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.

# 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;.
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 . 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 . Occupational exposures to diacetyl have been assessed in a variety of food production and flavoring facilities Martyny et al. 2008;NIOSH 2003aNIOSH , b, 2004aNIOSH , b, 2006NIOSH , 2007NIOSH , 2008aNIOSH , b, 2009NIOSH , 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 ; . However, current data indicate that acetoin is considerably less hazardous than diacetyl and it does not have the reactive -dicarbonyl group, which has been implicated in the toxicity of diacetyl and 2,3-pentanedione .
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 ), followed by the packaging area (2.8 ppm)  . 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 ]. Mean full-shift diacetyl air concentrations measured vi Occupational Exposure to Diacetyl and 2,3-Pentanedione at flavor manufacturing facilities ranged from 0.07 ppm to 2.73 ppm Martyny et al. 2008;NIOSH 2003aNIOSH , b, 2004aNIOSH , b, 2006NIOSH , 2007NIOSH , 2008aNIOSH , b, 2009NIOSH , 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 . 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 . In 2012, NIOSH conducted a health hazard evaluation (HHE) involving 75 current employees (88% participation) . 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 .
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 (FEV 1 /FVC) ratio and percent predicted FEV 1 (ppFEV 1 ) 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  and some flavored e-cigarettes . As extensively discussed in Chapter 3, increased prevalence of airway obstruction and decreased FEV 1 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
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;. 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 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  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 .
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.
1 Introduction

# 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 . 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.

# 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 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 andFEMA 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.

# 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 Ashley et al. 2008;Kanwal 2003;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 1 . Introduction as acetaldehyde, butyric acid, and acetoin, have been present in workforces with adverse health effects . 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.  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;.
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 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 . The observation of this case series led to the identification of another case of obliterative bronchiolitis in a separate facility . Since then, numerous cases of obliterative bronchiolitis have been observed in the microwave popcorn industry CDC 2002;Ezrailson 2002;NIOSH 2003NIOSH , 2004aNIOSH , b, 2006Parmet 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 .
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 . 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 . In 2010, California issued the first occupational standard for diacetyl .
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 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) . 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 .
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 .

# 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-  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 . It is not uncommon for odor threshold values reported in the literature to range over four orders of magnitude for the same chemical . Odor threshold variability can result from the source of data, the characteristics of human olfactory response, and the differences in experimental methodology . 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  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  (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  (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  (recognition threshold) Note: not a geometric mean because obtained from a single source.
15 ppb  (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  (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.  National Toxicology Program 2007];
(2) from 2,3-butanediol, by oxidative dehydrogenation of 2,3-butanediol over a copper or silver catalyst ;
(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 ; or, ( 4) from 1-hydroxyacetone (obtained by dehydrogenation of 1,2-propanediol), by the acid-catalyzed condensation of 1-hydroxyacetone with formaldehyde .
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 .
The compound 2,3-pentanedione is also naturally produced by fermentation and is recovered from dairy waste to be used as a flavoring ingredient . The chemical synthesis of 2,3-pentanedione is achieved in the following ways: (1) the condensation of lactic acid and an alkali metal lactate ; (2) the acid-catalyzed condensation of 1-hydroxyacetone with paraldehyde ; or (3) the oxidation of 2-pentanone with excess sodium nitrite and diluted hydrochloric acid in the presence of hydroxylamine hydrochloride .

# 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 .
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 . 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% .
Starter distillate, produced by fermenting milk with starter cultures, contains diacetyl in the 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 . 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 . 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) .
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 .

# 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 [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 .
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 .
Employers in the food manufacturing sector are generally small business owners with 89%  and 2,3-Pentanedione   9 1 . Introduction of establishments employing fewer than 100 employees and nearly 53% of establishments employing fewer than 10 employees . 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 .
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# Assessing Occupational
Exposure in Employees

# 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.

# 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.

# 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. 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, 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/m 3 ) 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 ) 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.

# OSHA Method 1016
OSHA Method 1016 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 . For a 10-liter sample, the RQL of 2,3-pentanedione is 9.3 ppb (38 g/m 3 ).

# OSHA Method PV2118
Superseded by OSHA Methods 1012 and 1013, OSHA Method PV2118  was developed as an air sampling method for diacetyl that uses two 150/75 mg silica gel sorbent tubes in series  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  allowing for greater sample capacity without breakthrough of diacetyl. Sample analysis remained unchanged.

# 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 .  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 . 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 ].

# 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 . 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 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 . 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) . 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.

# 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) .

# 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, 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 . As a result, modifications to the sampling methods are necessary to assess exposure to both vapor and dust.

# 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  are available for the collection and gravimetric analysis of airborne dust.

# 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.

# Bulk Liquids and Solids

# 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 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 ; 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 . 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.

# 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.

# 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 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.

# 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.

# 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 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.

# 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 .
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.

# Industrial Hygiene Surveys and Exposure Assessments
Several investigations have been completed by NIOSH and others within the flavoring and food production industries. Exposure 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 ]. An overview of diacetyl samples collected during multiple investigations is presented in Table 2-1.

# NIOSH Microwave Popcorn Production Exposure Assessments
NIOSH conducted health hazard evaluations at six microwave popcorn plants from 2000 to 2003 . 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 .
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.

# Other Microwave Popcorn Production Exposure Assessments
White et al.  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 . 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.

# 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 . Case histories showed severe fixed    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 , 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  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 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 . 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 . 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.

# Other Flavoring Manufacturing Exposure Assessments
In a study evaluating diacetyl exposures in 16 flavor manufacturing facilities, Martyny et al.
 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  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 (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 .

# 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. 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  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 . 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.

# 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 FEV 1 and a decreased FEV 1 to FVC ratio on spirometry testing. The magnitude of decline in FEV 1 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 . 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   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. 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 
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  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.   NIOSH 2009a], clinical bronchiolitis obliterans , bronchiolitis obliterans syndrome , and flavoring-related bronchiolitis obliterans . 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" . 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 . 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.

# 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 . Historically, bronchiolitis obliterans has been classified into two groups: proliferative bronchiolitis obliterans and constrictive bronchiolitis obliterans . 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 . BOOP was first described in 1985. Prior to this, many cases that matched the description for BOOP were classified as idiopathic bronchiolitis obliterans . The 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 . 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 .
Obliterative bronchiolitis (also referred to as constrictive bronchiolitis obliterans , constrictive bronchiolitis , and bronchiolitis obliterans ) 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 . In affected individuals, pulmonary function tests usually show airways obstruction and hyperinflation , but biopsy-confirmed cases may have normal or restrictive spirometry . Chest x-rays may be normal or show hyperinflation, peripheral attenuation of the vascular markings, and nodular or reticular opacities . 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 . 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 . Identification of the obliterative bronchiolitis lesion on lung biopsy may be difficult because of its patchy distribution , often requiring step-sectioning and special staining to identify airway walls . 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 . In comparison to proliferative bronchiolitis, obliterative bronchiolitis is generally unresponsive to corticosteroid medications and often progresses to more severe disease , although progression after exposure cessation is not characteristic of flavoring-related disease consistent with obliterative bronchiolitis ].
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 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 contemporary clinical criteria, including HRCT scans . 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) .
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 . 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 Ghanei et al. 2004a; .
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. 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 ]. 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.
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 ].

# 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 DL CO , chest x-rays, and a questionnaire 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) . 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. DL CO was normal in 96 of 103 employees tested, including all but one of those with airways obstruction.

# 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 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 . 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 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.  .

# 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 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.  .
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 . 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 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 FEV 1 .
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 FEV 1 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 FEV 1 from first to last survey in Group 2 packaging area employees . 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, . 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 ]. 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 ].
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 HHEs ]. Including the index plant, the three largest plants and one of the small plants had affected mixers NIOSH 2003bNIOSH , 2004a. 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 . 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 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 ].
NIOSH conducted analyses of aggregated data from the medical surveys conducted at the six microwave popcorn plants ].
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 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 ].

