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Effects of inhaled combined Benzene, Toluene, Ethylbenzene, and Xylenes

(BTEX): Toward an environmental exposure model

Article in Environmental Toxicology and Pharmacology · January 2021

DOI: 10.1016/j.etap.2020.103518

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 1

Effects of Inhaled Combined

Benzene, Toluene, Ethylbenzene, and Xylenes (BTEX):
Toward an Environmental Exposure Model

Cameron J. Davidson a, John H. Hannigan a,b,c,d, Scott E. Bowen a,d *

a
Department of Psychology,
b
Department of Obstetrics & Gynecology,
c
Merrill Palmer Skillman Institute for Child & Family Development,
d
Center for Urban Responses to Environmental Stressors
Wayne State University, Detroit, MI, USA

*Address correspondence to Dr. Scott E. Bowen

Department of Psychology
Wayne State University
5057 Woodward Ave
Detroit, MI, USA 48202
Email address: scott.bowen@wayne.edu

Running Title: Demands of an Animal Model of BTEX Exposure

Key Words: environmental toxicology, volatile organic solvents, animal models

This work was supported in part by a grant from the National Institute for Environmental Health
Sciences (P30 ES020957), and the Department of Psychology, Wayne State University.
 2

Abstract
Combined environmental exposures to the volatile organic compounds (VOCs) Benzene,
Toluene, Ethylbenzene, and Xylene (BTEX) pose clear risks to public health. Research into these
risks is under-studied even as BTEX levels in the atmosphere are predicted to rise. This review
focuses on the available literature using single- and combined-BTEX component inhaled solvent
exposures in animal models, necessarily also drawing on findings from models of inhalant abuse and
occupational exposures. Health effects of these exposures are discussed for multiple organ systems,
but with particular attention on neurobehavioral outcomes such as locomotor activity, impulsivity,
learning, and psychopharmacological responses. It is clear that animal models have significant
differences in the concentrations, durations and patterns of exposure. Experimental evidence of the
deleterious health and neurobehavioral consequences of exposures to the individual components of
BTEX were found, but these effects were typically assessed using concentrations and exposure
patterns not characteristic of environmental exposure. Future studies with animal models designed
appropriately to explore combined BTEX will be necessary and advantageous to discovering health
outcomes and more subtle neurobehavioral impacts of long-term environmental exposures.
 3

1. Introduction

Volatile Organic Compounds (VOCs) are ubiquitous and practically indispensable in

numerous household, commercial, and industrial products with substantial economic importance
(Bergman, 1979; Joshi and Adhikari, 2019). VOCs are also common byproducts of fossil fuel
combustion (NCEH, 2018). The alkylbenzenes Benzene, Toluene, Ethylbenzene, and Xylene(s)
(BTEX; Table 1) in particular are four solvent VOCs used heavily in industry and in the home as
chemical intermediates, solvents, fuel additives, and cleaners. BTEX are typically present together
at low levels in the air in urban areas, although local concentrations vary considerably depending on
geography, season, weather, and proximity to industry, refining and traffic (Huang et al., 2017;
McKenzie et al., 2012; Miller et al., 2012; Miri et al., 2016). Given their utility, prevalence and
economic importance, BTEX will likely remain persistent environmental contaminants in air for the
foreseeable future (Bolden et al., 2015).

Studies of environmental exposure capitalize on demographics and/or epidemiology, as well

as geospatial modeling, to assess the impact of air pollution and/or specific pollutants. These studies
are important for informing our understanding of the general impact of pollutants, but they are
limited in determining the specific impact of individual or mixed BTEX separate from other
environmental and population factors (e.g., water pollution, demographics, socioeconomic status,
nutrition, etc.; Bolden et al., 2015; Dehghani et al., 2018). Animal models and experimental designs
that control or eliminate confounding factors and that systematically manipulate exposures to mimic
environmental patterns would aid in understanding the neurobehavioral impact of combined BTEX.
Current models mimicking inhalation exposures typical of solvent abuse or occupational settings
have demonstrated negative health outcomes following exposure to the individual BTEX
components and their binary mixtures, but the whole mixture is under-studied in the laboratory.

BTEX exposures in any community pose a clear risk to public health (Montero-Montoya et
al., 2018; Webb et al., 2018). While there is experimental evidence of the deleterious health and
neurobehavioral consequences of exposures to the individual components of BTEX, these effects are
typically assessed using higher concentrations and exposure patterns that differ from environmental
exposure. With much less known about the specific combined effects of BTEX, there is a clear need
to examine the effects (and risks) of acute and chronic combined BTEX exposures at typical ambient
environmental levels (Billionnet et al., 2012). Here we review relevant research on environmental
 4

BTEX exposures in animal models, drawing on findings from models of inhalant abuse and
occupational exposures, and focusing on neurobehavioral outcomes. Environmental, occupational
and abuse exposures of volatilized BTEX components share the inhalation route of exposure, but
differ in the concentrations, durations, and patterns of exposure (Table 2). Even though inhalation of
combined BTEX components by people is common with environmental or occupational exposures,
there are currently limited animal models of these exposures. The results of research using abuse
and occupational models of inhaled VOC exposure will be used to inform an animal model of
combined environmental exposure to BTEX.

2. Environmental Burden

VOCs, particularly BTEX, are significant components of air pollution which is the fifth
leading risk factor for premature mortality in the world (HEI, 2019). Ninety percent of people
worldwide live in areas that exceed the World Health Organization (WHO) standards for air quality,
and half of those individuals live in areas not meeting the least stringent standards (HEI, 2019).
Environmental BTEX contribute significantly to poor air quality and pose a public health hazard
(Bolden et al., 2015; Dehghani et al., 2018; Huang et al., 2017; Miri et al., 2016). BTEX
components typically occur together in environmental and occupational settings in low to trace
amounts with relative concentrations, from greatest to least, of toluene > xylenes > ethylbenzene >
benzene (Badjagbo et al., 2010; Hamid et al., 2019) although benzene is more abundant than
ethylbenzene in some surveys (Carlsen et al., 2018). Benzene, toluene and xylenes consistently rank
in the top 20 chemicals produced annually in the United States (U.S.; NCEH, 2018). In 2018, U.S.
manufacturers produced approximately 750,000 tons of toluene, 203,000 tons of xylenes, 84,000
tons of benzene, and 46,000 tons of ethylbenzene (Table 1; EPA, 2018). While the majority of
BTEX in the U.S. are thought to be recycled, even the relatively small fractions of unrecovered
BTEX (B ~2.1%; T ~1.2%; E ~3% & X ~2.9%) result in substantial amounts being released into
local environments each year. Most unrecovered BTEX are released into air, ranging from 67% for
ethylbenzene to 90% for benzene (EPA, 2018). However, these numbers are for the U.S. only,
which has some of the more restrictive environmental protection laws. Environmental BTEX
releases are likely greater in other countries with weaker regulations and poorer enforcement (HEI,
2019).

3. Chemistry and Metabolism

 5

All BTEX components are structural variants of benzene with similar molecular weights and
varying in the number and position of attached methyl groups; ethylbenzene has one ethyl group (see
Table 1). BTEX, like all VOCs, have high vapor pressures and so distribute (dissipate) readily
through air at ambient temperatures. Several excellent reviews detail the absorption, distribution,
metabolism and clearance of each individual BTEX component (ATSDR, 2004, 2007a; 2007b, 2010,
2017). BTEX are absorbed rapidly through lungs, distributed throughout the body, and become
concentrated in highly vascularized organs (i.e., brain and liver) with regionally specific distribution
within the brain due to a greater affinity of more myelinated (lipid-rich) tissues (Aydin et al., 2002;
Bowen et al., 2007b; Ghantous and Danielsson, 1986; Rickert et al., 1979; Webb et al., 2018; Zhao
et al., 2019).

