DRAFT - DO NOT CITE OR QUOTE EPA/635/R-10/002 www.epa.gov/iris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 II of IV

Hazard Characterization

March 17, 2010

NOTICE

This document is an Inter-Agency Science Consultation draft . This information is distributed solely for the purpose of pre-dissemination peer re view under applicable information quality guidelines. It has not been formally disseminated by EPA. It does not represent and should not be construed to represent any Agency determination or policy. It is being circulated for review of its technical accuracy and science policy implic ations.

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 DRAFT—DO NOT CITE OR QUOTE II-ii DISCLAIMER 1

2 This document is a preliminary draft for review pu rposes only. This information is 3 distributed solely for the purpose of pre-dissemina tion peer review under applicable information 4 quality guidelines. It has not been formally disse minated by EPA. It does not represent and 5 should not be construed to represent any Agency det ermination or policy. Mention of trade 6 names or commercial products does not constitute en dorsement or recommendation for use. 7

8This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE II-iii CONTENTS—TOXICOLOGICAL REVIEW OF FORMALDEHYDE 1 (CAS No. 50-00-0) 2 3 4 LIST OF TABLES.....................................

................................................... ................................. xi LIST OF FIGURES ....................................

................................................... ............................... xx LIST OF ABBREVIATIONS AND ACRONYMS ................. .................................................. x xv 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 II-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 ...... ............................................... 3-31 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 II-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 ... ............................................... 4-32 4 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 ..... ................................................ 4-3 83 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 II-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 ........... ................................................ 4-4 08 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 II-vii CONTENTS (continued)

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

................................................... ............. 4-464 4.6.6.1. Cumulative Risk .......................... .................................................. 4 -464 4.6.6.2. Aggregate Exposure ....................... ............................................... 4-46 5 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 II-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 II-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 II-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 II-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 II-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 .............. ................................................4-1 48

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 II-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 II-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 II-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 ............... ................................................4-2 79

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 II-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) ................................4-3 44

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 II-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 II-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 II-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 II-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 II-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 II-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 II-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 ..................... ..............................................4-415

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 II-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 -109

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 II-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 II-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 II-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 II-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 II-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 II-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 II-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 II-xxxii 

### 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 4-1 4. HAZARD CHARACTERIZATION 1

2 3

4.1. HUMAN STUDIES 4 This chapter discusses epidemiologic studies of sit e-specific cancers and other adverse 5 health effects that may be caused by exposure to fo rmaldehyde. The primary focus is on the 6 literature describing inhalation exposure and its p otential carcinogenic and noncarcinogenic 7 health risks. In addition, oral, dermal, and ocula r exposures to formaldehyde are discussed. 8 The noncancer health effects section is organized b y endpoint, beginning with sensory 9 irritation and followed by pulmonary function, asth ma, respiratory tract pathology, immunologic 10 responses, neurological and behavioral responses, a nd, finally, developmental and reproductive 11

outcomes. 12 The carcinogenicity section is divided into two par ts, respiratory tract and non-respiratory 13 tract cancers. The first part discusses site-speci fic cancers that are chiefly located in the 14 respiratory tract where direct contact with formald ehyde occurs: nasopharyngeal cancers (NPCs), 15 nasal and paranasal cancers, other respiratory trac t cancers, and lung cancers. The second part 16 on non-respiratory tract cancer discusses those can cers at other sites with more distant exposure 17 to formaldehyde than respiratory epithelium—mainly, lymphohematopoietic (LHP) cancer, brain 18 and central nervous system (CNS) cancer, pancreatic cancer, and cancer at other sites. 19

20

4.1.1. Noncancer Health Effects 21

4.1.1.1. Sensory Irritation (Eye, Nose, Throat Irritation) 22 As a reactive gas, formaldehyde is a sensory irrita nt. Sensory irritation of the eyes and 23 respiratory tract by formaldehyde has been observed consistently in clinical and epidemiologic 24 studies in residential and occupational populations . Binding to sensory nerves at the portal of 25 entry (POE) results in direct sensory responses (e. g., detection of odor and tissue irritation) as 26 well as reflex responses to the sensory irritation and neurogenic sensitization. Reflex responses 27 result from CNS stimulation by the afferent sensory signals and include lacrimation, coughing, 28 sneezing, and bronchial constriction (BC). An addi tional reflex seen in rodents is reflex 29 bradypnea (RB) (also known as reflex apnea [RA]). Formaldehyde-induced sensory irritation 30 may be evident after acute exposures as well as in chronically exposed individuals. 31 Formaldehyde-induced neurogenic sensitization and a topy may result in lifelong health effects 32 from short-term or transient exposures. For this d iscussion, sensory irritation will include both 33 direct sensory response to formaldehyde exposure an d reflex responses (lacrimation, coughing, 34 sneezing, RB, and BC, and sensitization. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-2 Eye, nose, and throat irritation in response to for maldehyde inhalation exposure is well 1 documented (Doty et al., 2004). Broadly, studies e xamining these endpoints are either controlled 2 chamber studies with a defined population (e.g., he althy volunteers or sensitive individuals), 3 worker/student studies, or population (e.g., reside ntial) studies. Chamber studies, by design, are 4 acute studies, although some researchers have inves tigated repeated exposures. Occupational, 5 student, and residential exposures are generally lo nger duration, although there is variability in 6 exposure and duration among subjects. Endpoints in clude both local effects and reflex effects of 7 sensory irritation. The endpoints for assessing ir ritation include self-reporting of adverse 8 symptoms (e.g., pain, burning, itching) as well as objective measures of irritation (e.g., eye-blink 9 counts, lacrimation) (Doty et al., 2004). The foll owing review focuses on eye, nose, and throat 10 irritation but studies have documented other types of irritation, including dermal irritation 11

eczema and dermatitis. 12

13

4.1.1.1.1. Epidemiologic literature. A wide variety of epidemiologic studies have assess ed the 14 potential effects of exposure to formaldehyde on en dpoints, indicating sensory irritation of the 15 eye, nose, and throat. These studies generally inc lude three different types of exposure 16 populations: (1) Residents and visitors exposed to formaldehyde in homes and mobile buildings, 17 where formaldehyde is present from various sources, including building components, furniture 18 and home furnishings, heating and cooking combustio n as well as active and passive smoking; 19 (2) various occupational exposures from industrial processes related to wood products, furniture 20 making, and formaldehyde-based resins; and (3) anat omy students who are exposed under well21 defined conditions during academic courses where th ey are examining formaldehyde-preserved 22

cadavers. 23

24

4.1.1.1.1.1. Residential epidemiology. Among the residential epidemiology studies of 25 formaldehyde effects on sensory irritation, one of the strongest studies based on study design, 26 execution, analysis, and sample size was the observ ational study undertaken by Ritchie and 27 Lehnen (1987). In this cross-sectional study of ne arly 2,000 Minnesota residents living in 392 28 mobile and 494 conventional homes, personal data an d formaldehyde samples were collected 29 from residents that had responded to an offer by th e state health department to test homes for 30 formaldehyde. Technicians administered a symptom q uestionnaire to participating residents at 31 the time of formaldehyde sample collection. Reside nts were asked to close doors and windows 32 of their homes for 12 hours before testing was cond ucted, and a standardized collection protocol 33 was used for both sample collection and analysis. Measurements of formaldehyde exposure 34 were taken from two rooms of the home, usually the bedroom and living room, and were kept 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-3 refrigerated until analysis. Respondents were not aware of the results of the formaldehyde 1 analyses in their homes at the time they responded to the symptom questionnaire. The results 2 from Ritchie and Lehnen (1987) provide a clear dose -response relationship in the percentage of 3 residential occupants reporting eye, nose, and thro at irritation. Specifically, eye irritation 4 responses increase from 1–2% in homes with formalde hyde concentrations lower than 0.1 ppm 5 to 12–32% in homes with formaldehyde concentrations ranging from 0.1 to 0.3 ppm with 86– 6 93% of residents reporting . These effects were fo und in the same concentration range for 7 people living in either mobile (n = 851) or convent ional (n = 1,156) homes. Similar percentages 8 were found for nose/throat irritation. Reports of irritation were reported for smokers, passive 9 smokers, and nonsmokers with higher percentages of irritation among smokers, followed by 10 passive smokers and then nonsmokers. While the par ticipants in this study were self-selected 11 and not a random residential sample, a clear concen tration response was observed, and, even if 12 participants sought testing because they suspected that they were being exposed to formaldehyde 13 in their homes, they could not know the measured co ncentration of formaldehyde when reporting 14 their irritation symptoms, so recall bias cannot ex plain the concentration response. Neither can 15 confounding be an alternative explanation since the authors reported that formaldehyde was the 16 most important explanatory variable for all the sen sory irritation effects of the eye, nose, and 17

throat. 18 The results of an adverse association of sensory ir ritation with formaldehyde reported by 19 Ritchie and Lehnen (1987) are corroborated by Hanra han et al. (1984) who conducted a cross20 sectional survey by using a random sample of mobile homes from mobile home parks in 21 Wisconsin. Sixty-one teenage and adult residents p articipated. Health questionnaires were self22 administered by each occupant. Respondents were bl inded to the results of their home 23 formaldehyde vapor measurements, which were sampled from two rooms in the homes following 24 instruction to close windows, refrain from smoking, and turn off gas appliances for 30 minutes 25 prior to air sampling. Logistic regression analyse s were used to ascertain potential symptom risk 26 ratio dependency on each respondent’s age, smoking status, gender, and formaldehyde 27 concentration measures in the home. Formaldehyde c oncentrations ranged from 0.1 ppm to 28 0.8 ppm with a geometric mean of 0.16 ppm. Across this concentration range, a clear and 29 statistically significant concentration-response re lationship was reported in graphical form, 30 controlling for age, gender, and smoking status. A t 0.1 ppm, the regression model showed less 31 than 5% predicted prevalence of burning eyes. At 0 .2 ppm, the midpoint of the exposure 32 category in Ritchie and Lehnen (1987) that was repo rted to be the lowest adverse effect level for 33 eye irritation with 12–32% reporting eye irritation , the regression model of Hanrahan et al. 34 (1984) showed approximately 17.5% predicted prevale nce of burning eyes. The prevalence of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-4 burning eyes rose linearly to approximately 65% pre valence at 0.5 ppm, with some diminishment 1 in the rate of rise up to approximately 80% prevale nce at 0.8 ppm. While only 65 out of 208 2 randomly selected homes volunteered to complete the health questionnaires, the investigators 3 were able to complete home formaldehyde vapor measu rements on all the homes and reported 4 nearly an identical distribution of formaldehyde co ncentrations in participating and 5 nonparticipating homes. Demographic characteristic s of some of the non-respondents were 6 available and were reported as nearly identical to those of participants. There was no indication 7 of selection bias. Confounding is unlikely to expl ain such a strong concentration response. 8 These findings of associations of sensory irritatio n with residential exposures to 9 formaldehyde are further supported by studies that did not examine concentration response but 10 nonetheless assessed the association of formaldehyd e with sensory irritation. Similar findings to 11 those of Ritchie and Lehnen (1987) and Hanrahan et al. (1984) have been reported in other 12 residential studies of increased symptoms in associ ation with formaldehyde exposure (Liu et al., 13 1991; Thun et al., 1982; Dally et al., 1981). Dall y et al. (1981) collected data in 100 “complaint 14 structures” (65% mobile homes, 27% conventional hom es). Of these, 60% were from home 15 owners contacting the health department and 30% fro m physician referrals. Twenty percent of 16 the buildings had concentrations below the limit of detection (0.1 ppm), 20% had levels at or 17 above 0.81 ppm, and overall the concentrations rang ed from below detection to above 3 ppm 18 with an overall median of 0.35 ppm. The median lev els were 0.47 and 0.10 ppm for mobile and 19 conventional homes, respectively. No other contami nants were measured. Eye, nose, and throat 20 irritation were reported in a high percentage of oc cupants (eye irritation 68%, burning eyes 60%, 21 runny nose 60%, dry or sore throat 57%, cough 51%), but these were not reported as a function 22 of dose or home type. Thus, there was no control g roup to which rates of irritation could be 23 compared. However, symptoms reportedly stopped in 89% of occupants when they left the 24 “complaint structure.” The most recent residential study was performed on over 1,000 mobile 25 homes with 1,394 participants (Liu et al., 1991). Home formaldehyde concentration ranged from 26 below 0.01 to 0.46 ppm. Analyses used logistic reg ression to control for potential confounders. 27 Eye irritation was positively associated with forma ldehyde with a clear concentration response 28 demonstrated with cumulative exposure. During the summer and winter months, formaldehyde 29 exposure was associated with burning eyes. In the winter months, formaldehyde exposure was 30

associated with sore throat. There was no associat ion of formaldehyde exposure with cough or 31 running nose during either season. Liu et al. (199 1) also report a synergistic effect on irritation 32 by formaldehyde exposure and chronic disease preval ence. Thun et al. (1982) reported increased 33 symptoms of itchy skin and “wheezing and difficulty breathing” in residents in 395 homes 34 insulated with urea-formaldehyde foam relative to n earby homes without urea foam 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-5 formaldehyde insulation (UFFI); however, there were no measurements of formaldehyde 1 concentration taken in this study. 2 While not strictly a residential epidemiology study , Olsen and Dossing (1982) studied 3 occupational exposures within mobile and non-mobile daycare centers. They reported the mean 4 concentration in mobile and non-mobile day care cen ters were 350 (200–450) ppb and 65 (40– 5 90) ppb, respectively. Adverse eye, nose, and thro at irritation were significantly elevated in the 6 workers (n = 70) in the mobile units as compared wi th those in non-mobile units (n = 34). The 7 authors also state that a high percentage of worker s in the mobile day cares reported that the 8 symptoms disappeared after working hours; however, the authors did not report any such 9 percentages among those working in non-mobile units

. 10

11

4.1.1.1.1.2. Occupational epidemiology. Horvath et al. (1988) compared irritation symptoms 12 between 109 workers at a particleboard manufacturin g plant and 264 workers at food plants as a 13 control group. The mean 8-hour time-weighted avera ge (TWA) formaldehyde concentrations 14 between these two groups were 0.69 ppm (range 0.17– 2.93) and 0.05 ppm (range 0.03–0.12), 15 respectively. Eye, nose, and throat irritation wer e more common among the former group 16 (prevalence of symptoms during a work shift: throat sore or burning—test 22.0%, controls 3.9%; 17 cough—test 34.9%, controls 18.9%; burning of nose—t est 28.4%, controls 2.0%; stuffy nose— 18 test 33.9%, controls 14.2%; itching of nose—test 21 .1%, controls 7.9%; eyes burning or 19 watering—test 39.5%, controls 9.1%; eyes itching—te st 19.3%, controls 7.1%). 20 Similar results were reported for frequency of eye and nasal discomfort in a group of 21 workers involved in the manufacture of formaldehyde resins. These workers were exposed to a 22 mean concentration of 0.40 mg/m 3 . Alexandersson and Hedenstierna (1988) reported t hat the 23 frequency of eye, nose, and throat irritation was s ignificantly greater in 38 workers exposed to 24 formaldehyde and solvents in lacquers (average empl oyment duration 7.8 years) as compared 25 with 18 controls (nonexposed individuals working at the same factory). The frequency of eye 26 irritation was 65.8% among those exposed and 16.7% among controls. No controls reported 27 nose/throat irritation, but about 40% of those expo sed did. 28 A Swedish study conducted at a chemical plant found nasal and eye discomfort were 29 reported by 64 and 24%, respectively, of workers (n = 70) exposed to formaldehyde (range 0.05– 30 0.50 mg/m 3 with a mean of 0.26 mg/m 3 ) versus 25 and 6%, respectively, of the control g roup 31 made up of clerks from the local government (n = 36 ). In addition, the majority of workers 32 exposed to formaldehyde reported that their symptom s were relieved during weekends and 33 vacations (Holmström and Wilhelmsson, 1988). Anoth er study by the same authors 34 (Wilhelmsson and Holmström, 1992) reported similar results. In this study irritation prevalent 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-6 among 66 workers from a formaldehyde-producing plan t was compared with that seen among 1 36 community clerks. The workers were exposed to 0 .26 mg/m 3 of formaldehyde (range 0.05– 2 0.6 mg/m 3 ). The clerks were exposed to an average of 0.09 m g/m 3 . Nasal and eye discomfort 3 were reported at rates of 53 and 24%, respectively, among the workers. Among the community 4 clerks, 3 and 6%, respectively, reported discomfort

. 5 Holness and Nethercott (1989) reported significant increases in eye irritation (42 versus 6 21%) and nose irritation (44 versus 16%) among 84 f uneral service workers as compared with 7 38 controls (students and individuals from a servic e organization). The former group had been 8 actively embalming for approximately 10 years and h ad nearly twice the pack-years smoked as 9 the controls. The exposure concentration in both g roups was 0.36 and 0.02 ppm, respectively. 10

11

4.1.1.1.1.3. Epidemiology on laboratory students

. Several studies have monitored sensory 12 irritation in medical/physical therapy students exp osed to formaldehyde during anatomy courses. 13 These studies have particular advantages: the stude nt population generally has no former 14 occupational exposure, and, oftentimes, pre-class s urvey data serve as the control, providing a 15 better basis for assessing the effects of formaldeh yde exposure. 16 In a study of 24 formaldehyde-exposed anatomy stude nts (personal breathing zone 17 samples 0.73 ppm, range 0.49–0.93), the prevalence of eye irritation before the start of a cadaver 18 dissection class was 16%, while after the class, th e prevalence was 59%. The increase in eye 19 irritation was most pronounced, but increases were also observed in the prevalence of irritation 20 of the nose (21%) and throat (15%) (Kriebel et al., 1993). The authors also reported a tendency 21 for this increase in intensity between the beginnin g and end of class to diminish over the 22 10-week course, especially for eye irritation. How ever, although the intensity of the irritation 23 diminished, eye irritation was still present among the students after 10 weeks of intermittent 24 exposure. The report of increase in post- versus p re-class irritation symptoms in this study was 25 no greater for asthmatic students (n = 5) compared with non-asthmatic students. 26 Takahashi et al. (2007) showed that 143 medical stu dents reported various symptoms 27 (including eye and throat irritation) and that the percentage of students reporting symptoms 28 increased between the beginning and end of the cour se 2 months later. After the first day of 29 class, approximately 35% of students reported eye s oreness and about 15% reported throat 30 irritation. After the course ended, these rates we re close to 70% for eye soreness and slightly 31 above 40% for throat irritation. The reported aver age room formaldehyde concentration was 32 2.12 ppm (range 1.7–2.4), while the gas samplers wo rn on the students’ chests averaged 2.4 ppm 33 (range 1.8–3.8). Another study of students in an a natomy laboratory class in Japan (Takigawa et 34 al., 2005) measured formaldehyde concentrations and irritation symptoms before and after the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-7 installation of a ventilation system. This system reduced the median personal formaldehyde 1 exposure concentration from 2.7 to 0.72 ppm. Befor e installation of the ventilation system, the 2 students complained about exacerbation of all the s ensory irritation symptoms on average. The 3 increase in 8 out of 25 symptoms was significantly reduced after installing general ventilation 4 ( p < 0.05). After installation of the ventilation sys tem, a dose-dependent relationship with 5 formaldehyde was seen for irritated eyes but not fo r itchy nose. 6 Akbar-Khanzadeh et al. (1994) detected mean persona l area levels of formaldehyde at 7 1.24 ppm and a range of 0.1–2.94 ppm from personal air sampling devices. Almost 90% of the 8 students in this study reported eye irritation, 74% reported nose irritation, and close to 30% 9 reported throat irritation during or after exposure to formaldehyde during the laboratory period 10 after having completed at least 6 weeks of laborato ry sessions with formaldehyde exposure. In 11 addition, Uba et al. (1989) demonstrated that sympt oms of eye, nose, and throat irritation were 12 correlated with formaldehyde exposure among medical students by comparing students’ 13 responses on a questionnaire completed after a lab with formaldehyde exposure to a 14 questionnaire completed after a lab with no formald ehyde exposure. The authors compared 15 questionnaires completed prior to students’ first a natomy lab to a questionnaire completed 16 7 months later. Reports of cough were more frequen t after the 7 months. These students were 17 exposed to a mean level of 1.9 ppm (range 0.1–5.0) while dissecting (measured using portable 18 infrared spectrophotometer), and a TWA from all lab oratory activities ranged from below limits 19 of detection to 0.93 (measured using personal sampl ing devices in the students’ breathing zones). 20

21

4.1.1.1.2. Acute studies: controlled chamber exposures. Results from controlled human studies 22 demonstrate eye, nose, and throat irritation in ass ociation with formaldehyde exposure (Lang et 23 al., 2008; Yang et al., 2001; Krakowiak et al., 199 8; Kulle, 1993; Green et al., 1989, 1987; Kulle 24 et al., 1987; Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 25 1984; Bender et al., 1983; Anderson et al., 1983; W eber-Tschopp et al., 1977; Andersen, 1979; 26 Schuck et al., 1966). A key advantage of chamber s tudies is the ability to monitor and closely 27 control formaldehyde concentrations during exposure . However, chamber studies may also be 28 limited by other aspects of the study design, inclu ding small number of participants, use of 29 healthy volunteers, short exposure durations (a few minutes), and often studies were conducted 30 with only one exposure group and at relatively high concentrations (>1 ppm). The lack of 31 multiple exposure levels in many studies limits the understanding of exposure-response 32 relationships. Additionally, numerous reports that demonstrate multiple symptoms of eye, nose, 33 and throat irritation at levels at or above 1 ppm d id not explore lower levels of exposure and can 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-8 only be used for primary hazard identification (Yan g et al., 2001; Green et al., 1989, 1987; 1 Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 1984). 2 The National Aeronautical and Space Administration conducted experiments in closed3 environment living, including environmental monitor ing and air quality. James et al. (2002) 4 quantified air pollutants, including formaldehyde, during 30, 60, and 90-day tests in a closed 5 chamber study of a Lunar-Mars life support chamber. Unfortunately, the detection methods used 6 during the 30-day test were not sensitive enough to detect formaldehyde at levels below 2 7 mg/m 3 . Thus, badge samples were obtained in the 60-day and 90-day tests and provided greater 8 detection sensitivity (to 0.02 mg/m 3 ). Measured values of formaldehyde increased over time. In 9 the 60-day test, formaldehyde levels were well abov e accepted limits (data not shown). Health 10 effects data are limited since there were only four crew members. One crew member reported 11 eye and upper airway irritation at formaldehyde con centrations of 0.25 mg/m 3 (308 ppb) on day 12

15. It should also be noted that astronauts are ex ceptionally healthy individuals, and these data 13 should be interpreted carefully when determining ex pected health effects in the general 14 population. The experimenters determined that form aldehyde levels increased as temperature 15 increased. Formaldehyde was also linked to murals lining the chamber and was subsequently 16 removed before executing the 90-day study. Between days 0 and 60, formaldehyde levels 17 remained between 0.02 and 0.04 mg/m 3 , with one sharp peak that occurred at day three to 0.07 18 mg/m 3 . Between days 60 and 90, formaldehyde concentrati ons increased to 0.07 to 0.09 mg/m 3

. 19 The increase was attributed to an incomplete oxidat ion of methanol in a catalytic bed rather than 20 in excessive off-gassing of formaldehyde. No crew members reported any adverse effects in the 21 90-day study. 22 A few studies have been conducted that specifically address sensitive populations 23 (asthmatics) and/or individuals during exercise, wh ich can exacerbate asthma (further details of 24 these studies are in Section 4.1.1.3, Effects on As thmatics). In Sauder et al. (1986), 8-minute 25 bicycle exercise was completed multiple times durin g the exposure period (3 hours). However, 26 irritation symptoms were only reported after 2 hour s of exposure and do not address whether 27 changes occurred during the periods of exercise. O verall, reports of eye, nose, and throat 28 irritation increased with exposure to formaldehyde (3 ppm) compared with reports of irritation 29 with no exposure to formaldehyde. Green et al. (19 87) report that eye, nose, and throat irritation 30 symptoms were greater immediately after exercise du ring exposure to 3 ppm formaldehyde. 31 Additionally, the response levels were similar betw een asthmatic (n = 16) and non-asthmatic 32 (n = 22) subjects. Similar effects of exercise on certain symptoms, such as throat irritation, were 33 reported in 15 asthmatic subjects exposed to 2 ppm formaldehyde at rest and after exercise 34 (Witek et al., 1987). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-9 Kulle (1993) and Kulle et al. (1987) enrolled 19 he althy volunteers and exposed them to a 1 range of formaldehyde concentrations. At 2 ppm, 53 % reported mild or moderate eye irritation 2 (32% mild, 21% moderate). At 3 ppm, 100% of subjec ts exposed at this level (n = 9) reported 3 irritation. The reported increase in irritation wa s shown to correspond with increasing 4 formaldehyde concentration in a linear fashion. Mi ld nose/throat irritation was present among 5 37% of those exposed to 2 ppm of formaldehyde. Odo r detection was very similar to the 6 distribution seen for eye irritation. Nineteen sub jects performed light to moderate exercise while 7 exposed to 2 ppm; there was no increase in report o f eye irritation, but nose/throat irritation did 8 increase. The data were reanalyzed (Kulle, 1993), and thresholds for irritation were found to be 9 0.5–1 ppm for eye irritation and 1 ppm for nose/thr oat irritation. 10 Yang et al. (2001) reported that eight individuals exposed to varying levels of 11 formaldehyde (1.65, 2.99, and 4.31 ppm) had mild to moderate eye irritation during the 5-minute 12 exposures. The increase in irritation was detected at 30 seconds with exposure to 1.65 ppm of 13 formaldehyde. The highest severity ratings at this concentration occurred between 60 and 14 90 seconds. Frequency of eye blinking was also mea sured. The peak in blinking rate occurred 15 after about 1 minute of exposure and then decreased almost back to a normal rate after 5 minutes 16 of exposure. Higher formaldehyde concentrations we re associated with increased frequency of 17 blinking compared with the 1.65 ppm exposure. 18 Other studies have examined responses across multip le exposure levels. For example, 19 Weber-Tschopp et al. (1977) used two different meth ods of studying irritation resulting from 20 formaldehyde exposure. For one, they exposed subje cts (n = 33) to an increasing level of 21 formaldehyde (maximum exposure was 3.2 ppm). This design precluded evaluation of distinct 22 effects at different exposure levels. The research ers addressed this by examining another group 23 of subjects (n = 48) that were exposed to 0, 1, 2, 3, or 4 ppm five times for 90 seconds. Levels of 24

nasal and throat irritation for this discontinuous exposure were slightly higher than the irritation 25 levels reported among those with continuous exposur e. However, this was reversed for eye 26 irritation; those with continuous exposure reported higher levels of irritation than those with 27 discrete exposures. An objective measure, eye-blin king rate, was measured for those with 28 continuous exposure and was found to have a statist ically significant increase at 1.7 ppm. 29 Bender et al. (1983) conducted a study that enroll ed individuals who “responded” to 30 formaldehyde at 1.3 and 2.2 ppm and did not report irritation to the clean air control. They 31 found that, among these subjects, exposure to 1 ppm of formaldehyde (n = 27) resulted in the 32 reporting of eye irritation with a median response time of 78 seconds. Reports of irritation were 33 given as less than slightly irritating for formalde hyde concentrations of 0.3–0.9 ppm. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-10 Assessment of sensory irritation for pain and disco mfort often relies on self-reporting, 1 using symptom questionnaires and severity ratings ( e.g., mild, moderate, severe). In the case of 2 formaldehyde, subjective ratings of eye irritation correlate positively with eye-blinking 3 frequency (Lang et al., 2008). Lang et al. (2008) saw an increase in eye blinking after 4 195 minutes of exposure to formaldehyde at 0.5 ppm with four peak exposures of 1 ppm. After 5 this amount of time and formaldehyde exposure, ther e was also an increase in moderate eye 6 redness. Weber-Tschopp et al. (1977) reported that , among concentrations ranging from 0.03 to 7 3.2 ppm, eye-blinking frequency was increased at 1. 7 ppm; similarly Yang et al. (2001) reported 8 increased blinking at >1.5 ppm (the lowest concentr ation examined). There are studies that 9 suggest that psychological factors (e.g., anxiety) can impact the perception of irritation—and 10 perhaps more so at lower concentrations (Lang et al ., 2008; Ihrig et al., 2006; Dalton, 2003). 11 However, when Lang et al. (2008) controlled for moo d prior to exposure, subjective symptoms 12 of eye, nasal, and olfactory irritation were signif icantly related to exposure (0.5 ppm) 13 Schuck et al. (1966) performed a study that also ex amines self-reported eye irritation as 14 well as blinking rate. Fourteen individuals were e xposed to formaldehyde concentrations 15 ranging from 0 to 1 ppm. Increased irritation was reported with increasing formaldehyde 16 concentration. One subject, judged to be the least sensitive, was still able to detect formaldehyde 17 levels as low as 0.01 ppm. In addition, the author s examined the blinking rate of participants, 18 which they found was related to irritation intensit y. 19 Andersen (1979) and Anderson and Molhave (1983) rep orted on a controlled experiment 20 in which 16 individuals were exposed to varying lev els of formaldehyde for five hours and rated 21 their level of discomfort over the exposure period. Discomfort occurred within 1 hour at 22 formaldehyde exposure levels of 1 and 2 mg/m 3 (Andersen and Molhave, 1983) After 2 hours, 23 increasing discomfort was reported among the groups exposed to 0.3 and 0.5 mg/m 3 . Subject 24 reported that discomfort was mainly conjunctival ir ritation and dryness in the nose and throat. 25 Subjects complained at all four concentrations of f ormaldehyde: 0.3, 0.5, 1.0, and 2.0 mg/m 3 and 26 of 16 subjects, 3, 5, 15, and 15 subjects complaine d at each respective exposure concentration 27 (Andersen and Molhave, 1983). 28

Controlled chamber studies have also been conducted on various populations of 29 previously exposed individuals to determine if form aldehyde exposure potentiates an 30 individual’s response to acute exposures. Schachte r et al. (1987) reported on 15 laboratory 31 workers “frequently exposed to formaldehyde” (no qu antification of exposure is given; however, 32 the workers report being exposed for 1 to 7 days pe r week from a range of 1 to 21 years). Tests 33 performed at the start of the study found that thes e individuals had pulmonary function similar to 34 that seen in healthy individuals. The workers in t his study reported subjective measures of eye, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-11 nose, and throat irritation after 40 minutes of exp osure to 2 ppm of formaldehyde. However, the 1 2 ppm acute exposure in this study may be sufficien tly high to induce significant irritation in 2 most individuals. Krakowiak et al. (1998) reported that 10 asthmatics with occupational 3 exposure to formaldehyde (via formaldehyde solution s or pure gaseous formaldehyde) exhibited 4 similar symptom scores to healthy controls (never e xposed to formaldehyde in the workplace) 5 exposed to 0.4 ppm formaldehyde for 2 hours. The m ean symptom scores and standard 6 deviation (SD), which included information on sneez ing, rhinorrhea, mucosal edema, and 7 itching, were 4.6 ± 1.6 (mean ± SD) for asthmatics and 4.3 ± 1.2 for healthy subjects 8 immediately after inhalation. These dropped to 1.8 ± 1.2 and 1.2 ± 1.3, respectively, 4 hours 9 after the exposure. It is unclear if sensitive ind ividuals may not be represented in either of these 10 groups, as the workers were tolerating their exposu res during the work shift “healthy worker” 11 effect. However, residents (n = 9) exposed to form aldehyde in their homes, who complained 12 about adverse effects from the material, but with n o occupational exposure reported eye, nose, 13

and throat irritation at a similar rate as controls (individuals in homes without formaldehyde or 14 individuals in homes with formaldehyde but not repo rting adverse effects [n = 9]) after a 15 90-minute exposure to 1 ppm (Day et al., 1984). Th e number of individuals reporting eye 16 irritation, nasal congestion, and throat irritation were seven, three, and two among sensitive 17 individuals and eight, four, and three among contro ls, respectively. These individuals may be 18 considered a sensitive population since they had “p reviously complained of various 19 nonrespiratory effects from the UFFI in their homes ” (household concentrations unknown). 20

21

4.1.1.2. Pulmonary Function 22 Workers chronically exposed to formaldehyde have e xhibited signs of reduced lung 23 function consistent with BC, inflammation, or chron ic obstructive lung disease. Lung function 24 deficits have been reported both in pre-shift versu s post-shift measurements and as a result of 25 chronic exposures (Pourmahabadian et al., 2006; Her bert et al., 1994; Malaka and Kodama, 26 1990; Alexandersson and Hedenstierna, 1989; Alexand ersson et al., 1982). Decreases in 27 spirometric values, including vital capacity (VC), forced expiratory volume (FEV), forced vital 28 capacity (FVC), and FEV/FVC have been reported. Ch ronic studies also report increased 29 respiratory symptoms, such as cough, increased phle gm, asthma, chest tightness, and chest colds, 30 in exposed workers (Pourmahabadian et al., 2006; He rbert et al., 1994; Malaka and Kodama, 31 1990; Alexandersson and Hedenstierna, 1989; Alexand ersson et al., 1982). Similar findings 32 have been reported for low-level residential formal dehyde exposure, including decreased peak 33 expiratory flow (PEF) rates (Krzyzanowski et al., 1 990). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-12 Worker exposures that report cross-shift difference s in spirometric values are consistent 1 with formaldehyde-induced sensory irritation. Addi tionally, concordance has been reported 2 between subjective irritant response and measured c hanges in pulmonary function, further 3 supporting the possibility that cross-shift and sho rt-term evidence of BC may be a reflexive 4 response to sensory irritation. Absolute values fo r lung function parameters are likely to vary by 5 gender, age, height, and smoking status and are bes t compared when normalized to the expected 6 lung function based on these variables (Schoenberg et al., 1978). Individual variation can also be 7 addressed by each subject serving as his/her contro l with measurements taken before, during, and 8 after exposure. Analysis of the percent change in various parameters in this context may have 9 greater sensitivity to detect exposure-related chan ges in function. 10

In addition to individual variation in baseline lun g function, there is variation in bronchial 11 responsiveness. Reduced lung function parameters i n response to methacholine challenge is a 12 standard test for BC, and this can be used to defin e responsive, sensitive, or susceptible 13 individuals. Since formaldehyde-induced BC is meas ured with these lung function tests, 14 variability in bronchial responsiveness may impact interpretation of formaldehyde-induced 15 changes. Experiments with sensitive individuals ca n help address this question. However, 16 results need to be normalized in some way to accoun t for differences in responsiveness before 17 formaldehyde exposure. Researchers have in some ca ses excluded hyperresponsive individuals 18 or presented results as a proportion or percent of the unexposed value for each individual. 19 However, excluding sensitive individuals may bias r esults towards the null. 20 The American Thoracic Society (ATS) published an o fficial statement on what 21 constitutes an adverse health effect of air polluti on (ATS, 2000). According to the ATS 22 statement, exposure that increases the risk of an a dverse effect to the entire population can be 23 considered adverse, even though it may not increase the risk of any individual to an unacceptable 24 level. For example, a population of asthmatics cou ld have a distribution of lung function such 25 that no individual has a level associated with sign ificant impairment. Exposure to an air 26 pollutant could shift the distribution to lower lev els that still do not bring any individual to a 27 level that is associated with clinically relevant e ffects. However, this would be considered to be 28 adverse because individuals within the population w ould have diminished reserve function and 29 therefore would be at increased risk if affected by another agent. 30

31

4.1.1.2.1. Epidemiologic literature. The potential adverse effects of formaldehyde expos ure on 32 pulmonary function in humans can be examined on sev eral time scales of interest. The 33 epidemiologic literature supports the assessment of exposures among exposed anatomy medical 34 students where all participants have well-defined a nd similar duration of exposure (i.e., a 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-13 semester-long class) (Kriebel et al., 2001, 1993; A kbar-Khanzadeh and Mlynek, 1997; Akbar1 Khanzadeh et al., 1994; Uba et al., 1989; Fleisher, 1987), among individuals living or working in 2 buildings with formaldehyde exposure (Franklin et a l., 2000; Krzyzanowski et al., 1990; Main 3 and Hogan, 1983), and among workers (industrial, ma nufacture, mortuary, hospital staff, etc.) 4 (Ostojic et al., 2006; Herbert et al., 1994; Khamga onkar and Fulare, 1991; Malaka and Kodama, 5 1990; Nunn et al., 1990; Alexandersson and Hedensti erna, 1989; Holness and Nethercott, 1989; 6 Holmström and Wilhelmsson, 1988; Horvath et al., 19 88; Kilburn et al., 1985; Alexandersson et 7 al., 1982). 8 The observed effects in the previously unexposed an atomy students provide additional 9 information on acute exposures in two naïve populat ions (Kriebel et al., 2001, 1993) as well as 10 special insight into the intermediate stages of pos sible sensitization (Kriebel et al., 1993). 11 Kriebel and colleagues (1993) examined the pre-labo ratory and post-laboratory PEF in students 12 attending anatomy classes once per week. They foun d the strongest pulmonary response when 13 examining the average cross-laboratory decrement in PEF in the first 2 weeks of the study when 14 formaldehyde concentrations collected in the breath ing zones had a geometric average 15 concentration of 0.73 ppm. Overall, the students e xhibited a 2% decrement in PEF, while the 16 students with any history of asthma showed a 7.3% d ecrement in PEF. These findings of acute 17 decreases in PEF following students’ initial anatom y sessions were corroborated by the Kriebel 18 et al. (2001) study, which used a similar study des ign applied to another class of anatomy 19

students. 20 The Kriebel et al. (1993) study also shows how the acute effects of formaldehyde 21 exposure were altered following several weeks of we ekly episodic exposure. By the fifth week 22 of class, the pre- and post-laboratory measurements of PEF were no longer reflecting a clearly 23 demonstrated acute effect, but, following the seven th week of episodic exposure, both pre- and 24 post-laboratory PEF continued to drop steadily unti l the class adjourned after 10 weeks. While 25 the acute effects of formaldehyde exposure appeared to diminish after several weeks of exposure, 26

the intermediate effect across 9 weeks was a 24 L/m inute drop in PEF that was statistically 27 significant (

p < 0.01 after statistical control for random person effects, asthma, interaction 28 between time and asthma, and eye as well as nose sy mptoms of irritation). 29 Similar studies among medical students have been pe rformed. In one study, 34 exposed 30 and 12 control students completed pulmonary functio n tests before and after their work in the 31 laboratory (approximately 3 hours) (Akbar-Khanzadeh et al., 1994). The time-weighted average 32 exposure ranged from 0.07-2.94 ppm. More than 94% of the subjects were exposed to >0.3 ppm 33 and 31.7% were exposed to >0.5 ppm. Comparing pre- and post-exposures among the exposed 34 students, on average FVC decreased by 1.4%, FEV 3 decreased by 1.2%, FEV 1 /FVC increased by 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-14 1.6%, and FVC 25–75% increased 2.5%. These average percent changes in the control group are – 1 0.3, 1.30, 2.31, and 0.6%, respectively. The resea rchers also calculated correlation coefficients 2 by examining the relationship between lung function and formaldehyde concentration, but no 3 association was found. Akbar-Khanzadeh and Mlynek (1997) performed another study with 50 4 exposed students and 36 controls and reported a lar ger increase in lung function among controls 5 when compared with cases after 1–3 hours of exposur e (FVC 3.0 versus 0.9, FEV 1 4.1 versus 6 1.2, FEV 3 3.3 versus 0.8, forced expiratory flow during the middle of the FVC [FEF 25–75% ] 6.1 7 versus 0.7). These differences between cases and c ontrols remained for FEV 1 , FEV 3 , and FEF 25– 8

75% after 3 hours. 9 A study of 103 medical students was performed over a period of 7 months in which the 10 students were exposed to formaldehyde at a time wei ghted average of <1 ppm with peaks of 5 11 ppm during anatomy laboratory sessions (Uba et al., 1989). Twelve students were asthmatics. 12 Unlike the studies by Kriebel et al. (2001, 1993), these researchers did not find a change in 13 pulmonary function over the course of 7 months. Th e mean percent change for pulmonary 14 function before and after the exposure did change s lightly, with measures showing decreases in 15 function at the end of the laboratory session (meas urements taken at the 7-month time point: 16 FVC –0.79%, FEV 1 –0.48%, FEF 25–75% 0.07%, FEV 1 /FVC 0.24%). 17 Finally, Fleisher (1987) gave self-administered que stionnaires to medical students after 18 completing an anatomy laboratory session (formaldeh yde exposure measures as <1 ppm) and a 19 pathology/microbiology laboratory session (no forma ldehyde exposure). Over 8% of students 20 reported experiencing shortness of breath during th e laboratory with formaldehyde exposure, but 21 none of the students reported shortness of breath i n the laboratory session with no exposure. No 22 objective measurements of formaldehyde exposure wer e used. 23 Three studies have been performed that examine form aldehyde exposure from the 24 buildings in which individuals live or work. One s tudy included children 6–13 years of age and 25 measured the levels of formaldehyde in their homes. There was no association between FVC or 26 FEV and the indoor concentrations of formaldehyde, although there were signs of lower airway 27 inflammation as measured by levels of exhaled nitri c oxide (NO) in children exposed to average 28 formaldehyde levels ≥ 0.05 ppm (Franklin et al., 2000). Municipal employ ees with their children 29 (613 adults and 298 children) were randomly sampled in another study of home exposures 30 (Krzyzanowski et al., 1990). Residential exposures to formaldehyde were based on repeated 31 samples from each individual’s kitchen, living area , and bedroom. The average formaldehyde 32 concentration was 26 ppb, with a maximum sample val ue of 140 ppb. The majority of subjects 33 (83%) lived in homes with 2-week average concentrat ions below 40 ppb. Subjects’ peak 34 expiratory flow rates (PEFRs) were determined four times daily, in the morning, at noon, in the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-15 early evening, and before bed, for 2 weeks. A stat istically significant linear relationship between 1 increased formaldehyde exposure and decreased PEFR was reported in children but not adults. 2

All statistical models controlled for socioeconomic status, tobacco smoking (current active or 3 environmental tobacco smoking), and nitrogen dioxid e concentrations. Among adults, there was 4 a statistically significant nonlinear relationship with decreased morning PEFR for formaldehyde 5 concentration <40 ppb. 6 Main and Hogan (1983) reported on a group of indivi duals (n = 21) working in two 7 mobile trailers for 34 months and exposed to levels of formaldehyde ranging from 0.12 to 8 1.6 ppm (mean age 38 ± 9 years, 76% male, 19% nonsm okers). The control population was 9 comprised of individuals who did not work in the tr ailers (n = 18; mean age 30 ± 6, 50% male, 10 22% nonsmokers). There were no differences between the exposure and control groups’ percent 11 predicted FEV 1 or FVC regardless of smoking status. 12 Several studies allowed for the examination of pote ntial chronic effects of formaldehyde 13 exposure. These included an occupational study by Malaka and Kodama (1990) that reported 14 pre-shift pulmonary function as a percentage of exp ected among the formaldehyde exposed 15 compared with comparable people not exposed to form aldehyde. This study found that an 16 average 8-hour TWA formaldehyde exposure of 1.13 pp m from area samples was associated 17 with statistically significant decrements in FEV 1 , FEV 1 /FVC, and FEF 25–75% compared with a 18 referent population. The strongest response was fo r FEF 25–75%, which showed a 12% drop in 19 observed function compared with expected function i n the unexposed, but it is unclear how to 20 interpret the potential chronic adverse effect(s) w ith just the magnitude of the decrement and the 21 length of the average occupational tenure at this p lywood facility (6.5 years), which was not 22 reported by exposure status. 23 A study comparing oriented strand board workers (ex posed to formaldehyde) with oil/gas 24 field plant workers (not exposed to formaldehyde) d emonstrated a difference in pulmonary 25 function between the two groups (Herbert et al., 19 94). The groups were similar in regard to 26

measured FVC and FEV 1 (controlled for age, height, and smoking), but the workers exposed to 27 formaldehyde had lower FEV 1 /FVC. In addition, those exposed to formaldehyde s howed a 28

decrease in FVC and FEV 1 after their shift, with an average pre- and post-s hift difference of 29 47 mL (

p = 0.022) and 39 mL (

p = 0.044) for FVC and FEV 1 , respectively (however change 30 could not be compared with the controls of this stu dy because no post-shift measurements were 31 taken). Two other occupational studies found no as sociation between formaldehyde and lung 32 function (Holness and Nethercott, 1989; Horvath et al., 1988). One of these studies was 33 conducted among funeral workers and an unexposed co ntrol group (Holness and Nethercott, 34 1989). There was no difference in pulmonary functi on of the two groups at baseline. After 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-16 exposure, there was no change in lung function for the exposed or unexposed when comparing 1 lung function tests done immediately before and aft er an embalming procedure (for controls the 2 repeat measures were taken approximately 2–3 hours after the first measure) (changes in 3 percentage predicted FVC and FEV 1 were 0.88 ± 2.95 and –0.03 ± 2.40 for exposed and 1.13 ± 4 3.98 and 1.45 ± 4.43 for unexposed). Further analy sis showed no association between 5 formaldehyde levels and changes in lung function. Another study (Horvath et al., 1988) found 6 no differences in pre-shift pulmonary function betw een the exposed (workers at a particleboard 7 and molded products operation, formaldehyde measure d using individual monitors ranged from 8 0.17–2.93 ppm) and controls (workers from nearby fo od processing facilities, formaldehyde 9 measured using individual monitors ranged from 0.03 –0.12 ppm). However, the authors did find 10 a post-shift decline in FVC and FEV 1 among controls and FEV 1 and FEF 25–75% among workers 11 when using paired comparisons for each group. When assigning all controls a formaldehyde 12 exposure value of 0.05 ppm, there was a correlation detected in pre-and post-shift pulmonary 13 function changes and formaldehyde, though no specif ic details on regression analysis were 14 provided. 15 A study performed in India (Khamgaonkar and Fulare, 1991) examined individuals 16 working in anatomy and histopathology departments a nd exposed to formaldehyde (mean 17 1.00 ppm, range 0.036–2.27). Controls (individuals not working in laboratories with 18 formaldehyde) were exposed to an average of 0.102 p pm formaldehyde (range 0–0.52). Lung 19 function tests were performed on a Monday morning a fter days of no exposure in order to 20

examine chronic effects. The FVC and FEV 1 % of the exposed group, respectively, were 17.12 21 and 22.94% reduced compared with the control group (Khamgaonkar and Fulare, 1991); 22 however, while the pool of cases and controls were frequency-matched on age and gender, there 23 was no mention by the investigators of normalizing the pulmonary function metrics by gender 24 and height, which would have made for more appropri ate comparisons. Kilburn et al. (1985) 25 also demonstrated reduced pulmonary function (lower percent predicted FVC, FEV 1 , and 26

FEF 25-75% ) among workers occupationally exposed to formaldeh yde when compared with 27 individuals working at jobs without formaldehyde ex posure. 28 Two occupational studies found no association betwe en formaldehyde exposure and 29 deficits in pulmonary function (Ostojic et al., 200 6; Holmström and Wilhelmsson, 1988). 30 Ostojic et al. (2006) examined nonsmoking male heal th service professionals working in 31 pathoanatomic laboratories with 8 hours of formalde hyde exposure per day at an unspecified 32 concentration for at least 4 years (n = 16). The c ontrol group was comprised of sixteen age- and 33 stature-matched nonsmoking male controls. There wa s no difference in mean FVC or FEV 1 34 between exposed and controls. The researchers also examined values for diffusing lung capacity 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-17 for carbon monoxide and membrane diffusion capacity , which were similar between the exposed 1 and control groups. However, blood volume of pulmo nary capillaries was found to be higher in 2 the exposed group. Holmström and Wilhelmsson (1988 ) recruited individuals from a chemical 3 plant where formaldehyde and formaldehyde products were made (n = 70). Exposure levels 4 varied from 0.05–0.5 mg/m 3 . A control group was mostly comprised of clerks f or the local 5 government (n = 36). No difference in FEV% was det ected between the groups. Mean FVC was 6 lower than expected among the exposed group (expect ed values were based on age, sex, smoking 7 habits, height, and weight). This study went furth er and measured changes in pulmonary 8 function for those employed more than 5 years and r eported no signs of increasing restrictivity 9 after 5 years. No associations were seen and there was no correlation between pulmonary 10 function and cumulative dose of formaldehyde (Holms tröm and Wilhelmsson, 1988). 11 There have been only two studies that have reported on the longitudinal follow-up of 12 workers exposed to formaldehyde (Nunn et al., 1990; Alexandersson and Hedenstierna, 1989). 13 The Alexandersson and Hedenstierna (1989) investiga tion not only examined the acute effects of 14 exposure across shift but was able to do so among s ome of the same workers that had been 15 studied 5 years earlier (Alexandersson et al., 1982 ). Statistically significant decreases in 16

FEV 1 /FVC and FEF 25–75% were noted over the intervening 5 years in nonsmok ers after correction 17 for normal aging and reference lung function spirom etry values. The decrease in FEF 25–75% was 18 0.212 ± 0.066 L/second (mean ± SD) for each year of exposure and was highly significant ( p < 19 0.01). For comparison with the 12% drop in the sam e pulmonary metric reported by Malaka and 20 Kodama (1990) over an estimated 6.5 years, the extr apolated percentage decrease in FEF 25–75% 21 was computed for the Alexandersson and Hedenstierna (1989) study by using the reported yearly 22 decrement applied to the pre-shift values at the ti me of the initial study period. From the 23 predicted value of 4.57 L/second, a decrease of 0.1 68 L/second for each year of exposure 24 regardless of smoking status was calculated. For 6 .5 years of exposure, this would result in a 25 24% drop in FEF 25–75% . Formaldehyde concentrations were estimated at 0. 42 ppm in the first 26 Alexandersson et al. (1982) study and at 0.50 ppm i n the Alexandersson and Hedenstierna (1989) 27 study. The study by Nunn et al. (1990) assessed th e decrease in FEV 1 with age. The researchers 28

calculated the decrease in FEV 1 to be 42 mL/year among workers exposed to formalde hyde and 29 41 mL/year for workers who were not exposed to form aldehyde. Thus, they showed no 30 association between formaldehyde exposure and decre ased FEV 1

. 31 There are a few important limitations to consider i n these occupational studies of 32 formaldehyde exposure. First, an often-shared weak ness is the absence of data on, and 33 appropriate statistical control of, potential confo unding by occupational co-exposures. Also, 34 studies that did not report pre-shift pulmonary fun ction as a percentage of expected function 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-18 contribute less to an assessment of potential chron ic effects because, post-hoc, it is difficult to 1 calibrate the multiple pulmonary function data for cross-study comparison without knowledge of 2 the age, gender, smoking status, height, year of bi rth, etc., that are important determinants of the 3 pulmonary function metrics of concern. 4

5

4.1.1.2.2. Acute studies: controlled chamber exposures. Pulmonary effects of acute 6 formaldehyde exposure have been studied in both hea lthy volunteers and sensitive populations 7

under controlled conditions. Controlled chamber st udies have the advantage of measured 8 controlled exposures, but other factors can limit t he usefulness of the studies, especially when 9 study populations are small and there is high varia bility in the measured parameters. 10 In general, acute formaldehyde exposures (0.5–3 pp m) have not induced significant 11 pulmonary deficits in healthy, non-exercising volun teers (Kulle et al., 1987; Schachter et al., 12 1986; Witek et al., 1986; Day et al., 1984; Anderse n and Molhave, 1983). However, it is unclear 13 whether the data analysis in these reports had the statistical power to substantiate the small 14 deficits reported in occupational and student studi es. All four reports had relatively small study 15 groups of healthy individuals (n = 19 [Kulle et al. , 1987], n = 16 [Andersen and Molhave, 1983], 16 n = 15 [Schachter et al., 1986], n = 15 [Witek et a l., 1986], and n = 9 [Day et al., 1984]), and in 17 some cases the group was further divided by gender. Two studies report the absolute values of 18 the lung function parameters without adjustment to individual expected function or the 19 unexposed baseline for each individual (Kulle et al ., 1987; Andersen and Molhave, 1983). As 20 discussed, this decreases the power of the study to detect formaldehyde-induced changes in 21 pulmonary function. In contrast, Witek et al. (198 6) and Schachter et al. (1986) report lung 22 function as a percent of baseline (although not nor malized for age gender and height). Each 23 study showed an increase in FEV 1 in formaldehyde-exposed individuals at rest and in creases in 24 maximal expiratory flow (MEF) at 50% of expired vit al capacity (MEF50%) (Witek et al., 1986; 25 Schachter et al., 1986). However, in both reports the SDs of changes in lung function parameters 26 are quite large, nearly equaling the reported value and exceeding it in several cases. The absence 27 of normalized raw data, combined with large individ ual variation, limit the interpretation of these 28 studies. A small study (Day et al., 1984) that inc luded nine healthy individuals showed no 29 changes in FEV or FVC after 90 minutes of exposure to 1 ppm of formaldehyde. A more recent 30 study (Lang et al., 2008) of 21 healthy volunteers exposed to a range of formaldehyde 31 concentrations (0.0 to 0.15 or 0.3 ppm) reports no formaldehyde-related pulmonary deficits. 32 However, data were not shown, and it is unclear whe ther the authors compared absolute or 33 relative values of lung function and what variation in lung function was present in the study 34 population. Additionally, the authors did not prov ide the criteria used to gauge deficit of lung 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-19 function. If a clinically significant deficit was defined (e.g., 20%), then more subtle changes in 1 pulmonary function, as supported in other studies, would not have been reported. 2 Similar to these studies of healthy individuals, a cute controlled studies including 3 asthmatics also report no changes in pulmonary func tion associated with formaldehyde exposure 4 (Ezratty et al., 2007; Harving et al., 1990; Green et al., 1987; Sauder et al., 1987; Witek et al., 5 1987, 1986), including studies of individuals thoug ht to have formaldehyde-induced bronchial 6 asthma (Krakowiak et al., 1998); however, the numbe r of asthmatic individuals included in each 7 of these studies was small. (The details of these studies have been reported elsewhere in this 8 chapter. Briefly, the number of asthmatics in the study/total number of individuals in the study 9 are as follows: Ezratty et al. [2007]—12/12; Harvin g et al. [1990]—15/15; Green et al. [1987]— 10 16/38; Sauder et al. [1987]—9/9; Witek et al. [1987 ]—15/15; Witek et al. [1986]—15/30; 11 Krakowiak et al. [1998]—10/20]. The same is true f or individuals who are frequently exposed to 12 formaldehyde either at work (n = 15) (Schachter et al., 1987) or at home (n = 18) (Day et al., 13 1984). 14 Small but statistically significant deficits in pu lmonary function due to acute 15 formaldehyde exposure (2 or 3 ppm) have been report ed in healthy volunteers during exercise 16 (Green et al., 1989, 1987; Sauder et al., 1986; Sch achter et al., 1986). Although changes in lung 17 function parameters averaged over experimental grou ps were generally small, some individuals 18 exhibited clinically significant deficits, even aft er only 2 hours of exposure. Deficits in FEV 1 19

and FEF 25–75% in the first 30 minutes of a 2-hour exposure at 3 ppm formaldehyde were 2 and 20 7%, respectively. Changes in lung function were no t statistically significant after 60 and 21 180 minutes of exposure (Sauder et al., 1986), even when assessed as absolute rather than 22 relative measurements. Thirteen percent (5 of 38 s ubjects) demonstrated formaldehyde-induced 23 clinically significant deficits when exposed at 3 p pm during exercise (defined by Green et al. 24 (1987) as decrease in FEV 1 > 10% of control). 25

26

4.1.1.3. Asthma 27 A large number of studies have investigated the pot ential association between 28 formaldehyde exposure and a continuum of adverse he alth effects ranging from decrements in 29 pulmonary function to asthma. In general, epidemio logic studies of adults have reported varied 30 results between null findings and positive findings . However, The National Research Council 31 concluded in its report on Formaldehyde that, “Form aldehyde has been shown to cause bronchial 32 asthma in humans” (NRC, 1981), citing numerous stud ies demonstrating the induction of asthma 33 following exposure to formaldehyde (Hendrick and La ne, 1975, 1977; Laffont and Noceto, 1961; 34 Nova and Touraine, 1957; Paliard et al., 1949; Popa et al., 1969; Sakula, 1975; Schoenberg and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-20 Mitchell, 1975; Turiar, 1952; Vaughan, 1939). In a subsequent review article on formaldehyde 1

and the health effects that have been associated wi th it, Stenton and Hendrick (1994) reported on 2 formaldehyde and asthma in occupational settings an d starkly describe the “...first detailed case 3 report of formaldehyde asthma confirmed by specific inhalation challenge test occurring in a 4 nursing sister on a renal dialysis unit. Her sympt oms were suggestive of late asthmatic reactions 5 occurring 4 to 5 hours after heavy exposures. The occurrence of late reactions was confirmed in 6 a series of challenge tests that involved the paint ing of formalin onto cardboard pieces within a 7 confined space” (Stenton and Hendrick, 1994; Hendri ck 1997). The results of the challenge tests 8 are illustrated in Figure 4-1 . 9

10

11 12

13 14 Figure 4-1. Delayed asthmatic reaction following t he inhalation of 15 formaldehyde after “painting” 100% formalin for 20 minutes. Challenge 2 16 was premedicated with inhaled betamethasone 200 μg. 17 Source: (Stenton and Hendrick, 1994) 18

19 Five years later, the two nurses were re-challeng ed with the nurse who had left the 20 dialysis unit having no response to the subsequent challenge while the nurse who had remained 21 working in the unit developed mild late asthmatic r esponse with peripheral blood eosinophilia 22 (Stenton and Hendrick, 1994; Hendrick et al., 1982) . Stenton and Hendrick (1994) concluded 23 that these studies “provide clear evidence of forma ldehyde’s ability to induce asthma” but no 24 indication of the exposure concentrations to induce it. In a follow-up study of dialysis unit 25This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-21 staffers exposed to formaldehyde as a sterilizing a gent, 8/28 people reported respiratory 1 symptoms and a prolonged increase in circadian rhyt hm of peak expiratory flow rate was seen in 2 one subject (Hendrick and Lane, 1983) implying an i ncrease in airway responsiveness (Stenton 3 and Hendrick, 1994). It should be noted, however, that while there did appear to be a clear 4 response to formalin, it is not known what contribu tion to the response was attributable to 5 formaldehyde and what contribution might have been attributable to methanol. Further, while 6

the evidence of a causal association between formal dehyde and asthma is clear, the above studies 7

do not offer information on the concentrations at w hich adverse effects would expected in a 8 population. 9 There is at least one clinical study in humans that investigated whether exposure to a low 10 level of formaldehyde (500 μ g/m 3 ) would enhance inhaled allergen responses (Ezratty et al., 11 2007). Twelve subjects with intermittent asthma we re exposed to either formaldehyde or 12 purified air in a double-blind crossover study for 1 hour. Following exposure (8 hours), airway 13 responsiveness to methacholine challenge was measur ed. No significant effects on 14 methacholine-induced bronchial hyperresponsiveness (BHR) were detected due to formaldehyde 15

exposure. 16 Numerous epidemiologic studies have investigated ad verse effects in populations. 17 Decreased peak expiratory flow rates (PEFR) are an important component in the diagnosis of 18 asthma and there is evidence of formaldehyde-induce d decrements in PEFR (see Section 19 4.1.1.2). However, the diagnosis of asthma is both a more serious health condition and 20 diagnostically more complex than decreased PEFR alo ne and is evaluated here as a distinct 21 endpoint. A number of epidemiologic studies have i nvestigated the potential association 22 between formaldehyde exposure and a continuum of ad verse health effects from pulmonary 23

function to asthma. 24 The association between formaldehyde and asthma has been studied by examining 25 occupational exposures (Fransman et al., 2003; Mala ka and Kodama, 1990), school-related 26 exposures (Zhao et al., 2008; Smedje and Norback, 2 001; Norback et al., 2000) and residential 27 exposures (Matsunaga et al., 2008; Tavernier et al. , 2006; Gee et al., 2005; Delfino et al., 2003; 28 Rumchev et al., 2002; Garrett et al., 1999; Palczyn ski et al., 1999; Norback et al., 1995; 29 Krzyzanowski et al., 1990). The two occupational s tudies examined the respiratory health of 30 plywood workers (Fransman et al., 2003; Malaka and Kodama, 1990). The most recent of these 31 was conducted in New Zealand by Fransman et al. (20 03). Personal samples of formaldehyde 32 exposure were taken. The mean level of exposure wa s 0.08 mg/m 3 (65 ppb) and the majority of 33 samples were below the limit of detection which was reported to be 0.03 mg/m 3 (24 ppb). 34 Compared with those with low levels of formaldehyde exposure, workers with high levels of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-22 exposure were more likely to report having asthma ( OR=4.3 [95% CI]: 0.7–27.7]). The 1 association was not seen when examining formaldehyd e exposure and use of asthma medication. 2 The second study of plywood workers was completed i n Indonesia. Background levels of 3 formaldehyde ranged from 0.003 to 0.07 ppm. The hi ghest concentration of formaldehyde 4 detected in an air sample was in the particleboard unit (range 1.16 to 3.48 ppm). Asthma, which 5 was defined as “have you ever had an attack of whee zing that made you feel short of breath?”, 6 was found to be positively associated with formalde hyde exposure (Malaka and Kodama, 1990). 7 Studies of exposure to formaldehyde at schools have been performed in China (Zhao et 8 al., 2008) and in Sweden (Smedje and Norback, 2001) . In the study from China (Zhao et al., 9 2008), mean levels of formaldehyde were reported to be 2.3 μg/m 3 (range 1.0–5.0 μg/m 3 ) 10 indoors and 5.8 μg/m 3 (range 5.0–7.0 μg/m 3 ) outdoors. Cumulative asthma and daytime attacks 11

of breathlessness were found to be associated with outdoor formaldehyde levels. Neither of 12

these outcomes was associated with indoor concentra tions of formaldehyde; however, indoor 13

levels were found to be associated with nocturnal a ttacks of breathlessness. In Sweden (Smedje 14 and Norback, 2001), the levels of formaldehyde meas ured indoors were higher (mean 4, range 15 <5.0–72 μg/m 3 ). One difference between this Swedish study and t he study performed in China is 16 that the Swedish study examined the incidence of as thma over a 4-year period. This study did 17 not report an association between formaldehyde expo sure and the incidence of asthma (OR 1.2 18 [95% CI: 0.8–1.7]) among the whole study population . However, when the investigators 19 stratified on history of atopy, they reported that among children without a history of atopy, a new 20 diagnosis of asthma was significantly more likely a t higher concentrations of formaldehyde (OR 21 1.7 per 10 μg /m 3 [95% CI: 1.1–2.6]) and at higher total concentrati ons of mold (OR=4.7 per 10- 22 fold increased in total molds [95% CI: 1.2-18.4] in the classroom air. The finding for adverse 23 effects of formaldehyde and mold did not appear to control for the other exposure and no 24 information on the potential correlation between th e two exposures was provided. In order to 25 evaluate the potential for confounding of the repor ted formaldehyde association by the reported 26 mold association, the magnitude of effects must be compared on an appropriate scale since the 27 magnitude of an odds ratio depends on the magnitude of the change in exposure level that is 28 expected to produce increased risk. Standardizing the units to the reported geometric mean 29 standard deviation, the result for formaldehyde (GS M=2.3 μg /m 3 ) is OR 1 =1.13 per GSD and the 30

result for mold is OR 2 =1.02 for a comparison of risks at the GSM to 10*GS M and OR 3 =1.06 for 31

1 OR per GSD=exp[ln(OR per μg /m 3 )/ 10 μg /m 3 * 2.3 μg /m 3 ]=exp[ln(1.7)/10*2.3]=1.13

2 OR per GSD=exp[ln(OR per 10-fold increase)/ (9*GSM )*1.6 μg /m 3 ]=exp[ln(4.7)/162*1.6]=1.02

3 OR per GSD=exp[ln(OR per 10-fold increase)/ (9*Min imum)*1.6 μg /m 3 ]=exp[ln(4.7)/45*1.6]=1.06This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-23 a comparison of risks at the minimum value of total molds (5*10 3 /m 3 ) to 10*minimum. As it 1 appears that the magnitude of the formaldehyde effe ct is substantially stronger than that of the 2 mold effect (following standardization of exposure increment) it can be concluded that the 3 reported formaldehyde effect could not have been th e spurious result of uncontrolled 4 confounding by mold. 5 The results of studies measuring residential exposu re to formaldehyde and asthma are 6 varied, with some demonstrating an association and others finding no relationship. A recent 7 study (Matsunaga et al., 2008) found no association between 24-hour formaldehyde and 8 prevalence of asthma when pregnant women with an ex posure ≥ 47 ppb were compared to those 9 with exposure to <18 ppb. However, they did report at increased risk of atopic eczema. It 10 should be noted that this study did not assess the risk of incident asthma. A study utilizing self11 reported asthma prevalence as an outcome also found no association with levels of formaldehyde 12 (mean 25.9 μg/m 3 , range 2.0–66.8 μg/m 3 ) (Palczynski et al., 1999) although they did note that 13 the incidence of allergic diseases was highest in t he highest formaldehyde exposure group but 14 that the group was too small for statistical evalua tion. 15 A study performed by Tuthill (1984) measured formal dehyde exposure for children 16 grades K through 6 by using a combination of proxy variables. Overall, there was no 17 association, but some individual variables did show an increased risk. For example, the reported 18 risk ratio for having new construction or remodelin g performed in the house in the past 4 months 19 was 2.5 (95% CI: 1.7–3.9). The risk ratio for havi ng new or upholstered furniture in the house 20 (brought into the house within the past 4 months) w as 2.2 (95% CI: 1.2–3.9). 21 The study by Delfino et al. (2003) assessed whether ambient formaldehyde concentration 22 measured at a central monitoring site were associat ed with asthma symptoms. The study 23 examined 22 10–15 year olds with at least 1 year o f physician-diagnosed asthma and living in a 24 nonsmoking household. The mean levels of formaldeh yde were measured to be 7.21 ppb (range 25 4.27–14.02 ppb). There was a positive association between asthma symptom scores (comparing 26 children who report symptoms interfering with their daily activities versus those with no 27 symptoms or symptoms not great enough to affect the ir daily activities) and high current levels 28 of formaldehyde (OR 1.90 [95% CI: 1.13–3.19]). 29 Three studies (Tavernier et al., 2006; Gee et al., 2005; Garrett et al., 1999) were 30 performed by matching children with and without ast hma and comparing the levels of 31 formaldehyde in their homes. Gee et al. (2005) rep orted median formaldehyde levels of 0.03 32 ppm in living rooms and 0.04 ppm in bedrooms. Anal yses were limited to univariate 33 comparisons of formaldehyde levels for cases of exi sting asthma and controls without asthma. 34

The concentrations did not differ in a statisticall y significant manner. The study by Gee et al. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-24 (2005) was followed up with a more sophisticated an alysis of the same children in the same 1 homes. Tavernier et al. (2006) reiterated the earli er finding by Gee et al (2005) that 2 formaldehyde was not found to be associated with ex isting asthma. Tavernier et al. (2006) did 3 not report the measured levels of formaldehyde but gave the OR for the highest tertile of 4 exposure compared with the lowest tertile of exposu re as 0.99 (95% CI: 0.39–2.50). The width 5 of this confidence interval suggests that these fin dings would still be consistent with two-fold 6

increase in risk. 7 Garrett et al. (1999) reported on the risk of aller gy and asthma-like respiratory symptoms 8 due to formaldehyde exposure in a cross-sectional s urvey of households with children with (n = 9 53) or without (n = 88) doctor-diagnosed asthma. F ormaldehyde exposure was characterized by 10 4 seasonal in-home sampling events across the year for bedrooms and 4–day passive samples 11 collected in living rooms, kitchens and outdoors. Statistically significant linear trends for 12 increased risk of having asthma were seen with incr easing formaldehyde levels (p < 0.02); 13 however, the ORs for the association did not remain statistically significant after controlling for 14 parental allergy and asthma (exact ORs and 95% CIs not given). Garrett et al (1999) also 15 evaluated the prevalence and severity of allergic s ensitization to 12 common allergens and 16 reported increased prevalence with increasing forma ldehyde concentration in the home. The 17 respiratory symptom score was also increased and de monstrated a significant effect for 18 formaldehyde in a multiple regression after adjusti ng for multiple risk factors and interactions. 19 For the atopy and respiratory symptom endpoints, se verity/incidence was increased in the 20 medium (20–50 μg/m 3 ) and high (>50 μg/m 3 ) exposure groups relative to the low (<20 μg/m 3 ) 21 exposure group, based on the highest of four season al 4-day formaldehyde measurements in the 22 home. The associations between formaldehyde concen trations and severity of allergic 23 sensitization are clearly shown and further substan tiated with multivariate regression controlling 24 for potential confounders. In logistic regressions , both the prevalence and severity of allergic 25 sensitization to 12 common allergens increased with increasing formaldehyde concentration in 26 the home. The crude association for atopy with an increase in formaldehyde concentration per 27 10 μg/m 3 was OR=1.34 which increased when adjusted for paren tal asthma and gender to and 28 odds ratio of 1.42 per 10 μg/m 3 (95% CI: 0.99-2.04). Passive smoking, the presenc e of pets, 29 indoor nitrogen dioxide concentrations, airborne fu ngal spores and house-dust-mite allergens did 30

not influence the effect estimates and were unlikel y to be confounders. Additionally, a 31 calculated respiratory symptom score was increased and demonstrated a significant relationship 32 to increased formaldehyde concentration in a multip le linear regression after adjusting for 33 multiple risk factors and interactions. For each o f these endpoints, severity/incidence was 34 increased in the medium (20–50 μg/m 3 ) and high (>50 μg/m 3 ) exposure groups relative to the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-25 low (<20 μg/m 3 ) exposure group, based on the highest of four seas onal 4-day formaldehyde 1

measurements in the home. 2 Residential formaldehyde exposure was associated w ith an increased risk of asthma in a 3 population-based case-control study of 192 children aged 6 months to 3 years (Rumchev et al., 4 2002). The study, which comprises 88 cases of chi ldren discharged from the emergency 5 department of a children’s hospital in Perth, Austr alia, with a primary diagnosis of asthma and 6 104 controls, provides a positive exposure-response relationship. Seasonal in-home 7 formaldehyde measurements taken in the living room and subject’s bedroom were used to assess 8 exposure (8-hour passive sampler). The odds ratios (ORs) for risk of asthma by formaldehyde 9 exposure level category were adjusted for numerous risk factors both familial and environmental 10 including, familial history of asthma, age, sex, sm oking, presence of pets, and attributes of the 11 home. Of these, age, allergic sensitization to com mon allergens, and family history of allergy 12 were independent risk factors for asthma (ORs of 1. 09, 2.57, and 2.66, respectively). Categorical 13 analysis of the data indicates the ORs for asthma w ere increased in the two highest formaldehyde 14 exposure groups, reaching statistical significance for household exposures > 60 μg/m 3 (48 ppb) 15 (OR of 1.39). Analysis of the data with formaldeh yde as a continuous variable provides a 16 statistically significant increase in the risk of a sthma (3 % increase in risk per every 10 ug/m3 17 increase in formaldehyde level. All analyses contr olled for other indoor air pollutants, allergen 18 levels, relative humidity, and indoor temperature a s well as other risk factors. 19 A study of 202 households (mean formaldehyde level of 26 ppb) found that among 20 children aged 6–15 years old and exposed to environ mental tobacco smoke, the prevalence of 21

asthma was 45.5% for those with measured levels of formaldehyde in the kitchen >60 ppb. The 22 prevalence of asthma dropped to 15.1% for levels ≤ 40 ppb and 0% for 41–60 ppb. No trend in 23 asthma prevalence was seen for children who were no t exposed to environmental tobacco smoke 24 (Krzyzanowski et al., 1990). 25 Finally, a study by Norback et al. (1995) reported mean levels of formaldehyde were 29 26 μg/m 3 (range <5–110 μg/m 3 ) in the bedrooms of individuals experiencing noctu rnal 27 breathlessness compared with formaldehyde levels of 17 μg/m 3 (<5–60 μg/m 3 ) among those 28

without nocturnal breathlessness. The OR for this association was 12.5 (95% CI: 2.0–77.9) and 29 the effect was substantially stronger in magnitude than the associations observed for toluene, 30 terpenes and volatile organic compounds which makes confounding by those co-exposures 31 unlikely. 32 Formaldehyde has clearly been shown to be a cause of bronchial asthma and several 33 epidemiologic studies have identified causal eviden ce of an adverse effect of exposure on 34 pulmonary function and the incidence of asthma. Wh ile there are studies that did not find 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-26 associations, many of those were limited by their s tudy design, exposure measurement and the 1 definition of prevalent asthma as the health endpoi nt. 2

3

4.1.1.4. Respiratory Tract Pathology 4 Formaldehyde-induced respiratory tract pathology in cludes inflammation, rhinitis, goblet 5 cell hyperplasia, metaplastic changes, squamous cel l hyperplasia, and impaired mucociliary 6 transport. Formaldehyde may bind to the trigeminal nerve and trigger the release of neurogenic 7

mediators of inflammation that result in tissue ede ma, lacrimation, mucus production and 8 leukocyte infiltration. How much inflammation, hyp erplasia, and metaplastic change are due to 9 sensory irritation-induced inflammatory responses c ompared with formaldehyde-induced direct 10 cell damage cannot be distinguished. Increased muc us flow and metaplastic changes may 11 progress in relation to the concentration and durat ion of exposure to protect the underlying 12 tissue. When the exposure exceeds protective and d efensive mechanisms, permanent damage 13 results (Swenberg et al., 1983). Nonetheless, thes e changes serve as a sensitive indicator of 14 formaldehyde exposure, since they occur before gros s cellular damage and focal lesions 15 (Monticello et al., 1989), and potentially suggest a point at which the concentration and duration 16 of exposure exceed the protective nature of local r esponses (increased mucus flow, goblet cell 17 hyperplasia, squamous metaplasia, etc.) (Swenberg e t al., 1983). A number of human studies 18 have reported nasal lesions associated with exposur e to formaldehyde (Pazdrak et al., 1993; 19 Ballarin et al., 1992; Boysen et al., 1990; Holmstr öm et al., 1989c; Edling et al., 1988), while 20 other studies have documented changes in mucociliar y clearance and activity ( Holmström and 21 Wilhelmsson, 1988; Andersen and Molhave, 1983). Th ese studies are summarized below. 22

23

4.1.1.4.1. Nasal lesions. Ballarin et al. (1992) did a case-control study of 15 workers from a 24 plywood factory where urea-formaldehyde glue is use d. Mean levels of formaldehyde exposure 25 (8-hour average) were estimated to be 0.09, 0.1, an d 0.39 mg/m 3 in three regions of the facility 26 (sawmill, shearing press, and warehouse, respective ly). Nasal respiratory samples were 27 obtained. Stained cells were scored for histopatho logy. Cytology examination revealed 28 increased squamous metaplasia cells in 10 of 15 (67 %) factory workers (with an average severity 29 score of 2.3) compared with one of 15 (6%) controls (with an average histology severity score of 30 1.6). In addition, one formaldehyde exposed worker (n = 15) exhibited mild dysplasia and had 31 the highest severity score (3.0). Authors suggest that these results may be due to chronic 32 irritation of the nasal respiratory mucosa. This s mall study reported only incidence of lesions 33 and did not score based on severity of lesions. Th e lesion incidence was not reported in relation 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-27 to dose, so no dose-response relationship could be determined, precluding the establishment of a 1 point of departure (POD). 2 Holmström et al. (1989) collected nasal biopsy samp les from workers exposed to air or to 3 formaldehyde at a median concentration of 240 ppm. Nasal biopsy samples were scored on a 4 0–8 range with normal respiratory epithelium as 0 a nd carcinoma as 8. Observed histologic 5 changes included loss of cilia, goblet cell hyperpl asia, and cuboidal and goblet cell metaplasia 6 replacing normal columnar epithelium. The incidenc e associated with each histologic change 7 was not reported and cannot be compared between for maldehyde-exposed and control 8 individuals. Moreover, these biologically relevant changes were not analyzed independently in 9 the analysis. The mean scores were 1.56 (range, 0– 4) for the control group and 2.16 (range, 0–4) 10 for the formaldehyde-exposed group. Although the r ange of scores in the controls and 11 formaldehyde-exposed groups were the same (0–4), th e difference in mean scores (1.56 versus 12 2.16) was statistically significant ( p < 0.05); scores were worse in the formaldehyde-exp osed 13 group. The authors reported no correlation between the duration of exposure and histologic 14 changes and no correlation between smoking habits a nd biopsy scores. The loss of cilia, goblet 15 cell hyperplasia, and cuboidal and squamous cell me taplasia replacing the columnar epithelium 16 were increased in the group exposed to formaldehyde and is a biologically relevant change. This 17 study provides a lowest-observed-adverse-effect lev el (LOAEL) of 0.240 ppm for nasal 18 histopathology. 19 Edling et al. (1988) collected nasal biopsy samples from workers (n = 75) exposed to 20 formaldehyde at three plants (workers in two of the se plants were also exposed to wood dust) 21 compared with a referent group (n = 25). Concentra tions ranged from 0.1 to 1.1 mg/m 3 (TWA) 22 with peaks of 5 mg/m 3 . Nasal histology was scored from 0 to 8 by increa sing severity, from 23 normal respiratory epithelium (0) to carcinoma (8). A normal respiratory epithelium was noted 24 in 3 of 75 workers. A loss of cilia and goblet cel l hyperplasia (scores of 2) was reported in eight 25 workers. Mixed cuboid/squamous epithelium (metapla sia), stratified squamous epithelium, and 26 keratosis were reported in 58 of 75 workers (those with scores of 3, 4, and 5 were combined). 27 Dysplasia (score of 6) was reported in 6 of 75 form aldehyde-exposed workers. None of the 28 workers had lesions that warranted a histologic sco re higher than 6. Histologic scores did not 29 correlate with duration of exposure but could not b e confirmed due to poor reporting. Data from 30 the referent group were not included. A POD could not be determined from this study. 31 Boysen et al. (1990) collected nasal biopsy samples from workers exposed to air (n = 37) 32 or to formaldehyde (n = 37) and sometimes wood dust . The exposed workers were classified 33 into two exposure groups, 0.5–2 ppm and >2 ppm. Na sal biopsy samples were assessed by using 34 a histopathology score range of 0–5, based on the p athology of pseudostratified columnar 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-28 epithelium (0) to dysplasia (5). Mean pathology sc ores for the control were decreased compared 1 with the formaldehyde-exposed group (1.4 and 1.9, r espectively) but did not reach statistical 2 significance. Little quantitative pathology data w ere provided, although qualitative histology 3 revealed a range of observed effects from deciliate d epithelial cells to mixed stratified cuboidal, 4 squamous epithelium to dysplasia. None of the cont rol samples received histologic severity 5 scores of 4 or 5, indicating that keratinizing stra tified squamous epithelium and dysplasia were 6 not observed in controls. A wider variety of histo pathologic lesions were reported in exposed 7 workers compared with controls, and a greater numbe r of exposed workers had histologic 8 changes compared with controls. Incidence data for each type of histopathology were not 9 reported, but the authors wrote that the degree of metaplastic alterations was more pronounced 10 among the exposed workers. An upper range for the high concentration group (>2 ppm) was not 11 reported, and median concentrations were not provid ed. 12 Pazdrak et al. (1993) exposed human subjects (six men, three women) to 0.4 ppm 13 formaldehyde in a chamber for 2 hours. Approximate ly half of the subjects suffered from skin 14 hypersensitivity to formaldehyde, while the other s ubjects were healthy. An evaluation of nasal 15 lavage pretest and following formaldehyde exposure revealed that the hypersensitive and healthy 16 groups had similarly elevated eosinophil counts at 0 hours after exposure (from 17

