The New TLV for Endotoxins
What It Means for Exposure Assessment
BY JOHN P. SPRINGSTON AND CHERYL L. MARCHAM
Background: Aspergillus fumigatus.
Overlays: The types of endotoxins in one setting, such as an industrial compost plant, may be very different than those found in another setting, such as a pig farm.
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In June, the ACGIH Board of Directors approved and adopted a Threshold Limit Value (TLV) for airborne endotoxins of 90 endotoxin units per cubic meter of air (EU/m3). Now that there is an occupational exposure limit (OEL) for industrial hygienists to compare sample results against, it is inevitable that some individuals will begin collecting exposure samples without knowing what the results actually mean.
This article is intended to help practitioners better understand what endotoxins are, the wide variations in health effects that different endotoxins may elicit, and the significant limitations in measuring and assessing endotoxin exposures in varying environments. The discussion explains why the authors do not believe that the science currently supports a single TLV or other OEL for endotoxins. The opinions expressed in this article are ours alone and do not necessarily represent those of ACGIH, the ACGIH Bioaerosols Committee, AIHA, or The Synergist.
ENDOTOXINS AND OTHER BIOAEROSOLS
According to ACGIH, bioaerosols are a broad category of airborne particles and compounds that originate from living organisms. Bioaerosols encompass a wide range of biological particulates, including whole (and fragments of) fungi, bacteria, spores, pollen, viruses, and components of cell walls, such as bacterial endotoxins and fungal glucans. Additionally, bioaerosols can consist of primary and secondary metabolites and their byproducts; fragments and feces from insects, such as dust mites and cockroaches; and proteins found in the urine and saliva of certain pets and other furred animals, such as cats and rodents. Regarding potential health effects from exposure to bioaerosols, the risks depend on the specific microorganisms or agents involved, their viability and ability to become airborne and transported to susceptible individuals, and any concomitant exposures.
Exposure to bioaerosols can lead to a wide variety of adverse health effects, including infectious diseases, such as COVID-19, influenza, and tuberculosis, and noncommunicable conditions, such as allergies, asthma, and other chronic respiratory diseases. In certain cases, prolonged exposure to extreme levels of certain bioaerosols may even result in serious occupational diseases, such as hypersensitivity pneumonitis.
Despite decades of research on indoor mold and other bioaerosols, the development of OELs for most bioaerosols has been hampered by the wide diversity of biological agents and their varying effects on individuals; the often concurrent exposures to multiple bioaerosols and other, related or unrelated, chemical compounds; and the lack of epidemiological or toxicological data that establish clear dose-response relationships, even for carefully measured exposures to biological agents. In many cases, the lack of exposure data is due to the inadequacies, and sometimes extreme variabilities, of currently available sampling and analytical methods. Further complications include the challenges related to repeatable measurement of airborne contaminant concentrations (both collection and analytical methods) and inherent spatial and temporal variabilities in background concentrations.Figure 1. Cell wall structure of gram-negative bacteria. Source: BMG Labtech. Used with permission. Click or tap on the figure to view a larger version in your browser.
A BRIEF HISTORY OF ENDOTOXIN OELS
In 1998, the Dutch Expert Committee on Occupational Safety (DECOS) proposed an eight-hour health-based OEL for airborne endotoxins of 50 EU/m3 relative to an international reference standard endotoxin (RSE). This proposal was primarily based on a 1984 study published in the Annals of Internal Medicine that showed a no-observed effect level (NOEL) of 90 EU/m3 for selected sensitive healthy subjects in a mock six-hour work exposure to endotoxin-contaminated cotton dust. DECOS applied a safety factor of two to compensate for increased risks for certain groups of workers and to account for the possibility that endotoxins may have chronic pulmonary effects at levels lower than those that result in acute respiratory effects. The OEL was never adopted due to significant short-term economic and technical concerns.
In June 2000, the Social and Economic Council (SEC) of the Netherlands proposed a temporary legal limit of 200 EU/m3 over an eight-hour work shift. The proposal included a six-month transitional period to enable relevant industries to comply with the new limit. SEC also advised that, if feasible, the legal limit be lowered to 50 EU/m³ within two years. The introduction of the new limit was postponed, and, in July 2003, the proposal was withdrawn.
