vvEPA
EPA/635/R-17/331
IRIS Assessment Plan
www.epa.gov/iris
IRIS Assessment Plan for Nitrate and Nitrite
[CASRNs 14797-55-8 and 14797-65-0]
September 2017
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
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IRIS Assessment Plan for Nitrate and Nitrite
DISCLAIMER
This document is a preliminary draft for review purposes only. This information is
distributed solely for the purpose of pre-dissemination review under applicable information quality
guidelines. It has not been formally disseminated by EPA. It does not represent and should not be
construed to represent any Agency determination or policy. Mention of trade names or commercial
products does not constitute endorsement or recommendation for use.
This document is a draft for review purposes only and does not constitute Agency policy.
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IRIS Assessment Plan for Nitrate and Nitrite
CONTENTS
AUTHORS | CONTRIBUTORS v
1. INTRODUCTION 1
2. SCOPING AND INITIAL PROBLEM FORMULATION 2
2.1. BACKGROUND 2
2.2.SCOPING SUMMARY 3
2.3. PROBLEM FORMULATION 6
3. OVERALL OBJECTIVE, SPECIFIC AIMS AND DRAFT POPULATIONS, EXPOSURES,
COMPARATORS, AND OUTCOMES (PECO) FRAMEWORK 9
3.1. SPECIFIC AIMS 9
3.2. DRAFT PECO FRAMEWORK 10
3.3. KEY SCIENCE ISSUES 12
REFERENCES 13
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TABLES
Table 1. EPA program or regional office interest in an updated nitrate/nitrite assessment 4
Table 2. Nitrate/nitrite compounds considered for assessment 5
Table 3. Summary of the number of studies cited in ATSDR (2017) 6
Table 4. Draft PECO framework for the nitrate/nitrite assessment 10
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AUTHORS | CONTRIBUTORS
Assessment Team
Jenny Li (co-Assessment Manager)
Larissa Pardo (co-Assessment Manager)
Roman Mezencev
Susan Rieth
Ravi Subramaniam
U.S. EPA/ORD/NCEA
Executive Direction
Tina Bahadori
Mary Ross
Kris Thayer
Vincent Cogliano
Emma Lavoie
Samantha Jones
NCEA Center Director
NCEA Deputy Center Director
IRIS Division Director
IRIS Division Director (2010-2017)
Assistant Center Director for Scientific Support
NCEA Associate Director for Health (acting)
Contributors and Production Team
Vicki Soto
Dahnish Shams
Maureen Johnson
Project Management Team
This document is a draft for review purposes only and does not constitute Agency policy.
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1. INTRODUCTION
The Integrated Risk Information System (IRIS) Program is undertaking a reassessment of
the health effects of nitrate and nitrite. Nitrate and nitrite were included on the December 2015
IRIS Program multi-year agenda fhttps: //www.epa.gov/iris/iris-agenda] as chemicals having high
priority for assessment development, largely because of EPA Office of Water interest in an updated
health assessment
IRIS assessments provide high quality, publicly available information on the toxicity of
chemicals to which the public might be exposed. These assessments are not regulations, but
provide a critical part of the scientific foundation for decisions made in EPA program and regional
offices to protect public health.
Before beginning an assessment, the IRIS Program consults with EPA program and regional
offices to define the scope of the assessment, including the nature of the hazard characterization
needed, identification of the most important exposure pathways, and level of detail needed to
inform program and regional office decisions. Based on the scope defined by EPA, the IRIS Program
undertakes problem formulation activities to frame the scientific questions that will be the focus of
the assessment, which is conducted using systematic review methodology.
This document presents the draft assessment plan for nitrate and nitrite, including a
summary of the IRIS Program's scoping and initial problem formulation conclusions, objectives, and
specific aims of the assessment; draft PECO (Population, Exposure, Comparators, and Outcomes)
framework that outlines the evidence considered most pertinent to the assessment; and
identification of key areas of scientific complexity. Brief background information on uses and
potential for human exposure is provided for context. This document also discusses how EPA will
consider recent authoritative reviews, in particular, the Agency for Toxic Substances and Disease
Registry's Toxicological Profile for Nitrate and Nitrite (ATSDR. 2017) and the International Agency
for Research on Cancer monograph on nitrate and nitrate flARC. 20101. The assessment will
address both nitrate and nitrite together, as both are chemically related and metabolically linked,
and their biological effects are determined by conversion of nitrate to nitrite and vice versa.