# 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 . To assess for evidence of rapid lung function decline, the investigators identified employees with a progressive increase or decrease in FEV 1 of greater than 8% or 330 mL over 12 months among employees who participated in all three spirometry tests . 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 FEV 1 , 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 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. 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 Division of Labor collected 20-minute air samples that showed dust air concentrations of 20 mg/m 3 in an employee's breathing zone and 2.5 mg/m 3 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 FEV 1 " . 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 . 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.  . This prevalence was similar to that expected in comparison to national data from NHANES III . 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% . 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 . Some of the cases included in this analysis of California flavoring employee surveillance data were presented above in the descriptions of two NIOSH HHEs at California flavoring plants (Facilities B and C).

# 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 .

# 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  . NIOSH summarizes the evidence concerning spirometric restriction in flavoring-exposed employees in this section.  . 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 . 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 . 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.

# 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 . 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 ].
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    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  and Iranians following sulfur mustard exposure, in which the pathology included proliferative bronchiolitis . 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 , the much lower proportion of restrictive abnormalities may be explained by the prevailing understanding a decade ago that obliterative bronchiolitis is an obstructive disease.

# 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 . 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 FEV 1 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 . 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) . Average declines in percent predicted FEV 1 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.
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 . The study confirmed an excess risk of abnormal restrictive spirometry reported by NIOSH investigators with a similar prevalence ratio of  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.  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.  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.  paper omits report of average changes in FEV 1 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.
Ronk et al.  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.

# 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 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 . 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 . 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 .
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 FEV 1 decline is about 30 mL/year, and percent predicted FEV 1 does not usually change in the absence of disease because the predicted value is age corrected . Three of the affected former employees from the index microwave popcorn plant (Facility G) had declines in their FEV 1 percent of predicted of approximately 20% to 30% over approximately 2 years ]. NIOSH evaluated data from the eight NIOSH medical surveys at the index microwave popcorn plant for evidence of rapid lung function decline . The investigators chose as the criterion for rapid decline a decrease in FEV 1 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 FEV 1 greater than 8% or 330 mL should not be considered normal..." . 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 .
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 implementation of exposure controls ("Group 1"), 19 (22%) had FEV 1 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 FEV 1 of 600 mL, resulting in the employee leaving employment. This employee's FEV 1 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 FEV 1 to 39% of predicted FEV 1 . 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 FEV 1 declines of greater than 300 mL and/or 10% from their first to their last spirometry test . Of the 27 Group 1 employees who participated in all eight medical surveys, mean annualized decline in FEV 1 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 .  . 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 FEV 1 (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 . As indicated in the studies above, different approaches have been used by investigators over time to define excessive or rapid decline in FEV 1 . 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 criteria, all of which are discussed in Chapter 9, section 9.5.

# 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[NIOSH , 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 ; .
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 

# 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

# 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 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 FEV 1 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 , 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 .
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 NIOSH 1986;. Additionally, NIOSH documented rapid falls in lung function in exposed employees with initially normal spirometry at three plants . California public health surveillance showed that excessive FEV 1 decline occurred in employees in flavor manufacturing plants that participated in a preventive program attempting to lower flavoring exposures .
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 FEV 1 did not change significantly over time (consistent with an irreversible disease). However, employees hired after exposures were controlled had lower prevalences of respiratory symptoms and airways obstruction and higher mean percent predicted FEV 1 on their first medical survey than employees hired before exposures were controlled, and these findings did not change significantly over time NIOSH 2006]. Additionally, among 27 employees who participated in all eight NIOSH medical surveys from 2000 to 2003, annualized declines in FEV 1 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 . Similarly, the former employee index cases with clinical bronchiolitis obliterans had stable FEV 1 within about 2 years of exposure cessation ].
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 . 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 .
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 ]. 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) . 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[NIOSH , 2007a[NIOSH , 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 CDC 2007;Kim et al. 2010;NIOSH 1986NIOSH , 2007aNIOSH , 2008; . 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 .
Additional criteria to support a causal link between diacetyl exposure and severe lung 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 . 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 ]. 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) .
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 obstructionsome 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 (FEV 1 declines approximately 30 mL/year), in a subset of smokers lung function declines more rapidly (FEV 1 declines on average approximately 45-70 mL/year). An estimated 10%-15% of all smokers develop clinically important airflow obstruction . Smokers who experience rapid lung function decline will typically start to become short of breath once their FEV 1 falls below 60% of predicted; this usually occurs around age 50. Severe airways obstruction (e.g., FEV 1 less than 40% predicted) typically does not occur before 55-60 years of age . 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' DL CO measurements were normal. In airways obstruction due to smoking-related emphysema, DL CO 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 . 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.
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Schachter EN . Popcorn worker's lung. N Engl J Med 347( 5):360-361.
Schlesinger C, Meyer CA, Veeraraghavan S, Koss MN . Constrictive (obliterative) bronchiolitis: diagnosis, etiology, and a critical review of the literature. Ann Diagn Pathol 2( 5  Chemical and physical properties of diacetyl and 2,3-pentanedione are presented in section 1.4. As mentioned there, diacetyl is an -dicarbonyl. Endogenous -dicarbonyl compounds are among the reactive carbonyl species implicated in the formation of advanced glycation end products . Like other -dicarbonyl compounds, diacetyl is reactive, with a tendency to cause protein cross-links . The reactivity of the -dicarbonyl compounds is enhanced by electron attracting groups and decreased by electron donors ]. Thus, diacetyl is a reactive compound, but its alkyl components are electron donors that may somewhat decrease the reactivity of the adjacent carbonyl groups .
Diacetyl and related -dicarbonyl compounds can inactivate proteins, principally through reactions with the amino acid, arginine . The related -dicarbonyl flavoring, 2,3-pentanedione, has been reported to be even more reactive with arginine groups than diacetyl . Recently, in vitro studies indicate that diacetyl can cause -amyloid aggregates and covalently modify -amyloid at 5th arginine (Arg 5 ), although the in vivo relevance of this finding remains to be investigated . 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.

# 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 . Diacetyl imparts the flavor and aroma of butter to many common foods and drinks including butter, cheese, yogurt, beer, and wine . Diacetyl in food plays an important role in food preference .
While less extensively studied in foods than diacetyl, 2,3-pentanedione is a common flavoring in margarine and vegetable spreads . Roasted coffee also contains appreciable amounts of diacetyl . Because diacetyl is not a component of green coffee beans, it appears to be a product of the roasting process .
Bacteria and yeast produce diacetyl during fermentation . It can be produced by metabolism of an acetaldehyde-thiamine pyrophosphate complex in the presence of acetyl-coenzyme A . Microbes can also produce diacetyl from pyruvate in the presence of acetyl-coenzyme A .
Microbial culture conditions, such as pH, can influence the relative amount of diacetyl produced during fermentation . In addition, the steam distillate of several bacterial cultures grown on skim milk is known as "starter distillate" and is also considered a flavoring . Major components of some starter distillate samples include diacetyl and acetic acid . 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 .
Starter distillate can also contain 2,3-pentanedione as well as butyric acid, which inhibits the metabolism of diacetyl and 2,3-pentanedione . 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.

# 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 . Acetoin can be further reduced to 2,3-butanediol in an NADH-dependent manner . 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 . However, liver homogenates do not produce significant diacetyl from acetoin or 2, 3-butanediol . 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 . 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 . 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 . Inhibitors of DCXR are well described and include n-butyric acid, 2-furoic acid, benzoic acid, and nicotinic acid . 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 .
Occupational Exposure to Diacetyl and 2,3-Pentanedione  81 4 . Toxicology of Diacetyl and 2,3-Pentanedione 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.  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.
 Male and female Wistar-Han rats and B6C3F1 mice 2,3-Pentanedione 0, 50, 100 or 200 ppm 6 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  Male Sprague-Dawley 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.  In the rat kidney, DCXR is localized in the distal tubules and collecting ducts and colocalizes with carboxylmethyllysine, an advanced glycation end product . In the mouse kidney, DCXR is localized to the brush border of the proximal renal tubules . In the human prostate epithelial cells and in normal human skin, DCXR is associated with the cell membrane . In human skin, DCXR is located near the adhesion molecules, e-cadherin and -catenin .
Similarly, in the vascular endothelium in the dermis, DCXR localizes near intercellular junctions, suggesting a potential role for DCXR in cell adhesion . In addition, DCXR activity is present in the respiratory mucosa of the rat, with the highest activity in the olfactory epithelium . DCXR knockout mice are not well characterized phenotypically but are reported to be fertile . 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 . 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 -diketones .
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 . 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.