In 2010, DECOS suggested a health-based OEL of 90 EU/m3 as an eight-hour time-weighted average (TWA). This OEL was based on the 1984 study, a cross-sectional study of the chronic effects on the lung function of animal feed mill workers, and a five-year follow-up study of those workers. DECOS noted that, since the volunteers used in the original study had been selected because of their sensitivities to endotoxins, no further extrapolation or safety factor was necessary. The proposed OEL has never been adopted by the Netherlands.
The ACGIH TLV of 90 EU/m3 is based largely on the same studies that DECOS cited in its 2010 recommendation. The documentation for the TLV notes that “small differences in measurement approach can over- or under-estimate the true airborne concentration of endotoxin” and recommends using a specific consensus-based method—BS EN 14031:2021, Environmental Management - Environmental Performance Evaluation - Guidelines—to measure endotoxin concentrations.
ENDOTOXINS ARE NOT A SINGLE AGENT
Although researchers, OEHS professionals, and other scientists often use the singular term “endotoxin,” the substances classified as endotoxins vary greatly. Endotoxins are lipopolysaccharides (LPS) or, to a lesser extent, lipooligosaccharides (LOS) naturally contained within the outer membrane of the cell walls of gram-negative bacteria (GNB), as depicted in Figure 1. LPS and LOS serve a structural function in GNB and are the first line of defense against chemical and environmental threats, but GNB from different genera can produce different LPS, and even an individual bacterium can produce dissimilar LPS under different environmental conditions. Therefore, “endotoxin” represents a wide range of diverse chemical structures with molecular weights ranging from around 5,000 to 20,000 daltons that can elicit a variety of reactions and potential health effects in exposed individuals.
Much like mold spores, airborne endotoxins are ubiquitous in the environment. However, the specific types of endotoxins present in one setting, such as in a metalworking fluid environment, are likely to be quite different from those present in, say, a pig farm, which are likely very different than those found in composting facilities or, more importantly, homes or offices. Endotoxin chemistry in residences is much more limited than that found in most industrial or agricultural settings but is still highly variable depending on the presence of potential sources, such as pets, humidifiers, and indoor firewood storage, and the building’s location in an urban or rural environment.
Additionally, because of the extensive variety of other potentially harmful microorganisms or chemical compounds of biological origin that may be present, the risk from exposure will likely differ in different environments. Unlike the RSE typically used for calibration of laboratory analytical methods, endotoxins in environmental samples, such as air or settled dust, do not consist of a single substance. Industrial hygienists need to be aware of this fundamental source of measurement error and its potential ramifications for exposure and risk assessments.
HEALTH EFFECTS AND CONFOUNDING FACTORS
Endotoxins are potent stimuli of inflammatory and immune system responses. Numerous studies have reported on the adverse respiratory health effects of occupational exposure to endotoxins. Inhaled endotoxins prompt an inflammatory response in the lung, characterized by an influx of neutrophils and increased levels of cytokines. Systemic effects are most likely induced by the release of those cytokines into the blood. However, the specific sources of endotoxins and the nature of any concomitant exposures influence an individual’s response and any associated health effects. Because of the substantial structural variation in endotoxins from different GNB, the inflammatory potency to lung cells can vary by up to two orders of magnitude.
As previously noted, in addition to airborne endotoxins, individuals are simultaneously exposed to varying levels of other bioaerosols such as fungi, and the two levels are generally correlated. A 2013 paper published in Atmospheric Environment verified that higher levels of allergenic endotoxins occurred when microbial loading was higher. Co-exposure to dog and cat allergens has been found to increase the association of endotoxin exposure to both wheezing and asthma. In addition, individual susceptibility in humans appears to be genetically driven. Thus, the measurement of endotoxins in an exposure assessment may simply represent a proxy or substitute for other elements of the environmental microbiome, which contains a complex array of immune-active compounds and stimuli.
Endotoxins are believed to be an important contributor to occupational respiratory disease, particularly in agricultural settings and composting facilities. However, agriculture-based exposures are highly complex, and other agents in organic dust may also contribute to observed acute and chronic health effects. To add to the complexity, agricultural endotoxin exposures, which can routinely be well above 90 EU/m3, appear to have a protective effect against lung cancer in farmers and cotton textile mill workers. When reviewing studies that suggest endotoxin exposure as a potential cause of adverse respiratory effects, industrial hygienists need to consider both the kinds of endotoxins potentially present as well as other possible causative agents or confounding or additive effects.
