### 1 

# Policy Brief

An Updated Look at Mancozeb: Concerns about Farmworker Reproductive and Child Developmental Health

Joan Flocks, Jennifer Runkle, Jeannie Economos, Anne Dunlop and Jennifer Sass

### 2 A n Updated Look at Mancozeb:
Concerns a bout Farmworker R eproductive and Child Developmental Health

Executive summary

Mancozeb is a broad

- spectrum fungici de commonly used in agriculture worldwide. It is a member of the ethylene bisdithiocarbamate (EBDC) group of fungicides, which includes the related active in gredients maneb
and metiram. Currently, exposure to mancozeb is considered to pose minimal human health risks by most regulatory bodies . Despite mounting scientific evidence, the potential adverse re productive and human health developmental effects of manc ozeb

, especially in sensitive subpopulations (e.g., pregnant women, children, and agricultural workers), has not yet been fully considered in the registration and reregistration of this widely used fungicide.

We urge the EPA to systematically evaluate a nd consider the reproductive

, pregnancy , and fetal health impacts of the fungicide mancozeb in its upcoming Registration Review

. In particular, we urge the Agency to consider
the full risks

– including developmental and reproductive risks

- from exposure t o mancozeb experienced by vulnerable populations

, such as all children under the age of 21 and women who work in agricultur al operations (including in fields, nurseries, and ferneries) and who work with agro

- chemicals. The planned scheduled opening of the Registration Review docket for mancozeb is the third quarter of FY2015.

We make this request because current EPA policy does not reflect acknowledged data gaps and uncertainty about the health impacts of mancozeb . Our systematic review of mancozeb researc h indicates that the fungicide presents a risk of both reproductive and developmental harm to humans. Furthermore, recent cases of infants born with severe birth defects to farmworkers possibly exposed to mancozeb in violation of regulations should trigger the EPA to act in a precautionary manner by taking action to address the risk concerns from the use of
mancozeb.

The Problem

Our research with farmworkers of childbearing age demonstrates that : (1) an agricultural work environment characterized by mult iple stressors , including pesticide exposure, prolonged elevated temperatures, and musculoskeletal strain, can influence reproductive health, and (2) the farmworker c ommunity is profoundly

concern ed about the health risks associated with agricultural work, the scarcity of guidelines , information or regulations protecting the health of pregnant women in the workforce, and (3) the uncertainty regarding the impact of these exposures on pregnancy and child health. 1

, 2 

### One chemical that has been of particular co ncern in the farmworker community is mancozeb.

A systematic review of the relevant scientific literature related to mancozeb is provided in Appendix A.
Mancozeb is an ethylene bisdithiocarbamate (EBDC) fungicide with the degradate ethylenethiourea (ETU)

. ETU has known teratogenic, mutagenic, and carcinogenic risks

. The State of California and the Toxic Release Inventory (TRI) lists mancozeb as a carcinogen. The T RI also lists mancozeb as a developmental and reproductive toxicant . One study in rodents has r eported that mancozeb and ETU are capable of crossing the"""""""""""""""""""""""""""""""""""""""""""""""""""""" 1 Flocks J, Kelley M , Economos J, McCauley L. Female farmworkers’ perceptions of pesticide exposure and pregnancy health. J Immigr Minor Health 2012:14: 626

- 632.

2 Flocks J, Thien Mac V, Runkle J, Tovar

- Aguilar JA, Economos J, McCauley L. Female farmworkers’ perceptions of h eat

- related illness and pregnancy health. J Agromedicine 2013:18: 350

- 358.

### 3 placental barrier and can damage DNA and initiate tumors in fetal cells. 3 

### Mancozeb is also a suspected endocrine disruptor and is associated with both hyperthyroidism and hypothyroidism

. 3a,b



### From 2004

- 2006, six infants were born with a range of birth defects to farmworkers in southwest Florida. Of these, three cases were investigated by the Centers for Disease Control and Prevention in collaboration with state health departments in Florida and Nort h Carolina ( the two states in which the mothers worked harvesting
tomatoes for the same agricultural company ). In a report on the cases, mancozeb and ETU were implicated
because in two cases i t could be shown that the pregnant mothers had been exposed to t he fungicide in violation
of Restricted Entry Intervals. Also, the specific birth defects of the farmworkers’ infants resembled those observed in laboratory studies of rats exposed to mancozeb and ETU

. 4 

### This is concerning since o ur research
with female far mworkers in Central Florida revealed higher urinary metabolite levels of ETU compared with

non

- detect levels of ETU in other nationally representative women and Mexican Americans reported in the National Health and Nutrition Examination Survey (NHANES) , su ggesting the risk to women and their babies may be much more widespread than just these documented cases in Florida

. 5



### It is not just pregnant women and developing fetuses that are especially sensitive to pesticides, but al l worker s under the age of 21. Puberty is recognized as a critical or sensitive window of susceptibility to chemicals

, 6 

### particularly with respect to reproductive and endocrine disrupting effects. For example, exposure of rodents to high doses of some phthalates has been shown to delay onset of puberty while pubertal dosing of two rat species with the phthalate DEHP has been shown to d elay onset of puberty and cause testicular damage 7 

### and exposure to various chemicals can delay or accelerate onset of puberty in females. 8 

### Data primarily f rom laboratory animal
and cell culture studies have revealed more than 50 pesticides t hat have known or suspected endocrine disrupting properties on the female reproductive system. 9

, 10 

### These risks of reproductive harm are not fully
addressed in EPA’s mancoz eb registration process.

We recognize that for farmworkers to be adequately protected against occupational hazards, such as pesticides

,""""""""""""""""""""""""""""""""""""""""""""""""""""""

3 Shukla Y, Arora A. Transplacental carcinogenic potential of the carbamate fungicide mancozeb. J Environ Pathol Toxicol Oncol
2001:20: 127

– 131.

3a Goldner WS, Sandler DP, Yu F, Ho ppin JA, Kamel F, and Levan TD. Pesticide use and thyroid disease among women in the Agricultural Health Study. Am J Epidemiol 2010: 171 (4): 455

- 64.

3b Axelstad M, Boberg J, Nellemann C, et al. Exposure to widely used fungicide mancozeb causes thyroid hor mone disruption in rat dams but no behavioral effects in the offspring. Tox Sci. 2011: 120 (2): 439

- 46.

4 Calvert GM, Alarcon WA, Chelminski A, et al. Case report: three farmworkers who gave birth to infants with birth defects clos ely grouped in time and p lace

— Florida and North Carolina, 2004

– 2005. Environ Health Perspect 2007:115: 787

– 791.

5 Runkle JD, Tovar

- Aguilar JA, Economos E, Flocks J, Williams B, Muniz J, Semple M, and McCauley L. Pesticide r isk p erception
and b iomarkers of e xposure in Florida f emal e f armworkers. J Occup Environ Med 2013: 55: 1286

- 92.

6 Woodruff TJ, Carlson A, Schwartz JM, Giudice LC. Proceedings of the summit on environmental challenges to reproductive health
and f ertility: executive summary . Fertil Steril 2008:89: 281

- 300.

7 Noriega NC, Howdeshell KL, Furr J, Lambright CR, Wilson VS, Gray LE Jr. Pubertal administration of DEHP delays puberty, suppresses testosterone production, and inhibits reproductive tract development in male Sprague

- Dawley and Long

- Evans rats. Toxicol Sci 2009:11 1: 163

- 78.

8 Woodruff TJ, Carlson A, Schwartz JM, Giudice LC. Proceedings of the summit on environmental challenges to reproductive health
and f ertility: executive summary. Fertil Steril 2008:89: 281

- 300.

9 Bretveld RW, Thomas CMG, Scheepers PTJ, Zielhuis GA, Roeleveld N. Pesticide exposure: the hormonal function of the female reproductive system disrupted? Reprod Biol Endocrinol 2006: 4: 30.

10 Diamanti

- Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM

, Zoeller RT

, Gore AC . Endocrine

- disrupting chemicals: An endocrine society scientific statement. Endocr Rev 2009:30: 293

- 342

.

### 4 there must be meaningful regulation that is adequately and uniformly enforced. At present , pesticide regulations
are of ten ineffective or unenforced. Furthermore, existing regulations are often not based upon consideration of the reproductive health impacts and developmental outcomes, particularly for chronically exposed and
vulnerable populations such as farmworkers and t heir children . 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 reproduct ive health impacts in the reassessment of the currently registered uses of mancozeb. EPA must consider the growing evidence of human
health harm in its upcoming Registration Review docket for m ancozeb in 2015.

Regulatory History and Current Policy

Accor ding to the 2005 Reregistration Eligibility Decision (RED), 11 

### mancozeb was first registered in the U nited States in 1948 as a broad spectrum fungicide for use in agriculture, tur f management, and horticulture

. The
Mancozeb Task Force was formed in 1994 to r epresent the interests of the m ancozeb registrants, wh ich were then two companies: Rhom and Haas and E.I. DuPont De Nemours. The 2005 RED states that at that time the registrants included Dow AgroSciences, Griffin (now a DuPont subsidiary), and Cerexagri ( RED p. 3)

Mancozeb has demonstrated 50 years of fungicidal efficacy for roughly 400 different plant pathogens. It is commonly used on a variety of crops including tree fruits, vegetable crops, field crops, ornamental plants, and
sod farms. In 2005, the E PA reported that approximately 5.6 million pounds of mancozeb w ere used annually. The United States Geological Survey map and chart below show the estimated amount and distribution of agricultural use for mancozeb in 2009, the last year with confirmed crop data from the Census of Agriculture,
and a breakdown of crops on which mancozeb was used. As the map shows, mancozeb use is particularly heavy
in Florida and North Carolina

.

Source: U.S. Geological Survey, Pesticide Use Maps

– Mancozeb, http://water.usgs.gov/nawqa/pnsp/usage/maps/show_map.php?year=2011&map=MANCOZEB&hilo=L&disp=Mancozeb

. ( Accessed May 23, 2014 )

. 

### The 2005 RED states that m ancozeb and other EBDCs have been the subject of two special EPA reviews,"""""""""""""""""""""""""""""""""""""""""""""""""""""" 11 EPA 2005. Reregistration Eligibility Decision for mancozeb. Office of Prevention , Pesticides, and Toxic Substances. EPA 738

- R

04

- 012

### 5 initiated in 1977 and 1987

, because of particular health concerns, including developmental and thyroid effects, caused by their common degradate ETU . According to the RED

, th e final determination of the second special review concluded that the dietary risks for EBDCs exceeded the economic benefits for 11 fruit and vegetable food/feed crops , resulting in the cancellation of the use of mancozeb and other EBDC s on these specific crops ( RED pp. 2

- 3). The conclusion of the 2005 RED was to allow for continued use of mancozeb provided that certain risk mitigation measures were adopted and labels amended to reflect those measures. Certain uses of mancozeb, however, were voluntarily canceled, including its foliar use on cotton, use on residential lawns/turf,
and use on athletic fields. In the RED , the EPA raised several risk concern s and recognized data gap s for developmental, reproductive, and thyroid toxicity , including:

• Risk estimates wer e not provided for the following scenarios due to lack of worker exposure data: applicator using liquid dip for seed

- piece treatment, applicator using dusts for commercial or on farm seed treatment, and secondary handling for hand planting treated seed pie ces. These data will be required in the DCI for this RED ( RED p. 44)

• EPA intends to issue a DCI requiring submission of additional environmental fate data for mancozeb parent, the mancozeb complex, and the ETU degradate (RED p

. 53)

These above studies wo uld likely be very relevant to an evaluation of the exposures and resultant health risks to workers, including women and children in agriculture fields. It is not clear whether or not EPA ever received these data from the registrants, and, if received, wha t the data concluded.