# 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.

# 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 . 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 .

# 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 .
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 . Cutaneous sensitization by diacetyl and 2,3-pentanedione may be initiated through haptenation with proteins containing the amino acids lysine and arginine . Diacetyl is corrosive to the cornea of the eye using the Draise test ].

# 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 . 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 ]. 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 . 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 .
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 . 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 (R L ) and dynamic lung compliance (C dyn ) 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 . 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 . At both 200 and 400 ppm, diacetyl caused necrotizing rhinitis in mice that 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 . 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 ].
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 . 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 .
Oropharyngeal aspiration permits exposures that bypass the rodent nose and, hence, scrubbing at that site . 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 .
A subsequent study demonstrates development of obliterative bronchiolitis in rats after intratracheal instillation of diacetyl . 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 . A study demonstrated bronchial fibrosis in rats inhaling 150 or 200 ppm diacetyl for 2 weeks ].
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 caused a decrease in respiratory rate and an increase in the "time of break" between inhalation and exhalation, an indicator of sensory irritation . 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 . 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 . 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 ]. However, after 18 weeks of exposure, respiratory rates in mice inhaling 25 ppm diacetyl were significantly lower than in controls ].
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 . While this finding has generally been interpreted as being related to olfaction , the potential exists for toxicity to olfactory neurons that radiate into the olfactory bulb. Phagocytosis of olfactory nerve material and increases in Tnf 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 .
Although powdered butter flavoring can produce fewer vapors than liquid butter flavorings, the powders have a major respirable component .
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 m in diameter. Therefore, powders have the potential to reach the intrapulmonary airways .

# In Vitro Toxicology of Diacetyl and 2,3-Pentanedione
Diacetyl is mutagenic in the Salmonella typhimurium tester strains TA100 and TA104 . However, diacetyl also reacts with mutagenic heterocyclic amines and suppresses the mutagenicity of 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 . 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 . Additional studies on the genotoxicity of diacetyl have been reviewed in the background documents available online as part of the National Toxicology Program .
In isolated mitochondria, diacetyl closes the mitochondrial permeability transition pore and renders it insensitive to Ca 2+ . 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 . 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) . In addition, diacetyl can be metabolized by pig heart mitochondrial pyruvate kinase to form acetate and acetyl-CoA with a K m value of 0.46 mM. Diacetyl is also a competitive inhibitor of pyruvate metabolism by pyruvate dehydrogenase with a K i of 0.43 mM .
The isolated, perfused trachea system employing tracheas from unexposed guinea pigs has been used to investigate the effects of diacetyl in vitro ]. 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 increase in airway reactivity had been anticipated .
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 . 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) ]. The effects of diacetyl in isolated airways from naive animals does not involve the airway epithelium .
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 (V t , 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 V t further and decreased transepithelial resistance (R t , cm 2 ). R t 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  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 . Bioelectric responses of neurons also have been reported to be inhibited by 2,3-butanedione monixime .
2,3-Pentanedione is not mutagenic in Salmonella typhimurium strains TA98, TA100, TA102, or TA104 .
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 . Amphiregulin has previously been reported to play a role in pulmonary fibrosis, although the nature of that role is not fully understood .

# Toxicology of Inhaled Diacetyl Substitutes
Diacetyl is the compound largely responsible for the flavor of butter in butter . However, exposures in workplaces that make or use butter flavoring and emissions from heated butter flavoring involve multiple volatile compounds . Among the potential replacements for diacetyl, starter mix contains high concentrations of diacetyl . 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 ; .
Acetoin is structurally very similar to diacetyl but an -hydroxyketone in acetoin replaces the reactive -diketone implicated in the toxicity of diacetyl and 2,3-pentanedione . 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 . 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 -diketone, and diacetyl is a 4-carbon -diketone. Eighteen hours after a 6-hour inhalation exposure to 2,3-pentanedione (120, 240, 320, and 360 ppm), R L and C dyn in anesthetized rats were unaffected . 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 . 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 . 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 . Recently, more than 3500 genes were found to be upregulated in RNA isolated from the fibrotic bronchi of 2,3-pentanedione exposed rats . Some of the up-regulated genes were ones previously implicated in fibrosis, including transforming growth factor-2, interleukin-1, 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 .

# Diacetyl and 2,3-Pentanedione in Cigarette Smoke
A recent study suggests that mainstream tobacco smoke collected by a smoking machine contained significant amounts of diacetyl that varied with the smoking parameters set by different organizations.  2006;Polzin et al. 2007]. Other recent studies have identified diacetyl and 2,3-pentanedione in electronic cigarette liquids and aerosols .
In one study, after derivatization with 1,2phenylenediamine, quantification by GC-NPD, and confirmation by GC-MS, 301-433 g diacetyl/cigarette was measured in the total mainstream smoke withdrawn from each burning cigarette from 15 different commercial reference cigarettes . 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 g diacetyl/cigarette was measured from 41 different types of cigarettes . Thus, several studies suggest that significant diacetyl is present in mainstream cigarette smoke, although the concentrations vary . Finally, electronic cigarette liquids often contain diacetyl and 2,3-pentanedione .
The Pierce et al.  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 560fold 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, 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.
, these concentrations of diacetyl would be predicted to decrease FEV 1 in some individuals if inhaled for a working lifetime (see Chapter 5). Indeed, many smokers do demonstrate significant decreases in FEV 1 . Additionally, cigarette smoking is a major risk factor for chronic obstructive pulmonary disease, and a decrease in FEV 1 is a characteristic feature of this disease. In addition, bronchiolar fibrosis is part of the airway remodeling response that characterizes chronic obstructive pulmonary disease . Decreases in FEV 1 and fibrosis of bronchioles are features that also characterize obliterative bronchiolitis . However, smokers with chronic obstructive pulmonary disease have additional morphologic changes in their lungs, including emphysema, that are not seen in obliterative bronchiolitis . 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 , 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 , 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 FEV 1 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.

# 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 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 . Fourth, repeated inhalation exposure to 2,3-pentanedione causes fibrosis of intrapulmonary airways ]. 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 ]. 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 . 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 . 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 . 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 . 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 . 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 . 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 . Under resting conditions at an exposure concentration of 100 ppm, the rat nose and trachea are predicted to 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 . Because the respiratory epithelium of the terminal bronchiole of humans is believed to be the target tissue for the development of obliterative bronchiolitis , 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" . 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 .
Damage to the nose of rodents has recently been described for another agent implicated in causing obliterative bronchiolitis in humans, sulfur mustard , a chemical warfare agent. Obliterative bronchiolitis is considered a major cause of progressive respiratory disease in survivors of sulfur mustard exposure . 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 . 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 . 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.

