Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

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Mutation Research - Genetic Toxicology

and Environmental Mutagenesis
journal homepage: www.elsevier.com/locate/gentox

A critical review and meta-analysis of epidemiology studies of

occupationally exposed styrene workers evaluated for chromosomal
aberration incidence
James J. Collins a, *, Martha Moore b
a
Health and Human Sciences, Saginaw Valley State University, 7400 Bay Road University Center, Michigan 48710, United States
b
Martha M Moore LLC, 23 Alban Lane, Little Rock, Arkansas 72223, United States

A R T I C L E I N F O A B S T R A C T

Keywords: Chromosome aberrations in the peripheral blood lymphocytes of styrene exposed workers have been suggested as
Chromosome aberrations a potential early marker for cancer risk. We performed a critical review and abstracted data from all studies using
Styrene current chromosome aberration scoring criteria and providing at least a mean and standard deviation or standard
Bio-monitoring
error for the exposed and comparison groups. Using these data, we conducted a meta-analysis of occupational
Cancer risk
Occupational study
styrene exposed workers and incidence of chromosome aberrations. Our meta-analysis used the standardized
Meta-analysis mean difference as the summary statistic since all studies assess the same outcome but use different comparison
populations. The primary meta-analysis of the 20 comparisons of 505 styrene exposed workers to 532 com­
parison workers found a meta-mean difference of 0.361 (95 % CI − 0.084 to 0.807, random effects model), but
there was substantial lack of consistency across studies (I2 of 90.11, p-value <0.001, fixed effect model). Studies
with higher styrene exposures had lower mean standard differences compared to studies with lower styrene
exposures. While studies of styrene workers overall had a slight increase in chromosomal aberrations relative to
comparison groups, the lack of consistency across studies and the absence of an exposure response and other
limitations of the reviewed studies including inadequate exposure assessment, small numbers of participants per
study, and poorly matched exposed and comparison workers, we find insufficient evidence to support a
conclusion that styrene exposure increases chromosome aberration frequencies in styrene workers.

1. Introduction of styrene in their assessments. The recent IARC evaluation provided a

Styrene (C6H5CH = CH2, CAS number: 100-42-5) is primarily used
to produce polymers that are used in the production of plastic products.
More than 15,000 industrial plants in many countries produce or use
styrene in the manufacturing of various products [1]. Historically, the
highest occupational exposures to styrene in the air have been measured
in facilities manufacturing fiber-reinforced polymer composites. Styrene
exposures are generally lower in the production of styrene, polystyrene,
and styrene-based plastics and rubbers [2].
A recent evaluation by the International Agency for Research on
Cancer (IARC) concludes that styrene exposure is probably carcinogenic
to humans based on sufficient evidence in experimental animals but
limited evidence in humans [1]. The National Academy of Sciences
(NAS) and the National Toxicology program also reached similar con­
clusions [3,4]. Both IARC and the NAS considered genotoxicity studies
summary of the in vitro and in vivo studies that have been published for
styrene and styrene oxide exposures with an emphasis on the in vitro
studies that used human cells and an emphasis on studies of DNA ad­
ducts. The IARC review concludes that the in vitro studies provide evi­
dence that styrene metabolized to styrene oxide is genotoxic and causes
both gene mutation and cytogenetic effects. However, the IARC review
indicates that the in vivo rodent studies are “equivocal for cytogenetic
effects.” A recent critical review of the genotoxicity studies for styr­
ene/styrene oxide using current data interpretation criteria based on
Organization for Economic Cooperation and Development (OECD) ge­
netic toxicology test guidelines also concluded that styrene oxide is
genotoxic in vitro but the available in vivo rodent studies do not confirm
this genotoxicity [5]. The IARC review summarizes the occupational
human studies as providing evidence that styrene oxide derived DNA
adducts have been found in the blood and urine of exposed workers, but

* Corresponding author.
E-mail address: jjcollin@svsu.edu (J.J. Collins).

https://doi.org/10.1016/j.mrgentox.2020.503275
Received 21 May 2020; Received in revised form 15 October 2020; Accepted 16 October 2020
Available online 22 October 2020
1383-5718/© 2020 Elsevier B.V. All rights reserved.
J.J. Collins and M. Moore
that the studies for other indicators of genotoxicity provide “mixed”
results. The human studies evaluating chromosomal damage including
micronuclei and chromosome aberrations are briefly mentioned in the
IARC review and divided into those providing positive and those
providing negative results.
Workplace studies using genetic toxicology endpoints can be useful
to evaluate evidence of target tissue exposure and early biological effects
in humans and thus provide insight into human risk from occupational
exposures. Both chromosomal aberrations and micronuclei have been
used as genetic damage endpoints [6,7]. Thus, understanding whether
the positive genotoxic effects, including chromosomal damage, that
have been clearly demonstrated in vitro also occur in humans is impor­
tant to understanding the potential human risk from occupational sty­
rene exposure. It is noted that chromosomal damage has not been clearly
demonstrated to occur in rodents exposed by inhalation to high levels of
either styrene or styrene oxide.
There are three additional reviews on styrene exposure and chro­
mosomal aberrations. In 1996, Bonassi et al. undertook a re-analysis of
the published cytogenetic studies in occupationally exposed humans
[8]. The authors recognized that there was a lack of consistent findings
in these studies which they attributed to limited sample size, inadequate
exposure assessment and poor study design. They specifically stated that
it was not their goal to deal with an assessment of study quality, but
rather to determine if they could discern any general quantitative trend
between exposure to styrene and the cytogenetic endpoints. Their
strategy was to combine all the studies, in a quantitative review to
calculate a frequency ratio for the exposed compared to controls for each
of the cytogenetic endpoints. They also characterized the level of
exposure as either low or high for each study. Using a model to assess the
extent of the cytogenetic damage in relationship to the exposure, they
concluded that there was an association between chromosomal aberra­
tions and high styrene exposure. They concluded that the results for the
micronucleus endpoint were inconclusive.
In 1994, a critical review of human studies conducted using occu­
pationally styrene exposed individuals and evaluating cytogenetic end­
points (chromosome aberrations, micronuclei and sister chromatid
exchange), was conducted by Scott and Preston [9,10]. For their eval­
uation they considered the relationship between the positive and
negative findings in relation to the reported exposure levels. There were
52 human studies including some with exposure to multiple chemicals.
They concluded that, although the authors of 18 of the studies called
their results positive, the data presented in the publications were “not
compatible with the conclusion that styrene is responsible for the cy­
togenetic effects.” The listed reasons for this conclusion included: no
association between the degree of exposure and whether the response
was determined to be positive or negative, and no convincing evidence
that there was a dose-response relationship for the responses that were
called positive. They further concluded that any increases in cytogenetic
effects reported in some of the studies of styrene workers should be
attributed either to “inadequate investigations” or to exposures to other
genotoxic chemicals.
In 2005, Henderson and Speit published a critical review of the
published human studies evaluating gene mutation, micronuclei and
chromosomal aberrations [11]. Included in the publication is a review of
each of the studies. The authors concluded that there was no convincing
evidence that exposure to styrene results in “detectable levels” of so­
matic cell mutagenic damage in humans. Furthermore, they concluded
that the evidence for the induction of cytogenetic damage was less clear,
with most micronucleus studies showing a lack of induction and con­
flicting evidence for the induction of chromosomal aberrations.
While these critical reviews of studies for the genetic toxicology
endpoints provide insight, there have been no formal quantitative
analysis of the many available chromosomal aberration studies. A meta-
analysis of the studies provides an objective means to determine the
consistency of studies and to determine if any increase is may be due to
styrene exposure. If styrene exposure can induce a genotoxic effect, one
2
Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275
would expect consistency across studies and a relationship between the
level of a genotoxic effect with the level of exposure. A critical review
and meta-analysis of the published occupational styrene exposure
human studies that used current methodology for assessing the potential
for micronucleus induction was conducted recently [12]. The authors
concluded that there was a lack of consistency across the studies and an
equivocal finding concerning the relationship between the level of sty­
rene exposure and the micronucleus frequency. In the current study, we
conduct a similar critical review and a meta-analysis of the available
published studies using chromosome aberration as the measure of cy­
togenetic damage. We employ a meta-analysis along with a critical re­
view to assess the consistency across these studies and to assess study
differences that may be causing inconsistencies or results.
2. Material and methods
Search Strategy and Selection of Studies
This review and meta-analysis followed guidelines of PRISMA-P
(Preferred Reporting Items for Systematic Review and Meta-Analysis
Protocols) [13] and MOOSE (Meta-analysis of Observational Studies in
Epidemiology) [14]. Studies eligible for inclusion were chromosomal
aberrations evaluations among styrene exposed workers that were
published or identified from January 1, 1975 to February 29, 2020 in
PubMed (https://www.ncbi.nlm.nih.gov/pubmed/). Eligible studies
were searched in PubMed using two search strings: (styrene and human
chromosomal aberration) and (styrene and human chromosomal
aberrations).
We identified 133 publications in this search (see Fig. 1). All ab­
stracts found in the PubMed search were reviewed to identify those that
evaluated the ability of styrene to induce chromosomal aberrations in
humans. The review of the abstracts resulted in the elimination of 102
articles for one or more of the following reasons: (1) chromosome ab­
errations were not evaluated, (2) not a human study, (3) the publication
was only an abstract, (4) the publication was a review and did not
include primary data, or (4) the paper was not in English.
We both read and critically evaluated the 31 remaining studies
which were divided into those that could be used for the meta-analysis
and those that did not have the appropriate information to be included
in the meta-analysis. The first consideration for inclusion was whether
the data were presented in a way that included, or that we could
calculate, a mean and a standard deviation or standard error. The meta-
analysis evaluation requires this basic information and if it was not
Fig. 1. Flowchart of study selection.
J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

