v>EPA
EPA/635/R-16/163 Fb
www.epa.gov/iris
Toxicological Review of Ammonia
Noncancer Inhalation
[CASRN 7664-41-7]
Supplemental Information
September 2016
Integrated Risk Information System
National Center for Environmental Assessment
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC
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Supplemen tal Inform ation —Amm onia
DISCLAIMER
This document has been reviewed in accordance with U.S. Environmental Protection Agency
policy and approved for publication. Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
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Supplemen tal Inform ation —Amm onia
CONTENTS
APPENDIX A. ASSESSMENTS BY OTHER NATIONAL AND INTERNATIONAL HEALTH AGENCIES A-l
APPENDIX B. ADDITIONAL DETAILS OF LITERATURE SEARCH STRATEGY | STUDY SELECTION AND
EVALUATION B-l
APPENDIX C. INFORMATION IN SUPPORT OF HAZARD IDENTIFICATION AND DOSE-RESPONSE
ANALYSIS C-l
C.l. TOXICOKINETICS C-l
C.2. HUMAN STUDIES C-17
C.3. ANIMAL STUDIES INVOLVING INHALATION EXPOSURE C-41
C.4. ESTIMATING THE MEAN EXPOSURE CONCENTRATION IN THE HIGH-EXPOSURE
GROUP C-56
APPENDIX D. SUMMARY OF SAB PEER REVIEW COMMENTS AND EPA's DISPOSITION D-l
REFERENCES FOR APPENDICES R-l
iii
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Supplemen tal Inform ation —Amm onia
TABLES
Table A-l. Assessments by other national and international health agency assessments for
ammonia A-l
Table B-l. Literature search strings for computerized databases B-l
Table B-2. Processes used to augment the search of core computerized databases for ammonia B-6
Table B-3. Disposition of studies from the cleaning and hospital worker literature B-10
Table B-4. Electronic screening inclusion terms (and fragments) for ammonia B-14
Table B-5. Disposition of epidemiology studies identified in September 2015 literature search
update of core databases B-14
Table B-6. Evaluation of epidemiology studies summarized in Table 1-2 (industrial settings/respiratory
measures) B-16
Table B-7. Evaluation of epidemiology studies summarized in Table 1-3 (use in cleaning/disinfection
settings) B-25
Table B-8. Evaluation of epidemiology study summarized in Table 1-6 (industrial setting/serum
chemistry measures) B-32
Table C-l. Ammonia levels in exhaled breath of volunteers C-9
Table C-2. Symptoms and lung function results of workers exposed to different levels of TWA
ammonia concentrations C-18
Table C-3. The prevalence of respiratory symptoms and disease in urea fertilizer workers exposed
to ammonia C-20
Table C-4. Logistic regression analysis of the relationship between ammonia concentration and
respiratory symptoms or disease in exposed urea fertilizer workers C-20
Table C-5. Prevalence of respiratory symptoms and cross-shift changes in lung function among
workers exposed to ammonia in a urea fertilizer factory C-23
Table C-6. Comparison of lung function parameters in ammonia plant workers with controls C-24
Table C-7. Evidence pertaining to respiratory effects in populations exposed to ammonia in
agricultural settings with direct analysis of the relationship between ammonia exposure
and measured outcomes C-25
Table C-8. Evidence pertaining to respiratory effects in populations exposed to ammonia in
agricultural settings without direct analysis of the relationship between ammonia
exposure and measured outcomes C-30
Table C-9. Evidence pertaining to irritation effects and changes in lung function in controlled
human exposure studies C-32
Table C-10. Summary of histological changes observed in pigs exposed to ammonia for 6 weeks C-44
Table C-ll. Acute and short-term inhalation toxicity studies of ammonia in animals C-49
Table C-12. Frequency distribution of ammonia exposure from Holness et al. (1989) C-56
Table C-13. Observed and expected frequencies of ammonia exposure from Holness et al. (1989) ....C-57
FIGURES
Figure C-l. Glutamine cycle C-3
Figure C-2. The urea cycle showing the compartmentalization of its steps within liver cells C-4
Figure C-3. Histogram of Holness et al. (1989) doses C-58
Figure C-4. Histogram of mean exposures in high-exposure group (Holness et al., 1989) C-59
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Supplemental Information—Ammonia
ABBREVIATIONS
ADME absorption, distribution, metabolism, MRM
excretion NCEA
AEGL Acute Exposure Guideline Level
ACGIH American Conference of Governmental NH3
Industrial Hygienists NH4+
ALP alkaline phosphatase NIOSH
ATSDR Agency for Toxic Substances and
Disease Registry NOAEL
BCG bacillus Calmette-Guerin NRC
BMCL 95% lower bound on the benchmark ORD
concentration
BUN blood urea nitrogen PEF
CAAC Chemical Assessment Advisory PEFR
Committee PM
CAC cumulative ammonia concentration POD
CERCLA Comprehensive Environmental PPD
Response, Compensation, and Liability RfC
Act RfD
CFU colony forming unit RTECS
CI confidence interval
DAP diammonium phosphate SAB
EPA Environmental Protection Agency TLV
FEF forced expiratory flow TSCATS
FEVi forced expiratory volume in 1 second
FEVi% ratio of FEVi to FVC (FEVi/FVC) TWA
FVC forced vital capacity UF
HERO Health and Environmental Research UFa
Online UFh
HPV high production volume UFl
HPVIS high production volume information UFs
system
IgE immunoglobulin E UFd
IgG immunoglobulin G VEh
IOM Institute of Medicine
IRIS Integrated Risk Information System VEho
LC50 50% lethal concentration WOS
LD50 50% lethal dose
LOAEL lowest-observed-adverse-effect level
MLE maximum likelihood estimate
murine respiratory mycoplasmosis
National Center for Environmental
Assessment
ammonia
ammonium ion
National Institute for Occupational
Safely and Health
no-observed-adverse-effect level
National Research Council
EPA's Office of Research and
Development
peak expiratory flow
peak expiratory flow rate
particulate matter
point of departure
purified protein derivative
reference concentration
reference dose
Registry of Toxic Effects of Chemical
Substances
Science Advisory Board
Threshold Limit Value
Toxic Substance Control Act Test
Submissions Database
time-weighted average
uncertainty factor
interspecies uncertainty factor
intraspecies uncertainty factor
LOAEL to NOAEL uncertainty factor
subchronic-to-chronic uncertainty
factor
database deficiencies uncertainly factor
human occupational default minute
volume
human ambient default minute volume
Web of Science
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Supplemental Information—Ammonia
APPENDIX A. ASSESSMENTS BY OTHER NATIONAL
AND INTERNATIONAL HEALTH AGENCIES
Toxicity values and other health-related regulatory limits for ammonia that have been
developed by other national and international health agencies are summarized in Table A-l.
Table A-l. Assessments by other national and international health agency
assessments for ammonia
Organization
Toxicity value
Agency for Toxic Substances and Disease
Registry (ATSDR. 2004)a
Chronic inhalation MRL = 0.1 ppm (0.07 mg/m3)
Basis: Lack of significant alterations in lung function in chronically
exposed workers (Holness et al., 1989) and a composite UF of
30 (10 for human variability and a modifying factor of 3 for the lack
of reproductive and developmental studies).
National Advisory Committee for Acute
Exposure Guideline Levels for Hazardous
Substances (NRC, 2008)b
AEGL-1 (nondisabling) = 30 ppm (21 mg/m3) for exposures ranging
from 10 min to 8 hrs to protect against mild irritation
Basis: Mild irritation in human subjects (MacEwen et al., 1970)
AEGL-2 (disabling) = 220 ppm (154 mg/m3) for a 10-min exposure
to 110 ppm (77 mg/m3) for an 8-hr exposure
Basis: Irritation (eyes and throat; urge to cough) in human subjects
(Verberk, 1977)
AEGL-3 (lethal) = 2,700 ppm (1,888 mg/m3) for a 10-min exposure
to 390 ppm (273 mg/m3) for an 8-hr exposure
Basis: Lethalitv in the mouse (Kapeghian et al., 1982; MacEwen
and Vernot, 1972)
National Institute for Occupational Safety
and Health (NIOSH, 2015)°
REL established in 1992
REL = 25 ppm (18 mg/m3)d TWA for up to a 10-hr workday and a
40-hr work week
Basis: To project against respiratory and eye irritation. References
cited in support of the REL included review documents for the
years up to 1992; no specific reference served as the basis for the
REL.
Occupational Safety and Health
Administration (OSHA, 2006)e
PEL established in early 1970s
PEL for general industry = 50 ppm (35 mg/m3) TWA for an 8-hr
workday
Basis: The 1968 ACGIH TLV was promulgated as the OSHA PEL
soon after adoption of the Occupational Safety and Health Act in
1970. The ACGIH TLV from 1968 was intended to protect against
irritation of ammonia in humans; no specific reference served as
the basis for the 1968 TLV.
A-l
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Supplemental Information—Ammonia
Organization
Toxicity value
Food and Drug Admistration (FDA, 2011a, b)
Ammonium hydroxide: direct food substance affirmed as generally
recognized as safe (21 CFR 184.1139); substance generally
recognized as safe when used in accordance with good
manufacturing or feeding practices (21 CFR 582.1139).
aATSDR MRL = minimal risk level. An MRL is an estimate of daily human exposure to a hazardous substance that is
likely to be without an appreciable risk of adverse noncancer health effects over a specified route and duration of
exposure (http://www.atsdr.cdc.gov/mrls/index.asp; accessed 2/26/2016).
bAEGL = acute exposure guideline level. AEGLs are used by emergency planners and responders as guidance in
dealing with rare, usually accidental, releases of chemicals into the air and are calculated for exposure periods of
10 minutes, 30 minutes, 1 hour, 4 hours, and 8 hours. At concentrations above specificied levels, the general
population could experience the following: Level 1: notable discomfort, irritation, or certain asymptomatic non-
sensory effects; Level 2: ireversible or other serious, long-lasting adverse health effects or an impaired ability to
escape; and Level 3: life-threatening health effects or death (http://www.epa.gov/aegl/about-acute-exposure-
guideline-levels-aegls; accessed 2/26/2016).
cNIOSH REL = recommended exposure limit. An REL is an occupational exposure limit recommended by NIOSH to
OSHA for adoption as a permissible exposure limit. The REL is a level that NIOSH believes would be protective of
worker safety and health over a working lifetime if used in combination with engineering and work practice
controls, exposure and medical monitoring, posting and labeling of hazards, worker training, and personal
protective equipment (http://www.cdc.gov/niosh/npg/pgintrod.html; accessed 2/26/2016).
dNIOSH used slightly different ppm to mg/m3 conversion factors; TWA = time weighted average; UF = uncertainty
factor.
eOSHA PEL = permissible exposure limit; TLV = threshold limit value. PELs are legally enforceable occupational
standards (https://www.osha.gov/dsg/annotated-pels/; accessed 2/26/2016).
A-2
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Supplemental Information—Ammonia
APPENDIX B. ADDITIONAL DETAILS OF LITERATURE
SEARCH STRATEGY | STUDY SELECTION AND
EVALUATION
Table B-l. Literature search strings for computerized databases
Database
Query strings
Hits
PubMed
Period:
March 2013-
September 2015
Search date:
9/11/2015
((("Ammonia"[MeSH Terms] OR "ammonium hydroxide" [Supplementary
Concept]) AND (("ammonia/adverse effects"[MeSH Terms] OR
"ammonia/antagonists and inhibitors"[MeSH Terms] OR
"ammonia/blood"[MeSH Terms] OR "ammonia/cerebrospinal fluid"[MeSH
Terms] OR "ammonia/pharmacokinetics"[MeSH Terms] OR
"ammonia/poisoning"[MeSH Terms] OR "ammonia/toxicity"[MeSH Terms]
OR "ammonia/urine"[MeSH Terms]) OR ("hydroxides/adverse
effects"[MeSH Terms] OR "hydroxides/antagonists and inhibitors"[MeSH
Terms] OR "hydroxides/blood"[MeSH Terms] OR "hydroxides/cerebrospinal
fluid"[MeSH Terms] OR "hydroxides/pharmacokinetics"[MeSH Terms] OR
"hydroxides/poisoning"[MeSH Terms] OR "hydroxides/toxicity"[MeSH
Terms] OR "hydroxides/urine"[MeSH Terms]) OR
(("ammonia/metabolism"[MeSH Terms] OR
"hydroxides/metabolism"[MeSH Terms]) AND (animals[MeSH Terms] OR
humans[MeSH Terms])) OR (ci[Subheading] OR "environmental
exposure"[MeSH Terms] OR "endocrine system"[MeSH Terms] OR
"hormones, hormone substitutes, and hormone antagonists"[MeSH Terms]
OR risk[MeSH Terms] OR cancer[sb]) OR ((ammonia[majr] OR "ammonium
hydroxide"[Supplementary Concept]) AND (dose-response relationship,
drug[MeSH Terms] OR pharmacokinetics[MeSH Terms] OR
metabolism[MeSH Terms]) AND (humans[MeSH Terms] OR mammals[MeSH
Terms]))) OR ((Ammonia [Title] OR "Ammonium hydroxide"[Title] OR "Spirit
of hartshorn"[Title] OR Aquammonia[Title]) NOT medline[sb])) OR ((inhal*
OR (air OR breath OR exhal* OR respiration) OR (biological markers[MeSH
Terms] AND (air OR breath OR exhal* OR respiration)) OR ("air
pollutants"[MeSH Terms] AND (breath OR exhal*)) OR breath OR
(analysis[Subheading] AND breath) OR (respiration[MeSH Terms] OR breath
tests[MeSH Terms] OR exhalation[MeSH Terms])) AND (7664-41-7[rn] OR
1336-21-6[rn]))) AND (2013/03/01:3000[crdat] OR 2013/03/01:3000[mhda]
OR 2013/03/01:3000[edat])
1,473
B-l
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Supplemental Information—Ammonia
Database
Query strings
Hits
Period:
March 2012-
March 2013
Search date:
3/13/2013
("2012/03/26"[Date - Publication] : "3000"[Date - Publication]) AND
(("Ammonia"[MeSH Terms] OR "ammonium hydroxide" [Supplementary
Concept]) AND (("ammonia/adverse effects"[MeSH Terms] OR
"ammonia/antagonists and inhibitors"[MeSH Terms] OR
"ammonia/blood"[MeSH Terms] OR "ammonia/cerebrospinal fluid"[MeSH
Terms] OR "ammonia/pharmacokinetics"[MeSH Terms] OR
"ammonia/poisoning"[MeSH Terms] OR "ammonia/toxicity"[MeSH Terms]
OR "ammonia/urine"[MeSH Terms]) OR ("hydroxides/adverse
effects"[MeSH Terms] OR "hydroxides/antagonists and inhibitors"[MeSH
Terms] OR "hydroxides/blood"[MeSH Terms] OR "hydroxides/cerebrospinal
fluid"[MeSH Terms] OR "hydroxides/pharmacokinetics"[MeSH Terms] OR
"hydroxides/poisoning"[MeSH Terms] OR "hydroxides/toxicity"[MeSH
Terms] OR "hydroxides/urine"[MeSH Terms]) OR
(("ammonia/metabolism"[MeSH Terms] OR
"hydroxides/metabolism"[MeSH Terms]) AND (animals[MeSH Terms] OR
humans[MeSH Terms])) OR (ci[Subheading] OR "environmental
exposure"[MeSH Terms] OR "endocrine system"[MeSH Terms] OR
"hormones, hormone substitutes, and hormone antagonists"[MeSH Terms]
OR risk[MeSH Terms] OR cancer[sb]) OR ((ammonia[majr] OR "ammonium
hydroxide"[Supplementary Concept]) AND (dose-response relationship,
drug[MeSH Terms] OR pharmacokinetics[MeSH Terms] OR
metabolism[MeSH Terms]) AND (humans[MeSH Terms] OR mammals[MeSH
Terms]))) OR ((Ammonia [Title] OR "Ammonium hydroxide"[Title] OR "Spirit
of hartshorn"[Title] OR Aquammonia[Title]) NOT medline[sb]))
410
("2012/03/26"[Date - Publication] : "3000"[Date - Publication]) AND ((inhal*
OR (air OR breath OR exhal* OR respiration) OR (biological markers[MeSH
Terms] AND (air OR breath OR exhal* OR respiration)) OR ("air
pollutants"[MeSH Terms] AND (breath OR exhal*)) OR breath OR
(analysis[Subheading] AND breath) OR (respiration[MeSH Terms] OR breath
tests[MeSH Terms] OR exhalation[MeSH Terms])) AND (7664-41-7[rn] OR
1336-21-6[rn]))
50
Period:
March 2012-
March 2013
Search date:
9/10/2015a
((((((("Ammonia"[MeSH Terms] OR "ammonium hydroxide" [Supplementary
Concept]) AND (("ammonia/adverse effects"[MeSH Terms] OR
"ammonia/antagonists and inhibitors"[MeSH Terms] OR
"ammonia/blood"[MeSH Terms] OR "ammonia/cerebrospinal fluid"[MeSH
Terms] OR "ammonia/pharmacokinetics"[MeSH Terms] OR
"ammonia/poisoning"[MeSH Terms] OR "ammonia/toxicity"[MeSH Terms]
OR "ammonia/urine"[MeSH Terms]) OR ("hydroxides/adverse
effects"[MeSH Terms] OR "hydroxides/antagonists and inhibitors"[MeSH
Terms] OR "hydroxides/blood"[MeSH Terms] OR "hydroxides/cerebrospinal
fluid"[MeSH Terms] OR "hydroxides/pharmacokinetics"[MeSH Terms] OR
"hydroxides/poisoning"[MeSH Terms] OR "hydroxides/toxicity"[MeSH
Terms] OR "hydroxides/urine"[MeSH Terms]) OR
(("ammonia/metabolism"[MeSH Terms] OR
"hydroxides/metabolism"[MeSH Terms]) AND (animals[MeSH Terms] OR
humans[MeSH Terms])) OR (ci[Subheading] OR "environmental
exposure"[MeSH Terms] OR "endocrine system"[MeSH Terms] OR
"hormones, hormone substitutes, and hormone antagonists"[MeSH Terms]
OR risk[MeSH Terms] OR cancer[sb]) OR ((ammonia[majr] OR "ammonium
159
B-2
-------
Supplemental Information—Ammonia
Database
Query strings
Hits
hydroxide"[Supplementary Concept]) AND (dose-response relationship,
drug[MeSH Terms] OR pharmacokinetics[MeSH Terms] OR
metabolism[MeSH Terms]) AND (humans[MeSH Terms] OR mammals[MeSH
Terms]))) OR ((Ammonia [Title] OR "Ammonium hydroxide"[Title] OR "Spirit
of hartshorn"[Title] OR Aquammonia[Title]) NOT medline[sb]))) OR (((inhal*
OR (air OR breath OR exhal* OR respiration) OR (biological markers[MeSH
Terms] AND (air OR breath OR exhal* OR respiration)) OR ("air
pollutants"[MeSH Terms] AND (breath OR exhal*)) OR breath OR
(analysis[Subheading] AND breath) OR (respiration[MeSH Terms] OR breath
tests[MeSH Terms] OR exhalation[MeSH Terms])) AND (7664-41-7[rn] OR
1336-21-6[rn]))))))) AND «2012/03/26:2013/03/13[crdat] OR
2012/03/26:2013/03/13[mhda] OR 2012/03/26:2013/03/13[edat]) NOT
(2012/03/26:2013/03/13 [d p]))
No date limit
Search date:
3/26/2012
(("Ammonia"[MeSH Terms] OR "ammonium hydroxide" [Supplementary
Concept]) AND (("ammonia/adverse effects"[MeSH Terms] OR
"ammonia/antagonists and inhibitors"[MeSH Terms] OR
"ammonia/blood"[MeSH Terms] OR "ammonia/cerebrospinal fluid"[MeSH
Terms] OR "ammonia/pharmacokinetics"[MeSH Terms] OR
"ammonia/poisoning"[MeSH Terms] OR "ammonia/toxicity"[MeSH Terms]
OR "ammonia/urine"[MeSH Terms]) OR ("hydroxides/adverse
effects"[MeSH Terms] OR "hydroxides/antagonists and inhibitors"[MeSH
Terms] OR "hydroxides/blood"[MeSH Terms] OR "hydroxides/cerebrospinal
fluid"[MeSH Terms] OR "hydroxides/pharmacokinetics"[MeSH Terms] OR
"hydroxides/poisoning"[MeSH Terms] OR "hydroxides/toxicity"[MeSH
Terms] OR "hydroxides/urine"[MeSH Terms]) OR
(("ammonia/metabolism"[MeSH Terms] OR
"hydroxides/metabolism"[MeSH Terms]) AND (animals[MeSH Terms] OR
humans[MeSH Terms])) OR (ci[Subheading] OR "environmental
exposure"[MeSH Terms] OR "endocrine system"[MeSH Terms] OR
"hormones, hormone substitutes, and hormone antagonists"[MeSH Terms]
OR risk[MeSH Terms] OR cancer[sb]) OR ((ammonia[majr] OR "ammonium
hydroxide"[Supplementary Concept]) AND (dose-response relationship,
drug[MeSH Terms] OR pharmacokinetics[MeSH Terms] OR
metabolism[MeSH Terms]) AND (humans[MeSH Terms] OR mammals[MeSH
Terms]))) OR ((Ammonia [Title] OR "Ammonium hydroxide"[Title] OR "Spirit
of hartshorn"[Title] OR Aquammonia[Title]) NOT medline[sb])
13,012
Additional Search on Exhaled Breath
(inhal* OR (air OR breath OR exhal* OR respiration) OR (biological
markers[MeSH Terms] AND (air OR breath OR exhal* OR respiration)) OR
("air pollutants"[MeSH Terms] AND (breath OR exhal*)) OR breath OR
(analysis[Subheading] AND breath) OR (respiration[MeSH Terms] OR breath
tests[MeSH Terms] OR exhalation[MeSH Terms])) AND (7664-41-7[rn] OR
1336-21-6[rn])
1,600
B-3
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Supplemental Information—Ammonia
Database
Query strings
Hits
ToxLine
Period:
March 2012-
September 2015
Search date:
9/14/2015b
@or+( piscesqcorrection+"ammonia"+"ammonium hydroxide'VSpirit of
hartshorn"+"aquammonia"+@term+@rn+7664-41-7+@term+@rn+1336-
21-6)+@AND+@range+yr+2012+2015+@not+@org+pubmed+pubdart+
"nih+reporter"+tscats
33
Period:
March 2012-
March 2013
@AND+@OR+("7664-41-7"+"1336-21-6"+@TERM+@rn+7664-41-
7+@TERM+@rn+1336-21-6)+@na+ammon*+@RANGE+yr+2012+2013
100
Search date:
3/13/2013
No date limit
Search date:
3/26/2012
Searched via NLM (htto://toxnet.nlm.nih.gov/cgi-
bin/sis/htmlgen?TOXLINE): Limited to ammon* in title. This covered all
synonyms listed to both ammonia and ammonium hydroxide with the
exception of "spirit of hartshorn," which found no results when limited to
the title
2,417
TSCATS
TSCATS2: recent
notices
Search date:
9/15/2015
7664-41-7
1336-21-6
EPA receipt date: 01/01/2012-08/31/2015
0 TSCATS2
0 recent notices
TSCATS, TSCATS2,
TSCA: recent
notices
No date limit
7664-41-7
1336-21-6
50TSCATS1
7 TSCATS2
1 recent notice
Search date:
3/26/2012
Web of Science
Period:
March 2012-
September 2015
Search date:
9/10/2015
(TS="ammonia" OR TS="ammonium hydroxide" OR TS="Spirit of hartshorn"
ORTS="aquammonia") AND ((WC=("Toxicology" OR "Endocrinology &
Metabolism" OR "Gastroenterology & Hepatology" OR "Gastroenterology &
Hepatology" OR "Hematology" OR "Neurosciences" OR "Obstetrics &
Gynecology" OR "Pharmacology & Pharmacy" OR "Physiology" OR
"Respiratory System" OR "Urology & Nephrology" OR "Anatomy &
Morphology" OR "Andrology" OR "Pathology" OR "Otorhinolaryngology" OR
"Ophthalmology" OR "Pediatrics" OR "Oncology" OR "Reproductive Biology"
OR "Developmental Biology" OR "Biology" OR "Dermatology" OR "Allergy"
OR "Public, Environmental & Occupational Health") OR SU=("Anatomy &
Morphology" OR "Cardiovascular System & Cardiology" OR "Developmental
Biology" OR "Endocrinology & Metabolism" OR "Gastroenterology &
Hepatology" OR "Hematology" OR "Immunology" OR "Neurosciences &
Neurology" OR "Obstetrics & Gynecology" OR "Oncology" OR
"Ophthalmology" OR "Pathology" OR "Pediatrics" OR "Pharmacology &
3,691
B-4
-------
Supplemental Information—Ammonia
Database
Query strings
Hits
Pharmacy" OR "Physiology" OR "Public, Environmental & Occupational
Health" OR "Respiratory System" OR "Toxicology" OR "Urology &
Nephrology" OR "Reproductive Biology" OR "Dermatology" OR "Allergy"))
OR (WC="veterinary sciences" AND (TS="rat" ORTS="rats" ORTS="mouse"
ORTS="murine" ORTS="mice" ORTS="guinea" OR TS="muridae" OR
TS=rabbit* ORTS=lagomorph* ORTS=hamster* ORTS=ferret* OR
TS=gerbil* ORTS=rodent* ORTS="dog" ORTS="dogs" ORTS=beagle* OR
TS="canine" ORTS="cats" OR TS="feline" ORTS="pig" ORTS="pigs" OR
TS="swine" ORTS="porcine" ORTS=monkey* ORTS=macaque* OR
TS=baboon* ORTS=marmoset*)) OR (TS=toxic* AND (TS="rat" ORTS="rats"
ORTS="mouse" ORTS="murine" ORTS="mice" ORTS="guinea" OR
TS="muridae" ORTS=rabbit* ORTS=lagomorph* ORTS=hamster* OR
TS=ferret* OR TS=gerbil* OR TS=rodent* OR TS="dog" OR TS="dogs" OR
TS=beagle* ORTS="canine" ORTS="cats" ORTS="feline" ORTS="pig" OR
TS="pigs" ORTS="swine" OR TS="porcine" ORTS=monkey* OR
TS=macaque* ORTS=baboon* ORTS=marmoset*) OR (TS="child" OR
TS="children" ORTS=adolescen* ORTS=infant* ORTS="WORKER" OR
TS="WORKERS" ORTS="HUMAN" OR TS=patient* OR TS=mother OR
TS=fetal OR TS=fetus OR TS=citizens OR TS=milk OR TS=formula)) OR
TI=toxic*)
lndexes=SCI-EXPANDED, CPCI-S, CPCI-SSH, BKCI-S, BKCI-SSH, CCR-
EXPANDED, ICTimespan=2012-2015
(TS="ammonia" OR TS="ammonium hydroxide" OR TS="Spirit of hartshorn"
ORTS="aquammonia") AND (TS=breath ORTS=exhale* ORTS="expired air")
lndexes=SCI-EXPANDED, CPCI-S, CPCI-SSH, BKCI-S, BKCI-SSH, CCR-
EXPANDED, IC Timespan=2012-2015
125
Toxcenter
No date limit
Search date:
3/27/2012
((7664-41-7 OR 1336-21-6) not (patent/dt OR tscats/fs)) and (chronic OR
immunotox? OR neurotox? OR toxicokin? OR biomarker? OR neurolog? OR
pharmacokin? OR subchronic OR pbpk OR epidemiology/st,ct,it) OR acute
OR subacute OR Id50# OR Ic50# OR (toxicity OR adverse OR
poisoning)/st,ct,it OR inhal? OR pulmon? OR nasal? OR lung? OR respir? OR
occupation? OR workplace? OR worker? OR oral OR orally OR ingest? OR
gavage? OR diet OR diets OR dietary OR drinking(w)water OR (maximum
and concentration? and (allowable OR permissible)) OR (abort? OR
abnormalit? OR embryo? OR cleft? OR fetus? OR foetus? OR fetal? OR
foetal? OR fertil? OR malform? OR ovum OR ova OR ovary OR placenta? OR
pregnan? OR prenatal OR perinatal? OR postnatal? OR reproduc? OR steril?
OR teratogen? OR sperm OR spermac? OR spermag? OR spermati? OR
spermas? OR spermatob? OR spermatoc? OR spermatog? OR spermatoi?
OR spermatoi? OR spermator? OR spermatox? OR spermatoz? OR
spermatu? OR spermi? OR spermo? OR neonat? OR newborn OR
development OR developmental? OR zygote? OR child OR children OR
adolescen? OR infant OR wean? OR offspring OR age(w)factor? OR dermal?
OR dermis OR skin OR epiderm? OR cutaneous? OR carcinog? OR
cocarcinog? OR cancer? OR precancer? OR neoplas? OR tumor? OR tumour?
OR oncogen? OR lymphoma? OR carcinom? OR genetox? OR genotox? OR
mutagen? OR genetic(w)toxic? OR nephrotox? OR hepatotox? OR endocrin?
2,591
B-5
-------
Supplemental Information—Ammonia
Database
Query strings
Hits
OR estrogen? OR androgen? OR hormon?) AND (((biosis/fs AND py>1999
AND (hominidae/ct,st,it OR human/ct,st,it OR humans/ct,st,it OR
mammals/ct,st,it OR mammal/ct,st,it OR mammalia/ct,st,it)) OR ipa/fs OR
(caplus/fs AND 4-?/cc) OR ammonia/ti OR "ammonium hydroxide"/ti OR
"spirit of hartshorn"/ti OR aquammonia/ti)
Dupicates were removed; Biosis subfile results were date limited to avoid
extensive overlap with Toxline
Additional search on exhaled breath
(7664-41-7 OR 1336-21-6) AND (breath OR exhale? OR "expired air")
81
HERO
SQL statement
run on 3/14/13
select r.referencejd from tbl_reference r where r.sdelete = 'No' and (lower
(r.title) like '%ammonia%' or lower (r.title) like '%ammonium hydroxide%' or
lower (r.abstract) like '%ammonia%' or lower (r.abstract) like '%ammonium
hydroxide%') and r.year > 2011 and r.referencejd not in (select
referencejd from tbl_reference_project where projectjd = 36);
115
Search date:
3/27/2012
ammonia OR ammonium hydroxide
5,295
Combined
Reference Set
duplicates eliminated electronically
-28,000
aThis query expands the 2013 PubMed search from items published from March 2012 to March 2013 to all items
added to PubMed during that timeframe.
bThis query expands the 2013 Toxline search to include synonym searches for items not also appearing in the
PubMed database.
Table B-2. Processes used to augment the search of core computerized
databases for ammonia
System used
Key reference or source
Manual search of citations from
health assessment documents
ATSDR (2004). Toxicological profile for ammonia fATSDR Tox Profilel.
Atlanta, GA: U.S. Department of Health and Human Services, Public Health
Service. http://www.atsdr.cdc.gov/toxprofiles/tp.asp?id=ll&tid=2
NRC (2008). Acute exposure guideline levels for selected airborne
chemicals: Volume 6. Washington, DC: The National Academies Press.
httD://www.naD.edu/catalog.Dho?record id=12018
ACGIH (2001). Ammonia fTLV/BEIl. In Documentation of the threshold limit
values and biological exposure indices. Cincinnati, OH.