Metabolism of all BTEX components utilizes the cytochrome P-450 system, in particular
CYP2E1. The major urinary metabolites for benzene are phenol, muconic acid and S-
phenylmercapturic acid (Snyder and Hedli, 1996); for ethylbenzene, mandelic acid, phenylglyoxylic
acid and benzoic acid (Engstrom, 1984); for xylenes, methylhippuric acid (Langman, 1994), and for
toluene, hippuric acid (Duydu et al., 1999). Benzyl mercapturic acid, o-,m-,p-toluylmercapturic
acids and o-,m-,p-cresol are also minor metabolites of toluene that may be more sensitive markers of
toluene exposure than hippuric acid (Cosnier et al., 2013). The clearances of inhaled BTEX are
primarily through exhalation (Marchand et al., 2015). Otherwise, each BTEX component follows
first-order elimination kinetics, having both rapid and slow half-life phases. Typically, there is rapid
elimination from muscle and slower elimination from adipose tissue. As seen in Table 1, “rapid”
phase clearance rates of BTEX components vary but all have half-lives <1 hr while “slow” phase
rates range from ~12-13 hrs for benzene and toluene and up to ~20 hrs for ethylbenzene (Janasik et
al., 2008; Rickert et al., 1979).

The varying metabolism and clearance rates among BTEX components suggest that
simultaneous BTEX co-exposures could alter peak circulating levels of any one or all components.
Similar metabolic mechanisms for BTEX also implies that co-exposures could interfere with
metabolism of any one component, increasing half-lives, leading to higher and/or more persistent
tissue burdens, and elevated toxicity for each component. Pohl, et al. (2003) reviewed binary
interactions between BTEX components and concluded that concentration-dependent competitive
inhibition suggests additive neurotoxic actions at environmental levels (20 ppm). Evidence of
metabolic inhibition has been found in rats exposed to both toluene and xylene (Tardif et al., 1992).
 6

Co-exposure to BTEX would also be expected to increase the half-life of each component and result
in higher and more persistent levels of combined BTEX. Cosnier et al. (2014) also reported that in
rats exposed to toluene (50 ppm, 6 hrs/day, 5 days), co-exposure to xylenes (50 or 400 ppm)
increased circulating levels of toluene 2- to 3-fold after 5 days. However, Tardif and colleagues
reported that physiologically based pharmacokinetic (PBPK) modeling simulations suggested that
low environmental levels of BTEX might not result in biologically significant alterations of
metabolism (Haddad et al., 2001; Tardif et al., 1997; Tardif et al., 1995).

Higher circulating levels of BTEX for longer periods would theoretically increase the
toxicity of BTEX relative to each component. Interactions among components would also suggest
that exposure levels determined to be “safe” for one component may not apply when other
components are present, or when “acceptable” ambient exposure is maintained for more extended
periods (e.g., in the workplace or with environmental exposure). Few studies to date have examined
paired VOC components (e.g., TX, TE, EX, etc.) or more rarely, triple component exposures (e.g.,
TEX). In the absence of studies specifically testing the metabolism and clearance of combined
BTEX, judgments of risks based on metabolic interactions posed by environmental exposure will be
difficult and/or speculative.

4. Health Effects

This section describes health effects in general, based on clinical and epidemiological studies
and includes different types of VOC exposure. Section 6, below, on neurobehavioral effects focuses
on different models of VOC exposure, mostly in animals.

Lung

As inhalation is the main route of administration for BTEX, the lungs are the primary target
for injury. Epidemiological studies indicate that BTEX inhalation from occupational or
environmental sources can damage lung tissue (Cakmak et al., 2014). A recent investigation of
populations living near a coal chemical plant, a locale known to have higher levels of BTEX, found
increased incidences of chest inflammation in adults and decreased lung functioning in children
compared to a matched cohort living in a city farther from the plant (Chen et al., 2018). While acute
respiratory tract irritation in both humans and animals is a primary outcome for each BTEX
component at relatively low concentrations (≥ 16-33 parts per million [ppm]), Mogel et al. (2011)
 7

have reported that these irritations may be mediated more by benzene- and toluene-induced elevation
of prostaglandins in lung epithelial cells. Other researchers have shown positive correlations
between rates of acute asthmatic events and BTEX concentrations in urban outdoor air using 2-week
sampling periods across four seasons in different postal-codes spanning an international border
(Lemke et al., 2014). This link with increased exposure to BTEX was supported further by an
increased adjusted odds ratio (OR: 1.63) for physician-diagnosed asthma and, when undiagnosed,
increased odds for one or two wheezing attacks (OR: 1.68; Arif and Shah, 2007). Histological
analyses show lung damage after various exposures to toluene (1000 ppm, 5h/day, 5days/week for 4
weeks; Jacquot et al., 2006; 3000 ppm, 8h/day, 6days/week, 12 weeks; Kanter, 2011; 600 or 1,200
ppm, 6.5h/day, 5 days/week for 15 months; NTP, 1990; 600 or 800 ppm for 8 hrs one day; von
Oettingen et al., 1942), but not for ethylbenzene (2,000 ppm, 3 days -Toftgard and Nilsen, 1982)) or
benzene (≤ 100 ppm, 6 hrs/day, 5 days/week, for life -Snyder et al., 1978; Snyder et al., 1984). For
xylenes, lower concentrations (≤ 810 ppm, 13 weeks - Carpenter et al., 1975) showed no significant
effects while 2,000 ppm for 3 days decreased cytochrome P-450 levels (Toftgard and Nilsen, 1982).
These examples illustrate the wide variability in exposure parameters in the experimental literature.
While we are aware of no similar studies after combined BTEX, a recent analysis of individual
BTEX components identified potential health risks related to altered hydrophobic gas-liquid
interface properties in extracted porcine lungs (Zhao et al., 2019).

Kidney and Liver Dysfunction

Several reviews thoroughly detail adverse hepatic effects of BTEX components in both
animals and humans (see reviews ATSDR, 2007a; 2007b, 2010, 2017). For example, gas station
attendants exposed to vapors containing benzene, toluene, and xylenes were shown to have subtle,
subclinical or pre-pathologic changes in liver and kidney function (Neghab et al., 2015). The use of
strict inclusion/exclusion criteria as well as sound experimental precautions strengthened the
conclusions of this cross-sectional, human investigation. In addition, because the reported exposures
were below OSHA limits, these results may be relevant to environmental exposures. Animal studies
have also demonstrated kidney and/or liver damage after exposure to benzene (10-50 ppm for 5
hrs/day, for 2 weeks; El-Shakour et al., 2015), toluene (2000 ppm of various durations; Pyykko,
1983), ethylbenzene (100-1500 ppm for 6hrs/day, for 13 weeks; Zhang et al., 2010), and xylenes
(2000 ppm for 6hrs/day for 3 days; Toftgard and Nilsen, 1982). Because the kidney and liver are
 8

also susceptible to the toxic effects of BTEX tested at various concentrations for various durations,
the lack of an animal model of combined BTEX exposure is a significant gap in BTEX research.

Carcinogenicity

Benzene is a known Group 1 human carcinogen per a World Health Organization working
group, based on ample evidence from humans and animals, as well as supporting mechanistic studies
of genotoxicity (IARC, 2018; Loomis et al., 2017). Benzene produces acute non-lymphocytic
leukemia and myeloid leukemia and is also associated with increased risk for chronic lymphocytic
leukemia, non-Hodgkin’s lymphoma, and multiple myeloma in humans (IARC, 2018). There is also
evidence linking ambient (environmental) benzene and BTX (no “E”) exposure to human cancer
rates (Hamid et al., 2019; Warden et al., 2018). In mice also, co-exposed to intermittent benzene (50
ppm) and toluene (50 or 100 ppm) over a 13-day period showed significantly more erythrocyte
micronuclei, a marker of carcinogenic risk, than after benzene alone (Bird et al., 2010). While
ethylbenzene is not yet considered a human carcinogen, there is evidence that ethylbenzene-
associated reactive oxidative metabolites are linked to carcinogenesis (Midorikawa et al., 2004).
There is insufficient information on toluene and xylenes to determine carcinogenicity in humans
(Niaz et al., 2015).

