts of acetaldehyde, ethyl formate, ethyl acetate, acetone, ethyl alcohol, 

2-butanone, acetic acid, and acetoin.

Short-term exposure limit: Unless otherwise noted, the STEL is the 15-minute TWA exposure that 

shall not be exceeded at any time during a workday.

Supplied-air respirator system: An atmosphere-supplying respirator for which the source of breathing 

air is not carried by the user.

Tidal volume: The volume of air inhaled or exhaled during a single breath at rest.

Time-weighted average: indicates a time-weighted average concentration for up to a 10-hour work 

day during a 40-hour workweek.

Volatile organic compound: An organic chemical compound with high vapor pressure and low boiling point.
Occupational Exposure to Diacetyl and 2,3-Pentanedione xxvii

Acknowledgments

The following document was authored by a multidisciplinary team from six NIOSH divisions. 

The lead division was the Education and Information Division, Paul Schulte, PhD, Director, 

Lauralynn Taylor McKernan, ScD, CIH (Manager/Author), and R. Todd Niemeier, MS, CIH 

(Co-manager/Author).

The following individuals were the authors of the document:

Division of Applied Research 

and Technology

Kevin H. Dunn, MSEE, CIH

Alberto Garcia, MS

Deborah V.L. Hirst, PhD, PE

Fariba Nourian, BS

Robert P. Streicher, PhD

Jennifer L. Topmiller, MS

Respiratory Health Division

Kathleen B. Fedan, BS

Richard Kanwal, MD (former NIOSH)

Kathleen Kreiss, MD (retired NIOSH)

Greg J. Kullman, PhD, CIH (retired NIOSH)

Chris Piacitelli, MS, CIH (retired NIOSH)

Division of Surveillance, Hazard 

Evaluations, and Field Studies

Brian Curwin, PhD

Education and Information Division

David A. Dankovic, PhD

Ellen Galloway, MA

Stephen J. Gilbert, MS

Thomas J. Lentz, PhD

Lauralynn Taylor McKernan, ScD, CIH

R. Todd Niemeier, MS, CIH

Robert Park, MS

Randall Smith, MA

Christine Whittaker, PhD

Health Effects Laboratory Division

Jeff Fedan, PhD

Ann F. Hubbs, DVM, PhD, DACVP

National Personal Protective Technology 

Laboratory

Jay A. Parker, MS, CIH

Special appreciation is expressed to the following individuals for serving as independent, external 

peer reviewers and providing comments that contributed to the development of this document: 

Jonathan Borak, MD, Yale University

Harvey Checkoway, PhD, University of Washington

Mark Cullen, MD, Stanford University

Arthur Frank, MD, PhD, Drexel University

Warren Myers, PhD, West Virginia University

Yiliang Zhu, PhD, University of South Florida

Dale Hattis, PhD, Clark University

Sam Kacew, PhD, ATS, University of Ottawa

David Halton, PhD, CIH, CRSP, Private Consultant

John Howell, PhD, Private Consultant
Acknowledgments

xxviii Occupational Exposure to Diacetyl and 2,3-Pentanedione

Additional NIOSH contributors to the document include: 

Rachel Bailey, Devin S. Baker, Kenneth Brown, James Couch, Jean Cox-Ganser, Kristin Cummings, 

Daniel H. Echt, Gino Fazio, Ardith A. Grote, Paul Middendorf, John Piacentino, Stephanie M. 

Pendergrass, Nikki Romero, Rena Saito, Miriam Siegel, Brian Tift, Doug Trout, Christine Uebel, 

Abbas Virji, David Weissman, and Vanessa Williams.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 1

1 Introduction

1.1 Purpose

This document presents the criteria and 

components of a recommended standard 

necessary to reduce or eliminate significant 

risk of health impairment from exposure to 

diacetyl and 2,3-pentanedione and prevent 

flavorings-related lung disease. This document was developed in accordance with the 

Occupational Safety and Health Act of 1970 

[29 U.S.C. 669(a)(3); 29 U.S.C. 671(c)(1)]. 

This Act charges NIOSH with recommending 

occupational safety and health standards and 

developing criteria for toxic materials. These 

criteria are to describe exposures that are safe 

for various periods of employment, including 

but not limited to the exposures at which no 

employee will suffer diminished health, functional capacity, or life expectancy as a result of 

his or her work experience. 

The purpose of the criteria document is to 

evaluate the scientific literature concerning potential health effects, toxicology, risk 

assessment, engineering controls, work practices, personal protective equipment, and 

recommendations pertaining to diacetyl and 

2,3-pentanedione. The criteria document 

provides a basis for the REL for diacetyl and 

2,3-pentanedione, although compliance 

with this recommended standard is not the 

sole objective. The intended outcome of the 

document is to reduce occupational exposures to diacetyl and 2,3-pentanedione and 

thereby prevent flavorings-related lung disease 

through hazard guidance implementation. In 

their entirety, the RELs and the guidance are 

intended to help employers develop a more 

healthful work environment. The RELs and 

guidance will also provide useful information 

to help employees actively participate in their 

own protection.

1.2 Scope

This criteria document contains a review of relevant scientific information related to diacetyl 

and 2,3-pentanedione, and provides the rationale and criteria for establishing appropriate 

risk management recommendations. The basis 

for developing a criteria document on diacetyl and 2,3-pentanedione is described in this 

chapter. Chapter 2 provides an overview of 

studies conducted to characterize occupational 

exposure to diacetyl and 2,3-pentanedione. 

Chapter 3 describes the health effects observed 

in employees exposed to diacetyl and other 

flavoring compounds. Chapter 4 describes 

toxicology research from diacetyl and 2,3-pentanedione, while Chapters 5 and 6 describe the 

assessment of risk based on available human 

and animal data. Chapter 7 provides the basis 

for the RELs for diacetyl and 2,3-pentanedione. 

Chapter 8 describes procedures for informing employees about the safety of diacetyl 

and diacetyl substitutes as well as engineering 

interventions that could significantly reduce 

exposures when appropriately applied and 

fully operational. Also included in Chapter 8 

are recommendations for establishing globally harmonized system for classification and 

labelling (GHS) classifications for diacetyl and 

2,3-pentanedione based on the revised OSHA 
2 Occupational Exposure to Diacetyl and 2,3-Pentanedione

1 . Introduction

hazard communication standard. Additionally, 

recommendations for an effective respiratory 

protection program are provided. Chapter 9 

provides medical surveillance guidelines for 

the ongoing evaluation of the health status 

of employees. Chapter 10 describes the components of an effective exposure monitoring 

program and work practices that when implemented correctly, can reduce occupational 

exposures. Finally, Chapter 11 presents key 

research needs. 

This document results from a review of all 

relevant literature on diacetyl and 2,3-pentanedione, and describes selected studies 

which characterize exposures and discusses 

techniques shown to be effective in reducing 

those exposures. Published literature through 

October 2016 was used and extracted from 

databases including but not limited to PubMed, 

NIOSHTIC-2, Web of Knowledge, Toxline, 

and Chem Abstracts. The literature search was 

developed to identify critical scientific data 

relevant to workplace safety and health including physical and chemical properties, human 

health effects, laboratory testing, chemical 

toxicokinetics, toxicity, engineering controls, 

personal protective equipment and function, 

risk management, and modeling systems that 

are relevant to diacetyl and 2,3-pentanedione. The literature was searched using specific 

terminology for each scientific discipline. 

Evaluated data sources included peer reviewed 

journal articles, government publications, and 

peer reviewed data sources, high caliber professional practice manuals (i.e., ACGIH 2012 

and FEMA 2012) and high-quality information submitted to government dockets. In a few 

instances personal communications are cited 

where authors were contacted for additional 

clarification. The information that was identified in the comprehensive literature search was 

evaluated with considerations that included if 

the studies were peer-reviewed, if the data were 

generated with standardized protocols, if the 

exposure conditions were described in detail, 

confounders and existing information in peer 

reviewed journals. Specific studies pertaining 

to workplace exposure assessment, human 

health effects, and toxicology were specifically 

identified and are described in Chapters 2, 3, 

and 4 respectively. 

1.3 Background 

Diacetyl is one of the main components in 

butter flavoring that imparts a buttery taste, 

and it has been identified as a prominent volatile organic compound (VOC) in air samples 

from microwave popcorn plants and flavoring 

manufacturing plants [Akpinar-Elci et al. 2004; 

Ashley et al. 2008; Kanwal 2003; Kanwal et al. 

2006; Kanwal and Martin 2003; Martyny et al. 

2008; NIOSH 2004a; Parmet and Von Essen 

2002]. Diacetyl is used as a natural and artificial flavoring ingredient and aroma carrier in 

bakery products, dairy products, snack foods, 

and more. It is mainly used as a butter flavoring but is also used in the flavor formulation 

of a number of other flavors, including but 

not limited to strawberry, caramel, hazelnut, 

and butterscotch. It is also present as a natural 

byproduct in some fermented food products 

such as beer and roasted food products such 

as coffee. Occupational exposures in the flavoring and food production industries have been 

associated with respiratory disease, including 

obliterative bronchiolitis, an uncommon lung 

disease often characterized by fixed airways 

obstruction. Obliterative bronchiolitis refers 

to disease processes that show some degree 

of inflammation, narrowing, or obliteration 

of small airways (bronchioles) in the lung and 

is discussed in more detail in Chapter 3, specifically section 3.1.1. Although a causative 

relationship between diacetyl and respiratory 

disease has been observed, diacetyl may not be 

the only flavoring compound related to health 

impairment. Other flavoring ingredients such 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 3

1 . Introduction

as acetaldehyde, butyric acid, and acetoin, 

have been present in workforces with adverse 

health effects [Lockey et al. 1998; van Rooy 

et al. 2007]. In addition, new diacetyl substitutes with little or no toxicological information 

related to occupational safety and health are 

being used in production. 

Day et al. [2011] observed the flavoring compound 2,3-pentanedione in food production 

facilities. This compound has been used as 

a diacetyl substitute in many flavor manufacturing facilities because it has a related 

chemical structure and similar flavor properties 

to diacetyl. Published reports on the toxicity of 

2,3-pentanedione from experimental inhalation studies with rats indicate that exposure 

causes airway epithelial damage similar to 

that produced by diacetyl [Hubbs et al. 2012; 

Morgan et al. 2012]. 

No state or national registries are available to 

identify potential cases of obliterative bronchiolitis among employees. In 1985, two 

employees with fixed obstructive lung disease 

suggestive of obliterative bronchiolitis were 

observed in a facility where flavorings with 

diacetyl were made for the baking industry 

[Kreiss et al. 2002; NIOSH 1986]. Catastrophic 

fixed airways disease suggestive of obliterative 

bronchiolitis was observed in these two former 

mixing employees who were young nonsmokers with job tasks that involved blending 

corn starch and flour with various flavorings. 

Two additional employees who formerly had 

mixing responsibilities also had otherwise 

unexplained obstruction, whereas two current 

mixers were unaffected. A review of common 

ingredients listed diacetyl among other flavoring compounds. 

In the microwave popcorn industry, the first 

occurrences of obliterative bronchiolitis were 

observed in the year 2000 when eight employees formerly employed in a microwave popcorn 

facility were diagnosed with the disease [Kreiss 

et al. 2002]. The observation of this case series 

led to the identification of another case of 

obliterative bronchiolitis in a separate facility 

[Parmet 2002]. Since then, numerous cases of 

obliterative bronchiolitis have been observed in 

the microwave popcorn industry [Akpinar-Elci 

et al. 2004; CDC 2002; Ezrailson 2002; Kanwal 

et al. 2006; NIOSH 2003, 2004a, b, 2006; 

Parmet 2002; Schachter 2002]. In addition, a 

retrospective epidemiologic study found cases 

of obliterative bronchiolitis in employees who 

were employed in a diacetyl manufacturing 

plant with exposures to diacetyl, acetoin, acetic 

acid, and acetaldehyde [van Rooy et al. 2007]. 

In 2004 and 2006, two cases of obliterative 

bronchiolitis among employees who made 

food flavorings were reported to the California 

Department of Public Health (CDPH). An 

industry-wide public health investigation performed by CDPH, the California Occupational 

Safety and Health Administration (Cal/OSHA), 

and NIOSH initially found an additional five 

employees with severe, fixed obstructive lung 

disease [CDC 2007]. Outreach to the industry regarding the diacetyl hazard, including 

Cal/OSHA consultation site visits, prompted 

quick implementation of exposure controls and 

medical surveillance programs. A longer-term 

effort was focused on companies’ installation 

of effective engineering controls and further 

assessment of medical surveillance findings 

over time by CDPH and NIOSH. A crosssectional analysis of medical surveillance data 

from 16 companies confirmed the risk of lung 

disease among employees at companies using 

diacetyl [Kim et al. 2010]. In 2010, California 

issued the first occupational standard for 

diacetyl [California Code of Regulations. 

Title 8, §5197]. 

Employees within the flavoring production 

industry have complex exposures in terms of 

the physical form of the agents (vapors, mists, 

and airborne dusts) and the number of different chemicals used. Although thousands 
4 Occupational Exposure to Diacetyl and 2,3-Pentanedione

1 . Introduction

of flavoring compounds are in use, few have 

occupational exposure limits. The Flavor and 

Extract Manufacturers Association (FEMA) 

reports that of the more than 1,000 flavoring compounds considered to be potential 

respiratory irritants or hazards, only 46 have 

established OSHA permissible exposure limits 

(PELs) [FEMA 2012]. Given the lack of occupational exposure limits for most flavoring 

compounds, assessing workplace exposures and 

developing exposure control guidance are critical to help reduce the risk of flavorings-related 

lung disease. 

In 2010, California promulgated a regulation 

for occupational exposure to food flavorings 

containing diacetyl that requires installation of 

exposure controls to reduce exposures to the 

lowest feasible levels. In 2012, the American 

Conference of Governmental Industrial 

Hygienists (ACGIH) published a threshold 

limit value® of 0.010 ppm 8-hour TWA with a 

STEL of 0.020 ppm for diacetyl [ACGIH 2012]. 

In 2014, the European Commission published 

the Recommendation from the Scientific 

Committee on Occupational Exposure Limits 

of 0.02 ppm 8-hour TWA with a STEL of 0.10 

ppm for diacetyl [EU 2014]. 

1.4 Chemical and 

Physical Properties

The compound diacetyl has Chemical Abstract 

Service (CAS) number 431-03-8 and has 

several synonyms including 2,3-butanedione 

(International Union of Pure and Applied 

Chemistry nomenclature), 2,3-butadione, 

2,3-diketobutane, biacetyl, dimethyl diketone, 

and dimethylglyoxal. The compound 2,3-pentanedione has CAS number 600-14-6 and is 

also referred to by the name acetyl propionyl. 

Both diacetyl and 2,3-pentanedione are alpha 

diketones or vicinal diketones (also referred to 

less specifically as alpha dicarbonyls), which 

means that their molecular structures contain 

two carbonyl functional groups that are adjacent to one another, and the carbon molecules 

attached to the oxygen molecules are also 

attached to only carbon molecules. A listing 

of physical and chemical properties of diacetyl and 2,3-pentanedione and their molecular 

structures is presented in Table 1-1. 

The odor threshold of diacetyl and 2,3-pentanedione has been reported by many studies 

[Buttery et al. 1997; Hall and Andersson 1983; 

Leksrisompong et al. 2010; Nagata and Takeuchi 

1990; Sega et al. 1967]. It is not uncommon for 

odor threshold values reported in the literature 

to range over four orders of magnitude for the 

same chemical [AIHA 1989]. Odor threshold 

variability can result from the source of data, 

the characteristics of human olfactory response, 

and the differences in experimental methodology [AIHA 1989]. The following criteria were 

used to analyze the diacetyl and 2,3-pentandione odor threshold literature: (1) only primary 

odor threshold sources that were found in the 

literature and that were written in English were 

used; (2) only sources that clearly indicated the 

type of threshold being measured as a detection or recognition threshold were used; (3) 

sources that used a panel of at least five judges 

to account for the range of olfactory sensitivity 

in the population were used; and (4) sources 

that presented the different concentrations of 

odor samples in a way that eliminated olfactory fatigue were used. The geometric mean of 

the selected values was reported in Table 1-1 

[AIHA 1989]. 

1.4.1 How Diacetyl and 

2,3-Pentanedione are Prepared

Diacetyl can be synthesized chemically from 

four starting materials: (1) from methyl ethyl 

ketone, either by converting it into an isonitroso compound and then hydrolyzing 

with hydrochloric acid or by partial oxidation of methyl ethyl ketone over a copper or 

vanadium oxide catalyst [Aquila et al. 2001; 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 5

1 . Introduction

Table 1-1. Chemical and physical properties 

Property Diacetyl 2,3-Pentanedione

CAS # 431-03-8 600-14-6

Synonyms 2,3-butanedione; biacetyl; dimethyl diketone; dimethylglyoxal; 2,3-diketobutane 

[Merck and Co. Inc. 2006]

Acetylpropionyl [Lide 2008]

Molecular formula C4H6O2 C5H8O2 

Molecular weight 86.090 [Lide 2008] 100.117 [Lide 2008]

Molecular structure

H3C

CH3

O

O

H3C CH3

O

O

Density 0.9808 g/mL (18oC) [Lide 2008] 0.9565 g/mL (19oC) [Lide 2008]

Refractive index 1.3951 (20oC) [Lide 2008] 1.4014 (19oC) [Lide 2008]

Melting point −1.2oC [Lide 2008]; −2.4oC [IPCS 2009]; 

−4oC [Fischer Scientific 2007]

−52oC [Merck Chemicals 

International 2010]

Boiling point 88°C [Lide 2008]; 88°C [IPCS 2009] 108°C [Lide 2008]; 110oC–112oC [Merck 

Chemicals International 2010]; 112oC 

[Chem Service Inc. 1988]

Vapor density 3 3.45 

Vapor pressure 52.2 mm Hg (20°C) [Sigma Aldrich 2010] 21.4 mm Hg (20oC) [Merck Chemicals 

International 2010]

Saturated vapor 

concentration

[Perez 1991]

184 g/m3 (20oC); 246 g/m3

 (20oC) 117 g/m3 (20oC) 

Water solubility 200 g/L (25oC) [IPCS 2009] 60 g/L (15oC) [Merck Chemicals 

International 2010]

Flash point, closed cup 6°C [IPCS 2009]; 7°C [Sigma 

Aldrich 2010]

18oC [Merck Chemicals 

International 2010]

Autoignition 

temperature

365°C [IPCS 2009]; 345°C [Sigma 

Aldrich 2010]

265oC [Merck Chemicals 

International 2010]

Explosive limits in air 2.4% (V) – 13% (V) [IPCS 2009] 1.8% (V) – 10.9% (V) [Merck Chemicals 

International 2010]

Odor Quinine odor, vapors have chlorinelike odor [Merck and Co. Inc. 2006]; 

strong rancid, chlorine-like, butter-like 

[Fischer Scientific 2007]

Fruity/pleasant [Chem Service Inc. 1988] 

See footnotes at end of table. (Continued)
6 Occupational Exposure to Diacetyl and 2,3-Pentanedione

1 . Introduction

Property Diacetyl 2,3-Pentanedione

CAS # 431-03-8 600-14-6

Odor detection 

threshold 

0.27 ppb [Hall and Andersson 1983; 

Nagata and Takeuchi 1990] (odor 

measurement of diacetyl vapor-air mixtures). Note: geometric mean of two 

literature reported threshold values with 

a geometric standard deviation 

of 5.33 ppb. 

0.84 ppb [Buttery et al. 1997; 

Leksrisompong et al. 2010; Sega et al. 

1967] (odor measurement of diacetyl 

vapor in the headspace above aqueous 

solutions, diacetyl concentrations in 

solution converted to air concentrations 

using Henry’s Law constant). Note: geometric mean of three literature reported 

threshold values with a geometric standard deviation of 1.44 ppb. 

1.2 ppb [Lawless et al. 1994] (recognition 

threshold) Note: not a geometric mean 

because obtained from a single source.

15 ppb [Hall and Andersson 1983](odor 

measurement of 2,3-pentanedione 

vapor-air mixtures). Note: compared to 

1.4 ppb for diacetyl obtained from the 

same reference.¶

9.4 ppb [Buttery et al. 1997] (odor measurement of 2,3-pentanedione vapor in 

the headspace above aqueous solutions, 

2,3-pentanedione concentration in solution converted to an air concentration 

using Henry’s Law constant).Note: compared to 0.70 ppb for diacetyl obtained 

from the same reference.¶

Octanol/water partition 

coefficient, Log Pow

−1.34 [IPCS 2009] −0.85 [Illovo Sugar Limited 2010]

Henry’s Law Constant* 2.95 × 10-5 atm-m3/mol*; 1.75 × 10-5

atm-m3/mol at 25°C[Strekowski and 

George 2005]; 1.35 × 10-5 atm-m3/

mol at 25°C [Betterton 1991]; 1.75 × 

10-5 atm-m3/mol at 25°C [Snider and 

Dawson 1985] 

4.7 × 10-5 atm-m3/mol*

Appearance Yellowish green liquid [Merck and Co. 

Inc. 2006]; green-to-yellow liquid 

[IPCS 2009]

Dark yellow liquid [Lide 2008]; [yellow to 

green yellow liquid [OSHA 2014]

Electron impact mass 

spectrum, m/z (%)

43 (100%), 15 (34%), 86 (11%), 14 (10%), 

42 (7%), 13 (3%), 26 (2%), 29 (2%) 

[Nottingham University 1983]

43 (100%), 29 (69%), 57 (35%), 27 (30%), 

15 (26%), 100 (10%), 26 (9%), 14 (9%) 

[Nottingham University 1983]

Infrared spectrum 1715.6 cm-1, 1420.7 cm-1, 1353.2 cm-1, 

1115.5 cm-1, 537.2 cm-1 [Pouchert 1985]

2982.5 cm-1, 1715.1 cm-1, 1408.0 cm-1, 

1349.6 cm-1, 1094.2 cm-1, 908.7 cm-1, 

581.4 cm-1 [Pouchert 1985]

*

Estimated as a ratio of vapor pressure to water solubility

Table 1-1 (Continued). Chemical and physical properties 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 7

1 . Introduction

National Toxicology Program 2007]; (2) from 

2,3-butanediol, by oxidative dehydrogenation of 2,3-butanediol over a copper or silver 

catalyst [National Toxicology Program 2007]; 

(3) from acetoin (obtained by electrochemical 

oxidation of methyl ethyl ketone), by reacting 

acetoin with molecular oxygen in the presence 

of a copper oxide catalyst [Aquila et al. 2001]; 

or, (4) from 1-hydroxyacetone (obtained by 

dehydrogenation of 1,2-propanediol), by the 

acid-catalyzed condensation of 1-hydroxyacetone with formaldehyde [National Toxicology 

Program 2007]. 

Diacetyl is also a byproduct of fermentation. 

Natural diacetyl is used in the form of starter 

distillate, a concentrated flavor distillate, 

which may contain different concentrations of 

diacetyl depending on production conditions 

[Burdock 1997]. 

The compound 2,3-pentanedione is also naturally produced by fermentation and is recovered 

from dairy waste to be used as a flavoring 

ingredient [Miller et al. 1998]. The chemical 

synthesis of 2,3-pentanedione is achieved in 

the following ways: (1) the condensation of 

lactic acid and an alkali metal lactate [Miller 

et al. 1998]; (2) the acid-catalyzed condensation of 1-hydroxyacetone with paraldehyde 

[Lambrecht et al. 2004]; or (3) the oxidation 

of 2-pentanone with excess sodium nitrite and 

diluted hydrochloric acid in the presence of 

hydroxylamine hydrochloride [Burdock 1997].

1.5 Production Uses 

and Applications

The flavor manufacturing industry commonly 

uses diacetyl and 2,3-pentanedione during 

flavor formulation production. Flavor formulations are then sold to downstream users for the 

production of flavored food products. Flavored 

food production is the process of manufacturing food and beverage products that contain 

added flavor formulations or flavorings to 

enhance or modify the taste of the product. 

Examples of flavored food products include 

bakery products such as cake mixes, flour and 

margarines, dairy products such as cheese 

and yogurt, snack foods such as soft spreads 

and crackers, beverages such as soft drinks, in 

addition to candy, ice cream, frozen foods, and 

many other food and beverage products. The 

addition of concentrated flavorings including 

diacetyl is a cost effective way to impart the 

desired properties to manufactured food items. 

In flavor formulations, diacetyl and 2,3-pentanedione are typically found as components 

in liquid solutions but can also be added to 

powders in dry mixtures to create a solid particulate formulation. Many volatile compounds 

are also encapsulated in an amorphous carbohydrate, producing more stable products with 

more manageable properties. Encapsulated 

powder flavorings are often created with a spray 

dryer, which converts a slurry mixture into a 

powder in which the flavorings are surrounded 

by the powder instead of simply coating the 

powder. When the encapsulated powder 

comes into contact with moisture, the flavor is 

released quickly and completely [Ubbink and 

Schoonman 2002]. 

The percentage of diacetyl or 2,3-pentanedione 

in a particular flavor formulation varies widely 

depending upon the product and its use. In 

past years, microwave popcorn contained the 

highest proportion of diacetyl ranging from 1% 

to 25% diacetyl [Hallagan 2007]. The diacetyl 

content in flavor formulations has declined 

rapidly as many manufacturers have reduced or 

substituted diacetyl with other flavoring compounds with similar characteristics, such as 

2,3-pentanedione. Most confectionary flavors 

contain up to 1% diacetyl while marshmallow 

production uses up to 5% [Hallagan 2007]. 

Starter distillate, produced by fermenting milk 

with starter cultures, contains diacetyl in the 
8 Occupational Exposure to Diacetyl and 2,3-Pentanedione

1 . Introduction

range of 1% to 5% and is often used as a flavor 

enhancer in the dairy industry. Diacetyl is the 

major flavor component of starter distillate, 

constituting as much as 80% to 90% of the mixture’s organic flavor compounds [FDA 2009]. 

A NIOSH health hazard evaluation (HHE) at 

a modified dairy production company found 

concentrations of airborne diacetyl ranging up 

to 2.14 parts per million on a full-shift TWA 

basis [NIOSH 2009]. 

Diacetyl is also used as a chemical modifier of 

arginine residues in proteins in studying glycation (the nonenzymatic browning of foods or 

the nonenzymatic binding of sugar and protein 

molecules in the body) [Saraiva et al. 2006]. 

Other uses for diacetyl include reactant/starting 

material in chemical or biochemical reactions, 

analytical reagent, antimicrobial/preservative, 

electron stabilizing compound and modifier 

of radiation response for chemical and 

biological systems, and photoinitiator/photosensitizer in polymerizations [National 

Toxicology Program 1994]. 

1.6 Potential for Exposures 

It is difficult to quantify the number of employees directly involved with flavor manufacturing 

and more specifically having exposure to diacetyl or diacetyl substitutes in the United States. 

According to the Environmental Protection 

Agency (EPA) Non-Confidential Inventory 

Updating Report, diacetyl had an aggregate production volume between 10,000 

and 500,000 pounds in 2002 [EPA 2002]. 

The North American Industry Classification 

System (NAICS) category 311, the most relevant category, indicates nearly 1.5 million 

employees are employed in food manufacturing. Bureau of Labor Statistics and Department 

of Commerce data provide a breakdown of a 

portion of that number into categories shown 

in Table 1-2. According to the FEMA, whose 

members account for approximately 95% of all 

flavors produced in the United States, a total of 

6,520 employees work directly in flavor manufacturing or related laboratory activities in 

membership companies [Hallagan 2010].

Initial research concerning occupational exposure to diacetyl has focused on employees who 

directly produce flavorings or use them in the 

microwave popcorn industry. However, the 

employment figures for the food production 

industry suggest that some other employees have potential exposure to diacetyl and 

other food flavorings. For example, respiratory issues have been anecdotally reported for 

cheese production (Wisconsin), yogurt production (Ohio), and potato chip manufacturing 

[Alleman and Darcey 2002]. 

Employers in the food manufacturing sector 

are generally small business owners with 89% 

Table 1-2. Breakdown of employees in various categories of the food manufacturing industry

Category description No. of employees NAICS code Ref.

Bakeries and tortilla manufacturing 280,900 3118 [BLS 2008]

Other food manufacturing 164,100 3119 [BLS 2008]

Dairy product manufacturing 129,100 3115 [BLS 2008]

Sugar and confectionery product 

manufacturing

 70,800 3113 [BLS 2008]

Beverage industry 177,000 3121 [BLS 2008]
Occupational Exposure to Diacetyl and 2,3-Pentanedione 9

1 . Introduction

of establishments employing fewer than 100 

employees and nearly 53% of establishments 

employing fewer than 10 employees [United 

States Census Bureau 2004]. Industries that 

comprise food manufacturing can be found 

in every state in the United States; however, 

concentrations of specific industries are 

found in general geographic locations. For 

example, in 2004, 33% of the cheese manufacturing employees employed in the United 

States were in Wisconsin, and 20% of employees employed in the fruit and vegetable 

preservation industry were in California 

[BLS 2007]. 

There is increasing likelihood that various 

substances will be used as substitutes for 

diacetyl or 2,3-pentanedione. The potential 

for both employees’ exposure and disease from 

exposure to these substitutes still remains 

largely unstudied. 
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Occupational Exposure to Diacetyl and 2,3-Pentanedione 11

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 15

2 Assessing Occupational 

Exposure in Employees

2.1 Introduction

Measurement of diacetyl and 2,3-pentanedione 

exposure is helpful in preventing flavoringsrelated lung disease, even with complex 

flavorings formulations. Exposures to diacetyl 

and 2,3-pentanedione can be monitored using 

personal and area (environmental) air samples 

because the predominant route of exposure is 

inhalation. Results from air sampling can be 

compared with established criteria such as the 

NIOSH RELs. Measuring employees’ exposures to diacetyl or 2,3-pentanedione may 

help identify processes, locations, or tasks with 

exposures of concern; guide corrective actions 

such as engineering controls; identify improved 

work practices; and select appropriate respiratory protection.

This chapter discusses (1) available sampling 

and analytical techniques for monitoring 

diacetyl and 2,3-pentanedione vapor in the 

workplace; (2) techniques for measuring diacetyl and 2,3-pentanedione in airborne dust and 

bulk materials; (3) real-time techniques for 

measuring relevant airborne analytes and other 

flavoring compounds; and (4) results of some 

occupational exposure assessments by NIOSH 

and others of facilities that use diacetyl and 

2,3-pentanedione.

Many work environments have mixed exposures, with multiple chemical agents present. 

Although the primary focus of this criteria 

document is diacetyl and 2,3-pentanedione, 

other compounds can also be of concern. 

Depending upon the processes employed in 

a workplace, sampling should be conducted 

for agents of concern to maintain safe work 

environments. Common sampling and analytical methods to determine concentrations of 

diacetyl and 2,3-pentanedione are presented in 

Appendices A–E.

2.2 Time-integrated Air 

Sampling and Analytical 

Methods for Diacetyl and 

2,3-Pentanedione Vapor

Personal breathing zone sampling is the preferred approach for estimating employee 

exposure. For personal sampling, an employee 

wears the air sampling equipment, and the inlet 

to the collection medium is positioned within 

the employee’s breathing zone. Area sampling is 

performed for several purposes such as to evaluate exposure characteristics associated with an 

area or process, and to determine the efficiency 

of control systems. While the same sampling 

equipment may be used in some cases for both 

personal and area sampling, area sampling is 

stationary, in contrast to personal sampling, 

which allows for mobility by accompanying 

the employee throughout the sampling period. 

2.2.1 OSHA Methods 1012 and 1013 

In response to the need for longer sampling 

time periods with a lower limit of detection or 

reliable quantitation limit, the Occupational 

Safety and Health Administration (OSHA) 

validated two sampling and analytical methods, 

OSHA Method 1012 and OSHA Method 

1013, for diacetyl and acetoin in 2008 [OSHA 
16 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

2008a, b]. OSHA Method 1013 is for monitoring low ppm levels, while OSHA Method 1012 

is for monitoring ppb levels [OSHA 2008b].

These methods can be used for the simultaneous determination of diacetyl and acetoin. As of 

the publication of this document, these are the 

recommended methods for diacetyl. 

OSHA Methods 1012 and 1013 use two 600 

milligram (mg) sorbent tubes containing specially cleaned and dried silica gel (SKC Inc., 

Eighty Four, PA, Catalog no. 226-183) in series 

and air is sampled at a flow rate of 50 milliliters 

per minute (mL/min) for up to 180 minutes 

for determination of TWA concentrations, and 

a flow rate of 200 mL/min for 15 minutes for 

short-term concentration measurements. An 

opaque sampling tube protective cover should 

be used in conjunction with the sampler to 

prevent the glass sampling tube from breaking and to protect the sample from light, which 

can decompose diacetyl and acetoin. After sampling, the tubes should be separated, capped, 

and protected from light with aluminum foil 

or other opaque material. There is no requirement that samples be kept cold during shipping 

or storage.

OSHA Method 1013 has a reliable quantitation limit of 12 ppb (0.041 mg/m3

) diacetyl for 

a 9-liter sample, and samples are analyzed by 

gas chromatography using a flame ionization 

detector (GC-FID). OSHA Method 1012 has 

a nearly 10 times lower RQL of 1.3 ppb (4.57 

micrograms per meter cubed [μg/m3

]) diacetyl for a 9-liter sample, which is achieved by 

derivatizing diacetyl with 2 milligram per milliliter (mg/mL) O-(2,3,4,5,6-pentafluorobenzyl) 

hydroxylamine hydrochloride in the extraction 

solution and analyzing by gas chromatography 

using an electron capture detector (GC-ECD). 

An advantage of OSHA Method 1013 is that 

sample preparation can be performed in 

one hour, whereas the derivatization step of 

OSHA Method 1012 requires 36 hours. After 

samples have been extracted and analyzed 

using OSHA Method 1013, if needed (e.g., if 

sample concentration is not detectable), they 

can be derivatized and analyzed using OSHA 

Method 1012 to benefit from its lower detection capability. 

2.2.2 OSHA Method 1016

OSHA Method 1016 [OSHA 2010] can be used 

to measure 2,3-pentanedione concentrations. 

OSHA Method 1016 uses the same sampling 

media, sample collection procedure and analytical procedure as OSHA method 1013. However, 

OSHA Method 1016 allows for the simultaneous analysis of 2,3-pentanedione, diacetyl, and 

acetoin by using a different analytical column 

to optimize the analytical separation of these 

compounds. In addition, OSHA Method 1016 

requires samples to be shipped cold. If diacetyl 

and/or acetoin are not anticipated to be present, 

OSHA Method 1016 can be used to sample for 

an additional 20 minutes, or 200 minutes, at 50 

mL/min to determine TWA concentrations of 

2,3-pentanedione [OSHA 2010]. For a 10-liter 

sample, the RQL of 2,3-pentanedione is 9.3 ppb 

(38 μg/m3). 

2.2.3 OSHA Method PV2118 

Superseded by OSHA Methods 1012 and 1013, 

OSHA Method PV2118 [OSHA 2003] was 

developed as an air sampling method for diacetyl that uses two 150/75 mg silica gel sorbent 

tubes in series (SKC Cat. No. 226-10) at a recommended flow rate of 50 mL/min for one 

hour. In response to the limited capacity of this 

sampler in humid environments, a modified 

version of OSHA Method PV2118 was used 

by some practitioners in the field. The modified method uses larger 400/200 mg sorbent 

tubes packed with specially cleaned silica gel 

(SKC Cat. No. 226-10-03) allowing for greater 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 17

2 . Assessing Occupational Exposure in Employees

sample capacity without breakthrough of diacetyl. Sample analysis remained unchanged.

2.2.4 NIOSH Method 2557 

While no longer recommended for use, NIOSH 

developed NIOSH Method 2557 [NIOSH 1994] 

for measuring diacetyl vapor in air. It called for 

the collection of samples onto a 150/75 mg 

carbon molecular sieve sorbent tube (Cat. No. 

226-121, SKC Inc., Eighty Four, PA) at a flow 

rate between 10 and 200 mL/min for a sample 

volume between 1 and 10 liters. The method 

specifies that samples be stored cold and analyzed within 7 days of sampling. 

Until 2007, NIOSH Method 2557 was the predominant air sampling and analytical method 

for diacetyl used in the field, but it is no longer 

recommended for use [Ashley et al. 2008]. In 

2007, field and chamber investigations indicated that NIOSH Method 2557 was adversely 

affected by humidity, resulting in an underestimation of true diacetyl concentrations. To aid 

in the evaluation of sampling and analytical 

methods for diacetyl, a field comparison study 

between new and existing sampling collection 

methods was conducted [Ashley et al. 2008]. 

Side-by-side field samples were collected in 

flavor manufacturing facilities and analyzed 

according to NIOSH Method 2557, OSHA 

Method PV2118, and a modified version of 

OSHA Method PV2118. The results of this 

field work confirmed the tendency of NIOSH 

Method 2557 to underestimate the true concentration of diacetyl as humidity increases. 

However, no mathematical correlation was 

found in this data set which would produce 

an adjustment factor to allow for correction 

of results. 

As a result, NIOSH researchers collaborated 

with scientists at the OSHA Salt Lake Technical 

Center laboratory to study the effects of humidity on measured diacetyl air concentrations 

using NIOSH Method 2557. This laboratory has 

chamber facilities for the generation of known 

diacetyl air concentrations with the ability to 

control both temperature and relative humidity 

(RH). Controlled test atmospheres of diacetyl 

were generated and sampled through an array 

of sampling tubes at calibrated flow rates. Test 

atmospheres were controlled for diacetyl concentration, temperature, and relative humidity. 

Results indicated that diacetyl recoveries for 

NIOSH Method 2557 were affected by absolute 

humidity (AH), storage time of sample tube 

prior to extraction, and diacetyl air concentration. The study resulted in the development of a 

mathematical procedure to adjust diacetyl concentrations previously measured using NIOSH 

Method 2557. The procedure is presented in 

Appendix F and is also published elsewhere 

[Cox-Ganser et al. 2011]. 

2.2.5 Other Air Sampling Method(s) 

in Development

Because of current interest in occupational 

exposure to flavoring compounds, new 

methods continue to be developed for their 

measurement. At this time, however, none of 

these methods are validated.

A method is being developed by NIOSH 

to measure alpha-dicarbonyl compounds 

(such as diacetyl and 2,3-pentanedione) in 

air via derivatization with 1,2-phenylenediamine. This compound is known to react with 

alpha-dicarbonyl compounds to form stable 

quinoxaline derivatives [Rodrigues et al. 

1999]. In this method, air is sampled through 

a sorbent tube containing silica gel coated 

with 1,2-phenylenediamine at 0.1% by weight. 

Samples are extracted in the lab and extraction 

solutions analyzed by gas chromatographynitrogen/phosphorus detection (GC-NPD). A 

potential advantage of this method is greater 

sampling volume and sampling time without 

the breakthrough that would be experienced if 

sampling for an extended time with uncoated 

silica gel tubes. Experiments to date indicate 
18 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

no breakthrough of diacetyl, 2,3-pentanedione, 

or 2,3-hexanedione from the sampling tubes 

after passing 144 liters of air at 80% RH. This 

enables sampling for 8 hours without changing out sampling tubes. Another advantage is 

the high sensitivity of NPD detection, which 

will enable measurement of alpha-dicarbonyl 

compounds below the proposed REL for 

diacetyl of 5 ppb.

A new method for collecting air samples 

using evacuated canisters has been evaluated 

for several VOCs [LeBouf et al. 2012]. The 

450-milliliter canisters, which can be equipped 

with either instantaneous grab sampling 

attachments or restricted-flow controllers (for 

task-based or full-shift sampling), are suitable 

for collection of area and personal samples. 

The air samples are analyzed for VOCs using 

a preconcentrator/gas chromatography-mass 

spectrometry (GC-MS) system. At present, 

this canister method is in the process of being 

validated with three additional compounds, 

diacetyl, 2,3-pentanedione and 2,3-hexanedione, and is being reviewed for incorporation 

into the NIOSH Manual of Analytical Methods. 

A method for priority flavoring compounds 

is being investigated that utilizes a novel 

sampler—the helium diffusion sampler (HDS) 

[Entech Instruments Incorporated 2011]. The 

HDS collects a whole air sample for either 

short-term or full-shift sampling. The advantages of HDS are that no air sampling pump 

is required, there is no concern about breakthrough of the sample components, and there 

is minimal sample handling in the laboratory. A portion of the collected air sample is 

analyzed by a preconcentrator/GC-MS in the 

selected ion monitoring mode. Although HDS 

will not support limits of detection achieved 

by TD-GC-MS because of the relatively small 

air volume sampled (~20 mL), it may have 

adequate sensitivity to measure diacetyl at the 

proposed REL.

2.2.6 NIOSH Method 2549 – Qualitative 

Determination of Volatile 

Organic Compounds

To sample for diacetyl, 2,3-pentanedione, 

as well as a wide range of other flavoring 

VOCs, thermal desorption sorbent tubes 

can provide a high degree of sensitivity. This 

is because desorption of compounds from 

thermal desorption tubes does not involve 

dilution into an extraction solvent. Instead, 

compounds are thermally desorbed from the 

sampling tubes in a thermal desorption system. 

This technique is primarily used for qualitative 

screening purposes because of the ability of 

thermal desorption tubes to capture a diverse 

range of VOCs, but specific compounds can 

be quantified if corresponding standards are 

analyzed along with the samples. The thermal 

desorption tube is usually a stainless steel tube 

configured and filled with a single sorbent bed 

or multiple beds of various sorbents including carbonaceous materials, carbon molecular 

sieves, and/or porous polymers. The sorbents 

can be heated to high temperatures without 

breakdown or the generation of artifacts, so 

thermal desorption tubes can be cleaned and 

reused multiple times. The tubes are analyzed 

with a thermal desorber-GC-MS (TD-GC-MS) 

[NIOSH 1994]. 

2.3 Sampling for Diacetyl 

and 2,3-Pentanedione 

in Airborne Dust and in 

Bulk Materials 

Although diacetyl and 2,3-pentanedione are 

normally found in liquid form, they can also be 

encapsulated in or coated on a powder substrate. 

Air sampling for dust that may be generated 

during handling of powdered flavorings can be 

achieved by active sampling methods. During 

the sample collection, however, some of the 

diacetyl and 2,3-pentanedione may volatilize, 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 19

2 . Assessing Occupational Exposure in Employees

i.e., release from the dust particles and enter 

the vapor phase due to contact with moisture. 

In addition, environments in which dust is 

generated may also contain vapors of the flavoring compounds. Sorbent tubes used for the 

collection of vapor-phase diacetyl or 2,3-pentanedione cannot be used to adequately sample 

for dust at the low flow rates required by the 

tubes [OSHA 2008a]. As a result, modifications 

to the sampling methods are necessary to assess 

exposure to both vapor and dust. 

2.3.1 Size-Selective Air 

Sampling for Dust

Measurement of airborne dust particles according to their size (e.g. inhalable, thoracic, and 

respirable) can help to understand where they 

may deposit in the respiratory tract. Several 

types of sampling devices are available (e.g., 

inhalable dust samplers, impactors, cyclones, 

and sampling cassettes) to provide measurements of different size fractions of airborne 

dust. In most cases, dust is collected onto a filter, 

and the filter can be analyzed via gravimetric 

means to provide the mass of the dust. Filters 

should be hydrophobic in nature (e.g., polyvinyl chloride) in order to minimize collection 

of moisture. After being measured gravimetrically, filters can be analyzed for diacetyl and 

other compounds by the procedure described 

in section 2.3.2. Validated methods such as 

NIOSH Method 0500 for total dust and NIOSH 

Method 0600 for respirable dust [NIOSH 1994] 

are available for the collection and gravimetric 

analysis of airborne dust. 

2.3.2 Sampling for Diacetyl 

and 2,3-Pentanedione in 

Airborne Dust

A sampling and analytical method is being 

developed by NIOSH for the quantitative 

measurement of diacetyl, 2,3-pentanedione, 

and potentially other flavoring compounds 

in dust. A sampling cassette with a filter is 

used to collect airborne dust. The filter is then 

extracted in water and the aqueous solution is 

heated to promote the transfer of volatile components to the headspace above the solution. 

The headspace is sampled using a solid-phase 

microextraction (SPME) fiber. Headspace 

SPME involves an equilibrium process in which 

the volatile analytes establish equilibria between 

the sample solution, the headspace above the 

solution, and the polymer-coated fused silica 

fiber. The mechanism by which the analytes 

are extracted from the headspace is based on 

absorption of the analytes onto the fiber. The 

fiber is inserted directly into a GC-MS. The 

analytes are extraced from the fiber in the 

hot injection port and concentrated onto an 

analytical column. Because the entire sample 

collected on the fiber is introduced into the 

GC-MS instrument, as opposed to an aliquot 

of the sample for methods in which a solvent 

extract is used, lower detection limits can be 

achieved. This same procedure can be used to 

measure diacetyl, 2,3-pentanedione, and potentially other flavoring compounds in samples of 

bulk powders. 

2.3.3 Bulk Liquids and Solids 

2.3.3.1 Sample collection

Although the review of safety data sheets or 

other available product documentation may 

be helpful to identify flavor compounds and 

potential exposures, they are not always comprehensive or specific. Collection and analysis 

of bulk flavoring materials can be useful to 

identify and quantify chemical ingredients and 

guide exposure assessment strategies. Prior 

to collecting bulk samples, it is important to 

consider the physical state of the materials to 

be sampled (liquids, pastes, or powders), the 

need to sample opened or unopened containers, the sampling locations, the number of 

samples to collect, and the amount of sample 

to collect (often determined by requirements of 

the laboratory analysis). Bulk samples should 
20 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

be representative; in other words, they should 

be derived from a variety of sampling locations 

and obtained from multiple batches to capture 

any variability in the bulk materials used. 

When sampling, it is important to collect and 

transport the sample in a manner that does 

not contaminate or cross-contaminate the 

bulk materials. Only clean or unused sample 

containers that are compatible with the bulk 

materials sampled should be used. In general, 

glass containers are ideal because they will not 

react with most chemicals, but polyethylene or 

polypropylene containers may also be appropriate. A typical container is a 20-mL glass 

scintillation vial with a polytetrafluoroethylene 

(PTFE)-lined screw cap. Each container should 

be clearly labeled with information about the 

bulk sample including material sampled, 

company and product number, site of sampling, 

date of sampling, sample tracking number and 

any hazards or precautions to be taken when 

handling the bulk sample. 

After sampling, consideration should be given 

to preserve the integrity of the bulk samples 

during storage and shipping. For example, 

care should be taken to keep samples cold and 

protected from light if necessary. In addition, 

bulk materials should not be shipped together 

with air samples. Established Department of 

Transportation (DOT) and International Air 

and Transport Association (IATA) shipping 

regulations of hazardous materials and dangerous goods should be followed if hazardous 

materials are to be shipped. Materials that are 

considered hazardous for the purpose of transportation under the DOT regulations are listed 

in the hazardous materials table in Title 49 of 

the Code of Federal Regulations (CFR), Section 

172.101 [49 CFR 172.101]; materials that are 

considered dangerous goods for the purpose 

of shipping by air under IATA regulations are 

listed in the list of dangerous goods in IATA 

dangerous goods regulations, section 4.2 [IATA 

2012]. The DOT and IATA regulations guide 

the classification/identification and packaging of hazardous materials and the marking 

and labeling of shipping containers containing hazardous materials. If the materials to be 

shipped are known to be hazardous but the 

specific names of the materials are not found 

on either the DOT hazardous materials table 

or the IATA list of dangerous goods, then the 

materials must be classified into a hazard class 

according to section 3 of the IATA dangerous 

goods regulations handbook, and a proper 

shipping name must be assigned according to 

section 4 of the IATA dangerous goods regulations handbook. A person must be trained 

in DOT and IATA regulations and certified in 

order to mark a shipment as hazardous. If it is 

unknown whether the materials to be shipped 

are hazardous or not, then a person who is 

trained in DOT and IATA regulations should 

be consulted. 

2.3.3.2 Measurement of diacetyl or 

2,3-pentanedione content of 

bulk powders

The analytical procedure being developed for 

airborne dust samples described in section 

2.3.2 will also be used for analysis of bulk 

powder samples.

2.4 Real-time Techniques 

for Diacetyl and Other 

Flavoring Compounds 

Several analytical methods provide real-time 

or near real-time measurements of volatile 

compounds in air such as diacetyl and 2,3-pentanedione. These methods have the unique 

advantage of providing continuous exposure 

information over very short averaging periods 

that can be viewed as it is being generated 

during sampling or later if the instrument 

has data-logging capabilities. The abundance 

of measurement information provides valuable insight into variations in concentrations 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 21

2 . Assessing Occupational Exposure in Employees

throughout the sampling period as well as the 

short-term concentration peaks that can possibly be associated with their sources. While 

real-time monitoring instruments generally 

lack sufficient sensitivity and specificity for 

monitoring REL levels of diacetyl and 2,3-pentanedione, they can be useful for screening, 

identifying appropriate work practices, and 

to find leaks and “hotspots.” This information can be very useful in the development of 

exposure controls. 

2.4.1 Photoionization Detectors 

Photoionization detectors (PIDs) can be used to 

monitor VOC air concentrations in industrial 

work environments, including flavoring manufacturing facilities, and have become favored 

instruments for on-site monitoring because of 

ease of operation, reliability, versatility, cost, 

and response to a wide variety of substances. 

PID instruments measure the relative concentration of VOCs by passing the molecules of 

those compounds past an ultraviolet lamp that 

emits radiation over a narrow wavelength range 

in the ultraviolet region of the electromagnetic 

spectrum. Photons of ultraviolet radiation will 

form a molecular ion by removing an electron 

from orbit around that molecule, allowing for 

electronic detection of that ion, hence the name. 

The energy of the radiation emitted by the lamp 

is inversely proportional to its wavelength, 

and common PID lamps produce energy in 

the range from approximately 8 to 12 electron 

volts (eV). The amount of work required to 

form a molecular ion by removing an electron 

from orbit, a property known as ionization 

potential, varies by compound but for many 

hydrocarbons is in the range from 7 to 11 eV. 

Because nitrogen, oxygen, and many of the 

minor components of air (i.e., water vapor, 

carbon monoxide, carbon dioxide, argon) have 

ionization potentials significantly higher than 

12 eV, they are not ionized by the photons 

emitted from a PID. This property allows for 

the continuous monitoring of air to obtain an 

estimate of total hydrocarbon concentration.

PIDs respond to a broad range of VOCs and 

do not provide concentrations specific to any 

particular compound. They are often calibrated 

for isobutylene and can commonly detect total 

VOC concentrations from 1 to 2,000 ppm. 

Modern PIDs can be programmed to measure 

the concentration of VOCs at fixed time 

intervals and store these data for subsequent 

download to a computer. 

2.4.2 Infrared Analyzers 

The absorption of infrared (IR) radiation, while 

more commonly used as a qualitative tool, can 

also be used to quantify many substances by 

determination of response relative to known 

concentrations of that substance. Absorption 

of electromagnetic radiation in the IR region 

of the spectrum will produce transitions among 

vibrational and rotational states of the molecules absorbing that rotation. This absorption 

can only occur at wavelengths exactly matching the vibrational frequency of a chemical 

bond, and by selecting the proper analytical 

wavelength it is possible to obtain reasonable 

specificity in the compound being quantified.

Diacetyl can be detected and measured by using 

an IR gas analyzer such as the Thermo Electron 

MIRAN® “SapphIRe” (Thermo Fisher Scientific 

Inc., Waltham, MA), which is a portable directreading instrument that has the advantage 

of displaying real-time concentrations. The 

SapphIRe is a single beam IR spectrophotometer with a pathlength of 0.5 or 12.5 meters. 

It has a sample cell volume of 2.23 liters and 

a built-in pump that runs at approximately 

14 liters per minute. Single sample analyses 

are updated every 0.5 seconds. The detector is 

available with preloaded factory calibrations for 

over 100 gases, but because diacetyl is not in 

this standard library it should be set up for this 

application by the factory. The concentration 
22 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

range that can be measured is dependent on 

the compound in question. The high and low 

settings for the pathlength extend this range 

considerably. 

The predecessor model, the Foxboro/Wilks 

MIRAN 1A, has adjustable wavelength and 

pathlength controls and can be calibrated for 

gases or vapors using the closed loop system 

available. Many MIRAN 1A models are still in 

use in the field. The best wavelength for measuring diacetyl is about 9 micrometers. Neither 

water nor carbon dioxide should interfere significantly at that wavelength. The minimum 

detectable concentration should be less than 

0.5 ppm at the highest pathlength. 

Fourier transform infrared gas analyzer (FTIR) 

spectroscopy can be used to analyze a sample 

of gaseous molecules for both chemical composition and for the concentration of individual 

chemical constituents. In this analysis, chemical functional groups absorb IR radiation 

at specific, unique frequencies producing a 

characteristic spectrum of absorbed versus 

transmitted radiation. From this spectrum, 

identification and quantitation of the gas is 

possible. FTIR analysis can produce real-time 

quantitation of flavoring compounds in air providing chemical specific full-shift, partial-shift, 

and peak concentration measures although 

interferences can pose analytical difficulties in 

quantifying specific flavoring compounds in 

complex environments with multiple organic 

chemicals present.

2.4.3 Photoacoustic Spectroscopy 

(Infrared Absorbance) Techniques 

Because the absorption of infrared radiation 

produces transitions among vibrational states 

of molecules, the application of rapid pulses 

of IR photons at the proper wavelength can be 

used to produce pressure variations in the air 

surrounding the molecules absorbing that radiation. Those pressure variations can be detected 

as sound waves, the amplitude of which is proportional to the concentration of the analyte of 

interest. Using IR radiation and measuring this 

resultant amplitude to quantify an analyte is the 

technique of photoacoustic spectroscopy.

Diacetyl has been measured using the Innova 

photoacoustic infrared gas analyzers, which 

are direct-reading instruments that have the 

advantage of displaying real-time concentrations. Both personal and area concentrations 

were measured during tasks involving exposure to diacetyl in liquid and powder form and 

then 8-hour TWA exposures were calculated. 

The powder exposures only measured vapor 

released and did not include diacetyl adsorbed 

on the powder [Martyny et al. 2008].

Current available models of the photoacoustic 

analyzer are the 1314 and 1412, available from 

California Analytical Instruments, Inc., Orange, 

CA. The measurement system is based on photoacoustic infrared detection and provides the 

capability of measuring virtually any gas that 

absorbs in the infrared spectrum. Gas selectivity is achieved through the use of optical filters 

that provide both a means of detecting the gas 

of interest and compensating for interfering 

gases and water. Specifications on the unit indicate a dynamic range of 4 orders of magnitude 

and a repeatability of 1% of the measured value. 

The analyzer displays updated concentrations 

approximately every 30 seconds. The analyzer 

can be calibrated using diacetyl standards and 

can analyze diacetyl concentrations from the 

parts per billion range to hundreds or thousands of parts per million. 

2.5 Industrial Hygiene 

Surveys and Exposure 

Assessments 

Several investigations have been completed 

by NIOSH and others within the flavoring 

and food production industries. Exposure 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 23

2 . Assessing Occupational Exposure in Employees

conditions vary widely, depending upon 

site-specific parameters and the processes 

employed. Many diacetyl samples have been 

collected to evaluate occupational exposures in 

the workplace and are described below. When 

pertinent data on absolute humidity and time 

to sample extraction were available, measurements obtained using NIOSH Method 2557 

were subsequently corrected for the method’s 

tendency to underestimate [Cox-Ganser et 

al. 2011]. An overview of diacetyl samples 

collected during multiple investigations is presented in Table 2-1. 

2.5.1 NIOSH Microwave Popcorn 

Production Exposure 

Assessments 

NIOSH conducted health hazard evaluations 

at six microwave popcorn plants from 2000 

to 2003 [Kanwal et al. 2006]. In these facilities 

diacetyl-containing butter flavorings (liquids, 

pastes, or powders) were mixed with heated 

soybean oil in large heated mixing tanks. 

Salt and coloring were added to the flavoring 

mixture which was transferred to packaging 

lines and combined with kernel popcorn in 

microwaveable bags. Diacetyl concentrations 

were measured with NIOSH Method 2557 in 

multiple production locations using personal 

and area samples. 

In the plants, 29 area and 17 personal samples 

were collected in mixing areas, and 67 area and 

65 personal samples were collected in packaging areas. Humidity-corrected mean diacetyl air 

concentrations ranged from 0.63 to 57.2 ppm 

for area samples and from 0.035 to 1.33 ppm 

for personal samples in the mixing areas. In the 

packaging areas, mean concentrations ranged 

from 0.019 to 3.0 ppm for area samples and 

from 0.023 to 1.16 ppm for personal samples. 

In general, diacetyl concentrations were higher 

in the mixing rooms when the diacetyl-containing butter flavorings were heated. 

In 2010, a microwave popcorn company asked 

NIOSH to evaluate chemical constituents in 

eight liquid butter flavorings because their supplier did not identify chemical substitutes they 

were using in place of diacetyl [Boylstein 2012]. 

Quantitative GC-MS analysis showed acetoin 

in five samples, 2,3-pentanedione in four, and 

2,3-hexanedione in one, all at concentrations of 

0.5% or less by weight, except for one acetoin 

sample at 2%. The more sensitive semiquantitative headspace analysis with thermal detection 

tubes found diacetyl and acetoin in all samples, 

2,3-pentanedione in five, 2,3-hexanedione in 

one, and 2,3-heptanedione in one. 

2.5.2 Other Microwave Popcorn 

Production Exposure 

Assessments 

White et al. [2010] conducted a comprehensive, 

repeated exposure monitoring campaign at 

four microwave popcorn plants. A total of 639 

full shift diacetyl samples were collected during 

the day and night shifts in multiple production areas including all employees who worked 

in the slurry (mixing) room. In that study 

49% of 639 samples were below their limit of 

detection with the maximum measurement of 

11.72 ppm after correction for humidity [White 

et al. 2010]. Overall, exposures were higher for 

mixers compared to non-mixers and were consistent with diacetyl concentrations observed 

during previous NIOSH investigations. Diacetyl 

exposures declined substantially for mixers 

after the installation of engineering controls. 

2.5.3 NIOSH Flavoring Manufacturing 

Exposure Assessments

In 1985, NIOSH conducted a health hazard 

evaluation at a plant in Indiana that produced 

flavorings for the baking industry [NIOSH 

1986]. Case histories showed severe fixed 
See footnotes at end of table. (Continued)

24 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

Table 2-1. Multiple investigations of diacetyl in flavoring and food production industries

Diacetyl concentration in ppm (sample type)

Study Method* Location Arithmetic mean Geometric mean Range

Microwave popcorn plantsFacility G 

(first survey) 

[NIOSH 2006]

NIOSH 

Method 2557 (corrected)

Mixing roomPackaging areaQC labMaintenanceOther areas

57.2 (full-shift TWA)2.8 (full-shift TWA)0.8 (full-shift TWA)0.9 (full-shift TWA)< 0.15 (full-shift TWA)

_____

_____

Facilities 

G,J,K,L,N,O [Kanwal et al. 2006]

NIOSH 

Method 2557 (corrected)

Packaging areas (area samples)Packaging areas (personal samples)Mixing rooms/areas (area samples)Mixing rooms/areas (personal samples)

0.019–3.00.023–1.160.63–57.20.035–1.33

____

____

[White 2011] NIOSH 

Method 2557 (corrected)

MixersNon-mixers

0.119–2.704 (full shift)0.042–0.123 (full shift)

0.044–0.587 (full shift)< 0.001–0.019 

(full shift)

0.004–11.72 (full shift)0.004–1.984 (full shift)

Flavoring production plantsFacility B[NIOSH 2007a]

NIOSH 

Method 2557 (corrected)

Powdered flavoring production area 2.73 (full-shift TWA)

25.9 (partial shift)

__

204 (real-time peak)

_

Facility C[NIOSH 2008b]

OSHA Method 

PV2118

Liquid flavoring production areaPowdered flavoring production areaTask-based (pouring diacetyl)

0.46 (full-shift TWA)0.34 (full-shift TWA)

_

___

__

11 (10-minute peak)

Facility H[NIOSH 2008a]

OSHA Method 

PV2118

Liquid production room (area samples)Powder production room (area samples)

0.26 (full-shift TWA)0.07 (full-shift TWA)

__

__

NIOSH 

Method 2557

Liquid production (personal samples)Powder production (personal samples)

0.100.05

__

__
See footnotes at end of table. (Continued)

Occupational Exposure to Diacetyl and 2,3-Pentanedione 25

2 . Assessing Occupational Exposure in Employees

Diacetyl concentration in ppm (sample type)

Study Method* Location Arithmetic mean Geometric mean Range

Facility D[NIOSH 2009c]

OSHA Method 

PV2118

Starter distillate roomStarter distillate room (personal samples)Spray dry roomSpray dry room (personal samples)Flavors roomFlavors room (personal samples)Spray dry room, task-based (moving 

diacetyl between containers)

Spray dry room, task-based (cleaning 

barrel with hose)

________

1.06 (full-shift TWA)1.78 (full-shift TWA)1.07 (full-shift TWA)0.756 (full-shift TWA)0.171 (full-shift TWA)0.329 (full-shift TWA)

__

1.06 (full-shift TWA)1.78 (full-shift TWA)1.07 (full-shift TWA)0.756 (full-shift TWA)0.171 (full-shift TWA)0.329 (full-shift TWA)

__

Facility I[NIOSH 2011]

NIOSH 

Method 2557

Spray dryingSpray drying (personal samples)Other production areasOther production areas 

(personal samples)

____

0.169 (full-shift TWA)0.123 (full-shift TWA)0.375 (full-shift TWA)0.762 (full-shift TWA)

____

OSHA Method 

PV2118

Spray dryingSpray drying (personal samples)Coffee and tea areaLiquid compounding area 

(personal samples)

____

0.167 (full-shift TWA)0.182 (full-shift TWA)0.076 (full-shift TWA)1.900 (full-shift TWA)

____

Facility Q[NIOSH 2013a]

Canister Multiple locations (area samples)

_

_ No diacetyl detected

(< 0.0029)(instant to 3 hrs)

[Martyny et al. 

2008]

NIOSH 

Method 2557

All areas (personal samples) 2.48 (1–3 hours)

_ 0.01–60 (1–3 hours)

Diacetyl production[van Rooy 2007] Task specific

_

_ 0.6–83 (real-time peaks)

Table 2-1 (Continued). Multiple investigations of diacetyl in flavoring and food production industries
26 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

Diacetyl concentration in ppm (sample type)

Study Method* Location Arithmetic mean Geometric mean Range

Food productionFacility M

-popped corn

[NIOSH 2007b]

NIOSH 

Method 2557

All areasDirectly above heated popping oil

––

––

No diacetyl detected(< 

0.01)

0.14 (real-time peak)

Facility E

-bakery mix

[NIOSH 2009a]

OSHA Methods 

PV2118, 1013

All areas – – No diacetyl detected

Facilities F 

(3 sites)-cafeterias 

[NIOSH 2009b]

OSHA Method 

PV211

All areas – – No diacetyl detected

(LOD< 0.02)

Facility P

-cream cheese

[NIOSH 2013b]

OSHA Method 

1012

Cooking, filling, packaging 

(personal samples)

Cooking, filling, packaging (area samples)

––

––

0.0004–0.0083 (full shift)0.0003–0.0138 (full shift)

Facility U

-snack food

[NIOSH 2013c]

Canister Snack chip production (personal samples)

Snack chip production (area samples)

––

––

No diacetyl detected (LOD 

varied from < 0.0028– < 0.006) (2–3 hrs)

0.0014–0.0017 (instant)

Facility R-coffee[Bailey et al. 

2015; Duling et al. 2016]

OSHA Method 

1012

Warehouse (personal samples)Roasting room (personal samples)Grinding/packaging room 

(personal samples)

Flavoring room (personal samples)Other areas (personal samples)Warehouse (area samples)Roasting room (area samples)Grinding/packaging room (area samples)Flavoring room (area samples)Other areas (area samples)Packaging room (area sample)

0.008 (full-shift TWA)0.026 (full-shift TWA)0.093 (full-shift TWA)0.080 (full-shift TWA)0.007–0.081(fullshift TWA)0.011 (full-shift TWA)0.020 (full-shift TWA)0.104 (full-shift TWA)0.090 (full-shift TWA)0.004–0.062(fullshift TWA)

__________

––

___________

14.3 (15-minute)

*(corrected) indicates concentration values have been corrected for humidity and time to extraction effects_ no data collected

Table 2-1 (Continued). Multiple investigations of diacetyl in flavoring and food production industries
Occupational Exposure to Diacetyl and 2,3-Pentanedione 27

2 . Assessing Occupational Exposure in Employees

obstructive lung disease among employees in a 

mixing room. Data from previous air monitoring indicated a high dust concentration in the 

personal breathing zone of an employee during 

a mixing operation. Diacetyl was on a list of 

ingredients commonly used at this facility but 

airborne measurements of diacetyl or other flavoring compounds were not made. Although 

the investigators were unable to identify specific etiology at that time, they concluded that 

employees’ disease was most likely caused by 

some agent in the mixing room at the plant. 

NIOSH personnel conducted evaluations at 

three California flavoring manufacturing facilities where they measured exposures to diacetyl 

and other related compounds [NIOSH 2007a, 

2008a, b, c]. The objectives of these surveys 

included identifying common work tasks, 

plant processes, and procedures, as well as 

characterizing potential occupational exposures within the flavoring industry. Most of the 

data collected were from the liquid and powder 

production areas, with some information also 

coming from spray drying, preproduction, 

quality assurance, administration, and research 

and development locations. 

At one plant [NIOSH 2007a], the mean TWA 

diacetyl exposure, after NIOSH Method 2557 

humidity-based correction, from full-shift 

air sampling in the powdered flavoring production area was 2.73 ppm. Measurements 

made with partial-shift air sampling during 

the production of butter and vanilla powdered flavorings showed a diacetyl exposure of 

25.9 ppm. Employees’ real-time diacetyl exposures measured with an FTIR monitor during 

the packaging of these powders were as high 

as 204 ppm. At another plant [NIOSH 2008b], 

mean TWA diacetyl air concentrations from 

full-shift air sampling using modified OSHA 

Method PV2118 in November 2006 (area and 

personal samples combined) were 0.46 ppm in 

liquid flavoring production and 0.34 ppm in 

powdered flavoring production. A task-based 

personal air sample measured a diacetyl air 

concentration of 11 ppm when an employee 

poured diacetyl from a 55-gallon drum into 

multiple 5-gallon containers over a 10-minute 

period. Using modified OSHA Method PV2118 

for area air sampling at the other plant [NIOSH 

2008a], the mean full-shift concentration of 

diacetyl in the liquid production room was 0.26 

ppm, while in the powder production room it 

was 0.07 ppm. For personal samples that were 

collected with NIOSH Method 2557 and not 

corrected for humidity and time to extraction, 

the mean concentrations in liquid production 

and powder production rooms were 0.10 ppm 

and 0.05 ppm. This work also indicated high 

variability in concentrations of volatile organic 

compounds (as measured with a PID) and dust 

(as measured with personal dust monitors) 

with time. 

A health hazard evaluation was conducted at a 

facility in Wisconsin [NIOSH 2009c] that manufactured flavorings, modified dairy products, 

and bacterial additives. One of the flavoring 

products made at this plant was liquid starter 

distillate, a product of distillation of fermented 

milk stock, which contains about 4.5% diacetyl. 

Starter distillate and liquid diacetyl were used 

to make a variety of powdered (via spray drying 

processes) and liquid flavorings. NIOSH staff 

obtained 21 personal and 29 area air samples 

using modified OSHA Method PV2118 for 

diacetyl throughout the facility. They found 

the highest full-shift TWA concentrations in 

the starter distillate room (geometric mean of 

1.78 ppm for personal and 1.06 ppm for area 

samples), followed by the spray dry room (0.756 

and 1.07 ppm) and the flavors room (0.329 and 

0.171 ppm). In the spray dry room, FTIR realtime measurements indicated peak diacetyl 

concentrations up to 90 ppm in the employee’s 

breathing zone while dumping diacetyl from 

buckets to mixing tanks and while pumping 

diacetyl from a barrel into buckets. A peak 

exposure of about 18 ppm was measured in 
28 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

the breathing zone of an employee in the same 

room while cleaning a barrel with a water hose. 

Company air sampling data were obtained 

during a health hazard evaluation at an Indiana 

flavorings plant that used many ingredients, 

including diacetyl and starter distillate, in the 

batch production of a variety of liquid and 

powdered flavorings [NIOSH 2011]. Using 

NIOSH Method 2557 prior to the HHE 

request to measure diacetyl, they collected 22 

samples. The geometric mean full-shift TWA 

diacetyl concentration in spray drying operations was 0.123 ppm for personal samples and 

0.169 ppm for area samples, while in the other 

production areas, mean concentrations up to 

0.762 ppm and 0.375 ppm were measured for 

personal and area samples, respectively. Because 

of the problems with NIOSH Method 2557, 

these results were likely underestimations of 

the true concentrations. No data on humidity 

or time from collection to analysis was available, 

so no correction could be estimated. Subsequent 

measurements (45 personal and 71 area 

samples) by the company, after some control 

intervention, were collected using validated 

OSHA sampling Methods PV2118 and 1012 

for diacetyl. In the spray drying operations, the 

geometric mean for full-shift diacetyl personal 

samples was 0.182 ppm, and for area samples it 

was 0.167 ppm. The highest mean concentration in the other production areas was 1.900 

ppm for personal samples (liquid compounding area) and 0.076 ppm for area samples (coffee 

and tea area).

Another health hazard evaluation was performed at a flavorings plant in Kentucky that 

produced flavors, colors, and food and beverage 

ingredients used in the manufacture of consumer products [NIOSH 2013a]. Diacetyl was 

not found in use during the NIOSH air sampling 

survey. Using evacuated canisters, diacetyl and 

2,3-hexanedione were not detected in any of the 

instantaneous or 3-hour area air samples taken 

in several parts of the plant. 2,3-Pentanedione 

was detected in two area air samples taken in 

the liquid samples room. The detection limits 

ranged from 1.4 to 2.9 ppb for diacetyl, 1.5 to 

3.2 ppb for 2,3-pentanedione, and 1.7 to 3.6 ppb 

for 2,3-hexanedione. Of the two air samples that 

detected 2,3-pentanedione in the room, one was 

an instantaneous sample taken near a trash can 

for disposal of used pipettes while making a flavoring recipe and resulted in a level of 47 ppb. 

The other sample that detected 26 ppb 2,3-pentanedione was collected for 187 minutes in the 

center of the room. During the sampling period, 

several employees were preparing recipes, which 

included fruit and cheese flavors. 

2.5.4 Other Flavoring Manufacturing 

Exposure Assessments

In a study evaluating diacetyl exposures in 16 

flavor manufacturing facilities, Martyny et al. 

[Martyny et al. 2008] measured levels of that 

compound from the limit of detection (0.01 to 

0.18 ppm depending on sample duration) to 

as high as 60 ppm. Using a protocol designed 

to obtain measurements during worst-case 

exposures by collecting samples only during 

processes in which diacetyl was being used, 

181 personal and area samples were collected 

generally for 1 to 3 hours. Samples for diacetyl were collected and analyzed using NIOSH 

Method 2557 [NIOSH 1994] which was subsequently found to underestimate actual diacetyl 

concentrations. Without sampling environment absolute humidity information to make 

corrections, the results of this study likely 

underestimate true values. 

Results indicated personal exposures during 

the selected work processes ranged from <0.01 

to 60 ppm, with a mean of 2.48 ppm. Eighthour TWA concentrations were calculated with 

the assumption that there was no exposure to 

diacetyl during the unsampled 5 to 7 hours of 

a work shift. However real-time monitoring of 

airborne diacetyl vapor concentrations, made 

using a photoacoustic IR analyzer, indicated a 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 29

2 . Assessing Occupational Exposure in Employees

background of approximately 2 ppm diacetyl 

according to Figure 1 of that paper.

Data indicated that concentrations varied by 

process, with powder compounding having the 

highest mean and median diacetyl exposures. 

Martyny also concluded, “Compared with the 

microwave popcorn industry, there is wide 

variability in frequency and duration of use of 

diacetyl among flavor companies.” 

2.5.5 NIOSH Flavored Food Production 

Exposure Assessments 

NIOSH researchers conducted health hazard 

evaluations at food production facilities including a bakery mix production plant [NIOSH 

2009a], a popcorn popping plant [NIOSH 

2007b], three office building cafeterias [NIOSH 

2009b], a cream cheese manufacturing plant 

[NIOSH 2013b], a snack food production plant 

[NIOSH 2013c], and a coffee production plant 

[Bailey et al. 2015; Duling et al. 2016]. 

At the bakery mix production facility, employees combined liquid and powdered flavorings 

with flour, sugar, salt and other solid ingredients to produce baking mixes. For about a year 

up to July 2008, the plant used a buttermilk flavoring that contained 15% to 20% diacetyl and 

then began using a reformulated buttermilk 

flavoring that contained less than 1% diacetyl. 

The reformulated flavoring also contained the 

diacetyl substitute 2,3-pentanedione. Diacetyl 

was detected in qualitative screening air samples 

using NIOSH Method 2549 during industrial 

hygiene air sampling by NIOSH investigators 

in late September 2008, but the concentrations were too low to be detected in any of the 

9 personal or 10 area samples collected with 

the modified OSHA Method PV2118. Diacetyl 

was again not detectable in a second industrial hygiene survey in May 2009 when NIOSH 

investigators collected 13 personal and 11 

area air samples using OSHA Method 1013; 

however, one personal sample showed an air 

concentration of 2,3-pentanedione of 91 ppb 

(parts per billion parts air), and a corresponding 

area sample showed an air concentration of 78 

ppb. Nearly half of the samples detected 2,3-pentanedione in the air. Area air sampling using a 

method under development, in-tube derivatization with 1,2-phenylenediamine (section 2.2.5 

above), did not detect diacetyl in any of the 11 

samples, but it measured 2,3-pentanedione in 7 

samples, at concentrations ranging from 48 to 

95 ppb. The sample that showed an air concentration of 95 ppb was obtained in the same area 

where a sample obtained with OSHA Method 

1013 showed an air concentration of 78 ppb.

At the popcorn popping plant, neither the two 

personal nor the twelve area air samples found 

diacetyl concentrations above the minimum 

detectable concentration of 0.01 ppm using 

NIOSH Method 2557 during popcorn popping 

operations with butter-flavored oil. Diacetyl was 

detected in all three thermal desorption tube 

samples from the room with semiquantitative 

analyses (NIOSH Method 2549) but with very 

low abundances. A one-minute real-time concentration of 0.14 ppm diacetyl was measured 

with an FTIR monitor directly above the heated 

popping oil. 

At the three cafeterias, two of seven cooking oil 

products being used contained diacetyl. Neither 

diacetyl nor acetoin was found at or above the 

minimum detectable concentration (0.02 ppm) 

using the modified OSHA Method PV2118 to 

collect 20 personal and area air samples during 

grilling operations. 

At the cream cheese plant in 2011, several flavorings, including dairy, cheese, strawberry, 

blueberry, and smoke were found with headspace sampling to contain diacetyl with or 

without 2,3-pentanedione. Air sampling with 

OSHA Method 1012 during cooking, filling, 

and packaging of cream cheese made with 

some of those flavorings, measured area diacetyl concentrations (n=15 near full-shift) from 
30 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

0.3 to 13.8 ppb. The four highest concentrations were greater than 11 ppb: three of these 

were collected in a cooking area and the other 

in a filling area. Fourteen near full-shift personal 

diacetyl exposures ranged from 0.4 to 8.3 ppb, 

while six short-term samples collected mostly 

while ingredients were added to cook kettles 

ranged from 4.4 to 15.1 ppb. Fourteen area and 

six personal concentrations of 2,3-pentanedione measured with OSHA Method 1016 were 

all less than limits of detection (15.8 to 48.8 

ppb), as were two of the three also sampled with 

the more sensitive draft NIOSH method using 

1,2-phenylenediame-treated silica gel tubes (0.5 

ppb limit of detection) – the detectable concentration was 0.9 ppb while using smoke flavoring. 

Of six area samples collected alongside cleaning 

operations with evacuated canisters for 2,3-pentanedione (1.2 to 2.9 ppb limits of detection) 

and 2,3-hexanedione (1.5 to 3.6 ppb limits of 

detection), one measured 2,3-pentanedione at 

6.2 ppb and 2,3-hexanedione at 9.0 ppb during 

a nearly 3-hour cleaning procedure of cooking 

equipment containing strawberry cream cheese 

remnants while no cream cheese was being 

made in the room.

The snack food production plant applied powdered seasonings onto potato, corn, and tortilla 

chips after they were fried. Headspace analyses of bulk samples of seasonings found trace 

amounts of diacetyl, but no other alpha-diketone compounds, in four of the seven samples: 

barbeque, honey barbeque, cheddar sour 

cream, and chili cheese. Diacetyl, 2,3-pentanedione, and 2,3-hexanedione were not detected 

in the five 15- to 180-minute personal breathing zone evacuated canister air samples from 

processing line operators during nacho cheese 

tortilla chip production. The detection limits 

ranged from 2.8 to 6.0 ppb for diacetyl, 3.4 to 

7.2 ppb for 2,3-pentanedione, and 3.2 to 6.8 

ppb for 2,3-hexanedione. Although diacetyl was 

detected in three area samples collected instantaneously near the seasoning hopper, it was not 

quantifiable. Because it was found between the 

detectable level of 1.3 ppb and the quantifiable 

level of 4.3 ppb, the reported concentrations 

of 1.4 to 1.7 ppb are considered estimates. The 

area samples did not detect 2,3-pentanedione or 

2,3-hexanedione (detection limits of 1.5 and 1.6 

ppb, respectively). 

The coffee production plant produced flavored 

and unflavored whole bean and ground coffee. 

Full-shift area air samples collected for diacetyl 

with OSHA Method 1012 and for 2,3-pentanedione with OSHA Method 1016 had highest 

mean concentrations by location in the grinding/packaging room (103 ppb diacetyl, 63 ppb 

2,3-pentanedione), flavoring room (90 ppb 

diacetyl, 151 ppb 2,3-pentanedione), and the 

production offices (62 ppb diacetyl, 32 ppb 

2,3-pentanedione), which were located within 

the larger grinding/packaging room. These were 

followed by mean concentrations in the roasting 

room (20 ppb diacetyl, 6 ppb 2,3-pentanedione), green bean and finished goods warehouses 

(11 ppb diacetyl, <3 ppb 2,3-pentanedione), 

quality control room (8 ppb diacetyl, <3 ppb 

2,3-pentanedione), maintenance shop (7 ppb 

diacetyl, <3 ppb 2,3-pentanedione), and the 

nonproduction offices (4 ppb diacetyl, <3 ppb 

2,3-pentanedione). The flavoring room was 

under negative pressure with respect to the 

adjacent grinding/packaging room where unflavored roasted coffee was processed. 

Personal sample mean concentrations by 

location in the coffee plant were highest for 

employees working in the grinding/packaging 

room (93 ppb diacetyl, 53 ppb 2,3-pentanedione), flavoring room (80 ppb diacetyl, 122 ppb 

2,3-pentanedione), production offices (81 ppb 

diacetyl, 22 ppb 2,3-pentanedione), all over (59 

ppb diacetyl, 39 ppb 2,3-pentanedione), and 

housekeeping (54 ppb diacetyl, 18 ppb 2,3-pentanedione). These were followed by those in the 

roasting room (26 ppb diacetyl, 7 ppb 2,3-pentanedione), quality control room (24 ppb 

diacetyl, 11 ppb 2,3-pentanedione), warehouse 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 31

2 . Assessing Occupational Exposure in Employees

(8 ppb diacetyl, <3 ppb 2,3-pentanedione), and 

nonproduction offices (7 ppb diacetyl, <3 ppb 

2,3-pentanedione). 

The mean area concentrations on the grinding/packaging and flavoring room mezzanines, 

where roasted whole and ground bean storage 

hoppers were located, were higher than those 

measured on the main production levels of the 

rooms. A 15-minute short-term air sample collected at the open hatch of a grinding/packaging 

room mezzanine hopper holding unflavored 

ground coffee above an active packaging line 

measured concentrations of 14,300 ppb diacetyl 

and 13,800 ppb 2,3-pentanedione. The location 

of the sample was representative of the proximity of employees’ faces as they frequently and 

momentarily monitored coffee levels in the 

hoppers throughout their shift.

NIOSH also conducted a small industrywide 

study at some flavored food production facilities 

where diacetyl and other food flavorings were 

added to various food products. Seventy-four 

personal and 105 area samples were collected 

for diacetyl using OSHA Method 1013. With 

one exception where local exhaust ventilation 

was documented in some locations, no engineering controls were noted in any facility. Of 

the 179 total samples, 12 had detectable levels 

of diacetyl (LOD 0.5 – 1.0 ug/sample). The eight 

area samples ranged from 0.03 to 3.1 ppm, with 

three samples above 1 ppm (1.1, 2.1 and 3.1 

ppm). The four personal samples ranged from 

0.06 to 0.6 ppm [Curwin et al. 2015].

2.5.6 OSHA Site Visits Related to 

Diacetyl and Flavorings that 

Contain Diacetyl

Between January 2008 and January 2010, an 

OSHA contractor measured diacetyl exposure 

to employees in a series of 12 industrial hygiene 

surveys at various facilities that use (11 facilities) or manufacture (1 facility) formulated 

flavorings, including flavorings that contain 

diacetyl [Eastern Research Group 2008a, b, c, 

d, 2009a, b, c, d, e, 2010a, b, c]. In the first two 

surveys, conducted in January 2008, diacetyl 

was measured using OSHA Method PV2118. 

In the subsequent 10 surveys, OSHA Methods 

1012 and 1013 were used. At all facilities, visual 

observation was made of engineering controls 

in place at the various operations evaluated.

The measured range of diacetyl concentrations 

are presented in Table 2-2 below, along with the 

type of facility and synopsis of controls. Eastern 

Research Group returned to OSHA G Facility in 

2010 to remeasure airborne diacetyl concentrations following the installation of engineering 

controls and work-practice changes at that 

facility. In this follow-up study measurements 

were also made for 2,3-pentanedione in samples 

that contained diacetyl. 2,3-Pentanedione was 

not detected.

2.5.7 Other Exposure Assessments

Pierce et al. characterized diacetyl exposures 

that could potentially occur in a simulated small 

coffee shop during the preparation and consumption of unflavored coffee. Mean estimated 

8-hour TWA exposure concentrations ranged 

from 7 ppb to 13 ppb [Pierce et al. 2015]. 

Gaffney et al. evaluated exposures in a facility 

that roasts and grinds coffee beans. Results indicated that airborne concentrations of diacetyl 

and 2,3-pentanedione are similar to concentrations in food flavoring facilities [Gaffney 

et al. 2015].
32 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2 . Assessing Occupational Exposure in Employees

Table 2-2. Investigations of facilities using or producing diacetyl

 Facility Product Controls in place Diacetyl measurement range

OSHA A 

[ERG 2008a]

Coffee Dilution ventilation ND–54 ppb (TWA)

8 ppb (short-term sample)

OSHA B 

[ERG 2008b]

Commercial 

bakery 

Dilution ventilation,

LEV, process containment

ND–2703 ppb (TWA)

ND–1012 ppb (area)

OSHA C 

[ERG 2008c]

Seasoned snack 

product

Dilution ventilation; heat 

extraction for adjacent process

ND

OSHA D 

[ERG 2008d]

Baked snack food Secondary heat ventilation for 

heating and powder dumping

ND–164 ppb (TWA)

ND–139 ppb (short-term sample)

ND–111 ppb (area)

OSHA E 

[ERG 2009a]

Sauce production Engineering controls for other 

purposes, heat removal, etc.

ND–5.3 ppb (TWA)

ND–10.5 ppb (short-term sample)

ND–2.4 ppb (area)

OSHA F 

[ERG 2009b]

Low-calorie 

cracker

Canopy hoods in heated

Production process #1, dilution 

ventilation

ND–195.7 ppb (TWA)

ND–701.1 ppb (short-term sample)

ND–13.1 ppb (area)

OSHA G 

[ERG 2009c]

Buttered popcorn 

production

Before:

Heat extraction hoods, dilution 

ventilation

24.8–71.2 ppb (TWA)

466.8–2298.7 ppb (short-term sample) 

9.1–8660.2 ppb (area)

After:

Dilution ventilation; slot hood 

at tumbler; modified tank cover; 

work practice change

< 2.7–< 9.8 ppb (TWA)

< 10.4–98.8 ppb (short-term sample) 

2.7–5.4 ppb (area)

OSHA H 

[ERG 2009d]

Sour cream 

production

Exhaust ventilation for dust ND–32.4 ppb (TWA)

ND–138.6 ppb (short-term sample)

ND–4.6 ppb (area)

OSHA I 

[ERG 2009e]

Ice cream Controls for other purposes, 

immediate rinsing, cool temperature dilution ventilation

ND to 1.6 ppb (TWA)

ND (short-term sample)

ND (area)

OSHA J 

[ERG 2010a]

Cottage cheese Dilution ventilation ND–55.3 ppb (TWA)

32.3–317 ppb (short-term sample)

1.3–32.4 ppb (area)

OSHA K 

[ERG 2010b]

Food flavor 

production

Dilution ventilation;

hose from tank to floor drain

ND–2,990 ppb (TWA)

ND–12,373.1 ppb (short-term sample) 

29.1–381.5 (area)

OSHA L 

[ERG 2010c]

Retail bakery Dilution ventilation;

oven room heat extraction

ND–50.4 ppb (TWA)

ND–118.5 ppb (short-term sample)

ND–30.9 ppb (area)

ND: not detected

TWA: a sample concentration determined over a full work shift 

Short-term sample: a concentration measured for less than a full work shift
Occupational Exposure to Diacetyl and 2,3-Pentanedione 33

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production facility A—a coffee roaster. OSHA Docket 

No. 2008–0046–0068. By Eastern Research Group, Inc. 

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production facility E—sauce production. OSHA Docket 

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production facility G—buttered popcorn production 

(pre-popped). OSHA Docket No. 2008–0046–0081. By 

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to diacetyl and flavorings that contain diacetyl: food production facility J—dairy—cottage cheese production. 

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34 Occupational Exposure to Diacetyl and 2,3-Pentanedione

Eastern Research Group, Inc. [2010b]. Site visits related 

to diacetyl and flavorings that contain diacetyl: food production facility K—flavor production. OSHA Docket 

No. 2008–0046–0086. By Eastern Research Group, Inc. 

Lexington, MA.

Eastern Research Group, Inc. [2010c]. Site visits related 

to diacetyl and flavorings that contain diacetyl: food 

production facility L—a retail bakery. OSHA Docket 

No. 2008–0046–0085. By Eastern Research Group, Inc. 

Lexington, MA.

Entech Instruments Incorporated [2011]. HDSTM personal monitors. http://www.entechinst.com/pdf/2011%20

Updates/Seperate%20Sections/P78-81%20HDS%20

Personal%20Monitors.pdf. Date accessed: May 2011.

Gaffney SH, Abelmann A, Pierce JS, Glynn ME, Henshaw 

JL, McCarthy LA, Lotter JT, Liong M, Finley BL [2015]. 

Naturally occurring diacetyl and 2,3-pentanedione 

concentrations associated with roasting and grinding 

unflavored coffee beans in a commercial setting. Toxicol 

Rep 2: 1171–1181.

IATA [2012]. IATA Dangerous Goods Regulations. 

Montreal, Quebec, Canada: International Air 

Transport Association.

Kanwal R, Kullman G, Piacitelli C, Boylstein R, Sahakian 

N, Martin S, Fedan K, Kreiss K [2006]. Evaluation of 

flavorings-related lung disease risk at six microwave 

popcorn plants. J Occup Environ Med 48(2):149–157.

LeBouf RF, Stefaniak AB, Virji MA [2012]. Validation 

of evacuated canisters for sampling volatile organic 

compounds in healthcare settings. J Environ 

Monit 14(3):977–983.

Martyny JW, Van Dyke MV, Arbuckle S, Towle M, Rose 

CS [2008]. Diacetyl exposures in the flavor manufacturing industry. J Occup Environ Hyg 5(11):679–688.

NIOSH [1986]. Hazard evaluation and technical assistance report: International Bakers Services, Inc, South 

Bend, IN. By McConnell R, Hartle R. Cincinnati, OH: 

U.S. Department of Health and Human Services, 

Centers for Disease Control, National Institute for 

Occupational Safety and Health, NIOSH HETA Report 

No. 1985–0171–1710.

NIOSH [1994]. NIOSH manual of analytical methods 

(NMAM). 4th ed. Schlecht PC, O’Connor PF, eds. 

Cincinnati, OH: U.S. Department of Health and Human 

Services, Centers for Disease Control and Prevention, 

National Institute for Occupational Safety and Health, 

DHHS (NIOSH) Publication 94–113 (August, 1994); 

1st Supplement Publication 96–135; 2nd Supplement 

Publication 98–119; 3rd Supplement 2003–154.

NIOSH [2006]. Hazard evaluation and technical assistance report: Gilster-Mary Lee Corporation, Jasper, MO. 

By Kanwal R, Kullman G, Fedan K, Kreiss K. Cincinnati, 

OH: U.S. Department of Health and Human Services, 

Centers for Disease Control and Prevention, National 

Institute for Occupational Safety and Health, NIOSH 

HETA Report No. 2000–0401–2991.

NIOSH [2007a]. Hazard evaluation and technical assistance report: Carmi Flavor and Fragrance Company, Inc., 

Commerce, CA. By Kanwal R, Kullman G. Cincinnati, 

OH: U.S. Department of Health and Human Services, 

Centers for Disease Control and Prevention, National 

Institute for Occupational Safety and Health, NIOSH 

HETA Report No. 2006–0303–3043.

NIOSH [2007b]. Hazard evaluation and technical assistance report: Yatsko’s Popcorn, Sand Coulee, 

MT. By Kullman G, Sahakian N. Cincinnati, OH: U.S. 

Department of Health and Human Services, Centers for 

Disease Control and Prevention, National Institute for 

Occupational Safety and Health, NIOSH HETA Report 

No. 2006–0195–3044.

NIOSH [2008a]. Evaluating occupational exposures 

and work practices at Agilex Flavors, Inc. formerly Key 

Essentials, Inc., Rancho Santa Margarita, CA. A technical assistance report to the California/Occupational Safety 

and Health Administration. By McKernan L, Dunn K. 

Cincinnati, OH: U.S. Department of Health and Human 

Services, Centers for Disease Control and Prevention, 

National Institute for Occupational Safety and Health, 

NIOSH HETA Report No. 2006–0361–2.

NIOSH [2008b]. Hazard evaluation and technical assistance report: Gold Coast Ingredients, Inc., Commerce, 

CA. By Bailey R, McKernan L, Dunn K, Sahakian N, 

Kreiss K. Cincinnati, OH: U.S. Department of Health 

and Human Services, Centers for Disease Control and 

Prevention, National Institute for Occupational Safety 

and Health, NIOSH HETA Report No. 2007–0033–3074.

NIOSH [2008c]. In-depth survey report: evaluation of 

engineering controls for the mixing of flavoring chemicals: Gold Coast Ingredients, Inc., Commerce, CA. 

By Dunn K, Echt A, Garcia A. Cincinnati, OH: U.S. 

Department of Health and Human Services, Centers for 

Disease Control and Prevention, National Institute for 
References

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Occupational Safety and Health, NIOSH EPHB Report 

No. 322–11a.

NIOSH [2009a]. Hazard evaluation and technical assistance report. Report on an investigation of buttermilk 

flavoring exposures and respiratory health at a bakery 

mix production facility, General Mills, Los Angeles, CA. 

By Day G, Cummings K, Kullman G. Cincinnati, OH: 

U.S. Department of Health and Human Services, Centers 

for Disease Control and Prevention, National Institute for 

Occupational Safety and Health, NIOSH HETA Report 

No. 2008–0230–3096.

NIOSH [2009b]. Hazard evaluation and technical assistance report. Respiratory symptoms in workers at three 

commercial kitchens, Aramark, New York, NY. By 

Gaughan D, Boylstein R, Iossifova Y, Piacitelli C, Bailey 

R, Day G. Cincinnati, OH: U.S. Department of Health 

and Human Services, Centers for Disease Control 

and Prevention, National Institute for Occupational 

Safety and Health, NIOSH HETA Report No. 

2008–0125–0126–0127–3093.

NIOSH [2009c]. Hazard evaluation and technical assistance report: Chr. Hansen, Inc., New Berlin, WI. By 

Sahakian N, Kullman G, Dunn K, Kanwal R. Cincinnati, 

OH: U.S. Department of Health and Human Services, 

Centers for Disease Control and Prevention, National 

Institute for Occupational Safety and Health, NIOSH 

HETA Report No. 2007–0327–3083.

NIOSH [2011]. Hazard evaluation and technical assistance report: Lung function (spirometry) testing in 

employees at a flavorings manufacturing plant—Indiana. 

By Kreiss K, Piacitelli C, Cox-Ganser J. Morgantown, WV: 

U.S. Department of Health and Human Services, Centers 

for Disease Control and Prevention, National Institute for 

Occupational Safety and Health, NIOSH HETA Report 

No. 2008–0155–3131.

NIOSH [2013a]. Hazard evaluation and technical 

assistance report: An evaluation of respiratory health 

at a flavoring manufacturing facility—Kentucky. By 

Cummings K, Boylstein R, Stanton M, Piacitelli C. 

Morgantown, WV: U.S. Department of Health and 

Human Services, Centers for Disease Control and 

Prevention, National Institute for Occupational Safety 

and Health, NIOSH HETA Report No. 2012–0012–3192.

NIOSH [2013b]. Hazard evaluation and technical assistance report: Evaluation of respiratory concerns at a cream 

cheese manufacturing facility—New York. By Bailey R, 

Piacitelli C. Morgantown, WV: U.S. Department of Health 

and Human Services, Centers for Disease Control and 

Prevention, National Institute for Occupational Safety 

and Health, NIOSH HETA Report No. 2011–0102–3194.

NIOSH [2013c]. Hazard evaluation and technical assistance report: Evaluation of respiratory concerns at a snack 

food production facility—New York. By Cummings K, 

Piacitelli C, Stanton M, Bailey R. Morgantown, WV: U.S. 

Department of Health and Human Services, Centers for 

Disease Control and Prevention, National Institute for 

Occupational Safety and Health, NIOSH HETA Report 

No. 2011–0037–3172.

OSHA [2003]. OSHA Sampling and Analytical Methods: 

Diacetyl PV2118. http://www.osha.gov/dts/sltc/methods/

partial/t-pv2118/t-pv2118.html. Date accessed: June 2009.

OSHA [2008a]. OSHA Sampling and Analytical Methods: 

Acetoin/Diacetyl 1012. http://www.osha.gov/dts/sltc/

methods/validated/1012/1012.html. Date accessed: 

June 2009.

OSHA [2008b]. OSHA Sampling and Analytical Methods: 

Acetoin/Diacetyl 1013. http://www.osha.gov/dts/sltc/

methods/validated/1013/1013.html. Date accessed: 

June 2009.

OSHA [2010]. OSHA Sampling and Analytical Methods: 

2,3-Pentanedione 1016. http://www.osha.gov/dts/sltc/

methods/validated/1016/1016.html. Date accessed: 

July 2013.

Pierce JS, Abelmann A, Lotter JT, Comerford C, Keeton 

K, Finley BL [2015]. Characterization of naturally occurring airborne diacetyl concentrations associated with 

the preparation and consumption of unflavored coffee. 

Toxicol Rep 2:1200–1208.

Rodrigues JA, Barros AA, Rodrigues PG [1999]. 

Differential pulse polarographic determination of alphadicarbonyl compounds in foodstuffs after derivatization 

with o-phenylenediamine. J Agric Food Chem 

47(8):3219–3222.

van Rooy FG, Rooyackers JM, Prokop M, Houba R, Smit 

LA, Heederik DJ [2007]. Bronchiolitis obliterans syndrome in chemical workers producing diacetyl for food 

flavorings. Am J Respir Crit Care Med 176(5):498–504.

White KL [2011]. Correction to microwave popcorn 

plant diacetyl exposure data. J Occup Environ Hyg 8(4)

(Comment Letter):D25–26.

White KL, Heikkila K, Williams R, Levin L, Lockey JE, 

Rice C [2010]. Diacetyl exposures at four microwave 

popcorn plants. J Occup Environ Hyg 7(4):185–193.
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Occupational Exposure to Diacetyl and 2,3-Pentanedione 37

3 Effects of Exposure in Employees

Information on the effects on employees’ health 

of exposures to diacetyl and other flavoring compounds comes from case reports and 

case series and from cross-sectional and longitudinal medical and environmental surveys 

conducted at several flavoring and food manufacturing facilities (Table 3-1). NIOSH has 

conducted cross-sectional surveys as part of 

HHEs at six microwave popcorn plants where 

diacetyl-containing butter flavorings were 

used, at five flavoring manufacturing plants 

that used diacetyl and other flavoring compounds to produce different flavors for use in 

food products such as microwave popcorn, 

at a plant that used flavorings (including buttermilk flavoring) to produce baking mixes, 

and at three restaurants where grill cooks used 

butter-flavored oil. Academic researchers have 

also conducted studies at other food and flavoring manufacturing plants and at a chemical 

plant in the Netherlands that produced diacetyl. Surveillance with a longitudinal component 

has been conducted by NIOSH in two HHEs, 

by the California Department of Public Health, 

and by academic researchers.

At the time of most of these field investigations, 

which preceded the California diacetyl regulation implemented in December 2010, little 

2,3-pentanedione was being used for artificial 

butter flavoring. When food manufacturers 

began to request that diacetyl percentage be 

less than 1% of flavoring constituents, flavor 

manufacturers sometimes did not inform their 

clients of the substitution of 2,3-pentanedione 

and other diacetyl substitutes [Boylstein 2012; 

Day et al. 2011; NIOSH 2009b]. Accordingly, 

populations with 2,3-pentanedione exposure 

without previous diacetyl exposure are difficult 

to identify. Thus, illness attributable to 2,3-pentanedione alone has not been studied.

3.1 Obstructive Lung 

Disease Consistent with 

Obliterative Bronchiolitis 

The most significant health consideration for 

flavoring-exposed employees is the development of exertional dyspnea or findings 

consistent with obliterative bronchiolitis (also 

often called constrictive bronchiolitis, see 

discussion of terminology). Most textbooks 

characterize obliterative bronchiolitis as a rare 

disease with airways obstruction, defined by a 

decreased FEV1 and a decreased FEV1 to FVC 

ratio on spirometry testing. The magnitude of 

decline in FEV1 determines the severity of the 

disorder. However, three recent case series of 

biopsy-confirmed obliterative bronchiolitis 

document that many cases have normal spirometry and, when abnormal, the spirometric 

pattern can be restrictive, obstructive, or mixed 

restrictive and obstructive in nature [Ghanei et 

al. 2008; King et al. 2011; Markopoulou et al. 

2002]. Because of the historical assumption 

that obliterative bronchiolitis is an obstructive 

disease, the early NIOSH investigations focused 

on obstructive abnormalities. 

Airways obstruction can occur in diseases such 

as smoking-related COPD (including emphysema and chronic bronchitis) and in asthma. In 

emphysema, the airways obstruction is usually 
See footnotes at end of table. (Continued)

38 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

Table 3-1. Literature pertinent to flavoring health effects

Reference

Facility

NIOSH evaluated Study type(s), industry Contribution*

Akpinar-Elci et al. [2004] G Case report, 

MICROWAVE POPCORN MANUFACTURING

Nine former employees at the index plant exhibited moderate to very 

severe fixed airways obstruction; five of the cases were on lung transplant lists.

Bailey et al. [2015]

R Public health investigation,

FOOD PRODUCTION

Coffee processing employees had excess shortness of breath and 2.7-

fold risk of obstructive abnormalities; the group working in both high exposure areas (unflavored coffee grinding/packaging and flavoring room) had lower mean FEV1/FVC ratio and percent predicted mid-expiratory flow than employees without such exposure.

Cavalcanti et al. [2012] † Case report,

FOOD PRODUCTION

Four employees at a cookie manufacturing facility developed bronchiolitis within 1 to 3 years of employment.

Centers for Disease Control and 

Prevention (CDC) [2002]

G Public health investigation, 

MICROWAVE POPCORN MANUFACTURING

Eight cases of fixed obstructive lung disease resembling bronchiolitis obliterans among former employees at the index plant resulted 

in identification of excess risk for mixers compared to packaging employees, with no cases outside of microwave production.

CDC [2007] † Case report and public health 

investigation, FLAVORING MANUFACTURING

Two cases of work-related bronchiolitis obliterans from two different 

plants resulted in a public health surveillance effort identifying five additional cases of severe fixed obstruction in young, non-smoking employees who worked in flavor compounding or packaging.

CDC [2013]

R Case report,

FOOD PRODUCTION

Two employees in the flavoring room of a coffee roasting plant developed fixed airways obstructive disease and were diagnosed with 

obliterative bronchiolitis by biopsy.

Halldin et al. [2013] G Cohort mortality follow-up,

MICROWAVE POPCORN MANUFACTURING

Current and former employees studied by NIOSH in 2000–2003 had a 

4-fold increase in mortality coded as chronic obstructive pulmonary disease in an 11-year follow-up through late 2011.

Kanwal et al. [2006] G, J, K, L, N, O Summary of six plant 

surveys, MICROWAVE POPCORN MANUFACTURING

Synthesis of six cross-sectional NIOSH surveys identified an industrywide risk of fixed airways obstruction in five plants, one of which 

had mixing-area diacetyl exposures as low as 0.02 ppm. Mixers with longer work histories and packaging employees near nonisolated tanks of oil and flavorings had higher prevalences of respiratory symptoms and airways obstruction.
See footnotes at end of table. (Continued)

Occupational Exposure to Diacetyl and 2,3-Pentanedione 39

3 . Effects of Exposure in Employees

Reference

Facility

NIOSH evaluated Study type(s), industry Contribution*

Kanwal et al. [2011] G Intervention study, 

MICROWAVE POPCORN MANUFACTURING

Ventilation and isolation of flavor mixing at the index plant resulted 

in one to three orders of magnitude reduction in diacetyl air concentrations in different areas. Employees with high past exposures had stable chest symptoms, decreased mucous membrane and skin symptoms, and higher prevalence of rapid declines in lung function than employees hired after interventions began. These new employees had lower symptom prevalences and higher lung function, demonstrating that intervention resulted in improved health for new employees.

Kim et al. [2010] B,C† Cross-sectional industrywide public health investigation, FLAVORING 

MANUFACTURING

California flavoring employees had 2.7 times more severe airways 

obstruction than the general population. Risk factors for the 18 cases with obstruction among 467 employees were younger age, Hispanic ethnicity, liquid and powder production work, greater company diacetyl usage, and having a coworker with obstruction. Severity of obstruction was related to tenure. At least 12 employees had probable occupational fixed airways obstruction.

Kreiss et al. [2002] G Cross-sectional survey, 

MICROWAVE POPCORN MANUFACTURING

The 117 current employees at the index plant had 2.6 times the 

expected rates of respiratory symptoms and 3.3 times the expected rate of airways obstruction, with never-smokers having 10.8 times the expected rate. Quartile of cumulative exposure to diacetyl was related to the frequency and extent of airways obstruction.

Lockey et al. [2009] L† Longitudinal survey, 

MICROWAVE POPCORN MANUFACTURING

Study of 765 employees at four plants at two 6-month intervals 

showed significant FEV1 declines in mixers, who also had an 8-fold 

risk of obstructive abnormality. Cumulative diacetyl exposure of 0.8 ppm-yr was associated with an odds ratio of 9.2 for obstruction.

NIOSH [1986] A Cross-sectional survey, 

FLAVORING MANUFACTURING

Two young employees with no known risk factors developed severe, 

fixed obstructive lung disease suggestive of bronchiolitis obliterans within 1 year of employment.

NIOSH [2003a]

N Cross-sectional survey,

MICROWAVE POPCORN MANUFACTURING

Elevated prevalence of airways obstruction when compared to 

national rates; all observed obstruction was fixed.

Table 3-1 (Continued). Literature pertinent to flavoring health effects
See footnotes at end of table. (Continued)

40 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

Reference

Facility

NIOSH evaluated Study type(s), industry Contribution*

NIOSH [2004a] K Cross-sectional survey,

MICROWAVE POPCORN MANUFACTURING

A study of 157 employees in a plant having a mixing room employee 

previously diagnosed with fixed obstructive lung disease consistent with bronchiolitis obliterans found abnormal lung function in 6 of 13 mixers: 3 had fixed airways obstruction, and 3 had spirometric restriction.

NIOSH [2004b] L Cross-sectional survey,

MICROWAVE POPCORN MANUFACTURING

A survey of 205 employees at a plant with a mixer previously diagnosed with severe fixed obstructive lung disease consistent with 

bronchiolitis obliterans, identified 3 of 12 mixers and 5 of 110 packaging employees with fixed airways obstruction and normal diffusing capacity.

NIOSH [2006] G Eight cross-sectional surveys,

MICROWAVE POPCORN MANUFACTURING

Studies of 373 current employees over 2.75 years at the index plant 

determined that inhalation of butter flavoring compounds is a risk for occupational disease. Comparisons of employees hired before and after implementation of exposure controls document declines in the prevalence of eye, nose, and throat irritation. Large decreases in FEV1 (> 300 mL or 10%) were observed in 4 of 9 mixers; one young 

mixer lost 2,800 mL over 2.75 years.

NIOSH [2007a] B Two cross-sectional surveys,

FLAVORING MANUFACTURING

Two former employees and one current employee who made powdered flavorings (of 18 with current or previous production 

experience) had severe fixed obstructive lung disease consistent with bronchiolitis obliterans. Within months of the survey, one of these employees was diagnosed with bronchiolitis obliterans following biopsy.

NIOSH [2007b] M Cross-sectional survey,

FOOD PRODUCTION

All three employees in a popcorn popping business developed 

symptoms of airways disease during their tenure; all were lifetime nonsmokers. One of the employees had significant reversible airways obstruction with some clinical evidence suggesting possible bronchiolitis obliterans in addition to asthma.

Table 3-1 (Continued). Literature pertinent to flavoring health effects
See footnotes at end of table. (Continued)

Occupational Exposure to Diacetyl and 2,3-Pentanedione 41

3 

. Effects of Exposure in Employees

Reference

Facility

NIOSH evaluated Study type(s), industry Contribution*

NIOSH [2008] C Two cross-sectional surveys,

FLAVORING MANUFACTURING

One of 14 employees with production experience had severe fixed 

airways obstruction (subsequently confirmed as bronchiolitis obliterans), and an additional production employee developed mild fixed obstruction following the loss of 1 liter in FEV1 during a 4.5-month 

screening interval.

NIOSH [2009b] E Cross-sectional survey,

FOOD PRODUCTION

At a plant using a newly reformulated flavoring that included 2,3-pentanedione, no obstruction was identified in the 22 employees tested. 

Participants had higher than expected rates of shortness of breath, physician-diagnosed asthma, and a restrictive pattern on spirometry (four cases ranging from mild to moderately severe), compared to U.S. adults. Some participants reported symptoms with a workrelated pattern.

NIOSH [2009c] F Three cross-sectional surveys,

FOOD PREPARATION

Studies of employees at three sites found higher prevalences of spirometric restriction, wheeze, dyspnea on exertion, nasal and eye 

irritation, and nasal allergies when compared to national rates. Cooks were 3–4 times more likely to report work-related respiratory symptoms. Fixed airways obstruction identified in two employees did not appear to be work-related.

NIOSH [2009d] D Cross-sectional survey,

FLAVORING MANUFACTURING

This study of 34 employees found that bacterial products employees 

had higher prevalences of work-related eye symptoms and posthire skin problems than flavoring employees; both groups reported lower respiratory symptoms related to the substances they handled at work. One employee was identified with fixed airways obstruction and two employees with restriction on spirometry.

Table 3-1 (Continued). Literature pertinent to flavoring health effects
42 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

Reference

Facility

NIOSH evaluated Study type(s), industry Contribution*

NIOSH [2011] Kreiss [2014]

I Public health investigation,

FLAVORING MANUFACTURING

Review of company-supplied spirometry tests identified 39 (37%) 

employees with evidence of some abnormality (either the most recent spirometry test showed restriction or obstruction; and/or longitudinal spirometry showed excessive decline over time, with most recent spirometry values still within the normal range). Employees currently in areas with higher potential for flavorings exposure were 5.8 times more likely to have abnormal declines in FEV1 than 

employees in other areas.

NIOSH [2013]Cummings et al. [2014]

Q Cross-sectional survey,

FLAVORING MANUFACTURING

A survey of 367 employees at a plant that used thousands of chemicals 

found that employees that spent 1 hour or more daily in the production area had higher prevalences of spirometric abnormalities and low diffusing capacity. Employees with 7 or more years of tenure had lower mean values of percent predicted FEV1, FVC, and diffusing capacity.

van Rooy et al. [2007] † Case report, Crosssectional survey,

DIACETYL MANUFACTURING

Four cases of bronchiolitis obliterans syndrome ([BOS] previously 

attributed to chronic obstructive pulmonary disease [COPD] or asthma) were identified in screening of 103 process operators in a retrospective cohort of production plant employees.

van Rooy et al. [2009] † Cross-sectional 

survey, DIACETYL MANUFACTURING

Excess respiratory symptoms and asthma indices occurred among 

175 production plant employees compared to an internal reference group and a general population sample, with evidence of diacetyl exposure-response for FEV1.

* The health effects listed use the terminology stated in the original report or publication (e.g., fixed obstruction, bronchiolitis obliterans, bronchiolitis obliterans syndrome).† Referenced publication includes site(s) that NIOSH did not visit.

Table 3-1 (Continued). Literature pertinent to flavoring health effects
Occupational Exposure to Diacetyl and 2,3-Pentanedione 43

3 . Effects of Exposure in Employees

fixed (i.e., does not respond to bronchodilator 

medications), whereas in asthma, the airways 

obstruction is at least partially responsive to 

bronchodilators (reversible airways obstruction). Most employees who have developed 

obstructive lung disease while exposed to diacetyl and other flavoring compounds have had 

fixed airways obstruction. Additional medical 

tests in severely affected employees have generally revealed findings consistent with the 

irreversible obstructive lung disease obliterative bronchiolitis (discussed in detail in section 

3.1.2). Serial lung function testing with spirometry indicates that affected employees can 

experience very rapid lung function declines.

Obstructive lung disease in employees exposed 

to diacetyl and other flavoring compounds was 

first reported in employees in the microwave 

popcorn industry. Scientific publications that 

have reported on the occurrence and natural 

history of the illness have used different diagnostic terms including fixed obstructive lung 

disease [CDC 2002], popcorn worker’s lung 

[Schachter 2002], flavorings-related lung 

disease [Kanwal et al. 2006; NIOSH 2009a], 

clinical bronchiolitis obliterans [Kreiss et 

al. 2002], bronchiolitis obliterans syndrome 

[Akpinar-Elci et al. 2004], and flavoring-related 

bronchiolitis obliterans [Kreiss 2007]. Of the 

few surgical lung biopsies that have been performed in affected employees, some have been 

interpreted as showing evidence of “constrictive bronchiolitis” or “obliterative bronchiolitis” 

[Akpinar-Elci et al. 2004; Kanwal 2008]. The 

term fixed obstructive lung disease is the least 

specific of the terms. The term popcorn worker’s lung refers to the population of employees 

in which the disease was first identified. The 

term flavorings-related lung disease refers to 

the full spectrum of lung diseases that may be 

related to flavorings exposure and is not necessarily limited to obstructive conditions. The 

terms flavoring-related bronchiolitis obliterans, constrictive bronchiolitis, and obliterative 

bronchiolitis refer to pathologic findings of 

inflammation and fibrosis primarily involving 

the bronchioles, leading to irreversible airflow 

limitation. Terminology is complicated by the 

fact that, historically, researchers have applied 

the term “bronchiolitis obliterans” to different 

distinct disorders that involve the bronchioles 

[King 2003; King and Kinder 2008]. The terms 

clinical bronchiolitis obliterans and bronchiolitis obliterans syndrome refer to those who 

are thought to suffer from this pathologic 

condition based on clinical findings, but have 

not undergone lung biopsy for pathological 

confirmation. Additional discussion regarding diagnostic terminology in relation to the 

different recognized forms of bronchiolitis is 

included in section 3.1.1.

3.1.1 Bronchiolar Disease 

and Terminology

Bronchiolitis obliterans refers to disease processes that show some degree of inflammation, 

narrowing, or obliteration of small airways 

(bronchioles) in the lung [King 2003; King 

and Kinder 2008]. Historically, bronchiolitis 

obliterans has been classified into two groups: 

proliferative bronchiolitis obliterans and constrictive bronchiolitis obliterans [King 2003;

King and Kinder 2008]. The disorder known as 

bronchiolitis obliterans organizing pneumonia 

(BOOP) is included in the proliferative group. 

BOOP is characterized pathologically by intraluminal polyps in the respiratory bronchioles, 

alveolar ducts, and alveolar spaces accompanied 

by organizing pneumonia in the more distal 

parenchyma. Clinically it is usually associated 

with diffuse alveolar opacities on chest x-ray 

and computed tomography scan; pulmonary 

function testing may show a restrictive defect 

[King 2003; King and Kinder 2008]. BOOP 

was first described in 1985. Prior to this, many 

cases that matched the description for BOOP 

were classified as idiopathic bronchiolitis obliterans [King 2003; King and Kinder 2008]. The 
44 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

American Thoracic Society and the European 

Respiratory Society have recommended the use 

of the term cryptogenic organizing pneumonitis (COP) instead of BOOP to avoid confusion 

with the disease constrictive bronchiolitis obliterans [ATS and ERS 2002]. While proliferative 

bronchiolitis can be idiopathic (e.g., COP), 

known associations include collagen vascular 

diseases (e.g., systemic lupus erythematosus), 

acute infections (e.g., influenza, mycoplasma), 

organ transplantation, and aspiration pneumonitis. Proliferative bronchiolitis is generally 

responsive to corticosteroid medications and is 

usually reversible [King and Kinder 2008].

Obliterative bronchiolitis (also referred to 

as constrictive bronchiolitis obliterans [ATS 

and ERS 2002], constrictive bronchiolitis 

[Schlesinger et al. 1998; Visscher and Myers 

2006], and bronchiolitis obliterans [King 

2003; King and Kinder 2008]) is a rare disorder characterized by alterations in the walls 

of respiratory and membranous bronchioles 

that cause concentric narrowing or complete 

obliteration of the airway lumen, without 

involvement of the distal lung parenchyma 

by inflammation or organizing pneumonia 

[King 2003; King and Kinder 2008]. In affected 

individuals, pulmonary function tests usually 

show airways obstruction and hyperinflation 

[King and Kinder 2008], but biopsy-confirmed 

cases may have normal or restrictive spirometry [Ghanei et al. 2008; King et al. 2011;

Markopoulou et al. 2002]. Chest x-rays may 

be normal or show hyperinflation, peripheral attenuation of the vascular markings, and 

nodular or reticular opacities [King 2003]. The 

predominant finding of obliterative bronchiolitis on high-resolution computed tomography 

(HRCT) scan is heterogeneity of lung density 

due to mosaic perfusion and air trapping [King 

2003; King and Kinder 2008]. Other findings 

of bronchiolitis on HRCT scan include centrilobular thickening, bronchial wall thickening, 

bronchiolar dilatation, and the tree-in-bud 

pattern. Cylindrical bronchiectasis is frequently 

associated with obliterative bronchiolitis; scans 

with both inspiratory and expiratory views are 

helpful because expiratory views are important in assessing air trapping [King 2003]. 

Identification of the obliterative bronchiolitis 

lesion on lung biopsy may be difficult because 

of its patchy distribution [Estenne et al. 2002;

Schlesinger et al. 1998; Visscher and Myers 

2006], often requiring step-sectioning and 

special staining to identify airway walls [King 

2003; King and Kinder 2008]. The diagnosis is 

a multidisciplinary one involving a team with 

clinical, radiologic, and histopathologic expertise; HRCT evidence often replaces the need for 

surgical lung biopsy [King and Kinder 2008]. In 

comparison to proliferative bronchiolitis, obliterative bronchiolitis is generally unresponsive 

to corticosteroid medications and often progresses to more severe disease [King and Kinder 

2008], although progression after exposure cessation is not characteristic of flavoring-related 

disease consistent with obliterative bronchiolitis [Akpinar-Elci et al. 2004].

As mentioned previously and discussed in 

detail in the next section (3.1.2), the medical 

evaluations of employees who have developed 

lung disease during exposure to diacetyl and 

other flavoring compounds have generally 

revealed findings consistent with obliterative 

bronchiolitis. Because of concerns for patient 

welfare and the invasive nature and imperfect sensitivity of lung biopsy for diagnosing 

obliterative bronchiolitis, most patients have 

been diagnosed based upon clinical findings. 

Despite the small number of lung biopsies conducted, findings consistent with obliterative 

bronchiolitis have been identified in multiple 

flavorings-exposed patients [Akpinar-Elci et 

al. 2004; NIOSH 2007a]. Patients exposed to 

sulfur mustard gas are another patient population where obliterative bronchiolitis has been 

diagnosed in a small subfraction of the patients 

while other patients are diagnosed using 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 45

3 . Effects of Exposure in Employees

contemporary clinical criteria, including HRCT 

scans [Ghanei et al. 2004a; Ghanei et al. 2004b;

Ghanei et al. 2008; Rowell et al. 2009]. Other 

known causes of obliterative bronchiolitis 

include uncontrolled inhalation exposures to 

ammonia, chlorine, phosgene, nitrogen dioxide 

and sulfur dioxide, collagen vascular diseases 

(especially rheumatoid arthritis), infections, 

and organ transplantation (bone marrow, 

heart-lung, lung) [King and Kinder 2008].

Because of the difficulty of identifying the 

lesions of obliterative bronchiolitis on lung 

biopsy, and because the disease occurs 

commonly after heart-lung and lung transplants, in 1993 a committee sponsored by 

the International Society for Heart and Lung 

Transplantation proposed a clinical description 

for the disease termed bronchiolitis obliterans syndrome. The syndrome refers to graft 

deterioration secondary to persistent airflow 

obstruction as defined by pulmonary function changes with or without histolopathologic 

confirmation. Probable risk factors for BOS 

include acute graft rejection and cytomegalovirus pneumonitis [Estenne et al. 2002]. The 

term BOS has also been used in cases of obliterative bronchiolitis resulting from chemical 

injury and diagnosed using clinical criteria 

with or without biopsy [Akpinar-Elci et al. 

2004; Ghanei et al. 2004a; van Rooy et al. 2007].

Because the terminology used in the peerreviewed literature of flavorings-exposed 

employees has included several different 

accepted and frequently interchanged diagnostic terms, and indeed may have been influenced 

by the peer-review process itself, this criteria 

document sometimes provides the terms used 

in the cited papers and includes the criteria 

used in the patient evaluations.

3.1.2 Evidence from Field Studies

NIOSH first learned of the potential risk 

of obliterative bronchiolitis in microwave 

popcorn employees exposed to butter flavorings in August 2000 when they were asked by 

the Missouri Department of Health and Senior 

Services for technical assistance in investigating the occurrence of this illness in eight 

former employees (index cases) of a microwave popcorn plant (index Facility G)[CDC 

2002]. NIOSH reviewed medical records for 

these employees and in November 2000 conducted a medical survey of current and former 

employees of this plant. Survey results and 

medical records review for the eight index 

cases and a current employee with lung disease 

showed several findings consistent with obliterative bronchiolitis. All cases had moderate 

to very severe airways obstruction (FEV1s 

between 14.9% and 58.4% predicted), fixed 

in most cases; six of seven cases tested had 

increased residual volume consistent with air 

trapping. Diffusing capacity for carbon monoxide (DLCO) was normal initially in five of 

seven cases tested. All cases had chest x-rays 

that were normal or showed hyperinflation. 

All eight cases that had HRCT scans showed 

marked bronchial wall thickening and mosaic 

attenuation with air trapping; five cases also 

showed mild cylindrical bronchiectasis. In two 

of three cases that underwent lung biopsy, the 

reviewing pathologist reported findings that 

supported or were consistent with a diagnosis 

of bronchiolitis obliterans [Akpinar-Elci et al. 

2004]. These nine employees had developed 

a dry persistent cough, shortness of breath 

on exertion, and wheezing after a median of 

1.5 years of employment. At the time of 

symptom onset, five of the employees had 

been working in the room where butter flavorings, salt, and colorings were combined with 

heated soybean oil. The other four employees had been working in the adjacent room 

where the oil and flavoring mixture was combined with kernel popcorn in microwavable 

bags (packaging area). None of these employees were initially diagnosed by their personal 

physicians as having obliterative bronchiolitis. 
46 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

Initial diagnoses received by these employees 

included pneumonia, asthma, emphysema, 

bronchitis, COPD, hay fever, and sinusitis. Five 

of the employees had minimal smoking history. 

All nine employees had been prescribed oral 

corticosteroids, but none had improvement in 

lung function. Five of the employees had been 

placed on lung transplant waiting lists by their 

personal physicians [Akpinar-Elci et al. 2004].

3.1.2.1 Index plant lung function testing

The NIOSH medical survey at the index 

microwave popcorn plant (Facility G) in 

November 2000 included lung function testing 

with spirometry and DLCO, chest x-rays, and 

a questionnaire [Kreiss et al. 2002; NIOSH 

2006]. NIOSH compared the prevalences of 

respiratory symptoms, self-reported physiciandiagnosed asthma and chronic bronchitis, and 

airways obstruction on spirometry to data 

from the Third National Health and Nutrition 

Examination Survey (NHANES III) [CDC 

1996]. Of 135 current employees, 117 (87%) 

completed the questionnaire, and 97 (83%) of 

the survey participants worked in the microwave popcorn production areas of the plant. 