Review of the health effect literature for both chemicals in a single health assessment also follows
the approach taken by other health agencies (ATSDR. 2017: WHO. 2016: Health Canada. 2013:
TARC. 20101.
This document is a draft for review purposes only and does not constitute Agency policy.
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2. SCOPING AND INITIAL PROBLEM FORMULATION
2.1. BACKGROUND
Nitrate (NO3") and nitrite (NO2"), naturally occurring anions in the environment, play an
essential role in Earth's nitrogen cycle. Since 1950, human sources of reactive nitrogen into the
environment—released either intentionally (e.g., through fertilizer application) or unintentionally
(e.g., as a byproduct of fossil fuel combustion)—have increased substantially fFields. 2004], Nitrate
salts are mainly used as nitrogen fertilizers and in industrial explosives, fireworks, and glass
making; nitrites are largely used as preservatives for meat and fish curing and as color fixatives
flARC. 2010: PokornvL. 20061.
Nitrates account for most of the available total nitrogen in both ground and surface waters;
nitrite levels are generally low in both fDeSimone. 20091. According to monitoring data obtained
during EPA's second Six-Year Review of National Primary Drinking Water Regulations (U.S. EPA.
20091. nitrate was detected in approximately 70% of drinking water systems at a median
concentration of approximately 2-3 mg nitrate-nitrogen/L; maximum concentrations in ground
and surface waters were 99 and 49 mg nitrate-nitrogen/L, respectively. Nitrite was detected in
approximately 22% of drinking water systems at a median concentration of 0.02 mg nitrite-
nitrogen/L; maximum concentrations in ground and surface waters were 13 and 9 mg nitrate-
nitrogen/L, respectively fU.S. EPA. 20091. Human activities are responsible for increased levels of
nitrate in drinking water sources; DeSimone (20091 reported that nitrate concentrations greater
than 1 mg/L (as N) are levels in well water considered to result from the effects of human activities
in many parts of the United States. Populations served by private well water, especially shallow
wells in agricultural areas, may be exposed to nitrate at levels several times higher than those
served by public water systems fDeSimone. 2009: Ward. 20091.
The general population is exposed to nitrate in both drinking water and food. Vegetables
are the main source of exposure to ingested nitrate, with leafy vegetables comprising nearly 80% of
nitrate exposure in an average person's diet Other sources of dietary nitrate include cured
meats/fish, cereal grains, dairy products, and beer (ATSDR. 2017: IARC. 20101. In contrast to
nitrates, endogenous sources account for approximately 80% of all nitrites in the human body, as
5-8% of the total nitrate intake is converted into nitrite fWHO. 2016: Mensinga et al.. 20031.
Almost all exogenous exposure to nitrite comes from food, with relatively higher nitrite
concentrations found in cured meats (IARC. 20101. Drinking water is generally a minor source of
exposure to nitrite (IARC. 20101.
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The IRIS Program previously evaluated the oral health effects of nitrate and nitrite; oral
reference doses (RfDs) for nitrite1 and nitrate2 were posted to the IRIS database in 1987 and 1991,
respectively. EPA based these RfDs on surveys of clinical cases of methemoglobinemia in infants
associated with ingestion of nitrate-containing drinking water conducted in the early 1950s
(Walton. 1951: Bosch et al, 19501. Since 1987, a growing body of literature indicates potential
associations between nitrate/nitrite exposure and other noncancer health effects. Some
epidemiological studies also suggest an increased risk of cancer, especially gastric cancer,
associated with dietary nitrite exposure (ATSDR. 2017). Cancer risk associated with nitrate or
nitrite exposure is complicated by the fact that, under conditions of concurrent exposure to amines
or amides or low levels of antioxidants, endogenous nitrosation can occur, leading to the formation
of carcinogenic nitroso compounds (ATSDR. 2017:1 ARC. 20101. , concluded that
ingested nitrate or nitrite under conditions that result in endogenous nitrosation is probably
carcinogenic to humans (Group 2A).