Acute Toxicity

Acute dose-dependent toxicity from vapor exposure to each individual BTEX component has
been reported (Goldstein, 1988; Niaz et al., 2015; Tegeris and Balster, 1994; Win-Shwe and
Fujimaki, 2010). For example, a report of three workers fatally exposed to benzene notes that the
exposure was only a matter of minutes and yet caused second-degree chemical burns on the skin,
evidence that the concentration was very high (Avis and Hutton, 1993). While toluene abuse or
occupational exposure can result in lethality (e.g., Bowen et al., 1999; Garriott, 1992), these studies
typically have not reported concentrations or durations of toluene exposure. One study used the
blood toluene levels of a deceased painter (30.2 mg/L upon hospital admission) to estimate that the
fatal duration and concentration range was ~1 hr of exposure to 1800-2000 ppm (Hobara et al.,
2000). Additional indices of acute toxic exposures to BTEX have been reported, as those following
the British Petroleum (BP) oil spill in the Gulf States region in 2010 (Werder et al., 2019).
Individuals with higher average circulating BTEX levels (B: 0.09, T: 0.28, E: 0.06, X: 0.14 ng/ml)
reported increased incidences of dizziness, nausea, headaches, sweating, numbness, blurred vision
 9

and loss of coordination, as compared to geographically matched persons with lower circulating
BTEX blood levels (B: 0.03, T: 0.14, E: 0.04, X: 0.12 ng/ml; Werder et al., 2019). Werder et al.
(2019) attributed these effects predominantly to benzene with smaller associations for toluene.

5. Models of BTEX Exposure

Abuse Models

Most basic research studies of the neurobehavioral effects of BTEX have focused on the
individual components. Nearly all of those studies dealt with high levels of toluene, modeling abuse
or “glue-sniffing”/“huffing” for its intoxicating effects (Balster et al., 2009; Bowen et al., 2006;
Bowen and Cruz, 2014; Cruz, 2014; Cruz and Balster, 2013). Inhalant abuse is modeled by episodic
exposures to very high concentrations (e.g., toluene 1000 ppm to 30,000 ppm) for relatively brief
durations (10 to 30 mins), often repeated over several days, sometimes intermittently or irregularly
(see Table 2 and reviews, Bowen et al., 2006; Bowen et al., 2016; Howard et al., 2016; Howard et
al., 2017). VOC abuse has been modeled effectively in rodents with one or more daily exposures,
multiple exposures per week (with varying inter-session intervals), and up to several weeks’
exposure (e.g., Batis et al., 2010; Bowen et al., 2009a; Bowen and McDonald, 2009; O'Leary-Moore
et al., 2009). Animal models of VOC abuse allow experimental manipulation and/or control of
potential confounding factors that cannot be controlled or eliminated easily, if at all, in human case,
clinical or epidemiological studies. As far as we are aware, animal models of abuse have not
examined mixtures of BTEX.

Occupational Models

Exposure to VOCs in the workplace is regulated in the U.S. by the Occupational Safety and
Health Administration (OSHA), which sets limits defining “safe” concentrations to which workers
may be exposed legally over an 8-hour workday. The criteria for well-regulated, compliant
concentration limits over 8 hours define a maximum Time Weighted Average (TWA). OSHA also
sets maximum Short-Term Exposure Limits (STEL) that define the maximum concentrations to
which workers can be exposed safely for 10-15 min (Table 1). OSHA safety standards can be useful
in establishing exposure characteristics for valid animal models of occupational exposures to BTEX.
However, TWAs and STELs define exposures to individual solvents only, even though in
occupational settings exposure is often to mixtures, like BTEX. Animal models of occupational
 10

exposure also typically focus on exposure to a single VOC alone, although at relatively moderate
concentrations and longer exposures compared to abuse models (e.g., typically ~50 ppm to 800 ppm,
lasting up to 16 hrs/day). Valid occupational models often repeat episodes of exposure, typically for
more days than seen with abuse models. Occupational models explore both the acute and long-term
impacts of exposures gathered from both basic/preclinical (animal) and clinical (human) studies.
Animal models exist for occupational patterns of exposure to individual VOCs (Bird et al., 2010;
Chan et al., 1998; El-Shakour et al., 2015; Honma et al., 1983), although we are unaware of any
study examining mixtures of two or more individual BTEX components in such models.

Defining a Valid Environmental Exposure Model

Environmental exposures in human populations tend to be relatively constant at low

concentrations (e.g., toluene <50 ppm) for a given locale. Like occupational exposures,
environmental exposures are more “passive”, in that inhalation is incidental to location or activity, in
contrast to being actively, intentionally and deeply inhaled as in abuse. In contrast to abuse or
occupational exposures, environmental exposures in communities persist for extended periods
ranging from weeks, to seasons, to years, even to being comparatively constant for people living
near sources of VOCs (Miri et al., 2016). Exposures will vary as individuals move about each day
or week, as industrial activities and traffic fluctuate, and as seasons change. Mimicking
environmental patterns of exposure in a laboratory is necessarily limited in practice. It is not
feasible for a model to reflect such wide temporal variations in exposures. On the other hand, a
model need not use constant, long-term exposures to be valid. An environmental model limited to
combined BTEX and that does not include other common air-borne pollutants (e.g., other VOCs,
fine particulates, lead [Pb], NOx, SO2, etc.), may be considered “limited” as well, but would still be
quite valid. There are some studies of longer-term, low-level exposures to individual VOCs,
modeling either the workplace or environment (Ahmed et al., 2007; Gralewicz and Wiaderna, 2001;
Zhang et al., 2010), but we are aware of no studies of combined BTEX with such patterns of
exposure.

Cross-Model Considerations Regarding Inhalant “Dose”

Comparing across inhalation models is challenging because it is unclear how best to define
exposure in a precise way related to classical “dose.” In pharmacology, drug dose (exposure) is a
simple ratio of the amount of the drug administered divided by the organism’s body weight (e.g.,
 11

mg/kg), that is, defining what is put into an organism. In contrast, inhalation exposures are defined
by what the organism is put into, expressed as the concentration of the “drug” in the air breathed and
how long the organism breathes in that “drug-laden” air. The volume of air varies substantially
across exposure types. For example, the size of the bag a person is huffing in abuse, or the
workplace area in an occupational setting, or the local atmosphere in a community are not known
and would likely be unreliably measured, even if it were possible. In experimental settings, volume
can be defined precisely. An animal model could also account for ventilation rate (breaths per min)
and depth of inhalation (e.g., tidal volumes). While we are unaware of attempts to do so, ventilation
rate – which is a function of animal size – contributes to age differences in circulating levels of
inhaled VOCs (cf., Rodriguez et al., 2007).

It can be useful to construct a metric that allows comparisons across different exposure
models by considering concentration (e.g., ppm) and the duration of exposure (e.g., mins or hrs per
bout). Any temporal dimension should also account for the number of exposure bouts (e.g., times
per day and/or number of days). A crude “exposure metric” may be calculated by multiplying
concentration (ppm) by the total exposure time (e.g., mins X bouts per day X number of days) to
generate a standardized value with units like “ppm-mins.” Such a metric could be a valid basis for
comparisons of relative total “dose” across models. In this case, the exposure dynamics of each
model would explain differences in outcomes among models, differences to which. a calibrated
“dose” metric would be insensitive. These differences among models would then be defined
objectively by the concentration and the pattern/timing of exposure sessions, as duration and number
of bouts, and the intervals between daily bouts and between sessions, the characteristics of
environmental exposure. These factors influence inhalant exposure “momentum” that differs among
models and that might be reflected in peak circulating levels of a toxicant/pollutant.

In an initial exploration of just this sort of analysis, Batis et al. (2010) examined differences in
behavior across groups of rats that were matched in total time exposed to toluene but differed in the
exposure momentum. While the focus was toluene abuse, the pattern, or pace, of exposure varied
with the numbers of sessions, bout durations, and inter-bout intervals. Age-dependent, biphasic,
abuse-level, concentration-dependent shifts in the time course of locomotor activation were reported,
both during and after exposure, across three different patterns of exposure. This is consistent with
the notion that the pace of exposure to VOCs like BTEX components, as with persistent lower
 12

concentrations in environmental exposures, could produce complexly different behavioral responses

than seen with abuse or occupational exposures.