available, the study was eliminated. Second, uniform criteria have been Table 1
developed for scoring chromosomal aberrations. It is important to note Studies excluded from the meta-analysis and justification for their exclusion.
that gaps were once commonly included in the total number of aber­ Lead Author, Brief Study Description Justification for excluding
rations and for some of the earlier styrene publications their inclusion publication year the study from the meta-
impacts the overall positive or negative assessment. Since it is no longer analysis
considered appropriate to include gaps, for this meta-analysis, we only Meretoja et al. 1977 This group of investigators Data includes gaps,
used studies where the number of chromosomal aberrations without and Meretoja evaluated workers exposed aneuploid cells, polyploid
gaps and other non-standard events was reported or could be deter­ et al. 1978 to styrene in 3 plants in cells and decondensation
Finland and reported parts of from the normally scored
mined. There was also one study that used the FISH (fluorescent in situ the data in two publication. chromosome aberrations.
hybridization) rather than traditional scoring and was eliminated [15]. The study design and data Not possible to determine a
Third, peripheral blood lymphocytes are generally not in S-phase and so presentation are confusing mean and standard
the aberrations that are seen and scored occur when the cells are stim­ with some individuals deviation that does not
reported in both include these events.
ulated to divide in vitro as a part of the technical procedure required to
publications and some in
evaluate chromosomal aberrations in human studies. Because chromo­ only one publication. There
somal aberrations decrease in second generation cultured cells following were 18 exposed individuals
mitogen stimulation, a cell harvest time that maximizes the number of reported between the two
first division cells is critical for optimal results. The time post publications and 6 controls.
There were two sampling
PHA-stimulation that is currently used by most laboratories is 48 h. times (1976 and 1977) for
Several studies used culture times longer than 48 h with one study using some of the exposed
96 h [16]. This very long time would greatly decrease the number of individuals, but controls
cells with chromosome aberrations. Therefore, this study, the focus of were only sampled once
(1976). 100 metaphases
which was primarily methods development and not the assessment of
were scored per individual
styrene exposure and the induction of chromosome aberrations, was but, at least, at the second
eliminated. Fourth, it is important to score enough cells to obtain a sampling the scoring was not
reliable value for the actual frequency of chromosome aberrations. In blind (because there were no
2000, a World Health Organization International Programme on controls). The aberrant
chromosome frequencies are
Chemical Safety (WHO IPCS) workgroup published a set of guidelines,
among the highest reported
including essential study parameters, for the conduct and interpretation for occupationally styrene
of human studies utilizing genotoxicity endpoints [17]. Based on an workers. Aberrant
average background rate of about 1% cells with aberrations, IPCS rec­ lymphocytes included gaps,
aneuploid cells, polyploid
ommends that a minimum of 200 metaphases be scored per individual
cells, chromatid breaks,
[17]. The majority of the published studies for styrene scored only 100 chromosome breaks and
metaphases. There was one study that scored only 30 metaphases if the decondensation. A pie chart
culture was successful [18]. This publication was eliminated, but studies is presented showing the
scoring 100 or more cells were included. proportions of these various
events in the 1976 sampling
The application of the above outlined inclusion/exclusion criteria
and the 1977 sampling as
resulted in the elimination of 13 publications. Table 1 lists and provides well as in the 1976 sampling
some details for these 13 publications including the specific reason(s) for of the controls, but it is not
elimination. For the 18 publications that were included in the detailed possible to extract any
usable data from this for the
critical review and the meta-analysis, the following information was
meta-analysis.
extracted and is presented in Table 2) first author, 2) year of publication, Hogstedt et al. 1979 This was a small study of 6 The data are presented
3) study design, 4) country of study, 5) study subject overlap with other males working at a paper graphically with only a
studies, 6) number and description of the styrene exposed workers and factory in Sweden and 6 summary in the text. Only
the comparison population, 7) measurements of styrene exposures, 8) males matched on age and mean values presented for
sex who worked close by, but the exposed and controls.
blinding of exposure during slide coding and scoring, potential con­
with no chemical exposure. Not possible to determine a
founding factors, 9) the number of cells scored, 10) the mean chromo­ Styrene exposure was standard deviation.
somal aberrations levels and standard deviation for the exposed worker measured over several years.
and comparison group or groups, 11) the authors’ interpretation of the The workers were also
exposed to phthalic acid
major findings, and 12) our identified strengths and weaknesses of the
anhydride, maleic acid
study. anhydride, propylene glycol,
Study Assessment methyl ethyl ketone
A causal assessment was done for each study. We used some of the peroxide and cobalt salt.
guidelines from the Hill criteria to assess these studies [19]. Our eval­ Slides were blind-coded, and
200 cells were scored per
uation put most emphasis on consistency across studies, strength of the
individual. The authors
association, and exposure response. We also considered the potential for concluded that the workers
co-exposures and confounding exposures influencing the study out­ had a higher level of CA
comes. We also considered occupational exposure to styrene, overall (breaks = 6.9 per 100 cells)
risk, the consistency of findings across the studies, and the impact of than the controls (breaks =
2.5 per 100 cells). They also,
study characteristics on risk referred to as sensitivities, and the potential indicated that possible
for publication bias. exposure to chemicals other
styrene may have
3. Results contributed to the increase
in CA in the workers.
Watanabe et al. Eighteen workers from a Gaps were included in the
The results of the literature search and the selection of studies 1981 fiberglass reinforced plastic data presentation. Not
considered is described in Fig. 1. As mentioned previously, of the 133 (continued on next page)
published papers identified in the literature review, 102 were eliminated

