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 www.epa.gov/iris

TOXICOLOGICAL REVIEW OF

FORMALDEHYDE

INHALATION TOXICITY

(CAS No. 50-00-0) 

 

In Support of Summary Information on the 

Integrated Risk Information System (IRIS)

VOLUME I of IV 

Introduction, Background, 

and Toxicokinetics 

March 17, 2010

NOTICE

This document is an Inter-Agency Science Consultation review draft. This information is 

distributed solely for the purpose of pre-dissemination peer review under applicable information 

quality guidelines. It has not been formally disseminated by EPA. It does not represent and 

should not be construed to represent any Agency determination or policy. It is being circulated 

for review of its technical accuracy and science policy implications.

U.S. Environmental Protection Agency 

Washington, DC 
This document is a draft for review purposes only and does not constitute Agency policy.

I-ii DRAFT—DO NOT CITE OR QUOTE

DISCLAIMER

This document is a preliminary draft for review purposes only. This information is 

distributed solely for the purpose of pre-dissemination peer review under applicable information 

quality guidelines. It has not been formally disseminated by EPA. It does not represent and 

should not be construed to represent any Agency determination or policy. Mention of trade 

names or commercial products does not constitute endorsement or recommendation for use. 
This document is a draft for review purposes only and does not constitute Agency policy.

I-iii DRAFT—DO NOT CITE OR QUOTE

CONTENTS—TOXICOLOGICAL REVIEW OF FORMALDEHYDE

(CAS No. 50-00-0)

LIST OF TABLES......................................................................................................................... xi

LIST OF FIGURES .......................................................................................................................xx

LIST OF ABBREVIATIONS AND ACRONYMS.....................................................................xxv

FOREWORD ............................................................................................................................ xxxii

AUTHORS, CONTRIBUTORS, AND REVIEWERS............................................................ xxxiii

VOLUME I

1. INTRODUCTION .................................................................................................................. 1-1

2. BACKGROUND .................................................................................................................... 2-1

2.1. PHYSICOCHEMICAL PROPERTIES OF FORMALDEHYDE................................ 2-1

2.2. PRODUCTION, USES, AND SOURCES OF FORMALDEHYDE........................... 2-1

2.3. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE..................................... 2-4

2.3.1. Inhalation.......................................................................................................... 2-5

2.3.2. Ingestion ......................................................................................................... 2-10

2.3.3. Dermal Contact............................................................................................... 2-11

3. TOXICOKINETICS ............................................................................................................... 3-1

3.1. CHEMICAL PROPERTIES AND REACTIVITY ...................................................... 3-1

3.1.1. Binding of Formaldehyde to Proteins .............................................................. 3-1

3.1.2. Endogenous Sources of Formaldehyde ............................................................ 3-3

3.1.2.1. Normal Cellular Metabolism (Enzymatic)........................................ 3-3

3.1.2.2. Normal Metabolism (Non-Enzymatic).............................................. 3-5

3.1.2.3. Exogenous Sources of Formaldehyde Production............................. 3-5

3.1.2.4. FA-GSH Conjugate as a Method of Systemic Distribution .............. 3-6

3.1.2.5. Metabolic Products of FA Metabolism (e.g., Formic Acid).............. 3-6

3.1.2.6. Levels of Endogenous Formaldehyde in Animal and Human 

Tissues.............................................................................................. 3-6

3.2. ABSORPTION ............................................................................................................. 3-9

3.2.1. Oral................................................................................................................... 3-9

3.2.2. Dermal.............................................................................................................. 3-9

3.2.3. Inhalation.......................................................................................................... 3-9

3.2.3.1. Formaldehyde Uptake Can be Affected by Effects at the 

Portal of Entry ................................................................................ 3-10

3.2.3.2. Variability in the Nasal Dosimetry of Formaldehyde in 

Adults and Children........................................................................ 3-12

3.3. DISTRIBUTION ........................................................................................................ 3-13

3.3.1. Levels in Blood .............................................................................................. 3-13

3.3.2. Levels in Various Tissues............................................................................... 3-15

3.3.2.1. Disposition of Formaldehyde: Differentiating covalent 

Binding and Metabolic Incorporation ............................................ 3-16
This document is a draft for review purposes only and does not constitute Agency policy.

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CONTENTS (continued)

3.4. METABOLISM.......................................................................................................... 3-20

3.4.1. In Vitro and In Vivo Characterization of Formaldehyde Metabolism........... 3-20

3.4.2. Formaldehyde Exposure and Perturbation of Metabolic Pathways............... 3-23

3.4.3. Evidence for Susceptibility in Formaldehyde Metabolism............................ 3-24

3.5. EXCRETION.............................................................................................................. 3-25

3.5.1. Formaldehyde Excretion in Rodents.............................................................. 3-26

3.5.2. Formaldehyde Excretion in Exhaled Human Breath...................................... 3-27

3.5.3. Formaldehyde Excretion in Human Urine ..................................................... 3-31

3.6. MODELING THE TOXICOKINETICS OF FORMALDEHYDE AND DPX ......... 3-32

3.6.1. Motivation ...................................................................................................... 3-32

3.6.2. Species Differences in Anatomy: Consequences for Gas Transport and 

Risk................................................................................................................ 3-34

3.6.3. Modeling Formaldehyde Uptake in Nasal Passages ...................................... 3-40

3.6.3.1. Flux Bins ......................................................................................... 3-41

3.6.3.2. Flux Estimates................................................................................. 3-41

3.6.3.3. Mass Balance Errors........................................................................ 3-42

3.6.4. Modeling Formaldehyde Uptake in the Lower Respiratory Tract ................. 3-42

3.6.5. Uncertainties in Formaldehyde Dosimetry Modeling.................................... 3-44

3.6.5.1. Verification of Predicted Flow Profiles........................................... 3-44

3.6.5.2. Level of Confidence in Formaldehyde Uptake Simulations ........... 3-45

3.6.6. PBPK Modeling of DNA Protein Cross-Links (DPXs) Formed by 

Formaldehyde................................................................................................ 3-48

3.6.6.1. PBPK Models for DPXs.................................................................. 3-48

3.6.6.2. A PBPK Model for DPXs in the F344 Rat and Rhesus 

Monkey that uses Local Tissue Dose of Formaldehyde................. 3-50

3.6.6.3. Uncertainties in Modeling the Rat and Rhesus DPX Data.............. 3-51

3.6.7. Uncertainty in Prediction of Human DPX Concentrations............................ 3-53

VOLUME II

4. HAZARD CHARACTERIZATION....................................................................................... 4-1

4.1. HUMAN STUDIES...................................................................................................... 4-1

4.1.1. Noncancer Health Effects................................................................................. 4-1

4.1.1.1. Sensory Irritation (Eye, Nose, Throat Irritation)............................... 4-1

4.1.1.2. Pulmonary Function ........................................................................ 4-11

4.1.1.3. Asthma............................................................................................. 4-19

4.1.1.4. Respiratory Tract Pathology............................................................ 4-26

4.1.1.5. Immunologic Effects ....................................................................... 4-30

4.1.1.6. Neurological/Behavioral.................................................................. 4-42

4.1.1.7. Developmental and Reproductive Toxicity..................................... 4-45

4.1.1.8. Oral Exposure Effects on the Gastrointestinal Tract....................... 4-56

4.1.1.9. Summary: Noncarcinogenic Hazard in Humans............................. 4-56
This document is a draft for review purposes only and does not constitute Agency policy.

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CONTENTS (continued)

4.1.2. Cancer Health Effects..................................................................................... 4-57

4.1.2.1. Respiratory Tract Cancer................................................................. 4-57

4.1.2.2. Non-Respiratory Tract Cancer ........................................................ 4-84

4.1.2.3. Summary: Carcinogenic Hazard in Humans................................. 4-107

4.2. ANIMAL STUDIES................................................................................................. 4-109

4.2.1. Noncancer Health Effects............................................................................. 4-110

4.2.1.1. Reflex Bradypnea .......................................................................... 4-110

4.2.1.2. Respiratory Tract Pathology.......................................................... 4-120

4.2.1.3. Gastrointestinal Tract and Systemic Toxicity ............................... 4-201

4.2.1.4. Immune Function........................................................................... 4-216

4.2.1.5. Hypersensitivity and Atopic Reactions......................................... 4-225

4.2.1.6. Neurological and Neurobehavioral Function ................................ 4-250

4.2.1.7. Reproductive and Developmental Toxicity................................... 4-285

4.2.2. Carcinogenic Potential: Animal Bioassays.................................................. 4-324

4.2.2.1. Respiratory Tract........................................................................... 4-324

4.2.2.2. Gastrointestinal Tract .................................................................... 4-326

4.2.2.3. Lymphohematopoietic Cancer....................................................... 4-328

4.2.2.4. Summary........................................................................................ 4-335

4.3. GENOTOXICITY .................................................................................................... 4-335

4.3.1. Formaldehyde-DNA Reactions.................................................................... 4-335

4.3.1.1. DNA-Protein Cross-Links (DPXs)................................................ 4-336

4.3.1.2. DNA Adducts................................................................................ 4-341

4.3.1.3. DNA-DNA Cross-Links (DDXs).................................................. 4-343

4.3.1.4. Single Strand Breaks ..................................................................... 4-344

4.3.1.5. Other Genetic Effects of Formaldehyde in Mammalian Cells...... 4-345

4.3.2. In Vitro Clastogenicity ................................................................................. 4-345

4.3.3. In Vitro Mutagenicity................................................................................... 4-347

4.3.3.1. Mutagenicity in Bacterial Systems................................................ 4-347

4.3.3.2. Mutagenicity in Non-mammalian Cell Systems............................ 4-353

4.3.3.3. Mutagenicity in Mammalian Cell Systems ................................... 4-353

4.3.4. In Vivo Mammalian Genotoxicity ............................................................... 4-360

4.3.4.1. Genotoxicity in Laboratory Animals............................................. 4-360

4.3.4.2. Genotoxicity in Humans................................................................ 4-362

4.3.5. Summary of Genotoxicity ............................................................................ 4-370

4.4. SYNTHESIS AND MAJOR EVALUATION OF NONCARCINOGENIC

EFFECTS ................................................................................................................ 4-371

4.4.1. Sensory Irritation.......................................................................................... 4-376

4.4.2. Pulmonary Function ..................................................................................... 4-379

4.4.3. Hypersensitivity and Atopic Reactions........................................................ 4-382

4.4.4. Upper Respiratory Tract Histopathology ..................................................... 4-383

4.4.5. Toxicogenomic and Molecular Data that May Inform MOAs..................... 4-385

4.4.6. Noncancer Modes of Actions....................................................................... 4-387

4.4.7. Immunotoxicity ............................................................................................ 4-389
This document is a draft for review purposes only and does not constitute Agency policy.

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CONTENTS (continued)

4.4.8. Effects on the Nervous System .................................................................... 4-390

4.4.8.1. Irritant Threshold Detection .......................................................... 4-390

4.4.8.2. Behavioral Effects......................................................................... 4-391

4.4.8.3. Neurochemistry, Neuropathology, and Mechanistic Studies........ 4-392

4.4.8.4. Summary........................................................................................ 4-392

4.4.8.5. Data Gaps ...................................................................................... 4-393

4.4.9. Reproductive and Developmental Toxicity.................................................. 4-393

4.4.9.1. Spontaneous Abortion and Fetal Death......................................... 4-393

4.4.9.2. Congenital Malformations............................................................. 4-396

4.4.9.3. Low Birth Weight and Growth Retardation .................................. 4-396

4.4.9.4. Functional Development Outcomes (Developmental 

Neurotoxicity) .............................................................................. 4-397

4.4.9.5. Male Reproductive Toxicity.......................................................... 4-398

4.4.9.6. Female Reproductive Toxicity ...................................................... 4-399

4.4.9.7. Mode of Action.............................................................................. 4-400

4.4.9.8. Data Gaps ...................................................................................... 4-402

4.5. SYNTHESIS AND EVALUATION OF CARCINOGENICITY ............................ 4-402

4.5.1. Cancers of the Respiratory Tract.................................................................. 4-402

4.5.2. Lymphohematopoietic Malignancies........................................................... 4-408

4.5.2.1. Background.................................................................................... 4-408

4.5.2.2. All LHP Malignancies................................................................... 4-410

4.5.2.3. All Leukemia ................................................................................. 4-414

4.5.2.4. Subtype Analysis........................................................................... 4-418

4.5.2.5. Myeloid Leukemia......................................................................... 4-419

4.5.2.6. Solid Tumors of Lymphoid Origin................................................ 4-421

4.5.2.7. Supporting Evidence from Animal Bio-Assays for 

Formaldehyde-Induced Lymphohematopoietic Malignancies..... 4-423

4.5.3. Carcinogenic Mode(s) of Action.................................................................. 4-427

4.5.3.1. Mechanistic Data for Formaldehyde ............................................. 4-428

4.5.3.2. Mode of Action Evaluation for Upper Respiratory Tract 

Cancer (Nasopharyngeal Cancer, Sino-Nasal)............................. 4-439

4.5.3.3. Mode(s) of Action for Lymphohematpoietic Malignancies.......... 4-446

4.5.4. Hazard Characterization for Formaldlehyde Carcinogenicity ..................... 4-453

4.6. SUSCEPTIBLE POPULATIONS............................................................................ 4-454

4.6.1. Life Stages.................................................................................................... 4-454

4.6.1.1. Early Life Stages ........................................................................... 4-455

4.6.1.2. Later Life Stages............................................................................ 4-459

4.6.1.3. Conclusions on Life-Stage Susceptibility ..................................... 4-459

4.6.2. Health/Disease Status................................................................................... 4-460

4.6.3. Nutritional Status.......................................................................................... 4-461

4.6.4. Gender Differences....................................................................................... 4-462

4.6.5. Genetic Differences...................................................................................... 4-462
This document is a draft for review purposes only and does not constitute Agency policy.

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CONTENTS (continued)

4.6.6. Co-Exposures ............................................................................................... 4-464

4.6.6.1. Cumulative Risk ............................................................................ 4-464

4.6.6.2. Aggregate Exposure ...................................................................... 4-465

4.6.7. Uncertainties of Database............................................................................. 4-465

4.6.7.1. Uncertainties of Exposure ............................................................. 4-465

4.6.7.2. Uncertainties of Effect................................................................... 4-466

4.6.8. Summary of Potential Susceptibility............................................................ 4-467

VOLUME III

5. QUANTITATIVE ASSESSMENT: INHALATION EXPOSURE....................................... 5-1

5.1. INHALATION REFERENCE CONCENTRATION (RfC) ........................................ 5-2

5.1.1. Candidate Critical Effects by Health Effect Category ..................................... 5-3

5.1.1.1. Sensory Irritation of the Eyes, Nose, and Throat .............................. 5-3

5.1.1.2. Upper Respiratory Tract Pathology................................................... 5-5

5.1.1.3. Pulmonary Function Effects.............................................................. 5-6

5.1.1.4. Asthma and Allergic Sensitization (Atopy) .................................... 5-10

5.1.1.5. Immune Function............................................................................. 5-16

5.1.1.6. Neurological and Behavioral Toxicity ............................................ 5-17

5.1.1.7. Developmental and Reproductive Toxicity..................................... 5-25

5.1.2. Summary of Critical Effects and Candidate RfCs.......................................... 5-33

5.1.2.1. Selection of Studies for Candidate RfC Derivation ........................ 5-33

5.1.2.2. Derivation of Candidate RfCs from Key Studies............................ 5-40

5.1.2.3. Evaluation of the Study-Specific Candidate RfC............................ 5-66

5.1.3. Database Uncertainties in the RfC Derivation ............................................... 5-69

5.1.4. Uncertainties in the RfC Derivation............................................................... 5-72

5.1.5. Previous Inhalation Assessment..................................................................... 5-74

5.2. QUANTITATIVE CANCER ASSESSMENT BASED ON THE NATIONAL 

CANCER INSTITUTE COHORT STUDY.............................................................. 5-74

5.2.1. Choice of Epidemiology Study ...................................................................... 5-75

5.2.2. Nasopharyngeal Cancer.................................................................................. 5-76

5.2.2.1. Exposure-Response Modeling of the National Cancer 

Institute Cohort............................................................................... 5-76

5.2.2.2. Prediction of Lifetime Extra Risk of Nasopharyngeal Cancer 

Mortality......................................................................................... 5-79

5.2.2.3. Prediction of Lifetime Extra Risk of Nasopharyngeal Cancer 

Incidence ........................................................................................ 5-81

5.2.2.4. Sources of Uncertainty .................................................................... 5-83

5.2.3. Lymphohematopoietic Cancer ....................................................................... 5-88

5.2.3.1. Exposure-Response Modeling of the National Cancer 

Institute Cohort............................................................................... 5-88
This document is a draft for review purposes only and does not constitute Agency policy.

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CONTENTS (continued)

5.2.3.2. Prediction of Lifetime Extra Risks for Hodgkin Lymphoma 

and Leukemia Mortality ................................................................. 5-91

5.2.3.3. Prediction of Lifetime Extra Risks for Hodgkin Lymphoma 

and Leukemia Incidence................................................................. 5-93

5.2.3.4. Sources of Uncertainty .................................................................... 5-95

5.2.4. Conclusions on Cancer Unit Risk Estimates Based on Human Data............. 5-99

5.3. DOSE-RESPONSE MODELING OF RISK OF SQUAMOUS CELL 

CARCINOMA IN THE RESPIRATORY TRACT USING ANIMAL DATA...... 5-102

5.3.1. Long-Term Bioassays in Laboratory Animals............................................. 5-104

5.3.1.1. Nasal Tumor Incidence Data ......................................................... 5-104

5.3.1.2. Mechanistic Data ........................................................................... 5-105

5.3.2. The CIIT Biologically Based Dose-Response Modeling............................. 5-106

5.3.2.1. Major Results of the CIIT Modeling Effort .................................. 5-111

5.3.3. This Assessment’s Conclusions from Evaluation of Dose-Response 

Models of DPX Cell-Replication and Genomics Data, and of BBDR 

Models for Risk Estimation......................................................................... 5-111

5.3.4. Benchmark Dose Approaches to Rat Nasal Tumor Data ............................. 5-118

5.3.4.1. Benchmark Dose Derived from BBDR Rat Model and Flux 

as Dosimeter................................................................................. 5-118

5.3.4.2. Comparison with Other Benchmark Dose Modeling Efforts........ 5-125

5.3.4.3. Kaplan-Meier Adjustment............................................................. 5-128

5.3.4.4. EPA Time-to-Tumor Statistical Modeling .................................... 5-129

5.4. CONCLUSIONS FROM THE QUANTITATIVE ASSESSMENT OF 

CANCER RISK FROM FORMALDEHYDE EXPOSURE BY INHALATION .. 5-133

5.4.1. Inhalation Unit Risk Estimates Based on Human Data................................ 5-133

5.4.2. Inhalation Unit Risk Estimates Based on Rodent Data................................ 5-133

5.4.3. Summary of Inhalation Unit Risk Estimates................................................ 5-135

5.4.4. Application of Age-Dependent Adjustment Factors (ADAFs).................... 5-136

5.4.5. Conclusions: Cancer Inhalation Unit Risk Estimates .................................. 5-137

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND 

DOSE-RESPONSE............................................................................................................... 6-1

6.1. SUMMARY OF HUMAN HAZARD POTENTIAL................................................... 6-1

6.1.1. Exposure........................................................................................................... 6-1

6.1.2. Absorption, Distribution, Metabolism, and Excretion ..................................... 6-1

6.1.3. Noncancer Health Effects in Humans and Laboratory Animals...................... 6-4

6.1.3.1. Sensory Irritation............................................................................... 6-4

6.1.3.2. Respiratory Tract Pathology.............................................................. 6-5

6.1.3.3. Effects on Pulmonary Function ......................................................... 6-8

6.1.3.4. Asthmatic Responses and Increased Atopic Symptoms.................... 6-9

6.1.3.5. Effects on the Immune System........................................................ 6-10

6.1.3.6. Neurological Effects........................................................................ 6-11

6.1.3.7. Reproductive and Developmental Effects....................................... 6-12
This document is a draft for review purposes only and does not constitute Agency policy.

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CONTENTS (continued)

6.1.3.8. Effects on General Systemic Toxicity............................................. 6-13

6.1.3.9. Summary.......................................................................................... 6-14

6.1.4. Carcinogenicity in Human and Laboratory Animals ..................................... 6-14

6.1.4.1. Carcinogenicity in Humans............................................................. 6-14

6.1.4.2. Carcinogenicity in Laboratory Animals.......................................... 6-20

6.1.4.3. Carcinogenic Mode(s) of Action..................................................... 6-21

6.1.5. Cancer Hazard Characterization..................................................................... 6-24

6.2. DOSE-RESPONSE CHARACTERIZATION ........................................................... 6-25

6.2.1. Noncancer Toxicity: Reference Concentration (RfC).................................... 6-25

6.2.1.1. Assessment Approach Employed .................................................... 6-25

6.2.1.2. Derivation of Candidate Reference Concentrations........................ 6-25

6.2.1.3. Adequacy of Overall Data Base for RfC Derivation....................... 6-26

6.2.1.4. Uncertainties in the Reference Concentration (RfC) ...................... 6-29

6.2.1.5. Conclusions ..................................................................................... 6-32

6.2.2. Cancer Risk Estimates.................................................................................... 6-32

6.2.2.1. Choice of Data................................................................................. 6-32

6.2.2.2. Analysis of Epidemiologic Data...................................................... 6-33

6.2.2.3. Analysis of Laboratory Animal Data .............................................. 6-36

6.2.2.4. Extrapolation Aporoaches............................................................... 6-37

6.2.2.5. Inhalation Unit Risk Estimates for Cancer...................................... 6-41

6.2.2.6. Early-Life Susceptibility ................................................................. 6-41

6.2.2.7. Uncertainties in the Quantitative Risk Estimates............................ 6-42

6.2.2.8. Conclusions ..................................................................................... 6-45

6.3. SUMMARY AND CONCLUSIONS ......................................................................... 6-45

REFERENCES ............................................................................................................................R-1

VOLUME IV

APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

COMMENTS AND DISPOSITIONS ............................................................. A-1

APPENDIX B: SIMULATIONS OF INTERINDIVIDUAL AND ADULT-TO-CHILD 

VARIABILITY IN REACTIVE GAS UPTAKE IN A SMALL SAMPLE 

OF PEOPLE (Garcia et al., 2009).....................................................................B-1

APPENDIX C: LIFETABLE ANALYSIS.................................................................................C-1

APPENDIX D: MODEL STRUCTURE & CALIBRATION IN CONOLLY ET AL. 

(2003, 2004)..................................................................................................... D-1

APPENDIX E: EVALUATION OF BBDR MODELING OF NASAL CANCER IN THE 

F344 RAT: CONOLLY ET AL. (2003) AND ALTERNATIVE 

IMPLEMENTATIONS.....................................................................................E-1

APPENDIX F: SENSITIVITY ANALYSIS OF BBDR MODEL FOR FORMALDEHYDE 

INDUCED RESPIRATORY CANCER IN HUMANS....................................F-1
This document is a draft for review purposes only and does not constitute Agency policy.

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CONTENTS (continued)

APPENDIX G: EVALUATION OF THE CANCER DOSE-RESPONSE MODELING 

OF GENOMIC DATA FOR FORMALDEHYDE RISK ASSESSMENT ..... G-1

APPENDIX H: EXPERT PANEL CONSULTATION ON QUANTITATIVE 

EVALUATION OF ANIMAL TOXICOLOGY DATA FOR 

ANALYZING CANCER RISK DUE TO INHALED FORMALDEHYDE .. H-1
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES

Table 2-1. Physicochemical properties of formaldehyde ...........................................................2-2

Table 2-2. Ambient air levels by land use category ...................................................................2-6

Table 2-3. Studies on residential indoor air levels of formaldehyde (non-occupational) .......... 2-8

Table 3-1. Endogenous formaldehyde levels in animal and human tissues and body fluids .....3-8

Table 3-2. Formaldehyde kinetics in human and rat tissue samples ........................................3-21

Table 3-3. Allelic frequencies of ADH3 in human populations...............................................3-25

Table 3-4. Percent distribution of airborne [14C]-formaldehyde in F344 rats..........................3-26

Table 3-5. Apparent formaldehyde levels in exhaled breath of individuals attending a 

health fair, adjusted for methanol and ethanol levels which contribute to the 

detection of the protonated species with a mass to charge ratio of 31 reported 

as formaldehyde (m/z = 31)....................................................................................3-29

Table 3-6. Measurements of exhaled formaldehyde concentrations in the mouth and nose, 

and in the oral cavity after breath holding in three healthy male laboratory 

workers...................................................................................................................3-30

Table 3-7. Extrapolation of parameters for enzymatic metabolism to the human....................3-53

Table 4-1. Cohort and case-control studies of formaldehyde cancer and NPC........................4-59

Table 4-2. Case-control studies of formaldehyde and nasal and paranasal cancer...................4-71

Table 4-3. Epidemiologic studies of formaldehyde and pharyngeal cancer (includes 

nasopharyngeal cancer)..........................................................................................4-78

Table 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancers..........4-98

Table 4-5. Respiratory effects of formaldehyde-induced reflex bradypnea in various 

strains of mice ......................................................................................................4-112

Table 4-6. Respiratory effects of formaldehyde-induced reflex bradypnea in various 

strains of rats ........................................................................................................4-113

Table 4-7. Inhaled dose of formaldehyde to nasal mucosa of F344 rats and B6C3F1 

mice exposed to 15 ppm.......................................................................................4-116

Table 4-8. Exposure regimen for cross-tolerance study .........................................................4-117
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES (continued)

Table 4-9. Summary of formaldehyde effects on mucociliary function in the upper 

respiratory tract ....................................................................................................4-127

Table 4-10. Concentration regimens for ultrastructural evaluation of male CDF rat 

nasoturbinates.......................................................................................................4-129

Table 4-11. Enzymatic activities in nasal respiratory epithelium of male Wistar rats 

exposed to formaldehyde, ozone, or both.............................................................4-130

Table 4-12. Lipid analysis of lung tissue and lung gavage from male F344 rats exposed 

to 0, 15, or 145.6 ppm formaldehyde for 6 hours.................................................4-138

Table 4-13. Formaldehyde effects on biochemical parameters in nasal mucosa and lung 

tissue homogenates from male F344 rats exposed to 0, 15, or 145.6 ppm 

formaldehyde for 6 hours.....................................................................................4-139

Table 4-14. Mast cell degranulation and neutrophil infiltration in the lung of rats 

exposed to formaldehyde via inhalation...............................................................4-140

Table 4-15. Summary of respiratory tract pathology from inhalation exposures to 

formaldehyde—short term studies .......................................................................4-143

Table 4-16. Location and incidence of respiratory tract lesions in B6C3F1 mice 

exposed to formaldehyde......................................................................................4-146

Table 4-17. Formaldehyde effects (incidence and severity) on histopathologic changes in 

the noses and larynxes of male and female albino SPF Wistar rats exposed to 

formaldehyde 6 hours/day for 13 weeks ..............................................................4-148

Table 4-18. Formaldehyde-induced nonneoplastic histopathologic changes in male 

albino SPF Wistar rats exposed to 0, 10, or 20 ppm formaldehyde 

and examined at the end of 130 weeks inclusive of exposure..............................4-149

Table 4-19. Formaldehyde-induced nasal tumors in male albino SPF Wistar rats 

exposed to formaldehyde (6 hours/day, 5 days/week for 13 weeks) and 

examined at the end of 130 weeks inclusive of exposure ....................................4-150

Table 4-20. Formaldehyde effects on nasal epithelium for various concentration-bytime products in male albino Wistar rats..............................................................4-153

Table 4-21. Rhinitis observed in formaldehyde-treated animals; data pooled for male 

and female animals..............................................................................................4-154
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES (continued)

Table 4-22. Epithelial lesions found in the middle region of nasoturbinates of 

formaldehyde-treated and control animals; data pooled for males and 

females.................................................................................................................4-155

Table 4-23. Cellular and molecular changes in nasal tissues of F344 rats exposed to 

formaldehyde.......................................................................................................4-156

