DRAFT-DO NOT CITE OR QUOTE EPA/635/R-10/002 www.epa.gov/iris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-dissemina tion peer review under applicable information quality guidelines. It has not been formally disse minated by EPA. It does not represent and should not be construed to represent any Agency det ermination or policy. It is being circulated for review of its technical accuracy and science po licy implications.

U.S. Environmental Protection Agency Washington, DCThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-ii DISCLAIMER

This document is a preliminary draft for review pu rposes only. This information is distributed solely for the purpose of pre-dissemina tion peer review under applicable information quality guidelines. It has not been formally disse minated by EPA. It does not represent and should not be construed to represent any Agency det ermination or policy. Mention of trade names or commercial products does not constitute en dorsement or recommendation for use.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-iii CONTENTS—TOXICOLOGICAL REVIEW OF FORMALDEHYDE (CAS No. 50-00-0)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 Product ion............................. 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 Anim al 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 Ef fects at the Portal of Entry .................................... ............................................ 3-10 3.2.3.2. Variability in the Nasal Dosimetry of For maldehyde 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: Differentiat ing covalent Binding and Metabolic Incorporation................ ............................. 3-16This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-iv CONTENTS (continued)

3.4. METABOLISM ...................................

................................................... .................... 3-20 3.4.1. In Vitro and In Vivo Characterization of Fo rmaldehyde Metabolism............ 3-20 3.4.2. Formaldehyde Exposure and Perturbation of M etabolic 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 Bre ath...................................... 3-27 3.5.3. Formaldehyde Excretion in Human Urine...... ................................................ 3-3 1 3.6. MODELING THE TOXICOKINETICS OF FORMALDEHYDE A ND DPX.......... 3-32 3.6.1. Motivation .................................

................................................... .................. 3-32 3.6.2. Species Differences in Anatomy: Consequence s for Gas Transport and Risk...............................................

................................................... .............. 3-34 3.6.3. Modeling Formaldehyde Uptake in Nasal Passa ges....................................... 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 R espiratory Tract.................. 3-42 3.6.5. Uncertainties in Formaldehyde Dosimetry Mod eling .................................... 3-44 3.6.5.1. Verification of Predicted Flow Profiles.. ......................................... 3-44 3.6.5.2. Level of Confidence in Formaldehyde Uptak e Simulations............ 3-45 3.6.6. PBPK Modeling of DNA Protein Cross-Links (D PXs) 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 Rhe sus DPX Data.............. 3-51 3.6.7. Uncertainty in Prediction of Human DPX Conc entrations ............................ 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 Irr itation)................................ 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.................. ................................................ 4-4 2 4.1.1.7. Developmental and Reproductive Toxicity.. ................................... 4-45 4.1.1.8. Oral Exposure Effects on the Gastrointest inal Tract ....................... 4-56 4.1.1.9. Summary: Noncarcinogenic Hazard in Humans ............................. 4-56This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-v CONTENTS (continued)

4.1.2. Cancer Health Effects......................

................................................... ............ 4-57 4.1.2.1. Respiratory Tract Cancer................. ................................................ 4-5 7 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 Toxic ity................................ 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... ................................................ 4-3 24 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 ..................... ................................................ 4-3 44 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 System s............................ 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 NONCARCINOG ENIC 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 I nform MOAs ..................... 4-385 4.4.6. Noncancer Modes of Actions .................

................................................... ... 4-387 4.4.7. Immunotoxicity .............................

................................................... ............ 4-389This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-vi 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 Mecha nistic 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 (Developm ental 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......................... ................................................ 4-4 19 4.5.2.6. Solid Tumors of Lymphoid Origin.......... ...................................... 4-421 4.5.2.7. Supporting Evidence from Animal Bio-Assay s for Formaldehyde-Induced Lymphohematopoietic Malignanci es...... 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 Respi ratory 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 C arcinogenicity...................... 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 ........................

................................................... ........... 4-462This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-vii CONTENTS (continued)

4.6.6. Co-Exposures ...............................

................................................... ............. 4-464 4.6.6.1. Cumulative Risk .......................... .................................................. 4 -464 4.6.6.2. Aggregate Exposure....................... ................................................ 4-4 65 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 R fCs.......................................... 5-33 5.1.2.1. Selection of Studies for Candidate RfC De rivation......................... 5-33 5.1.2.2. Derivation of Candidate RfCs from Key Stu dies ............................ 5-40 5.1.2.3. Evaluation of the Study-Specific Candidat e RfC ............................ 5-66 5.1.3. Database Uncertainties in the RfC Derivatio n ............................................... 5-6 9 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 N ATIONAL 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 Nationa l Cancer Institute Cohort................................... ............................................ 5-76 5.2.2.2. Prediction of Lifetime Extra Risk of Naso pharyngeal Cancer Mortality.......................................... ............................................... 5-79 5.2.2.3. Prediction of Lifetime Extra Risk of Naso pharyngeal 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 Nationa l Cancer Institute Cohort................................... ............................................ 5-88This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-viii CONTENTS (continued)

5.2.3.2. Prediction of Lifetime Extra Risks for Ho dgkin Lymphoma and Leukemia Mortality ............................. .................................... 5-91 5.2.3.3. Prediction of Lifetime Extra Risks for Ho dgkin Lymphoma and Leukemia Incidence............................. .................................... 5-93 5.2.3.4. Sources of Uncertainty ................... ................................................. 5- 95 5.2.4. Conclusions on Cancer Unit Risk Estimates B ased on Human Data............. 5-99 5.3. DOSE-RESPONSE MODELING OF RISK OF SQUAMOUS CE LL CARCINOMA IN THE RESPIRATORY TRACT USING ANIMAL DAT A ...... 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 M odeling.............................. 5-106 5.3.2.1. Major Results of the CIIT Modeling Effort ................................... 5-111 5.3.3. This Assessment’s Conclusions from Evaluati on of Dose-Response Models of DPX Cell-Replication and Genomics Data, a nd of BBDR Models for Risk Estimation......................... ................................................ 5-1 11 5.3.4. Benchmark Dose Approaches to Rat Nasal Tumo r Data ............................. 5-118 5.3.4.1. Benchmark Dose Derived from BBDR Rat Mode l and Flux as Dosimeter ....................................... .......................................... 5-118 5.3.4.2. Comparison with Other Benchmark Dose Mode ling 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 INHALATIO N... 5-133 5.4.1. Inhalation Unit Risk Estimates Based on Hum an Data................................ 5-133 5.4.2. Inhalation Unit Risk Estimates Based on Rod ent Data ................................ 5-133 5.4.3. Summary of Inhalation Unit Risk Estimates .. .............................................. 5-135 5.4.4. Application of Age-Dependent Adjustment Fac tors (ADAFs) .................... 5-136 5.4.5. Conclusions: Cancer Inhalation Unit Risk Es timates................................... 5-137

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HA ZARD 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 E xcretion ..................................... 6-1 6.1.3. Noncancer Health Effects in Humans and Labo ratory 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 ... .................................... 6-12This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-ix 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 Ani mals...................................... 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 Concent rations ........................ 6-25 6.2.1.3. Adequacy of Overall Data Base for RfC Der ivation ....................... 6-26 6.2.1.4. Uncertainties in the Reference Concentrat ion (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 Es timates ............................ 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 PU BLIC

COMMENTS AND DISPOSITIONS.......................... .................................... A-1 APPENDIX B: SIMULATIONS OF INTERINDIVIDUAL AND ADU LT-TO-CHILD

VARIABILITY IN REACTIVE GAS UPTAKE IN A SMALL SAMPL E OF PEOPLE (Garcia et al., 2009).................... .................................................B- 1 APPENDIX C: LIFETABLE ANALYSIS ....................

................................................... ..........C-1 APPENDIX D: MODEL STRUCTURE & CALIBRATION IN CONOL LY ET AL.

(2003, 2004).......................................

................................................... ........... D-1 APPENDIX E: EVALUATION OF BBDR MODELING OF NASAL C ANCER 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-1This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-x 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 QUANTITAT IVE

EVALUATION OF ANIMAL TOXICOLOGY DATA FOR ANALYZING CANCER RISK DUE TO INHALED FORMALDEHYDE.. . H-1This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xi LIST OF TABLES

Table 2-1. Physicochemical properties of formaldeh yde................................................ ............ 2-2

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

................................................... ................. 2-6

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

Table 3-1. Endogenous formaldehyde levels in anima l 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 po pulations .......................................... ..... 3-25

Table 3-4. Percent distribution of airborne [ 14 C]-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 leve ls 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 co ncentrations in the mouth and nose,

and in the oral cavity after breath holding in thre e healthy male laboratory workers ............................................

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

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

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

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

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

nasopharyngeal cancer) .............................

................................................... ..........4-78

Table 4-4. Epidemiologic studies of formaldehyde a nd lymphohematopoietic cancers...........4-98

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

strains of mice ....................................

................................................... ...............4-112

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

strains of rats ....................................

................................................... .................4-113

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

mice exposed to 15 ppm.............................

................................................... .......4-116

Table 4-8. Exposure regimen for cross-tolerance st udy................................................ ..........4-117This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xii LIST OF TABLES (continued)

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

respiratory tract..................................

................................................... ................4-127

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

nasoturbinates.....................................

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

Table 4-11. Enzymatic activities in nasal respirat ory 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 pa rameters 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 se verity) on histopathologic changes in

the noses and larynxes of male and female albino SP F Wistar rats exposed to formaldehyde 6 hours/day for 13 weeks.............. .................................................4- 148

Table 4-18. Formaldehyde-induced nonneoplastic his topathologic 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 e xposure..............................4-149

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

exposed to formaldehyde (6 hours/day, 5 days/week f or 13 weeks) and examined at the end of 130 weeks inclusive of expos ure ....................................4-150

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

................................................... 4-153

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

and female animals.................................

................................................... ..........4-154This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xiii LIST OF TABLES (continued)

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

formaldehyde-treated and control animals; data pool ed for males and females............................................

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

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

formaldehyde.......................................

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

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

cerebral cortex of male Wistar rats exposed to form aldehyde 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 measurement s 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 exp osed to inhaled formaldehyde and wood dust for 104 weeks ........... ............................................4-166

Table 4-30. Histopathologic changes (including tum ors) 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 n asal 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 Kern s et al. (1983) bioassay ...........................................

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

Table 4-33. Incidence and location of nasal squamo us 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, tr achea, and free lung cells isolated

from male Wistar rats after inhalation exposures to formaldehyde.....................4-194This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xiv LIST OF TABLES (continued)

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

cell count, basal membrane length, proliferation ce lls, and two measures of cell proliferation, LI and ULLI, in male F344 rats . .............................................4-196

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

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

Table 4-38. Cell population and surface area estim ates 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 cel l proliferation in the upper

respiratory tract..................................

................................................... ...............4-202

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

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

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

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

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

MNNG and NaCl in male Wistar rats exposed to formal dehyde (0.5% formalin) in drinking water for 8 weeks............ ..................................................4 -212

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

reported in male and female Sprague-Dawley rats exp osed to formaldehyde in drinking water at different ages... .............................................4-214

Table 4-44. Incidence of hemolymphoreticular neopl asia 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 7 001) .................................4-215

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

B6C3F1 mice after a 3 week, 15-ppm formaldehyde exp osure ..........................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 det ermining 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 ...................

................................................... .4-226This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xv LIST OF TABLES (continued)

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

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

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

inhalation, topical application, or footpad injecti on............................................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 swe lling responses and skin mRNA

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

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

sensitization due to formaldehyde in experimental a nimals................................4-251

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

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

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

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

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

male and female LEW.1K rats ........................

................................................... .4-263

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

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

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

dams exposed via inhalation from mating through ges tation..............................4-289

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

male Wistar rats...................................

................................................... .............4-298

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

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

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

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

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

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

formaldehyde-treated mice ..........................

................................................... .....4-302This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xvi LIST OF TABLES (continued)

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

gestation..........................................

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

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

abnormal sperm after oral administration of formald ehyde to male Wistar rats........................................

................................................... .................4-305

Table 4-66. Effect of formaldehyde on spermatogeni c 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 exposu re of male rats to formaldehyde...........4-308

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

exposure studies ...................................

................................................... ............4-311

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

studies............................................

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

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

exposure studies ...................................

................................................... ............4-318

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

studies............................................

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

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

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

studies............................................

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

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

lymphoma...........................................

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

Table 4-76. Formaldehyde-DNA reactions (DPX format ion) ............................................... 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 formaldehyd e.................................................. ............4-348

Table 4-81. Summary of mutagenicity of formaldehyd e in bacterial systems.......................4-350This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xvii 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 scien ce 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 stu dies which evaluated the

incidence of all LHP cancers in formaldehyde-expose d populations (ICD-8 Codes: 200-209) and all leukemias (ICD-8 Cod es: 204-207).................4-412

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

disease groupings within the broad category of all lymphohematopoetic malignancies.......................................

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

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

leukemia sub-types .................................

................................................... ..........4-420

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

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

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

lymphoma...........................................

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

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

Sprague-Dawley rats................................

................................................... .........4-425

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

exposure...........................................

................................................... .................4-469This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xviii LIST OF TABLES (continued)

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

animal studies ....................................

................................................... ................5-19

Table 5-2. Effects of formaldehyde exposure on com pletion 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 formal dehyde RfC development by

health endpoint category...........................

................................................... ..........5-36

Table 5-5. Adjustment for nonoccupational exposure s 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 f rom 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-li near 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. EC 0005 , LEC 0005 , and inhalation unit risk estimates for NPC mortal ity from

formaldehyde exposure based on the Hauptmann et al. (2004) log-linear trend analyses for cumulative exposure.............

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

Table 5-11. EC 0005 , LEC 0005 , and inhalation unit risk estimates for NPC incide nce 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 metric s.........................................5-90

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

from NCI trend test models .........................

................................................... .......5-90

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

of continuous exposure to formaldehyde............. ..................................................5 -91This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xix LIST OF TABLES (continued)

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

continuous exposure to formaldehyde................

................................................... 5-91

Table 5-16. EC 0005 , LEC 0005 , and inhalation unit risk estimates for Hodgkin ly mphoma

mortality from formaldehyde exposure based on Beane Freeman et al. (2009) log-linear trend analyses for cumulative exp osure.....................................5-93

Table 5-17. EC 005 , LEC 005 , and inhalation unit risk estimates for leukemia m ortality

from formaldehyde exposure based on Beane Freeman e t al. (2009) log-linear trend analyses for cumulative exposure .. ..............................................5-93

Table 5-18. EC 0005 , LEC 0005 , and inhalation unit risk estimates for Hodgkin

lymphoma incidence from formaldehyde exposure, base d on Beane Freeman et al. (2009) log-linear trend analyses for cumulative exposure.............5-94

Table 5-19. EC 005 , LEC 005 , and inhalation unit risk estimates for leukemia i ncidence

from formaldehyde exposure based on Beane Freeman e t al. (2009) log-linear trend analyses for cumulative exposure .. ..............................................5-94

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

Table 5-21. Calculation of combined cancer inciden ce unit risk estimate at 0.1 ppm ..........5-100
Table 5-22. Summary of tumor incidence in long-ter m bioassays on F344 rats....................5-105
Table 5-23. BMD modeling of unit risk of SCC in th e human respiratory tract ...................5-125
Table 5-24. Formaldehyde-induced rat tumor inciden ces................................................ ......5-128
Table 5-25. Human benchmark extrapolations of nasa l tumors in rats using

formaldehyde flux and DPX..........................

................................................... ...5-134
Table 5-26. Summary of inhalation unit risk estima tes ................................................ .........5-135
Table 5-27. Total cancer risk from exposure to a c onstant formaldehyde exposure

level of 1 μg/m 3 from ages 0–70 years .............................. ..................................5-137
Table 6-1. Summary of candidate Reference Concentr ations (RfC) for co-critical studies.....6-27
Table 6-2. Effective concentrations (lifetime cont inuous 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........................................

................................................... ..................6-42This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xx LIST OF FIGURES

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

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

Figure 2-2. Locations of hazardous air pollutant m onitors............................................ ............2-5

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

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

Figure 3-1. Formaldehyde-mediated protein modifica tions.............................................. .........3-2

Figure 3-2. 3 H/ 14 C ratios in macromolecular extracts from rat tissue s following exposure

to 14 C and 3 H-labeled formaldehyde (0.3, 2, 6, 10, 15 ppm)..... .........................3-18

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

(GSH-dependent)....................................

................................................... .........3-20

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

................................................... ......................3-22

Figure 3-5. Scatter plot of formaldehyde concentra tions 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 r at, rhesus monkey, and human................3-36

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

simulations with water-dye studies .................

................................................... 3-37

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

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

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

inspiratory flow rates.............................

................................................... ..........3-39

Figure 3-10. Single-path model simulations of surf ace 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 e t al., 1990) or in rats in vivo............................................

................................................... ..................3-45

Figure 3-12. Formaldehyde-DPX dosimetry in the F34 4 rat .............................................. .....3-47

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

“painting” 100% formalin for 20 minutes............ ..............................................4-20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxi 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 o riented to the left)............................... .......4-121

Figure 4-4. Main components of the nasal respirato ry 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 sect ion levels chosen for morphometry

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

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

formaldehyde.......................................

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

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

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

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

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

Figure 4-10. Effect of formaldehyde exposure on ce ll 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/m 3 carbon black, or both ............................. ..................4-223

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

following formaldehyde exposure in guinea pigs afte r OVA sensitization and OVA challenge ......................................

................................................... ........4-235

Figure 4-13. OVA-specific IgG1 (1B) in formaldehyd e-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 wee k 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 Wis tar rats............................4-239This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxii LIST OF FIGURES (continued)

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

infiltration (Panel A) and goblet cell proliferatio n (Panel B) after Der f challenge in the nasal mucosa of male ICR mice afte r sensitization and challenge..........................................

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

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

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

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

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

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

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

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

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

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

after termination of 10-minute formaldehyde exposur e ...................................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 t he 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 forma ldehyde-exposed mice .........................4-274

Figure 4-27. Mortality corrected cumulative incide nces 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 rat s exposed to formaldehyde for

24 months ..........................................

................................................... ............4-332

Figure 4-30. Cumulative leukemia incidence in fema le 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.. ....................................4-334This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxiii LIST OF FIGURES (continued)

Figure 4-32. DNA-protein cross-links (DPX) and thy midine 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 formaldehyd e exposure and the risk of

lymphohematopoietic malignancy..................... ...............................................4-41 5

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

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

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

human colon carcinoma cells and in (B) human umbili cal 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 lymphocy tes 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 ex posed to formaldehyde in the high

exposure index category with 8-hour time weighted a verage 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 concent rations. ..............................5-41

Figure 5.5. Odds ratios for physician-diagnosed as thma in children associated with inhome formaldehyde levels in air ...................

................................................... .5-45

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

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

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

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

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

formaldehyde exposures and sensory irritation (eye irritation) ..........................5-53This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxiv LIST OF FIGURES (continued)

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

formaldehyde exposures and sensory irritation (burn ing 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 pharmacok inetic and pharmacodynamic

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

Figure 5-12. Fit to the rat tumor incidence data u sing 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, resul ting in 20 flux bins ..........5-120

Figure 5-14. Distribution of cells at risk across flux bins in the F344 rat nasal lining..........5-1 20

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

two BBDR models....................................