NIOSH (2015). NIOSH pocket guide to chemical hazards: Ammonia.
http://www.cdc.gov/niosh/npg/npgd0028.html
OSHA (2006). Table Z-l: Limits for air contaminants. Occupational safetv
and health standards, subpart Z, toxic and hazardous substances. (OSHA
standard 1910.1000, 29 CFR). Washington, DC: U.S. Department of Labor.
htto://www.osha.gov/Dls/oshaweb/owadiso.show document?o table=ST
ANDARDS&d id=9992
B-6
-------
Supplemental Information—Ammonia
System used
Key reference or source
FDA (2011a) Direct food substances affirmed as generally recognized as
safe (GRAS): Ammonium hydroxide, 21 CFR § 184.1139.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm ?f
r= 184.1139
FDA (2011b) Substances generally recognized as safe: General purpose
food additives: Ammonium hydroxide, 21 CFR § 582.1139.
http://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm ?f
r=582.1139
Manual search of citations from key
studies in cleaning and hospital
worker literature
Dumas et al. (2012). Occupational exposure to cleaning products and
asthma in hospital workers. Occup Environ Med 69: 883-889.
http://dx.doi.org/10.1136/oemed-2012-100826
Zock et al. (2010). Update on asthma and cleaners (Reviewl. Curr Opin
Allergy Clin Immunol 10:114-120.
http://dx.doi.org/10.1097/ACI.0b013e32833733fe
Mirabelli et al. (2007). Occupational risk factors for asthma among nurses
and related healthcare professionals in an international study. Occup
Environ Med 64: 474-479. http://dx.doi.org/10.1136/oem.2006.031203
Web of Science forward search
(performed in 2013 and updated in
2015)
Kennedv et al. (2000). Development of an asthma specific iob exposure
matrix and its application in the epidemiological study of genetics and
environment in asthma (EGEA). Occup Environ Med 57: 635-641.
http://dx.doi.Org/10.1136/oem.57.9.635
Search of online chemical
assessment-related websites
Combination of CASRN and synonyms searched on the following websites:
Period:
No limit- March 2012; updated
2012-August2015
Search date:
9/10/2015
ATSDR (http://www.atsdr.cdc.gov/substances/index.asp)
CalEPA (Office of Environmental Health Hazard Assessment)
(http://www.oehha.ca.gov/risk.html)
eChemPortal (includes: ACToR, AGRITOX, CCR, CCR DATA, CESAR, CHRIP,
ECHA CHEM, EnviChem, ESIS, GHS-J, HPVIS, HSDB, HSNO CCID, INCHEM,
J-CHECK, JECDB, NICNAS PEC, OECD HPV, OECD SIDS IUCUD, SIDS UNEP, UK
CCRMP Outputs, EPA IRIS, EPA SRS)
(http://www.echemportal.org/echemportal/participant/page.actionPpagel
D=9)
EPA Acute Exposure Guideline Levels
(http://www.epa.gov/aegl/access-acute-exposure-guideline-levels-aegls-
values#chemicals)
EPA - IRIS Assessments (http://cfpub.epa.gov/ncea/iris2/atoz.cfm)
EPA NSCEP (http://www.epa.gov/nscep)
EPA Science Inventory (http://cfpub.epa.gov/si/)
Federal Docket (www.regulations.gov)
Health Canada First Priority List Assessments (http://www.hc-
sc.gc.ca/ewh-semt/pubs/contaminants/psll-lspl/index-eng.php)
Health Canada Second Priority List Assessments (http://www.hc-
sc.gc.ca/ewh-semt/pubs/contaminants/psl2-lsp2/index-eng.php)
IARC (http://monographs.iarc.fr/ENG/Monographs/PDFs/index.php)
IPCS INCHEM (http://www.inchem.org/)
NAS via NAP (http://www.nap.edu/)
B-7
-------
Supplemental Information—Ammonia
System used
Key reference or source
NCI (htto://www.cancer.gov)
NCTR
(httD://www.fda.gov/AboutFDA/CentersOffices/OC/OfficeofScientificandM
edicalPrograms/NCTR/default.htm)
NIEHS (htto://www. niehs.nih.gov/)
NIOSHTIC 2 (htto://www2a.cdc.gov/nioshtic-2/)
NTP - RoC, status, results, and management reports
(https://ntp.niehs.nih.gov/)
WHO assessments - CICADS, EHC
(http://www.who.int/ipcs/assessment/en/)
Period:
No limit-August 2015
Search date:
9/10/2015
ACGIH (htto://www.acgih.org/home.htm)
AICS (htto://www.nicnas.gov.au/regulation-and-comoliance/aics/aics-
search-oage)
AIHA: WEELs (httos://www.aiha.org/get-
involved/AIHAGuidelineFoundation/WEELs/Documents/2011WEELValues.
odf); ERPGs (httos://www.aiha.org/get-
involved/AIHAGuidelineFoundation/EmergencvResoonsePlanningGuideline
s/Documents/2014%20ERPG%20Values.Ddf)
CalEPA Drinking Water Notification Levels
(htto://www.swrcb.ca.gov/drinking water/certlic/drinkingwater/Notificati
onLevels.shtml)
CalEPA Office of Environmental Health Hazard Assessment: OEHHAToxicity
Criteria Database (htto://www.oehha.ca.gov/tcdb/index.aso):
Biomonitoring California-priority and designated chemicals
(httD://biomonitoring. ca.gov/chemicals/chemical-index); OEHHA Fact
Sheets (http://www.oehha.ca.gov/public info/facts/index.html); Non-
cancer health effects Table-RELs
(http://www.oehha.ca.gov/air/allrels.html); Cancer Potency Factors-
Appendix A and AppendixB
(httD://www.oehha.ca.gov/air/hot SDOtsAsd052909.html)
CHRIP (htto://www.safe.nite.go.io/english/db.html)
CPSC (htto://www.cosc.gov)
ECHA Chem (httoV/echa.eurooa.eu/)
Environment Canada - Search entire site
(httD://www.ec.gc.ca/default.asD?lang=En&n=ECD35C36)
EPA HPVIS (htto://iava.eoa.gov/chemview) - Limit output selection to High
Production Volume Information System;
EPA OPP Pesticide Chemical Search
(httD://iasDub.eDa.gov/aDex/Desticides/f?D=chemicalsearch:l)
FDA (htto://www.fda.gov/)
Health Canada: Toxic Substances Managed Under CEPA
(httD://www.ec.gc.ca/toxiaues-toxics/Default.asD?lang=En&n=98E80CC6-
1); Final Assessments (httD://www.ec.gc.ca/lcDe-
ceDa/default.asD?lang=En&xml=09F567A7-BlEE-lFEE-73DB-
8AE6C1EB7658); Draft Assessments (httD://www.ec.gc.ca/lcDe-
ceDa/default.asD?lang=En&xml=6892C255-5597-C162-95FC-
4B905320F8C9); Health Canada Drinking Water Documents
(http://www.hc-sc.gc.ca/ewh-semt/pubs/water-eau/index-
eng.php#tech doc)
B-8
-------
Supplemental Information—Ammonia
System used
Key reference or source
NICNAS - PEC only covered by eChemPortal
(htto://www. nicnas.gov.au/chemical-information)
NIOSH (htto://www. cdc.gov/niosh/tooics/)
NRC - AEGLs via NAP (htto://www. nao.edu/)
OECD HPV (htto://webnet. oecd.org/hov/ui/Search.asox)
OSHA
(htto://www.osha.gov/dts/chemicalsamDling/toc/toc chemsamD.html)
RTECS (httD://ccinfoweb.ccohs.ca/rtecs/search.html)
ACGIH = American Conference of Governmental Industrial Hygienists; ACToR = Aggregated Computational
Toxicology Resource; AEGL = Acute Exposure Guideline Level; AICS = Australian Inventory of Chemical Substances;
AIHA = American Industrial Hygiene Association; ATSDR = Agency for Toxic Substances and Disease Registry;
CalEPA = California Environmental Protection Agency; CASRN = Chemical Abstract Service Registry Number;
CCID = Chemical Classification Information Database; CCR = Canadian Categorization Results;
CCRMP = Coordinated Chemicals Risk Management Programme Publications; CEPA = Canadian Environmental
Protection Act; CESAR = Canada's Existing Substances Assessment Repository; CHRIP = Chemical Risk Information
Platform; CICAD = Comision Interamericana para el Control del Abuso de Drogas (Inter-American Drug Abuse
Control Commission); CPSC = Consumer Product Safety Commission; ECHA = European Chemicals Agency;
EHC = Environmental Health Criteria; EPA = Environmental Protection Agency; ERPG = Emergency Response
Planning Guidelines; FDA = Food and Drug Administration; HPV = High Production Volume; HPVIS = High
Production Volume Information System; HSDB = Hazardous Substances Data Bank; HSNO = Hazardous Substances
and New Organisms; IARC = International Agency for Research on Cancer; IPCS = International Programme on
Chemical Safety; IRIS = Integrated Risk Information System; IUCLID = International Uniform Chemical Information
Database; J-CHECK = Japan CHEmicals Collaborative Knowledge; JECDB = Japan Existing Chemical Data Base;
NAP = National Academies Press; NAS = National Academy of Sciences; NCI = National Cancer Institute;
NCTR= National Center for Toxicological Research; NICNAS = National Industrial Chemicals Notification and
Assessment Scheme; NIEHS = National Institute for Environmental Health Sciences; NIOSH = National Institute for
Occupational Safety and Health; NIOSHTIC = National Institute for Occupational Safety and Health Technical
Information Center; NRC = National Research Council; NSCEP = National Service Center for Environmental
Publications; NTP = National Toxicology Program; OECD = Organisation for Economic Cooperation and
Development; OEHHA = Office of Environmental Health Hazard Assessment; OPP = Office of Pesticide Programs;
OSHA = Occupational Safety and Health Administration; PEC = Priority Existing Chemical; REL = Reference
Exposure Level; RTECS = Registry of Toxic Effects of Chemical Substances; RoC = Report on Carcinogens;
SIDS = Screening Information Data Set; SRS = Substance Registry Services; UNEP = United Nations Environment
Programme; WEEL = Workplace Environmental Exposure Level; WHO = World Health Organization
B-9
-------
Supplemental Information—Ammonia
Table B-3. Disposition of studies from the cleaning and hospital worker
literature
Studies selected for full text review
Review of
full text or
abstract?
Disposition based
on
inclusion/exclusion
criteria in Table LS-1
References identified by manual backward search of seminal studies identified in March 2013 and forward
search of Kennedy et al. (2000) performed in 2013
Kogevinas et al. (2007). Exposure to substances in the workplace and
new-onset asthma: an international prospective population-based study
(ECRHS-II). Lancet 370: 336-341. http://dx.doi.org/10.1016/S0140-
6736(07)61164-7
Full-text
Exclude
No ammonia-specific
data
Mirabelli et al. (2007). Occupational risk factors for asthma among nurses
and related healthcare professionals in an international study. Occup
Environ Med 64: 474-479. http://dx.doi.org/10.1136/oem.2006.031203
Full-text
Exclude
No ammonia-specific
data
Zock et al. (2007). The use of household cleaning spravs and adult
asthma: An international longitudinal study. Am J Respir Crit Care Med
176: 735-741. http://dx.doi.org/10.1164/rccm.200612-17930C
Full-text
Include
Zock et al. (2009). Domestic use of hypochlorite bleach, atopic
sensitization, and respiratory symptoms in adults. J Allergy Clin Immunol
124: 731-738. http://dx.doi.Org/10.1016/i.iaci.2009.06.007
Abstract
Exclude
No ammonia-specific
data
Orriols et al. (2006). Reported occupational respiratory diseases in
Catalonia. Occup Environ Med 63: 255-260.
http://dx.doi.org/10.1136/oem.2005.022525
Abstract
Exclude
No ammonia-specific
data
Cherrv et al. (2009). Data linkage to estimate the extent and distribution
of occupational disease: new onset adult asthma in Alberta, Canada. Am J
Ind Med 52: 831-840. http://dx.doi.org/10.1002/aiim.20753
Full-text
Exclude
No ammonia-specific
data
Mazurek et al. (2008). Work-related asthma in the educational services
industry: California, Massachusetts, Michigan, and New Jersey, 1993-
2000. Am J Ind Med 51: 47-59. http://dx.doi.org/10.1002/aiim.20539
Abstract
Exclude
No ammonia-specific
data
Obadia et al. (2009). Relationships between asthma and work exposures
among non-domestic cleaners in Ontario. Am J Ind Med 52: 716-723.
http://dx.doi.org/10.1002/aiim.20730
Abstract
Exclude
No ammonia-specific
data
Lvnde et al. (2009). Cutaneous and respiratory svmptoms among
professional cleaners. Occup Med (Lond) 59: 249-254.
http://dx.doi.org/10.1093/occmed/kap051
Abstract
Exclude
No ammonia-specific
data
Massin et al. (2007). Respiratory svmptoms and bronchial responsiveness
among cleaning and disinfecting workers in the food industry. Occup
Environ Med 64: 75-81
Full-text
Exclude
Quarternary ammonia
de Fatima Macaira et al. (2007). Rhinitis and asthma svmptoms in non-
domestic cleaners from the Sao Paulo metropolitan area, Brazil. Occup
Environ Med 64: 446-453. http://dx.doi.org/10.1136/oem.2006.032094
Full-text
Exclude
Ammonium
B-10
-------
Supplemental Information—Ammonia
Studies selected for full text review
Review of
full text or
abstract?
Disposition based
on
inclusion/exclusion
criteria in Table LS-1
Medina-Ramon et al. (2006). Short-term respiratory effects of cleaning
exposures in female domestic cleaners. Eur Respir J 27: 1196-1203.
http://dx.doi.org/10.1183/09031936.06.00085405
Full-text
Include
Bernstein et al. (2009). Evaluation of cleaning activities on respiratory
symptoms in asthmatic female homemakers. Ann Allergy Asthma
Immunol 102: 41-46. http://dx.doi.org/10.1016/S1081-1206(10)60106-8
Full-text
Exclude
No ammonia-specific
data
Delclos et al. (2007). Occupational risk factors and asthma among health
care professionals. Am J Respir Crit Care Med 175: 667-675.
http://dx.doi.org/10.1164/rccm.200609-13310C
Full-text
Exclude
No ammonia-specific
data
Arif et al. (2009). Occupational exposures and asthma among nursing
professionals. Occup Environ Med 66: 274-278.
http://dx.doi.org/10.1136/oem.2008.042382
Full-text
Exclude
No ammonia-specific
data
Liss et al. (2011). Work-related asthma in health care in Ontario. Am J Ind
Med 54: 278-284. http://dx.doi.org/10.1002/aiim.20935
Abstract
Exclude
No ammonia-specific
data
Pechter et al. (2005). Work-related asthma among health care workers:
surveillance data from California, Massachusetts, Michigan, and New
Jersey, 1993-1997. Am J Ind Med 47: 265-275.
http://dx.doi.org/10.1002/aiim.20138
Full-text
Exclude
No ammonia-specific
data
Arif and Delclos (2012). Association between cleaning-related chemicals
and work-related asthma and asthma symptoms among healthcare
professionals. Occup Environ Med 69: 35-40.
http://dx.doi.org/10.1136/oem.2011.064865
Full-text
Include
Vizcava et al. (2011). A workforce-based studv of occupational exposures
and asthma symptoms in cleaning workers. Occup Environ Med 68: 914-
919. http://dx.doi.org/10.1136/oem.2010.063271
Full-text
Include
Quirce and Barranco (2010). Cleaning agents and asthma. J Investig
Allergol Clin Immunol 20: 542-550.
Full-text
Exclude
Review article
(references checked;
no new references
identified)
Chan-Yeung and Malo (1994). Aetiological agents in occupational asthma.
Eur Respir J 7: 346-371.
http://dx.doi.org/10.1183/09031936.94.07020346
Full-text
Exclude
Review article
Medina-Ramon et al. (2005). Asthma, chronic bronchitis, and exposure to
irritant agents in occupational domestic cleaning: A nested case-control
study. Occup Environ Med 62: 598-606.
http://dx.doi.org/10.1136/oem.2004.017640
Full-text
Include
Le Moual et al. (2012). Domestic use of cleaning spravs and asthma
activity in females. Eur Respir J 40:1381-1389.
http://dx.doi.org/10.1183/09031936.00197611
Full-text
Exclude
No ammonia-specific
data (Ammonia
B-ll
-------
Supplemental Information—Ammonia
Studies selected for full text review
Review of
full text or
abstract?
Disposition based
on
inclusion/exclusion
criteria in Table LS-1
analyzed as part of
"Factor 3", with
decalcifiers, acids,
stain removers, and
sprays for carpets,
rugs and curtains)
Ghosh et al. (2013). Asthma and occupation in the 1958 birth cohort.
Thorax 68: 365-371. http://dx.doi.org/10.1136/thoraxinl-2012-202151
Full-text
Exclude
No ammonia-specific
data
Lemiere et al. (2012). Occupational risk factors associated with work-
exacerbated asthma in Quebec. Occup Environ Med 69: 901-907.
http://dx.doi.org/10.1136/oemed-2012-100663
Full-text
Include
References identified in September 2015 update of forward search of Kennedv et al. (2000)
Beach et al. (2012). Estimating the extent and distribution of new-onset
adult asthma in British Columbia using frequentist and Bayesian
approaches. Ann Occup Hyg 56: 719-727.
http://dx.doi.org/10.1093/annhvg/mes004
Full-text
Exclude
No ammonia-specific
data
Casas and Nemerv (2014). Irritants and asthma. Eur Respir J 44: 562-564.
http://dx.doi.org/10.1183/09031936.00090014
Full-text
Exclude
Editorial
Christensen et al. (2013a). Occupational exposure during pregnancy and
the risk of hay fever in 7-year-old children. Clinical Respiratory Journal 7:
183-188. http://dx.doi.Org/10.llll/i.1752-699X.2012.00300.x
Full-text
Exclude
No ammonia-specific
data
Christensen et al. (2013b). Maternal occupational exposure to
asthmogens during pregnancy and risk of asthma in 7-year-old children: a
cohort studv. BMJ Open 3. http://dx.doi.org/10.1136/bmiopen-2012-
002401
Full-text
Exclude
No ammonia-specific
data
Dumas et al. (2014a). Occupational irritants and asthma: an Estonian
cross-sectional study of 34 000 adults. Eur Respir J 44: 647-656.
http://dx.doi.org/10.1183/09031936.00172213
Full-text
Exclude
No ammonia-specific
data
Dumas et al. (2013). Work related asthma. A causal analysis controlling
the healthy worker effect. Occup Environ Med 70: 603-610.
http://dx.doi.org/10.1136/oemed-2013-101362
Full-text
Exclude
No ammonia-specific
data
Dumas et al. (2014b). Cleaning and Asthma Characteristics in Women. Am
J Ind Med 57: 303-311. http://dx.doi.org/10.1002/aiim.22244
Full-text
Exclude
No ammonia-specific
data
Henneberger et al. (2015). Occupational exposures associated with severe
exacerbation of asthma. Int JTuberc Lung Dis 19: 244-250.
http://dx.doi.org/10.5588/iitld.14.0132
Full-text
Exclude
No ammonia-specific
data
B-12
-------
Supplemental Information—Ammonia
Studies selected for full text review
Review of
full text or
abstract?
Disposition based
on
inclusion/exclusion
criteria in Table LS-1
Jeebhav et al. (2014). Risk factors for nonwork- related adult- onset
asthma and occupational asthma: a comparative review. Curr Opin
Allergy Clin Immunol 14: 84-94.
http://dx.doi.Org/10.1097/ACI.0000000000000042
Full-text
Exclude
Review article
(references checked;
no new references
identified)
Kellberger et al. (2014). Predictors of work-related sensitisation, allergic
rhinitis and asthma in early work life. Eur Respir J 44: 657-665.
http://dx.doi.org/10.1183/09031936.00153013
Full-text
Exclude
No ammonia-specific
data
Koehoorn et al. (2013). Population-based surveillance of asthma among
workers in British Columbia, Canada. Chronic Dis Inj Can 33: 88-94.
Full-text
Exclude
No ammonia-specific
data
Le Moual et al. (2014). Occupational exposures and uncontrolled adult-
onset asthma in the European Community Respiratory Health Survey II.
Eur Respir J 43: 374-386. http://dx.doi.org/10.1183/09031936.00034913
Full-text
Exclude
No ammonia-specific
data
Lillienberg et al. (2013). Occupational Exposure and New-onset Asthma in
a Population-based Study in Northern Europe (RHINE). Ann Occup Hyg 57:
482-492. http://dx.doi.org/10.1093/annhvg/mes083
Full-text
Exclude
No ammonia-specific
data
Lillienberg et al. (2014). Exposures and asthma outcomes using two
different job exposure matrices in a general population study in northern
Europe. Ann Occup Hyg 58: 469-481.
http://dx.doi.org/10.1093/annhvg/meu002
Full-text
Exclude
No ammonia-specific
data
Lindstrom et al. (2013). Middle-Aged Men With Asthma Since Youth The
Impact of Work on Asthma. J Occup Environ Med 55: 917-923.
http://dx.doi.org/10.1097/JOM.0b013e31828dc9c9
Full-text
Exclude
No ammonia-specific
data
Mirabelli et al. (2012). Occupation and three-vear incidence of respiratory
symptoms and lung function decline: the ARIC Study. Respir Res 13.
http://dx.doi.org/10.1186/1465-9921-13-24
Full-text
Exclude
No ammonia-specific
data
Thilsing et al. (2012). Chronic rhinosinusitis and occupational risk factors
among 20- to 75-year-old Danes-A GA(2) LEN-based study. Am J Ind Med
55:1037-1043. http://dx.doi.org/10.1002/aiim.22074
Full-text
Exclude
No ammonia-specific
data
B-13
-------
Supplemental Information—Ammonia
Table B-4. Electronic screening inclusion terms (and fragments) for ammonia
(gastrointestinal OR gastro-intestinal OR digestive tract OR stomach* OR (gastric AND (mucosa* OR cancer* OR
tumor* OR tumour* OR neoplas*)) OR (ammoni*[title] AND intestin*[title or keyword]) OR genotox* OR
(genetic* AND toxic*) OR ames assay* OR ames test* OR aneuploid* OR chromosom*[title] OR clastogen* OR
cytogen* OR dominant lethal OR genetic*[title] OR genotox* OR hyperploid* OR micronucle* OR mitotic* OR
mutagen*[title] OR mutat*[title] OR recessive lethal OR sister chromatid OR ((kidney* OR renal) AND (toxic* OR
poisoning OR adverse OR congestion OR calcif*)) OR nephrotox* OR ((spleen* OR splenic) AND (toxic* OR
poisoning OR adverse OR congestion OR enlarged)) OR absorption OR distribution OR metabolism[title or
keywords] OR excret* OR PBPK OR toxicokinetic* OR pharmacokin* OR exhal* OR breath OR (expired AND air)
OR (respiratory AND (irritation OR symptom* or disease* OR adverse OR chemically induced)) OR lung* OR
(pulmonary AND (irritation* OR function*)) OR FVC OR Forced vital capacity OR Forced expiratory volume OR FEV
OR FEV1 OR inflammation OR congest* OR edema* OR hemorrhag* OR discharge* OR phlegm* OR cough* OR
wheez* OR dyspnea OR bronchitis OR pneumonitis OR asthma* OR nose OR nasal OR throat OR trachea* OR
bronchial OR airway* OR (chest AND tightness) OR epithelium* OR epithelia* OR immune OR immun*[title] OR
antibod* OR antigen* OR autoimmun* OR cytokine* OR granulocyte* OR interferon OR interleukin* OR
leukocyte* OR lymph* OR lymphocyt* OR monocyt* OR immunosupress* OR immunotox* OR (immun* AND
toxic*) OR hypersensitivity OR ((dermal OR skin) AND lesion*) OR erythema* OR host resistance OR ((bacterial OR
bacteria) AND coloniz*) OR T cell* OR T-Lymphocyte* OR thymocyte* OR ((liver* OR hepatic) AND (function* OR
congest* OR toxic* OR poisoning OR adverse)) OR hepatotox* OR fatty liver OR clinical chemistry OR adrenal OR
((heart* OR cardiac) AND (toxic* OR adverse OR poisoning)) OR cardiotox* OR myocardium OR myocardial OR
lacrimation OR ocular OR (eye* AND discharge*) OR opacity OR blood pH OR neurotransmitter* OR (amino acid*
AND brain) OR reproduct*[title] OR reproductive OR developmental[title or keywords] OR terato* OR
(ammoni*[title] AND (abort* OR cleft* OR embryo* OR fertilit* OR fetal OR fetus* OR foetal OR foetus* OR
gestation* OR infertilit* OR malform* OR neonat* OR newborn* OR ova OR ovaries OR ovary OR ovum OR
perinatal OR placenta* OR postnatal OR pregnan* OR prenatal OR sperm OR sterilit* OR zygote*)) NOT
(hyperammon* OR ammonemia OR ammonaemia OR hepatic coma OR liver failure OR (reye AND syndrome) OR
((hepatic OR liver OR portosystemic OR portal-systemic) AND (encephalopathy OR cirrhosis)) OR fish OR fishes OR
carp OR catfish OR crayfish OR jellyfish OR daphnia OR shrimp OR frog OR frogs OR amphibians OR bivalve OR
bivalves OR clam OR Crustacea OR crustaceans)
Table B-5. Disposition of epidemiology studies identified in September 2015
literature search update of core databases
Epidemiology study
Review of
full text or
abstract?
Disposition
Folletti et al. (2014). Asthma and rhinitis in cleaning workers: a
systematic review of epidemiological studies. J Asthma 51: 18-28.
htto://dx.doi.org/10.3109/02770903.2013.833217
Full-text
Exclude
Review article
Siracusa et al. (2013). Asthma and exposure to cleaning products - a
European Academy of Allergy and Clinical Immunology task force
consensus statement. Allergy 68:1532-1545.
httD://dx.doi.org/10.1111/all. 12279
Full-text
Exclude
Review article
Casas et al. (2013). Use of household cleaning products, exhaled nitric
oxide and lung function in children [Letter], Eur Respir J 42: 1415-1418.
http://dx.doi.org/10.1183/09031936.00066313
Full-text
Include
B-14
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Supplemental Information—Ammonia
Hovland et al. (2013). Longitudinal lung function decline among workers
in a nitrate fertilizer production plant. Int J Occup Environ Health 19:119-
126. http://dx.doi.org/10.1179/2049396713Y.0000000025
Full-text
Exclude
Extremely low
ammonia
concentrations
(maximum
concentration of
0.1 mg/m3) and
mandatory respiratory
protection. Not
expected to be
informative for
evaluating
relationships between
ammonia exposure
and health effects
Hovland et al. (2014). Longitudinal decline in pulmonarv diffusing
capacity among nitrate fertilizer workers. Occup Med (Lond) 64: 181-187.
http://dx.doi.org/10.1093/occmed/kqtl74
Full-text
Exclude
No ammonia
concentrations
reported
Loftus et al. (2015). Ambient ammonia exposures in an agricultural
community and pediatric asthma morbidity. Epidemiology 26: 794-801.
htto://dx.doi.org/10.1097/EDE.0000000000000368
Full-text
Include
Nemer et al. (2015). Airwav inflammation and ammonia exposure among
female Palestinian hairdressers: Across-sectional study. Occup Environ
Med 72: 428-434. httD://dx.doi.org/10.1136/oemed-2014-102437
Full-text
Include
Ulvestad et al. (2014). Short-term lung function decline in tunnel
construction workers. Occup Environ Med 72:108-113.
httD://dx.doi.org/10.1136/oemed-2014-102262
Full-text
Exclude
No analysis of
ammonia-specific
exposures;
confounding of
respiratory effects by
silica exposure
B-15
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Supplemental Information—Ammonia
Table B-6. Evaluation of epidemiology studies summarized in Table 1-2 (industrial settings/respiratory
measures)
Comments
Study setting/
Outcome
Consideration of
regarding potential
Reference
participant selection
Exposure parameters
measured
confounding
Statistical analysis
major limitations
Respiratory symptoms
Nemer et al.
Palestine, laboratory at
Ammonia air
Modified version
Other hair salon
Statistical software
Device used for
(2015)
Hebron University; cross-
concentrations
of a standardized
exposures known to
was used to
exposure
sectional study of female
measured in 13 salons
respiratory
cause irritation,
calculate arithmetic
measurements had
hairdressers in 13 hair
using an
questionnaire
inflammation, or
means and SDs for
limited specificity for
salons from 10/2012 to
electrochemical sensor
from the American
other respiratory
exposure data and
measuring ammonia
03/2013
instrument (direct
Thoracic Society,
effects (such as
outcome variables
relative to other gases
Exposed: n = 33
reading device) affixed
including
persulfates) were not
(potential false
nonsmoking female
to one hairdresser in
questions on
measured
positives from other
hairdressers (age
each salon; sample
respiratory
gases); potential
19-50 yrs; mean 38 yrs);
duration 45-305 min;
symptoms (chest
Factors potentially
selection bias in
selected from a cohort of
concentration range
tightness,
predicting ammonia
control group due to
200 hairdressers studied
0-202 mg/m3; duration
shortness of
exposure, including
differences in
previously (every sixth
variation due to the
breath, coughing,
size of salon, number
recruitment (self-
participant from a list
variation in the number
wheezing, phlegm
of hairdressers at
selected based on
sorted by salon name was
of customers serviced
production during
work and number of
interest in the study)
invited to participate)
the past 12 mo
customers, tasks
or workload; small
Controls: n = 35;
Limited specificity for
and doctors'
being done (coloring,
sample size and only a
nonsmoking female
measuring ammonia
diagnosed
bleaching, cutting,
single measurement
students from Hebron
compared to other
asthma)
spraying), were
of ammonia at each
University (n = 27) and staff
gases
evaluated
salon (which may not
(n = 8); age of all controls
have been
18-49 yrs, mean 24 yrs;
No adjustment for
representative of
recruited through
smoking since all
salon exposures)
advertisements
participants were
nonsmokers
B-16
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Supplemental Information—Ammonia
Comments
Study setting/
Outcome
Consideration of
regarding potential
Reference
participant selection
Exposure parameters
measured
confounding
Statistical analysis
major limitations
Rahman et
Bangladesh, urea fertilizer
Personal airborne levels
Respiratory
Nitrogen dioxide
Fisher's exact test;
Study population and
al. (2007)
factory; cross sectional
of ammonia exposure
symptoms (five-
(measured by Drager
repeated excluding
design: "healthy"
study
by two direct-reading
point scale for
tubes) was below
33 current smokers
workers; long
Exposed: n = 88
methods: Drager
severity over last
detection limit in all
or workers with
duration—potential
(24 ammonia plant workers
diffusion tube and
shift), based on
areas (urea plant,
history of previous
for lack of complete
and 64 urea plant workers)
Drager PAC III
Optimal Symptom
ammonia plant, and
respiratory disease
ascertainment of
Controls: n = 25
monitoring instrument0;
Score
administration area);
effect
Exposed: production
one worker per day per
Questionnaire)
other workplace
operators in ammonia (low
measure
exposures not
Differences in
exposure; 24 out of
assessed
exposure
63 workers participated)3
Correlation between
measurement
and urea (high exposure,
methods; r = 0.80, but
Exposure analysis
methods (Drager
64 out of 77 workers
higher absolute values
adjusted for current
diffusion tube and
participated)15 plants,
(by 4-5-fold) using
smoking and duration
Drager PAC III
5-9 out of 15-19 per shift
Drager diffusion tubes;0
monitoring
selected; excluded if
concentrations based
instrument)
planned to have <4-hr work
on PAC III monitoring:
considered a
day; mean age ~40 yrs,
Low-exposure group
limitation for
mean duration ~18 yrs;
(ammonia plant):
quantitation of
never smoked ~52%.
6.9 ppm (4.9 mg/m3)
exposure-response
Controls: from
High-exposure group
relationship but not a
administration building,
(urea plant): 26.1 ppm
limitation for hazard
4-7/d over 5 d selected
(18.5 mg/m3)
identification due to
Mean age ~43 yrs, mean
uncertainty in the
duration ~16 yrs; never
absolute value, but
smoked ~72%.
not the relative
ranking, of exposure
B-17
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Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure parameters
Outcome
measured
Consideration of
confounding
Statistical analysis
Comments
regarding potential
major limitations
Ballal et al.
(1998)
Saudi Arabia, two urea
fertilizer factories; cross-
sectional study; all males
Exposed: n = 161
Factory A: n = 84
Factory B: n = 77
Controls: n = 355
Exposed: 20% of workers
selected (systematic
sample representing
different workplaces using
payroll lists); 100%
participation rate; mean
age 30 yrs, mean duration
51.8 mo; never smoked
~59%
Controls: administrative
staff from other companies
in the area (same sampling
system as exposed);
participation rate 100%
Mean age 34 yrs, mean
duration 73 mo; never
smoked ~49%
Area monitors (three
sets in each work
section taken at least
3 mo apart, mean
16 measures per set);
spectrophotometric
absorption measure
Computed geometric
mean concentration per
section and cumulative
ammonia concentration
(a function of both
exposure intensity and
duration of service)
assigned to each worker
Prevalence of
respiratory
symptoms and
conditions based
on the British
Medical Research
Council
questionnaire
Authors stated no
other pollutants in
workplace; stratified
or adjusted for
smoking
Contingency tables
(stratified by
smoking); logistic
regression of
exposure measures,
adjusted for
duration, smoking
(yes, no)
Study population and
design: "healthy"
workers; long
duration—potential
for lack of complete
ascertainment of
effect
Holness et
al. (1989)
Canada, sodium carbonate
(soda ash) production
plant; cross-sectional study
Exposed: n = 58
Controls: n = 31
Exposed: 52 of 64 available
production workers (82%)
and 6 maintenance
workers; all males; mean
Airborne levels of
ammonia (mean =
6.5 mg/m3 for exposed;
mean = 0.2 mg/m3 for
controls) using NIOSH-
recommended protocol
for personal sampling
and analysis (measured
Prevalence of self-
reported
symptoms and
conditions
obtained through
questionnaire
based on
American Thoracic
Adjusted for smoking
(pack-yrs); other
workplace exposures
were not assessed,
but study authors
note a high level of
control of exposures
in the plant
Comparison
between groups by
logistic regression;
also analyzed by
three categories of
exposure
Study population and
design: "healthy"
workers; long
duration—potential
for lack of complete
ascertainment of
effect
B-18
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Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure parameters
Outcome
measured
Consideration of
confounding
Statistical analysis
Comments
regarding potential
major limitations
age 39 yrs, mean duration
14.4 yrs, nonsmokers ~29%
Controls from stores and
office workers in the plant;
excluded if previous
ammonia exposure
Participation rate not
reported; mean age 43 yrs,
mean duration 12.2 yrs;
nonsmokers ~39%
Indication of self-selection
of exposed out of
workplace based on atopy
(lower prevalence of hay
fever)
over one work-shift per
person, mean 8.4 hrs)
Society
questionnaire
Relatively small
sample size—potential
of not being able to
detect a difference
between controls and
exposed when one
might exist
Low exposure
concentrations-
potential that an
effect level may not
have been reached
Lung function
Nemer et al.
(2015)
Palestine, laboratory at
Hebron University; cross-
sectional study of female
hairdressers in 13 hair
salons from 10/2012 to
03/2013
Exposed: n = 33
nonsmoking female
hairdressers (age
19-50 yrs; mean 38 yrs);
selected from a cohort of
200 hairdressers studied
previously (every sixth
participant from a list
Ammonia air
concentrations
measured in 13 salons
using an electro-
chemical sensor
instrument (direct
reading device) affixed
to one hairdresser in
each salon; sample
duration 45-305 min;
concentration range
0-202 mg/m3; duration
variation due to the
variation in the number
of customers serviced
Lung function test
performed
according to
American Thoracic
Society/European
Respiratory
Standards
guidelines using a
PC spirometer;
data adjusted for
age, height, and
BMI
Other hair salon
exposures known to
cause irritation,
inflammation, or
other respiratory
effects (such as
persulfates) were not
measured
Factors potentially
predicting ammonia
exposure, including
size of salon, number
of hairdressers at
work and number of
customers, tasks
Linear regression
used to assess the
relationship
between ammonia
exposure and lung
function
Device used for
exposure
measurements had
limited specificity for
measuring ammonia
relative to other gases
(potential false
positives from other
gases); potential
selection bias in
control group due to
differences in
recruitment (self-
selected based on
interest in the study)
or workload; small
B-19
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Supplemental Information—Ammonia
Comments
Study setting/
Outcome
Consideration of
regarding potential
Reference
participant selection
Exposure parameters
measured
confounding
Statistical analysis
major limitations
sorted by salon name was
Limited specificity for
being done (coloring,
sample size and only a
invited to participate)
measuring ammonia
bleaching, cutting,
single measurement
Controls: n = 35;
compared to other
spraying), were
of ammonia at each
nonsmoking female
gases
evaluated
salon (which may not
students from Hebron
have been
University (n = 27) and staff
No adjustment for
representative of
(n = 8); age of all controls
smoking since all
salon exposures)
18-49 yrs, mean 24yrs;
participants were
recruited through
nonsmokers
advertisements
Rahman et
Bangladesh, urea fertilizer
Personal airborne levels
Spirometry by
Nitrogen dioxide
Paired t-tests
Study population and
al. (2007)
factory; cross-sectional
of ammonia exposure
standard protocol,
(measured by Drager
compared cross
design: "healthy"
study
by two direct-reading
beginning and end
tubes) was below
shift differences in
workers; long
Exposed: n = 88
methods: Drager
of shift
detection limit in all
lung function within
duration-potential for
(24 ammonia plant workers
diffusion tube and
areas (urea plant,
and between plants;
lack of complete
and 64 urea plant workers);
Drager PAC III
ammonia plant, and
analyses repeated
ascertainment of
production operators in
monitoring instrument0;
administration area);
excluding workers
effect
ammonia (low exposure;
one worker per day per
other workplace
with previous
24 out of 63 workers
measure
exposures not
respiratory
Differences in
participated)3 and urea
assessed
diseases; multiple
exposure
(high exposure, 64 out of
Correlation between
linear regression
measurement
77 workers participated)15
methods; r = 0.80, but
Exposure analysis
analyzed exposure
methods (Drager
plants, 5-9 out of
higher absolute values
adjusted for current
level and change in
diffusion tube and
15-19 per shift selected.
(by 4-5-fold) using
smoking and duration
lung function for
Drager PAC III
Excluded if planned to have
Drager diffusion tubes;0
n = 23 with both
monitoring
<4-hr work day; mean age
concentrations based
concurrent measure
instrument)
~40 yrs, mean duration
on PAC III monitoring:
considered a
~18 yrs; never smoked
Low-exposure group
limitation for
~52%
(ammonia plant):
6.9 ppm (4.9 mg/m3)
High-exposure group
(urea plant): 26.1 ppm
(18.5 mg/m3)
quantitation of
exposure-response
relationship but not a
limitation for hazard
identification due to
B-20
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Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure parameters
Outcome
measured
Consideration of
confounding
Statistical analysis
Comments
regarding potential
major limitations
uncertainty in the
absolute value, but
not the relative
ranking, of exposure
Ali et al.
(2001)
Saudi Arabia, urea fertilizer
factory; cross-sectional
study (appears to be same
as Factorv A in Ballal et al.
(1998)
Exposed: n = 73
Controls: n = 348
Exposed: 20% of workers
selected (systematic
sample representing
different workplaces using
payroll lists); 95%
participation rate; mean
age 30 yrs, mean duration
51.8 mo; nonsmokers ~49%
Controls: administrative
staff from four industrial
groups (same sampling
system as exposed);
participation rate 98%
Mean age 34 yrs;
nonsmokers ~42%
Ammonia concentration
in air determined by
sampling pump with a
flow rate of 1 L/min for
4 hrs for each
measurement and
spectrophotometry
(i.e., by absorption
techniques and
comparison to a
standard)
Computed cumulative
ammonia concentration
(a function of both
exposure level and
duration of service)
assigned to each
worker, dichotomized
to high and low at
50 mg/m3-yrs
Spirometry by
standard protocol,
morning
measurement,
three or more
replicates
Stratified by smoking
status
T-tests and Chi-
square tests for
comparisons
between groups
and by exposure
level among
exposed
Study population and
design: "healthy"
workers; long
duration—potential
for lack of complete
ascertainment of
effect
Bhat and
Ramaswamv
(1993)
Mangalore; fertilizer
chemical plant; cross
sectional study
Exposed: n = 91
Controls: n = 68
No measurement of
exposure made
Spirometry by
standard protocol,
three replicates
with highest
reading retained
for calculation
All smokers excluded
from study; other
workplace exposures
not assessed
Paired t-test for
comparisons
between exposed
and controls
Study population and
design: "healthy"
workers; long
duration—potential
for lack of complete
B-21
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Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure parameters
Outcome
measured
Consideration of
confounding
Statistical analysis
Comments
regarding potential
major limitations
Exposed: 30 urea plant
workers, 30 DAP plant
workers, and 31 ammonia
plant workers; sex of
workers not reported; age,
sex, height, weight, and
duration of exposure were
recorded but not reported;
duration of exposure
dichotomized into two
groups (up to 10 yrs and
>10 yrs); smokers excluded
Controls: people having
comparable body surface
area chosen from the same
socio-economic status and
sex; smokers excluded; no
other information provided
on participant selection
ascertainment of
effect
Holness et
al. (1989)
Canada, sodium carbonate
(soda ash) production
plant; cross-sectional study
Exposed: n=58
Controls: n=31
Exposed: 52 of 64 available
production workers (82%)
and 6 maintenance
workers; all males, mean
age 39 yrs, mean duration
14.4 yrs; nonsmokers ~29%
Controls from stores and
office workers in the plant;
excluded if previous
Airborne levels of
ammonia
(mean = 6.5 mg/m3 for
exposed;
mean = 0.2 mg/m3 for
controls) using NIOSH-
recommended protocol
for personal sampling
and analysis (measured
over one work-shift per
person, mean 8.4 hrs)
Spirometry by
standard protocol,
beginning and end
of shift,
3-6 replicates,
each worker
measured on
2 test days
Adjusted for smoking
(pack-yrs); other
workplace exposures
not assessed
Baseline lung
function compared
between groups
using linear
regression,
adjusting for age,
height, and pack-yrs
(linear regression).