The remaining 20 survey participants worked 

in areas where butter flavorings were not 

used such as plain kernel popcorn packaging, offices, warehouse, and outside receiving. 

The prevalences of respiratory and systemic 

symptoms, mucous membrane irritation, and 

skin irritation were higher among employees 

in microwave popcorn production areas than 

in other areas. Among all survey participants, 

the prevalences of chronic cough and shortness of breath when hurrying on level ground 

or walking up a slight hill were 2.6 times higher 

than expected; the prevalence of wheezing was 

three times higher than expected. The prevalences of self-reported physician-diagnosed 

asthma and chronic bronchitis were 1.8 and 2.1 

times higher than expected, respectively. Of the 

116 employees who underwent spirometry, 21 

had airways obstruction, 3.3 times higher than 

expected. Airways obstruction in nonsmokers 

was 10.8 times higher than expected, and only 

two employees with airways obstruction had 

a significant response to administered bronchodilator. Five of six employees in the quality 

control (QC) laboratory had airways obstruction; these employees popped up to 100 bags 

of microwave popcorn in microwave ovens per 

employee per 8-hour work shift. Of the 115 

survey participants who had an x-ray, 111 had 

no abnormalities, two had evidence of emphysema, one had saber-sheath tracheal narrowing 

attributable to COPD or tracheal stenosis, and 

one had focal upper-zone scarring and atelectasis at the left lung base. DLCO was normal in 96 

of 103 employees tested, including all but one 

of those with airways obstruction.

3.1.2.2 Index plant environmental survey

In addition to the cross-sectional medical 

survey, NIOSH conducted a detailed environmental survey at the index microwave popcorn 

plant (Facility G) in November 2000 [Kanwal et 

al. 2011; NIOSH 2006]. The predominant VOC 

in the air of the plant was the butter flavoring 

compound diacetyl. All measurements above 

detectable limits (except where noted otherwise 

below) were subsequently corrected for underestimation inherent to NIOSH Method 2557 

related to absolute humidity and days to extraction [Cox-Ganser et al. 2011]. The relative 

humidity and temperature measurements used 

for correction were available from in-facility 

area-specific and shift-specific measurements 

during all sampling, and sample-specific days 

to extraction were supplied by the laboratory. 

The mixing room had the highest mean air 

concentration of diacetyl (57.2 ppm); the next 

highest mean air concentration of diacetyl 

was in the packaging area for machine operators (2.8 ppm). The mean air concentration 

of diacetyl in the QC laboratory was 0.8 ppm, 

and for maintenance it was 0.9 ppm. The much 

higher prevalence of airways obstruction in 

QC employees, despite much lower average 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 47

3 . Effects of Exposure in Employees

air concentrations of diacetyl, may reflect an 

enhanced risk of peak flavoring exposures 

when microwaved bags of popcorn product 

were opened; peak exposures were also likely 

present in maintenance employees and mixers. 

Mean diacetyl air concentrations in other plant 

areas were less than 0.15 ppm. 

These area-specific diacetyl concentrations 

and work history data provided by employees 

on the medical survey questionnaire were used 

to calculate estimated cumulative exposure 

to diacetyl for each survey participant. When 

survey participants were grouped into quartiles 

of increasing estimated cumulative exposure 

to diacetyl (corrected for underestimation by 

NIOSH Method 2557), the prevalence of any 

airways obstruction on spirometry was 14.3% 

in the lowest exposure quartile, 6.7% in the next 

lowest quartile, and 27.6% in the highest two 

exposure quartiles (statistically significant; P 

for trend = 0.04). The prevalences of abnormal 

spirometry, whether obstructed or restricted or 

mixed, by quartile were 21.4% for cumulative 

exposures < 0.82 ppm-yr; 16.7% for cumulative exposures between 0.82 and < 6.4 ppm-yr; 

34.5% for cumulative exposures between 6.4 

and < 19.2 ppm-yr; and 37.9% for cumulative 

exposures > 19.2 ppm-yr (statistically significant; P for trend = 0.04). Lung function as 

indicated by the average percent of predicted 

FEV1 on spirometry was 93.5%, 95.8%, 86.5% 

and 84.3% in the lowest to highest quartiles (P

for trend = 0.03) [Kreiss et al. 2002].

3.1.2.3 Findings of index plant 

follow-up surveys

NIOSH conducted seven follow-up medical 

and eight follow-up environmental surveys at 

the index microwave popcorn plant (Facility 

G) from 2001 to 2003 [Kanwal et al. 2011;

NIOSH 2006]. These surveys were conducted 

to follow employee symptoms and lung function over time as exposures decreased with 

the implementation of engineering controls. 

NIOSH recommended a respiratory protection program for mixing room employees to 

minimize their exposures while engineering 

controls were being implemented; this program 

was initiated at the time of the November 2000 

NIOSH survey. Starting in February 2001, 

the company began implementing several 

engineering controls to decrease air concentrations of flavoring compounds in the mixing 

room, the main source of air contaminants in 

the plant. An exhaust fan was installed in an 

outer wall of the mixing room to move contaminated air from this room to the outdoors 

and to maintain this room under negative air 

pressure relative to the rest of the plant. An 

air lock was installed at the entrance to the 

mixing room to further isolate the room from 

the rest of the plant. Local exhaust ventilation 

of the air space (headspace) above the contents 

of the heated flavoring tanks and the mixing 

tank in which flavorings are mixed into heated 

soybean oil was accomplished via ducts connecting the tank lids to the wall exhaust fan. A 

pump was installed to facilitate closed transfer 

of heated butter flavorings into the mixing tank. 

In 2002, the company constructed and began 

using a new mixing room that was more isolated from the packaging area than the original 

mixing room. In the packaging area, additional 

general dilution ventilation was implemented 

in 2001 along with local exhaust ventilation for 

seven heated holding tanks located on a mezzanine above the packaging lines that contained 

soybean oil and butter flavoring mixtures transferred via pipes from the mixing room. The 

entire mezzanine was walled off from the packaging area in 2003. Additional general dilution 

ventilation was also implemented in the QC 

laboratory in 2001. In 2003, all microwave 

ovens were eventually moved into a separate 

“popping room” adjacent to the QC laboratory 

with additional exhaust ventilation.

Compared to the mean diacetyl air concentrations NIOSH measured in November 2000, 
48 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

concentrations measured in November 2001 

were approximately 96% lower in the mixing 

room, 85% lower in the microwave popcorn 

packaging machine operator area, and 51% 

lower in the QC laboratory. After the implementation of a new, more isolated mixing room 

in fall 2002, mean diacetyl air concentrations 

in the microwave popcorn packaging machine 

operator area further declined to less than 

quantifiable limits (~0.004 ppm) in January 

2003 [Kanwal et al. 2011].

In their analyses of data from the eight NIOSH 

medical surveys at Facility G from November 

2000 to August 2003, NIOSH compared health 

outcomes in microwave popcorn production 

employees hired after the implementation 

of exposure controls to health outcomes in 

employees who had been working at the plant 

prior to the implementation of controls [Kanwal 

et al. 2011]. For these analyses, investigators 

classified employees according to their hire date 

as follows: “Group 1” consisted of employees 

who were already working at the plant at the 

time of the November 2000 survey (i.e., before 

exposure controls were implemented), and 

“Group 2” consisted of employees who started 

work at the plant after the November 2000 

survey (i.e., after exposure controls were implemented and exposures had declined). Because 

of a high turnover rate among employees hired 

after the November 2000 survey, participation 

in more than one medical survey was much 

higher in Group 1 (100 of 146 [68%] Group 

1 survey participants) than in Group 2 (86 of 

227 [38%] Group 2 survey participants). Mean 

length of employment for Group 1 survey participants was approximately 6 years, compared 

to 6 months for Group 2 survey participants. 

For all Group 1 microwave popcorn production employees who participated in one of the 

last two surveys in February 2003 and August 

2003 and in an earlier survey, NIOSH compared symptoms and lung function on their 

first survey to their last survey results. Most 

Group 2 employees who participated in more 

than one survey worked in the packaging 

area. Therefore, for all Group 2 packaging area 

employees who participated in more than one 

survey, investigators compared symptoms and 

lung function on their first survey to their last 

survey results. In Group 1, the only statistically significant change in symptom prevalence 

over time was a decline in reported eye, nose, 

or throat irritation. There were no statistically 

significant changes in the prevalence of airways 

obstruction or in mean percent predicted FEV1. 

Based on data from employees’ first surveys, 

packaging area employees in Group 2 had 

lower prevalences of respiratory symptoms 

and airways obstruction on spirometry, and 

mean percent predicted FEV1 was significantly 

higher compared to packaging area employees 

in Group 1. All these differences were statistically significant except for usual cough. There 

were no statistically significant changes in the 

prevalences of symptoms, airways obstruction, 

or mean percent predicted FEV1 from first to 

last survey in Group 2 packaging area employees [Kanwal et al. 2011]. Of interest is that 47% 

of all employees with abnormal spirometry 

tested by NIOSH (in Groups 1 and 2) were 

asymptomatic. 

NIOSH conducted a mortality study on 

Facility G employees based on Social Security 

Administration vital status determination as of 

November 30, 2011 [Halldin et al. 2013]. The 

cohort consisted of employees with potential 

flavoring exposure: 356 current employees 

who had participated in any of the eight 

NIOSH cross-sectional medical surveys from 

November 2000 through August 2003 and 

155 former employees tested by NIOSH at the 

county health department during that time 

period. There were 15 decedents altogether, not 

significantly different from the 17.39 expected. 

However, there were five COPD-associated 

multiple causes of death (International 

Classification of Diseases [ICD]-10 codes 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 49

3 . Effects of Exposure in Employees

J40-J44) coded among four decedents, for 

a standardized mortality ratio (SMR) of 4.3 

(95% confidence interval [CI] 1.40–10.04). 

There is no specific ICD-10 code for obliterative bronchiolitis, so it is likely that death from 

the condition would be coded using a COPD 

classification code. Consistent with this, the 

specific code J44 “other COPD” was assigned 

as a multiple cause of death for the four decedents (0.98 expected; SMR = 4.10, 95% CI 

1.12–10.49). Three of the four COPD-coded 

deaths occurred among former employees and 

employees employed before the company began 

to implement interventions to reduce diacetyl 

exposure (Group 1 above).

3.1.2.4 Other NIOSH microwave popcorn 

health hazard evaluations

NIOSH conducted HHEs that included crosssectional medical and environmental surveys at 

five other microwave popcorn plants (Facilities 

J, K, L, N, and O) from 2001 to 2003 [NIOSH 

2003a, b, c, 2004a, b]. These plants and the 

index plant (Facility G) were similar with 

regard to some production and exposure characteristics; however, there were some important 

differences as well [Kanwal et al. 2006]. The 

similarities in production and exposure characteristics at the six microwave popcorn plants 

evaluated by NIOSH were as follows:

(1) At each plant, one to three employees per 

work shift (i.e., mixers) measured butter 

flavorings (liquids, pastes, and powders) in 

open containers such as 5-gallon buckets 

and poured the flavoring into heated 

soybean oil in large (e.g., 500-gallon) 

heated mixing tanks, most of which had 

loose-fitting lids.

(2) Most mixers did not use respirators. Only 

one mixer at one plant reported consistent 

use of a respirator with organic vapor cartridges during mixing tasks.

(3) Mixers added salt and coloring to the oil 

and flavoring mixture, which was then 

transferred by pipes to nearby packaging 

lines to be combined with kernel popcorn 

in microwaveable bags.

(4) Employees on the packaging lines operated 

the packaging machines and facilitated 

the placement of the finished product into 

cartons and boxes.

In most plants, QC employees popped product 

in microwave ovens that were usually located 

in a separate QC laboratory. Other employees 

were located in warehouse and office areas. In 

separate areas of some plants, employees also 

packaged plain kernel popcorn in plastic bags 

without oil or flavorings. The six microwave 

popcorn plants differed in size as follows: 

(1) Two small plants (Facilities J and O) had 

fewer than 15 employees, one or two 

mixing tanks, and one packaging line.

(2) One medium-sized plant (Facility N) had 

approximately 50 employees, one mixing 

tank, three holding tanks for heated oil and 

butter flavoring mixtures, and three packaging lines.

(3) The three largest plants (Facilities G, K, 

and L) had more than 100 employees, five 

or more tanks, and seven or more packaging lines.

In some plants, flavoring-mixing activities and 

tanks were in a separate room adjacent to the 

packaging area. In other plants, some or all 

tanks of heated oil and flavoring were adjacent 

to or were inadequately isolated from the packaging lines [Kanwal et al. 2006].

In addition to the employees with findings consistent with bronchiolitis obliterans at the index 

microwave popcorn plant, employees with fixed 

airways obstruction and air trapping on HRCT 

scans consistent with obliterative bronchiolitis 

were identified at four of the other five microwave popcorn plants where NIOSH conducted 
50 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

HHEs [Kanwal et al. 2006]. Including the 

index plant, the three largest plants and one of 

the small plants had affected mixers [AkpinarElci et al. 2004; NIOSH 2003b, 2004a, b]. Like 

the index plant, the medium-sized plant had 

affected packaging area employees. At both of 

these plants, packaging area employees worked 

near tanks of heated oil and butter flavorings 

[NIOSH 2003a, 2006]. The biopsies of three of 

the six employees who underwent lung biopsy 

at the medium-sized plant were reported by the 

reviewing pathologists as having findings consistent with bronchiolitis obliterans [Kanwal et 

al. 2006; NIOSH 2003a]. Compared to mean 

diacetyl air concentrations measured at the 

index microwave popcorn plant, mean corrected diacetyl air concentrations at the other 

five microwave popcorn plants were lower: 0.02 

to 0.83 ppm in the packaging areas and 0.63 to 

1.54 ppm in the mixing rooms/areas [Kanwal 

et al. 2006].

NIOSH conducted analyses of aggregated data 

from the medical surveys conducted at the six 

microwave popcorn plants [Kanwal et al. 2006]. 

Only the data from the first survey at the index 

microwave popcorn plant were aggregated with 

the data from the surveys at the other plants. 

Compared to employees who had never worked 

as mixers, employees who had at least one day 

of experience mixing butter flavorings into 

heated soybean oil had statistically significant 

(P < 0.05) higher prevalences of respiratory 

symptoms and a statistically significant lower 

mean percent predicted FEV1. Compared to 

mixers with 12 months or less experience, 

mixers with more than 12 months experience 

had higher prevalences of respiratory symptoms (shortness of breath was statistically 

significant) and airways obstruction on spirometry. Mean percent predicted FEV1 was 82% in 

mixers with more than 12 months experience 

compared to 95% in mixers with 12 months or 

less experience (P = 0.004). The same pattern 

of higher prevalences of respiratory symptoms 

and worse lung function in ever mixers (who 

had ever worked at least one day mixing flavorings in oil) and in mixers with more than 

12 months experience was still evident after 

index plant data were excluded from the 

analyses [Kanwal et al. 2006]. Compared to 

packaging area employees at plants where tanks 

of heated oil and butter flavorings were isolated from the packaging lines, packaging area 

employees at plants where tanks were adjacent 

to or inadequately isolated from the packaging lines had higher prevalences of respiratory 

symptoms and airways obstruction on spirometry and lower mean percent predicted FEV1 

(29% vs. 10% for wheezing, P = 0.001; 14% 

vs. 5% for airways obstruction, P = 0.06; P > 

0.05 for all other comparisons). Of 27 packaging area employees with airways obstruction at 

plants where tanks were adjacent to or inadequately isolated from the packaging lines, 21 

of 23 who were administered a bronchodilator 

had fixed airways obstruction. After excluding 

index plant data from the analyses, packaging area employees in plants where tanks were 

adjacent to or inadequately isolated from the 

packaging lines still had higher prevalences of 

airways obstruction (11.5% vs 5.5%; not statistically significant) and wheezing (25% vs 

10.7%, P = 0.01) compared to packaging area 

employees at plants where tanks were isolated. 

The prevalences of other respiratory symptoms 

were similar in both groups. The findings across 

the six plants suggested that those employee 

groups with peak exposures, sometimes with 

relatively low average exposures, had higher 

prevalences of chest symptoms or pulmonary 

function abnormalities than those employees 

without intermittent high exposures [Kanwal 

et al. 2006].

3.1.2.5 Results of private surveys

A large food company hired private consultants 

to conduct medical and environmental surveys 

at the company’s four microwave popcorn 

plants [Lockey et al. 2009; White et al. 2010]. 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 51

3 . Effects of Exposure in Employees

One of the company’s plants, Facility L, was 

among the six microwave popcorn plants 

evaluated by NIOSH. A mixer at this plant 

had developed severe airways obstruction 

and other findings consistent with obliterative bronchiolitis. The investigators conducted 

spirometry tests three times at each plant from 

February 2005 through January 2006. During 

this time, 765 full-time employees worked at 

the four plants. Four employees were not tested 

because of significant cardiovascular disease 

or pneumonia, and four had unusable tests. 

The investigators excluded from subsequent 

analyses the test results of 11 office employees and 21 employees with a history of asthma 

that began prior to employment and who were 

taking asthma medications. The investigators 

classified employees into five groups for data 

analyses: (1) non-mixers (i.e., employees in 

the packaging line area, warehouse, or shipping/receiving areas), (2) mixers with mixing 

experience before the company implemented 

mandatory use of powered air-purifying respirators (PAPRs) with an assigned protection 

factor of 25 for mixers in April 2003, (3) mixers 

who only had mixing experience after implementation of mandatory use of PAPRs, (4) 

mechanics and supervisors who spent more 

than 30 minutes per month in the mixing 

room, and (5) quality assurance employees who 

popped approximately 50 bags of microwave 

popcorn per day. The investigators identified 

the following statistically significant associations from their data analyses:

(1) Work as a mixer before the implementation of mandatory PAPR use was associated 

with a decrease in the FEV1 percent of predicted of 6.1% for non-Asian males and 

11.8% for Asian males, in comparison to 

employees with no mixing room or quality 

assurance employment (P = 0.03 and P = 

0.02, respectively).

(2) Having a cumulative diacetyl exposure 

greater than or equal to 0.8 ppm-yrs was 

associated with a decrease in the FEV1

percent of predicted of 10.3% for non-Asian 

and 12.7% for Asian males, compared to 

having a cumulative diacetyl exposure less 

than 0.8 ppm-years.

(3) Among non-Asian males, work as a mixer 

before the implementation of mandatory 

PAPR use was associated with an 8-fold 

increased risk of airways obstruction 

(95% CI 2.26–29.24), and work as a mixer 

after the implementation of mandatory 

PAPR use was associated with a 5.7-fold 

increased risk of airways obstruction (95% 

CI 1.23–26.24).

(4) Having a cumulative diacetyl exposure 

greater than or equal to 0.8 ppm-yrs was 

associated with airways obstruction (odds 

ratio 9.2, 95% CI 2.29–36.75).

To assess for evidence of rapid lung function 

decline, the investigators identified employees 

with a progressive increase or decrease in FEV1

of greater than 8% or 330 mL over 12 months 

among employees who participated in all three 

spirometry tests [Lockey et al. 2009]. They 

found no association between current diacetyl 

exposure (less than 0.05 ppm or greater than/

equal to 0.05 ppm) and a short-term persistent increase or decrease in FEV1, adjusted for 

pack-years of smoking and body mass index. 

Of 39 mixers with mixing experience before the 

implementation of mandatory PAPR use, five 

had airways obstruction. Three of the five had 

bronchodilator administered, and all three had 

a bronchodilator response. Three of the five had 

HRCT scans; two of the scans showed air trapping on the expiratory view. The investigators 

concluded that, “The contribution of exposure 

to butter flavouring with diacetyl to these clinical findings is uncertain.” Three mixers who 

began mixing after the implementation of mandatory PAPR use were found to have airways 

obstruction. Preplacement spirometry was 

not available for these individuals. One of the 

three employees had pre-existing asthma, and 
52 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

the other two had long smoking histories (24 

and 63 pack-years, respectively). The investigators concluded that the airways obstruction in 

these three individuals was likely due to asthma 

and smoking but could not rule out the possibility that short-term exposure to diacetyl 

contributed to the airways obstruction when 

respirators had not been used 100% of the time. 

Analyses of 6 years of spirometric follow-up of 

these four plant cohorts are pending.

3.1.2.6 Field studies at flavoring 

manufacturing plants

Employees at several flavoring manufacturing plants have developed severe fixed airways 

obstruction and other findings consistent with 

obliterative bronchiolitis [Kanwal 2008]. The 

first known publicly available report of bronchiolitis obliterans in flavoring manufacturing 

employees is a 1986 report of a NIOSH HHE 

at Facility A that manufactured flavors for the 

baking industry [NIOSH 1986]. At this plant, 

two young previously healthy male employees 

(28 and 30 years old; nonsmokers) who prepared batches of flavorings developed severe 

fixed obstructive lung disease within 7 months 

of employment. Each employee developed progressive shortness of breath on exertion and 

nonproductive cough 4 to 5 months after starting work. Pulmonary function testing within 

1 to 2 months of symptom onset revealed an 

FEV1 of 1.2 and 0.7 liters, respectively, in the 

two employees. NIOSH reported that one 

employee had a “mild” response to bronchodilators and the other had a “minimal” response. 

Neither employee showed significant improvement in lung function within 1 to 2 years after 

they stopped working at the plant. Diffusing 

capacity was initially normal in both employees, and chest x-rays were normal or showed 

hyperinflation. NIOSH concluded that, even 

without pathological confirmation, the clinical 

picture was more compatible with bronchiolitis obliterans than with emphysema. One of 

the two employees was relocated to work in 

the loading dock but eventually left the job 11 

months after starting work at the plant because 

of shortness of breath. The other employee left 

the job when he was identified with severe fixed 

airways obstruction 5 months after starting 

work at the plant in the same job. Two current 

mixers with 5 to 6 years of experience were 

asymptomatic and had normal lung function 

on spirometry. Two other former mixers (36 

and 38 years old) had asymptomatic airways 

obstruction on spirometry. One had moderately 

severe airways obstruction and a normal chest 

x-ray; the other had mild airways obstruction, 

normal DLCO, and a normal chest x-ray. Both 

were former smokers.

At the time of the NIOSH HHE at Facility A, 

mixers produced flavors by mixing liquid flavor 

compounds into dextrose and corn starch 

powder in large blenders. This included using 

both 300-pound and 500-pound capacity “day 

mixers” (ribbon blenders), and a 1,500-pound 

capacity Littleford Mixer [NIOSH 1986]. 

Employees used approximately 200 Food and 

Drug Administration (FDA)-approved flavor 

compounds to produce different flavors. A list 

of commonly used ingredients at this plant 

included diacetyl. A supplied-air respirator system had been installed several months 

before the first employee to develop severe 

fixed airways obstruction had started work. 

Management had required employees to wear 

respirators when weighing or adding the flavors 

or base ingredients to the mixers. However, 

employees did not always wear respirators 

during clean-up activities where exposure to 

powdered flavors was possible. NIOSH concluded that it was probable that some agent 

in the mixing room produced severe fixed 

obstructive lung disease in two employees. 

They did not identify a specific etiologic agent, 

but suspected an airborne agent because the 

lung was the only affected organ and because 

air sampling by the Indiana Division of Labor 

had revealed high dust exposures. The Indiana 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 53

3 . Effects of Exposure in Employees

Division of Labor collected 20-minute air 

samples that showed dust air concentrations 

of 20 mg/m3

 in an employee’s breathing zone 

and 2.5 mg/m3 inside the hood of an employee’s 

supplied-air respirator. NIOSH analyzed bulk 

ingredient samples for levels of proteolytic 

enzymes and endotoxin. They did not identify 

proteolytic activity in any of the samples; endotoxin levels were “below levels seen in other 

workplaces where endotoxin has been associated with large decrements in FEV1” [NIOSH 

1986]. Air sampling for specific flavoring compounds was not conducted.

A cluster of cases consistent with obliterative 

bronchiolitis among production employees at a 

flavoring manufacturing company was reported 

by Dr. James Lockey at the 2002 American 

Thoracic Society International Conference 

[Lockey et al. 2009]. After identification of an 

index case of biopsy-documented bronchiolitis 

obliterans at this plant, a survey of the workforce identified an additional four employees 

with clinical findings consistent with obliterative bronchiolitis. All five employees with 

these findings had normal spirometry tests at 

the start of employment. These employees went 

on to develop moderate to severe fixed airways 

obstruction. For 4 to 5 years after cessation of 

exposure to flavoring compounds, the affected 

employees had no further declines in their 

lung function.

In 2007, the California Department of Public 

Health reported that seven flavoring manufacturing employees from four California plants 

had severe fixed airways obstruction [CDC 

2007]. NIOSH conducted HHEs that included 

cross-sectional medical and environmental 

surveys at two of these plants (Facilities B and 

C) [NIOSH 2007a, 2008]. Facility B produced 

liquid and powdered flavorings; powdered flavorings were produced by combining liquid 

flavoring compounds such as diacetyl with 

powder ingredients in ribbon blenders. Out 

of a workforce of 36 at the time of the NIOSH 

survey, 12 worked in the flavoring production 

room. Before July 2006, management provided 

production employees with 3M® N95 filteringfacepiece respirators for voluntary use. In 2005, 

a 42-year-old production employee who had 

worked for 7 years primarily making powdered 

flavorings developed cough and progressive 

shortness of breath. Medical tests conducted 

by this employee’s personal physicians revealed 

the following: fixed airways obstruction with 

an FEV1 of 0.55 liters (18% of predicted) on 

spirometry, an HRCT scan of the chest that 

showed small areas of patchy ground-glass 

opacities in the lungs, a follow-up computed 

tomography (CT) scan that revealed a small 

amount of scarring in the right lower lobe 

and lingula (part of the left lung) and resolution of the ground-glass opacities, and an open 

lung biopsy that was interpreted as showing 

peribronchial fibrosis and some granulomas. 

An occupational pulmonary medicine physician who evaluated this employee favored 

a diagnosis of bronchiolitis obliterans over 

hypersensitivity pneumonitis. This employee 

stopped working at the plant in December 2005 

because of severe cough and shortness of breath 

on exertion. In the July 2006 NIOSH medical 

survey, spirometry testing in this employee 

again showed severe fixed airways obstruction 

(FEV1 of 0.54 liters; 21% of predicted). Another 

former employee and a current employee who 

had worked in powdered flavoring production also had severe fixed airways obstruction 

on NIOSH spirometry tests. The FEV1 was 

1.11 liters (32% of predicted) for the former 

employee and 0.78 liters (23% of predicted) for 

the current employee. The current employee 

with severe airways obstruction reported a past 

history of asthma but said that he was asymptomatic when he began working at the plant. He 

reported the onset of difficulty breathing within 

2 weeks of starting work in powdered flavoring 

production. He had been relocated to the warehouse just before the NIOSH survey because 

of severe shortness of breath on exertion. 
54 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

An open lung biopsy was interpreted by the 

reviewing pathologist as showing bronchiolitis 

obliterans. An additional current production 

employee was found to have mild restriction 

on spirometry; the rest of the medical survey 

participants (31 of 36 current employees and 

three former employees) had normal spirometry tests [NIOSH 2007a].

NIOSH conducted an HHE at a second flavoring manufacturer (Facility C) over several 

visits to the plant from October 2006 to July 

2007 [NIOSH 2008]. This plant produced 

liquid and powdered flavorings (encapsulated and nonencapsulated powders) and 

colors. Nonencapsulated powdered flavorings 

were produced by combining liquid flavoring compounds such as diacetyl with powder 

ingredients in ribbon blenders. Encapsulated 

powdered flavorings were produced by drying a 

slurry (a mixture of powdered and liquid ingredients) in a spray dryer. With encapsulated 

powder flavors, volatile flavor ingredients such 

as diacetyl are enclosed within an encapsulant 

material to decrease volatility. Out of a workforce of 47 at the time of the NIOSH survey, 12 

were production employees. Forty-one employees participated in the first NIOSH medical 

survey conducted from October 30, 2006, to 

November 1, 2006. Of 41 employees tested, 3 

had abnormal spirometry: a laboratory/QC 

employee had mild restriction, a flavoring production employee had borderline obstruction, 

and an employee in the warehouse with several 

years of experience in flavoring production 

had severe fixed airways obstruction. This last 

employee had started working at the plant in 

powdered flavoring production in 1995 at age 

26. He used an N95 filtering facepiece respirator from 1995 to 1999 and then started using 

a full-face, negative-pressure, air-purifying 

respirator; he was not fit-tested for either respirator. Because of respiratory symptoms, he 

was reassigned to liquid flavoring production 

in 2000. In April 2006, he was reassigned to the 

warehouse. His personal physician diagnosed 

chronic rhinitis in 2003 and acute bronchitis in 

2004. A spirometry test in March 2005 showed 

severe fixed airways obstruction (FEV1 20% 

of predicted). In May 2005, a pulmonologist 

diagnosed bronchiectasis of unknown etiology 

based on HRCT scan of the chest. The employee 

was hospitalized twice for his lung condition. 

NIOSH spirometry testing in October 2006 

showed severe fixed airways obstruction (FEV1 

17.9% of predicted). On follow-up spirometry 

testing by NIOSH at the plant in March 2007 

his FEV1 was 20.7% of predicted. The flavoring 

employee who had borderline airways obstruction on NIOSH testing in October 2006 was 

found to have mild fixed airways obstruction 

in March 2007; his FEV1 had dropped approximately one liter (percent predicted FEV1

declined from 86% to 64%).

NIOSH performed an HHE in 2007 that 

included a medical and environmental survey 

at a flavoring manufacturer (Facility D) in 

Wisconsin [NIOSH 2009d]. At the time of the 

HHE, this plant manufactured flavors, colors, 

and bacterial blends used as silage inoculants 

and probiotics. One of the flavor products 

produced at this plant is starter distillate, a 

diacetyl-containing distillate of a milk stock 

produced from fermented dairy cultures. The 

diacetyl concentration in this distillate was 

4.5%. Other flavor products made at this plant 

included powdered encapsulated starter distillates and other butter flavors produced by spray 

drying, and other liquid flavors. The NIOSH 

medical survey included a questionnaire, spirometry testing, and methacholine challenge 

testing (to identify airways hyperresponsiveness as occurs in asthma). Of 40 employees in 

production areas, the quality control laboratory, 

the warehouse, and in maintenance who were 

invited to participate in the medical survey, 34 

agreed to participate. Of these 34 employees, 

15 worked in jobs where they could potentially 

be exposed to flavoring-related compounds 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 55

3 . Effects of Exposure in Employees

including diacetyl. Of 10 former employees 

who had worked in flavoring production areas 

and were invited to participate in the medical 

survey, three agreed to participate. Of the 15 

current employees with jobs in which they 

could potentially be exposed to flavoringrelated compounds including diacetyl, one 

employee with a pre-employment history of 

asthma was found to have mild fixed airways 

obstruction mixed with restriction. NIOSH 

recommended that this employee pursue additional medical evaluation to look for further 

evidence of obliterative bronchiolitis or another 

illness; follow-up results were not available 

to NIOSH. In addition to the employee with 

mild fixed airways obstruction (mixed with 

restriction), two employees had restrictive 

abnormalities. Of the 15 employees with potential exposures to flavoring-related compounds, 

five reported having currently active physiciandiagnosed asthma. All five were diagnosed 

with asthma before starting work at the plant; 

no employees reported recurrence after hire of 

pre-existing asthma that had been inactive for 2 

or more years prior to hire. Two of 11 employees with normal spirometry who underwent 

methacholine challenge testing were found 

to have airways hyperreactivity. Both of these 

employees had physician-diagnosed asthma 

before coming to work at the plant.

In 2012, NIOSH conducted a cross-sectional 

medical survey at a flavoring company (Facility 

Q) in Kentucky in which two former employees 

had received physician diagnoses of obliterative 

bronchiolitis [Cummings et al. 2014; NIOSH 

2013]. Of 357 employees with spirometry, 13 

had obstruction (of whom 2 of 10 responded 

to bronchodilator), 15 had restriction, and 

2 had mixed obstruction and restriction. 

The prevalences of abnormal spirometry 

were not elevated in relation to NHANES III 

expected rates, adjusted for age, sex, race/

ethnicity, smoking status, and body mass 

index. However, participating employees had 

statistically significant excesses of wheeze in 

the last 12 months, sinusitis or sinus problems 

in the last 12 months, phlegm on most days 

for three consecutive months during the year, 

a diagnosis of hay fever, a lifetime diagnosis 

of asthma, and current asthma, when compared to the U.S. adult population. Shortness 

of breath was twice as common in those with 7 

or more year’s tenure, and remained significant 

in a model adjusted for age and smoking status. 

Work-related breathing trouble, wheeze, nasal 

symptoms, sinusitis, eye symptoms, and cough 

were all statistically significantly increased 

in employees currently using flavoring compounds compared to remaining employees, and 

these work-related symptoms remained significantly associated with flavoring compound use 

in models adjusted for age and smoking status. 

Participating employees who spent an hour or 

more daily in production areas had twice the 

prevalence of any spirometric abnormality 

and three times the prevalence of low diffusing capacity than other participants. Mean lung 

function parameters (expressed as percent predicted) were significantly lower in participants 

with tenure of 7 or more years and those who 

spent one or more hours daily in production 

areas. Differences in lung function could not 

be explained by age, smoking status, or employment at another flavoring plant, and persisted 

in analyses stratified by ever having been in 

production. The association of symptoms and 

lung function parameters with exposure indices 

suggested that they resulted from workplace 

exposures. Diacetyl was not used in the plant 

during the NIOSH air sampling, but 2,3-pentanedione was detected in two air samples 

collected with evacuated canisters in a liquid 

compounding room where fruit and cheese 

flavor recipes were being prepared.

3.1.2.7 Lung disease in flavoring 

manufacturing employees

The California Department of Public Health 

provided information on a flavoring production 
56 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

employee who developed bronchiolitis obliterans while working at another California 

flavoring plant [California Department of 

Public Health 2007; CDC 2007]. This employee 

primarily prepared powdered flavorings by 

pouring “diacetyl and other liquid ingredients 

through a hole on the blender lid.” He started 

working at the plant in October 2001 at the age 

of 27. Two years later he developed progressive shortness of breath on exertion, decreased 

exercise tolerance, intermittent wheezing, 

left-sided chest pain, and a productive cough. 

In November 2003, his physician prescribed 

antibiotics and bronchodilators for suspected 

bronchitis and allergic rhinitis. He stopped 

working in January 2004, but his shortness of 

breath continued to worsen. An HRCT scan of 

his chest showed cylindrical bronchiectasis in 

the lower lobes, with scattered peribronchial 

ground-glass opacities. Spirometry in April 

2004 showed severe fixed airways obstruction (FEV1 28% of predicted). Lung volume 

measurements showed severe air trapping. 

Diffusing capacity was normal. A follow-up 

HRCT with inspiratory and expiratory views in 

October 2004 showed central peribronchiolar 

thickening with central airway dilatation and 

subtle areas of mosaic attenuation scattered 

throughout the lungs, predominantly in the 

right lower lobe [CDC 2007].

In 2006, Cal/OSHA and the California 

Department of Public Health developed a lung 

disease prevention program for employees of 

California flavoring manufacturing plants. In 

analyses of aggregated medical surveillance 

data (questionnaire and spirometry results) 

from 467 employees at 16 companies who 

had usable questionnaires and acceptable spirometry tests, 18 employees (3.9%) from six 

companies with 315 participating employees 

had airways obstruction [Kim et al. 2010]. 

This prevalence was similar to that expected 

in comparison to national data from NHANES 

III [CDC 1996]. However, the distribution by 

severity of obstruction was highly skewed, with 

six mild cases, seven moderate, one severe, and 

the remaining four very severe. The prevalence 

of severe and very severe obstruction combined 

was 2.7 times higher than expected overall (95% 

CI 1.2–6.4) and 15 times higher than expected 

in employees less than 40 years old (95% CI 

5.1–44.1). Sixteen obstructed cases worked in 

four companies using ≥ 800 pounds of diacetyl annually compared to two obstructed cases 

in companies using less diacetyl (prevalence 

of 5.3% versus 1.2%), for an odds ratio (OR) 

of 4.5 (95% CI 1.03–19.9). The prevalence of 

obstruction in employees currently doing any 

production task was 4.5% compared to 2.0% 

in production support employees (laboratory technicians/scientists, quality control 

technicians, maintenance/repair employees, 

warehouse employees, and truck drivers) and 

2.3% in office employees. Of the 18 employees 

with obstruction, 14 currently worked in production, two worked in production support 

(one had just moved from production because 

of dyspnea), one with previous production 

experience currently worked in the office, 

and one could not be classified. Tenure was 

statistically significantly higher in employees 

with moderate or worse obstruction than in 

employees with mild obstruction (1.5 versus 

9.0 years; P = 0.02). Half of the 18 employees 

with obstruction reported no chest symptoms 

(five of six employees with mild obstruction 

and four of seven with moderate obstruction). 

Of the 13 with documented postbronchodilator spirometry, 12 had fixed obstruction 

(including all four with severe or very severe 

obstruction). Of the 12 of 18 with obstruction 

who had medical evaluation results submitted to the California Department of Public 

Health, eight were diagnosed by their physicians to have either bronchiolitis obliterans 

(one biopsy-confirmed) or fixed obstruction 

related to flavorings; all eight had moderate to 

very severe disease [Kim et al. 2010]. Some of 

the cases included in this analysis of California 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 57

3 . Effects of Exposure in Employees

flavoring employee surveillance data were presented above in the descriptions of two NIOSH 

HHEs at California flavoring plants (Facilities 

B and C).

3.1.2.8 Lung disease in diacetyl 

production employees

Lung disease consistent with obliterative bronchiolitis was reported among employees of a 

plant in the Netherlands that produced diacetyl [van Rooy et al. 2007]. From 1960 through 

2003 when diacetyl production ceased, 206 

employees had potentially been exposed to 

diacetyl at this plant. Of 196 employees still 

alive, 175 consented to participate in a medical 

survey conducted by Dutch investigators. The 

survey included a questionnaire, spirometry, 

and review of medical files of the Occupational 

Health Service. Employees with possible 

airways obstruction on screening spirometry 

were referred for additional medical evaluation including an HRCT scan with inspiratory 

and expiratory views. Of the 175 survey participants, 102 worked as “process operators.” 

The other participants worked in other jobs 

such as the quality control laboratory, “technical service,” management, research and 

development, and logistics. Four employees 

were found to have fixed airways obstruction. 

One of these four employees (with a predicted 

FEV1 of 72%) refused further evaluation. FEV1

percent predicted in the other three employees, all process operators, ranged from 35% to 

42%. All three employees had evidence of air 

trapping on HRCT scan expiratory views. One 

of these three employees underwent a thoracoscopic lung biopsy that did not show evidence 

of obliterative bronchiolitis. Two of these three 

employees were nonsmokers who had initially 

been diagnosed with COPD; the third employee 

(with a 14 pack-year smoking history) had initially been diagnosed with COPD and asthma. 

Two of these three employees developed shortness of breath on exertion within a year or 

two of starting work at the plant at ages 45 

and 39 years. The other employee developed 

shortness of breath at age 52, 14 years after 

starting work. A fourth employee (process 

operator; nonsmoker) with severe fixed 

airways obstruction and findings compatible 

with obliterative bronchiolitis on HRCT scan 

was identified among survey nonparticipants 

after the survey. During production of diacetyl, employees were also potentially exposed 

to acetoin, acetaldehyde, and acetic acid. The 

diacetyl plant was one of several in operation 

at the production site; all process employees 

also worked at other chemical plants at the 

production site. The investigators noted that 

“Among the gaseous chemicals identified in 

the plants, only ammonia and chlorine were 

of potential concern for bronchiolitis obliterans, but none of the cases reported having had 

significant exposure to these agents” [van Rooy 

et al. 2007]. Regarding diacetyl exposures, 26 

area samples (82–219 minutes) and 4 personal 

task-based samples (33–90 minutes) were taken 

between 1995 and 2003. Many jobs were not 

sampled. These data were insufficient for quantitative risk assessment over the period of plant 

operation from 1960 to 2003.

The investigators who evaluated the workforce of the diacetyl-producing plant in the 

Netherlands compared respiratory symptom 

and asthma prevalence among male employees 

to data from the Dutch section of the European 

Community Respiratory Health Survey [van 

Rooy et al. 2009]. Compared to the Dutch 

European Community Respiratory Health 

Survey population, the diacetyl plant workforce had significantly higher prevalences of 

continuous trouble with breathing, daily cough, 

and asthma attacks. Compared to a minimally 

exposed internal comparison group, process 

operators (including the three with severe 

fixed airways obstruction and evidence of air 

trapping on HRCT scan expiratory views who 

were identified in the medical survey [van Rooy 

et al. 2007]) and quality control laboratory 
58 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

employees reported ever trouble with breathing significantly more often. Operators also 

reported significantly more shortness of 

breath in the last year. Spirometry test results 

for the 149 white male diacetyl plant employees did not differ significantly from the Dutch 

European Community Respiratory Health 

Survey population after adjusting for smoking 

history. The investigators were not able to demonstrate an exposure-response relationship 

between relative cumulative exposure to diacetyl and FEV1. However, they had previously 

demonstrated an average 292 mL decrement 

in FEV1 in process operators in comparison to 

a minimally exposed internal reference group 

[van Rooy et al. 2007].

Available information on TWA and peak 

exposures to diacetyl in flavoring and diacetyl 

manufacturing plants where employees have 

developed findings consistent with obliterative 

bronchiolitis indicates that employees’ exposures in these plants may have been similar to 

employees’ exposures at microwave popcorn 

plants. At one flavoring plant [NIOSH 2007a], 

the mean TWA diacetyl exposure from fullshift air sampling in the powdered flavoring 

production area was 2.73 ppm. Measurements 

made with partial-shift air sampling during 

the production of butter and vanilla powdered 

flavorings showed a diacetyl exposure of 25.9 

ppm. Employees’ real-time diacetyl exposures 

during the packaging of these powders were 

as high as 204 ppm. At a second flavoring 

plant [NIOSH 2008], mean TWA diacetyl air 

concentrations from full-shift air sampling in 

November 2006 (area and personal samples 

combined) were 0.46 ppm in liquid flavoring 

production and 0.34 ppm in powdered flavoring production. A task-based personal air 

sample measured a diacetyl air concentration 

of 11 ppm when an employee poured diacetyl 

from a 55-gallon drum into multiple 5-gallon 

containers over a 10-minute period. At the 

diacetyl production plant in the Netherlands 

where Dutch investigators identified four 

former employees with severe fixed airways 

obstruction and evidence of air trapping on 

HRCT scan expiratory views, task-specific 

diacetyl exposures ranged from 3 to 396 mg/

m3

 (0.6 ppm to 83 ppm) during discharge of 

diacetyl from a reactor vessel into containers 

[van Rooy et al. 2007]. The measured diacetyl 

exposures at these three plants are comparable to exposures (corrected for absolute 

humidity and days to extraction) measured at 

the six microwave popcorn plants evaluated 

by NIOSH. In the mixing room at the index 

microwave popcorn plant (Facility G), the 

mean TWA diacetyl air concentration from 

area samples in November 2000 was 57.2 ppm. 

At the three other microwave popcorn plants 

where mixers developed findings consistent 

with obliterative bronchiolitis, TWA diacetyl 

exposures from personal samples were 0.31 

ppm, 0.69 ppm, and 1.33 ppm [NIOSH 2003b,

2004a, b]. Real-time measurements at one of 

these plants showed that a mixer’s diacetyl 

exposures increased up to 80 ppm to 120 ppm 

when he added liquid butter flavorings to a 

mixing tank [NIOSH 2004a].

3.1.2.9 Other food production 

case reports

In addition to cases consistent with obliterative bronchiolitis in flavoring manufacture, 

diacetyl manufacture, and microwave popcorn 

production, case reports have surfaced in 

other food production industries in which 

flavorings are introduced into food products. 

In cookie manufacture with artificial butter 

flavoring in Brazil, four cases of bronchiolitis 

were described in young men, aged 24 to 27 

years, who had worked between 1 and 3 years 

handling flavorings in preparation of cookie 

dough [Cavalcanti et al. 2012]. One of the 

four had confirmation of bronchiolitis obliterans on open lung biopsy, and the remaining 

three were diagnosed on the basis of consistent 

chest symptoms (cough, progressive dyspnea, 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 59

3 . Effects of Exposure in Employees

and wheezing); moderate to severe mixed 

obstructive and restrictive spirometry; abnormal chest CT findings of lung hyperinflation, 

air trapping, bronchial thickening, and mosaic 

perfusion; and persistence of spirometric findings in 4 years of follow up. Two cases had 

partial response to bronchodilators and one 

case had ground-glass opacity.

In a coffee production plant, two cases have 

biopsy confirmation of obliterative bronchiolitis among employees with artificial flavorings 

exposure in the production of roasted coffee 

beans and ground coffee [CDC 2013]. An 

additional three cases from the same plant 

were reported by Dr. Jeffrey Levin in an 

abstract at the 2013 American Thoracic Society 

International Conference. In 2012, NIOSH 

conducted an HHE at this coffee production 

plant involving 75 current employees (88% 

participation)[Bailey et al. 2015]. Excluding 

the five sentinel former employees (all neversmokers under age 42), standardized morbidity 

ratios were elevated 1.6-fold for shortness of 

breath and 2.7-fold for obstructive spirometric 

abnormalities. The sum of diacetyl and 2,3-pentanedione air concentrations were equivalent in 

the flavoring room and in the grinding/packaging area of unflavored coffee. The subgroup of 

employees who currently worked in both coffee 

flavoring and grinding/packaging of unflavored 

coffee had significantly lower mean FEV1/FVC 

ratio and percent predicted mid-expiratory flow 

than employees without such exposure. In addition to the sentinel former employee cases, six 

current employees had abnormalities suggestive 

of obliterative bronchiolitis and five additional 

employees had suspect work-related asthma. 

NIOSH investigators could not separate risks 

of employees in unflavored coffee grinding/

packaging from risks of flavored coffee production because most employees’ exposures were 

similar during their work tenure.

3.2 Restrictive Spirometry 

in Flavoring-exposed 

Workforces 

NIOSH work on flavoring-related lung disease 

concentrated on obstructive spirometric 

abnormalities between 2000 and 2008 because 

the classic textbook description of obliterative 

bronchiolitis described an obstructive disease. 

NIOSH included employees with mixed 

obstructive and restrictive spirometry among 

the obstructed because NIOSH assumed that 

air trapping explained their decreases in forced 

vital capacity. Had NIOSH added employees 

with mixed obstructive and restrictive disease 

to those with pure spirometric restriction, 

NIOSH would have had excesses of restriction 

similar to those of obstruction in comparison 

to general population prevalences in some field 

investigations. NIOSH now has evidence from 

several investigators outside of the flavoring 

lung disease field that the clinical spectrum 

of biopsy-confirmed obliterative bronchiolitis includes both restrictive spirometry and 

normal spirometry, as well as those with fixed 

obstructive spirometry [Ghanei et al. 2008;

King et al. 2011; Markopoulou et al. 2002]. 

NIOSH summarizes the evidence concerning 

spirometric restriction in flavoring-exposed 

employees in this section.

Spirometric restriction is defined as a FVC 

below the lower limit of normal and an FEV1/

FVC ratio that is normal. Lung diseases involving scarring (fibrosis), and inflammation of 

the interstitium or alveolar spaces commonly 

are accompanied by spirometric restriction. 

Examples of these lung diseases are hypersensitivity pneumonitis, pneumoconioses, and 

BOOP in which bronchiolar changes extend 

into the alveolar spaces. Non-pulmonary 

causes of spirometric restriction are poor 

effort or incomplete exhalation maneuvers, 

obesity, and neuromuscular weakness. Low 

lung volumes such as total lung capacity 
60 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

and low residual volume support pulmonary 

causes of spirometric restriction, but normal 

lung volumes do not rule out lung pathology 

accounting for spirometric restriction [Boros 

et al. 2004].

The proportions of abnormal spirometry that 

were restrictive in the three case series of 

biopsy-documented constrictive bronchiolitis 

are instructive. In dyspneic U.S. soldiers, 3 of 

38 soldiers had restriction (2 with low diffusing 

capacity), 2 had obstruction (1 with low diffusing capacity), and one had mixed restriction 

and obstruction (with low diffusing capacity). 

The remaining soldiers had normal spirometry 

and lung volumes, although 19 had low diffusing capacity [King et al. 2011]. Of 15 cases of 

chronic dyspnea and cough following sulfur 

mustard exposure 20 years previously, 13 had 

normal spirometry, one had restriction, and 

one had obstruction; all had pathologic evidence of bronchiolar disease. The cases with 

biopsy-documented constrictive bronchiolitis 

all had normal spirometry, and the two with 

the abnormal spirometry had chronic cellular 

bronchiolitis [Ghanei et al. 2008]. Of 19 cases 

of biopsy-documented obliterative bronchiolitis, six had normal spirometry (although 

2 had isolated gas trapping), 11 had obstruction, one had restriction, and one had a mixed 

pattern [Markopoulou et al. 2002]. This last 

case series originated from a clinical referral center without common exposures. These 

pathologic case series suggest two conclusions. 

First, abnormal spirometry is insensitive to 

pathologic obliterative bronchiolitis that results 

in symptoms warranting clinical evaluation. 

Second, the finding of restriction in populations 

with cases of fixed airways obstruction consistent with obliterative bronchiolitis is likely to 

be part of the spectrum of obliterative bronchiolitis, although the differential diagnosis in 

individual employees requires investigation.

3.2.1 Index Plant Findings 

Regarding Restriction

Among the former employees who developed 

findings consistent with obliterative bronchiolitis while working at the index microwave 

popcorn plant (Facility G), lung function tests 

in one employee showed a reduced total lung 

capacity and reduced residual volume in addition to airways obstruction. These reduced 

lung volumes indicate that this employee 

had restrictive lung disease as well as airways 

obstruction [Akpinar-Elci et al. 2004]. This 

former employee also had a low carbon monoxide diffusing capacity and was unusual among 

the former employee cases in having some 

reversibility after ceasing employment at the 

microwave popcorn [Akpinar-Elci et al. 2004].

In the first cross-sectional survey of the index 

plant (Facility G), 10 of 116 employees had 

isolated abnormal FVC, of whom 7 had low 

total lung capacity; 11 employees had isolated 

airways obstruction. An additional 10 employees had both low FVC and airways obstruction, 

for a total of 21 of 116 employees having any 

restrictive spirometric pattern. None of those 

with any restriction had radiologic interstitial abnormalities. When the prevalence of 

any restrictive abnormality was examined by 

cumulative exposure quartile (using exposure 

estimates corrected for humidity and time to 

extraction), a trend for exposure response relationship was evident: From lowest to highest 

exposure quartile, the prevalence of any restriction was 10.7%, 13.3%, 20.7%, and 24.1% 

(P = 0.08). During follow up of these plant 

employees, one employee with rapidly falling 

pulmonary functions in a restrictive pattern 

underwent open lung biopsy. The pathology 

report documented caseating lung granulomas around airways, but grossly normal areas 

of lung were not sampled for examination of 

possible obliterative bronchiolitis. Cultures 

and stains for microorganisms did not yield an 

infectious etiology, and the physician concluded 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 61

3 . Effects of Exposure in Employees

Table 3-2. Distribution of spirometric abnormalities in flavoring-exposed employees by facility, 

reference, facility type, and type of spirometric abnormality

Spirometric 

abnormalities

Restrictive 

abnormalities

Facility 

NIOSH 

evaluated Reference Facility type

Current 

employees 

tested

N

%

N

%*

Obstructive 

abnormalities

Mixed obstructive 

and restrictive 

abnormalities

G Kreiss et al. [2002] Microwave popcorn 116 31 26.7 10 32.3 11 10 

N NIOSH [2003a] Microwave popcorn 35 7 20.0 2 28.6 2 3

K NIOSH [2004a] Microwave popcorn 86 13 15.1 7 53.8 3 3

L NIOSH [2004b] Microwave popcorn 205 29 14.1 10 34.5 12 7

G NIOSH [2006] Microwave popcorn 368 75 20.4 36 48.0 24 15

B NIOSH [2007a] Flavor manufacturer 34 2 5.9 1 50.0 0 1

C NIOSH [2008] Flavor manufacturer 41 3 7.3 1 33.3 1 1

D NIOSH [2009d] Flavor manufacturer 28 3 10.7 2 66.7 0 1

E NIOSH [2009b] Food production 22 4 18.2 4 100.0 0 0

F NIOSH [2009c] Food preparation 104 20 19.2 15 75.0 4 1

B,C† Kim et al. [2010] Flavor manufacturer 467 59 12.6 41 69.5 10 8

I NIOSH [2011] Flavor manufacturer 106 34 32.1 30 88.2 3 1

G Halldin et al. [2013] Microwave popcorn 356 57 16.0 27 47.4 16 14

155‡ 50 32.3 23 46.0 10 17

Q NIOSH [2013] Flavor manufacturer 357 30 8.4 15 50.0 13 2

R Bailey et al [2015] Food production 69 7 10.2 2 2.9 5 0

*Percentage of abnormal pulmonary function tests (PFTs) classified as restriction†Referenced publication includes site(s) that NIOSH did not visit‡Former employees
62 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

that the abnormalities were related to occupational flavoring exposures [Kreiss 2012].

3.2.2 NIOSH Findings of Restrictive 

Spirometry at Other Microwave 

Popcorn Plants

Most microwave popcorn plant populations 

surveyed cross-sectionally by NIOSH had 

similar proportions of restrictive, obstructive, and mixed abnormalities among those 

employees with abnormal pulmonary functions (Table 3-2). In the three large microwave 

popcorn plants (Facilities G, K, and L), the 

restrictive proportion of abnormal spirometry 

ranged from 32.3% to 53.8%. These proportions 

are similar to those cited in two case series of 

biopsy-documented constrictive bronchiolitis, 

which were 50% in the case of U.S. soldiers in 

Iraq and Afghanistan [King et al. 2011] and 

Iranians following sulfur mustard exposure, 

in which the pathology included proliferative 

bronchiolitis [Ghanei et al. 2008]. In the three 

large microwave popcorn plants, the proportion 

of mixed restrictive and obstructive spirometry 

in those with abnormal spirometry was similar 

to the proportion with pure obstructive and 

pure restrictive abnormalities. In the consecutive clinical case series [Markopoulou et al. 

2002], the much lower proportion of restrictive 

abnormalities may be explained by the prevailing understanding a decade ago that obliterative 

bronchiolitis is an obstructive disease.

3.2.3 NIOSH Findings of Restrictive 

Spirometry at Flavoring 

Manufacturing Plants

As in the microwave popcorn investigations, 

flavoring manufacturing workforces with cases 

consistent with obliterative bronchiolitis have 

also had employees with restrictive spirometry, 

with proportions of restriction among those 

with abnormal spirometry ranging from 28.6% 

to 88.2% (Table 3-2).

NIOSH found an unusually high prevalence 

of a restrictive spirometric pattern among 

production employees at a flavoring manufacturing plant (Facility I) in Indiana [NIOSH 

2011]. Among the 106 employees with interpretable spirometry test results obtained 

by the company, 30 (28%) had a restrictive 

pattern (22 with a mild abnormality, six with 

a moderate abnormality, one with a moderately severe abnormality, and one with a severe 

abnormality). In addition, three employees 

had obstructive abnormalities, and one had 

a very severe mixed abnormality. Combining 

all spirometric abnormalities with those with 

only excessive decline in FEV1 in the subset of 

employees with serial abnormalities, 39 (37%) 

employees had abnormal findings. In comparison to the U.S. general population, the 

employee prevalence of restrictive spirometric abnormalities was 3.8 times higher than 

expected, after adjustment for race, ethnicity, 

sex, age, smoking status, and body mass index. 

NIOSH later detected an error in abstraction of 

smoking information from company spirometry reports and corrected this comparison 

to 3.7 [Kreiss 2014]. NIOSH also found evidence of rapid lung function decline in this 

workforce (section 3.3) with a 7.0-fold risk of 

excessive decline in the subgroup of production 

employees with higher potential for flavorings 

exposure (later corrected to 5.8) [Kreiss 2014]. 

Average declines in percent predicted FEV1

and FVC for the employees with four annual 

measurements were in a pattern consistent with 

the evolution of restrictive lung disease. As in 

other flavoring plants, chemical exposures 

were diverse, although diacetyl was used nearly 

daily. Personal samples of diacetyl obtained 

by the company using NIOSH Method 2557 

(uncorrected for absolute humidity and days to 

extraction) ranged to 0.76 ppm and area measurements to 10.2 ppm. Company samples in 

2008–2009 using OSHA methods (not requiring correction) ranged to 1.9 ppm for personal 

and 2.9 ppm for area samples. 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 63

3 . Effects of Exposure in Employees

A company-sponsored re-analysis of Facility I 

spirometry data reported finding that no flavoring compounds, including diacetyl, had 

produced an increased risk of abnormal spirometric findings or longitudinal changes in 

spirometry [Ronk et al. 2013]. The study confirmed an excess risk of abnormal restrictive 

spirometry reported by NIOSH investigators 

with a similar prevalence ratio of 3.3 (95% CI 

2.2–4.6) in comparison to the general population reflected in NHANES III. The authors 

offered the inadequacy of the NHANES III 

study population as a comparison group, despite 

adjusting for age, sex, and body mass index, 

because the national data were largely drawn 

from urban centers, and the authors alleged 

that the flavoring employees in a large city in 

Indiana were largely agrarian. As an alternative comparison group, the authors described 

the employee group with lower potential 

for flavoring exposure as an internal control 

group with no or minimal exposure, also 

referring to them as an administrative group. 

However, all employees in the medical surveillance program were in production areas, and 

company data documented measurable diacetyl in all production areas, including worrisome 

measurements in packaging which was classified in the NIOSH health hazard evaluation 

as having lower potential for exposure. Thus, 

the similar distribution of abnormal restrictive 

spirometry across the production workforce, 

without regard to higher and lower potential 

for flavoring exposure, remained unexplained 

and cannot be attributed to misclassification 

of lung disease by spirometry, variable quality 

spirometry, or body habitus, also mentioned by 

the authors. The most likely explanation for the 

3.3–3.7 increased odds for restrictive disease in 

the Facility I workforce is that risk for workrelated abnormality existed across both groups 

of production area employees in comparison to 

the national predicted estimate. 

The Ronk et al. [2013] study conclusion that 

none of the flavoring compounds caused workrelated spirometric abnormalities hinges on 

absence of association of pulmonary function 

abnormalities or decrements in employees with 

tenure in higher potential for flavoring exposure areas. The authors explain the difference 

in findings between their “negative” study and 

the NIOSH findings of work-related spirometric abnormalities by a NIOSH methodologic 

flaw in not taking account of correlated measures of serial lung functions. However, the 

authors misrepresent NIOSH analyses in 

which the outcome variables were the slopes 

of spirometric changes, expressed as mL/

year, based on linear regression as a smoothing function. NIOSH also used categorical 

outcomes of excessive spirometric decline. 

Neither of these NIOSH outcomes reflected 

correlated serial data. In addressing serial 

(correlated) spirometry measures, Ronk et al. 

[2013] used generalized estimating equation 

modeling, which is a reasonable approach. 

However, the authors chose an exchangeable 

correlation structure, which assumes that the 

variation between any two measures is equal; 

this assumption would not appear appropriate for pulmonary function test measures at 

varying intervals. Measures taken at a 6-month 

interval would likely be more correlated than 

measures at several-year intervals, as occurred 

in the Facility I spirometry data set. The generalized estimating equation models assume 

that cumulative tenure is linearly related to the 

change in spirometry measures, which may 

not be the case in a short-latency health effect 

as has occurred in flavoring-exposed employees. The Ronk et al. [2013] paper omits report 

of average changes in FEV1 and FVC per year 

in their model without workplace covariates, 

which might have indicated unusually high 

average decrements per year. NIOSH had found 

that the average FVC decline in the employee 

population was 108 mL/year, about 3.5-fold the 

expected decline of approximately 30 mL/year. 
64 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

Ronk et al. [2013] separately modeled tenure 

in work areas with higher potential for exposure and tenure in liquid compounding with 

the apparent assumption that the remainder 

of the plant population had zero tenure (exposure), which is simply false. In particular, the 

liquid compounding tenure model ignores 

tenure in other higher potential for exposure 

jobs, which would clearly result in no associations with their work parameters. In contrast, 

the simpler NIOSH analyses of decline in lung 

function by areas with higher and lower potential for flavoring exposure demonstrated that 

both average declines and excessive decline 

differed between the two groups of production employees in statistically significant ways. 

These simple methods were not affected by correlated measurements. 

A subsequent Indiana Occupational Safety 

and Health Administration (IOSHA) compliance investigation of Facility I reported 

hydrogen sulfide exposures above the NIOSH 

level immediately dangerous to life and health 

of 100 ppm [IOSHA 2012]. Hydrogen sulfide 

can result in obliterative bronchiolitis. IOSHA 

measured high concentrations of diacetyl (well 

above the proposed recommended exposure 

limit) in the packaging area that NIOSH had 

classified as lower potential for exposure. Thus, 

the diversity of exposures encountered by 

employees in this flavoring facility precluded 

identifying a specific cause(s) of the excess 

lung disease. However, the burden of likely 

occupational disease, reflected in the excess of 

restrictive spirometry and excessive annualized decline in spirometry, requires control of 

flavoring vapors, flavoring-related particulates, 

and hydrogen sulfide.

NIOSH found a high prevalence of a restrictive 

pattern on spirometry among employees at a 

plant (Facility E) where production employees 

combined liquid and powdered flavorings with 

flour, sugar, salt and other solid ingredients to 

produce baking mixes [NIOSH 2009b]. Of 41 

employees, 23 (including 18 of 27 production 

employees) participated in a NIOSH medical 

survey that included spirometry testing. Of 22 

employees with interpretable spirometry results, 

four (18%) had a restrictive pattern. All other 

spirometry tests were normal. The prevalence 

of restriction was approximately three times 

greater than expected compared to U.S. general 

population data from NHANES III [CDC 

1996]. From June 2007 through May 2008, the 

company had used a buttermilk flavoring that 

contained 15% to 20% diacetyl. The company 

began using a reformulated buttermilk flavoring that contained less than 1% diacetyl in July 

2008. The reformulated buttermilk flavoring 

contained 2,3-pentanedione, a diacetyl substitute that contains an additional methyl group. 

Use of the buttermilk flavoring was reported to 

be infrequent. In an industrial hygiene survey 

conducted by NIOSH from September 30, 

2008, to October 2, 2008, diacetyl was detected 

qualitatively in screening air samples obtained 

with thermal desorption tubes and analyzed 

with gas chromatography/mass spectrometry 

according to NIOSH Method 2549. However, 

the diacetyl air concentrations were too low 

to be quantified or detected with the modified 

OSHA Method PV2118. In a second industrial 

hygiene survey conducted by NIOSH in May 

2009, air sampling with OSHA Method 1013 

again did not reveal detectable or quantifiable 

concentrations of diacetyl; however, one personal sample showed an air concentration of 

2,3-pentanedione of 91 ppb, and a corresponding area sample showed an air concentration of 

78 ppb. Area air sampling with an additional 

method under development, in-tube derivatization with 1,2-phenylenediamine (section 2.2.5 

above), did not detect diacetyl but did show 

2,3-pentanedione in several areas, at concentrations ranging from 48 to 95 ppb. The sample 

that showed an air concentration of 95 ppb 

was obtained in the same area where a sample 

obtained with OSHA Method 1013 showed an 

air concentration of 78 ppb [Day et al. 2011].
Occupational Exposure to Diacetyl and 2,3-Pentanedione 65

3 . Effects of Exposure in Employees

In 2008 NIOSH conducted an HHE of three cafeterias located at three different office buildings 

in New York City (Facilities F) [NIOSH 2009c]. 

The HHE request was motivated by concern 

about diacetyl in butter-flavored cooking oils 

used on grill surfaces. Laboratory analyses of 

bulk samples of butter and two samples of one 

brand of cooking oil used at the three facilities 

did reveal diacetyl. Air samples obtained by 

NIOSH at the three facilities showed that air 

concentrations of diacetyl were below the limit 

of detection (0.02 ppm). NIOSH conducted a 

medical survey that included a questionnaire 

and spirometry tests. Approximately 80% of the 

workforce at the three facilities participated in 

the medical survey (116 of 141 employees completed the questionnaire; 104 of 111 employees 

who underwent spirometry testing had a valid 

test). Five employees (5%) had airways obstruction, and two of these five employees had 

fixed obstruction. Both employees with fixed 

obstruction had started work at their current 

facility after butter-flavored cooking products 

were no longer in use. All five employees with 

obstruction denied having ever worked as professional cooks. Fifteen employees (14%) had 

restriction on spirometry, for a prevalence 

that was twice as high as expected compared 

to general population data from NHANES III 

[CDC 1996]. Five of the 15 had body mass 

indices over 30. Only three of the 15 reported 

cooking experience, and 13 reported cleaning experience. Compared to employees who 

did not cook at work, employees who reported 

cooking among their job duties were twice as 

likely to report asthma-like symptoms; more 

than three times as likely to report shortness of 

breath after exercise, cough, and work-related 

wheezing; approximately five times more likely 

to report work-related shortness of breath 

following exercise; and more than twice as 

likely to report work-related nasal symptoms. 

Employees who reported cleaning among their 

job duties were three times more likely to report 

asthma-like symptoms or shortness of breath 

while hurrying on level ground or walking up 

a slight hill than employees who did not clean 

at work. Employees who reported cleaning hot 

surfaces at work were almost four times more 

likely to report shortness of breath following 

exercise than those who had not cleaned hot 

surfaces at work.

In these field investigations in microwave 

popcorn production, flavoring production, 

and food preparation, clinical evaluations of 

employees with spirometric restriction are 

unavailable, with the exception of the two 

employees at Facility G [Akpinar-Elci et al. 

2004; Kreiss 2012]. One reason for the absence 

of pathophysiologic data is the previous focus 

of NIOSH investigators and clinicians on 

obstructive lung disease. Although the evolving literature now documents that obliterative 

bronchiolitis can manifest with normal or 

restrictive spirometry as well, NIOSH did 

not examine evidence for work-relatedness 

of restrictive disease and FEV1 decline until 

reporting the findings in 2011 from medical 

surveillance data for flavoring Facility I [Kreiss 

2014; NIOSH 2011]. A published case report 

exists of BOOP in an employee with exposure to spices and flavorings in making snack 

foods [Alleman and Darcey 2002] which has 

resulted in permanent impairment 10 years 

later [NIOSH unpublished data]; the role of 

flavorings in this case with restriction remains 

unclear. Because obstructive abnormalities are 

insensitive for pathologic obliterative bronchiolitis, future work should evaluate dyspnea and 

any spirometric abnormalities.

3.3 Rapid Lung 

Function Decline

Indirect and direct evidence shows that employees exposed to flavoring-related compounds 

can experience excessive lung function decline, 

whether within the normal range of spirometry 

or in those with either restrictive or obstructive 
66 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

spirometric abnormalities. Indirect evidence 

comes from reviews of medical records and 

work histories of flavoring-exposed employees who developed obliterative bronchiolitis. 

In a case series summarizing the eight affected 

former employees and one additional current 

employee at the index microwave popcorn 

plant (Facility G), the median length of employment prior to symptom onset was 1.5 years; the 

median duration of employment was 2 years 

[Akpinar-Elci et al. 2004]. At a company that 

manufactured flavors for the baking industry 

(Facility A), two flavoring production employees developed respiratory symptoms and severe 

fixed airways obstruction within 7 months 

of starting work at the plant [NIOSH 1986]. 

Although these employees did not have baseline spirometry tests before they began working 

with flavorings, it is unlikely that their lung 

function was already significantly decreased 

when they started work. Production jobs such 

as preparing the oil and flavoring mixture for 

microwave popcorn production and mixing 

liquid and powder flavor ingredients in flavoring manufacture often require the employee to 

lift 50- to 100-pound containers. It is unlikely 

that employees could have performed such 

tasks if their lung function was already severely 

compromised when they started work. Some 

affected employees stopped working when they 

could no longer do the job because of severe 

shortness of breath on exertion, while others 

were relocated to less strenuous jobs [NIOSH 

1986, 2007a, 2008]. Severe airways obstruction 

as seen in obliterative bronchiolitis is rare in 

the general population. Data from NHANES 

III show that, among individuals less than 

50 years old (including both smokers and 

never-smokers), the prevalence of obstruction 

with an FEV1 less than 40% of predicted is 0.1% 

(1 in 1,000 people) [CDC 1996].

Direct evidence that employees exposed to 

flavoring-related compounds can experience rapid lung function decline comes from 

exposed employees who have had serial spirometry tests. Normal average FEV1 decline 

is about 30 mL/year, and percent predicted 

FEV1 does not usually change in the absence of 

disease because the predicted value is age corrected [Redlich et al. 2014]. Three of the affected 

former employees from the index microwave 

popcorn plant (Facility G) had declines in 

their FEV1 percent of predicted of approximately 20% to 30% over approximately 2 years 

[Akpinar-Elci et al. 2004]. NIOSH evaluated 

data from the eight NIOSH medical surveys 

at the index microwave popcorn plant for evidence of rapid lung function decline [Kanwal 

et al. 2011]. The investigators chose as the criterion for rapid decline a decrease in FEV1 of 

300 mL and/or 10% from an employee’s initial 

(baseline) spirometry test to the employee’s last 

spirometry test. This criterion was similar to a 

threshold developed based on a study of coal 

miners evaluated over time with spirometry of 

high technical quality in which the researchers 

concluded that “when healthy working males 

perform spirometry according to American 

Thoracic Society standards, a yearly decline in 

FEV1 greater than 8% or 330 mL should not 

be considered normal...” [Wang and Petsonk 

2004]. The sensitive criterion used by the investigators, who did not annualize declines, was 

chosen because of the potential severity of the 

irreversible health outcome and the high technical quality of the pulmonary function tests, 

which allows for a sensitive cutpoint. For their 

analysis of the data from the surveys at the 

index microwave popcorn plant, investigators 

excluded survey participants with fewer than 

three interpretable spirometry tests because 

interpretation of change over time based 

on only two tests is less reliable [Pellegrino 

et al. 2005].

Of the 88 survey participants who participated 

in three or more NIOSH medical surveys at 

the index microwave popcorn plant (Facility 

G) and had started working there prior to the 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 67

3 . Effects of Exposure in Employees

implementation of exposure controls (“Group 

1”), 19 (22%) had FEV1 declines of greater than 

300 mL and/or 10% from their first to their 

last spirometry test. Four of these 19 employees had worked at some point in the mixing 

room, including one employee who experienced a 1,300-mL decline from the first test 

in November 2000 to the next test 5 months 

later; the next spirometry test 4 months after 

the second test showed an additional decline 

in FEV1 of 600 mL, resulting in the employee 

leaving employment. This employee’s FEV1

continued to fall after leaving employment, 

with a total fall of 2,800 mL over 2.75 years, 

representing a decline from 96% of predicted 

FEV1 to 39% of predicted FEV1. In comparison 

to survey participants who began working at 

the plant before the company started implementing exposure controls, only 3 (7%) of 41 

survey participants with three or more spirometry tests who were hired after the company 

began implementing controls (“Group 2”) had 

FEV1 declines of greater than 300 mL and/or 

10% from their first to their last spirometry 

test [Kanwal et al. 2011]. Of the 27 Group 1 

employees who participated in all eight medical 

surveys, mean annualized decline in FEV1 in 

the first year of follow-up was 144 mL per year. 

Annualized decline in the second year of follow-up fell to 40 mL per year as exposures were 

controlled, and the annualized decline fell to 

22 mL per year in the third year of follow-up, 

a rate of decline consistent with normal agingrelated lung function decline [Kreiss 2007].

NIOSH identified rapid lung function decline at 

a flavoring plant where a production employee 

had developed severe fixed airways obstruction 

[NIOSH 2008]. Another flavoring production 

employee at this plant had borderline airways 

obstruction on his first spirometry test, which 

is defined as a normal FEV1 with a FEV1/FVC 

ratio below the lower limit of normal. This 

employee was found to have mild fixed airways 

obstruction on his second test 5 months later; 

his FEV1 had declined approximately one liter 

in the 5 months between tests.

NIOSH found evidence of excessive lung 

function decline among flavoring production employees at a flavoring manufacturing 

company (Facility I) in Indiana [NIOSH 2011]. 

Diacetyl was used nearly daily in the plant and 

was measured in the air in many areas of the 

plant. In the course of an HHE at this facility, 

NIOSH reviewed results of spirometry tests 

obtained by the company on 112 production 

employees. Interpretable spirometry results 

were available for 106 current and former 

production employees. NIOSH compared 

the results of each employee’s spirometry test 

to reference values based on U.S. population 

data on healthy nonsmokers from NHANES 

III [Hankinson et al. 1999]. The investigators calculated changes in FEV1 over time for 

70 employees with more than one spirometry test result. To assess abnormal excessive 

declines in FEV1, they determined the average 

within-person variation in FEV1 to be 5%. 

Using spirometry longitudinal data analysis 

(SPIROLA), a NIOSH freeware program that 

adjusts for data quality (within-person variation) and length of follow up [NIOSH 2010], 

NIOSH found that 19% (13) of employees 

with serial spirometry had excessive decline 

in FEV1 based on a 12.4% longitudinal decline 

supplemented by a reference decline of 30 mL/

year. Five of the 13 still had spirometry values 

within the normal range despite their excessive declines. Employees currently working in 

areas with higher potential of flavorings exposure had 7.0-fold odds (later corrected to 5.8) 

[Kreiss 2014] of having excessive FEV1 decline 

(95% CI 1.3–38.2, corrected to 1.2–28.8) in 

comparison to employees who were not currently working in areas with higher potential 

for exposure. The areas with higher potential 

for flavorings exposure included dry blend, 

extract and distillation, liquid compounding, process flavors, and spray dry areas. The 
68 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

employees in these areas had 2.8 times greater 

average annual declines in FEV1 than employees in other areas. The 18 production employees 

who had annual tests for 4 years (2006–2009) 

had average changes in their percent predicted 

FEV1 and FVC measurements that declined in 

parallel with stable FEV1/FVC ratios suggestive of an average tendency toward evolution of 

restrictive spirometry. Historical measurements 

of diacetyl and other flavoring compounds were 

insufficient to evaluate quantitative exposureresponse relations. NIOSH also found a high 

prevalence (28%) of a restrictive pattern on spirometry tests in this workforce (section 3.2.3).

Company-sponsored re-analyses of the longitudinal spirometry data using generalized 

estimating equation models were interpreted 

as not showing any exposure-related declines 

in longitudinal spirometry measures [Ronk 

et al. 2013]. However, as noted earlier, the 

paper used an internal control group of production employees with diacetyl exposure as 

a control group, assumed zero tenure (reflecting zero exposure) for subgroups in the lower 

potential for exposure “control” group, and an 

exchangeable correlation structure for modeling that is not suitable for differing intervals 

of spirometric measures. See section 3.2.3 for 

further details.

The California Department of Public Health 

received serial spirometry test data for 416 

flavor manufacturing employees administered from 2004 until early 2009, of whom 

9.6% (40) had abnormal FEV1 decline [Kreiss 

et al. 2012]. Abnormal FEV1 decline rates (per 

person-month of follow up) were greater at 

companies using ≥ 800 lbs/year diacetyl than 

at companies using lesser amounts (7.3 versus 

3.0 per 1,000 person-months, P = 0.01) and 

greater in companies previously shown to have 

four-person clusters of spirometric obstruction than at companies with no or only one 

employee with obstruction. Using only high 

quality serial spirometry data on a subset of 

289 employees, 21 (7.3%) had abnormal decline 

using the 4% within-individual variation that 

characterized this subset [NIOSH 2010]. Only 

one of the 21 had airways obstruction; this 

employee lost 23.9% (−980 mL) of his baseline FEV1 over 25 months. Only five of the 21 

employees had abnormal restrictive spirometry 

on one or more tests, three of whom developed 

restriction on their last test. The remaining 15 

employees with excessive FEV1 decline were 

within the normal range of FEV1. The greatest annualized FEV1 decline in the group with 

good quality data was −2534 mL/year (−1700 

mL in 8 months), and the average annualized 

FEV1 loss in this group was −85 mL/year. The 

mean FEV1 change for employees in companies 

using ≥ 800 lbs/yr of diacetyl was −113.6 mL/yr 

compared to −51.6 mL/yr in companies using 

less diacetyl (P = 0.06).

Other investigators have examined rapid 

declines in flavoring-exposed or diacetylexposed employees. The bronchiolitis obliterans 

syndrome cases identified in the Dutch diacetyl 

manufacturing plants had accelerated declines 

in FEV1, with one case having an annualized 

decline of 175 mL/year from 1995 to 2003 [van 

Rooy et al. 2007]. In contrast, in a microwave 

popcorn manufacturing cohort studied over 

12 months, no relationship was demonstrated 

between current exposure level (dichotomized 

at 0.05 ppm) and an abnormal decrease in FEV1

(found in 7% of employees using a criterion of 

a greater than 320 mL or 8% decline over one 

year), adjusted for pack-years of smoking and 

body mass index [Lockey et al. 2009].

As indicated in the studies above, different 

approaches have been used by investigators 

over time to define excessive or rapid decline 

in FEV1. These include percentage decline with 

various criteria, absolute decline with various 

criteria, normative population-based criteria 

for longitudinal limits of decline over various 

time intervals, and spirometry quality-adjusted 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 69

3 . Effects of Exposure in Employees

criteria, all of which are discussed in Chapter 9, 

section 9.5.

3.4 Asthma

At the index microwave popcorn plant and 

at one of the other five microwave popcorn 

plants that NIOSH evaluated (Facilities G and 

L), the prevalence of self-reported physiciandiagnosed asthma was approximately two 

times higher than expected [NIOSH 2004b,

2006]. This suggests the possibility that some 

employees exposed to diacetyl and other flavoring compounds may be at increased risk for 

asthma (reversible airways obstruction) while 

others might be at risk for obliterative bronchiolitis (fixed airways obstruction). However, few 

of the survey participants with airways obstruction at these two plants who were administered 

a bronchodilator medication had a significant 

response (i.e., their airways obstruction was 

fixed); therefore, it is possible that some of 

these individuals had a different lung disease 

and that asthma may have been a misdiagnosis. 

Some employees at microwave popcorn plants 

and flavoring plants who were initially diagnosed with asthma were ultimately found to 

have fixed airways obstruction and other findings consistent with obliterative bronchiolitis 

[Akpinar-Elci et al. 2004; van Rooy et al. 2007].

It is possible that individuals with pre-existing asthma may experience an exacerbation 

of their asthma due to the irritant properties 

of diacetyl or similar vapors. Many asthmatics react nonspecifically with bronchospasm 

to strong odors. Diacetyl has been reported to 

be a sensitizer in a rodent local lymph node 

assay, and other diketones, including 2,3-pentanedione, 2,3-hexanedione, 3,4-hexanedione 

and 2,3-heptanedione, have similar potency 

as sensitizers [Anderson et al. 2013]. Some 

aldehydes found in flavoring manufacturing plants are sensitizers. If sensitization to 

diacetyl or another chemical were to occur in 

a susceptible individual, that individual might 

develop allergic-type asthma, with diacetyl 

exposure triggering airways obstruction and 

respiratory symptoms. In the coffee manufacturing plant investigation (Facility R), evidence 

for occupational asthma among current and 

former employees consisted of sensitization to 

coffee and castor bean antigens known to be 

exposures in the industry, and exacerbation of 

asthma was reported in relation to roasting area 

smoke and dusts [Bailey et al. 2015].

NIOSH conducted an HHE at a small plant 

(Facility M) where employees popped popcorn 

in heated oil and applied flavorings (including butter flavorings) prior to packaging 

[Sahakian et al. 2008]. Before 2002, they had 

used diacetyl-containing salt, and they used 

butter-flavored oil at the time of the survey. 

All three employees (lifelong nonsmokers) 

who had ever worked at the company developed respiratory disease while working there. 

One former employee had a mixed pattern of 

airways obstruction and restriction on spirometry; the airways obstruction was responsive to 

administered bronchodilator. This employee 

eventually died as a result of his respiratory 

disease. “Status asthmaticus with acute cardiopulmonary arrest” was listed as the primary 

diagnosis on the hospital discharge summary. 

Of the two other employees who had symptoms 

of asthma, one had an FEV1 that improved by 

480 mL (11%) and an FVC by 510 mL (8%) 

within the normal ranges after bronchodilator administration. The other employee had 

abnormal airways resistance of 322% of predicted; 19% improvement of the mid-maximal 

forced expiratory flow after bronchodilator; 

and improvement in FEV1 of 6% after bronchodilator. While employed at the plant, all 

three employees experienced worsening of 

their respiratory symptoms on the days they 

worked. HRCT scans of the chest showed 

findings suggesting possible bronchiolitis obliterans in the employee who died and in one of 
70 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

the other two employees. Air sampling results 

indicated that aldehydes were the predominant 

type of VOC in the plant air during production 

processes. Air samples obtained with thermal 

desorption tubes and analyzed with gas chromatography/mass spectrometry according to 

NIOSH Method 2549 showed that diacetyl was 

present in the plant air. However, the 2-hour 

and 4-hour diacetyl concentrations were less 

than the minimal detectable concentrations of 

0.02 and 0.01 ppm respectively with NIOSH 

Method 2557 [NIOSH 2007b].

3.5 Mucous Membrane 

Irritation (Eye, 

Upper Respiratory)

Eye, nose, and throat irritation has been frequently reported by employees in NIOSH 

medical surveys at microwave popcorn plants 

and flavoring manufacturing plants. At the 

index microwave popcorn plant (Facility 

G), among employees who started work in 

microwave popcorn production prior to the 

implementation of exposure controls, approximately 65% reported eye, nose, or throat 

irritation on their first medical survey. Only 

33% of these employees reported eye, nose, 

or throat irritation on their last survey after 

exposures had declined. Microwave popcorn 

packaging area employees who started work 

after exposures had declined had a similar 

lower prevalence of irritant mucosal symptoms (25%) [NIOSH 2006]. At the two small 

microwave popcorn plants NIOSH evaluated 

(Facilities J and O), most employees reported 

eye and/or nasal irritation [NIOSH 2003b, c]. 

At one of these two plants (Facility J), several 

employees developed severe eye irritation and 

blurred vision when the company started using 

a new butter flavoring [NIOSH 2003b]. After 

the company stopped using the new flavoring and halted production for several days, the 

employees’ eye problems resolved. At one of the 

large microwave popcorn plants NIOSH evaluated (Facility K), management implemented 

use of full-facepiece respirators for mixing 

room employees soon after the company began 

producing microwave popcorn (before the 

respiratory hazard from butter flavoring vapors 

had been recognized), because these employees 

experienced severe eye irritation from butter 

flavoring vapors [NIOSH 2004a]. However, 

employees did not wear respirators consistently at all times during which they might be 

exposed [NIOSH 2004a]. At another microwave 

popcorn plant evaluated by NIOSH (Facility L), 

83% of employees in the mixing room reported 

nasal irritation [NIOSH 2004b]. All laboratory 

and warehouse employees who participated in 

the NIOSH medical survey at a flavor manufacturer (Facility B) reported post-hire nasal 

irritant symptoms; 80% of employees in the 

production room and the laboratory reported 

post-hire eye irritation [NIOSH 2007a]. Of 

employees who had ever worked in production 

at another flavor manufacturer (Facility C), 

93% reported post-hire eye irritation [NIOSH 

2008]. One employee reported eye burning 

from exposure to diacetyl and starter distillate 

during a NIOSH survey at a third flavoring producer (Facility D) [NIOSH 2009d].

3.6 Dermatologic Effects

Of the former employees who developed findings consistent with obliterative bronchiolitis 

while working at the index microwave popcorn 

plant (Facility G), one employee also developed 

a severe skin rash [Akpinar-Elci et al. 2004]. 

The employee developed thick keratotic plaques 

and fissures of the palms and soles, associated 

with dystrophic fingernails. Skin punch biopsy 

revealed mild acanthosis and spongiosis with 

focal superficial epidermal necrosis and an 

associated subepidermal dense lymphohistiocytic infiltrate. Patch testing showed early 

and late reactions to two butter flavorings and 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 71

3 . Effects of Exposure in Employees

late reactions to six other butter flavorings, all 

used in the plant. This employee’s dermatitis 

improved when he stopped work.

Prevalences of reported post-hire skin problems at microwave popcorn plants and flavoring 

plants have ranged from 12% at one of the six 

microwave popcorn plants (Facility N) NIOSH 

evaluated [NIOSH 2003a] to 36% among production employees at a flavoring plant (Facility 

B). Post-hire skin problems were reported by 

60% of employees who primarily made liquid 

flavorings at this plant [NIOSH 2007a].

3.7 Discussion

Medical evaluations of employees who have 

developed progressive shortness of breath 

while working at several microwave popcorn 

plants and flavoring plants have shown findings consistent with the severe irreversible 

lung disease obliterative bronchiolitis. Some 

affected employees have experienced extremely 

rapid declines in lung function, with severe 

airways obstruction occurring within several 

months of the start of exposure to flavoring 

compounds [Akpinar-Elci et al. 2004; NIOSH 

1986]. Whether restrictive lung disease is part 

of the spectrum of obliterative bronchiolitis in flavoring-exposed employees remains 

incompletely evaluated, although restrictive 

spirometry has been a common finding; in one 

plant, excessive FEV1 declines in a restrictive 

pattern appear to be associated with potential 

for flavorings exposure. Employees as young 

as 22 years old have been affected by obstructive disease. Some affected employees have 

been placed on lung transplant waiting lists by 

their physicians because of the severity of their 

disease [Akpinar-Elci et al. 2004], and some flavoring-exposed employees have received lung 

transplants. The findings from investigations 

and studies conducted at multiple plants have 

revealed a link between exposure to diacetyl 

and risk for severe occupational lung disease. 

These findings meet the criteria that are often 

used to determine if the results of multiple 

studies indicate that an exposure is the likely 

cause of specific health effects [Gordis 1996;

Hill 1965].

The first of these criteria is temporality: the 

exposure precedes disease development. 

Evidence of this comes from the many instances 

where initially asymptomatic diacetyl-exposed 

employees developed progressive shortness 

of breath within months of starting work and 

then were found to have severe fixed airways 

obstruction [Kreiss et al. 2002; NIOSH 1986;

van Rooy et al. 2007]. Additionally, NIOSH 

documented rapid falls in lung function in 

exposed employees with initially normal spirometry at three plants [NIOSH 2006, 2008,

2011]. Lockey et al. reported at the 2002 

American Thoracic Society International 

Conference that five flavoring employees who 

developed moderate or severe fixed airways 

obstruction had normal spirometry at the start 

of employment [Lockey et al. 2009]. California 

public health surveillance showed that excessive FEV1 decline occurred in employees in 

flavor manufacturing plants that participated 

in a preventive program attempting to lower 

flavoring exposures [Kreiss et al. 2012].

Temporality requires the exposure to precede 

disease development, and the inverse is that 

new disease cases should decline in a population with cessation of exposure, an evaluation 

by intervention or “experiment”. Follow-up 

medical and environmental surveys at the index 

microwave popcorn plant (Facility G) revealed 

evidence of decreased lung disease risk with 

control of exposures. In employees hired before 

exposures were controlled, the prevalences of 

respiratory symptoms and airways obstruction and mean percent predicted FEV1 did 

not change significantly over time (consistent 

with an irreversible disease). However, employees hired after exposures were controlled had 

lower prevalences of respiratory symptoms 
72 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

and airways obstruction and higher mean 

percent predicted FEV1 on their first medical 

survey than employees hired before exposures 

were controlled, and these findings did not 

change significantly over time [Kanwal et al. 

2011; NIOSH 2006]. Additionally, among 27 

employees who participated in all eight NIOSH 

medical surveys from 2000 to 2003, annualized 

declines in FEV1 improved from 144 mL per 

year to 40 mL per year to 22 mL per year, the 

last being consistent with normal aging-related 

lung function decline [Kreiss 2007]. Similarly, 

the former employee index cases with clinical 

bronchiolitis obliterans had stable FEV1 within 

about 2 years of exposure cessation [AkpinarElci et al. 2004].

Another criterion is strength of the association: the magnitude of the apparent health risk 

due to the exposure. In analyses of data from 

the initial NIOSH medical survey at the index 

microwave popcorn plant (Facility G), the 

prevalence of airways obstruction among nonsmoking current employees was approximately 

11 times higher than expected in comparison to 

national data from NHANES III. It was approximately three times higher than expected in 

older smokers [Kreiss et al. 2002]. In analyses 

of California flavoring employee surveillance 

data, the prevalence of severe airways obstruction was approximately three times higher 

than expected among all employees compared 

to national data. The prevalence in employees 

less than 40 years old was 15 times higher than 

expected [Kim et al. 2010].

The criterion of replication of findings (and 

strength of the association) between diacetyl 

exposure and development of severe occupational lung disease is apparent in the number 

of plants where employees have been affected 

and the number of production employees 

in these plants. The six microwave popcorn 

plants NIOSH evaluated represent a large 

segment of the microwave popcorn industry in 

the United States. Employees who developed 

findings consistent with obliterative bronchiolitis at these plants prepared the mixture of 

butter flavorings and soybean oil (“mixers”) 

or worked nearby in the packaging area. Four 

of the six microwave popcorn plants NIOSH 

evaluated had affected mixers [Kanwal et al. 

2006]. Each of these plants had one to three 

mixers per work shift at the time of the NIOSH 

HHEs. The occurrence of multiple cases of 

severe airways obstruction in such a small job 

category (approximately 20 mixers across the 

six plants) is far greater than expected when 

compared to the U.S. population prevalence 

of severe airways obstruction from NHANES 

III data (0.1%, or 1 in 1,000, in individuals 

less than 50 years old, including smokers and 

never-smokers) [CDC 1996]. A similar magnitude of risk exists in some flavoring companies. 

At least six flavoring production employees 

developed findings consistent with obliterative 

bronchiolitis at three flavoring plants (Facilities 

A, B, and C) where NIOSH conducted medical 

surveys. There were approximately 30 production employees across these three plants at 

the time of the NIOSH HHEs [NIOSH 1986,

2007a, 2008].

Consistency is also supported by the occurrence 

of lung disease consistent with obliterative 

bronchiolitis in diacetyl-exposed employees in 

at least eight flavoring manufacturing plants, 

a diacetyl production plant, a cookie manufacturing plant, and a coffee production plant 

[Akpinar-Elci et al. 2004; CDC 2007; Kanwal et 

al. 2006; Kim et al. 2010; NIOSH 1986, 2007a,

2008; van Rooy et al. 2007]. Private consultants 

who conducted medical and environmental surveys at four microwave popcorn plants 

owned by one large food company also found 

in their data analyses that a history of working 

as a mixer and higher cumulative exposure to 

diacetyl were associated with decreased lung 

function [Lockey et al. 2009]. 

Additional criteria to support a causal link 

between diacetyl exposure and severe lung 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 73

3 . Effects of Exposure in Employees

disease include biologic plausibility, doseresponse relationship, and consideration of 

alternate explanations. Biologic plausibility is 

supported by experimental studies of diacetyl 

toxicity summarized in Chapter 4.

NIOSH found evidence of a dose-response 

relationship (i.e., worse lung disease or more 

employees affected with higher diacetyl exposure) in analyses of medical survey data from 

the index microwave popcorn plant (Facility 

G) and in analyses of aggregated data from 

medical surveys at the index plant and five 

additional microwave popcorn plants. The 

analyses of data from the initial survey at the 

index plant showed an increasing prevalence 

of abnormal spirometry with increasing quartiles of estimated cumulative diacetyl exposure 

[Kreiss et al. 2002]. Analyses of aggregated data 

from surveys at the six microwave popcorn 

plants showed higher prevalences of respiratory symptoms and worse lung function in 

mixers with more than 12 months experience 

and in packaging area employees at plants 

where heated tanks of oil and flavorings were 

not adequately isolated, compared to less 

exposed comparison groups [Kanwal et al. 

2006]. Additional evidence of a dose-response 

relationship was found in analyses of California 

flavoring employee surveillance data. An analysis of obstruction by amount of plant diacetyl 

use showed that there were 16 employees with 

obstruction in four companies that used more 

than 800 pounds of diacetyl annually compared 

to two employees with obstruction in companies that used less diacetyl (prevalence of 5.3% 

versus 1.2%), for an OR of 4.5 (95% CI 1.03–

19.9) [Kim et al. 2010].

In diacetyl-exposed employees with severe 

fixed airways obstruction and other findings 

of obliterative bronchiolitis, a consideration of 

alternate explanations should take into account 

the fact that while obstructive lung diseases 

such as asthma and smoking-related emphysema are common in the general population, 

severe airways obstruction is rare, especially in 

young individuals. Asthma is characterized by 

episodes of reversible airways obstruction—

some individuals with severe or inadequately 

treated asthma can develop fixed airways 

obstruction. However, asthma does not appear 

to be a possible explanation for cases of severe 

lung disease among diacetyl-exposed employees for the following reasons:

(1) Most affected employees denied having any 

pre-existing lung disease or symptoms at 

the start of exposure.

(2) Once shortness of breath developed, it did 

not improve when employees were away 

from the workplace as would be expected 

in employees with occupational asthma 

(either new onset asthma or exacerbation 

of pre-existing asthma).

(3) Employees’ illnesses did not improve when 

they took medications for asthma such as 

bronchodilators and corticosteroids.

(4) Most employees did not have a significant 

response to administration of bronchodilators in any of their spirometry tests (i.e., 

airways obstruction was fixed). 

While some diacetyl-exposed employees who 

developed severe lung disease were smokers, 

the natural history of smoking-related disease 

and the results of medical evaluations of 

affected employees make it unlikely that the 

cases of severe fixed airways obstruction 

among diacetyl-exposed employees are smoking-related. Compared to the normal decline 

in lung function that occurs with aging (FEV1

declines approximately 30 mL/year), in a subset 

of smokers lung function declines more rapidly 

(FEV1 declines on average approximately 45–70 

mL/year). An estimated 10%–15% of all smokers 

develop clinically important airflow obstruction 

[Ryu and Scanlon 2001]. Smokers who experience rapid lung function decline will typically 

start to become short of breath once their FEV1

falls below 60% of predicted; this usually occurs 
74 Occupational Exposure to Diacetyl and 2,3-Pentanedione

3 . Effects of Exposure in Employees

around age 50. Severe airways obstruction (e.g., 

FEV1 less than 40% predicted) typically does not 

occur before 55–60 years of age [Wise 2008]. 

Several diacetyl-exposed employees developed severe fixed airways obstruction while 

still in their 20s and 30s. Any smoking history 

among these affected employees (as well as in 

affected employees younger than 50) would not 

explain their severe fixed airways obstruction. 

Additional evidence against smoking as a cause 

of severe lung disease in these employees is the 

fact that most employees’ DLCO measurements 

were normal. In airways obstruction due to 

smoking-related emphysema, DLCO is reduced.

Obliterative bronchiolitis is known to occur 

as a result of a variety of infections, exposures, 

or nonpulmonary diseases. Examples include 

overexposure to highly irritating gases or 

vapors such as chlorine, ammonia, and nitrogen 

oxides or in association with connective tissue 

diseases such as systemic lupus erythematosus 

and rheumatoid arthritis, or in organ transplant 

recipients. The diacetyl-exposed employees who 

developed severe fixed airways obstruction 

did not have histories or medical evaluation 

findings to suggest that they had developed 

obliterative bronchiolitis from another exposure or medical condition. Airways obstruction 

can also occur due to diseases that affect other 

airways besides the bronchioles such as bronchiectasis or upper airway lesions [Ryu and 

Scanlon 2001]. However, individuals with 

airways obstruction from such other causes 

typically have characteristic history, physical 

exam, and medical test findings that usually 

serve to reveal the nature of the illness (e.g., 

copious sputum in someone with bronchiectasis or evidence of upper airway obstruction on 

spirometry). Such findings were not apparent 

in diacetyl-exposed employees who developed 

severe fixed airways obstruction.

Investigations of severe lung disease consistent 

with obliterative bronchiolitis among diacetylexposed employees have provided substantial 

evidence of a causal relationship between diacetyl exposure and development of this disease. 

Exposure preceded disease development, and 

lung disease risk decreased with control of 

exposures. Analyses of data from workplace 

medical and environmental surveys revealed 

a strong, consistent association of the disease 

with diacetyl manufacture, use of diacetyl in 

flavoring production, and use of diacetyl-containing butter flavorings in microwave popcorn 

production. The investigations have also shown 

evidence of a dose-response effect, and animal 

and other laboratory studies have provided 

evidence of biologic plausibility. Medical evaluations of affected employees did not identify 

alternative explanations for their illness besides 

their workplace exposure to diacetyl and other 

flavoring compounds. Accordingly, the criteria 

for interpreting epidemiologic associations as 

causal have all been met by the body of investigation presented in this criteria document for a 

recommended standard.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 75

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Centers for Disease Control and Prevention, National 

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HETA Report No. 2008–0155–3131.

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Boylstein R, Stanton M, Piacitelli C. Morgantown, WV: 

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for Occupational Safety and Health, NIOSH HETA No. 

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Casaburi R, Coates A, van der Grinten CP, Gustafsson 

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Eschenbacher WL, Von Essen SG, Sigsgaard T, Weissman 

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Ronk CJ, Hollins DM, Jacobsen MJ, Galbraith DA, 

Paustenbach DJ [2013]. Evaluation of pulmonary function within a cohort of flavorings workers. Inhalation 

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COPD, asthma, and many imitators. Mayo Clin Proc 

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 79

4 Toxicology of Diacetyl and 

2,3-Pentanedione

4.1 Chemistry 

and Metabolism

Chemical and physical properties of diacetyl and 2,3-pentanedione are presented in 

section 1.4. As mentioned there, diacetyl is 

an α-dicarbonyl. Endogenous α-dicarbonyl 

compounds are among the reactive carbonyl species implicated in the formation of 

advanced glycation end products [Nakagawa 

et al. 2002a; Wondrak et al. 2002]. Like other 

α-dicarbonyl compounds, diacetyl is reactive, 

with a tendency to cause protein cross-links 

[Miller and Gerrard 2005]. The reactivity of 

the α-dicarbonyl compounds is enhanced by 

electron attracting groups and decreased by 

electron donors [Roberts et al. 1999]. Thus, 

diacetyl is a reactive compound, but its alkyl 

components are electron donors that may 

somewhat decrease the reactivity of the adjacent carbonyl groups [Roberts et al. 1999]. 

Diacetyl and related α-dicarbonyl compounds 

can inactivate proteins, principally through 

reactions with the amino acid, arginine [Epperly 

and Dekker 1989; Mathews et al. 2010; Saraiva 

et al. 2006]. The related α-dicarbonyl flavoring, 2,3-pentanedione, has been reported to be 

even more reactive with arginine groups than 

diacetyl [Epperly and Dekker 1989]. Recently, 

in vitro studies indicate that diacetyl can cause 

β-amyloid aggregates and covalently modify 

β-amyloid at 5th arginine (Arg5), although the 

in vivo relevance of this finding remains to be 

investigated [More et al. 2012b]. Thus, existing 

studies indicate that diacetyl is a reactive compound that can modify proteins and suggest 

that diacetyl-associated protein modification 

may occur in vivo.

4.1.1 Diacetyl and 2,3-Pentanedione 

in Food

Diacetyl and 2,3-pentanedione have a long 

history as components of food, suggesting 

that exposures can occur in diverse workplaces. They occur as natural products in 

many foods [Jiang et al. 2013; Majcher and 

Jelen 2005; Majcher et al. 2013; RinconDelgadillo et al. 2012; Santos et al. 2013]. 

Diacetyl imparts the flavor and aroma of 

butter to many common foods and drinks 

including butter, cheese, yogurt, beer, and 

wine [Jang et al. 2013; Rincon-Delgadillo et 

al. 2012]. Diacetyl in food plays an important 

role in food preference [Liggett et al. 2008]. 

While less extensively studied in foods than 

diacetyl, 2,3-pentanedione is a common flavoring in margarine and vegetable spreads 

[Rincon-Delgadillo et al. 2012]. Roasted coffee 

also contains appreciable amounts of diacetyl 

[CDC 2013; Daglia et al. 2007a; Daglia et al. 

2007b]. Because diacetyl is not a component of 

green coffee beans, it appears to be a product 

of the roasting process [Daglia et al. 2007a]. 

Bacteria and yeast produce diacetyl during 

fermentation [Chuang and Collins 1968]. It 

can be produced by metabolism of an acetaldehyde-thiamine pyrophosphate complex in 

the presence of acetyl-coenzyme A [Speckman 

and Collins 1968]. Microbes can also produce 

diacetyl from pyruvate in the presence of acetyl-coenzyme A [Chuang and Collins 1968]. 
80 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

Microbial culture conditions, such as pH, can 

influence the relative amount of diacetyl produced during fermentation [Garcia-Quintans 

et al. 2008; St-Gelais et al. 2009]. In addition, 

the steam distillate of several bacterial cultures 

grown on skim milk is known as “starter distillate” and is also considered a flavoring [FASEB 

1980; FDA 1983]. Major components of some 

starter distillate samples include diacetyl and 

acetic acid [FASEB 1980; Rincon-Delgadillo 

et al. 2012]. A recent study demonstrated 

that diacetyl remains a frequent component 

of commercial starter distillate samples, and 

diacetyl concentrations exceeded 20 mg/g in 

one sample [Rincon-Delgadillo et al. 2012]. 

Starter distillate can also contain 2,3-pentanedione as well as butyric acid, which inhibits 

the metabolism of diacetyl and 2,3-pentanedione [Morris and Hubbs 2009; Nakagawa 

et al. 2002a; Rincon-Delgadillo et al. 2012]. 

Thus, diacetyl and 2,3-penanedione can occur 

naturally in food, may be added to food as flavorings, may be produced during the roasting 

process, and can be anticipated components 

when starter distillate is added as a flavoring. 

4.1.2 Metabolism in Mammalian Cells

In the rat and hamster liver, diacetyl is metabolized principally by reduction to acetoin in an 

enzymatic reaction catalyzed by dicarbonyl/Lxylulose reductase (DCXR), the enzyme is 

also known as diacetyl reductase, with either 

nicotinamide adenine dinucleotide (NADH) 

or nicotinamide adenine dinucleotide phosphate (NADPH) as coenzymes [Nakagawa 

et al. 2002a; Otsuka et al. 1996; Sawada et 

al. 1985]. Acetoin can be further reduced 

to 2,3-butanediol in an NADH-dependent 

manner [Otsuka et al. 1996]. This diacetyl 

reductase activity is higher in the liver than 

in the kidney, and kidney activity is higher 

than in the brain. However, after oral administration of diacetyl, the levels of acetoin are 

much higher in the brain than in the kidney 

or liver. 2,3-Butanediol accumulates in liver, 

kidney, and brain after the administration 

of diacetyl, acetoin, or 2,3-butanediol. Oral 

administration of acetoin and, to a lesser 

extent, 2,3-butanediol, in rats also causes 

diacetyl accumulation in the liver and brain 

[Otsuka et al. 1996]. However, liver homogenates do not produce significant diacetyl 

from acetoin or 2,3-butanediol [Otsuka et 

al. 1996]. Thus, the metabolic interconversion of the 4-carbon compounds, acetoin, 

diacetyl, and 2,3-butanediol occurs in mammalian systems in vivo and in vitro but the 

full spectrum of metabolic pathways remains 

incompletely investigated.

As mentioned above, in mammalian cells, the 

predominant metabolic pathway for diacetyl is 

catalyzed by DCXR, a tetrameric protein that 

is comprised of four subunits, each 244 amino 

acids long [El-Kabbani et al. 2005; Ishikura et 

al. 2001; Nakagawa et al. 2002a; Sawada et al. 

1985]. In addition to the reductive metabolism 

of diacetyl, DCXR catalyzes the metabolism of 

several other dicarbonyl compounds, including 

2,3-pentanedione, 2,3-hexanedione, 2,3-heptanedione, and 3,4-hexanedione [Nakagawa 

et al. 2002a]. In addition, DCXR catalyzes the 

reductive metabolism of a number of monosaccharides, including L-xylulose, and plays 

a role in the glucuronic acid/uronate cycle of 

glucose metabolism as well as the metabolism of carbonyl compounds [Carbone et al. 

2005; El-Kabbani et al. 2005; Nakagawa et al. 

2002a]. Inhibitors of DCXR are well described 

and include n-butyric acid, 2-furoic acid, 

benzoic acid, and nicotinic acid [Carbone et 

al. 2005; Nakagawa et al. 2002a]. At least one 

of these DCXR inhibitors, n-butyric acid, is 

well absorbed in the nose, and its presence in 

vapor mixtures causes small but significant 

decreases in diacetyl absorption in the nasal 

mucosa and, thereby, leaves more diacetyl 

in the vapor stream of the nasal airways for 

delivery to the lung [Morris and Hubbs 2009]. 
See footnotes at end of table. (Continued)

Occupational Exposure to Diacetyl and 2,3-Pentanedione 81

4 . Toxicology of Diacetyl and 2,3-Pentanedione

Table 4-1. Experimental respiratory toxicology studies

Reference Test subject Exposure Effects of exposure

Fedan et al. [2006] In vitro preparations of guinea 

pig trachea

Diacetyl1 mM3 mM10 mM30 mM

Direct effect of diacetyl:Very weak tracheal contractions with threshold at 1 mM, 

relaxation at exposures above 3 mM.

Diacetyl effect on response to intraluminal (mucosal) 

methacholine:

4-hour perfusion with 3 mM diacetyl increased methacholine 

reactivity 10 times; 10 mM diacetyl completely inhibited the methacholine response.

Depolarization of transepithelial potential difference at 3 and 

10 mM.

Decrease in transepithelial potential difference at 10 mM.

Gloede et al. 

[2011]

F344 rats Respiratory uptake of diacetyl in F344 rats was used 

to validate a model of respiratory tract uptake of diacetyl

At a given inhaled diacetyl dose, the predicted dose to the 

bronchiolar epithelium of a lightly exercising employee is predicted to be more than 40 times the dose to the bronchiole of a rat.

Describes a low affinity, high capacity and a high affinity, low 

capacity pathway for diacetyl metabolism in the rat respiratory epithelium. The high affinity pathway is inhibited by sodium benzoate, indicating that it is DCXR.

[Goravanahally et 

al. 2014]

Male SpragueDaley Rats 6-hr diacetyl inhalation with sacrifice 18–20 hr post-exposure

0 ppm25 ppm249 ppm346 ppm

Diacetyl increased substance P positive neurons in the jugular 

ganglia in a dose-dependent manner. 

PGP9.5 nerve fiber density was decreased in foci with denuded 

tracheal epithelium after inhaling 249 or 346 ppm diacetyl. Substance P-positive nerve fiber density was increased in foci of intact epithelium adjacent to denuded epithelium.

Hubbs et al. [2002] Male SpragueDawley rats Control (n=16) 0 ppm

Diacetyl exposures were to vapors of a complex 

mixture of diacetyl-containing butter flavoring

Exposure for 6 hr:Low constant exposure (n=6)203 ppm of the diacetyl component

Airway epithelial damage with necrosis and inflammation in 

nose
See footnotes at end of table. (Continued)

82 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

Reference Test subject Exposure Effects of exposure

Middle constant exposure (n=3)285 ppm of the diacetyl component

Airway epithelial damage with necrosis and inflammation in 

nose and lungs (bronchi)

High constant exposure (n=3)352 ppm of the diacetyl component

Airway epithelial damage with necrosis and inflammation in 

nose and lungs (bronchi) 

High pulsed exposure (n=3)371 ppm (range 72–940) of the diacetyl component

Airway epithelial damage with necrosis and inflammation in 

nose and lungs (bronchi and bronchioles) 

Hubbs et al. [2008] Male SpragueDawley rats Control (n=18) 0 ppm Diacetyl inhalation for 6 hr:

99.3 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

the nose (1/6)

198.4 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

nose (6/6)

294.6 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

nose (6/6) and bronchi (2/6)

Four ~15 min diacetyl inhalation pulses in 6-hr 

(TWA):

122 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

nose (2/6)

225 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

nose (6/6) and trachea (2/6)

365 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

the nose (6/6), trachea (6/6) and bronchi (6/6)

Continuous diacetyl inhalation exposure for 6 hr to 

match pulse TWA:

120 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

nose (5/6) and bronchus (1/6)

224 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

nose (6/6), trachea (5/6) and bronchus (1/6)

356 ppm (n=6) Airway epithelial damage with necrosis and inflammation in 

nose (6/6), trachea (6/6) and bronchi

Table 4-1 (Continued). Experimental respiratory toxicology studies
See footnotes at end of table. (Continued)

Occupational Exposure to Diacetyl and 2,3-Pentanedione 83

4 . Toxicology of Diacetyl and 2,3-Pentanedione

Reference Test subject Exposure Effects of exposure

Single ~15 min pulse exposure1949 ppm (92.9 6 hr TWA) (n=6)

Airway epithelial damage with necrosis and/or inflammation 

in nose (3/6)

Hubbs et al. [2012] Male SpragueDawley rats 6-hr inhalation exposures to air, 2,3-pentanedione (PD) or diacetyl; sacrifice 18–20 hr after exposure. 

Six exposure groups:Air111.7 ± 0.12 ppm PD241.2 ± 0.15 ppm PD318.4 ± 0.17 ppm PD354.2 ± 0.20 ppm PD240.3 ± 0.26 ppm diacetyl

Apoptosis and necrosis of respiratory and transitional 

epithelium of the nose in all 2,3-pentanedione- and diacetylexposed rats; necrosis of olfactory neuroepithelium and necrotizing tracheitis in diacetyl exposed rats and rats inhaling ≥ 241.2 ppm 2,3-pentanedione; necrotizing bronchitis in rats inhaling ≥ 318.4 ppm 2,3-pentanedione

6-hr inhalation exposures to air or 319 (317.9 to 

318.9) ppm PD. Air exposed rats sacrificed at 18 hr post-exposure; 2,3-pentanedione exposed rats were sacrificed at 3 different post-exposure time points: 

0–2 hr post-exposure12–14 hr post-exposure18–20 hr post-exposure

In the proximal nose, severity of damage increased between 

the 0–2 hr post-exposure and 12–14 hr post-exposure, suggesting delayed toxicity; disorganization of the neuroepithelium with a loss of the organized expression of DCXR at the air/olfactory neuroepithelium interface; activation of caspase-3 in olfactory axons

Larsen et al. 

[2009]

Male BALB/cJ mice Diacetyl inhalation exposures to 191, 334, 79, 

or 1154 ppm

Pulmonary irritation at 790 and 1154 ppm, decrease in respiratory rate, increase in “time of break”

Challenge after acute exposure555 ppm

Higher diacetyl concentrations at the acute exposure reduced 

sensitivity to challenge exposure, lower acute exposure increased sensitivity; challenge exposure did not alter lung inflammation

Morgan et al. 

[2008]

Male C57BI/6 mice Subacute diacetyl inhalation, whole body 6 hr/day, 

5 days

0 ppm200 ppm Necrosis and inflammation in mucosa of the nose and larynx

400 ppm Necrosis and inflammation in the mucosa of the nose, larynx 

and bronchi

Table 4-1 (Continued). Experimental respiratory toxicology studies
See footnotes at end of table. (Continued)

84 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

Reference Test subject Exposure Effects of exposure

Intermittent diacetyl inhalation, nose only0, 100, 200, or 400 ppm1 hr/day, 5 days/week, 2 weeks

Chronic-active inflammation in the nose of all diacetyl 

exposed mice (100, 200 and 400 ppm), squamous metaplasia of respiratory epithelium of the nose (1/5 at both 100 and 200 ppm); 4/4 at 400 ppm; necrosis and ulceration of respiratory epithelium of the nose at 400 ppm (3/4); atrophy of olfactory epithelium of the nose (1/4 at 100 ppm and 2/2 at 400 ppm; lymphocyte infiltrates around bronchi in the lung (4/5 at 100 ppm; 5/5 at 200 and 400 ppm)

0, 100, 200, or 400 ppm1 hr/day, 5 days/week, 4 weeks

Chronic-active inflammation in the nose (2/5 at 100 ppm; 5/5 

at 200 and 400 ppm); squamous metaplasia of the respiratory epithelium of the nose (1/5 at 100 ppm; 5/5 at 200 and 400 ppm). Atrophy of olfactory epithelium (1/5 at 200 ppm; 5/5 at 400 ppm). Lymphocytic infiltrates around bronchi (2/3 at 100 ppm; 5/5 at 200 and 400 ppm).

0 or 1200 ppm15 min twice a day, 5 days/week, 4 weeks

Chronic-active inflammation in the nose (5/5); necrosis and 

ulceration of the respiratory epithelium of the nose (3/5); squamous metaplasia of the respiratory epithelium of the nose (5/5); atrophy of the olfactory epithelium (4/5); both peribronchial and peribronchiolar lymphocytic infiltrates (5/5)

Subchronic inhalation, whole body0, 25, 50, or 100 ppm 

6 hr/day, 5 days/week,6 weeks

Necrosis and ulceration of the respiratory epithelium of the 

nose (2/5 at 50 ppm and 5/5 at 100 ppm); squamous metaplasia of the respiratory epithelium of the nose (1/4 at 25 ppm; 3/5 at 50 ppm; 5/5 at 100 ppm); atrophy of the olfactory epithelium (3/5 at 50 and 1/5 at 100 ppm); peribronchial lymphocytic inflammation (3/5 at 25 ppm; 5/5 at 50 ppm; 5/5 at 100 ppm); denudation and atrophy of bronchial epithelium (5/5 at 100 ppm).

Table 4-1 (Continued). Experimental respiratory toxicology studies
See footnotes at end of table. (Continued)

Occupational Exposure to Diacetyl and 2,3-Pentanedione 85

4 . Toxicology of Diacetyl and 2,3-Pentanedione

Reference Test subject Exposure Effects of exposure

0, 25, 50, or 100 ppm 

6 hr/day, 5 days/week,12 weeks

Necrosis and ulceration of the respiratory epithelium of the 

nose (1/5 at 50 ppm and 5/5 at 100 ppm); squamous metaplasia of the respiratory epithelium of the nose (2/5 at 25 ppm; 4/5 at 50 ppm; 5/5 at 100 ppm); atrophy of the olfactory epithelium (5/5 at 100 ppm); peribronchial lymphocytic infiltrates (2/5 at 25 ppm; 4/5 at 50 ppm; 5/5 at 100 ppm); denudation and attenuation of bronchial epithelium (5/5 at 100 ppm)

Oropharyngeal aspiration of diacetyl0, 100, 200, or 400 mg/kg, single dose4 days post-aspiration

Foci of fibrohistiocytic proliferation with little or no inflammation present at the junction of the terminal bronchioles and 

alveolar ducts (400 mg/kg)

Morgan et al. 

[2012]

Male and female 

Wistar-Han rats and B6C3F1 mice

2,3-Pentanedione0, 50, 100 or 200 ppm6 hr/day, 5 days/week,Up to 12 exposures

In rats, 12-day 2,3-pentanedione exposure cause necrosis of 

respiratory epithelium and atrophy of olfactory neuroepithelium in the nose; intramural and intraluminal fibrosis of intrapulmonary airways. 

In rats, 5 or 10 days of inhaling 200 ppm 2,3-pentanedione 

increased bronchoalveolar lavage fluid concentrations of monocyte chemotactic protein-1, monocyte chemotactic protein-3, C-reactive protein, fibroblast growth factor-9, and fibrinogen.

In mice, 12-day 2,3-pentanedione exposure caused necrosis 

of nasal turbinates, suppurative exudate and atrophy of olfactory neuroepithelium in the nose; inflammation in intrapulmonary airways with necrosis, ulceration and regeneration at 200 ppm exposure. 

Morris and Hubbs 

[2009]

Male SpragueDawley rats Diacetyl 100 or 300 ppm in airstream flows of 

100–400 mL/min

A hybrid computational fluid dynamic-physiologically based 

pharmacokinetic model (CFD-PBPK) was used to extrapolate diacetyl and butyric acid uptake in rat airways epithelium to human airways epithelium. The CFD-PBPK suggests that nasal injury in rats can predict a risk to deep lung tissue in humans. Diacetyl damages airway epithelium when it reaches a critical concentration in the target cells; the CFD-PBPK indicates that more diacetyl will be absorbed in the nose of rats than in humans. Butyric acid is shown to shift diacetyl absorption from the nose and trachea into deeper lung tissue.

Table 4-1 (Continued). Experimental respiratory toxicology studies
86 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

Reference Test subject Exposure Effects of exposure

Palmer et al. 

[2011]

Male SpragueDawley Rats 200 μl of a 188 mg/mL diacetyl solution by intratra- cheal instillation (125 mg/kg) Initial airway epithelial injury was followed by the develop- ment of obliterative bronchiolitis

Zaccone et al. 

[2013]

In vitro preparation 

of rat trachea after exposure of animals

Diacetyl6-hr inhalation exposures to 100, 200, 300, or 

360 ppm

(n=6–11)

Small increase in basal airway compliance at 360 ppmIncrease in reactivity at 320 and 360 ppm

2,3-Pentanedione6-hr inhalation exposures to 120, 240, 320, or 

360 ppm

(n=5–8)

No effect on basal airway resistance or complianceIncrease in reactivity at 320 and 360 ppm

In vivo exposure 

of male SpragueDawley rats

Diacetyl6-hr inhalation exposures to 100, 200, 300, or 

360 ppm

Decrease in reactivity at 360 ppm

2,3-Pentanedione6-hr inhalation exposures to 120, 240, 320, or 

360 ppm

(n=4–10)

Decrease in reactivity at 240 and 320 ppm

Table 4-1 (Continued). Experimental respiratory toxicology studies
Occupational Exposure to Diacetyl and 2,3-Pentanedione 87

4 . Toxicology of Diacetyl and 2,3-Pentanedione

In the rat kidney, DCXR is localized in 

the distal tubules and collecting ducts and 

colocalizes with carboxylmethyllysine, an 

advanced glycation end product [Nakagawa 

et al. 2002a]. In the mouse kidney, DCXR is 

localized to the brush border of the proximal 

renal tubules [Nakagawa et al. 2002a]. In the 

human prostate epithelial cells and in normal 

human skin, DCXR is associated with the cell 

membrane [Cho-Vega et al. 2007a, b; ChoVega et al. 2007b]. In human skin, DCXR is 

located near the adhesion molecules, e-cadherin and β-catenin [Cho-Vega et al. 2007b]. 

Similarly, in the vascular endothelium in 

the dermis, DCXR localizes near intercellular junctions, suggesting a potential role 

for DCXR in cell adhesion [Cho-Vega et al. 

2007b]. In addition, DCXR activity is present 

in the respiratory mucosa of the rat, with the 

highest activity in the olfactory epithelium 

[Morris and Hubbs 2009]. DCXR knockout 

mice are not well characterized phenotypically 

but are reported to be fertile [Nakagawa et al. 

2002b]. People without DCXR excrete pentose 

in their urine but are otherwise believed to be 

healthy, indicating that DCXR and the major 

diacetyl metabolic pathway are not essential 

for life [Flynn 1955; Lane and Jenkins 1985]. 

Importantly, the metabolism of diacetyl is not 

exclusively by DCXR. For example, aldo-keto 

reductase 1C15 is a newly-described aldoketo reductase expressed in rat lung that can 

metabolize α-diketones [Endo et al. 2007]. 

Recently, a low affinity, high capacity and a 

high affinity, low capacity pathway for diacetyl 

metabolism have been described in the respiratory tract of the rat [Gloede et al. 2011]. The 

high affinity pathway was inhibited by sodium 

benzoate indicating that it is DCXR. The low 

affinity pathway is not believed to play a major 

role at diacetyl concentrations associated with 

most occupational exposures.

4.2 In Vivo and in Vitro 

Toxicology Studies

Diacetyl and 2,3-pentanedione may be consumed in food, the vapors may be inhaled, and 

direct skin contact is possible. In vivo studies 

have modeled these routes of human exposure.

Table 4-1 summarizes key respiratory toxicology findings for diacetyl and 2,3-pentanedione 

through 2013. Studies of acute oral toxicity 

have used gavage exposures in rats. Based upon 

gavage administration of a 20% diacetyl solution in water, the LD50 for a single oral dose of 

diacetyl is estimated to be 3 g/kg in female rats 

and 3.4 g/kg in male rats [Colley et al. 1969].

4.2.1 In Vivo Toxicology of Orally 

Administered Diacetyl

Subchronic (90-day) gavage administration 

of 540 mg diacetyl/kg/day caused multiple 

changes in exposed rats, including decreased 

body weight, increased water consumption, 

increased adrenal weight, increased relative 

kidney and liver weights (in females absolute 

kidney and liver weights were also increased), 

decreased blood hemoglobin concentration 

and gastric ulceration [Colley et al. 1969]. No 

adverse effects were noted at the next highest 

dose level, which was 90 mg/kg/day. On a mg/

kg basis, the 90 mg/kg/level was estimated to 

be roughly 500-fold greater than the estimated 

human maximum daily intake of diacetyl from 

foods consumed at that time, with 50 ppm 

diacetyl being the highest estimated concentration in any food [Colley et al. 1969].

4.2.2 Effects of Topically Applied 

Diacetyl and 2,3-Pentanedione 

in Vivo

Sensitization following topical application of 

diacetyl is predicted on the basis of results of a 

murine local lymph node assay [Anderson et al. 