................................................... .....5-124

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

Monticello et al. (1996) data on tumor incidence sh owing BMC 10 and BMCL 10 ................................................... ................................................... ......5-127

Figure 5-17. EPA multistate Weibull modeling: nas al 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........ ........................................5-132This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxv LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industria l 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 Regis try 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 concentratio n BMCR binuclated micronucleated cell ratefluoresce BMD benchmark dose BMDL 95% lower bound on the benchmark doseThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxvi 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 Preventio n 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 CO 2 carbon dioxide COEHHA California Office of Environmental Health Ha zard Assessment CREB cyclic AMP responsive element binding protein s 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 assayThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxvii 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 fa ctor 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 transfera se 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 CancerThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxviii 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 K m Michaels-Menton constant KM Kaplan-Meier LD 50 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 levelThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxix 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 Researc h Council/Agriculture and Resource

Management Council of Australia and New Zealand NNK nitrosamine nitrosamine 4-(methylnitrosamino)- 1-(3-pyridyl)-butanone N 6 -hmdA N 6 -hydroxymethyldeoxyadenosine N 4 -hmdC N 4 -hydroxymethylcytidine N 2 -hmdG N 2 -hydroxymethyldeoxyguanosine NICNAS National Industrial Chemicals Notification a nd Assessment Scheme NIOSH National Institute for Occupational Safety an d 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 volumeThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxx 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 ind ex RA reflex apnea RANTES regulated upon activation, normal T–cell exp ressed and secreted RB reflex bradypnea RBC red blood cells RD 50 exposure concentration that results in a 50% redu ction 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 dehydrogena se SEER Surveillance, Epidemiology, and End Results SEM standard error of the mean SEN sensitizer SH sulfhydryl SHE Syrian hamster embryo SLMA spontaneous locomotor activityThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxxi LIST OF ABBREVIATIONS AND ACRONYMS (continued)

SMR standardized mortality ratio SNP single nucleotide polymorphism SOD superoxide dismutase SOMedA N 6 -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 fo r EuropeThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxxii FOREWORD

The purpose of this Toxicological Review is to prov ide 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 comprehen sive 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, unc ertainties, 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 (20 2) 566-1676 (phone), (202) 566-1749 (fax), or hotline.iris@epa.gov (email address).This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxxiii AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGERS

John E. Whalan, D.A.B.T. 1
EPA-ORD-NCEA

Danielle DeVoney, PhD, D.A.B.T., PE 2
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 a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxxiv 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-NCEAThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxxv AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)

CONTRACTOR SUPPORT

The literature search and preliminary drafts of th is document as well as support for editing and formatting were provided by Oak Ridge I nstitute for Science and Education (ORISE), Oak Ridge Associated Universities (ORAU), Departmen t of Energy, under Interagency Agreement (IAG) Project No. 03-18. The ORISE indiv iduals 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 respon se model evaluation was provided by ENVIRON International Corporation of Monroe, Louisi ana (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 8This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxxvi 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 Environm ental 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 ToxicsThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE I-xxxvii 

### This page intentionally left blank.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 1-1 1. INTRODUCTION 1 2 3 This document presents background information and j ustification for the Integrated Risk 4 Information System (IRIS) Summary of the hazard and dose-response assessment of 5 formaldehyde. IRIS Summaries may include oral refe rence dose (RfD) and inhalation reference 6 concentration (RfC) values for chronic and other ex posure durations, and a carcinogenicity 7

assessment. 8 The RfD and RfC, if derived, provide quantitative i nformation for use in risk assessments 9 for health effects known or assumed to be produced through a nonlinear (presumed threshold) 10 mode of action. The RfD (expressed in units of mg/ kg-day) is defined as an estimate (with 11 uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human population 12 (including sensitive subgroups) that is likely to b e without an appreciable risk of deleterious 13 effects during a lifetime. The inhalation RfC (exp ressed in units of mg/m 3 ) is analogous to the 14 oral RfD, but provides a continuous inhalation expo sure estimate. The inhalation RfC considers 15 toxic effects for both the respiratory system (port al of entry [POE]) and for effects peripheral to 16 the respiratory system (extrarespiratory or systemi c effects). Reference values are generally 17 derived for chronic exposures (up to a lifetime), b ut may also be derived for acute ( ≤ 24 hours), 18 short-term (>24 hours up to 30 days), and subchroni c (>30 days up to 10% of lifetime) exposure 19 durations, all of which are derived based on an ass umption of continuous exposure throughout 20 the duration specified. Unless specified otherwise , the RfD and RfC are derived for chronic 21 exposure duration. 22 The carcinogenicity assessment provides information on the carcinogenic hazard potential 23 of the substance in question and quantitative estim ates of risk from oral and inhalation exposure 24 may be derived. The information includes a weight- of-evidence judgment of the likelihood that 25 the agent is a human carcinogen and the conditions under which the carcinogenic effects may be 26 expressed. Quantitative risk estimates may be deri ved from the application of a low-dose 27 extrapolation procedure. If derived, the oral slop e factor is a plausible upper bound on the 28 estimate of risk per mg/kg-day of oral exposure. S imilarly, an inhalation unit risk is a plausible 29 upper bound on the estimate of risk per μ g/m 3 air breathed. 30 Development of these hazard identification and dose -response assessments for 31 formaldehyde has followed the general guidelines fo r risk assessment as set forth by the National 32 Research Council (NRC) (1983). EPA Guidelines and Risk Assessment Forum Technical Panel 33 Reports that may have been used in the development of this assessment include the following: 34 Guidelines for the Health Risk Assessment of Chemic al Mixtures (U.S. EPA, 1986a), Guidelines 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 1-2 for Mutagenicity Risk Assessment (U.S. EPA, 1986b), Recommendations for and Documentation 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 A ssessment (U.S. EPA, 1995), 6 Guidelines for Reproductive Toxicity Risk Assessmen t (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 A ssessment of Chemical 10
Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference Concen tration 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 Environment al Exposures to Children (U.S. EPA, 15 2006b). 16 The literature search strategy employed for this co mpound was based on the Chemical 17 Abstracts Service Registry Number (CASRN) and at le ast one common name. Any pertinent 18 scientific information submitted by the public to t he 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 b een considered in this assessment. 21
22This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-1 2. BACKGROUND 1 2 3 This chapter provides an overview of the physical a nd chemical characteristics of 4 formaldehyde. Also provided in this chapter are a description of the production, uses, and 5 sources of formaldehyde and information regarding e nvironmental levels and human exposure. 6 A description of the toxicokinetics and toxicodynam ic processes involved in formaldehyde 7 toxicity for the inhalation, oral, and dermal route s can be found in Chapter 3 (Toxicokinetics). 8 9 2.1. PHYSICOCHEMICAL PROPERTIES OF FORMALDEHYDE 10 Formaldehyde (CASRN 50-00-0) is the first of the se ries of aliphatic aldehydes and is a 11 gas at room temperature. Its molecular structure i s depicted in Figure 2-1. It is noted for its 12 reactivity and versatility as a chemical intermedia te. It readily undergoes polymerization, is 13 highly flammable, and can form explosive mixtures w ith air. It decomposes at temperatures 14
above 150 ° C. 15

16 Figure 2-1. Chemical structure of formaldehyde. 17 18 19 At room temperature, pure formaldehyde is a colorle ss gas with a strong, pungent, 20 suffocating, and highly irritating odor. Formaldeh yde is readily soluble in water, alcohols, ether, 21 and other polar solvents. A synopsis of its physic ochemical properties is given in Table 2-1. 22
23 2.2. PRODUCTION, USES, AND SOURCES OF FORMALDEHYDE 24 Formaldehyde has been produced commercially since t he early 1900s and, in recent years, 25 has been ranked in the top 25 highest volume chemic als produced in the U.S. (National 26 Toxicology Program [NTP], 2002). In 2003, 4.33 mil lion metric tons of formaldehyde were 27 produced in the U.S. (Global Insight, 2006). In 20 00, worldwide formaldehyde production was 28 estimated to be 21.5 million metric tons, (Internat ional Agency for Research on Cancer [IARC], 29 2006). 30 

# C O

H HThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-2 Table 2-1. Physicochemical properties of formaldeh yde 1 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 K ow –0.75 to 0.35 Henry’s law constant 3.4 × 10 –7 atm-m 3 /mol at 25 ° C 2.2 × 10 –2 Pa-m 3 /mol at 25°C Conversion factors (25 o C, 760 mm Hg) 1 ppm = 1.23 mg/m 3 (v/v) 1 mg/m 3 = 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 a ir Autoignition temperature 300 ° C Solubility Very soluble in water; soluble in alcoho ls, ether, acetone, benzene Reactivity Reacts with alkalis, acids and oxidizers 3 Sources: American Conference of Governmental Indus trial Hygienists (ACGIH) (2002); 4 International Programme on Chemical Safety (IPCS) ( 2002); Agency for Toxic Substances and 5 Disease Registry (ATSDR) (1999); Gerberich and Seam an (1994); Walker (1975). 6 7 8 Formaldehyde is a chemical intermediate used in the production of plywood adhesives, 9 abrasive materials, insulation, foundry binders, br ake linings made from phenolic resins, surface 10 coatings, molding compounds, laminates, wood adhesi ves made from melamine resins, phenolic 11 thermosetting, resin curing agents, explosives made from hexamethylenetetramine, urethanes, 12 lubricants, alkyd resins, acrylates made from trime thylolpropane, plumbing components from 13 polyacetal resins, and controlled-release fertilize rs made from urea formaldehyde concentrates 14This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-3 (IPCS, 1989). Formaldehyde is used in smaller quan tities for the preservation and embalming of 1 biological specimens. It is also used as a germici de, an insecticide, and a fungicide, as well as an 2 antimicrobial agent in soaps, shampoos, hair prepar ations, deodorants, lotions (e.g., suntan lotion 3 and dry skin lotion), makeup, and mouthwashes, and is present in hand cream, bath products, 4 mascara and other eye makeup, cuticle softeners, na il creams, vaginal deodorants, and shaving 5 cream (IPCS, 2002; ATSDR, 1999). 6 Formaldehyde is commonly produced as an aqueous so lution called formalin, which 7 usually contains about 37% formaldehyde and 12–15% methanol. Methanol is added to formalin 8 to slow polymerization that leads eventually to pre cipitation as paraformaldehyde. 9 Paraformaldehyde has the formula (CH 2 O) n where n is 8 to 100. It is essentially a solid fo rm of 10 formaldehyde and therefore has some of the same use s as formaldehyde (Kiernan, 2000). When 11 heated, paraformaldehyde sublimes as formaldehyde g as. This characteristic makes it useful as a 12 fumigant, disinfectant, and fungicide, such as for the decontamination of laboratories, 13 agricultural premises, and barbering equipment. Lo ng-chain polymers (e.g., Delrin plastic) are 14 less inclined to release formaldehyde, but they hav e a formaldehyde odor and require additives to 15 prevent decomposition (U.S. EPA, 2008). 16 The major sources of anthropogenic emissions of for maldehyde are motor vehicle 17 exhaust, power plants, manufacturing plants that pr oduce or use formaldehyde or substances that 18 contain formaldehyde (i.e., adhesives), petroleum r efineries, coking operations, incineration, 19 wood burning, and tobacco smoke. Among these anthr opogenic sources, the greatest volume 20 source of formaldehyde is automotive exhaust from e ngines not fitted with catalytic converters 21 (NEG, 2003). The Toxic Release Inventory (TRI) dat a for 2007 show total releases of 22 21.9 million pounds with about half to the air and half to underground injection (EPA TRI 23 Explorer, http://www.epa.gov/triexplorer/) (U.S. EP A, 2009a). 24 Formaldehyde is formed naturally in the lower atmo sphere during the oxidation of 25 hydrocarbons (i.e., methane and terpene), which rea ct with hydroxyl radicals and ozone to form 26 formaldehyde and other aldehydes, as intermediates in a series of reactions that ultimately lead to 27
the formation of carbon monoxide and carbon dioxide , hydrogen and water. Formaldehyde can 28 also be formed in a variety of other natural proces ses, such as decomposition of plant residues in 29 the soil, photochemical processes in sea water, and forest fires (National Library of Medicine 30 [NLM], 2001). 31 Formaldehyde emitted to the ambient air primarily r eacts with photochemically generated 32 hydroxyl radicals in the troposphere or undergoes d irect photolysis (IPCS, 2002). Overall, half33 lives for formaldehyde in air can vary considerably under different conditions. Estimates for 34 atmospheric residence time in several U.S. cities r anged from 0.3 hours under conditions typical 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-4 of a rainy winter night to 250 hours under conditio ns typical of a clear summer night (assuming 1 no reaction with hydroperoxyl radicals). Given the generally short daytime residence times for 2 formaldehyde, there is limited potential for long-r ange transport (IPCS, 2002). In cases where 3 organic precursors are transported long distances, however, secondary formation of formaldehyde 4 may occur far from the anthropogenic sources of the precursors. 5 Formaldehyde is released to water from the discharg es of both treated and untreated 6 industrial wastewater from its production and from its use in the manufacture of formaldehyde7 containing resins (ATSDR, 1999). Formaldehyde is a lso a possible drinking-water disinfection 8 by-product from the use of ozone and/or hydrogen pe roxide. In water, formaldehyde is rapidly 9 hydrated to form a glycol, and the equilibrium favo rs the glycol. 10
11
2.3. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 12 General population exposure to formaldehyde can occ ur via inhalation, ingestion and 13 dermal contact. Each of these pathways and associa ted media levels are discussed below. 14 Formaldehyde exposure can also occur occupationally via three main scenarios: 15
16

• The production of aqueous solutions of formaldehyde (formalin) and their use in the 17 chemical industry (e.g., for the synthesis of vario us resins, as a preservative in medical 18 laboratories and embalming fluids, and as a disinfe ctant). 19

• Release from formaldehyde-based resins in which it is present as a residue and/or through 20 their hydrolysis and decomposition by heat (e.g., d uring the manufacture of wood 21 products, textiles, synthetic vitreous insulation p roducts, and plastics). In general, the use 22 of phenol–formaldehyde resins results in much lower emissions of formaldehyde than 23
those of urea- based resins. 24

• The pyrolysis or combustion of organic matter (e.g. , in engine exhaust gases or during 25 firefighting) (IARC, 2006). 26
27 Industries with the greatest potential for exposure include health services, business 28 services, printing and publishing, manufacture of c hemicals and allied products, manufacture of 29 apparel and allied products, manufacture of paper a nd allied products, personal services, 30 machinery (except clerical), transport equipment, a nd furniture and fixtures (IARC, 1995). 31
32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-5 2.3.1. Inhalation 1 The most current ambient air monitoring data for fo rmaldehyde come from EPA’s air 2 quality system database (EPA’s AirData Web site: ht tp://www.epa.gov/air/data/index.html) (U.S. 3 EPA, 2009b). These data have been collected from a wide variety of sources, including state and 4 local environmental agencies, but have not been col lected from a statistically based survey. The 5 most recent data, for the year 2007, come from 188 monitors located in 33 states as shown in 6 Figure 2-2 (U.S. EPA, 2008). The annual means for these monitors range from 0.7–45.03 μ g/m 3 7 (0.56–36.31 ppb) and have an overall average of 3.4 4 μ g/m 3 (2.77 ppb). The annual means are 8 derived by EPA by averaging all available daily dat a from each monitor. Table 2-2 shows a 9 breakout of the data by land use category based on the annual means from each monitor for 2005, 10 2006, and 2007. The land use is established on the basis of the most prevalent land use within 11 0.25 miles of the monitor. The mobile category (la nd near major highways or interstates such 12 that it is primarily impacted by mobile sources) ha s the highest mean levels, and agricultural 13
lands have the lowest. 14 15

16 Figure 2-2. Locations of hazardous air pollutant m onitors. 17 Dasgupta et al. (2005) measured formaldehyde levels in 5 U.S. cities during 1999 - 2002. 18 Samples were collected over approximately a one mon th period in the spring or summer. 19 Mean levels were 5.05 ppb in Nashville, TN; 7.96 p pb in Atlanta, GA; 4.49 ppb in 20 Houston, TX; 3.12 ppb in Philadelphia, PA; and 2.6 3 in Sydney, FL. 21This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-6 Table 2-2. Ambient air levels by land use category 1 2 Formaldehyde Exposure by Category a Agriculture Commercial Forest Industrial Mobile b 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/m 3

. 4

b “Mobile” is ambient air in locations primarily impa cted by mobile sources. 5 6 Source: AirData for 2005, 2006, and 2007 (U.S. EPA , 2009b). 7 8 9 Under the National-Scale Air Toxics Assessment (NAT A) program, EPA has conducted 10 an emissions inventory for a variety of hazardous a ir pollutants (HAPs), including formaldehyde 11 (U.S. EPA, 2006c). The NATA uses the emissions inv entory data to model nationwide air 12 concentrations/exposures (U.S. EPA, 2006c). The re sults of the 1999 ambient air concentration 13 modeling for formaldehyde suggest that county media n air levels range from 0 to 6.94 μg/m 3 (0 – 14 5.59 ppb) with a national median of 0.56 μg/m 3 (0.45 ppb) (see Figure 2-3). Similar results were 15 found for the year 2002: county concentrations rang ed from 0.12 to 9.17 μg/m 3 (0.097 – 7.38 16 ppb) with median of 0.78 μg/m 3 (0.63 ppb). NATA has not provided updated concent ration 17 maps for 2002. The 1999 map shows the highest leve ls in the far west and northeastern regions 18 of the U.S. While these modeling results can be us eful, it is important to consider their 19 limitations. Some of the geographical differences result from differences in methods used by 20 states supplying the data. For example, the high l evels indicated for Idaho result from the large 21 amount of wood burned during forest fires and the r elatively high emission factor that Idaho uses 22 (compared with other states) to estimate formaldehy de emissions from forest fires. A 23 comparison of modeling results from NATA to measure d values at the same locations is 24 presented in U.S. EPA (2006c). For 1999, it was fo und that formaldehyde levels were 25 underestimated at 76% of the sites (n = 68). One p ossible reason why the NATA results appear 26 low compared to measurements is that the modeling h as not accounted for secondary formation 27 of formaldehyde in the atmosphere. 28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-7 1 2 Figure 2-3. Modeled ambient air concentrations bas ed on 1999 emissions. 3 4 5 In general, ambient levels of formaldehyde in outdo or air are significantly lower than 6 those measured in the indoor air of workplaces or r esidences (ATSDR, 1999; IARC, 1995). 7 Indoor sources of formaldehyde in air include volat ilization from pressed wood products, carpets, 8 fabrics, insulation, permanent press clothing, late x paint, and paper bags, along with emissions 9 from gas burners, kerosene heaters, and cigarettes (NLM, 2001). In general, the major indoor air 10 sources of formaldehyde can be described in two way s: 1) those sources that have the highest 11 emissions when the product is new with decreasing e mission over time, as with the first set in the 12 examples above; and 2) those sources that are reocc urring or frequent such as the second set of 13 examples above. Gilbert et al. (2006) studied 96 h omes in Quebec City, Canada and found 14
elevated levels in homes with new wood or melamine furniture purchased within the previous 12 15 months. A summary of indoor data is provided in Ta ble 2-3. Results vary depending on housing 16 characteristics and date of study. 17This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-8 1 Table 2-3. Studies on residential indoor air level s of formaldehyde (nonoccupational)

Citation No. of

Samples Target Population/House Type Mean (μg/m 3 ) Range (μg/m 3 )
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/m 3 3 4 5 Salthammer et al. (2010) present a thorough review of formaldehyde sources and levels 6
found in the indoor environment. Based on an exami nation of international studies carried out in 7 2005 or later they conclude that the average exposu re of the population to formaldehyde is 20 to 8This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-9 40 μ g/m 3 under normal living conditions. They used the dia gram shown in Figure 2-4 to 1 summarize data they found on the range of formaldeh yde air concentrations (in ppb) in different 2
environments. 3

4 Figure 2-4. Range of formaldehyde air concentratio ns (ppb) in different 5 environments. Source: Salthammer et al. (2010) 6 7 8 Data on formaldehyde levels in outdoor and indoor a ir 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 a nd four urban sites sampled in the period 11 1990–1998. A Monte Carlo analysis applied to the p ooled data (n = 151) was used to estimate 12 the distribution of time-weighted 24-hour air expos ures. This study suggested that mean levels in 13 outdoor air were 3.3 μg/m 3 (2.7 ppb) and mean levels in indoor air were 35.9 μg/m 3 (29.2 ppb) 14 (Health Canada and Environment Canada, 2001). The simulation analysis also suggested that 15 general population exposures averaged 33–36 μg/m 3 (27–30 ppb). 16 Since the early to mid 1980s, manufacturing process es and construction practices have 17 been changed to reduce levels of indoor formaldehyd e 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 Emissi ons from Composite Wood Products; 20 http://www.arb.ca.gov/regact/2007/compwood07/fro-fi nal.pdf) has limited the amount of 21 0.1 1 10 100 1000

Remote Air

Rural Air

Urban Air

Normal Indoor Air

Polluted Indoor Air

Extreme ConditionsThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-10 formaldehyde that can be released by specific compo site wood products (i.e., hardwood plywood, 1 particle board, and medium density fiberboard) sold , supplied, or manufactured for use in 2
California. For this reason the mean indoor air lev els presented by Health Canada and 3 Environment Canada (2001) (based on samples collect ed from 1989–1995) may overestimate 4 current levels. In addition, the Canadian indoor a ir data may overestimate formaldehyde levels in 5 U.S. homes, because many residential homes in Canad a use wood burning stoves more 6 frequently and have tighter construction (due to co lder winters), leading to less dilution of indoor 7 emissions. The outdoor air levels, however, appear to have remained fairly constant over recent 8 years, and the median outdoor level from the Canadi an study (2.8 μg/m 3 ) (2.3 ppb) is very 9 similar to the median of the U.S. monitoring data ( 2.83 μg/m 3 ) (2.3 ppb) in 1999