42 × 10 3 cells/mL to 72 × 10 3 cells/mL for healthy subjects [ p < 0.05] and from 18

39 × 10 3 cells/mL to 69 × 10 3 cells/mL for hypersensitive subjects [ p < 0.05]). Similar 19 eosinophil levels were also seen in both groups at 3 and 18 hours. Both groups had equivalent 20 increases in lavage albumin and total protein level s following exposure, but basophil counts were 21 unchanged. Based on evidence of formaldehyde-induc ed inflammation, these data provide a 22 LOAEL of 0.4 ppm for nasal histopathology. 23

24

4.1.1.4.2. Mucociliary clearance. In addition to abnormal nasal histopathology, chang es in 25 mucociliary clearance were also observed in some of these studies at similar exposure 26 concentrations. The mucociliary apparatus is an im portant barrier to infection and exogenous 27 agents and, thus, is considered as a potential adve rse effect. These effects may be due to direct 28 interaction of formaldehyde with the mucus itself o r to SI-induced inflammation in the nasal 29 tissue that affects mucus production and creation o f an effective mucosal barrier. 30 Andersen and Molhave (1983) reviewed five controlle d human studies, one of which 31 (Andersen and Lundqvist, 1974) examined mucus flow rate in 16 individuals acutely exposed to 32 0, 0.3, 0.5, 1, or 2 ppm formaldehyde for 4–5 hours in a chamber. Mucus flow rate was 33

decreased in the anterior and middle third of the c iliated mucosa at 0.3 ppm, but statistical 34 significance was not determined. This study includ ed smokers and nonsmokers. The small 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-29 sample size, potential confounder effect from smoki ng, and lack of dose-response relationship 1 preclude the establishment of a POD. 2 Holmström and Wilhelmsson (1988) demonstrated reduc ed mucociliary clearance and 3 nasal mucosal swelling in 70 workers exposed to a m edian formaldehyde concentration of 4 0.21 ppm, compared with a referent group of store c lerks (n = 36) and was further averaged over 5 years of exposure. Mucosal swelling and mucociliar y activity was measured in the nasal 6 turbinates. The authors also reported symptoms not only during the weekdays, but also over 7 weekends and vacation periods. Formaldehyde-expose d subjects self-reported significantly more 8 nasal discomfort, eye discomfort, deeper airway dis comfort, and frequent headache than the 9 referent group. Groups exposed to formaldehyde had more pronounced mucosal swelling (10.7 10 nasal resistance score) compared with the reference group (6.5 nasal resistance score). This 11 difference persisted when data were normalized for differential nasal congestion in the subjects. 12 Decreased mucociliary activity was seen in 3% of co ntrols and 20% of formaldehyde-exposed 13 subjects and reached statistical significance ( p < 0.05). It is not clear whether impaired 14 mucociliary clearance was a consequence of altered cell morphology or increased mucus 15 viscosity. These data provide a LOAEL of 0.21 ppm based on impaired mucociliary clearance. 16 Thus, mild nasal epithelial lesions observed in formaldeh yde-exposed workers have been 17 observed consistently at levels of about 0.20 ppm t o about 2 ppm (Boysen et al., 1990; 18 Holmström et al., 1989; Edling et al., 1988). Of t hese, Holmström et al. (1989) and Edling et al. 19 (1988) do not appear to be confounded by exposure t o wood dust. Nasal biopsy pathology from 20 formaldehyde-exposed workers is consistent with irr itant and reactive properties of 21 formaldehyde (Ballarin et al., 1992; Boysen et al., 1990; Holmström et al., 1989; Edling et al., 22 1988; Berke, 1987). Moreover, these findings are s upported by results from animal toxicity and 23 pharmacokinetic and anatomical airflow studies, ind icating that, at concentrations less than 24 1 ppm, inhaled formaldehyde gas does not reach lowe r regions of the respiratory tract. Of the 25 available human studies that evaluated histopatholo gy, Holmström and Wilhelmsson (1988) 26 appears to be the most robust and sensitive. The s tudy was carefully designed and included a 27 large sample of formaldehyde-exposed subjects who w ere considered separately from workers 28 exposed to combinations of exposures (formaldehyde and wood dust). Study subjects had been 29 exposed to formaldehyde regularly for many years. The authors reported not only weekday 30 exposures but effects reported on weekends and on v acation. Total exposure was carefully 31 calculated and averaged. The data were controlled for potential confounders, such as smoking. 32 The endpoint of reduced mucociliary clearance has b een substantiated by Andersen and Molhave 33 (1983) and Holmström et al. (1989). Animal studies have also reported formaldehyde-induced 34 changes on the nasal mucosa and are highlighted in Section 4.2.1.2. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-30 4.1.1.5. Immunologic Effects 1 Numerous studies have examined the immunologic resp onses of individuals exposed to 2 formaldehyde. This section will discuss four speci fic areas related to immunotoxicity after 3 exposure to formaldehyde: increased upper respirato ry tract (URT) infections, systemic immune 4 dysfunction, sensitization and atopy, and productio n of formaldehyde-protein complexes. Some 5 studies report increased incidence of URT infection s after exposure to formaldehyde (Lyapina et 6 al., 2004; Krzyzanowski et al., 1990; Holness and N ethercott, 1989). This effect appears to 7 occur independently of systemic immune changes and may be due to damage to the mucosal 8 barrier, thus facilitating pathogen access. A numb er of studies have investigated the hypothesis 9 that formaldehyde may induce systemic immunomodulat ion (Ohtani et al., 2004a, b; Erdei et al., 10 2003; Thrasher et al., 1990, 1987; Pross et al., 19 87). Some studies have also evaluated immune 11 system effects by investigating the role of reactiv e oxygen species (ROS) from respiratory burst 12 associated with immune cells (Lyapina et al., 2004; Gorski et al., 1992) and by assessing 13 chromosomal damage in immune cells (Orsière et al., 2006; Yu et al., 2005). In addition to the 14 effects of formaldehyde on asthmatics and the poten tial for formaldehyde exposure to exacerbate 15 asthmatic responses, reviewed in Section 4.1.1.2, n umerous studies have investigated whether 16 formaldehyde may directly induce sensitization and atopic responses by measuring 17 immunoglobulin E (IgE) levels associated with forma ldehyde exposure (Ohmichi et al., 2006; 18 Vandenplas et al., 2004; Doi et al., 2003; Baba et al., 2000; Palczynski et al., 1999; Krakowiak et 19 al., 1998; Wantke et al., 1996a, b; Liden et al., 1 993; Salkie, 1991; Grammer et al., 1990; 20 Kramps et al., 1989). Findings are largely negativ e and suggest that formaldehyde-induced IgE 21 production is not likely. Lastly, studies have inv estigated the production of formaldehyde22 specific antibodies, formaldehyde-albumin complexes , and formaldehyde-heme complexes (Kim 23 et al., 2001; Carraro et al., 1997; Grammer et al., 1993, 1990; Dykewicz et al., 1991; Thrasher et 24 al., 1990). Heme complex formation is not a strict immunologic endpoint but may trigger 25 antibody formation and thus it will be discussed in this section. This section will thus summarize 26 the human studies that have specifically addressed the increased incidence of URT infections, 27 immunotoxic endpoints, atopy and sensitization, and formation of formaldehyde-heme and 28 formaldehyde-albumin complexes. 29

30

4.1.1.5.1. Increased URT infections. Diverse studies have investigated the possibility t hat 31 formaldehyde exposure leads to increased URT infect ions (Lyapina et al., 2004; Krzyzanowski 32 et al., 1990; Holness and Nethercott, 1989). Lyapi na et al. (2004) studied 29 workers who were 33 occupationally exposed occupationally to formaldehy de for an average of 12.7 years through 34 contact with carbamide-formaldehyde glue. The mean values of the average shift concentrations 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-31 of formaldehyde in the application of carbamide-for maldehyde glue to be 0.71 ppm TWA with a 1 range of 0.32 to 1.57 ppm. The workers were divide d into two subgroups, one (n = 12) that 2 suffered from either a long history (with clinical findings) of chronic mucous inflammation of 3 the URT with multiple relapses and a second group ( n = 17) whose URT inflammations were 4 short, acute, and predominantly viral. Twenty-one healthy subjects served as controls. A 5 statistically significant association of self-repor ted chronic bronchitis and decreased resistance to 6 URT infection was reported in all the exposed worke rs compared with controls ( p = 0.02). Of 7 the workers, 41% had a history of chronic respirato ry infection and frequent long-lasting 8 infectious inflammatory relapses (group 1a). Anoth er group (group 1b) consisted of 17 exposed 9 workers, 12 of whom had no history of recurrent vir al infections of the URT. There was a 10 statistically significant association of frequency and duration of inflammatory relapses between 11 groups 1a and 1b. No dates were provided regarding when these measurements were made or 12 over what period of time they were calculated. 13 Krzyzanowski et al. (1990) measured formaldehyde le vels in homes and recorded, by 14 way of a questionnaire, health histories from adult and child residents. Formaldehyde levels 15 were reported from samples taken for two 1-week per iods in various rooms of the home (kitchen, 16 living room, subject’s bedroom). The average forma ldehyde level was 26 ppb in 202 homes, and 17 levels were stratified into homes with exposure lev els below 40 ppb, between 40 and 60 ppb, and 18 above 60 ppb. Incidences of doctor-diagnosed chron ic bronchitis were more prevalent in 19 children (under age 15) living in homes with higher formaldehyde (>60 ppb) readings in the 20 kitchen (

p < 0.001). This effect was more pronounced ( p < 0.001) in children simultaneously 21 exposed to environmental tobacco smoke. The preval ence of chronic cough was also increased 22 in adults living in homes with measurable levels of formaldehyde, but data were not shown. 23 Holness and Nethercott (1989) assessed chronic bron chitis in 87 funeral workers, where the 24 average formaldehyde exposure was reported at 0.38 ± 0.19 ppm. Chronic bronchitis was 25 observed in 20 funeral workers (n = 87) exposed to formaldehyde compared with 3 cases of 26 chronic bronchitis in nonexposed referent controls (n = 38). 27 These studies suggest that exposure to formaldehyde may be associated with increased 28

incidence of chronic bronchitis. The mechanism for this association has not been elucidated. 29 Pathogens may gain access to the URT via a compromi sed mucosal barrier, as has been shown in 30 histopathology studies (Section 4.1.1.4). 31

32

4.1.1.5.2. Immune function. A number of studies have evaluated the ability of f ormaldehyde to 33 induce systemic immunotoxic effects (Ohtani et al., 2004a, b; Erdei et al., 2003; Thrasher et al., 34 1990, 1987; Pross et al., 1987). Some studies have reported altered innate immune responses 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-32 associated with formaldehyde exposure (Erdei et al. , 2003), while others have noted adaptive 1 immune response suppression associated with formald ehyde exposure (Thrasher et al., 1990, 2 1987) and changes associated with alterations to a predominant T—lymphocyte helper 2 (Th2) 3 pattern (Ohtani et al., 2004a, b). In contrast, Pr oss et al. (1987) did not observe formaldehyde4 associated changes in systemic immune function. 5 Erdei et al. (2003) found that Haemophilus influenzae humoral biomarker (H.in.IgG), 6 Klebsiella pneumoniae biomarker (K.pn.IgG), and elevated monocyte concen trations were 7 significantly associated with high formaldehyde con centrations in asthmatic children, compared 8 with nonsensitive children. Briefly, Erdei et al. (2003) compared the immune system responses 9 in 9- to 11-year-old Hungarian school children whos e respiratory systems were immunologically 10 compromised (chronic respiratory disease, asthma) a nd normal children who were exposed to 11 indoor air pollutants, including formaldehyde. In the homes of the children with the highest 12 levels of pollutants, 49.3% of formaldehyde measure ments exceeded the Hungarian indoor 13 standard of 0.01 ppm, while 20% exceeded the World Health Organization’s (WHO’s) suggested 14 indoor level of 0.09 ppm. The authors excluded fro m consideration all measurements that 15 exceeded WHO’s air quality guidelines in one uniden tified city to prevent a “city-related bias,” 16 since these measurements occurred entirely in that city. The average formaldehyde 17 concentration in the 123 homes tested was 14 ppm wi th a range of 0.5 to 46 ppm. H.in.IgG and 18 K.pn.IgG were significantly associated with high fo rmaldehyde concentrations ( p < 0.013 and 19

p < 0.049, respectively) in sensitive children compar ed with nonsensitive children. These 20 markers were also correlated with high levels of ni trogen dioxide, the number of cigarettes 21 smoked, and exposure to paint, volatile organic com pounds, and solvents. Additionally, indoor 22 formaldehyde exposure was significantly associated with increased monocyte concentrations 23 ( p < 0.017) that are important to the innate immune re sponse (inflammation) in diseased tissue. 24

The authors concluded that the elevation of immune biomarkers in sensitive children with 25 respiratory disease is likely the result of high co ncentrations of toxic indoor air pollutants, 26 including formaldehyde. 27 Thrasher et al. (1987) assessed the effects of form aldehyde exposure on cellular 28 immunity and antibody formation in eight exposed an d eight unexposed individuals. The 29 exposed group consisted of three males and five fem ales. Seven of the exposed individuals had 30 resided in mobile homes for periods ranging from 2 to 7 years; the eighth was a laboratory 31 worker who resided in a newly decorated, energy-eff icient apartment. Air monitoring in four of 32 the homes revealed formaldehyde vapor concentration s ranging from 0.07 to 0.55 ppm. Venous 33 blood samples were collected from all subjects and T- and B-cells were counted and monitored 34 for blastogenesis. When IgG and IgE antibodies to formaldehyde were monitored in serum, no 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-33 IgE antibodies to formaldehyde were detected in exp osed or control subjects. IgG antibody titers 1 in exposed subjects ranged from 1:8 to 1:256 but es sentially were undetected (1:4) in seven of 2 the controls. T- and B-cell numbers were significa ntly lower ( p < 0.05) in mobile home residents 3 (48 and 12.6%, respectively) compared with those of control subjects (65.9 and 14.75%, 4 respectively). As determined by incorporation of 3 H-thymidine into 48-hour unaltered 5 lymphocytes, phytohemagglutinin-stimulated T- and B -cell blastogenesis was significantly 6 depressed (

p < 0.01) in cells of mobile home residents compared with those of control subjects 7 (17,882 and 28,576 cpm, respectively). Thrasher et al. (1987) concluded that exposure to 8 formaldehyde decreases the proportion of peripheral T cells. 9 In a later study, Thrasher et al. (1990) evaluated five groups of subjects with varying 10 levels and durations of formaldehyde exposure. The groups consisted of (1) asymptomatic 11 chiropractic students exposed during anatomy classe s (controls with only intermittent exposure 12 to formaldehyde), (2) mobile home residents, (3) of fice workers, (4) patients with multiple 13 symptoms who had been removed from the source of fo rmaldehyde for at least a year, and 14 (5) occupationally exposed patients. All groups we re assessed for immunologic function via 15 white cell, lymphocyte, and T-cell counts, T-helper /suppressor ratios and B-cell counts. When 16 compared with controls (chiropractic students), the patient groups had significant elevations in 17 formaldehyde antibody titers and B-cell titers. 18 Ohtani et al. (2004a, b) reported effects of exposu re to formaldehyde and diesel exhaust 19 particles on cytokine production by human monocyte- derived dendritic cells (MoDCs) and 20

T cells in vitro. Dendritic cells were stimulated with CD40 ligand and interferon (IFN)- γ , T cells 21 with anti-CD3/CD28 antibodies. Cytokine proteins a nd mRNA levels were measured in 22 supernatants by enzyme-linked immunosorbent assay ( ELISA) and polymerase chain reaction 23 (PCR), respectively. Formaldehyde and diesel exhau st particles significantly increased tumor 24 necrosis factor (TNF)- α levels and suppressed interleukin (IL)-12p40 prote in and mRNA levels 25 in MoDCs. The same treatment suppressed protein sy nthesis and mRNA expression of IFN- γ 26

and IL-10 in T cells. The authors concluded that t heir findings support a role of formaldehyde 27 and diesel exhaust particles in altering the immune response to a Th2-dominant pattern that 28 furthers allergic inflammation. Further details, s uch as exposure concentrations and 29 experimental protocols, are not available. 30 In contrast, Pross et al. (1987) concluded that for maldehyde does not induce altered 31 immune activity. The authors evaluated the immunol ogic response of asthmatic subjects 32 exposed to UFFI off-gas products. Subjects consist ed of 23 individuals with a history of 33 asthmatic symptoms attributed to UFFI and 4 individ uals with asthma unrelated to UFFI off-gas 34 products. All subjects were exposed in an environm ental chamber according to the following 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-34 sequence: (1) room air (placebo) for 30 minutes; (2 ) 1 ppm formaldehyde gas for 3 hours; (3) 1 UFFI particles (4 μ m diameter, 0.5 particles/mL) for 3 hours, commenci ng 48 hours after 2 formaldehyde gas exposure; and (4) UFFI off-gas pro ducts for 3 hours, commencing 48 hours 3 after UFFI particle exposure. There was a signific ant increase in the percentage and absolute 4 number of eosinophils and basophils in the subjects who lived in UFFI homes but no differences 5 between exposure groups with respect to lymphocyte subpopulations either before or after UFFI 6 exposure. However, when T8 suppressor cells were c ounted, values in the UFFI-exposed group 7 pre-exposure and postexposure, a small but statisti cally significant ( p < 0.01) increase in T8 cell 8 count was observed. The biological significance of this increase in T8 cell count in exposed 9 asthmatics is not known. Pross et al. (1987) concl uded that short-term exposure to formaldehyde 10 was not immunosuppressive and did not result in sys temic immune reactivity. 11 Respiratory burst from immune cells creates ROS tha t can incur further cellular damage. 12 Several studies have evaluated, either directly or indirectly, the potential role of ROS as potential 13 mediators of formaldehyde-associated effects, parti cularly those caused by immune cells. Gorski 14 et al. (1992) measured chemiluminescence resulting from the release of free radicals from 15 granulocytes of healthy and formaldehyde-sensitive subjects. Thirteen subjects with contact 16 dermatitis who were occupationally exposed to forma ldehyde and five healthy volunteers 17 participated in the study. All underwent skin-pric k tests for common allergens as well as a 18 histamine inhalation provocation test. Subjects we re exposed to 0.5 mg/m 3 (0.41 ppm) 19 formaldehyde for 2 hours, and the PEFR was measured immediately before exposure, after 60 20 and 120 minutes of exposure, and 6 and 21 hours aft er completion of exposure. Peripheral blood 21 granulocyte chemiluminescence was measured in the p resence of luminol. Free radical 22 production was increased significantly within 30 mi nutes of beginning the exposure in subjects 23 with allergic dermatitis and remained elevated for 24 hours compared with baseline values. 24 Gorski et al. (1992) concluded that granulocyte che miluminescence did not increase in healthy, 25 formaldehyde-exposed patients but was diagnostic fo r formaldehyde-sensitive patients. These 26 results also suggest a putative role for oxidative damage associated with formaldehyde exposure, 27 particularly in sensitized individuals. 28 Lyapina et al. (2004) also reported effects of form aldehyde exposure on neutrophil 29 respiratory burst activity (NRBA), the capacity of polymorphonuclear leukocytes to produce 30 reactive oxygen radicals in response to chemical or microbial stimuli using flow cytometry. 31 Briefly, Lyapina et al. (2004) studied 29 workers w ho were occupationally exposed to 32 formaldehyde for an average of 12.7 years through c ontact with carbamide-formaldehyde glue 33 with a mean value of the average shift concentratio n of formaldehyde reported as 0.71 ppm 34 TWA with a range of 0.32 to 1.57 ppm. The workers were divided into two subgroups, one (n = 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-35 12) that suffered from either a long history (with clinical findings) of chronic mucous 1 inflammation of the URT with multiple relapses, and a second group (n = 17) whose URT 2 inflammations were short, acute, and predominantly viral. Twenty-one healthy subjects served 3 as controls. A suite of hematological tests and fl ow cytometric analysis for respiratory burst 4 activity were performed. Although no significant d ifference was observed in the spontaneous 5 and stimulated NRBA (median percentage of oxidizing cells) between the 29 exposed workers 6 with URT inflammation and the healthy controls (0.8 3 versus 1.35, respectively), a separate 7 comparison of the NRBA of 12 workers with chronic, repeating URT infections and 17 workers 8 with short, infrequent episodes of URT inflammation s was significant (0.45 versus 1.00, p = 9 0.037). When the NRBA of the group with chronic UR T infections (n = 12) was separately 10 compared with that of the healthy controls (n = 21) , the results were also significant (0.45 versus 11 1.35, p = 0.012). Individuals with chronic URT infections have reduced NRBA that could be 12 due to formaldehyde exposure. Neutrophils respond to tissue damage or local invasion of 13 microorganisms and act to phagocytize foreign cells . If neutrophilic activity is hampered or 14 altered by formaldehyde exposure, then the ability to fight infection will be diminished, leading 15 to prolonged infection. However, no dose-response pattern of formaldehyde exposure could be 16 determined from this study. 17 Other investigators have reported chromosomal dama ge in immune cells due to 18 formaldehyde (Orsière et al., 2006; Yu et al., 2005 ). Yu et al. (2005) evaluated chromosomal 19 damage in lymphocytes from 151 exposed and nonexpos ed workers from a plywood factory 20 detected by comet assay. The authors reported that chromosomal damage was statistically 21 elevated in lymphocytes from formaldehyde-exposed w orkers compared with controls. 22 However, no information on exposure duration or lev els was provided. Orsière et al. (2006) 23 studied DNA damage in lymphocytes from 59 hospital employees with formaldehyde exposures 24 from pathology and anatomy laboratories in five hos pitals. Controls were 37 workers from the 25 same hospitals, matched on gender, age, and smoking habits, with no known exposure to 26 genotoxic agents. Study participations were exclud ed if workers had a history of radio- or 27 chemotherapy or had used therapeutic medications th at were known to be mutagenic. 28 Occupational exposure was determined through 15-min ute and 8-hour personal air sampling 29 during a typical workday. Mean formaldehyde concen trations were 2 ppm (range: <0.1– 30 20.4 ppm) for 15-minute sampling and 0.1 ppm (range : <0.1–0.7 ppm) for 8-hour sampling. No 31 change in DNA damage was found between the beginnin g and end of the workday among 32 exposed workers (3.9 ± 0.6 versus 3.6 ± 0.5 relativ e light units/ng DNA). However, exposed 33 workers had significant elevations in the binucleat ed micronucleated cell rate (BMCR) per 34 1,000 cells compared with controls (16.9 ± 9.3 vers us 11.1 ± 6.0%; p < 0.001), but BMCR did 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-36 not appear to be correlated with formaldehyde conce ntration. Linear regression analysis showed 1 that the effect for exposure remained after adjusti ng for gender, age, smoking, and drinking 2 habits. For 18 exposed and 18 control workers who underwent cytokinesis-blocked 3 micronucleus assay (CBMA) combined with fluorescent in situ hybridization (FISH) with pan4 centromeric DNA probe, results showed that the freq uency of micronuclei (MN) containing only 5 one centromere (C1+MN) was elevated among the expos ed compared with unexposed workers 6 (11.0 ± 6.2% versus 3.1 ± 2.4%; p < 0.001). The effect of exposure remained signifi cant after 7 controlling for gender, age, smoking, and drinking habits. Results from Yu et al. (2005) and 8 Orsière et al. (2006) suggest that formaldehyde exp osure may promote chromosomal damage 9 leading to micronucleated lymphocytes. 10 Compromised lymphocyte function may significantly contribute to altered immune 11 status. The mechanism underlying this effect has n ot been elucidated. 12

13

4.1.1.5.3. Sensitization and atopy. Numerous studies have documented formaldehyde-induc ed 14 exacerbation of asthmatic responses (see Section 4. 1.1.2). The mechanism of this effect has not 15 been clarified and has led investigators to assess the potential for formaldehyde to directly induce 16 formation of formaldehyde-specific antibodies, lead ing to allergic responsiveness. One case 17 report showed systemic allergic reactions (e.g., an aphylaxis) to formaldehyde in a patient 18 undergoing hemodialysis (Maurice et al. [1986] refe renced in Thrasher et al. [1990]). Some 19 studies have evaluated the potential association offormaldehyde-specific IgE in already20 sensitized individuals (Baba et al., 2000; Palczyns ki et al., 1999). Other studies have 21 investigated whether formaldehyde can directly indu ce IgE in nonsensitized individuals. Most of 22 the studies have not identified presence of formald ehyde-specific IgE (Ohmichi et al., 2006; 23 Krakowiak et al., 1998; Grammer et al., 1993, 1990; Kramps et al., 1989; Thrasher et al., 1987) 24 and are summarized below. A few studies (Vandenpla s et al., 2004; Doi et al., 2003; Liden et 25 al., 1993) reported positive IgE against formaldehy de, associated with exposure, but the IgE 26 titers were either transient (Vandenplas et al., 20 04) or were positive in a small subset of 27 previously sensitized subjects (2 of 15) (Liden et al., 1993). Doi et al. (2003) detected IgE 28 against formaldehyde in two asthmatic children (out of 122 asthmatic children), but the response 29 severity did not correlate with exposure level. 30 Palczynski et al. (1999) evaluated whether exposure to formaldehyde might facilitate 31 specific sensitization to common allergens. The st udy population was comprised of residents of 32 apartments built in 1989–1990. Only households wit h children from 5–15 years were eligible for 33 the study. A random sample of 120 apartments was s elected in which lived a total of 465 34 persons aged 5–65 years. Individual demographic ch aracteristics and medical histories were 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-37 determined by questionnaire. Residents were tested , using the skin-prick method, for allergen 1 response to a variety of materials, such as househo ld dust, pollens, and feathers. Total serum IgE 2 levels were measured, and the presence of formaldeh yde-specific IgE antibodies was determined. 3 Measured mean levels of formaldehyde were 21.05 ± 8 .94 ppb. No significant relationship 4 between respiratory allergy prevalence and indoor e xposure to formaldehyde was detected. 5 Significant increases in serum IgE levels were foun d in children exposed to both environmental 6 tobacco smoke and formaldehyde. 7 Baba et al. (2000) investigated whether production of formaldehyde-specific IgE could be 8 detected in adult asthmatics. Formaldehyde exposur e levels were not documented. 9 Formaldehyde-IgE was detected in two asthmatic pati ents (n = 80), one male and one female, but 10 the titer of IgE did not parallel the severity of t he asthmatic responses and could not be linked to 11 formaldehyde exposure. Thus, formaldehyde-specific IgE-mediated allergy was rare in adult 12

chronic asthmatics. 13 Several studies have examined serum for formaldehyd e-specific IgE antibodies in groups 14 of formaldehyde-exposed humans (Ohmichi et al., 200 6; Krakowiak et al., 1998; Wantke et al., 15 1996a, b; Salkie, 1991; Grammer et al., 1990; Kramp s et al., 1989). While formaldehyde16 specific IgE was reported in one study (Wantke et a l., 1996a), results from most other studies 17 failed to find a consistently strong association be tween formaldehyde-specific IgE or IgG 18 antibodies in groups of formaldehyde-exposed humans

. 19 Wantke et al. (1996a) detected elevated levels of f ormaldehyde-specific IgE in 24 of 62 20 8-year-old children who were students in three part icleboard-paneled classrooms in which the 21 estimated formaldehyde air concentrations were 0.07 5, 0.069, and 0.043 ppm. In a health 22 survey, the children reported headaches (29/62), fa tigue (21/62), dry nasal mucosa (9/62), rhinitis 23 (23/62), cough (15/62), and nosebleeds (14/62). Th e number of children with symptoms in each 24 classroom decreased with decreasing formaldehyde co ncentration (49, 47, and 24, respectively, 25 for the 0.075, 0.069, and 0.043 ppm classrooms). H owever, the investigators reported that 26 elevated levels of specific IgE did not correlate w ith the number and severity of symptoms. 27

When the children were evaluated after 3 months in a new school that did not have particleboard 28 paneling and had lower ambient formaldehyde concent rations (0.029, 0.023, and 0.026 ppm), the 29 number of children reporting symptoms decreased sig nificantly from earlier figures, and, when 30 measured in 20 of the children, the mean serum leve ls of formaldehyde-specific IgE declined 31 significantly compared with pre-moving mean levels. 32 In contrast, a study by Krakowiak et al. (1998) mea sured serum IgE levels in asthmatic 33 and healthy subjects as part of a larger study to c haracterize the mechanism of 34 formaldehyde-induced nasal and bronchial response i n asthmatic subjects with suspected 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-38 formaldehyde allergy. Ten subjects reported to hav e formaldehyde rhinitis and asthma and 1 10 healthy subjects underwent a 2-hour inhalation c hallenge in an exposure chamber with 2 formaldehyde at a concentration of 0.5 mg/m 3 (0.41 ppm). Formaldehyde-specific serum IgE 3 antibodies were measured, and cellular, biochemical , and mediator changes were assessed in 4 nasal lavage before, immediately after, and at 4 an d 24 hours after challenge. Challenges with 5 formaldehyde caused only transient symptoms of rhin itis in both groups. Furthermore, none of 6 the subjects thought to have occupational asthma de veloped clinical symptoms of bronchial 7 irritation. No specific IgE antibodies to formalde hyde were detected in persons with 8 occupational exposure to formaldehyde. No differen ces in the nasal response to formaldehyde 9 were found between subjects reported to have occupa tional allergic respiratory diseases and 10 healthy subjects (

p > 0.05). The study showed that inhaled formaldehy de at a level as low as 11 0.5 mg/m 3 did not induce a specific allergic response either in the upper or in the lower part of 12 the respiratory tract. In addition, it demonstrate d that there was no difference in nasal response 13 to formaldehyde between asthmatic subjects occupati onally exposed to formaldehyde and 14 healthy subjects. 15 Similarly, formaldehyde-specific IgE antibodies wer e detected in only 1 serum sample 16 (out of 86) from four groups of formaldehyde-expose d subjects (Kramps et al., 1989). The 17 subject with detected formaldehyde-specific IgE dis played allergic symptoms. The groups 18 included (1) 28 subjects living or working in place s with formaldehyde-containing construction 19 materials (e.g., chipboard) and estimated formaldeh yde concentrations ranging from 0.08 to 20 0.37 ppm, (2) 18 occupationally exposed subjects fr om an anatomy laboratory and in other 21 unspecified industries where air concentrations wer e not measured, (3) 12 hospital attendants 22 who worked with formaldehyde-sterilized hemodialysi s equipment, and (4) 28 hemodialysis 23 patients coming into contact with equipment sterili zed with formaldehyde. Other subjective 24 symptoms, such as headache, eye irritation, and res piratory complaints, were reported by 25 24/28 subjects in the construction material group a nd confirm that formaldehyde is an irritant 26 (reviewed in Section 4.1.1.1). Durations of exposu re or length of employment were not reported 27 for the subjects in this study. 28 Grammer et al. (1990) studied the immunologic natur e of formaldehyde sensitivity in 29 37 workers who were examined by a group of physicia ns in response to complaints of 30 formaldehyde-related illness. Air sampling of form aldehyde ranged from 0.003 to 0.078 ppm, 31 but specific levels were not tied to specific workp lace areas. Blood samples were collected and 32 assayed for IgE and IgG activity against formaldehy de. None of the workers had IgG activity 33 against formaldehyde. No IgE antibodies were detec ted in the other 32 workers. The authors 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-39 concluded that there was no evidence of an immunolo gically mediated response to formaldehyde 1 in this group of workers. 2 Formaldehyde-specific IgE was not detected in any o f a group of 45 medical students 3

before or after the students attended a 4-week anat omy dissecting course (Wantke et al., 1996b). 4

Estimates of ambient air concentrations of formalde hyde ranged from 0.059 to 0.219 ppm (0.124 5 ± 0.05 ppm; mean ± SD). However, the survey reveal ed frequencies of irritation symptoms that 6 were consistent with other studies (e.g., itching o f the skin in 33/45 students, headache in 15/45, 7 and burning eyes in 13/45). 8 Similarly, Ohmichi et al. (2006) were unable to cor relate formaldehyde exposure with 9 specific IgE production among eight students attend ing a gross anatomy laboratory. 10 Formaldehyde exposure was estimated to range from 0 .33 to 1.47 ppm during the laboratory 11 sessions. The sample size was small, and IgE level s varied substantially (ranging from <19 to 12 >5,000 international units/mL). Compared with IgE levels taken 90 minutes prior to the start of 13 the first session, IgE levels measured shortly afte r the last session and up to 23 days following 14 the last session showed no association with exposur e. 15 Salkie (1991) investigated the prevalence of formal dehyde-specific IgE in practicing 16 pathologists who complained of formaldehyde sensiti vity. Exposure levels were not reported. 17 Serum samples were assayed for total IgE and formal dehyde-specific IgE. Of the 46 subjects, 18 29 self-reported atopy that was confirmed in 12 sub jects by positive IgE. Moreover, 29 subjects 19 complained of formaldehyde-specific sensitivity. H owever, zero subjects had formaldehyde20 specific IgE, and there was no evidence that atopic individuals were more sensitive to 21 formaldehyde than non-atopic individuals. The auth ors noted that atopic individuals may have 22 selectively reduced their exposure to formaldehyde. 23 Vandenplas et al. (2004) evaluated a case study of a 31-year-old male who was 24 accidentally exposed to formaldehyde for 2 hours. The exposure level was not provided. The 25 subject had smoked a pack of cigarettes a day for 1 3 years and was admitted to the emergency 26 room for asthmatic symptoms. Eight days following exposure, increased levels of 27 formaldehyde-specific IgE antibodies were detected but could not be detected in subsequent 28

assessments. 29 A clinical study by Liden et al. (1993) evaluated I gE-specific antibodies against 30 formaldehyde in 23 subjects who had previously test ed positive for skin sensitization by skin 31 prick test. Subjects were exposed to formaldehyde by skin patch (1% formaldehyde in water). 32 Ten of the subjects were classified as atopic Tho ugh 15 of 23 of the sensitized subjects were 33 also sensitive to formaldehyde applied by skin patc h, formaldehyde-IgE was positive in 2 of 15 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-40 individuals who were not classified as atopic. No dose-response relationship could be 1 determined from the study design of this study. 2 Doi et al. (2003) conducted a clinical study in 155 children of which 122 were 3 asthmatics. No specific exposure to formaldehyde w as documented. IgE against formaldehyde 4 was determined in blood. Formaldehyde-specific IgE was found in two asthmatic children. 5 Thus, while several studies have documented formald ehyde-specific IgE, the occurrence is rare 6 and may be transient. Asthmatic children may be mo re predisposed to form formaldehyde7 specific IgE than non-atopic individuals or adults. The formation of formaldehyde-specific IgE 8 is quite rare. 9

10

4.1.1.5.4. Formaldehyde-albumin and formaldehyde-heme complexe s

. Numerous studies have 11 shown that formaldehyde can bind to blood proteins as formaldehyde-heme and formaldehyde12 human serum albumin (formaldehyde-HSA) complexes (C arraro et al., 1997; Grammer et al., 13 1993, 1990; Dykewicz et al., 1991; Thrasher et al., 1990). Kim et al. (2001) failed to identify 14 IgE against formaldehyde-HSA complexes in one case- control subject following industrial 15 occupational exposure to formaldehyde. These compl exes may serve to traffic formaldehyde 16 throughout the bloodstream and throughout the body. While formaldehyde may be too small to 17 engender an immune response, these complexes may be able to trigger formaldehyde-protein18 specific antibodies, leading to an immune response, including sensitization. 19 Thrasher et al. (1990) evaluated five groups of sub jects as follows with varying levels 20 and durations of formaldehyde exposure: asymptomati c chiropractic students exposed during 21 anatomy classes (controls with only intermittent ex posure to formaldehyde), mobile home 22 residents, office workers, patients with multiple s ymptoms who had been removed from the 23 source of formaldehyde for at least a year, and occ upationally exposed patients. All groups were 24 assessed for production of antibodies against forma ldehyde-HSA. The level of autoantibodies 25 was significantly elevated in patients exposed long -term to formaldehyde. From the data, 26 Thrasher et al. (1990) concluded that exposure to f ormaldehyde stimulates IgG antibody 27 production to formaldehyde-HSA. 28 Grammer et al. (1990) studied the immunologic natur e of formaldehyde sensitivity in 29 37 workers who were examined by a group of physicia ns in response to complaints of 30 formaldehyde-related illness. Air sampling of form aldehyde ranged from 0.003 to 0.078 ppm, 31 but specific levels were not tied to specific workp lace areas. Blood samples were collected and 32 assayed for IgE and IgG activity against formaldehy de and formaldehyde-HSA. None of the 33 workers had IgG activity against formaldehyde. Fiv e workers had IgE against both HSA alone 34 and against formaldehyde-HSA complexes. No IgE ant ibodies were detected in the other 32 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-41 workers. The authors concluded that there was no e vidence of an immunologically mediated 1 response to formaldehyde in this group of workers. 2 Grammer et al. (1993) described the evaluation of a worker with bronchospasm 3 symptoms caused by formaldehyde exposure. The work er was evaluated by means of ELISA, 4 cutaneous tests, and methacholine and formaldehyde inhalation challenges. The ELISA showed 5 that the worker had positive IgE and IgG titers to formaldehyde-HSA. The worker also had a 6 positive cutaneous test for formaldehyde-HSA but a negative methacholine challenge at 25 7 mg/mL and negative formaldehyde inhalation challeng es at exposure concentrations of 0.3, 1, 3, 8 and 5 ppm for 20 minutes. The worker might have de veloped a positive response if a higher 9 concentration of formaldehyde had been used for the challenge, but it is more probable that the 10 worker’s symptoms were not caused by immunologicall y mediated asthma. 11 Dykewicz et al. (1991) evaluated whether IgE or IgG antibodies to formaldehyde were 12 related to formaldehyde exposure or to respiratory symptoms arising from such an exposure. 13 The authors studied 55 potentially exposed subjects (hospital histology technicians, internal 14 medicine residents, pathology residents, current sm okers, and persons with known workplace 15 exposure to formaldehyde) and compared them to cont rols with no history of formaldehyde 16 exposure. Reported workplace formaldehyde concentr ations were 0.2–0.64 ppm for pathology 17 residents, 0.64 ppm for histology technicians, and 0.6–11 ppm for miscellaneous formaldehyde 18 exposure scenarios. Workplace air concentrations w ere not measured for the other occupations. 19 Occupational exposure to formaldehyde averaged 12.4 5 years for histology technicians, 20 0.38 years for medical residents, 3.21 years for pa thology residents, and 18.34 years for five 21 subjects exposed to formaldehyde in miscellaneous w orkplaces. Blood samples were analyzed 22 for IgE and IgG reactivity with formaldehyde-HSA co mplexes. Three subjects had IgE against - 23 HSA; these three and two others had low levels of a nti-formaldehyde-HSA IgG. The presence of 24 IgG and IgE antibodies to formaldehyde was not clea rly related to formaldehyde exposure or 25 pack-years of smoking. One subject had both IgE an d IgG antibodies and also suffered from eye 26 and respiratory symptoms when exposed to formaldehy de at his workplace. However, the 27 authors concluded that they could not establish a r elationship between IgE and IgG levels and 28 formaldehyde exposure. This study has several limi tations. First, the volunteers (hospital 29 workers) may not be representative of exposed worke rs in the general population. One of the 30 exposure groups comprised cigarette smokers. Altho ugh the study focused on formaldehyde 31 antibodies, which would be unaffected by the other chemicals, respiratory symptoms among 32 smokers would reflect exposures to the constituents of smoke. Dykewicz et al. (1991) concluded 33 that immunologically mediated asthma caused by form aldehyde is extremely rare and may not 34

exist at all. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-42 Carraro et al. (1997) reported development of a rel iable assay to effectively measure 1 formaldehyde-HSA complexes in smokers, ex-smokers, and nonsmokers. A correlation between 2 formaldehyde-HSA antibodies and smoking status was detected. This study did not correlate 3 formaldehyde exposure and formaldehyde-HSA antibodi es. 4 Given that formaldehyde is a sensory irritant that is particularly bothersome to 5 individuals with compromised lung function or asthm a, numerous studies have assessed the 6 ability of formaldehyde to induce immunotoxic effec ts. Some studies have documented 7

increased rates of chronic bronchitis and URT infec tions associated with exposure to 8 formaldehyde, which suggests a possible immunomodul atory effect. However, of the numerous 9 articles that have investigated systemic immunomodu latory effects due to formaldehyde 10 (Lyapina et al., 2004; Gorski et al., 1992; Thrashe r et al., 1990, 1988, 1987; Pross et al., 1987), 11 few have reported significant immune modulation rel ated to formaldehyde exposure. Significant 12 decreases in specific adaptive immune cell populati ons do not appear correlated to formaldehyde 13 exposure (Erdei et al., 2003; Gorski et al., 1992; Thrasher et al., 1990, 1987; Pross et al., 1987). 14 Thus, the tendency for increased infection rates as sociated with formaldehyde may not be related 15 to altered immune function. Perhaps altered mucoci liary clearance and disturbed mucosal barrier 16 may provide greater access for pathogens and result in greater infection rates. Moreover, 17 formaldehyde has been associated with exacerbation of asthmatic or atopic responses, 18 particularly in sensitized individuals. However, t his effect does not appear to occur by increased 19 IgE or formaldehyde-specific IgE levels (Ohmichi et al., 2006; Palczynski et al., 1999; 20 Krakowiak et al., 1998; Wantke et al., 1996b; Salki e, 1991; Grammer et al., 1990; Kramps et al., 21 1989). Thus, formaldehyde-associated enhanced alle rgic responses does not appear to be due to 22 direct induction of sensitization and may not occur via an immunologic mechanism. Lastly, the 23 formation of formaldehyde-heme and formaldehyde-HSA complexes has been well documented 24 (Grammer et al., 1993, 1990; Dykewicz et al., 1991; Thrasher et al., 1990) and may serve as a 25 biomarker of exposure (Carraro et al., 1997). More over, these complexes may provide a means 26 by which formaldehyde travels throughout the bloods tream and may drive antibody formation 27 that may lead to immune activation. 28

29

4.1.1.6. Neurological/Behavioral 30 There is some suggestion of neurological impairment in humans following occupational 31 exposure to formaldehyde; the data are limited and the results from several studies are potentially 32 confounded by exposure to other solvents. Two stud ies of histology technicians with 33 occupational exposure to formaldehyde and other sol vents found neurological deficits and poorer 34 performance on neurocognitive tests associated with formaldehyde exposure (Kilburn et al., 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-43 1987, 1985). In another study, Kilburn and Warshaw (1992) found no change from initial 1 performance, for as long as 4 years, in follow-up e valuations of histology technicians with 2 continuing exposure to formaldehyde. In a prelimin ary report from a prospective study, 3 Weiskopf et al. (2009) found a strong association b etween duration of formaldehyde exposure 4 and death from amyotrophic lateral sclerosis (ALS). In a controlled exposure study, Bach et al. 5 (1990) found that, when workers with chronic formal dehyde exposure were challenged with an 6 acute formaldehyde exposure, they exhibited poorer performance on some neurocognitive tests 7 compared with workers without chronic exposure unde rgoing the same acute challenge 8 conditions. In another controlled exposure study, Lang et al. (2008) found equivocal changes in 9 reaction time following an acute exposure. 10

11

4.1.1.6.1. Epidemiological studies. Kilburn et al. (1985) reported that a group of 76 f emale 12 histology technicians displayed statistically signi ficantly greater frequencies of neurobehavioral 13 deficits (lack of concentration and loss of memory, disturbed sleep, impaired balance, variations 14 in mood, and irritability), than did a referent gro up of 56 unexposed female clerical workers. 15 The technicians had been employed from 2 to 37 year s (mean 12.8 years). Analysis of 16 workplace air samples indicated the presence of sev eral solvents, ranging from 0.2 to 1.9 ppm for 17 formaldehyde, 3.2 to 102 ppm for xylene, 2 to 19.1 ppm for chloroform, and 8.9 to 12.6 ppm for 18 toluene. Subsequently, Kilburn et al. (1987) admin istered a battery of 10 tests to 305 female 19 histology technicians to assess various aspects of cognitive and motor function. The researchers 20 analyzed the results by regression analysis with ag e, years of smoking, and hours per day of 21 exposure to formaldehyde and other solvents as expl anatory variables. Increased daily hours of 22 exposure to formaldehyde were significantly correla ted with decreased performance in several 23 tests (including several types of memory, dexterity , and balance), whereas hours of daily 24 exposure to other solvents were only correlated wit h decreased performance in a single memory 25 test. In a later prospective study of performance, 318–494 histology technicians were tested in a 26 battery of neurobehavioral tests, and testing for a subset of subjects was repeated yearly for up to 27 4 years. No statistically significant decrement in performance was found when initial test results 28 were compared with retest results to evaluate effec ts of continuing occupational exposure to 29 formaldehyde (or other solvents) or possible effect s of aging (Kilburn and Warshaw, 1992). 30 Kilburn (1994) later reported that three anatomists and one railroad worker, occupationally 31 exposed to airborne formaldehyde for 14–30 years, e ach showed impaired performance on 32 several neurobehavioral tests (e.g., choice reactio n time, abnormal balance, digit symbol, and 33 perceptual motor speed). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-44 Weisskopf et al. (2009) evaluated the association b etween chemical exposure and death 1 from ALS, using the cohort of 987,229 people from t he prospective Cancer Prevention Study II 2 of the American Cancer Society. From 1989–2004, 1, 156 deaths from ALS were identified from 3 mortality records from the National Death Index. E xposure assessment occurred prior to follow4 up and was based on a questionnaire; participants w ere asked about current exposure to 12 types 5 of chemicals and whether they had been regularly ex posed in the past. After controlling for a 6 number of potentially confounding factors (includin g age, sex, smoking status, military service, 7 education, alcohol intake, occupation, vitamin use, and exposure to other chemicals), it was 8 found that exposure to formaldehyde for a known dur ation was statistically significantly 9 associated with increased risk of death from ALS ( p < 0.0001) with a relative risk (RR) of 2.47 10 (95% CI: 1.58–3.86) based on 22 deaths. Weisskopf et al. (2009) reported that the association 11 had a strongly significant dose-response relationsh ip, with increased duration of exposure 12 associated with increased RR of ALS mortality with a reported p value for continuous trend of 13 0.0004. Multivariate adjusted rate ratios were 1.5 for known formaldehyde exposures less than 14 4 years, 2.1 for 4–10 years, and 4.1 for >10 years. Although the authors indicated that these 15 results need independent verification, the results of this study of the nearly one million people 16 followed for 15 years is unlikely to be biased due to its longitudinal design. 17

18

4.1.1.6.2. Controlled exposure studies. Bach et al. (1990) examined whether cognitive and 19 motor performance of humans responded acutely to fo rmaldehyde exposure and whether 20 previous chronic exposure to formaldehyde affected the responses observed following acute 21 exposure. Thirty-two men with at least 5 years of occupational exposure to formaldehyde and 22 29 matched controls were exposed to formaldehyde at concentrations of 0.04, 0.21, 0.48, or 23 1.10 ppm for 5.5 hours. During the exposure period , symptoms were assessed by using a 24 standardized questionnaire, and subjects were evalu ated in four tests designed to estimate several 25 aspects of cognitive function. Testing was perform ed once prior to exposure and twice during 26 the exposure period. The authors noted that the ty pical dose-related symptoms of respiratory 27 irritation were not seen in this study. Previously unexposed subjects reported more headaches, 28 “heavy head,” and physical tiredness than the expos ed workers. In both occupationally exposed 29 and unexposed subjects, decreased performance in an addition test was significantly correlated 30 with increasing concentration of formaldehyde. Com pared with previously unexposed subjects, 31 occupationally exposed subjects showed significantl y decreased performance in three other tests, 32 although the effect was not dose related. The stud y did not adjust for several potential 33 confounders, including prior exposure to other chem ical agents, and the age and health status of 34

the cases and controls. Authors concluded that the ir data indicated that acute exposure to 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-45 formaldehyde might cause acute effects on CNS funct ions but that more investigation was 1 needed to verify their results. 2 In a study evaluating chemosensory irritation, Lang et al. (2008) assessed possible 3 changes in reaction time during an acute (4-hour) e xposure to formaldehyde concentrations 4 between 0–0.5 ppm (some exposure sessions also incl uded short concentration peaks of up to 5 1 ppm) with or without a masking agent (ethyl aceta te). Twenty-one healthy volunteers were 6 exposed once per day to each of 10 different exposu re combinations in random order (for a total 7 of 10 sessions per subject). Reaction time was tes ted before and after each exposure session. 8 Significant increases in reaction time were seen at 0.3 ppm formaldehyde, with or without 9 masking agent, but not at 0.5 ppm. The significanc e of these findings is unclear. 10 Performance of 16 healthy volunteers on addition, m ultiplication, and card punching 11 tasks was measured by Andersen and Molhave (1983) b efore and during a 5-hour exposure to 12 formaldehyde at concentrations up to 2 mg/m 3 . The authors reported that formaldehyde 13 exposure had no effect on performance, but results were not presented. 14

15

4.1.1.6.3. Summary. The limited information currently available from hu man studies does not 16 permit a definitive conclusion regarding an associa tion between formaldehyde exposure and 17 human neurotoxicity. There is, however, sufficient information to raise a serious concern for this 18 type of effect, and additional studies are needed. 19 20

4.1.1.7

. Developmental and Reproductive Toxicity 21 Epidemiologic studies suggest a convincing relation ship between occupational exposure 22 to formaldehyde and adverse reproductive outcomes i n women. Several of these studies deal 23 with spontaneous abortion following maternal occupa tional formaldehyde exposure (Taskinen et 24 al., 1999, 1994; John et al., 1994; Seitz and Baron , 1990; Hemminki et al., 1985, 1982; Axelsson 25 et al., 1984), but not all reported a significant a ssociation between exposure and spontaneous 26 abortion. A study of fecundability found an increa se in time to pregnancy among female 27 workers exposed to formaldehyde (Taskinen et al., 1 999). Three studies that examined the effect 28 of occupational exposures on the incidence of conge nital malformation produced mixed results 29 (Dulskiene and Gražulevi č iene, 2005; Taskinen et al., 1994; Hemminki et al., 1985). A 30 population-based, semi-ecologic study found an asso ciation between atmospheric formaldehyde 31 exposure and low birth weight (Gražulevi č iene et al., 1998). 32

33

4.1.1.7.1. Spontaneous abortion. Several epidemiologic studies report a relationship between 34 occupational exposure to formaldehyde and increases in risk of spontaneous abortion following 35 maternal occupational formaldehyde exposure (Taskin en et al., 1999, 1994; John et al., 1994; 36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-46 Seitz and Baron, 1990; Axelsson et al., 1984). Inc reased RRs were in the range of 1.7 to more 1 than 3.0. However, other studies (Hemminki et al., 1985, 1982) found no association between 2 occupational formaldehyde exposure and spontaneous abortion. Paternal occupational exposure 3 to formaldehyde was not related to spontaneous abor tion (Lindbohm et al., 1991). 4 The earliest report of an association between spont aneous abortion and formaldehyde 5 exposure comes from a Swedish cohort study of femal e laboratory workers (Axelsson et al., 6 1984). Subjects were women born in 1935 or later a nd worked in a university laboratory during 7 1968–1979. There were 745 women who responded to a mailed questionnaire (response rate = 8 95%), 556 of whom reported on 1,180 pregnancies tha t resulted in 997 births. Exposure to 9 formaldehyde was estimated based on answers to the questionnaires. Formaldehyde exposure 10 was reported only in connection with 10 pregnancies , of which 5 went to term, 3 were reported 11 as miscarriages, and 2 were terminated by induced a bortion. Excluding the latter, the 12 spontaneous abortion rate among women exposed to fo rmaldehyde in the first trimester was 3/8 13 (37.5%) compared with 14/148 (9.5%) in the populati on of laboratory workers not exposed to 14 any solvent in the first trimester. 15 While not computed by the authors, the OR can be ca lculated as 5.7 (95% CI: 1.2–26.6). 16 The exposure assessment on which this result is bas ed was methodologically weak but unlikely 17 to be a source of bias. Given the exploratory natu re of this study, potential confounders were not 18 controlled for, but no other co-exposure was more s trongly related to the increased risk of 19 miscarriage, so this result is not likely to be exp lained by confounding. Selection bias is also an 20 unlikely explanation given the high participation r ate. However, although this association of an 21 increased risk of pregnancy loss with formaldehyde exposure is statistically significant, the CI is 22 wide and chance may be a possible explanation for t his finding. 23 In a 1988 Health Hazard Evaluation, the National In stitute for Occupational Safety and 24 Health (NIOSH) investigated complaints of adverse r eproductive outcomes at a plant where 25 work pants were cut and sewn with a fabric that was treated with a resin that releases 26 formaldehyde (Seitz and Baron, 1990). In a NIOSH l aboratory, the fabric released 163 to 1,430 27

μ g of formaldehyde/gram of cloth. TWA personal brea thing space formaldehyde levels ranged 28 from trace to 0.46 ppm, while workstation values ra nged from 0.32 to 0.70 ppm. The 29 investigators studied the outcomes by using a maile d questionnaire. The response rate for 30 current employees was 98%. There were 296 pregnanc ies among a cohort of 188 women. The 31 investigators found increased rates of spontaneous abortion, premature birth, and congenital 32 malformations. The crude rate of spontaneous abort ion was 21% among women working at the 33 plant while pregnant (4 of 19 pregnancies), 15% amo ng women employed elsewhere while 34 pregnant (11 of 71 pregnancies), and 5% among women at home while pregnant (10 of 206 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-47 pregnancies). The investigators did not explain ho w workers employed elsewhere or at home 1 during pregnancy were categorized compared with cur rent workers, nor did they calculate RRs. 2 As calculated from data presented in Table 5 of the monograph, the crude OR (not corrected for 3 multiple observations per woman) for those pregnant while currently working at the plant 4 compared with all others was 3.2 (95% CI: 0.8–12). There were also excess congenital 5 malformations (13 versus 2%) and premature births ( 13 versus 4%) among the live births (both 6 based on two births each in the exposed group) from the women who were pregnant while 7 employed at the textile plant compared with women w ho stayed at home. After the NIOSH 8 investigation, changes were made in the plant to im prove ventilation. 9 Because the report provides insufficient details of the methodology and the fact that there 10 was no personal exposure classification in this stu dy, it is difficult to validate the findings in thi s 11 report. The results did not take into account othe r potential risk factors for spontaneous abortion 12 or correct for multiple pregnancies per woman. Fur thermore, the marked differences between 13 the “home” and “work” pregnancies were difficult to interpret. 14 A cohort study of effects of paternal occupational exposures in Finland by Lindbohm et 15 al. (1991) found that exposure to formaldehyde had little effect on the rate of spontaneous 16 abortions among 99,186 pregnancies listed in the na tional hospital discharge register. The 17 analysis was limited to births/spontaneous abortion s in 1976 and from May 1980 to April 1982. 18 Spontaneous abortion incidence came from the hospit al discharge register and data from 19 outpatient clinics. There were 808 pregnant wives among potentially formaldehyde-exposed 20 fathers. Exposure to formaldehyde was based on emp loyment information listed in the Finnish 21 1980 census. Compared with pregnancies among wives of unexposed spouses, the age and 22 socioeconomic level-adjusted ORs were 1.1 for low p aternal exposure to formaldehyde and 1.0 23 for moderate to high paternal exposure. Paternal o ccupational exposures to ethylene oxide, 24 gasoline/benzene, and rubber industry chemicals wer e associated with spontaneous abortion. 25 The authors hypothesized that the mode of action (M OA) for spontaneous abortion following 26 male exposure to chemicals is genetic damage to ger m cells. 27 The indirect exposure assessment was a substantial limitation of this study. Some 28 confounders in a study of this type could not be co ntrolled for (smoking history, previous 29 spontaneous abortions, alcohol use), and census dat a could not provide completely accurate 30 information, potentially masking associations betwe en paternal formaldehyde exposure and 31 spontaneous abortion. 32 A case-control study by Taskinen et al. (1994) of e ffects of maternal occupational 33 exposure to chemicals used in laboratories in Finla nd indicated that exposure to formalin, which 34 is a 37% aqueous solution of formaldehyde, was rela ted to an increased risk of spontaneous 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-48 abortion. The investigators studied subjects from payrolls of Finnish state-employed laboratory 1 workers, the laboratory workers’ union, and a regis ter of workers occupationally exposed to 2 carcinogens. These records were cross-referenced w ith the hospital discharge register. The 3 investigators selected women who had a single spont aneous abortion during the period 1973– 4 1986 and two controls who had delivered a baby with out malformations. The final sample size 5

was 208 cases and 329 controls after refusals and o ther exclusions. The response rate was 6

82.4%. 7 Information on occupational exposure, health status , medication, contraception, and 8 pregnancy history came from mailed questionnaires. Industrial hygienists’ construction of an 9 exposure index was based on the subjects’ descripti ons of their work assignments, use of 10 solvents including estimates of quantity used, and use of a fume hood. ORs were adjusted for 11 employment, smoking, alcohol consumption, parity, p revious miscarriage, birth control failure, 12 febrile disease during pregnancy, and other organic solvents found in laboratory work. 13 Spontaneous abortion was associated with 3–5 days p er week of formalin exposure (OR 3.5 14 [95% CI: 1.1–11.2]). A contemporaneous study of fo rmaldehyde concentrations in similar 15 Finnish workplaces (pathology and/or histology labo ratories) reported workroom air to range 16 from 0.01 to 7 ppm with a mean of 0.45 ppm formalde hyde (Heikkilä et al., 1991 [as cited in 17 Taskinen et al., 1994]) and that the highest exposu res occurred during emptying of sample 18 containers, dish washing, and preparation of formal dehyde solution. 19 Although the results of this study indicate an incr eased risk between spontaneous 20 abortion and exposure to formaldehyde/formalin, the women were also exposed to several 21 chemicals concurrently, of which toluene (OR 4.7 [9 5% CI: 1.4–15.9]) and xylene (OR 3.1 [95% 22 CI: 1.3–7.5]) were also significantly associated wi th the incidence of spontaneous abortion. 23 However, the investigators reported that the women were more likely to be co-exposed to 24 formalin and xylene, which would make confounding b y toluene less likely, and, since xylene 25 was not as strongly associated with the outcome as was formaldehyde, it too is unlikely to fully 26 explain the reported relationship between formaldeh yde and increased risk of spontaneous 27 abortion. While it is possible that exposure miscl assification may have occurred because of the 28 indirect assessment of workplace chemical exposure, an overall conclusion is that, since the 29 exposure assessment was conducted by industrial hyg ienists, it is unlikely that this form of bias 30 will have impacted the results of the study to any great extent. 31 In a U.S. study (John et al., 1994), the results of a case-control study of cosmetologists 32 also supported an association between spontaneous a bortion and the use of formaldehyde-based 33 disinfectants. The study population came from the 1988 North Carolina cosmetology license 34 registry. Women on this list who were 22–36 years of age were screened to find those who were 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-49 recently pregnant. The cases were full-time cosmet ologists who experienced a spontaneous 1 abortion before gestational week 20 during 1983–198 8. The most recent spontaneous abortion 2

was used as the reference case. Controls were full -time cosmetologists who delivered a live 3 infant during the same time period. 4 Information was based on mailed questionnaires. Wo men were not told the purpose of 5 the study in order to avoid selection and recall bi as. Of 8,356 women who received the 6 screening questionnaires, 72.5% responded. Of thos e, 1,696 qualified for the study and 73.6% 7 completed a more detailed questionnaire. Among the m, 96 women were “absolutely sure” they 8 had a spontaneous abortion and qualified as cases. There were 1,058 live births that qualified as 9 controls. Exposure assessment included identificat ion of disinfectants used as well as types of 10 chemicals used on hair, use of gloves, hours worked , number of procedures involving chemicals, 11 and use of manicure products. Presence of formalde hyde in the cosmetology profession in 12 general was confirmed in two NIOSH hazard reports ( Almaguer and Klein, 1991; Almaguer and 13 Blade, 1990). ORs were adjusted for age, smoking, pregnancy characteristics, other jobs, hours 14 worked, education (cosmetology school), hours stand ing per week, number of chemical 15 procedures per week, hair dyes per week, bleachings per week, permanents per week, use of 16 gloves, beauty salon characteristics, and use of al cohol or formaldehyde disinfectants. 17 An elevated OR of 2.1 (95% CI: 1.0–4.3) was reporte d with the use of formaldehyde18 based disinfectants adjusted for maternal character istics and other workplace exposures. Other 19 chemical exposures were also associated with sponta neous abortion, including number of 20 chemical services per week, hair dyes, bleaches, an d alcohol

- based disinfectants. Strengths of 21 this study include adjustment for important confoun ding risk factors for spontaneous abortion, 22 detailed collection of interview-based information, a favorable response rate, and the fact that the 23 index population had a high likelihood of formaldeh yde exposure. These data provide overall 24 support for an association between formaldehyde exp osure and spontaneous abortion. 25 In a retrospective cohort study by Taskinen et al. (1999) of female woodworkers in 26 Finland, exposure to formaldehyde was associated wi th delayed conception and spontaneous 27 abortion. The subjects, recruited from a woodworke rs’ union and other businesses involving 28 wood processing, were linked to a national register of births. Women were included if they were 29 born between 1946 and 1975, had a live birth at age 20–40 years during 1985–1995, had worked 30 in the wood processing industry for at least 1 mont h, and had first employment in the wood 31 processing industry beginning at least 6 months bef ore the index pregnancy. The first pregnancy 32 that fulfilled the above criteria was the index pre gnancy. There were 1,094 women with these 33 criteria. Information about personal characteristi cs, pregnancies, and exposures were collected 34 from mailed questionnaires for which the response r ate was 64%. After other exclusions 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-50 (primarily infertility history, unknown time to pre gnancy, and contraceptive failure), the final 1 sample included 602 women. 2 Estimates of mean daily exposure to formaldehyde we re based on measurements taken at 3 the women’s factories of employment during the earl y 1990s. Where measurements were 4 unavailable, measurements from equivalent industrie s were used. An exposure index 5 representing a TWA exposure was established for eve ry person in the study based on the 6 concentration of workplace formaldehyde multiplied by the proportion of the workday exposed 7 to formaldehyde. The investigators categorized TWA formaldehyde exposure into categories of 8 low (mean of 18 ppb), medium (mean of 76 ppb), and high (mean of 219 ppb) exposure. 9 Time-to-pregnancy data were analyzed by a discrete proportional hazards regression 10 procedure with, as the outcome, a fecundability den sity ratio (FDR), in which a ratio of average 11 incidence densities of pregnancies for exposed wome n was compared with that of the employed, 12 unexposed women. As explained by Taskinen et al. ( 1999), an FDR significantly below unity 13 suggests that conception was delayed. The age-, em ployment-, smoking-, alcohol consumption-, 14 parity-, and menstrual irregularity-adjusted FDR wa s 0.64 (95% CI: 0.43–0.92) for women 15 exposed to high formaldehyde levels compared with t he unexposed controls, indicating that there 16 was a substantial delay in time to conception in th is group of women. Among a subset of women 17 with high exposure who did not use gloves, the FDR was even lower (0.51 [95% CI: 0.28–0.92]), 18 suggesting that these results might be explained in part through dermal contact with 19 formaldehyde or might indicate an individual’s fail ure to follow appropriate precautions, which 20 might have increased inhalation exposures in other ways. Exposure to solvents, wood dust and 21 other dusts, and phenols was not associated with de creased fecundability. 22 The investigators further conducted an analysis of the risk of spontaneous abortion after 23 carefully including only women who had the same wor kplace during the year of the spontaneous 24 abortion as they had during the beginning of the ti me-to-pregnancy period. Spontaneous 25 abortion was associated with formaldehyde exposure in the low exposure group (OR = 2.4 [95% 26 CI: 1.2–4.8]), in the medium exposure group (OR = 1 .8 [95% CI: 0.8–4.0]), and in the high 27 exposure group (OR = 3.2 [95% CI: 1.2–8.3]). Endom etriosis was also associated with the 28 highest formaldehyde level (OR = 4.5 [95% CI: 1.0–2 0.0]). 29 This study by Taskinen et al. (1999) was a well-des igned population-based case-control 30 study that appears to have been well executed and a ppropriately analyzed. The study population 31 of Finnish women was well defined and adequately se lected so as to allow for meaningful 32 comparisons of health effects between individuals w ith different levels of exposure to 33 formaldehyde. The participation rate was 64%, whic h is low enough to raise a concern about the 34 potential for selection bias. However, the authors noted that selection bias has not influenced the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-51 results of other reproductive epidemiology studies reporting results on smoking, irregular 1 menstruation, and earlier miscarriages, which are k nown to lengthen the time to pregnancy 2 (Bolumar et al., 1996; Sallmén et al., 1995; Baird and Wilcox, 1985). Furthermore, there is no 3 evidence to support conjecture that an individual’s decision to participate in this study would be 4 differential with respect to their workplace formal dehyde exposures while being non-differential 5 with respect to the other exposures of interest, in cluding organic solvents, wood dust, and 6 phenols. Since the women who chose to participate in this study were not likely to be aware of 7 the specific hypotheses under investigation, nor co uld they have known the formaldehyde 8 exposures that were independently estimated by an i ndustrial hygienist, selection bias is not a 9 likely explanation for the findings of adversity. 10 Data on pregnancy history, including spontaneous ab ortions, were collected by 11 questionnaire. Spontaneous abortion is the most co mmon adverse outcome of pregnancy (Klein 12 et al., 1989), and retrospective self-report of spo ntaneous abortion has been found to match well 13 with prospectively collected reproductive histories (Wilcox and Horney, 1984). Many 14 spontaneous abortions, however, are missed with sel f-reporting with the magnitude likely 15 exceeding 25%, but only rarely do women self-report false positive events (Wilcox and Horney, 16 1984). The effect of such an undercount is to caus e a bias towards the null when the likelihood 17 of undercounting is unrelated to formaldehyde expos ure. The implication is that the observed 18 association of increased risk of spontaneous aborti on associated with occupational exposure to 19 formaldehyde may be an underestimation of the true risk. 20 Two studies (Hemminki et al., 1985, 1982) specifica lly assessed the effects of 21 formaldehyde exposure and reported no significant i ncrease in the risk of spontaneous abortion. 22 Hemminki and colleagues (1982) conducted a retrospe ctive cohort study of nurses who were 23 potentially exposed to chemical sterilizing agents, including formaldehyde, ethylene oxide, and 24 glutaraldehyde. The risk of having a spontaneous a bortion among the women on the sterilizing 25 staff was compared with that among the control popu lation of nursing auxiliaries whom the 26 supervisory nurses thought to be unexposed to the c hemical sterilizing agents during the previous 27 three decades. However, no measurements of the che mical sterilizing agents were taken. 28 Information about exposure to chemical sterilizing agents was obtained from the supervising 29 nurses. When the women were conducting sterilizing procedures during their pregnancies, the 30 frequency of spontaneous abortion was 15.1% compare d with 4.6% for the nonexposed 31 pregnancies among the sterilizing staff. The incre ased frequency of spontaneous abortion 32 correlated with exposure to ethylene oxide but not with exposure to glutaraldehyde or 33 formaldehyde. The investigators reported that ethy lene oxide concentrations have been 34 measured in many sterilizing units in Finnish hospi tals; 8-hour weighted mean concentrations 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-52 have ranged from 0.1 to 0.5 ppm with peak concentra tions up to 250 ppm (measurements by the 1 Finnish Institute of Occupational Health) (Hemminki et al, 1982). No measurements of 2 glutaraldehyde concentrations were available. Hemm inki et al. (1982) reported that exposure to 3 formaldehyde in the sterilization units may be mini mal, particularly when gas chambers are used. 4 The range of formaldehyde concentrations measured i n sterilizing units has been reported as 5 0.03–3.5 ppm. 6 It is not clear that the unexposed women who serve d as controls were an appropriate 7 comparison group to the sterilizing staff. The inv estigators reported that, among the sterilizing 8 staff, those women who were unexposed during pregna ncy experienced a rate of spontaneous 9 abortion of 4.6% but that, among the comparison pop ulation of nursing auxiliaries who were 10 presumed to be unexposed, the rate of spontaneous a bortion was 10.5%. Had the nursing 11 auxiliaries been an appropriate comparison group, i t would be expected that their rate of 12 spontaneous abortion would be similar to the unexpo sed sterilizing staff. Given this anomaly in 13 study design and the unknown concentrations of form aldehyde exposure that were assessed as 14 positive or negative by supervisory nurses regardin g occupational exposures in the previous 15 30 years, it is concluded that this report of no as sociation between formaldehyde exposure and 16 the risk of spontaneous abortion does not temper th e conclusion that formaldehyde exposure has 17 been shown to increase the risk of spontaneous abor tion. 18 A second study by the same lead author (Hemminki e t al., 1985) used a different study 19 design to reassess the hypothesis that chemical exp osures common in the field of nursing could 20 be risk factors for spontaneous abortion. This cas e-control study found no increase in the risk of 21 spontaneous abortion associated with exposure to fo rmaldehyde. The head nurses at each 22 hospital were asked by the investigators whether ea ch case or control had been exposed to 23 formaldehyde during a given 3-month period correspo nding to the first trimester of a study 24 participant’s pregnancy during 1973–1979. Formalde hyde exposure was assessed as positive or 25 negative for either use as a sterilizing agent or u se of sterilized instruments. The reported crude 26 OR for formaldehyde exposure was 0.6; no CIs were p rovided. From the data reported in 27 Table 2 in Hemminki et al. (1985), the unadjusted O R and its CI can be computed post hoc as 28 OR (0.70 [95% CI: 0.28–1.73]). The authors acknowl edged that the study failed to distinguish 29 between sterilizing work and the use of sterilized instruments, where only very small exposures 30 could be expected. Given the likelihood of extreme exposure misclassification and the 31 presentation of only crude results without control of potential confounding for formaldehyde, 32 these results do not appear to be exculpatory of a true causal association between formaldehyde 33 exposure and the risk of spontaneous abortion. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-53 A meta-analysis of formaldehyde exposure and sponta neous abortion was conducted by 1 Collins et al. (2001b). However, the published res ults should be interpreted with caution. This 2 meta-analysis included one very large null study of paternal formaldehyde exposure along with 3 seven studies of maternal exposure. The two null s tudies by Hemminki et al. (1985, 1982) were 4 also included without consideration of the potentia lly extreme exposure misclassification that 5 may have attenuated any true adverse effect. Never theless, the overall reported meta-analytic 6 RR for parental formaldehyde exposure based on eigh t maternal and paternal exposure studies 7 was 1.6 (95% CI: 0.9–2.7). For case-control studie s the RR was 1.8 (95% CI: 0.7–4.8), and for 8 cohort studies the RR was 1.7 (95% CI: 1.2–2.3). C ollins et al. (2001b) argued that the method 9 of exposure evaluation may have influenced the obse rved results; they stated that several of the 10 studies whose exposures were based on the investiga tor’s judgment were likely misclassified, 11 which may have obscured the true relationship, whil e other studies that assessed exposure based 12 on self-reporting could have suffered from recall b ias. They report that RRs were higher for 13 studies based on self-reported exposures (RR = 1.9 [95% CI: 1.3–2.6]) than those based on 14 objective exposure assessments (RR = 1.5 [95% CI: 0 .6–3.7]) and suggested that this difference 15 might reflect recall bias in the exposure assessmen t. However, for recall bias to have been 16 operable in these studies, the women who provided s elf-reported data on pregnancy history and 17 occupational exposure would have had to appreciate that the hypothesis of interest was the 18 specific effect of formaldehyde on the risk of spon taneous abortion. In the specific case of the 19 study by Taskinen and colleagues (1999), the invest igator also looked at the effects of other 20 exposures, such as organic solvents, dust, and phen ols, and did not report adverse effects. It is 21 therefore unlikely that the women providing exposur e data were doing so in a manner indicative 22 of recall bias. If the supposition of non-differen tial misclassification error in exposure is indeed 23 correct, the observed results of the meta-analysis would likely have been biased towards the null. 24 Therefore, the true RR for maternal formaldehyde co uld be higher than Collins et al. (2001b) 25 reported and would likely be statistically signific ant. Had the study of paternal exposure been set 26 aside, the meta-analysis almost surely would have s hown a statistically significant increase in the 27 risk of spontaneous abortion associated with matern al formaldehyde exposure. This single study 28 reported a null finding based on exposure assessmen t from census records of employment, and, 29 as the largest of the studies in the meta-analysis, it contributed the greatest weight. 30 Lastly, Collins and coworkers (2001b) suggested tha t there were potential confounding 31 factors in each of the workplaces that might have p roduced the observed findings of increased 32 risk of spontaneous abortion associated with formal dehyde. While each of these occupational 33 studies focused on women who were co-exposed to for maldehyde and other chemicals, the 34 occupational groups were quite different and had di fferent sets of co-exposures. The 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-54 woodworkers in the Taskinen et al. (1999) study wer e potentially co-exposed to organic solvents 1 related to painting and lacquering, dusts, and phen ols, none of which was shown to be an 2 independent predictor of adverse risk. The cosmeto logists studied by John et al. (1994) were 3 co-exposed to hair dyes, bleach, alcohol-based disi nfectants, and chemicals specific to services, 4 such as fingernail sculpturing, but, in analyses th at were specifically adjusted for other work 5 exposures and their potentially confounding effects , the investigators reported an OR of 2.1 6 (95% CI: 1.0–4.3) for the use of formaldehyde-based disinfectants. The laboratory workers 7 studied by Axelsson et al. (1984) were potentially co-exposed to a wide range of solvents, but the 8 miscarriage rate was highest among those exposed to formaldehyde, and, for a potential 9 confounder to entirely explain an observed effect o f another exposure, it must be more strongly 10

associated with the adverse outcome. 11 It does not appear that the collective results of f ormaldehyde exposures associated with 12 increased risk of spontaneous abortion—often in spi te of exposures being crudely measured— 13 can be explained by information bias or confounding

. 14 The findings by Taskinen et al. (1999) of reduced f ertility and increased risk of 15 spontaneous abortion are internally consistent and coherent with other reports of increased risk 16 of pregnancy loss associated with exposure to forma ldehyde (John et al., 1994; Taskinen et al., 17 1994; Seitz and Baron, 1990; Axelsson et al., 1984) . Absent evidence of alternative explanation 18 for these findings, it is concluded that exposure t o formaldehyde is associated with pregnancy 19 loss and diminished fertility. 20

21

4.1.1.7.2. Congenital malformations. Only three studies have reported on the epidemiolog ic 22 evidence of an association between formaldehyde exp osure and the risks of births having 23 congenital malformations. In the earliest study by Hemminki et al. (1985), the investigators 24 presented an analysis of 34 congenital malformation s from the Finnish Register of Congenital 25 Malformations and compared them with a group of 95 controls from those used in the larger 26 study. An association was found between formaldehy de exposure and malformations based on 27 three exposed cases (OR = 1.8). 28 The case-control study by Taskinen et al. (1994) of effects of occupational exposure to 29

chemicals used in laboratories in Finland examined the potential effects of exposure to formalin 30 on both spontaneous abortions and congenital malfor mation. The investigators reported on a 31 study of 36 laboratory workers with a child registe red in the Finnish Register of Congenital 32

Malformations and 105 controls. There was no assoc iation between formalin and congenital 33

malformations. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-55 A Lithuanian study (Dulskiene and Gražulevi č iene, 2005) for which only a brief 1 summary is available in English investigated the ri sk of congenital heart malformations as a 2 result of exposure to 43 different agents. The num ber of births included in the study was not 3 given in the English abstract. Exposure to residen tial ambient formaldehyde concentrations of 4 >2.42 μg/m 3 (0.002 ppm) was associated with a 24% increase in the risk of congenital heart 5 malformations (OR = 1.24 [95% CI: 0.81–2.07]). The details of this study are unavailable in 6 English translation, making it impossible to critic ally analyze details, such as co-exposure and 7 other possible confounders. 8

9

4.1.1.7.3. Low birth weight. A case-control study by Gražulevi č iene et al. (1998) examined the 10 association of low birth weight (<2,500 grams) and air pollutants, including formaldehyde, 11 particulates, sulfur dioxide, lead, ozone, and nitr ogen dioxide, measured in 12 areas in the city of 12 Kaunas, Lithuania. This city has conducted environ mental pollutant measurements since 1993, 13 and the investigators classified formaldehyde expos ure based on the area of residence of the 14 study subjects. Formaldehyde levels in the 12 dist ricts of Kaunas in 1994 ranged from 1.36 to 15 5.28 μg/m 3 (0.0011–0.0043 ppm), with a citywide average of 3. 14 μg/m 3 (0.0026 ppm). 16 Information on infants came from a birth registry. There were 244 cases of low birth weight and 17 4,089 normal controls born in 1994. Personal data came from record-based prenatal interviews, 18 and pregnancy data came from hospital records. 19 The crude RR of low birth weight among women expose d to the highest airborne 20 formaldehyde level was 1.68 (95% CI: 1.24–2.27). A fter adjustment for age, occupation, 21 hazardous work, education, marital status, smoking, hypertension, and other air pollutants, the 22 OR was still elevated but no longer statistically s ignificant (OR 1.37 [95% CI: 0.90–2.09]). 23 Although formaldehyde exposure was the only single air pollutant associated with low birth 24 weight, factors such as smoking, marital status, an d pregnancy-related factors had more of an 25 impact on birth weight. Total suspended particulat es (OR 2.58 [95% CI: 1.34–4.99]) and 26 hazardous work (OR 2.62 [95% CI: 1.12–6.10]), which was not defined by the authors, were also 27 related to low birth weight. 28 Aside from studies of birth weight deficits from to bacco smoke and occupational 29 exposure, the literature on exposure to ambient air pollutants to support the investigators’ 30 hypothesis is limited. The strength of the associa tion between total suspended particulates and 31 low birth weight supports the idea that incidence o f birth weight <2,500 grams may be related to 32 atmospheric pollution, although this finding may no t be specific to formaldehyde. Because of 33 the large number of variables evaluated in the anal ysis, large fluctuations in the atmospheric 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-56 formaldehyde measurements, co-exposure to other pol lutants, and geographic variability of low 1 birth weight, it is difficult to estimate the impac t of formaldehyde alone on low birth weight. 2