Table 4-24. Percent body weight gain and concentrations of iron, zinc, and copper in 

cerebral cortex of male Wistar rats exposed to formaldehyde via inhalation 

for 4 and 13 weeks...............................................................................................4-158

Table 4-25. Zinc, copper, and iron content of lung tissue from formaldehyde-treated 

male Wistar rats...................................................................................................4-158

Table 4-26. Total lung cytochrome P450 measurements of control and formaldehydetreated male Sprague-Dawley rats.......................................................................4-159

Table 4-27. Cytochrome P450 levels in formaldehyde-treated rats ......................................4-160

Table 4-28. Summary of respiratory tract pathology from inhalation exposures to 

formaldehyde, subchronic studies.......................................................................4-162

Table 4-29. Histopathologic findings and severity scores in the naso- and 

maxilloturbinates of female Sprague-Dawley rats exposed to inhaled 

formaldehyde and wood dust for 104 weeks.......................................................4-166

Table 4-30. Histopathologic changes (including tumors) in nasal cavities of male 

Sprague-Dawley rats exposed to inhaled formaldehyde or HCl alone and 

in combination for a lifetime ...............................................................................4-170

Table 4-31. Summary of neoplastic lesions in the nasal cavity of f344 rats exposed to 

inhaled formaldehyde for 2 years........................................................................4-173

Table 4-32. Apparent sites of origin for the SCCs in the nasal cavity of F344 rats 

exposed to 14.3 ppm of formaldehyde gas in the Kerns et al. (1983) 

bioassay ...............................................................................................................4-174

Table 4-33. Incidence and location of nasal squamous cell carcinoma in male F344 

rats exposed to inhaled formaldehyde for 2 years...............................................4-175

Table 4-34. Summary of respiratory tract pathology from chronic inhalation exposures 

to formaldehyde...................................................................................................4-183

Table 4-35. Cell proliferation in nasal mucosa, trachea, and free lung cells isolated 

from male Wistar rats after inhalation exposures to formaldehyde ....................4-194
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES (continued)

Table 4-36. The effect of repeated formaldehyde inhalation exposures for 3 months on 

cell count, basal membrane length, proliferation cells, and two measures of 

cell proliferation, LI and ULLI, in male F344 rats..............................................4-196

Table 4-37. Formaldehyde-induced changes in cell proliferation and (ULLI) in the nasal 

passages of male F344 rats exposed 6 hours/day................................................4-198

Table 4-38. Cell population and surface area estimates in untreated male F344 rats and

regional site location of squamous cell carcinomas in formaldehyde 

exposed rats for correlation to cell proliferation rates.........................................4-199

Table 4-39. Summary of formaldehyde effects on cell proliferation in the upper 

respiratory tract ...................................................................................................4-202

Table 4-40. Summary of lesions observed in the gastrointestinal tracts of Wistar rats 

after drinking-water exposure to formaldehyde for 4 weeks ..............................4-206

Table 4-41. Incidence of lesions observed in the gastrointestinal tracts of Wistar rats 

after drinking-water exposure to formaldehyde for 2 years................................4-209

Table 4-42. Effect of formaldehyde on gastroduodenal carcinogenesis initiated by 

MNNG and NaCl in male Wistar rats exposed to formaldehyde (0.5% 

formalin) in drinking water for 8 weeks..............................................................4-212

Table 4-43. Summary of benign and malignant gastrointestinal tract neoplasia 

reported in male and female Sprague-Dawley rats exposed to 

formaldehyde in drinking water at different ages ...............................................4-214

Table 4-44. Incidence of hemolymphoreticular neoplasia reported in male and 

female Sprague-Dawley rats exposed to formaldehyde in drinking water 

from 7 weeks old for up to 2 years (experiment BT 7001).................................4-215

Table 4-45. Battery of immune parameters and functional tests assessed in female 

B6C3F1 mice after a 3 week, 15-ppm formaldehyde exposure ..........................4-218

Table 4-46. Summary of the effects of formaldehyde inhalation on the mononuclear 

phagocyte system (MPS) in female B6C3F1 mice after a 3-week, 15 ppm 

formaldehyde exposure ( 6 hours/day, 5 days/week)..........................................4-219

Table 4-47. Formaldehyde exposure regimens for determining the effects of 

formaldehyde exposure on pulmonary S. aureus infection.................................4-221

Table 4-48. Summary of immune function changes due to inhaled formaldehyde 

exposure in experimental animals.......................................................................4-226
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES (continued)

Table 4-49. Study design for guinea pigs exposed to formaldehyde through different 

routes of exposure: inhalation, dermal, and injection .........................................4-232

Table 4-50. Sensitization response of guinea pigs exposed to formaldehyde through 

inhalation, topical application, or footpad injection............................................4-233

Table 4-51. Cytokine and chemokine levels in lung tissue homogenate supernatants in 

formaldehyde-exposed male ICR mice with and without Der f sensitization.....4-240

Table 4-52. Correlation coefficients among ear swelling responses and skin mRNA 

levels in contact hypersensitivity to formaldehyde in mice ................................4-249

Table 4-53. Summary of sensitization and atopy studies by inhalation or dermal 

sensitization due to formaldehyde in experimental animals ...............................4-251

Table 4-54. Fluctuation of behavioral responses when male AB mice inhaled 

formaldehyde in a single 2-hour exposure: effects after 2 hours........................4-259

Table 4-55. Fluctuation of behavioral responses when male AB mice inhaled 

formaldehyde in a single 2-hour exposure: effects after 24 hours......................4-259

Table 4-56. Effects of formaldehyde exposure on completion of the labyrinth test by 

male and female LEW.1K rats ............................................................................4-263

Table 4-57. Summary of neurological and neurobehavioral studies in inhaled 

formaldehyde in experimental animals ...............................................................4-279

Table 4-58. Effects of formaldehyde on body and organ weights in rat pups from 

dams exposed via inhalation from mating through gestation..............................4-289

Table 4-59. Formaldehyde effects on Leydig cell quantity and nuclear damage in adult 

male Wistar rats...................................................................................................4-298

Table 4-60. Formaldehyde effects on adult male albino Wistar rats.....................................4-299

Table 4-61. Formaldehyde effects on testosterone levels and seminiferous tubule 

diameters in Wistar rats following 91 days of exposure .....................................4-300

Table 4-62. Effects of formaldehyde exposure on seminiferous tubule diameter and 

epithelial height in Wistar rats following 18 weeks of exposure ........................4-302

Table 4-63. Incidence of sperm abnormalities and dominant lethal effects in 

formaldehyde-treated mice..................................................................................4-302
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES (continued)

Table 4-64. Body weights of pups born to beagles exposed to formaldehyde during 

gestation...............................................................................................................4-303

Table 4-65. Testicular weights, sperm head counts, and percentage incidence of 

abnormal sperm after oral administration of formaldehyde to male 

Wistar rats............................................................................................................4-305

Table 4-66. Effect of formaldehyde on spermatogenic parameters in male Wistar rats 

exposed intraperitoneally ....................................................................................4-306

Table 4-67. Incidence of sperm head abnormalities in formaldehyde-treated rats................4-307

Table 4-68. Dominant lethal mutations after exposure of male rats to formaldehyde ..........4-308

Table 4-69. Summary of reported developmental effects in formaldehyde inhalation 

exposure studies ..................................................................................................4-311

Table 4-70. Summary of reported developmental effects in formaldehyde oral exposure 

studies..................................................................................................................4-317

Table 4-71. Summary of reported developmental effects in formaldehyde dermal 

exposure studies ..................................................................................................4-318

Table 4-72. Summary of reported reproductive effects in formaldehyde inhalation 

studies..................................................................................................................4-319

Table 4-73. Summary of reported reproductive effects in formaldehyde oral studies..........4-322

Table 4-74. Summary of reported reproductive effects in formaldehyde intraperitoneal 

studies..................................................................................................................4-323

Table 4-75. Summary of chronic bioassays which address rodent leukemia and 

lymphoma............................................................................................................4-329

Table 4-76. Formaldehyde-DNA reactions (DPX formation)...............................................4-340

Table 4-77. Formaldehyde-DNA reactions (DNA adduct formation)...................................4-343

Table 4-78. Formaldehyde-DNA interactions (single stranded breaks)................................4-344

Table 4-79. Other genetic effects of formaldehyde in mammalian cells...............................4-346

Table 4-80. In vitro clastogenicity of formaldehyde .............................................................4-348

Table 4-81. Summary of mutagenicity of formaldehyde in bacterial systems......................4-350
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES (continued)

Table 4-82 Mutagenicity in mammalian cell systems...........................................................4-355

Table 4-83. Genotoxicity in laboratory animals....................................................................4-361

Table 4-84. MN frequencies in buccal mucosa cells of volunteers exposed to 

formaldehyde.......................................................................................................4-364

Table 4-85. MN and SCE formation in mortuary science students exposed to 

formaldehyde for 85 days....................................................................................4-364

Table 4-86. Incidence of MN formation in mortuary students exposed to formaldehyde 

for 90 days...........................................................................................................4-365

Table 4-87. Multivariate regression models linking genomic instability/occupational 

exposures to selected endpoints from the MN assay...........................................4-369

Table 4-88. Genotoxicity measures in pathological anatomy laboratory workers and

controls................................................................................................................4-370

Table 4-89. Summary of human cytogenetic studies.............................................................4-372

Table 4-90. Summary of cohort and case-control studies which evaluated the 

incidence of all LHP cancers in formaldehyde-exposed populations 

(ICD-8 Codes: 200-209) and all leukemias (ICD-8 Codes: 204-207) ................4-412

Table 4-91. Secondary analysis of published mortality statistics to explore alternative 

disease groupings within the broad category of all lymphohematopoetic 

malignancies........................................................................................................4-419

Table 4-92. Summary of studies which provide mortality statistics for myeloid 

leukemia sub-types..............................................................................................4-420

Table 4-93. Summary of mortality statistics for Hodgkin’s lymphoma, lymphoma and 

multiple myeloma from cohort analyses of formaldehyde exposed workers......4-422

Table 4-94. Summary of chronic bioassays which address rodent leukemia and 

lymphoma............................................................................................................4-424

Table 4-95. Incidence of lymphoblastic leukemia and lymphosarcoma orally dosed in 

Sprague-Dawley rats ...........................................................................................4-425

Table 4-96. Available evidence for susceptibility factors of concern for formaldehyde 

exposure...............................................................................................................4-469
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF TABLES (continued)

Table 5-1. Points of departure (POD) for nervous system toxicity in key human and 

animal studies .......................................................................................................5-19

Table 5-2. Effects of formaldehyde exposure on completion of the labyrinth test by 

male and female LEW.1K rats .............................................................................5-23

Table 5-3. Developmental and reproductive toxicity PODs including duration 

adjustments – key human study.............................................................................5-31

Table 5-4. Summary of candidate studies for formaldehyde RfC development by 

health endpoint category .......................................................................................5-36

Table 5-5. Adjustment for nonoccupational exposures to formaldehyde ...............................5-64

Table 5-6. Summary of reference concentration (RfC) derivation from critical study and 

supporting studies..................................................................................................5-68

Table 5-7. Relative risk estimates for mortality from nasopharyngeal malignancies 

(ICD-8 code 147) by level of formaldehyde exposure for different 

exposure metrics....................................................................................................5-78

Table 5-8. Regression coefficients from NCI log-linear trend test models for NPC 

mortality from cumulative exposure to formaldehyde ..........................................5-79

Table 5-9. Extra risk estimates for NPC mortality from various levels of continuous 

exposure to formaldehyde ....................................................................................5-80

Table 5-10. EC0005, LEC0005, and inhalation unit risk estimates for NPC mortality from 

formaldehyde exposure based on the Hauptmann et al. (2004) log-linear 

trend analyses for cumulative exposure ................................................................5-81

Table 5-11. EC0005, LEC0005, and inhalation unit risk estimates for NPC incidence from 

formaldehyde exposure based on the Hauptmann et al. (2004) trend 

analyses for cumulative exposure..........................................................................5-82

Table 5-12. Relative risk estimates for mortality from Hodgkin lymphoma 

(ICD-8 code 201) and leukemia (ICD-8 codes 204–207) by level of 

formaldehyde exposure for different exposure metrics.........................................5-90

Table 5-13. Regression coefficients for Hodgkin lymphoma and leukemia mortality 

from NCI trend test models...................................................................................5-90

Table 5-14. Extra risk estimates for Hodgkin lymphoma mortality from various levels 

of continuous exposure to formaldehyde ..............................................................5-91
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LIST OF TABLES (continued)

Table 5-15. Extra risk estimates for leukemia mortality from various levels of 

continuous exposure to formaldehyde...................................................................5-91

Table 5-16. EC0005, LEC0005, and inhalation unit risk estimates for Hodgkin lymphoma 

mortality from formaldehyde exposure based on Beane Freeman et al. 

(2009) log-linear trend analyses for cumulative exposure ....................................5-93

Table 5-17. EC005, LEC005, and inhalation unit risk estimates for leukemia mortality 

from formaldehyde exposure based on Beane Freeman et al. (2009) 

log-linear trend analyses for cumulative exposure................................................5-93

Table 5-18. EC0005, LEC0005, and inhalation unit risk estimates for Hodgkin 

lymphoma incidence from formaldehyde exposure, based on Beane 

Freeman et al. (2009) log-linear trend analyses for cumulative exposure ............5-94

Table 5-19. EC005, LEC005, and inhalation unit risk estimates for leukemia incidence 

from formaldehyde exposure based on Beane Freeman et al. (2009) 

log-linear trend analyses for cumulative exposure................................................5-94

Table 5-20. Calculation of combined cancer mortality unit risk estimate at 0.1 ppm...........5-100

Table 5-21. Calculation of combined cancer incidence unit risk estimate at 0.1 ppm..........5-100

Table 5-22. Summary of tumor incidence in long-term bioassays on F344 rats...................5-105

Table 5-23. BMD modeling of unit risk of SCC in the human respiratory tract...................5-125

Table 5-24. Formaldehyde-induced rat tumor incidences.....................................................5-128

Table 5-25. Human benchmark extrapolations of nasal tumors in rats using 

formaldehyde flux and DPX................................................................................5-134

Table 5-26. Summary of inhalation unit risk estimates.........................................................5-135

Table 5-27. Total cancer risk from exposure to a constant formaldehyde exposure 

level of 1 μg/m3 from ages 0–70 years................................................................5-137

Table 6-1. Summary of candidate Reference Concentrations (RfC) for co-critical studies....6-27

Table 6-2. Effective concentrations (lifetime continuous exposure levels) predicted 

for specified extra cancer risk levels for selected formaldehyde-related 

cancers...................................................................................................................6-36

Table 6-3. Inhalation unit risk estimates based on epidemiological and experimental 

animal data.............................................................................................................6-42
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LIST OF FIGURES

Figure 2-1. Chemical structure of formaldehyde.......................................................................2-1

Figure 2-2. Locations of hazardous air pollutant monitors........................................................2-5

Figure 2-3. Modeled ambient air concentrations based on 1999 emissions..............................2-7

Figure 2-4. Range of formaldehyde air concentrations (ppb) in different environments..........2-9

Figure 3-1. Formaldehyde-mediated protein modifications......................................................3-2

Figure 3-2. 3

H/14C ratios in macromolecular extracts from rat tissues following exposure 

to 14C and 3

H-labeled formaldehyde (0.3, 2, 6, 10, 15 ppm)..............................3-18

Figure 3-3. Formaldehyde clearance by ALDH2 (GSH-independent) and ADH3 

(GSH-dependent)................................................................................................3-20

Figure 3-4. Metabolism of formate..........................................................................................3-22

Figure 3-5. Scatter plot of formaldehyde concentrations measured in ppb in direct breath 

exhalations (x axis) and exhaled breath condensate headspace (y axis)............3-31

Figure 3-6. Reconstructed nasal passages of F344 rat, rhesus monkey, and human ...............3-36

Figure 3-7. Illustration of interspecies differences in airflow and verification of CFD 

simulations with water-dye studies....................................................................3-37

Figure 3-8. Lateral view of nasal wall mass flux of inhaled formaldehyde simulated in 

the F344 rat, rhesus monkey, and human...........................................................3-38

Figure 3-9. CFD simulations of formaldehyde flux to human nasal lining at different 

inspiratory flow rates..........................................................................................3-39

Figure 3-10. Single-path model simulations of surface flux per ppm of formaldehyde 

exposure concentration in an adult male human ................................................3-43

Figure 3-11. Pressure drop vs. volumetric airflow rate predicted by the CIIT CFD 

model compared with pressure drop measurements made in two hollow 

molds (C1 and C2) of the rat nasal passage (Cheng et al., 1990) or in rats 

in vivo.................................................................................................................3-45

Figure 3-12. Formaldehyde-DPX dosimetry in the F344 rat...................................................3-47

Figure 4-1. Delayed asthmatic reaction following the inhalation of formaldehyde after 

“painting” 100% formalin for 20 minutes..........................................................4-20
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LIST OF FIGURES (continued)

Figure 4-2. Formaldehyde effects on minute volume in naïve and formaldehydepretreated male B6C3F1 mice and F344 rats...................................................4-115

Figure 4-3. Sagittal view of the rat nose (nares oriented to the left) .....................................4-121

Figure 4-4. Main components of the nasal respiratory epithelium........................................4-122

Figure 4-5. Decreased mucus clearance and ciliary beat in isolated frog palates 

exposed to formaldehyde after 3 days in culture..............................................4-126

Figure 4-6. Diagram of nasal passages showing section levels chosen for morphometry 

and autoradiography in male rhesus monkeys exposed to formaldehyde ........4-135

Figure 4-7. Formaldehyde-induced cell proliferation in male rhesus monkeys exposed to 

formaldehyde....................................................................................................4-136

Figure 4-8. Formaldehyde-induced lesions in male rhesus monkeys exposed to formaldehyde 

..........................................................................................................................4-137

Figure 4-9. Frequency and location by cross-section level of squamous metaplasia in 

the nasal cavity of F344 rats exposed to formaldehyde via inhalation ............4-172

Figure 4-10. Effect of formaldehyde exposure on cell proliferation of the respiratory 

mucosa of rats and mice ...................................................................................4-190

Figure 4-11. Alveolar MP Fc-mediated phagocytosis from mice exposed to 5 ppm 

formaldehyde, 10 mg/m3 carbon black, or both ...............................................4-223

Figure 4-12. Compressed air in milliliters as parameter for airway obstruction 

following formaldehyde exposure in guinea pigs after OVA sensitization and 

OVA challenge .................................................................................................4-235

Figure 4-13. OVA-specific IgG1 (1B) in formaldehyde-treated sensitized guinea pigs 

prior to OVA challenge ....................................................................................4-235

Figure 4-14. Anti-OVA titers in female Balb/C mice exposed to 6.63 ppm 

formaldehyde for 10 consecutive days, or once a week for 7 weeks...............4-236

Figure 4-15. Vascular permeability in the tracheae and bronchi of male Wistar rats 

after 10 minutes of formaldehyde inhalation ...................................................4-238

Figure 4-16. Effect of select receptor antagonists on formaldehyde-induced vascular 

permeability in the trachea and bronchi of male Wistar rats............................4-239
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LIST OF FIGURES (continued)

Figure 4-17. The effects of formaldehyde inhalation exposures on eosinophil 

infiltration (Panel A) and goblet cell proliferation (Panel B) after Der f 

challenge in the nasal mucosa of male ICR mice after sensitization and 

challenge...........................................................................................................4-241

Figure 4-18. NGF in BAL fluid from formaldehyde-exposed female C3H/He mice 

with and without OA sensitization ...................................................................4-243

Figure 4-19. Plasma Substance P levels in formaldehyde-exposed female C3H/He 

mice with and without OVA sensitization .......................................................4-244

Figure 4-20. Motor activity in male and female rats 2 hours after exposure to 

formaldehyde expressed as mean number of crossed quadrants ± SEM .........4-256

Figure 4-21. Habituation of motor activity was observed in control rats during the 

second observation period (day 2, 24 hours after formaldehyde exposure).....4-257

Figure 4-22. Motor activity was reduced in male and female LEW.1K rats 2 hours 

after termination of 10-minute formaldehyde exposure...................................4-258

Figure 4-23. The effects of the acute formaldehyde (FA) exposures on the 

ambulatory and vertical components of SLMA ...............................................4-260

Figure 4-24. Effects of formaldehyde exposure on the error rate of female LEW.1K 

rats performing the water labyrinth learning test .............................................4-264

Figure 4-25. Basal and stress-induced trunk blood corticosterone levels in male 

LEW.1K rats after formaldehyde inhalation exposures...................................4-269

Figure 4-26. NGF production in the brains of formaldehyde-exposed mice.........................4-274

Figure 4-27. Mortality corrected cumulative incidences of nasal carcinomas in the 

indicated exposure groups................................................................................4-325

Figure 4-28. Leukemia incidence in Sprague-Dawley rats exposed to formaldehyde 

in drinking water for 2 years ............................................................................4-330

Figure 4-29. Unscheduled deaths in female F344 rats exposed to formaldehyde for 

24 months.........................................................................................................4-332

Figure 4-30. Cumulative leukemia incidence in female F344 rats exposed to 

formaldehyde for 24 months ............................................................................4-333

Figure 4-31. Cumulative incidence or tumor bearing animals for lymphoma in 

female mice exposed to formaldehyde for 24 months......................................4-334
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF FIGURES (continued)

Figure 4-32. DNA-protein cross-links (DPX) and thymidine kinase (tk) mutants in 

TK6 human lymphoblasts exposed to formaldehyde for 2 hours ....................4-357

Figure 4-33. Developmental origins for cancers of the lymphohematopoietic system .........4-409

Figure 4-34A. Association between peak formaldehyde exposure and the risk of 

lymphohematopoietic malignancy ...................................................................4-415

Figure 4-34B. Association between average intensity of formaldehyde exposure and 

the risk of lymphohematopoietic malignancy ..................................................4-416

Figure 4-35. Effect of various doses of formaldehyde on cell number in (A) HT-29 

human colon carcinoma cells and in (B) human umbilical vein epithelial cells 

(HUVEC) .........................................................................................................4-433

Figure 4-36. Integrated MOA scheme for respiratory tract tumors.......................................4-446

Figure 4-37. Location of intra-epithelial lymphocytes along side epithelial cells in 

the human adenoid............................................................................................4-450

Figure 5-1. Change in number of additions made in 10 minutes following formaldehyde 

exposure at 32, 170, 390, or 890 ppb .................................................................5-21

Figure 5-2. Effects of formaldehyde exposure on the error rate of female LEW.1K rats 

performing the water labyrinth learning test......................................................5-24

Figure 5-3. Fecundity density ratio among women exposed to formaldehyde in the high 

exposure index category with 8-hour time weighted average formaldehyde 

exposure concentration of 219 ppb ....................................................................5-27

Figure 5-4. Estimated reduction in peak expiratory flow rate (PEFR) in children in 

relation to indoor residential formaldehyde concentrations...............................5-41

Figure 5.5. Odds ratios for physician-diagnosed asthma in children associated with inhome formaldehyde levels in air .......................................................................5-45

Figure 5-6. Prevalence of asthma and respiratory symptom scores in children associated 

with in-home formaldehyde levels.....................................................................5-48

Figure 5-7. Prevalence and severity of allergic sensitization in children associated 

with in-home formaldehyde levels.....................................................................5-49

Figure 5-8. Positive exposure-response relationships reported for in-home 

formaldehyde exposures and sensory irritation (eye irritation) .........................5-53
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF FIGURES (continued)

Figure 5-9. Positive exposure-response relationships reported for in-home 

formaldehyde exposures and sensory irritation (burning eyes) .........................5-54

Figure 5-10. Age-specific mortality and incidence rates for myeloid, lymphoid, and 

all leukemia ........................................................................................................5-98

Figure 5-11. Schematic of integration of pharmacokinetic and pharmacodynamic 

components in the CIIT model.........................................................................5-110

Figure 5-12. Fit to the rat tumor incidence data using the model and assumptions in 

Conolly et al. (2003).........................................................................................5-112

Figure 5-13. Spatial distribution of formaldehyde over the nasal lining, as 

characterized by partitioning the nasal surface by formaldehyde flux to 

the tissue per ppm of exposure concentration, resulting in 20 flux bins..........5-120

Figure 5-14. Distribution of cells at risk across flux bins in the F344 rat nasal lining..........5-120

Figure 5-15. MLE and upper bound (UB) added risk of SCC in the human nose for 

two BBDR models............................................................................................5-124

Figure 5-16. Replot of log-probit fit of the combined Kerns et al. (1983) and 

Monticello et al. (1996) data on tumor incidence showing BMC10 and 

BMCL10 ............................................................................................................5-127

Figure 5-17. EPA multistate Weibull modeling: nasal tumor dose response .......................5-131

Figure 5-18. Multistage Weibull model fit ............................................................................5-132

Figure 5-19. Multistage Weibull model fit of tumor incidence data compared with 

KM estimates of spontaneous tumor incidence................................................5-132
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygienists

ADAF age-dependent adjustment factors

ADH alcohol dehydrogenase

ADS anterior dorsal septum

AIC Akaike Information Criterion

AIE average intensity of exposure

AIHA American Industrial Hygiene Association

ALB albumin

ALDH aldehyde dehydrogenase

ALL acute lymphocytic leukemia

ALM anterior lateral meatus

ALP alkaline phosphatase

ALS amyotrophic lateral sclerosis

ALT alanine aminotransferase

AML acute myelogenous leukemia

AMM anterior medial maxilloturbinate

AMPase adenosine monophosphatase

AMS anterior medial septum

ANAE alpha-naphthylacetate esterase

ANOVA analysis of variance

APA American Psychiatric Association

ARB Air Resources Board

AST aspartate aminotransferase

ATCM airborne toxic control measure

ATP adenosine triphosphate

ATPase adenosine triphosphatase

ATS American Thoracic Society

ATSDR Agency for Toxic Substances and Disease Registry

AUC area under the curve

BAL bronchoalveolar lavage

BALT bronchus associated lymphoid tissue

BBDR biologically based dose response

BC bronchial construction

BCME bis(chloromethyl)ether

BDNF brain-derived neurotrophic factor

BEIR biologic effects of ionizing radiation

BfR German Federal Institute for Risk Assessment

BHR bronchial hyperresponsiveness

BMC benchmark concentration

BMCL 95% lower bound on the benchmark concentration

BMCR binuclated micronucleated cell ratefluoresce

BMD benchmark dose

BMDL 95% lower bound on the benchmark dose
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

BMR benchmark response

BN Brown-Norway

BrdU bromodeoxyuridine

BUN blood urea nitrogen

BW body weight

CA chromosomal aberrations

CalEPA California Environmental Protection Agency

CAP College of American Pathologists

CASRN Chemical Abstracts Service Registry Number

CAT catalase

CBMA cytokinesis-blocked micronucleus assay

CBMN cytokinesis-blocked micronucleus

CDC U.S. Centers for Disease Control and Prevention

CDHS California Department of Health Services

CFD computational fluid dynamics

CGM clonal growth model

CHO Chinese hamster ovary

CI confidence interval

CIIT Chemical Industry Institute of Toxicology

CLL chronic lymphocytic leukemia

CML chronic myelogenous leukemia

CNS central nervous system

CO2 carbon dioxide

COEHHA California Office of Environmental Health Hazard Assessment

CREB cyclic AMP responsive element binding proteins

CS conditioned stimulus

C × t concentration times time

DA Daltons

DAF dosimetric adjustment factor

DDX DNA-DNA cross-links

DEI daily exposure index

DEN diethylnitrosamine

Der f common dust mite allergen

DMG dimethylglycine

DMGDH dimethylglycine dehydrogenase

DNA deoxyribonucleic acid

DOPAC 3,4-dihydroxyphenylacetic acid

DPC / DPX DNA-protein cross-links 

EBV Epstein-Barr virus

EC effective concentration

ED effective dose

EHC Environmental Health Committee

ELISA enzyme-linked immunosorbent assay
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