Exposure via inhalation is typically not standardized to body weight, which would contribute
to error variance and limit the ability to detect true effects. Since body size is correlated with
ventilation rate, tidal volumes and body composition (esp., fat), controlling for body weight would
be necessary to calculate inhalant exposure properly as “dose” (Rodriguez et al., 2007). Similarly, it
is also important to consider sex and age (or developmental stage) as factors in calculating exposure.
For example, keeping exposure parameters (i.e., concentration, duration) constant in preclinical
models would impose different exposure burdens on males versus females of the same age. One
way to mitigate this potential confound is to use sex as a factor and body weight as a covariate
during analyses (Ernstgard et al., 2003). These factors and measures could also serve as proxies for
physiological differences noted above (e.g., respiration rate, tidal volume). The complexity of
inhalant “dose”, subsequent complications of combined exposure calculations, as well as sex and
developmental differences are considered below under Potential Confounds.

6. Neurobehavioral Effects

Studies of VOCs like the components of BTEX have assessed wide-ranging behaviors
including activity, motor function, indices of impulsivity and anxiety, learning and memory, and
responses to reward. Prior studies largely dealt with single components, usually toluene, and almost
exclusively using abuse models of exposure. The relatively few papers reporting these outcomes,
and others, in animals using designs that approximate environmental exposures are summarized in
Table 3; none use combined BTEX.

Coordination and Locomotor Activity

Abuse patterns/levels of exposure to toluene produce bi-phasic, dose-dependent changes in

locomotor activity. Concentrations of toluene <6000 ppm increase activity while higher
concentrations inhibit activity and can produce sedation (Apawu et al., 2015; Batis et al., 2010;
Bowen and Balster, 1998; Bowen et al., 2010). Disruptions in motor function during and following
abuse patterns of toluene exposure include decreases in rearing, poor righting reflex, poor forelimb
grip strength, poor motor coordination, and abnormalities in gait and landing foot splay (Tegeris and
 13

Balster, 1994). The acute administration of each BTEX component individually disrupted
coordination in rodents within 5 min after exposure (Tegeris and Balster, 1994).

Some studies have demonstrated differences in the bi-phasic locomotor-activating effects

among the individual BTEX components (Armenta-Resendiz et al., 2019; Tegeris and Balster, 1994)
and among the isomers of xylene (Molnár et al., 1986). For example, Molnár et al. (1986; Fig. 1)
reported that the locomotor activity of rats peaked at ~1000 ppm for ethylbenzene and toluene
following a 3- or 4-hr exposure. The increase in activity for ethylbenzene was quite modest and a
high degree of motor incoordination was reported for toluene. In contrast, peaks in activity after p-
xylene (~1500 ppm) and benzene (~4000 ppm) were larger and accompanied by tremor. Also, for
benzene, the increases at the peak concentration were significant even after only a 1-hr exposure.
The m- and o-xylenes caused only minimal increases in locomotor activity but still showed bi-phasic
responses with reduced activity at the highest concentration tested (~≥2000 ppm). Individual BTEX
components also differed in the average concentration at which the rats slept (i.e., a “minimum
narcotic concentration”) with p-xylene the most potent (1940 ppm) and toluene and benzene the least
(3590 ppm & 5940 ppm, respectively; Molnár et al., 1986). These concentration- and exposure
duration-dependent differences in locomotor activation among individual VOCs could make it
difficult to predict how motor function would be affected by combined BTEX exposure. The results
from Molnár et al (1986) suggest a simple additive interaction may operate for increasing locomotor
activity, particularly at lower concentrations of each VOC.

There are also studies of locomotor activity and coordination after inhalation patterns of
toluene exposure that may be considered representative of occupational and/or environmental
exposures. Berenguer et al. (2003) used a rat model of “sub-chronic” toluene exposure (40 ppm for
8 to 12 hrs/day for 6 days/week for 16 weeks), levels designed to be below “occupational exposure
standards.” They reported no differences in spontaneous locomotor activity but decreased rearing in
the toluene-exposed group. They also reported an increased sensitivity to toluene-induced loss-of-
righting (i.e., “narcosis”) after the “sub-chronic” toluene exposure. Other investigations of chronic,
intermittent inhaled toluene (10,000 ppm, 1 hr/day, 3 days/week, 4 weeks) in adolescent rats did not
report altered motor learning in the rotarod task approximately one week following toluene exposure
(Dick et al., 2013).
 14

Effects of long exposures (6 h/day, 5 days/week for 4 weeks) to lower level (80 ppm) toluene
inhalation (6 h/day, 5 days/week for 4 weeks) produce a subtle but long-lasting impact on motor
activity (von Euler et al., 2000). This group reported only non-significant “trends” toward increased
locomotion and rearing in an open field 4 weeks after exposure; although the fact that 4 rats were
tested in the same apparatus at the same time may have been a confounding factor. Yet there was a
significant impairment in the ability to walk narrow planks with correct hind paw placement in the
rats with prior toluene exposure (von Euler et al., 2000). In earlier studies, this same pattern of
toluene exposure did not change spontaneous activity 3 or 17 days after exposure, or after 40-ppm
toluene (von Euler et al., 1991; von Euler et al., 1994; von Euler et al., 1993).

While there is evidence that individual components of BTEX affect some behaviors (e.g.,
locomotion, narcosis, tremor, coordination), it is unknown how these behaviors might be impacted
by combined exposure. We hypothesize that exposure to BTEX mixtures may cause a total
concentration-response shift of the peak “to the left” for locomotor activity and loss of coordination
compared to any individual component. It is also possible that there would be a cross-sensitization
among individual components, for example, in a loss-of-righting response.

Anxiety and Impulsivity

Brief abuse-like toluene exposures tend to decrease anxiety-like behaviors in several tasks,
including open field, elevated plus maze, light-dark box, marble burying, and forced swim task,
when animals are tested after cessation of exposure (Batis et al., 2010; Bouchatta et al., 2016; Bowen
et al., 2018; Kouzoupis et al., 2010). The differences in behavior during versus after exposures to
abuse patterns of toluene have been interpreted in terms of intoxication and withdrawal,
respectively. For example, toluene (2,000 ppm to 6,000 ppm) showed diazepam-like anxiolytic
effects in the elevated plus-maze (Bowen et al., 1996) and marble burying tasks (Lopez-Rubalcava et
al., 2000). A 30-min exposure to toluene (1,000 ppm to 4,000 ppm) decreased immobility in a
forced swim task, similar to antidepressant drugs, suggesting toluene reduces “depression-like” or
“anxiety-like” behaviors in rodents (Cruz et al., 2009). High levels of toluene (10,000 ppm to
30,000 ppm) or diazepam (2.5 & 5 mg/kg) re-instated lever responses that were previously
suppressed by punishment, responses also interpreted to indicate a reduction in anxiety (Geller et al.,
1983). Bowen et al. (2018) also reported increases in anxiety-like behaviors in rats during the
immediate withdraw period (24-72 hrs) after a continuous 24-hr exposure to 5,000 ppm toluene.
 15

This increase in anxiety-like behavior was ameliorated with an acute re-exposure to toluene 30 min
prior to testing, suggestive of withdrawal (Bowen et al., 2018).

Impulsive-like behaviors were decreased during and increased after abuse-level toluene
exposure. Effects on impulsivity may last longer than on anxiety-like behaviors. For example,
1,000-ppm toluene produced significant increases in impulsivity in a “waiting for reward task.” Rats
that had been exposed previously to toluene had more “resets” than non-exposed rats in a fixed-ratio
(FR) schedule of reinforcement indicating increased impulsivity (Bowen and McDonald, 2009). In a
resident intruder task with mice, Bouchatta et al. (2016) reported that chronic exposure to Norlatex®
glue, containing toluene (18.29%), xylenes (10.57%), 1,3,5-trimethyl benzene (7.90%), as well as
alkane hydrocarbons (e.g., decanes & nonanes - 17.54%), increased latency to attack and decreased
the number of attacks. These results were interpreted as decreased impulsivity during glue
exposure. When assessed one week after that exposure, the mice attacked more quickly and more
often than the previous week during glue exposure than mice not previously exposed, indicating
greater impulsivity during this post-exposure/withdrawal period (Bouchatta et al., 2016). There is
no basis to judge how combined exposure to BTEX may have differentially affected impulsivity
compared to any component alone.