2013; Anderson et al. 2007; Roberts et al. 1999]. 
88 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

On the basis of the results of the local lymph 

node assay and immune cell phenotyping, it 

is suggested that diacetyl and 2,3-pentanedione function as T-cell mediated chemical 

sensitizers [Anderson et al. 2013]. Cutaneous 

sensitization by diacetyl and 2,3-pentanedione may be initiated through haptenation with 

proteins containing the amino acids lysine and 

arginine [Roberts et al. 1999]. Diacetyl is corrosive to the cornea of the eye using the Draise 

test [Sugai et al. 1990]. 

4.2.3 Toxicology of Inhaled Diacetyl 

in Vivo

In rats, acute exposures to diacetyl or diacetylcontaining butter flavoring vapors cause necrosis 

in the epithelial lining of nasal and pulmonary 

airways. Rats inhaling vapors of butter flavoring that contained diacetyl developed multifocal 

necrotizing bronchitis one day after a 6-hour 

exposure. The mainstem bronchus was the most 

affected intrapulmonary airway. However, nasal 

airways were more affected than intrapulmonary 

airways. Necrosuppurative rhinitis was seen in 

rats inhaling butter flavoring vapors at concentrations of butter flavoring that did not cause 

damage in intrapulmonary airways [Hubbs 

et al. 2002]. As a single agent acute exposure 

in rats, a 6-hour diacetyl inhalation exposure 

caused epithelial necrosis and inflammation in 

bronchi at concentrations of > 294.6 ppm and 

caused epithelial necrosis and inflammation in 

the trachea and larynx at concentrations of ≥224 

ppm [Hubbs et al. 2008]. The airway epithelial 

damage in rats inhaling 356 ppm was remarkable, with an average pathology score of 9.5 on 

a 10-point scale in the nasopharyngeal duct and 

larynx, while damage in the tracheal epithelium 

averaged 8.7 on a 10-point scale. In a pattern 

reminiscent of airway damage from butter flavoring vapors, diacetyl causes greater damage to 

nasal airways than to intrapulmonary airways 

[Hubbs et al. 2008]. The data from the National 

Toxicology Program 90-day inhalation study are 

available online and was used for the NIOSH 

animal-based risk assessment (see Chapter 6). 

Airway damage in mice one day after diacetyl 

inhalation was found to correlate with markers 

of increased protein turnover, implicating 

protein damage in the etiology of diacetylinduced airway damage [Hubbs et al. 2016].

Eighteen hours after a 6-hour exposure to 

inhaled diacetyl (100, 200, 300, or 360 ppm), in 

anesthetized rats 360 ppm elevated slightly lung 

resistance and dynamic compliance [Zaccone et 

al. 2013]. Subsequent inhalation of methacholine 

aerosol (0.3–10 mg/mL) revealed that airway 

reactivity was decreased after exposure to diacetyl at 360 ppm. It had been predicted, based on 

extensive epithelial damage noted after diacetyl 

inhalation, that reactivity to inhaled methacholine would be increased. Eighteen hours after a 

6-hour exposure to inhaled 2,3-pentanedione 

(120, 240, 300, or 360 ppm), in anesthetized 

rats basal lung resistance (RL) and dynamic lung 

compliance (Cdyn) were not affected.

Following inhalation of 346 ppm diacetyl by 

rats, foci in the trachea that demonstrate epithelial denudation also appear to have loss 

of sensory nerves, as reflected in a decreased 

density of PGP9.5-immunoreactive nerve fibers. 

However, in the epithelium adjacent to denuded 

foci, increased numbers of nerve fibers contain 

immunoreactive substance P, a neuropeptide 

important in neurogenic airway inflammation. 

In addition, the number of substance P immunoreactive neurons increase in the ganglia 

supplying the trachea in a dose-dependent 

manner in exposed rats. These findings suggest 

that diacetyl-induced airway epithelial damage 

may be accompanied by changes in the sensory 

nerves [Goravanahally et al. 2014]. In mice, 

inhaling diacetyl at concentrations of 200 or 

400 ppm for 6 hours/day for up to 5 days causes 

respiratory tract changes similar to those seen in 

rats inhaling diacetyl or butter flavoring vapors 

[Morgan et al. 2008]. At both 200 and 400 ppm, 

diacetyl caused necrotizing rhinitis in mice that 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 89

4 . Toxicology of Diacetyl and 2,3-Pentanedione

was most prominent in the front portion of the 

nose. At 400, but not at 200 ppm, the olfactory 

epithelium demonstrated vacuolar degeneration and apoptosis. Necrotizing laryngitis was 

consistently observed in all mice inhaling 400 

ppm diacetyl, while only one mouse inhaling 

200 ppm diacetyl had comparable necrotizing 

laryngitis, but erosive laryngitis was present in 9 

of 10 mice inhaling the 200 ppm concentration.

Exposing mice to diacetyl for only 1 hour/day at 

100, 200, or 400 ppm diacetyl, 5 days per week, 

for 2 to 4 weeks rather than 6 hours/day for 

the same number of days eliminated epithelial 

necrosis in mice inhaling 200 ppm diacetyl and 

decreased the severity of epithelial necrosis in 

mice inhaling 400 ppm diacetyl. Lymphocytic 

inflammation was seen around the bronchi in 

some mice inhaling 100 ppm and in all mice 

inhaling 200 or 400 ppm diacetyl [Morgan et al. 

2008]. Exposing mice to diacetyl for 15 minutes 

per day at 1,200 ppm diacetyl, 5 days/week for 

2 weeks also caused lymphocytic infiltrates 

around bronchi, and lymphocytic infiltrates 

extended deeper into the lung, reaching the 

level of the preterminal bronchioles [Morgan et 

al. 2008].

Subchronic, 12-week, diacetyl inhalation for 6 

hours/day, 5 days/week caused significant histopathologic changes in mice at all concentrations 

studied. Peribronchial lymphocytic infiltrates 

were seen at terminal sacrifice at 12 weeks in all 

subchronically-exposed mice inhaling 100 ppm 

diacetyl and in some mice inhaling 25 or 50 ppm 

diacetyl. In mice inhaling 100 ppm diacetyl, 

bronchial epithelial changes included denudation, attenuation, and hyperplasia [Morgan et al. 

2008]. Chronic active nasal inflammation was 

seen in all mice inhaling 50 or 100 ppm and in 

four of five mice inhaling 25 ppm diacetyl for 12 

weeks, an exposure that also caused minimal to 

mild lymphocytic bronchitis in two of five mice. 

This suggests that the no observable adverse 

effect level in mice for subchronic inhalation 

may be less than 25 ppm diacetyl. 

Butyric acid caused a small but significant 

reduction in nasal uptake of diacetyl in the rat 

nose, and, thereby, increased the diacetyl exposure to the lung due to a reduced “scrubbing” 

effect [Morris and Hubbs 2009].

Oropharyngeal aspiration permits exposures 

that bypass the rodent nose and, hence, scrubbing at that site [Foster et al. 2001; Rao et al. 

2003]. A single aspiration exposure to 400 

mg/kg diacetyl produced a fibrohistiocytic 

response at the bronchioloalveolar junction of 

mice after 4 days. While oropharyngeal aspiration of diacetyl delivers a high bolus dose of 

diacetyl, the unusual fibrohistiocytic response 

could suggest that the smallest airways may be 

particularly susceptible to diacetyl-induced epithelial injury and fibrosis [Morgan et al. 2008]. 

A subsequent study demonstrates development 

of obliterative bronchiolitis in rats after intratracheal instillation of diacetyl [Palmer et al. 

2011]. In this model, diacetyl-induced obliterative bronchiolitis was associated with abnormal 

repair of the injured bronchiolar epithelium. 

The reports of the induction of obliterative 

bronchiolitis and obliterative bronchiolitis-like 

changes in the deep lung of laboratory animals 

following aspiration of diacetyl are important 

because no prior animal model of obliterative 

bronchiolitis existed, and it is a technique that 

bypasses the rodent nose, which CFD-PBK 

models have demonstrated to absorb more 

diacetyl than will be absorbed in the upper 

airways of employees (section 4.2.6). However, 

as noted in the study, the very large single dose 

used in these studies may have limitations for 

the use of single exposure intratracheal instillations for risk assessment purposes [Palmer 

et al. 2011]. A study demonstrated bronchial 

fibrosis in rats inhaling 150 or 200 ppm diacetyl 

for 2 weeks [Morgan et al. 2016]. 

Pulmonary function changes have been investigated in mice after acute or subchronic diacetyl 

exposure. In mice, acute 2-hour diacetyl inhalation at concentrations from 191 to 1154 ppm 
90 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

caused a decrease in respiratory rate and an 

increase in the “time of break” between inhalation and exhalation, an indicator of sensory 

irritation [Larsen et al. 2009]. In addition, acute 

diacetyl inhalation in mice caused decreases in 

tidal volume and mid-expiratory flow rate. Mice 

previously exposed to high diacetyl concentrations were less sensitive to the sensory irritation 

effects of a diacetyl challenge exposure, while 

mice previously exposed to low diacetyl concentrations were more sensitive to a diacetyl 

challenge exposure. Extrapolation of the mouse 

dose-response relationship to humans suggested 

no sensory irritation to warn employees during 

acute diacetyl exposures at concentrations less 

than 20 ppm [Larsen et al. 2009]. As mentioned 

earlier, a recent study suggests that acute diacetyl 

inhalation exposures can actually increase the 

number of substance P-positive neurons in the 

jugular ganglia and the number of nerve fibers 

containing substance P in the epithelium adjacent to sites of epithelial damage, but decreases 

the sensory innervation at the actual sites of 

greatest epithelial damage [Goravanahally et 

al. 2014]. As a group, these studies suggest dysregulation of airway sensory innervation and 

responses. Additional studies support the potential for diacetyl to alter pulmonary function in 

exposed rodents. Mice inhaling 100 ppm diacetyl for 12 weeks had concentration-dependent 

decreases in respiratory rate and minute volume 

after 3 and 6 weeks of exposure; mice inhaling 

50 ppm diacetyl had decreased respiratory rates 

after 6 weeks exposure, but pulmonary function 

improved with time with continued exposure 

at these concentrations [Morgan et al. 2008]. 

However, after 18 weeks of exposure, respiratory rates in mice inhaling 25 ppm diacetyl were 

significantly lower than in controls [Morgan 

et al. 2008].

The effects of diacetyl inhalation may not be 

limited to the respiratory tract. Inhaling 2,500 

ppm diacetyl for 45 minutes increased 2-deoxyglucose uptake in foci in the posterior portion of 

the rat brain olfactory bulb [Johnson et al. 2007]. 

While this finding has generally been interpreted as being related to olfaction [Johnson 

et al. 2007], the potential exists for toxicity to 

olfactory neurons that radiate into the olfactory 

bulb. Phagocytosis of olfactory nerve material 

and increases in Tnfα mRNA were recently 

demonstrated in the olfactory bulb of mice one 

day after a 6-hr diacetyl inhalation exposure. By 

immunofluorescence, the multifunctional scaffolding protein sequestosome-1 accumulated 

in the olfactory bulb of these mice and often 

congregated in the microglial cells that contained phagocytized olfactory neuronal material 

[Hubbs et al. 2016]. 

Although powdered butter flavoring can produce 

fewer vapors than liquid butter flavorings, the 

powders have a major respirable component 

[Boylstein et al. 2006; Rigler and Longo 2010]. 

If powdered butter flavorings are substituted for 

liquid butter flavorings, diacetyl and 2,3-pentanedione vapor concentrations may well be 

below exposure limits. In particular, encapsulated flavoring powders are designed to contain 

diacetyl or 2,3-pentanedione vapors. However, 

inhalable particulates can be deposited in the 

nose, the conducting airways, and deep lung. 

No peer reviewed studies have investigated the 

potential for encapsulated flavorings to release 

diacetyl and/or 2,3-pentanedione directly to the 

target cells lining airways. However, a recent 

study indicates that more than a quarter of particulates in flavoring powders are less than 10 

μm in diameter. Therefore, powders have the 

potential to reach the intrapulmonary airways 

[Rigler and Longo 2010].

4.2.4 In Vitro Toxicology of Diacetyl 

and 2,3-Pentanedione

Diacetyl is mutagenic in the Salmonella 

typhimurium tester strains TA100 and TA104 

[Kim et al. 1987; Marnett et al. 1985]. However, 

diacetyl also reacts with mutagenic heterocyclic amines and suppresses the mutagenicity of 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 91

4 . Toxicology of Diacetyl and 2,3-Pentanedione

heterocyclic amines in Salmonella typhimurium

tester strain TA98. Diacetyl also enhances chromosome loss by proprionitrile in Saccharomyces 

cerevisiae. Recently, diacetyl in the presence 

of human S9 demonstrated a high degree of 

mutagenicity in a mouse lymphoma mutation assay [Whittaker et al. 2008]. Consistent 

with these findings, recent in vitro studies 

of direct interactions between diacetyl and 

single-stranded oligonucleotides under acellular conditions indicate that diacetyl can form 

adducts with 2-deoxyguanosine [More et al. 

2012a]. Additional studies on the genotoxicity 

of diacetyl have been reviewed in the background documents available online as part of 

the National Toxicology Program [National 

Toxicology Program 2007].

In isolated mitochondria, diacetyl closes the 

mitochondrial permeability transition pore 

and renders it insensitive to Ca2+ [Eriksson 

et al. 1998]. This effect of diacetyl occurs at 

concentrations that could occur in tissues of 

diacetyl-exposed individuals, with half-maximal inhibition of the mitochondrial transition 

reported to be at a diacetyl concentration of 

1 mM [Eriksson et al. 1998]. This concentration has been shown to have pharmacological 

activity in airways and has been modeled to be 

achieved in the airway wall after inhalation (see 

below). The effect of diacetyl on the mitochondrial permeability transition pore appears to 

be caused by arginine modification (see above) 

[Eriksson et al. 1998]. In addition, diacetyl can 

be metabolized by pig heart mitochondrial 

pyruvate kinase to form acetate and acetyl-CoA 

with a Km value of 0.46 mM. Diacetyl is also a 

competitive inhibitor of pyruvate metabolism 

by pyruvate dehydrogenase with a Ki of 0.43 

mM [Sumegi et al. 1982]. 

The isolated, perfused trachea system employing tracheas from unexposed guinea pigs has 

been used to investigate the effects of diacetyl in vitro [Fedan et al. 2006]. In this model, 

the direct, potentially toxic effects of the agent 

on epithelium may be examined. Agents such 

as diacetyl may be applied to the epithelium 

(mucosal surface) or separately to the smooth 

muscle (serosal surface) of the trachea while 

measuring contractile or relaxant responses of 

the airway smooth muscle. An advantage of this 

model is that the effects of the diketone do not 

involve an inflammatory response, inasmuch as 

the trachea has been removed from the animal 

and there is no source for the recruitment of 

inflammatory cells into the wall of the airway.

In unstimulated tracheas, diacetyl or 2,3-pentanedione applied to the mucosal surface in 

concentrations 1 mM and higher dissolved in 

a physiological salt solution elicited small contractions; in concentrations higher than 3 mM 

(i.e., 10 and 30 mM), contractions to diacetyl 

and 2,3-pentanedione were followed by relaxations. The relaxation responses were larger 

than the contractile responses. Exploring these 

phenomena further, adding the flavorings to the 

mucosa of tracheas that were first contracted 

with methacholine, a bronchoconstrictor 

agonist, resulted in full relaxation of the smooth 

muscle over the same range of diacetyl concentrations. These findings indicate that diacetyl is 

a weak contractile agent when applied to the 

epithelial surface, but that it is capable of eliciting strong relaxant responses. Thus, a diversity 

of responses in the airway that depend on the 

diacetyl concentration is produced.

Investigation of inhaled diacetyl effects (60, 

100, 200, 300, and 360 ppm) and inhaled 

2,3-pentanedione effects (120, 240, 320, and 

360 ppm) on reactivity of tracheas removed 

from exposed rats and studied in the isolated, 

perfused trachea model showed that reactivity 

to methacholine applied to the mucosal surface 

was increased slightly after inhalation of 300 

and 360 ppm diacetyl and 320 and 360 ppm 

2,3-pentanedione. Based on epithelial damage 

in airways after exposure to diacetyl, a larger 
92 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

increase in airway reactivity had been anticipated [Zaccone et al. 2013]. 

Diacetyl inhalation elicits substantial histopathologic changes to airway epithelium, 

including denudation and necrosis (section 

4.2.3). Commonly, damage to respiratory epithelium leads to airway hyperreactivity. For 

example, after ozone inhalation, airway reactivity of guinea pigs to inhaled methacholine 

is increased; likewise, reactivity to methacholine applied to the mucosa of isolated, 

perfused trachea is also increased [Fedan et 

al. 2000]. Incubation of perfused trachea with 

diacetyl dissolved in a physiological salt solution and applied to the mucosal surface led to 

no effect (1 mM diacetyl), an approximately 

10-fold increase in reactivity to methacholine 

(3 mM), or full suppression of contraction to 

methacholine (10 mM) [Fedan et al. 2006]. The 

effects of diacetyl in isolated airways from naïve 

animals does not involve the airway epithelium 

[Zaccone et al. 2013]. 

Damage to the epithelium after diacetyl inhalation suggests that epithelial ion transport and 

electrical resistance could be affected by the diketone. In rat tracheal segments investigated in 

vitro with Ussing chambers, diacetyl dissolved 

in physiological salt solution at 3 mM decreased 

transepithelial potential difference (Vt, mV), 

indicative of a decrease in electrogenic ion 

transport and/or an effect on paracellular ion 

transport involving tight junctions, whereas 10 

mM diacetyl reduced Vt further and decreased 

transepithelial resistance (Rt, Ωcm2). Rt is an 

index of tight junction permeability. Thus, ion 

transport and epithelial integrity are affected 

directly by diacetyl.

The diacetyl concentrations observed to 

affect tracheal diameter and elicit bioelectric 

responses, i.e., 1 to 3 mM, are within the range 

estimated to occur in the rat tracheal mucosa 

after diacetyl inhalation. Using a CFD-PBK 

model, Morris and Hubbs [Morris and Hubbs 

2009] calculated that inhalation levels of 100, 

200, and 300 ppm diacetyl could yield concentrations in the mucosa of 1.1 to 1.2, 2.3 to 2.5, 

and 3.7 to 3.8 mM diacetyl, respectively. This 

suggests that some or all of the observed in 

vitro effects may occur in the airways during 

vapor inhalation.

The mechanism(s) of the effects of diacetyl 

on trachea in vitro are not known at present. 

However, a related structure, 2,3-butanedione 

monixime, has been reported to inhibit contraction of smooth muscle, perhaps as a result 

of inhibiting phosphorylation of myosin light 

chains [Lizarraga et al. 1998; Siegman et al. 

1994; Stowe et al. 1997; Waurick et al. 1999]. 

Bioelectric responses of neurons also have been 

reported to be inhibited by 2,3-butanedione 

monixime [Lizarraga et al. 1998].

2,3-Pentanedione is not mutagenic in 

Salmonella typhimurium strains TA98, TA100, 

TA102, or TA104 [Aeschbacher et al. 1989; 

Marnett et al. 1985].

A recent study demonstrates that in vitro

diacetyl exposure increases shedding of the 

epidermal growth factor ligand, amphiregulin. These findings are further supported by 

evidence that amphiregulin transcripts and 

protein are also increased in an in vivo model 

of obliterative bronchiolitis induced by repeated 

intratracheal instillation of diacetyl [Kelly et 

al. 2014]. Amphiregulin has previously been 

reported to play a role in pulmonary fibrosis, 

although the nature of that role is not fully 

understood [Zhou et al. 2012].

4.2.5 Toxicology of Inhaled 

Diacetyl Substitutes 

Diacetyl is the compound largely responsible 

for the flavor of butter in butter [FASEB 1980; 

FDA 1983]. However, exposures in workplaces 

that make or use butter flavoring and emissions from heated butter flavoring involve 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 93

4 . Toxicology of Diacetyl and 2,3-Pentanedione

multiple volatile compounds [Boylstein et al. 

2006; Kullman et al. 2005]. Among the potential 

replacements for diacetyl, starter mix contains 

high concentrations of diacetyl [FASEB 1980; 

FDA 1983]. Another chemical that adds the 

flavor of butter to food is acetoin, and it was 

present along with diacetyl in many of the 

workplaces where obliterative bronchiolitis 

occurred in employees who make or use diacetyl [Kullman et al. 2005; van Rooy et al. 2007]. 

Acetoin is structurally very similar to diacetyl 

but an α-hydroxyketone in acetoin replaces 

the reactive α-diketone implicated in the toxicity of diacetyl and 2,3-pentanedione [Hubbs 

et al. 2012]. In a National Toxicology Program 

(NTP) 90-day study on acetoin, the chosen 

maximum exposure level (generally representing the maximum tolerated dose) was 800 ppm, 

whereas in the NTP 90-day diacetyl study the 

maximum exposure level was 100 ppm (Chapter 

6). In 90-day inhalation exposures, diacetyl 

produced statistically significant respiratory 

tract lesions from exposures as low as 25 ppm, 

in both rats and mice (Chapter 6). Statistically 

significant histopathology changes in the 25 

ppm diacetyl exposures were nasal respiratory 

epithelial metaplasia (in both male and female 

rats), nasal olfactory epithelial degeneration in 

female rats; nasal respiratory epithelial necrosis 

(in male and female mice) plus chronic inflammation and respiratory epithelial hyperplasia 

of the larynx in female mice (Chapter 6). In 

addition acetoin (150 ppm), after inhalation 

for 6 hours by rats, had no effect on reactivity 

to inhaled methacholine while inhaled acetic 

acid (27 ppm for 6 hours), another component 

of flavoring mixture, increased airway reactivity to methacholine [Zaccone et al. 2013]. Thus, 

current data, while limited, indicate that acetoin 

is considerably less hazardous than diacetyl.

2,3-Pentanedione is structurally very similar 

to diacetyl because it is a 5-carbon α-diketone, 

and diacetyl is a 4-carbon α-diketone. Eighteen 

hours after a 6-hour inhalation exposure to 

2,3-pentanedione (120, 240, 320, and 360 ppm), 

RL and Cdyn in anesthetized rats were unaffected 

[Zaccone et al. 2013]. Subsequently, airway 

reactivity to inhaled methacholine aerosol was 

decreased after inhalation of 120, 240, and 

320 ppm 2,3-pentanedione. 2,3-Pentanedione 

affected methacholine reactivity more than 

diacetyl. Airway hyperreactivity to methacholine had been anticipated, in view of the 

epithelium damage caused by the flavoring. 

Following inhalation exposure of rats to these 

same concentrations of 2,3-pentanedione 

and perfusion of the trachea in vitro, reactivity to mucosally-applied methacholine was 

increased by 240, 320, and 360 ppm 2,3-pentanedione [Zaccone et al. 2013]. The magnitude 

of this effect surpassed that caused by diacetyl 

inhalation. 

Morphologic data suggest that 2,3-pentanedione can cause airway epithelial damage similar 

to the damage caused by diacetyl [Hubbs et al. 

2012; Morgan et al. 2012; Morgan et al. 2016]. 

Rats repeatedly inhaling 2,3-pentanedione at 

concentrations ≥ 150 ppm for up to 2 weeks 

develop fibrosis of intrapulmonary airways, 

a morphologic change similar to obliterative 

bronchiolitis in humans [Morgan et al. 2016]. 

Recently, more than 3500 genes were found to 

be upregulated in RNA isolated from the fibrotic 

bronchi of 2,3-pentanedione exposed rats 

[Morgan et al. 2015]. Some of the up-regulated 

genes were ones previously implicated in fibrosis, including transforming growth factor-β2, 

interleukin-1α, interleukin-18, interleukin-33, 

and fibronectin. In addition, at high exposure 

concentrations, messenger RNA changes were 

noted in the brain of rats after acute 2,3-pentanedione inhalation [Hubbs et al. 2012]. 

4.2.6 Diacetyl and 2,3-Pentanedione in 

Cigarette Smoke

A recent study suggests that mainstream 

tobacco smoke collected by a smoking machine 
94 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

contained significant amounts of diacetyl that 

varied with the smoking parameters set by different organizations. The average for seven types 

of cigarettes ranged between 285 μg diacetyl/

cigarette and 42.8 μg 2,3-pentanedione/cigarette for the International Organization for 

Standardization (ISO) parameters to 778 μg 

diacetyl/cigarette and 83.5 μg 2,3-pentanedione/

cigarette by the Health Canada Intensive (HCI) 

parameters. In mainstream cigarette smoke, the 

calculated diacetyl concentration ranged from 

250 ppm with ISO parameters to 361 ppm with 

HCI parameters, while the concentration of 

2,3-pentanedione ranged from 32.2 ppm with 

ISO parameters to 50.1 ppm with HCI parameters [Pierce et al. 2014]. Unfortunately, the 

analytical technique used in the study (high performance liquid chromatography with ultraviolet 

detection analyses of 2,4-dintrophenylhydrazine 

derivatives) is not especially selective and would 

be prone to interferences in complex mixtures. 

Nevertheless, two other recent studies support 

the presence of significant diacetyl in mainstream cigarette smoke [Fujioka and Shibamoto 

2006; Polzin et al. 2007]. Other recent studies 

have identified diacetyl and 2,3-pentanedione in electronic cigarette liquids and aerosols 

[Allen et al. 2016; Farsalinos et al. 2015].

In one study, after derivatization with 1,2-

phenylenediamine, quantification by GC-NPD, 

and confirmation by GC-MS, 301–433 μg 

diacetyl/cigarette was measured in the total 

mainstream smoke withdrawn from each 

burning cigarette from 15 different commercial reference cigarettes [Fujioka and 

Shibamoto 2006]. However, that study did 

not use a smoking machine to simulate actual 

smoking behavior, so that the amount of diacetyl observed may not be representative of the 

amount produced under realistic smoking conditions. In another study, using GC/MS and a 

smoking machine with ISO parameters, a range 

of 12.7–145 μg diacetyl/cigarette was measured 

from 41 different types of cigarettes [Polzin 

et al. 2007]. Thus, several studies suggest that 

significant diacetyl is present in mainstream cigarette smoke, although the concentrations vary 

[Fujioka and Shibamoto 2006; Pierce et al. 2014; 

Polzin et al. 2007]. Finally, electronic cigarette 

liquids often contain diacetyl and 2,3-pentanedione [Allen et al. 2016; Farsalinos et al. 2015].

The Pierce et al. [2014] report is the first to calculate diacetyl concentrations in mainstream 

cigarette smoke in ppm. However, it should be 

noted that the calculated concentration in ppm 

such as the reported average concentration of 

250 ppm diacetyl using the ISO parameters is 

actually not comparable to workplace exposures [Pierce et al. 2014]. Using ISO parameters 

an average of 0.3255 liters of mainstream cigarette smoke was inhaled, but this would not be 

the only air inhaled by a smoker. Roughly 6.75 

liters per minute (L/min) of air is inhaled by 

the average person at rest, while 19.5 L/min is 

inhaled under conditions of light work [ICRP 

1975]. Assuming that all of the air an employee 

inhales contains the concentration of diacetyl 

measured in the workplace, it is critical to recognize that the mainstream smoke of a smoker 

is not the only source of air; they are also breathing air with much lower diacetyl concentrations. 

Assuming that the other air breathed by smokers 

does not contain diacetyl, the workplace equivalent exposure concentration for a smoker during 

the smoking time period is roughly 190- to 560-

fold lower than the concentration measured in 

the mainstream cigarette smoke using the ISO 

parameters of one 0.035 L puff/min (6.75/0.035 

is 193 and 19.5/.035 is 557). Using the ISO 

parameters, this would be an average equivalent 

of a workplace concentration of 0.45 to 1.3 ppm 

for diacetyl and 58 to 170 ppb for 2,3-pentanedione during the smoking process. If the smoking 

machine parameters of 9.3 minutes/cigarette 

are used to calculate the duration of exposure, a 

smoker who smokes one pack of 20 cigarettes/

day spends 186 minutes a day smoking, but the 

employee is working for 8 hours. Therefore, 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 95

4 . Toxicology of Diacetyl and 2,3-Pentanedione

the 8-hour time-weighted average equivalent 

concentration for the smoker is 2.6-fold lower 

to reflect the time period they are not exposed, 

which would be 170 to 500 ppb for diacetyl and 

22 to 65 ppb for 2,3-pentanedione, depending 

upon exercise level. 

Calculations of workplace equivalent exposures are similar using total mass of diacetyl 

and 2,3-pentanedione reported by Pierce et al. 

[Pierce et al. 2014]. The total mass of diacetyl 

and 2,3-pentanedione in an average cigarette 

measured with ISO parameters was 285 and 

42.8 μg, respectively [Pierce et al. 2014]. For 

a one pack a day smoker that results in 5,700 

μg of diacetyl and 856 μg of 2,3-pentanedione 

inhaled each day. If that mass was contained in 

the amount of air inhaled in a work day under 

light exercise conditions, at 25°C and 760 torr, 

the one pack/day smoker inhales the approximate equivalent of an occupational exposure to 

169 ppb diacetyl and 22 ppb 2,3-pentanedione 

during an 8-hour work day. 

Although it is important to recognize that breathing patterns differ between cigarette smokers 

or electronic cigarette users (“vapers”) and 

employees, which may affect the pharmacokinetics of inhaled diacetyl and 2,3-pentanedione 

[Hubbs et al. 2015], these concentrations of 

diacetyl would be predicted to decrease FEV1

in some individuals if inhaled for a working 

lifetime (see Chapter 5). Indeed, many smokers 

do demonstrate significant decreases in FEV1

[Barnes 2004; Fletcher and Peto 1977; Wright 

et al. 1987; Xu et al. 1994]. Additionally, cigarette smoking is a major risk factor for chronic 

obstructive pulmonary disease, and a decrease 

in FEV1 is a characteristic feature of this disease. 

In addition, bronchiolar fibrosis is part of the 

airway remodeling response that characterizes chronic obstructive pulmonary disease 

[Kim et al. 2008; Sohal et al. 2013]. Decreases 

in FEV1 and fibrosis of bronchioles are features 

that also characterize obliterative bronchiolitis 

[Schlesinger et al. 1998]. However, smokers with 

chronic obstructive pulmonary disease have 

additional morphologic changes in their lungs, 

including emphysema, that are not seen in obliterative bronchiolitis [Snider 2003]. While it has 

been hypothesized that the failure to diagnose 

diacetyl-induced obliterative bronchiolitis as a 

cigarette smoker-associated disease suggests that 

diacetyl does not cause obliterative bronchiolitis 

in exposed employees [Pierce et al. 2014], the 

data when corrected for the actual corresponding occupational exposure concentrations may 

instead suggest the hypothesis that diacetyl and 

related reactive carbonyl compounds in cigarettes could potentially contribute to chronic 

obstructive pulmonary disease. Because chronic 

obstructive pulmonary disease is a leading cause 

of morbidity and mortality [Halbert et al. 2006; 

Lopez et al. 2006], the role of diacetyl and other 

reactive carbonyl compounds in cigarette smoke 

in contributing to chronic obstructive pulmonary disease is a worthy topic for future studies. 

As extensively discussed in Chapter 3, airway 

obstruction and decreased FEV1 can, nevertheless, be identified in smokers who are exposed 

to diacetyl. Most importantly, because diacetyl 

causes obstructive lung disease and because 

smoking causes obstructive lung disease, the 

presence of diacetyl in cigarette smoke does not 

diminish the need to control diacetyl exposures 

in employees. In fact, it highlights the greater 

risks occurring in employees who are exposed to 

diacetyl in the workplace and who also smoke.

4.2.7 Relevance of Diacetyl Animal 

Studies to Humans

Four converging lines of evidence support 

the relevance of diacetyl inhalation studies 

in rats and mice to humans. First, diacetyl inhalation causes damage to respiratory 

epithelium in rats and mice. This finding is 

important because injury to the respiratory 

epithelium of the deep lung is the accepted 

cause for obliterative bronchiolitis. Second, 

dosimetry calculations indicate that diacetyl 
96 Occupational Exposure to Diacetyl and 2,3-Pentanedione

4 . Toxicology of Diacetyl and 2,3-Pentanedione

concentration in respiratory epithelium of the 

human deep lung under working conditions 

may be much higher than diacetyl concentrations in laboratory animals. Third, another 

organic compound, sulfur mustard, implicated 

in causing obliterative bronchiolitis in humans, 

produces a similar pattern of predominantly 

nasal injury in rats exposed by nose-only inhalation [Weber et al. 2010]. Fourth, repeated 

inhalation exposure to 2,3-pentanedione 

causes fibrosis of intrapulmonary airways 

[Morgan et al. 2012; Morgan et al. 2016]. Each 

of these findings supports the conclusion that 

with appropriate dosimetry studies, damage to 

the respiratory epithelium of the upper airways 

of rodents should be considered when evaluating risk for humans.

Animal exposure studies have revealed that the 

upper airways of rodents are sensitive to flavoring-induced toxicity, whereas the lower airways 

of humans are most affected by these agents. 

Importantly, diacetyl exposures in rodents 

caused extensive damage to the respiratory epithelium lining the nose and the trachea [Hubbs 

et al. 2008; Morgan et al. 2008]. The cell types 

that are injured in the nose and trachea in 

rodents are very similar to the respiratory epithelium lining the airways of the deep lung of 

humans that are involved pathophysiologically 

in the development of obliterative bronchiolitis [Borthwick et al. 2009; King 1989]. In 

addition, the bronchi were damaged at high 

concentrations in acute exposures and at 

lower concentrations in subchronic exposures 

in mice. Thus, inhalation toxicology studies 

showed that diacetyl could damage respiratory 

epithelium, providing biological plausibility 

for its etiologic role in obliterative bronchiolitis. Indeed, at the time of the first inhalation 

toxicology studies of diacetyl, no accepted 

cause of obliterative bronchiolitis in humans 

had been demonstrated to cause obliterative 

bronchiolitis in rodents. Recently, repeated 

inhalation exposures to 2,3-pentanedione 

have been shown to cause fibrosis of intrapulmonary airways in rats, demonstrating a 

pathologic change in the rodent model that 

is very similar to obliterative bronchiolitis in 

humans [Morgan et al. 2012]. Interpretation 

of the species difference in the anatomic location of diacetyl-induced damage to respiratory 

epithelium may be explained by species differences in respiratory tract anatomy, breathing 

patterns, and diacetyl dosimetry.

Rodents are obligate nasal breathers while 

humans are oronasal breathers. Inhaled air 

may bypass the human nose, particularly 

during exertion [Conolly et al. 2004]. In addition, the rodent nasal passageways and the 

rodent trachea are much narrower than the 

corresponding human nasal passageways and 

trachea. Small airway diameter increases the 

percentage of the airstream that is in contact 

with the mucous layer, increases resistance, and 

thereby increases mucosal absorption of watersoluble vapors [Frederick et al. 1998; Morris 

1997]. Thus, the dimensions of the rodent nose 

predict much greater absorption of diacetyl 

vapors in the rodent than in the human nose. 

However, the surface area of the airways within 

the human lung is 100 times greater than the 

surface area of airways in the rat lung [Mercer 

et al. 1994]. These anatomic differences predict 

that, at a given exposure concentration, the 

mucosa in the rodent nose receives a much 

higher diacetyl dose than does the human 

nose and that the human lung receives a much 

higher dose than the rodent lung.

To investigate these dosimetry predictions, 

a CFD-PBPK model of diacetyl uptake was 

developed [Morris and Hubbs 2009]. The 

CFD-PBPK model also predicted greater intrapulmonary diacetyl concentrations in the lung 

of humans than in rats at a given exposure concentration, especially during mouth breathing 

[Morris and Hubbs 2009]. Under resting conditions at an exposure concentration of 100 

ppm, the rat nose and trachea are predicted to 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 97

4 . Toxicology of Diacetyl and 2,3-Pentanedione

remove 39% of the inhaled diacetyl, while the 

trachea of a mouth breathing human would 

remove 4% of the inhaled diacetyl. This same 

study suggests the potential importance of 

nasal lesions in rats for predicting pulmonary 

toxicity in humans [Morris and Hubbs 2009]. 

Because the respiratory epithelium of the terminal bronchiole of humans is believed to be 

the target tissue for the development of obliterative bronchiolitis [King 1989], and because 

diacetyl doses reaching the respiratory epithelium in the nose of rodents can be similar to 

diacetyl doses reaching the respiratory epithelium of the deep lung in humans, it may be 

appropriate to consider toxicity to the respiratory epithelium lining the nose of rodents 

in evaluating the risk of diacetyl to mouthbreathing employees. In addition, butyric acid, 

which is a component of some butter flavorings, caused a small but statistically significant 

reduction in nasal uptake of diacetyl in the rat 

nose, and thereby increased the diacetyl exposure to the lung due to a reduced “scrubbing 

effect” [Morris and Hubbs 2009]. A subsequent 

CFD-PBK model indicates that with low levels 

of exercise that could occur in the workplace, 

diacetyl dose to the bronchiolar epithelium of 

humans may be more than 40-fold greater than 

the dose received by the bronchiolar epithelium 

of experimentally exposed rodents [Gloede 

et al. 2011].

Damage to the nose of rodents has recently 

been described for another agent implicated in causing obliterative bronchiolitis in 

humans, sulfur mustard [bis(2-chloroethyl)

sulfide], a chemical warfare agent. Obliterative 

bronchiolitis is considered a major cause of 

progressive respiratory disease in survivors of 

sulfur mustard exposure [Ghanei 2004a,b,c; 

Ghanei et al. 2008; Rowell et al. 2009]. Noseonly inhalation exposures of F344 rats to sulfur 

mustard caused severe mucosal damage in 

the rat nose but the changes in the lung were 

absent or minimal [Weber et al. 2010]. When 

the nose was bypassed using intubation with 

tubing lined by Teflon®, sulfur mustard did 

indeed cause necrosis of the epithelium lining 

the proximal airways [Weber et al. 2010]. This 

suggests that sulfur mustard, an accepted 

cause of obliterative bronchiolitis in humans, 

causes a similar pattern of injury to the pattern 

observed with diacetyl at different levels of the 

respiratory tract of rodents. Thus, predominantly nasal injury has been seen in rodent 

inhalation studies with organic agents implicated in causing obliterative bronchiolitis.

4.3 Conclusions

Inhalation toxicity studies in rats and mice, and 

in vitro studies in guinea pig tracheal preparations, indicate that diacetyl-containing butter 

flavoring vapors can damage airway epithelium 

and cause inflammation in the respiratory tract 

after acute or subchronic exposure. In addition, in vivo local lymph node assays indicate 

that diacetyl is a sensitizer, and in vitro studies 

indicate that diacetyl is mutagenic. Diacetyl can 

react with arginine residues causing structural 

changes in proteins that influence the function of the altered proteins. These functional 

changes in proteins include changes in enzyme 

activity and the mitochondrial permeability 

pore. Pharmacologic studies in vitro indicate 

that diacetyl can alter ion transport and reduce 

epithelial integrity. CFD-PBPK modeling indicates that diacetyl concentrations in the deep 

airways of humans may be higher than those 

in laboratory rodents, explaining the tendency 

for diacetyl-induced airway damage to be more 

anterior in the respiratory tract of rodents than 

in humans. Most recently, studies of the related 

α-diketone, 2,3-pentanedione, suggest that the 

airway toxicity of diacetyl may be shared with 

other structurally related, α-diketones, and that 

inhalation of either diacetyl and 2,3-pentanedione can cause airway fibrosis in rats. 
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Occupational Exposure to Diacetyl and 2,3-Pentanedione 99

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 105

5 Quantitative Risk Assessment Based 

on Employee Data

Taken together, the human and animal studies 

provide a compelling case for the respiratory 

toxicity of diacetyl and 2,3-pentanedione, and 

potentially other alpha diketones used in butter 

flavorings, of which diacetyl is the most thoroughly studied. The clinical experience and 

employee population studies have revealed a 

clear association between diacetyl exposure 

and diminishing respiratory capacity that has 

been shown in some cases to become manifest 

as obliterative bronchiolitis. The expanding 

animal research on diacetyl clearly describes 

pathological changes specific to this compound 

that provide an ample mechanistic basis for 

anticipating respiratory disease in humans. In 

this chapter, a risk assessment is presented that 

begins with the established premise that diacetyl causes irreversible respiratory damage. The 

analyses presented are designed specifically to 

describe that causal relationship for the purpose 

of predicting risk in working populations, not 

to prove that a causal relationship exists. Thus 

statistical significance is less important than 

insights provided into the nature of the relationship between diacetyl and diminishing 

respiratory capacity.

Other potentially reactive or toxic compounds 

can be present in association with diacetyl in 

flavoring applications, such as acetoin or acetaldehyde. An NTP 90-day study on acetoin 

is in progress [National Toxicology Program 

2013b] but the chosen maximum exposure 

level (generally representing the maximum 

tolerated dose) is 800 ppm whereas in the NTP 

90-day diacetyl study the maximum exposure 

level is 100 ppm [National Toxicology Program 

2013a]. This implies a considerably lower level 

of potency for acetoin toxicity. Furthermore, 

in the population on which the risk assessment was based, acetoin concentrations were 

an order of magnitude lower than diacetyl 

levels. Acetaldehyde is less consistently associated with diacetyl and is often below the limit 

of detection. 

The goal of this chapter is first to present a 

numerical estimate of the risk of developing 

respiratory disease due to occupational exposure to diacetyl using standard epidemiological 

methods. This estimate is based on statistical 

models that describe the relationship between 

exposure to diacetyl and the development of 

impaired lung function in a known population of exposed employees. Exposure-response 

modeling requires making assumptions about 

the exposures of the persons studied over the 

course of their working lifetime, and about the 

mathematical form of the exposure-response 

relationship. Using these models, a further 

goal was to estimate an exposure level below 

which there would be a relatively low risk. 

One approach that is used (benchmark dose) 

is to estimate what additional proportion of a 

known population would have abnormal lung 

function if their past exposure corresponded 

to a lifetime of working at some specific exposure level. Another approach estimates how 

many new cases of abnormal lung function 

would develop over a lifetime (excess lifetime 

risk) as a result of working at various exposure 

levels. Finally, the various methods are used to 

develop a range of plausible risk estimates for 

occupational exposure to diacetyl.
106 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

Although diacetyl causes obliterative bronchiolitis, a debilitating and potentially fatal 

condition, it may be associated with a spectrum of disorders. Clinical observations present 

a picture of largely obstructive disease with a 

combination of reduced FEV1 and FEV1/FVC 

ratio. However, it may also cause restrictive ventilatory impairment, characterized by reduced 

FEV1 and normal FEV1/FVC ratio [AkpinarElci et al. 2004; Kreiss 2007; Lockey et al. 2009]. 

FEV1 is the most commonly used outcome variable to assess lung function impairment caused 

by hazardous agents, regardless of the specific 

nature of impairment (obstructive or restrictive or combined). American Thoracic Society/

European Respiratory Society (ATS/ERS) recommendations are to use FEV1 to assess the 

severity of any type of spirometric abnormality 

[Pellegrino et al. 2005]. The health effects outcomes of diacetyl exposure that NIOSH used 

in this risk assessment therefore included (1) 

reductions in FEV1 (which would be seen in 

either obstruction or restriction), (2) reductions in FEV1/FVC (a measure more specific 

to obstruction), and (3) onset of cases defined 

by symptoms in employees whose FEV1 and/

or FEV1/FVC are below their lower limits of 

normal, conditions that plausibly would include 

cases of developing obliterative bronchiolitis.

5.1 Methods: Study 

Population, Exposure 

Assessment, and 

Outcomes

5.1.1 Study Population 

Six NIOSH HHEs conducted at workplaces 

producing microwave popcorn with diacetyl 

exposures were reviewed for possible use in 

risk assessment [NIOSH 2003a, b, c, 2004a, 

b, 2006]. Two of the HHEs had very small 

workforces involved in popcorn production 

(< 10 per shift) [NIOSH 2003b, c]. Four were 

determined to have the potential to provide 

sufficient work history, environmental 

assessment, and outcome information 

(pulmonary function) to support modeling 

of exposure response: Company G [NIOSH 

2006], Company L [NIOSH 2004b], Company 

K [NIOSH 2004a], and Company N [NIOSH 

2003a] (Table 5-1). In three of these HHEs a 

single episode of environmental and health 

outcomes assessment was conducted, but for 

Company G [NIOSH 2006] nine different 

surveys (eight with spirometry assessments) 

were performed, providing the possibility 

of a longitudinal analysis. With estimates of 

Table 5-1. Study populations from NIOSH health hazard evaluations

Name G K N L

Number of surveys 9* 1 1 1

Total workforce at survey 135–165 193 48 313

Workforce evaluated (%) 363†

 (73–91) 157 (81) 35 (73) 206 (66)

Date of survey Nov 2000–Jul 2003 Jul 2002 Nov 2002 Mar 2003

Start date for diacetyl use 1-Jul-1986 1-Jul-1988 1-Jul-1986 1-Jan-1994

Source: NIOSH health hazard evaluations

*Nine exposure assessments and eight medical evaluations were performed.

†

Number of unique employees evaluated one or more times
Occupational Exposure to Diacetyl and 2,3-Pentanedione 107

5 . Quantitative Risk Assessment Based on Employee Data

the diacetyl exposure response, standard risk 

assessment procedures can be applied.

5.1.2 Environmental Assessment and 

Exposure Estimation

For workplace environmental assessments, 

the HHE surveys generally collected full-shift 

personal breathing zone and area diacetyl air 

samples using NIOSH Method 2557. This sampling identified a number of air contaminants 

in addition to diacetyl and acetoin (Table 5-2), 

including acetaldehyde. Problems in diacetyl 

sample determination with NIOSH Method 

2557 related to humidity at the time of sampling and the elapsed time to sample extraction 

were subsequently uncovered. NIOSH 

researchers worked extensively to understand 

this problem and derive an appropriate correction for estimating diacetyl levels [Cox-Ganser 

et al. 2011]. This correction, based on absolute 

humidity and time to extraction, was applied 

to the diacetyl exposure levels above the limit 

of detection (LOD) as measured in the selected 

HHEs. For other chemical exposures in microwave popcorn production determined using 

NIOSH Method 2557, such as acetoin, no 

humidity/extraction correction was needed. 

For laboratory diacetyl determinations below 

the LOD, the sample value was set equal to 

LOD/2. For determinations above the LOD 

but below the limit of quantification (LOQ), 

the actual diacetyl determination (corrected) 

was used. At Company K, 44 out of 60 samples 

(personal and area) were < LOD. At Company 

L, 4 out of 125 samples were < LOD and at 

Company G, 105 out of 262 personal and 146 

out of 346 area samples were < LOD. 

The characterization of historical exposures 

was limited by the absence of air sampling prior 

to the NIOSH HHEs. In the case of Company 

L, engineering modifications had been 

implemented prior to the NIOSH exposure 

assessment, including adjustment of factory air 

pressures to reduce migration of diacetyl from 

the mixing areas. In contrast, the situation at 

Company N at the time of the assessment had 

not changed over time. Over the course of the 

nine evaluations at Company G a dramatic 

downward trend in diacetyl air concentrations 

was observed, reflecting implementation of 

NIOSH recommendations and consultations 

for controlling exposures. However, it is not 

known what changes, if any, may have occurred 

prior to the first assessment. The NIOSH exposure assessment for Company K found diacetyl 

airborne concentrations to be quite low and 

similar to Company L airborne concentrations. Company K had taken exposure control 

steps, including provision of powered, airpurifying respirators for diacetyl mixers, soon 

after the introduction of microwave production 

Table 5-2. Numbers of air samples for diacetyl and acetoin from 

health hazard evaluation environmental assessments

Company

Diacetyl Acetoin

Personal Area Personal Area

N 20 12 20 12

K 30 30 30 31

L 76 49 76 49

G 262 346 270 314
108 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

following an outbreak of eye irritation. NIOSHmeasured diacetyl exposure levels for key 

process locations showed considerable variation across the four selected HHE sites with 

higher levels at Company G and Company N 

(Table 5-3). The generally lower airborne concentrations at Company K and Company L may 

have occurred because the mixing operations at 

those two plants were isolated from the production areas unlike the situations at Companies 

G and N. 

Mean diacetyl exposures for the Company 

K, L, and N populations were calculated 

classifying by department and job (Appendix 

G, Tables G.1–G.3) based on the corrected air 

concentrations of diacetyl. The most extensive 

and representative diacetyl exposure data and 

the largest body of respiratory outcomes data 

were available from the HHE at the Company 

G microwave popcorn plant [Kullman et al. 

2005; NIOSH 2006]. This population had 

the largest number of air samples, over nine 

surveys, and based on their inquiries the HHE 

investigators determined that no significant 

control changes had been implemented prior 

to the first survey. For Company G, with 

repeated environmental assessments between 

November 2000 and July 2003 (2.7 years), to 

estimate employees’ diacetyl exposures over 

time within department/job combinations, a 

job exposure matrix (JEM) was constructed 

through collaboration between NIOSH and 

OSHA (Appendix H). Plant job titles were 

aggregated into eight exposure categories based 

on work and environmental similarities(Table 

5-4) [Corn and Esmen 1979]. Starting with the 

humidity- and time-to-extraction-corrected 

personal breathing zone sample concentrations 

(in parts per million), means were calculated 

for the cells in the JEM (Appendix G, Table 

G.4). Arithmetic means of personal samples 

are the preferred measure of central tendency 

for estimating cumulative exposure in 

Table 5-3. Arithmetic mean air concentrations (ppm) 

of diacetyl in major processes at four sites 

Company

Mixing Production Quality control Maintenance

n, nND Mean (SD) n, nND Mean (SD) n, nND Mean (SD) n, nND Mean (SD)

Personal samples

N 1,0 0.79 (0.00) 7,0 0.740 (0.640) 2,0 0.250 (0.014) 2,0 0.160 (0.066)

K 5,1 0.31 (0.41) 7,5 0.040 (0.079) 3,3 0.003 (0.001) 3,2 0.020 (0.030)

L 10,0 1.15 (0.74) 36,0 0.028 (0.016) 5,0 0.034 (0.019) 6,1 0.014 (0.008)

G 25,1 2.36 (3.92) 112,34 0.490 (0.900) 20,4 0.366 (0.390) 17,9 0.080 (0.126)

Area samples

N 2,0 1.03 (0.45) 2,0 0.620 (0.140) 2,0 0.320 (0.140) 0,0 —

K 2,1 2.42 (3.42) 7,5 0.032 (0.052) 3,3 0.002 (0.000) 2,1 0.037 (0.048)

L 6,0 1.54 (0.91) 23,0 0.028 (0.015) 6,0 0.018 (0.011) 3,0 0.019 (0.014)

G 50,1 16.8 (31.6) 123,29 1.050 (1.870) 24,10 0.253 (0.370) 17,11 0.122 (0.327)

Abbreviations: n = total number of samples, nND = number of non-detect samples, SD = standard deviation,—indicates lack of data

Note: Means are for corrected concentrations and given in parts per million (ppm); not all jobs fall within the four process 

categories displayed.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 109

5 . Quantitative Risk Assessment Based on Employee Data

chronic disease investigations [Smith 1992]. 

However, for the first industrial hygiene survey 

(November 2000), only area samples were 

collected. For this survey, personal-sample 

equivalents to the area samples were estimated 

using area and personal sampling data from 

surveys 2 and 3 for the higher-exposed jobs, 

and using other procedures for samples with 

the lower values (Appendix H, Table H.3). 

Unique exposure time periods were developed 

for each of the eight exposure categories to 

reflect impact of the exposure control changes 

implemented at the plant from November 

2000 to July 2003. Within the time periods for 

each JEM exposure category, exposures were 

assumed to be constant. Exposure estimates in 

the JEM were assigned to employees based on 

their history of jobs performed, job duration, 

and the calendar time period. For work history 

prior to the first industrial hygiene survey, 

exposure estimates from the first time period 

were used. For some employees such as those 

in the mixers exposure category, the measured 

personal diacetyl exposure was adjusted for the 

use of respirators in selected exposure periods 

(Appendix H). 

Problems in the retrospective exposure assessment for diacetyl include (1) uncertainty 

over when diacetyl was introduced and on 

the extent of its use as a flavoring component 

over time (and therefore on employee exposure levels), (2) variation in diacetyl content 

across different product lines over time, (3) 

the relative presence of diacetyl as a vapor vs. 

mist, adsorbed to powders or encapsulated, 

and (4) seasonal variation in the role of natural 

ventilation. Cumulative exposure and other 

exposure metrics were calculated starting at 

the dates when diacetyl was estimated to have 

first been used in regular production at the four 

plants: Company K (July 1, 1988), Company L 

(January 1, 1994), Company G (July 1, 1986), 

and Company N (July 1, 1986). These dates are 

uncertain, particularly for Company N.

5.1.3 Work History

The employees studied were current employees 

at time of survey except at Company G where 

some former employees were also examined. 

All results presented are for current employees 

except at Company G where, due to repeated 

pulmonary testing over months or years, initially current employees could become former 

employees at a subsequent survey. Participation 

was voluntary and generally quite high among 

current employees (66%–91%) (Table 5-1). 

Work history was routinely collected in 

HHEs by employee interview and consisted of 

Table 5-4. Exposure categories used for constructing job exposure matrix at Company G 

Exposure category Jobs included in exposure category

Warehouse Warehouse 

Maintenance Maintenance 

Outside processing/office Outside processing and office 

Polyethylene line Polyethylene packer and polyethylene stacker 

Microwave mixing Microwave mixer 

Microwave packaging line Machine operator, packer, stacker, supervisor, and inventory control

Bag print Bag print 

Quality control Quality control 
110 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

successive periods in specific department and 

job title assignments with corresponding beginning and ending dates. Gaps in employment 

were treated as unexposed and not included in 

duration-of-exposure measures.

5.1.4 Outcomes

Reported symptoms and PFT results defined 

the HHE outcomes. A medical questionnaire 

was administered that included standard ATS 

items on respiratory health [Ferris 1978] as 

well as dermal symptoms, allergies, detailed 

smoking history, and questions on other 

exposures and protective equipment used. 

Sustained-symptom onset dates were also 

collected. Spirometry testing was performed 

following ATS guidelines [Ferris 1978]. The 

predicted and lower limit of normal (LLofN) 

values for FEV1, FVC and FEV1/FVC were 

calculated using prediction equations produced 

from NHANES III [Hankinson et al. 1999]. 

The lower limit of normal has been defined by 

ATS as approximately the lower 5th percentile 

of ventilatory function within the nonsmoking 

general population classified by age, sex, race, 

and height.

For risk assessment purposes employees’ 

percent of predicted values for FEV1 (pp FEV1 ) 

and actual FEV1/FVC ratios were the outcomes 

modeled as continuous variables. In identifying 

possible developing obliterative bronchiolitis 

cases, a classification of pulmonary impairment 

was defined based on FEV1 and/or FEV1/

FVC being less than their respective LLofN. 

This discrete outcome, onset of impairment, 

was analyzed by modeling incidence rates. 

Obliterative bronchiolitis is thought of as 

largely irreversible obstruction; reversibility 

of obstructive changes was assessed in these 

HHEs using bronchodilator medication for 

individuals with FEV1/FVC and FEV1 less 

than their respective LLofNs. However, 57% 

of the cases defined using FEV1 at Company G 

were not tested for reversibility, and only one 

of the cases tested was reversible (increases 

in FEV1 of at least 200 mL and 12%). Thus 

there was a substantial residual deficit after 

bronchodilation. Therefore cases were defined 

without regard to reversibility. The classification 

of cases was not based on clinical diagnoses 

because the systematic medical data collected 

in the HHEs were limited to the questionnaire 

and spirometry tests. A complete diagnostic 

work-up of probable obliterative bronchiolitis 

cases is not routinely performed in NIOSH 

HHEs, but full disclosure of individual test 

results and recommendations for referral are 

provided to participating employees. 

5.2 Methods: Analysis of 

Exposure Response

5.2.1 Exposure Metrics

The most appropriate measure of past diacetyl 

exposure for predicting health consequences 

is not known and hence was determined by 

assessment of the statistical fit of models using 

different exposure terms. Cumulative exposure 

(time summation of concentration, cum(DA)) 

was the starting choice for exposure metric, but 

dose-rate effects were examined by calculating 

the time summation of the square root or 

square of diacetyl concentration corresponding 

respectively to diminishing and increasing 

marginal responses to increasing exposure 

intensity (dose-rate effects) as follows:

cum(DA) = Σi (DA), cum(DA0.5) = Σi

(DA0.5) , and cum(DA2.0) = Σi (DA2.0) where 

the summation was over calendar days.

Transformed cumulative exposures as the 

square root, square, or logarithm were evaluated as were duration of exposure and average 

exposure concentration (cumulative exposure/

duration of exposure). Peak exposures were not 

available from full-shift (8-hour) TWA concentrations although selected jobs had been 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 111

5 . Quantitative Risk Assessment Based on Employee Data

analyzed using a real-time method (FTIR) to 

assess time-variability. 

5.2.2 Models of Percent Predicted FEV1

and FEV1/FVC

The spirometry determinations, (1) ppFEV1

and, (2) FEV1/FVC, were analyzed as continuous 

outcomes in multiple linear regression models. 

Terms in the models included gender, ethnicity 

(Hispanic/Non-Hispanic), race (African 

American/Other), ever-smoked, pack-years 

and pack-years squared as of the date of testing. 

Pack-years squared permits some nonlinearity 

in the smoking response as might occur with 

survival or susceptibility effects. Models of 

FEV1/FVC included age (centered at 40). 

Known potential confounders were retained 

in models regardless of statistical significance 

according to good epidemiologic practice. 

Models were assessed using overall model R2

as well as the P value for exposure metric terms. 

In the case of Company G with repeated survey 

outcomes, the last recorded spirometry was 

used for analyses unless stated otherwise. In 

models of ppFEV1, the expected intercept in the 

absence of exposure or employment selection 

effects would be 100 (in nonsmokers). Models 

were fit using PROC REG in SAS 9.2 [SAS 

Institute Inc. 2008]. 

To make full use of the serial spirometry 

determinations at Company G, a longitudinal 

analysis of ppFEV1 was performed in which 

exposure metrics were calculated from time 

of first diacetyl exposure up to the time of 

each successive spirometry determination. 

This analysis included employees with two or 

more spirometry results. All employees were 

active at their first survey but could have left 

employment prior to a subsequent survey. These 

models were fit using PROC MIXED in SAS 9.2 

[SAS Institute Inc. 2008] with random effects 

permitted for individual employee’s intercepts 

and exposure responses. A second set of metrics 

was calculated with exposure cumulation 

starting at the time of an employee’s first survey 

and used in a subsidiary longitudinal analysis 

along with the full cumulative exposure metric. 

This analysis permitted a test of homogeneity, 

i.e., (1) for exposure effects before and after the 

first survey and (2) for possible survivor bias.

Pooled analyses were conducted for two plant 

populations (Company K, L) with similar 

reported average exposures and estimated 

exposure responses. A plant effect was 

introduced to allow for systematic differences 

between the two sites, and there was a test of 

heterogeneity in the exposure effects. 

5.2.3 Models of the Incidence of 

Pulmonary Obstruction

For analyses of onset of discrete adverse effect 

outcomes, conducted for the Company G 

population (n=361), three case-definitions of 

pulmonary impairment were applied: 

(1) FEV1 < LLofN; n=36

(2) FEV1/FVC < LLofN; n=27

(3) FEV1 < LLofN and FEV1/FVC 

< LLofN; n=19

Definitions 2 and 3 represent definitions more 

specific to obstruction. For the combined 

Company K and L populations, the case 

definition used for determining onset of 

pulmonary impairment was: FEV1 < LLofN 

(n=25). The definitions more specific to 

obstruction produced too few cases for 

meaningful analysis with the combined 

Company K and L populations.

In identifying cases, a date of onset for a 

condition resulting in impairment and possibly 

representing early obliterative bronchiolitis 

was estimated as the average of the dates on 

which the employee reported the start of 

one or more continuing symptoms (cough, 

wheezing, shortness of breath, tightness of 

chest or phlegm, based on questionnaire items), 
112 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

provided those symptom dates were after their 

date of first exposure to diacetyl. The average 

date was chosen over the first date to be more 

robust for recalled dates. If no symptom date 

existed, then date of onset was set to the date 

of the first case-qualifying spirometry result 

(n=12, case definition 1; n=4, case definition 3 

for Company G) unless this was the employee’s 

first survey in which case the employee was 

excluded from analysis (n=42, case definition 

1; n=21, case definition 3 for Company G). 

These excluded employees may have had onset 

of impairment prior to exposure but, according 

to the participating HHE clinicians, may also 

have included asymptomatic cases caused by 

diacetyl exposure with unknown onset date.

The incidence of new cases was modeled using 

Poisson regression [Checkoway et al. 2004]. 

This method produces an estimate of the 

background rate needed for a life-table-based 

calculation of excess lifetime risk. Observation 

time was compiled beginning with date first 

exposed to diacetyl. Models were fit using 

PROC COUNTREG in SAS 9.2 [SAS Institute 

Inc. 2008] and model fit assessed with the 

likelihood ratio test. Employment duration 

and the other covariates (age, gender, smoking) 

were included in these models. This design 

has potential bias leading to underestimated 

rates arising from the departure of affected 

employees more often than others with similar 

exposure from employment. Incident cases 

are available for analysis only if the individual 

remains in employment until, and chooses to 

participate in, a spirometry-medical survey. 

5.3 Results: Exposure 

Response

5.3.1 Cross-Sectional Pulmonary 

Function Changes

Multiple regression analyses for the Company 

G population (the largest group, n=361, with 

the most extensive exposure assessment) 

controlling for gender, ethnicity, and smoking, 

revealed statistically significant declining 

ppFEV1 for all diacetyl exposure metrics, with 

Cum(DA) (p=10-6) and (p=4×10-7) 

performing considerably better than exposure 

duration alone, and with average exposure 

to diacetyl [Avg(DA)] and Cum(DA2.0) 

performing less well than duration (Table 5-5). 

The estimate for the exposure-response with 

Cum(DA) was a 0.50 reduction in ppFEV1 for 

each ppm-year of cumulative exposure (Tables 

5-5, 5-6). (After 1 year at 1 ppm an employee’s 

ppFEV1, starting at 100, would be predicted to 

be 99.5.) For FEV1/FVC the percent reduction 

with 1 ppm-yr DA was 0.16. 

Seventy-nine percent of the cross-sectional 

study population (n=286) had duration of 

employment of < 4 yr and 49% had less than 6 

months, reflecting a high workforce turnover 

rate. Models restricted to < 4 yr duration 

produced considerably larger effect estimates; 

for ppFEV1: −1.07 (vs. −0.50) and for FEV1/

FVC: −0.87 (vs. −0.16) (Table 5-5). With < 

4 yr, the metric was a less strong 

predictor than Cum(DA).

In the models with the better predicting 

exposure metrics, gender and ethnicity 

(possible indicators of differential healthy 

employee selection) were unimportant 

predictors. Ever-smoking was associated 

with an increase in ppFEV1 but cumulative 

smoking, in pack-years, predicted a decline 

in ppFEV1 (implying that, initially, smokers 

may be healthier than nonsmokers); both 

effects were statistically significant (Table 5-6). 

Regression models based on the first Company 

G spirometry determination rather than the 

last yielded similar estimates of diacetyl effects 

(data not shown). The metric cumulative square 

root of diacetyl concentration was a slightly 

stronger predictor of spirometry changes 

than simple cumulative exposure (Table 5-6), 

implying that if there is any dose-rate effect it is 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 113

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-5. Multiple regression models for percent predicted FEV1 and 

FEV1/FVC: various diacetyl exposure metrics for Company G

Percent predicted FEV1 FEV1/FVC (expressed as percent)

Exposure metric R2 Int est t P R2 Int est t P

All (n=361)

Avg(DA) 0.128 94.99 −1.77 2.41 0.0167 0.348 76.88 −1.26 −3.58 0.004

Cum(DA2.0) 0.142 94.62 −0.081 3.41 0.0007 0.338 76.19 −0.032 −2.77 0.0059

(Cum(DA))2.0 0.148 94.76 −0.012 3.76 0.0002 0.334 76.08 −0.0036 −2.31 0.021

Duration 0.161 97.17 −0.964 4.43 9×10−6 0.333 76.78 −0.256 −2.12 0.035

Cum(DA) 0.169 95.95 −0.500 4.83 10−6 0.342 76.62 −0.164 −3.06 0.0024

Cum(DA0.5) 0.172 96.38 −0.843 4.95 7×10−7 0.339 76.71 −0.258 −2.87 0.0044

(Cum(DA))0.5 0.174 97.34 −2.77 5.04 4×10−7 0.346 77.25 −0.981 −3.41 0.0007

(Cum(DA0.5))0.5 0.176 98.25 −3.70 5.16 2×10−7 0.344 77.49 −1.24 −3.24 0.0013

Less than 4 yr exposure duration (n=286)

Cum(DA) 0.095 100.24 −1.069 −2.48 0.014 0.321 77.28 −0.872 −4.34 1.4×10−5

(Cum(DA))0.5 0.087 100.76 −2.57 −1.93 0.054 0.314 77.90 −2.47 −3.98 6×10−5

Int = intercept; est = effect estimate for exposure metric; t = t-statistic for estimate; P = P value

Avg(DA) − time-weighted average exposure = cum(DA)/duration

Cum(DA) = cumulative exposure = Σi (DA)

Cum(DA0.5) = Σi (DA0.5)

Cum(DA2.0) = Σi (DA2.0)

Model = ppFEV1 = α + βsex + γHispanic + δBlack + εsmoker + θpackyrs + Φpackyrs 2

 + η(exposure metric)

Model = FEV1/FVC = α + βsex + βAge + γHispanic + δBlack + εsmoker + θpackyrs + Φpackyrs 2

 + η(exposure metric)

Table 5-6. Multiple regression models for percent predicted FEV1

and best-fitting diacetyl exposure metrics for Company G

Cum(DA) (Cum(DA))0.5 Cum(DA0.5) (Cum(DA0.5))0.5

R2 = 0.169 R2 = 0.174 R2 = 0.172 R2 = 0.176

β P β P β P β P

Intercept 95.95 — 97.34 — 96.38 — 98.25 —

Female −0.386 0.82 0.092 0.96 −0.306 0.86 0.078 0.96

Hispanic 1.99 0.40 1.42 0.55 1.70 0.47 1.18 0.62

Black 8.58 0.45 7.78 0.49 8.30 0.46 7.15 0.52

Smoke_ever 7.29 0.0020 6.86 0.0038 6.88 0.004 6.49 0.0063

Packyrs −0.571 0.0008 −0.562 0.0009 −0.560 0.0009 −0.558 0.0009

Packyr2 0.0024 0.36 0.0024 0.34 0.0025 0.32 0.0025 0.31

Exposure metric −0.500 10−6 −2.77 4×10−7 −0.843 7×10−7 −3.70 2×10−7
114 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

probably negative—higher exposures have less 

than proportional association with decreases in 

spirometry. Results for Company N, based on a 

small number (n=35) of employees and only 20 

breathing-zone air samples, are not presented 

but were generally comparable to Company 

G results.

The best-predicting exposure metric depended 

on the HHE population analyzed (Tables 5-7, 

5.8). In predicting FEV1/FVC the R-square 

values were consistently larger compared 

with the ppFEV1 regressions but the exposure 

effects were sometimes less significant. For 

Company G, Avg(DA) and were the 

better predictors of FEV1/FVC; for Company 

K, Cum(DA) was best while for Company L, 

Avg(DA), and Cum(DA2.0) were 

all equivalent better predictors. For ppFEV1, 

model fit at Company K was strongest for 

(Cum(DA))2.0, however, Cum(DA) provided a 

similar fit. For Company L, Avg(DA) was the 

strongest predictor of ppFEV1. In the pooled 

analysis of the Company K and Company L 

plants, the differences in exposure response 

(heterogeneity) between the plants for ppFEV1

and FEV1/FVC were highly significant for 

the better predicting metrics Cum(DA) and 

(Cum(DA)2

 (Tables 5-9, 5-10). The pooled 

regression estimate for the Cum(DA) metric 

corresponded to a decline in ppFEV1 of 4.22 

per ppm-yr of cumulative exposure (Table 5-9), 

almost an order of magnitude higher than the 

Company G estimated decline in ppFEV1 of 

0.50 per ppm-yr of cumulative exposure (Table 

5-6) but with very different estimates for the 

individual plants. For plant K, the estimated 

fall in ppFEV1 per ppm-yr was 7.83 while for 

Plant L the decrease in ppFEV1 was 2.70 (= 

−7.83+5.13) per ppm-yr. At these two plants, 

many of the environmental samples collected 

were below the limit of detection for diacetyl, 

and the HHE environmental assessments were 

cross-sectional and not necessarily reflective of 

exposures prior to the survey date. This may 

explain the divergence in optimum exposure 

metrics compared with the Company G 

results. For example, if jobs with the highest 

exposures had been given priority for control 

interventions, then the subsequently measured 

levels would underestimate most of the jobs 

previously having the highest levels. Therefore, 

an exposure metric like Cum(DA2.0), which 

gives greater weight to high values, might 

better predict spirometry changes than 

Cum(DA), as was observed at Company K 

for ppFEV1 and FEV1/FVC, and at Company 

L for FEV1/FVC (Table 5-5). Because of the 

inconsistencies between them and less certain 

exposure histories, the results for Company 

K and Company L were not the final basis for 

the NIOSH risk assessment for diacetyl which, 

instead, relied on the Company G findings.

By far the highest exposures at Company G were 

among mixers (Table 5-3) raising the possibility 

that the observed losses in pulmonary function 

could be limited to that group. To examine this 

question, the basic multiple regression models 

(Table 5-6) were applied to the population at 

Plant G from which all employees who were 

ever mixers had been excluded. The result was 

slightly stronger estimates of the DA effect both 

for (1) the linear cumulative exposure term 

(β=0.61 vs. 0.50 for full population; R2

=0.182 

vs. 0.169, resp.) and (2) the square root of 

cumulative exposure (β=3.02 vs. 2.75 for full 

population; R2

=0.182 vs. 0.173, respectively) 

(results not shown).

Another concern was the possibility of a 

diacetyl-smoking interaction, with smoking 

possibly enhancing the harmful effect of 

diacetyl. Models for ppFEV1 and FEV1/

FVC including interaction terms (products 

of the diacetyl exposure metrics with the 

ever-smoking and pack-yrs terms) yielded 

statistically significant protective effects of 

ever-smoking on the linear and square root 

cumulative diacetyl exposures and small, 

mostly insignificant, additive interactions for 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 115

5 

. Quantitative Risk Assessment Based on Employee Data

Table 5-7. Preliminary regression model results for percent predicted FEV1 and FEV1/FVC at Companies K, L, and G

Duration Avg(DA) Cum(DA0.5) Cum(DA) (Cum(DA))0.5

% pred. FEV1

Company n β R2 P β R2 P β R2 P β R2 P β R2 P

K 161 −0.31 0.189 0.20 −21.7 0.217 0.01 −5.26 0.306 < 10−4 −7.77 0.322 < 10−7 −14.3 0.286 10−6

L 215 −0.35 0.098 0.31 −18.6 0.157 0.0001 −3.57 0.135 0.0018 −3.56 0.138 0.0012 −9.15 0.146 0.0004G 361 −0.96 0.161 < 10−5 −1.77 0.128 0.02 −0.84 0.172 < 10−6 −0.50 0.169 10−6 −2.77 0.174 < 10−6

FEV1/FVC (expressed as percent)

K 161 −0.28 0.323 0.068 −13.8 0.357 0.0009 −2.99 0.430 < 10−7 −4.30 0.449 < 10−7 −8.24 0.420 < 10−7

L 215 −0.069 0.139 0.70 −9.92 0.222 < 10−5 −2.14 0.197 < 104 −2.16 0.213 < 10−5 −5.26 0.212 < 10−5

G 358 −0.26 0.333 0.035 −1.26 0.348 0.0004 −0.26 0.339 0.0044 −0.16 0.342 0.0024 −0.98 0.346 0.0007

β = parameter estimate for diacetyl exposure metricR2 = R-squared measure of multiple regression model fit 

P = P value for exposure metric effect
116 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-8. Preliminary regression model results for percent predicted FEV1

and FEV1/FVC with quadratic exposure metrics at Companies K, L, and G

Cum(DA2.0) (Cum(DA))2.0

% pred. FEV1

Company n β R2 P β R2 P

K 161 −8.79 0.300 < 10−6 −1.36 0.325 < 10−7

L 215 −2.64 0.136 0.0017 −0.40 0.122 0.010

G 361 −0.08 0.142 0.0007 −0.01 0.148 0.0002

FEV1/FVC

n β R2 P β R2 P

K 161 −4.93 0.432 < 10−7 −0.79 0.415 < 10−4

L 215 −1.54 0.215 < 10−5 −0.25 0.195 < 10−4

G 358 −0.032 0.338 0.0059 −0.004 0.334 0.021

β = parameter estimate for diacetyl exposure metric

R2 = R-squared measure of multiple regression model fit 

P = P value for exposure metric effect

Table 5-9. Pooled analyses with Company L and Company K populations: % pred. FEV1

Duration Avg(DA) (Cum(DA))0.5 Cum(DA) (Cum(DA))2.0

R2 = 0.129 R2 = 0.171 R2 = 0.191 R2 = 0.188 R2 = 0.165

β P β P β P β P β P

Intercept: K 99.07 — 97.92 — 99.82 — 97.95 — 97.24 —

Intercept: deviation* −2.80 0.1240 −1.19 0.47 −1.11 0.49 −1.51 0.35 −1.50 0.36

Exposure: pooled −0.309 0.1092 −17.6 < 10−5 −10.4 < 10−7 −4.22 < 10−7 −0.47 < 10−5

R2 = 0.129 R2 = 0.172 R2 = 0.197 R2 = 0.208 R2 = 0.202

Intercept: K 99.07 — 98.14 — 101.00 — 98.95 — 98.19 —

Intercept: deviation −2.81 0.28 −1.51 0.38 −3.05 0.13 −2.83 0.090 −2.52 0.12

Exposure: K −0.309 0.18 −21.8 0.0061 −14.31 < 10−5 −7.83 < 10−7 −1.37 < 10−7

Exposure: deviation* 0.001 0.99 5.35 0.55 6.36 0.10 5.13 0.0025 1.11 < 10−4

*

Deviation from Company K estimate by Company L

β = parameter estimate for diacetyl exposure metric

P = P value for exposure metric effect
Occupational Exposure to Diacetyl and 2,3-Pentanedione 117

5 . Quantitative Risk Assessment Based on Employee Data

the pack-years × diacetyl metric terms (P = 

0.04 – 0.25 depending on exposure metric and 

outcome; data not shown). In the absence of the 

smoking interaction terms, the diacetyl effects 

in non-smokers are somewhat underestimated 

and overestimated in smokers.

Acetoin is another exposure in the popcorn 

flavoring environment (typically present with 

diacetyl in flavoring additive packages), and its 

presence was strongly associated with diacetyl 

(corr = 0.85) at Plant G, but it is not subject 

to the humidity degradation problem in air 

sampling. In response to concerns that the 

corrected historical exposure measurements 

for diacetyl were inaccurate, NIOSH repeated 

models of exposure-response relationships 

using acetoin measures. Applying to acetoin the 

procedure used for constructing the exposure 

matrix for diacetyl resulted in estimated acetoin 

concentrations over employees’ work histories. 

Multiple linear regressions predicting percent 

of predicted FEV1 based on acetoin exposure 

metrics produced the same pattern of results 

as observed with diacetyl and with almost 

identical model fit. For the metric square root 

of cumulative exposure, the R2 observed was 

0.1743 and 0.1737, respectively, for acetoin and 

diacetyl; the t-statistics for the exposure terms 

were 5.09 and 5.06 respectively. In microwave 

production jobs at Plant G, the mean DA concentration over all sampling surveys, combining 

both area and personal samples, was 3.4 ppm 

compared with 0.28 ppm for acetoin determinations from the same air samples. Because 

there is little support for acetoin itself playing 

a major role other than as surrogate for diacetyl in pulmonary toxicity, and because acetoin 

was present at much lower concentrations, this 

result supports the validity of the diacetyl exposure assessment and subsequent findings, but 

also implies that the diacetyl effects are being 

underestimated as a result of misclassification, 

otherwise the diacetyl effects would produce a 

stronger model fit than acetoin. 

Table 5-10. Pooled analyses with Company L and Company K populations: 

FEV1/FVC (expressed as percent)

Duration Avg(DA) (Cum[DA])0.5 Cum(DA) (Cum[DA])2.0

R2 = 0.209 R2 = 0.226 R2 = 0.289 R2 = 0.292 R2 = 0.268

β P β P β P β P β P

Intercept: K 81.19 — 80.42 — 81.37 — 80.28 — 79.88 —

Intercept: deviation* −1.37 0.14 0.795 0.37 0.264 0.76 0.541 0.53 0.588 0.51

Exposure: pooled −0.227 0.044 −10.8 < 10−7 −6.27 < 10−7 −2.65 < 10−7 −0.315 < 10−7

R2 = 0.213 R2 = 0.267 R2 = 0.295 R2 = 0.307 R2 = 0.299

Intercept: K 81.83 — 80.53 — 81.98 — 80.73 — 80.33 —

Intercept: deviation −2.62 0.048 0.963 0.30 −1.24 0.23 −1.11 0.21 −1.07 0.22

Exposure: K −0.316 0.016 −12.8 .0011 −8.35 < 10−7 −4.29 < 10−7 −0.749 < 10−7

Exposure: deviation* 0.275 0.18 2.64 0.55 3.32 0.081 2.33 .0051 0.534 < 10−4

*

Deviation from Company K estimate by Company L

β = parameter estimate for diacetyl exposure metric

P = P value for exposure metric effect
118 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

5.3.2 Longitudinal Analyses of ppFEV1

at Company G

Longitudinal mixed effect models of ppFEV1

(where individual intercepts and responses 

are treated as random effects) show smaller 

effects for both Cum(DA) and 

exposure metrics (Table 5-11, models 1 and 2) 

compared to the analyses based on the FEV1 at 

last survey (Tables 5-7, 5-8); the effects remain 

statistically significant. Differences in the 

effects of employees’ exposures accruing from 

their initial evaluation (their first survey) until 

the current survey, compared to all exposures 

prior to the current survey, using either the 

metric Cum(DA) or , were small and 

not statistically significant (P > 0.7) (Table 5-11, 

models 3 and 4). This supports the conclusion 

that bias arising from cases preferentially 

leaving employment prior to the first survey is 

not different from that following the first survey 

when exposures were declining, suggesting that 

the bias in estimating the decline in ppFEV1 is 

not large.

5.3.3 Incidence of Pulmonary 

Impairment at Company G

Poisson regression analysis with the log-linear 

specification was applied to model incidence 

rates adjusted for gender, age, and smoking 

(race and ethnicity were not important predictors). The original sentinel cases of obliterative 

bronchiolitis reported from this plant were not 

present in this study population. For the first 

definition of case (FEV1< LLofN, n=36), excluding (a) candidate cases for which no qualifying 

date of onset was available and (b) subjects 

with missing smoking data, left 314 subjects 

for analysis. Increasing duration of exposure 

or diacetyl cumulative exposure (Cum(DA)) 

both predicted diminishing onset (Table 5-12, 

models 1 and 2). Model fit improved with both 

terms in the model but the duration effect 

remained negative. Other diacetyl metrics performed similarly (Table 5-12) with avg(DA) and 

cum(DA) providing the best fit (largest Δ-2lnL, 

smallest LRT P value). The negative duration 

term implies diminishing background rate with 

increasing duration. 

Using case definition 2, (FEV1/FVC< LLofN, 

n=27) the same pattern was observed, with 

the negative duration effect (P=0.0004) and 

positive cumulative exposure effect (P=.00003) 

now highly statistically significant despite a 

smaller number of cases (Table 5-13, model 

3). With the most stringent case definition 3, 

(FEV1< LLofN and FEV1/FVC< LLofN), the 

negative duration effect (P=0.023) and the 

cumulative exposure effect (P=.016) remained 

statistically significant now with 19 cases (Table 

5-14, model 3). In this model, smoking effects 

were not statistically significant, and age and 

sex were marginally significant (Table 5-15). 

Three other metrics yielded strong associations 

based on likelihood ratio test, particularly 

 and (Avg(DA)) (both P=.003) 

although with average exposure, duration was 

no longer significant (Table 5-14). 

5.3.4 Evidence of Variable 

Susceptibility to Diacetyl Effects

When the joint distribution of cases by 

exposure duration and cumulative exposure 

was examined (case definition 1; all jobs 

had exposures > 0.0), the pattern suggested 

the possible presence of a low-risk survivor 

population or variable susceptibility. For 

example, there were five cases in the cell 

with lowest duration and lowest exposure 

and another five cases in a different cell with 

comparable person-years of observation (89 

years) in the highest exposure category and 2 

to 4 years duration (Tables 5-16, 5-17). Thus 

similar rates were observed despite the greater 

than tenfold difference in cumulative exposure. 

Of the 36 cases, 22 occurred in the first 4 years 

of exposed employment, which encompassed 

about 80% of the study population. The 

rapid onset of this disease has been reported 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 119

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-11. Longitudinal analyses of percent predicted FEV1

at Company G using random effects models 

Random: ID Random: ID, DA-effect

Model no. β t P β t P

1 Cum(DA) −0.438 −4.49 0.0001 −0.427 −2.90 0.016

2 Cum(DA)0.5 −1.82 −3.35 0.007 −1.99 −3.07 0.012

3 Cum(DA) −0.436 −4.23 0.002 −0.437 −4.25 0.002

Cum(DA) since first survey −0.0309 −0.07 0.95 −0.110 −0.18 0.86

4 Cum(DA)0.5 −2.59 −4.82 0.0007 −2.60 −4.83 0.0007

Cum(DA)0.5 since first survey 0.718 0.37 0.71 0.766 0.34 0.74

Note: Cum(DA) is calculated up to each survey of an employee (two or more are in the analysis). Cum(DA) since first survey is calculated 

from an employee’s first survey up to each subsequent survey. 

β = parameter estimate for diacetyl exposure metric

t = t-statistic for exposure metric effect

P = P value for exposure metric effect

Table 5-12. Company G incidence rate models: exposure metrics 

(case definition 1: FEV1 < LLofN; n=36)

Model 

no. Metric

Intercept

baseline rate

Effect

estimate

RR

10yr @ 1 ppm

RR

5yr @ 2 ppm Δ − 2lnL Wald P LRT P

1 Duration −8.61 −0.081 0.92 0.96 0.0 0.14 —

2 Cum(DA) −8.96 −0.0002 1.00 1.00 — 0.84 —

3 Duration −8.59 −0.162 0.063

Cum(DA) 0.040 1.49 1.49 1.80 0.17 0.18

4 Duration −8.57 −0.166 0.17

Cum(DA0.5) 0.067 1.95 1.60 0.74 0.41 0.39

5 Duration −8.76 −0.016 0.071

(Cum(DA))0.5 0.220 2.01 2.01 1.64 0.21 0.20

6 Duration −8.87 −0.086 0.13

Avg(DA) 0.161 1.17 1.38 1.94 0.14 0.16

Baseline rate: as Log(rate); per day

LRT = likelihood ratio test

Model = rate = exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + Φpackyrs2 + ηcum(dur) + μcum(DA) ) (model 3)

RR = relative rate, at 1 or 2 ppm, or 1 year (duration)

Δ − 2lnL = change in − 2 × ln(likelihood), relative to model 1)
120 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-13. Company G incidence rate models: exposure metrics 

(case definition 2: FEV1/FVC < LLofN; n = 27)

Model

no. Metric

Intercept

baseline rate

Effect

estimate

RR

10yr 

@ 1 ppm

RR

5yr 

@ 2 ppm Δ − 2lnL Wald P LRT P

1 Duration −9.37 −0.081 — — — 0.20 —

2 Cum(DA) −10.04 0.028 1.32 1.32 — 0.19 —

3 Duration −9.54 −0.416 0.0004

Cum(DA) 0.140 4.06 4.06 14.55 0.00003 0.0001

4 Duration −9.40 −0.972 0.0003

Cum(DA0.5) 0.580 330. 60.4 15.75 0.0002 0.00007

5 Duration −10.05 −0.386 0.003

(Cum(DA))0.5 0.750 10.7 10.7 11.23 0.001 0.0008

6 Duration −10.04 −0.085 0.20

Avg(DA) 0.338 1.40 1.96 7.61 0.002 0.006

Baseline rate: as Log(rate); per day

LRT = likelihood ratio test

Model − rate = exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + Φpackyrs2 + ηcum(dur) + μcum(DA) ) (model 3)

RR = relative rate, at 1 or 2 ppm, or 1 year (duration)

Δ − 2lnL = change in − 2 × ln(likelihood), relative to model 1)

Table 5-14. Company G incidence rate models: exposure metrics 

(case definition 3: FEV1 < LLofN and FEV1/FVC < LLofN; n=19)

Model 

no. Metric

Intercept

baseline rate

Effect

estimate

RR

10yr @ 1 ppm

RR

5yr @ 2 ppm Δ − 2lnL Wald P LRT P

1 Duration −9.60 −0.085 — — 0.0 0.23 —

2 Cum(DA) −10.2 0.0124 1.13 1.13 — 0.60 —

3 Duration −9.61 −0.300 0.023

Cum(DA) 0.090 2.46 2.46 5.31 0.016 0.021

4 Duration −9.51 −0.555 0.036

Cum(DA0.5) 0.316 23.7 9.37 5.50 0.041 0.020

5 Duration −10.3 −0.411 0.0085

(Cum(DA))0.5 0.804 12.7 12.7 8.76 0.005 0.003

6 Duration −10.6 −0.088 0.24

Avg(DA) 0.468 1.60 2.55 8.75 0.001 0.003

Baseline rate: as Log(rate); per day

LRT = likelihood ratio test

Model = rate = exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + Φpackyrs2 + ηcum(dur) + μcum(DA) ) (model 3)

RR = relative rate, at 1 or 2 ppm, or 1 year (duration)

Δ − 2lnL = change in − 2 × ln(likelihood), relative to model 1)
Occupational Exposure to Diacetyl and 2,3-Pentanedione 121

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-15. Company G incidence rate model with duration and cum(DA) 

(case definition 3: FEV1 < LLofN and FEV1/FVC < LLofN; n = 19)

Parameter Estimate SE t Wald P

Intercept −9.61 0.647 −14.85 < 0.0001

Ind:female 0.845 0.518 1.63 0.10

Age − 40 0.051 0.028 1.82 0.068

(Age − 40)2 −0.0019 0.0022 −0.86 0.39

Smoke_ever −0.232 0.913 −0.25 0.80

Packyrs 0.012 0.068 0.17 0.86

Packyrs2 0.0003 0.0011 0.29 0.77

Duration −0.300 0.132 −2.27 0.023

Cum(DA) 0.090 0.037 2.41 0.016

rate = exp(α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2) + θpackyrs + Φpackyrs2 + ηcum(dur) + μcum(DA) ) 

Model likelihood ratio test for cum(DA), LRT = 5.306, P = 0.021

t = t-statistic for exposure metric effect

Table 5-16. Company G: observed cases (case definition 1: 

FEV1 < LLofN) by duration and cumulative diacetyl

Duration (yrs)

Observed cases

cumulative diacetyl exposure (ppm-yrs)

< 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 5 2 0 0 0 7

0.5 < 1.0 3 0 1 0 0 4

1.0 < 2.0 2 0 0 2 1 5

2.0 < 4.0 1 0 0 0 5 6

≥ 4.0 1 0 0 1 12 14

All 12 2 1 3 18 36

Table 5-17. Company G: person-yrs (case definition 1: 

FEV1 < LLofN) by duration and cumulative diacetyl

Person-yrs

cumulative diacetyl exposure (ppm-yrs)

Duration (yrs) < 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 89.0 29.6 0.3 0.1 0.0 119.0

0.5 < 1.0 27.2 26.7 16.9 0.7 1.2 72.7

1.0 < 2.0 23.5 10.2 12.4 39.0 10.7 95.8

2.0 < 4.0 14.9 4.7 7.1 14.2 88.8 129.7

≥ 4.0 25.2 16.0 1.7 9.4 222.1 274.5

All 179.8 87.2 38.5 63.5 322.9 691.8
122 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-18. Company G: baseline rate (case definition 1: 

FEV1 < LLofN) by duration and cumulative diacetyl

 Predicted baseline rate (cum. exp. = 0)

cumulative diacetyl exposure (ppm-yrs)

Duration 

(yrs) < 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 0.061 0.063 0.039 0.032 — 0.061

0.5 <1.0 0.057 0.059 0.056 0.053 0.043 0.057

1.0 < 2.0 0.054 0.045 0.059 0.057 0.046 0.054

2.0 < 4.0 0.044 0.034 0.046 0.048 0.045 0.045

≥ 4.0 0.024 0.011 0.032 0.024 0.022 0.022

All 0.053 0.049 0.054 0.050 0.029 0.041

— indicates no person-time in stratum

Based on Table 5-12, model 3

[Akpinar-Elci et al. 2004; CDC 2007; Israel 

et al. 2009; Kreiss et al. 2002; NIOSH 2006, 

2008]. Examination of onset graphically 

(data not shown) also suggested that many 

cases arose after relatively short employment 

duration. A similar pattern was exhibited in 

the 46 cases (defn 1) identified among former 

employees (no longer employed at the time 

of their first survey) (data not shown). The 

predicted baseline incidence (from the model 

with diacetyl exposure set = 0) in the same 

array (Table 5-18) has an elevated level in the 

early years of employment, falling from 0.061 

(6.1% per year) in the first 6 months, to 0.022 

(2.2% per year) after 4 years. Dividing the 

model-predicted total rate by a fixed baseline 

rate of 0.022 yields a rate ratio that appears to 

be systematically elevated at < 4 years vs. >= 

4 years durations of exposure (employment 

after 1986) and at high cumulative exposures 

(Table 5-19). The same situation was observed 

in the pooled Company K and Company L 

populations using the first case definition. Out 

of 25 cases, 20 occur in the < 4 yr duration 

strata (Table 5-20), with elevated rate ratios 

predicted for low durations and high exposures 

(Table 5-21). With the third case definition in 

the Company G population, the same pattern 

is observed but now with fewer cases (n=19 

vs. 36) and 9 out of 19 in the < 4 yr duration 

group (Table 5-22). The predicted rate ratios 

relative to the long-duration baseline rate are 

again elevated at both low duration and high 

cumulative exposures (Table 5-23).

In the loglinear Poisson regression models 

using a (negative) duration term, the excess 

cases at short duration are actually being 

treated as part of the background rate, i.e., 

not attributable to diacetyl exposure. On the 

suspicion that susceptibility was declining 

with duration because low-risk individuals are 

remaining longer in employment, a different 

Poisson regression model was fit. Using a 

linear relative rate specification, this model 

included a term intended to capture excess 

risk arising from diacetyl exposures (1) in an 

unknown portion of the population declining 

with time that has higher susceptibility or (2) 

due to individual susceptibility declining with 

duration of exposure. An exponential decline 

was assumed and halflives of 0.5, 1, and 2 years 

were evaluated. Using case definition 3, a model 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 123

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-19. Company G: rate ratio (case definition 1: 

FEV1 < LLofN) by duration and cumulative diacetyl

Predicted rate ratio (relative to fixed baseline: 0.022) 

cumulative diacetyl exposure (ppm-yrs)

Duration (yrs) < 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 2.77 3.00 1.96 1.64 — 2.82

0.5 <1.0 2.59 2.82 2.77 2.86 2.50 2.73

1.0 < 2.0 2.50 2.18 2.96 3.00 2.82 2.77

2.0 < 4.0 2.00 1.64 2.32 2.55 2.86 2.68

≥ 4.0 1.09 0.55 1.59 1.27 2.00 1.77

All 2.41 2.32 2.68 2.64 2.27 2.36

— indicates no person-time in stratum

Based on Table 5-13, model 3: predicted rate divided by 0.022 from Table 5-18: dur > 4yrs

Table 5-20. Companies K and L pooled: cases (case definition 1: 

FEV1 < LLofN) by duration and cumulative diacetyl 

Observed cases 

cumulative diacetyl exposure (ppm-yrs)

Duration (yrs) < 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 4 0 1 0 0 5

0.5 <1.0 2 1 0 0 0 3

1.0 < 2.0 0 3 0 1 0 4

2.0 < 4.0 0 0 3 4 1 8

≥ 4.0 0 0 2 0 3 5

All 6 4 6 5 4 25

Table 5-21. Companies K and L pooled: rate ratio (case definition 1: 

FEV1 < LLofN) by duration and cumulative diacetyl

Predicted rate ratio (relative to fixed baseline: 0.004); cumulative diacetyl exposure (ppm-yrs)

Duration (yrs) < 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 7.53 9.11 9.23 7.59 — 7.71

0.5 <1.0 6.59 6.04 8.13 8.73 — 6.73

1.0 < 2.0 5.66 5.12 4.84 7.43 13.12 5.74

2.0 < 4.0 4.03 3.00 3.28 4.94 16.10 4.34

≥ 4.0 1.27 0.62 1.42 1.18 6.44 1.93

All 4.96 2.65 2.80 3.34 7.57 3.94

— indicates no person-time in stratum

predicted rate divided by 0.004
124 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-23. Company G: rate ratio (case definition 3: 

FEV1 < LLofN and FEV1/FVC < LLofN) by duration and cumulative diacetyl

Predicted rate ratio (relative to baseline: 0.0046)

cumulative diacetyl exposure (ppm-yrs)

Duration (yrs) < 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 5.39 6.54 1.91 1.15 — 5.67

0.5 <1.0 4.39 6.22 6.57 7.70 5.39 5.59

1.0 < 2.0 4.26 3.72 7.00 7.63 6.98 6.22

2.0 < 4.0 2.54 4.43 4.70 5.61 7.74 6.63

≥ 4.0 0.83 0.85 3.15 1.57 5.11 4.33

All 4.22 4.89 5.85 6.17 5.85 5.35

— indicates no person-time in stratum

Based on Table 5-14, model 3: predicted rate divided by 0.0046

Table 5-22. Company G: cases (case definition 3: 

FEV1 < LLofN and FEV1/FVC < LLofN) by duration and cumulative diacetyl 

Observed cases

cumulative diacetyl exposure (ppm-yrs)

Duration (yrs) < 0.5 0.5 < 2.0 2.0 < 3.0 3.0 < 5.0 ≥ 5.0 All

< 0.5 2 1 0 0 0 3

0.5 <1.0 0 0 1 0 0 1

1.0 < 2.0 1 0 0 1 1 3

2.0 < 4.0 0 0 0 0 2 2

≥ 4.0 0 0 0 0 10 10

All 3 1 1 1 13 19
Occupational Exposure to Diacetyl and 2,3-Pentanedione 125

5 . Quantitative Risk Assessment Based on Employee Data

with a term of the form [Avg(DA)]2×exp(−0.69 

× Duration), i.e., halflife of 1 year and squared 

average exposure, produced a significant fit 

(LRT=7.97, 2df, p=.0186; Table 5-24, model 3) 

with the two exposure terms being considerably 

stronger predictors than in models with either 

one alone (Table 5-24, models 1–3). Of the 

choices examined for parameters in the shortduration risk term, the best fit occurred with 

a halflife of 2.0 years and squared average 

exposure (LRT=9.52, 2df, p=.0086; Table 

5-24, model 4; Table 5-25). In this model, the 

estimated rate ratio for 1.0 pack-year of smoking 

(with no diacetyl exposure) relative to a very 

low baseline rate was 17.7 and, for 1.0 ppm-yr 

of diacetyl exposure in the “low-risk” group 

(with duration >4 years and no smoking), the 

rate ratio was 12.3; the initial high risk (at start 

of exposure, zero duration, and no smoking) 

rate ratio at 1 ppm diacetyl was 69.8. A similar 

result was obtained with case definitions 1 and 

2 (data not shown) although, for case definition 

1, the exposure parameter estimates were not 

statistically significant. 

The relative fit of various incidence-rate model 

specifications (case definition 3) indicates that, 

for a single metric, the average prior exposure

metric fits best, but considerable improvement 

comes with an added duration term (Table 

5-26, models 1–3 vs. 4–6). The best fit was for 

(a) square root of cumulative exposure with 

duration term (loglinear relative rate model 5), 

and for (b) cumulative exposure and the term 

for a high-risk subpopulation (linear relative 

rate model 10). 

5.3.5 Interpretation of 

Modeling Results 

Multiple linear regression models of continuous 

spirometry outcomes at Company G reveal that 

both cum(DA) and are the preferred 

predictors of FEV1 decline based on model fit. 

Average exposure was the weakest predictor of 

ppFEV1. Subsidiary analyses indicate that (1) a 

dose-rate effect, if present, is small and negative 

(i.e., effects are not limited to high exposures); 

(2) bias arising from possible removal of earlier 

cases was probably small, and (3) the bias introduced by the correction procedure addressing 

degradation of diacetyl air samples is also small 

although possibly resulting in underestimation of the diacetyl effect. Evidence for non 

uniform susceptibility includes the somewhat 

superior prediction by compared 

to Cum(DA) which may be a reflection of a 

reduced response in the population at longer 

durations of exposure. 

In the modeling of incidence, fewer cases 

met the third case definition than the first or 

second (19 vs. 36, 27) due to the requirement 

that both ppFEV1 and FEV1/FVC be less than 

their LLofN. This was consistent with some 

restriction as was observed in regression 

models of FVC (data not shown). Using the 

third case definition, the estimated baseline 

rate is very small (Table 5-24, models 3, 

4); baseline annual rate = 0.007% per year 

(365.25× exp(−15.48)=0.00007), indicating 

that virtually all cases were attributable to 

either diacetyl exposure or smoking. The 

strong association with the term representing 

short duration of exposure supports the 

conjecture that the population with “normal” 

susceptibility was declining by about half with 

each 2 years of exposure duration. Although 

average diacetyl exposure by itself is a strong 

predictor of incidence, as with prediction of 

FEV1/FVC, this appears to be an artifact of 

changing population susceptibility and has little 

biological plausibility as a risk factor itself.

The existence of a changing population composition with respect to susceptibility poses 

a challenge for predicting excess cases over a 

45-year working lifetime because the composition of the population with respect to 

the factor(s) conveying risk is unknown and 

workforce turnover continually introduces a 

higher-risk segment into employment.
126 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-24. Incidence rate models using linear relative rate model with term for transient high-risk 

group (shortdur(DA)) at Company G (case definition 3: FEV1 < LLofN and FEV1/FVC < LLofN)

Model no. Parameter Estimate RR LRT P value

1 −2ln(L) = 353.53 

intercept −10.9 Baseline rate = 6.5×10−3

smoke_ever −0.879 0.42

Ind:female 1.097 2.30

age − 40 0.029 1.03

(age − 40)2 −0.002 0.998

packyrs 0.273 1.27

cum(DA) 0.156 1.16 1.74 0.187

2 −2ln(L) = 350.77 

Intercept −10.5 Baseline rate = 9.9×10−3

smoke_ever −0.714 0.49

Ind:female 0.915 2.50

age − 40 0.043 1.04

(age − 40)2 −0.002 0.998

packyrs 0.155 1.16

shortdur(DA) 0.536 1.54 4.50 0.034

3 −2ln(L) = 347.27

intercept −15.5 Baseline rate = 6.8×10-5

smoke_ever −0.795 0.45

Ind:female 1.019 2.77

age − 40 0.037 1.04

(age − 40)2 −0.002 0.998

packyrs 21.16 22.2

cum(DA) 16.42 17.4 3.50 0.061

shortdur(DA) 79.60 80.6 6.26 0.012

4 −2ln(L) = 345.75

intercept −15.5 Baseline rate = 6.9×10−5

smoke_ever −0.683 0.51

Ind:female 0.967 2.63

age − 40 0.041 1.04

(age − 40)2 −0.002 0.998

packyrs 17.71 18.7

cum(DA) 12.29 13.3 2.19 0.139

shortdur(DA) 69.82 70.8 7.78 0.0053

LRT = likelihood ratio test

General model:

Rate = {exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

)}{1 + θpackyrs + σhr(DA) + μcumDA} 

Baseline rate (cases/P − Yr): 365.25exp(intercept)

RR − @ 1 pack − yr, 1 ppm at day 1 (hr(DA)), 1 ppm-yr (cum(DA))

Models 2,3: shortdur(DA) = [Avg(DA)]2

exp(−0.693dur), for halflife = 1.0 yr; LRT for exposure terms = 7.97 (2 df)

Model 4: shortdur(DA) = [Avg(DA)]2

exp(−0.693dur/2), for halflife = 2.0 yr; LRT for exposure terms = 9.52 (2 df)
Occupational Exposure to Diacetyl and 2,3-Pentanedione 127

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-25. Likelihood ratio tests and P values for choices of constants defining shortdur(DA) 

variable at Company G (case definition 3: FEV1 < LLofN and FEV1/FVC < LLofN)

LRT for cum(DA) and shortdur(DA) terms (p)

Half-life, b

1.0 2.0

 Avg(DA) 5.93 (0.052) 6.37 (0.019)

 (Avg(DA))2.0 7.97 (0.019) 9.52 (0.0086)

LRT = likelihood ratio test, 2df 

shortdur(DA) = (Avg Exp) a

 × e−0.693dur/b

Table 5-26. Relative fit of selected model specifications for incidence rate 

(case definition 3: FEV1 < LLofN and FEV1/FVC < LLofN)

Model 

no. Rate model Intercept Deviance

Loglinear models (multiplicative exposure terms)

 1 exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + μcum(DA) ) −10.21 354.89

 2 exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + μ(cum(DA) ) 0.5) −10.83 354.52

 3 exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + μavg(DA) ) −11.14 346.41

 4 exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + ηdur + μcum(DA) ) −9.66 348.19

 5 exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + ηdur + μ(cum(DA) )0.5) −10.39 344.87

 6 exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

) + θpackyrs + ηdur + μavg(DA) ) −10.64 344.93

Linear relative rate models (additive exposure terms)

 7 {exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

))}{1 + θpackyrs + μcum(DA) } −10.93 353.53

 8 {exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

))}{1 + θpackyrs + μ(cum(DA))0.5 } −15.36 352.04

 9 {exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

))}{1 + θpackyrs + μavg(DA) } −15.37 348.93

10 {exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

))}{1 + θpackyrs + μcum(DA) + 

σshortdur(DA)} 

−15.47 345.75

11 {exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

))}{1 + θpackyrs + μ(cum(DA))0.5 + 

σshortdur(DA)} 

−14.46 346.99

12 {exp( α + βsmoker + γsex + δ(age − 40) + ε(age − 40)2

))}{1 + θpackyrs + μavg(DA) + 

σshortdur(DA)} 

−15.21 346.17

Smaller deviance = better fit

shortdur(DA) ~ [DA]2exp(−0.693dur/2) – for half-life = 2.0 yr
128 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

In a population with relatively uniform 

response to diacetyl exposure (uniform 

susceptibility), the early new cases resulting 

from diacetyl exposure would in general 

constitute individuals who were already very 

close to their LLofN. For a given age and height, 

this subpopulation is proportional to the height 

of the FEV1 distribution at the LLofN (Figure 

5.1). With increasing cumulative exposure the 

FEV1 distribution would be shifted toward 

lower values and the segment at immediate 

risk of falling below the LLofN would be 

increasing as long as the mean (mode or peak) 

of the shifted distribution remains above 

LLofN. This is not what was observed; initially 

the rate of new cases is generally larger and 

declines with increasing duration or cumulative 

exposure (Tables 5-19, 5-21), implying variable 

susceptibility, i.e., most individuals in the 

exposed population are losing FEV1 much faster 

than those with longer duration of exposure.

5.4 Human Data-based 

Assessment of Risks

Using the impairment findings from Company 

G NIOSH employed two approaches for assessing risk of diacetyl exposures. The first was the 

benchmark dose procedure, which is appropriate for cross-sectional population surveys with 

continuous health outcomes, and the second 

was calculation of excess lifetime risk, a life-table 

procedure which accounts for competing risks 

using a model for the rate of onset of a discrete 

outcome. In these calculations, three risk estimates were derived: for a life-time exposure 

(45 yr) and also for 2.5- and 10-year exposures 

Figure 5-1. New cases expected from a hypothetical population with uniform susceptibility 

to diminishing percent predicted FEV1 with increasing cumulative exposure to diacetyl

Cumulative DA (ppm-yr)

Population density

40 60 80 100 120

0.0 0.01 0.02 0.03

0 ppm-yr

5 ppm-yr

10 ppm-yr

15 ppm-yr

20 ppm-yr

Lower limit of normal

New cases: 0–5 ppm-yr

New cases: 10–15 ppm-yr
Occupational Exposure to Diacetyl and 2,3-Pentanedione 129

5 . Quantitative Risk Assessment Based on Employee Data

(more typical employment durations and 

implying a larger workforce ever exposed). The 

nominal standard for acceptable risk used was 

one per thousand excess risk of impairment, a 

standard choice used in OSHA regulation for 

chronic diseases. 

5.4.1 Benchmark Dose

5.4.1.1 Methods

For continuously distributed respiratory 

endpoints such as FEV1, the benchmark 

dose approach permits estimation of excess 

prevalence of impairment as a function of prior 

exposure history [Bailer et al. 1997; Clewell et 

al. 2003; Crump 1995; Park et al. 2006]. On the 

basis of regression models and population data 

on the distribution of FEV1 from NHANES III 

[CDC and NCHS 2011], the proportions of 

the workforce predicted to be impaired after 

working at specified exposure levels can be 

calculated. Unlike animal-based studies where 

exposures are in discrete levels, the analyses 

here utilized continuously distributed exposure 

metrics and a linear statistical model which 

made unnecessary the point-of-departure 

procedure commonly used in benchmark 

dose calculations. This method, however, does 

require specification of what degree of deficit 

constitutes impairment and the maximum 

increase in impairment prevalence that is 

considered acceptable, which are policy choices. 

The exposure resulting in a maximum allowable 

increase in impairment over some time period 

is called the benchmark dose (BMD).

5.4.1.2 Risk assessment with percent 

predicted FEV1 and FEV1/FVC

With the conventional benchmark dose 

procedure, the excess prevalence of an adverse 

condition is calculated using an exposureresponse relationship derived from modeling. 

With the linear regression result for percent 

predicted FEV1 and Cum(DA) (coef.=−0.50, 

Table 5-6), the excess prevalence after 2.5, 10, or 

45 years of exposure for falling below (1) 60% 

of predicted, or (2) the 5th percentile of normal, 

was calculated as a function of exposure level 

(Table 5-27). Given these two pulmonary 

impairments, a 1/1000 excess prevalence after 

45 years was found for diacetyl exposures 

(BMDs, central tendency estimates) of about 

0.04, and 0.007 ppm diacetyl, respectively. 

Using the exposure metric, , which 

better predicts ppFEV1 in the full population, 

substantially lower BMDs result (data not 

shown); 1/1000 excess risk for impairment at 

the 5th percentile after 45 years occurs with a 

diacetyl exposure concentration of less than 

0.0001 ppm vs. 0.007 with the Cum(DA) metric 

(Table 5-27). These lower BMDs result from 

the increasing (negative) slope of the exposure 

response with diminishing exposure metric. 

Although better captures the risk 

of initially employed employees, extrapolation 

to decreasing durations with this nonlinear 

metric could introduce considerable error. 

For this reason NIOSH chose Cum(DA) over

 as the basis for risk assessment using 

the BMD procedure. In addition, to address 

the same issue for early exposures, the BMD 

was also calculated based on results from the 

< 4 yr population (Table 5-28) but also for a 

45 yr. working lifetime. The resulting excess 

prevalence estimates were about double those 

based on the full population.

For impairment defined in relation to LLofN 

as opposed to some fixed threshold such as 

the 5th percentile of ppFEV1, the BMD procedure is less direct because LLofN is specific to 

age, height, gender and race. The distribution 

of various functions of FEV1 and LLofN, such 

as FEV1/LLofN or (FEV1– LLofN)/(ppFEV1 – 

LLofN) are not readily specifiable. An alternate 

approach was taken: in the NHANES population [CDC and NCHS 2011], the cumulative 

exposure (Cum(DA)) that would reduce an 

individual’s FEV1 to their LLofN was calculated 

using the exposure-response estimates from the 
130 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

preferred regression models of ppFEV1 (coef.=-

0.50, -1.07 (< 4 yr); Table 5-6). The prevalence 

of individuals predicted to be below their 

LLofN was then calculated in the NHANES III 

population as a function of exposure over 2.5, 

10 or 45 years. This “empirical” BMD procedure 

(using the empirical, nonparametric distribution of the NHANES population) yielded 

BMDs for both FEV1 and FEV1/FVC for the 

full population and for < 4 yr (Table 5-29). For 

FEV1 below the LLofN (FEV1) the BMD values 

were similar to those calculated the traditional 

way for ppFEV1 in relation to impairment at the 

5th percentile of normal; the excess prevalence 

after 45 years at 0.01 ppm diacetyl was 2.5/1000 

and 1.5/1000, respectively (Tables 5-27, 5-29). 

BMDs for FEV1/FVC below the LLofN (FEV1/

FVC) were comparable to those for FEV1 (Table 

5-29). In the pooled Company K and Company 

L population, where reported exposures were 

lower than at Company G, the estimated 

1/1000 BMDs for 45 yr were much lower: for 

FEV1, 0.0005 ppm and FEV1/FVC, 0.0004 ppm 

(Table 5-30). Using the less satisfactory, average

exposure, Avg(DA), as the predicting metric in 

the Company G population, the excess prevalence was estimated to be considerably lower 

(Table 5-31), and of course, did not depend on 

Table 5-27. Benchmark dose, based on exposure response 

with cum(DA) for full population at Company G 

Percent of predicted FEV1 (ppFEV1)

Excess prevalence of impairment (per thousand)

Diacetyl

ppm

Model-predicted 

ppFEV1

< 60% of 

predicted

< 5th 

percentile

< 60% of 

predicted

< 5th

percentile

< 60% of 

predicted

< 5th 

percentile

2.5 yr 10 yr 45 yr 2.5 yr 10 yr 45 yr

1.0 98.8 95.0 77.5 1.4 8.8 7.4 42.4 126.7 366.8

0.5 99.38 97.5 88.8 0.7 4.3 3.0 18.7 27.9 126.7

0.2 99.75 99.00 95.5 0.3 1.7 1.1 6.9 6.4 37.2

0.1 99.88 99.50 97.8 0.2 0.9 0.5 3.4 2.7 16.6

0.05 99.94 99.75 98.9 0.1 0.4 0.3 1.7 1.2 7.8

0.02 99.98 99.90 99.55 0.1 0.2 0.1 0.7 0.5 3.0

0.01 99.99 99.95 99.78 0.1 0.1 0.0 0.3 0.2 1.5

0.005 99.99 99.98 99.89 0.1 0.1 0.0 0.2 0.1 0.7

0.002 100.0 99.99 99.96 0.0 0.1 0.0 0.1 0.0 0.3

0.001 100.0 100.0 99.98 0.0 0.0 0.0 0.0 0.0 0.1

0.0005 100.0 100.0 99.99 0.0 0.0 0.0 0.0 0.0 0.1

0.0002 100.0 100.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0

0.0001 100.0 100.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0

Baseline prevalence for < 60% of predicated = 0.0053, for <5th percentile = 0.0498
Occupational Exposure to Diacetyl and 2,3-Pentanedione 131

5 . Quantitative Risk Assessment Based on Employee Data

duration of work. The 1/1000 BMD for FEV1, 

was correspondingly higher: 0.05 ppm diacetyl. 

5.4.2 Excess Lifetime Risk for 

Pulmonary Impairment

5.4.2.1 Methods

Using the life-table approach as implemented 

in the Biological Effects of Ionizing Radiation 

IV report [Committee on the Biological Effects 

of Ionizing Radiation 1988] together with the 

observed exposure-response relationship from 

models of incidence rate, one can estimate the 

excess numbers of cases of diacetyl-associated 

impairment that would occur as a result of 

lifetime exposures at various concentrations. 

This method assumes irreversibility and 

removes incident cases from the population 

at risk with increasing age along with deaths 

arising from the usual causes in the general 

population. Although typical applications of the 

excess lifetime risk calculation are for deaths 

arising from chronic diseases, the method 

can be applied to incidence of an irreversible 

condition provided a baseline incidence rate 

for the condition is known and an estimate 

of the exposure-related incidence rate ratio is 

available. In this analysis, Poisson regression 

Table 5-28. Benchmark dose, based on exposure response with cum(DA) for 

duration less than 4 yrs at Company G 

Percent of predicted FEV1 (ppFEV1)

Excess prevalence of impairment (per thousand)

Diacetyl

ppm

Model-predicted 

ppFEV1

< 60% of 

predicted

< 5th 

percentile

< 60% of 

predicted

< 5th

percentile

< 60% of 

predicted

< 5th

percentile

2.5 yr 10 yr 45 yr 2.5 yr 10 yr 45 yr

1.0 97.3 89.3 51.9 3.3 19.9 25.3 116.5 691.2 872.2

0.5 98.7 94.7 76.0 1.5 9.3 8.1 45.6 148.7 403.3

0.2 99.47 97.9 90.4 0.5 3.6 2.5 15.5 20.9 100.1

0.1 99.73 98.9 95.2 0.3 1.8 1.1 7.3 7.0 39.9

0.05 99.87 99.47 97.6 0.1 0.9 0.5 3.6 2.9 17.7

0.02 99.95 99.79 99.04 0.1 0.3 0.2 1.4 1.0 6.6

0.01 99.97 99.89 99.52 0.0 0.2 0.1 0.7 0.5 3.2

0.005 99.99 99.95 99.76 0.0 0.1 0.1 0.3 0.2 1.6

0.002 99.99 99.98 99.90 0.0 0.0 0.0 0.1 0.1 0.6

0.001 100.0 99.99 99.95 0.0 0.0 0.0 0.1 0.0 0.3

0.0005 100.0 99.99 99.98 0.0 0.0 0.0 0.0 0.0 0.2

0.0002 100.0 100.0 99.99 0.0 0.0 0.0 0.0 0.0 0.1

0.0001 100.0 100.0 100.0 0.0 0.0 0.0 0.0 0.0 0.0

Baseline prevalence for < 60% of predicated = 0.0055, for <5th percentile = 0.0500
132 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

models formed the basis of the calculation 

(Table 5-24, model 4, and the related model 

without terms for smoking), with the model 

intercept describing the baseline risk. 

5.4.2.2 Risk assessment: excess 

lifetime risk

A national life-table constructed from Social 

Security data [SSA 2005] was used. The 

surviving population (living but not yet a case) 

was calculated annually assuming exposure 

starts at age 20 and ceases at age 65, for a 45-yr 

exposure. For 2.5 and 10 years of exposure, the 

life-table exposures start at age 20. Because 

smoking information was used in modeling, 

several variants for lifetime risk are presented 

(Table 5-32). For example, at 0.01 ppm diacetyl, 

using an incidence rate model (case definition 3) 

that ignores smoking determinants, the excess 

lifetime risk (analogous to excess prevalence 

in the BMD approach) was 3.2/1000. With the 

model that includes smoking determinants, 

the excess lifetime risk at 0.01 ppm diacetyl for 

nonsmokers was 11.2/1000, while for smokers 

(one pack/day) it was 2.2/1000. Smokers have 

a smaller lifetime risk because (1) smoking 

reduces the amount of additional impairment 

caused by diacetyl over and above that caused 

by smoking, (2) smoking is a strong competing 

Table 5-29. Empirical benchmark dose, FEV1 and FEV1/FVC based on exposure response with 

cum(DA) for all employees and for those < 4 yr duration, at Company G

Empirical BMD 

Excess prevalence < lower limit of normal (per thousand)

FEV1 all FEV1 duration < 4 yr FEV1/FVC all

FEV1/FVC 

duration< 4 yr

DA

ppm 2.5 yr 10 yr 45 yr 2.5 yr 10 yr 45 yr 2.5 yr 10 yr 45 yr 2.5 yr 10 yr 45 yr

1.0 13.9 68.1 532.5 31.2 188.7 879.4 7.3 30.4 220.5 41.3 287.4 890.8

0.5 6.8 28.9 202.9 14.8 74.5 573.8 3.8 14.1 82.4 19.2 103.0 806.7

0.2 2.6 10.5 58.7 5.9 23.9 161.6 2.4 6.4 27.4 7.5 32.3 243.7

0.1 1.3 5.5 25.7 2.8 11.6 64.1 1.1 3.4 12.1 4.0 15.2 90.1

0.05 0.5 2.6 12.3 1.4 5.9 27.8 0.6 2.4 6.8 2.8 7.5 36.4

0.02 0.2 1.1 4.8 0.4 2.3 10.0 0.4 0.9 3.2 1.1 3.5 13.3

0.01 0.2 0.4 2.5 0.2 1.2 5.3 0.2 0.4 2.1 0.6 2.5 7.0

0.005 0.1 0.2 1.3 0.2 0.4 2.6 0.1 0.4 1.0 0.4 1.1 3.7

0.002 0.1 0.2 0.4 0.1 0.2 1.2 0.1 0.2 0.4 0.2 0.4 2.4

0.001 0.1 0.1 0.2 0.1 0.2 0.4 0.1 0.1 0.3 0.1 0.4 1.0

0.0005 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.1 0.2 0.1 0.2 0.6

0.0002 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.4

0.0001 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.1 0.2
Occupational Exposure to Diacetyl and 2,3-Pentanedione 133

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-30. Empirical benchmark dose based on pooled Company K and Company L populations

Empirical BMD—pooled Company K, L populations

excess prevalence (per thousand)

Diacetyl

ppm

FEV1

< LLofN

FEV1

< 60 %

FEV1/FVC

< LLofN

10 yr 45 yr 10 yr 45 yr 10 yr 45 yr

1.0 854.2 892.5 550.5 994.7 877.5 898.1

0.5 491.4 892.5 108.5 994.4 550.9 898.1

0.2 134.8 823.6 16.7 443.4 135.3 863.9

0.1 54.9 428.0 5.86 84.7 52.0 471.2

0.05 23.6 158.4 2.45 20.4 24.3 160.6

0.02 9.00 48.2 0.88 5.09 9.09 45.6

0.01 4.41 21.2 0.42 2.17 5.29 21.9

0.005 2.29 9.88 0.21 1.00 3.09 10.1

0.002 0.88 4.15 0.08 0.38 1.15 4.50

0.001 0.35 1.94 0.04 0.18 0.71 3.00

0.0005 0.18 1.15 0.02 0.09 0.44 1.32

0.0002 0.09 0.18 0.00 0.03 0.18 0.35

0.0001 0.09 0.35 0.00 0.01 0.18 0.62

Table 5-31. Empirical BMD for exposure response based on 

average diacetyl estimated in Company G population

Average

diacetyl 

ppm

Excess prevalence < lower limit

of normal per thousand

FEV1 FEV1/FVC

1.0 19.23 23.5

0.5 9.18 10.9

0.2 3.88 4.94

0.1 1.59 3.00

0.05 0.97 1.50

0.02 0.35 0.71

0.01 0.18 0.35

0.005 0.09 0.18

0.002 < 0.09* 0.18

0.001 < 0.09 0.09

0.0005 < 0.09 < 0.09

0.0002 < 0.09 < 0.09

0.0001 < 0.09 < 0.09

*

Method unable to resolve risks below this level
134 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

cause for becoming a case, and, (3) smoking 

was assumed to continue after age 65.

A number of investigations have observed 

that declining pulmonary function is a risk 

factor for mortality independent of other possibly associated risk factors such as age, sex, 

smoking, and body mass index – BMI. Three 

such studies investigated rate of decline in 

pulmonary function as a predictor of mortality [Mannino and Davis 2006; Mannino et al. 

2006; Rodriguez et al. 1994] and five others predicted mortality using current FEV1 [Bang et al. 

1993; Hole et al. 1996; Ryan et al. 1999; Sabia 

et al. 2010; Schunemann et al. 2000; Sin et al. 

2005]. Three studies provide estimates of rate 

ratios (RRs) that can be applied to a life-table 

analysis of excess lifetime risk [Bang et al. 1993; 

Ryan et al. 1999; Schunemann et al. 2000]. The 

estimates range from 1.010 to 1.019 per percent 

decline in FEV1 in men, and from 1.01 to 1.025 

in women. Assuming a RR of 1.015 per percent 

decline in FEV1, and using the exposure 

response for FEV1 from the full population and 

from the < 4 yr group, a life-table analysis produced estimates of excess lifetime risk (Table 

5-33) that were comparable (fortuitously) to 

those based on the incidence of pulmonary 

impairment, e.g., FEV1 falling below LLofN 

(Table 5-32). These estimates of excess mortality are the result of a generic effect of declining 

FEV1 on mortality not specific to obliterative 

Table 5-32. Excess lifetime risk based on incidence rate model (case definition 3) 

with term for short duration group at Company G 

Excess lifetime risk (per thousand)

2.5 yr 10 yr 45 yr

Diacetyl

ppm

Model

a

Model b 

for nonsmokers

Model b 

for 

smokers Model a

Model b 

for nonsmokers

Model b 

for 

smokers Model a

Model b 

for nonsmokers

Model 

b for 

smokers

1.0 31.9 110.4 22.5 99.0 320.9 69.5 248.8 659.5 164.6

0.5 15.7 56.0 11.2 51.3 175.7 36.3 140.7 424.0 94.2

0.2 6.2 22.6 4.5 21.0 74.3 14.9 60.8 199.9 41.0

0.1 3.1 11.3 2.2 10.5 37.9 7.5 31.2 105.8 21.1

0.05 1.5 5.7 1.1 5.3 19.1 3.8 15.8 54.5 10.7

0.02 0.6 2.3 0.4 2.1 7.7 1.5 6.4 22.2 4.3

0.01 0.3 1.1 0.2 1.1 3.9 0.8 3.2 11.2 2.2

0.005 0.2 0.6 0.1 0.5 1.9 0.4 1.6 5.6 1.1

0.002 0.1 0.2 0.0 0.2 0.8 0.2 0.6 2.2 0.4

0.001 0.0 0.1 0.0 0.1 0.4 0.1 0.3 1.1 0.2

0.0005 0.0 0.1 0.0 0.1 0.2 0.0 0.2 0.6 0.1

0.0002 0.0 0.0 0.0 0.0 0.1 0.0 0.1 0.2 0.0

0.0001 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.1 0.0

case definition 3: FEV1 < LLof N and FEV1/FVC < LLof N

Model a: no smoking terms in model of case incidence (Table 5-24, model 4)

Model b: smoking terms in model of case incidence (Table 5-24, model 4) but risk calculated separately for nonsmokers and 

smokers: 1 pack/day
Occupational Exposure to Diacetyl and 2,3-Pentanedione 135

5 . Quantitative Risk Assessment Based on Employee Data

bronchiolitis. This generic effect would not 

adequately predict mortality proceeding from 

advancing obliterative bronchiolitis disease 

itself with high exposures to diacetyl.