3

4.1.1.7.4. Summary. Although all studies on potential developmental tox icity of formaldehyde 4 have limitations and do not uniformly report positi ve results, the associations between 5 spontaneous abortion, delayed conception, or reprod uctive outcomes and formaldehyde exposure 6 in multiple studies cannot be dismissed, because se veral studies report concordant findings 7 across several populations and study methodologies. The results of most of the studies with 8 positive findings were adjusted for many potentiall y confounding factors that may be related to 9 spontaneous abortion and infertility, including smo king and alcohol use, pregnancy and 10 reproductive history, and other chemical exposures. 11 The association between fertility and formaldehyde (Taskinen et al., 1999) stands out 12 because of its strong quantitative statistical anal ysis, adequate sample size, and rigorous exposure 13 assessment. This study was designed to specificall y assess the effect of formaldehyde on 14 reproductive outcomes. Furthermore, it was the onl y study with an exposure assessment based 15 on quantitative measurements from the subject’s wor kplace. Moreover, the investigators 16 conducted a multivariable survival analysis that ap proximates a longitudinal life table or person17 year analysis while simultaneously adjusting for im portant confounders. The findings were 18 strengthened by statistically significant associati ons between formaldehyde and spontaneous 19

abortion and endometriosis. The fact that the use of gloves may reduce the reproductive effect of 20 formaldehyde supports the dose-response relationshi p in this study, and the lack of an association 21 between time to pregnancy and any other workplace e xposures strengthens the specificity of 22 formaldehyde effects. The results also support ass ociations reported between formaldehyde and 23 increased risk of spontaneous abortion because subf ertility and spontaneous abortion are 24 biologically linked (subclinical pregnancy losses a re increased among women with fertility 25 problems) (Gray and Wu, 2000; Hakim et al., 1995), and both subfertility and spontaneous 26 abortion may be related to sensitivity to environme ntal agents (Correa et al., 1996). 27

28

4.1.1.8

. Oral Exposure Effects on the Gastrointestinal Tract 29 No human epidemiology studies exist to determine a n association between oral exposure 30 of formaldehyde and adverse health effects in the g astrointestinal (GI) tract. 31

32

4.1.1.9. Summary: Noncarcinogenic Hazard in Humans 33 Formaldehyde has clearly and consistently been show n to be a potent sensory irritant 34 with a variety of adverse health effects. Eye, nos e, and throat irritation as a result of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-57 formaldehyde exposure has been documented in a wide range of epidemiologic studies. Workers 1 chronically exposed to formaldehyde have exhibited signs of reduced lung function consistent 2 with BC, inflammation, or chronic obstructive lung disease. A well-conducted residential 3 epidemiology study has convincingly shown a concent ration response for decreased pulmonary 4 function among children with increased formaldehyde exposures. Several cross-sectional studies 5

have described associations between increased conce ntrations of formaldehyde and increased 6 prevalence of asthma. However, two case-control st udies that focused on risk factors for the 7 initial physician diagnosis of asthma, which is ind icative of atopic switching, have also shown 8 concentration-dependent adverse effects associated with formaldehyde exposure. 9 Results of research on the effects of formaldehyde on tissue histology suggest that 10 formaldehyde is also responsible for reduced mucoci liary clearance and the induction of 11 histopathologic lesions in the nose. In addition, there is evidence of neurological impairment in 12 several studies of formaldehyde-exposed histology t echnicians, but confounding exposures to 13

other neurotoxic solvents and inconsistent results prevent drawing definitive conclusions 14 concerning the neurotoxicity of formaldehyde from t hese studies. 15 Finally, there is epidemiologic evidence that forma ldehyde is associated with adverse 16 reproductive outcomes. Four of six occupational st udies found an increased risk of spontaneous 17 abortion among formaldehyde-exposed women. Results of other studies suggested associations 18 among formaldehyde and congenital malformations, lo w birth weight, and endometriosis. The 19 strongest evidence of an association between formal dehyde and an adverse reproductive outcome 20 came from a well-conducted study of infertility in women employed in the wood processing 21 industry. This study found a greater than threefol d increased risk of spontaneous abortion, a 22 nearly 50% decrease in a measure of delayed concept ion indicating reduced fertility, and 23 increased time to pregnancy associated with average daily formaldehyde exposures of 0.15– 24 1 ppm. 25

26

4.1.2. Cancer Health Effects 27

4.1.2.1. Respiratory Tract Cancer 28

4.1.2.1.1. NPC. NPC is a very rare form of cancer. The incidence i s less than 1 per 100,000 29 persons throughout most parts of the world. The mo st common form of NPC arises from the 30 epithelial cells lining the nasopharynx. This pres entation constitutes between 75 and 100% of all 31 NPCs. There are two types, squamous cell carcinoma (SCC) and nonkeratinizing carcinoma. In 32 the U.S., the 5-year survival rate for NPC is about 25% (Burt et al., 1992). Certain risk factors 33 have been implicated in its etiology, including Eps tein-Barr virus (EBV), wood dust and 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-58 particles applied to wood in its treatment, exhaust fumes, occupational smoke, and nitrosamines. 1 The epidemiologic studies of NPC are summarized in Table 4-1. 2

3

4.1.2.1.1.1. Cohort studies. The International Agency for Research on Cancer (IA RC) reported 4 on eleven cohort studies of formaldehyde-exposed in dustry workers (Marsh et al., 2002, 1996, 5 1994; Andjelkovich et al., 1995; Gardner et al., 19 93; Bertazzi et al., 1989, 1986; Stayner et al., 6 1988; Blair et al., 1987, 1986; Edling et al., 1987 ) and results from eight cohort studies of 7 professional workers (Hall et al., 1991; Hayes et a l., 1990; Stroup et al., 1986; Harrington and 8 Oakes, 1984; Levine et al., 1984; Walrath and Fraum eni, 1984, 1983; Friedman and Ury, 1983) 9 (IARC, 2006). 10 Several of these studies measured exposure to forma ldehyde at 10 production facilities 11

that contributed to a cohort that has been studied by Blair et al. (1987, 1986) and Hauptmann et 12 al. (2004). The National Cancer Institute (NCI) co nducted a mortality study of solid tumors 13 among a cohort of 25,619 workers who were employed in these 10 plants that produced or used 14 formaldehyde in the U.S. before 1966 (Blair et al., 1987, 1986). Subjects were followed to 15 January 1, 1980, accruing approximately 600,000 per son-years of follow-up. Hauptmann et al. 16 (2004) updated the cohort to December 31, 1994 and reported a significant excess risk of NPC in 17 exposed workers based on U.S. population death rate s (standardized mortality ratio [SMR] = 2.1 18 [95% CI: 1.05–4.21]). In addition to the SMR based on an external comparison population, RRs 19 were presented based on internal comparisons of sim ilar workers in order to minimize potential 20 selection bias due to the well-known healthy worker effect (HWE). For NPC, RRs increased 21 with several different exposure metrics, including average exposure intensity, cumulative 22 exposure, highest peak exposure, and duration of ex posure to formaldehyde ( p values for tests 23 for trends were 0.066, 0.025, <0.001, and 0.147, re spectively). These results were based on 24 primary data analyses of the health and exposure da ta collected by the NCI, according to their 25 research protocol and analyzed accordingly. As suc h, the reported statistical p values may be 26 appropriately interpreted as showing that these wor kers were at increased risk of NPC associated 27 with exposure to formaldehyde. These NCI investiga tions controlled for potential selection bias 28 due to the HWE and for several potential confounder s, including calendar year, age, sex, race, 29 and pay category. There was no evidence of any dif ferential measurement error that could have 30 produced the observation of a spurious association. Any non-differential measurement error 31 would likely have led to an observed effect of form aldehyde that was less than that which would 32

otherwise have been observed in the absence of meas urement error. 33This document is a draft for review purposes only a nd does not constitute Agency policy 4-59 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)OverallNonexposed SMR1.56(95% CI: 0.39–23) (2)Exposed SMR2.10(95% CI: 1.05–21) (8)Peak exposure (ppm)0 RRb1.00(95% CI: NS) (2)>0 to <2.0 N/A(95% CI: NS) (0)2.0 to <4.0 N/A(95% CI: NS) (0)4.0 or greater 1.83(95% CI: NS) (7)Trend p < 0.001Average intensity of exposure (ppm)0 RRb1.00(95% CI: NS) (2)≤0.5 N/A(95% CI: NS) (0)0.5 to <1.0 0.38(95% CI: NS) (1)1.0 or greater1.67(95% CI: NS) (6)Trend p = 0.066Cumulative exposure (ppm-years)0 RRb2.40(95% CI: NS) (2)>0 to <1.5 1.00(95% CI: NS) (3)1.5 to <5.5 1.19(95% CI: NS) (1)5.5 or more 4.14(95% CI: NS) (3)Trend p = 0.025Duration (years)0 RRb1.77(95% CI: NS) (2)>0 to <5 1.00(95% CI: NS) (4)5 to <15 0.83(95% CI: NS) (1)15 or more 4.18(95% CI: NS) (2)Hauptmann et al. (2004) Retrospective cohortmortality study of 25,619workers employed at 10formaldehyde plants in theU.S. followed from eitherplant start-up or firstemployment through 1994.The 10 plants producedformaldehyde (3 plants),molding compounds(3 plants), photographicfilm (2 plants), plywood(1 plant), and formaldehyderesins (6 plants).Exposure estimatesabasedon job titles, tasks, visitsto plants by studyindustrial hygienists, andmonitoring datameasurements. Peakexposure = short-termexcursions >8-hour TWAformaldehyde intensityand knowledge of jobtasks. Workerscontributed pre-exposureperson time to nonexposedcategory. RRs were fromPoisson regressionmodels, using a 15-yearlag to account for tumorlatency.Trend p = 0.147Cohort studyOverallMarsh et al. (2002) Retrospective cohortmortality study of 7,328workers hired up to 1984Worker-specific exposureafrom job exposure matrixbased on availableU.S. SMR4.94(95% CI: 1.99–10) (7)This document is a draft for review purposes only a nd does not constitute Agency policy 4-60 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)County SMR5.00(95% CI: 2.01–10) (7)Short-term worker (<1 year)SMR5.35(95% CI: 1.46–14) (4)Long-term worker (1 or more years)SMR4.59(95% CI: 0.95–13) (3)Year of hire1941−1946 SMR(0)1947−1956 SMR8.13(95% CI: 2.98–18) (6)1957 or later SMR2.63(95% CI: 0.07–15)Cumulative exposure (ppm-years) countyUnexposed SMR(0)0 to <0.004 SMR3.97(95% CI: 0.10–22) (1)0.004–0.219 SMR5.89(95% CI: 1.22–17) (3)0.22+ SMR7.51(95% CI: 1.55–22) (3)Average exposure (ppm) countyUnexposed SMR(0)0 to <0.03 SMR2.41(95% CI: 0.06–13) (1)0.03–0.159 SMR15.30(95% CI: 4.16–39) (4)0.16+ SMR4.13(95% CI: 0.50–15) (2)Duration of exposure to >0.2 ppm (years)Unexposed SMR3.01(95% CI: 0.36–11) (2)0 to <1 SMR4.81(95% CI: 0.58–17.4) (2)1–9 SMR4.04(95% CI: 0.10–22.51) (1)10+ SMR27.60(95% CI: 3.34–100) (2)Duration of exposure to≥0.7 ppm (years)Unexposed SMR3.64(95% CI: 0.99–9.31) (4)<1 SMR9.51(95% CI: 1.15–34.4) (2)and followed until 1998 inone plant from Hauptmannet al. (2004). Mortality wascompared with death ratesin two Connecticut countiesand the U.S.sporadic sampling datafrom 1965−1987, jobdescriptions, and verbaljob descriptions by plantpersonnel and industrialhygienists. Exposuresranked on a 7-point scalewith exposure rangeassigned to each rank.17% of jobs validated withcompany monitoring data;remaining 83% based onprofessional judgment.Assumed pre-1965exposure levels same aspost-1965 levels.1+ SMR11.07(95% CI: 0.28–61.67) (1)This document is a draft for review purposes only a nd does not constitute Agency policy 4-61 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)Nested case-control analysiscDuration of exposure to >0.2 ppm (years)Unexposed OR 1.00(8)0 to <1 OR 1.13(95% CI: 0.24–5.29) (6)1–9 OR 1.38(95% CI: 0.18–9.03) (3)10+ OR 9.49(95% CI: 0.55–701) (5)Duration of exposure to≥0.7 ppm (years)Unexposed OR 1.00(16)<1 OR 0.52(95% CI: 0.08–2.45) (4)Marsh et al. (2002) A nested case-controlanalysis of all pharyngealcancer cases also conductedwith four controls randomlyselected from cohort andmatched on age, year ofbirth, race, and sex.Conditional logistic modelused for nested case-controlanalysis.1+ OR 1.11(95% CI: 0.06–11.3) (2)OverallHayes et al. (1990) Proportionate mortalitycohort study of 4,046 U.S.male embalmers and funeraldirectors who died between1975 and 1985.Exposure presumed.PMR 2.16(4)Overall SPIR1.3(95% CI: 0.03–3.2) (4)Hansen and Olsen (1995) Proportionate incidencestudy of 2,041 men withcancer who died between1970 and 1984, identifiedfrom the Danish CancerRegistry and matched withthe Danish SupplementaryPension Fund, whoselongest work experienceoccurred at least 10 yearsbefore the cancer diagnosis.The SPIR measured theproportion of cases of NPCin formaldehyde-associatedcompanies relative to theproportion of cases of NPCamong all employees inDenmark.Linked companies throughtax records to the nationalDanish Product Register.This document is a draft for review purposes only a nd does not constitute Agency policy 4-62 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)Men OR 0.7(95% CI: 0.3–1.7)Women OR 2.6(95% CI: 0.3–22)Olsen et al. (1984) Case-control study of 314cases of NPC fromDanishCancer Registry linked tothe Registry during 1970–1982. Three controls/casesampled with cancer of thecolon, rectum, breast, andprostate by age, sex, andyear of diagnosis of cases.Employment historiesafter 1964 from filesmaintained by DanishCancer Registry evaluatedby industrial hygienists.Length of exposure (years)<15 RRd2.7(95% CI: 1.1–6.6)15 or more 1.2(95% CI: 0.5–3.2)Length of exposure lagged 10 years (years)<15 RRd1.6(95% CI: 0.7–3.8)15 or more 2.1(95% CI: 0.7–6.2)Years since first exposure<25 RRd1.3(95% CI: 0.6–3.2)25 or more 2.9(95% CI: 1.1–7.6)Age at first exposure (years)<25 RRd2.7(95% CI: 1.1–6.6)West et al. (1993) Case-control study of 104non-Chinese incident NPCcases from the PhilippineGeneral Hospital matchedwith 104 hospital and 101community controls.Personal interview,including job history.Industrial hygienistsblinded to case-controlstatus reviewed and ratedjobs as likely or unlikelyto be exposed. Analysisby length of exposure,length of exposure lagged10 years, time since firstexposure, and age at firstexposure, based on date ofinterview or death.25 or older 1.2(95% CI: 0.5–3.3)This document is a draft for review purposes only a nd does not constitute Agency policy 4-63 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)Exposure levelsI ORe1.0(95% CI: 0.6–1.7)II 1.3(95% CI: 0.7–2.4)III 1.4(95% CI: 0.6–3.1)IV 2.3(95% CI: 0.9–6.0)Roush et al. (1987) Population-based casecontrol study of 173 malecases from the ConnecticutTumor Registry who died ofany cause from 1935–1975.605 male controls randomlyselected from state deathcertificates during sametime period.Four categories: I,probably exposed most ofworking life; II,probablyexposed most of workinglife and probably exposed20+ years before death;III,probably exposed mostof working life andprobably to high level insome year; IV,probablyexposed most of workinglife and probably exposedto high level 20+ yearsbefore death.IntensityLow ORf1.2(95% CI: 0.5–3.3)Medium/high 1.4(95% CI: 0.4–4.7)No. years exposed1–9 ORf1.2(95% CI: 0.5–3.1)10 or more 1.6(95% CI: 0.4–5.8)Exposure score A: no lag5–19 ORf0.9(95% CI: 0.2–3.2)20 or more 2.1(95% CI: 0.6–7.8)Exposure score B: 15-year lag5–19 ORf1.7(95% CI: 0.5–5.7)20 or more 2.1(95% CI: 0.4–10)Vaughan et al. (1986a) Population-based casecontrol study of 27incidence cases of NPC(during 1980–1983) from a13-county area (WashingtonState Cancer SurveillanceSystem) and 552 matchedcontrols from random digitdialing in same area, foroccupational exposures.Interview-basedinformation on lifetimeoccupational exposure toformaldehyde with cases,next of kin, and controls.Exposure from availablehygiene data, NIOSH andother data, and NCI jobexposure linkage system.Exposure levels based oninvestigator’s judgment.Exposure score A:weighted sum of no. yearsspent per job (weight =estimated formaldehydelevel). B: weighted sumof no. years spent per jobwith 15-year lag (latency).This document is a draft for review purposes only a nd does not constitute Agency policy 4-64 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)Years of residence in mobile home1–9 ORg2.1(95% CI: 0.7–6.6)10 or more 5.5(95% CI: 1.6–19)Years of exposure to particleboard1–9 ORg1.4(95% CI: 0.5–3.4)10 or more 0.6(95% CI: 0.2–2.3)Exposure sourceOccupation only ORg1.7(95% CI: 0.5–5.7)Mobile home only 2.8(95% CI: 1.0–7.9)Vaughan et al. (1986b) Population-based casecontrol study of 27incidence cases on NPC(during 1980–1983) from a13-county area (WashingtonState Cancer SurveillanceSystem) and 552 matchedcontrols from random digitdialing in same area, forresidential exposures.No direct measurements.Interview informationfrom cases/controls ornext of kin: residence inpast 50 years, use ofparticleboard or plywood,and lifetime occupationaland chemical exposurehistory.Both 6.7(95% CI: 1.2–39)Possible, probable, or definite exposure (61 cases,76 controls)Ever ORh1.6(95% CI: 1.0–2.8)Duration (years)1–5 ORh0.9(95% CI: 0.4–2.1)6–17 1.9(95% CI: 0.9–4.4)18 or more 2.7(95% CI: 1.2–6.0)Trend p = 0.014Cumulative exposure (ppm-years)0.05–0.40 ORh0.9(95% CI: 0.4–2.0)0.41–1.10 1.8(95% CI: 0.8–4.1)≥1.10 3.0(95% CI: 1.3–6.6)Trend p = 0.033Probable or definite exposure (27 cases, 30 controls)Vaughan et al. (2000)Ever ORh2.1(95% CI: 1.1–4.2)Duration (years)1–5 ORh2.0(95% CI: 0.8–5.0)6–17 3.3(95% CI: 0.9–12)18 or more 1.6(95% CI: 0.5–5.6)Population-based casecontrol study of 196incident epithelial NPCpatients identified from 5U.S. cancer registries from1987–1993 matched with244 controls from randomdigit dialing in the samegeographic regions.Interviewed for lifetimeoccupational and chemicalexposure. Exposureestimates by industrialhygienist withoutknowledge of casecontrols status.Probability of exposure:definitely not or unlikely(<10%); possible (≥10%and <50%); probable(>50% and <90%); anddefinite≥90%). Jobs withpotential exposureassigned estimatedconcentration levels basedon TWA: low (<10 ppm),moderate (≥10 and<50 ppm), and high(≥50 ppm).Trend p = 0.069This document is a draft for review purposes only a nd does not constitute Agency policy 4-65 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)Cumulative exposure (ppm-years)0.05–0.40 ORh1.9(95% CI: 0.7–4.9)0.41–1.10 2.6(95% CI: 0.7–9.5)≥1.10 2.2(95% CI: 0.7–7.0)Trend p = 0.13Definite exposure (10 cases, 2 controls)Ever ORh13.3(95% CI: 2.5–70)Ever exposed RRi1.4(95% CI: 0.9–2.2)Duration (years)≤10 RRi1.3(95% CI: 0.69–2.3)>10 RRi1.6(95% CI: 0.91–2.9)Trend p = 0.08EBV posjEver exposed RRi2.7(95% CI: 1.2–6.2)≤10 RRi2.8(95% CI: 0.8–9.7)>10 2.6(95% CI: 0.9–7.7)Cumulative exposure (average intensity-years)<25 RRi1.3(95% CI: 0.7–2.4)≥25 RRi1.5(95% CI: 0.9–2.7)Trend p = 0.10EBV posj<25 RRi4.0(95% CI: 0.9–17)≥25 RRi2.2(95% CI: 0.8–5.8)Years since 1st exposure<20 RRi2.3(95% CI: 1.0–5.8)Hildesheim et al. (2001) Population-based casecontrol study of 375incident cases from twoTaiwanese hospitalsbetween 7/15/91 and12/31/94. 325 controlscame from a random sampleof households from anational householdregistration system andwere age, sex, and area-ofresidence matched. Tumorswere histologicallyconfirmed. All subjectswere tested for the EBV.Exposure metrics werestratified by seropositivity.In-person interviewscollected information onrisk factors and job historyfor jobs held >1 year,including length of timejob held, type of industry,and tasks, tools, andmaterials used on the job.Industrial hygienistassigned StandardIndustry Classification/Standard OccupationalClassification codes tojobs, assigning each aprobability and intensityof exposure on a 0–9scale. Exposure metricswere duration, averageintensity (intensity scale),average probability(probability scale),cumulative (averageintensity), years since 1stexposure, and age at 1stexposure. Analysis of≥20 RRi1.2(95% CI: 0.8–2.0)This document is a draft for review purposes only a nd does not constitute Agency policy 4-66 DRAFT—DO NOT CITE OR QUOTETable 4-1. Cohort and case-control studies of formaldehyde cancer and NPCReference Study design Exposure assessment Results;statistical significance (number observed deaths)EBV posh<25 RRi2.3(95% CI: 0.5–10)≥25 RRi2.8Age at 1st exposure<20 RRi1.3(95% CI: 0.8–2.0)≥20 RRi3.4(95% CI: 0.9–12)EBV posj<25 RRi2.6(95% CI: 1.1–6.5)nonkeratinizing orundifferentiated tumorsyielded similar results asoverall analysis.≥25 RRi3.1(95% CI: 0.4–24)aExposure estimates by Hauptmann et al. (2004) were10 times higher than those of Marsh et al. (2002).bAdjusted for calendar year, age, sex, race, and paycategory (salaried versus wage).cResults for cumulative and average intensity of exposure are not included here because condition logistic regression produces unstable estimates for thissmallnumber of cases.dAdjusted for years since first exposure to dust andexhaust fumes.eAdjusted for age at death, year at death, and availability of occupational information (Roush et al.,1987).fAdjusted for cigarette smoking, alcohol consumption, gender, and age.gAdjusted for ethnic origin and cigarette smoking.hAdjusted for age, sex, race, SEER site, cigarette usage, proxy status, and education.iAdjusted for age, sex, education, and ethnicity.jEBV seropositives defined as positive for one of the following anti-EBV antibodies known to be associated with NPC: viral capsid antigen IgA, EBV nuclearantigen 1 IgA, early antigen IgA, DNA binding protein IgG, and anti-DNase IgG.N/A = not applicable, NS = not significant, PMR = proportionate mortality ratio.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-67 Following these reports of increased risk of NPC as sociated with formaldehyde exposure, 1 a series of post hoc analyses of similar data were undertaken by Marsh and coworkers (Marsh et 2 al., 2007a, b, 2002, 1996; Marsh and Youk, 2005). Briefly, these studies focused on the specific 3 findings from a single plant in the NCI cohort (Wal lingford, Connecticut) that generated the 4 majority of the NPC cases. 5 In the most recent subsequent report, Marsh et al. (2007a) continues to argue against a 6 formaldehyde-NPC association. Although earlier rep orts speculated on anecdotal evidence that 7 the statistically significant excess risk of NPC ob served at the Wallingford, Connecticut, plant 8 reflected the influence of unmeasured nonoccupation al risk factors associated with employment 9 outside the plant, the new report (Marsh et al., 20 07a) suggests that occupational or hobby10 related work in silversmithing may have confounded the observed effect of formaldehyde on the 11 increased risk of NPC. In this report, Marsh et al . (2007a) show that their subjectively assessed 12 work in silversmithing is strongly associated with NPC. While the reported ORs are indeed quite 13 high, the estimates are extremely unstable and it i s not clear how many a priori hypotheses were 14 tested for statistical significance. There are no citations of an association between silversmithing 15 exposures and NPC in the medical literature. Marsh and coworkers mention that there was 16 concordance of silver manufacturing history in the Wallingford, Connecticut, area. If 17 silversmithing exposures are indeed independent ris k factors for NPC, it would be expected that 18 the rates of NPC in the surrounding counties with h istorical silver-related exposures would be 19 elevated. However they are not increased, as evide nced by the comparability of the increased 20 rates of NPC among the plant workers compared with both the national and local county rates 21 that were very similar (Marsh et al., 2007a). The comparable rates indicate the counties’ rates of 22 NPC were very similar to the national rates and wea ken an association between silversmithing 23 and NPC. Given the many post hoc reexaminations of alternative hypotheses to explain the 24 original NCI findings, it is more likely that silve rsmithing is an artifactual confounder. 25

26

4.1.2.1.1.2. Professional cohort studies. Two cohort studies of professional groups, such as 27 anatomists, pathologists, embalmers, and funeral di rectors, examined the risk of NPC and 28 formaldehyde exposure. In general, measurements of formaldehyde concentrations were not 29 available in studies of professionals but are gener ally below 1 ppm (IARC, 1995; Korczynski, 30 1994; Stewart et al., 1992; Moore and Ogrodnik, 198 6). Hayes et al. (1990) reported an excess 31 risk of NPC among male professional embalmers and f uneral directors, based on 4 deaths with 32 1.9 expected based on age, gender, and calendar-yea r-specific proportions of deaths in the U.S. 33 population. Hansen and Olsen (1995) studied male D anish cancer patients employed in 34 companies in which formaldehyde was used or produce d. Only a slight excess risk of NPC was 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-68 found, based on 4 cases with 3.2 expected (standard ized proportionate incidence ratio [SPIR] 1 1.3). Hansen and Olsen (1995) also reported on a s ignificantly elevated risk of sinonasal cancer. 2

3

4.1.2.1.1.3. Case-control studies. Five case-control studies (West et al., 1993; Roush et al., 4 1987; Vaughan et al., 1986a, b; Olsen et al., 1984) reviewed by IARC in 1995 provided evidence 5 of excess risks of NPC due to formaldehyde. Most o f these studies report significant and 6 nonsignificant elevations in risk of NPC in the ran ge of 1.5–3.0, with some higher than 5.0. In 7 its report, IARC (1995) concluded that, taking the data as a whole, formaldehyde appears to have 8 a causal role in the induction of NPC, recognizing that the conclusion is based on small numbers 9

of cancer cases. 10

Three case-control studies have been conducted sinc e the 1995 IARC report. Armstrong 11 et al. (2000) found no association between formalde hyde exposure and NPC (adjusted OR = 0.71 12 [95% CI: 0.34–1.43]), controlling for wood dust and industrial heat. Using data from the 13 Surveillance Epidemiology and End Results (SEER) pr ogram, Vaughan et al. (2000) found an 14 OR for ever-exposed persons of 3.1 (95% CI: 1.0–9.6 ) among cases of epithelial NPC, 15 suggesting differences in the etiology of cancers a t this site. There was a trend of increasing risk 16 of NPC with increasing duration of exposure and cum ulative exposure, controlling for wood dust 17 exposure. Finally, Hildesheim et al. (2001) found that exposure to formaldehyde produced 18 modest risk elevations for duration of exposure (OR = 1.6 for 10 years or less and 1.2 for over 19 10 years of exposure), for cumulative exposure (ORs were 1.3 for <25 years of exposure and 1.5 20 for 25+ years of exposure), and for years since fir st exposure. Among those with EBV, the OR 21 was 2.7 (95% CI: 1.2–6.2) for ever-exposed persons. The risk was significantly higher among 22 exposed persons whose work history was within the l ast 10 years (OR = 4.7 [95% CI: 1.1–20.0]) 23 and for those followed 20+ years after exposure (OR = 2.8 [95% CI: 1.1–7.6]). 24

25

4.1.2.1.1.4. Summary of NPC studies. Findings from the large NCI cohort studies of NPC r isk 26 due to formaldehyde exposure clearly show a consist ent pattern of increased risk with increased 27 exposures. Post hoc reanalyses have challenged the interpretation of these findings but have not 28 been able to dispute the reported excess in NPC mor tality (Marsh et al., 2007a, b, 2002, 1996; 29 Marsh and Youk, 2005). The major questions that ha ve been raised by Marsh and coworkers 30 highlight the observation that the NPC findings app ear to depend on the results of 1 of the 10 31 plants that made up the NCI cohort. While it is th eoretically possible for coexposures at that 32 plant or among those workers to act as potential co nfounders or modifiers of the observed effect 33 of formaldehyde on increased risk of NPC, there is no solid evidence of such a relationship that 34 would outweigh or supersede the reported adverse ef fects of formaldehyde exposure. While all 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-69 of the cohort members at the Wallingford, Connectic ut, plant were also exposed to particulates, 1 the NCI investigators did observe a dose-response r elationship with formaldehyde among 2 individuals with high particulate exposures, thereb y strengthening the causal interpretation of the 3 formaldehyde relationship with an increased risk of NPC. The described association of a 4 potential occupational relationship with silversmit hing and NPC has no basis in the medical 5 literature and is inconsistent with the supposition that this activity is common in the locality of 6 Wallingford, Connecticut, but has not been associat ed with increased rates of NPC in 7 surrounding New Haven County (Connecticut). Marsh and coworkers did report significantly 8 increased rates of pharyngeal cancer (including NPC s) among workers from the Wallingford 9 plant compared with both the county and national ra tes. It is more plausible that the observed 10 association at the Wallingford plant reflects highe r formaldehyde exposures than at other plants. 11 The exposure levels at Plant 2 were even higher tha n at the Wallingford plant and were 12

associated with a fivefold increase in risk associa ted with NPC, even though this was based on a 13 single observed case and was not significant. 14

In addition to the evidence from the NCI cohort stu dies, modest additional evidence is 15 found in the professional cohort studies of Hayes e t al. (1990) and Hansen and Olsen (1995). 16 The rarity of the disease and difficulties in obtai ning valid and reliable historical exposure 17

estimates are substantial limitations of these coho rt studies. Further evidentiary support comes 18

from the results of several case-control studies th at support an increased risk of NPC from 19 exposure to formaldehyde. The studies of Vaughan e t al. (2000) and Hildesheim et al. (2001) 20 provide evidence of an association of NPC with expo sure to formaldehyde. Vaughan et al. 21 (2000) found a dose-response relationship of NPC wi th increasing exposure to formaldehyde, as 22 did Hildesheim et al. (2001). These studies, in ge neral, are easier to conduct and may provide 23 more statistical power for a specific level of risk estimate than do cohort studies. 24

25

4.1.2.1.2. Nasal and paranasal cancer 26

4.1.2.1.2.1. Case-control studies. Eight case-control studies were evaluated in the 19 95 IARC 27 monograph regarding the risk of nasal cavity and ac cessory sinuses from exposure to 28 formaldehyde (Luce et al., 1993; Roush et al., 1987 ; Hayes et al., 1986; Olsen and Asnaes, 1986; 29 Vaughan et al., 1986a, b; Brinton et al., 1984; Ols en et al., 1984). Of three studies that identified 30 a cell type, two reported a positive finding of sin onasal cancer (Hayes et al., 1986; Olsen and 31 Asnaes, 1986). One of the positive studies did not report any exposure to the potentially 32 confounding influence of wood dust, while the other two did report an adjustment for exposure 33 to wood dust. Of the remaining five studies where a cell type was not identified, only Roush et 34 al. (1987) and Olsen et al. (1984) found positive r esults. The remaining studies (Vaughan et al., 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-70 1986a, b; Brinton et al., 1984) from the 1995 IARC monograph did not find associations between 1 exposure and sinonasal cancer. Study details of th e epidemiologic studies of nasal and paranasal 2 cancer are summarized in Table 4-2. Vaughan et al. (1986b) matched 53 sinonasal cancer 3 patients to 552 controls. Potential residential ex posure to formaldehyde was estimated by 4 utilizing residence in a mobile home with or withou t the presence of UFFI or particleboard or 5 plywood as a surrogate for exposure. The authors f ound an OR of 1.5 for sinonasal cancer in 6 subjects reporting residence of 10 or more years in a mobile home with UFFI before diagnosis. 7 A higher OR (1.8) was reported for less than 10 yea rs of mobile home residence. However, 8 because actual formaldehyde levels in the subjects’ mobile homes are unknown, the exposure 9 estimates are, at best, imprecise surrogates that t ypically have the effect of attenuating any true 10 risk. In Vaughan et al. (1986a), the same cases an d controls were examined for occupational 11 exposures to formaldehyde, but no increase in risk of nasal or paranasal cancer was reported. 12 More recently, Luce et al. (2002) pooled data from 12 case-control studies. Combined, 13

these studies had 195 adenocarcinomas and 432 SCCs of the sinonasal passages compared with 14 3,136 controls. The authors reported a significant increase in the risk of sinonasal 15 adenocarcinoma in men (adjusted OR = 3.0 [95% CI: 1 .5–5.7]; 91 cases) and in women (adjusted 16 OR = 6.2 [95% CI: 2.0–19.7]; 5 cases) with a high p robability of exposure to formaldehyde. For 17 SCCs, the ORs were more modest: OR = 1.2 in men and OR = 1.5 in women for a high 18 probability of exposure to formaldehyde. In an ana lysis of 11 formaldehyde-exposed cases of 19 sinonasal adenocarcinomas who were not exposed to w ood dust, there was an elevated risk in 20 men (OR = 1.9; 3 cases) and a significantly increas ed risk in women (OR = 11.1 [95% CI: 3.2– 21 38.0]; 5 cases) with a high probability of exposure to formaldehyde. Limitations of these studies 22

were the lack of information about the actual level s or intensity of exposure to formaldehyde, 23 exposure to multiple occupational carcinogens, and the small number of cases in some 24 subgroups. In spite of those limitations, which ge nerally obfuscate the observation of a true 25 underlying effect, these studies identified effects of formaldehyde that were statistically 26 significant predictors of sinonasal cancers. 27

28This document is a draft for review purposes only a nd does not constitute Agency policy 4-71 DRAFT—DO NOT CITE OR QUOTETable 4-2. Case-control studies of formaldehyde and nasal and paranasal cancerReference Study design Exposure assessment Results,statistical significance (number of cases)Squamous cell carcinoma/lymphoepitheliomaEver vs. neverFormaldehyde only RR2.0 (95% CI: 0.7–5.9)Formaldehyde + wooddustRR1.6 (95% CI: 0.8–3.3)10 or more years since first exposureFormaldehyde only RR1.4 (95% CI: 0.3–6.4)Formaldehyde + wooddust RR1.8 (95% CI: 0.7–4.4)AdenocarcinomaEver vs. NeverFormaldehyde only RR7.0 (95% CI: 1.1–44)Formaldehyde + wooddust RR40.0 (95% CI: 22–71)10 or more years since first exposureFormaldehyde only RR9.5 (95% CI: 1.6–58)Olsen and Asnaes(1986)Case-control study ofhistologically confirmed casesof squamous cellcarcinoma/lymphoepitheliomaof the sinonasal cavities andparanasal cancers in 215 menand adenocarcinomas of thesinonasal cavities andparanasal cancers in 39 menmatched with 2,465 controlswith other cancers from theDanish Cancer Registry,1970–1982.Employment historiesafter 1964 from filesmaintained by DanishCancer Registryestimated by industrialhygienists.Formaldehyde + wooddust RR44.0 (95% CI: 22–88)Industrial hygienist AAny exposure RR 3.0 (90% CI: 1.3–6.4)Moderate exposure 2.7 (90% CI: 1.0–7.2)High exposure 3.1 (90% CI: 0.9–10.0)Industrial hygienist BAny exposure RR 1.9 (90% CI: 1.0–3.6)Moderate exposure 1.4 (90% CI: 0.5–3.4)High exposure 2.4 (90% CI: 1.1–5.1)Hayes et al. (1986) Case-control study of 91 menwith SCC of the nasal cavityand paranasal sinuses, fromclinical records of six medicalinstitutions in the Netherlands.195 controls from living anddeceased males frommunicipal residence registries,from 1978–1981.Cases selected fromclinical records of sixinstitutions in theNetherlands. 91 malepatients and 195controls from living anddeceased males frommunicipal residenceregistries with little orno exposure to wooddust. Industrialhygienists evaluated jobhistories according toprobability of exposurebased on job records.This document is a draft for review purposes only a nd does not constitute Agency policy 4-72 DRAFT—DO NOT CITE OR QUOTETable 4-2. Case-control studies of formaldehyde and nasal and paranasal cancerReference Study design Exposure assessment Results,statistical significance (number of cases)Exposure levels Sinonasal cancerI ORa0.8 (95% CI: 0.5–1.3)II 1.0 (95% CI: 0.5–1.8)III 1.0 (95% CI: 0.5–2.2)IV 1.5 (95% CI: 0.6–3.9)Roush et al. (1987) Population-based case-controlstudy of 198 male cases ofsinonasal cancer from theConnecticut Tumor Registrywho died of any cause in1935–1975. Controls were605 males dying inConnecticut during the sametime period, randomly selectedfrom state death certificates.Occupations from citydirectories andevaluation of job byindustrial hygienist whoclassed exposure into I,probably exposed tosome level most ofworking life; II,probably exposed tosome level most ofworking life andprobably exposed tosome level 20+ yearsbefore death; III,probably exposed tosome level most ofworking life andprobably exposed to ahigh level in someyears; IV, probablyexposed to some levelmost of working lifeand probably exposedto a high level 20+years before death.AdenocarcinomabPossible exposure ORc1.28 (95% CI: 0.16–10)Probable/definite exposureAverage level≤2ORc4.15 (95% CI: 0.96–18)>25.33 (95% CI: 1.28–22)Duration (years)≤20ORc1.03 (95% CI: 0.18–5.77)Luce et al. (1993)Case-control study of menwith sinonasal cancer(histologically confirmed), 77with adenocarcinoma, 59 withsquamous cell carcinomas,and 25 tumors of other types,matched with 409 controlsfrom 27 French hospitals andIndustrial hygienistestimation based on jobhistories from personalinterviews. Subjectswere broken out into noexposure, possibleexposure, orprobable/definite>206.86 (95% CI: 1.69–28)This document is a draft for review purposes only a nd does not constitute Agency policy 4-73 DRAFT—DO NOT CITE OR QUOTETable 4-2. Case-control studies of formaldehyde and nasal and paranasal cancerReference Study design Exposure assessment Results,statistical significance (number of cases)Cumulative level (years)≤30ORc1.13 (95% CI: 0.19–6.95)30–60 2.66 (95% CI: 0.38–19)>606.91 (95% CI: 1.69–28)Age 1st exposed (years)≤15ORc9.99 (95% CI: 1.85–54)16–20 4.12 (95% CI: 0.95–18)>202.74 (95% CI: 0.58–13)Date 1st exposed (years)After 1954 ORc6.02 (95% CI: 1.18–31)Before 1954 4.26 (95% CI: 1.06–17)Other cell type carcinomabPossible exposure ORc0.81 (95% CI: 0.15–4.36)Probable/definite exposureAverage level≤2ORc1.67 (95% CI: 0.51–5.42)>23.04 (95% CI: 0.95–9.7)Duration (years)≤20ORc2.82 (95% CI: 0.94–8.4)>201.62 (95% CI: 0.48–5.51)Cumulative level (years)≤30ORc2.18 (95% CI: 0.65–7.31)>302.21 (95% CI: 0.73–6.73)Age 1st exposed (years)≤20ORc2.03 (95% CI: 0.63–6.54)>202.36 (95% CI: 0.76–7.33)Date 1st exposed (years)After 1954ORc0.48 (95% CI: 0.05–4.35)from lists of names suppliedby patients.exposure. Thoseclassed asprobable/definiteexposure furthercategorized into threelevels of frequency ofexposure during anormal workweek: 1 =<5% of the time; 2 = 5–30% of the time; and 3= >30% of the time.Concentration wascategorized into 3levels: low (<0.0 ppm);medium (0.1–1 ppm);and high (>1 ppm).The exposure index =concentration ×frequency. Cumulativelevel = sum of exposureindices. Average level= cumulativelevel/duration andranged from 1 to 9.Nearly all cases had hadwood dust exposure.Before 19543.27 (95% CI: 1.15–9.33)AdenocarcinomaHigh probability of exposureLuce et al. (2002) Pooled analysis of 195adenocarcinomas and 432squamous cell carcinomas ofJob exposure matrixbased on interview datadeveloped for pooledMen ORd3.0 (95% CI: 1.5–5.7)This document is a draft for review purposes only a nd does not constitute Agency policy 4-74 DRAFT—DO NOT CITE OR QUOTETable 4-2. Case-control studies of formaldehyde and nasal and paranasal cancerReference Study design Exposure assessment Results,statistical significance (number of cases)Women ORe6.2 (95% CI: 2.0–20)Squamous cell carcinomaHigh probability of exposureMen ORe1.2 (95% CI: 0.8–1.8)the sinus/nasal cavity matchedwith 3,136 controls from 12case-control studies.analysis. Industrialhygiene data used todevelop indices ofexposure. 11formaldehyde casesreported no exposure towood dust.Women ORe1.5 (95% CI: 0.6–3.8)Overall male and female RR 0.35 (95% CI: 0.1–1.8)Residence in mobile homeORf0.6 (95% CI: 0.2–1.7)Years of exposure to particleboard1 to 9 ORf1.8 (95% CI: 09–3.8)10 or more 1.5 (95% CI: 0.7–3.2)Brinton et al. (1984) Case-control study of 160patients with cancer of thenasal cavity and paranasalsinuses from four NorthCarolina and Virginiahospitals matched with 290hospital controls with otherconditions, based onoccupational exposures.Interview data on jobhistory. Estimation ofexposure based onindustry type. Onlytwo cases employed inindustry associated withformaldehyde. Therewere no deaths in thehigh exposure category.MenFormaldehyde onlyEver exposed RR 2.8 (95% CI: 1.8–4.3) (33)RR 3.1 (95% CI: 1.8–5.3) (23)Exposure to wood dust andformaldehydeEver exposed RR 3.5 (95% CI: 2.2–5.6) (28)Olsen et al. (1984) Case-control study of 488cases of nasal cancer linked tothe Danish Cancer Registryduring 1970–1982. Controlswere individuals with cancerof the colon, rectum, breast,and prostate. Three controlsper case were selected for thesame distributions of age, sex,and year of diagnosis as cases.Employment historiesafter 1964 from filesmaintained by DanishCancer Registryestimated by industrialhygienists.1stexposure >10 years ormore before diagnosis RR 4.1 (95% CI: 0.2.3–7.3) (20)Overall SPIR2.3 (95% CI: 1.3–4.0) (13)Low formaldehyde SPIR0.8 (95% CI: 0.02–4.4) (1)SPIR3.0 (95% CI: 1.4–5.7) (9)Formaldehyde, no wooddustSPIR5.0 (95% CI: 0.5–13) (2)Hansen and Olsen(1995)Proportionate incidence studyof 2,041 men with sinonasalcancer who died between 1970and 1984 identified from theDanish Cancer Registrymatched with the DanishLinked companiesthrough tax records tonational Danish ProductRegister, wherecompanies must reportamount ofUnknown SPIR1.0 (95% CI: 0.03–6.1) (1)This document is a draft for review purposes only a nd does not constitute Agency policy 4-75 DRAFT—DO NOT CITE OR QUOTETable 4-2. Case-control studies of formaldehyde and nasal and paranasal cancerReference Study design Exposure assessment Results,statistical significance (number of cases)Supplementary Pension Fundwhose longest workexperience occurred at least 10years before the cancerdiagnosis. The measure ofrisk was the SPIR, whichmeasured the proportion ofcases of sinonasal cancer informaldehyde-associatedcompanies relative to theproportion of cases ofsinonasal cancer among allemployees in Denmark.formaldehyde used peryear.Overall SMR 0.87 (95% CI: 0.11–3.14) (2)Coggon et al. (2003) Cohort mortality study of14,014 men employed in 6factories of the chemicalindustry in Great Britain fromperiods during whichformaldehyde was produced.Cohort followed through2000. Sinonasal cancermortality SMRs based onEnglish and Welsh age andcalendar-year-specificmortality rates.Exposures assessmentbased on dataabstracted fromcompany records. Eachjob categorized asbackground, low,moderate, high, orunknown levels. Foranalysis of sinonasalcancer, no gradient usedbecause of smallnumber of observedcases.aAdjusted for age at death, year at death, and availability of occupational information.bAll had medium to high exposure to wood dust.cAdjusted for age and exposure to glues and adhesives.dAdjusted for age and study.eAdjusted for age, study, and cumulative exposure todust.fAdjusted for cigarette smoking, alcohol consumption, gender, and age.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-76 4.1.2.1.2.2. Cohort studies of nasal and paranasal cancer. IARC (1995) also reported the 1 results of several cohort studies of professional a nd industrial workers for nasal and paranasal 2 cancer (Andjelkovich et al., 1995; Gardner et al., 1993; Hall et al., 1991; Hayes et al., 1990; 3 Bertazzi et al., 1989; Edling et al., 1987; Blair e t al., 1986; Stroup et al., 1986; Harrington and 4 Oakes, 1984; Levine et al., 1984; Walrath and Fraum eni, 1984, 1983; Friedman and Ury, 1983). 5 Only a few studies reported any cases of sinonasal cancer. No cases of this type of cancer were 6 reported in any of the studies of professional work ers examined by the IARC. Only 2 cases (2.2 7 expected) were reported by Blair et al. (1986), and only 1 case (1.7 expected) was reported by 8 Gardner et al. (1993). The likelihood of finding t his rare tumor type in a long-term cohort study 9

is low. 10 Three subsequent cohort studies reported on nasal a nd paranasal cancer. Hansen and 11 Olsen (1995), in a proportional incidence study, fo und a significantly increased risk of sinonasal 12 cavity cancer (SPIR = 2.3 [95% CI: 1.3–4.0]; 13 obs erved) in 265 Danish industries, where 13 2,041 of 91,182 cancer patients had at least 10 yea rs of continuous formaldehyde-related work 14 experience before diagnosis. Coggon et al. (2003), in a cohort study of 14,014 employees in six 15 chemical factories in Great Britain, found only 2 d eaths from sinonasal cancer (2.3 expected 16 based on national death rates in Great Britain). F inally, Hauptmann et al. (2004) evaluated the 17

sinonasal cancer risk in the NCI cohort and found t hree cases (SMR = 1.19 [95% CI: 0.38–3.68]) 18 among those with a 15-year lag period. 19

20

4.1.2.1.2.3. Summary of nasal and paranasal cancers. The pooled case-control study of Luce 21 et al. (2002) provides strong evidence of an associ ation between formaldehyde exposure and 22

increased risk of sinonasal adenocarcinoma. The co hort studies may not have had sufficient 23 statistical power to show an association, and studi es that did not distinguish cancer type may 24 have aggregated a truly causal relationship with a noncausal relationship with SCC. In summary, 25 there appears to be increased risk of sinonasal can cer associated with formaldehyde exposure 26 with or without exposure to wood dust. The effect appears to be stronger when the risk is 27 stratified by cancer type with higher risks of aden ocarcinoma compared with SCC. Taken 28 together with the NPC findings in the neighboring t issue, it is concluded that there is evidence of 29 higher risks of sinonasal cancer associated with ex posure to formaldehyde. 30

31 4.1.2.1.3. Other respiratory tract cancers

. Of six cohort studies of buccal/pharynx cancer in 32 studies of professionals reviewed by IARC (Hayes et al., 1990; Logue et al., 1986; Stroup et al., 33 1986; Levine et al., 1984; Walrath and Fraumeni, 19 84, 1983), no evidence of a risk associated 34 with exposure to formaldehyde was reported (see Tab le 4-3). In studies of industrial worker 35 cohorts where buccal/pharynx cancer was examined (A ndjelkovich et al., 1995; Stayner et al., 36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-77 1988; Blair et al., 1986), only one (Stayner et al. , 1988) reported an excess risk of death from this 1 tumor (SMR = 3.4, based on four deaths in a cohort of 6,741 white women). Three case-control 2 studies (Merletti et al., 1991; Vaughan et al., 198 6a, b) did not find an association between oral 3 cavity, oropharyngeal, and hypopharyngeal cancers a nd formaldehyde exposure. However, 4 Merletti et al. (1991) found an elevated OR of 1.8 associated with probable or definite exposure 5 to formaldehyde in a study of 86 patients matched w ith 373 controls. There was no risk of 6 laryngeal cancer associated with formaldehyde in a case-control study (Wortley et al., 1992) of 7 235 patients with laryngeal cancer and 547 controls . The OR in that study was 1.0 (adjusted for 8 age, smoking, drinking, and level of education). I ARC (1995) concluded that there was little 9 evidence of an increased risk of laryngeal cancer. 10

11

4.1.2.1.3.1. Cohort studies of other respiratory tract cancers. Hansen and Olsen (1995) 12 reported a 10% increase in the risk of cancer of th e buccal cavity and pharynx (SPIR = 1.1) in 13 their proportional incidence study of Danish worker s. Marsh et al. (1996) reported no excess 14 risk of buccal cavity cancer cases (SMR = 1.31) bas ed on U.S. rates and no excess based on state 15 mortality rates (SMR = 1.0). For oropharyngeal can cer, the SMR was 1.84 (based on two cases), 16 the SMR for hypopharyngeal cancer was 1.41 (based o n one case), and the SMR for laryngeal 17 cancer was 1.47 (based on six cases). The latter r isks were elevated even when SMRs were 18 derived from Connecticut mortality rates. 19 The Marsh et al. (2002) update also derived elevate d risk estimates for oropharyngeal, 20 hypopharyngeal, and pharyngeal-unspecified cancers. The SMRs ranged from 2.11 to 2.25 21

based on U.S. rates and 1.52 to 1.89 based on count y rates. When combined with NPC 22 International Classification of Death (ICD) codes 1 46–149 to increase statistical power, the total 23 pharyngeal cancer SMRs were significant based on U. S. death rates (SMR = 2.63, n = 22, p < 24 0.01) and county death rates (SMR = 2.23, n = 22, p < 0.01) and remained significant for both 25 short-term (less than 1 year) and long-term exposur es. Furthermore, using the exposure 26 estimates of Marsh et al. (1996), both cumulative a nd average exposure to formaldehyde resulted 27 in elevated SMRs, some of which were significant fo r pharyngeal cancer. Coggon et al. (2003) 28 identified 14 cases of cancer of the larynx (13.1 e xpected) in their cohort of formaldehyde29 exposed factory workers. Pinkerton et al. (2004) f ound an excess risk of mortality from buccal 30 cavity cancer (SMR = 1.33, four deaths observed) an d a deficit of the risk of pharyngeal cancer 31 (SMR = 0.64, three observed). Since the number of observed deaths was small and the risk 32 estimates were subject to much variation for both s tudies, no conclusions about cancer risk can 33

be drawn. 34This document is a draft for review purposes only a nd does not constitute Agency policy 4-78 DRAFT—DO NOT CITE OR QUOTETable 4-3. Epidemiologic studies of formaldehyde and pharyngeal cancer (includes nasopharyngeal cancer)Reference Study design Exposure assessment Results,statistical significance (number observed deaths for cohort study)Cohort studyOverallU.S. SMR 2.63(95% CI: 1.65–3.98) (22)County SMR 2.23(95% CI: 1.40–3.38) (22)Short-term worker (<1 year)SMR 2.35(95% CI: 1.22–4.11) (12)Long-term worker (1 or more years)SMR 2.10(95% CI: 1.01–3.86) (10)Cumulative exp. (ppm-years) countyUnexposed SMR 1.24(95% CI: 0.15–4.49) (2)>0 to <0.004 SMR 3.31 (95% CI: 1.22–7.21) (6)0.004–0.219 SMR 2.06(95% CI: 0.83–4.24) (7)0.22+ SMR 2.30(95% CI: 0.92–4.73) (7)Average Exposure (ppm) countyUnexposed SMR 1.24(95% CI: 0.15–4.49) (2)>0 to <0.03 SMR 2.02(95% CI: 0.74–4.40) (6)0.03–0.159 SMR 3.82(95% CI: 1.54–7.88) (7)0.16+ SMR 2.03(95% CI: 0.82–4.19) (7)Exposure to≤0.2 ppmSMR 1.72(95% CI: 0.74–3.39) (8)Exposure to >0.2 ppm SMR 2.68(95% CI: 1.46–4.49) (14)Exposure to≤0.7 ppmSMR 2.12(95% CI: 1.21–3.45) (16)Nested case-control analysisCumulative exp. (ppm-years)<0.004 OR 1.00(8)0.004–0.219 OR 0.71(95% CI: 0.20–2.43) (7)0.22+ OR 0.79(95% CI: 0.18–3.20) (7)Average exposure (ppm)<0.03 OR 1.00(8)0.03–0.159 OR 1.71(95% CI: 0.47–6.10) (7)0.16+ OR 0.99(95% CI: 0.27–3.55) (7)Exposure to >0.2 ppm OR 1.35(95% CI: 0.45–4.25) (14)Marsh et al. (2002) Retrospective cohortmortality study of 7,328workers hired up to 1984and followed until 1998 inone plant from Blair et al.(1986, 1987) andHauptmann et al. (2004).Mortality was comparedwith death rates in twoConnecticut counties andU.S. A nested case-controlanalysis was also conductedwith 4 controls matched onage, year of birth, race, andsex randomly selected fromcohort. Conditional logisticmodel was used for nestedcase-control analysis.Worker-specific exposuresfrom job exposure matrixwere based on availablesporadic sampling data from1965–1987, job descriptions,and verbal job descriptionsby plant personnel andindustrial hygienists.Exposures were then rankedon a 7-point scale. Anexposure range was assignedto each rank. 17% of jobsvalidated with companymonitoring data, remaining83% based on professionaljudgment. Pre-1965 levels offormaldehyde were assumedto be the same as post-1965levels.Exposure to >0.7 ppm OR 1.60(95% CI: 0.15–9.77) (6)This document is a draft for review purposes only a nd does not constitute Agency policy 4-79 DRAFT—DO NOT CITE OR QUOTETable 4-3. Epidemiologic studies of formaldehyde and pharyngeal cancer (includes nasopharyngeal cancer)Reference Study design Exposure assessment Results,statistical significance (number observed deaths for cohort study)Overall SMR 1.55(95% CI: 0.87–2.56) (15)High exposure SMR 1.91(95% CI: 0.70–4.17) (6)Coggon et al. (2003)Cohort mortality study of14,014 chemical workersemployed in 6 Britishfactories.Based on data abstractedfrom company records. Eachjob was categorized as havingbackground, low, moderate,high, or unknown levels offormaldehyde.Shangina et al.(2006)Multicentered, hospitalbased case-control study infour European countries;men only. Cancer cases: 34hypopharyngeal; 316laryngeal. Controls: 728hospital patients withvarious conditions.Exposures determined bylocal industrial hygienists,chemists, and physicians.Coding was established andstandardized. Categorieswere developed for 73agents; frequency wasestimated as the proportion oftime a worker was exposed.Linear trends were examinedfor duration in years,weighted duration in hours,and cumulative exposure.Laryngeal cancer:FormaldehydeEver vs. neverHighest cumulative(>22,700 mg/m3-hours)vs. lowestTests of trends:Years exposedCumulative exposureORORp= 0.06p= 0.071.683.12(95% CI: 0.85–3.31)(95% CI: 1.23–7.91)This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-80 Hauptmann et al. (2004) combined URT cancers (cance rs of the salivary gland, mouth, 1 nasopharynx, nasal cavity, and larynx). For averag e intensity of exposure (AIE), the RR was 2 1.69 for the medium exposure category (0.5 to <1.0 ppm) and 2.21 ( p < 0.05) for the high 3 exposure category (>1.0 ppm). For peak exposure, t he RR was 1.24 for the medium exposure 4 category (2.0 to <4.0 ppm) and 1.65 for the high ex posure category (>4.0 ppm). For cumulative 5 exposure, the RR was 1.92 for the medium exposure c ategory (1.5 to <5.5 ppm) but 0.86 in the 6 high exposure category (>5.5 ppm-years). The dose trends for these analyses, while suggestive 7 for average and peak exposures, were not statistica lly significant. 8

9

4.1.2.1.3.2. Case-control studies of other respiratory cancers. Gustavsson et al. (1998) 10 conducted a case-control study of 545 cases of SCC of the oral cavity, oropharynx, hypopharynx, 11 larynx, and esophagus, frequency-matched by age and region with 641 controls. Regression 12 analyses among 545 male cases showed elevated but n onsignificant risks of SCC of the oral 13 cavity (OR = 1.28), esophagus (OR = 1.90), and lary nx (RR = 1.45) associated with 14 formaldehyde exposure. However, several of the car cinoma types were statistically significantly 15 associated with exposure to welding fumes, polyarom atic hydrocarbons, asbestos, and metal 16

dust. 17 In a case-control study, Laforest et al. (2000) exa mined 201 patients with squamous cell 18 hypopharyngeal cancer and 296 patients with squamou s cell laryngeal cancer, who were matched 19

to 296 controls with cancers of other sites in 15 F rench hospitals. Adjusting for potential 20 confounders, the OR of hypopharyngeal cancer in pat ients with a high probability of exposure to 21 formaldehyde was 3.78 (95% CI: 1.50–9.49). The ORs were significantly increased with both 22 exposure durations and high cumulative level of exp osure. 23 Marsh et al. (2002) conducted a nested case-control study of the 22 pharyngeal cancer 24 deaths in the Wallingford, Connecticut, plant cohor t. Each of the pharyngeal cancer deaths was 25 matched on race, sex, age, and year of birth to fou r controls from the cohort. Twenty of the 26 22 cases were exposed to formaldehyde, yielding an OR of 3.04 after adjustment for smoking 27 and year of hire. There was little or no associati on of pharyngeal cancer incidence in these 28 workers with either average or cumulative exposure, based on the exposure estimates in this 29 study. There was a suggested trend of increasing O R with increasing duration of exposure for 30 any formaldehyde exposure as well as for formaldehy de exposure >0.2 ppm. The results of this 31 nested case-control analysis are inconclusive becau se of its low statistical power and 32 questionable exposure estimates, which differed sub stantially from those estimated by the NCI 33 (see Section 4.1.1.1). In addition, the relatively flat dose-response curve in the nested case- 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-81 control study contradicts the positive dose-respons e curves reported (particularly for NPCs) in 1 the same study, based on SMRs derived from county a nd U.S. death rates in the cohort analysis. 2 Shangina et al. (2006) conducted a multicentered ca se-control study in Europe and Russia 3 of 34 cases of hypopharyngeal cancer, 316 cases of laryngeal cancer, and 728 controls. With 4 regard to formaldehyde exposure, a nonsignificant p ositive association was found for laryngeal 5 cancer (OR = 1.68, 95% CI: 0.85–3.31). Trends over increasing exposure were found for 6 duration of exposure in years ( p = 0.06) and for cumulative exposures ( p = 0.07). The 7 investigators reported an OR of 3.12 (95% CI: 1.23– 7.91) for the highest cumulative exposure 8 group (>22,700 mg/m 3 -hours) compared with the unexposed group. 9

10

4.1.2.1.3.3. Summary of other respiratory cancers. The evidence for a compound-specific 11 effect on the risk of buccal/pharynx, oral cavity, oropharynx, hypopharynx, and laryngeal 12 cancers as a result of exposure to formaldehyde is minimal. Only the study by Laforest et al. 13 (2000) and, to a lesser extent, that by Shangina et al. (2006) provided evidence of an association 14 between formaldehyde and these tumors. However, ev en the study of Laforest et al. (2000) had 15 major limitations that made the evidence of an asso ciation suggestive at best. 16

17

4.1.2.1.4. Lung Cancer. None of the cohort studies of workers in specific professions indicated 18 excess risks of lung cancer. Of the professional s tudies reviewed, the RRs range from an 19 extremely low value (SMR) of 0.2 in Hall et al. (19 91), based on nine deaths, to an RR 20 (proportional mortality ratio) of 1.1, based on 70 lung cancer deaths in Walrath and Fraumeni 21 (1983). 22

23

4.1.2.1.4.1. Industrial worker cohort studies of lung cancer. Evidence of a relationship 24 between formaldehyde exposure and lung cancer is co nflicting, with some studies showing 25 modest increases while others show significant defi cits in risk. There is, at best, only weak 26 evidence from several studies to suggest that expos ure to formaldehyde is associated with lung 27

cancer. 28 Several industrial cohort studies (Andjelkovich et al., 1995; Bertazzi et al., 1989, 1986; 29 Stayner et al., 1988; Edling et al., 1987) reported no significant excess risks of lung cancer from 30 exposure to formaldehyde. No consistent associatio n between formaldehyde exposure and lung 31 cancer was found in several reports of the NCI 10-p lant cohort originally investigated by Blair et 32 al. (1987, 1986). Hauptmann et al. (2004) gives th e most recent report on this cohort, which has 33 been studied in part or in its entirety by several others (Marsh et al., 1994, 1992a, b; Sterling and 34 Weinkam, 1994, 1989a, b, 1988; Robins et al., 1988; Liebling et al., 1984; Fayerweather et al., 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-82 1983; Wong, 1983; Marsh, 1982). Marsh et al. (1996 ) reported small but significantly increased 1 risks of respiratory cancer in males at one plant f rom the cohort. The SMRs were 1.22 based on 2 U.S. rates, 1.34 based on Connecticut rates, and 1. 28 based on county rates. 3 Similarly, Gardner et al. (1993) and Acheson et al. (1984) found a significant but modest 4 association between lung cancer and formaldehyde ex posure (SMR = 1.2 [95% CI: 1.1–1.4]). In 5 workers hired after 1964, the SMR was 1.1. No tren ds by level or duration of exposure were 6 found. Pinkerton et al. (2004) and Stayner et al. (1988) studied a cohort of 11,030 workers in 7 three garment plants and found an SMR of 1.1 for lu ng cancer. Some studies have found modest 8 elevations in risk of lung cancer with formaldehyde exposure, some of which were significant. 9 Coggon et al. (2003) updated the Gardner et al. (19 93) study of industrial workers. By taking 10 data from all six factories together, results showe d a statistically significant excess risk of lung 11 cancer in the high-exposure category when compared with British national mortality rates (SMR 12 = 1.58 [95% CI: 1.40–1.78]) and to local mortality rates (SMR = 1.28 [95% CI: 1.13–1.44]). 13 Callas et al. (1996) reanalyzed the cumulative expo sure of 279 lung cancer cases among white 14 male workers from the NCI study, which comprised 80 % of the NCI cohort (Blair et al., 1986). 15 The analysis revealed modest RRs of 1.46, 1.27, and 1.38 for lung cancer in the cumulative 16 exposure categories 0.05 to 0.5, 0.51 to 5.5, and g reater than 5.5 ppm-years, respectively. None 17 of these RRs were significant. Finally, Matanoski (1991) reported a significant deficit in the risk 18 of respiratory cancer (SMR = 0.56 [95% CI: 0.44–0.7 0]; 77 observed) in pathologists 19 presumably exposed to formaldehyde based on U.S. mo rtality rates. 20

21

4.1.2.1.4.2. Case-control studies. Several case-control studies of lung cancer (Partan en et al., 22 1990; Gerin et al., 1989; Bond et al., 1986; Coggon et al., 1984; Fayerweather et al., 1983; 23 Anderson et al., 1982) showed no excess lung cancer risk associated with potential exposure to 24 formaldehyde when analyzed by length of exposure, i ntensity, and potential exposure 5, 10, or 25 15 years before death or by combinations of these f actors. By contrast, Coggon et al. (1984) 26 reported a statistically significant increase in ri sk of lung cancer among male patients with any 27 potential exposure to formaldehyde based on occupat ions listed on death certificates (SMR = 1.5 28 [95% CI: 1.2–1.8]). 29 De Stefani et al. (2005) conducted a case-control s tudy of 338 adenocarcinomas of the 30 lung in male patients admitted to four Montevideo h ospitals from 1994 to 2000. The highest 31 ORs were for smoking (6.0 [95% CI: 3.3–11]) and for former smokers (4.0 [95% CI: 2.1–7.3]). 32 In addition, three agents (i.e., asbestos, silica d ust, and formaldehyde) indicated significant 33 excess risks of lung adenocarcinoma after adjusting for smoking history. A significant exposure- 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-83 duration relationship was found for formaldehyde fo r 21+ years of exposure (OR = 3.0 [95% CI: 1 1.6–5.8]; p = 0.004 for trend). 2

3

4.1.2.1.4.3. Summary of lung cancer. The evidence for an association between formaldehyd e 4 and lung cancer is limited. Only one study has fou nd a statistically significant effect (Coggon et 5 al., 2003). However, there may be other explanatio ns rather than exposure to formaldehyde for 6

this association

. Except for the findings of De Stefani et al. (2005) , other studies of lung cancer 7 and exposure to formaldehyde have not supported thi s finding, including several well-done 8 cohort studies that were specifically designed to e valuate lung cancer. Until the Coggon et al. 9 (2003) study of British formaldehyde workers is rep licated or reevaluated to determine the cause 10 of the excessive lung cancer risk, evidence from th at study alone is insufficient at this time to 11 support an association between lung cancer and form aldehyde exposure. 12

13

4.1.2.1.5. Summary of respiratory tract cancers. Recent studies of NPC continue to support an 14 association with exposure to formaldehyde even at l ow levels. In some studies, the association 15 between formaldehyde and NPC persisted even when ad justed for the effect of potential 16 confounders (Hauptmann et al., 2004). Data from so me reports have suggested a dose-response 17 relationship (Hauptmann et al., 2004; Marsh et al., 2002; Vaughan et al., 2000). 18 The risk of NPC was significantly elevated among in dustrial workers with cumulative 19 exposure, average exposure, and peak exposure to fo rmaldehyde (Hauptmann et al., 2004). The 20 studies of the single Wallingford plant by Marsh et al. (2002, 1996, 1994) and Marsh and Youk 21 (2005) also revealed a dose-response trend, althoug h the absolute exposure level estimates were 22 much lower. The relatively flat dose-response curv e seen in the nested case-control study by 23 Marsh et al. (2002) of all pharyngeal cancers was i nconsistent with the positive dose-response 24 curves reported in the same paper based on county a nd U.S. death rates, particularly for NPC. 25 Also of interest was the finding of a statistically significant increase in the risk of NPC in 26 formaldehyde-exposed workers who were seropositive for EBV in the Hildesheim et al. (2001) 27 study. 28 The pooled analysis by Luce et al. (2002) provides evidence of a relationship of sinonasal 29 cancer, particularly adenocarcinoma, with formaldeh yde. However, as with some of the studies 30 of NPC, the findings are potentially confounded by concurrent exposure to wood dust. When 31 wood dust exposure was adjusted for in the analysis , the resulting risks were still positive but 32 based on small numbers and, as a result, subject to much variability. The more recent studies 33 continued to reveal small significant and nonsignif icant associations among cancers of 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-84 buccal/pharynx, oral cavity, hypopharynx, and laryn x and exposure to formaldehyde. However, 1 the estimates were always based on small numbers. 2 A recent study that reported statistically signific ant lung cancer in association with a high 3 level of exposure to formaldehyde was conducted by Coggon et al. (2003). The investigators 4 suggested that unknown lifestyle factors, including smoking, could be responsible for the 5 finding. Despite the results of their analysis, th e authors were unconvinced that formaldehyde 6 was the agent responsible for the elevation in lung cancer risk. However, De Stefani et al. (2005) 7 also reported a statistically significant risk of l ung adenocarcinoma in formaldehyde-exposed 8 hospital patients even when smoking was controlled for in their analyses. 9 In all studies of formaldehyde and lung cancer, smo king remains an important 10 confounder and possibly an effect modifier. Residu al confounding of smoking or other 11 respiratory exposures (e.g., wood dust or chemical or particular exposures) must always be 12

considered. 13

14

4.1.2.2. Non-Respiratory Tract Cancer 15

4.1.2.2.1. LHP cancers

. Cancers of the hematopoietic system include lymphos arcoma, 16 reticulosarcoma, Hodgkin’s disease, non-Hodgkin’s d isease, multiple myeloma, and all types of 17 leukemia, including lymphoid and myeloid. Virtuall y all of the studies of LHP cancers and 18 formaldehyde are cohort studies and are divided int o two groups: professional and industrial. 19 Several of the studies of professional groups were reviewed in an IARC (1995) monograph and 20 are briefly discussed in the next section regarding their findings on cancer of the LHP system. 21 One case-control study of non-Hodgkin’s lymphoma is discussed at the end of this section. 22

23

4.1.2.2.1.1. Professional cohort studies. Several cohort studies have been undertaken by 24 professional groups (i.e., anatomists, pathologists , embalmers, and funeral directors) because 25 their careers are likely to bring them into contact with formaldehyde. Some studies have 26 reported an increase in the risk of myelogenous leu kemia and other LHP cancers (see Table 4-4). 27 A few of the increased risks were statistically sig nificant. None of the studies of professionals 28 have used personal exposure measurements of formald ehyde or other chemicals, making 29 specificity for any single exposure difficult to de termine. 30 Harrington and Shannon (1975) conducted a cohort mo rtality study of 2,079 British 31 pathologists (1955–1973) and 12,944 British medical laboratory technicians (1963–1973). When 32 compared with death rates for England and Wales, th e all-cause SMR for the pathologists was 33 0.60 versus 0.67 for the laboratory technicians. T here was a significant increase in the risk of 34 lymphatic and hematopoietic neoplasia (SMR 2.0; 8 o bserved with 3.3 expected; p < 0.01) 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-85 among male pathologists. However, the SMR for tech nicians was only 0.6 (3 observed). The 1 low SMRs suggest that these professionals have a he althier profile compared with the British 2 population. No actual exposure estimates are avail able. 3 Harrington and Oakes (1984) expanded the above stud y to include 2,307 male and 413 4 female pathologists and laboratory technicians. Mo rtality was only examined from 1973 until 5 1980; deaths that occurred before 1974 were not inc luded in the update. The SMR for leukemia 6 was 0.91 in men and 9.26 (based on one case) in wom en. Although the earlier LHP cancer 7 deaths were not included in this analysis, the inve stigators say in their conclusion that their 8 previous suggestion of an increase in certain lymph atic neoplasia was not confirmed in the 9 present study because of small numbers. The except ionally low SMRs suggest that this group of 10 professionals enjoyed a healthier lifestyle compare d with the British population as a whole. Just 11 as in the earlier studies of these professionals, n o exposure estimates are available. 12 Hall et al. (1991) expanded the above study by incl uding the newest members of the 13 Royal College of Pathologists. The cohort totaled 4,512 individuals, although only 3,069 males 14 and 803 females were included in the analysis. The reasons for this discrepancy were not 15 specified, although the authors mentioned that an u nknown number of expected deaths for 16 Northern Irish and female Scottish pathologists wer e not calculated, 32 pathologists were lost in 17 follow-up, and cause of death was unknown for 9 ind ividuals. Follow-up was extended from 18 1980 to 1986. Mortality was enumerated from 1974 t o 1987, a period of time that differed from 19

both of the earlier studies described above. There were statistically not significant excess risks 20 for lymphatic and hematopoietic cancer (SMR 1.44; 1 0 observed) and leukemia (SMR 1.52; 21 4 observed) for both sexes combined, based on morta lity rates in England and Wales. 22 Separately, there was 1 female death in the lymphat ic and hematopoietic cancer category (0.57 23 expected). The most striking observation in this s tudy is that, despite the low cancer mortality 24 (SMR 0.45 for all cancer; 53 observed but 118.19 ex pected), there was still an excess (but not 25 statistically significant) risk of hematopoietic ca ncers. This finding of an extremely low risk for 26 all cancers suggests that population death rates ma y not be appropriate as a referent group—for 27 example, the SMRs for lung cancer (0.19) and nonneo plastic respiratory diseases (0.23) were 28 significantly decreased, suggesting a lower prevale nce of smoking among the pathologists 29 compared with the general population of England and Wales. However, the finding of a possibly 30 increased risk of LHP cancers should be analyzed fu rther by selecting a more appropriate 31 reference population (another professional group wi thout exposure to formaldehyde) or by 32 utilizing internal comparisons. 33 Walrath and Fraumeni (1983) conducted a proportiona te mortality study of all embalmers 34

and funeral directors licensed in the state of New York between 1902 and 1980 who were known 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-86 to have died between 1925 and 1980. The investigat ors requested death certificates for 1,678 1 persons but received only 1,263 (75%). The investi gators restricted their analysis to 1,132 2

males. The distribution of the causes of death was compared with the age-, race-, and calendar3 year-specific proportions of deaths for each cause among the male U.S. population. Duration of 4 exposure was approximated by time since first licen se. While the methodology could not be 5 applied in all calculations because of data gaps, e xcess risks were found for lymphatic and 6 hematopoietic cancers, with a proportionate mortali ty ratio (PMR) of 1.2 (observed 25), and for 7 leukemia, with a PMR of 1.32 and proportionate canc er mortality ratio (PCMR) of 1.19 8 (12 observed). The PMRs were not affected when the estimates were stratified by latency 9 (<35 years or 35 years since first license) or by a ge at first license. Because the cause of death 10 could not be determined for nearly 25% of the study group, the risk estimates could be 11 underestimated. The metrics, PMR, and PCMR are not stochastic processes. An increase in one 12 cause would produce decreases in all the other caus es. 13 Using the proportionate mortality method, Walrath a nd Fraumeni (1984) studied 1,007 14 deceased white male embalmers, members of the Calif ornia Bureau of Funeral Directing and 15 Embalming, whose deaths occurred between 1925 and 1 980. The decedents had to have been 16 licensed to practice between 1916 and 1978. For ly mphatic and hematopoietic cancer, the PMR 17 was 1.22 (19 deaths observed). For leukemia alone, the PMR was 1.75 and significant 18 (12 deaths observed, p < 0.05). Among embalmers licensed for 20 years or longer, the risk of 19 leukemia increased and was also significant (PMR 2. 21; 8 observed; p < 0.05). But this study, 20 like the study of New York embalmers, had the same limitations discussed above. The 21 investigators did not provide information on the nu mber of embalmers for whom no cause of 22

death could be found. 23 Levine et al. (1984) conducted a cohort mortality s tudy of 1,477 male Ontario 24

undertakers first licensed between 1928 and 1957 an d followed until the end of 1977. Out of 25 359 subjects who had died, there were 8 deaths from lymphatic and hematopoietic cancers 26 compared with 6.5 expected. Additionally, there we re 4 deaths from leukemia versus 27 2.5 expected. Because death rates were not availab le for Ontario before 1950, person-years and 28

deaths before 1950 could not be counted. No actual exposure estimates are available for these 29

undertakers. 30 Stroup et al. (1986) conducted an historic cohort m ortality study of 2,317 men who were 31

members of the American Association of Anatomists b etween 1888 and 1969. The investigators 32 derived SMRs from the U.S. white male population an d used members of the American 33 Psychiatric Association (APA) as a comparison group . Vital status was ascertained between 34 1925 and 1979. Women were excluded from analysis b ecause of the small numbers. Only 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-87 738 deaths were observed versus 1,133.9 expected, b ased on U.S. death rates (SMR 0.65), 1 possibly indicating a sizable HWE. However, a slig ht increase in the risk of lymphatic and 2 hematopoietic cancers (SMR 1.2; 18 observed) and th e risk of leukemia (SMR 1.5; 10 observed) 3 was evident. A significant increase in the risk of brain cancer (SMR 2.7; 10 observed; p < 0.05) 4 was also reported. When the leukemia analysis was restricted to the myeloid type, the SMR 5 increased to 8.8, based on five deaths ( p < 0.05). The analysis using the APA group was 6

restricted to deaths that occurred between 1900 and 1969. This restriction removed five 7 leukemia deaths and person-years from the analysis because they likely died after 1969. Because 8 of this, there were only 3 leukemia deaths versus 3 .6 expected, based on APA death rates. The 9 investigators concluded that the etiological agent had not been definitively identified, mentioning 10 that a wide range of solvents, stains, and preserva tives, including formaldehyde, are used to 11 prepare biological specimens. 12 Logue et al. (1986) conducted a cohort study of mal e radiologists and pathologists 13 registered with the Radiation Registry of Physician s and the College of American Pathologists 14 (CAP) between 1962 and 1977. Although the main foc us was on determining mortality in 15 radiologists from exposure to ionizing radiation, m ortality was also ascertained for pathologists 16 alone. To derive SMRs, expected deaths were the su m of the products of person-years times 17 death rates for both cohorts during the follow-up p eriod in white males only. However, there 18 were no exposure measurements, and the SMRs were no t adjusted for calendar time. Of 5,585 19 members of the CAP, 496 had died by December 31, 19 77. Although the SMR was 0.48 for 20 pathologists for cancer of the lymphatic and hemato poietic system, for the more specific 21 category of leukemia and aleukemia the SMR was 1.06 (neither was significant). For 22 radiologists, the SMRs were 0.78 and 1.55, respecti vely, also not significant. Cause of death 23

could not be determined for 8% of the deaths. Alth ough age-adjusted rates for leukemia were 24 also calculated for each cohort, they were only use d for comparison between the two separate 25 professional groups. 26 Hayes et al. (1990) conducted a proportionate morta lity study of 3,649 deceased white 27

and 397 deceased nonwhite U.S. male embalmers and f uneral directors who had died between 28 1975 and 1985, using records from local licensing b oards, state funeral directors’ associations in 29 32 states and the District of Columbia, the Nationa l Funeral Directors’ Association, and state 30 offices of vital statistics (n = 894). Expected de aths by cause were derived from 5-year age- and 31 calendar-year-specific proportions of deaths among appropriate race groups from the U.S. 32 population. No measured exposure data were availab le. A PCMR would be derived by 33 excluding noncancer causes of death. Statistically significant excesses in hematopoietic and 34 lymphatic cancers were found in white (PMR 1.31 [95 % CI: 1.06–1.59]; 100 observed) and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-88 nonwhite (PMR 2.41 [95% CI: 1.35–3.97]; 15 observed ) embalmers and funeral directors. The 1 combined PMR was 1.39 (95% CI: 1.15–1.63). The exc ess risk was higher for myeloid 2 leukemia (ML) (PMR 1.61 [95% CI: 1.02–2.41]; 23 obs erved) and for other unspecified 3 leukemias (PMR 2.08 [95% CI: 1.21–3.34]; 17 observe d) in white males. The risks were 4 elevated in nonwhite males based on only a few case s (PMR 1.33 [95% CI: 1.10–1.60]; 5 4 observed). 6 Matanoski (1991) conducted a study of 6,111 male pa thologists for NIOSH. Members of 7 the cohorts were part of an earlier unpublished stu dy. Twenty-nine thousand psychiatrists were 8 used as a comparison group. Both samples were sele cted from the membership rolls of 9 professional associations. A total of 3,787 pathol ogists died between 1940 and 1978. Women 10 were excluded from the analysis. Of the population of psychiatrists, 4,788 died by 1980. U.S. 11 age- and calendar-time-specific death rates from 19 25 were used to develop SMRs. Separate 12 SMRs were based on psychiatrists’ death rates. The risk of hematopoietic cancer (excluding 13 Hodgkin’s disease) was elevated (SMR 1.25; 57 obser ved) based on U.S. white males. For 14 leukemia, the SMR was 1.35 (31 observed). The SMR for leukemia among psychiatrists was 15 0.83 (35 observed). Compared with leukemia in psyc hiatrists, the SMR for pathologists was 16 1.68 (95% CI: 1.14–2.38). The SMR for other lympha tic cancers was 1.53 (16 observed) and for 17 LHP cancer 1.22 (64 observed). Comparing the patho logists’ death rates to those of psychiatrists 18 could be thought to have greater validity than if d eath rates for the U.S. population as a whole 19 had been used, because of shared socioeconomic circ umstances and access to medical care 20 between the two professional groups. Differences i n access to health care might have been 21 greater for subjects in the earlier part of the stu dy, because improved diagnosis and medical care 22 for LHP cancers became more broadly available later in the study period. By using SMRs based 23 on U.S. death rates, which include those who do not have adequate access to medical care, the 24 difference between expected and observed deaths wou ld be reduced. This is less likely to occur 25 when one professional group is compared with anothe r professional group, assuming 26 psychiatrists and pathologists have equal access to care. 27