Learning and Memory

Effects on learning and memory have been reported for some of the BTEX components.
Deficits have been found in acquisition, recall/recognition, long-term memory, reversal learning and
shifting reinforcement contingencies. For example, Ghosh and colleagues (1987) demonstrated a
concentration-dependent depression in the rate of lever pressing for a 5% sucrose solution on a FR
schedule during relatively lower concentrations of p-xylene exposure (113 ppm to 430 ppm; Ghosh
et al., 1987). Changes in performance over 2-hr sessions implied tolerance to these effects of p-
xylene. Others reported bi-phasic concentration-dependent responses using a Differential
Reinforcement of Low rates of responding 10 sec (DRL10) operant schedule to higher
concentrations of xylenes (1,400 ppm - 7,000 ppm). Exposures to single and mixed xylene isomers
increased responses after 1,400 ppm and 2,400 ppm, while the highest concentration (7,000 ppm)
significantly decreased/disrupted DRL10 response rates and resulted in “gross ataxia and
prostration” of the mice (Moser et al., 1985).
 16

Gralewicz & Wiaderna (2001) reported a doubling in the number of trials for rats to reach an
inhibitory avoidance criterion two weeks after the last in a series of m-xylene exposures (100 ppm, 6
hrs/day, 5 days/week, for 4 weeks). In an earlier study, Gralewicz et al. (1995) reported memory
deficits in Y-maze after 100-ppm or 1,000-ppm m-xylene, levels modeling occupational exposure (6
hr/day, 5 days/week, 12 weeks). Maze completion times were nearly doubled for rats exposed to m-
xylene compared to controls (Gralewicz et al., 1995). Gralewicz et al. (1995) also found differences
in the number of perseveration errors in a radial arm maze. While m-xylene-exposed rats (100 ppm
& 1000 ppm) had fewer total errors than non-exposed controls, the main difference was in omission
errors: exposed rats did not explore each of the arms of the maze. By the 4th and 5th sessions, there
were concentration-dependent increases in omission errors in m-xylene-exposed animals (Gralewicz
et al., 1995).

Concentration- and operant schedule-dependent changes in rates of reinforcement for lever

pressing by mice have been reported. Toluene exposures ≤ 2000 ppm produced decreased Fixed
Interval (FI) responding (Bowen and Balster, 1998). Higher toluene concentrations (≥ 3200 ppm)
decreased responding under both FR and FI schedules (Bowen and Balster, 1998; Moser and Balster,
1986). Huerta-Rivas et al. (2012) showed that both acute and repeated exposures to abuse levels of
toluene (1000 ppm to 6000 ppm) in Wistar rats decreased short- and long-term memory in a novel-
object-recognition task. There were also deficits in both acquisition and retention of a step-through
inhibitory avoidance task assessing memory after acute (30 min) and chronic (30 min/twice a day/10
days) toluene exposure (1000 ppm to 6000 ppm). Toluene exposure also affects appetitive
responding for food reward. In animals trained for food reinforcement in a visual signal detection
task, acute abuse-like toluene exposures (1200 ppm - 2400 ppm) resulted in fewer correct responses
and delayed response times (Oshiro et al., 2007). Delayed acquisition of instrumental responding for
sucrose was also found after chronic intermittent toluene (10,000 ppm) exposure (Dick et al., 2013).
Differences in these appetitive tasks demonstrate a general dysfunction in learning after toluene
exposure due to altered motivation, or reward processing, or reward valuation.

Performance has also been compromised in maze- and spatial-learning tasks following
toluene exposure. In one study, adolescent or adult rats (PND25 or PND90) were exposed to toluene
vapors each day for 40 days (Bikashvili et al., 2012). Although unspecified, the concentration was
very high because exposure lasted only “until they fell over” in 3 to 5 mins. Over 10 days of testing
after exposure, the toluene-exposed groups took significantly longer and made more errors
 17

traversing a “multi-branch maze” than non-exposed groups. Performance deficits were seen in
young rats immediately after ending toluene exposure and in adult rats immediately and 3 months
after toluene exposure (Bikashvili et al., 2012).

In a different learning and memory task, Callan et al. (2017) showed that rats exposed
prenatally to toluene (8,000 ppm or 12,000 ppm) demonstrated no impairment of initial learning
ability or short-term recall. However, there was impaired performance during reversal learning and
probe trials after acquisition for the highest toluene concentration (12,000 ppm). In a separate study
of adolescent rats, Gmaz and colleagues (2012) found that repeated toluene (5,000 ppm) exposure
had no effect on the acquisition of goal platform location in a Morris Water Maze task. However, the
first performance of reversal trials in the maze was negatively affected. With repeated reversal
trials, rats were no different from unexposed controls. This suggests cognitive inflexibility and
differences in learning trajectories following toluene exposure (Gmaz et al., 2012).

However, chronic intermittent toluene (10,000 ppm, 1 hr/day, 3 days/week, 4 weeks) in

adolescent rats did not affect hippocampal-dependent spatial learning in a Morris maze beginning 3
days after exposure ended (Dick et al., 2013). Spatial learning assessed following lower-level (80
ppm), longer-term exposure toluene inhalation (6 h/day, 5 days/week for 4 weeks) found no
difference in swim speed or escape latency over 4 training days, but there was a significant increase
in time spent in the goal quadrant during a probe trial, suggesting not a difference in learning but in
perseverance (von Euler et al., 2000). While effects of combined BTEX exposures have not been
tested in the Morris Maze, the combination of Formaldehyde, Benzene, Toluene, and Xylene (FBTX)
has been. FBTX negatively affected performance for escape latency and number of errors after
environmental-like exposures to very low concentrations of each (e.g., ≤0.81 ppm) for 2 hrs per day
for 90 consecutive days (Wang et al., 2014).

Brain
The individual components of BTEX can have potent, brain region-specific influences on
multiple neurotransmitters, with changes in opposing directions. Zhvania and colleagues (2012)
investigated a potential neural mechanism underlying certain behavioral effects of toluene exposure
by assessing the impact on hippocampal structure and composition. Repeated short-duration toluene
exposures (2000 ppm, 3-4 min/day, 40 days) in adolescent (PN28-PN32) and adult (PN70-PN75)
rats were sufficient to alter hippocampal cytoarchitecture. Specifically, toluene-exposed adolescent
 18

and adult rats had lower CA1 hippocampal cell counts immediately after exposure and after a 90-day
abstinence period. For the CA3 region, only the toluene-exposed adolescent rats showed lower cell
counts immediately following toluene exposure, with both groups showing lower cell counts
following the 90-day abstinence period. Additionally, the significant differences observed between
rats exposed in adolescence or as adults indicates that the structural hippocampal changes are
partially dependent upon the age of exposure.

Honma et al. (1983) reported that occupational-like toluene exposure for 30 days increased
dopamine levels in rat striatum and decreased serotonin (5-HT) in cortex and hippocampus in a
concentration-dependent fashion (200 ppm - 800 ppm). In contrast, the same exposure pattern for
xylenes had the opposite effects: decreasing striatal dopamine while increasing cortical and
hippocampal 5-HT. Further, toluene decreased, and xylenes increased norepinephrine in the cortex
and hippocampus. Occupation-like exposure to toluene (80 ppm for 6 hrs/day, 5 days/week for 4
weeks) increased dopamine D2 receptor binding characteristics (i.e., Bmax) by 30% to 40% in
striatum (von Euler et al., 1994), but not D3 receptors in caudate putamen (Hillefors-Berglund et al.,
1995; von Euler et al., 2000). This group also reported that this type of exposure resulted in a 6-10%
reduction of the cerebral cortex, particularly the parietal cortex (von Euler et al., 1994). Similar
exposure to xylenes did not affect D2 receptor binding (Hillefors-Berglund et al., 1995).