Left: Endotoxins can be found in metalworking fluid environments. Right: 3-D illustration of mold.
The specific types of endotoxins present in one setting are likely very different from those found in others.
BARRIERS TO ANALYSIS AND ASSESSMENT
Unlike the quantitative concentration units familiar to industrial hygienists, such as parts per million (ppm) and milligrams per cubic meter (mg/m3), endotoxin units are relative. EUs compare the activity of an environmental sample in the Limulus amebocyte lysate (LAL) assay against an RSE extract derived from the bacterium Escherichia coli O113:H10. The assay is based on the reaction between endotoxins and the lysate of the blood cells of horseshoe crabs (Limulus polyphemus). When endotoxins contact the LAL reagent, they trigger a series of biochemical reactions that release a clotting protein called coagulase. The amount of coagulase produced is proportional to the amount of endotoxins present in the sample. This test was originally developed to detect the presence of pyrogens, or fever-inducing endotoxins, in injectable drugs and certain clinical applications. The U.S. Food and Drug Administration (FDA) and the U.S. Pharmacopeia (USP) have validated methods for testing injectable medicines and vaccines and other medical applications, but these methods have not been validated for the detection of endotoxins in environmental samples.
While highly sensitive, the LAL assay is nonspecific. It reports the presence of any substance that triggers the same coagulase release as endotoxins do. As a result, the assay may detect other types of microbial toxins or compounds, such as β-D-glucans, that can activate the same clotting pathway. Because it cannot distinguish between endotoxins and other substances that produce similar reactions, the LAL assay can lead to false positives.
These variations in results, attributable to the cross-reactivity of other environmental substances as well as inter-laboratory variability, are sources of measurement error. In addition, the results of exposure assessments are influenced by the method of sample collection and measurement, sample storage conditions, the extraction method employed, the assay reagents used, and the analytical method itself. Even slight differences in methodology make it difficult to compare results from different laboratories.
Because endotoxins are ubiquitous in the environment, including in laboratories themselves, the LAL assay requires extensive quality assurance and quality control (QA/QC) measures. To obtain the most reliable data, the extraction and preparation for the assay needs to be performed within a biological safety cabinet to avoid contamination from the ambient air. Even when sampling from environments with extremely high endotoxin levels, such as wastewater treatment facilities, serial dilution of the samples can introduce significant error, so careful processing of the samples is still required. Additionally, all materials used in the sampling and analytical procedures, including pipettes, weighing boats, centrifuge tubes, and even the gloves worn by laboratory personnel, must be rendered free of endotoxins.
ADDITIONAL CHALLENGES
The greatest challenge associated with applying an OEL for endotoxins is illustrated by findings from the National Health and Nutrition Examination Survey (NHANES) endotoxins study. Responsiveness to endotoxins was found to be mediated by concurrent exposures to cat and dog allergens, climate region, ambient air pollution, and environmental tobacco smoke. Given the wide range of chemical structures and potential environments in which exposure can occur, the responsiveness to endotoxins and subsequent health effects vary with the specific types of endotoxins one is exposed to, the presence of other potentially additive, synergistic, or confounding co-exposures, and variations in individual susceptibility due to genetic differences.
Other significant challenges include the lack of a standard protocol for the sampling and analysis of endotoxins in environmental samples, inefficient extraction of endotoxins from dust samples, inadequate assay QA/QC, the cross-reactivity of other environmental substances that may also be present in the samples, and inter-laboratory variability that can be an order of magnitude or more.
It is our opinion that, given these and other limitations, implementation of a single health-based OEL for mixtures of endotoxins that can vary so greatly in inflammatory potency and health impacts depending on the exposure source and types of bacteria present, remains challenging and is not well supported by currently available data. While setting an endotoxin OEL that is applicable to only a specific industry, such as for cotton textile mill workers, may be more feasible, it would still be subject to many of the same challenges and limitations discussed in this article.
JOHN P. SPRINGSTON, MS, CIH, CSP, FAIHA, is a technical consultant for Atlas.
CHERYL L. MARCHAM, PhD, CIH, CSP, CHMM, FAIHA, is an associate professor and program coordinator at Embry-Riddle Aeronautical University.
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