. 10 Even though formaldehyde levels in construction mat erials have declined, indoor 11 inhalation concerns still persist. For example, re cent studies have measured formaldehyde levels 12 in mobile homes. ATSDR (2007) reported on air samp ling 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 level s in closed trailers averaged 1,250 ± 828 15 μg/m 3 (mean ± standard deviation [SD]) (1.04 ± 0.69 ppm) , with a range of 12–4,390 μg/m 3 16 (0.01–3.66 ppm). The levels decreased to an averag e of 468 ± 324 μg/m 3 (0.39 ± 0.27 ppm), 17 with a range of 0.00–1,960 μg/m 3 (0.00–1.63 ppm) when the air conditioning was turn ed on. 18 Levels also decreased to an average of 108 ± 96 μg/ m 3 (0.09 ± 0.08 ppm), with a range of 12– 19 588 μg/m 3 (0.01–0.49 ppm) when the windows were opened. ATS DR (2007) found an 20 association between temperature and formaldehyde le vels; higher temperatures were associated 21 with higher formaldehyde levels in trailers with th e windows closed. They also noted that 22 different commercial brands of trailers yielded dif ferent 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 h omes (“trailers”) (CDC, 2008). At the time of 26 the study, sampled trailers were in use as temporar y shelters for Louisiana and Mississippi 27 residents displaced by hurricanes Katrina and Rita. The geometric mean level of formaldehyde 28 in sampled trailers was 95 μg/m 3 (77 ppb), and the range was 3.7–730 μg/m 3 (3–590 ppb). 29
30 2.3.2. Ingestion 31
Limited U.S. data indicate that concentrations in d rinking water may range up to 32 approximately 10 μg/L in the absence of specific co ntributions 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 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-11 concentration (5 μg/L) was judged to be a reasonabl e estimate of the average formaldehyde in 1 Canadian drinking water. Concentrations approachin g 100 μg/L were observed in a U.S. study 2 assessing the leaching of formaldehyde from domesti c 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 for maldehyde 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 h as been used as a bacteriostatic agent in 8 some foods, such as cheese (IARC, 1995). There hav e been no systematic investigations of 9 levels of formaldehyde in a range of foodstuffs tha t 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 f ood are highly variable. In the few studies of 12 the formaldehyde content of foods in Canada, the co ncentrations were within a range of 13 <0.03–14 mg/kg (Health Canada and Environment Canad a, 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 (1 992) 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 t he 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 group s, Owen et al. (1990) calculated that annual 28 consumption of dietary formaldehyde results in an i ntake 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 materials, 33 such as some paper products, fabrics, and cosmetics . For example, nail hardeners contain 34 formalin, and cosmetics products such as hand cream s and suntan lotions contain formaldehyde- 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 2-12 releasing agents (but not formaldehyde) as preserva tives. Generally, though, dermal contact is 1 more of a concern in occupations that involve handl ing concentrated forms of formaldehyde, 2 such as those occurring in embalming and chemical p roduction. 3
4This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-1 3. TOXICOKINETICS 1 2 3 3.1. CHEMICAL PROPERTIES AND REACTIVITY 4 Formaldehyde (HCHO) is the smallest aldehyde (30 g /mol) and is a gas at room 5 temperature. It is highly water soluble and reacti ve. In water, less than 0.1% of formaldehyde 6 exists unhydrated, with the majority reported to be in the hydrated form, methylene glycol 7 (CH 2 (OH) 2 ) (Priha et al., 1996). Formaldehyde reacts readil y with high and low molecular 8 weight biological constituents. 9
10 3.1.1. Binding of Formaldehyde to Proteins 11 Formaldehyde is a reactive molecule that is likely to react with both low molecular 12 weight cellular components (e.g., reduced glutathio ne[GSH]) as well as high molecular weight 13 components. Unlike deoxyribonucleic acid (DNA), wh ich has some additional barriers to 14 exposure (i.e., nucleus), extracellular and intrace llular proteins are obvious targets for interacting 15 with formaldehyde. Formaldehyde is a well-known cr oss-linking agent that is used in the 16 fixation of tissues, preparation of vaccines, and s tudy of protein-protein interactions (Metz et al., 17 2006). However, the exact nature of the protein mo difications used for these purposes is not yet 18 fully characterized (Metz et al., 2006, 2004). Fig ure 3-1 provides a general reaction scheme for 19 formaldehyde-mediated modifications of amino acids. In step 1, formaldehyde reacts with 20 primary N-terminal amines to form a labile methylol adduct. This adduct can undergo 21 dehydration (step 2) to form an imine, or Schiff ba se (

− N=CH 2 ). Metz et al. (2004) examined the 22 types of formaldehyde-protein reactions that are li kely to occur in vivo by synthesizing several 23 identical polypeptides with one varying amino acid (X) within the sequence VEL X VLL 24 (V=valine, E=glutamate, L=Leucine, X=varying amino acid). Several peptides with reactive 25 amino acids did not exhibit modifications, suggesti ng that the peptide sequence/structure affects 26 the ability of formaldehyde to react with amino aci ds. Peptides that were modified indicated 27 formation of methylol adducts (Figure 3-1, step 1) or a mixture of methylol and imine adducts 28 (Figure 3-1, step 2). 29This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-2 1 2 Figure 3-1. Formaldehyde-mediated protein modifica tions. 3 4 Note: Formaldehyde reacts with primary N -terminal amines to form a methylol 5 adduct [1], which increases the molecular weight by 30 Da ( ∆ m). This labile 6 adduct can rearrange to form an amine, or Schiff ba se [2], that results in an 7 increase in MW of 12 Da. Schiff bases can react wi th certain amino acids to form 8 intra- or intermolecular methylene bridges [3]. Th e two amino acids depicted in 9 step 3 may be within the same protein or possibly f rom two different proteins. 10 11 Source: Metz et al. (2004). 12 13 14 Mucus is composed of water, electrolytes, polysacch arides, and about 0.5% soluble 15 proteins (Priha et al., 1996; Bogdanffy et al., 198 7). Bogdanffy et al. (1987) showed that 16 although human nasal mucus can bind 70% of 100 mM f ormaldehyde, irreversible binding of 17 [ 14 C]-formaldehyde to serum albumin (the major protein in mucus) was shown to be insignificant 18 after a 1-hour incubation. Irreversible binding (5 0% or more) did not occur until after about 7 19 hours of incubation. These data suggest that the p rotein content of mucus may not provide a 20 significant formaldehyde irreversible sink. Noneth eless, the solubility of formaldehyde in mucus 21 along with mucus flow and ingestion likely indicate that much of the inhaled dose is removed— 22 perhaps as much as 42% in rodents (IARC, 2005; Schl osser, 1999). 23This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-3 In general, formaldehyde interacts with proteins. Studies carried out in cell culture media 1 containing serum and formaldehyde have shown that s uch mixtures are quite labile. For 2 example, during a 60-minute incubation of formaldeh yde with complete cell media (i.e., with 3 fetal calf serum) at 38 ° C, gas chromatography-mass spectrometry (GC-MS) exh ibited very 4 different peak profiles at different points during the incubation (Proctor et al., 1986). In contrast

, 5 GC-MS chromatograms of cell media containing formal dehyde but no serum proteins appeared 6 relatively unchanged throughout the incubation. Co mpared to cell culture medium alone, 7 complete media were considered to provide a more su itable model for the hypothetical 8 interactions that formaldehyde could undergo in viv o (including perhaps blood). 9
10 3.1.2. Endogenous Sources of Formaldehyde 11 Endogenous formaldehyde is produced through a) nor mal cellular metabolism through 12 enzymatic or non-enzymatic reactions, and also as a detoxification product of xenobiotics during 13
cellular metabolism: 14
15
3.1.2.1. Normal Cellular Metabolism (Enzymatic) 16 Formaldehyde is produced during normal metabolis m of methanol, amino acids (e.g., 17 glycine, serine, methionine), choline, dimethylglyc ine, and methylamine and through the folate18 dependent endogenous one-carbon pool etc. 19 i) One of the endogenous sources for formaldehyde production is methanol, 20 formed during normal cellular metabolism. However, this fraction may also be derived 21 through consumption of fruits, vegetables and alcoh ol (Shelby et al . 2004; IPCS, 1997). 22 In studies conducted with healthy humans whose diet was devoid of methanol-containing 23 or methanol-generating foods (such as cereals conta ining aspartame, a precursor of 24 methanol) and who abstained from alcohol consumptio n, the background blood levels of 25 methanol range from 0.25–4.7 mg/L (Reviewed in Shel by et al 2004 [CERHR]). 26 Methanol is metabolized to formaldehyde predominant ly by hepatic alcohol 27 dehydrogenase-1 (ADH1) in primates and by ADH1 and catalase (CAT) in rodents, 28 ADH1 requiring nicotinamide adenine dinucleotide (N AD + ) as a cofactor. 29 ii) Dimethylglycine (DMG), one of the byproducts of choline metabolism 30 endogenously present in the body, is an indirect so urce of endogenous formaldehyde. 31 Two specific dehydrogenases, i) dimethylglycine deh ydrogenase (DMGDH) which 32 converts DMG to sarcosine (methylglycine) and ii) s arcosine dehydrogenase (SDH) 33 which converts sarcosine to glycine, have been show n to non-covalently bind to the folate 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-4 enzyme, tetrahydrofolate (THF). Further, these deh ydrogenases form “active 1 formaldehyde” by removing the 1-carbon groups from THF (Binzak et al., 2000). 2 iii) Another source of endogenous formaldehyde is methylamine (MA), an 3 intermediary component of the metabolism of adrenal ine, sarcosine, creatine, lecithin, 4 and other dietary sources (Yu and Zuo 1996). The e nzyme semicarbozole-sensitive 5 amine oxidase (SSAO), predominantly present in the plasma membrane of endothelial 6 smooth muscle cells and in circulating blood, conve rts methylamine to formaldehyde, 7 hydrogen peroxide and ammonia. The formaldehyde th us released has been shown to 8 cause endothelial injury eventually leading to athe rosclorosis (Kalasz, 2003). Yu et al. 9 (1997) have shown that adrenaline, released in the body as a response to stress, is known 10 to be deaminated by the enzyme monoamine oxidase, w ith further conversion of 11 methylamine to formaldehyde by SSAO (Yu et al . 199 7). Creatine is another precursor 12 for methylamine which is metabolized by SSAO to for m formaldehyde. It has been 13 shown that short-term, high-dose dietary supplement ation of creatine in healthy humans 14 causes a significant increase in urinary methylamin e and formaldehyde levels (Poortmans 15 et al., 2005). 16 iv) Endogenous formaldehyde is also a constituent of the one-carbon pool, a 17
network of interrelated biochemical reactions that involve the transfer of one-carbon 18 groups from one compound to another (usually the tr ansfer of the hydroxymethyl group 19 of serine to tetrahydrofolic acid). 20
21 Tyihak et al. (1998) have demonstrated that formal dehyde, but not the methyl radical or 22 methyl cation, is involved in the enzymatic transme thylation and demethylation reactions, and 23 suggested the presence of a formaldehyde cycle in c ells for the production and removal of 24 formaldehyde utilizing the transfer through methion ine  S-adenosylmethionine  S-adenosyl25 homocysteine  homocysteine (Tyihak et al., 1998). However, these studies did not clearly show 26 whether the formaldehyde released in this cycle is in free or bound form. 27 Formaldehyde has been shown to be produced in norm al and leukemic leukocytes from 28
N 5 -methyl-THF by enzymatic degradation (Thorndike an d Beck, 1977). This is a two-step 29 reaction involving 1) enzymatic conversion of the m ethyl-THF to formaldehyde followed by 2) 30 nonenzymatic reaction of formaldehyde with an amine . Thorndike and Beck (1977) showed that 31 leukocyte (granulocyte and lymphocyte) cell extract s from normal individuals and patients with 32 chronic lymphocytic leukemia (CLL) or chronic myelo cytic leukemia (CML) incubated with 14 C33 methyl-THF and saturating amounts of tryptamine pro duced free formaldehyde which is 34 detected as its corresponding carboline derivative formed with tryptamine. These results 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-5 demonstrate the activity of the enzyme N 5 , N 10 -methylene THF reductase which oxidizes N 5

- 1 methyltetrahydrofolate to N 5 , N 10 methylene THF. The authors noted that the enzyme levels 2 were in the order of normal granulocytes < normal l ymphocytes < granulocytes from a CML 3 individual < lymphocytes from a CLL individual (Tho rndike and Beck, 1977), suggesting 4 increased activity of formaldehyde producing enzyme in leukemic cells compared to normal 5 leukocytes. Overall, formaldehyde might be a bypro duct as well as an intermediary product in 6
several of these reactions. 7
8
3.1.2.2. Normal Metabolism (Non-Enzymatic) 9 i) Formaldehyde can also be formed non-enzymatical ly by the spontaneous reaction of 10 methanol with hydroxyl radicals, wherein cellular h ydrogen peroxide is the precursor for 11 hydroxyl radicals generated through Fenton reaction (Cederbaum and Qureshi, 1982). 12
13 ii) Another mechanism of nonenzymatic production o f formaldehyde is through lipid 14 peroxidation of polyunsaturated fatty acids (PUFA) (Shibamoto, 2006; Slater, 1984). In this 15 mechanism, reactive oxygen species (ROS) generated during oxidative stress abstract a hydrogen 16 atom from a methylene group of polyunsaturated fatt y acids (PUFA) in cell membranes causing 17 autooxidation of lipids with the eventual productio n of free radicals (e.g., peroxy radical). It is 18
known that a certain level of oxidative stress and lipid peroxidation does occur in normal 19 individuals, and these cellular metabolic processes are likely to contribute to endogenous 20 formaldehyde production. 21
22
3.1.2.3. Exogenous Sources of Formaldehyde Production 23 Microsomal cytochrome P450 enzymes catalyze oxidat ive demethylation of N-, O- and S24 methyl groups of xenobiotic compounds whereby forma ldehyde is produced as a primary 25 product, which is subsequently incorporated into th e one-carbon pool by reacting with 26 tetrahydrofolic acid or is oxidized to formate (Dah l and Hadley, 1983; Heck et al., 1982). Also, 27 some special peroxidases, such as peroxide-dependen t horseradish peroxidase enzymatically 28 catalyze xenobiotics to generate formaldehyde in th e body. In particular, an ethyl peroxide29 dependent horseradish peroxidase has been shown to act on N,N -dimethylaniline and produce 30 equimolar amounts of N -methylaniline and formaldehyde (Kedderis and Holle nberg, 1983). 31 The tobacco-specific nitrosamine nitrosamine 4-(met hylnitrosamino)-1-(3-pyridyl)-butanone 32 (NNK) is another source of formaldehyde. It has be en shown that formaldehyde is also produced 33 during the methyl hydroxylation of NNK by rat liver microsomes (Castonguay et al., 1991). 34
Also recent studies have demonstrated the formation of formaldehyde-DNA adducts in NNK- 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-6 treated rats using a highly sensitive liquid chroma tography-electrospray ionization-tandem mass 1 spectrometry with selected reaction monitoring (Wan g et al., 2007), suggesting formation of 2 formaldehyde from nitrosamines. Cigarette smoke is also a source of exogenously produced 3 methylamine which is converted to formaldehyde by S SAO (Yu, 1998). 4
5
3.1.2.4. FA-GSH Conjugate as a Method of Systemic Distributi on 6 Formaldehyde is primarily metabolized by alcohol d ehydrogenase (ADH3) which uses 7 the formaldehyde-glutathione hemiacetal adduct as t he substrate. Sanghani et al. (2000) have 8 shown that due to high circulating concentrations ( 50-fold) of glutathione in human blood, the S9 (hydroxymethyl)glutathione (HMGSH) adduct, the none nzymatic product of formaldehyde with 10 glutathione is the major form of formaldehyde seen in vivo (Sanghani et al., 2000). It is likely 11 that the reversibly bound HMGSH may be transported to different tissues through circulation, 12 but, specific experimental evidence is lacking. 13
14
3.1.2.5. Metabolic Products of FA Metabolism (e.g., Formic A cid) 15 Formate is converted to carbon dioxide (CO 2 ) in rodents predominantly by a folate16 dependent enzyme pathway (Dikalova et al., 2001). Formate is also oxidized to CO 2 and water 17 by a minor pathway involving catalase located in ra t liver peroxisomes (Waydhas et al., 1978; 18 Oshino et al., 1973). In the folate-dependent path way, tetrahydrofolate (THF)-mediated 19
oxidation of formate and the transfer of one-carbon compounds between different derivatives of 20
THF has been described. 21 Endogenous levels of formate also will be affected by dietary intake of methanol22 producing or methanol-containing diets since methan ol is initially converted to formaldehyde and 23 eventually metabolized to formate. It has been sh own in several studies in human subjects who 24 were restricted on consuming methanol producing die ts, aspartame or alcohol, that the 25 endogenous blood concentrations of formate ranged f rom 3.8 to 19.1 mg/L (Shelby et al 2004 26 [CERHR]). The biological half life of formic acid is 77-90 min (Owen et al., 1990b). The levels 27 of formate in the urine of unexposed individuals ra nge from 11.7 to 18 mg/L (Boeniger, 1987). 28 One source of formic acid intake is through diet wh ich ranges from 0.4 to 1.2 mg per day 29 (Boeniger 1987). The half life for plasma formate is ~30 minutes or longer (Boeniger 1987). 30
31
3.1.2.6. Levels of Endogenous Formaldehyde in Animal and Hum an Tissues 32 Heck et al. (1982) estimated that endogenous level s of formaldehyde (free as well as 33 bound) in rats ranged from 0.05 to 0.5 μmole/g (1.5 -15 μg/g) of wet tissue as analyzed by the 34 stable isotope dilution with GC-MS method (Heck et al., 1982). Although the levels of free 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-7 formaldehyde cannot be measured due to their high r eactivity and short half life, they were 1 calculated by Heck et al. (1985) using an indirect method. They added a molar excess of GSH or 2 THF to the test tube containing formaldehyde in aqu eous solution enabling complete binding. 3 When estimated, they observed that the amount of fo rmaldehyde detected was equal to the total 4 amount added to the reaction suggesting that the fo rmaldehyde measured contained both free and 5 bound forms. Further, they calculated the free for maldehyde concentration using the dissociation 6
constant of the HMGSH adduct and cellular concentra tion of GSH. Human formaldehyde 7 dehydrogenase has been shown to have a dissociation constant of 1.5 mM for the formaldehyde8 GSH hemithioacetal adduct (Uotila and Koivusalo 197 4), while the folate enzyme product 9
N 5 ,N 10 -methylene-THF has a dissociation constant of 30 mM (Kallen and Jencks 1966b; a). This 10 could be evaluated using the Michaels-Menton consta nt (K m ) of formaldehyde dehydrogenase for 11 the GSH adduct (~4 μM at 25 o C), whereby they calculated the free formaldehyde l evel to be 12 around 3-7 μM or 1-2% of the total formaldehyde as measured by GC-MS in rat tissues (Heck 13 1982). 14 Cascieri and Clary (1992) estimated the total body content of formaldehyde in human 15 body based on the following assumptions. For an ind ividual with an average body wt of 70 kg 16 and with body fluids accounting for 70% of body wei ght, total formaldehyde content is 17 distributed in ~49 kg of body mass or 49 L of body f luids, owing to the water solubility and 18 uniform distribution of formaldehyde in body fluids . It has been shown that the average blood 19 concentration (mean ± S.E.) of formaldehyde in unex posed rats and humans was 2.24 ± 0.07 and 20 2.61 ± 0.14 μg/g of blood, respectively (Heck et al ., 1985), and in unexposed rhesus monkeys it 21 was 2.42 ± 0.09 μg/g of blood (Casanova et al., 198 8), overall giving an average of 22 approximately 2.5 ppm (2.5 mg/L) formaldehyde acros s the species. All these studies used 23 pentafluorophenyl hydrazine derived formaldehyde us ing GC-MS analysis (Table 3-1). 24 Assuming these values, the body content of total fo rmaldehyde is 122.5 mg (49 L x 2.5 mg/L) or 25 1.75 mg/kg body wt at any given time. Formaldehyde given intravenously to rhesus monkeys has 26 been shown to have a half life of ~1.5 min in blood, wherein formaldehyde in blood was 27 measured by the dimedone method (McMartin et al., 1 979). Using this information Cascieri and 28 Clary (1992) calculated that the human body generat es approximately 40.83 mg/min [(122.5 mg 29 /2 x 1.5] of formaldehyde. Biotransformation of fo rmaldehyde to carbon dioxide in the liver 30 alone has been estimated at 22 mg/minute (Owen et a l., 1990a). 31 Free formaldehyde is detected in body fluids and t issues using dimedone (Szarvas et al., 32 1986) or 2,4-dinitrohenyhydrazine (DNPH) or pentafl uoropheyl hydrazine (PFPH) derivative 33 (Heck et al., 1985) or as a fluorescent derivative (Luo et al . 2001) as trapping agent and detected 34 by analytical techniques such as thin-layer chromat ography (TLC), high-performance liquid 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-8 chromatography (HPLC) and gas-chromatography mass s pectrometry (GC-MS). Data from 1 several studies is summarized in Table 3-1. Using 14 C-labeled dimedone, a chemical which 2 condenses with free formaldehyde forming a product termed “formaldemethone” enabling 3 radiometric detection, Szarvas et al (1986) estimat ed the levels of endogenous formaldehyde in 4 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 . 5 1986). 6
7 Table 3-1. Endogenous formaldehyde levels in anima l and human tissues 8 and body fluids 9 10 Tissue Method Detected as Formaldehyde levels Refer ence 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 PFPHderivative 1.5 - 15 ppm

(0.05-0.5 μmole/g) Heck et al 1982a

Blood GC-MS with select ion

monitoring As PFPHderivative 2.24 ± 0.07 ppm

(2.24 ± 0.07 μg/g) Heck et al 1985

Blood GC-MS with select ion

monitoring As PFPHderivative 2.61 ± 0.14 ppm

(2.61 ± 0.14 μg/g) Heck et al 1985

Plasma Reverse phase HPLCfluorescent detection As 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 PFPHderivative 2.42 ± 0.09 ppm

(2.42 ± 0.09 μg/g) Casanova et al 1988

Plasma Radiometric method As formaldemethone adduct 0.4 - 0.6 ppm

(0.4 - 0.6 μg/mL) Szarvas et al 1986

Urine Radiometric method As formaldemethone adduct 2.5 - 4.0 ppm

(2.5 - 4.0 μg/mL) Szarvas et al 1986 11 Values in the parenthesis, originally cited in the references, are converted to parts per million (ppm ) as indicated. 12 PFPH, pentafluorophenyl hydrazone derivative; DNPH, dinitrophenyl hydrazine; GC-MS, gas-chromatography mass 13 spectrometry; HPLC, high performance liquid chromat ography. 14 15 16 Hileman (1984) reported that the endogenous levels of metabolically derived 17 formaldehyde will be in the range of 3-12 ng/g of t issue (Hileman 1984). So for an average 70 18 kg individual, the endogenous level of metabolicall y derived formaldehyde would be 210 μg to 19 840 μg (3-12 ng/g x 1000 g x 70). 20
21This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-9 3.2. ABSORPTION 1
3.2.1. Oral 2 Oral absorption of [ 14 C]-formaldehyde (7 mg/kg) in rats resulted in 40% e limination as 3