3
J.J. Collins and M. Moore
Table 1 (continued )
Lead Author, Brief Study Description
publication year
boat factory and a polyester-
resin board factory in Japan
were evaluated. Thirteen
controls were defined as
unexposed and were age and
sex matched. Cell
proliferation was measured
by comparing cells in M1,
M2 and M3. Slides were
coded. The number of cells
scored varied from a low of
17 for one of the controls to
50, leading one to speculate
that the culture conditions
may have been inadequate
to obtain sufficient
metaphases. The number of
cells scored was
unacceptably small. The
metaphases scored were
from second division cells.
The authors concluded that
there was no difference
between the workers and the
controls.
Camurri et al. 1983, These authors reported data
and Camurri et al. in two publications. The
1984. 1984 publication includes
some of the data from the
1983 publication. Overall,
individuals from 9 plants
(ranging from 3 to 10
workers per plant) in Italy
were studied. The methods,
particularly for participant
selection are not well
described. The
characteristics of the
controls is not described, but
it appears that they may
have been workers in some
of the plants. While the
methods state that 100 cells
were scored, the actual
reported number varied
from a low of 20 in one of the
controls and a high of 182 in
one of the styrene workers;
in many cases fewer than
100 cells were scored. The
range of CA values for the
exposed workers is 19− 54%;
the CA values for the control
groups is 2− 13%. The
authors concluded that there
were more CAs in the styrene
workers than the controls.
Hansteen et al. A glass-fiber-reinforced
1984 polyester plant was
evaluated in Norway.
Breathing zone evaluated for
weekly exposure. Low
exposure group (n = 11)
mean 7.5 ppm (range: 2− 13
ppm). High exposure group
(n = 7) mean 22.3 ppm
(range of 14− 44 ppm). It
was noted that all workers
had exposure levels below
the recommended level of 50
ppm. Urinary MA and PGA
presented graphically in
comparison with the
monitored air levels. Specific
Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275
Table 1 (continued )
Justification for excluding
the study from the meta-
analysis
possible to determine a
mean and standard
deviation that does not
include gaps.
This study must be
eliminated from the meta-
analysis because the number
of CAs reported is
extraordinarily high. It is
unclear whether gaps are
included in the total, but
from the values, it appears
that they were included. Not
possible to determine a
mean and standard
deviation that does not
include gaps. The variable
number of scored cells
indicates that there were
likely cell culture problems
and therefore, the overall
quality of the study must be
questioned. In addition, the
workers were also exposed
to organic peroxides,
solvents and dyes.
The CA data were reported
by group (low exposure,
high exposure and controls).
Only the means and the
ranges were reported. It is
not possible to determine a
standard deviation.
4
Lead Author, Brief Study Description Justification for excluding
publication year the study from the meta-
analysis
values per individual not
reported. The sum of the
metabolites was generally
correlated with the air levels
of styrene. There was no
exposure evaluated in the
control group. The control
group (n = 9) were sex, age
and smoking habit matched
and were office workers.
There was no difference
between the workers and
controls when the numbers
of CAs without gaps were
compared; there was a
difference when gaps were
included. Exposed: Low
exposure group: mean 1.1
(range 0− 4) CA/100 cells.
High exposure group: 1.4
(range 0− 2) CA/100 cells.
Workers overall: mean 1.2
(range 0− 4) CA/100 cells.
Comparisons: mean: 1.7
(range 0− 3) CA/100 cells.
Hogstedt 1984 Thirty-eight plastic factory The very long culture time
workers and 20 mechanical means that many/most of
factory workers in Sweden the cells scored would be
were evaluated. The age of past the first cell division. If
the 38 workers ranged from aberrations were induced,
19− 63 years (mean 38.7) they would have been lost,
and the styrene exposure resulting in much lower
ranged from 1 to 40 ppm assay sensitivity. Therefore,
(mean 13 ppm). This paper is this study was excluded.
a methods development
publication for the
micronucleus assay. The
focus of the paper was not to
evaluate the association
between styrene exposure
and CAs (or MNs). The cells
were treated with hypotonic
saline and 100 cells scored. It
appears that CAs were
scored in the same 96 -h
cultures that were used for
the micronucleus endpoint.
Individual results are
presented for both CA and
MN. The results of the
aberration analysis are
discussed in relation to the
micronucleus data. The
styrene exposed group
displayed a weak, but
statistically significant,
correlation between the MN
and CA frequencies.
Workers exposed to styrene
at two factories in the Czech
Republic were evaluated.
Both external and internal
exposure evaluated. External
Exposure: Factory 1:
manufacturing polystyrene Only the mean CA values
Pohlová and Sram containers for the food are reported. It is not
1985 industry lamination machine possible to determine a
operators had air levels of standard deviation.
151.6 mg/m3 (range
14–982.8) & manual
operations levels of 69.1 mg/
m3 (range 5.6− 377.5).
Factory 2: making boats and
plastic slides for children. air
(continued on next page)
J.J. Collins and M. Moore
Table 1 (continued )
Lead Author, Brief Study Description
publication year
levels of 96− 548 mg/m3 and
other operations 39− 60 mg/
m3.
Biomonitored Exposure:
Urinary MA in the workers:
Factory 1: ranged from 35 to
510 μg/L at the first
sampling time and between
88 to 972 μg/L at the second
sampling. Factory 2 ranged
from 40 to 1140 μg/L first
sampling and 200 to 3000
μg/L at the second sampling.
CA results:
Exposed: Factory 1:
Sampling time 1 (N = 36):
1.38 %. Sampling time 2 (N
= 34): Factory 2: 1.41 %.
Sampling time 1 (N = 22):
1.72% Sampling time 2 (N =
19): 2.81%.
Comparisons: Factory 1: a
single sampling time (N =
19) 1.26 %. Factory 2:
Sampling time 1 (N = 22):
1.36 % Sampling time 2 (N =
17): 1.88 %.
The authors concluded that
there was no increase in the
CA levels in workers
occupationally exposed to
the air styrene
concentrations used in the
study.
Jablonicka et al.
1988
Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275
Table 1 (continued )
Justification for excluding
the study from the meta-
analysis
Eleven female workers (ages
30− 55) exposed to styrene
during the lamination of
sport utensils, boats and
containers in
Czechoslovakia were
compared to 11 “healthy
persons”. The control group
was selected to be
comparable in age, social
habits and their working
environment, except that
they were not exposed to
styrene. It was noted that
the workers were almost
continuously exposed to
styrene vapors and had
direct skin contact with
styrene. Air concentrations
in the working area
averaged 253 mg/m3 and
ranged from 118− 582 mg/
m3. The workers did not use
PPE. The worker urinary
MA and PGA ranged
between 214− 711 mL/
μmole creatinine and
50–175 mL/μmole
creatinine, respectively.
There was no exposure
evaluation for the controls.
CA results: the mean CA of
the workers was 1.27% and
the mean CA for the
comparison group was
1.36%. The authors
concluded that there was no
difference in the CA
frequencies in the workers
and the controls.
5
Lead Author, Brief Study Description Justification for excluding
publication year the study from the meta-
analysis
The data were
presented as an
overall mean
number of CAs
per group. It is
not possible to
determine a
standard
deviation.
Forni et al. 1988 Styrene workers (n = 40 Only a mean was reported.
males) in two factories It is not possible to
located in the Milan determine a standard
hinterland in Italy. The first deviation.
factory was a manufacturer
of reinforced plastic
laminates and insulating
polymers. Exposure was high
prior to 1978 with TWA
ranging from 123− 249 mg/
m3. After 1978 the TWA was
1.7 to 17 mg/m3. The second
factory engaged in plastic
boat manufacture. After
1978 the TWA ranged from
41− 198 mg/m3. The study
was conducted in
1985− 1986. Urinary MA
was indicated as measured
but was not reported. The
external control group (N =
40 males) were matched for
age (within 5 years),
smoking habit and lived in
the same industrialized area.
The controls had no know
exposure to chromosome
damaging agents. There was
no exposure assessment for
the control workers. The
authors concluded that there
was a difference between
workers and controls. The
mean (no data on the
variability) for workers in
Factory 1 was 2.3% and for
controls was 1.6%. For
Factory 2 the workers were
2.5% and the controls 1.5%.
Oberheitmann et al. Fourteen boat builders were Because the FISH method
2001 compared to 7 employees of was used for scoring CAs, it
a wood manufacturing is not possible to compare
industry training center in these data with CAs scored
Germany. The methods were using the standard scoring
not described, however CAs method.
were evaluated using
florescent in situ
hybridization (FISH). The
authors concluded that there
was a slightly higher number
of exchange-type aberrations
in the exposed individuals
than in the controls. This
difference was not
statistically significant.
Helal and Elshafy Forty male plastic factory The number of cells scored
2012 workers were compared to (30, if the culture was
50 male administrative successful) is not sufficient
workers from the same to provide a useful estimate
factory in Egypt. of CAs. Therefore, this study
Lymphocytes were isolated cannot be included in the
after 3 days of whole blood meta-analysis.
culture. 30 cells were scored
(if the culture was
successful). They included
(continued on next page)
J.J. Collins and M. Moore
Table 1 (continued )
Lead Author, Brief Study Description Justification for excluding
publication year the study from the meta-
analysis
gaps, “iso-gaps” and “iso-
breaks” in the scoring. The
authors report a statistical
difference between the
workers and the controls.
because they were not done on humans, were only abstracts, had no
relevant data for chromosomal aberrations, were review papers or were
not in English. Thirty-one publications were identified which measured
chromosomal aberrations among workers exposed to styrene.
Table 1 lists and provides some details for the 13 publications which
had one or more deficiencies which preclude inclusion in the meta-
analysis. The specific reason(s) for eliminating these publications are
included in this Table and are briefly described here. The Finnish
workers’ study of 3 plants reported in two publications could not be
included because there was no way to separate out and exclude gaps,
aneuploid cells, polyploid cells and decondensation from the normally
scored chromosomal aberrations [20,21]. The results of the study in
Sweden by Hogstedt et al. are presented only graphically and we could
not determine the standard deviations of the exposed and the compar­
ison gruops [22]. The study conducted in Japan by Watanabe et al. was
eliminated because the metaphases scored were second division cells
and gaps were included in the chromosomal aberrations [23]. Two
publications reported on individuals from 9 Italian reinforced plastics
plants (ranging from 3− 10 workers per plant). The reported number of
chromosomal aberrations in both the workers and the controls was
extraordinarily high indicating that gaps were included in the scoring
[24,25]. Hansteen reported a study of a glass-fiber-reinforced polyester
plant conducted in Norway and reported chromosomal aberration data
only as a mean for the low exposure, high exposure and control groups
[26]. It was not possible to determine a standard deviation for the three
groups. A study conducted by Hogstedt in Sweden was a methods paper
for evaluating micronuclei and while chromosomal aberrations are
scored there are no means or standard deviations presented for exposed
and control workers and there was insufficient detail in methods used
for chromosomal aberrations, except it was clear the scoring was done at
96 h of culture [27]. Pohlová and Srám examined workers exposed to
styrene at two factories in the Czechoslovakia [24]. While the differ­
ences in chromosomal aberrations were reported for exposed workers
and controls, the standard deviations for each group were not reported.
Jablonicka et al. examined 11 female workers exposed to styrene during
the lamination of sport utensils, boats and containers in Czechoslovakia
compared to 11 “healthy persons” [28]. Only the mean with no standard
deviations of the chromosomal aberration frequencies for each group
were presented. The chromosomal aberration frequencies of 40 styrene
workers in two factories located in the Milan hinterland in Italy were
compared with 40 controls matched on age by Forni et al. [29]. The
mean chromosomal aberration frequencies for each group were re­
ported, however, the standard deviations were not. Fourteen boat
builders were compared to 7 employees of a wood manufacturing in­
dustry training center in Germany by Oberheitmann et al. [15]. It is not
possible to compare these data with chromosomal aberrations scored
using the standard scoring method because the FISH method was used
for scoring chromosomal alterations. A study done by Helal and Elshafy
in Egypt of 40 styrene exposed workers and 50 controls found differ­
ences in chromosomal aberrations for gaps, “iso-gaps” and “iso-breaks”,
however, only 30 cells were scored per individual if the culture was
successful [18]. Of the studies eliminated, one study reached no con­
clusions about whether their study determined styrene exposures were
related to chromosomal aberration frequencies [20], 6 studies found a
positive relationship [15,18,22,24,27,29], and 4 studies found a nega­
tive relationship [23,26,28,30].
6
Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275
Eighteen publications including 21 groups of workers with styrene
exposures compared to a comparison group were selected for the meta-
analysis and are summarized in Table 2 [31–48]. When multiple pub­
lications were available on single study, only the first publication is
listed in Table 2. The study of Thiess and Fleig [32] is also referenced in
two additional studies [33,34]. The exposures in the Watanabe et al.
study [37] were also discussed in another study [23]. The study of
Artuso et al. does not present data for the total exposed population but
rather for high and low styrene exposures [43]. The study of Sorsa et al.
does not present all exposed workers but rather high and low styrene
exposed lamination workers and high and low exposed “other workers”
[48]. We treat each of these sub categories of these two studies as if it
were separate studies in the meta-analysis.
All studies included in the meta-analysis were cross-sectional, but 7
of these studies used some form of matching on one or more of these
characteristics potentially associated with variability in the level of
chromosomal aberrations [31,35–37,41–43].
Most of the studies selected for the meta-analyses provided estimates
of exposure to styrene including air monitoring or biomonitoring and in
some cases both. Studies or subgroups of studies were assigned to the
high styrene exposed category when either the mean air concentration
exceeded the ACGIH 2003 standard of 20 ppm (87 mg/m3) or 400 mg/g
of mandelic plus phenyglyoxylic acids (MA + PGA) creatinine for bio­
monitored exposures [49]. We converted industrial hygiene air moni­
toring reported in mg/m3 to ppm. We divided the studies and sometimes
exposure subgroups within the studies into low if the mean or median
styrene exposure based on air monitoring estimates were below ACGIH
standard and high if they exceeded this standard. Because there was no
exposure data presented in the Biro et al. study, we could not classify it
as either high or low exposure. The study of Anwar and Shamy only
reported biomonitoring results and we converted the results to ppm
using the study of Choi et al. [50]. Seven studies were wholly classified
in the high category [35–37,39,41,46,47]. Also included in the high
styrene exposed category was part of the Thiess and Fleig study [32],
part of Tomanin et al. study [42], part of Anwar and Shamy [31], part of
the Artuso et al. study [43] and part of the Sorsa et al. study [48]. The
low styrene exposed group included all remaining studies or parts of
studies. Fig. 2 shows the range of industrial hygiene monitoring for
styrene exposure from the each of the studies on Table 1. Studies are
shown in chronological order and studies or parts of studies are identi­
fied based on our classification as low or high exposure. Three of the
very early studies show a larger range of exposures compared to later
studies. Almost all studies, including those with high exposure mean/­
median values, have exposure ranges that include some low (below 20
ppm) exposure values. Likewise, some of the low exposure studies have
ranges that extend above 20 ppm. This overlap between the study ranges
indicates that our classification into low and high exposure most likely
has limitations.
Six studies examined exposure response among their study subjects
as shown in Table 2. Fleig examined three manufacturing plants, two of
which, the polystyrene plant and the monostyrene plants, had relatively
high styrene exposures [32–34]. This study employed a cross-sectional
unmatched study design to examine three manufacturing plants. The
polystyrene plant with 12 workers and the monostyrene plant with 14
workers reported exposures from 50 to 300 ppm. The styrene
manufacturing plant had relatively low styrene exposure levels with
exposures less than 1 ppm with some occasional excursions up to 70 ppm
among 5 workers [32–34]. The monostyrene plant had chromosomal
aberrations of 5.4 %, the polystyrene plant 1.9 % and the styrene
manufacturing plant 1.1 %. The comparison group from the same fac­
tory not exposed to styrene had chromosomal aberrations levels of 1.9
%. Andersson et al. examined workers at a boat manufacturing plant and
divided workers into low and high exposed groups based on air moni­
toring by job [36]. The low exposed group of 22 workers had mean air
exposure of 31.5 ppm (range 1.4–283) and the high exposed group of 14
workers had a mean of 276.8 ppm (range 163.2–365.2). The comparison
J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2
Studies included in the Meta-Analysis.
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

(1) Fleig 1978; Exposed: 3 different External Exposure: Peripheral blood All males of many Low Exposure Cell cultures were grown for
Unmatched Plants: 1) styrene lymphocyte cultures ages. Participants 1) Styrene 70− 72 hours. This would result
Cross- 1) styrene manufacturing plant grown for 70− 72 h, screened for manufacturing plant: in loss of sensitivity as many
sectional; manufacturing plant; making reinforced 100 metaphases occupational Exposed: n = 5, mean cells would be in second
Germany n = 5, ages from resins and corrugated scored per individual. exposure to any other = 1.6, SD = 1.1% division. The number of
33− 53 (mean = 42.8) sheets: Exposure less agent know to induce (range 0-3%) Defined workers in plant 1 (n = 5) is
and Exposure than 1.0 ppm with chromosome as low exposure very low. There was some
duration from 14–25 occasional excursions aberration, However, Comparison: Data for exposure to other chemicals.
years (mean = 21.6). of 70 ppm. (classified smoking status not the controls only Smoking status was not
2) polystyrene plant; as Low Exposure) reported. Co- presented as a mean = reported.
n = 12, ages from 2) polystyrene plant exposures to 2.1%indicated in text
22− 62 (mean = 44.9) making glass methylene chloride, that the range was
and exposure reinforced epoxy acetone and other between 1− 9%.
duration from 3− 39 moldings: Working solvents, some 2) polystyrene plant:
years (mean = 20.3). area concentrations of exposed to epoxide Exposed: N = 12,
3) processing styrene evaluated 8 resins and hardeners. mean = 1.917%,
unsaturated polyester times in 9 different Also, some exposure (calculated) SD =
resins (monostyrene); areas. Range from to styrene oxide. 0.9% (range = 0− 3%).
n = 14, 3 plants, ages 0.02 ppm to 46.92 3) processing
from 29− 52 (mean = ppm. Only 2 of the 9 unsaturated polyester
41) & exposure areas show any values resins plant:
duration from 2− 24 above 1 ppm. Exposed:
years (mean = 7.9) Exposure at various N = 14, Mean = 5.4 +
Comparisons: n = 20 points in plant 2.2 % (range =
from the same factory 50− 300 ppm. 3− 10%)
not exposed. Mean (classified as High High Exposed Group
age 46.6; not known Exposure) Polystyrene and
to be exposed to 3) processing monostyrene plants
styrene or any other unsaturated polyester combined (calculated)
chromosome resins plants making N = 26, mean = 3.769;
damaging agent (e.g. reinforced epoxy SD = 2.438 (Defined
clerical staff). resins: indicated as as high)
measured but the data All 3 Exposed Groups
were not included. N = 31, mean = 3.419,
Exposure at various SD = 2.405.
points in plant Comparison: Data for
50− 300 ppm. the 20 controls only
(classified as High presented as a mean =
Exposure) 2.1%. indicated in the
Biomonitored text that the range was
Exposure: urinary between 1− 9%.
MA Data for 12 of the
1) styrene controls. Mean =
manufacturing plant: 1.916 + 1.37 % (range
only in 2 of 5 = 0− 4%).
individuals; values of Comparison: Data for
19 and 40 mg/L urine. the controls only
2) polystyrene presented as a mean =
manufacturing plant: 2.1%. indicated in the
measured in 8 of 12 text that the range was
individuals; range between 1− 9%.
<5− 100 mg/L urine. Authors’
3) processing interpretation: No
unsaturated polyester induction of CA for the
resins plants: all styrene manufacturing
individuals; range and processing plants.
102–>1500 The levels of MA were
urine. low (low exposure).
Exposure among There was a different
comparisons: in the CA % for
Not measured workers in the plants
processing
unsaturated polyester
resins.
(2) Thiess, Exposed: Individuals External Exposure: Peripheral Some of the controls Exposed: N = 24, Cells were grown for 70− 72
1980; Cross- from two different Measured June, July lymphocyte cultures were plant mean = 1.917%, SD = hours, thus resulting in many
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7
J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