2.2. SCOPING SUMMARY
During scoping, the IRIS Program met with EPA program and regional offices that had
interest in an updated IRIS assessment for nitrates and nitrites to discuss specific assessment
needs. Table 1 provides a summary of input from this outreach.
1https://cfpub.epa.gov/ncea/iris/iris documents/documents/subst/0078 summarv.pdf
2https://cfpub.epa.gov/ncea/iris/iris documents/documents/ subst/0076 summarv.pdf
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Table 1. EPA program or regional office interest in an updated nitrate/nitrite
assessment
Program or
regional office
Oral
Inhalation
Statutes/ regu lations
Description of
authority/ regu lation
Anticipated
uses/interest
Office of Water
Safe Drinking Water
Act (SDWA) -
Section 1412
EPA must review each
national primary drinking
water regulation at least
once every six years and
revise them, if appropriate.
Six-year review of
the National Primary
Drinking Water
regulations.
Region 5a
At the discretion of the state,
nitrate levels, not to exceed
20 mg/L, may be allowed in a
non-community water
system if the supplier of
water demonstrates to the
satisfaction of the State that:
(1) Such water will not be
available to children under 6
months of age; and
(2) The non-community
water system is meeting the
public notification
requirements under
§141.209, including
continuous posting of the
fact that nitrate levels
exceed 10 mg/L and the
potential health effects of
exposure; and
(3) Local and state public
health authorities will be
notified annually of nitrate
levels that exceed 10 mg/L;
and
(4) No adverse health effects
shall result.
Evaluation of special
provision of the
NPDW regulation [40
CFR 141.11(d)]
allowing, at the
discretion of the
state, non-
community water
systems to exceed
the nitrate MCL.
aRegion 5 serves Illinois, Indiana, Michigan, Minnesota, Ohio, Wisconsin, and 35 tribes.
1 The Office of Water regulates nitrates and nitrites under the National Primary Drinking
2 Water Regulations (40 CFR141,142); the current maximum contaminant levels (MCLs) for nitrate
3 and nitrite, promulgated in 1991, are 10 mg/L and 1 mg/L (as nitrogen), respectively (40 CFR
4 141.62; 56 FR 3594, January 30,1991). An updated health assessment of nitrate and nitrite will be
5 considered in the next Six-Year Review cycle for National Primary Drinking Water regulations. A
6 provision of the current regulation [40 CFR 141.11(d)] allows, at the discretion of the state,
7 noncommunity water systems to exceed the nitrate MCL up to 20 mg/L if the supplier can
8 demonstrate that the water will not be available to children under 6 months of age and that no
9 adverse health effects will result. The availability of more recent health effect literature published
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since 1991 raises questions about whether the current MCLs for nitrate and nitrite and the
provision allowing exceedance of the nitrate MCL up to 20 mg/L provide adequate health
protection for the general population (all life stages).
This assessment will address inorganic forms of nitrate and nitrite and will specifically
consider health effect information for the compounds included in Table 2. These salts are highly
soluble in water and dissociate under environmental conditions; in solution, they exist as ions
fATSDR, 20171. Because the cations are not expected to introduce significant differences in the
toxicity of the different salts, toxicity findings from all five compounds are considered relevant to an
assessment of nitrate and nitrite toxicity. These five compounds listed in Table 2 are the most
common nitrate and nitrite salts in the environment (ATSDR. 20171. These compounds were also
the subject of two recent health assessments of nitrate and nitrite fATSDR. 2017:1 ARC. 20101. The
decision to develop the assessment of nitrate/nitrite using health effect information for these five
compounds was also based on known general population exposure to these five compounds and
availability of epidemiological or toxicological information. Specifically, ammonium nitrate is a
leading nitrogen fertilizer, and for this reason, has been used in toxicological studies as a
component of "California mixture" and "Iowa mixture." These two mixtures are representative of
groundwater contamination by fertilizers and pesticides and used for simulations of environmental
exposures to pesticides mixtures. Sodium nitrate, sodium nitrite, potassium nitrate, and potassium
nitrite are used as food additives to cure meats. The National Toxicology Program (NTP) has
assessed the toxicities of sodium nitrate and sodium nitrite in animal toxicology and carcinogenicity
studies.