14 C-carbon dioxide ( 14 CO 2 ), with 10% excretion in urine, 1% excretion in fec es, and much of the 4 remaining 49% retained within the carcass, presumab ly due to metabolic incorporation (IARC, 5 1995; Buss et al., 1964). 6
7
3.2.2. Dermal 8 Jeffcoat et al. (1983) reported on the disposition of various doses of [ 14 C]-formaldehyde 9 dermally administered to rats, guinea pigs, and mon keys. Very little (<1% of the applied dose) of 10 the radiolabel was found in the major organs excise d during necropsy. As noted by the authors, 11 the disposition of formaldehyde when administered v ia the dermal route was markedly different 12 to that observed when the compound was administered intravenously or intraperitoneally. In the 13 latter cases, there was much evidence of metabolic activity, and substantial portions of the load 14 were expired as CO 2 . The difference appeared to be the result of a re action of dermally applied 15 formaldehyde with macromolecules at or near the ski n surface or of its evaporation. In general, 16 portions of the load that succeed in entering the c irculation probably do so bound to 17 macromolecules or by incorporation of the radiolabe l via the one-carbon pool. Likewise, Bartnik 18 et al. (1985) who applied [ 14 C]-formaldehyde to the shaved backs of rats conclud ed that the 19 overwhelming majority of the formaldehyde load rema ined sequestered in the outer layers of skin 20 at or near the site of application. At the end of the various measurements, approximately 70% of 21 the dose was found in the treated skin, with a mark ed localization of the remaining radioactivity 22 in the uppermost layers. This fraction of the load was considered to be permanently sequestered, 23 most likely as a result of irreversible binding to macromolecular components. 24
25
3.2.3. Inhalation 26 Studies indicate that the majority of inhaled forma ldehyde is absorbed in the upper 27 respiratory tract (URT) but that the extent of the scrubbing in this region varies significantly 28 across species. In dogs, nearly 100% of nasally in haled formaldehyde is absorbed (Egle, Jr., 29 1972). Lower respiratory tract (LRT) studies desig ned to collect formaldehyde via a tube 30
inserted into the lower trachea revealed that nearl y 95% of formaldehyde was absorbed during 31 the first pass through the upper respiratory tract (Egle, Jr., 1972), an effect observed with 32 multiple ventilation rates. The rat nasal passages also scrub nearly all of the inhaled 33 formaldehyde (on average ~97%) (Morgan et al., 1986) . In computational dosimetry modeling 34 based on anatomically realistic representation of t he human nasal airways from a single 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-10 individual, approximately 90% of inhaled formaldehy de was predicted to be absorbed in the nose 1 at resting inspiration. As the inspiratory rate in creased, this fraction decreased to about 70% at 2 light exercise and to 58% at heavy exercise conditi ons (see Figure 1 in Kimbell et al. [2001b]). 3 The normal human breathing mode during heavy exerci se is oronasal (with ~54% of airflow 4 being oral) (ICRP 66, 1994). Consequently, it is e stimated that during heavy exercise breathing 5 (50 L/min) the flux of formaldehyde into tissue (or rate of mass transported per mm 2 of tissue 6 surface area) in the first six to eight generations of the tracheobronchial airways is comparable to 7 that in the nasal region (Overton et al., 2001). 8 It is important to note that the computer simulatio ns mentioned above are based on 9 anatomical representations of a single individual. Significant anatomical variations occur in 10 human nasal airways. For example, the nasal volume s of 10 adult nonsmoking subjects between 11 18 and 50 years of age in a study in the U.S. varie d between 15 and 60 mL (Santiago et al., 12 2001), and disease states can result in considerabl e further variation (Singh et al., 1998). 13 Species differences in kinetic factors have been ar gued to be the key determinants of 14 species-specific lesion distributions for formaldeh yde and other reactive inhaled gases. Airway 15 geometry is an important determinant of inhaled-for maldehyde dosimetry in the respiratory tract 16 and its differences across species. These issues w ill be discussed in a later section on dosimetry 17 modeling. 18
19
3.2.3.1. Formaldehyde Uptake Can Be Affected by Effects at t he Portal of Entry 20 Certain formaldehyde-related effects have the poten tial to modulate its uptake and 21 clearance. The mucociliary apparatus of the upper respiratory tract is the first line of defense 22 against airborne toxins. Comprising a thick mucus layer (epiphase), hydrophase, and a ciliated 23 epithelium, the mucociliary apparatus may entrain, neutralize, and remove particulates and 24 airborne chemicals from inspired air. As reviewed by Wolfe (1986), airborne pollutants and 25 reactive gases have been shown to decrease mucus fl ow rates in several animal models (Mannix 26 et al., 1983; Iravani, 1974; Carson et al., 1966; D alhamn, 1956; Cralley, 1942). Degradation in 27 the continuity or function of this mucociliary appa ratus could result in a lower clearance of 28 inhaled pollutants at the portal of entry. 29 Morgan et al. (1983) first reported defects in muco ciliary function in F344 rats exposed to 30 15 ppm formaldehyde 6 hours/day for 1–9 days. Muco stasis occurred in several regions in all 31 rats after a single 15 ppm exposure. Ciliastasis o ccurred with greater frequency and across more 32 regions of the nasoturbinate in subsequent days of exposure. The authors observed that 33 mucostasis preceded ciliastasis in most cases, and vigorous ciliary activity was noted in areas 34 without mucus flow. Morgan et al. (1984a) also stu died formaldehyde effects on the mucociliary 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-11 apparatus of isolated frog palates in vitro. Mucos tasis was evident as mucus became stiff and 1 eventually rigid with increasing formaldehyde conce ntration and time of exposure. Ciliary beat 2 continued even after mucostasis, but ciliastasis ul timately occurred when exposure reached 4 and 3 9 ppm. 4 When a rodent is exposed to an irritant, its inhale d dose and pattern of deposition can be 5 profoundly affected by reflex bradypnea, a protecti ve reflex seen in rodents but not in humans. 6 Reflex bradypnea can occur when the trigeminal nerv e is exposed to a sufficient concentration of 7 an irritant, such as formaldehyde. It is manifest as markedly decreased activity or prostration, 8 reduced metabolism, hypothermia (as much as 5oC), s ignificantly reduced respiratory rate and 9 minute volume, and altered blood and brain chemistr y. Because of their small size, rodents are 10 able to rapidly lower their metabolism and body tem perature and therefore their oxygen demand. 11 The consequence is that their inhaled dose of an ir ritating chemical is dramatically lowered. 12 Reflex bradypnea is quantified as the RD 50 , which is the concentration of a chemical that res ults 13 in a 50% decrease in respiratory rate. It can take as much as two hours for rodents to fully 14 recover from the effects of reflex bradypnea. The clinical manifestations of reflex bradypnea can 15 easily be misconstrued as toxicity. None of the st udies described in this assessment took into 16 account the fact that reflex bradypnea may have con founded the results. Reflex bradypnea is 17 discussed in depth in section 4.2.1.1. 18 Sensory irritation studies suggest that formaldehyd e activates the trigeminal nerve by 19 activating nociceptors through the modification of receptor amino acids, possibly including thiol 20 groups. Cassee et al. (1996) measured sensory irri tation to formaldehyde, acetaldehyde, and 21 acrolein in male Wistar rats, following a 30-minute nose-only exposure. Formaldehyde and 22 acrolein elicited similar responses, whereas acetal dehyde was far less irritating. The authors 23 suggested that the differences in sensitivity to th e aldehydes might be explained by differences in 24 physicochemical properties and by regional differen ces in activities of detoxifying enzymes for 25 each chemical. In addition, it has been suggested that acetaldehyde might interact with sensory 26 nerves via an amino group (Steinhagen and Barrow, 1 984), whereas the receptor-binding site for 27 formaldehyde and acrolein is believed to be a thiol group. Differential binding sites for sensory 28 irritants in the trigeminal nerve have been reporte d (Nielsen, 1991). 29 Sensory irritation effects are discussed in depth i n Chapter 4 but are noted here because 30 stimulation of the trigeminal nerve by formaldehyde can result in significantly lower pulmonary 31 ventilation, and formaldehyde exposure in rodents a t concentrations that approach the RD 50 . 32 Barrow et al. (1983) have estimated the “inhaled do se” equivalent to an exposure concentration 33 of 15 ppm in mice and rats used in the chronic form aldehyde bioassays by Kerns et al. (1983) and 34 Monticello and Morgan (1994). Their results indica te that, because mice are observed to 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-12 decrease their minute volume by approximately 75% a s compared to 45% in rats, a twofold 1 greater inhaled dose would be expected in rats vers us mice. This difference may be relevant to 2 the increased incidence of squamous cell carcinoma of the nasal cavity in F344 rats as compared 3 to B6C3F1 mice. Chang et al. (1983) estimated a re duction of 25% in the minute volume of 4 F344 rats. Yokley et al. (2008) have recently publ ished a model that accounts for physiological 5 changes in ventilation rate induced by sensory irri tation in rats. Thus, the “standard” minute 6 volumes used for rats and mice need to be adjusted downward when calculating dosimetric 7 adjustment factors for extrapolation of adverse eff ects to humans (Thompson et al., 2008). This 8 question is further discussed in the section on mod eling the dosimetry. 9 Another effect that modulates dosimetry is the dyn amic tissue remodeling of nasal 10 airways that occurs as a consequence of exposure to reactive gases. For example, formaldehyde 11 dosimetry is influenced by the occurrence of squamo us metaplasia, an adaptive tissue conversion 12 to squamous that occurs in nasal epithelium exposed to toxic levels of formaldehyde. The 13 metaplasia has been observed to occur in rats at ex posure concentrations of 3 ppm and higher 14 (Kimbell et al., 1997). Squamous epithelium is kno wn to absorb considerably less formaldehyde 15 than other epithelial types (Kimbell et al., 1997). Overall, the highest flux levels of 16 formaldehyde in the simulations of the rat nose in Kimbell et al. (2001a) are estimated in the 17 region just posterior to the nasal vestibule. A co nsequence of squamous metaplasia would be to 18 “push” the higher levels of formaldehyde flux towar d the more distal regions of the nose 19 (Kimbell et al., 1997). Subramaniam et al. (2008) discussed this issue further in the context of 20 uncertainties in the modeling of formaldehyde dosim etry. 21
22
3.2.3.2. Variability in the Nasal Dosimetry of Formaldehyde in Adults and Children 23 Garcia et al. (2009) used computational fluid dynam ics to study human variability in the 24 nasal dosimetry of reactive, water-soluble gases in 5 adults and 2 children, aged 7 and 8 years 25 old. They considered two model categories of gases, corresponding to maximal and moderate 26 absorption at the nasal lining. We focus here only on the “maximum uptake” simulations in 27 Garcia et al. (2009). In this case, the gas was con sidered so highly reactive and soluble that it was 28 reasonable to assume an infinitely fast reaction of the absorbed gas with compounds in the airway 29 lining. Although such a gas could be reasonably con sidered to represent formaldehyde, these 30 results cannot be fully utilized to inform quantita tive estimates of formaldehyde dosimetry (and it 31 does not appear to have been the intent of the auth ors either). This is because the same boundary 32 condition corresponding to maximal uptake was appli ed on the vestibular lining of the nose as 33 well as on the transitional and transitional epithe lial lining on the rest of the nose. This is not 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-13 appropriate for formaldehyde as the lining on the n asal vestibule is made of keratinized 1 epithelium which is considerably less absorbing tha n the rest of the nose (Kimbell et al. 2001). 2 The Garcia et al. (2009) study and the results of t heir analyses have been further 3 described and evaluated in Appendix 3-1. Overall up take efficiency, average flux (rate of gas 4 absorbed per unit surface area of the nasal lining) and maximum flux levels over the entire nasal 5 lining did not vary substantially between adults (1 .6-fold difference in average flux and much 6 less in maximum flux), and the mean values of these quantities were comparable between adults 7 and children. These results are in agreement with c onclusions reached by Ginsberg et al. (2005) 8 that overall extrathoracic absorption of highly and moderately reactive and soluble gases 9 (corresponding to category 1 and 2 reactive gases a s per the scheme in USEPA [1994]) is similar 10 in adults and children. On the other hand, Figure 6 A of the paper (reproduced as Figure A in 11 Appendix 3-1), shows significant interhuman variabi lity in flux values at specific points on the 12 nasal walls. The local flux of formaldehyde varies among individuals by a factor of 3 to 5 at 13 various distances along the septal axis of the nose

. 14
15
3.3. DISTRIBUTION 16
3.3.1. Levels in Blood 17 Inhalation studies in several species indicate that exposure to formaldehyde does not 18
result in elevated levels in blood. These studies were carried out over a wide range of exposure 19 concentrations and durations. Rats exposed to 14 p pm formaldehyde for 2 hours exhibited no 20 increase in blood formaldehyde levels [2.25 ± 0.07 μg/(g blood) in treated animals compared 21 with 2.24 ± 0.07 μg/(g blood) in control animals] w hen measured by GC-MS using a stable 22 isotope dilution technique (Heck et al., 1985, 1982 ). Similarly, mean formaldehyde blood levels 23 in humans (n = 6) exposed to 1.9 ppm formaldehyde f or 40 minutes in a walk-in chamber (2.77 ± 24 0.28 μg/g blood) were not statistically different f rom measurements in the same population 25 before exposure (mean of 2.61 ± 0.14 μg/g) (Heck an d Casanova-Schmitz, 1984). The 26 variability in the levels was large. At the indivi dual level, the data showed both increase and 27 decrease in blood levels relative to pre-exposure l evels, which was attributed by the authors as 28 plausibly due to temporal variations in baseline le vels in humans, particularly since the 29 experiment did not control food intake prior to exp osure. Studies in rhesus monkeys have 30 revealed endogenous formaldehyde levels (2.4 μg/g b lood) comparable to humans and that levels 31 were also unaltered following exposure to 6 ppm for maldehyde via inhalation 6 hours/day for 32 4 weeks, measurements being taken at both 7 minutes and 45 hours post final exposure 33 (Casanova et al., 1988). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-14 It is important to keep in mind that the GC-MS meth od is not capable of detecting 1 irreversibly bound formaldehyde; for example, forma ldehyde levels detected by this method, 2 even in the anterior nasal mucosa of rats exposed t o 6 ppm of formaldehyde, were not elevated 3 over control levels. Furthermore, the GC-MS method does not differentiate between free and 4 reversibly bound adducts of formaldehyde (Heck et a l., 1982). Thus, measured levels represent 5 total formaldehyde concentration that includes free formaldehyde as well as reversibly bound 6
adducts. Based on the known Michaelis-Menten const ant, Km, for formaldehyde dehydrogenase 7 with respect to the GSH adduct formation, Heck et a l. (1982) estimated under certain 8 assumptions that free formaldehyde comprised only a bout 1–2% of the total formaldehyde 9 measured by their method. Furthermore, as shown by Metz et al. (2006, 2004), formaldehyde 10 reactions with primary amino and thiol groups can, in a second step, react with many other amino 11 acids to form stable methylene bridges. Presumably , such reactions would not be detectable by 12 using the methods employed by Heck et al. (1982). 4 Thus, the limited interpretation of GC-MS 13 measurements of blood levels suggests that formalde hyde does not appreciably reach the blood, 14 is rapidly metabolized or interacts with macromolec ules when it escapes metabolism, or is 15
otherwise undetected. 16 Results from an earlier experiment using radiolabel ed formaldehyde in rats are consistent 17
with the conclusion based on the GC-MS measurements of no appreciable increase in blood 18 levels of formaldehyde. Following a 6-hour exposur e of F344 rats to 15 ppm of [ 14 C]- 19 formaldehyde (Heck et al., 1983), the concentration s of 14 C in the nasal mucosa were 28-fold 20 higher than those in the blood. The observed half- life of the terminal phase of the radioactivity 21 was long (55 hours); on the other hand, it is known that the half-life of free formaldehyde in the 22 rat blood is very short. Therefore, the authors co ncluded that the radioactivity was likely due to 23 modification of macromolecules or metabolic incorpo ration rather than slow metabolic clearance 24 of formaldehyde. The terminal decline of the radio activity in the packed cell fraction of the 25
blood was much slower and observed to be consistent with incorporation into erythrocytes. 26 In the same paper, Heck et al. (1983) report on the similarity in the pharmacokinetics of 27 radiolabeled formaldehyde and radiolabeled formate in the rat blood, supporting their hypothesis 28 that oxidation of formaldehyde to formate and subse quent incorporation of this compound 29 through one-carbon metabolism were major factors in the disposition of formaldehyde. Studies 30 by Gottschling et al. (1984) have also established that the main product of metabolic clearance of 31

4 Additionally, note that, although Heck et al. (198 2) demonstrated that formaldehyde concentration can be accurately measured from glutathione and tetrahydro folate adducts, similar experiments were not perfor med by using protein samples or cellular extracts (i.e., in the presence of various amino acids). In addition, sta ndard curves for predicting formaldehyde concentration in tissues we re generated in aqueous solutions rather than biolo gical samples.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-15 formaldehyde is formate, which is either further me tabolized to CO 2 and water, incorporated into 1 the one-carbon pool, and/or eliminated in the urine as a sodium salt at about 13 mg/L urine. 2
3
3.3.2. Levels in Various Tissues 4 The radiolabeling studies indicated high levels of 14 C in the rat nasal mucosa (equivalent 5
concentrations of 14 C-formaldehyde in the nasal mucosa of rats naïvely exposed to 15 ppm 6

14 C-formaldehyde were 2,148 ± 255 nmol/g compared wit h 76 ± 11 nmol/g in plasma). In 7 contrast, the GC-MS studies did not detect elevated formaldehyde in this region. This is not to 8 be interpreted as a discrepancy, because the radiol abeling study did not distinguish among 9 radiolabeled species and thus the measured radioact ivity could potentially be free or bound 10 formaldehyde, formate, or any [ 14 C] metabolically incorporated into macromolecules. 11 In concurrent studies, Casanova-Schmitz et al. (198 4) resolved the question as to whether 12 the higher [ 14 C] levels in the nasal mucosa were a consequence of GSH depletion and a 13 subsequent reduction in GSH-dependent clearance of formaldehyde. An important result in these 14 studies was that there was no significant differenc e in labeling in either the nasal mucosa or in 15 plasma between naïve F344 rats and those pre-expose d to unlabeled 15 ppm formaldehyde 16 6 hours/day for the 9 previous days. These finding s indicated little or no apparent effect on the 17 disposition of formaldehyde following short-term ex posure to relatively high levels of 18 formaldehyde. In contrast, Farooqui et al. (1986) reported decreases in GSH in several tissues 19 3 hours after a sublethal I.P. injection of formald ehyde but not after 6 and 9 hours. Taken 20 together, these data suggest that formaldehyde expo sure does not result in long-term alterations 21 in cellular GSH levels and that repeated inhalation exposure does not alter the dosimetry to the 22 bloodstream or formaldehyde body burden. 23 Heck et al. (1983) determined the 14 C concentrations in different tissues in the F344 r at 24 body by exposing rats in a head-only chamber to var ious concentrations (5–24 ppm) of 25 radiolabeled formaldehyde for 6 hours. (Concentrati ons of 14 C in internal organs and tissues 26 relative to that in plasma did not appear to vary m uch as exposure concentrations increased; 27 therefore only averages over the concentration rang e were reported.) Except for the esophagus, 28 levels in the heart, spleen, lung, intestines, live r, and kidney were 1–3 times higher relative to 29 that in plasma. Labeling in the esophagus was high (fivefold relative to plasma). The authors 30 attributed this relatively higher dose to mucocilia ry action in the nose and trachea. The data also 31 indicate that the brain, testes, and erythrocytes a ppear to have about threefold lower 14 C levels 32 than plasma. Pre-exposure to formaldehyde (for 9 d ays) did not alter the measured radioactivity 33 in the nasal mucosa or plasma. Thus, it was conclu ded that the single exposure findings may also 34 be qualitatively extended to chronic exposures. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-16 The total radiolabel measured in the bone marrow (f emur) of F344-rats exposed for 6 1 hours to 0.3–15 ppm of radiolabeled formaldehyde in the Casanova et al. (1984) experiment was 2 high (generally within a factor of 0.5 of the total labeling in the nasal respiratory mucosa). Nearly 3
half of the 14 C was contained in the DNA in this tissue presumabl y on account of the high rate of 4 cell turnover in the bone marrow, indicating that t he carbon derived from 14 C-formaldehyde was 5 utilized for DNA synthesis (Casanova-Schmitz et al. , 1984). 6 Chang et al. (1983) described visceral labeling (vi a autoradiography) in rats, following 7 exposure to 15 ppm [ 14 C]-formaldehyde 6 hours/day for 4 days. The author s attributed this 8 labeling to mucociliary clearance and grooming-rela ted ingestion of formaldehyde. 9 In summary, following exposure to radiolabeled form aldehyde, the radioactivity was very 10 high in the nasal mucosa but was also extensively d istributed to various tissues. In particular, 11 levels in the bone marrow were high. On the other hand, formaldehyde levels in the blood 12 measured by GC-MS were not significantly elevated. Thus, the authors considered it unlikely 13
that the elevated 14 C in various tissues was due to free formaldehyde. Instead, these levels were 14 thought to arise from either rapid metabolic incorp oration or formation of covalent adducts or 15 incorporation via carboxylation reactions of the 14 CO 2 formed during metabolism. 16 The data presented thus far in this section illustr ate that measuring the distribution of the 17 absorbed formaldehyde based on 14 C-radiolabeling and GC-MS studies alone is problema tic 18 because it is difficult to resolve (through these s tudies) whether it is free, reversibly bound, 19 irreversibly bound, formate, one-carbon pool, etc. This is of significance with regard to 20 understanding the availability of the absorbed form aldehyde. More indirect methods had to be 21 developed to further examine the disposition of for maldehyde; however, as discussed below, the 22 interpretation of these approaches may also not be straightforward. 23
24
3.3.2.1. Disposition of Formaldehyde: Differentiating Covale nt Binding and Metabolic 25 Incorporation 26 The motivation in presenting this section is twofo ld, as follows: 27
1. As concluded above, subsequent studies were necessa ry to ascertain whether measured 28 radiolabeling in different experiments was due to f ormaldehyde adducts or incorporation 29 of [ 14 C] one-carbon units of formaldehyde into macromolec ules via the one-carbon pool. 30
2. DNA protein cross-links (DPXs) formed by formaldehy de (covalently bound in this case) 31 have been regarded as a surrogate dose metric for t he intracellular concentration of 32 formaldehyde (Hernandez et al., 1994; Casanova et a l., 1991, 1989). This is particularly 33 relevant because of the nonlinear dose response for DPX formation due to saturation of 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-17 enzymatic defenses at high concentrations (Casanova et al., 1991, 1989). Thus, the 1 ability to measure DPX is an important development. 2
3 An important question is whether the formaldehyde d isposed in the form of DPX is 4 detected in remote tissues. A set of elegant but c omplex experiments involving dual isotope 5 labeling ( 14 C and 3 H) was carried out to this end by the Heck and Casa nova-Schmitz and their 6 coworkers. Casanova-Schmitz et al. (1984) and Casa nova-Schmitz and Heck (1983) used dual 7 isotope labeling of formaldehyde as a way to partia lly distinguish between formaldehyde adducts 8 formation and metabolic incorporation. In separate experiments, F344 rats were exposed to 3 H9
and 14 C- formaldehyde at different exposure concentration s (0.3–15.0 ppm), and the 3 H/ 14 C ratios 10 of different phases of DNA were measured. Only the highlights of the results and significant 11 issues are presented here. The overall conclusions from these experiments were as follows: 12
13