sectional work sites. Pilot plant and August 1978. Air grown for 70− 72 h. maintenance 1.381 %; range 0− 5% cells being in 2 or more
matched on and laboratory for levels and personal 100 metaphases workers. None of the Comparisons: N = 24, divisions. This results in lack of
age and process development breathing area scored. Samples were controls were mean = 1.541%, SD = sensitivity. Note that the
years of (n = 24). Ages ranging from 0.7 ppm process exposed to radiation 1.215%; range 0− 4% workers with the “higher”
exposure; 23− 59. to 178 ppm (mean simultaneously and or genotoxic Authors’ levels of aberrations did not
Germany Comparisons: 58.1 ppm) in the pilot coded. suspected chemicals. Interpretation: no have higher levels of urinary
Employees of the plant and 1.0 Smoking habits, evidence that MA. 3 of the 4 were from the
Occupational ppm–11.5 ppm (mean alcohol intake, viral exposure to styrene at laboratory where the exposure
Medicine and Health 6.0 ppm) in the diseases and drug use an average level of 58 to styrene was only between
Protection laboratory. Parallel were determined but ppm induces CA. 1− 11.5 ppm. No exposure
Department and plant measurements using a no data were assessment for the controls.
maintenance workers Drager tube reported. There is no Smoking status not reported.
(not exposed to monostyrene 10/a information on the
styrene) (24 and 50/a found ages of the controls.
individuals). individual values Sex of the
(pinpoint measures) participants are not
of <10 to 400 ppm. reported for either
Average styrene group. Unclear how
concentration 58.1 well the exposed and
ppm. No styrene oxide comparison groups
was detected. are matched.
(classified as High
Exposure)
Biomonitored
Exposure: Urinary
MA measured in all
but 2 of the workers.
19 workers had levels
between 0− 80 mg/L
urine; 3 workers
between 130− 32 mg/
L. Data were
presented for 4
workers who had
“higher” levels of
aberrations. MA levels
in these 4 individuals
were 50, 30, 20 & 30
mg/L urine.
Exposure among
comparisons: not
mentioned.
(3) Andersson, Exposed: External Exposure: Blood samples were Health histories were Note results are CA Culture conditions may have
1980; Cross- (39 males, 36 were Boat manufacture taken in 1978. taken on the day of per 100 cells. been suboptimal based on the
sectional evaluated for with fiberglass Lymphocyte cultures blood draw, but (Table 4) comment that cells were grown
matched on chromosome reinforced plastic. grown for 66− 68 h in results not reported. Exposed: Total (N = longer to obtain enough
age and sex; aberrations). Ages Breathing zone; order to get enough Controls matched 36); mean = 7.9: SD = dividing cells to score. Some
Sweden ranged from 20− 58. measured several dividing cells to with the exposed 2.2 CA/100 cells. Low cells were in second division.
25 smokers. times and for different score; many cells in based on age. exposure group (N = The presence of dicentrics and
Comparisons: processes in the boat second division, 100 Authors mention 22): mean = 8.0 + 2.3 rings from both the exposed
Workers in the same making production metaphases exposure to other CA/100 cells. High and control groups, calls the
plant, but not between 1973 and evaluated from coded chemicals in the exposure group (N = scoring criteria into question.
exposed, worked in 1978. slides. Results were workplace, but do 14): mean = 7.8 + 2.1 These types of events have not
the assembly shop, For some workers a reported as not specify. Smoking CA/100 cells. been observed in in vitro
the workshop or cumulated exposure chromatid status not reported. Comparisons: N = 37: styrene studies (Henderson and
office (41 M, 37 were was estimated. Based aberrations (breaks, mean = 3.2, SD = 1.9 Speit, 2005). The exposure
evaluated for on calculated minutes and others) CA/100 cells assessment was an estimate
chromosome accumulated dose and chromosomal Authors’ over several years, therefore,
aberrations). Ages over the period from aberrations Interpretation: not necessarily reflective of the
ranged from 18− 65. 1973− 1978, low and (dicentrics/rings and Authors conclude that exposure level at time of
12 smokers. high exposure groups acentric fragments). there was a difference sampling. No difference in the
were established. The Results reported as between the styrene observed between the low
low group (N = 23) aberrations per 100 exposed workers and exposure and high exposure
was estimated as 31.5 cells. controls. They saw no groups. Authors mention
ppm (range 1.4–65.0) difference in the level potential exposure to other
and high group (N = of CAs between the chemicals. Data presented for
16) as 276.8 ppm low exposure and high smokers and non-smokers
(range 163.2− 365.2) exposure groups. indicate that the 25 control
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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

Both low and high Within the low individuals that were non-
grouping classified exposure group, they smokers had a higher mean
as high indicate an increase in level of CAs per 100 cells than
Biomonitored the frequency of CA the smokers (3.64 + 2.03 vs.
Exposure: not with increasing 2.33 + 1.15). For the exposed
evaluated exposure to styrene (r workers the means were
Exposure among = 0.576). No dose similar. Non-smokers (N = 11)
comparisons: response within the mean: 8.36 + 2.57 vs smokers
Unclear whether any high exposure group. (N = 25) mean: 7.68 + 2.01.
measurements were
made
(4) Watanabe, Exposed: Two External Exposure: Whole blood cultures, The workers were Table 1. Exposed: N = Small number of
1983; Cross- factories (18 males; Two boat building 24 h after initiation of stated as not having 18; mean = 1.12%; SD individuals—particularly in the
sectional 10 smokers). Ages factories. Personal culture BrdUrd was exposure to other = 1.13%; range 0− 3% control group. There is a wide
frequency between 16− 71 with exposure levels added. After 50 h industrial chemicals. Comparisons: N = 6; age range for the workers
matched on a mean of 46.7. estimated to be culture cells All participants were mean = 1.07%; SD = (between 16− 71). Stated that
age; Japan Comparisons: 40− 50 ppm TWA. harvested and interviewed for their 0.65%; range 0–2% the controls were age matched
Individuals not These data were “about” 100 M1 cells occupational and Authors’ but that would be difficult to
exposed to styrene (6 referenced from scored using blind medical histories (no Interpretation: accomplish for such a small
males; 3 smokers). another publication coded slides. data reported except A marginal difference number (6) of controls; ages not
Indicated as age by the same research for smoking). between the exposed presented for controls. Large
matched but the group. Smoking was a and controls for CAs in range in the levels of
specific data not (classified as High potential first-division biomarkers in the workers.
presented. Exposure) confounder. The data metaphases when gaps Smoking is a confounder. There
Biomonitored are presented by were included but no was no association between the
Exposure: Workers: individual and the difference when gaps level of urinary biomarkers of
Urinary MA ranged average daily were excluded. Cell exposure and the %CAs. In fact,
from 0− 1041 μg/mL. number of cigarettes proliferation was the 6 workers with the highest
Mean = 332 + 397. presented by inhibited in the %CAs (2− 3%) had relatively
Urinary PGA ranged individual. styrene exposed low levels of MA (0− 466 μg/
from 1 to 358 μg/mL. workers. mL) and PGA (1− 111 μg/mL).
Mean = 76 + 98.
Exposure among
comparisons: Not
evaluated
(5) Nordenson, Exposed: (15 males; External Exposure: Lymphocyte cultures Individual data Note results were Small numbers; Gaps were also
1984; Cross- 4 smokers). Ages Factory using grown for 64− 68 h. presented for age, presented as CA per scored and the authors stated
sectional ranged from 21− 61. polyester reinforced 200 cells scored. smoking status, 200 cells—We that there was no difference in
with no Comparisons: glass fiber, coating duration of exposure calculated the mean the frequency of gaps between
matching; Nearby industry, no and spraying styrene to styrene. It was and SD the workers and the controls.
Sweden occupational compounds. Air noted that workers Calculated from
exposure to styrene or concentration mean were also exposed to Table 1
other solvents, office was 24 ppm. acetone but not to Exposed: N = 15;
clerks and salesman (classified as Low other solvents. mean = 0.800; SD =
(13 males; 3 Exposure) 1.014; CA/100 cells
smokers). Ages range Biomonitored Comparisons: N = 13;
from 26− 53. Exposure: Urinary mean = 0.692; SD =
MA reported as “low” 1.251; CA/100 cells
below 1 mmol/L and Authors’
none above 2 mmol/L Interpretation: No
in the workers. No difference in the
individual data number of CAs either
presented. with or without gaps
Exposure among in the workers and
comparisons: not controls.
evaluated
(6) Maki- Exposed: (21 External Exposure: Lymphocyte cultures Workers and controls Table 1 and text Small number of subjects, no
Paakkanen, workers; 15 smokers). Reinforced plastic grown for 50 h, 100 matched for sex and Exposed: N = 21; exposure assessment of the
1987; Cross- Mean age of the workers. Individual metaphases scored smoking. All subjects mean = 3.0; SD = controls. A relatively large
sectional smokers was 33.2 and exposure for 10 of the were interviewed for 0.3%. number in both groups were
with no mean age of the non- workers ranged from vaccinations, recent Comparisons: N = 21; smokers. Long term exposure
matching; smokers was 39.3 7.8 to 60.5 ppm in air viral infections, drug mean¼3.7; SD = 0.5 assessment in the workers. Air
Finland with an overall mean with a mean of 22.5 and alcohol use and Authors’ levels could occasionally be as
age of 35. ppm. occupational Interpretation: high as 630 mg/m3. This would
Comparisons: Office Biomonitored exposures. The No difference between exceed the highest
workers (21 controls; Exposure: Urinary authors indicate no workers and controls. recommended average
15 smokers). Mean MA (determined for differences in the exposure level of 210 mg/m3 in
age of the smokers, all workers) ranged groups in these
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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

non-smokers and from 0 to 7 mM /L factors—data not place in Finland at the time of

