Environment International 99 (2017) 29–42

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Environment International

journal homepage: www.elsevier.com/locate/envint

Review article

A systematic review of Mancozeb as a reproductive and

developmental hazard
Jennifer Runkle a,d,⁎, Joan Flocks b, Jeannie Economos c, Anne L. Dunlop d
a
Cooperative Institute for Climate and Satellites-North Carolina, North Carolina State University, Asheville, NC, USA
b
Center for Governmental Responsibility, Levin College of Law, University of Florida, Gainesville, FL, USA
c
Farmworker Association of Florida, Apopka, FL, USA
d
Nell Hodgson Woodruff School of Nursing, Emory University, Atlanta, GA, USA

a r t i c l e i n f o a b s t r a c t

Article history: Background: The potential adverse reproductive and developmental effects of Mancozeb, especially in sensitive
Received 4 May 2016 subpopulations, have not been fully reviewed for this widely used fungicide.
Received in revised form 3 November 2016 Objective: To review the experimental and epidemiologic evidence for the association between exposure to
Accepted 3 November 2016 Mancozeb and reproductive and developmental health outcomes using an adaptation of the National Toxicology
Available online 23 November 2016
Program's Office of Health Assessment and Translation (OHAT) systematic review framework.
Data sources: Four databases (PubMed, TOXNET, Web of Science, Google Scholar) were searched for published
Keywords:
Mancozeb
studies on Mancozeb. Of 403 identified articles, 30 met our inclusion criteria for systematic review.
Reproductive health Results: Results from in vitro studies provide evidence that Mancozeb may indirectly disrupt or impair reproduc-
Developmental toxicant tion at the cellular level and should be regarded as a reproductive toxicant. Animal studies confirm reproductive
Office of Health Assessment and Translation and developmental toxicity in mammals and suggest that males chronically exposed to Mancozeb experience
systematic review framework significant changes in physiological, biochemical, and pathological processes that may lead to infertility. Epidemi-
Pesticide registration review ological studies were limited to indirect methods of exposure assessment and examined the effect of fungicides
more broadly during pre-conception, pregnancy, and birth, yielding mixed results.
Conclusions: High confidence ratings from in vitro and animal studies, in combination with moderate confidence
ratings from epidemiologic studies employing indirect methods of exposure assessment, provide evidence that
Mancozeb should be regarded as a suspected developmental hazard and a presumed reproductive hazard in
humans. More population-based studies linking direct measures and/or biomarkers of exposure to adverse effects
on male and female fertility, as well as in utero and early life development, are needed to improve the quality of
the evidence base concerning the human reproductive and developmental consequences of Mancozeb exposure.
© 2016 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2. Materials and methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.1. Search strategy and study selection . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30
2.2. Evaluation strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 31
3. Results . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.1. In vitro experimental studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.2. Animal studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3. Reproductive & developmental endpoints . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3.1. Females. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
3.3.2. Males. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.4. Epidemiologic studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
3.4.1. Preconception . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37
4. Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
4.1. Summary of findings . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40

⁎ Corresponding author at: Cooperative Institute for Climate and Satellites-North Carolina, North Carolina State University, 151 Patton Avenue, Room 500, Asheville, NC 28801-5001, USA.
E-mail address: jrrunkle@ncsu.edu (J. Runkle).