Table 2. Nitrate/nitrite compounds considered for assessment
Compound
Chemical formula
CAS Number
Ammonium nitrate
NH4NO3
6484-52-2
Sodium nitrate
NaNOs
7631-99-4
Sodium nitrite
NaN02
7632-00-0
Potassium nitrate
KNOb
7757-79-1
Potassium nitrite
KNO2
7758-09-0
Assessment of the health effects of nitrate and nitrite following inhalation and dermal
routes of exposure will not be included in the scope of this assessment. Inhalation and dermal
exposures to nitrate or nitrite in the general population (i.e., populations not exposed
occupationally, such as factory and fertilizer workers) are expected to be negligible compared to
oral exposure fATSDR. 20171. Focusing on the health effects associated with oral exposure to
nitrate and nitrite is consistent with the needs of EPA programs and regional offices.
Based on input received during scoping, the IRIS assessment will include evaluation of both
noncancer and cancer human health hazards associated with ingested nitrate and nitrite. Because
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the association between nitrate/nitrite and methemoglobinemia has been well established (Ward
et al. 2005: Walton. 19511. the assessment of this outcome will focus primarily on the quantitative
relationship between nitrate/nitrite exposure and methemoglobinemia. For cancer, EPA will
develop a qualitative assessment of the carcinogenic potential of nitrate and nitrite, and will
explore the feasibility of developing a quantitative assessment based in part on consistency of
effects observed across studies and the availability of data that support dose-response analysis.
EPA anticipates that a quantitative cancer assessment will be particularly challenging, given the
influence of concurrent exposure to dietary sources of nitrosatable compounds and antioxidants,
conflicting results across studies, and design limitations in a number of epidemiological studies that
have investigated associations between nitrate or nitrite exposure and cancer at different sites.
2.3. PROBLEM FORMULATION
A preliminary literature survey was performed using health assessments produced by other
federal, state, and international health agencies fATSDR. 2017: WHO. 2016: Health Canada. 2013:
I ARC. 2010: IPCS. 2005: Cal/EPA. 20001 to identify noncancer and cancer health outcomes for
which possible association with exposure to nitrate/nitrite has been investigated.
In particular, EPA relied on the AT SDR (20171 Toxicologicctl Profile for Nitrate and Nitrite, as
the most recent authoritative health agency assessment, to identify the pertinent health effect
literature through 2016.3 Because AT SDR (20171 updated the comprehensive review of the cancer
epidemiological literature provided in I ARC (20101 (i.e., literature published up to approximately
2007), the IARC monograph also was used to identify the cancer literature. The numbers of animal
and human studies cited in ATSDR (20171 and IARC (20101 by health effect category were tallied as
a measure of the extent to which the association between a given health effect and nitrate/nitrite
exposure has been investigated (see Table 3).
Table 3. Summary of the number of studies cited in ATSDR f 2 PIT'S3
Health effect
Human studies
Animal studies
Occupational
epidemiology studies
General population
epidemiology studies
Controlled exposure
studies
Case reports and
case series reports
Chronic
Subchronic
Short-term
Acute
Multigenerational
Gestational
Cancer
60+b
18
Hematological
25+
3
10+
4
6
3
1
Developmental
14
2
6
3ATSDR conducted their literature search through May 2016.