EPA U.S. Environmental Protection Agency

ERPG emergency response planning guideline

ET ethmoid turbinates

FALDH formaldehyde dehydrogenase

FDA U.S. Food and Drug Administration

FDR fecundability density ratio

FEF forced expiratory flow

FEMA Federal Emergency Management Agency

FEV1 forced expiratory volume in 1 second

FISH fluorescent in situ hybridization

FSH follicle-stimulating hormone

FVC forced vital capacity

GALT gut-associated lymphoid tissue

GC-MS gas chromatography-mass spectrometry

GD gestation day

GI gastrointestinal

GO gene ontology

G6PDH glucose-6-phosphate dehydrogenase

GPX glutathione peroxidase

GR glutathione reductase

GM-CSF granulocyte macrophage-colony-stimulating factor

GSH reduced glutathione

GSNO S-nitrosoglutathione

GST glutathione S-transferase

HAP hazardous air pollutant

Hb hemoglobin

HCl hydrochloric acid

HCT hematocrit

HEC human equivalent concentration

5-HIAA 5-hydroxyindoleacetic acid

hm hydroxymethyl

HMGSH S-hydroxymethylglutathione

HPA hypothalamic-pituitary adrenal

HPG hypothalamo-pituitary-gonadal

HPLC high-performance liquid chromatography

HPRT hypoxanthine-guanine phosphoribosyl transferase

HR high responders

HSA human serum albumin

HSDB Hazardous Substances Data Bank

Hsp heat shock protein

HWE healthy worker effect

I cell initiated cell

IARC International Agency for Research on Cancer
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

ICD International Classification of Diseases

IF interfacial

IFN interferon

Ig immunoglobulin

IL interleukin

I.P. intraperitoneal

IPCS International Programme on Chemical Safety

IRIS Integrated Risk Information System

Km Michaels-Menton constant

KM Kaplan-Meier

LD50 median lethal dose

LDH lactate dehydrogenase

LEC 95% lower bound on the effective concentration

LED 95% lower bound on the effective dose

LHP lymphohematopoietic

LI labeling index

LM Listeria monocytogenes

LMS linearized multistage

LLNA local lymph node assay

LOAEL lowest-observed-adverse-effect level

LPS lipopolysaccharide

LR low responders

LRT lower respiratory tract

MA methylamine

MALT mucus-associated lymph tissues

MCH mean corpuscular hemoglobin

MCHC mean corpuscular hemoglobin concentration

MCS multiple chemical sensitivity

MCV mean corpuscular volume

MDA malondialdehyde

MEF maximal expiratory flow

ML myeloid leukemia

MLE maximum likelihood estimate

MMS methyl methane sulfonate

MMT medial maxilloturbinate

MN micronucleus, micronuclei

MNNG N-methyl-N-nitro-N-nitrosoguanidine

MOA mode of action

MoDC monocyte-derived dendritic cell

MP macrophage

MPD multistage polynomial degree

MPS mononuclear phagocyte system

MRL minimum risk level
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

mRNA messenger ribonucleic acid

MVE-2 Murray Valley encephalitis virus

MVK Moolgavkar, Venzon, and Knudson

N cell normal cell

NaCl sodium choride

NAD+ nicotinamide adenine dinucleotide

NADH reduced nicotinamide adenine dinucleotide

NALT nasally associated lymphoid tissue

NATA National-Scale Air Toxics Assessment

NCEA National Center for Environmental Assessment

NCHS National Center for Health Statistics

NCI National Cancer Institute

NEG Nordic Expert Group

NER nucleotide excision repair

NGF nerve growth factor

NHL non-Hodgkin’s lymphoma

NHMRC/ARMCANZ National Health and Medical Research Council/Agriculture and Resource 

Management Council of Australia and New Zealand

NNK nitrosamine nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-butanone

N6

-hmdA N6-hydroxymethyldeoxyadenosine

N4-hmdC N4-hydroxymethylcytidine

N2

-hmdG N2-hydroxymethyldeoxyguanosine

NICNAS National Industrial Chemicals Notification and Assessment Scheme

NIOSH National Institute for Occupational Safety and Health

NLM National Library of Medicine

NMDA N-methyl-D-aspartate

NO nitric oxide

NOAEL no-observed-adverse-effect level

NPC nasopharyngeal cancer

NRBA neutrophil respiratory burst activity

NRC National Research Council

NTP National Toxicology Program

OR odds ratio

OSHA Occupational Safety and Health Administration

OTS Office of Toxic Substances

OVA ovalbumin

PBPK physiologically based pharmacokinetic

PC Philadelphia chromosome

PCA passive cutaneous anaphylaxis

PCMR proportionate cancer mortality ratio

PCNA proliferating cell nuclear antigen

PCR polymerase chain reaction

PCV packed cell volume
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

PECAM platelet endothelial cell adhesion molecule

PEF peak expiratory flow

PEFR peak expiratory flow rates

PEL permissible exposure limit

PFC plaque-forming cell

PG periglomerular

PHA phytohemagglutinin

PLA2 phospholipase A2

PI phagocytic index

PLM posterior lateral meatus

PMA phorbol 12-myristate 13-acetate

PMR proportionate mortality ratio

PMS posterior medial septum

PND postnatal day

POD point of departure

POE portal of entry

PTZ pentilenetetrazole

PUFA polyunsaturated fatty acids

PWULLI population weighted unit length labeling index

RA reflex apnea

RANTES regulated upon activation, normal T–cell expressed and secreted

RB reflex bradypnea

RBC red blood cells

RD50 exposure concentration that results in a 50% reduction in respiratory rate

REL recommended exposure limit

RfC reference concentration

RfD reference dose

RGD regional gas dose

RGDR regional gas dose ratio

RR relative risk

RT reverse transcriptase

SAB Science Advisory Board

SCC squamous cell carcinoma

SCE sister chromatid exchange

SCG sodium cromoglycate

SD standard deviation

SDH succinate dehydrogenase; sarcosine dehydrogenase

SEER Surveillance, Epidemiology, and End Results

SEM standard error of the mean

SEN sensitizer

SH sulfhydryl

SHE Syrian hamster embryo

SLMA spontaneous locomotor activity
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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

SMR standardized mortality ratio

SNP single nucleotide polymorphism

SOD superoxide dismutase

SOMedA N6-sulfomethyldeoxyadenosine

Sp1 specificity protein

SPIR standardized proportionate incidence ratio

SSAO semicarbozole-sensitive amine oxidase

SSB single strand breaks

STEL short-term exposure limit

TBA tumor bearing animal

TH T-lymphocyte helper

THF tetrahydrofolate

TK toxicokinetics

TL tail length

TLV threshold limit value

TNF tumor necrosis factor

TP total protein

TRI Toxic Release Inventory

TRPV transient receptor potential vanilloid

TWA time-weighted average

TZCA thiazolidine-4-carboxylate

UCL upper confidence limit

UDS unscheduled DNA synthesis

UF uncertainty factor

UFFI urea formaldehyde foam insulation

ULLI unit length labeling index

URT upper respiratory tract

USDA U.S. Department of Agriculture

VC vital capacity

VOC volatile organic compound

WBC white blood cell

WDS wet dog shake

WHO World Health Organization

WHOROE World Health Organization Regional Office for Europe
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FOREWORD

The purpose of this Toxicological Review is to provide scientific support and rationale 

for the hazard and dose-response assessment in IRIS pertaining to chronic inhalation exposure to 

formaldehyde. It is not intended to be a comprehensive treatise on the chemical or toxicological 

nature of formaldehyde.

In Chapter 6, Major Conclusions in the Characterization of Hazard and Dose Response,

EPA has characterized its overall confidence in the qualitative and quantitative aspects of hazard 

and dose response by addressing knowledge gaps, uncertainties, quality of data, and scientific 

controversies. The discussion is intended to convey the limitations of the assessment and to aid 

and guide the risk assessor in the ensuing steps of the risk assessment process.

For other general information about this assessment or other questions relating to IRIS, 

the reader is referred to EPA’s IRIS Hotline at (202) 566-1676 (phone), (202) 566-1749 (fax), or 

hotline.iris@epa.gov (email address).
This document is a draft for review purposes only and does not constitute Agency policy.

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AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGERS

John E. Whalan, D.A.B.T.1

EPA-ORD-NCEA

Danielle DeVoney, PhD, D.A.B.T., PE2

EPA-ORD-NCEA

AUTHORS

Thomas Bateson, PhD

EPA-ORD-NCEA

Susan Euling, PhD

EPA-ORD-NCEA

Jennifer Jinot, PhD

Susan Makris, PhD

EPA-ORD-NCEA

Kathleen Raffaele, PhD

EPA-ORD-NCEA

John Schaum, PhD

EPA-ORD-NCEA

Ravi Subramaniam, PhD

EPA-ORD-NCEA

Suryanarayana Vulimiri, PhD

EPA-ORD-NCEA

CONTRIBUTORS

Gillian Backus, PhD 3

Stanley Barone, PhD

EPA-ORD-NCEA

 1 Chemical Manager since July 2003.

2 Chemical Manager since June 2009

3 Separated from the Agency prior to final revisions to document.
This document is a draft for review purposes only and does not constitute Agency policy.

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AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)

David Bayliss, PhD 3

EPA-ORD-NCEA

Ted Berner, PhD

EPA-ORD-NCEA

David Bussard

EPA-ORD-NCEA

David Farrar, PhD

EPA-ORD-NCEA

John Fox, PhD

EPA-ORD-NCEA

EPA-ORD-NCEA

Karen Hogan, PhD

EPA-ORD-NCEA

Rosemarie Hakim, PhD 3

EPA-ORD-NCEA

Babashaheb Sonawane, PhD

EPA-ORD-NCEA

Chad Thompson, PhD 3

EPA-ORD-NCEA

Larry Valcovic, PhD 3

EPA-ORD-NCEA

John J. Vandenberg, PhD

EPA-ORD-NCEA

Lisa Vinikoor, PhD

EPA-ORD-NCEA

Paul White, PhD

EPA-ORD-NCEA
This document is a draft for review purposes only and does not constitute Agency policy.

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AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)

CONTRACTOR SUPPORT

The literature search and preliminary drafts of this document as well as support for 

editing and formatting were provided by Oak Ridge Institute for Science and Education 

(ORISE), Oak Ridge Associated Universities (ORAU), Department of Energy, under 

Interagency Agreement (IAG) Project No. 03-18. The ORISE individuals who contributed to 

this effort include Sheri Hester, George Holdsworth, Bobette D. Nourse, Wanda Olson, and Lutz 

W. Weber.

Assistance with the biologically based dose response model evaluation was provided by 

ENVIRON International Corporation of Monroe, Louisiana (subcontractors to ORISE; Project 

No. 03-18). The primary scientists involved in the work were Kenny S. Crump and Cynthia Van 

Landingham.

INTERNAL EPA REVIEWERS

Daniel Axelrad, PhD

Office of Policy, Economics, and Innovation

Iris Camacho, PhD

Office of Pollution Prevention and Toxics

Christina Cinalli, PhD

Office of Pollution Prevention and Toxics

Rebecca Edelstein, PhD

Office of Pollution Prevention and Toxics

Ernest Falke, PhD

Office of Pollution Prevention and Toxics

Stiven Foster, PhD

Office of Solid Waste and Emergency Response

Greg Fritz

Office of Pollution Prevention and Toxics

Susan Griffin, PhD

EPA Region 8
This document is a draft for review purposes only and does not constitute Agency policy.

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AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)

Timothy Leighton, PhD

Office of Pesticide Programs

Elizabeth Margosches, PhD

Office of Pollution Prevention and Toxics

Timothy McMahon, PhD

Office of Pesticde Programs

Julie Migrin-Sturza, PhD

Office of Policy, Economics, and Innovation

Greg Miller, PhD

Office of Children’s Health Protection and Environmental Education 

Deirdre Murphy, PhD

Office of Air and Radiation

Marion Olson, PhD

EPA Region 2

Andrea Pfahles-Hutchens, PhD

Office of Pollution Prevention and Toxics

Jennifer Seed, PhD

Office of Pollution Prevention and Toxics
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1 1. INTRODUCTION

2

3

4 This document presents background information and justification for the Integrated Risk 

5 Information System (IRIS) Summary of the hazard and dose-response assessment of 

6 formaldehyde. IRIS Summaries may include oral reference dose (RfD) and inhalation reference 

7 concentration (RfC) values for chronic and other exposure durations, and a carcinogenicity 

8 assessment. 

9 The RfD and RfC, if derived, provide quantitative information for use in risk assessments 

10 for health effects known or assumed to be produced through a nonlinear (presumed threshold) 

11 mode of action. The RfD (expressed in units of mg/kg-day) is defined as an estimate (with 

12 uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human 

13 population (including sensitive subgroups) that is likely to be without an appreciable risk of 

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3 14 ) is 

15 analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The 

16 inhalation RfC considers toxic effects for both the respiratory system (portal of entry [POE]) and 

17 for effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference 

18 values are generally derived for chronic exposures (up to a lifetime), but may also be derived for 

19 acute (≤24 hours), short-term (>24 hours up to 30 days), and subchronic (>30 days up to 10% of 

20 lifetime) exposure durations, all of which are derived based on an assumption of continuous 

21 exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are 

22 derived for chronic exposure duration.

23 The carcinogenicity assessment provides information on the carcinogenic hazard 

24 potential of the substance in question and quantitative estimates of risk from oral and inhalation 

25 exposure may be derived. The information includes a weight-of-evidence judgment of the 

26 likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic 

27 effects may be expressed. Quantitative risk estimates may be derived from the application of a 

28 low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on 

29 the estimate of risk per mg/kg-day of oral exposure. Similarly, an inhalation unit risk is a 

plausible upper bound on the estimate of risk per μg/m3 30 air breathed. 

31 Development of these hazard identification and dose-response assessments for 

32 formaldehyde has followed the general guidelines for risk assessment as set forth by the National 

33 Research Council (NRC) (1983). EPA Guidelines and Risk Assessment Forum Technical Panel 

34 Reports that may have been used in the development of this assessment include the following: 

35 Guidelines for the Health Risk Assessment of Chemical Mixtures (U.S. EPA, 1986a), Guidelines 

36 for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Recommendations for and Documentation 
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1 of Biological Values for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for 

2 Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Interim Policy for Particle Size and 

3 Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, 1994a), Methods for Derivation of 

4 Inhalation Reference Concentrations and Application of Inhalation Dosimetry (U.S. EPA, 

5 1994b), Use of the Benchmark Dose Approach in Health Risk Assessment (U.S. EPA, 1995), 

6 Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 1996), Guidelines for 

7 Neurotoxicity Risk Assessment (U.S. EPA, 1998), Science Policy Council Handbook: Risk 

8 Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance Document (U.S. 

9 EPA, 2000b), Supplementary Guidance for Conducting Health Risk Assessment of Chemical 

10 Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference Concentration 

11 Processes (U.S. EPA, 2002a), Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), 

12 Supplemental Guidance for Assessing Susceptibility from Early-Life Exposure to Carcinogens 

13 (U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 2006a), and A 

14 Framework for Assessing Health Risks of Environmental Exposures to Children (U.S. EPA, 

15 2006b).

16 The literature search strategy employed for this compound was based on the Chemical 

17 Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent 

18 scientific information submitted by the public to the IRIS Submission Desk was also considered 

19 in the development of this document. The relevant literature was reviewed through April, 2009, 

20 but some criticial literature after this date has been considered in this assessment.

21
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1 2. BACKGROUND

2

3

4 This chapter provides an overview of the physical and chemical characteristics of 

5 formaldehyde. Also provided in this chapter are a description of the production, uses, and 

6 sources of formaldehyde and information regarding environmental levels and human exposure. 

7 A description of the toxicokinetics and toxicodynamic processes involved in formaldehyde 

8 toxicity for the inhalation, oral, and dermal routes can be found in Chapter 3 (Toxicokinetics). 

9

10 2.1. PHYSICOCHEMICAL PROPERTIES OF FORMALDEHYDE

11 Formaldehyde (CASRN 50-00-0) is the first of the series of aliphatic aldehydes and is a 

12 gas at room temperature. Its molecular structure is depicted in Figure 2-1. It is noted for its 

13 reactivity and versatility as a chemical intermediate. It readily undergoes polymerization, is 

14 highly flammable, and can form explosive mixtures with air. It decomposes at temperatures 

15 above 150°C.

16

17 Figure 2-1. Chemical structure of formaldehyde.

18

19

20 At room temperature, pure formaldehyde is a colorless gas with a strong, pungent, 

21 suffocating, and highly irritating odor. Formaldehyde is readily soluble in water, alcohols, ether, 

22 and other polar solvents. A synopsis of its physicochemical properties is given in Table 2-1. 

23

24 2.2. PRODUCTION, USES, AND SOURCES OF FORMALDEHYDE

25 Formaldehyde has been produced commercially since the early 1900s and, in recent 

26 years, has been ranked in the top 25 highest volume chemicals produced in the U.S. (National 

27 Toxicology Program [NTP], 2002). In 2003, 4.33 million metric tons of formaldehyde were 

28 produced in the U.S. (Global Insight, 2006). In 2000, worldwide formaldehyde production was 

29 estimated to be 21.5 million metric tons, (International Agency for Research on Cancer [IARC], 

30 2006).

31

C O

H

H
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1 Table 2-1. Physicochemical properties of formaldehyde

2

Name Formaldehyde

International Union for Pure and Applied 

Chemistry name

Formaldehyde

Synonyms Formic aldehyde

Methanal

Methyl aldehyde

Methylene oxide

Oxomethane

Oxymethylene

Chemical Abstracts Service Index name Formaldehyde

CASRN 50-00-0

Formula HCHO

Molecular weight 30.03

Density Gas: 1.067 (air = 1)

Liquid: 0.815 g/mL at –20°C

Vapor pressure 3,883 mm Hg at 25°C

Log Kow –0.75 to 0.35

Henry’s law constant 3.4 × 10–7 atm-m3 /mol at 25°C

2.2 × 10–2 Pa-m3/mol at 25°C

Conversion factors (25oC, 760 mm Hg) 1 ppm = 1.23 mg/m3 (v/v)

1 mg/m3 = 0.81 ppm (v/v)

Boiling point –19.5°C at 760 mm Hg

Melting point –92°C

Flash point 60°C; 83°C, closed cup for 37 %, methanol-free 

aqueous solution; 50°C closed cup for 37% 

aqueous solution with 15% methanol

Explosive limits 73% upper; 7% lower by volume in air

Autoignition temperature 300°C

Solubility Very soluble in water; soluble in alcohols, ether, 

acetone, benzene

Reactivity Reacts with alkalis, acids and oxidizers

3

4 Sources: American Conference of Governmental Industrial Hygienists (ACGIH) (2002); 

5 International Programme on Chemical Safety (IPCS) (2002); Agency for Toxic Substances and 

6 Disease Registry (ATSDR) (1999); Gerberich and Seaman (1994); Walker (1975).

7

8

9 Formaldehyde is a chemical intermediate used in the production of plywood adhesives, 

10 abrasive materials, insulation, foundry binders, brake linings made from phenolic resins, surface 

11 coatings, molding compounds, laminates, wood adhesives made from melamine resins, phenolic 

12 thermosetting, resin curing agents, explosives made from hexamethylenetetramine, urethanes, 

13 lubricants, alkyd resins, acrylates made from trimethylolpropane, plumbing components from 

14 polyacetal resins, and controlled-release fertilizers made from urea formaldehyde concentrates 
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1 (IPCS, 1989). Formaldehyde is used in smaller quantities for the preservation and embalming of 

2 biological specimens. It is also used as a germicide, an insecticide, and a fungicide, as well as an 

3 antimicrobial agent in soaps, shampoos, hair preparations, deodorants, lotions (e.g., suntan lotion 

4 and dry skin lotion), makeup, and mouthwashes, and is present in hand cream, bath products, 

5 mascara and other eye makeup, cuticle softeners, nail creams, vaginal deodorants, and shaving 

6 cream (IPCS, 2002; ATSDR, 1999).

7 Formaldehyde is commonly produced as an aqueous solution called formalin, which 

8 usually contains about 37% formaldehyde and 12–15% methanol. Methanol is added to formalin 

9 to slow polymerization that leads eventually to precipitation as paraformaldehyde. 

10 Paraformaldehyde has the formula (CH2O)n where n is 8 to 100. It is essentially a solid form of 

11 formaldehyde and therefore has some of the same uses as formaldehyde (Kiernan, 2000). When 

12 heated, paraformaldehyde sublimes as formaldehyde gas. This characteristic makes it useful as a 

13 fumigant, disinfectant, and fungicide, such as for the decontamination of laboratories, 

14 agricultural premises, and barbering equipment. Long-chain polymers (e.g., Delrin plastic) are 

15 less inclined to release formaldehyde, but they have a formaldehyde odor and require additives 

16 to prevent decomposition (U.S. EPA, 2008). 

17 The major sources of anthropogenic emissions of formaldehyde are motor vehicle 

18 exhaust, power plants, manufacturing plants that produce or use formaldehyde or substances that 

19 contain formaldehyde (i.e., adhesives), petroleum refineries, coking operations, incineration, 

20 wood burning, and tobacco smoke. Among these anthropogenic sources, the greatest volume 

21 source of formaldehyde is automotive exhaust from engines not fitted with catalytic converters 

22 (NEG, 2003). The Toxic Release Inventory (TRI) data for 2007 show total releases of 

23 21.9 million pounds with about half to the air and half to underground injection (EPA TRI 

24 Explorer, http://www.epa.gov/triexplorer/) (U.S. EPA, 2009a).

25 Formaldehyde is formed naturally in the lower atmosphere during the oxidation of 

26 hydrocarbons (i.e., methane and terpene), which react with hydroxyl radicals and ozone to form 

27 formaldehyde and other aldehydes, as intermediates in a series of reactions that ultimately lead 

28 to the formation of carbon monoxide and carbon dioxide, hydrogen and water. Formaldehyde 

29 can also be formed in a variety of other natural processes, such as decomposition of plant 

30 residues in the soil, photochemical processes in sea water, and forest fires (National Library of 

31 Medicine [NLM], 2001).

32 Formaldehyde emitted to the ambient air primarily reacts with photochemically generated 

33 hydroxyl radicals in the troposphere or undergoes direct photolysis (IPCS, 2002). Overall, half34 lives for formaldehyde in air can vary considerably under different conditions. Estimates for 

35 atmospheric residence time in several U.S. cities ranged from 0.3 hours under conditions typical 
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1 of a rainy winter night to 250 hours under conditions typical of a clear summer night (assuming 

2 no reaction with hydroperoxyl radicals). Given the generally short daytime residence times for 

3 formaldehyde, there is limited potential for long-range transport (IPCS, 2002). In cases where 

4 organic precursors are transported long distances, however, secondary formation of 

5 formaldehyde may occur far from the anthropogenic sources of the precursors.

6 Formaldehyde is released to water from the discharges of both treated and untreated 

7 industrial wastewater from its production and from its use in the manufacture of formaldehyde8 containing resins (ATSDR, 1999). Formaldehyde is also a possible drinking-water disinfection 

9 by-product from the use of ozone and/or hydrogen peroxide. In water, formaldehyde is rapidly 

10 hydrated to form a glycol, and the equilibrium favors the glycol.

11

12 2.3. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE

13 General population exposure to formaldehyde can occur via inhalation, ingestion and 

14 dermal contact. Each of these pathways and associated media levels are discussed below. 

15 Formaldehyde exposure can also occur occupationally via three main scenarios:

16

17 • The production of aqueous solutions of formaldehyde (formalin) and their use in the 

18 chemical industry (e.g., for the synthesis of various resins, as a preservative in medical 

19 laboratories and embalming fluids, and as a disinfectant). 

20 • Release from formaldehyde-based resins in which it is present as a residue and/or 

21 through their hydrolysis and decomposition by heat (e.g., during the manufacture of 

22 wood products, textiles, synthetic vitreous insulation products, and plastics). In general, 

23 the use of phenol–formaldehyde resins results in much lower emissions of formaldehyde 

24 than those of urea- based resins. 

25 • The pyrolysis or combustion of organic matter (e.g., in engine exhaust gases or during 

26 firefighting) (IARC, 2006). 

27

28 Industries with the greatest potential for exposure include health services, business 

29 services, printing and publishing, manufacture of chemicals and allied products, manufacture of 

30 apparel and allied products, manufacture of paper and allied products, personal services, 

31 machinery (except clerical), transport equipment, and furniture and fixtures (IARC, 1995).

32
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1 2.3.1. Inhalation

2 The most current ambient air monitoring data for formaldehyde come from EPA’s air 

3 quality system database (EPA’s AirData Web site: http://www.epa.gov/air/data/index.html) 

4 (U.S. EPA, 2009b). These data have been collected from a wide variety of sources, including 

5 state and local environmental agencies, but have not been collected from a statistically based 

6 survey. The most recent data, for the year 2007, come from 188 monitors located in 33 states as 

7 shown in Figure 2-2 (U.S. EPA, 2008). The annual means for these monitors range from 0.7–

45.03 μg/m3 (0.56–36.31 ppb) and have an overall average of 3.44 μg/m3 8 (2.77 ppb). The annual 

9 means are derived by EPA by averaging all available daily data from each monitor. Table 2-2 

10 shows a breakout of the data by land use category based on the annual means from each monitor 

11 for 2005, 2006, and 2007. The land use is established on the basis of the most prevalent land use 

12 within 0.25 miles of the monitor. The mobile category (land near major highways or interstates 

13 such that it is primarily impacted by mobile sources) has the highest mean levels, and 

14 agricultural lands have the lowest.

15

16

17 Figure 2-2. Locations of hazardous air pollutant monitors.

18 Dasgupta et al. (2005) measured formaldehyde levels in 5 U.S. cities during 1999 - 2002. 

19 Samples were collected over approximately a one month period in the spring or summer. 

20 Mean levels were 5.05 ppb in Nashville, TN; 7.96 ppb in Atlanta, GA; 4.49 ppb in 

21 Houston, TX; 3.12 ppb in Philadelphia, PA; and 2.63 in Sydney, FL.
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1 Table 2-2. Ambient air levels by land use category

2

Formaldehyde Exposure by Categorya

Agriculture Commercial Forest Industrial Mobileb Residential

Number of data points 17 166 19 61 16 282

Mean ± standard deviation 2.08 ± 0.98 3.26 ± 2.76 2.79 ± 2.17 6.28 ± 14.45 6.84 ± 7.28 2.75 ± 1.71

Minimum 0.34 0.20 0.40 0.14 2.02 0.17

Maximum 4.34 20.61 7.33 74.72 23.39 12.35

3 a

Values are μg/m3 4 . b 5 “Mobile” is ambient air in locations primarily impacted by mobile sources.

6

7 Source: AirData for 2005, 2006, and 2007 (U.S. EPA, 2009b).

8

9

10 Under the National-Scale Air Toxics Assessment (NATA) program, EPA has conducted 

11 an emissions inventory for a variety of hazardous air pollutants (HAPs), including formaldehyde 

12 (U.S. EPA, 2006c). The NATA uses the emissions inventory data to model nationwide air 

13 concentrations/exposures (U.S. EPA, 2006c). The results of the 1999 ambient air concentration 

modeling for formaldehyde suggest that county median air levels range from 0 to 6.94 μg/m3 14 (0 

– 5.59 ppb) with a national median of 0.56 μg/m3 15 (0.45 ppb) (see Figure 2-3). Similar results 

were found for the year 2002: county concentrations ranged from 0.12 to 9.17 μg/m3 16 (0.097 –

7.38 ppb) with median of 0.78 μg/m3 17 (0.63 ppb). NATA has not provided updated concentration 

18 maps for 2002. The 1999 map shows the highest levels in the far west and northeastern regions 

19 of the U.S. While these modeling results can be useful, it is important to consider their 

20 limitations. Some of the geographical differences result from differences in methods used by 

21 states supplying the data. For example, the high levels indicated for Idaho result from the large 

22 amount of wood burned during forest fires and the relatively high emission factor that Idaho uses 

23 (compared with other states) to estimate formaldehyde emissions from forest fires. A 

24 comparison of modeling results from NATA to measured values at the same locations is 

25 presented in U.S. EPA (2006c). For 1999, it was found that formaldehyde levels were 

26 underestimated at 76% of the sites (n = 68). One possible reason why the NATA results appear 

27 low compared to measurements is that the modeling has not accounted for secondary formation 

28 of formaldehyde in the atmosphere.
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1

2

3 Figure 2-3. Modeled ambient air concentrations based on 1999 emissions.

4

5

6 In general, ambient levels of formaldehyde in outdoor air are significantly lower than 

7 those measured in the indoor air of workplaces or residences (ATSDR, 1999; IARC, 1995). 