Unpaired t-tests
compared change in
lung function over
workshift between
groups; percent
Study population and
design: "healthy"
workers; long
duration—potential
for lack of complete
ascertainment of
effect
Relatively small
sample size—potential
of not being able to
detect a difference
between controls and
B-22
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Supplemental Information—Ammonia
Comments
Study setting/
Outcome
Consideration of
regarding potential
Reference
participant selection
Exposure parameters
measured
confounding
Statistical analysis
major limitations
ammonia exposure.
predicted lung
exposed when one
Participation rate not
function at baseline
might exist
reported; mean age 43 yrs,
and change in lung
mean duration 12.2 yrs;
function also
Low exposure
nonsmokers ~39%
analyzed by three
concentrations-
Indication of self-selection
categories of
potential that an
of exposed out of
exposure
effect level may not
workplace based on atopy
have been reached
(lower prevalence of hay
fever)
Sputum, exhaled NO (eNO) and blood parameters
Nemer et al.
Palestine, laboratory at
Ammonia air
Sputum collected;
Other hair salon
Median regression
Device used for
(2015)
Hebron University; cross-
concentrations
total cell count
exposures known to
used to compare
exposure
sectional study of female
measured in 13 salons
and cell viability;
cause irritation,
inflammatory cell
measurements had
hairdressers in 13 hair
using an electro-
differentiate cell
inflammation, or
levels in the
limited specificity for
salons from 10/2012 to
chemical sensor
counts
other respiratory
sputum, eNO levels,
measuring ammonia
03/2013
instrument (direct
effects (such as
and blood
relative to other gases
reading device) affixed
Exhaled NO (eNO)
persulfates) were not
parameters
(potential false
Exposed: n = 33
to one hairdresser in
measured using
measured
between
positives from other
nonsmoking female
each salon; sample
the NIOX MINO
hairdressers and
gases); potential
hairdressers (age
duration 45-305 min;
device (flow rate
Factors potentially
control group
selection bias in
19-50 yrs; mean 38 yrs);
concentration range
50 mL/sec), in
predicting ammonia
control group due to
selected from a cohort of
0-202 mg/m3; duration
accordance with
exposure, including
differences in
200 hairdressers studied
variation due to the
manufacturer's
size of salon, number
recruitment (self-
previously (every sixth
variation in the number
protocol and
of hairdressers at
selected based on
participant from a list
of customers serviced
American Thoracic
work and number of
interest in the study)
sorted by salon name was
Society
customers, tasks
or workload; small
invited to participate)
Limited specificity for
recommendations;
being done (coloring,
sample size and only a
Controls: n = 35;
measuring ammonia
eNO data adjusted
bleaching, cutting,
single measurement
nonsmoking female
compared to other
for height and age
spraying), were
of ammonia at each
students from Hebron
gases
evaluated
salon (which may not
University (n = 27) and staff
have been
B-23
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Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure parameters
Outcome
measured
Consideration of
confounding
Statistical analysis
Comments
regarding potential
major limitations
(n = 8); age of all controls
18-49 yrs, mean 24yrs;
recruited through
advertisements
Blood samples
analyzed for a
complete blood
count; blood
parameters
adjusted for BMI
and age
No adjustment for
smoking since all
participants were
nonsmokers
representative of
salon exposures)
aAmmonia plant workers checked temperature, pressure, and concentration of ammonia and checked the pumps, prepared solutions, and checked the
revolutions per minute of various motors. These are considered the low-exposure group.
bUrea plant workers purged solution and washed pipelines, operated various pumps, and washed and cleaned the cooling fluidized bed in the production area.
These are considered the high-exposure group.
cBased on communication with technical support at Drager Safety Inc. (Bacom and Yanosky, 2010), the Environmental Protection Agency (EPA) considered the
PAC III instrument to be a more sensitive monitoring technology than the Drager tubes. Therefore, more confidence is attributed to the PAC III air
measurements of ammonia for the Rahman et al. (2007) study.
BMI = body mass index; DAP = diammonium phosphate; SD = standard deviation
B-24
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Supplemental Information—Ammonia
Table B-7. Evaluation of epidemiology studies summarized in Table 1-3 (use in cleaning/disinfection settings)
Comments
regarding
Study setting/
Outcome
Consideration of
Statistical
potential major
Reference
participant selection
Exposure measure
measured
confounding
analysis
limitations
Casas et al.
Menorca, Spain; population-
Interviewer-led
Questionnaires on
Models adjusted for
Multivariate
Sample size was
(2013)
based, cross-sectional birth
questionnaire on the
wheezing, asthma,
sex, age, maternal
linear
relatively small
cohort study; recruitment
frequency of use of
treatment, and
education, parental
regression
(n = 46 for ammonia
during pregnancy; 432 infants
10 different cleaning
allergies were
smoking indoors,
models were
use) and may have
were enrolled; 295 individuals
products (bleach,
administered by
asthma medication,
developed for
limited power;
completed the 10-yr follow up
ammonia, polishes or
mother from birth to
season of
FeNO, FVC, and
exposure to
visit and the cleaning products
waxes, acids, solvents,
age 10 yrs; at ages
respiratory test
FEVito predict
cleaning products
questionnaire and performed
furniture sprays, glass
10-13 yrs, FeNO and
measurement, and
log-transformed
was assessed by
the FeNO and/or lung function
cleaning sprays, degreasing
lung function tests
height and weight
FeNO
parental report;
test
sprays, air freshening
were carried out
(lung function
concentration
over-reporting the
sprays and air freshening
measurement only)
and non-
use of cleaning
35% of recruited population
plug-ins)
transformed
products or changes
were excluded because
Measurements of
levels of FVC
in behavior related
information on use of cleaning
The means of the reported
indoor volatile
and FEVi
to their use was
products and/or respiratory
days of use per week
organic compounds
possible
tests not available
(never = 0, <1 d/wk = 0.5,
1-3 d/wk = 2 and 4-7 d/wk
or home inspections
were not performed
46 individuals reported use of
= 5.5) for each product
ammonia
were summed providing a
score ranging from 0 (no
exposure)to 55 (exposed
to all 10 products used
4-7 d/wk)
Dumas et al.
France; nested case-control
Exposure to specific agents
Asthma attack,
Adjusted for age
Products
(2012)
study of adult asthma cases
based on three methods
respiratory
and smoking status.
analyzed if
recruited from pulmonary
(ever exposed, based on all
symptoms or asthma
Additional
>5 exposed
clinics in 1991-1995; follow-
jobs held at least 3 mo):
treatment in the last
adjustment for BMI
cases.
up in 2003-2007
Self-report: two job
exposure questionnaire
12 mo (based on
tested
Analyses
stratified by sex
B-25
-------
Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure measure
Outcome
measured
Consideration of
confounding
Statistical
analysis
Comments
regarding
potential major
limitations
Drawn from the
modules for health care
standardized
Association with
(small sample
Epidemiological study on the
workers (including
questionnaire)
ammonia stronger
size for men so
Genetics and Environment in
frequency of use of specific
than that seen with
focused on
Asthma (EGEA) study
products); possible
bleach (odds ratios
women)
(included first degree relatives
underestimate of
1.87 and 0.93,
of cases and population
exposure
respectively, for
Familial
control group)
Expert assessment:
ammonia and
dependence in
Study base = 1,355: included if
hospital workers
bleach)
data accounted
had occupation data, excluded
(probability, frequency,
for by
if asthma at baseline or and
intensity; 18 products)
generalized
missing data on smoking
Asthma-specific job
estimating
Selected if ever worked in
exposure matrix
equations
hospital (exposure group) and
(22 agents) with expert
referent group
review
Hospital workers:
Control group: "Never
179 (43 men, 136 women)
exposed to cleaning/
Referent group:
disinfecting products"
545 (212 men, 333 women)
based on each of the
Smoking history and age
methods described above,
similar for women; smoking
plus expert review of
history similar for men (but
additional (broader)
mean age approximately 5 yrs
information from main
higher in hospital workers)
occupation questionnaire
Possible "healthy worker"
bias, with underestimation of
associations from movement
out of jobs or avoidance of
specific jobs by affected
individuals
B-26
-------
Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure measure
Outcome
measured
Consideration of
confounding
Statistical
analysis
Comments
regarding
potential major
limitations
Arif and
Delclos
(2012)
United States (Texas); survey
of 3,650 licensed health care
professionals (physicians,
nurses, respiratory therapists,
occupational therapists)
Response rate 66% (3,650 out
of 5,600)
For longest job held:
frequency of use of specific
products (never/once a
month, at least once a
week, more than once a
day, every day) (for
2,049 of the 3,650,
current/most recent job
was longest held job)
For all jobs: ever been in
contact with list of
28 products at least once a
month for a period of
>6 mo (ammonia part of
general cleaning factor in
factor analysis)
Four outcomes,
based on structured
questionnaire
Work Related
Asthma Symptoms
(WRAS):
wheezing/whistling
at work or shortness
of breath at works
that gets better away
from work or worse
at work
Work Related
Asthma (WRA): same
as above and
physician-diagnosed
asthma (n = 74)
Work exacerbated
asthma (WEA): onset
before began work
(n = 41)
Occupational asthma
(OA): onset after
began work (n = 33)
Adjusted for age,
sex, race/ethnicity,
BMI, seniority,
atopy, and smoking
status
Multinomial
logistic
regression with
four asthma
outcome
categories:
WRAS, WEA,
OA, and none
Oversampling
nurses and
physicians was
accounted for
with post-
stratification
weights
Limited exposure
assessment (i.e.,
"ever exposed")
Lemiere et
al. (2012)
Quebec; case-control study.
Workers with work-related
asthma (WRA) seen at two
tertiary care centers; WRA
based on specific inhalation
challenges (SIC); reversible
airflow limitation or airway
Structured interview about
last/current job (including
job title, tasks, machines,
materials), work
environment, protective
equipment; this
information used in
Diagnoses made
based on reference
tests
OA if specific
inhalation challenge
test was positive
(n = 67)
Assessed
confounding effects
of age, smoking,
occupational
exposure to heat,
cold, humidity,
dryness, and
Logistic
regression
B-27
-------
Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure measure
Outcome
measured
Consideration of
confounding
Statistical
analysis
Comments
regarding
potential major
limitations
hyper-responsiveness
(provocative concentration of
methacholine) inducing a 20%
fall in FEVi <8 mg/mL
Controls: non-work related
asthma seen at same clinics
but symptoms did not worsen
at work
Total n = 153 (33 controls,
120 WRA)
conjunction with other
material (e.g., technical
and material safety data
sheets, occupational
hygiene literature,
databases, and web sites)
for expert review and
classification of exposure
to 41 specific agents,
blinded to case status
Semiquantitative estimate
(low =1, medium = 2,
high = 3) for intensity,
frequency, and confidence
WEA if specific
inhalation test was
negative but
symptoms worsened
at work (n = 53)
physical strain; not
included in final
models because
none acted as
confounders of
exposures under
study
Vizcava et
al. (2011)
Barcelona, Spain; survey of
1,018 cleaning services to find
companies willing to
participate; 286 (28%) not
eligible (no longer in
business); 37 agreed to
participate (number workers
ranged from 6 to >1,000).
4,993 questionnaires
distributed by company
representatives to employees;
950 (19%) completed;
33 excluded because of
missing data
Total n = 917; two companies
completed non-responder
Standardized
questionnaire about
cleaning tasks and
products used in the last
year
Reference group = never
cleaners AND current
cleaners who had not used
bleach, degreasers, multi-
purpose cleaners, glass
cleaners, perfumed
products, air fresheners,
mop products,
hydrochloric acid,
ammonia, polishes or
Current asthma
based on structured
questionnaire (in
past 12 mo, woken
by an attack of
shortness of breath,
had an attack of
asthma, or currently
taking any asthma
medications
(including inhalers,
aerosols or tablets)
Asthma score: Sum of
"yes" answers to five
questions on asthma
symptoms in last
Adjusted for age,
country of birth
(Spanish versus non-
Spanish), sex, and
smoking status
Asthma: logistic
regression
Asthma score:
negative
binomial
regression (to
account for
over-dispersion
in the data)
Exposure
assessment limited
(use in past year; no
frequency data)
B-28
-------
Supplemental Information—Ammonia
Comments
regarding
Study setting/
Outcome
Consideration of
Statistical
potential major
Reference
participant selection
Exposure measure
measured
confounding
analysis
limitations
survey (sex, age, nationality,
waxes, solvents, or carpet
12 mo (wheeze with
job position); no major
cleaners in the last year
breathlessness,
differences with responders;
woken up with chest
selection bias unlikely
tightness, attack of
shortness of breath
at rest, attack of
shortness of breath
after exercise, woken
by attack of
shortness of breath)
Zock et al.
Europe (22 sites in
At follow-up, standardized
Incident (since
Adjusted for sex,
Incident asthma
Referent group
(2007)
10 countries); longitudinal
interview about use of
baseline survey)
age, smoking,
and wheeze:
included some
study
15 cleaning products in the
current asthma,
employment in a
log-binomial
exposure (to the
Random population sample,
home (frequency never,
defined by asthma
cleaning job during
regression
product, and to
ages 20-44 yrs (the European
<1 d/wk, 1-3 d/wk,
attack or nocturnal
follow-up, and study
Incident
other products);
Community Respiratory
4-7 d/wk)
shortness of breath
center; hetero-
physician
could under-
Health Survey), 9-yr follow-up
Reference group: did not
in the past 12 mo
geneity by center
diagnosed
estimate risk;
period
use the product or used
or current use of
also assessed
asthma: Cox
although it is an
Excluded 764 individuals with
<1 d/wk
medication for
Correlations among
proportional
incident study, the
asthma at baseline
asthma
products generally
hazards
exposure
Analysis limited to individuals
Incident physician-
weak (Spearman rho
regression, with
information was
reporting doing the cleaning
diagnosed asthma,
<0.3)
date on onset
collected at follow-
or washing in their home
defined as above
defined as
up, so may not
(n = 3,503)
with confirmation by
a physician and
information on age
or date of first attack
Incident (since
baseline survey)
current wheeze,
defined as wheezing
reported date
of first attack
Referent
category = used
product never
or <1d/wk
reflect pre-disease
patterns (if practices
changed because of
symptoms) or could
be influenced by
knowledge of
outcome
B-29
-------
Supplemental Information—Ammonia
Comments
regarding
Study setting/
Outcome
Consideration of
Statistical
potential major
Reference
participant selection
Exposure measure
measured
confounding
analysis
limitations
or whistling in the
chest in last 12 mo
when not having a
cold
Medina-
Cornelia, Spain; 2-wk diary
2-Wk diary recorded daily
Respiratory
Adjusted for
Respiratory
Pulmonary function
Ramon et al.
and pulmonary function
use of cleaning products
symptoms based on
respiratory
symptom
measured by
(2006)
study, 2001-2002
and cleaning tasks
2-wk daily diary
infection, use of
scores
participant;
Female domestic cleaners
(checklist of cleaning
(seven symptoms,
maintenance
dichotomized
validation of
aged 31-66 yrs with a history
exposures, number of
five-point intensity
medication, and
as >2 and <2 for
method not
of obstructive lung disease,
hours cleaning in each
scale); summed score
age; daily number of
use in logistic
reported—potential
recruited from participants in
house)
for upper respiratory
cigarettes smoked,
regression
for knowledge of
a nested case-control study
symptoms (blocked
yrs of employment
exposure to affect
based on population survey
nose, throat
in domestic
PEF analysis
reporting of
from 2000-2001 (see Medina-
irritation, watery
cleaning,
based on night
symptoms
Ramon et al. (2005), below)
eyes) and lower
and/or weekly
time and the
Selected if reported current
respiratory
working hours in
next morning
asthma symptoms or chronic
symptoms (chest
domestic cleaning
values; linear
bronchitis in 2000-2001
tightness, wheezing,
also assessed and
regression
survey (standard definitions)
shortness of breath,
included as
Excluded if illiterate or unable
cough)
necessary
to complete diary (n = 57)
PEF measured with
80 met eligibility criteria;
mini-Wright peak
51 (64%) completed diary
flow meter (with
Participants and
training and written
nonparticipants similar except
instructions);
for higher prevalence of
measured morning,
bronchial hyper-
lunchtime, and night
responsiveness and shorter
(three measurements
duration of domestic cleaning
each; highest
employment among
recorded)
responders
B-30
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Supplemental Information—Ammonia
Reference
Study setting/
participant selection
Exposure measure
Outcome
measured
Consideration of
confounding
Statistical
analysis
Comments
regarding
potential major
limitations
Occupational asthma
based on analysis of
PEF patterns by
occupational asthma
system (OASYS)
Medina-
Ramon et al.
(2005)
Cornelia, Spain; nested case-
control study in 2001-2002 of
650 cleaning workers drawn
from population-based survey
in 2000-2001, 4,521 women
ages 30-65 yrs
Cases: 160 identified, 117 still
employed in domestic
cleaning, 87 (74%) agreed to
participate, 40 met final case
definition
Controls: 386 identified,
281 still employed in domestic
cleaning, 194 (69%) agreed to
participate, 155 met final
control definition
Job-specific questionnaire
for cleaning workers,
frequency of use of
22 specific products
(times/wk, mo, or yr);
summed across each home
and personal home and
divided into two groups
(cut-point = 12 times/yr).
Also assessed accidental
exposures (e.g., spills)
Measurements taken in
10 cleaning sessions to
obtain data on exposure to
chlorine and ammonia
during specific tasks and
with specific products
(ammonia used in kitchen
cleaning; median
0.6-6.4 ppm; peaks
>50 ppm)
Case based on
asthma and/or
bronchitis at both
assessments
Asthma = asthma
attack or being
woken by attack or
shortness of breath
in past 12 mo
Chronic bronchitis =
regular cough or
regular bringing up
phlegm for at least
3 mo each year
Controls: no history
of respiratory
symptoms in
preceding year and
no asthma at either
assessment
Correlations among
tasks/products
reported to be
generally weak (but
specific values for
ammonia and other
products not
reported)
Multivariate model
adjusted for age
tertile and smoking
status (but results
for ammonia in this
model only reported
as "not statistically
significant;" no
information on
effect estimate/
variability)
Logistic
regression
Results of adjusted
model not reported
in detail, but
confounding
unlikely major factor
if correlations weak
FeNO = fraction of exhaled nitric oxide; FEVi = forced expiratory volume in 1 second; FVC = forced vital capacity; PEF = peak expiratory flow
B-31
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Supplemental Information—Ammonia
Table B-8. Evaluation of epidemiology study summarized in Table 1-6 (industrial setting/serum chemistry
measures)
Comments
Study setting/
Exposure
Outcome
Consideration of
regarding major
Reference
participant selection
parameters
measured
confounding
Statistical analysis
limitations
Abdel Hamid
Egypt, urea fertilizer
No direct
Fasting blood
No information on
Type of statistical
Study population and
and El-
production plant; cross-
measurement of
sample for AST,
exposure to other
test not reported
design: "healthy"
Gazzar
sectional study
ammonia exposure;
ALT (measures of
contaminants; no
(EPA assumes to be
workers; long
(1996)
Exposed: n = 30
blood urea was
liver function),
information on
t-test); data
duration—potential
Controls: n = 30
used as a surrogate
hemoglobin,
smoking status
presented as group
for lack of complete
Exposed: workers selected
measure (ammonia
catalase enzyme
means ± SD, with
ascertainment of
randomly (process not
is detoxified mainly
activity as
p-value
effect
described); mean age 36 yrs,
through the
mediator of cell
mean duration 12 yrs
formation of urea in
membrane
Lack of information on
Controls from administrative
the liver)
permeability, and
smoking, and alcohol
departments with no known
Mean (± SD) mg/dL
serum monoamine
use—potential for
history of ammonia exposure;
(p < 0.01)
oxidase enzyme
possible confounding
matched to exposed by age,
Exposed:
activity as
for liver function
educational status, and
31.9 (±7.6)
mediator of
measures; uncertain
socioeconomic status; mean
Controls:
effects on nervous
effect on enzyme
age 35 yrs
20.3 (±5.1)
The reliability of
blood urea and
correlation with
ammonia exposure
not reported
system
measures
ALT = alanine aminotransferase; AST = asparate aminotransferase
B-32
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Supplemental Information—Ammonia
APPENDIX C. INFORMATION IN SUPPORT OF
HAZARD IDENTIFICATION AND DOSE-RESPONSE
ANALYSIS
C.l. TOXICOKINETICS
C.l.l. Absorption
Inhalation Exposure
A study in volunteers1 indicated that ammonia is almost completely retained in the nasal
mucosa (83-92%) during short-term acute exposure (i.e., up to 120 seconds) over a wide exposure
range (40-354 mg/m3) (Landahl and Herrmann. 1950). Longer-term acute exposure
(10-27 minutes) to 354 mg/m3 ammonia resulted in lower retention (4-30%), with expired breath
concentrations of 247-283 mg/m3 observed by the end of the exposure period f Silverman etal..
1949). suggesting saturation of absorption into the nasal mucosa. Nasal and pharyngeal irritation,
but not tracheal irritation, suggests that ammonia is retained in the upper respiratory tract
Unchanged levels of blood urea nitrogen (BUN), nonprotein nitrogen, urinary urea, and urinary
ammonia following these acute exposures are evidence of low absorption into the blood.
Data in rabbits and dogs provide supporting evidence for high-percentage nasal retention,
resulting in a lower fraction of the inhaled dose reaching the lower respiratory tract (Egle. 1973:
Dalhamn. 1963: Boyd etal.. 19441. Continuous exposure of rats to ammonia at concentrations up to
23 mg/m3 for 24 hours did not result in statistically significant increases in blood ammonia levels,
whereas exposures to 219-818 mg/m3 resulted in significantly increased blood concentrations of
ammonia within 8 hours of exposure initiation, indicating a potential for systemic absorption of
inhaled ammonia (Schaerdel etal.. 19831.
Gastrointestinal Contributions to Systemic Ammonia
Ammonia as ammonium ion (NH4+) is endogenously produced in the human intestines
through the use of amino acids as an energy source (glutamine deamination) fTavlor and Curthovs.
2004: Mcfarlane Anderson et al.. 19761 and by bacterial degradation of nitrogenous compounds
from ingested food (Romero-Gomez etal.. 2009). About 99% of the ammonia produced in the
lrThe human toxicokinetic studies cited in this section did not provide information on the human subjects'
research ethics procedures undertaken in the studies; however, there is no evidence that the conduct of the
research was fundmentally unethical or significantly deficient relative to the ethical standards prevailing at
the time the research was conducted.
C-l
-------
Supplemental Information—Ammonia
intestines is systemically absorbed. Evidence suggests that fractional absorption of ammonia
increases as the lumen pH increases, and that transport occurs at lower pH levels (absorption has
been detected at a pH as low as 5) fCastell and Moore. 1971: Mossberg and Ross. 19671. NH |+
absorbed from the gastrointestinal tract travels via the hepatic portal vein directly to the liver
where, in healthy individuals, most of it is converted to urea and glutamine.
C.1.2. Distribution
The range of mean ammonia concentrations in humans as a result of endogenous
production was reported as 0.1-0.6 |J.g/mL in arterial blood and 0.2-1.7 |J.g/mL in venous blood
fHuizenga et al.. 19941. More recent sources provide values for the normal range of blood ammonia
of 0.1-0.8 |J.g/mL (venous blood) and 0.15-0.45 [ig/mL.2 Given its importance in amino acid
metabolism, the urea cycle, and acid-base balance, ammonia is homeostatically regulated to remain
at low concentrations in the blood. At normal physiological blood pH, 98.3% of total ammonia is
present as NHi\ and 1.7% as NH3 fWeiner and Verlander. 20131.3
Ammonia is present in fetal circulation. In vivo studies in several animal species and in
vitro studies of human placenta suggest that ammonia is produced within the uteroplacenta and
released into the fetal and maternal circulations (Bell etal.. 1989: lohnson et al.. 1986: Hauguel et
al.. 1983: Meschia et al.. 1980: Remesar etal.. 1980: Holzman et al.. 1979: Holzman etal.. 1977:
Rubaltelli and Formentin. 1968: Luschinskv. 19511. lozwik etal. f 2 0 0 51 reported that ammonia
levels in human fetal blood (specifically umbilical arterial and venous blood) at birth were
1.0-1.4 [ig/mL, compared to 0.5 |J.g/mL in the mothers' venous blood. DeSanto etal. f 19931
similarly collected human umbilical arterial and venous blood at delivery and found that umbilical
arterial ammonia concentrations (0.51-5.9 [ig/mL) from 15-17 caesarian section deliveries,
intended to better represent in utero values were significantly higher than venous concentrations
(0.43-5.13 [ig/mL). There was no correlation between umbilical ammonia levels and gestational
age (range of 25-43 weeks of gestation; vaginal and cesarean section deliveries). In sheep, the
uteroplacental tissue is the main site of ammonia production, with outputs of ammonia into both
the uterine and umbilical circulations (lozwik etal.. 19991. In late-gestation pregnant sheep that
were catheterized to allow measurement of ammonia exposure to the fetus, concentrations of
ammonia in umbilical arterial and venous blood and uterine arterial and venous blood ranged from
approximately 0.39 to 0.60 |J.g/mL (lozwik etal.. 2005: lozwik etal.. 19991.
2University of Rochester Medical Center, Health Encyclopedia: Ammonia,
h ttps://www, urmc. rochester.edu/encyclopedia/content.aspx?ContentTvpel D=167&ContentlD=ammonia.
accessed 1/19/2016, and U.S. National Library of Medicine. Medline Plus. Ammonia blood test.
https://www.nlm.nih.gov/medlineplus/encv/article/0035Q6.htm. accessed 1/19/2016.
3The relative amounts of NH4+ and NH3 are determined by pH. For every 0.3 pH unit change, the amount of
NH3 changes in parallel by 100% (i.e., atpH 7.7, the total ammonia present as NH3 is 3.4%, and atpH 7.1,
0.85%). The amount of NH4+ changes in the opposite direction by an equivalent absolute amount (decreases
1.7% to 96.7% at pH 7.7, and increases 0.85% to 99.15% at pH 7.1) (Weiner and Verlander. 20131.
C-2
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Supplemental Information—Ammonia
Ammonia is present in human breast milk as one of the sources of nonprotein nitrogen
(Atkinson et al.. 19801.
Little information on the distribution of inhaled ammonia was found in the available
literature. Information on the distribution of endogenously produced ammonia suggests that any
ammonia absorbed through inhalation would be distributed to all body compartments via the
blood, where it would be used in protein synthesis as a buffer, reduced to normal concentrations by
urinary excretion, or converted by the liver to glutamine and urea (Takagaki etal.. 19611. Rats
inhaling 212 mg/m3 ammonia 6 hours/day for 15 days exhibited increased blood ammonia (200%)
and brain glutamine (28%) levels at 5 days of exposure, but not at 10 or 15 days (Manninen etal..
19881. demonstrating transient distribution of ammonia to the brain.
C.1.3. Metabolism/Endogenous Production of Ammonia
Ammonia is produced endogenously by catabolism of amino acids by glutamate
dehydrogenase or glutaminase primarily in the liver, renal cortex, and intestines, but also in the
brain and heart (Souba. 19871. In skeletal muscle, ammonia may be produced by metabolism of
amino acids or adenosine monophosphate via adenylate deaminase. Ammonia is metabolized to
glutamine via glutamine synthetase in the glutamine cycle (Figure C-l), or incorporated into urea as
part of the urea cycle as observed in the hepatic mitochondria and cytosol (Figure C-2) fNelson and
Cox. 20081 before entering the systemic circulation. Van de Poll et al. f20081 reported that the liver
removes an amount of ammonia from circulation equal to the amount added by the intestines at
metabolic steady state, indicating that the gut does not contribute significantly to systemic
ammonia release. However, when hepatic function is disrupted (see Section 1.3.2, Susceptible
Populations and Lifestages), intestine-derived ammonia may reach the systemic circulation (Van de
Poll etal.. 2008: Romero-Gomez etal.. 20041.
Glutamate
glutaminase
(in liver
mitochondria)
glutamine
synthetase
Glutamine
y-Glutamyl
isphate
glutamine
synthetase
Pi NH4+
Adapted from: Nelson and Cox (2008).
Figure C-l. Glutamine cycle.
C-3
-------
Supplemental Information—Ammonia
CO, + NH,
h2o
2 ATP
2ADP J
P,4^
3H+
Mitochondrial^
Matrix
Carbamoyl
phosphate
synthase I
Carbamoyl
phosphate pi
u
Ornithine . . Citrulline
Ornithine
transcarbamoylase
Urea 1
H,0-
Ornithine
A
Citrulline
ATP
Arginase Argininosuccinate y* Aspartate
V synthase A
4 ^AMP+ PPj
Arginine Argininosuccinate
Argininosuccinate
lyase
Cytosol
F umarate
Adapted from: Nelson and Cox (2008).
Figure C-2. The urea cycle showing the compartmentalization of its steps
within liver cells.
Ammonia generated in the renal proximal tubule cells can be eliminated via the kidneys
fWeiner and Verlander. 2013: Kim. 20091. While renal elimination via the kidney is a major
contributor to ammonia homeostasis, the kidneys are themselves a source of ammonia. Renal
ammonia is derived from the utilization of glutamate as an energy source by the renal proximal
tubule cells and in the maintenance of the acid-base balance (Weiner and Verlander. 2013: Kim.
20091. The fact that the sum of urinary ammonia and renal vein ammonia substantially exceeds
renal arterial ammonia delivery fWeiner and Verlander. 20111 indicates that that the kidney adds
ammonia to the body. This is demonstrated in studies of patients with renal artery stenosis where
the concentrations of ammonia in the renal vein are slightly higher than those in systemic
circulation (Olde-Damink etal.. 20021.
Ammonia can also be produced in the gastrointestinal tract. The enzymatic activity of
glutaminase, which produces ammonia, is high in the gastrointestinal tract Such enzymatic activity
in the small intestines is approximately 4-fold that found in the large intestine mucosa flames etal..
19981. While bacterial content of the gut may contribute to circulating levels of ammonia, results
C-4
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Supplemental Information—Ammonia
from studies with germ-free animals suggest that hyperammonemia can be produced without
bacterial involvement (Nance and Kline. 1971: Warren and Newton. 19591.
In addition to the production of endogenous ammonia from the liver, kidneys, and intestine,
exercising skeletal muscle liberates ammonia by deamination of adenosine monophosphate.
Ammonia produced from the skeletal muscle is also effectively incorporated into glutamine in these
cells before entering the circulation fHuizenga et al.. 19961. Under conditions of prolonged
exercise, skeletal muscle may derive as much as 10% of its energy from amino acid metabolism
(Graham and MacLean. 19921.
Given its important metabolic role, ammonia exists in a homeostatically regulated
equilibrium in the body. In particular, free ammonia has been shown to be homeostatically
regulated to remain at low concentrations, with 95-98% of body burden existing in the blood (at
physiological pH) as NH|+ fda Fonseca-Wollheim. 1995: Souba. 19871. Two studies in rats
fManninen et al.. 1988: Schaerdel etal.. 19831 provide evidence that exposure to environmental
ammonia at concentrations <18 mg/m3 do not measurably alter blood ammonia concentrations.
Schaerdel etal. (19831 exposed rats to ammonia for 24 hours at concentrations of 11, 23, 219, or
818 mg/m3. Exposure to 11 and 23 mg/m3 ammonia did not statistically significantly increase
blood ammonia concentrations after 24 hours; the difference between pre- and postexposure
concentrations ranged from -0.58 to 0.006 millimoles/L whole blood. Concentrations
>219 mg/m3 caused an exposure-related increase in blood ammonia, but blood ammonia levels at
12- and 24-hour sampling periods were lower than at 8 hours (increase at 8 hours of
0.192-0.244 millimoles/L compared to pre-exposures levels), suggesting compensation by
increasing ammonia metabolism. Any changes in blood gas (pC>2, pCC>2, pH) and liver microsomal
activity (ethylmorphine-N-demethylase, cytochrome P450) were small and not associated with
environmental ammonia concentrations, suggesting no measureable effect of short-term
environmental ammonia exposure on the parameters measured in this study. In rats inhaling
18 mg/m3 ammonia 6 hours/day for 5,10, or 15 days fManninen etal.. 19881. blood ammonia
levels (0.021-0.057 millimoles/L) were not statistically significantly different from controls
(0.032-0.043 millimoles/L). Rats inhaling 212 mg/m3 exhibited statistically significantly
increased levels of blood ammonia (3-fold) at 5 days of exposure, but not at 10 or 15 days. Brain
ammonia levels did not differ from controls at either exposure concentration. Blood glutamine (at
212 mg/m3) and brain glutamine (at 18 and 212 mg/m3) on day 5 were increased over control, but
were no longer elevated on days 10 and 15. The return of blood ammonia and blood and brain
glutamine levels to control levels within days is consistent with metabolic adaptation, and these
data suggest that animals have the capacity to handle high concentrations of inhaled ammonia.
Various disease states can affect the rate of glutamine uptake and catabolism and thereby
affect the blood and tissue levels of ammonia. Acute renal failure can result in increased renal
glutamine consumption and ammonia production with a decreased capability of eliminating urea in
the urine f Souba. 19871. Abnormally elevated levels of breath ammonia (and corresponding
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increases in plasma urea) are indicative of end-stage renal failure (Davies etal.. 19971. Both acute
(e.g., fulminant hepatitis) and chronic (e.g., end-stage liver failure; hepatic cirrhosis) liver disease
may result in decreased ureagenesis and increased levels of ammonia in blood (hyperammonemia),
leading to increased uptake into the brain and the onset of hepatic encephalopathy. The increased
metabolic alkalosis associated with hepatic encephalopathy may result in a shift in the NH4VNH3
ratio in the direction of ammonia, which may pass through the blood-brain barrier more effectively
than ammonium (Katavama. 2004). In patients with liver cirrhosis and acute clinical hepatic
encephalopathy, the mean net metabolic flux of [13N]-ammonia from the blood into the brain was
3-5-fold higher in patients with cirrhosis than healthy controls; cerebral trapping of ammonia was
primarily attributable to increased blood ammonia fKeiding et al.. 2010: Keiding et al.. 20061.
S0rensen et al. (2009) demonstrated greater unidirectional clearance of ammonia from the blood to
brain cells than metabolic clearance of ammonia from the blood, both in healthy controls and in
cirrhotic patients with and without hepatic encephalopathy.
C.1.4. Elimination
Ammonia is excreted by the kidneys as urea. Elimination of ammonia in the kidney involves
specific proteins mediating transport of NH3 and NH4+. For example, in the proximal tubule, the
apical Na+/H+ exchanger, NHE-3, preferentially secretes NH4+. The Rhesus glycoproteins, Rh B
glycoprotein (Rhbg) and Rh C glycoprotein (Rhcg), are ammonia transporters in the distal tubule
and collecting duct (Weiner and Verlander. 2011: Bishop etal.. 2010: Lee etal.. 2010: Lee etal..
2009: Han etal.. 2006: Handlogten etal.. 2005). Angiotensin II is one of the factors that modulates
ammonia release from renal proximal tubule cells fNagami and Warech. 1992: C ho banian and lulin.
1991). Diseases and conditions that increase angiotensin II may thus increase production and
decrease elimination of ammonia (Agrovannis etal.. 1998).