http://dx.doi.org/10.1016/j.envint.2016.11.006
0160-4120/© 2016 Elsevier Ltd. All rights reserved.
30 J. Runkle et al. / Environment International 99 (2017) 29–42
5. Future research directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 41
1. Introduction
Mancozeb, an ethylene bisdithiocarbamate (EBDC) fungicide with
the degradate ethylenethiourea (ETU), was first registered in the United
States in 1948 as a broad spectrum fungicide (EPA, 2005). Mancozeb has
since demonstrated nearly seventy years of fungicidal efficacy in a wide
range of agricultural and industrial applications, including use as a fun-
gicide in major agricultural crops (e.g., potato, tomato, grapevine, and
citrus) for roughly 400 different plant pathogens. Mancozeb is currently
registered as a general use pesticide by the Environmental Protection
Agency (EPA). According to a recent industry analysis, Mancozeb exhib-
ited the fastest growing production volume accounting for N 20% of the
global fungicide market in 2014 (Fungicides Market, 2015). Mancozeb
production is forecasted to continue to grow faster than normal by the
early 2020's due to a low purchase price, increasing global demand for
fruits and vegetables, and continued non-selective fungicidal efficacy
(Fungicides Market, 2015). In the United States, the estimated use of
Mancozeb has remained relatively stable since 2010 ranging from 4.2
and 7.2 million pounds per year for vegetable and fruit crops and or-
chards and grapes, respectively (U.S. Geological Survey, n.d).
Routes of exposure among the general public primarily involve lim-
ited dietary exposure through the consumption of contaminated pro-
duce (e.g., tomatoes, potatoes, citrus fruits) or drinking water.
Workplace exposure to Mancozeb occurs among workers who produce
the chemical and among agricultural workers following dermal contact,
inhalation of dusts or fine spray, or accidental/incidental ingestion, as in
eating or smoking without prior handwashing. Mancozeb is rapidly me-
tabolized by the body and has been characterized as having low-acute
toxicity in animal studies (Ellenhorn and Barceloux, 1988). However,
exposure to this fungicide has been linked to a wide range of environ-
mental health hazards including neurotoxic effects and Parkinson-like
symptoms (Zhou et al., 2004) and sensitization in vulnerable popula-
tions (e.g., women and children), including thyroid hormone disruption
exhibited in women chronically exposed (Goldner et al., 2010) and
disregulations in fetal brain development (Nordby et al., 2005).
Mancozeb and other EBDCs have been the subject of two special EPA
reviews, initiated in 1977 and 1987 because of particular health con-
cerns, including developmental and thyroid effects caused by the com-
mon degradate ETU (EPA, 2005). In the 2005 Reregistration Eligibility
decision (RED), the EPA raised several risk concerns and recognized
data gaps for developmental, reproductive, and thyroid toxicity in re-
sponse to exposure to Mancozeb. Since 2005, the EPA has issued final
rules on tolerances for residues of Mancozeb on various crops including
almonds, cabbage, lettuce, peppers, and broccoli in 2011(40 CFR Part
180 2011 ed.) and walnuts and tangerines in 2013(40 CFR Part 180
2013 ed.). In these final rules, the EPA continued to acknowledge certain
data gaps for Mancozeb and ETU, especially regarding the impact of ex-
posure on the developing thyroid and reproductive system. The 2013
final rule acknowledged results demonstrating an association between
Mancozeb exposure in rat and rabbit studies and maternal mortality,
spontaneous abortion, thyroid effects, maternal body weight gain dec-
rements, and decreased pup body weight. The EPA also acknowledged
fetal malformations, including hydrocephaly and domed head, observed
in rat and rabbits exposed to ETU.
The longer-term toxicity of the fungicide Mancozeb and its metabo-
lite, ETU, includes known endocrine disruptive, teratogenic, mutagenic,
and carcinogenic risks (EPA, 2005; WHO, 1988). Recent toxicological
evidence has shown lasting genotoxic and pre-malignant changes in
human ovarian and immune cells following exposure to Mancozeb
elevating concerns of potential cancer and reproductive health risks in
exposed human populations (Paro et al., 2012; Srivastava et al., 2012).
Experiments conducted on rodents have established that Mancozeb
and ETU are capable of crossing the placental barrier with large poten-
tial to disrupt reproductive performance, cause DNA damage, and
initiate tumors in fetal cells (Cecconi et al., 2007; Shukla and Arora,
2001). Belpoggi et al. (2002) demonstrated the multipotent carcinogen-
ic potential of Mancozeb following long-term exposure in rats.
Mancozeb is also a suspected endocrine disruptor associated with
both hyperthyroidism and hypothyroidism (Axelstad et al., 2011;
Goldner et al., 2010).
Growing evidence raises unresolved questions regarding the link be-
tween routine use of Mancozeb and human reproductive and develop-
mental consequences. To-date no systematic review has evaluated the
current state of scientific evidence linking occupational and environ-
mental exposure to this fungicide to associated adverse reproductive
health endpoints during susceptible windows of exposure, including
preconception, prenatal, or early-life developmental periods. There is a
general consensus among clinicians and scientists concerning the ro-
bustness of the scientific literature linking exposure to certain environ-
mental chemicals to reproductive and developmental harms (ACOG,
2013), yet regulatory bodies have not adequately considered the poten-
tial adverse reproductive health effects in the registration and reregis-
tration of pesticides. At the time of writing this manuscript, the US
EPA has initiated the registration review for Mancozeb (case no.
0643). Registered pesticides are subject to recurrent review as required
by the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) and as
amended by the Food Quality Act (FQPA) of 1996 and the Pesticide Reg-
istration Improvement (PRIA) Act of 2003 (7 U.S.C. sec. 136, et seq.). The
intent behind the registration review process is to examine evidence
from accumulating scientific studies and reevaluate the risks to
human health and the environment of a registered pesticide.
We reviewed the literature for notable experimental and human
health studies that link environmental or occupational exposure to
Mancozeb to adverse reproductive and developmental health effects.
Existing regulations are not often based upon consideration of the risk
to human reproduction and development, particularly for chronically
exposed and vulnerable populations, such as farmworkers and their
children. We urge regulatory agencies to implement similar systematic
review procedures that closely examine the scientific evidence on re-
productive health endpoints into their risk assessment and decision-
making process for registered chemicals.
2. Materials and methods
2.1. Search strategy and study selection
The systematic review utilized the PubMed database (www.ncbi.
nlm.nih.gov), Google Scholar (www.google.com/scholar), TOXNET da-
tabase (www.toxnet.nlm.nih.gov), and Web of Science database from
1950 through August of 2016 using the following search terms:
“Mancozeb” or “dithiocarbamates” or “ethylene thiourea (ETU)” and
(1) “reproductive toxicity” or “reproductive effect” or “infertility” or
“sperm quality;“ (2) or “developmental toxicity” or “developmental ef-
fects”; (3) or “pregnancy” or “pregnancy effects” or “birth outcomes” or
“birth defects”; (4) “in utero exposure” or “chronic exposure” or “envi-
ronmental exposure” or “occupational exposure”; and (5) “endocrine
disrupting” or “endocrine disruptor”. Additionally, we performed
 J. Runkle et al. / Environment International 99 (2017) 29–42
manual reviews of the reference lists from relevant peer-reviewed re-
search and review articles.
For experimental studies, we reviewed in vitro and in vivo studies
that included the oral or injected administration of Mancozeb and rele-
vant reproductive (e.g., pre-conception, pregnancy) and developmental
(e.g., birth, early perinatal period) cellular, toxicological or health end-
points. We identified epidemiologic studies that measured Mancozeb
exposure using environmental monitoring, biomarkers, or indirect
measures (e.g., occupation, work-related activities, surveys, and farm
production) and restricted our search to the following distinct sensitive
periods of development: preconception, intrauterine or prenatal, and
postnatal or early infancy exposure timing periods (Halfon and
Hochstein, 2002). The exclusion criteria used in this review were: 1)
no peer-reviewed original research (e.g., exclusion of editorial, review
articles); 2) lack of reproductive or developmental health effects; 3)
no data on Mancozeb exposure; and 4) non-English articles. All articles
included in the final review related to environmental and occupational
exposure to Mancozeb, broadbase exposure to the dithiocarbamate
class of fungicides, or mixture effects involving Mancozeb, and the asso-
ciation with reproductive or early developmental health effects. Fig. 1
provides an overview of the study selection process.
2.2. Evaluation strategy
We adapted the National Toxicology Program's Office of Health As-
sessment and Translation (OHAT) systematic review framework
(Rooney et al., 2014) to evaluate Mancozeb as a reproductive and
Fig. 1. Flow diagram of the experimental and epidemiologic study selection process.
31
development toxicant. The OHAT approach is comprised of seven
steps: 1) identify the problem and develop the review protocol, 2)
search for and select studies for inclusion, 3) extract data from studies,
4) assess the quality or risk of bias of individual studies, 5) rate the con-
fidence in the body of evidence, 6) translate the confidence ratings into
levels of evidence, and 7) integrate evidence from different literatures
(human, animal, and in vitro studies) to develop hazard identification
conclusions (Rooney et al., 2014). Article review was performed by
two investigators using a standardized data abstraction form. The ab-
straction form consisted of three parts: I. Classification information de-
scribing the study design, type of study (e.g., human, animal) and
primary outcome measures; II. Descriptive information detailing the
study/intervention, evaluation of study characteristics, measurement
of outcome, and results; and III. Study quality describing the execution
of the study methodology (e.g., confounding, exposure characterization,
outcome assessment).
The internal validity of each study was assessed using the OHAT Risk
of Bias Rating Tool (NTP, 2015). Each review investigator was asked to
answer 11 risk-of-bias questions using a 4-point scale ranging from
low and high risk-of-bias options (see Table 1). Discrepancies
pertaining to confidence ratings between reviewers were resolved
using consensus. In particular, confidence ratings were downgraded
when the reviewers noted the following key study design features: is-
sues with internal validity, inconsistent results (e.g., variation in the di-
rection or magnitude of point estimates across studies, no overlap
between confidence intervals), and imprecision. Alternatively, confi-
dence ratings were upgraded for example when results were consistent
32 J. Runkle et al. / Environment International 99 (2017) 29–42

Table 1 Lin and Garry (2000) showed Mancozeb exposure induced cytotoxic
Summary risk-of-bias domain assessment for experimental animal and human observa- events and apoptotic patterns of cell death at dose levels of 10 and
tional studies included in the review.
50 μg/ml at 24 h of exposure in MCF-7 breast cancer cell lines, while
Risk of bias domain Experimental animal Human observational
ETU produced chemical induction of cell death at much higher concen-
Selection bias ++ + trations (1000 μg/ml) during a 24 hour treatment time. The administra-
Performance bias ++ + – tion of Mancozeb (Dithane M-45, 80% Mancozeb) did not result in
Attrition/exclusion bias ++ + increased embryonic injury or a higher incidence in developmental ab-
Detection bias ++ + – normality in chicken embryos, but when mixed with copper sulfate
Selective reporting + + (0.1% applied concentration) showed significant embryonic mortality
++ Definitely low risk of bias: compared to individual doses (Fejes et al., 2002). Another study exam-
There is direct evidence of low risk–of–bias practices
ining the effect of mixtures demonstrated that single low-dose expo-
+ Probably low risk of bias:
There is indirect evidence of low risk–of–bias practices OR it is deemed that sure to Mancozeb (0.003 μg/ml) induced developmental injury in
deviations from low risk–of–bias practices for these criteria during the study murine pre-implementation embryos and a pesticide mixture (includ-
would not appreciably bias results, including consideration of direction and
magnitude of bias ing Mancozeb) reduced development and increased apoptosis in
– Probably high risk of bias: mural embryos (Greenlee et al., 2004).
There is indirect evidence of high risk–of–bias practices OR there is insufficient
information (e.g., not reported or “NR”) provided about relevant risk–of–bias
Finally, two recent studies demonstrate evidence for Mancozeb as a
practices reproductive and developmental toxicant. Paro et al. (2012) used gran-
Definitely high risk of bias:
–– ulosa cells from mice and from women undergoing assisted reproduc-
There is direct evidence of high risk–of–bias practices
Source: Adapted from OHAT Risk Tool, 2015 (NTP, 2015). tive procedures to examine the effects of increasing Mancozeb
exposure (0.001, 0.01, 0.1, and 1 μg/ml). Cells from mice exposed to
across study designs (e.g., across study types, population groups), a Mancozeb showed time- and dose-dependent changes in morphology,
large magnitude of effect was observed, or when evidence for a dose-re- migration patterns, decreased levels of p53, and no changes in apopto-
sponse was observed. sis. Human ovarian follicle cells showed morphology changes and re-
Confidence ratings were assigned to individual study designs (e.g., ductions in p53 expression following exposure to increasing
animal studies were considered separately from human observational concentrations of Mancozeb. This study demonstrated that Mancozeb
studies) and were translated into a level of evidence for establishing a damages the somatic cells by inducing a premalignant-like status in
credible link between Mancozeb exposure and a reproductive or devel- both mice and human granulosa cells. Kjeldsen et al. (2013) examined
opmental health outcome (“high”, “moderate,” “low,” “evidence of no the in vitro effects of Mancozeb on androgen receptor (AR) function in
health effect,” and “inadequate evidence”, see Table 2). Lastly, all evi- human breast carcinoma MVLN cells and hamster ovary CHO-K1 cells
dence from observational human, experimental animal, and in vitro and observed the highest inhibitory effect on AR activity for the lowest
studies was integrated to develop a hazard identification conclusion dose of Mancozeb, suggesting a mechanistic link between Mancozeb as
(see Table 3). an endocrine disruptor and sexual health disorders in males.