In addition to the studies above, t he following g uideline stu d i es were identified as missing, and required as part of a DCI (RED p p. 101

- 102):

Toxicology: 870.6200 Acute neurotoxicity Occupational Exposure 875.1100 Dermal exposure m onitoring, outdoor (potato seed piece treatment with liquids and dusts) 875.1300 Inhalation exposure monitoring, outdoor (potato seed piece treatment with liquids and dusts) Toxicology 870.3700 Developmental toxicity study in rabbits
870.3800 2 Generation Reproductive Toxicity Study 870.6300 Developmental neurotoxicity study Special study Comparative thyroid toxicity study in young and adult rats

The RED states that EPA “intends to issue a separate product

- specific data call

- in (PDCI) outlining specific
da ta requirements” (RED p. 103). These above studies are directly and critically relevant to an evaluation of the exposures and resultant health risks to women and children

– including underage workers

- in agriculture fields.
It is not clear whether or not EPA ever received these data from the registrants, and, if received, what the data
concluded regarding developmental and reproductive risks to humans from mancozeb.

Since 2005, the EPA has issued final rules on tolerances for residues of mancozeb on vari ous crops including almonds, cabbage, lettuce, peppers, and broccoli in 2011 12 

### and walnuts and tangerines in 2013. 13 

### In these final"""""""""""""""""""""""""""""""""""""""""""""""""""""" 12 Mancozeb; Pesticide Tolerances. 40 CFR Part 180. 2013 ed.

13 Mancozeb; Pesticide Tolerances. 40 CFR Part 180. 2011 ed.

### 6 rules, EPA continued to acknowledge certain gaps for mancozeb and ETU, especially regarding impact on the developing thyroid a nd reproductive system s

.

The 2013 final rule acknowledge d that maternal mortality, clinical signs, spontaneous abortion, thyroid effec t s, body weight gain decrements, and decreased pup body weight were observed in rat and rabbit studies with
mancozeb. It acknowledge d fetal malformations, hydrocephaly, and domed head were observed in rat and rabbit
studies with ETU.

For mancozeb and ETU, th e EPA retained the 10X FQPA safety factor for women of childbearing age and for
children less than six years old . Ye t, despite the data gap and uncertainty, the EPA determined that children
older than six would be adequately protected if the FQPA safety factor were reduced to 1X , even though these are the years of puberty in which hormonal disruption could render childr en and youth most vulnerable

. It is not clear how EPA made that determination, given the substantial data gaps and lack of relevant information, along with scientific evidence that pregnant women and children under age 21 exposed occupationally to pesticid es represent vulnerable and inadequately

- protect ed populations.

For vulnerable populations, such as farmworkers, who are more frequently and intensely exposed to agrochemicals, the risk associated with data gaps and uncertainties is multiplicative. The EP A has seemingly recognized these inherent risks for farmworkers. In the 2005 RED, the EPA stated: “ Based on its evaluation of mancozeb, the Agency has determined that mancozeb products, unless labeled and used as specified in this document, would present r isks inconsistent with FIFRA. Accordingly, should a registrant fail to implement any of the risk mitigation measures identified in this document, the Agency may take regulatory action to address the risk concerns from the use of mancozeb.” It is not clear to us whether or not the registrants fully implemented the label requirements to mitigate risk, or if not, whether any Agency regulatory action was taken.

Systematic Review of Research on Mancozeb

The e vidence summarized in our review of thirty

- six studi es suggests that mancozeb presents a risk of both reproductive and developmental harm to humans

. Data from i n vitro studies provide strong evidence that mancozeb may indirectly disrupt or impair reproduction at the cellular level and should be regarded as a reproductive toxicant

. Animal studies also provide strong evidence that mancozeb may induce reproductive and developmental toxicity in mammals. A summary of the data is below, and is presented in more detail in the systematic review in Appendix A

.

• In vi tro studies provide strong evidence in support of mancozeb as a reproductive toxicant, with over a decade of research demonstrating high endocrine disrupting potential, significant cytotoxic and pro

apoptotic patterns at 10 and 50 ug/ml between 24 and 48 h ours, developmental injury in murine pre

implementation embryos, oxidative and genotoxic damage associated with chronic exposure, premalignant status in somatic cells of mammalian ovarian follicles, and dose

- dependent anti

androgenic effects likely involve d in fetal disruption of male sexual development.

• Early in vivo studies failed to find a significant effect on reproduction for female rats exposed to the maximum tolerated dose of ETU before and during gestation (80 mg/kg/day); however, significant morph ological fetal abnormalities, with the fetal brain most commonly affected, were detected at doses showing no maternal toxicity or fetal mortality in rat offspring.

• Several studies documented a reduction in reproductive potential in female animals at highe r levels of exposure, while mixture studies revealed that prenatal exposure to a low dose pesticide mixture containing mancozeb may lead to adverse developmental toxicity in F1 generations including disruptions in the developing mouse cerebellar cortex, lo ng

- term delayed effects at dose levels where single pesticides showed no effects, and genital malformations in male rat pups.

### 7 • Animal studies also confirm ed reproductive and developmental toxicity in mammals and suggest ed significant changes in physiologic al, biochemical, and pathological processes involved in normal reproduction may lead to infertility in males chronically exposed to mancozeb.

• Epidemiological studies examining the effect of mancozeb on reproduction were mixed, but results suggest ed that m ancozeb may interfere with normal reproductive functioning of women persistently exposed (either occupationally or indirectly by spouse’s occupation) and thereby may delay or complicate pregnancy.

• F ew population

- based studies have examined the association between exposure to mancozeb and adverse reproductive or developmental outcomes.

Policy Recommendations

In summary, high confidence ratings from in vitro and animal studies, in combination with moderate confidence ratings from human health studies, prov ide sufficient evidence that mancozeb should be regarded as a presumed developmental and reproductive hazard in humans

. We urge the EPA to systematically consider the reproductive , pregnancy, and developmental health impacts of m ancozeb. T he chronic exposu re experienced in vulnerable population s , such as farmworkers and children

, to m ancozeb makes this matter urgent. The planned scheduled opening of the Registration Review docket for m ancozeb is the third quarter of FY2015.

The EPA has previously acknowle dged data gaps in its consideration of mancozeb toxicity. To address th is, the Agency should clearly identify the data for which it issued a DCI and when it expected to receive that data, a
check

- list of the studies that were submitted to EPA in response t o the DCI, a link to the Data Evaluation Record (DER)

, and a short summary of what the data results were. EPA should furthermore indicate if there are any outstanding studies that have not been received, or that were received but were found to be unaccepta ble as guideline studies.

In light of the acknowledged data gaps, we urge the agency to r econsider the 1X FQPA safety factor for
children over six. T he inconsistent safety factor pattern makes little sense in the face of continuing uncertainties
about the health impact s of mancozeb and ETU

, especially for vulnerable populations. Currently, girls and boys ages 6

- 13 receive a 1X safety factor, whereas girls and women ages 13

- 49 receive a 10X safety factor. Yet, children under age 21 should not be evaluated a s adults. Puberty is recognized as a critical or sensitive window of susceptibility to chemicals 14 

### particularly with respect to reproductive and endocrine disrupting effects. For example, exposure of rodents to high doses of some phthalates has been shown to delay onset of puberty while pubertal dosing of two rat species with the phthalate DEHP has been shown to delay onset of puberty and cause testicular damage 15 

### and exposure to various chemicals can delay or accelerate onset of puberty in females. 16 

### Data pr imarily from laboratory animal and cell culture studies have shown over 50 pesticides to have known or suspected endocrine disrupting properties on the female reproductive system. 17

, 18 

### Studies have reported that""""""""""""""""""""""""""""""""""""""""""""""""""""""

14 Woodruff TJ, Carlson A, Schwartz JM, Giudice LC. Proceedings of the summit on environmental challenges to reproductive health
and f ertility: executive summary . Fertil Steril 2008:89: 281

- 300.

15 Noriega NC, Howdeshell KL, Furr J, Lambright CR, Wilson VS, Gray LE Jr. Pubertal administration of DEHP delays puberty, suppresses testoster one production, and inhibits reproductive tract development in male

. Sprague

- Dawley and Long

- Evans rats. Toxicol Sci 2009:111: 163

- 78.

16 Woodruff TJ, Carlson A, Schwartz JM, Giudice LC. Proceedings of the summit on environmental challenges to reproductive health
and f ertility: executive summary . Fertil Steril 2008:89: 281

- 300.

17 Bretveld RW, Thomas CMG, Scheepers PTJ, Zielhuis GA, Roeleveld N. Pesticide exposure: the hormonal function of the female reproductive system disrupted? Reprod Biol Endocrinol 2006: 4: 30.

18 Diamanti

- Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM

, Zoeller RT

, Gore AC . Endocrine

- disrupting chemicals: An endocrine society scientific statement. Endocr Rev 2009:30: 293

- 342.

### 8 dioxin causes abnormal breast development in p re

- pubertal girls, the plastics

- component Bisphenol A (BPA) has shown to cause precocious puberty in girls, the pesticide endosulfan has been reported to affect pubertal boys by slowing down the timing of reproductive maturation, and the fungicides vincloz olin and iprodione have been shown in pubertal male rats to impair normal puberty hormone levels. 19

, 20

, 21

, 22 

### EPA has not adequately addressed the potential risks for reproductive harm from childhood exposures to mancozeb. Until this is done, the full 10X FQPA factor should be retained for all children, male and female, under the age of 21.