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Health effect
Human studies
Animal studies
Occupational
epidemiology studies
General population
epidemiology studies
Controlled exposure
studies
Case reports and
case series reports
Chronic
Subchronic
Short-term
Acute
Multigenerational
Gestational
Endocrine (thyroid)
6
1
4
3
1
Gastrointestinal
1
1
7
5
1
Other systemic toxicity0
10
2
1
1
Neurological and sensory
2
6
1
1
1
Metabolic disease (type 1
diabetes)
8
Reproductive
3
1
2
3
1
Hepatic
3
2
Cardiovascular
1
1
3
Dermal and ocular
1
Renal
1
Immunological
Musculoskeletal
Respiratory
aThe numbers represent the numbers of studies that investigated a particular health effect, not the number of
studies that identified a positive association with exposure to nitrate or nitrite. If a journal article or report
included, for example, a study in both rats and mice, it was counted as two studies. Health effects are listed
generally in decreasing order of the number of studies that investigated that effect.
bMore than 50 epidemiological studies that examined the association between nitrate/nitrite intake and cancer
were cited in the 2010 IARC Monograph (1ARC, 2010); an additional 13 selected cohort and case-control studies
published after IARC conducted the literature search were identified in the ATS PR (2017) Toxicological Profile.
cBody weight.
2 Based on the preliminary literature survey (i.e., the secondary sources used to develop
3 Table 3), EPA anticipates conducting a systematic review for the following health effect categories:
4 • Cancer
5 • Hematological effects
6 • Developmental effects
7 • Thyroid effects (endocrine effect)
8 • Type 1 diabetes (metabolic effect)
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• Reproductive effects
For these health effect categories, the available epidemiology and experimental animal
studies are likely to be sufficient for drawing conclusions about human hazard. Of the studies
identified for other health effect categories (e.g., gastrointestinal, hepatic, and nervous system)
positive findings generally were reported in only a few studies, some with study design deficiencies,
and generally were inconsistent Therefore, EPA anticipates that a systematic review for health
effect categories other than the six identified above will not be undertaken unless additional
evidence of a positive association is discovered upon review of references identified during the
comprehensive literature search. Such determinations will involve evaluation of the body of
evidence from both new and previously identified studies, taking into consideration factors such as
study quality, directness or relevance of the experimental model, nature of the endpoints examined,
and consistency across studies.
The preliminary literature survey revealed several studies reporting potential association
between nitrate/nitrite exposure and beneficial cardiovascular outcomes. Because IRIS
assessments focus on the adverse effects associated with exposure to environmental chemicals, a
systematic review of the potential beneficial outcomes to the cardiovascular system associated with
the intake of nitrate or nitrite will not be included in this assessment.
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3. OVERALL OBJECTIVE, SPECIFIC AIMS AND DRAFT
POPULATIONS, EXPOSURES, COMPARATORS,
AND OUTCOMES (PECO) FRAMEWORK
The overall objective of this assessment is to identify adverse health effects and
characterize exposure-response relationships for these effects of nitrate and nitrite to support
development of toxicity values. This assessment will use systematic review methods to evaluate
the epidemiological and toxicological literature for these chemicals, including consideration of
relevant mechanistic evidence. The evaluations conducted in this assessment will be consistent
with relevant EPA guidance.4 The systematic review protocol will be disseminated after review of
the draft assessment plan and will reflect changes made to the specific aims and PECO framework
in response to public input
3.1. SPECIFIC AIMS
• Identify epidemiological (i.e., human) and toxicological (i.e., experimental animal) literature
reporting effects of exposure to nitrate and nitrite as outlined in the PECO framework.
Chapter 3 (Health Effects) of ATSDR's Toxicological Profile of Nitrate and Nitrite f AT SDR.
20171 and the IARC monograph on nitrate and nitrate flARC. 20101 will serve as starting
points to identify PECO-relevant evidence published through 2016. A literature search
update will be performed to identify new health effect references for nitrate/nitrite
published since completion of the literature searches conducted by ATSDR and IARC.
Health outcome studies cited in ATSDR (20171 and IARC (20101 will be combined with the
literature search results from the updated database search and screened for PECO
relevance.
• Use an iterative approach to determine which mechanistic studies are most important to
summarize, based on factors such as robustness of the evidence in humans and animals,
likelihood to impact evidence synthesis conclusions for human health, and directness or
relevance of the model systems for understanding potential human health hazards. When
summarizing individual mechanistic studies is not critical, this information will generally be
summarized by relying on other published authoritative sources, such as public health
agency reports and expert review articles.