Ammonia is also eliminated through the skin through sweat production or possibly due to
direct diffusion of systemic plasma NH |+ fSchmidt et al.. 2 0131.
Additionally, ammonia is eliminated in the expired air of all humans fManolis. 1983).
Exhalation serves as a clearance mechanism. Several investigators specifically measured ammonia
in breath exhaled from the nose (Schmidtetal.. 2013: Solga etal.. 2013: Smith etal.. 2008: Larson et
al.. 1977). Smith etal. (2008) reported median ammonia concentrations of 0.059-0.078 mg/m3 in
exhaled breath from the nose of three healthy volunteers (with samples collected daily over a
4-week period); these concentrations were similar to, or slightly higher than, the mean laboratory
air level of ammonia of 0.056 mg/m3 reported in this study. In another study of 20 healthy
volunteers, the mean ammonia concentration in exhaled breath from the nose was 0.032 mg/m3
(range: 0.0092-0.1 mg/m3) fSchmidt etal.. 2013). Larson etal. (1977) reported that the median
concentration of ammonia collected from air samples exhaled from the nose ranged from 0.013 to
0.046 mg/m3. One sample collected from the trachea via a tube inserted through the nose of one
subject was 0.029 mg/m3—a concentration within the range of that found in breath exhaled
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through the nose (Larson et al.. 19771. Solga etal. (2013) reported 0.682 mg/m3 ammonia in
expired breath of a single subject during "mouth-closed breathing."
Higher and more variable ammonia concentrations were reported in breath exhaled from
the mouth or oral cavity than in breath exhaled from the nose. In studies that reported ammonia in
breath samples from the mouth or oral cavity, ammonia concentrations were commonly found in
the range of 0.085-2.1 mg/m3 fSchmidt et al.. 2 013: Solga etal.. 2013: Smith etal.. 2008: Spanel et
al.. 2007a. b; Turner etal.. 2006: Diskin etal.. 2003: Smith etal.. 1999: Norwood etal.. 1992: Larson
etal.. 19771 and were strongly correlated with saliva pH fSchmidt etal.. 20131. These higher
concentrations are largely attributed to the production of ammonia by bacterial degradation of food
protein in the oral cavity or gastrointestinal tract fTurner etal.. 2006: Smith etal.. 1999: Vollmuth
and Schlesinger. 19841. This source of ammonia in breath was demonstrated by Smith etal. (19991.
who observed elevated ammonia concentrations in the expired air of six healthy volunteers
following the ingestion of a protein-rich meal.
Other factors that can affect ammonia levels in breath exhaled from the mouth or oral cavity
include diet, oral hygiene, age, living conditions, and disease state. Norwood etal. (19921 reported
decreases in baseline ammonia levels (0.085-0.905 mg/m3) in exhaled breath following tooth
brushing (<50% depletion), a distilled water oral rinse (<50% depletion), and an acid oral rinse
(80-90% depletion). Solga etal. (2013) similarly reported decreased ammonia in the expired
breath of a single subject following rinses with water, hydrogen peroxide, and Coca-Cola®, and an
increase with Mylanta®, which has a basic pH. These findings are consistent with ammonia
generation in the oral cavity by bacterial and/or enzymatic activity. Several investigators have
reported that ammonia in breath from the mouth and oral cavity increases with age (Spanel etal..
2007a. b; Turner etal.. 2006: Diskin etal.. 2003). with ammonia concentrations increasing on
average about 0.1 mg/m3 for each 10 years of life fSpanel etal.. 2007al. Turner etal. f2 0061
reported that the age of the individual accounted for about 25% of the variation observed in mean
breath ammonia levels, and the remaining 75% was due to factors other than age. Certain disease
states can also influence ammonia levels in exhaled breath. Large increases in the breath
concentration of ammonia were measured in patients in renal failure (Spanel et al.. 2007a: Davies
etal.. 1997). These studies are further described in Table C-l.
Because ammonia measured in samples of breath exhaled from the mouth or oral cavity can
be generated in the oral cavity and may thus be substantially influenced by diet and other factors,
ammonia levels measured in mouth or oral cavity breath samples do not likely reflect systemic
(blood) levels of ammonia. Ammonia concentrations in breath exhaled from the nose appear to
better represent levels at the alveolar interface of the lung and are thought to be more relevant to
understanding systemic levels of ammonia (Schmidt et al.. 2013: Smith etal.. 2008). That said, the
amount of ammonia that equilibrates between the endogenous lung metabolic pool and alveolar air
is likely to be small even under hyperammonemic conditions. In a study that measured the amount
of label in exhaled air of anesthetized rats administered an intravenous dose of [13N] ammonia
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(Cooper and Freed. 20051. trace amounts of label could be detected in the expired breath over a
5-minute period, whereas approximately 30% of the administered dose passed through the lungs
within seconds, with most of the blood-derived ammonia in the rat lung incorporated into
glutamine.
In evaluating measures of ammonia in expired air, it is important to recognize that ammonia
in ambient air is the source of some of the ammonia in exhaled breath. Studies of ammonia in
exhaled breath (see Table C-l) were conducted in environments with measureable levels of
ambient (exogenous) ammonia rather than in ammonia-free environments, and it has been
established that concentrations of certain trace compounds in exhaled breath are correlated with
their ambient concentrations fSpanel etal.. 20131. Spanel etal. T20131 determined that the
concentration of ammonia in inhaled breath could account for approximately 70% of the ammonia
in exhaled breath. It is likely that ammonia concentrations in exhaled breath, and particularly from
the nose, would be lower if the inspired air were free of ammonia.
Ammonia has also been detected in the expired air of animals. Whittaker et al. (20091
observed a significant association between ambient ammonia concentrations and increases in
exhaled ammonia in stabled horses. Analysis of endogenous ammonia levels in the expired air of
rats showed concentrations of 0.007-0.250 mg/m3 (mean = 0.06 mg/m3) f Barrow and Steinhagen.
19801. Larson etal. (19801 reported ammonia concentrations measured in the larynx of dogs
exposed to sulfuric acid ranging between 0.02 and 0.16 mg/m3 following mouth breathing and
between 0.04 and 0.16 mg/m3 following nose breathing.
Physiologically Based Pharmacokinetic Models
No physiologically based pharmacokinetic models have been developed for ammonia. An
expanded one-compartment toxicokinetic model in rats was developed by Diack and Bois (20051.
which used physiological values to represent first-order uptake and elimination of inhaled
ammonia (and other chemicals). The model is not useful for dose-response assessment of ammonia
because: (1) it cannot specify time-dependent amounts or concentrations of ammonia in specific
target tissues, (2) it has not been verified against experimental data for ammonia, glutamate, or
urea levels in tissues, and (3) it does not support extrapolation of internal doses of ammonia
between animals and humans.
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Table C-l. Ammonia levels in exhaled breath of volunteers
Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
Breath samples from the nose and trachea
Single test subject (no
information on age,
health status)
Subject exhaled into breath
sampler for at least 10 sec
while maintaining constant
exhalation flow rate of
50 mL/sec (maintained via
orifice in breath sampler);
ammonia in exhaled breath
measured for open and
closed mouth breathing and
with a water rinse (closed
breathing only); 11 breath
collections over
10 consecutive work days
Mean pre-rinse baseline
concentration of breath ammonia
(mg/m3):
Mouth closed: 0.682 (± 0.315)
Post-rinse (water) concentration
of breath ammonia:
Mouth closed: 0.119 (± 0.062)
Continuous
wave (CW)
distributed
feedback
quantum
cascade laser
(DFB-QCL)
based sensor
coupled to
breath
sampling device
measuring both
mouth pressure
and real-time
concentration
of carbon
dioxide
Rinsing the mouth with water
significantly lowered the amount
of breath ammonia exhaled
Solga et al.
(2013)
20 healthy volunteers
(13 males and 7 females
aged 22-61 yrs)
Subjects fasted overnight
and refrained from exercise
in the morning before
sampling; samples collected
between 8 and 11 AM; end-
tidal breath samples
collected from the nose;
subjects breathed
continuously into the
sampling piece for 3-5 min
to obtain stable sample;
Concentrations in exhaled breath
from the nose (mg/m3):
Range = 0.0092-0.10
Mean = 0.032 (95% CI:
0.021-0.042)
Median = 0.024
Concentrations following acidic
mouth wash (mg/m3):
Range = 0.011-0.027
Commercial
cavity ring-
down
spectrometer
Ammonia concentrations were
down to 0.0004 g/m3 in outdoor
air, 0.002-0.004 mg/m3 in indoor
air, and 0.006-0.007 mg/m3 in
indoor air in the presence of
humans
Schmidt et
al. (2013)
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Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
samples also collected after
an acidic mouth wash
Mean = 0.016 (95% CI:
0.014-0.018)
Median = 0.015
Three healthy male
volunteers (>30 yrs of
age)
Ammonia levels measured in
nose-exhaled breath of test
subjects each morning about
2 hrs after eating a regular
breakfast; samples collected
daily over a 4-wk period
Volunteer A = 0.0728 ±
0.000848 mg/m3
Volunteer B =
0.0777 ± 0.000919 mg/m3
Volunteer C =
0.0587 ± 0.000848 mg/m3
(median ammonia levels
estimated as geometric mean ±
geometric SD)
SI FT-MS
analysis
Mean ambient air level of
ammonia was 0.056 ±
0.0071 mg/m3
The authors indicated that
ammonia measured in mouth-
exhaled breath may be
generated in the oral cavity and
suggested that concentrations in
nose-exhaled breath may better
represent systemic conditions
(such as metabolic disease)
Smith et al.
(2008)
Sixteen healthy subjects
(9 males aged 25-63 yrs
and 7 females aged
23-41 yrs); subgroups
tested were all male
Breath samples collected
during quiet nose breathing,
and direct sampling during a
deep inspiration followed by
breath-holding with the
glottis closed
Ammonia concentrations ranged
from 0.013 to 0.046 mg/m3
during nose breathing (median
0.025 mg/m3) (five male
subjects), and 0.029 mg/m3 from
an air sample collected from the
trachea (collected from a tube
inserted into one male subject's
nose and into the trachea)
Chemi-
luminescence
Larson et
al. (1977)
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Supplemental Information—Ammonia
Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
Breath samples from the mouth and oral cavity
Single test subject (no
information on age or
health status)
Subject exhaled into breath
sampler for at least 10 sec
while maintaining constant
exhalation flow rate of
50 mL/sec (maintained via
orifice in breath sampler);
ammonia in exhaled breath
measured for open and
closed-mouth breathing and
with different rinses (water,
hydrogen peroxide,
Mylanta®, Coca Cola®);
11 breath collections over
10 consecutive work days
Mean pre-rinse baseline
concentration of breath ammonia
(mg/m3):
Mouth open: 0.719 (± 0.291)
Post-rinse (water) concentration
of breath ammonia:
Mouth open: 0.121 (± 0.057)
CW DFB-QCL
based sensor
coupled to
breath
sampling device
measuring both
mouth pressure
and real-time
concentration
of carbon
dioxide
Rinsing the mouth with water
and the two acidic rinses
significantly lowered the amount
of breath ammonia exhaled (by
~50-75%). The basic rinse,
Mylanta®, significantly increased
breath ammonia (by ~40%)
In trials with different rinses, the
study subject breathed without
direction as to the mode of
breathing; EPA assumed that this
included mouth breathing
Solga et al.
(2013)
20 healthy volunteers
(13 males and 7 females
aged 22-61 yrs)
Subjects fasted overnight
and refrained from exercise
in the morning before
sampling; samples collected
between 8 and 11 AM; end-
tidal breath samples
collected from the mouth;
subjects breathed
continuously into the
sampling piece for 3-5 min
to obtain stable sample;
samples also collected after
an acidic mouth wash
Concentrations in exhaled breath
from the mouth (mg/m3):
Range = 0.28-1.5
Mean = 0.55 (95% CI: 0.42-0.68)
Median = 0.49
Concentrations following acidic
mouth wash (mg/m3):
Range = 0.010-0.027
Mean = 0.015 (95% CI:
0.014-0.018)
Median = 0.015
Commercial
cavity ring-
down
spectrometer
Ammonia concentrations were
down to 0.0004 mg/m3 in
outdoor air, 0.002-0.004 mg/m3
in indoor air, and
0.006-0.007 mg/m3 in indoor air
in the presence of humans
Schmidt et
al. (2013)
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Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
Three healthy male
volunteers (>30 yrs of
age)
Ammonia levels measured in
mouth-exhaled breath and in
the closed mouth cavity of
test subjects each morning
about 2 hrs after eating a
regular breakfast; samples
collected daily over a 4-wk
period
Via mouth:
Volunteer A = 0.769 ±
0.000919 mg/m3
Volunteer B =
0.626 ±0.000919 mg/m3
Volunteer C =
0.604 ±0.000919 mg/m3
Via oral cavity:
Volunteer A =
1.04 ± 0.000990 mg/m3
Volunteer B =
1.52 ± 0.00106 mg/m3
Volunteer C =
1.31 ± 0.000919 mg/m3
(median ammonia levels
estimated as geometric mean ±
geometric SD)
SI FT-MS
analysis
Mean ambient air level of
ammonia was 0.056 ±
0.0071 mg/m3
The authors indicated that
ammonia measured in mouth-
exhaled breath may be
generated in the oral cavity and
suggested that concentrations in
nose-exhaled breath may better
represent systemic conditions
(such as metabolic disease)
Smith et al.
(2008)
Four healthy children
(two males and two
females, 4-6 yrs old)
Thirteen senior
volunteers (11 males and
2 females, 60-83 yrs old);
four had type-2 diabetes
mellitus with onset at
ages between 50 and
70 yrs, and controlled by
diet
Breath samples collected in
morning at least 1 hr after
breakfast and at least 1 hr
prior to lunch; each
volunteer performed two
exhalation/inhalation cycles
(both about 5-10 sec in
duration)
Children = 0.157-0.454 mg/m3
Seniors = 0.224-1.48 mg/m3
SI FT-MS
analysis
Ammonia breath levels
significantly increased with age
Some seniors reported diabetes
Measured ammonia level in
breath reported for each subject
Spanel et
al. (2007a)
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Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
All subjects had their
regular breakfast without
any specific restrictions
Twenty-six secondary
school students (10 males
and 16 females, 17-18 yrs
old and one 19 yrs old)
Three sequential breath
exhalations collected over
5 min following the students
listening to a 1-hr
presentation (at least 1 hr
following breakfast and
before lunch); alveolar
portion measured (identified
using humidity)
Median values reported for:
17-yr-olds = 0.165 mg/m3
18-yr-olds = 0.245 mg/m3
SI FT-MS
analysis
Significant differences in
ammonia levels in exhaled
breath between 17- and 18-yr-
olds (p < 10"8) were reported
(statistical test not reported)
Soanel et
al. (2007b)
Thirty healthy volunteers
(19 males and 11 females,
24-59 yrs, 28 Caucasian,
1 African, and 1 mixed
race); volunteers were
instructed to maintain
their normal daily
routines and to not rinse
out their mouths prior to
providing a breath sample
Breath samples collected in
the morning prior to lunch at
approximately weekly
intervals for about 6 mo;
some volunteers provided
samples more frequently
than others; 480 samples
collected and analyzed for
ammonia
Geometric mean and geometric
SD = 0.589 ± 0.00114 mg/m3
Median = 0.595 mg/m3
Range = 0.175-2.08 mg/m3
SI FT-MS
analysis
Ammonia breath levels were
shown to increase with age
Background levels in the testing
laboratory were typically around
0.28 mg/m3
Turner et
al. (2006)
Five subjects (two
females, three males; age
range 27-65 yrs)
Breath samples collected
between 8 and 9 AM in three
sequential breath
exhalations on multiple days
(12-30 d) over the course of
1 mo
Ammonia concentrations were
0.298-1.69 mg/m3
SI FT-MS
analysis
Differences in ammonia breath
levels between individuals were
significant (p < 0.001; ANOVA
test)
Diskin et
al. (2003)
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Supplemental Information—Ammonia
Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
Six normal nonsmoking
male volunteers
(24-61 yrs old), fasted for
12 hrs prior to testing
Baseline breath sample
obtained; breath samples
collected 20, 40, and 60 min
and 5 hrs following the
ingestion of a liquid protein-
calorie meal
Premeal levels were
0.2-0.4 mg/m3;
Postmeal levels at 30 min were
0.1 mg/m3 increasing to
maximum values at 5 hrs of
0.4-1.3 mg/m3
SI FT-MS
analysis
A biphasic response in breath
ammonia concentration was
observed after eating
(Smith et
al., 1999)
Fourteen healthy,
nonsmoking subjects (age
range 21-54 yrs)
performed one or more
of the following hygiene
maneuvers:
(1) acidic oral rinse
(pH 2.5)
(2) tooth brushing
followed by acidic oral
rinse
(3) tooth brushing
followed by distilled
water rinse
(4) distilled water rinse
Subjects fasted for 8 hrs prior
to baseline measurement,
refrained from oral hygiene
after their most recent meal,
refrained from heavy
exercise for 12 hrs, and had
no liquid intake for several
hours; initial breath
ammonia was measured
between 8 and 10 AM, then
subjects performed one or
more of the hygiene
measures listed (at 30-min
intervals for a total 90-min
period; samples collected
over 5 min)
Baseline levels varied from
0.085 to 0.905 mg/m3
Nitrogen oxide
analyzer with
an ammonia
conversion
channel (similar
to chemi-
luminescence)
An 80-90% depletion of volatile
ammonia emissions was seen
within 10 min of acid rinsing;
<50% depletion of ammonia was
seen following tooth brushing or
distilled water rinse; gaseous
ammonia levels increased after
all rinse procedures over time
Norwood
et al.
(1992)
Sixteen healthy subjects
(nine males aged
25-63 yrs and seven
females aged 23-41 yrs);
subgroups tested were all
male
Breath samples collected
during quiet mouth
breathing
Ammonia concentrations ranged
from 0.029 to 0.52 mg/m3 during
mouth breathing (median of
0.17 mg/m3)
Chemi-
luminescence
The oral cavity appeared to be a
source of breath ammonia; no
attempt was made to control the
diet of subjects or standardize
the interval between the last
meal and the measurement
Larson et
al. (1977)
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Supplemental Information—Ammonia
Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
Breath samples: source (nose/mouth/oral cavity) not specified
Sixteen healthy,
nonsmoking subjects
(4 females and 12 males,
29 ± 7 yrs); no significant
differences in mean age,
height, weight, BMI, or
time since last oral intake;
10 subjects tested in each
experiment
Experiment 1: single whole-
breath samples collected
from each subject (same
samples immediately
reanalyzed within <10 sec to
assess instrument specific
variability)
Experiment 2: three repeat
breath samples collected
from each subject (to
evaluate intra-subject
differences); this experiment
evaluated differences based
on standardization of
expiratory pressure and flow
Experiment 3: two mixed
breath samples and two bag
alveolar breath samples
collected in short succession
from each subject
Experiment 1:
0.843 ± 0.0601 mg/m3
(median ± measurement error)
Experiment 2:
Nonstandardized = 0.712 ±
0.130 mg/m3 (median ± SD)
Standardized =
1.01 ± 0.113 mg/m3 (median ± SD)
Experiment 3:
Mixed = 0.860 ± 0.585 mg/m3
(median ± SD)
Alveolar = 0.920 ± 0.559 mg/m3
(median ± SD)
SI FT-MS
analysis
This study
established
that SIFT-MS
analysis is
reliable and
repeatable
Relatively small number of
healthy subjects used
Did not address the breath of
those with disease
Intra- and inter-day repeatability
were not investigated
Boshier et
al. (2010)
Eight healthy subjects
(average age
39.8 ±9.6 yrs)
Subjects fasted for 6 hrs prior
to samples being collected;
subjects breathed normally
into collection device for
5 min
Mean breath ammonia = 0.35 ±
0.17 mg/m3
Fiber optic
sensor
This study measured ammonia
levels in healthy volunteers
compared to Helicobacter pylori
positive individuals (five
subjects) (data not shown); the
experiment also included a
challenge with a 300 mg urea
capsule to evaluate the urease
activity of healthy versus
infected individuals (data not
Kearnev et
al. (2002)
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Test subjects
Breath samples
Levels of ammonia in exhaled
breath
Methods
Comments
Reference
shown); the authors concluded
that breath ammonia
measurement may be feasible as
a diagnostic test for H. pylori
Three groups of children
were used as test
subjects:
(1) 68 asthmatic children
residing in a National Park
in the mountains (mean
age 10 yrs, 48 boys,
20 girls)
(2) 52 asthmatic children
in an urban area (mean
age 9 yrs, 35 boys,
17 girls)
(3) 20 healthy children
from the same urban area
as a control group (mean
age 10 yrs, 12 boys,
8 girls)
Subjects performed a 5-sec
breath-hold and exhaled
slowly into collection device
Asthmatic children from National
Park = 0.0040 ± 0.0033 mg/m3
Asthmatic urban children:
Mean NHs = 0.0101 ±
0.00721 mg/m3
Urban children control group:
Mean NHs = 0.0105 ±
0.00728 mg/m3
Chemi-
luminescence
Both groups of asthmatic
children had some subjects on
glucocorticoids, often combined
with histamine antagonists
and/or b2 agonists, while others
were left untreated; ammonia
concentrations in exhaled breath
appeared to be correlated with
exposure to urban air
Giroux et
al. (2002)
ANOVA = analysis of variance; BMI = body mass index; CI = confidence interval; SD = standard deviation; SIFT-MS = selected ion flow tube mass spectrometry
C-16
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C.2. HUMAN STUDIES
More detailed summaries are provided of epidemiology studies of workers in industrial
exposure settings that examined respiratory parameters; information from these studies was used
as the basis for the reference concentration (RfC).
C.2.1. Occupational Studies in Industrial Worker Populations
Holness et ah (19891
Holness etal. (1989) conducted a cross-sectional study of workers in a soda ash (sodium
carbonate) plant4 who had chronic, low-level exposure to ammonia. The cohort consisted of
58 workers and 31 controls from stores and office areas of the plant All workers were males
(average age 43 years), and the average exposure duration for the exposed workers at the plant
was 12 years. The mean time-weigh ted average (TWA) ammonia exposure of the exposed group
based on personal sampling over one work shift (mean sample collection time 8.4 hours) was
9.2 ppm (6.5 mg/m3) compared to 0.3 ppm (0.2 mg/m3) for the control group. The average
concentrations of ammonia to which workers were exposed were determined using the procedure
recommended by the National Institute for Occupational Safety and Health (NIOSH), which involves
the collection of air samples on sulfuric acid-treated silica gel adsorption tubes fNIOSH. 19791.
No statistically significant differences were observed in age, height, years worked,
percentage of smokers, or pack-years smoked for exposed versus control workers. Exposed
workers weighed approximately 8% (p < 0.05) more than control workers. Information regarding
past occupational exposures, working conditions, and medical and smoking history, as well as
respiratory symptoms and eye and skin complaints, was obtained by means of a questionnaire that
was based on an American Thoracic Society questionnaire fFerris. 19781. Each participant's sense
of smell was evaluated at the beginning and end of the work week using several concentrations of
pyridine (0.4, 0.66, or 10 ppm). Lung function tests were conducted atthe beginning and end of the
work shift on the first and last days of their work week (four tests administered). Differences in
reported symptoms and lung function between groups were evaluated using the actual exposure
values with age, height, and pack-years smoked as covariates in linear regression analysis. Exposed
workers were grouped into three exposure categories (high, >12.5 ppm [>8.8 mg/m3]; medium,
6.25-12.5 ppm [4.4-8.8 mg/m3]; and low, <6.25 ppm [<4.4 mg/m3]) for analysis of symptom
reporting and lung function data.
Endpoints evaluated in the study included sense of smell, prevalence of respiratory
symptoms (cough, bronchitis, wheeze, dyspnea, and others), eye and throat irritation, skin
problems, and lung function parameters (forced vital capacity [FVC], forced expiratory volume in
4At this plant, ammonia, carbon dioxide, and water were the reactants used to form ammonium bicarbonate,
which in turn was reacted with salt to produce sodium bicarbonate and subsequently processed to form
sodium carbonate. Ammonia and carbon dioxide were recovered in the process and reused.
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1 second [FEVi], FEVi/FVC, forced expiratory flow [FEF50], and FEF75). No statistical differences in
the prevalence of respiratory irritation or eye irritation were evident between the exposed and
control groups (Table C-2).
Table C-2. Symptoms and lung function results of workers exposed to
different levels of TWA ammonia concentrations
Parameter
Ammonia concentration
Control
0.2 mg/m3
Exposed
<4.4 mg/m3
Exposed
4.4-8.8 mg/m3
Exposed
>8.8 mg/m3
Symptom
Cough
3/31 (10)a
6/34 (18)
1/12 (8)
2/12 (17)
Sputum
5/31 (16)
9/34 (26)
3/12 (25)
1/12 (8)
Wheeze
3/31 (10)
5/34 (15)
1/12 (8)
0/12 (0)
Chest tightness
2/31 (6)
2/34 (6)
0/12 (0)
0/12 (0)
Shortness of breath
4/31 (13)
3/34 (9)
1/12 (8)
0/12 (0)
Nasal complaints
6/31 (19)
4/34 (12)
2/12 (17)
0/12 (0)
Eye irritation
6/31 (19)
2/34 (6)
2/12 (17)
1/12 (8)
Throat irritation
1/31 (3)
2/34 (6)
1/12 (8)
1/12 (8)
Skin problems
2/31 (6)
10/34* (29)
1/12 (8)
1/12 (8)
Lung function (% predicted)
FVC
98.6
96.7
96.9
96.8
FEVi
95.1
93.7
93.9
95.3
FEFso
108.4
106.9
106.2
111.2
FEF75
65.2
71.0
67.8
78.8
aNumber affected/number examined. The percentage of workers reporting symptoms is indicated in parentheses.
^Significantly different from controls, p < 0.05, by Fisher's exact test performed for this review.
Source: Holness et al. (1989).
There was a statistically significant increase (p < 0.05) in the prevalence of skin problems in
workers in the lowest exposure category (<4.4 mg/m3) compared to controls; however, the
prevalence was not increased among workers in the two higher exposure groups. Workers also
reported that exposure at the plant had aggravated specific symptoms including coughing,
wheezing, nasal complaints, eye irritation, throat discomfort, and skin problems. Odor detection
threshold and baseline lung functions were similar in the exposed and control groups. No changes
in lung function were demonstrated over either work shift (days 1 or 2) or over the work week in
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the exposed group compared with controls. No relationship was demonstrated between chronic
ammonia exposure and baseline lung function changes either in terms of the level or duration of
exposure. Study investigators noted that this finding was limited by the lack of adequate exposure
data collected over time, precluding development of a meaningful index accounting for both level
and length of exposure. Based on the lack of exposure-related differences in subjective
symptomatology, sense of smell, and measures of lung function, EPA identified the high-exposure
category (>8.8 mg/m3) as the no-observed-adverse-effect level (NOAEL). A lowest-observed-
adverse-effect level (LOAEL) was not identified for this study.
Ballal et al. (1998)
Ballal etal. (1998) conducted a cross-sectional study of male workers at two urea fertilizer
factories in Saudi Arabia5. The cohort consisted of 161 exposed subjects (84 from Factory A and
77 from Factory B) and 355 unexposed controls. Workers in Factory A were exposed to air
ammonia levels of 2-130 mg/m3 and workers in Factory B were exposed to levels of 0.02-7 mg/m3.
Mean duration of employment was 51.8 months for exposed workers and 73.1 months for controls.
Exposure levels were estimated by analyzing a total of 97 air samples collected over 8-hour shifts
close to the employee's work site. The prevalence of respiratory symptoms and diseases was
determined by administration of a questionnaire. The authors stated that there were no other
chemical pollutants in the workplace that might have affected the respiratory system. Smoking
habits were similar for exposed workers and controls.
In Factory A, the relative risks for respiratory symptoms (cough, phlegm, wheezing,
dyspnea) were elevated in smokers, whereas in Factory B, all relative risks were nonsignificant.
The prevalence rate of hemoptysis (coughing up blood) was higher in Factory A (relative risk = 4.1,
95% confidence interval [CI] 1.63-10.28) than in Factory B (relative risk = 0.47, 95% CI 0.06-3.66),
although chest roentgenograms showed no specific pulmonary changes. Stratifying the workers by
ammonia exposure levels (above or below the American Conference of Governmental Industrial
Hygienists [ACGIH] threshold limit value [TLV] of 18 mg/m3) showed that those exposed to
ammonia concentrations higher than the TLV had 2.2-4-fold higher relative risks for cough,
phlegm, wheezing, dyspnea, and asthma than workers exposed to levels below the TLV (Table C-3).
The relative risk for wheezing was also elevated among those exposed to ammonia levels at or
below the TLV. Distribution of symptoms by cumulative ammonia concentration (CAC,
mg/m3-years) also showed 2-4.8-fold higher relative risk for all of the above symptoms among
those with higher CAC (T able C-3). Results of the logistic regression analysis showed that ammonia
concentration was significantly related to cough, phlegm, wheezing with and without shortness of
breath, and asthma (Table C-4).
5The process of fertilizer production involved synthesis of ammonia from natural gas, followed by reaction of
the ammonia and carbon dioxide to form ammonium carbamide, which was then converted to urea.
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Table C-3. The prevalence of respiratory symptoms and disease in urea
fertilizer workers exposed to ammonia
Respiratory
symptom/disease
Relative risk (95% CI)
Exposure category
CACa (mg/m3-yrs)
ACGIH TLV
(18 mg/m3)
(n = 17)
<50
(n = 130)
>50
(n = 30)
Cough
0.86 (0.48-1.52)
3.48 (1.84-6.57)
0.72 (0.38-1.35)
2.82 (1.58-5.03)
Wheezing
2.26 (1.32-3.88)
5.01 (2.38-10.57)
1.86 (1.04-3.32)
5.24 (2.85-9.52)
Phlegm
0.79 (0.43-1.47)
3.75 (1.97-7.11)
0.63 (0.31-1.26)
3.03 (1.69-5.45)
Dyspnea
1.13 (0.62-2.04)
4.57 (2.37-8.81)
1.19 (0.66-2.17)
2.59 (1.25-5.36)
Chronic bronchitis
1.43 (0.49-4.19)
2.32 (0.31-17.28)
0.61 (0.13-2.77)
5.32 (1.72-16.08)
Bronchial asthma
1.15 (0.62-2.15)
4.32 (2.08-8.98)
1.22 (0.66-2.28)
2.44(1.10-5.43)
Chronic bronchitis and
bronchial asthma
2.57 (0.53-12.59)
6.96 (0.76-63.47)
1.82 (0.31-10.77)
8.38 (1.37-45.4)
aOne missing value.
Source: Ballal et al. (1998).
Table C-4. Logistic regression analysis of the relationship between ammonia
concentration and respiratory symptoms or disease in exposed urea fertilizer
workers
Respiratory symptom/disease
Odds ratio (95% CI)
Cough
1.32 (1.08-1.62)*
Phlegm
1.36 (1.10-1.67)*
Shortness of breath with wheezing
1.26 (1.04-1.54)*
Wheezing alone
1.55 (1.17-2.06)*
Dyspnea on effort
0.83 (0.68-1.02)
Diagnosis of asthma
1.33 (1.07-1.65)*
*p < 0.05.
Source: Ballal et al. (1998).
Ali etal. (2001)
Results from limited spirometry testing of workers from Factory A were reported in a
follow-up study f Ali etal.. 20011. The lung function indices measured in 73 ammonia workers and
348 control workers included FEVi and FVC. Prediction equations for these indices were developed
for several nationalities (Saudis, Arabs, Indians, and other Asians), and corrected values were
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expressed as the percentage of the predicted value for age and height Workers with cumulative
exposure >50 mg/m3-years had significantly lower FEVi% predicted (7.4% decrease, p < 0.006)
and FVC% predicted (5.4% decrease, p < 0.030) than workers with cumulative exposure
<50 mg/m3-years. A comparison between symptomatic and asymptomatic exposed workers
showed that FEVi% predicted and FEVi/FVC% were significantly lower among symptomatic
workers (9.2% decrease in FEVi% predicted, p < 0.001, and 4.6% decrease in FEVi/FVC%,
p < 0.02).6
Rahman etal. (2007)
Rahman etal. (2007) conducted a cross-sectional study of workers at a urea fertilizer
factory in Bangladesh that consisted of an ammonia plant and a urea plant The exposed group
consisted of 24 participants of the 63 operators in the ammonia plant and 64 participants of the
77 operators in the urea plant; 25 individuals from the administration building served as a control
group. Mean duration of employment exceeded 16 years in all groups. Personal ammonia
exposures were measured by two different methods (Drager PAC III and Drager tube) in five to nine
exposed workers per day for 10 morning shifts in the urea plant (for a total of 64 workers) and in
five to nine exposed workers per day for 4 morning shifts from the ammonia plant (for a total of
24 workers). Four to seven volunteer workers per day were selected from the administration
building as controls, for a total of 25 workers over a 5-day period. Questionnaires were
administered to inquire about demographics, past chronic respiratory disease, past and present
occupational history, smoking status, respiratory symptoms (cough, chest tightness, runny nose,
stuffy nose, and sneezing), and use of protective devices. Lung function tests (FVC, FEVi, and peak
expiratory flow rate [PEFR]) were administered preshift and postshift (8-hour shifts) to the 88
exposed workers after exclusion of workers who had planned to have less than a 4-hour working
day; lung function was not tested in the control group. Personal ammonia exposure and lung
function were measured on the same shift for 28 exposed workers. Linear multiple regression was
6Table 3 of Ali etal. (20011 provided a comparison of pulmonary function indices for exposed workers and
controls. FVC% predicted was statistically significantly higher than the control group; FEVi% predicted and
FEVi/FVC% were not Based on comparison of values for exposed workers in Tables 3, 4, and 5 of the paper,
EPA concluded that the value for FVC% in the exposed group (Table 3) was likely an error. FVC% predicted
for all exposed workers (n = 73) in Table 3 was 105.65. Tables 4 and 5 provided values for FVC% predicted
for exposed workers subdivided two different ways: (1) exposed workers with cumulative exposures
<50 mg/m3-years (105.64; n = 45) and >50 mg/m3-years (100.22; n = 28) (Table 4), and (2) exposed
workers that were symptomatic (102.23; n = 33) and asymptomatic (104.58) (n = 40) (Table 5). The values
for the exposed workers when subdivided (either by cumulative exposure or by presence/absence of
symptoms) should bracket the FVC% predicted value for all exposed workers (105.65). This was not the
case in either instance. Two of the authors of the study were contacted (email from Dr. H.O. Ahmed to
S. Rieth, EPA, on May 14, 2013; email from Dr. S.G. Ballal to S. Rieth, EPA, on May 9, 2013); both reported that
the original data were no longer available. Given concerns about the pulmonary function values in Table 3,
only evidence from Tables 4 and 5 of the Ali et al. (20011 study were considered in this assessment.
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used to analyze the relationship between workplace and the percentage cross-shift change in FEVi
(AFEVi%) while adjusting for current smoking.
Mean exposure levels at the ammonia plant determined by the Drager tube and Drager
PAC III methods were 25.0 and 6.9 ppm (17.7 and 4.9 mg/m3), respectively; the corresponding
means in the urea plant were 124.6 and 26.1 ppm (88.1 and 18.5 mg/m3) f Rah man etal.. 20071.
Although the Drager tube measurements indicated ammonia levels about 4-5 times higher than
levels measured with the PAC III instrument, there was a significant correlation between the
ammonia concentrations measured by the two methods (p = 0.001). No ammonia was detected in
the control area using the Drager tube (concentrations less than the measuring range of
2.5-200 ppm [1.8-141 mg/m3]). The study authors observed that their measurements indicated
only relative differences in exposures between workers and production areas, and that the validity
of the exposure measures could not be evaluated based on their results. Based on an evaluation of
the two monitoring methods and communication with technical support at Drager Safety Inc.
(Bacom and Yanoskv. 2010). EPA considered the PAC III instrument to be a more sensitive
monitoring technology than the Drager tubes. Therefore, the PAC III air measurements were
considered the more reliable measurement of exposure to ammonia for the Rahman etal. (2007)
study.
The prevalence of respiratory irritation and a decrease in lung function were higher in the
urea plant than in the ammonia plant or in the administration building. Comparison between the
urea plant and the administration building showed that cough and chest tightness were statistically
higher in the former; a similar comparison of the ammonia plant and the administration building
showed no statistical difference in symptom prevalence between the two groups (Table C-5).