5.5 Sensitivity Analyses and 

Alternate Hypotheses

NIOSH conducted sensitivity analyses to the 

impact of various parameters, variables and 

assumptions on risk estimates. NIOSH evaluated many different statistical models and 

procedures using continuous and discrete 

outcomes based on different definitions of 

impairment, different exposure metrics, and 

data from different plants. For Company G, 

the risk estimates are similar for the different 

modeling approaches and the diacetyl levels 

estimated for a given level of lifetime prevalence or risk are generally pretty close, within 

an order of magnitude.

Models where percent predicted FEV1 or FEV1/

FVC were used as the response to occupational 

diacetyl exposure showed declines in 

pulmonary function with increasing exposure, 

no matter which exposure metric was used. 

Similarly, when models looking at the incidence 

rate of pulmonary impairment, defined three 

different ways, were compared, the same 

pattern was observed revealing an unexpected 

elevation of effect in the low duration group 

compared to long durations. Exposures in 

mixers by themselves were shown not to 

account for the declining respiratory measures 

in the Company G population, and smoking 

did not exacerbate the diacetyl effects (actually 

Table 5-33. Excess lifetime risk of mortality due to 

FEV1 deficit arising from 45 yrs diacetyl exposure

Diacetyl 

ppm

FEV1 effect based on 

full population

FEV1 effect based on 

duration < 4 yr

1.0 221.6 392.1

0.5 121.1 234.1

0.2 51.2 105.0

0.1 26.1 54.6

0.05 13.2 27.8

0.02 5.30 11.3

0.01 2.65 5.66

0.005 1.33 2.84

0.002 0.53 1.14

0.001 0.27 0.57

0.0005 0.13 0.28

0.0002 0.05 0.11

0.0001 0.03 0.06

Based on multiple regression model of fall in percent predicted FEV1 with diacetyl exposure (0.5% per ppm-yr diacetyl) and on published estimates of all-cause mortality 

dependence on FEV1 after controlling for age, sex, BMI, smoking, and various cardiovascular risk factors (1.5% increase in mortality rate per 1% decline in FEV1)
136 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

was protective). Alternate formulations such as 

for dose-rate, comparing exposure effects preand post-first survey, comparing prediction 

based on diacetyl vs. acetoin, a surrogate for 

diacetyl, all supported the final choices utilized 

in the risk assessment. 

On the question of exposure uncertainties 

prior to the NIOSH surveys, particularly the 

date when widespread diacetyl exposures 

commenced at Company G, analyses specifying different years for the start of exposures 

suggested that the optimum starting year was 

about 1994 instead of 1986, but this assumption had only a small impact on the estimated 

exposure response because most employees 

surveyed were hired after 1994.

In constructing the exposure matrix for the 

plants studied, the decision was made not to 

apply the humidity correction for air samples 

below the LOD. To determine if this choice 

affected the analytical results, analyses were 

repeated having applied the correction to 

all air samples. The resulting difference in 

parameter estimate for the model of percent 

predicted FEV1 with the cumulative exposure 

term was very small: −0.500 vs. −0.499. For the 

metric, square root of cumulative exposure, 

the parameter estimate is slightly larger 

when samples < LOD are corrected: −2.77 

(uncorrected) vs. −2.82 (corrected). For the 

models of FEV1/FVC there was no change. 

Therefore there was no impact on risk estimates 

which were based on these parameter estimates.

Several alternate explanations were considered 

for the apparent variability in susceptibility:

(1) The proportion of Hispanic employees 

was higher among the short duration 

cases: Hispanics also comprised a higher 

proportion among recent hires and 

the cross-sectional surveys tended to 

reflect more recent employees due to 

high turnover. 

(2) Bias from candidate cases lacking styptom 

onset: using the date of their first qualifying 

spirometry would tend to increase rather 

than decrease the estimate of duration of 

exposure until onset and thus would not 

account for the short-duration cases. 

(3) Recall bias on symptom onset: employees 

with fast onset probably estimated symptom 

onset in relation to hire date, which is generally precisely known, not in relation to 

survey date. For example, an employee with 

3 years employment probably would recall 

that symptoms began after about 6 months 

on the job, not 2.5 years ago. 

(4) Jobs with peak exposures would favor an 

early onset: this would happen only if the 

cumulative exposure metric was underestimating the relevant exposure. This could 

occur with a positive dose-rate effect, but 

what was observed was, if anything, a 

negative dose-rate effect (Table 5-5) where 

summing the square root of air concentrations over time was a much better predictor 

than summing the square of concentrations. Serious exposure misclassification 

could cause a pattern indistinguishable 

from variable susceptibility; employees 

whose exposures were substantially underestimated would appear to respond more 

strongly (faster) with adverse health effects 

and conversely for employees whose exposures are overestimated. However, the “high 

risk” cases were not largely associated with 

specific job groups such as mixers or quality 

control; many came from the general production line, and excluding mixers did 

not reduce affect estimates. Undoubtedly 

misclassification was present but a systematic discrepancy in risk by a factor of 10, 

as observed between the short and long 

duration groups and others arising from 

misclassification is implausible.

In summary, these sensitivity analyses 

substantiated the parameters, variables, and 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 137

5 . Quantitative Risk Assessment Based on Employee Data

assumptions used in the final risk assessment 

and provide confidence in the risk estimates.

5.6 Discussion

The NIOSH HHE investigations in popcorn 

manufacturing were not specifically designed 

for quantitative risk assessment and have 

limitations in terms of unknown selection of 

study subjects and limited historical exposure 

information. Nonetheless, these observations of 

diacetyl-exposed employees have proved useful 

for risk assessment. The likelihood that the 

Company G population represents a survivor 

cohort together with the relatively high 

participation rate implies that underestimation 

of effects has probably resulted. Further 

underestimation has resulted from exclusion 

of asymptomatic cases in the analyses of 

incidence. Acting against bias from selection of 

a surviving population and missing cases is the 

possibility that participants may have included 

a more than representative proportion of cases. 

However, the high participation rate (~80%) 

limits this potential participation bias. 

The exposure metric, average exposure, which 

is simply the cumulative exposure divided 

by duration of exposure (employment 

duration since start of diacetyl use) was a 

strong predictor of pulmonary impairment in 

some analyses. It is implausible that average 

exposure, in a homogeneous population, would 

predict impairment without consideration of 

duration. Rather, a more credible explanation 

for the association of impairment with average 

exposure is the changing composition of 

the population over time since exposures 

began. The more responsive individuals 

leaving the population sooner than others 

would diminish the apparent importance of 

cumulative exposure. Thus average exposure 

might predict impairment, but it could be very 

population-specific depending on duration 

of observation and how the particular plant 

population changed over time, and would not 

be a generalizable exposure response. For this 

reason average exposure was not utilized in the 

risk assessment procedures.

Appropriate in the risk assessment and 

development of the REL for diacetyl is 

consideration that the health effects should 

be viewed in the complementary contexts of 

an individual employee’s risk of impairment 

which is the clinician’s measure of impact, 

and the risk incurred by the population 

of employees with diacetyl exposure. The 

American Thoracic Society, in a statement 

on the effects of air pollution, concluded that 

shifts in the respiratory health of a population, 

resulting from some exposure, that diminish 

individual reserve function, are adverse “even 

in the absence of the immediate occurrence 

of frank illness” [ATS 2000]. In the clinical 

context, if an employee’s FEV1/FVC is less 

than 0.7 (or FEV1 less than or equal to 80%), 

that would be considered mild COPD [GOLD 

2011]. Similarly, if diacetyl exposure decreases 

the mean pulmonary function of the exposed 

population by some small increment, this too 

could be considered an adverse event [ATS 

2000]. 

The health significance of small spirometry 

changes, such as a 1% decline in FEV1 after 2 

years of exposure at 1 ppm diacetyl, depends 

partly on whether such changes are early 

indications of lung pathology that eventually 

would manifest as obliterative bronchiolitis. 

In studies of obliterative bronchiolitis arising 

from lung transplantation, unrelenting 

irreversible FEV1 decrements are observed 

that ultimately lead to the diagnosis of 

obliterative bronchiolitis and fatal disease 

[Heng et al. 1998]. However, incomplete 

knowledge concerning the natural history of 

obliterative bronchiolitis development with 

diacetyl exposure is a limitation in the present 

risk assessment. Not only is risk for mortality 

increased, as estimated in this risk assessment, 
138 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

quality of life is degraded [Ferrer et al. 2002] 

and risk is increased for cardiovascular disease 

and progressive respiratory disease [Cullen 

et al. 1983; Ebi-Kryston et al. 1989; Knuiman 

et al. 1999; Kuller et al. 1989; Schroeder et 

al. 2003; Wise 2006]. The decrease in FEV1

predicted after working for 10 years in diacetyl 

exposures of 0.2 ppm (about 1% loss) is 

comparable to changes observed in children, a 

more vulnerable population, exposed to levels 

of air pollution that lead to clinical impairment 

in later life [Gauderman et al. 2004]. 

Variation in susceptibility poses issues for risk 

assessment. If less-susceptible individuals are 

remaining in employment longer, the estimated 

exposure response for long durations when 

applied to a hypothetical population of 1,000 

employees employed 45 years, will generate 

excess risk values that understate the true risk 

of a workforce that turns over more often. 

All of the risk assessment procedures used 

here assume some degree of low-dose linearity, 

with effects diminishing proportionally with 

decreasing exposure levels that are held 

constant over 10 or 45 years. Model linearity 

was observed particularly after limiting the 

population to < 4 yr duration. Moreover a 

significant fraction of career-average exposures 

fell below 0.01 ppm (17% of employees) a factor 

of only 2.0 higher than the proposed REL. Thus 

low-dose extrapolation was limited. Below 0.01 

ppm, there can be some significant departure 

from linearity although diversity in response 

would tend to favor linearity to lower levels 

[Clewell and Crump 2005; National Research 

Council 2009].

5.7 Conclusion 

Excess prevalence (BMD) and lifetime risk 

estimates variously derived for 45 years of 

diacetyl exposure were similar, based on 

Company G analyses (Table 5-34). Impairment 

has been defined here as pulmonary function 

falling below the lower limit of normal. The 

BMD estimates for excess prevalence of FEV1 

impairment are within a factor of 2.0 of the lifetable estimates of excess lifetime risk (1) using 

case definitions 2 and 3 (ignoring smoking) 

and (2) for excess mortality. Excess risk of 

1/1,000 corresponds to approximately 0.001–

0.005 ppm diacetyl (3.5-17.5 μg/m3) in the full 

Company G population. NIOSH has selected 

Company G risk estimates as the basis for a 

recommended REL because Company G had 

the most extensive and representative diacetyl 

exposure data and largest body of respiratory 

outcomes data. In the pooled Company K-L 

population, determined by NIOSH to be a 

less adequate basis for risk assessment, the 

benchmark dose analysis for 1/1,000 excess risk 

corresponds to approximately 0.0004–0.0005 

ppm diacetyl. Diacetyl exposures predicted to 

result in various levels of risk are displayed in 

Table 5-35. 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 139

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-34. Risk assessment synthesis: excess prevalence or

lifetime risk (per thousand) for 45-yr exposure to diacetyl

Diacetyl

ppm

Method

BMD—Excess prevalence of impairment 

(per thousand)

Life-table—Excess lifetime risk 

(per thousand)

FEV1

(LLofN)

FEV1/FVC

(LLofN)

FEV1

(LLofN)

< 4 yrs

FEV1/FVC

(LLofN)

< 4 yrs

Incidence

case defn 2

Incidence

case defn 3

All-cause mortality

All < 4 yr

0.05 12.3 6.8 27.8 36.4 20.7 15.8 13.2 27.8

0.02 4.8 3.2 10.0 13.3 8.4 6.4 5.3 11.3

0.01 2.5 2.1 5.3 7.0 4.2 3.2 2.7 5.7

0.005 1.3 1.0 2.6 3.7 2.1 1.6 1.3 2.8

0.004 1.1 0.8 1.9 3.4 1.7 1.3 1.1 2.3

0.003 0.6 0.6 1.5 3.0 1.3 1.0 0.8 1.7

0.002 0.4 0.4 1.2 2.4 0.9 0.6 0.5 1.1

0.001 0.2 0.3 0.4 1.0 0.4 0.3 0.3 0.6

case definition 2: FEV1/FVC < LLofN 

case definition 3: FEV1 < LLofN and FEV1/FVC < LLofN 

BMD: Based on empirical benchmark dose procedure, the predicted number of individuals with FEV1 or FEV1/FVC < lower limit of normal that would be prevalent in a population of 1000 with 45 yr exposure

Excess Lifetime Risk: Based on life-table analysis, the predicted number of new cases in a population of 1,000 starting with exposure at age 

20 through 65, until age 85 

1/1000 risk exposures in bold; based on rate model not including smoking determinants.
140 Occupational Exposure to Diacetyl and 2,3-Pentanedione

5 . Quantitative Risk Assessment Based on Employee Data

Table 5-35. Risk assessment synthesis: diacetyl exposure levels

(ppm) over 45 yrs predicting excess prevalence or lifetime risk 

Method 

Life-table: Excess lifetime risk 

BMD: Excess prevalence impairment Case onset, definition 3 Mortality

Excess

risk

FEV1

(LLofN)

FEV1/FVC

(LLofN)

FEV1

(LLofN)

< 4 yrs

FEV1/FVC

(LLofN)

< 4 yrs all

for nonsmokersfor 

smokers all < 4 yr

1/10 0.30 0.60 0.15 0.11 0.30 0.10 0.50 0.40 0.20

1/100 0.04 0.08 0.02 0.02 0.03 0.01 0.05 0.04 0.02

1/1000 0.004 0.005 0.002 0.001 0.003 0.0009 0.005 0.004 0.002

1/10000 0.0004 0.0005 0.0002 0.0001 0.0002 0.00009 0.0004 0.0004 0.0002

1/100000 0.00004 0.00005 0.00002 0.00001 0.00002 0.000009 0.00004 0.00004 0.00002

case definition 3: FEV1 < LLofN and FEV1/FVC < LLofN 

BMD: Based on benchmark dose procedures, the exposure for 45 yr predicted to confer the specified excess prevalence of FEV1 or FEV1/

FVC < lower limit of normal 

Excess Lifetime Risk: Based on life-table analysis, the exposure at age 20 through 65 predicted to confer the specified excess life-time risk 

< 4 yrs: analyses based on population with < 4 yr exposure to DA, thought to be less affected by healthy employee survivor effect

1/1000 risk exposures in bold.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 141

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 145

6 Quantitative Risk Assessment Based 

on Animal Data

6.1 Introduction

6.1.1 Diacetyl

Dose-response data for diacetyl toxicity in 

laboratory animals are available, and there are 

limited but useful animal data on the toxicity 

of 2,3-pentanedione. Although the NIOSH 

REL for diacetyl is based on the analysis of 

human data described in Chapter 5, NIOSH 

has assessed the animal data for diacetyl 

to determine whether they are consistent 

with the human data. For 2,3-pentanedione, 

NIOSH has conducted a comparative potency 

analysis, comparing the toxicity of inhaled 

2,3-pentanedione to that of diacetyl. These 

quantitative risk assessments are described 

below. NIOSH interpretation of the findings 

and implications for occupational exposure 

recommendations for diacetyl are described 

below and in Chapter 7. 

Laboratory animal studies designed to evaluate 

the effects of exposure to butter flavoring 

vapor or of diacetyl alone have demonstrated a 

relationship between exposure and respiratory 

effects. In rats exposed by inhalation to 

butter flavoring vapor for 6 hours (diacetyl 

concentrations ranged from 203 to 352 ppm), 

rhinitis (at the lowest exposure concentration) 

and bronchitis (at the higher two exposure 

concentrations) were observed one day after 

exposure [Hubbs et al. 2002]. In a follow-up 

study rats were exposed by inhalation to 

diacetyl (intermittently or continuously for up 

to 6 hours), which resulted in various adverse 

respiratory effects including epithelial necrosis 

and inflammation in the nose, larynx, trachea, 

and bronchi [Hubbs et al. 2008]. The nasal 

region was observed to be the most sensitive. 

Morgan et al. [2008] reported similar adverse 

respiratory effects in mice exposed by inhalation 

to diacetyl for up to 12 weeks. Adverse nasal 

and lung effects were observed with the latter 

found in the bronchial, peribronchial, and 

peribronchiolar regions. 

The NTP has issued findings from a 90-day 

inhalation study of diacetyl in both mice and 

rats [National Toxicology Program 2011]. 

Adverse effects were observed in the nose, 

larynx, trachea, and bronchi in mice and rats. 

Because the 2011 NTP study had the longest 

exposure durations among all experimental 

animal studies, included two species, and used 

more animals per dose group than the Morgan 

et al.[2008] study, it was used in the doseresponse analysis to BMDs, the lower bound 

on the BMDs (BMDLs), and corresponding 

human equivalent concentrations (HECs), as 

discussed below.

6.1.2 2,3-Pentanedione 

Histopathological data from repeatedexposure inhalation toxicology studies with 

2,3-pentanedione were first published in 2012, 

but are limited to 2-week exposures using 

small numbers of animals [Morgan et al. 2012]. 

Although these data are limited, it is possible to 

compare the toxicity produced by 2,3-pentanedione to that produced by diacetyl under similar 

conditions, and thus estimate the potency of 

2,3-pentanedione relative to diacetyl. Therefore, 
146 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

the limited toxicological data for 2,3-pentanedione are not used directly to establish a REL 

for 2,3-pentanedione, but only to develop an 

estimate of the toxic potency of 2,3-pentanedione relative to that of diacetyl. Like diacetyl, 

2,3-pentanedione is a reactive alpha-dicarbonyl 

compound that can damage protein [Epperly 

and Dekker 1989; Morgan et al. 2016]. In 

acute inhalation studies, 2,3-pentanedione has 

respiratory epithelial toxicity comparable to 

diacetyl [Hubbs et al. 2012]. Recently, bronchial 

fibrosis has been documented in rats inhaling 

either 2,3-pentanedione or diacetyl for 2 weeks 

[Morgan et al., 2016].

6.2 Methods

6.2.1 Data

6.2.1.1 Diacetyl

The response data that were analyzed were 

obtained from the experimental study reported 

by the NTP [2011]. Male and female WistarHan rats and male and female B6C3F1 hybrid 

mice were exposed to diacetyl vapors at 

concentrations of 6.25, 12.5, 25, 60, and 100 

ppm, 6 hours per day, 5 days per week, for 13 

weeks. The microscopic evaluations of tissues 

from the larynx, lung, nose, and trachea 

described whether or not one or more lesions 

were detected, the types of lesions that were 

detected, and the assignment of a numeric 

score describing the lesion’s severity on an 

ordinal scale (1-minimal, 2-mild, 3-moderate, 

4-marked) for each type that was detected. 

Descriptions of the types of lesions observed 

among rats and mice that were considered for 

this analysis are given in Tables 6-1 and 6-2, 

respectively.

6.2.1.1 2,3-Pentanedione

The results of a 2-week inhalation study of 

2,3-pentanedione toxicity were reported by 

Morgan et al. [2012]. Individual animal data 

Table 6-1. Respiratory system lesions 

observed in rats exposed to diacetyl 

that were considered for this analysis

Tissue Response

Larynx Inflammation, Chronic Active

Larynx Epithelium, Necrosis

Larynx Respiratory Epithelium, Hyperplasia

Larynx Respiratory Epithelium, Metaplasia, 

Squamous

Larynx Respiratory Epithelium, Regeneration 

(Females only)

Larynx Squamous Epithelium, Hyperplasia*

Lung Infiltration Cellular, Histiocyte

Lung Inflammation, Eosinophil or Acute

Lung Bronchiole, Epithelium, Hyperplasia

Lung Bronchus, Inflammation, Chronic 

(Males only)

Lung Bronchus, Epithelium, Hyperplasia†

Lung Bronchus, Epithelium, Necrosis

Lung Bronchus, Epithelium, Regeneration

Nose Inflammation, Suppurative

Nose Lymphoid Tissue, Hyperplasia

Nose Olfactory Epithelium, Atrophy

Nose Olfactory Epithelium, Degeneration

Nose Olfactory Epithelium, Metaplasia, 

Respiratory

Nose Olfactory Epithelium, Necrosis

Nose Respiratory Epithelium, Hyperplasia

Nose Respiratory Epithelium, Metaplasia, 

Squamous

Nose Respiratory Epithelium, Necrosis

Nose Turbinate, Atrophy

Trachea Inflammation, Chronic Active

Trachea Epithelium, Regeneration

Trachea Epithelium, Hyperplasia

Trachea Epithelium, Metaplasia, Squamous

Trachea Epithelium, Necrosis

*

Includes two males classified as having mild “Squamous 

Epithelium, Hyperplasia, Atypical”

†

Includes three males and four females classified as having 

mild “Bronchus, Epithelium, Hyperplasia, Atypical”
Occupational Exposure to Diacetyl and 2,3-Pentanedione 147

6 . Quantitative Risk Assessment Based on Animal Data

from this study were graciously provided for 

this analysis by Dr. Daniel Morgan, National 

Institute for Environmental Health and 

Safety (NIEHS) (personal communication 

to Dr. Lauralynn Taylor McKernan, NIOSH, 

November 30, 2010). These data describe the 

pathological responses of male and female 

Wistar-Han rats and B6C3F1 mice exposed to 

2,3-pentanedione by inhalation for 6 hours per 

day, 5 days per week, for 2 weeks plus 2 days. 

The exposure concentrations were 0 ppm, 50 

ppm, 100 ppm, and 200 ppm, with six animals 

per dose group; nasal, tracheal, and pulmonary endpoints were assessed. The tissue and 

pathological endpoints that could be modeled 

successfully for both 2,3-pentanedione and 

diacetyl (for comparative purposes) are listed 

in Table 6-3.

In addition to the 13-week NTP bioassay data 

described above for diacetyl, the 2,3-pentanedione data were also compared to data for 

diacetyl from Morgan et al. [2008]. These data 

describe the pathological responses of male 

C57Bl/6 mice exposed to diacetyl by inhalation 

for 6 hours per day, 5 days per week, for either 6 

or 12 weeks. The exposure concentrations were 

0 ppm, 25 ppm, 50 ppm, and 100 ppm, with 

five animals per dose group. Nasal, tracheal, 

and pulmonary endpoints similar to those 

examined in the 2,3-pentanedione study were 

assessed. In addition to the data in the Morgan 

et al. [2008] publication, tables of individual 

Table 6-2. Respiratory system lesions observed in mice exposed 

to diacetyl that were considered for this analysis

Tissue Response Tissue Response

Larynx Inflammation, Chronic Active Nose Olfactory Epithelium, Atrophy

Larynx Epithelium, Necrosis Nose Olfactory Epithelium, Metaplasia, 

Respiratory

Larynx Respiratory Epithelium, Hyperplasia Nose Respiratory Epithelium, Metaplasia, 

Squamous

Larynx Respiratory Epithelium, Metaplasia, 

Squamous*

Nose Respiratory Epithelium, Necrosis

Larynx Respiratory Epithelium, Regeneration Nose Respiratory Epithelium, Regeneration¶

Larynx Squamous Epithelium, Hyperplasia† Nose Turbinate, Atrophy

Lung Bronchus, Inflammation, Chronic Trachea Inflammation, Chronic Active

Lung Bronchus, Epithelium, Hyperplasia‡ Trachea Epithelium, Degeneration or Regeneration**

Lung Bronchus, Epithelium, Regeneration§ Trachea Epithelium, Hyperplasia

Nose Inflammation, Suppurative Trachea Epithelium, Metaplasia, Atypical Squamous

*

Includes lesions classified as “Respiratory Epithelium, Metaplasia, Atypical Squamous”

†

Includes lesions classified as “Squamous Epithelium, Hyperplasia, Atypical”

‡

Includes lesions classified as “Bronchus, Epithelium, Hyperplasia, Atypical”

§

One male classified as having a minimal “Bronchus, Epithelium, Degeneration” lesion was pooled with 10 other males having a 

regenerative response.

¶

One male and two females classified as having a “Respiratory Epithelium, Degeneration” lesion were pooled with 20 other males, 

and 20 other females having the regenerative response.

**Seven males and seven females had only the regenerative response, and 12 males and 11 females had only the degenerative 

response.
148 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

animal’s responses were provided by Dr. Daniel 

Morgan, NIEHS (personal communication to 

Dr. Christine Sofge, NIOSH, November 18, 

2008, and November 20, 2008).

6.2.2 Analytical approach

An empirical approach based on parametric 

regression modeling of the ordinal response 

data was adopted to maximize the information 

available for analysis from the limited numbers 

of rodents* in order to assess the potency of 

diacetyl to increase risk and to assess the relative potency of the two chemicals.

6.2.2.1 Benchmark concentration analysis 

for rats exposed to diacetyl

The assessment of the potency of diacetyl to 

increase risk employed the benchmark dose 

approach that was originally proposed for risk 

assessment of non-cancer responses by Crump 

[1984]. It provides a general framework that 

accommodates a range of responses including 

responses observed on dichotomous†, ordinal, 

and continuous scales. It has received extensive 

*

5 ≤ n ≤10 rodents were used per species-sex-exposure 

group. 

†

Dichotomous responses are often referred to as 

quantal responses.

development over the past three decades, and it 

has become an accepted approach for risk 

assessment [EPA 2012]. Benchmark concentration (BMC) estimates for the pathological 

endpoints listed in Table 6-1 (for rats) were 

based on modeling of the exposure concentrations and the associated pathology. In order to 

avoid the loss of information inherent in dichotomizing ordinal response data, a categorical 

regression procedure for ordinal data was used 

to estimate benchmark concentrations. 

Categorical regression has been previously used 

in the analysis of toxicological data with multiple levels of severity [Guth et al. 1997; Haber et 

al. 2001]. The severity scores‡ for each tissue and 

type of lesion were assumed to be samples from 

a multinomial distribution following a complementary§

 cumulative logistic model fitted 

separately for each species and sex as follows:

where

Yci denotes the corresponding severity 

score of the ith rodent exposed to concentration, concc,

j Є element of {observed severity scores 

excluding zero} for the corresponding 

tissue and type of lesion,

Pr(Yci≥j)denotes the expected proportion of response score Yci greater 

than or equal to j, each αj is an 

unknown real-valued parameter 

with αj' < αj for j'> j, and β is an 

unknown real-valued parameter 

describing the slope of the effect of 

concentration on the logit scale.

‡

When no evidence of the lesion being modeled was 

detected a severity score of zero (0) was assigned.

§

The term complementary discerns this model from an 

equivalent cumulative logistic model of Pr(Yci≥j) .

Table 6-3. Pathological endpoints associated 

with exposure to 2,3-pentanedione that were 

modeled in this analysis

Tissue Description of response

Lung Bronchus, Inflammation, Chronic

Lung Bronchus, Epithelium, Regeneration

Nose Inflammation, Suppurative

Nose Olfactory Epithelium, Atrophy

Nose Respiratory Epithelium, Metaplasia

Nose Respiratory Epithelium, Necrosis

Nose Respiratory Epithelium, Regeneration
Occupational Exposure to Diacetyl and 2,3-Pentanedione 149

6 . Quantitative Risk Assessment Based on Animal Data

The logistic model is based on the logit transformation above which maps the range of 

expected response proportions, 0<p<1, to 

(−∞, ∞); hence, models defined in terms of the 

transform constrain the expected proportions 

to the appropriate range. It is readily parameterized so that this form of the systematic 

relation applies under varying conditions that 

are consistent with biological considerations 

including the redefinition of the response categories by merging them [McCullagh 1980]; this 

specifically includes merging them to form the 

dichotomous responses more familiar to toxicology while preserving the interpretations of 

the model parameters thereby facilitating its 

application. The method of maximum likelihood was applied in order to fit¶

 the model, 

and a likelihood ratio (LR) test for a (non-null) 

dose-response was performed. Adequacy of 

the fit was assessed by performing two statistical tests, i.e., a score test for separate slopes (a 

slope for each unique value of j) and a LR test 

for an unrestricted multinomial distribution. 

The null distribution of the statistic of each 

test was approximated by its asymptotic chisquare distribution. For those models having 

a significant dose-response (P<0.05) and an 

adequate fit (P>0.05) on both tests, BMCs were 

estimated corresponding to the concentrations 

that increased expected proportions by 0.10 

over controls** for severity scores of 1+ (lesion 

was at least minimal) and 2+ (lesion exceeded 

minimal severity). Ninety-five percent confidence intervals for the BMC were calculated 

from percentiles of 200,000 samples of the 

asymptotic multivariate normal distribution 

of the MLE of the model parameters††; both 

a two-sided 95% confidence interval and a 

¶

The Logistic procedure of SASTM 9.3 was used. **(i.e., a benchmark response of 0.10 for “added risk”)

††The function, rmvnorm, of Splus with mean=MLE 

and covariance matrix=estimate of Cov(MLE) 

was used.

lower one-sided 95% confidence limit (BMCL) 

were estimated.

6.2.2.2 Benchmark concentration analysis 

for mice exposed to diacetyl

Benchmark concentration estimates for the 

pathological endpoints listed in Table 6-2 (for 

mice) were developed as described above for 

the rat data; however, an analysis of the residual 

errors of the fitted models provided substantial 

evidence against the model for the data on mice 

(Figure 6-1). 

These residuals have mean equal to zero asymptotically if the linear-in-concentration model is 

correct. However, the distribution of the residuals of Figure 6-1 is shifted above zero at 50 

ppm corresponding to underprediction and the 

distribution is shifted below zero corresponding to overprediction at 100 ppm. Figure 6-1 

provides support for making a modification 

of the dose-response model in a manner that 

allows for a reduction of the rate of increase of 

the response at high doses. Because mice are 

able to substantially alter their breathing rates 

in a dose-dependent manner when exposed 

[Larsen et al. 2009; Morgan et al. 2008] the 

model of the data for mice was modified to 

include a quadratic dose term to allow it to 

more closely fit the data in the high-dose region 

of the dose-response relationship; this term was 

parameterized to represent a directly proportional relationship of the change in breathing 

rate with concentration relative to the breathing 

rate of the controls. The resulting estimate for 

male mice exposed to diacetyl was compared 

with corresponding ventilation measurements provided by Dr. Daniel Morgan, NIEHS 

(personal communication to Randall Smith, 

NIOSH, June 5, 2014). In addition, two parameters allowing for adjustment of the intercepts 

of each sex and a third parameter allowing for 

adjustment of the effect of exposure were added 

to the model to account for the varying durations of these studies. This model was further 
150 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

extended to incorporate the comparative 

potency analysis of 2,3-pentanedione relative 

to diacetyl and incorporated an allowance for 

the responses of each mouse to be correlated by 

including random effects. It is described below 

in section 6.2.2.7. 

6.2.2.3 Extrapolation of rodent benchmark 

concentrations to humans

Extrapolation of rodent BMCs to humans 

was based on a PBPK/CFD model for diacetyl 

[Gloede et al. 2011; Morris and Hubbs 2009]. 

The Gloede et al. [2011] extension of the Morris 

and Hubbs [2009] model predicts tissue concentrations of diacetyl for mucosal surfaces 

in the nose, trachea, bronchi, and bronchioles 

of rats and humans exposed to 1 ppm diacetyl. Nose-breathing and mouth-breathing 

humans are considered, as well as the effects 

of light exercise as might be expected to occur 

in the workplace. The Gloede et al. [2011] 

model assumes mouth breathing during light 

0 20 40 60 80 100

-6 -4 -2 0 2

4

Concentration (ppm)

Pearson residual ( mice )

Symbol Lesion score 

Triangle Minimal 

Square Mild 

Circle Moderate

 

Color Gender 

 Red Females 

 Blue Males 

Figure 6-1. Pearson residuals of complementary cumulative logistic 

models with linear effect of concentration fitted to data on mice. The 

points have been slightly jittered horizontally to improve resolution.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 151

6 . Quantitative Risk Assessment Based on Animal Data

exercise conditions. For extrapolation purposes, an 8-hour work day was considered to 

consist of 2.5 hours of sedentary exposure and 

5.5 hours of light exercise, as described by the 

International Commission on Radiological 

Protection (ICRP) human respiratory tract 

model [ICRP 1994]. The ICRP model assumes 

20 breaths per minute and a tidal volume of 

1,250 mL for light exercise and 12 breaths per 

minute and a tidal volume of 625 mL for sedentary sitting, for a total inhalation volume of 

9.6 m3 in an 8-hour work day. Therefore, to 

extrapolate from rodents to humans, the BMC 

estimates described above were adjusted by a 

weighted average of the rat:human ratios of the 

predicted tissue concentrations for a particular 

anatomical region, under sedentary and light 

exercise conditions. The Gloede et al. [2011] 

estimates incorporating tissue metabolism 

(Vmax for the rat, and Kcat for humans) were 

used, because local metabolism is predicted 

to impact significantly on the local tissue concentration [Gloede et al. 2011] (Table 3). For 

example, the predicted tissue diacetyl concentration for the proximal tracheal mucosa 

of a rat exposed to 1 ppm diacetyl is 0.33 μM, 

while the predicted tissue concentration for the 

same anatomical region is 1.4 μM in a sedentary nose-breathing human and 2.5 μM in a 

mouth-breathing exercising human. The rat 

BMCs based on pathological changes to this 

anatomical region were divided by a factor of 

(1.4 μM * 2.5 hours + 2.5 μM * 5.5 hours)/(0.33 

μM * 6 hours), or 8.71. The factor of 6 hours 

in the denominator adjusts for the 6-hour/day 

duration of the experimental exposures, as 

compared to the 8-hour workday assumed for 

occupational exposures. Gloede et al. [2011] 

did not report tissue concentration estimates 

for the larynx; BMC extrapolation for this 

region was based on the tissue concentrations 

estimated for the proximal trachea. Gloede et 

al. [2011] reported tissue concentrations for 

both mainstem and small bronchi, and BMC 

extrapolation for bronchial endpoints were 

based on the mean of the rat:human ratios of 

tissue concentrations for mainstem bronchi 

and small bronchi. Rat to human scaling for 

the alveoli was based on the estimated fractional penetration of diacetyl through the 

bronchioles in the Gloede et al. PBPK model, 

provided by Dr. John Morris, University of 

Connecticut (personal communication to 

Dr. David A. Dankovic, NIOSH, November 

8, 2012). The rat:human extrapolation factors 

used are shown in Table 6-4.

6.2.2.4 Extrapolation of BMCs and BMCLs 

from the mouse to the rat

Because no PBPK model for diacetyl exposures 

in the mouse is currently available, the rat PBPK 

model [Gloede et al. 2011] was extended to the 

mouse using the EPA reference concentration 

(RfC) methodology [EPA 1994]. In the RfC 

methodology, the deposition and uptake 

of volatile chemicals are estimated from a 

combination of chemical characteristics (i.e., 

reactivity and solubility) and the physiological 

characteristics of the relevant species (i.e., 

minute ventilation and the surface area of 

the relevant portion of the respiratory tract). 

Diacetyl is classified as a “category 1” gas 

in the RfC methodology because of its high 

water solubility. Category 1 gases are not 

expected to reach the pulmonary region in 

high concentration, but rather to be deposited 

primarily in the upper respiratory tract and 

the tracheobronchial region. This is consistent 

with the behavior of diacetyl in the Gloede et 

al. [Gloede et al. 2011] PBPK model, so that 

the classification of diacetyl as a category 1 gas 

appears to be appropriate.

Interspecies dosimetric adjustments via the RfC 

methodology are based on an estimate of the 

regional gas dose ratio (RGDR). The RGDR 

estimates the ratio of gas deposition with a 

given respiratory tract region in the two species 

being compared. 
152 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

For the ET region, the RGDR is calculated [EPA 

1994], eqn. 4-18, as:

where: 

VE = minute volume (mL/min = cm3/min)

SA = surface area (cm2

)

ET = a subscript denoting the extrathoracic region

A, B = subscripts denoting experimental animal and target species, 

respectively

For the TB region, the RGDR is calculated 

[EPA 1994], eqn. 4-22, as:

where:

VE = minute volume (mL/min = cm3

/min)

SA = surface area (cm2

)

TB = a subscript denoting the tracheobronchial region

ET = a subscript denoting the extrathoracic region

A, B = subscripts denoting experimental animal and target species, 

respectively

Table 6-4. Factors for rodent-to-human extrapolation of airway tissue 

concentrations of diacetyl, based on Gloede et al. [2011]

Species Human Human (light work) Human (light work)

Breathing via nose mouth mouth nose + mouth nose + mouth

rest/exercise rest rest exercise rest + exercise* rest + exercise*

Human-to-rat ratio† Human-to-mouse ratio‡

Proximal nose 1.6 0 0 0.67 0.3

Proximal trachea 4.2 6.1 7.6 8.7 2.7

Mainstem bronchi 10.0 14.0 21.0 23.0 7.3

Small bronchi 7.2 10.0 32.2 32.0 10.0

Average bronchi§ 8.6 12.0 26.6 28.0 8.7

Bronchioles 5.0 7.3 40.9 40.0 12.0

Alveoli¶ 4.69 — 15.0 15.7 4.9

“Light work” was estimated to be a combination of 2.5 hours at rest, with nasal breathing, plus 5.5 hours of exercise, with mouth 

breathing, per 8-hour work day; this was compared to a 6-hour/day exposure for rodents in the experimental studies.

†

Rat-to-human scaling based on the overall catalytic rate, Kcat, in Gloede et al. [2011]Table 3, except as noted below for alveoli.

‡

Mouse-to-human scaling assuming mouse is 2.4 times as sensitive as the rat for nasal effects and 3.2 times as sensitive for tracheobronchial effects, based on the regional gas dose ratio (see section 6.2.2.4)

§

“Average bronchi” = arithmetic mean of values for mainstem and small bronchi

¶

Rat to human scaling for the alveoli was based on the estimated fractional penetration of diacetyl through the bronchioles in the 

Gloede et al. PBPK model.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 153

6 . Quantitative Risk Assessment Based on Animal Data

The values assumed for VE and SA, and the 

resulting RGDR values for mouse-to-rat extrapolation, are shown in Table 6-5. The rat VE value 

is based on data from Gloede et al. [2011], and 

the mouse VE was taken from Morgan et al. 

[2008]. The SA values are from EPA [1994].

The RGDR is used to adjust a point of departure 

(POD), i.e., a BMC or BMCL in the laboratory 

species to an equivalent concentration in the 

target species as follows:

PODBEC = PODA * RGDR

where:

PODBEC = POD equivalent concentration 

in the target species;

PODA = POD in the experimental species; 

and

RGDR = Species A-to-species B regional 

gas dose ratio for the appropriate 

region of the respiratory tract. 

Although the RGDR is typically used to develop 

human equivalent concentrations from experimental animal data, in this case it is used to 

develop a rat equivalent concentration for a 

point of departure estimated from experimental data in the mouse. The Gloede et al. [2011] 

PBPK model is then used to extrapolate from 

the rat equivalent concentration to a human 

equivalent concentration.

6.2.2.5 Duration adjustment and final 

human equivalent concentration 

conversions

Adjustment for the daily duration of exposure 

(6 hours/day for the NTP experimental study 

vs. 8 hours/day assumed for occupational 

exposures) is included in the PBPK modelbased extrapolation from rodents to humans, 

as described in section 6.2.2.2 above; therefore, 

no additional adjustment for exposure hours 

per day is needed. The experimental exposure 

protocol of five exposures per week matches 

the assumed occupational exposure pattern, 

so that no adjustment for days exposed per 

week is required in extrapolating from animals 

to humans. Occupational exposures may take 

place for an entire working lifetime, which 

is assumed to be up to 45 years in duration. 

Ideally, the datasets used for quantitative risk 

assessment of occupational exposures to toxicants would include data from 2-year rodent 

bioassays; however, in this case the available 

data are limited to exposures of 13 weeks or 

less. An 8-fold dosimetric adjustment (104 

weeks/13 weeks) could be considered in order 

to account for this discrepancy; however, 

this appears to be unnecessary for diacetyl. 

Table 6-5. Calculation of RGDR for mouse-to-rat extrapolation

Species VE

*

 (mL/min) URT SA† (cm2) TB SA‡ (cm2) URT RGDR§ TB RGDR¶

Rat 264.0 15 22.5 — —

Mouse 128.5 3 3.5 2.4 3.2

*

Minute volume ventilation

†

Upper respiratory tract surface area

‡

Tracheobronchial surface area

§

Mouse-to-rat regional gas dose ratio for the upper respiratory tract

¶

Mouse-to-rat regional gas dose ratio for the tracheobronchial region
154 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

This conclusion is based on the analysis of 

Allen [2009a], who concluded that the 6- and 

12-week mouse experiments had response rates 

that could be modeled together (i.e., the duration of the experiment could be ignored) for all 

the lesions analyzed; there did not appear to be 

a progression toward higher rates of response 

or more severe responses when the exposure 

level remained the same but the duration of 

exposure was increased from 6 to 12 weeks. 

However, because of the small number of 

animals used in this study, the power to detect 

differences between the 6-week and 12-week 

experiments is limited. As a consequence of 

the limited duration of the experimental studies 

and the limited ability to detect differences 

between the responses at 6 and 12 weeks, the 

possibility of increased toxicity with lifetime 

exposure cannot be entirely ruled out. This possibility was addressed through the application 

of an uncertainty factor (UF) – discussed below 

– rather than a dosimetric adjustment.

6.2.2.6 Application of uncertainty factors

The HECs are estimates of frankly toxic 

exposure levels, and must be adjusted by the 

application of UFs to allow for uncertainty 

in animal-to-human extrapolation, interindividual variability, and less than lifetime 

exposure. In general, these UFs are assumed to 

be 10-fold for animal-to-human extrapolation 

and another 10-fold for interindividual variability. The animal-to-human extrapolation can 

be subdivided into a factor of 4 for pharmacokinetics and a factor of 2.5 for interspecies 

variability in susceptibility [WHO 1994]. In this 

case, the interspecies pharmacokinetic factor is 

replaced by the use of the Gloede et al. [2011] 

pharmacokinetic model, leaving an interspecies 

UF of 2.5. The UF for interindividual variability can be subdivided into two factors of √10, 

or 3.2, one for interindividual variability in 

pharmacokinetics and the other for interindividual variability in susceptibility [WHO 1994]. 

Because the toxicity of diacetyl occurs at the 

point of contact with respiratory tract mucosa 

there is relatively little opportunity for interindividual variability in pharmacokinetics, and 

so the first subfactor is not applied. However, 

interindividual variability in susceptibility 

to toxicity cannot be ruled out; therefore, a 

factor of 3.2 is applied. In addition, a factor of 

3 is applied for conversion from subchronic to 

chronic exposure. When the three factors (3.2-

fold for interindividual variability, 2.5-fold 

for interspecies variability, and 3-fold for subchronic to chronic) are multiplied, the resulting 

total UF is 24. 

6.2.2.7 Joint analysis of the data on 

mice from the diacetyl and 

2,3-pentanedione bioassays

To avoid the loss of information inherent in 

dichotomizing ordinal data the severity scores 

of each type of lesion observed among nasal 

and lung tissues were modeled as having been 

sampled from conditional multinomial distributions given the unobserved random effects 

associated with each mouse described by the 

following family of complementary cumulative 

logistic models:

= αsjr(t) + ubskci + ωs∙τbskci

+fbskctiβsjr(t) {m(s, k, concbskci, t, τbskci; θsr(t), 

φskt, γs)} ∙ concbskci ,

where 

αsjr(t) + uskci + ωs∙τbskci describes effects in 

the absence of exposure, 

fbskctiβsjr(t) {m(s, k, concbskci, t, τbskci; θsr(t), 

φskt, γs)} ∙ concbskci

 describes effects of exposure 

and 

b indexes the bioassay study

s indexes sex,
Occupational Exposure to Diacetyl and 2,3-Pentanedione 155

6 . Quantitative Risk Assessment Based on Animal Data

k = 0 ←→ 2,3-pentanedione exposure 

and k = 1 ←→ diacetyl exposure, 

bkc identifies the exposure group and 

concbkc is the corresponding exposure 

concentration, 

i = 1, ..., nbskc indicates each of the mice 

within the exposure group identified 

by bskc and concbskci denotes the corresponding exposure concentration,

r(t) identifies the response lesion, r nested 

within tissue, t, (lung or nasal),

Ybskcr(t)i is the response variable that is 

integer-valued based on the assigned 

severity score and it ranges over 

{0,1,2,3} for all response lesions‡‡

except necrosis of the respiratory epithelium of the nose where the range 

was {0,1,2},

Pr(Ybskcir(t) ≥ j) represents the expected 

proportion having response severity 

score greater than or equal to j for j Є 

{1, ..., max (Ybskcr(t)i )}, 

αsjt(r) denotes the intercept parameters 

for lesion r(t) which are subject to 

constraints 

αs2t(r)−αs1t(r)=∆αs2<0 and

αs3t(r)−αs2t(r)=∆αs3<0 thus ensuring§§

αs3t(r)<αs2t(r)<αs1t(r),

ubskci~N(0, σ 2

su) is a normally distributed 

random effect associated with the 

i

th mouse of bskc; likelihood ratio 

tests of null values of the variance 

‡‡When no evidence of the lesion being modeled was 

detected a severity score of zero (0) was assigned.

§§Hence, the requirement that 

r(Ykcit(r) ≥ 3) < Pr(Ykcit(r) ≥ 2) < Pr(Ykcit(r) ≥ 1) is satisfied 

for the controls.

parameters, σ 2

us , were performed¶¶

and subject to being incorporated 

into the model.

ωs∙τbskcirepresents an adjustment to 

the intercepts allowing for effects 

associated with the longer durations quantified by τbskci of the 

diacetyl studies described by the 

unknown parameter, ωs ,

βsjr(t) are slope parameters for the effect 

exposure to 2,3-pentanedione, which 

are subject to constraints 

βs2t(r) −βs1t(r) = ∆βs2 ≤ 0 and 

βs3t(r) −βs2t(r) = ∆βs3 ≤ 0 thus ensuring***

βs3r(t) ≤βs2r(t) ≤βs1r(t). 

A test of ∆βs2 =∆βs3 = 0 was performed‡‡

and subjected to incorporation.

The slope parameters are subject to 

modification by the multiplicative 

function, 

m(s, k, concbskci, t, τbskci; 0sr(t), φskt, γs)

=[1+γs∙τbskci][1+I(k=1)∙(0sr(t)−1) + φskt 

∙ concbskci]

where the factor, 

[1+γs∙τbskci],describes an adjustment 

for the longer durations of the 

diacetyl studies parameterized 

by γs>−1∕max(τbskci); however, the 

assumption, γs = γ, was imposed 

because information was absent from 

female mice on this parameter, the 

diacetyl indicator, I(k=1)=1, when 

k=1 and I(k=1)=0 when k=0, 0sr(t) are 

parameters describing the potency of 

diacetyl relative to 2,3-pentanedione 

¶¶Whenever the fitted values of the parameters were 

null, i.e., 0, the test statistic −2 log(Likelihood ratio) =

 0 and the test was deemed to be nonsignificant.

***Hence, the requirement that Pr(Ykcit(r) ≥ 3) < 

Pr(Ykcit(r) ≥ 2) < Pr(Ykcit(r) ≥ 1) is satisfied globally.
156 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

at low doses for {r(t)}; the hypothesis, 

0sr(t) = 0s , was tested and subject to 

being incorporated into the model, 

and

φskt allows for an adjustment for a 

quadratic effect of concentration that 

may be attributed to directly proportional changes in respiratory ventilation with concentration where φskt

is the constant of proportionality in 

units of controls’ ventilation; thus φskt

describes the change relative to controls. The hypothesis, φsk,lung =φsk,nose 

=φsk,was tested and subject to being 

incorporated into the model.

fbskcti is one of a pair of lognormally distributed random effects (one effect per 

tissue indicated by t) of the i

th mouse 

of exposure group bskc acting multiplicatively on the effect of dose. Thus, 

an allowance for multiplicative variations from mouse to mouse by tissuespecific positive factors acting on the 

magnitudes of the slope parameters 

was incorporated. Each fbskcti was 

modeled as having unit expectation 

and variance (e σ 2

st−1); thus, the variance of log (fbskcti) = σ 2

st , t = 1, 2 for the 

lung and nose, respectively, together 

with an associated covariance parameter σs12 . The hypothesis that lognormal random effects are independent 

was examined by testing σs12 = 0 and 

was subject to being incorporated. 

Furthermore, the hypothesis that 

only one lognormal random effect 

for each mouse was necessary, i.e., 

fbskc1i = fbskc2i was tested and subject to 

being incorporated.

Model development proceeded by sequentially 

fitting a series of nested models of increasing 

complexity with all random effects omitted. 

This was advantageous for obtaining initial estimates of the fixed effects parameters for fitting 

a corresponding model that included random 

effects. Models were fitted by the method of 

maximum likelihood; the likelihoods of models 

containing unobserved random effects were 

obtained by integrating out these effects using 

adaptive Gaussian quadrature as described by 

Pinheiro and Bates [1995]. Likelihood ratio 

tests were performed to test hypotheses about 

model parameters and associated P values 

were based on the chi-square approximation 

to −2log (LR). Evidence against incorporating 

the previously described restrictions on model 

parameters was deemed significant if the P

value of the corresponding test was less than 

0.05 for selecting the model on which to base 

the estimation of relative potency parameters 

and benchmark concentrations. 

The model selected for estimation of relative 

potencies and BMCs contained three lognormal 

random effects parameters and 53 fixed-effects 

parameters; it had the following form:

= αsjr(t)+ωs∙τbskci+fbskctiβsr(t) {m(s, k, 

concbskci, t, τbskci; θsr(t), φsk, γ)}∙concbskci

where m(s, k, concbskci, t, τbskci; θsr(t), φsk, γ)

=[1+γ∙τskci] [1+I(k=1)∙(θsr(t)−1) +φsk ∙ 

conckci]

i.e., this model was simplified by incorporating 

the following:

Null values of the variance parameters, σ 2

us

[intercept random effects omitted],

∆βs3 = ∆βs2 = 0→βs3r(t) = βs2r(t) = βs1r(t) = βsr(t)

[single 2,3-pentanedione slope parameter 

for each sr(t)],
Occupational Exposure to Diacetyl and 2,3-Pentanedione 157

6 . Quantitative Risk Assessment Based on Animal Data

Separate relative potency parameters, 0sr(t)

were retained since significant evidence against the hypothesis 0sr(t)

= 0s was obtained; hence, 0sr(t) βsr(t)

describes the corresponding diacetyl 

slope for each sr(t),

φsk,lung = φsk,nose =φsk [quadratic effect independent of tissue], 

MLE(σ 2

st) = 0 for lognormal random effects 

of nasal responses of female mice was 

replaced by nullifying this parameter,

The adequacy of a single lognormal 

random effect was rejected,

Independence of the lognormal random 

effects for lung and nasal tissues of 

male mice [implied by acceptance of 

σs12 =0 ] was assumed.

The model was coded and fitted using the 

NLMixed procedure of SASTM 9.3. At least two 

lines of evidence provided support that the 

algorithm for fitting the model converged to a 

solution for the parameters that was a unique 

optimum as follows: (1) The Hessian matrix of 

the fit was positive definite††† and (2) exploration of the likelihood surface in a neighborhood 

of the solution via examination of likelihood 

profiles supported its optimality in all cases 

that were examined. Hence, this evidence supports the identifiability of the parameters of 

the model with these data suggesting that the 

model is not overparameterized.

The fit of the model was assessed by calculating 

grouped‡‡‡ Pearson residuals conditional on the 

†††NLMixed minimizes –log(L) and it provides a 

warning if its criteria for a positive definite Hessian is 

not satisfied; no such warning was given.

‡‡‡The term “grouped” is to clarify that they are based 

on summing the observed and fitted expectations and 

variances over the mice within each treatment group 

defined by each unique combination of b x k x s x c.

empirical Bayes estimates of the random effects 

 for each tissue-response as follows: 

where the fitted expectations and variances 

of each mouse were based on the associated 

binomial distribution of a factoring of the 

conditional multinomial likelihood into its conditionally independent binomial components 

corresponding to the “outcomes” (Y≥1 |f ) , 

(Y≥2|Y≥1,f ), and (Y≥3|Y≥2,f ).

Furthermore, a saturated fixed-effects model 

with random effects omitted was compared 

to the selected model by examination of 

twice the difference of log(Likelihood) values 

relative to the difference in the number of 

parameters. Finally, an ad hoc procedure was 

applied wherein binomial deviance residuals 

corresponding to factoring the multinomial 

likelihood of the corresponding 53 parameter 

model (with random effects omitted) into a 

product of conditional binomial terms were 

used to estimate a factor for adjusting the 

width of the confidence intervals analogous 

to an adjustment for overdispersion because 

the model-based confidence intervals may 

be too narrow if the model is incorrect. Twosided 95% confidence limits with and without 

adjustment were calculated from application 

of a normal approximation to the natural logarithms of the relative potencies and the BMCs 

associated with a 10% benchmark response for 

additional risk.§§§

§§§i.e., Pr(Yskcr(t) ≥ j | conc=BMC jskr(t), fskcti = 1)−Pr(Yskcr(t)

≥ j | conc = 0, fskcti = 1) = 0.10.
158 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

6.2.2.8 Benchmark concentration analysis 

using quantal models

To explore the impact of the categorical regression procedure described above on the BMC 

estimates for diacetyl, the data for the pathological endpoints listed in Table 6-1 (for rats) and 

Table 6-2 (for mice) were also dichotomized, 

and alternative benchmark concentration estimates were developed using quantal modeling 

and model averaging. Any response of minimal 

or greater severity was treated as a positive 

response, and the model averaging procedure 

was based on fitting the multistage, Weibull, 

and log-probit models, as described by Wheeler 

and Bailer [2007]. Only datasets with two or 

more partial response groups were modeled. 

The benchmark response rate was set at 10%, 

and the resulting BMC and BMCL estimates 

are shown in Table 6-9. Only models with an 

average-model P value of 0.05 or greater were 

considered to fit the data adequately.

6.3 Results

6.3.1 Diacetyl

BMC and BMCL estimates based on diacetyl 

toxicity in rats and mice were developed 

as described in sections 6.2.2.1 and 6.2.2.7, 

respectively. Not all of the pathological 

endpoints listed in Tables 6-1 and 6-2 could 

be adequately modeled. The rat endpoints that 

could be modeled adequately according to the 

criteria listed in section 6.2.2.1 (a score test for 

separate slopes and a likelihood ratio test for 

an unrestricted multinomial distribution) are 

shown in Table 6-6. Mouse endpoints that could 

be modeled adequately by the criteria described 

in section 6.2.2.7 are shown in Tables 6-7 and 

6-8. The associated ventilation coefficient¶¶¶ of 

diacetyl among males was −0.378 ± 0.0582 and 

among females it was −0.530 ± 0.357.

¶¶¶Estimate of φs,diacetyl ± Model-based standard error 

per 100 ppm. 

The BMC and BMCL estimates were 

extrapolated to HECs as described in sections 

6.2.2.2 – 6.2.2.4, and the HECs were converted 

to candidate REL values by the application of 

UFs as described in section 6.2.2.5. The BMC/

BMCL values for rats, and their corresponding 

HEC and candidate REL values are shown in 

Table 6-6. The BMC/BMCL values for mice, 

and their corresponding HEC and candidate 

REL values are shown in Tables 6-7 and 6-8; 

the BMCL values in Table 6-7 have not been 

adjusted for overdispersion, while the BMCL 

values in Table 6-8 have been adjusted for 

overdispersion. Scatter plots of the 359 grouped 

Pearson residuals calculated from the data on 

mice indicated they were positively skewed 

at low concentrations and negatively skewed 

at high concentrations. Hence, they were not 

approximately normally distributed, which 

is to be expected given the discrete nature of 

the response data and the small numbers of 

mice in each treatment group (5 ≤ n ≤ 10). 

Although evidence of systematic departures 

of the residuals was not apparent, 13 of the 

residuals indicated deviations from the fit of the 

joint model of diacetyl and 2,3-pentanedione 

by more than three standard errors (not 

shown). Although less than one such residual 

deviation would be expected for normally 

distributed residuals the observation of 13 such 

deviations seems suggestive that extraneous 

variations may be present and motivated our 

having increased the widths of model-based 

confidence limits by the application of an 

overdispersion factor of 1.61 for adjusting the 

model-based standard errors. 

Overall, the BMCs range from 16.8–68 ppm 

diacetyl, and the BMCLs range from 10–49.9 

ppm diacetyl. After interspecies pharmacokinetic adjustments based on the Gloede et al. 

[2011] model, the human-equivalent BMCL 

values (BMCL_HECs) range from 1.4–95.8 

ppm diacetyl, and the BMCL candidate REL 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 159

6 . Quantitative Risk Assessment Based on Animal Data

values (after the application of uncertainty 

factors) range from 0.06–4.0 ppm diacetyl.

6.3.1.1 Sensitivity analysis

As a sensitivity analysis, alternative BMC 

and BMCL values were also derived for the 

NTP [2011] diacetyl study by dichotomizing 

the data, fitting quantal models, and model 

averaging, as described in section 6.2.2.8. The 

model average BMCs ranged from 14.6–78 

ppm, with BMCLs of 2.4–57.9 ppm. The model 

average BMCs and BMCLs were extrapolated to 

humans as described above for the categoricalregression derived BMCs/BMCLs. The 

BMCLHEC values ranged from 0.9–54.3 ppm, 

and the BMCLREL values ranged from 0.04–

2.26. As shown in Table 6-9, if the candidate 

RELs were derived from the quantal modeling 

rather than categorical regression, the lowest 

candidate REL value would be reduced from 

0.06 ppm to 0.04 ppm.

Another assumption made in this risk assessment is that toxicity observed in mice can be 

scaled to rats using the EPA [1994] RfC methodology to estimate a mouse-to-rate respiratory 

dose ratio, or RGDR. It was assumed that this 

extrapolation is best performed on the basis of 

measured values of respiratory ventilation, as 

opposed to estimating respiratory ventilation 

on the basis of body weight. As detailed above 

in section 6.2.2.4, use of the measured respiratory ventilation rates leads to RGDRs of 2.4 

for upper-respiratory toxicity and 3.2 for lower 

respiratory toxicity. The impact of the decision 

to use measured respiratory rates in the RGDR 

calculation was evaluated by a comparison to 

the RGDRs which would be obtained using 

the default RfC methodology, based on body 

weights, and described in EPA [1994]. Using the 

EPA [1994] default methodology, in which the 

respiratory ventilation rate is estimated from 

the animal biody weight, results in RGDRs of 

1.15 for upper respiratory tract effects and 1.5 

for lower respiratory tract effects. Therefore the 

mouse-to-rat scaling factor would be approximately halved, and as shown in Table 6-9 the 

lowest candidate REL value would be reduced 

to 0.03 ppm, based on chronic bronchial 

inflammation in the female mouse lung.

A key assumption made in this risk assessment 

is that the Gloede et al. [2011] PBPK model is 

the most appropriate method for extrapolating 

from rats to humans. A possible alternative 

would be to use the EPA [1994] RfC 

methodology to estimate animal-to-human 

scaling factors, based on the RGDR. Measured 

respiratory ventilation values are available for 

mice and rats, as used in section 6.2.2.4, and 

the human occupational respiration rate can 

be assumed to be 20 L/min. Using these values 

and the EPA [1994] procedures for category 1 

gases, the estimated RGDRs for rat-to-human 

extrapolation are 0.18, 1.9, and 2.1 for the 

upper respiratory tract, the tracheobronchial 

region, and the pulmonary tract, respectively. 

Corresponding values for mouse-to-human 

extrapolation are 0.43, 5.9, and 6.9 for the upper 

respiratory tract, the tracheobronchial region, 

and the pulmonary tract, respectively. These 

RGDRs would replace the Gloede et al. PBPK 

model for extrapolating from rats to humans, 

and would result in candidate RELs ranging 

from 0.15–16.1 ppm for BMCs, and from 0.10–

14.3 ppm for BMCLs. The lowest candidate REL 

derived using the RGDR method would be 0.10 

ppm, as opposed to 0.06 ppm using the Gloede 

et al. [2011] model. The endpoints yielding the 

lowest alternative candidate REL values from 

the sensitivity analysis are shown in Table 6-9, 

along with the lowest of the candidate RELs 

from the main analysis, for comparison.
160 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

Table 6-6. Benchmark concentration* (BMC and BMCL) estimates, human-equivalent concentrations (HECs), 

and candidate recommended exposure limits based on toxicity in rats exposed to diacetyl

Sex Tissue Response

Separate 

slope

P value†

Likelihood 

ratio

P value‡

BMC 

(ppm)

BMCL 

(ppm)

Animal-tohuman PK 

factor

BMCHEC 

(ppm)

BMCLHEC 

(ppm) UF

BMCREL 

(ppm)

BMCLREL 

(ppm)

M Lung Infiltration cellular, 

histiocyte

0.49 0.45 43 30 15.70 2.7 1.9 24 0.11 0.08

M Lung Inflammation, eosinophil or acute 0.055 0.35 29 22 15.70 1.8 1.4 24 0.08 0.06

M Nose Olfactory epithelium, 

degeneration

0.59 0.95 20 13 0.66 30.3 19.7 24 1.26 0.82

M Nose Olfactory epithelium, metaplasia, 

respiratory

0.67 0.78 41 27 0.66 62.1 40.9 24 2.59 1.70

M Nose Olfactory epithelium, 

necrosis

0.23 0.62 27 19 0.66 40.9 28.8 24 1.70 1.20

M Trachea Epithelium, 

hyperplasia

0.28 0.81 68 47 8.70 7.8 5.4 24 0.33 0.23

F Nose Inflammation, 

suppurative

0.12 0.59 22 15 0.66 33.3 22.7 24 1.39 0.95

F Nose Lymphoid tissue, 

hyperplasia

0.83 0.20 23 18 0.66 34.8 27.3 24 1.45 1.14

F Nose Turbinate, atrophy 0.42 >0.99 36 24 0.66 54.5 36.4 24 2.27 1.52

*The benchmark concentration is based on a 10% benchmark response for a minimal or greater level of severity. †Chi-square test 

P value for separate slopes for severity scores; 

P > 0.05 considered to indicate an adequate model fit by this criterion.

‡Chi-square test 

P value for a likelihood ratio test for an unrestricted multinomial distribution; 

P > 0.05 considered to indicate an adequate model fit by this criterion.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 161

6 

. Quantitative Risk Assessment Based on Animal Data

Table 6-7. Benchmark concentration* (BMC and BMCL) estimates, human-equivalent concentrations, and candidate 

recommended exposure limits based on toxicity in mice exposed to diacetyl; BMCLs not adjusted for overdispersion

Sex Tissue Response

BMC 

(ppm)

BMCL 

(ppm)

Animal-tohuman PK 

factor

BMCHEC

(ppm)

BMCLHEC

(ppm) UF

BMCREL

(ppm)

BMCLREL

(ppm)

M Lung Bronchus, inflammation, chronic 41.8 27.4 8.7 4.8 3.1 24 0.20 0.13

M Lung Bronchus, epithelium, regeneration 54.2 38.1 8.7 6.2 4.4 24 0.26 0.18M Nose Inflammation, suppurative 30.5 24.7 0.28 109.0 88.2 24 4.54 3.68M Nose Olfactory epithelium, atrophy 32.3 23.0 0.28 115.5 82.1 24 4.81 3.42M Nose Respiratory epithelium, metaplasia, squamous 26.5 19.2 0.28 94.8 68.6 24 3.95 2.86M Nose Respiratory epithelium, necrosis 36.0 26.8 0.28 128.5 95.9 24 5.35 4.00M Nose Respiratory epithelium, regeneration 40.2 23.5 0.28 143.7 83.9 24 5.99 3.50F Lung Bronchus, inflammation, chronic 19.4 15.3 8.7 2.2 1.8 24 0.09 0.08F Lung Bronchus, epithelium, regeneration 56.1 49.9 8.7 6.5 5.7 24 0.27 0.24F Nose Inflammation, suppurative 27.0 22.9 0.28 96.5 81.7 24 4.02 3.40

F Nose Olfactory epithelium, atrophy 22.0 17.2 0.28 78.5 61.4 24 3.27 2.56

F Nose Respiratory epithelium, metaplasia, squamous 21.8 17.8 0.28 77.7 63.7 24 3.24 2.65

F Nose Respiratory epithelium, necrosis 16.8 12.2 0.28 59.8 43.5 24 2.49 1.81

F Nose Respiratory epithelium, regeneration 18.7 13.4 0.28 66.6 47.8 24 2.78 1.99

*The benchmark concentration is based on a 10% benchmark response for a minimal or greater level of severity.
162 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

Table 6-8. Benchmark concentration* (BMC and BMCL) estimates, human-equivalent concentrations, and candidate 

recommended exposure limits based on toxicity in mice exposed to diacetyl; BMCLs adjusted for overdispersion

Sex Tissue Response

BMC 

(ppm)

BMCL 

(ppm)

Animal-tohuman PK 

factor

BMCHEC

(ppm)

BMCLHEC

(ppm) UF

BMCREL 

(ppm)

BMCLREL 

(ppm)

M Lung Bronchus, inflammation, chronic 41.8 21.2 8.7 4.8 2.4 24 0.20 0.10M Lung Bronchus, epithelium, regeneration 54.2 30.8 8.7 6.2 3.5 24 0.26 0.15M Nose Inflammation, suppurative 30.5 21.7 0.28 109.0 77.6 24 4.54 3.23M Nose Olfactory epithelium, atrophy 32.3 18.7 0.28 115.5 66.7 24 4.81 2.78M Nose Respiratory epithelium, metaplasia, 

squamous

26.5 15.8 0.28 94.8 56.3 24 3.95 2.35

M Nose Respiratory epithelium, necrosis 36.0 22.5 0.28 128.5 80.2 24 5.35 3.34M Nose Respiratory epithelium, regeneration 40.2 16.9 0.28 143.7 60.5 24 5.99 2.52F Lung Bronchus, inflammation, chronic 19.4 13.3 8.7 2.2 1.5 24 0.09 0.06F Lung Bronchus, epithelium, regeneration 56.1 46.4 8.7 6.5 5.3 24 0.27 0.22F Nose Inflammation, suppurative 27.0 20.7 0.28 96.5 73.9 24 4.02 3.08

F Nose Olfactory epithelium, atrophy 22.0 14.8 0.28 78.5 52.9 24 3.27 2.20F Nose Respiratory epithelium, metaplasia, 

squamous

21.8 15.8 0.28 77.7 56.5 24 3.24 2.35

F Nose Respiratory epithelium, necrosis 16.8 10.0 0.28 59.8 35.9 24 2.49 1.50F Nose Respiratory epithelium, regeneration 18.7 10.9 0.28 66.6 39.0 24 2.78 1.63

*The benchmark concentration is based on a 10% benchmark response for a minimal or greater level of severity.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 163

6 

. Quantitative Risk Assessment Based on Animal Data

Table 6-9. Alternate benchmark concentration (BMC and BMCL) estimates, human-equivalent concentrations, 

and candidate recommended exposure limits developed as a sensitivity analysis for key risk assessment assumptions

Species Sex Tissue Response

BMC

(ppm)

BMCL

(ppm)

Animalto-human

PK factor

BMCHEC

(ppm)

BMCLHEC

(ppm) UF

BMCREL

(ppm)

BMCLREL

(ppm)

Rat Male Lung Eosinophilic inflammation* 29 22 15.7 1.8 1.4 24 0.08 0.06

Mouse Female Lung Bronchus, inflammation, chronic† 19.4 13.3 8.7 2.2 1.5 24 0.09 0.06Mouse Female Larynx Chronic inflammation‡ 16 2.4 2.7 5.9 0.9 24 0.25 0.04Mouse Male Lung Bronchus, inflammation, chronic§ 19.4 13.3 18.7 1.0 0.7 24 0.04 0.03Rat Male Nose Olfactory epithelium, degeneration¶ 20 13 0.18** 3.6 2.3 24 0.15 0.10

*From main analysis, Table 6-6, for comparison to alternate estimates.†From main analysis, Table 6-8, for comparison to alternate estimates.‡Alternative analysis based on quantal modeling.§Alternative analysis using respiratory ventilation rates based on body weight, rather than measured values.¶Alternative analysis using EPA [1994] RfC methodology rather than the Gloede et al. [2011] PBPK model. **The animal-to-human PK factor shown here is the RGDR for the rat nose, which in EPA methodology is applied to the BMC/BMCL as a multiplicative factor. This is unlike the 

PBPK-derived PK factors above, which are applied as divisors for the BMC/BMCL values.
164 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

6.3.2 2,3-Pentanedione

The ventilation coefficient  of 2,3-pentanedione among male mice was −0.312 ± 0.0139 

and among females it was −0.182 ± 0.0530. The 

relative potency estimates (diacetyl/2,3-pentanedione) are shown in Table 6-10, below, and 

range from 0.81–7.3, depending on sex and the 

specific endpoint evaluated. A relative potency 

of 1.00 indicates that the two compounds have 

equal toxic potency for the endpoints examined; 

a relative potency less than 1.00 indicates that 

2,3-pentanedione is more toxic than diacetyl, 

while a relative potency greater than 1.00 indicates that 2,3-pentanedione is less toxic than 

diacetyl. Model-based 95% confidence limits 

range from 0.55–14, and the overdispersionadjusted confidence limits range from 0.44–21. 

These estimates suggest that the potency of 

diacetyl was significantly greater than that of 

2,3-pentanedione among female mice for these 

responses. However, one source of contribution to these estimates among females is that 

their fitted ventilation coefficient of diacetyl exposure was 2.9-fold of the coefficient 

fitted for 2,3-pentanedione exposure; thus, 

the observed responses were associated with 

substantially less diacetyl having been inhaled 

thereby increasing its fitted potency relative to 

2,3-pentanedione. In contrast the corresponding value among males was 1.2. Furthermore, 

all seven estimates among females depended 

on the modeling assumption that the exposure duration parameter was identical to that 

of males and results of profiling the likelihood 

(not shown) illustrated that this dependence 

was unidimensional, i.e., the seven relative 

potency estimates for the females varied in 

unison with the duration parameter, whereas 

this was not the case for the seven parameter 

estimates of the males. Hence, the interpretation of the relative potency estimates among 

females warrants a substantially larger degree 

** **Estimate of φs,PD ± Model-based standard error per 

100 ppm

of caution. Although the majority of the relative 

potency estimates among male mice are greater 

than 1.0, suggesting that 2,3-pentanedione may 

be somewhat less toxic than diacetyl, two of the 

seven relative potency estimates (for olfactory 

epithelial atrophy and respiratory epithelial 

degeneration in the nasal tissues of male mice) 

are less than 1.0. In addition to these endpoints, 

the overdispersion-adjusted lower confidence 

limit estimates of relative potency for necrosis of the nasal respiratory epithelium, chronic 

bronchial inflammation and bronchial epithelial regeneration are also less than 1.0. Hence, 

these results suggest that equal or greater toxic 

potency for 2,3-pentanedione relative to diacetyl cannot be ruled out on the basis of currently 

available data.

6.4 Discussion

6.4.1 Diacetyl

6.4.1.1 Modeling issues in BMC estimation 

for diacetyl

Categorical regression modeling for diacetyl 

BMC estimation was initially conducted as 

described in section 6.2.2.1 for rat and mouse 

data. However, it was noted that the mouse 

models showed systematic overprediction of 

the observed response at the highest exposure 

concentrations. Mice are well known to 

exhibit reduced respiration when exposed 

to respiratory irritants [Alarie and Stokinger 

1973], including diacetyl [Larsen et al. 2009]. 

Reduced respiratory rate and reduced minute 

volume have been observed in male mice 

exposed to diacetyl [Morgan et al. 2008]. 

Speculatively, reduced respiration at high 

exposure concentrations may contribute to 

the attenuation of response noted in the high 

exposure groups, relative to a model where 

the effects of exposure are proportional to 

concentration. A strategy was therefore 

employed of modifying the model structure by 

including a quadratic dose term parameterized 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 165

6 . Quantitative Risk Assessment Based on Animal Data

Table 6-10. Relative potency estimates for diacetyl relative to 2,3-pentanedione, on the basis of data in male and female mice

Sex Response

Relative potency 

(diacetyl/

2,3-pentanedione)

Lower confidence 

limit*(model-based)

Upper 

confidence limit*

(model-based)

Lower 

confidence limit 

(OD-adjusted)†

Upper 

confidence limit 

(OD-adjusted)**

F Bronchus, inflammation, chronic 3.7 2.0 6.7 1.4 9.6F Bronchus, epithelium, regeneration 4.0 2.3 7.0 1.7 9.8F Nasal inflammation, suppurative 4.7 3.0 7.4 2.2 9.8F Olfactory epithelium, atrophy 2.0 1.4 2.9 1.1 3.7F Nasal respiratory epithelium, 

Metaplasia, squamous

7.3 3.8 14 2.5 21

F Nasal respiratory epithelium, 

necrosis

3.5 2.2 5.3 1.7 6.9

F Nasal respiratory epithelium, 

regeneration

2.9 1.6 5.3 1.1 7.7

M Bronchus, inflammation, chronic 1.4 1.1 1.7 0.94 2.0M Bronchus, epithelium, regeneration 1.3 1.1 1.6 0.95 1.8M Nasal inflammation, suppurative 1.6 1.3 1.9 1.2 2.1M Olfactory epithelium, atrophy 0.89 0.70 1.1 0.60 1.3M Nasal respiratory epithelium, metaplasia, squamous 1.5 1.2 1.8 1.0 2.1M Nasal respiratory epithelium, 

necrosis

1.4 1.0 1.9 0.84 2.2

M Nasal respiratory epithelium, regeneration 0.81 0.55 1.2 0.44 1.5

*The upper and lower confidence limits form a 95% confidence limit for the relative potency estimate.†Upper and lower confidence limits after adjusting for overdispersion, as described in section 6.2.2.7.
166 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

to represent directly proportional changes of 

ventilation with concentration in modeling the 

mouse data, which allowed sufficient model 

flexibility to accommodate the attenuation of 

response seen in the high-dose mouse data. The 

resulting coefficients of ventilation for nasal 

and lung tissues within each sex and exposure 

chemical were homogeneous and subsequently 

pooled. Furthermore, the coefficients of male 

mice for each chemical were similar and the 

diacetyl coefficient was consistent with the 

observations of minute volume by Morgan et 

al. [2008]. However, the coefficients of the two 

chemicals for the females were substantially 

dissimilar. The seven tissue responses of 

each mouse were jointly analyzed because 

they were governed by the same ventilation 

coefficient. To account for correlations among 

the responses, random effects were included in 

the model thereby utilizing all of the data for 

the estimation of parameters common to all 

responses. However, the increased complexity 

of the model in combination with the small 

sample sizes and discrete responses presented 

challenges for assessing its fit. Residuals 

were calculated conditional on estimates 

of the random effects but interpretations 

of these residuals based on their having an 

approximately normal distribution appeared 

to be problematic because a systematic 

relationship between their skewness and 

concentration was apparent. However, our 

interpretation of these residuals as providing 

evidence of deviations exceeding modelbased predictions is prudent and motivated 

the increase of the widths of the confidence 

intervals. However, these modifications were 

not necessary in modeling the rat data, and 

were not included in the models developed for 

BMC estimation with the rat data.

In the current analysis, BMC estimates for 

diacetyl, based on categorical regression modeling, range from 16.8–68 ppm diacetyl, and 

the BMCL estimates range from 10–49.9 ppm 

diacetyl (Tables 6-6, 6-7, and 6-8). For comparison, alternative BMC estimates based on a 

quantal modeling range from 14.6–78 ppm, and 

quantal model BMCL estimates range from 2.4–

57.9 ppm. Although the central BMC estimates 

were similar for the quantal and categorical 

modeling approaches, some of the quantal 

model BMCL estimates are several-fold lower 

than any obtained using categorical modeling. 

It is possible that this result may be due to the 

inclusion of additional information — response 

severity, as well as incidence — in the categorical regression modeling approach, leading 

to narrower confidence limits in comparison 

to the quantal modeling results. Additional 

sensitivity analyses explored the sensitivity of 

the toxicologically-based risk assessment for 

diacetyl to basing the mouse-to-rat extrapolation on allometrically-scaled respiration rates 

rather than measured values, and to basing the 

animal-to-human extrapolation on RfC methodology [EPA 1994] rather than the Gloede et 

al. [2011] PBPK model. As described in section 

6.3.1.1, varying these assumptions would have 

relatively modest effects on the toxicologicallybased REL estimate for diacetyl. As shown in 

Table 6-9, the lowest candidate REL values from 

the various sensitivity analyses are all within a 

factor of ±2 of the candidate REL values from 

the main analysis, suggesting that the value of 

the toxicologically-based candidate REL is not 

strongly dependent on these assumptions.

6.4.1.2 Comparison with other 

toxicologically-based risk 

assessments

The numerical values of BMD estimates for 

diacetyl are not all directly comparable, even 

when based on a common response rate of 

10%, because of variations in the dose units 

used (ppm concentration versus regional 

penetration versus tissue concentration). The 

occupational exposure limits (OELs) developed 

by the various authors are directly comparable, 

but depend in part on assumptions regarding 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 167

6 . Quantitative Risk Assessment Based on Animal Data

uncertainty factors, which may vary between 

studies. In contrast, the HEC estimates derived 

in this analysis can be directly compared to the 

HEC estimates that have been developed in 

prior risk assessments. 

Earlier toxicologically-based risk assessments 

of diacetyl [Allen 2009; Maier 2010] have 

been based on the 6-week and 12-week mouse 

study of Morgan et al. [2008], rather than the 

more extensive subchronic study conducted 

by the NTP [2011]. Because the NTP [2011] 

subchronic study included data from both mice 

and rats and included both more dose levels and 

more animals per dose group than the Morgan 

et al. [2008] study, the NTP [2011] diacetyl 

study was chosen as the basis for risk assessment 

in this document. However, comparison of the 

current risk assessment findings to the results 

of the earlier risk assessments is instructive. The 

HECs derived in prior diacetyl risk assessments 

are summarized in Table 6-11.

The BMC10 HEC estimates in the current study 

span a range of 1.8–143.7 ppm, compared to 

the range of 4.5–61 ppm reported in prior 

diacetyl risk assessments. The BMCL10 HEC 

estimates in the current study span a range of 

1.4–95.9 ppm, compared to the range of 1.3–10 

ppm reported in prior diacetyl risk assessments. The wider range of HEC estimates in the 

current study, as compared to prior analyses, is 

partially due to the application of animal-tohuman dosimetry estimates from the Gloede et 

al. [2011] PBPK/CFD model, which was published subsequent to the prior risk assessments 

and was, obviously, not available to prior risk 

assessors. In addition, the current study has the 

benefit of a more extensive toxicological data 

base for diacetyl because of publication of the 

NTP [2011] subchronic inhalation study, and 

therefore includes data from more pathological 

endpoints than the prior analyses did.

Maier et al. [2010] conducted a risk assessment 

for diacetyl for the purpose of deriving an OEL. 

This risk assessment was based on the mouse 

pilot study data of Morgan et al. [2008], using 

BMD methodology. The authors concluded 

that the most sensitive endpoint in the mouse 

Table 6-11. HECs (ppm atmospheric concentration) corresponding to 10% BMDs 

and 10% BMDLs reported in prior diacetyl risk assessments

Study Endpoint Dose measure

BMD10

HEC (ppm)

BMDL10

HEC (ppm)

Current study, categorical 

regression modeling

Various

(Tables 6-6, 6-7, and 6-8)

Tissue concentration 1.8 – 143.7 1.4 – 95.9

Current study, quantal 

modeling

Various

(Table 6-9)

Tissue concentration 3.1 – 95.7 0.9 – 54.3

Maier et al. [2010] Peribronchial inflammation Regional penetration 6.5 1.8

Allen [2009a] Nasal inflammation Regional penetration 61.0 10.4

Allen [2009a] Nasal inflammation Tissue concentration 4.5 3.0

Allen [2009a] Peribronchial inflammation Regional penetration 38.6 8.3

Allen [2009a] Peribronchial inflammation Tissue concentration 5.1 1.3

TERA [IDFA 2008] Peribronchial inflammation Regional penetration 9.0 2.0
168 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

was peribronchial lymphocytic inflammation. 

The authors estimated a BMDL10 of 1.98 ppm 

diacetyl, which they converted to a HEC of 1.8 

ppm, rounded to 2 ppm. The authors concluded 

that a total UF of 10 was appropriate, yielding 

in an OEL of 0.2 ppm.

A toxicologically-based quantitative risk assessment for diacetyl was conducted by Bruce C. 

Allen in the reports titled “A Quantitative Risk 

Assessment for Diacetyl Based on Respiratory 

Tract Lesions in Mice” [Allen 2009a] and 

“Report on Model Averaging Analysis and 

Results for Diacetyl Mouse Data Sets” [Allen 

2009b] prepared under OSHA contract number 

DOLQ059622303 (2009) Task Order 50. These 

reports served as the basis for the toxicologically-based diacetyl risk assessment in the draft 

NIOSH criteria document for diacetyl in 2011 

but have been supplanted in the current document by an analysis of more recent data. A 

summary of the risk assessment extracted from 

these reports is included here, for comparison 

to the current toxicologically-based quantitative risk assessment. 

The [Allen 2009a] quantitative risk assessment 

was based on an analysis of adverse respiratory 

effects in mice exposed to diacetyl by inhalation for up to 12 weeks [Morgan et al. 2008]. 

Adverse nasal and lung effects were observed 

with the latter found in the peribronchial, bronchial, and peribronchiolar regions. The Morgan 

et al. [2008] study was used to derive BMDs, 

BMDLs, and corresponding HECs, as discussed 

below. The responses analyzed were those most 

relevant to longer-term exposures, i.e., those 

from the subchronic portion of the study that 

included constant exposures of 25, 50, and 100 

ppm for 6 hours/day, 5 days/week, for either 

6 or 12 weeks. The 6- and 12-week data were 

pooled for the final analysis, based on a likelihood ratio test that indicated that the 6- and 

12-week results were not significantly different. 

A variety of dosimetric adjustments were considered in extrapolating the results from mice 

to humans. The most significant of these adjustments was the choice of dose metrics, either 

“regional penetration” (based on the percentage 

of diacetyl reaching a given portion of the respiratory tract), or “tissue concentration” (based 

on the Morris and Hubbs [2009] PBPK model). 

Because the choice of dose metrics has a significant impact on the HEC, and it is not clear 

which dose metric is preferable, HECs derived 

using both dose metrics are reported in Table 

6-11. An assessment completed by Toxicology 

Excellence for Risk Assessment (TERA) [IDFA 

2008] also utilized the dose-response data 

of Morgan et al. [2008], and estimated HECs 

based on BMDLs for 10% risk, comparable to 

those estimated in the current analysis. TERA 

excluded the nasal lesions from consideration 

prior to their analysis, stating that the evidence 

of upper respiratory symptoms in humans 

exposed to diacetyl was inconsistent and that 

those symptoms lacked reliable concentrationresponse information. In contrast, the current 

assessment assumes that the dose-response 

relationship in a test species, rather than the 

lesion site, is the best criterion for choosing 

which endpoints to model for quantitative risk 

estimation. Thus, the current analysis assumes 

that site concordance is not a requirement 

because once the dose has been adequately 

adjusted (and ideally, once toxicodynamic 

considerations have been carefully considered), a valid dose-response relationship at any 

respiratory tract site/lesion in a test species is 

a reasonable basis for characterizing human 

risk. Additionally, exact site concordance across 

species would not be expected after exposure to 

diacetyl because of the differences in deposition 

of the chemical within the respiratory tracts of 

rodents and humans, as indicated by the PBPK 

model of Gloede et al. [2011]. The Gloede et 

al. [2011] model indicates that a much higher 

percentage of inhaled diacetyl reaches the bronchial and bronchiolar regions in humans than 

in rodents which provides a basis for the findings that diacetyl toxicity is observed primarily 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 169

6 . Quantitative Risk Assessment Based on Animal Data

in the upper respiratory tract of rodents and 

the lower respiratory tract of humans. TERA 

[IDFA 2008] estimated HECs using the EPA 

default methods [EPA 1994] modified by the 

PBPK/CFD model predictions of Morris and 

Hubbs [2009]. However, rather than using the 

relationships between the default and CFDmodel-predicted scrubbing factors to define 

a mouse-specific estimate of airway scrubbing of diacetyl, they assumed that mice were 

exactly like the CFD-modeled rats (i.e., used 

the CFD model predictions for the rats as if 

they were equally relevant to mice). The TERA 

[IDFA 2008] risk assessment did not consider 

light exercise conditions, as may occur in the 

workplace, as these were not incorporated 

into the PBPK/CFD modeling of Morris and 

Hubbs [2009]. Moreover, for the effective dose 

(regional penetration) measure calculated 

by TERA, the default mouse ventilation rates 

were used. As discussed above in regard to the 

Allen [2009a] risk assessment, the experimentally measured ventilation rates for the Morgan 

et al. [2008] study were substantially greater 

than the EPA default values (by a factor of 3 

to 5), and this would have a major impact on 

the HEC estimates (TERA’s estimates would be 

about 3 to 5 times greater, because the major 

effect of changing the ventilation rate is on 

the effective dose measure, VE/SA, rather than 

the scrubbing).

TERA’s analysis resulted in estimates of 

HECs that were 9 and 2 ppm, corresponding 

to the estimated BMD(10) and BMDL(10), 

respectively, from their dose-response 

analysis of the peribronchial inflammation 

endpoint from Morgan et al. [2008]. The 

TERA assessment suggested that a composite 

uncertainty factor of 10 should be used to 

adjust those HECs downward to an OEL. 

That factor of 10 was the product of a factor 

of 3 for interspecies differences and another 

factor of 3 for human variability [IDFA 2008]. 

These factors of 3 are well-accepted uncertainty 

factors commonly used by EPA and others in 

risk assessment. Their recommended OEL was 

therefore 0.2 ppm (as an 8-hour TWA). 

6.4.2 2,3-Pentanedione

Toxic potency estimation for 2,3-pentanedione 

is constrained by both the limited numbers of 

animals that have been tested and the differing exposure durations used in the diacetyl 

and 2,3-pentanedione studies. The currently 

available histopathological data for repeated 

exposures to 2,3-pentanedione are limited to a 

single study involving exposures of 2 weeks + 2 

days (totaling 12 exposures per animal), in both 

rats and mice. The rat data and female mouse 

data for diacetyl are limited to a single 13-week 

study [National Toxicology Program 2011], so 

that no data on the relationship of toxicity to 

duration of exposure are available for the rat 

or the female mouse. For male mice, limited 

data are available from the 6- and 12-week 

exposures reported by Morgan et al. [2008]. 

Although no mouse studies are available that 

closely approximate the 2 week + 2 day exposure protocol used in the 2,3-pentanedione 

study, the 6-, 12-, and 13-week diacetyl data on 

male mice were used to estimate an adjustment 

to predict what the toxicity of diacetyl would 

have been in a study of the same duration as 

the 2,3-pentanedione study. Although a small 

increase of toxicity with exposure duration was 

fitted it was retained in the model even though 

it was not significant in order to account for it as 

a source of variation in obtaining the standard 

errors of the seven relative potency estimates††††

of each sex. The resulting relative potency estimates suggest that 2,3-pentanedione may have 

equal or greater toxic potency than diacetyl for 

five of the seven responses of male mice from 

††††For those readers acquainted with the concept of 

Stein estimation for adjusting a set of three or more 

estimates an application of a criterion of Bock [1975] 

to the covariance matrix of each set did not support 

making them. 
170 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

Table 6-10. Although the responses of Table 

6-10 superficially suggest that 2,3-pentanedione is or seems to be less toxic than diacetyl 

to female mice, these estimates are sensitively 

dependent on the assumption that the parameter for exposure duration is identical to that 

of males. Furthermore, there is a complete lack 

of information from these studies for assessing 

this assumption and profiling the likelihood 

indicated that the relative potency estimates 

of the female mice were substantially sensitive to this parameter whereas this did not 

hold for the estimates of the males. Hence, it 

would be prudent to refrain from concluding 

that 2,3-pentanedione is less toxic than diacetyl 

to female mice on the basis of the estimates of 

Table 6-10. 

Recent data support the conclusion that 

2,3-pentanedione should be used cautiously 

in the workplace and exposures to 2,3-pentanedione should be minimized. Rats (but 

not mice) develop intramural and intraluminal airway fibrosis following exposure to 

either diacetyl or 2,3-pentanedione [Morgan 

et al. 2016]. This lesion shares many features 

with obliterative bronchiolitis of humans, the 

condition that originally brought medical 

attention to employees exposed to diacetyl. A 

2-week inhalation exposure of 150 or 200 ppm 

to either diacetyl or 2,3-pentanedione could 

produce bronchial fibrosis in rats [Morgan et 

al. 2016]. This finding suggests that 2,3-pentanedione causes airway fibrosis comparable 

to diacetyl at equal exposure concentrations. 

Because no chronic or subchronic studies 

of 2,3-pentanedione are currently available 

and the number of rats in the 2-week exposure is low, it is not possible to quantitatively 

assess the toxicity of 2,3-pentanedione relative to diacetyl for producing airway fibrosis. 

However, these data do suggest that it would 

be prudent to treat 2,3-pentanedione as at 

least equally toxic as diacetyl until additional 

toxicological data become available on the 

toxic potency of 2,3-pentanedione.

6.5 Conclusions 

Pathological lesions produced by inhalation 

exposure to diacetyl and 2,3-pentanedione have 

been assessed using categorical regression techniques and benchmark dose estimation. For 

diacetyl a CFD/PBPK model is available for 

both rats and humans that allows rodent BMC 

and BMCL estimates to be extrapolated directly 

to human exposures. The results of this exercise indicate that the most sensitive endpoint 

in terms of estimated human toxicity is that 

associated with eosinophilic inflammation in 

the male rat lung. The HEC associated with this 

endpoint is 1.8 ppm, with a 95% lower-bound 

estimate of 1.4 ppm (Table 6-6). Application of 

a 24-fold uncertainty factor to the lower-bound 

HEC leads to a candidate REL of 0.06 ppm, or 

60 ppb diacetyl. The estimated human toxicity 

based on chronic bronchial inflammation in 

the female mouse lung is very similar to the ratbased estimate (Table 6-8), and also leads to a 

candidate REL of 0.06 ppm or 60 ppb. If human 

data on the toxicity of diacetyl were not available, these estimates could serve as the bases for 

REL development for diacetyl. Because human 

data do exist and are sufficient for derivation 

of an REL, the toxicologically-based candidate 

RELs should be viewed as complementary to 

the epidemiologically-based REL. Because the 

toxicologically-based REL is within an order of 

magnitude of the epidemiologically-based REL 

it supports the epidemiologically-based REL. 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 171

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Number DOLQ05622303.

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accessed: August 2014.

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validated hybrid computational fluid dynamics-physiologically based pharmacokinetic model for respiratory 

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application to inhalation dosimetry of diacetyl. Toxicol 

Sci 123(1):231–246.

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Jones WG [2002]. Necrosis of nasal and airway epithelium in rats inhaling vapors of artificial butter flavoring. 

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regression analysis of toxicity data. Comments on toxicology 7(5-6):437–452.

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D, Friend S, Schwegler-Berry D, Mercer RR, Reynolds JS, 

Hubbs AF, Goldsmith WT, Kashon ML, Frazer D, Mercer 

RR, Battelli LA, Kullman GJ, Schwegler-Berry D, Friend 

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Pathol 36(2):330–344.

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radiological protection. ICRP Publication 66 Ann 

ICRP 24(1–3).

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Acute airway effects of diacetyl in mice. Inhal Toxicol 

21(13):1123–1128.

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[2010]. Evaluation of concentration–response options 

for diacetyl in support of occupational risk assessment. 

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Morgan DL, Flake GP, Kirby PJ, Palmer SM [2008]. 

Respiratory toxicity of diacetyl in C57BL/6 mice. Toxicol 

Sci 103(1):169–180.

Morgan DL, Jokinen MP, Price HC, Gwinn WM, Palmer 

SM, Flake GP [2012]. Bronchial and bronchiolar fibrosis 

in rats exposed to 2,3-pentanedione vapors: implications for bronchiolitis obliterans in humans. Toxicol 

Pathol 40(3):448–465.

Morgan DL, Jokinen MP, Johnson CL, Price HC, Gwinn 

WM, Bousquet RW, Flake GP [2016]. Chemical reactivity 

and respiratory toxicity of the alpha-diketone flavoring 

agents: 2,3-butanedione, 2,3-pentanedione, and 2,3-hexanedione. Toxicol Pathol 44(5):763–783.
172 Occupational Exposure to Diacetyl and 2,3-Pentanedione

6 . Quantitative Risk Assessment Based on Animal Data

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diacetyl and butyric acid, two components of butter flavoring vapors. Toxicol Sci 108(1):173–183.

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 173

7 Basis of the Recommended Standards 

for Diacetyl and 2,3-Pentanedione

In the Occupational Safety and Health Act of 

1970 (Public Law 91–96), Congress mandated 

that NIOSH develop and recommend criteria for identifying and controlling workplace 

hazards that may result in occupational illness 

or injury. In fulfilling this mandate, NIOSH has 

reviewed the relevant human and/or animal 

data to assess the health effects of diacetyl and 

2,3-pentanedione; assessed the risks of occupational exposure; characterized anticipated 

employee exposures; and developed recommended criteria for exposure limits, exposure 

monitoring, engineering and work practice 

controls, and medical monitoring. 

The basis for the RELs is described in this 

chapter. The primary objective of the recommendations for diacetyl is to reduce loss of 

lung function associated with diacetyl exposure because diacetyl (and potentially related 

diones) has been shown to cause potentially 

fatal obliterative bronchiolitis in employees. 

The NIOSH REL for 2,3-pentanedione would 

be identical to that for diacetyl but is slightly 

higher based upon the limitations of the analytical method.

7.1 Health Effect Studies of 

Employees Exposed 

to Diacetyl

As detailed in Chapter 3, medical evaluations 

showed that employees exposed to diacetyl developed progressive shortness of breath 

while working at several microwave popcorn 

plants and flavoring plants, findings consistent 

with the severe irreversible lung disease obliterative bronchiolitis. Obliterative bronchiolitis, 

sometimes characterized by spirometric abnormality, has been described in employees in 

the microwave popcorn and flavor-manufacturing industries [CDC 2002, 2007; Kanwal 

et al. 2006]. Some affected employees have 

experienced extremely rapid declines in lung 

function, with severe airways obstruction in 

some cases occurring within several months of 

the start of exposure to flavoring compounds 

[Akpinar-Elci et al. 2004; NIOSH 1986]. 

Employees as young as 22 years old have been 

affected. Some affected employees have been 

placed on lung transplant waiting lists by their 

physicians because of the severity of their 

disease [Akpinar-Elci et al. 2004]. The findings 

from investigations and studies conducted at 

multiple plants presented in Chapter 3 have 

established a link between exposure to diacetyl 

and risk for severe occupational lung disease. 

These findings meet the standard criteria used 

to determine causation: that an exposure is the 

likely cause of specific health effects [Gordis 

1996; Hill 1965]. Investigations of severe lung 

disease consistent with obliterative bronchiolitis among diacetyl-exposed employees have 

provided clear evidence of a causal relationship 

between diacetyl exposure and development of 

this disease.

7.2 Toxicological Studies 

of Diacetyl 

In rats, acute exposures to diacetyl or diacetylcontaining butter flavoring vapors cause 
174 Occupational Exposure to Diacetyl and 2,3-Pentanedione

7 . Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione

necrosis in the epithelial lining of nasal and pulmonary airways. Rats inhaling vapors of butter 

flavoring that contained diacetyl developed 

multifocal necrotizing bronchitis one day after 

a 6-hour exposure. The mainstem bronchus 

was the most affected intrapulmonary airway. 

However, nasal airways were more affected than 

intrapulmonary airways. Necrosuppurative rhinitis was seen in rats inhaling butter flavoring 

vapors at concentrations that did not cause 

damage in intrapulmonary airways [Hubbs 

et al. 2002]. As a single agent acute exposure 

in rats, diacetyl caused epithelial necrosis and 

inflammation in bronchi at concentrations of 

>290 ppm and caused epithelial necrosis and 

inflammation in the trachea and larynx at concentrations of ≥220 ppm [Hubbs et al. 2008]. In 

a pattern similar to that of airway damage from 

diacetyl-containing butter flavoring vapors, 

diacetyl causes greater damage to nasal airways 

than to intrapulmonary airways in rats [Hubbs 

et al. 2008].

In mice, inhaling diacetyl at concentrations 

of 200 or 400 ppm for 6 hours/day for up to 5 

days caused respiratory tract changes similar to 

those seen in rats inhaling diacetyl or diacetylcontaining butter flavoring vapors [Morgan et 

al. 2008]. Subchronic diacetyl inhalation caused 

significant histopathological changes in mice 

at all concentrations studied. Peribronchial 

lymphocytic infiltrates were seen at terminal sacrifice at 12 weeks in all subchronically 

exposed mice inhaling 100 ppm diacetyl and 

in some mice inhaling 25 or 50 ppm diacetyl. 

Using a CFD-PBPK model, the rodent pathologic changes, though at higher regions in 

the respiratory tract, were consistent with the 

human bronchiolar pathology once differential nasal scrubbing, size of airway, and target 

organ doses were accounted for [Gloede et al. 

2011; Morris and Hubbs 2009]. In rats in which 

nasal scrubbing was bypassed by administering 

a single dose of 125 mg/kg diacetyl via intratracheal instillation, histopathological alterations 

characteristic of bronchiolitis obliterans ensued, 

including damage to airway epithelium [Palmer 

et al. 2011]. 

NIOSH concludes that the toxicological 

responses to diacetyl observed in animal 

studies support the conclusions of the epidemiologically-based risk assessment for this 

compound. The animal-based risk assessment 

presented in Chapter 6 further corroborates 

the epidemiologic assessment by demonstrating a causal link between diacetyl exposure 

and respiratory health effects and by showing 

a clear dose-response relationship in exposed 

animals as was observed in employees exposed 

to diacetyl in the epidemiologic assessment. 

7.3 Quantitative Risk 

Assessment for Deriving 

the Recommended 

Exposure Limit

NIOSH has reviewed the literature on diacetyl toxicology and exposures in the workplace 

and subsequently conducted a quantitative 

risk assessment. Results from this comprehensive review demonstrate a causal relationship 

between diacetyl exposure and development of 

severe occupational lung disease. The quantitative risk assessment used to derive the REL 

was based solely on human (employee) data, 

but the results were informed and supported 

by animal risk assessments. On the basis of a 

quantitative risk assessment of data collected 

in a series of NIOSH health hazard evaluations 

(full description in Chapter 5), NIOSH has 

concluded that employee exposure to diacetyl 

is associated with a reduction in lung function. 

Specifically, a statistically significant exposureassociated reduction in the FEV1/FVC ratio 

and percent predicted FEV1 and an exposureassociated incidence of obstructive lung disease 

were observed. NIOSH quantified these exposure-response relationships and determined the 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 175

7 . Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione

exposure levels that correspond to a variety of 

risks (Chapter 5, Table 5-35). Excess lifetime 

risks in the range of 1:1,000 corresponded to 

working lifetime diacetyl exposure of approximately 5 ppb. Once the risks were characterized, 

NIOSH examined the analytical methods 

(OSHA Methods 1012 and 1016) and available 

engineering controls and determined that they 

supported establishing an REL at that level. 

7.4 Objectives

The NIOSH objective in establishing RELs for 

diacetyl and 2,3-pentanedione is to reduce 

the risk of respiratory impairment (decreased 

lung function) and the severe irreversible lung 

disease obliterative bronchiolitis associated 

with occupational exposure to these compounds. In addition, maintaining exposures 

below the RELs will help prevent other adverse 

health effects including but not limited to irritation of the skin, eyes, and respiratory tract in 

exposed employees. The recommendation to 

limit exposure to diacetyl and 2,3-pentanedione is based upon data from human and animal 

studies and the quantitative risk assessment, 

however, additional considerations included 

sampling and analytical feasibility and the 

achievability of engineering controls.

A variety of risk estimates were evaluated and 

presented in Chapter 5. NIOSH has historically targeted excess risks predicted to be in the 

range of approximately 1 per 1,000 in establishing RELs (see Chapter 5, Tables 5-34, 5-35 for 

risk estimates). In occupational exposure to 

diacetyl, the ultimate health effect of concern is 

obliterative bronchiolitis, a debilitating, sometimes fatal, and irreversible effect. The goal 

is to prevent the respiratory impairment that 

precedes the appearance of obliterative bronchiolitis. There are validated analytical methods 

that can be used to effectively measure employee 

exposures at the selected level. Additionally, 

information from site visits indicates that the 

REL is achievable with engineering controls 

where diacetyl is used or handled [Eastern 

Research Group 2009; Kanwal et al. 2011].

7.5 Recommended 

Exposure Limits

7.5.1 Recommended Exposure Limit 

for Diacetyl

On this basis, NIOSH recommends a REL of 5 

ppb for diacetyl (as a TWA for up to 8 hours/

day during a 40-hour workweek). NIOSH has 

determined that employees exposed to diacetyl 

at this level for 8 hours a day, 40 hours a week 

for a 45-year working lifetime should have no 

more than a 1/1,000 excess risk of lung function 

falling below the lower limit of normal due to 

diacetyl exposure. 

To ensure that employee exposures are routinely below the REL for diacetyl, NIOSH also 

recommends using an action level (AL) of 2.6 

ppb with the exposure monitoring program 

to ensure that all control efforts (engineering controls, medical surveillance, and work 

practices) are in place and working properly. 

When exposures exceed the AL, employers 

should take corrective action (determine the 

source of exposure, identify methods for controlling exposure) to ensure that exposures 

are maintained below the REL. NIOSH has 

concluded that the use of an AL in conjunction with periodic monitoring of employee 

exposures (described in Chapter 10) will help 

protect employees.

NIOSH is also recommending a STEL for diacetyl of 25 ppb for a 15-minute time period. The 

establishment of a short-term exposure limit 

is based on the concern that peak exposures 

may have greater toxicity than the same total 

dose spread out over a longer period of time. 

Some limited evidence of this type of dose-rate 

effect is available in animal studies [Hubbs et 

al. 2008]. On the basis of general industrial 
176 Occupational Exposure to Diacetyl and 2,3-Pentanedione

7 . Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione

hygiene principles, the STEL, which is five 

times the REL, would serve to reduce peak 

exposures and tend to reduce overall employee 

exposures to diacetyl. The selection of a STEL 

that is five times the REL is based upon past 

precautionary practice [Federal Register 1997]. 

In the absence of a STEL in workplaces complying with the NIOSH REL for diacetyl of 5 

ppb TWA, employees could theoretically be 

exposed to 2,400 ppb diacetyl for 1 minute or 

480 ppb for 5 minutes in an 8-hour day with 

no additional exposure the remaining part of 

their 8-hour shift. The STEL for diacetyl of 25 

ppb would limit those exposures to a possible 

peak of 375 ppb for 1 minute and 75 ppb for 

5 minutes and should prevent acute irritation 

from brief high exposures. 

7.5.2 Recommended Exposure Limit 

for 2,3-Pentanedione

2,3-Pentanedione, which has been used as 

a substitute for diacetyl, is also of concern 

because of structural similarities with diacetyl and because animal studies show similar 

toxicity for the respiratory tract [Hubbs et al. 

2012; Morgan et al. 2012; Morgan et al. 2016]. 

Morphologic data suggest that 2,3-pentanedione can cause airway epithelial damage similar 

to the damage caused by diacetyl [Hubbs et al. 

2012; Morgan et al. 2012; Morgan et al. 2016]. 

Rats repeatedly inhaling 2,3-pentanedione at 

concentrations ≥ 150 ppm for up to 2 weeks 

develop fibrosis of intrapulmonary airways, 

a morphologic change similar to obliterative 

bronchiolitis in humans [Morgan et al. 2016]. 

Recently, more than 3500 genes were found 

to be upregulated in RNA isolated from the 

fibrotic bronchi of 2,3-pentanedione exposed 

rats [Morgan et al. 2015]. Some of the up-regulated genes were ones previously implicated 

in fibrosis, including transforming growth 

factor-β2, interleukin-1α, interleukin-18, 

interleukin-33, and fibronectin. In addition, at 

high exposure concentrations, messenger RNA 

changes were noted in the brain of rats after 

acute 2,3-pentanedione inhalation [Hubbs et 

al. 2012]. 

The toxic potency of the two materials appears 

to be comparable in mice exposed by inhalation (see Chapter 6, section 2 for a full 

discussion). Given the structural similarity 

between diacetyl and 2,3-pentanedione and the 

evidence published, NIOSH would prefer to 

recommend an identical REL for diacetyl and 

2,3-pentanedione. However, OSHA Method 

1016, the validated analytical method available for 2,3-pentanedione, can only reliably 

quantify 2,3-pentanedione at concentrations 

9.3 ppb and above. Therefore the NIOSH 

REL for 2,3-pentanedione, while informed by 

the toxicological potential, is based upon the 

limitations of the analytical method and is 

established at 9.3 ppb. This REL for 2,3-pentanedione will result in a residual risk of lung 

disease similar to diacetyl, but may be higher. 

It does not imply that 2,3-pentanedione is safer 

than diacetyl. Because the REL is established at 

the reliable quantitation level, no AL is established for 2,3-pentanedione.

Because of their structural similarity, concerns 

for short-term exposures to 2,3-pentanedione 

also apply. Accordingly, a STEL for 2,3-pentanedione is established at 31 ppb (i.e., the 

lowest concentrations the method can sample 

accurately during a 15-minute time period). 

The NIOSH REL for 2,3-pentanedione of 9.3 

ppb and STEL of 31 ppb would limit exposures to a possible peak of 465 ppb for 1 

minute and 93 ppb for 5 minutes. Because 

of the concern for potential dose-rate effects, 

NIOSH recommends STELs for diacetyl and 

2,3-pentanedione to reduce peak exposures to 

employees. 

Maintaining diacetyl and 2,3-pentanedione concentrations at or below the RELs 

and STELs requires the implementation of 

a comprehensive safety and health program 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 177

7 . Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione

that includes engineering controls, exposure 

monitoring, routine medical surveillance, and 

employee training in good work practices. 

Specific recommendations for these components can be found in Chapters 2, 8, 9, and 10 

of this document.

7.6 Rationale for the 

Recommended 

Exposure Limit

The recommendation to limit occupational 

exposures to diacetyl to an 8-hour TWA of 5 

ppb is based on data from human quantitative risk assessment with additional rationale 

provided by animal toxicological studies. 

From the human studies, 5 ppb represents 

a reasonable summary of estimates from 

several concordant approaches to risk assessment. Although smoking affects the excess 

lifetime risk estimates, a full treatment for the 

purpose of developing separate REL recommendations on smoking status would require 

including interactions between smoking and 

diacetyl exposure histories for which NIOSH 

believes there is insufficient historical information and statistical power to implement. 

Furthermore, there is no precedent for developing standards that are specific to smoking 

status. NIOSH also recommends an AL of 2.6 

ppb to help protect employees from exposure 

to diacetyl above the 5 ppb REL and a STEL 

of 25 ppb to limit peak exposures and protect 

against dose-rate effects. Engineering controls 

and work practices are available to control 

diacetyl exposures below the REL (and the 

AL) in workplaces. OSHA Method 1012 is a 

validated analytical method that can be used 

to effectively measure employee exposures 

to diacetyl. Establishing the recommended 

exposure limits for diacetyl is consistent 

with the mission of NIOSH mandated in the 

Occupational Safety and Health Act of 1970. 

7.7 Controlling Diacetyl 

and 2,3-Pentanedione 

Exposures in the 

Workplace

In general, many industries have implemented 

engineering controls to reduce exposure and 

risk of disease among their employees. Many 

of the processes where diacetyl and 2,3-pentanedione are manufactured, handled, or used 

are similar to other industries and may allow 

for common approaches to reducing employee 

exposure. These processes include blending, 

mixing, and handling of flavoring compounds 

in liquid and powder form. A 3-year study 

of a microwave popcorn production facility showed that the use of exposure controls 

can dramatically reduce diacetyl concentrations in mixing rooms and for all production 

employees [Kanwal et al. 2011]. As a result 

of the implementation of exposure controls, 

average combined personal and area diacetyl 

air concentrations declined an order of magnitude in the mixing room (from 57.2 ppm to 

2.88 ppm) while concentrations in the quality 

control laboratory (from 0.82 ppm to < LOD) 

and packaging area (from 2.76 ppm to < LOD 

for machine operators) declined to below 

detectable limits. These interventions included 

providing general room exhaust ventilation 

to the mixing room and local exhaust ventilation for the heated flavoring and mixing tanks. 

Closed transfer processes were implemented 

through the installation of a pump to transfer 

heated butter flavorings from the holding tanks 

to oil/flavor mixing tanks. The building of an 

enclosure for all oil/flavor holding tanks and 

installing local exhaust ventilation on all tanks 

further reduced exposures to employees in the 

packaging area of this plant. In the final survey 

conducted following the implementation of 

all engineering and process controls, personal 

diacetyl exposures for all employees/job categories in the plant were below detectable limits 
178 Occupational Exposure to Diacetyl and 2,3-Pentanedione

7 . Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione

with the exception of mixers which ranged 

from below the LOD to 12.6 ppm.

The design concepts required for working with 

hazardous materials include specification of 

general ventilation, local exhaust ventilation, 

maintenance, cleaning and disposal, personal 

protective equipment, exposure monitoring, and medical surveillance [Naumann et 

al. 1996]. Bag emptying, bag filling, charging 

tanks, benchtop weighing and handling, and 

drum filling and emptying are a few of the 

production processes of concern. Other more 

specialized processes (for example, candy 

panning, a process in which candy pieces in 

a rotating drum are sprayed with chocolate or 

other flavoring compounds; coffee roasting; 

commercial fry-cooking) may also result in 

employee exposure. Special attention should 

be given to manual handling of flavoring 

compounds, particularly in heated processes, 

and when spraying flavoring compounds. 

Research on food industry practices has led 

to the development of engineering controls 

that may help reduce employee exposure to 

diacetyl, 2,3-pentanedione, and other chemicals. Chapter 8 describes engineering controls 

for the industries where diacetyl is handled or 

used within products. Table 8-2 in Chapter 8 

provides a summary of NIOSH evaluated engineering control efficiencies for the mixing of 

food flavorings. 

Although many job categories can be effectively 

controlled to levels below the REL, tasks associated with transfer of diacetyl may continue 

to pose risk to the employees even following 

the implementation of controls. For example, 

mixers may continue to be exposed at levels 

above the REL when handling butter flavorings and from tank emissions. However, these 

exposures can be reduced through the implementation of closed transfer systems and local 

exhaust ventilation approaches discussed in 

Chapter 8. NIOSH acknowledges that the frequent use of personal protective equipment, 

including respirators, may be required for some 

employees who handle diacetyl, 2,3-pentanedione, diacetyl-containing flavorings or flavored 

products. The frequent use of PPE may be 

required during job tasks for which (1) airborne 

concentrations of diacetyl or 2,3-pentanedione 

(e.g., pouring, mixing, packaging) above the 

REL exist, (2) the airborne concentration of 

diacetyl or 2,3-pentanedione is unknown or 

unpredictable, and (3) job tasks are associated 

with highly variable airborne concentrations 

because of environmental conditions or the 

manner in which the job is performed. In all 

work environments where diacetyl, 2,3-pentanedione, diacetyl-containing flavorings or 

flavored products are found, control of exposure through engineering controls should be 

the highest priority.

7.8 Hazards Associated with 

Diacetyl Substitutes

Much has been made of the possible removal/

substitution of diacetyl and 2,3-pentanedione from the flavor manufacturing or food 

production industries. A health benefit from 

substitution can only be realized if the substitute is safer than diacetyl or 2,3-pentanedione. 

However, the current knowledge on toxicity of 

available substitutes is limited; few if any have 

OELs, and therefore exposure to substitutes 

should be controlled.

There is reason to think that, like diacetyl, 

other alpha-dicarbonyl compounds would 

have a tendency to cause protein cross-links 

[Miller and Gerrard 2005]. The reactivity of 

the alpha-dicarbonyl compounds is enhanced 

by electron-attracting groups and decreased 

by electron donors [Roberts et al. 1999]. 

Alpha-dicarbonyl compounds can inactivate 

proteins, principally through reactions with 

the amino acid, arginine [Epperly and Dekker 

1989; Saraiva et al. 2006]. The related alphadicarbonyl flavoring, 2,3-pentanedione, has 
Occupational Exposure to Diacetyl and 2,3-Pentanedione 179

7 . Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione

been reported to be even more reactive with 

arginine groups than diacetyl [Epperly and 

Dekker 1989]. 

While the focus of this document is on diacetyl and 2,3-pentanedione, NIOSH has concern 

about other flavoring substitutes with structures similar to diacetyl or moieties that are 

biologically active and capable of producing 

similar toxic effects as diacetyl. Therefore, 

NIOSH recommends that such exposures also 

be considered and controlled to concentrations 

as low as possible, taking into account potential 

additive effects of flavoring compounds.

The guidance recommendations presented in 

Chapter 8 regarding control of exposures are 

applicable not only to diacetyl and 2,3-pentanedione, but also to their substitutes and other 

flavorings and flavoring compounds used in this 

industry. The control of exposures is discussed 

in detail in Chapter 8, but several LEV systems 

described have been shown to be particularly 

effective in controlling diacetyl and would be 

expected to work well for similar compounds. 

Ventilated backdraft workstations used for 

small batch mixing have been evaluated in two 

field studies conducted in flavoring production 

plants. The field studies showed reductions in 

exposure of 90%–97% when performing mixing 

tasks using these stations [NIOSH 2008a]. Also, 

the use of controls to reduce employee exposure during pouring and mixing of ingredients 

in a commercial mixer has been evaluated in 

a flavoring production plant [NIOSH 2008b]. 

The use of LEV at the mixing tank helps to 

maintain the vessel at a negative pressure and 

contain evaporative emissions. NIOSH evaluated the impact of a ventilated tank lid on the 

exposure of an employee during the mixing of 

a food flavoring [NIOSH 2008b]. The use of 

the ventilated tank lid resulted in a reduction 

of approximately 76% exposure. Most of the 

exposure during the evaluated mixing process 

was attributed to tasks performed outside of 

the hood. Ventilated tank lids have also been 

recommended by the British Health and Safety 

Executive (HSE) to contain vapors during the 

mixing of liquids with other liquids or solids 

[Health and Safety Executive 2003]. 

7.9 Summary

The following points summarize the relevant 

information used as the basis for the NIOSH 

recommendation for limiting occupational 

exposure to diacetyl and 2,3-pentanedione:

■ Airborne exposures to diacetyl and 

2,3-pentanedione have been characterized as potentially hazardous based on a 

review of the available literature regarding 

both human exposure and animal studies. 

■ Human health and animal data indicate 

a causal relationship between diacetyl 

exposure and development of obliterative 

bronchiolitis. Studies show a progressive shortness of breath for employees at 

several microwave popcorn plants and 

flavoring plants as well as employees 

who have experienced rapid declines in 

lung function. 

■ Rats repeatedly inhaling 2,3-pentanedione at concentrations ≥ 150 ppm for up 

to 2 weeks develop fibrosis of intrapulmonary airways, a morphologic change 

similar to obliterative bronchiolitis in 

humans. Inhalation studies on mice produced similar results.

■ Risk assessment using data from both 

animal and inhalation human studies 

indicates that a diacetyl REL of 5 ppb as 

a TWA for up to 8 hours/day during a 

40-hour workweek would be appropriate 

to achieve a 1/1,000 excess lifetime risk. 

Further, NIOSH recommends a STEL 

of 25 ppb to limit peak exposures and 

protect against dose-rate effects. An AL 

of 2.6 ppb is recommended to ensure that 

employee exposures are routinely below 
180 Occupational Exposure to Diacetyl and 2,3-Pentanedione

7 . Basis of the Recommended Standards for Diacetyl and 2,3-Pentanedione

the REL for diacetyl and to ensure that 

all control efforts (engineering controls, 

medical surveillance, and work practices) 

are in place and working properly. 

■ Given evidence that 2,3-pentanedione can cause airway epithelial 

damage and the structural similarity of 

2,3-pentanedione to diacetyl, NIOSH 

recommends a 2,3-pentanedione REL 

of 9.3 ppb as a TWA for up to 8 hours/

day during a 40-hour workweek. The 

REL for 2,3-pentanedione is based upon 

the reliable quantitation limit for the 

analytical method and does not imply 

that 2,3-pentanedione is of lower toxicity 

than diacetyl. Further, NIOSH recommends a STEL of 31 ppb to limit peak 

exposures on the same basis of analytic 

method limitation.

■ Data gathered on diacetyl exposure demonstrated that engineering controls and 

work practices currently available can 

control diacetyl exposures below the 

REL. A validated analytical method can 

be used to effectively measure employee 

exposures at these levels.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 181

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[2002]. Fixed obstructive lung disease in workers at a 

microwave popcorn factory—Missouri, 2000–2002. 

MMWR 51(16):345–347.

CDC (Centers for Disease Control and Prevention) 

[2007]. Fixed obstructive lung disease among workers 

in the flavor-manufacturing industry—California, 2004–

2007. MMWR 56(16):389–393.

Eastern Research Group, Inc. [2009]. Site visits related 

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No. 1985–0171–1710.

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and work practices at Agilex Flavors, Inc. formerly Key 

Essentials, Inc., Rancho Santa Margarita, CA. A technical 

assistance report to the California/Occupational Safety 

and Health Administration. By McKernan L, Dunn K. 

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engineering controls for the production of liquid and 

powder flavoring chemicals. Kerry Ingredients and 

Flavours, Inc., formerly Mastertaste, Inc., Commerce, 

CA. By Dunn K, Echt A, Couch J, Myers D. Cincinnati, 

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Kirby PJ, Foley JF, Gwinn WM, Morgan DL [2011]. 

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 183

8 Hazard Prevention and Control 

of Exposures to Diacetyl and 

2,3-Pentanedione

8.1 Introduction

Employee exposure to air contaminants can 

best be reduced by a combination of efforts to 

minimize air contaminant generation through 

good work practices and to control emissions at 

their source through process changes or engineering controls. Traditionally, a hierarchy of 

controls has been used to determine how to 

implement feasible and effective controls. One 

representation of this hierarchy can be summarized as follows:

■ Elimination/Substitution

■ Engineering controls

■ Administrative controls (including 

work practices) 

■ Personal protective equipment 

The idea behind this hierarchy is that the 

control methods at the top of the list are potentially more effective, protective, and economical 

(in the long run) than those at the bottom. 

Following the hierarchy normally leads to the 

implementation of inherently safer systems 

where the risk of illness or injury has been substantially reduced.

The first item in the hierarchy is elimination/

substitution. The intention of eliminating a 

flavoring or other chemical in the workplace 

is to remove the exposure by removing the 

source. Similarly, the goal of substitution is to 

substitute a flavoring or chemical with another 

of lower toxicity. The removal of diacetyl and 

2,3-pentanedione from the flavor manufacturing or flavoring industries would be practical 

only with the substitution of an alternative 

butter flavor chemical, which is currently being 

done in some situations. However, the current 

knowledge on toxicity of available substitutes 

is limited, and exposure to substitutes may also 

need to be controlled. Therefore, elimination 

and substitution may not provide a feasible 

control and are not discussed in detail. The 

recommendations that follow are applicable not 

only to diacetyl and 2,3-pentanedione, but also 

to other flavorings and flavoring compounds 

used in this industry. 

Engineering controls, as discussed below, are 

mechanical techniques for removing contaminants from the workplace. For instance, local 

exhaust ventilation can be used to capture and 

remove emissions from a hazardous or nuisance 

source. A major advantage of this type of system 

is that, when properly designed, it requires 

minimal user effort or training. 

Work practices are procedures followed by 

employers and employees to control hazards in 

the workplace. The use of good work practices, 

incorporated into the facility’s standard operating procedures, can help reduce exposures to 

diacetyl, 2,3-pentanedione, and other flavoring 

compounds while at the same time maximizing 

efficiency and product quality. Work practices 

include housekeeping and cleaning, storage and 

use procedures, work clothes, labels and postings, hazard training, and procedures for use 

of engineering controls. NIOSH has recently 

published additional engineering and work 

practice control guidance for employees who 

are exposed to diacetyl [NIOSH 2015].
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

184 Occupational Exposure to Diacetyl and 2,3-Pentanedione

The use of respirators, a form of PPE, is discussed because this control, while not favored, 

is in common use in some facilities. As the 

discussion demonstrates, considerable effort 

is required in the proper selection and use 

of respiratory protection in the workplace. 