• Conduct study evaluations (risk of bias and sensitivity) for individual epidemiological and
toxicological studies. Studies with critical deficiencies will be considered uninformative and
will not be considered further.
4EPA guidance documents: http://www.epa.gov/iris/basic-information-about-integrated-risk-information-
svstem# guidance /
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1 •
2
3 •
4
5 •
6
7
8 •
9
10
11
12
13 •
14
15 •
16
17
18
19
20 3X DRAFT PECO FRAMEWORK
21 A PECO (Populations, Exposures, Comparators, and Outcomes) framework is used as an aid
22 to focus the research question(s), search terms, and inclusion/exclusion criteria in a systematic
23 review. The draft PECO framework for nitrate and nitrite (Table 4) was based on (1) nomination of
24 the chemical for assessment, (2) discussions with scientists in EPA program and regional offices to
25 determine the scope of the assessment that will best meet Agency needs, and (3) preliminary
26 review of the health effects literature for nitrate and nitrite (primarily reviews and authoritative
27 health assessment documents) to identify the major health hazards associated with exposure to
28 nitrate and nitrite and key areas of scientific complexity.
Table 4. Draft PECO framework for the nitrate/nitrite assessment
PECO element
Evidence
Population
Human: Anv population and life stage (e.g.. children, general population, occupational, or high
exposure from an environmental source). As children younger than 6 months of age appear to be
a susceptible population, this population will be emphasized during review. The following study
designs will be considered most informative: controlled exposure, cohort, case-control, cross-
sectional, and ecological. Note: Case reports and case series will be tracked during study screening
but are not the primary focus of this assessment. They may be retrieved for full-text review and
subsequent evidence synthesis if no or few more informative study designs are available. Case
Extract data on relevant health outcomes from epidemiological and toxicological studies
included based on the study evaluation.
Synthesize the evidence across studies, assessing similar health outcomes using a narrative
approach or meta-analysis (if appropriate).
For each health outcome, express confidence in conclusions from across studies (or sub-sets
of studies) within human and animal evidence streams, evaluating each evidence stream
(human and animal) separately.
For each health outcome, integrate results across evidence streams (human and animal) to
conclude whether a substance is hazardous to humans. Identify and discuss issues
concerning potentially susceptible populations and life stages. Biological support provided
from mechanistic studies and non-mammalian model systems will be considered based on
the iterative prioritization approach outlined in the PECO framework.
Derive toxicity values, as supported by the available data. For nitrate and nitrite anions
separately, consider deriving RfDs and, if feasible, a cancer slope factor.
Characterize uncertainties and identify key data gaps and research needs such as
limitations of the evidence base, limitations of the systematic review, and consideration of
dose relevance and pharmacokinetic differences when extrapolating findings from higher-
dose animal studies to lower levels of human exposure.
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PECO element
Evidence
reports also can be used as supportive information to establish biologic plausibility for some target
organs and health outcomes.
Animal: Nonhuman mammalian animal species (whole organism) of anv life stage (including
preconception, in utero, lactation, peripubertal, and adult stages).
Nonmammalian model svstems/in vitro/in silico: Nonmammalian model svstems (e.g.. fish.
amphibians, birds, Caenorhabditis elegans); human or animal cells, tissues, or biochemical reactions
(e.g., ligand-binding assays) with in vitro exposure regimens; bioinformatics pathways of disease
analysis; or high-throughput screening data. These studies are tagged during title and abstract
screening, and an iterative approach is used to prioritize their inclusion for full-text retrieval and
evidence synthesis based on likelihood to impact evidence synthesis conclusions for human health.3
Exposure
Exposure to specific nitrate/nitrite compounds including (CASRN): ammonium nitrate (6484-52-5),
potassium nitrate (7757-79-1), potassium nitrite (7758-09-0), sodium nitrate (7631-99-4), sodium
nitrite (7632-00-0), inorganic nitrate/nitrite in drinking water and inorganic nitrate/nitrite in foods.