# 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 -diketone, 2,3-pentanedione, suggest that the airway toxicity of diacetyl may be shared with other structurally related, -diketones, and that inhalation of either diacetyl and 2,3-pentanedione can cause airway fibrosis in rats.
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# References
Aeschbacher HU, Wolleb U, Loliger J, Spadone JC, Liardon R . Contribution of coffee aroma constituents to the mutagenicity of coffee. Food Chem Toxicol 27( 4 Colley J, Gaunt IF, Lansdown AB, Grasso P, Gangolli SD . Acute and short-term toxicity of diacetyl in rats. Food Cosmet Toxicol 7( 6):571-580.
Conolly RB, Kimbell JS, Janszen D, Schlosser PM, Kalisak D, Preston J, Miller FJ . Human respiratory tract cancer risks of inhaled formaldehyde: dose-response predictions derived from biologically-motivated computational modeling of a combined rodent and human dataset. Toxicol Sci 82( 1 Eriksson O, Fontaine E, Bernardi P . Chemical modification of arginines by 2,3-butanedione and phenylglyoxal causes closure of the mitochondrial permeability transition pore. J Biol Chem 273( 20 Larsen ST, Alarie Y, Hammer M, Nielsen GD . Acute airway effects of diacetyl in mice. Inhal Toxicol 21( 13 Majcher MA, Klensporf-Pawlik D, Dziadas M, Jelen HH . Identification of aroma active compounds of cereal coffee brew and its roasted ingredients. J Agric Food Chem 61( 11):2648-2654.
Morris JB, Hubbs AF . Inhalation dosimetry of diacetyl and butyric acid, two components of butter flavoring vapors. Toxicol Sci 108( 1  the diacetyl exposure response, standard risk assessment procedures can be applied.

# 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 . 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  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  chronic disease investigations . 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.
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.

# 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%) (  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.

# Outcomes
Reported symptoms and PFT results defined the HHE outcomes. A medical questionnaire was administered that included standard ATS items on respiratory health ] 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 ]. The predicted and lower limit of normal (LLofN) values for FEV 1 , FVC and FEV 1 /FVC were calculated using prediction equations produced from NHANES III [Hankinson et al. 1999 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) =  i (DA), cum(DA 0.5 ) =  i (DA 0.5 ) , and cum(DA 2.0 ) =  i (DA 2.0 ) 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 analyzed using a real-time method (FTIR) to assess time-variability. To make full use of the serial spirometry determinations at Company G, a longitudinal analysis of ppFEV 1 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  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.

# 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:
(  -6 ) and (p=410 -7 ) performing considerably better than exposure duration alone, and with average exposure to diacetyl  and Cum(DA 2.0 ) performing less well than duration (Table 5-5). The estimate for the exposure-response with Cum(DA) was a 0.50 reduction in ppFEV 1 for each ppm-year of cumulative exposure (Tables 5-5, 5-6). (After 1 year at 1 ppm an employee's ppFEV 1 , starting at 100, would be predicted to be 99.5.) For FEV 1 /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 ppFEV 1 : 1.07 (vs. 0.50) and for FEV 1 / 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 ppFEV 1 but cumulative smoking, in pack-years, predicted a decline in ppFEV 1 (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  The best-predicting exposure metric depended on the HHE population analyzed (Tables 5-7, 5.8). In predicting FEV 1 /FVC the R-square values were consistently larger compared with the ppFEV 1 regressions but the exposure effects were sometimes less significant. For Company G, Avg(DA) and were the better predictors of FEV 1 /FVC; for Company K, Cum(DA) was best while for Company L, Avg(DA), and Cum(DA 2.0 ) were all equivalent better predictors. For ppFEV 1 , 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 ppFEV 1 . In the pooled analysis of the Company K and Company L plants, the differences in exposure response (heterogeneity) between the plants for ppFEV 1 and FEV 1 /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 ppFEV 1 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 ppFEV 1 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 ppFEV 1 per ppm-yr was 7.83 while for Plant L the decrease in ppFEV 1 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(DA 2.0 ), which gives greater weight to high values, might better predict spirometry changes than Cum(DA), as was observed at Company K for ppFEV 1 and FEV 1 /FVC, and at Company L for FEV 1 /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 (=0.61 vs. 0.50 for full population; R 2 =0.182 vs. 0.169, resp.) and ( 2) the square root of cumulative exposure (=3.02 vs. 2.75 for full population; R 2 =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 ppFEV 1 and FEV 1 / 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   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 FEV 1 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 R 2 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.  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 ppFEV 1 is not large.

# Incidence of Pulmonary
Impairment at Company G

# 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          CDC 2007;Israel et al. 2009;NIOSH 2006NIOSH , 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 (        with a term of the form  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. . 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).

# 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 FEV 1 decline based on model fit. Average exposure was the weakest predictor of ppFEV 1 . 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 ppFEV 1 and FEV 1 /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 FEV 1 /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.    (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.

# Benchmark Dose

# Methods
For continuously distributed respiratory endpoints such as FEV 1 , the benchmark dose approach permits estimation of excess prevalence of impairment as a function of prior exposure history . On the basis of regression models and population data on the distribution of FEV 1 from NHANES III , 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).

# Risk assessment with percent predicted FEV 1 and FEV 1 /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 FEV 1 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 ppFEV 1 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 ppFEV 1 , the BMD procedure is less direct because LLofN is specific to age, height, gender and race. The distribution of various functions of FEV 1 and LLofN, such as FEV 1 /LLofN or (FEV 1 -LLofN)/(ppFEV 1 -LLofN) are not readily specifiable. An alternate approach was taken: in the NHANES population , the cumulative exposure (Cum(DA)) that would reduce an individual's FEV 1 to their LLofN was calculated using the exposure-response estimates from the preferred regression models of ppFEV 1 (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 FEV 1 and FEV 1 /FVC for the full population and for < 4 yr (Table 5 -29). For FEV 1 below the LLofN (FEV 1 ) the BMD values were similar to those calculated the traditional way for ppFEV 1 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, . BMDs for FEV 1 /FVC below the LLofN (FEV 1 / FVC) were comparable to those for FEV 1 (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 FEV 1 , 0.0005 ppm and FEV 1 /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  Baseline prevalence for < 60% of predicated = 0.0053, for <5th percentile = 0.0498 duration of work. The 1/1000 BMD for FEV 1 , was correspondingly higher: 0.05 ppm diacetyl.

# Excess Lifetime Risk for Pulmonary Impairment

# Methods
Using the life-table approach as implemented in the Biological Effects of Ionizing Radiation IV report  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        Rodriguez et al. 1994] and five others predicted mortality using current FEV 1 . Three studies provide estimates of rate ratios (RRs) that can be applied to a life-table analysis of excess lifetime risk . The estimates range from 1.010 to 1.019 per percent decline in FEV 1 in men, and from 1.01 to 1.025 in women. Assuming a RR of 1.015 per percent decline in FEV 1 , and using the exposure response for FEV 1 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., FEV 1 falling below LLofN (Table 5-32). These estimates of excess mortality are the result of a generic effect of declining FEV 1 on mortality not specific to obliterative    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 FEV 1 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 FEV 1 /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 assumptions used in the final risk assessment and provide confidence in the risk estimates.

# 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" . In the clinical context, if an employee's FEV 1 /FVC is less than 0.7 (or FEV 1 less than or equal to 80%), that would be considered mild COPD . 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 .
The health significance of small spirometry changes, such as a 1% decline in FEV 1 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 FEV 1 decrements are observed that ultimately lead to the diagnosis of obliterative bronchiolitis and fatal disease . 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, quality of life is degraded  and risk is increased for cardiovascular disease and progressive respiratory disease . The decrease in FEV 1 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 .
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 .

# 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 FEV 1 impairment are within a factor of 2.0 of the life-    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 . 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 ]. The nasal region was observed to be the most sensitive.  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 . 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  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.

# 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 . 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, 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 . Recently, bronchial fibrosis has been documented in rats inhaling either 2,3-pentanedione or diacetyl for 2 weeks ].

# Methods

# Data

# Diacetyl
The response data that were analyzed were obtained from the experimental study reported by the NTP . Male and female Wistar-Han rats and male and female B6C3F 1 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.

# 2,3-Pentanedione
The results of a 2-week inhalation study of 2,3-pentanedione toxicity were reported by Morgan et al. . Individual animal data  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 . 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  publication, tables of individual 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.