Berenguer et al. (2003) devised a pattern of “sub-chronic” toluene exposure in rats intended
to mimic “indoor air pollution … at home and/or at work.” Using 40-ppm toluene exposures that
lasted 48 or 56 hours twice per week over 16 weeks, they reported significant increases in
DA/DOPC ratios in putamen and NAc but not hippocampus or prefrontal cortex. Cintra and
colleagues (1999) using a similar toluene exposure model (40 or 80 ppm, 6 hrs/day, 4 weeks)
reported a significant increase in dopamine levels in toluene-exposed rats (80 ppm) with no
DA/DOPAC changes. Prior single exposures to VOCs (i.e., toluene or “benzine” - a petroleum
distillate not to be confused with benzene) in rats, led to a concentration- and time-dependent shift in
lever pressing for electrical intracranial self-stimulation (ICSS) of the median forebrain bundle
(Bespalov et al., 2003). Finally, Yavich and Zvartau (1994) compared the effects of toluene alone
on ICSS with the effects of a solvent mixture containing 25% toluene and other VOCs (i.e., 37%
benzine, 31% ethyl acetate, 7% methylene chloride). Toluene alone (3,600 ppm to 14,400 ppm)
produced a biphasic concentration-response shift, increasing, and then slightly decreasing ICSS
rates. In contrast, the mixture with toluene had a significantly greater effect over the same
 19

concentration range, doubling the increase in ICSS at 7,200 ppm and dramatically reducing ICSS
rate at 14,000 ppm compared to toluene alone (Yavich and Zvartau, 1994). These solvents are not
BTEX and it is difficult to extrapolate “dose-response” from a single component to a mixture, but
these findings are consistent with the hypothesis that a combination of VOCs can exacerbate
neurobehavioral impact relative to a single VOC. These are examples of the kinds of neural changes
following various patterns and levels of VOC exposures, including some combinations of solvents,
but not BTEX. A systematic appraisal of effects on brain function would be served by an animal
model of combined environmental BTEX exposure.

Neuro-Psychopharmacological Challenges

Shifts in dose-response curves for neural and/or behavioral responses to pharmacological

challenges after VOC exposure(s) indicate functional changes in brain function. Shifts have been
reported following abuse and occupational patterns of VOC exposure in several neurotransmitter
systems, and sometimes in different directions. Altered responses have been reported to challenges
with the VOC itself (sensitization/desensitization) as well as to other drugs of abuse even long after
the exposure (cross-sensitization/cross-tolerance). For example, “sub-chronic” low-level toluene
exposure (40 ppm) produced significant sensitization to loss-of-righting reflex upon exposure to
higher concentrations of toluene (3200 ppm to 3560 ppm; Berenguer et al., 2003). Repeated
exposures to toluene (>2000 ppm and < 8000 ppm) sensitized animals to the locomotor-stimulating
effects of toluene (Bowen et al., 2007a).

The dose-response to toluene can also be shifted by co-administration of

monoamine/catecholamine agonists or antagonists. Von Euler and colleagues (1994) reported that
17 days after an occupational pattern of toluene exposure (80 ppm for 6 hrs/day, 5 days/week for 4
weeks), with no difference in baseline activity, there was a significant increase in apomorphine-
induced locomotor activity and “motility” compared to the non-exposed group. A longer pattern of
low-level toluene exposure (6 h/day, 5 days/week for 4 weeks, 80 ppm) increased sensitivity to
apomorphine-induced locomotion and sometimes rearing (von Euler et al., 1991; von Euler et al.,
1994; von Euler et al., 1993). Cintra et al. (1999) reported that the contralateral rotational behavior
induced by apomorphine was significantly decreased after exposure to 80-ppm toluene (6 hrs/day, 4
weeks). Duncan et al. (2014) reported that chronic intermittent exposure to abuse levels of toluene
(10,000 ppm) decreased behavioral activation to amphetamine 3 or 4 days after exposure and to
 20

MK801 20 days after exposure. In contrast, Beyer et al. (2001) found increases in both locomotor
activity and dopamine concentrations in the nucleus accumbens (NAc) following an acute cocaine
challenge 1 or 4 days after toluene exposure in adult rats, indicating cross-sensitization. In another
study, when toluene inhalation (3000 ppm, 40 min) and cocaine administration (20 mg/kg, i.p.) were
simultaneous, the combination produced a super-additive response, with an 802% increase in
dopamine within NAc compared to the 450% increase reported following cocaine alone (Gerasimov
et al., 2002). Previous results from our lab demonstrated that exposure to abuse-like toluene (2000
ppm or 4000 ppm, 30 min/day, 5 days) during periadolescence decreased dose-dependent behavioral
activation to cocaine 4 or 12 days after the last toluene exposure. This suggests that peri-adolescent
exposure to toluene may desensitize responses to stimulants (Davidson, 2018). This same
investigation also demonstrated that prior toluene exposure during peri-adolescence did not
significantly impact behavioral responses to a cumulative ethanol challenge in early adulthood
(Davidson, 2018). Dick et al. (2014) reported similar findings for ethanol in that repeated toluene
exposure during adolescence did not significantly impact voluntary ethanol consumption in
adulthood. These studies of brain function and responses to drug challenge differ in species (mouse
vs. rat), which and how much VOC is tested, and pattern of exposure. Despite the differences in
models and outcomes, in general the evidence supports the idea that prior toluene and other VOC
exposures alter brain neurotransmitter levels, brain function, and responses to other drugs of abuse.

7. Other Behavioral and Medical Outcomes

The research reviewed above focuses on outcomes associated with exposures to individual
components of BTEX although the impact of combined BTEX exposure is largely unknown. The
behaviors affected in rodents tend to be relatively simple. However, there are also associations of
BTEX exposure with complex developmental neurobehavioral and cognitive disorders, such as
attention deficit hyperactivity disorder (ADHD) in epidemiological studies of human populations
(e.g., Dellefratte et al., 2019; Forns et al., 2018; Till et al., 2001), as well as medical complications
reviewed briefly above, as well as asthma (e.g., Bolden et al., 2015; Lemke et al., 2014). Generally,
most studies of this kind have addressed air pollution, including fine particulates, greenhouse gases,
and polycyclic aromatic hydrocarbons, and have not focused on BTEX. The myriad possible
constituents of air pollution, including other VOCs, can all contribute substantially to outcomes.
There are also important and substantial interactions among many of these exposures, BTEX and
other VOCs included, as well as other risk factors like poverty or racial disparities (e.g., Symanski et
 21

al., 2009). These complexities, which impact wide-ranging outcomes, are why we argue that
understanding the effects of combined environmental BTEX per se requires a preclinical model.

8. Conclusions: Demands of an Environmental Model of BTEX Exposure

Sustained environmental exposures to VOCs pose a constant risk to public health. The
potential public health risks of environmental levels of combined BTEX exposure, in particular,
appear well appreciated (Bolden et al., 2015; Dehghani et al., 2018), although not always not well
characterized. Our goal here is to describe what is necessary for a useful animal model of
environmental exposures to combined VOCs, in particular BTEX. Animal research investigating
environmental patterns of exposure to even single VOCs are relatively rare. There are few published
studies using a combination of VOCs at any pattern/level of exposure (Haddad et al., 2001; Haddad
et al., 2000; Haddad et al., 1999; Wang et al., 2014), and there are none assessing the combination of
BTEX with environmental patterns. It is clear from our review that little is known about effects of
combined environmental BTEX exposures in animals. It is also clear that several features important
to any inhalation model must be incorporated properly when developing an environmental model of
BTEX exposure. The inherent complexity of a living being’s response to BTEX seem to require the
use of an animal model to better explore the human experience. The advantages of an environmental
model under controlled laboratory conditions include being able to vary systematically those critical
features defining risk exposure: the components, combinations, concentrations and patterns/timing.
The almost incalculable variety of potential exposure patterns that may exist in the “real world” is a
challenge that need not be mimicked perfectly for animal models to be quite useful.

We explored the ways that knowledge of the neurobehavioral effects of abuse and
occupational patterns of exposure to individual or combined components of BTEX might help
inform development of a valid preclinical model of environmental exposure. We believe that the
comparatively rich research literatures with other models demonstrate clearly the importance of
well-defined concentrations and timing (duration, rate, pace) that define risk “patterns” of exposure.
However, it is also clear that findings from abuse or occupational models, whether to single or the
rare multiple VOCs, cannot substitute for the long-term, low-level, patterns of mixed exposures
characterizing the experience of people living in urban communities and near industrial sites where
BTEX is prevalent. This is the first “gap” in the literature. Models are not interchangeable because
the “momentum” of exposure, a proxy for “dose,” varies substantially, both quantitatively and
 22

qualitatively. The considerable differences in BTEX concentrations, multiplied by durations and

repetitions, and even breathing dynamics, limit extrapolation among models. Therefore,
development of new preclinical models using patterns of exposure that seek to mimic environmental
exposures is clearly necessary.