8 Indoor sources of formaldehyde in air include volatilization from pressed wood products, 

9 carpets, fabrics, insulation, permanent press clothing, latex paint, and paper bags, along with 

10 emissions from gas burners, kerosene heaters, and cigarettes (NLM, 2001). In general, the major 

11 indoor air sources of formaldehyde can be described in two ways: 1) those sources that have the 

12 highest emissions when the product is new with decreasing emission over time, as with the first 

13 set in the examples above; and 2) those sources that are reoccurring or frequent such as the 

14 second set of examples above. Gilbert et al. (2006) studied 96 homes in Quebec City, Canada 

15 and found elevated levels in homes with new wood or melamine furniture purchased within the 

16 previous 12 months. A summary of indoor data is provided in Table 2-3. Results vary 

17 depending on housing characteristics and date of study.

18
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1

Table 2-3. Studies on residential indoor air levels of formaldehyde (nonoccupational)

Citation

No. of 

Samples Target Population/House Type Mean (μg/m3)

Range 

(μg/m3)

Gold et al., 1993 Complaint homes

Older conventional homes

<60 24 - 960

Hare et al., 1996 Newly built homes 91

Hare et al., 1996 30 days after installing pressed wood 42 - 540

Gammage and 

Hawthorne, 1985

>1200

131

>500

260

Homes with UFFI

Homes without UFFI

Complaint mobile homes

Newer mobile homes

Older mobile homes

60 - 144

30 - 84

120 - 1080

1032

300

12 - 4080

12 - 204

0 - 5040

Hawthorne et al., 

1986 a,b

18

11

11

40

Conventional homes 0-5 yr

Conventional homes 5-15 yr

Conventional homes >15 yr

Conventional homes overall

96

48

36

72 24 - 480

U.S.EPA, 1987 560 Noncomplaint, conventional, 

randomly selected

Noncomplaint, mobile homes, 

randomly selected

32-109

109-744

6-576

12-3480

Health Canada and 

Environment 

Canada, 2001

151 Residential (Canadian) noncomplaint 

homes

35 ?-148

Zhang et al., 1994 

a,b

6 Residential, carpeted, non-smoking 

homes

66 42-89

Gilbert et al., 2006 96 Residential (Canadian) 29.5 9.6 – 90.0

Shah and Singh, 

1988

315 Residential & commercial 59 23-89

Stock, 1987 43 Conventional homes 84 96-216

Krzyzanowski et al., 

1990

202 Conventional homes 31

2

Note: 1 ppb = 1.2 μg/m3 3

4

5

6 Salthammer et al. (2010) present a thorough review of formaldehyde sources and levels 

7 found in the indoor environment. Based on an examination of international studies carried out in 

8 2005 or later they conclude that the average exposure of the population to formaldehyde is 20 to

40 μg/m3 9 under normal living conditions. They used the diagram shown in Figure 2-4 to 
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1 summarize data they found on the range of formaldehyde air concentrations (in ppb) in different 

2 environments.

3

4 Figure 2-4. Range of formaldehyde air concentrations (ppb) in different 

5 environments. Source: Salthammer et al. (2010)

6

7

8 Data on formaldehyde levels in outdoor and indoor air were collected under Canada’s 

9 National Air Pollution Surveillance program (IPCS, 2002; Health Canada and Environment 

10 Canada, 2001). The effort included four suburban and four urban sites sampled in the period 

11 1990–1998. A Monte Carlo analysis applied to the pooled data (n = 151) was used to estimate 

12 the distribution of time-weighted 24-hour air exposures. This study suggested that mean levels 

in outdoor air were 3.3 μg/m3 (2.7 ppb) and mean levels in indoor air were 35.9 μg/m3 13

14 (29.2 ppb) (Health Canada and Environment Canada, 2001). The simulation analysis also 

suggested that general population exposures averaged 33–36 μg/m3 15 (27–30 ppb).

16 Since the early to mid 1980s, manufacturing processes and construction practices have 

17 been changed to reduce levels of indoor formaldehyde emissions (ATSDR, 1999). A 2008 law 

18 enacted by the California Air Resource Board (CARB. 2008, Final Regulation Order: Airborne 

19 Toxic Control Measure to Reduce Formaldehyde Emissions from Composite Wood Products; 

20 http://www.arb.ca.gov/regact/2007/compwood07/fro-final.pdf) has limited the amount of 

21 formaldehyde that can be released by specific composite wood products (i.e., hardwood 

0.1 1 10 100 1000

Remote Air

Rural Air

Urban Air

Normal Indoor Air

Polluted Indoor Air

Extreme Conditions
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1 plywood, particle board, and medium density fiberboard) sold, supplied, or manufactured for use 

2 in California. For this reason the mean indoor air levels presented by Health Canada and 

3 Environment Canada (2001) (based on samples collected from 1989–1995) may overestimate 

4 current levels. In addition, the Canadian indoor air data may overestimate formaldehyde levels 

5 in U.S. homes, because many residential homes in Canada use wood burning stoves more 

6 frequently and have tighter construction (due to colder winters), leading to less dilution of indoor 

7 emissions. The outdoor air levels, however, appear to have remained fairly constant over recent 

years, and the median outdoor level from the Canadian study (2.8 μg/m3 8 ) (2.3 ppb) is very 

similar to the median of the U.S. monitoring data (2.83 μg/m3 9 ) (2.3 ppb) in 1999.

10 Even though formaldehyde levels in construction materials have declined, indoor 

11 inhalation concerns still persist. For example, recent studies have measured formaldehyde levels 

12 in mobile homes. ATSDR (2007) reported on air sampling in 96 unoccupied trailers provided by 

13 the Federal Emergency Management Agency (FEMA) used as temporary housing for people 

14 displaced by Hurricane Katrina. Formaldehyde levels in closed trailers averaged 1,250 ± 828 

μg/m3 (mean ± standard deviation [SD]) (1.04 ± 0.69 ppm), with a range of 12–4,390 μg/m3 15

(0.01–3.66 ppm). The levels decreased to an average of 468 ± 324 μg/m3 16 (0.39 ± 0.27 ppm), 

with a range of 0.00–1,960 μg/m3 17 (0.00–1.63 ppm) when the air conditioning was turned on. 

Levels also decreased to an average of 108 ± 96 μg/m3 18 (0.09 ± 0.08 ppm), with a range of 12–

588 μg/m3 19 (0.01–0.49 ppm) when the windows were opened. ATSDR (2007) found an 

20 association between temperature and formaldehyde levels; higher temperatures were associated 

21 with higher formaldehyde levels in trailers with the windows closed. They also noted that 

22 different commercial brands of trailers yielded different formaldehyde levels.

23 In December 2007 and January 2008, the Centers for Disease Control and Prevention 

24 (CDC) measured formaldehyde levels in a stratified random sample of 519 FEMA-supplied 

25 occupied travel trailers, park models, and mobile homes (“trailers”) (CDC, 2008). At the time of 

26 the study, sampled trailers were in use as temporary shelters for Louisiana and Mississippi 

27 residents displaced by hurricanes Katrina and Rita. The geometric mean level of formaldehyde 

in sampled trailers was 95 μg/m3 (77 ppb), and the range was 3.7–730 μg/m3 28 (3–590 ppb). 

29

30 2.3.2. Ingestion

31 Limited U.S. data indicate that concentrations in drinking water may range up to 

32 approximately 10 μg/L in the absence of specific contributions from the formation of 

33 formaldehyde by ozonation during water treatment or from leaching of formaldehyde from 

34 polyacetyl plumbing fixtures (IPCS, 2002). In the absence of other data, one-half this 

35 concentration (5 μg/L) was judged to be a reasonable estimate of the average formaldehyde in 
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1 Canadian drinking water. Concentrations approaching 100 μg/L were observed in a U.S. study 

2 assessing the leaching of formaldehyde from domestic polyacetal plumbing fixtures, and this 

3 concentration was assumed to be representative of a reasonable worst case (IPCS, 2002).

4 Formaldehyde is a natural component of a variety of foodstuffs (IARC, 1995; IPCS, 

5 1989). However, foods may be contaminated with formaldehyde as a result of fumigation (e.g., 

6 grain fumigation), cooking (as a combustion product), and release from formaldehyde resin7 based tableware (IARC, 1995). Also, the compound has been used as a bacteriostatic agent in 

8 some foods, such as cheese (IARC, 1995). There have been no systematic investigations of 

9 levels of formaldehyde in a range of foodstuffs that could serve as a basis for estimation of 

10 population exposure (Health Canada and Environment Canada, 2001). According to the limited 

11 available data, concentrations of formaldehyde in food are highly variable. In the few studies of 

12 the formaldehyde content of foods in Canada, the concentrations were within a range of 

13 <0.03–14 mg/kg (Health Canada and Environment Canada, 2001). Data on formaldehyde levels 

14 in food have been presented by Feron et al. (1991) and IPCS (1989) from a variety of studies, 

15 yielding the following ranges of measured values:

16

17 • Fruits and vegetables: 3–60 mg/kg

18 • Meat and fish: 6–20 mg/kg

19 • Shellfish: 1–100 mg/kg

20 • Milk and milk products: 1–3.3 mg/kg

21

22 Daily intake of formaldehyde was estimated by IPCS (1989) to be in the range of 1.5–

23 14 mg for an average adult. Similarly, Fishbein (1992) estimated that the intake of formaldehyde 

24 from food is 1–10 mg/day but discounted this on the belief that it is not available in free form. 

25 Although the bioavailability of formaldehyde from the ingestion of food is not known, it is not 

26 expected to be significant (ATSDR, 1999). Using U.S. Department of Agriculture (USDA) 

27 (1979) consumption rate data for various food groups, Owen et al. (1990) calculated that annual 

28 consumption of dietary formaldehyde results in an intake of about 4,000 mg or approximately 

29 11 mg/day.

30

31 2.3.3. Dermal Contact

32 The general population may have dermal contact with formaldehyde-containing 

33 materials, such as some paper products, fabrics, and cosmetics. For example, nail hardeners 

34 contain formalin, and cosmetics products such as hand creams and suntan lotions contain 

35 formaldehyde-releasing agents (but not formaldehyde) as preservatives. Generally, though, 
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1 dermal contact is more of a concern in occupations that involve handling concentrated forms of 

2 formaldehyde, such as those occurring in embalming and chemical production.

3
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3-1 DRAFT—DO NOT CITE OR QUOTE

1 3. TOXICOKINETICS

2

3

4 3.1. CHEMICAL PROPERTIES AND REACTIVITY

5 Formaldehyde (HCHO) is the smallest aldehyde (30 g/mol) and is a gas at room 

6 temperature. It is highly water soluble and reactive. In water, less than 0.1% of formaldehyde 

7 exists unhydrated, with the majority reported to be in the hydrated form, methylene glycol 

8 (CH2(OH)2) (Priha et al., 1996). Formaldehyde reacts readily with high and low molecular 

9 weight biological constituents. 

10

11 3.1.1. Binding of Formaldehyde to Proteins 

12 Formaldehyde is a reactive molecule that is likely to react with both low molecular 

13 weight cellular components (e.g., reduced glutathione[GSH]) as well as high molecular weight 

14 components. Unlike deoxyribonucleic acid (DNA), which has some additional barriers to 

15 exposure (i.e., nucleus), extracellular and intracellular proteins are obvious targets for interacting 

16 with formaldehyde. Formaldehyde is a well-known cross-linking agent that is used in the 

17 fixation of tissues, preparation of vaccines, and study of protein-protein interactions (Metz et al., 

18 2006). However, the exact nature of the protein modifications used for these purposes is not yet 

19 fully characterized (Metz et al., 2006, 2004). Figure 3-1 provides a general reaction scheme for 

20 formaldehyde-mediated modifications of amino acids. In step 1, formaldehyde reacts with 

21 primary N-terminal amines to form a labile methylol adduct. This adduct can undergo 

22 dehydration (step 2) to form an imine, or Schiff base (−N=CH2). Metz et al. (2004) examined 

23 the types of formaldehyde-protein reactions that are likely to occur in vivo by synthesizing 

24 several identical polypeptides with one varying amino acid (X) within the sequence VELXVLL 

25 (V=valine, E=glutamate, L=Leucine, X=varying amino acid). Several peptides with reactive 

26 amino acids did not exhibit modifications, suggesting that the peptide sequence/structure affects 

27 the ability of formaldehyde to react with amino acids. Peptides that were modified indicated 

28 formation of methylol adducts (Figure 3-1, step 1) or a mixture of methylol and imine adducts 

29 (Figure 3-1, step 2). 
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3-2 DRAFT—DO NOT CITE OR QUOTE

1

2

3 Figure 3-1. Formaldehyde-mediated protein modifications. 4

5 Note: Formaldehyde reacts with primary N-terminal amines to form a methylol 

6 adduct [1], which increases the molecular weight by 30 Da (∆m). This labile 

7 adduct can rearrange to form an amine, or Schiff base [2], that results in an 

8 increase in MW of 12 Da. Schiff bases can react with certain amino acids to form 

9 intra- or intermolecular methylene bridges [3]. The two amino acids depicted in 

10 step 3 may be within the same protein or possibly from two different proteins.

11

12 Source: Metz et al. (2004).

13

14

15 Mucus is composed of water, electrolytes, polysaccharides, and about 0.5% soluble 

16 proteins (Priha et al., 1996; Bogdanffy et al., 1987). Bogdanffy et al. (1987) showed that 

17 although human nasal mucus can bind 70% of 100 mM formaldehyde, irreversible binding of 

[

14 18 C]-formaldehyde to serum albumin (the major protein in mucus) was shown to be insignificant 

19 after a 1-hour incubation. Irreversible binding (50% or more) did not occur until after about 7 

20 hours of incubation. These data suggest that the protein content of mucus may not provide a 

21 significant formaldehyde irreversible sink. Nonetheless, the solubility of formaldehyde in mucus 

22 along with mucus flow and ingestion likely indicate that much of the inhaled dose is removed—

23 perhaps as much as 42% in rodents (IARC, 2005; Schlosser, 1999).
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1 In general, formaldehyde interacts with proteins. Studies carried out in cell culture media 

2 containing serum and formaldehyde have shown that such mixtures are quite labile. For 

3 example, during a 60-minute incubation of formaldehyde with complete cell media (i.e., with 

4 fetal calf serum) at 38°C, gas chromatography-mass spectrometry (GC-MS) exhibited very 

5 different peak profiles at different points during the incubation (Proctor et al., 1986). In contrast, 

6 GC-MS chromatograms of cell media containing formaldehyde but no serum proteins appeared 

7 relatively unchanged throughout the incubation. Compared to cell culture medium alone, 

8 complete media were considered to provide a more suitable model for the hypothetical 

9 interactions that formaldehyde could undergo in vivo (including perhaps blood).

10

11 3.1.2. Endogenous Sources of Formaldehyde

12 Endogenous formaldehyde is produced through a) normal cellular metabolism through 

13 enzymatic or non-enzymatic reactions, and also as a detoxification product of xenobiotics during 

14 cellular metabolism: 

15

16 3.1.2.1. Normal Cellular Metabolism (Enzymatic)

17 Formaldehyde is produced during normal metabolism of methanol, amino acids (e.g., 

18 glycine, serine, methionine), choline, dimethylglycine, and methylamine and through the folate19 dependent endogenous one-carbon pool etc. 

20 i) One of the endogenous sources for formaldehyde production is methanol, 

21 formed during normal cellular metabolism. However, this fraction may also be derived 

22 through consumption of fruits, vegetables and alcohol (Shelby et al . 2004; IPCS, 1997). 

23 In studies conducted with healthy humans whose diet was devoid of methanol-containing 

24 or methanol-generating foods (such as cereals containing aspartame, a precursor of 

25 methanol) and who abstained from alcohol consumption, the background blood levels of 

26 methanol range from 0.25–4.7 mg/L (Reviewed in Shelby et al 2004 [CERHR]). 

27 Methanol is metabolized to formaldehyde predominantly by hepatic alcohol 

28 dehydrogenase-1 (ADH1) in primates and by ADH1 and catalase (CAT) in rodents, 

ADH1 requiring nicotinamide adenine dinucleotide (NAD+ 29 ) as a cofactor. 

30 ii) Dimethylglycine (DMG), one of the byproducts of choline metabolism 

31 endogenously present in the body, is an indirect source of endogenous formaldehyde. 

32 Two specific dehydrogenases, i) dimethylglycine dehydrogenase (DMGDH) which 

33 converts DMG to sarcosine (methylglycine) and ii) sarcosine dehydrogenase (SDH) 

34 which converts sarcosine to glycine, have been shown to non-covalently bind to the 
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1 folate enzyme, tetrahydrofolate (THF). Further, these dehydrogenases form “active 

2 formaldehyde” by removing the 1-carbon groups from THF (Binzak et al., 2000). 

3 iii) Another source of endogenous formaldehyde is methylamine (MA), an 

4 intermediary component of the metabolism of adrenaline, sarcosine, creatine, lecithin, 

5 and other dietary sources (Yu and Zuo 1996). The enzyme semicarbozole-sensitive 

6 amine oxidase (SSAO), predominantly present in the plasma membrane of endothelial 

7 smooth muscle cells and in circulating blood, converts methylamine to formaldehyde, 

8 hydrogen peroxide and ammonia. The formaldehyde thus released has been shown to 

9 cause endothelial injury eventually leading to atherosclorosis (Kalasz, 2003). Yu et al. 

10 (1997) have shown that adrenaline, released in the body as a response to stress, is known 

11 to be deaminated by the enzyme monoamine oxidase, with further conversion of 

12 methylamine to formaldehyde by SSAO (Yu et al . 1997). Creatine is another precursor 

13 for methylamine which is metabolized by SSAO to form formaldehyde. It has been 

14 shown that short-term, high-dose dietary supplementation of creatine in healthy humans 

15 causes a significant increase in urinary methylamine and formaldehyde levels (Poortmans 

16 et al., 2005).

17 iv) Endogenous formaldehyde is also a constituent of the one-carbon pool, a 

18 network of interrelated biochemical reactions that involve the transfer of one-carbon 

19 groups from one compound to another (usually the transfer of the hydroxymethyl group 

20 of serine to tetrahydrofolic acid). 

21

22 Tyihak et al. (1998) have demonstrated that formaldehyde, but not the methyl radical or 

23 methyl cation, is involved in the enzymatic transmethylation and demethylation reactions, and 

24 suggested the presence of a formaldehyde cycle in cells for the production and removal of 

25 formaldehyde utilizing the transfer through methionine  S-adenosylmethionine  S-adenosyl26 homocysteine  homocysteine (Tyihak et al., 1998). However, these studies did not clearly 

27 show whether the formaldehyde released in this cycle is in free or bound form. 

28 Formaldehyde has been shown to be produced in normal and leukemic leukocytes from 

N5 29 -methyl-THF by enzymatic degradation (Thorndike and Beck, 1977). This is a two-step 

30 reaction involving 1) enzymatic conversion of the methyl-THF to formaldehyde followed by 2) 

31 nonenzymatic reaction of formaldehyde with an amine. Thorndike and Beck (1977) showed that 

32 leukocyte (granulocyte and lymphocyte) cell extracts from normal individuals and patients with 

33 chronic lymphocytic leukemia (CLL) or chronic myelocytic leukemia (CML) incubated with 

14 34 C-methyl-THF and saturating amounts of tryptamine produced free formaldehyde which is 

35 detected as its corresponding carboline derivative formed with tryptamine. These results 
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3-5 DRAFT—DO NOT CITE OR QUOTE

demonstrate the activity of the enzyme N5, N10-methylene THF reductase which oxidizes N5 1 -

methyltetrahydrofolate to N5

, N10 2 methylene THF. The authors noted that the enzyme levels 

3 were in the order of normal granulocytes < normal lymphocytes < granulocytes from a CML 

4 individual < lymphocytes from a CLL individual (Thorndike and Beck, 1977), suggesting 

5 increased activity of formaldehyde producing enzyme in leukemic cells compared to normal 

6 leukocytes. Overall, formaldehyde might be a byproduct as well as an intermediary product in 

7 several of these reactions.

8

9 3.1.2.2. Normal Metabolism (Non-Enzymatic)

10 i) Formaldehyde can also be formed non-enzymatically by the spontaneous reaction of 

11 methanol with hydroxyl radicals, wherein cellular hydrogen peroxide is the precursor for 

12 hydroxyl radicals generated through Fenton reaction (Cederbaum and Qureshi, 1982). 

13

14 ii) Another mechanism of nonenzymatic production of formaldehyde is through lipid 

15 peroxidation of polyunsaturated fatty acids (PUFA) (Shibamoto, 2006; Slater, 1984). In this 

16 mechanism, reactive oxygen species (ROS) generated during oxidative stress abstract a hydrogen 

17 atom from a methylene group of polyunsaturated fatty acids (PUFA) in cell membranes causing 

18 autooxidation of lipids with the eventual production of free radicals (e.g., peroxy radical). It is 

19 known that a certain level of oxidative stress and lipid peroxidation does occur in normal 

20 individuals, and these cellular metabolic processes are likely to contribute to endogenous 

21 formaldehyde production.

22

23 3.1.2.3. Exogenous Sources of Formaldehyde Production

24 Microsomal cytochrome P450 enzymes catalyze oxidative demethylation of N-, O- and 

25 S-methyl groups of xenobiotic compounds whereby formaldehyde is produced as a primary 

26 product, which is subsequently incorporated into the one-carbon pool by reacting with 

27 tetrahydrofolic acid or is oxidized to formate (Dahl and Hadley, 1983; Heck et al., 1982). Also, 

28 some special peroxidases, such as peroxide-dependent horseradish peroxidase enzymatically 

29 catalyze xenobiotics to generate formaldehyde in the body. In particular, an ethyl peroxide30 dependent horseradish peroxidase has been shown to act on N,N-dimethylaniline and produce 

31 equimolar amounts of N-methylaniline and formaldehyde (Kedderis and Hollenberg, 1983). 

32 The tobacco-specific nitrosamine nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-butanone 

33 (NNK) is another source of formaldehyde. It has been shown that formaldehyde is also 

34 produced during the methyl hydroxylation of NNK by rat liver microsomes (Castonguay et al., 

35 1991). Also recent studies have demonstrated the formation of formaldehyde-DNA adducts in 
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3-6 DRAFT—DO NOT CITE OR QUOTE

1 NNK-treated rats using a highly sensitive liquid chromatography-electrospray ionization-tandem 

2 mass spectrometry with selected reaction monitoring (Wang et al., 2007), suggesting formation 

3 of formaldehyde from nitrosamines. Cigarette smoke is also a source of exogenously produced 

4 methylamine which is converted to formaldehyde by SSAO (Yu, 1998).

5

6 3.1.2.4. FA-GSH Conjugate as a Method of Systemic Distribution

7 Formaldehyde is primarily metabolized by alcohol dehydrogenase (ADH3) which uses 

8 the formaldehyde-glutathione hemiacetal adduct as the substrate. Sanghani et al. (2000) have 

9 shown that due to high circulating concentrations (50-fold) of glutathione in human blood, the S10 (hydroxymethyl)glutathione (HMGSH) adduct, the nonenzymatic product of formaldehyde with 

11 glutathione is the major form of formaldehyde seen in vivo (Sanghani et al., 2000). It is likely 

12 that the reversibly bound HMGSH may be transported to different tissues through circulation, 

13 but, specific experimental evidence is lacking.

14

15 3.1.2.5. Metabolic Products of FA Metabolism (e.g., Formic Acid)

16 Formate is converted to carbon dioxide (CO2) in rodents predominantly by a folate17 dependent enzyme pathway (Dikalova et al., 2001). Formate is also oxidized to CO2 and water 

18 by a minor pathway involving catalase located in rat liver peroxisomes (Waydhas et al., 1978; 

19 Oshino et al., 1973). In the folate-dependent pathway, tetrahydrofolate (THF)-mediated 

20 oxidation of formate and the transfer of one-carbon compounds between different derivatives of 

21 THF has been described. 

22 Endogenous levels of formate also will be affected by dietary intake of methanol23 producing or methanol-containing diets since methanol is initially converted to formaldehyde 

24 and eventually metabolized to formate. It has been shown in several studies in human subjects 

25 who were restricted on consuming methanol producing diets, aspartame or alcohol, that the 

26 endogenous blood concentrations of formate ranged from 3.8 to 19.1 mg/L (Shelby et al 2004 

27 [CERHR]). The biological half life of formic acid is 77-90 min (Owen et al., 1990b). The levels 

28 of formate in the urine of unexposed individuals range from 11.7 to 18 mg/L (Boeniger, 1987). 

29 One source of formic acid intake is through diet which ranges from 0.4 to 1.2 mg per day 

30 (Boeniger 1987). The half life for plasma formate is ~30 minutes or longer (Boeniger 1987). 

31

32 3.1.2.6. Levels of Endogenous Formaldehyde in Animal and Human Tissues

33 Heck et al. (1982) estimated that endogenous levels of formaldehyde (free as well as 

34 bound) in rats ranged from 0.05 to 0.5 μmole/g (1.5-15 μg/g) of wet tissue as analyzed by the 

35 stable isotope dilution with GC-MS method (Heck et al., 1982). Although the levels of free 
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1 formaldehyde cannot be measured due to their high reactivity and short half life, they were 

2 calculated by Heck et al. (1985) using an indirect method. They added a molar excess of GSH or 

3 THF to the test tube containing formaldehyde in aqueous solution enabling complete binding. 

4 When estimated, they observed that the amount of formaldehyde detected was equal to the total 

5 amount added to the reaction suggesting that the formaldehyde measured contained both free and 

6 bound forms. Further, they calculated the free formaldehyde concentration using the 

7 dissociation constant of the HMGSH adduct and cellular concentration of GSH. Human 

8 formaldehyde dehydrogenase has been shown to have a dissociation constant of 1.5 mM for the 

9 formaldehyde-GSH hemithioacetal adduct (Uotila and Koivusalo 1974), while the folate enzyme 

product N5

,N10 10 -methylene-THF has a dissociation constant of 30 mM (Kallen and Jencks 1966b; 

11 a). This could be evaluated using the Michaels-Menton constant (Km) of formaldehyde 

dehydrogenase for the GSH adduct (~4 μM at 25o 12 C), whereby they calculated the free 

13 formaldehyde level to be around 3-7 μM or 1-2% of the total formaldehyde as measured by GC14 MS in rat tissues (Heck 1982).

15 Cascieri and Clary (1992) estimated the total body content of formaldehyde in human 

16 body based on the following assumptions. For an individual with an average body wt of 70 kg 

17 and with body fluids accounting for 70% of body weight, total formaldehyde content is 

18 distributed in ~49 kg of body mass or 49 L of body fluids, owing to the water solubility and 

19 uniform distribution of formaldehyde in body fluids. It has been shown that the average blood 

20 concentration (mean ± S.E.) of formaldehyde in unexposed rats and humans was 2.24 ± 0.07 and 

21 2.61 ± 0.14 μg/g of blood, respectively (Heck et al., 1985), and in unexposed rhesus monkeys it 

22 was 2.42 ± 0.09 μg/g of blood (Casanova et al., 1988), overall giving an average of 

23 approximately 2.5 ppm (2.5 mg/L) formaldehyde across the species. All these studies used 

24 pentafluorophenyl hydrazine derived formaldehyde using GC-MS analysis (Table 3-1). 

25 Assuming these values, the body content of total formaldehyde is 122.5 mg (49 L x 2.5 mg/L) or 

26 1.75 mg/kg body wt at any given time. Formaldehyde given intravenously to rhesus monkeys 

27 has been shown to have a half life of ~1.5 min in blood, wherein formaldehyde in blood was 

28 measured by the dimedone method (McMartin et al., 1979). Using this information Cascieri and 

29 Clary (1992) calculated that the human body generates approximately 40.83 mg/min [(122.5 mg 

30 /2 x 1.5] of formaldehyde. Biotransformation of formaldehyde to carbon dioxide in the liver 

31 alone has been estimated at 22 mg/minute (Owen et al., 1990a).