3.2. Animal studies

3. Results
Fifteen experimental studies in mice, rats, and rabbits published be-
3.1. In vitro experimental studies tween 1973 and 2012 met our inclusion criteria (Fig. 1, Table 5). In all
fifteen studies, Mancozeb was either administered orally, injected, or
Five in vitro studies published between 2000 and 2012 met our in- delivered directly via gavage for lengths of time ranging from 30 days
clusion criteria (Fig. 1, Table 4). All five in vitro studies examined the re- to 1 or more years. While each study included in the review measured
productive toxicity of Mancozeb. The media used in four of these studies exposure to Mancozeb, several studies simultaneously examined expo-
was mammalian cells. All in vitro studies included in this review provide sure to other chemical agents including: maneb, probineb, endosulfan,
evidence that Mancozeb may indirectly disrupt or impair reproduction and phosphamidon. Four studies assessed the independent effects of
at the cellular level. exposure to Mancozeb and then as a mixture with copper oxychloride
(one study), paraquat (one study), and procymidone, epoxicanazole,
Table 2 tebuconazole, and prochloraz (three studies). Duration of exposure
Confidence rating for a health effect given strengths and weaknesses of a collection of and dose ranges in female studies (thirteen studies) were categorized
animal and human studies. as: (1) 15 to 30 before gestational days (lowest dose: 500 mg/kg/day
Level of confidence for health effect and highest dose: 800 mg/kg/day); (2) 1 to 30 gestational days (lowest
Developmental toxicant Reproductive toxicant
Experimental animal studies High Moderate dose: 5 mg/kg/day and highest dose: 1300 mg/kg/day); and (3) postna-
Observational human studies tal days (lowest dose: 6.25 mg/kg/day and highest dose: 500 mg/kg/
Cohort Low Moderate/low
Case–control Low Low
day). For studies examining only males (n = 3), length of experiment
Cross–sectional Low Moderate/low time ranged from 5 to 360 days, with daily doses ranging from 10 to
Case series Very low ––
1500 mg/kg.
++ ++ High in the association between exposure to the substance and the
confidence outcome. The true effect is highly likely to be reflected in the 3.3. Reproductive & developmental endpoints
apparent relationship.

+ ++ Moderate in the association between exposure to the substance and the 3.3.1. Females
confidence outcome. The true effect may be reflected in the apparent
relationship. Two early experimental studies dating back to the 1970s examined
the teratogenic effects of Mancozeb. Khera (1973) was one of the first
++ Low in the association between exposure to the substance and the
confidence outcome. The true effect may be different from the apparent studies to investigate prenatal survival and congenital defects in rats
relationship. and rabbits following Mancozeb exposure. Results indicated no signifi-
cant effect on reproduction for female rats exposed to the maximum tol-
+ Very low in the association between exposure to the substance and the erated dose of ETU (80 mg/kg/day) before and during gestation.
confidence outcome. The true effect is highly likely to be different from the
apparent relationship. However, significant morphological fetal abnormalities, with the fetal
brain most commonly affected, were detected at doses showing no
 J. Runkle et al. / Environment International 99 (2017) 29–42 33

Table 3 skeletal malformations in rat offspring. More internal malformations

Translation schema for level of confidence from animal and human studies into hazard compared to external malformations were observed in the group re-
identification conclusion.
ceiving the highest doses of Mancozeb (1330 mg/kg); while pups in
Hazard identification the low-dose group (380 mg/kg/day) exhibited significant declines in
Level of confidence: animal & Not fetal weight.
human studies Known Presumed Suspected classifiable Following a twenty-year experimental study gap, a collection of an-
High X X imal studies examined the effect of Mancozeb exposure on reproductive
Moderate X X and pregnancy health in female rats with mixed results. While an earlier
Low X study (Castro et al., 1999) observed no change in the number of estrous
Very low X cycles following treatment with Mancozeb, several subsequent studies
documented a significant decline in the number of estrous cycles,
healthy follicles and number of corpora lutea, as well as a significant
maternal toxicity or fetal death in rat offspring. While increased resorp- changes in the duration of proestrus, estrus, and metestus, with
tion sites and reduced brain weight at 80 mg/kg of daily exposure were N600 mg/kg/day Mancozeb treatment (700 mg/kg/day and 800 mg/
noted for female rabbits, rabbit offspring showed no sensitivity to kg/day) (Baligar and Kaliwal, 2001, 2004; Mahadevaswami et al.,
Mancozeb at any dose level and no teratogenic effects on developing 2000). Significant increases in thyroid weight were also observed in
brains in utero. Larsson et al., 1976 reported significant internal and all rats treated with Mancozeb, with the exception of the experimental

Table 4
In vitro studies (five studies) examining exposure to Mancozeb and reproductive and developmental outcomes.

Source Type of cell/tissue Compound (daily dose) Incubation Outcomes Confidencea

Lin and Garry (2000) MCF-7 breast cancer Herbicides: Test chemicals were added
Mancozeb produced dose-dependent toxic +++
cell line 2,4-D LV4 (0.1–10 μg/ml) to cell culture within 2 to 4
effects in cells (50–70% decrease in cell number
2,4-D Amine (1–10 μg/ml) h of incubation and cultured was observed at the highest concentration
2,4-Dichlorophenoxyacetic for 72 h in media tested).
acid, isooctyl ester (1–10 Following 24 and 48 h of testing, Mancozeb
μg/ml) demonstrated an apoptotic pattern of cell death
2,4-Dichlorophenoxyacetic at lower concentrations (10 and 50 μg/ml).
acid ETU caused programmed cell death at a much
Roundup (1–10 μg/ml) higher concentration of exposure (1000 μg/ml).
Glyphosate (0.228–2.28
μg/ml)
Fungicides:
Triphenyltin acetate
Mancozeb (reagent grade)
(10–50 μg/ml)
Manzate (commercial
grade, 75% Mancozeb)
Tilt (reagent grade)
Tilt (commercial grade,
42% propiconazole)
Fejes et al. (2002) 288 chicken embryos 80% Mancozeb containing 19 days Mancozeb exposure did not result in an increase ++
formulation (Dithane in toxic effects to the embryo or an increase in
M-45) the incidence of developmental abnormalities in
Copper-sulfate (0.1% chicken embryos.
applied concentration) Increased mortality was observed in embryos
exposed to a combination mixture of Mancozeb
and copper-sulfate.
Greenlee et al. (2004) Mice embryos Several agrochemicals 96 h Low-levels of Mancozeb exposure generated +++
were tested at low-doses increases in developmental injury at the
in the study. pre-implementation stage.
Mancozeb (0.003 μg/ml) Fungicide mixture (including Mancozeb)
reduced development to mouse blastocyst and
increased apoptosis during blastocyst formation.
Paro et al. (2012) Mouse granulosa cells Mancozeb (increasing 1, 24, and 36 h Time- and dose-dependent changes in ++++
and human granulosa concentrations (0.001–1 morphology, migration patterns, decreased
cells obtained from μg/ml)) levels of p53, and no change in apoptosis were
women undergoing observed in mouse granulosa cells exposed to
assisted reproductive Mancozeb.
therapy Human ovarian follicle cells demonstrated
changes in morphology and reduction in p53
expression following exposure to increasing
concentrations of Mancozeb.
Kjeldsen et al. (2013) Human breast carcinoma 14 pesticides were tested 48 h Mancozeb exposure resulted in dose-dependent ++++
MVLN cells and hamster Mancozeb concentrations suppression or reduction in androgen receptor
ovary CHO-K1 cells tested singly: (AR) transactivation activity.
LOEC (M): 1 × 10−5 At the lowest dose, Mancozeb exposure showed
MOEC (M): 1 × 10–5% of the highest inhibitory effect on androgenic
solvent control (0.02% hormone levels.
DMSO): 45
a
Confidence ratings in body of evidence ratings: High confidence (++++) in the association between exposure to Mancozeb and the outcome. The true effect is highly likely to be
reflected by the apparent relationship; Moderate confidence (+++) in the association between exposure to Mancozeb and the outcome. The true effect is may be reflected in the appar-
ent relationship; Low confidence (++) in the association between exposure to Mancozeb and the outcome. The true effect is likely to be different than the apparent relationship; and Very
low confidence (+) in the association between exposure to Mancozeb and the outcome. The true effect is highly likely to be different than the apparent relationship.
34 J. Runkle et al. / Environment International 99 (2017) 29–42