Preshift measurement of FVC, FEVi, and PEFR did not differ between urea plant and ammonia plant
workers. Significant cross-shift reductions in FVC and FEVi were reported in the urea plant (2 and
3%, respectively, p < 0.05), but not in the ammonia plant. When controlled for current smoking, a
significant decrease in AFEVi% was observed in the urea plant (p < 0.05). Among 23 workers with
concurrent measurements of ammonia and lung function on the same shift, ammonia exposure and
years working in the factory were correlated with a cross-shift decline in FEVi. EPA identified a
NOAEL of 4.9 mg/m3 and a LOAEL of 18.5 mg/m3 in the Rahman et al. (2007) study based on
increased prevalence of respiratory symptoms and a decrease in lung function.
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Table C-5. Prevalence of respiratory symptoms and cross-shift changes in
lung function among workers exposed to ammonia in a urea fertilizer factory
Parameter
Ammonia plant
(4.9 mg/m3)a
Urea plant
(18.5 mg/m3)a
Administration building
(concentration not
determined)13
Respiratory symptoms
Cough
4/24 (17%)c
18/64 (28%)*
2/25 (8%)
Chest tightness
4/24 (17%)
21/64 (33%)*
2/25 (8%)
Stuffy nose
3/24 (12%)
10/64 (16%)
1/25 (4%)
Runny nose
1/24 (4%)
10/64 (16%)
1/25 (4%)
Sneeze
0/24 (0%)
14/64 (22%)
2/25 (8%)
Lung function parameters (cross-shift percentage change)d,e
FVC
0.2 ±9.3
(Pre-shift: 3.308;
Post-shift: 3.332)
-2.3 ±8.8
(Pre-shift: 3.362;
Post-shift: 3.258)
No data
FEVi
3.4 ± 13.3
(Pre-shift: 2.627;
Post-shift: 2.705)
-1.4 ±8.9
(Pre-shift: 2.701;
Post-shift: 2.646)
No data
PEFR
2.9 ± 11.1
(Pre-shift: 8.081;
Post-shift: 8.313)
-1.0 ± 16.2
(Pre-shift: 7.805;
Post-shift: 7.810)
No data
aMean ammonia concentrations measured by the Drager PAC III method.
Concentrations in the administration building were rejected by study authors due to relatively large drift in the
zero levels.
"Values are presented as incidence (prevalence expressed as a percentage).
Calculated as ([postshift - preshift]/preshift) x 100.
eValues are presented as mean ± SD.
^Statistically significant (p < 0.05) by Fisher's exact test, comparing exposed workers to administrators.
Source: Rahman et al. (2007).
Bhat and Ramaswamv (1993)
A cross-sectional study of workers exposed to fertilizer chemicals in a plant in Mangalore,
India f Bhat and Ramaswamv. 19931 showed significant reduction in lung function parameters
(PEFR/minute and FEVi) compared to a control group. The exposed group consisted of 91 workers
who underwent lung function testing and included 3 0 urea plant workers, 3 0 diammonium
phosphate (DAP) plant workers, and 31 ammonia plant workers. The control group included
68 people having comparable body surface area who were chosen from the same socioeconomic
status and sex. All smokers were excluded from the study to avoid the effect of smoking on lung
function. Other workplace exposures were not assessed. The duration of exposure was
dichotomized into two groups (<10 and >10 years), but no exposure measurements were made.
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Lung function parameters (FVC, FEVi, and PEFR/minute) were measured by a standard
spirometry protocol for all workers in the study, and the highest of three replicates were retained
for calculation. A comparison of FVC, FEVi, and PEFR/minute was made between controls and
fertilizer workers as a whole and also between controls and urea workers, DAP workers, and
ammonia workers individually. The ammonia plant workers showed a significant decrease in FEVi
(p < 0.05) and PERF/minute (p < 0.001) when compared to controls, but no significant decrease in
FVC (Table C-6). PEFR/minute, a measure of airflow in the bronchi, was reduced in all plant
workers (urea, DAP, and ammonia), indicating that these fertilizer chemicals affected the larger
airways. The reduction of FEVi, a measure of the amount of air that can be exhaled in 1 second, in
ammonia plant workers suggested that ammonia can enter into the smaller bronchioles and cause
bronchospasm. NOAEL and LOAEL values were not identified by the authors of this study or by
EPA due to the lack of exposure concentration measurements in this study.
Table C-6. Comparison of lung function parameters in ammonia plant
workers with controls
Parameter
Controls (n = 68)
(mean ± standard error)
Ammonia Plant (n = 31)
(mean ± standard error)
FVC
3.43 ±0.21
3.19 ±0.07
FEVi
2.84 ±0.10
2.52 ±0.1*
PEFR/min
383.3 ±7.6
314 ± 19.9**
^Significantly different from controls (p < 0.05); paired t-test.
**Significantly different from controls (p < 0.001); paired t-test.
Source: Bhat and Ramaswamv (19931.
C.2.2. Studies of Populations in Agricultural Settings (Livestock Farmers/Populations in Close
Proximity to Animal Feeding Operations)
Several studies have investigated respiratory health and other outcomes related to
ammonia exposure in agricultural settings. Some of these studies have also demonstrated
respiratory effects associated with exposure to other air constituents (e.g., respirable dust,
endotoxin). Ammonia exposure was associated with a decrease in lung function measures in six of
the eight studies fLoftus etal.. 2015: Monso etal.. 2004: Donham et al.. 2000: Reynolds etal.. 1996:
Donham etal.. 1995: Preller etal.. 1995: Zeida etal.. 1994: Heederik etal.. 1990) examining this
outcome (Table C-7). Six of these studies addressed confounding in some way; four of these studies
controlled for co-exposures (e.g., endotoxin, dust, disinfectants) (Melbostad and Eduard. 2001:
Reynolds etal.. 1996: Donham etal.. 1995: Preller etal.. 1995). one study noted only weak
correlations (i.e., Spearman r < 0.20) between ammonia and dust or endotoxin f Donham etal..
20001. and one study observed associations with ammonia but not with endotoxin or dust
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measures fHeederik etal.. 19901. Two studies did not address confounding fMonso etal.. 2004:
Zeida etal.. 19941. and one study noted a lack of analysis for other potential confounders fLoftus et
al.. 201 SI.
The studies that controlled for co-exposures (e.g., endotoxin, dust, disinfectants) fMelbostad
and Eduard. 2001: Reynolds etal.. 1996: Donham etal.. 1995: Preller etal.. 19951. noted only weak
correlations (i.e., Spearman r < 0.20) between ammonia and dust or endotoxin f Donham etal..
20001. or observed associations with ammonia but not with endotoxin or dust measures (Heederik
etal.. 19901 are the studies that EPA considered to be methodologically strongest (see Literature
Search Strategy | Study Selection and Evaluation section). In summary, this set of studies provides
relatively consistent evidence of an association between ammonia exposure and reduced lung
function in studies of populations in agricultural settings, accounting for endotoxin and dust
Some of these studies in agricultural settings also included analyses of respiratory
outcomes in relation to exposure, based on ammonia measurements. The studies analyzing
prevalence of respiratory symptoms (including cough, phlegm, wheezing, chest tightness, and eye,
nasal, and throat irritation) in relation to ammonia provide generally negative results (Melbostad
and Eduard. 2001: Preller etal.. 1995: Zeida etal.. 19941. Two other studies reported an increased
prevalence of respiratory symptoms in pig farmers fChoudat et al.. 1994: Crook etal.. 19911. The
authors of these studies measured air ammonia, but did not include a direct analysis of respiratory
symptoms in relation to ammonia (Table C-8). One study found no relationship between reported
asthma symptoms or medication use for asthma and ammonia exposure (Loftus etal.. 20151.
Table C-7. Evidence pertaining to respiratory effects in populations exposed
to ammonia in agricultural settings with direct analysis of the relationship
between ammonia exposure and measured outcomes
Study reference and design
Results
Lung function
Monso et al. (2004)
105 never-smoking farmers (84 males, 21 females)
working inside animal confinement buildings; sampled
from the European Farmers' Study; mean age 45 yrs
Exposure: Area samples (confinement building, morning)
Median
Ammonia 10 ppm (7 mg/m3)
Total dust 5.6 mg/m3
Total endotoxin 687.1 units/m3
Outcome: Lung function (standard spirometry, before and
after shift; chronic obstructive pulmonary disease (COPD)
defined as FEVi <70 (n = 18; 17%).
COPD, odds ratio (95% CI), by quartile of ammonia (1st
and 2nd groups = referent)
ppm OR (95%CI)
0-10 1.0 (referent)
>10-17 0.73 (0.17,3.20)
>17-60 1.32 (0.34,5.12)
Adjusted for age, gender, and types of farming.
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Study reference and design
Results
Donham et al. (2000) (United States. Iowa)
257 poultry workers (30% women, 70% men); 150 controls
(42% women, 58% men; postal workers and electronics
plant)
Exposure: Personal samples (workshift)
Mean
Ammonia 18.4 ppm (13 mg/m3)
Total dust 6.5 mg/m3
Respirable dust 0.63 mg/m3
Total endotoxin 1,589 EU/m3 (0.16 ng/m3)
Respirable endotoxin 58.9 EU/m3 (0.006 ng/m3)
Outcome: Lung function (standard spirometry, before and
after work shift)
OR (95% CI) for 3% or greater cross-shift decline in
FEVi, by quartile of ammonia
ppm OR (95%CI)
>0-<5 1.88 (0.68,5.14)
5-<12 1.93 (0.72,5.17)
12-<25 4.25 (1.60, 11.2)
>25 2.45 (0.88,6.85)
Adjusted for age, years worked in poultry industry,
gender, smoking status, education.
In linear regression, ammonia was statistically
significant predictor of 5% decline in FEF25-75
(p = 0.045; Beta not reported).
Correlations between ammonia and other exposures
relatively weak (Spearman r < 0.20).
Revnolds et al. (1996) (United States. Iowa)
151 men >18 yrs of age employed at swine farms and
spent time in swine confinement buildings (mean years of
employment = 12.4); a farm comparison group
(nonconfinement production) was included (number not
given). Follow-up studv of Donham et al. (1995).
Exposure: Personal samples (workshift)
Geometric mean (Time 2)
Ammonia 5.15 ppm (4 mg/m3)
Total dust 3.45 mg/m3
Respirable dust 0.26 mg/m3
Total endotoxin 176.12 EU/m3
Respirable endotoxin 11.86 EU/m3
Ammonia levels similar at Time 1 (5.65 ppm), but total
dust and respirable dust higher at Time 1 than Time 2
Outcome: Lung function (standard spirometry, before and
after work shift at two times, 2 yrs apart (same season)
Correlation between cross-shift decline in FEVi and
ammonia: Spearman r = 0.18 (p < 0.05); strongest for
0-6 and 10-13 yrs duration
Predictive model relating ammonia to cross-shift
change in FEVi developed at baseline was
corroborated by Time 2 data; dust and endotoxin did
not add to the significance of ammonia as predictor
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Study reference and design
Results
Donham et al. (1995) (United States. Iowa)
201 men >18 yrs of age employed at swine farms and
spent time in swine confinement buildings (mean years of
employment = 9.6); a farm comparison group
(nonconfinement production) was included (number not
provided)
Exposure: Personal samples
Geometric mean
Ammonia 5.64 ppm (4 mg/m3)
Total dust 4.53 mg/m3
Respirable dust 0.23 mg/m3
Total endotoxin 202.35 EU/m3
Respirable endotoxin 16.59 EU/m3
Outcome: Lung function (standard spirometry, before shift
and then after a minimum of 2 hrs of exposure)
Ammonia was significant predictor of cross-shift
decline in lung function (included with age, duration,
smoking, total dust, respirable dust, and total
endotoxin in the models, as well as interaction terms)
Positive correlations were associated with changes in
lung function and exposure to total dust, respirable
dust, respirable endotoxin, and ammonia; dust was
related to all lung function measures; ammonia
results more variable across measures and duration
strata—strongest for 7-9 yrs duration); exposure to
ammonia concentrations of >7.5 ppm (5 mg/m3) were
predictive of a >3% decrease in FEVi
Heederik et al. (1990) (Nethelands)
27 pig farmers (mean age of 29 yrs; 43% current smokers)
Exposure: Area samples, used in conjunction with
duration of specific tasks to calculate an individual
exposure measure
Mean
Ammonia 5.6 mg/m3
Total dust 1.57 mg/m3
Total endotoxin 24 ng/m3
Outcome: Lung function (standard spirometry, before and
after work shift, taken on Monday, Tuesday, and Friday)
Change (mL) in cross-shift lung function per 5 mg/m3
increase in ammonia
Beta (SE) (p-value)
FVC -3 (35)
FEVi -112 (38) (<0.05)
MMEF -330 (131) (<0.05)
PEF -170 (335)
MEF75 -505 (300) (<0.05)
MEFso -404 (215) (<0.05)
MEF25 -70 (179)
Results from Tuesday measures presented; other days
reported to be similar patterns but not as strong; no
association between dust or endotoxins with the lung
function variables
Lung function and respiratory symptoms
Loftus et al. (2015) (United States)
Animal feeding operations; health and environmental data
collected from AFARE (Aggravating Factors of Asthma in a
Rural Environment) project
n = 59 asthmatic children enrolled (inclusion criteria:
school-age, no serious illness other than asthma);
n = 51 (exposed) participated in the study (86.4%
participation rate)
Associations between FEVi% and estimated ammonia
concentrations measured at the nearest neighbor
monitors
Point estimates and 95% CI of FEVi%*
Entire cohort Subjects within 1 km
(n = 51) (n = 23)
1-d lag -3.8% (0.2, 7.3) -6.0% (0.4, 12.5)*
2-d lag -3.0% (0.5, 5.8) -6.3% (2.3, 10.0)
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Supplemental Information—Ammonia
Study reference and design
Results
Exposure: 14 ammonia monitoring devices located outside
the home of a subset of the participants throughout study
area
24-Hr ammonia concentrations ranged from 0.0002 to
0.238 mg/m3; median ammonia concentration measured
at each site ranged from 0.0029 to 0.0727 mg/m3; annual
average ammonia concentrations in study region was
0.019 mg/m3
Outcome: Lung function (FEVi) measurements, twice daily
by child given instructions for proper use according to
American Thoracic Society guidelines; asthma symptoms
(nighttime waking, shortness of breath, limitation of
activities, wheezing, and morning asthma symptom) and
medication use (frequency of use of short-acting
bronchodilator)
*Point estimates and 95% CI represent changes
associated with an IQR increase in 24-hr average
ammonia (25 ng/m3); FEVi% indicates forced
expiratory volume in 1 sec as percent of predicted
*This value was estimated from Figure 3 in Loftus et al.
(2015)
Odds of specific asthma symptoms associated
with estimated weekly ammonia
Symptom or medication use OR (95% CI)*
Limitation of activities 1.1 (0.79,1.4)
Wheezing 0.99 (0.77,1.3)
Nighttime waking 0.92 (0.76,1.3)
Shortness of breath 1.1 (0.86,1.3)
Symptoms worse in morning 0.88 (0.75,1.0)
Use of short-acting "relief 0.97 (0.82,1.2)
medication
*OR is for report of any symptom/medication use in
week prior associated with an IQR increase in weekly
ammonia (18 ng/m3).
Preller et al. (1995) (Netherlands)
194 swine farmers (94 with chronic respiratory symptoms,
100 without symptoms); 106 with complete data for lung
function analysis.
Exposure: Personal samples (two workshifts; winter and
summer)
Mean
Ammonia 2 mg/m3
Total dust 2.7 mg/m3
Total endotoxin 112 ng/m3
Long-term average exposure derived based on measured
values and model based on farm characteristics and tasks
Outcome: Lung function (standard spirometry, single
measure); standardized questionnaire for respiratory
symptoms
Association between ammonia and lung function
(n = 106)
Beta (SE) (p-value)
FVC(I) -0.05 (0.13) (0.36)
FEVi (1) -0.27 (0.13) (0.022)
MMEF (l/s) -0.68 (0.23) (0002)
PEF (l/s) -0.77 (0.43) (0.039)
Adjusted for age, height, smoking, endotoxin,
disinfection variables
Stronger patterns seen in symptomatic group (n = 55);
no association with respiratory symptoms (chronic
cough, chronic phlegm, wheezing, shortness of
breath, chest tightness)
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Supplemental Information—Ammonia
Study reference and design
Results
Zeida et al. (1994)
54 male swine producers (mean age = 36.3 yrs; mean
duration of employment = 10.7 yrs)
Exposure: Area samples
Correlation coefficients (Spearman r) with ammonia
with hr/d interaction
Ammonia
Total dust
Respirable dust
Total endotoxin
Mean
11.3 ppm (8 mg/m3)
2.93 mg/m3
0.13 mg/m3
11,332 units/m3
FVC (%
predicted)
FEVi (%
predicted)
FEVi/FVC
FEF (%
predicted)
0.18
0.18
0.00
0.08
-0.13
-0.16
-0.06
-0.09
Exposure measures categorized into tertiles (cut-points
10.2 and 12.7 ppm) for some analyses.
Outcome: Lung function (standard spirometry, single
measure); respiratory symptoms-based on standardized
questionnaire (cough, phlegm, chest wheeze, chest
tightness)
Adjusted for age, height, and smoking
Some symptoms associated with ammonia
exposure—hrs/d interaction but it is difficult to
distinguish these effects from the other exposures
and interactions in the analyses (particularly
endotoxin)
Respiratory symptoms (without lung function measures)
Melbostad and Eduard (2001)
Survey of 8,482 farmers and spouses; exposure study
conducted in 102 farmers
Exposure: personal samples
Ammonia
Total dust
Total endotoxin
Fungal spores
Bacteria
Range
0-8.2 ppm (0-6 mg/m3)
0.4-5.1 mg/m3
500-28,000 EU/m3
0.02-2.0 x 106/m3
0.2-48 x 106/m3
Outcome: Respiratory symptoms (standard
questionnaire); eye, nose, and throat irritation, cough,
chest tightness, and wheezing
Negative correlation (r = -0.64) with total symptom
prevalence
EU = endotoxin unit (10 EU/ng); IQR = interquartile range; MEF = maximum expiratory flow; MMEF = maximum
midexpiratory flow; PEF = peak expiratory flow; SE = standard error
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Supplemental Information—Ammonia
Table C-8. Evidence pertaining to respiratory effects in populations exposed
to ammonia in agricultural settings without direct analysis of the relationship
between ammonia exposure and measured outcomes
Study reference and design
Results
Lung function and respiratory symptoms
Crook et al. (1991) (Scotland)
29 swine farmers (from 12 farms); 48 electronic workers
(controls for IgE/IgG serum analysis only)
Exposure: Area samples of 20 pig houses were monitored
for dust and ammonia concentrations over a working
shift every 4 wks over a 24-wk period lasting from July to
December; aerobiological analysis was conducted once in
six pig houses
Mean
Ammonia3 1.50-13.23 ppm
(1-9 mg/m3)
Total dusta b 1.66-21.04 mg/m3
Airborne microorganisms 105-107 CFU/m3
Airborne endotoxin 1.9-28.5 ng/m3
Outcome: Lung function (FEVi, FVC); respiratory
symptoms (standard questionnaire); serum
measurements of IgG and IgE antibodies specific to pig
skin, pig urine, and pig feed components
Impaired lung function (decreased FEVi and FVC) was
observed in 3/29 swine farmers (quantitative values
not reported)
Respiratory symptoms
Incidence
Nasal/eye irritation 20/29
Cough 15/29
Wheeze/chest tightness 13/29
Any respiratory complaint 23/29
The study authors suggested that the presence of IgE
in some farmers with wheeze (and absence in
asymptomatic farmers) may indicate the involvement
of an allergic response in these farmers, rather than a
respiratory response to ammonia exposure
Choudat et al. (1994) (France)
102 male swine farmers who worked at least half-time in
a swine confinement building (mean age 39.7 yrs; mean
duration of employment of 15.7 yrs); 51 male dairy
farmers (mean age 40.1 yrs; mean duration of
employment of 20.3 yrs); and 81 male dairy industry
workers (referents; mean age 38.5 yrs; mean duration of
employment of 15.7 yrs)
Exposure: Area samples in 28 swine confinement
buildings from six farms
Number of
samples Mean
Ammonia 48 8.5 mg/m3
Total dust 21 2.41 mg/m3
Inspirable particles 28 1.82 mg/m3
Respirable fraction 24 0.17 mg/m3
Carbon dioxide 28 1,000-5,000 ppm
(1,800-9,000 mg/m3)
No significant differences in baseline lung function
observed between groups
Prevalence (%) of bronchial hyperreactivity to
methacholine
Swine Dairy Dairy
farmers farmers industry
Responders (>10% 17.9* 35.6f 6.7
decrease in FEVi)
Responders (>15% 6.3 17.8* 4.0
decrease in FEVi)
Prevalence (%) of respiratory symptoms (in general)
Swine Dairy Dairy
farmers farmers industry
Morning cough 13.3* 10.4 3.8
Diurnal cough 13.St 6.2 1.3
Fits of coughing 24.0** 22.9* 9.0
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Supplemental Information—Ammonia
Study reference and design
Results
Personal samples in swine farmers (n = 4)
Mean
Morning phlegm 10.2 16.7 7.7
Chest tightness 3.1 10.4* 1.3
Sneezing 29.6 25.2 19.2
Ammonia 3.23 mg/m3
Inspirable particles 3.63 mg/m3
Prevalence (%) of respiratory symptoms at work
Swine Dairy Dairy
farmers farmers industry
Airborne exposure levels were not measured in dairy
farm or industry buildings; study authors noted that dairy
workers do not work in confinement buildings. The
percentage of smokers in the pig and dairy farm groups
(28.4 and 27.4%, respectively) was significantly lower
than in the referent group (44.4%).
Outcome: Lung function tests (FEVi, FVC, PF) before and
after bronchial responsiveness (methacholine challenge);
respiratory symptoms (standard questionnaire)
Fits of coughing 24.5f 8.3 5.1
Sneezing 21.4* 10.4 9.0
*p < 0.05;
fp < 0.01;
fp < 0.001 (compared with dairy industry referents)
No significant differences in the prevalence of
wheezing, shortness of breath, or rhinitis (in general
or at work) between pig or dairy farmers and dairy
industry workers
aMean concentrations were higher in winter due to decreased ventilation.
bMean concentrations were higher in pig houses using restricted feeding systems.
CFU = colony forming unit; IgF = immunoglobin F; IgG = immunoglobic G; PF = peak flow rate
C.2.3. Controlled Human Inhalation Exposure Studies
Controlled exposure studies conducted with volunteers to evaluate irritation effects and
changes in lung function following acute inhalation exposure to ammonia are summarized in
Table C-9.
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Supplemental Information—Ammonia
Table C-9. Evidence pertaining to irritation effects and changes in lung
function in controlled human exposure studies3
Study reference and design
Results
Lung function
Sieurdarson et al. (2004)b (United States, Iowa)
Six healthy volunteers (two males, four females; 25-45
yrs old) and eight volunteers with mild asthma (four
males, four females; 18-52 yrs old)
Exposure: Volunteers were exposed to ammonia, grain
dust, or ammonia + dust for 30-min sessions in an
exposure hood with 1 wk between different exposure
scenario sessions; a nose-clip was used to ensure mouth
breathing
Exposure levels:
Ammonia
Total dust
Respirable fraction
Endotoxin content
16-20 ppm (11-14 mg/m3)
4 mg/m3
1 mg/m3
4 ng/m3
Outcome: Lung function (FEVi, DLCO, exhaled NO) before
and after exposure, post-exposure bronchial
responsiveness (methacholine challenge)
Ammonia-only
No significant changes in lung function in healthy or
asthmatic subjects
Ammonia + dust or dust-only
Significantly decreased FEVi and increased
bronchial hyperreactivity were observed in
asthmatic subjects post-exposure. No significant
changes were observed in DLCO or exhaled NO in
asthmatic subjects. No significant changes in lung
function were observed in healthy subjects.
Cormier et al. f2000)b (Canada)
Eight healthy male volunteers (23-28 yrs old)
FEVi and FVC values were significantly decreased
after exposure in each of the eight swine confinement
Exposure: Volunteers were exposed for 4 hrs to ambient
air in eight swine confinement buildings with 1 wk
between different site exposures
Area samples in eight confinement buildings:
buildings, but values were not significantly correlated
with any airborne exposures.
Pearson's correlation coefficients (p-values) between
airborne exposures and changes in lung function
Mean
Range
AFEVi
AFVC
Ammonia
20.7 ppm
(14.6 mg/m3)
2.80-38.55 ppm
(1.98-27.25 mg/m3)
Ammonia
-0.29 (0.49)
-0.22 (0.60)
Total dust
3.54 mg/m3
2.20-5.62 mg/m3
Total dust
0.07 (0.87)
-0.24 (0.57)
Bacteria
4.25 x 105
CFU/m3
1.67 x 105-
9.29 x 105 CFU/m3
Bacteria
0.36 (0.38)
0.40 (0.32)
Endotoxin
404 EU/m3
215-596 EU/m3
Endotoxin
-0.01 (0.99)
-0.07 (0.87)
Mold
883 CFU/m3
138-1,805 CFU/m3
Mold
0.30 (0.47)
0.19 (0.66)
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Supplemental Information—Ammonia
Study reference and design
Results
Outcome: Lung function (FEVi, FVC) before and after
exposure; post-exposure bronchial responsiveness
(methacholine challenge); nasal lavage levels of white
blood cells and IL-8
Bronchial responsiveness was increased in
3/64 measurements; this increase was significant only
in swine confinement building 2 (lowest ammonia
concentration, second highest mold concentration; all
other airborne values were mid-range).
Nasal lavage levels of total white blood cells,
neutrophils, and IL-8 were significantly or near-
significantly (p = 0.06) increased after exposure in
each of the eight swine confinement buildings (data
presented graphically); the only significant correlation
between airborne exposure and nasal lavage
endpoints was a significant positive correlation
between endotoxin level and IL-8 (correlation
coefficient = 0.72; p-value = 0.05).
Lung function and irritation effects
Petrova et al. (2008)b (United States, Pennsylvania)
25 healthy volunteers (mean age 29.7 yrs) and
15 mild/moderate persistent asthmatic volunteers (mean
age 29.1 yrs)
Exposure: Volunteers were exposed to 20 dilution steps
of ammonia (2-500 ppm [1-354 mg/m3]) via a nasal
cannula and/or a specially fitted set of googles for up to
1.5 hrs; two separate sessions were conducted, separated
by at least 48 hrs
Outcome: Lung function (FEVi) before, during, and after
exposure; subjective reporting of odor intensity,
annoyance, and irritation threshold (with or without
velopharyngeal [VP] closure manipulation to isolate the
throat from the nasal passages by raising the soft palate)
No significant changes in lung function were observed
for healthy or asthmatic subjects during or after
exposure
Reported irritation thresholds in ppm (mg/m3)
Exposure Asthmatic Healthy
Nasal 167 (116) 179 (125)
Ocular 133 (93) 127 (88)
Combined (VP 94 (65) 87 (61)
open)
Combined (VP 77 (54) 102 (71)
closed)
Odor intensity, irritation, and annoyance scores
during combined ocular and nasal exposure were not
significantly different between healthy volunteers
and asthmatics at personal threshold concentrations
or two dilution steps above threshold.
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Supplemental Information—Ammonia
Study reference and design
Results
Sundblad et al. (2004)b (Sweden)
12 healthy volunteers (7 females, 5 males; mean age 25
yrs)
Exposure: Volunteers were exposed to each of the
following concentrations in randomized order during
three separate exposures in inhalation chamber: 0, 5, and
25 ppm (0, 4, and 18 mg/m3). Exposure duration was 3
hrs, in which 50% of the time was spent resting and 50%
exercising on a stationary bike (alternating every 30 min);
exposures were separated by at least 1 week.
Outcome: Lung function (VC, TLC, FEVi, PEF, exhaled NO)
and questionnaire for irritation and respiratory effects
(0-100 mm visual analogue scale) before, during, and 7
hrs after exposure; post-exposure bronchial
responsiveness (methacholine challenge); determination
of total cell and cytokine (IL-6, IL-8) concentration in nasal
lavage fluid
No significant changes in lung functions or bronchial
responsiveness were observed for healthy or
asthmatic subjects during or after exposure
Change in symptom rating during exposure compared
with pre-exposure rating
Exposure in ppm (mg/m3)
0
5(4)
25 (18)
Eye irritation
-0.5
3.6*
14.8*
Nose
irritation
-4.7
3.4
15.3*
Throat/airway
irritation
-2.9
1.2
14.2*
Breathing
difficulty
-1.2
2.3
12.2*
Solvent smell
0.2
38.1*
61.8*
*p < 0.05
No significant changes in total cell concentration or
IL-8 concentration were observed in nasal lavage
fluid. IL-6 in lavage fluid was below the level of
detection
Cole et al. H977)c (United Kingdom)
18 healthy servicemen volunteers (mean age 24.1 yrs)
Exposure: Exposure to ammonia for half-day sessions
during exercise on a cycle ergometer; exercise sessions in
ambient air the day before and the day after acted as
controls (two separate studies were conducted)
Ammonia concentrations in exposure chamber samples:
Mean
Percent change in lung function during exercise +
ammonia exposure, compared with exercise +
ambient air
Exposure in mg/m3
Study 1, morning session
Study 1, afternoon session
Study 2, morning session
Study 2, afternoon session
71 mg/m3
144 mg/m3
106 mg/m3
235 mg/m3
0
71
106
144
235
VE45
-
-4
-8*
*
O
1
1
-6*
vt30
-
2
3*
-9*
-8*
/R30
-
-2
-3*
10*
8*
Outcomes: Lung function endpoints during exercise:
exercise cardiac frequency at 45 mmol Ch/min (/C45);
ventilation minute volume at 45 mmol Ch/min (VE45);
exercise tidal volume at ventilation volume of 30 L/min
(Vt3o); and mean respiratory frequency at a ventilation
volume of 30 L/min (/R30); irritation effects (subjective
reporting)
*p < 0.05
Subjective complaints during ammonia exposure
included "a prickling sensation in the nose and slight
dryness of the mouth," but incidence data for these
self-reported symptoms were not reported.
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Supplemental Information—Ammonia
Study reference and design
Results
Ferguson et al. H977)c (United States, New Jersey)
five male and one female volunteers (24-46 yrs old); no
previous occupational exposure to ammonia
Exposure: Volunteers were exposed to 25, 50, or 100
ppm (18, 35, and 71 mg/m3) ammonia for 2-6 hrs/d, 1
d/wk over 6 wks; occasional brief exposure to
150-200 ppm (106-141 mg/m3) were reported (note that
exposure durations were inconsistent across exposure
levels)
Exposure in ppm (mg/m3)d
25 (18) 50 (35) 100 (71)
Study authors reported that lung function was not
impaired with exposure at any concentration.
Incidence of physician-reported eye, nose, or throat
irritations (per total number of observations)
Exposure in ppm (mg/m3)
Incidence (%)
0(pre-
exposure)
4/45
(9%)
25
50
100
(18)
(35)
(71)
2/78
22/198
11/84
(3%)
(11%)
(13%)
Group A
Wks 1, 4
Wks 2, 5
Wks 3, 6
(n = 2)
(2 hrs/d)
(4 hrs/d)
(6 hrs/d)
Group B
NA
Wks 1-6
NA
(n = 2)
(6 hrs/d)
Group C
Wk 3
Wk 2,5
Wk 1
(n = 2)
(2 hrs/d)
(4 hrs/d)
(6 hrs/d)
Wk 4
Wk 6
(6 hrs/d)
(2 hrs/d
Volunteers did not make any subjective complaints at
concentrations <100 ppm. All subjects reported mild
eye, nose, and throat irritation after brief exposures
>150 ppm.
Outcome: Lung function (FEVi, FVC) tests were
conducted by subjects; irritation effects were evaluated
by a physician looking at eyes and mucosa of the nose
and throat before, during, and after exposure (note that
the study authors did not indicate whether or not pre-
exposure lung function measurements were performed)
Verberk U977)c (Netherlands)
16 volunteers; 8 were considered experts (knew effects of
ammonia from literature; 7 males, 1 female; 29-53 yrs),
8 were non-science students and considered non-experts
(not familiar with effects of ammonia; 6 males, 2 females;
18-30 yrs)
Exposure: Volunteers were exposed to 50, 80,110, and
140 ppm ammonia (35, 57, 78, and 99 mg/m3) for 2 hrs in
an exposure chamber at 1-wk intervals
Outcome: Lung function (VC, FEVi, FIVi) before and after
exposure; irritation effects during exposure (subjective
reporting on scale of 1-5); post-exposure bronchial
responsiveness (histamine challenge)
VC, FEVi, or FIVi were within 10% of pre-exposure
values in all subjects
Incidence of subjects reporting at least one symptom
(smell, irritation of eyes, nose, throat, and/or urge to
cough) with score >3/5 (= nuisance) after 30 min of
exposure
Exposure in ppm (mg/m3)
50(35) 80(56) 110(77) 140(98)
Expert 2/8 2/8 5/8 7/8
Non-expert 5/8 7/8 7/8 8/8
All non-experts found 140 ppm "unbearable" and left
the exposure chamber within 2 hrs; all experts
tolerated 140 ppm for the full 4-hr exposure
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Supplemental Information—Ammonia
Study reference and design
Results
Silverman et al. (1949)° (United States. Massachusetts)
7 adult male volunteers
Exposure: Volunteers were exposed to 500 ppm
(354 mg/m3) for 15-30 min via nose and mouth breathing
mask
Outcome: Lung function (respiratory rate, minute volume
before, during, and after exposure, irritation effects
(subjective reporting)
Respiratory rate and minute volume were increased
by 50-250% during exposure, compared with pre-
exposure values; elevated minute volumes during
exposure showed cyclic variation (~25% decrease
from peak values every 4-7 min)
Subjective complaints included excessive lacrimation
(2/7), nasal irritation (5/7), and nasal and throat
irritation lasting up to 24 hrs after exposure (2/7)
Irritation effects (without lung function measures)
Smeets et al. (2007)b (Netherlands)
24 healthy female volunteers (mean age 29.9 yrs)
Exposure: Volunteers were exposed to a concentration
series (maximum of 2 sec per concentration, 30-60-sec
break between exposures) via static or dynamic nasal
exposure through fitted nosepieces (separate airstreams
to each nostril); each subject was exposed twice by each
method over a 2-wk period
Exposure range
ppm (mg/m3)
Static 1.23 x 10-6-341.95
(0.87 x 10"6-241.76)
Dynamic 0.10-615.38
(0.07-435.07)
Outcome: Odor and irritation threshold
Reported thresholds (geometric mean) in ppm
(mg/m3)
Static Dynamic
Odor 2.56 (1.81) 2.62 (1.85)
Irritation 31.69 (22.4) 60.92 (43.07)
Values determined via static or dynamic methods
were not statistically significantly different
Ihrig et al. (2006)b (Germanv)
10 healthy male volunteers exposed to ammonia
regularly at the workplace (mean age 33 yrs) and 33
healthy male volunteers unfamiliar with the smell of
ammonia (naive; mean age 29 yrs)
Exposure: Volunteers were exposed to 0,10, 20, and
50 ppm (0, 7,14, and 35 mg/m3) for 4 hrs/d (d 1, 2, 3, and
5 of study, respectively) in an exposure chamber; on d 4,
volunteers were exposed to 20 ppm for 4 hrs with two
peak 30-min exposures at 40 ppm (14 + 28 mg/m3)
Outcome: Irritation effects during exposure (standardized
questionnaire with rating scale of 1-5); data shown
graphically
Mean intensity ratings for irritation in naive
volunteers (but not in workers) significantly increased
with increasing exposure level during exposure; mean
intensity ratings were <2 in all groups ("somewhat
irritative")
The increased ratings were driven primarily by
olfactory symptom; at 50 ppm, naive volunteers had
an average rating of between 3 ("rather much") and
4 ("considerably" irritative), compared with a rating
of ~2 in workers
Douglas and Coe (1987)c (England)
Unspecified number of volunteer subjects
Exposure: Volunteers were exposed to a concentration
series via tight fitting goggles (up to 15 sec) or
mouthpiece (10 inhaled breaths while wearing nose clip);
one concentration was tested per day; concentrations
used and duration of experiment were not specified
Outcome: Ocular and pulmonary irritation threshold
Reported thresholds in ppm (mg/m3)
Lachrymatory 55 (39)
Bronchoconstriction 85 (60)
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Supplemental Information—Ammonia
Study reference and design
Results
MacEwen et al. (1970)b (United States. Ohio)
6 male volunteers (mean age 31 yrs)
Exposure: Volunteers were exposed to 30 and 50 ppm
(21 or 35 mg/m3) for 10 min in a head-only inhalation
chamber
Outcome: Self-reported ocular and nasal irritation; odor
intensity
Mean rating
Exposure in ppm (mg/m3)
30 (21) 50 (35)
Eye/nasal irritation 0.4 1.5
(scale 0-4)
Odor intensity 3.6 4.0
(scale 0-5)
3Kalandarov et al. (1984), a study of the effect of ammonia on the adrenocortical system in 20 volunteer subjects,
was eliminated from further consideration because of concerns regarding ethical conduct of the study, including
the absence of information on ethical procedures followed and statement of informed consent by volunteers, and
lack of clarity about the reported exposures (reported as 17-37 days in a sealed chamber),
investigators reported the use of ethical standards involving informed consent by volunteers and/or study
approval by an Institutional Review Board or other ethics committee.