• Labeling in the nasal mucosa was due to both covale nt binding and metabolic 14 incorporation. 15

• DPX was formed at 2 ppm and greater concentrations in the respiratory mucosa. 16

• In the bone marrow, formaldehyde did not bind coval ently to bone marrow 17 macromolecules at any exposure concentration. The labeling of bone marrow 18 macromolecules was found to be entirely due to meta bolic incorporation and not due to 19 covalent binding. 20
21 Macromolecules such as DNA and protein can be isola ted from tissue homogenates by 22 extraction into three phases: an organic phase cons isting of proteins, an aqueous phase consisting 23 of only double-stranded DNA, and an interfacial pha se consisting of both DNA and protein. 24 Single-stranded (but not double-stranded) DNA was p articularly likely to form adducts. DNA 25 from this interfacial phase can be further purified and has been shown to consist of DPXs 26 (Casanova-Schmitz and Heck, 1983). Because both [ 14 C]-formaldehyde and [ 3 H]-formaldehyde 27 can become incorporated into DNA and protein metabo lically as well as by cross-linking, the 28

3 H/ 14 C ratio in such cross-linked material should be hig her than in material that primarily 29 contains metabolically incorporated formaldehyde. Figure 3-2 shows the labeling of tissue from 30 the nasal respiratory mucosa and bone marrow (dista l femur) in rats exposed to [ 14 C]- 31 formaldehyde and [ 3 H]-formaldehyde vapor. 32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-18 1 2 Figure 3-2. 3 H/ 14 C ratios in macromolecular extracts from rat tissue s 3 following exposure to 14 C- and 3 H-labeled formaldehyde (0.3, 2, 6, 10, 4 15 ppm). 5 6 Note that the small yield of interfacial (IF) phase from bone marrow tissue precluded 7 further analysis; this is prima facie evidence for the lack of significant DPXs in this 8 tissue. 9 10 Source: Casanova-Schmitz et al. (1984a). 11
12 13 In the nasal mucosa the interfacial phase has a sig nificantly higher 3 H/ 14 C ratio than the 14 material in the aqueous phase. This suggests that interfacial DNA has significantly more 3 H, a 15 phenomenon likely explained by additional [ 3 H]-formaldehyde molecules present as DPXs prior 16
to extraction. The amount of interfacial DNA was f ound to have a clear dose response. These 17 cross-links were also judged to be due to exogenous formaldehyde. Likewise, the organic phase 18
of the nasal mucosa showed a similar increase in 3 H/ 14 C ratio at higher concentrations, a result 19
that could be attributed to various inter- and intr a-protein adducts (Metz et al., 2004; Trezl et al., 20 2003; Skrzydlewska, 1996). 21 In contrast, analysis of macromolecules at the dist al femur location presents a different 22 pattern (Figure 3-2, part B). First, the interfaci al phase was not detected during extraction, 23 suggesting that there were few or no DPXs to be det ected. Second, there was no increase in 24

3 H/ 14 C ratio in the organic (i.e., protein) phase as a f unction of dose. Therefore, it was 25 concluded that either radiolabeled formaldehyde or formate reached the distal site and was 26 subsequently incorporated into macromolecules. Acc ording to the mechanistic interpretation of 27 these studies, the quantity plotted on the ordinate in Figure 3-2 (the ratio of 3 H/ 14 C between the 28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-19 tissue and the exposure gas) should approach unity as metabolism becomes saturated and more 1 adduct formation occurs, particularly for protein. Indeed, this is what is observed (see 2 Figure 3-2, part A). In contrast, there is no dose effect in the femur, suggesting that the labeling 3 at all doses in that tissue may be due to metabolic incorporation and not due to the parent 4 formaldehyde. 5 (Note: These data were originally shown in the abse nce of an analysis of isotope effects 6 on covalent binding and metabolism. Subsequent stu dies determined that [ 3 H]-formaldehyde is 7 oxidized less rapidly than [ 14 C]-formaldehyde and unlabeled formaldehyde. This s uggests that 8
the 3 H/ 14 C ratio, and therefore the amount of formaldehyde c ovalently bound to tissue, is likely 9 overestimated because more [ 3 H]-formaldehyde remains unmetabolized, i.e., free t o bind [Heck 10 and Casanova, 1987]. The authors hypothesized that this overestimate was relatively greater at 11 the lower concentrations.) 12 Similar results were obtained in GSH-depleted rats (Casanova and Heck, 1987). Again, 13 these authors observed a dose-dependent increase in the 3 H/ 14 C ratio in the interfacial DNA and 14 organic fractions of disrupted cells of the respira tory and olfactory mucosa and no such increases 15 in bone marrow. Interestingly, at 10 ppm exposure (only), GSH-depleted rats exhibited a higher 16

3 H/ 14 C ratio in the organic phase than did normal rats. Casanova and Heck (1987) posited that 17 much of the covalent binding at 6 ppm and lower was due to binding to extracellular proteins, 18 whereas the higher 3 H/ 14 C ratio in GSH-depleted rats at 10 ppm was due to m ore intracellular 19 binding. 20 In their first experiment to measure DPX concentrat ions, Casanova-Schmidt et al. (1984) 21 and Casanova and Heck (1987) used the dual isotope method ( 3 H/ 14 C) mentioned above. In this 22 experiment, DPX was observed only at formaldehyde c oncentrations ≥ 2 ppm. Subsequently, 23 Casanova et al. (1989) developed a more sensitive m ethod using high-performance liquid 24 chromatography (HPLC) for measuring DPX. In this m ethod, tissue homogenates were digested 25 with a proteolytic enzyme and extracted with a phen olic solvent. DPX was detected in the nasal 26 mucosa of rats at formaldehyde concentrations as lo w as 0.3 ppm. This method was also used to 27 measure DPX in the nasal region, the larynx, trache a and carina, and major intrapulmonary 28 airways (airway diameters > 2mm) of rhesus monkeys exposed for 6 hours to 0.7, 2.0 and 6.0 29 ppm of formaldehyde. DPX was detected in the nose ( including the nasopharynx) at all 30 concentrations and at 2.0 and 6.0 ppm in the larynx , trachea, carina and other lower airways. 31 However, DPX was not detectable in the bone marrow of these monkeys at any concentration. 32 Overall, Heck and Casanova-Schmitz and their cowork ers interpreted the results of these 33 various experiments to mean that inhaled formaldehy de could not reach distant sites in the body. 34 It may be noted in this context that Shaham et al. [1996] reported elevated DPX levels in the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-20 white blood cells of laboratory workers exposed to formaldehyde. These data are further 1 reported in Chapter 4.) 2
3
3.4. METABOLISM 4 Formaldehyde is primarily metabolized by glutathion e-dependent formaldehyde 5 dehydrogenase (FALDH) and aldehyde dehydrogenases ( ALDHs). Numerous studies now 6 recognize FALDH as a member of the alcohol dehydrog enase (ADH) family, specifically ADH3 7 (Thompson et al., 2009; Liu et al., 2004, 2001; Hed berg et al., 2003; Høøg et al., 2003; and the 8 references in each of these). The remainder of thi s report will refer to FALDH as ADH3. 9
10
3.4.1. In Vitro and In Vivo Characterization of Fo rmaldehyde Metabolism 11 Formaldehyde is oxidized to formate by two metaboli c pathways (Figure 3-3). The first 12 pathway involves conversion of free formaldehyde to formate by the so-called low-K m (K m = 400 13 μM) mitochondrial aldehyde dehydrogenase-2 (ALDH2). The second pathway involves a two14 enzyme system that converts glutathione-conjugated formaldehyde (S-hydroxymethylglutathione 15 [HMGSH]) to the intermediate S-formylglutathione, w hich is subsequently metabolized to 16 formate and GSH by S-formylglutathione hydrolase. 17 18

19 20 Figure 3-3. Formaldehyde clearance by ALDH2 (GSH-i ndependent) and 21 ADH3 (GSH-dependent). 22 The K m value for ALDH2 and free formaldehyde is about 400 μM (Teng et al., 23 2001), whereas the K m value for HMGSH and ADH3 is 6.5 μM (Uotila and 24 Koivusalo, 1974a,b). The ADH-mediated reactions ar e reversible in the presence 25 of excess reduced nicotinamide adenine dinucleotide (NADH). 26 Source: Adapted from Teng et al. (2001). 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-21 Though ADH3 is rate limiting in this second pathway , the affinity of HMGSH for ADH3 1 (K m = 6.5 μM) is about 100-fold higher than that of fr ee formaldehyde for ALDH2. In addition 2 to the kinetic properties, this member of the ADH g ene family (Høøg et al., 2003, 2001; Liu et 3 al., 2001; Jornvall et al., 2000; Estonius et al., 1996) appears to be ubiquitously expressed in 4 organ tissues (Molotkov et al., 2002; Ang et al., 1 996a, b), exhibits cytoplasmic and nuclear 5 localization (Fernandez et al., 2003), and is the m ost abundant ADH family member in the liver 6 and brain (Galter et al., 2003). 7
In vitro studies have examined the clearance of for maldehyde in several human and rat 8 tissues (Table 3-2). Examination of formaldehyde m etabolism in the rat nasal and olfactory 9 mucosa indicates nearly identical pharmacokinetics in the rat liver on a per mg of cell lysate basis 10 (Casanova-Schmitz et al., 1984b). Similar results have been obtained in the absence of GSH, 11 where other ALDH family members oxidize formaldehyd e, albeit with significantly lower 12 affinity (i.e., higher K m ). Hedberg et al. (2000) demonstrated that human b uccal tissue lysate 13 kinetics are in close agreement with those reported for purified human liver ADH3 (Uotila and 14 Koivusalo, 1974a). Additionally, micro-array analy sis indicates that these cells express far more 15 ADH3 and S-formylglutathione hydrolase than ALDH1 o r ALDH2 (Hedberg et al., 2001a). The 16 results of Ovrebo et al. (2002) are not easily comp ared with the other studies in Table 3-2 17
because these studies were in intact cell cultures. However, it is apparent that the 18 pharmacokinetic values in these human cells are com parable to intact rat liver cells. 19
20 Table 3-2. Formaldehyde kinetics in human and rat tissue samples 21 22

Source K m (μM) V max (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 explants a 5,100 3.3 Ovrebo et al. (2002) Human bronchial epithelial a 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 These studies were carried out in intact cells by m easuring the formation of formate. This likely exp lains the nearly 23 1,000-fold increase in apparent K m , since much of the formaldehyde was likely to be b ound extracellularly. The 24 remaining studies used either purified enzyme or ce ll lysates (as indicated) and measured the formatio n of NADH. 25

b n.d. = not determined. 26This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-22 The data in Table 3-3 along with data indicating th e ubiquity of ADH3, indicate that 1 many human tissues and cells, particularly in the r espiratory tract, appear to exhibit significant 2 capacity to metabolize formaldehyde. Molecular bio logy techniques have demonstrated the 3 importance of ADH3 in formaldehyde clearance. For example, ADH-knockout studies have 4 shown that the median lethal dose (LD 50 ) values for formaldehyde in wild type, ADH1 −/−

, 5
ADH3 −/− , and ADH4 −/− mice strains were 0.200, 0.175, 0.135, and 0.190 g /kg, respectively 6 (Deltour et al., 1999). Although the statistical s ignificance was not reported, the data indicate 7 that deletion of ADH3 increases the sensitivity of mice to formaldehyde. 8 The pharmacokinetics of formate are complex. Forma te can undergo adenosine 9 triphosphate (ATP)-dependent addition to tetrahydro folate (THF), which can carry either one or 10 two one-carbon groups. Formate can conjugate with THF to form N 10 -formyl-THF and its 11
isomer N 5 -formyl-THF, both of which can be converted to N 5 ,N 10 -methenyl-THF and 12 subsequently to other derivatives that are ultimate ly incorporated into DNA and proteins via 13 biosynthetic pathways (Figure 3-4). 14 15

16 17 Figure 3-4. Metabolism of formate. 18 Note: 1, formyl-THF synthetase; 2, formyl-THF dehy drogenase. 19 20 Source: Adapted from Black et al. (1985). 21 22 23 Elevated levels of formate in urine have been detec ted following inhalation of methanol 24 or formate under certain conditions (Liesivuori and Savolainen, 1987), although the 25 interpretation of this finding is unclear. There i s also evidence that formate generates CO 2 – 26This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-23 radicals and can be metabolized to CO 2 via catalase and via the oxidation of N 10 -formyl-THF 1 (Dikalova et al., 2001, and references therein). T he significance of formate in formaldehyde 2 toxicity is unclear. Black et al. (1985) reported that hepatic tetrahydrofolate levels in monkeys 3 are 60% of those in rats and that primates are far less efficient in clearing formate than are rats 4 and dogs. Studies involving [ 14 C]-formate suggest that about 80% is exhaled as 14 CO 2 , 2–7% is 5 excreted in the urine, and about 10% undergoes meta bolic incorporation (Hanzlik et al., 2005, 6 and references therein). Mice deficient in formyl- THF dehydrogenase exhibit no change in LD 50 7 (via I.P. dose) for methanol or in oxidation of hig h doses of formate (Cook et al., 2001). It has 8 been suggested that rodents efficiently clear forma te via folate-dependent pathways, peroxidation 9 by catalase, or an unknown third pathway. Converse ly, primates do not appear to exhibit such 10 capacity and are more sensitive to metabolic acidos is following methanol poisoning (Cook et al., 11 2001). 12
13 3.4.2. Formaldehyde Exposure and Perturbation of M etabolic Pathways 14 The enzyme ADH3 has received renewed attention in r ecent years because of new 15
functions that have been attributed to it. ADH3 is central to the metabolism of formaldehyde; 16 however, exposure to formaldehyde in turn alters th e activity of ADH3 (in multiple dose17 dependent ways), thereby leading to perturbation of critical metabolic pathways. These are 18 briefly mentioned below (refer to cited papers for details). 19
1. Exposure to formaldehyde increases cell replication . These proliferating epithelial and 20 inflammatory cells are rich in both the messenger r ibonucleic acid (mRNA) and protein 21 of ADH3 (Nilsson et al., 2004; Hedberg et al., 2000 ). Studies in the rodent lung suggest 22 that increases in ADH3 in such cells dramatically a lter the biology of other important 23 ADH3 substrates that are involved in protein modifi cation and cell signaling (Que et al., 24 2005). 25
2. ADH3 also participates in the oxidation of retinol and long-chain primary alcohols, as 26 well as the reduction of S-nitrosoglutathione (GSNO ) (Staab et al., 2009; Thompson et 27 al., 2009; Hedberg et al., 2003; Høøg et al., 2003; Molotkov et al., 2002; Liu et al., 2001; 28 Jornvall et al., 2000; Jensen et al., 1998). The a ctivity of ADH3 toward some of these 29 substrates has been shown to be significantly incre ased in the presence of formaldehyde. 30 Staab et al. (2009) showed that (in cultured cells) GSNO can accelerate ADH3-mediated 31 formaldehyde oxidation and, likewise, that formalde hyde increases ADH3-mediated 32 GSNO reduction nearly 25-fold. The following effec ts may be noted with regard to the 33 relevance of such perturbations. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-24 a. GSNO is an endogenous bronchodilator and reservoir of nitric oxide (NO) activity 1 (Jensen et al., 1998). Details on the ADH3-mediate d reduction of GSNO are 2 shown in Thompson and Grafstrom (2008). 3
b. ADH3 is implicated in playing a central role in reg ulating bronchiole tone and 4 allergen-induced hyperresponsiveness (Gerard, 2005; Que et al., 2005). 5

c. As concluded by California Environmental Protection Agency (CalEPA) (2008), 6 “the dysregulation of NO by formaldehyde [in this m anner] helps to explain the 7 variety and variability in the toxic manifestations following formaldehyde 8
inhalation.” 9
10 3.4.3. Evidence for Susceptibility in Formaldehyde Metabolism 11 Teng et al. (2001) provided evidence that inhibitio n of ADH1, ALDH2, and ADH3 has 12 significant impact on formaldehyde toxicity. The a uthors speculated that deficiencies in any of 13 these enzymes would confer an increased susceptibil ity to formaldehyde toxicity (Teng et al., 14 2001). Polymorphism in ALDH2 has been shown to hav e implications in human risk 15 assessment, specifically with regard to acetaldehyd e metabolism (Ginsberg et al., 2002). It is 16 worth noting, however, that Teng et al. (2001) only demonstrated the importance of ALDH2 in 17 rat hepatocytes with formaldehyde concentrations of 2.5 mM and greater. Since this 18 concentration is fivefold greater than the 0.5 mM K m for free formaldehyde, ALDH2 19 involvement is not unexpected at such high concentr ations. Teng et al. (2001) also demonstrated 20 the importance of ADH1 in driving the reverse react ion (i.e., formaldehyde to methanol) by 21 coadministration of NADH-generators. This would ha ve the effect of prolonging the life of 22 formaldehyde by continuous recycling. This is not surprising, given that many ADH reactions 23 are reversible. However, levels of nicotinamide ad enine dinucleotide (NAD + ) are normally much 24 higher than NADH. 25 To date, two studies have reported polymorphisms in ADH3, using the new 26
nomenclature. 5 ADH3 transcription appears to be regulated by spe cificity protein (Sp1), with a 27 minimal promoter located at positions −34 to +61. The reported polymorphisms in ADH3 28 involve four base-pair substitutions in the promote r region and no polymorphisms in the coding 29 region (Hedberg et al., 2001b). The three polymorp hisms include −197/−196 (GG → AA), −79 30 (G → A), and +9 (C → T). The genotype frequencies are shown in Table 3- 3. Of these alleles, the 31 +9 (C → T) polymorphism (in the putative Sp1 minimal promot er region) reduced transcriptional 32