# 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.

# 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 . development over the past three decades, and it has become an accepted approach for risk assessment . 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 . 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 Y ci denotes the corresponding severity score of the i th rodent exposed to concentration, conc c , j  element of {observed severity scores excluding zero} for the corresponding tissue and type of lesion, Pr( Y ci j)denotes the expected proportion of response score Y ci greater than or equal to j , each  j is an unknown real-valued parameter with  j'  j, and  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(Y ci j) . lower one-sided 95% confidence limit (BMCL) were estimated.

# 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  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 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.

# Extrapolation of rodent benchmark concentrations to humans
Extrapolation of rodent BMCs to humans was based on a PBPK/CFD model for diacetyl Morris and Hubbs 2009]. The Gloede et al.  extension of the Morris and Hubbs  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.  model assumes mouth breathing during light  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 . 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 m 3 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.  estimates incorporating tissue metabolism (V max for the rat, and K cat for humans) were used, because local metabolism is predicted to impact significantly on the local tissue concentration  (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 M, while the predicted tissue concentration for the same anatomical region is 1.4 M in a sedentary nose-breathing human and 2.5 M 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 M - 2.5 hours + 2.5 M - 5.5 hours)/(0.33 M - 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.  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.  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.

# 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  was extended to the mouse using the EPA reference concentration (RfC) methodology . 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.  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.
For the ET region, the RGDR is calculated , eqn. 4-18, as:
where:
V E = minute volume (mL/min = cm 3 /min) SA = surface area (cm 2 ) 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 , eqn. 4-22, as:
where:
V E = minute volume (mL/min = cm 3 /min) SA = surface area (cm 2 ) 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 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.
The values assumed for V E and SA, and the resulting RGDR values for mouse-to-rat extrapolation, are shown in Table 6-5. The rat V E value is based on data from Gloede et al. , and the mouse V E was taken from . The SA values are from EPA .
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:
POD BEC = POD A - RGDR where: POD BEC = POD equivalent concentration in the target species; POD A = 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.  PBPK model is then used to extrapolate from the rat equivalent concentration to a human equivalent concentration.  Mouse-to-rat regional gas dose ratio for the upper respiratory tract  Mouse-to-rat regional gas dose ratio for the tracheobronchial region This conclusion is based on the analysis of Allen , 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.

# 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 . In this case, the interspecies pharmacokinetic factor is replaced by the use of the Gloede et al.  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 .
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 (3.2fold for interindividual variability, 2.5-fold for interspecies variability, and 3-fold for subchronic to chronic) are multiplied, the resulting total UF is 24.

# 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:
=  sjr(t) + u bskci +  s  bskci +f bskcti  sjr(t) {m(s, k, conc bskci , t,  bskci ;  sr(t) ,  skt ,  s )} - conc bskci ,
where  sjr(t) + u skci +  s  bskci describes effects in the absence of exposure, f bskcti  sjr(t) {m(s, k, conc bskci , t,  bskci ;  sr(t) ,  skt ,  s )} - conc bskci describes effects of exposure and b indexes the bioassay study s indexes sex, k = 0  2,3-pentanedione exposure and k = 1  diacetyl exposure, bkc identifies the exposure group and conc bkc is the corresponding exposure concentration, i = 1, ..., n bskc indicates each of the mice within the exposure group identified by bskc and conc bskci denotes the corresponding exposure concentration, r(t) identifies the response lesion, r nested within tissue, t, (lung or nasal), Y bskcr(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(Y bskcir(t)  j) represents the expected proportion having response severity score greater than or equal to j for j  {1, ..., max (Y bskcr(t)i )},  sjt(r) denotes the intercept parameters for lesion r(t) which are subject to constraints  s2t(r)  s1t(r) = s2 <0 and  s3t(r)  s2t(r) = s3 <0 thus ensuring    s3t(r) < s2t(r) < s1t(r) , u bskci ~N(0,  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(Y kcit(r)  3) < Pr(Y kcit(r)  2) < Pr(Y kcit(r)  1) is satisfied for the controls. at low doses for {r(t)}; the hypothesis, 0 sr(t) = 0 s , was tested and subject to being incorporated into the model, and  skt allows for an adjustment for a quadratic effect of concentration that may be attributed to directly proportional changes in respiratory ventilation with concentration where  skt is the constant of proportionality in units of controls' ventilation; thus  skt describes the change relative to controls. The hypothesis,  sk,lung =  sk,nose =  sk , was tested and subject to being incorporated into the model. f bskcti 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 f bskcti was modeled as having unit expectation and variance (e  2 st 1); thus, the variance of log (f bskcti ) =  2 st , t = 1, 2 for the lung and nose, respectively, together with an associated covariance parameter  s12 . The hypothesis that lognormal random effects are independent was examined by testing  s12 = 0 and was subject to being incorporated. Furthermore, the hypothesis that only one lognormal random effect for each mouse was necessary, i.e., f bskc1i = f bskc2i 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 . 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: 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  was assumed.
=  sjr(t) + s  bskci +f bskcti  sr(t) {m(s, k, conc bskci , t,  bskci ;  sr(t) ,  sk , )}conc bskci where m(s, k, conc bskci , t,  bskci ;  sr(t) ,  sk , ) =  i.e.,
The model was coded and fitted using the NLMixed procedure of SAS TM 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" (Y1 | f ) , (Y2|Y1,f ), and (Y3|Y2,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(Y skcr(t)  j | conc=BMC jskr(t) , f skcti = 1)Pr(Y skcr(t)  j | conc = 0, f skcti = 1) = 0.10.

# 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 . 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.

# Results

# 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 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.  model, the human-equivalent BMCL values (BMCL_HECs) range from 1.4-95.8 ppm diacetyl, and the BMCL candidate REL values (after the application of uncertainty factors) range from 0.06-4.0 ppm diacetyl.

# Sensitivity analysis
As a sensitivity analysis, alternative BMC and BMCL values were also derived for the NTP  diacetyl study by dichotomizing the data, fitting quantal models, and model averaging, as described in section 6. Another assumption made in this risk assessment is that toxicity observed in mice can be scaled to rats using the EPA  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 . Using the EPA  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.  PBPK model is the most appropriate method for extrapolating from rats to humans. A possible alternative would be to use the EPA  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  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.  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.    Alternative analysis using respiratory ventilation rates based on body weight, rather than measured values.  Alternative analysis using EPA  RfC methodology rather than the Gloede et al.  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.

# 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  s,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

# 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 , including diacetyl . Reduced respiratory rate and reduced minute volume have been observed in male mice exposed to diacetyl ]. 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 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 . 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  rather than the Gloede et al.  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.

# 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 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  have been based on the 6-week and 12-week mouse study of , rather than the more extensive subchronic study conducted by the NTP . Because the NTP  subchronic study included data from both mice and rats and included both more dose levels and more animals per dose group than the  study, the NTP  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 BMC 10 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 BMCL 10 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.  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  subchronic inhalation study, and therefore includes data from more pathological endpoints than the prior analyses did.
Maier et al.  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 , using BMD methodology. The authors concluded that the most sensitive endpoint in the mouse   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  quantitative risk assessment was based on an analysis of adverse respiratory effects in mice exposed to diacetyl by inhalation for up to 12 weeks . Adverse nasal and lung effects were observed with the latter found in the peribronchial, bronchial, and peribronchiolar regions. The  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  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)  also utilized the dose-response data of , 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. . The Gloede et al.  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 in the upper respiratory tract of rodents and the lower respiratory tract of humans. TERA  estimated HECs using the EPA default methods  modified by the PBPK/CFD model predictions of Morris and Hubbs . 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  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 . 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  risk assessment, the experimentally measured ventilation rates for the  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, V E /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 . 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 . 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).

# 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 , 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 .
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  to the covariance matrix of each set did not support making them.  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 ]. 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 ]. 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.

# 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.
Morris JB, Hubbs AF . Inhalation dosimetry of diacetyl and butyric acid, two components of butter flavoring vapors. Toxicol Sci 108( 1 Wheeler MW, Bailer AJ . Properties of model-averaged BMDLs: a study of model averaging in dichotomous response risk estimation. Risk Anal 27(3):659-670. WHO . Environmental Health Criteria 170. Assessing human health risks of chemicals: Derivation of guidance values for health-based exposure limits. International Programme on Chemical Safety, Geneva, Switzerland.
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.