28

4.1.2.2.1.2. Industry worker cohort studies. This section discusses updated industrial worker 29

studies that show associations between LHP cancer a nd formaldehyde. The studies by Marsh et 30 al. (1994), Blair et al. (1986), and Acheson et al. (1984) and the later update by Gardner et al. 31 (1993) provide estimates of exposure to formaldehyd e. The remaining studies generally rely 32 either on duration of exposure (number of years in the job) as a surrogate (Pinkerton et al., 2004) 33 or provide no exposure assessment. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-89 Marsh et al. (1994), in an early study of the Walli ngford plant, which is also part of the 1 Hauptmann et al. (2004, 2003) and Blair et al. (198 6) studies, found SMRs of 0.89 and 0.91, 2 based on U.S. and county death rates, respectively (25 observed deaths). The authors did not 3 further discuss this cancer site until after Hauptm ann et al. (2003) was published. Blair et al. 4 (1986) reported on 4,396 deaths from all causes in the 10 formaldehyde-associated factories that 5 made up the NCI cohort of 26,561 workers employed b efore January 1, 1966. There was little 6 evidence of an association with LHP system cancer ( SMR 0.91; 56 observed) in exposed white 7 men, who dominated the cohort. 8 Hauptmann et al. (2003) updated the cohort mortalit y study of Blair et al. (1986) that 9 consisted of predominantly the same (25,619) worker s from 10 plants. The primary focus of this 10 analysis was cancer of the LHP system, including le ukemia. The description and demographics 11 of the current study are the same as those reported by Blair et al. (1986) and Stewart et al. 12 (1986). In the current update, follow-up was exten ded through December 31, 1994. The 13 additional 15 years of follow-up increased the numb er of deaths from 4,349 to 8,486. Exposures 14 were not updated for the 4% of workers still in exp osed jobs in 1980, but eliminating exposure 15 estimates for these workers did not change the resu lts since exposures received after this date 16

were considered so low as to contribute little to t he analysis by the authors. 17 Peak exposure categories were defined as nonexposed , low (0.1–1.9 ppm), medium (2.0– 18 3.9 ppm), and high (4.0 ppm or greater). Average i ntensity categories of exposure were defined 19 as nonexposed, low (0.1–0.4 ppm), medium (0.5 to <0 .9 ppm), and high ( ≥ 1.0 ppm). Cumulative 20 exposure was defined as nonexposed, low (0.1–1.4 pp m-years), medium (1.5–5.4 ppm-years), 21 and high ( ≥ 5.5 ppm-years). Duration of exposure was defined a s 0, 0.1–4.9 years, 5.0– 22 14.9 years, and ≥ 15 years. The median TWA exposure level was 0.45 p pm, range 0.01– 23 4.25 ppm. Only 2.6% of the workers had average exp osure intensities of 2 ppm or higher, and 24 14.3% had peak exposures of 4 ppm or higher. A tot al of 3,201 workers had no exposure. The 25 median duration in formaldehyde-exposed jobs was 2 years. The median TWA intensity for 26 formaldehyde exposure was 0.5 ppm among exposed wor kers. 27 A Poisson regression model was stratified for calen dar year, age, sex, race, and pay 28 category (salary/wage). A minimum latency period o f 2 years between exposure and death from 29 a potentially exposure-related LHP cancer was assum ed by the investigators to prevent the 30 inclusion of exposures not likely to contribute to the development of LHP cancer because of their 31 timing. Other lag times were evaluated that did no t improve the regression models. 32 There were 2,099 cancer deaths. Hauptmann et al. ( 2003) reported that mortality from all 33 causes, all cancers, and LHP malignancies were sign ificantly lower among the unexposed (SMRs 34 0.77, 0.65, and 0.62, respectively). Among the exp osed, the SMRs were 0.95, 0.90, and 0.80, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-90 respectively, for mortality from all causes, all ca ncers, and LHP malignancies. These SMRs in 1 part reflect the HWE caused by using external U.S. population death rates as a referent. 2 Unexposed workers also may have differed from the e xposed workers in other ways. The 3 remaining analyses used internal comparisons, avoid ing the HWE. The referent group in this 4 analysis was the low exposure group rather than the unexposed group, because nonexposed 5 workers, who are primarily managers, secretaries, a nd other non-production personnel, were 6 considered likely to have different socioeconomic c haracteristics than workers in the production 7

areas. 8 Statistically significant positive associations wer e found for LHP malignancies and 9 leukemia, particularly myeloid, in certain higher e xposure categories in comparison with 10 employees in the lowest exposure categories (Table 4-4). In the highest peak exposure level, the 11 RR for LHP malignancies was 1.87 (95% CI: 1.27–2.75 ; 64 observed) and for ML was 3.46 12 (95% CI: 1.27–9.43; 14 observed) compared with empl oyees in the low exposure peak level. For 13 workers with high peak exposure levels, the RR for LHP malignancies was 1.71 (95% CI: 14 1.14-2.58; 49 observed) and 2.43 (95% CI: 0.81–7.25 ) for ML. The trend tests for slope were 15 highly statistically significant for both LHP malig nancies ( p < 0.002) and ML (

p < 0.009). 16 Significant results for LHP cancers were also seen with the average intensity exposure 17 metric. RRs were 1.63 ( p < 0.05) and 1.50 (

p < 0.05) for the medium and high categories, 18 respectively. The risk of ML was also significantl y increased (RR = 2.49) in the highest 19 exposure category. In contrast, Hauptmann et al. ( 2003) did not find statistically significant 20 associations of formaldehyde with LHP cancer, eithe r by cumulative exposure or years of 21 duration. However, there were positive association s for leukemia (RR = 1.39) and ML (RR = 22 1.35) when exposure was 15 years or longer. 23 The authors concluded that formaldehyde may cause l eukemia, particularly ML, in 24 humans. However, because results from other studie s were inconsistent, they suggested caution 25 in drawing definite conclusions. A biological basi s for the significant excess risk of LHP cancer 26 remains unclear. The authors pointed out several s tudies that indicate changes that are consistent 27 with chromosomal changes in formaldehyde-exposed pe rsons, such as increased frequencies of 28 (MN (He et al., 1998; Kitaeva et al., 1996; Suruda et al., 1993), sister chromatid exchanges 29 (SCEs) (Shaham et al., 2002, 1997; Yager et al., 19 86), chromosomal aberrations (CAs) (He et 30 al., 1998; Bauchinger and Schmid, 1985), and DNA-pr otein cross-links (DPXs) (Shaham et al., 31 1997, 1996a) in peripheral lymphocytes of humans ex posed to formaldehyde. 32 Hauptmann et al. (2003) identified 11 suspected car cinogens used at the plants: 33 antioxidants (unspecified), asbestos, carbon black, dyes and pigments, hexamethylenetetramine, 34 melamine, phenol, plasticizers, urea, wood dust, an d benzene. Some workers were employed as 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-91 chemists and laboratory technicians. The investiga tors did not find substantial changes in the 1 risk estimates after adjusting for exposure to thes e substances or for working as a chemist or 2 laboratory technician. They also eliminated the 58 6 benzene-exposed persons from their 3 analysis and found similar results (benzene is a kn own human leukemogen). Smoking was not 4 likely to explain an increased risk of leukemia in this cohort, because no increase was seen for 5 smoking-related diseases, including lung cancer. T he cohort consisted predominantly of males 6 (88%). Strengths include the fact that the cohort was large and there was a long period of 7 follow-up that was 96.6% successful. Internal anal yses eliminated the HWE. One potential 8

limitation that could lead to an underestimate of r isks is the 3.4% or 866 lost to follow-up. 9 The study by Hauptmann et al. (2003) has been criti cized extensively by several experts 10 representing the formaldehyde industry (Tarone and McLaughlin, 2005; Casanova et al., 2004; 11 Cole and Axten, 2004; Collins, 2004; Collins and Li neker, 2004). Most of the same criticisms 12 have been repeated in other critiques by the above- mentioned authors and have been addressed in 13 the discussions concerning the details of the metho dology. However, a few new issues have 14 arisen from these critiques, as follows. One issue pertains to a concern that person-years at risk 15 of death may have been assigned wrongly to the high est “peak” category of exposure for the 16 duration of the study period. For example, there i s inconsistency in the fact that only 4% of the 17 original cohort (Blair et al., 1986) had average ex posures equaling or exceeding 2 ppm yet 45% 18 of the person-years were assigned to the peak expos ure category. Average exposures are time19 weighted exposures that can have brief excursions o ver 4 ppm and still average 2 ppm or less. 20 Only for the peak exposure surrogate were person-ye ar values assigned to the peak category 21 following the exposure, because it is a test for th e possibility that biological changes could have 22 been initiated from that brief high exposure that m ight increase the risk of cancer. If these 23 genetic changes are irrevocable, then the risk of c ancer could be increased and subsequent person 24 × years should be assigned to that higher risk cate gory. 25 According to Casanova et al. (2004), the assignment of peak exposures in the Hauptmann 26 et al. (2003) study was questionable because they w ere based on professional judgement. 27 However, there are adequate grounds for hypothesizi ng that the assignment of peak exposure 28 was completed before determination of vital status and cause of death. It is always possible that 29 some subjects may be subject to misclassification. Hauptmann et al. (2003) chose this metric 30 partly because it more closely resembled the exposu re that embalmers and pathologists may have 31 received from formaldehyde. This same criticism co uld be said about the Coggon et al. (2003) 32 study as well. 33 Hauptmann et al. (2003) have also been criticized b ecause the metric “cumulative 34 exposure” was not significant and did not show a tr end. No adequate explanation has been given 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-92 by Hauptmann et al. (2003) except that it is possib le that this metric is not as sensitive for this 1 agent. Duration of exposure was only weakly associ ated with a trend of increasing risk. After 2 15 years’ duration, there appeared to be a slight i ncrease in ML based on 10 cases (RR = 1.35 3 [95% CI: 0.56–3.24]). For all leukemia the risk in workers who were exposed 15 or more years 4 was somewhat higher (RR = 1.39 [95% CI: 0.78–2.49]) based on 22 cases. 5

Another of the criticisms from these authors discus sed the lack of a biologically plausible 6 explanation for how leukemia could result from expo sure to formaldehyde when there appears to 7 be no recognizable indication of the presence of fo rmaldehyde in excessive quantities in the 8 blood of animals or any associated metabolites in e xperimental research animals. Hauptmann et 9 al. (2004b) responded that there is evidence that g enotoxic effects can be detected in vivo in the 10 bone marrow of rats and in human peripheral lymphoc ytes. 11 Stayner et al. (1988) conducted a cohort study of 1 1,030 workers (82% female) followed 12 from 1955 or the beginning date of exposure through 1982 in three garment factories. Personnel 13 records from three garment manufacturing facilities , one in Pennsylvania and two in Georgia, 14

were used to assemble a cohort of workers who attai ned a minimum of 3 months of exposure 15 after the introduction of formaldehyde into these f acilities. Formaldehyde resins were used to 16 treat fabrics, beginning in 1955 and 1959. Althoug h formaldehyde levels were available on a 17 subset of the employees from monitoring data availa ble from surveys completed in 1981 and 18 1984, they were not used in this analysis. Instead , the results were stratified by duration and 19 latency. SMRs were based on U.S. population mortal ity rates. Based on six cases, the SMRs for 20 leukemia were 2.43 and 3.81 among workers with 20 o r more years since first exposure or at 21 least 10 years of exposure, respectively. In their conclusions, the authors suggested that, 22 although the numbers of deaths from LHP cancers wer e small, the risks were related to duration 23 and latency. 24 Pinkerton et al. (2004) updated the Stayner et al. (1988) study by adding 16 years of 25 follow-up. No new exposure information was added. The mean TWA exposure in 1981–1984 26 for the three plants was 0.15 ppm. No additional i nformation regarding earlier industrial hygiene 27 data was available, although the authors stated tha t the levels of exposure to formaldehyde were 28 greater in the years before 1980. Stayner et al. ( 1988) cited independent studies of exposure 29 levels in similar garment factories in the 1960s th at seemed to indicate that the formaldehyde 30 levels during that period ranged from 0.9 to 2.7 pp m (Blejer and Miller, 1966) in one garment 31 manufacturing area. Another report (Shipkovitz, 19 66) of 10-minute personal exposure samples 32 indicated a range from 0.3 to 2.7 ppm in eight garm ent plants. In another study (Ahmad and 33 Whitson, 1973), the levels ranged from 2 to 10 ppm. Goldstein (1973) calculated that 34 concentrations in the cutting rooms of garment plan ts dropped from 10 ppm in 1968 to less than 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-93 2 ppm in 1973 because of an improvement in the resi n treating process. The authors assumed 1 that exposure ceased in 1981 and 1983. This produc ed an underestimate of exposure based on 2 duration of employment for about 11% of the cohort who were still actively employed after those 3 dates. Stayner et al. (1988) speculated that the r isks of cancer of the buccal cavity, leukemia, and 4 other LHP neoplasia may have been due to exposure t o the highest potential formaldehyde levels 5 in the industry between 1955 and 1962, because the resin used to treat permanent press fabrics 6 still contained a relatively large amount of formal dehyde. 7 The SMRs were derived from age-, race-, and calenda r-time-adjusted U.S. mortality 8 rates. The analysis was repeated using Georgia or Pennsylvania mortality rates. In addition to 9 the primary analysis of the underlying cause of dea th, the analysis used all causes listed on the 10 death certificates to evaluate multiple cause morta lity. As a referent for this, the analysis relied 11 on multiple cause death rates available since 1960 from the National Death Index maintained by 12

the U.S. Centers for Disease Control and Prevention (CDC). 13 Altogether, 608 cancer deaths were observed. The S MR for all cancer was 0.89 (95% CI: 14 0.82–0.97). The overall SMR for leukemia was 1.09 (24 deaths) and 1.44 (15 deaths) for ML. 15 After 10 years of exposure, the risk for ML was 2.1 9. Exposure prior to 1963 was associated 16 with a risk of 1.61. Among garment workers followe d for 20 or more years from initial 17 exposure, the SMR was significantly elevated for ML (1.91; p < 0.05; 13 deaths), as was the 18 SMR for multiple cause leukemia (1.92 [95% CI: 1.08 –3.17]; 15 deaths) in the subgroup with 19 10 or more years of exposure to formaldehyde and wh o were followed for 20 or more years after 20 first exposure. The multiple cause mortality for M L for this subgroup of workers was also 21 significant (SMR 2.55 [95% CI: 1.10–5.03]; 8 deaths ). 22 The study by Stayner et al. (1988) has only limited power to detect excess risks of rare 23 cancers, such as NPC and nasal cancer (13 and 16%, respectively). Limitations to the 24 interpretations of the findings include a lack of a ny monitoring data before 1981, particularly 25 during the critical time period 1955 to 1962, and l ack of personal exposure estimates for any 26 members of the cohort. The possibility exists that misclassification may still be present because 27 the intensity of exposure to formaldehyde decreased as improvements were made in the resin 28 systems used to treat fabrics (e.g., a person who w orked 5 years beginning in 1955 might have 29 been subject to greater exposure than a person who worked 5 years beginning in 1993). 30 However, workers from the 1950s and 1990s were both placed in the same category of having 31 worked fewer than 10 years. The median duration of exposure was 3.3 years. Work histories 32 were not updated in the follow-up study; however, t he low or background exposure levels that 33 probably existed after 1981 were not likely to cont ribute substantially to the risk of cancer. The 34 use of mortality data to estimate risk, when the ca se fatality rate was less than 100% for most 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-94 cancer sites evaluated, could potentially produce a n underestimate of the actual risk. Despite 1 these limitations, this study provides additional e vidence of an association between leukemia, 2 especially ML, and formaldehyde in comparison with the general population. 3 Gardner et al. (1993) reported that the risk of leu kemia was not statistically significant 4 (SMR 0.9) based on 15 deaths among workers employed before 1965. Only four leukemia 5 deaths were observed after 1964 through 1989, produ cing an SMR of 0.9. 6 When Coggon et al. (2003) updated the above cohort study of 14,014 men first employed 7 before 1965 in six factories by adding 11 additiona l years of follow-up (ending December 31, 8 2000), no increase in the risk of leukemia or relat ed cancers of the hematopoietic system was 9 reported, either in the entire cohort (SMR 0.91; 31 observed) or in the group with the highest 10 formaldehyde exposure (>2 ppm) (SMR 0.71; 8 observe d). Similar results were obtained for 11 Hodgkin’s disease, non-Hodgkin’s lymphoma, and mult iple myeloma. No other cancers of the 12 hematopoietic system were evaluated, and no additio nal analyses were performed to assess a 13 possible leukemia risk. However, the main finding from this study was a marked association of 14 lung cancer with formaldehyde (discussed in the lun g cancer section). This study’s main focus 15 was respiratory disease, lung cancer, and stomach c ancer, not LHP cancers. For cancers of the 16 LHP system, there was neither latency evaluation no r internal comparisons. The HWE is also 17 potentially a problem. 18 Andjelkovich et al. (1995) studied a cohort of 3,92 9 male iron foundry workers 19 potentially exposed to formaldehyde between January 1, 1960, and December 31, 1989, in which 20 127 cancer deaths had occurred during the observati on period. An industrial hygienist, after 21 reviewing work histories, categorized formaldehyde exposure into four levels corresponding to 22 the approximate midpoint of the ranges: none, low ( 0.05 ppm), medium (0.55 ppm), and high 23 (1.5 ppm) for exposure to formaldehyde. Boundaries of these exposure categories were not 24 given. The authors warned that the assignment of e xposure levels was not perfect because 25 “subjective judgment had to be applied in many inst ances.” SMRs were based on U.S. male 26 mortality rates, but actual ranges were not specifi ed. The authors also compared the exposed to 27 2,032 nonexposed workers from the same company. Th e population-based SMR for 28 hematopoietic cancer in the exposed population was 0.59 (based on seven observed deaths). For 29 leukemia the SMR was 0.43, based on two deaths. Th ere were no other analyses for leukemia or 30 LHP cancers in this study. Because of the uncertai nty about workers’ true formaldehyde 31 exposure, there was no analysis by level of exposur e, duration, or latency. There were also very 32 few LHP cancers in the cohort. Thus, these results neither support nor refute an association of 33 formaldehyde exposure with LHP cancers. The main f ocus of this paper was on lung cancer risk. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-95 Bertazzi et al. (1989, 1986), in a cohort mortality study, followed 1,330 male workers 1 from 1959 through 1986 at a formaldehyde resin plan t in Italy. The workers had to have been 2 employed for at least 30 days at the plant sometime between 1959 and 1980 to be included in the 3 study. Their mortality was compared with national and local rates adjusted for age and calendar 4 time period. No individual exposure estimates were available, but mean levels were estimated to 5 be between 0.2 and 3.8 mg/m 3 (0.16 and 3.1 ppm) during the period 1974–1979. T he authors 6 found an SMR of 2.01 (five deaths observed) for can cer of the lymphatic and hematopoietic 7 system. The study’s limitations included incomplet e work histories, small numbers of deaths, 8 and a follow-up period that may not have been suffi cient to allow for a latency period for the 9 development of LHP cancers. As before, the results neither support nor refute an association of 10 formaldehyde exposure with LHP cancers. 11 Edling et al. (1987) reported on the incidence of d isease in a cohort of 521 blue collar 12 Swedish workers in plants where abrasives bound wit h formaldehyde resins were manufactured. 13 Formaldehyde levels ranged from 0.1 to 1.0 mg/m 3 (0.08–0.8 ppm). The workers in the cohort 14 were employed between 1955 and 1983, and incidence rates were calculated from 1958 through 15 1981. There were only 24 total cancer cases (28.5 expected) of which 2 (1.0 expected) were 16 lymphomas and 2 (0.5 expected) were multiple myelom as. Expected cases were determined 17 through the Swedish National Cancer Register. No o ther LHP cancers were observed. This 18 study lacked the power to detect any significant as sociations between LHP cancer and exposure 19 to formaldehyde. 20 Dell and Teta (1995) conducted a cohort mortality s tudy of 5,932 male employees of a 21 New Jersey plastics manufacturing, research, and de velopment facility. The workers, who had 22 been employed during the period 1946–1967, were fol lowed-up for an average of 32 years. 23 SMRs were based on U.S. and New Jersey mortality ra tes. Hourly workers (n = 3,853) were 24 analyzed separately from the 2,079 salaried employe es. Although no excess risk was evident for 25 hematopoietic cancer in hourly workers (SMR 0.93; 2 8 observed), there was an SMR of 1.69 26 (95% CI: 1.07–2.53; 23 observed) among salaried wor kers. This association was further 27 narrowed to mainly research and development workers (eight leukemia deaths observed with 28 three expected, for an SMR of 2.67). No common exp osure was found when work history 29 records were examined. The decedents were mostly a ssociated with process development in two 30 research pilot plants, where chemical engineers, la b technicians, and plant operators executed 31 small-scale product development. Although notebook s referred to benzene and toluene solvents, 32 no definite connection was made with formaldehyde o r any of the solvents. No ambient air 33 measurements of formaldehyde were available. The f indings cannot be assumed to be due to 34 formaldehyde exposure because of the presence of ot her potential leukemogens. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-96 Blair et al. (1993) conducted a study that evaluate d the risk of non-Hodgkin’s lymphoma 1 from exposure to formaldehyde. This was a populati on-based, case-control, interview-based 2 study of 1,867 white males of whom 622 cases had th e disease and 1,245 were controls. 3 Subjects had lived in Iowa and Minnesota between 19 80 and 1983. This study was exploratory 4 and designed to find associations with any environm ental exposures and non-Hodgkin’s 5 lymphoma. Subjects or next of kin were interviewed to determine what exposures the cases and 6 controls may have received based on agricultural ex posures, work histories, medical conditions, 7 and family history. Extra effort was made to colle ct information about occupation, industrial 8 exposures, and other selected exposures. The analy sis revealed an OR of 1.2 for exposure to 9 formaldehyde. Similar associations were found for metals and other substances in the study. 10 This study, because it did not select cases and con trols from a population with possible 11 formaldehyde exposure, could not detect specific re lationships between formaldehyde and 12 non-Hodgkin’s lymphoma. 13

14

4.1.2.2.1.3. Summary of non-respiratory tract cancers. The Hauptmann et al. (2003) study 15 appears to provide the strongest evidence of an ass ociation for ML in particular. Statistically 16 significant positive associations were found for LH P malignancies and leukemia, particularly 17 ML, in certain higher exposure categories in compar ison with employees in the lowest exposure 18 categories. In the highest peak exposure level, th e RR for LHP malignancies was 1.87 (95% CI: 19 1.27–2.75; 64 observed) and for ML was 3.46 (95% CI : 1.27–9.43; 14 observed) compared with 20 employees in the low-exposure peak level. For work ers with high-peak exposure levels, the RR 21 for LHP malignancies was 1.71 (95% CI: 1.14–2.58; 4 9 observed) and 2.43 (95% CI: 0.81–7.25) 22 for ML. The trend tests for slope were highly stat istically significant for both LHP malignancies 23 ( p < 0.002) and ML (

p < 0.009). Significant results for LHP cancers were also seen with the 24 average intensity exposure metric. RRs were 1.63 ( p < 0.05) and 1.50 (

p < 0.05) for the medium 25 and high categories, respectively. The risk of ML was also significantly increased (RR = 2.49) 26 in the highest exposure category. In contrast, res ults showed no associations of formaldehyde 27 with LHP cancer, either by cumulative exposure or y ears of duration. 28 Additional support linking LHP cancer and formaldeh yde comes from a study of garment 29 workers (Pinkerton et al., 2004) and studies of pat hologists and other medical workers exposed 30 to formaldehyde (Matanoski, 1991; Blair et al., 199 0; Hayes et al., 1990; Stroup et al., 1986; 31 Walrath and Fraumeni, 1984, 1983; Harrington and Sh annon, 1975). Hayes et al. (1990) and 32 Stroup et al. (1986) also reported significant exce ss risks of ML. 33 Several reports have challenged the association bet ween LHP cancer and formaldehyde 34 (Casanova et al., 2004; Cole and Axten, 2004; Colli ns, 2004; Collins and Lineker, 2004; Coggon 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-97 et al., 2003; Casanova and Heck, 1987; Heck et al., 1985). These papers argue that a biological 1 explanation for the excess risk of LHP cancer or le ukemia remains unclear. Absent a plausible 2 MOA by which formaldehyde could cause these cancers , many investigators have been unable to 3 accept the reported increased risks identified in t he epidemiologic literature. Some researchers 4 have argued against the biological plausibility of formaldehyde-induced lymphoreticular cancers 5 based solely on the assumption that formaldehyde as a reactive gas does not penetrate past the 6 POE. This argument is relevant to diseases for whi ch transformation of stem cells in the bone 7 marrow is essential. However, cancers that arise f rom more mature cells present outside of the 8 bone marrow compartment cannot be dismissed with th is argument. Although often grouped for 9 analysis, the lymphohemoreticular system cancers re present many distinct malignancies that may 10 arise from discrete cell types in different tissues throughout the body. For example, acute 11 lymphocytic leukemia (ALL) is believed to arise fro m the transformation of a lymphoid stem cell 12 in the bone marrow, resulting in a blood-borne leuk emia of immature cells of the lymphoid cell 13 line. However, if transformation occurs in a matur e lymphocyte (e.g., post-germinal center B 14 cell), a chronic lymphocytic leukemia (CLL) results . Although etiologically different, these 15 cases would both be lymphocytic leukemia. When con sidering biological plausibility of an 16 exogenous agent increasing the incidence of ALL, bo ne marrow toxicity would be expected. 17 However, when considering the biological plausibili ty of CLL, bone marrow toxicity would not 18 be essential. Mutation or epigenetic changes attai ned in the mature cell may be passed on to 19 daughter cells during response to antigen and event ually lead to transformation. So the 20 etiologies of these two leukemias need not be simil ar. In contrast, a non-Hodgkin’s lymphoma 21

results from transformation of a mature B or T cell resulting in a solid tumor. The etiology of 22 this cancer is actually similar to CLL. A recent r eclassification of lymphoid malignancies by the 23 WHO designates adult B-cell leukemias and lymphomas as the same disease, with ALL as a 24 separate disease. Therefore, mortality analysis by ICD code and the standard groupings of those 25 codes does not reflect the biology of the cancers. 26 Considering the whole class of LHP cancers, there i s a range of biological plausibility for 27 an agent whose primary action is at the POE. Acute leukemias (ALL and acute myelogenous 28 leukemia [AML]), believed to arise from transformat ion of stem cells in the bone marrow, are 29 less plausible, although trafficking of stem cells to different tissues would be an alternative 30 etiology for exogenous compounds acting at the POE. In contrast CLL, lymphomas, multiple 31 myelomas (from plasma B cells), and unspecified can cers may involve an etiology in peripheral 32 tissues to include cells, cell aggregates, germinal centers, and lymph nodes. An association of 33 these cancers to an exogenous agent acting at the P OE is biologically plausible. 34

35This document is a draft for review purposes only a nd does not constitute Agency policy 4-98 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)PathologistsAll cause mortality SMR0.60LHP cancers SMR2.0p< 0.01 (8)Hodgkin’s disease SMR1.4 (1)Leukemia SMR0.6 (1)TechniciansAll cause mortality SMR0.67LHP cancers SMR0.5(3)Hodgkin’s disease SMR–(0)Harrington andShannon (1975)Cohort mortality study of 2,079pathologists and laboratorytechnicians from the RoyalCollege of Pathologists and thePathological Society of GreatBritain from 1955–1973. Thecomparison population camefrom national mortality data.Presumed exposure toformaldehyde tissuefixative.Leukemia SMR0.5(1)All causesMen SMR0.56Women SMR0.99LeukemiaMen SMR0.91 (90% CI: 0.05–4.29) (1)Women SMR9.26 (90% CI: 0.47–43.9) (1)Other LHP cancersMen SMR0.53 (90% CI: 0.03–2.54) (1)Harrington andOakes (1984)Cohort mortality study of 2,720pathologists from the RoyalCollege of Pathologists and thePathological Society of GreatBritain from 1974–1980. Vitalstatus obtained from the census, anational health registry, and othersources. SMRs developed fromthe English, Scottish, Irish, andWelsh populations.Presumed exposure toformaldehyde tissuefixative.Women SMR– – (0)All cause mortalityMen SMR0.43 (176)Women SMR0.65 (18)Hodgkin’s disease SMR1.21 (95% CI: 0.03–6.71) (1)All cancersSMR1.44 (95% CI: 0.69–2.63) (10)SMR1.52 (95% CI: 0.41–3.89) (4)Hall et al. (1991) Cohort mortality study of 4,512pathologists from the RoyalCollege of Pathologists and thePathological Society of GreatBritain from 1974–1987. Vitalstatus obtained from the census, anational health registry, and othersources. SMRs developed fromthe English and Welshpopulations.Presumed exposure toformaldehyde tissuefixative.LeukemiaLevine et al. (1984) Cohort mortality study of 1,477 Presumed exposure toAll LHP cancers SMR1.24 (8)This document is a draft for review purposes only a nd does not constitute Agency policy 4-99 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)Leukemia SMR1.60 (4)male Ontario undertakers firstlicensed 1928–1957, followedfrom 1950 to 1977. SMRsdeveloped from Ontariomortality rates.formaldehyde tissuefixative.All cause mortality SMR0.65 (95% CI: 0.60–0.70) (738)All LHP cancersSMR1.2 (95% CI: 0.7–2.0) (18)Lymphosarcoma andreticulosarcomaSMR0.7 (95% CI: 0.1–2.5) (2)Hodgkin’s disease SMR– – (0)Leukemia SMR1.5 (95% CI: 0.7–2.7) (10)Stroup et al. (1986) Cohort mortality study of 2,317white male members of theAmerican Association ofAnatomists from 1888 to 1969who died 1925–1979. SMRsdeveloped using U.S. populationmortality rates.Presumed exposure toformaldehyde tissuefixative.Other lymphatic SMR2.0 (95% CI: 0.7–4.4) (6)SMR0.48 (NR)LHP cancer other thanleukemiaLeukemia SMR1.06 (NR)Logue et al. (1986) Cohort mortality study of 4,485pathologists who were membersof the College of AmericanPathologists, 1962–1972,followed for mortality through1977. SMRs developed fromU.S. population mortality rates.Presumed exposure toformaldehyde tissuefixative.All cancer SMR0.78 (95% CI: 0.71–0.85) (508)SMR1.25 (95% CI: 0.95–1.62) (57)All LHP cancersSMR1.31 (95% CI: 0.66–2.35) (11)Lymphosarcoma andreticulosarcomaHodgkin’s disease SMR 0.36 (95% CI: 0.04–1.31) (2)Leukemia SMR1.35 (95% CI: 0.92–1.92) (31)Matanoski (1991) Cohort mortality study of 6,111male pathologists frommembership rolls of theAmerican Medical Association1912–1950. Mortality wasfollowed through 1978. SMRsdeveloped from U.S. populationwhite male mortality rates.Presumed exposure toformaldehyde tissuefixative.Other lymphatic SMR1.54 (95% CI: 0.82–2.63) (13)All LHP cancersExposed SMR0.80 (95% CI: 0.69–0.94) (161)Unexposed SMR0.62 (95% CI: 0.39–1.00) (17)Hauptmann et al.(2003)Retrospective cohort mortalitystudy of 25,619 workersemployed at 10 formaldehydeplants in the U.S. followed fromeither the plant start-up or firstExposure estimatesbased on job titles,tasks, visits to plants bystudy industrialhygienists, andPeak exposure (ppm)employment through 1994. monitoring data through0 RR1.08 (95% CI: 0.60–1.94) (17)This document is a draft for review purposes only a nd does not constitute Agency policy 4-100 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)0.1–1.9 RR1.00Reference value(48)2.0 to <4.0 RR1.71 (95% CI: 1.14–2.58) (49)4.0 or greater RR1.87 (95% CI: 1.27–2.75) (64)Trend p = 0.002Average exposure (ppm)0 RR0.91 (95% CI: 0.52–1.59) (17)0.1–0.4 RR1.00Reference value(81)0.5 to <1.0 RR1.63 (95% CI: 1.11–2.37) (42)1.0 or greater RR1.50 (95% CI: 1.01–2.24) (38)Trend p = 0.050Cumulative exposure(ppm-years)0 RR0.74 (95% CI: 0.42–1.30) (17)0.1–1.4 RR1.00Reference value(94)1.5 to 5.4 RR0.79 (95% CI: 0.52–1.21) (29)5.5 or greater RR1.03 (95% CI: 0.70–1.52) (38)Trend p = 0.157LeukemiaPeak exposure (ppm)0 RR0.78 (95% CI: 0.25–2.43) (4)0.1–1.9 RR1.00Reference value(16)2.0 to <4.0 RR2.04 (95% CI: 1.04–4.01) (20)4.0 or greater RR2.46 (95% CI: 1.31–4.62) (29)Trend p = 0.001Average exposure (ppm)0 ppm RR0.56 (95% CI: 0.19–1.66) (4)0.1–0.4 RR1.00Reference value(32)0.5 to <1.0 RR1.52 (95% CI: 083–2.79) (16)SMRs calculated using sex-, age-, race-, and calendar-yearspecific U.S. mortality rates.RRs estimated using Poissonregression stratified by calendaryear, age, sex, and race; adjustedfor pay category.1980. Peak exposuredefined as short-termexcursions exceedingthe 8-hour TWAformaldehyde intensityand knowledge of jobtasks. Exposures to 11other compounds wereidentified. Workerscontributed preexposure person-time tononexposed category.Poisson regressionmodels used a 2-yearlag to account for tumorlatency.1.0 or greater RR1.68 (95% CI: 0.91–3.08) (17)This document is a draft for review purposes only a nd does not constitute Agency policy 4-101 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)Trend p = 0.193Cumulative exposure (ppm-years)0 RR0.48 (95% CI: 0.16–1.42) (4)0.1–1.4 RR1.00Reference value(35)1.5–5.4 RR0.90 (95% CI: 0.47–1.73) (13)5.5 or greater RR1.14 (95% CI: 0.63–2.07) (17)Trend p = 0.183Hodgkin’s diseasePeak exposure (ppm)0 RR0.51 (95% CI: 0.06–4.52) (1)0.1–1.9 RR1.00Reference value(5)2.0 to <4.0 RR3.45 (95% CI: 0.98–12.2) (7)4.0 or greater RR3.35 (95% CI: 0.97–11.6) (8)Trend p = 0.014Average exposure (ppm)0 RR0.46 (95% CI: 0.05–3.93) (1)0.1–0.4 RR1.00Reference value(7)0.5 to <1.0 RR4.70 (95% CI: 1.61–13.8) (8)1.0 or greater RR3.12 (95% CI: 0.91–10.7) (5)Trend p = 0.022Cumulative (ppm-years)0 RR0.29 (95% CI: 0.04–2.34) (1)0.1–1.4 RR1.00Reference value(12)1.5–5.4 RR1.35 (95% CI: 0.45–3.99) (5)5.5 or greater RR1.17 (95% CI: 0.31–4.46) (3)Trend p = 0.037MLPeak exposure (ppm)0 RR0.67 (95% CI: 0.12–3.61) (2)0.1 to 1.9 RR1.00Reference value(6)2.0 to <4.0 RR2.43 (95% CI: 0.81–7.25) (8)4.0 or greater RR3.46 (95% CI: 1.27–9.43) (14)This document is a draft for review purposes only a nd does not constitute Agency policy 4-102 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)Trend p = 0.003Average exposure (ppm)0 RR0.41 (95% CI: 0.08–1.95) (2)0.1 to 0.4 RR1.00Reference value(14)0.5 to <1.0 RR1.15 (95% CI: 0.41–3.23) (5)1.0 or greater RR2.49 (95% CI: 1.03–6.03) (9)p = 0.086Cumulative (ppm-years)0 RR0.32 (95% CI: 0.07–1.51) (2)0.1-1.4 RR1.00Reference value(17)1.5-5.4 RR0.57 (95% CI: 0.19–1.73) (4)5.5 or greater RR1.02 (95% CI: 0.40–2.55) (7)Trend p = 0.123All LHP cancers SMR0.97 (95% CI: 0.74–1.26) (59)SMR0.85 (95% CI: 0.28–1.99) (5)Lymphosarcoma andreticulosarcomaHodgkin's disease SMR0.55 (95% CI: 0.07–1.98) (2)Other lymphatic SMR0.97 (95% CI: 0.64–1.40) (28)Leukemia SMR1.09 (95% CI: 0.70–1.62) (24)Mortality since 1960Lymphocytic leukemia SMR0.60 (95% CI: 0.12–1.75) (3)ML SMR1.44 (95% CI: 0.80–2.37) (15)10+ years ofexposureSMR2.19 NS (8)SMR1.91p> 0.05 (13)20+ years since 1stexposureMultiple cause leukemiaSMR1.92 (95% CI: 1.08–3.17) (15)Pinkerton et al.(2004)Cohort mortality study of 11,098workers in 3 garment plantsexposed≥3 months afterformaldehyde was introduced.Women comprised 81.7% of thecohort. Vital status was followedthrough 1998. SMRs werecalculated by using sex-, age-,race-, and calendar-year-specificU.S. mortality rates. Multiplecause SMRs were derived fromall contributing causes fromdeath certificates.Data for 549 randomlyselected employees in 5departments in 1981 and1984 used to estimateoverall exposure levels.Levels presumed to be0.09–0.20 ppm.10+ years ofexposure and 20+years since 1stexposureThis document is a draft for review purposes only a nd does not constitute Agency policy 4-103 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)Multiple cause MLSMR2.02 (95% CI: 1.13–3.34) (15)20+ years since 1stexposure10+ years ofexposure and 20+years since 1stexposureSMR2.55 (95% CI: 1.10–5.03) (8)Non-Hodgkin's lymphomaOverall SMR0.98 (95% CI: 0.67–1.39) (31)High exposureSMR0.89 (95% CI: 0.41–1.70) (9)LeukemiaOverall SMR0.91 (95% CI: 0.62–1.29) (31)High exposure SMR0.71 (95% CI: 0.31–1.39) (8)Multiple myelomaOverall SMR0.86 (95% CI: 0.48–1.41) (15)Coggon et al. (2003) Cohort mortality study of 14,014men employed in 6 factories ofthe chemical industry in GreatBritain from periods duringwhich formaldehyde wasproduced. Cohort mortalityfollowed from 1941 through2000. SMRs based on Englishand Welsh age- and calendaryear-specific mortality rates.Exposure assessmentbased on data abstractedfrom company records.Jobs categorized asbackground, low,moderate, high, orunknown levels.High exposure SMR1.18 (95% CI: 0.48–2.44) (7)SMR0.59 (95% CI: 0.23–1.21) (7)All LHP cancersAndjelkovich et al.(1995)Cohort mortality study of 3,929automotive industry iron foundryworkers exposed from 1960–1987 and followed through 1989.SMRs calculated using sex-, age-, race-, and calendar-yearspecific U.S. mortality rates.Exposure assessmentbased on review ofwork histories by anindustrial hygienist.Leukemia SMR0.43 (95% CI: 0.05–1.57) (2)Bertazzi et al. (1986) Cohort mortality study of 1,330male workers in an Italian resinplant. Subjects were employedany time between 1959 and 1980for at least 30 days. Vital statusfollowed through 1986. SMRscalculated using sex-, age-, race-,and calendar-year-specificnational and local mortality rates.Exposure assessmentbased on reconstructionof work history.All LHP cancers SMR2.01 (5)This document is a draft for review purposes only a nd does not constitute Agency policy 4-104 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)Lymphoma SMR2.0 (95% CI: 0.2–7.2) (2)Edling et al. (1987) Cohort mortality and incidencestudy of 521 Swedish workers inan abrasive production plant withat least 5 years of employmentbetween 1955 and 1983,followed through 1983.Exposure level of1–5 μg/m3.Multiple myeloma SMR4.0 (95% CI: 0.5–14) (2)All LHP cancersHourly workers SMR0.93 (28)Salaried workers SMR1.69 (23)LeukemiaHourly workersSMR0.98 (12)Dell and Teta (1995) Cohort mortality study of 5,932male employees of a New Jerseyplastics manufacturing, researchand development facility.Examination of workhistories to identify jobswhere formaldehydewas involved.Salaried workersSMR1.98 (11)All LHP cancersPMR1.15 (21)Lymphosarcoma and reticulosarcomaPMR1.08 (4)Hodgkin’s diseasePMR1.0 (2)Other lymphatic lymphomaPMR1.18 (5)LeukemiaWalrath andFraumeni (1983)Proportionate mortality cohortstudy of 1,132 white maleembalmers licensed to practicebetween 1902 and 1980 in NewYork who died between 1925and 1980 identified fromregistration files. Deaths werecompared with age-, race-, andcalendar-year-expected numbersof deaths from the U.S.population.No directmeasurements.Presumed exposure toformaldehyde tissuefixative.PMR1.32 (10)All LHP cancersPMR1.22 (19)Lymphosarcoma and reticulosarcomaPMR0.97 (3)Hodgkin’s diseasePMR– (0)Other lymphatic lymphomaWalrath andFraumeni (1984)Proportionate mortality cohortstudy of 1,007 white maleembalmers from the CaliforniaBureau of Funeral Directing andEmbalming who died between1925 and 1980. Deaths werecompared with age- andcalendar-year-expected numbersNo directmeasurements.Presumed exposure toformaldehyde tissuefixative.PMR1.33 (4)This document is a draft for review purposes only a nd does not constitute Agency policy 4-105 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)LeukemiaPMR1.75p< 0.05 (12)Licensed <20 years PMR1.24 (4)of deaths from the U.S.population.Licensed≥20 yearsPMR2.21p< 0.05 (8)All LHP cancersPMR1.39 (95% CI: 1.15–1.63) (115)RaceWhite PMR1.31 (95% CI: 1.06–1.59) (100)Nonwhite PMR2.41 (95% CI: 1.35–3.97) (15)Occupation on death certificateEmbalmer PMR1.23 (95% CI: 0.78–1.85) (23)Funeral director PMR1.56 (95% CI: 1.23–1.94) (78)Other PMR1.30 (95% CI: 0.67–2.28) (12)Age at death<60 PMR1.35 (95% CI: 0.88–1.98) (26)60–74 PMR1.72 (95% CI: 1.33–2.19) (66)≥75PMR1.16 (95% CI: 0.74–1.74) (23)Hodgkin’s diseasePMR0.72 (95% CI: 0.15–2.10) (3)Non-Hodgkin’s lymphomaPMR1.26 (95% CI: 0.87–1.76) (34)Lymphosarcoma and reticulosarcomaPMR1.12 (95% CI: 0.58–1.96) (12)Multiple myelomaPMR1.37 (95% CI: 0.84–2.12) (20)Other lymphatic lymphomaPMR1.35 (95% CI: 0.85–2.01) (22)Lymphatic leukemiaPMR0.74 (95% CI: 0.29–1.53) (7)MLPMR1.57 (95% CI: 1.01–2.34) (24)Hayes et al. (1990)Proportionate mortality cohortstudy of 3,649 deceased whiteand 397 deceased nonwhite U.S.male embalmers and funeraldirectors, derived from licensingboards and funeral directorassociations in the 32 states andthe District of Columbia.Occupation was confirmed ondeath certificate. Deaths werecompared with age- andcalendar-year-expected numbersof deaths from the U.S.population.No directmeasurements.Presumed exposure toformaldehyde tissuefixative.Other leukemiaThis document is a draft for review purposes only a nd does not constitute Agency policy 4-106 DRAFT—DO NOT CITE OR QUOTETable 4-4. Epidemiologic studies of formaldehyde and lymphohematopoietic cancersReference Study design Exposure assessmentResults, statistical significance (number of observed deaths for cohortstudy)PMR2.28 (95% CI: 1.39–3.52) (20)Non-Hodgkin’slymphoma(formaldehydeexposure)ORa1.2 (95% CI: 0.9–1.7)Blair et al. (1993) Population-based case-controlstudy of 622 white men withLHP cancers. Cancers selectedfrom Iowa and Minnesota cancersurveillance networks diagnosedbetween 10/80 and 9/82. 1,245matched controls for living casesselected by random digit dialingif younger than age 65 and fromMedicare records if 65 or older.Study focused on agriculturalexposures.Personal interviews ofsubjects or next of kinincluded job histories,agricultural exposures,and chemical exposures.Job titles used to createjob exposure matrix.Industrial hygienistestimated probabilityand intensity ofexposures to largenumbers of substances.Funeral service worker ORa2.1 (95% CI: 0.5–7.9) (6)aAdjusted for age, state, smoking, family history ofmalignant proliferative disease, agricultural exposure to pesticides, use of dye, and direct/surrogate responseto interview.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-107 4.1.2.2.2. Brain and CNS cancer

. Several studies of professional groups discussed ea rlier 1 investigated brain and other CNS cancers among thos e exposed to formaldehyde on the job. 2 Several of these studies found that exposure increa sed risk two to three times among exposed 3 professionals (Hall et al., 1991; Stroup et al., 19 86; Walrath and Fraumeni, 1984), while others 4 found modest or no increase in risk (Hayes et al., 1990; Levine et al., 1984; Walrath and 5 Fraumeni, 1983). 6 None of the industrial cohort worker mortality stud ies of exposure to formaldehyde found 7 a clear relationship between formaldehyde exposure and risk of brain or CNS cancer (Pinkerton 8 et al., 2004; Coggon et al., 2003; Andjelkovich et al., 1995; Gardner et al., 1993; Stayner et al., 9 1988; Blair et al., 1987, 1986). To date, no case- control studies of brain and CNS cancer have 10 been completed. In the Hauptmann et al. (2004) stu dy, the authors reported that no clear 11

association was seen for cancer of the brain and CN S and exposure to formaldehyde. 12

13

4.1.2.2.3. Pancreatic and other cancers. Two studies (Kernan et al., 1999; Dell and Teta, 19 95) 14 have found increases in the risk of pancreatic canc er in association with possible exposure to 15 formaldehyde. Collins et al. (2001a) conducted a m eta-analysis of fourteen studies (Kernan et 16 al., 1999; Andjelkovich et al., 1995; Hansen and Ol sen, 1995; Gardner et al., 1993; Hall et al., 17 1991; Matanoski, 1991; Hayes et al., 1990; Gerin et al., 1989; Stayner et al., 1988; Blair et al., 18 1986; Stroup et al., 1986; Levine et al., 1984; Wal rath and Fraumeni, 1984, 1983) and found a 19 small increase in risk (RR = 1.1 [95% CI: 1.0–1.3])

. 20

Other sites that have been examined are stomach can cer (Coggon et al., 2003) (SMR = 21 1.47; p < 0.05), intraoccular melanoma (Holly et al., 1996 ) (OR = 2.9 [95% CI: 1.2–7.0]), and 22 thyroid cancer among women (Wong et al., 2006) (OR = 8.33 [95% CI: 1.16–60.0]; 2 cases). 23 However, without further substantiation, it is diff icult to infer causation based on these isolated 24

results alone. 25

26

4.1.2.3. Summary: Carcinogenic Hazard in Humans 27 The weight of the epidemiologic evidence at this ti me supports a link between 28 formaldehyde exposure and NPC in humans. This conc lusion is based on the longitudinal cohort 29 study of Hauptmann et al. (2004) as well as the cas e-control studies of NPC and formaldehyde 30 exposure completed by Hildesheim et al. (2001), Vau ghan et al. (2000), and several additional 31

case-control studies described in the text. With t he exception of Hauptmann et al. (2004), most 32

of the other cohort studies found little or no incr eased risk of NPC from exposure to 33 formaldehyde. However, Hauptmann et al. (2004) emp loyed different exposure metrics and 34 based their analyses on conservative internal compa risons that limited the potential for the HWE 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-108 to obscure true effects. The case-control studies that provide additional evidence of an 1 association between NPC and formaldehyde have more power and generally rely on better 2 diagnoses of NPC. Better ascertainment of histolog ic types of tumors can sometimes also be 3 obtained if the cases are taken from cancer registr ies. The NPC risk is also supported by 4 experimental evidence in animals in which formaldeh yde induces nasal cancers (Section 4.2.2). 5 Since the physiology of the rat nasal passage is so mewhat different from that of humans, it is not 6 possible to obtain a direct site-specific correspon dence between the species. However, in both 7 species, the tumors are found within the same area of the URT where maximum exposure can be 8 expected to occur. 9 Several researchers have challenged the conclusion of a relationship between 10 formaldehyde and NPC. Those critical of the link a rgue that, given the wide variability in results 11 across studies and competing explanations, conclusi ons about any link from the existing studies 12 are premature. The difficulty in attaining consens us on whether formaldehyde influences the risk 13

of NPC in humans arises from several limitations in herent in epidemiologic methods and 14 exposure assessment, as well as from the characteri stics of the disease. The most prominent of 15 these limitations are the rarity of the cancer and imprecise estimates of exposure. Because NPC 16 is a very rare cancer with an incidence of less tha n 1 per 100,000, it is difficult to obtain precise 17 estimates of risk from cohort studies. Although ca se-control studies are better suited for 18 studying rare conditions, they are limited in obtai ning valid and precise exposure assessments. A 19 further problem with exposure assessment is isolati ng formaldehyde exposure from other 20 potential chemical or particulate exposures that ma y influence risk of NPC. Imprecise exposure 21 assessment and the inability to isolate formaldehyd e exposure from other exposures are largely 22

the bases on which Marsh and coworkers have challen ged the NCI cohort study (Marsh et al., 23 2007a, b, 2002, 1996; Marsh and Youk, 2005). Marsh and coworkers (Marsh et al., 2007a) show 24 that subjectively assessed exposure to silversmithi ng is tentatively associated with NPC. Given 25 that there were no prior citations of an associatio n between silversmithing exposures and NPC in 26 the medical literature and given the many post hoc reexaminations of alternative hypotheses to 27 explain the original NCI findings, it is more likel y that silversmithing is an artifactual potential 28

confounder. 29 It may be expected that, without new approaches for obtaining more accurate and precise 30 estimates of exposure, further follow-up of current cohorts and future epidemiologic studies of 31 formaldehyde and NPC will face the same limitations and criticisms found with existing studies. 32 These limitations notwithstanding, the epidemiologi c studies reviewed here represent what may 33 be currently discernable about a formaldehyde-NPC l ink in humans by using rigorous 34 observational methods. As such, concluding any inf luence of formaldehyde must be made on the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-109 weight of all human and animal evidence in the face of known and expected limitations in study 1 design and exposure assessment. 2 The results of two well-designed cohort studies fou nd a positive association between 3 formaldehyde-exposed professionals, such as patholo gists, embalmers, and funeral directors, and 4 LHP cancer, particularly ML. The largest cohort st udy of formaldehyde has the most extensive 5 exposure assessment (Blair et al., 1986; Stewart et al., 1986), and the cohort was followed for a 6 median duration of 35 years (Hauptmann et al., 2003 ). By using cumulative exposure measures 7 not previously used and by using internal compariso n groups, significant increases in the risk of 8 cancer of the LHP system, particularly ML, were rep orted. This study demonstrated that 9 formaldehyde was a risk factor for LHP cancers, ind ependent of other risk factors, such as 10 benzene and smoking. Hauptmann et al. (2003) found statistically significant dose trends for 11 peak exposure and AIE. Pinkerton et al. (2004) als o found a significant increase in the risk of 12 ML in garment workers 20 years after their initial exposure and in workers with 10 or more years 13 of exposure. Additionally, several studies of path ologists, embalmers, and other medical 14 workers reported greater numbers of observed deaths from leukemia than expected although 15 many studies of these groups suffer from a substant ial HWE based on comparisons with external 16 death rates. Two of these studies, Hayes et al. (1 990) and Stroup et al. (1986), also report a 17 significantly excess risk of ML in embalmers, funer al directors, and anatomists. 18 There is a range of biological plausibility for an agent whose primary action is at the 19 POE. Acute leukemias (ALL and AML), believed to ar ise from transformation of stem cells in 20 the bone marrow, are less plausible. In contrast c hronic lymphatic leukemia, lymphomas, 21 multiple myelomas (from plasma B cells), and unspec ified cancers may involve an etiology in 22 peripheral tissues to include cells, cell aggregate s, germinal centers, and lymph nodes. An 23 association of these cancers to an exogenous agent acting at the POE is biologically plausible. 24

It is the conclusion of this assessment that the we ight of the epidemiologic evidence at 25 this time supports a link between formaldehyde expo sure and carcinogenicity in humans. 26

27

4.2. ANIMAL STUDIES 28 This section discusses the available laboratory ani mal data on the toxicity of inhalation, 29 oral, and dermal exposures to formaldehyde. A comp rehensive database of laboratory animal 30 studies is available for formaldehyde, including nu merous 2-year bioassays by both the 31 inhalation and oral exposure routes and dermal appl ication studies. Although a large portion of 32 the literature reports studies focused on toxic eff ects at the site of contact or POE, general 33 systemic effects as well as neurobehavioral effects , reproductive and developmental effects, 34 immunologic changes, and sensitization are well rep resented in the literature as well. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-110 RB is a reduction in ventilation rate, minute volum e, and other physiological parameters 1 experienced by rodents exposed to an irritant/react ive gas. Although humans and nonhuman 2 primates do not exhibit the same change in respirat ory rate, these studies are included in Section 3

4.2.1.1 in order to better understand the effects o n RB in interpreting rodent studies presented in 4 the balance of the chapter. Additionally, although binding to the trigeminal nerve and 5 subsequent downstream events do not result in pulmo nary changes in humans, the mechanism 6 itself plays a role in understanding other adverse health effects observed in humans. 7 The available data for the three exposure routes co nfirm direct formaldehyde-induced 8 toxicity in tissues present at the POE. These obse rvations are consistent with the 9 physicochemical characteristics, reactivity, and me tabolic pathways of formaldehyde as 10 discussed in Chapter 3. Indications of cell damage , cell proliferation, and inflammatory 11 responses are similar for each route of exposure, t herefore effects at the POE for inhalation and 12 oral exposures are described first (Sections 4.2.1. 2 and 4.2.1.3, respectively). Given the well13

established nature of these health effects and the wealth of literature for inhalation exposures, 14 complete study summaries for respiratory tract effe cts are provided. Studies are organized by 15 study duration—acute, subchronic and chronic—where some of the chronic bioassays were 16 designed to address carcinogenic potential. Sectio n 4.2.2 pulls together the findings of chronic 17 studies across the routes of exposure to evaluate t he carcinogenic potential of formaldehyde 18

exposure. 19 Although a majority of the oral and inhalation stud ies focus on health effects at the 20 POE—respiratory tract and GI tract—the general syst emic toxicity of formaldehyde is addressed 21 where it was integral to the study. Therefore, bod y weight and organ weight changes, gross 22 pathology, organ histopathology outside of the POE, blood and urine chemistry, and other 23 biochemical measures may be included in these study summaries. An overview of general 24 systemic findings is provided in Section 4.4 for al l routes of exposure. 25 Studies addressing immune function, neurobehavioral effects, sensitization, and 26 reproductive and developmental effects are addresse d across routes of exposure. The specialized 27 nature of these studies requires discrete treatment , and inclusion of data across routes of 28 exposure allows for a synthesis of the available in formation. 29

30

4.2.1. Noncancer Health Effects 31

4.2.1.1. Reflex Bradypnea 32 Reflex bradypnea (RB), which is believed to be a pr otective response, is often observed 33 in rodents exposed to reactive gases. It is primar ily characterized by marked decreases in 34 activity, respiratory rate, body temperature, and m etabolic rate. RB is not seen in humans and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-111 nonhuman primates. An understanding of the RB is i mportant to the interpretation of many of 1 the animal bioassays examining formaldehyde-induced health effects. Of chief concern is that 2 the physiological effects of RB, described below, m ay interfere with appropriate interpretation of 3 adverse effects noted with formaldehyde exposure. It is important to distinguish between an 4 effect directly related to RB versus formaldehyde e xposure. Additionally the effects of RB may 5 mask or alter formaldehyde-induced health effects. Secondly, differential respiratory effects of 6 RB due to species and strain will result in differe ntial inhaled doses at the same exposure level. 7 This needs to be considered both when comparing the results of animal studies and in 8 extrapolation to humans. Finally, although humans do not experience RB, the mechanism of RB 9 as a reflex response to trigeminal nerve stimulatio n assists in understanding human health related 10 to localized and reflex responses due to trigeminal nerve stimulation. 11 Irritant gases have been shown to decrease body tem perature, heart rate, and blood 12 pressure as well as alter blood chemistry in rodent s (Pauluhn, 2003, 1996; Jaeger and Gearhart, 13 1982). Because of their small size, mice can rapid ly lower their body temperatures and thus their 14 metabolic rate and ventilation rate. The hypotherm ia that results from RB can directly affect 15 nearly all biological processes (Gordon et al., 200 8). Formaldehyde exposure can dramatically 16 lower ventilation rate and reduce body temperature in mice by as much as 4°C, and it has been 17 posited that decreased oxygen supply is likely to h ave profound effects on organisms with 18 substantial oxygen demands (Jaeger and Gearhart, 19 82). The effects of RB are reversible, 19 though it may take several minutes to several hours to return to pre-exposure conditions 20 (Pauluhn, 1996; Jaeger and Gearhart, 1982). 21 The literature on sensory irritation is broad; many studies have investigated species 22 differences, dose response relationships, tolerance , and cross-tolerance to other sensory irritants 23 (see Tables 4-5 and 4-6). This discussion focuses on the changes in respiratory rate and minute 24 volume during formaldehyde exposure. Sensory irrit ation is often quantified as the statistically 25 derived exposure concentration that results in a 50 % reduction in respiratory rate (RD 50 ) in 26 rodents (ASTM, 2000; Kane et al., 1979). Kane and Alarie (1977) evaluated various aspects of 27 sensory irritation, including establishing the RD 50 , exploring the reproducibility of response, 28 investigating the effect of tracheal cannulation, a nd determining the potential for tolerance with 29 repeated exposure or pre-exposure in male Swiss-Web ster mice, caused by formaldehyde and 30

acrolein. The RD 50 was established by exposing four mice for 10 minut es at each concentration 31 across a range representing approximately 10 to 80% reduction in respiration and calculated by 32 using least squares regression. The RD 50 and its 95% CI for formaldehyde were calculated to be 33 3.1 (2.1–4.7) ppm (3.8 [2.58–5.77] mg/m 3 ). The tracheal cannulation experiments demonstrat ed 34 that the effect on respiratory rate was caused by U RT sensory irritation. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-112 Table 4-5. Respiratory effects of formaldehyde-ind uced reflex bradypnea in 1 various strains of mice 2 3

Species/strain No./ group Treatment a Respiratory effects Reference

Male SwissWebster mice 4 Duration: 10 minutes. Exposure: up to 100 ppm. RD 50 a = 3.1 ppm (95% CI: 2.1–4.7). Kane and Alarie (1977)

Male SwissWebster mice 8 Duration: 3 hours/day for 3 days. Exposure: 0.52, 0.44, 1.16, 1.83, 3.10, 5.35, 5.60, and 11.2 ppm. RD 50 = 3.4 ppm (95% CI: 2.4–4.7). Kane and Alarie (1977)

Male SwissWebster mice 4 Duration: 10 minutes (head only). Exposure: up to 10 ppm. RD 50 = 3.2 ppm (95% CI: 2.1–4.7). Steinhagen and Barrow (1984)

Male Swiss OF 1 mice 6 Single 5-minute exposure to four unspecified concentrations. RD 50 = 5.3 ppm. De Ceaurriz et al. (1981)

Male B6C3F1 mice 4 Duration: 10 minutes (head only). Exposure: Range up to 10 ppm. RD 50 = 4.9 ppm (95% CI: 3.9–6.4). Steinhagen and Barrow (1984)

Male B6C3F1 mice 4 Duration: 10 minutes (head only). Exposure: up to 15 ppm Pretreatment: 2, 6, or 15 ppm 6 hours/day for 4 days. Naïve mice: RD 50 = 4.4 ppm (95% CI: 0.9–5.0)

Pretreated mice: RD 50 = 4.3 ppm (95% CI: 3.4–5.5). Chang et al. (1981); Barrow et al. (1983)

Male C57BL6/F1 mice 3 Whole-body exposure for up to 2 hours. After 1.25 hours: Tidal volume reduced by 33%; 68% reduction in respiratory frequency; CO 2 production reduced by 50%; %; body temperature dropped from 37.8 to 34.7°C. Jaeger and Gearhart (1982)

4

a Exposure concentration that results in a 50% reduct ion in respiratory rate. 5This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-113 Table 4-6. Respiratory effects of formaldehyde-ind uced reflex bradypnea in various 1 strains of rats 2 3

Species/strain No./ group Treatment Respiratory effects Reference

Male Crl-CD rats 4 RD 50 = 13.8 ppm. Male Wistar rats 4 30-minute nose-only exposure to a range of formaldehyde concentrations. RD 50 = 10.0 ppm. Cassee et al. (1996)

Male F344 rats 4 Duration: 10 minutes (head only). Exposure: up to 56 ppm. Pretreatment:2, 6, or 15 ppm 6 hours/day for 4 days Naïve rats: RD 50 = 13.1 ppm (95% CI: 10.6–17.5)

Pretreated rats: RD 50 = 10.8 ppm (95% CI: 7.6–16.9) Chang et al. (1981); Barrow et al. (1983)

Male F344 rats 4 Single 10-minute head-only exposure to a range of concentrations.

Pretreatment: 15 or 28 ppm formaldehyde or 10 ppm chlorine. Baseline RD 50 = 31.7 ppm.Pre-exposure to formaldehydeinduced tolerance at 28 ppm (RD 50 = 20.2 ppm) but not 15 ppm. Pre-exposure to chlorine-induced tolerance to formaldehyde (RD 50 ranged from 64.5 to 115 ppm, depending on exposure duration). Chang and Barrow (1984)

Male F344 rats ND 10 minute exposure to acrolein or acetaldehyde (head only).

Pre-exposed to formaldehyde at 15 ppm for 6 hours/day for 9 days. Pre-exposure to formaldehydeinduced tolerance: Acetaldehyde (RD 50 = 2,991 ppm in naive versus 10,601 ppm in preconditioned animals) Acrolein (RD 50 = 6 ppm in naïve versus 29.6 ppm in preconditioned animals). Babiuk et al. (1985)

Male Charles Rivers CD rats 3 Whole-body exposure for up to 2 hours. After 0.7 hours: Tidal volume reduced by 22%; 20% reduction in respiratory frequency; CO 2 production unaffected. Jaeger and Gearhart (1982)

4 5 Across the literature there is fairly good agreemen t on RD 50 values for various strains of 6 mice (Table 4-5), ranging from 3.1 ppm in male Swis s-Webster mice to 4.9 ppm in male 7 B6C3F1 mice. Rats are less sensitive, with RD 50 values ranging from 10 ppm in male Wistar 8 rats to 31.7 ppm in male F344 rats. No reported RD 50 for female rodents exposed to 9 formaldehyde exists. 10 Jaeger and Gearhart (1982) evaluated the effect of formaldehyde on respiratory rate, tidal 11 volume, minute volume, carbon dioxide (CO 2 ) production (exhaled to air) as a reflection of to tal 12 metabolism, and core body temperature in male Charl es River CD rats and male C57BL6/F1 13This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-114 mice. Animals (three/concentration) were exposed t o 15 ppm (18.4 mg/m 3 ) formaldehyde for up 1 to 2 hours. Mice exhibited a greater decrease in r espiratory frequency and minute volume 2 compared with the rats. CO 2 production and body temperature were also affected to a greater 3 extent in the mice (Table 4-5). The authors postul ated that the decreased body temperature in 4 mice would likely lead to decreased biologic action of formaldehyde in the tissue. 5

6 4.2.1.1.1. Tolerance. Tolerance is defined as an increase in the concentr ation required to elicit 7 the same degree of RB response and was evaluated by Kane and Alarie (1977). In the first set of 8 experiments, mice (four/concentration) were exposed 3 hours/day for 4 days at the concentration 9 associated with either a 30 or 50% decrease in resp iratory frequency (specific concentrations not 10 given) (Kane and Alarie, 1977). Naïve animals serv ed as controls for each day. The maximum 11 response increased with each additional day of expo sure, and the diminution of response that was 12 typically exhibited after 60 minutes of exposure in naïve animals was markedly delayed. In the 13 second set of experiments, mice were exposed to a f ormaldehyde concentration at one-tenth the 14

RD 50 (i.e., 0.3 ppm) 3 hours/day for 3 days. On the fo urth day the animals underwent a similar 15 exposure protocol to identify the concentration tha t resulted in an RD 50 , following the above 16 protocol. No change in the RD 50 was demonstrated. Both of these experiments indic ate no 17 change in tolerance with either type of pretreatmen t in Swiss-Webster mice. 18 Chang and Barrow (1984) tested whether tolerance wo uld develop in male F344 19 (CDF[F344]Crl/Br) rats exposed to formaldehyde. Ex posure to formaldehyde at 15 ppm 20 (18.4 mg/m 3 ) for 6 hours/day, 5 days/week failed to induce tol erance. However, tolerance was 21 observed following exposure to 28 ppm (34.4 mg/m 3 ) formaldehyde for 4 days. The 22 concentration-response curve in these animals was s ignificantly different than that of naïve 23 animals, with an increase in the RD 50 estimate for this exposure duration from 31.7 to 7 0.2 ppm. 24

25

4.2.1.1.2. Cross-species differences in inhaled dose. Formaldehyde-induced RB lowers both 26 respiratory rate and tidal volume and thus reduces the inhaled dose of formaldehyde at a given 27 exposure concentration. Chang et al. (1983) and Ba rrow et al. (1983) evaluated the species 28

differences and the effective inhaled dose between rats and mice, since mice seem to be more 29 sensitive to formaldehyde-induced RB and do not exh ibit tolerance as shown in F344 rats. 30 Groups (four/concentration) of male F344 rats and m ale B6C3F1 mice were exposed to 31 formaldehyde concentration ranges of 6.2–48 ppm (7. 6–59 mg/m 3 ) or 0.78–14.0 ppm (0.96– 32 17.2 mg/m 3 ), respectively, for 10 minutes. Pretreated animal s used in the tolerance experiments 33 were exposed to formaldehyde at 2, 6, or 15 ppm (2. 45, 7.36, or 18.4 mg/m 3 ) 6 hours/day for 34 4 days prior to determination of the RD 50 and concentration response across the same ranges. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-115 A concentration-dependent decrease in respiratory r ate was seen in both naïve and 1 pretreated rats during formaldehyde exposure. Tole rance (defined as a decrease in respiratory 2 rate followed by a subsequent return to control val ues) occurred after 4 minutes of exposure and 3 was more pronounced at concentrations above 4 ppm. Concentration-response relationships 4 were very similar for naïve and pretreated rats, an d the RD 50 s were similar for both groups 5 (naïve = 13.1 ppm [95% CI: 10.6–17.5]; pretreated = 10.8 ppm [95% CI: 7.6–16.9 ]). In 6 contrast, naïve or pretreated mice did not develop tolerance during exposures. An examination 7 of concentration-response relationships for mice sh owed similar RD 50 values (naïve = 4.4 ppm 8 [95% CI: 0.9–5.0 ] and pretreated = 4.3 ppm [95% CI : 3.4–5.5]) compared with rats, although 9 the slopes of the concentration-response regression s were statistically different (Figure 4-2). 10

11

12 13 Figure 4-2. Formaldehyde effects on minute volume in naïve and formaldehyde14 pretreated male B6C3F1 mice and F344 rats. 15 16 Source: Redrawn from Chang et al. (1983). 17 18 19 Exposure of naïve or pretreated rats resulted in an increased (compensatory) tidal 20 volume. However, the increase in tidal volume did not compensate entirely for the decrease in 21 ventilation rate and was only concentration depende nt in pretreated rats. Comparison of tidal 22 volume from naïve and pretreated mice exposed to fo rmaldehyde showed a slight increase in 23 naïve animals but a decrease in pretreated ones. T he effect of formaldehyde exposure on tidal 24 1 1 0

100

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0 0 2 4 6 8 10 120 240 3 6 0 0 2 4 6 8 1 0 1 2 0 2 4 0 360 B 6 C 3 F 1 M i c e F 3 4 4 R a t s

N a ï v e (6 p p m) P r e t r eat e d ( 6 p p m) N a ï v e (15 p p m) P r e t r eat e d ( 1 5 p p m)

Ti me (mi n .) d u r i n g e x p o su r eThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-116 volume was concentration dependent in both groups o f mice. These results indicate that tidal 1 volume does not compensate entirely for the decreas e in respiratory rate and that the 2 compensation is slightly greater in rats than in mi ce. 3 These studies (Barrow et al., 1983; Chang et al., 1 983) showed that B6C3F1 mice sustain 4 RB, whereas F344 rats develop tolerance more readil y both during exposure and with 5 pretreatment. Thus, these results suggest that the rat may be the more sensitive species for the 6 effects of inhaled formaldehyde due in part to the difference in sensitivity between mice and rats 7 as evidenced by an RD 50 of 4.9 versus 31.7 ppm and the ability of rats to develop tolerance while 8 mice appear to sustain RB. Barrow et al. (1983) us ed the results of these experiments to estimate 9 an inhaled dose equivalent to the exposure concentr ation of 15 ppm for the strains of mice and 10 rats used in the chronic formaldehyde bioassays by Kerns et al. (1983) and Monticello and 11 Morgan (1994) described in Section 4.1.2 as follows : 12

13 Inhaled dose ( μ g/min-cm 2 ) = 14 HCHO concentration (μg/L) × minute volume (L/min) 15 Nasal cavity surface area (cm 2 ) (5-1) 16

17 As shown in Table 4-7, because mice were observed t o be able to decrease their minute 18 volume by approximately 75% as compared with 45% in rats, a twofold higher inhaled dose 19 would be expected in rats versus mice. This differ ence may be relevant to the increased 20 incidence of SCC in the nasal cavity seen in F344 r ats when compared with B6C3F1 mice. 21

22 Table 4-7. Inhaled dose of formaldehyde to nasal m ucosa of F344 rats and 23 B6C3F1 mice exposed to 15 ppm 24 25

Parameter F344 rats B6C3F1 mice

HCHO concentration (μg/L) 18.4 18.4 Minute volume (L/min) 0.114 0.012 URT surface area (cm 2 ) 13.44 2.89 Inhaled dose (μg/min/cm 2 ) 0.156 0.076 26 Source: Barrow et al. (1983). 27 28 29 4.2.1.1.3. Cross-tolerance. Cross-tolerance of chemically-induced reflex respon ses has been 30 examined in several systems in order to better unde rstand the specificity and nature of the 31 interaction of reactive chemicals (such as formalde hyde with chlorine) with the trigeminal nerve 32 involved in the RB. Development of cross-tolerance to formaldehyde following preexposure to 33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-117 chlorine or to chlorine following preexposure to fo rmaldehyde was shown to be a function of the 1 duration of the pretreatment in male F344 rats (Cha ng and Barrow, 1984) (Table 4-8). A 7-day 2 recovery period resulted in only a slight loss of c ross-tolerance from a 4-day pre-exposure to 3 either chlorine or formaldehyde (data not shown). The cross-tolerance between formaldehyde 4 and chlorine demonstrated in the Chang and Barrow ( 1984) study suggests that these chemicals 5 may act via a common mechanism and may involve the trigeminal nerve. In rats, cross-tolerance 6 was induced after chlorine exposure but not after f ormaldehyde exposure, which suggests that 7 the trigeminal nerve may have different reactive si tes that are differentially activated, depending 8

on the stimulus. 9

10

Table 4-8. Exposure regimen for cross-tolerance st udy 11 12

Chlorine RD 50 Pre-exposure FA-pretreated Naïve

1 day 22.6 ppm 4 days 16.8 ppm Formaldehyde 15 ppm

6 hours/day 10 days 64.5 ppm 10.9 ppm

Formaldehyde RD 50 Pre-exposure Cl-pretreated Naive

1 day 64.5 ppm 4 days 66 ppm Chlorine 10 ppm

6 hours/day 10 days 115 ppm 31.7

13 Source: Chang and Barrow (1984). 14 15 16 Babiuk et al. (1985) evaluated the potential for fo rmaldehyde pretreatment to cause cross17 tolerance with various other inhaled aldehydes, inc luding acetaldehyde and acrolein. Male F344 18 rats were pretreated with 15 ppm (18.4 mg/m 3 ) formaldehyde 6 hours/day for 9 days and 19 challenged on the 10 th day with the second aldehyde for 10 minutes at var ious concentrations 20 (four rats/concentration) to establish an RD 50 . Exposure to acetaldehyde and acrolein, the two 21 smallest molecules in the series of aldehydes teste d, resulted in cross-tolerance. The RD 50 and 22 its 95% CI for acetaldehyde were estimated at 2,991 (95% CI: 2,411–3,825) ppm in the naïve 23 rats, and this was increased by approximately 3.5-f old to 10,601 (95% CI: 7,902–15,442) ppm in 24 the rats pretreated with formaldehyde. With acrole in, the RD 50 increased approximately fivefold, 25 from 6.0 (95% CI: 3.5–18.1) ppm to 29.6 (95% CI: 15 .6–93.0) ppm. Cross-tolerance with 26 formaldehyde has only been demonstrated with acetal dehyde, acrolein, and chlorine (Babiuk et 27 al., 1985; Chang and Barrow, 1984), suggesting that it is not a generalized phenomenon. 28 Whether the phenomenon of tolerance involves modula tion of specific trigeminal nerve 29 receptors or whether it results from less specific chemical injury of the nasal mucosa has not 30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-118 been determined. For example, different mechanisms lead to stimulation of the trigeminal nerve 1 and are likely to control the decrease in respirato ry rate. In particular, acetaldehyde might 2 interact with sensory nerves via an amino group (St einhagen and Barrow, 1984; Schauenstein et 3 al., 1977), whereas the receptor-binding site for f ormaldehyde and acrolein is believed to be a 4 thiol group. Furthermore, different binding sites exist on the trigeminal nerve for different 5 irritants (Nielsen, 1991). Thus, Bos et al. (1992) concluded that the data on tolerance or 6 “desensitization” versus “sensitization” (as define d strictly on the basis of the respiratory apneic 7 response) may be the result of adaptation or revers ible/irreversible adverse changes. The 8 mechanisms underlying sensitization or desensitizat ion are not well characterized. 9

10

4.2.1.1.4. Formaldehyde binding and activation of trigeminal n erve afferent activity. Kane 11 and Alarie (1978) evaluated the effect of 11 combin ations of acrolein and formaldehyde on 12 respiratory rate in outbred specific-pathogen-free male Swiss-Webster mice. Exposure 13 concentrations ranged from 0.12–8.97 ppm (0.28–21 m g/m 3 ) for acrolein and 0.37–9.73 ppm 14 (0.45–11.9 mg/m 3 ) for formaldehyde. The data were evaluated using a simple model of 15 competitive antagonism. Comparing the observed and predicted responses indicated no apparent 16 differences, and paired t-tests showed no statistic al significance. The authors concluded that 17 acrolein and formaldehyde acted at the same recepto r site and acted as competitive antagonists 18 when exposure occurred simultaneously. 19 Kulle and Cooper (1975) investigated the effects of formaldehyde on trigeminal nerve 20 afferent activity in adult male Sprague-Dawley rats . The authors isolated both the ethmoid and 21 nasopalatine branches of the trigeminal nerve and r ecorded afferent signaling as electrical 22 activity while reactive gases (formaldehyde, ozone, and amyl alcohol) were passed through the 23 nasal passages of the anesthetized animals. The au thors reported that both branches of the 24 trigeminal nerve responded similarly to all three c hemicals, and they therefore conducted the 25 balance of their experiments on the nasopalatine br anch of the nerve. Nerve response was 26 calculated as the difference between exposed and co ntrol activity, and the threshold for a positive 27 response was arbitrarily defined as an increase of 0.1 spikes per second. The sensory threshold 28 was determined by extrapolation from the measured n erve response to a range of formaldehyde 29 concentrations (0.5–2.5 ppm) or ozone (5.0–29 ppm) for an exposure duration of 2 minutes. 30 Amyl alcohol exposure (0.3–10.0 ppm) lasted for 25 seconds. Threshold was arbitrarily defined 31 as an increase of 0.1 spikes per second. The mean thresholds were 0.25 ppm for formaldehyde, 32 5.0 ppm for ozone, and 0.30 ppm for amyl alcohol, s uggesting that the trigeminal nerve is highly 33 sensitive to formaldehyde and amyl alcohol compared with ozone exposure. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-119 In a second set of experiments, Kulle and Cooper (1 975) investigated the effects of 1 prolonged formaldehyde-exposure on the odor respons e to amyl alcohol. Rats were pre-exposed 2 to a series of amyl alcohol concentrations (0.3, 0. 7, 1.0, 3.3, 6.7, or 10.0 ppm [1.08, 2.52, 3.6, 3 11.9, 24, or 36 mg/m 3 ]) then a 1-hour continuous formaldehyde exposure ( 0, 0.5, 1.0, 1.5, or 4 2.0 ppm [0, 0.61, 1.23, 1.84, or 2.45 mg/m 3 ]). There was a progressive decrease in odor 5 response to amyl alcohol with increasing stimulus o f formaldehyde concentration ( p < 0.01, 6 analysis of variance [ANOVA]). The response to for maldehyde concentration was described by 7 a power function Y = 0.741 × X 1.47 , where X is the formaldehyde concentration. The e ffects of 8 exposure to 2.0 ppm were similar, regardless of whe ther it was presented immediately as a 9 separate exposure or as the final concentration of a progressively increasing series. The response 10 to amyl alcohol did not fully recover within the 1- hour extended recovery period. Thus, it 11 appeared that the afferent function depression was not due to receptor adaptation or insufficient 12 time for formaldehyde diffusion away from receptor sites. 13 In an attempt to elucidate the basis of the differe ntial effects of various types of 14 aldehydes on sensory irritation, Tsubone and Kawata (1991) recorded the afferent activity of the 15 surgically isolated ethmoidal nerve (a branch of th e trigeminal nerve) during delivery of 0.32– 16 4.7 ppm (0.39–5.77 mg/m 3 ) formaldehyde, 0.18–7.2 ppm (0.41–16.5 mg/m 3 ) acrolein, and 134– 17 2,232 ppm (241–4,021 mg/m 3 ) acetaldehyde into the cannulated URT of male Wist ar rats 18 (six/aldehyde) at a flow rate of 200 mL/minutes for 22 seconds. Only one aldehyde was used in 19 each animal and each exposure was repeated two to f our times at different concentrations. The 20 activity of the nerve was recorded as the number of electrical discharges for a total period of 21 100 seconds, including pre-inhalation (30 second), inhalation (22 second), and post-inhalation 22 (48 second) periods. Nitrogen was used as the cont rol gas and as the vehicle to dilute the 23 aldehyde gases in order to not interfere with the g as chromatography used to analyze the 24 exposures. The vapor concentrations associated wit h a 50% increase in nerve activity over the 25 level of control gas were calculated as approximate ly 1.8, 1.2, and 908 ppm for formaldehyde, 26 acrolein, and acetaldehyde, respectively. These re sults are consistent with the findings of 27 Steinhagen and Barrow (1984) and the hypothesis tha t the differences in RD 50 are due to 28 differences in chemical reactivity in the tissue. 29 In summary, RB is a phenomenon observed in rodents exposed to reactive gases, 30 believed to be a protective response to the irritan t properties of the gas. In comparative studies, 31 rats appear more sensitive to irritant gases since they have a more pronounced RB response 32 compared with mice at a given concentration of form aldehyde and because the dose required to 33 elicit a bradypneic response is higher in rats than in mice. Interestingly, only rats appear to 34 develop tolerance to irritant gases, while mice sus tain an RB response. When formaldehyde 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-120 exposure is studied in concert with other reactive gases like chlorine and other aldehydes like 1 acetaldehyde and acrolein, cross-tolerance develope d. However, the mechanism underlying this 2 response is unknown. It is thought that RB may occ ur as a result of stimulation of the trigeminal 3 nerve. Thus, although RB appears to be a phenomeno n specific to rodents, the mechanism by 4 which it occurs, trigeminal nerve stimulation, may be applicable to understanding MOAs in other 5 species, such as primates and humans, particularly in regard to sensitization. 6