5 Other epidemiologic studies investigating links be tween ADH3 and oral cancer use the older nomenclatu re and thus refer to Class I ADH (i.e., ADH1) enzymes.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-25 activity twofold in in vitro reporter gene experime nts. According to Hedberg et al. (2001b), no 1 studies have demonstrated differences in ADH3 enzym e activity in humans. More recently, 2 single nucleotide polymorphisms in ADH3 have been r eported to be associated with childhood 3 risk of asthma, although the functional relevance o f these polymorphisms has not been published 4 (Wu et al., 2007). 5
6 Table 3-3. Allelic frequencies of ADH3 in human po pulations 7 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 Source: Adapted from Hedberg et al. (2001b). 10 11 12 Alterations in THF pathways may also have an impact on formaldehyde toxicity. These 13 could result from polymorphisms in various enzymes or differences in folate intake and 14 absorption. Species differences in tetrahydrofolat e levels (Black et al., 1985) are thought to play 15 a role in the differential responses to methanol ac ross species. Cook et al. (2001) speculate that 16 rats have redundant pathways for formate clearance that may be absent or less efficient in 17 primates. 18
19
3.5. EXCRETION 20 The main product of metabolic clearance of formalde hyde is formate, which is further 21
metabolized to CO 2 and water, incorporated into the one-carbon pool, o r eliminated in the urine. 22 There is also some evidence that formaldehyde is pr esent in exhaled breath; however, it is 23 unclear whether this originates from endogenous sou rces, or is simply a function of ambient 24 formaldehyde dissolved in fluids lining POEs. The following sections describe first experiments 25 in laboratory species and then available data in hu mans. Broadly, these studies address two 26 important questions that may be of relevance for ri sk assessment. First, it may be of interest to 27 know what levels of formaldehyde are exhaled for co mparison with inhaled levels, and whether 28 there is any relationship between external exposure and exhaled levels. Second, there are recent 29 studies that have attempted to relate genetic polym orphisms and changes in gene transcription 30 level to levels of putative urinary formaldehyde bi omarkers. 31
32
33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-26 3.5.1. Formaldehyde Excretion in Rodents 1 Heck et al. (1983) determined the relative contribu tions of various excretion pathways in 2 F344 rats following inhalation exposure to formalde hyde. Table 3-4 indicates that the relative 3 excretion pathways were independent of exposure con centration (at least between 0.63 and 4 15 ppm). Nearly 40% of inhaled [ 14 C]-formaldehyde appeared to be eliminated via expir ation, 5 probably as CO 2 (it should be recalled that nearly 100% of inhaled formaldehyde is absorbed). 6 Within 70 hours of a 6-hour exposure to formaldehyd e, about 17 and 5% were eliminated in the 7 urine and feces, respectively. Nearly 40% of inhal ed [ 14 C]-formaldehyde remained in the 8 carcass, presumably due to metabolic incorporation. 9
10 Table 3-4. Percent distribution of airborne [ 14 C]-formaldehyde in F344 rats 11 12 Concentration of formaldehyde (ppm) 0.63 13.1 Source 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 Values are means ± standard deviations (n = 4). 14 15 Source: Heck et al. (1983). 16
17 18 Mashford and Jones (1982) examined elimination path ways of formaldehyde in rats 19 exposed by I.P. injection. Urine and exhaled gases were collected from rats exposed to 4 or 20 40 mg/kg [ 14 C]-formaldehyde. At 48 hours postinjection, 82 and 78% of the radiolabel were 21
exhaled as 14 CO 2 , whereas exhaled [ 14 C]-formaldehyde was not detected. Mashford and Jon es 22 (1982) also further identified the urinary metaboli tes. Five hours after injection of the higher 23 dose, formate was determined to comprise 80% of the urinary metabolites. The authors were 24 unable to detect cysteine derivatives observed in o ther studies (see below) in the urine of these 25 rats prior to or after formaldehyde exposure. The authors stated that if formaldehyde were to be 26 excreted in urine containing cysteine, then thiazol idine-4-carboxylate (TZCA) would likely be 27 produced. They speculated that species differences in urinary compounds may produce 28 formaldehyde conjugates (or artifacts). 29 Hemminki (1982) reacted formaldehyde and acetaldehy de with cysteine, 30 N-acetylcysteine, and GSH and found that formaldehy de reacted most rapidly with cysteine to 31 form TZCA. Similarly, acetaldehyde reacted prefere ntially with cysteine, albeit slower than 32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-27 formaldehyde, to form a thiazolidine derivative. H owever, when each aldehyde was 1 administered I.P. (10% formaldehyde, 50% acetaldehy de), thioether concentrations (nmol/mol 2 creatinine) significantly increased in the 24 and 4 8 hour urine of acetaldehyde-treated rats but not 3 formaldehyde-treated rats. These data suggest that formaldehyde is not appreciably excreted in 4 urine and thus cysteine conjugates are not likely t o represent formaldehyde exposure. 5 Most recently, Shin et al. (2007) attempted to show that formaldehyde inhalation 6 increased urinary TZCA levels in Sprague-Dawley rat s. Treated rats were exposed to 3.1 and 7 38.1 ppm formaldehyde for 6 hours/day for 2 weeks, and urine was collected for 3 days. The 8 TZCA level in four control rats was 0.07 ± 0.02 mg/ L, whereas levels in the 3 and 38 ppm 9 groups were 0.18 ± 0.045 and 1.01 ± 0.36, respectiv ely. Notably, the concentrations in the four 10 highest exposed animals (0.71, 0.70, 1.20, and 1.43 ppm) exhibited a nearly twofold range. 11 However, these comparisons are confounded if the ex posures have any influence on urine 12 production and urine cysteine levels. The study doe s not provide any data that might allow one to 13
examine this issue. 14
15 3.5.2. Formaldehyde Excretion in Exhaled Human Bre ath 16 Several human and animal studies have attempted to measure the concentration of 17 formaldehyde in exhaled breath. None of the human studies were designed to distinguish 18 between exogenous (room air) and endogenous (system ic) formaldehyde in exhaled breath. In 19 order to discern whether endogenous formaldehyde is excreted into the lungs, test subjects must 20 breathe formaldehyde-free air. Because subjects we re breathing room air, which contained 9-10 21 ppb formaldehyde in two studies and unspecified con centrations in two other studies, it is 22 impossible to ascertain whether there was any endog enous formaldehyde in their exhaled breath. 23 One study demonstrates that the formaldehyde concen tration is lower in exhaled breath than in 24 inhaled breath (Cáp et al., 2008). Also, none of t he human studies investigated whether there is 25 any relationship between exhaled formaldehyde level s and food intake, life stage, smoking, or 26
health status. This assessment identifies a critic al research need for further studies on the 27 measurement of exhaled formaldehyde. 28 Proton transfer reaction mass spectrometry (PTR-MS) has been applied to measure trace 29 compounds in exhaled breath including volatile orga nics and specifically formaldehyde. The 30
basic method of PTR-MS is based on the transfer of protons from H 3 0 + to gases in exhaled breath 31 and the in-line monitoring of products where gases are tentatively identified by the mass to 32 charge ratio ( m/z ) where a m/z of 31 is consistent with protonated formaldehyde ( Hansel et al., 33 1995; Lindinger et al., 1998). It is important to note that reaction products from methanol and 34 ethanol may also produce fragments with an m/z ratio of 31 (Kusch et al., 2008). Up to 1% of the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-28 mass of ethanol and methanol in exhaled breath may be detected with an m/z ratio of 31 and thus 1 be identified as formaldehyde, so accurate quantita tion of formaldehyde should adjust for this 2 contribution (Špan ĕ l and Smith, 2008). Selected ion flow tube mass sp ectrometry (SIFT-MS) is 3 an application of PTR-MS developed for real-time an alysis of trace gases in breath (Smith and 4 Špan ĕ l, 2005; Špan ĕ l and Smith, 2007). 5 Moser et al. (2005) measured levels of 179 volatile organic compounds (VOCs) in the 6 exhaled breath of 344 individuals. This study was not designed to ascertain whether exhaled 7 formaldehyde is of endogenous origin, but rather to demonstrate that proton transfer reaction8 mass spectrometry can be used as a new method for r apid screening of large collectives for risk 9 factors (e.g., smoking behavior), potential disease biomarkers, and ambient air characterization. 10 The study was conducted at a health fair. The test subjects had a mean age of 61.6 years; 63% 11 were males and 14% were smokers. Samples of room a ir were collected and evaluated in parallel 12 with exhaled breath samples. The authors note that formaldehyde was detected in room air, but 13 did not report the levels; rather they stated that the background concentrations were negligible. 14 Of the 179 volatile organic compounds measured, dat a were reported for 14, including 15 formaldehyde and formic acid. The formaldehyde lev els in exhaled breath spanned from 1.2 to 16 72.7 ppb with a median of 4.3 ppb and 75 th percentile of 6.3 ppb. No explanation was provide d 17 for this wide range in values, and there was no dis tinction of the data by sex, age, health, or 18 smoking status. 19 Because the test subjects were breathing ambient in door air which contained an 20 unspecified concentration of formaldehyde, it is im possible to ascertain whether exhaled 21 formaldehyde was from an endogenous or exogenous so urce, or both. Moser et al. also note that 22 significant differences in exhaled breath compositi on could be found between smokers and non23 smokers for 32 of the 179 chemicals measured, but t he 32 chemicals were not named and no 24 substantiating data were provided. Formaldehyde ma y have been among these 32 chemicals 25 since it is a major component of cigarette smoke (N LM, 2001). 26 The report by Moser et al. does not provide the lim it of detection for any of the 27 compounds measured or details of the analytical met hod. The minimum formaldehyde level 28 reported in exhaled breath was 1.23 ppb. The metho d employed by Moser et al. does not adjust 29 the detection of apparent formaldehyde ( m/z ) by accounting for the contribution of methanol an d 30
ethanol. The levels of methanol and ethanol in exh aled breath were reported, however. Based on 31 1% of the mass of these chemicals contributing to t he m/z = 31 in the PTR-MS method (Špan ĕ l 32 and Smith, 2008), Table 3-5 demonstrates that the h ighest formaldehyde levels reported may 33 have been an artifact of high methanol and ethanol in exhaled breath. Because the in-room 34 formaldehyde concentrations were not reported, it i s unknown how much of this formaldehyde 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-29 represents formaldehyde levels in inhaled air. Add itionally, since these samples were simple 1 exhaled breath samples and not SIFT-MS samples, it is impossible to distinguish between air 2 which reached the pulmonary region versus air which only entered the upper airways. 3
4 Table 3-5. Apparent formaldehyde levels in exhaled breath of individuals 5 attending a health fair, adjusted for methanol and ethanol levels which 6 contribute to the detection of the protonated speci es with a mass to charge 7 ratio of 31 reported as formaldehyde ( m/z = 31) 8 9

Chemical Minimum 25 th percentile Median 75 th percentile 97.5 th

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 Source: Moser et al. (2005). 11 12 13 Turner et al. (2008) compared levels of volatile co mpounds in exhaled breath to levels in 14 emissions from the skin in five males (3 in their m id 20s, and the others 42 and 49 years old). 15 The subjects fasted overnight, and measurements wer e taken before and after ingesting 75 g of 16 glucose. The source of the inhaled air was laborat ory air which contained an unreported 17 concentration of formaldehyde. Formaldehyde was no t detected in the exhaled breath of any 18 subjects. The limit of detection was 5 ppb or bett er. 19 Wang et al. (2008) measured the concentrations of f ormaldehyde and 9 other chemicals in 20 the exhaled breath of three healthy male laboratory workers. The limit of quantification for 21 formaldehyde was not reported. A series of measure ments were taken in the nose and mouth, 22 and also in the oral cavity during breath holding. Table 3-6 presents the median formaldehyde 23 levels and geometric standard deviations for the th ree subjects. The authors noted that 24 formaldehyde in exhaled breath was at a level somew hat lower than the ambient air 25 concentration, but they could not be certain of its origin. 26
27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-30 Table 3-6. Measurements of exhaled formaldehyde co ncentrations in the 1 mouth and nose, and in the oral cavity after breath holding in three healthy 2 male laboratory workers 3 4

Subject Formaldehyde (median ppb / σ g) 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 Source: Wang et al. (2008). 6 7 8 Cáp et al. (2008) evaluated relationships between v olatile organic compounds measured 9
in exhaled breath and exhaled breath condensate. E xhaled breath condensate consists of 10 aerosolized particles of airway lining fluid evolve d from the airway wall by turbulent airflow that 11 serve as seeds for substantial water vapor condensa tion, which then serves to trap water soluble 12 volatile gases. This study also attempted to ascer tain whether the source of each compound was 13 endogenous or exogenous. According to the publishe d article and electronic communication 14 with Dr. Patrik Špan ĕ l, a co-author for this study, the limit of quantif ication was 3 ppb or better. 15 Measurements of formaldehyde in the direct exhaled breath of 34 subjects (25 to 62 years; 11 16 males; 2 smokers) varied from 0 to 12 ppb with a me an of 2 ppb and a median of 1 ppb. 17 Measurements of formaldehyde in exhaled breath cond ensate ranged from 0 to 12 ppb with a 18 mean of 2 ppb and a median of 0 ppb. Two smokers e xhaled formaldehyde concentrations of 0 19 and 3 ppb. The scatter plot in Figure 3-5 shows th at most subjects exhaled formaldehyde 20 concentrations of 0-3 ppb (x axis), which was consi derably lower than the ambient air 21 concentration of 9.6 ±1.5 ppb. The authors conclud ed that the exhaled concentrations were 22 influenced by exogenous levels in room air, which w ere not controlled. Dr. Špan ĕ l offered two 23 plausible explanations for why exhaled formaldehyde levels are lower than inhaled levels. He 24 noted that some subjects were tested within several minutes of entering the laboratory from 25 outdoors, so they may not have had time to acclimat e to the higher indoor air concentration. 26 Another explanation is that a substantial portion o f inhaled formaldehyde, which is highly 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-31 reactive, was retained in the respiratory tract and thus not exhaled; that is, 9.6 ppb of 1 formaldehyde was inhaled but only a mean of 2 ppb w as exhaled. 2 In this and other human studies, there was no adjus tment for an artifact in the analytical 3 method that makes it impossible to distinguish betw een formaldehyde and reaction products for 4 1% of exhaled methanol and ethanol because they hav e the same mass to charge ratio ( m/z = 31). 5 In fact, the concentration of methanol and ethanol that is misidentified as formaldehyde exceeds 6 the reported concentrations of exhaled formaldehyde . Thus, it is highly likely that the actual 7 exhaled formaldehyde concentration in Cáp et al. (2 008) was significantly lower than 2 ppb, and 8 that there was little or no endogenous formaldehyde in the exhaled breath. This would be 9 consistent with an animal study in which Mashford a nd Jones (1982) detected no exhaled 10 formaldehyde in rats injected I.P. with 40 mg/kg [ 14 C]-formaldehyde. Over 48 hours, 78% of the 11 radioactivity was exhaled as 14 CO 2 and 11% was excreted in the urine as formate, N12 (hydroxymethyl)urea, N,N’-bis-(hydroxymethyl)urea, and possibly polymethylene urea. In 13 summary, there are insufficient data at this time t o confidently establish a concentration of 14 formaldehyde in exhaled breath that can be attribut ed to endogenous sources. 15
16

17 Figure 3-5. Scatter plot of formaldehyde concentra tions measured in ppb in 18 direct breath exhalations (x axis) and exhaled brea th condensate headspace 19 (y axis)

. 20 21 Source: Cáp et al. (2008). 22 23 24 3.5.3. Formaldehyde Excretion in Human Urine 25 Gottschling et al. (1984) examined urinary formic a cid in 35 veterinary students. 26 Personal monitoring badges were worn and returned a fter class, and urine samples were taken 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-32 prior to class and within 2 hours after the class. Mean exposure levels were about 100 ppb. 1 Baseline averages of urinary formic acid (as a sodi um salt) were 12.47 mg/L and ranged from 2 2.43 to 28.38 mg/L among subjects. Postexposure fo rmate levels were slightly elevated but were 3 not statistically significant. Moreover, formate l evels decreased in several individuals relative to 4 pre-exposure levels. The authors concluded that va riability in urinary formate may mask any 5 changes and that monitoring formate within 2 hours of exposure is not informative. It is worth 6 noting, however, that interpretation of this findin g is confounded due to the fact that diet was not 7 controlled and because no markers for urinary norma lization were employed (Boeniger, 1987). 8 Boeniger (1987) reviewed previously published data on formate in urine (some of which 9 were in German). In one occupational study, worker s were exposed to an average formaldehyde 10 exposure of 1.28 mg/m 3 over a 6-hour work shift. This implies an average intake of 6 mg; 6 11 Boeniger reported a range of 2.5 to 13 mg. However , the original study reported that post-shift 12 formate levels were 152 mg/L, whereas the levels we re only 24 mg/L 6 days later (no exposure). 13 Considering that only a small percentage of inhaled formaldehyde would be excreted in urine, it 14 is unclear how (or whether) formaldehyde exposure, with the highest total dose of 13 mg, could 15 be responsible for the observed increase. 16 In the previously described study by Shin et al. (2 007), human urine samples were shown 17 to contain TZCA, although variability was not repor ted. A subsequent study reported that urine 18 TZCA levels were higher in individuals living in ne wer apartments (0.18 ± 0.121 mg/g 19 creatinine) as compared to older apartments (0.097 ± 0.040 mg/g creatinine) (Li et al., 2007) 7

. 20
The authors cited this as evidence that TZCA is a u rinary marker for formaldehyde exposure, 21 even though TZCA levels were not correlated to meas ured (or estimated) formaldehyde 22 exposures. The individuals also differed significa ntly in age (21.5 vs. 28.6, p = 0.053) and 23 differed in smoking percentage (10 vs. 27%). Clear ly these two studies do not establish a 24 relationship between human formaldehyde exposure an d urine TZCA levels. 25
26
3.6. MODELING THE TOXICOKINETICS OF FORMALDEHYDE A ND DPX 27
3.6.1. Motivation 28 Airway geometry is expected to be an important dete rminant of inhaled formaldehyde 29 dosimetry in the respiratory tract and its differen ces across species. The uptake of formaldehyde 30 in the upper respiratory tract is highly nonhomogen ous and spatially localized and exhibits strong 31 species differences. Species differences in kineti c factors have been argued to be the key 32

6 1.28 mg/m 3 / 1,000L/m 3 × 13.8L/minute × 60 minutes/hour × 6 hours.