32 Free formaldehyde is detected in body fluids and tissues using dimedone (Szarvas et al., 

33 1986) or 2,4-dinitrohenyhydrazine (DNPH) or pentafluoropheyl hydrazine (PFPH) derivative 

34 (Heck et al., 1985) or as a fluorescent derivative (Luo et al . 2001) as trapping agent and detected 

35 by analytical techniques such as thin-layer chromatography (TLC), high-performance liquid 
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1 chromatography (HPLC) and gas-chromatography mass spectrometry (GC-MS). Data from 

several studies is summarized in Table 3-1. Using 14 2 C-labeled dimedone, a chemical which 

3 condenses with free formaldehyde forming a product termed “formaldemethone” enabling 

4 radiometric detection, Szarvas et al (1986) estimated the levels of endogenous formaldehyde in 

5 human blood plasma to be 0.4-0.6 μg/mL and in human urine to be 2.5-4 μg/mL (Szarvas et al . 

6 1986). 

7

8 Table 3-1. Endogenous formaldehyde levels in animal and human tissues 

9 and body fluids

10

Tissue Method Detected as Formaldehyde levels Reference

Not 

specified Not specified Not specified

0.003-0.012 ppm

(3-12 ng/g) Hileman 1984

Not 

specified

GC-MS with stable 

isotope dilution method

As PFPHderivative1.5 - 15 ppm

(0.05-0.5 μmole/g) Heck et al 1982a

Blood

GC-MS with select ion 

monitoring

As PFPHderivative2.24 ± 0.07 ppm

(2.24 ± 0.07 μg/g) Heck et al 1985

Blood

GC-MS with select ion 

monitoring

As PFPHderivative2.61 ± 0.14 ppm

(2.61 ± 0.14 μg/g) Heck et al 1985

Plasma

Reverse phase HPLCfluorescent detectionAs product of 

ampicillin

1.65 ppm

(1.65 μg/mL) Luo et al 2001

Heart 

perfusate HPLC As DNPH adduct

0.089 - 0.126 ppm 

(2.98 - 4.21 nmol/mL) Shibamoto 2006

Blood

GC-MS with select ion 

monitoring

As PFPHderivative2.42 ± 0.09 ppm

(2.42 ± 0.09 μg/g)

Casanova et al 

1988

Plasma Radiometric method

As formaldemethone adduct0.4 - 0.6 ppm

(0.4 - 0.6 μg/mL) Szarvas et al 1986

Urine Radiometric method

As formaldemethone adduct2.5 - 4.0 ppm

(2.5 - 4.0 μg/mL) Szarvas et al 1986

11

12 Values in the parenthesis, originally cited in the references, are converted to parts per million (ppm) as indicated.

13 PFPH, pentafluorophenyl hydrazone derivative; DNPH, dinitrophenyl hydrazine; GC-MS, gas-chromatography mass 

14 spectrometry; HPLC, high performance liquid chromatography.

15

16

17 Hileman (1984) reported that the endogenous levels of metabolically derived 

18 formaldehyde will be in the range of 3-12 ng/g of tissue (Hileman 1984). So for an average 70 

19 kg individual, the endogenous level of metabolically derived formaldehyde would be 210 μg to 

20 840 μg (3-12 ng/g x 1000 g x 70).

21
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1 3.2. ABSORPTION

2 3.2.1. Oral

Oral absorption of [14 3 C]-formaldehyde (7 mg/kg) in rats resulted in 40% elimination as 

14C-carbon dioxide (14 4 CO2), with 10% excretion in urine, 1% excretion in feces, and much of the 

5 remaining 49% retained within the carcass, presumably due to metabolic incorporation (IARC, 

6 1995; Buss et al., 1964).

7

8 3.2.2. Dermal

Jeffcoat et al. (1983) reported on the disposition of various doses of [14 9 C]-formaldehyde 

10 dermally administered to rats, guinea pigs, and monkeys. Very little (<1% of the applied dose) 

11 of the radiolabel was found in the major organs excised during necropsy. As noted by the 

12 authors, the disposition of formaldehyde when administered via the dermal route was markedly 

13 different to that observed when the compound was administered intravenously or 

14 intraperitoneally. In the latter cases, there was much evidence of metabolic activity, and 

15 substantial portions of the load were expired as CO2. The difference appeared to be the result of 

16 a reaction of dermally applied formaldehyde with macromolecules at or near the skin surface or 

17 of its evaporation. In general, portions of the load that succeed in entering the circulation 

18 probably do so bound to macromolecules or by incorporation of the radiolabel via the onecarbon pool. Likewise, Bartnik et al. (1985) who applied [14 19 C]-formaldehyde to the shaved 

20 backs of rats concluded that the overwhelming majority of the formaldehyde load remained 

21 sequestered in the outer layers of skin at or near the site of application. At the end of the various 

22 measurements, approximately 70% of the dose was found in the treated skin, with a marked 

23 localization of the remaining radioactivity in the uppermost layers. This fraction of the load was 

24 considered to be permanently sequestered, most likely as a result of irreversible binding to 

25 macromolecular components.

26

27 3.2.3. Inhalation

28 Studies indicate that the majority of inhaled formaldehyde is absorbed in the upper 

29 respiratory tract (URT) but that the extent of the scrubbing in this region varies significantly 

30 across species. In dogs, nearly 100% of nasally inhaled formaldehyde is absorbed (Egle, Jr., 

31 1972). Lower respiratory tract (LRT) studies designed to collect formaldehyde via a tube 

32 inserted into the lower trachea revealed that nearly 95% of formaldehyde was absorbed during 

33 the first pass through the upper respiratory tract (Egle, Jr., 1972), an effect observed with 

34 multiple ventilation rates. The rat nasal passages also scrub nearly all of the inhaled 

35 formaldehyde (on average ~97%) (Morgan et al., 1986). In computational dosimetry modeling 
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1 based on anatomically realistic representation of the human nasal airways from a single 

2 individual, approximately 90% of inhaled formaldehyde was predicted to be absorbed in the nose 

3 at resting inspiration. As the inspiratory rate increased, this fraction decreased to about 70% at 

4 light exercise and to 58% at heavy exercise conditions (see Figure 1 in Kimbell et al. [2001b]). 

5 The normal human breathing mode during heavy exercise is oronasal (with ~54% of airflow 

6 being oral) (ICRP 66, 1994). Consequently, it is estimated that during heavy exercise breathing 

(50 L/min) the flux of formaldehyde into tissue (or rate of mass transported per mm2 7 of tissue 

8 surface area) in the first six to eight generations of the tracheobronchial airways is comparable to 

9 that in the nasal region (Overton et al., 2001). 

10 It is important to note that the computer simulations mentioned above are based on 

11 anatomical representations of a single individual. Significant anatomical variations occur in 

12 human nasal airways. For example, the nasal volumes of 10 adult nonsmoking subjects between 

13 18 and 50 years of age in a study in the U.S. varied between 15 and 60 mL (Santiago et al., 

14 2001), and disease states can result in considerable further variation (Singh et al., 1998). 

15 Species differences in kinetic factors have been argued to be the key determinants of 

16 species-specific lesion distributions for formaldehyde and other reactive inhaled gases. Airway 

17 geometry is an important determinant of inhaled-formaldehyde dosimetry in the respiratory tract 

18 and its differences across species. These issues will be discussed in a later section on dosimetry 

19 modeling. 

20

21 3.2.3.1. Formaldehyde Uptake Can Be Affected by Effects at the Portal of Entry

22 Certain formaldehyde-related effects have the potential to modulate its uptake and 

23 clearance. The mucociliary apparatus of the upper respiratory tract is the first line of defense 

24 against airborne toxins. Comprising a thick mucus layer (epiphase), hydrophase, and a ciliated 

25 epithelium, the mucociliary apparatus may entrain, neutralize, and remove particulates and 

26 airborne chemicals from inspired air. As reviewed by Wolfe (1986), airborne pollutants and 

27 reactive gases have been shown to decrease mucus flow rates in several animal models (Mannix 

28 et al., 1983; Iravani, 1974; Carson et al., 1966; Dalhamn, 1956; Cralley, 1942). Degradation in 

29 the continuity or function of this mucociliary apparatus could result in a lower clearance of 

30 inhaled pollutants at the portal of entry. 

31 Morgan et al. (1983) first reported defects in mucociliary function in F344 rats exposed 

32 to 15 ppm formaldehyde 6 hours/day for 1–9 days. Mucostasis occurred in several regions in all 

33 rats after a single 15 ppm exposure. Ciliastasis occurred with greater frequency and across more 

34 regions of the nasoturbinate in subsequent days of exposure. The authors observed that 

35 mucostasis preceded ciliastasis in most cases, and vigorous ciliary activity was noted in areas 
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1 without mucus flow. Morgan et al. (1984a) also studied formaldehyde effects on the mucociliary 

2 apparatus of isolated frog palates in vitro. Mucostasis was evident as mucus became stiff and 

3 eventually rigid with increasing formaldehyde concentration and time of exposure. Ciliary beat 

4 continued even after mucostasis, but ciliastasis ultimately occurred when exposure reached 4 and 

5 9 ppm.

6 When a rodent is exposed to an irritant, its inhaled dose and pattern of deposition can be 

7 profoundly affected by reflex bradypnea, a protective reflex seen in rodents but not in humans. 

8 Reflex bradypnea can occur when the trigeminal nerve is exposed to a sufficient concentration of 

9 an irritant, such as formaldehyde. It is manifest as markedly decreased activity or prostration, 

10 reduced metabolism, hypothermia (as much as 5oC), significantly reduced respiratory rate and 

11 minute volume, and altered blood and brain chemistry. Because of their small size, rodents are 

12 able to rapidly lower their metabolism and body temperature and therefore their oxygen demand. 

13 The consequence is that their inhaled dose of an irritating chemical is dramatically lowered. 

14 Reflex bradypnea is quantified as the RD50, which is the concentration of a chemical that results 

15 in a 50% decrease in respiratory rate. It can take as much as two hours for rodents to fully 

16 recover from the effects of reflex bradypnea. The clinical manifestations of reflex bradypnea can 

17 easily be misconstrued as toxicity. None of the studies described in this assessment took into 

18 account the fact that reflex bradypnea may have confounded the results. Reflex bradypnea is 

19 discussed in depth in section 4.2.1.1.

20 Sensory irritation studies suggest that formaldehyde activates the trigeminal nerve by 

21 activating nociceptors through the modification of receptor amino acids, possibly including thiol 

22 groups. Cassee et al. (1996) measured sensory irritation to formaldehyde, acetaldehyde, and 

23 acrolein in male Wistar rats, following a 30-minute nose-only exposure. Formaldehyde and 

24 acrolein elicited similar responses, whereas acetaldehyde was far less irritating. The authors 

25 suggested that the differences in sensitivity to the aldehydes might be explained by differences in 

26 physicochemical properties and by regional differences in activities of detoxifying enzymes for 

27 each chemical. In addition, it has been suggested that acetaldehyde might interact with sensory 

28 nerves via an amino group (Steinhagen and Barrow, 1984), whereas the receptor-binding site for 

29 formaldehyde and acrolein is believed to be a thiol group. Differential binding sites for sensory 

30 irritants in the trigeminal nerve have been reported (Nielsen, 1991).

31 Sensory irritation effects are discussed in depth in Chapter 4 but are noted here because 

32 stimulation of the trigeminal nerve by formaldehyde can result in significantly lower pulmonary 

33 ventilation, and formaldehyde exposure in rodents at concentrations that approach the RD50. 

34 Barrow et al. (1983) have estimated the “inhaled dose” equivalent to an exposure concentration 

35 of 15 ppm in mice and rats used in the chronic formaldehyde bioassays by Kerns et al. (1983) 
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1 and Monticello and Morgan (1994). Their results indicate that, because mice are observed to 

2 decrease their minute volume by approximately 75% as compared to 45% in rats, a twofold 

3 greater inhaled dose would be expected in rats versus mice. This difference may be relevant to 

4 the increased incidence of squamous cell carcinoma of the nasal cavity in F344 rats as compared 

5 to B6C3F1 mice. Chang et al. (1983) estimated a reduction of 25% in the minute volume of 

6 F344 rats. Yokley et al. (2008) have recently published a model that accounts for physiological 

7 changes in ventilation rate induced by sensory irritation in rats. Thus, the “standard” minute 

8 volumes used for rats and mice need to be adjusted downward when calculating dosimetric 

9 adjustment factors for extrapolation of adverse effects to humans (Thompson et al., 2008). This 

10 question is further discussed in the section on modeling the dosimetry.

11 Another effect that modulates dosimetry is the dynamic tissue remodeling of nasal 

12 airways that occurs as a consequence of exposure to reactive gases. For example, formaldehyde 

13 dosimetry is influenced by the occurrence of squamous metaplasia, an adaptive tissue conversion 

14 to squamous that occurs in nasal epithelium exposed to toxic levels of formaldehyde. The 

15 metaplasia has been observed to occur in rats at exposure concentrations of 3 ppm and higher 

16 (Kimbell et al., 1997). Squamous epithelium is known to absorb considerably less formaldehyde 

17 than other epithelial types (Kimbell et al., 1997). Overall, the highest flux levels of 

18 formaldehyde in the simulations of the rat nose in Kimbell et al. (2001a) are estimated in the 

19 region just posterior to the nasal vestibule. A consequence of squamous metaplasia would be to 

20 “push” the higher levels of formaldehyde flux toward the more distal regions of the nose 

21 (Kimbell et al., 1997). Subramaniam et al. (2008) discussed this issue further in the context of 

22 uncertainties in the modeling of formaldehyde dosimetry.

23

24 3.2.3.2. Variability in the Nasal Dosimetry of Formaldehyde in Adults and Children

25 Garcia et al. (2009) used computational fluid dynamics to study human variability in the 

26 nasal dosimetry of reactive, water-soluble gases in 5 adults and 2 children, aged 7 and 8 years 

27 old. They considered two model categories of gases, corresponding to maximal and moderate 

28 absorption at the nasal lining. We focus here only on the “maximum uptake” simulations in 

29 Garcia et al. (2009). In this case, the gas was considered so highly reactive and soluble that it 

30 was reasonable to assume an infinitely fast reaction of the absorbed gas with compounds in the 

31 airway lining. Although such a gas could be reasonably considered to represent formaldehyde, 

32 these results cannot be fully utilized to inform quantitative estimates of formaldehyde dosimetry 

33 (and it does not appear to have been the intent of the authors either). This is because the same 

34 boundary condition corresponding to maximal uptake was applied on the vestibular lining of the 

35 nose as well as on the transitional and transitional epithelial lining on the rest of the nose. This is 
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1 not appropriate for formaldehyde as the lining on the nasal vestibule is made of keratinized 

2 epithelium which is considerably less absorbing than the rest of the nose (Kimbell et al. 2001). 

3 The Garcia et al. (2009) study and the results of their analyses have been further 

4 described and evaluated in Appendix 3-1. Overall uptake efficiency, average flux (rate of gas 

5 absorbed per unit surface area of the nasal lining) and maximum flux levels over the entire nasal 

6 lining did not vary substantially between adults (1.6-fold difference in average flux and much 

7 less in maximum flux), and the mean values of these quantities were comparable between adults 

8 and children. These results are in agreement with conclusions reached by Ginsberg et al. (2005) 

9 that overall extrathoracic absorption of highly and moderately reactive and soluble gases 

10 (corresponding to category 1 and 2 reactive gases as per the scheme in USEPA [1994]) is similar 

11 in adults and children. On the other hand, Figure 6A of the paper (reproduced as Figure A in 

12 Appendix 3-1), shows significant interhuman variability in flux values at specific points on the 

13 nasal walls. The local flux of formaldehyde varies among individuals by a factor of 3 to 5 at 

14 various distances along the septal axis of the nose. 

15

16 3.3. DISTRIBUTION

17 3.3.1. Levels in Blood

18 Inhalation studies in several species indicate that exposure to formaldehyde does not 

19 result in elevated levels in blood. These studies were carried out over a wide range of exposure 

20 concentrations and durations. Rats exposed to 14 ppm formaldehyde for 2 hours exhibited no 

21 increase in blood formaldehyde levels [2.25 ± 0.07 μg/(g blood) in treated animals compared 

22 with 2.24 ± 0.07 μg/(g blood) in control animals] when measured by GC-MS using a stable 

23 isotope dilution technique (Heck et al., 1985, 1982). Similarly, mean formaldehyde blood levels 

24 in humans (n = 6) exposed to 1.9 ppm formaldehyde for 40 minutes in a walk-in chamber (2.77 ± 

25 0.28 μg/g blood) were not statistically different from measurements in the same population 

26 before exposure (mean of 2.61 ± 0.14 μg/g) (Heck and Casanova-Schmitz, 1984). The 

27 variability in the levels was large. At the individual level, the data showed both increase and 

28 decrease in blood levels relative to pre-exposure levels, which was attributed by the authors as 

29 plausibly due to temporal variations in baseline levels in humans, particularly since the 

30 experiment did not control food intake prior to exposure. Studies in rhesus monkeys have 

31 revealed endogenous formaldehyde levels (2.4 μg/g blood) comparable to humans and that levels 

32 were also unaltered following exposure to 6 ppm formaldehyde via inhalation 6 hours/day for 

33 4 weeks, measurements being taken at both 7 minutes and 45 hours post final exposure 

34 (Casanova et al., 1988). 
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1 It is important to keep in mind that the GC-MS method is not capable of detecting 

2 irreversibly bound formaldehyde; for example, formaldehyde levels detected by this method, 

3 even in the anterior nasal mucosa of rats exposed to 6 ppm of formaldehyde, were not elevated 

4 over control levels. Furthermore, the GC-MS method does not differentiate between free and 

5 reversibly bound adducts of formaldehyde (Heck et al., 1982). Thus, measured levels represent 

6 total formaldehyde concentration that includes free formaldehyde as well as reversibly bound 

7 adducts. Based on the known Michaelis-Menten constant, Km, for formaldehyde dehydrogenase 

8 with respect to the GSH adduct formation, Heck et al. (1982) estimated under certain 

9 assumptions that free formaldehyde comprised only about 1–2% of the total formaldehyde 

10 measured by their method. Furthermore, as shown by Metz et al. (2006, 2004), formaldehyde 

11 reactions with primary amino and thiol groups can, in a second step, react with many other 

12 amino acids to form stable methylene bridges. Presumably, such reactions would not be 

detectable by using the methods employed by Heck et al. (1982).4

17 Results from an earlier experiment using radiolabeled formaldehyde in rats are consistent 

18 with the conclusion based on the GC-MS measurements of no appreciable increase in blood 

levels of formaldehyde. Following a 6-hour exposure of F344 rats to 15 ppm of [14 19 C]-

formaldehyde (Heck et al., 1983), the concentrations of 14 20 C in the nasal mucosa were 28-fold 

21 higher than those in the blood. The observed half-life of the terminal phase of the radioactivity 

22 was long (55 hours); on the other hand, it is known that the half-life of free formaldehyde in the 

23 rat blood is very short. Therefore, the authors concluded that the radioactivity was likely due to 

24 modification of macromolecules or metabolic incorporation rather than slow metabolic clearance 

25 of formaldehyde. The terminal decline of the radioactivity in the packed cell fraction of the 

26 blood was much slower and observed to be consistent with incorporation into erythrocytes. 

13 Thus, the limited 

14 interpretation of GC-MS measurements of blood levels suggests that formaldehyde does not 

15 appreciably reach the blood, is rapidly metabolized or interacts with macromolecules when it 

16 escapes metabolism, or is otherwise undetected. 

27 In the same paper, Heck et al. (1983) report on the similarity in the pharmacokinetics of 

28 radiolabeled formaldehyde and radiolabeled formate in the rat blood, supporting their hypothesis 

29 that oxidation of formaldehyde to formate and subsequent incorporation of this compound 

30 through one-carbon metabolism were major factors in the disposition of formaldehyde. Studies 

31 by Gottschling et al. (1984) have also established that the main product of metabolic clearance of 

 4 Additionally, note that, although Heck et al. (1982) demonstrated that formaldehyde concentration can be 

accurately measured from glutathione and tetrahydrofolate adducts, similar experiments were not performed by using

protein samples or cellular extracts (i.e., in the presence of various amino acids). In addition, standard curves for 

predicting formaldehyde concentration in tissues were generated in aqueous solutions rather than biological samples.
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1 formaldehyde is formate, which is either further metabolized to CO2 and water, incorporated into 

2 the one-carbon pool, and/or eliminated in the urine as a sodium salt at about 13 mg/L urine.

3

4 3.3.2. Levels in Various Tissues

The radiolabeling studies indicated high levels of 14 5 C in the rat nasal mucosa (equivalent 

concentrations of 14 6 C-formaldehyde in the nasal mucosa of rats naïvely exposed to 15 ppm 

14 7 C-formaldehyde were 2,148 ± 255 nmol/g compared with 76 ± 11 nmol/g in plasma). In 

8 contrast, the GC-MS studies did not detect elevated formaldehyde in this region. This is not to 

9 be interpreted as a discrepancy, because the radiolabeling study did not distinguish among 

10 radiolabeled species and thus the measured radioactivity could potentially be free or bound 

formaldehyde, formate, or any [14 11 C] metabolically incorporated into macromolecules.

12 In concurrent studies, Casanova-Schmitz et al. (1984) resolved the question as to whether 

the higher [14 13 C] levels in the nasal mucosa were a consequence of GSH depletion and a 

14 subsequent reduction in GSH-dependent clearance of formaldehyde. An important result in 

15 these studies was that there was no significant difference in labeling in either the nasal mucosa or 

16 in plasma between naïve F344 rats and those pre-exposed to unlabeled 15 ppm formaldehyde 

17 6 hours/day for the 9 previous days. These findings indicated little or no apparent effect on the 

18 disposition of formaldehyde following short-term exposure to relatively high levels of 

19 formaldehyde. In contrast, Farooqui et al. (1986) reported decreases in GSH in several tissues 

20 3 hours after a sublethal I.P. injection of formaldehyde but not after 6 and 9 hours. Taken 

21 together, these data suggest that formaldehyde exposure does not result in long-term alterations 

22 in cellular GSH levels and that repeated inhalation exposure does not alter the dosimetry to the 

23 bloodstream or formaldehyde body burden.

Heck et al. (1983) determined the 14 24 C concentrations in different tissues in the F344 rat 

25 body by exposing rats in a head-only chamber to various concentrations (5–24 ppm) of 

radiolabeled formaldehyde for 6 hours. (Concentrations of 14 26 C in internal organs and tissues 

27 relative to that in plasma did not appear to vary much as exposure concentrations increased; 

28 therefore only averages over the concentration range were reported.) Except for the esophagus, 

29 levels in the heart, spleen, lung, intestines, liver, and kidney were 1–3 times higher relative to 

30 that in plasma. Labeling in the esophagus was high (fivefold relative to plasma). The authors 

31 attributed this relatively higher dose to mucociliary action in the nose and trachea. The data also 

indicate that the brain, testes, and erythrocytes appear to have about threefold lower 14 32 C levels 

33 than plasma. Pre-exposure to formaldehyde (for 9 days) did not alter the measured radioactivity 

34 in the nasal mucosa or plasma. Thus, it was concluded that the single exposure findings may 

35 also be qualitatively extended to chronic exposures.
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1 The total radiolabel measured in the bone marrow (femur) of F344-rats exposed for 6 

2 hours to 0.3–15 ppm of radiolabeled formaldehyde in the Casanova et al. (1984) experiment was 

3 high (generally within a factor of 0.5 of the total labeling in the nasal respiratory mucosa). 

Nearly half of the 14 4 C was contained in the DNA in this tissue presumably on account of the high 

rate of cell turnover in the bone marrow, indicating that the carbon derived from 14 5 C6 formaldehyde was utilized for DNA synthesis (Casanova-Schmitz et al., 1984). 

7 Chang et al. (1983) described visceral labeling (via autoradiography) in rats, following 

exposure to 15 ppm [14 8 C]-formaldehyde 6 hours/day for 4 days. The authors attributed this 

9 labeling to mucociliary clearance and grooming-related ingestion of formaldehyde.

10 In summary, following exposure to radiolabeled formaldehyde, the radioactivity was very 

11 high in the nasal mucosa but was also extensively distributed to various tissues. In particular, 

12 levels in the bone marrow were high. On the other hand, formaldehyde levels in the blood 

13 measured by GC-MS were not significantly elevated. Thus, the authors considered it unlikely 

that the elevated 14 14 C in various tissues was due to free formaldehyde. Instead, these levels were 

15 thought to arise from either rapid metabolic incorporation or formation of covalent adducts or 

incorporation via carboxylation reactions of the 14 16 CO2 formed during metabolism. 

17 The data presented thus far in this section illustrate that measuring the distribution of the 

absorbed formaldehyde based on 14 18 C-radiolabeling and GC-MS studies alone is problematic 

19 because it is difficult to resolve (through these studies) whether it is free, reversibly bound, 

20 irreversibly bound, formate, one-carbon pool, etc. This is of significance with regard to 

21 understanding the availability of the absorbed formaldehyde. More indirect methods had to be 

22 developed to further examine the disposition of formaldehyde; however, as discussed below, the 

23 interpretation of these approaches may also not be straightforward.

24

25 3.3.2.1. Disposition of Formaldehyde: Differentiating Covalent Binding and Metabolic 

26 Incorporation

27 The motivation in presenting this section is twofold, as follows: 

28 1. As concluded above, subsequent studies were necessary to ascertain whether measured 

29 radiolabeling in different experiments was due to formaldehyde adducts or incorporation 

of [14 30 C] one-carbon units of formaldehyde into macromolecules via the one-carbon pool. 

31 2. DNA protein cross-links (DPXs) formed by formaldehyde (covalently bound in this case)

32 have been regarded as a surrogate dose metric for the intracellular concentration of 

33 formaldehyde (Hernandez et al., 1994; Casanova et al., 1991, 1989). This is particularly 

34 relevant because of the nonlinear dose response for DPX formation due to saturation of 
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1 enzymatic defenses at high concentrations (Casanova et al., 1991, 1989). Thus, the 

2 ability to measure DPX is an important development.

3

4 An important question is whether the formaldehyde disposed in the form of DPX is 

5 detected in remote tissues. A set of elegant but complex experiments involving dual isotope 

labeling (14C and 3 6 H) was carried out to this end by the Heck and Casanova-Schmitz and their 

7 coworkers. Casanova-Schmitz et al. (1984) and Casanova-Schmitz and Heck (1983) used dual 

8 isotope labeling of formaldehyde as a way to partially distinguish between formaldehyde adducts 

formation and metabolic incorporation. In separate experiments, F344 rats were exposed to 3 9 Hand 14C- formaldehyde at different exposure concentrations (0.3–15.0 ppm), and the 3

H/14 10 C 

11 ratios of different phases of DNA were measured. Only the highlights of the results and 

12 significant issues are presented here. The overall conclusions from these experiments were as 

13 follows:

14

15 • Labeling in the nasal mucosa was due to both covalent binding and metabolic 

16 incorporation.

17 • DPX was formed at 2 ppm and greater concentrations in the respiratory mucosa. 

18 • In the bone marrow, formaldehyde did not bind covalently to bone marrow 

19 macromolecules at any exposure concentration. The labeling of bone marrow 

20 macromolecules was found to be entirely due to metabolic incorporation and not due to 

21 covalent binding.

22

23 Macromolecules such as DNA and protein can be isolated from tissue homogenates by 

24 extraction into three phases: an organic phase consisting of proteins, an aqueous phase consisting 

25 of only double-stranded DNA, and an interfacial phase consisting of both DNA and protein. 

26 Single-stranded (but not double-stranded) DNA was particularly likely to form adducts. DNA 

27 from this interfacial phase can be further purified and has been shown to consist of DPXs 

(Casanova-Schmitz and Heck, 1983). Because both [14C]-formaldehyde and [3 28 H]-formaldehyde 

29 can become incorporated into DNA and protein metabolically as well as by cross-linking, the 

3

H/14 30 C ratio in such cross-linked material should be higher than in material that primarily 

31 contains metabolically incorporated formaldehyde. Figure 3-2 shows the labeling of tissue from 

the nasal respiratory mucosa and bone marrow (distal femur) in rats exposed to [14 32 C]-

formaldehyde and [3 33 H]-formaldehyde vapor. 