overall control group urine. Mean of 1.6 ± presented. the study.
was 39.8. 0.4. (Exposures to
styrene were low).
Urinary MA levels had
been monitored over
6 years with a mean of
the averages of 21
measurements 2.04 +
0.4 mM/L urine and a
range of 0.00− 7.25
mM/L urine.
(classified as High
Exposure)
Exposure among
comparisons: not
evaluated
(7) Hagmar, Exposed: (19 males & External Exposure: Whole blood cultures Smoking Table 3 Potential confounding
1989; Cross- 1 female; 6 smokers). Glass-reinforced grown for 48 h. 100 Acetone, methylene Exposed: N = 11. occupational exposure. Small
sectional Mean age 40.5 (range plastics plant. Mean cells scored per chloride (both 1.2% + 1.6 (range number of individuals after
with no 18− 64) concentration during individual. Poor reported as “low 0− 3). many of the samples could not
matching; Comparisons: (22 1985− 1986 was 12.8 preparation resulted exposures”. The Comparisons: N = 15. be scored. No measure of
Sweden males; 11 smokers). ppm. Ranges during in 9 of the workers measured range for 1.5% + 2.1 (range internal dose in either workers
Mean age 42.3 (range this period were from and 7 controls not acetone in 0− 5). or controls. Smoking may
23− 61). 1 to 37.7 ppm. Noted scored. (5 of the 11 1985–1986 was 15- Authors’ confound the interpretation.
that the Swedish workers who were 137 mg/m3. The Interpretation: The was a large age range in
occupational limit at evaluated were range for methylene Authors report no both workers and controls,
this time was 25.3 smokers and 9 of the chloride was 0-48 statistically significant unclear as to the range of ages
ppm. Workers used controls were mg/m3. Of the difference between for the individuals evaluated
respiratory protection smokers). exposed, 13 had exposed and controls. for CAs.
during spraying radiographic
operations and when examinations during
working with the previous year and
polyester inside the three regularly took
tank, otherwise they prescription drugs.
did not use protection. Of the controls 12
Biomonitored had been exposed to
Exposure: 43 urine radiographic exams
samples measured MA and 5 regularly took
which averaged 128 prescription drugs.
(range 6− 317)
(classified as Low
Exposure)
Exposure among
comparisons: Gelatin
production plant; no
occupational styrene
exposure. (“no
significant chemical
exposure”)
(8) Maki- Exposed: 17 workers; External Exposure: Lymphocyte cultures Factors that were Table 1 and text Small number of subjects. No
Paakkanen, mean age = 33.0; Reinforced plastics grown for 50 h. 100 considered “to Exposed: N = 17; actual external measure of
1991; Cross- Exposure length = 6.7 plant metaphases scored. exclude the effect of mean = 3.0%; SD = exposure-estimated from the
sectional years; Smoking status Not measured but Cultures from such variables on the 2.1%. biomarker data. No measure of
with (11 smokers, 6 non- estimated to be about workers and controls results”: age, sex, Comparisons: N = 17; exposure for the controls. No
matching on smokers). 68.9 ppm of styrene were processed at the smoking, recent viral mean¼3.1%; SD = assessment of confounding
smoking; Comparisons: 17 based on the styrene same time. Slides infections, 1.5%. exposures in controls. A high
Finland; workers at research glycol levels in blood. were blind coded and vaccinations, alcohol Authors’ percentage in both groups were
institute; mean age (classified as High scored by a single and drug intake, Interpretation: smokers.
34.9; Smoking (11 Exposure) individual. health status, Authors report a
smokers, 6 non- Biomonitored exposure to possible difference between
smokers). Exposure: Urinary mutagenic non-smoking workers
MA (mmol/L) = 9.4 + chemicals. The sex of and non-smoking
6.4 (range 1− 21.5): the workers and controls. However,
smokers = 11.0 + 6.3 controls is not this was only the case
(range 1− 21.5), non- presented and when gaps were
smokers = 6.5 + 6.0 information on included. There was
(range 1− 16.6). smoking and other
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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

Blood styrene glycol factors is not no difference when

(mmol/L) = 2.5 + 1.5 reported. gaps were excluded.
(range <0.6− 6.3):
smokers = 2.9 + 1.6
(range <0.6− 6.3),
non-smokers = 1.8 +
1.3 (range 0.9− 4.0)
Exposure among
comparisons: not
evaluated
(9,101,112) Exposed: 32 small External Exposure: It is unclear how Age and smoking. Table 3 The paper included a much
Sorsa 1991; workshops building The mean TWA 8 h many of the workers Age was associated Laminators high: N = larger number of workers than
Cross- boats selected to concentration of evaluated for CA with CA. 13, mean = 0.7, SD = were included in the CA part of
sectional; no include various styrene in the were in each of the 0.4 the study. A relatively large
matching: production methods personal air was 43 occupational groups. Laminators low: N = number of workers, but unclear
Finland (N = 39 workers). ppm (range 1− 182 100 metaphases 15, mean = 0.6, SD = as to how many of the workers
Controls: 31 workers ppm) for the scored. 0.5 evaluated for CA were in each
not exposed to laminators Classified Other workers high: N of the occupational groups.
styrene. Urinary as high = 6, mean = 1.0, SD =
metabolites and 11 ppm (range 1− 133 0.2
respiratory zone ppm) for the other Other workers low: N
styrene levels were workers. Classified as = 5, mean = 0.4, DS =
evaluated and low. 0.3
presented as means Biomonitored Control: N = 31, mean
and standard Exposure: All = 0.8, SD = 0.5
deviations. laminators: urinary Authors
MA + PGA = 2,2 Interpretation: The
mmol/l. authors conducted a
Controls: No regression analysis for
measurement of any potential dose-
styrene exposure. response relationship
and reported the
response to be
negative. They also
calculated an exposure
index based on time
working in reinforced
plastic work, the type
of job and the
measured mandelic
acid concentrations.
Laminators and other
workers were
separated into two
exposure bands and
compared with
controls. CA were not
associated with
styrene exposures.
(13) Tomanin, Exposed: Two plants: External Exposure: 2 Whole blood cultures Participants were Table 2 with Small number of participants
1992; Cross- Factory 1: small plants with grown for 48 h. Slides evaluated for calculations when the results were
sectional fiberglass tank Reinforced plastics were blind coded and previous exposure to Exposed: Factory 1: N evaluated separately for the
with production (7 males; applications; scored by a single other genotoxic = 7; mean = 1.43%; two factories. Exposure not
matching on 3 smokers). Mean age personal air monitors individual. An agents, alcohol and SD = 1.72% (range assessed in the controls. While
age, sex and 29.4 (range 20− 37). concentration: average of 100 drug intake, 0–5%). Factory 2: N = the % CAs were higher in the
smoking; For the 3 smokers: g/ Factory 1: range metaphases scored diagnostic X-ray, 11; mean = 3.02 %; SD exposed from the 2nd plant, the
Italy day tobacco mean = 4.8− 23.0 ppm. per individual. recent viral = 2.28% (range of controls for that plant had
17 (range 10− 20). (classified as Low Because of technical infections and 0− 6%). Total: N = 18; relatively low levels of %
Factory 2: fiberglass Exposure) problems CAs could vaccinations (data mean = 2.399%; SD = aberrations. The authors
boats (5 males & 7 Factory 2: range not be evaluated in 1 not reported). 2.176% (range 0–6%). presented their historical
females; 7 smokers). 112− 435 mg/m3. worker from factory Individual data Comparisons: control data for CAs. Based on
Mean age 37.2 (range (classified as High 2. presented for sex, Factory 1: N = 7; mean results from 99 subjects, the
22− 49). For the 7 Exposure) age, tobacco use per = 1.43%; SD = 0.98%; 99% confidence limit of their
smokers: g/day Biomonitored day, length of range (0− 3 %). historical controls was 2.7%
tobacco mean = 14 Exposure: Urinary exposure, urinary Factory 2: N = 11; with a mean of 0.69%. A high
(range 8− 20). MA: Factory 1: mean MA, and CAs. mean = 0.82%; SD = percentage of individuals from
Exposure length: = 186 + 114 mg/g Controls matched to 0.75% (range of the second plant in both groups
Factory 1: mean 9 creatinine. range cases on sex, age and 0− 2%. Total: N = 18; were smokers. The workers and
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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

(range 1− 18) years. 46− 345 mg/g smoking habits. Note mean = 1.056%; SD = controls were matched not just
Factory 2: mean 6.2 creatinine. Factory 2: that in factory 1 the 0.8726% (range for the number of smokers but
(range 1.5− 15) years. mean = 725 + 329 workers were all 0–3%). also for tobacco used per day.
Comparisons: mg/g creatinine range male and the controls Authors’
“healthy workers” not 423− 1325 mg/g were 5 males & 2 Interpretation: There
exposed to known creatinine. females. In factory 2 was no difference in
genotoxicants; Exposure among workers were 5 males CA between workers
Matched on sex, age comparisons: not & 7 females and the and controls for
and smoking habits mentioned controls were 4 males factory 1 but there was
Factory 1: (5 males & & 8 females. For both a difference for factory
2 females; 3 smokers). factories the workers 2. This correlated with
Age mean 31.1 (range and controls were the fact that factory 2
25− 36). For the 3 matched for amount had a higher worker
smokers: g/day of tobacco used per exposure compared to
tobacco mean = 16 day. factory 1. There was
(range 8− 20). no impact of smoking
Factory 2: (4 males & on the CA results, but
8 females; 7 smokers). the authors note the
Mean age: 36.9 small number of
(range 26− 48). For individuals making
the 7 smokers: g/day this evaluation
tobacco mean = 16 difficult.
(range 10− 25).
(14) Anwar, Exposed: 18 males; External Exposure: Two cultures per Matched on sex, age Table 2 This study is important because
1995; Cross- mean working years Workers employed in blood sample, grown and smoking habits Exposed: N = 18; all individuals were non-
sectional = 14.33 (range = plant manufacturing for 48 h, 50 to 100 (none reported ever mean = 4.00%; SD = smokers. The data are
with 5− 22); Age = 40.61 reinforced plastics; air cells scored smoking). The 3.85% (range 0− 13) presented by individual. It
matching on (range = 31− 55); all monitoring not depending upon controls were stated Comparisons: N = 18; should be noted that this paper
age, sex and reported being non- mentioned. quality of to be healthy and not mean = 1.44 %; SD = includes data from a pilot
smoking; smokers. Biomonitored preparation. Slides exposed to known 2.83 % (range 0− 5) exposure study and therefore
Egypt; Comparisons: 18 Exposure: Urinary were coded and genotoxicants. Calculated from the exposure data presented in
males matched samples collected on scored by a single Table 2 using this summary is only from the
administrative Wednesday individual. It should biomonitored results individuals who were
workers; mean afternoon. Urinary be noted that the Low exposure evaluated for CA. The control
working years not MA: mean = 328.44 + blood samples for the N = 15, mean = 3.933, individuals, who worked in the
reported; age = 41.06 266.21 (range = controls were taken a SD = 4.200 same plant as the “styrene
(range = 26− 55); all 145− 1204) mg/g week after the Matched control exposed” workers, had low
reported being non- creatinine. Urinary samples for the N = 15, mean = 1.267, levels of styrene exposure (but
smokers. thioethers (used as a workers. SD = 1.830 much less than the workers).
. biomarker for High Exposure While these controls showed
electrophilic N = 3, mean = 4.333, exposure to styrene, the CA
compounds): mean = SD = 1.527 levels reported are in line with
83.71 + 40.33 (range Control Group (not higher than) the CA levels
= 36.9− 202) μmol/ N = 3, mean = 2.333, from controls in other studies.
mmol creatinine. SD = 0.577 No significant correlation
(individual Authors’ found with urinary MA level,
biomonitoring data interpretation: urinary thioether level and CAs
in Table 2 in report Urinary MA was The number of individuals in
used to classify significantly higher in the study was small. Very large
workers as high or workers than controls. variability in the urinary MA in
low exposure. 15 in Authors concluded the workers. Scored only
low exposure group that there was a between 50− 100 cells based on
and 3 in high difference in the the “quality of the
exposure group) frequency of preparation”. The fact that
Exposure among chromosome preparation quality would
controls: Urinary aberrations between restrict the number of cells
samples collected on workers and controls. scored is problematic and casts
Wednesday There were no doubt on the results. No dose
afternoon. Urinary correlations between response was observed.
MA: mean = 50.09 + the duration of Because all individuals were
12.84 (range = exposure, the level of non-smokers, smoking was not
21.9− 91.7) mg/g urinary thioether, a confounder.
creatinine. Urinary urinary MA and the
thioethers (used as a level of CAs.
biomarker for
electrophilic
compounds): mean =
38.32 + 2.28 (range =
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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