Table 5
In vivo studies (fifteen studies) examining exposure to Mancozeb and reproductive and developmental outcomes.

Experimental
Source animal n Compound (route) Daily dose Duration Outcomes Ratinga

Khera (1973) Nulliparous 209 ethylenethiourea (ETU) (oral) 0, 5, 10, 20, 40, or 80 Rats: Group I: Prenatal survival in rats. ++++
adult Wistar rats mg/kg/day 21–42 days There were no observed changes
female rats & 33 before gestation in the number of viable fetuses
New Zealand rabbits and gestation or fetal deaths in rats exposed to
White rabbits Days 1–15; any dose of ETU.
Group II: 6–15 High doses (80 mg/kg) in Group
gestation days 2 and
and moderate doses (40 mg/kg) of
Group III: ETU in Groups 1 and 3 were
gestation days associated with reductions in
7–20 fetal weight.
Rabbits: Daily administration of ETU at
30 gestation days the highest dose (80 mg/kg)
before and during pregnancy
had no significant effect.
Teratogenicity in rats.
ETU caused developmental
malformations in pups at doses
that generated no change in
maternal toxicity or fetal death,
including substantial changes in
jaw, tongue, eyelid, short or
absent tail malformations. The
fetal brain was the most
frequently impacted organ.
Prenatal survival in rabbits.
At the highest concentration of
ETU exposure (80 mg/kg),
exposed rabbits demonstrated
an increase in the number of
resorption sites (including dead
fetuses) and reductions in brain
weight.
Teratogenicity in rabbits.
There were no skeletal
abnormalities or fetal brain
developmental effects observed
in rabbits treated with any dose
of ETU.
Larsson et al. (1976) NMRI mice and 56 Oral dose administered of Three dose levels: 18 gestation days Mice: +++
Sprague-Dawley mice maneb, Mancozeb, and 400, 770, or 1420 There were no observed adverse
rats 56 rats propineb mg/kg maneb maternal effects across the
380, 730, or 1330 different doses of Mancozeb
mg/kg Mancozeb (380, 730, or 1330 mg/kg).
400, 760, or 2300 Reductions in fetal weight were
not observed in the exposed
mg/kg propineb
group with the exception of
reductions in fetal weight among
mothers treated with the lowest
dosage of Mancozeb (380
mg/kg).
Rats:
Following exposure to medium
(730 mg/kg) and high (1330
mg/kg) doses of Mancozeb,
significant malformations and
excessive bleeding were
observed in 25% of the alive
fetuses. More internal
malformations compared to
external malformations were
observed in fetuses dosed with
the highest level of Mancozeb
(1330 mg/kg).
Khan and Sinha (1996) Males of Swiss 56 Endosulfan, phosphamidon, Pesticides: 35 days A decrease in mean sperm ++++
albino mice and Mancozeb (oral) 3, 6 or 1000 counts and sperm head
Vitamin C (injection) mg/kg/day abnormalities was observed in
Vitamin C: experimental groups exposed to
10 (lower dose), 20 any of the three individual
(middle dose) and pesticides, including Mancozeb.
40 mg/kg bw/day Abnormal head size was more
(higher dose) commonly observed in the
sperm of Mancozeb-treated
group compared to mice treated
 J. Runkle et al. / Environment International 99 (2017) 29–42 35

Table 5 (continued)

Experimental
Source animal n Compound (route) Daily dose Duration Outcomes Ratinga

with the other pesticides.

Kackar et al. (1997) Male albino rats 200 Mancozeb in peanut oil (oral) 500, 1000, and 1500 30, 60, 90, 180, All dosages of Mancozeb in rats ++++
mg/kg/day and 360 days chronically exposed for 1 year
resulted in outcomes of
poisoning in rats.
Mortality events were more
frequent in rats in the acute
phase of exposure (days 0–90)
compared to longer-term
exposure to Mancozeb (days
90–360).
Rats exposed to higher doses of
Mancozeb (1000 and 1500
mg/kg/day) at a longer duration
(180 and 360 days) showed
changes in testes pathology, and
these changes were associated
with a decrease in sperm count.
Castro et al. (1999) Wister rats 120 Mancozeb (mixed into diet) 0, 2000 or 3.000 ppm Group A: Mancozeb exposure was not +++
gestation days associated with changes in
1–6, estrous cycles during pregnancy,
Group B: maternal weight gain, nor the
gestation days number of live pup births.
6–15. Offspring exhibited post-natal
delays in physical development
and subtle genetic abnormalities.
Mahadevaswami et al. Female Wistar 36 Mancozeb (oral) 500, 600, 700, and 15 before Rats receiving 700 and 800 ++++
(2000) virgin rats 800 mg/kg/day gestation days mg/kg/day doses of Mancozeb
demonstrated a decrease in
ovary enlargement. Low dose
exposure (500 mg/kg/day)
showed no significant changes in
the number or duration of
estrous cycles.
Following exposure to increasing
concentrations of Mancozeb
(600, 700, and 800 mg/kg/day),
significant increases were
observed in the number and
duration of estrous cycles. The
highest exposed group (800
mg/kg/day) demonstrated a
decline in healthy follicle counts
and a simultaneous increase in
defective ovarian follicles.
Baligar and Kaliwal Wister rats 40 Mancozeb (oral) 500, 600, 700 and 30 days Rats treated with Mancozeb +++
(2001) (virgin) 800 mg/kg/day demonstrated significant
declines in the number of
estrous cycles and healthy
follicles, as well as changes in the
duration of proestrus, estrus and
metestrus phases.
Rats exposed to Mancozeb at
concentrations N500 mg/kg/day
showed a significant increase in
thyroid weight.
Ksheerasagar and Male Swiss Mancozeb (oral) 800 mg/kg/day 5, 10, 20 and 30 Dose-dependent and ++++
Kaliwal (2003) albino mice days time-dependent decreases in
average weight of testes were
observed in mice treated with
Mancozeb. Prolonged exposure
to Mancozeb resulted in
increases in thyroid and thymus
weight.
Baligar and Kaliwal Female virgin 70 Mancozeb (75% wettable 700 mg/kg/day 5, 10, 20 or 30 Rats dosed with Mancozeb for 5, +++
(2004) albino rats powder) dissolved in olive oil before gestation 10, and 20–30 days showed
(oral) days significant changes in diestrus,
estrous cycles, and a decline in
the number of healthy follicles
and an increase in atretic
follicles.
Miranda-Contreras Pregnant mice 160 Paraquat [PQ] and Mancozeb Group I: saline, Pregnant mice: Body weight ++++
et al. (2005) (NMRI) [MZ] (injection) Group II: PQ (10 gestation days Significant reductions were
mg/kg), 1–30, pup day observed in body weight (27%)