EPA should also consider the occupational health of vulnerable populations, such as farmworkers in the case of mancozeb, as part of its risk assessments. Farmworkers experie nce multiple stressors in addition to pesticides, including poverty, lack of sanitation, heat stress from outdoor field work, lack of access to health care, poor housing conditions, risk of pesticide drift into farmworker housing, lack of stability of home life, separation from family, rape and sexual assault risks, and other significant issues. Because of economic instability, it is not uncommon for farmworker women to continue working while pregnant, sometimes well into the third trimester
and our researc h has indicated that there are no special workplace accommodations made for pregnant farmworkers, who risk direct contact to pesticide residues or drift. 23 

### Furthermore, some low

- income
farmworkers do not have access to childcare

, and farmworkers have been k nown to bring their children to the workplace with them. Farmworkers are paid by a piece rate system and the resulting production demands often means they do not have or take the time to practice occupational safety behavior such as frequent handwashing,

w earing adequate protective clothing, or replacing defective equipment. 24

, 25 

### Sometimes workers are not provided with proper PPE and/or decontamination supplies by their employer and must provide it themselves. 26 

### Despite federal regulations, farmworkers often lack access to basic information about pesticides applied at their
worksites. W idespread lack of compliance and inadequate enforcement of current Worker Protection Standards means that many farmworkers receive no or minimal pesticide health and safety trai ning or that information about pesticides applied is not being posted correctly. 27 

### Language barriers can impede the understanding of workers of the risks posed by the pesticides around which they are working, including those who mix, handle, and/or apply t he pesticides. Pesticide labels are generally in English only. Even in the best scenarios in which"""""""""""""""""""""""""""""""""""""""""""""""""""""" 19 Cowin PA, Gold E, Aleksova J, O'Bryan MK, Foster PM, Scott HS, Risbridger GP. Vinclozolin exposure in utero induces postpubertal prostatitis and reduces sperm production via a reversible hormone

- regulated mechanis m. Endocrinology 2010: 151:783

- 92.

20 Blystone CR, Lambright CS, Cardon MC, Furr J, Rider CV, Hartig PC, Wilson VS, Gray LE Jr. Cumulative and antagonistic effects of a mixture of the antiandrogens vinclozolin and iprodione in the pubertal male rat. Toxico l Sci 2009:111:179

- 88.

21 Roy JR, Chakraborty S, Chakraborty TR. Estrogen

- like endocrine disrupting chemicals affecting puberty in humans

-- a review. Med
Sci Monit 2009:15:RA137

- 45.

22 Schoeters G, Den Hond E, Dhooge W, van Larebeke N, Leijs M. Endocrine d isruptors and abnormalities of pubertal development.
Basic Clin Pharmacol Toxicol 2008:102:168

- 75.

23 Runkle JD, Tovar

- Aguilar JA, Economos E, Flocks J, Williams B, Muniz J, Semple M, and McCauley L. Pesticide r isk p erception
and b iomarkers of e xposure in Florida f emale f armworkers. J Occup Environ Med 2013: 55: 1286

- 92.

24 Mayer B, Flocks J, and Monaghan P. The role of employers and supervisors in promoting pesticide safety behavior among Florida
farmworkers. Am J Ind Med 2010:53:814

- 24.

25 Runkle JD, Tova r

- Aguilar JA, Economos E, Flocks J, Williams B, Muniz J, Semple M, and McCauley L. Pesticide r isk p erception
and b iomarkers of e xposure in Florida f emale f armworkers. J Occup Environ Med 2013: 55: 1286

- 92.

26 Flocks J, Kelley M, Economos J, McCauley L. Fema le farmworkers’ perceptions of pesticide exposure and pregnancy health. J Immigr Minor Health 2012:14: 626

- 32.

27 Flocks J, Monagha n P, Albrecht S, Bahena A. Florida f armworkers’ p erceptions and l ay k nowledge of o ccupational p esticides

. J Commun Health 200 7: 32:181

- 94.

### 9 an employer conscientiously complies with all existing regulations, current WPS training requirements do not require information about reproductive health o r risks posed by take

- home exposures, al though this is being considered under the proposed regulations current ly open for public comment.

In 2009, the Committee on Improving Risk Analysis Approaches Used by the U.S. EPA, part of the National
Research Cou ncil (NRC), emphasized the need for cumulative risk assessments

, as defined by EPA, “that include combined risks posed by aggregate exposure to multiple agents or stressors; aggregate exposure includes all routes, pathways, and sources of exposure to a giv en agent or stressor. Chemical, biologic, radiologic, physical, and psychologic stressors are considered in this definition.” 28 

### The NRC report points out that, “although cumulative risk assessment has been used in various contexts, there has been little co nsideration of nonchemical stressors, vulnerability, and background risk factors” and recommends EPA seriously consider adopting the recommendations of a NRC report on phthalates 29 

### that “addresses issues related to the framework
within which dose

- response a ssessment can be conducted in the context of simultaneous exposures to multiple
stressors.” The NRC report also emphasize s that “variability in human susceptibility has not received sufficient or consistent attention in many EPA health risk assessments... gr eater attention to susceptibility in practice is needed, particularly for specific population groups that may have greater susceptibility because of their age, ethnicity, or socioeconomic status.” 30 

### EPA should implement the recommendations of these NRC rep orts with mancozeb and other pesticide assessments.

Finally, m ore detail is needed for EPA’s assessment of endocrine effects of mancozeb. EPA should describe how the agency will move forward on its analysis of endocrine disrupting effects and specificall y address how it
consider s non

- linear dose responses and adverse health effects occurring at extremely low doses (e.g. parts per trillion) for which there is a growing body of scientific evidence. For example, pesticides, at extremely low levels, can inter fere with the proper functioning of hormone systems including estrogen, androgens, thyroid, and the pituitary/hypothalamic axis. When exposures, even at very low levels, coincide with critical windows of development, they may be more potent than higher dos e exposures, or the low dose exposure may otherwise

exert a non

- traditional dose

- response curve. The consequences of exposure to an endocrine disruptor may also be delayed with additive or synergistic effects from a myriad of chemical exposures. Da ta prima rily from laboratory animal and cell culture studies have shown that more than 50 pesticides have known or suspected endocrine disrupting properties on the female reproductive system. 31

, 32

### """"""""""""""""""""""""""""""""""""""""""""""""""""""

28 National Research Council. Science and Decisions: Advancing Risk Assessment . Washington, DC: The National Academies Press, 2009, 478 pages.

29 National Research Council. Phthalates and Cumulative Risk Assessment: The Task Ahead . Washington, D C: The National Academies Press, 2008, 208 pages.

30 National Research Council. Science and Decisions: Advancing Risk Assessment . Washington, DC: The National Academies Press, 2009, 478 pages.

31 Bretveld RW, Thomas CMG, Scheepers PTJ, Zielhuis GA, Roeleveld N. Pesticide exposure: the hormonal function of the female reproductive system disrupted? Reprod Biol Endocrinol 2006:4: 30.

32 Diamanti

- Kandarakis E, Bourguignon JP, Giudice LC, Hauser R, Prins GS, Soto AM

, Zoeller RT

, Gore AC . Endocrine

- disrupting chemicals: An endocrine society scientific statement. Endocr Rev 2009:30: 293

- 342

.

### 10 Appendix A: Systematic Review Findings

In Vitro Studies. S even in vitro studies published between 2000 and 2012 met our inclusion criteria (Figure 1, Table 1). We found seven in vitro studies in which the reproductive toxicity of mancozeb was examined. The
media for most of these studies was mammalian cells. All of these studies provide evidence that mancozeb may indirectly disrupt or impair reproduction at the cellular level.

Lin et al. 1 

### showed mancozeb induced cytotoxicity and apoptotic patterns of cell death at doses of 10 and 50 ug/ml between 24 and 48 hours of exposure, while ETU produced apoptotic induction at much higher concentrations (1000 ug/ml). A later study examining adrenocortical steriodogenic cells of rainbow trout demonstrated significant impairment of cortisol secretion at low

- doses (50 ug/ml) and showed mancozeb as the highest ranked endocrine disru ptor based on LC50/EC50 values. 2 

### Mancozeb exposure did not result in increased
embr yo toxicity or a higher incidence in developmental abnormality in chicken embryos, but when mixed with copper sulfate showed significant embryomortality compared to individual doses. 3 

### Another mixture study revealed that single exposure to mancozeb induced developmental injury in murine pre

- implemenation embryos and a fungicide mixture (including mancozeb) reduced blastocyst development and increased apoptosis. 4



### Calviello et al. 5 

### studied the DNA damaging and proapoptotic effects of mancozeb in RAT

- 1 fibroblasts and peripheral blood mononucleated cells (PBMC) isolated from Wistar rats at one and four hours post

- exposure. Following one hour of exposure to 500 ug/ml of mancozeb, fibroblast cells produced dose

- dependent induction of single strand breaks and elevated levels of DNA o xidation and reactive oxygen studies. After four hours,
mancozeb induced dose

- dependent apoptosis in fibroblasts and PBMC cells.

Finally, two recent studies demonstrate mancozeb should be regarded as a reproductive and developmental
toxicant. Paro et al . examined the toxic effects of mancozeb exposure on granulosa cells from mice and women undergoing assisted reproductive procedures. 6 

### Granulosa cells from mice exposed to mancozeb showed time

and dose

- dependen t changes in morphology, an acquired ability to migrate, decreased levels of p53, but no changes in apoptosis. Human granulosa cells showed dose

- dependent morphology changes and reduced p53 expression post

- exposure to mancozeb. This study demonstrates that mancozeb damages the somatic cells of mammalian ovarian follicles by inducing a premalignant

- like status in both mice and human granulosa cells. In 2013, Kjeldsen et al. 7 

### demonstrated the in vitro effects of mancozeb on the androgen receptor (AR) function in human breast carcinoma MVLN cells and hamster ovary CHO

- K1 cells. Results showed concentration

dependent effects on the agonist

- induced AR transactivity, with mancozeb showing the highest inhibitory effect, suggesting a link between mancozeb a s an AR disruptor and disorders of male sexual health. Because

m ancozeb has been shown to cross

- placental barriers in mice, it is likely that mancozeb can disrupt fetal reproductive development in males.

Overall, in vitro studies provide strong evidence that mancozeb is a reproductive or endocrine disrupting
xenobiotic.

Animal Studies. Fifteen experimental studies in mice , rats, rabbits

, and pigeons published between 1973 and 2012 met our inclusion criteria (Figure 1, Table 2). In all fifteen studies

, m ancozeb was either administered orally in foo d or delivered directly into the stomach (gavage) for lengths of time ranging from 30 days to 1 or more years. While each study included in the review measured individual exposure to mancozeb, several studies
si multaneously examined exposure to other chemical agents including: maneb, probineb, endosulfan, and phosphamidon. Four studies assessed independent exposure to mancozeb and then as a mixture with copper oxycloride ( one study ), paraquat ( one study ), and pr ocymidone, epoxicanazole, tebuconazole, and prochloraz ( two studies ). Duration of exposure and dose ranges in female studies ( twelve studies ) were categorized as: (1)
15 to 30 before gestational days (lowest dose: 500 mg/kg/day and highest dose: 800 mg/kg/ day); (2) 1 to 30

### 11 gestational days (lowest dose: 5 mg/kg/day and highest dose: 1,300 mg/kg/day); and (3) postnatal days (lowest dose: 6.25 mg/kg/day and highest dose: 500 mg/kg/day). For studies examining only males (n=3), length of experiment time ranged from 5 to 360 days, with daily doses ranging from 10 to 1,500 mg/kg. Details are
below.