Mixture studies for animal and in vitro studies will be included if they have an arm with a
nitrate/nitrite compound only.
Human and animal: Exposure routes to be considered are anv oral exposures. Where possible,
exposures will be assessed separately for drinking water and dietary nitrate/nitrite. Other exposure
routes, including inhalation, dermal, or injection, will be tracked during title and abstract as
"supplemental information."
Nonmammalian model svstems/in vitro/in silico: Exposure via growth or assav medium.
Comparator
Human: A comparison or reference population exposed to lower levels (or no exposure/exposure
below detection levels) of nitrate/nitrite or to nitrate/nitrite for shorter periods.
Animal: Quantitative exposure versus lower or no exposure with concurrent vehicle control group.
Nonmammalian model svstems/in vitro/in silico: Quantitative exposure versus lower or no
exposure with concurrent vehicle control group.
Outcomes
All health outcomes (both cancer and noncancer). For methemoglobinemia, only studies that
inform the quantitative relationship between nitrate/nitrite exposure and methemoglobinemia will
be included. In general, endpoints related to clinical diagnostic criteria, disease outcomes,
histopathological examination, or other apical/phenotypic outcomes will be prioritized for
evidence synthesis over outcomes such as biochemical measures. As discussed above, EPA
anticipates that a systematic review for health effect categories other than the six identified above
(hematological, thyroid, type 1 diabetes, development, reproduction, and cancer) will not be
undertaken unless a significant amount of new evidence is found upon review of references
identified during the comprehensive literature search.
aNote: An iterative approach is used to prioritize evidence from nonmammalian model systems (e.g., fish,
amphibians, birds, C. elegans), in vitro, in silico, and other types of mechanistic studies based on likelihood to
impact evidence synthesis conclusions for human health. Evidence from these studies will be tagged preliminarily
during title/abstract screening as "Other Informative Studies" or "Supplemental Information" according to hazard
categories or types of mechanistic outcomes/pathways. These studies are prioritized for full-text retrieval and
evidence synthesis to focus on those studies most important to summarize, based on factors such as robustness
of the evidence in humans and animals, directness or relevance of the model systems, and concentrations tested.
For example, if robust epidemiological or nonhuman mammalian evidence is available, the need to conduct a
thorough assessment of individual nonmammalian and mechanistic studies could be diminished unless
controversial issues need to be resolved, e.g., issues related to applicability of animal evidence to humans or the
shape of the dose-response relationship at low exposure levels.
This document is a draft for review purposes only and does not constitute Agency policy.
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//?/S Assessment Plan for Nitrate and Nitrite
3.3. KEY SCIENCE ISSUES
Based on the preliminary literature survey, the following key scientific issues were
identified that warrant evaluation in the assessment.
• Nitrate and nitrite are generated endogenously as part of the nitrate-nitrite-nitric oxide
cycle that controls the availability of nitric oxide, which is a signaling molecule involved in
the regulation of both physiological and pathological processes. The roles of endogenous
versus exogenous nitrate and nitrite in toxicity, particularly methemoglobinemia in infants,
have been debated in the scientific literature.
• Several susceptible populations and life stages have been identified for
methemoglobinemia. These include infants under 6 months of age; individuals with higher-
than-normal gastric pH; individuals with glucose-6-phosphate dehydrogenase or NADH
(nicotinamide adenine dinucleotide (NAD) + hydrogen)-dependentmethemoglobin
reductase deficiency; individuals with diseases such as anemia, cardiovascular disease, lung
disease, and sepsis; individuals with abnormal hemoglobin species including
carboxyhemoglobin, sulfhemoglobin, and sickle hemoglobin.
• A physiologically based pharmacokinetic (PBPK) model for simulating the kinetics of
methemoglobinemia formation after oral exposure to nitrate in adults is available
(Zeilmaker et al, 2010: Zeilmaker et al, "19961 and needs to be evaluated for its potential to
inform interspecies variability in the dose-response assessment
• Previously published assessments by th Canada ("20131. ATSDR (20171.1 ARC ("20101.
and V 3161 and newer animal and epidemiological studies published after 2014 raise
the following issues related to cancer risk:
° Risk associated with intake of nitrates, nitrites, or both from cured meats, vegetables,
and drinking water could differ because of co-occurrence with antioxidants (e.g.,
vitamin C, vitamin E) in vegetables, amines in fish and meats, and calcium in drinking
water. Consequently, risks associated with dietary intake, intake by drinking water, and
total intake may need to be assessed separately.