21.5− 63.4) μmol/

mmol creatinine.
(1516) Artuso, Exposed: (50 males, Boat building Samples were taken All participants Table 3: Data are # of Large study. Cultures were
1995; Cross- 45.4% smokers) fiberglass reinforced over a period of two completed a CA per 100 cells with grown for 72 h which means
sectional Two exposure groups plastics months and questionnaire for standard error; that many cells were in second
with evaluated for CA (low External exposure: alternatively from personal data, Convert SEM to SD. (or even third) division. There
frequency n = 23 and high n = Breathing zone. exposed and controls. working activity, Exposed: High was no assessment of internal
matching on 23). Workers who spent at Whole blood cultures. recent illnesses, X- exposure group: N = exposure for the workers and
age and Comparisons: least 20% time gel Metaphases rays within the past 5 23; mean 4.0; SD = no exposure assessment for the
smoking; Residents of the same coating exposed to a harvested after 72 h years, use of alcohol, 2.514 CA per 100 controls. Diagnostic irradiation
Italy area not range of 80.6− 319.3 culture. Two different drugs, coffee and cells. Low exposure might be a confounder; the % of
occupationally ppm. Workers labs involved in the smoking habits. Note group: N = 22; mean = people with diagnostic X-rays
exposed to spending at least 50% scoring. One lab in that 60.9% of the 2.76; SD = 1.9183. CA was high—there is no data to
genotoxicants (55 time laminating, Florence received high exposure group per 100 cells. clarify the timing of these X-
males; 45.1% rolling and assembly samples from 13 were smokers. 90% Comparisons: N = 34; rays in relation to the time of
smokers). exposed to a range workers and 13 of the controls and mean = 2.13; SD = blood sampling. Smoking may
between 19.8–151.7 controls. A lab in 70% of the workers 1.5744CA per 100 be a confounder. Taking
ppm. Workers with Genoa received had diagnostic cells. samples over a period of time
other activities in the samples from 42 irradiation within the Authors’ and shipping to two different
laminating area controls and 37 past 5 years interpretation: labs where there was a total of 3
exposed to a range exposed. The slides Authors report a scorers is not an ideal design.
between 0− 27.6 ppm. were blind coded and statistical difference There were more people who
23 workers in the two there were 2 scorers between controls and had diagnostic X-rays in the
higher exposure in the Genoa lab and the high exposure control group than the worker
groups were classified 1 scorer in the group. They also group and there were more
as the high exposure Florence lab. 150 report a positive dose smokers in the high exposure
group and 23 workers cells were scored for response trend. The group than the low exposure
in the low exposure everyone for number of CAs group. The authors note that
group. chromosome reported was higher this difference in smoking was
Workers in Gel aberrations. Four for both controls and at least partially compensated
coating (classified as cultures from the workers in the slides for when the number of
High Exposure) workers and four scored in lab 1 than in cigarettes smoked per day was
Workers in other from the controls lab 2. The data are not factored in. The authors
activities (classified “failed for lack of presented by lab, but concluded that smoking,
as Low Exposure) proper growth the authors state that alcohol use and diagnostic X-
Internal exposure: stimulation”. Thus, this did not alter the rays were not associated with
Not evaluated there were 46 observation that CAs CA level. However, styrene
Exposure in workers and 51 were higher in the exposure was.
unexposed controls for which high exposure group
comparisons: not CAs were scored. than the controls.
evaluated
(17) Exposed: 3 groups External exposure: Whole blood cultures All individuals Unclear as to whether The exposure level for group 1,
Smorovska, based on exposure: Mean workplace grown for 48 h. Slides interviewed for the Data are CAs per based on both external and
1999; Cross- (1) high exposure: levels by exposure were coded. 100 smoking habits, 100 cells (as stated in internal evaluation was much
sectional hand laminators (17 Group 1: 45.7 ppm ± metaphases scored alcohol consumption the footnote to the higher than that for the other
with no W; 3 smokers; mean 23.2, per individual. and medications. The table) or as % cells two groups. There was
matching; age of 40). (2) (classified as High Scoring was done by high exposure group with aberrations—as measurable styrene in the
Netherlands medium exposure: Exposure) two cytogeneticists. was all female, with indicated in the text. blood and exhaled air for the
sprayers (10 M & 2 F; Group 2: 12.6 ppm ± only 3 smokers. The Tables 1 and 2 controls and these were not
7 smokers; mean age 5.3. other two exposure Exposed: (High) n = dramatically different than the
of 38). (3) low (classified as Low groups had both 17, mean = 3.75, Sd = internal exposure measures in
exposure: Exposure) males and females. 1.13, (low)n = 12, the two lower exposure groups.
maintenance workers Group 3: 6.3 ppm ± The control group mean = 2.50, SD = There was no dose response for
(5 M & 10 F; 6 5.8. was close to an even 0.85, (Low) n = 15, the 3 different levels of
smokers; mean age of (classified as Low number of males and mean = 3.27, SD = exposure. There was a
36). Exposure) females. Overall the 0.70, Total n = 44, statistical difference between
Comparisons: Clerks Internal exposure: individual exposure mean = 3.27, SD = the exposed and the unexposed
in the same factory (8 Blood levels of styrene groups were not well 1.02 for each of the exposure level
M & 11 F; 8 smokers; (μg/L). By exposure matched to the Comparisons: groups as compared to the
mean age of 40). group: (1) 2098.2 ± controls in terms of Controls: n = 19, mean controls. The authors noted
1330.8, (2) 81.3 ± male/female and = 1.37, SD = 0.83. that the proliferative response
80.3, (3) 84.5 ± 81.9. smoker/non-smoker. Authors’ of the stimulated T-
Styrene in exhaled air interpretation: lymphocytes was significantly
(μg/L): by exposure Although there was no suppressed in the styrene
group: (1) 87.1 ± dose response, there exposed individuals
53.9, (2) 2.5 ± 3.6, (3) was a statistical
7.0 ± 6.4. difference between the
Exposure in
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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

unexposed workers and the

comparisons: No controls.
external exposure.
Internal exposure:
Blood levels of styrene
(μg/L): 39.0 ± 84.
Styrene in exhaled air
(μg/L): 1.3 ± 3.2.
(18) Biro, Exposed: 10 workers External exposure: Whole blood grown All individuals were Data presented as % The exposed & the control
2002; Cross- exposed to styrene in Not evaluated for 50 h. Slides were interviewed for aberrant cells + S.E. group were not well matched
sectional a refinery (8 males; 8 Internal exposure: blind coded. 100 smoking and Table 1 with regard to number male/
with no smokers; mean age = Not evaluated metaphases scored drinking habits, age, Exposed: N = 10, female and smoking status. No
matching; 42). Exposure in per individual. medications, medical mean = 3.8, SD = 1.0. exposure assessment. Higher
Hungary Comparisons: unexposed and work histories. Comparisons: N = 25, proportion of smokers in the
25 Individuals comparisons: Not The groups are not mean = 1.8, SD = 0.4 styrene group than the control
defined as “not evaluated well matched for # of Authors’ group. The study was designed
exposed” (5 males; 5 individuals, male/ interpretation: to evaluate the relationship
smokers; mean age = female and smoking Although the means between immunotoxicity of
38). status. were different, the several solvents (including
difference was not styrene) and genotoxicity
statistically endpoints (including CA).
significant.
(1920) Exposed: 86 Workers External exposure: 100 cells evaluated Exposed workers and Tables 1 and 2 One of the larger studies. Data
Vodicka, at 3 reinforced plastic Workplace styrene for chromosome comparisons were of Exposed: tables contain substantial
2004; Cross- plants (A, B & C). measured using aberrations the same Overall workers: N = information—presented as
sectional mean age = 36.5; 25 F personal dosimeter on socioeconomic 86, mean = 2.3%, SD group means. The exposed vs
with no & 61 M; 44 smokers. the day of sampling. background. On = 0.6%, control groups are not well
matching; Plant A = 35 (20 All exposed: 81.3 mg/ average the exposed Plant A: N = 35, mean matched by smoking status,
Czech males); mean age = m3 measured for 73 workers were 8.7 = 2.5 %, SD = 1.6 %. gender and age. There is no
Republic; 36.8; 15 smokers; individuals years younger than Plant B: N = 31, mean exposure assessment of the
years of employment Plant A: (29 the controls & there = 2.3 %, SD= + 1.6 %. Regional Hygienic Station
= 3.4. individuals) 25.8 ppm were more men in the Plant C: N = 20, mean employees (this group has the
Plant B = 31 M; mean + 13.2. exposed 71% vs. = 2.0 %, SD = 1.4 % highest level of aberrations).
age = 38.0; 16 Plant B: (27 52%, more smokers Comparisons:
smokers; years of individuals) 10.8 ppm (51 vs 19%) and for Maintenance worker
employment = 5.6. + 9.6. the smokers the controls: N = 16, mean
Plant C = 20 (10 Plant C: (17 workers had smoked = 1.7%, SD = 0.9%,
males); mean age = individuals) 18.9 ppm about twice as many Regional hygienic
33.7; 13 smokers; + 10.1. cigarettes during controls: N = 26, mean
years of employment (Classified as Low their lifetime than = 3.2%, SD = 2.0%.
= 2.5 + 1.9. Exposure) the controls. The Authors’
Comparisons: 42 Internal exposure: workers and controls interpretation:
workers from a from Styrene in blood and were not well The group with the
Regional Hygiene metabolites in urine matched. highest mean level of
Station and plant B collected at the end of CAs was the Regional
maintenance workers. shift on the day of Hygiene Station
Regional Hygiene sampling. All control group. The
Station: 26 (6 males); exposed: blood group with the lowest
mean age = 42.8; 6 styrene = 0.56 + 0.43 level was the Plant
smokers. mg/L (N = 78). MA + control group. In the
Plant B maintenance PGA mean = 496.9 + worker groups, Plant
workers: 16 males; 402.2 mg/g creatinine A had the highest level
mean age = 48.5; 2 (N = 80). of CAs but the level
smokers. Plant A. Blood was not statistically
styrene: 0.71 + 0.47 different than the
mg/L (N = 34). MA + other groups. CA
PGA = 797.5 + 418.9 frequencies correlated
mg/g creatinine. (N = positively with age.
33).
Plant B. Blood
styrene: 0.40 + 0.31
mg/L (N = 27). MA +
PGA: 269.9 + 180.0
mg/g creatinine (N =
31).
Plant C: Blood
styrene: 0.50 + 0.44
mg/L (N = 17). MA +
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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 2 (continued )
(Study Description of Cases Exposure to styrene Technical details, Potential Mean chromosome Comments
Number), First and controls for cases including length of culture, confounders aberration levels, +SD
author, external exposure, coding and scoring of considered expressed as % cells
publication biomonitored slides with aberrations
year, type of exposure, and unless noted
study, location exposure among otherwise. N for cases
controls and controls. Authors’
interpretation and
major findings