Reproductive & developmental endpoints

Females. Two early experimental studies dating back to the 1970s examined the teratogenic effects of mancozeb. Khera investiga ted prenatal survival and teratogenicity in rats and rabbits. 8 

### Results indicated no significant effect on reproduction for female rats exposed to the maximum tolerated dose of ETU before and during gestation (80 mg /kg/day). However, significant morphological fetal abnormalities, with the fetal brain most commonly affected, were detected at doses showing no maternal toxicity or fetal mortality in rat offspring. While increased resorption sites and reduced brain weigh t at 80 mg/kg/day were noted for female rabbits, rabbit offspring showed no sensitivity to mancozeb at any dose level and no teratogenic effects on developing brains.
Larsson et al. 9 

### reported profound internal and skeletal malformations in rat offspring. For the highest level of
mancozeb exposure (1330 mg/kg/day), the frequency of internal malformations was markedly higher than that
of external malformations. The low

- dose group (380 mg/kg/day) resulted in significant reductions in fetal weight for exposed mice. Fo llowing a twenty

- year gap, a collection of animal studies examined the effect of mancozeb exposure on reproductive and pregnancy health in female rats with mixed results. While an earlier study 10 

### showed no change in the number of estrous cycles post

- mancozeb exposure, several studies documented a reduction in the number of estrous cycles, duration of proestrus, estrus, and metestus, healthy follicles and
number of co rpora lutea with greater than 600 mg/kg/day mancozeb treatment (700 mg/kg/day and 800 mg/kg/day). 11

- 13 

### A significant increase in thyroid weight was also observed in all rats treated with mancozeb

, except for the gro up treated with 500 mg/kg/d. 11,12 

### High prenatal doses of mancozeb (500 mg/kg) resulted in reduced maturity and fertilizability of oocytes in females of the F1 generation. 14 

### These studies mark the first report on the effects of mancozeb on reproductive potential in animals

.

Whole animal studies examining prenatal exposure to mancozeb report significant adverse effects . Axelstad et
al. 15 

### observed toxic effects in rat dams treated with high doses of mancozeb (150 mg/kg/d) including severe weight loss and temporary paralysis in hind limbs. Results also showed a dose

- depende nt decline in T4 levels for all dams exposed to mancozeb (50 mg/kg, 100 mg/kg, and 150 mg/kg) on gestation day fifteen . Four mixture studies included in the review revealed that prenatal exposure to a low dose pesticide mixture containing mancozeb may lead to adverse developmental toxicity including disruptions in the developing mouse
cerebellar cortex 16 

### , long

- term delayed effects at dose levels where single pesticides showed no effects 17 

### , and genital malformations in male rat pups. 18



### Males . Experimental studies showed significant declines in male reproductive functioning including, spermatoxic e ffects in mice, and biochemical and structural alterations in testes of rats and mice following chronic exposure. Khan & Sinha 19 

### observed decreased sperm counts and significantly higher sperm head abnormalities in all three groups exposed to mancozeb (3, 6, or 1,000 mg/kg/day). Abnormal spermatic head size occurred most frequently in mice treated with mancozeb. A long

- term experimental study 20 

### in male rats resulted in higher mortality

- re lated acute effects in all groups fed mancozeb. During months 5 through 12, male rats demonstrated prolonged reproductive effects at higher doses of mancozeb (1,000 and 1,500 mg/kg/d) including a small increase in testes weight and reduction in epididymis size. Animals in the high dose group (1,500 mg/kg/d) demonstrated temporal changes in pathology for seminiferous tubules, with pronounced damage in epithelial cells of epididymis resulting in reduced sperm count. Ksheerasagar & Kaliwal 21 

### also found
time

- dependent declines in weight of testes and other organs (liver, kidneys, and spleen), as well as, si gnificant increases in thyroid and thymus weight of treated mice.

### 12 Animal studies provide strong evidence for acute toxicity in mammals and suggest significant changes in physiological , biochemical, and pathological processes involved in normal reproducti on may lead to infertility in males chronically exposed to mancozeb.

Human Health Studies. Fourteen epidemiologic studies published between 2001 and 2007 met our inclusion criteria (Figure 1, Table 3.) The majority of studies included used a case

- control ( six studies ) and cross

sectional design ( four studies ), with the exception of one case study and three cohort studies ( one prospective study and two retrospective studies ). We observed substantial differences in mancozeb exposure assessment.
Most studies assessed exposure indirectly, using pesticide use questionnaires or database registries ( eleven
studies ) . Three studies examining Italian vineyard workers obtained individual measures of recent mancozeb exposure by measuring urinary metabolite levels of E TU. 22

- 24



### Pre

- conception

. Studies examin ing the fertility effects of mancozeb or other members of the ethylene bisdithiocarbamate (EBDC) group of fungicides, which includes the related active ingredients maneb and

m etiram

, were mixed. Farr et al. 25 

### found that women who reported using mancozeb or maneb were four times more likely to experience longer menstrual cycles and two times more likely to have a missed a menstrual cycle compared to unexposed women living on farms in Iowa or North Carolin a. In a case study of three women working in similar agricultural industries around the same time, two of the women were exposed to mancozeb during the first trimester, and all three women gave birth to infants with severe malformations.

Mancozeb is cons idered an endocrine disruptor. While Rull et al. observed no apparent effect on neural tube defects when mancozeb was applied within 1,000 meters of maternal residences, authors found an increased
odds ratio of 1.5 and 2.0 of having a baby with a neural tu be defect or anencephaly, respectively

, among pregnant women living within 1,000 meters of where two or more endocrine disrupting chemicals were applied. 26



### Some studies report on the fertility impacts of fungicide exposures generally, but do not identify which fungicides the subjects were exposed to. A trend in longer time to pregnancy was observed in female flower production workers exposed to fungicides year

- round. 27 

### In a n early study, Arbuckle et al. observed elevated
risk of late

- term spontaneous abortions among farm couples exposed to fungicides and double the risks of spontaneous abortions in older women. 28 

### A later study demons trated no difference in birth rate for families of fungicide applicators compared to applicators using herbicides and other agrochemicals (2.85 vs 2.80). Garry et
al. 29 

### examined spouses of pesticide applicators in the Red River Valley and found an association between personal use of pesticides and an increased risk of fetal lo ss. This study also observed an increased frequency of miscarriages for summer, fall, and winter combined among spouses of pesticide applicators who worked with fungicides.

A modest risk of fetal death during the first and second trimesters , especially th e 3 rd 

### and 4 th 

### month of pregnancy, was found in women exposed to thio carbamates during pregnancy. 30 

### In this study, mancozeb was assigned to the carbamate/thiocarbamate pesticide class. One study showed an increased risk of birth defects following parental exposure to fungicides during pregnancy, including an increa sed risk of anencephaly in children of
mothers work ing in agriculture during the three months before conception and whose fathers also applied pesticides at work

. 31



### These results provide some evidence that mancoze b is a reproductive toxicant. Mancozeb exposure may interfere with normal reproductive functioning of women persistently exposed (either occupationally or indirectly by spouse’s occupation) and thereby may delay or complicate pregnancy. Very few population

- based
studies have examined the association between exposure to mancozeb and adverse reproductive or developmental outcomes.

### 13 Figure 1. Flow diagram of study selection process for systematic review

.

Overview of Systematic Review Protocol ( Adapted f rom National Toxicology Program Office of Health Assessment and Translation approach for systematic review and evidence integration from literature

- based health assessments)

1. Draft protocol strategy

a. Discuss lit erature search strategy b. Determine criteria fo r selection of studies relevant to review question

c. Discuss data abstraction components (risk of bias assessment and confidence in evidence)
2. Search strategy

a. Decide on search criteria b. Search literature

c. Read abstracts and select studies to include in review; obtain full paper
3. Data abstraction

a. Develop and test data abstraction form
b. Extract data & assess quality of study using OHAT criteria (BPA example)

c. Assess confidence in studies
4. Translate confidence ratings into level of evidence for health effect

### 14 Table 1

. In vitro studies (seven studies) examining exposure to Mancozeb and reproductive and developmental outcomes.
Source Type of
cell/tissue Compound (Daily Dose) Incubation Outcomes Confidence*

Lin & Garry, 2000 1 MCF

- 7 breast
cancer cell line Herbicides : 2,4

- D LV4 (0.1

- 10 ug/ml) 2,4

- D Amine (1

- 10 ug/ml) 2,4

- Dic hlorophenoxyacetic acid, isooctyl ester (1

- 10 ug/ml) 2,4

- Dichlorophenoxyacetic acid Roundup (1

- 10 ug/ml) Glyphosate (0.228

- 2.28 ug/ml)

Fungicides : Triphenyltin acetate Mancozeb (reagent grade) (10

- 50 ug/ml) Manzate (commercial grade, 75% mancozeb)
Til t (reagent grade) Tilt (commercial grade, 42% propiconazole) Test chemicals
added within 2 to 4
hours of incubation
and cultured for 72
h ou r s in media Three fungicides (triphenyltin, mancozeb,and Tilt) induced cytotoxicity over the concentration range tes ted ( 50

– 70% decrease in cell number at the highest concentration tested )

Triphenyltin (4.1 ug/ml) and mancozeb (at 10 and 50 μ g/ml) showed an apoptotic pattern of cell death between 24 and
48hrs.

ETU induced apoptosis at a much higher concentration (100 0 ug/ml). +++

Bisson et al. 2002 2 Adrenocortical
cells of rainbow

trout Atrazine (0.0005, 0.05, 5, 500 uM)

Diazinon (0.05, 0.5, 5, 50, and 500 uM)

Endosulfan (0.05, 0.5, 5, 50, and 500 uM)

Mancozeb (0.5, 5, 50, 500 and 5,000 uM) 60 minutes Mancozeb exposed cells showed significant effects on cortisol secretion and on viability.

• Significant impairment of
cortisol secretion and no sig nificant reduction of viability
at a concentration of 500 u M.

• S ignificant reduction of cortisol
secretion (no reduction in viability reporte d ) a t 50 u M of
mancozeb

.

• Mancozeb had the highest
LC50/EC50 ratio *

.

* The ratio LC50/ EC50 for mancozeb
was 16

— a concentration about 16

- fold higher was required to kill 50% of cells than the concentration required to impair
50% of cortisol secretion. ++++

### 15 Fejes et al. 2002 3 288 chicken embryos 80% mancozeb containing formulation (Dithane M

- 45)

Copper

- sulfate (0.1% applied concentration) 19 days Mancozeb did not increase embroytoxicity or result in an increased
incidence in developmental
abnormalities.

Increased embryomortality was observed in embryos exposed to both mancozeb and copper

- sulfate. ++

Greenlee et al. 2004 4 Mice embryos Several agrochemicals were tested at
low

- doses in the study.