This controlled-exposure study did not provide information on the human subjects research ethics procedures
undertaken in the study; however, there is no evidence that the conduct of the research was fundamentally
unethical or significantly deficient relative to the ethical standards prevailing at the time the research was
conducted.
dFor the 25- and 50-ppm levels, locations were occupational work sites with reportedly stable ammonia levels; the
100 ppm location was a temporary exposure chamber. However, no monitoring data for ammonia
concentrations were presented.
DLCO = diffusion capacity of the lung for carbon monoxide; /C45 = exercise cardiac frequency at 45 mmol
02/minute; FIVi = forced inspiratory volume during 1 second;/R3o = mean respiratory frequency at a ventilation
volume of 30 L/minute; IL-6 = interleukin-6; IL-8 = interleukin-8; NO = nitric oxide; TLC = total lung capacity;
TV = tidal volume; VC = vital capacity; VE45 = ventilation minute volume at 45 mmol 02 /minute; Vt3o = exercise
tidal volume at ventilation volume of 30L/minute
Twelve healthy volunteers exposed to 4 and 18 mg/m3 ammonia on three different
occasions for 1.5 hours in an exposure chamber while exercising on a stationary bike reported
discomfort in the eyes and odor detection at 4 mg/m3 (Sundblad etal.. 20041. Eye irritation was
also shown to increase in a concentration-dependent manner in 16 volunteers exposed to ammonia
for 2 hours in an exposure chamber at concentrations of 50, 80,110, and 40 ppm (35, 57, 78, and
99 mg/m3); ammonia concentrations of 99 mg/m3 caused severe and intolerable irritation
(Verberk. 19771. The lachrymatory threshold was determined to be 39 mg/m3 in volunteers
exposed to ammonia gas inside tight-fitting goggles for an acute duration of up to 15 seconds
(Douglas and Coe. 19871. In contrast, exposures to up to 35 mg/m3 ammonia gas did not produce
severe lacrimation in seven volunteers after 10 minutes in an exposure chamber, although
increased eye erythema was reported (MacEwen etal.. 19701. Exposure to 354 mg/m3 of ammonia
gas for 30 minutes through a masked nose and throat inhalation apparatus resulted in two of seven
volunteers reporting lacrimation and two of seven reporting nose and throat irritation that lasted
up to 24 hours after exposure (Silverman et al.. 19491.
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Supplemental Information—Ammonia
Petrova et al. (2008) investigated irritation threshold differences between 25 healthy
volunteers and 15 mild-to-moderate persistent asthmatic volunteers exposed to ammonia via the
eyes and nose at concentrations of 1-354 mg/m3 for durations lasting up to 2.5 hours. Irritation
threshold, odor intensity, and annoyance were not significantly different between the two groups.
The nasal and eye irritation thresholds were reported to be 91 and 124 mg/m3, respectively.
Smeets etal. f20071 investigated odor and irritation thresholds for ammonia vapor in 24 healthy
female volunteers at concentrations of 0.02-435 mg/m3. This study found a mean odor detection
threshold of 2 mg/m3 and a mean irritation threshold of 22 or 43 mg/m3, depending on the
olfactometry methodology followed (static versus dynamic, respectively). Irritation thresholds may
be higher in people who have had prior experience with ammonia exposure flhrig et al.. 20061.
Thirty male volunteers who had not experienced the smell of ammonia and 10 male volunteers who
had regular workplace exposure to ammonia were exposed to ammonia vapors at concentrations of
0, 7,14, and 35 mg/m3 on 5 consecutive days (4 hours/day) in an exposure chamber; an additional
group was exposed to 14 mg/m3 plus two peak exposures to 28 mg/m3 for 30 minutes. Volunteers
in the group familiar with the smell of ammonia reported fewer symptoms than the nonhabituated
group, but at a concentration of 14 mg/m3, there were no differences in perceived symptoms
between the groups. However, the perceived intensity of symptoms was concentration-dependent
in both groups, but was only significant in the group of volunteers not familiar with ammonia
exposure flhrig et al.. 20061. Ferguson etal. f!9771 reported habituation to eye, nose, and throat
irritation in six male and female volunteers after 2-3 weeks of exposure to ammonia
concentrations of 18, 35, and 71 mg/m3 during a 6-week study (6 hours/day, 1 time/week).
Continuous exposure to even the highest concentration tested became easily tolerated with no
general health effects occurring after acclimation.
Several studies evaluated lung functions following acute inhalation exposure to ammonia.
Volunteers exposed to ammonia (lung only) through a mouthpiece for 10 inhaled breaths of gas
experienced bronchioconstriction at a concentration of 60 mg/m3 fDouglas and Coe. 19871:
however, there were no bronchial symptoms reported in seven volunteers exposed to ammonia at
concentrations of 21, 35, and 64 mg/m3 for 10 minutes in an exposure chamber (MacEwen etal..
1970). Similarly, 12 healthy volunteers exposed to ammonia on three separate occasions to 4 and
18 mg/m3 for 1.5 hours in an exposure chamber while exercising on a stationary bike did not have
changes in bronchial responsiveness, upper airway inflammation, exhaled nitric oxide levels, or
lung function as measured by vital capacity and FEVi fSundblad etal.. 2004). In another study,
18 healthy servicemen volunteers were placed in an exposure chamber for 3 consecutive half-day
sessions. Exposure to ammonia at concentrations of 50-344 mg/m3 occurred on the second
session, with sessions 1 and 3 acting as controls (Cole etal.. 1977). The no-effect concentration was
determined to be 71 mg/m3. Exercise tidal volume was increased at 106 mg/m3, but then
decreased at higher concentrations in a concentration-dependent manner fCole etal.. 19771.
Decreased FEVi and FVC were reported in eight healthy male volunteers exposed to a mean
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airborne ammonia concentration of 15 mg/m3 in swine confinement buildings for 4 hours at
I-week intervals; however, swine confinement buildings also include confounding exposures to
dust, bacteria, endotoxin, and molds, thereby making measurement of effects due to ammonia
uncertain in this study fCormier et al.. 20001.
Differences in lung function between healthy and asthmatic volunteers exposed to ammonia
were evaluated in several studies. There were no changes in lung function as measured by FEVi in
25 healthy volunteers and 15 mild/moderate persistent asthmatic volunteers after ocular and nasal
exposure to 1-354 mg/m3 ammonia at durations lasting up to 2.5 hours fPetrova etal.. 20081. In
another study, six healthy volunteers and eight mildly asthmatic volunteers were exposed to
II-14 mg/m3 ammonia, ammonia and dust, and dust alone for 30-minute sessions, with 1 week
between sessions fSigurdarson etal.. 20041. There were no significant changes in lung function as
measured by FEVi in the healthy volunteers for any exposure. A decrease in FEVi was reported in
asthmatics exposed to dust and ammonia, but not to ammonia alone; similarly, increased bronchial
hyperreactivity was reported in asthmatics after exposure to dust and ammonia, but not to
ammonia alone. Exposure to dust alone caused similar effects, suggesting that dust was responsible
for decreased lung function fSigurdarson etal.. 20041.
In summary, controlled human exposure studies demonstrate that eye irritation can occur
following acute exposure to ammonia at concentrations as low as 4 mg/m3. Irritation thresholds
may be higher in people who have had prior experience with ammonia exposure, and habituation to
eye, nose, and throat irritation occurs over time. Lung function was not affected in workers acutely
exposed to ammonia concentrations as high as 71 mg/m3. Studies comparing the lung function of
asthmatics and healthy volunteers exposed to ammonia do not suggest that asthmatics are more
sensitive to the lung effects of ammonia.
C.2.4. Case Reports of Human Exposure to Ammonia
Inhalation is the most frequently reported route of exposure and cause of morbidity and
fatality, and often occurs in conjunction with dermal and ocular exposures. Acute effects from
inhalation have been reported to range from mild to severe, with mild symptoms consisting of nasal
and throat irritation, sometimes with perceived tightness in the throat (Price and Watts. 2008:
Prudhomme etal.. 1998: Weiser and Mackenroth. 1989: Yang etal.. 1987: O'Kane. 1983: Ward etal..
1983: Caplin. 19411. Moderate effects are described as moderate to severe pharyngitis;
tachycardia; frothy, often blood-stained sputum; moderate dyspnea; rapid, shallow breathing;
cyanosis; some vomiting; transient bronchospasm; edema and some evidence of burns to the lips
and oral mucosa; and localized to general rhonchi in the lungs (Weiser and Mackenroth. 1989: Yang
etal.. 1987: O'Kane. 1983: Ward etal.. 1983: Couturier etal.. 1971: Caplin. 19411. Severe effects
include second- and third-degree burns to the nasal passages, soft palate, posterior pharyngeal
wall, and larynx; upper airway obstruction; loss of consciousness; bronchospasm; dyspnea;
persistent, productive cough; bilateral diffuse rales and rhonchi; production of large amounts of
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mucous; pulmonary edema; marked hypoxemia; local necrosis of the lung; deterioration of the
whole lung; and fatality. Delayed effects of acute exposure to high concentrations of ammonia
include bronchiectasis; bronchitis; bronchospasm/asthma; dyspnea upon exertion and chronic
productive cough; bronchiolitis; severe pulmonary insufficiency; and chronic obstructive
pulmonary disease fOrtiz-Puiols etal.. 2014: Lalic etal.. 2009: Leduc etal.. 1992: Bernstein and
Bernstein. 1989: Flurvetal.. 1983: Ward etal.. 1983: Stroud. 1981: Close etal.. 1980: Taplinetal..
1976: Walton. 1973: Kass etal.. 1972: Slot. 19381
Respiratory effects were also observed following chronic occupational exposure to
ammonia. After 18 months and 1 year on the job, respectively, two men developed cough, chest
tightness, and wheezing, typically after 2-6 hours from the beginning of each work day, but not on
weekends or holidays. In another case, progressive deterioration of the clinical condition of a
68-year-old male was documented for 4 years, and development of diffuse interstitial and severe
restrictive lung disease was reported following long-term repetitive occupational exposure to
ammonia at or above the odor recognition level of 3-50 ppm fBrautbar etal.. 20031. Lee et al.
(1993) reported a case of a 39-year-old man who developed occupational asthma 5 months after
beginning a job requiring the polishing of silverware. The room in which he worked was poorly
ventilated. The product used contained ammonia and isopropyl alcohol, and the measured
ammonia concentration in the breathing zone when using this product was found to be
6-11 mg/m3.
Acute dermal exposure to anhydrous (liquid) ammonia and ammonia vapor has resulted in
caustic burns of varying degrees to the skin and eyes. There are numerous reports of exposures
from direct contact with anhydrous ammonia in which first-, second-, and third-degree burns
occurred over as much as 50% of the total body surface (Lalic etal.. 2009: Pirjavec etal.. 2009:
Arwood etal.. 19851. Frostbite injury has also been reported in conjunction with exposure to
sudden decompression of liquefied ammonia, which is typically stored at -33°F (George etal..
2000: Sotiropoulos etal.. 1998: Arwood etal.. 19851. However, direct contact is not a prerequisite
for burn injury. Several reports have indicated that burns to the skin occurred with exposure to
ammonia gas or vapor. Kass etal. (19721 reported one woman with chemical burns to her
abdomen, left knee, and forearm and another with burns to the feet when exposed to anhydrous
ammonia gas released from a derailed train in the vicinity. Several victims at or near the scene of
an overturned truck that had been carrying 8,000 gallons of anhydrous ammonia were reported as
having second- and third-degree burns over exposed portions of the body f Burns etal.. 1985: Close
etal.. 1980: Hatton etal.. 19791. In a case involving a refrigeration leak in a poorly ventilated room,
workers located in an adjacent room reported a "burning skin" sensation fde la Hoz et al.. 19961.
while in another case involving the sudden release of ammonia from a pressure valve in a
refrigeration unit, one victim received burns to the leg and genitalia (O'Kane. 19831.
In addition to the skin, the eyes are particularly vulnerable to ammonia burns due to the
highly water-soluble nature of the chemical and the ready dissociation of ammonium hydroxide to
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release hydroxy! ions. When ammonia or ammonia in solution has been splashed or sprayed into
the face (accidently or intentionally), immediate effects include temporary blindness,
blepharospasm, conjunctivitis, corneal burns, ulceration, edema, chemosis, and loss of corneal
epithelium (George etal.. 2000: Helmers etal.. 1971: High man. 1969: McGuiness. 1969: Levy etal..
1964: Abramovicz. 19251. The long-term effects included photophobia, progressive loss of
sensation, formation of bilateral corneal opacities and cataracts, recurrent corneal ulcerations,
nonreactive pupil, and gradual loss of vision (Yang etal.. 1987: Kass etal.. 1972: Helmers etal..
1971: Highman. 1969: Osmond and Tallents. 1968: Levy etal.. 1964: Abramovicz. 19251. White et
al. (20071 reported a case with acute bilateral corneal injury that developed into bilateral uveitis
with stromal vascularization and stromal haze and scarring, and pigmented keratic precipitates
that resulted in legal blindness. An increase in intraocular pressure, resembling acute-angle closure
glaucoma, was reported by Highman T19691 following ammonia intentionally sprayed into the eyes
during robbery attempts.
C.3. ANIMAL STUDIES INVOLVING INHALATION EXPOSURE
Anderson et al. (1964)
Anderson et al. Q9641 exposed a group of 10 guinea pigs (strain not given) and 10 Swiss
albino mice of both sexes continuously to 20 ppm (14 mg/m3) ammonia vapors for up to 6 weeks
(anhydrous ammonia, purity not reported). Controls (number not specified) were maintained
under identical conditions except for the exposure to ammonia. An additional group of six guinea
pigs was exposed to 50 ppm (35 mg/m3) for 6 weeks. The animals were observed daily for
abnormal signs or lesions. At termination, the mice and guinea pigs were sacrificed (two per group
at 1, 2, 3, 4, and 6 weeks of exposure), and selected tissues (lungs, trachea, turbinates, liver, and
spleen) were examined for gross and microscopic pathological changes. No significant effects were
observed in animals exposed for up to 4 weeks, but exposure to 14 mg/m3 for 6 weeks caused
darkening, edema, congestion, and hemorrhage in the lung. Exposure of guinea pigs to 35 mg/m3
ammonia for 6 weeks caused grossly enlarged and congested spleens, congested livers and lungs,
and pulmonary edema.
Coon et al. (1970)
Coon etal. (19701 exposed groups of male and female Sprague-Dawley and Long-Evans rats,
male and female Princeton-derived guinea pigs, male New Zealand rabbits, male squirrel monkeys,
and purebred male beagle dogs to 0,155, or 770 mg/m3 ammonia for 8 hours/day, 5 days/week for
6 weeks (anhydrous ammonia, >99% pure). The investigators stated that a typical loaded chamber
contained 15 rats, 15 guinea pigs, 3 rabbits, 3 monkeys, and 2 dogs. Blood samples were taken
before and after the exposures for determination of hemoglobin concentration, packed erythrocyte
volume, and total leukocyte counts. Animals were routinely checked for clinical signs of toxicity. At
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termination, sections of the heart, lung, liver, kidney, and spleen were processed for microscopic
examination in approximately half of the surviving rats and guinea pigs and all of the surviving dogs
and monkeys. Sections of the brain, spinal cord, and adrenals from dogs and monkeys were also
retained, as were sections of the thyroid from the dogs. The nasal passages were not examined in
this study.
Exposure to 155 mg/m3 ammonia did not result in any deaths or adverse clinical signs of
toxicity in any of the animals. Hematological values were within normal limits for the laboratory
and there were no significant gross alterations in the organs examined. Microscopic examination
showed evidence of focal pneumonitis in the lung of one of three monkeys. Exposure to 770 mg/m3
caused initial mild to moderate lacrimation and dyspnea in rabbits and dogs. However, these
clinical signs disappeared by the second week of exposure. No significant alterations were
observed in hematology tests or upon gross or microscopic examinations at the highest dose.
However, consistent nonspecific inflammatory changes (not further described) that were more
extensive than in control animals (incidence not reported) were observed in the lungs from rats
and guinea pigs in the high-dose group.
Coon etal. (1970) also exposed rats (15-51/group) continuously to ammonia (anhydrous
ammonia, >99% pure) at 0, 40,127, 262, 455, or 470 mg/m3 for 90-114 days. Fifteen guinea pigs,
three rabbits, two dogs, and three monkeys were also exposed continuously under similar
conditions to ammonia at either 40 or 470 mg/m3. No significant effects were reported in any
animals exposed to 40 mg/m3 ammonia. Exposure of rats to 262 mg/m3 ammonia caused nasal
discharge in 25%; nonspecific circulatory and degenerative changes in the lungs and kidneys were
also demonstrated (not further described, incidence not reported), which the authors stated were
difficult to relate to ammonia inhalation. A frank effect level at 45 5 mg/m3 was observed due to
high mortality in the rats (50/51). Thirty-two of 51 rats died by day 25 of exposure; no
histopathological examinations were conducted in these rats. Exposure to 470 mg/m3 caused death
in 13/15 rats and 4/15 guinea pigs and marked eye irritation in dogs and rabbits. Dogs
experienced heavy lacrimation and nasal discharge, and corneal opacity was noted in rabbits.
Hematological values did not differ significantly from controls in animals exposed to 470 mg/m3
ammonia. Histopathological evaluation of animals exposed to 470 mg/m3 consistently showed
focal or diffuse interstitial pneumonitis in all animals and alterations in the kidneys (calcification
and proliferation of tubular epithelium), heart (myocardial fibrosis), and liver (fatty change) in
several animals of each species (incidence not reported). The study authors did not determine a
NOAEL or LOAEL concentration from this study. EPA identified a NOAEL of 262 mg/m3 and a
LOAEL of 455 mg/m3 based on nonspecific inflammatory changes in the lungs and kidneys in rats
exposed to ammonia for 90 days.
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Stombauah etal. (1969)
Stombaugh etal. (1969) exposed groups of Duroc pigs (9/group) to measured
concentrations of 12, 61,103, or 145 ppm ammonia (8, 43, 73, or 103 mg/m3) continuously for
5 weeks (anhydrous ammonia, purity not reported). Endpoints evaluated included clinical signs,
food consumption (measured 3 times/week), weight gain (measured weekly), and gross and
microscopic examination of the respiratory tract at termination. A control group was not included.
In general, exposure to ammonia reduced food consumption and body weight gain, but because a
control group was not used, it could not be determined whether this reduction was statistically
significant. Food efficiency (food consumed/kg body weight gain) was not affected. Exposure to
>73 mg/m3 ammonia appeared to cause excessive nasal, lacrimal, and mouth secretions and
increased the frequency of cough (incidence data for these effects were not reported). Examination
of the respiratory tract did not reveal any significant exposure-related alterations. The study
authors did not identify a NOAEL or LOAEL concentration from this study.
Doia and Willouahbv (1971)
Doig and Willoughbv (1971) exposed groups of six specific-pathogen-free derived Yorkshire
Landrace pigs to 0 or 100 ppm ammonia (0 or 71 mg/m3) continuously for up to 6 weeks. The
mean concentration of ammonia in the control chamber was 8 ppm (6 mg/m3). Additional groups
of pigs were exposed to similar levels of ammonia as well as to 0.3 mg/ft3 of ground corn dust to
simulate conditions on commercial farms. Pigs were monitored daily for clinical signs and changes
in behavior. Initial and terminal body weights were measured to determine body weight gain
during the exposure period. Blood samples were collected prior to the start of each experiment and
at study termination for hematology (packed cell volume, white blood cell, differential leukocyte
percentage, and total serum lactate dehydrogenase). Two pigs (one exposed and one control) were
necropsied at weekly intervals, and tracheal swabs for bacterial and fungal culture were taken.
Histological examination was conducted on tissue samples from the lung, trachea, and bronchial
lymph nodes.
During the first week of exposure, exposed pigs exhibited slight signs of conjunctival
irritation including photophobia and excessive lacrimation. These irritation effects were not
apparent beyond the first week. Measured air concentrations in the exposure chambers increased
to more than 150 ppm (106 mg/m3) on two occasions. Doig and Willoughby (1971) reported that,
at this concentration, the signs of conjunctival irritation were more pronounced in all pigs. No
adverse effects on body weight gain were apparent. Hematological parameters and gross pathology
were comparable between exposed and control pigs. Histopathology revealed epithelial thickening
in the trachea of exposed pigs and a corresponding decrease in the numbers of goblet cells (see
Table C-10). Tracheal thickening was characterized by thinning and irregularity of the ciliated
brush border and an increased number of cell layers. Changes in bronchi and bronchioles,
characterized as lymphocytic cuffing, were comparable between exposed and control pigs.
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Similarly, intraalveolar hemorrhage and lobular atelectasis were common findings in both exposed
and control pigs. Pigs exposed to both ammonia and dust exhibited similar reactions as those pigs
exposed only to ammonia, although initial signs of conjunctival irritation were more severe in these
pigs, and these pigs demonstrated lesions in the nasal epithelium similar to those observed in the
tracheal epithelium of pigs exposed only to ammonia.
Table C-10. Summary of histological changes observed in pigs exposed to
ammonia for 6 weeks
Duration of exposure
(wks)
Thickness of tracheal epithelium
(pm)
Number of tracheal goblet cells (per
500 pm)
Control
71 mg/m3 NH3
Control
71 mg/m3 NH3
1
15.7
21.0
13.6
24.0
2
20.4
29.3
22.7
10.3
3
20.4
36.6
18.9
7.3
4
21.8
36.2
18.3
10.7
5
19.3
33.2
20.2
10.0
6
18.9
41.6
20.0
1.3
Mean ± SD
19.4 ±2.1
32.9 ±7.2
18.9 ±3.0
10.6 ±7.5
Source: Doig and Willoughbv (1971).
Doig and Willoughbv (1971) concluded that ammonia exposure at 71 mg/m3 may be
detrimental to young pigs. The authors suggested that although the structural damage to the upper
respiratory epithelium was slight, such changes may cause severe functional impairment. The
study authors did not identify a NOAEL or LOAEL concentration from this study. EPA identified a
LOAEL of 71 mg/m3 based on damage to the upper respiratory epithelium. A NOAEL could not be
identified from this single-concentration study.
Broderson etal. (1976)
Broderson et al. f 19761 exposed groups of Sherman rats (5/sex/dose) continuously to 10 or
150 ppm ammonia (7 or 106 mg/m3, respectively) for 75 days (anhydrous ammonia, purity not
reported). The 7 mg/m3 exposure level represented the background ammonia concentration
resulting from cage bedding that was changed 3 times/week. The 106 mg/m3 concentration
resulted from cage bedding that was replaced occasionally, but never completely changed. F344
rats (6/sex/group) were exposed to ammonia in an inhalation chamber at concentrations of 0 or
250 ppm (177 mg/m3) continuously for 35 days. Rats were sacrificed atthe end of the exposure
period, and tissues were prepared for histopathological examination of nasal passages, middle ear,
trachea, lungs, liver, kidneys, adrenal, pancreas, testicle, mediastinal lymph nodes, and spleen.
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Histopathological changes were observed in the nasal passage of rats exposed to
106 mg/m3 for 75 days (from bedding) or 177 mg/m3 for 35 days (inhalation chamber). Nasal
lesions were most extensive in the anterior portions of the nose compared with posterior sections
of the nasal cavity. The respiratory and olfactory mucosa was similarly affected with a 3-4-fold
increase in the thickness of the epithelium. Pyknotic nuclei and eosinophilic cytoplasm were
observed in epithelial cells located along the basement membrane. Epithelial cell hyperplasia and
formation of glandular crypts were observed, and neutrophils were located in the epithelial layer,
the lumina of submucosal glands, and the nasal passages. Dilation of small blood vessels and edema
were observed in the submucosa of affected areas. Collagen replacement of submucosal glands and
the presence of lymphocytes and neutrophils were also observed. No histopathological alterations
were seen in control rats (7 mg/m3 from bedding or 0 mg/m3 from the inhalation chamber).
Broderson et al. f 19761 did not identify a NOAEL or LOAEL from this study. EPA identified a NOAEL
of 7 mg/m3 and a LOAEL of 106 mg/m3 based on nasal lesions in rats exposed to ammonia (from
bedding) for 75 days.
Gaafar etal. (1992)
Gaafar etal. (1992) exposed 50 adult male white albino mice under unspecified conditions
to ammonia vapor derived from a 12% ammonia solution (air concentrations were not reported)
for 15 minutes/day, 6 days/week for up to 8 weeks. Twenty-five additional mice served as
controls. Starting the fourth week, 10 exposed and 5 control mice were sacrificed weekly.
Following sacrifice, the nasal mucosa was removed and examined histologically. Frozen sections of
the nasal mucosa were subjected to histochemical analysis (succinic dehydrogenase, nonspecific
estrase, acid phosphatase, and alkaline phosphatase [ALP]). Histological examination revealed a
progression of changes in the nasal mucosa of exposed rats from the formation of crypts and
irregular cell arrangements at 4 and 5 weeks; epithelial hyperplasia, patches of squamous
metaplasia, and loss of cilia at 6 weeks; and dysplasia in the nasal epithelium at 7 weeks. Similar
changes were exaggerated in the nasal mucosa of rats sacrificed at 8 weeks. Neoplastic changes
included a carcinoma in situ in the nostril of one rat sacrificed at 7 weeks, and an invasive
adenocarcinoma in one rat sacrificed at 8 weeks. Histochemical results revealed changes in
succinic dehydrogenase, acid phosphatase, and ALP in exposed mice compared to controls
(magnitude of change not reported), especially in areas of the epithelium characterized by
dysplasia. Succinic dehydrogenase and acid phosphatase changes were largest in the superficial
layer of the epithelium, although the acid phosphatase reaction was stronger in the basal and
intermediate layers in areas of squamous metaplasia. The presence of ALP was greatest in the
goblet cells from the basal part of the epithelium and basement membrane.
In summary, Gaafar etal. (1992) observed that ammonia exposure induces histological
changes in the nasal mucosa of male mice that increase in severity over longer exposure periods.
Corresponding abnormalities in histochemistry suggest altered cell metabolism and energy
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production, cell injury, cell proliferation, and possible chronic inflammation and neoplastic
transformation. The study authors did not determine a NOAEL or LOAEL concentration from this
study. EPA did not identify a NOAEL or LOAEL because air concentrations were not reported in the
study.
Done etal. (2005)
Done etal. (2005) continuously exposed groups of 24 weaned pigs of several breeds in an
experimental facility to atmospheric ammonia at 0, 0.6,10,18.8, or 37 ppm (0, 0.4, 7,13.3, or
26 mg/m3) and 1.2, 2.7, 5.1, or 9.9 mg/m3 inhalable dust for 5 weeks (16 treatment combinations).
The concentrations of ammonia and dust used were representative of those found commercially. A
split-plot design was used in which one dust concentration was allocated to a "batch" (which
involved five lots of 24 pigs each) and the four ammonia concentrations were allocated to the four
lots within that batch. The fifth lot served as a control. Each batch was replicated.
2 x [4 dust concentrations x 4 ammonia concentrations + 4 controls] = 40 lots total
In total, 960 pigs (460 males and 500 females) were used in the study; 560 pigs were given
postmortem examinations. Blood was collected from 15 sows before the start of the experiment
and tested for porcine reproductive and respiratory syndrome virus and swine influenza. Five
sentinel pigs were sacrificed at the start of each batch, and lung, nasal cavity, and trachea, together
with material from any lesions, were examined postmortem and subjected to bacteriological
examination.
Postmortem examination involved examination of the pigs' external surfaces for condition
and abnormalities, examination of the abdomen for peritonitis and lymph node size, internal gross
examination of the stomach for abnormalities, and gross examination of the nasal turbinates,
thorax, larynx, trachea, tracheobronchial lymph nodes, and lung. Pigs were monitored for clinical
signs (daily), growth rate, feed consumption, and feed conversion efficiency (frequency of
observations not specified). After 37 days of exposure, eight pigs from each lot were sacrificed.
Swabs of the nasal cavity and trachea were taken immediately after death for microbiological
analysis, and the pigs were grossly examined postmortem. On day 42, the remaining pigs were
removed from the exposure facility and transferred to a naturally ventilated building for a recovery
period of 2 weeks. Six pigs from each lot were assessed for evidence of recovery and the remaining
10 pigs were sacrificed and examined postmortem.
The pigs in this study demonstrated signs of respiratory infection and disease common to
young pigs raised on a commercial farm (Done etal.. 2005). The different concentrations of
ammonia and dust did not have a significant effect on the pathological findings in pigs or on the
incidence of pathogens. In summary, exposure to ammonia and inhalable dust at concentrations
commonly found at pig farms was not associated with an increase in the incidence of respiratory or
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other disease. The study authors did not identify a NOAEL or LOAEL concentration from this study.
EPA identified a NOAEL of 26 mg/m3, based on the lack of respiratory or other disease following
exposure to ammonia in the presence of respirable dust.
Weatherbv (1952)
Weatherbv f 19521 exposed a group of 12 guinea pigs (strain not reported) to a target
concentration of 170 ppm (120 mg/m3) 6 hours/day, 5 days/week for up to 18 weeks (anhydrous
ammonia, purity not reported). The actual concentration measured in the exposure chamber
varied between 140 ppm (99 mg/m3) and 200 ppm (141 mg/m3). A control group of six guinea
pigs was exposed to room air. All animals were weighed weekly. Interim sacrifices were conducted
at intervals of 6 weeks (four exposed and two control guinea pigs), and the heart, lungs, liver,
stomach and small intestine, spleen, kidneys, and adrenal glands were removed for microscopic
examination; the upper respiratory tract was not examined.
No exposure-related effects were observed in guinea pigs sacrificed after 6 or 12 weeks of
exposure. However, guinea pigs exposed to ammonia for 18 weeks showed considerable
congestion of the spleen, liver, and kidneys, and early degenerative changes in the adrenal gland.
The most severe changes occurred in the spleen and the least severe changes occurred in the liver.
The spleen of exposed guinea pigs contained a large amount of hemosiderin, and kidney tubules
showed cloudy swelling with precipitated albumin in the lumens and some urinary casts
(cylindrical structures indicative of disease). The incidence of histopathological lesions was not
reported. EPA identified the ammonia concentration of 120 mg/m3 to be a LOAEL based on
congestion of the spleen, liver, and kidneys and early degenerative changes in the adrenal gland. A
NOAEL could not be identified in this single-concentration study.
Curtis et al. (1975)
Curtis etal. Q9751 exposed groups of crossbred pigs (4-8/group) to 0, 50, or 75 ppm
ammonia (0, 35, or 53 mg/m3) continuously for up to 109 days (anhydrous ammonia, >99.9%
pure). Endpoints evaluated included clinical signs and body weight gain. At termination, all pigs
were subjected to a complete gross examination, and representative tissues from the respiratory
tract, the eye and its associated structures, and the visceral organs (not specified) were taken for
subsequent microscopic examination. Weight gain was not significantly affected by exposure to
ammonia, and the results of the evaluations of tissues and organs were unremarkable. The
turbinates, trachea, and lungs of all pigs were classified as normal. The study authors did not
identify a NOAEL or LOAEL from this study. EPA identified a NOAEL of 53 mg/m3 based on the
absence of effects occurring in pigs exposed to ammonia; a LOAEL was not identified from this
study.
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C.3.1. Reproductive/Developmental Studies
Diekman etal. (1993)
Diekman et al. Q9931 reared 80 crossbred gilts (young female pigs) in a conventional
grower from 2 to 4.5 months of age; pigs were exposed naturally during that time to Mycoplasma
hyopneumoniae and Pasteurella multocida, which cause pneumonia and atrophic rhinitis,
respectively. At 4.5 months of age, the pigs were transferred to environmentally regulated rooms
where they were exposed continuously to a mean concentration of ammonia of 7 ppm (range,
4-12 ppm) (5 mg/m3; range, 3-8.5 mg/m3) or 35 ppm (range, 26-45 ppm) (25 mg/m3; range,
18-32 mg/m3) for 6 weeks f Diekman etal.. 19931. A control group was not included in this study.
The low concentration of ammonia was obtained by the flushing of manure pits weekly and the
higher concentration of ammonia was maintained by adding anhydrous ammonia (purity not
reported) to manure pits that were not flushed. After 6 weeks of exposure, 20 gilts from each group
were sacrificed, and sections of the lungs and snout were examined for gross lesions. In addition,
the ovaries, uterus, and adrenal glands were weighed. The remaining 20 gilts/group were mated
with mature boars and continued to be exposed to ammonia until gestation day 30, at which time
they were sacrificed. Fetuses were examined for viability, weight, and length, and the number of
corpora lutea were counted.
Gilts exposed to 25 mg/m3 ammonia gained less weight than gilts exposed to 5 mg/m3
during the first 2 weeks of exposure (7% decrease, p < 0.01), but growth rate recovered thereafter.
Mean scores for lesions in the lungs and snout were not statistically different between the two
exposure groups, and there were no differences in the weight of the ovaries, uterus, or adrenals.
Age at puberty did not differ significantly between the two groups, but gilts exposed to 25 mg/m3
ammonia weighed 7% less (p < 0.05) at puberty than those exposed to 5 mg/m3. In gilts that were
mated, conception rates were similar between the two groups (94.1 versus 100% in low versus
high exposure, respectively). At sacrifice on day 30 of gestation, body weights were not
significantly different between the two groups. In addition, there were no significant differences
between the two groups regarding percentage of lung tissue with lesions and mean snout grade.
Number of corpora lutea, number of live fetuses, and weight and length of the fetuses on day 30 of
gestation were not significantly different between treatment groups. Diekman etal. (1993) did not
identify NOAEL or LOAEL concentrations for maternal or fetal effects in this study. EPA did not
identify NOAEL or LOAEL values from this study due to the absence of a no-ammonia control group
and due to confounding exposures to bacterial and mycoplasm pathogens.
C.3.2. Acute and Short-term Inhalation Toxicity Studies
Table C-ll provides information on animal studies of acute and short-term inhalation
exposure to ammonia.
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Table C-ll. Acute and short-term inhalation toxicity studies of ammonia in animals
Animal
Ammonia
concentration
(mg/m3)
Duration
Parameters
examined
Results
Reference
Rats
Female Porton rats
(16/group)
0 or 141
Continuous
exposure for 4,
8, or 12 d
Histology of the trachea
4 d: transitional-stratified appearance of the
epithelium
8 d: gross change with disappearance of cilia
and stratification on luminal surface
12 d: increased epithelial thickness
Gamble and Clough
(1976)
Male OFA rats
(27/group)
0 or 354
Continuous
exposure for
1-8 wks
Body weight, organ
weights, airway structure,
cell population, alveolar
macrophages
No deaths occurred; decreased food
consumption and body weight gain; increased
lung and kidney weights; at 3 wks, nasal
irritation and upper respiratory tract
inflammation, but no effect on lower airways;
slight decrease in alveolar macrophages; no
histopathological effects seen at 8 wks,
suggesting adaptation to exposure
Richard et al. (1978a)
Male and female
Wistar rats
(5/sex/group)
9,898-37,825; no
mention of control
group
10, 20, 40, or
60 min
Clinical signs, pathology,
LCso
Eye irritation, eye and nasal discharge,
dyspnea; hemorrhagic lungs on necropsy;
10-min LC50 = 28,492 mg/m3
20-min LC50 = 20,217 mg/m3
40-min LC50 = 14,352 mg/m3
60-min LC50 = 11,736 mg/m3
Aooelman et al.