34
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1

2

Figure 3-2. 3

H/14 3 C ratios in macromolecular extracts from rat tissues 

following exposure to 14C- and 3 4 H-labeled formaldehyde (0.3, 2, 6, 10, 

5 15 ppm).

6

7 Note that the small yield of interfacial (IF) phase from bone marrow tissue precluded 

8 further analysis; this is prima facie evidence for the lack of significant DPXs in this 

9 tissue.

10

11 Source: Casanova-Schmitz et al. (1984a). 

12

13

In the nasal mucosa the interfacial phase has a significantly higher 3H/14 14 C ratio than the 

material in the aqueous phase. This suggests that interfacial DNA has significantly more 3 15 H, a 

phenomenon likely explained by additional [3 16 H]-formaldehyde molecules present as DPXs prior 

17 to extraction. The amount of interfacial DNA was found to have a clear dose response. These 

18 cross-links were also judged to be due to exogenous formaldehyde. Likewise, the organic phase 

of the nasal mucosa showed a similar increase in 3H/14 19 C ratio at higher concentrations, a result 

20 that could be attributed to various inter- and intra-protein adducts (Metz et al., 2004; Trezl et al., 

21 2003; Skrzydlewska, 1996). 

22 In contrast, analysis of macromolecules at the distal femur location presents a different 

23 pattern (Figure 3-2, part B). First, the interfacial phase was not detected during extraction, 

24 suggesting that there were few or no DPXs to be detected. Second, there was no increase in

3

H/14 25 C ratio in the organic (i.e., protein) phase as a function of dose. Therefore, it was 

26 concluded that either radiolabeled formaldehyde or formate reached the distal site and was 

27 subsequently incorporated into macromolecules. According to the mechanistic interpretation of 

these studies, the quantity plotted on the ordinate in Figure 3-2 (the ratio of 3H/14 28 C between the 
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1 tissue and the exposure gas) should approach unity as metabolism becomes saturated and more 

2 adduct formation occurs, particularly for protein. Indeed, this is what is observed (see 

3 Figure 3-2, part A). In contrast, there is no dose effect in the femur, suggesting that the labeling 

4 at all doses in that tissue may be due to metabolic incorporation and not due to the parent 

5 formaldehyde.

6 (Note: These data were originally shown in the absence of an analysis of isotope effects 

on covalent binding and metabolism. Subsequent studies determined that [3 7 H]-formaldehyde is 

oxidized less rapidly than [14 8 C]-formaldehyde and unlabeled formaldehyde. This suggests that 

the 3H/14 9 C ratio, and therefore the amount of formaldehyde covalently bound to tissue, is likely 

overestimated because more [3 10 H]-formaldehyde remains unmetabolized, i.e., free to bind [Heck 

11 and Casanova, 1987]. The authors hypothesized that this overestimate was relatively greater at 

12 the lower concentrations.)

13 Similar results were obtained in GSH-depleted rats (Casanova and Heck, 1987). Again, 

these authors observed a dose-dependent increase in the 3H/14 14 C ratio in the interfacial DNA and 

15 organic fractions of disrupted cells of the respiratory and olfactory mucosa and no such increases 

16 in bone marrow. Interestingly, at 10 ppm exposure (only), GSH-depleted rats exhibited a higher 

3

H/14 17 C ratio in the organic phase than did normal rats. Casanova and Heck (1987) posited that 

18 much of the covalent binding at 6 ppm and lower was due to binding to extracellular proteins, 

whereas the higher 3H/14 19 C ratio in GSH-depleted rats at 10 ppm was due to more intracellular 

20 binding. 

21 In their first experiment to measure DPX concentrations, Casanova-Schmidt et al. (1984) 

and Casanova and Heck (1987) used the dual isotope method (3

H/14 22 C) mentioned above. In this 

23 experiment, DPX was observed only at formaldehyde concentrations ≥2 ppm. Subsequently, 

24 Casanova et al. (1989) developed a more sensitive method using high-performance liquid 

25 chromatography (HPLC) for measuring DPX. In this method, tissue homogenates were digested 

26 with a proteolytic enzyme and extracted with a phenolic solvent. DPX was detected in the nasal 

27 mucosa of rats at formaldehyde concentrations as low as 0.3 ppm. This method was also used to 

28 measure DPX in the nasal region, the larynx, trachea and carina, and major intrapulmonary 

29 airways (airway diameters > 2mm) of rhesus monkeys exposed for 6 hours to 0.7, 2.0 and 6.0 

30 ppm of formaldehyde. DPX was detected in the nose (including the nasopharynx) at all 

31 concentrations and at 2.0 and 6.0 ppm in the larynx, trachea, carina and other lower airways. 

32 However, DPX was not detectable in the bone marrow of these monkeys at any concentration. 

33 Overall, Heck and Casanova-Schmitz and their coworkers interpreted the results of these 

34 various experiments to mean that inhaled formaldehyde could not reach distant sites in the body. 

35 It may be noted in this context that Shaham et al. [1996] reported elevated DPX levels in the 
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3-20 DRAFT—DO NOT CITE OR QUOTE

1 white blood cells of laboratory workers exposed to formaldehyde. These data are further 

2 reported in Chapter 4.)

3

4 3.4. METABOLISM

5 Formaldehyde is primarily metabolized by glutathione-dependent formaldehyde 

6 dehydrogenase (FALDH) and aldehyde dehydrogenases (ALDHs). Numerous studies now 

7 recognize FALDH as a member of the alcohol dehydrogenase (ADH) family, specifically ADH3 

8 (Thompson et al., 2009; Liu et al., 2004, 2001; Hedberg et al., 2003; Høøg et al., 2003; and the 

9 references in each of these). The remainder of this report will refer to FALDH as ADH3. 

10

11 3.4.1. In Vitro and In Vivo Characterization of Formaldehyde Metabolism

12 Formaldehyde is oxidized to formate by two metabolic pathways (Figure 3-3). The first 

13 pathway involves conversion of free formaldehyde to formate by the so-called low-Km

14 (Km = 400 μM) mitochondrial aldehyde dehydrogenase-2 (ALDH2). The second pathway 

15 involves a two-enzyme system that converts glutathione-conjugated formaldehyde 

16 (S-hydroxymethylglutathione [HMGSH]) to the intermediate S-formylglutathione, which is 

17 subsequently metabolized to formate and GSH by S-formylglutathione hydrolase.

18

19

20

21 Figure 3-3. Formaldehyde clearance by ALDH2 (GSH-independent) and 

22 ADH3 (GSH-dependent).

23 The Km value for ALDH2 and free formaldehyde is about 400 μM (Teng et al., 

24 2001), whereas the Km value for HMGSH and ADH3 is 6.5 μM (Uotila and 

25 Koivusalo, 1974a,b). The ADH-mediated reactions are reversible in the presence 

26 of excess reduced nicotinamide adenine dinucleotide (NADH).

27 Source: Adapted from Teng et al. (2001).

28
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1 Though ADH3 is rate limiting in this second pathway, the affinity of HMGSH for ADH3 

2 (Km = 6.5 μM) is about 100-fold higher than that of free formaldehyde for ALDH2. In addition 

3 to the kinetic properties, this member of the ADH gene family (Høøg et al., 2003, 2001; Liu et 

4 al., 2001; Jornvall et al., 2000; Estonius et al., 1996) appears to be ubiquitously expressed in 

5 organ tissues (Molotkov et al., 2002; Ang et al., 1996a, b), exhibits cytoplasmic and nuclear 

6 localization (Fernandez et al., 2003), and is the most abundant ADH family member in the liver 

7 and brain (Galter et al., 2003). 

8 In vitro studies have examined the clearance of formaldehyde in several human and rat 

9 tissues (Table 3-2). Examination of formaldehyde metabolism in the rat nasal and olfactory 

10 mucosa indicates nearly identical pharmacokinetics in the rat liver on a per mg of cell lysate 

11 basis (Casanova-Schmitz et al., 1984b). Similar results have been obtained in the absence of 

12 GSH, where other ALDH family members oxidize formaldehyde, albeit with significantly lower 

13 affinity (i.e., higher Km). Hedberg et al. (2000) demonstrated that human buccal tissue lysate 

14 kinetics are in close agreement with those reported for purified human liver ADH3 (Uotila and 

15 Koivusalo, 1974a). Additionally, micro-array analysis indicates that these cells express far more 

16 ADH3 and S-formylglutathione hydrolase than ALDH1 or ALDH2 (Hedberg et al., 2001a). The 

17 results of Ovrebo et al. (2002) are not easily compared with the other studies in Table 3-2 

18 because these studies were in intact cell cultures. However, it is apparent that the 

19 pharmacokinetic values in these human cells are comparable to intact rat liver cells.

20

21 Table 3-2. Formaldehyde kinetics in human and rat tissue samples

22

Source Km (μM)

Vmax

(nmol/mg protein × min) Reference

Purified human liver ADH3 6.5 2.77 ± 0.12 Uotila and Koivusalo (1974a, b)

Rat olfactory mucosa (+ GSH) 2.6 ± 0.5 1.77 ± 0.12 Casanova-Schmitz et al. (1984b)

Rat olfactory mucosa (− GSH) 647 ± 43 4.39 ± 0.14 Casanova-Schmitz et al. (1984b)

Rat respiratory mucosa (+ GSH) 2.6 ± 2.6 0.90 ± 0.24 Casanova-Schmitz et al. (1984b)

Rat respiratory mucosa (− GSH) 481 ± 88 4.07 ± 0.35 Casanova-Schmitz et al. (1984b)

Rat liver (+ GSH) 5.0 ± 1.9 2.0 ± 0.3 Casanova-Schmitz et al. (1984b)

Human bronchial explantsa 5,100 3.3 Ovrebo et al. (2002)

Human bronchial epitheliala 1,400 6.1 Ovrebo et al. (2002)

Rat hepatocytes

a 1,250 4.2 Ovrebo et al. (2002)

Human buccal tissue (+ GSH) 11 ± 2 2.9 ± 0.6 Hedberg et al. (2000)

Human buccal tissue (− GSH) 360 ± 90 1.2 ± 0.7 Hedberg et al. (2000)

Human keratinocytes n.d.b 14.5 ± 1.8 Hedberg et al. (2000)

Human fibroblasts n.d. 17.9 ± 1.4 Hedberg et al. (2000) a 23 These studies were carried out in intact cells by measuring the formation of formate. This likely explains the nearly 

24 1,000-fold increase in apparent Km, since much of the formaldehyde was likely to be bound extracellularly. The 

25 remaining studies used either purified enzyme or cell lysates (as indicated) and measured the formation of NADH. b 26 n.d. = not determined.

27
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1 The data in Table 3-3 along with data indicating the ubiquity of ADH3, indicate that 

2 many human tissues and cells, particularly in the respiratory tract, appear to exhibit significant 

3 capacity to metabolize formaldehyde. Molecular biology techniques have demonstrated the 

4 importance of ADH3 in formaldehyde clearance. For example, ADH-knockout studies have 

shown that the median lethal dose (LD50) values for formaldehyde in wild type, ADH1−/− 5 , 

ADH3−/−, and ADH4−/− 6 mice strains were 0.200, 0.175, 0.135, and 0.190 g/kg, respectively 

7 (Deltour et al., 1999). Although the statistical significance was not reported, the data indicate 

8 that deletion of ADH3 increases the sensitivity of mice to formaldehyde. 

9 The pharmacokinetics of formate are complex. Formate can undergo adenosine 

10 triphosphate (ATP)-dependent addition to tetrahydrofolate (THF), which can carry either one or 

two one-carbon groups. Formate can conjugate with THF to form N10 11 -formyl-THF and its 

isomer N5

-formyl-THF, both of which can be converted to N5,N10 12 -methenyl-THF and 

13 subsequently to other derivatives that are ultimately incorporated into DNA and proteins via 

14 biosynthetic pathways (Figure 3-4).

15

16

17

18 Figure 3-4. Metabolism of formate. 

19 Note: 1, formyl-THF synthetase; 2, formyl-THF dehydrogenase.

20

21 Source: Adapted from Black et al. (1985). 

22

23

24 Elevated levels of formate in urine have been detected following inhalation of methanol 

25 or formate under certain conditions (Liesivuori and Savolainen, 1987), although the 

interpretation of this finding is unclear. There is also evidence that formate generates CO2

– 26
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radicals and can be metabolized to CO2 via catalase and via the oxidation of N10 1 -formyl-THF 

2 (Dikalova et al., 2001, and references therein). The significance of formate in formaldehyde 

3 toxicity is unclear. Black et al. (1985) reported that hepatic tetrahydrofolate levels in monkeys 

4 are 60% of those in rats and that primates are far less efficient in clearing formate than are rats 

and dogs. Studies involving [14C]-formate suggest that about 80% is exhaled as 14 5 CO2, 2–7% is 

6 excreted in the urine, and about 10% undergoes metabolic incorporation (Hanzlik et al., 2005, 

7 and references therein). Mice deficient in formyl-THF dehydrogenase exhibit no change in LD50

8 (via I.P. dose) for methanol or in oxidation of high doses of formate (Cook et al., 2001). It has 

9 been suggested that rodents efficiently clear formate via folate-dependent pathways, 

10 peroxidation by catalase, or an unknown third pathway. Conversely, primates do not appear to 

11 exhibit such capacity and are more sensitive to metabolic acidosis following methanol poisoning 

12 (Cook et al., 2001).

13

14 3.4.2. Formaldehyde Exposure and Perturbation of Metabolic Pathways

15 The enzyme ADH3 has received renewed attention in recent years because of new 

16 functions that have been attributed to it. ADH3 is central to the metabolism of formaldehyde; 

17 however, exposure to formaldehyde in turn alters the activity of ADH3 (in multiple dose18 dependent ways), thereby leading to perturbation of critical metabolic pathways. These are 

19 briefly mentioned below (refer to cited papers for details).

20 1. Exposure to formaldehyde increases cell replication. These proliferating epithelial and 

21 inflammatory cells are rich in both the messenger ribonucleic acid (mRNA) and protein 

22 of ADH3 (Nilsson et al., 2004; Hedberg et al., 2000). Studies in the rodent lung suggest 

23 that increases in ADH3 in such cells dramatically alter the biology of other important 

24 ADH3 substrates that are involved in protein modification and cell signaling (Que et al., 

25 2005).

26 2. ADH3 also participates in the oxidation of retinol and long-chain primary alcohols, as 

27 well as the reduction of S-nitrosoglutathione (GSNO) (Staab et al., 2009; Thompson et 

28 al., 2009; Hedberg et al., 2003; Høøg et al., 2003; Molotkov et al., 2002; Liu et al., 2001; 

29 Jornvall et al., 2000; Jensen et al., 1998). The activity of ADH3 toward some of these 

30 substrates has been shown to be significantly increased in the presence of formaldehyde. 

31 Staab et al. (2009) showed that (in cultured cells) GSNO can accelerate ADH3-mediated 

32 formaldehyde oxidation and, likewise, that formaldehyde increases ADH3-mediated 

33 GSNO reduction nearly 25-fold. The following effects may be noted with regard to the 

34 relevance of such perturbations.
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1 a. GSNO is an endogenous bronchodilator and reservoir of nitric oxide (NO) 

2 activity (Jensen et al., 1998). Details on the ADH3-mediated reduction of GSNO 

3 are shown in Thompson and Grafstrom (2008).

4 b. ADH3 is implicated in playing a central role in regulating bronchiole tone and 

5 allergen-induced hyperresponsiveness (Gerard, 2005; Que et al., 2005).

6 c. As concluded by California Environmental Protection Agency (CalEPA) (2008), 

7 “the dysregulation of NO by formaldehyde [in this manner] helps to explain the 

8 variety and variability in the toxic manifestations following formaldehyde 

9 inhalation.”

10

11 3.4.3. Evidence for Susceptibility in Formaldehyde Metabolism

12 Teng et al. (2001) provided evidence that inhibition of ADH1, ALDH2, and ADH3 has 

13 significant impact on formaldehyde toxicity. The authors speculated that deficiencies in any of 

14 these enzymes would confer an increased susceptibility to formaldehyde toxicity (Teng et al., 

15 2001). Polymorphism in ALDH2 has been shown to have implications in human risk 

16 assessment, specifically with regard to acetaldehyde metabolism (Ginsberg et al., 2002). It is 

17 worth noting, however, that Teng et al. (2001) only demonstrated the importance of ALDH2 in 

18 rat hepatocytes with formaldehyde concentrations of 2.5 mM and greater. Since this 

19 concentration is fivefold greater than the 0.5 mM Km for free formaldehyde, ALDH2 

20 involvement is not unexpected at such high concentrations. Teng et al. (2001) also demonstrated 

21 the importance of ADH1 in driving the reverse reaction (i.e., formaldehyde to methanol) by 

22 coadministration of NADH-generators. This would have the effect of prolonging the life of 

23 formaldehyde by continuous recycling. This is not surprising, given that many ADH reactions 

are reversible. However, levels of nicotinamide adenine dinucleotide (NAD+ 24 ) are normally 

25 much higher than NADH.

26 To date, two studies have reported polymorphisms in ADH3, using the new 

nomenclature.5

 5 Other epidemiologic studies investigating links between ADH3 and oral cancer use the older nomenclature and 

thus refer to Class I ADH (i.e., ADH1) enzymes.

27 ADH3 transcription appears to be regulated by specificity protein (Sp1), with a 

28 minimal promoter located at positions −34 to +61. The reported polymorphisms in ADH3 

29 involve four base-pair substitutions in the promoter region and no polymorphisms in the coding 

30 region (Hedberg et al., 2001b). The three polymorphisms include −197/−196 (GG→AA), −79 

31 (G→A), and +9 (C→T). The genotype frequencies are shown in Table 3-3. Of these alleles, the 

32 +9 (C→T) polymorphism (in the putative Sp1 minimal promoter region) reduced transcriptional 
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1 activity twofold in in vitro reporter gene experiments. According to Hedberg et al. (2001b), no 

2 studies have demonstrated differences in ADH3 enzyme activity in humans. More recently, 

3 single nucleotide polymorphisms in ADH3 have been reported to be associated with childhood 

4 risk of asthma, although the functional relevance of these polymorphisms has not been published 

5 (Wu et al., 2007).

6

7 Table 3-3. Allelic frequencies of ADH3 in human populations

8

Allele frequencies (%)

Population, n AA-197/-196 GG-197/-196 A-79 G-79 T+9 C+9

Chinese, 83 22 78 100 – – 100

Spanish, 95 41 59 62 38 – 100

Swedish, 96 47 53 67 33 1.5 98.5

9

10 Source: Adapted from Hedberg et al. (2001b).

11

12

13 Alterations in THF pathways may also have an impact on formaldehyde toxicity. These 

14 could result from polymorphisms in various enzymes or differences in folate intake and 

15 absorption. Species differences in tetrahydrofolate levels (Black et al., 1985) are thought to play 

16 a role in the differential responses to methanol across species. Cook et al. (2001) speculate that 

17 rats have redundant pathways for formate clearance that may be absent or less efficient in 

18 primates.

19

20 3.5. EXCRETION

21 The main product of metabolic clearance of formaldehyde is formate, which is further 

22 metabolized to CO2 and water, incorporated into the one-carbon pool, or eliminated in the urine. 

23 There is also some evidence that formaldehyde is present in exhaled breath; however, it is 

24 unclear whether this originates from endogenous sources, or is simply a function of ambient 

25 formaldehyde dissolved in fluids lining POEs. The following sections describe first experiments 

26 in laboratory species and then available data in humans. Broadly, these studies address two 

27 important questions that may be of relevance for risk assessment. First, it may be of interest to 

28 know what levels of formaldehyde are exhaled for comparison with inhaled levels, and whether 

29 there is any relationship between external exposure and exhaled levels. Second, there are recent 

30 studies that have attempted to relate genetic polymorphisms and changes in gene transcription 

31 level to levels of putative urinary formaldehyde biomarkers. 

32

33
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1 3.5.1. Formaldehyde Excretion in Rodents

2 Heck et al. (1983) determined the relative contributions of various excretion pathways in 

3 F344 rats following inhalation exposure to formaldehyde. Table 3-4 indicates that the relative 

4 excretion pathways were independent of exposure concentration (at least between 0.63 and 

15 ppm). Nearly 40% of inhaled [14 5 C]-formaldehyde appeared to be eliminated via expiration, 

6 probably as CO2 (it should be recalled that nearly 100% of inhaled formaldehyde is absorbed). 

7 Within 70 hours of a 6-hour exposure to formaldehyde, about 17 and 5% were eliminated in the 

urine and feces, respectively. Nearly 40% of inhaled [14 8 C]-formaldehyde remained in the 

9 carcass, presumably due to metabolic incorporation. 

10

Table 3-4. Percent distribution of airborne [14 11 C]-formaldehyde in F344 rats

12

Source

Concentration of formaldehyde (ppm)

0.63 13.1

Distribution (%)a

Expired air 39.4 ± 1.5 41.9 ± 0.8

Urine 17.6 ± 1.2 17.3 ± 0.6

Feces 4.2 ± 1.5 5.3 ± 1.3

Tissues and carcass 38.9 ± 1.2 35.2 ± 0.5

13 a 14 Values are means ± standard deviations (n = 4).

15

16 Source: Heck et al. (1983).

17

18

19 Mashford and Jones (1982) examined elimination pathways of formaldehyde in rats 

20 exposed by I.P. injection. Urine and exhaled gases were collected from rats exposed to 4 or 

40 mg/kg [14 21 C]-formaldehyde. At 48 hours postinjection, 82 and 78% of the radiolabel were 

exhaled as 14CO2, whereas exhaled [14 22 C]-formaldehyde was not detected. Mashford and Jones 

23 (1982) also further identified the urinary metabolites. Five hours after injection of the higher 

24 dose, formate was determined to comprise 80% of the urinary metabolites. The authors were 

25 unable to detect cysteine derivatives observed in other studies (see below) in the urine of these 

26 rats prior to or after formaldehyde exposure. The authors stated that if formaldehyde were to be 

27 excreted in urine containing cysteine, then thiazolidine-4-carboxylate (TZCA) would likely be 

28 produced. They speculated that species differences in urinary compounds may produce 

29 formaldehyde conjugates (or artifacts). 

30 Hemminki (1982) reacted formaldehyde and acetaldehyde with cysteine, 

31 N-acetylcysteine, and GSH and found that formaldehyde reacted most rapidly with cysteine to 

32 form TZCA. Similarly, acetaldehyde reacted preferentially with cysteine, albeit slower than 
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1 formaldehyde, to form a thiazolidine derivative. However, when each aldehyde was 

2 administered I.P. (10% formaldehyde, 50% acetaldehyde), thioether concentrations (nmol/mol 

3 creatinine) significantly increased in the 24 and 48 hour urine of acetaldehyde-treated rats but 

4 not formaldehyde-treated rats. These data suggest that formaldehyde is not appreciably excreted 

5 in urine and thus cysteine conjugates are not likely to represent formaldehyde exposure. 

6 Most recently, Shin et al. (2007) attempted to show that formaldehyde inhalation 

7 increased urinary TZCA levels in Sprague-Dawley rats. Treated rats were exposed to 3.1 and 

8 38.1 ppm formaldehyde for 6 hours/day for 2 weeks, and urine was collected for 3 days. The 

9 TZCA level in four control rats was 0.07 ± 0.02 mg/L, whereas levels in the 3 and 38 ppm 

10 groups were 0.18 ± 0.045 and 1.01 ± 0.36, respectively. Notably, the concentrations in the four 

11 highest exposed animals (0.71, 0.70, 1.20, and 1.43 ppm) exhibited a nearly twofold range. 

12 However, these comparisons are confounded if the exposures have any influence on urine 

13 production and urine cysteine levels. The study does not provide any data that might allow one 

14 to examine this issue. 

15

16 3.5.2. Formaldehyde Excretion in Exhaled Human Breath

17 Several human and animal studies have attempted to measure the concentration of 

18 formaldehyde in exhaled breath. None of the human studies were designed to distinguish 

19 between exogenous (room air) and endogenous (systemic) formaldehyde in exhaled breath. In 

20 order to discern whether endogenous formaldehyde is excreted into the lungs, test subjects must 

21 breathe formaldehyde-free air. Because subjects were breathing room air, which contained 9-10 

22 ppb formaldehyde in two studies and unspecified concentrations in two other studies, it is 

23 impossible to ascertain whether there was any endogenous formaldehyde in their exhaled breath. 

24 One study demonstrates that the formaldehyde concentration is lower in exhaled breath than in 

25 inhaled breath (Cáp et al., 2008). Also, none of the human studies investigated whether there is 

26 any relationship between exhaled formaldehyde levels and food intake, life stage, smoking, or 

27 health status. This assessment identifies a critical research need for further studies on the 

28 measurement of exhaled formaldehyde.

29 Proton transfer reaction mass spectrometry (PTR-MS) has been applied to measure trace 

30 compounds in exhaled breath including volatile organics and specifically formaldehyde. The 

basic method of PTR-MS is based on the transfer of protons from H30+ 31 to gases in exhaled 

32 breath and the in-line monitoring of products where gases are tentatively identified by the mass 

33 to charge ratio (m/z) where a m/z of 31 is consistent with protonated formaldehyde (Hansel et al., 

34 1995; Lindinger et al., 1998). It is important to note that reaction products from methanol and 

35 ethanol may also produce fragments with an m/z ratio of 31 (Kusch et al., 2008). Up to 1% of 
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1 the mass of ethanol and methanol in exhaled breath may be detected with an m/z ratio of 31 and 

2 thus be identified as formaldehyde, so accurate quantitation of formaldehyde should adjust for 

3 this contribution (Španĕl and Smith, 2008). Selected ion flow tube mass spectrometry (SIFT4 MS) is an application of PTR-MS developed for real-time analysis of trace gases in breath 

5 (Smith and Španĕl, 2005; Španĕl and Smith, 2007).

6 Moser et al. (2005) measured levels of 179 volatile organic compounds (VOCs) in the 

7 exhaled breath of 344 individuals. This study was not designed to ascertain whether exhaled 

8 formaldehyde is of endogenous origin, but rather to demonstrate that proton transfer reaction9 mass spectrometry can be used as a new method for rapid screening of large collectives for risk 

10 factors (e.g., smoking behavior), potential disease biomarkers, and ambient air characterization. 

11 The study was conducted at a health fair. The test subjects had a mean age of 61.6 years; 63% 

12 were males and 14% were smokers. Samples of room air were collected and evaluated in 

13 parallel with exhaled breath samples. The authors note that formaldehyde was detected in room 

14 air, but did not report the levels; rather they stated that the background concentrations were 

15 negligible. Of the 179 volatile organic compounds measured, data were reported for 14, 

16 including formaldehyde and formic acid. The formaldehyde levels in exhaled breath spanned 

from 1.2 to 72.7 ppb with a median of 4.3 ppb and 75th 17 percentile of 6.3 ppb. No explanation 

18 was provided for this wide range in values, and there was no distinction of the data by sex, age, 

19 health, or smoking status. 

20 Because the test subjects were breathing ambient indoor air which contained an 

21 unspecified concentration of formaldehyde, it is impossible to ascertain whether exhaled 

22 formaldehyde was from an endogenous or exogenous source, or both. Moser et al. also note that 

23 significant differences in exhaled breath composition could be found between smokers and non24 smokers for 32 of the 179 chemicals measured, but the 32 chemicals were not named and no 

25 substantiating data were provided. Formaldehyde may have been among these 32 chemicals 

26 since it is a major component of cigarette smoke (NLM, 2001). 

27 The report by Moser et al. does not provide the limit of detection for any of the 

28 compounds measured or details of the analytical method. The minimum formaldehyde level 

29 reported in exhaled breath was 1.23 ppb. The method employed by Moser et al. does not adjust 

30 the detection of apparent formaldehyde (m/z) by accounting for the contribution of methanol and 

31 ethanol. The levels of methanol and ethanol in exhaled breath were reported, however. Based 

32 on 1% of the mass of these chemicals contributing to the m/z = 31 in the PTR-MS method 

33 (Španĕl and Smith, 2008), Table 3-5 demonstrates that the highest formaldehyde levels reported 

34 may have been an artifact of high methanol and ethanol in exhaled breath. Because the in-room 

35 formaldehyde concentrations were not reported, it is unknown how much of this formaldehyde 
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1 represents formaldehyde levels in inhaled air. Additionally, since these samples were simple 

2 exhaled breath samples and not SIFT-MS samples, it is impossible to distinguish between air 

3 which reached the pulmonary region versus air which only entered the upper airways.