7

4.2.1.2. Respiratory Tract Pathology 8 The database for evaluating the POE toxicity in the respiratory tract of inhaled 9 formaldehyde is robust, with well-designed studies that span a duration range of a few hours to 10 chronic 2-year bioassays. Toxicity testing has bee n performed in various species, including 11 mice, rats, hamsters, guinea pigs, dogs, and nonhum an primates. Although a few studies include 12 examination of tissues outside of the URT, the majo rity of studies focus on changes in cell 13 proliferation and cell pathology in the nasal mucos a. Both mice and rats are well-defined animal 14 models with standard histologic sections establishe d to evaluate various regions of the nasal 15 passages, divided into Levels 1 to 5 and illustrate d in Figure 4-3. Pathology of the nasal mucosa 16 will be discussed with reference to these sections, and the region examined will be stipulated 17 (e.g., nasoturbinates, maxilloturbinates, or ethmoi d turbinates [ETs]). Additionally, pathology of 18 the respiratory epithelium will be distinguished fr om effects on the olfactory epithelium, 19 although the nature of the lesions is similar. 20

21

4.2.1.2.1. Mucociliary clearance

. The mucociliary apparatus of the URT is the first l ine of 22 defense against airborne toxicants. Comprising a t hick mucus layer (epiphase), hydrophase, and 23 ciliated epithelium, the mucociliary apparatus may entrain, neutralize, and remove particulates 24 and airborne chemicals from inspired air (Figure 4- 4). The mucus serves to entrain or neutralize 25 and remove exogenous agents from the nasal epitheli um (e.g., particles, reactive chemicals). As 26 reviewed by Kim et al. (2003), the nasal mucus cont ains proteins, glycoprotein, and lipids but is 27 primarily water (95%) and is propelled along by mov ement of the underlying cilia. Degradation 28 in the continuity or function of the mucociliary ap paratus, which provides protection to the nasal 29 epithelium, would result in higher levels of gases and particles reaching the nasal epithelium 30 itself and greater penetration of chemicals into th e respiratory tract. Therefore, breakdown and 31 disruption of mucociliary function are adverse effe cts, since a key bodily defense to exogenous 32 agents (including infectious agents) is damaged. 33

34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-121 1 Figure 4-3. Sagittal view of the rat nose (nares o riented to the left). 2 3 Note: The figure shows the normal distribution of n asal mucosae and the section 4 levels used in contemporary histopathology (Brennem an et al., 2000; Mery et al., 5 1994). Sections 1, 2, 4, and 5 correspond to Level s I, II, III, and IV as proposed 6 by Young (1981). S = squamous, T/R = transitional/ respiratory, O = olfactory 7

mucosa. 8 9 Source: Brenneman et al. (2000). 10 11This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-122 1 Figure 4-4. Main components of the nasal respirato ry epithelium. 2 3 Note: OM = osmiophilic membrane; EP = epiphase; HY = hypophase; Cl = cilia; 4 MV = microvilli; CJ = cell junction; CC = ciliated cell; NCC = non-ciliated cell; 5 GC = goblet cell; NE = nerve; GL = gland; BV = bloo d vessel; ECS = 6 extracellular space; BM = basement membrane. 7 8 Source: Morgan et al. (1986d). 9 10 Mucus flow slows upon formaldehyde exposure, despit e an increase in the ciliary 11 beat of the underlying epithelial cells, which prop el the mucus across the nasal epithelium 12 (Morgan et al., 1986a, c, d; 1983). These findings are consistent with other studies since 13 airborne pollutants and reactive gases have been sh own to decrease mucus flow rates in 14 several animal models (Mannix et al., 1983; Iravani , 1974; Carson et al., 1966; Dalhamn, 15 1956; Cralley, 1942). In addition to slowing flow, the mucus layer has been observed 16 breaking up as it floats on the epiphase, creating gaps in the epiphase and revealing the 17 hydrophase below (Morgan et al., 1986c, d). Formal dehyde reacts with glycoproteins in 18 the mucus of the epiphase, creating cross-links bet ween these large molecules; this is 19 believed to increase the viscosity of the mucus. 20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-123 In their first experiments, Morgan et al. (1983) de scribe progressive mucostasis (slowing 1 of mucous flow) and ciliastasis (disruption of cili ary beat) with increasing days of exposure to 2 formaldehyde in male F344 rats (15 ppm; 6 hours/day for 1, 2, 4, or 9 days). Ciliastasis occurred 3 with greater frequency and across more regions of t he nasoturbinate with subsequent days of 4 exposure. After 9 days, mucostasis was recorded in all but two regions evaluated. Although the 5 severity and time course of these changes varied ac ross regions of the nose, the process followed 6 a similar pattern: decreased flow, increased ciliar y action, mucostasis, and ciliastasis. Since the 7 formaldehyde-induced deficits in mucociliary functi on increased with days of exposure, activity 8 did not fully recover between exposures (18 hours) (Morgan et al., 1983). Therefore, the 9 severity and extent of adverse effects are dependen t on both the concentration of exposure and 10 duration (in this case, days of repeated exposures)

. 11 In subsequent studies, Morgan et al. (1986c) examin ed the exposure-response 12 relationship of formaldehyde effects on mucociliary function and functional recovery 18 hours 13 after exposure ceased. Exposure regimens similar t o the above experiment included additional 14 exposure concentrations (0.5, 2, and 6 ppm) and an additional time point of 15 days duration. 15 Exposure at 2 and 6 ppm resulted in the same progre ssion of effects on mucus flow and ciliary 16 beat. Considering both severity and extent of effe cts a clear exposure-response relationship was 17 demonstrated. Additionally, within each exposure g roup, effects progressed both in severity and 18 extent by duration of exposure to formaldehyde (fro m 1 to 4, 9, and 15 days of exposure) 19 (Morgan et al., 1986c). 20 Flow and ciliary beat were not reduced, but rather increased, in epithelium from rats 21 exposed to 0.5 ppm formaldehyde. Mucus flow in 2 o f 10 areas assessed was clearly increased 22 (275 and 200% of controls) after 4 days of exposure to 0.5 ppm formaldehyde. Two other 23 epithelial regions showed a similar trend (150% of controls), but this change was not statistically 24 significant. Interestingly, measurements made in c orresponding areas after 9 days of exposure 25 did not show an increase, and measurements in one r egion were reduced to 37% of control. 26 Although it is not known whether the observed incre ase in mucus flow rate is a subtle indication 27 of an adaptive response to a low level irritant, th e increase appears to be transient. It is not 28

known if flow rate would continue to decrease below control levels for repeated exposures at 29 0.5 ppm for longer than 9 days. 30 The regions affected at 15 ppm generally included t he lateral aspects of the nasoturbinate 31 and both the dorsal and medial aspects of the maxil loturbinate. In general there was an anterior 32 to posterior effect with increasing concentration a nd time. Additionally, impaired mucociliary 33 function was more extensive with greater concentrat ion and length of exposure. Nasal lesions 34 were seen on the nasal epithelium and correlated wi th those areas where some inhibition of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-124 ciliary function was measured. Areas without mucus flow but that still retained ciliary function 1 did not develop epithelial lesions. Morgan et al. (1986c) reported “coagulated mucus,” viewed 2 as a “continuous membrane” over the epithelium afte r 6 hours of exposure to 15 ppm 3 formaldehyde. Minor cell damage and infiltrating n eutrophils and monocytes were also seen in 4 these areas. The coagulated mucus was not seen in similarly exposed rats that were allowed 5 18 hours of recovery before sacrifice. However, ci liated cells were damaged, and there was a 6 greater presence of neutrophils and macrophages (MP s) after this recovery period. The authors 7 noted that, as the exposure continued, these areas exhibited increased signs of inflammation and 8 epithelial damage, eventually resulting in “severe degenerative changes.” 9 Morgan et al. (1986a) refined their study design to implement a nose-only exposure to 10 formaldehyde in order to better examine the progres sion of changes in mucociliary function 11 during short-term exposure, allowing examination of mucus flow immediately following 12 exposure. Three F344 rats/group were exposed to 15 ppm (18.4 mg/m 3 ) formaldehyde for 10, 13 20, 45, or 90 minutes or 6 hours. Two groups of ra ts were exposed to 2 ppm to determine a no 14

effect level for 90 minutes or 6 hours. The extent and severity of mucostasis and ciliastasis seen 15 after a 6-hour 15 ppm (18.4 mg/m 3 ) formaldehyde exposure and a 1-hour recovery perio d were 16 similar to the earlier study (Morgan et al., 1986a) , indicating that similar exposure conditions 17 were reached with this nose-only apparatus. Cilias tasis and mucostasis were both less severe and 18 less extensive in a time-dependent manner and at th e earlier time points of 10, 20, 45, and 19 90 minutes. Significant recovery was seen in mucoc iliary function by allowing a 1-hour 20 recovery between exposure and sacrifice. Regions o f both the nasal septum and lateral wall, 21

which exhibited no mucus flow when examined immedia tely after a 6-hour exposure, had 22 measurable flow after the 1-hour recovery period. Similar recovery was seen at all durations of 23 exposure. No decreases in mucociliary function wer e seen after exposure for either 90 minutes 24 or 6 hours at 2 ppm formaldehyde. However, given e vidence of recovery (Morgan et al., 1986a) 25

and the time taken to dissect and view the tissues ex vivo may have obscured more subtle effects. 26 To assess more immediate effects on mucociliary app aratus, Morgan et al. (1984a) have 27 examined formaldehyde effects on the mucociliary ap paratus of isolated frog palates. This 28 system allowed observation of mucociliary function during exposure. Unexposed frog palates 29 were covered by a continuous sheet of mucus of vari able thickness, which was observed to flow 30 in streams across the palette, exhibiting a wave-li ke form in some areas of the epiphase. The 31 authors reported particle movement in a lower, less viscous layer that was consistent with a less 32 viscous underlying hydrophase, similar to that desc ribed in rat mucosa. The basal mucus flow 33 rate was 0–4 mm/minute, with localized ciliary acti vity. Short periods of increased mucus flow 34 were associated with seemingly spontaneous increase s in ciliary beat. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-125 Formaldehyde exposure resulted in an initial increa se in ciliary beat and mucus flow rate 1 in all palates exposed at 1.37, 4.36, and 9.58 ppm formaldehyde (but not 0.23 ppm). With 2 increasing formaldehyde concentration and time of e xposure, mucostasis was evident as mucus 3 became stiff and eventually rigid. Ciliary beat co ntinued after mucostasis was reached until 4 palates were exposed to 4.36 and 9.48 ppm formaldeh yde, when ciliastasis was reached. The 5 time course to peak mucus flow rate, mucostasis, an d ciliastasis was concentration dependent, 6

with mucostasis reached in less than 3 minutes at 9 .48 ppm. In contrast, increased mucus flow 7 peaked at 8 minutes in palettes exposed at 1.52 ppm formaldehyde, which, though declining, 8

remained above basal levels after 25 minutes with n o mucostasis or ciliastasis noted at this level. 9 Fló-Neyret et al. (2001) demonstrated reduced mucoc iliary clearance and decreased 10 frequency of ciliary beats by using a similar isola ted frog palette mucociliary apparatus. 11 However the palates were exposed by formaldehyde in the Ringer’s solution in which the palates 12 were placed (0, 1.25, 2.5, or 5 ppm). Also, mucus was removed from the palettes and did not 13 come into direct contact with the formaldehyde. De spite these differences, formaldehyde caused 14 mucociliary clearance to decrease in a time- and co ncentration-dependent manner; mucostasis 15 occurred after 60 minutes of exposure to 5 ppm form aldehyde (Figure 4-5). Ciliary beat was 16 decreased in a time-dependent manner at 2.5 and 5 p pm exposure but increased at 1.25 ppm 17 formaldehyde (Figure 4-5). Reduced mucociliary cle arance at 2.5 and 5 ppm was consistent with 18 the reduced ciliary beat. However, clearance decre ased at 1.25 ppm formaldehyde, where there 19 was an apparent increase in ciliary beat. The auth ors suggest this may be a result of disrupting 20 the harmonic movement of the cilia, impairing effec tive mucociliary clearance. Based on study 21 results, the authors hypothesize that changes in ci liary beat, including excitation at lower 22 exposures, are likely to be a direct effect of form aldehyde on epithelial cells or other cellular 23 components of the mucosa. 24 In summary, numerous studies have identified impair ed mucociliary clearance activity 25 associated with formaldehyde exposure (Table 4-9). Although low-dose and short-term 26 exposures first increase ciliary beat, impaired muc us flow, slowed ciliary beat, and eventual 27

mucostasis and ciliastasis have been demonstrated i n both in vivo and in vitro exposure systems. 28

These effects are both concentration and duration d ependent and can be seen in as few as 29 15 minutes from exposure. Repeated inhalation expo sures in rats indicate the effect does not 30 fully recovery in an 18-hour period between exposur es, contributing to greater impairment over 31 extended periods of exposure. 32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-126 1

2

3

4

5

6

7

8

9 Figure 4-5. Decreased mucus clearance and ciliary beat in isolated frog palates 10 exposed to formaldehyde after 3 days in culture. 11 12 Source: Fló-Neyret et al. (2001). 13 14 Morgan et al. (1983) suggested that the initial sti mulation of ciliary activity may be a 15 defensive response to the irritant gas, possibly in dicating some penetration of formaldehyde to 16 the underlying epithelial cells. Later effects of mucostasis may be a result of cross-linking of 17 mucus glycoproteins by formaldehyde, creating a rig id mucus that is not able to flow even with a 18 rigorous ciliary beat. It is unknown if the eventu al cessation of ciliary beat is a result of 19 compound-related effects on ciliated epithelium as formaldehyde diffuses through the mucus or 20

an indirect effect associated with mucostasis. How ever, in vitro experiments by Fló-Neyret et al. 21 (2001) indicate that formaldehyde in solution, supp orting isolated frog palates without mucus, 22 resulted in the same sequence of effects, including increased ciliary beat at the lowest exposure. 23 These data suggest a role of formaldehyde beyond it s ability to form protein cross-links in 24 mucociliary proteins. 25

26

4.2.1.2.2. Cellular pathology . This section summarizes studies that have investiga ted cellular 27 pathology in the URT and in the lung. Below, full study descriptions are provided for both 28 short-term and subchronic duration studies (includi ng, where appropriate, how cell proliferation 29 relates to the observed formaldehyde-induced pathol ogy). 30

31This document is a draft for review purposes only a nd does not constitute Agency policy 4-127 DRAFT—DO NOT CITE OR QUOTETable 4-9. Summary of formaldehyde effects on mucociliary function in the upper respiratory tractSpecies NaTreatmentMeasure ofmucociliaryfunction Summary of results by location ReferenceMale F344rats10 15 ppm formaldehyde6 hours/day for 1, 2, 4,or 9 daysMucus flow andciliary beatMucostasis in regions 2, 3, 4, 5, and 8 for all rats after a singledose.Mucostasis in all but two regions evaluated byday 9. Ciliastasisfollowed mucostasis.Morgan et al.(1983)Male F344rats6 0, 0.5, 2, 6, or 15 ppmformaldehyde6 ourshours/day for 1,4, 9, or 15 daysMucus flow andciliary beat andhistopathologicanalysisFlow or ciliary beat were increased at 0.5 ppm.After1After 1 day, slowed or halted mucociliary flow at 15 ppm after 6hours.After 9 days, slowed or halted mucociliary flow decreased or completelystopped in all nasal regions evaluated.Regions affected included lateral aspect of the nasoturbinate and dorsal andmedial aspects of maxilloturbinate.Morgan et al.(1986c)Male F344rats3 pergroup15 ppm formaldehydefor 10, 20, 45, or90 minutes or 6 hoursMucus flow andciliary beatCiliastasis and mucostasis increased in a time- andconcentration-dependentmanner, with maximal response at 6 hours.Significant recovery was observed when a 1-hour recovery period occurredbetween exposure and sacrifice.Morgan et al.(1986a)Isolated frogpalatesNotstated0.23, 1.37, 4.36, or9.58 ppmformaldehydeMucus flow ratesand histopathologyCiliary beat and mucus flow increased from baselineat 1.37, 4.36, and9.58 ppm.Over time, mucus became rigid, and ciliastasis occurredMorgan et al.(1984a)Isolated frogpalates4 0, 1.25, 2.5, and 5 ppmformaldehyde every 15minutes for 60 minutesMucociliaryclearance and ciliarybeatCiliary beat decreased in a time-dependent manner at 2.5 and 5,0 ppm butwas increased at 1.25 ppm.Mucostasis occurred after 60 minutes at 5 ppm.Fló-Neyret etal. (2001)N = number of animals in study.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-128 4.2.1.2.2.1. Nasal pathology short-term studies. Inhalation of formaldehyde for a few hours has 1 been shown to result in damage of the nasal mucosa, depending on the exposure concentration. 2 Bhalla et al. (1991) observed changes in cell morph ology in male Sprague-Dawley rat nasal 3 epithelia after a single 4-hour exposure to 10 ppm (12.3 mg/m 3 ) formaldehyde. Three exposed 4 rats were sacrificed 1 hour and 24 hours after expo sure, with two control rats at each time point. 5 Noses were fixed, decalcified, and sliced along the midsagittal plane through the nasal septum. 6 The exposed turbinates were examined by scanning el ectron microscopy. Transverse sections 7 through the hard palate, at the level of the incisi ve papillae, were prepared for light microscopy 8 from similarly exposed rats (n = 10). The authors provided detailed descriptions of cell epithelial 9 organization in untreated rat turbinates and change s observed in formaldehyde-treated rats, as set 10 forth below. No statistical analysis was provided. 11 Scanning electron microscope examination of nasotur binates showed increased mucus, 12 erythrocyte infiltration, swelling of microvillus c ells, and some cell separation in formaldehyde13 treated rats. Nasoturbinates examined 1 day after exposure showed greater effects, including cell 14 damage, matted cilia, and blebbing of cell membrane s. Damage to microvillus cells of the 15 maxilloturbinate included deformed cilia, cell swel ling and rupture, and lack of typical microvilli 16 on the cell margins. As in the nasoturbinates, dam age was more marked 24 hours after exposure. 17 The epithelium of the ETs exhibited less cell damag e than in the nasal and maxillary regions, 18 with the slight lesions noted in the upper (ET1) po rtion and little to no damage noted on the mid 19 and lower (ET2 and ET3) regions. Examination of tr ansverse tissue sections revealed swollen 20 goblet cells and stretched epithelial cells that fo rmed an epithelial lining approximately 40% 21 taller than the lining seen in control rats. There was also a patchy loss of ciliated cells in the 22 respiratory epithelium, where columnar cells were p resent. 23 Buckley et al. (1984) investigated the respiratory tract lesions associated with several 24 sensory irritants. As part of this investigation, male Swiss-Webster mice were exposed to 25 3.13 ppm (3.85 mg/m 3 ) formaldehyde 6 hours/day for 5 days. A total of nine chemicals were 26 tested in parallel. The report indicates there wer e 24–34 mice in each group, although not 27

detailed for each chemical. One-half of the treatm ent group and unexposed controls were 28 sacrificed immediately after the last exposure. Th e remaining exposed mice were sacrificed 29 72 hours later. The head, trachea, and lungs were fixed and heads decalcified. Five sections 30 were taken of each nose at levels equivalent to sta ndard levels 2–6 (Figure 4-3) and were 31 examined by light microscopy. Details on lung and trachea sections were not given. 32 Formaldehyde induced lesions in the respiratory epi thelium of exposed mice, including 33 inflammation, exfoliation, erosion, ulceration, nec rosis, and squamous metaplasia. The section 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-129 level for these effects was not given. No effects were reported in the squamous epithelium, 1 olfactory epithelium, trachea, or lungs of formalde hyde-exposed mice. 2 Monteiro-Riviere and Popp (1986) evaluated damage t o the respiratory epithelium due to 3 acute formaldehyde exposures. Male F344 (CDF [F344 ]/CrlBr) rats (three to five per group) 4 were exposed at 0.5, 2.0, 6.0, or 15 ppm (0.62, 2.5 , 7.4, or 18.5 mg/m 3 ) formaldehyde 5 6 hours/day for either 1, 2, or 4 days. Rats were sacrificed either immediately after exposure or 6 18 hours later (Table 4-10). After fixation and de calcification, blocks of tissue were collected 7

from transverse sections of the skull. The first b lock of tissue, 1 μm thick, was taken just 8 posterior of the incisor teeth. The second block w as taken halfway between the first block and 9 the incisive papillae. The dorsal nasal conchae, l ateral wall, and ventral nasal conchae were 10 microdissected, postfixed, and viewed by transmissi on electron microscopy (Monteiro-Riviere 11 and Popp, 1986). 12

13 Table 4-10. Concentration regimens for ultrastruct ural evaluation of male 14 CDF rat nasoturbinates 15 16 Formaldehyde a,b Duration Time of sacrifice Observations 0.5 ppm (3) 6 hours for 1 day

6 hours for 4 days 18 hours later No lesions. Altered ciliary configuration. 2.0 ppm (3) 6 hours for 1 day

6 hours for 4 days 18 hours later No lesions. Altered ciliary configuration. 6 ppm (5 each group) 6 hours for 1 day

6 hours for 1 day 6 hours for 2 days 6 hours for 4 days Immediately

18 hours later Focal lesions on dorsal and nasal conchae and

lateral wall. Severity of lesions increased with exposure duration.

15 ppm (5 each group) 6 hours for 1 day

6 hours for 2 days 18 hours later Focal lesions on dorsal and nasal co nchae and lateral wall. Severity of lesions increased with exposure duration. Severity of lesions increased with concentration. 17

a Number of exposed rats is shown in parentheses. 18

b Five control rats were examined for each experiment

. 19 Source: Monteiro-Riviere and Popp (1986). 20 21 No lesions were observed at either 0.5 or 2.0 ppm f ormaldehyde for either 1 day or 22 4 days, evaluated 18 hours after exposure. However , an unusual altered ciliary configuration, 23 including blebbing of the cell membrane, was observ ed in almost all formaldehyde-treated rats, 24 whereas it was only “occasionally noted” in control rats. Focal lesions in the dorsal and ventral 25 conchae and lateral wall were seen in rats exposed at 6 and 15 ppm for 1 day and sacrificed 26 immediately after exposure. These lesions included cytoplasmic and autophagic vacuoles, loss 27 of microvilli, and hypertrophy. Lesions increased in severity with both exposure concentration 28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-130 and duration. Neutrophil infiltration and intercel lular edema were seen after 1 day at 6 and 1 15 ppm. Nonkeratinized squamous metaplasia was not ed after 4 days at 6 and 15 ppm in treated 2 rats. Cell death and sloughing were noted after on ly 2 days of exposure at 6 ppm formaldehyde. 3 As described above, Cassee and Feron (1994) examine d the effects of intermittent 4 exposure to formaldehyde (3.5 ppm [4.3 mg/m 3 ]), ozone (0.44 ppm [0.86 mg/m 3 ]), or a 5 combination of the two on changes to the rat nasal epithelium. Exposure occurred through six 6 consecutive 12-hour cycles in which rats were expos ed for 8 hours and then not exposed for a 7 further 4 hours. Rats were weighed before the firs t and after the last exposure periods and 8 sacrificed immediately after the last exposure. To collect tissue for biochemical analysis, skulls 9 were split sagittally and the respiratory epitheliu m collected. Tissues from six rats were pooled 10 and homogenized to enable the measurement of glutat hione (GSH) and the activities of the 11 following enzymes: glutathione S-transferase (GST), glutathione peroxidase (GPX), glucose-6- 12 phosphate dehydrogenase (G6PDH), glutathione reduct ase (GR), alcohol dehydrogenase (ADH), 13 and formaldehyde dehydrogenase (FALDH). The remain ing heads were fixed, decalcified, and 14 sectioned (standard cross sections [Figure 4-3]). 15 All groups, including controls, lost weight during the course of treatment. Rats exposed 16 to formaldehyde, ozone, or both lost more weight th an controls ( p < 0.05, p < 0.01, and 17

p < 0.001, respectively). Formaldehyde treatment alo ne increased GPX from 48.6 to 18 64.0 μmole/minute-mg protein ( p < 0.05) (Table 4-11). Formaldehyde exposure, in co njunction 19 with ozone, decreased GST from 490 to 389 μmole/min ute-mg protein ( p < 0.05). No other 20 enzyme activities or tissue GSH levels were affecte d by formaldehyde exposure. 21

22 Table 4-11. Enzymatic activities in nasal respirat ory epithelium of male 23 Wistar rats exposed to formaldehyde, ozone, or both 24 25

Enzyme Controls a Formaldehyde (3.5 ppm) Ozone (0.4 ppm) Both b ADH 2.66 (0.99) 3.53 (0.13) 3.40 (0.33) 2.42 (0.61) GST 490 (32) 494 (24) 514 (4) 389 (28) c GPX 48.6 (4.3) 64.0 (7.9) c 55.6 (2.0) 54.5 (0.3) G6PDH 58.9 (7) 60.8 (4.7) 65.8 (1.0) 45.5 (6.8) GR 275 (16) 288.2 (16) 279 (17) 236 (14) FALDH 0.77 (0.03) 0.68 (0.04) 0.68 (0.07) 0.80 (0.0 8) 26

a Values shown are the means and SDs of three measure ments of a pooled sample. Units are 27

μ mole/minute/mg of cytosolic protein. 28

b Rats were exposed intermittently, 12-hour cycles of 8 hours exposed and 4 hours unexposed, for 3 days. 29

c Different from control, p < 0.05. 30 31 Source: Cassee and Feron (1994). 32 33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-131 Formaldehyde-exposed rats exhibited lesions in the nasal epithelium at levels 2 and 3 of 1 the nose, with effects slightly more severe in leve l 2. Lesions observed include necrosis, 2 hyperplasia accompanied by squamous metaplasia, and rhinitis. Exposure to formaldehyde in 3 the presence of ozone resulted in more severe squam ous metaplasia (statistics not given). These 4 findings are similar to those of Monteiro-Riviere a nd Popp (1986), indicating that single or 5 repeated exposures can result in cell damage and de ath. Cell death and increased cell 6 proliferation were seen here after 3 days of repeat ed exposures to 3.5 ppm formaldehyde. While 7 no increases were seen in olfactory epithelium, fra nk necrosis, squamous metaplasia, and 8 hyperplasia of both ciliated and nonciliated epithe lium were noted at level 2 and 3. 9 Javdan and Taher (2000) exposed male and female alb ino Wistar rats (five/group) at 0, 2, 10 or 5 ppm (0, 2.5, or 6.2 mg/m 3 ) formaldehyde 8 hours/day for either 3 or 30 days. Transverse 11

tissue sections at the base of the incisive teeth a nd the first palatine folds were examined by light 12 microscopy. Lesions reported after 3 days of expos ure to 2 ppm formaldehyde included chorion 13 congestion, cell disarrangement, squamous hyperplas ia, atypical mitosis, and epithelial 14 hyperplasia. Similar lesions were seen after 30 da ys but were more severe. Effects at 5 ppm 15 formaldehyde included goblet cell proliferation, ol factory epithelial hyperplasia, calcified 16 regions, and an abscess on the chorion. These lesi ons were more severe after 30 days of 17

exposure. 18 Kamata et al. (1996a, b) conducted several high-dos e studies by inhalation in rats. 19 Specifically they exposed male F344 rats to 0, 128. 4, or 294.5 ppm (0, 158, or 362 mg/m 3 ) 20 formaldehyde for 6 hours (Kamata et al., 1996a). I n a subsequent study in the same laboratory, 21 male F344 rats were exposed to either 0, 15, or 145 ppm (0, 18.5, or 178 mg/m 3 ) formaldehyde 22 nose only for 6 hours (Kamata et al., 1996b). Cong estion was noted in the nasal cavities of 23 formaldehyde-exposed rats and was more severe at 14 5.6 ppm (Kamata et al., 1996b). Rats 24 exposed to 15 ppm formaldehyde had lesions in the n asal turbinate and trachea (not detailed) 25 (Kamata et al., 1996b). A slight hypersecretion of mucus was noted in the tracheal epithelium in 26 the absence of histopathologic changes. Rats expos ed to 145.6 ppm had more dramatic lesions 27 that penetrated more deeply into the respiratory tr act. Hyperkeratosis of the squamous 28 epithelium was found at level 1 of the nasal cavity . Hypersecretion, desquamation, and irregular 29 mucosal epithelium were seen in levels 2, 3, 4, and 5 of the nasal cavity, with more severe 30 changes noted in the nasal septum. Increased secre tion and desquamation of mucosal cells 31 occurred in the trachea, and a slight hyperplasia o f the alveolar wall was noted in rats exposed to 32 145.6 ppm formaldehyde (Kamata et al., 1996b) 33 Hester et al. (2003) carried out a transcriptional analysis of the nasal epithelium of male 34 F344 rats 24 hours after nasal instillation of 40 μ L of 400 mM formaldehyde. Immediately after 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-132 sacrifice, cells were harvested from the nasal cavi ty for RNA extraction. The authors found 1 several phase I and II enzymes, indicative of oxida tive stress, to be elevated. They also reported 2 the greatest increase in inflammatory genes, such a s iNOS and neuropeptides. In an effort to 3 phenotypically link any gene changes to pathology, Hester et al. (2003) noted that this exposure 4 scenario has been demonstrated to induce regenerati ve hyperplasia with minimal cytotoxicity. In 5 this regard, they observed no significant change in nine genes involved in three apoptotic 6 pathways. 7 In an expansion of their earlier study, Hester et a l. (2005) carried out a transcriptional 8 analysis of the nasal epithelium of male F344 rats that had been exposed to formaldehyde by 9 nasal instillation for a single exposure, 5 days of exposures, or 28 days of exposure. In addition, 10 this study also attempted to characterize the compa rative toxicity of glutaraldehyde with 11 structurally similar formaldehyde (van Birgelen et al., 2000). Thus, four animals per group were 12 instilled with 40 μL of deionized water (control gr oup), 40 μL of 400 mM formaldehyde, or 13 40 μL of 20 mM glutaraldehyde. Phenotypically, bot h aldehydes induced similar 14 histopathologic changes. 15 Both aldehydes induced similar changes in DNA repai r and apoptotic pathways initially, 16 but the patterns of gene changes were different aft er about 5 days of exposure. Eight genes were 17 differentially expressed between formaldehyde and g lutaraldehyde that indicated different 18 pathways for DNA repair, including recombination, b ase excision repair, and nucleotide excision 19 repair. Within this group, replication protein 70 and DNA excision repair ERCC1 showed a 20 twofold induction by formaldehyde compared with glu taraldehyde. Since both of these genes 21 and their products function by recognizing and remo ving damaged DNA bases, Hester et al. 22 (2005) hypothesized that formaldehyde-exposed cells may remove damaged bases more 23 efficiently than glutaraldehyde-exposed cells 24

25

4.2.1.2.2.2. Lung pathology: short-term studies. In addition to nasal pathology, several 26 researchers specifically investigated formaldehyde- induced effects in the trachea, bronchi, and 27 pulmonary tissues of the deep respiratory tract in a variety of species (Lino dos Santos Franco et 28 al., 2006; Kamata et al., 1996a, b; Schreibner et a l., 1979; Ionescu et al., 1978). 29 Ionescu et al. (1978) described progressive damage in pulmonary tissue of adult male 30 rabbits exposed to an aerosol of 3% formaldehyde so lution 3 hours/day for up to 50 days 31 (method of aerosol generation or particle size were not provided). An equivalent air 32 concentration was not reported and cannot be derive d from the information given. Animals were 33 sacrificed at several time points (3, 7, 15, 20, 30 , and 50 days), and fragments of the caudal lobes 34 of both lungs were taken to examine bronchi (intrap ulmonary and distal) and lung parenchyma. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-133 Enzymatic activity was characterized in frozen sect ions for β -galactosidase, adenosine 1 triphosphatase (ATPase), adenosine monophosphatase (AMPase), lactate dehydrogenase (LDH), 2 malate dehydrogenase, succinate dehydrogenase (SDH) , acid phosphatase, Tween-60 esterase, 3 naphthol-AS-D-acetate esterase, proline oxidase, hy droxyproline epimerase, leucyl 4 aminopeptidase, and β -glucuronidase. A portion of the lung was fixed an d sectioned and viewed 5 by light microscopy to determine changes in cell po pulations and tissue pathology. 6 In addition, biochemical analysis revealed that enz ymatic activity of β -galactosidase, 7 ATPase, AMPase, LDH, malate dehydrogenase, and SDH were all unchanged by formaldehyde 8 exposure across the course of treatment (Ionescu et al., 1978). The activities of several enzymes 9 were increased through the course of exposure, incl uding acid phosphatase, Tween-60 esterase, 10 naphthol-AS-D-acetate esterase, proline oxidase, an d hydroxyproline epimerase. Although no 11 details were reported, the authors described the ch anges as progressive, with the increase in 12 proline oxidase and hydroxyproline epimerase seen o nly in the second half of the treatment 13 course. The activities of two enzymes, leucyl amin opeptidase and β -glucuronidase, were 14 observed to decrease rapidly (time frame not provid ed) (Ionescu et al., 1978) 15 Histologic changes in the lung tissue were noted af ter only 3 days of exposure and were 16 generally progressive throughout the course of trea tment. Early changes in the bronchial 17 epithelium included increased mucus secretion, hype rplasia, and hypertrophy of epithelial cells. 18 Lymphocyte infiltration was noted in many areas, an d a limited thickening of the alveolar walls 19 was reported after 3 days of exposure. Epithelial cell lesions, thickening of the alveolar, and 20 infiltration of lymphocytes increased as exposure c ontinued. Mucus cells increased as much as 21 40% after 40 days of treatment. After 40 days of t reatment, Ionescu et al. (1978) observed 22 “destructive and fibrotic lesions” and provided a d etailed description of progressive lesions. 23 Schreiber et al. (1979) also examined histologic ch anges in lung tissue after high 24 formaldehyde exposures. Syrian golden hamsters (34 , sex not stated) were exposed to 250 ppm 25 (308 mg/m 3 ) formaldehyde 1 hour/day for 1, 2, 5, or 15 days. Five hamsters in each treatment 26 group were sacrificed 2 days after exposure was end ed. Three hamsters in each group were 27 sacrificed 1, 2, or 6 weeks after exposure ended to determine if formaldehyde-induced changes 28 regressed over time. Tracheal washing was carried out to collect cytologic samples in each 29 animal prior to sacrifice. Samples were fixed, sta ined, and examined by light microscopy. 30 Lungs and tracheae were removed en bloc and fixed, and 20, 1 μm thick cross sections were 31 taken (location not detailed). The remaining respi ratory tissue was sectioned at 200 μm 32 intervals. Sections were stained and viewed by lig ht microscopy. 33 Abnormal epithelial cells were found in tracheal wa shings from formaldehyde-exposed 34 hamsters. Schreibner et al. (1979) described cells with lobulated nuclei and a coarse chromatin 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-134 pattern, especially in cells showing signs of degen eration (e.g., vacuolization of nuclei and 1 cytoplasm) (Schreiber et al., 1979). Cell number a nd damage were not quantified, and there was 2 no discussion of the effects of exposure duration o n treatment, if any, on these observations. 3 Tracheal washing was normal 2 and 6 weeks after the end of exposure, indicating that the 4 cytological changes were reversible (Schreiber et a l., 1979). 5 Formaldehyde exposure caused multifocal lesions in the mucociliary epithelium in the 6 trachea and larger bronchi. Dysplastic and poorly differentiated squamous metaplastic foci 7 replaced ciliated epithelium (Schreiber et al., 197 9). Abnormal nuclear membranes, tonofibrils 8 around the nuclei, the appearance of nucleoli, and heterochromatin condensation were distinct in 9 the formaldehyde-treated hamsters. These changes, observed 2 days after formaldehyde 10 exposure, were reversible over time and not seen 2 and 6 weeks later. 11 Because of the similarity of form and physiology of rhesus monkey URTs to the human 12 respiratory tract, the effects of short-term formal dehyde exposure were evaluated in both nasal 13 and lung tissue in these monkeys by Monticello et a l. (1989). Male rhesus monkeys (nine/group) 14 were exposed to 6 ppm formaldehyde (7.4 mg/m 3 ) 6 hours/day for 5 days/week for either 1 or 15 6 weeks. Control animals were exposed to the same regimen of filtered air for 1 week. Monkeys 16 were weighed during the course of exposure and obse rved for clinical signs of irritation or 17 sickness. Monkeys were intravenously injected with [ 3 H]-thymidine 18 hours after the last 18 formaldehyde treatment to evaluate induced cell pro liferation. Sections of the nasal passages, 19 trachea, larynx, lung carina, and duodenum were pro cessed for histoautoradiography. Tissues 20 fixed and sectioned for examination by light micros copy included nose, adrenal, sternum (bone 21 marrow), duodenum, esophagus, eyes, gallbladder, he art, kidney, liver, lymph nodes, pancreas, 22 stomach, spleen, and tongue. The nose was cut into a series of transverse sections, 3 μm thick, 23 and sections from five levels were examined (Figure 4-6). Lung lobes were trimmed 24 midsagittally and sectioned with care to include ai rway bifurcations. Sections of the nasal 25 passages, trachea (cross section), larynx (cross se ction), lung carina (frontal section), and 26 duodenum were also processed for histoautoradiograp hy. 27 There were no significant changes in body weight ov er the course of the experiment. 28 Oronasal breathing was noted in the first 15 minute s of formaldehyde exposure (Monticello et 29 al., 1989). Monkeys did experience eye irritation (mild lacrimation and conjunctival hyperemia) 30 during exposure. 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-135 1 Figure 4-6. Diagram of nasal passages, showing sec tion levels chosen for 2 morphometry and autoradiography in male rhesus monk eys exposed to 3 formaldehyde. 4 5 Source: Redrawn from Monticello et al. (1989). 6 7 Formaldehyde-related lesions were reported in the n asal passages, tracheas, and in the 8 larynx of treated animals (Figure 4-7) (Monticello et al., 1989). Nasal epithelium from treated 9 animals exhibited many of the histologic lesions de scribed in rodent studies, including loss of 10 goblet cells, loss of cilia, epithelial hyperplasia , squamous metaplasia, and neutrophilic 11 inflammatory response in the respiratory epithelium . The lesions were more severe after 12 6 weeks of exposure and were present over a greater percentage of the epithelium compared with 13 the 1-week exposure group ( p < 0.05) (Figure 4-7). 14

15This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-136 

### B 25

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### 1 WK EXP 6 WK EXP CONTROL

A) B)

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2 Figure 4-7. Formaldehyde-induced cell proliferatio n in male rhesus monkeys 3 exposed to formaldehyde 4 5 Note: Animals were exposed to 6 ppm formaldehyde 6 hours/day, 5 days/week 6 for 1 or 6 weeks. Bar graph depicting mean labelin g indices for the respiratory 7 epithelium at levels B−E. A: One-week exposure gro up. B: Six-week exposure 8 group. *Statistically different from controls ( p ≤ 0.05). Statistically different 9 from 1-week exposure group ( p ≤ 0.05). 10 11 Source: Redrawn from Monticello et al. (1989). 12 13 14 There was a distinct anterior to posterior gradient in both 1-week and 6-week treatment 15 groups in which the anterior regions had a higher p ercentage of impacted epithelium (Monticello 16 et al., 1989). However, the longer duration exposu re produced significantly more lesions in the 17 larynx and trachea compared with those observed aft er only 1 week of exposure ( p < 0.05). No 18 formaldehyde-related lesions were reported for the epithelium of the maxillary sinus, a structure 19 not present in rodents. Labeling indices (LIs) fro m the histoautoradiograms indicated increased 20 cell proliferation in transitory, respiratory, and olfactory epithelial cells after the 6-week 21This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-137 formaldehyde exposure (Figure 4-8) (Monticello et a l., 1989). Similar trends were seen after 1 only 1 week but were statistically significant only in the respiratory epithelium. Although 2 increased proliferation in the trachea and carina w as statistically significant after 1 week of 3 exposure, the greater increases seen after 6 weeks of exposure, compared with controls, were not 4 statistically significant. A small sample size (n = 3) and high variability may have contributed to 5 the lack of statistical significance. Monticello e t al. (1989) noted that increased cell proliferatio n 6 was seen in locations with minimal histologic chang es, indicating proliferation may be a more 7 sensitive predictor of adverse health effects of fo rmaldehyde exposure. 8

9

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10 11 Figure 4-8. Formaldehyde-induced lesions in male r hesus monkeys exposed 12 to formaldehyde. 13 14 Note: Animals were exposed to 6 ppm formaldehyde 6 hours/day, 5 days/week 15 for 1 or 6 weeks. Bar graph showing levels B–E of the nasal passages and the 16 larynx/trachea (L/T), depicting percent surface are a with formaldehyde-induced 17 lesions. Morphometry of level A was excluded due t o the similarity of normal 18 features of transitional epithelium to formaldehyde -induced lesions in the 19 respiratory epithelium. A: One-week exposure group . B: Six-week exposure 20

group. 21 *Statistically different from controls ( p ≤ 0.05). 22 | Statistically different from 1-week exposure grou p ( p ≤ 0.05). 23 Source: Redrawn from Monticello et al. (1989). 24

25 There are two reports in the literature assessing c hanges in pulmonary tissues after acute 26 formaldehyde exposures (Kamata et al., 1996a, b). Kamata et al. (1996a) exposed male F344 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-138 rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m 3 ) formaldehyde for 6 hours. Lung lavage 1 samples were collected and the fluid analyzed for t he lipids, free cholesterol, phosphatidyl 2 ethanolamine, phosphatidyl choline, sphingomyelin, and triglyceride. 3 The bronchoalveolar lavage (BAL) was analyzed for t riglycerides, cholesterol, and 4 phosphatidyl choline. As in the first experiment ( Kamata et al., 1996a), triglyceride 5 concentration was reduced in the lavage of treated animals, in this case, to 16% of controls in 6 lavage in those rats exposed to 145.6 ppm formaldeh yde (Table 4-12). Cholesterol concentration 7 was unchanged and phosphatidyl choline was increase d to 220% of that of control rats as a result 8 of exposure to 145.6 ppm formaldehyde. However, BA L lipids were unchanged in 15 ppm 9 exposed rats. Triglycerides were reduced in unwash ed lung tissue from formaldehyde-treated 10 rats in a concentration-dependent manner and free f atty acids were reduced in rats exposed to 11 145.6 ppm formaldehyde. Neither triglyceride nor s phingomyelin was detected in lung lavage 12 fluid from the high treatment group. 13

14 Table 4-12. Lipid analysis of lung tissue and lung lavage from male F344 15 rats exposed to 0, 15, or 145.6 ppm formaldehyde fo r 6 hours 16 17

Control a 15 ppm a 145 ppm a

Lung tissue Free fatty acids (mg/g lung) 3.30 (0.7) 3.11 (1.23) 1.41 (0.63) b Triglyceride (mg/g lung) 1.55 (0.23) 0.74 (0.14) c 0.62 (0.17) c Cholesterol (mg/g lung) 1.72 (0.10) 1.41 (0.25) 1.1 6 (0.55) Phosphatidyl ethanolamine (mg/g lung) 7.41 (1.81) 7 .46 (2.28) 5.49 (1.78) Phosphatidyl choline (mg/g lung) 11.0 (1.49) 9.65 ( 3.21) 7.53 (3.52) Sphingomyelin (mg/g lung) 3.44 (0.75) 3.13 (1.28) 2 .51 (0.95) Lung lavage Triglyceride (mg/lung) 0.31 (0.10) 0.24 (0.09) 0.05 (0.02) c Cholesterol (mg/lung) 0.04 (0.01) 0.04 (0.01) 0.04 (0.01) Phosphatidyl choline (mg/lung) 0.66 (0.23) 0.84 (0. 35) 1.45 (0.31) c 18

a SD given in parentheses. 19

b Significant difference from controls ( p < 0.05). 20

c Significant difference from controls ( p < 0.01). 21 22 Source: Kamata et al. (1996b). 23 24 Concentration-dependent decreases were seen in nonp rotein sulfhydryl (SH) groups and 25 lipooxygenase in nasal mucosa homogenate and nonpro tein SH groups in lung tissue 26 homogenate (Table 4-13). Increases in both lipooxy genase and LDH activities were found in 27 lung tissue homogenate from formaldehyde-exposed ra ts. 28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-139 Table 4-13. Formaldehyde effects on biochemical pa rameters in nasal 1 mucosa and lung tissue homogenates from male F344 r ats exposed to 0, 15, 2 or 145.6 ppm formaldehyde for 6 hours 3 4

Control a 15 ppm a 145 ppm a

Nasal mucosa b Nonprotein SH groups ( μ M/g tissue) c 1.64 (0.50) 1.29 (0.28) 0.73 (0.21) f Lipid peroxides ( μ M/g tissue) 118 (23) 71 (16) f 59 (18) f Glucose-6-dehydrogenase (U/g tissue) d 1.96 (0.10) 1.87 (0.07) 2.07 (0.13) Lung e Nonprotein SH groups ( μ M/g tissue) c 1.83 (0.18) 1.70 (0.11) 1.29 (0.28) f Lipid peroxides ( μ M/g tissue) 72 (8) 95 (15) f 93 (8) g Glutathione reductase (U/g tissue) d 0.42 (0.25) 0.25 (0.05) 0.22 (0.05) Lactate dehydrogenase (U/g tissue) d 77.37 (9.28) 88.69 (7.66) 93.62 (4.99) f 5

a SD given in parentheses. 6

b 5 or 10% nasal mucosa homogenates. 7

c nmol malonaldehyde/g tissue. 8

d Units per gram tissue. 9

e 20% lung homogenates. 10

f Significant difference from controls ( p < 0.01). 11

g Significant difference from controls ( p < 0.05). 12 13 Source: Kamata et al. (1996b). 14 15 16 Lino dos Santos Franco et al. (2006) studied the ef fects of inhaled formaldehyde on lung 17 injury and changes in airway reactivity in rats. T he extent of local and systemic inflammation 18 was assessed by changes in leukocyte counts in BAL fluid, blood, bone marrow, and spleen. 19 Changes of reactivity of isolated tracheae and intr apulmonary bronchi in response to 20 methacholine were monitored in response to formalde hyde exposure. The authors exposed male 21 Wistar rats to formaldehyde generated from a 1% sol ution of formalin. However, they provided 22 insufficient information for the exposure concentra tion to be determined. Groups of six animals 23 were exposed to formaldehyde for either 0, 30, 60, or 90 minutes on 4 consecutive days. All 24 experiments were carried out 24 hours after the fin al exposure. 25 The authors reported a significantly increased numb er of leukocytes in the BAL fluid of 26 animals exposed to formaldehyde via inhalation. Th e effect reached a maximum for the longer 27 exposure duration (90 minutes). Compared with cont rols, rats exposed to formaldehyde 28 90 minutes/day for 4 days also displayed an increas e in the number of total blood leucocytes 29 (1.4 ± 0.06 × 10 4 versus 0.8 ± 0.01 × 10 4 cells/mm 3 ). These values are means ± standard error of 30 the mean (SEM) for six animals/group. The effect a ppeared to reflect changes in the 31 mononuclear cell population (1.1 ± 0.02 × 10 4 versus 0.6 ± 0.003 × 10 4 cells/mm 3 ) rather than 32 peripheral blood neutrophils (0.2 ± 0.003 × 10 4 cells/mm 3 in test animals and controls). There 33 was also an apparently compound-related increase in the total cell count in the spleen 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-140 (112.7 ± 4.4 × 10 6 versus 94.2 ± 5.5 × 10 6 cells). However, a change in the number of cells 1

eluted from bone marrow did not reach statistical s ignificance (54.6 ± 1.3 × 10 6 versus 2

45.0 ± 4.3 × 10 6 cells). Lino dos Santos Franco et al. (2006) prov ided data on dose-dependent 3 changes in methacholine-induced contractions in iso lated tracheae and bronchi obtained from 4 formaldehyde-exposed and control rats. Although th e maximal contractile response induced by 5 methacholine in tracheae of formaldehyde-treated ra ts was unchanged compared with controls, 6 contractions in isolated bronchi were significantly weaker than those observed in controls. 7 The authors examined the effect of formaldehyde inh alation on rat lung mast cells. 8 Degranulation and significant neutrophil infiltrati on were features of the response to 9 formaldehyde (Table 4-14). 10

11 Table 4-14. Mast cell degranulation and neutrophil infiltration in the lung of 12 rats exposed to formaldehyde via inhalation 13 14

Treatment group Mast cell degranulation (cells/mm 2 ) a Neutrophil infiltration (cells/mm 2 ) a

Controls 0 b 0.3 ± 0.2 Formaldehyde-exposed 2.0 ± 0.4 c 5.2 ± 1.7 c 15

a Values are means ± SEM; n = 6. 16

b 4.2 ± 0.6 cells/mm 2 intact mast cells were found in the lungs of contr ols. 17

c No statistical analysis was provided by the authors for these changes. 18 19 Source: Lino dos Santos Franco et al. (2006). 20 21 22 Selected pharmacological agents were used to explor e the mechanism by which exposure 23 to formaldehyde might have brought about the observ ed lung infiltration and bronchial 24 hyporesponsiveness. Lino dos Santos Franco et al. (2006) provided data showing that separate 25 pretreatment of the animals with compound 48/80, so dium cromoglycate (SCG), and 26 indomethacin reduced the formaldehyde effect on neu trophil release into BAL but had no effect 27 on mononuclear cell counts. Compound 48/80 and SCG also reversed the formaldehyde-induced 28 reduction in bronchial response to methacholine, bu t indomethacin had the opposite effect 29 (causing an additional decrease in bronchial respon siveness). In broad terms, these findings 30 were thought to implicate mast cells as a possible mediator of the toxicological effects of 31 formaldehyde. Histologically, a significantly incr eased number of degranulated mast cells were 32 evident in the pulmonary tissue of rats that were e xposed to formaldehyde. 33 Lino dos Santos Franco et al. (2006) also examined the regulatory role of NO on 34 formaldehyde-induced bronchial activity. Nitrites generated by cultured cells of BAL from 35 formaldehyde-treated rats increased about threefold compared with those from controls. 36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-141 However, pretreatment with the NO synthase inhibito r, N-nitro-L-arginine methyl ester, 1 prevented the formaldehyde-induced bronchial hypore sponsiveness to methacholine but had no 2 effect on pulmonary leukocyte recruitment. These d ata implicate the existence of distinct 3 mechanisms for the induction of lung inflammation v ersus bronchial hyporeactivity. Further 4 support for this concept came from an experiment in which rats were pretreated with capsaicin to 5 examine the involvement of sensory fibers in lung i nflammation and the bronchial 6 hyporesponsiveness induced by formaldehyde inhalati on. Although the treatment did not 7 influence formaldehyde-induced bronchial hyporespon siveness to methacholine, the number of 8 leukocytes recovered in the BAL fluid were reduced compared with those of rats exposed to 9 formaldehyde alone. 10

11

4.2.1.2.2.3. Extrapulmonary effects: short-term studies

. Kamata et al. (1996a) exposed male 12 F344 rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m 3 ) formaldehyde for 6 hours. In 13 addition, blood samples were monitored for hematolo gy and clinical chemistry parameters, 14 including red blood cell (RBC) count, hemoglobin (H b), packed cell volume (PCV), mean 15 corpuscular volume (MCV), mean corpuscular hemoglob in concentration (MCHC), white blood 16 cell (WBC) count, and plasma levels of total protei n (TP), albumin (ALB), blood urea nitrogen 17 (BUN), glucose, phospholipids, triglycerides, total cholesterol, cholinesterase, and LDH. Male 18 rats exposed to 294.5 ppm formaldehyde had increase d RBC count, Hb, hematocrit (HCT), 19 MCV, and serum glucose ( p < 0.05) compared with controls (Kamata et al., 1996 a). There were 20

concentration-related decreases in serum measures o f TP, ALB, and phospholipids ( p < 0.05). 21 BUN was decreased in rats exposed to 128.4 ppm but increased in the higher treatment group 22 ( p < 0.05). Phospholipid analysis of the lung surfact ant indicated a decrease in the production in 23 formaldehyde-treated animals ( p < 0.05). Total free cholesterol, phosphatidyl etha nolamine, and 24 phosphatidyl choline were reduced to 60, 55, and 38 % of controls for rats treated with 294.5 ppm 25 formaldehyde (

p < 0.05). Sphingomyelin was reduced to 32% of contr ols in the low treatment 26 group (

p < 0.05). 27 In a subsequent study in the same laboratory (Kamat a et al., 1996b), male F344 rats were 28 exposed to either 0, 15, or 145 ppm (0, 18.5, or 17 8 mg/m 3 ) formaldehyde nose only for 6 hours 29 (Kamata et al., 1996b). Fifteen animals were treat ed at each level and separated into subgroups 30

of five animals each for tissue collection and the determination of other endpoints. Blood 31 samples were collected from one subgroup to determi ne such hematological and clinical 32 chemistry parameters as RBC count, Hb, PCV, MCV, MC HC, WBC count, and plasma levels of 33 TP, ALB, BUN, glucose, phospholipids, triglycerides , total cholesterol, LDH, alkaline 34 phosphatase (ALP), aspartate aminotransferase (AST) , alanine aminotransferase (ALT), and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-142 G6PDH. BAL was collected from five animals of each group and analyzed for phospholipids. 1 Lung homogenate from five animals in each treatment group was analyzed for nonprotein SH 2 groups, lipid peroxides, and total lipids. The 20, 000 × g supernatant of the lung homogenate was 3 assayed for the activities of GR, G6PDH, and LDH. Similarly, nonprotein SH groups and lipid 4 peroxidase were measured in homogenates of excised nasal mucosa. At autopsy, organs (brain, 5 heart, lung, liver, kidney, spleen, and testis) wer e weighed and tracheae and nasal turbinates 6 examined. After fixation and decalcification, five sections across the nose were taken, 7 corresponding to standard sections 1–5 (Figure 4-3)

. 8 Several blood parameters were affected after these acute exposures. The WBC count was 9 slightly increased, from 4.7 × 10 3 cells/mm 3 in control rats to 5.1 × 10 3 cells/mm 3 and 6.1 × 10 3 10

cells/mm 3 at 15 and 145.6 ppm formaldehyde, respectively (Ka mata et al., 1996b). Serum levels 11 of AST and LDH decreased in an apparent concentrati on-dependent manner (AST 68 and 54% 12 of controls and LDH 48 and 28% of controls, respect ively). Serum levels of G6PDH and ALT 13 were decreased similarly across exposure groups at 45 and 78% of controls, respectively. 14 A synopsis of respiratory pathology findings follow ing short-term exposure to 15 formaldehyde is presented in Table 4-15. 16

17

4.2.1.2.2.4. Nasal pathology : subchronic studies. In a study by Maronpot et al. (1986), female 18 and male B6C3F1 mice (10/group) were exposed at 0, 2, 4, 10, 20, or 40 ppm (0, 2.46, 4.92, 19 12.3, 24.6, or 49.2 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for 13 week s. Clinical 20 observations were made daily, and mice were weighed weekly. At autopsy, tissue sections from 21 each organ system (approximately 50 tissues per mou se) were fixed, stained, and examined by 22 light microscopy. Noses were fixed, decalcified, a nd transversely trimmed at three levels: the 23 incisor teeth, midway between the incisor teeth and first molar teeth, and the second molar teeth 24 (corresponding to sections 2, 3, and 4 in Figure 4- 3). 25 Although control mice gained weight, mice exposed t o 40 ppm formaldehyde lost weight 26 during the 13-week exposures. Expressed by the aut hors as a percent of weight gain in controls, 27 the weight losses were −235% in males and −168.6% i n females. Early mortality for both male 28 and female mice exposed to 40 ppm was 80%. Althoug h gross and histochemical effects in 29 excised pieces from each organ system were evaluate d, endometrial hypoplasia in mice treated 30 with 40 ppm was the only effect noted outside the r espiratory system. The authors considered 31 this effect secondary to the observed respiratory t ract lesions and frank toxicity at 40 ppm 32 formaldehyde. 33

34This document is a draft for review purposes only a nd does not constitute Agency policy 4-143 DRAFT—DO NOT CITE OR QUOTETable 4-15. Summary of respiratory tract pathologyfrom inhalation exposures to formaldehyde—short-term studiesSpecies/strainNo./groupTreatment Respiratory effects LOAEL/NOAEL ReferenceNasal pathologyMale SpragueDawley rats3 Single 4-hour exposure to10 ppm formaldehyde.Marked histopathologic changes to the nasoturbinates,maxilloturbinates, ethmoidal turbinates, and gobletandmicrovillus cells.LOAEL = 10 ppm. Bhalla et al.(1991)Male SwissWebster mice24–34 0 or 3.13 ppm formaldehyde6 hours/day for 5 days.Histopathologic lesions to the respiratory epithelium,including inflammation, exfoliation, erosion,ulceration, necrosis, and squamous metaplasia.LOAEL = 3.13 ppm. Buckley et al.(1984)Male F344 rats 3–5 0, 0.5, 2, 6, or 15 ppm6 hours/day for 1, 2, or 4 days.Histopathologic lesions to the nasal conchae, lateralwall, and ventral nasal conchae.NOAEL = 2 ppm forfocal lesions. Somechanges in ciliaryconfiguration wereevident at all exposures.Monteiro-Riviereand Popp (1986)Male Wistar rats 20 0 or 3.5 ppm formaldehydethrough six consecutive 12-hourcycles in which rats wereexposed for 8 hours; 10 wereunexposed for 4 hours.The activity of GPX was increased in respiratoryepithelium homogenates. The nasal respiratoryepithelium showed frank necrosis.LOAEL = 3.5 ppm. Cassee and Feron(1994)Male F344 rats 5 0, 6, or 15 ppm[14C]-formaldehyde 6 hours/dayfor a single day (naïve group).A pretreated group was exposedto 6 or 15 ppm formaldehyde6 hours/day for 4 days prior to[14C]-formaldehyde exposure.Cellular necrosis to the nasal epithelium. 10.05%cellular proliferation.LOAEL = 6 ppm. Chang et al.(1983)Male and femaleWistar rats5/sex 0, 2, or 5 ppm formaldehyde8 hours/day for 3 or 30 days.Cell disarrangement, squamous hyperplasia, atypicalmitosis, and epithelial hyperplasia.NOAEL = 2 ppm. Javdan and Taher(2000)Male F344 rats 15 0, 15, or 145.6 ppmformaldehyde for a single 6-hourexposure.Histopathologic lesions in the nasal turbinates andtracheaLOAEL = 15 ppm. Kamata et al.(1996b)Male rhesusmonkeys9 0 or 6 ppm formaldehyde6 hours/day for 1 or 6 weeks.[3H]-thymidine was injectedprior to sacrifice.Histopathologic lesions, including loss of goblet cells,loss of cilia, epithelial hyperplasia, squamousmetaplasia, and neutrophilic inflammation.LOAEL = 6 ppm. Monticello et al.(1989)This document is a draft for review purposes only a nd does not constitute Agency policy 4-144 DRAFT—DO NOT CITE OR QUOTETable 4-15. Summary of respiratory tract pathologyfrom inhalation exposures to formaldehyde—short-term studiesSpecies/strainNo./groupTreatment Respiratory effects LOAEL/NOAEL ReferenceTracheal and lung pathologySyrian goldenhamsters (sexunstated)5 0 or 250 ppm 1 hour/day for 1,2, 5, or 15 days.Abnormal cells in tracheal lavage, an effect that wasreversed on cessation of treatment.LOAEL = 250 ppm. Schreiber (1979)Male rabbits(strain unstated)ND Aerosol generated from a 3%formaldehyde solution3 hours/day for up to 50 days(air concentration unknown).Necrosis of the bronchi and lung parenchyma.Increased activities of acid phosphatase, Tween-60esterase, naphthol-AS-D-acetate esterase, prolineoxidase, and hydroxyproline epimerase. Reducedactivities of leucyl aminopeptidase andβ-glucuronidase. Adverse histopathologic changes.ND. Ionescu et al.(1978)Male F344 rats 6 0, 128.4, or 294.5 ppm for asingle 6-hour exposure.Phospholipid content was reduced in lung surfactant,for example, sphingomyelin to 43% of controls in thelow-concentration group.LOAEL = 128.4 ppm. Kamata et al.(1996a)Male F344 rats 15 0, 15, or 145.6 ppmformaldehyde for a single 6-hourexposure.Biochemical changes in lung homogenates. Alteredlipid content of BAL in high concentration rats.LOAEL = 15 ppm. Kamata et al.(1996b)Male Wistar rats 6 Aerosol generated from a 1%formalin solution 0, 30, 60, or90 minutes/day on 4 consecutivedays (air concentrationunknown).Increased leukocyte count in bronchoalveolar fluid.Degranulation of mast cells and increased neutrophilinfiltration.ND. Lino dos SantosFranco et al.(2006)Extrapulmonary effectsMale F344 rats 6 0, 128.4, or 294.5 ppm for asingle 6-hour exposure..LOAEL = 128.4 ppm. Kamata et al.(1996a)Male F344 rats 15 0, 15, or 145.6 ppmformaldehyde for a single 6-hourexposure..LOAEL = 15 ppm. Kamata et al.(1996b)ND = not determined; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effectlevel.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-145 While no statistical comparison was provided, respi ratory tract lesions showed an 1 increased incidence with concentration, as well as an increased distribution throughout the 2 respiratory tract (Table 4-16). No lesions were se en in the nasal cavity, larynx, trachea, or lung 3 of control mice or mice treated with 2 ppm formalde hyde. Minimal squamous metaplasia in the 4 nasal cavity was noted in 1 of 10 male mice treated with 4 ppm formaldehyde, but none were 5 observed in the female mice. However, squamous met aplasia was observed in all mice in the 6 higher treatment groups (10, 20, and 40 ppm). Lesi ons became more severe and penetrated more 7 deeply into the respiratory tract as exposure conce ntration increased. Where lesions were present 8 in the nasal cavities of all mice exposed to 10 ppm , similar lesions were reported in the larynx 9 and trachea of some animals exposed to 20 ppm and a ll animals exposed to 40 ppm 10 formaldehyde. Mice exposed to 40 ppm formaldehyde exhibited lesions as deep as the lung, 11 including squamous metaplasia, submucosal fibrosis inflammation, and epithelial hyperplasia. 12 The findings of Maronpot et al. (1986) indicated a no-observed-adverse-effect level 13 (NOAEL) of 4 ppm and a LOAEL of 10 ppm in mice, bas ed on squamous metaplasia in the 14 nasal epithelium. Although a LOAEL of 10 ppm was o bserved, there was 80% mortality for 15 both sexes at 40 ppm, indicating a very narrow rang e between the first observed adverse health 16

effects and frank effect concentrations in mice for this 13-week treatment. 17 In a study by Woutersen et al. (1987), male and fem ale albino SPF Wistar rats (10/group) 18 were exposed to 0, 1, 10, or 20 ppm (0, 1.23, 12.3, or 24.6 mg/m 3 ) formaldehyde 6 hours/day, 19 5 days/week for 13 weeks. Rats were checked daily and weighed weekly. Three longitudinal 20 sections of lungs, trachea, and larynx and six stan dard cross sections of the nose were taken for 21 microscopic examination. Two rats per exposure gro up were similarly treated for 3 days and 22 sacrificed 18 hours later, and nasoturbinates were dissected to measure cell proliferation. 23 Woutersen et al. (1987) noted that the majority of the dose-dependent increases in cell 24 proliferation seen at section level 3 after 3 days of repeated 6-hour exposures to 10 and 20 ppm 25 (12.3 and 24.6 mg/m 3 ) formaldehyde occurred in areas of the epithelium showing “clear 26 squamous metaplasia and hyperplasia.” Cell prolife ration rates in metaplastic epithelium of 27 29.5 and 33.2% were much higher than the 1.4 to 2.8 % proliferation in the visibly unaffected 28 respiratory epithelium from rats exposed at 10 ppm formaldehyde. Although there was a slight 29 trend towards increased cell proliferation in the v isibly unaffected epithelium of exposed animals 30 compared with unexposed controls, the majority of i ncreased cell proliferation resulting from 31 exposure to 10 and 20 ppm formaldehyde was attribut ed to the metaplastic epithelium 32 (Woutersen et al., 1987). 33This document is a draft for review purposes only a nd does not constitute Agency policy 4-146 DRAFT—DO NOT CITE OR QUOTETable 4-16. Location and incidence of respiratorytract lesions in B6C3F1 mice exposed toformaldehydeControl 2 ppm 4 ppm 10 ppm 20 ppm 40 ppmLocation of respiratorytract lesionsMale Female Male Female Male Female Male Female Male Female Male FemaleNasal cavitySquamous metaplasiaSeropurulent inflammation–a–––––––1/10–––10/104/1010/10–10/1010/1010/108/1010/1010/1010/1010/10LarynxSquamous metaplasia––––––––6/93/910/107/8TracheaSquamous metaplasiaEpithelial hyperplasiaSeropurulent inflammationSubmucosal fibrosis––––––––––––––––––––––––––––1/10–––3/104/10––5/102/10––10/102/108/109/1010/10---5/105/10Lung (Bronchus)Squamous metaplasiaInflammationSubmucosal fibrosis––––––NDbNDNDNDNDNDNDNDNDNDNDND––––––––––––4/103/102/103/102/10–aDash indicates no lesions recorded in that treatment group.bND = no data.Source: Maronpot et al. (1986).This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-147 Statistically significant increases were seen in fo cal respiratory epithelial hyperplasia and 1

keratinization in both male and female rats at the highest treatment level (20 ppm) (Table 4-17). 2 Male rats also had statistically significant increa ses in observed respiratory epithelial squamous 3 metaplasia, focal olfactory epithelial thinning, an d rhinitis. Both male and female rats treated 4 with 10 ppm formaldehyde showed statistically signi ficant increases in squamous metaplasia, 5 hyperplasia, and keratinization of the respiratory epithelium (Woutersen et al., 1987). 6 Disarrangement of the respiratory epithelium was on ly significantly increased in female rats, but 7 this change was observed at both the 10 and 20 ppm treatment levels. Although some lesions 8 were observed in animals treated with 1 ppm formald ehyde, their incidences were not 9 statistically significant and the findings were equ ivocal. 10 Feron et al. (1988) examined recovery of formaldehy de-induced nasal lesions after 11 subchronic exposures. Male albino SPF Wistar rats (50–55/group) were exposed to 0, 10, or 12 20 ppm (0, 12.3, or 24.6 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for either 4, 8, or 13 13 weeks. All groups were observed for a total of 130 weeks, including treatment and recovery. 14 Rats were weighed weekly for the first 13 weeks and monthly thereafter. Rats (five/group) were 15 sacrificed immediately after the end of exposure (4 , 8, or 13 weeks). The balance of the rats 16 were sacrificed after 130 weeks, inclusive of expos ure time. At sacrifice, noses were fixed and 17 sectioned by using standard section levels. 18 Formaldehyde exposure (20 ppm) was associated with reduced body weight throughout 19 the exposure period (4, 8, or 13 weeks). However, body weight in these groups matched that of 20 controls after 8, 40, and 100 weeks, respectively. Rats exposed to 10 ppm for 8 or 12 weeks had 21 slightly decreased body weight (further details not given). 22 Nonneoplastic lesions were reported in the nasal mu cosa of rats exposed to either 10 or 23 20 ppm formaldehyde and examined immediately after exposure was discontinued (4, 8, or 24 13 weeks). Lesions increased in severity with both exposure duration and concentration (details 25 of severity and incidence were not provided). Rhin itis, hyperplasia, and squamous metaplasia of 26 the respiratory epithelium were seen in rats from b oth dose groups, but changes in olfactory 27 epithelia were only seen in rats exposed to 20 ppm, where cell disruption, thinning of the 28 epithelium, and simple cuboidal or squamous metapla sia were also reported. Changes in the 29 dorsomedial region, at the junction of the respirat ory and olfactory epithelium, were similar to 30 those seen in the olfactory epithelium of rats expo sed to 20 ppm formaldehyde. A similar 31 concentration- and duration-dependent increase in h istopathologic changes in nasal epithelium 32 was observed after the full 130 weeks, which includ ed 126, 122, or 117 weeks of recovery for 33 the three duration groups, 4, 8, and 13 weeks, resp ectively (Table 4-17). 34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-148 Table 4-17. Formaldehyde effects (incidence and se verity) on histopathologic 1 changes in the noses and larynxes of male and femal e albino SPF Wistar rats 2 exposed to formaldehyde 6 hours/day for 13 weeks 3 4 Concentration of formaldehyde (ppm) 0 1 10 20 0 1 10 20 Respiratory epithelium Severity Males Females Slight – a

– – – – – – 3 Moderate – – – 5 b

– – – 4 Diffuse squamous metaplasia Severe – – – 5 b

– – – 3 Very slight – 1 – – – – 1 – Slight – 1 6 b

– – – 7 c

– Focal squamous metaplasia Moderate – – 4 – – – 2 – Very slight – – 1 1 – – 2 1 Slight – – 6 b 7 c – 1 6 b 6 b Focal hyperplasia

Moderate – – 1 – – – – – Very slight – – 1 – – – 2 1 Slight – – 3 – – 1 6 b 6 b Focal disarrangement

Moderate – – 1 – – – – – Very slight – 2 6 b 1 – – 6 b 6 b Slight – – 3 6 b

– – 2 4 Focal keratinization

Moderate – – – 1 – – – – Olfactory epithelium Slight – – – 2 – – – 2 Moderate – – – 1 – – – 2 Focal thinning

Severe – – – 5 b

– – – 2 Slight – – – 4 – – – 3 Focal squamous metaplasia Moderate – – – 4 – – – 1 Very slight – – – 1 – – – – Focal keratinization Slight – – – 2 – – – – Rhinitis – 2 5 b 10 c

– – 3 2 Larynx Very slight – – – 3 – NE d NE – Slight – – – 1 – NE NE – Squamous metaplasia

Moderate – – – 1 – NE NE – Keratinization Slight – – – 2 – NE NE – 5

a Dash indicates no lesions reported. 6

b Different from control, p < 0.05. 7

c Different from control, p < 0.01. 8

d NE = not evaluated. 9 10 Source: Woutersen et al. (1987). 11 12 13 Feron et al. (1988) did not provide a direct compar ison among lesions reported at the 14

interim sacrifice and terminal sacrifice after the extended recovery period. However, similar 15 lesions were reported after the recovery period, in cluding focal hyperplasia and stratified 16 squamous metaplasia of the respiratory epithelium, stratified cuboidal or squamous metaplasia in 17 the dorsomedial area, and replacement of olfactory epithelium. The incidence and severity of 18This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-149 these lesions in rats exposed to 20 ppm formaldehyd e were statistically different from control 1 animals, regardless of exposure duration (Table 4-1 8). 2

3 Table 4-18. Formaldehyde-induced nonneoplastic his topathologic changes in 4 male albino SPF Wistar rats exposed to 0, 10, or 20 ppm formaldehyde 5 (6 hours/day, 5 days/week) and examined at the end of 130 weeks inclusive of 6

exposure 7 8

4 Weeks 8 Weeks 13 Weeks Formaldehyde, ppm 0 10 20 0 10 20 0 10 20 Total noses examined 44 44 45 45 44 43 45 44 44 Respiratory epithelium focal hyperplasia Very slight 0 0 0 0 1 3 0 5 a 2 Slight 0 3 8 b 2 2 12 b 1 6 14 b

Moderate 0 0 1 0 1 0 0 0 4 Respiratory epithelium focal stratified squamous metaplasia Very slight 3 6 14 b 8 16 17 a 2 10 a 2 Slight 4 2 19 b 2 1 20 b 3 18 b 26 b

Moderate 0 2 3 0 0 2 1 5 14 b

Severe 0 0 0 0 0 0 0 0 1 Respiratory/olfactory epithelium stratified cuboidal or squamous metaplasia 0 0 4 0 0 17 b 0 2 23 b

Rhinitis 7 7 18 a 4 6 22 a 8 11 23 b Olfactory epithelium replacement by respiratory epithelium and regeneration Very slight 0 0 0 0 0 2 0 0 1 Slight 1 0 6 0 0 14 b 0 0 12 b

Moderate 0 0 1 0 0 3 0 0 12 b

Severe 0 0 0 0 0 1 0 0 1 9

a Significantly different from control, p < 0.05. 10

b Significantly different from control, p < 0.01. 11 12 Source: Feron et al. (1988). 13 14 Although a slight increase in changes to the olfact ory epithelium and dorsomedial area 15 was seen in rats treated with 20 ppm formaldehyde f or only 4 weeks, these differences were 16 significant and more severe in the 8- and 13-week t reatment groups. Replacement of olfactory 17 epithelium by respiratory epithelium was described as slight after 8 weeks of exposure and slight 18 to moderate after 13 weeks of exposure in the 20 pp m treatment groups. Therefore, 19 formaldehyde-induced lesions were not resolved afte r a considerable nonexposure recovery 20 period of up to 126 weeks (Feron et al., 1988). 21 Feron et al., (1988) derived a correlation between the development of nonneoplastic 22 changes in nasal epithelium and the development of nasal tumors as a result of these subchronic 23 formaldehyde exposures. Two SCCs were reported in rats exposed to 10 ppm formaldehyde but 24This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-150 were not considered to be formaldehyde related beca use of their locations (nasolacrimal duct, 1 incisor tooth). Six tumors were observed in the 20 ppm, 13-week exposure group (Table 4-19) 2

of which three of the tumors were SCCs similar to t hose observed as a result of chronic 3 formaldehyde exposure. Two polypoid adenomas also were reported in rats exposed to 20 ppm 4 formaldehyde. Feron et al. (1988) concluded that s ubchronic exposures to 20 ppm formaldehyde 5 could result in an increase in nasal tumors, an eff ect that followed observation of cellular 6 proliferation. 7

8 Table 4-19. Formaldehyde-induced nasal tumors in m ale albino SPF Wistar 9 rats exposed to formaldehyde (6 hours/day, 5 days/w eek for 13 weeks) and 10 examined at the end of 130 weeks inclusive of expos ure 11 12 Tumor type 0 ppm 10 ppm 20 ppm

No. of rats exposed for 4 weeks 44 44 45 Polypoid adenoma 0 0 1 a SCC 0 0 1 No. of rats exposed for 8 weeks 45 44 43 Polypoid adenoma 0 0 1 a SCC 2 1 1 No. of rats exposed for 13 weeks 45 44 44 SCC 0 1 3 a Cystic squamous cell carcinoma 0 0 1 Carcinoma in situ 0 0 1 a Ameloblastoma 0 0 1 13

a Tumor considered to be associated with formaldehyde exposure. 14 15 Source: Feron et al. (1988). 16 17 18 A companion study from the same laboratory examined the effects of lower concentration 19 formaldehyde exposures (Zwart et al., 1988). Male and female albino Wistar rats (50/group) 20 were exposed to 0, 0.3, 1, or 3.0 ppm (0, 0.37, 1.2 , or 3.7 mg/m 3 ) formaldehyde 6 hours/day, 21 5 days/week for 13 weeks. Body weight, general con dition, and behavior were recorded weekly. 22 No effects of formaldehyde exposure on body weight changes were noted, and growth was 23 considered comparable among different exposure grou ps and controls. Rats were sacrificed 24 during week 14, and noses were fixed and sectioned (exact time after exposure ended not given). 25