(continued on next page)

36 J. Runkle et al. / Environment International 99 (2017) 29–42

Table 5 (continued)

Experimental
Source animal n Compound (route) Daily dose Duration Outcomes Ratinga

Group III: MZ (30 12–20 following 30 days of prenatal

mg/kg) or exposure to PQ + MZ mixture;
Group IV: the while the PQ-only group
combination of PQ demonstrated a 21% reduction in
and MZ (10 mg/kg body weight and the MZ
PQ + 30 mg/kg MZ) only-exposed treatment group
showed a 17% decrease in body
weight.
Among mice prenatally exposed
to chronic doses of MZ and PQ +
MZ, significant declines in neuro
transport chemicals were
observed during pup brain
development. MZ-only exposed
mice demonstrated a decline in
inhibitory neurotransmitters
during brain development.
Rossi et al. (2006) Swiss CD1 25 Mancozeb in sesame oil (oral) 50 and 500 mg/kg Gestation day 2 to Mice treated with either low (50 +++
female mice pup day 20 mg/kg) or high (500 mg/kg)
doses of Mancozeb
demonstrated no significant
change in mean number of
oocytes from F1 generation
compared to untreated mice.
High doses of Mancozeb resulted
in a significant decline in eggs
during ovulation and an
important decline in
fertilizability.
Axelstad et al. (2011) Wister rats 64 Mancozeb in corn oil (oral) 0, 50, 100, or 150 Gestation day 7 to Dams treated with high doses of ++++
(litters) mg/kg/day pup day 16 Mancozeb (150 mg/kg bw/day)
resulted in significant weight
loss and hind limb paralysis.
Dose-dependent declines were
observed in total thyroxine
(thyroid hormone) levels in
dams for all three treatment
groups.
Hass et al. (2012) Time-mated 198 Procymidone, epoxiconazole, Procymidone: 12.5 Gestation day Among rats treated with ++++
nulliparous, tebuconazole, Mancozeb, and and 50 mg/kg/day; 7–21, pup day Mancozeb, there was no
young adult prochloraz (gavage) Epoxiconazol: 3.75 1–16 significant effect on gestation
female Wistar and 15 mg/kg/day; length, maternal or pup body
rats Tebuconazole: 12.5 weight gain.
and 50 mg/kg/day; Rats exposed to the highest
Mancozeb: 6.25 and mixture concentrations
25 mg/kg/day; and exhibited significantly longer
Prochloraz: 8.75 and gestation periods. Exposure to
35 mg/kg/day mixture concentrations at single
(see Table 1 in Hass doses of pesticides below
et al. (2012) for NOAELS adversely impacted
pesticide mixture male sexual development and
doses) gestation length.
Jacobsen et al. (2012) Time-mated 198 Procymidone, epoxiconazole, Procymidone: 12.5 Dams were Exposure to pesticide mixtures ++++
nulliparous, tebuconazole, Mancozeb, and and 50 mg/kg/day; treated by gavage resulted in important changes in
young adult prochloraz (gavage) Epoxiconazole: 3.75 each day from reproductive organ weight,
female Wistar and 15 mg/kg/day; gestation day 7 to sperm count, semen quality, and
rats Tebuconazole: 12.5 pup day 16 spatial learning.
and 50 mg/kg/day;
Mancozeb: 6.25 and
25 mg/kg/day;
Prochloraz: 8.75 and
35 mg/kg/day
Mixture:
0, 14.6 (8.3% of
NOAEL), 29.2 (17% of
NOAEL) or 43.8 (25%
of NOAEL)
mg/kg/day of the
mixture of the 5
pesticides
Jacobsen et al. (2010) Time-mated 80 Procymidone, epoxiconazole, Pix-ratios: Dams were Exposure to pesticide mixtures ++++
nulliparous, tebuconazole, Mancozeb, and 25%, 50%, 75%, 100%, treated by gavage demonstrated substantial joint
young adult prochloraz (gavage) 125% each day from effects resulting in severe
female Wistar *Pesticide mixture Procymidone: 12.5, gestation day 7 to impaired parturition and high
rats compositions (i.e., Pmix-100%) 25.0, 37.5, 50.0, 62.5 day before perinatal pup mortality;
 J. Runkle et al. / Environment International 99 (2017) 29–42
Table 5 (continued)
Experimental
Source animal n Compound (route)
were chosen based on the
individual doses of each
pesticide that caused no effect
on pregnancy length and pup
survival
Pesticide mixture totals:
25%: 43.75,
50%: 87.5,
75%: 131.25, 100%: 175.0,
125%: 218.75
BG = before gestation, GD = gestation day, PD = postnatal day, mg/kg/day = mg/kg body weight/day, NF = not found.
a
Confidence ratings in body of evidence ratings: High confidence (++++) in the association between exposure to Mancozeb and the outcome. The true effect is highly likely to be
reflected by the apparent relationship; Moderate confidence (+++) in the association between exposure to Mancozeb and the outcome. The true effect is may be reflected in the appar-
ent relationship; Low confidence (++) in the association between exposure to Mancozeb and the outcome. The true effect is likely to be different than the apparent relationship; and Very
low confidence (+) in the association between exposure to Mancozeb and the outcome. The true effect is highly likely to be different than the apparent relationship.
group that received 500 mg/kg/day (Baligar and Kaliwal, 2001, 2004).
High prenatal doses of Mancozeb (500 mg/kg) resulted in reduced ma-
turity and fertilizability of oocytes in females of the F1 generation (Rossi
et al., 2006). These studies mark the first report on the effects of
Mancozeb on reproductive potential in animals.
Whole animal studies investigating prenatal exposure to Mancozeb
report important adverse developmental effects. Axelstad et al. (2011)
observed toxic effects in rat dams treated with high doses of Mancozeb
(150 mg/kg/day) including severe weight loss and temporary paralysis
in hind limbs. Results also showed a concentration-dependent decline
in T4 levels for all dams treated with Mancozeb (50 mg/kg, 100 mg/
kg, and 150 mg/kg) on gestation day fifteen. Five mixture studies in-
cluded in the review revealed that prenatal exposure to a low dose pes-
ticide mixture containing Mancozeb may lead to adverse
developmental endpoints including disruptions in pup brain develop-
ment (Miranda-Contreras et al., 2005), long-term developmental de-
layed effects at dose levels where single pesticides showed no effects
(Jacobsen et al., 2012), and malformed genitalia in male rat pups
(Hass et al., 2012). One pesticide mixture study examining the endo-
crine disrupting effects for a range of doses demonstrated a lengthened
gestational period at any dose of exposure and birth complications
resulting in significant increases in perinatal deaths among offspring
at pesticide mixture totals of 25% or more (Jacobsen et al., 2010).
Study findings also observed adverse postnatal impacts on sexual differ-
entiation in male offspring, including decreased anogenital distance,
nipple retention, and gonadal malformations, and female offspring, in-
cluding increased anogenital distance, at individual doses of each pesti-
cide that previously had shown no effect on gestation length or
offspring survival (Jacobsen et al., 2010).
3.3.2. Males
Experimental studies showed significant declines in male reproduc-
tive functioning in animals treated with Mancozeb including
spermatoxic effects in mice and biochemical and structural alterations
in testes of rats and mice following chronic exposure. Khan and Sinha
(1996) observed decreased sperm counts and significantly higher
sperm head abnormalities in all three groups exposed to Mancozeb (3,
6, or 1000 mg/kg/day). Abnormal spermatic head size occurred most
frequently in mice treated with Mancozeb. A long-term experimental
study in male rats resulted in higher mortality-related acute effects in
all groups fed Mancozeb (Kackar et al., 1997). Male rats undergoing
prolonged exposure (180 to 360 days) to higher doses of Mancozeb
(1000 and 1500 mg/kg/day) demonstrated changes in testes pathology
and associated important reductions in sperm counts (Kackar et al.,
1997). Animals in the high dose group (1500 mg/kg/day) demonstrated
temporal changes in pathology for seminiferous tubules, with
37
Daily dose Duration Outcomes Ratinga
mg/kg/day; expected birth increased anogenital distance
Epoxiconazole: 3.75, (GD21) was observed in female offspring
7.5, 11.25, 15.0, Two range
18.75 mg/kg/day; finding studies
Tebuconazole: 12.5, conducting 2
25.0, 37.5, 50.0, 62.5 months apart
mg/kg/day; (i.e., study 1,
Mancozeb: 6.25, study 2)
12.5, 18.75, 25.0,
31.25 mg/kg/day;
Prochloraz: 8.75,
17.50, 26.25, 35.0,
43.75 mg/kg/day
pronounced damage in epithelial cells of epididymis resulting in re-
duced sperm count. Ksheerasagar and Kaliwal (2003) also found time-
dependent declines in weight of testes and other organs (liver, kidneys,
and spleen), as well as, significant increases in thyroid and thymus
weight of treated mice.
3.4. Epidemiologic studies
Ten epidemiologic studies published between 2001 and 2007 met
our inclusion criteria (Fig. 1, Table 6). The majority of observational
studies included in the review used a case-control (three studies) and
cross-sectional design (four studies), with the exception of one case
study and two cohort studies (one prospective study and one retrospec-
tive). We observed significant variations in Mancozeb exposure assess-
ment between studies; however, all ten studies assessed exposure
indirectly using pesticide use questionnaires or database registries.
3.4.1. Preconception
A few human studies investigated the effects of Mancozeb on fertil-
ity specifically, while other studies more broadly examined exposure to
other members of the ethylene bisdithiocarbamate (EBDC) group with
active ingredients maneb and metiram) yielding mixed results. Farr et
al. (2004) found that women who reported using Mancozeb or maneb
were more likely to experience a longer menstrual cycle (OR 4.7, 95%
CI: 1.3–16.6) or a missed menstrual cycle (OR 2.5, 95% CI: 1.0–5.9) com-
pared to unexposed women living on farms in Iowa or North Carolina. In
a case study of three women working in similar agricultural industries
around the same time, two of the women were potentially exposed to
Mancozeb during the first trimester, and all three female agricultural
workers gave birth to infants with severe malformations (Calvert et
al., 2007). Rull et al. (2006) observed no apparent effect on neural
tube defects when Mancozeb was applied within 1000 m of maternal
residences (OR 0.7, CI: 0.2–1.9).
Several human health studies report on the fertility impacts of pop-
ulation-based exposure to fungicides as a class of pesticides more
broadly, but do not characterize exposure to Mancozeb specifically. In
an early study, Arbuckle et al. (2001) observed elevated risk of late-
term spontaneous abortions (12–19 weeks) among farm couples ex-
posed to fungicides (OR 1.4, CI: 0.9–2.1); whereby fungicides the risk
of spontaneous abortions doubled among women 35 years of age and
older (OR 2.4 CI: 1.05–5.9) (Arbuckle et al., 2001). A later study demon-
strated no difference in birth rate for families of fungicide applicators
compared to applicators using herbicides and other agrochemicals
(2.85 vs. 2.80 children per family) (Garry et al., 2002b). Garry et al.,
2002a showed an increased risk in fetal loss among spouses of pesticide
applicators in the Red River Valley who reported personal use
38 J. Runkle et al. / Environment International 99 (2017) 29–42