PGA: 307.8 + 271.9

mg/g creatinine (N =
16).
(High exposure
based on internal
exposure)
Exposure in
unexposed
comparisons: RHS:
not measured.
Plant B maintenance
workers: blood
styrene = 0.07 + 0.06
mg/L (N = 10). MA +
PGA mean = 42.1 +
15.5 mg/g creatinine
(N = 14).
(21) Migliore, Exposed: 95 persons A high proportion of Chromosome Participants Table 4 One of the largest studies. No
2006; Cross- (76 males) in the workers used PPE aberrations were interviewed for Exposed: N = 72, exposure assessment of the
sectional fiberglass reinforced such as masks and evaluated in whole personal, mean = 2.4%, SD = controls. A large percentage of
with no plastics, 13 different gloves. blood lymphocyte occupational and 0.2% both groups were smokers; CA
matching; plants (10 in Tuscany External Exposure: cultures for 72 medical history as Unexposed: N = 89, frequency data were presented
Tuscany and and 3 in Parma); Age TWA of airborne workers and 89 well as lifestyle. mean = 2.5%, SD = for smokers (N = 72; 2.8; 0.2%
Parma, Italy. = 35.4; Smoking styrene measured for controls. Whole blood Recent exposure 0.2% CAs) & non-smokers (N = 89;
status: 51 smokers workers (26 males & cultures grown for 44 (defined as within 3 Authors’ 2.1; 0.2% CA). There was a
and 44 non-smokers; 19 females) from the h. 100 metaphases months) or current Interpretation: difference (p = 0.02) in the
Mean # of cigarettes Parma cohort using scored per individual use of medicinal There was no frequency of CAs in the smokers
per day = 9.3; Years passive-diffusive using coded slides products was used to significant difference vs the non-smokers. There was
of employment = samplers during the and one scorer. exclude potential between the workers no difference between the
11.5 (range <1− 34). work shift. Air subjects. The controls and controls on chromosome aberration levels
Unexposed: concentrations 8.5 were selected from chromosome in workers vs controls.
Residents of the same ppm ± 0.9 (range the same geographic aberrations. The
areas as the exposed. 0.5–122.9). areas as the workers. authors did report a
98 healthy volunteers Internal Exposure: positive correlation
(67 males) with no Urine collected at the between the frequency
history of end of shift. MA + of CAs and airborne
occupational styrene PGA: mean 300.0 ± styrene and some of
exposure: Age = 36.6; 338.2 (range the urinary
Smoking status: 41 10.2–1856) mg/g biomarkers of
smokers & 57 non- creatinine. exposure. The authors
smokers; mean # of High Exposure cited this as indicating
cigarettes per day = For the Parma cohort that CAs were
5.9. additional urinary associated with
biomarkers evaluated. exposure level.
PHEMAs: mean 0.9
(range 0.01–3.29)
mg/g creatinine. 4-
VPTs: mean 1.9
(range 0.1–7.74) mg/
g creatinine.
Exposure in
Unexposed
Comparison: Not
measured.

Abbreviations: mandelic acid (MA; phenyglyoxylic acids (PGA; personal protective equipment (PPE; time-weighted average TWA.

group which consisted of 37 employees in the same plant but not
working in the high exposed areas had average chromosomal aberra­
tions levels of 3.2 per 100 cells. The low exposed workers had chro­
mosomal aberrations of 8.0 per 100 cells, and the high exposed workers
a chromosomal aberrations level of 7.8 per 100 cells. While both
exposed groups of workers had chromosomal aberrations greater than
the control group, there was no difference between low and high
exposed workers. Sorsa et al. studied laminators and “other workers”
and presented monitoring data indicating that laminators had higher
exposures than other workers [48]. The authors conducted a regression
analysis of the exposure categories, smoking and age, and found no as­
sociation between chromosomal aberrations and styrene exposures.
Tomanin et al. examined two plants with workers making fiberglass with
one plant having higher exposures than the other [42]. Factory 1 in this
study reported personal air monitoring results of 4.8–23.0 ppm among 7
workers and factory 2 reported 25.7–100.0 ppm among 11 workers.
Factory 1 reported chromosomal aberrations levels 1.43 per 100 cells
compared to a matched comparison group of 1.43 per 100 cells. Factory
2 reported chromosomal aberrations levels of 3.02 per 100 cells and the
control group of 0.82 per 100 cells. The authors concluded that there

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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

difference indicates that the styrene exposed workers had a higher

percent of chromosomal aberrations than the control group. Compre­
hensive Meta-Analysis (CMA) software of Biostat, Inc. (Englewood, NJ,
USA) was used to estimate both fixed-effects and random-effects meta-
standardized mean difference between exposed and comparison pop­
ulations with 95 % confidence intervals (CI). Evaluation of study het­
erogeneity included comparison of meta-mean differences estimates
from fixed-effects models, as well as calculation of the I2 statistic, which
assesses the percentage of between-study variance in differences that is
due to study heterogeneity rather than to chance. Sensitivity analyses,
designed to evaluate potential sources of heterogeneity on meta-RR re­
sults, stratified by exposure levels of styrene, and matched versus un­
matched cross-sectional study designs. Evidence for publication bias was
assessed by inspecting funnel plot symmetry using the approach of Egger
et al. [51]. The study of Vodicka et al. presented an internal (within the
same plant) and an external (outside the plant) comparison group [46].
Fig. 2. Range of exposures in parts per million (ppm) for studies categorized as
We examined the meta-analysis with using each of the comparison
low and high. groups, but used the external comparison group as the definitive control
because it was larger than the internal comparison group and eliminated
the possibility that workers within the plant may have been exposed to
was no difference in chromosomal aberrations between workers and
low levels of styrene.
lower exposed group, factory 1, but there were more chromosomal ab­
Fig. 3 is the forest plot for the meta-mean differences for the 20
errations in the workers with higher exposures, factory 2. Anwar and
comparisons included in the meta-analysis. Of the 20 comparisons, 12
Shamy examined workers in the reinforced plastics industry.32 We
are positive where the chromosomal aberrations frequencies are higher
divided workers based on the reported biomonitoring levels from
in the exposed than in the comparison. Assessing statistical significance,
Table 2 of that study. The 15 workers in the low exposure group had
7 of the comparisons represented statistically significant increases in
mean urinary mandelic acid (MA) level of 233.7 and chromosomal ab­
chromosomal aberrations in the styrene exposed workers and the com­
errations levels of 3.9 per 100 cells compared to matched controls of 1.3
parison groups and 3 studies reported statistically significant decreases
per 100 cells. The 3 workers in the high exposure group had mean MA
in chromosomal aberrations among styrene exposed worker. Table 3
levels of 802.3 and chromosomal aberrations levels of 4.3 per 100 cells
presents overall meta-analysis of the 20 comparisons for 505 styrene
compared to matched controls of 2.3 per 100 cells. Thus, both the low
exposed workers and 532 comparisons. A meta-mean difference for the
and high exposed group had higher chromosomal aberrations levels
random effects model is 0.361 (95 % CI − 0.084 to 0.807) and for the
than the matched controls. The authors also report that there was no
fixed effect model is 0.209 (95 % CI 0.073 to 0.345). The fixed effect
correlation between MA levels and chromosomal aberrations levels.
model is used as the definitive summary of these studies because they
Artuso et al. examined workers in the boat building industry and divided
are performed on differing populations, with differing characteristics,
workers into high and low exposure to styrene based on air monitoring.
over different times. The fixed effects model produced a p-value for
[43] The 23 workers spending time in high exposed areas such as gel
heterogeneity of <0.001 and an I2 of 90.114 indicating significant het­
coating, and laminating were considered high exposures. Air monitoring
erogeneity among studies. This analysis for the Vodicka study uses the
in gel coating ranged from 80.6− 319.3 ppm and in lamination ranged
external comparison group [46]. When the internal comparison group is
from 19.8 to 151.7 ppm. The 22 workers not spending significant time in
used from the Vodicka study, the results are similar. We also examined
these areas were classified as low exposure ranging from 0 to 27.6 ppm.
the exposure levels in these studies. The more highly exposed styrene
The exposed workers were frequency matched with controls on age and
workers produced a meta-mean difference of 0.407 (95 % CI − 0.168 to
smoking. The 23 workers with high exposure had a mean of 4.00
0.982 random effects model) compared to the lower styrene exposed
chromosomal aberrations per 100 cells. The 22 workers with low
workers of 0.494 (95 % CI − 0.089 to 1.077 random effects model). The
exposure had a mean of 2.76 chromosomal aberrations per 100 cells.
matched cross-sectional studies had a meta-mean difference of 0.699 (95
The 34 control workers had a mean of 2.13 chromosomal aberrations per
100 cells. The authors report a significant difference between controls
and the high exposure group and report a positive dose response trend
based on individual exposure estimates and chromosomal aberrations.
Overall, 4 of the studies found no or an equivocal exposure response [26,
32,36,48], and 3 studies found some evidence of an exposure response
[31,42,43].
All the studies included in the meta-analysis report scoring at least
100 metaphases or “about 100 metaphases.” The study of Artuso et al.
scored 150 and the study of Nordensen and Beckman scored 200 [38,
43].
Meta-analysis
Meta-analysis was done on mean differences between workers
exposed to styrene and comparison groups with little or no occupational
exposure to styrene. The standardized mean difference was used as the
summary statistic since all studies assess the same outcome but use
different comparison populations. Thus, it is necessary to standardize
the results of the studies to a uniform scale before they are combined.
The standardized mean difference expresses the size of the effect of
styrene exposed workers to comparison persons in each study relative to
the variability observed in that study. A positive standardized mean Fig. 3. Meta-Analysis of Ever Exposed to Styrene Standardized Differences in
Means for Chromosome Aberrations.

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J.J. Collins and M. Moore Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275

Table 3
Meta-analysis of model specifications.
Meta- Sensitivity Studies included Exposed Comparison1 Meta-standard 95 % confidence I2 (p
analysis mean difference limit heterogeneity)
model

1, random All using external comparison [1–19,21] 505 532 0.361 − 0.084 to 0.807
effects group from Vodika, 2004
1, fixed All using external comparison [1–19,21] 505 532 0.209 0.073− 0.345 90.114 (0.000)
effects group from Vodika, 2004
2, random All using internal comparison [1–18,20,21] 505 522 0.411 − 0.027 to 0.849
effects group from Vodika, 2004
2, fixed All using internal comparison [1–18,20,21] 505 522 0.293 0.155− 0.432 89.480 (0.000)
effects group from Vodika, 2004
3, random High Exposure Part of [1,2–4,6,8], part of [9,10, 382 361 0.407 − 0.168 to 0.982
effects 12,13] part of [15,19,21]
3, fixed High Exposure Part of [1,2–4,6,8], part of [9,10, 382 361 0.121 − 0.038 to 0.280 91.641 (0.000)
effects 12,13] part of [15,19,21]
3, random Low Exposure Part of [1,5,7], part of [9,11,14, 105 202 0.494 − 0.089 to 1.077
effects 15], part of [17,18]
3, fixed Low Exposure Part of [1,5,7], part of [9,11,14, 105 202 0.464 0.209− 0.719 79.910 (0.000)
effects 15], part of [17,18]
4, random Matching [2–4,8–12] 176 188 0.699 0.172− 1.226
effects
4, fixed Matching [2–4,8–12] 176 188 0.743 0.521− 0.964 81.877 (0.000)
effects
4, random No Matching [1,5–7,13–19,21] 243 318 0.218 − 0.464 to 0.146
effects
4, fixed No Matching [1,5–7,13–19,21] 243 318 − 0.042 − 0.280 to 0.146 91.215 (0.000)
effects
1
The same control group in studies [1,3,10,12–14,20] are used for exposed subgroup comparisons.