Finally, the protection of skin, eyes, and face is 

also discussed.

8.2 Engineering Controls

Currently, there is no model or standard guidance for engineering controls for flavoring and 

food production processes. If it is not possible 

to eliminate toxic compounds from the workplace or replace them with less toxic substances, 

then the use of engineering controls and work 

practices to minimize exposures is the next 

level of controls for the necessary reduction 

of exposure.

8.2.1 General Considerations

A properly designed supply air ventilation 

system can provide plant ventilation, building 

pressurization, and exhaust air replacement. 

When LEV is installed in production areas, it 

is important to consider the need for replacement air. In general, it is necessary to balance 

the amount of exhausted air with a nearly 

equal amount of supply air. Without replacement air, uncontrolled drafts will exist at doors, 

windows, and other openings; doors become 

difficult to open because of the high pressure 

difference; and exhaust fan performance may 

degrade. Good supply air design consists of 

ducted supply with air discharge registers about 

10 feet above floor level [ACGIH 2013].

Controls need to be fitted to individual 

processes by each plant and cannot be a “onesize-fits-all” approach. Controls need to be 

evaluated after installation. Evaluations should 

be completed to quantify exposures after controls have been implemented to ensure that 

target goals have been achieved. It is important 

to confirm that the LEV system is operating as 

designed by periodically measuring exhaust 

airflows. A standard measurement, hood static 

pressure, provides important information on 

the hood performance, because any change 

in airflow results in a change in hood static 

pressure. For hoods designed to prevent exposures to hazardous airborne contaminants, the 

ACGIH Operation and Maintenance Manual 

recommends the installation of a fixed hood 

static pressure gauge [ACGIH 2007].

In addition to routine monitoring of the hood 

static pressure, additional system checks should 

be completed periodically to ensure adequate 

system performance, including smoke tube 

testing, hood slot/face velocity measurements, 

and duct velocity measurements using an anemometer. These system evaluation tasks should 

become part of a routine preventive maintenance schedule to check system performance. 

It is important to note that the collection and 

release of air contaminants may be regulated; 

companies should contact agencies responsible 

for local air pollution control to ensure compliance with emissions requirements when 

implementing new or revised engineering 

controls. 

To minimize exposure and reduce the risk of 

flavoring-related lung disease, a few standard 

precautions should be followed in areas where 

flavoring-related exposures may occur:

■ Isolate rooms where flavorings or flavoring compounds are handled from the 

rest of the plant with walls, doors, or 

other barriers.

■ Maintain flavoring mixing rooms and 

other areas where flavorings are handled 

under negative air pressure relative to 

the rest of the plant. Check status with 

airflow indication equipment such as a 

smoke tube.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 185

■ Install hood static pressure gauges 

(manometers) near hoods to provide 

a way to verify proper hood performance. Check pressure frequently to 

ensure that the system is operating properly compared to baseline. Check hood 

face velocities and capture velocities 

frequently to ensure that system is performing as designed.

■ Ensure that employees are properly 

trained on the use of the controls if 

using proximity switches for fan activation. Consider installing a control “on/

off ” light to indicate the status of the 

exhaust fan. 

■ Place hoods away from doors, windows, 

air supply registers, and aisles when possible to reduce the impact of cross drafts. 

■ Provide supply air to production rooms 

to replace most of the exhausted air.

■ Direct exhaust air discharge stacks away 

from air intakes, doors, and windows.

■ Inspect hoods and enclosures for signs 

of damage or leaks (rust/corrosion, 

open access doors, etc.) and obstructions (paper, gloves, rags, etc.). Where 

possible, use screens to prevent foreign 

objects from being pulled into the system 

through openings (slots, hood faces, etc.).

8.2.2 Primary Production Processes 

and Controls

The food and flavoring production industries 

have several primary processes that may result 

in increased potential for employee exposure 

to diacetyl, 2,3-pentanedione and other flavoring compounds. These may be grouped, from 

an exposure standpoint, into a few general 

categories including production operations, 

packaging operations, cleaning, and maintenance operations [Eastern Research Group 

2008b]. Employees in each of these job categories may potentially be exposed to flavoring 

compounds, including diacetyl and 2,3-pentanedione. Table 8-1 displays a list of job 

categories and work activities associated with 

these manufacturing processes. For each activity, the section of this document that discusses 

relevant exposure control and the figure(s) 

at the end of this chapter that shows relevant 

LEV systems are indicated. Other job categories 

may potentially be exposed to flavoring compounds. These include supervisory personnel, 

laboratory and quality controls personnel, and 

cleaning and maintenance personnel. When 

these personnel are in production areas, they 

should comply with recommended control 

procedures and wear appropriate PPE posted 

for that specific area. Additional considerations 

Table 8-1. Controls for job categories and major activities 

in the food and flavor production industries

Job category Major activities See section See figure(s)

Production operator Benchtop weighing and handling

Charging/filling tanks and mixers

Bag dumping/emptying

Drum filling and emptying

8.2.2.1

8.2.2.2

8.2.2.3

8.2.2.4

8-1, 8-2, 8-3, 8-4

8-5, 8-6, 8-7, 8-8

8-9

8-10, 8-11

Packaging personnel Bag filling

Drum filling and emptying

8.2.2.5

8.2.2.4

8-12, 8-13

Quality assurance/

quality control personnel

Benchtop weighing and handling 8.2.2.1.1 8-4
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

186 Occupational Exposure to Diacetyl and 2,3-Pentanedione

may be necessary for the maintenance job category, specifically for intermittent tasks such as 

filter change out. 

Many different industries have implemented 

engineering controls to reduce exposure and 

risk of disease among their employees. Many 

of the processes used in the flavoring and 

food manufacturing industries are similar to 

those of other industries and may allow for 

common approaches to reducing employee 

exposure. These processes include blending, 

mixing, and handling of flavoring compounds 

in liquid and powder form. The design concepts 

required for working with hazardous materials include specification of general ventilation, 

LEV, maintenance, cleaning and disposal, PPE, 

exposure monitoring, and medical surveillance [Naumann et al. 1996]. Bag emptying, bag 

filling, charging tanks, benchtop weighing and 

handling, and drum filling and emptying are 

a few of the production processes of concern. 

Other more specialized processes (for example, 

candy panning, a process in which candy pieces 

in a rotating drum are sprayed with chocolate 

or other flavoring compounds) may also result 

in employee exposure. Special attention should 

be given to manual handling of flavoring compounds, particularly in heated processes, and 

when spraying flavoring compounds.

Research into various food industries has led 

to the development of potential engineering 

controls to help reduce employee exposure to 

diacetyl, 2,3-pentanedione and other chemicals. 

The following sections describe the primary 

production processes used in the food and 

flavoring industries and discuss engineering 

controls that can be used to minimize employee 

exposure to diacetyl, 2,3-pentanedione and 

other potential airborne hazards.

8.2.2.1 Benchtop weighing and handling

Small-scale weighing and handling of ingredients are common tasks used in flavoring 

production, bakeries, dairy production, and 

snack food manufacturing. The tasks of weighing out dry and wet food ingredients can lead 

to employee exposure primarily through the 

scooping, pouring, and dumping of these 

materials. Studies in bakeries have shown that 

the employees exposed to dusts, commonly 

from flour, are those who perform mixing 

and weighing tasks [Elms et al. 2003]. In addition, a recent survey at a commercial bakery 

showed that mixer operators were exposed to 

diacetyl when they measured and added an 

artificial butter flavor to a dough mixer [Eastern 

Research Group 2008a]. Because weighing and 

pouring are often performed on a benchtop 

workstation, the addition of slotted backdraft 

ventilation for both the bench and the weighing 

area is recommended. This approach can also 

be applied to larger-scale operations.

The application of engineering controls to 

reduce employee exposure to chemicals during 

mixing and weighing has been evaluated in 

flavoring production. In flavoring production facilities, compounders measure and 

pour flavoring compounds on a bench and 

then transfer these mixtures to open tanks for 

liquid flavoring production or to blenders used 

for powdered flavoring production. The use 

of ventilated backdraft workstations, adapted 

from welding bench designs available in the 

ACGIH Industrial Ventilation Design Manual 

(Figure 8-1) has been evaluated by NIOSH in 

two field studies conducted in flavoring production plants [ACGIH 2013].

Ventilated back-draft workstations used for 

small batch mixing have been evaluated in 

two field studies conducted in flavoring production plants (Figure 8-2). These stations 

were designed to maintain an air velocity of 

100–150 feet per minute (fpm) at the face of the 

enclosure. The field studies showed reductions 

in exposure of 90%–97% when performing 

mixing tasks using these stations [NIOSH 

2008c, d]. The key design parameters are to 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 187

enclose as much of the activity as possible and 

to use properly sized exhaust slots to maintain a uniform air velocity across the face of 

the station. 

Other groups have also produced designs that 

may be amenable to the control of exposure 

during benchtop mixing and weighing activities. The HSE has developed a series of control 

approaches based on common processes in a 

variety of industries. One approach is similar 

to the one evaluated by NIOSH in flavoring 

facilities and recommends a control velocity of 

100–200 fpm (0.5–1 meters per second [m/s]) 

at the face of the workstation when working 

with flour improvers (Figure 8-3) [Health and 

Safety Executive 2003i]. 

The selection of proper control velocity should 

be made on the basis of the material being 

used (powder versus liquid), plant conditions 

(background drafts), and momentum of contaminant source (pouring versus spraying or 

vigorous mixing). The use of baffles on the 

side and top of these workstations to better 

enclose the process provides improved control 

and minimizes the deleterious effects of cross 

drafts on contaminant control. Plastic curtains 

can provide reasonable enclosure while allowing improved access to the bench area. The 

proper positioning of these workstations away 

from doors, windows, air supply registers, and 

aisle ways will also help to reduce the impact of 

cross drafts. 

8.2.2.1.1 Laboratory chemical hoods

Laboratory personnel will typically perform 

benchtop weighing and handling of flavorings 

in a chemical fume hood. A properly designed 

and maintained chemical fume hood can offer 

significant employee protection if used properly. There are many different hood designs, but 

the most common categories are the conventional or constant-flow hood, the bypass hood, 

and the variable air volume constant-velocity 

hood. The constant-flow hood is the oldest and 

simplest chemical hood design. The exhaust fan 

induces a constant volumetric airflow moving 

through the sash opening. For this hood design, 

the face velocity is lowest when the sash is wide 

open; when the sash is lowered the face velocity increases. The bypass hood maintains a 

constant hood face velocity and incorporates 

a bypass grille above the sash opening. When 

the sash is wide open it blocks the bypass 

grille, allowing all of the air to flow through 

the hood opening. As the sash is lowered, it 

uncovers increasingly greater amounts of the 

bypass grille, allowing increasing amounts of 

air to flow through this alternative path. If it 

is designed and operated properly, the amount 

of air flowing through the bypass grille is just 

sufficient to maintain a constant face velocity. 

Typically, however, this constant velocity can 

be maintained over a certain part of the sash’s 

total range. The constant-velocity hood uses a 

control system to detect the sash position, face 

velocity and system pressure, and change the 

fan motor speed or other mechanism, such as 

mechanical dampers, to increase the airflow 

when the sash is raised and decrease it when 

the sash is lowered, thus maintaining a constant 

face velocity. 

All chemical hoods have certain common 

design elements, including an exhaust fan to 

move air through the hood, a moving sash, 

exhaust slots, and a horizontal work surface 

(Figure 8-4). The sash can be designed to move 

in either a vertical or a horizontal direction. A 

crucial performance element for any chemical 

hood is the face velocity, defined as the average 

air velocity at the face of the hood at the sash 

opening. Maintaining a constant, minimum 

face velocity provides confidence that operations and hazardous agents within the hood 

will be contained. The current consensus of 

the literature is that the average face velocity 

for a laboratory chemical hood should be in 

the range of 80–120 fpm [Burgess et al. 2004]. 

The flow control system on a constant-velocity 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

188 Occupational Exposure to Diacetyl and 2,3-Pentanedione

hood should be adjusted to give a face velocity in this range. Each chemical hood should 

be clearly marked with the proper hood sash 

location that will give the desired face velocity; depending on the hood design, this could 

be a single location or a range of locations. 

Containment verification using tracer gases 

to provide quantitative data and smoke testing 

to visualize airflow patterns is recommended 

when the hood is installed, when substantial 

changes are made to the ventilation system, 

and periodically as part of a preventive maintenance program. In addition to the face velocity, 

it is important that the airflow be distributed 

evenly across the hood face. ANSI/AIHA Z9.5 

[2003] recommends that variations of velocity 

across the hood face should be within ±20% of 

the average face velocity; however, some laboratories select a stricter standard of ±10%.

8.2.2.2 Charging/filling tanks and mixers

The addition of solid and liquid ingredients 

into tanks and other mixing vessels can cause 

exposure to dusts and vapors due to the displacement of air in the vessel. Medical and 

environmental surveys conducted in the 

microwave popcorn manufacturing industry 

have shown that employees who mixed butter 

flavorings into heated soybean oil had the 

highest exposures to diacetyl and the highest 

risk of developing severe irreversible lung 

disease [Kanwal et al. 2006]. These employees 

measured out artificial butter flavoring in open 

containers and poured the flavoring into heated 

mixing tanks filled with oil. Real-time monitoring of a mixer at one plant measured a diacetyl 

peak of more than 80 ppm over several minutes 

as he poured flavorings into the mixing tank 

[Kanwal et al. 2006]. NIOSH investigations at 

a plant where many exposed employees developed severe lung disease also showed that the 

implementation of LEV for heated tanks of oil 

and flavorings and general dilution ventilation 

for production areas reduced diacetyl concentrations. As a result of the implementation of 

exposure controls, average personal diacetyl air 

concentrations declined in the mixing room, 

from 57.2 ppm to 2.88 ppm [Kanwal et al. 

2011]. Exposures to diacetyl were also recorded 

at a plant that produced flavorings and other 

products in employees who added flavors to 

mixing and spray dryer feed tanks while the 

tanks were being filled. One employee who 

was adding diacetyl-containing starter distillate 

and starch to a spray dryer slurry feed tank was 

exposed to elevated levels of volatile organic 

compounds including diacetyl for a sustained 

period of time [NIOSH 2009]. In addition, elevated concentrations of volatile contaminants 

were measured as an employee poured diacetylcontaining starter distillate from a collection 

vessel into a bulk container. 

The use of controls to reduce employee exposure during pouring and mixing of ingredients 

in a commercial mixer has been evaluated in 

a flavoring production plant [NIOSH 2008d]. 

The implementation of LEV at the mixing tank 

helps to maintain the vessel at a negative pressure and contain evaporative emissions. NIOSH 

evaluated the impact of a ventilated tank lid on 

the exposure of an employee during the mixing 

of a food flavoring (Figure 8-5) [NIOSH 2008d]. 

The use of the ventilated tank lid resulted in a 

reduction of approximately 76% compared to 

the same operation without the ventilated tank 

lid. However, most of the exposure during the 

evaluated mixing process was attributed to tasks 

performed outside of the hood. Ventilated tank 

lids have also been recommended by the HSE 

to contain vapors during the mixing of liquids 

with other liquids or solids [Health and Safety 

Executive 2003e]. A NIOSH laboratory study of 

different mixing tank hood designs for a 4 foot 

diameter tank showed that capture efficiencies 

above 90% were possible for all hoods and configurations at an exhaust flow rate of 200 cubic 

feet per minute (cfm) with a crossdraft of 100 

fpm or less [Hirst et al. 2014]. 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 189

Another approach evaluated by NIOSH at a flavoring manufacturing facility was the use of a 

ventilated mixing booth. This booth allows a 

large portable mixing tank to be rolled inside 

so that chemical vapors emitted during pouring 

and mixing of flavoring compounds in the 

tank are captured and exhausted (Figure 8-6). 

However, the booth provides some flexibility 

and can also be used for other production tasks 

such as large pouring and product packaging 

activities. The use of slots across the booth 

plenum helps evenly distribute the flow across 

the height and width of the booth. A field study 

showed hood capture efficiencies of greater 

than 95% based on tracer gas tests [Dunn et al. 

2008]. An important design consideration is to 

make the booth deep enough to fully contain 

the process.

Other approaches to controlling exposure 

during filling of mixing vessels and tanks 

include the use of a simple exhaust hood near 

the opening of fixed tanks. This approach is 

highlighted in the HSE Control Approach 

210, titled “Charging Reactors and Mixers 

from a Sack or Keg” (Figure 8-7) [Health and 

Safety Executive 2003a]. This design calls for 

the use of a local exhaust hood near the tank 

opening with an inward velocity of at least 200 

fpm. Another design provided by the HSE and 

ACGIH for mixers and tanks includes the use 

of rim exhausts placed around the edge of the 

mixer/tank. These designs take the shape of 

an annular slotted hood, which pulls air away 

from employees as they add ingredients or 

operate the mixer (Figure 8-8) [ACGIH 2013; 

Health and Safety Executive 2003f]. An annular 

exhaust provides a semicircular ventilation ring 

around the edge of the tank to capture contaminants as they evaporate or are displaced 

during pouring/mixing. Typical rim exhausts, 

however, are limited in the area where they can 

provide adequate capture velocity and should 

not be used to capture contaminants beyond 

approximately 24 inches from the hood face 

[Goodfellow and Tähti 2001]. 

8.2.2.3 Bag dumping/emptying

Manual handling of solid powders is a process 

used in many industries, including food and flavoring production. The opening and dumping 

of bags of powdered ingredients is commonly 

performed by employees in the production of 

flavorings, dairy products, snack foods, and 

in baked goods. Typically, an employee cuts 

open bags of material (e.g., 50-pound bags) 

and dumps the ingredients into a hopper, and 

then stacks or disposes of the empty bags. In 

powdered flavoring production, these hoppers 

are commonly outfitted onto blenders used to 

load the base starch ingredient for dry flavor 

blends. In snack food production, they may 

be used to load spices and flavors for application to the product via open drum coaters just 

before packaging. These open-ended devices 

typically are used to coat larger, more irregularly shaped materials such as cereal flakes or 

expanded snacks. Coatings may be applied as 

a slurry or as a dry mix following spray application of oil or lecithin. The drums rotate as 

the flavoring is being applied to allow for even 

coverage of the snacks. This process can cause 

employee exposure to the powdered flavoring; 

a case of bronchiolitis obliterans organizing 

pneumonia was reported in a spice process 

technician whose primary responsibility was to 

manually dump spices from bags into a slurry 

for application to potato chips [Alleman and 

Darcey 2002]. 

Technology used to control dusts during bag 

dumping has been in place for many years. 

The standard control—a ventilated bag dump 

station—consists of a hopper outfitted with an 

exhaust ventilation system to pull dusts away 

from employees as they open and dump bags 

of powdery materials. The designs for these 

devices are available from several sources of 

industrial ventilation guidance. The HSE has 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

190 Occupational Exposure to Diacetyl and 2,3-Pentanedione

developed a control approach for a ventilated 

station for emptying bags of solid materials. 

The control includes the specification of a face 

velocity of 200 fpm (1.0 m/s) and includes a 

waste bag collection chute (Figure 8-9) [Health 

and Safety Executive 2003g]. 

Research into the effectiveness of these types 

of devices has shown that they can effectively 

reduce employee exposure to dust and vapors. 

A review of commercially available units 

showed that their use controlled dust levels to 

1–2 mg/m3 [Heitbrink and McKinnery 1986]. 

However, dust contamination on the surface of 

the bag and handling or disposal of bags caused 

increased employee exposure. An integral pass 

through to a bag disposal chute or compactor will help reduce dust exposure resulting 

from bag handling. Further studies in mineral 

processing plants showed that the use of an 

overhead air supply also significantly decreased 

employee exposure [Cecala et al. 1988]. 

The ACGIH Ventilation Manual also has two 

designs that are applicable to the control of 

powder materials during bag dumping. Design 

plate VS-15-20, Toxic Material Bag Opening, is 

similar in design to the HSE station described 

above but recommends a slightly higher control 

velocity of 250 fpm at the face of the station 

opening. In addition, Design plate VS-50-10, 

Bin and Hopper Ventilation, requires a hood 

face velocity of 150 fpm. In general, higher 

velocities may be needed to adequately capture 

dusts in a plant environment. Air velocities 

around 200 fpm into the hood should provide 

reasonable contaminant removal for these 

operations [ACGIH 2013]. 

8.2.2.4 Drum filling and emptying

In some cases, manually operated and powered 

pumps have been used to transfer liquids from 

barrels to mixing and feed tanks. Although 

the use of these devices can reduce exposure 

by reducing the amount of open handling, 

care should be taken when filling and emptying drums of flavoring compounds. The use 

of ventilation at the barrel opening has been 

recommended for capture of vapors during 

transfer of chemicals. The HSE has developed 

two engineering control approaches for drum 

filling and emptying (Figure 8-10) [Health and 

Safety Executive 2003b, c]. For drum filling, the 

guidance recommends the use of an annular 

exhaust hood around the interface between the 

drum and feed pipe (at the bung hole). The recommended airflow is a minimum of 100 fpm 

across the drum cap/bung hole. The use of a 

pump to move flavoring compounds or finished 

flavorings for packaging may provide a preferable “closed transfer” approach [Health and 

Safety Executive 2003b]. For flammable liquids, 

suitable fans and equipment as well as appropriate grounding schemes should be used to 

prevent the buildup and discharge of static electricity. The ACGIH Ventilation Manual also has 

developed a design plate with several different 

implementation options based on the process 

(Figure 8-11) [ACGIH 2013]. In all cases, 

grounding and bonding requirements should 

be met to prevent sparks and explosions when 

transferring flammable liquids [NFPA 2007]. 

8.2.2.5 Bag filling

The process by which bags are filled with 

products is typically done by flavor manufacturers and other producers of powder materials. 

Powder flavorings are typically mixed with 

industrial blenders or produced by a spray 

drying process. For the blending process, a 

powdered starch or other carbohydrate is combined with a liquid or paste flavoring agent. 

When the blending is completed, the powder 

product may be discharged into a bulk tote 

or packaged into smaller containers. In the 

spray drying process, a mixture of liquid and 

powder ingredients (slurry) is sprayed within 

a large sealed tank. Heat within the tank dries 

the slurry droplets, leaving a powder as the 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 191

finished product. This powder is then collected 

and packaged in product containers.

Studies conducted at flavoring production 

facilities have shown that intermittent peak 

exposures to dust and flavoring volatile ingredients occur when powder products are being 

packaged following blending or spray drying 

[NIOSH 2007, 2008a, b, 2009]. The use of a ventilated collar-type hood around the discharge 

point can help minimize employee exposure 

to dust and vapors. The HSE has developed a 

control approach for an exhaust hood for the 

filling of bags with solid materials. The control 

includes the specification of a ventilated enclosure around the powder discharge outlet and 

has applicability to the filling of smaller product 

bags as well as intermediate bulk containers 

(Figure 8-12) [Health and Safety Executive 

2003d, h]. This design guidance recommends 

an air velocity of 200 fpm (1.0 m/s) into the 

enclosure. The ACGIH Industrial Ventilation 

Manual, Design plate VS-15-02, Bag Filling, 

is similar in design to the HSE exhaust hood 

but specifies an overall hood exhaust flow of 

400–500 cfm for nontoxic dust or 1,000–1,500 

cfm for toxic dust with a maximum inward air 

velocity of 500 fpm [ACGIH 2013]. 

In addition to ventilation solutions, other dust 

control approaches have been used in a variety 

of industries and should be applicable for food 

and flavoring production. For example, an 

inflatable seal can be used to create a dust tight 

seal on the discharge outlet of an industrial 

blender (Figure 8-13). The outlet spout can be 

fitted with an inflatable seal that prevents dust 

from escaping during the bag filling process. 

The seal inflates during the product transfer 

from the blender to the packaging bag (providing the seal) and deflates once the transfer 

is completed to allow removal of the packaging bag. These systems are available on many 

commercially available bulk bag filling systems 

[Hirst et al. 2002]. 

Another system that can be used is the continuous liner system. Polypropylene liners 

are often used when products are discharged 

from the industrial blenders into the final 

product container. In this operation, a sleeve 

of polypropylene liners is stowed around the 

circumference of the discharge outlet. The first 

liner, the bottom having been sealed, is pulled 

down into the overpack (usually a 5-gallon 

bucket or a cardboard box). Product is discharged into the liner through a butterfly valve 

on the blender outlet. Once full, the top of the 

first liner sleeve is closed with tape or a fastener, or it is heat sealed and cut. The product 

is sealed within the poly-lined container, and 

a new sealed poly-liner is pulled down to start 

discharge into the next container. This continuous process seals off the primary leak paths for 

dust during unloading of an industrial blender 

or other equipment. These systems are commonly used in the pharmaceutical industry 

and may provide effective alternatives to traditional local exhaust ventilation control systems 

for food and flavoring production. 

8.2.3 Summary of Capture Efficiencies 

of Control Approaches

Producing flavorings and flavored foods 

involves a variety of steps. These processes 

require the handling and manipulation of 

flavorings and flavoring compounds, which 

have been shown to be a point of exposure 

for employees. Table 8-2 shows the capture 

efficiencies of those controls which have been 

evaluated by NIOSH in the laboratory or in 

flavoring manufacturing plants and discussed 

in this chapter. These controls have shown to 

be effective at reducing potential employee 

exposure by 90% or greater across the wide 

range of processes and tasks commonly seen 

in flavoring and flavored food production. 

However, for some tasks, this may not be 

enough to reach the exposure control goals. 

When implementing engineering controls, it 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

192 Occupational Exposure to Diacetyl and 2,3-Pentanedione

is important to use a tiered approach, which 

includes reducing the emissions at the source 

through containment, process modifications, or 

local exhaust ventilation as well as using facility 

provisions such as pressurization schemes. 

These approaches should be used in conjunction with those described below including 

administrative controls and the use of personal 

Table 8-2. Summary of efficiencies for controls evaluated for 

the mixing of food flavorings by NIOSH

Process Control Evaluation 

% Reduction 

(vs. no control) Source

Benchtop 

Weighing 

and Handling

Slotted exhaust hood/

workstation enclosure

Simulated mixing/weighing 

with alcohol

97 NIOSH 2008d

Slotted exhaust hood/

worktable

Simulated mixing/weighing 

with alcohol

89–100 NIOSH 2008c, 

Dunn et al. 2008

Bag Dumping/

Emptying

Bag dump/slotted exhaust 

around perimeter

Dumping of 50 lb dextrose 

bags

96 NIOSH 2008d

Bag Filling Simple exterior exhaust 

hood

Discharge of dextrose from 

blender into 15 gallon 

containers

97 NIOSH 2008d

Simple exterior exhaust 

hood

Scooping/packaging of 

dextrose into 15 gallon 

containers

64 NIOSH 2008d

Charging/

Filling Tanks 

and Mixers

Ventilated tank lid Preparation of a food flavor 

in a large mixing tank

76 NIOSH 2008d

Dome hood–1.5 inch 

gap/200 cfm EX/100 

fpm CD

Tracer gas emission from 

mixing tank

99 Hirst et al. 2014

Ventilated hinged lid/200 

cfm EX/100 fpm CD

Tracer gas emission from 

mixing tank

98 Hirst et al. 2014

Slot hood open/200 cfm 

EX/100 fpm CD

Tracer gas emission from 

mixing tank

96 Hirst et al. 2014

Slotted back draft booth Tracer gas emission from

mixing tank 97–98 NIOSH 2008c, 

Dunn et al. 2008

CD = crossdraft

EX = exhaust flow rate
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 193

protective equipment. 

8.3 Administrative Controls

Work practices, an administrative control, are 

procedures followed by employers and employees to control hazards in the workplace. The use 

of good work practices, incorporated into the 

facility’s standard operating procedures, can 

help reduce exposures to diacetyl, 2,3-pentanedione, and other flavoring compounds 

while at the same time maximizing efficiency 

and product quality. Work practices include 

housekeeping and cleaning, storage and use 

procedures, work clothes, labels and postings, 

hazard training, and procedures for use of 

engineering controls, many of which are discussed here.

The emission of the volatile components in 

each flavoring mixture can be minimized by 

preventing spillage. To the extent possible, 

containers used to mix and store flavoring 

compounds should be covered when not in use. 

This practice will minimize the evaporation of 

chemicals into the workplace air and minimize 

likelihood of inadvertent spills. Manual handling of chemicals also provides a potentially 

significant source of employee exposures and 

emissions. Use of closed transfer processes, 

where feasible, significantly reduces exposure. Also, slow careful pouring/handling of 

chemicals can reduce splashing, spillage, and 

exposure during this activity [Boylstein et al. 

2006]. Reduction in spills and elimination of 

leakage from vessels aid in reducing the overall 

emission of chemicals into the workplace and 

lower employee exposure.

8.3.1 Good Housekeeping Practices

An organized, clean workplace enables faster 

and easier production, improves quality assurance, and reduces the potential for slips, trips, 

and falls. It is important to maintain good 

general housekeeping practices so that leaks, 

spills, and other process integrity problems are 

readily detected and corrected. Proper practices 

regarding spills include:

■ Allowing only individuals wearing appropriate PPE who are properly trained, 

equipped, and authorized for response to 

enter the affected area until the cleanup 

has been completed and the area properly ventilated.

■ Using high-efficiency particulate air 

(HEPA)-filtered vacuums, wet sweeping, or a properly enclosed wet vacuum 

system for cleaning up dust that contains 

diacetyl or 2,3-pentanedione. Dust should 

be treated as dust containing diacetyl and 

not as nuisance dust.

■ Cleaning work areas regularly with 

HEPA-filtered vacuums or with wet 

sweeping methods to minimize the accumulation of dust.

■ Cleaning up spills promptly.

■ Limiting accumulations of liquid or solid 

materials on work surfaces, including 

floors, to reduce contamination of products and the work environment.

8.3.2 Closed Transfers, Containers, 

and Processes

Because of the volatile nature of diacetyl, 

2,3-pentanedione and other flavoring compounds, proper handling to limit the duration 

of exposure to vapors is essential. The use of 

closed vessels and closed transfer procedures is 

one technique to promote proper handling. To 

limit exposure time: 

■ Avoid open pouring, measuring, and 

transfer of diacetyl, 2,3-pentanedione,

and other flavoring compounds on the 

FEMA priority list whenever possible 

[FEMA 2012].
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

194 Occupational Exposure to Diacetyl and 2,3-Pentanedione

■ Add diacetyl, 2,3-pentanedione and other 

priority chemicals into tanks last, when 

possible, to minimize the time during 

which volatilization can occur.

■ Keep tanks and containers of flavoring compounds/ingredients sealed at 

all times.

■ Maintain and use volatile flavoring compounds at the lowest possible temperature 

within the manufacturers’ recommended 

temperature range for each chemical to 

minimize volatility.

■ Use appropriate personal protective 

equipment during cleaning of diacetylcontaining vessels. 

Some manufacturing processes may be enclosed 

to keep airborne diacetyl, 2,3-pentanedione, 

and other priority flavoring compounds contained and separated from employees by:

■ Isolating mixing and other high-exposure 

processes from the rest of the workplace

■ Maintaining the isolated work areas 

under negative air pressure

■ Ensuring that employees take special precautions and if necessary use appropriate 

PPE on entry into production work areas 

where diacetyl, 2,3-pentanedione, and 

other flavoring compounds are handled

When production processes that utilize flavorings or flavoring compounds are not enclosed 

or contained, employees performing other 

work tasks in the vicinity should be informed 

and required to use appropriate PPE to prevent 

incidental exposures.

8.3.3 Hygiene Procedures

Good personal hygiene is important to limit 

not only inhalation exposures to diacetyl, 

2,3-pentanedione, and other flavoring compounds, but also exposure from ingestion and 

dermal absorption. Important hygiene considerations include:

■ Employers should not allow employees to 

smoke, eat, or drink in work areas where 

diacetyl, 2,3-pentanedione, and other flavoring compounds are used.

■ Employers should provide appropriate PPE to protect the employees from 

dermal exposure during normal work 

activities. Examples include gloves, chemical resistant arm sleeves, and aprons.

■ Employees should wash their hands and 

exposed skin before eating, drinking, 

or smoking.

8.3.4 Reduced Process Temperatures 

for Priority Flavoring Compounds

To minimize volatilization, the temperature 

of diacetyl, 2,3-pentanedione, and other flavoring compounds in heated tanks should be 

maintained as low as production processes 

will allow, even when closed systems are used. 

Employers should make sure that:

■ All temperature-related equipment such 

as thermometers and automatic shutoff mechanisms are regularly checked 

to ensure that they are in good working order. 

■ Tank thermometers and thermostats are 

calibrated at least monthly or as recommended by the manufacturer. 

■ Employees take periodic manual 

temperature readings with a stem thermometer inserted just below the surface 

of the heated agents or with an infrared thermometer.

8.3.5 Cleaning Practices for Equipment 

and Tools

Where possible, cold water should be used to 

clean out tanks and blenders to reduce the volatilization of chemicals into plant air. Employees 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 195

who are involved in cleaning or are working 

nearby should use appropriate PPE including 

respiratory protection, eye, and skin protection.

8.3.6 Limit Access to Priority 

Flavoring Compounds 

Employers should structure work tasks to minimize the amount of time employees spend near 

priority chemicals and production processes 

that involve these chemicals. Employers should 

limit access to areas where diacetyl, 2,3-pentanedione, or other flavoring compounds are 

used to only those employees who are essential 

to the process or operation. These areas should 

be clearly marked with signage.

8.3.7 Informing Employees about 

the Hazard

8.3.7.1 Safety and health programs

Employers should establish a comprehensive 

safety and health program for all employees who 

are performing any activity, such as manufacturing, using, handling, or disposing of diacetyl 

or 2,3-pentanedione, that involves exposure 

to these compounds or mixtures that include 

these compounds. This program should include 

training on workplace hazards, monitoring of 

airborne diacetyl and 2,3-pentanedione levels, 

and medical surveillance of employees exposed 

to these compounds or mixtures that include 

these compounds. All containers of food flavorings fall under the labeling requirements of the 

OSHA hazard communication standard (HCS) 

unless they are covered under the Federal Food, 

Drug and Cosmetic Act or the Virus-SerumToxin Act of 1913 [29 CFR 1910.1200 (b)(5)].

Employee training should include information outlined in the OSHA HCS in the section 

titled “Employee Information and Training” [29 

CFR 1910.1200 (h)(3)]. This includes information about diacetyl and 2,3-pentanedione and 

mixtures containing these compounds to which 

employees are exposed, explanation of safety 

data sheets and label elements, appropriate 

routine and emergency handling procedures, 

and recognition of the adverse health effects 

of exposure to these compounds, as well as 

other training requirements outlined in the 

OSHA HCS. 

OSHA revised the HCS to align with the 

United Nations Globally Harmonized System 

of Classification and Labeling of Chemicals 

(GHS) in March 2012. This revision provides 

detailed criteria for hazard classification as 

well as new label elements (pictograms, signal 

words, hazard statements, and precautionary 

statements) and establishes a standardized 

safety data sheet (SDS) format. An SDS (formerly known as a material safety data sheet 

or MSDS) is a form that communicates the 

dangers of hazardous chemicals and mixtures and guidance for safe use. As of June 1, 

2015, OSHA will require that SDSs adhere to 

a uniform format and include 16 sections that 

require specific information for the chemical 

or mixture listed on the SDS. More information on SDSs can be found on the OSHA HCS 

website at https://www.osha.gov/dsg/hazcom/

index.html. Employers should be aware of the 

changes, requirements, phase-in dates, and 

compliance effective dates of the revised HCS 

standard. OSHA has provided additional information on the phase-in requirements and dates 

for the transition to the revised HCS on their 

website at http://www.osha.gov/dsg/hazcom/

index.html. 

8.3.7.2 GHS classifications of diacetyl and 

2,3-pentanedione 

NIOSH has provided the following classification and labeling recommendations for diacetyl 

(Table 8-3) and 2,3-pentanedione (Table 8-4) 

according to the hazard classification and labeling elements outlined in the OSHA hazard 

communication standard [29 CFR 1910.1200]. 

These classifications are based on the health 

hazard criteria presented in Appendix A, and 
See footnotes at end of table. (Continued)

196 Occupational Exposure to Diacetyl and 2,3-Pentanedione

8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Table 8-3. Hazard classifications of diacetyl 

Health hazards*

GHS endpoint Hazard category Rationale [reference] Pictogram Hazard phrase Signal word†

Acute toxicity Category 2, inhalation Estimated 4 hr LC50 is 441 ppm based on 6 

hr exposure of 294.6 ppm in rats. Further explanation of this adjustment is provided in section 8.3.7.2.1 [Hubbs et al. 2008] 

Fatal if inhaled Danger

Serious eye 

damage/ eye irritation 

Category 1, serious eye 

damage 

0.1 ml of diacetyl in rabbits produced severe 

eye irritation with non-reversible effects after 21 days [Sugai et al. 1990]

Causes serious 

eye damage

Danger

Skin sensitization Category 1B, skin sensitizer EC3 values ranged from 11.3%–17.9% 

in mice via local lymph node assay [Anderson et al. 2011; Anderson et al. 2013; Roberts et al. 1999]

May cause an 

allergic skin reaction

Warning

Specific target 

organ toxicitysingle exposure‡

Category 1 Epithelial necrosis and inflammation in the 

trachea and larynx in rats at 224 ppm 

[Hubbs et al. 2008]

Causes damage 

to the respiratory system if inhaled

Danger

Specific target organ 

toxicity- repeated exposure‡

Category 1

Peribronchial lymphocytic infiltrates in mice 

at 25 ppm [Morgan et al. 2008; National Toxicology Program 2011]. Several case studies, public health investigations, and a cohort mortality follow-up study link exposure to flavorings containing diacetyl to fixed airway obstruction [Akpinar-Elci et al. 2004; Cavalcanti et al. 2012; CDC 2002, 2007, 2013; Halldin et al. 2013]. 

Causes damage 

to respiratory system through prolonged or repeated exposure if inhaled

Danger
Occupational Exposure to Diacetyl and 2,3-Pentanedione 197

8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Health hazards*

GHS endpoint Hazard category Rationale [reference] Pictogram Hazard phrase Signal word†

Physical hazards*

Flammable liquid Category 2 6°C [IPCS 2009]; 7°C [Sigma Aldrich 2010]

Highly flammable liquid 

and vapor

Danger

*Precautionary statements for the health and physical hazard classifications presented can be found in Appendix C of the hazard communication standard [29 CFR 1910.1200].†Appendix C of the hazard communication standard [29 CFR 1910.1200] provides several precedence rules regarding the application of pictograms and signal words as well as rules 

for combining or omitting hazard and precautionary statements. These precedence rules save space on the label and improve readability.

‡NIOSH recommends that these GHS classifications should appear on product labels and SDSs when found in mixtures below the specific cut-off values/concentration limits that 

are provided in the hazard communication standard [29 CFR 1910.1200]. See section 8.3.7.3 below for further information.

Table 8-3 (Continued). Hazard classifications of diacetyl 
See footnotes at end of table. (Continued)

198 Occupational Exposure to Diacetyl and 2,3-Pentanedione

8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Table 8-4. Hazard classifications of 2,3-pentanedione 

Health hazards*

GHS endpoint Hazard category Rationale [reference] Pictogram Hazard phrase Signal word†

Acute toxicity Category 2, inhalation Estimated 4 hr LC50 is 441 ppm based 

on 6 hr exposure of 294.6 ppm. Further explanation of this adjustment is provided in section 8.3.7.2.1 [Hubbs et al. 2012; Morgan et al. 2012]

Fatal if inhaled Danger

Skin sensitization Category 1B, skin 

sensitizer

EC3 value 15.4 % in mice via local lymph 

node assays 

May cause an allergic 

skin reaction

Warning

Specific target 

organ toxicitysingle exposure‡

Category 1 6-hour inhalation study in rats at 112 ppm 

caused necrotizing rhinitis. At 120 ppm exposure for 6 hrs, decreased airway activity to methacholine aerosol was observed.

[Hubbs et al. 2012]. After 120, 240, and 320 

ppm exposure for 6 hr, decreased airway activity to methacholine aerosol was observed [Zaccone et al. 2013].

Causes damage to the 

respiratory system if inhaled

Danger

Specific target 

organ toxicityrepeated exposure‡

Category 1 Inhalation exposure study (6 hours/day, 5 

days/week, for 2 weeks) in rats caused damage to airway epithelium, potential disruption to underlying basement membrane, and statistically significant influx of neutrophils in BALF at 200 ppm. In mice, the same repeat dose exposure regimen caused nasal turbinate necrosis (100 ppm) and laryngeal lesions (50 ppm). [Morgan et al. 2012]

Causes damage to respiratory system through 

prolonged or repeated exposure if inhaled

Danger
Occupational Exposure to Diacetyl and 2,3-Pentanedione 199

8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Table 8-4 (Continued). Hazard classifications of 2,3-pentanedione 

Health hazards*

GHS endpoint Hazard category Rationale [reference] Pictogram Hazard phrase Signal word†

Physical hazards

Flammable liquid Category 2 [Chem Service Inc. 1988; Merck Chemicals 

International 2010]

Highly flammable liquid 

and vapor

Danger

*Precautionary statements for the health and physical hazard classifications presented can be found in Appendix C of the hazard communication standard [29 CFR 1910.1200].

*Precautionary statements for the health and physical hazard classifications presented can be found in Appendix C of the hazard communication standard [29 CFR 1910.1200].

†Appendix C of the hazard communication standard [29 CFR 1910.1200] provides several precedence rules regarding the application of pictograms and signal words as well as rules 

for combining or omitting hazard and precautionary statements. These precedence rules save space on the label and improve readability.

‡NIOSH recommends that these GHS classifications should appear on product labels and SDSs when found in mixtures below the specific cut-off values/concentration limits that 

are provided in the hazard communication standard [29 CFR 1910.1200]. See section 8.3.7.3 below for further information.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

200 Occupational Exposure to Diacetyl and 2,3-Pentanedione

physical hazard criteria presented in Appendix 

B of the hazard communication standard [29 

CFR 1910.1200]. These classifications are based 

on the data from employee investigations 

(Chapter 3) and from experimental toxicology 

studies (Chapter 4). OSHA has provided guidance on hazard communication for diacetyl and 

food flavorings that contain diacetyl [OSHA 

2013] on the basis of the previous version of 

the HCS, but that guidance does not address 

some of the requirements in the revised HCS 

based on GHS.

8.3.7.2.1 Further justification of acute inhalation 

toxicity for diacetyl and 2,3-pentanedione

The GHS classification for acute inhalation 

toxicity, category 2 for diacetyl is based upon 

rat acute inhalation studies of diacetyl and 

2,3-pentanedione [Hubbs et al. 2012; Hubbs 

et al. 2008]. In the diacetyl study, the histopathology changes seen in rats exposed for 6 

hours to a time-weighted average of 294.6 to 

365 ppm diacetyl would be predicted to cause 

death if the animals had been observed for a 

longer time period. In exposures conducted in 

this concentration range, the severity scores in 

the airway epithelium of trachea, larynx, and 

multiple sections of nose had an average score 

of 7.5 to 9.5 on a scale of 1 to 10 (with 10 being 

most severe). Damage to airway epithelium 

is the accepted underlying cause for obliterative bronchiolitis in man, which causes human 

morbidity and mortality [King 1989]. The 

importance of extrapulmonary airway injury 

in the rodents to human risk assessment is discussed in the toxicology section. 

In the 2,3-pentanedione inhalation study in 

rats, clinical observations documented that no 

clinical signs were present immediately after the 

6 hour inhalation exposures to 318 or 354 ppm 

but respiratory signs were present in more than 

half of the rats at 18 hours post-exposure, when 

the rats were sacrificed [Hubbs et al. 2012].

While both of these inhalation studies were not 

intended to produce lethality, contemporary 

laboratory animal studies frequently use early 

indicators of impending mortality rather than 

actual mortality for studies of lethality [Stokes 

2002]. The presence of extensive respiratory 

epithelial damage in 100% of the rats at exposures of approximately 294.6 ppm or greater 

for 6 hours in both of these studies and timedependent progressive respiratory clinical signs 

are considered a humane endpoint for use in 

place of mortality. In this case, expert scientific 

judgment needs to be used to determine the 

LC50 because of the humane considerations. 

Because all rats had high pathology scores 

after inhaling 294.6 ppm or higher, NIOSH 

concludes that the LC50 based on a 4-hour 

exposure would be 441 ppm (the 4-hr equivalent of 294.6 ppm) or less. After inhaling 100 to 

120 ppm diacetyl for 6 hours, histopathology 

changes were limited to the first nasal section 

and single exposures at this concentration did 

not suggest potential acute lethality. Similarly, 

after inhaling 111 ppm 2,3-pentanedione for 6 

hours, rats did not have clinical signs and significant histopathology changes were limited to 

the first two nasal sections. This equates to a 

GHS acute inhalation toxicity category 2 classification (>100 and <500 ppm) for both diacetyl 

and 2,3-pentanedione.

8.3.7.3 Classifying mixtures containing 

diacetyl and 2,3-pentanedione

The HCS indicates that mixtures that contain 

compounds that require classification and 

labeling can be evaluated under a set of bridging principles if no toxicological data are 

available for the mixture itself. These bridging 

principles can be applied when there is “sufficient data on both the individual ingredients 

and similarly tested mixtures to adequately 

characterize the hazards of the mixture” [29 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 201

CFR 1910.1200.A.0.5]. If these bridging principles cannot be applied, the HCS provides 

specific cut-off values/concentration limits 

that are specified for each health hazard class 

and category. Most of these specific cut-off 

values/concentration limits are either ≥0.1% 

or ≥1%, under which mixtures containing classified compounds should be labeled 

accordingly. However, a few endpoints have 

different specific cut-off value/concentration limits specified. For most of the chemical 

hazards for which NIOSH made classifications 

(Tables 8-3 and 8-4), the specific cut-off values/

concentration limits specified by the HCS are 

≥1%. Exceptions include the hazard category 

for “serious eye damage/eye irritation” (≥3%) 

and for “flammable liquids,” for which the 

HCS does not have a cut-off value/concentration limit. If these mixtures contain classified 

compounds below the specified HCS cut-off 

values/concentration limits, classification 

and labeling of those mixtures are not usually 

required. However, the standard indicates that 

“while the adopted cut-off values/concentration limits adequately identify the hazard for 

most mixtures, there may be some that contain 

hazardous ingredients at lower concentrations 

than the specified cut-off values/concentration 

limits that still pose an identifiable hazard [29 

CFR 1910.1200.A.0.4.3.1]. As explained below, 

this is an important consideration for mixtures 

containing diacetyl and 2,3-pentanedione. 

Cal/OSHA provided industrial hygiene monitoring results from a Flavor Industry Safety and 

Health Evaluation Program evaluation in 2006 

and 2007 at a food flavoring manufacturer for 

the production of vanilla dry blend product 

[Widess 2013]. In this evaluation, a task-based 

personal breathing zone sample concentration of diacetyl collected over 19 minutes 

ranged from 3.5 to 5 ppm during dispensing of dry powder containing 0.14% diacetyl 

by weight. If a TWA exposure was calculated 

over an 8-hour work shift, assuming no other 

diacetyl exposure during the work shift, the 

8-hour TWA exposure would have been 0.2 

ppm, which exceeds the NIOSH 8-hour TWA 

REL (0.005 ppm). The exposure in the Flavor 

Industry Safety and Health Evaluation Program 

evaluation also exceeds the NIOSH STEL for 

diacetyl (0.025 ppm). In a NIOSH evaluation 

at a wholesale flavors and colors manufacturer, 

a task sample was collected when an employee 

was packaging dairy based flavoring into small 

containers over 33 minutes. Diacetyl comprised 

less than 1% of the total dairy flavored powder 

formulation. If a TWA exposure was calculated 

over an 8-hour work shift, assuming no other 

diacetyl exposure, the 8-hour TWA exposure 

would have been 0.33 ppm, which also exceeds 

the NIOSH 8-hour TWA REL [NIOSH 2008a]. 

Additionally, a laboratory-based study also 

identified emissions of diacetyl from natural 

butter and butter flavor powders, pastes, and 

liquid products in a laboratory environment 

[Rigler and Longo 2010]. Determinations show 

that even in the butter flavoring containing the 

lowest amount of diacetyl in the bulk flavoring (1.01% by weight), heating this flavoring to 

37.5°C released vapor concentrations of diacetyl as high as 13.67 ppm. This suggests that 

even if diacetyl is present in bulk concentrations of <1%, vapor concentrations of diacetyl 

could greatly exceed the NIOSH REL and 

STEL. NIOSH does not have data to confirm 

this same relationship between concentrations 

in bulk mixture and air for 2,3-pentanedione. 

Although the vapor pressure of 2,3-pentanedione (21.4 mm Hg at 20°C) is lower than diacetyl 

(52.2 mm Hg at 20°C) and will not volatilize 

as readily as diacetyl at room temperature, 

the initial boiling point of 2,3-pentanedione 

(108°C) suggests that it is still a volatile organic 

compound [EPA 2013] that can readily enter 

the vapor phase upon heating, leading to 

employee exposures.

The data presented in this criteria document strongly suggest that diacetyl and 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

202 Occupational Exposure to Diacetyl and 2,3-Pentanedione

2,3-pentanedione are toxic to the respiratory 

system at very low vapor concentrations. For 

this reason, NIOSH recommends that flavoring 

mixtures that contain diacetyl or 2,3-pentanedione should be provided on product labels and 

SDSs at concentrations below the default GHS 

mixture cutoff points. Specifically, NIOSH recommends labeling at concentrations that under 

the anticipated conditions of use could generate 

vapors exceeding the NIOSH REL and/or STEL. 

In these cases the labels and SDSs should carry 

the pictogram, hazard phrase, signal word, and 

precautionary statements for the specific target 

organ toxicity-single exposure and specific target 

organ toxicity-repeated exposure endpoints. If 

specific cut-off values can be established otherwise, this recommendation does not need to 

be followed.

Regarding the nonrespiratory endpoints under 

which diacetyl and 2,3-pentanedione have 

been classified by NIOSH (Tables 8-3 and 8-4), 

NIOSH does not have any data to suggest that 

mixtures containing these compounds in concentrations less than the specific cutoff values/

concentration limits specified by the HCS are 

hazardous. This includes the acute toxicity, skin 

corrosion/irritation, serious eye damage/eye irritation, skin sensitization, and flammable liquid

endpoints for diacetyl, and acute toxicity and 

flammable liquid endpoints for 2,3-pentanedione. NIOSH recommends that manufacturers 

carefully evaluate whether mixtures containing 

these compounds below the cut-off values/concentration limits specified in the HCS should 

be labeled. 

The Flavor and Extract Manufacturers 

Association has recommended that several 

flavoring substances, including diacetyl and 

2,3-pentanedione, should include the following label warning if they are present in 

compounded flavors (including liquid and 

dry or powdered mixtures) in any concentration if they will be heated during processing 

[FEMA 2012]:

WARNING – This flavor may pose an inhalation 

hazard if improperly handled. Please contact 

your workplace safety officer before opening 

and handling, and read the MSDS. Handling 

of this flavor that results in inhalation of fumes, 

especially if the flavor is heated, may cause severe 

adverse health effects.

FEMA has also recommended that this same 

warning should be used for containers of neat 

substances such as diacetyl and 2,3-pentanedione as well as other “high priority” substances 

listed in the FEMA guidance document. 

Additionally, FEMA has recommended that all 

containers of compounded flavors (liquid and 

dry or powdered) or natural flavoring complexes that contain diacetyl, 2,3-pentanedione 

or other flavoring substances in concentrations of >1.0% should be labeled with the above 

warning [FEMA 2012]. It is of note that the 

use of the word “warning” in the FEMA text is 

inconsistent with the specific criteria for its use 

and application as a signal word in the HCS. 

NIOSH recommends removal of the word 

“warning” when using the FEMA text (see 

section 8.3.7.4 for details)

8.3.7.4 Labeling and posting

To communicate hazard information effectively 

to employees, employers should:

■ Post appropriate labeling on all flavoring product containers according to the 

HCS requirements [29 CFR 1910.1200]. 

In this document, NIOSH is providing 

the recommended label elements, including signal word, hazard statements, and 

pictograms, that should be included for 

labeling of diacetyl and 2,3-pentanedione on SDSs and labels for shipping 

containers [see Tables 8-3 and 8-4]. The 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 203

precautionary statements that are also 

required can be found in Appendix C to 

the HCS [29 CFR 1910.1200]. NIOSH 

also recommends that mixtures containing diacetyl or 2,3-pentanedione at any 

concentration that could generate vapors 

that could exceed the NIOSH REL and/or 

STEL carry the pictogram, hazard phrase, 

and signal word for the specific target 

organ toxicity-single exposure and specific 

target organ toxicity-repeated exposure

classifications until it can be demonstrated that mixtures containing these 

compounds in concentrations less than 

the specific cut-off values/concentration 

limits specified by HCS are not harmful.

■ Place the following warning, as recommended by FEMA [FEMA 2012], on 

containers of compounded flavors that 

contain diacetyl, 2,3-pentanedione, or 

other flavoring substances identified in 

Table 1 of the FEMA document, in any 

concentration if the flavors are to be 

heated: This flavor may pose an inhalation hazard if improperly handled. 

Please contact your workplace safety 

officer before opening and handling, and 

read the MSDS. Handling of this flavor 

that results in inhalation of fumes, especially if the flavor is heated, may cause 

severe adverse health effects. Note: While 

NIOSH agrees with the content of the 

italicized text above, the word “warning,” 

which appears in the FEMA guidance 

document, was not included because it is 

inconsistent with the specific criteria for 

its use and application as a signal word in 

the HCS. NIOSH recommends that the 

word “warning” should not be included 

on hazard statements containing diacetyl or 2,3-pentanedione, as this word has 

specific meaning and conflicts with standardized HCS signal word terminology.

■ Post warning labels and signs describing 

the health risks associated with flavoring compound exposures at entrances to 

work areas and inside work areas where 

diacetyl, 2,3-pentanedione, or other flavoring compounds are used.

■ Post warning labels and signs describing 

any needs for PPE in the work area. 

■ Post the statement “Wear Respiratory 

Protection in this Area” if respiratory protection is required.

■ Print all labels and warning signs in 

English and in the predominant language 

of employees who do not read English.

■ Verbally inform employees about the 

hazards and instructions printed on the 

labels and signs if they are unable to 

read them.

■ Follow the requirements of the HCS 

for classifying and labeling diacetyl,

2,3-pentanedione, and other flavoring 

compounds. NIOSH recommends that 

development of SDSs and labels should 

occur as soon as possible given the importance of warning users about exposures 

of diacetyl and 2,3-pentanedione above 

the REL and or STEL. The OSHA website 

has additional information on the hazard 

communication standard at http://www.

osha.gov/dsg/hazcom/index.html.

8.3.7.5 Training

Employees should receive training as mandated 

by the HCS [29 CFR 1910.1200]. As part of the 

training, employers should also:

■ Inform all potentially exposed employees, including temporary and contract 

employees, about diacetyl or 2,3-pentanedione-associated health risks such 

as acute toxicity, skin irritation and 

sensitization, eye irritation or damage, 

respiratory disease, and flammability hazards.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

204 Occupational Exposure to Diacetyl and 2,3-Pentanedione

■ Train employees to report to management any eye or skin problems that may 

be associated with exposure to flavoring 

compounds and any persistent or worsening respiratory symptoms such as cough, 

shortness of breath, or wheezing.

■ Train employees to recognize hazardous situations.

■ Inform employees about practices or 

operations that may generate airborne 

diacetyl or 2,3-pentanedione concentrations above the REL and or STEL 

(e.g., mixing).

■ Establish procedures for reporting 

hazards and giving feedback about 

actions taken to correct them.

■ Train employees in the proper use and 

maintenance of implemented engineering 

controls to protect them from hazardous exposures.

■ Train employees in the proper use and 

maintenance of PPE.

■ Inform employees about other flavoring 

compounds that may pose occupational 

exposure hazards.

8.4 Respiratory Protection

Respirators should not be used as the primary 

means of controlling employee exposures to 

inhalation hazards for routine operations. 

Whenever possible, engineering and work 

practice control techniques discussed above 

should be used. Respirators may be needed and 

can be used during the implementation of engineering controls and work practices, during 

some short-duration maintenance procedures, 

and during emergencies. Respirators should be 

used for exposure situations when engineering 

controls cannot reduce exposures to concentrations below the REL.

Employers need to monitor work processes 

to accurately determine exposure levels of 

airborne chemicals. Respiratory protection 

should be provided when that assessment indicates exposures may exceed the NIOSH REL 

of 5 ppb TWA or 25 ppb STEL for diacetyl; 

when exposures may exceed the NIOSH REL 

of 9.3 ppb TWA or 31 ppb STEL for 2,3-pentanedione; when occupational exposure limits 

of other chemicals may be exceeded; or when 

exposures of concern to diacetyl substitutes 

without OELs occur. When respiratory protection is used, employers need to establish a 

written respiratory protection program that 

meets the requirements of the OSHA respiratory protection standard 29 CFR 1910.134. The 

program should be administered by a suitably 

trained program administrator and updated to 

reflect changes in workplace conditions that 

affect respirator use [29 CFR 1910.134]. 

A respiratory protection program should 

include the following elements:

■ Procedures for selecting respirators for 

use in the workplace.

■ Medical evaluations of employees 

required to use respirators.

■ Fit testing procedures for tight-fitting respirators.

■ Procedures for proper use of respirators 

in routine and reasonably foreseeable 

emergency situations.

■ Procedures and schedules for cleaning, disinfecting, storing, inspecting, 

repairing, discarding, and otherwise 

maintaining respirators.

■ Procedures to ensure adequate air quality, 

quantity, and flow of breathing air for 

atmosphere-supplying respirators

■ Training for employees in the respiratory 

hazards to which they are potentially 

exposed during routine and emergency situations.

■ Training for employees in the proper use 

of respirators, including putting on and 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 205

removing them, any limitations on their 

use, and their maintenance.

■ Procedures for regularly evaluating the 

effectiveness of the program.

If an air-purifying respirator with cartridge/

canister for the protection against gases and 

vapors does not have an end-of-service-life 

indicator, then the employer is required to 

implement a cartridge/canister change schedule 

based on objective information that will ensure 

that the cartridges/canisters are changed before 

the end of their service life, according to the 

OSHA respiratory protection standard which 

was revised in 1998. The revised OSHA respiratory protection standard removed the previous 

method of determining the end of a cartridge’s 

service life by using warning properties such as 

odor and irritation. A cartridge’s useful service 

life is how long it provides adequate protection 

from the harmful chemicals in the air which are 

identified in the respirator approval. A change 

schedule to establish the time period for replacing respirator cartridges and canisters is the 

part of the written respirator program that is 

used to determine how often cartridges should 

be replaced. Data and information relied upon 

to establish the schedule should be included 

in the respirator program. The use of warning 

properties such as odor and irritation cannot be 

used as the sole basis for determining change 

schedules. However, respirator users should 

be trained to understand that they should 

leave the area if abnormal odor or irritation is 

experienced. The respirator should be checked 

to see if the odor or irritation is evidence that 

respirator cartridges need to be replaced or the 

respirator facepiece needs adjustment for better 

face seal fit. 

The following table indicates which types 

of respirators are recommended for use 

against diacetyl and 2,3-pentanedione and 

the maximum use concentrations for diacetyl 

and 2,3-pentanedione, calculated using the 

OSHA-assigned protection factors for each type 

of respirator listed [29 CFR 1910.134 (d)(3)(i)

(A)]. For escape, use a gas mask with a full facepiece and OV-P100 canisters or self-contained 

breathing apparatus. All of the air-purifying 

respirators listed in Table 8-5 are equipped with 

combination organic vapor/P100 or organic 

vapor/high efficiency filter cartridges, which 

are capable of protecting wearers against both 

vapor and particulate hazards.

All respirators selected for use should be 

approved by NIOSH under the provisions of 

42 CFR Part 84, as required by OSHA regulations. The current listing of NIOSH certified 

respirators can be found in the NIOSH Certified 

Equipment List, which is available on the 

NIOSH website [NIOSH 2010].

Selection of a specific respirator within a given 

class of recommended respirators depends on 

the particular situation; this choice should be 

made only by qualified personnel. There is no 

formal certification requirement for a respiratory protection program manager. Employee 

activity and employee location in a hazardous environment need to be considered in 

respirator selection, as well as the time period 

of use, and the type of respirator application, 

such as for routine, nonroutine, emergency or 

rescue use.

Additional information on the selection and 

use of respirators can be found in the NIOSH 

Respirator Selection Logic [NIOSH 2004].

8.5 Dermal, Eye, and 

Face Protection

Diacetyl can cause skin and eye irritation. 

Chemical resistant gloves or sleeves or other 

appropriate protection for exposed skin 

should be used when handling liquid, paste, 

or powdered flavoring compounds containing diacetyl that could cause dermal injury 
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

206 Occupational Exposure to Diacetyl and 2,3-Pentanedione

[29 CFR 1910.138]. It is important to select 

the most appropriate chemical resistant glove 

for the application and to determine how 

long it can be worn, and whether it can be 

reused. Procedures should be implemented 

to ensure that the gloves are replaced before 

breakthrough occurs. NIOSH recommends 

that before purchasing gloves or other protective clothing, the employer should refer to 

the SDS from the manufacturer of the diacetyl and 2,3-pentanedione being used, and /or 

request documentation from the glove or protective clothing manufacturer that the gloves 

meet the appropriate test standard(s) for the 

hazard(s) anticipated, and to request any glove 

and protective clothing breakthrough time data 

against diacetyl and 2,3-pentanedione that may 

be available from these sources. Tight-fitting 

Table 8-5. OSHA assigned protection factors and maximum use concentrations of respirators 

for diacetyl and 2,3-pentanedione

Type of respirator

OSHA assigned 

protection factor

Maximum use 

concentration 

for diacetyl*

Maximum use 

concentration for 

2,3-pentanedione*

Full facepiece air purifying, w/OV-P100 

cartridge(s) or canister(s)

50 0.25 ppm

(250 ppb)

0.46 ppm

(460 ppb)

PAPR, full facepiece w/OV-HE cartridge(s) 

or canister(s)

1,000 5 ppm

(5,000 ppb)

9.3 ppm

(9,300 ppb)

PAPR, hood or helmet w/OV-HE 

cartridge(s) or canister(s)

25/1,000† 0.12/5 ppm

(120/5,000 ppb)

0.23/9.3 ppm

(230/9,300 ppb)

PAPR, loose fitting facepiece w/OV-HE 

cartridge(s) or canister(s)

25 0.12 ppm

(120 ppb)

0.23 ppm

(230 ppb)

SAR, continuous flow mode or 

pressure- demand mode or other 

positive- pressure mode, full facepiece

1,000 5 ppm

(5,000 ppb)

9.3 ppm

(9,300 ppb)

SAR, hood or helmet 25/1,000† 0.12/5 ppm

(120/5,000 ppb)

0.23/9.3 ppm

(230/9,300 ppb)

SAR, loose fitting facepiece 25 0.12 ppm

(120 ppb)

0.23 ppm

(230 ppb)

SCBA, pressure-demand or other positivepressure mode (e.g. open/closed circuit), 

full facepiece or hood/helmet

10,000 50 ppm 93 ppm

PAPR = Powered air-purifying respirator

SAR = Supplied air respirator

OV-HE = Organic vapor-high efficiency particulate

SCBA = Self-contained breathing apparatus 

*

Maximum use concentrations will be lower than shown when those concentrations are equal to or exceed immediately dangerous 

to life and health levels. 

†

The employer should have evidence provided by the respirator manufacturer that testing of these respirators demonstrates performance at a level of protection of 1,000 or greater to receive an assigned protection factor (APF) of 1,000. Absent such evidence, 

these respirators receive an APF of 25.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 207

chemical goggles, used in conjunction with a 

face shield or other appropriate eye and face 

protection should also be used. 

Eye and face protection should be provided 

when there is a hazard from flying particles, 

molten metal, liquid chemicals, acids or caustic 

liquids, chemical gases or vapors, or potentially 

injurious light radiation. OSHA regulations at 

29 CFR 1910.133 contain the specific requirements. Protective eye and face devices 

purchased after July 5, 1994, should comply 

with ANSI Z87.1-1989, “American National 

Standard Practice for Occupational and 

Educational Eye and Face Protection,” which is 

incorporated by reference in the OSHA regulations [29 CFR 1910.133]. The ANSI standard 

was revised in 2010 [ANSI 2010]. The current 

edition also includes respirators that cover the 

eyes and face as approvable under the standard.

Goggles for chemical splash should be used 

for eye protection for employees with potential exposures to diacetyl, 2,3-pentanedione, or 

food flavorings containing these compounds 

who are not also required to wear a respirator with a full facepiece, hood, or helmet. Face 

shields can also be used in conjunction with 

goggles to shield the wearer’s face, or portions 

thereof, in addition to the eyes for protection 

from liquid splash. Face shields should be worn 

only in conjunction with spectacles and goggles, 

as required by ANSI Z87.1-2010 [ANSI 2010]. 

A face shield with a polyethylene terephthalate 

visor should provide good chemical resistance 

against diacetyl, 2,3-pentanedione, or food flavorings containing these compounds.

Gloves and protective clothing such as aprons 

made from butyl rubber, TeflonTM, or TychemTM 

are effective in reducing skin contact with 

ketones to prevent skin irritation [OSHA 2013]. 

Diacetyl and 2,3-pentanedione are diketones 

and certain food flavorings containing either 

may contain other ketones or diketones. Glove 

suppliers should be contacted to ensure that 

appropriate glove materials are selected for the 

specific chemicals involved [OSHA 2002].

An analysis should be performed on each 

operation involving diacetyl, 2,3-pentanedione, or other food flavoring compounds to 

assess the potential exposures and to establish 

specific guidance about when to use skin, eye, 

and face protection.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

208 Occupational Exposure to Diacetyl and 2,3-Pentanedione

W

Baffles are

desirable

Maximum plenum velocity

1/2 slot velocity

12”

24”

Slots-size for 2000 feet

per minute

45o

 taper angle

100 feet per 

minute

Figure 8-1. Welding ventilation bench hood*

*

VS-90-01, From ACGIH, Industrial Ventilation: A Manual of Recommended Practice for Design, 26th Edition. 

Copyright 2009. Reprinted with permission
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 209

Figure 8-2. Ventilated small batch mixing workstation
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

210 Occupational Exposure to Diacetyl and 2,3-Pentanedione

0.5 to 1 

meters per 

second

(100 to 200 fpm)

Figure 8-3. Benchtop ventilation for weighing and 

handling powders 0.5 to 1 m/s = 100 to 200 fpm*

*

Contains public sector information published by the Health and Safety Executive and licensed under the Open 

Government License v1.0.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 211

Figure 8-4. Schematic of a laboratory chemical fume hood*

*

Reprinted from SEFA 1-2002, Laboratory Fume Hoods: Recommended Practices[SEFA 2002].

Auxiliary Air Plenum 

( Optional )

Bypass Front Panel

Aerodynamic Fascia

Sash

Services

Hood Exterior

Airfoil Sill

Storage Cabinet

STORAGE

Work

Surface

Slots

Liner

Baffle

Light 1000

EXHAUST

EXIT

SELF
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

212 Occupational Exposure to Diacetyl and 2,3-Pentanedione

Figure 8-5. Mixing vessel with a ventilated hinged tank lid
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 213

Figure 8-6. Ventilated booth for large batch mixing
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

214 Occupational Exposure to Diacetyl and 2,3-Pentanedione

Exhaust

Exhaust

Minimum airflow

1 meters per second

Charging port

200 feet per minute

Figure 8-7. Charging reactors and mixers from a sack or keg*, 1 m/s = 200 fpm

*

Contains public sector information published by the Health and Safety Executive and licensed under the Open 

Government License v1.0.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 215

Exhaust

Figure 8-8. Annular exhaust for capturing dusts/vapors from mixers
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

216 Occupational Exposure to Diacetyl and 2,3-Pentanedione

Exhaust

1.0 meters per 

second minimum 

airflow

Waste bag collection Open grill work shelf

Figure 8-9. Ventilated bag dumping/emptying station*

1.0 m/s = 200 fpm

*

Contains public sector information published by the Health and Safety Executive and licensed under the Open 

Government License v1.0.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 217

SIDE VIEW

OVERHEAD VIEW

Exhaust

Feed pipe

Figure 8-10. Annular exhaust for capturing vapors during drum filling*

*

Contains public sector information published by the Health and Safety Executive and licensed under the Open 

Government License v1.0
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

218 Occupational Exposure to Diacetyl and 2,3-Pentanedione

Figure 8-11. Ventilation design options for capturing vapors during drum filling*

*

VS-15-01, From ACGIH, Industrial Ventilation: A Manual of Recommended Practice for Design, 26th Edition. 

Copyright 2009. Reprinted with permission.

Close clearance

D + 12”

D

45°

1” slot

4” min.

2 Q = 100 cfm/ft barrel top (minimum)

Minimum duct velocity = 3500 fpm

h = 1.78 VPs

 + 0.25 VP e d he

2 Q = 150 cfm/ft of open face area

Minimum duct velocity = 3500 fpm

= 0.25 VPd (45°) taper)

Feed spout

4” min. dia. Exhaust duct

45°

Flex duct

Q = 50 cfm × drum diam. (ft)

Minimum duct velocity = 3500 fpm

h = 0.25 VP e d

45°

Q = 300–400cfm

Minimum duct velocity = 3500 fpm

h = 0.25 VP e d
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

Occupational Exposure to Diacetyl and 2,3-Pentanedione 219

Feed hopper

Exhaust

Partial

enclosure

around filling

head

Bag clamp

Grid area to

catch spillages

Figure 8-12. Ventilation for bag filling*

*

Contains public sector information published by the Health and Safety Executive and licensed under the Open

Government License v1.0.
8 . Hazard Prevention and Control of Exposures to Diacetyl and 2,3-Pentanedione

220 Occupational Exposure to Diacetyl and 2,3-Pentanedione

Figure 8-13. Dust control during bag filling operation
Occupational Exposure to Diacetyl and 2,3-Pentanedione 221

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Evaluation of the hypersensitivity potential of alternative 

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[abstract]. Toxicologist 120(2):520.

Anderson SE, Franko J, Wells JR, Lukomska E, Meade 

BJ [2013]. Evaluation of the hypersensitivity potential of alternative butter flavorings. Food Chem 

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Hygiene Association Publication No. ANSI Z9.5–2002.

Boylstein R, Piacitelli C, Grote A, Kanwal R, Kullman G, 

Kreiss K [2006]. Diacetyl emissions and airborne dust 

from butter flavorings used in microwave popcorn production. J Occup Environ Hyg 3(10):530–535.

Burgess WA, Ellenbecker MJ, Treitman RD [2004]. 

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Hoboken, NJ: Wiley-Interscience.

Cavalcanti ZD, Albuquerque Filho AP, Pereira CA, 

Coletta EN [2012]. Bronchiolitis associated with 

exposure to artificial butter flavoring in workers 

at a cookie factory in Brazil. Jornal Brasileiro 

DePneumologia 38(3):395–399.
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microwave popcorn factory—Missouri, 2000–2002. 

MMWR 51(16):345–347.

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[2007]. Fixed obstructive lung disease among workers 

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coffee-processing facility—Texas, 2008-2012. MMWR 

62(16):305–307.

Cecala AB, Volkwein JC, Daniel JH [1988]. Reducing bag 

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 225

9 Medical Monitoring and Surveillance of 

Exposed Employees

Despite attempts to control exposure to diacetyl, 

2,3-pentanedione, and similar flavoring compounds, some employees may develop health 

effects as a result of insufficient control, additive 

effects, intermittent peak exposures, susceptibility, unmeasured flavoring compounds in 

powdered form, or unrecognized hazardous 

exposures. Medical monitoring and surveillance 

of employees exposed to diacetyl and similar 

flavoring compounds are important, as these 

employees are at risk of rapidly developing 

severe irreversible lung disease. The rapid onset 

and progression of diacetyl-related lung disease 

requires that more frequent medical monitoring 

evaluations be done than for slowly progressive occupational lung diseases such as silicosis 

and coal worker’s pneumoconiosis. The most 

important component of an effective medical 

monitoring program for an employee exposed 

to diacetyl and similar flavoring compounds is to 

carefully follow spirometry test results over time, 

comparing current to past test results to identify 

excessive declines in lung function [California 

Department of Public Health 2012]. Spirometry 

tests must be of high quality to allow valid interpretation of lung function changes over time. 

This chapter provides information on how to 

conduct effective medical monitoring of these 

employees. The chapter also provides examples 

that illustrate how medical surveillance can 

identify workplace risk factors.

Medical Monitoring

Medical monitoring of employees, sometimes 

called medical screening, involves periodic 

medical follow up for early detection of workrelated disease. The intended benefit of early 

detection is to identify disease in early stages 

when steps can still be taken to prevent progression from pre-clinical to clinical disease 

or from milder to more symptomatic disease. 

This approach is called secondary prevention 

because it attempts to ameliorate or at least 

halt the progression of health effects that have 

already occurred. Evidence of early disease identified through medical monitoring serves as a 

sentinel event or warning that other employees 

might be at risk for the same exposures and outcomes. This warning should stimulate efforts to 

evaluate the workplace to identify possible risk 

factors for exposures that can be controlled. 

Systematic evaluation and use of medical monitoring data obtained from individual employees 

to better protect a population of employees is 

an important component of the overall medical 

surveillance program. This approach contributes 

to the goal of primary prevention, to prevent 

disease from developing in other employees. 

Medical Surveillance

The systematic analysis of aggregated results 

over time constitutes medical (epidemiologic) 

surveillance of trends in symptoms or functional 

changes that can be assessed in relationship to 

jobs, tasks, and exposures [Silverstein 1990]. 

For medical monitoring to serve surveillance 

purposes, a formal process should be in place 

to assure that data from a screened employee 

population is evaluated in aggregate at regular 

intervals. Epidemiologic analysis of medical 
9 . Medical Monitoring and Surveillance of Exposed Employees

226 Occupational Exposure to Diacetyl and 2,3-Pentanedione

results and questionnaire and/or administrative data to evaluate for possible risk factors 

for disease can result in understanding what 

actions need to be prioritized to decrease the 

risk of subsets of employees and can document 

the effectiveness of interventions over time in 

preventing flavoring-related health effects. 

9.1 Medical Monitoring 

Program Director

The medical monitoring program director 

should be a licensed physician with training 

and experience in identifying and preventing occupational lung disease. This is because 

flavoring-related lung disease can progress 

rapidly and have grave consequences, so it is 

important to assure that the medical monitoring program director can quickly evaluate 

clinical data and make medical judgments 

about appropriate diagnostic and therapeutic 

measures, including medical removal. This 

individual (hereafter referred to as “the medical 

monitoring program director”) should ensure 

that the monitoring program collects high 

quality data, including relevant questionnaire 

data and high quality spirometry tests that 

adhere to ATS/ERS technical guidelines for spirometry [Miller et al. 2005], or the most recent 

equivalent guidelines. The medical monitoring program director should also ensure that 

medical monitoring data is appropriately 

evaluated for surveillance purposes, including evaluation of aggregated results to identify 

risk factors and opportunities to better prevent 

flavoring-related lung disease. 

The employer should ensure that the medical 

monitoring program director is familiar with 

the natural history of flavoring-related lung 

disease and is knowledgeable about operating 

a spirometry program that maintains high test 

accuracy, precision and validity. The employer 

should provide the following to the medical 

monitoring program director: 

■ A copy of the NIOSH Alert, “Preventing 

Lung Disease in Workers Who Use or 

Make Flavorings” [NIOSH 2003]; 

■ A copy of this criteria document; 

■ A description of work areas, job categories, and work tasks;

■ A description of any personal protective 

equipment to be used by employees; and

■ Results of any environmental sampling 

related to potential flavorings exposures.

9.2 Employees to Include in 

the Medical Monitoring 

Program

All permanent, temporary, and contract 

employees who work in or enter areas where 

diacetyl, 2,3-pentanedione, or similar flavoring compounds or products that contain these 

compounds are used or produced should be 

included in the medical monitoring program. 

Employees who work in or enter these areas for 

a total of 40 or more hours per year should be 

included in the medical monitoring program. 

In addition to production employees, employees who are periodically exposed such as 

supervisors, warehouse employees, laboratory employees, quality assurance/control 

employees, shipping and receiving employees, 

maintenance employees, janitorial employees, 

and office employees should also be included in 

the program, as employees with lung function 

abnormalities were identified in nonproduction 

jobs during several NIOSH HHE investigations [Kanwal et al 2006; Kanwal et al 2011]. 

Employees with past experience in such jobs 

or performing such duties should be included 

in the monitoring program for one year and 

longer if abnormalities are present [California 

Department of Public Health 2012].

To achieve the intent of primary and secondary prevention, employers have an interest in 
9 . Medical Monitoring and Surveillance of Exposed Employees

Occupational Exposure to Diacetyl and 2,3-Pentanedione 227

attaining a high rate of employee participation 

in regular medical monitoring. Voluntary participation should be encouraged at a time and 

place convenient to employees and should be 

provided at no cost to employees.

9.3 Medical Monitoring 

Program Elements

The medical monitoring evaluation should 

include a questionnaire to obtain health and 

exposure information and spirometry to assess 

lung function. The questionnaire data from all 

employees in a medical monitoring program 

should be entered into a database along with 

spirometry results for use in epidemiologic 

analyses for medical surveillance. These analyses may reveal associations between health 

outcomes and exposure variables such as work 

tasks and practices that can be addressed to 

decrease lung disease risk (see section 9.9).

9.3.1 Questionnaire

The purpose of the questionnaire is to obtain 

standardized information on demographics, work history, exposures, personal risk 

factors such as smoking and health history. 

The medical monitoring program director can 

use information from the questionnaire when 

assessing the employee at each evaluation. 

Because employees with biopsy-documented 

obliterative bronchiolitis may have normal 

spirometry, chest symptoms such as exertional shortness of breath merit attention as 

suggestive of an occupational lung condition 

requiring employee education and follow up. 

Similarly, persons with abnormal spirometry, 

despite absent chest symptoms, may have occupational lung disease requiring attention.

Work history questions should allow employees to correctly indicate the specific job titles 

they have held at their current employer. For 

each job title, the questionnaire should collect 

information on specific work tasks and practices that may affect the employee’s exposure 

to diacetyl and similar flavoring compounds. 

For example, for an employee whose job 

requires direct handling of diacetyl-containing 

flavorings, specific questions might address 

how often a particular task is performed, the 

amounts of flavorings used, whether open or 

closed containers of flavorings are used, and 

whether respiratory protection is used, including the type of respirator used and when it is 

worn. To help the medical monitoring program 

director develop appropriate questions on jobs 

and exposures, the employer should provide 

the medical monitoring program director with 

the specific job titles of potentially exposed 

employees, a description of the work tasks for 

each job that may be associated with potential 

for exposure to diacetyl and similar flavoring 

compounds, and the types of personal protective equipment (e.g., respirators) and other 

measures that employees have available to them 

to minimize exposures in each job. A visit to 

the plant by the medical monitoring program 

director to view the production process may 

provide additional useful information for questionnaire development.

The questionnaire should contain questions on 

the presence or absence of respiratory symptoms such as shortness of breath on exertion, 

cough, and wheezing; respiratory illnesses such 

as asthma, emphysema, chronic bronchitis, and 

COPD; and the dates of diagnosis. Additional 

questions might inquire about work-related 

nasal, ocular, and dermal symptoms. The 

American Thoracic Society Respiratory 

Symptom Questionnaire [Ferris 1978] or the 

NHANES III questionnaire [CDC 1994] can 

provide standardized questions. Examples of 

questions NIOSH has used in HHE medical 

surveys of flavoring-exposed employees can be 
9 . Medical Monitoring and Surveillance of Exposed Employees

228 Occupational Exposure to Diacetyl and 2,3-Pentanedione

found in NIOSH HHE reports at http://www.

cdc.gov/niosh/hhe/.

While respiratory symptom information 

is important in the assessment of employees exposed to diacetyl and similar flavoring 

compounds or products that contain these 

compounds, the medical monitoring program 

director should not conclude that an employee’s 

exposures are below harmful levels solely by the 

absence of respiratory symptoms. Employees 

may not experience respiratory symptoms early 

in the course of excessive lung function decline. 

NIOSH medical surveys of flavoring-exposed 

employees have identified airways obstruction [Kreiss et al. 2002] and excessive declines 

in lung function [NIOSH 2008] in employees 

who did not report respiratory symptoms. 

Similarly, about half of the employees with 

airways obstruction found in surveillance of 

California flavoring manufacturing employees had no chest symptoms [Kim et al. 2010]. 

Absence of symptoms does not negate the need 

for clinical differential diagnosis and evaluation 

of employees with spirometric abnormalities.

The medical monitoring program director 

should counsel employees identified as having 

pre-existing lung disease on their initial evaluation regarding the potential risks of working 

in areas where they may be exposed to diacetyl 

and other flavoring compounds. The medical 

monitoring program director should also 

explain that it may be hard to determine the 

relative contributions of work exposures vs. 

pre-existing lung disease to any future abnormal lung function declines. Such employees 

should also be referred to their personal 

physician for additional evaluation and recommendations regarding potential exposure to 

these substances.

9.3.2 Spirometry

Every employee in the medical monitoring 

program should have a spirometry test at each 

evaluation irrespective of respiratory symptom 

status. Evaluation of lung function over time 

is the most important component of medical 

monitoring for identifying possible workrelated lung disease in employees exposed to 

diacetyl and similar flavoring compounds (see 

section 9.6). High quality spirometry tests 

are necessary to allow the medical monitoring program director to correctly interpret the 

results and make appropriate recommendations 

to the employee and the employer. Accurate 

spirometry measurements depend on four 

key elements: (1) a trained technician who 

can obtain valid test results, (2) a reliable and 

accurate spirometer, (3) an approved testing 

protocol, and (4) a spirometry quality assurance 

program directed by a laboratory supervisor or 

the medical monitoring program director.

9.3.2.1 Persons administering the 

spirometry examination

Each person administering spirometry 

examinations should successfully complete 

a NIOSH-Approved Spirometry Training 

Course (information at http://www.cdc.gov/

niosh/topics/spirometry/training.html) or 

equivalent and maintain valid certificates. The 

medical monitoring program director may 

also benefit from this training. The ATS/ERS 

[Miller et al. 2005] and the American College 

of Occupational and Environmental Medicine 

(ACOEM) [Townsend 2011] endorse the 

content of NIOSH-approved spirometry training and also recommend refresher training 

for spirometry technicians. Both the ATS/ERS 

and ACOEM recommend ongoing review of 

spirometry tests for quality after training to 

identify and correct any aspects of the technician’s performance that have resulted in poor 

quality tests. The medical monitoring program 

director should provide for ongoing review of 

test quality and feedback to technicians about 

opportunities for improvement. The combination of initial training, refresher training, 

electronic feedback from spirometers during 
9 . Medical Monitoring and Surveillance of Exposed Employees

Occupational Exposure to Diacetyl and 2,3-Pentanedione 229

testing, and ongoing review of test quality 

with timely feedback to technicians can help 

a program achieve a high proportion of technically acceptable spirometry tests [Redlich 

et al. 2014]. Certification of acceptable spirometry test administration is an additional 

means of addressing quality concerns (National 

Board for Respiratory Care [NBRC 2016]; 

American Association for Respiratory Care 

[AARC 2011]).

9.3.2.2 Spirometer specifications

Spirometry testing equipment should meet the 

ATS/ERS guidance for standardization of spirometry or most recent equivalent [Miller et 

al. 2005], specifications for spirometer accuracy and precision, and real-time display size 

and content. Written verification from a third 

party testing laboratory (not the manufacturer 

or distributor) that the model of spirometer 

being used has successfully passed its validation 

checks as required by the most current ATS/

ERS protocol should be requested from the spirometer manufacturer.

9.3.2.3 Spirometry testing protocol and 

reporting information

Administration of spirometry tests should 

follow the ATS/ERS guidance for standardization of spirometry or most recent equivalent 

Testing 

Procedures

1. Spirometer calibration checks should be performed using a currently calibrated (per 

manufacturer recommendations) 3-liter syringe on each day of testing [Miller et al. 

2005]. A copy of the spirometer calibration report should be maintained in either 

electronic or hard copy form.

2. Spirometry should be performed in the same documented position (either sitting or 

standing) during the baseline and all subsequent tests.

3. A minimum of three forced exhalation maneuvers producing “acceptable curves” on 

the spirometry report should be characterized by the following:

■ Lack of hesitation (back-extrapolation volume should be less than 5% of FVC 

or 150 mL, whichever is larger)

■ No cough in the first second of the maneuver

■ No evidence of airflow cessation, variable effort, leak, obstructed mouthpiece, 

positive or negative zero flow error(s), or extra breath(s)

■ Acceptable end-of-test criteria (≤ 25 mL increase in volume for 1 second or a 

maneuver longer than 15 seconds)

4. Less than 150 mL difference between the two highest FVC measurements and the 

two highest FEV1 measurements is the goal. 

Spirometry

Predicted Values

If spirometry software allows a choice of predicted values, NHANES III or the most 

recent equivalent should be used [Hankinson et al. 1999] as they are based on a large 

sample of the U.S. population. Because predicted values are not available from NHANES 

III for Asian people born in the United States, these predicted values may be estimated by multiplying the NHANES III Caucasian predicted values for FEV1 and FVC by 

0.88 [Hankinson et al. 2010; Redlich et al. 2014]. In the future, it will be preferable to 

use Asian-specific equations for predicted values, such as from NHANES Plus data, 

when they are available. If spirometry software does not include lower limits of normal 

values, the spirometry reference value calculator at http://www.cdc.gov/niosh/topics/

spirometry/RefCalculator.html can be used to calculate lower limits of normal for 

NHANES III reference values. 

Figure 9-1. Spirometry guidelines for testing procedures and interpretation
9 . Medical Monitoring and Surveillance of Exposed Employees

230 Occupational Exposure to Diacetyl and 2,3-Pentanedione

[Miller et al. 2005]. These guidelines outline the 

criteria to follow to ensure overall test results 

are valid (Figure 9-1). The technician should 

be able to view real-time testing displays as 

specified in the most recent ATS/ERS spirometry standardization. On-site back-up of the 

results should include spirometry test reports 

and retention of all spirometry test results 

in printed or electronic format. Spirometry 

test reports for the employee’s health record 

should contain, at a minimum, the employee’s 

age, height, sex, race, and weight; numerical values and volume-time and flow-volume 

spirograms for at least the three best valid expiratory maneuvers; normal reference value set 

used; employee position during testing (standing or sitting); dates of test and last calibration 

check; ambient temperature and barometric 

pressure (volume spirometers); and the technician’s unique identification number or initials. 

The name, postal mailing and contact e-mail 

addresses, and telephone and fax numbers 

of the facility completing the spirometry test 

results and forms should also be recorded.

9.3.2.4 Spirometry quality assurance

A comprehensive spirometry quality assurance program is necessary to minimize the 

rate of invalid test results. This program should 

include all of the following components: 

instrumentation calibration checks, automated 

maneuver and test session quality checks, and 

ongoing monitoring of test quality. Testing personnel should be fully familiar with and adhere 

to the current ATS/ERS guidelines for instrument calibration check procedures. Calibration 

check procedures should include daily (day of 

testing) leak checks (for volume spirometers) 

and volume accuracy checks (performed at different speeds of injection for flow spirometers) 

and according to the frequency established by 

the current ATS/ERS spirometry standardization statement. Instrument calibration check 

records should be maintained by the provider 

for as long as the related employees’ medical 

reports are maintained. Spirometer software 

should automatically perform quality assurance checks on expiratory maneuvers during 

each spirometry testing session. Messages 

should alert the technician to maneuver acceptability errors and test session nonrepeatability. 

Each spirometry test session should have the 

goal of obtaining three acceptable with two 

repeatable forced expiratory maneuvers, as 

defined by the current ATS/ERS spirometry 

standardization statement. Because all spirometry software packages are not able to identify 

all the possible technical errors encountered 

during testing, NIOSH developed a poster 

that provides guidance to identify and correct 

common testing errors and improve spirometry test quality [NIOSH 2011a]. This document 

has been translated into several languages and 

can be accessed at http://www.cdc.gov/niosh/

docs/2011-135/. Providers should utilize physicians or other qualified healthcare professionals 

with expertise in evaluation and interpretation 

of spirometry to conduct ongoing monitoring of test quality. Determination of quality 

requires review of the flow-volume and volume-time curves for each acceptable maneuver 

and comparison of the two highest FEV1 and 

FVC measurements [Townsend 2011]. When 

suboptimal quality tests with potential for 

improvement are identified, the reviewing 

physician or other appropriate healthcare 

professional should provide feedback to the 

appropriate technician(s) along with specific 

suggestions for improvement. Some studies 

have found evidence that providing regular 

feedback to technicians improves test quality 

and decreases variability. In two studies where 

extensive feedback was provided to technicians 

on the quality of their tests, the investigators 

found lower measures of variability for their 

test measurements than in other studies where 

extensive feedback to technicians was not provided [Enright et al. 1991; Malmstrom et al. 

2002]. In these studies, the technicians received 

immediate feedback from the spirometry device 
9 . Medical Monitoring and Surveillance of Exposed Employees

Occupational Exposure to Diacetyl and 2,3-Pentanedione 231

on the acceptability of a forced exhalation 

maneuver and on the overall quality of the test. 

The investigators also provided ongoing review 

of the quality of their tests and gave feedback to 

the technicians; additional technician training 

was provided as needed. Test quality in these 

studies was graded using an A, B, C, D, F scale. 

In a study of a workplace spirometry testing 

program, use of a new spirometer that provided 

technicians with feedback during the test led to 

increases in the mean FEV1 and mean FVC of 

the study group, compared to use of an older 

spirometer without feedback capability [Banks 

et al. 1996].

With poor quality tests, some employees’ 

results that are truly normal may be considered 

abnormal, and employers may incur costs for 

lost work time in follow-up testing and clinical 

evaluation. In addition, employees may suffer 

needless worry, risks of unnecessary medical 

tests, and may be subject to workplace discrimination or even job loss. An example of an 

incorrect interpretation due to a poor quality 

test is the finding of a restrictive abnormality 

because the test subject did not exhale long 

enough during the maneuver; this results in a 

falsely low FVC. High quality spirometry tests 

are also necessary for comparison of spirometry 

results over time, an important consideration 

for flavoring-exposed employees. Low quality 

spirometry has greater variability in test results; 

over time, decreased precision may cause the 

medical monitoring program director to incorrectly identify whether an employee has had an 

excessive decline in lung function from one test 

to the next.

In reviewing the quality of spirometry tests 

performed for employers by private healthcare providers, NIOSH has identified instances 

where the quality of most tests was poor and 

thus not useful for assessing lung function 

changes over time [Kanwal et al. 2011; Kreiss et 

al. 2012; NIOSH 2004b, 2006]. High quality spirometry minimizes the variability in the results 

caused by technical aspects (i.e., how the test 

was conducted) so that changes in spirometry 

measurements over time reflect true changes 

in lung function more accurately. In California 

public health surveillance, only one of 13 commercial providers of surveillance spirometry 

for flavoring employees who reported results 

to the California Department of Public Health 

met a minimum quality criterion of 80% of 

test sessions with FEV1 of good quality [Kreiss 

et al. 2012]. Employers of flavoring-exposed 

employees should be aware of the characteristics of high quality spirometry programs so 

they can evaluate the quality of spirometry 

services offered by medical providers, monitor 

performance, and take corrective actions if necessary. OSHA and NIOSH have published an 

information sheet on spirometry for employers 

[NIOSH 2011b]. 

9.4 Frequency of Medical 

Monitoring Evaluations

Newly hired employees and current employees 

should have baseline evaluations before they 

are allowed to work in or enter areas as previously described where they may be exposed 

to diacetyl, 2,3-pentanedione, or similar flavoring compounds. Employees in the medical 

monitoring program should be evaluated with 

a questionnaire and spirometry every 6 months 

due to the potentially rapid development of 

flavoring-related lung disease [Redlich et al. 

2014]. If an employee exposed to diacetyl or 

similar flavoring compounds is identified as 

likely having lung disease from this exposure, 

then all employees who perform similar job 

tasks or have a similar or greater potential for 

exposure should be evaluated every 3 months. 

More frequent evaluation (every 3 months) 

is also appropriate for employees with excessive decline in FEV1 and similarly exposed 

employees. Identification of flavoring-related 

lung disease or excessive FEV1 decline should 
9 . Medical Monitoring and Surveillance of Exposed Employees

232 Occupational Exposure to Diacetyl and 2,3-Pentanedione

also trigger an environmental assessment to 

identify and correct potential sources of hazardous exposures. Although interpretation of 

excessive decline is challenging for short intervals between testing because of measurement 

error, the increased numbers of tests may facilitate improvement of spirometry quality and 

increasing monitoring physicians’ confidence 

in trends that may be occurring. The 3-month 

schedule should be maintained until factors 

that may have led to excessive exposure have 

been corrected and 12 months have passed 

during which no additional employees with 

likely flavoring-related lung disease are identified. Employees should be instructed to report 

to their occupational health service or supervisor any new persistent or worsening shortness 

of breath, cough, wheezing, or other respiratory 

symptoms that last more than 6 weeks. Such 

employees should be immediately evaluated by 

the medical monitoring program director. All 

employees who have been in the monitoring 

program should have a final evaluation at the 

end of employment [California Department of 

Public Health 2012].

9.5 Reporting Medical 

Monitoring Results

The medical monitoring program director or designee should review and interpret 

questionnaire and spirometry results, including assessing spirometry quality. During 

an employee’s scheduled visit for a medical 

monitoring program evaluation, the medical 

monitoring program director or designee 

should inquire about the employee’s knowledge of the potential risk from exposure to 

diacetyl, 2,3-pentanedione, or similar flavoring 

compounds and of how to minimize the risk. 

The medical monitoring program director or 

designee should educate employees as needed 

[California Department of Public Health 2012], 

and encourage employees to report any new 

persistent respiratory symptoms to their supervisor or the monitoring physician. At the end 

of each evaluation visit or as soon as possible 

thereafter, the medical monitoring program 

director should provide the employee with a 

written report describing the following items:

■ The results of any medical tests performed 

on the employee

■ The medical monitoring program director’s opinion regarding any abnormalities 

detected during the evaluation and recommendations for further evaluation 

and treatment

■ Whether or not the employee has any 

detected medical condition which 

would place the employee at increased 

risk to health from exposure to diacetyl, 2,3-pentanedione, or similar 

flavoring compounds

■ Recommendations, if necessary, for 

reducing the employee’s exposure to 

diacetyl, 2,3-pentanedione, or similar 

flavoring compounds

■ Any recommended limitation upon 

the employee’s use of personal protective equipment.

The medical monitoring program director should inform the employer in writing of 

the following:

■ Any recommendations for limiting 

the employee’s workplace exposures 

(e.g., reducing exposure to diacetyl, 

2,3-pentanedione, or similar flavoring 

compounds by removal, or limitations 

of the employee’s duties or activities) or 

on the employee’s use of personal protective equipment

■ A statement that the physician has 

informed the employee of the results of 

the medical examination and any medical 

conditions that require further evaluation 

or treatment.
9 . Medical Monitoring and Surveillance of Exposed Employees

Occupational Exposure to Diacetyl and 2,3-Pentanedione 233

The specific condition, issue, or concern 

resulting in recommendations for limiting the 

employee’s exposure to diacetyl, 2,3-pentanedione, or similar flavoring compounds or on the 

employee’s use of personal protective equipment should not be specified in the write-up to 

the employer without the employee’s consent. 

Also, any aspect of the employee’s medical 

history that has no bearing on whether the 

employee should continue to work in areas 

where diacetyl, 2,3-pentanedione, or similar 

flavoring compounds are used should not be 

revealed to the employer. A copy of the medical 

monitoring program director’s written opinion 

provided to the employer should also be provided to the employee.

9.6 Early Identification of 

Affected Employees

Early recognition of employees with lung 

disease due to exposure to diacetyl, 2,3-pentanedione, or similar flavoring compounds 

is essential to prevent rapid progression to 

severe irreversible disease. Identifying affected 

employees will also stimulate prevention efforts 

so that risk to other employees is minimized. 

The most effective means for identifying 

affected employees early is careful evaluation 

of results of serial spirometry tests of employees in the medical monitoring program. 

Symptom reports alone are not a reliable indicator of early disease, as many employees with 

early disease will be asymptomatic. However, 

symptom reports of exertional shortness of 

breath can reflect pathologic obliterative bronchiolitis even when spirometry remains normal 

[Kreiss 2013].

At each evaluation of an employee in the 

medical monitoring program, the medical 

monitoring program director should compare 

the results of the current spirometry test to 

the baseline (pre-exposure) test, or to the test 

with the highest values if post-hire spirometry 

values were higher than at baseline. The most 

important finding that may indicate development of lung disease from exposure to 

diacetyl, 2,3-pentanedione, or similar flavoring compounds is an abnormal decline in the 

FEV1. An employee’s longitudinal test results 

may reveal an abnormal decline in FEV1 compared to baseline even when each individual 

test value is found to be normal because it is 

above the LLofN calculated from the reference population [Townsend et al 2011; Kreiss 

et al. 2012; Redlich et al. 2014]. While such 

test results might not meet the criteria for an 

abnormality such as airways obstruction or 

spirometric restriction, an abnormal decline 

in FEV1 may indicate early disease in this case 

and should be further evaluated. Additionally, 

any new abnormality on spirometry compared 

to baseline should prompt further evaluation. 

Flavoring-exposed employees with obstructive abnormalities (FEV1/FVC ratio and FEV1

less than the LLofN) need additional medical 

tests to assess whether they have obliterative 

bronchiolitis. Employees with restrictive abnormalities (FVC less than LLofN and normal 

FEV1/FVC ratio) also need additional medical 

tests to differentiate between nonlung causes 

and lung causes of spirometric restriction, 

including obliterative bronchiolitis [Ghanei 

et al. 2008; King et al. 2011; Markopoulou 

et al. 2002].

The criteria for an abnormal excessive decline 

in the FEV1 depend on the quality of the spirometry tests performed as part of the medical 

monitoring program and the time period of 

follow-up [Redlich et al. 2014]. ATS/ERS and 

ACOEM have stated that a decline in FEV1

over one year should exceed 15% before being 

considered clinically meaningful [Pellegrino 

et al. 2005; Townsend 2011]. By this criterion, 

someone with a baseline FEV1 of 4 liters would 
9 . Medical Monitoring and Surveillance of Exposed Employees

234 Occupational Exposure to Diacetyl and 2,3-Pentanedione

have to experience a decline of at least 600 mL 

for the results to be considered abnormal. 

Because lung disease caused by flavorings can 

progress rapidly, it is useful to identify those 

potentially at risk before so much lung function is lost [Kreiss et al. 2002; NIOSH 2006, 

2007]. Some studies indicate that when ATS/

ERS criteria for spirometry quality are followed 

and high standards of quality are achieved, a 

threshold less than 15% can indicate an abnormally rapid decline in FEV1 in a year. In a study 

that used data from a spirometry surveillance 

program for coal miners, Wang and Petsonk 

[2004] found that the 5th percentile for FEV1

declines over 6 months in all employees studied 

was 320 mL (7.8%). In stable employees (those 

employees whose FEV1 slope over 5 years was 

less than 90 mL/year), it was 300 mL (7.1%). 

In healthy employees (those employees without 

symptoms or methacholine responsiveness over 

5 years), it was 280 mL (6.5%). The quality of 

spirometry data in this study reflected a withinperson variation of 3% that is rarely achievable. 

Within-person variation of 6% is typical for spirometry programs, and an assumption of that 

level of variability was used by ATS to develop 

its recommendation for using 15% loss of FEV1

as a threshold [Redlich et al. 2014]. 

In another study that used data with a withinperson variation of 4% from a spirometry 

surveillance program for thousands of employees at a large chemical company, Wang et al. 

[2006] found that the 5th percentile values 

for FEV1 decline for testing at one-year intervals were 380 mL (10.4%) in men and 280 mL 

(10.6%) in women. These studies suggest that 

in a medical monitoring program that follows 

ATS/ERS criteria and achieves high quality spirometry, an FEV1 decline of 10% or higher in 

one year or less can be considered abnormal and 

used as a threshold for further medical evaluation of the employee. ACOEM now accepts 

this 10% criterion after allowing for expected 

average annual loss due to aging in high risk 

settings when the relationship between longitudinal results and endpoint disease is clear, 

as in flavoring-exposed employees [Townsend 

2011]. Lower quality spirometry programs have 

the disadvantage of only being able to detect 

larger declines in FEV1 as abnormal.

NIOSH has developed a computer program, 

SPIROLA, to help spirometry programs 

measure their within-person variation in 

FEV1 as a measure of the precision of spirometry obtained by the spirometry providers (an 

indication of spirometry quality across the 

providers’ programs). SPIROLA also provides 

a longitudinal limit of decline (LLD) for each 

individual tested, a threshold for determining 

abnormal loss of FEV1 that is adjusted for the 

quality of the provider’s spirometry program 

[NIOSH 2010]. The LLD allows the spirometry provider to determine if an individual’s 

serial spirometry results suggest an excessive 

decline in lung function and allows higher 

quality programs to identify smaller changes 

in lung function as abnormal (http://www.

cdc.gov/niosh/topics/spirometry/spirola-software.html). The advantage of using relative 

lower LLD and 5th percentile approaches 

over the 15% criterion in flavorings-exposed 

microwave popcorn employees has been demonstrated [Chaisson et al. 2010]. 

9.7 Continuity of Medical 

Monitoring

Employers may change medical providers of 

medical monitoring services. Employers should 

ensure that prior medical monitoring program 

directors transfer medical monitoring records, 

including spirometry tests and questionnaires, 

to new medical monitoring program directors. 

If necessary to gain access, employers or new 

providers should ask employees to sign releases 

allowing new providers to obtain previous 

medical monitoring and surveillance records 

from previous provider(s). 
9 . Medical Monitoring and Surveillance of Exposed Employees

Occupational Exposure to Diacetyl and 2,3-Pentanedione 235

9.8 Tests Used in Medical 

Monitoring 

9.8.1 Spirometry

The first step in evaluating an employee whose 

medical monitoring spirometry test shows 

either an excessive decline in FEV1 (even 

if individual test results are still above the 

LLofN) or a new abnormality (e.g., obstructive, 

restrictive, or mixed spirometric abnormality) 

compared to baseline is to repeat the test within 

one month to confirm the change. If the repeat 

spirometry test confirms an excessive decline 

in FEV1 or other abnormality, the employee 

should be referred for more extensive pulmonary function tests (PFTs) (described below). 

The medical monitoring program director may 

request these and other necessary tests or refer 

the employee to a pulmonary medicine physician at no cost to the employee. 

9.8.2 Other Pulmonary Function Tests

The referred employee should receive complete 

PFTs that include spirometry with an assessment of bronchodilator response, DLCO, and 

static lung volumes. Most employees who have 

developed lung disease while being exposed to 

diacetyl and similar flavoring compounds have 

not had a response to bronchodilator [AkpinarElci et al. 2004; Kim et al. 2010]. In other words, 

they had fixed airways obstruction with an 

FEV1 and/or FVC increase less than 12% and 

200 mL after bronchodilator) [Pellegrino et al. 

2005]. DLCO in affected employees with airways 

obstruction has usually been normal, although 

some individuals with advanced disease have 

had a low DLCO [Akpinar-Elci et al. 2004]. 

Lung volume measurements have shown a 

normal or elevated total lung capacity (TLC) 

and an increased residual volume, consistent 

with air trapping [Akpinar-Elci et al. 2004]. 

Individuals with moderate to severe airways 

obstruction may have a mixed obstructive/

restrictive (reduced FEV1, FEV1/FVC ratio, and 

FVC) pattern of spirometry because air trapping decreases the FVC. The actual underlying 

physiology can be clarified by determining 

lung volumes.

9.8.3 High-resolution Computerized 

Tomography

Employees found to have fixed airways obstruction or other abnormalities on complete PFTs 

should have additional evaluation with a highresolution computerized tomography (HRCT) 

scan of the chest with inspiratory and expiratory views. Heterogeneous air trapping during 

expiration has been the most common finding 

in flavoring-exposed employees with fixed 

airways obstruction. Other common findings 

include cylindrical bronchiectasis, bronchial 

wall thickening, and a mosaic pattern of attenuation. Centrilobular nodules may also be seen 

[Cox et al. 2014]. Patchy ground glass opacities have been observed less commonly. These 

findings may not be present despite obliterative 

bronchiolitis documented by biopsy [King et 

al. 2011]. HRCTs have not been systematically 

performed in flavoring-exposed employees 

with restrictive pulmonary function abnormalities or with excessive FEV1 declines within the 

normal range of FEV1. Specialist consideration 

of the diagnostic utility of this test is suggested.

9.8.4 Lung Biopsy

It is not routinely necessary to obtain a lung 

biopsy to diagnose obliterative bronchiolitis in 

employees exposed to diacetyl or 2,3-pentanedione when spirometry and HRCT results are 

consistent with the diagnosis. While some physicians might desire biopsy confirmation, it is 

important to recognize that the patchy nature of 

obliterative bronchiolitis and lack of familiarity 

of some pathologists with the techniques necessary to identify bronchiolar lesions may prevent 

identification of the disease on biopsy. HRCT 

has become the method of choice for assessing 
9 . Medical Monitoring and Surveillance of Exposed Employees

236 Occupational Exposure to Diacetyl and 2,3-Pentanedione

bronchiolar morphology, often replacing surgical lung biopsy [King and Kinder 2008]. 

Physicians caring for another population at 

high risk for obliterative bronchiolitis, lung 

transplant patients, use a similar noninvasive 

approach. Obliterative bronchiolitis commonly occurs after patients receive a lung 

transplant. Because this disease is difficult to 

identify on biopsy, the International Society 

for Heart and Lung Transplantation developed 

a clinical description for the disease termed 

bronchiolitis obliterans syndrome. The syndrome refers to graft deterioration secondary 

to persistent airflow obstruction as defined by 

pulmonary function changes with or without 

biopsy confirmation [Estenne et al. 2002]. The 

term bronchiolitis obliterans syndrome has also 

been applied to flavoring-exposed employees 

without surgical lung biopsies [Akpinar-Elci et 

al. 2004; van Rooy et al. 2007], but may lead 

to confusion because flavoring-related obliterative bronchiolitis differs in natural history 

from post-transplant bronchiolitis obliterans 

syndrome, which is relentlessly progressive.

There are some situations, described in the 

next section, where lung biopsy is appropriate for diagnosis. To obtain adequate tissue 

for diagnosis, a thoracoscopic or open lung 

biopsy should be obtained. Obtaining wedge 

biopsies from multiple lobes is recommended, 

as this approach increases the diagnostic yield 

[Devakonda et al. 2010]. Transbronchial lung 

biopsies are not useful for evaluating clinical 

obliterative bronchiolitis in employees exposed 

to diacetyl, 2,3-pentanedione, or similar 

compounds. 

9.8.5 Determining Diagnosis 

Responsible for Lung Disease

Determination of the diagnosis responsible 

for lung disease in an employee exposed to 

diacetyl, 2,3-pentanedione, or similar flavoring compounds should take into account the 

changes identified in medical monitoring 

spirometry tests, the results of complete PFTs 

and of HRCT scans of the chest, the course of 

the employee’s illness over time, and medical, 

work, and personal risk factor history. 

In an exposed employee with evidence of 

clinical obliterative bronchiolitis on PFTs or 

HRCT scans and no other identifiable cause 

for the disease, biopsy is not necessary. The 

noninvasive clinical findings alone are sufficient to conclude that an exposed employee 

likely has clinical obliterative bronchiolitis 

and should no longer be exposed to diacetyl, 2,3-pentanedione, or similar flavoring 

compounds. When clinically apparent 

lung disease occurs in several employees 

at a particular plant, the need for biopsy 

confirmation in each employee is usually 

unnecessary. 

When HRCT is normal in dyspneic employees, particularly if the PFTs are restrictive or 

normal, lung biopsy has a role. Some medical 

surveys of flavoring-exposed employees have 

revealed an increased prevalence of an isolated restrictive pattern on spirometry (i.e., 

without concurrent airways obstruction), 

but static lung volume measurements of TLC 

and biopsies have not been available in these 

studies to confirm restrictive lung disease 

[Kreiss 2012; NIOSH 2009, 2011c]. The evidence for restrictive and normal pulmonary 

functions in obliterative bronchiolitis is in 

patients exposed to other lung hazards, such 

as sulfur mustard gas and in U.S. soldiers 

serving in Iraq and Afghanistan, some of 

whom had sulfur dioxide exposure. Despite 

evidence from three biopsy-confirmed case 

series of obliterative bronchiolitis [Ghanei 

et al. 2008; King et al. 2011; Markopoulou 

et al. 2002], many pulmonary and occupational medicine specialists are not aware 

of the range of spirometric findings in this 

disease and may be reluctant to diagnose 

obliterative bronchiolitis in patients with 
9 . Medical Monitoring and Surveillance of Exposed Employees

Occupational Exposure to Diacetyl and 2,3-Pentanedione 237

spirometric restriction or normal spirometry 

without pathologic confirmation. Employees 

who develop restrictive abnormalities or 

who have excessive parallel FEV1 and FVC 

declines should have assessment of lung 

volumes, diffusing capacity, and HRCT to 

differentiate between restrictive lung disease 

and other causes of restrictive spirometric 

patterns. Further evaluation of restrictive 

lung disease for a specific diagnosis should 

be pursued as clinically appropriate and 

may require biopsy. Case reports of pathologic findings in dyspneic flavoring-exposed 

employees with restrictive or normal spirometry will be of interest in further guidance for 

clinicians responsible for the lung health of 

such employees.

The evaluating physician should exclude 

alternative causes of respiratory disease such 

as work-related asthma (new onset asthma 

or exacerbation of pre-existing asthma). An 

employee with no past asthma history who 

experiences post-hire recurrent respiratory symptoms and has airways obstruction 

responsive to bronchodilator on PFTs 

(reversible airways obstruction) may have 

new onset asthma due to workplace exposures. If an employee with asthma symptoms 

does not have changes over time on medical 

monitoring spirometry, a methacholine or 

mannitol challenge test may be necessary 

to determine if the employee has airways 

hyperresponsiveness as occurs in asthma. 

Worsening symptoms in an employee with 

pre-existing asthma may be due to exposure 

to diacetyl, similar flavoring compounds, or 

other agents in the workplace [Sahakian et 

al. 2008]. An important consideration for 

diacetyl-exposed employees with worsening 

pre-existing asthma or new onset reversible 

airways obstruction is that this may actually 

reflect early disease that may ultimately progress to clinical obliterative bronchiolitis. An 

employee at a California flavoring plant who 

had stable pre-existing asthma (no symptoms at time of hire) developed progressive 

shortness of breath and was found to have 

severe fixed airways obstruction on PFTs; 

a lung biopsy showed evidence of bronchiolitis obliterans [NIOSH 2007]. Employees 

with worsening pre-existing asthma or new 

onset reversible airways obstruction should 

be evaluated with an HRCT scan of the 

chest to determine if findings consistent 

with clinical obliterative bronchiolitis are 

present. However, because HRCT abnormalities may be insensitive in detecting early 

or mild disease, such asthmatic employees 

require careful and frequent follow-up [King 

et al. 2011].

An employee exposed to diacetyl, 2,3-pentanedione, or similar flavoring compounds 

who has normal pre-exposure spirometry 

and subsequently develops fixed airways 

obstruction and has evidence of air trapping on complete PFTs or on HRCT scan, or 

has an excessive decline in FEV1 and whose 

pulmonary function does not improve after 

exposure cessation, likely has clinical obliterative bronchiolitis due to this exposure. 

In exposed employees who smoke, fixed 

airways obstruction should not be attributed 

to smoking if there is no evidence of emphysema on medical tests. Clinically significant 

emphysema occurs in a subset of smokers 

after many years of smoking; it is uncommon in smokers less than 50 years old [Wise 

2008]. In middle-aged and older smoking 

employees, work history, clinical course, 

and medical tests are important in attempting to differentiate between smoking-related 

COPD and flavoring-related obstruction. 

Smoking explains about 80% of COPD in the 

United States, with about 15% attributable to 

work exposures. Smoking diacetyl-exposed 

employees appear to have lower excess risk 

of obstruction than never-smoking flavoringexposed employees [Kreiss et al. 2002].
9 . Medical Monitoring and Surveillance of Exposed Employees

238 Occupational Exposure to Diacetyl and 2,3-Pentanedione

9.9 Response to 

Identification of Workrelated Lung Disease 

Employees with abnormalities identified on 

medical monitoring spirometry should be 

counseled about the risks of further exposure 

and that removal from exposure is prudent 

because of the irreversibility of the disease, 

short latency, and often rapid progression. 

Employees who receive a diagnosis of flavoringrelated lung disease or who have findings on 

medical evaluation that indicate likely clinical 

obliterative bronchiolitis or other lung disease 

due to workplace exposures should be placed 

on work restrictions to prevent any further 

exposure to flavoring compounds or other 

substances in the workplace that may cause 

their lung disease to worsen. Personal protective equipment is the least effective means for 

controlling employee exposures. The proper 

use of personal protective equipment requires 

a high level of employer and employee involvement and commitment to be effective. The use 

of respiratory protection is not equivalent to 

removal from exposures because employees 

may still be exposed due to incomplete compliance, selection of an inappropriate respirator, or 

respirator malfunction [California Department 

of Public Health 2012]. If possible, employers 

should offer affected employees the opportunity to transfer to available jobs in work areas 

that have minimal or nonexistent exposures. 

Such employees should retain seniority, wages, 

and benefits.

Employers of an employee with confirmed or 

likely flavorings-related lung disease should 

arrange for an industrial hygiene evaluation of 

the plant areas where the employee had been 

assigned. The evaluation may identify aspects 

of the production process or work practices 

where control strategies can be implemented 

to minimize exposures. This may prevent additional employees from developing work-related 

lung disease. Medical monitoring evaluations of 

employees in these areas should increase in frequency from every 6 months to every 3 months, 

with a return to 6-month intervals after factors 

that may have led to excessive exposure have 

been corrected and 12 months have passed 

during which no additional employees with 

likely flavoring-related lung disease are identified (see section 9.4).

When informed, employers should record 

all flavoring-related lung disease cases in the 

OSHA Form 300 Logs of Work-Related Injuries 

and Illnesses.

9.10 Medical Surveillance 

Analyses

A workplace assessment conducted after identification of a sentinel case of work-related lung 

disease may reveal sources of uncontrolled 

exposures from particular aspects of production processes and work practices that can be 

improved to prevent other employees from 

becoming affected. However, this approach may 

not identify all such risk factors for hazardous 

exposure in a given workplace. Additional risk 

factors may be identified through a medical 

monitoring and surveillance program, which 

includes the use of epidemiologic techniques 

for analyses of aggregated data obtained from 

evaluations of all employees in a medical monitoring program. Such analyses show trends 

and distributions of health outcomes by exposure variables such as work area, job category, 

and work task. In some instances, the results 

of such analyses may provide early evidence 

of risk factors that can be addressed before 

employees develop significant lung disease. 

Because production processes and work practices in manufacturing plants that use diacetyl 

or similar flavoring compounds or products 

that contain these compounds vary from 

plant to plant, medical surveillance may also 

allow identification of risk factors unique to 
9 . Medical Monitoring and Surveillance of Exposed Employees

Occupational Exposure to Diacetyl and 2,3-Pentanedione 239

a particular plant. For these reasons, systematic evaluation of medical monitoring data is 

an important component of medical monitoring and surveillance programs for employees 

exposed to diacetyl or similar flavoring compounds. If the medical monitoring program 

director is not able to conduct such analyses, 

the employer or medical monitoring program 

director should arrange for consultants with 

expertise in epidemiology to undertake this 

task. Two examples below show how medical 

surveillance can help to identify lung disease 

risk factors in the workplace.

Example 1. At the plant where microwave 

popcorn employees were first identified as being 

at risk for severe fixed airways obstruction 

consistent with clinical obliterative bronchiolitis from exposure to butter flavoring vapors 

(index facility G), four known affected former 

employees had worked in the mixing room as 

mixers of oil and butter flavorings, and four 

other affected former employees had worked 

on the packaging lines near the mixing room. 

A medical survey of current employees showed 

that the prevalence of airways obstruction on 

NIOSH spirometry tests was 3.3 times higher 

than expected in comparison to U.S. population data, a finding that was consistent with 

the known disease in former employees. The 

environmental assessment showed that air concentrations of the butter flavoring compound 

diacetyl were highest in the mixing room. The 

next highest exposures were in the packaging 

line area because of contamination from the 

mixing room, which was not isolated from the 

rest of the plant. Diacetyl air concentrations in 

other parts of the plant were lower. Analyses of 

the medical and environmental data showed a 

dose-response relationship between abnormal 

spirometry and quartiles of estimated cumulative exposure to diacetyl [Kanwal et al. 2011; 

Kreiss et al. 2002; NIOSH 2006].

Additional analyses of the medical survey 

data revealed an unexpected finding: Among 

current employees, the highest prevalence of 

airways obstruction was found in QC laboratory employees, five of six (83%) of whom had 

airways obstruction [Kreiss et al. 2002]. These 

employees popped approximately 100 bags of 

microwave popcorn in microwave ovens per 

8-hour shift. The mean time-weighted average 

diacetyl air concentration in the QC laboratory was 0.8 ppm compared to approximately 

57.2 ppm in the mixing room and 2.8 ppm for 

machine operators in the packaging line area. 

QC laboratory employees may be at risk for 

lung disease because they experience intermittent peak exposures to vapors of diacetyl from 

microwave popcorn bags during and after 

popping in microwave ovens; mixers experience similar intermittent peaks when they add 

butter flavorings to tanks of heated oil [NIOSH 

2003]. Another possible explanation is that the 

much higher temperatures that occur in microwave popping (compared with the temperatures 

in heated tanks of oil and butter flavorings) 

increase the volatilization of other chemicals. 

QC laboratory employees’ exposures may be 

substantially different from those of other production employees; diacetyl air concentrations 

alone may not be a satisfactory predictor of risk 

for these employees. Because of this evidence of 

risk to QC laboratory employees, NIOSH recommended implementing exposure controls 

in the QC laboratory in addition to the mixing 

room and packaging line area [Kanwal et al. 

2011; NIOSH 2006].

In evaluations at five other microwave popcorn 

plants, NIOSH found evidence of affected 

mixers in four plants and evidence of affected 

packaging line employees in one plant [Kanwal 

et al. 2006]. No other plant had an elevated 

prevalence of airways obstruction in QC 

employees. Fewer bags of microwave popcorn 

were popped per employee per day in those 

plants, and the mean time-weighted average 

diacetyl air concentrations in the QC laboratories were lower than at index facility G.
9 . Medical Monitoring and Surveillance of Exposed Employees

240 Occupational Exposure to Diacetyl and 2,3-Pentanedione

Example 2. At a microwave popcorn plant 

where a young mixing room employee 

developed moderately severe fixed airways 

obstruction and other findings consistent with 

clinical obliterative bronchiolitis, management 

had put a mandatory respirator use policy for 

mixing room employees in place soon after the 

company first started production. In addition 

to using respirators, the company had also ventilated and isolated the mixing room from the 

rest of the plant and had local exhaust ventilation for tanks of heated oil and butter flavorings. 

Butter flavorings were handled in open containers as they were at other microwave popcorn 

plants. The respirators used were full facepiece 

respirators with organic vapor cartridges and 

particulate filters. Included in the questionnaire 

that NIOSH administered to current employees during a medical survey at the plant were 

questions about respirator use for the following 

work tasks: (1) weighing or handling open containers of flavorings, (2) pouring flavorings into 

tanks in the mixing room, (3) pouring other 

ingredients into tanks in the mixing room, (4) 

checking the levels in the tanks, and (5) other 

duties in the mixing room. Thirteen current 

employees reported ever having worked as 

a mixer; six had abnormal lung function on 

NIOSH spirometry tests. The reported percentages of time these employees used respirators 

during these activities ranged from 0% to 100%. 

The median reported percentage of time was 

20% for all activities, except for those where 

other ingredients (not flavorings) were poured 

into tanks in the mixing room where the 

median was 50% [NIOSH 2004a]. These results 

showed that employees were not fully compliant with management’s respirator use policy; 

management was able to address this problem 

through employee education and enforcement 

of the policy. Had the company become aware 

of this problem earlier by regularly collecting and evaluating information on respirator 

use during medical monitoring evaluations, it 

could have increased compliance with respirator use and thus minimized some employees’ 

exposures to butter flavoring compounds. 

(Before 2001 when NIOSH informed microwave popcorn companies of the risk of severe 

lung disease to employees exposed to butter flavorings, the company had been unaware of the 

respiratory toxicity potential of diacetyl. The 

company had implemented a mandatory respirator use policy for mixing room employees 

many years earlier to prevent severe eye irritation that employees had experienced when 

handling certain flavorings.)

Thus, analysis of population data generated by 

medical monitoring and surveillance programs 

plays an important role in primary prevention 

by helping employers of flavoring-exposed 

employees to recognize and take steps to characterize and correct hazardous conditions. 