7 This study is described in greater detail in Chapte r 5.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-33 determinants of species-specific lesion distributio ns for formaldehyde and other reactive inhaled 1 gases. In the first subsection here, the advantage gained in the quantitative risk assessment by 2 modeling these differences in the upper respiratory tract is explained. Such a model was 3 constructed by using computational fluid dynamic (C FD) methods for modeling airflow and 4 regional formaldehyde uptake in the rat, rhesus mon key, and human nose by scientists at the 5 Chemical Industries Institute of Toxicology (CIIT) Centers for Health Research (presently the 6 Hamner Institutes of Health Sciences). While frank effects were seen only in the upper 7 respiratory tract in rodents, mild lesions were als o present in the major bronchiolar region of the 8 rhesus monkey. Therefore, with regard to extrapola tion of cancer risk from animal bioassays to 9 humans, the upper and lower human respiratory tract should both be considered potentially at 10 risk of developing formaldehyde-induced squamous ce ll carcinoma. Therefore, formaldehyde 11 dose to the entire human respiratory tract human re spiratory tract was modeled in order to 12 develop a dose-response relationship that considere d the entire respiratory tract. Accordingly, in 13 the second subsection results of modeling airflow and regional formaldehyde uptake in the 14 human lower respiratory tract by Overton et al. (20 01) are provided. Unsteady effects were 15 argued to be insignificant at resting breathing rat es, and therefore steady-state inspiratory flow 16
was assumed. Since these models have been describe d in various reports and publications, the 17 technical details are consigned to appendices or th e literature is referenced. 18 The fluid dynamics modeling in the respiratory trac t comprises two steps (Kimbell et al. 19 2001): airflow through the lumen and formaldehyde u ptake by the lining of the respiratory tract. 20 Flow streamlines in the CFD simulations agreed reas onably well with experimentally observed 21 patterns in casts of the rat, monkey, and human nas al passages as well as with measurements of 22 velocity taken in hollow molds of the human nose. Pressure drop as a function of volumetric 23 flow also compared well with measurements made in r ats. Unlike the airflow simulations, no 24 validation of the regional formaldehyde uptake simu lations (that is, the spatial distribution of 25 uptake) was possible. (It was possible to compare s imulations of overall uptake with 26 experimentally observed values.) In this assessment , several indirect qualitative and quantitative 27 lines of evidence were relied on to provide general confidence in the formaldehyde regional 28 uptake profile in the F344 rat nasal passages. In addition to the agreement mentioned earlier with 29 respect to airflow profiles, this evidence includes general agreement between measured and 30 predicted levels of formaldehyde DPXs (in the “high -tumor” regions) when simulations of 31 airflow and regional formaldehyde uptake were used as input to a physiologically based 32 pharmacokinetic (PBPK) model for DPX kinetics (Cohe n-Hubal et al. 1997). Such indicators are 33 not available for the simulation of uptake patterns in the human. With regard to modeling the 34 lower respiratory tract, calculations in Overton et al. (2001) are based on an idealized 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-34 representation of airways in the human lung and of air flow through them (referred to in the 1 literature as “single-path” models). Overton et al. (2001) did not attempt to validate their 2 simulations. However, the following observation may be noted in support of their model. In the 3 case of the deposition of coarse and fine particula te matter, single-path models have traditionally 4 provided a reasonably accurate representation of th e average deposition in a given generation of 5 the lung airways (i.e. airways at a given depth in the lung) for a normal human population. 6
There were mass balance errors in the CFD calculat ions (Kimbell et al. 2001). Mass 7 balance errors associated with formaldehyde uptake into tissue ranged from less than 14% for the 8 rat, monkey, and human at resting minute volume to approximately 27% at the highest 9 inspiratory flow rates of 31.8 and 37 L/minute in t he human. 10 If DPX formation and cell proliferation are driven by formaldehyde flux, then these 11 quantities may also be expected to exhibit site and species differences, thus arguing for linking 12 these quantities to the modeled formaldehyde flux ( Conolly et al. 2000). 13
The third subsection evaluates PBPK models for DPX kinetics in the F344 rat and rhesus 14 monkey, using CFD simulations of formaldehyde flux to the nasal lining as input (Klein et al., 15 2009; Subramaniam et al., 2007; Conolly et al., 200 0), and discusses their uncertainties. This 16
subsection further discusses issues related to the scaling of the animal models to the human. 17 This assessment uses internal dose metrics comput ed by using the models described in 18 this section so as to derive more accurate human eq uivalent concentrations from the animal 19 bioassays than would be obtained by averaging over the respiratory surface area. The strengths 20
and uncertainties associated with the data and the models and their relevance to the hypothesized 21 mode of action are discussed in some length. 22
23 3.6.2. Species Differences in Anatomy: Consequence s for Gas Transport and Risk 24 As discussed earlier, formaldehyde is highly reacti ve and water soluble (categorized as a 25 category 1 gas), thus its absorption in the mucus l ayer and tissue lining of the upper respiratory 26 tract is known to be significant. The regional inh aled dose of formaldehyde to the respiratory 27 tract of a given species depends on the amount of f ormaldehyde delivered by inhaled air, the 28 absorption characteristics of the nasal lining, and reactions in the tissue. The amount delivered 29 by inhaled air is a function of the major airflow p atterns, air-phase diffusion, and absorption at 30 the airway-epithelial tissue interface. The dose o f formaldehyde to the epithelial tissue, which is 31 different from the amount delivered, depends on the amount absorbed at the airway-tissue 32 interface, water solubility, mucus-to-tissue phase diffusion, and chemical reactions, such as 33 hydrolysis, protein binding, and metabolism. It ha s been argued strongly that species differences 34 in these kinetic factors are determinants of specie s-specific lesion distributions for formaldehyde 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-35 and other inhaled gases (Moulin et al., 2002; Bogda nffy et al., 1999; Ibanes et al., 1996; 1 Monticello et al., 1996; Monticello and Morgan, 199 4; Morgan et al., 1991). 2 Because of the convoluted nature of the airways in the upper respiratory tract, the 3 absorption of such gases in the upper respiratory t ract is highly nonhomogeneous. There are 4 large differences across species in the anatomy of the upper respiratory tract (Figure 3-6) and in 5 airflow patterns (Figure 3-7). Therefore, as shown in the simulations in Figure 3-8, it may be 6 expected that the uptake patterns and thus risk due to inhaled formaldehyde will also show strong 7 species dependence. Morgan et al. (1991) concluded that airflow-driven dosimetry plays a 8 critical role in determining the site specificity o f various formaldehyde-induced responses, 9 including tumors, in the nose of the F344 rat. The convoluted geometry of the airway passages in 10 the upper respiratory tract, as seen from the cross sections of the nose in Figure 3-6, renders an 11 idealized representation of fluid flow and uptake p rofiles almost impossible. For these reasons, 12 Kimbell et al. (1993), Kepler et al. (1998), and Su bramaniam et al. (1998) developed 13 anatomically realistic finite-element representatio ns of the noses of humans, F344 rats, and 14 rhesus monkeys. These representations were subsequ ently used in physical and computational 15 models (Figure 3-6). This assessment utilizes dosi metry derived from these representations. 16 An accurate calculation of species differences in f ormaldehyde dosimetry in the upper 17 respiratory tract is important to the extrapolation problem for another reason. The upper 18 respiratory tract in rats is an extremely efficient scrubber of reactive gases (97% uptake) (Morgan 19 et al., 1986), thereby protecting the lower respira tory tract from gaseous penetration. On the 20 other hand, there is considerably more fractional p enetration of formaldehyde into the lower 21 respiratory tract of the rhesus monkey than in the rat (see Figure 3-8). Therefore, an accurate 22 determination of scrubbing in the upper respiratory tract is important to delineate species 23 differences in the level of risk to the lower respi ratory tract. Thus, in the case of the rhesus 24 monkey, the model by Kepler et al. (1998) included the trachea. Because the human model also 25 had to address the potential for oronasal breathing , an idealized single-path model of the lower 26 respiratory tract was attached to a model of the up per respiratory tract (Overton et al., 2001). It i s 27 important to note that the models mentioned above r epresent nasal passages reconstructed from a 28 single individual from each species (Kimbell et al. , 2001a, b; Conolly et al., 2000; CIIT, 1999; 29 Subramaniam et al., 1998). This is discussed later in the context of i ntraspecies variability. 30 The highly localized nature of uptake patterns show n in Figure 3-8 means that averaging 31 uptake over the entire nasal surface area would dil ute the regional dose over areas where 32 response was observed and that an extrapolation bas ed on such averaging would clearly not be 33

accurate. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-36 Figure 3-6. Reconstructed nasal passages of F344 r at, rhesus monkey, and 1 human. 2 3 Note: Nostril is to the right, and the nasopharynx is to the left. Right side shows 4 the finite element mesh. Left-hand side shows trac ings of airways obtained from 5 cross sections of fixed heads (F344 rat and rhesus monkey) and magnetic 6 resonance image sectional scans (humans). Aligned cross sections were 7 connected to form a three-dimensional reconstructio n and finite-element 8 computational mesh. Source: Adapted from Kimbell et al. (2001a). Additional 9 images provided courtesy of Dr. J.S. Kimbell, CIIT Hamner Institutes. 10This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-37 1 2 Figure 3-7. Illustration of interspecies differenc es in airflow and verification 3 of CFD simulations with water-dye studies. 4 Note: Panels A and B show the simulated airflow pat tern versus water-dye 5 streams observed experimentally in casts of the nas al passages of rats and 6 monkeys, respectively. Panel C shows the simulated inspiration airflow pattern, 7 and the histogram depicts the simulated axial veloc ities (white bars) vs. 8 experimental measurements made in hollow molds of t he human nasal passages. 9 Dye stream plots were compiled for the rat and monk ey over the physiological 10 range of inspiration flow rates. Modeled flow rate s in humans were 15 L/minute. 11 Source: Adapted from Kimbell et al. (2001a). 12This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-38 1

F344 Rat

Rhesus Monkey

Human Key pmol/(mm 2 -hr-ppm)

2000

1500

1000

500

0

2 Figure 3-8. Lateral view of nasal wall mass flux o f inhaled formaldehyde 3 simulated in the F344 rat, rhesus monkey, and human

. 4 5 Note: Nostrils are to the right. Simulations were exercised in each species at 6 steady-state inspiration flow rates of 0.576 L/minu te in the rat, 4.8 L/minute in the 7 monkey, and 15 L/minute in the human. Flux was con toured over the range from 8 0–2,000 pmol/(mm 2 -hour-ppm) in each species. 9 10 Source: Kimbell et al. (2001a). 11 12 13 Another factor to consider in the extrapolation is that monkeys and humans are oronasal 14 breathers while rats are obligate nose-only breathe rs. Thus, for humans and monkeys, oronasal 15 or oral breathing implies a significantly higher up take in the lower respiratory tract. It is known 16 that a significant fraction of the human population breathes normally through the mouth. Finally, 17 activity profiles are also determinants of extracti on efficiency (see Figure 3-9) and of breathing 18 route (Niinimaa et al., 1981). Given the fact that formaldehyde-induced lesions were observed as 19This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-39 far down the respiratory tract as the first bifurca tion of the lungs in exposed monkeys, the entire 1 human respiratory tract should be considered when e xtrapolating data from rats. 2
3

4
5 Figure 3-9. CFD simulations of formaldehyde flux t o human nasal lining at 6 different inspiratory flow rates. 7 8 Note: Right lateral view. Uptake is shown for the non-squamous portion of the 9 epithelium. The front portion of the nose (vestibu le) is lined with keratinized 10 squamous epithelium and is expected to absorb relat ively much less 11 formaldehyde. 12 13 Source: Kimbell et al. (2001b). 14 15This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-40 3.6.3. Modeling Formaldehyde Uptake in Nasal Passa ges 1
CIIT scientists chose the F344 strain of the rat si nce it was assumed to be anatomically 2 representative of its species and because it is wid ely used experimentally, most notably in 3 bioassays sponsored by the National Toxicology Prog ram and at CIIT. The approximate 4 locations of squamous, mucus-coated, and nonmucus e pithelial cells were mapped onto the 5 reconstructed nasal geometry of the CFD models. Ta ken together, these regions of nonmucus 6 and mucus-coated cells comprise the entire surface area of the nasal passages (see original papers 7 and CIIT [1999] for further details on reconstructi on and morphometry). Types of nasal 8 epithelium overlaid onto the geometry of the models were assumed to be similar in 9 characteristics across all three species (rat, monk ey, and human) except for thickness, surface 10 area, and location. Species-specific mucosal thick ness, surface area, and location were estimated 11 from the literature, from the documentation of the CFD models, or by direct measurements 12 (Conolly et al., 2000; CIIT, 1999). The nasal pass ages of all three species were assumed to have 13 a continuous mucus coating over all surfaces except specific areas in the nasal vestibule. As 14 discussed at the beginning of this chapter, formald ehyde hydrolyzes in water and reacts readily 15 with a number of components of nasal mucus. Absorp tion rates of inhaled formaldehyde by the 16 nasal lining were therefore assumed to depend on wh ere the epithelial lining is coated by mucus 17
and where it is not. 18 To calculate an airflow rate that would be comparab le among species, the amount of 19 inspired air (tidal volume, V T ) was divided by the estimated time involved in inh alation (half the 20 time a breath takes, or (1/2)(1/[breathing frequenc y, f]). Thus, an inspiratory flow rate was 21
calculated to be 2V T f, or twice the minute volume. Predicted flux valu es represent an average of 22 one nasal cycle. Minute volumes were allometricall y scaled to 0.288 L/minute for a 315 g rat 23 from data given by Mauderly (1986). Simulations we re therefore carried out at 0.576 L/minute 24
for the rat. 25 The fluid dynamics modeling in the respiratory trac t comprises two steps: modeling the 26 airflow through the lumen (solution of Navier-Stoke s equations) and modeling formaldehyde 27 uptake by the respiratory tract lining (solution of convective-diffusion equations for a given 28 airflow field). Details of these simulations, incl uding boundary conditions for air flow and mass 29 transfer, are provided in Kimbell et al. (2001a, b, 1993) and Subramaniam et al. (1998). 30 Formaldehyde absorption at the airway-to-epithelial tissue interface was assumed to be 31 proportional to the air-phase formaldehyde concentr ation adjacent to the nasal lining layer in 32 monkeys and humans (see the original paper [Kimbell et al., 2001a] for a more detailed 33
elaboration of the calculations for these coefficie nts). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-41 Because formaldehyde is highly water soluble and re active, Kimbell at al. (2001a) 1 assumed that absorption occurred only during inspir ation. Thus, for each breath, flux into nasal 2 passage walls (rate of mass transport in the direct ion perpendicular to the nasal wall per mm 2 of 3 the wall surface) was assumed to be zero during exh alation, with no backpressure to uptake built 4 up in the tissues. Overton et al. (2001) estimated the error due to this assumption to be small, 5 roughly an underestimate of 3% in comparison to cyc lic breathing. Also, this assumption is the 6
same as that used in default methods for reference concentration determination and has been used 7 in other PBPK model applications to describe nasal uptake (Andersen and Jarabek, 2001). 8
9
3.6.3.1. Flux Bins 10 A novel contribution of the CIIT biologically motiv ated dose-response model is that cell 11 division rates and DPX concentrations are driven by the local concentration of formaldehyde. 12 These were determined by partitioning the nasal sur face by flux, resulting in 20 “flux bins.” 13 Each bin was comprised of elements (not necessarily contiguous) of the nasal surface that receive 14 a particular interval of formaldehyde flux per ppm of exposure concentration (Kimbell et al., 15 2001a). The spatial coordinates of elements compri sing a particular flux bin were fixed for all 16 exposure concentrations, with formaldehyde flux in a bin scaling linearly with exposure 17 concentration (ppm). Thus, formaldehyde flux was e xpressed as pmol/(mm 2 -hour-ppm). 18
19
3.6.3.2. Flux Estimates 20 Formaldehyde flux was estimated for the rat, monkey , and human over the entire nasal 21 surface and over the portion of the nasal surface t hat was lined by nonsquamous epithelium. 22 Formaldehyde flux was also estimated for the rat an d monkey over the areas where cell 23 proliferation measurements were made (Monticello et al., 1991, 1989) and over the anterior 24 portion of the human nasal passages that is lined b y nonsquamous epithelium. Figure 3-8 shows 25 the mass flux of inhaled formaldehyde to the latera l wall of nasal passages in the F344 rat, rhesus 26 monkey, and human (Kimbell et al., 2001a). 27 Maximum flux estimates for the entire upper respira tory tract were located in the mucus28 coated squamous epithelium on the dorsal aspect of the dorsal medial meatus near the boundary 29 between nonmucus and mucus-coated squamous epitheli um in the rat, at the anterior or rostral 30 margin of the middle turbinate in the monkey, and i n the nonsquamous epithelium on the 31 proximal portion of the mid-septum near the boundar y between squamous and nonsquamous 32 epithelium in the human (see Kimbell et al. [2001b] for tabulations of comparative estimates of 33 formaldehyde flux across the species). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-42 The rat-to-monkey ratio of the highest site-specifi c fluxes in the two species was 0.98. In 1 the rat, the incidence of formaldehyde-induced squa mous cell carcinomas in chronically exposed 2 animals was high in the anterior lateral meatus (Mo nticello et al., 1996). Flux predicted per ppm 3 in this site and flux predicted near the anterior o r proximal aspect of the inferior turbinate and 4 adjacent lateral walls and septum in the human were similar, with a rat-to-human ratio of 0.84. 5
6
3.6.3.3. Mass Balance Errors 7 Overall uptake of formaldehyde was calculated as 10 0% x (mass entering nostril − mass 8 exiting outlet)/(mass entering nostril). Mass bala nce errors for air, 100% x (mass of air entering 9 nostril − mass exiting outlet)/(mass entering nostr il), and inhaled formaldehyde, 100% x (mass 10 entering nostril − mass absorbed by airway walls − mass exiting outlet)/(mass entering nostril), 11
were calculated. Mass balance errors associated wi th simulated formaldehyde uptake from air 12 into tissue ranged from less than 14% for the rat, monkey, and human at 7.4 and 15 L/minute to 13 approximately 27% at the highest inspiratory flow r ates of 31.8 and 37 L/minute (Kimbell et al., 14 2001b). Kimbell et al. (2001b) corrected the simul ation results for these errors by evenly 15 distributing the lost mass over the entire nasal su rface. 16
17 3.6.4. Modeling Formaldehyde Uptake in the Lower R espiratory Tract 18 Lesions were observed in the lower respiratory trac t of rhesus monkeys exposed to 6 ppm 19 formaldehyde. Therefore it is appropriate to consi der the human lower respiratory tract as 20 potentially at risk for formaldehyde-induced cancer . Accordingly, fluid flow and formaldehyde 21 uptake in the lower respiratory tract were also mod eled for the human in the CIIT approach by 22 using dosimetry estimates for the human lower respi ratory tract. 23 The single-path idealization of the human lung anat omy captures the geometrical 24 characteristics of the airways for a given lung dep th, and of airflow through these airways, in an 25 average, homogeneous sense. For particulates, this has provided a reasonable representation of 26 the average deposition in a given generation of the lung airways for a normal human population. 27 The one-dimensional model by Weibel (1963) is gener ally considered adequate unless the fluid 28 dynamics at airway bifurcations need to be explicit ly modeled. However, such an idealization of 29 the lung geometry has been successfully used in var ious models for the dosimetry of ozone and 30 particulate and fibrous matter. Most likely, the l ung geometries of the susceptible population, 31 such as those with chronic obstructive pulmonary di sease, would depart significantly from the 32 geometry described in Weibel (1963). Unlike the ac curate representation of the nasal anatomy 33 used in the CFD modeling, the lung geometry is idea lized in the CIIT approach as a typical path 34 Weibel geometry. This captures the lung structure in an average, homogeneous sense for a given 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-43 lung depth. The single-path model used to calculat e formaldehyde uptake in the human 1 respiratory tract (Overton et al., 2001; CIIT, 1999 ) applied a one-dimensional equation of mass 2 transport to each generation of an adult human symm etric, bifurcating Weibel-type respiratory 3 tract anatomical model, augmented by an upper respi ratory tract. The detailed CFD modeling of 4 the upper respiratory tract was made consistent wit h the upper respiratory tract in the single-path 5 model by requiring that the one-dimensional version of the nasal passages have the same 6 inspiratory air-flow rate and uptake during inspira tion as the CFD simulations for four daily 7 human activity levels. The reader is referred to O verton et al. (2001) for further details of the 8

simulations. Results most relevant to this assessm ent are shown in Figure 3-10. 9 10 Figure 3-10. Single-path model simulations of surf ace flux per ppm of 11 formaldehyde exposure concentration in an adult mal e human. 12 13 Source: Overton et al. (2001). 14 15 16 The primary predictions of the model, as shown in F igure 3-10, were that more than 95% 17 of the inhaled formaldehyde would be retained and f ormaldehyde flux in the lower respiratory 18 tract would increase for several lung airway genera tions from that in the posterior-most segment 19 of the nose and then decrease rapidly, resulting in almost zero flux to the alveolar sacs. 20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-44 Overton et al. (2001) modeled uptake at higher insp iratory rates, including those at 1 50 L/minute of minute volume (well beyond levels wh ere the oronasal switch occurs in the 2 normal nasal breathing population). At these rates Figure 3-8 indicates that formaldehyde flux in 3 the mouth cavity is comparable (but a bit less) to that occurring in the nasal passages. Overton et 4 al. (2001) did not model uptake in the oral cavity at minute volumes less than 50 L/minute. This 5
would be of interest because mouth breathers form a large segment of the population. 6 Furthermore, at concentrations of formaldehyde wher e either odor or sensory irritation becomes a 7 significant factor, humans are likely to switch to mouth breathing even at resting inspiration. At 8 a minute volume of 50 L/minute, Overton et al. (200 1) assumed, citing Niinimaa et al. (1981), 9 that 0.55 of the inspired fraction is through the m outh. Therefore, based on the results in 10 Figure 3-8, it is not unreasonable to assume that f or mouth breathing conditions at resting or light 11 exercise inspiratory rates, average flux across the human mouth lining would be comparable to 12 the average flux across the nasal lining computed i n Kimbell et al. (2001a, b). 13
14 3.6.5. Uncertainties in Formaldehyde Dosimetry Mod eling 15
3.6.5.1. Verification of Predicted Flow Profiles 16 The simulated streamlines of steady-state inspirati on airflow predicted by the CFD model 17 agreed reasonably well with experimentally observed patterns of water-dye streams made in casts 18 of the nasal passages for the rat and monkey as sho wn in panels A and B in Figure 3-7. The 19 airflow velocity predicted by CFD model simulations of the human also agreed well with 20
measurements taken in hollow molds of the human nas al passages (panel C, Figure 3-8) (Kepler 21 et al., 1998; Subramaniam et al., 1998; Kimbell et al., 1997, 1993). However, the accuracy and 22 relevance of these comparisons are limited. The pr ofiles were verified by video analysis of dye 23 streak lines in the molds of rats and rhesus monkey s, although this method is reasonable for only 24 the major airflow streams. 25 Plots of pressure drop vs. volumetric airflow rate predicted by the CFD simulations 26 compared well with measurements made in rats in viv o (Gerde et al., 1991) and in acrylic casts 27 of the rat nasal airways (Cheng et al., 1990) as sh own in Figure 3-11. This latter comparison 28 remains qualitative due to differences among the si mulation and experiments as to where the 29 outlet pressure was measured and because no tubing attachments or other experimental apparatus 30 were included in the simulation geometry. The simu lated pressure drop values were somewhat 31 lower, possibly due to these differences. 32 Inspiratory airflow was assumed to be constant in t ime (steady state). Subramaniam et al. 33 (1998) considered this to be a reasonable assumptio n during resting breathing conditions based 34
on a value of 0.02 obtained for the Strouhal number . Unsteady effects are insignificant when this 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-45 number is much less than one. However, this assump tion may not be reasonable for light and 1 heavy exercise breathing scenarios. 2
3