% CI 0.172− 1.226 random effects model) compared to the unmatched
cross-sectional studies of 0.218 (95 % CI − 0.464 to 0.146 random effects
model). There was significant heterogeneity in all comparisons.
4. Discussion
This critical review examines several dimensions of a quality study
including study design, measurement of chromosomal aberrations
endpoints, risk estimation of positive effect, study size, measurement of
workplace exposure in both the “exposed” and presumably “unexposed”
individuals, and use of a valid comparison group for assessing increase
in chromosomal aberrations among exposed workers. Appropriate se­
lection and matching of the comparison group to the exposed group is of
key importance and was evaluated in our review.
There are several co-exposures or potential confounding factors they
may be important to consider when examining chromosomal aberra­
tions including genetic differences, lifestyle factors, diet, exercise, co-
morbidities, exposure to X-rays, drug usage, occupational and home
use of other chemicals, age, gender as well as laboratory variation [17].
Two study designs have been used in studies to examine the effects of
exposure on genotoxic outcomes, the cross-sectional study and the
longitudinal cohort study. The cross-sectional study relies on the selec­
tion of a comparison group that would in theory consist of the same
individuals in the exposed group had they not been exposed. Since it is
impossible for the same person to be exposed and unexposed simulta­
neously, an exposed group and a comparison group are selected for the
study. This design can be improved by matching on key co-exposures
and potential confounding factors. The longitudinal cohort design has
also been used occasionally, though rarely, where exposure and the
genotoxic outcome are measured on the same individual several times
before, during and after exposure. A longitudinal cohort study has the
advantage of identifying relationships not related to co-exposures and
potential confounding factors and thus is viewed as a superior design to
the cross-sectional study.
The complete assessment of all possible styrene exposures for study
groups for both the workers and the controls is an important factor in a
human study [17]. In the absence of a full exposure evaluation, it should
not be assumed that the workers were not exposed to chemicals other
than styrene. It should also not be assumed, particularly when the
controls worked in the same factory, that the control individuals were
not exposed to styrene. Biomarkers of internal exposure, such as urinary
metabolites mandelic acid (MA) and phenylglyoxylic acid (PGA), mea­
sure recent styrene exposure levels [52]. Urinary thioether, which in­
dicates exposure to electrophilic compounds, but is not specific for
styrene, has also been used in human styrene studies [31]. For workers,
exposure duration, exposure pattern and exposure levels should all be
evaluated. The measurement of peak exposure is particularly important
because this may result in overloaded defense mechanisms which may
be responsible for producing genotoxic effects. The timing of the expo­
sure assessment and obtaining the biological sample is also important
with appropriate timing dependent upon the endpoint being measured.
There are several areas in which the studies of chromosomal aber­
rations among styrene exposed workers fail to meet some key elements
recommended in the IPCS document for monitoring genotoxic events for
potential carcinogens [17]. All the studies but one reported some mea­
surement of styrene exposure. The study of Biro et al. did not report
styrene exposure monitoring results because it focus was on other
chemicals [45]. In addition, the styrene exposed workers in the Brio
et al. study had a much higher percentage of smokers and males than the
comparison group. Eight studies reported both air monitoring and bio­
monitoring in at least part of their study locations [32,35,37–40,42,44,
46,47]. However, only 2 studies measured exposure levels among the
comparison groups making it difficult to determine if they did have
workplace styrene exposure [31,44]. Most of the styrene worker studies
for chromosomal aberrations scored only 100 cells per individual while
200 or more are recommended [17]. Also, 7 of the 20 studies used a
matched cross-sectional design to address potential confounding from
age, sex or smoking [31,35–37,41–43]. However, only 2 studies
matched on all three potential confounders [31,42]. Only 4 of the 20
studies indicated that the slides were blinded to the scorer [41–43, [45].
Furthermore, most studies had a very small number of subjects. This
factor makes it impossible to draw definitive conclusions from a single
study or even a small group of studies.
There were four criteria we relied on to determine if workplace
styrene exposure was associated with increased levels of chromosomal
aberrations. The first criterion was strength of the association. Is

17
J.J. Collins and M. Moore
workplace styrene exposure related to chromosomal aberrations? In the
meta-analysis, we did observe an increase in chromosomal aberrations
among styrene exposed workers overall. Second, was there an exposure
response? We found from the meta-analysis that the studies with the
higher exposure to styrene had lower chromosomal aberration levels
than the studies with lower styrene exposure. Further, while individu­
ally some studies report an exposure response, most did not. High
exposure levels were not associated with high chromosomal aberrations
frequencies. Third, could co-exposures or confounding exposures be
influencing the risks observed? We found that the studies which
matched on potential confounding factors reported a higher level of
chromosomal aberrations than the studies that did use matching. Since
the matched studies would most likely be a better design than the un­
matched studies at addressing potential confounding, our findings
indicate that confounding exposures such as age, sex and smoking may
be important considerations for causal assessment in future studies.
Fourth, is there consistency of findings across studies? There was
considerable heterogeneity in all models indicating that the findings
were not consistent across studies. Two outlier studies capture the
extend of this lack of consistency. The study of Biro [45] had the highest
standardized mean difference of 3.2 (95 % CI 2.2–4.3) and the study of
Maki-Paakkanen 1987 [39] had the lowest standardized mean differ­
ence of -0.1.7 (95 % CI − 2.4 to − 1.00.)
The conclusions from our current review and meta-analysis shows
agreement with the 2004 critical review of Henderson and Speit [11]
and the 1996 review of Scott and Preston [9] where both reviews
conclude most studies show no clear evidence that styrene exposure in
workers results in increases in chromosomal aberrations. Our conclu­
sions, however, are different than the quantitative modeling of 1996
study of Bonassi and colleagues which found an increase of chromo­
somal aberrations among workers with high styrene exposures [8]. This
study, however, was several years ago and thus did not include many of
the recent studies included in our review. The recent IARC monograph
including many of the same human studies in our review and analysis
concludes that while there is evidence of styrene oxide derived DNA
adducts in the blood and urine of exposed workers the results with other
indicators of genotoxicity were “mixed” [1]. Supplementary Table 1 lists
all of the publications that either we identified or that were included in
the IARC review of human epidemiology studies examining chromo­
somal aberrations. There were three publications that IARC included
that we did not include. Smejkalova et al. [53] and Huang et al. [54]
were published in Czech and Chinese, respectively. The English abstract
for the Smejkalova et al. study was not clearly written and was difficult
to understand key details of that study. The English abstract for the
Huang et al. paper indicates an evaluation of styrene exposed in­
dividuals but does not mention any unexposed controls. We were unable
to obtain the Dolmierski et al. paper [55]. The IARC review comment
concerning the Dolmierski et al. paper was that there was a small control
group (n = 2) and the controls were apparently younger compared with
the exposed subjects. Clearly, none of these three publications included
data that could be used for the meta-analysis. The 31publications that
we evaluated included 4 studies that were not mentioned in the IARC
review [31,37,45,47]. All but one of these were included in the
meta-analysis [37]. While the IARC assessment was a hazard identifi­
cation based on the human and experimental data, we performed a risk
assessment based only on the human studies which measured risk
chromosomal aberrations and styrene exposures. While our
meta-analysis did find an increase in chromosomal aberrations among
styrene workers overall, the findings were not consistent across studies
and there was lack of evidence of an exposure response.
In addition to the “standard” chromosomal aberrations studies which
were the focus of our meta-analysis, we note that Kolstad et al. reported
a nested case-referent study in which 12 of 19 myeloid leukemia patients
who worked in the Danish reinforced plastics industry and similar in­
dustries showed clonal chromosomal aberrations [56]. Fifteen of the 19
myeloid leukemia patients had been employed in the reinforced plastics
18
Mutation Research - Genetic Toxicology and Environmental Mutagenesis 861-862 (2021) 503275
industry and four were presumably not occupationally exposed to sty­
rene. Of the four individuals not exposed to styrene, one had a clonal
chromosomal aberrations expansion and three had normal karyotypes.
Five of the individuals, all of whom were employed in the
fiber-reinforced polymer composites industry, had translocations
involving chromosomes 9 and 22; an additional six individuals had
other types of clonal aberrations. One of the reinforced plastics industry
employees had a normal karyotype. The authors indicate that the
number of patients is small, the myeloid leukemias included both
chronic myeloid and acute myeloid, and the clonal chromosomal aber­
rations may simply be present because they are a part of the etiology of
the disease. Despite these limitations, they conclude that “the results
suggest that styrene may cause leukemia through a clastogenic effect.”
However, based upon the small number of people and the multiple other
possible explanations for the observed results, we conclude that this
study does not provide information as to whether styrene can cause
somatic cell chromosome damage in humans.
5. Conclusions
As we have outlined, there are important limitations in the published
studies examining chromosomal aberrations in styrene exposed workers.
The small size and quality of the studies, the imprecision of the exposure
estimates for exposed workers, lack of exposure assessment for most
controls, the scoring of an insufficient number of cells to provide reliable
chromosomal aberrations frequencies, the use of cross-sectional design
with the poorly matched cases and controls in most studies may have
produced the lack of consistency across studies. A major limitation of the
studies, we reviewed is the use of a cross-sectional design because of the
many potential co-exposures and potential confounders in the compar­
ison group. The limitations were partially addressed in some of the
studies by matching on characteristics, but this does not guarantee a
valid comparison as in most cases the number of exposed and compar­
ison individuals is relatively small. A longitudinal design with both
chromosomal aberrations measured before, during and after workplace
styrene exposures occurred would be the ideal study design as each
person in the study would serve as their own comparison for co-
exposures and potential confounders. This study design would include
internal and external measurement of workplace styrene exposures
several times during the conduct of the study. A large longitudinal study
would provide important additional information on the potential rela­
tionship between workplace styrene exposure and chromosomal
aberrations.
We found some evidence of higher chromosomal aberration levels
among styrene exposed workers but no consistent evidence of an
exposure response in the present study. Our evaluation indicates that the
risks across studies are inconsistent and thus is insufficient to support the
notion that styrene exposure increase chromosomal aberrations in
occupationally exposed workers. New, much larger, and better designed
studies would include cross-sectional matching, a preference for longi­
tudinal study designs, better exposure assessment and scoring of a suf­
ficient number of metaphases to provide reliable chromosomal
aberrations frequencies with appropriate power to detect an effect.
Declaration of Competing Interest
We wish to draw the attention of the Editor to the following facts
which may be considered as potential conflicts of interest and to sig­
nificant financial contributions to this work. Dr. Collins and Dr. Moore
report that they have each received funding from the Styrene Research
and Information Center (SIRC), a group with company members that
may be affected by the research reported in the enclosed paper. SIRC
was provided an opportunity to review the manuscript prior to sub­
mission and their comments regarding clarity of presentation were
considered. The scientific opinions and critical evaluation of the litera­
ture are those exclusively of the authors. Dr. Collins worked as an
J.J. Collins and M. Moore
epidemiologist until 2014 for the Dow Chemical Company, a company
with interest in styrene. In his capacity, he was deposed several times for
litigation against Dow for several chemical exposures but not styrene.
Since 2014, he has been employed by Saginaw Valley State University in
Michigan and has provided consulting for several companies and in­
dustry groups. Dr. Moore is currently a principle at Martha M Moore
LLC, and has been employed by Ramboll US Corporation, since 2013 and
has provided consultation for several companies and industry groups.
Prior to that, she worked for the US Food and Drug Administration as the
Director of the Division of Genetic and Molecular Toxicology at the
National Center for Toxicological Research.
We confirm that the manuscript has been read and approved by all
named authors and that there are no other persons who satisfied the
criteria for authorship but are not listed. We further confirm that the
order of authors listed in the manuscript has been approved by both of
us. We confirm that we have given due consideration to the protection of
intellectual property associated with this work and that there are no
impediments to publication, including the timing of publication, with
respect to intellectual property. In so doing we confirm that we have
followed the regulations of our institutions concerning intellectual
property.
We understand that the Corresponding Author is the sole contact for
the Editorial process (including Editorial Manager and direct commu­
nications with the office). He is responsible for communicating with the
other author about progress, submissions of revisions and final approval
of proofs. We confirm that we have provided a current, correct email
address which is accessible by the Corresponding Author and which has
been configured to accept email from jjcollin@svsu.edu.
Appendix A. Supplementary data
Supplementary material related to this article can be found, in the
online version, at doi:https://doi.org/10.1016/j.mrgentox.2020.50
3275.
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