Table 6
Epidemiologic studies (ten studies) examining exposure to Mancozeb and reproductive and developmental outcomes.

Source Designa Population Mancozeb assessment Outcome Confidenceb

Arbuckle et al. RCo Farm couples in the Ontario Family
(2001) Health Study (N = 2110 women)
Bell et al. (2001) CaCo Cases = 319 neonatal deaths within
24 h of birth and controls = 611
normal live births in 10 California
counties in 1984.
Garry et al. (2002a) CrSe 1070 licensed pesticide applicators
(males) from five counties in Red
River Valley, Minnesota, USA (n =
851 were married or in a relationship) among applicators. Data regarding current applied pesticide or where the mother had
Authors combined data about pesticide Preconception exposure to thiocarbamates +++
exposure collected about four pesticide (OR = 1.8; 95% CI, 1.1–3.0), glyphosate (OR
classes (herbicides, insecticides, fungicides = 1.7; 95% CI, 1.0–2.9), fungicides (OR =
and miscellaneous) examining farm 1.4; 95% CI, 0.9–2.1), and the miscellaneous
operators (i.e., husbands and wives) to class of pesticides (OR = 1.5; 95% CI, 1.0–2.4)
construct a history of monthly agricultural were associated with elevated risks of late
and residential pesticide use. spontaneous abortions (12–19 weeks).
Preconception exposure to both fungicides
and herbicides doubled the relative risk of
spontaneous abortions compared to
woman exposed only to fungicides (OR =
2.0; 95% CI, 1.1–3.5).
Exposure to fungicides doubled the risk of
spontaneous abortion compared to those
not exposed (OR = 2.4; 95% CI, 1.0–5.9) in
older women (age 35+). There was no
increased risk of spontaneous abortion
observed in women younger than age 35.
The public land survey system was used to There was no strong association between ++
map local maternal address. Pesticide pesticide classes and fetal death,
exposure was determined by linking the regardless of trimester of exposure.
maternal address and pesticide application There were weakly elevated risks of fetal
reports from the California Pesticide Use death after applications of halogenated
Report database. hydrocarbons (Hazard ratios (HR) of 1.3
Five pesticide categories were examined: (95% confidence interval (CI): 1.0, 1.8);
phosphates, carbamates, pyrethroids, carbamates/thiocarbamates (HR of 1.3,
halogenated hydro-carbons, and 95% CI: 1.0, 1.8); estrogenic pesticides (HR
endocrine disruptors. of 1.4, 95% CI: 0.8, 2.5); and carbamate
Mancozeb was assigned to the acetylcholinesterase inhibitors (HR 1.4,
carbamates/thiocarbamates class. 95% CI: 1.0, 1.8) during the second
trimester.
There was a moderately increased risk of
fetal death after exposure to halogenated
hydrocarbons (HR of 1.4, 95% CI: 1.0, 2.0)
during the fourth and fifth months of
pregnancy, with similar results for
exposure to carbamate and carbamate
inhibitors during the third and fourth
months of pregnancy.
Marginal increases were observed in HRs
following exposure to carbamates during
the first and second trimesters. A monthly
analysis demonstrated an increased risk
only after exposure during the third and
fourth months of gestation.
Pesticide use Paternal pesticide exposure +++
Phone interviews were conducted to Of 540 respondent households with
gather information on pesticide use children where the biologic father had
and past pesticide use were gathered, been pregnant, 25% of women experienced
including product names and the number a miscarriage. More miscarriages were
of days/year spent applying pesticides. reported among women whose spouses
A follow-up questionnaire at 6-months had applied herbicides, insecticides, and
was used to record applicator use by fungicides than any other pesticide
pesticide class (herbicides, insecticides, application group. Significant increases in
fumigants, and fungicides), acreage fetal loss were observed after use of
treated, type of crop, and use of personal organotin (OR = 1.55; CI 1.01–2.37)
protective gear. The initial and follow-up and/or use of EBDC (ethylene
questionnaires included an overlap period bisdithiocarbamate) fungicides (OR =
that was used to validate pesticide class. 1.77; CI 1.11–2.83) such as Mancozeb,
when compared to nonuse of fungicides.
Maternal pesticide exposure
Only 36 of 379 women who responded
applied or mixed pesticides. For these
women, mixing, loading and applying
pesticides was a significant risk factor
(fetal loss/pregnancy OR = 1.81; CI
1.04–3.12), with 30% experiencing fetal
loss compared to 21% of women who did
not mix or apply pesticides.
Miscarriage and season
The number of first trimester miscarriages
in spring was significantly elevated for (1)
any pesticide class and (2) herbicide use
but no fungicide use when compared to all
 J. Runkle et al. / Environment International 99 (2017) 29–42 39