Six standard cross sections were examined for each animal by light microscopy, anterior to 26 posterior. Noses were fixed and decalcified, and s ix standard cross sections were taken and 27 developed. 28 No formaldehyde-related lesions were reported in th e respiratory epithelium at section 29 level 3 after 13 weeks of formaldehyde exposure (0. 1 ppm, 1 ppm, or 3 ppm). Signs of 30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-151 inflammation (rhinitis, sinusitis, mononuclear cell infiltrates) were observed in 1 formaldehyde-treated rats, but there was no concent ration-response relationship (data not 2 provided). Formaldehyde-related pathology in the a nterior part of level 2 epithelium was 3 reported in 37/50 males and 21/50 female rats expos ed to 3.0 ppm for 13 weeks. Both 4 keratinized and unkeratinized squamous metaplasia w ere present, and disarranged cells and 5 hyperplastic respiratory epithelium were found in t he transitional zone between squamous and 6 pseudostratified epithelium at level 2. Foci of ke ratinized squamous epithelium, glandularization 7 of goblet cells, and deciliated epithelium were obs erved by electron microscopy in anterior 8 sections of level 2 of rats exposed to 3 ppm formal dehyde. Epithelial cells with irregularly 9 shaped and strongly indented nuclei were described at level 2 in animals exposed to 0.3 and 10 1 ppm formaldehyde and were considered to be disarr anged as well at 3 ppm formaldehyde 11

exposures. 12 Although early cell proliferation at level 3 corres ponded to basal cell hyperplasia at 13 3 days, neither effect persisted for the course of the exposure. The authors speculate that this is 14 an indication of an adaptive response, perhaps thro ugh increased function of the mucociliary 15 apparatus present at level 3. In contrast, the ear ly changes at section level 2 were less dramatic 16 but persisted through 13 weeks, including clear for maldehyde-related pathology. 17 Concentration times time (C × t) issues have been investigated for histopathology as well 18 as for cellular proliferation, outlined above. Spe cifically, Wilmer et al. (1989, 1987) compared 19

the effects of 8-hour continuous and 8-hour intermi ttent formaldehyde exposure in two studies. 20 Fifty male albino Wistar rats (10/group) were expos ed to different exposure regimens to achieve 21 similar compound-related C × t products. A C × t product of 40 ppm-hours (49.2 mg/m 3 -hours) 22 was attained by an 8-hour exposure to 5 ppm (6.2 mg /m 3 ) or a 4-hour exposure to 10 ppm (12.3 23 mg/m 3 ) (Wilmer et al., 1987). Similarly, an 80 ppm-hour s (98.4 mg/m 3 -hours) C × t product was 24 attained from continuous 10 ppm exposure or intermi ttent 20 ppm (24.6 mg/m 3 ) exposure. Rats 25 were exposed to one of these regimens 8 hours/day f or either 3 days (two/group) or 4 weeks 26 (eight/group). Eighteen hours after exposure ended , rats were injected with [ 3 H]-thymidine and 27

sacrificed 2 hours later. Noses were fixed and dec alcified, and six standard cross sections were 28 taken and developed. 29 Thinning and disarrangement of the respiratory epit helium, squamous metaplasia, basal 30 cell hyperplasia, and rhinitis were seen in formald ehyde-treated rats. Lesions were most severe 31 in group 4 (20 ppm intermittent). Groups 2 and 3 h ad similar lesions (10 ppm intermittent and 32 continuous). Rats in group 1 had mild lesions. Fo rmaldehyde concentration was the major 33 determinate in severity of nasal lesions. Formalde hyde effects were less severe in group 1 than 34 in group 3, even though the C × t product was the s ame, indicating concentration rather than 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-152 duration or cumulative exposure correlates to sever ity. Epithelial lesions in group 3 rats were 1 similar among rats exposed to 10 ppm, regardless of duration (groups 2 and 3). 2 In a follow-up study, Wilmer et al. (1989) assessed both cellular proliferation and 3 histologic lesions in Wistar rats exposed to formal dehyde in groups that differed by 4 concentration and time. Group A served as a contro l group (0 ppm). Group B was exposed to 5 1 ppm for 8 hours, group C to 2 ppm for 8 hours, gr oup D to 2 ppm for 4 hours (30 minutes for 6 8 hours), and group E to 4 ppm for 4 hours (30 minu tes for 8 hours). The experimental design 7 and cellular proliferation results are illustrated in Table 4-20. Intermittent exposures at 2 and 4 8 ppm resulted in formaldehyde-related histopathologi c lesions similar to those reported by Zwart 9 et al. (1988). Disarrangement and squamous metapla sia in respiratory epithelium were observed 10 at 4 ppm (Table 4-20). Disarrangement, nest-like i nfolds, goblet cell hyperplasia, and rhinitis 11 were observed at 2 ppm. Rats exposed continuously for 8 hours at 2 ppm formaldehyde had 12 fewer lesions than rats intermittently exposed to 2 ppm and were not statistically different from 13 controls. Although lesions were noted in rats give n the continuous 1 ppm, 8-hour treatment, 14 their incidence was not significantly different fro m the controls (Table 4-20). It should be noted 15 that the control rats in this study were reported t o have a higher frequency of lesions than 16 controls in two previous studies from this laborato ry employing the same techniques (Zwart et 17 al., 1988; Woutersen et al., 1987). For example, l esions noted in the respiratory epithelium of 25 18 control rats included 13 disarrangements, 13 basal cell hyperplasia, and 5 each of goblet cell 19 hyperplasia, nest-like infolds, and squamous metapl asia. This is in contrast to the data of 20 Woutersen et al. (1987), who reported no lesions in the respiratory epithelium of 20 control rats 21 (male and female). Although Zwart et al. (1988) di scussed inflammatory lesions in control rats, 22

no mention was made of the other scored lesions in control animals. Overall, Wilmer et al. 23 (1989) reported clear adverse effects at 2 ppm form aldehyde, resulting from intermittent 24 exposure for 8 hours/day, 5 days/week for 13 weeks. The indication of no effects at 1 ppm and 2 25 ppm continuous exposure should be considered with s ome caution, given the unusual incidence 26

of lesions in the control animals. 27 The results reported by Wilmer et al. (1989, 1987) indicate a greater influence of 28 concentration, rather than exposure regimen (contin uous versus intermittent) on formaldehyde 29 toxicity. However, these studies were conducted as repeated 8-hour exposure regimens over a 30 course of days or weeks. Therefore both regimens a llowed for a 16-hour recovery time before 31 the next reexposure and do not represent a true con tinuous exposure. This research group has 32 speculated that defensive adaptation of the nasal m ucosa may include the function of the 33 mucociliary apparatus (Feron et al., 1989). Morgan et al. (1986a) have shown formaldehyde 34 effects on mucus flow and ciliary beat in F344 rats to result from hourly exposures to 15 ppm 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-153 formaldehyde. However, effects seen in repeated 8- hour exposures may not correspond to those 1 effects resulting from shorter duration exposures t o higher formaldehyde concentrations. 2 3 Table 4-20. Formaldehyde effects on nasal epitheli um for various 4 concentration-by-time products in male albino Wista r rats 5 6

Exposure regimen (number of animals) A (25) B (22) C (24) D (23) E (25) 0 ppm 1 ppm 2 ppm 2 ppm 4 ppm Respiratory epithelium at crosssection level 2 8-Hour continuous 8-Hour

continuous 8-Hour

intermittent 8-Hour

intermittent Disarrangement Focal Diffuse 12 1 4

1 8

0 3 a

15 b 8

11 c Necrosis Focal Diffuse 4 0 3

0 0

0 2

2 3

2 Basal cell hyperplasia Focal Diffuse 9 4 4

0 6

0 11

4 10

11 Squamous metaplasia Focal 5 0 1 7 16 c

Keratinization 0 0 1 0 3

Nest-like infolds Focal Diffuse 5 0 4

3 11

1 14 c

0 7

1 Goblet cell hyperplasia Focal Diffuse 0 5 1

2 1

8 2

13 b 1

10

Rhinitis 3 2 3 16 c 8 7

a

p < 0.05, compared with group A. 8

b

p < 0.001, compared with group A. 9

c

p <0.01, compared with group A. 10 11 Source: Wilmer et al. (1989). 12 13 Rusch et al. (1983a, b) performed a comparative stu dy of formaldehyde effects on the 14 nasal epithelium in F344 rats, Syrian golden hamste rs, and cynomolgus monkeys. Groups of 15 animals were exposed at 0, 0.2, 1, or 3 ppm (0, 0.2 5, 1.2, or 3.7 mg/m 3 ) formaldehyde 16 22 hours/day, 7 days/week for 26 weeks. Six male m onkeys, 10 male and 10 female hamsters, 17 and 20 male and 20 female rats were exposed at each exposure level. The experiment was run in 18 two trials, each with its own control group: trial 1 at 0.2 or 1 ppm and trial 2 at 3 ppm. Animals 19 were weighed weekly and physically assessed (detail s not given). At sacrifice, organ weights 20 were recorded for the kidney, adrenals, heart, and liver. Tissue sections of the lung (4), trachea, 21 and nasal turbinates (4) of each animal were examin ed by light microscopy (section locations not 22This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-154 given). Additionally, sections were examined by el ectron microscopy for rats in the control and 1 1 ppm treatment groups (five rats per group). 2 Body weights of both male and female rats in the 3 ppm treatment group were depressed 3 by 20% between week 2 and the end of the 26-week ex posure. Absolute liver weights were 4 decreased in these animals as well (26% lower in ma les and 12% lower in females, p < 0.05). 5 This decrease in liver weight remained significant for male rats when normalized for body 6 weight (a ratio of 2.9 in treated versus 3.16 in co ntrols) but not for female rats. No significant 7 body weight or organ weight changes were seen in ha msters or monkeys. Increased incidences 8 of congestion (36/156), hoarseness (32/156), and na sal discharge (62/156) were observed in 9 monkeys in the 3.0 ppm treatment group versus no ho arseness or congestion and only five 10 observations of nasal discharge in 156 observations for control monkeys. Increased nasal 11 congestion was noted in the two lower treatment gro ups of monkeys: 30/156 and 12 45/156 observations, respectively, versus 9/156 obs ervations in nasal discharge in the controls. 13 The authors reported an increase in nasal discharge and lacrimation in treated hamsters but no 14 increases in symptoms in rats. However, observatio ns of adverse symptoms in the control rats 15 were greater than 10% on some measures. 16 Rhinitis increased in rats in the 3 ppm treatment g roup, and the incidence in controls was 17 notable (Table 4-21). All groups of monkeys showed some rhinitis, and no treatment effects 18 were observed in either monkeys or hamsters. Monke ys and rats in the high treatment group 19 (3 ppm) had a greater incidence of lesions in the n asoturbinate epithelium (Table 4-22). Rusch 20 et al. (1983a, b) noted that most lesions were mild to moderate but were “somewhat more 21 severe” in the high treatment group. Hamsters did not exhibit a similar increase, with few 22 lesions noted in the nasal epithelium. Overall, th ese studies show a clear increase in adverse 23 health effects at 3 ppm for rats and monkeys, with no adverse effects seen in hamsters at this 24 treatment level or rats and monkeys at the lower co ncentrations (0.2 ppm and 1 ppm). 25

26 Table 4-21. Rhinitis observed in formaldehyde-trea ted animals; data pooled for 27 male and female animals 28 29

F344 rats Cynomolgus monkeys Syrian golden hamsters Trial 1: I, Control II, 0.2 ppm III, 1 ppm 17/38 14/39 14/38 4/6 4/6 5/6 0/14 0/4 0/11 Trial 2: IV, Control V, 3 ppm 12/40

25/39 2/6 4/6 0/9 2/16 30 Source: Rusch et al. (1983a, b). 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-155 Table 4-22. Epithelial lesions found in the middle region of nasoturbinates of 1 formaldehyde-treated and control animals; data pool ed for males and 2 females 3 4

F344 rats Cynomolgus monkeys Syrian golden hamsters

Basal cell hyperplasia Squamous

metaplasia/ hyperplasia Squamous metaplasia/

hyperplasia Nasal epithelium Trial 1: I, Control II, 0.2 ppm III, 1 ppm 0/38

0/38 0/36 2/38

1/38 3/36 0/6

0/6 1/6 No lesions noted

Trial 2: IV, Control V, 3 ppm 4/39

25/37 3/39

23/37 0/6

6/6 No lesions noted

5 Source: Rusch et al. (1983a, b). 6 7 8 Andersen et al. (2008) examined the effect of form aldehyde exposure at several 9 concentrations and durations. This study comprised histopathology and cell proliferation data, as 10 well as genomic analyses at Level II of the nasal c avity. Toxicogenomics analysis was 11 performed only at Level II because this was the reg ion where the most severe lesions have been 12 reported in chronic bioassays (Andersen et al., 200 8; Monticello et al., 1991; Kerns et al., 1983). 13 More specifically, Andersen et al. (2008) stated th at the histopathologic and cell proliferation 14 effects at Levels II and III (with similar tissue s tructure) (Monticello et al., 1991) provided 15 phenotypic anchoring for the genetic analysis. Tab le 4-23 summarizes many of the broad 16 phenotypic findings. 17 The primary conclusions of this study with regard to the histopathology and cell 18 proliferation are as follows: 19

• The presence of inflammatory cell infiltrates in th e nasal epithelial tissue of F344 rats is 20 highly variable and provides no coherent pattern wi th dose or duration at levels below 21 6 ppm. 22

• Hyperplasia was observed following exposure to ≥ 2 ppm. 23

• Metaplasia was observed at 6 ppm on day 5, but not before or after. 24

• Cell proliferation (as measured by labeling indices ) was significantly elevated in 25 Levels I–III at 6 ppm on day 5 and Level I on day 1 5, leading to the conclusion that 26 significant changes in cell proliferation may not o ccur at exposures to ≤ 2 ppm. 27

• A significant decrease in cell density was observed at Level I in animals exposed to 28 6 ppm formaldehyde for 15 days, which was posited t o be related to tissue remodeling in 29 response to this concentration. 30This document is a draft for review purposes only a nd does not constitute Agency policy 4-156 DRAFT—DO NOT CITE OR QUOTETable 4-23. Cellular and molecular changes in nasal tissues of F344 rats exposed to formaldehydeD1 D1R D5 D6 D6R D15ppmResponse00.72615 0 0.726150 0.7 2 6 1500.7261500.726150 0.7 2 6 15I 01 68– 4 2 17– 1 1 5 8 –52 47–61 37–3 1 0 5 –H 00 00– 0 1 38– 0 0 3 8 –00 18–00 28–0 0 2 7 –M 00 00– 0 0 00– 0 0 0 7 –00 00–00 00–0 0 0 0 –P1 39±9 37±1565±40 155±89a79±55 56±37 51±44 119±38aP2 – – –a– – – –P3 – – –a– – – –CD 321±30336±64377±141400±61362±61340±57321±37293±53bG –0 142745– 0 00– – 0 15 28 ––0 09––– –––– 0 0 54D = day; R = recovery.I = infiltrations (number out of 8 total animals);H = hyperplasia (number/8); M = metaplasia (number/8).P1–P3 = proliferation at levels I–III (ULLI).CD = cell density (cells/mm) at Level I.G = genes significantly altered at Level II of nasal epithelial tissue.aSignificantly elevated ULLI and LI at Level I on day 5 or significantly elevated lLI lat Level I on day 15; indexawithout numerical value indicates significantincreases in ULLI in all subregions of Levels II and III at day 5.bStatistically significant difference from control (p< 0.05).Source: Andersen et al. (2008).This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-157 Based on their analysis of the microarray data, An dersen et al. (2008) concluded that no 1 genes were significantly altered by exposure to 0.7 ppm from 1 to 15 days. Exposure to 2 ppm 2 primarily resulted in gene changes at 5 days of exp osure, but not thereafter. One gene was 3 significantly increased on day 1, but the authors d id not identify that gene. At 6 and 15 ppm, 4 42 and 745 genes were altered at day 1, respectivel y. After 5 days, gene changes were only 5 observed at 6 ppm (15 ppm was not examined after da y 1). These findings support conclusions 6 reached by their laboratory in an earlier analysis. Thus, the primary conclusion in the Andersen 7 et al. (2008) study is that genomic changes, includ ing those suggestive of mutagenic effects, did 8 not temporally precede or occur at lower doses than phenotypic changes in the tissue. The 9 implications of this finding will be examined later in Section 4.5. 10

11

4.2.1.2.2.5. Lung pathology: subchronic studies. Studies have also investigated the ability for 12 formaldehyde to induce pathology in the trachea, br onchi, and lung tissue. These studies have 13 reported tracheal tissue changes, lung inflammation , necrosis, changes to the biochemistry of 14 BAL fluid and lung surfactant in a variety of speci es. Özen et al. (2003a) noted changes in zinc 15 concentration in the lung tissue following exposure for formaldehyde. Dallas et al. (1989) and 16 Dinsdale et al. (1993) observed changes in P450 enz yme activity in the lung associated with 17 formaldehyde exposure. 18 Özen et al. (2003a) measured zinc, copper, and iron content in lung tissue from 19 formaldehyde-exposed Wistar rats. Adult male rats were exposed to 0, 5, or 15 ppm (0, 6.2, or 20 18.5 mg/m 3 ) formaldehyde 8 hours/day, 5 days/week for either 4 or 13 weeks. Rats were 21 checked daily and weighed weekly. At sacrifice, ra ts were autopsied and examined for gross 22 pathological changes. Lung tissue was homogenized and analyzed for zinc, copper, and iron. 23 Body weight gain was depressed in all treatment gro ups in a concentration-dependent 24 manner (

p < 0.001) (Table 4-24). Formaldehyde-exposed rats c onsumed less food and water 25 than controls and showed unsteady breathing, increa sed nose cleaning, excessive licking, 26 frequent sneezing, and nasal mucosa hemorrhages. S ignificant decreases were seen in the zinc 27 content of lungs after either 5 or 10 ppm formaldeh yde exposure (Table 4-25). Copper content 28 was unchanged from controls in all treatment regime ns, whereas iron content was increased after 29 4 weeks of 5 ppm exposure and after 13 weeks of eit her 5 or 10 ppm formaldehyde exposure 30 (Özen et al., 2003a). 31

32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-158 Table 4-24. Percent body weight gain and concentra tions of iron, zinc, and 1 copper in cerebral cortex of male Wistar rats expos ed to formaldehyde via 2 inhalation for 4 and 13 weeks 3 4 Exposure (mg/m 3 ) Weight gain (%) a Zinc (mg/kg) a Copper (mg/kg) a Iron (mg/kg) a 4-week data 0 20.11 ± 2.87 120 ± 6.03 4.60 ± 0.42 25.07 ± 2.83 6.1 7.27 ± 1.49 e 130 ± 7.26 c 5.60 ± 0.50 b 23.00 ± 2.32 12.2 5.24 ± 1.52 e 185 ± 10.36 e 5.80 ± 0.60 d 22.14 ± 1.95 b

13-week data 0 60.53 ± 7.84 123 ± 6.22 4.67 ± 0.38 24.92 ± 2.84 6.1 38.41 ± 2.53 e 155 ± 7.94 e 5.41 ± 0.56 c 22.00 ± 2.41 12.2 25.87 ± 1.32 e 163 ± 6.03 e 6.10 ± 0.73 e 21.00 ± 1.96 b 5

a Values are means ± SDs (n = 7). 6 7 Statistical significance of differences versus cont rols, as calculated by the authors: 8

b p < 0.05. c

p < 0.02. d

p < 0.002. e

p < 0.001. 9 10 Source: Özen et al. (2003b). 11 12 13 Table 4-25. Zinc, copper, and iron content of lung tissue from formaldehyde14 treated male Wistar rats 15 16

Concentration Duration a Zinc b,c Copper b,c Iron b,c

0 ppm Control 20.7 (1.6) 0.39 (0.05) 12.5 (0.8) 5 ppm 4 weeks 16.1 (1.3) d 0.32 (0.07) 12.9 (1.0) 10 ppm 4 weeks 13.8 (1.2) e 0.36 (0.04) 17.5 (1.3) e

0 ppm Control 20.0 (1.6) 0.39 (0.05) 12.7 (0.4) 5 ppm 13 weeks 15.3 (1.4) e 0.37 (0.04) 17.9 (1.1) e 10 ppm 13 weeks 13.0 (1.1) e 0.39 (0.05) 22.4 (1.4) e 17

a Rats were exposed 8 hours/day, 5 days/week for the number of weeks indicated. 18

b Concentrations are expressed as moles/mg of tissue, wet basis. 19

c Values are means (n = 7); SDs shown in parentheses. 20

d p < 0.005, compared with controls, as calculated by a uthors. 21

e p < 0.001, compared with controls, as calculated by t he authors. 22 23 Source: Özen et al. (2003a). 24 25 There are two reports of lung cytochrome P450 level s after formaldehyde exposure. The 26 first report by Dallas et al. (1989) describes conc entration- and duration-dependent changes in P450 27 levels. Male Sprague-Dawley rats were exposed at 0 , 0.5, 3.0, or 15 ppm (0, 0.62, 3.7, or 18.5 28 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for 1 day, 4 days, 12 weeks, or 24 weeks. There 29 were six rats in each exposure group, but the exper iment was run in two parts, with three rats in 30 each subgroup. Rats were sacrificed after 1 day, 4 days, 12 weeks, or 24 weeks of exposure, and 31 liver microsomes were prepared. TP and P450 conten t were determined on each sample. 32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-159 Average P450 levels in control groups ranged from 1 7–76 pmol P450/mg protein. 1 However, no P450 was detected in lung from formalde hyde-treated animals after 1 day of 2 exposure, with a method detection limit of approxim ately 10 pmol P450/mg protein. In contrast, 3 P450 levels were elevated significantly above contr ols in a concentration-dependent manner after 4 4 days of formaldehyde exposure (Table 4-26). Alth ough P450 levels remained elevated in some 5 experimental groups after 12 and 24 weeks of exposu re, results were variable and less dramatic. 6

7 Table 4-26. Total lung cytochrome P450 measurement s of control and 8 formaldehyde-treated male Sprague-Dawley rats 9 10 1 Day a,b 4 Days 12 Weeks 24 Weeks Formaldehyde Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 0 ppm 17 (6) 44 (13) 39 (11) 23 (3) 29 (10) 19 (2 3) 76 (49) 18 (11) 0.5 ppm ND ND 103 (52) 137 (14) e 87 (11) d 35 (7) 172 (12) c 38 (9) 3.0 ppm ND ND 357 (10) e 278 (100) e 91 (10) d 67 (34) 92 (103) 30 (15) 15 ppm ND ND 362 (38) e 334 (4) e 130 (2) e 56 (6) 151 (9) 48 (7) c 11

a Rats were exposed 6 hours/day, 5 days/week for the duration shown. 12

b Cytochrome P450 expressed as pmol P450/mg of protei n. Values are means (SDs) (n = 3). 13

c Different from control, p < 0.05. 14

d Different from control, p < 0.01. 15

e Different from control, p < 0.001, as calculated by the authors. 16 ND = not detected above the limit of detection, app roximately 10 pmol/mg protein. 17 18 Source: Dallas et al. (1989). 19 20 21 A later study by Dinsdale et al. (1993) attempted t o confirm the increase in P450 levels 22 reported by Dallas et al. (1989). In their first e xperiment, Dinsdale et al. (1993) treated male 23 Sprague-Dawley rats at approximately 10 ppm (12.3 m g/m 3 ) formaldehyde 6 hours/day for 24 4 days. The formaldehyde vapor was generated from formalin by a concentric jet atomizer. For 25 the second experiment, Dinsdale et al. (1993) simil arly exposed rats to formaldehyde, but the gas 26 was generated by the thermal depolymerization of pa raformaldehyde as was done by Dallas et al. 27 (1989). The concentration of P450 and activity of several P450 isozymes were measured in lung 28 microsomes (pentoxyresorufin O-dealkylase, benzylox yresorufin O-dealkylase, ethoxyresorufin 29 O-dealkylase, and 2-aminofluorene N-hydroxylation). ALP and γ -glutamyl transpeptidase 30 activity were measured in BAL fluid collected from each animal. No changes were seen in BAL 31 enzyme activity or the activity of lung microsomes for the P450 substrates tested. Cytochrome 32 P450 levels were unchanged in experiment 1, where f ormaldehyde was generated from formalin. 33 Cytochrome P450 levels were increased in experiment 2 with formaldehyde generated from 34 paraformaldehyde (Table 4-27). 35

36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-160 Table 4-27. Cytochrome P450 levels in formaldehyde -treated rats 1 2 Experiment 1 (formalin) a Experiment 2

(paraformaldehyde) a Group (nmol/mg protein) Control 82 ± 30 85 ± 5 10 ppm formaldehyde 73 ± 27 125 ± 23 b 3

a Values are means ± SDs (n = 3–5). 4

b Different from controls, p < 0.05. 5 6 Source: Dinsdale et al. (1993). 7 8 4.2.1.2.2.6. Extrapulmonary toxicity: subchronic studies. Several studies have investigated 9 toxicity in organs other than those associated with the respiratory tract. An earlier cross-species 10 study examined changes in lung tissue resulting fro m continuous exposure (Coon et al., 1970). 11 Animals were exposed to 3.7 ppm (4.6 mg/m 3 ) formaldehyde for 90 days. Five species of 12 animals were studied: male and female Sprague-Dawle y and Long-Evans derived rats (15), male 13 and female Princeton-derived guinea pigs (15), male New Zealand albino rabbits (3), male 14 squirrel monkeys ( Saimiri sciureus ) (3), and purebred male beagle dogs (2). Blood sa mples were 15 taken for Hb concentration, HCT, leukocyte counts, and serum levels of BUN, AST, ALT, ALP, 16 and LDH. Sections of heart, lung, liver, kidney, a nd spleen were fixed and examined from each 17 species (details of method not provided). Brain, s pinal cord, and adrenal tissue also were 18 examined in monkeys and dogs as well as thyroid fro m dogs. Liver and kidney sections were 19

stained for reduced nicotinamide adenine dinucleoti de, lactate, isocitrate, and β -hydroxybutyrate. 20

Tissue sections of the nasal mucosa were not examin ed in this study. 21 Hematological parameters were unaffected by formald ehyde treatment. The lung tissue 22 of all species exhibited interstitial inflammation after 90 days of formaldehyde exposure 23 (detailed description not provided). Formaldehyde- treated rats and guinea pigs also had focal 24 chronic inflammation in heart and kidney tissue sec tions. However, the authors were uncertain 25 whether the observed changes to heart and kidney we re due to formaldehyde exposure. 26 As mentioned above, Woutersen et al. (1987) exposed male and female albino SPF 27 Wistar rats (10/group) to 0, 1, 10, or 20 ppm (0, 1 .23, 12.3, or 24.6 mg/m 3 ) formaldehyde 28 6 hours/day, 5 days/week for 13 weeks. Rats were c hecked daily and weighed weekly. During 29 week 13, blood samples were taken for Hb, PCV, RBC count, and a differential count of 30 leukocytes. Urine samples were also analyzed. At sacrifice, blood samples were analyzed for 31 ALB, creatinine, glucose, TP, BUN, and the enzyme a ctivities (AST, ALT, and ALP). GSH and 32 protein content were determined in liver homogenate s. Organs were examined and weighed: 33 adrenals, brain, heart, kidneys, liver, lungs, ovar ies, pituitary, spleen, testes, thymus, and thyroid

. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-161 No gross pathological changes were seen upon autops y, but body weights decreased in 1 both male and female rats at the 20 ppm treatment l evel. Of the organs weighed, 6 of 11 had 2 significantly increased relative rates in male rats exposed to 20 ppm formaldehyde. Relative 3 brain weight was increased in female rats at the sa me treatment level (Woutersen et al., 1987). 4 Clinical chemistry parameters of liver and kidney f unction and hematological parameters 5 were also measured after the 13-week treatment by W outersen et al. (1987). Compared with 6 those of controls, activities of AST, ALT, and ALP were significantly elevated in plasma from 7 the 20 ppm treated male rats (by 124, 132, and 126% , respectively; p < 0.05). Total plasma 8 protein was reduced to 95% of controls in the same animals. Although there was an observed 9 increase in BUN in male rats treated with 1 ppm, th is was not considered a treatment effect. 10 Furthermore, no statistically significant differenc es were seen for these parameters in female rats 11 at any concentration level (Woutersen et al., 1987)

. 12 Sul et al. (2007) exposed Sprague-Dawley rats to 0, 5, and 10 ppm formaldehyde for 13 6 hours/day (5 days/week) for 2 weeks and collected lung samples for tissue damage and 14 genomic analysis. According to their results, 21 g enes were altered in a dose-dependent manner 15 by microarray analysis; 2 were up regulated and 19 were down regulated in the lung tissue of 16 animals exposed to formaldehyde. However, six of t he nine genes further analyzed by PCR did 17 not show dose dependency (authors did not comment). Although the authors briefly describe the 18 functions and potential implications for changes in the expression of some of the altered genes, 19 there is no discussion of the relationship between these altered genes (i.e., there is no pathway 20 analysis). 21 In 2006, Im et al. (2006) published a proteomic an alysis using the same exposure 22 protocols (possibly using the same animals as in th e Sul et al. [2007] study, although neither 23 study makes reference to the other). Im et al. (20 06) examined DNA damage in lymphocytes 24 and liver tissues, as well as protein and lipid oxi dation in plasma and liver samples. Similar to 25 changes reported in the lung (discussed elsewhere), using two-dimensional electrophoresis and 26 matrix-assisted laser desorption ionization time-of -flight mass spectrometry, the authors also 27 reported dose-dependent changes in the levels of 32 proteins in plasma (19 up, 13 down). None 28 of the changes in plasma proteins correspond to the changes in lung reported by Sul et al. (2007). 29 Again, no pathway analysis was provided. Interesti ngly, Im and colleagues (2006) also 30 demonstrated a dose-dependent increase in plasma IL -4 and dose-dependent decrease in IFN γ , 31 perhaps indicative of Th-2-mediated inflammatory re sponse. An overview of formaldehyde 32 exposure-related pathology in the respiratory syste m of laboratory animals is presented in 33

Table 4-28. 34This document is a draft for review purposes only a nd does not constitute Agency policy 4-162 DRAFT—DO NOT CITE OR QUOTETable 4-28. Summary of respiratory tract pathologyfrom inhalation exposures to formaldehyde, subchronic studiesSpecies/strainNo./group TreatmentaRespiratory effects LOAEL/NOAEL ReferenceNasal pathologyB6C3F1 mice(male andfemale)10 0, 2, 4, 10, 20, or 40 ppmformaldehyde 6 hours/day,5 days/week for 13 weeksMinimal squamous metaplasia in 1 of 10 mice(4 ppm). Squamous metaplasia observed in all miceat 10 and 20 ppm.NOAEL = 4 ppm Maronpot et al.(1986)SPF Wistar Rats(male andfemale)10 0, 1, 10, or 20 ppmformaldehyde 6 hours/day, 5days/week for 13 weeksIncreased respiratory epithelial hyperplasia andkeratinization at 20 ppm; squamous metaplasia at10 ppm in males and females.NOAEL = 1 ppm Woutersen et al.(1987)SPR Wister rats(male)50–55 0, 10, or 20 ppm formaldehydefor 6 hours/day, 5 days/weekfor 4, 8, or 13 weeksRhinitis, hyperplasia, and squamous metaplasia inrespiratory epithelium at all doses (number of weeksnot specified).Squamous metaplasia of olfactory epithelium at20 ppm (number of weeks not specified)NOAEL = 1 ppm Feron et al. (1988)Wistar rats (maleand female)50 0, 0.3, 1, or 3.0 ppmformaldehyde 6 hours/day,5 days/week for 13 weeksKeratinized and non-keratinized squamousmetaplasia in level 2 epithelium in 37/50 male and21/50 female rats at 3 ppm for 13 weeks.NOAEL = 1 ppm Zwart et al. (1988)Wistar rats (male) 10 40 ppm-hours (8 hours at5 ppm, 4 hours at 10 ppm) or80 ppm hours (10 ppmcontinuous or 20 ppmintermittently)Thinning and disarrangement of respiratoryepithelium, squamous metaplasia, most severe in20 hours intermittent exposureNOAEL = 10 ppm Wilmer et al.(1987)Wistar rats (male) 10 0, 8, or 16 ppm, given eithercontinuously or intermittentlyDisarrangement and squamous metaplasia at 4 ppm.Continuous exposure yielded less severe lesionsthan intermittent exposureLOAEL = 8 ppm Wilmer et al.(1989)F344 rats (maleand female),Syrian goldenhamsters (maleand female),cynomolgusmonkeys20 rats,10hamsters,6 monkeys0.0.2, 1, or 3 ppm22 hours/day, 7 days/week,26 weeksRats: rhinitis at 3 ppm, increased incidence of nasallesions at 3 ppm.Monkeys: rhinitis at all doses, increased incidenceof nasal lesions at 3 ppm.Hamsters: no significant nasal lesions.NOAEL = 1 ppm Rusch et al.(1983a, b)Tracheal and lung pathologyWistar rats (male) 6 0, 5, 15 ppm for 8 hours/day,5 days/week, 4 or 13 weeksSignificant decreases in zinc content in lung, copperunchanged, iron increased in lung.LOAEL = 5 ppm Özen et al. (2003)This document is a draft for review purposes only a nd does not constitute Agency policy 4-163 DRAFT—DO NOT CITE OR QUOTETable 4-28. Summary of respiratory tract pathologyfrom inhalation exposures to formaldehyde, subchronic studiesSpecies/strainNo./group TreatmentaRespiratory effects LOAEL/NOAEL ReferenceSprague-Dawleyrats (male)6 but n = 5in sometrials0, 0.5, 3.0, 15 ppm 6hours/day, 5 days/week, for 1day, 4 days, 12 weeks, 24weeksIncreased P450 levels after 4 days at 3 ppm. NOAEL= 0.5 ppm Dallas et al.(1989)Sprague-Dawleyrats (male)5 0 or 10 ppm 6 hours/day,4 days using both formalin andparaformaldehydeP450 levels increased at 10 ppm only in groupstreated with paraformaldehyde.LOAEL = 10 ppm Dinsdale et al.(1993)Extrapulmonary effectsRats and guineapigs15 3.7 ppm for 90 days Focal chronic inflammation in heart and kidneytissue.LOAEL = 3.7 ppm Coon et al. (1970)SPF Wistar rats(male andfemale)10 0, 1, 10, or 20 ppm6 hours/day, 5 days/week for13 weeksRelative brain weight increased in female rats at20 ppm; increased AST, ALT, ALP in plasma at20 ppm.NOAEL = 10 ppm Woutersen et al.(1987)This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-164 4.2.1.2.3. Chronic inhalation bioassays. The respiratory pathology observed in chronic 1 bioassays is consistent with the subchronic studies . As exposure concentration and duration of 2 exposure are increased, the pathology becomes more severe and penetrates more deeply into the 3 respiratory tract. These effects are progressive o ver time. Tumors are reported in several 4 bioassays, primarily SCCs. Experimental results re garding both the severity of respiratory tract 5 pathology as well as the tumor incidence vary by sp ecies strain and experimental design. As 6 discussed above rodents experience RB, and species differences in respiratory and physiological 7 depression would result in differences in absorbed dose in the respiratory tract, given the same 8 exposure concentration (Chang and Barrow, 1983). A dditionally, differences in the nasal 9 architecture result in species-dependent variation of formaldehyde absorption (flux) within the 10 respiratory tract (see Section 3.4). Therefore, ch ronic studies are discussed by species for greater 11 clarity. 12

13

4.2.1.2.3.1. Mice. Early experiments by Horton et al. (1963) subjected mice (C3H, sex 14 unspecified) to extreme formaldehyde concentrations (0, 0.05, 0.1, and 0.2 mg/L or 41–163 ppm) 15 in an attempt to simulate lung pathology reported i n humans exposed to cigarette smoke. The 16 mice were exposed 1 hour/day, 3 days a week for up to 35 weeks. The authors did not note the 17 effects of RB or provide any information on patholo gy of the URT. There was a clear increase 18 in histologic changes in the tracheobronchial epith elium by exposure, including basal-cell 19 hyperplasia, stratification squamous cell metaplasi a and atypical metaplasia. Subsequent 20 exposures to various combinations of formaldehyde a nd coal tar did result in squamous cell 21 tumors. The findings of Horton et al. (1963) sugge st a role for formaldehyde in lung cancer 22 under some conditions. However, the exposure desig n and early deaths in the treatment groups 23 severely limit the usefulness of these data in huma n health risk assessment. 24 In a comprehensive study conducted by Swenberg et al. (1980) (also reported in Kerns et 25 al. [1983]) in conjunction with Chemical Industry I nstitute of Toxicology (CIIT) and Battelle 26 Columbus Laboratories, male and female C57BL/6 × C3 H F 1 (B6C3F 1 ) mice (approximately 27 120/sex/concentration) were exposed to 0, 2.0, 5.6, or 14.3 ppm (0, 2.45, 6.87, or 17.5 mg/m 3 ) 28 formaldehyde 6 hours/day, 5 days/week for 24 months . This exposure period was followed by 29 up to 6 months of nonexposure to evaluate recovery. Interim sacrifices were conducted at 6, 12, 30 18, 24, 27, and 30 months (due to unscheduled death s, no male mice were sacrificed at 18 or 31 27 months). Exposure generation was accomplished b y sublimation of paraformaldehyde, and 32 exposures were conducted in whole-body chambers. D etailed sectioning and examination of the 33 nasal passages were conducted at each interim sacri fice, beginning at 12 months, and for all 34 unscheduled deaths. Gross organ pathology was note d for all animals and complete 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-165 histopathologic examination was conducted on all an imals in the control and high-exposure 1 groups. There were no differences in survival in a ny exposure group compared with controls. 2 Generally, poor survival in all groups of male mice was attributed to fighting and infections of 3 the urogenital tract associated with group housing; 78, 77, 81, and 82 unscheduled deaths were 4 recorded before 24 months in the 0, 2.0, 5.6, and 1 4.3 ppm treatment groups, respectively ( 5 n = 119, 120, 120, and 119 males, respectively). A fter the interim sacrifices (6 and 12 months) 6 only 17–22 male mice survived to the 24-month sched uled sacrifice. Female mice had much 7 greater survival with only 30, 34, 19, and 34 unsch eduled deaths prior to the 24-month sacrifice. 8 The authors did not note the effects of RB in mice, although the RD 50 for a 10-minute exposure 9 for male B6C3F1 mice has been reported at 4.9 ppm a nd 4.4 ppm (Steinhagen and Barrow, 1984; 10 Chang et al., 1981). 11

The first examination of the nasal cavities was co nducted at the 12-month interim 12

sacrifice. Inflammation in the nasal turbinates wa s evident in mice in the 2 and 6 ppm treatment 13 groups (14/20 and 18/20, respectively), including a denitis of the nasal lacrimal duct, lacrimal 14 duct, and vomeronasal gland. Inflammation was not present in mice exposed at 15 ppm, 15 although serous rhinitis was seen in 4 of 20 animal s. At 18 months, mice exposed at 2 and 16 6 ppm no longer exhibited adenitis in the nasoturbi nates. Epithelial dysplasia was evident in 4 of 17 20 mice at 6 ppm exposure. Mice in the high-exposu re group had significantly greater nasal 18 pathology; epithelial dysplasia and squamous metapl asia were reported in 18/19 and 17/19 19 female mice, respectively, exposed to 15 ppm. Afte r 24 months, squamous epithelial hyperplasia 20 of the nasolacrimal duct (29/45) and atrophy of the olfactory epithelium (18/45) were also noted 21 in animals from the high-exposure group (male and f emale) (Battelle Columbus Laboratories, 22 1981). Similar pathology was reported in only a sm all fraction of mice exposed at 2 and 6 ppm 23 (5/48 and 11/60, respectively). 24 Three months after cessation of exposure, only ni ne female mice were available for 25 sacrifice, but within this small sample the data su ggested recovery of nasal lesions: epithelial 26 dysplasia (4/9), squamous metaplasia (2/9), atrophy of the olfactory epithelium (1/9), and 27 squamous epithelial hyperplasia of the nasolacrimal duct (1/9) (Battelle Columbus Laboratories, 28 1981). 29

Of the 17 male mice that survived to 24 months in the 14.3 ppm exposure group, 2 had 30 SCC in the nasal cavity ( p < 0.05). Of the two tumor-bearing mice, one exhibi ted significant 31 epithelial pathology, including rhinitis, dysplasia , squamous metaplasia, and hyperplasia. 32 Squamous metaplasia of the nasolacrimal duct was th e only related pathology reported for the 33 second mouse. No SCCs were found in female mice, a lthough 48 mice survived to 24 months. 34 The authors reported no other formaldehyde-related tumors. However, comparisons were based 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-166 on summary tables by organ. Although lymphomas wer e analyzed by organ and site (e.g., 1 increase in salivary gland lymphoma considered sepa rately from mandibular lymphoma), later 2 reanalysis of lymphoma in female mice, based on tum or-bearing animals (TBAs), does indicate 3 an association with formaldehyde exposure. 4

5

4.2.1.2.3.2. Rats. Holmström et al. (1989a) evaluated co-exposure of i nhaled formaldehyde 6 with wood dust in 16 female Sprague-Dawley rats/gro up. Rats were exposed in whole-body 7 chambers for 6 hours/day, 5 days/week for 104 weeks to formaldehyde alone at 12.4 ± 1.1 ppm 8 (15.21 ± 1.35 mg/m 3 ), wood dust alone (25 mg/m 3 ), or both wood dust (25 mg/m 3 ) and 9 formaldehyde (12.7 ± 1.0 ppm) or to room air as the control. The wood dust was generated from 10 grinding of beech. Microscopic measurements of the wood particles indicated that 11 approximately 70% had a geometric diameter of about 10 μ m, while 10–20% were about 5 μ m or 12

less. Animals were sacrificed at 104 weeks and his topathology was performed on five transverse 13 sections of the nasal cavity (Figure 4-3) and the l ungs (not otherwise specified). 14 There were no differences in mortality among the gr oups at any time during the study 15 period. Rats exposed to formaldehyde were reported to have exhibited yellow discoloration of 16 the fur, and many displayed eye irritation. Formal dehyde exposure, with and without wood dust, 17 induced squamous metaplasia, keratinization, and dy splasia of the nasal epithelium (Table 4-29). 18

19 Table 4-29. Histopathologic findings and severity scores in the naso- and 20 maxilloturbinates of female Sprague-Dawley rats exp osed to inhaled 21 formaldehyde and wood dust for 104 weeks 22 23

Treatment Pronounced

squamous metaplasia Pronounced

squamous metaplasia with keratinization Pronounced

squamous metaplasia with presence of dysplasia Sum of rats

with pronounced metaplasia and/or dysplasia CCSCC Histologic scores at the level of naso- and maxilloturbinates (mean ± SD) Formaldehyde group (n = 16) 7 2 1 10 1 2.25 ± 1.73 aFormaldehydewood dust group (n = 15) 7 1 4 12 0 2.6 ± 1.88 a Wood dust group (n = 15) 0 0 0 0 0 1.86 ± 0.83 b Control group (n = 15) 0 0 0 0 0 1.07 ± 0.70 24

a p < 0.01. 25

b p < 0.05. 26 27 Source: Holmström et al. (1989a). 28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-167 Among the five levels of the nasal cavity that were examined, Holmström et al. (1989a) 1 presented findings for the naso- and maxilloturbina tes since formaldehyde-induced tumors had 2 been associated with this level (Morgan et al., 198 6a, b). The data also suggested an effect of 3 wood dust on formaldehyde-induced nasal pathology, with a slightly higher histologic score and 4 greater incidence of dysplasia than formaldehyde ex posure alone. One SCC (1/16) occurred in 5 the group exposed to formaldehyde only but not in t he group exposed to formaldehyde and wood 6 dust. Microscopic examination of the lungs reveale d that emphysema (diagnostic criteria not 7 specified) was more prevalent in both groups expose d to wood dust compared with the control 8 group (

p < 0.05). There was no significant difference in pu lmonary epithelial histopathology 9 among the groups. 10 Tobe et al. (1985) also evaluated F344 rats (32/gro up) exposed to inhaled formaldehyde 11 for 28 months. Exposures were for 6 hours/day, 5 d ays/week to formaldehyde concentrations of 12 0, 0.3, 2, and 14 ppm (0, 0.37, 2.45, and 17.2 mg/m 3 ). Fourteen of 32 rats (44%) in the high 13 concentration group developed nasal SCCs, compared with none in the other exposed groups and 14 the control group. Tobe et al. (1985) reported inc reased rhinitis, hyperplasia, and squamous 15 metaplasia of the nasal respiratory epithelium, inc luding in the low-exposure group (0.3 ppm.) 16 However, some level of rhinitis, hyperplasia, and m etaplasia were also present in controls. 17 Without a more complete report, it is unknown wheth er or not the pathology reported at 0.3 ppm 18 was a formaldehyde-related effect. 19 Kamata et al. (1997) evaluated the effects of inhal ed formaldehyde in male F344 20 (F344/DuCrj) rats (32/group) exposed for 28 months. Formaldehyde exposure was generated by 21 metering 37% formalin (containing 10% methanol) int o a sprayer in a glass bottle and diluting 22

with room air. Concentration in the chamber was mo nitored twice daily by the acetyl acetone 23 method. Exposures were for 6 hours/day, 5 days/wee k at nominal formaldehyde concentrations 24 of 0, 0.3, 2.0, and 15 ppm (0, 0.37, 2.45, and 18.4 mg/m 3 ). Actual levels were 0, 0.3 ± 0.07, 2.17 25 ± 0.32, and 14.85 ± 2.22 ppm (mean ± SD). Rats in the 0 ppm group were given methanol to 26 inhale at the same concentration (4.2 ppm) as the 1 5 ppm group. A room control no-exposure 27 group was also included in the study. All animals were observed for clinical signs once a day 28 during the study. Body weights and food consumptio n were recorded weekly. Five animals per 29 group, randomly selected at the end of 12, 18, and 24 months, and all surviving animals at 30 28 months were sacrificed for hematological measure ments (Hb, RBCs, PCV, MCV, mean 31 corpuscular hemoglobin [MCH], MCHC, and WBCs), bioc hemical determinations (TP, ALB, 32 BUN, ALP, AST, ALT, glucose, albumin/globulin ratio , phospholipids, triglycerides, and total 33 cholesterol), and pathological examinations. Wet w eights were taken on brain, heart, lungs, 34 liver, kidneys, spleen, testes, and adrenal gland o f each rat. Histopathology was performed on all 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-168 moribund or dead animals and those at specified sac rifices on all gross lesions and the following 1 tissues: pituitary, thyroid, nasal cavity, trachea, esophagus, stomach, small and large intestines, 2 prostate gland, urinary bladder, muscle, femur, sci atic nerve, spinal cord, and mesenteric lymph 3 nodes. Histopathologic sections of the nose were o btained from five anatomical levels, but these 4 did not correspond to the typical levels taken in o ther bioassays. Most notably, section level B 5 was anterior and not posterior to the incisor teeth . The incidence data for nasal histopathology 6 were not reported with respect to section level loc ation, with the exception that the 7 nonproliferative lesions and tumors reported were d escribed to occur predominantly at levels B 8

and C. 9

Yellow discoloration of the coats occurred in anima ls exposed at the 2 and 15 ppm levels. 10 Significant decreases in body weight and food consu mption were observed in the high 11 concentration (15 ppm) group throughout the exposur e period, and elevated mortality was noted 12 at 28 months (88.3 versus 31.8% in controls). The first death occurred after 6 versus 18 months 13 in the control group. Other effects noted in the 1 5 ppm exposure group include decreased 14 triglycerides, reduced liver weight (both relative and absolute), and increased relative adrenal 15 weights. 16 Treatment-related macroscopic and histopathologic f indings were limited to the nasal 17 cavity. Squamous cell metaplasia was reported in a ll treatment groups: 16% (0.3 ppm), 37.5% 18 (2 ppm), and 91% (15 ppm) of exposed rats. Epithel ial hyperplasia was similarly present in 12.5, 19 22, and 91% of the animals, respectively. Since a no-effect level could not be determined, the 20 authors reported benchmark doses (BMDs) of 0.25 and 0.24 ppm for squamous cell metaplasia 21 and epithelial hyperplasia (10% response.). Additi onal lesions only occurring in the 15 ppm 22 dose group were papillary hyperplasia (2/32), SCC ( 13/32), squamous cell papilloma (3/32), and 23 sarcoma (1/32). The majority of the tumors were lo cated at levels B and C of the nasal cavity. 24 Albert et al. (1982) and Sellakumar et al. (1985) r eported on a set of lifetime studies 25 performed in male Sprague-Dawley rats to evaluate t he effects of inhaled formaldehyde alone 26 and in combination with hydrochloric acid (HCl). R ats were exposed 6 hours/day, 5 days/week 27 for life. In the first experiment (Albert et al., 1982), 8-week-old male inbred Sprague-Dawley 28 rats (n = 99) were exposed to a mixture of 10 ppm ( 12.3 mg/m 3 ) HCl and 14 ppm (17.2 mg/m 3 ) 29 formaldehyde, and there were two control groups: ai r-sham and untreated (n = 50). 30 Bis(chloromethyl)ether (BCME), a known animal carci nogen (Albert et al., 1975; Kuschner et 31 al., 1975; Figueroa et al., 1973; Laskin et al., 19 71), is formed when formaldehyde and HCl are 32

mixed. BCME concentrations were estimated at about 1 ppb in the formaldehyde-HCl mixed 33 exposures, based on levels in the mixing chamber. Complete necropsies were conducted when 34 animals died naturally or were killed when moribund . Histologic sections were taken from the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-169 nasal cavity, larynx, trachea, pulmonary lobes, liv er, bladder, kidney, spleen, and other organs 1 with gross pathologic alterations. 2 Exposure to the mixed gases (formaldehyde-HCl-BCME) induced nasal lesions, 3 including epithelial hyperplasia (71%), squamous me taplasia (64%), squamous papilloma (3%), 4 and SCC (25%) (Albert et al., 1982). Although a fe w squamous metaplasia were noted in the 5 larynx, trachea, and bronchi, these lesions were al so noted in controls. Mortality in exposed rats 6 was significantly increased over controls and was a pproximately 30% when the first carcinoma 7 was reported (233 days). Mortality in exposed rats rose quickly to approximately 60% after the 8 first year of exposure. Therefore, the authors use d a life-table method to calculate a mortality9 corrected cumulative incidence, reporting a correct ed cumulative incidence of 77% at 720 days 10 after first exposure. 11 In the second experiment performed in this laborato ry (Sellakumar et al., 1985; Albert et 12 al., 1982), Sprague-Dawley rats were similarly expo sed to HCl (10 ppm) alone, formaldehyde 13 alone (15 ppm), or a combination of both. The comb ination exposure was generated in two 14 different ways to better understand the influence o f BCME formation on study results: premixed 15 at high concentrations and gases fed separately int o the inlet air supply at the target 16 concentrations. BCME concentration measured by a g as chromatography/mass spectrometry 17 method in the premixed chamber varied between 0.1 a nd 0.4 ppb. Cage-side observations and 18 necropsy procedures were as described in Albert et al. (1982) with the exception of the histologic 19 preparation of the head. The head was cut transver sely into four tissue blocks, and sections were 20

taken from the face of each. 21 Animals exposed to formaldehyde alone and formaldeh yde-HCl (premixed or non22 premixed) showed a marked decrease in body weight a fter 16 weeks. After 32 weeks rats 23 exposed to the premixed formaldehyde-HCl (with BCME ) had higher mortality compared with 24 the other mixed gas exposures ( p < 0.05). Nasal pathology was similar among rats e xposed to 25 formaldehyde alone or the mixed gases (Table 4-30). Desquamation of respiratory epithelial 26 cells was reported in the respiratory epithelium th at covers the nasomaxillary turbinates and the 27 nasal septum (approximately section levels 2 and 3) . Olfactory epithelium in the ET frequently 28 showed an inflammatory reaction with seropurulent e xudate filling the lumen. Squamous 29 metaplasia and hyperplasia were reported in the lar ynx and trachea in all treatment groups. 30 Tumors arose primarily from the nasomaxillary turbi nates and nasal septum. The SCCs 31 were predominantly moderate to well differentiated, with excessive amounts of keratin occluding 32 the lumen, killing the animals by asphyxiation. St atistical comparisons by the log rank test (Peto 33 test) showed that tumor incidence was increased in the premixed formaldehyde-HCl combined 34 exposure group over formaldehyde alone or the combi ned formaldehyde-HCl (not premixed). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-170 There were no significant differences in the latenc y among groups, with the average latency 1 varying from 603 to 645 days. Rats exposed to HCl exposure alone did not develop tumors. 2 The esthesioneuroepithelioma is a unique tumor type observed with a high incidence in 3 an earlier inhalation study of rats exposed to BCME (Kuschner et al., 1975), suggesting that the 4 higher incidence of nasal tumors observed in the pr emixed-combined formaldehyde-HCl5 exposure group may have been due to BCME (Krimsky, 1986) since this premixed protocol was 6 the one most likely to generate BCME. Sellakumar e t al. (1985) refuted this assertion, stating 7 that this singular tumor occurred in the absence of other changes in the ethmoid region or in the 8 lungs where BCME was also demonstrated to cause tum ors. Furthermore, exposure was 9 approximately one-tenth the cumulative dose in the Kuschner et al. (1975) study that was 10 associated with a single similar tumor. Sellakumar et al. (1985) attributed the higher incidence 11 in the premixed-combination group to traces of othe r alkylating agents (not BCME) that could 12

have been formed. The results demonstrate that ani mals exposed to either a combination of 13 formaldehyde-HCl or to formaldehyde alone develop n asal tumors, principally SCCs, at about 14 the same frequency, indicating that HCl plays littl e or no role in the carcinogenicity of inhaled 15 formaldehyde. 16

17 Table 4-30. Histopathologic changes (including tum ors) in nasal cavities of 18 male Sprague-Dawley rats exposed to inhaled formald ehyde or HCl alone 19 and in combination for a lifetime 20 21

Observation Premixed HCl-HCHO Non-premixed

HCl-HCHO HCHO HCl Air Colony Number of animals examined 100 100 100 99 99 99 Rhinitis 74 75 74 81 72 70 Epithelial or squamous hyperplasia 54 53 57 62 51 4 5 Squamous metaplasia 64 68 60 9 5 6 Polyp or papilloma 13 11 10 0 0 0 SCC 45 27 38 0 0 0 Adenocarcinoma 1 2 0 0 0 0 Mixed carcinoma 0 0 1 0 0 0 Fibrosarcoma 1 0 1 0 0 0 Esthesioneuroepithelioma 1 0 0 0 0 0 Larynx Hyperplasia 11 22 21 22 2 2 Squamous metaplasia 10 15 4 0 0 0 Trachea Hyperplasia 18 32 21 26 6 2 Squamous metaplasia 9 8 7 0 0 0 22 Source: Sellakumar et al. (1985). 23 24 25This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-171 In a companion study to the chronic mouse study des cribed above (Kerns et al., 1983; 1 Swenberg et al., 1980), groups of F344 rats (approx imately 120/sex/concentration) were exposed 2 to 0, 2.0, 5.6, or 14.3 ppm (0, 2.45, 6.87, or 17.5 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week 3 for 24 months. This exposure period was followed b y up to 6 months of nonexposure to evaluate 4 recovery. Interim sacrifices were conducted at 6, 12, 18, 24, 27, and 30 months. Study 5 parameters and methods were as described above. 6 Formaldehyde exposure increased mortality of both m ale and female rats in all treatment 7 groups (

p < 0.05 for 6 and 15 ppm groups). Severe treatment -related mortality was seen at the 8 highest exposure group beginning at 12 months with only 30% surviving to the 24 months. 9 There were no alterations in clinical chemistry, ne urofunctional, or ophthalmological 10 measurements considered to be related to formaldehy de exposure. A concentration-dependent 11 increase in yellow discoloration of the hair coat w as observed. This discoloration dissipated over 12 the 3-month postexposure period. Rats in the highe st-concentration group were dyspneic 13 ( p < 0.01) and emaciated ( p < 0.05) and had many facial swellings that on close r examination 14 were revealed to be carcinomas protruding through t he nasal cavity. Neoplastic lesions in the 15 URT were first observed clinically at day 358 in fe males and day 432 in males. 16 Macroscopically, these lesions originated in the an terior portion of the nasal cavity and, in a few 17 instances, extended into the ETs. 18 Figure 4-9 shows the frequency of squamous metaplas ia by location in the noses of rats 19 sacrificed at various time points along the 2-year exposure period. Histopathologic lesions were 20 confined to the nasal cavity and proximal trachea i n concentration-dependent fashion. The 21 morphologic diagnosis of squamous metaplasia was us ed to designate zones of altered 22 epithelium that were characterized by the presence of a well-differentiated germinal layer 23 (stratum germinativum) and superficial layers of ep ithelium (stratum spinosum and stratum 24 corneum). Keratin was produced only in areas of sq uamous metaplasia. Epithelial dysplasia was 25 detected earlier than squamous metaplasia and was c haracterized by a mucosa that had 26 undergone a transition from nonciliated simple cubo idal to one that was several cells thick and 27 squamoid with an organization and polarity of the i ndividual cells that had changed from vertical 28 to horizontal with respect to the basement membrane . Similar histomorphologic changes have 29 also been called basal cell hyperplasia and epiderm oid metaplasia (e.g., Albert et al. [1982]). 30 Figure 4-9 clearly illustrates that concentration i s a dominant determinant of lesion distribution. 31 At low concentrations the lesions occur only in the most anterior region (cross-section level 1). 32 At 5.6 ppm, the squamous metaplasia in levels 1, 2, and 3 was also associated with purulent 33 rhinitis and epithelial dysplasia. At the highest concentration, the lesions progress to the more 34 distal URT, with lesions evident in level 5 and no difference in the incidence at level 1 or 2 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-172 across the various sacrifice times. Statistically significant ( p < 0.05) regression of the lesion was 1

evident at most locations at the 27-month sacrifice (3 months postexposure) (e.g., level 1 in the 2 2 ppm group, all levels of the 5.6 ppm group, and l evels 4 and 5 of the 14.3 ppm group). 3

4

6 12 18 24 27 Months Months Months Months Months 100

80

60

40

20

100

80

60

40

20

100

80

60

40

20

Location of squamous metaplasia in nasal cavity

Level I Level II Level III Level IV Level V 

## A

B

C

5 Figure 4-9. Frequency and location by cross-sectio n level of squamous 6 metaplasia in the nasal cavity of F344 rats exposed to formaldehyde via 7 inhalation. 8 9 Note: Exposure concentrations were 2.0 ppm (A), 5.6 ppm (B), or 14.3 ppm (C). 10 Nasal cavity levels 2, 3, 4, and 5 were not evaluat ed at the 6- and 12-month 11 interim sacrifices in the 14.3 ppm exposure group. 12 13 Source: Redrawn from Kerns et al. (1983). 14 15This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-173 Furthermore, progression of lesions distally to the lower respiratory tract (LRT) occurred 1 only in the high concentration group. Tracheal pat hology observed at 18 months included 2 multifocal areas of minimal to mild epithelial hype rplasia, epithelial dysplasia, or squamous 3 metaplasia. There were no significant tracheal les ions present in the 0, 2.0, or 5.6 ppm exposure 4 groups, and tracheal lesions were not observed duri ng the postexposure period in the 14.3 ppm 5

exposure group. 6 Table 4-31 provides the summary data of all neoplas tic lesions in the nasal cavity of 7 exposed rats. The adjusted cumulative incidence ra tes of SCC in male and female rats from the 8 14.3 ppm exposure group at 24 months were 67 and 87 %, respectively. In this group, the 9 formation of zones of squamous metaplasia with zone s of squamous epithelial hyperplasia and 10 increased keratin production appeared to precede ar eas of squamous papillary hyperplasia with 11 foci of cellular atypia. More advanced lesions inc luded carcinoma in situ and invasive SCC of 12 the nasal turbinates. The neoplasia were extremely osteolytic and were associated with excessive 13 keratin production and mild to severe purulent rhin itis. In many animals from the high-exposure 14 group (with or without carcinoma), the excessive ac cumulation of keratin and inflammatory 15

exudates within the lumen of the URT caused severe dyspnea and death. Polypoid adenomas 16 were also observed in eight rats (four/sex) from th e low-exposure group, six male rats from the 17 intermediate-exposure group, and six rats (five mal es, one female) from the high-exposure group 18 in level 1, 2, or 3. One control male rat had a si milar lesion. When adjusted and unadjusted data 19 were analyzed, no significant differences were obse rved in pair-wise analyses; however, a 20 significant adjusted trend ( p < 0.05) was reported for male rats. There was no e vidence of 21 progression from polypoid adenoma to SCC. 22

23 Table 4-31. Summary of neoplastic lesions in the n asal cavity of F344 rats 24 exposed to inhaled formaldehyde for 2 years 25 26

Formaldehyde (ppm) Sex No. of nasal

cavities evaluated SCC Nasal carcinoma Undifferentiated

carcinoma or

sarcoma Carcinosarcoma Polypoid

adenoma Osteochondroma M 118 0 0 0 0 1 1 0 F 114 0 0 0 0 0 0

M 118 0 0 0 0 4 0 2.0 F 118 0 0 0 0 4 0

M 119 1 0 0 0 6 0 5.6 F 116 1 0 0 0 0 0

M 117 51 1 a 2 a 1 4 0 14.3 F 115 52 1 0 0 1 0 27

a One rat in this group also had an SCC. 28 29 Source: Kerns et al. (1983). 30 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-174 Morgan et al. (1986b) performed an additional analy sis of the slides and tissues 1 from the Kerns et al. (1983) study to more precisel y determine the location of each tumor 2

recorded. Additional sections were cut from the ex isting tissue blocks if a full slide set 3 (i.e., five sections) was unavailable for each anim al. For each animal, the location of 4 each tumor was recorded on diagrams of the cross se ction of the nose, and an attempt to 5 determine the site of origin was made based on the center of the tumor mass. The results 6 for each case were assigned an accuracy rating that was based on the degree of 7 confidence that the pathologist had in the designat ed site of origin. Results for SCCs are 8

shown in Table 4-32. 9

10 Table 4-32. Apparent sites of origin for the SCCs in the nasal cavity of F344 11 rats exposed to 14.3 ppm of formaldehyde gas in the Kerns et al. (1983) 12 bioassay 13 14 Total SCC (%) a

Sex Accuracy

rating Number of

animals Area 1 b Area 2 b Area 3 b Area 4 b Unable to determine Male High

Low 36

25 56

56 28

20 14

8 3

0 NA

16 Female High

Low 45

15 62

47 27

33 7

13 4

0 NA

7

Totals 121 57 26 10 3 4 15

a Rounded to nearest whole number. 16

b Area 1 = lateral aspect of the nasoturbinate and ad jacent lateral wall; Area 2 = midventral septum; Ar ea 3 = dorsal 17 septum and roof of dorsal meatus; Area 4 = dorsal a nd lateral aspect of the maxilloturbinate. 18 NA = not applicable. 19 20 Source: Morgan et al. (1986b). 21 22 23 In the 14.3 ppm exposure group, 98/103 rat noses ha d adequate numbers and quality of 24 slides for mapping the SCC distribution. Single ne oplasia were present in 80 (40/sex), while 25 multiple neoplasia were present in 9 males (21 neop lasia) and 9 females (20 neoplasia). The 26 results were similar for cases with high or low acc uracy. For example, more than half (57%) of 27

the SCCs occurred on the lateral side of the nasotu rbinate and adjacent lateral wall at the front of 28 the nose (levels 1 and 2); approximately 25% were l ocated on the midventral nasal septum 29 (levels 2 and 3); and about 10% were on the dorsal septum and roof of the dorsal meatus 30 (levels 1, 2, and 3). A small number (3%) were fou nd on the maxilloturbinate (levels 2 and 3), 31 which only involved the medial aspect. All other r egions of the nose where SCC was found were 32

considered to be involved as a result of invasion f rom one or more of the above sites. There 33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-175 were two tumors in the 5.6 ppm group: one male had a single neoplasm on the ventral nasal 1 septum (level 3) while a female had an SCC from the lateral aspect of the maxilloturbinate to the 2 adjacent lateral wall (level 2). 3 On the basis of the morphology of 19 small neoplasi a in this study and in additional work 4 described below (Morgan, 1997; Monticello et al., 1 996), it was further concluded that the SCCs 5 arose from the epithelium lining the airway and not from the underlying glandular epithelium. 6 This mapping procedure and that of Monticello et al . (1996) described below were in good 7 concordance and showed a clear site specificity; mo st of the SCC arose in the anterior lateral 8 meatus (ALM) (57%), which is lined by transitional epithelium, and the midventral nasal septum 9 (26%), which is lined by respiratory epithelium (Mo rgan, 1997). 10 The CIIT performed a second bioassay on inhaled for maldehyde in 9-week-old male 11 F344 (CDF[F344]/CrlBr) rats (Monticello et al., 199 6). The rats were exposed 6 hours/day, 5 12 days/week for 24 months to 0, 0.7, 2, 6, 10, and 15 ppm (0, 0.86, 2.45, 7.36, 12.3, and 13 18.4 mg/m 3 ) formaldehyde. Study objectives were to repeat th e Kerns et al. (1983) bioassay, 14 better defining the concentration response relation ship and to seek a correlation between 15

localized data on tumor sites and concomitant cell proliferation assays. Histopathology was 16 performed on six cross-section levels of the nasal cavity on every animal of an unscheduled 17

death and all those of the terminal sacrifice after 24 months. The distribution of lesions for each 18 individual animal was recorded onto epithelial maps of the nasal cavity at 30 selected levels 19 designed to permit accurate localization (Mery et a l., 1994). Cell proliferation was measured in a 20 subset of animals (five per treatment group) at 3, 6, 18, and 24 months of exposure in each of the 21 nasal regions to which tumors were mapped (Table 4- 33). 22

23 Table 4-33. Incidence and location of nasal squamo us cell carcinoma in male 24 F344 rats exposed to inhaled formaldehyde for 2 yea rs 25 26

Nasal location

Formaldehyde concentration (ppm) No. of

nasal cavities examined Anterior

lateral meatus Posterior

lateral meatus Anterior

midseptum Posterior

midseptum Anterior

dorsal septum Anterior

medialmaxilloturbinate Maxillary

sinus No. of

animals with SCC a

0 90 0 0 0 0 0 0 0 0 0.7 90 0 0 0 0 0 0 0 0 2 96 0 0 0 0 0 0 0 0 6 90 1 0 0 0 0 0 0 1 10 90 12 2 0 0 0 0 0 20 15 147 17 9 8 1 3 4 0 69 27

a Total number of animals with SCCs, including those too large to allocate and those located in a site n ot listed in this 28 table. 29 Source: Monticello et al. (1996). 30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-176 Yellow discoloration of the fur, a consistent respo nse to formaldehyde in rats, was 1 observed in the rats exposed to 10 and 15 ppm forma ldehyde. There were numerous premature 2 deaths in the 15 ppm exposure group, resulting in s ignificantly decreased survival relative to 3 controls (18.8 versus 35.7%; p < 0.001). Survival was higher in the three lowest exposure 4 groups and statistically comparable to controls in the 10 ppm exposure group (35.7 versus 5 31.3%, respectively). 6 Control animals showed no histopathologic evidence of disease in the nasal passages. 7 Buccal cavity SCC, not associated with the nasal ca vity, was present in 2 of 90 control animals. 8 This was considered an incidental finding and withi n the spontaneous incidence range reported 9

for this strain of rat. Buccal SCCs were observed in three animals at 15 ppm and in one animal 10 at 2 ppm. All other neoplastic responses in the re spiratory tract were confined to the nose and 11 considered to have originated from the epithelium l ining the nasal airways. The nasal neoplasia 12 included SCCs and polypoid (transitional) adenomas and were similar in morphologic 13

characteristics to those described in the Kerns et al. (1983) chronic bioassay. The incidence of 14 nasal SCCs by location is summarized in Table 4-33, which demonstrates a clear concentration15 response relationship. No SCCs occurred in the two lowest exposure groups or in the controls. 16 One nasal rhabdomyosarcoma and two nasal adenocarci nomas were reported in animals in the 17 highest treatment groups. 18 Regional analysis indicated that the SCCs arose in nasal regions lined with transitional or 19 respiratory epithelium and were most common in the lateral meatus and the midseptum (Table 20 4-33). Within the lateral meatus and mid-septum, t here was clear evidence of a higher tumor 21 incidence rate in the anterior sample site ( p = 0.001 and 0.02, respectively). Smaller numbers o f 22 SCCs were observed on the medial aspect of the maxi lloturbinate and the dorsal septum and on 23 the posterior lateral wall and lining of the nasoph aryngeal meatus (data not shown). No SCCs 24 were observed in the maxillary sinus, with the exce ption of one animal exposed to 15 ppm that 25

had a small tumor in the wall of the ostium of this sinus. Tumor rates across the seven nasal 26 epithelial sites are presented in Table 4-33. Ther e was an increasing tumor response between the 27 10 and 15 ppm exposure groups in all sites, except in the ALM. The SCC rates at 10 and 15 ppm 28 were virtually identical (13.3 and 11.6%, respectiv ely), which is probably attributable to the 29 occurrence of many large neoplasia in the lateral m eatus site that were not suitable and not 30 counted in the analysis. 31 The nonlinear tumor response is mirrored by a highl y nonlinear response in cell 32 proliferation measured after 3, 6, 12, and 18 month s of exposure. Significant treatment-induced 33 responses in cell proliferation indices at these ti me points were only observed at the two highest 34 exposure concentrations (10 and 15 ppm). Other tre atment-induced lesions, predominantly 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-177 epithelial hypertrophy, hyperplasia, squamous metap lasia, and mixed inflammatory cell 1 infiltrate, were also most severe at these two expo sure concentrations. Significant distortion and 2

destruction of the nasoturbinate architecture occur red in many animals exposed to 15 ppm. 3 Nasal turbinate adhesions and olfactory degeneratio n (usually confined to the walls of the 4 anterior dorsal medial meatus) also occurred in ani mals exposed to 10 and 15 ppm. Lesions in 5 the 6 ppm exposure group were limited to focal squa mous metaplasia in the anterior regions. 6 As discussed briefly above, small numbers of polypo id adenomas were also induced by 7 formaldehyde exposure and were similar in acinar-li ke structure and location to those in the 8 Kerns et al. (1983) bioassay. No polypoid adenomas occurred in the control animals or in the 9 0.7, 2, or 6 ppm exposure groups. A clear concentr ation response was observed in the 10 and 10 15 ppm exposure groups. Five of 90 animals (5.6%) in the 10 ppm exposure group and 14 of 11 147 animals (9.5%) in the 15 ppm exposure group had a polypoid adenoma. Most of these 12 polypoid adenomas (79%) were located in or adjacent to the lateral meatus. The significance of 13

these tumors for risk assessment remains to be dete rmined (Morgan, 1997). 14 Appelman et al. (1988) studied the effects of bilat eral intranasal electrocoagulation 15 damage on susceptibility to inhaled formaldehyde in male SPF Wistar (Cpb: WU) rats. Rats 16 were exposed 6 hours/day, 5 days/week for 13 or 52 weeks to 0, 0.1, 1.0, or 10 ppm (0, 0.12, 17 1.23, or 12.3 mg/m 3 ) formaldehyde. These concentrations were chosen b ecause the various 18 short-term studies performed in the same laboratory (described in Section 4.2.1.2) showed that 19 formaldehyde was noncytotoxic to the nasal mucosa a t levels of 0.3, 1.0, and 2.0 ppm, slightly 20 cytotoxic at 3 and 4 ppm, and strongly cytotoxic at 10 and 20 ppm (Zwart et al., 1988; Wilmer et 21 al., 1987; Woutersen et al., 1987). Furthermore, b ecause nasal tumors have only been found at 22 exposure concentrations that also induced severe de generative, hyperplastic, and metaplastic 23 changes in the nasal epithelium (Griesemer et al., 1985; Squire and Cameron, 1984), Feron et al. 24 (1984) and the investigators at the TNO-CIVO Toxico logy and Nutrition Institute postulated that 25 formaldehyde at a subcytotoxic concentration was on ly a very weak initiator without promoting 26 activity. Appelman et al. (1988) used an electroco agulation method in this study to evaluate if 27 damage to the mucosa followed by compensatory cell proliferation might render the epithelium 28 vulnerable to subcytotoxic levels of formaldehyde. One-half of the rats used in the study 29 (10/group) were damaged bilaterally and then subjec ted to the first 6-hour exposure to 30 formaldehyde approximately 20–26 hours after the el ectrocoagulation procedure. Ten 31 undamaged rats/group were also exposed at each conc entration for either 13 or 52 weeks. 32 Histopathologic examination included six standard c ross-section levels in the nose; livers of all 33 rats killed at 14 weeks and of all control and 10 p pm exposed rats killed in week 53; larynges, 34 tracheas, and lungs of all rats of the control and 10 ppm exposed rats killed in week 53; and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-178 organs and tissues of control and 10 ppm exposed ra ts with an undamaged nasal mucosa killed in 1

week 53. 2

Yellow discoloration of the fur occurred in all ani mals of the two highest exposure 3 groups. Growth retardation was observed in the ani mals killed with or without damaged noses 4 after 2 weeks of exposure to 10 ppm formaldehyde. No toxicologically significant findings in 5 the body weights or organ weights of any animals in the other exposure groups were observed. 6 No relevant differences between groups were found i n any of the hematological or urinary 7 parameters with the exception of frequent oliguria ( p < 0.05) in the top exposure group without 8 nasal coagulation and killed in week 53. Three-way ANOVA revealed a significant increase in 9 TP content of the liver in rats with damaged noses as compared with rats with undamaged noses, 10 and there was a significant negative correlation be tween the formaldehyde exposure level and TP 11 in these same rats. Hepatic GSH was positively cor related with both nasal damage and age of 12 the animals. No treatment-related gross findings w ere observed in animals sacrificed at either 14 13 or 53 weeks except for yellow discoloration of the fur in rats exposed at the two highest 14 concentrations. No changes observed in the larynx, trachea, lungs, liver, or other tissues 15 evaluated were regarded as related to formaldehyde. 16 Few nasal lesions were noted in intact rats exposed at 0.1 or 1 ppm for either 13 or 52 17 weeks (n = 10/group). Focal squamous metaplasia wa s noted in a single animal exposed at 18 1 ppm for 13 weeks. Rats exposed at 10 ppm formald ehyde demonstrated clear pathology in the 19 respiratory epithelium progressing from 13 to 52 we eks, including squamous metaplasia, basal 20 cell hyperplasia, and focal rhinitis. Additionally , focal nest-like infolds of the epithelium were 21 present in 4 of 10 rats at 52 weeks, and minor chan ges to the olfactory epithelium were noted 22 (thinning/disarrangement and focal basal cell hyper plasia.) 23 All rats with damaged nasal passages exhibited simi lar minor pathology of the respiratory 24 epithelium at 13 and 52 weeks (squamous metaplasia, focal basal cell hyperplasia, and focal 25 rhinitis). Formaldehyde-related effects were note at 52 weeks, where the squamous metaplasia 26 of the respiratory epithelium was no longer noted i n controls (versus 13 weeks) but was clearly 27 present in all formaldehyde-treatment groups, inclu ding progression from focal to diffuse lesions 28 (at 1 and 10 ppm) and keratinization (3/10 and 4/10 at 0.1 ppm and 10 ppm, respectively). The 29 formaldehyde effects on the respiratory epithelium were much more severe in rats with damaged 30 nasal passages, with all animals demonstrating thin ning and disarrangement of the olfactory 31 epithelium and 8 of 10 rats exhibiting “loosely arr anged submucosal tissue.” Squamous 32 metaplasia and focal rhinitis of the olfactory epit helium were seen in less than half of the 33 formaldehyde-treated rats with damage. No changes in the olfactory epithelium due only to 34 electrocoagulation were encountered. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-179 The most notable effects of nasal damage from elect rocoagulation were the ones at the 1 highest formaldehyde exposure (10 ppm) on the olfac tory epithelium. Damage to the respiratory 2 epithelium also occurred more posteriorly in rats w ith damaged noses. Since electrocoagulation 3 often induced damage that included partial or compl ete loss of turbinates and septal perforation, 4 a likely explanation for the posterior distribution of the damage is an abnormal airflow pattern. 5 This gross damage to the nasal structure may have a lso disrupted normal mucous production and 6 flow. Therefore, formaldehyde-induced pathology ap pearing deeper in the nasal passages, 7 including the respiratory epithelium, may be due to formaldehyde penetrating more deeply into 8 the nasal passages and resulting in greater tissue doses in these areas. 9 Woutersen et al. (1989) conducted a lifetime study in parallel to the 1-year study 10 described above for Appelman et al. (1988). Male W istar rats with nasal damage induced by 11 electrocoagulation (60/group) or without nasal dama ge (30/group) were exposed 6 hours/day, 12 5 days/week to the same concentrations as in the pr evious study (0, 0.1, 1.0, and 10 ppm) for 13 28 months or for 3 months followed by a 25-month ob servation period. The general condition 14 and behavior of the animals were checked daily. Bo dy weight, organ weight, and gross 15 pathology were evaluated as described for Appelman et al. (1988). Histopathologic examination 16

was conducted on all animals at the standard six cr oss sections (see Figure 4-3). 17 No remarkable findings on behavior were observed ex cept for yellowing of the fur in 18 animals at the two highest concentrations. There w ere no relevant differences in mortality (data 19 not shown). Growth retardation was observed relati ve to controls in animals with or without 20 damaged noses exposed to 10 ppm from day 14 onward. Body weights were generally slightly 21 lower in formaldehyde-exposed animals with an intac t nasal mucosa and slightly higher in 22 exposed animals with damaged noses than in the corr esponding controls. 23 The effects of formaldehyde exposure on the respira tory and olfactory epithelium after 24 28 months of exposure were similar to those reporte d for 52 weeks exposure (Appelman et al., 25 1988): rhinitis, squamous metaplasia with some kera tinization of the respiratory epithelium, and 26 thinning/disarrangement and slight squamous metapla sia of the olfactory epithelium at the 27 10 ppm exposure. Effects attenuated from the anter ior to posterior sections (I–II, III, IV, and V– 28 VI). A low incidence of olfactory epithelium repla ced by respiratory epithelium (<10%) and 29 vacuolation and atrophy of olfactory cells (<10%) w as reported, this in part may be due to the 30 larger study size (30 rats per group versus 10). S quamous metaplasia in levels I–II of the 31 respiratory epithelium at 10 ppm was the only treat ment-related pathology remaining in rats 32 exposed for 3 months followed by a 25-month recover y period. 33 Similarly, as reported by Appelman et al. (1988), r ats with noses damaged by 34 electrocoagulation did demonstrate increased pathol ogy of the respiratory epithelium. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-180 Formaldehyde exposure at 10 ppm exacerbated these c hanges, and effects were noted in more 1 posterior sections than in rats without nasal damag e (levels III, IV, and V). Olfactory pathology 2 was also greater in formaldehyde-treated rats: basa l cell hyperplasia, replacement of olfactory 3 epithelium by respiratory epithelium (10–20% at lev el III and <10% at level IV). Although the 4 incidences are low, there is some evidence that eff ects on the olfactory epithelium may be 5 increased at the lower formaldehyde exposures (0.1 and 1 ppm.) Analysis of the number of 6 animals with olfactory pathology would be helpful t o better understand the potential of low-level 7 formaldehyde effects on these less frequent lesions . Interestingly, the recovery of the olfactory 8 and respiratory epithelium seen in rats with undama ged nasal cavities after a 25-month recovery 9 period was not evident in rats with damaged noses. Formaldehyde-exposure effects are only 10 present at the 10 ppm exposure for the respiratory epithelium (squamous metaplasia, basal cell 11 hyperplasia), and the formaldehyde-related effects on the olfactory epithelium 12 (thinning/disarrangement, basal cell hyperplasia, a nd replacement by respiratory epithelium) are 13 seen at 0.1 and 1.0 ppm as well. 14 A single SCC, 1 out of 30 rats, was found in each 2 8-month formaldehyde-treatment 15 group (1/26, 1/28, and 1/26, respectively) but not in any control animals (n = 52). SCCs were 16 also noted in rats with noses damaged by electrocoa gulation (1/54, 1/58, 0/56, and 15/58 for 17 control rats and the formaldehyde-treatment groups, respectively). These data clearly indicate a 18 synergistic effect of high formaldehyde exposure an d nasal damage on the formation of SCCs in 19 rats. One adenosquamous carcinoma and one adenocar cinoma were also reported as increasing 20 the frequency to 17/58 for all tumors. Additionall y SCC was present in two rats in the 0.1 and 21 1 ppm 3-month exposure groups with damaged noses on ly, although only one SCC was reported 22 in the 10 ppm 3-month groups with and without damag ed noses. Rats not surviving to 23 28 months are included in these results, as well as the histopathology reported above. Since no 24 mortality data are reported, it should be noted tha t the incidence of both nasal lesions and tumors 25 are not controlled for early deaths. 26 In total, 30 tumors were examined from this study. In general, the tumors (26/30 or 87%) 27 were SCCs, and 69% (18/26) of these clearly origina ted from the respiratory epithelium lining 28 the septum or nasal turbinates. The eight other SC Cs, derived from the epithelium lining the 29 nasolacrimal duct, were seen in connection with sev ere odontodystrophy and periodontitis or 30 might have originated from the skin or salivary gla nds. Four remaining rats bearing a nasal 31 tumor developed a small polypoid adenoma located on the nasoturbinate, an adenocarcinoma 32 originating from the olfactory epithelium, an adeno squamous carcinoma of the respiratory 33 epithelium lining the septum or turbinates, or a ca rcinoma in situ of epithelium in the 34

nasolacrimal duct. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-181 4.2.1.2.3.3. Hamsters . Dalbey (1982) examined the effects of inhaled fo rmaldehyde alone for a 1 lifetime or combined with diethylnitrosamine (DEN) in an initiation-promotion study design 2 using male Syrian golden hamsters. For the first e xperiment, hamsters were exposed at either 0 3 or 10 ppm (0 or 12.3 mg/m 3 ) formaldehyde in whole body chambers 5 hours/day, 5 days/week 4 for a lifetime (132 controls, 88 exposed). Histopa thologic evaluations were carried out on two 5 transverse sections of the nasal turbinates (otherw ise not specified), longitudinal sections of 6 larynx and trachea, and all lung lobes cut along th e bronchus prior to embedding. In the 7 formaldehyde-only (10 ppm) experiment, mortality wa s increased relative to unexposed controls 8 ( p < 0.05). No tumors and little evidence of toxicit y to the nasal epithelium were observed. 9 There was no increase in rhinitis. Epithelial hype rplasia and metaplasia were increased in 10 formaldehyde-treated animals (5% incidence) versus none observed in controls. 11 The second set of experiments by Dalbey (1982) exam ined interaction of formaldehyde 12 exposure on tumor formation from DEN administered s ubcutaneously. The five treatment 13 groups included: (1) controls (n = 50); (2) formald ehyde only (n = 50); (3) DEN 0.5 mg, once 14 per week for 10 weeks (n = 100); (4) formaldehyde e xposure for life with DEN injection for the 15 first 10 weeks given 48 hours after formaldehyde ex posure (n = 27); and (5) DEN injection for 16 10 weeks, followed by formaldehyde exposure for lif e (n = 23). In all groups hamsters were 17 exposed at 30 ppm formaldehyde 5 hours/day, once a week. Histopathologic examinations were 18

conducted as above. 19 Although weekly exposures to formaldehyde alone (30 ppm once a week) did not 20 influence mortality, treatment with DEN alone signi ficantly ( p < 0.05) increased mortality above 21 that of untreated controls, and mortality was furth er elevated ( p < 0.05) in the two groups 22 exposed to both DEN and formaldehyde compared with DEN alone. No respiratory tract tumors 23

were observed in untreated animals or those receivi ng only formaldehyde. DEN treatment alone 24 resulted in a high incidence (77%) of tumors (nasal , larynx, trachea, and lung). Formaldehyde 25 pre- or posttreatment did not further increase the number of TBAs-. All tumors observed were 26 classified as adenomas. Formaldehyde pretreatment nearly doubled the number of tumors per 27 animal in the trachea (but not lung or larynx) ( p < 0.05). This increase in tumors initiated by 28 DEN given 48 hours after formaldehyde exposure sugg ests a role of formaldehyde- induced 29 changes in the respiratory tract in tumor promotion (e.g., cell proliferation and inflammation). 30