Recognition can require epidemiologic evaluation of medical monitoring, population, and 

environmental data. It is therefore important 

for employers to ensure that this applied epidemiology is provided as part of the medical 

monitoring and surveillance program.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 241

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 245

10 Exposure Monitoring in 

Occupational Safety and 

Health Programs

Employers should develop and implement 

comprehensive occupational safety and health 

programs to prevent occupational injuries, 

illnesses, and deaths. To be successful, safety 

and health programs should be developed and 

implemented as part of an employer’s management system, with strong management 

commitment, employee involvement, and 

occupational safety and health expertise. A 

safety and health program designed to protect 

employees from the adverse effects of exposure 

to diacetyl, 2,3-pentanedione, and other flavoring compounds should include mechanisms to 

identify all risk factors for exposure to flavoring 

substances. Just as medical monitoring is part 

of an overall occupational safety and health 

program, so is exposure monitoring. Exposure 

monitoring should be conducted whenever 

there is workplace exposure to diacetyl or 

2,3-pentanedione. 

10.1 Exposure Monitoring 

Program Goals

A workplace exposure monitoring program 

should have clear, stated goals [Mulhausen 

and Damiano 1998]. Site-specific exposure 

assessment strategies should be developed to 

accomplish each of these goals: (1) to determine 

employee exposure to diacetyl, 2,3-pentanedione, and other flavoring compounds used in 

the workplace; (2) to evaluate the effectiveness 

of work practices and engineering controls; and 

(3) to facilitate selection of appropriate personal protective equipment, if needed. Each of 

these goals requires a different sample strategy 

with different parameters (see Section 10.2). 

In addition to routine monitoring of airborne 

contaminant concentrations, the monitoring strategy should assess the effectiveness 

of engineering controls, work practices, PPE, 

training, and other factors in controlling exposures to flavoring compounds. The monitoring 

program should also identify areas or tasks that 

are associated with higher exposures to flavoring compounds where additional control efforts 

and/or sampling are needed. The program 

should also determine how changes in production (processes, chemicals and other substances 

used, and products) affect employee exposures. 

10.2 Exposure Monitoring 

Program Elements

Proper measurement of contaminants in the 

environment involves a variety of program elements. The sampling and analytical methods 

referred to in this chapter include an outline of 

tested and validated procedures that produce 

statistically reliable data when used in the 

manner prescribed. Several of the more significant elements of a monitoring program are 

described below [Gross and Pechter 2002; Milz 

et al. 2003; Soule 2000].

Where possible, a written sampling strategy 

or protocol should be developed prior to sampling; this protocol should guide all aspects 

of the sampling process. The protocol should 

contain a description of (1) the objectives of 

sampling, (2) what to sample, (3) whom and 

where to sample, (4) how to sample, (5) when to 
10 . Exposure Monitoring in Occupational Safety and Health Programs

246 Occupational Exposure to Diacetyl and 2,3-Pentanedione

sample, (6) how long to sample, (7) how many 

samples to collect, and (8) how to handle, store, 

and ship samples [Gross and Pechter 2002; Milz 

et al. 2003; Soule 2000]. A walk-through survey 

or preliminary worksite visit is often useful in 

developing the sampling strategy [Jennison et 

al. 1996] and knowledge of the data-keeping 

system to be used to store and retrieve subsequent information can also have an effect. 

The sampling strategy should be developed to 

facilitate data analysis and interpretation for the 

specific exposure assessment goal.

10.2.1 Objectives of Sampling

Sampling as part of an exposure monitoring program for diacetyl, 2,3-pentanedione, 

and other flavoring substances has several 

objectives. Often, this sampling is part of a comprehensive assessment to identify and quantify 

exposure hazards throughout a designated 

plant or work area to protect employees’ health. 

The frequency of monitoring will depend on 

the purpose and rationale of the sampling campaign. Specific sampling objectives can include:

(1) Characterizing (qualitatively or quantitatively) the flavoring compounds present in 

workplace air or in bulk materials

(2) Ensuring compliance with existing OELs

(3) Assessing the effectiveness of engineering 

controls, work practices, PPE, training, or 

other methods used for exposure control

(4) Identifying areas, tasks, or jobs with higher 

exposures that require additional exposure control

(5) Evaluating exposures related to production 

process changes and from changes in products made or materials used

(6) Evaluating specific high risk job categories 

to ensure that exposures do not exceed 

exposure standards or guidelines

(7) Measuring exposures of employees who 

report symptoms or illnesses

Sampling can also be used to assess any fugitive emissions from plant processes into the 

surrounding community.

Exposure monitoring should be conducted 

by qualified professionals. The sampling 

strategy should provide an opportunity to 

determine each employee’s exposure, either 

by direct measure using personal breathing 

zone samples or through reasonable estimates 

based on the sampling of similar work tasks or 

jobs. Sampling strategies that group employees according to exposure zones, uniform job 

titles, or functional job categories have been 

used in some industries to reduce the number 

of required samples while increasing the confidence that all employees at similar risk will 

be identified [Mulhausen and Damiano 1998]. 

Area sampling may also be useful in exposure 

monitoring for determining sources of airborne 

contaminants and assessing the effectiveness of 

engineering controls.

When sampling to determine whether 

employee exposures are below an OEL, a compliance sampling strategy, and/or a “focused 

strategy,” that targets employees perceived 

to have the highest exposure concentrations 

may be more useful than random sampling. A 

focused strategy is most efficient for identifying exposures above the OEL if maximum-risk 

employees and time periods are accurately 

identified. Focused sampling may help identify 

short-duration tasks involving high airborne 

concentrations that could result in elevated 

exposures over a full work shift and also tasks 

that result in exposures over the STEL.

10.2.2 What to Sample (Specific 

Agents and Physical States)

Because flavorings can consist of many 

chemicals in addition to diacetyl and 2,3-pentanedione, deciding what to sample often 

requires preliminary knowledge of the specific flavoring compounds being produced or 
10 . Exposure Monitoring in Occupational Safety and Health Programs

Occupational Exposure to Diacetyl and 2,3-Pentanedione 247

used, or that are present in flavorings or other 

food ingredients used in the workplace, and 

the known exposure hazards posed by each. 

Information on possible food and flavoring 

compounds present in workplace air can be 

obtained from reviews of product ingredient 

lists, flavor or food recipes, SDSs, and other 

information provided by the employer or flavor 

manufacturer [Gross and Pechter 2002]. In 

the flavor manufacturing industry, the recipe 

for each flavoring indicates the chemicals, 

solvents, and other ingredients used in the 

formulation. In the food manufacturing industry, this information may be available directly 

from the company or from SDSs for all flavorings and other ingredients used, although 

some flavoring SDSs do not list all potentially 

hazardous chemicals that may be present. 

Additional information may be needed from 

the flavoring manufacturers. Often, qualitative 

characterization may be useful prior to quantitative measurement to better guide the selection 

of substances to measure in the workplace. A 

review of any past exposure assessment reports 

from the target workplace or similar workplaces, may also be helpful in selecting which 

agents to sample. In either case, a list of substances to which employees will potentially be 

exposed should be developed to help determine 

which of those compounds are the most critical 

to sample [Mulhausen and Damiano 1998]. In 

instances where a company has stopped using 

diacetyl and 2,3-pentanedione in a flavor or 

food product, this list should include the butter 

flavor substances substituted for diacetyl or 

2,3-pentanedione. Determining which chemicals to sample and measure should be based 

upon the chemical, physical, and toxicological 

properties as well as the chemical quantities in 

use. For example, industry reference materials may provide helpful information on which 

flavoring compounds to use or avoid [FEMA 

2012]. Other databases that might prove helpful 

may include but are not limited to National 

Library of Medicine (Hazardous Substances 

Data Bank and ChemIDplus Lite, Agency 

for Toxic Substances and Disease Registry 

(Toxicological Profiles), U.S. Environmental 

Protection Agency (Superfund Chemical Data 

Matrix). Diacetyl, 2,3-pentanedione, and other 

flavoring compounds can be present in air as 

solids, liquids, gases/vapors, or a combination 

of these. The physical state of the flavoring 

compound in air influences decisions about 

sampling [NIOSH 1977]. 

10.2.3 Whom and Where to Sample

Selecting whom or where to sample depends 

in part on the sampling objectives as previously described. When sampling to determine 

whether employee exposures are below existing OELs, a focused or compliance sampling 

strategy that targets employees perceived to 

have the highest exposures may be more efficient than other strategies if maximum-risk 

employees and time periods can be accurately identified. Focused sampling, including 

personal breathing zone sampling, may also 

help identify short-duration tasks involving 

high flavoring compound concentrations that 

could result in peak exposures or contribute to 

elevated exposures over a full work shift. The 

sampling protocol should include sampling 

during the production of foods or flavorings 

with higher diacetyl, 2,3-pentanedione, or 

other food flavoring content. Sampling considerations include (1) distance from a diacetyl, 

2,3-pentanedione, or flavoring compound 

exposure source; (2) employee mobility; (3) 

air movement patterns; (4) specific tasks or 

work patterns; (5) individual work habits; and 

(6) exposure controls [NIOSH 1977]. When 

a sampling strategy is selected that groups 

employees according to similar exposure 

potential, uniform job titles, or functional job 

categories, the industrial hygienist should select 

at random a predetermined number of employees from each group for personal air sampling 
10 . Exposure Monitoring in Occupational Safety and Health Programs

248 Occupational Exposure to Diacetyl and 2,3-Pentanedione

to represent the exposures of those groups 

[Mulhausen and Damiano 1998; NIOSH 1977]. 

Area sampling may be useful for determining 

sources of airborne contaminants and identifying the worst-case chemical concentrations 

in various locations or processes. Selection of 

which employees or work locations should be 

sampled can help to characterize (confirm or 

refute) suspected areas of potential concern. 

10.2.4 How to Sample

A variety of methods are available to sample for 

diacetyl, 2,3-pentanedione, or other food and 

flavoring substances. These include (1) gas and 

vapor air methods, (2) methods to sample particulates in air, (3) direct reading and real-time 

methods for gases/vapors and for particulates, 

(4) evacuated container sampling methods, 

(5) particle size distribution methods, (6) bulk 

air methods, and (7) bulk material methods. 

Selecting appropriate sampling and analytical methods and using professionally accepted 

techniques maximize the validity of measurements of flavoring compounds in the work 

environment. While the state of the art in 

measuring diacetyl and 2,3-pentanedione 

continues to evolve, the methods with the 

most veracity at the time of publication of 

this document are OSHA Methods 1012 and 

1013 for diacetyl and OSHA Method 1016 for 

2,3-pentanedione.

Some sampling and analytical methods for 

diacetyl, 2,3-pentanedione, and other flavoring compounds published by NIOSH at http://

www.cdc.gov/niosh/nmam/ and by OSHA at 

http://www.osha.gov/dts/sltc/methods/index.

html are described in detail in Chapter 2 of 

this document and are presented in Appendices 

A–E. These methods include recommendations on sampling media, flow rate, duration, 

storage, shipment, sampling and analytical 

equipment, and procedures. A typical protocol 

for measuring diacetyl and 2,3-pentanedione is 

presented in Appendix I.

To minimize the likelihood of inaccurate 

results, sampling equipment should be maintained in reliable working order through 

proper care and maintenance. All equipment 

should be regularly inspected and cleaned; 

sampling pumps should be calibrated before 

and after each use. Because differences in pressure drop across the sampler affect flow rate, 

each sampling pump should be precalibrated 

and postcalibrated with the specific type of 

sampling media used for sampling. 

Careful record keeping in the field is also 

important. A detailed description of the work 

tasks conducted and the processes and materials involved is essential. Pertinent information 

such as sampling location, job category or task, 

air temperature, relative humidity, and possible interfering compounds in air should be 

documented. To avoid confusion in the laboratory, samples should be carefully labeled 

and accompanied by accurate paperwork. The 

exact sampling duration should be known 

to accurately calculate the sampled volume. 

Determining the sampling duration from the 

recorded start and stop times assumes that the 

pump functions consistently over the entire 

sampling period. Occasional spot checks to 

verify proper sampler operation should be 

made throughout the sampling period.

Personnel performing field sampling should 

not overlook quality assurance procedures. The 

field sampling parameters, such as calibration 

checks and accurate timing, often affect precision and accuracy of the final result more than 

the measurement’s parameters. Field personnel 

should devote time to learning the sampling 

and analytical methods and sampling equipment operation procedures prior to arriving 

at the sampling site. These methods usually 

specify the sampling media to be used, the 

correct flow rate and sample volume, as well as 
10 . Exposure Monitoring in Occupational Safety and Health Programs

Occupational Exposure to Diacetyl and 2,3-Pentanedione 249

special precautions of sample handling, shipping, and possible interferences.

Because many modern analytical techniques 

are extremely sensitive, care should be taken 

to avoid contaminating field samples. Samples 

should not be stored or shipped with bulk 

materials that might spill or otherwise contaminate the field samples. The glassware or 

other containers used in sampling and shipping 

should be cleaned as recommended in the analytical method. For many sampling methods, 

the analytical laboratory requires submission of 

a specific number of blank samples with each 

set of samples to be analyzed; this number of 

samples is specific to the method. Blanks are 

used to mitigate the potential for unrecognized 

contamination due to media or sample handling [NIOSH 1994]. The two types of sample 

blanks are field blanks and media blanks. Field 

blanks are unopened new samplers or media 

taken to the sampling site and handled in 

every way like the actual samples, except that 

no air is drawn through them. Media blanks 

are simply unopened new samplers or media 

that are submitted to the laboratory with the 

samples (these blanks are not usually taken to 

the field). Additional blind field blanks, labeled 

as field samples, should be sent along with the 

field samples as a further check on the analysis. 

Another occasionally used quality control practice is to include spiked samples—samples with 

known amounts of flavoring substance added—

along with the other field samples sent to the 

laboratory for analysis. These spiked samples 

are often prepared by a separate laboratory and 

then included with the other field samples sent 

to the analytical laboratory. They are labeled as 

field samples so that the analytical laboratory 

is blinded to their identity as spiked samples.

The variety of types of direct-reading methods 

available for monitoring specific gases and 

vapors, as well as general contaminant concentration, is large and expanding. Detector 

tubes (short-term and long-term), also referred 

to as colorimetric indicator tubes, are widely 

used sampling devices for obtaining immediate, quantitative measures of gas or vapor 

concentrations in air. Also, aerosol monitors, integrating passive monitors for certain 

gases, and portable instrumentation for gas 

chromatography or infrared spectroscopy, 

are becoming more commonly used for measuring exposures to flavoring compounds 

[ACGIH 2001; Soule 2000]. Many direct-reading instruments now used for personal or area 

measurements have evolved from laboratory 

or process control instruments. These types of 

monitoring techniques have significant advantages, although to date none of these methods 

has been validated for monitoring diacetyl, 

2,3-pentanedione, or other flavoring compounds in the work environment.

10.2.5 When to Sample

Because of the considerable variation in 

exposure during the production of food or 

flavoring products, individuals conducting air 

sampling should coordinate with plant management to ensure that sampling is conducted 

when food or flavoring products of particular interest are being manufactured. Sampling 

several products or production runs may be 

necessary to better characterize exposures. 

Additionally, some products may be produced 

infrequently, and production schedules may 

change rapidly, so the timing of sampling can 

be challenging. Exposure monitoring should 

be conducted whenever changes in production 

processes, controls, work practices, or other 

conditions indicate a potential change in exposure conditions.

In order to determine compliance with STEL 

criteria, sampling should be done during 

tasks that are considered likely to produce 

the highest short-term exposures. A series 

of sequential or overlapping samples can be 

taken for 15-minute intervals to determine the 

maximum exposures.
10 . Exposure Monitoring in Occupational Safety and Health Programs

250 Occupational Exposure to Diacetyl and 2,3-Pentanedione

10.2.6 How Long to Sample

In general, TWA exposures should be determined by collecting samples over a full work 

shift, for comparison with OELs and other 

toxicological data. Information on allowable 

sampling duration is given in validated sampling and analytical methods; depending on 

the method, in some instances it is necessary to 

collect multiple shorter-term samples to obtain 

an integrated full work-shift sample. Work 

shifts that exceed 8 hours require extended 

sampling duration. 

When the potential for exposure to diacetyl, 

2,3-pentanedione, or flavoring compounds 

is sporadic throughout a work shift, shortterm or task-based sampling may be needed 

to replace or supplement full-shift sampling. 

Short-term samples for diacetyl and 2,3-pentanedione can be collected for 15 minutes in 

duration. Data from these short-term measurements and other task-based sampling can 

provide valuable perspective on task-based 

exposures and on the effectiveness of various 

control techniques. They can also be used to 

evaluate exposures relative to a short-term 

exposure limit [Milz et al. 2003] such as the 

STEL values recommended for diacetyl and 

2,3-pentanedione.

10.2.7 How Many Samples to Collect

The numbers of samples to collect is important 

in that it relates to the confidence that can be 

placed in the exposure estimate. The number 

of samples needed for an accurate and reliable 

exposure assessment depends on the purpose 

of the sampling, the number of processes, 

work tasks or jobs to be evaluated, the variability inherent in the measured contaminant 

concentrations, sampling and analytical variability, and other factors. In most instances, 

time and budget constraints are major factors 

determining sample size. Statistical methods 

are available for calculating the minimum 

sample size needed to characterize a maximum 

risk employee exposure subgroup or to achieve 

a set degree of statistical confidence in the representativeness of an exposure measurement 

[NIOSH 1977, 1994; Snedecor and Cochran 

1967; Soule 2000]. Recently, exposure control 

banding and Bayesian decision analysis have 

been used to help support exposure assessment decisions with limited sample numbers 

[Hewett et al. 2006].

10.2.8 Sample Handling, Storage, 

and Shipment

Following sampling, appropriate sample handling, storage, and shipping methods should be 

used. Some flavoring compound analytes such 

as diacetyl are light sensitive and should be 

protected from light during sample collection 

and stored in the dark prior to analysis. Many 

volatile flavoring substance analytes should 

be stored and shipped refrigerated to ensure 

sample stability; this necessitates access to 

field refrigeration dedicated to sample storage. 

Some flavoring substance analytes/methods 

may have requirements for timely analysis or 

desorption to ensure analyte stability. Working 

closely with the analytical laboratory before 

sampling to determine the handling, storage, 

and shipping methods required for each 

analyte is advised. An American Industrial 

Hygiene Association or other accredited analytical laboratory should analyze collected 

samples. Consulting with the analytical laboratory before sampling to ensure that the 

measurement methods available can meet the 

defined sampling needs is essential. 

10.3 Outcomes of Exposure 

Monitoring

10.3.1 Interpretation

As stated above, a monitoring strategy should 

assess the effectiveness of various methods used 
10 . Exposure Monitoring in Occupational Safety and Health Programs

Occupational Exposure to Diacetyl and 2,3-Pentanedione 251

to control airborne flavoring substance concentrations and to identify areas or tasks that are 

associated with higher exposures to flavoring 

substances. A common technique for evaluating the effectiveness of controls is to compare 

the outcome of environmental measurements 

made prior to the installation of those controls 

with measurements made following that installation. A control technique can be judged, for 

example, to be 50% efficient if the post-installation contaminant concentration is half of the 

pre-installation concentration.

The TWA and STEL measurements of exposure 

to flavoring substances, made with the collection of personal breathing zone air samples, 

can be used to assess employees’ exposures 

relative to an OEL. As discussed in the section 

of this document describing the development 

of the RELs, an 8-hour TWA measurement in 

excess of 5 ppb diacetyl or 9.3 ppb 2,3-pentanedione indicates that the employee in question 

was at a greater risk of developing occupationally induced illness. A 15-minute short-term 

exposure in excess of 25 ppb diacetyl or 31 ppb 

2,3-pentanedione during task based personal 

sampling would be interpreted similarly. 

If monitoring indicates that exposures have 

increased over past measurements, or exposures exceed the selected OELs, a thorough 

investigation of controls to identify problems 

and guide remedial actions is needed. Regular 

routine monitoring (e.g., yearly) will help 

ensure the continued effectiveness of controls. 

Employers should monitor employees in such a 

fashion that he has a high degree of confidence 

that a very high percentage of actual daily 

exposures are below the REL. In statistical 

terms, the employer should try to attain 95% 

confidence that no more than 5% of employee 

days are over the REL.

10.3.2 Notification of Employees

Employers should establish procedures for 

the timely notification of employees of their 

environmental monitoring results or results 

that represent their work group, any identified exposure hazards, and any subsequent 

actions taken based on this monitoring to 

reduce their exposures. Employees should be 

informed about any products or processes 

that may generate high concentrations of 

diacetyl, 2,3-pentanedione, or other flavoring compounds and any PPE and changes in 

work practices needed in response. Employers 

should ensure that employees understand this 

information and their role in helping to maintain a healthful workplace. Information should 

be conveyed in English and other languages as 

needed to ensure that all employees receive 

and comprehend this information. 
252 Occupational Exposure to Diacetyl and 2,3-Pentanedione

References

ACGIH (American Conference of Governmental 

Industrial Hygienists) [2001]. Air-sampling instruments 

for evaluation of atmospheric contaminants, 9th ed. 

Cincinnati, OH: American Conference of Governmental 

Industrial Hygienists.

FEMA [2012]. Respiratory health and safety in the 

flavor manufacturing workplace. In: The flavor and 

extract manufacturers association of the United States. 

Washington, DC: p. 28

Gross ER, Pechter E [2002]. Evaluation. In: Plog BA, ed. 

Fundamentals of industrial hygiene. 5th ed. National 

Safety Council, pp. 487–519.

Hewett P, Logan P, Mulhausen J, Ramachandran G, 

Banerjee S [2006]. Rating exposure control using Bayesian 

decision analysis. J Occup Environ Hyg 3(10):568–581.

Jennison EA, Kullman GJ, Parker JE [1996]. Site visits. In: 

Harber P, Schenker M, Balmes J, eds. Occupational and 

environmental respiratory disease. New York: Mosby, 

pp. 140–147.

Milz SA, Conrad RG, Soule RD [2003]. Principles of 

evaluating worker exposure. In: DiNardi SR, ed. The 

occupational environment: its evaluation, control, and 

management. Fairfax, VA: The American Industrial 

Hygiene Association, pp. 116–128.

Mulhausen JR, Damiano J [1998]. A strategy for assessing and managing occupational exposures, 2nd ed. 

Fairfax, VA: American Industrial Hygiene Association, 

pp. 15–56.

NIOSH [1977]. Occupational exposure sampling strategy manual. Cincinnati, OH: U.S. Department of Health, 

Education, and Welfare, Center for Disease Control, 

National Institute for Occupational Safety and Health, 

DHEW (NIOSH) Publication No. 77–173.

NIOSH [1994]. NIOSH manual of analytical methods 

(NMAM). 4th ed. Schlecht PC, O’Connor PF, eds. 

Cincinnati, OH: U.S. Department of Health and Human 

Services, Centers for Disease Control and Prevention, 

National Institute for Occupational Safety and Health, 

DHHS (NIOSH) Publication 94–113 (August, 1994); 

1st Supplement Publication 96–135; 2nd Supplement 

Publication 98–119; 3rd Supplement 2003–154.

Snedecor GW, Cochran WG [1967]. Statistical methods. 

Ames, IA: The Iowa State University Press, pp. 111–119.

Soule RD [2000]. Workplace sampling and analysis. In: 

Patty’s industrial hygiene. 5th ed. New York: John Wiley 

and Sons, pp. 265–315.
Occupational Exposure to Diacetyl and 2,3-Pentanedione 253

11 Research Needs

In this chapter, knowledge gaps pertaining 

to diacetyl, 2,3-pentanedione and flavoringinduced lung disease are identified. General 

areas of need include environmental research 

to better measure and control exposures to flavoring substances, clinical and field studies on 

the epidemiology of flavoring-induced diseases, 

research related to personal protective equipment, and toxicological studies concerning the 

etiology of flavoring-related diseases. 

Research is needed to characterize exposures 

associated with various job tasks in the food 

production industries and to develop and validate control measures to reduce exposures to 

potentially harmful substances. This research 

should address questions such as:

■ Can a more sensitive analytical method 

be developed for 2,3-pentanedione that is 

comparable to the sensitivity and lower 

limit of quantification for diacetyl? Can 

more sensitive analytical methods be 

developed for other flavoring compounds? 

■ How does one effectively measure exposure to airborne particulate for diacetyl 

and other flavoring compounds? Would 

sampling and analytical methods be 

influenced by particle size? 

■ Can a real-time, portable sampling device 

be developed that will allow for both fullshift and peak exposure measurements 

for diacetyl and other flavoring agents?

■ Is canister sampling with GC-MS 

analysis comparable to thermal desorption GC-MS for flavoring volatile 

organic compounds?

■ What are appropriate variability estimates 

for occupational exposures in food production facilities?

■ What jobs have peak flavoring exposures 

that may be pertinent to health risks?

■ What are the major food production processes involving flavorings that require 

engineering controls?

■ What are the exposures for the downstream employees in food production 

processes or workplaces?

■ What work practice interventions most 

effectively reduce employee exposure?

Clinical and field research studies should 

address such questions as:

■ Is the asthma excess in flavoring employees a misdiagnosis of fixed obstruction 

or part of the range of flavoring-related 

diseases or their natural history?

■ What flavoring or other chemicals are 

responsible for the increased prevalence in 

restrictive spirometry seen in one flavoring manufacturing employee population? 

■ To what extent does the spectrum of 

diacetyl-related lung disease include 

restrictive lung disease?

■ Because obliterative bronchiolitis can 

be present pathologically with normal 

spirometric measures, should exposed 

employees with exertional shortness of 

breath be removed from further flavoring exposure or followed more intensively 

by clinicians until the natural history 

becomes clear? 
11 . Research Needs

254 Occupational Exposure to Diacetyl and 2,3-Pentanedione

■ What is the natural history of flavoringinduced illness with continuing exposures, and with cessation of exposure?

■ Do biomarkers of flavoring exposure or 

lung injury exist that could be used in 

employee screening or diagnosis?

■ Are there genetic or epigenetic markers 

for susceptibility for diacetyl-related 

respiratory effects?

■ Can longitudinal examination of spirometry in flavoring-exposed employees 

for excessive declines be effective in 

primary and secondary prevention of 

lung impairment in flavoring employees? 

What minimum quality requirements in 

spirometry equipment, technician performance, interpretation, and physician 

follow-up are necessary for flavoringexposed employee medical surveillance 

to be effective?

■ Can the effectiveness of a proposed 

standard, given the limitations of risk 

assessment, be substantiated by employee 

medical surveillance?

■ Should flavoring-exposed employees undertake their personal medical 

surveillance with peak flow meters or 

portable spirometers?

■ Could mortality studies of flavoring 

employees elucidate other potential 

flavoring-related risks such as kidney toxicity or burden of respiratory mortality?

■ What is the prevalence of increased respiratory morbidity in employees making 

scented candles, hard candies, snack 

foods, dairy products, baked goods, e-cigarettes, fragrances, etc.?

■ What nonflavoring, volatile chemicals have similar inhalation toxicity for 

employees in industries already shown 

to have excess obstructive lung disease 

in population-based studies such as 

NHANES or in clusters of obliterative 

bronchiolitis in specific industries, such 

as plastic-reinforced glass fibers in boat 

building or in U.S. soldiers returning 

from Iraq and Afghanistan? 

Research needs have been identified in the 

area of respirators and other personal protective equipment that will continue to have an 

important role in employee protection.

■ What methodology should be used for 

respirator selection for mixed chemical environments?

■ What gloves should be used in the workplace and how frequently should they 

be changed?

■ What guidance can be provided regarding 

change-out schedules for organic vapor 

cartridges used in flavoring production 

in mixed chemical environments?

■ What are the end-of-service indicators for 

respirators used in mixed environments?

Unanswered questions about the mechanisms 

of health effects of diacetyl and 2,3-pentanedione, include the following:

■ What are the chronic respiratory toxicological and pathophysiological effects of 

diacetyl inhalation?

■ Can a CFD-PBPK model be developed 

for diacetyl or 2,3-pentanedione absorption during chronic exposure?

■ What are the roles of metabolism and 

adhesion molecules in diacetyl or 

2,3-pentanedione toxicity?

■ Do biomarkers of flavoring exposure or 

lung injury exist that could be used in 

employee screening or diagnosis?

■ Are there genetic or epigenetic markers 

for susceptibility for diacetyl-related 

respiratory effects?

■ What is the role of protein damage in 

diacetyl or 2,3-pentanedione toxicity?
11 . Research Needs

Occupational Exposure to Diacetyl and 2,3-Pentanedione 255

■ What is the role of immunotoxicity in 

diacetyl or 2,3-pentanedione toxicity? 

■ What is the relationship between the 

chemical structure of diacetyl and 2,3-pentanedione and pulmonary toxicity?

■ Do nonflavoring volatile workplace chemicals implicated in causing obstructive 

lung disease have mechanistic similarities 

to diacetyl or 2,3-pentanedione?

■ Can structure-activity relationships be 

developed that predict the airway toxicity of volatile compounds?

■ Do inhalation-related and lung transplant-associated obliterative bronchiolitis 

share common mechanisms of injury?

■ What role do other components of butter 

flavoring play on diacetyl-induced respiratory tract injury?

■ What is the respiratory toxicity of substitutes (or other systems’ toxicity) for 

diacetyl and 2,3-pentanedione?

■ What are the pathophysiological mechanisms of acute and chronic diacetyl or 

2,3-pentanedione toxicity?

■ What characteristics of butter flavoring 

powder contribute to airway injury?

■ Can in vitro models of acute and 

chronic exposures to flavorings be developed to provide information useful to 

risk assessment?

■ What is the relative respiratory toxicity of 

flavoring vapors compared to powders? 

■ Do mixed exposures of alpha-diketone 

flavorings have different airway epithelial 

toxicity from single agents in rodents?

■ What laboratory tests are the best predictors of flavoring compounds that cause 

airway injury in humans? 

■ What is the role of diacetyl and other 

reactive carbonyl compounds in cigarette smoke (both tobacco cigarettes and 

e-cigarettes) in contributing to chronic 

obstructive pulmonary diseases? 

■ At what steady-state concentration 

of diacetyl, or above what cumulative 

exposure to diacetyl, does a fulminant, 

progressive pathological process initiate as opposed to a regular, constant, 

relatively lower rate of deterioration (as 

usually reflected in pulmonary function 

or structural changes)? Does this accelerated decline continue after cessation 

of exposure?
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Appendix A
 

OSHA PV2118 (Diacetyl)
 
 

Diacetyl 

Related Information: Chemical Sampling - J2iiJ.reM 

Method no.: PV2118 

Control no.: T-PV2118-01-0301-CH 

Target concentration: 25 ppm ( 88 mg/m3) 

Procedure: Samples are collected by drawing a known volume of air through two silica gel sampling tubes 

connected in series. Samples are extracted with ethyl alcohol: water (95:5) and analyzed by GC using 

a flame ionization detector (FID). 

Recommended sampling time 

and sampling rate: 60 min at 0.05 L/min (3 L) 

Reliable quantitation limit: 0.28 ppm (1.00 mg/m3) 

Special requirements: Samples are collected on two silica gel tubes in series. The second tube is used as a backup for the 

first tube. Samples should be protected from the light after sampling. 

Status of method: Partially evaluated method. This method has been subjected to established evaluation procedures of 

the Method Development Team and is presented for information and trial use. 

Date: January 2003 (revised September 2006) 

Chemist: Yogi C. Shah 

1. General Discussion 

1.1 Background 

1.1.1 History 

Chromatography Team 

Industrial Hygiene Chemistry Division 

OSHA Salt Lake Technical Center 

The purpose of this evaluation was to develop a sampling procedure for diacetyl that gave a better 

storage stability than did the NIOSH Method 2557, which used SKC Anasorb CMS as the sampling 

medial .The NIOSH method requires that the samples be refrigerated immediately after sampling, and the 

analysis be performed within 7 days. A more stable sampling media was desired for OSHA samples. The 

following media were tested at SLTC but all gave poor storage stability: coconut shell charcoal Lot 2000, 

4-tert-butylcatechol coated charcoal, XAD-7, and OVS-7. Silica gel tubes (150mg/75 mg) were tried next 

and had an average storage recovery of 94.9% for samples stored at room temperature for 14 days. A 

sampling train of two silica gel tubes in series was necessary because a significant amount of the diacetyl 

was found on the smaller, backup section of the first tube in the retention study. A second tube in series 

insures that all of the sample will be collected on the sampling train. The desorbing solvent of 95:5 ethyl 

alcohol:water with 0.25 μL/mL p-cymene internal standard gave an average recovery of 99.1 % over the 

concentration range of 26.5 to 529 μg of diacetyl. 

1.1.2 Toxic Effects.2 (This section is for information only and should not be taken as the basis of OSHA 

policy.) 

In 2002, the CDC published a report in the Morbidity and Mortality Weekly Report (MMWR) on employee 

exposures at a microwave popcorn factory in Missouri. A group of former employees had developed fixed 

airways obstructive lung disease. All eight had a respiratory illness that resembled a rare lung disease 

called bronchiolitis obliterans. Some of the cases had such severe illness they were candidates for lung 

transplants. The main volatile organic chemical (VOC) found in the workplace atmospheres was diacetyl, 

which was used in a mixture of heated soybean oil, salt and flavorings to impart a butter flavoring to the 

popcorn. During NIOSH's investigation of the facility, diacetyl was chosen as a marker compound for voe exposure. The MMWR publication reported that the geometric mean air concentration of diacetyl was 18 

ppm in the room where the mixing tank was located, 1.3 ppm in the packaging area, and 0.02 in other 

areas of the plant. Of the eight former employees with severe respiratory illness, four were mixers and 

four worked in packaging. The report concluded that "workers exposed to flavorings at microwave 

popcorn factories are at risk for developing fixed obstructive lung disease." 

Appendix A – OSHA PV2118 (Diacetyl) 

258 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

1.1. 3 Workplace exposurel-1 

Diacetyl is a naturally occurring chemical in bay and other oils, beer, butter, coffee, vinegar, and other 

food products. It is an artificial flavoring which adds the flavor of butter, cream or creaminess, and 

butterscotch. 

1.1.4 Physical properties and other descriptive information:i,1 

CAS number: 

RTECS number: 

melting point 

appearance: 

odor: 

autoignition 

temperature: 

solubility: 

synonyms: 

structure: 

431-03-8 IMIS: D740~ 

EK2625000 molecular weight 86.09 

-3DC boiling point: 88°C 

green-yellow liquid molecular formula: CHO 

characteristic flashpoint: 6°C buttery 

density (g/ml): 0.99 

365°C 

ether; alcohol; acetone. DMSO 

2,3 -butanedione; 2,3 -diketobutane; dimethyl diketone; 

dimethylglyoxal 

Jy 

0 

This method was evaluated according to the OSHA SLTC "Evaluation Guidelines For Air Sampling Methods Utilizing Chromatographic 

Analysis"fi. The Guidelines define analytical parameters, specify required laboratory tests, statistical calculations and acceptance 

criteria. The analyte air concentrations throughout this method are based on the recommended sampling and analytical parameters. 

Air concentrations listed in ppm are referenced to 25°c and 101.3 kPa (760 mmHg). 

1.2 Detection Limit of the Overall procedure (DLOP) and Reliable Quantitation Limit (RQL) 

The DLOP is measured as mass per sample and expressed as equivalent air concentrations, based on the recommended 

sampling parameters. Ten samplers were spiked with equal descending increments of analyte, such that the highest 

sampler loading was 3.7 μg diacetyl. This is the amount spiked on a sampler that would produce a peak approximately 

3 times the response for a sample blank. These spiked samplers were analyzed with the recommended analytical 

parameters, and the data obtained used to calculate the required parameters (standard error of estimate and slope) for 

the calculation of the DLOP. The slope was 13.89 and the SEE was 41.82. The RQL is considered the lower limit for 

precise quantitative measurements. 

Table 1.2 

Detection Limit of the Overall 

Procedure for Diacetyl 

Mass per sample 

(μg) 

0.00 

1.3 

1.58 

1.85 

2.11 

2.38 

2.64 

2.90 

3.17 

3.43 

3.70 

area counts 

(μV-s) 

293 

504 

631 

658 

676 

700 

734 

759 

788 

816 

838 

~ 

~ 

! 

0 

0 

~ "' 

950 

soo 

650 

500 

350 

D LO P RO L 

zoo'--~~-'-~~~~~~-'-~~---' 

o 

Figure 1.2.1 Plot of data to determine the DLOP/RQL for 

diacetyl. (Y=139X+349) 

Appendix A – OSHA PV2118 (Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 259
 

RQL is determined from the regression line parameters obtained for the calculation of the DLOP, providing 75% to 

125% of the analyte is recovered. The DLOP and RQL were 0.902 μg and 3.01 μg respectively 

Below is chromatogram of the RQL level. 

2. Sampling Procedure 

a .am 

a .DJS 

S" 

~ a .aJa ~ 

~ 

c 

0 

Q. 

~ 

~ 

a .a2s 

"' 

a .a2a 

a .a 1 s 

6 .2 6 .I 6.6 6 .8 

Tin• 1!n lo ) 

Figure 1.2.2 Chromatogram of the diacetyl standard near RQL 

(key: (1) diacetyl). 

1 .a 

All safety practices that apply to the work area being sampled should be followed. The sampling equipment should be attached to the 

worker in such a manner that it will not interfere with work performance or safety. 

2.1 Apparatus 

2.1.1 Samples are collected using a personal sampling pump calibrated, with the sampling device 

attached, to within ±5% of the recommended flow rate. 

2.1.2 Silica gel tubes: glass tube with both ends flame sealed, 70 mm x 6-mm i.d. containing 2 sections 

of 20/40 mesh silica gel separated by a 2-mm portion of urethane foam. The adsorbing section contains 

150 mg of silica gel, the backup section 75 mg. A 3-mm portion of urethane foam is placed between the 

outlet end of the tube and the backup section. A plug of silane-treated glass wool is placed in front of the 

front section( SKC No. 226-10) tubes or equivalent was used in this evaluation. 

2.2 Reagents 

None required. 

2.3 Technique 

2.3.1 Immediately before sampling, break off the ends of the flame-sealed tube to provide an opening 

approximately half the internal diameter of the tube. Wear eye protection when breaking ends. Use tube 

holders to minimize the hazard of broken glass. All tubes should be from the same lot. 

2.3.2 Connect two tubes in series to the sampling pump with flexible tubing. The smaller sections of the 

silica gel tubes should be positioned nearer the sampling pump. The tube closer to the pump is used as a 

backup. A minimum amount of tubing is used to connect the two sampling tubes together. Position the 

sampling pump, tube holder and tubing so they do not impede work performance or safety. 

2.3.3 Draw the air to be sampled directly into the inlet of the tube holder. The air being sampled is not to 

be passed through any hose or tubing before entering the sampling tube. 

2.3.4 After sampling for the appropriate time, remove the adsorbent tube and seal it with plastic end 

caps. Seal each sample end-to-end with an OSHA-21 form as soon as possible. 

2.3.5 Submit at least one blank sample with each set of samples. Handle the blank sample in the same 

manner as the other samples except draw no air through it. 

2.3.6 Record sample air volumes (liters), sampling time (minutes) and sampling rate (ml/min) for each 

sample, along with any potential interferences on the OSHA-91A form. 

Appendix A – OSHA PV2118 (Diacetyl) 

260 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

2.3.7 Submit the samples to the laboratory for analysis as soon as possible after sampling. If delay is 

unavoidable, store the samples at refrigerator temperature. Ship any bulk samples separate from the air 

samples. 

2 .4 Extraction efficiency 

The extraction efficiency was determined by liquid-spiking silica gel tubes with diacetyl at 0.1 to 2 times the target 

concentration. These samples were stored overnight at ambient temperature and then extracted for 30 minutes with 

occasional shaking and analyzed. The mean extraction efficiency over the studied range was 99.1 %. The wet extraction 

efficiency was determined at the target concentration by liquid spiking the analyte on the front, larger, section of the 

first silica gel tube of the sampling train of two silica gel tubes in series, and drawing 3 L humid air (absolute humidity 

of 15.9 mg/L of water, about 80% relative humidity at 22.2oq through them. The mean recovery for the wet samples 

was 100.2 % 

Table 2.4 

Extraction Efficiency (%) of Diacetyl 

level sample number 

xtarget μg per 2 3 4 5 mean con en sample 

0.1 26.5 105.0 105.0 105.8 100.3 100.8 103.4 

0.25 66.5 110.4 98.6 100.5 97.7 100 101.4 

0.5 133 91.0 90.8 90.8 90.6 95.1 91.7 

1.0 265 98.8 100.3 99.1 98.9 99.6 99.3 

2.0 529 100.8 101.3 99.2 98.7 99.6 99.9 

1.0 (wet) 265 104.2 101.6 99.6 93.3 102.2 100.2 

2. 5 Retention efficiency 

Six silica gel tubes were spiked with 0.265 mg (25.ppm) of diacetyl and allowed to equilibrate for 6 h at room 

temperature in a drawer .. The spiked tubes were placed in series with a second unspiked silica gel tube and had 3 L 

humid air (absolute humidity of 15.9 mg/L of water, about 80% relative humidity at 22.2oq pulled through them at 

0.05 L/min. The samples were extracted and analyzed. The mean retention recovery was 94.3%. There was no analyte 

found on the backup section of any of the tubes. 

Table 2.5 

Retention Efficiency(%) of Diacetyl 

sample number 

section 2 3 4 5 6 mean 

front 94.3 94.1 96.8 93.7 93.8 93.1 94.3 (a+b) 

rear (a) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 

rear (b) 0.0 0.0 0.0 0.0 0.0 0.0 0.0 

total 94.3 94.1 96.8 93.7 93.8 93.1 94.3 

2.6 Sample storage 

Nine silica gel tubes were spiked with 0.265 mg (25.ppm) of diacetyl and allowed to equilibrate for 6 h at room 

temperature in a drawer. The tubes were placed in series with a second unspiked silica gel tube and had 3 L humid air 

(absolute humidity of 15.9 mg/L of water, about 80% relative humidity at 22.2°C) pulled through them at 0.05 L/min. 

Three samples were analyzed immediately, and the rest were sealed and stored at room temperature in a drawer. 

Three more were analyzed after 7 days of storage and the remaining three after 14 days of storage. The amounts 

recovered indicate good storage stability for the time period studied. 

Appendix A – OSHA PV2118 (Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 261
 

Table 2.6 

Storage Test for Diacetyl (% Recovery) 

time (days) 

0 

7 

14 

2.7 Recommended air volume and sampling rate. 

sample number 

2 3 

99.4 96.9 96.2 

94.5 97.7 95.0 

97.2 92.8 94.8 

mean 

97.5 

95.7 

94.9 

Based on the data collected in this evaluation, 3-l air samples should be collected at a sampling rate of 0.05 l/min for 

60 minutes. 

2.8 Interferences (sampling) 

2.8.1 There are no known compounds that will severely interfere with the collection of diacetyl. 

2.8.2 Suspected interferences should be reported to the laboratory with submitted samples. 

3. Analytical Procedure 

Adhere to the rules set down in your Chemical Hygiene Plan. Avoid skin contact and inhalation of all chemicals and review all 

appropriate MSDSs. 

3.1 Apparatus 

3.1.1 A gas chromatograph equipped with an FID. For this evaluation, an Agilent 6890 Plus gas 

Chromatograph equipped with a 7683 Automatic Sampler was used. 

3.1.2 A GC column capable of separating diacetyl from the desorption solvent, internal standard and any 

potential interferences. A 60-m x 0.32-mm i.d. capillary DBWAX with a 0.5-μm df (J&W Scientific) was 

used in the evaluation. 

3.1.3 An electronic integrator or some other suitable means of measuring peak areas. A Waters 

Millennium32 Data System was used in this evaluation. 

3.1.4 Amber glass vials with poly(tetrafluoroethylene)-lined caps. For this evaluation 2-ml vials were 

used. 

3.1.5 A dispenser capable of delivering 1.0 ml of desorbing solvent to prepare standards and samples. If 

a dispenser is not available, a 1.0-ml volumetric pipet may be used. 

3.1.7 Volumetric flasks - 10-ml and other convenient sizes for preparing standards. 

3.1.8 Calibrated 10-μl syringe for preparing standards. 

3.2 Reagents 

3.2.1 Diacetyl, Reagent grade. Aldrich 99% (lot 09122TS BO) was used in this evaluation. 

3.2.2 Ethyl alcohol, USP grade 190 proof. Aaper (lot 98G23BB) was used for this evaluation. 

3.2.3 p-Cymene, Reagent grade. Aldrich 99% (lot 306PZ) was used in this evaluation. 

3.2.4 The extraction solvent was 0.25 μl/ml p-cymene in ethyl alcohol:water (95:5). 

3.2.5 GC grade nitrogen, air, and hydrogen. 

3.3 Standard preparation 

3.3.1 Prepare working analytical standards by injecting micro liter amounts of diacetyl into volumetric 

flasks containing the extraction solvent. An analytical standard at a concentration of 0.530 mg/ml (5.3 

μL/10 ml) is equivalent to 50 ppm based on a 3-l air volume. Stock standards were stored in amber vials 

at refrigerated temperature for stability. 

Appendix A – OSHA PV2118 (Diacetyl) 

262 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

3.3.2 Bracket sample concentrations with working standard concentrations. If sample concentrations are 

higher than the concentration range of prepared standards, prepare and analyze additional standards, at 

least as high a concentration as the highest sample, to ascertain the linearity of response, or dilute the 

sample with extracting solvent to obtain a concentration within the existing standard range. The range of 

standards used in this study was from 0.00132 to 0.60 mg/ml analyze additional standards, at least as 

high a concentration as the highest sample, to ascertain the linearity of response, or dilute the sample 

with extracting solvent to obtain a concentration within the existing standard range. The range of 

standards used in this study was from 0.00132 to 0.60 mg/ml. 

3.4 Sample preparation 

3.4.1 Remove the plastic end caps from the sample tubes and carefully transfer both adsorbent sections 

from front tube and each section of backup tube to separate labeled 2-ml amber glass vials. Discard the 

glass tube and glass wool plug. 

3.4.2 Add 1.0 ml of extraction solvent to each vial using the same dispenser as used for preparation of 

standards. 

3.4.3 Immediately seal the vials with poly(tetrafluoroethylene)-lined caps. 

3.4.4 Place vials on shaker and agitate for 60 minutes. 

3.5 Analysis 

3.5.1 Analytical conditions. 

GC conditions 

injector: 

detector: 

run time: 

column gas flow: 

septum purge: 

injection size: 

200°c 

2so0 c 

16 min 

2.5 ml/min (hydrogen) 

1.9 ml/min (hydrogen) 

1.0 μl (10: 1 split) 

column: 60-m x 0.32-mm i.d. capillary DBWAX (0.5-um df) 

column temperatures: so0 c for 6 min, 1s°C/min to 1so0 c final time 3 min 

retention times: 

FID conditions 

hydrogen flow: 

air flow: 

makeup flow: 

5.51 min ethyl alcohol, 6.48 min diacetyl, 12.46 min p-cymene 

30ml/min 

400 ml/min 

25 ml/ min (nitrogen) 

15 

S' 1D 

€-

0 

e- as 

"' 

1 

. 

2 

l J 

10 2 0 

Tlm~ (min) 

Figure 3.5.1 A chromatogram of 268 μg/ml diacetyl in 95:5 

ethyl alcohol:water with 0.25 μI of p-cymene as internal 

standard. (Key: (1) ethyl alcohol, (2) diacetyl, (3) impurity, 

and (4) p-cymene). 

Appendix A – OSHA PV2118 (Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 263
 

3.5.2 Peak areas are measured by an integrator or other suitable means. 

3.5.3 An internal standard (ISTD) calibration method is used. A calibration curve can be constructed by 

plotting !STD-corrected response of standard injections versus micrograms of analyte per sample. Bracket 

the samples with freshly prepared analytical standards over a range of concentrations 

3.6 Interferences (analytical) 

~ D.6 x1cf 

~ 

!! 

8 

.. 

"' <( 

a.zx1rf 

"'" 

Figure 3.5.3 Calibration curve of diacetyl. 

(Y = 696 x - 336) 

maa 

3.6.1 Any compound that produces a GC response and has a similar retention time as the analyte is a 

potential interference. If any potential interferences were reported, they should be considered before 

samples are extracted. Generally, chromatographic conditions can be altered to separate an interference 

from the analyte. 

3.6.2 When necessary, the identity or purity of an analyte peak may be confirmed by mass spectrometry 

or by another analytical procedure. The mass spectrum in Figure 3.6.2 was from the NIST spectral library. 

mam ,, 

saaa 

~ oaaa 

~ .Q 

'""" <( 

2aaa ,. 

.11 a ... 

8 6 

I a :ZS sa '"" mil 

Figure 3.6.2 Mass spectrum of diacetyl. 

3.6.3 Calculations 

The amount of analyte per sampler is obtained from the appropriate calibration curve in terms of 

micrograms per sample, uncorrected for extraction efficiency. This total amount is then corrected by 

subtracting the total amount (if any) found on the blank. The air concentration is calculated using the 

following formulas. 

M where CMis concentration by weight (mg/m3) 

CM M is micro grams per sample 

VEE V is liters of air sampled 

EE is extraction efficiency, in decimal form 

VMCM where Cv is concentration by volume (ppm) 

Cv VM is molar volume ate 25°C=24.46 

Mr CM is concentration by weight 

Mr is molecular weight = 86.09 

Appendix A – OSHA PV2118 (Diacetyl) 

264 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

4. Recommendations for Further Study 

Several other tests need to be performed to make this a validated method. 

1. NIOSH Method 2557. 

2. Simoes E., et al. (2002) "Fixed Obstructive Lung Disease in Workers at a Microwave Popcorn Factorv - Missouri" 2000-2002. 

MMWR 51(16):345-347. 

3. O'Neil, M., The Merck Index, 13th ed., Merck & Co. Inc.: Whitehouse Station, NJ, 2001, p 522. 

4. Lewis, R., Sax's Dangerous Properties of Industrial Materials, 10th ed., Vol. 2, John Wiley & Sons, New York, 2000, p 595. 

5. OSHA Chemical Sampling Guide. 

6. Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M. C. Evaluation Guidelines For Air Sampling Methods Utilizing 

Chromatographic Analysis; OSHA Salt Lake Technical Center, U.S. Department of Labor: Salt Lake City, UT, 1999. 

Appendix A – OSHA PV2118 (Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 265
This page intentionally left blank. 
Appendix B
 

OSHA 1012 (Acetoin and Diacetyl)
 
 

Method no.: 

Control no.: 

Target concentration: 

OSHA PEL: 

ACG/H TLV: 

Procedure: 

Recommended sampling time 

and sampling rate: 

Reliable quantitation limit: 

Standard error of 

estimate at the target 

concentration: 

Special requirements: 

Status of method: 

November 2008 

1012 

Aceto in 

Diacetyl 

T-1012-FV-01-0811-M 

0.05 ppm (7WA) (0.18 mglm2 acetoin 

0. 05 ppm (TWA) (0. 18 mglm ) diacetyl 

none acetoin 

none diacetyl 

none acetoin 

none diacetyl 

Samples are collected by drawing workplace air through two tubes 

containing specially cleaned and dried silica gel connected in series. 

Samples are extracted and derivatized with a solution of 95:5 ethyl 

a/cohol:water containing 2 mg/mL of 0-(2, 3, 4, 5, 6-pentafluorobenzyl) 

hydroxy/amine hydrochloride (PFBHA) and analyzed by gas 

chromatography using an electron capture detector (GC-ECD). 

180 min at 0.05 Umin (9.0 L) (7WA) 

15 min at 0.2 Umin (3 L) (short term) 

1.49 ppb (5.37 μglm2 acetoin 

1.30 ppb (4.57 μglm) diacetyl 

5. 06% acetoin 

5. 11 % diacetyl 

Protect samplers from the light during and after sampling with aluminum 

foil or opaque tape. 

Evaluated method. This method has been subjected to the established 

evaluation procedures of the OSHA Salt Lake Technical Center Methods 

Development Team. 

Methods Development Team 

Industrial Hygiene Chemistry Division 

OSHA Salt Lake Technical Center 

Sandy UT 84070-6406 

1of34 

Mary E. Eide 

T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

268 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

1. General Discussion 

For assistance with accessibility problems in using figures and illustrations presented in this 

method, please contact Salt Lake Technical Center (SL TC) at (801) 233-4900. This procedure 

was designed and tested for internal use by OSHA personnel. Mention of any company name 

or commercial product does not constitute endorsement by OSHA. 

1.1 Background 

1. 1. 1 History 

On September 24, 2007 OSHA issued a Hazard Communication Guidance for Diacetyl 

and Food Flavorings Containing Diacetyl1 in which diacetyl was identified as an 

indicator compound for hazardous exposures found at plants packaging microwave 

popcorn. This was based on Health Hazard Evaluations performed by NIOSH which 

found the occurrence of severe lung disease in some employees at microwave popcorn 

packaging plants and flavorings manufacturing facilities. In three NIOSH Health Hazard 

Evaluation reports, acetoin and diacetyl are listed as major constituents of butter 

flavoring and they were used as indicators of exposure to butter flavoring vapors. 2

'

3

'

4 

OSHA has a partially validated method for diacetyl, PV2118, which recommends the 

use of two standard sized silica gel tubes in series to collect diacetyl at 0. 05 Umin for 1 

hour. 5 There were three reasons a new method was needed: 1) the reliable quantitation 

limit of PV2118 is 0.28 ppm which is higher than the target concentration of 0.05 ppm 

for this method; 2) a new medium was needed to enable the industrial hygienist to 

sample for a longer sampling time and take fewer samples; and 3) to allow acetoin and 

diacetyl to be sampled and analyzed together. The new medium used in this method is 

a tube packed with specially cleaned and dried silica gel (600 mg) with a glass wool 

plug and a glass fiber filter in front of the dried silica gel bed (this medium is referred to 

as dried silica gel in this method). It was necessary to specially dry the silica gel to 

obtain a higher capacity because of the amount of water already present on the silica 

gel in the currently commercially available tubes. The dried silica gel tube can be used 

to sample diacetyl for up to 1.5 times longer than the currently available silica gel tube. 

There was not a capacity problem with acetoin. The powder and liquid formulated 

forms of acetoin and diacetyl may contain oily compounds and other base materials 

such as maltodrextin. These materials could affect the extraction of acetoin and 

diacetyl from the silica gel. The glass fiber filter in the tube serves only to trap these 

materials before they enter the silica gel bed. Retention studies using a powder 

containing acetoin and diacetyl showed that the acetoin and diacetyl can be stripped off 

the powder and collected on the silica gel, especially when sampling high humidity air. 

(Section 4.9) 

Hazard Communication Guidance for Diacetyl and Food Flavorings Containing Diacetyl, 2007. U.S. Department of Labor, 

Occupational Safety and Health Administration Web site. http:llwww.osha.gov/dsglguidanceldiacetyl-guidance.html 

(accessed 311712008). 2 HETA 2001-0474-2943 American Pop Corn Company, 2004. Centers for Disease Control and Prevention, The National Institute 

for Occupational Safety and Health Web site. http:llwww.cdc.gov/nioshlhhelreports/pdfs/2001-047 4-2943.pdf (accessed 

311512008). 3 HETA 2002-0408-2915 Agrilink Foods Popcorn Plant, 2003. Centers for Disease Control and Prevention, The National Institute 

for Occupational Safety and Health Web site. http:llwww.cdc.gov/nioshlhhelreports/pdfs/2002-0408-2915.pdf (accessed 

311512008). 

HETA 2003-0112-2949 ConAgra Snack Foods, 2004. Centers for Disease Control and Prevention, The National Institute for 

Occupational Safety and Health Web site. http:llwww.cdc.gov/nioshlhhelreports/pdfs/2003-0112-2949.pdf (accessed 

311512008). 

Shah, Y. C. OSHA Diacetyl (OSHA Method PV2118), 2003. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http:llwww.osha.gov!dts/sltclmethods/partiallt-pv2118/t-pv2118.html (accessed 311712008). 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 269
 

To obtain adequate sensitivity for this method, it was necessary to derivatize the 

acetoin and diacetyl. 2,4-Dinitrophenyl hydrazine (DNPH) was the first derivatizing 

agent tried, but DNPH can react with both ketone and a-hydroxy ketones6

, and while it 

initially formed unique derivatives of acetoin and diacetyl by reacting with the first 

ketone group, it eventually reacted also with the alcohol group on acetoin and the 

second ketone group on diacetyl, forming the same derivative. In EPA Method 556. 1 

0-pentafluorobenzyl hydroxy_lamine hydrochloride (PFBHA) was used to derivatize 

ketone and aldehyde groups. 7 Unique derivatives of acetoin and diacetyl are formed by 

reacting them with PFBHA. The first ketone group on diacetyl reacts within four hours 

with PFBHA, but the second ketone group takes 36 hours to reach completion. Acetoin 

reacts within 3 hours. In this method, samples are extracted and derivatized in an 

extraction solution containing PFBHA. This is accomplished by first rotating the 

samples for 60 min and then allowing the samples to stand at room temperature for an 

additional 36 hours for the derivatization reaction to reach completion. 

F~F 0-N 

~ck\CH, OH 

Figure 1.1.1.1. The reaction of acetoin with PFBHA to form the acetoin-PFBHA 

derivative. 

*

F ,,.NH2 0 

• HCI 

F 

F~F 

~F H 0 

I NXCH, 

H C "N 3 I 

:qHP ~ F 

~I F 

F 

Figure 1.1.1.2. The reaction of diacetyl with PFBHA to form the diacetyl-PFBHA 

derivative. 

This method is designed for low air concentrations of acetoin, diacetyl, and potential 

interferences. If high exposures are anticipated, use OSHA Method 10138 or increase 

6 Smith, M., March, J.; March's Advanced Organic Chemistry: Reactions, Mechanisms, and Structure, 5th ed.; John Wiley & Sons 

Inc.: New York, 2001, p 1193. 

EPA Method 556.1 Determination of Carbonyl Compounds in Drinking Water by Fast Gas Chromatography, 1999. U.S. 

Environmental Protection Agency Web site. http:llwww.epa.gov/safewaterlmethods/pdfs/methods/met556_ 1.pdf (accessed 

311712008). 

Simmons, M., Hendricks, W. Acetoin Diacetyl (OSHA Method 1013), 2008. U.S. Department of Labor, Occupational Safety and 

Health Administration Web site. http:llwww.osha.gov!dts/sltclmethods/validatedl1013!1013.html (accessed 111112008). 

3 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

270 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

the amount of PFBHA in the extraction solution to ensure complete derivatization. 

Samples extracted by OSHA Method 1013 can be derivatized and analyzed by this 

method to detect lower concentrations. 

1.1.2 Toxic effects (This section is for information only and should not be taken as the basis 

of OSHA policy.) 

NIOSH Health Hazard Evaluations (HHE) of microwave popcorn manufacturing plants 

found fixed airway obstruction, in some cases, consistent with bronchiolitis obliterans in 

some employees. 9 Acetoin, diacetyl, acetic acid, acetaldehyde, and 2-nonanone were 

amongst the chemicals found by NIOSH in several popcorn manufacturing plants. 10 

Diacetyl was found to be present in all workplaces where the bronchiolitis obliterans 

was observed, and acetoin was found in some of the workplaces. Animal toxicology 

studies were performed by NIOSH with diacetyl, or butter flavorings containing diacetyl. 

Respiratory tract damage, including necrosis of the nasal and tracheal epithelium, and 

death were reported in rodents exposed to diacetyl, and butter flavorings containing 

diacetyl, at an air concentration of approximately 200 ppm of diacetyl for 6 hours. Mice 

exposed to 200 and 400 ppm diacetyl via inhalation for 6 hours per day over 5 days 

had the following health effects: death, acute necrotizing rhinitis, and erosive or 

necrotizing laryngitis. Mice exposed to 200 and 400 milligrams per kilogram (mg/kg) 

diacetyl via oropharyngeal aspiration for 6 hours per day over 5 days had bronchiolar 

fibrosis and death. Rats exposed to butter flavoring vapors containing 300 ppm 

diacetyl for 6 hours had epithelial injury in the nasal passages and pulmonary 

airways. 11 

1. 1.3 Workplace exposure 

Workers are exposed to acetoin and diacetyl in various manufacturing processes. 

Acetoin and diacetyl are natural flavorings that are also synthesized for use in odor and 

flavor manufacturing. 12' 

13 Acetoin and diacetyl are found in tobacco smoke, vapors 

from garbage, vapors from liquid and solid animal wastes, exhaust emissions from 

petroleum based fuels, vapors from moldy buildings, charcoal production, vapors from 

latex-polyurethane backed carpet, and as chemical reagents and in chemical 

reactions. 14 Diacetyl is also used as an anti-microbial preservative, modifier of radiation 

responses for chemical and biological systems, and as a photoinitializer in 

polymerization of plastics. 

Occupational exposure to acetoin and diacetyl in microwave popcorn manufacturing 

has been studied since the first reported case of severe obstructive lung disease in 

2000. 15 NIOSH identified acetoin and diacetyl as useful indicator compounds that can 

Hazard Communication Guidance for Diacetyl and Food Flavorings Containing Diacetyl, 2007. U.S. Department of Labor, 

Occupational Safety and Health Administration Web site. http:llwww.osha.gov/dsglguidanceldiacetyl-guidance.html 

(accessed 311712008). 1

° Flavorings-Related Lung Disease: Health Hazard Evaluations. Centers for Disease Control and Prevention, The National 

Institute for Occupational Safety and Health Web site. http:llwww.cdc.gov/nioshltopicslflavoringslhhe-eval.html (accessed 

311712008). 11 Hazard Communication Guidance for Diacetyl and Food Flavorings Containing Diacetyl, 2007. U.S. Department of Labor, 

Occupational Safety and Health Administration Web site. http:llwww.osha.gov/dsglguidanceldiacetyl-guidance.html 

(accessed 311712008). 12 Fenarolli's Handbook of Flavor Ingredients, 5th ed.; Burdock, G.A.; CRC Press; Boca Raton, FL, 2005, p 11. 13 Fenarolli's Handbook of Flavor Ingredients, 5th ed.; Burdock, G.A.; CRC Press; Boca Raton, FL, 2005, p 411. 14 Chemical Information Review Document for Artificial Butter Flavoring and Constituents Diacetyl (GAS No. 431-03-8) and Acetoin 

(GAS No. 513-86-0), 2007. Department of Health and Human Services, National Toxicology Program Web site. 

http:llntp.niehs.nih.govlntplhtdocs!Chem_Background!ExSumpdfl Artificial_butter_flavoring.pdf (accessed 311712008). 15 HETA 2000-0401-2991 Gilster-Mary Lee Corporation, 2000. Centers for Disease Control and Prevention, The National Institute 

for Occupational Safety and Health Web site. http:llwww2a.cdc.govlhhelselect.asp?PjtName=40422&bFlag=1&ID=1 

(accessed 311712008). 

4 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 271
 

be used to represent exposure to butter flavorings. Areas of concern were the flavor 

production rooms, mixing/blending rooms, packaging/production rooms, rooms where 

the mixing tanks were located, maintenance and cleaning operations, and quality 

control labs. 16 

Acetoin is used as an aroma carrier and as a flavor ingredient to impart a creamy taste 

in fragrances and flavorings. 17 Acetoin annual use in food and flavors manufacturing in 

2004 was 34,000 pounds. Acetoin is used as a flavor ingredient for butter, milk, yogurt, 

and strawberry flavors. The FDA maximum allowable concentration for acetoin in 

beverages is 5 ppm, and in food is 50 ppm. Acetoin is naturally found in fresh apple, 

cooked apple, leek, cooked leek, corn, honey, cocoa, butter, roasted coffee, cheeses, 

yogurt, milk, wines, beer, fermented tea, scallops, crowberry, quince, and other 

sources. Acetoin is used in manufacturing alcoholic beverages, baked goods, 

breakfast cereals, cheese, chewing gum, condiments and relishes, confections and 

frostings, fats and oils, frozen dairy products, fruit juices, gelatins and puddings, gravies 

and mixes, hard candy, imitation dairy products, meat products, milk products, 

nonalcoholic beverages, grains, reconstituted vegetables, seasonings and flavorings, 

snack foods, soft candy, soups, and sweet sauce. 

Diacetyl is used as a fragrance and flavor ingredient to give products a buttery or 

creamy odor and flavor. 18 Diacetyl annual use in food and flavor manufacturing in 2004 

was 153,500 pounds. The FDA maximum allowable concentration for diacetyl in 

beverages is 5 ppm, and in food is 50 ppm. Diacetyl naturally occurs in butter, milk 

products, yogurt, grains, meat, wines, beer, oils of pine, oil of angelica, oils of lavender 

and other flowers, many flowers, raspberries, strawberries, citrus, ligonberry, guava, 

cabbage, peas, tomato, vinegar, cheeses, chicken, beef, mutton, pork, cognac, 

whiskies, tea, and coffee. Diacetyl is used in manufacturing as a flavoring in alcoholic 

beverages, baked goods, cheese, chewing gum, fats and oils, frozen dairy products, 

gelatins and puddings, gravies, hard candy, soft candy, imitation dairy, meat products, 

milk products, nonalcoholic beverages, and snack foods. 

1. 1.4 Physical properties and other descriptive information 

acetoin19'

20'21 

Acetoin is found as the liquid monomer and the solid dimer. The pure monomer forms 

the dimer at room temperature. The monomer can be formed from the dimer by 

heating, distilling, or by dissolving in water or other solvents. 

16 HETA 2001-0474-2943 American Pop Corn Company, 2001. Centers for Disease Control and Prevention, The National 

Institute for Occupational Safety and Health Web site. 

http:llwww2a.cdc.govlhhelselect.asp?PjtName=36271&bFlag=O&ID=2 (accesed 311712008). 17 Fenarolli's Handbook of Flavor Ingredients, 5th ed.; Burdock, G.A.; CRC Press; Boca Raton, FL, 2005, p 11. 18 Fenarolli's Handbook of Flavor Ingredients, 5th ed.; Burdock, G.A.; CRC Press; Boca Raton, FL, 2005, p 411. 19 Budavari, S., Ed; The Merck Index, 13th ed.; Merck & Co. Inc.: Whitehouse Station, NJ, 2001; p 68. 20 Material Safety Data Sheet: Acetoin, Chemwatch, Victoria, Australia (accesed 3117108). 21 Acetoin MSDS. SigmaAldrich Web site. http:llwww.sigmaaldrich.com!cataloglsearch!ProductDetail!ALDRICHIA 17951 

(accessed 311712008). 

5 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

272 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

synonyms: 

IMIS22: 

GAS number: 

boiling point: 

melting point: 

density: 

molecular weight: 

flash point: 

autoignition 

temperature: 

appearance: 

acetyl methyl carbinol; 2,3-butanolone; 2-butanone, 3-hydoxy-; 

2-butanol-3-one; dimethylketol; y-hydroxy-{3-oxobutane; 

3-hydroxybutan-2-one; 3-hydroxy-2-butanone; 1-hydroxyethyl 

methyl ketone; methyl acetyl carbinol 

A624 

513-86-0 (monomer); 23147-57-1 (dimer/3 

148 °C (298 °F) (monomer) 

15 °C (59 °F) (monomer); 90 °C (194 °F) (dimer) 

1.005 glmL @20120 (monomer) 

88. 11 (monomer) 

50.6 °C (123 °F) (closed cup) (monomer) 

370 °C (773.8 °F) 

clear to light yellow liquid (monomer); light cream to light yellow 

crystals (dimer) 

vapor density: > 1 (air = 1) 

molecular formula: C4H80 2 (monomer); C8H160 4 (dimer) 

odor: pleasant buttery odor 

solubility: soluble in water; miscible with alcohol; sparingly soluble in ether and 

petroleum ether 

reactive hazards: acetoin is light sensitive 24 (Section 4.9) 

structural formula: 

(monomer) 

structural formula: 

( acetoin-PFBHA derivative) 

F~~ F I ~ 0-....N 

F H NCH3 

F H CH 3 

OH 

22 Acetoin (OSHA Chemical Sampling Information), 2007. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http:llwww.osha.gov!dts/chemicalsamplingldata!CH_217010.html (accessed 311712008). 23 CID: 90884 Acetyl Methyl Carbinol Dimer, 2008. Department of Health and Human Services, National Institutes of Health, 

National Center for Biotechnology Information. http:llpubchem.ncbi.nlm.nih.govlsummarylsummary.cgi?cid= 

90884&1oc=ec_rcs (accessed 311712008). 24 Material Safety Data Sheet: Acetoin, 2008. The Good Scents Company Web site. http://www.thegoodscentscompany.com 

lmsds/md102388.html (accessed 311712008). 

6 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 273
 

diacetyf5,26,21,2a 

synonyms: biacetyl; 2,3-butanedione; 2,3-butadione; 2,3-diketobutane; 

dimethyldiketone; dimethylglyoxal; glyoxal, dimethyl-; 

IMIS29: 0740 

GAS number: 431-03-8 

boiling point: 88 °C (190 °F) 

melting point: 3-4 °C (37.4-39.2 °F) 

density: 0.99 glmL@ 15115 

molecular weight: 86.09 

vapor pressure: 7 kPa @ 20 °C 

flash point: 26. 7 °C (80 °F) (closed cup) 

appearance: yellow to yellow-green liquid 

vapor density: 3 (air = 1) 

molecular formula: C4H60 2 

odor: butter in lower concentrations, quinone odor or chlorine-like odor in 

higher concentrations 

solubility: 4 parts water; miscible with alcohol, ether 

reactive hazards: diacetyl is light sensitive (Section 4.9); vapors may ignite when 

pouring or pumping due to static electricity 

autoignition 

temperature: 285 °C (545 °F) 

structural formula: 

structural formula: 

(diacetyl-PFBHA derivative) 

F~~ F 

FI .o o ..... N WF F 

H \ N --....;::: 

F H CH\)...__ ~ 'o H \ ~3 l/ .0 F 

CH3 F 

F 

25 The Merck Index, 13th ed.; Budavari, S., Ed.; Merck & Co. Inc.: Whitehouse Station, NJ, 2001; p 522. 26 Material Safety Data Sheet: Diacetyl, Chemwatch, Victoria, Australia (accessed 311712008). 27 Material Safety Data Sheet: 2, 3-Butanedione, 2007. Fisher Scientific Web site. https:llfscimage.fishersci.comlmsds/03275.htm 

(accessed 311712008). 28 Material Safety Data Sheet: 2,3-Butanedione, 2007. Chem Service Inc Web site. http:llwww.chemservice.com/msds/ 

msds_detail.asp?catnum=0-816 (accessed 311712008). 29 Diacetyl (OSHA Chemical Sampling Information), 2007. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http:llwww.osha.gov!dts/chemicalsamplingldata!CH_231710.html (accessed 311712008). 

7 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

274 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

This method was evaluated according to the OSHA SL TC "Evaluation Guidelines for Air Sampling 

Methods Utilizing Chromatographic Analysis"30• The Guidelines define analytical parameters, specify 

required laboratory tests, statistical calculations, and acceptance criteria. The analyte air concentrations 

throughout this method are based on the recommended sampling and analytical parameters. Air 

concentrations in ppm are referenced to 25 °C and 101.3 kPa (760 mmHg). 

1.2 Limit defining parameters 

1.2.1 Detection limit of the analytical procedure 

The detection limit of the analytical procedure is 0.17 pg for acetoin and 0.11 pg for 

diacetyl. These are the amounts of analyte that will give a detector response that is 

significantly different from the response of a reagent blank. (Section 4. 1) 

1.2.2 Detection limit of the overall procedure 

The detection limit of the overall procedure is 14.5 ng (0.447 ppb or 1.61 μg/m3

) for 

acetoin and 12.3 ng (0.389 ppb or 1.37 μg/m3

) for diacetyl. These are the amounts of 

analyte spiked on the sampler that will give detector responses that are significantly 

different from the responses of the respective sampler blanks. (Section 4.2) 

1.2.3 Reliable quantitation limit 

The reliable quantitation limit is 48.4 ng (1.49 ppb or 5.37 μg/m3

) for acetoin and 41.1 

ng (1.30 ppb or 4.57 μg/m3

) for diacetyl. These are the amounts of analyte spiked on 

the samplers that will give detector responses that are considered the lower limits for 

precise quantitative measurements. (Section 4.2) 

1.2.4 Instrument calibration 

The standard error of estimate is 0.019 μg/sample for acetoin over the range of 0.41 to 

3.28 μg/sample. The standard error of estimate is 0.052 μg/sample for diacetyl over 

the range of 0.40 to 3.16 μglsample. This range corresponds to 0.25 to 2 times the 

TWA target concentration. (Section 4.3) 

1.2.5 Precision 

The precision of the overall procedure at the 95% confidence level for the ambient 

temperature 18-day storage test at the target concentration from dried silica gel tubes 

was ±9.9% for acetoin and ±10.0% for diacetyl. These each include an additional 5% 

for sampling pump variability. (Section 4.4) 

1.2.6 Recovery 

The recoveries of acetoin and diacetyl from samples used in the 18-day storage test 

remained above 98.4% for acetoin and 98. 0% for diacetyl when the samples were 

stored at 23 °C. (Section 4. 5) 

30 Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M.; Evaluation Guidelines For Air Sampling Methods 

Utilizing Chromatographic Analysis, 1999. U.S. Department of Labor, Occupational Safety and Health Administration Web 

site. http://www.osha .govldts/sltclmethods/chromguidelindex.html (accessed 311512008). 

8 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 275
 

1.2. 7 Reproducibility 

Six samples were collected from a controlled test atmosphere and submitted for 

analysis by the OSHA Salt Lake Technical Center. The samples were analyzed 

according to a draft copy of this procedure after being stored at 4 °C for 20 days and at 

-12 °C for an additional 19 days. No individual sample result deviated from its 

theoretical value by more than the precision reported in Section 1.2.5. (Section 4.6) 

2. Sampling Procedure 

All safety practices that apply to the work area being sampled should be followed. The sampling 

equipment should be attached to the worker in such a manner that it will not interfere with work 

performance or safety. 

2. 1 Apparatus 

Samples are collected with two tubes in series. The tubes consist of 110-cm x 7-mm o.d. glass 

sampling tubes packed with one section (600 mg) of specially cleaned and dried silica gel. 

From the front to back, the sampler consists of a silane-treated glass wool plug, glass fiber 

filter, 600 mg specially cleaned silica gel, and a second silane-treated glass wool plug. The 

silica gel should be cleaned and dried as described in Appendix A of OSHA Method 1013. 31 

The tubes used in this evaluation were labeled front and back tube. The front tube is 

connected to the back tube with a piece of tubing to form the sampling train. For this evaluation 

commercially prepared sampling tubes containing the specially dried silica gel were purchased 

from SKC, Inc. (Catalog no. 226-183, lot no. CPM112907-001). 

Samples are collected using a personal sampling pump calibrated, with the sampling device 

attached, to within ±5% of the recommended flow rate. 

Use aluminum foil, opaque tape, or a tube holder, such as SKC, Inc. Cover D (catalog no. 244-

290), to protect samples from light. 

2.2 Reagents 

None required 

2.3 Technique 

Immediately before sampling, break off both ends of the flame-sealed tube to provide an 

opening approximately half the internal diameter of the tube. Wear eye protection when 

breaking the tube. Use tube holders to minimize the hazard of broken glass and to protect 

tubes from light exposure during sampling. All tubes should be from the same lot. 

A sampling train is created by attaching two tubes in series with a small section of tubing so 

that the front opening of the back tube is close to the back opening of the front tube. The front 

of each tube contains glass wool followed by a glass fiber filter, and the back of the tube 

contains only the glass wool. 

The back tube is used as a back-up and is positioned nearest the sampling pump. Attach the 

tube holder to the sampling pump so that the adsorbent tube is in an approximately vertical 

position with the inlet in the breathing zone. Position the sampling pump, tube holder, and 

tubing so they do not impede work performance or safety. Use a tube holder or wrap the tubes 

31 Simmons, M., Hendricks, W. Acetoin Diacetyl (OSHA Method 1013), 2008. U.S. Department of Labor, Occupational Safety and 

Health Administration Web site. http:llwww.osha.gov!dtslsltclmethodslvalidated/1013!1013.html (accessed 111112008). 

9 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

276 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

in aluminum foil to insure that both sampling tubes are protected from light exposure. Light will 

decompose the acetoin and diacetyl. 

Draw the air to be sampled directly into the inlet of the tube holder. The air being sampled is 

not to pass through any hose or tubing before entering the sampling tube. 

After sampling for the appropriate time, remove the sampling train, separate the tubes, and seal 

each tube with plastic end caps. Wrap each tube in aluminum foil or opaque tape, and then 

seal each sample end-to-end with a Form OSHA-21 seal as soon as possible. 

Submit at least one blank sample with each set of samples. Handle the blank sample in the 

same manner as the other samples except draw no air through it. 

Record sample air volumes (liters), sampling time (minutes), and sampling rate (Umin) for each 

sample, along with any potential interferences on the Form OSHA-91A. 

Submit the samples to the laboratory for analysis as soon as possible after sampling. As a 

precaution, store the samples at refrigerator temperature if a delay in shipment is unavoidable. 

Ship any bulk samples separate from the air samples. 

2.4 Sampler capacity (Section 4. 7) 

The sampling capacity was determined using test atmospheres containing the analytes. The 

concentrations of the test atmospheres were: 0.101 ppm (0.365 mg/m3

) acetoin, and 0.101 ppm 

(0.355 mg/m3

) diacetyl with an average relative humidity (RH) of 80% at 23 °C. The samples 

were collected at 0.05 Umin. The 5% breakthrough air volumes were determined to be 12.1 L 

for diacetyl and greater than 24 L for acetoin. 

There was no acetoin or diacetyl on the back-up tube when a 15 min sample was taken at 0.2 

Umin. The 5% breakthrough air volumes for a flow rate of 0.2 Umin were determined to be 

11.98 L for diacetyl and greater than 13 L for acetoin. 

2. 5 Extraction efficiency (Section 4. 8) 

It is the responsibility of each analytical laboratory to determine the extraction efficiency of the 

analyte from the media because the adsorbent material, internal standard, reagents and 

laboratory techniques may be different than those listed in this evaluation and influence the 

results. 

The mean extraction efficiencies from dry silica gel over the range of RQL to 2 times the target 

concentration were: 102.0% (0.022 to 3.28 μg/sample) for acetoin and 97.6% (0.01 to 3.16 

μg/sample) for diacetyl. The extraction efficiency was not affected by the presence of water. 

Extracted samples remain stable for at least 24 h. 

2. 6 Recommended sampling time and sampling rate 

Sample with dried silica gel tubes for up to 180 min at 0.05 Umin (9 L) to collect TWA (longterm) samples, and for 15 min at 0.2 Umin (3 L) to collect short-term samples. 

When short-term samples are collected, the air concentration equivalent to the reliable 

quantitation limit becomes larger. For example, the reliable quantitation limits for dried silica gel 

tubes for a 15 min sample taken at 0.2 Umin are 0.0044 ppm (0.016 mg/m3

) for acetoin and 

0.0042 ppm (0.015 mglm3

) for diacetyl. 

10 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 277
 

2. 7 Interferences, sampling (Section 4.9) 

Retention efficiency 

The mean retention efficiency was 96. 7% for acetoin and 96. 9% for diacetyl when dried silica 

gel tubes containing 0.819 μg of acetoin and 0.808 μg of diacetyl were allowed to sample 6. 75 

L of contaminant-free air having an average relative humidity of 80% at 23 °C. (Section 4.9) 

Low humidity 

The ability of dried silica gel tubes to collect the analytes from a relatively dry atmosphere was 

determined by sampling an atmosphere containing two times the target concentration and at an 

average relative humidity of 20% RH at 23 °C. The mean recoveries (% of theoretical) were 

98. 7% for acetoin and 98.5% for diacetyl. (Section 4.9) 

Low concentration 

The ability of dried silica gel tubes to collect the analytes at low concentrations was tested by 

sampling an atmosphere at 0. 1 times the target concentration with at an average relative 

humidity of 80% RH at 23 °C. The mean recoveries (% of theoretical) were 99.0% for acetoin 

and 98.4% for diacetyl. (Section 4.9) 

Sampling interference 

The ability of dried silica gel tubes to collect the analyte when other potential interferences are 

present was tested under two separate series of tests. The first test was an atmosphere similar 

to ones found at some popcorn manufacturing plants consisting of acetoin and diacetyl at the 

target concentration with an interference mixture of acetaldehyde, acetic acid, and methyl ethyl 

ketone at an average humidity of 80% at 23 °C. All three of these interferences can react with 

PFBHA. The concentrations of the analytes in this test atmosphere were: 0.051 ppm (0.184 

mglm3

) acetoin and 0.051 ppm (0.180 mg/m3) diacetyl, 1.01 ppm (1.82 mglm3) acetaldehyde, 

1.05 ppm (2.58 mglm3

) acetic acid, and 1.02 ppm (3.01 mglm3) methyl ethyl ketone. Three 

samplers had contaminated air drawn through them at 0. 05 Umin for 180 min. All of the 

samples were immediately analyzed. The mean recoveries (% of theoretical) were: acetoin 

97.9% and diacetyl 98.2%. 

The second series of tests was with acetoin and diacetyl at the target concentration and each 

of the interferences listed above individually at their PEL concentration following the guidelines 

in SL TC "Evaluation Guidelines for Air Sampling Methods Utilizing Chromatographic Analysis"32• 

The concentrations of these interferences are much higher than would normally be expected in 

a food or flavoring manufacturing workplace. The PFBHA extraction solution needed to be 

modified to 18 mg/mL PFBHA (72. 1 μmoles/mL) to insure that there was enough PFBHA to 

derivatize all the analytes. These interferences and acetoin react fully within 4 hours of 

extraction, but the diacetyl requires 36 hours to fully react. These three test atmospheres each 

contained the one of the followinq concentrations of interference: 190 ppm (350 mg/m3

) 

acetaldehyde, 9.49 ppm (23.3 mglm) acetic acid, or 190 ppm (560 mglm3

) methyl ethyl ketone. 

These three compounds were chosen because they can collect onto the dried silica gel tubes 

and can react with the PFBHA. For each test, three sampling trains had contaminated air (air 

containing the analytes and an interference) drawn through them at 0.05 Umin for 180 min for 

each test. All of the samples were immediately analyzed. The average recoveries (% of 

theoretical) with 190 ppm acetaldehyde were 97.8% for acetoin and 95.5% for diacetyl. The 

average recoveries (% of theoretical) with 9.49 ppm acetic acid were 97.3% for acetoin and 

32 Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M. Evaluation Guidelines For Air Sampling Methods 

Utilizing Chromatographic Analysis, 1999. U.S. Department of Labor, Occupational Safety and Health Administration Web 

site. http://www.osha .govldtslsltclmethodslchromguidelindex.html (accessed 311512008). 

11 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

278 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

98. 2% for diacetyl. The average recoveries (% of theoretical) with 200 ppm methyl ethyl ketone 

were 98.4% for acetoin and 97. 6% for diacetyl. These interferences were not a sampling 

interference, but under normal sample analysis, these levels of interferences would be 

analytical interferences. (Section 4. 9) 

Light 

Acetoin and diacetyl are light-sensitive. The interference of light during sampling was tested 

using three foil-wrapped sampling trains and three uncovered sampling trains. An atmosphere 

containing twice the target concentration at an average relative humidity of 78% at 23 °C was 

sampled for 180 min at 0. 05 Umin, and the samples were extracted that day. The average 

recovery for acetoin of the foil-wrapped samplers was 98.5% and the uncovered samplers had 

an average recovery of 93. 9%. The average recovery for diacetyl of the foil-wrapped samplers 

was 98.9% and the uncovered samplers had an average recovery of 94.3%. An additional 

three sampling trains were collected at the same time, and were protected from the light by 

aluminum foil. After collection, these samplers had the foil removed and were placed on the 

counter at ambient temperature under room light. These samples were analyzed 24 h after 

sampling during which they were exposed to the room light for 14 of the 24 h. The average 

recoveries were 81.3% for acetoin and 80.0% for diacetyl. Light is a significant interference; 

therefore, both tubes in the sampling train need to be covered by aluminum foil or opaque tape 

during and after sampling. (Section 4.9) 

Powder form 

The powder form of acetoin and diacetyl tested consisted of starch coated with acetoin and 

diacetyl. Three tests were performed on this powder. The first consisted of a sampling train of 

a pre-weighed PVC filter in a conical cassette in series with two dried silica gel tubes. The two 

dried silica gel tubes were used to collect any vapors of acetoin and diacetyl which would strip 

off from the powder. Known amounts of the powder were placed onto the PVC filter, and 9 L of 

air at an average relative humidity of 78% at 22 °C were pulled through the sampling trains at 

0.05 Umin. The recovery of acetoin and diacetyl on the pre-weighed PVC filters was 0% to 

1.9% for acetoin and 0% to 2.3% for diacetyl. The recovery on the dried silica gel tubes was 

96.6% for acetoin and 97.8% for diacetyl. The acetoin and diacetyl recoveries were calculated 

from the percentages obtained from analysis of the powder and the amounts of powder 

weighed out. The second and third tests consisted of a sampling train of two dried silica gel 

tubes in series, with the powder spiked on the front glass wool of the front tube. The two tests 

had 9 L of air drawn through the sampling trains at 0.05 Umin, the first test used air at an 

average relative humidity of 20% at 22 °C, and the other test used air at an average relative 

humidity of 78% at 22 °C. At 20% RH most of the acetoin and diacetyl were found on the front 

glass wool and glass fiber filter, but at 78% RH most of the acetoin and diacetyl were found on 

the dried silica gel beds. These tubes can collect particulates, but cannot be used as a 

particulate sampler at 0.05 Umin. (Section 4.9) 

3. Analytical Procedure 

Adhere to the rules set down in your Chemical Hygiene Plan33. Avoid skin contact and inhalation of all 

chemicals and review all MSDSs before beginning this analytical procedure. 

3. 1 Apparatus 

Gas chromatograph equipped with an electron capture detector. An Agilent Model 6890 GC 

equipped with a Chemstation, an automatic sample injector, and a μ-electron capture detector 

(μECO) was used in this evaluation. 

33 Occupational Exposure to Hazardous Chemicals in Laboratories. Code of Federal Regulations, Part 1910.1450, Title 29, 2003. 

12 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 279
 

A GC column capable of separating the PFBHA derivatives of acetoin and diacetyl from the 

PFBHA extraction solution, potential interferences, and internal standard. A 30-m x 0.32-mm 

i.d. fused silica capillary column (DB-5 0.25-μm df) (Agilent Technologies, Santa Clara CA) was 

used in this evaluation. 

An electronic integrator or other suitable means of measuring GC detector response. A Waters 

Empower 2 Data System was used in this evaluation. 

Amber glass vials with PTFE-lined caps. Amber 2 and 4-mL vials were used in this evaluation. 

A dispenser capable of delivering 2.0 mL of PFBHA extraction solution to prepare standards 

and samples. If a dispenser is not available, 2. 0-mL volumetric pipettes can be used. 

Class A volumetric flasks of appropriate sizes such as 10-mL and other convenient sizes for 

preparing standards. 

Calibrated 10-μL syringe for preparing standards. 

Micro-analytical balance capable of weighing at least 0. 001 mg. An Ohaus Galaxy 160D was 

used in this evaluation. 

Rotator. A Fisher Rota Rack was used to extract the samples. 

3. 2 Reagents 

Acetoin, [GAS no. 513-86-0}, reagent grade or better. Acetoin used in this evaluation was 

99+% (lot no. 05025DH) purchased from Sigma-Aldrich (Milwaukee, WI). 

Diacetyl, [GAS no. 431-03-8], reagent grade or better. Diacetyl used in this evaluation was 

97% (lot no. 10815TD) purchased from Sigma-Aldrich (Milwaukee, WI). 

Ethyl alcohol, [GAS no. 64-17-5}, 95% vlv (190 proof) A.C.S. Spectrophotometric grade. Ethyl 

alcohol used in this evaluation was 95% (lot no. 80513970) purchased from Acros (Morris 

Plains, NJ). 

0-(2,3,4,5,6-pentafluorobenzyl)hydroxylamine hydrochloride, [GAS no. 57981-02-9] (PFBHA), 

reagent grade or better. PFBHA used in this evaluation was 99+% (lot no. 1242759 54706063) 

purchased from Fluka, a subsidiary of Sigma-Aldrich (Milwaukee, WI). 

4-Bromobenzylbromide, [GAS no. 589-15-1], reagent grade or better. 4-Bromobenzylbromide 

used in this evaluation was 98% (lot no. A0251708) purchased from Acros (Morris Plains, NJ). 

DI water, 18 Mo-cm. A Barnstead NanoPure Diamond system was used to purify the water for 

this evaluation. 

The PFBHA extraction solution used for this evaluation consisted of 20 μg/mL 

4-bromobenzylbromide in the 95:5 ethyl alcohol:water with 2 mg/mL PFBHA. The 

4-bromobenzylbromide was added to 95:5 ethyl alcohol:water as an internal standard. Other 

internal standards can be used provided they are fully tested. Store this solution in a tightly 

sealed container in a refrigerator that does not contain solutions of aldehydes, acids, or 

ketones. This solution can absorb formaldehyde, other aldehydes, ketones, and acids out of 

the air. These compounds will react with the PFBHA, decreasing the amount available to react 

with acetoin or diacetyl. This solution can be stored in the refrigerator for 1 week. 

13 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

280 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

3. 3 Standard preparation 

Prepare stock solution of acetoin and diacetyl in water. Acetoin is usually sold as the dimer, 

which will disassociate in water to the monomer as the solid dimer dissolves. This stock 

solution will remain stable for four weeks if stored in an amber bottle in the refrigerator. 34 

Freshly prepare analytical standards from the stock solutions for each analysis. These 

analytical standards are prepared for each of the analytes by injection of micro/iter amounts of 

a stock solution into 2-mL volumetric flasks and diluting with the PFBHA extraction solution over 

a concentration range of 0.02 to 6 μg/sample. For example: a target concentration standard of 

1. 60 μg/sample acetoin and 1. 56 μg/sample diacetyl was prepared by injecting 16 μL of a stock 

solution containing 0.10 μg/mL acetoin and 0.10 μUmL (0.0975 μg/mL) diacetyl in water into a 

2-mL volumetric flask containing about 1. 75 mL of PFBHA extraction solution and then diluting 

to the mark with PFBHA extraction solution (this is equivalent to 0.80 μg/mL acetoin or 0.049 

ppm based on a 2-mL extraction and 9 L air volume, and 0. 78 μg/mL diacetyl or 0. 049 ppm 

based on a 2-mL extraction and 9 Lair volume). Standards must be allowed to react with the 

PFBHA at room temperature for 36 hours. 

Bracket sample concentrations with standard concentrations. If upon analysis, sample 

concentrations fall outside the range of prepared standards, prepare and analyze additional 

standards to confirm instrument response, or dilute high samples with PFBHA extraction 

solution and reanalyze the diluted samples. 

3.4 Sample preparation 

Remove the plastic end caps from the sample tube and carefully transfer the section of the 

adsorbent from each tube into separate 4-mL amber vials. Normally the front glass wool plug 

and glass fiber filter are discarded. If the industrial hygienist requests the analysis, the front 

glass wool plug and the glass fiber filter should be placed into a separate 4-mL amber vial. 

Discard the glass tubes and back glass wool plugs. 

Add 2. 0 mL of PFBHA extraction solution to each vial and immediately seal the vials with 

PTFE-lined caps. 

Place the samples on a mechanical rotator and rotate at approximately 40 rpm for 60 min. Do 

not use a shaker to extract samples, as the recoveries will be lower. 

Allow the samples to stand at room temperature for an additional 36 hours for the derivatization 

reaction to reach completion. 

Transfer each solution from the 4-mL vial to a labeled amber 2-mL glass autosampler vial and 

seal with a PTFE-lined cap. 

If more sensitivity is desired for samples prepared by OSHA Method 101335, they can be 

derivatized by the PFBHA solution and analyzed by GC-ECD. The samples in OSHA 1013 are 

extracted with 2 mL 95:5 ethyl alcohol:water. The samples can be derivatized by the following 

procedure: add 0.5-mL of sample and 0.5-mL of PFBHA extraction solution into a labeled 2-mL 

vial, and react for 36 hours, and then analyze by GC-ECD following the analytical conditions in 

this method. Standards prepared by OSHA Method 1013 are derivatized following the same 

procedure. The RQL will be a factor of 2 higher due to this dilution of the samples. 

34 Simmons, M., Hendricks, W., Acetoin Diacetyl (OSHA Method 1013), 2008. U.S. Department of Labor, Occupational Safety and 

Health Administration Web site. http:llwww.osha.gov!dts/sltclmethods/validatedl1013!1013.html (accessed 111112008). 35 Simmons, M., Hendricks, W., Acetoin Diacetyl (OSHA Method 1013), 2008. U.S. Department of Labor, Occupational Safety and 

Health Administration Web site. http:llwww.osha.gov!dts/sltclmethods/validatedl1013!1013.html (accessed 111112008). 

14 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 281
 

Vi' 

:;;. 

2' 

~ 

<:: 

::i 

0 

(.) 

"' @ 

<( 

3. 5 Analysis 

4x106 

3x106 

2x106 

1x106 

3.5.1 Analytical conditions: 

GC conditions: 

column: 

injector: 

detector: 

initial 100 °C, hold 1 

min, program at 5 

°Clmin to 200 °C, 

hold O min 

250°C 

250°C 

run time: 20 min 

column gas flow: 3. 0 mUmin 

(hydrogen) 

column mode: constant pressure 

column pressure: 6. 8 psi 

injection size: 1.0 μL (40:1 split) 

column: 30-m x 0.32-mm i.d. 

capillary column (DB-5 

df= 0.25 μm) 

retention times: 0. 85 min ethyl alcohol 

1.44 min PFBHA 

S' 

,§_ 

Sl 

<:: 

0 

~ 

& 

1 2 5 

150 

100 34 

50 

0 J '\. I l11 II I I I .1 

0 5 10 15 20 

Time (min) 

Figure 3. 5.1. A chromatogram of the PFBHA 

derivatives of 1.60 μg!sample acetoin and 1.56 

μg!sample diacetyl in the extraction solution. 

(Key: (1) ethyl alcohol; (2) PFBHA; (3) 4-

bromobenzylbromide (/STD); (4) acetoinPFBHA; and (5) diacetyl-PFBHA; all other 

peaks are from PFBHA and its breakdown 

products) 

4. 60 min 4-bromobenzylbromide 

ECO conditions: 

makeup flow: 

5. 04 min acetoin-PFBHA 

16. 75 min diacetyl-PFBHA 

40mUmin 

(nitrogen) 

Peak areas are measured with an integrator or other suitable means. 

3.5.2 An internal standard (/STD) calibration method is used. A calibration curve can be 

constructed by plotting response of standard injections versus micrograms of analyte 

per sample. Bracket the samples with freshly prepared analytical standards over the 

range of concentrations. 

8x106 

Acetoin Calibration Curve Diacetyl Calibration Curve 

6x106 

Vi' 

:;;. 

2' 

~ 

<:: 4x106 ::i 

0 

(.) 

"' @ 

<( 

2x106 

o~~~~~~~~~~~~~~~~ o~~~~~~~~~~~~~~~~ 

0 2 3 

Mass (μg} per Sample 

Figure 3.5.2.1. Calibration curve for acetoin. 

(y = 9.16E5x + 1.44E4) 

4 

15 of 34 

0 2 3 

Mass (μg) per Sample 

Figure 3.5.2.2. Calibration curve for diacetyl. 

(y = 1.97E6x + 4.59E4) 

4 

T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

282 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

3. 6 Interferences (analytical) 

Any compound that produces a GC-ECD response and has a similar retention time as the 

analyte is a potential interference. If any potential interferences were reported, they should be 

considered before samples are extracted. Generally, chromatographic conditions can be 

altered to separate an interference from the analyte. 

3. 7 Calculations 

The amount of analyte per sampler is obtained from the appropriate calibration curve in terms 

of micrograms of analyte per sample, uncorrected for extraction efficiency. The front amount 

found is then corrected by subtracting the total amount (if any) found on the front blank. The 

back amount found is then corrected by subtracting the total amount (if any) found on the back 

blank. The amount found on the back dried silica gel tube is added to the front tube for the total 

loading on each sample. The back-up tube is analyzed separately to determine the extent of 

analyte saturation to determine if breakthrough occurred. Even though the analytes are 

analyzed as the PFBHA derivatives and the calibration and results are as the amount of 

analyte. The air concentration is calculated using the following formulas. 

4. Backup data 

M = {Mfront - Mfront blank} + {Mback - Mback blank] 

where M 

Mrront 

Mback 

Mrront blank 

Mblank 

is total micrograms per sample 

is micrograms found on front tube 

is micrograms found on back tube 

is micrograms found on front blank tube 

is micrograms found on back blank tube 

where CM is concentration by weight (mglm3

) 

M is micrograms per sample 

V is liters of air sampled 

EE is extraction efficiency, in decimal form 

where Cv is concentration by volume (ppm) 

VM is 24.46 (molar volume at NTP) 

CM is concentration by weight (mg/m3

) 

Mr is molecular weight of analyte 

(acetoin = 88.11 and diacetyl = 86.09 

General background information about the determination of detection limits and precision of the 

overall procedure is found in the "Evaluation Guidelines for Air Sampling Methods Utilizing 

Chromatography Analysis". 36 The Guidelines define analytical parameters, specify required laboratory 

tests, statistical calculations, and acceptance criteria. 

4. 1 Detection limit of the analytical procedure (DLAP) 

The DLAP is measured as the mass of analyte introduced onto the chromatographic column. 

Ten analytical standards were prepared with equally descending increments with the highest 

standard containing 97.9 ng/mL acetoin, and for diacetyl the highest standard was 95.5 ng/mL. 

36 Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M. Evaluation Guidelines For Air Sampling Methods Utilizing 

Chromatographic Analysis, 1999. U.S. Department of Labor, Occupational Safety and Health Administration Web site. 

http:llwww.osha.gov!dts/sltclmethods/chromguidelindex.html (accessed 311512008). 

16 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 283
 

These are the concentrations that would produce peaks at least 10 times the response of a 

reagent blank near the elution time of the analyte. These standards, and the reagent blank 

were analyzed with the recommended analytical parameters (1-μL injection with a 40:1 split), 

and the data obtained were used to determine the required parameters (slope and standard 

error of estimate) for the calculation of the DLAP. For acetoin, the slope and standard error of 

estimate, respectively, were 3818 and 219. For diacetyl, the slope and standard error of 

estimate, respectively, were 9595 and 366. 

Table 4.1.1 

Detection Limit of the Analytical Procedure 

for Acetoin 

concentration 

(nglmL) 

0 

9.79 

19.6 

29.4 

39.2 

49.0 

58.7 

68.5 

78.3 

88.1 

97.9 

mass on 

column (pg) 

0 

0.245 

0.490 

0.735 

0.980 

1.23 

1.47 

1.71 

1.96 

2.20 

2.45 

Table 4.1.2 

area counts 

(μV•s) 

0 

863 

1679 

2588 

3443 

4167 

5301 

6084 

7465 

8098 

9529 

Detection Limit of the Analytical Procedure 

for Diacety/ 

concentration 

(nglmL) 

0 

9.55 

19.1 

28.7 

38.2 

47.8 

57.3 

66.9 

76.4 

86.0 

95.5 

mass on 

column (pg) 

0 

0.238 

0.478 

0.718 

0.955 

1.20 

1.43 

1.67 

1.91 

2.15 

2.39 

area counts 

(μV•s) 

0 

2824 

5099 

7020 

9587 

11701 

13790 

15745 

18523 

20511 

23882 

Vi' 

:;;. 

2' 

.l!l 

§ 

0 

(.) 

"' @ 

<( 

Vi' 

:;;. 

2' 

.l!l 

§ 

0 

(.) 

"' @ 

<( 

10000 

Acetoin 

SEE= 219 

8000 DLAP=017pg 

6000 

4000 

2000 

DLAP 0 

0 05 1.0 1.5 2.0 2.5 

Mass (pg} Injected onto Column 

Figure 4.1.1. Plot of data to determine the DLAP 

for acetoin. (y = 3818x - 202) 

25000 Diacetyl 

SEE= 366 

20000 DLAP=011pg 

15000 

10000 

5000 

DLAP 

0 

0 05 1.0 1.5 2.0 2.5 

Mass (pg} Injected onto Column 

Figure 4.1.2. Plot of data to determine the DLAP 

for diacetyl. (y = 9595x + 238) 

4.2 Detection limit of the overall procedure (DLOP) and reliable quantitation limit (RQL) 

DLOP is measured as mass per sample and expressed as equivalent air concentrations, based 

on the recommended sampling parameters. Ten samplers were spiked with equally 

descending increments of analyte. The highest amount is the amount spiked on the sampler 

that would produce a peak approximately 10 times the response of a sample blank. These 

spiked samplers and the sample blank were analyzed with the recommended analytical 

parameters, and the data obtained used to calculate the required parameters (slope and 

standard error of estimate) for the calculation of the DLOP. For acetoin, the slope and 

standard error of estimate, respectively, were 46.9 and 227. For diacetyl, the slope and 

17 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

284 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

standard error of estimate, respectively, were 121 and 497. For acetoin, the DLOP was 14.5 

ng and the RQL was 48.4 ng. For diacetyl, the DLOP was 12.3 ng and the RQL was 41.1 ng. 

Table 4.2.1 

Detection Limit of the Overall 

Procedure for Acetoin 

mass per sample area counts 

(ng) (!:!: V•s) 

0 0 

19.6 866 

39.2 1901 

58.7 2927 

78.3 3421 

97.9 4158 

117 5543 

137 6002 

157 7399 

176 8221 

196 9373 

Table 4.2.2 

Detection Limit of the Overall 

Procedure for Diacety/ 

mass per sample 

(ng) 

0.0 

19.1 

38.2 

57.4 

76.4 

95.5 

115 

134 

153 

172 

191 

area counts 

(!:!: V•s) 

0 

2758 

5554 

7690 

10101 

11743 

13988 

15701 

18651 

21621 

23995 

10000 

Acetoin 

SEE=227 

DLOP = 14 5 ng 8000 RQL = 48.4 ng 

Vi' 

::;. 

2' 6000 ~ 

<:: 

"' 8 4000 (lJ 

@ 

<( 

2000 

DLOP RQL 0 

0 50 100 150 200 

Mass per Sample (ng) 

Figure 4.2.1. Plot of data to determine the 

DLOPIRQL for acetoin. (y = 46. 9x - 63.1) 

Vi' 

::;. 

25000 Diacetyl 

SEE= 497 

DLOP = 12.3 ng 

20000 RQL = 41.1 ng 

2' 15000 

~ 

§ 

8 10000 

~ <( 

5000 

DLOP RQL o~~~~~~~~~~~~~~~~ 

0 50 100 150 200 

Mass per Sample (ng) 

Figure 4.2.2. Plot of data to determine the 

DLOPIRQL for diacetyl. (y = 121 x + 407) 

The RQL is considered the lower limit for precise quantitative measurements. It is determined 

from the regression line parameters obtained for the calculation of the DLOP, providing 75% to 

125% of the analyte is recovered. The RQLs are listed in Table 4.2.3. 

Table 4.2.3 

Reliable Quantitation Limits 

analyte ng ppb μglm3 EE 

acetoin 48.4 1.49 537 102.3 

diacetyl 41.1 1.30 4.57 97.3 

EE = extraction efficiency 

18 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 285
 

S' 

§_ 

Q) 

~ 

~ 

& 

6 

4 4 

2 S' 

§_ 

Q) 

2 "' 2 <:: 

0 

~ 

& 

0 0 

v-..... 

-2 -2 

4.7 4.9 5.1 5.3 5.5 16.0 16.2 16.4 16.6 16.8 17.0 

Time (min) Time (min) 

Figure 4.2.3. A chromatogram of the RQL of 

acetoin. (Key: (1) acetoin-PFBHA, (2) 

interference) 

Figure 4.2.4. A chromatogram of the RQL of 

diacetyl. (Key: (1) diacetyl-PFHBA) 

4.3 Instrument calibration 

4.4 

The standard error of estimate was determined from the linear regression of data points from 

standards over a range that covers 0.25 to 2 times the TWA target concentration. Calibration 

curves were constructed and shown in Section 3. 5. 2 from the three injections each of five 

standards. The standard errors of estimates were 0.019 μg for acetoin and 0.052 μg for 

diacetyl. 

Table 4.3.1 

Instrument Calibration for Acetoin 

standard concn area counts 

(μglsample) (μV·s) 

0.41 367186 360667 370276 

0.82 759141 752935 771533 

1.64 1550965 1559979 1538639 

2.46 2318162 2277568 2290341 

3.28 2993893 2999180 2959244 

Table 4.3.2 

Instrument Calibration for Diacety/ 

standard concn area counts 

(μglsample) (μV·s) 

0.40 818644 817236 817895 

0.79 1658619 1654024 1658622 

1.58 3140780 3142807 3140857 

2.37 4604360 4645231 4644018 

3.16 6349382 6315236 6309791 

Precision (overall procedure) 

The precision at the 95% confidence level is obtained by multiplying the standard error of 

estimate by 1.96 (the z-statistic from the standard normal distribution at the 95% confidence 

level). In Section 4.5, 95% confidence intervals are drawn about their respective regression 

lines in the storage graph figures. The precisions of the overall procedure were obtained from 

the ambient temperature 18 day storage tests were ±9.9% for acetoin and ±10.0% for diacetyl. 

19 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

286 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

~ 

c 

~ 

8 

(!) 

a:: 

4. 5 Storage test 

100 

75 

50 

25 

0 

0 

Storage samples for acetoin and diacetyl were prepared using dried silica gel tubes from 

controlled test atmospheres using the recommended sampling conditions. The concentrations 

were 0.051 ppm (0.184 mg!m3

) acetoin and 0.050 ppm (0.180 mg/m3) diacetyl at an average 

relative humidity of 80% at 23 °C. Thirty-three storage samples were prepared. Three 

samples were analyzed on the day of generation. Fifteen of the tubes were stored at reduced 

temperature (4 °C) and the other fifteen were stored in a closed drawer at ambient temperature 

(about 23 °C). At 3 to 4-day intervals, three samples were selected from each of the two 

storage sets and analyzed. Recoveries are not corrected for extraction efficiency. 

time 

(days) 

0 100.4 

4 99.1 

7 99.5 

10 100.5 

14 97.9 

18 98.5 

time 

(days) 

0 100.2 

4 99.3 

7 99.8 

10 97.3 

14 99.7 

18 98.7 

: 0 

-

Acetoin Ambient Storage 80% RH 

y = -0.0824x + 99.9 

Std Error of Estimate = 5. 06% Qt;OL ;~:4-~ +11 nr-1n:: fl~I 

5 10 

Storage Time (Days) 

Table 4.5.1 

Storage Test for Acetoin at 80% RH 

ambient storage refrigerated storage 

recovery (%) recovery (%) 

98.5 101.1 

100.3 98.9 100.1 100.4 98.6 

99.1 98.6 98.9 99.7 100.8 

98.8 99.4 98.5 100.1 99.9 

99.3 98.3 99.9 99.3 98.6 

99.3 97.6 99.8 98.3 99.1 

Table 4.5.2 

Storage Test for Diacety/ at 80% RH 

ambient storage refrigerated storage 

recovery (%) recovery (%) 

100.4 98.2 

100.1 98.1 99.4 100.1 97.3 

98.7 97.2 100.3 99.3 97.1 

99.8 98.9 97.5 100.0 99.8 

99.1 97.6 99.7 98.9 96.6 

97.7 96.8 98.6 97.7 96.5 

: -~ 

100 - 0 

v - ; - ~ 

~ 75 

c 

~ 

8 50 (!) 

a:: 

25 Acetoin Refrigerated Storage 80% RH 

y = -0.0512 x + 100 

Std Error of Estimate = 5. 07% 

+Cl qo.i; 0 

0 

95% Confidence Limits= +(1 961(5.071 = ±9.9% 

15 20 5 10 15 

Storage Time (Days) 

-

-

20 

Figure 4. 5.1. Ambient storage test for acetoin at 

80%RH. 

Figure 4.5.2. Refrigerated storage test for acetoin at 

80% RH. 

20 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 287
 

~ 

c 

~ 

8 

(!) 

a:: 

100 

75 

50 

25 

0 

0 

- Q A ~ Q 

- " " - ~ 

Diacetyl Ambient Storage 80% RH 

y = -0.0846 x + 99.5 

Std Error of Estimate = 5. 11 % 

95% Confidence Limits= ±(1 961(5.111 = ±10.0% 

5 10 15 

Storage Time (Days) 

~ 

c 

~ 

8 

(!) 

a:: 

20 

100 

75 

50 

25 

0 

0 

- A n A A 

u u u u " 

Diacetyl Refrigerated Storage 80% RH 

y = -0.0951 x + 99.6 

Std Error of Estimate = 5. 15% 

95% Confidence Limits= ±(1.961(5.151 = ±10.1% 

5 10 15 20 

Storage Time (Days) 

Figure 4.5.3. Ambient storage test for diacetyl at 

80% RH. 

Figure 4. 5.4. Refrigerated storage test for 

diacetyl at 80% RH. 

Storage studies were also performed using tubes packed with 4001200 mg sections of dried 

silica gel, at an average relative humidity of 22% RH at 23 °C to determine the effects of low 

humidity on storage and on migration. The concentrations were 0.051 ppm (0.184 mg/m3

) 

acetoin and 0. 050 ppm (0. 180 mg/m3

) diacetyl. Thirty-three storage samples were prepared. 

Three samples were analyzed on the day of generation. At 3 to 4-day intervals, three samples 

were selected from each of the two storage sets and analyzed. Fifteen of the tubes were 

stored at reduced temperature (4 °C) and the other fifteen were stored in a closed drawer at 

ambient temperature (about 23 °C). At 22% RH ambient and refrigerated storage samples 

showed no migration for acetoin or diacetyl. Recoveries are not corrected for extraction 

efficiency. 

time 

(days) 

0 

4 

7 

10 

14 

17 

time 

(days) 

0 

4 

7 

10 

14 

17 

100.2 

99.9 

98.2 

99.9 

98.9 

99.2 

100.4 

99.9 

99.6 

99.9 

99.7 

99.0 

Table 4.5.3 

Storage Test for Acetoin at 22% RH 

ambient storage refrigerated storage 

recovery (%) recovery (%) 

99.8 97.9 

97.4 98.4 

100.5 96.9 

97. 7 97.1 

99.4 96.8 

97.3 95.7 

Table 4.5.4 

100.1 

99.7 

99.4 

98.2 

96.2 

97.4 

98.8 

97.7 

99.9 

98.7 

Storage Test for Diacety/ at 22% RH 

ambient storage refrigerated storage 

recovery (%) recovery (%) 

97.1 98.5 

98.2 97.0 99.5 100.1 

98.8 97.1 99.9 98.7 

98.1 96.9 99.8 98.9 

96.5 98.4 99.5 98.0 

98.0 95.7 98.1 99.3 

99.6 

97.5 

100.3 

96.9 

99.3 

97.3 

97.4 

97.0 

96.8 

96.3 

21of34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

288 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

~ 

c 

~ 

8 

(!) 

a:: 

100 

75 

50 

25 

0 

0 

c " n " n ~ " - v 0 

Acetoin Ambient Storage 22% RH 

y = -0.0880 x + 99.2 

Std Error of Estimate = 5. 17% 

95% Confidence Limits= ±(1.96)(5.17) = ±10.1% 

5 10 15 20 

Storage Time (Days) 

~ 

c 

~ 

8 

(!) 

a:: 

100 

75 

50 

25 

0 

0 

- C> A n v - " v 0 u 

Acetoin Refrigerated Storage 22% RH 

y = -0.0670x + 99.3 

Std Error of Estimate = 5. 15% 

95% Confidence Limits= +f1 961f5.151 = +10.1% 

5 10 15 20 

Storage Time (Days) 

Figure 4. 5. 5. Ambient storage test for acetoin at 

22% RH. 

Figure 4. 5. 6. Refrigerated storage test for acetoin at 

22% RH. 

~ 

c 

~ 

8 

(!) 

a:: 

100 

75 

50 

25 

0 

0 

0 A 0 C> C> 

" v " 0 u 

Diacetyl Ambient Storage 22% RH 

y = -0. 0535x + 98. 7 

Std Error of Estimate = 5. 18% 

95% Confidence Limits= ±(1 96)(5.18) = ±10.2% 

5 10 15 20 

Storage Time (Days) 

~ 

c 

~ 

8 

(!) 

a:: 

100 

75 

50 

25 

0 

0 

A 0 A c C> 

v v v " 0 

Diacetyl Refrigerated Storage 22% RH 

y = -0.0549 x + 99 

Std Error of Estimate = 5. 16% 

95% Confidence Limits= ±(1 96)(5.16) = ±10.1% 

5 10 15 20 

Storage Time (Days) 

Figure 4.5. 7. Ambient storage test for diacetyl at 

22% RH. 

Figure 4.5.8. Refrigerated storage test for 

diacetyl at 22% RH. 

At the beginning of this method, the SKC 226-183 tubes were available as a 4001200 mg tube. 

Migration studies showed that it would be necessary to use two tubes in series, so subsequent 

tubes were packed as a single 600 mg tube. A 600 mg section makes it easier for the analyst 

to prepare the samples for extraction. Migration occurs when the analyte equilibrates between 

the two sections of the tube after collection. There is more migration with higher humidities, 

due to the higher amounts of water collected. Using 4001200 mg dried silica gel tubes, at 80% 

RH acetoin showed no migration but the diacetyl refrigerated samples at day 18 showed a 

4. 5% migration and ambient showed 15. 2% migration. Based on these results, a single 

4001200 mg dried silica gel tube should not be used for sampling. 

22 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 289
 

day 

4 

7 

10 

14 

18 

Table 4.5.5 

Migration of Diacetyl on 4001200 mg Dried Silica Gel Tube 

Sampled at 0. 05 Umin for 180 min from 0. 05 ppm Atmosphere 

ambient refrigerated 

400mg 

% of total found 

96.1 

96.0 

94.4 

93.4 

92.9 

91.5 

89.1 

91.3 

90.9 

88.0 

87.8 

86.0 

81.2 

82.7 

83.7 

200 mg 400 mg 200 mg 

% of total found % of total found % of total found 

a2 9~4 ao 

4.1 100.1 0.0 

3.7 97.3 0.0 

5.4 100.3 0.0 

5.8 99.3 0.0 

5.7 97.1 0.0 

a2 97.5 ao 

8.5 100.0 0.0 

8.0 99.8 0.0 

11.7 97.9 1.8 

11.3 97.3 1.6 

11.6 95.7 0.9 

17.5 86.9 4.2 

15.0 85.5 4.5 

13.1 83.2 4.8 

4. 6 Reproducibility 

Six samples were prepared from a controlled test atmosphere at the target concentration at an 

average relative humidity of 78% at 23 °C. The samples were submitted to the OSHA Salt 

Lake Technical Center for analysis, along with a draft copy of this method. The samples were 

analyzed after being stored at 4 °C for 20 days and at-12 °C for an additional 19 days. Sample 

results were corrected for extraction efficiency. No sample result for acetoin or diacetyl had a 

deviation greater than the precision of the overall procedure determined in Section 4.4. 

Table 4.6.1 Table 4.6.2 

Reproducibility Data for Acetoin Ree_roducibilit'!.. Data for Diacet'tf 

theoretical recovered recovery deviation theoretical recovered recovery deviation 

(μglsame_le) (μglsame_le) (%) (%) (gg/same_le) (μg/sam e_le) (%) (%) 

1.62 

1.65 

1.67 

1.66 

1.69 

1.64 

1.59 98.1 -1.9 1.62 1.53 94.4 -5.6 

1.53 92.7 -7.3 1.64 1.48 90.2 -9.8 

1.54 92.2 -7.8 1.60 1.49 92.5 -7.5 

1.56 94.0 -6.0 1.61 1.50 93.2 -6.8 

1.64 97.0 -3.0 1.66 1.53 92.2 -7. 8 

1.51 92.1 -7.9 1.62 1.50 92.6 -7.4 

Samples that are prepared and analyzed by OSHA Method 101337 can be derivatized and reanalyzed by this method to detect lower levels. The following samples were prepared from a 

controlled test atmosphere at 0.51 ppm (0.184 mglm3

) acetoin and 0.50 ppm (0.180 mglm3) 

diacetyl at 74% RH and 24 °C. They were submitted for analysis by OSHA Method 1013 and 

then reanalysis by OSHA Method 1012. The average acetoin recovery of samples analyzed by 

OSHA Method 1013 was 99.3% and by OSHA Method 1012 was 97.1%. The average diacetyl 

recovery of samples analyzed by OSHA Method 1013 was 98.9% and by OSHA Method 1012 

was 96.6%. 

37 Simmons, M., Hendricks, W., Acetoin Diacetyl (OSHA Method 1013), 2008. U.S. Department of Labor, Occupational Safety and 

Health Administration Web site. http:llwww.osha.gov!dts/sltclmethods/validatedl1013!1013.html (accessed 111112008). 

23 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

290 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

4.7 

Table 4.6.3 

Samples for Acetoin Analyzed by OSHA Method 1013 and Then by OSHA Method 1012 

OSHA Method 1013 GC-FID OSHA Method 1012 GC-ECD 

theoretical 

(μglsample) 

16.5 

16.4 

16.6 

15.9 

16.5 

16.3 

recovered 

(μglsample) 

16.4 

16.2 

16.3 

16.1 

16.1 

16.4 

recovery 

(%) 

99.4 

98.8 

98.2 

101.3 

97.6 

100.6 

deviation 

(%) 

-0.6 

-1.2 

-1.8 

+1.3 

-2.4 

+0.6 

Table 4.6.4 

recovered 

(μglsample) 

16.2 

16.0 

16.1 

15.6 

15.8 

15.6 

recovery 

(%) 

98.2 

97.6 

97.0 

98.1 

95.8 

95.7 

deviation 

(%) 

-1.8 

-2.4 

-3.0 

-1.9 

-4.2 

-4.3 

Samples for Diacety/ Analyzed by OSHA Method 1013 and Then by OSHA Method 1012 

OSHA Method 1013 GC-FID OSHA Method 1012 GC-ECD 

theoretical recovered recovery deviation recovered recovery deviation 

(μg!sample) (μg!sample) (%) (%) (μg!sample) (%) (%) 

16.0 15.9 99.4 -0.6 15.6 97.5 -1.8 

15.7 15.4 98.1 -1.9 15.1 96.2 -2.4 

15.8 15.5 98.1 -1.9 15.1 95.6 -3.0 

15.6 15.8 101.3 +1.3 15.2 97.4 -1.9 

15.7 15.2 96.8 -3.2 15.0 95.5 -4.2 

15.9 15.8 99.4 -0.6 15.5 97.5 -4.3 

Sampler capacity 

The sampling capacity of the front tube of two dried silica gel tubes in series was tested by 

sampling from a dynamically generated test atmosphere with an average relative humidity of 

81% at 23°C at concentrations of 0.101 ppm (0.365 mg/m3

) acetoin, and 0.101 ppm (0.355 

mg/m3

) diacetyl. The second tube in the sampling train was changed at 1 h intervals for the 

first 3 hours then at 0. 5 hour intervals for the rest of the sampling. The dried silica gel tube 

sampling trains were used to sample at approximately 0.05 Umin (each air volume listed below 

uses that specific tube's flow rate). The presence of analyte on the second tube was defined 

as breakthrough. The percentage of the amount found on the second tube of the total 

concentration is the % breakthrough. The % breakthrough was plotted versus the air volume 

sampled to determine the 5% breakthrough air volumes. The 5% breakthrough air volume for 

diacetyl was 12. 1 L. The recommended air volume is 80% of the breakthrough air volume 

which is 9. 68 L. Acetoin had no breakthrough after samples were collected for up to 8 hours. 

Table 4. 7.1 

Capacity Test for Diacety/ on Dried Silica Gel Tubes at 0.101 ppm 

sampling train 1 sampling train 2 sampling train 3 

air volume % BT air volume % BT air volume % BT 

2.71 0.0 2.80 0.0 2.78 0.0 

5.51 0.0 5.69 0.0 5.67 0.0 

8.36 0.0 8.64 0.0 8.60 0.0 

9.69 0.0 10.0 0.0 9.97 0.0 

12.0 5.2 12.6 27.8 12.5 20.3 

%BT= % breakthrough 

24 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 291
 

~ 20 

..c: 

g> 

e 

:£ 

"" 

@ 10 CXl 

Diacetyl Capacity at 0.101 ppm 

with a Flow Rate of 0. 05 Umin 

Air Volume (L) 

Figure 4. 7.1. Five percent breakthrough test for 

diacetyl from a 0. 101 ppm atmosphere, with a flow 

rate of 0. 05 Umin. 

A capability of collection at higher flow rates with a 15 minute short term sample was tested for 

breakthrough. A test atmosphere was dynamically generated with an average relative humidity 

of 79% at 23 °C at concentrations of 0.101 ppm (0.365 μglm3

) acetoin and 0.101 ppm (0.355 

mg/m3

) diacetyl. A sampling train consisting of two dried silica gel tubes (4001200 mg) in series 

was used to test the capacity. Three sampling trains at each flow rate of 0.1 Umin or 0.2 Umin 

were tested. There was no acetoin or diacetyl on the second tube of any of the sampling trains. 

Since the short term sampling may be a time of higher exposure, two higher concentrations 

were also tested. The first was 0.541 ppm (1.95 mg/m3

) acetoin and 0.506 ppm (1.78 mg/m2 

diacetyl and a relative humidity of 79% at 23 °C. The second was 23.2 ppm (83.5 mg/m) 

acetoin and 22.4 ppm (78. 8 mg/m3

) diacetyl at an average relative humidity of 79% at 23 °C. In 

all of these tests there was no acetoin or diacetyl on the back-up tube of the sampling train. 