# 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 . 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 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 ]. 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 . 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.

# Toxicological Studies of Diacetyl
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 that did not cause damage in intrapulmonary airways . 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 . 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 ].
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 . 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 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

# 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 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 ]. On the basis of general industrial 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 .
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 . Morphologic data suggest that 2,3-pentanedione can cause airway epithelial damage similar to the damage caused by diacetyl . 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 . Recently, more than 3500 genes were found to be upregulated in RNA isolated from the fibrotic bronchi of 2,3-pentanedione exposed rats . Some of the up-regulated genes were ones previously implicated in fibrosis, including transforming growth factor-2, interleukin-1, 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 .
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 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.

# 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.

# 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 . 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 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 . 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.

# 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 . The reactivity of the alpha-dicarbonyl compounds is enhanced by electron-attracting groups and decreased by electron donors . Alpha-dicarbonyl compounds can inactivate proteins, principally through reactions with the amino acid, arginine . The related alphadicarbonyl flavoring, 2,3-pentanedione, has been reported to be even more reactive with arginine groups than diacetyl .
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 . 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 . 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 .

# 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: 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. 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.

# 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.

# 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 .
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 .
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.
 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.).

# 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 . 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 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 . 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.

# 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 . 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 . 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 .
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 . The key design parameters are to 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 ) at the face of the workstation when working with flour improvers (Figure 8-3) .
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.

# 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 . The flow control system on a constant-velocity 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  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%.

# 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 ]. 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 . 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 . 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 . 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 . 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) . 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 . 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 .
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 . 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) . 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

# 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 .
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 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) .
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/m 3 . 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 .
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.

# 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 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 . 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 .
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 .
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.

# 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 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 protective equipment.

# Administrative Controls
Work practices, an administrative control, are procedures followed by employers and employees to control hazards in the workplace. 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 . Reduction in spills and elimination of leakage from vessels aid in reducing the overall emission of chemicals into the workplace and lower employee exposure.

# 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.

# 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.

# 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 who are involved in cleaning or are working nearby should use appropriate PPE including respiratory protection, eye, and skin protection.

# 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.

# Informing Employees about the Hazard

# 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-Serum-Toxin Act of 1913 .
Employee training should include information outlined in the OSHA HCS in the section titled "Employee Information and Training" . This includes information about diacetyl and 2,3-pentanedione and mixtures containing these compounds to which employees are exposed, explanation of safety   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 Cavalcanti et al. 2012;CDC 2002CDC , 2007CDC , 2013Halldin et al. 2013]. Precautionary statements for the health and physical hazard classifications presented can be found in Appendix C of the hazard communication standard [29 CFR 1910[29 CFR .1200.  Appendix C of the hazard communication standard [29 CFR 1910[29 CFR .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[29 CFR .1200. See section 8.3.7.3 below for further information.    [29 CFR 1910[29 CFR .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[29 CFR .1200.  Appendix C of the hazard communication standard [29 CFR 1910[29 CFR .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[29 CFR .1200. See section 8.3.7.3 below for further information.
physical hazard criteria presented in Appendix B of the hazard communication standard  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. The GHS classification for acute inhalation toxicity, category 2 for diacetyl is based upon rat acute inhalation studies of diacetyl and 2,3-pentanedione . 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 . 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 .
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 . 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.

# 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  8-3 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[29 CFR .1200. As explained below, this is an important consideration for mixtures containing diacetyl and 2,3-pentanedione. 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 . 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)

# 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[29 CFR .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 . The precautionary statements that are also required can be found in Appendix C to the HCS [29 CFR 1910[29 CFR .1200 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)

# 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 . 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 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. Diacetyl and 2,3 . The ANSI standard was revised in 2010 . The current edition also includes respirators that cover the eyes and face as approvable under the standard.

# Occupational Exposure to
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. 1-20101- [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 . 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 .
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.          CDC (Centers for Disease Control and Prevention) . Fixed obstructive lung disease among workers in the flavor-manufacturing industry -California, 2004.
CDC .

# 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 .
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

# 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 , 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" ;  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.

# 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 .
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 .
To achieve the intent of primary and secondary prevention, employers have an interest in 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.

# 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).

# 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   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  and excessive declines in lung function  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 . 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.

# 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.

# Spirometer specifications
Spirometry testing equipment should meet the ATS/ERS guidance for standardization of spirometry or most recent equivalent , 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.

# 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 . 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 FEV 1 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  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 FEV 1 and FVC by 0.88 . 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 / spirometry/RefCalculator.html can be used to calculate lower limits of normal for NHANES III reference values.   ]. 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.  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 . In these studies, the technicians received immediate feedback from the spirometry device 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 FEV 1 and mean FVC of the study group, compared to use of an older spirometer without feedback capability .
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 Kreiss et al. 2012;NIOSH 2004bNIOSH , 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 FEV 1 of good quality . 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 .

# 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 . 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.

# 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 , 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.
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.

# 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 .
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 FEV 1 . An employee's longitudinal test results may reveal an abnormal decline in FEV 1 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 . While such test results might not meet the criteria for an abnormality such as airways obstruction or spirometric restriction, an abnormal decline in FEV 1 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 (FEV 1 /FVC ratio and FEV 1 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 FEV 1 /FVC ratio) also need additional medical tests to differentiate between nonlung causes and lung causes of spirometric restriction, including obliterative bronchiolitis .
The criteria for an abnormal excessive decline in the FEV 1 depend on the quality of the spirometry tests performed as part of the medical monitoring program and the time period of follow-up . ATS/ERS and ACOEM have stated that a decline in FEV 1 over one year should exceed 15% before being considered clinically meaningful Townsend 2011]. By this criterion, someone with a baseline FEV 1 of 4 liters would 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 NIOSH 2006NIOSH , 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 FEV 1 in a year. In a study that used data from a spirometry surveillance program for coal miners, Wang and Petsonk  found that the 5th percentile for FEV 1 declines over 6 months in all employees studied was 320 mL (7.8%). In stable employees (those employees whose FEV 1 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 FEV 1 as a threshold .
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.  found that the 5th percentile values for FEV 1 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 FEV 1 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 . Lower quality spirometry programs have the disadvantage of only being able to detect larger declines in FEV 1 as abnormal.
NIOSH has developed a computer program, SPIROLA, to help spirometry programs measure their within-person variation in FEV 1 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 FEV 1 that is adjusted for the quality of the provider's spirometry program . 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 (. 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 .

# 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).
Occupational Exposure to Diacetyl and 2,3-Pentanedione 235 The first step in evaluating an employee whose medical monitoring spirometry test shows either an excessive decline in FEV 1 (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 FEV 1 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.

# 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 Kim et al. 2010]. In other words, they had fixed airways obstruction with an FEV 1 and/or FVC increase less than 12% and 200 mL after bronchodilator) . DL CO in affected employees with airways obstruction has usually been normal, although some individuals with advanced disease have had a low DL CO .
Lung volume measurements have shown a normal or elevated total lung capacity (TLC) and an increased residual volume, consistent with air trapping ]. Individuals with moderate to severe airways obstruction may have a mixed obstructive/ restrictive (reduced FEV 1 , FEV 1 /FVC ratio, and FVC) pattern of spirometry because air trapping decreases the FVC. The actual underlying physiology can be clarified by determining lung volumes.

# 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 . Patchy ground glass opacities have been observed less commonly. These findings may not be present despite obliterative bronchiolitis documented by biopsy . HRCTs have not been systematically performed in flavoring-exposed employees with restrictive pulmonary function abnormalities or with excessive FEV 1 declines within the normal range of FEV 1 . Specialist consideration of the diagnostic utility of this test is suggested.