We had affirmed above how critical the concentrations of VOCs and the patterns of exposure
are to defining any model of inhalation exposure. It is impractical to suggest that nothing short of
continuous, even lifetime environmental exposure would be an experimentally valid animal model,
and yet very long duration exposures to relatively low concentrations should be approximated. In
fact, the demand for lower concentrations of VOCs in an environmental model requires longer
exposures than in abuse or occupational models. Yet, any non-continuous temporal pattern of
exposure in a lab setting aimed at modeling the environment would be no more or less a limitation
on the model than other factors, such as species differences, that require qualification in interpreting
results.

Obviously, a second critical “gap” in the literature on neurobehavioral outcomes is the

limited animal research on combined BTEX, especially with environmental patterns of exposure. As
noted, a major conclusion of this review is that prior research with other models is insufficient to
extrapolate effects of environmental BTEX exposure. While a valid model demands combined
exposures, one challenge is to avoid the “rabbit hole” of parametric designs. Three or more
concentrations of two, three or four chemicals would rapidly expand an experiment to unattainable
proportions. A related challenge is to determine the proper control/comparison groups for combined
BTEX exposures, especially if interactions among components are relevant. There should of course
always be a non-exposed, 0-ppm, baseline group. It is an open question about which other
comparison group(s) are necessary in an environmental BTEX model. It would likely be
unreasonably complex to include each individual VOC (B, T, E & X), and perhaps it would be
unreasonably simplistic to have only one (T). It would be as unreasonable to include groups for all
possible pairs (BT, BE, BX, TE, TX, EX), and/or triads (BTE, BTX, BEX, TEX). If only some
groups are used, which few would be required to support statistical comparisons or judgements
about the relative risk for deleterious consequences of BTEX? Though interactions among acute
binary combinations of BTEX components are catalogued (ASTDR, 2004, e.g., tables 2.1 and 2.6-
2.10), we believe that experimental designs to assess interactions among components may be an
unnecessary complication in an environmental model than systematically varying BTEX levels
 23

together. This is because BTEX occur together in the environment, from the same and related
sources, and their total concentrations between regions and seasons tend to vary as a whole without
substantially altering relative proportions and rank orders among them (e.g., Mohammadi et al.,
2020). For example, the proportions among air-borne BTEX component concentrations are
remarkably consistent across 20 cities around the world, even as those cities ranged 69-fold in the
total amount of combined BTEX in the local airsheds (Carlsen et al., 2018).

Potential Confounds

Sex Differences: Men and women can differ in VOC/BTEX exposure and susceptibility.
Males had been more likely to abuse solvents (Aydin et al., 2002; Johnston et al., 2019), although
that difference had lessened as more females began “huffing” (SAMHSA, 2007a, b, 2008). Sex-
differences in employment in a population can influence occupational exposure to specific VOCs
(i.e., relatively more males in automobile upkeep/repair, construction and manufacturing (Badjagbo
et al., 2010; Moro et al., 2017) and more females in salons and similar settings (Lamplugh et al.,
2019)). While differences between sexes in environmental exposures may be less likely, any animal
model demands consideration of sex differences in susceptibility to the effects of VOCs. Berenguer
et al. (2003) reported greater toluene-induced “narcosis” in males than in females after a prior “sub-
chronic” toluene exposure. Effects of prenatal toluene exposure on motor coordination and
performance in learning/memory tasks may be more pronounced in female than in male offspring
(Hass et al., 1999; Hass et al., 1994). In contrast, prenatal toluene shifted dose-response curves for
locomotion to an amphetamine challenge in male but not female rats (Bowen et al., 2007c).

There is evidence of sex differences in BTEX metabolism. For example, women have a
higher metabolic rate for benzene than men have (Brown et al., 1998; Moro et al., 2017). Women
exhaled significantly more m-xylene than men, whereas equivalently exposed males had
significantly higher levels of urinary m-xylene metabolites than females, when adjusted for body
weight (Ernstgard et al., 2003). Sex differences in responses to BTEX may also be mediated by
endocrine disruption. For example, limited occupational exposure to toluene can impact on the
luteal phase of menstruation (Luderer et al., 1999). Fecundity was reported to be affected in women
but not men exposed to occupational levels of toluene and/or xylenes (Plenge-Bonig and Karmaus,
1999). Sex differences in effects of combined BTEX are largely unknown. New environmental
models of BTEX exposure would offer an opportunity to explore these differences.
 24

Developmental Differences. How young or old an organism is when exposed matters.

Exposure to higher levels of air pollutants, including BTEX, has been associated with lower birth
weight in humans (Aguilera et al., 2010; Aguilera et al., 2009). Animal models have demonstrated
that gestational exposure to the individual BTEX components can be toxic to both the dam and
offspring, and that fetal toxicity tends to be seen at lower levels than maternal toxicity (Hannigan
and Bowen, 2010; Hudak and Ungvary, 1978; Roberts et al., 2007; Saillenfait et al., 2003). Prenatal
exposures to ethylbenzene, individual xylene isomers or combined isomers in technical xylenes,
showed small, concentration-dependent reductions in maternal food intake and weight gain late in
pregnancy, and reduced fetal weight, but no teratogenic outcomes, with minor differences among the
VOCs (Saillenfait et al., 2003). This group also showed similar concentration-dependent effects of
toluene or ethylbenzene inhalation in pregnant rats on maternal and fetal weight, and the effects of
both were exacerbated by combined exposure to butyl acetate, again with no increase in
malformations (Saillenfait et al., 2007).

Susceptibility to and expression of neurobehavioral effects of individual BTEX components

appear to differ with age or stage of development. As discussed, Zhvania et al. (2012) reported that
40 days of toluene exposure (2000 ppm; 3-4 min/day) beginning at adolescence led to greater
decreases in hippocampal cell counts than exposure beginning in adulthood. We examined effects of
various single or repeated abuse-like exposures to toluene (2,000 ppm to 16,000 ppm, for 15 or 20
min, twice a day) at three developmental periods in rats, adolescence (PN28), young adult (PN44),
and adult (>PN70). While locomotor activity increased in all ages, older groups showed greater
increases in locomotor activity compared to the younger groups (Batis et al., 2010; Bowen et al.,
2007a). There are also enduring, concentration-dependent effects of prenatal exposure to abuse-
level toluene on growth, maturation and behavior (Bowen et al., 2005; Bowen and Hannigan, 2006;
Bowen et al., 2009b; Callan, 2015; Hannigan and Bowen, 2010; Jones and Balster, 1997). Together
these studies suggest that age of the animal during exposure is a critical factor for toluene impact and
should be considered in the creation of preclinical environmental BTEX exposure models.

Summary

This review of the effects of inhaled volatile organic solvents (VOCs) focused on current
literature on exposures to Benzene, Toluene, Ethylbenzene, and Xylenes, collectively “BTEX.”
 25

Primarily through research with animal models, evidence for the effects of single-BTEX component
inhalation exposure demonstrates that each of these VOCs can negatively affect health and
neurobehavioral outcomes. These outcomes include poor coordination, elevated locomotor activity,
measures indicative of anxiety and impulsivity, and compromised learning and memory.
Neurochemical and psychopharmacological studies also show that exposure to BTEX components
affects the brain in ways that likely underlie the behavioral effects. However, this research
predominantly used patterns of exposure modeling solvent abuse and occupational settings. There
are very few animal research studies with individual BTEX exposures at levels and patterns
consistent with environmental exposure. This made extrapolating from abuse and occupational
models necessary. Further, there are essentially no animal studies of the effects of combined BTEX
exposure, in abuse, occupational or environmental models. The aim of this review was to inform an
environmental model based on prior research. However, the great differences among the models in
concentrations, durations and lengths exposures means that the risk of environmental exposure to
BTEX cannot be estimated reliably from research with other models. A valid animal model of
environmental BTEX demands the low concentrations of combined BTEX exposures for extended
periods characteristic of those experienced by human communities. It is clear that assessing the
unique health and neurobehavioral impact of combined environmental BTEX exposures, distinct
from cofounding exposures to other pollutants, including other VOCs, and risk factors (e.g., poverty,
pre-existing conditions) common in exposed communities, requires an animal model that
approximates well the characteristics of environmental patterns of combined exposures.
 26

Table 1: Components Table

Average Annual Metabolic Half-Life
Component OSHA Permissible
Other Names Structure Production and Release (by phase)
Exposure Limits
(U.S.)
Benzene benzol, annulene Production 2009-2018: 169.5 Rapid: 0.70 hrs
C6H6 TWA: 1 ppm million lbs.
MW : 78.11 Slow: 13 hrs
STEL: 5 ppm Waste (air) 2009-2018:
MD: 0.8765 3.2 million lbs.