Mancozeb (.003 ug/mL) 96 hours Mancozeb induced developmental injury in pre

- implementation embryos

Fungicide mixture (including mancozeb) reduced development to blastocyst and increased apoptosis +++

Calv i ello et al. 2006 5 Wistar rats :

1) RAT

- 1
fibroblasts

2) Peripheral
blood
mononucleated cells (PBMC) Mancozeb and Zineb (concentrations ranging from 125 to 500 ng/ml) 1 hour and 4 ho urs At 1 hour of mancozeb exposure (500ng/mL):

• Cells produced dose

- dependent induction of single strand breaks
in RAT

- 1

• Increased levels of markers of
DNA oxidation and reactive oxygen species in RAT

- 1

• PBMC less responsive than
RAT

- 1 to oxidative insult Af

ter 4 hours of exposure, results show ed
dose

- dependent:

• Oxidative effect and prooxidant
action of Mancozeb

• Apoptosis in PBMC and RAT

- 1
cells ++++

Paro et al. 2012 6 Mouse granulosa
cells and human granulosa cell s from women undergoing
assisted reproductive therapy Mancozeb (increasing concentrations (0.001

- 1 ug/ml) 1, 24, and 36 hours Mouse granulosa cells exposed to
mancozeb showed time

- and dose

dependent changes in morphology, acquired ability to migrate, decr eased level of p53, and no change in apoptosis

Human granulosa cells showed dose

dependent changes in morphology and reduction in p53 expression post

mancozeb exposure. ++++

Kjeldsen et al. 2013 7 human breast
carcinoma
MVLN cells and 14 pesticides were tested

Mancozeb concentrations tested singly: 48 hours When tested as a single compound, mancozeb exposure showed
concentration

- dependent antia ndrogenic ++++

### 16 hamster ova ry CHO

- K1 cells LOEC (M): 1 x 10

- 5 MOEC (M): 1 x 10

- 5 % of Solvent control (0.02% DMSO): 45 effects on agonist

- induced AR transactivity.

At the lowest tested dose inducing the maximum effect (MOEC), mancozeb showed the highest inhibitory effect in the form of an agonist

- reduced response.

* 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#th e#outcome.#The#true#effect#is# may#be reflected#in#the# apparent#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 confiden ce (+)#in#the#association#between#exposure#to#mancozeb#and#the#outcome.#The#true#effect#is# highly#likely#to#be different#than#the#apparent# relationship.##

Table 2. In vivo studies ( fifteen studies ) examining exposure to Mancozeb and reproductive and devel opmental outcomes. Source Experimental
Animal n Compound (route) Daily Dose Duration Outcomes Rating*

Khera 1976 8 Nulliparous adult
Wistar female rats
& New Zealand
White rabbits 209 rats
33 rabbits Mancozeb (o ral) 0, 5, 10, 20, 40, or 80 mg/kg/day Rats: Group I : 21

- 42 days BG and
GD 1

- 15; Group II : 6

- 15
GD and Group III : GD
7

- 20

Rabbits:
30 GDs Prenatal survival in rats

. Numbers of corpora lutea, live fetuses,
and fetal death at all doses were similar

to c ontrols in all the three experiments.

Doses of 80 mg/kg in Group 2 and 40 mg/kg in Groups 1 and 3 reduced the mean fetal weight as compared to matching control groups.

ETU at the maximum tolerated doses administered before and during gestation had no s ignificant effect on any reproductive parameter.

Teratogenicity in rats

. ETU was teratogenic in rats at doses that produced no apparent maternal toxicity or fetal lethality.

Significant morphological fetal abnormalities were observed (including jaw, to ngue, eyelids, short or absent tail malformations); fetal brain appeared to be the most commonly affected organ. ++++

### 17 Prenatal survival in rabbits

. Trend observed for (i) increased frequency of resorption sites (including dead fetuses), and (ii) reduced brai n weigh at 80 mg/kg.

Teratogenicity in rabbits

. No skeletal abnormalities observed in
animals treated with mancozeb. Rabbit
fetal brain was insensitive to teratogenic effects at any dose level.

Larsson et al. 1976 9 Three

- month

- old
NMRI mice and virgin female Sprague

- Dawley

rats 56 mice

56 rats Maneb, Mancozeb,
and Propineb (oral) Mice received 400, 770, or 1420 mg/kg
Maneb

380, 730, or 1330 mg/kg
M ancozeb

400, 760, or 2300 mg/kg
P ropineb 18 GDs Mouse : No adverse maternal effects or fetal
effects we re observed for the different dosages of maneb or mancozeb.

Fetal weight was not reduced in the experimental group compared to control groups except in the fetuses from
mothers treated with the lowest dose of mancozeb on day 9.

Rat : Gross malformations occurred in all surviving fetuses after treatment with
the medium and high dosages of maneb and in 25% of those exposed to the highest dose of mancozeb.

Superficial hemorrhages were found in the 2 highest mancozeb dosage groups.

F requency of internal malformations

w as extremely high compared with that
of external malformations for highest
dose of mancozeb

. +++

Khan & Sinha,
1996 19 males of Swiss
albino mice 56 Endosulfan, Phosphamidon, and Mancozeb (oral)

Vitamin C (injection) Pesticides: 3, 6 or 1000 mg/kg/day

Vi tam in C: 35 days Significant decreases in mean sperm counts in each of the three pesticide

treated groups compared to controls

.

All three categories of sperm head ++++

### 18 10 (lower dose), 20 (middle dose) and 40 mg/kg body wt/day (higher dose) abnormalities increas ed significantly ( compared to control ) in the groups treated with each of the three pesticides.

Sperm with abnormal head size were
most common in the mancozeb

- treated

group.

Kacker et al. 1997 20 Male albino rats 200 Mancozeb in peanut oil (oral) 500, 1,000,
and 1,500 mg/kg/day 30, 60, 90, 180, and 360 days Mancozeb at all doses produced signs of poisoning ( diarrhea , dyspnea, salivation, nasal bleeding, hi nd limb paralysis).

Mortality was more pronounced during days 0

- 90 as compared to days 90

- 360.

Higher doses (1,000 and 1,500 mg/kg/d) over a period of 180 and 360 days produced a slight increase in relative weight of testes and a decrease in epididymis.

Testes of rats dosed with mancozeb (1,500 mg/kg/d) produced time dependent pathological changes in
seminiferous tubules and showed damaged epithelial cells in the tubules of epididymis with declines in sperm

count.

Increased activity of LDH, ALP and
de creased activity of ACP and SDH in testes and epididymis

. ++++

Roperto & Galati
1998 32 nonmigratory pigeons 10 mixture of 30%
mancozeb and 15%

copper ox ychloride -- Longterm

exposure over
years Pi geons living in control environment: No apparent optical and ultrastructural
lesions were found in the trachea or lungs via histopathology, scanning EM,

or x

- ray. The metallic components of the fungicidal mixture (Cu and Zn) were not detected in the lung s via spectrometer.

Pigeons living in the exposed
environment: Severity of the histopathological lesions +++

### 19 was dependent on age.

Intimal proliferation of the intra

parabronchial arteries and scattered foci
of fibrosis were also evident as was depositi on of Zn and Cu particles throughout lungs.

Castro et al. 1999 10 Wister rats 120 Mancozeb (mixed into diet) 0, 2000 or 3.000 ppm Group A : GD
1

- 6,

Group B : GD
6

- 15, Mancozeb exposure did not interfere with estrous cycle in pregnant females, maternal weight gain, nor percenta ge of birth viability of pups.

P ups of mancozeb exposed dams experienced d elays in physical development.

P renatal mancozeb exposure also resulted in developmental motor alterations in pups. +++

Mahadevaswami et
al. 2000 13 Female Wistar virgin rats 36 Mancozeb (oral) 500, 600, 700,
and 800 mg/kg/day 15 BG days Treatment with 700 and 800 mg/kg /day
mancozeb showed a decrease in ovarian hypertrophy.

No significant change in the number of estrous cycles and duration of each phase of the estrous cycle with 500 mg/kg/day mancozeb.

Decreased number of estrous cycles, duration of proestrus, estru s, and metestrus with significant increase in the duration of the diestrus phase with > 600 mg/kg/day mancozeb treatment.

Decreased number of healthy follicles
with increase in the number of atretic follicles at higher doses of mancozeb.

No significant changes in the body and organ weight with any dose level of
mancozeb. ++++

### 20 Treatment with 600, 700, and 800 mg/kg/day mancozeb showed a significant decrease in the levels of protein, glycogen, total lipid, phospholipid, and neutral lipid in the liver, uterus, and ovary, with the exception of liver total lipid and uterine glycogen.

Mancozeb treatment reduced the number of healthy follicles with a
concomitant increase in the number of
atretic follicles.

Baligar & Kaliwal
2001 12 Wister rats (virgin) 40 Mancozeb (oral) 500, 600, 700
and 800 mg/kg/d 30 days Significant decrease in the number of estrous cycle, duration of proestrus,
estrus and metestrus with concomitant
increase in duration of diestrus in all

mancoze b tre ated groups.

S ignificant decrease in the number of healthy follicles and increase in atretic
follicles in all mancozeb treated groups.

Reduced number of corpora lutea and size of ovary in high dose s of mancozeb
treated rats.

S ignificant increase in thy roid weight in all mancozeb treated rats except in 500 mg/kg/d dosage group. +++

Ksheerasagar &
Kaliwal 2003 21 Male Swiss
albino mice Mancozeb (oral) 800 mg/kg/day 5, 10, 20 and 30 days Weight of testes decreased significantly with increasing durations of pesticide
treatment in mice.

Increasing durations of mancozeb resulted in a significant dec rease in the weight of liver, kidneys and spleen.

Significant increase in the thyroid and thymus weight in duration dependent ++++

### 21 manner.

Baligar & Kaliwal
2004 11 Female virgin
albino rats 70 Mancozeb (75% wettable powder) dissolved in
olive oil (oral) 700 mg/kg/d 5, 10, 20 or 30 BG days Mancozeb treatment for 1) 5 days

- significant increase in the
duration of diestrus and decrease
in state II and total number of healthy follicles. 2) 10 days

- significant increase in the number of estrous cycles and
du ration of estrous, with increase in diestrus; decrease in stages I, II, and IV and in total number of healthy follicles. 3) 20 and 30 days

- number of estrous cycles and duration of proestrus, estrus, and metestrus were significantly decreased, with
increase i n duration of diestrus;
increase in atretic follicles and thyroid weight +++

Miranda

- Contreras
et al. 2005 16 Pregnant NMRI
mice 160 Paraquat [PQ] and Mancozeb [MZ] (injection) Group I : saline,

Group II : PQ (10 mg/kg),

Group III : MZ (30 mg/kg) or

Group IV : the
combination of PQ and MZ (10 mg/kg PQ + 30 mg/kg MZ) Pregnant mice:
GD 1

- 30

Dams: PD 12
and PD 20 Body Weight Second postnatal week: Significant reductions in body weight were found in mice exposed t o PQ + MZ

After 1 month:

• marked decline (27%) in body weight as compared with the control group (permanent effects of prenatal exposure to PQ + MZ on the physical growth of the animals).

• PQ

- exposed group showed significant declines 21% and
17% for the MZ

- exposed
animals

Pups showed significant reductions in excitatory amino acid neurotransmitters during cerebellar cortex development of prenatally exposed mice to chronic doses of MZ and PQ + MZ. ++++

### 22 In MZ

- exposed important variations in the developmental variation in Gly levels compared to control were registered: a 36% reduction at P3, a
150% increment at P15 and a 43% fall
at P30.