# 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 236 Occupational Exposure to Diacetyl and 2,3-Pentanedione bronchiolar morphology, often replacing surgical lung biopsy .
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 . The term bronchiolitis obliterans syndrome has also been applied to flavoring-exposed employees without surgical lung biopsies ; , 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 . Transbronchial lung biopsies are not useful for evaluating clinical obliterative bronchiolitis in employees exposed to diacetyl, 2,3-pentanedione, or similar compounds.

# 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 , 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 spirometric restriction or normal spirometry without pathologic confirmation. Employees who develop restrictive abnormalities or who have excessive parallel FEV 1 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 ]. 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 . 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 .
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 FEV 1 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 . 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 .
Occupational Exposure to Diacetyl and 2,3-Pentanedione

# 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 . 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.

# 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 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 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 . 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 . 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 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 . 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.
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% . 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. 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.

# Exposure Monitoring Program Goals
A workplace exposure monitoring program should have clear, stated goals . 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)

# 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 .
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 246Occupational 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 . A walk-through survey or preliminary worksite visit is often useful in developing the sampling strategy  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.

# 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 .
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.

# 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 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 . 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 . 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 . Other databases that might prove helpful may include but are not limited to National Library of Medicine (Hazardous Substances to represent the exposures of those groups .
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.

# 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 . 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 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 . 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 addedalong 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 . 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.

# 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.

# 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  such as the STEL values recommended for diacetyl and 2,3-pentanedione.

# 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 . Recently, exposure control banding and Bayesian decision analysis have been used to help support exposure assessment decisions with limited sample numbers .  As stated above, a monitoring strategy should assess the effectiveness of various methods used 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.

# 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.

# 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.

# 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 L/mL p-cymene internal standard gave an average recovery of 99.1 % over the concentration range of 26.5 to 529 g 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 fi xed 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 mi xers and four worked in packaging. The report concluded that "workers exposed to flavorings at microwave popcorn factories are at risk for developing fi xed obstructive lung disease." 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 g 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.  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 g and 3.01 g respectively Below is chromatogram of the RQL level.

# .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  tubes or equivalent was used in this evaluation.

# Reagents
None required .

# 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.
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.
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.

# .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 %   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.

# 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.

# 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-m 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 Millennium 32 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.  3.2.4 The extraction solvent was 0.25 l/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 L/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.
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.  (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

# 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. To obtain adequate sensitivity for this method, it was necessary to derivatize the acetoin and diacetyl. 2, was the first derivatizing agent tried, but DNPH can react with both ketone and a-hydroxy ketones 6, 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. This method is designed for low air concentrations of acetoin, diacetyl, and potential interferences. If high exposures are anticipated, use OSHA Method 1013 8 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 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.  ; 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 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.  lmsds/md102388.html (accessed 311712008

# 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.

# 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, , to protect samples from light.

# Reagents
None required

# 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.  ) 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.
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 g/sample) for acetoin and 97.6% (0.01 to 3.16 g/sample) for diacetyl. The extraction efficiency was not affected by the presence of water.
Extracted samples remain stable for at least 24 h.
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 (long term) 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/m 3
) for acetoin and 0.0042 ppm (0.015 mglm 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 g of acetoin and 0.808 g 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 mglm 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 moles/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/m 3 ) acetaldehyde, 9.49 ppm (23.3 mglm) acetic acid, or 190 ppm (560 mglm 3 ) 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.;Elskamp, C.;Rose, M. Evaluation Guidelines For Air Sampling Methods Utilizing Chromatographic Analysis, 1999. U.S. Department of Labor, Occupational Safety and Health Administration Web site.  .govldtslsltclmethodslchromguidelindex.html (accessed 311512008). 11 of 34 T-1012-FV-01-0811-M 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)

# Analytical Procedure
Adhere to the rules set down in your Chemical Hygiene Plan
. Avoid skin contact and inhalation of all chemicals and review all MSDSs before beginning this analytical procedure.

# 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 -electron capture detector (ECO) was used in this evaluation.
33 Occupational Exposure to Hazardous Chemicals in Laboratories. Code of Federal Regulations, Part 1910.1450, Title 29, 2003.    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.
.l!l  0 (.) "' @ <(

# Vi'
.l!l  0 (.) "' @   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
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 g for acetoin and 0.052 g for diacetyl.  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!m 3 ) acetoin and 0.050 ppm (0.180 mg/m 3 ) 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.     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/m 3)
acetoin and 0. 050 ppm (0. 180 mg/m 3 ) 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.    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.   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 glm 3) acetoin and 0.101 ppm (0.355 mg/m 3 ) 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 ) 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.

# 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 g/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 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.

# 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.

# of 34 T-1012-FV-01-0811-M
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. 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).
Material Safety Data Sheet: Diacetyl, Chemwatch, Victoria, Australia (accesed 311712008). Sheet: 2,3-Butanedione, https:llfscimage.fishersci.comlmsds/03275.htm (accessed 311712008). Material Safety Data Sheet: 2,3-Butanedione, http:llwww.chemservice.com/msds/msds_detail.asp?catnum=0-816 (accessed 311712008).

# Material Safety Data

# 31of34
T-1012-FV-01-0811-M

# . 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. This procedure, Method 1013, was streamlined for monitoring low ppm levels, and Method 1012 6 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 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 1012 7 , Section 4.9).
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. 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.

# 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.
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.

# 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)

# 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.

# 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, .
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.

# Reagents
None required

# 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.
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.
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/m 3 or 0.99 ppm) and diacetyl (3.55 mg/m 3 or 1.01 ppm) with an average relative humidity of 40% at 34 C (absolute humidity of 14.8 mg/L H 2 0). 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.)
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 g 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 g 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.
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/m 3 ) for acetoin and 0.035 ppm (0.12 mg/m 3 ) for diacetyl when 3 Lare collected.
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 g of acetoin and 8.1 g 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 H 2 0). Samples were collected at a sampling rate of 0.05 L/min.

# 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 H 2 0). 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 H 2 0). 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 H 2 0). 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/m 3 of 2-nonanone and 1.88 mg/m 3 of 2,3-pentanedione. The test atmosphere had an average relative humidity of 38% at 34 C (absolute humidity of 14.1 mg/L H 2 0). 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 H 2 0). 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.

# 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.

# 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-m df) capillary column was used in this evaluation.
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.

# 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.

# 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. The Guidelines define analytical parameters, specify required laboratory tests, statistical calculations and acceptance criteria.
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 g/sample acetoin and 1 .05 g/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-L 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. 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.  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 g of acetoin per sample and 0.96 g 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-L 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 g acetoin per sample (0.0031 ppm or 0.011 mg/m 3 for a TWA sample).  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 g diacetyl per sample (0.0034 ppm or 0.012 mg/m 3 for a TWA sample).  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
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 g/sample for acetoin and 0.82 g/sample for diacetyl. 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. 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 H 2 0). 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.

# 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.
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/m 3 or 0.99 ppm) and diacetyl (3.55 mg/m 3 or 1.01 ppm) with an average relative humidity of 40% at 34 C (absolute humidity of 14.8 mg/L H 2 0). 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.   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. 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.

# 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.
Interferences (sampling) Retention The ability of the sampler to retain acetoin and diacetyl was tested by sampling from a dynamically generated test atmosphere of acetoin (3.67 -----------------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/m 3 or 1.13 ppm) and diacetyl (4.03 mg/m 3 or 1 .14 ppm) with an average relative humidity of 8% at 33 C (absolute humidity of 2.82 mg/L H 2 0). 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/m 3 or 0.0515 ppm) and diacetyl (0.175 mg/m 3 or 0.0497 ppm) with an average relative humidity of 42% at 33 C (absolute humidity of 14.8 mg/L H 2 0). 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/m 3 or 0.0514 ppm) and diacetyl (0.175 mg/m 3 or 0.0497 ppm) with an average relative humidity of 42% at 33 C (absolute humidity of 14.8 mg/L H 2 0). 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/m 3 (0.45 ppm) of acetoin, 1.56 mg/m 3 (0.44 ppm) of diacetyl, 2.59 mg/m 3 (0.44 ppm) of 2-nonanone and 1.88 mg/m 3 (0.44 ppm) of 2,3-pentanedione with an average relative humidity of 38% at 34 C (absolute humidity of 14.1 mg/L H 2 0). 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 2,3pentanedione. 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/m 3 or 0.53 ppm) and diacetyl (1.87 mg/m 3 or 0.53 ppm) with an average relative humidity of 40% at 35 C (absolute humidity of 15.6 mg/L H 2 0). The samples were collected at a sampling rate of 0.05 L/min for 3 hours. Nine of 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 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. The internal standard, 3-pentanone, was stable for up to 9 days in both the clear and ambient vials.