(1982)
Male CrkCOBS CD
Sprague-Dawley
rats (8/group)
11, 23, 219, and 818;
arterial blood
collected prior to
exposure served as
control
24 hrs
Clinical signs, histology,
blood pH, blood gas
measurement
No clinical signs of toxicity, no histologic
differences in tracheal or lung sections, no
change in blood pH or pCC>2, minor changes in
pCh
Schaerdel et al.
(1983)
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Animal
Ammonia
concentration
(mg/m3)
Duration
Parameters
examined
Results
Reference
Male CrhCOBS CD
Sprague-Dawley
rats (14/group)
3,17, 31,117, and
505; arterial blood
collected prior to
exposure served as
control
3 and 7 d
Hepatic cytochrome P450
content and
ethylmorphine-
N-demethylase activity
No dose-related change in P450 content or
enzyme activity
Schaerdel et al.
(1983)
Male Long-Evans
rats (4/group)
70 and 212; results
were compared to
"control," but it was
not clear if the
authors were
referring to historical
or concurrent
controls
6 hrs
Clinical signs, behavioral
observation
Decreased running; decreased activity
Tepper et al. (1985)
Female Wistar rats
(5/group)
0,18, or 212
6 hrs/d for 5,
10, or 15 d
Blood ammonia, urea,
glutamine, and pH; brain
ammonia, glutamine;
histopathology of lungs,
heart, liver, and kidneys
(light and electron
microscopy)
Brain and blood glutamine increased; slight
acidosis (i.e., decreased blood pH) at
212 mg/m3; lung hemorrhage observed in
some exposed rats
Manninen et al.
(1988)
Female Wistar rats
(5/group)
0,18, or 212
6 hrs/d for 5 d
Plasma and brain
ammonia and amino acid
analysis
Increase in brain and plasma glutamine
concentrations; increased brain/plasma ratio
of threonine
Manninen and
Savolainen (1989)
Female albino rats
(8/group)
0, 848-1,068
3 hrs
Mortality, respiratory
movement, and O2
consumption
No deaths reported; inhibition of external
respiration and decreased O2 consumption
Reiniuk et al. (2007)
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Supplemental Information—Ammonia
Animal
Ammonia
concentration
(mg/m3)
Duration
Parameters
examined
Results
Reference
Male Sprague-
Dawley rats
(number/group not
given)
Air concentration not
given; ammonia
vapor added to
inspiratory line of
ventilator; controls
exposed to same
volume of room air
20 sec
Activity of upper thoracic
spinal neurons
Lower airway irritation, activation of vagal
pulmonary afferents and upper thoracic spinal
neurons receiving pulmonary sympathetic
input
Qin et al. (2007a);
Qin et al. (2007b)
Male rats
(10/group)
0, 848-1,068 at the
beginning and end of
the exposure period
3 hrs
Oxygen consumption
Decreased O2 consumption
Reiniuk et al. (2008)
Male Wistar rats
(4/group)
0, 92-1,243; the
preexposure period
was used as the
control for each
animal
45 min
Airway reflexes by the
changes in respiratory
patterns elicited by
ammonia in either dry,
steam-humidified, or
aqueous aerosol-
containing atmospheres
Ammonia-induced upper respiratory tract
sensory irritation is not affected to any
appreciable extent by wet atmospheres (with
or without aerosol) up to 1,243 mg/m3
Li and Pauluhn (2010)
Mice
Mice (20/group,
species, sex not
specified)
6,080-7,070; no
controls
10 min
LCso
LC50 = 7,056 mg/m3
Silver and McGrath
(1948)
Male Swiss albino
mice (4/group)
5,050-20,199; no
controls
30-120 min
LC50
LC50 (30 min) = 15,151 mg/m3
Hilado et al. (1977)
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Animal
Ammonia
concentration
(mg/m3)
Duration
Parameters
examined
Results
Reference
Albino mice (sex
not specified;
6/dose)
Air concentration not
measured; results
were compared to
"control," but it was
not clear if the
authors were
referring to historical
or concurrent
controls
Continuously
for 2 or 5 d
Regional brain
metabolism (cerebral
cortex, cerebellum,
brainstem); monoamine
oxidase, enzymes of
glutamate and gamma-
aminobutyric acid (GABA)
metabolism, and (Na+-K+)-
ATPase; amino acid levels
in the brain
Altered activities of monoamine oxidase,
glutamate decarboxylase, ALT, GABA-
transaminase, and (Na+-K+)-ATPase; increased
alanine and decreased glutamate
Sadasivudu et al.
(1979); Sadasivudu
and Radha Krishna
Murthv (1978)
Male Swiss-
Webster mice
(4/group)
Concentrations not
given; baseline levels
established prior to
exposure
10 min
Reflex decrease in
respiratory rate was used
as an index of sensory
irritation; RDso = the
concentration associated
with a 50% decrease in
the respiratory rate
RDso = 214 mg/m3
Kane et al. (1979)
Male albino ICR
mice (12/dose)
0-3,436
1 hr(14-d
follow-up)
Clinical signs, body
weight, organ weight,
histopathology, LCso
Eye and nose irritation, dyspnea, ataxia,
seizures, coma, and death; decreased body
weight and increased liver to body weight
ratio in mice surviving to 14 d; effects in the
lung included focal pneumonitis, atelectasis,
and intralveolar hemorrhage; liver effects
included hepatocellular swelling and necrosis,
vascular congestion; LC50 = 2,990 mg/m3
Kaoeghian et al.
(1982)
Male Swiss-
Webster mice
(16-24/group)
0 or 216
6 hrs/d for 5 d
Respiratory tract
histopathology
Lesions in the nasal respiratory epithelium
(moderate inflammation, minimal necrosis,
exfoliation, erosion, or ulceration); no lesions
in trachea or lungs
Bucklev et al. (1984)
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Animal
Ammonia
concentration
(mg/m3)
Duration
Parameters
examined
Results
Reference
Male albino ICR
mice (12/dose)
0, 954, 3,097, or
3,323
4 hrs
Hexobarbital sleeping
time, microsomal protein
content, liver microsomal
enzyme activity
Increased hexobarbital sleeping time
(3,097 mg/m3), increased microsomal protein
content and aminopyrene-N-deethylase and
aniline hydroxylase activities (3,323 mg/m3)
Kaoeghian et al.
(1985)
Male albino ICR
mice (12/dose)
0, 81, or 233
4 hrs/d for 4 d
Microsomal protein
content, liver microsomal
enzyme activity
No dose-dependent effects on microsomal
enzymes
Kaoeghian et al.
(1985)
Male Swiss mice
(6/dose)
71 and 212; data
collected during the
2 d separating each
ammonia exposure
served as the control
baseline
6 hrs
Clinical signs, behavioral
observation
Decreased running, decreased activity
Teooer et al. (1985)
Mice (sex not
specified; 4/group)
3, 21, 40, or 78,
lowest measured
concentration was
the nominal control
group
2d
Responses to
atmospheric ammonia in
an environmental
preference chamber with
four chambers of
different concentrations
of ammonia
No distinguishable preference for, or aversion
to, different ammonia concentrations
Green et al. (2008)
Male 0F1 mice
(4/group)
0, 92-1,243; the
preexposure period
was used as the
control for each
animal
45 min
Airway reflexes by the
changes in respiratory
patterns elicited by
ammonia in either dry,
steam-humidified, or
aqueous aerosol
containing atmospheres
Ammonia-induced upper respiratory tract
sensory irritation is not affected to any
appreciable extent by wet atmospheres (with
or without aerosol) up to 1,243 mg/m3
Li and Pauluhn (2010)
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Animal
Ammonia
concentration
(mg/m3)
Duration
Parameters
examined
Results
Reference
Rabbits
Female New
Zealand White
rabbits (7-9/dose)
0, 35, or 71
2.5-3.0 hrs
Lung function
Decreased respiratory rate at both
concentrations
Mavan and Merilan
(1972)
Rabbits (species,
sex, number/dose
not specified)
0, 707-14,140
15-180 min
Lung function, death
Bradycardia at 1,768 mg/m3; arterial pressure
variations and blood gas modifications
(acidosis indicated by decreased pH and
increased pCCh) at 3,535 mg/m3; death
occurred at 4,242 mg/m3
Richard et al. (1978b)
New Zealand White
rabbits (sex not
specified; 16 total;
8/dose)
Peak concentrations:
24,745-27,573;
concurrent controls
tested
4 min
Lung function, heart rate,
blood pressure, blood
gases
Lung injury was evident after 2-3 min
(decreased pC>2 increased airway pressure)
Sioblom et al. (1999)
Cats
Mixed breed stray
cats (sex not
specified; 5/group)
0 or 707
10 min
Lung function, lung
histopathology on 1, 7,
21, and 35 d
postexposure
Lung function deficits were correlated with
lung histopathology; acute effects were
followed by chronic respiratory dysfunction
(secondary bronchitis, bronchiolitis, and
bronchopneumonia)
Dodd and Gross
(1980)
Pigs
Young pigs (sex not
specified; 2/group)
0, 35, 71, or 106
Continuous
exposure for
4 wks
Clinical signs, food
consumption, body
weight, gross necropsy,
organ weight,
histopathology
Lethargy and histopathological alterations in
the tracheal and nasal epithelium were
observed at 71 and 106 mg/m3; decreased
body weight occurred at all concentrations
(7-19% decrease from control)
Drummond et al.
(1980)
Male and female
Belgian Landrace
pigs (4/group)
0, 18, 35, or 71
6 d
Clinical signs, body
weight, lung function
Lethargy and decreased body weight gain (all
concentrations); no effect on lung
microvascular hemodynamics or permeability
Gustin et al. (1994)
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Animal
Ammonia
concentration
(mg/m3)
Duration
Parameters
examined
Results
Reference
Belgian Landrace
pigs (sex not
specified; 4/group)
0, 18, 35, or 71
6 d
Clinical signs, body
weight, neutrophil count,
and albumin in nasal
lavage fluid
Nasal irritation (increased neutrophils in nasal
lavage fluid) and decreased body weight gain
at all concentrations
Urbain et al. (1994)
Landrace-Yorkshire
pigs (sex not
specified; 4/group)
0 or 42
15 min/d for
8 wks
Thromboxane A2 (TXA2),
leukotriene C4 (LTC4),
and prostaglandin (PGI2)
production
Significant increases in TXA2 and LTC4; no
significant effect on PGI2 production
Chaung et al. (2008)
Hybrid gilts (White
synthetic Pietrain,
white Duroc,
Landrace, Large
White)
(14 pigs/group)
<4 (control) or 14
15 wks
Salivary Cortisol, adrenal
morphometry, body
weight, food conversion
efficiency, general health
scores, play behavior;
reaction to light and noise
intensity tested
concurrently
Decreased salivary Cortisol; larger adrenal
cortices; less play behavior; no measurable
impact on productivity or physiological
parameters
O'Connor et al.
(2010)
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C.4. ESTIMATING THE MEAN EXPOSURE CONCENTRATION IN THE HIGH-
EXPOSURE GROUP
To estimate the mean exposure concentration in the high-exposure group, the exposure
concentration was assumed to follow the lognormal distribution. This assumption is reasonable
given the typically skewed nature of chemical exposures. The frequency distribution provided in
Holness etal. f 19891 was used to estimate the parameters (log-scale mean and standard deviation)
of the lognormal distribution that best fit the data. This frequency distribution is provided in
Table C-12.
Table C-12. Frequency distribution of ammonia exposure from Holness et al.
fl989)
Exposure group
Interval of exposures
(mg/m3)
Interval of exposures
(ppm)
Number of exposed
workers
Low
0-4.4
0-6.25
34
Medium
4.4-8.8
6.25-12.5
12
High3
8.8-17.7
12.5-25
9
>17.7
>25
3
aEPA divided the high-exposure group into two subgroups based on the statement in Holness et al. (1989): "Three
workers were exposed to TWA concentrations of ammonia in excess of 25 ppm, the current exposure guideline."
The lognormal parameter estimates were obtained by applying the maximum likelihood
method to this frequency distribution. Using the estimated distribution defined by these parameter
estimates, the estimated mean exposure in the high-exposure group and 95% lower confidence
bound on this mean were calculated:
mean exposure estimate = 17.9 mg/m3
95% lower confidence bound = 13.6 mg/m3
Using Pearson's chi-square goodness-of-fit test, the fit of the estimated lognormal
distribution to the frequency distribution was determined to be plausible (p-value = 0.49). Details
on the estimation methods and goodness-of-fit test are provided in the remainder of this section.
All calculations were done in R, version 3.1.2; for the code used, please see U.S. EPA (20161.
Documentation of the estimation of the mean ammonia concentration in high-exposure group
from Holness et al (1989)
Assuming that the data are lognormal, the log-scale mean [i and standard deviation a were
estimated using the frequency distribution in Table C-12 and the mean exposure was subsequently
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estimated. For ease of calculation, the distribution was parametrized using a = — \i/o and b =
1 /a, and the likelihood function was written in terms of a and b. Generalizing the data grouping
into four intervals with nonrandom interval limits, it was assumed that tt, t2, t3, and t4 represent
the number of workers in the low and medium exposure groups and the two high (8.8-17.7 and
>17.7 mg/m3) exposure groups, respectively. The log-likelihood function of a and b is given by:
4
Ł(a, b; t) = I tj log [<5(a + b ¦ log Xj) - <5(a + b ¦ log x^)],
i=i
where x0 = 0, x4 = oo, and O is the cumulative distribution function (CDF) of the standard normal
distribution. The log-likelihood was maximized by finding the roots of its first derivatives with
respect to a and b, using the function 'nleqslv' in R ('nleqslv' package). The resulting parameter
estimates were a = —1.23, b = 0.970, and thus, the log-scale mean and standard deviation of the
estimated lognormal distribution were fl = —a/b = 1.27, a = 1/b = 1.03. Using these parameter
estimates, the mean of the high exposure group is calculated from the following formula (from
p. 241 of lohnson etal. T1994I with r = 1 to represent the 1st moment).
^ / 8.8) = exp (/2 + —J t _ = 17.9 mg/m3,
where y ISittH.
u a
To test the adequacy of the estimated lognormal distribution as a model of the frequency
data from Holness et al. f!989I a Pearson's chi-square goodness-of-fit test was conducted. Here,
the observed frequencies were set equal to the interval frequencies listed in Table C-12, and the
expected frequencies were calculated under the lognormal assumption with log-scale mean fl and
standard deviation a. The observed and expected frequencies are listed in Table C-13.
Table C-13. Observed and expected frequencies of ammonia exposure from
Holness etal. (1989)
Exposure group
Interval of exposures
(mg/m3)
Observed frequencies
Expected frequencies
Low
0-4.4
34
33.8
Medium
4.4-8.8
12
13.2
High3
8.8-17.7
9
7.5
>17.7
3
3.5
aEPA divided the high-exposure group into two subgroups based on the statement in Holness et al. (1989): "Three
workers were exposed to TWA concentrations of ammonia in excess of 25 ppm, the current exposure guideline."
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The results of the test were
xl = 0.467, p-value = 0.49,
where the degrees of freedom of the test statistic were equal to (number of intervals - number of
estimated parameters estimated - 1) = 1. Because the p-value of the test was >0.05, the lognormal
fit was determined not to be inadequate for this dataset It should be noted that because of the low
degrees of freedom, the power of this test is very low.
Figure C-3 presents a histogram of the data in Table C-12 with the superimposed estimated
lognormal density.
o
CM —
0 5 10 15 20 25 30 35
Exposure
Figure C-3. Histogram of Holness etal. (1989) doses.
To obtain a 95% lower confidence bound on the mean of the high exposure group,
10,000 bootstrap samples were randomly selected from the lognormal distribution with log-scale
mean and standard deviation equal to (/Ł, a) = (1.27,1.03) from the original sample. The estimated
mean exposure in the high-exposure group was calculated for each bootstrap sample using the
same method as for the original sample. Specifically, each bootstrap sample was grouped into the
four exposure intervals listed in Table C-12, the maximum likelihood estimates (MLEs) of the log-
scale mean and standard deviation were calculated based on the group frequencies, and the
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estimated mean exposure in the high exposure group was calculated based on these MLEs using the
mean formula presented above. The 95% lower confidence bound was set equal to the 5th
percentile of the 10,000 high-exposure group mean estimates:
95% lower confidence bound = 13.6 mg/m3
As expected, a histogram of these means revealed high skewness, with 28 means ranging
from 50 to 181 mg/m3 and the remaining means less than 50 mg/ m3. Figure C-4 is a histogram of
the estimated mean exposures from the bootstrap samples. To alleviate the bunching of data points
on the low end, the 28 means that exceeded 50 mg/m3 were omitted from the histogram.
o
o
no
CM
o
o
o
CM
Hi 20 30 40 50
Mean exposure
Figure C-4. Histogram of mean exposures in high-exposure group (Holness et
al.. 19891.
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APPENDIX D. SUMMARY OF SAB PEER REVIEW
COMMENTS AND EPA's DISPOSITION
The draft Toxicological Review of Ammonia, dated August 2013, underwent a formal
external peer review in accordance with Environmental Protection Agency (EPA) guidance on peer
review (U.S. EPA. 20061. This peer review was conducted by the Chemical Assessment Advisory
Committee (CAAC) Augmented for the Integrated Risk Information System (IRIS) Ammonia
Assessment (CAAC Ammonia panel) of EPA's Science Advisory Board (SAB). An external peer
review workshop was held on July 14-16, 2014. Public teleconferences of the CAAC Ammonia
panel were held on December 17 and 19, 2014, to discuss the Panel's draft review report The SAB
held a public teleconference on June 8, 2015 to conduct a quality review of the draft peer review
report. The final report of the SAB was released in August 2015 (U.S. EPA. 2015).
The SAB was tasked with providing feedback in response to charge questions related to the
hazard identification and dose-response assessment of ammonia, as well as EPA's implementation
of recommendations of the National Research Council (NRC) for improving the development of IRIS
assessments. A summary of the SAB's major recommendations, and EPA's responses to these
recommendations, follows and is organized by charge question. In addition, the SAB offered
editorial suggestions to improve the clarity of specific portions of the text; changes in response to
these editorial suggestions were incorporated in the Toxicological Review as appropriate and are
not included below in the summary of major SAB recommendations.
The SAB generally commended EPA for progress in implementing NRC's recommendations
and the new document structure for IRIS toxicological reviews. The SAB concurred with the
selection of the study used to derive the inhalation reference concentration (RfC), with respiratory
effects as the critical effect, and with the application of uncertainty factors (UFs), but offered
recommendations related to the identification of the point of departure (POD) for the RfC. Changes
to the POD resulted in an increase in the RfC from 0.3 to 0.5 mg/m3 (see Charge Questions E2 and
E3). In response to SAB recommendations on the evaluation of the toxicity of ingested ammonia,
the scope of this assessment was revised to contain evaluation of the toxicity of inhaled ammonia
only. An evaluation of ammonia's oral toxicity will be conducted as a separate assessment in order
to expand the evaluation to include a systematic review of the ammonium salts literature (see
Charge Question Dl).
Charge Question 1: NRC f20111 indicated that the introductory section of IRIS assessments
needed to be expanded to describe more fully the methods of the assessment. NRC stated
that they were "not recommending the addition of long descriptions of EPA guidelines to the
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introduction, but rather clear, concise statements of criteria used to exclude, include, and
advance studies for derivation of [toxicity values]." Please comment on whether the new
Preamble provides a clear, concise, useful and objective description of the guidance and
methods that EPA uses in developing IRIS assessments.
Comment: The SAB commended EPA for the progress made thus far in implementing the NRC's
recommendations for the IRIS Program. The Panel observed that the Preamble is a "work in
progress" that goes a long way to providing a clear, concise, useful, and objective summary of the
complex set of guidance and methods that EPA uses in developing IRIS assessments. The SAB
recommended that the Preamble should make clear that it does not establish new policy and that it
is generic and some elements are not necessarily applicable to the ammonia assessment Other
specific recommendations for this assessment related to the Preamble included the following:
• Section 6 (Selection of studies for derivation of toxicity values) would benefit from
elaboration and the addition of citations to relevant EPA guidance documents. EPA should
clarify how the factors used to select the studies for the derivation of toxicity values are
balanced against each other or against other factors not listed.
• EPA should confirm that all relevant guidance documents are included.
• EPA should describe the process for peer reviewing articles not previously peer reviewed.
• EPA should clarify which "ethical standards" are considered (page xvi, lines 3-5).
• EPA should consider whether assessments should provide ranges for typical levels of
exposure or intake for comparison to estimated doses or concentrations.
• The statement on page xx, line 26-30 needs to be revised such that the scientific quality of
studies is foremost in assessing credibility.
• The role of NRC (2014) and NRC (2011) in the IRIS protocol development process should be
mentioned.
Response: The IRIS program has substantially revised the Preamble based on: (1) experience with
implementing the new document structure and systematic review procedures after the ammonia
assessment was submitted for SAB review in 2013; (2) recommendations from SAB reports on the
draft assessments for ammonia and trimethylbenzenes; and (3) comments from EPA's program and
regional offices, other federal agencies and the Executive Office of the President, and the public.
The revised Preamble reflects recommendations for a shorter section, and some
information previously in the Preamble is now discussed in the Toxicological Review
(e.g., literature searching, screening, and study evaluation) or in the upcoming IRIS Handbook of
Operating Procedures for Systematic Review of Environmental Health Hazards ("IRIS Handbook")
being developed by the IRIS Program. The Preamble begins with a new statement that it
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summarizes general principles and systematic review procedures. Section 1 now states that"[t]his
Preamble summarizes and does not change... EPA guidance," addressing the SAB recommendation
that EPA make clear that the Preamble does not establish new policy. In place of summaries of
specific citations to EPA guidance documents, the Preamble directs users to links to relevant
guidance documents on the IRIS Program website. In response to the SAB recommendation that
the Preamble clarify that it is generic and that some elements are not necessarily applicable to the
ammonia assessment, Section 9 of the Preamble states that"[t]he Preface also identifies
assessment-specific approaches that may differ from the general approaches outline in this
Preamble." New text in the Preface of the ammonia assessment describes features of the
assessment that differ from those outlined in the Preamble. Finally, with a shorter, refocused
Preamble, some of the text that was the subject of specific SAB recommendations no longer appears
in the Preamble. Several of the specific SAB recommendations, including identification of relevant
EPA guidance documents, reference to implementation of NRC recommendations, and extensive
consideration of study quality as part of IRIS procedures for systematic review, are addressed in the
upcoming IRIS Handbook.
Charge Question 2: NRC (20111 provided comments on ways to improve the presentation of
steps used to generate IRIS assessments and indicated key outcomes at each step, including
systematic review of evidence, hazard identification, and dose-response assessment. Please
comment on the new IRIS document structure and whether it will increase the ability for the
assessments to be more clear, concise, and easy to follow.
The SAB observed that the new format used for the ammonia assessment is a refreshing
improvement over the old format, and evidence of EPA's commitment to a stepwise
implementation of the NRC's recommendations for systematic review. The SAB further observed
that the ammonia assessment has not fully implemented the systematic review envisioned by the
NRC, but that the NRC/Institute of Medicine (IOM) approach is not a directive and is expected to
need modification to address issues that EPA faces as implementation progresses. The SAB
anticipated that refinements would be forthcoming in future assessments. Specific
recommendations offered by the SAB related to the ammonia assessment are summarized below.
Comment: A clearer statement of how the main text reviews are intended to be different from the
appendix summaries should be provided.
Response: A statement describing how the synthesis of health effects information in the main text
relates to the study summaries provided in appendices in the Supplemental Information was added
to the beginning of Section 1.2, Synthesis of Evidence.
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Comment: The SAB observed that the bulk of study descriptions was presented in appendix
summaries, and that it was cumbersome to refer back and forth between the main text and
Supplemental Information when looking for specific details. The SAB suggested that hyperlinks
between the main text and Supplemental Information be added to facilitate referring between the
two documents.
Response: Ammonia health effect studies that appear in the Supplemental Information were
developed as part of the draft assessment that used the "old" toxicological review format, i.e., the
format used by the IRIS Program before implementing the NRC recommendations for improving the
structure of the toxicological review. Based on experience with the new document structure after
the ammonia assessment was released for peer review, separate study summaries will not be
included in the Supplemental Information in the future. For historical reasons, the EPA retained
previously developed study summaries, without hyperlinks, for this assessment only.
Comment: A more detailed description and evaluation of the principal study, Holness etal. (1989).
should be provided in the main assessment.
Response: The description of the Holness etal. (1989) study was expanded as described further in
response to recommendations under Charge Question CI.
Comment: EPA should continue to work on efficiently summarizing and presenting data through
tables and figures. It would be helpful to indicate study quality in the tables and figures, or present
only studies that met clearly stated minimal criteria. By way of example, the SAB recommended
that EPA tag the Anderson et al. f 19641 study in Figure 1-1 as a weak study or omit the study from
the figure.
Response: EPA is continuing to work on efficiently and transparently summarizing health effects
evidence in tables and graphs; these changes will be reflected in future IRIS assessments. These
changes include increased use of graphics to summarize health effect data and results of the
systematic review of study evaluation for epidemiology studies. EPA is also exploring alternative
approaches for documenting study quality, including the addition of study quality information to
evidence tables. EPA notes that some methodologic features relevant to study quality (e.g., number
of exposure groups, group sizes) are summarized in the current ammonia evidence tables.
The evaluation of animal toxicity studies of ammonia was revised to provide a more explicit
framework by which individual studies were evaluated, including considerations related to test
animals, experimental design, exposure characterization, endpoint evaluation, and results
presentation (see Literature Search Strategy | Study Selection and Evaluation). Text documenting
the outcome of this evaluation was added, including discussion of the limitations of the Anderson et
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al. (1964) study. The representation of this study in the evidence tables was revised to more
accurately reflect the number of animals used. Anderson et al. (1964) was retained in the evidence
tables and in the exposure-response array, but was given less weight in the synthesis of evidence,
along with other studies with similar limitations.
Comment: Consideration should be given to moving appropriate kinetic or absorption/
distribution/metabolism/elimination (ADME) information into the main text from the appendices if
it is used in selection and weighing of studies, RfC/reference dose (RfD) derivation, or other key
steps in the assessment.
Response: A new section (Section 1.1, Overview of Chemical Properties and Toxicokinetics) moves
important information from the Supplemental Information document to the main document In
addition, an overview of key toxicokinetic information that provides useful context for evaluating
the health effects of ammonia was provided in this new section. More detailed information on
ammonia toxicokinetics was retained in the Supplemental Information.
Charge Question 3: NRC f20111 states that "all critical studies need to be thoroughly
evaluated with standardized approaches that are clearly formulated" and that
"strengthened, more integrative and more transparent discussions of weight of evidence are
needed." NRC also indicated that the changes suggested would involve a multiyear process.
Please comment on EPA's success thus far in implementing these recommendations.
Comment: The SAB observed that the ammonia assessment is "an excellent first step" in addressing
NRC's recommendations, although there is "still terrain to cover." The NRC recommended that a
standardized approach be adopted to provide more transparency and clarity for future
assessments.
Response: The NRC anticipated that implementing their recommendations would be a multiyear
process. EPA is continuing to make progress in fully implementing systematic review methods in
new IRIS assessments that are in the problem formulation or early draft development steps. This
includes the consistent application of study exclusion/inclusion criteria, methods to systematically
evaluate study quality, and transparent integration of evidence. Assessments further along in the
IRIS process, such as the ammonia assessment, incorporated elements of systematic review
methods, as well as other improvements such as streamlining the document structure and
increased incorporation of tables, figures, and exposure-response arrays. Future assessments will
reflect greater implementation of systematic review.
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Charge Question 4: EPA solicited public comments on the draft IRIS assessment of ammonia
and has revised the assessment to respond to the scientific issues raised in the comments. A
summary of the public comments and EPA's responses are provided in Appendix G of the
Supplemental Information to the Toxicological Review of Ammonia. Please consider in your
review whether there are scientific issues that were raised by the public as described in
Appendix G that may not have been adequately addressed by EPA.
The SAB noted that, in general, EPA adequately and appropriately addressed the scientific
issues raised by public commenters, and provided adequate scientific justification for the Agency's
conclusions. Specific public comments that the SAB considered deserved further attention are
summarized below.
Comment: EPA should attempt to obtain data from Dr. Holness in order to determine a
representative exposure concentration from the no-observed-adverse-effect level (NOAEL) study
group, and then elaborate their response to this recommendation.
Response: EPA contacted the office of Dr. Linn Holness at St Michael's Hospital in Toronto, Canada,
in February 2015 and learned that no original data from the study were retained. In the absence of
individual subject data, EPA re-analyzed the findings in the published paper to calculate a central
estimate of the high-exposure group (see further discussion in response to recommendations
related to the RfC under Charge Question E2).
Comment: EPA should consider expanding Appendix A to include other U.S. and international
exposure guidelines (e.g., Threshold Limit Values [TLVs] and Acute Exposure Guideline Level-1
[AEGL-1] values), including their definition, purpose, and links to the assessments that explain the
rationale for the guidelines and chemical-specific documentation that supports them.
Response: Table A-l in Appendix A was expanded to include more information and links to toxicity
values developed by other national and international health agencies.
Charge Question Al: Please comment on whether the conclusions have been clearly and
sufficiently described for purposes of condensing the Toxicological Review information into
a concise summary.
The SAB observed that the Executive Summary was too vague and unclear in some of the
subsections. The SAB specifically recommended the following.
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Comment: A section should be included at the beginning of the Executive Summary that provides
information on the chemistry of ammonia, ammonium, and ammonium salts and the rationale for
excluding or including ammonium salts.
Response: A brief summary of the chemical properties of ammonia was added to the Executive
Summary. The scope of this assessment was revised to include the inhalation route of exposure
only. A full evaluation of the complexities associated with ingestion of ammonium salts will be
considered in a separate assessment (see Charge Question Dl).
Comment: The discussion of noncancer effects from inhalation exposure should be placed before
the discussion of oral exposures if an RfD is not derived, and the first sentence of the noncancer oral
section should indicate that an oral RfD was not derived.
Response: As indicated above, an oral RfD will be considered in a separate assessment (see Charge
Question Dl).
Comment: A brief discussion of the weight of evidence of critical epidemiology studies should be
provided by adding descriptors for the nature of effects measured (e.g., self-reported versus clinical
examination) and a brief discussion of how each key epidemiology study used for RfC derivation
controlled for potential confounding effects of co-exposures to other chemicals or particulate
matter that might cause similar respiratory effects.
Response: The Executive Summary was revised by providing information on outcome
measurement (e.g., self-report), magnitude of lung function changes, and potential co-exposures.
Comment: Description of the evidence that ammonia may act as a cancer promoter should be
expanded.
Response: As indicated below under Charge Question C3, carcinogenicity will be addressed in a
separate assessment on the oral route of exposure.
Comment: EPA should consider including parts of the discussion of the actual study data relevant
for asthmatics as a susceptible population from Section 1.3.2.
Response: A brief summary of the nature and extent of the evidence for asthmatics as a susceptible
population was added to the Executive Summary.
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Comment: In the gray summary box of the Executive Summary, EPA should indicate that there is
inadequate information to evaluate the carcinogenicity of ammonia or to derive an oral RfD for
ammonia.
Response: Evaluation of the toxicity of ammonia via oral exposure, including carcinogenicity, will be
addressed in a separate assessment (see Charge Questions C3 and Dl).
Charge Question Bl: The process for identifying and selecting pertinent studies for
consideration in developing the assessment is detailed in the Literature Search
Strategy/Study Selection and Evaluation section. Please comment on whether the literature
search approach, screening, evaluation, and selection of studies for inclusion in the
assessment are clearly described and supported. Please comment on whether EPA has
clearly identified the criteria (e.g., study quality, risk of bias) used for the selection of studies
to review and for the selection of key studies to include in the assessment. Please identify
any additional peer-reviewed studies from the primary literature that should be considered
in the assessment of noncancer and cancer health effects of ammonia.
Comment: The SAB observed that, overall, the literature search approach, screening, evaluation,
and selection of studies for inclusion in the assessment were fairly well described and supported,
and incorporated elements of systematic review; however, several areas needed further
clarification and strengthening. The SAB encouraged EPA to incorporate and implement
recommendations from both NRC reports as much as reasonably possible given time constraints.
The SAB recognized that some of the weaknesses regarding the application of literature search and
evaluation protocols identified by the panel may reflect EPA's progress in implementing past and
more recent NRC recommendations, or insufficient clarity as to the extent and mechanisms for their
application in the ammonia assessment. The SAB recommended that EPA accelerate the
development of standardized, detailed literature search and evaluation protocols specific to IRIS
objectives. Specific recommendations of the SAB related to literature search and study selection
follow in the comments below.
Response: As already noted, future assessments will reflect greater implementation of systematic
review. IRIS assessments that are currently in the problem formulation or early draft development
steps will include the development and application of protocols for literature searching, literature
screening, and evaluating studies, and transparent documentation of the results of the literature
search, literature screening, and study evaluation. The literature search strategy section of the
ammonia assessment was revised to more transparently present the approach for study
identification and screening. (See responses to specific recommendations below for more
information on enhancements to documentation of the literature search strategy for ammonia.)
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Comment: The list of databases included in the literature search should be expanded. The SAB
agreed with EPA's objective of using the literature supporting the Agency for Toxic Substances and
Disease Registry (ATSDR) Toxicological Profile to reduce unnecessary duplication of effort across
agencies, but stated that it was unclear if and to what extent ATSDR's literature search strategy
incorporated principles of systematic review. As such, ATSDR's literature search should not be
deemed directly transferrable to the EPA's assessment without further clarification.
Response: The reference list in ATSDR's Toxicological Profile (ATSDR. 20041 was examined to
ensure that the search using on-line databases did not miss any health effect studies. In addition,
ATSDR's Toxicological Profile for Ammonia was used, in particular, to identify toxicokinetic studies.
The literature on ammonia toxicokinetics is extensive because of ammonia's importance in nitrogen
homeostasis and acid-base balance. ATSDR's Toxicological Profile was used to facilitate the
identification of key toxicokinetic literature. Use of ATSDR's Toxicological Profile in the literature
search section was clarified.
The initial literature search had included other databases and resources (e.g., EPA's Office of
Pesticide Program chemical search, Organization of Economic Co-operation and Development's
High Production Volume (HPV) chemical database, EPA's High Production Volume Information
System (HPVIS), and the National Institute for Occupational Safety and Health (NIOSH) Registry of
Toxic Effects of Chemical Substances database) to augment the search of the core computerized
databases (PubMed, Toxline, Toxic Substance Control Act Test Submissions [TSCATS] database,
Health and Environmental Research Online [HERO], Web of Science [WOS], and Toxcenter), but
failed to include documentation of these databases and resources. Documentation of these
searches was added to the Supplemental Information (see Appendix B, Table B-2), and reference to
the search of these databases/resources was added to the Literature Search Strategy | Study
Selection and Evaluation section.
Comment: The SAB recommended that inclusion/exclusion criteria be made more transparent, to
provide insight as to why some apparently relevant publications were not included or cited (e.g..
Mirabelli et al.. 20071. In addition, the SAB encouraged EPA to consider publications beyond March
2013 fe.g.. Hovland etal.. 20141.
Response: The literature search section was rewritten to more clearly describe the approach used
to identify and screen the ammonia literature. Figure LS-1 (literature search and screening flow
diagram) was revised, adding a table (Table LS-1) of inclusion and exclusion criteria used to screen
studies. Also included was a modified set of criteria for the post-SAB literature search update.
Discussion of the focused search of literature on cleaning and hospital workers was moved from the
Supplemental Information to the main document, and documentation of this search was added to
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the Supplemental Information. Also included in this table is a disposition of each study based on
inclusion/exclusion criteria. As noted in this table, the study by Mirabelli etal. (2007) was
previously identified, but excluded because of a lack of ammonia-specific data. An updated
literature search was conducted in September 2015. Eight new epidemiology studies were
retrieved in the literature search update and screen; five of the eight were excluded from further
consideration because they were reviews that did not contain primary data or were determined to
be uninformative. The three studies added to the assessment did not substantively changed
conclusions about ammonia hazard. No new animal toxicity studies were identified in the literature
search update. Documentation of the post-SAB updated literature search, including the disposition
of epidemiological studies identified in this updated search, was added to the Supplemental
Information (Table B-5).