Table 4.7.2 

15 min Capability to Sample at 0. 2 Umin from an Atmosphere of 0.101 ppm 

Acetoin and 0.101 ppm Diacety/ 

flow rate 

(Umin/ 

0.1 

0.1 

0.1 

0.2 

0.2 

0.2 

front tube 

(%) 

98.6 

99.4 

99.9 

99.2 

98.5 

97.7 

back tube 

(%) 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

25 of 34 

diacetvl 

front tube back tube 

(%) (%) 

99.4 0.0 

98.7 0.0 

99.1 0.0 

99.5 0.0 

98.4 0.0 

99.8 0.0 

T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

292 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Table 4.7.3 

15 min Capability to Sample at 0. 2 Umin from an Atmosphere of 0. 541 ppm 

Acetoin and 0.506 ppm Diacety/ 

0.1 

0.1 

0.1 

0.2 

0.2 

0.2 

front tube 

(%) 

99.7 

99.0 

98.8 

99.3 

97.9 

99.5 

back tube 

(%) 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

Table 4. 7.4 

diacetvl 

front tube back tube 

(%) (%) 

99.9 0.0 

98.4 0.0 

97.9 0.0 

99.4 0.0 

98.9 0.0 

99.0 0.0 

15 min Capability to Sample at 0.2 Umin from an Atmosphere of 23.2 ppm Acetoin 

and 22.4 ppm Diacety/ 

flow rate 

(Umin/ 

0.1 

0.1 

0.1 

0.2 

0.2 

0.2 

front tube 

(%) 

98.6 

99.4 

99.0 

99.9 

97.5 

98.1 

back tube 

(%) 

0.0 

0.0 

0.0 

0.0 

0.0 

0.0 

diacetvl 

front tube back tube 

(%) (%) 

99.6 0.0 

98.7 0.0 

97.3 0.0 

99.6 0.0 

99.0 0.0 

97.8 0.0 

A capacity test at 0.2 Umin was performed at two test air concentrations, 0.101 ppm (0.365 

mg/m3

) acetoin and 0.101 ppm (0.355 mg/m3) diacetyl at an average relative humidity of 78% 

air at 22 °C; and 23.2 ppm (83.5 mg/m3

) acetoin and 22.4 ppm (78.8 mg/m3) diacetyl at relative 

humidity of 77% at 22 °C. There was no acetoin on the back-up tube after 13.9 L was sampled. 

The 5% breakthrough air volume for diacetyl with 0.101 ppm atmosphere was 11.98 L, and with 

a 22.4 ppm atmosphere was 11.64 L. 

Table 4.7.5 

Capacity Test for Diacetyl on Dried Silica Gel Tubes 

at a Flow Rate of 0. 2 Umin and 0.101 ppm 

sampling train 1 sampling train 2 sampling train 3 

air volume % BT air volume % BT air volume % BT 

5.98 0.0 5.95 0.0 6.03 0.0 

7.97 0.0 7.94 0.0 8.04 0.0 

9.97 0.0 9.92 0.0 10.05 0.0 

10.96 0.7 10.91 0.0 11.06 1.4 

11.96 5.4 11.90 3.4 12.06 8.8 

12.95 26.4 12.90 22.7 13.07 35.1 

%BT= % breakthrough 

26 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 293
 

~ ..c: 

g> 

e 

:£ 

"" ., 

~ 

CXl 

40 

30 

20 

10 

Table 4.7.6 

Capacity Test for Diacetyl on Dried Silica Gel Tubes 

at a Flow Rate of0.2 Umin and 22.4 ppm 

sampling train 1 sampling train 2 sampling train 3 

air volume % BT air volume % BT air volume % BT 

6.15 0.0 5.94 0.0 6.06 0.0 

8.20 0.0 7.92 0.0 8.08 0.0 

10.25 0.0 9.90 0.0 10.10 0.0 

11.28 2.1 10.89 0.6 11.11 1.2 

12.30 17.2 11.88 5.1 12.12 10.5 

13.33 48.5 12.87 24.1 13.13 40.5 

%BT= % breakthrough 

50 

Diacetyl Capacity at 0.101 ppm Diacetyl Capacity at 22. 4 ppm 

with a Flow Rate of 0. 2 Umin with a Flow Rate of 0. 2 Umin 

40 

~ ..c: 30 g> 

e 

:£ 

"" 20 @ 

CXl 

10 

O"----~~~~~~~~~~""""'----+-~~~----.J 

0 5 10 15 

Air Volume (L) 

Figure 4. 7.2. Five percent breakthrough test for 

diacetyl from a 0. 101 ppm atmosphere, with a flow 

rate of0.2 Umin. 

Air Volume (L) 

Figure 4. 7.3. Five percent breakthrough test for 

diacetyl from a 22.4 ppm atmosphere, with a flow 

rate of 0.2 Umin. 

4. 8 Extraction efficiency and stability of extracted samples 

The extraction efficiency is dependent on the extraction solvent as well as the internal standard. 

The extraction solvent used for this evaluation consisted of 95:5 ethyl alcohol:water with 2 

mg/mL PFBHA and 20 μg/mL 4-bromobenzyl bromide. Other extraction solvents or internal 

standards may be used provided that the new extraction solution or internal standard is tested. 

The new extraction solvent or internal standard should be tested as described below. 

Extraction efficiency 

The extraction efficiencies of acetoin and diacetyl were determined by liquid-spiking four dried 

silica gel tubes, at each concentration level, with the analyte from the RQL to 2 times the target 

concentration. These samples were stored overnight at ambient temperature and then 

analyzed. The samples need to be extracted on a rotator for 1 hour, and then allowed to set at 

room temperature for 36 hours. Do not use a shaker as recoveries will be much lower (Table 

4.8.3). The mean extraction efficiency over the working range from the RQL to 2 times the 

target concentration is 102.0% for acetoin and 97.6% for diacetyl. The extraction efficiency for 

the wet samplers and samplers extracted on the shaker were not included in the overall mean 

because it would bias the results. The test of wet samplers was performed to determine if the 

amount of water that would collect under high humidity conditions at the recommended air 

volume would affect the extraction efficiency. Wet samplers were prepared by sampling humid 

27 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

294 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

air having an average relative humidity of about 80% at 23 °C for 180 minutes at 0. 05 Umin 

and then liquid-spiking the sampler with the analyte. The dried silica gel tube (600 mg) collects 

140 mg water at 78% RH and 23 °C when sampled for 9 L. 

Table 4.8.1 

Extraction Efficiency (%) of Acetoin 

level same.le number mean 

x target μg per 2 3 4 

concn sameJe 

RQL 0.022 104.2 102.1 101.2 101.6 102.3 

0.25 0.41 103.7 102.3 102.1 100.8 102.2 

0.5 0.82 100.7 102.4 101.1 100.9 101.3 

1.0 1.64 102.3 100.5 103.3 103.5 102.4 

1.5 2.46 102.6 103.1 100.6 100.8 101.8 

2.0 3.28 103.0 103.3 101.6 100.4 102.1 

1.0 (wet) 1.64 101.1 102.9 103.1 102.2 102.3 

Table 4.8.2 

Extraction Efficiency (%) of Diacetyl 

level same.le number mean 

x target μg per 2 3 4 

concn sameJe 

RQL 0.02 96.7 95.7 97.8 98.9 97.3 

0.25 0.40 97.5 98.0 99.1 98.5 98.3 

0.5 0.79 98.5 96.8 99.4 98.0 98.2 

1.0 1.58 96.9 95.3 96.4 95.4 96.0 

1.5 2.37 99.9 95.9 96.5 97.8 97.5 

2.0 3.16 97.1 99.6 99.9 97.5 98.5 

1.0 (wet) 1.58 98.1 96.6 95.8 97.1 96.9 

Table 4.8.3 

Extraction Efficiency (%) of Acetoin and Diacetyl at 1. 0 x Target Concentration Using a Shaker 

same.le number 

analyte μg per 2 3 4 mean sam le 

acetoin 1.64 87.5 88.8 90.1 87.7 88.5 

diacetyt 1.58 82.6 81.9 85.5 84.3 83.6 

Stability of extracted samples 

The stability of extracted samples was investigated by reanalyzing the target concentration 

samples 24 h after initial analysis. After the original analysis was performed, two autosampler 

vials were recapped with new septa while the remaining two retained their punctured septa. 

The samples were reanalyzed with fresh standards. The average percent change was +O. 7% 

for acetoin and +1.6% for diacetyl when samples were resealed with new septa and -1.1% for 

acetoin and +0.3% for diacetyl when samples retained their punctured septa. Each septum 

was punctured 5 times for each analysis. The test was performed at room temperature. 

28 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 295
 

Table 4.8.4 

Stability of Extracted Samples for Acetoin 

punctured septa replaced punctured septa retained 

initial after one difference initial after one difference 

(%) day (%) (%) (%) day (%) (%) 

102.3 101.5 -0.8 103.3 101.9 -1.4 

100.5 102.7 +2.2 103.5 102.7 -0.8 

101.4 

(mean) 

102.1 +0.7 103.4 

Table 4.8.5 

(mean) 

102.3 

Stability of Extracted Samples for Diacety/ 

punctured septa replaced punctured septa retained 

-1.1 

initial after one difference initial after one difference 

(%) day (%) (%) (%) day (%) (%) 

96.9 98.3 +1.4 96.4 95.1 -1.3 

95.3 97.1 +1.8 95.4 97.3 +1.9 

96.1 

(mean) 

97.7 +1.6 95.9 

(mean) 

96.2 +0.3 

4.9 Interferences (sampling) 

Retention 

The ability of a dried silica gel tube to retain the analytes after they have been collected was 

tested by using a test atmosphere having an average relative humidity of 80% at 23 °C. The 

test atmosphere was dynamically generated at 0.101 ppm (0.364 mg/m3

) acetoin, and 0.102 

ppm (0.359 mg/m3

) diacetyl. Six samplers had contaminated air drawn through them at 0.05 

Umin for 45 min. Sampling was discontinued and three samples set aside. The generation 

system was flushed with contaminant-free air. Sampling resumed with the other three samples 

having contaminant-free air drawn through them at 0.05 Umin for 135 min and then all six 

samplers were analyzed. The mean recoveries for the samples in the second set divided by 

the first set were: 96. 7% for acetoin, and 96.9% for diacetyl. 

set 

first 

second 

second/first 

Table 4.9.1 

Retention of Acetoin 

percent recovery 

1 2 3 

99.5 100.4 98.9 

95.0 96.8 97.0 

Low humidity 

mean 

99.6 

96.3 

96.7 

set 

first 

second 

second/first 

Table 4.9.2 

Retention of Diacetyl 

percent recovery 

1 2 3 

100.2 99.9 98.1 

96.3 97.4 95.3 

mean 

99.4 

96.3 

96.9 

The ability of dried silica gel tubes to collect the analytes from a relatively dry atmosphere was 

tested by using a test atmosphere having an average relative humidity of 20% at 23 °C. The 

test atmosphere was dynamically generated at 0.101 ppm (0.364 mg/m3

) acetoin and 0.102 

ppm (0.359 mg/m3

) diacetyl. Three samplers had contaminated air drawn through them at 0.05 

Umin for 180 min. All of the samples were immediately analyzed. The recoveries (% of 

theoretical) for acetoin were: 97.0%, 101.4%, and 97.8%; and for diacetyl were: 98.3%, 96.8%, 

and 100.3%. 

29 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

296 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Low concentration 

The ability of dried silica gel tubes to collect the analytes from a low concentration atmosphere 

was tested by using a test atmosphere at 0. 1 times the target concentration having an average 

relative humidity of 80% at 23 °C. The test atmosphere was dynamically generated at 0.0051 

ppm (0.0184 mglm3

) acetoin and 0.0051 ppm (0.0180 mglm3) diacetyl. Three samplers had 

contaminated air drawn through them at 0. 05 Umin for 180 min. All of the samples were 

immediately analyzed. The recoveries (% of theoretical) for acetoin were: 99.8%, 99.9%, and 

97.2%, and fordiacetyl were: 97.3%, 98.1%, and 99.8%. 

Sampling interference 

The ability of dried silica gel tubes to collect the analytes from an atmosphere containing 

interferences was tested under two different sets of conditions. The first set of conditions was a 

test atmosphere of 0.051 ppm (0.0184 mglm3

) acetoin and 0.051 ppm (0.0180 mglm3) diacetyl 

and an interference mixture of 1.01 ppm (1.82 mg/m3

) acetaldehyde, 1.05 ppm (2.58 mg/m3) 

acetic acid, and 1.02 ppm (3.01 mg/m3

) methyl ethyl ketone at an average humidity of 80% at 

23 °C. These lower concentrations were chosen for two reasons: they are similar to some of 

the concentrations found in plants manufacturing microwave popcorn, and all of these 

compounds will be derivatized by the PFBHA; therefore, there would be enough PFBHA in 

solution to derivatize all of the analytes that were collected (8.01 μmole/mL PFBHA). The 

recoveries (% of theoretical) of acetoin and diacetyl were: 95.4%, 98. 5%, and 99. 7% for acetoin 

and 95.8%, 98.9%, and 99.8% for diacetyl. There was no analyte on the backup tube of the 

two dried silica gel tubes in series for any of the tests. 

The second series of tests was with acetoin and diacetyl at the target concentration and each of 

the interferences listed above individually at their PEL concentration following the guidelines in 

SL TC "Evaluation Guidelines for Air Sampling Methods Utilizing Chromatographic Analysis"38• 

The concentrations of these interferences are much higher than would normally be expected in 

a food or flavoring manufacturing workplace. These three compounds were chosen as 

interferences because they collect on the dried silica gel tubes and react with the PFBHA. The 

extraction solution needed to be modified to 18 mg/mL PFBHA (72. 1 μmoles/mL) to insure that 

there was enough PFBHA in solution to derivatize all the analytes. These three atmospheres 

each contained acetoin and diacetr,1 with one of the following concentrations of the interference 

mixture in it: 194 ppm (350 mg/m) acetaldehyde, 9.49 ppm (23.3 mg/m3

) acetic acid, or 190 

ppm (560 mg/m3

) methyl ethyl ketone. Three samplers had contaminated air drawn through 

them at 0. 05 Umin for 180 min for each test. All of the samples were immediately analyzed. 

The recoveries (% of theoretical) of acetoin and diacetyl with 190 ppm acetaldehyde were: 

99.8%, 95.9%, and 97.7% for acetoin and 97.2%, 93.5%, and 95.7% for diacetyl. The 

recoveries (% of theoretical) of acetoin and diacetyl with 9.49 ppm acetic acid were: 95.3%, 

97. 7%, and 98.9% for acetoin and 95.5%, 99.3%, and 99.8% for diacetyl. The recoveries(% of 

theoretical) of acetoin and diacetyl with 190 ppm methyl ethyl ketone were: 96. 7%, 98. 7%, and 

99.9% for acetoin and 95.8%, 97.8%, and 99.3% for diacetyl. There was no analyte found on 

the backup tube of the two dried silica gel tubes in series for any of the tests. These 

interferences were not a sampling interference, but under normal sample analysis, these levels 

of interferences would be an analytical interference. 

Light 

Diacetyl and acetoin are light-sensitive. 39'

40'41'

42 The interference of light during sampling was 

tested using three foil-wrapped sampling trains and three uncovered sampling trains. An 

38 Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M. Evaluation Guidelines For Air Sampling Methods 

Utilizing Chromatographic Analysis, 1999. U.S. Department of Labor, Occupational Safety and Health Administration Web 

site. http://www.osha .govldts/sltclmethods/chromguidelindex.html (accessed 311512008). 39 Material Safety Data Sheet: Acetoin, http:llwww.thegoodscentscompany.com/msds/md102388.html (accessed 311712008). 

30 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 297
 

atmosphere containing twice the target concentration at an average humidity of 78% at 23 °C 

was sampled for 180 min at 0. 05 Umin, and the samples were extracted that day. 

Table 4.9.3 

Ught Interference During Sampling 

acetoin diacetvl 

tube foil wrapped uncovered foil wrapped uncovered 

# recovery (%) recovery (%) recovery (%) recovery (%) 

1 98.9 93.7 97.8 93.3 

2 97.0 92.6 98.9 94.6 

3 99. 5 95.4 99. 9 95. 0 

mean 98.5 93.9 98.9 94.3 

An additional three sampling trains were collected at the same time, and were protected from 

the light by aluminum foil. After collection, these samplers had the foil removed and were 

placed on the counter at ambient temperature under room light. These samples were analyzed 

24 h after sampling during which they were exposed to the room light for 14 of the 24 h, and the 

recoveries were 80. 7%, 84. 7%, and 78.5% for acetoin and 79.3%, 82.4%, and 78.4% for 

diacetyl. 

Powder form 

The powder form of acetoin and diacetyl tested consisted of starch coated with acetoin and 

diacetyl. Three tests were performed on this powder. The first consisted of a sampling train of 

a pre-weighed (tared) PVC filter in a conical cassette in series with two dried silica gel tubes. 

Two dried silica gel tubes were used to collect any vapors of acetoin and diacetyl which would 

be stripped off of the powder. Known amounts of the powder were placed onto the PVC filter, 

and 9 L of air at an average relative humidity of 78% RH and 22 °C were pulled through the 

sampling trains at 0. 05 Umin. The recovery of acetoin and diacetyl on the pre-weighed PVC 

filters was 0% to 1.9% for acetoin and 0% to 2.3% for diacetyl, with larger amounts found on 

the PVC filters that were spiked with larger amounts of powder. Most of the acetoin and 

diacetyl was stripped from the starch and collected on the dried silica gel tubes. The average 

recovery found on the dried silica gel tubes was 96. 6% for acetoin and 97. 8% for diacetyl 

(Table 4.9.4). The acetoin and diacetyl theoretical weights were calculated from the 

percentages obtained from analysis of the powder and the amounts of the powder weighed out. 

The second and third tests consisted of a sampling train of two dried silica gel tubes in series, 

with the powder spiked on the front glass wool of the front tube. The two tests had 9 L air 

drawn through the sampling trains at 0.05 Umin, the first test used air at an average relative 

humidity of 20% at 22 °C, and the other test used air at an average relative humidity of 78% at 

22 °C. At 20% RH most of the acetoin and diacetyl were found on the front glass wool and 

glass fiber filter, but at 78% RH most of the acetoin and diacetyl were found on the dried silica 

gel beds. The sampling trains with 78% RH air drawn through them had the highest amounts 

of acetoin and diacetyl on the glass wool and filter on the tube spiked with the highest amount 

of powder, which may be due to the size of the clump of powder weighed out (Table 4. 9. 5 and 

4.9.6). 

40 Material Safety Data Sheet: Diacetyl, Chemwatch, Victoria, Australia (accesed 311712008). 41 Material Safety Data Sheet: 2,3-Butanedione, https:llfscimage.fishersci.comlmsds/03275.htm (accessed 311712008). 42 Material Safety Data Sheet: 2,3-Butanedione, http:llwww.chemservice.com/msds/msds_detail.asp?catnum=0-816 (accessed 

311712008). 

31of34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

298 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Table 4.9.4 

% Recovery of Acetoin and Diacetyl from Powder on Tared PVC Filters in a Conical Cassette in Series with 

Dried Silica Gel Tubes with 78% RH Air Sam led 

acetoin diacetvt 

amount powder theoretical PVC front back silica gel theoretical PVC filter front back silica gel 

of powder weight weight filter tube tube recovery weight (μg) tube tube recovery 

(μg) found (μg) (μg) (μg) (μg) (%) (μg) (μg) (μg) (%) 

1130 1082 18.1 0.0 18.0 0.0 99.4 29.4 0.0 28.0 0.0 95.2 

2110 2021 33.8 0.6 32.1 0.0 95.0 54.9 1.0 53.1 0.0 96.7 

2960 2856 47.4 0.9 46.3 0.0 97.7 77.0 1.8 75.9 0.0 98.6 

2940 2809 47.0 0.3 45.0 0.0 95.7 76.4 0.8 75.7 0.0 99.1 

1310 1265 21.0 0.2 20.5 0.0 97.6 34.1 0.6 34.0 0.0 99.7 

1010 964 16.2 0.0 15.3 0.0 94.4 26.3 0.0 25.6 0.0 97.3 

Table 4.9.5 

% Recover of Acetoin and Diacet I from Powder S iked on Dried Silica Gel Tubes with 20% RH Air Sam led 

acetoin diacetvt 

amount theoretical front front front back silica gel theoretical front front front back silica gel 

of weight glass glass tube tube recovery weight glass glass tube tube recovery 

powder (μg) wool and wool (μg) (μg) (%) (μg) wool and wool (μg) (μg) (%) 

(μg) filter and filter filter and filter 

(μg) recovery (μg) recovery 

% % 

1080 17.3 16.7 96.5 0.0 0.0 0.0 28.1 26.3 93.6 1.1 0.0 3.9 

1240 19.8 19.5 98.5 0.0 0.0 0.0 32.2 30.1 93.5 1.5 0.0 4.7 

1750 28.0 27.4 97.9 0.0 0.0 0.0 45.5 42.8 94.1 1.8 0.0 4.0 

2080 33.3 32.1 96.4 0.0 0.0 0.0 54.1 50.2 92.8 2.3 0.0 4.3 

2240 35.8 34.5 96.4 0.5 0.0 1.4 58.2 53.4 91.8 2.8 0.0 4.8 

2380 38.1 36.7 96.3 0.7 0.0 1.8 61.9 55.8 90.1 3.6 0.0 5.8 

Table 4.9.6 

% Recover of Acetoin and Diacet I from Powder S iked on Dried Silica Gel Tubes with 78% RH Air Sam led 

acetoin diacetvt 

amount theoretical front front front back silica gel theoretical front front front back silica gel 

of weight glass glass tube tube recovery weight glass glass tube tube recovery 

powder (μg) wool wool and (μg) (μg) (%) (μg) wool and wool (μg) (μg) (%) 

(μg) and filter filter filter and filter 

(μg) recovery (μg) recovery 

(%) (%) 

1220 19.5 0.0 0.0 19.1 0.0 97.9 31.7 0.0 0.0 30.9 0.0 97.5 

1760 28.2 0.0 0.0 26.9 0.0 95.4 45.8 0.0 0.0 44.2 0.0 96.5 

1070 17.1 0.0 0.0 16.9 0.0 98.8 27.8 0.0 0.0 27.5 0.0 98.9 

1590 25.4 0.0 0.0 24.9 0.0 98.0 41.3 0.0 0.0 40.9 0.0 99.0 

2030 32.5 0.0 0.0 32.4 0.0 99.7 52.8 0.0 0.0 52.5 0.0 99.4 

5020 80.3 0.7 0.9 79.4 0.0 98.9 130.5 2.2 1.7 129.9 0.0 99.5 

4. 10 Qualitative analysis 

When necessary, the identity or purity of an analyte peak can be confirmed by GC-mass 

spectrometry or by another analytical procedure. The mass spectra of the acetoin-PFBHA and 

diacetyl-PFBHA derivative were determined by analyzing an analytical standard on an Agilent 

6890 with a 5973 mass selective detector using a 30-m x 0.25-mm i.d. fused silica capillary 

column (DB-1-MS 0.25-μm df) capillary column at a temperature program of 50 °C, hold 2 min, 

program at 10 °C/min up to 180 °C hold 10 min, with injection port at 240 °C and mass 

spectrometer at 250 °C. 

32 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 299
 

10000 

181 

8000 

1l 6000 <:: 

{'l 240 § 

.Q 

<( 4000 

2000 45 58 

,,, 1l 1l 

85 I 99 ;,11 16 195 I ~ I. 282 0 

0 100 200 300 

mlz 

Figure 4.10.1. Mass spectrum of acetoin-PFBHA derivative. 

10000 ~--------------~ 

181 

8000 

Q) 

() 6000 <:: 

(lJ 

'tl 

<:: 

"' .Q 

<( 4000 

2000 

279 446 476 42 99_ 161 195 274 0 IL I I I 

0 100 200 300 400 500 

mlz 

Figure 4.10.2. Mass spectrum of diacetyl-PFBHA derivative. 

4.11 Generation oftest atmospheres 

The test atmosphere of acetoin and diacetyl was generated from a water solution. 

The following apparatus was placed in a walk-in hood. The acetoin and diacetyl vapors were 

generated by pumping the solution, using the lsco pump, through a short length of 0. 53-mm 

uncoated fused silica capillary tubing into a vapor generator where it was heated and 

evaporated into the dilution air stream (Figure 4. 11 ). The vapor generator consisted of a 15-cm 

length of 5-cm diameter glass tubing with a side port for introduction of the capillary tubing. 

The glass tube of the vapor generator was wrapped with heating tape to evaporate the 

chemicals. The humidity, temperature, and volume of the dilution stream of air were regulated 

by use of a Miller Nelson Flow-Temperature-Humidity controller. The test atmosphere passed 

into a glass mixing chamber (76-cm x 30-cm) from the vapor generator, and then into a glass 

exposure chamber (76-cm x 20-cm). Active samplers were attached to glass tubes extending 

from the exposure chamber. The humidity and temperature were measured at the exit of the 

exposure chamber with an Omega Digital Thermo-hygrometer. 

33 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

300 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Generation of test atmospheres required extra heating of the air stream to vaporize the acetoin. 

The temperature and humidity were measured after the air had exited the sampling chamber. 

The air stream cooled as it passed from the mixing chamber to the sampling chamber and then 

out the exit. While the air coming out of the exit was 23 °C and 80% RH, the temperature 

measured in the front of the sampling chamber was 30 °C and 54% RH, giving similar absolute 

humidities of 16.4 mg/L H20. 

exit 

lsco 

pump 

exposure chamber 

active samplers 

vapor 

generator 

Miller Nelson 

mixing 

chamber 

Figure 4.11. The test atmosphere generation and 

sampling apparatus. 

34 of 34 T-1012-FV-01-0811-M 

Appendix B – OSHA 1012 (Acetoin and Diacetyl) 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 301
This page intentionally left blank. 
Appendix C
 

Acetoin Diacetyl 1013
 
 

Method no.: 

Control no.: 

Target concentration: 

OSHA PEL: 

ACGIH TLV: 

Procedure: 

Recommended sampling 

time and sampling rate: 

Reliable quantitation limit: 

Standard error of estimate 

at the target concentration: 

Special requirement: 

Status of method: 

September 2008 

1013 

Aceto in 

Diacetyl 

T-1013-FV-01-0809-M 

0.5 ppm (1.80 mg/m3

) acetoin 

0.5 ppm (1.76 mg/m3

) diacetyl 

none for acetoin 

none for diacetyl 

none for acetoin 

none for diacetyl 

Samples are collected by drawing workplace air through two sampling 

tubes, containing specially dried and cleaned silica gel, connected in 

series. Samples are extracted with ethyl alcohol:water (95:5) and 

analyzed by gas chromatography (GC) using a flame ionization 

detector (FID). 

180 min at 0.05 Umin (9 L) (TWA) 

15 min at 0.2 Umin (3 L) (short term) 

0.011 ppm (0.039 mg/m3

) acetoin 

0.012 ppm (0.041 mg/m3

) diacetyl 

5.7% acetoin 

5.2% diacetyl 

Protect samples from light exposure during sampling, shipping and 

analysis. 

Evaluated method. This method has been subjected to the established 

evaluation procedures of the Methods Development Team. 

Methods Development Team 

Industrial Hygiene Chemistry Division 

OSHA Salt Lake Technical Center 

Sandy UT 84070-6406 

1of25 

Michael Simmons 

Warren Hendricks 

T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

304 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

1 . General Discussion 

For assistance with accessibility problems in using figures and illustrations presented in this method, 

please contact the Salt Lake Technical Center (SL TC) at (801) 233-4900. This procedure was 

designed and tested for internal use by OSHA personnel. Mention of any company name or 

commercial product does not constitute endorsement by OSHA. 

1.1 Background 

1.1.1 History 

In 2003 OSHA issued Method PV21181 for sampling and analysis of diacetyl using two 

silica gel sorbent tubes (150/75 mg) in series. PV2118 has a recommended sampling 

volume of 3 L and a reliable quantitation limit of 3 ~g (0.28 ppm). In 2003 NIOSH 

issued Method 25572 for diacetyl and Method 2558 for acetoin. Both methods use 

Anasorb CMS sorbent (150/75 mg) tubes, can sample up to 10 L of air and have a limit 

of detection for acetoin of 1 μg and 0.6 μg for diacetyl. These two methods use slightly 

different acetone/methanol extraction solvents and were not optimized for simultaneous 

analysis of both analytes. In 2008 a note was placed on NIOSH Method 2557 

indicating that high humidity is a sampling interference that results in underestimation of 

the true concentration. 

In September of 2007, OSHA published a Hazard Communication Guidance 

Document4 and a Safety and Health Information Bulletin on Respiratory Disease 

among Employees in Microwave Popcorn Processing Plants5 for diacetyl. Due to the 

increasing concern of workplace exposure to diacetyl, two new sampling and analytical 

methods were validated that permitted longer sampling times and had lower 

quantitation limits than PV2118. The new methods were also validated for acetoin 

because it has been found in facilities in which diacetyl was in use. 

This procedure, Method 1013, was streamlined for monitoring low ppm levels, and 

Method 10126 was optimized for ppb levels. Both methods use two 600 mg silica gel 

sorbent tubes in series. Both methods have a recommended sampling time of 3 hours 

(9 L) and both use the same solvent for sample extraction. However, in Method 1012, 

acetoin and diacetyl are derivatized using 0-pentafluorobenzyl hydroxylamine 

hydrochloride. This derivatization results in a reliable quantitation limit approximately 

10 times less than Method 1013. The disadvantage of derivatizing acetoin and diacetyl 

is that the derivatization step requires 36 hours; whereas, with this method sample 

preparation can be performed in 1 hour. Also, samples extracted and analyzed 

according to this procedure can then be derivatized and analyzed using Method 1012, if 

needed. 

The silica gel used in the sampler for this method, and for Method 1012, has been 

specially cleaned and dried as described in Appendix A. It was found that sampler 

1 Shah, Y. C. Diacetyl (OSHA Method PV2118), 2003. U.S. Department of Labor, Occupational Safety and Health Administration 

Web site. http://www.osha.gov/dts/sltc/methods/partial/t-pv2118/t-pv2118.html (accessed July 2008). 2 Pendergrass, S. M. Diacetyl (NIOSH Method 2557), 2003. Centers for Disease Control and Prevention, National Institute of 

Occupational Safety and Health Web site. http://www.cdc.gov/niosh/nmam/pdfs/2557.pdf (accessed July 2008). 3 Pendergrass, S. M. Acetoin (NIOSH Method 2558), 2003. Centers for Disease Control and Prevention, National Institute of 

Occupational Safety and Health Web Site. http://www.cdc.gov/niosh/nmam/pdfs/2558.pdf (accessed July 2008). 4 Hazard Communication Guidance for Diacetyl and Food Flavorings Containing Diacetyl, 2007. U.S. Department of Labor, 

Occupational Safety and Health Administration Web site. http://www.osha.gov/dsg/guidance/diacetyl-guidance.html 

(accessed July 2008). 5 Respiratory Disease Among Employees in Microwave Popcorn Processing Plants, 2007. U.S. Department of Labor, Occupational 

Safety and Health Administration Web site. http://www.osha.gov/dts/shib/shib092107.html (accessed July 2008). 6 Eide, M. Acetoin and Diacetyl (OSHA Method 1012), 2008. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http://www.osha.gov/dts/sltc/methods/validated/1012/1012.html (accessed September 2008). 

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capacity for diacetyl was not based on analyte concentration but limited by the amount 

of water remaining on the silica gel after cleanup and on the amount of water collected 

during sampling. In other words, the silica gel tube acts as a chromatography column 

and water elutes the collected diacetyl. By removing as much water as possible from 

the silica gel prior to sampling, the sampling volume for diacetyl can be increased 

because the time required to saturate the silica gel during sampling increases. Diacetyl 

was also found to gradually migrate within the sampling tube during storage resulting in 

the need to use a second tube in series during sampling in order to detect 

breakthrough. Acetoin has no capacity or migration issues on silica gel at the 

recommended sampling volume. 

The powder and liquid formulated forms of acetoin and diacetyl may contain oily 

compounds and other base materials such as maltodrextin. These materials could 

affect the extraction of acetoin and diacetyl from the silica gel. The sampler contains a 

front glass wool plug followed by a glass fiber filter that serves only to trap any of these 

materials before they enter the silica gel bed. Retention studies using a powder 

containing acetoin and diacetyl showed the acetoin and diacetyl can be stripped off the 

powder and collected on the silica gel. These studies demonstrate that the glass fiber 

filter is not an efficient collector for diacetyl and acetoin, and will not normally be 

analyzed (see OSHA Method 10127

, Section 4.9). 

1.1.2 Toxic effects (This section is for information only and should not be taken as the basis 

of OSHA policy.) 

Exposure to acetoin may result in skin, eyes, nose and throat irritation. 8 

Exposure to diacetyl "liquid or vapors can cause irritation to the skin, eyes, nose, and 

throat". "Animals exposed to diacetyl experienced damage to the nose and upper 

airways, including severe damage to cells lining the respiratory tract" and "NIOSH has 

reported that employees exposed to butter flavorings containing diacetyl are at risk of 

developing occupational lung diseases". 9 

Diacetyl, and to some extent acetoin, may be responsible for the occurrence of a rare 

and potentially fatal lung disease, bronchiolitis obliterans, among workers in microwave 

popcorn manufacturing plants and flavor manufacturing plants. 10 Symptoms of 

bronchiolitis obliterans include cough, shortness of breath with exertion, and spirometry 

test results showing fixed airways obstruction. 11 

Aceto in and diacetyl are used in the production of powdered flavorings. 12 These 

powdered flavorings may provide a means to deliver the substances deep into the 

lungs of exposed workers, however, the significance of this form of exposure is 

presently unknown. 13 

7 Eide, M. Acetoin and Diacetyl (OSHA Method 1012), 2008. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http://www.osha.gov/dts/sltc/methods/validated/1012/1012.html (accessed September 2008). 8 Acetyl Methyl Carbinol (Chemical Sampling Information), 2007. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http://www.osha.gov/dts/chemicalsampling/data/CH 21701 O.html (accessed July 2008). 9 Hazard Communication Guidance for Diacetyl and Food Flavorings Containing Diacetyl, 2007. U.S. Department of Labor, 

Occupational Safety and Health Administration Web site. http://www.osha.gov/dsg/guidance/diacetyl-guidance.html 

(accessed July 2008). 10 van Rooy, F.; et al. Bronchiolitis Obliterans Syndrome in Chemical Workers Producing Diacetyl for Food Flavoring. Am. J. Grit. 

Care Med. 2007, 176 (5), 498-504. 11 Kanwal, R. Bronchiolitis obliterans in workers exposed to flavoring chemicals. Curr Opin Pulm Med. 2008, 14 (2), 141-6. 12 Kanwal, R.; Kullman, G. Report on Severe Fixed Obstructive Lung Disease in Workers at a Flavoring Manufacturing Plant Health 

Hazard Evaluation Report #2006-0303-3043, 2007. Centers for Disease Control and Prevention, National Institute of 

Occupational Safety and Health Web site. http://www.cdc.gov/niosh/hhe/reports/pdfs/2006-0303-3043.pdf (accessed July 

2008) pp 11-13. 13 Boylstein, R. J.; et al. Diacetyl Emissions and Airborne Dust from Butter Flavorings Used in Microwave Popcorn Production. J. 

Occup. Environ. Hyg. 2006, 3 (10), 530-535. 

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306 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

1.1.3 Workplace exposure 

Acetoin has a somewhat creamy taste and a woody yogurt odor. It is used as an 

ingredient in yogurt, butter, milk and strawberry flavors. It occurs naturally in foods 

such as wines, chesses, fruits and vegetables. 14 Occupational exposures can occur by 

inhalation or skin contact in locations where it is produced, used as a food additive, or 

used to produce flavorings or aromas. 

Diacetyl has a strong butter odor in dilute form and a chlorine-quinone odor when 

concentrated. It is used as an ingredient to produce a butter flavor in many foods and 

beverages. It occurs naturally in alcoholic and nonalcoholic beverages, dairy products, 

fruits, plants, vegetables, meats, and natural aromas. 15 Like acetoin, occupational 

exposures to diacetyl can occur by inhalation or skin contact in locations where it is 

produced, used as a food additive, or used to produce flavorings or aromas. 

Recently, occupational exposure to butter flavorings in the production of microwave 

popcorn and in other industries has received much publicity. NIOSH has identified 

acetoin and diacetyl as useful indicator compounds that can be used to represent 

exposure to butter flavorings. 16 Areas of special concern include flavor production 

rooms, areas where mixing/blending operations occur, packing/packaging operations, 

areas where flavors are handled openly, rooms where mixing tanks are located, quality 

control laboratories, and maintenance and cleaning operations. 17 ' 

18 

1.1.4 Physical properties and other descriptive information 

Acetoin 19 ' 20 

Acetoin occurs as the liquid monomer and the solid dimer. The monomer can be 

formed from the dimer by dissolving in water or other solvents. 

synonyms: 

IMIS21 : 

CAS number: 

boiling point: 

melting point: 

density: 

molecular weight: 

flash point: 

appearance: 

molecular formula: 

acetyl methyl carbinol; 2,3-butanolone; dimethylketol; yhydroxy-f3-oxobutane; 1-hydroxyethyl methyl ketone 

A624 

513-86-0 (monomer) 

148 °C (298 °f) @ 760 mmHg (monomer) 

15 °C (59 °f) (monomer); 91 °C (196 °f) (dimer) 

1.005 (g/ml@ 25 °C ) (monomer) 

88.11 (monomer) 

46.7 °C (116 °f) (closed cup) (monomer) 

Pale yellow to colorless as liquid or solid 

C4H80 2 (monomer); C8H160 4 (dimer) 

14 Burdock, G. A. Fenaroli's Handbook of Flavor Ingredients, 5th ed.; CRC Press: Boca Raton, FL, 2005; pp 11-12. 15 Burdock, G. A. Fenaroli's Handbook of Flavor Ingredients, 5th ed.; CRC Press: Boca Raton, FL, 2005; pp 411-412. 16 Kanwal, R.; Boylstein, R. J.; Piacitelli, C. NIOSH Health Hazard Evaluation Report #2001-0474-2943, 2004. Centers for Disease 

Control and Prevention, National Institute of Occupational Safety and Health Web site. 

http://www.cdc.gov/niosh/hhe/reports/pdfs/2001-047 4-2943.pdf (accessed July 2008) pp 8-9. 17 Kanwal, R. Bronchiolitis obliterans in workers exposed to flavoring chemicals. Curr Opin Pulm Med. 2008, 14 (2), 141-6. 18 Kreiss, K. Flavoring-related bronchiolitis obliterans. Curr Opin Allergy Clin lmmunol 2007, 7 (2), 162-167. 19 The Merck Index; 1ih ed.; Budavari, S., Ed.; Merck & Co. Inc.: Whitehouse Station, NJ, 1996; p 12. 20 Material Safety Data Sheet: Acetoin, 2008. The Good Scents Company Web site. 

http://www.thegoodscentscompany.com/msds/md102388.html (accessed July 2008). 21 Acetyl Methyl Carbinol (Chemical Sampling Information), 2007. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http://www.osha.gov/dts/chemicalsampling/data/CH 21701 O.html (accessed June 2008). 

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solubility: 

structural formula: 

Diacetvl 22 '

23 

synonyms: 

IMIS24 : 

CAS number: 

boiling point: 

melting point: 

density: 

molecular weight: 

vapor pressure: 

flash point: 

appearance: 

vapor density: 

molecular formula: 

odor: 

solubility: 

autoignition 

temperature: 

structural formula: 

miscible with water and alcohol; sparingly soluble in ether 

and petroleum ether 

biacetyl; 2,3-butanedione; 2,3-butadione; 2,3-diketobutane; 

dimethyl diketone; dimethylglyoxal; glyoxal, dimethyl-; 

2,3-diketobutane 

0740 

431-03-8 

88 oc (190 Of) 

3-4°C (37.4-39.2 °f) 

0.99 (g/ml@ 15/15) 

86.09 

7 kPa@20°C 

26.7 °C (80 °f) (closed cup) 

yellow to yellow-green liquid 

3(air=1) 

C4H502 

quinone odor in higher concentrations, butter in lower 

concentrations 

4 parts water; miscible with alcohol, ether 

285 oc (545 Of) 

22 The Merck Index; 1 ih ed.; Budavari, S., Ed.; Merck & Co. Inc.: Whitehouse Station, NJ, 1996; p 503. 23 Material Safety Data Sheet: Diacetyl, 2007. Chemwatch; Victoria, Australia (accessed March 2008). 24 Diacetyl (Chemical Sampling Information), 2007. U.S. Department of Labor, Occupational Safety and Health Administration Web 

site. http://www.osha.gov/dts/chemicalsampling/data/CH 23171 O.html (accessed 2008). 

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308 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

This method was evaluated according to the OSHA SL TC "Evaluation Guidelines for Air Sampling 

Methods Utilizing Chromatographic Analysis" 25 • The Guidelines define analytical parameters, specify 

required laboratory tests, statistical calculations and acceptance criteria. The analyte air concentrations 

throughout this method are based on the recommended sampling and analytical parameters. Air 

concentrations in ppm are referenced to 25 °C and 101.3 kPa (760 mmHg). 

1 .2 Limit defining parameters 

1.2.1 Detection limit of the analytical procedure 

The detection limit of the analytical procedure is 0.017 ng for acetoin and 0.033 ng for 

diacetyl. These are the amount of analytes that will give a detector response that is 

significantly different from the response of a calibration blank. (Section 4.1) 

1.2.2 Detection limit of the overall procedure 

The detection limit of the overall procedure for acetoin is 0.10 μg per sample (0.0031 

ppm or 0.011 mg/m3

) and 0.11 μg per sample for diacetyl (0.0034 ppm or 0.012 

mg/m3

). These are the amounts spiked onto the sampler that will give a detector 

response that is significantly different from the response of a sampler blank. (Section 

4.2) 

1.2.3 Reliable quantitation limit 

The reliable quantitation limit for acetoin is 0.35 μg per sample (0.011 ppm or 0.039 

mg/m3 for a TWA sample) and 0.37 μg per sample for diacetyl (0.012 ppm or 0.041 

mg/m3 for a TWA sample). These are the amounts spiked onto the sampler that will 

give a detector response that is considered the lower limit for precise quantitative 

measurements. (Section 4.2) 

1.2.4 Instrument calibration 

The standard error of estimate is 0.42 μg for acetoin over the range of 3.73 μg to 31.0 

μg. The standard error of estimate is 0.82 μg for diacetyl over the range of 3.58 μg to 

29.9 μg. These ranges correspond to approximately 0.25 to 2 times the target 

concentration. (Section 4.3) 

1.2.5 Precision 

The precision of the overall procedure at the 95% confidence level for the ambient 

temperature 18-day storage test (at the target concentration) is ±11.2% for acetoin and 

±10.1 % for diacetyl. These include an additional 5% for sampling pump variability. 

(Section 4.4) 

1.2.6 Recovery 

The recovery from samples used in a 18-day storage test remained above 88.5% for 

acetoin and 102. 7% for diacetyl when the samples were stored at ambient temperature. 

(Section 4.5) 

25 Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M. C. Evaluation Guidelines For Air Sampling Methods 

Utilizing Chromatographic Analysis, 1999. U.S. Department of Labor, Occupational Safety and Health Administration 

Web site. http://www.osha.gov/dts/sltc/methods/chromguide/chromguide.pdf (accessed November 2007). 

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1.2.7 Reproducibility 

Six samples collected from a controlled test atmosphere were submitted for analysis by 

the OSHA Salt Lake Technical Center. The samples were analyzed according to a 

draft copy of this procedure after 20 days of storage at refrigerated temperature. No 

individual sample result deviated from its theoretical value by more than the precision 

reported in Section 1 .2.5. (Section 4.6) 

2. Sampling Procedure 

All safety practices that apply to the work area being sampled should be followed. The sampling 

equipment should be attached to the worker in such a manner that it will not interfere with work 

performance or safety. 

2.1 Apparatus 

Sampler: glass tube with both ends flame sealed, 110-mm x 7-mm i.d., containing a glass fiber 

filter and 1 section of 20140 mesh silica gel. From front to back, the sampling tube consists of a 

silane-treated glass wool plug, a glass fiber filter to collect particulate, 600 mg of silica gel and 

a second plug of silane-treated glass wool. The silica gel should be cleaned and dried as 

described in Appendix A. Sampling tubes are available for purchase through SKC, Inc. (cat. no. 

226-183). 

Samples are collected using a personal sampling pump calibrated, with the sampling device 

attached, to within ±5% of the recommended flow rate. 

Use aluminum foil or a tube cover, such as SKC, Inc Tube Cover D (cat. no. 224-290), to 

protect samples from light. 

2.2 Reagents 

None required 

2.3 Technique 

Immediately before sampling, break the ends off of two flame-sealed glass tubes to provide an 

opening approximately half the internal diameter of the tube. Wear eye protection when 

breaking ends. Use a tube holder to minimize the hazard of broken glass and to protect 

samplers from light exposure during sampling. All tubes should be from the same lot. 

Connect the two silica gel sampling tubes in series, using the least amount of flexible tubing as 

possible between the sampling tubes, and then connect to a sampling pump with flexible tubing. 

The filter in the silica gel tubes should be positioned away from the sampling pump. The tube 

closer to the pump is used as a backup. Use a tube cover or wrap sampling tubes in aluminum 

foil to insure that both sampling tubes are protected from light exposure. Place the sampling 

tubes in a vertical position with the inlet in the breathing zone and position the sampling pump 

and tubing so they do not impede work performance or safety. 

Draw air directly into the inlet of the sampler. The air being sampled should not pass through 

any hose or tubing before entering the sampler. 

After sampling for the appropriate time, disconnect the tubes from the pump tubing and seal 

each tube with plastic end caps. Separately wrap each tube in aluminum foil and seal end-to 

end with a Form OSHA-21. 

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310 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Submit at least one blank sample with each set of samples. Handle the blank sample in the 

same manner as the other samples except draw no air through it. 

Record sample air volume (L), sampling time (min) and sampling rate (L/min) for each sample, 

along with any potential interferences on the Form OSHA-91A. 

Submit the samples to the laboratory for analysis as soon as possible after sampling. If a delay 

is unavoidable, store the samples in a refrigerator. Ship any bulk samples separate from the air 

samples. 

2.4 Sampler capacity (Section 4.7) 

The sampling capacity of the front tube was tested by sampling a dynamically generated test 

atmosphere of acetoin (3.58 mg/m3 or 0.99 ppm) and diacetyl (3.55 mg/m3 or 1.01 ppm) with an 

average relative humidity of 40% at 34 °C (absolute humidity of 14.8 mg/L H20). The samples 

were collected at a sampling rate of approximately 0.05 L/min for 270 min. The 5% 

breakthrough sampling time was determined to be 248 min for diacetyl. No breakthrough was 

observed for acetoin. (Note: In order to volatilize acetoin the test atmosphere generation 

conditions were modified slightly for this method evaluation as described in the second 

paragraph of Section 4.11.) 

2.5 Extraction efficiency (Section 4.8) 

It is the responsibility of each analytical laboratory to determine the extraction efficiency 

because the adsorbent material, reagents and laboratory techniques may be different than 

those listed in this evaluation and influence the results. 

The mean extraction efficiency for acetoin from dry silica gel over the range of RQL to 2 times 

the target concentration (0.33 to 31.0 μg per sample) was 92.9%. The extraction efficiency was 

not affected by the presence of water. 

The mean extraction efficiency for diacetyl from dry silica gel over the range of RQL to 2 times 

the target concentration (0.38 to 29.9 μg per sample) was 99.6%. The extraction efficiency was 

not affected by the presence of water. 

Extracted samples remain stable for at least 72 hr. 

2.6 Recommended sampling time and sampling rate 

Sample for up to 180 min at 0.05 L/min (9 L) to collect TWA (long-term) samples. 

Sample for up to 15 min at 0.2 L/min (3 L) to collect short-term samples. 

When short-term samples are collected, the air concentration equivalent to the reliable 

quantitation limit becomes larger. For example, the reliable quantitation limit is 0.032 ppm (0.12 

mg/m3

) for acetoin and 0.035 ppm (0.12 mg/m3) for diacetyl when 3 Lare collected. 

2.7 Interferences, sampling (Section 4.9) 

Retention efficiency 

The retention efficiency for all samples was 100.6% of theoretical for acetoin and 96.6% for 

diacetyl, when samplers containing approximately 8.3 μg of acetoin and 8.1 μg of diacetyl were 

allowed to sample 6.75 L of contaminant-free air having an average relative humidity of 40% at 

35 °C (absolute humidity of 15.6 mg/L H20). Samples were collected at a sampling rate of 0.05 

L/min. 

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 311
 

Low humidity 

The collection efficiency for all samples was 100.7% of theoretical for acetoin and 101.5% for 

diacetyl, when the samplers were used to sample a test atmosphere containing two times the 

target concentration having an average relative humidity of 8% at 33 °C (absolute humidity of 

2.82 mg/L H20). Samples were collected at a sampling rate of 0.05 Umin for 180 min. 

Low concentration 

The collection efficiency for all samples was 91.8% of theoretical for acetoin and 95.6% for 

diacetyl, when the samplers were used to sample a test atmosphere containing approximately 

0.1 times the target concentration having an average relative humidity of 42% at 33 °C 

(absolute humidity of 14.8 mg/L H20). Samples were collected at a sampling rate of 0.05 

Umin for 180 min. 

The collection efficiency for all samples when taking short term samples was 106% of 

theoretical for acetoin and 90.6% for diacetyl, when the samplers were used to sample a test 

atmosphere containing approximately 0.1 times the target concentration having an average 

relative humidity of 42% at 33 °C (absolute humidity of 14.8 mg/L H20). Samples were 

collected at a sampling rate of0.2 Umin for 15 min. 

Sampling interference 

The collection efficiency for all samples was 95.5% of theoretical for acetoin and 101.8% for 

diacetyl, when the sampler was used to sample a test atmosphere containing approximately 

one times the target concentration of acetoin and diacetyl and 2.59 mg/m3 of 2-nonanone and 

1.88 mg/m3 of 2,3-pentanedione. The test atmosphere had an average relative humidity of 

38% at 34 °C (absolute humidity of 14.1 mg/L H20). Samples were collected at a sampling rate 

of 0.05 Umin for 181 min. 

Sampler exposure to light, particularly sunlight, during sampling will result in degradation of 

both acetoin and diacetyl. The recovery for all samples was 67 .0% of theoretical for acetoin and 

6.43% for diacetyl, when the sampler was used to sample a test atmosphere containing 

approximately one times the target concentration of acetoin and diacetyl and then exposed to 3 

h of direct sunlight (samples were covered during sampling). The test atmosphere had an 

average relative humidity of 40% at 35 °C (absolute humidity of 15.6 mg/L H20). Samples were 

collected at a sampling rate of 0.05 Umin for 180 min. See Section 4.9 for data on other light 

tests performed. 

3. Analytical Procedure 

Adhere to the rules set down in your Chemical Hygiene Plan26 . Avoid skin contact and inhalation of all 

chemicals and review all appropriate MSDSs. 

3.1 Apparatus 

A gas chromatograph equipped with an FID. For this evaluation an Agilent Technologies 6890 

Plus Gas Chromatograph equipped with a 7683 Automatic Sampler and an Agilent tapered, 

deactivated, split, low pressure drop liner with glass wool (catalog no. 5183-4647). 

A GC column capable of separating acetoin and diacetyl from the desorption solvent, internal 

standard and any potential interferences. A Restek 60-m x 0.32-mm i.d. Rix-Volatiles (1.5-μm 

df) capillary column was used in this evaluation. 

26 Occupational Exposure to Hazardous Chemicals in Laboratories. Code of Federal Regulations, Part 1910.1450, Title 29, 2003. 

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312 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

An electronic integrator or other suitable means of measuring GC detector response. Waters 

Empower 2 Data System was used in this evaluation. 

A dispenser capable of delivering 2.0 ml of desorbing solvent to prepare standards and 

samples. If a dispenser is not available, a 2.0-ml volumetric pipet can be used. 

Amber glass vials with PTFE-lined caps. For this evaluation 2 and 4-ml vials were used. 

Calibrated 10-μl and 25-μl syringes for preparing standards. 

Water purifier. A Barnstead NANOpure Diamond system was used to produce 18.0 Mn-cm DI 

water in this evaluation. 

Water bath. A Precision Scientific (5 - 100 °C range) water bath was used in this evaluation. 

A mechanical rotator. A Fisher Roto-Rack was used in this evaluation. 

Class A 1-l volumetric flasks. 

Class A 1-ml and 5-ml volumetric pipets. 

3.2 Reagents and Standards 

Acetoin (C4H80 2), [CAS no. 513-86-0]. The acetoin (lot no. 05025DH) used in this evaluation 

was purchased from Sigma Aldrich (Milwaukee, WI). 

Diacetyl (C4H60 2), [CAS no. 431-03-8]. The diacetyl used in this evaluation was 97+% (lot no. 

17823lD) purchased from Sigma Aldrich (Milwaukee, WI). 

DI water, 18.0 Mn-cm. 

Ethyl Alcohol [CAS no. 64-17-5]. The ethyl alcohol used in this evaluation was 95% v/v (190 

proof) A.C.S. spectrophotometric grade (lot no. 80513920) purchased from Acros Organics 

(Morris Plains, NJ). 

3-Pentanone [Cas no. 96-22-0]. The 3-pentanone used in this evaluation was 99+% (lot no. 

HR 00231 KF) purchased from Aldrich (Milwaukee, WI). 

The extraction solvent used for this evaluation consisted of 0.007 μL/ml 3-pentanone in 95% 

v/v ethyl alcohol. The 3-pentanone was added to the ethyl alcohol as an internal standard 

(ISTD). 

3.3 Standard preparation 

Prepare a concentrated stock standard of acetoin and diacetyl in 18.0 Mn-cm DI water and 

store in an amber vial or bottle. (Note: Acetoin is usually obtained as the solid dimmer and will 

convert back to the monomer when dissolved in water.) Acetoin will slowly dissolve in water, 

however, this process can be accelerated by placing the solution in a 60 °C water bath for 10 

min. Refrigerate the stock standard when not in use and remake once a month. 

Prepare working analytical standards by injecting micro liter amounts of the concentrated stock 

standard into amber 4-ml vials containing 2 ml of the extraction solvent delivered by the same 

dispenser used to extract samples. For example, to prepare a target level standard (16.25 

μg/sample acetoin and 15.86 μg/sample diacetyl) , inject 13 μl of a stock standard containing 

1 .25 μg/μl acetoin and 1.22 μg/μl diacetyl into 2-ml of extraction solvent. Transfer working 

standards to 2-ml amber glass autosampler vials. 

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Occupational Exposure to Diacetyl and 2,3-Pentanedione 313
 

Bracket sample concentrations with standard concentrations. If upon analysis, sample 

concentrations fall outside the range of prepared standards, prepare and analyze additional 

standards to confirm instrument response, or dilute high samples with extraction solvent and 

reanalyze the diluted samples. 

3.4 Sample preparation 

Remove the plastic end caps from the front sample tube and carefully transfer the silica gel to a 

4-ml amber glass vial. The sampling tube and the back of the glass fiber filter should be 

carefully inspected to insure that all the silica gel is transferred into the 4-ml vial. Remove the 

plastic end caps from the backup tube and carefully transfer the silica gel to a second 4-ml 

amber glass vial. If the industrial hygienist requests analysis of the front glass fiber filter, which 

is not normally analyzed, place the front glass wool plug and filter from the front tube into a third 

4-ml vial. If analysis of filter is not requested then discard the front glass wool plug and filter. 

Discard the glass tubes and back glass wool plugs and back glass fiber filter. 

Add 2.0 ml of extraction solution to each vial and immediately seal with PTFE-lined caps. 

Note: The use of an extraction solution or internal standard other than that specified in Section 

3.2 should not be used unless a full extraction efficiency study is performed using both dry and 

wet media as described in Section 4.8. 

Place the 4-ml vials on a mechanical rotator and rotate at approximately 40 rpm for 60 min. 

Transfer the extraction solution in each 4-ml vial to a 2-ml amber glass autosampler vial and 

seal with a PTFE-lined cap. 

Analyze samples for acetoin and diacetyl as described in Section 3.5. 

Note: If after analysis lower detection limits are needed samples can be derivatized and 

analyzed according to Section 3.4 of OSHA Method 101227 . 

3.5 Analysis 

3.5.1 Analytical conditions 

GC conditions 

column 

temperature: Initial 60 °C, hold 4 min; ramp at 15 °C/min to 135 °C, hold 0 min; 

ramp at 60 °C/min to 250 °C, hold 4 min 

zone 

temperatures: 240 °C (injector); 250 °C (detector) 

run time: 14.75 min 

column mode: constant pressure 

column 

pressure: 

initial column 

gas flow: 

injection size: 

14 psi 

3.3 ml/min (hydrogen) 

1.0 μL (2:1 split) 

27 Eide, M. Acetoin and Diacetyl (OSHA Method 1012), 2008. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http://www.osha.gov/dts/sltc/methods/validated/1012/1012.html (accessed September 2008). 

11of25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

314 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

50000 

(j) 40000 

:> 

2' 

.l!l 30000 c 

:J 

0 

() 

"' 20000 Q) 

.;c 

10000 

0 

0 

column: Restek 60-m x 0.32-mm i.d. capillary Rix-Volatiles (df = 1.5-μm) or 

equivalent 

inlet liner: Agilent 5183-464 7 or equivalent 

retention times: 5.2 min (diacetyl) 

FID conditions 

hydrogen flow: 

air flow: 

nitrogen 

makeup flow: 

> 5 

Q) 

:!! 

0 

c. 

"' Q) 

Cl:'. 

25 

20 

15 

10 

5 

8.1 min (acetoin) 

7 .5 min (ISTD) 

40 ml/min 

450 ml/min 

45 ml/min 

0 

~ 

~ 

" 0 "' 

Time (min) 

0 

f-

~ 

~ 

~ ::J 

~ 

Figure 3.5.1. Chromatogram obtained at target 

concentrations with recommended conditions. 

3.5.2 Calibration 

An internal standard calibration method is used. A calibration curve can be constructed 

by plotting !STD-corrected response of standard injections versus micrograms of 

analyte per sample. Bracket the samples with freshly prepared analytical standards 

over the range of concentrations. 

40000 

30000 

(j) 

], 

.l!l 

c 

:J 20000 0 

() 

"' ~ <( 

10000 

0 

10 20 30 0 10 20 

Mass (μg acetoin) per sample Mass (μg diacetyl) per sample 

Figure 3.5.2.1. Calibration curve of Figure 3.5.2.2. Calibration curve of 

acetoin. (Y = 1678X- 389) diacetyl. (Y = 1320X - 625) 

12 of 25 T-1013-FV-01-0809-M 

30 

Appendix C – Acetoin Diacetyl 1013
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 315
 

3.6 Interferences (analytical) 

3.6.1 Any compound that produces an FID response and has a similar retention time as the 

analytes or internal standard is a potential interference. If any potential interferences 

were reported, they should be considered before samples are extracted. Generally, 

chromatographic conditions can be altered to separate an interference from the 

analyte. 

3.6.2 When necessary, the identity of an analyte peak may be confirmed with additional 

analytical data (Section 4.12). 

3.7 Calculations 

The amount of analyte per sampler is obtained from the appropriate calibration curve in terms 

of micrograms per sample, uncorrected for extraction efficiency. The back tube is analyzed 

primarily to determine the extent of sampler saturation. If any analyte is found on the back tube, 

it is added to the amount on the front tube. This total amount is then corrected by subtracting 

the total amount (if any) found on the blank. The air concentration is calculated using the 

following formulas. 

Total micrograms per sample of analyte is 

M = [Mrront - Mbtank] + [Mback - Mbtank] 

Concentration by weight of analyte (mg/m3

) is 

M 

CM=-­

EEV 

Concentration by volume of analyte (ppm) is 

4. Backup data 

where 

M is total μg per sample 

Mrront is total μg found on front tube 

Mback is total μg found on back tube 

Mbtank is total μg found on blank tube 

where 

CM is concentration by weight (mg/m3

) 

M is total μg per sample 

EE is extraction efficiency in decimal form 

V is L of air sampled 

where 

Cv is concentration by volume (ppm) 

CM is concentration by weight (mg/m3

) 

VM is molar volume at NTP (24.46 L/mole) 

Mr is molecular weight (88.1 for acetoin, 86.09 

for diacetyl) 

General background information about the determination of detection limits and prec1s1on of the 

overall procedure is found in the "Evaluation Guidelines for Air Sampling Methods Utilizing 

13 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

316 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Chromatography Analysis" 28 . The Guidelines define analytical parameters, specify required 

laboratory tests, statistical calculations and acceptance criteria. 

4.1 Detection limit of the analytical procedure (DLAP) 

The DLAP is measured as mass of analyte introduced onto the chromatographic column. Ten 

analytical standards were prepared with equally descending increments with the highest 

standard containing 1.1 O μg/sample acetoin and 1 .05 μg/sample diacetyl. This is the 

concentration that would produce a peak approximately 10 times the response of a calibration 

blank. These standards, and the calibration blank were analyzed with the recommended 

analytical parameters (1-μL injection with a 2:1 spit), and the data obtained were used to 

determine the required parameters (standard error of estimate and slope) for the calculation of 

the DLAP. For acetoin values of 5171 and 30 were obtained for the slope and standard error of 

estimate respectively. The DLAP for acetoin was calculated to be 0.017 ng acetoin. 

Table 4.1.1 1600 

Detection Limit of the Analytical 

Procedure for Acetoin 

concentration mass on area counts 1200 

(μg/sample) column (μV·S) '"" n ], 

0.000 0.000 0 ~ 800 

0.110 0.028 157 0 

() 

0.220 0.055 224 "' 0.330 0.083 386 ~ <( 

0.440 0.110 515 400 

0.550 0.138 738 

0.660 0.165 818 

0.770 0.193 998 0 DLAP 0 0.1 0.2 0.3 

0.880 0.220 1117 Mass (ng acetoin) Injected onto Column 0.990 0.248 1248 

1.100 0.275 1414 Figure 4.1.1. Plot of data to determine the DLAP for 

acetoin. (Y = 5171X - 19.9) 

For diacetyl values of 4325 and 47 were obtained for the slope and standard error of estimate 

respectively. The DLAP for diacetyl was calculated to be 0.033 ng diacetyl. 

Table 4.1.2 

Detection Limit of the Analytical 

Procedure for Diacetyl 

concentration mass on area counts 

(μg/sample) column (μV·S) 

0.000 

0.191 

0.287 

0.382 

0.478 

0.573 

0.669 

0.764 

0.860 

0.955 

1.051 

n 

0.000 

0.048 

0.072 

0.096 

0.120 

0.143 

0.167 

0.191 

0.215 

0.239 

0.263 

0 

155 

201 

350 

417 

590 

615 

706 

877 

1043 

1089 

1200 

900 

'"" ], 

~ 600 

0 

() 

"' ~ <( 300 

DLAP 

0.1 0.2 

Mass (ng diacetyl) Injected onto Column 

Figure 4.1.2. Plot of data to determine the DLAP for 

diacetyl. (Y = 4325X - 62) 

28 Burright, D.; Chan, Y.; Eide, M.; Elskamp, C.; Hendricks, W.; Rose, M. C. Evaluation Guidelines For Air Sampling Methods 

Utilizing Chromatographic Analysis, 1999. U.S. Department of Labor, Occupational Safety and Health Administration 

Web site. http://www.osha.gov/dts/sltc/methods/chromguide/chromguide.pdf (accessed November 2007). 

14 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 317
 

4.2 Detection limit of the overall procedure (DLOP) and reliable quantitation limit (RQL) 

The DLOP is measured as mass per sample and expressed as equivalent air concentrations, 

based on the recommended sampling parameters. Ten samplers were spiked with equally 

descending increments of acetoin and diacetyl, such that the highest sampler loading was 

equivalent to 1.10 μg of acetoin per sample and 0.96 μg of diacetyl per sample. This is the 

amount spiked on a sampler that would produce a peak approximately 10 times the response 

of a calibration blank. These spiked samplers, and the sample blank were analyzed with the 

recommended analytical parameters (1-μL injection with a 2:1 spit), and the data obtained were 

used to determine the required parameters (slope and standard error of estimate) for the 

calculation of the DLOP. For acetoin values of 1029 and 36 were obtained for the slope and 

standard error of estimate respectively. The DLOP was calculated to be 0.10 μg acetoin per 

sample (0.0031 ppm or 0.011 mg/m3 for a TWA sample). 

Table 4.2.1 

Detection Limit of the Overall Procedure for 

Aceto in 

mass per sample 

(μg/sample) 

0.000 

0.110 

0.220 

0.330 

0.440 

0.550 

0.660 

0.770 

0.880 

0.990 

1.100 

area counts 

(μV·s) 

0 

119 

226 

316 

432 

517 

605 

771 

848 

1057 

1150 

'Vi' 800 

~ 

~ 0 

() 

~ 400 

<( 

DLOP RQL 

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 1.1 

Mass per Sample (ug) Acetoin 

Figure 4.2.1. Plot of data to determine the 

DLOP/RQL for acetoin. (Y = 1029X - 16.8) 

For diacetyl values of 1241 and 46 were obtained for the slope and standard error of estimate 

respectively. The DLOP was calculated to be 0.11 μg diacetyl per sample (0.0034 ppm or 

0.012 mg/m3 for a TWA sample). 

Table 4.2.2 

Detection Limit of the Overall Procedure for 

mass per sample 

(μg/sample) 

0.000 

0.096 

0.191 

0.287 

0.382 

0.478 

0.573 

0.669 

0.764 

0.860 

0.955 

Diacetyl 

area counts 

(μV·s) 

0 

118 

214 

357 

515 

623 

744 

916 

864 

1043 

1208 

1000 

"' :> 

2' 

.l!l 

c 

:J 

0 

() 500 

"' ~ 

<( 

DLOP RQL 

0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1.0 

Mass per Sample (ug) Diacetyl 

Figure 4.2.2. Plot of data to determine the 

DLOP/RQL for diacetyl. (Y = 1241X- 7.3) 

The RQL is considered the lower limit for precise quantitative measurements. It is determined 

from the regression line parameters obtained for the calculation of the DLOP, providing 75% to 

15 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

318 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

125% of the analyte is recovered. The RQL for acetoin is 0.35 μg per sample (0.011 ppm or 

0.039 mg/m3 for a TWA sample). Recovery at this concentration is 102%. The RQL for diacetyl 

is 0.37 μg per sample (0.012 ppm or 0.041 mg/m3 for a TWA sample). Recovery at this 

concentration is 93.5%. 

Acetoin 6.95 

Diacetyl 

> 7.1 > 

5 5 Q) 

:!! 

0 

c. 

"' Q) 

ill 

c 

0 

a. 6.85 

"' Cl:'. 7.0 ~ 

7.9 8.0 8.1 8.2 

Time (min) 

Figure 4.2.3. Chromatogram of acetoin at 

the RQL. 

4.3 Instrument calibration 

8.3 

6.75 

5.0 5.1 5.2 5.3 5.4 

Time (min) 

Figure 4.2.4. Chromatogram of diacetyl at 

the RQL. 

5.5 

The standard error of estimate was determined from the linear regression of data points from 

standards over a range that covers approximately 0.25 to 2 times the target concentration. 

Calibration curves for acetoin and diacetyl were constructed and are shown in Section 3.5.2 

from the three injections of five standards. The standard error of estimate is 0.42 μg/sample for 

acetoin and 0.82 μg/sample for diacetyl. 

Table 4.3.1 

Acetoin Instrument Calibration 

standard concn 

(μg/sample) 

3.73 

8.69 

16.1 

23.6 

31.0 

5782 

14230 

26940 

38318 

52053 

area counts 

(μV·s) 

6047 

14168 

26458 

39021 

51292 

4.4 Precision (overall procedure) 

6004 

14323 

26198 

39714 

52127 

Table 4.3.2 

Diacetyl Instrument Calibration 

standard concn area counts 

(μg/sample) ( μV·s) 

3.58 4242 4347 

8.36 10205 10350 

15.5 20275 19361 

22.7 28772 29121 

29.9 39287 38363 

4352 

10373 

19540 

29255 

39653 

The precision at the 95% confidence level is obtained by multiplying the standard error of 

estimate by 1.96 (the z-statistic from the standard normal distribution at the 95% confidence 

level). In Section 4.5, 95% confidence intervals are drawn about their respective regression 

lines in the storage graph figures. For acetoin the precision of the overall procedure of ±11.2% 

was obtained from the standard error of estimate of 5.73% in Figure 4.5.1. For diacetyl the 

precision of the overall procedure of ±10.1 % was obtained from the standard error of estimate 

of 5.15% in Figure 4.5.3. The precision includes an additional 5% for sampling error. 

16 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 319
 

4.5 Storage test 

time 

(days) 

0 

4 

7 

11 

14 

18 

120 

80 

~ 

~ Q) 

> 0 

u 

Q) 

0:: 40 

Storage samples for acetoin and diacetyl were prepared by collecting samples from a controlled 

test atmosphere using the recommended sampling conditions. The concentration of acetoin 

and diacetyl were at the target concentration with an average relative humidity of 41 % at 34 °C 

(absolute humidity of 15.2 mg/L H20). Thirty-three storage samples were prepared. Three 

samples were analyzed on the day of generation. Fifteen of the samples were stored at 

reduced temperature (3 °C) and the other fifteen were stored in a closed drawer at ambient 

temperature (about 21 °C). At 3-4 day intervals, three samples were selected from each of the 

two storage sets and analyzed. Sample results were not corrected for extraction efficiency. 

Table 4.5.1 

Storage Test for Acetoin 

ambient storage refrigerated storage 

recovery(%) recovery(%) 

86.9 87.7 89.8 86.9 87.7 89.8 

83.1 92.0 88.3 88.0 86.1 87.4 

91.8 85.1 90.4 95.3 90.0 94.0 

89.1 92.3 90.9 90.6 91.4 92.1 

90.9 88.5 91.5 90.7 88.5 91.9 

86.5 85.5 86.1 91. 7 87.6 89.9 

0 8 8 0 

~ 0 

Acetoin Ambient Storage 

Y = -0.0139X + 88.8 

Std Error of Estimate= 5.73% 

95% Confidence Limits= ±(1.96)(5. 73) = ±11.2% 

Table 4.5.2 

Storage Test for Diacetyl 

time 

(days) 

ambient storage refrigerated storage 

recovery(%) recovery(%) 

~ 

~ Q) 

> 0 

u 

Q) 

0:: 

0 

4 

7 

11 

14 

18 

120 

80 

40 

100.5 99.9 100.7 100.5 99.9 100.7 

98.6 100.9 100.3 97.4 96.2 98. 7 

102.6 100.9 101.2 101.5 98.8 100.9 

102.7 104.8 101.6 101.9 101.9 102.4 

101.9 101.0 102.7 100.2 98.8 103.2 

101.1 103.8 101.9 100.7 98.4 102.8 

~ .. Q 

!! 0 I 

Acetoin Refrigerated Storage 

Y = 0.122X + 88.9 

Std Error of Estimate = 5.57% 

95% Confidence Limits= ±(1.96)(5.57) = ±10.9% 0 

0 10 15 0 0 10 15 

Storage Time (Days) Storage Time (Days) 

Figure 4.5.1. Ambient storage test for acetoin. Figure 4.5.2. Refrigerated storage test for acetoin. 

120 120 

Q Q ;; ;; " " 0 

II v " ' 

80 80 

40 40 

Diacetyl Ambient Storage Diacetyl Refrigerated Storage 

Y = 0.135X + 100.3 Y = 0.101X + 99.4 

Std Error of Estimate = 5.15% Std Error of Estimate = 5.33% 

95% Confidence Limits= ±(1.96)(5.15) = ±10.1 % 95% Confidence Limits= ±(1.96)(5.33) = ±10.4% 0 0 0 10 15 0 10 15 

Storage Time (Days) Storage Time (Days) 

Figure 4.5.3. Ambient storage test for diacetyl. Figure 4.5.4. Refrigerated storage test for diacetyl. 

17 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

320 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

4.6 Reproducibility 

Six samples were prepared by collecting them from a controlled test atmosphere similar to that 

which was used in the collection of the storage samples. The samples were submitted to the 

OSHA Salt Lake Technical Center for analysis along with a draft copy of this method. The 

samples were analyzed after being stored for 20 days at refrigerated temperature (about 3 °C). 

Sample results were corrected for extraction efficiency. No sample result for acetoin and 

diacetyl had a deviation greater than the precision of the overall procedure determined in 

Section 4.4. 

Table 4.6.1 

Reproducibility Data for Acetoin 

Table 4.6.2 

Reproducibility Data for Diacetyl 

theoretical 

(μg/sample) 

recovered recovery 

(μg/sample) (%) 

deviation 

(%) 

theoretical 

(μg/sample) 

recovered recovery 

(μg/sample) (%) 

deviation 

(%) 

16.3 

16.4 

16.1 

15.8 

16.1 

16.6 

17.3 106.1 

15.8 96.3 

16.8 104.3 

15.2 96.2 

15.7 97.5 

16.0 96.4 

6.1 

-3.7 

4.3 

-3.8 

-2.5 

-3.6 

15.9 

15.9 

15.7 

15.4 

15.7 

16.2 

16.6 104.4 

16.3 102.5 

16.5 105.1 

15.8 102.6 

16.0 101.9 

16.6 102.5 

4.4 

2.5 

5.1 

2.6 

1.9 

2.5 

4.7 Sampler capacity 

test 

no. 

2 

3 

The sampling capacity of the front tube was tested by sampling from a dynamically generated 

test atmosphere at 2 times the target concentration of acetoin (3.58 mg/m3 or 0.99 ppm) and 

diacetyl (3.55 mg/m3 or 1.01 ppm) with an average relative humidity of 40% at 34 °C (absolute 

humidity of 14.8 mg/L H20). The samples were collected at a sampling rate of 0.05 L/min. 

Backup tubes were placed in-line behind the front tube and were changed regularly after the 

initial collection of 225 min. Breakthrough for diacetyl was observed after sampling 12.4 L. No 

breakthrough was observed for acetoin even after sampling for 265 min. The recommended 

sampling time is 3 h. 

Table 4.7 90 

Breakthrough of Diacetyl 80 

air vol sampling downstream breakthrough 

(L) time concn (%) 70 

(min) (mg/m3

) 

~ 60 

11.1 225 0.00 0.00 .<::: 

11.8 240 0.00 0.00 g> 50 

12.1 245 0.00 0.00 e 

.<::: 40 

12.3 250 0.00 0.00 ~ • 0 

"' 12.6 255 0.06 1.55 ~ 30 []J 

12.8 260 0.24 6.68 20 

13.0 265 0.61 17.2 12.4 L 10 

12.0 225 0.00 0.00 ----------------------------~ 0 

12.7 240 0.22 6.32 0 10 

13.0 245 0.49 13.8 Air Volume (L) 

15 

13.3 250 0.90 25.3 Figure 4.7. Five percent breakthrough air volume for 

13.5 255 1.36 38.3 diacetyl. 

13.8 260 1.86 52.3 

14.1 265 2.05 57.7 

11.6 225 0.00 0.00 

12.4 240 0.25 7.04 

12.6 245 0.66 18.7 

12.9 250 1.32 37.0 

13.1 255 1.96 55.1 

13.4 260 2.36 66.5 

13.6 265 2.96 75.8 

18 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 321
 

4.8 Extraction efficiency and stability of extracted samples 

The extraction efficiency is dependent on the extraction solvent as well as the internal standard. 

Other extraction solvents or internal standards may be used provided that the new extraction 

solution or internal standard is tested. The new extraction solvent or internal standard should 

be tested as described below. 

Extraction efficiency 

The extraction efficiency of acetion and diacetyl was determined by liquid spiking four samplers, 

at each concentration level, with the analytes from the RQL to 2 times the target 

concentrations. These samples were stored overnight at ambient temperature and then 

analyzed. The mean extraction efficiency over the working range of the RQL to 2 times the 

target concentration is 92.9% for acetoin. The extraction efficiency for the wet samplers was 

not included in the overall mean because it would bias the results. 

Table 4.8.1 

Extraction Efficiency(%) of Acetoin 

level sam12le number 

x target μg acetoin 2 3 4 mean 

concn per sample 

RQL 0.33 94.0 96.5 97.4 96.7 96.2 

0.25 3.73 90.5 87.8 90.1 90.5 89.7 

0.5 8.69 90.2 92.4 94.6 95.6 93.2 

1.0 16.2 93.2 93.7 91.9 92.6 92.8 

1.5 23.6 92.3 93.6 93.5 92.0 92.8 

2.0 31.0 92.7 93.8 92.7 92.5 92.9 

1.0 (wet) 16.2 96.8 94.5 95.3 95.0 95.4 

The mean extraction efficiency over the working range of the RQL to 2 times the target 

concentration is 99.6% for diacetyl. The extraction efficiency for the wet samplers was not 

included in the overall mean because it would bias the results. 

Table 4.8.2 

Extraction Efficiency (%) of Diacetyl 

level sam12le number 

x target μg diacetyl 2 3 4 mean 

concn per sample 

RQL 0.38 94.1 97.5 101.2 89.9 95.7 

0.25 3.58 96.8 97.9 99.3 98.4 98.1 

0.5 8.36 101.8 100.4 101.9 101.6 101.4 

1.0 15.5 98.0 101.4 100.2 101.7 100.3 

1.5 22.7 100.9 102.2 101.4 100.5 101.2 

2.0 29.9 100.9 101.2 100.7 100.4 100.8 

1.0 (wet) 15.5 97.8 97.3 97.2 99.7 98.0 

Stability of extracted samples 

The stability of extracted samples was investigated by reanalyzing the target concentration 

samples 24 h and 72 h after initial analysis. After each analysis was performed, two vials were 

recapped with new septa while the remaining two retained their punctured septa. The samples 

were reanalyzed with fresh standards. Samples were stored at ambient temperature and each 

septum was punctured 4 times for each analysis. 

19 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

322 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

The average percent change for acetoin samples after 24 h was +0.5% for samples that were 

resealed with new septa and +0.5% for those that retained their punctured septa. The test was 

performed at room temperature (about 21 °C). 

Table 4.8.3 

24 Hour Stability of Extracted Samples for Acetoin 

12unctured se12ta re12laced 12unctured se12ta retained 

initial after difference initial after difference 

(%) one day (%) (%) one day (%) 

% % 

93.2 93.1 -0.1 91.9 92.9 +1.0 

93.7 94.7 +1.0 92.6 92.5 -0.1 

(mean) (mean) 

93.4 93.9 +0.5 92.2 92.7 +0.5 

The average percent change for acetoin samples after 72 h was -1.8% for samples that were 

resealed with new septa and -0.9% for those that retained their punctured septa. 

Table 4.8.4 

72 Hour Stability of Extracted Samples for Acetoin 

12unctured se12ta re12laced 12unctured se12ta retained 

initial after difference initial after difference 

(%) one day (%) (%) one day (%) 

% % 

93.2 91.5 -1.7 91.9 91.3 -0.6 

93.7 91.8 -1.9 92.6 91.3 -1.3 

(mean) (mean) 

93.4 91.6 -1.8 92.2 91.3 -0.9 

The average percent change for diacetyl after 24 h was +0.4% for samples that were resealed 

with new septa and -1.4% for those that retained their punctured septa. The test was 

performed at room temperature (about 21 °C). 

Table 4.8.5 

24 Hour Stability of Extracted Samples for Diacetyl 

12unctured se12ta re12laced 12unctured se12ta retained 

initial after difference initial after difference 

(%) one day (%) (%) one day (%) 

% % 

98.0 99.0 +1.0 100.2 99.5 -0.7 

101.4 101.2 -0.2 101. 7 99.7 -2.0 

(mean) (mean) 

99.7 100.1 +0.4 101.0 99.6 -1.4 

The average percent change for diacetyl samples after 72 h was +1.0% for samples that were 

resealed with new septa and -0.8% for those that retained their punctured septa. 

Table 4.8.6 

72 Hour Stability of Extracted Samples for Diacetyl 

12unctured se12ta re12laced 12unctured se12ta retained 

initial after difference initial after difference 

(%) one day (%) (%) one day (%) 

% % 

98.0 99.8 +1.8 100.2 100.7 +0.5 

101.4 101.5 +0.1 101.7 99.7 -2.0 

(mean) (mean) 

99.7 100.6 +1.0 101.0 100.2 -0.8 

20 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 323
 

4.9 Interferences (sampling) 

Retention 

Table 4.9.1 

Retention Efficiency(%) of Acetoin 

set no. 1 2 3 

first 

second 

93.6 

94.0 

92.5 

95.6 

99.8 

98.1 

The ability of the sampler to retain acetoin 

and diacetyl was tested by sampling from a 

dynamically generated test atmosphere of 

acetoin (3.67 mg/m3 or 1.02 ppm) and 

diacetyl (3.58 mg/m3 or 1.02 ppm) with an 

average relative humidity of 40% at 35 °C second/first (absolute humidity of 15.6 mg/L H20). Six 

samplers had contaminated air drawn 

through them at 0.05 L/min for 45 min. 

Sampling was discontinued and three 

samples set aside (first set). The generation 

system was flushed with contaminant-free air. 

Sampling resumed with the other three 

Table 4.9.2 

Retention Efficiency(%) of Diacetyl 

set no. 1 2 3 

first 108.0 103.0 108.5 

second 102.4 102.3 103.9 

samples having contaminant-free air drawn second/first 

mean 

95.3 

95.9 

100.6 

mean 

106.5 

102.9 

96.6 

through them at 0.05 L/min for 135 min and -----------------

then all six samplers were analyzed. The mean of the samples in the second set had retained 

100.6% for acetoin and 96.6% for diacetyl of the mean collected by the first three samples. 

Low humidity 

The ability of the sampler to collect acetoin and diacetyl from a relatively dry atmosphere was 

tested by sampling from a dynamically generated test atmosphere of acetoin (4.06 mg/m3 or 

1.13 ppm) and diacetyl (4.03 mg/m3 or 1 .14 ppm) with an average relative humidity of 8% at 33 

°C (absolute humidity of 2.82 mg/L H20). Three samplers had contaminated air drawn through 

them at 0.05 L/min for 180 min. All of the samples were immediately analyzed. The samplers 

collected 103.0%, 96.9% and 102.2% of theoretical for acetoin and 96.7%, 106.6% and 101.2% 

of theoretical for diacetyl. 

Low concentration 

The ability of the sampler to collect acetoin and diacetyl at low concentrations was tested by 

sampling from a dynamically generated test atmosphere of 0.1 times the target concentration of 

acetion (0.185 mg/m3 or 0.0515 ppm) and diacetyl (0.175 mg/m3 or 0.0497 ppm) with an 

average relative humidity of 42% at 33 °C (absolute humidity of 14.8 mg/L H20). Three 

samplers had contaminated air drawn through them at 0.05 L/min for 180 min. All of the 

samples were immediately analyzed. The samplers collected 93.9%, 91.5% and 89.9% of 

theoretical for acetoin and 92.8%, 97.4% and 96. 7% of theoretical for diacetyl. 

The ability of the sampler to collect acetoin and diacetyl at low concentrations when taking 

short term samples was tested by sampling from a dynamically generated test atmosphere of 

0.1 times the target concentration of acetion (0.185 mg/m3 or 0.0514 ppm) and diacetyl (0.175 

mg/m3 or 0.0497 ppm) with an average relative humidity of 42% at 33 °C (absolute humidity of 

14.8 mg/L H20). Three samplers had contaminated air drawn through them at 0.2 L/min for 15 

min. All of the samples were immediately analyzed. The samplers collected 103.8%, 104.1 % 

and 110.0% of theoretical for acetoin and 88.1 %, 89.2% and 94.4% of theoretical for diacetyl. 

Interferences 

The ability of the sampler to collect acetoin and diacetyl was tested when other potential 

interferences are present by sampling an atmosphere containing 1.63 mg/m3 (0.45 ppm) of 

acetoin, 1.56 mg/m3 (0.44 ppm) of diacetyl, 2.59 mg/m3 (0.44 ppm) of 2-nonanone and 1.88 

mg/m3 (0.44 ppm) of 2,3-pentanedione with an average relative humidity of 38% at 34 °C 

21 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

324 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

(absolute humidity of 14.1 mg/L H20). Three samplers had contaminated air drawn through 

them at 0.05 L/min for 181 min. All of the samples were immediately analyzed. The samplers 

collected 93.2%, 96.5% and 96.8% of theoretical for acetoin and 100.6%, 100.6% and 104.1% 

of theoretical for diacetyl. Selection of 2-nonanone as a potential interference was based on its 

common use in butter flavorings used in microwave popcorn manufacturing facilities 29 . 2,3-

Pentanedione was selected because it has been suggested as a possible replacement for 

diacetyl. (Note: The GC retention time of 2-nonanone was 14.4 min and 7.4 min for 2,3-

pentanedione. For this test the GC column temperature program was slightly changed to Initial 

60 °C, hold 4 min; ramp at 15 °C/min to 225 °C, hold 0 min; ramp at 60 °C/min to 250 °C, hold 4 

min to allow for the elution of 2-nonanone.) 

Light 

The possibility of light 

degradation was tested for 

both acetoin and diacetyl on 

the sampling medium and in 

the extraction solution. For 

the sample medium test 12 

samples were collected by 

sampling from a dynamically 

generated test atmosphere 

of acetoin (1.92 mg/m3 or 

0.53 ppm) and diacetyl (1.87 

mg/m3 or 0.53 ppm) with an 

average relative humidity of 

40% at 35 °C (absolute 

humidity of 15.6 mg/L H20). 

The samples were collected 

at a sampling rate of 0.05 

L/min for 3 hours. Nine of 

Table 4.9.3 

Sampler Light Exposure Test for Acetoin 

sam12le number 

tl'.12e of sam12ler light ex12osure 2 3 

no light exposure 94.0 97.3 92.0 

3h ambient light exposure during 95.0 91.3 96.0 

sampling 

24h direct fluorescent light exposure 92.5 86.4 87.1 

after sampling, none during sampling 

3h direct sunlight exposure after 79.7 63.5 63.7 

sampling, none during sampling 

Table 4.9.4 

Sampler Light Exposure Test for Diacetyl 

sam12le number 

tl'.12e of sam12ler light ex12osure 2 3 

no light exposure 95.4 97.6 96.8 

3h ambient light exposure during 98.0 94.9 95.8 

mean 

94.4 

94.1 

88.7 

67.0 

mean 

96.6 

96.2 

the samples were covered sampling 

with aluminum foil during 24h direct fluorescent light exposure 88.4 86.1 86.0 86.8 

sampling and three were not after sampling, none during sampling 

covered. The three samples 3h direct sunlight exposure after 5.68 7.08 6.52 6.43 

not covered and three of the sampling, none during sampling 

covered samples were ------------------------

immediately analyzed after sampling. Three of the covered samples were placed under a 

fluorescent lamp for 24 h and the reaming three were placed outside in direct sunlight for three 

hours before analyzing. The samples covered during sampling and immediately analyzed after 

sampling had mean recoveries of 94.4% of theoretical for acetoin and 96.6% for diacetyl. The 

samples not covered during sampling and immediately analyzed after sampling had mean 

recoveries of 94.1 % of theoretical for acetoin and 96.2% for diacetyl. The samples covered 

during sampling and then exposed to fluorescent light for 24 h before analysis had mean 

recoveries of 88.7% of theoretical for acetoin and 86.8% for diacetyl. The samples covered 

during sampling and then exposed to sunlight for 3 h before analysis had mean recoveries of 

67.0% of theoretical for acetoin and 6.43% for diacetyl. This data clearly indicates that the 

sampler should be protected from exposure to light. 

To test the possibility of light degradation on extracted samples nine analytical standards at the 

target concentration were prepared. Six of the standards were placed in 2-ml amber glass 

vials and three were placed in 2-ml clear glass vials. Three of the amber vials, along with the 

29 Kanwal, R.; Boylstein, R. J.; Piacitelli, C. NIOSH Health Hazard Evaluation Report #2001-0474-2943, 2004. Centers for Disease 

Control and Prevention, National Institute of Occupational Safety and Health Web site. 

http://www.cdc.gov/niosh/hhe/reports/pdfs/2001-0474-2943.pdf (accessed July 2008) p 46. 

22 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 325
 

clear glass vials were stored on the autosampler tray during the entire test while the other three 

amber vials were stored in the refrigerator when not being analyzed. All nine standards were 

analyzed eight times over a 10 day period with none of the septa being replaced during the 

test. With the exception of diacetyl in clear vials, acetoin and diacetyl did not degrade. This 

data clearly indicates that extracted samples should be protected from exposure to light. This 

data also indicates that acetoin and diacetyl are stable in the extraction solution for up to 9 days 

as long as they are stored in amber vials. 

Table 4.9.5 Table 4.9.6 

Extracted Sample Light Exposure Test for Extracted Sample Light Exposure Test for 

Aceto in Diacetl'.I 

mean of 3 peak areas mean of 3 peak areas 

day clear vials amber vials amber vials day clear vials amber vials amber vials 

(ambient) (ambient) (refrigerated) (ambient) (ambient) (refrigerated) 

0 24226 23552 23485 0 20537 19789 19640 

1 24232 23642 23535 1 19037 19667 19716 

2 23693 23232 22932 2 17814 19301 19336 

3 23455 23376 23383 3 16289 19354 19723 

4 23765 23137 23050 4 15703 19026 19304 

7 24191 23973 23280 7 14603 19687 19577 

8 23734 22969 22684 8 13328 18509 19026 

9 24245 23740 23309 9 12408 19324 19606 

The internal standard, 3-pentanone, was stable for up to 9 days in both the clear and ambient 

vials. 

4.10 Diacetyl migration within sampling tubes 

In the majority of solid sorbent sampling tubes used by Table 4.1 o 

OSHA the sampling bed and the backup bed of sorbent Ambient Storage Diacetyl Migration 

are placed in the same sampling tube. For diacetyl this Test 

was not possible due to the migration of diacetyl within time diacetyl found on backup 

the sampling tube during storage. To demonstrate (days) section(%) 

migration fifteen tubes were packed with 600 mg of silica O 0.00 0.00 0.00 

gel and a backup section of 200 mg silica gel separated 4 3.o7 0.54 0.82 

with a glass wool plug. These fifteen tubes were used to 1

7

1 ~:~~ ~1 ; i:~~ collect samples from a dynamically generated test 14 9.63 11.9 13.0 

atmosphere of acetoin (3.35 mg/m3 or 0.93 ppm) and 

diacetyl (3.17 mg/m3 or 0.90 ppm) with an average relative humidity of 42% at 33 °C (absolute 

humidity of 14.8 mg/L H20). The samples were collected at a sampling rate of 0.05 L/min for 3 

hours. Three samples were analyzed on the day of generation and the other twelve were 

stored in a closed drawer at ambient temperature (about 21 °C). At 3-4 day intervals, three 

additional samples were analyzed. After 14 days up to 13.0% of diacetyl was found to have 

migrated from the front to the back section of the modified sampling tube. Acetoin did not 

migrate within the sampling tube. 

4.11 Generation of test atmospheres 

A test atmosphere generator, as diagramed in Figure 4.11, was set up in a walk-in hood. 

House air was dried and then humidified and regulated using a Miller Nelson Model 401 FlowTemperature-Humidity Control System. A measured flow (typically 10 μL per min) of an acetoin 

and diacetyl water solution was pumped through a 0.53-mm uncoated fused silica capillary tube 

into the inlet manifold, using a Series D ISCO Syringe Pump with Controller, and mixed with 

dilution air (typically 100 liters per min) coming from the Miller Nelson Control System. The inlet 

manifold was heated by wrapping it in heat tape, regulated with a variable autotransformer, in 

order to insure vaporization of acetoin. The acetoin and diacetyl gas mixture then flowed 

continuously into the mixing chamber (76-cm x 15-cm) and then into the sampling chamber 

23 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

326 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

(56-cm x 9.5-cm). Samples were collected 

through sampling ports on the sampling 

chamber. Temperature and humidity were 

measured near the exit of the sampling 

chamber using an Omega Digital Thermohygrometer model RH411. 

With the exception of low humidity tests 

OSHA normally generates test atmospheres 

at an average relative humidity of 80% at 22 

°C resulting in an absolute humidity of 15.5 

mg/L H20. Due to the use of heat tape on 

the inlet manifold, used as mentioned above 

to insure the vaporization of acetoin, the test 

atmosphere generation temperature for this 

evaluation was typically around 34 °C at the 

sampling chamber outlet, 37 °C in the 

middle of the sampling chamber, 45 °C at 

Sampling Chamber 

Sampling ports 

1. Mixing chamber 

2. ISCO syringe pump 

3. Drierite desiccant 

4. Temp/Humidity probe 

5. Heat tape around 

inlet manifold 

House air 

in 

the sampling chamber inlet and 86 °C at Figure 4.11. Diagram of apparatus used to 

the mixing chamber inlet. In order to generate test atmospheres. 

maintain a humidity of 15.5 mg/L H20 at 34 

°C the relative absolute humidity was adjusted to approximately 41 %. 

4.12 Qualitative analysis 

g 

j 

<( 

When necessary, the identity or purity of an analyte peak can be confirmed by GC-mass 

spectrometry or by another analytical procedure. The mass spectra in Figure 4.12.1 and 4.12.2 

are taken from the NIST spectral library. 

10000 10000 43 45 

8000 8000 

6000 g 6000 

4000 15 

j 

<( 4000 

2000 2000 88 

86 29 

11 II I 

73 

0 ,,,, 11 0 0 20 40 60 80 100 0 20 40 60 80 

m/z m/z 

Figure 4.12.1. Mass spectrum of diacetyl. Figure 4.12.2. Mass spectrum of acetoin. 

24 of 25 T-1013-FV-01-0809-M 

100 

Appendix C – Acetoin Diacetyl 1013
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 327
 

Appendix A 

A.1 Silica gel preparation 

For this evaluation sampling tubes were custom made by SKC, Inc. and are now available for 

purchase through SKC, Inc. (cat. no. 226-183). 

Below are instructions on how the silica gel is prepared for the sampling tubes used in this 

evaluation. 

A.1 .1 Apparatus 

Tube furnace and quartz process tube. A Lindberg model 55035 tube furnace and 1-inch 

diameter quartz process tube were used in this evaluation. 

Nitrogen gas. 

A.1 .2 Silica Gel 

Washed 20/40 mesh silica gel with 30 angstrom pore size (washed silica gel can be 

purchased from SKC, Inc.). A description of a washing procedure for silica gel can be 

found in the appendix of NIOSH 790330 . 

A.1 .3 Preparation of silica gel 

Insert a quartz wool plug in a 1-inch diameter quartz process tube, followed by 50 g of 

washed silica gel and a second quartz wool plug to hold the silica gel in place. 

Place the process tube in a tube furnace and set the temperature to 180 °C. Continually 

purge the process tube with nitrogen at a rate of about 0.5 L/min. Allow the silica gel to 

dry in the tube furnace for 4 hours. 

After 4 hours allow the process tube to cool while continuing to purge the tube with 

nitrogen. Once the silica gel is cool, remove one of the quartz wool plugs, and transfer 

silica gel into an airtight container. 

30 Cassinelli, M. E. Acids, Inorganic (NIOSH Method 7903), 1994. Centers for Disease Control and Prevention, National Institute of 

Occupational Safety and Health Web Site. http://www.cdc.gov/niosh/nmam/pdfs/7903.pdf (accessed July 2008). 

25 of 25 T-1013-FV-01-0809-M 

Appendix C – Acetoin Diacetyl 1013
 

328 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
Appendix D
 

2, 3-Pentanedione
 
 

Method no.: 

Version: 

Target concentration: 

Procedure: 

Recommended sampling time 

and sampling rate: 

Reliable quantitation limit: 

Standard error of estimate 

at the target concentration: 

Special requirements: 

Status of method: 

July 2010 

2,3-Pentanedione 

1016 

1.0 

0.5 ppm (2.05 mg/m3

) (TWA) 

Active samples are collected by drawing workplace air through specially 

dried silica gel tubes with personal sampling pumps. Samples are 

extracted with 95:5 ethyl alcohol:water and analyzed by gas 

chromatography using a flame ionization detector (GC-FID). 

200 min at 50 ml/min (10.0 L) (TWA); 15 min at 0.2 L/min (3 L) (short 

term) 

180 min at 50 ml/min (9.0 L) (TWA); 15 min at 0.2 L/min (3 L) (short 

term) if sampling for acetoin and diacetyl along with 2,3-pentanedione 

9.3 ppb (38 μg/m3) 

10.1% 

Protect samplers from the light exposure during sampling, shipping, and 

analysis. Samples should be kept cold and shipped cold to the lab as 

soon as possible after sampling, preferably by overnight or express 

shipping. Samples should be analyzed within 17 days of sampling. 

Fully validated method. This method has been subjected to the 

established validation procedures of the Methods Development Team. 

Methods Development Team 

Industrial Hygiene Chemistry Division 

OSHA Salt Lake Technical Center 

Sandy UT 84070-6406 

1 of 17 

Mary E. Eide 

Appendix D – 2, 3-Pentanedione 

330 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

1. General Discussion 

For assistance with accessibility problems in using figures and illustrations presented in this 

method, please contact Salt Lake Technical Center (SL TC) at (801) 233-4900. These 

procedures were designed and tested for internal use by OSHA personnel. Mention of any 

company name or commercial product does not constitute endorsement by OSHA. 

1.1 Background 

1.1.1 History 

OSHA is concerned about workplace exposure to 2,3-pentanedione because it is a 

butter flavoring agent that is sometimes substituted for diacetyl. 1 2,3-Pentanedione is 

chemically similar to diacetyl and may have similar toxicological properties. 2 This work 

was performed because OSHA has no sampling and analytical method for 2,3-

pentanedione and none was found in a literature review. 

One of the main objectives of this work was to enable OSHA CSHOs to monitor 

workplace exposure to diacetyl, acetoin and 2,3-pentanedione simultaneously on the 

same sample. Because of the similarities of the chemicals, it was decided to validate 

existing sampling and analytical methodology specified in OSHA Method 10133 for 2,3-

pentanedione. That method requires sampling with two commercially available silica 

gel tubes connected in series. This method specifies a different GC column than 

specified in Method 1013 in order to optimize the analytical separation. The reliable 

quatitation limits for acetoin and diacetyl cited in OSHA Method 1013 were confirmed 

with the GC column used in this validation. 

1.1.2 Toxic effects (This section is for information only and should not be taken as the basis 

of OSHA policy.) 

2,3-Pentanedione is moderately toxic by ingestion, a skin irritant, and can cause eye 

and respiratory tract irritation. 4 The oral LD50 in rats is 3000 mg/kg. The skin irritation 

test in rabbits showed moderate irritation for an exposure of 500 mg/24h. Studies 

exposing rats to 118, 241, 318, or 354 ppm 2,3-pentanedione for 6 hours showed 

epithelial changes in the airways which increased with increasing air concentrations 

with necrosuppurative tracheitis in the rats exposed to 354 ppm. 5 This epithelial cell 

damage was found to progress post-exposure in rats sacrificed a day later. These 

epithelial changes included degeneration, apoptosis, necrosis, and neutrophilic 

inflammation. 

News Watch, Diacetyl. The Synergist. March 2010. American Industrial Hygiene Association Web site. http://www.aihasynergistdigital.org/aihasynergist/201003#pg39 (accessed August 2010). 

Hubbs, A.F.; Mosely, A.E.; Goldsmith, WT.; Jackson, M.C.; Kashan, M.L.; Battelli, L.A.; Schwegler-Berry, D.; Goravanahally, 

M.P.; Frazer, D.; Fedan, J.S.; Kreiss, K.; and Castranova, V. Airway Epithelial Toxicity of the Flavoring Agent, 2,3-

Pentanedione. Toxicologist [CD-ROM] 2010, 114, 319. 

Simmons, M., Hendricks, W. Acetoin and Diacetyl (OSHA Method 1013), 2008. U.S. Department of Labor, Occupational Safety 

and Health Administration Web site. https://www.osha.gov/dts/sltc/methods/validated/1013/1013.html (accessed December 

2009). 4 Sax's Dangerous Properties of Industrial Materials, 101

h ed.; Vol. 3, Lewis, R.J. Ed.; John Wiley & Sons; New York, 2000, p 2843. 

Hubbs, A.F.; Mosely, A.E.; Goldsmith, WT.; Jackson, M.C.; Kashan, M.L.; Battelli, L.A.; Schwegler-Berry, D.; Goravanahally, 

M.P.; Frazer, D.; Fedan, J.S.; Kreiss, K.; and Castranova, V. Airway Epithelial Toxicity of the Flavoring Agent, 2,3-

Pentanedione. Toxicologist [CD-ROM] 2010, 114, 319. 

2of17 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 331
 

1.1.3 Workplace exposure 

2,3-Pentanedione is a natural flavorant and odorant that is also synthesized for use in 

odor and flavor manufacturing. 6 It is used to give products a buttery, nutty, cheesy, 

fruity, toasted, chocolate, or caramel taste. It also gives products a buttery, fruity, and 

caramel odor. There can be as much as 58 ppm in food flavorings, and up to 0.08% in 

fragrances. 

2,3-Pentanedione is used as a solvent for cellulose acetate, paints, inks, lacquers, as a 

starting material for dyes, pesticides and pharmaceuticals, and as a photoinitializer for 

photo-reactive dyes. 7 

1.1.4 Physical properties and other descriptive information8

'

9

' 

10 

synonyms: 

IMIS11 : 

boiling point: 

density: 

flash point: 

appearance: 

autoignition 

acetyl propanal; acetyl propionyl; 13,y-dioxopentane; beta, gammadioxopentane; 2,3-pentadione 

P110 CAS number: 600-14-6 

110-112 °C (230-234 °F) melting point: -52 °C (-62 °F) 

0.957 g/ml @25 °C molecular weight: 100.12 

19 °C (66 °F) (open cup) molecular formula: CsHs02 

yellow to yellow-green liquid lower explosive limit: 1.8% (by volume) 

temperature: 265 °C (509 °F) 

solubility: 66.7 g/L water; miscible with alcohol, fixed oils, propylene glycol 

odor: butter-like in dilute concentration, quinone-like in high concentration 

reactive hazards: light sensitive (Section 4.9); vapors are highly flammable and may 

ignite when pouring or pumping due to static electricity 

structural formula of 2,3-pentanedione 

0 

/lf)l 

0 

This method was validated according to the OSHA SL TC "Guidelines for Air Sampling Methods Utilizing 

Chromatographic Analysis" 12 • The Guidelines define analytical parameters, specify required laboratory 

tests, statistical calculations, and acceptance criteria. The analyte air concentrations throughout this 

method are based on the recommended sampling and analytical parameters. Air concentrations in ppm 

are referenced to 25 °C and 760 mmHg (101.3 kPa). 

6 Fenarolli's Handbook of Flavor Ingredients, 5th ed.; Burdock, G.A.; CRC Press; Boca Raton, FL, 2005, p 1495. 

2,3-Pentanedione, Chemicalland21 Website. http://chemicalland21.com/lifescience/foco/2,3-PENTANEDIONE.htm (accessed 

February 2010). 8 Sax's Dangerous Properties of Industrial Materials, 1 oth ed.; Vol. 3, Lewis, R.J.; John Wiley & Sons; New York, 2000, p 2843. 

Lewis, R. J. Sr., Ed. Hawley's Condensed Chemical Dictionary, 14th ed.; Van Nostrand Reinhold Co.: New York, 2001, p 14. 10 3-Pentanedione(600-14-6) Chemical Book Web site. http://www.chemicalbook.com/ProductMSDSDetailCB6166470_EN.htm 

(accessed 1/27/2010). 11 2,3-Pentanedione (OSHA Chemical Sampling Information), 2010. U.S. Department of Labor, Occupational Safety and Health 

Administration Web site. http://www.osha.gov/dts/chemicalsampling/data/CH_260240.html, (accessed 1/5/2010). 12 Eide, M.; Hendricks, W.; Simmons, M. Guidelines For Air Sampling Methods Utilizing Chromatographic Analysis, 2010. U.S. 

Department of Labor, Occupational Safety and Health Administration Web site. 

http://www.osha.gov/dts/sltc/methods/chromguide/chromguide.html (accessed January 2010). 

3of17 

Appendix D – 2, 3-Pentanedione 

332 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

2. Sampling Procedure 

All safety practices that apply to the work area being sampled should be followed. The sampling 

equipment should be attached to the worker in such a manner that it will not interfere with work 

performance or safety. 

2.1 Apparatus 

Samples are collected with 110-cm x 7-mm o.d. glass sampling tubes packed with a single 

section (600 mg) of specially cleaned and dried silica gel. The section is held in place with 

glass wool and with a glass fiber filter in the front and glass wool at the back. A sampling train 

is prepared by placing two tubes in series. For this validation, commercially prepared sampling 

tubes were purchased from SKC, Inc. The two tubes are identical, but SKC labels the tubes as 

"Part A" which is the front tube and as "Part B" which is the back tube (Catalog no. 226-183, lot 

no. 6148). 

Use an opaque tube holder, such as SKC, Inc. Tube Cover D (cat. no. 224-290) to cover the 

sampling train during sampling. If the tube holder is not opaque, wrap the sampler with 

aluminum foil. Light can decompose collected 2,3-pentanedione. 

Samples are collected using a personal sampling pump calibrated to within ±5% of the 

recommended flow rate with the sampling device in-line. 

2.2 Reagents 

None required 

2.3 Technique 

Immediately before sampling, break off both ends of the flame-sealed tube to provide an 

opening approximately half the internal diameter of the tube. Wear eye protection when 

breaking ends. Use sampling tube holders to minimize the hazard to the worker from the 

broken ends of the tubes and to minimize the potential of glass shards entering the foodstuffs. 

All tubes should be from the same lot. 

A sampling train is prepared by attaching a Part A tube in front of and in series with a Part B 

tube, with both glass fiber filters facing forward. 

The Part B tube in the sampling train is used as a back-up and is positioned nearest the 

sampling pump. Attach the tube holder (with the adsorbent tube sampling train) to the 

sampling pump so that the sampling train is in an approximately vertical position with the inlet 

facing down in the worker's breathing zone during sampling. Position the sampling pump, tube 

holder and tubing so they do not impede work performance or safety. 

Draw the air to be sampled directly into the inlet of the tube holder. The air being sampled is 

not to be passed through any hose or tubing before entering the sampling tube. 

Sample for up to 200 min at 50 ml/min (10 L) to collect TWA (long-term) samples. If acetoin 

and/or diacetyl are anticipated to be present, sample for up to 180 min at 50 ml/min (9 L) to 

collect TWA (long-term) samples. 

Sample for 15 min at 0.2 L/min (3 L) to collect short-term samples. 

After sampling for the appropriate time, remove the sampling train, separate the tubes, and cap 

each tube with plastic end caps. Separately wrap each tube in aluminum foil and seal each 

tube end-to end with a Form OSHA-21 as soon as possible. 

4of17 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 333
 

Submit at least one blank sample with each set of samples. Handle the blank sample in the 

same manner as the other samples except draw no air through it. 

Record sample air volumes (L), sampling time (min), and sampling rate (ml/min) for each 

sample, along with any potential interferences on the Form OSHA-91A. 

Submit the samples to the laboratory for analysis as soon as possible after sampling, preferably 

by overnight or express shipping. If delay is unavoidable, store the samples in a refrigerator. 

Ship samples cold to laboratory, such as shipping with frozen plastic ice packs in a cooler. 

Ship any bulk samples separate from the air samples. 

3. Analytical Procedure 

Adhere to the rules set down in your laboratory's Chemical Hygiene Plan 13 (for instance OSHA SL TC 

adheres to: "The OSHA SL TC Chemical Hygiene Plan"). Avoid skin contact and inhalation of all 

chemicals and review all MSDSs before beginning this analytical procedure. Follow all applicable 

quality assurance practices established in your internal quality system (for instance OSHA SL TC 

follows: "The OSHA SL TC Quality Assurance Manual"). 

3.1 Apparatus 

Gas chromatograph equipped with an FID. An Agilent 6890 GC System equipped with a 

Chemstation, an automatic sample injector, and an Agilent tapered, deactivated, split, low 

pressure drop injection port liner with glass wool (catalog no. 5183-4647) was used in this 

validation. 

A GC column capable of separating 2,3-pentanedione from the extraction solvent, potential 

interferences, and internal standard. A DB-1 60-m x 0.32-mm i.d. (5-μm df) capillary column 

was used in this validation. 

An electronic integrator or other suitable means of measuring GC detector response. A Waters 

Empower 2 Data System was used in this validation. 

Amber glass vials with PTFE-lined caps. Two and 4-ml vials were used in this validation. 

A dispenser capable of delivering 2.0 ml of extraction solvent to prepare standards and 

samples. If a dispenser is not available, 2.0-ml volumetric pipettes can be used. 

Class A volumetric flasks - 10-ml and other convenient sizes for preparing standards. 

Calibrated syringe - 25-μL and other convenient sizes for preparing standards. 

Rotator. A Fisher Roto Rack was used to extract the samples in this validation. 

3.2 Reagents 

DI water, 18.0 MQ-cm. A Barnstead NanoPure Diamond system was used to purify the water 

in this validation. 

Ethyl Alcohol, [CAS no. 64-17-5]. The ethyl alcohol:water solution used in this validation was 

95% v/v (190 proof) A.C.S. spectrophotometric grade (lot no. 80513920) purchased from Acros 

Organics (Morris Plains, NJ). Do not use absolute alcohol or denatured alcohol in this method. 

13 Occupational Exposure to Hazardous Chemicals in Laboratories. Code of Federal Regulations, Part 1910.1450, Title 29, 2003. 

5of17 

Appendix D – 2, 3-Pentanedione 

334 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

2,3-Pentanedione [CAS no. 600-14-6]. The 2,3-pentanedione used in this validation was 97% 

(lot no. 29598lJ) purchased from Aldrich (Milwaukee, WI). 

3-Pentanone [CAS no. 96-22-0]. The 3-pentanone used in this validation was 99+% (lot no. HR 

00231 KF) purchased from Aldrich (Milwaukee, WI). 

The extraction solvent used for this validation consisted of 0.007 μL/ml 3-pentanone in 95% v/v 

ethyl alcohol/water. The 3-pentanone was added to the ethyl alcohol as an internal standard 

(ISTD). 

3.3 Standard preparation 

(Note: Store all standards in amber glass bottles and vials) 

Prepare concentrated stock standards in water at 1.021 mg/ml (1 .021 μg/μl) by injecting 11 μl 

of neat 2,3-pentanedione into water in a 10-ml volumetric flask and diluting to the mark. This 

stock standard will remain stable for two weeks if stored in an amber bottle in the refrigerator. 

When using refrigerated stock standards, be sure to allow the standards to warm to room 

temperature and then shake them vigorously before use. Prepare analytical standards by 

injecting microliter amounts of concentrated stock standards into 2-ml volumetric flasks 

containing about 1.75 ml of extraction solvent and then diluting with extraction solvent over a 

concentration range of 0.1 to 20 μg/ml (0.2 to 40 μg/2 ml). For example: a target 

concentration standard of 20.4 μg/sample was prepared by injecting 20 μl of the stock 

standard into a 2-ml flask containing about 1.75 ml of extraction solvent and then diluting to 

the mark with extraction solvent (10.2 μg/ml or 0.5 ppm based on a 2-ml extraction volume 

per sample and 10 lair volumes). 

Bracket sample concentrations with standard concentrations. If upon analysis, sample 

concentrations fall outside the range of prepared standards, prepare and analyze additional 

standards to confirm instrument response, or dilute high samples with extraction solvent and 

reanalyze the diluted samples. 

3.4 Sample preparation 

(Note: prepare all samples in amber glass vials) 

Remove the plastic end caps from the front sample tube and carefully transfer the silica gel to a 

labeled 4-ml amber glass vial. The sampling tube and the back of the glass fiber filter should 

be carefully inspected to ensure that all the silica gel is transferred into the 4-ml vial. Remove 

the plastic end caps from the backup tube and carefully transfer the silica gel to a second 

labeled 4-ml amber glass vial. If the industrial hygienist requests analysis of the front glass 

fiber filter, which is not normally analyzed, place the front glass wool plug and filter from the 

front tube into a third 4-ml vial. If analysis of filter is not requested then discard the front glass 

wool plug and filter. Discard the glass tubes and back glass wool plugs and back glass fiber 

filter. 

Add 2.0 ml of extraction solvent to each vial and immediately seal the vials with PTFE-lined 

caps. 

Immediately place the vials on a rotator for 60 min. Transfer the sample into autosampler vials 

for analysis. 

3.5 Analysis 

3.5.1 Gas chromatographic conditions (these conditions are different from OSHA Method 

1013 to obtain better separation of the 2,3-pentanedione peak from the 3-pentanone 

internal standard peak). 

6of17 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 335
 

100 

75 

l 50 

~ 0 

c. 

"' Q) 25 Cl:'. 

0 

0 

GC conditions 

oven temperature: 

injector temperature: 

detector temperature: 

run time: 

column: 

column mode: 

initial column gas flow: 

column pressure: 

injection size: 

inlet liner: 

retention times: 

FID conditions: 

hydrogen flow: 

air flow: 

nitrogen makeup flow: 

1 

_A 

" 5 10 

Time (min) 

2 

initial 60 °C, hold 4 min, program at 10 °C/min to 150 °C, hold 

5 min, 20 °C/min to 200 °C hold 1 min 

240°C 

250 °C 

total time is 21.5 min, data is collected for 15 min, the excess 

time is to clear the column 

60-m x 0.32-mm i.d. DB-1 capillary column (df = 5-μm) 

constant pressure 

1.8 ml/min (hydrogen) 

9.4 psi 

1.0 μL (2:1 split) 

Agilent 5183-464 7 or equivalent 

13.2 min 2,3-pentanedione 

13.5 min 3-pentanone 

40 ml/min 

450 ml/min 

40 ml/min 

> 

80 

1 

3 

4 

2 

3 s I 5 

Q) 40 :!! 

0 

c. 

"' Q) 

Cl:'. 

0 ~~ - __}_ 

15 0 5 10 15 

Time (min) 

Figure 3.5.1.1. A chromatogram of 20.4 μg/sample 

2,3-pentanedione. (Key: 1) ethyl alcohol; 2) 2,3-

pentanedione; and 3) 3-pentanone.) 

Figure 3.5.1.2. A chromatogram of 20.4 μg/sample 

2,3-pentanedione, 15.8 μg/sample acetoin, and 15.6 

μg/sample diacetyl. (Key: 1) ethyl alcohol; 2) diacetyl; 3) 

2,3-pentanedione; 4) 3-pentanone; and 5) acetoin.) 

3.5.2 An internal standard (ISTD) calibration method is used. A calibration curve can be 

constructed by plotting !STD-corrected response of standard injections versus 

micrograms of analyte per sample. Bracket the samples with freshly prepared 

analytical standards over the range of concentrations. 

7of17 

Appendix D – 2, 3-Pentanedione 

336 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

60000 

(/) 

~ 40000 (/) 

c 

::J 

0 

0 

"' ~ 

<( 20000 

0 

0 10 20 30 40 

Mass (μg) per Sample 

Figure 3.5.2.1. Calibration curve for 2,3-pentanedione. 

(y = 1535x - 295) 

3.6 Interferences (analytical) 

3.6.1 Any compound that produces a GC response and has a similar retention time as the 

analyte or internal standard is a potential interference. If potential interferences were 

reported, they should be considered before samples are extracted. Generally, 

chromatographic conditions can be altered to separate interferences from the analyte. 

3.6.2 When necessary, the identity of an analyte peak can be confirmed with additional 

analytical data or procedures (Section 4.10). 

3.7 Calculations 

The amount of analyte per sample is obtained from the appropriate calibration curve in terms of 

micrograms per sample, uncorrected for extraction efficiency. The second tube is analyzed 

primarily to determine the extent of sampler saturation. If any analyte is found on the back 

tube, it is added to the amount on the front tube. If more than 20% of the total amount is found 

on the back tube, report that the sampler may have been saturated on the Form OSHA-91 B. 

This total amount is then corrected by subtracting the total amount (if any) found on the blank. 

The air concentration is calculated using the following formulas. 

4. Method Validation 

where CM is concn by weight (mg/m3

) 

M is micrograms per sample 

V is liters of air sampled 

EE is extraction efficiency in decimal form 

where Cv is concn by volume (ppm) 

CM is concn by weight (mg/m3

) 

VM is 24.46 (molar volume at NTP) 

Mr is molecular weight of analyte 

(2,3-pentanedione = 100.12) 

General instruction for the laboratory validation of OSHA sampling and analytical methods that employ 

chromatographic analysis is presented in "Validation Guidelines for Air Sampling Methods Utilizing 

8of17 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 337
 

Chromatography Analysis" 14 . These Guidelines detail required validation tests, show examples of 

statistical calculations, list validation acceptance criteria, and define analytical parameters. Air 

concentrations listed in ppm are referenced to 25 °C and 760 mmHg (101.3 kPa). 

4.1 Detection limit of the analytical procedure (DLAP) 

The DLAP is measured as mass of analyte introduced into the chromatographic column. Ten 

analytical standards were spiked with equally descending increments of analyte. The highest 

amount is the amount spiked on the sampler that would produce a peak approximately 10 times 

the response of a reagent blank at or near the retention time of the analyte. The standards and 

the reagent blank were analyzed with the recommended analytical parameters (1-μL injection 

with a 2:1 split). The data obtained were used to determine the required parameters (standard 

error of estimate and slope) for the calculation of the DLAP. The slope and standard error of 

estimate, respectively, were 6.62 and 62.2. The DLAP was calculated to be 28 pg. 

Table 4.1 

Detection Limit of the Analytical Procedure 

concn 

(ng/ml) 

0 

51 

102 

153 

204 

255 

306 

357 

408 

460 

511 

mass on 

column (pg) 

0 

26 

51 

77 

102 

128 

153 

179 

204 

230 

256 

area counts 

(μV•s) 

0 

160 

367 

556 

618 

883 

949 

1084 

1281 

1547 

1784 

1500 

if) 

~ (/) 1000 c 

::J 

0 

0 

11! "' 

<( 

500 

0 

0 100 200 

Mass (pg) Injected onto Column 

Figure 4.1. Plot of data to determine the DLAP (y = 

6.62x - 7.12). 

4.2 Detection limit of the overall procedure (DLOP) and reliable quantitation limit (RQL) 

The DLOP is measured as mass per sample and expressed as equivalent air concentrations, 

based on the recommended sampling parameters. Ten samplers were spiked with equally 

descending increments of analyte. The highest amount is the amount spiked on the sampler 

that would produce a peak approximately 10 times the response of a sample blank at or near 

the retention time of the analyte. The spiked samplers, and the sample blank were analyzed 

with the recommended analytical parameters, and the data obtained used to determine the 

required parameters (slope and standard error of estimate) for the calculation of the DLOP. For 

2,3-pentanedione values of 1597 and 61.2 were obtained for the slope and standard error of 

estimate respectively. The DLOP was calculated to be 0.11 μg (2.7 ppb or 11 μg/m3

). 

14 Eide, M.; Hendricks, W.; Simmons, M. Guidelines For Air Sampling Methods Utilizing Chromatographic Analysis. 

https://www.osha.gov/dts/sltc/methods/chromguide/chromguide.pdf, OSHA Salt Lake Technical Center, U.S. Department of 

Labor: Salt Lake City, UT, 201 O (accessed January 2010). 

9of17 

Appendix D – 2, 3-Pentanedione 

338 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Table 4.2 

Detection Limit of the Overall Procedure 

mass per sample 

(μg) 

area counts 

(μV•s) 

1500 

0.00 '"" 

0.10 

0.20 

0.31 

0.41 

0.51 

0.61 

0.71 

0.82 

0.92 

1.02 

0 

153 

349 

545 

599 

848 

930 

1063 

1217 

1485 

1731 

:> 

2' 

~ 0 

0 

"' ~ 

<( 

1000 

500 

0 

0 

DLOP RQL 

0.4 0.8 1.2 

Mass per Sample (μg) 

Figure 4.2.1. Plot of data to determine the DLOP/RQL (y 

= 1597x - 3. 74). 

The RQL is considered the lower limit for precise quantitative measurements. It is determined 

from the regression line parameters obtained for the calculation of the DLOP, providing 75% to 

125% of the analyte is recovered. The RQL for 2,3-pentanedione is 0.38 μg per sample (9.3 

ppb or 38 μg/m3 for a TWA sample). Recovery at this concentration is 97.9%. 

When short-term samples are collected, the air concentration equivalent to the reliable 

quantitation limit becomes larger. For example, the reliable quantitation limit for the 

recommended sampler is 31 ppb (127 μg/m3

) when 3 Lis sampled. 

-3.0 

-4.5 

> 5 

Q) 

:!! -6.0 0 

c. 3 "' Q) 

Cl:'. 

-7.5 

-9.0 13 14 

Time (min) 

Figure 4.2.2. A chromatogram of the RQL of 

2,3-pentanedione. (Key: 1) 2,3-pentanedione; 2) 3-

pentanone; and 3) interferant.) 

4.3 Precision of the analytical method 

The precision of the analytical method was measured as the mass equivalent to the standard 

error of estimate determined from the linear regression of data points from standards over a 

range that covers 0.1 to 2 times the TWA target concentration for the sampler. A calibration 

curve was constructed and shown in Section 3.5.2 from the three injections each of five 

standards. The standard error of estimate was 0.49 μg. 

10of17 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 339
 

x target concn 

(μg/sample) 

area counts 

(μV·s) 

0.1 x 

2.04 

3175 

3189 

3091 

Table 4.3 

Instrument Calibration 

0.5x 1.0x 

10.2 20.4 

15104 31328 

15132 30963 

15094 31087 

1.5 x 

30.6 

46035 

45869 

46183 

2.0 x 

40.8 

62945 

63015 

62497 

4.4 Storage stability test 

~ 

~ Q) 

> 

0 

" Q) 

Cl:'. 