# Diacetyl migration within sampling tubes
In the majority of solid sorbent sampling tubes used by  (3.17 mg/m 3 or 0.90 ppm) with an average relative humidity of 42% at 33 C (absolute humidity of 14.8 mg/L H 2 0). 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.
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 Flow Temperature-Humidity Control System. A measured flow (typically 10 L 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 (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 Thermo hygrometer model RH411. 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.

# 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

# 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 2,3pentanedione 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 1013
3 for 2,3pentanedione. 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 LD 50 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.
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.

# 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.

# 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,  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.

# Reagents
None required

# 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. The extraction solvent used for this validation consisted of 0.007 L/ml 3-pentanone in 95% v/v ethyl alcohol/water. The 3-pentanone was added to the ethyl alcohol as an internal standard (ISTD).

# 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 g/l) by injecting 11 l 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 g/ml (0.2 to 40 g/2 ml). For example: a target concentration standard of 20.4 g/sample was prepared by injecting 20 l 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 g/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.

# 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. (Key: 1) ethyl alcohol; 2) 2,3pentanedione; and 3) 3-pentanone.) 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).

# 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.
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-L 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.   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 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 g per sample (9.3 ppb or 38 g/m 3 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 g/m 3 ) when 3 Lis sampled.

# 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 g.  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/m 3) 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%.

# 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.
Two dried silica gel tubes in series  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.

# 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.

# 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 2,3pentanedione in the test atmosphere was 0.501 ppm (2.05 mg/m 3 ) 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.  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/m 3 ) 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).
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# 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.

# 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.
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. 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/m
3 ) of 2,3pentanedione 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/m 3 ) of 2,3pentanedione 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/m 3 ) 2,3-pentanedione at 80% relative humidity and 23 C and two interferences whose concentrations were 0.51 ppm (1.82 mg/m 3 ) acetoin, and 0.51 ppm (1.78 mg/m 3 ) 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. 2,3pentanedione at an average humidity of 80% at 23C 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 foil wrapped samplers had the foil removed and were exposed to 3 h of sunlight before analysis 15of17 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.  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. 1. Sampler: Thermal sampling tube, %" s.s. tube, multi-bed sorbents capable of trapping organic compounds in the C 3 -C 16 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-L, 10-L (liquid); 100-L, 500-L (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. 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 . 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 L; liquid standards, 0.1 to 2 L) 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.

# INTRODUCTION
D iacetyl (2,, a dike tone 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-3 l Laboratory animal studies have documented that diacetyl alone has toxic properties that are similar to the effects of exposure to diacetyl-containing artifi cial butter flavoring mixtures.C 4 - 5 l 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.C 6 -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   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 De cember 2009 during four 1-week periods and one 2-week period of tests. We collected a total of 964 CMS tube sam ples during 80 tests, with relative humidity (RH) levels rang ing from 16 to 92% and temperatures of 22.4 to 33.8C giving absolute humidity (AH) levels ranging from 3.5 to 22.5 mg H 2 0/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. Sam ples were collected using sampling flow rates of 50 or 150 cc/min to investigate any effect on diacetyl recovery associ ated with differences in sampling flow rate. The test atmo sphere 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 H 2 0/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 in vestigated 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 H 2 0/L air (RH= 17, 36, and 78%, respectively, at 25.7C). 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 post sampling, the remaining liquid portion (without sorbent mate rial) 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 H 2 0/L air. The samples were extracted and analyzed 1,4,7,10,16, and 35 days post sampling. 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. Re analysis of these samples was completed on Days 2, 5, 13, and 34 post-extraction.

# 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 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 di acetyl 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 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 recov ery by plotting percent diacetyl recovered against either RH (Figure 2a) in% or AH (Figure 2b) in mg H 2 0/L air using data from samples collected from a target diacetyl concentration of 5 ppm at target temperatures of 25C and 32C. 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 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. 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 re covered 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 8 1 represents the horizontal asymptote on the right hand 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; 8 3 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
In our models, percent recovered diacetyl was the Y vari able, 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 non linear model for the data overall; this model took into ac count differences in the 4-parameter values for the individual logistic models. We found that 8 1 was well approximated (R 2 = 0.99) by a linear function of target concentration Co   We entered this form of the equation (Eq. 3) into the nonlin ear 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 (b 0 , m 1 , 8 2 , 8 3 , and 8 4 ) 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 R 2 (amount of total variability in the data accounted for by the model) for the overall model was 0.91. The R 2 for the 150 cc/min model was 0.93, and the R 2 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 3predicts 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   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 calcu lated 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.

# 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 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 , 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 three parameter first-order decay functions for the coefficients for the intercept, the AH term and the AH 2 term of the quadratic function based on the diacetyl concentrations. This gave esti mates 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 R 2 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  where 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(C 0 , AH)g(C 0 , AH, t) Co ( 5)
Full Model + h(Co)AH + f3(Co)AH 2 )(t -l)]
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 concentra tion, we solved Eq. 5 for C 0 . Using Eqs. 3 and 4, Eq. 5 can be rewritten as: AH, t) where Since this is a nonlinear equation for C 0 , it is necessary to use an iterative procedure to find C 0 . Initially, Eq. 6 is solved for Co using the quadratic formula with the dependence of g on Co ignored: b 2 + 4 a Ceca, ~H, t))
aC6 + bCo -( c ) = 0 (6) g(Co,
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+ 1 l b2 + 4a ( 0 c
) g(C 0 ' , AH, t) cg+1) = ------------ 2a -b+(8)
It is necessary to start the procedure with an initial Co (i.e., c6 1 \ 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 H 2 0/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 H 2 0/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 H 2 0 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 c6 1 l in an iterative procedure using the equation:
cCi+l) - c 0 - (Cl ) g C 0 ', 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 correc tion 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 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 H 2 0/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 extrapo lations 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 concentra tions of 1.0 ppm or less where the full model does not con verge; 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 hu mid air rather than our actively sampled tubes using a test atmosphere. As our results showed, early extraction mini mized 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 ap-preciable 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 concen tration 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 chem icals 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 meth ods indicated a high correlation, which increases our con fidence 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 stabil ity 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 previ ously studied by NIOSH and others will contribute to ongoing efforts to understand the health risk associated with occu pational 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 al lows measurements from historical diacetyl samples collected and analyzed using NIOSH Method 2557, which 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.

# 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 NIOSH 2006]. Details of the JEM construction are described below.

# 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 . 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 personal equivalent diacetyl exposures.

# 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 . 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 . 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= . 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 ofreplacing LOD measurements with a value ofLOD/2 was used . 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.

# 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.

# 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 . We assumed, based on survey observations and questionnaire responses, that mixers spent, on average, about 4 hours per shift 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. 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. 3Outside Processing I Office Use the mean of the area sample diacetyl concentrations from survey 1.
Polyethylene Line Use the mean of the area sample diacetyl concentrations from survey 1.
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.
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.
Microwave ovens moved into popping room in quality control lab 14 -18, 2003)    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. 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.
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.
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. 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.
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 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 (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. 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.
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 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,. 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 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 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/m 3 ) for diacetyl and 9.3 ppb (38 g/m 3 ) 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 .
- 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 .
- 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 .

# 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 . 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 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.