Toluene methylbenzene, Production 2009-2018: 1,550 Rapid: 0.88 hrs

C7H8 toluol, TWA: 200 ppm million lbs.
MW : 92.14 methylbenzol, Slow: 12.9 hrs
STEL: 500 ppm Waste (air) 2009-2018:
MD: 0.867 phenylmethane 16.8 million lbs.
Ethylbenzene ethylbenzol, Production 2009-2018: 92.8 Rapid: 0.69 hrs
C8H10 phenylethane, TWA: 100 ppm million lbs.
MW : 106.17 aethylbenzol Slow: 19.2 hrs
STEL: 125 ppm Waste (air) 2009-2018:
MD: 0.866 2.2 million lbs.
p-Xylene

Production 2009-2018: 406.7

Xylenes m-Xylene
million lbs.
C8H10 dimethylbenzene, TWA: 100 ppm Rapid: 0.84 hrs
Waste (air) 2009-2018:
MW : 106.16 methyltoluene, 11.4 million lbs. Slow: 10.9 hrs
MD: 0.861 xylenen, xylol STEL: 150 ppm

o-Xylene
 27

Table 1. Components of BTEX: For each molecule, various names with its formula, Molecular Weight MW (g/mol), and molecular
density MD (g/mL), and Structure. OSHA safety limits are specified as Time Weighted Average (TWA) calculated for an 8-
hour workday, and as maximum Short-Term Exposure Limit (STEL), the legally defined safe maximum concentration for a
10-15 min exposure. Average production and air waste per year over a ten year period (2009-2018) is reported. Lastly,
reports of both the rapid and slow half life of each component. Adapted in part from Joshi and Adhikari (2019) and EPA
(2018).
 28

Table 2: Types of VOC Exposure Studies

Concentrations Exposure Temporal

Model Type
(approximate range) Durations Patterns

Environmental Low Concentrations Very long Relatively

(<50 ppm) (weeks – months) constant

Occupational Moderate Concentrations Typical workday Intermittent,

(>50 ppm to <~800 ppm) (8 hrs) repeated Long duration

Abuse High Concentrations Short duration Intermittent,

(>1,000 ppm to ~30,000 ppm) (1-2 hrs) repeated Short duration

Table 2. Exposure characteristics typical of the different research models

 29

Table 3. Summary of Outcomes after Environment-Like Exposures

BTEX Exposure Sex, Strain, &
Paper Significant Outcome(s) Non-significant Outcome(s)
Component(s) Pattern(s) Species
Ahmed et al., Toluene 6 hrs/day, 5 Female In hippocampus, upregulated NMDA No changes in NMDA subunit
2007 (50 ppm) days/wk, 6 or 12 C3H/HeN subunit NR2B, and ↑induction of NR2A or CREB-2
wks Mice CaMKIV, CREB-1 and FosB/DeltaFosB
Berenguer et Toluene 8 to 12 hrs/day for Male and ↑ DA/DOPAC ratio in putamen and No DA changes in prefrontal
al., 2003 (40 ppm) 6 days/wk for 16 Female nucleus accumbens (both males & cortex or hippocampus
wks Sprague- females)
Dawley Rats ↑ Rearing

↑ Susceptibility to toluene-induced
narcosis
↑ 5-HTP accumulation in
hippocampus and medial prefrontal
cortex in females – ↓ in males
Cintra et al., Toluene 6 hrs/day, 4 wks Male ↑ DA levels in substantia nigra and No DOPAC or DOPAC/DA
1999 (40 or 80 ppm) Sprague- neostriatum at 80 ppm after 6-OH DA ratio changes for 80 ppm;
Dawley Rats lesion No DA, DOPAC or DOPAC/DA
changes at 40 ppm

↓ 6-OH DA-induced contralateral No effect in 6-OHDA-induced

rotation behavior at 80 ppm contralateral rotation at 40
ppm

El-Shakour et Benzene 5 hrs/day, 5 Male Wistar Liver and kidney damage (↑markers
al., 2015 (10, 30 & 50 days/wk, for 2 wks Rats of oxidative stress)
ppm)
Gralewicz & m-Xylene 6 hrs/day, 5 Male Wistar ↑ Spontaneous locomotor activity (25 No effect on body weight
Wiaderna, 2001 (100 ppm) days/wk, for 4 wks Rats days after exposure);
↓ Passive avoidance learning (39-48
days after exposure);
↑ Hot-plate paw-lick latencies 24 h
after foot shock (51 days after
exposure);
 30

↑ Trials to criterion in inhibitory

avoidance task (54-60 days after
exposure)
Gralewicz et al., m-Xylene 6 hrs/day, 5 Male ↑ Perseveration and omission errors No effect on body weight;
1995 (100 ppm) days/wk, 12 wks LOD-Wist in radial arm maze; No changes in
Rats ↓ maturation of spontaneous electroencephalographic
neocortical spike and wave discharges arousal

Hillefors- Toluene 6 hrs/day, 5 Male ↓ Weight of caudate-putamen and No effect on body weight or
Berglund et al., (40 or 80 ppm) days/wk, 4 wks Sprague- limbic areas; total brain wet weight
1995 Dawley Rats ↓ [3H]raclopride-binding in caudate-
putamen at 80 ppm
Xylenes No effect on body or total
(40 or 80 ppm) brain wet weight;
No effect on DA measures

von Euler et al., Toluene 6 hrs/day, 5 Male ↓ Hind-paw placement during plank No effect on acquisition,
2000 (80 ppm) days/wk for 4 wks Sprague- walking: escape latency, or swim
Dawley Rats ↑ Time in goal quadrant on probe speed in Morris Maze
trial in Morris Water Maze;

6-10% ↓ in parietal cortex volume No changes in DA D(3)

receptor binding metrics

von Euler et al., Toluene 6 hrs/day, 5 Male ↑ Serum prolactin levels

1994 (80 ppm) days/wk for 4 wks Sprague-
(tested 17 days Dawley Rats ↑ Apomorphine-induced locomotion No effect on rearing
after exposure) and motility

↑ DA D2 receptor binding;
 31

von Euler et al., Toluene 6 hrs/day, 5 Male ↑ Escape latency during acquisition No effect on spontaneous
1993 (80 ppm) days/wk for 4 wks Sprague- training and retention in a Morris locomotion, motility or
Dawley Rats Water Maze rearing

↑ Apomorphine-induced locomotion
and motility
↑ DA D2 receptor numbers in
striatum

Wang et al., Combined 2 hrs/day, 90 Male ↑ Escape latency and errors in Morris
2014 Formaldehyde, consecutive days Kunming Water Maze
Benzene, Mice
Toluene, & ↓ Grip strength
Xylene (FBTX)
(≤0.81 ppm ↑ Levels of reactive oxygen species;
each) ↓ Neurotransmitter metabolism and
expression of NMDA receptors.

Zhang et al., Ethylbenzene 6 hrs/day for 13 Male and ↑ Kidney damage (↑ apoptosis in
2010 (100 ppm) wks Female renal tubular epithelial cells)
Sprague-
Dawley Rats

Table 3. Studies with low-level, relatively long-term exposures consistent with an environmental exposure model. Inclusion criteria
were animal studies testing BTEX components at ≤ 100 ppm and ≥ 2 weeks of exposure. These studies are reviewed in the text.
This does not exhaust the literature. Note there are no studies assessing all BTEX, and only one study (Wang, et al., 2014)
combined B, T and X, but that mixture also included formaldehyde.
 32

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