Comment: The exclusion of ammonium salts should be supported by a thorough and systematic
review of the relevant literature. If a systematic search was done, EPA should indicate this clearly
in the description of search criteria and in Appendix C of the Supplemental Information. The SAB
suggested that the rationale for excluding ammonium salts could be buttressed by adding data on
50% lethal concentration (LC50) and 50% lethal dose (LD50) values for various ammonium salts to
show the variability in response.
Response: The scope of the current assessment was revised to include inhalation only. A systematic
review of the ammonium salts literature will be conducted as part of a separate oral assessment
(see also Charge Question Dl).
Comment: The description of studies in Appendix C (previously Appendix E in the revised external
review draft) should be made uniform across all types of studies. The SAB also noted that it would
be useful to provide hyperlinks between citations and Appendix C (previously Appendix E in the
revised external review draft) summaries in electronic versions of the assessment and supporting
information.
The outline for describing key study characteristics according to the criteria and major
limitations (as listed in Tables D-2 to D-4) that was applied to studies of health care/cleaning and
livestock farming settings (pp. xlii-xliii) should also be applied to the industrial studies. The SAB
also recommended that the outline for the narrative be made uniform, with attention paid to
describing the range of different co-exposures present in the various types of study settings.
Response: The formats of the summary tables of human evidence in the Supplemental Information
(Tables C-7, C-8, and C-9 in Appendix C; previously Appendix E in the revised external review draft)
were revised to be consistent Based on experience gained with the new structure for IRIS
toxicological reviews, such detailed study summaries will not be provided in future IRIS
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assessments, and the EPA retained previously developed study summaries, without hyperlinks, in
this assessment (see also response under Charge Question 2).
The evaluations of studies of industrial settings, health care/cleaning settings, and
agricultural settings in the Literature Search Strategy | Study Selection and Evaluation section were
based on the same key study characteristics. To make it clearer that these same characteristics
were evaluated across all studies, subheadings corresponding to each evaluation aspect
(e.g., participant selection, exposure parameters, outcome measurements, confounding) were
added to the evaluation of studies in cleaning and agricultural settings consistent with the
evaluation of industrial studies. Information on potential co-exposures is summarized in the
evaluation of individual epidemiology studies in Tables B-6 to B-8 in Appendix B and is discussed in
the study evaluation section and in the synthesis of effects of ammonia on the respiratory system
(Section 1.2.1).
Comment: The potential contribution to ammonia exposure from cigarette smoke and the varying
levels of ammonia in tobacco and cigarette smoke should be described. The panel specifically cited
Seeman and Carchman (2008).
Response: A brief description of the potential contribution of ammonia exposure from tobacco
smoke and the varying levels of ammonia in tobacco and cigarette smoke was added to the
discussion of confounding in the Literature Search Strategy | Study Selection and Evaluation
section. As discussed in this section, potential confounding by smoking of ammonia-containing
tobacco or by inhaling tobacco smoke was not considered to be a major limitation of the
occupational epidemiology studies because smoking as a potential confounder was adequately
addressed in the studies that examined effects on the respiratory system.
Comment: The criteria by which EPA determines the acceptability of studies and the significance of
specific study limitations should be clarified. The SAB recommended including a summary of the
consistency of exposures, confounders, and outcomes across categories of studies, including
relevant findings from the epidemiology studies.
Response: A discussion of what constitutes major and minor limitations was added to the Literature
Search Strategy | Study Selection and Evaluation section in the Considerations for Evaluation of
Epidemiology Studies subsection.
EPA considered the consistency of findings across three categories of studies (industrial,
cleaner, and agricultural settings) that differed in population characteristics, level and pattern of
exposure, and potential confounders as adding strength to the evidence for an association between
respiratory effects and ammonia exposure. Rather than add this observation to the Literature
Search Strategy | Study Selection and Evaluation section, discussion of the consistency in
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respiratory findings across different categories of studies was added to the synthesis of evidence of
ammonia as a respiratory hazard (Section 1.2.1) and to the Executive Summary.
As discussed in response to recommendations under Charge Question 2, the critical
evaluation of animal toxicity studies of ammonia was revised by providing a more explicit
framework by which individual studies were evaluated (e.g., considerations related to test animals,
experimental design, exposure characterization, endpoint evaluation, and results presentation).
EPA is developing approaches to systematic evaluation and documentation of study quality, and
these will be reflected in future assessments.
Comment: EPA should clarify why requests for additional data from the public were not extended
beyond 2009.
Response: Federal Register notices specifically soliciting public input on ammonia were published
in 2007 and in 2009. In addition, EPA encourages the public to submit information throughout the
assessment development process for all IRIS assessments. For example, the announcement of the
2012 IRIS agenda (77 FR 26751, May 7, 2012) reiterated that the public may submit information on
any chemical substance at any time. The text in the literature search section was revised to indicate
that the request for data from the public was broader than the two Federal Register notices
published in 2007 and 2009.
Charge Question CI: A synthesis of the evidence for ammonia toxicity is provided in
Chapter 1, Hazard Identification. Please comment on whether the available data have been
clearly and appropriately synthesized for each toxicological effect. Please comment on
whether the weight of evidence for hazard identification has been clearly described and
scientifically supported.
The SAB stated that the scientific evidence for respiratory effects is sufficiently robust to
support the conclusion that ammonia induces respiratory effects in humans and animals.
Recommendations related to improving the synthesis of evidence were as follows.
Comment: The SAB recommended more precise documentation of how evaluation criteria were
applied to individual studies and ultimately integrated into the weight-of-evidence analysis, and
suggested that these revisions be included in tabular summaries. Additionally, the SAB
recommended including a more detailed description of Holness etal. fl9891 in support of the RfC,
and a brief summary of acute and short-term studies that identify ammonia as an irritant and
toxicant to the upper respiratory tract (and the eye).
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Response: In the peer review draft of the assessment, specific methodological features of individual
epidemiology studies were systematically evaluated (including selection of study participants,
outcome measurement, exposure parameters, confounding, and statistical analysis) (see Literature
Search Strategy | Study Selection and Evaluation section). Documentation of the evaluation of
animal toxicity studies was expanded in the Study Selection and Evaluation section by adding
Table LS-3, which provides the framework used to evaluate individual animal studies, and text that
reflects the application of this framework (including considerations related to test animals,
experimental design, exposure, endpoint evaluation, and results presentation) to the ammonia
literature.
Section 2.1.1 was revised to provide more detailed descriptions of the Holness etal. f 19891
study and the three other cross-sectional studies that provided information useful for evaluating
the relationship between chronic ammonia exposure and respiratory effects, as well as further
discussion of key strengths and limitations in the individual studies considered for quantitative
analysis for the RfC. The evidence pertaining to ammonia as a respiratory tract irritant following
acute exposure is discussed in Section 1.2.1 under "Respiratory Symptoms."
Comment: The SAB recommended that the biological bases for tolerance/adaptation be considered
as part of the evaluation, and discussed in the context of exposure to ambient ammonia (NH3) gas.
The integration of tolerance into the evaluation should be differentiated from "healthy worker"
issues or independent host factors also known to influence the response and sensitivity to inhalable
irritants. Three papers on ammonia tolerance were identified for consideration: Von Essen and
Romberger (2003). Lavinka etal. (2009). andPetrova etal. (2008).
Response: Section 2.1.4, Uncertainties in the Derivation of the Reference Concentration, was revised
to include a discussion of the potential for underestimation of response to ammonia in the general
population based on findings in worker-exposed populations as a result of development of
tolerance and "healthy worker" bias. The discussion of potential for developing tolerance following
repeated exposure to ammonia relied on studies by Ihrigetal. (2006) and Ferguson etal. (1977).
two papers that specifically addressed habituation to ammonia. The contribution of Von Essen and
Romberger (2003). Lavinka etal. (2009). and Petrova et al. (2008) in examining tolerance to
ammonia was limited compared to Ihrigetal. f20061 and Ferguson et al. f 19771. As a result, these
papers were not included for the following reasons: (1) Von Essen and Romberger (2003) focused
on adaptation of workers to repeated exposure to endotoxin in swine confinement barns, and did
not specifically address effects of ammonia; (2) Lavinka etal. (2009) examined the natural lack of
neuropeptides in naked mole-rats as a mechanism for adaptation to a subterranean environment
with high levels of ammonia; this paper was considered less relevant than the available human
studies; and (3) Petrova etal. f20081 evaluated the irritation potential of ammonia in asthmatics
and healthy volunteers, but did not examine habituation to ammonia in either population.
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Comment: The SAB recommended that gastrointestinal effects of ammonia be re-examined as part
of a more integrated evaluation of the in vivo biological properties of ammonia (e.g., Bodega et al.
f 199311.
Response: The evidence for an association between ammonia exposure and gastrointestinal effects
will be re-examined as part of a separate health assessment of ingested ammonia (see Charge
Question Dl).
Charge Question C2: Does EPA's hazard assessment of noncancer human health effects of
ammonia clearly integrate the available scientific evidence (i.e., human, experimental
animal, and mechanistic evidence) to support the conclusion that ammonia poses a potential
hazard to the respiratory system?
Comment: The SAB observed that the scientific evidence supporting the conclusion that ammonia
poses a potential hazard to the respiratory system was well-integrated. However, the SAB
recommended expanding the evaluation of the chemical reactions and ammonia generation that
may impact gastrointestinal endpoints and their impact on the decision not to derive an RfD.
Response: As noted above, EPA agrees with expanding the evaluation of ammonia's oral toxicity to
include a systematic review of the ammonium salts literature. This will be conducted as a separate
assessment (see Charge Question Dl).
Charge Question C3: Does EPA's hazard assessment of the carcinogenicity of ammonia
clearly integrate the available scientific evidence to support the conclusion that under EPA's
Guidelines for Carcinogen Risk Assessment (U.S. EPA. 20051. there is "inadequate
information to assess the carcinogenic potential" of ammonia?
Comment: The SAB stated that the scientific evidence supported the conclusion that there is
inadequate information to assess the carcinogenic potential of ammonia, and agreed that the
evidence presented by Tsuiii etal. Q9931 suggesting that ammonia exhibits tumor-promoting
properties is limited. The SAB recommended that the EPA expand on the strengths and weaknesses
of the following two relevant lines of evidence: (1) an epidemiologic study regarding promoter
influences Fang etal. (20111: and (2) an animal study reporting increased numbers of
adenocarcinomas following exposure to ammonium acetate via intra-rectal infusions fClinton et al..
19881.
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Response: Information on the carcinogenic potential of ammonia comes from oral exposure studies.
Therefore, the assessment of ammonia carcinogenicity, including the two studies identified for
consideration by the SAB, will be addressed in a separate assessment of the health effects of
ingested ammonia (see Charge Question Dl).
Charge Question Dl: Please comment on whether the rationale for not deriving an RfD is
scientifically supported and clearly described (see Section 2.1). Please comment on whether
data are available to support the derivation of an RfD for ammonia. If so, please identify
these data.
Comment: The SAB offered the following recommendations related to EPA's decision not to derive
an RfD:
• EPA should thoroughly re-evaluate the publications to determine if they should continue to
exclude ammonium salts from the IRIS assessment, or explicitly expand the scope of the
assessment to include the ammonium ion with ammonia. The rationale and presentation of
data to support their conclusions need to be strengthened.
• EPA should evaluate the relevant toxicity studies of ammonium salts as studies that could
additionally inform consideration of gastrointestinal effects and to determine if they offer
valuable information for the derivation of an RfD. In particular, the panel pointed to the
study by Lina and Kuiipers (2004). which included a CI- control.
• A decision to address ammonium salts would require further evaluation of the RfC and the
impact of the inhalation of ammonium-containing airborne particulate matter.
Response: EPA agrees with expanding the evaluation of ammonia's oral toxicity to include a
systematic review of the ammonium salts literature. This will be conducted as a separate
assessment Specific SAB recommendations related to the evaluation of the health effects of
ingested ammonia will be addressed in this separate assessment
Response to the SAB recommendation to evaluate the impact of inhaling ammonium-
containing airborne particulate matter on the RfC is addressed in response to comments on the RfC
(see response to recommendations under Charge Question El).
Charge Question D2: As described in the Preface, data on ammonia salts were not
considered in the identification of effects of the derivation of an RfD for ammonia and
ammonium hydroxide because of concerns about the potential impact of the counter ion on
toxicity outcomes. Please comment on whether the rationale for this decision is
scientifically supported and clearly described.
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Comment: The SAB recommended that the rationale for the decision not to derive an RfD be better
supported and more clearly detailed, especially given lack of clarity about the chemistry of
ammonia/ammonium, and given the existence of at least one study of ammonium that appears to
have adequately controlled for the possible toxicity of the counter ion (Lina and Kuiipers. 20041.
Response: As discussed in response to recommendations provided under Charge Question Dl, this
will be addressed in a separate assessment.
Charge Question El: Please comment on whether the evaluation and selection of studies and
effects for the derivation of the RfC is scientifically supported and clearly described (see
Section 2.2.1). Please identify and provide the rationale for any other studies or effects that
should be considered.
The SAB observed that the evaluation of studies was clearly described in the supplementary
materials and concisely summarized in the main assessment Specific comments and
recommendations related to study selection and evaluation for deriving the RfC were the following.
Comment: A better description of the controlled human studies should be provided and the
rationale for their exclusion should be strengthened.
Response: Section 2.1.1 was expanded to include the rationale for notusing controlled human
exposure studies for dose-response analysis, i.e., that the short exposure durations used in these
studies (15 seconds to 6 hours) make them inappropriate for evaluating the effects of chronic
exposure to ammonia.
Comment: Further discussion of the potential implications of reversibility and long-term
attenuation of effects through acclimatization and/or the healthy worker effect (e.g., self-selected
attrition due to respiratory symptoms) should be added.
Response: As noted under Charge Question CI, Section 2.1.4, Uncertainties in the Derivation of the
Reference Concentration, was revised to include a discussion of the potential for underestimation of
response to ammonia in the general population as a result of development of tolerance and "healthy
worker" bias in worker-exposed populations.
Comment: The EPA should elaborate on its rationale for the selection of self-reported respiratory
symptoms and small subclinical changes in lung function measures as "adverse" health outcomes.
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Response: Discussion of the use of self-reported respiratory symptoms and small subclinical
changes in lung function measures as adverse health outcomes was expanded in Section 2.1.1 by
referring to what the American Thoracic Society considers as adverse respiratory health effects in
the context of air pollution. These considerations distinguish between lung function changes that
may be clinically significant at the individual level and those that may be significant at the
population level. Small changes in the distribution of pulmonary function can result in a proportion
of the exposed population shifted down into the lower "tail" of the pulmonary function distribution.
Comment: It was unclear if the quality of the Holness etal. (1989) study overrode other factors
listed in the Preamble for selection of a key study, especially considering that the Ballal etal. f 19981
and Rahman et al. (2007) studies could be used to derive benchmark doses, which the Preamble
indicates is preferred over the NOAEL/LOAEL approach. The role in study selection of any
differences in outcome measures and of confounding controls among these studies was also
unclear.
Response: Documentation of the factors considered in evaluating the quality of individual
epidemiologic studies is provided in Appendix B, Tables B-6 to B-8, and discussed in the study
evaluation section. For dose-response analysis, EPA determined that the overall coherence in the
set of industrial studies of ammonia supported derivation of an RfC. Factors considered in selecting
the NOAEL from Holness etal. (1989) as the basis for the RfC included exposure characterization,
outcome measures, and potential for confounding. Specifically, the Holness etal. (1989) study was
selected over the studies by Ballal etal. (1998) and Rahman et al. (2007) because of higher
confidence in measurement of ammonia exposure, evaluation of both respiratory symptoms and
lung function parameters, smaller potential for co-exposures to other workplace chemicals, and the
fact that the estimated NOAEL for respiratory effects was the highest of the NOAELs estimated from
the candidate principal studies. Section 2.1.1 was revised to provide a more transparent discussion
of considerations weighed in selecting studies for dose-response analysis (in particular differences
in outcome measures and control for confounding).
Comment: A brief discussion of the possible deleterious effects of airborne particulate ammonia
should be added to the assessment based on a recent study fPaulotand lacob. 20141 that found that
ammonia gas emanating from farming practices can form aerosols that adversely affect human
health.
Response: Mention of Paulot and lacob (2014) was added to the Preface in the context of ammonia
from agricultural sources as a contributor to fine inorganic particular matter (PM2.5). Paulot and
lacob f 20141 used a chemical transport model to estimate the impact of U.S. agricultural sources of
ammonia (NH3) on the concentration of fine inorganic particulate matter (PM2.5) present in the
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atmosphere as ammonium-sulfate-nitrate salts. These authors examined the health benefits that
could be achieved by reducing NH3, SO2, and NOx emissions and thereby reducing PM2.5 mass, but
did not investigate the health effects of airborne particulate ammonia itself.
A growing body of literature has attempted to identify whether individual components of
PM are more strongly associated with morbidity or mortality compared to particulate matter (PM)
mass alone. This literature was evaluated in EPA's Integrated Science Assessment for Particulate
Matter (U.S. EPA. 2009b). which reviews the extensive literature on sources of PM and components
that react to produce PM, atmospheric chemistry and transport models, exposure, and health
effects. Based on an evaluation of studies of various components and sources of PM, including the
ammonium ion (NH4+), the 2009 Integrated Science Assessment for PM concluded that "many
constituents of PM can be linked with differing health effects and the evidence is not yet sufficient
to allow differentiation of those constituents or sources that are more closely related to specific
health outcomes" (U.S. EPA. 2009b). Thus, the PM literature does not support analysis of NH4+ as a
component of PM and health outcomes. Further, literature on particulate ammonia other than as a
contributor to PM was not identified.
Given the fact that the literature on airborne particulate ammonium is limited to ammonia
as a source of PM2.5, a topic covered in the scientific review that supports the PM National Ambient
Air Quality Standard, and the lack of evidence to support associations between specific constituents
of PM (including NH4+) and health outcomes as per U.S. EPA f2009bl consideration of the health
effects of airborne particulate ammonia was not added to the IRIS assessment of ammonia. An
updated Integrated Science Assessment for PM is under development and the study by Paulot and
lacob f 20141 will be considered in the context of that review.
Charge Question E2: The NOAEL/LOAEL approach was used to identify the point of
departure (POD) for derivation of the RfC (see Section 2.2.2). Please comment on whether
this approach is scientifically supported and clearly described.
The SAB observed that the approach for RfC derivation was reasonable and clearly
described, but offered the following recommendations.
Comment: EPA should attempt to obtain individual-level data and/or the mean/median exposure
concentrations for the high-exposure group from Dr. Holness in order to identify a better supported
POD. The SAB suggested that EPA consider using a central estimate (i.e., mean or median) of the
high-exposure group ammonia concentration rather than the minimum. If individual data are
unavailable, EPA should consider whether there is sufficient information available in the Holness et
al. (1989) study to estimate the mean concentration for the high-exposure group (e.g., assuming a
lognormal or other skewed distribution for the measured concentrations). The SAB noted that the
Holness etal. f 19891 study should be used whether the individual data are obtained or not
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Response: EPA was informed by the office of Dr. Linn Holness (call from Susan Rieth, U.S. EPA, to
Charmaine Clayton, administrative assistant to Dr. Holness, St Michael's Hospital, Center for
Research Expertise in Occupational Health, Toronto, Canada, February 11, 2015) that no original
data from the study were retained. In the absence of individual subject data, the frequency
distribution information provided in Holness etal. f 19891 was used to estimate the parameters of
the lognormal distribution that best fit the data. This frequency distribution is provided in
Table C-12 in Appendix C. Assuming a lognormal distribution for the measured concentrations,
EPA estimated the mean concentration for the high-exposure group (17.9 mg/m3). The 95% lower
confidence bound on the mean exposure concentration, or 13.6 mg/m3, was used as the POD for
deriving the RfC to reflect the statistical uncertainty around the estimate of the mean.
Comment: The SAB recommended clarifying and strengthening the evidence that supports the idea
that the reported respiratory and lung function effects of ammonia result from cumulative exposure
rather than acute exposure. The SAB observed that some support is provided in Table 3 of the
Ballal etal. T19981 study.
Response: As discussed in response to recommendations provided in Appendix B of the SAB report
(Comments on the Supplemental Information), the study by Rahman et al. f20071 provides
evidence of contributions from both immediate (acute) exposure and length of exposure
(cumulative exposure) to ammonia's respiratory effects. In addition, Ballal etal. (1998) found a
significant correlation between respiratory symptoms (cough, phlegm, and wheezing) and duration
of service (a proxy for exposure duration). Section 2.1.2 was revised to acknowledge the potential
contribution of both immediate (acute) exposure and length of exposure to ammonia's respiratory
effects. In the absence of clear evidence that respiratory effects in occupationally-exposed
populations are an acute response, and given evidence for the contribution of exposure duration
(cumulative exposure) to the respiratory effects of ammonia, the standard adjustment to
continuous exposure was applied.
Comment: The SAB recommended that the source of exposure values and the rationale for their use
be clarified. Specifically, the SAB suggested that EPA clarify the assumed inhalation rates of
10 m3/8-hour workday and 20 m3/24-hour day, noting that inhalation rates provided in the
assessment differ from those referenced in the 2011 Exposure Factors Handbook fU.S. EPA. 20111.
If a breathing rate of 20 m3/day is meant to be an upper bound, EPA should cite its data source and
discuss whether incorporation of this aspect of inter-individual pharmacokinetic variability at the
NOAEL determination stage has implications for later selection of an uncertainty factor (UF).
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Response: The ratio of the workday to daily average inhalation rate of 10 m3/20 m3 (or 0.5) was
retained, but the reference to support the value was corrected to U.S. EPA (1994). Methods for
Derivation of Inhalation Reference Concentrations and Application of Inhalation Dosimetry. The
inhalation rate values are consistent with inhalation rates from a 2009 study conducted by U.S. EPA
(2009c) and as cited in the 2011 Exposure Factors Handbook fU.S. EPA. 20111. Section 2.1.2 was
revised to include a discussion of the consistency in inhalation rates between U.S. EPA T19941 and
U.S. EPA (2009c). By using average values for both occupational and daily inhalation rates, there
should be no significant implications for interindividual uncertainty or for the selection of the
intraspecies UF of 10.
Charge Question E3: Please comment on the rationale for the selection of the uncertainty
factors (UFs) applied to the POD for the derivation of the RfC (see Section 2.2.3). Are the UFs
appropriate based on the recommendations described in Section 4.4.5 of A Review of the
Reference Dose and Reference Concentration Processes (U.S. EPA. 20021. and clearly
described? If changes to the selected UFs are proposed, please identify and provide
scientific support for the proposed changes.
Comment: The SAB observed that the selection of UFs was appropriate, clearly described, and
consistent with the 2002 EPA recommendations.
Response: No response needed.
Charge Question Fl: Quantitative cancer estimates were not derived for ammonia because
of inadequate information. Please comment on whether the rationale for not deriving
quantitative cancer estimates for ammonia is scientifically supported and clearly described
(see Section 2.3). Please comment on whether data are available to support a quantitative
cancer assessment. If so, please identify these data.
Comment: The SAB agreed with EPA's conclusion that the existing data are inadequate to reach a
conclusion on the carcinogenicity of ammonia, and thus, it would not be scientifically justified to
develop quantitative cancer risk estimates for this chemical. The SAB further observed that the
rationale for not deriving quantitative cancer estimates was described clearly and supported
scientifically.
Response: As noted in response to recommendations under Charge Question C3, the assessment of
ammonia carcinogenicity will be addressed in a separate assessment of the health effects of
ingested ammonia.
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Charge Question Gl: Ammonia is produced endogenously and has been detected in the
expired air of healthy volunteers. Please comment on whether the discussion of endogenous
ammonia in Section 2.2.4 (currently 2.1.4) of the Toxicological Review is scientifically
supported and clearly described.
Comment: The SAB considered the description of endogenous ammonia production to be generally
appropriate. The panel recommended providing a clearer understanding of the pathways for
ammonia generation and the health effects associated with increased ammonia levels, and
expanding the section to include all sources of endogenous ammonia.
The SAB provided information on the relationship (or lack of relationship) between
endogenous ammonia, concentrations of ammonia in inhaled, expired, and alveolar air, the lung
metabolic pool of ammonia, and ammonia in the oral cavity. The panel observed that the
concentration of ammonia in the mouth is not a major contributor to either the systemic or inhaled
concentration of ammonia. The panel also observed that exhaled ammonia concentrations are
likely higher than inhaled concentrations even for mouth breathers, much as exhaled CO2 is higher
than inhaled CO2. The panel recommended that the assessment clearly state that exhalation of air
and ammonia is a clearance mechanism of an otherwise toxic contaminant.
Response: A summary of the pathways for the production of endogenous ammonia in Appendix C,
Section C.1.3 (Metabolism/Endogenous Production of Ammonia) was expanded. The discussion of
disease states that can lead to hyperammonemia was expanded in Section 1.3.2 (Susceptible
Populations and Lifestages).
The endogenous ammonia discussion in Section 2.1.4, Uncertainties in the Derivation of the
Reference Concentration, provides a comparison of the range of ammonia concentrations in
exhaled breath with the RfC, noting that ammonia in exhaled breath has, under certain conditions,
been measured at concentrations that exceed the RfC. The intent of this uncertainty section was to
provide context for this comparison, and not to be a broad review of endogenous ammonia and its
sources. The section was rewritten to clarify the objective of the section, focusing on the following
points:
• Ammonia is produced endogenously; one route of elimination is exhalation.
• Ammonia concentrations exhaled through the mouth are higher than concentrations
exhaled through the nose. Concentrations in the nose generally do not exceed the RfC,
better represent levels at the alveolar interface of the lung, and are thought to be more
relevant to understanding systemic levels of ammonia.
• Concentrations in breath cannot be correlated with blood ammonia concentrations or with
previous exposure to environmental (ambient) concentrations of ammonia.
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• The exhalation of ammonia is a clearance mechanism for a product of metabolism that is
otherwise toxic in the body at sufficiently high concentrations. Exhaled concentrations may
be higher than inhaled concentrations, particularly when compared to exhaled air from the
mouth or oral cavity, although ammonia concentrations in exhaled air from the respiratory
tract are generally lower than the RfC.
In addition, the title of the section was changed to "Comparison of Exhaled Ammonia to the
RfC" to more transparently identify the uncertainty addressed in this section. Section C.1.3 in
Appendix C was revised to include an expanded discussion of sources of endogenous ammonia and
the relationship between ammonia concentrations in different internal compartments. A reference
to Appendix C for further information on endogenous ammonia was included in the uncertainties
discussion.
Comment: EPA should consider including (in Section 2.1.4 and the Executive Summary) ammonia
concentration ranges for typical indoor and ambient air to provide context for the potential
contributions of endogenously generated ammonia to NH3 inhalation doses, and for placing the RfC
in the context of expected concentrations in non-industrial, residential, and office indoor
environments, and in outdoor air.
Response: Concentration ranges for ammonia in indoor and ambient air were added to the Preface
and the Executive Summary. EPA considers these sections to be more appropriate locations for
background information on ammonia, including typical air concentrations, than Section 2.1.4.
Appendix B of the SAB Report
In Appendix B of their report, the SAB provided a number of specific recommendations for
changes to improve the clarity or accuracy of the Toxicological Review; these changes were adopted
by EPA in revising the Toxicological Review. Summaries of these specific recommendations and
responses to these recommendations are not addressed further in this appendix. Specific
recommendations pertaining to the evaluation of oral health effects data, derivation of the oral RfD,
and evaluation of cancer data will be considered in a separate assessment of the health effects of
ammonia following oral exposure and are not further addressed in this appendix. Other changes
made in response to SAB comments that were not already addressed as recommendations under
charge questions to the SAB are summarized below.
Comments on the Executive Summary and Toxicological Review of Ammonia
• The section of the Preface that described major uses of ammonia was expanded to include a
summary of the major sources of ammonia exposure. Information on ammonia
concentrations in ambient air based on measurements from the National Atmospheric
Deposition Program's Ammonia Monitoring Network was added to the Preface and
Executive Summary.
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• Evidence for effects on the adrenal gland and kidney, based largely on inhalation studies,
was reconsidered. EPA concluded that the evidence of possible effects of ammonia on the
adrenal gland (i.e., one guinea pig study fWeatherbv. 19521 that was limited in design and
reporting) was insufficient to evaluate hazard. Findings of effects on the kidney come from
three inhalation studies in multiple animal species; these studies, all from the toxicological
literature published between 1952 and 1970, were also limited in design and reporting. For
example, none of the three studies provided incidence of histopathologic lesions, and
characterization of lesions in the Weatherbv T19521 study (e.g., "congestion of the kidneys)
was non-specific. The summary of evidence for an association between inhaled ammonia
and effects on the kidney was revised to more clearly describe these limitations in the
evidence.
Comments on the Supplemental Information
• Appendix C, Section C.l.l, Absorption, was revised to more accurately describe absorption
of ammonia from the intestines. More current references were added to address the SAB
comment that the better quality data suggest/support that the small intestine also
contributes to intestinal ammoniagenesis via the use of amino acids as an energy source.
• Normal blood ammonia levels from more recent sources were added to the text in
Appendix C, Section C.1.2, Distribution; the statement pertaining to blood ammonia levels
based on papers from the older ammonia literature (i.e., Conn (1972). Brown et al. (1957))
was deleted in light of the lower reliability of assays used at that time.
• Appendix C, Section C.1.2, Distribution, was updated to include the relative amounts of NH4+
and NH3 at physiological pH as reported by Weiner and Verlander f20131 (see footnote 3).
• The text in Appendix C, Section C.1.3, Metabolism/Endogenous Production of Ammonia, was
revised to clarify that intestinal ammonia production can exceed hepatic metabolism
capacity, leading to increased blood ammonia levels, under conditions of abnormal liver
function.
• Appendix C, Sections C.1.3, Metabolism/Endogenous Production of Ammonia, and C.1.4,
Distribution, were revised to more accurately describe the kidney's role in the production
and elimination of ammonia, noting that the kidneys actually add ammonia to the body, as
renal vein ammonia content exceeds renal artery ammonia content.
• Appendix C, Section C.1.4, Distribution, was revised to more accurately characterize the
mechanisms of ammonia elimination. As noted by the SAB, characterization of renal
ammonia transport is highly complex, involving proteins such as the ammonia transporter
proteins Rhbg and Rhcg, and is beyond the scope of this assessment. Citations for recent
review papers were added to provide readers with a source of more detailed information.
Selection of the RfC
• Findings related to hemoptysis in Ballal etal. (1998) were added to the summary in
Appendix C, Section C.2.1.
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The findings in Table 3 of Ali etal. (2001) were further evaluated in response to SAB
comments on the value of the forced expiratory volume in 1 second (FEVi)%. Forced vital
capacity (FVC)% predicted was statistically significantly higher in the exposed workers than
in the control group; FEVi% predicted was approximately 1.5% higher in the exposed
workers than the control, but the difference was not statistically significant and was not
considered consistent with a beneficial effect of exposure. Comparison of the values for
FVC% predicted in Tables 3, 4, and 5 of the paper suggests that the value for FVC%
predicted of 105.65 in Table 3 may be incorrect. The basis for this determination was
added to the summary of the Ali etal. (2001) study in the Supplemental Information. Given
the concerns regarding the FVC% predicted value in Table 3, only study results from Tables
4 and 5 of the Ali etal. (2001) study were presented in the Toxicological Review.
Results from the Rahman etal. (2007) study were re-evaluated in response to SAB
observations comparing pre-shift values between the ammonia and urea plants in this
study. EPA agreed with the SAB that results in Table 5 of Rahman et al. f2007) provide
evidence of an immediate effect of ammonia exposure on lung function. Specifically, mean
preshift FVC and FEVi values in ammonia and urea plant workers were similar (suggesting
similar lung function in low- and high-exposure workers upon arrival at work), and cross-
shift changes in FVC and FEVi in the urea plant workers (i.e., the more highly-exposed
workers) were statistically significantly decreased. However, other findings from the
Rahman etal. (2007) study suggest contributors to lung function changes other than daily
(immediate) exposure. The study authors applied a multiple regression model to data from
23 workers (from both the ammonia and urea plants) with concurrent measurements of
ammonia exposure and lung function; both the concentration of ammonia and duration of
exposure (using years of employment as a proxy for duration) contributed to percentage
cross-shift decrease in FEVi% (AFEVi%) (Table 6). Rahman etal. f2007) reported that each
year of work in a production section was associated with a decrease in AFEVi% of 0.6%.
These findings were added to Table 1-2. It should be noted that a limitation of the multiple
regression analysis was the failure to explore the age parameter, since there was a high
correlation between age and years of work (Pearson correlation coefficient 0.97). The
evidence from Rahman etal. (2007) for contributions of both immediate exposure and
length of exposure to ammonia's respiratory effects was discussed in Section 2.1.2 in the
context of adjustment of noncontinuous (occupational) exposure to continuous (general
population) exposure in deriving the RfC.
Chapter 2 was revised to include bulleted summaries of the studies considered for dose-
response analysis, with a focus on the contribution of each to the understanding of the dose-
response relationship between ammonia exposure and respiratory effects.
In response to other SAB comments on Section 2.2.1, the summary of outcomes in Rahman
etal. (2007) was expanded to include the magnitude of cross-shift decline in FEVi and FVC
in the high-exposure group; support for self-reported respiratory symptoms as well
accepted outcomes for evaluating respiratory health was provided by reference to the
American Thoracic Society guidelines and EPA's Methods for Derivation of Inhalation
Reference Concentrations and Application of Inhalation Dosimetry; and consideration of
potential co-exposures as they relate to selection of studies for dose-response analysis was
added.
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Other Comments
• Discussion of a focused literature search of studies of cleaning and hospital workers,
undertaken to address a new area of research identified during the 2013 literature search
update, was added to the Literature Search Strategy | Study Selection and Evaluation
section. More detailed documentation of the focused search was included in Appendix B of
the Supplemental Information, Table B-3.
Appendix C of the SAB Report
In Appendix C of their report, the SAB provided suggestions for additional studies relevant
to selection of the RfD, neurotoxic effects from exposure to ammonia, and endogenous production
of ammonia. Specific recommendations pertaining to the oral RfD will be considered in a separate
assessment of the health effects of ammonia following oral exposure and are not further addressed
in this appendix. Observations pertinent to inhaled ammonia were considered in revising this
assessment Specific SAB recommendations were addressed as follows:
Comment: The SAB recommended further development of the discussion regarding measurement
of ammonia in exhaled air and how it may impact the RfC, and the relevance to hyperammonemia,
ingested ammonia, or long-term exposure to gaseous ammonia.
The SAB recommended that, in addition to the cited references that evaluated the
relationship between ammonia concentration in exhaled breath and systemic ammonia levels (i.e.,
Schmidt etal.. 2013: Smith etal.. 2008: Larson etal.. 1977). the recent paper by Solga etal. (2013)
should also be cited; these authors found that the amount of ammonia in expired air was influenced
by temperature of the breath sample and breath analyzer, the pH of a mouth rinse, and open versus
closed mouth breathing.
Response: Discussion of ammonia levels in exhaled breath and the relationship of exhaled ammonia
to the RfC in Section 2.1.4 was revised as discussed in response to Charge Question Gl.
Hyperammonemia has not been associated with exposure to ammonia at environmental
concentrations. The discussion of ammonia in exhaled breath in cases of disease states resulting in
hyperammonemia is included in Appendix C, Section C.1.4, Ammonia Elimination. As the SAB
observed in addressing Charge Question Gl, correlating prior chronic exposure with alveolar
concentrations is challenging. This point was added to the Toxicological Review in Section 2.1.4,
Comparison of Exhaled Ammonia to the RfC.
A summary of the paper by Solga etal. (2013) was added to Table C-l; discussion of the
findings from this study were added to Appendix C, Section C.1.4. Because this study involved a
single volunteer and did not report ambient ammonia concentrations, this study did not contribute
substantially to the existing discussion of ammonia in expired air.
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