Table 6 (continued)

Source Designa Population Mancozeb assessment Outcome Confidenceb

other seasons, but no peak in the

Garry et al. (2002b) CrSe Families of licensed pesticide
applicators in Red River Valley,
Minnesota, USA (N = 536 couples)
Farr et al. (2004) CrSe Women living on farms in Iowa and
North Carolina (N = 3103)
Idrovo et al. (2005) CrSe Female flower production workers in
Bogota, Colombia (N = 2085).
Nordby et al. (2005) RCo National registers in Norway
(1925–1971), identifying female (n = were collected from ag-censuses and
105,403) and male farmers (n =
131,243) and their children (n = 300, included in the analysis as proxies for
805).
Lacasana et al. CaCo 151 cases of anencephaly of N20
(2006) (paired) weeks' gestation
151 controls born in three Mexican
states
Phone survey on current and past
pesticide use in agriculture with 6 month
follow-up interview to validate pesticide
class.
Class of pesticide
Herbicides
Insecticides
Fungicides
Fumigants
Pesticide exposure questions assessed
lifetime use of any of 50 examined
pesticides.
An exposure matrix was constructed
based on history of work in flower
production.
Farm production and fungal forecasts data
weather stations. These measures were
Mancozeb exposure.
A questionnaire obtained information on
lifetime occupational history, history of
agricultural work, and whether or not the
participant directly handled any pesticide.
frequency of miscarriages during the
spring was observed among applicator
spouses who applied herbicides with
fungicides compared to application of
herbicides without fungicides.
There was an increased frequency of
first-trimester miscarriages during
summer, fall, and winter seasons for
applicator spouses when compared to
non-applicator spouses.
There was a significant reduction in male
births (25%) among applicators who
applied herbicides, insecticides, and
fungicides and among those applicators
who applied Mancozeb and/or
triphenyltin fungicides.
There was an increased frequency of male ++
births in families with pesticide applicator
fathers (excluding fungicides) compared
to families with non-applicator fathers.
There was an increased frequency of male
children born with birth defects compared
to female children (M/F sex ratio = 1.8)
when no fungicides were applied.
If fungicides were applied by the male
partner, far fewer male children with birth
defects were born (M/F sex ratio = 0.57, p
= 0.02).
No difference was observed in birth rate
for families of fungicide applicators
compared to applicators of herbicides and
other products (2.85 vs. 2.80 children per
family).
Carbamate pesticides use was associated ++
with increased odds of long menstrual
cycles (OR = 2.1, 95% CI: 1.1–3.7).
Women who used Mancozeb or maneb
had 4.7 times the odds of experiencing
long cycles (95% CI: 1.3–16.6) and 2.5
times the odds of a missed menstrual cycle
(95% CI: 1.0–5.9) compared to those who
had never used pesticides.
A sub-analysis excluding female licensed
applicators and spouses who had not likely
used hormonally active pesticides in the
last year showed elevated odds of
prolonged menstrual cycles, missed cycles,
and intermenstrual bleeding among
women exposed to lindane, atrazine, or
Mancozeb or maneb compared to
unexposed women.
A trend in longer time to pregnancy (TTP) ++
was observed in women engaged in any
type of wage work.
A positive dose-response relationship
between duration of work in flower
production and TTP was observed.
Number of neural tube defects (131 cases, +++
prevalence 12.8/10,000 births) was
moderately associated with potato
cultivation (PR 1.6, 95% CI 1.1–2.3) and
paternal work of N500 h/year (PR 1.6, 95%
CI 1.1–2.5).
Mancozeb exposure was not associated
with thyroid cancer.
An increased risk of anencephaly (OR = ++
4.57, 95% CI 1.05 to 19.96) was observed
among children of mothers who worked in
agriculture during the periconception
period.
Risk of fathering an anencephalic child
was greater for pesticide applicators no
matter whether application occurred in
the acute risk period (ARP) or not (OR =

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40 J. Runkle et al. / Environment International 99 (2017) 29–42

Table 6 (continued)

Source Designa Population Mancozeb assessment Outcome Confidenceb

2.50, 95% CI 0.73 to 8.64; and OR = 2.03,

95% CI 0.58 to 7.08, respectively).
Pesticides most often used by case parents
in decreasing order of frequency were:
permethrin (pyrethroid), methamidophos
(organophosphate), methyl parathion
(organophosphate), atrazine (triazine),
2,4-dichlorophenoxyacetic acid
(chlorinated phenoxy), chlorpyrifos
(organophosphate), Mancozeb
(dithiocarbamate), picloram (pyridine),
dimethoate (organophosphate), and
carbofuran (carbamate).
Pesticides most often used by control
parents in decreasing order of frequency
were: methyl parathion
(organophosphate), methamidophos
(organophosphate),
2,4-dichloro-phenoxyacetic acid
(chlorinated phenoxy), chlorpyrifos
(organophosphate), and monocrotophos
(organophosphate).
Rull et al. (2006) CaCo Infants with neural tube defects Exposure to restricted use pesticides was Conventional logistic regression: ++
(NTDs) (n = 928) and normal formed examined by using a geographic measure 1) Single-pesticide models:
controls (n = 1263) born in California linking pesticide-use reports from the OR: 0.7 95% CI: 0.2, 1.9
(1987–1991). California Department of Pesticide 2) Multiple-pesticide model:
Regulation with land-use survey maps of OR: 0.5 95% CI: 0.1, 2.3
crops from the Public Land Survey System. Hierarchical logistic regression:
A physiochemical category for 1) Multiple-pesticide model:
Dithiocarbamate and pesticide category OR: 0.5 95% CI: 0.2, 1.4
for Mancozeb were included in the Increased odds of 1.5 (95% CI: 1.2, 2.0) for
analysis. any NTDs observed among maternal
Mancozeb was classified as an endocrine residences within 1000 m of any applied
disruptor. endocrine disrupting pesticide compared
to no exposure to endocrine disruptors.
The effect of exposure to 2 or more
endocrine disrupting chemicals applied
within 1000 m of maternal residences
resulted in twice the odds of having a baby
born with anencephaly compared to
babies in the no exposure group (95% CI:
1.3, 3.0).
Calvert et al. (2007) CaS Three cases of mothers and their North Carolina Department of Agricultural All three mothers unknowingly worked +
infants born with congenital and Consumer Services (NCDACS) and the during REIs in tomato fields during critical
abnormalities within 8 weeks of each Florida Department of Agriculture and gestational periods for the developmental
other. All the mothers worked for the Consumer Services (FLDACS) provided system that was ultimately affected in
same tomato grower. maternal exposure information that their infants. Cases 1 and 2 were
included pesticide application and worker potentially exposed to Mancozeb during
assignment records. the maximal sensitivity period.
a
Design—Co: Cohort; RCo: retrospective cohort; CaCo: case-control; CrSe: cross-sectional; and CaS: case series/case report.
b
Confidence ratings in body of evidence ratings: High confidence (++++) in the association between exposure to Mancozeb and the outcome. The true effect is highly likely to be
reflected by the apparent relationship; Moderate confidence (+++) in the association between exposure to Mancozeb and the outcome. The true effect is may be reflected in the appar-
ent relationship; Low confidence (++) in the association between exposure to Mancozeb and the outcome. The true effect is likely to be different than the apparent relationship; and Very
low confidence (+) in the association between exposure to Mancozeb and the outcome. The true effect is highly likely to be different than the apparent relationship.