° Susceptible populations, such as postmenopausal women (Inoue-Choi et al. 20151
Hones et al. 20161. appear to display increased risk associated with intake of
nitrate/nitrite.
° Populations vary significantly in the ability to reduce salivary nitrate by oral bacteria
(e.g., actinomyces and veilonella) (Bryan and Petrosino, 20171. For example, patients
with migraines were shown to have higher abundance of nitrate, nitrite, and nitric oxide
reductase genes in their oral bacterial metagenome (Gonzalez et al. 20161. In contrast,
the use of antiseptic mouthwashes appears to deplete nitrate-reducing oral bacteria and
affect some nitrite-mediated biological processes fKapil et al. 20131. Individuals from
some subgroups might be able to convert more nitrate to nitrite and consequently
produce more carcinogenic N-nitroso derivatives.
This document is a draft for review purposes only and does not constitute Agency policy.
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REFERENCES
ATSDR (Agency for Toxic Substances and Disease Registry). (2017). Toxicological profile for nitrate
and nitrite [ATSDR Tox Profile], Atlanta, GA: U.S. Department of Health and Human Services,
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Bosch. HM: Rosenfield. AB: Huston. R: Shipman. HR: Woodward. FL. (1950). Methemoglobinemia
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Bryan. NS: Petrosino. IF. (2017). Nitrate-reducing oral bacteria: Linking oral and systemic health.
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Gonzalez. A: Hyde. E: Sangwan. N: Gilbert. TA: Viirre. E: Knight. R. (2016). Migraines are correlated
with higher levels of nitrate-, nitrite-, and nitric oxide-reducing oral microbes in the
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Ward. MH. (2015). Nitrate and nitrite ingestion and risk of ovarian cancer among
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Beane Freeman. LE: Silverman. DT: Ward. MH. (2016). Nitrate from drinking water and diet
This document is a draft for review purposes only and does not constitute Agency policy.
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and bladder cancer among postmenopausal women in Iowa. Environ Health Perspect 124:
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Kapil. dar. SM: Pearl. V: Lundberg. 10: Weitzberg. E: Ahluwalia. A. (2013). Physiological role
for nitrate-reducing oral bacteria in blood pressure control. Free Radic Biol Med 55: 93-100.
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Mensinga. TT: Speijers. GI: Meulenbelt. I. (2003). Health implications of exposure to environmental
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Pokorny L. M. aturana I. B..ortle WH. (2006). Sodium Nitrate and Nitrite. In Kirk-Othmer
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Walton. G. (1951). Survey of literature relating to infant methemoglobinemia due to nitrate-
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htto i / /dx.d oi. or g ,/d oi: 1.986.
Ward. MH: Dekok. TM: Levallois, P: Brende [is. G: Nolan. BT: Vanderslice. 1: Epidemiology.
ISfE. (2005). Workgroup report: Drinking-water nitrate and health-recent findings and
research needs. Environ Health Perspect 113: 1607-1614.
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Ward. MH. (2009). Too much of a good thing? Nitrate from nitrogen fertilizers and cancer [Review],
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WHO (World Health Organization). (2016). Nitrate and nitrite in drinking-water: Background
document for development of WHO guidelines for drinking-water quality.
(WHO/FWC/WSH/16.52). Geneva, Switzerland.
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janl7.pdf.
Zeilmaker. MI: Meulenbi wtbover. 1M: Slob. W. (1996). Safety assessment of nitrate:
Mathematical modeling of nitrite formation in man and its application in the risk
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116: 323-335. http:// dx.doi.org/10.1093/toxsci/kfa093.
This document is a draft for review purposes only and does not constitute Agency policy.
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