4 Figure 3-11. Pressure drop vs. volumetric airflow rate predicted by the CIIT 5 CFD model compared with pressure drop measurements made in two hollow 6 molds (C1 and C2) of the rat nasal passage (Cheng e t al., 1990) or in rats 7 in vivo (Gerde et al., 1991). 8 9 Source: Kimbell et al. (1997). 10 11 12 3.6.5.2. Level of Confidence in Formaldehyde Uptake Simulati ons 13 Unlike the airflow simulations, it was not possible to evaluate the formaldehyde uptake 14 calculations directly. Since the mass transfer bou ndary conditions were set by fitting overall 15 uptake to the average experimental data for various exposure concentrations, it was not possible 16 to independently verify even the overall uptake val ues with empirical data. This assessment has 17 relied on several indirect qualitative and quantita tive lines of evidence listed below to provide 18 general confidence in the uptake profile for the F3 44 rat nasal passages, as modeled in CIIT 19 (1999), when gross averages are considered over cer tain regions of the nasal lining. 20 In an earlier simulation, where the nasal walls wer e set to be infinitely absorbing of 21 formaldehyde, uptake of inhaled formaldehyde in the upper respiratory tract was predicted to be 22 90% in the rat for simulations corresponding to the resting minute volume in the F344 rat. This 23 estimate compared reasonably well with the range of 91–98% observed by Morgan et al. (1986a). 24This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-46 Morgan et al (1991) showed general qualitative corr espondence between the main routes 1 of flow and lesion distribution induced by formalde hyde in the rat nose. In their initial work with 2 a CFD model that represented a highly reactive and soluble gas, Kimbell et al. (1993) described 3 similarities in computed regional mass flux pattern s and lesion distribution due to formaldehyde. 4
When the results from this work in the coronal sec tion immediately posterior to the vestibular 5 region were considered, simulated flux levels over regions such as the medial aspect of the 6 maxilloturbinate and the adjacent septum (where les ions were seen) were an order of magnitude 7 higher than over other regions, such as the nasotur binate (where lesions were not seen). 8 8 The results of a PBPK model by Cohen-Hubal et al. ( 1997) provide a reasonable level of 9 confidence in regional uptake simulations for the F 344 rat when gross averages over nasal sites 10 are carried out. Cohen-Hubal et al. (1997) linked the CFD dosimetry model for formaldehyde to 11 a PBPK model for formaldehyde-DPX concentration in the F344 rat. This PBPK model was 12 calibrated by optimizing the model to combined DPX data from high-tumor incidence and low13 tumor incidence regions of the rat nose that were o btained in separate experiments by Casanova 14 et al. (1991, 1989). These data were obtained at 0 .3, 0.7, 2.0, 6.0, and 10 ppm for both regions 15 and also at 15 ppm for the high-tumor region. Mode l prediction of DPX concentrations for the 16 high-tumor region only compared well with the exper imental data, including 15 ppm for which 17
the model had not been calibrated. This is shown i n Figure 3-12. 18 The CFD simulations do not model reflex bradypnea, a protective reflex seen in rodents 19 but not in humans. As discussed at length in secti ons 3.2.3.1 and 4.2.1.1, it is reasonable to 20 expect a range of 25% (Chang et al., 1983) to 45% ( Barrow et al., 1983) decrease in minute 21 volume in F344 rats at the exposure concentration o f 15 ppm. Explicit omission of this effect in 22 the modeling is, however, not likely to be a source of major uncertainty in the modeled results for 23 uptake of formaldehyde in the rat nose for the foll owing reason. The CFD model for the F344 rat 24 was calibrated to fit the overall experimental resu lt for formaldehyde uptake in the F344 rat at 15 25 ppm exposure concentration. This was carried out b y adjusting the mass transfer coefficient used 26 as boundary condition on the absorbing portion of t he nasal lining. Thus, the reflex bradypnea 27 occurring in those experimental animals is phenomen ologically factored into the value used for 28 the boundary condition. Nonetheless, some error in the localized distribution of uptake patterns 29 may be expected, even if the overall uptake is repr oduced correctly. Furthermore, since the same 30

8 However, this 1993 CFD model differed somewhat fro m the subsequent model by Kimbell et al. (2001a) us ed in this assessment. In the 1993 model, the limiting m ass-transfer resistance for the gas was assumed to be in the air phase; that is, the concentration of formaldehyde w as 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 vesti bule was considered to be non-absorbing. Unfortunately, Kim bell at al. (2001a) did not report on correspondenc es between flux patterns and lesion distribution.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-47 value for the mass transfer coefficient was used in human simulations (as obtained from 1 calibration of the rat model), there is additional uncertainty in the modeled human flux estimates. 2 This issue was not addressed by Kimbell et al. (20 01a, b), Conolly et al. (2004), or Schlosser et 3 al. (2003), and we are unable to assess the extent of this error more accurately. 4

5 Figure 3-12. Formaldehyde-DPX dosimetry in the F34 4 rat. 6 Panel A: calibration of the PBPK model using data f rom high and low tumor 7 incidence sites. Panel B: model prediction compare d against data from high 8 tumor incidence site. Dashed line in panel A shows the extrapolation outside the 9 range of the calibrated data. 10 Source: Cohen-Hubal et al. (1997). 11This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-48 3.6.6. PBPK Modeling of DNA Protein Cross-Links (D PXs) Formed by Formaldehyde 1
3.6.6.1. PBPK Models for DPXs 2 As can be seen from the previous sections, measurin g the distribution of the absorbed 3 formaldehyde and identifying its form have proven d ifficult. Because of the high reactivity of 4 formaldehyde, rapid metabolism of formaldehyde, and complexity of formate clearance, dose 5 surrogates (or biomarkers) of exposure have been us ed to characterize the extent of absorption 6 and distribution of formaldehyde. As with other so luble and reactive gases, typical PBPK 7 models that predict steady-state blood concentratio ns are not useful for predicting formaldehyde 8 dosimetry at this time. As noted previously, inhal ation exposure to formaldehyde has not been 9 shown to increase blood formaldehyde levels. Thus, most modeling efforts for formaldehyde 10 have focused on disposition at the site of contact. 11 As discussed earlier, the concentration of DPXs for med by formaldehyde has been treated 12 as a surrogate for the tissue dose of formaldehyde in earlier efforts by Casanova et al. (1991) and 13 in EPA’s efforts to update its health assessment of formaldehyde (Hernandez et al., 1994). These 14 efforts used data from rats and rhesus monkeys (Cas anova et al., 1991, 1989). Using DPXs in 15 this manner allowed the incorporation of both clear ance and metabolism of formaldehyde and the 16 incorporation of the effect of saturation on detoxi fication of formaldehyde at higher doses. 17 Calculation of the average DPX concentration from t hese data was seen as a surrogate for the 18 area under the curve (AUC) of the reactive formalde hyde species in the epithelium. Based on 19 these data, Casanova et al. (1991) developed a PBPK model for predicting DPXs in these species 20 and for extrapolating to the human. 21 The Casanova et al. (1991) model consists of three anatomical compartments representing 22 different parts of the upper respiratory tract of t he rhesus monkey. The results indicated a 10-fold 23 difference in DPX formation between rats and monkey s, due primarily to species differences in 24 minute volume and differing quantities of DNA in th e nasal mucosa. Casanova et al. (1991) then 25 developed a monkey/rat scaling factor for these par ameters by taking the ratio of nasal mucosa 26 tissue between the two species, a determinant that was proportional to the total body weight 27 differences between the two species. Using these s caling factors in their model, the authors’ 28 predictions in monkey (based on the rat data) were in close agreement with observed DPXs in 29 monkey, particularly at higher formaldehyde concent rations. However, the model overpredicted 30 DPX formation in the monkey at lower formaldehyde c oncentrations. Subsequent rat-human and 31 monkey-human scaling results predicted much lower D PX formation in man. Again, the values 32 obtained at lower concentrations may have been over predicted, as was the case for rat-monkey 33 extrapolation. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-49 Georgieva et al. (2003) developed a model for the u ptake and disposition of formaldehyde 1 in the rat nasal lining. This model was designed t o predict the distribution of formaldehyde in 2 the nasal mucosa. The model indicated that, at 6 p pm exposure, a steady-state elevation of 15– 3 20 μM formaldehyde would be achieved within 30 seco nds. Furthermore, this same elevation 4 was predicted when the exposure was 6 ppm formaldeh yde for 60 minutes. Given that human 5 blood formaldehyde levels are predicted to be about 100 ± 15 μM (Heck et al., 1985) and 6 assuming that blood formaldehyde concentration is r oughly equivalent to the concentration 7 predicted at the basement membrane of the epitheliu m, this model predicts roughly a 15–20% 8 increase in blood formaldehyde. However, it should be noted that a 40-minute inhalation 9 exposure of humans to 1.99 ppm formaldehyde did not lead to a measurable increase in blood 10 formaldehyde (Heck et al., 1985). 11 Franks (2005) published a mathematical model for pr edicting the disposition of 12 formaldehyde in the human nasal mucosa and blood. The calculated concentrations of 13 formaldehyde in the mucus, the epithelium, and the blood attained steady-state profiles within a 14 few seconds of exposure. The increase of the forma ldehyde concentration in the blood was 15 predicted to be insignificant compared with the exi sting pre-exposure levels in the body: an 16 increase of 0.00044 mg/L in blood formaldehyde foll owing exposure to 1.9 ppm formaldehyde 17 for up to 8 hours. The model described formaldehyd e concentration gradients across the mucus, 18 epithelial, and submucosal compartments in the huma n nose. Transport of formaldehyde was 19 governed by the following processes: diffusional (i n the mucus); a combination of diffusional, 20 two first order terms representing intrinsic reacti vity of formaldehyde and binding to DNA, and 21 Michaelis-Menten kinetics representing enzymatic me tabolism (in the epithelial layer); a first22 order term representing non-enzymatic removal gover ned by the blood perfusion rate (in the 23 submucosal compartment). The model used the values for the first order reaction rate constants 24 and the Michaelis-Menten parameters (V max and K m ) estimated by Conolly et al. (2000) in their 25 model for extrapolating the rat and rhesus monkey d ata to the human. The modeling in Franks 26 (2005) was not calibrated or validated against expe rimental data, but the predictions of negligible 27 penetration of free formaldehyde to the blood are q ualitatively in agreement with the conclusions 28 in Heck et al. (1985). 29 Following the efforts by Casanova and co-workers, C ohen-Hubal et al. (1997), Conolly et 30 al. (2000), and Georgieva et al. (2003) developed m odels that linked local formaldehyde flux 31 from CFD models to DPX predictions. The focus here will be on the Conolly et al. (2000) effort 32 for the following two reasons: it explicitly incorp orates regional formaldehyde dosimetry in the 33 nasal lining by using results from CFD modeling of airflow and gas uptake and it brings data 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-50 across species (rat and rhesus monkey) to bear on m odel calibration, such a situation being 1 relatively rare in chemical health risk assessments

. 2
3
3.6.6.2. A PBPK Model for DPXs in the F344 Rat and Rhesus Mo nkey that Uses Local Tissue 4 Dose of Formaldehyde 5 In earlier risk assessment efforts (Hernandez et al ., 1994; Casanova et al., 1991; U.S. 6 EPA, 1991b), the average DPX concentration was cons idered a surrogate tissue dose metric for 7 the AUC of the reactive formaldehyde species. Cono lly et al. (2003) assigned a more specific 8 role for DPXs, treating local DPX concentration as a dose surrogate indicative of the intercellular 9 concentration of formaldehyde, leading to formaldeh yde-induced mutations. These authors 10
indicated that it was not known whether DPXs direct ly induced mutations (Conolly et al., 2003; 11 Merk and Speit, 1998). This is discussed in detail in the mode-of-action sections in this 12 document. The Conolly et al. (2000) model for the disposition of inhaled formaldehyde gas and 13 DPX in the rat and rhesus nasal lining is relativel y simple in terms of model structure because it 14 consists of a single well-mixed compartment for the nasal lining as follows: 15
1. Formaldehyde flux to a given region of the nasal li ning is provided as input to the 16 modeling and is obtained in turn as the result of a CFD model. This flux is defined as the 17 amount of formaldehyde delivered to the nasal linin g per unit time per unit area per ppm 18
of concentration in the air in a direction transver se to the airflow. It is locally defined as a 19 function of location in the nose and the inspirator y flow rate and is linear with exposure 20
concentration. 21
2. The clearance of formaldehyde from the tissue is mo deled as follows: 22

a. a saturable pathway representing enzymatic metaboli sm of formaldehyde, which 23 is primarily by formaldehyde dehydrogenase (involvi ng Michaelis-Menten 24 parameters V max and K m ) 25
b. a separate first-order pathway, which is assumed to represent the intrinsic 26 reactivity of formaldehyde with tissue constituents (rate constant k f ) 27

c. first-order binding to DNA that leads to DPX format ion (rate constant k b ) 28
3. The clearance or repair of this DPX is modeled as a first order process (rate constant 29
k loss ). 30
31
DPX data 32 DPX concentrations were estimated from a study by C asanova et al. (1994) in which rats 33 were exposed 6 hours/day, 5 days/week, plus 4 days for 11 weeks to filtered air (naive) or to 0.7, 34 2, 6, or 15 ppm (0.9, 2.5, 7.4, or 18 mg/m 3 ) formaldehyde (pre-exposed). On the 5th day of th e 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-51 12th week, the rats were then exposed for 3 hours t o 0, 0.7, 2, 6, or 15 ppm 14 C-labeled 1 formaldehyde (with pre-exposed animals exposed to t he same concentration as during the 2 preceding 12 weeks and 4 days). The animals were s acrificed and DPX concentrations 3
determined at two sites in the nasal mucosa. Conol ly et al. (2000) used these naive rat data to 4 develop a PBPK model that predicted the time-course of DPX concentrations as a function of 5 formaldehyde flux at these sites. 9 6
7
3.6.6.3. Uncertainties in Modeling the Rat and Rhesus DPX Da ta 8
3.6.6.3.1. Half-life of DPX repair. In the development of the PBPK model for DPXs, Cono lly 9 et al. (2000) assumed a value of 6.5 × 10 –3 minute –1 for k loss , the first-order rate constant for the 10 clearance (repair) of DPXs, such that the DPXs pred icted at the end of a 6-hour exposure to 11 15 ppm were reduced to exactly the detection limit for DPXs in 18 hours (the period between the 12 end of 1 day’s 6-hour exposure and the beginning of the next). This determination of rapid 13 clearance was based on an observation by Casanova e t al. (1994) that the DPX concentrations 14 observed in the pre-exposed animals were not signif icantly higher than those in naïve animals (in 15 which there was no significant DPX accumulation). However, in vitro data (Quievryn and 16 Zhitkovich, 2000) indicate a much slower clearance, with an average k loss of 17
9.24 × 10 –4 minute –1

. 18 Subramaniam et al. (2007) examined the Casanova et al. (1994) data and argued that 19 there was a significantly decreased (~ 40%) level of DPXs in high tumor regions of pre-exposed 20 animals vs. naive animals at 6 and 15 ppm and that the weight of the tissues dissected from those 21 regions increased substantially, indicating a thick ening of the tissues. After testing the outcome 22 of changing the tissue thickness in the PBPK model for DPXs, it was apparent to these authors 23 that such a change alone could not account for the dramatic reduction in DPX levels after pre24 exposure, even with the higher value of k loss used by Conolly et al. (2000). Therefore, in addi tion 25 to the gross increase in tissue weight, these data indicated either an induction in the activity of 26 enzymes that remove formaldehyde (aldehyde and form aldehyde dehydrogenase) or other 27 changes in the biochemical properties of the highly exposed tissue that must have occurred. 28 Given such a change, Subramaniam et al. (2007) conc luded that the experimental results in 29 Casanova et al. (1994) were consistent with the sma ller experimental value of k loss indicated by 30 the Quievryn and Zhitkovich (2000) data. In partic ular, they argued that if V max increased with 31 exposure (in a tissue region- and dose-specific man ner), then it was possible to explain the naïve 32

9 Note that Conolly et al. (2000) stated that they u sed the pre-exposed data, but this statement appear s to be in error (see Subramaniam et al. [2007]).This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-52 vs. pre-exposed data of Casanova et al. (1994), wit h the value of k loss effectively measured in 1 vitro by Quievryn and Zhitkovich (2000). Furthermo re, this value was measured directly, rather 2 than obtained by indirect interpretation of measure ments made at only two time points where 3 significant changes in the tissue had occurred. Th erefore, Subramaniam et al. (2007) considered 4
the use of this lower value for k loss to be more appropriate. Consequently, they re-imp lemented 5 and re-optimized the Conolly et al. (2000) model wi th this modification and found that the fit so 6
obtained to the acute DPX data was excellent. The re-implemented model will be used in this 7 assessment, and more details can be found in Subram aniam et al. (2007). 8 It should be noted that this slower DPX repair rate was obtained in an in vitro study by 9 using human cell lines that were transformed and im mortalized. However, it appears that DPX 10 repair in normal cells would be even slower. When non-transformed freshly purified human 11 peripheral lymphocytes were used instead, the half- life for DPX repair was about 50% longer 12 than in the cultured cells (Quievryn and Zhitkovich , 2000). 13
14
3.6.6.3.2. Statistical uncertainty in parameter estimates and extrapolation

. Klein et al. (2009) 15 developed methods for deriving statistical inferenc es of results from PBPK models. They used 16 the Conolly et al. (2000) model for this purpose, s pecifically because of the sparse time-course 17 information in the above DPX data. However, they u sed the value of k loss deduced from 18 Quievryn and Zhitkovich (2000) and fitted the model simultaneously to both the rat and rhesus 19 monkey data, as opposed to the sequential fitting i n Conolly et al. (2000). They found that the 20 predicted DPX concentrations were extremely sensiti ve to V max and tissue thickness as was also 21 concluded by Georgieva et al. (2003) and Cohen-Huba l et al. (1997). K m was seen to be 22 substantially different across species, a finding t hat was attributed plausibly to the involvement of 23 more than one enzyme (Klein et al., 2009; Georgieva et al., 2003). Klein et al. (2009) concluded 24 that the two efforts (Conolly et al. [2000] vs. Kle in et al. [2009]) resulted in substantially 25 different predictions outside the range of the obse rved data over which the models were 26
calibrated. 27 The differences between these models occur in spite of the fact that both methods use all the 28 available DPX data in both species and the same mod el structures. At the 0.1 ppm exposure 29 concentration, in general these authors obtained th ree- to fourfold higher DPX concentrations 30 averaged over a 24-hour period after exposure. Fur thermore, the standard deviations in Klein et 31 al. (2009) for V max and K m were an order of magnitude higher and that for k f was 35-fold lower 32 than the corresponding standard deviations reported in Conolly et al. (2000). The relatively 33 larger standard deviation for k f resulted in this parameter becoming negative in Co nolly et al. 34 (2000) at even half the standard deviation below th e maximum likelihood estimate (MLE) value. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-53 Note that, at a negative value of k f , formaldehyde would be produced as opposed to bein g cleared 1 through its intrinsic reactivity. 2 Klein et al. (2009) concluded that these “remarkabl e differences outside the range of the 3 observed data suggest caution in the use of these m odels in a predictive sense for extrapolating to 4 human exposures.” 5
6 3.6.7. Uncertainty in Prediction of Human DPX Conc entrations 7 Conolly et al. (2000) used both the rat and rhesus monkey data to predict human DPX 8
concentrations and constructed a PBPK model for the rhesus monkey along similar lines as for 9 the F344 rat. In the rhesus monkey model, they mai ntained the same values of k b , k loss , and k f

as 10 in the rat model but optimized the values of V max and K m against the rhesus monkey data from 11 Casanova et al. (1994). The rat and rhesus monkey parameters were then used to construct a 12 human model (see Conolly et al. [2000] for a more d etailed report of implementing the rhesus 13 monkey model and the extrapolations to the situatio n in humans). 14 For the human, the model used the value of K m obtained in the rhesus monkey model and 15 the epithelial thickness averaged over three region s of the rhesus monkey nose. The maximum 16 rate of metabolism, V max , which was estimated independently for the rat and rhesus monkey by 17 fitting to the DPX data available for these species , was then extrapolated to the human by 18 assuming a power law scaling with body weight (BW) (i.e., V max = a × BW b ), and the coefficient 19 “a” and exponent “b” were derived from the independ ently estimated values of (V max ) RAT and 20 (V max ) MONKEY . Table 3-7 gives the values of V max and K m in the Conolly et al. (2000) 21 extrapolation. 22 23 Table 3-7. Extrapolation of parameters for enzymat ic metabolism to the 24 human 25 26 Parameter F344 rat Rhesus monkey Human

V max (pmol/min-mm 3 ) 1,008.0 91.0 15.7
K m (pmol/mm 3 ) 70.8 6.69 6.69 27 Source: Conolly et al. (2000). 28 29 30 The extent of mechanistic data across species, as a vailable in this case, is rarely seen with 31 other chemicals, and the above scale-up procedure w as an attempt to use both the rodent and 32 primate DPX data. However, allometric relationship s across species are generally based on 33 regressing data from multiple species and usually m ultiple sources of data points. Thus, the 34 empirical strength of a power law derived by using two data points (F344 rat and rhesus monkey) 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 3-54 is extremely weak for use as an allometric relation ship that can then be used to extrapolate to the 1
human. 2 The following observations indicate the high level of uncertainty in the values of the 3 parameters V max and K m in the Conolly et al. (2000) models for predicting DPXs. First, K m 4 varies by an order of magnitude across the rat and monkey models but is then considered 5 invariant between the monkey and human models (Cono lly et al., 2000). Second, the values in 6 Conolly et al. (2000) for V max /K m , the low-dose limit of the rate of enzymatic metab olism, is 7 roughly similar between the rat and monkey but lowe r by a factor of six in the human. 8 Another factor that can substantially influence the above extrapolation of DPXs in the 9 human is that Conolly et al. (2000) assumed the tis sue to be a well-mixed compartment with 10 regard to formaldehyde interaction with DNA and use d the amount of formaldehyde bound to 11 DNA per unit volume of tissue as the DPX dose metri c. Considering formaldehyde’s highly 12 reactive nature, the concentrations of formaldehyde and DPX are likely to have a sharp gradient 13 with distance into the nasal mucosa (Georgieva et a l., 2003). Given the interspecies differences 14 in tissue thickness, there is consequent uncertaint y as to whether DPX per unit volume or DPX 15 per unit area of nasal lining is the more appropria te dose metric to be used in the extrapolation. 16 In particular, it may be assumed that the cells at risk for tumor formation are only those in the 17 epithelium and that measured DPX data (in monkeys a nd rats) are an average over the entire 18 tissue thickness. Since the epithelial DPXs in mon keys (and presumably humans) would then be 19 more greatly “diluted” by lower levels of DPX forma tion that occur deeper into the tissue than in 20 rats, it could be predicted that the ratio of epith elial to measured DPXs in monkeys and humans 21 would be much higher than the ratio in rats. 22
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# End of Volume I 24