120 

90 

60 

Storage samples for 2,3-pentanedione were prepared by sampling a dynamically generated 

controlled test atmosphere using the recommended sampling parameters. The concentration 

of 2,3-pentanedione in the test atmosphere was 0.501 ppm (2.05 mg/m3

) and the relative 

humidity was 80% at 23 °C. Thirty-three storage samples were prepared. Three samples were 

analyzed on the day of generation. Fifteen of the tubes were stored at reduced temperature (4 

°C) and the other fifteen were stored in a closed drawer at ambient temperature (about 23 °C). 

At 3 to 4-day intervals, three samples were selected from each of the two storage sets and 

analyzed. Sample results are not corrected for extraction efficiency. Results for the ambient 

storage test decreased by more than 10% which is a significant uncorrectable bias that must be 

avoided, therefore, samples should be stored in a refrigerator until analyzed, and analysis 

should be completed within two weeks of sampling. Recovery is determined from the 

regression line and the maximum change allowed by OSHA methods development guidelines 

is ±10%. 

Table 4.4 

Storage Test for 2,3-Pentanedione 

time ambient storage refrigerated storage 

(days) recovery(%) recovery(%) 

0 95.1 96.7 97.7 95.1 96.7 97.7 

4 94.4 93.2 95.4 96.6 97.5 95.9 

7 91.2 93.0 94.4 97.1 94.8 96.2 

10 87.8 86.9 91.4 92.5 94.3 93.0 

14 85.1 84.3 86.6 90.8 92.5 93.2 

17 82.4 85.0 83.9 91.4 89.5 92.7 

120 

a e ~ 0 

e - - ~ 

v 90 v 

~ 

~ Q) 

> 60 0 

" Q) 

Cl:'. 

30 2,3-Pentadione Ambient Storage 

y = -0.802x + 97.2 

30 2,3-Pentanedione Refrigerated Storage 

y = -0.359x + 97.4 

Std Error of Estimate = 5.23% 

95% Confidence Limits= ±(1.96)(5.23%) = ±10.3% 

o~~~~~~~~~~~~~~~~ 

0 5 10 15 20 

Storage Time (Days) 

0 

0 

Std Error of Estimate = 5.16% 

95% Confidence Limits= ±(1.96)(5.16%) = ±10.1 % 

5 10 15 

Storage Time (Days) 

20 

Figure 4.4.1. 

pentanedione. 

Ambient storage test for 2,3- Figure 4.4.2. Refrigerated storage test for 2,3-

pentanedione. 

11 of 17 

Appendix D – 2, 3-Pentanedione 

340 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

4.5 Precision (overall procedure) 

The precision of the overall procedure at the 95% confidence level is obtained by multiplying 

the standard error of estimate by 1.96 (the z-statistic from the standard normal distribution at 

the 95% confidence level). Ninety-five percent confidence intervals are drawn about the 

regression lines in the storage stability figures shown in Section 4.4. 

4.5.1 Two dried silica gel tubes in series (SKC 226-183) 

The precision at the 95% confidence for the refrigerated temperature (4 °C) 17-day 

storage test was± 10.1 %. It contains an additional 5% for sampling pump error. 

4.5.2 Recovery 

The recovery of 2,3-pentanedione from samples used in a 17-day storage test 

remained above 91 .3% when samples were stored at 4 °C. 

4.6 Reproducibility 

Six samples were prepared by sampling a dynamically generated controlled test atmosphere 

similar to that used in the collection of the storage samples. The concentrations of 2,3-

pentanedione in the test atmosphere was 0.501 ppm (2.05 mg/m3

) at 78% relative humidity and 

23 °C. The samples were submitted to the OSHA Salt Lake Technical Center for analysis. The 

samples were analyzed after being stored at 4 °C for 4 days. Sample results were corrected for 

extraction efficiency. No sample result had a deviation greater than the precision of the overall 

procedure determined in Section 4.4. 

4.7 Sampler capacity 

theoretical 

(μg/sample) 

20.5 

20.4 

21.0 

20.5 

21.0 

23.0 

Table 4.6 

Reproducibility Data 

recovered recovery 

(μg/sample) (%) 

20.0 97.6 

19.4 95.1 

19.8 94.3 

19.9 97.1 

20.3 96.7 

22.5 97.8 

deviation 

(%) 

-2.4 

-4.9 

-5.7 

-2.9 

-3.3 

-2.2 

The sampling capacity of the front tube of the recommended air sampler (two dried silica gel 

tubes in series) was tested by sampling a dynamically generated controlled test atmosphere 

containing 2,3-pentanedione at two times the target concentration (1.01 ppm or 4.10 mg/m3

) 

and 80% relative humidity at 23 °C. The samples were collected at 50 ml/min. The second 

tube in the sampling train was changed at 3 h then at 0.25 h intervals for the rest of the 

sampling. The presence of analyte on the second tube was defined as breakthrough. The 

percentage of the amount found on the second tube in relation to the concentration of the test 

atmosphere was defined as % breakthrough. The % breakthrough was plotted versus the air 

volume sampled to determine breakthrough air volumes. Breakthrough is considered to have 

occurred when the effluent from the active sampler contains a concentration of analyte that is 

5% of the upstream concentration. The 5% breakthrough air volume for 2,3-pentanedione was 

12.5 L. The recommended air volume is 80% of the breakthrough air volume which is 10 L 

(200 min sampled at 50 ml/min). 

12of17 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 341
 

Table 4.7 

Breakthrough of 2,3-Pentanedione From Front 

Sameling Tube of Recommended Air Sameler 

air sampling downstream breaktest vol time concn through 

no. (L) (min) mg/m3 (%) 

1 9.27 180 0 0.0 

10.8 210 0 0.0 

11.6 225 0 0.0 

12.4 240 0.19 4.63 

13.2 255 2.32 56.6 

2 9.06 180 0 0.0 

10.6 210 0 0.0 

11.3 225 0 0.0 

12.1 240 0.22 5.36 

12.8 255 0.67 16.3 

3 8.69 180 0 0.0 

10.1 210 0 0.0 

10.9 225 0 0.0 

11.6 240 0 0.0 

12.3 255 0.18 4.59 

13.0 270 1.29 31.5 

60 

~ 40 

.c 

Ol 

::J 

e 

~ 

"' 20 ~ 

m 

5% Breakthrough= 12.5 L 

10 15 

Air Volume (L) 

Figure 4. 7. Five percent breakthrough air volume for 

2,3-pentanedione. 

4.8 Extraction efficiency and stability of extracted samples 

The extraction efficiency is affected by the extraction solvent, the internal standard, the 

sampling medium, and the technique used to extract the samples. Other reagents and 

techniques than described in this method can be used provided they are tested as specified in 

the guidelines. 15 

Extraction efficiency 

The extraction efficiency of 2,3-pentanedione was determined by liquid-spiking four front 

sampling tubes of the recommended air sampler at each concentration level. These samples 

were stored overnight at ambient temperature and then analyzed. The overall mean extraction 

efficiency over the working range of 0.1 to 2 times the target concentration was 97.6%. The 

presence of water had no significant effect on extraction efficiency. The extraction efficiencies 

for the RQL and for the wet samplers are not included in the overall mean. Wet media were 

prepared by sampling humid air (78% RH at 23 °C) for 200 min at 50 ml/min. The data 

obtained are shown in Table 4.8.1. 

15 Eide, M.; Hendricks, W.; Simmons, M. Guidelines for Air Sampling Methods Utilizing Chromatographic Analysis; OSHA Web site. 

http://www.osha.gov/dts/sltc/methods/chromguide/chromguide.pdf (accessed 2/24/2010). 

13of17 

Appendix D – 2, 3-Pentanedione 

342 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

initial 

% 

96.6 

96.0 

96.3 

Table 4.8.1 

Extraction Efficiency (%) of 2, 3-Pentanedione 

level sam12le number 

x target μg per 

concn sample 1 2 3 4 mean 

0.1 2.05 98.2 97.1 96.6 98.8 97.7 

0.25 5.12 97.2 98.1 95.4 96.1 96.7 

0.5 10.3 98.4 95.9 97.4 97.6 97.3 

1.0 20.5 96.6 96.0 97.3 98.5 97.1 

1.5 30.8 98.5 98.1 98.9 96.8 98.1 

2.0 40.1 97.4 99.3 99.0 98.4 98.5 

RQL 0.4 98.4 96.5 97.7 99.0 97.9 

1.0 (wet) 20.5 95.3 97.8 96.2 95.0 96.1 

Stability of extracted samples 

The stability of extracted samples was examined by reanalyzing the target concentration 

samples 24, 48, and 72 h after the initial analysis. After the original analysis was performed 

two vials were recapped with new septa which were replaced after each analysis. The 

remaining two vials retained their punctured septa throughout this test. All samples were 

allowed to stand in the autosampler tray at 22 °C. The samples were reanalyzed with freshly 

prepared standards. Diff is the difference between the initial analysis and the subsequent 

analysis. Each septum was punctured 5 times for each analysis. The data obtained are shown 

in Table 4.8.2. 

Table 4.8.2 

Stabilit of Extracted Sam les for 2,3-Pentanedione 

12unctured se12ta re12laced 12unctured se12ta retained 

24 h diff 48 h diff 72 h diff initial 24 h diff 48 h diff 72 h diff 

% % % % % % % % % % % % % 

96.4 -0.2 96.0 -0.6 95.3 -1.3 97.3 98.7 +1.4 97.7 +0.4 95.0 -2.3 

95.0 -1.0 94.8 -1.2 94.3 -1.7 98.5 97.3 -1.2 95.9 -2.6 96.4 -2.1 

(mean) (mean) 

95.7 -0.6 95.4 -0.9 94.8 -1.5 97.9 98.0 +0.1 96.8 -1.1 95.7 -2.2 

4.9 Sampling interferences 

The tested sampling interferences had no significant effect on the ability of the recommended 

sampler to collect or retain 2,3-pentanedione when the samples were protected from exposure 

to light. 

Retention 

Retention was tested by sampling a dynamically generated controlled test atmosphere 

containing two times the target concentration (1 ppm or 4.1 mg!m3) of 2,3-pentanedione at 80% 

relative humidity and 23 °C. The test atmosphere was sampled with the recommended 

sampler at 50 ml/min for 50 min. After 50 min sampling was discontinued and the samplers 

were separated into two sets of 3 samplers each. The generation system was flushed with 

contaminate-free air. Contaminant-free air is laboratory conditioned air at known relative 

humidity and temperature but without any added chemical except water. Sampling was 

resumed with a set of three samples and contaminant-free air at 80% RH and 23 °C was 

14of17 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 343
 

sampled at 50 ml/min for 150 min and then all six samplers were analyzed. The data obtained 

are shown in Tables 4.9.1. 

Table 4.9.1 

Retention of 2,3-Pentanedione 

recovery(%) 

set 2 3 

first 98.4 100.5 98.2 

second 97.7 96.8 95.1 

second/first 

Low humidity 

mean 

99.0 

96.5 

97.5 

The effect of low humidity was tested by sampling a dynamically generated controlled test 

atmosphere containing two times the target concentration (1 ppm or 4.1 mg/m3

) of 2,3-

pentanedione at 20% relative humidity and 23 °C. The test atmosphere was sampled with 

three of the recommended samplers at 50 ml/min for 200 min. All of the samples were 

immediately analyzed. Sample results were 98.8%, 99.1%, and 97.4% of theoretical. 

Low concentration 

The effect of low concentration was tested by sampling a dynamically generated controlled test 

atmosphere containing 0.1 times the target concentration (0.05 ppm or 0.205 mg/m3

) of 2,3-

pentanedione at 80% relative humidity and 23 °C. The test atmosphere was sampled with 

three of the recommended samplers at 0.05 ml/min for 200 min. All of the samples were 

immediately analyzed. Sample results were 98.7%, 97.0%, and 95.8% of theoretical. 

Chemical interference 

The ability of the recommended sampler to collect 2,3-pentanedione was tested when other 

potential interferences are present by sampling an atmosphere containing 0.5 ppm (2.05 

mg/m3

) 2,3-pentanedione at 80% relative humidity and 23 °C and two interferences whose 

concentrations were 0.51 ppm (1.82 mg/m3

) acetoin, and 0.51 ppm (1.78 mg/m3) diacetyl. The 

test atmosphere was sampled with three of the recommended samplers at 50 ml/min for 200 

min. All of the samples were immediately analyzed. Sample results for 2,3-pentanedione were 

97.1 %, 96.3%, and 95.5% of theoretical. 

Light 

2,3-pentanedione is lightsensitive. The interference of 

light during sampling was 

tested using nine foil-wrapped 

samplers and three unwrapped samplers. An 

atmosphere containing 0.5 

ppm (2.05 mg/m3

) 2,3-

pentanedione at an average 

Table 4.9.2 

Effect of Light Exposure While Sampling 

type of sampler light exposure 

no light exposure 

200 min room light 

24 h fluorescent 

3 h sunlight 

97.5 

95.1 

90.7 

39.6 

sample number 

2 3 

98.0 99.1 

96.8 97.9 

91.3 89.0 

42.9 44.6 

mean 

98.2 

96.6 

90.3 

42.4 

humidity of 80% at 23°C was sampled for 200 minutes at 50 ml/min. The three foil-wrapped 

and three unwrapped samples were analyzed immediately and the average recovery for the foil 

wrapped was 98.2% and the un-wrapped sampler average recovery was 96.6%. Three of the 

foil-wrapped samplers had the foil removed after sampling and were exposed to fluorescent 

room lights for 24 h before analysis and had an average recovery of 90.3%. The last three foilwrapped samplers had the foil removed and were exposed to 3 h of sunlight before analysis 

15of17 

Appendix D – 2, 3-Pentanedione 

344 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

and had an average recovery of 42.4%. This data clearly indicates that the sampler should be 

protected from exposure to light. 

To test the possibility of light degradation on extracted samples nine analytical standards at the 

target concentration were prepared. Six of the standards were placed in 2-ml amber glass vials 

and three were placed in 2-ml clear glass vials. Three of the amber vials, along with the clear 

glass vials were stored on the autosampler tray during the entire test while the other three 

amber vials were stored in the refrigerator when not being analyzed. All nine standards were 

analyzed eight times over a 10 day period with none of the septa being replaced during the 

test. The standards in clear vials degraded significantly, but standards in amber vials did not 

degrade. This data clearly indicates that extracted samples should be protected from exposure 

to light. The internal standard, 3-pentanone was stable for up to 9 days in both the clear and 

ambient vials. The data obtained is shown in Table 4.9.3. 

Table 4.9.3 

Extracted Sample Light Exposure Test 

of 2,3-Pentanedione 

mean of peak areas from 3 vials 

day clear vials amber vials amber vials 

ambient (ambient) (refrigerated) 

0 31456 31502 31435 

1 29007 31003 31354 

2 27183 30961 31269 

3 25072 30839 31178 

4 24193 30709 31073 

7 22056 30423 30834 

8 20502 30389 30805 

9 19584 30355 30793 

4.10 Qualitative analysis 

When necessary, the identity or purity of 

an analyte peak can be confirmed by GCmass spectrometry or by another 

analytical procedure. 

The mass spectrum of 2,3-pentanedione 

shown in Figure 4.10 was obtained by 

analysis on an Agilent 7890A GC System 

with a 5975 Mass Selective Detector. 

10000 

8000 

§ 6000 

"' § 

~ 4000 

29 

27 

GC/MS conditions 

oven temperature: 

2000 

0 

0 

15 

J 25 

43 

57 

50 75 

m/z 

initial 35 °C, 

hold 5 min, 

program at 1 O 

°C/min to 270 

°C, hold 0 min 

Figure 4.10. Mass spectrum of 2,3-pentanedione. 

injector temperature: 

transfer line temperature: 

run time: 

240 °C 

250 °C 

29 min 

1.0 ml/min (helium) 

0.5 μL (splitless) 

column gas flow: 

injection size: 

column: 30-m x 0.25-mm i.d. DB-5 capillary column (df = 0.25 μm) 

16of17 

100 

I. 100 

Appendix D – 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 345
 

retention times: 

MS conditions 

MS source temperature: 

MS quad temperature: 

Mass range: 

3.8 min 2,3-pentanedione 

4.2 min 3-pentanone 

230 °C 

150 °C 

12-250 amu 

4.11 Generation of test atmospheres 

The following apparatus was placed in a 

walk-in hood. The test atmospheres 

were generated by pumping low 

microliter volumes of a solution 

containing 2,3-pentanedione in water 

with an ISCO precision LC pump 

through a short length of 0.53-mm 

uncoated fused silica capillary tubing 

into a vapor generator where it was 

heated and evaporated into the dilution 

air stream (Figure 4.11 ). The vapor 

generator consisted of a 15-cm length 

of 5-cm diameter glass tubing with a 

side port for introduction of the capillary 

tubing. The vapor generator was heated 

with a variable voltage controlled 

heating tape to evaporate the 2,3-

ISCO 

pump 

exposure chamber 

active samplers 

vapor 

generator 

Miller Nelson 

mixing 

chamber 

pentanedione. The humidity, Figure 4.11. The test atmosphere generation and 

temperature, and volume of the dilution sampling apparatus. 

air were regulated by use of a Miller 

Nelson Flow-Temperature-Humidity controller. The test atmosphere passed into a glass mixing 

chamber (76-cm x 30-cm) from the vapor generator, and then into a glass exposure chamber 

(76-cm x 20-cm). Active samplers were attached to glass ports extending from the exposure 

chamber. The humidity and temperature were measured at the exit of the exposure chamber 

with an Omega Digital Thermo-hygrometer. The theoretical concentrations were calculated 

from the ISCO pump flow rate, the concentration of the 2,3-pentanedione solution, and the air 

flow volumes. The theoretical concentrations were used throughout this validation. 

17of17 

Appendix D – 2, 3-Pentanedione 

346 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
Appendix E
 

Volatile Organic Compounds (Screening) 2549
 
 

FORMULA see Table 1 MW: see Table 1 CAS: see Table 1 RTECS: see Table 1 

METHOD: 2549, Issue 1 EVALUATION: PARTIAL Issue 1: 15 May 1996 

OSHA: 

NIOSH: varies with compound 

ACGIH: 

SYNONYMS:VOCs; See individual compounds in Table 1 

SAMPLER: 

FLOW RATE: 

VOL-MIN: 

-MAX: 

SHIPMENT: 

SAMPLE 

STABILITY: 

BLANKS: 

RANGE STUDIED: 

BIAS: 

SAMPLING 

THERMAL DESORPTION TUBE 

(multi-bed sorbent tubes containing 

graphitized carbons and carbon molecular 

sieve sorbents [See Appendix]) 

0.01 to 0.05 Umin 

1 L 

6L 

Ambient in storage containers 

Compound dependent (store@ -10 °C) 

1 to 3 per set 

ACCURACY 

not applicable 

not applicable 

OVERALL PRECISION $,r): not applicable 

ACCURACY: not applicable 

PROPERTIES: 

TECHNIQUE: 

ANALYTE: 

DESORPTION: 

INJECTION 

VOLUME: 

See Table 1 

MEASUREMENT 

THERMAL DESORPTION, GAS 

CHROMATOGRAPHY, MASS 

SPECTROMETRY 

See Table 1 

Thermal desorption 

Defined by desorption split flows (See 

Appendix) 

TEMPERATURE-DESORPTION: 300 °C for 10 min. 

-DETECTOR (MS): 280 °c 

-COLUMN: 35 °C for 4 min; 8 °C/min 

CARRIER GAS: 

COLUMN: 

CALIBRATION: 

RANGE: 

ESTIMATED LOO: 

PRECISION ~,): 

Helium 

to 150 °C, 15 °C/min to 

300 °C 

30 meter DB-1, 0.25-mm ID, 1.0-μm 

film, or equivalent 

Identification based on mass spectra 

interpretation and computerized library 

searches. 

not applicable 

100 ng per tube or less 

not applicable 

APPLICABILITY:This method has been used for the characterization of environments containing mixtures of volatile organic compounds 

(See Table 1 ). The sampling has been conducted using multi-bed thermal desorption tubes. The analysis procedure has been able to 

identify a wide range of organic compounds, based on operator expertise and library searching. 

INTERFERENCES: Compounds which coelute on the chromatographic column may present an interference in the identification of each 

compound. By appropriate use of background subtraction, the mass spectrometrist may be able to obtain more representative spectra 

of each compound and provide a tentative identity (See Table 1 ). 

OTHER METl-DDS: Other methods have been published for the determination of specific compounds in air by thermal desorption/gas 

chromatography [1-3]. One of the primary differences in these methods is the sorbents used in the thermal desorption tubes. 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

348 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

REAGENTS: 

1. Air, dry 

2. Helium, high purity 

3. Organic compounds of interest for mass spectra 

verification (See Table 1 ).* 

4. Solvents for preparing spiking solutions: carbon 

disulfide (low benzene chromatographic grade), 

methanol, etc.(99+% purity) 

* See SPECIAL PRECAUTIONS 

EQUIPMENT: 

1. Sampler: Thermal sampling tube, %" s.s. tube, 

multi-bed sorbents capable of trapping organic 

compounds in the C3-C16 range. Exact sampler 

configuration depends on thermal desorber 

system used. See Figure 1 for example. 

2. Personal sampling pump, 0.01 to 0.05 L/min, with 

flexible tubing. 

3. Shipping containers for thermal desorber 

sampling tubes. 

4. Instrumentation: thermal desorption system, 

focusing capability, desorption temperature 

appropriate to sorbents in tube (-300 °C), and 

interfaced directly to a GC-MS system. 

5. Gas chromatograph with injector fitted with 1/4" 

column adapter, 1/4" Swagelok nuts and Teflon 

ferrules (or equivalent). 

6. Syringes: 1-μL, 10-μL (liquid); 

100-μL, 500-μL (gas tight) 

7. Volumetric Flasks, 10-ml. 

8. Gas bulb, 2 L 

SPECIAL PRECAUTIONS:Some solvents are flammable and should be handled with caution in a fume 

hood. Precautions should be taken to avoid inhalation of the vapors from solvents as well. Skin contact 

should be avoided. 

SAMPLING: 

NOTE: Prior to field use, clean all thermal desorption tubes thoroughly by heating at or above the 

intended tube desorption temperature for 1-2 hours with carrier gas flowing at a rate of at least 

50 ml/min. Always store tubes with long-term storage caps attached, or in containers that 

prevent contamination. Identify each tube uniquely with a permanent number on either the 

tube or tube container. Under no circumstances should tape or labels be applied directly to 

the thermal desorption tubes. 

1. Calibrate each personal sampling pump with a representative sampler in line. 

2. Remove the caps of the sampler immediately before sampling. Attach sampler to personal sampling 

pump with flexible tubing. 

NOTE: With a multi-bed sorbent tube, it is extremely important to sample in the correct direction, from 

least to maximum strength sorbent. 

3. For general screening, sample at 0.01 to 0.05 L/min for a maximum sample volume of 6 L. Replace 

caps immediately after sampling. Keep field blanks capped at all times. Tubes can act as diffusive 

samplers if left uncapped in a contaminated environment. 

4. Collect a "humidity test" sample to determine if the thermal adsorption tubes have a high water 

background. 

NOTE: At higher sample volumes, additional analyte and water (from humidity) may be collected on 

the sampling tube. At sufficiently high levels of analyte or water in the sample, the mass 

spectrometer may malfunction during analysis resulting in loss of data for a given sample. 

5. Collect a "control" sample. For indoor air samples this could be either an outside sample at the same 

location or an indoor sample taken in a non-complaint area. 

6. Ship in sample storage containers at ambient temperature. Store at -10 °C. 

SAMPLE PREPARATION: 

7. Allow samples to equilibrate to room temperature prior to analysis. Remove each sampler from is 

storage container. 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 349
 

8. Analyze "humidity test" sampler first to determine if humidity was high during sampling (step 10). 

9. If high humidity, dry purge the tubes with purified helium at 50 to 100 ml/min for a maximum of 3 Lat 

ambient temperature prior to analysis .. 

10. Place the sampler into the thermal desorber. Desorb in reverse direction to sampling flow. 

CALIBRATION AND QUALITY CONTROL: 

11. Tune the mass spectrometer according to manufacturer's directions to calibrate. 

12. Make at least one blank run prior to analyzing any field samples to ensure that the TD-GC-MS system 

produces a clean chromatographic background. Also make a blank run after analysis of heavi~ 

concentrated samples to prevent any carryover in the system. If carryover is observed, make additional 

blank runs until the contamination is flushed from the thermal desorber system. 

13. Maintain a log of thermal desorber tube use to record the number of times used and compounds found. 

If unexpected analytes are found in samples, the log can be checked to verify if the tube may have been 

exposed to these analytes during a previous sampling use. 

14. Run spiked samples along with the screening samples to confirm the compounds of interest. To 

prepare spiked samples, use the procedure outlined in the Appendix . 

MEASUREMENT: 

15. See Appendix for conditions. MS scan range should cover the ions of interest, typically from 20 to 300 

atomic mass units (amu). Mass spectra can either be identified by library searching or by manua 

interpretation (see Table 1). In all cases, library matches should also be checked for accurate 

identification and verified with standard spikes if necessary. 

EVALUATION OF METHOD: 

The method has been used for a number of field screening evaluations to detect volatile organic compounds. 

Estimate of the limit of detection for the method is based on the analysis of spiked samples for a number of 

different types of organic compounds. For the compounds studied, reliable mass spectra were collected at a 

level of 100 ng per compound or less. In situations where high levels of humidity may be present on the 

sample, some of the polar volatile compounds may not be efficiently collected on the internal trap of the 

thermal desorber. In these situations, purging of the samples with 3 L of helium at 100 ml/min removed the 

excess water and did not appreciably affect the recovery of the analytes on the sample. 

REFERENCES: 

[1] Health and Safety Executive [1992]. MOHS 72 - Volatile organic compounds in air. Methods for the 

determination of hazardous substances. HMSO: London: ISBN 0-11-885692-8. 

[2] McCaffrey CA, Maclachlan J, Brookes Bl [1994]. Adsorbent tube evaluation for the preconcentration 

of volatile organic compounds in air for analysis by gas chromatography-mass spectrometry. Analyst 

119:897-902. 

[3] Bianchi AP, Varney MS [1992]. Sampling and analysis of volatile organic compounds in estuarine air 

by gas chromatography and mass spectrometry. J. Chromatogr. 643:11-23. 

[4] EPA [1984]. Environmental Protection Agency Air Toxics Method T01. Rev. 1.0 (April, 1984): Method 

for the determination of volatile organic compounds in ambient air using Tenax(R) adsorption and gas 

chromatography/mass spectrometry (GC/MS), Section 13. 

METHOD WRITTEN BY: 

Ardith A. Grote and Eugene R. Kennedy, Ph.D., NIOSH, DPSE 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

350 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

TABLE 1. COMMON VOLATILE ORGANIC COMPOUNDS WITH MASS SPECTRAL DATA 

Compound CAS# Empirical MW• BPb VPC@25 °C Characteristic 

/Synonyms RTE CS Formula (DC) mm Hg kPa Ions, m/z 

Aromatic Hydrocarbons 

Benzene 71-43-2 CsHs 78.11 80.1 95.2 12.7 78* 

/benzol CY1400000 

Xylene 1330-20-7 CaH10 106.7 91, 106*, 105 

/dimethyl benzene ZE2100000 

a-xylene 144.4 6.7 0.9 

m-xylene 139.1 8.4 1.1 

p-xylene 138.4 8.8 1.2 

Toluene 108-88-3 C1Hs 92.14 110.6 28.4 3.8 91, 92* 

/toluol XS5250000 

Aliphatic Hydrocarbons 

n-Pentane 109-66-0 CsH12 72.15 36.1 512.5 68.3 43, 72*, 57 

RZ9450000 

n-Hexane 110-54-3 CsH14 86.18 68.7 151.3 20.2 57, 43, 86*,41 

/hexyl-hydride MN9275000 

n-Heptane 142-82-5 C1H1s 100.21 98.4 45.8 6.1 43, 71, 57, 

Ml7700000 100*,41 

n-Octane 111-65-9 CaH18 114.23 125.7 14.0 1.9 43, 85, 114*, 57 

RG8400000 

n-Decane 124-18-5 C10H22 142.29 174 1.4 0.2 43,57,71,41, 

/decyl hydride HD6500000 142* 

Ketones 

Acetone 67-64-1 C3H60 58.08 56 266 35.5 43, 58* 

/2-propanone AL3150000 

2-Butanone 78-93-3 C4H80 72.11 79.6 100 13 43, 72* 

/methyl ethyl ketone EL6475000 

Methyl isobutyl ketone 108-10-1 CsH120 100.16 117 15 2 43, 100*, 58 

/MIBK, hexane SA9275000 

Cyclohexanone 108-94-1 C6H100 98.15 155 2 0.3 55, 42, 98*, 69 

/cyclohexyl ketone GW1050000 

Alcohols 

Methanol 67-56-1 CHPH 32.04 64.5 115 15.3 31, 29, 32* 

/methyl alcohol PC1400000 

Ethanol 64-17-5 C2H50H 46.07 78.5 42 5.6 31, 45, 46* 

/ethyl alcohol KQ6300000 

lsopropanol 67-63-0 C3Hy0H 60.09 82.5 33 4.4 45, 59, 43 

/1-methyl ethanol NT8050000 

Butanol 71-36-3 C4H90H 74.12 117 4.2 0.56 56, 31, 41, 43 

/butyl alcohol E01400000 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 351
 

Compound CAS# Empirical MW• BPb VPC@25 °C Characteristic 

/Synonyms RTE CS Formula (DC) mm Hg kPa Ions, m/z 

Glycol Ethers 

Butyl cellosolve 111-76-2 CsH14 02 118.17 171 0.8 0.11 57, 41, 45, 75, 

/2-butoxyethanol KJ8575000 87 

Diethylene glycol ethyl 111-90-0 CsH1403 134.17 202 0.08 0.01 45, 59, 72, 73, 

ether /Carbitol KK8750000 75, 104 

Phenolics 

Phenol 108-95-2 C6H50H 94.11 182 47 0.35 94*, 65,66, 39 

/hydroxybenzene SJ3325000 

Cresol 1319-77-3 C7HyOH 108.14 108*, 107, 77, 

G05950000 79 

2-methylphenol 95-48-7 190.9 1.9 0.25 

3-methylphenol 108-39-4 202.2 1.0 0.15 

4-methylphenol 106-44-5 201.9 0.8 0.11 

Chlorinated Hydrocarbons 

Methylene chloride 75-09-2 CH2Cl2 84.94 40 349 47 86*, 84,49, 51 

/dichloromethane PA8050000 

1, 1, 1-Trichloroethane 71-55-6 CCl3CH3 133.42 75 100 13.5 97, 99, 117, 

/methyl chloroform KJ2975000 119 

Perchloroethylene 127-18-4 CCl3CCl3 236.74 187 0.2 <0.1 164*, 166, 168, 

/hexachloroethane KX3850000 (subl) 129, 131, 133, 

94, 96 

0-,p- C6H4Cl2 147.0 146*, 148, 111, 

Dichlorobenzenes 113, 75 

/1,2-dichlorobenzene 95-50-1 172-9 1.2 0.2 

CZ4500000 

/1,4- 106-46-7 173.7 1.7 0.2 

dichlorobenzene CZ4550000 

1, 1,2-Trichloro-1,2,2- 76-13-1 CCl2FCCIF2 187.38 47.6 384 38 101, 103, 151, 

trifluoroethane KJ4000000 153, 85, 87 

/Freon 113 

Terpenes 

d-Limonene 5989-27-5 C10H1s 136.23 176 1.2 68, 67, 93, 121, 

OS8100000 136* 

Turpentine (Pinenes) 8006-64-2 C10H1s 136.23 156 to 4@ 93, 121, 136*, 

170 20° 91 

a-pinene 80-56-8 156 

~-pinene 127-91-3 165 

Aldehydes 

Hexanal 66-25-1 CsH120 100.16 131 10 1.3 44, 56, 72, 82, 

/caproaldehyde MN7175000 41 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

352 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Compound 

/Synonyms 

Benzaldehyde 

/benzoic aldehyde 

Nonanal 

/pelargonic aldehyde 

Acetates 

Ethyl acetate 

/acetic ether 

Butyl acetate 

/acetic acid butyl 

ester 

Amyl acetate 

/banana oil 

other 

Octamethylcyclotetrasiloxane 

• Molecular Weight 

b Boiling Point 

c Vapor Pressure 

*Indicates molecular ion 

APPENDIX 

CAS# Empirical 

RTE CS Formula 

100-52-7 C1H120 

CU4375000 

124-19-6 C9H180 

RA5700000 

141-78-6 C4Ha02 

AH5425000 

123-86-4 CsH1202 

AF7350000 

628-63-7 C1H1402 

AJ1925000 

556-67-2 CaH2404Si4 

GZ4397000 

MW• BPb VPC@25 °C Characteristic 

(DC) mm Hg kPa Ions, m/z 

106.12 179 1.0 0.1 77, 105, 106*, 

51 

142.24 93 23 3 43, 44, 57, 98, 

114 

88.1 77 73 9.7 43, 88*, 61, 70, 

73, 45 

116.16 126 10 1.3 43, 56, 73, 61 

130.18 149 4 0.5 43, 70, 55, 61 

296.62 175 281,282, 283 

Multi-bed sorbent tubes: Other sorbent combinations and instrumentation/conditions shown to be equivalent 

may be substituted for those listed below. In particular, if the compounds of interest are known, specifc 

sorbents and conditions can be chosen that work best for that particular compound(s). The tubes that have 

been used in NIOSH studies with the Perkin Elmer ATD system are %" stainless steel tubes, and are shown 

in the diagram below: 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 353
 

Figure 1 

Direction of Desorption Flow 

Carbopack Y 

40/60 Mesh 

90 mg 

Carbopack B 

40/60 Mesh 

115 mg 

Silanized Glass Wool 

Direction of Sample Flow 

Carboxen 1003 

40/60 Mesh 

150 mg 

CarbopackTM and CarboxenTM adsorbents are available from Supelco, Inc. 

Preparation of spiked samples Spiked tubes can be prepared from either liquid or gas bulb standards. 

Liquid standards: Prepare stock solutions by adding known amounts of analytes to 10-ml volumetric 

flasks containing high purity solvent (carbon disulfide, methanol, toluene). Solvents are chosen based 

on solubility for the analytes of interest and ability to be separated from the analytes when 

chromatographed. Highly volatile compounds should be dissolved in a less volatile solvent. For most 

compounds, carbon disulfide is a good general purpose solvent, although this will interfere with early 

eluting compounds. 

Gas bulb standards: Inject known amounts of organic analytes of interest into a gas bulb of known 

volume filled with clean air [4]. Prior to closing the bulb, place a magnetic stirrer and several glass beads 

are placed in the bulb to assist in agitation after introduction of the analytes. After injection of all of the 

analytes of interest into the bulb, warm the bulb to 50 °C and place it on a magnetic stirring plate and stir 

for several minutes to ensure complete vaporization of the analytes. After the bulb has been stirred and 

cooled to room temperature, remove aliquots from the bulb with a gas syringe and inject into a sample 

tube as described below. 

Tube spiking Fit a GC injector with a%" column adapter. Maintain the injector at 120 °C to assist in 

vaporization of the injected sample. Attach cleaned thermal desorption tubes to injector with%" 

Swagelok nuts and Teflon ferrules, and adjust helium flow though the injector to 50 ml/min. Attach the 

sampling tube so that flow direction is the same as for sampling. Take an aliquot of standard solution 

(gas standards 100 to 500 μL; liquid standards, 0.1 to 2 μL) and inject into the GC injector. Allow to 

equilibrate for 10 minutes. Remove tube and analyze by thermal desorption using the same conditions 

as for field samples. 

lnstrumentation:Actual media, instrumentation, and conditions used for general screening of unknown 

environments are as follows: Perkin-Elmer ATD 400 (automated thermal desorption system) interfaced 

directly to a Hewlett-Packard 5980 gas chromatograph/HP5970 mass selective detector and data system. 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

354 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

A TD conditions: 

Tube desorption temperature: 300°C 

Tube desorption time: 10 min. 

Valve/transfer line temperatures: 150°C 

Focusing trap: Carbopack B/Carboxen 1000, 60180 mesh, held at 27°C during tube desorption 

Focusing trap desorption temperature: 300°C 

Desorption flow: 50-60 ml/min. 

Inlet split: off 

Outlet split: 20 ml/min. 

Helium: 10 PSI 

GC conditions: 

DB-1 fused silica capillary column, 30 meter, 1-μm film thickness, 0.25-mm l.D. 

Temperature program: Initial 35°C for 4 minutes, ramp to 100°C at 8°/min., then ramp to 300°C at 

15°/min, hold 1-5 minutes. 

Run time: 27 min. 

MSD conditions: 

Transfer line: 280°C 

Scan 20-300 amus, El mode 

EMV: set at tuning value 

Solvent delay: 0 min. for field samples; if a solvent-spiked tube is analyzed, a solvent delay may be 

necessary to prevent MS shutdown caused by excessive pressure. 

NIOSH Manual of Analytical Methods (NMAM), Fourth Edition, 5/15/96 

Appendix E – Volatile Organic Compounds (Screening) 2549
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 355
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Appendix F
 

Correcting Diacetyl Concentrations from Air 


Samples Collected with NIOSH Method 2557
 
 

Journal of Occupational and Environmental Hygiene, 8: 59-70 

ISSN: 1545-9624 print I 1545-9632 online 

DOI: 10.1080/15459624.2011.540168 

Correcting Diacetyl Concentrations from Air Samples 

Collected with NIOSH Method 2557 

Jean Cox-Ganser, 1 Gary Ganser,2 Rena Saito, 1 Gerald Hobbs,3 

Randy Boylstein, 1 Warren Hendricks,4 Michael Simmons,4 Mary Eide,4 

Greg Kullman, 1 and Chris Piacitelli 1 

1Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, 

Morgantown, West Virginia 2Department of Mathematics, West Virginia University, Morgantown, West Virginia 3Department of Statistics, West Virginia University, Morgantown, West Virginia 4Salt Lake Technical Center, Occupational Safety and Health Administration, Sandy, Utah 

Diacetyl (2,3-butanedione), a diketone chemical used to 

impart a buttery taste in many flavoring mixtures, has been 

associated with bronchiolitis obliterans in several industrial 

settings. For workplace evaluations in 2000-2006, National 

Institute for Occupational Safety and Health (NIOSH) investigators used NIOSH Method 2557, a sampling and analytical 

method for airborne diacetyl utilizing carbon molecular sieve 

sorbent tubes. The method was subsequently suspected to progressively underestimate diacetyl concentrations with increasing sampling site humidity. Since underestimation of worker 

exposure may lead to overestimation of respiratory health risk 

in quantitative exposure-effect analyses, correction of the diacetyl concentrations previously reported with Method 2557 is 

essential. We studied the effects of humidity and sample storage 

duration on recovery of diacetyl from experimental air samples 

takenfrom a dynamically generated controlled test atmosphere 

that allowed control of diacetyl concentration, temperature, 

relative humidity, sampling duration, and sampling flow rate. 

Samples were analyzed with Method 2557, and results were 

compared with theoretical test atmosphere diacetyl concentration. After fitting nonlinear models to the experimental data, we 

found that absolute humidity, diacetyl concentration, and days 

of sample storage prior to extraction affected diacetyl recovery 

as did sampling flow rate to a much smaller extent. We derived 

a mathematical correction procedure to more accurately estimate historical workplace diacetyl concentration based on 

laboratory-reported concentrations of diacetyl using Method 

2557, and sample site temperature and relative humidity (to 

calculate absolute humidity), as well as days of sample storage 

prior to extraction in the laboratory. With this correction procedure, quantitative risk assessment for diacetyl can proceed 

using corrected exposure levels for air samples previously collected and analyzed using NIOSH Method 2557 for airborne 

diacetyl. 

Keywords correction equation, diacetyl, humidity effect, sample 

storage effect 

Correspondence to: Jean Cox-Ganser, Field Studies Branch, Division of Respiratory Disease Studies, National Institute for Occupational Safety and Health, Centers for Disease Control and Prevention, 

1095 Willowdale Road, MS 2800, Morgantown, WV 26505-2888; 

e-mail: jjc8@cdc.gov. 

The findings and conclusions in this report are those of the authors 

and do not necessarily represent the views of the National Institute for 

Occupational Safety and Health or Occupational Safety and Health 

Administration. 

INTRODUCTION 

D iacetyl (2,3-butanedione, CAS no. 431-03-8), a diketone chemical used to impart a buttery taste in many 

flavoring mixtures, has been associated with severe respiratory 

disease in several different occupational settings, including 

microwave popcorn manufacturing, flavoring production, and 

diacetyl manufacturing_Cl-3l Laboratory animal studies have 

documented that diacetyl alone has toxic properties that are 

similar to the effects of exposure to diacetyl-containing artificial butter flavoring mixtures.C4•

5l The Occupational Safety and 

Health Administration (OSHA) is in the process ofrulemaking 

on occupational exposure to diacetyl. 

National Institute for Occupational Safety and Health 

(NIOSH) researchers developed and published an analytical 

method, NIOSH Method 2557, to measure airborne diacetyl in 

the workplace.C6

-

7

l This method specifies air sample collection 

through carbon molecular sieve (CMS) sorbent tubes, followed 

by extraction with acetone/methanol (99: 1) and analysis by 

gas chromatography with flame ionization detection (GC/FID) 

within 7 days of sampling. Subsequent to the use of this 

sampling method in several workplace investigations, NIOSH 

researchers found that the method appeared to progressively 

Journal of Occupational and Environmental Hygiene February 2011 59 

Appendix 2 Page 1 Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

358 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

underestimate diacetyl concentrations with increasing sampling site humidity as compared with OSHA Method PV2118. CS) 

Silica gel is used as the collection medium in the OSHA 

method. C

9l NIOSH Method 2557 should not be used to measure 

airborne diacetyl in future studies. 

We studied the effect of humidity on measured diacetyl air 

concentrations using NIOSH Method 2557 with the aim of 

developing a means for mathematically correcting previously 

obtained measurements of airborne diacetyl. In addition, we 

investigated sample storage stability over time because we 

were aware that some previously obtained field samples had 

been analyzed beyond the method's specified 7-day maximum 

storage duration. 

METHODS 

Protocol 

The initial objective of our experiments was to determine if 

sampling site humidity affects diacetyl recovery in air samples 

and, if so, to develop a mathematical procedure to correct 

existing diacetyl air sampling data from previous workplace 

studies for those effects. NIOSH and OSHA investigators 

conducted a total of 6 weeks of tests during five visits by 

NIOSH investigators to the OSHA Salt Lake Technical Center 

(SLTC) laboratory. During the first week of tests, we started 

to investigate the effect of humidity and sampling flow rate, as 

well as the homogeneity of diacetyl mixing in the dynamically 

generated controlled test atmosphere. During the second and 

third weeks, we investigated effects of temperature, sampling 

duration, sampling flow rate, and test atmosphere diacetyl 

concentration on diacetyl recovery. 

Based on results of the first 3 weeks of tests, during the 

following 2-week test period, we ran tests to further evaluate 

the effect of test atmosphere diacetyl concentration. In addition, during that 2-week test period we studied sample storage 

stability using a single test atmosphere diacetyl concentration. 

Based on the sample storage stability results, we further evaluated the test atmosphere diacetyl concentration effect during 

a final week of tests. Since we found an effect of sample 

storage duration on diacetyl recovery, which was dependent 

on both humidity and test atmosphere diacetyl concentration, 

the primary objective was extended to include this effect in the 

mathematical correction procedure. 

During each of the five visits, we also collected a number of 

samples using OSHA Method PV2118 (OSHA 1013CIO) was 

used once it became available) to compare with test atmosphere 

diacetyl concentration. 

Test Atmosphere Generation 

Test atmospheres of diacetyl were generated at the OSHA 

SLTC laboratory by pumping an aqueous diacetyl solution 

(approximately 1 to 100% diacetyl depending on target concentration), using a syringe pump (Series D; Teledyne Isco 

Inc., Lincoln, Neb.), through a short length of 0.53 mm diameter uncoated fused silica capillary tubing into a vapor 

generator where it was heated and evaporated into a dilution 

Syringe 

pump 

Glass exposure chamber 

Sampling ports Glass 

Heated 

vapor 

generator 

mixing 

chamber 

Flow/temperature/RH 

controller 

FIGURE 1. Test atmosphere generation apparatus. 

airstream (Figure 1). The vapor generator, a 20 cm length of 

3 cm diameter glass tubing with a side port for introduction of the capillary tubing, was wrapped with heating tape 

to evaporate the solution. Humidity, temperature, and volume of the dilution stream of air were regulated by use of a 

flow-temperature-humidity control system (Model HCS-401; 

Miller-Nelson Instruments Inc., Pleasanton, Calif.). 

The diacetyl-laden air passed from the vapor generator into 

a glass mixing chamber (76 cm length x 15 cm diameter) and 

then into a glass exposure chamber (76 cm length x 8 cm 

diameter). Eighteen evenly spaced glass tube sampling ports 

extended from the exposure chamber: nine from the bottom 

and nine from a side. The temperature and relative humidity 

were measured at the exit of the exposure chamber with a 

digital thermo-hygrometer (Model RH-411; Omega Engineering, Inc., Stamford, Conn.). The test atmosphere generation 

apparatus was located in a walk-in hood. Theoretical test 

atmosphere concentrations of diacetyl were derived using mass 

flow calculations. These calculations used syringe pump flow 

rate, chamber airflow rate, and diacetyl concentration in the 

aqueous solution. 

Sampling Procedure 

CMS sorbent tubes (Anasorb CMS 226-121; SKC, Eighty 

Four, Pa.) and pairs (in series) of SKC Model 226-183 silica 

gel sorbent tubes were attached to the sampling ports, and the 

test atmosphere was pulled via vacuum through the sorbent 

tubes with sampling flow rate controlled by adjustable orifices. 

For each test, flow through each sorbent tube was pre- and postcalibrated with a flowmeter (Model 4100; TSI, Inc., Shoreview, 

Minn.). After sampling, the sorbent tubes were immediately 

capped, wrapped in foil, and placed on ice packs in a cooler 

along with blank sorbent tubes. The coolers were shipped 

nightly via express mail to a NIOSH-contracted analytical 

laboratory, where the sorbent tubes were extracted on arrival on 

Day 1 after sampling (or later, as directed for a few sets of CMS 

tubes used for storage stability experiments) and analyzed by 

GC/FID. 

60 Journal of Occupational and Environmental Hygiene February 2011 

Appendix 2 Page 2 Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 359
 

TABLE I. Test Atmosphere Conditions and Sample Numbers 

Actual Diacetyl Actual Target Concentration Actual AH RH Actual Sampling Sampling Number 

Target Diacetyl Mean Range Range (mg Range Temperature Duration Flow Rate of 

Concentration (ppm) (ppm) (ppm) H20/L air) (%) Range (

0

C) (hr) (cdmin) Samples! 

Humidity Test Samples 

0.2 0.23 0.23-0.24 4.69-19.12 21-81 23.9-26.2 4,8 50, 150 87 

0.5 0.58 0.56-0.60 3.51-19.26 17-91 22.6-26.3 2,4,8 50, 150 107 

1.0 1.1 1.1 6.99-14.92 29-62 25.8-26.0 2 50, 150 41 

5.0 5.5 5.0-5.9 3.65-22.50 17-92 22.8-27.0, 2,4,8 50, 150 373 

31.9-33.8 

25 24.8 24.5-25.7 3.57-19.06 16-92 22.4-26.1 2 50, 150 109 

Stability Test SamplesB 

0.5 0.57 0.57-0.58 3.51-18.17 17-91 22.6-23.3 4,8 50 54 

5.0 5.6 5.6-5.7 3.65-18.67 17-92 22.8-25.7 2 50 107 

25 25.0 24.9-25.1 3.57-18.66 18-92 22.4-22.8 2 50 53 

A Number of samples used in equation development analyses. 

8 Nine each of the 0.5 and 25 ppm samples and 18 of the 5.0 ppm samples were used in both humidity and storage stability analyses. 

Sampling Test Conditions 

Samples were collected between January 2008 and December 2009 during four 1-week periods and one 2-week 

period of tests. We collected a total of 964 CMS tube samples during 80 tests, with relative humidity (RH) levels ranging from 16 to 92% and temperatures of 22.4 to 33.8°C 

giving absolute humidity (AH) levels ranging from 3.5 to 

22.5 mg H20/L air and with diacetyl concentrations ranging 

from 0.23 to 25.7 ppm. Samples were collected over 2, 4, 

or 8 hr to test for differences in diacetyl recovery due to 

sampling duration or because of limit of detection (LOD) 

concerns during tests at low diacetyl concentrations. Samples were collected using sampling flow rates of 50 or 150 

cc/min to investigate any effect on diacetyl recovery associated with differences in sampling flow rate. The test atmosphere conditions and sample numbers are summarized in 

Table I. 

Over the five visits, we collected 134 silica gel samples at 

a flow rate of 50 cc/min during 43 of the 2-hr tests. These 

samples were collected with an AH range of 3.57 to 22.50 mg 

H20/L air and diacetyl concentrations from 0.56 to 25.7 ppm. 

Sample Storage Stability Tests 

In total, storage stability of diacetyl both in the sampling 

tubes (in-tube) and after extraction from the tubes was investigated using 214 samples (Table I). In the first set of 

experimental conditions, six sets of triplicate samples were 

collected at 50 cc/min from a 5.7 ppm diacetyl test atmosphere 

at each of three AH levels: 3.97, 8.59, and 18.67 mg H20/L 

air (RH= 17, 36, and 78%, respectively, at 25.7°C). Samples 

were sent overnight on ice to the analytical laboratory, where 

they were extracted and analyzed according to NIOSH Method 

2557 for diacetyl 1, 4, 7, 10, 13, and 16 days post-sampling. 

All samples were stored in a refrigerator until the scheduled 

day of extraction. 

After analysis of the first set of samples on Day 1 postsampling, the remaining liquid portion (without sorbent material) of each sample was split into two new vials and one stored 

at room temperature and the other refrigerated. These samples 

underwent further stability testing via re-analysis 1, 2, 5, and 

11 days post-extraction. New septum caps were placed on each 

vial after each analysis, and freshly prepared standards were 

used for each re-analysis. To investigate diacetyl concentration 

effect on storage stability, during the final week of tests, six 

sets of triplicate samples each were collected from 0.57, 5.6, 

and 25.0 ppm diacetyl test atmospheres at each of three mean 

AH levels: 3.6, 8.5, and 18.5 mg H20/L air. The samples 

were extracted and analyzed 1, 4, 7, 10, 16, and 35 days postsampling. When splitting the samples for the extract storage 

stability tests, equal portions of the sorbent material were 

placed into the two vials with the liquid to better simulate 

treatment of field samples as directed in Method 2557. Reanalysis of these samples was completed on Days 2, 5, 13, and 

34 post-extraction. 

Data Analyses 

Statistical analysis was carried out using JMP V.8 software 

(SAS Institute, Cary, N.C.). We used the nonlinear modeling 

platform to calculate the parameter coefficients for the correction model. Details of models used in the JMP nonlinear 

platform are discussed in the Results section. We used analysis 

of variance modeling to investigate effects of sampling port 

position, sampling duration, and sampling flow rate on percent 

diacetyl recovered. 

Journal of Occupational and Environmental Hygiene February 2011 61 

Appendix 2 Page 3 Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

360 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

RESULTS 

0 f 964 CMS samples collected, 717 were used in humidity 

effect analyses (extraction Day 1 after sampling), 214 

were used in sample storage stability analyses (36 of these were 

used in both analyses), 42 samples from 1 day of tests were 

excluded due to excessive analytical laboratory variability (the 

mean coefficient of variation for that day's tests was 73% as 

compared with a range of 3% to 23% for other days), 13 were 

excluded due to greater than 5% changes in sampling flow 

rate during the tests, 3 were excluded due to errors during 

sampling, 1 had missing data from the analytical laboratory, 

1 outlier (greater than 300% recovery) was excluded, and 9 

(a) 

100 

~ • 

~ 90 

80. t • I ~ I• 

• • 70 • ~ • • • ~ 60 • 'I' Q) • 1 6 • u 50 • ~ • ~ 40 • I Q) 

• u • Cll 

• 

• 

I 0 30 I . ~· ·I •• 20 

10 

0 

10 20 30 40 

samples collected at low concentration and high humidity were 

excluded because of nondetectable diacetyl. 

Of the 134 silica gel samples, 121 that had matching CMS 

sample groups during 39 tests were used in the comparison 

analyses. 

Effects of Sampling Port Position, Sampling 

Duration, and Sampling Flow Rate 

During the first week of tests, homogeneous mixing of diacetyl in the exposure chamber was investigated, and analysis 

of variance indicated no significant effects of sampling port 

position on diacetyl recovery. An analysis of variance model 

using data from the first three laboratory visits (n = 448) with 

•25°C 

032°C 

• i ~ ' • • 

• • • 50 60 70 80 

RH(%) 

(b) 

100 

90' 9 80 ij 

• 11· •25°C 

• • •32°C 

~ 70 : . .~ 60 • !J Q) 

6 

u • • 50 ~ • • ~ 40 • 

' Q) 

u • • Cll 

0 30 ''I • I 20 • •• I I • • I • 10 

• • 0 

2 4 6 8 10 12 14 16 18 20 22 24 

AH (mg H20 IL air) 

FIGURE 2. Plots for 5.0 ppm target diacetyl concentration data from 25°C and 32°C showing diacetyl recovery in terms of (a) RH and (b) AH. 

62 Journal of Occupational and Environmental Hygiene 

Appendix 2 Page 4 

February 2011 

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Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 361
 

percent diacetyl recovery as the outcome variable and AH, test 

atmosphere diacetyl concentration, target sampling flow rate, 

and sampling duration as the predictor variables indicated a 

significant (p = 0.0042) effect of sampling flow rate, with 

percent diacetyl recovery being higher for the 150 cc/min 

sampling flow rate than for 50 cc/min. The magnitude of the 

effect was not large; the adjusted means (least squares means) 

for 150 cc/min and 50 cc/min were 44.9 and 40.3% diacetyl 

recovery, respectively. In this model there was no significant 

effect for sampling duration (p = 0.89), with adjusted means of 

42.3, 41.8, and 43. 7% diacetyl recovery for sampling durations 

of 2, 4, and 8 hr, respectively. 

Absolute Humidity Effect-Model for Data from 

Samples Extracted on Day 1 After Sampling 

We investigated the effect of temperature on diacetyl recovery by plotting percent diacetyl recovered against either RH 

(Figure 2a) in% or AH (Figure 2b) in mg H20/L air using data 

from samples collected from a target diacetyl concentration of 

5 ppm at target temperatures of 25°C and 32°C. We calculated 

AH from RH and temperature (Tc) using Eq. 1, which we 

derived from a National Weather Service approximation for 

humidity calculations in surface observations.01l 

( 

17.67Tc ) 13.25RHexp 

AH= Tc+ 243.50 (

1) 

Tc+ 273.15 

As seen in Figure 2, the substantial difference in diacetyl 

recovery for the two temperatures was removed when humidity 

was expressed as AH. Thereafter, we modeled the percent 

recovered diacetyl in terms of AH. 

120 

110 • to 

100 

90 

~ 0 80 >-

Qj 

70 6 

u 

~ 60 :;, 

Q5 

u 50 Cll 

0 40 

30 

20 

10 

0 

2 4 6 8 10 12 

Using the JMP model library of nonlinear functions, we 

visually determined that the 4-parameter logistic function was 

suitable to describe the sigmoidal relationship of percent recovered diacetyl with humidity, for samples extracted on Day 1 

after sampling. The 4-parameter logistic model has parameters 

81, 82, 83, and 84, each of which has graphical meaning. The 

parameter 81 represents the horizontal asymptote on the righthand side of the graph where humidity is at the highest level; 8 2 

represents the horizontal asymptote on the left-hand side of the 

graph where humidity is at the lowest level; 83 is the "slope" 

or the shape parameter; and 8 4 is the humidity at which 50% 

of the maximal response is observed. The general equation in 

terms of the 4-parameter logistic model is: 

82-81 

y = 81 + --------

1 + exp[83 (X - 84)] (2) 

In our models, percent recovered diacetyl was the Y variable, and humidity was the X variable. 

We fitted separate 4-parameter logistic models to the data 

for each of the target test atmosphere diacetyl concentrations 

(0.2, 0.5, 5.0, and 20 ppm). We had too few levels of AH for the 

1.0 ppm test atmosphere diacetyl concentration to adequately 

fit the 4-parameter logistic model. Figure 3 shows the separate 

4-parameter logistic models for percent recovered diacetyl vs. 

AH as fitted through the overall test data (for both sampling 

flow rates combined). 

Using information from these models, we created one nonlinear model for the data overall; this model took into account differences in the 4-parameter values for the individual 

logistic models. We found that 81 was well approximated 

(R2 = 0.99) by a linear function of target concentration Co 

14 16 

- 25 ppm curve 

-- 5.0 ppm curve 

-- 0.5 ppm curve 

----· 0.2 ppm curve 

• 25 ppm 

5.0 ppm 

+ 1.0 ppm 

0.5 ppm 

0.2 ppm 

18 20 22 24 

AH (mg H20 IL air) 

FIGURE 3. Four-parameter logistic models fitted to test data. Note: Data are insufficient to create a logistic curve for 1.0 ppm. 

Journal of Occupational and Environmental Hygiene 

Appendix 2 Page 5 

February 2011 

Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

63 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

362 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

TABLE II. Parameter Coefficients for the Overall 

Diacetyl Correction Equation and for Two Sampling 

Flow Rates 

Sampling Flow Rate 

Parameter Overall SOcdmin 150 cc/min 

bo 6.91166 5.85971 8.32618 

mi 1.69272 1.70372 1.68917 

82 101.31390 101.06329 101.81000 

83 0.72068 0.70943 0.73539 

84 8.22607 8.18808 8.26746 

q 0.05362 0.05362 0.05362 

0.41384 0.41384 0.41384 

-0.00589 -0.00589 -0.00589 

u -0.01719 -0.01719 -0.01719 

v 0.30359 0.30359 0.30359 

w 0.00558 0.00558 0.00558 

x 0.00153 0.00153 0.00153 

y 0.26802 0.26802 0.26802 

z -0.00002 -0.00002 -0.00002 

(i.e., 81 = b0 + m1 Co. where b0 is the intercept and m1 is 

the slope) and so substituted this linear function into the 4-

parameter logistic function using the values for bo and m1 

as starting values for the overall model. Since the other parameters showed variability but no trend with levels of Co. 

we used the arithmetic means of the separate model 82 , 83 , 

and 8 4 parameters for the four target test atmosphere diacetyl 

100 

90 

~ 80 

>- 70 

Q; 

6 60 u 

~ 

~ 50 Q) 

u 

ro 40 

0 

30 

20 

10 

0 

concentrations as the overall model starting values for these 

three parameters. We expressed percent recovered diacetyl 

(lOOc/ Co. where c is the recovered concentration reported 

by the laboratory) by rewriting Eq. 2 as follows: 

lOOc 

Percent recovered diacetyl = --= h(C0 , AH) = bo +mi Co 

Co 

82 - bo - mi Co 

+ 1 + exp[8 3(AH - 84 )] (J) 

We entered this form of the equation (Eq. 3) into the nonlinear fitting platform for a fit through all the data (including the 

data for a test atmosphere diacetyl concentration of 1.0 ppm). 

We repeated the fit through the data stratified by sampling flow 

rate. The final values for the parameters (b0 , m1, 82 , 8 3, and 

84 ) both overall and for the two sampling flow rates are given 

in Table II (the table also contains parameter values for the 

effect of in-tube storage as described below). The R2 (amount 

of total variability in the data accounted for by the model) 

for the overall model was 0.91. The R2 for the 150 cc/min 

model was 0.93, and the R2 for the 50 cc/min model was 0.90. 

Figure 4 shows how Eq. 3 describes the pattern of diacetyl 

recoveries for a range of concentrations both overall and for 

the two sampling flow rates and illustrates that the effect of 

sampling flow rate was not large. 

Equation 3 predicts that at a concentration of approximately 

56 ppm, the diacetyl recovery would be approximately 100% 

at all AH values (this was similar for the overall model and 

the 50 and 150 cc/min models). At diacetyl concentrations 

above these values, the model predicts diacetyl recoveries of 

higher than 100% across the range of AH values, which does 

not represent a real-life solution. Predicted diacetyl recoveries 

150cc/min 

Overall 

50 cc/min 

2 4 6 8 10 12 14 16 18 20 22 24 

AH (mg H20 IL air) 

FIGURE 4. Model for data from samples extracted on Day 1 after sampling (Eq. 3) applied at three diacetyl concentrations using parameter 

coefficients (bo, m1, 82, 83, 84 ) for the whole data set (overall) and the two sampling flow rates used in the experiment. 

64 Journal of Occupational and Environmental Hygiene 

Appendix 2 Page 6 

February 2011 

Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 363
 

~ 30 E 

0.. 

2: 

§ 25 

~ § 20 

8 

~ 15 

16 

~ 10 

I 

(/) 

0 5 

(b) 35 

E' 30 0.. 

2: 

c 

0 25 

~ 20 § 

8 

~ 15 Q) 

16 

'O 10 <( 

I 

(/) 

0 5 

5 10 15 20 25 30 

Test atmosphere diacetyl concentration (ppm) 

5 10 15 20 25 30 

Corrected NIOSH diacetyl concentration (ppm) 

FIGURE 5. Comparison of diacetyl sampling results obtained using OSHA method (a) to calculated test atmosphere concentration, 

and (b) to corrected values of the diacetyl sampling results using 

NIOSH Method 2557. 

from Eq. 3 for very low diacetyl concentrations do not have 

the same problem since, mathematically, in the limit as the 

diacetyl concentration goes to zero, the recoveries range from 

approximately 100% to approximately 7% as AH goes from 

low to high. 

OSHA Silica Gel Sample Results 

Diacetyl concentrations from the 121 silica gel samples 

taken at a number of AH conditions were quite similar to 

the calculated test atmosphere concentrations (Figure 5a) and 

were not affected by AH. Using the model (Eq. 3) we calculated the corrected diacetyl concentrations from the matched 

NIOSH Method 2557 CMS samples, and as shown in Figure 

5b, we found a strong linear relationship with the silica gel 

results. 

(a) 80 

~ 60 "Cl 

E 40 0 

.;: 

2:- 20 Q) 

> 8 

E! 

c 

§ -20 

E! -40 ~ 

i:5 -60 'd!-

-80 

0 10 

(b) 

120 

::::l 100 (/) 

E! 

~ 80 

"Cl 

E 

0 60 .;: 

2:-

Q) 

> 40 8 

Q) 

a: 

'd!- 20 

0 

0 10 

•--Ambient 

o ········· Refrigerated 

.I"! 

• 

20 30 40 

Days post-sampling 

· ... ~ ...... 

~ 

0- 0.57ppm(AH=3.51) 

D ---- (AH=B.47) 

I> (AH=18.17) 

e-- 25.0ppm (AH=3.57) 

• --- (AH=B.63) 

.a.········ (AH=18.66) 

············· ························· 

20 30 

Days post-sampling 

FIGURE 6. (a) Extract stability: Samples extracted and analyzed 

on Day 1 post-sampling and the remaining sorbent material and 

extract kept at ambient or refrigerated temperature before further 

analyses on subsequent days. Lines represent regressions. (b) Intube stability: Samples extracted and analyzed on the same day. 

Curves were created from Eq. 4. 

Model for Effect of In-Tube Storage 

Plots of extract storage and in-tube storage stabilities are 

shown in Figure 6. Samples stored as extracts, either with or 

without sorbent material, under refrigerated conditions were 

stable, having less than 2 % loss at each of the three All levels 

overnearly 40 days of storage (1.4% at 7 days and 1.7% at 38 

days). Under ambient conditions, the loss was 2.9% at 7 days 

and 11.0% at 38 days. In contrast, plots of diacetyl recovered 

by number of days of in-tube storage (i.e., days from sampling 

to extraction) indicated decreased recovery over time, with 

the changes over time showing dependence on both AH and 

diacetyl concentration. For a given diacetyl concentration, 

diacetyl losses over time were greater with increasing AH. 

For a given AH, diacetyl losses over time were greater with 

decreasing concentration. 

To model in-tube storage effects, we used first-order decay 

functions to estimate decay constants for the 12 combinations 

of diacetyl concentrations and AH. We normalized the diacetyl 

recovery data by dividing the diacetyl recovery data by the 

Journal of Occupational and Environmental Hygiene February 2011 65 

Appendix 2 Page 7 Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

364 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

mean for recovery on Day 1 after sampling and included (t-1) 

in the first-order decay functions (see below). The first-order 

decay model is given by: Y =(starting amount) exp[-k(t-1)], 

where starting amount = 1 for normalized data, t = days from 

sampling to extraction, and k is the decay constant. 

We substituted functions of AH and diacetyl concentration 

for the decay constants (k). This was accomplished in two 

steps. In Step 1, we fitted quadratic functions to the k values 

for the three target diacetyl concentration (0.5 ppm, 5 ppm, and 

25 ppm) curves of k vs. AH. In Step 2, we substituted threeparameter first-order decay functions for the coefficients for 

the intercept, the AH term and the AH2 term of the quadratic 

function based on the diacetyl concentrations. This gave estimates for the nine coefficients (q, r, s, u, v, w, x, y, and z) in 

the model (as shown below). In a final step, the values of the 

nine parameters were used as starting values to get a fit of this 

nonlinear model through the full set of in-tube storage data. 

The R2 for this model was 0.90. The coefficients are given in 

Table II. The form of the nonlinear model for the effect of 

in-tube storage was: 

Normalized recovery= g(Co, AH, t) = exp[-(f1(Co) 

where 

Full Model 

+ h(Co)AH + f3(Co)AH2)(t - l)] 

(4) 

f1(Co) = qexp( - rCo) + s 

h(Co) = uexp( - vCo) + w 

f3(Co) = xexp( - yC0 ) + z 

The full model can be conceptualized in two steps. First, the 

AH, the recovered diacetyl concentration (c), and the number 

of days from sampling to extraction (t) are used to predict the 

recovered diacetyl concentration on Day 1 of extraction after 

sampling. Second, this predicted diacetyl value and AH is used 

to predict the corrected concentration. The full model for the 

percent of diacetyl recovered is: 

lOOc 

Percent recovered diacetyl = -- = h(C0 , AH)g(C0 , AH, t) Co 

(5) 

where his given by Eq. 3 and g is given by Eq. 4. 

Since, in practice, the values of c, AH, and tare known, and 

the value of Co is the predicted corrected diacetyl concentration, we solved Eq. 5 for C0 . Using Eqs. 3 and 4, Eq. 5 can be 

rewritten as: 

aC6 + bCo - ( c ) = 0 (6) g(Co, AH, t) 

where 

Since this is a nonlinear equation for C0 , it is necessary to 

use an iterative procedure to find C0 . Initially, Eq. 6 is solved 

for Co using the quadratic formula with the dependence of g 

on Co ignored: 

b

2 

+ 4a Ceca, ~H, t)) Co= ______ 2_a ____ _ 

-b+ 

(7) 

(Note: The other solution for Eq. 6 using the quadratic formula 

yields a nonphysical negative value for Co since a > 0 and 

b > 0.) c In Eqs. 6 and 7 the value of ( ) is the estimate 

g(Co, AH, t) 

for the diacetyl concentration corrected for days to extraction 

after sampling. 

To solve Eq. 7, an iterative procedure is used with the i 

value cgl used to calculate the Ci+ 1) value cg+ 1l 

b2 + 4a ( 0 c ) 

g(C0' , AH, t) 

cg+1) = ------------

2a 

-b+ 

(8) 

It is necessary to start the procedure with an initial Co (i.e., 

c61

\ We found the procedure robust to the choice of starting 

value and suggest the use of c (the recovered concentration 

reported by the laboratory). 

The sequence of solutions is then calculated until two 

consecutive values for Co are identical to a chosen number of 

decimal places (convergence). We tested the model for regions 

of convergence using theoretical recovered concentrations ( c) 

from 0.001to70 ppm, AH from 2 to 25 mg H20/L air, and days 

to extraction from 1 to 36. We found that convergence occurred 

for all concentrations above 1.0 ppm. For lower concentrations, 

convergence occurred whenever AH was less than 14.5 mg 

H20/L air and days to extraction were fewer than 9. The region 

of convergence improved from a concentration of0.001 to 1.0 

ppm. At 1.0 ppm, convergence occurred whenever AH was 

less than 21 mg H20 IL air and days to extraction were fewer 

than 17. 

As discussed above, for values of Co > 56 ppm, the Day 1 

model does not yield real-life solutions. For these values, only 

the model for effect of days to extraction should be applied and 

we predict the concentration of diacetyl for Day 1 of extraction 

after sampling. If the converged value as calculated above is> 

56 ppm, use it as the starting value c61l in an iterative procedure 

using the equation: 

cCi+l) - c 

0 - (Cl ) g C0

', AH, t 

(9) 

The sequence of solutions is then calculated until two 

consecutive values for Co are identical to a chosen number 

of decimal places. Figure 7 is a flow diagram of the correction procedure as described above. When corrected diacetyl 

concentrations fall between 0.23 and 25.7 ppm, which were 

the lowest and highest diacetyl test atmosphere concentrations 

66 Journal of Occupational and Environmental Hygiene February 2011 

Appendix 2 Page 8 Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 365
 

2a 

Corrected 

concentration in 

interpolated region 

i=i + 1 

i=i + 1 

Corrected 

concentration in 

extrapolated region 

Yes 

FIGURE 7. Flow diagram of the correction procedure that begins with known values of recovered diacetyl concentration (c), absolute humidity 

(AH) during sampling, and the number of days from sampling to extraction (t), and ends with the corrected diacetyl concentration in either the 

interpolated or extrapolated region. As explained in the text, there are some conditions of no convergence. 

Journal of Occupational and Environmental Hygiene 

Appendix 2 Page 9 

February 2011 

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Occupational and Environmental Hygiene. 

67 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

366 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

(a) 3.5 (b) 

16 

E c=0.02 ppm 

0.. 3.0 

E: - t= 1 day 

c - t = 7 days 0 

·.i:; 2.5 - t = 14 days 

g )K no convergence c 

(]) 2.0 u 

c 

0 

u 

E' c= 1.0 ppm 0.. 14 

E: - t = 1 day c 12 - t = 7 days 0 

·.i:; - t = 14 days g 

c 10 (]) 

u 

c 

0 8 u -c 1.5 -c 

~ u 

~ 1.0 0 

u 

~ 6 u 

~ 

0 4 u II II 

u0 0.5 u 

0.0 0+-~~~~~~~~~~~~~~~~~~----1 

4 6 8 10 12 14 16 18 20 22 24 2 4 6 8 10 12 14 16 18 20 22 24 

AH (mg H20 /Lair) AH (mg H20 /Lair) 

(c) 

E c= 20 ppm 80 0.. 

E: - t= 1 day 

- t = 7 days c 70 0 

·.i:; t = 14 days 

....-. t = 14 days, C0

g >56 60 

c 

(]) 

u 

c 50 

0 

u 

-c 40 

~ u 30 ~ 

0 

u 20 II 

uo 10 

0 

2 4 6 8 10 12 14 16 18 20 22 24 

AH (mg H20 / Lair) 

FIGURE 8. Predicted diacetyl concentrations using the model at selected laboratory-reported (recovered) concentrations of (a) 0.02 ppm, (b) 

1.0 ppm, and (c) 20 ppm. 

used in our experiments, we consider the corrections to be 

within the interpolated range and have the most confidence in 

these values. Figure 8 shows diacetyl concentrations predicted 

by our models for a number of different conditions. We chose 

three laboratory-reported diacetyl concentrations (c) of 0.02, 

1.0, and 20 ppm over a wide range of AH and days from 

sampling to extraction (t) that should represent possible field 

conditions. We see that both changes in AH and t substantially 

affect the value of the corrected concentration. In Figure 8a, 

we indicate a point where nonconvergence begins for c = 0.02 

ppm and AH= 19 on the 14 days from sampling to extraction 

curve. In Figure 8c, we indicate a region where Co > 56, which 

is only corrected for days from sampling to extraction. 

DISCUSSION 

From experimental test atmosphere work, we have created 

a procedure that allows historical diacetyl concentration 

data from analysis of samples using NIOSH Method 2557 to 

be corrected to more accurately estimate historical workplace 

airborne diacetyl concentrations. This correction procedure 

provides a means for applying these diacetyl concentration estimates in planned quantitative risk assessment relating health 

effects observed among workers to their diacetyl exposure. In 

addition, it will allow for a better understanding of historical 

workplace concentrations of diacetyl that will give insight for 

exposure control strategies. Use of this correction procedure 

68 Journal of Occupational and Environmental Hygiene 

Appendix 2 Page 10 

February 2011 

Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 367
 

requires laboratory-reported concentrations of diacetyl in ppm 

(samples collected and analyzed using NIOSH Method 2557), 

temperature and RH (to calculate AH) conditions at the time 

of sampling, and the number of days from sample collection 

to sample extraction for analysis. We give overall parameter 

values for the full model, as well as for sampling flow rates 

of 50 and 150 cc/min (Table II). Since the effect of sampling 

flow rate was not large, investigators have the option of using 

the overall parameter values, especially if their historical data 

were collected at sampling flow rates other than 50 and 150 

cc/min. 

A strength of this work was the use of a controlled test 

atmosphere to simulate historical field survey conditions where 

airborne diacetyl was sampled together with humid air. By 

using two target temperatures with similar ranges of RH, we 

were able to show that both temperature and RH had an effect 

on diacetyl recovery and that using AH (mg H20/L air) was the 

key variable to connect the correlation between temperature 

and concentration. This finding extends the work ofMcKernan 

and colleagues, CS) who were unable to separate the effect of 

temperature and RH in their field-based work. By running tests 

with several different test atmosphere diacetyl concentrations 

over a wide range from 0.23 to 25.7 ppm, we were able to 

observe differences in diacetyl recovery related to theoretical 

diacetyl concentration. We found a large difference in diacetyl 

recovery between the test atmosphere diacetyl concentration 

of about 25 ppm and all the lower concentrations, especially 

at the higher AH values. The final correction equation predicts 

that humidity would no longer have an effect on diacetyl 

recovery at approximately 56 ppm, but we have no empirical 

data to test this prediction. Corrected diacetyl concentrations 

that lie outside our test atmosphere range represent extrapolations of the models, and we have less confidence in these 

concentrations. 

We do not suggest the use of the correction procedure 

with historical concentration data below the limit of detection 

(LOD), for which concentration may have been estimated 

(e.g., using LOD/2 or LOD/,J2°). It is not possible to know 

if the workplace diacetyl concentration was indeed below the 

LOD or if the losses due to humidity and days from sampling 

to extraction in the laboratory caused the sample value to 

be below the LOD. We did find some regions of AH and 

days from sampling to extraction for recovered concentrations of 1.0 ppm or less where the full model does not converge; however, such conditions should not occur often in the 

field. 

Our storage stability test findings were contrary to the 

NIOSH Method 2557 specification of good stability for 7 

days from sampling to analysis.(7) This may have been due 

to the fact that storage stability tests completed during method 

development used spiked sampling tubes without using humid air rather than our actively sampled tubes using a test 

atmosphere. As our results showed, early extraction minimized further sample loss, especially when the samples were 

refrigerated in accordance with the method, which means 

that delays in analysis after extraction should not cause appreciable loss. A limitation of our work is that we did not 

collect in-tube storage data for all the tests to determine the 

effect of AH on sample recovery, but we did collect data 

for three target test atmosphere diacetyl concentrations and 

three target AH values. Thus, we estimated the effect of AH 

and the effect of in-tube storage on different data sets and 

combined the two models mathematically to create the final 

model. 

Our correction equations accounted for about 90% of the 

variability in the experimental data by taking into account 

the effects of AH, test atmosphere diacetyl concentration, 

sampling flow rate, and days of in-tube storage. The variability 

seen in the data at any combination of AH and diacetyl concentration values has a number of sources, including variability 

in keeping test atmosphere conditions constant, variability 

in sampling flow rates during the tests, sampling duration 

differences (although not found significant), and analytical 

laboratory variability. 

Our test atmosphere experiments used no flavoring chemicals besides diacetyl. In field situations, diacetyl may occur 

together with other chemicals in the air. Any effect of these 

mixtures on the diacetyl recovery using NIOSH Method 2557 

might not be accounted for with our correction procedure. 

Comparison between corrected diacetyl concentrations and 

the results from side-by-side samples taken with OSHA methods indicated a high correlation, which increases our confidence in the applicability of the correction method. Despite 

the limitations, the correction procedure enables more accurate 

quantitative risk assessment now under way for regulatory 

guidance on occupational exposure to diacetyl. Representative 

exposures in the flavoring manufacturing industry are difficult 

to assess because of short-duration batch production methods 

in which hour-to-hour and day-to-day variations in diacetyl 

exposures is expected in workplaces where scores of different 

kinds of flavorings are manufactured. Hence, relative stability of diacetyl exposures in microwave popcorn production 

facilities offers the advantage of less potential for exposure 

misclassification. 

However, without appropriate correction, the systematic 

underestimation of true diacetyl exposures in the 2000-2006 

historical data would lead to overestimation of health risk 

associated with diacetyl exposure. Accordingly, use of our 

correction procedure to recalculate the historical exposure 

estimates from microwave popcorn production facilities previously studied by NIOSH and others will contribute to ongoing 

efforts to understand the health risk associated with occupational exposure to diacetyl. Our experimental work may 

also motivate further research exploring the mechanism by 

which analyte recovery from CMS sorbent may be affected by 

sampling site humidity for a variety of analytes. 

CONCLUSIONS 

W e have developed a mathematical procedure that allows measurements from historical diacetyl samples 

collected and analyzed using NIOSH Method 2557, which 

Journal of Occupational and Environmental Hygiene February 2011 69 

Appendix 2 Page 11 Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

368 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

may be biased low, to be adjusted for a more accurate exposure 

assessment. In addition to the historical laboratory-reported 

diacetyl concentrations, this correction procedure requires data 

on AH (determined from temperature and RH measurements) 

during sampling and on the number of days between sample 

collection and laboratory extraction of the sampling tubes. 

NIOSH Method 2557 should not be used to measure airborne 

diacetyl in future studies. 

REFERENCES 

1. Kreiss, K., A. Gomaa, G. Kullman, et al.: Clinical bronchiolitis 

obliterans in workers at a microwave-popcorn plant. N. Engl. J. Med. 

347:330-338 (2002). 

2. Kanwal, R.: Bronchiolitis obliterans in workers exposed to flavoring 

chemicals. Cun Opin. Pulm. Med. 14:141-146 (2008). 

3. Van Rooy, F.G., J.M. Rooyackers, M. Prokop, et al.: Bronchiolitis obliterans syndrome in chemical workers producing diacetyl 

for food flavorings. Am. J. Respir. Crit. Care Med. 176(5):498-504 

(2007). 

4. Hubbs, A., L. Battelli, R. Mercer, et al.: Inhalation toxicity of the 

flavoring agent, diacetyl (2,3-butanedione ), in the upper respiratory tract 

of rats. Toxicol. Sci. 78(Suppl 1): 438-439 (2004). 

5. Hubbs, A.F., W.T. Goldsmith, M.L. Kashon, et al.: Respiratory 

toxicologicpathology of inhaled diacetyl in Sprague-Dawley rats. Toxicol. 

Pathol. 36:330-344 (2008). 

6. Pendergrass, S.M.: Method development for the determination of 

diacetyl and acetoin at a microwave popcorn plant. Environ. Sci. Technol. 

38(3):858-861 (2004). 

7. "Diacetyl: Method 2557. NIOSH Manual of Analytical Methods 

(NMAM)." [Online] Available at http://www.cdc.gov/niosh/nmam/ 

pdfs/2557.pdf (Accessed May 21, 2010). 

8. McKernan, L.T., E. Burroughs, J. Deddens, et al.: Analytical 

performance criteria: Field evaluation of diacetyl sampling and analytical 

methods. J. Occup. Environ. Hyg. 5:Dll 1-Dl 16 (2008). 

9. "Diacetyl: Method PV2118. OSHA Sampling and Analytical Methods." 

[Online] Available at http://www.osha.gov/dts/sltc/methods/partial/tpv2118/t-pv2118.html (Accessed May 21, 2010). 

10. "Diacetyl: Method 1013. OSHA Sampling and Analytical Methods." [Online] Available at http://www.osha.gov/dts/sltc/methods/ 

validated/101311013.html (Accessed May 21, 2010). 

11. Bolton, D.: The computation of equivalent potential temperature. Mon. 

Wea. Rev. 108:1046-1053 (1980). 

70 Journal of Occupational and Environmental Hygiene February 2011 

Appendix 2 Page 12 Reprinted with permission of the Journal of 

Occupational and Environmental Hygiene. 

Appendix F – Correcting Diacetyl Concentrations from Air Samples Collected with NIOSH Method 2557
 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 369
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Appendix G
 

JEM Tables for Four Plants
 
 

JEM Tables for Four Plants 

The mean diacetyl vapor concentrations estimated for the three plants with a single NIOSH 

survey are shown in Tables A3.1 -A3.3. For the fourth plant, Company G, time-dependent 

exposure levels were estimated in the NIOSH-OSHA JEM collaboration (Table A3.4). All 

Method 2557 measurements were corrected for temperature, humidity, and days to extraction. 

Table A3.1 Diacetyl exposure matrix for health hazard evaluation: Company N 

Department 

Maintenance office 

Microwave popcorn line 

Mixing, measuring room 

Packing area 

Poly line 

Quality control area 

Stencil area 

Warehouse 

Job n ppm diacetyl 

Maintenance I mechanic 2 0.164 

Bag placer 2 0.696 

Machine operator 3 1.160 

Packer I Stacker 2 0.143 

(area) 2 0.621 

Tank mixer 0.794 

(area) 2 1.032 

Packer I Stacker 2 0.121 

(area) 2 0.159 

Poly line worker 2 0.235 

(area) 2 0.182 

Quality control worker 2 0.250 

(area) 2 0.320 

Stenciler 2 0.045 

(area) 2 0.024 

Fork lift operator 2 0.005 

Table A3.2 Diacetyl exposure matrix for health hazard evaluation: Company K 

Department 

41-A Entire Area 

41-A Blending Room (Mixing) 

41-A Carton I Tray 

Job 

Micro Pdn-Manager/Supvr 

Micro Pdn-Sanitation/Cleaning 

Micro Production-Mixer 

41-A Filler Side 

2 

N 

7 

ppm diacetyl 

0.003 

0.003 

0.913 

0.002 

Appendix G – JEM Tables for Four Plants 

372 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Micro Pdn-MainUMechanic 0.054 

Micro Pdn-Production Worker 5 0.039 

41-A Case I Pallet Micro Production-Stacker 5 0.002 

41-A Filler Side 41-A Case/Pallet 8 0.038 

Plant-Maintenance/Mechanic 0.003 

Plant-Other 0.002 

41-A Lab 41-B Warehouse 5 0.003 

41 Warehouse Plant-Other 0.268 

41-B Warehouse Micro Pdn-Forklift Operator 0.002 

Plant-Other 0.002 

41 & 41-B Warehouses Micro Production-Stacker 0.002 

41-Microwave Pdn Building Micro Pdn-MainUMechanic 2 0.037 

Micro Pdn-Manager/Supvr 3 0.003 

Micro Production-Mixer 0.002 

41-B Warehouse 0.003 

Micro Pdn-Production Worker 0.002 

Plant-Other 0.003 

Office Building Office/Management/Sales 2 0.002 

Poly Production Building Poly-Production 2 0.309 

Poly-QC 2 0.002 

Ambient Ambient 2 0.003 

Pre-mix Area Micro Production-Mixer 3 0.043 

Appendix G – JEM Tables for Four Plants 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 373
 

Table A3.3 Diacetyl exposure matrix for health hazard evaluation: Company L 

Department Job N ppm diacet~I 

Press Room Press room worker 10 0.015 

Slurry Room (Mixing) Supervisor 3 0.723 

Mixer 13 1.426 

Packaging Supervisor 8 0.020 

Phaser operator 10 0.026 

Case packer operator 10 0.026 

Cartoner operator 13 0.031 

Palletizer operator 9 0.033 

Line sanitation 3 0.024 

Forklift operator 6 0.036 

QA QA monitor 11 0.025 

Warehouse Supervisor 0.033 

Warehouse worker 7 0.035 

Forklift operator 0.037 

Main Office Office worker 6 0.025 

Maintenance Press room worker 0.011 

Supervisor 0.012 

All over plant 2 0.006 

Other 5 0.021 

Ambient Outside 0.003 

Table A3.4 Diacetyl exposure matrix for health hazard evaluation: Company G 

Department Job Period ppm diacetyl Start Date End Date 

Microwave Production Oil mixing 9.713 7/1/1986 2/11/2001 

Mixing room 2 2.509 2/12/2001 4/5/2001 

3 0.245 4/6/2001 9/6/2002 

4 0.006 9/7/2002 8/15/2003 

Office Office 0.009 7/1/1986 8/15/2003 

Office Lab technician/quality 0.335 7/1/1986 2/11/2001 

control 

2 0.250 2/12/2001 5/21/2001 

3 0.123 5/22/2001 81812002 

4 0.034 81812002 3/8/2003 

4 

Appendix G – JEM Tables for Four Plants 

374 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

5 0.007 3/9/2003 8/15/2003 

Warehouse Warehouse 1 0.557 7/1/1986 2/11/2001 

2 0.017 2/12/2001 8/15/2003 

Warehouse/Microwave Bag checker and bag 1.613 7/1/1986 2/11/2001 

machine operator 

Production 2 0.947 2/12/2001 5/21/2001 

3 0.053 5/22/2001 9/6/2002 

4 0.003 9/7/2002 8/15/2003 

Polypropylene Supervisor, machine 0.047 7/1/1986 2/11/2001 

operator, line packer, 

line stacker 2 0.020 2/12/2001 8/15/2003 

Microwave Production Maintenance 1.145 7/1/1986 2/11/2001 

and All Over 2 0.294 2/12/2001 5/21/2001 

3 0.054 5/22/2001 9/6/2002 

4 0.002 91712002 8/15/2003 

Microwave Production Supervisor, machine 2.668 7/1/1986 2/11/2001 

opr, do-boy opr,line 

packer, line stacker, 2 0.672 2/12/2001 5/21/2001 

inventory control, 

fill-in on line, box folder 3 0.343 5/22/2001 9/6/2002 

4 0.003 9/7/2002 8/15/2003 

Microwave Production Lab technician/quality 1.312 7/1/1986 2/11/2001 

control 

Quality Control Lab 2 0.974 2/12/2001 5/21/2001 

3 0.467 5/22/2001 81812002 

4 0.108 81812002 3/8/2003 

5 0.002 3/9/2003 8/15/2003 

Microwave Production Line packer/machine 1 5.016 7/1/1986 2/11/2001 

operator/mix er 

2 1.284 2/12/2001 4/5/2001 

3 0.530 4/6/2001 5/21/2001 

4 0.311 5/22/2001 9/6/2002 

5 0.004 91712002 8/15/2003 

Outside I Yard Maintenance and 1 0.009 7/1/1986 8/15/2003 

outside processing 

All Over I Float Supervisor and janitor 2.068 7/1/1986 2/11/2001 

2 0.685 2/12/2001 4/5/2001 

3 0.402 4/6/2001 5/21/2001 

4 0.165 5/22/2001 81812002 

5 0.105 8/9/2002 9/6/2002 

6 0.026 9/7/2002 3/8/2003 

7 0.009 3/9/2003 8/15/2003 

All Over I Float Exterminator 1 0.009 7/1/1986 8/15/2003 

Microwave and Line packer, line stacker 1.358 7/1/1986 2/11/2001 

on both lines 

Polypropylene 2 0.346 2/12/2001 5/21/2001 

5 

Appendix G – JEM Tables for Four Plants 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 375
 

Production 

6 

3 

4 

0.182 

0.012 

512212001 9/6/2002 

9/712002 8/15/2003 

Appendix G – JEM Tables for Four Plants 

376 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
Appendix H
 

Development of a Job Exposure Matrix 

for Company G 
 

1.0 Development of a Job Exposure Matrix for Company G 

1.1 Overview 

To estimate worker exposures for risk assessment, we developed a job exposure matrix (JEM) 

containing estimates of the average 8-hour, time-weighted average (TWA) exposure levels for 

diacetyl vapor in parts per million parts air (ppm). This JEM includes estimates for eight major 

job categories with selected time periods specific for each job category to reflect changes in 

processes and engineering controls over time. The exposure levels presented in the JEM are 

based on diacetyl air sampling data collected during nine industrial hygiene surveys conducted 

by NIOSH industrial hygienists between November 2000 and July 2003 at a microwave popcorn 

plant in Missouri [Kreiss et al. 2002; Kullman et al. 2005; NIOSH 2006]. Details of the JEM 

construction are described below. 

1.2 Industrial Hygiene Surveys 

A total of nine industrial hygiene surveys were conducted over a period of 4 years from 2000 to 

2003. The sampling was typically conducted during the day shift, because this shift presented the 

opportunity to sample all job categories. However, samples were also collected from second and 

third shifts, but not routinely. The dates for these industrial hygiene surveys are presented in 

Table A4.l. 

Personal breathing zone (PBZ) and area diacetyl samples were collected during these surveys 

using NIOSH Method 2557. These measurements were subsequently adjusted to account for 

interferences due to humidity and sample storage [Cox-Ganser et al. 2011]. During all surveys 

except the first, full-shift PBZ samples were collected from workers performing typical tasks 

representative of each of the major job categories. In addition, concurrent full-shift area samples 

were taken throughout the plant from locations where workers would typically spend their time. 

The PBZ sample measurements were used to develop the exposure estimates for the eight job 

categories in the JEM. In some instances where personal diacetyl samples were not collected, for 

example during the first survey, area samples were used to obtain estimates of personalequivalent diacetyl exposures. 

1.3 Creation of Job Categories and Estimation of Arithmetic Means 

For the purpose of developing exposure estimates for the JEM, plant job titles were aggregated 

into eight job categories based primarily on work and environmental similarities with respect to 

potential for diacetyl exposures [Com and Esmen 1979]. These eight categories are listed in 

Table A4.2 along with the jobs that comprise each category. 

Arithmetic means (AM) using PBZ samples were calculated for the cells in the JEM as the AMs 

are the preferred measure of central tendency for estimating cumulative exposure in chronic 

disease investigations [Smith 1992]. Few PBZ measurements were collected for most job 

categories in each of the nine surveys (range: n= 1-6) except for the job category of Microwave 

line (range n= 11-18). Moreover, a large fraction of the PBZ measurements were below the limit 

of detection (LOD) for most job categories (>50%) especially during surveys 6-9, except for the 

job categories of microwave packaging line, quality control and microwave mixing. Thus 

because of the small sample size and large fractions of LOD data, a simple substitution method 

2 

Appendix H – Development of a Job Exposure Matrix for Company G 

378 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

ofreplacing LOD measurements with a value ofLOD/2 was used [Ganser and Hewett 2010]. 

The calculation of the arithmetic mean exposures by the different time periods is described in 

detail in the next section on "Creation of Exposure Periods." 

As noted earlier, PBZ diacetyl samples were not collected during survey 1 and had to be 

estimated from personal and area samples collected during subsequent surveys (i.e. surveys 2-9). 

A hierarchical approach was used to estimate the PBZ exposures for survey 1 depending on the 

job category and the availability and fraction of personal or area measurements below the LOD. 

To start with, for jobs categories with sufficient personal and area samples in surveys 2-9, a 

prediction model was used to estimate personal exposures from area exposure measurements 

(e.g. microwave mixers, microwave line, quality control). For job categories with small sample 

size and/or large fraction of measurements below the LOD for surveys 2-9, the arithmetic mean 

of the area samples from survey 1 was assigned to personal estimates for survey 1, assuming a 

ratio of 1 for personal to area measurements (e.g., warehouse, outside/office, polyethylene line). 

For jobs with no area measurements in survey 1 (e.g., bag print) or the area measurements were 

not representative of personal measurements (maintenance), exposure estimates from similar 

jobs in survey 1 were assigned. The detailed approach to calculate personal-equivalent diacetyl 

exposure for each job category for the first survey is described in Table A4.3. 

1. 4 Creation of Exposure Periods 

After estimating the personal-equivalent exposures for survey 1, arithmetic means were 

calculated for the different time periods. Unique exposure time periods were developed for each 

of the eight job categories to reflect impact of the exposure control changes implemented at the 

plant between November 2000 to July 2003. Table A4.4 lists these exposure control changes 

according to the time of implementation. These exposure time periods varied by job categories 

since some control changes would have a greater impact on some job categories than others. In 

addition, the fraction of LOD samples for job categories during the different surveys also 

impacted the creation of time periods. Surveys for which a large fraction of the measurements for 

a job category were below the LOD were combined into one time period. For example, for 

warehouse, 50%-100% of personal measurements were below the LOD for surveys 3-9, hence 

these surveys were combined into one time period. For the selection of time periods for the job 

categories, the LOD patterns were consistent with the implementation of controls. The detailed 

approach used to create the time periods for each job category is described in Table A4.5. Thus a 

JEM was created consisting of 8 job categories and 2-5 time periods spanning the time duration 

from November 2000 to July 2003. 

1. 5 Adjustment for Respirator Use 

The JEM created as described above was based on samples collected from workers breathing 

zone and did not account for respirator use by workers. However, during survey 4 and onward, 

workers in microwave mixing were using respirators and the JEM estimates were adjusted in the 

appropriate time periods to reflect the PPE use. Thus for the mixers during time periods 3 and 4, 

we adjusted the measured personal diacetyl exposure for the use of respirators. During these time 

periods, mixers used respiratory protection while in the mixing room and these respirators 

included either a P APR or air-line respirator with a loose fitting hood; both types of respirators 

have an applied protection factor (APF) of 25 [NIOSH 2004]. We assumed, based on survey 

observations and questionnaire responses, that mixers spent, on average, about 4 hours per shift 

3 

Appendix H – Development of a Job Exposure Matrix for Company G 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 379
 

in the mixing room in respiratory protection. Because respirators were required in the mixing 

room by plant management, we assumed that mixers wore respirators at all times while in the 

mixing room and did not wear these respirators when outside the mixing room and in the 

microwave packaging room. During these time periods, the mixers' desk was located in the 

microwave packaging room near packing line 1 so we further assumed that, when not in 

respirators in the mixing room, mixers would be in the microwave packaging area and receive 

diacetyl exposures consistent with those personal exposures measured in microwave packaging. 

The mixer personal samples were taken outside of the respirator and would reflect both mixing 

and packaging exposure components. Accordingly, to adjust mixer exposures to diacetyl for the 

use of respirator, we (1) determined the mixing room exposure component from the combined 

mixing and packing line diacetyl concentration as reflected in the personal sample (back 

calculated) and (2) applied a protection factor of 25 to the mixing room component of the mixers 

exposure. 

To determine the mixing room (A) personal diacetyl exposure from the combined mixing and 

packaging (C) concentration measured by personal sampling we applied the following equation: 

C = (4A(mixing) + 4B(packaging))/8; solving for A gives, A= 2C -B 

Where A= the mixing room personal exposure component in ppm, B =the packaging room 

personal exposure component in ppm, and C = the measured mixer personal exposure in ppm 

reflecting both mixing room and packaging room components. 

To correct the mixers exposure for the use of respiratory protection, we used the following 

equation: 

CR= 1h (A/25 + B) where CR= respirator adjusted mixer diacetyl exposure in ppm, A= personal 

mixer diacetyl exposure in the mixing room in ppm and B = personal diacetyl exposure in the 

microwave packaging area in ppm. 

This adjusted diacetyl concentration in ppm was applied to mixers for time periods 3 and 4 to 

adjust for the use of respiratory protection by mixers while in the mixing room. 

References 

Com M, Esmen NA [1979]. Workplace exposure zones for classification of employee exposures 

to physical and chemical agents. Am Ind Hyg Assoc J 40(1):47-57. 

Cox-Ganser J, Ganser G, Saito R, Hobbs G, Boylstein R, Hendricks W, Simmons M, Eide M, 

Kullman G, Piacitelli C [2011]. Correcting diacetyl concentrations from air samples collected 

with NIOSH method 2557. J Occup Environ Hyg 8(2):59-70. 

Ganser GH, Hewett P [2010]. An accurate substitution method for analyzing censored data. J 

Occup Environ Hyg 7( 4):233-244. 

Kreiss K, Gomaa A, Kullman G, Fedan K, Simoes EJ, Enright PL [2002]. Clinical bronchiolitis 

obliterans in workers at a microwave-popcorn plant. N Engl J Med 347(5):330-338. 

4 

Appendix H – Development of a Job Exposure Matrix for Company G 

380 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Kullman G, Boylstein R, Jones W, Piacitelli C, Pendergrass S, Kreiss K [2005]. Characterization 

ofrespiratory exposures at a microwave popcorn plant with cases of bronchiolitis obliterans. J 

Occup Environ Hyg 2(3):169-178. 

NIOSH [2004]. NIOSH Respirator Selection Logic. By Bollinger N. Cincinnati, OH: U.S. 

Department of Health and Human Services, Centers for Disease Control and Prevention, 

National Institute for Occupational Safety and Health, DHHS (NIOSH) Publication No. 2005-

100. 

NIOSH [2006]. Hazard evaluation and technical assistance report: Gilster-Mary Lee 

Corporation, Jasper, MO. By Kanwal R, Kullman G, Fedan K, Kreiss K. Cincinnati, OH: U.S. 

Department of Health and Human Services, Centers for Disease Control and Prevention, 

National Institute for Occupational Safety and Health, NIOSH HETA Report No. 2000-0401-

2991. 

Smith TJ [ 1992]. Occupational exposure and dose over time: limitations of cumulative exposure. 

Am J Ind Med 21(1):35-51. 

5 

Appendix H – Development of a Job Exposure Matrix for Company G 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 381
 

Table A4. l Industrial Hygiene Survey Dates 

Survey Survey Dates 

1 November 11 - 18, 2000 

2 Jan 17 - 19, 2001 

3 April 1 - 4, 2001 

4 September 4 - 8, 2001 

5 November 6 - 8, 2001 

6 March 18 - 21, 2002 

7 August 11 - 16, 2002 

8 January 27 - 31, 2003 

9 July 14 - 18, 2003 

Table A4.2 Exposure Categories used for JEM 

# Exposure Cate2ory Jobs Included in Exposure Cate2ory 

1 Warehouse Warehouse Job Category 

2 Maintenance Maintenance Job Category 

3 Outside Processing I Office Outside Processing & Office Job Categories 

4 Polyethylene Line Polyethylene Packer and Polyethylene Stacker Job 

Categories 

5 Microwave Mixing Microwave Mixer Job Category 

6 Microwave Packaging Line Microwave Job Categories: Machine operator, Packer, 

Stacker, Supervisor, and Inventory Control 

7 Bag Print Bag Print Job Category 

8 Quality Control Quality Control Job Category 

Table A4.3 Procedures Used for Estimating Personal Equivalent Diacetyl Exposures 

for Survey 1 

6 

Appendix H – Development of a Job Exposure Matrix for Company G 

382 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

# Exposure Category Procedures used by Exposure Category 

1 Warehouse Use the mean of the area sample diacetyl concentrations 

from survey 1. 

2 Maintenance Calculate ratio of the survey 3 diacetyl mean for personal 

samples to the average of survey 3 diacetyl mean from 

personal samples for polyethylene, mixer, and microwave 

packaging line job categories. Apply this ratio to the 

average of the same three groups from survey 1 after they 

have been converted to personal equivalent exposures. 

3 Outside Processing I Office Use the mean of the area sample diacetyl concentrations 

from survey 1. 

4 Polyethylene Line Use the mean of the area sample diacetyl concentrations 

from survey 1. 

5 Microwave Mixing Model personal to area diacetyl concentrations from 

surveys 2-9 and apply model to survey 1 area samples to 

determine personal equivalent diacetyl exposures. 

6 Microwave Packaging Line Model personal to area diacetyl concentrations from 

surveys 2-9 and apply model to survey 1 area samples to 

estimate personal equivalent exposures. 

7 Bag Print Use the average of the personal equivalent diacetyl 

exposures for survey 1 from the microwave packaging line 

and warehouse job categories. (Note: there were no bag 

print area diacetyl samples for survey 1) 

8 Quality Control Model personal to area diacetyl concentrations from 

surveys 2-9 and apply model to survey 1 area samples to 

determine personal equivalent diacetyl exposures. 

7 

Appendix H – Development of a Job Exposure Matrix for Company G 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 383
 

Table A4.4. Dates of exposure control changes and NIOSH industrial hygiene surveys. 

Date Event 

Cross-Sectional Industrial Hygiene Survey, Respiratory protection training by NIOSH (November 11-18, 

2000) 

Engineering Control Technology Survey (January 17 - 19, 2001) 

February 12, 2001 Exhaust fan installed in oil and flavoring mixing room 

February 2001 Heated liquid flavoring tanks (2) vented to exhaust fan 

Pump installed for closed transfer of flavorings between holding and 

March 29, 2001 mixing tanks 

Follow-up Survey (April 2-5, 2001) 

Mixers supplied with powered air-purifying respirators and respirator 

training. Respirators available to workers in other microwave production 

April 6, 2001 areas on voluntary basis 

Local exhaust ventilation installed for 2 of 7 oil tanks on mezzanine. 

(Note, tanks were initially vented into packaging area air until September 

May 22, 2001 2001) 

June 6, 2001 Flavoring storage cabinets completed for storing bulk flavorings 

July 16, 2001 Temperature control installed on one flavoring tank 

Tempered, outside supply air intake system completed, providing 

August 7, 2001 replacement air for microwave popcorn production areas 

Follow-up Survey (September 4 - 8, 2001) 

September 11, 2001 Exhaust fan installed in quality control lab 

September 18, 2001 Fresh air intake installed in quality control lab 

September 21-30, 2001 Completion of local exhaust ventilation for all mezzanine oil tanks 

November 2, 2001 Flavoring transfer pump installed for 5-gallon containers 

November 2, 2001 Air lock installed outside of mixing room 

Follow-up Survey (November 6 - 8, 2001) 

Follow-up Survey (March 18 - 21, 2002) 

Started use of supplied-air respirators for mixers in mixing room and 

mezzanine (air-purifying respirators with organic vapor cartridges and 

August 2, 2002 particulate filters had been used prior to this.) 

Microwave ovens and testing counter in quality control lab enclosed with 

August 9, 2002 plastic curtain 

Follow-up Survey (August 11 - 16, 2002) 

September 7, 2002 Started using new mixing room (ventilation incomplete) 

September 30, 2000 Discontinued use of one paste butter flavoring 

October 1, 2002 New mixing room wall exhaust fan operational 

Follow-up Survey (January 27 - 31, 2003) 

March 9, 2003 Enclosure of tanks on mezzanine completed 

April 10, 2003 Air-handler functional on mezzanine 

New exhaust fan operational in quality control lab (in new "popping 

April 15, 2003 room") 

April 15, 2003 2 additional exhaust fans (for mezzanine and mix room) 

8 

Appendix H – Development of a Job Exposure Matrix for Company G 

384 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

Microwave ovens moved into popping room in quality control lab 

14 - 18, 2003) 

Table A4.5 JEM Exposure Time Periods by Exposure Category 

# Exposure Cateeory Time Periods 1 

1 Warehouse: 

Time 1 (Surveys 1 & 2 sampling results): Reflects exposures before major control changes 

that would impact warehouse worker exposures. Warehouse workers would go into 

microwave packaging, primarily on fork-lifts to remove finished product, and could 

receive higher, packaging area exposures accordingly. 

Time 2 (Surveys 3 - 9 sampling results): Reflects the control changes implemented in the 

microwave mixing room in February of 2001 including the addition of exhaust ventilation, 

closed transfer of liquid flavorings, and flavor tank ventilation. These mixing room control 

changes impacted warehouse diacetyl exposures since warehouse workers would enter 

microwave production area daily. Additionally, in August of 2001, the installation of an 

outside supply air intake system provided clean, tempered air into the warehouse area and 

makeup air for microwave production axial wall exhaust fans. This allocation of time 

periods was also selected since a majority of warehouse exposures to diacetyl were nondetectable since April of 2001. 

2 Maintenance: 

Time 1 (Surveys 1 & 2 sampling results): Reflects exposures before major control changes 

that would impact microwave production exposures, including maintenance worker 

exposures since maintenance workers would work on the production lines as well as in the 

microwave mixing room. 

Time 2 (Survey 3 sampling results): Reflects the control changes implemented in the 

microwave mixing room in February of 2001 including the addition of exhaust ventilation, 

closed transfer of liquid flavorings, and flavor tank ventilation. These mixing room control 

changes would affect maintenance worker exposures since they would work in mixing and 

microwave production and there was a maintenance office located in microwave 

production. 

Time 3 (Surveys 4 - 7 sampling results): Reflects the control changes including the 

installation of an outside supply air intake system providing clean, tempered air into the 

warehouse, the completion of LEV ventilation on mezzanine flavor holding tanks and the 

air-lock installation on the mixing room. All these microwave mixing and production 

control changes would impact maintenance workers since they would work in these 

production areas. Also, maintenance exposures during this time period were still primarily 

above the LOD. 

9 

Appendix H – Development of a Job Exposure Matrix for Company G 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 385
 

Time 4 (Surveys 8 & 9 sampling results): Reflects the control changes including first use 

of enclosure of the mezzanine tanks. Additionally, maintenance exposures during this time 

period were largely below detectable limits. 

3 Outside Processing I Office Workers: 

Time I (Surveys I - 9 sampling results): Reflects low, predominantly non-detectable 

exposures for workers who were outside (outside processing) or normally away from 

microwave mixing and production operations. 

4 Polyethylene Line: 

Time I (Surveys I & 2 sampling results): Reflects polyethylene line worker exposures 

before major control changes in the microwave production area that could impact 

polyethylene line workers; although exposures in this category were low by comparison to 

microwave production lines, there were some detectable diacetyl exposures in personal and 

area samples for polyethylene line workers during this time period so a separate time 

period was used. While the polyethylene lines were located away from the microwave 

production area, there was some potential for exposure in this work group prior to control 

changes. 

Time 2 (Survey 3-9 sampling results): Reflects the first control changes implemented in the 

microwave mixing room (including the addition of exhaust ventilation, closed transfer of 

liquid flavorings, and flavor tank ventilation) plus all subsequent changes. After the first 

implementation of exposure control in microwave mixing and production areas, exposure 

among polyethylene line workers were largely below detectable limits. 

5 Microwave Mixers: 

Time I (Survey I & 2 sampling results): Reflects exposures before major control changes 

that would impact microwave mixer exposures. 

Time 2 (Survey 3 sampling results): This time period reflects the first control changes 

implemented in the microwave mixing room including the addition of exhaust ventilation, 

closed transfer of liquid flavorings, and flavor tank ventilation. During this time, the mixers 

desk was moved outside of the mixing room as an administrative control. This time period 

was also before the use of powered air purifying respirators (PAPRs) or air-line respirators. 

Time 3 (Surveys 4 - 7 sampling results): Reflects significant control changes for mixing 

workers including first use of P APR respirators in April of 200 I as well as subsequent use 

of supplied-air respirators in August of 2002 which would have significantly reduced mixer 

exposures. Both types of respiratory protection employed loose-fitting hoods with an 

applied protection factor (APF) of 25. (See description below on procedures used to 

estimate mixers exposure adjusting for respirator use). Other significant changes during 

this time period would include the addition of an outside supply- air system which 

provided clean, tempered make-up air for microwave production and mixing room air 

10 

Appendix H – Development of a Job Exposure Matrix for Company G 

386 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

exhaust fans. 

Time 4 (Survey 8 & 9 sampling results): Reflects the first use of the new mixing room and 

the addition of new mixing room exhaust fans. This time period also reflects enclosure of 

the mezzanine area reducing microwave packaging exposures outside the mixing room 

below quantifiable or detectable levels; this would affect mixers exposures when in the 

microwave packaging area and not in respiratory protection in the mixing room. 

6 Microwave Line: 

Time 1 (Surveys 1 & 2 sampling results): Reflects exposures before major control changes 

that would impact microwave line exposures. 

Time 2 (Survey 3 sampling results): Reflects the first control changes implemented in the 

microwave mixing room including the addition of exhaust ventilation, closed transfer of 

liquid flavorings, and flavor tank ventilation which would impact microwave line 

exposures since the mixing room was adjacent and open to the mixing lines. 

Time 3 (Surveys 4 - 7 sampling results): Reflects several control changes including the 

installation of an outside supply air intake system providing clean, tempered air for 

microwave production area exhaust fans. This period also reflects completion of LEV 

ventilation on mezzanine flavor holding tanks and air-lock installation isolating the mixing 

room from packaging areas. 

Time 4 (Surveys 8 & 9 sampling results): Reflects the first use of the new mixing room 

and the addition of new mixing room exhaust fans. This period also reflects enclosure of 

the mezzanine area reducing microwave production exposures outside the mixing room 

below quantifiable or detectable levels. 

7 Bag Printing: 

Time 1 (Survey 1 & 2 sampling results): Reflects exposures before major control changes 

that would impact bag printing exposures due to close proximity to the microwave 

production lines. Also, when the bag printing operations were shut down, bag print workers 

would often work on the microwave production lines. 

Time 2 (Survey 3 sampling results): Reflects the first control changes implemented in the 

microwave mixing room including the addition of exhaust ventilation, closed transfer of 

liquid flavorings, and flavor tank ventilation. These control changes would impact bag 

printing exposures since the bag print lines were located in the warehouse just outside a 

large open doorway into microwave production; additionally, when the bag printing 

operations were shut down, bag print workers would often work on the microwave 

production lines. 

Time 3 (Surveys 4 - 7 sampling results): Reflects several control changes including the 

installation of an outside supply air intake system providing clean, tempered air into the 

11 

Appendix H – Development of a Job Exposure Matrix for Company G 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 387
 

warehouse and subsequently for microwave production area exhaust fans. This period also 

reflects completion of LEV ventilation on mezzanine flavor holding tanks and air-lock 

installation isolating the mixing room from packaging areas. 

Time 4 (Surveys 8 & 9 sampling results): Reflects the first use of the new mixing room 

and the addition of new mixing room exhaust fans. This period also reflects enclosure of 

the mezzanine area reducing microwave production exposures outside the mixing room 

below quantifiable or detectable levels. 

8 Quality Control: 

Time I (Surveys I & 2 sampling results): Reflects exposures before major control changes 

that would impact microwave quality control exposures. 

Time 2 (Survey 3 sampling results): Reflects the first control changes implemented in the 

microwave mixing room including the addition of exhaust ventilation, closed transfer of 

liquid flavorings, and flavor tank ventilation. These changes would impact quality control 

exposures since the quality control room opened into the microwave production area. Also, 

the quality control room was generally under negative pressure relative to the microwave 

production room at this time. 

Time 3 (Surveys 4 - 6 sampling results): Reflects installation of an exhaust fan and fresh 

air intake in the quality control lab. It also reflects several changes that would impact 

quality control worker exposures through reduction of diacetyl concentrations in the 

microwave mixing and production areas including the installation of an outside supply air 

intake system providing clean, tempered air into the warehouse and subsequently for 

microwave production area exhaust fans; installation of a mixing room air-lock; and 

ventilation of mezzanine flavor holding tanks. 

Time 4 (Surveys 7 & 8 sampling results): Reflects the enclosure of the microwave ovens 

in the quality control lab. 

Time 5 (Survey 9 sampling results): Reflects the relocation of all microwave ovens into a 

separate, ventilated room. This step reduced quality control exposures below detectable 

levels. Other control changes to the microwave production area during this period reduced 

microwave production exposures below detectable levels further impacting quality control 

exposures. 

12 

Appendix H – Development of a Job Exposure Matrix for Company G 

388 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
Appendix I
 

Typical Protocol for Collecting Air Samples 

for Diacetyl and 2, 3-Pentanedione 
 

Typical protocol for collecting air samples for diacetyl and 2, 3-pentanedione. 

While the elements of a well-designed exposure monitoring program are discussed in Chapter 

10, the details of a typical sampling protocol for determination of airborne concentrations of 

diacetyl and 2, 3-pentanedione vapor are described here. This protocol, which is based on OSHA 

Method 1012 (Appendix 2 - A), is available at 

http://www.osha.gov/dts/sltc/methods/validated/1012/1012.html. The same air sampler is 

specified in OSHA Method 1012 and 1013 for diacetyl, and in OSHA Method 1016 for 2,3-

pentanedione. It consists of two silica gel tubes connected in series using the least amount as 

possible flexible tubing. Each tube contains a single 600-mg section of specially cleaned and 

dried silica gel with a glass-wool plug and a glass fiber filter at front of the tube before the silica 

gel, and another glass wool plug at the end of the tube. This method is selected because it has 

greater sensitivity than OSHA Method 1013. Method 1012 requires the use of an ethanol 

solution containing a derivatizing reagent to extract and chemically derivative diacetyl in the 

samples. 

Preparation 

Before entering the work area all members of the sampling team should be made aware of any 

requirements for safety equipment such as hair nets, respirators, or safety shoes, and possess all 

necessary equipment and training, including respirator certification if needed. Procedures and 

schedules should be coordinated with the analytical laboratory to assure compatibility of 

procedures and availability of personnel to process samples in a timely manner. 

All sampling equipment and supplies should be prepared in advance. Equipment may include 

battery powered personal sampling pumps capable of operating in the appropriate flow rate range 

and pressure drop, chargers for those pumps, sample holders of a size compatible with the 

sampling media, and flexible tubing to connect pumps and sample holders. In this protocol 

diacetyl and 2, 3-pentanedione vapor samples are collected with two silica gel sorbent tubes in 

series (SKC Inc., Eighty Four, PA, Catalog no. 226-183). The front tube is connected to the back 

tube with a piece of tubing to form the sampling train. If the sample holders are not opaque, these 

sorbent tubes should be wrapped in foil or opaque tape during and after sampling to prevent 

2 

Appendix I – Typical Protocol for Collecting Air Samples for Diacetyl and 2, 3-Pentanedione 

390 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

exposure to light. Each sampling tube should be marked with a unique identification number, 

either before or after sampling. This information is entered in the field data sheet along with the 

corresponding pump ID, calibrated flow rate, and other information. A useful convention is to 

mark each of the two tubes of a sample with the same initial identifier, then add an "f' for the 

front tube and an "r" for the rear tube. 

Sampling trains should be assembled and calibrated with sampling media in line, and this 

sampling media should not be used for any other purpose. Nominal sampling rates for this 

method are 0.05 Lpm for 180-minute TWA samples and 0.2 Lpm for 15-minute STEL samples. 

Calibrated flow rate for each pump should be recorded on a field data sheet with an identification 

code for that pump. A supply of belts, clips, tape, and miscellaneous tools should be available to 

attach the sampling trains to workers to minimize interference or safety concerns with their jobs. 

Collection 

To collect samples for the full work shift, the sampling team should be prepared to place 

sampling trains on the workers as they begin their shifts. Workers and locations to be evaluated 

should have been previously identified from knowledge of the tasks to be performed and the 

compounds to be used. A common practice in selecting sampling locations is to choose tasks 

anticipated to produce the greatest level of exposure to diacetyl or 2, 3-pentanedione and to 

sample the workers conducting those tasks or collect area samples in those areas. This allows for 

the greatest likelihood of obtaining samples above the limit of detection for the analytical 

method, and assumes that if exposure is controlled so that the highest exposures are within 

allowable limits then all exposures are within those limits. 

Immediately before sampling, break off both ends of the flame-sealed tube to provide an opening 

approximately half the internal diameter of the tube. Attach the tube holder to the worker so that 

the adsorbent tube is in an approximately vertical position with the inlet in the breathing zone. 

Position the sampling pump, tube holder, and tubing so they do not impede work performance or 

safety. As each sampling train is placed and started, the start time should be noted on the field 

data sheet for that pump, along with the name or other identifier of the person wearing that 

pump, job title or a description of tasks, and location within the work facility. Sampling site 

3 

Appendix I – Typical Protocol for Collecting Air Samples for Diacetyl and 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 391
 

temperature, relative humidity, barometric pressure, and any other relevant observations should 

be recorded on field data sheets throughout the duration of sampling. Members of the sampling 

team should rotate among the sampling locations during the collection of samples. They should 

occasionally check all sampling devices, observe workers tasks, and note observations on the 

field data sheet. The use of personal protective equipment and other safety and health controls, 

ventilation, and all other salient observations should also be noted. Attempt to determine through 

observation and discussions if workers are engaging in "normal operations." 

OSHA states a reliable quantitation limit of 1.3 ppb ( 4.57 ug/m3

) for diacetyl and 

9.3 ppb (38 μg/m3

) for 2, 3-pentanedione with a 180-minute sample duration and a flow rate of 

0.05 lpm (or 15 minutes at 0.2 lpm). If the shift being sampled is 8 hours long, three samples 

approaching 180 minutes would be acceptable to obtain a TWA analyte concentration. These 

samples should be able to quantify diacetyl and 2, 3-pentanedione at the REL of 5 ppb) TWA or 

25 ppb) STEL without exceeding the breakthrough capacity of the sorbent media. 

Sampling Surveys 

Employers shall conduct exposure monitoring surveys to ensure that worker exposures 

(measured by full-shift samples) do not exceed the REL, either on a time weighted or short term 

basis. Because adverse respiratory health effects may occur at the REL, it is desirable to achieve 

lower concentrations whenever possible. When workers are potentially exposed to airborne 

flavoring compounds, employers shall conduct exposure monitoring surveys as follows: 

• Collect representative personal samples over the entire work shift [NIOSH 1977]. 

• Perform periodic sampling at least annually and whenever any major process change 

takes place or whenever another reason exists to suspect that exposure concentrations 

may have changed. 

• Collect all routine personal samples in the breathing zones of the workers. 

• If workers are exposed to concentrations above the REL, perform more frequent exposure 

monitoring as engineering changes are implemented and until at least two consecutive 

samples indicate that exposures no longer exceed the REL [NIOSH 1977]. 

4 

Appendix I – Typical Protocol for Collecting Air Samples for Diacetyl and 2, 3-Pentanedione 

392 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
 

• Notify all workers of monitoring results and of any actions taken to reduce their 

exposures. 

• When developing an exposure sampling strategy, consider variations in work and 

production schedules as well as the inherent variability in most area sampling [NIOSH 

1995]. 

Focused sampling 

When sampling to determine whether worker exposures to diacetyl or 2, 3-pentanedione are 

below the REL, a focused sampling strategy may be more practical than a random sampling 

approach. A focused sampling strategy targets workers perceived to be exposed to the highest 

concentrations of a hazardous substance [Leidel and Busch 1994]. This strategy is most efficient 

for identifying exposures above the REL if maximum-risk workers and time periods are 

accurately identified. Short tasks involving high concentrations of airborne vapors could result in 

elevated exposure over full work shifts. 

Area sampling 

Area sampling may be useful in exposure monitoring to determine sources of airborne diacetyl or 

2, 3-pentanedione, and to assess the effectiveness of engineering controls. 

Post-collection 

After sampling for the appropriate time, remove the sampling train, record stop time, and remove 

equipment to an uncontaminated area where you can separate the tubes, and seal each tube with 

plastic end caps. Although tubes were protected from light during sampling, it is also necessary 

to wrap each tube in aluminum foil or opaque tape making sure that the sample identification 

number is observable. Samples should be shipped cold (preferable via an overnight carrier) to 

the accredited analytical laboratory using a "six-pack" cooler and frozen ice packs (Blue Ice) or 

similar means to refrigerate samples. Submit blank samples as discussed with the laboratory with 

5 

Appendix I – Typical Protocol for Collecting Air Samples for Diacetyl and 2, 3-Pentanedione 

Occupational Exposure to Diacetyl and 2,3-Pentanedione 393
 

each set of samples. Handle the blank samples in the same manner as the other samples except 

draw no air through them. 

Measure the air flow rate through each sampling train (using surrogate sampling media in line, 

not the actual sample), record this post-sampling flow rate. Determine total sampling time 

(minutes), mean sampling flow rate, and sample volumes (liters). Place sampling pumps on 

charge for reuse in required. 

Submit the samples to the laboratory for analysis as soon as possible after sampling. As a 

precaution, store the samples at refrigerator temperature if a delay in shipment is unavoidable. 

Ship any bulk samples separate from the air samples. 

References: 

Leidel NA, Busch KA [1994]. Statistical design and data analysis requirements. In: Harris RL, 

Cralley L V, eds. Patty's industrial hygiene and toxicology. 3rd ed. Volume 3, Part A New York, 

NY: John Wiley and Sons, Inc., pp. 453-582 

NIOSH [1977]. Occupational exposure sampling strategy manual. Cincinnati, OH: U.S. 

Department of Health, Education, and Welfare, Center for Disease Control and Prevention, 

National Institute for Occupational Safety and Health, DHEW (NIOSH) Publication No. 77-173. 

NIOSH [1995]. NIOSH criteria for a recommended standard: occupational exposure to respirable 

coal mine dust. Cincinnati, OH: U.S. Department of Health and Human Services, Public Health 

Service, Centers for Disease Control and Prevention, National Institute for Occupational Safety 

and Health, DHHS (NIOSH) Publication No. 95-106. 

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Appendix I – Typical Protocol for Collecting Air Samples for Diacetyl and 2, 3-Pentanedione 

394 Occupational Exposure to Diacetyl and 2,3-Pentanedione 
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