MZ exposed groups showed general trend of reduction in inhibitory neurotransmitters during cerebellar cortex development.

Locomotor activity Mice prenatally exposed to MZ were more active by 3.1 fold compared to controls; at P30 locomotor activity was significantly lower than controls for all three experimental groups.

P renatal exposure to either PQ or MZ or
the combinatio n of both may lead to alterations to the chronology and magnitude of synaptic transmission in developing mouse cerebellar cortex

.

Rossi et al. 2006 14 Swiss CD1 female
mice 25 Mancozeb in sesame oil (oral) 50 and 500 mg/kg GD 2 to PD 20 No significant difference in m ean number of oocytes from F1 generation treated with either low (50 mg/kg) or high (500 mg/kg) doses of mancozeb compared to controls.

P roduction of MII

- arrested oocytes in F1 gener ation was slightly, although significantly, reduced in mice exposed to high d oses (500 mg/kg) of man

cozeb; exposure to low doses (50mg/kg)
was ineffective.

82% of the oocytes derived from control or low dose treatment groups formed male and female pronu clei +++

### 23 71 % of oocytes derived from the high
dose treatment
Axelstad et al.
2011 15 Wister rats 64 (litters) Mancozeb in corn oil (oral) 0, 50, 100, or
150 mg/kg/d GD 7 to PD16 Doses of 150 mg/kg bw/day and above
caused toxic effects in the dosed dams ( e.g., severe weight loss and transient paralysis of the hind limbs)

T4 levels showed a dose

- dependent and significant decline in dams from all three dose g roups on gestational day 15 ( offspring T4 levels, thyroid weights and histology were unaffected on postnatal day 16 )

. ++++

Hass et al. 2012 18 Time

- mated nulliparous, young
adult female Wistar

rats 198 procymidone, epoxiconazole, tebuconazole, mancozeb, and prochlo raz (gavage) p rocymidone:
12.5 and 50 mg/kg/day;

epoxiconazol : 3.75 and 15 mg/kg/day;

tebuconazole:
12.5 and 50 mg/kg/day;

mancozeb:
6.25 and 25 mg/kg/day;
and

prochloraz:
8.75 and 35 mg/kg/day GD 7

- 21, PN
1

- 16 No significant effect on gestation len gth, maternal or pup body weight gain for rats exposed to mancozeb.

Gestation length was significantly increased in the two highest mixture groups compared to controls.

Nipple retention in male offspring was significantly increased in the groups exposed to the mixture and in the groups

expose d to the high dose of mancozeb,
tebuconazole and b oth doses of procymidone.

One male in each of the groups dosed
with mancozeb showed malformation of
the seminal vesicle.

All pesticides except for mancozeb
exhibited AR antagonism in vitro.

Combined developmental exposure to endocrine disrupting pesticides at dose levels below NOAELs for the single pesticides caused adverse effects on male sexual development and gestation length

. ++++

Jacobsen et al. Time

- mated 198 procymidone, Procymidone: Dams were One animal from each of the mancozeb ++++

### 24 2012 17 nulliparous, young
adult female Wistar

rats epoxiconazole, tebuconazole, mancozeb, and prochloraz (gavage) 12.5 and 50 mg/kg/day;

epoxiconazole
: 3.75 and 15 mg/kg/day;

tebuconazole:
12.5 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 dosed daily by gavage, from GD 7 to pup day (PD) 16 groups and one animal from the low dose mixture group had more than double thyroid weight co m

- pared to
controls.

S ignificant increase in the number of animals with follicles dominated by columnar to cuboidal epithelium was seen in mancozeb exposed animals compared to the control group (observed in 4 of 9 animals from the high dose mancozeb grou p compared to 1 of 17 control animals).

Significantly higher activity level s were observed in females fr om the high dose mancozeb group.

** Note: Lowest dose of each pesticide
was similar to the dose included in the highest mixture dose and then highest dose of the single pesticides was 4 times higher, corresponding to 25% of
NOAEL

BG=Before#Gestation,# GD=Gestation#day,#PD=Postnatal#day,# mg/kg/d=# mg/kg#body#weight/day ,#NF=not#found

* Confidence#ratings#in#body#of#evidence#ratings:# High%confiden ce (++++)#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#out come.#The#true#ef fect#is# may#be reflected#in#the# apparent#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#associat ion#between#exposure#to#mancozeb#and#the#outcome.#The#true#effect#is# highly#likely#to#be different#than#the#apparent# relationship.##

### 25 Table 3. Epidemiologic studies (fourteen studies) examining exposure to Mancozeb and reproductive and developmental out comes.
¶ 

### Design

— Co: Cohort; RCo: Retrospective Cohort; CaCo: Case

- Control; CrSe: Cross

- sectional; and CaS: Case Series/Case report.
Source Design ¶ 

### Population Mancozeb Assessment Outcome Confidence*
Arbuckle et al.,
2001 28 RCo Farm couples in the Ontario Family Health Study (N= 2,110 women ) Pooled pesticide exposure
information from the farm operator, husband, and wife to construct a history of monthly agricultural and residential pesticide use.

Four pesticide cl asses: 1) Herbicide, 2) Insecticide, 3) Fungicide and 4) Miscellaneous.

**Note: Mancozeb was not directly mentioned or measured in the study Preconception exposure to thiocarbamates (OR = 1.8; 95% CI,
1.1

– 3.0), glyphosate (OR = 1.7; 95% CI, 1.0

– 2.9), fungicides (OR = 1.4; 95% CI, 0.9

– 2.1), and the miscellaneous class of pesticides (OR = 1.5; 95% CI, 1.0

– 2.4) were associated with elevated risks of late spontaneous abortions (12

- 19 weeks).

Exposure to both fungicides and herbicides before conception
dou bled the risk relative 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 having a spontaneous abortion compared to those not exposed (OR = 2.4; 95% CI, 1.0

– 5.9) i n older women (age 35+). No increased risk was observed in

younger women. +++

Bell et al., 2001 30 CaCo Cases =319
neonatal deaths
within 24 hours of
birth and controls = 611 normal live
births in 10
California counties
in 1984. County maps to locate the township, range, and section
for each maternal address (mater nal TRS) was determined by using the public land survey system. Pesticide exposure was determined by linking the maternal TRS to the township, range, and section of each pesticide application located in the
California Pesticide Use Report database.

Five c ategories were selected for analysis: phosphates, carbamates, pyrethroids, halogenated hy dro

- carbons, and endocrine disruptors. Mancozeb was assigned to the
Carbamates/thiocarbamates
class. Pesticide classes were not strongly associated with fetal death, regardless of trimester of exposure.

Weakly elevated risks were observed for applications of halog enated hydrocarbons, carbamates/thiocarbamates, es trogenic pesticides, and carba mate acetylcholinesterase inhibitors during the second trimester, with HRs of 1.3 (95 percent confidence interval (CI): 1.0, 1.8), 1.3 (95 percent CI: 1.0, 1.8), 1.4 (95 percent CI: 0.8, 2.5), and 1.3 (95 percent CI: 1.0,1.8), respectively.

Modestly increased risk of fetal death was observed when exposure to halogenated hydrocar bons occurred during the fourth and fifth months of gestation, with HRs of 1.4 (95 percent CI: 1.0, 2.0) for both months. Results were similar for exposure to carbamates and carbamate inhibitors during the third and fourth months of gestation.

Slightly el evated HRs were observed for exposure to carbamates during the first and second trimesters of gestation, the monthly analysis showed that risk increased only for exposure during the third and fourth months of gestation. ++

Colosio et al.,
2002 22 CaCo (pilot) Italian vineyard workers (N=13) First urine spot sample collected in the morning pre

Urinary ETU was significantly increased at the end of shift (2.5,
0.5

– 95.2 m g/g creatinine) in workers compared with baseline ++

### 26 and controls (N=13) mancozeb application. Dermal exposure assessment captured morning in which
mancozeb mixture was

prepa red and applied by workers (pads were placed on clothes and skin)

* assessment of the exposure was performed in the absence of previous applications of
mancozeb and/or other pesticides levels.

End

- shift urinary ETU levels were higher in operators using open tractors (n=7) than in those using closed tractors (n=5) (16.2 vs.
2.4 m g/g creatinine), but not statistically significant.

End

- shift urinary ETU was positively correlated with dermal exposure to mancozeb determined both over the clothes and on the
skin

.

Garry et al.,
2002 29 CrSe 1,070 licensed pesticide applicators (males), 851 were
married or had a marriage

- like relationship, residing in five
counties in Red River Valley, Minnesota, USA Pest icide use Each certified pesticide applicator was initially inter

viewed by phone to obtain current and past pesticide use with specific attention to product name and the number of days per year applied.

6 months follow

- up questionnaire to document

com mon pesticide use by pesticide class, acreage treated, type of crop, and use of personal protective gear. Overlap between the two questionnaires was intentional to validate use of pesticides by class (herbicides, insecticides, fumigants, and fungicides). M ale Pesticide Use, Fetal Loss, and Miscarriages

* Sample: 540 farm families with children, where the applicator was the biologic father or whose spouse had been pregnant in the
relation ship, participated and gave de tailed pesticide use data.

Among couples who had children or who had been pregnant, one in four women had a miscarriage.

Spouses of pesticide applicators who reported use of herbicides, insecticides, and fungicides had more miscarriages than any other pesticide application group.

Specific Fun gicide Use
T he unadjusted odds ratio for specific use of organotin (OR = 1.55; CI 1.01

– 2.37) and/or use of EBDC (ethylene bisdithiocarbamate) fungicides (OR = 1.77; CI 1.11

– 2.83) such as mancozeb demonstrates a significantly increased frequency of
fetal lo ss compared to nonuse of fungicides by applicators.

Maternal Pesticide Exposure *Sample: Only 36 of 379 women respondents applied or mixed pesticides.

Personal use of pesticides, (e.g., mixing, loading and pesticide applications ) by the female spouse, w as a significant risk factor for fetal loss (fetal loss/pregnancy OR = 1.81; CI 1.04

– 3.12).

30% had a fetal loss compared to 21% of those women who did not mix or apply pesticides.

The number of fetal losses per pregnancy is significantly elevated +++

### 27 in t hose who did mix or apply pesticides (15% vs. 8.9%).

Miscarriage and Season # of first trimester miscarriages in spring was significantly elevated compared to all other seasons.

Spouses of applicators who appl ied fungicides and other pesticides
show ed n o peak in the miscarriage frequency in spring.

Frequency of first

- trimester miscarriages was significantly higher in summer, fall, and winter combined for the spouses of applicators of fungicides compared to applicator families whose male spouse did not apply fungicides.

S ignificant shift in the sex ratio of children born to the spouse of appliers who appli ed fungicides

.

• 25% reduction in male births in the children of applicators who appl ied herbicides, insecticides, and fungicides or among those applica tors who appl ied mancozeb and/or triphenyltin fungicides was significant.