4

5 Table 3-5. Apparent formaldehyde levels in exhaled breath of individuals 

6 attending a health fair, adjusted for methanol and ethanol levels which 

7 contribute to the detection of the protonated species with a mass to charge 

8 ratio of 31 reported as formaldehyde (m/z = 31)

9

Chemical Minimum

25th

percentile Median

75th

percentile

97.5th

percentile Maximum

Methanol (m/z = 31) 13.367 106.227 161.179 243.185 643.614 1536.499

1% of methanol 

predicted as m/z = 31 0.13 1.06 1.61 2.43 6.44 15.36

Ethanol 11.583 23.1 34.664 64.24 549.24 9779.768

1% of ethanol predicted 

as m/z = 31 0.12 0.23 0.35 0.64 5.49 97.80

Mass of m/z = 31 

attributable to methanol 

and ethanol

0.25 1.29 1.96 3.07 11.93 113.16

Mass of m/z = 31 

reported as 

formaldehyde

1.23 3.1 4.26 6.33 39.8 72.7

10

11 Source: Moser et al. (2005).

12

13

14 Turner et al. (2008) compared levels of volatile compounds in exhaled breath to levels in 

15 emissions from the skin in five males (3 in their mid 20s, and the others 42 and 49 years old). 

16 The subjects fasted overnight, and measurements were taken before and after ingesting 75 g of 

17 glucose. The source of the inhaled air was laboratory air which contained an unreported 

18 concentration of formaldehyde. Formaldehyde was not detected in the exhaled breath of any 

19 subjects. The limit of detection was 5 ppb or better. 

20 Wang et al. (2008) measured the concentrations of formaldehyde and 9 other chemicals 

21 in the exhaled breath of three healthy male laboratory workers. The limit of quantification for 

22 formaldehyde was not reported. A series of measurements were taken in the nose and mouth, 

23 and also in the oral cavity during breath holding. Table 3-6 presents the median formaldehyde 

24 levels and geometric standard deviations for the three subjects. The authors noted that 

25 formaldehyde in exhaled breath was at a level somewhat lower than the ambient air 

26 concentration, but they could not be certain of its origin. 

27

28
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1 Table 3-6. Measurements of exhaled formaldehyde concentrations in the 

2 mouth and nose, and in the oral cavity after breath holding in three healthy 

3 male laboratory workers

4

Subject

Formaldehyde

(median ppb / σg)

A Mouth 5 / 2.3

Nose 7 / 2.1

Oral cavity 5 / 2.3

B Mouth 7 / 2.3

Nose 5 / 2.1

Oral cavity 6 / 1.9

C Mouth 4 / 2.5

Nose 6 / 1.9

Oral cavity 6 / 1.9

Laboratory air 9.6 ±1.5

5

6 Source: Wang et al. (2008).

7

8

9 Cáp et al. (2008) evaluated relationships between volatile organic compounds measured 

10 in exhaled breath and exhaled breath condensate. Exhaled breath condensate consists of 

11 aerosolized particles of airway lining fluid evolved from the airway wall by turbulent airflow 

12 that serve as seeds for substantial water vapor condensation, which then serves to trap water 

13 soluble volatile gases. This study also attempted to ascertain whether the source of each 

14 compound was endogenous or exogenous. According to the published article and electronic 

15 communication with Dr. Patrik Španĕl, a co-author for this study, the limit of quantification was 

16 3 ppb or better. Measurements of formaldehyde in the direct exhaled breath of 34 subjects (25 to 

17 62 years; 11 males; 2 smokers) varied from 0 to 12 ppb with a mean of 2 ppb and a median of 1 

18 ppb. Measurements of formaldehyde in exhaled breath condensate ranged from 0 to 12 ppb with 

19 a mean of 2 ppb and a median of 0 ppb. Two smokers exhaled formaldehyde concentrations of 0 

20 and 3 ppb. The scatter plot in Figure 3-5 shows that most subjects exhaled formaldehyde 

21 concentrations of 0-3 ppb (x axis), which was considerably lower than the ambient air 

22 concentration of 9.6 ±1.5 ppb. The authors concluded that the exhaled concentrations were 

23 influenced by exogenous levels in room air, which were not controlled. Dr. Španĕl offered two 

24 plausible explanations for why exhaled formaldehyde levels are lower than inhaled levels. He 

25 noted that some subjects were tested within several minutes of entering the laboratory from 

26 outdoors, so they may not have had time to acclimate to the higher indoor air concentration. 

27 Another explanation is that a substantial portion of inhaled formaldehyde, which is highly 
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1 reactive, was retained in the respiratory tract and thus not exhaled; that is, 9.6 ppb of 

2 formaldehyde was inhaled but only a mean of 2 ppb was exhaled. 

3 In this and other human studies, there was no adjustment for an artifact in the analytical 

4 method that makes it impossible to distinguish between formaldehyde and reaction products for 

5 1% of exhaled methanol and ethanol because they have the same mass to charge ratio (m/z = 31). 

6 In fact, the concentration of methanol and ethanol that is misidentified as formaldehyde exceeds 

7 the reported concentrations of exhaled formaldehyde. Thus, it is highly likely that the actual 

8 exhaled formaldehyde concentration in Cáp et al. (2008) was significantly lower than 2 ppb, and 

9 that there was little or no endogenous formaldehyde in the exhaled breath. This would be 

10 consistent with an animal study in which Mashford and Jones (1982) detected no exhaled 

formaldehyde in rats injected I.P. with 40 mg/kg [14 11 C]-formaldehyde. Over 48 hours, 78% of the 

radioactivity was exhaled as 14 12 CO2 and 11% was excreted in the urine as formate, N13 (hydroxymethyl)urea, N,N’-bis-(hydroxymethyl)urea, and possibly polymethylene urea. In 

14 summary, there are insufficient data at this time to confidently establish a concentration of 

15 formaldehyde in exhaled breath that can be attributed to endogenous sources. 

16

17

18 Figure 3-5. Scatter plot of formaldehyde concentrations measured in ppb in 

19 direct breath exhalations (x axis) and exhaled breath condensate headspace 

20 (y axis). 

21

22 Source: Cáp et al. (2008).

23

24

25 3.5.3. Formaldehyde Excretion in Human Urine

26 Gottschling et al. (1984) examined urinary formic acid in 35 veterinary students. 

27 Personal monitoring badges were worn and returned after class, and urine samples were taken 
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1 prior to class and within 2 hours after the class. Mean exposure levels were about 100 ppb. 

2 Baseline averages of urinary formic acid (as a sodium salt) were 12.47 mg/L and ranged from 

3 2.43 to 28.38 mg/L among subjects. Postexposure formate levels were slightly elevated but were 

4 not statistically significant. Moreover, formate levels decreased in several individuals relative to 

5 pre-exposure levels. The authors concluded that variability in urinary formate may mask any 

6 changes and that monitoring formate within 2 hours of exposure is not informative. It is worth 

7 noting, however, that interpretation of this finding is confounded due to the fact that diet was not 

8 controlled and because no markers for urinary normalization were employed (Boeniger, 1987). 

9 Boeniger (1987) reviewed previously published data on formate in urine (some of which 

10 were in German). In one occupational study, workers were exposed to an average formaldehyde 

exposure of 1.28 mg/m3 over a 6-hour work shift. This implies an average intake of 6 mg;6

17 In the previously described study by Shin et al. (2007), human urine samples were shown 

18 to contain TZCA, although variability was not reported. A subsequent study reported that urine 

19 TZCA levels were higher in individuals living in newer apartments (0.18 ± 0.121 mg/g 

creatinine) as compared to older apartments (0.097 ± 0.040 mg/g creatinine) (Li et al., 2007)

11

12 Boeniger reported a range of 2.5 to 13 mg. However, the original study reported that post-shift 

13 formate levels were 152 mg/L, whereas the levels were only 24 mg/L 6 days later (no exposure). 

14 Considering that only a small percentage of inhaled formaldehyde would be excreted in urine, it 

15 is unclear how (or whether) formaldehyde exposure, with the highest total dose of 13 mg, could 

16 be responsible for the observed increase. 

7

26

20 . 

21 The authors cited this as evidence that TZCA is a urinary marker for formaldehyde exposure, 

22 even though TZCA levels were not correlated to measured (or estimated) formaldehyde 

23 exposures. The individuals also differed significantly in age (21.5 vs. 28.6, p = 0.053) and 

24 differed in smoking percentage (10 vs. 27%). Clearly these two studies do not establish a 

25 relationship between human formaldehyde exposure and urine TZCA levels. 

27 3.6. MODELING THE TOXICOKINETICS OF FORMALDEHYDE AND DPX

28 3.6.1. Motivation

29 Airway geometry is expected to be an important determinant of inhaled formaldehyde 

30 dosimetry in the respiratory tract and its differences across species. The uptake of formaldehyde 

31 in the upper respiratory tract is highly nonhomogenous and spatially localized and exhibits 

32 strong species differences. Species differences in kinetic factors have been argued to be the key 

 6 1.28 mg/m3 / 1,000L/m3 × 13.8L/minute × 60 minutes/hour × 6 hours. 7 This study is described in greater detail in Chapter 5.
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1 determinants of species-specific lesion distributions for formaldehyde and other reactive inhaled 

2 gases. In the first subsection here, the advantage gained in the quantitative risk assessment by 

3 modeling these differences in the upper respiratory tract is explained. Such a model was 

4 constructed by using computational fluid dynamic (CFD) methods for modeling airflow and 

5 regional formaldehyde uptake in the rat, rhesus monkey, and human nose by scientists at the 

6 Chemical Industries Institute of Toxicology (CIIT) Centers for Health Research (presently the 

7 Hamner Institutes of Health Sciences). While frank effects were seen only in the upper 

8 respiratory tract in rodents, mild lesions were also present in the major bronchiolar region of the 

9 rhesus monkey. Therefore, with regard to extrapolation of cancer risk from animal bioassays to 

humans, the upper and lower human respiratory tract should both be considered potentially

19 The fluid dynamics modeling in the respiratory tract comprises two steps (Kimbell et al. 

20 2001): airflow through the lumen and formaldehyde uptake by the lining of the respiratory tract. 

21 Flow streamlines in the CFD simulations agreed reasonably well with experimentally observed 

22 patterns in casts of the rat, monkey, and human nasal passages as well as with measurements of 

23 velocity taken in hollow molds of the human nose. Pressure drop as a function of volumetric 

24 flow also compared well with measurements made in rats. Unlike the airflow simulations, no 

25 validation of the regional formaldehyde uptake simulations (that is, the spatial distribution of 

26 uptake) was possible. (It was possible to compare simulations of overall uptake with 

27 experimentally observed values.) In this assessment, several indirect qualitative and quantitative 

28 lines of evidence were relied on to provide general confidence in the formaldehyde regional 

29 uptake profile in the F344 rat nasal passages. In addition to the agreement mentioned earlier 

30 with respect to airflow profiles, this evidence includes general agreement between measured and 

31 predicted levels of formaldehyde DPXs (in the “high-tumor” regions) when simulations of 

32 airflow and regional formaldehyde uptake were used as input to a physiologically based 

33 pharmacokinetic (PBPK) model for DPX kinetics (Cohen-Hubal et al. 1997). Such indicators 

34 are not available for the simulation of uptake patterns in the human. With regard to modeling the 

35 lower respiratory tract, calculations in Overton et al. (2001) are based on an idealized 

10 at 

11 risk of developing formaldehyde-induced squamous cell carcinoma. Therefore, formaldehyde 

12 dose to the entire human respiratory tract human respiratory tract was modeled in order to 

13 develop a dose-response relationship that considered the entire respiratory tract. Accordingly, in 

14 the second subsection results of modeling airflow and regional formaldehyde uptake in the 

15 human lower respiratory tract by Overton et al. (2001) are provided. Unsteady effects were 

16 argued to be insignificant at resting breathing rates, and therefore steady-state inspiratory flow 

17 was assumed. Since these models have been described in various reports and publications, the 

18 technical details are consigned to appendices or the literature is referenced.
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1 representation of airways in the human lung and of air flow through them (referred to in the 

2 literature as “single-path” models). Overton et al. (2001) did not attempt to validate their 

3 simulations. However, the following observation may be noted in support of their model. In the 

4 case of the deposition of coarse and fine particulate matter, single-path models have traditionally 

5 provided a reasonably accurate representation of the average deposition in a given generation of 

6 the lung airways (i.e. airways at a given depth in the lung) for a normal human population. 

7 There were mass balance errors in the CFD calculations (Kimbell et al. 2001). Mass 

8 balance errors associated with formaldehyde uptake into tissue ranged from less than 14% for the 

9 rat, monkey, and human at resting minute volume to approximately 27% at the highest 

10 inspiratory flow rates of 31.8 and 37 L/minute in the human. 

11 If DPX formation and cell proliferation are driven by formaldehyde flux, then these 

12 quantities may also be expected to exhibit site and species differences, thus arguing for linking 

13 these quantities to the modeled formaldehyde flux (Conolly et al. 2000). 

14 The third subsection evaluates PBPK models for DPX kinetics in the F344 rat and rhesus 

15 monkey, using CFD simulations of formaldehyde flux to the nasal lining as input (Klein et al., 

16 2009; Subramaniam et al., 2007; Conolly et al., 2000), and discusses their uncertainties. This 

17 subsection further discusses issues related to the scaling of the animal models to the human.

18 This assessment uses internal dose metrics computed by using the models described in 

19 this section so as to derive more accurate human equivalent concentrations from the animal 

20 bioassays than would be obtained by averaging over the respiratory surface area. The strengths 

21 and uncertainties associated with the data and the models and their relevance to the hypothesized 

22 mode of action are discussed in some length.

23

24 3.6.2. Species Differences in Anatomy: Consequences for Gas Transport and Risk

25 As discussed earlier, formaldehyde is highly reactive and water soluble (categorized as a 

26 category 1 gas), thus its absorption in the mucus layer and tissue lining of the upper respiratory 

27 tract is known to be significant. The regional inhaled dose of formaldehyde to the respiratory 

28 tract of a given species depends on the amount of formaldehyde delivered by inhaled air, the 

29 absorption characteristics of the nasal lining, and reactions in the tissue. The amount delivered 

30 by inhaled air is a function of the major airflow patterns, air-phase diffusion, and absorption at 

31 the airway-epithelial tissue interface. The dose of formaldehyde to the epithelial tissue, which is 

32 different from the amount delivered, depends on the amount absorbed at the airway-tissue 

33 interface, water solubility, mucus-to-tissue phase diffusion, and chemical reactions, such as 

34 hydrolysis, protein binding, and metabolism. It has been argued strongly that species differences 

35 in these kinetic factors are determinants of species-specific lesion distributions for formaldehyde 
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1 and other inhaled gases (Moulin et al., 2002; Bogdanffy et al., 1999; Ibanes et al., 1996; 

2 Monticello et al., 1996; Monticello and Morgan, 1994; Morgan et al., 1991).

3 Because of the convoluted nature of the airways in the upper respiratory tract, the 

4 absorption of such gases in the upper respiratory tract is highly nonhomogeneous. There are 

5 large differences across species in the anatomy of the upper respiratory tract (Figure 3-6) and in 

6 airflow patterns (Figure 3-7). Therefore, as shown in the simulations in Figure 3-8, it may be 

7 expected that the uptake patterns and thus risk due to inhaled formaldehyde will also show 

8 strong species dependence. Morgan et al. (1991) concluded that airflow-driven dosimetry plays 

9 a critical role in determining the site specificity of various formaldehyde-induced responses, 

10 including tumors, in the nose of the F344 rat. The convoluted geometry of the airway passages 

11 in the upper respiratory tract, as seen from the cross sections of the nose in Figure 3-6, renders an 

12 idealized representation of fluid flow and uptake profiles almost impossible. For these reasons, 

13 Kimbell et al. (1993), Kepler et al. (1998), and Subramaniam et al. (1998) developed 

14 anatomically realistic finite-element representations of the noses of humans, F344 rats, and 

15 rhesus monkeys. These representations were subsequently used in physical and computational 

16 models (Figure 3-6). This assessment utilizes dosimetry derived from these representations.

17 An accurate calculation of species differences in formaldehyde dosimetry in the upper 

18 respiratory tract is important to the extrapolation problem for another reason. The upper 

19 respiratory tract in rats is an extremely efficient scrubber of reactive gases (97% uptake) 

20 (Morgan et al., 1986), thereby protecting the lower respiratory tract from gaseous penetration. 

21 On the other hand, there is considerably more fractional penetration of formaldehyde into the 

22 lower respiratory tract of the rhesus monkey than in the rat (see Figure 3-8). Therefore, an 

23 accurate determination of scrubbing in the upper respiratory tract is important to delineate 

24 species differences in the level of risk to the lower respiratory tract. Thus, in the case of the 

25 rhesus monkey, the model by Kepler et al. (1998) included the trachea. Because the human 

26 model also had to address the potential for oronasal breathing, an idealized single-path model of 

27 the lower respiratory tract was attached to a model of the upper respiratory tract (Overton et al., 

28 2001). It is important to note that the models mentioned above represent nasal passages 

29 reconstructed from a single individual from each species (Kimbell et al., 2001a, b; Conolly et al., 

30 2000; CIIT, 1999; Subramaniam et al., 1998). This is discussed later in the context of 

31 intraspecies variability.

32 The highly localized nature of uptake patterns shown in Figure 3-8 means that averaging 

33 uptake over the entire nasal surface area would dilute the regional dose over areas where 

34 response was observed and that an extrapolation based on such averaging would clearly not be 

35 accurate.
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1 Figure 3-6. Reconstructed nasal passages of F344 rat, rhesus monkey, and 

2 human. 

3

4 Note: Nostril is to the right, and the nasopharynx is to the left. Right side shows 

5 the finite element mesh. Left-hand side shows tracings of airways obtained from 

6 cross sections of fixed heads (F344 rat and rhesus monkey) and magnetic 

7 resonance image sectional scans (humans). Aligned cross sections were 

8 connected to form a three-dimensional reconstruction and finite-element 

9 computational mesh. Source: Adapted from Kimbell et al. (2001a). Additional 

10 images provided courtesy of Dr. J.S. Kimbell, CIIT Hamner Institutes.
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1

2

3 Figure 3-7. Illustration of interspecies differences in airflow and verification 

4 of CFD simulations with water-dye studies.

5 Note: Panels A and B show the simulated airflow pattern versus water-dye 

6 streams observed experimentally in casts of the nasal passages of rats and 

7 monkeys, respectively. Panel C shows the simulated inspiration airflow pattern, 

8 and the histogram depicts the simulated axial velocities (white bars) vs. 

9 experimental measurements made in hollow molds of the human nasal passages. 

10 Dye stream plots were compiled for the rat and monkey over the physiological 

11 range of inspiration flow rates. Modeled flow rates in humans were 15 L/minute.

12 Source: Adapted from Kimbell et al. (2001a).

13
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1

F344 Rat

Rhesus Monkey

Human

Key

pmol/(mm2-hr-ppm)

2000

1500

1000

500

0

2

3 Figure 3-8. Lateral view of nasal wall mass flux of inhaled formaldehyde 

4 simulated in the F344 rat, rhesus monkey, and human.

5

6 Note: Nostrils are to the right. Simulations were exercised in each species at 

7 steady-state inspiration flow rates of 0.576 L/minute in the rat, 4.8 L/minute in the 

8 monkey, and 15 L/minute in the human. Flux was contoured over the range from 

0–2,000 pmol/(mm2 9 -hour-ppm) in each species.

10

11 Source: Kimbell et al. (2001a).

12

13

14 Another factor to consider in the extrapolation is that monkeys and humans are oronasal 

15 breathers while rats are obligate nose-only breathers. Thus, for humans and monkeys, oronasal 

16 or oral breathing implies a significantly higher uptake in the lower respiratory tract. It is known 

17 that a significant fraction of the human population breathes normally through the mouth. 

18 Finally, activity profiles are also determinants of extraction efficiency (see Figure 3-9) and of 

19 breathing route (Niinimaa et al., 1981). Given the fact that formaldehyde-induced lesions were 
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1 observed as far down the respiratory tract as the first bifurcation of the lungs in exposed 

2 monkeys, the entire human respiratory tract should be considered when extrapolating data from 

3 rats.

4

5

6

7 Figure 3-9. CFD simulations of formaldehyde flux to human nasal lining at 

8 different inspiratory flow rates.

9

10 Note: Right lateral view. Uptake is shown for the non-squamous portion of the 

11 epithelium. The front portion of the nose (vestibule) is lined with keratinized 

12 squamous epithelium and is expected to absorb relatively much less 

13 formaldehyde.

14

15 Source: Kimbell et al. (2001b). 

16

17
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1 3.6.3. Modeling Formaldehyde Uptake in Nasal Passages

2 CIIT scientists chose the F344 strain of the rat since it was assumed to be anatomically 

3 representative of its species and because it is widely used experimentally, most notably in 

4 bioassays sponsored by the National Toxicology Program and at CIIT. The approximate 

5 locations of squamous, mucus-coated, and nonmucus epithelial cells were mapped onto the 

6 reconstructed nasal geometry of the CFD models. Taken together, these regions of nonmucus 

7 and mucus-coated cells comprise the entire surface area of the nasal passages (see original 

8 papers and CIIT [1999] for further details on reconstruction and morphometry). Types of nasal 

9 epithelium overlaid onto the geometry of the models were assumed to be similar in 

10 characteristics across all three species (rat, monkey, and human) except for thickness, surface 

11 area, and location. Species-specific mucosal thickness, surface area, and location were estimated 

12 from the literature, from the documentation of the CFD models, or by direct measurements 

13 (Conolly et al., 2000; CIIT, 1999). The nasal passages of all three species were assumed to have 

14 a continuous mucus coating over all surfaces except specific areas in the nasal vestibule. As 

15 discussed at the beginning of this chapter, formaldehyde hydrolyzes in water and reacts readily 

16 with a number of components of nasal mucus. Absorption rates of inhaled formaldehyde by the 

17 nasal lining were therefore assumed to depend on where the epithelial lining is coated by mucus 

18 and where it is not.

19 To calculate an airflow rate that would be comparable among species, the amount of 

20 inspired air (tidal volume, VT) was divided by the estimated time involved in inhalation (half the 

21 time a breath takes, or (1/2)(1/[breathing frequency, f]). Thus, an inspiratory flow rate was 

22 calculated to be 2VTf, or twice the minute volume. Predicted flux values represent an average of 

23 one nasal cycle. Minute volumes were allometrically scaled to 0.288 L/minute for a 315 g rat 

24 from data given by Mauderly (1986). Simulations were therefore carried out at 0.576 L/minute 

25 for the rat.

26 The fluid dynamics modeling in the respiratory tract comprises two steps: modeling the 

27 airflow through the lumen (solution of Navier-Stokes equations) and modeling formaldehyde 

28 uptake by the respiratory tract lining (solution of convective-diffusion equations for a given 

29 airflow field). Details of these simulations, including boundary conditions for air flow and mass 

30 transfer, are provided in Kimbell et al. (2001a, b, 1993) and Subramaniam et al. (1998). 

31 Formaldehyde absorption at the airway-to-epithelial tissue interface was assumed to be 

32 proportional to the air-phase formaldehyde concentration adjacent to the nasal lining layer in 

33 monkeys and humans (see the original paper [Kimbell et al., 2001a] for a more detailed 

34 elaboration of the calculations for these coefficients).
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1 Because formaldehyde is highly water soluble and reactive, Kimbell at al. (2001a) 

2 assumed that absorption occurred only during inspiration. Thus, for each breath, flux into nasal 

passage walls (rate of mass transport in the direction perpendicular to the nasal wall per mm2 3 of 

4 the wall surface) was assumed to be zero during exhalation, with no backpressure to uptake built 

5 up in the tissues. Overton et al. (2001) estimated the error due to this assumption to be small, 

6 roughly an underestimate of 3% in comparison to cyclic breathing. Also, this assumption is the 

7 same as that used in default methods for reference concentration determination and has been 

8 used in other PBPK model applications to describe nasal uptake (Andersen and Jarabek, 2001). 

9

10 3.6.3.1. Flux Bins

11 A novel contribution of the CIIT biologically motivated dose-response model is that cell 

12 division rates and DPX concentrations are driven by the local concentration of formaldehyde. 

13 These were determined by partitioning the nasal surface by flux, resulting in 20 “flux bins.” 

14 Each bin was comprised of elements (not necessarily contiguous) of the nasal surface that 

15 receive a particular interval of formaldehyde flux per ppm of exposure concentration (Kimbell et 

16 al., 2001a). The spatial coordinates of elements comprising a particular flux bin were fixed for 

17 all exposure concentrations, with formaldehyde flux in a bin scaling linearly with exposure 

concentration (ppm). Thus, formaldehyde flux was expressed as pmol/(mm2 18 -hour-ppm).

19

20 3.6.3.2. Flux Estimates

21 Formaldehyde flux was estimated for the rat, monkey, and human over the entire nasal 

22 surface and over the portion of the nasal surface that was lined by nonsquamous epithelium. 

23 Formaldehyde flux was also estimated for the rat and monkey over the areas where cell 

24 proliferation measurements were made (Monticello et al., 1991, 1989) and over the anterior 

25 portion of the human nasal passages that is lined by nonsquamous epithelium. Figure 3-8 shows 

26 the mass flux of inhaled formaldehyde to the lateral wall of nasal passages in the F344 rat, rhesus 

27 monkey, and human (Kimbell et al., 2001a).

28 Maximum flux estimates for the entire upper respiratory tract were located in the mucus29 coated squamous epithelium on the dorsal aspect of the dorsal medial meatus near the boundary 

30 between nonmucus and mucus-coated squamous epithelium in the rat, at the anterior or rostral 

31 margin of the middle turbinate in the monkey, and in the nonsquamous epithelium on the 

32 proximal portion of the mid-septum near the boundary between squamous and nonsquamous 

33 epithelium in the human (see Kimbell et al. [2001b] for tabulations of comparative estimates of 

34 formaldehyde flux across the species).
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1 The rat-to-monkey ratio of the highest site-specific fluxes in the two species was 0.98. In 

2 the rat, the incidence of formaldehyde-induced squamous cell carcinomas in chronically exposed 

3 animals was high in the anterior lateral meatus (Monticello et al., 1996). Flux predicted per ppm 

4 in this site and flux predicted near the anterior or proximal aspect of the inferior turbinate and 

5 adjacent lateral walls and septum in the human were similar, with a rat-to-human ratio of 0.84.

6

7 3.6.3.3. Mass Balance Errors

8 Overall uptake of formaldehyde was calculated as 100% x (mass entering nostril − mass 

9 exiting outlet)/(mass entering nostril). Mass balance errors for air, 100% x (mass of air entering 

10 nostril − mass exiting outlet)/(mass entering nostril), and inhaled formaldehyde, 100% x (mass 

11 entering nostril − mass absorbed by airway walls − mass exiting outlet)/(mass entering nostril), 

12 were calculated. Mass balance errors associated with simulated formaldehyde uptake from air 

13 into tissue ranged from less than 14% for the rat, monkey, and human at 7.4 and 15 L/minute to 

14 approximately 27% at the highest inspiratory flow rates of 31.8 and 37 L/minute (Kimbell et al., 

15 2001b). Kimbell et al. (2001b) corrected the simulation results for these errors by evenly 

16 distributing the lost mass over the entire nasal surface. 

17

18 3.6.4. Modeling Formaldehyde Uptake in the Lower Respiratory Tract

19 Lesions were observed in the lower respiratory tract of rhesus monkeys exposed to 6 ppm 

20 formaldehyde. Therefore it is appropriate to consider the human lower respiratory tract as 

21 potentially at risk for formaldehyde-induced cancer. Accordingly, fluid flow and formaldehyde 

22 uptake in the lower respiratory tract were also modeled for the human in the CIIT approach by 

23 using dosimetry estimates for the human lower respiratory tract.