of pesticides (OR 1.81; CI 1.04–3.12) and use of EBDC (ethylene
bisdithiocarbamate) fungicides (e.g., Mancozeb) (OR 1.77, CI: 1.11–
2.83). This study also observed an increase in first-trimester miscar-
riages for summer, fall, and winter combined among spouses of pesti-
cide applicators who worked with fungicides compared to spouses
who did not use fungicides (p = 0.05).
Women exposed to thiocarbamates during pregnancy in 10 Califor-
nia counties exhibited a modest risk of fetal death during the first and
second trimesters, especially the third (OR 1.4 CI 1.0–1.9) and fourth
month (OR 1.4, CI 0.9–1.9) of pregnancy (Bell et al., 2001). In this
study, Mancozeb was 1 of 22 chemicals assigned to the carbamate/
thiocarbamate pesticide class. One paired case-control study in central
Mexico observed an increased risk of anencephalic infants among
mothers working in agriculture during the three months before concep-
tion (OR 4.5 CI: 1.05–19.96) (Lacasana et al., 2006). Furthermore, fathers
who applied pesticides three months before conception experienced 2.5
times the risk of an anencephalic child compared to non-agricultural
workers (CI 0.73–8.64) (Lacasana et al., 2006). Mancozeb was one of
several pesticides (e.g., chlorpyrifos, methyl parathion,
methamidophos, Mancozeb, 2,4-D, and atrazine) used by male applica-
tors associated with the anencephaly cases that were suspected to have
adverse reproductive health consequences.
4. Discussion
4.1. Summary of findings
The evidence summarized in our review of thirty in vitro, animal,
and epidemiological studies suggests that a range of high and low
doses of exposure to Mancozeb individually or in a mixture of multiple
pesticide results in significant reductions or impairment in reproduction
and developmental risk to humans either occupationally or residentially
exposed and these associated risks should be strongly considered in the
registration review of Mancozeb (Table 2). While the OHAT framework
 J. Runkle et al. / Environment International 99 (2017) 29–42
does not yet incorporate in vitro studies, we relied on results from these
cell culture studies to either upgrade or downgrade evidence collected
from animal and human studies. Data from in vitro studies provide a
high to moderate level of confidence that Mancozeb may indirectly dis-
rupt or impair reproduction at the cellular level and should be regarded
as a reproductive toxicant. High confidence ratings from in vitro and an-
imal studies, in combination with low to moderate confidence ratings
from human health studies provided sufficient evidence that Mancozeb
should be regarded as a presumed reproductive hazard to humans and
as a suspected developmental hazard to humans.
Animal studies provided high to moderate confidence for reproduc-
tive and developmental toxicity in mammals exposed to Mancozeb,
with over a decade of research demonstrating high endocrine
disrupting potential, including developmental injury in murine pre-im-
plementation embryos, oxidative and genotoxic damage associated
with chronic exposure, premalignant status in somatic cells of mamma-
lian ovarian follicles, and dose-dependent anti-androgenic effects likely
involved in fetal disruption of male sexual development. Several exper-
imental animal studies documented a reduction in reproductive poten-
tial in female animals at higher levels of exposure, while mixture studies
revealed that prenatal exposure to low doses of pesticide mixture con-
taining Mancozeb may lead to adverse developmental toxicity in F1
generations. Changes in the developing mouse cerebellar cortex, devel-
opmental delays at dose levels where single pesticides showed no ef-
fects, and genital malformations in male rat pups were just a few of
the developmental consequences of Mancozeb exposure demonstrated
in our review.
Results from human observational studies provided a moderate to
low level of confidence for Mancozeb as a reproductive toxicant and a
low level of confidence for the chemical as a developmental toxicant. Ep-
idemiological studies examining the effect of Mancozeb at preconcep-
tion were mixed, but overall results suggest that Mancozeb may
interfere with the normal reproductive functioning of women persis-
tently exposed (either occupationally or indirectly by spouse's occupa-
tion) and thereby may delay or complicate pregnancy.
5. Future research directions
Our review identified a gap in population-based health studies, es-
pecially recent studies conducted within the last five to seven years, ex-
amining the association between occupational or environmental
exposure to Mancozeb and adverse reproductive or developmental out-
comes. In particular, an important limitation of the evidence base is a
lack of longitudinal cohort studies examining a wide range of types
(e.g., individual pesticide versus pesticides in a mixture, chronic versus
acute exposure, preconception versus early childhood exposure pe-
riods) and levels (e.g., low doses versus high doses) of exposure to
Mancozeb - including those that may occur to workers in agricultural
and factory settings that utilize or produce Mancozeb, and/or the family
members of such workers - and the association with adverse reproduc-
tive and developmental health outcomes in workers and their families.
To better improve the quality of the evidence base, and thereby better
elucidate the role of Mancozeb exposure in reproductive and develop-
mental health, studies are needed that employ direct measures of expo-
sure and/or biomarker data to accurately assess both low dose
exposures and cumulative exposures, and study designs that link expo-
sure data to adverse reproductive health outcomes and other develop-
ment health endpoints during critical windows of development. In
particular, occupational health studies of exposure to Mancozeb in
farmworker populations are critically important to better understand
the reproductive health consequences of chronic exposure, particularly
in conjunction with additive effects of other chemical and environmen-
tal exposures that may be occurring simultaneously.
The combination of potential for teratogenic effects and high expo-
sure levels during preconception or among potentially pregnant farm-
workers merits careful review. The EPA recognized a gap in reliable
41
data documenting the potential for Mancozeb and ETU to cause repro-
ductive harm by retaining the 10-fold FQPA safety factor (10 ×) for
women of childbearing age and for children less than six years old (40
CFR Part 190 2013 ed.). Accurate exposure and risk characterization of
Mancozeb as a potential reproductive toxicant is increasingly becoming
a priority in the scientific community, yet regulatory bodies are lagging
in their consideration of the potential reproductive health impacts fol-
lowing chronic exposure in the reassessment of the currently registered
uses of Mancozeb.
Limited research has been performed on the use of systematic re-
view frameworks to inform regulatory decision-making in chemical
risk assessment, especially as it pertains to environmental and occupa-
tional health and exposure risks to commonly used fungicides, as well
as how to rate the evidence across multiple streams of data from cellu-
lar, animal, and human studies. This paper provides a useful case study
on the systematic application of the NTP OHAT framework to evaluate
the evidence base for Mancozeb as a reproductive hazard and inform
regulatory and public policy decision-making. We urge the EPA to con-
sider the growing evidence of reproductive harm in its registration re-
view of Mancozeb.
Currently there are an estimated 740 registration review cases open
and the EPA plans to review at least 70 chemicals every year until 2017.
The EPA initiates a registration review case by opening the docket for
public comment. The agency is in the beginning stages of implementing
a systematic review approach into their chemical risk assessment and
regulatory decision-making process. This is an especially timely issue
given that the EPA's Integrated Risk Information System (IRIS) program
recently convened their third systematic review workshop in December
2015 to examine recent methodological advancements in reviewing dif-
ferent streams of scientific evidence (epidemiology, toxicology, and
mechanistic studies). While the adoption and use of systematic reviews
in chemical risk assessment is a promising approach in advancing a
standardized methodology for the evaluation of risk and transparent
decision-making, one key challenge is the time burden and tremendous
effort in conducting these types of reviews. Environmental health scien-
tists are uniquely poised to assist the EPA and other regulatory agencies
by providing systematic reviews during comment periods and imple-
mentation at other points in the chemical risk assessment process for
a more thorough characterization of health risks for chemicals undergo-
ing the registration review process, as well as increases the legitimacy,
efficiency, and transparency behind chemical risk assessment, deci-
sion-making, and policy for Mancozeb and other commonly used
pesticides.
Acknowledgement
This work was supported in part by the University of California San
Francisco Program for Reproductive Health and the Environment's
Reach the Decision Makers Fellowship 2013–2014.
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Jennifer Runkle owns and is employed as a consultant by Khloë Solutions, LLC. She partic-
ipated in this research during her time as a postdoctoral fellow at Emory University. Nei-
ther Khloë Solutions nor anyone connected to litigation provided funding for this review.
The other authors declare they have no actual or potential competing financial interests.