31

4.2.1.2.3.4. Summary

. Chronic rodent studies of inhalation exposure to fo rmaldehyde provide a 32 consistent picture of the agent’s toxicity—especial ly on the URT—on which most studies focus. 33 All three species tested—hamsters, mice, and rats—h ad some degree of hyperplastic and 34 metaplastic change in the nasal passages. The path ology defined in acute and subchronic 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-182 exposures is similarly described in chronic studies , where progression, severity, and presence in 1 more posterior sections of the nose increase with b oth the concentration and duration of 2

exposure. 3 Pathology of the respiratory epithelium includes r hinitis, goblet cell hyperplasia, 4 pseudoepithelial cell hyperplasia, squamous metapla sia, and dysplasia (see Table 4-34). At 5 higher exposures and longer durations of exposure, similar effects are seen on the olfactory 6 epithelium, present further into the nasal passages . In addition to hyperplasia and squamous 7 metaplasia, thinning and disarrangement of the olfa ctory epithelium noted and, in a few cases, 8 cell damage and replacement of olfactory epithelium with respiratory epithelium appear, 9 including loss of sensory cells (Woutersen et al., 1989; Kerns et al., 1983; Battelle Columbus 10 Laboratories, 1981). 11 Clear species differences in the severity of lesio ns are present. Although the bioassays in 12 mice, hamsters, and rats do represent similar expos ure concentrations and duration of exposure, 13 hamsters exhibit little pathology and rats (three s trains tested) exhibit gross toxicity and even 14 increased mortality. Mice similarly exposed exhibi t a range of effects on the respiratory 15 epithelium but not near the severity seen in rats. Many factors may contribute to these observed 16 species differences. As Chang and Barrow (1983) re ported, the increased RB of mice seems to 17 be protective of POE damage in comparison to that o f rats. The reduced ventilation rate and 18 minute volume of rodents in the presence of a react ive gas can reduce the effective delivered 19 dose at the same exposure concentration (Chang and Barrow, 1983). Additionally, as illustrated 20 in the computational fluid dynamic (CFD) modeling ( see Section 3.5), there are species 21

differences in nasal architecture that influence ar eas of formaldehyde absorption or flux into the 22 tissue. Localized differences in mucus flow and pr oduction as well as metabolic enzymes have 23 also been posited as having roles in differential t oxicity of formaldehyde on the URT (see 24 Chapter 3). 25 Formaldehyde-induced tumors were present in expose d rats and mice and primarily 26 involved SCCs later in life (Kamata et al., 1997; T obe et al., 1985; Kerns et al., 1983; Swenberg 27 et al., 1980). Although exposure of male Syrian ha msters to either 10 or 30 ppm did not result in 28 formaldehyde-induced nasal tumors, a classic initia tion-promotion assay with DEN-induced 29 tumor formation did indicate that formaldehyde incr eased the tumor burden per animal, where 30 DEN induced tumors in 77% of the animals (Dalbey, 1 982). This study suggests a role for 31 promotion in the observed carcinogenicity of formal dehyde. Less clear are the implications of 32 the synergistic effect of formaldehyde exposures an d gross damage to the respiratory epithelium 33 by electrocoagulation on tumor formation (Woutersen et al., 1989). 34

35This document is a draft for review purposes only a nd does not constitute Agency policy 4-183 DRAFT—DO NOT CITE OR QUOTETable 4-34. Summary of respiratory tract pathologyfrom chronic inhalation exposures to formaldehydeSpecies/strainNo./group TreatmentaRespiratory effectsNoncancerLOAEL/NOAEL ReferenceChronic bioassaysMiceC3H mice (sexunstated)60 0, 41, 82, or 163 ppm1 hour/day, 3 days/week for upto 35 weeks.Low- and mid-group mice thenexposed at either 122 or244 ppm during weeks 35–70.Pathology:Histologic changes in the tracheobronchialepithelium by exposure, including basal-cellhyperplasia, stratification squamous cell metaplasia, andatypical metaplasia.Carcinogenicity:No SCC formation was evident inmice exposed to formaldehyde alone.LOAEL = 41 ppmNo evidence ofcarcinogenicityHorton et al. (1963)Male and femaleB6C3F1 mice120/sex 0, 2, 5.6, or 14.3 ppm6 hours/day, 5 days/week for 24months.The protocol featured a 6-monthrecovery period. Interimsacrifices occurred at 6, 12, 18,24, and 30 months.Pathology:Rhinitis; hyperplasia, dysplasia, andsquamous metaplasia of the nasal epithelium; atrophy ofthe olfactory epithelium; glandular adenitis andnasolacrimal duct hyperplasia and metaplasia.Carcinogenicity:Nasal SCC in male mice at 24 months(2/17). No SCC in female mice.LOAEL = 2 ppmEvidence ofcarcinogenicitySwenberg et al.(1980); Kerns et al.(1983); CIIT(1982) ; BattelleColumbusLaboratories (1981)RatsFemale SpragueDawley rats16 0 or 12.4 ppmformaldehyde ± wood dust6 hours/day, 5 days/week for104 weeks.Pathology:Squamous metaplasia and dysplasia.Carcinogenicity:One of 16 rats exposed toformaldehyde alone developed SCCs.LOAEL = 12.4 ppmSupport forcarcinogenicityHolmström et al.(1989a)Male and femaleF344 rats32/sex 0, 0.3, 2, or 14 ppm6 hours/day, 5 days/week for28 months.Pathology:Increased rhinitis, hyperplasia, andsquamous metaplasia of the nasal respiratory epitheliumCarcinogenicity:Nasal SCCs in high concentrationrats (44%).LOAEL = 0.3 ppmSupport forcarcinogenicityTobe et al. (1985)Male F344 rats 32 0, 0.3, 2, or 15 ppm6 hours/day, 5 days/week for28 months.Pathology:Squamous cell metaplasia and epithelialhyperplasia.Carcinogenicity:SCC (13/32), squamous cellpapilloma (3/32), and sarcoma (1/32).LOAEL = 0.3 ppmBMD10= 0.24 ppmEvidence ofcarcinogenicityKamata et al. (1997)This document is a draft for review purposes only a nd does not constitute Agency policy 4-184 DRAFT—DO NOT CITE OR QUOTETable 4-34. Summary of respiratory tract pathologyfrom chronic inhalation exposures to formaldehydeSpecies/strainNo./group TreatmentaRespiratory effectsNoncancerLOAEL/NOAEL ReferenceMale SpragueDawley rats100 0 or 15 ppm 6 hours/day,5 days/week for life.Pathology:Squamous metaplasia, epithelialhyperplasia, and polyps/papillomas.Carcinogenicity:SCCs formed in the nasomaxillaryturbinates and nasal septum (25%).LOAEL = 15 ppmEvidence ofcarcinogenicityAlbert et al. (1982);Sellakumar et al.(1985)Male and femaleF344 rats120/sex 0, 2, 5.6, or 14.3 ppm6 hours/day, 5 days/week for24 months. The protocolfeatured a 6-month recoveryperiod. Interim sacrificesoccurred at 6, 12, 18, 24, and30 months.Pathology:Lesions of the nasal cavity were theprimary effects, including squamous metaplasia andepithelial dysplasia, hyperkeratosis, goblet cellhyperplasia, and rhinitis.Salivary gland: atrophy, squamous metaplasia, andsialadenitis.Carcinogenicity:SCCs were evident in the nasalcavity of high concentration rats, plus some polypoidadenomas.LOAEL = 2 ppmEvidence ofcarcinogenicitySwenberg et al.(1980); Kerns et al.(1983); CIIT (1982);Battelle ColumbusLaboratories (1981);Morgan et al.(1986b)Male F344 rats 90 and 150controls0, 0.7, 2, 6, 10, or 15 ppm6 hours/day, 5 days/week for24 months.Pathology:Olfactory degeneration, squamousmetaplasia, epithelial hypertrophy and hyperplasia,andmixed inflammatory cell infiltrate.Carcinogenicity:SCCs and polypoid adenomas in thenasal cavityLOAEL = 2 ppmEvidence ofcarcinogenicityMonticello et al.(1996)Male SPF Wistarrats10 0, 0.1, 1, or 10 ppm6 hours/day, 5 days/week for 13or 52 weeks.An electrocoagulation methodwas applied to damage thenoses of 1⁄2 of each study group.Pathology:Formaldehyde-induced focal changes to therespiratory and olfactory epithelium, including rhinitis,hyperplasia, and metaplasia (10 ppm).In rats with damaged noses: squamous metaplasia oftherespiratory epithelium increased at all formaldehydeexposures. Pathology of the olfactory epitheliumincreased at the 10 ppm exposure.Carcinogenicity:No tumors noted; 1-year studyLOAEL = 0.1 ppmin rats with damagednasal passagesNOAEL = 1 ppm forrats with intact nosesAppelman et al.(1988)This document is a draft for review purposes only a nd does not constitute Agency policy 4-185 DRAFT—DO NOT CITE OR QUOTETable 4-34. Summary of respiratory tract pathologyfrom chronic inhalation exposures to formaldehydeSpecies/strainNo./group TreatmentaRespiratory effectsNoncancerLOAEL/NOAEL ReferenceMale Wistar rats 30 (withoutnasaldamage),60 (withnasaldamage)0, 0.1, 1, and 10 ppm6 hours/day, 5 days/week for 28months or for 3 months with a25-month observation period.An electrocoagulation methodwas applied to damage the nasalcavity.Pathology:Intact noses: squamous metaplasia in thehigh concentration group exposed for 28 months anddegeneration of the olfactory epithelium.Changes were more severe in animals with damagednoses .Carcinogenicity:SCCs developed in 15/60 rats withdamaged noses exposed at 10 ppm. In other groups,theincidence of nasal tumors was low irrespective of thestate of nasal damage.NOAEL = 1 ppmEvidence ofcarcinogenicityWoutersen et al.(1989)HamstersMale Syriangolden hamsters88 treated132controls.0 or 10 ppm formaldehyde5 hours/day, 5 days/week forlife.Pathology:Increased mortality. Epithelial hyperplasiaand metaplasia increased in formaldehyde-treatedanimals (5% incidence)Carcinogenicity:No tumors reported.LOAEL = 10 ppmNo evidence ofcarcinogenicityDalbey (1982)Male Syriangolden hamsters50 0 or 30 ppm5 hours/day, 1 day/week for life± injections with 0.5 mg DEN.Pathology:Increased mortality in conjunction withDEN—above DEN-only treated animals. Respiratorypathology not reported.Carcinogenicity:Only hamsters receiving DENdeveloped tumors (77%, adenomas). There was anincrease in the number of tumors per TBAs in thetrachea of animals exposed to formaldehyde 48 hoursprior to DEN (but no increase in TBAs).LOAEL = 30 ppm.Evidence forformaldehyde as apromoterDalbey (1982)This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-186 4.2.1.2.4. Summary of respiratory pathology. The progressive pathology of the nasal passages 1 from inhalation exposure to formaldehyde is well do cumented, especially in rodents (rats and 2 mice) (see Tables 4-9, 4-15, 4-28, 4-34). Although there are species differences in tissue dose 3 (Section 3.4) due to variations in nasal architectu re and breathing patterns, the nature and 4 progression of the pathology is fairly well conserv ed across species, including nonhuman 5 primates. The observed formaldehyde-induced pathol ogy includes disruption of the mucociliary 6 apparatus, rhinitis ( s erous and purolent), hyperplasia (cell proliferatio n), metaplasia (transition of 7 cell type), dysplasia (disarrangement of cells), ne st-like infolds and invaginations of the 8 epithelium, thinning of the epithelial layer and fo cal to diffuse lesions, atrophy of the olfactory 9 epithelium, thickening and keratinization (usually of squamous metaplasia), tumors (adenoma, 10 sarcoma, carcinoma) (Section 4.2.2). 11 Progression of lesions can be viewed as progression from the anterior to posterior 12 sections of the nasal cavity or as a progression in severity of lesions at a particular location (e.g.

, 13 level or region) of the nasal passages. In both ca ses, progression is evident with increasing 14 exposure concentration and with increasing duration of exposure (Kamata et al., 1997; 15 Monticello et al., 1996; Morgan et al., 1986b; Taka hashi et al., 1986; Sellakumar et al., 1985; 16 Kerns et al., 1983; Albert et al., 1982). The data suggest that concentration and duration of 17 exposure do not act in a simply cumulative manner ( e.g., C × t). Additionally the influence of 18 concentration, duration, and repeated exposure may be different for various effects. For 19 example, some lesions may be transient (e.g., low-e xposure cell proliferation), others may have a 20 threshold and vary little after that (e.g., rhiniti s). Additionally, as the nasal epithelium responds 21 with both adaptive and adverse epithelial changes, the absorption of formaldehyde into the tissue 22 at that location may be reduced. As respiratory ep ithelium transitions to squamous metaplasia, 23 the effective tissue dose of formaldehyde increases posterior to these lesions. As barriers to 24 formaldehyde flux into the tissue develop (e.g., sq uamous metaplasia, keratinization), 25 formaldehyde penetrates more deeply into the nasal passages (Kimbell et al., 2006). Therefore, 26 although both concentration and duration of exposur e do effect the adverse effect, the 27 relationship is difficult to define and in fact may be different for various adverse effects. 28 Respiratory histopathology has been commonly report ed in response to exposure to 29 formaldehyde in rats and mice (Lino dos Santos Fran co et al., 2006; Javden and Taher, 2000; 30 Kamata et al., 1996a, b; Cassee and Feron, 1994; Bh alla et al., 1991; Monteiro-Riviere and Popp, 31 1986; Buckley et al., 1984; Chang et al., 1983), ra bbits (Ionescu et al., 1978), hamsters 32 (Schreibner et al., 1979), and rhesus monkeys (Mont icello et al., 1989). The histopathologic 33 lesions ranged from inflammation to ulceration, nec rosis, and metaplasia that occurred in nasal 34 turbinates, maxilloturbinates, and goblet and micro villus cells (Bhalla et al., 1991). These effects 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-187 were observed at a variety of doses (e.g., 10 ppm f or 4 hours, 3.13 ppm for 6 hours for 1, 2, or 1 4 days, 6 or 15 ppm). Wilmer et al. (1989, 1987) a ssessed whether a dose and time-dependent 2 interaction (C × t) is associated with histopatholo gic lesions. Results indicated that 3 concentration, rather than duration or cumulative e xposure, correlates best with severity of 4 lesions (Wilmer et al., 1989, 1987). 5 Histopathologic lesions and changes to biochemistry have been reported in the lung as 6 well, though these effects were observed following a high dose of formaldehyde. In addition, 7 changes in clinical chemistry, P450 expression and activity in lung tissue, and gene expression 8 that is phenotypically anchored to the observed res piratory pathology have been reported. 9 Extrapulmonary effects have also been noted, includ ing changes in liver chemistry, relative brain 10 weight, and focal, chronic inflammation in the hear t and kidney. Most of these changes occurred 11 at exposures of 20 ppm, and those that occurred at lower formaldehyde exposures (3.7 ppm) 12 could not be strictly correlated with formaldehyde exposure. 13 Some researchers have reported formaldehyde-induced effects in the pulmonary region in 14 rats, mice, and rabbits. Kamata et al. (1996a) obs erved reduced lipid content of pulmonary 15 surfactant in rats exposed to 128.4 or 294.5 ppm fo rmaldehyde. Kamata et al. (1996b) reported 16 biochemical changes in lung homogenates and altered lipid content of BAL at 145.6 ppm 17 formaldehyde. Lino dos Santos Franco et al. (2006) observed increased leukocytes (and 18 neutrophils) and degranulated mast cells recovered in BAL fluid (concentration of 1% formalin 19 not provided). In rabbits, Ionescu et al. (1978) o bserved frank necrosis of lung parenchyma after 20 aerosol inhalation of 3% formalin for 3 hours/day f or 50 days (concentration of formaldehyde 21 not provided). These pulmonary effects may be due to frank toxicity resulting from the high 22 dose of formaldehyde used in these studies. 23 Several recent toxicogenomics studies have assessed gene expression changes in nasal 24 and lung tissue in animals and in humans by using i n vivo and in vitro approaches. Hester et al. 25 (2005, 2003) documented changes in gene expression associated with DNA repair and apoptosis 26 in nasal tissue from male rats after a single insti llation of formaldehyde. Other gene expression 27 changes were observed in those genes related to xen obiotic metabolism and in cell cycle and 28 repair. These preliminary results provide an initi al basis for forming a phenotypically anchored 29 set of gene expression changes associated with expo sure to formaldehyde and may assist in 30 determining the underlying MOA, as will be discusse d in Section 4.5. Sul et al. (2007) 31 investigated gene expression genes in lung tissue f rom formaldehyde-exposed rats. Yang et al. 32 (2005) performed a proteomics analysis by using lun g tissue extracted from formaldehyde33 exposed rats. Two studies used human tracheal cell lines to investigate formaldehyde-induced 34 gene expression changes in vitro (Lee et al., 2008, 2007). However, the relevance of these 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-188 findings to actual exposures remains unknown. In t otal, toxicogenomics studies hold promise, 1 but they must be interpreted with caution until res ults can be replicated and phenotypically 2 linked to observable changes. 3 Thus, formaldehyde-induced respiratory pathology ha s been commonly described in the 4 nasal passages and includes cellular proliferation, mucociliary function, and histopathologic 5 lesions. Pulmonary effects have been documented as well but at high doses. The nasal 6 pathology may occur as a result of both concentrati on and duration components of exposure. 7

8

4.2.1.2.5. Cell proliferation. Formaldehyde-induced cell proliferation has been de monstrated 9 under range of exposure conditions in vivo and in v itro as well (Chapter 3). Formaldehyde10 induced mitogenesis may be a primary effect (as dem onstrated in the in vitro work) or secondary 11 to adaptive responses and tissue remodeling (Swenbe rg et al., 1983). This section provides a 12 comprehensive discussion of formaldehyde effects on cell proliferation in the epithelial tissues in 13 the respiratory tract. The majority of the work di scussed investigates cell proliferation with 14 in vivo labeling of proliferating cells, although a dditional methods, such as flow-cytometry, have 15 been employed in some instances. 16 Swenberg et al. (1986) conducted a series of experi ments in rodents to assess cell 17 proliferation in the nasal mucosa after formaldehyd e inhalation. Radiolabeled thymidine 18 [ 3 H]-thymidine was injected intraperitoneally (I.P.) into male F344 rats and B6C3F1 mice after 19 formaldehyde exposure to assess the extent of in vi vo incorporation into proliferating cells. Two 20 hours later, animals were sacrificed and the nasal passages were fixed, embedded, and sectioned 21 to examine the nasal mucosa. Slides were exposed f or 12 weeks and developed to identify cells 22 that incorporated the radiolabeled thymidine. The percentage of labeled cells, as indicated by the 23 presence of five or more grains over the nucleus, w as determined by visual count. A total of 24 4,000 or 1,500 cells were counted per section for r ats and mice, respectively. 25 The first set of studies reported by Swenberg et al . (1986) compared the dose response of 26 rats and mice. Animals were exposed to 0, 0.5, 2, 6, or 15 ppm (0, 0.61, 2.45, 7.36, or 27 18.4 mg/m 3 ) formaldehyde 6 hours/day for 3 days. Tritiated t hymidine for cell labeling was 28 injected 2 hours after the end of exposure. No cha nge in the percentage of labeled cells was seen 29 after 0.5 or 2 ppm formaldehyde exposure. However, the nasal passages of rats exposed at 6 and 30 15 ppm showed 10- to 20-fold increases over control s in LI at level 2. A similar cell 31 proliferation response was seen in mice treated wit h 15 ppm formaldehyde, although no increase 32 over control was seen in mice exposed to 6 ppm form aldehyde. These findings are consistent 33

with other data that indicate rats are more sensiti ve to formaldehyde exposure than mice. This 34 may be due to differences in the reflex apneic resp onse between the two species. As discussed in 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-189 Section 4.2.1.1, mice maintain decreases in minute volume in response to formaldehyde, which 1

results in a lower overall effective internal dose to the mice. 2 Comparing cell proliferation rates after 2 versus 1 8 hours of exposure, Swenberg et al. 3 (1986) found that the longer exposure duration gave twice the cell proliferation rates after 4 repeated exposures. Therefore, these researchers c onducted a second dose-response study to 5 examine cell proliferation 18 hours after exposure instead of the shorter exposure duration. The 6 dose-response study varied dose as well as duration of treatment. Rats were exposed 6 hours/day 7 to either 0.5, 2, or 6 ppm (0.61, 2.45, or 7.36 mg/ m 3 ) formaldehyde over periods of 1, 3, or 9 8 days. Formaldehyde exposure at 0.5, 2, or 6 ppm fo r 1 day increased cell proliferation in the 9 nasal epithelium. However, these increases were tr ansient, and cell proliferation was not 10 increased after 3 or 9 days of exposure to 0.5 ppm or 2 ppm formaldehyde. Although still 11 elevated after a 3-day exposure to 6 ppm formaldehy de, cell proliferation returned to control 12 values after 9 days of exposure to 6 ppm formaldehy de (Swenberg et al., 1986). Therefore, 13 although concentration is a major determinant of ce ll proliferation, duration of exposure also 14 influenced formaldehyde-induced cell proliferation in the nasal epithelium. 15 Swenberg et al. (1986) directly tested the effects of cumulative exposure versus 16

concentration for both mice and rats. Animals were treated with one of three regimens, resulting 17 in the same C × t product: 3 ppm × 12 hours, 6 ppm × 6 hours, or 12 ppm × 3 hours, each 18 exposure resulting in 36 ppm-hours. The animals we re exposed once a day for either 3 or 9 days. 19 Tritiated thymidine was injected 18 hours after exp osure to label of proliferating cells. Tissue 20 sections from levels 1 and 2 of the nasal passages were examined in each case, and the 21 percentage of cells labeled was reported as the per centage of proliferating cells (Figure 4-10). 22 Cell proliferation at level 1 in the nasal cavity w as much greater than at level 2 for all C × 23 t combinations of formaldehyde exposure in both mic e and rats (Figure 4-10). The authors noted 24 that level 1 is more anterior and lacks significant defense from the mucociliary apparatus, which 25 may account for the observed greater sensitivity to formaldehyde. At all C × t exposure 26 products, 3 days of exposure resulted in greater ce ll proliferation than 9 days of exposure. This 27 was true for both species and for both examined lev els of the nasal cavity. The decrease in cell 28 proliferation by day 9 is consistent with data on r ats labeled 18 hours postexposure (Swenberg et 29 al., 1986). 30

31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-190 1

30

20

10

0

MICE RATS MICE RATS 10

5

0

CONTROL 3 x 12 6 x 6 12 x 3

15

10

5

0

MOUSE RAT 3

2

1

0

MICE RATS (a) (b)

(d) (c) 2

3 Figure 4-10. Effect of formaldehyde exposure on ce ll proliferation of the 4 respiratory mucosa of rats and mice. 5 6 Note: a and b are data following 3 days of exposure; c and d are for 9 days of 7

exposure. a and c are from level 1 (most anterior); b and d are from level 2. 8 [ 3 H]-thymidine was administered 18 hours after the la st exposure. 9 10 Source: Swenberg et al. (1986). 11 12 13 When comparing C × t exposures for a single species and location, the findings are more 14 complex. Cell proliferation in level 2 of the nasa l passages appeared to be more dependent on 15 concentration than on duration or cumulative exposu re, with the strongest response seen for 16 12 ppm formaldehyde in combination with the shortes t exposure period, 3 hours (Figure 4-10). 17 This pattern was observed in both rats and mice aft er 3 days of exposure and in rats after 9 days 18 of exposure. No increases in cell proliferation at level 2 were seen for any C × t combination in 19 mice after 9 days. In contrast, increases in cell proliferation at level 1 of the nasal passages were 20 not strictly concentration dependent. After a 3-da y exposure, no clear differences were seen 21 among different C × t treatments for either mice or rats, suggesting cumulative exposure may be 22This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-191 the important metric. Therefore, it may be conclud ed that cell proliferation for level 1 of the 1 nasal passages, where there is less protection of t he epithelium, is influenced by concentration, 2 time, and duration of exposure. Cell proliferation at level 2 appeared to be more dependent on 3 concentration than time of exposure (Swenberg et al ., 1986). 4 Cassee and Feron (1994) reported a qualitative incr ease in histochemical staining for 5 proliferating cell nuclear antigen (PCNA) in the re spiratory epithelium of the nasoturbinates, 6 maxilloturbinates, septum, and lateral wall at leve ls 2 and 3 of rat nasal passages after repeated 7 exposures to 3.5 ppm (4.29 mg/m 3 ) formaldehyde 22 hours/day for 3 days. While no i ncreases 8 were seen in olfactory epithelium, frank necrosis, squamous metaplasia, and hyperplasia of both 9 ciliated and nonciliated epithelium were noted at t hese section levels. 10 Quantitative cell proliferation studies have been c onducted by several researchers in the 11 same laboratory (Reuzel et al., 1990; Wilmer et al. , 1989; Zwart et al., 1988; Wilmer et al., 1987; 12 Woutersen et al., 1987) (Summary Table 4-39). Thes e studies build off of those of Swenberg et 13 al. (1986), who labeled proliferating cells with [ 3 H]-thymidine in assessing cell proliferation 14 within the nasal mucosa. The studies, all performe d in male albino Wistar rats and using a 15 similar experimental design, provide the basis for comparing different exposure levels and dose 16 regimens across studies. Wilmer et al. (1987) demo nstrate a concentration-dependent increase in 17 cell proliferation after 3 days of repeated 8-hour exposures at 5, 10, or 20 ppm (6.13, 12.3, or 18 24.6 mg/m 3 ) formaldehyde, regardless of continuous versus int errupted exposure conditions 19 (2.83, 8.87, and 19.8 versus 0.86% proliferation in controls). Similar trends were seen when the 20 repeated continuous exposures were extended for 4 w eeks, but cell proliferation was not 21 maintained at the same levels. As observed by Swen berg et al. (1986), these results suggest that 22 duration of repeated exposures may be an important determinant of cell proliferation rates. 23 Woutersen et al. (1987) reported that the majority of the dose-dependent increases in cell 24 proliferation seen at section level 3 after 3 days of repeated 6-hour exposures to 10 and 20 ppm 25 (12.3 and 24.6 mg/m 3 ) formaldehyde occurred in areas of the epithelium showing “clear 26 squamous metaplasia and hyperplasia.” Cell prolife ration rates in metaplastic epithelium of 27 29.5 and 33.2% were much higher than the 1.4 to 2.8 % proliferation in the visibly unaffected 28 respiratory epithelium from rats exposed at 10 ppm formaldehyde. Although there was a slight 29 trend towards increased cell proliferation in the v isibly unaffected epithelium of exposed animals 30 compared with unexposed controls, the majority of i ncreased cell proliferation resulting from 31 exposure to 10 and 20 ppm formaldehyde was attribut ed to the metaplastic epithelium. 32 Similarly, dose-dependent increases in cell prolife ration seen at level 3 after 3 days of 33 repeated 6-hour exposures at 0.3, 1, and 3 ppm (0.3 7, 1.23, and 3.68 mg/m 3 ) formaldehyde 34 ( p < 0.001) corresponded to focal basal cell hyperplas ia and loss of cilia (Woutersen et al., 1987). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-192 No necrosis or focal erosion was noted at these lev els of formaldehyde exposure. Cell 1 proliferation was not sustained at this location, a nd no lesions were noted after 13 weeks of 2 repeated 6-hour exposures. The authors hypothesize d that defensive mechanisms, such as the 3 mucociliary apparatus, may have provided greater pr otection of the mucosa at level 3. Swenberg 4 et al. (1986) drew a similar conclusion when evalua ting extended exposures, suggesting that 5 more posterior sections had a greater adaptive abil ity than those anterior sections with little 6 mucociliary function. Both Woutersen et al. (1987) and Swenberg et al. (1986) reported 7 sustained cell proliferation and development of les ions in the more anterior cross section. 8 Repeated exposures to 3 ppm formaldehyde (6 hours/d ay) resulted in significant increases in cell 9 proliferation in the epithelial cells at level 2, w ith accompanying disarrangement, focal 10 hyperplasia, and squamous metaplasia (Woutersen et al., 1987). Although no cell death was 11 observed at level 2 when viewed by light microscopy , “strongly indented and disarranged nuclei” 12 were seen by electron microscopy, which may be cons istent with apoptosis (Woutersen et al., 13 1987). However, later work in the same laboratory indicated no increased cell proliferation at 14 levels 2 or 3 in male Wistar rats exposed to formal dehyde at 1 or 2 ppm (1.23 and 2.45 mg/m 3 ) 15 (8-hour repeated exposures for 3 days or 13 weeks) and only minimal response in rats exposed at 16 4 ppm formaldehyde (interrupted 8-hour exposures fo r 3 days or 13 weeks) (Wilmer et al., 17 1989). 18 Reuzel et al. (1990) published the only report in w hich formaldehyde effects on cell 19 proliferation were studied for longer daily exposur e durations: 22 hours/day versus 6– 20 8 hours/day. Male Wistar rats were exposed to form aldehyde, ozone, or the combination of the 21 two 22 hours/day for 3 consecutive days. The conce ntrations of formaldehyde were 0.3, 1.0, or 22 3.0 ppm (0.37, 1.23, or 3.68 mg/m 3 ). Rats were injected with [ 3 H]-thymidine 2 hours rather than 23 18 hours after the last exposure. Cell proliferati on was quantified by enumerating the percentage 24

of labeled cells in fixed and stained tissue sectio ns. Cell proliferation on the nasoturbinates, 25 maxilloturbinates, lateral wall, and septum at leve ls 2 and 3 were quantified and reported 26 separately. Cell proliferation was increased at al l locations in level 2 at 3 ppm formaldehyde 27 exposure (

p < 0.05) but not at 0.3 or 1 ppm exposures (Summary Table 4-39). Whereas 28 proliferation of cells in the nasoturbinate, maxill oturbinate, and septum was nearly undetectable 29 in control animals, 4, 5, and 3% proliferation was reported after repeated 22-hour exposures to 3 30 ppm formaldehyde. Basal proliferation in the later al wall was greater than in other areas, 31 approximately 1% increasing to 6% after exposure to 3 ppm formaldehyde. Although basal 32 levels of cell proliferation were slightly higher i n all areas of level 3, formaldehyde had no 33 significant effects on cell proliferation in the le vel 3 areas evaluated. There was a slight trend fo r 34 increases at 3 ppm, but all proliferation rates wer e below 1%. Exposure to 3 ppm formaldehyde 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-193 also damaged the respiratory epithelium at levels 2 and 3, where cell disarrangement and 1 hyperplastic and metaplastic lesions were reported. 2 Roemer et al. (1993) investigated the effects of fo rmaldehyde exposure on cell 3 proliferation in the trachea and lung in addition t o nasal mucosa. Male Sprague-Dawley rats 4 were exposed head only to 2, 6, or 20 ppm (2.45, 7. 36, or 24.5 mg/m 3 ) formaldehyde 6 hours/day 5 for either 1 or 3 days. Proliferating cells were l abeled with 5-bromodeoxyuridine (BrdU), the 6 label injected 16–22 hours after formaldehyde expos ure ended. Free lung cells were harvested 7 by tracheal lavage, and the majority of isolated ce lls were MPs (>97%). Epithelial cells were 8 isolated from the nasal and tracheal mucosa by diss ection, physical disaggregation, and enzyme 9 treatment to release epithelial cells. All cells w ere fixed and stained with fluorescent dyes to 10 detect BrdU and total DNA. Flow cytometry was used to determine the percentage of BrdU11 labeled cells as a measure of cell proliferation. Cells undergoing unscheduled DNA synthesis 12 (e.g., DNA repair) were excluded by cell cycle anal ysis. 13 The proportion of BrdU-labeled cells from the nose and trachea increased two- to 14 threefold above control values after a single 6-hou r exposure to formaldehyde (Table 4-35). The 15 lowest effective dose for increased cell proliferat ion was 2 ppm for nose and tracheal cell 16 proliferation (

p < 0.05). However, increased proliferation in the nasal mucosa at the lowest dose 17 was transient, returning to control levels after a 3-day exposure. Cell proliferation remained 18 increased in the nasal mucosa after exposure to 6 o r 10 ppm (7.36 or 12.3 mg/m 3 ) formaldehyde 19 for 3 days. In contrast, proliferation of tracheal cells appeared to be reduced as a result of a 20 3-day exposure to 2 or 6 ppm formaldehyde. A simil ar trend was seen in free lung cells, but the 21 differences were not statistically significant. 22 The flow cytometry employed by Roemer et al. (1993) allowed for subtle changes in 23 proliferation rates to be measured with good discri mination. However, the method of cell 24 isolation did not allow examination of proliferatio n rates in discrete regions of the mucosa, 25 which may have attenuated the magnitude of the resp onse. Additionally, proliferation rates 26 represent a mix of cell types that were not separat ed in this analysis, making the findings difficult 27 to interpret. This may be especially noteworthy in the free lung cells that were reportedly 28 primarily MPs. 29

30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-194 Table 4-35. Cell proliferation in nasal mucosa, tr achea, and free lung cells 1 isolated from male Wistar rats after inhalation exp osures to formaldehyde 2 3 1 Day a Control 2 ppm 6 ppm 20 ppm

Nose 1.3 b 2.4 c 3.7 c 2.7 Trachea 1.2 3.1 c 2.1 c 2.8 Lung d 1.8 2.6 3.3 3.1 3 Days Control 2 ppm 6 ppm 20 ppm Nose 1.3 1.4 2.5 c 2.3 c

Trachea 1.2 0.3 c 0.6 c 2.5 c Lung 1.8 2.2 2.4 5.1 4

a Exposures were 6 hours/day. 5

b Proliferation is measured as the percent of BrdU-la beled cells. 6

c Statistically different from controls ( p < 0.05). 7

d The majority of free lung cells were MPs (97%). 8 9 Source: Roemer et al. (1993). 10 11 12 Monticello et al. (1990) investigated whether chang es in cell proliferation rate correlated 13 with areas of cell injury or with areas that develo ped tumors due to formaldehyde exposures by 14 using a unique metric of cell proliferation. They hypothesized that treatment-related effects on 15 cell populations could influence the apparent cell proliferation measured as LI, even though no 16 proliferative effect had occurred. For example, ce ll death could give an apparent increased 17 proliferation as a LI (% cells proliferating) by re ducing the total number of cells present. This 18 would be especially true for a stratified epitheliu m, where the number of basal cells in active 19 proliferation may not change but cells above the ba sal layer might die or slough off, thereby 20 reducing the overall number of population of cells counted. The unit length labeling index 21 (ULLI) metric was developed to normalize proliferat ion rates against length of basal membrane 22 rather than cell population. However, application of a ULLI to the pseudostratified epithelium of 23 the nasal mucosa introduced additional complexities . First, undamaged mucosa has a single 24 layer of epithelial cells that have the capability for cell proliferation. Second, cells only become 25 layered in response to cell damage as a protective measure. Therefore, the total cells present and 26 the linear cell density should be considered, as we ll as the number and density of proliferating 27 cells, in developing an understanding of the prolif erative response of these tissues to toxic insult. 28 Monticello et al. (1990) directly compared the appa rent effects of formaldehyde exposure 29 on cell proliferation when quantified as an LI or a s a ULLI. Male F344 rats were divided into 30 groups (n = 6) and exposed to 0, 2, 6, or 15 ppm (0 , 2.45, 7.36, or 18.4 mg/m 3 ) formaldehyde 31 6 hours/day, 5 days/week for 12 weeks. Rats were a dministered [ 3 H]-thymidine continuously for 32 the last 5 days of exposure by surgically implanted osmotic pumps. After sacrifice, nasal 33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-195 passages were fixed, and sections from standard lev el 3 were prepared for examination. Cell 1 proliferation was quantified at the midseptum and t he lateral meatus at this level. Basement 2 membrane length, total number of cells present, and number of labeled proliferating cells were 3

recorded for each location. Each of these areas al so was scored for the presence of nasal lesions. 4 The formaldehyde-related lesions included epithelia l hyperplasia, squamous metaplasia, 5

and acute inflammation. These lesions were most se vere in animals exposed to 15 ppm, mild at 6 6 ppm, but absent at 2 ppm. Cell proliferation, me asured either as LI or ULLI, was increased in 7 the level 3 septum and lateral meatus after 13 week s of exposure to 15 ppm formaldehyde but 8 not to 6 or 2 ppm (Table 4-36). There was a slight increase in both cell number and labeled cells 9 in the lateral meatus of rats exposed to 6 ppm form aldehyde, but both measures of proliferation 10 were unchanged from controls. The increased prolif eration in the lateral meatus at 15 ppm was 11 entirely due to an increased number of labeled cell s. Total cells were unchanged at 15 ppm; 12 therefore, both LIs demonstrated a similar increase over control. In addition to increased labeled 13 cells in the septum at 15 ppm, total cells were inc reased from 470 to 640 ( p < 0.05). Where the 14 total cells and linear cell density were increased, the ULLI was proportionally increased over the 15

LI. These observations are consistent with the dev elopment of squamous metaplasia and 16 hyperplasia seen at 15 ppm. However, while both LI and ULLI showed an eightfold increase in 17 cell proliferation in the lateral meatus, they gave different results in the septum where cell 18 number was increased by formaldehyde treatment. LI increased 19-fold and ULLI 25-fold with 19 repeated exposures to 15 ppm formaldehyde. Althoug h these data are based on only 5–6 20 animals/group, and only in an extended study, the r esults suggest that the ULLI and LI may not 21 be proportional under all conditions studied. In s imilar experiments the LI and ULLI provided 22 different indices of proliferation in the olfactory epithelium after methyl bromide exposure 23 (Monticello et al., 1990). Methyl bromide exposure decreased cell number/mm of basement 24 membrane in a time-dependent manner, and the LI and ULLI were not proportional across these 25 changes. At day 3 there was an increase in labeled cells but a decrease in total cells; therefore, 26 the LI was increased greater than 20-fold, where th e ULLI was only increased eightfold. The 27 authors endeavored to explain why the ULLI and LI y ielded different findings. Where ULLI is a 28 more time-efficient method of assessing cell prolif eration, the authors suggested that 29 representative areas should be quantified by LI to better understand the nature of increased 30

ULLI. 31

32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-196 Table 4-36. The effect of repeated formaldehyde in halation exposures for 1 3 months on cell count, basal membrane length, prol iferation cells, and two 2 measures of cell proliferation, LI and ULLI, in mal e F344 rats 3 4 Formaldehyde exposure level (6 hours/day, 5 days/week for 3 months) 0 ppm 2 ppm 6 ppm 15 ppm Lateral meatus Total cells BM length (mm) b

Cells/mm BM Labeled cells

LI ULLI 1,800 ± 100 12.7 ± 0.6 150 ± 5 130 ± 10

7.2% c 10.2 cells/mm d 1,800 ± 150

11.9 ± 0.5 150 ± 10 130 ± 20

7.2% 10.9 cells/mm 2,300 a ± 1700 13.4 ± 0.3 170 ± 10 210 ± 30

9.1% 15.7 cells/mm 1,900 ± 160

11.6 ± 0.7 150 ± 5 1,400 ± 130

73.7% 120.7 cells/mm Septum Total cells BM length (mm) Cells/mm BM Labeled cells

LI ULLI 470 ± 20 2.9 ± 0.1 160 ± 10 20 ± 1

4.3% 6.9 cells/mm 460 ± 30

2.7 ± 0.1 170 ± 10 40 ± 10

8.7% 14.8 cells/mm 470 ± 20

2.9 ± 0.1 160 ± 3 10 ± 2

2.1% 3.45 cells/mm 640 a ± 20

2.9 ± 0.1 220 a ± 10 250 ± 50

39% 86.2 cells/mm 5

a Different from control, p < 0.05. 6

b BM is basal membrane length in mm. 7

c Calculated from group averages: LI = (labeled cells )/total cells 8

d Calculated from group averages: ULLI = (labeled cel ls)/BM length 9 10 Source: Monticello et al. (1990). 11 12 13 Monticello et al. (1990) reported similar results i n a contemporary abstract; although 14 treatment groups were slightly different than in th e above experiments, the findings were similar. 15 Rats were exposed to 0, 0.7, 2, 6, 10, or 15 ppm (0 , 0.86, 2.45, 7.36, 12.3, or 18.4 mg/m 3 ) 16 formaldehyde 6 hours/day for 4 days, 6 weeks, or 3 months. ULLIs were determined in the 17 septum and lateral meatus (methods not detailed). It is not stated whether [ 3 H]-thymidine 18 labeling was carried out by injection or continuous infusion. Significant increases in cell 19 proliferation were reported after repeated exposure s to 6, 10, and 15 ppm for 4 days and 6 weeks. 20 After 3 months of exposure, cell proliferation was still increased in rats exposed to 10 and 21 15 ppm formaldehyde. The authors noted that, altho ugh increased cell proliferation was seen at 22 earlier time points, sustained increased cell proli feration was only seen at 10 and 15 ppm, which 23 they considered the clearly carcinogenic doses. 24 Monticello et al. (1991) applied the ULLI measureme nts in evaluating formaldehyde 25 effects on cell proliferation after short-term and subchronic repeated exposures. Six male F344 26 rats/group were exposed to 0, 0.7, 2, 6, 10, or 15 ppm (0, 0.86, 2.45, 7.36, 12.3, or 18.4 mg/m 3 ) 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-197 formaldehyde 6 hours/day for 1, 4, or 9 days and fo r 6 weeks, using a 5 days/week regimen. 1 Rats were injected with [ 3 H]-thymidine 18 hours postexposure to label prolife rating cells. All 2 animals were sacrificed 2 hours later. Nasal passa ges were fixed, and sections from levels 2 and 3 3 were prepared for examination. Cell proliferatio n was quantified for three locations in level 2 4 (specifically, the lateral meatus, midseptum, and m edial aspect of the maxilloturbinate) and for 5 two regions of level 3 (the lateral wall and midven tral septum). Each of these areas also was 6 scored for the presence of nasal lesions. 7 As discussed above, proliferating cells were visual ly identified by the number of grains 8 over the nucleus, 10 grains indicating a proliferat ing cell. Cell proliferation was quantified as the 9 number of proliferating cells per length of basemen t membrane (cells/mm) and reported as a 10 ULLI. The report does not indicate the length of m embrane viewed for each section as an 11 indication of how representative the counts are for each region. Lesions associated with 12 formaldehyde exposure may change the density of cel ls/mm of basement membrane (Monticello 13 et al., 1990). Areas of disarranged cells, erosion , metaplasia, or layering of epithelial cells may 14 exhibit different cell profiles. These processes w ould alter cell density, and therefore the ULLI, 15 independent of differential proliferation rates. A s such, it is not expected to be proportional to 16 cell proliferation rates across conditions that hav e the potential to change cell density 17 (Monticello et al., 1990). 18 No formaldehyde-induced epithelial lesions or incre ases in the ULLI were seen in rats 19 exposed to 0.7 or 2.0 ppm formaldehyde, regardless of duration (Table 4-37). Formaldehyde20 induced lesions were present in all regions of the nasal epithelium after exposures to 10 and 21 15 ppm formaldehyde, regardless of duration (Montic ello et al., 1991). Incidence and severity of 22

the lesions increased with concentration and durati on of treatment and were correlated to areas 23 with increased cell proliferation. Rats exposed to 6 ppm formaldehyde developed lesions in the 24 level 2 nasal passages, where the ULLI was clearly elevated, but not in the deeper level 3 25 passages. For example, no formaldehyde-related les ions were seen at the lateral meatus and 26 septum of level 3 at 1, 4, and 9 days of repeated e xposure at 6 ppm, although cell proliferation 27

was increased. This transient increase in ULLI ret urned to near-control levels after 6 weeks of 28 repeated exposure (Table 4-37). Monticello et al. (1991) suggested that cell proliferation is a 29 more sensitive indicator of cellular response and n ot necessarily dependent on cellular necrosis. 30

31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-198 Table 4-37. Formaldehyde-induced changes in cell p roliferation (ULLI) in 1 the nasal passages of male F344 rats exposed 6 hour s/day 2 3 Exposure concentration

Location a 0 ppm 0.7 ppm 2 ppm 6 ppm b 10 ppm b 15 ppm b

Level 2: lateral meatus 1 day 4 days 9 days 6 weeks 2.16 1.46 1.44 0.91 1.31

1.37 1.20 0.88 2.36

1.72 1.73 1.36 16.9 b

30.5 b

23.5 b

14.4 b 11.2 b

20.9 b

28.6 b

23.9 b 12.7 b

25.8 b

24.6 b

28.7 b Level 2: midseptum 1 day 4 days 9 days 6 weeks 1.08 1.03 1.09 0.41 1.01

0.97 0.80 0.24 1.69

0.67 0.97 0.68 3.85

10.0 b

10.9 b

2.10 17.9 b

26.1 b

19.6 b

21.4 b 16.7 b

29.1 b

29.1 b

25.9 b

Level 2: medial maxilloturbinate 1 day 4 days 9 days 6 weeks 2.49 1.36 1.38 1.02 1.75

1.54 0.80 1.21 2.81

1.09 1.48 1.11 18.15 b

25.03 b

22.54 b

16.32 b 5.9

20.3 b

21.0 b

26.1 b 5.3

19.4 b

28.7 b

25.1 b

Level 3: lateral meatus 1 day 4 days 9 days 6 weeks 1.83 1.10 1.36 0.98 1.72

1.27 1.40 0.91 2.46

1.09 1.74 0.86 7.53 b,,c

8.77 b,

, c 7.35 b, c 2.08 14.5 b

20.0 b

30.6 b

24.2 b 16.4 b

30.8 b

40.4 b

34.8 b Level 3: midseptum 1 day 4 days 9 days 6 weeks 3.02 2.81 1.68 2.18 1.74

3.09 1.06 1.54 2.39

1.43 1.43 2.57 4.20

9.22 b,

, c 9.50 b,

, c 2.58 24.4 b

18.7 b

28.6 b

14.0 b 19.3 b

34.4 b

32.5 b

27.5 b 4

a ULLI is expressed as the number of labeled cells/mm of basement membrane. 5

b Indicates significantly different from control, p < 0.05. 6

c Indicates a location where epithelial lesions were not seen by light microscopy. 7 8 Source: Monticello et al. (1991). 9 10 11 The sustained cell proliferation at the lateral mea tus and midseptum in rats exposed to 10 12 and 15 ppm formaldehyde, locations where SCCs are k nown to arise, supports a role for 13 compensatory cell proliferation in tumor developmen t. However, Monticello et al. (1991) noted 14 that regional differences in sustained cell prolife ration do not always correspond to the 15 occurrence of nasal tumors, primarily SCCs, in form aldehyde-exposed rats. Where sustained 16 cell proliferation has been demonstrated in the med ial maxilloturbinate (MMT) at level 2 17 (Monticello et al., 1991), SCCs have not been found to originate in this area at similar exposures 18 (Monticello et al., 1996; Woutersen et al., 1989). Monticello et al. (1991) suggested that the 19 findings of Bermudez and Allen (1984), indicating t hat the epithelial cells of the maxilloturbinate 20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-199 are more resistant to the genotoxic effects of DEN, support the possibility that differences in 1 regional tissue susceptibility may contribute to si te specificity of formaldehyde-related SCCs. 2 Monticello et al. (1996) further explored the corre lation between measures of cell 3 proliferation and tumor site by modifying the ULLI to take into consideration the total number of 4 cells in a region that may be subject to increased cell proliferation. The population weighted 5 ULLI (PWULLI) is the product of the expected number of cells on a three-dimensional surface 6

in the nasal mucosa and the ULLI of a cross section of that surface. For this series of 7 experiments, six male F344 rats/group were exposed to 0, 0.7, 2, 6, 10, or 15 ppm (0, 0.86, 2.45, 8 7.36, 12.3, or 18.4 mg/m 3 ) formaldehyde for up to 24 months with interim sac rifices at 3, 6, 12, 9 and 18 months. Before each interim sacrifice [ 3 H]-thymidine was continuously injected for the 10 last 5 days of exposure through a surgically implan ted pump. Nasal passages were prepared, and 11 six standard sections were taken and developed as a bove for [ 3 H]-thymidine-labeled cells. 12 Stained tissue sections were viewed in order to map all nasal tumors. A ULLI was determined 13 for each region (details not provided). The total cell population of each nasal region was 14

estimated from control animals sacrificed at 3 mont hs (Table 4-38). Cell profiles were counted 15 across 0.5 mm of basement membrane length at two lo cations for each region (site not specified). 16 Total cells per region were estimated from these co unts and the modeled surface area expected in 17 each region (Fluid Dynamics Analysis Package versio n 7.0). It is unclear if one or more rats 18 were used to quantify cell population. Cell counts and variability were not reported. 19

20 Table 4-38. Cell population and surface area estim ates in untreated male 21 F344 rats and regional site location of squamous ce ll carcinomas in 22 formaldehyde-exposed rats for correlation to cell p roliferation rates 23 24

SCC incidence c

Nasal region Total cells

(number) a Area

(mm 2 ) b Cell density

(cell/mm 2 ) 10 ppm 15 ppm Anterior lateral meatus 976,000 59.5 16,400 12 17 Anterior midseptum 184,000 10.5 17,500 0 1 Anterior dorsal septum 128,000 3.84 33,300 0 3

Anterior medial maxilloturbinate 104,000 7.63 13,600 0 4

Posterior lateral meatus 508,000 38.1 13,300 2 9 Posterior midseptum 190,000 10.8 17,600 0 1 Maxillary sinus 884,000 38 23,300 0 0 Region not specified c -- -- -- 6 25 25

a Total cell number determined in unexposed rats as a product of representative cell counts and expected surface area 26 of the region. 27

b Modeled surface area of the defined region by FDIP version 7.0. 28

c The number of animals bearing a tumor located in th e region. Animals were exposed 6 hours/day for 24 months 29 prior to sacrifice. 30 Source: Monticello et al. (1996). 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-200 ULLIs were quantified by region of the nasal passag es in order to correlate with regional 1 localization of tumors. For example, the anterior midseptum included cells from the midseptum 2 from approximately standard section levels 2 to 3. An anterior to posterior pattern of 3 formaldehyde effects, especially differences in cel l proliferation rates, has been well established. 4 As such, cell proliferation rates would be expected to vary across the nasal regions used in this 5 analysis. Areas considered to possibly be preneopl astic were not quantified for this work. 6 Monticello et al. (1996) reported increased ULLIs i n the ALM and the MMT of rats exposed to 7 10 or 15 ppm at all time points (3, 6, 12, and 18 m onths) but provided no indication of variability 8 or a statistical analysis, making it difficult to d etermine where true differences may exist. Some 9 caution should be used in interpreting the ULLI cou nts assigned for each region. 10 The PWULLI was calculated by multiplying the report ed ULLIs by the calculated cell 11 populations by region. SCC incidence by region had a greater correlation to the calculated 12 PWULLI than the ULLI, R 2 = 0.88 versus R 2 = 0.46, respectively. The authors noted that the 13

relative lack of correlation with the ULLI was infl uenced by findings at the maxilloturbinate 14 where cell proliferation was high but SCC incidence was low. Other tumor types were not 15 included in the analysis (polypoid adenomas, adenoc arcinomas, and rhabdomyosarcomas). 16 Additionally, 54 of the SCC tumors could not be acc urately localized and were excluded from 17 the analysis, resulting in exclusion of 30 and 39% of animals with SCCs in the 10 and 15 ppm 18 treatment groups, respectively. The authors cautio ned that the absence of these data might have 19 skewed the regional analysis of tumor location. Al though the purpose of weighting the ULLIs 20 by total population of cells available in each regi on is to better represent the chance of a tumor 21 arising in each region, the cancer incidence was re presented by the number of animals, not the 22 number of tumors, per region. Based on the exclusi on of location data (up to 40% of the 23 animals), lack of variability and significance repo rted for the ULLI for cell counts, and SCC 24 incidence considered by animal rather than by tumor , the significance of a greater correlation by 25 PWULLI versus ULLI is of questionable value. 26 Monticello et al. (1989) also assessed formaldehyde -induced cell proliferation and 27 regional site location of lesions in the respirator y tract of rhesus monkeys (see Section 4.2.1.2.2.2 28 for a full study description). LIs from the histoa utoradiograms indicated increased cell 29 proliferation in transitory, respiratory, and olfac tory epithelial cells after the 6-week 30 formaldehyde exposure. Similar trends were seen af ter only 1 week but were statistically 31 significant only in the respiratory epithelium. Al though increased proliferation in the trachea and 32 carina was statistically significant after 1 week o f exposure, the greater increases seen after 33 6 weeks of exposure were not statistically signific ant. A small sample size (n = 3) and high 34 variability may have contributed to the lack of sta tistical significance. The authors noted that 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-201 increased cell proliferation was seen in locations with minimal histologic changes, indicating 1 proliferation may be a more sensitive predictor of adverse health effects of formaldehyde 2 exposure. Table 4-39 provides a summary of formald ehyde-induced cell proliferation data. 3

4

4.2.1.3. Gastrointestinal Tract and Systemic Toxicity 5 As with inhalation, the POE is thought to be the pr incipal target tissue in response to oral 6 exposure. A concentration-dependent pattern of tox icity longitudinally down the GI tract has 7 been observed upon oral exposure. Some evidence (T il et al., 1989, 1988) suggests that, with 8 regard to oral exposure, duration in addition to co ncentration is important in the development of 9 toxicity. 10 Formalin and paraformaldehyde were used to dose ani mals in oral toxicity studies. 11 Formalin contains 12–15% methanol as a preservative to inhibit the polymerization of 12 formaldehyde and subsequent precipitation as parafo rmaldehyde (Kiernan, 2000). The presence 13 of methanol in formalin may confound the results of a formaldehyde study. Methanol has been 14 shown to be a developmental and neurologic toxin (e .g., Degitz et al. [2004a, b]; Rogers et al. 15 [2004, 2002]; Weiss et al. [1996]; Sharpe et al. [1 982]). Oral dosing with paraformaldehyde is 16 preferred because it allows for the preparation of methanol-free formaldehyde in the laboratory 17 by dissolving paraformaldehyde in slightly basic wa ter. 18

19

4.2.1.3.1. Short-term and subchronic studies. Til et al. (1988) evaluated the oral toxicity of 20 formaldehyde and acetaldehyde in a subacute study i n Wistar (Cpb:WU; Wistar random) rats. 21 Groups of rats (10/sex/dose) were exposed to parafo rmaldehyde dissolved in drinking water at 0, 22 5, 25, and 125 mg/kg-day for 4 weeks. The control group was comprised of 20 rats of each sex. 23 To account for potential effects of decreased water consumption in treated animals, an additional 24 control group of 10 male and 10 female rats was giv en drinking water in an amount equal to the 25 amount of liquid consumed by the group given the hi ghest dose. Examination of the GI tract was 26 performed in all dose groups and included the tongu e, esophagus, and stomach. Histopathology 27 for the other tissues was performed on high-dose an d control animals. 28

29This document is a draft for review purposes only a nd does not constitute Agency policy 4-202 DRAFT—DO NOT CITE OR QUOTETable 4-39. Summary of formaldehyde effects on cell proliferation in the upper respiratory tractSpecies NaTreatmentbMeasure of cellproliferationSummary of results by locationcReferenceMale F344rats;maleB6C3F1miceNRd0.5, 2, 6, or 15 ppm6 hours/day for3 daysLI: percent labeled cellson tissue sections(3H-thymidine I.P.d2 hours postexposure)Level 2: Rats exhibited greater increased cell proliferation than mice.No increase seen in rats or mice at 0.5 or 2.0 ppm.No increase seen in mice at 6 ppm, but rats had 20-fold increase inproliferation.10- to 20-fold increase seen in both rats and miceat 15 ppm.Swenberg etal. (1986)Male F344ratsNR0.5, 2, or 6 ppm6 hours/day1, 3, or 9 daysLI: percent labeled cellson tissue sections (3Hthymidine I.P. 18 hourspostexposure)Level 2: Transient increase in cell proliferation on day 1 at 0.5 and 2.0 ppm.Increase in cell proliferation on days 1, 3, and 9by 6 ppm.Swenberg etal. (1986)Male F344rats;maleB6C3F1miceNR3 ppm for 12 hours,6 ppm for 6 hours, or12 ppm for 3 hours3 or 9 daysLI: percent labeled cellson tissue sections (3Hthymidine I.P. 18 hourspostexposure for 2 hours)Level 1:3 days: Greater increased proliferation in rats than mice. Increasessimilar for various concentrations yielding the same C × t product.9 days: Mice exhibited duration-dependent increasesin proliferation,inverse to concentration for constant C × t.Level 23 days: Concentration-dependent increase in cell proliferation.9 days: Concentration-dependent increase in cell proliferation in rats; noincrease in mice.Swenberg etal. (1986)Male F344rats4–515 ppm6 hours/day1 or 5 daysLI: percent labeled cellson tissue sections (3Hthymidine I.P. 18 hourspostexposure for 2 hours)Level 2: Increase in cell proliferation in respiratory epithelium,nasoturbinates, maxilloturbinates, and lateral wall.1 day: 5.51fversus 0.43% in controls5 days: 10.1%fChang et al.(1983)eMaleBC3F1mice4–515 ppm6 hours/day1 or 5 daysLI: percent labeled cellson tissue sections (3Hthymidine I.P. 18 hourspostexposure for 2 hours)Level 2: Increase in cell proliferation in respiratory epithelium,nasoturbinates, maxilloturbinates, and lateral wall.1 day: 2.14fversus 0.27% in controls5 day: 3.42%fChang et al.(1983)eMalealbinoWistar rats5d3.5 ppm8 hours, twice a dayfor 3 daysQualitative staining forPCNA on tissue sectionsLevels 2 and 3: Increase in cell proliferation in respiratory epithelium,nasoturbinates, maxilloturbinates, septum, and lateral wall.Cassee andFeron (1994)eThis document is a draft for review purposes only a nd does not constitute Agency policy 4-203 DRAFT—DO NOT CITE OR QUOTETable 4-39. Summary of formaldehyde effects on cell proliferation in the upper respiratory tractSpecies NaTreatmentbMeasure of cellproliferationSummary of results by locationcReferenceMalealbinoWistar rats30, 5, or 10 ppm8 hours/daycontinuously for3 days or 4 weeks, or0, 10, or 20 ppm8 hours/dayintermittentgfor 3 days or 4 weeksLI: percent labeled cellson tissue sections (3Hthymidine I.P.18 hourspostexposure for 2 hours)Section level not stipulated in report.3 days: 0.86% in controls2.83%fat 5 ppm continuous8.87%fat 10 ppm continuous9.80%fat 10 ppm interrupted19.8%fat 20 ppm interrupted4 weeks: 0.68% in controls1.33% at 5 ppm continuous8.85%hat 10 ppm continuous3.41%fat 10 ppm interrupted13.9%fat 20 ppm interruptedWilmer et al.(1987)eMalealbinoWistar rats20, 1, 10, or 20 ppm6 hours/day for3 daysLI: percent labeled cells(18 hour postexposure exvivo3H-thymidine labeledexcised mucosa)Level 3Metaplastic epithelium: increased proliferation31.4% at 10 ppm, 37.6% at 20 ppmVisibly unaffected respiratory epithelium1.6% in controls2.6% at 10 ppm, 2.8% at 20 ppmWoutersen etal. (1987)eMalealbinoWistar rats50, 1, or 2 ppm8 hours/daycontinuously for3 days or 4 weeks,or 0, 2, or 4 ppm8 hours/dayintermittentgfor 3 days or 4 weeksLI: percent labeled cellson tissue sections (3Hthymidine I.P. 18 hourspostexposure for 2 hours)Level 23 days: No change from controls0.60% in controls0.34%at 1 ppm continuous0.61%at 2 ppm continuous0.29%at 2 ppm interrupted0.58%at 4 ppm interrupted4 weeks: no change from controls1.03% in controls0.81%at 1 ppm continuous0.91%at 2 ppm continuous1.16%at 2 ppm interrupted2.86%at 4 ppm interruptedWilmer et al.(1989)eMale andfemalealbinoWistar rats50, 0.3, 1, 3 ppm6 hours/day,5days/weekfor 3 days or13 weeks.LI: percent labeled cellson tissue sections (3Hthymidine I.P. 18 hourspostexposure for 2 hours)Level 2: Increased cell proliferation at days 3 and13 weeks (p< 0.001).Level 3: Transient dose-dependent increase at 1 and3 ppm;only seen at day 3(p< 0.001).Note: Results pooled by sex. Data shown graphically on log-normal scale.Zwart et al.(1988)eMaleWistar rats5 0, 0.3, 1, or 3 ppm22 hours/day for3 daysLI: percent labeled cellson tissue sections (2 hourspostexposure ex vivo3Hthymidine-labeled excisedmucosa)Level 2: 3 ppm increased cell proliferation in nasoturbinates,maxilloturbinates, septum, and lateral wall (p< 0.05).Level 3: No significant increases in cell proliferation.Reuzel et al.(1990)This document is a draft for review purposes only a nd does not constitute Agency policy 4-204 DRAFT—DO NOT CITE OR QUOTETable 4-39. Summary of formaldehyde effects on cell proliferation in the upper respiratory tractSpecies NaTreatmentbMeasure of cellproliferationSummary of results by locationcReferenceMaleSpragueDawleyrats3–50, 2, 6, or 20 ppm6 hours/day for1 or 3 daysLI: percent labeled cellsby flow cytometry(5-bromodeoxyuridine I.P.18 hours postexposure for2 hours)Respiratory and olfactory epithelial cells.1 day: 1.3% in controls2.4% at 2 ppmf3.7% at 6 ppmf2.7% at 20 ppmfTracheal epithelial cells1 day: 1.2% in controls3.1% at 2 ppmf2.1% at 6 ppm2.8% at 20 ppmfFree lung cells (>97% MPs):no significant change.3 days:1.4% at 2 ppm2.5% at 6 ppmf2.3% at 20 ppmf3 days:0.3% at 2 ppmf0.6% at 6 ppmf2.5% at 20 ppmfRoemer et al.(1993)Male F344rats6 0.7, 2, 6, 10, or15 ppm6 hours/day,5 days/week for 1, 4,or 9 days or 6 weeksULLI (unit length LI) (3Hthymidine I.P. 18 hourspostexposure for 2 hours)Level 3No increases in cell proliferation at 0.7 or 2ppm.Level 4ULLI increases in locations without lesions at6 ppm.Increases in ULLI at all locations at 10 and 15 ppm.Monticello etal. (1991)Malerhesusmonkeys36 ppm6 hours/day for1 or 6 weeksLI: percent labeled cellson tissue sections (3Hthymidine I.P. 18 hourspostexposure for 2 hours)Nasal passages: Duration-dependent increase in cellproliferation at all levels(B–E) in transitional, respiratory, and olfactory epithelium.Increased cell proliferation in areas with minimallesions.Larynx: trend for increased proliferationTrachea: increased cell proliferation1 week : 1.14 versus 0.55% in controls6 weeks: 3.73%Carina of trachea: increased cell proliferation.1 week: 1.34 versus 0.43% in controls6 weeks: 3.60%Respiratory bronchioles: no increase in proliferation.Monticello etal. (1989)aN = number of animals per treatment group.bTreatment is given as the concentration of formaldehyde, duration of exposure each day, and length ofthe experiment in days and weeks.cStandard section levels of the nasal passages as shown in Figure 4-3 are given for experiments in ratsor mice.dNR = not reported; I.P. = intraperitoneally.eStudy is described in full in section 4.2.1.2.2.4..fDifferent from control,p< 0.05.gIntermittent exposures were 30 minutes per hour for8 hours.hData from one animal only.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-205 The rats appeared to be healthy throughout the stud y, and no effects on growth occurred 1 despite significant decreases in food and water int ake that occurred at the high dose (125 mg/kg2 day). Yellow discoloration of the fur occurred in the rats on the high dosage from week 3 3 onward. There were no significant changes in hemat ology among the exposed groups except for 4 slight (not statistically different) increases in P CVs in the water-restricted group and in high-dose 5 males. The high-dose groups of the formaldehyde ex posed and in the water-restricted controls 6 had slightly increased urine density, but again thi s was not statistically significant. Plasma TP 7

and ALB levels were decreased in the males of the h ighest dose group. No changes in organ 8 weights occurred except for relative kidney weights that were slightly increased in the females of 9 the high-dose group. Gross pathological findings w ere restricted to the GI tract and revealed a 10 thickening of the limiting ridge of the forestomach in all animals exposed at the highest dose that 11 was accompanied by a yellowish discoloration of the mucosa. These latter changes were not 12 observed in the acetaldehyde-exposed animals. Trea tment-related histopathologic changes were 13 seen in the GI tract only. Slight (8/20) or modera te (12/20) focal hyperkeratosis of the 14 forestomach and slight focal atrophic gastritis occ urred in animals of the high-dose groups only 15 (Table 4-40). One female had moderate focal papill omatous hyperplasia. No histopathologic 16 changes were observed in any animals of the lower-d ose groups. The study established a 17 LOAEL and NOAEL for epithelial changes in the GI tr act of male and female Wistar rats 18 exposed to formaldehyde in drinking water at 125 mg /kg-day and 25 mg/kg-day, respectively. 19 Johannsen et al. (1986) performed a subchronic stud y by using rats and dogs exposed to 20 paraformaldehyde dissolved in drinking water. Grou ps of albino Sprague-Dawley rats (15/sex) 21 were administered the equivalent of 0, 50, 100, or 150 mg/kg-day in their drinking water for 22 91 consecutive days. Pure-bred beagle dogs (four/s ex/group) were fed a diet with added aqueous 23 formaldehyde to approximate 0, 50, 75, or 100 mg/kg -day. Dogs were observed daily and rats at 24 frequent intervals for behavioral reactions. Body weights and food and water intake were 25 recorded on a weekly basis in both species. Hemato logy (HCT, Hb, total and differential 26 leukocyte counts), clinical chemistry (blood sugar, BUN, ALP, AST and ALT in dogs only), and 27 urine analyses (color, appearance, pH, specific gra vity, sugar, protein, and microscopic elements) 28

were evaluated in 10 male and 10 female rats select ed from each test group and in all dogs. 29 Organ weights were recorded for the adrenals, gonad s, hearts, kidneys, livers, lungs, and thyroids 30 in each species. Histopathology was performed on a set of over 20 or 30 tissues and organs from 31 rats or dogs, respectively, in the high-dose and co ntrol groups only. 32

33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-206 Table 4-40. Summary of lesions observed in the gas trointestinal tracts of 1 Wistar rats after drinking-water exposure to formal dehyde for 4 weeks 2 3 Formaldehyde (mg/kg-day) 0 5 25 125 Number of male rats examined Type of lesion 20 10 10 10 Focal hyperkeratosis of forestomach Very slight 3 0 0 0 Slight 1 0 0 4 Moderate 0 0 0 6 Focal gastritis Slight 0 0 0 2 Moderate 0 0 0 1 Dilated fundic glands (single or a few) 0 0 0 0 Submucosal mononuclear cell infiltrate 0 0 0 1 Number of female rats examined Type of lesion 20 10 10 10 Focal hyperkeratosis of forestomach Very slight 6 0 0 2 Slight 0 0 0 2 Moderate 0 0 0 6 Focal gastritis Very slight 0 0 0 1 Slight 0 0 0 1 Moderate 0 0 0 1 Focal papillomatous hyperplasia 0 0 0 1 Polymorphonuclear leukocytic infiltration 0 0 0 1 4 Source: Til et al. (1988). 5 6 7 No deaths or abnormal reactions were observed in ei ther species. Significant reductions 8 in weight gain were observed in dogs of both sexes at 100 mg/kg-day, in rats of both sexes at 9 150 mg/kg-day, and in male rats at 100 mg/kg-day of formaldehyde. There was a dose-related 10 decrease in liquid consumption of both sexes in rat s given formaldehyde, but there was no 11

overall difference in mean food intake or feed effi ciency, so the reductions in body weight gain 12 were considered to be systemic effects. Dogs admin istered formaldehyde had reduced food 13 consumption and feed efficiency at all doses tested . No significant effects on hematology, 14 clinical chemistry, or urine analyses were observed in either species. No effects in either species 15 were reported on organ weights. The GI mucosa in b oth species was reported to appear normal 16 with no indication of irritation. This study sugge sts a NOAEL of 150 mg/kg-day in Sprague17 Dawley rats and of 100 mg/kg-day in beagle dogs for formaldehyde in drinking water. 18

Differences in the results for the rats with those reported in other studies (Til et al., 1989, 1988; 19 Tobe et al., 1989) may be due to strain differences or duration of the exposure. The dog may be 20 a more sensitive species than the rat based on thes e results and on those of 2-week pilot studies. 21This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-207 4.2.1.3.2. Chronic bioassays: oral exposure to formaldehyde

. The same laboratory that tested 1 formaldehyde and acetaldehyde in a 4-week study (Ti l et al., 1988) performed a chronic bioassay 2 with formaldehyde in drinking water. Til et al. (1 989) administered paraformaldehyde dissolved 3 in drinking water to Wistar rats (Cpb: WU; Wistar r andom) (70/sex/dose). Interim sacrifices 4 (10/sex/dose) were performed at 12 and 18 months. Formaldehyde was administered in drinking 5 water to provide target doses of 0, 5, 25, and 125 mg/kg-day. The mean formaldehyde doses 6 administered were 0, 1.2, 15, or 82 mg/kg-day for m ales and 0, 1.8, 21, or 109 mg/kg-day for 7 females. Concentrations were adjusted weekly for t he first 12 weeks based on dose estimates 8 derived from body weight and liquid consumption dat a. Such adjustments were made every 9 4 weeks from weeks 12 to 52 and kept constant. Fre sh solutions of the test concentrations were 10 prepared weekly and stored at 15oC. 11 Endpoints examined included daily observations for condition and behavior, body weight 12 at weekly intervals for the first 12 weeks and then every 4 weeks thereafter, liquid intake weekly, 13 and food intake weekly for the first 12 weeks and t hen every 2 weeks for the remainder of the 14 study. Samples of blood were taken for hematologic al and clinical chemistry analyses on weeks 15 26 and 103. Analysis of blood glucose and urine pH , density, and volume was performed on 16 samples at weeks 27, 52, 78, and 104. Pooled urine samples were also evaluated for glucose, 17 occult blood, ketones, urobilinogen, and bilirubin in samples at weeks 27 and 104. Weights of 18 all major organs were recorded at interim sacrifice s and at term. Gross and histopathologic 19 examinations were carried out on all major tissues of the rats in the high-dose and control 20 groups. The livers, lungs, stomach, and noses were examined in all rats. Additionally, the 21 adrenals, kidneys, spleens, testes, thyroids, ovari es, pituitaries, and mammary glands (for 22 females) were examined in all sacrificed animals at weeks 53 and 79 and at term. 23 The general health and behavior of the rats were no t affected in any of the formaldehyde24 exposed groups. Slight yellowing of the fur did oc cur in the animals exposed at the mid and high 25 doses from week 3 onward. The mean body weights we re decreased in the males from week 1 26 and in the females from week 24 onward. At the hig h dose, liquid consumption was significantly 27 decreased in both sexes, and food intake was signif icantly decreased in the males. There were no 28 toxicologically significant effects on hematologica l, urinary, or clinical chemistry parameters. 29 Decreases in absolute heart, liver, and testis (mal es) weights were attributed to lower body 30 weights. Relative kidney weights were increased in females of the high-dose group, and relative 31 brain weights were increased in both sexes of the h igh-dose group. Relative testis weight was 32 increased in males. Treatment-related changes in g ross pathology were restricted to the 33 forestomach. Histopathologic examinations at the t wo interim sacrifices and final sacrifice 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-208 revealed GI tract changes. Renal changes were obse rved in the high-dose group at final 1

sacrifice. There was no indication of treatment-re lated effects in other tissues. 2 As shown in Table 4-41, significant histopathology in the GI tract was limited to the 3 forestomach and stomach of rats in the high-dose gr oups. Some progression with duration of 4 exposure may have occurred by week 105 because GI l esions were observed in the lower dose 5 groups at this time point, whereas none were observ ed in these groups at interim sacrifices. The 6 histopathologic changes included papillary epitheli al hyperplasia in the forestomach that was 7 frequently accompanied by hyperkeratosis on the lim iting ridge or its vicinity. The mucosa 8 showed an irregular layer of hyperplastic basal cel ls, but no atypical nuclei or other subcellular 9 structures were observed. Chronic atrophic gastrit is occurred to varying degrees in the stomachs 10 of all high-dose rats. In some cases the inflammat ory process involved the entire mucosa and 11

was seen to extend to the whole muscularis mucosae and met the criteria for ulceration. 12 Histologic examination also showed that the inciden ce and degree of renal papillary 13 necrosis was increased in animals of the high-dose groups at the terminal sacrifice. This change 14 was located at the tip of the papilla and was chara cterized by patchy necrosis of interstitial cells, 15 capillaries, and loops of Henle. There was no evid ence of a dose-related response in chronic 16 nephropathy. The incidence of chronic nephropathy was lower in the males of the high-dose 17 group than in controls. In females, the incidence was slightly higher in the test groups than in 18 controls but only achieved statistical significance at the lowest dose. It is likely that the decreas e 19 in liquid intake incurred in the high-dose groups c ontributed to the increased incidence and 20 degree of renal papillary necrosis observed in the high-dose animals because dehydration has 21 been shown to enhance its production by various ana lgesics. 22 The results of this chronic bioassay indicated that formaldehyde is cytotoxic to the 23 epithelial mucosa of the nonglandular (forestomach) and glandular stomach with a LOAEL of 82 24 and 109 mg/kg-day and a NOAEL of 15 and 21 mg/kg-da y in males and females, respectively. 25 The findings provided no evidence of carcinogenicit y in either the GI tract or systemic sites for 26 formaldehyde administered in drinking water to Wist ar rats at doses as high as 82 mg/kg-day. 27