24 The single-path idealization of the human lung anatomy captures the geometrical 

25 characteristics of the airways for a given lung depth, and of airflow through these airways, in an 

26 average, homogeneous sense. For particulates, this has provided a reasonable representation of 

27 the average deposition in a given generation of the lung airways for a normal human population. 

28 The one-dimensional model by Weibel (1963) is generally considered adequate unless the fluid 

29 dynamics at airway bifurcations need to be explicitly modeled. However, such an idealization of 

30 the lung geometry has been successfully used in various models for the dosimetry of ozone and 

31 particulate and fibrous matter. Most likely, the lung geometries of the susceptible population, 

32 such as those with chronic obstructive pulmonary disease, would depart significantly from the 

33 geometry described in Weibel (1963). Unlike the accurate representation of the nasal anatomy 

34 used in the CFD modeling, the lung geometry is idealized in the CIIT approach as a typical path 

35 Weibel geometry. This captures the lung structure in an average, homogeneous sense for a given 
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1 lung depth. The single-path model used to calculate formaldehyde uptake in the human 

2 respiratory tract (Overton et al., 2001; CIIT, 1999) applied a one-dimensional equation of mass 

3 transport to each generation of an adult human symmetric, bifurcating Weibel-type respiratory 

4 tract anatomical model, augmented by an upper respiratory tract. The detailed CFD modeling of 

5 the upper respiratory tract was made consistent with the upper respiratory tract in the single-path 

6 model by requiring that the one-dimensional version of the nasal passages have the same 

7 inspiratory air-flow rate and uptake during inspiration as the CFD simulations for four daily 

8 human activity levels. The reader is referred to Overton et al. (2001) for further details of the 

9 simulations. Results most relevant to this assessment are shown in Figure 3-10.

10 

11 Figure 3-10. Single-path model simulations of surface flux per ppm of 

12 formaldehyde exposure concentration in an adult male human.

13 

14 Source: Overton et al. (2001). 

15 

16 

17 The primary predictions of the model, as shown in Figure 3-10, were that more than 95% 

18 of the inhaled formaldehyde would be retained and formaldehyde flux in the lower respiratory 

19 tract would increase for several lung airway generations from that in the posterior-most segment 

20 of the nose and then decrease rapidly, resulting in almost zero flux to the alveolar sacs.
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1 Overton et al. (2001) modeled uptake at higher inspiratory rates, including those at 

2 50 L/minute of minute volume (well beyond levels where the oronasal switch occurs in the 

3 normal nasal breathing population). At these rates Figure 3-8 indicates that formaldehyde flux in 

4 the mouth cavity is comparable (but a bit less) to that occurring in the nasal passages. Overton et 

5 al. (2001) did not model uptake in the oral cavity at minute volumes less than 50 L/minute. This 

6 would be of interest because mouth breathers form a large segment of the population. 

7 Furthermore, at concentrations of formaldehyde where either odor or sensory irritation becomes 

8 a significant factor, humans are likely to switch to mouth breathing even at resting inspiration. 

9 At a minute volume of 50 L/minute, Overton et al. (2001) assumed, citing Niinimaa et al. (1981), 

10 that 0.55 of the inspired fraction is through the mouth. Therefore, based on the results in 

11 Figure 3-8, it is not unreasonable to assume that for mouth breathing conditions at resting or 

12 light exercise inspiratory rates, average flux across the human mouth lining would be 

13 comparable to the average flux across the nasal lining computed in Kimbell et al. (2001a, b). 

14

15 3.6.5. Uncertainties in Formaldehyde Dosimetry Modeling

16 3.6.5.1. Verification of Predicted Flow Profiles

17 The simulated streamlines of steady-state inspiration airflow predicted by the CFD model 

18 agreed reasonably well with experimentally observed patterns of water-dye streams made in 

19 casts of the nasal passages for the rat and monkey as shown in panels A and B in Figure 3-7. 

20 The airflow velocity predicted by CFD model simulations of the human also agreed well with 

21 measurements taken in hollow molds of the human nasal passages (panel C, Figure 3-8) (Kepler 

22 et al., 1998; Subramaniam et al., 1998; Kimbell et al., 1997, 1993). However, the accuracy and 

23 relevance of these comparisons are limited. The profiles were verified by video analysis of dye 

24 streak lines in the molds of rats and rhesus monkeys, although this method is reasonable for only 

25 the major airflow streams.

26 Plots of pressure drop vs. volumetric airflow rate predicted by the CFD simulations 

27 compared well with measurements made in rats in vivo (Gerde et al., 1991) and in acrylic casts 

28 of the rat nasal airways (Cheng et al., 1990) as shown in Figure 3-11. This latter comparison 

29 remains qualitative due to differences among the simulation and experiments as to where the 

30 outlet pressure was measured and because no tubing attachments or other experimental apparatus 

31 were included in the simulation geometry. The simulated pressure drop values were somewhat 

32 lower, possibly due to these differences.

33 Inspiratory airflow was assumed to be constant in time (steady state). Subramaniam et al. 

34 (1998) considered this to be a reasonable assumption during resting breathing conditions based 

35 on a value of 0.02 obtained for the Strouhal number. Unsteady effects are insignificant when 
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1 this number is much less than one. However, this assumption may not be reasonable for light 

2 and heavy exercise breathing scenarios.

3

4

5 Figure 3-11. Pressure drop vs. volumetric airflow rate predicted by the CIIT 

6 CFD model compared with pressure drop measurements made in two hollow 

7 molds (C1 and C2) of the rat nasal passage (Cheng et al., 1990) or in rats 

8 in vivo (Gerde et al., 1991).

9

10 Source: Kimbell et al. (1997).

11

12

13 3.6.5.2. Level of Confidence in Formaldehyde Uptake Simulations

14 Unlike the airflow simulations, it was not possible to evaluate the formaldehyde uptake 

15 calculations directly. Since the mass transfer boundary conditions were set by fitting overall 

16 uptake to the average experimental data for various exposure concentrations, it was not possible 

17 to independently verify even the overall uptake values with empirical data. This assessment has 

18 relied on several indirect qualitative and quantitative lines of evidence listed below to provide 

19 general confidence in the uptake profile for the F344 rat nasal passages, as modeled in CIIT 

20 (1999), when gross averages are considered over certain regions of the nasal lining.

21 In an earlier simulation, where the nasal walls were set to be infinitely absorbing of 

22 formaldehyde, uptake of inhaled formaldehyde in the upper respiratory tract was predicted to be 

23 90% in the rat for simulations corresponding to the resting minute volume in the F344 rat. This 

24 estimate compared reasonably well with the range of 91–98% observed by Morgan et al. 

25 (1986a).
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1 Morgan et al (1991) showed general qualitative correspondence between the main routes 

2 of flow and lesion distribution induced by formaldehyde in the rat nose. In their initial work 

3 with a CFD model that represented a highly reactive and soluble gas, Kimbell et al. (1993) 

4 described similarities in computed regional mass flux patterns and lesion distribution due to 

5 formaldehyde. When the results from this work in the coronal section immediately posterior to 

6 the vestibular region were considered, simulated flux levels over regions such as the medial 

7 aspect of the maxilloturbinate and the adjacent septum (where lesions were seen) were an order 

8 of magnitude higher than over other regions, such as the nasoturbinate (where lesions were not 

seen).8

10 The results of a PBPK model by Cohen-Hubal et al. (1997) provide a reasonable level of 

11 confidence in regional uptake simulations for the F344 rat when gross averages over nasal sites 

12 are carried out. Cohen-Hubal et al. (1997) linked the CFD dosimetry model for formaldehyde to 

13 a PBPK model for formaldehyde-DPX concentration in the F344 rat. This PBPK model was 

14 calibrated by optimizing the model to combined DPX data from high-tumor incidence and low15 tumor incidence regions of the rat nose that were obtained in separate experiments by Casanova 

16 et al. (1991, 1989). These data were obtained at 0.3, 0.7, 2.0, 6.0, and 10 ppm for both regions 

17 and also at 15 ppm for the high-tumor region. Model prediction of DPX concentrations for the 

18 high-tumor region only compared well with the experimental data, including 15 ppm for which 

19 the model had not been calibrated. This is shown in Figure 3-12.

9 

20 The CFD simulations do not model reflex bradypnea, a protective reflex seen in rodents 

21 but not in humans. As discussed at length in sections 3.2.3.1 and 4.2.1.1, it is reasonable to 

22 expect a range of 25% (Chang et al., 1983) to 45% (Barrow et al., 1983) decrease in minute 

23 volume in F344 rats at the exposure concentration of 15 ppm. Explicit omission of this effect in 

24 the modeling is, however, not likely to be a source of major uncertainty in the modeled results 

25 for uptake of formaldehyde in the rat nose for the following reason. The CFD model for the 

26 F344 rat was calibrated to fit the overall experimental result for formaldehyde uptake in the F344 

27 rat at 15 ppm exposure concentration. This was carried out by adjusting the mass transfer 

28 coefficient used as boundary condition on the absorbing portion of the nasal lining. Thus, the 

29 reflex bradypnea occurring in those experimental animals is phenomenologically factored into 

30 the value used for the boundary condition. Nonetheless, some error in the localized distribution 

31 of uptake patterns may be expected, even if the overall uptake is reproduced correctly. 

 8 However, this 1993 CFD model differed somewhat from the subsequent model by Kimbell et al. (2001a) used in 

this assessment. In the 1993 model, the limiting mass-transfer resistance for the gas was assumed to be in the air 

phase; that is, the concentration of formaldehyde was set to zero at the airway lining. Furthermore, this same 

boundary condition was used on the nasal vestibule as well, while, in the more recent model, the vestibule was 

considered to be non-absorbing. Unfortunately, Kimbell at al. (2001a) did not report on correspondences between 
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1 Furthermore, since the same value for the mass transfer coefficient was used in human 

2 simulations (as obtained from calibration of the rat model), there is additional uncertainty in the 

3 modeled human flux estimates. This issue was not addressed by Kimbell et al. (2001a, b), 

4 Conolly et al. (2004), or Schlosser et al. (2003), and we are unable to assess the extent of this 

5 error more accurately.

6

7 Figure 3-12. Formaldehyde-DPX dosimetry in the F344 rat.

8 Panel A: calibration of the PBPK model using data from high and low tumor 

9 incidence sites. Panel B: model prediction compared against data from high 

10 tumor incidence site. Dashed line in panel A shows the extrapolation outside the 

11 range of the calibrated data.

12 Source: Cohen-Hubal et al. (1997). 

13 

flux patterns and lesion distribution.
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1 3.6.6. PBPK Modeling of DNA Protein Cross-Links (DPXs) Formed by Formaldehyde

2 3.6.6.1. PBPK Models for DPXs

3 As can be seen from the previous sections, measuring the distribution of the absorbed 

4 formaldehyde and identifying its form have proven difficult. Because of the high reactivity of 

5 formaldehyde, rapid metabolism of formaldehyde, and complexity of formate clearance, dose 

6 surrogates (or biomarkers) of exposure have been used to characterize the extent of absorption 

7 and distribution of formaldehyde. As with other soluble and reactive gases, typical PBPK 

8 models that predict steady-state blood concentrations are not useful for predicting formaldehyde 

9 dosimetry at this time. As noted previously, inhalation exposure to formaldehyde has not been 

10 shown to increase blood formaldehyde levels. Thus, most modeling efforts for formaldehyde 

11 have focused on disposition at the site of contact. 

12 As discussed earlier, the concentration of DPXs formed by formaldehyde has been 

13 treated as a surrogate for the tissue dose of formaldehyde in earlier efforts by Casanova et al. 

14 (1991) and in EPA’s efforts to update its health assessment of formaldehyde (Hernandez et al., 

15 1994). These efforts used data from rats and rhesus monkeys (Casanova et al., 1991, 1989). 

16 Using DPXs in this manner allowed the incorporation of both clearance and metabolism of 

17 formaldehyde and the incorporation of the effect of saturation on detoxification of formaldehyde 

18 at higher doses. Calculation of the average DPX concentration from these data was seen as a 

19 surrogate for the area under the curve (AUC) of the reactive formaldehyde species in the 

20 epithelium. Based on these data, Casanova et al. (1991) developed a PBPK model for predicting 

21 DPXs in these species and for extrapolating to the human. 

22 The Casanova et al. (1991) model consists of three anatomical compartments 

23 representing different parts of the upper respiratory tract of the rhesus monkey. The results 

24 indicated a 10-fold difference in DPX formation between rats and monkeys, due primarily to 

25 species differences in minute volume and differing quantities of DNA in the nasal mucosa. 

26 Casanova et al. (1991) then developed a monkey/rat scaling factor for these parameters by taking 

27 the ratio of nasal mucosa tissue between the two species, a determinant that was proportional to 

28 the total body weight differences between the two species. Using these scaling factors in their 

29 model, the authors’ predictions in monkey (based on the rat data) were in close agreement with 

30 observed DPXs in monkey, particularly at higher formaldehyde concentrations. However, the 

31 model overpredicted DPX formation in the monkey at lower formaldehyde concentrations. 

32 Subsequent rat-human and monkey-human scaling results predicted much lower DPX formation 

33 in man. Again, the values obtained at lower concentrations may have been overpredicted, as was 

34 the case for rat-monkey extrapolation.
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1 Georgieva et al. (2003) developed a model for the uptake and disposition of 

2 formaldehyde in the rat nasal lining. This model was designed to predict the distribution of 

3 formaldehyde in the nasal mucosa. The model indicated that, at 6 ppm exposure, a steady-state 

4 elevation of 15–20 μM formaldehyde would be achieved within 30 seconds. Furthermore, this 

5 same elevation was predicted when the exposure was 6 ppm formaldehyde for 60 minutes. 

6 Given that human blood formaldehyde levels are predicted to be about 100 ± 15 μM (Heck et al., 

7 1985) and assuming that blood formaldehyde concentration is roughly equivalent to the 

8 concentration predicted at the basement membrane of the epithelium, this model predicts roughly 

9 a 15–20% increase in blood formaldehyde. However, it should be noted that a 40-minute 

10 inhalation exposure of humans to 1.99 ppm formaldehyde did not lead to a measurable increase 

11 in blood formaldehyde (Heck et al., 1985).

12 Franks (2005) published a mathematical model for predicting the disposition of 

13 formaldehyde in the human nasal mucosa and blood. The calculated concentrations of 

14 formaldehyde in the mucus, the epithelium, and the blood attained steady-state profiles within a 

15 few seconds of exposure. The increase of the formaldehyde concentration in the blood was 

16 predicted to be insignificant compared with the existing pre-exposure levels in the body: an 

17 increase of 0.00044 mg/L in blood formaldehyde following exposure to 1.9 ppm formaldehyde 

18 for up to 8 hours. The model described formaldehyde concentration gradients across the mucus, 

19 epithelial, and submucosal compartments in the human nose. Transport of formaldehyde was 

20 governed by the following processes: diffusional (in the mucus); a combination of diffusional, 

21 two first order terms representing intrinsic reactivity of formaldehyde and binding to DNA, and 

22 Michaelis-Menten kinetics representing enzymatic metabolism (in the epithelial layer); a first23 order term representing non-enzymatic removal governed by the blood perfusion rate (in the 

24 submucosal compartment). The model used the values for the first order reaction rate constants 

25 and the Michaelis-Menten parameters (Vmax and Km) estimated by Conolly et al. (2000) in their 

26 model for extrapolating the rat and rhesus monkey data to the human. The modeling in Franks 

27 (2005) was not calibrated or validated against experimental data, but the predictions of 

28 negligible penetration of free formaldehyde to the blood are qualitatively in agreement with the 

29 conclusions in Heck et al. (1985).

30 Following the efforts by Casanova and co-workers, Cohen-Hubal et al. (1997), Conolly et

31 al. (2000), and Georgieva et al. (2003) developed models that linked local formaldehyde flux 

32 from CFD models to DPX predictions. The focus here will be on the Conolly et al. (2000) effort 

33 for the following two reasons: it explicitly incorporates regional formaldehyde dosimetry in the 

34 nasal lining by using results from CFD modeling of airflow and gas uptake and it brings data 
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1 across species (rat and rhesus monkey) to bear on model calibration, such a situation being 

2 relatively rare in chemical health risk assessments.

3

4 3.6.6.2. A PBPK Model for DPXs in the F344 Rat and Rhesus Monkey that Uses Local Tissue 

5 Dose of Formaldehyde

6 In earlier risk assessment efforts (Hernandez et al., 1994; Casanova et al., 1991; U.S. 

7 EPA, 1991b), the average DPX concentration was considered a surrogate tissue dose metric for 

8 the AUC of the reactive formaldehyde species. Conolly et al. (2003) assigned a more specific 

9 role for DPXs, treating local DPX concentration as a dose surrogate indicative of the 

10 intercellular concentration of formaldehyde, leading to formaldehyde-induced mutations. These 

11 authors indicated that it was not known whether DPXs directly induced mutations (Conolly et 

12 al., 2003; Merk and Speit, 1998). This is discussed in detail in the mode-of-action sections in 

13 this document. The Conolly et al. (2000) model for the disposition of inhaled formaldehyde gas 

14 and DPX in the rat and rhesus nasal lining is relatively simple in terms of model structure 

15 because it consists of a single well-mixed compartment for the nasal lining as follows: 

16 1. Formaldehyde flux to a given region of the nasal lining is provided as input to the 

17 modeling and is obtained in turn as the result of a CFD model. This flux is defined as the 

18 amount of formaldehyde delivered to the nasal lining per unit time per unit area per ppm 

19 of concentration in the air in a direction transverse to the airflow. It is locally defined as 

20 a function of location in the nose and the inspiratory flow rate and is linear with exposure 

21 concentration.

22 2. The clearance of formaldehyde from the tissue is modeled as follows:

23 a. a saturable pathway representing enzymatic metabolism of formaldehyde, which 

24 is primarily by formaldehyde dehydrogenase (involving Michaelis-Menten 

25 parameters Vmax and Km)

26 b. a separate first-order pathway, which is assumed to represent the intrinsic 

27 reactivity of formaldehyde with tissue constituents (rate constant kf)

28 c. first-order binding to DNA that leads to DPX formation (rate constant kb) 

29 3. The clearance or repair of this DPX is modeled as a first order process (rate constant 

30 kloss).

31

32 DPX data

33 DPX concentrations were estimated from a study by Casanova et al. (1994) in which rats 

34 were exposed 6 hours/day, 5 days/week, plus 4 days for 11 weeks to filtered air (naive) or to 0.7, 

2, 6, or 15 ppm (0.9, 2.5, 7.4, or 18 mg/m3 35 ) formaldehyde (pre-exposed). On the 5th day of the 
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12th week, the rats were then exposed for 3 hours to 0, 0.7, 2, 6, or 15 ppm 14 1 C-labeled 

2 formaldehyde (with pre-exposed animals exposed to the same concentration as during the 

3 preceding 12 weeks and 4 days). The animals were sacrificed and DPX concentrations 

4 determined at two sites in the nasal mucosa. Conolly et al. (2000) used these naive rat data to 

5 develop a PBPK model that predicted the time-course of DPX concentrations as a function of 

formaldehyde flux at these sites.9

7

6 

8 3.6.6.3. Uncertainties in Modeling the Rat and Rhesus DPX Data

9 3.6.6.3.1. Half-life of DPX repair. In the development of the PBPK model for DPXs, Conolly 

et al. (2000) assumed a value of 6.5 × 10–3 minute–1 10 for kloss, the first-order rate constant for the 

11 clearance (repair) of DPXs, such that the DPXs predicted at the end of a 6-hour exposure to 

12 15 ppm were reduced to exactly the detection limit for DPXs in 18 hours (the period between the 

13 end of 1 day’s 6-hour exposure and the beginning of the next). This determination of rapid 

14 clearance was based on an observation by Casanova et al. (1994) that the DPX concentrations 

15 observed in the pre-exposed animals were not significantly higher than those in naïve animals (in 

16 which there was no significant DPX accumulation). However, in vitro data (Quievryn and 

17 Zhitkovich, 2000) indicate a much slower clearance, with an average kloss of 

9.24 × 10–4 minute–1 18 .

19 Subramaniam et al. (2007) examined the Casanova et al. (1994) data and argued that 

20 there was a significantly decreased (~ 40%) level of DPXs in high tumor regions of pre-exposed 

21 animals vs. naive animals at 6 and 15 ppm and that the weight of the tissues dissected from those 

22 regions increased substantially, indicating a thickening of the tissues. After testing the outcome 

23 of changing the tissue thickness in the PBPK model for DPXs, it was apparent to these authors 

24 that such a change alone could not account for the dramatic reduction in DPX levels after pre25 exposure, even with the higher value of kloss used by Conolly et al. (2000). Therefore, in 

26 addition to the gross increase in tissue weight, these data indicated either an induction in the 

27 activity of enzymes that remove formaldehyde (aldehyde and formaldehyde dehydrogenase) or 

28 other changes in the biochemical properties of the highly exposed tissue that must have occurred. 

29 Given such a change, Subramaniam et al. (2007) concluded that the experimental results in 

30 Casanova et al. (1994) were consistent with the smaller experimental value of kloss indicated by 

31 the Quievryn and Zhitkovich (2000) data. In particular, they argued that if Vmax increased with 

32 exposure (in a tissue region- and dose-specific manner), then it was possible to explain the naïve 

 9 Note that Conolly et al. (2000) stated that they used the pre-exposed data, but this statement appears to be in error 

(see Subramaniam et al. [2007]).
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1 vs. pre-exposed data of Casanova et al. (1994), with the value of kloss effectively measured in 

2 vitro by Quievryn and Zhitkovich (2000). Furthermore, this value was measured directly, rather 

3 than obtained by indirect interpretation of measurements made at only two time points where 

4 significant changes in the tissue had occurred. Therefore, Subramaniam et al. (2007) considered 

5 the use of this lower value for kloss to be more appropriate. Consequently, they re-implemented 

6 and re-optimized the Conolly et al. (2000) model with this modification and found that the fit so 

7 obtained to the acute DPX data was excellent. The re-implemented model will be used in this 

8 assessment, and more details can be found in Subramaniam et al. (2007).

9 It should be noted that this slower DPX repair rate was obtained in an in vitro study by 

10 using human cell lines that were transformed and immortalized. However, it appears that DPX 

11 repair in normal cells would be even slower. When non-transformed freshly purified human 

12 peripheral lymphocytes were used instead, the half-life for DPX repair was about 50% longer 

13 than in the cultured cells (Quievryn and Zhitkovich, 2000).

14

15 3.6.6.3.2. Statistical uncertainty in parameter estimates and extrapolation. Klein et al. (2009) 

16 developed methods for deriving statistical inferences of results from PBPK models. They used 

17 the Conolly et al. (2000) model for this purpose, specifically because of the sparse time-course 

18 information in the above DPX data. However, they used the value of kloss deduced from 

19 Quievryn and Zhitkovich (2000) and fitted the model simultaneously to both the rat and rhesus 

20 monkey data, as opposed to the sequential fitting in Conolly et al. (2000). They found that the 

21 predicted DPX concentrations were extremely sensitive to Vmax and tissue thickness as was also 

22 concluded by Georgieva et al. (2003) and Cohen-Hubal et al. (1997). Km was seen to be 

23 substantially different across species, a finding that was attributed plausibly to the involvement 

24 of more than one enzyme (Klein et al., 2009; Georgieva et al., 2003). Klein et al. (2009) 

25 concluded that the two efforts (Conolly et al. [2000] vs. Klein et al. [2009]) resulted in 

26 substantially different predictions outside the range of the observed data over which the models 

27 were calibrated. 

28 The differences between these models occur in spite of the fact that both methods use all the 

29 available DPX data in both species and the same model structures. At the 0.1 ppm exposure 

30 concentration, in general these authors obtained three- to fourfold higher DPX concentrations 

31 averaged over a 24-hour period after exposure. Furthermore, the standard deviations in Klein et 

32 al. (2009) for Vmax and Km were an order of magnitude higher and that for kf was 35-fold lower 

33 than the corresponding standard deviations reported in Conolly et al. (2000). The relatively 

34 larger standard deviation for kf resulted in this parameter becoming negative in Conolly et al. 

35 (2000) at even half the standard deviation below the maximum likelihood estimate (MLE) value. 
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1 Note that, at a negative value of kf, formaldehyde would be produced as opposed to being 

2 cleared through its intrinsic reactivity. 

3 Klein et al. (2009) concluded that these “remarkable differences outside the range of the 

4 observed data suggest caution in the use of these models in a predictive sense for extrapolating to 

5 human exposures.”

6

7 3.6.7. Uncertainty in Prediction of Human DPX Concentrations

8 Conolly et al. (2000) used both the rat and rhesus monkey data to predict human DPX 

9 concentrations and constructed a PBPK model for the rhesus monkey along similar lines as for 

10 the F344 rat. In the rhesus monkey model, they maintained the same values of kb, kloss, and kf as 

11 in the rat model but optimized the values of Vmax and Km against the rhesus monkey data from 

12 Casanova et al. (1994). The rat and rhesus monkey parameters were then used to construct a 

13 human model (see Conolly et al. [2000] for a more detailed report of implementing the rhesus 

14 monkey model and the extrapolations to the situation in humans).

15 For the human, the model used the value of Km obtained in the rhesus monkey model and 

16 the epithelial thickness averaged over three regions of the rhesus monkey nose. The maximum 

17 rate of metabolism, Vmax, which was estimated independently for the rat and rhesus monkey by 

18 fitting to the DPX data available for these species, was then extrapolated to the human by 

assuming a power law scaling with body weight (BW) (i.e., Vmax = a × BWb 19 ), and the coefficient 

20 “a” and exponent “b” were derived from the independently estimated values of (Vmax)RAT and 

21 (Vmax)MONKEY. Table 3-7 gives the values of Vmax and Km in the Conolly et al. (2000) 

22 extrapolation.

23

24 Table 3-7. Extrapolation of parameters for enzymatic metabolism to the 

25 human

26

Parameter F344 rat Rhesus monkey Human

Vmax (pmol/min-mm3) 1,008.0 91.0 15.7

Km (pmol/mm3) 70.8 6.69 6.69

27

28 Source: Conolly et al. (2000).

29

30

31 The extent of mechanistic data across species, as available in this case, is rarely seen with 

32 other chemicals, and the above scale-up procedure was an attempt to use both the rodent and 

33 primate DPX data. However, allometric relationships across species are generally based on 

34 regressing data from multiple species and usually multiple sources of data points. Thus, the 

35 empirical strength of a power law derived by using two data points (F344 rat and rhesus 
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1 monkey) is extremely weak for use as an allometric relationship that can then be used to 

2 extrapolate to the human.

3 The following observations indicate the high level of uncertainty in the values of the 

4 parameters Vmax and Km in the Conolly et al. (2000) models for predicting DPXs. First, Km

5 varies by an order of magnitude across the rat and monkey models but is then considered 

6 invariant between the monkey and human models (Conolly et al., 2000). Second, the values in 

7 Conolly et al. (2000) for Vmax/Km, the low-dose limit of the rate of enzymatic metabolism, is 

8 roughly similar between the rat and monkey but lower by a factor of six in the human.

9 Another factor that can substantially influence the above extrapolation of DPXs in the 

10 human is that Conolly et al. (2000) assumed the tissue to be a well-mixed compartment with 

11 regard to formaldehyde interaction with DNA and used the amount of formaldehyde bound to 

12 DNA per unit volume of tissue as the DPX dose metric. Considering formaldehyde’s highly 

13 reactive nature, the concentrations of formaldehyde and DPX are likely to have a sharp gradient 

14 with distance into the nasal mucosa (Georgieva et al., 2003). Given the interspecies differences 

15 in tissue thickness, there is consequent uncertainty as to whether DPX per unit volume or DPX 

16 per unit area of nasal lining is the more appropriate dose metric to be used in the extrapolation. 

17 In particular, it may be assumed that the cells at risk for tumor formation are only those in the 

18 epithelium and that measured DPX data (in monkeys and rats) are an average over the entire 

19 tissue thickness. Since the epithelial DPXs in monkeys (and presumably humans) would then be 

20 more greatly “diluted” by lower levels of DPX formation that occur deeper into the tissue than in 

21 rats, it could be predicted that the ratio of epithelial to measured DPXs in monkeys and humans 

22 would be much higher than the ratio in rats.

23

24 End of Volume I