Garry et al.,
2002 33 CrSe Families of licensed pesticide applicators in Red River Valley, Minnesota, USA (N=536 couples) Phone survey on current and past pesticide use in agriculture with 6 month
follow

- up interview to validate pesticide class. Class of pesticide
Herbicides

Chlorophenoxy Oxyphenoxy Sulfonylurea
Carbanilate Bromophenol
Benzothiazole
Nitroaniline
Inse cticides

Organophosphate Synthetic pyrethroids
Carbamate Fungicides

Organotin Ethylene bisdithiocarbamate
Triazole In families whose male partner did not apply fungicides, increased frequency of males born com pared to men who appl ied fungicide products

.

Increased frequency of male children born with birth defects than female children (M/F sex ratio = 1.8) when no fungicides were applied.

If fungicides were applied by the male partner, far fewer male
childre n with birth defects were born (M/F sex ratio = 0.57, p = 0.02).

No difference 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)

. ++

### 28 Fumigants

Phosphide
Other
Sallme n et al.,
2003 34 Co Finnish greenhouse
worker: employers (N=419) and employees (N=14,733) Exposure to pesticides was
assessed on the basis of questionnaire information and data gathered from the employers Reduced fecundabi lity in exposed greenhouse workers not wearing proper personal protection equipment (FDR 0.67 95%CI: 0.33

- 1.35).

0.92 (85%CI: 0.45

- 1.88) and 0.77 (95%CI 0.46

- 1.29) for high exposure, moderate, and low exposure groups compared to unexposed greenhouse worke rs.

Fertility was comparable between exposed workers who used PPE and unexposed greenhouse workers ++

Farr et al., 2004 25 CrSe Women living on
farms in Iowa and
North Carolina (N=3,103) pesticide exposure questions assessed lifetime use of any pesticides and hormonally active pest icides

women were asked, in their lifetime, had they ever mixed or applied any of 50 different pesticides Carbamate pesticides use associated with increased odds of long menstrual cycles (OR = 2.1) and decreased odds of irregular cycles (OR = 0.38).

Wome n who used mancozeb or maneb had four times the odds of experiencing long cycles and two times the odds of missed periods as women who had never used pesticides.

No other outcomes were associated with the use of mancozeb or
maneb.

A subanalysis excludin g female licensed applicators and spouses who had not likely used hormonally active pesticides in the last year results showed elevated odds of long cycles, missed periods, and intermenstrual bleeding among women exposed to lindane, atrazine, or mancozeb o r maneb compared to unexposed women.

* Models adjusted for age, body mass index, and current smoking

status

. 

### ++

Corsini et al.,
2005 24 CaCo 13 Vineyard
workers and 13 matched healthy
controls in Northern Italy W orkers : urine and blood samples were collected before the beginning of the spraying season, prior to any exposure to pesticides, and at the end of the work shift of the last day of Mancozeb application.

Controls : urine and blood samples were collected on ly Urinary ETU metabolite results: Significant increase in urine ETU concentration among applicators in post

- exposure compared to baseline values and controls samples.

No difference observed in baseline levels of urine ETU in workers
or controls.

Blood cell counts: Significant increase in CD19+ cells and decrease in CD25+ cells 

### +++

### 29 once in the morning at the same time of the year of
workers. were detected in follow

- up blood samples compared to controls.

Significant reduction in eosinophil and serum IgE in w orkers post

exposure

.

Significant increase in the proliferative response to phorbol myristate acetate plus ionomycin (PMA + ionomycin) was observed in the post

- exposure group compared to controls and
baseline

.

Significant decrease observed in LPS

- induced TNF

- a release post

exposure in workers.

Idrovo et al.,
2005 27 CrSe Female flower production workers in Bogota,
Colombia (N= 2,085 ). Exposure matrix constructed
based on work in flowers and time in agricultur e; exposure was categorized as low, medium, and high.

*Exposure to pesticides is

year

- round and fungicides (especial ly dithiocarbamates such as pro pineb and mancozeb) are more commonly used for 1 out of 5
industries. An estimated 5.7%
of industries used mancozeb. A trend in longer time to pregnancy (TTP) was observed in women engaged in any type of waged work.

Positive dose

- response relationship between duration of work in flower production and TTP. ++

Nordby et al.,
2005 35 RCo National registers in Norway, identifying female ( n= 105 403 ) and
male farmers ( n= 131, 243 ) and their
children ( n=300

, 805 ) born in 1925

- 1971

. Data on farm production and fungal forecasts (humid and temperate weather conditions)
in 1973

– 19 90, obtained from agricultural censuses and meteorological measurement stations, respectively, served as the mancozeb exposure
indicators. 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 >500 hours/year (PR 1.6, 95% CI
1.1

– 2.5).

319 thyroid cancer cases were identified:

1) 141 in female farmers (incidence 10.2/100 000 person years ),

2) 79 in male farmers (incidence 3.2/100 000 person

- years),

3) 99 in offspring (female and male incidence 3.4 and 0.6/100 000 person

- years).

Mancozeb exposure was not associated with thyroid cancer. +++

Lascana et al.,
2006 31 CaCo (paired) 151 cases of anencephaly of > Questionnaire obtaining
information on: Children of mothers who worked in agriculture in the ARP (acute risk period), that is periconceptional period, had a greater risk of +++

### 30 20 wks’ gestation

151 controls born
in three Mexican

states 1) lifetime occupational activities (start/end dates of job);

2) time involved in agricultural work, application of pesticides, and/or other
activit ies with potential exposure to these substances (manufacture, formulation, sale, or distribution of pesticides; work in fruit or vegetable warehouses; pest control work or gardening);

3) direct handling of pesticides
and time periods involved.

. Work in a gri culture was categorized as non agricultural, agricultural, and applicators. anencephaly (OR = 4.57, 95% CI 1.05 to 19.96 ).

Risk of fathers having a child with anencephaly was greater in those who applied pesticides irrespective of whether it was done in
the periconceptonal or the non

- acute risk period (OR = 2.50, 95%CI 0.73 to 8.64; and OR= 2.03, 95% CI 0.58 to 7.08,

resp ectively).

Pesticides most used by the parents of the cases in decreasing order of frequency were: permethrin (pyrethroid), methamidophos (organophosphate ), methyl parathion (orga nophosphate), atrazine (triazine), 2,4

- dichlorophenoxyacetic acid (chlorinat ed phenoxy), chlorpyrifos (organophosphate), mancozeb (dithiocarbamate)

, picloram (pyridine), dimetho ate (organophosphate), and carbofuran (carbamate); pesti cides most used by the parents of the controls in decreasing order of frequency were: methyl parath ion (organophosphate), methamidophos (organophosphate), 2,4

- dichloro

- phenoxyacetic acid (chlorinated phenoxy), chlorpyrifos (organophosphate), and monocrotophos (organophosphate).

Rull et al., 2006 26 CaCo In fants with neural
tube defects (NTDs) (n=928)
and normal formed controls (n=1263)
delivered in
California between
1987 and 1991 Potential exposures to specific
restricted

- use agricultural pesticides were evaluated by using a geographic metric
based on link ing pesticide

- use reports from the California Department of Pesticide Regulation with land

- use survey maps of crops from the Public Land Survey System.

A physiochemical category for Dithiocarbamate and pesticide category for Mancozeb were
included in the analysis.
Mancozeb was classified as an endocrine disruptor. Effect estimates for mancozeb applied within 1,000 meters of maternal residences on neural tube defects, California, 1987

- 1991
Cases: 6/731
Controls: 11/940

Conventional logistic regression : 1) Si ngle

- pesticide models:
OR : 0.7 95%CI : 0.2, 1.9 2) Multiple

- pesticide model:
OR : 0.5 95%CI : 0.1, 2.3

Hierarchical logistic regression : 1) Multiple

- pesticide model:
OR : 0.5 95%CI : 0.2, 1.4

E xposure to any endocrine disruptors applied within 1,000 m of

materna l residences resulted in an increased odds of 1.5 (95%CI: 1.2, 2.0) for any NTDs compared to no exposure of any endocrine disruptors.

The effect of exposure to 2+ endocrine disruptors applied within 1,000 meters of maternal residences resulted in 2 times the odds of having a baby born with anencephaly compared to babies in the no ++

### 31 exposure group (95%CI: 1.3, 3.0). Calvert et al.,
2007 36 CaS Three mothers and
their infants born with congenital
abnormalities
with in 8 weeks of
one another and
whose mother
worked for the

same tomato

grower Exposure information was
obtained from North Carolina Department of Agricultural
and Consumer Services (NCDACS) and the Florida Department of Agriculture and
Consumer Services (FL DACS), which obtained pesticide application and worker assignment records from the grower. Maternal Exposure History
Case 1 : The period for limb development is 24

– 36 days after fertilization. During this period, this child’s mother worked in
violation o f the REI for up to 4 days involving exposure to several pesticides, including mancozeb.

Case 2 : During gestational days 14

– 57, this child’s mother worked in violation of the REI for up to 8 days. On seven of these days, the pesticides applied to the fie lds where the mother worked included methamidophos. In addition, on gestational days 7 and 10, the mother worked in fields when an REI was possibly in effect (mancozeb on both days, and abamectin and methylpyrrolidone on day 7). The mother has three other living children, none of whom are known to have birth defects. This mother also had one previous stillbirth but without obvious birth
defects.

Case 3 : During gestational days 14

– 59, the mother worked in violation of REIs for up to 10 days. On eight of the se days, the REI for methamidophos was in effect on some of the fields where the mother worked. Abamectin and methylpyrrolidone were applied to some of the fields on two other days, but the mother may have worked in those fields before the applications wer e made. The mother had two previous pregnancies. One pregnancy 3 years earlier involved a malformed fetus and ended in miscarriage. The mother could not recall her employment or whether she had any toxic exposures during that pregnancy. The other previous pregnancy resulted in a normal child.

Birth Outcomes
Case 1 : This infant was born with tetraamelia (absence of all four limbs).

Case 2 : This infant was born with mild Pierre Robin syndrome (micrognathia, high arched palate, and mild persistent palatine rugae).

Case 3 : This infant had multiple severe malformations including cleft lip and palate, imperforate anus, solitary kidney, vertebral anomalies, dysplastic low

- set ears, and ambiguous genitalia.

Cases 1 and 2 were potentially exposed to mancozeb d uring the +

### 32 maximal sensitivity period.

There is evidence to suggest that each mother was exposed to pesticides during the maximal sensitivity period for the organ system/structure that was affected.

During the period of organogenesis (approximately 14

– 59 days after fertilization) when birth defects are most likely to o ccur, all
three mothers unknow ingly worked in tomato fields that were
under an REI because the fields were r ecently treated with pesticides.
Colosio et al.,
2007 23 CaCo Ninety

- three subjects entered the study: 48 vineyard workers intermittently exposed to mancozeb (14
from Pavia and 34 from Trento) and 45 control subjects (11 from Pavia and 34 from Trento) not exposed to
known sources of pesticides in Northern Italy. Recent exposure to mancozeb was assessed by measuring urinary ETU.

Immunotoxic effects of mancozeb were studied using
several blood cellular and serum parameters: complete
and differential blood cell

counts , lymphocyte subpopulations,

a glycoproteins, erythrocyte sedimentation rate (ESR), complement 3 and 4 fractions (C3, C4), antinuclear (ANA), antismooth muscle (SMA) and antimitochondrial (AMA) autoantibodies, and serum immunoglobulins (IgG1, IgG4, IgM, IgA, and total and specific IgE to most common allergens).

Three time points: 1) T0 baseline

— before start of seasonal use of mancozeb; 2) T30

-- exposure to mancozeb for 30 days; and 3) T45

-- 40

- 45 days. Only time point 1 (T0) and 3 (T45)
were obtained for controls. Urinary ETU T0 : A low but detectable amount of ETU observed in vineyard
workers and controls at baseline

.

T30 : Significant increase of urinary ETU in agricultural workers following exposure confirmed that absorption of mancozeb took place during application;

P ost exposure urinary ETU exceeded the reference value of 5 mg /

g creatinine.

Blood
T0 : Higher prevalence of cold or flu symptoms, lower percentage of monocytes, higher absolute count of T lymphocytes, CD4 and natural killer cells, and lower plasma levels of I gA and IgM in
workers. These differences did not continue at T30 or T45.

At T0, the agricultural workers showed lower plasma levels of IgA and IgM in comparison with controls.

Within agricultural workers a significant increase of serum
concentratio ns of IgA and IgM was observed after exposure.

No biologically relevant differences were observed between

groups (farmworker vs control) or at different times in other serum parameters.

No statistically significant differences were observed in
farmwork er compared to control groups in the total or specific IgE
levels. ++ +

### 33 ¶ Design

— Co:#Cohort; RCo:#Retrospective#Cohort; CaCo:#Case

Y Control;#CrSe:#Cross

Y sectional;#and#CaS:#Case#Series/Case#report.

* Confidence#ratings#in#body#of#evidence#ratings:# High%confid ence (++++)#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#out come.#The#true# effect#is# may#be reflected#in#the# apparent#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#associ ation#between#exposure#to#mancozeb#and#the#outcome.#The#true#effect#is# highly#likely#to#be different#than#the#apparent# relationship.##

### 34 References
1. Lin N, Garry VF. In vitro studies of cellular and molecular developmental toxicity of adjuvants herbicides, and fungicides commonly used in Red River Valley, Minnesota. Journal of Toxicology and
Environmental Health

- Part A. Jul 28 2000;60(6):423

- 439.
2. Bisson M, Hontela A. Cytotoxic and endocrine

- disrupting potential of atrazine, diazin on, endosulfan, and mancozeb in adrenocortical steroidogenic cells of rainbow trout exposed in vitro. Toxicology and applied pharmacology. Apr 15 2002;180(2):110

- 117.
3. Fejes S, Budai P, Varnagy L, Molnar T, Szabo R, Fancsi T. Toxicity of a mancozeb conta ining fungicide formulation and CU

- sulphate to chicken embryos after administration as single compounds or in combination. 2002;67(2):105

- 109.
4. Greenlee AR, Ellis TM, Berg RL. Low

- dose agrochemicals and lawn

- care pesticides induce developmental toxicity in murine preimplantation embryos. Environmental Health Perspectives

. 2004;112(6):703.
5. Calviello G, Piccioni E, Boninsegna A, et al. DNA damage and apoptosis induction by the pesticide
Mancozeb in rat cells: Involvement of the oxidative mechanism. Toxic ology and applied pharmacology. Mar 1 2006;211(2):87

- 96.
6. Paro R, Tiboni GM, Buccione R, et al. The fungicide mancozeb induces toxic effects on mammalian granulosa cells. Toxicology and applied pharmacology. Apr 15 2012;260(2):155

- 161.
7. Kjeldsen LS, Gh isari M, Bonefeld

- Jorgensen EC. Currently used pesticides and their mixtures affect the function of sex hormone receptors and aromatase enzyme activity. Toxicology and applied pharmacology. Oct 15 2013;272(2):453

- 464.
8. Khera KS. Ethylenethiourea: teratog enicity study in rats and rabbits. Teratology. Jun 1973;7(3):243

- 252.
9. Larsson KS, Arnander C, Cekanova E, Kjellberg M. Studies of teratogenic effects of the dithiocarbamates maneb, mancozeb, and propineb. Teratology. Oct 1976;14(2):171

- 183.
10. Castro V L, Tambasco AJ, Paraiba LC, Tambasco DD. Cytogenetic and teratological effects of mancozeb pre natal exposure on rats. Brazilian Archives of Biology and Technology. 1999;42(2):127

- 134.
11. Baligar PN, Kaliwal BB. Morphometric analysis of follicular growth and biochemical constituents in albino rats exposed to mancozeb. J Basic Clin Physiol Pharmacol. 2004; 15(3

- 4):241

- 62.
12. Baligar PN, Kaliwal BP. Induction of Gonadal Toxicity to Female Rats after Chronic Exposure to
Mancozeb. Industrial Health. 2001;39: 235

- 243.
13. Mahadevaswami MP, Jadaramkunti UC, Hiremath MB, Kaliwal BB. Effect of mancozeb on ovarian compensatory hypertrophy and biochemical constituents in hemicastrated albino rat. Reproductive toxicology. Mar

- Apr 2000;14(2):127

- 134.
14. Rossi G, Bucc ione R, Baldassarre M, Macchiarelli G, Palmerini MG, Cecconi S. Mancozeb exposure in vivo impairs mouse oocyte fertilizability. Reproductive Toxicology. 2006;21(2):216

- 219.
15. Axelstad M, Boberg J, Nellemann C, et al. Exposure to the Widely Used Fungicide Mancozeb Causes Thyroid Hormone Disruption in Rat Dams but No Behavioral Effects in the Offspring. Toxicological
Sciences. Apr 2011;120(2):439

- 446.
16. Miranda

- Contreras L, Davila

- Ovalles R, Benitez

- Diaz P, Pena

- Contreras Z, Palacios

- Pru E. Effects of

pren atal paraquat and mancozeb exposure on amino acid synaptic transmission in developing mouse
cerebellar cortex. Brain research. Developmental brain research. Nov 7 2005;160(1):19

- 27.
17. Jacobsen PR, Axelstad M, Boberg J, et al. Persistent developmental tox icity in rat offspring after low dose exposure to a mixture of endocrine disrupting pesticides. Reproductive Toxicology. Sep 2012;34(2):237

- 250.
18. Hass U, Boberg J, Christiansen S, et al. Adverse effects on sexual development in rat offspring after low
d ose exposure to a mixture of endocrine disrupting pesticides. Reproductive Toxicology. Sep 2012;34(2):261

- 274.

### 35 19. Khan PK, Sinha SP. Ameliorating effect of vitamin C on murine sperm toxicity induced by three pesticides (endosulfan, phosphamidon and mancoz eb). Mutagenesis. 1996 ; 11 :33

- 36

. 20. Kackar R, Srivastava MK, Raizada RB. Induction of gonadal toxicity to male rats after chronic exposure
to mancozeb. Industrial Health. Jan 1997;35(1):104

- 111.
21. Ksheerasagar RL, Kaliwal BB. Temporal effects of mancoz eb on testes, accessory reproductive organs
and biochemical constituents in albino mice. Environmental Toxicology and Pharmacology. Dec 2003;15(1):9

- 17.
22. Colosio C, Fustinoni S, Birindelli S, et al. Ethylenethiourea in urine as an indicator of exposure to mancozeb in vineyard workers. Toxicology letters. Aug 5 2002;134(1

- 3):133

- 140.
23. Colosio C, Fustinoni S, Corsini E, et al. Changes in serum markers indicative of health effects in vineyard workers following exposure to the fungicide mancozeb: an Itali an study. Biomarkers : biochemical indicators of exposure, response, and susceptibility to chemicals. Nov

- Dec 2007;12(6):574

- 588.
24. Corsini E, Birindelli S, Fustinoni S, et al. Immunomodulatory effects of the fungicide Mancozeb in agricultural workers. T oxicology and applied pharmacology. Oct 15 2005;208(2):178

- 185.
25. Farr SL, Cooper GS, Cai J, Savitz DA, Sandler DP. Pesticide Use and Menstrual Cycle Characteristics among Premenopausal Women in the Agricultural Health Study. American Journal of Epidemio logy. December 15, 2004;160(12):1194

- 1204.
26. Rull RP, Ritz B, Shaw GM. Neural tube defects and maternal residential proximity to agricultural pesticide applications. Am J Epidemiol. Apr 15 2006;163(8):743

- 753.
27. Idrovo AJ, Sanin LH, Cole D, et al. Time to first pregnancy among women working in agricultural production. International archives of occupational and environmental health. Jul 2005;78(6):493

- 500.
28. Arbuckle TE, Lin Z, Mery LS. An exploratory analysis of the effect of pesticide exposure on the risk of spontaneous abortion in an Ontario farm population. Environmental Health Perspectives

. Aug 2001;109(8):851

- 857.
29. Garry VF, Harkins M, Lyubimov A, Erickson L, Long L. R eproductive Outcomes in the Women of the Red River Valley of the North. I. Th e Spouses of Pesticide Applicators: Pregnancy loss

, age at menarche

, and exposures to pesticides

. Journal of Toxicology and Environmental Health, Part A. 2002;65(11):769

- 786.
30. Bell EM, Hertz

- Picciotto I, Beaumont JJ. Case

- Cohort Analysis of Agricultural Pesticide Applications
near Maternal Residence and Selected Causes of Fetal Death. American Journal of Epidemiology. October 15, 2001;154(8):702

- 710.
31. Lacasana M, Vazquez

- Grameix H, Borja

- Aburto VH, et al. Maternal and paternal occupational exposure

to agricultural work and the risk of anencephaly. Occupational and environmental medicine. Oct 2006;63(10):649

- 656.
32. Roperto F, Galati D. Exposure of nonmigratory pigeons to mancozeb: A sentinel model for humans. Journal of Toxicology and Environmental He alth

- Part A. Jul 24 1998;54(6):459

- 466.
33. Garry VF, Harkins ME, Erickson LL, Long

- Simpson LK, Holland SE, Burroughs BL. Birth defects, season of conception, and sex of children born to pesticide applicators living in the Red River Valley of Minnesota, US A. Environmental H ealth P erspectives. Jun 2002;110 Suppl 3:441

- 449.
34. Sallmen M, Liesivuori J, Taskinen H, et al. Time to pregnancy among the wives of Finnish greenhouse
workers. Scandinavian journal of work, environment & health. Apr 2003;29(2):85

- 93.
3 5. Nordby KC, Andersen A, Irgens LM, Kristensen P. Indicators of mancozeb exposure in relation to thyroid cancer and neural tube defects in farmers' families. Scandinavian journal of work, environment
& health. Apr 2005;31(2):89

- 96.
36. Calvert GM, Alarcon WA, Chelminski A, et al. Case report: three farmworkers who gave birth to infants with birth defects closely grouped in time and place

- Florida and North Carolina, 2004

- 2005. Environmental Health Perspectives

. May 2007;115(5):787

- 791.