28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-209 Table 4-41. Incidence of lesions observed in the g astrointestinal tracts of 1 Wistar rats after drinking-water exposure to formal dehyde for 2 years 2 3 Incidence of lesions with formaldehyde dose (mg/kg-day) a Males Females 0 1.2 15 82 0 1.8 21 109 Week 53 Number of rats examined b 9 10 10 10 10 10 10 9 Forestomach Focal papillary epithelial hyperplasia 0 0 0 7 0 0 0 5 Glandular stomach Chronic atrophic gastritis 0 0 0 10 c 0 0 0 9 c Focal ulceration 0 0 0 3 0 0 0 1 Focal mononuclear cell infiltrate 1 0 3 0 2 0 0 0 Atypical glandular hyperplasia 0 0 0 0 0 0 0 1 Week 79 Number of rats examined 10 10 10 10 10 9 10 10 Forestomach Focal papillary epithelial hyperplasia 2 1 1 8 1 0 1 9 Glandular stomach Chronic atrophic gastritis 0 0 0 10 c 0 0 0 10 c Focal ulceration 0 0 0 2 0 0 0 0 Focal squamous metaplasia 0 0 0 1 0 0 0 0 Submucosal inflammatory cell infiltrate 1 2 0 0 0 0 0 0 Focal mononuclear cell infiltrate 0 1 0 0 1 1 0 0 Glandular dilation 2 4 4 1 2 2 4 0 Week 105 Number of rats examined b 47 45 44 47 48 49 47 48 Forestomach Focal papillary epithelial hyperplasia 1 2 1 45 c 1 0 2 45 c Focal hyperkeratosis 2 6 4 24 c 3 5 3 33 c Focal ulceration 1 1 1 8 0 0 2 5 Focal acanthosis 1 0 2 1 0 0 0 1 Focal basic cell hyperplasia 0 1 1 0 1 0 0 0 Diverticulum 0 0 1 0 0 0 0 0 Exophytic papilloma 0 1 0 0 1 0 0 0 Glandular stomach Chronic atrophic gastritis 0 0 0 46 c 0 0 0 48 c Focal ulceration 0 0 0 11 c 0 0 0 10 c Glandular hyperplasia 0 1 0 20 c 0 0 0 13 c Mineralization 3 2 1 0 0 0 0 0 Focal inflammatory cell infiltrate 5 3 2 0 2 3 1 0 4

a Incidence in rats that died or were killed when mor ibund during the experiment or were killed at 5 week 53, 79, or 105. 6

b A few rats were lost because of advanced autolysis. 7

c The values differ significantly (Fisher’s exact tes t) from the control value ( p < 0.001). 8 9 Source: Til et al. (1989). 10 11This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-210 Tobe et al. (1989) performed a chronic toxicity stu dy of Wistar rats (Slc:Wistar) exposed 1 to paraformaldehyde dissolved in drinking water. G roups of 20 male and 20 female rats were 2 given formaldehyde solution in their drinking water at concentrations of 0, 0.02, 0.10, and 0.50% 3 for 24 months. Interim sacrifices of six randomly chosen rats from each group were performed 4

after 12 and 18 months. Based on the estimated ave rage amount of water intake and body 5 weight, the actual doses of formaldehyde in either sex were reported to be 0, 10, 50, and 6 300 mg/kg-day. Fresh test solutions were prepared twice each week. The rats were observed 7 daily for the entire study. Body weights and water and diet intake were measured once weekly 8 or biweekly. Hematology (RBC, WBC, and Hb) and ser um clinical chemistry (TP, ALB, BUN, 9 uric acid, total cholesterol, inorganic phosphorous , ALP, AST, and ALT) were made at each 10 necropsy. Organ weights were measured for the brai n, heart, lung, liver, kidney, spleen, adrenal, 11 testis or ovary, pituitary, and thyroid. These org ans and the stomach, small and large intestine, 12 pancreas, uterus, lymph nodes, and all tumors were examined histopathologically. 13 The general condition of animals in the high-dose g roup was poor with significantly 14 reduced body weight gain as well as intake of water and diet. An increase in mortality was also 15 observed in this group. Some clinical chemistry pa rameters were altered in this group. No 16 significant changes in absolute or relative organ w eights were observed. Mortality was 100% in 17 the high-dose group by 24 months. At the 12-month sacrifice, hyperplasia of the squamous 18 epithelium with or without hyperkeratosis was obser ved in the forestomach of all high-dose 19 animals (12/12). Basal cell hyperplasia with growt h into the submucosa was also observed in 20 most cases (10/12). Erosions and/or ulcers with su bmucosal inflammatory cell infiltrates were 21 observed in the glandular stomach of most rats (10/ 12). Regenerative changes of the glandular 22 epithelium (glandular hyperplasia) were noticed in most cases (10/12) along the limiting ridge of 23

the fundic mucosa. No lesions were observed in the glandular stomach at the 50 mg/kg-day 24 dose, and forestomach hyperplasia was observed in o nly one of six males and in one of eight 25

females at 18 and 24 months. No lesions in either the forestomach or glandular stomach were 26 observed in rats treated at 10 mg/kg-day. 27 This study corroborates the Til et al. (1989) study and shows that the main targets for 28 formaldehyde toxicity administered by drinking wate r to rats are the forestomach and glandular 29 stomach. Although the lesions observed at the 50 m g/kg-day were minimal in this study, Tobe et 30 al. (1989) designated the NOAEL at 10 mg/kg-day, fu rther supporting the NOAEL of 15 mg/kg31 day from the Til et al. (1989) study. 32 Takahashi et al. (1986) studied the effects of form aldehyde in an initiation-promotion 33 model of stomach carcinogenesis in male outbred Wis tar rats (Shizuoka Laboratory Center, 34 Shizuoka). Rats (n = 17) were given 100 mg/L of N- methyl-N1-nitro-N-nitrosoguanidine 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-211 (MNNG) in drinking water and a diet supplemented wi th 10% sodium chloride (NaCl) for the 1 first 8 weeks as an initiation phase. This was fol lowed by 0.5% formalin (which contains 12– 2 15% methanol) in drinking water for 32 weeks as the promotion phase of the protocol. A 3 comparison group (n = 10) was given stock water and diet without any supplementation for the 4 first 8 weeks followed by 0.5% formalin in drinking water for 32 weeks. Animals were observed 5 daily and weighed once every 4 weeks. Small pieces of the stomach and other tissues in the 6 peritoneal cavity were fixed for histopathologic ex amination. 7 Body weight gain was reduced by exposure to MNNG wi th sodium chloride, and 8 formaldehyde exposure during the promotion phase ex acerbated this effect. Histopathologic 9 investigations were restricted to the GI tract. Fo rmaldehyde was shown to statistically increase 10

the incidence of lesions in the forestomach and sto mach in the animals initiated with MNNG 11 with NaCl as compared with controls receiving no in itiation (Table 4-42). Increases in 12 papilloma in the forestomach, adenomatous hyperplas ia in the fundus, and adenocarcinoma in 13 the pylorus were observed. Histopathology in the a nimals receiving formaldehyde alone during 14 weeks 9 through 32 showed an increase in forestomac h papillomas but with no lesions in the 15 glandular stomach (Table 4-42). The adenomatous hy perplasia were defined as proliferative, 16 noninvasive mucosal lesions, and the adenocarcinoma s were defined as well differentiated and 17 composed of typical glandular structures, demonstra ting a tubular pattern and cellular or 18 structural atypism without metastasis. No definiti on of criteria for papilloma diagnosis was 19 provided. The findings in this study are inconsist ent with those of Til et al. (1989), who found 20 no evidence of carcinogenicity in a 2-year bioassay at comparable concentrations (assuming 37% 21 formaldehyde in formalin results in 0.19% formaldeh yde in this study). As discussed above, the 22 differences may be due to differences in the strain s of rat or in the diagnostic criteria. The lack 23 of more than one test concentration precludes dose- response analysis of this study and provides 24 only a stand-alone LOAEL of 0.2% formaldehyde in dr inking water. The lack of consumption 25 data precludes an estimation of dose in mg/kg-day. 26This document is a draft for review purposes only a nd does not constitute Agency policy 4-212 DRAFT—DO NOT CITE OR QUOTETable 4-42. Effect of formaldehyde on gastroduodenal carcinogenesis initiated by MNNG and NaCl in maleWistar rats exposed to formaldehyde (0.5% formalin)in drinking water for 8 weeksNo MNNG initiation prior to 8-week oral exposure toformaldehyde (0.5% formalin in drinking water)Glandular stomach tumorsFundus Pylorus DuodenumGastroduodenalcarcinomaForestomachpapillomasAdenocarcinomaAdenomatoushyperplasia AdenocarcinomaPreneoplastichyperplasia AdenocarcinomaControl 0% 0% 0% 0% 0% 0% 0%Formaldehyde 0% 80%a0% 0% 0% 0% 0%MNNG initiation (100 mg/L in drinking water for 8 weeks) prior to 8-week oral exposure to formaldehyde(0.5% formalin in drinking water)Glandular stomach tumorsFundus Pylorus DuodenumGastroduodenalcarcinomaForestomachpapillomasAdenocarcinomaAdenomatoushyperplasia AdenocarcinomaPreneoplastichyperplasia AdenocarcinomaControl 13.3% 0% 0% 0% 3.3% 23.3% 10.0%Formaldehyde 29.4% 88.2%a0% 88.2%a23.5%b41.2% 5.9%aSignificantly different from control animals with MNNG initiation,p< 0.01.bSignificantly different from control animals with MNNG initiation,p< 0.05.Source: Takahashi et al. (1986).This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-213 Soffritti et al. (1989) administered formaldehyde i n drinking water to Sprague-Dawley 1 rats at different ages. Formaldehyde was produced via the Formox process, which yields carbon 2 monoxide, dimethyl ether, and small quantities of C O 2 and formic acid. In one experiment 3 denoted as BT 7001, rats (50/sex/dose) that were 7 weeks old were administered 0, 10, 50, 100, 4 500, 1,000, or 1,500 mg/L for 104 weeks. As is usu al for experiments carried out at the 5 Ramazzini Foundation, all animals were maintained u ntil natural death at which point they were 6 necropsied and examined histopathologically. In ex periment BT 7005, 25-week-old breeder rats 7 or offspring (beginning postnatal day [PND] 12) wer e provided drinking water with 0 or 2,500 8 mg/L formaldehyde for 104 weeks. Fluid and food co nsumption were measured weekly for the 9 first 13 weeks and then every 2 weeks thereafter. Individual body weights were recorded for the 10 first 13 weeks and then every 2 weeks thereafter. Histopathology was routinely performed on all 11 major tissues, including oral and nasal cavities, t he GI tract (esophagus, stomach, and intestines 12 [4 levels]), primary and secondary lymph organs (th ymus, spleen, subcutaneous lymph nodes, 13 mesenteric lymph nodes, mediastinal lymph nodes, an d femur [bone marrow]), head and face 14 bones, and other organ systems (liver, kidney, blad der, reproductive, and various glands). 15 Noncancer health effects were not reported. 16 No GI neoplasia were observed in any of the control rats (experiments BT 7001 and BT 17 7005). Historical controls for the BT experimental colony (n = 5,259) indicate an incidence of 18 approximately 1% for benign neoplasia (papillomas a nd acanthomas) and an incidence of 0.19% 19 for malignant stomach neoplasia (adenomas, SCCs, an d adenocarcinomas, fibrosarcomas, and 20 leiomyosarcomas taken together). Therefore, the si ze of the control groups (18–50 rats/sex) 21 makes detection of background neoplasia unlikely. Similarly, one or two tumors noted in a 22 treatment group (n = 18–50) would represent an appa rent increase in these relatively rare tumors. 23 Although stomach and intestinal tumors were found i n rats exposed to formaldehyde in drinking 24 water, the low incidence makes it difficult to disc ern any dose-response effect for individual 25 neoplasia. The authors report formaldehyde-induced GI tract neoplasia to include benign tumors 26 (papillomas and acanthomas of the forestomach and a denomas) and malignant tumors 27 (adenocarcinomas and leiomyosarcomas). The majorit y of malignant tumors were present in the 28 duodenum, jejunum, and ileum. Benign and malignant neoplasia were consistently noted in the 29 two highest exposure groups: 1500 mg/mL formaldehyd e in experiment BT 7001 and 30 2,500 mg/mL in experiment BT 7005 (Table 4-43). Co mparison of overall GI neoplasia in 31 breeder and offspring rats of experiment BT 7005 su ggests that rats exposed beginning on 32 PND 12 had a greater incidence of malignant tumors. However, it should be noted that, although 33 there may be an apparent increase in overall tumors , summing across sites and locations is 34 needed before a response can be seen. Even then, s everal data points are based on a single 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-214 observed tumor. However, nonspecific MOAs, such as mutagenicity and regenerative 1 proliferation, would be expected to act on all cell types at the POE, and summing tumor types 2 may have some utility. These results constitute a weak positive result for cancer due to oral 3 exposure to formaldehyde. 4

5 Table 4-43. Summary of benign and malignant gastro intestinal tract 6 neoplasia reported in male and female Sprague-Dawle y rats exposed to 7 formaldehyde in drinking water at different ages 8 9

Experiment Dose Sex Total benign tumors Total malignant

tumors All tumors

M 1.08% 0.31% 1.4% Historical controls Not

applicable F 0.97% 0.41% 1.4%

M – a

– – Control

F – – –

M – 2% b 2% b 1,000 mg/mL

F 2% b – 2% b

M 4% 6% 10% BT 7001 (7 weeks old)

1,500 mg/mL

F 6% – 6%

M – – – Control

F – – –

M – 5.6% b 5.6% b BT 7005 breeders (25 weeks old) 2,500 mg/mL

F 5.6% b – 5.6% b

M – – – Control

F – – –

M 5.6% 8.3% 13.9% BT 7005 offspring (PND 12) 2,500 mg/mL

F – 21.6% 21.6% 10

a Dash indicates no tumors reported. An incidence wa s not reported. 11

b Percentage is based on the observation of a single neoplasm. 12 13 Source: Soffritti et al. (1989). 14 15 Oral exposure to formaldehyde resulted in a dose-de pendent increase in all 16 hemolymphoreticular neoplasia in both male and fema le rats in experiment BT 7001 (Table 17 4-44) (Soffritti et al., 1989). The most frequent neoplasia noted were lymphoblastic leukemias 18 and lymphomas. The authors combined lymphoblastic leukemias and lymphosarcomas for 19 analysis and summing across sites. This analysis i s appropriate since there is broad consensus 20 that “neoplasms presenting as solid tumors and thos e presenting with blood and marrow 21 involvement are biologically the same disease but w ith different clinical presentations,” as stated 22 in the recent WHO reclassification of hematological malignancies (Harris et al., 2000b). 23 Inspection of the data tables suggests that the onl y treatment-related effects occurred at 1,000 24This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-215 and 1,500 mg/L. The reported incidence of lymphoid neoplasia and of all hemolymphoreticular 1 neoplasia in rats exposed to the vehicle (methyl al cohol, 15 mg/L) was similar to results reported 2 in rats exposed to 50 or 100 mg/L formaldehyde in d rinking water. Soffritti et al. (1989) 3 provided no statistical analysis of the data. Alth ough the authors report a similar increase in 4 experiment BT 7005, the apparent 5% increase is rep resentative of a single animal in a treatment 5 group of 18 and may not represent a true increase, the study’s usefulness for 6 hemolymphoreticular neoplasia being somewhat limite d by study size. 7

8 Table 4-44. Incidence of hemolymphoreticular neopl asia reported in male 9 and female Sprague-Dawley rats exposed to formaldeh yde in drinking 10 water from 7 weeks old for up to 2 years (experimen t BT 7001) 11 12

Treatment Sex Lymphoid neoplasia (%) Other leukemias (%) All leukemias and

lymphomas (%)

M 4 – a 4 Control F 1 2 3

M 10 – 10 Vehicle control F 2 4 6

M 2 – 2 10 mg/mL F 4 – 4

M 10 – 10 50 mg/mL F 8 – 8

M 8 2 10 100 mg/mL F 4 4 8

M 12 4 16 500 mg/mL F 4 4 8

M 12 – 12 1,000 mg/mL F 10 4 14

M 22 – 22 1,500 mg/mL F 10 4 14 13

a Dash indicates no neoplasm was reported. 14 15 Source: Adapted from Soffritti et al. (1989). 16 17 18 The study of Soffritti et al. (1989) does provide q ualitative corroboration of evidence 19 from other studies that observed formaldehyde toxic ity in the forestomach and stomach. 20 However, the dosages required to induce such lesion s in this study were higher than in other 21 studies. Soffritti et al. (1989) is the only anima l study of oral exposure to formaldehyde that 22 reports an increase in lymphoblastic leukemia or ly mphosarcoma. These occurred at the two 23 highest doses of 1,000 and 1,500 mg/L used in the s tudy of animals exposed from 7 weeks of age 24 (experiment BT 7001). 25

26This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-216 4.2.1.3.3. Summary of toxicity in the GI tract. Short-term and subchronic exposures to 1 formaldehyde via drinking water for 4 weeks yielded slight to moderate histopathologic lesions 2 (focal hyperkeratosis) at 125 mg/kg-day in male and female Wistar rats, as well as slight focal 3 gastritis and submucosal infiltrate in one to two a nimals of both sexes (Til et al., 1988). No 4 histopathologic lesions were noted in albino Spragu e-Dawley rats or beagle dogs that received 5 oral doses of formaldehyde in drinking water for 91 days (Johannsen et al., 1986). In both 6 studies, decreases in weight gain were noted in exp osed animals compared with controls. 7 In a chronic drinking water study, Til et al. (1989 ) reported that formaldehyde is 8 cytotoxic to the epithelial mucosa of the nonglandu lar (forestomach) and glandular stomach with 9 a LOAEL of 82 and 109 mg/kg-day and a NOAEL of 15 a nd 21 mg/kg-day in males and female 10 Wistar rats, respectively. The findings provided n o evidence of carcinogenicity in either the GI 11 tract or systemic sites for formaldehyde administer ed in drinking water to Wistar rats at doses as 12 high as 82 mg/kg-day. The incidence and degree of renal papillary necrosis was increased in 13 animals of the high-dose groups at the terminal sac rifice (Til et al., 1989). Findings by Tobe et 14 al. (1989) corroborate the Til et al. (1989) study and show that the main targets for formaldehyde 15 toxicity administered by drinking water to rats are the forestomach and glandular stomach. 16 Takahashi et al. (1986) studied the effects of form aldehyde in an initiation-promotion model of 17 stomach carcinogenesis in male outbred Wistar rats (Shizuoka Laboratory Center, Shizuoka, 18 Japan). In contrast to Til et al. (1989), Takahash i et al. (1986) found increases in incidence of 19 papilloma in the forestomach, adenomatous hyperplas ia in the fundus, and adenocarcinoma in 20 the pylorus in a 2-year bioassay at comparable conc entrations (assuming 37% formaldehyde in 21 formalin results in 0.19% formaldehyde in this stud y). Soffritti et al. (1989) administered 22 formaldehyde in drinking water to Sprague-Dawley ra ts at different ages. Rats (50/sex/dose, age 23 7 weeks) were administered 0, 10, 50, 100, 500, 1,0 00, or 1,500 mg/L formaldehyde in drinking 24 water for 104 weeks. The authors reported formalde hyde-induced GI tract neoplasia that 25 included benign tumors (papillomas and acanthomas o f the forestomach and adenomas) and 26 malignant tumors (adenocarcinomas and leiomyosarcom as), albeit at a relatively low incidence 27 after summing across sites and locations. Interest ingly, oral exposure to formaldehyde resulted 28 in a dose-dependent increase in all hemolymphoretic ular neoplasia in both male and female rats 29 (Soffritti et al., 1989). The most frequent neopla sia noted were lymphoblastic leukemias and 30 lymphomas. 31

32

4.2.1.4. Immune Function 33 Leach et al. (1983) documented potential immunomodu latory effects of formaldehyde 34 inhalation exposure. F344 rats were exposed nose o nly to formaldehyde 6 hours/day, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-217 5 days/week for up to 30 days. The target concentr ations for exposure were 0, 3, 16, 61, and 1 99 ppm formaldehyde (0, 3.7, 19.7, 75.0, and 122 mg /m 3 ). Body weight and food consumption 2 were recorded, and blood samples for standard hemat ology and immune assays were collected 3 (details not given). Immune measures referenced in clude in vitro lymphocyte transformation, 4 hemagglutination assays, and enumeration of B cells , WBCs, and RBCs. No effects were seen at 5 3 ppm formaldehyde. However, dose-dependent respon ses were reported for weight loss, 6 decreased food consumption, increased WBCs, increas ed segmented neutrophils and nucleated 7 RBCs, and decreased ability to produce antibodies t o sheep RBCs. The results of the 8 lymphocyte transformation assay were inconsistent, with a 25–30% reduction in stimulation after 9 exposure to 99 ppm but an initial stimulation seen after 16 and 61 ppm exposures. Further 10 details were not available, making it difficult to determine if these reported immunomodulatory 11 effects may have been, in part or in full, secondar y to effects on the URT. Subchronic exposures 12 at 61 and 99 ppm formaldehyde would be expected to result in frank toxic effects in mice (see 13 Section 4.2.1). However, these findings suggest po ssible immunomodulatory effects due to 14 formaldehyde exposure and require further explorati on. 15 Dean et al. (1984) investigated the effects of form aldehyde exposure on a range of 16

indicators of immune function. Female B6C3F1 mice were exposed to 15 ppm formaldehyde 17 (18.4 mg/m 3 ) 6 hours/day, 5 days/week for 3 weeks. Three tria ls were run with a total of 255 18 formaldehyde-treated mice. Body and organ weights were recorded at sacrifice for control and 19 formaldehyde-exposed mice (10 per group). Measures of host susceptibility, cell-mediated 20 immunity MP function, and antibody reactions were c onducted 2 to 6 days after the end of 21 exposure (Table 4-45). Lymphocyte subsets, spleen cellularity, bone marrow cellularity, and 22 progenitor cell subsets were enumerated. Host susc eptibility and delayed type hypersensitivity 23 were measured in vivo. Lymphocyte proliferation, n atural killer cell activity, phagocytosis, 24 hydrogen peroxide production, and IgM plaque-formin g cells (PFCs) were measured ex vivo 25

after in vivo stimulation in some cases (Table 4-45). 26 Body weight, organ weights and cellularity, progeni tor cell populations, blood cell 27 counts, and differentials were unchanged in formald ehyde-treated mice (Dean et al., 1984). 28 Circulating blood monocytes were decreased in treat ed mice, which may be a reflection of the 29 local inflammatory response expected in the nasal e pithelium (Dean et al., 1984). However, 30 there was no corresponding decrease in peritoneal M Ps. There was a trend, but no statistical 31 significance, for decreased spleen weight, cellular ity, and B cell precursors (87, 83, and 78% of 32 controls, respectively). The mean body weight of f ormaldehyde-treated mice was 21.1 versus 33 20.9 g in control mice, and thymus and spleen weigh ts were not normalized by body weight. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-218 Table 4-45. Battery of immune parameters and funct ional tests assessed in 1 female B6C3F1 mice after a 3 week, 15-ppm formaldeh yde exposure 2 (6 hours/day, 5 days/week) 3 4 Immune function Model Challenge Metric Tumor resistance PYB6 sarcoma cells Subcutaneous i njection, followed by skin palpation to track tumor development Tumor resistance 16F10 melanoma cells Lung tumor b urden determined by [ 125 I]UdR incorporation Host susceptibility

Bacterial resistance Listeria monocytogenes Survival after challenge

Delayed type hypersensitivity Keyhole limpet

hemocyanin Radiometric index of delayed hypersensitivity responses Lymphocyte proliferation T-cell mitogen, PHA a

B-cell mitogen, LPS b (ex vivo) Ex vivo proliferation, 3 days, measured by [ 3 H]-thymidine incorporation Lymphocyte subsets None Percentage of cells positiv e for cell surface markers (Thy-1, Mac-1, Lyt-1) Cell-mediated immunity

Natural killer cell

activity Yac-1 target cells

( 51 Cr labeled) (ex vivo) % cytotoxicity by 51 Cr release

Phagocytosis Sheep RBCs

( 51 Cr labeled) (ex vivo) 51 Cr incorporation as a measure of RBCs phagocytized MP function (both resident and MVE-1 elicited MP) Hydrogen peroxide

production Pharmacologic

stimulation (ex vivo) H 2 O 2 release in culture

Humoral cell immunity Antibody PFC

responses, IgM PFCs Sheep RBCs,

TVF-LPS, or TNFFicoll Plaques formed

Bone marrow cellularity (femur) None Cell enumeration by a Coulter

counter Granulocyte-MP progenitors None Cell enumeration by a Coulter

counter Progenitor cells

B-cell precursors None Clonogenic assay 5

a T-cell mitogen, phytohemagglutinin (PHA-P). 6

b B-cell mitogen, lipopolysaccharide ( Escherichia coli ). 7 8 Source: Dean et al. (1984). 9 10 11 All indicators of natural killer cell function, cel l-mediated immunity, and humoral 12 immunity in formaldehyde-treated mice were unchange d from controls (Dean et al., 1984). 13 Phagocytic capacity of both resident and elicited p eritoneal MPs was unchanged by 14 formaldehyde treatment. However, hydrogen peroxide production in elicited peritoneal MPs was 15 significantly increased in formaldehyde-treated mic e, 78 versus 42 nmol/mg protein ( p < 0.05) 16 (Dean et al., 1984). 17 As shown in Table 4-46, several indicators of host resistance in the female B6C3F1 mice 18 were increased after formaldehyde exposure (Dean et al., 1984). Tumor mass and pulmonary 19This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-219 foci after B16F10 melanoma cell challenge were sign ificantly reduced in formaldehyde-treated 1 mice, indicating improved tumor immunity ( p < 0.05). However, following PYB6 sarcoma cell 2 challenge, formaldehyde-treated mice had a 7.1% tum or incidence versus 11.1% in controls, 3 which was not statistically different. Mortality d ue to Listeria monocytogenes (LM) was 4 decreased from 70 to 30% ( p < 0.05). Because resistance to LM is primarily MP dependent, the 5 authors speculated that this enhanced resistance mi ght be due in part to increased bactericidal 6 activity as was also suggested by increased hydroge n peroxide production ex vivo in elicited 7 peritoneal MPs from female mice (Dean et al., 1984)

. 8

9 Table 4-46. Summary of the effects of formaldehyde inhalation on the 10 mononuclear phagocyte system (MPS) in female B6C3F1 mice after a 11 3-week, 15 ppm formaldehyde exposure (6 hours/day, 5 days/week) 12 13 In vivo indicators of MPS Metric Formaldehyde effec t Circulating monocytes Decreased a CMF progenitor cells No change a Resident peritoneal MP No change a,b Cellularity

Elicited peritoneal MP No change a,b

LM Increased resistance a B16F10 tumor challenge Increased resistance a In vivo test of host resistance

PYB6 tumor challenge No significant increase a Ex vivo indicators of MPS Cell type/activation Form aldehyde effect Resident, no PMA c None detected a,b

Resident, with PMA None detected a,b

Elicited, no PMA d None detected a,b H 2 O 2 production

Elicited, with PMA Increased a,b Resident No change a Phagocytosis Elicited No change a

Resident Decreased b Elicited No change b Resident No change b Assessment of MP maturation Leucine aminopeptidase content

Elicited No change b Acid phosphatase content Resident No change b Elicited No change b Binding of tumor cells Resident No change b Lysing of tumor cells Elicited Increased at mid-ran ge target-to-effector cell ratio b 14

a Dean et al. (1984). 15

b Adams et al. (1987). 16

c Phorbol 12-myristate 13-acetate (PMA). 17

d Peritoneal MPs were elicited with the pyran copolym er Murray Valley encephalitis virus (MVE-2). 18 19 Sources: Adams et al. (1987); Dean et al. (1984). 20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-220 Overall, the observations of increased hydrogen per oxide production and increased host 1 resistance in peritoneal MPs distant from the POE s uggest that formaldehyde has an effect on the 2 mononuclear phagocyte system (MPS). The authors po stulated that this effect may be indirect, 3 due in part to the tissue inflammatory response in the URT or a direct systemic effect on the 4 MPS by formaldehyde exposure (Dean et al., 1984). Subsequent studies by the same researchers 5 explored the possibility of systemic effects of for maldehyde exposure on MPS function and 6 maturation stage (Adams et al., 1987). Female B6C3 F1 mice were exposed to 15 ppm 7 (18.4 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for 3 weeks , as before (Adams et al., 8 1987). Both resident and Murray Valley encephaliti s virus (MVE-2)-elicited peritoneal MPs 9 were examined for hydrogen peroxide production, enz ymatic activity, phagocytic ability, 10 binding, and lysis of tumor cells (Adams et al., 19 87). 11 Similar to the findings of Dean et al. (1984), form aldehyde treatment increased hydrogen 12 peroxide production almost twofold in MVE-2 elicite d peritoneal MPs (Adams et al., 1987). As 13 summarized in Table 4-46, no treatment differences were seen in phagocytic ability in either 14 resident or elicited MPs (Adams et al., 1987). Res ident peritoneal MPs from formaldehyde15 treated mice were not different in their ability to bind or lyse tumor cells. Although 16 formaldehyde treatment did not increase the ability of elicited MPs to bind tumor cells, lysis of 17 the target cells (P815 tumor cells) was increased f rom 28 to 37% by formaldehyde treatment but 18 only at the midrange target-to-effector-cell ratio tested in the assay ( p < 0.05) (Adams et al., 19 1987). Although this is statistically significant, the authors questioned the biological 20 significance of this result since it was not observ ed at all three target cell ratios tested. However

, 21 an increase in tumor cell lysis in vitro would be c onsistent with the in vivo increased tumor 22 resistance previously reported (Dean et al., 1984). The in vitro lysis response curve suggests that 23 assay conditions may result in a maximum cytolysis near 40%. If so, any treatment effects on 24 lysis would be difficult to discern at higher effec tor cell ratios. 25 Jakab (1992) investigated the effect of formaldehyd e exposure on the alveolar MPs and 26 resistance to respiratory infections. The first se t of experiments assessed bactericidal activity by 27 directly quantifying the pulmonary bacterial loadin g after exposure to Staphylococcus aureus

. 28 White female Swiss mice were exposed to formaldehyd e after bacterial infection (regimens A 29 and C), before bacterial infection (regimen B), or before and after infection (regimen D) 30 (Table 4-47). 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-221 Table 4-47. Formaldehyde exposure regimens for det ermining the effects of 1 formaldehyde exposure on pulmonary S. aureus infection 2 3

Pre-infection treatment Postinfection treatment Res ults

Regimen A None 4 hours 0, 1, 5, 10, or 15 ppm a 15 ppm, increased bacterial loading Regimen B 18 hours

0, 0.5, or 1 ppm

b None No effect

Regimen C None 4 hours 0, 0.5, or 1 ppm No effect

Regimen D 18 hours

0, 0.5, or 1 ppm 4 hours 0, 0.5, or 1 ppm 1 ppm, increased bacterial loading 4

a 0, 1.2, 6.2, 12.3, or 18.5 mg/m 3 formaldehyde. 5

b 0, 0.62, or 1.2 mg/m 3 formaldehyde. 6 7 Source: Jakab (1992). 8 9 10 For regimen A, mice were exposed to 0, 1, 5, 10, or 15 ppm (0, 1.2, 6.2, 12.3, or 11 18.5 mg/m 3 ) formaldehyde. For regimens B–D, mice were expose d to 0, 0.5, or 1 ppm (0, 6.2, or 12 1.2 mg/m 3 ) formaldehyde. A 30-minute exposure to an infecti ous aerosol of S. aureus deposited 13

2 × 10 5 staphylococci in the lungs. Bacterial loading was determined in homogenized lung tissue 14 by culturing diluted aliquots for an estimate of ba cteria present immediately after loading and 4 15 hours later. Bacterial loading was expressed as a percentage change between control and 16 formaldehyde-exposed animals. Mice exposed to 15 p pm formaldehyde for the 4 hours 17 following bacterial infection (regimen A) had appro ximately an 8% increase in bacteria, 18 indicating decreased host resistance ( p = 0.006) (Jakab, 1992) (Table 4-47). Mice receivin g 19 lower concentrations of formaldehyde following bact erial infection did not have increased 20 pulmonary bacterial loading. Pre-infection exposur e to 0.5 or 1.0 ppm did not change bacterial 21 loading 4 hours after infection (regimen B). Howev er, combining an 18-hour pre-infection 22 formaldehyde exposure with a 4-hour postinfection 1 ppm formaldehyde exposure increased 23 pulmonary bacterial loading by approximately 6.5% ( p < 0.05). This effect was not seen with 24 only a 0.5 ppm pre- and posttreatment regimen. Inc reased bacterial loading indicates that 25 formaldehyde exposure (regimens A and D) reduced pu lmonary bacterial resistance. This is in 26 apparent contradiction to the findings of increased host resistance by Dean et al. (1984). 27 However, there are important differences between th e studies. The studies by Jakab (1992) are 28 acute studies examining effects at the respiratory tract where direct effects are possible. 29 Additionally, in some cases, the exposures were con current with bacterial infection, and it is 30 difficult to distinguish the potential for formalde hyde effects directly on the mucociliary 31 apparatus as a barrier to infection. 32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-222 A second set of experiments in the same report (Jak ab, 1992) examined the effects of 1 co-exposure to formaldehyde and carbon black on pul monary infection with S. aureus. The 2 particle size distribution of the carbon black aero sol was less than a 5 μ m aerodynamic diameter 3 and, therefore, 98% respirable. Female Swiss mice were exposed nose only to formaldehyde and 4 carbon black. Experiments were run at two target c oncentrations: (1) 2.5 ppm (3.1 mg/m 3 ) 5 formaldehyde and 3.5 mg/m 3 carbon black or (2) 5 ppm (6.2 mg/m 3 ) formaldehyde and 6 10 mg/m 3 carbon black. Co-exposure was given either for 4 hours after a 30-minute S. aureus 7 infection or 4 hours/day for 4 days as a pretreatme nt prior to S. aureus infection. Bacterial 8 loading was determined 0 and 4 hours after the S. aureus infection to assess bacterial survival. 9 Formaldehyde-carbon black co-exposure did not alter bacterial survival either as a pretreatment 10 or posttreatment to bacterial exposure. However, t his exposure regimen was not run for 11 formaldehyde or carbon black separately, and the 4 hours/day for 4 days pretreatment was not 12 included in the formaldehyde alone experiments (Tab le 4-47). 13 Jakab (1992) also assessed the phagocytic activity of alveolar MPs collected by lavage at 14 various time points after formaldehyde, carbon blac k, or co-exposure. Female Swiss mice were 15 co-exposed to 5 ppm (6.2 mg/m 3 ) formaldehyde and 10 mg/m 3 carbon black 4 hours/day for 16 4 days. Mice were sacrificed and alveolar MPs harv ested 1, 3, 5, 25, and 40 days after exposure. 17 Mice exposed only to formaldehyde or carbon black w ere sacrificed 3, 10, 25, and 40 days after 18 exposure. Fc-receptor-mediated phagocytosis was as sessed ex vivo by using sensitized sheep 19 RBCs. The phagocytic index (PI) was reported as th e total number of RBCs in 100 MPs. 20 Neither formaldehyde nor carbon black exposure alon e significantly changed the PI (Jakab, 21 1992). These findings are consistent with the firs t co-exposure experiment, since no changes in 22 PI were seen immediately after exposure. However, co-exposure did decrease the PI of alveolar 23 MPs in a time-dependent manner, with maximal decrea se to less than 70% of controls by 25 days 24 after exposure (Figure 4-11). Decreases in the PI reflect changes in both the percentage of 25 phagocytic MPs and the number of RBCs phagocytized (Jakab, 1992). The PI recovered to 26 control levels by 40 days postexposure. 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-223 1

100 110

90

80

70

60

50

0 10 20 30 40

Days after last exposure HCHO

Carbon

Carbon + HCHO

2 3 Figure 4-11. Alveolar MP Fc-mediated phagocytosis from mice exposed to 4 5 ppm formaldehyde, 10 mg/m 3 carbon black, or both. 5 6 Note: Exposure was 4 hours/day for 4 days. Each va lue represents the mean ± 7 SEM of five determinations. 8 9 Source: Redrawn from Jakab (1992). 10 11 12 Holmström et al. (1989b) evaluated the effects of l ong-term formaldehyde exposure on 13 antibody production. Female Sprague-Dawley rats we re exposed to 12.6 ppm formaldehyde 14 (15.5 mg/m 3 ) 6 hours/day, 5 days/week for 22 months. Body wei ght, tumor incidence, and 15 pathology were reported elsewhere (Holmström et al. , 1989b). Rats were given a subcutaneous 16 injection of pneumococcal polysaccharide antigens o r tetanus toxoid 21 to 25 days prior to 17 sacrifice. The two vaccines chosen represent T-cel l-dependent and T-cell-independent antigens, 18 respectively. Antibody titers (IgG and IgM) were d etermined prior to vaccination and at 19 sacrifice. Formaldehyde treatment had no effect on antibody titers either before or after 20 vaccination (Holmström et al., 1989b). 21

22

4.2.1.4.1. Summary of formaldehyde effects on immune functio n. Although there were initial 23 reports of systemic immunomodulation attributed to formaldehyde exposure (Leach et al., 1983), 24 formaldehyde effects on measures of humoral and cel l-mediated immunity were not confirmed 25 by Dean et al. (1984). The authors did report incr eased host resistance to both tumor and 26 bacterial tumor challenges after a 3-week exposure to 15 ppm formaldehyde. An increased 27Phagocytic index (% of control)Phagocytic index (% of control)This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-224 resistance to these challenges, presented distal to the site of formaldehyde exposure 1 (administered subcutaneously or intravenously), sug gests a systemic effect of formaldehyde 2 exposure. In addition, increased host resistance a nd hydrogen peroxide release from peritoneal 3 MPs were reported and confirmed (Adams et al., 1987 ; Dean et al., 1984). Chronic 4 inflammation and tissue damage to the respiratory m ucosa expected with formaldehyde exposure 5 may result in an up regulation of the MPS and there fore increase host immunity. It is unclear if 6 this response would be specific to formaldehyde or similar to enhancement of immune function 7

seen with chronic inflammation. 8 Jakab (1992) demonstrated decreased pulmonary resis tance to bacterial infection where 9 animals were exposed to 15 ppm formaldehyde immedia tely after bacterial loading or when they 10 were given an 18-hour pre-exposure to formaldehyde followed by 1 ppm formaldehyde exposure 11 after bacterial loading. The authors speculated th at formaldehyde may directly act on pulmonary 12 MPs, reducing their effectiveness. However, Jakab (1992) showed that there was no change in 13 Fc-mediated phagocytosis of alveolar MPs immediatel y after formaldehyde exposures. 14 Degradation of the protective mucus layer and possi ble epithelial cell damage may contribute to 15 more effective bacterial infection in the presence of formaldehyde without a direct action on MP 16 function. As mentioned above, degradation of the m ucus layer may result in a more potent 17 inoculation and therefore higher bacterial loading. 18 Although neither formaldehyde nor carbon black alon e impacted Fc-mediated 19 phagocytosis of alveolar MPs, Jakab (1992) demonstr ated that there was decreased Fc-mediated 20 phagocytosis after formaldehyde and carbon black co -exposure. Carbon black may have acted as 21 a carrier for formaldehyde, allowing higher levels of formaldehyde to be delivered more deeply 22 into the lungs than would be seen with formaldehyde alone. 23 Formaldehyde is known to break down the mucus layer protecting the respiratory tract, 24 allowing exposure of the underlying epithelium (Mor gan et al., 1986a, c, d). Additionally, 25 formaldehyde can directly induce tissue inflammatio n through sensory irritation via substance P 26 from the trigeminal nerve (Fujimaki et al., 2004a). These actions together could contribute to 27 some of the observed effects on immune response att ributed to formaldehyde exposures. 28 Degradation of the protective mucus layer would mak e antigens more available to the immune 29 system. It has been shown that direct application of an antigen to the nasal associated lymph 30 tissue, bypassing the mucus layer, is a more effect ive delivery of antigen (Hou et al., 2002). 31 Therefore, increased availability of these antigens to the immune system may in part explain 32 observed increased antibody production seen against ovalbumin (OVA) or common dust mite 33 allergen (Der f) during formaldehyde exposure (Sada kane et al., 2002; Riedel et al., 1996; 34 Tarkowski and Gorski, 1995). Neurogenic inflammati on may also contribute to more efficient 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-225 antigen processing and presentation by activation o f resident MPs. These factors are consistent 1 with the observation that formaldehyde exposures do not affect antibody production to antigens 2 administered outside of the respiratory tract, even after chronic exposures (Holmström et al., 3 1989b). 4 This effect was initially observed several days aft er exposure was ended with maximal 5 suppression seen 25 days after a 4-day formaldehyde exposure. The delayed onset of this 6 response, however, suggests an effect beyond the PO E effects observed at the time of exposure. 7 Table 4-48 presents a summary overview of the effec ts of formaldehyde on immune function in 8 laboratory animals. 9

10

4.2.1.5. Hypersensitivity and Atopic Reactions 11 Adverse reactions in humans exposed to formaldehyde in the workplace and homes have 12 been reported, which are consistent with an allergi c response or a chemical sensitivity (see 13 Section 4.1.1 for details). Rashes and skin reacti ons are reported in some individuals after 14 dermal exposures, and in some cases exacerbation of asthma is reported after inhalation of 15 formaldehyde. However, the reports of human reacti ons do not allow a clear determination of 16 whether this sensitization is immunogenic or neurog enic in origin. Formaldehyde-induced 17 sensitization may have both neurogenic and immunolo gic components. Numerous animal 18

studies have been conducted in order to understand the potential for sensitization to 19 formaldehyde. Although hypersensitivity and allerg ic sensitization are often considered solely 20 immunologic in origin, neurogenic mechanisms may re sult in bronchial hypersensitivity and 21 increased immunologic sensitization. Therefore, th e animal studies regarding formaldehyde22 induced sensitization are evaluated discretely in o rder to examine these etiologic possibilities. 23 Classically, hypersensitivity is characterized as a n immune response to an antigen, 24 resulting in an inflammatory reaction that itself d amages the tissues or is otherwise harmful 25 (Kuby, 1991). These reactions may be localized, as in topical dermatitis, or systematic, as in 26 anaphylactic shock from an allergen. Hypersensitiv ity can be mediated by a humoral immune 27 response or by a cell-mediated immune response. Fo ur classes of hypersensitivity are generally 28 recognized that differ in their immune system compo nents and functions. Although a single 29 agent (e.g., penicillin) may induce all four types of hypersensitivity, it is more usual for an agent 30 to primarily induce one form of hypersensitivity. 31This document is a draft for review purposes only a nd does not constitute Agency policy 4-226 DRAFT—DO NOT CITE OR QUOTETable 4-48. Summary of immune function changes dueto inhaled formaldehyde exposure in experimental animalsSpeciesNo./groupTreatmentaObservationsLOAEL/NOAELReferenceF344 rats 8 0, 3, 16, 61, 99 ppm6 hours/day,5 days/week for4 weeksNo effects at 3 ppm. Mixed results at higher dosesthat were notconsistent.NAbLeach et al.(1983)B6C3F1mice(female)10 15 ppm 6 hours/day,5 days/week for3 weeksIncreased H2O2production, and increased host resistance to tumorformation, but other immune parameters unchanged.LOAEL 15 ppm Dean et al.(1984)B6C3F1mice(female)PooledMPs froma numberof mice15 ppm 6 hours/day,5 days/week for3 weeksIncreased H2O2production in MVE-2-elicited peritoneal MPs. LOAEL15 ppm Adams et al.(1987)WhiteSwiss mice(female)18 0, 1, 5, 10, or50 ppm for 18 hoursbefore and/or4 hours after a30-minute exposureto bacterial infection(S. aureus)Combining an 18-hour pre-exposure to formaldehyde with 4-hourpostexposure to formaldehyde increased bacterial loading at 1 ppm by6.5%.LOAEL 1 ppm Jakab (1992)_WhiteSwiss mice(female)18 5 ppm (2.6 mg/m3)formaldehyde and10 mg/m3carbonblack 4 hours/day for4 daysPhagocytic index was decreased by co-exposure to formaldehyde andcarbon black but not by either insult alone.NA Jakab (1992)SpragueDawley rats(female)5 12.6 ppm6 hours/day,5 days/week,22 monthsFormaldehyde treatment had no effect on antibody titers either beforeor after vaccination with pneumococcal polysaccharide antigen ortetanus toxoid.NA Holmström etal. (1989b)NA = not applicable.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-227 Chemical sensitivity generally implies a neurogeni cally induced sensitization (Meggs, 1 1995). A chemical may directly interact with senso ry nerves, releasing mediators that trigger 2 inflammation, such as substance P (a tachykinin). Repeated exposure to the same chemical is 3 hypothesized to potentiate neurogenic inflammation (Meggs, 1995). The resulting signs of tissue 4 inflammation may be similar to immunogenic inflamma tion, but there would be no requirement 5 that the immune system recognize the chemical as an antigen for this type of response. 6 Therefore, a chemical may induce one or more clinic al signs of atopic asthma without a type 1, 7 IgE-mediated hypersensitivity response. One form o f sensitivity, , directly affects sensory nerve 8 endings, resulting in neurogenic inflammation and i s a well-known health effect attributed to 9 formaldehyde. Neurogenic responses may result from the direct and acute interaction of the 10 chemical with sensory nerve ending receptors of the trigeminal nerve that may lead to persistent 11 rhinitis and an asthma-like reactive airway dysfunc tion syndrome that may develop after short12 term human exposures (Brooks et al., 1985). Thus, there is evidence to suggest that neurogenic 13 inflammation may contribute to observed increases i n formaldehyde-induced airway 14 hyperresponsiveness and atopic responses. The avai lable animal studies that have investigated 15 formaldehyde-induced airway hyperresponsiveness and atopic responses are summarized below. 16

17

4.2.1.5.1. Inhalation studies in experimental animals. This section summarizes animal studies 18 informing the role of formaldehyde-induced chemical sensitization. The symptoms of 19 sensitization (atopy, airway hyperresponsiveness) a re frequently associated with immunologic 20 markers (cytokine production, leukocyte infiltratio n histamine release, and antibody production) 21 but may be mediated by neurogenic sensory irritatio n, principally by activation of the trigeminal 22 nerve (see Section 4.1.1.1 for a discussion of sens ory irritation). The animal studies that 23 illuminate these neurogenic and immunologic respons es are discussed outside of the classic 24 neurotoxicology and immunotoxicology study summary sections to allow synthesis of these data. 25 Sensitization to chemical exposure by inhalation of ten manifests as an allergic or 26 asthmatic response as characterized by BC or BHR. This sensitization may be a result of 27 immune involvement, as in the case of hypersensitiv ity, or a neurogenic sensitization, where a 28 chemical may directly stimulate inflammation. Asth ma is a specific manifestation of IgE29 mediated hypersensitivity, characterized by BHR and airway inflammation, resulting in lower 30 airway obstruction (Fireman, 2003; Kuby, 1991). In asthma, an allergen capable of cross-linking 31 membrane-bound IgE on mast cells initiates immunoge nic inflammation resulting in an influx of 32 eosinophils, neutrophils, and lymphocytes. Mediato rs of BC, including histamine, eicosanoids, 33 and bradykinin (Kuby, 1991), are released during th is process. Prior exposure to the allergen can 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-228 increase allergen-specific IgE, potentiating the al lergic reaction; this is immunogenic 1

sensitization. 2 Biagini et al. (1989) evaluated the effect of a sin gle pulmonary exposure of formaldehyde 3 on pulmonary mechanics, including BC. The research ers chose cynomolgus monkeys known to 4 be hyperreactive to methacholine (acetyl- β -methacholine chloride), which is a direct-acting 5 stimulant of BC (Cain, 2001). Measures of pulmonar y mechanics included pulmonary flow 6 resistance; dynamic compliance; PEFR; FVC; FEV; FEF 25–75% , and 50% of VC; and FEFs 7 normalized for VC. Nine cynomolgus monkeys were ex posed to increasing levels of 8 methacholine for 1 minute at 10-minute intervals (0 , 0.125, 0.5, 2, and 8 mg/mL) as an aerosol 9 (0.065 mL/minute with a mean aerodynamic diameter o f 1.0–1.5 μ m). Pulmonary mechanics 10 were measured to establish each monkey’s response t o methacholine. Methacholine challenge, 11 as the positive control, increased pulmonary flow r esistance at increasing levels of methacholine 12 (0.125, 0.5, 2, and 8 mg/mL) to 196 ± 16, 285 ± 57, 317 ± 64, and 461 ± 120 % of baseline 13 levels, respectively. After a 2-week recovery peri od, each methacholine-sensitized monkey was 14 exposed to 2.5 ppm formaldehyde (generated from for malin, 15% methanol) for 10 minutes. 15 Measures of pulmonary function were performed at 2, 5, and 10 minutes after exposure. 16 Formaldehyde exposure increased pulmonary flow resi stance from 11.3 ± 1.4 cm H 2 O 17 prior to formaldehyde exposure to 16.1 ± 2.1, 16.9 ± 2.8, and 20.0 ± 3.4 cm H 2 O at 2, 5, and 18 10 minutes after 2.5 ppm formaldehyde exposure (wit h 142, 150, and 177 % change, 19 respectively). All other measures of formaldehyde- induced pulmonary mechanics were not 20 significantly different from controls. Increased p ulmonary flow resistance, a measure of 21 increased BC, was induced by formaldehyde challenge in previously sensitized mice. However, 22 the differences between methacholine challenge and formaldehyde challenge were not 23 statistically significant. Although both formaldeh yde challenge and methacholine challenge 24 increased pulmonary flow resistance, there was no c orrelation between individual methacholine 25 responsiveness and the magnitude of effect after fo rmaldehyde exposure ( p > 0.1). Therefore, 26 although formaldehyde exposure stimulated BC simila rly to a known direct stimulating agent, 27 formaldehyde may not work through the same site of action as methacholine. 28 Swiecichowski et al. (1993) assessed pulmonary resi stance and airway reactivity due to 29 formaldehyde exposure alone and in response to incr easing doses of acetylcholine chloride (a 30 direct-acting BC agent) after formaldehyde exposure in vivo. Male Hartley guinea pigs (eight 31 per group) were exposed at 0.86, 3.4, 9.4, or 31.1 ppm (1.1, 4.2, 11.6, or 38.3 mg/m 3 ) 32 formaldehyde for 2 hours or at 0.11, 0.31, 0.59, or 1.05 ppm (0.14, 0.38, 0.73, or 1.29 mg/m 3 ) 33 formaldehyde for 8 hours. Total pulmonary resistan ce increased after 2 hours formaldehyde 34 exposure at 9.4 and 31.1 ppm and reached similar pe ak resistance at the end of the exposure 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-229 period. This effect was rapidly reversible, with v alues returning to baseline within 30 minutes 1 after exposure. Although 2-hour exposures at 3 and 1 ppm did not increase pulmonary 2 resistance, 8-hour exposures at 0.3 and 1 ppm did i ncrease pulmonary resistance to similar levels 3 as the 2-hour exposure at 30 ppm. The results indi cate that both concentration and exposure time 4 impacted the measured increase in pulmonary resista nce. However, a simple multiplicative 5 model (e.g., C × t) does not adequately represent t he effects observed. It is noted that an 8-hour 6 exposure at 1 ppm (8 ppm-hours), reached approximat ely the same pulmonary resistance as 2 7 hours at 9.4 ppm (19 ppm-hours). This may in part be due to a maximum practical increase in 8 pulmonary resistance in the animals. Conversely, t here was no effect at 3 ppm for 2 hours 9 (6 ppm-hours), although significant increase in pul monary resistance was recorded after an 10 8-hour exposure at 0.3 ppm (2.4 ppm-hours). Formal dehyde does not appear to exert its effects 11 via a classic C × t paradigm. Exposure concentrati on, however, did seem to impact recovery 12

time. 13 In addition, specific pulmonary resistance and air way reactivity to increasing doses of 14 intravenous acetylcholine chloride, a direct respir atory stimulant, were measured immediately 15 after formaldehyde exposure for up to 60 minutes. Formaldehyde-induced airway 16 hyperreactivity was defined as a decrease in the le vel of acetylcholine chloride needed to 17 produce twice the basal specific resistance (effect ive dose [ED] 200 ). The dose of acetylcholine 18 chloride required to double the specific pulmonary resistance (ED 200 ) and airway reactivity was 19 decreased in animals exposed for 2 hours to formald ehyde. When the duration was extended to 8 20 hours of formaldehyde exposure, the effective dose of formaldehyde required to elicit a doubled 21 pulmonary resistance (ED 200 ) in the presence of acetylcholine chloride was dec reased to 22 1.07 ppm. Lower ED 200 s were recorded in formaldehyde-treated animals. T his indicates that 23 less acetylcholine was needed to produce BC when fo rmaldehyde was present. Thus, 24 formaldehyde can exacerbate BHR. Additionally the formaldehyde-induced effect increased 25 with duration of exposure, indicating that time as well as exposure concentration are factors in 26 the magnitude of the response. Directly induced in creases in airway hyperreactivity peaked 1 27 hour after exposure and persisted 6 hours after exp osure. 28 In a second set of experiments, male Hartley guinea pigs were treated for 8 hours at 29 3.4 ppm (4.2 mg/m 3 ) in order to measure airway hyperreactivity ex viv o (Swiecichowski et al., 30 1993). After formaldehyde exposure, tracheae were excised and mounted in tissue baths, where 31 tracheal contraction was measured in response to di rect application of acetylcholine and then 32 carbachol. Tracheae from similarly exposed guinea pigs were fixed and sectioned for histologic 33 examination and were assessed for signs of inflamma tion. Formaldehyde exposure did not 34 increase ex vivo tracheal constriction and suggests that changes in airway reactivity were 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-230 produced due to both local humoral changes and neur al reflexes. However, no changes in 1 epithelial cell morphology or influx of inflammator y cells were recorded even up to 4 days after 2 formaldehyde exposure ended. The authors speculate d on possible MOAs for BHR, such as the 3 role of an irritant receptor or altered epithelial cell biochemistry. It may be that the window of 4 acute inflammation occurred early in the exposure p rotocol and was resolved by the time of first 5 measurement, after 8 hours of exposure. The absenc e of inflammatory markers may argue 6 against a classic type 1 sensitivity. 7 The binding of an allergen to receptor bound IgE tr iggers degranulation of mast cells and 8 basophils, releasing mediators of type 1 hypersensi tivity, including the histamine responsible for 9 BC. Brown Norway (BN) rats are known for their hig h capacity for IgE production and airway 10 hyperresponsiveness in response to allergens or oth er chemicals; they have often been used as a 11 model of allergic respiratory disease. Ohtsuka et al. (1997) compared the effects of 12 formaldehyde exposure on the nasal epithelium of F3 44 and BN rats. If the formaldehyde13 induced inflammatory response in the nasal epitheli um is IgE mediated, BN rats would be 14 expected to display more severe effects of formalde hyde exposure than F344 rats. Both strains 15 of age- and sex-matched rats were exposed to formal dehyde aerosol for 3 hours/day, 5 16 days/week for 2 weeks. The aerosol was generated from a 1% formaldehyde solution by a two17 fluid atomizer, and formaldehyde level was maintain ed at 2 mg (1% sol.)/L (approximately 18 16 ppm or 20 mg/m 3 ), by adjusting the flow rate for formaldehyde solu tion to the atomizer. 19 During the course of exposure, the following clinic al signs were monitored: abnormal 20 respiration, stridor wheezing, nasal discharge, and sneezing. Rats were weighed weekly. Two 21 days postexposure, rats were sacrificed and tissues from the head, trachea, and lungs were fixed 22

and sectioned. Transverse sections were taken at t he following palatal landmarks from three 23 animals: level 1 (lateral edge of incisor teeth), l evel 2 (between incisive papilla and the first 24 palatal ridge), and level 3 (on the second upper mo lar). The nasal septa of the remaining two 25 animals were revealed for examination by electron m icroscopy. 26 Formaldehyde-treated F344 rats showed less body wei ght gain over the 2-week 27 treatment, resulting in lower body weight at week 1 and week 2 than F344 controls ( p < 0.05 and 28 0.01). Body weights of formaldehyde-treated BN rat s were unchanged from BN controls. The 29 authors observed fewer clinical signs of respirator y irritation in the formaldehyde-exposed BN 30 rats compared with formaldehyde-exposed F344 rats, such as abnormal respiration (three versus 31 five) and nasal discharge (three versus five). His tologic analysis of lung and trachea tissues 32 revealed no distinct signs of inflammation in eithe r strain. Formaldehyde exposure induced cell 33 damage in URT tissues. Epithelial cell damage was milder and impacted a smaller portion of the 34 URT in BN rats compared with F344 rats. Squamous m etaplasia were present in the respiratory 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-231 epithelium (levels 1 and 2) in both strains in form aldehyde-treated rats. However, a distinct 1 keratinized layer was noted in level 1 epithelium o f F344 rats, and the extent of lesions in level 2 2 respiratory epithelium was much greater than that s een in BN rats. Additionally, the olfactory 3 epithelium (level 2) in formaldehyde-exposed F344 r ats exhibited degeneration, necrosis, and 4 desquamation not seen in BN rats. Mild squamous me taplasia was noted in level 3 of the 5 respiratory epithelium in the treated F344 rats but not the treated BN rats. No pulmonary 6 function measurements were taken, and, thus, no dir ect comparison in BHR or BC between BN 7 and F344 rats in response to formaldehyde can be ma de. It appears that BN rats are more 8 resistant to formaldehyde-induced cell damage than are F344 rats, despite the fact that BN rats 9 are known to be IgE responders. These results sugg est that IgE responsiveness may be 10 protective of formaldehyde-induced cell damage, or IgE may not play a role at all. The authors 11

note that their earlier research indicated the BN r ats have well-developed submucosal glands and 12 speculate that greater mucus flow may be partly res ponsible for the greater resistance of BN rats 13 to the histologic signs of formaldehyde toxicity. 14 In a subsequent study in the same laboratory, Ohtsu ka et al. (2003) compared histology 15 and cytokine profiles in the nasal mucosa of formal dehyde-treated F344 and BN rats. 16 Formaldehyde aerosol was generated as above and rat s (nine per group) were exposed 17 3 hours/day for 5 days to approximately 16 ppm of f ormaldehyde (20 mg/m 3 ). Clinical signs 18 were recorded daily, and monitored respiratory para meters included abnormal respiration, stridor 19 wheezing, nasal discharge, and sneezing. Tissue se ctions of the nose (five rats per group) were 20 prepared for light microscopy as above: transverse sections at levels 1, 2, and 3. Th-1 cytokines 21 (IFN- γ , IL-2) and Th2 cytokines (IL-4 and IL-5) were dete rmined from the whole nasal mucosa 22 in four rats of each treatment group. 23 As expected, lesions and neutrophilic infiltration were more severe in F344 24 formaldehyde-exposed rats compared with treated BN rats. In addition, lesions were observed in 25 all three levels of epithelium examined in F344 rat s and impacted both respiratory and olfactory 26 epithelium. Mucosal lesions in formaldehyde-treate d BN rats impacted the respiratory 27 epithelium of levels 1 and 2 only. Changes in form aldehyde-induced cytokine mRNA 28 expression were modest in both strains. Th-1-relat ed cytokines (IFN- γ, Il-2) in formaldehyde29 treated BN rats were significantly decreased compar ed with control BN rats. A similar, although 30 not statistically significant, decrease in Th-2 cyt okines (IL-4, IL-5) was observed in 31 formaldehyde-treated BN rats compared with unexpose d BN rats. There were no treatment 32 differences in either Th-1 or Th-2 cytokine express ion in formaldehyde-treated F344 rats 33 compared with unexposed F344 rats. The modest chan ges in cytokine profile reported in 34 formaldehyde-treated BN rats were not consistent wi th type 1 hypersensitivity since type 1 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-232 hypersensitivity reactions generally result in incr eased Th-2 cytokines. The mRNA expression 1 results were not corroborated with protein levels a nd may not have been captured at their peak 2 expression levels. 3 Lee et al. (1984) evaluated the potential for forma ldehyde to act as a sensitizing agent 4 through different routes of exposure in guinea pigs . The inhalation studies will be highlighted 5 here. Dermal exposure and associated contact sensi tivity results will be discussed in the dermal 6 exposure section (4.2.1.5.2). Three groups of male English smooth-haired guinea pigs 7 (four/group) were exposed via inhalation to either 6 or 10 ppm (7.4 or 12.3 mg/m 3 ) 8 formaldehyde 6 hours/day for 5 consecutive days. D epending on the group, animals were then 9 subjected to bronchial provocation challenge with 2 or 4 ppm formaldehyde on day 7 or days 7, 10 22, and 29 after exposure (see Table 4-49 for clari fication). 11

12 Table 4-49. Study design for guinea pigs exposed t o formaldehyde through 13 different routes of exposure: inhalation, dermal, a nd injection 14 15

Formaldehyde

exposure Bronchial provocation challenge Skin test Blood drawn for antibody titer Group I—Inhalation 6 ppm formaldehyde a

, days 1–5 Day 7 2 ppm formaldehyde a for 1 hour Day 9 Day 14

Group II—Inhalation 10 ppm formaldehyde, days 1–5 Day 7 2 ppm formaldehyde for 1 hour Day 9 Day 14

Group III—Inhalation 10 ppm formaldehyde, days 1–5 Days 7, 22, and 29 4 ppm formaldehyde for 4 hours Day 31 Day 14

Group IV—Dermal 100 μ L formalin, days 1 and 3 Day 22 2 ppm formaldehyde for 1 hour 4 ppm formaldehyde for 4 hours Day 7 Day 14

Group V—Injection 37 mg formaldehyde with Freund’s adjuvant Day 19 2 ppm formaldehyde Day 7 Day 14

16 Source: Lee et al. (1984). 17 18 19 Dermal and injection groups are shown for compariso n. Pulmonary hypersensitivity was 20 assessed by measuring respiratory rate and tidal vo lume in response to exposure to 2 ppm 21 formaldehyde challenge for 1 hour, 2 days postexpos ure for all three groups, and additional 22 measurements were taken 22 and 29 days postexposure for group III. Blood was drawn to 23 characterize IgE antibodies to formaldehyde in a pa ssive cutaneous anaphylaxis (PCA) assay. 24 Respiratory rate was measured following initial for maldehyde exposure and again after bronchial 25 challenge with formaldehyde. 26This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-233 Respiratory rate (exhibited as a pause during expir ation) was depressed by 45% following 1 exposure to 10 ppm formaldehyde during the first ho ur of exposure. During the first hour of 2 exposure, decreased respiratory rate was accompanie d by a pause during expiration that has been 3 categorized as RB and indicated sensory irritation. The decreased respiratory rate is consistent 4 with URT sensory irritation and induction of the tr igeminal (neurogenic) reflex (Lee et al., 5 1984). After the first hour of exposure, decreased respiratory rate was characterized by a pause 6 between breaths, which is similar to the breathing pattern seen in mice exposed to formaldehyde 7 via tracheal cannula (Alarie, 1981). This suggests a separate effect of formaldehyde on the LRT 8 after deep penetration of formaldehyde and suggests pulmonary irritation (Lee et al., 1984). 9 However, subsequent bronchial provocation challenge with either 2 or 4 ppm 10 formaldehyde for either 1 or 4 hours failed to elic it immediate or delayed-onset respiratory 11 sensitization (Table 4-50). Respiratory rates were reported as being within ±20% of pre12 challenge levels (data not shown) and did not refle ct statistical significance (Lee et al., 1984). 13 Moreover, increased respiratory sensitivity was not observed in animals that had received an 14 emulsification of formaldehyde and Freund’s complet e adjuvant by injection. Only two to four 15 animals given formaldehyde injections in the presen ce of Freund’s complete adjuvant developed 16 a low titer of antibodies to formaldehyde (Lee et a l., 1984). 17

18 Table 4-50. Sensitization response of guinea pigs exposed to formaldehyde 19 through inhalation, topical application, or footpad injection 20 21 Exposure route Pulmonary sensitization Dermal sensi tization Antibody production

Inhalation 6 ppm (Group I) 10 ppm (Group II) 10 ppm (Group III) 0/4 0/4 0/4 0/4 0/4 2/4 0/4 0/4 0/4 Topical 0/8 8/8 0/8 Injection 0/4 4/4 2/4 22 Source: Lee et al. (1984). 23 24 25 Thus, inhalation exposure to 6 or 10 ppm formaldehy de (8 hours/day for 5 days) followed 26 by bronchial challenge with 2 or 4 ppm formaldehyde failed to result in respiratory sensitivity 27 defined as greater than 20% change in respiratory r ate. Second, for animals that received an 28 injection of formaldehyde with Freud’s adjuvant, it was not effective in inducing pulmonary 29 sensitivity. While neither inhaled formaldehyde ch allenge nor injected formaldehyde and 30 Freud’s adjuvant emulsion were effective in produci ng pulmonary sensitivity, this study relied 31 on increased respiratory rate as an indication of h yperresponsiveness and may not be an accurate 32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-234 measure of hyperresponsiveness. Thus, overall, con clusions are uninformative due to study 1 design flaws. 2 Riedel et al. (1996) tested the effects of formalde hyde inhalation on the development of 3 sensitization to a known allergen. Female Perlbrig ht-white Dunkin-Hartley guinea pigs (12 per 4 group) were exposed to 0, 0.13, or 0.25 ppm formald ehyde 8 hours/day for 5 consecutive days. 5 On day 5, the animals were sensitized to the common model allergen, OVA, in a 3-minute, head6 only exposure to an aerosol of a 5% OVA solution. A booster sensitization with OVA occurred 7 on day 19. A compressor nebulizer with an output r ate of 0.75 mL/minute generated the aerosol. 8 Particle size ranged from 0.5 to 5.0 μ m. On day 26, bronchial provocation testing was co nducted 9 with 1% OVA challenge (aerosol). Blood samples wer e taken and anti-OVA IgG antibodies 10 were quantified by ELISA. Significant airway obstr uction was defined as an increase in 11 compressed air in the lung that cannot be expired. Three guinea pigs were exposed to 12 formaldehyde (0.20 ppm) or clean air for 5 days. I mmediately after exposure, lung and tracheal 13 tissues were fixed for histologic and morphometric evaluation. Wall thickness of bronchial and 14 alveolar septa was measured systematically with a m icroscope-digitizing-table set. 15 Significant airway obstruction as measured by compr essed air was seen in 3 of 12 16 controls, 8 of 12 0.13 ppm-exposed, and 10 of 12 0. 25 ppm-treated animals after OVA challenge. 17 The average airway obstruction was increased after 0.25 ppm (mean = 0.35 mL, p < 0.01) but not 18 after 0.13 ppm formaldehyde exposure. However, ind ividual response to OVA sensitization was 19 highly varied, and animals exhibiting a 10-fold inc rease in obstruction (measured as compressed 20 air) were seen in both treatment groups (0.13 and 0 .25 ppm). Even at the lower exposure (0.13 21 ppm), biologically significant responses were seen in individuals (Figure 4-12). 22 Specific anti-OVA antibodies (IgG1 class) were not detected in animals prior to 23

sensitization or in control-treated animals after s ensitization . Measurable anti-OVA antibodies 24 were elevated in 3 of 12 (at 0.13 ppm) and 6 of 12 (at 0.25 ppm) formaldehyde-treated guinea 25 pigs after sensitization (Figure 4-13). The averag e anti-OVA titer for the high-dose group was 26 significantly higher than for controls ( p < 0.05). The individual responses at the 0.13 ppm 27 exposure level indicate that, although the average group OVA titer may not have reached 28 statistical significance, there was a measureable b iological response in three individuals. These 29 results indicate that formaldehyde exposure can sen sitize previously naïve (non-sensitized) 30

animals to OVA. 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-235 

Controls FA 0.13 ppm FA 0.25 ppm C A ( m l )

1

0,8

0,6

0,4

0,2

0 1 2 Figure 4-12. Compressed air in milliliters as para meter for airway 3 obstruction following formaldehyde exposure in guin ea pigs after OVA 4 sensitization and OVA challenge

. 5 6 Note: CA = compressed air; FA = formaldehyde;

― = median; ** = p < 0.01. 7 8 Source: Redrawn from Riedel et al. (1996). 9 10

+++ 11 12

13 14 Figure 4-13. OVA-specific IgG1 (1B) in formaldehyd e-treated sensitized 15 guinea pigs prior to OVA challenge. 16 17 Note: EU = experimental units; FA = formaldehyde;

― = median; ** = p < 0.01. 18 19 Source: Redrawn from Riedel et al. (1996). 20 21 22 C o n t r o l s F A 0

. 1 3 p p m F A 0

. 2 5 p p m  1

. 0 0 0

1 0 0

1 0 A n t i

- O V

A

- l g G 1 [ E U ]This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-236 The only significant, treatment-related histologic change was bronchial edema, with 1 thickening of the bronchial wall in formaldehyde-ex posed animals compared with non-treated 2 animals subjected to OVA sensitization and subseque nt OVA challenge. Bronchial walls were 3

measured as 40.9 ± 2.5 versus 28.2 ± 1.2 μ m. No signs of inflammation in the bronchial mucos a 4

were seen with this edema. 5 Tarkowski and Gorski (1995) exposed female Balb/C m ice to 0 or 6.63 ppm (0 or 6 2 mg/m 3 ) formaldehyde for either 6 hours/day for 10 days o r 6 hours/day once a week for 7 7 weeks. All mice were sensitized intranasally to OVA for 10 days or once a week for 7 weeks. 8 IgE anti-OVA titers were determined from sera colle cted from four mice every 8 days (1 day 9 after OVA booster) by PCA. A parallel experiment t o compare the role of the route of 10

administration was conducted with I.P. rather than intranasal sensitization (1 μ g OVA once every 11 7 days). 12 OVA titers increased similarly in control mice and mice exposed to formaldehyde once a 13 week (Figure 4-14). In contrast, mice exposed to f ormaldehyde 6 hours/day for 10 consecutive 14 days at the beginning of the experiment had increas ed anti-OVA beginning after the fourth OVA 15 sensitization, which continued to increase through seven doses of OVA to a peak of 70 PCA 16 units (

p < 0.01) (Figure 4-14). Anti-OVA IgE titers were s ignificantly different between 17 formaldehyde-treated and nonexposed mice. 18

19

20

Figure 4-14. Anti-OVA titers in female Balb/C mice exposed to 6.63 ppm 21 formaldehyde for 10 consecutive days or once a week for 7 weeks. 22 23 Note: = control mice;  = formaldehyde once a week × 7;  = formaldehyde 24 10 days. 25 Source: Redrawn from Tarkowski and Gorski (1995). 26This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-237 Intraperitoneal sensitization to OVA was much more effective than intranasal 1 sensitization, resulting in titers as high as 1,000 after 4 weeks. However, there were no 2

differences between controls and animals treated wi th formaldehyde via the I.P. route of 3 exposure. Thus, formaldehyde administered intranas ally 6 hours/day for 10 days may facilitate 4 the sensitization to allergens. These changes were not observed when formaldehyde was 5 administered intranasally once a week for 10 weeks or via I.P. injection (Tarkowski and Gorski, 6 1995). The authors speculate that formaldehyde may increase permeability of respiratory 7 epithelium and destruction of immunologic barriers. Thus the respiratory tract may become 8 vulnerable to inhaled allergens after formaldehyde exposure (Tarkowski and Gorski, 1995). 9 Ito et al. (1996) conducted three experiments to ex amine the effects of acute 10 formaldehyde exposure on bronchoconstriction and th e mediators of vascular permeability. 11 Male Wistar rats (five to eight per group) were exp osed to 0, 2, 5, 15, or 45 ppm (0, 2.5, 6.2, 12 18.5, or 55.4 mg/m 3 ) formaldehyde for 10 minutes. Baseline pulmonary insufflation and blood 13 pressure were determined prior to formaldehyde expo sure and monitored throughout the 14 experiment. Vascular leakage was measured by injec tion of Evans blue dye prior to the 15 experiment and determining extravasation 5 minutes postexposure. Briefly, lungs were perfused 16 with 0.9% saline through an aortic cannula. The lo wer portion of the trachea and main bronchi 17 were removed, and the Evans blue dye remaining was determined and expressed as ng dye/g 18 tissue. A second experiment was conducted to deter mine if dye leakage continued to increase 19 after exposure. Seven rats were exposed to 15 ppm formaldehyde for 10 minutes, as above. 20 Evans blue dye was injected 5 minutes postexposure, and tissues were perfused and excised 21 15 minutes later. The final experiment was conduct ed to determine the effect of certain receptor 22 agonists on the formaldehyde-induced microvascular leakage. Ten groups of Wistar rats (four to 23 seven per group) were exposed to 15 ppm formaldehyd e and injected with Evans blue dye, as 24 before. However, each receptor agonist under test or saline sham was injected 4–5 minutes prior 25 to the 10-minute formaldehyde exposure. Agonists t ested included tachykinin NK 1 receptor 26 antagonist (CP-99,994) at 1, 3, or 6 mg/kg; a brady kinin B 2 receptor antagonist (HOE 140) at 27 0.65 mg/kg; and a histamine H 1 receptor antagonist (ketotifen) at 1 mg/kg. 28 Formaldehyde exposure did not change pulmonary insu fflation pressure or blood 29 pressure. Formaldehyde increased vascular permeabi lity in a concentration-dependent manner in 30

both the trachea and main bronchi for the first 5 m inutes after exposure, as measured by Evans 31 blue dye extravasation (Ito et al., 1996) (Figure 4 -15). Vascular permeability was not increased 32 by formaldehyde exposure from 5 to 15 minutes poste xposure (experiment 2). Administration of 33

a selective NK 1 receptor antagonist (CP-99,994) inhibited the form aldehyde-induced vascular 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-238 permeability, reducing Evans dye extravasation to c ontrol levels at the 3 and 6 mg/kg doses 1 (Figure 4-16). 2

3

  

   200

100

0 2 5 15 45 2 5 15 45 Room Air Formaldehyde

(ppm) Formaldehyde (ppm) Trachea Main Bronchi

4 5 Figure 4-15. Vascular permeability in the trachea and bronchi of male 6 Wistar rats after 10 minutes of formaldehyde inhala tion. 7 8 Note: Vascular permeability was tested by an increa se in Evans blue dye 9 extravasation in the tissue. Solid bars: formaldeh yde; open bars: room air, n = 7. 10 Values are the means ± SEM of five to seven animals . * p < 0.05 and †

p < 0.01 11 versus room-air-exposed group (Williams’ test). 12 13 Source: Redrawn from Ito et al. (1996). 14 15This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-239 1

200

100

0

Formaldehyde Formaldehyde Sham Sham Trachea Main Bronchi

 

 

 

     

2 3 Figure 4-16. Effect of select receptor antagonists on formaldehyde-induced 4 vascular permeability in the trachea and bronchi ma le of Wistar rats. 5 6 Note: Vascular permeability was tested by an increa se in Evans blue dye 7 extravasation. Rats were treated i.v. with 1, 3, o r 6 mg/kg CP-99,996 (open bars), 8 0.65 mg/kg HOE 140 (hatched bars), 1 mg/kg ketotife n (solid bars), or vehicle 9 (shaded bars) before formaldehyde challenge. Sham: animals were exposed to the 10 sham gas for 15 ppm formaldehyde (10 minutes) after pretreatment with 0.9% 11 saline (0.5 mL/kg i.v.). Data are the means ± SEM of six to seven rats/group. 12

*

p < 0.05 versus sham-stimulated group (unpaired Stude nt’s t test or Welch’s 13 test). †

p < 0.05. ††

p < 0.01 versus 0.9% saline-pretreated, formaldehyde14 exposed control group (Williams’ test). 15 16 Source: Redrawn from Ito et al. (1996). 17 18 Neither the bradykinin B 2 nor histamine H 1 receptor agonists affected formaldehyde19 induced vascular permeability (Ito et al., 1996). Therefore, the immediate effect of 20 formaldehyde exposure on vascular permeability is m ediated, at least in part, through the NK 1 21 receptor but does not seem to require the B 2 or H 1 receptors. This implies a role for tachykinins 22 in formaldehyde-induced vascular permeability. The se findings suggest a neurogenic 23 inflammatory response because the tachykinins are r eleased from sensory nerve endings in the 24 trachea and bronchi, whereas bradykinin is released from mast cells. 25 Sadakane et al. (2002) investigated the effects of formaldehyde exposure on airway 26 inflammation caused by Der f. Two groups of male o utbred ICR mice (18/group) were exposed 27 to an aerosol of 0.5% formaldehyde solution produce d by an ultrasonic nebulizer for 15 minutes, 28 once a week for 4 weeks. Two groups were similarly treated but exposed to saline aerosol only. 29 Details of the aerosol generation and resulting mag nitude of exposure were not given. One 30 group each of control and formaldehyde-exposed mice was sensitized to Der f by an injection 1 31 day prior to formaldehyde exposure (1.5 mg/animal). The same groups were challenged with 32This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-240 intratracheal instillation of Der f (10 μ g/animal) after 4 weeks. Three days after allergen 1 challenge, mice were sacrificed and blood plasma an d lung tissue were collected. Blood plasma 2 was analyzed for Der f-specific immunoglobulins (Ig G1 and IgE). Lungs from nine mice in each 3 treatment group were homogenized, and Th1 cytokine IL-2, Th2 cytokines IL-4 and IL-5, 4 granulocyte macrophage-colony-stimulating factor (G M-CSF), and the “chemokine regulated 5 upon activation, normal T-cell expressed and secret ed” (RANTES) protein levels were quantified 6 in the supernatant via ELISA. Lungs from nine mice in each group were fixed, sectioned, and 7 stained to evaluate eosinophil infiltration, lympho cyte infiltration, goblet cell proliferation, and 8 localization of RANTES in the airway epithelium. 9 Der f-specific IgG1 was present in blood plasma of sensitized mice but was unchanged 10 by formaldehyde exposure (Sadakane et al., 2002). IgE was too low to titer. IL-2 and GM-CSF 11 were undetected in lung homogenate supernatant, and Il-4 was unchanged by sensitization or 12 formaldehyde exposure. However, RANTES was increas ed by both formaldehyde exposure and 13 allergen sensitization and challenge (Table 4-51). These increases were more pronounced but 14 less than additive for formaldehyde-exposed, allerg en-sensitized mice. IL-5 was increased by 15 allergen but unaffected by formaldehyde exposure on ly. However, formaldehyde exposure 16 potentiated the IL-5 increase seen with allergen ch allenge. 17

18

Table 4-51

. Cytokine and chemokine levels in lung tissue homoge nate 19 supernatants in formaldehyde-exposed male ICR mice with and without 20 Der f sensitization 21 22 Group Formaldehyde Der f GM-CSF IL-2 IL-4 IL-5 RANT ES

1 - - ND a ND 68.1 ± .9 4.4 ± 0.3 200.1 ± 19.7 2 + - ND ND 59.5 ± 4.3 4.1 ± 0.2 390.6 ± 37.4 b

3 - + ND ND 70.7 ± 4.9 13.6 ± 1.6 c,e 479.6 ± 80.0 c

4 + + ND ND 62.3 ± 5.8 21.6 ± 2.7 c,e,f 593.3 ± 58.2 c,d 23

a None detected. 24

b p < 0.05 from control. 25

c p < 0.001 from control. 26

d p < 0.05 from Group 2. 27

e p < 0.001 from Group 2. 28

f p < 0.001 from Group 3. 29 30 Source: Sadakane et al. (2002). 31 32 33 Der f sensitization and challenge increased eosinop hil infiltration into the interstitium 34 around the bronchi and bronchioles as well as goble t cell proliferation in the bronchial 35 epithelium (Figure 4-17). Formaldehyde exposure ex acerbated the eosinophilic and goblet cell 36 responses to a challenge dose of Der f ( p < 0.05) (Sadakane et al., 2002). Formaldehyde-ind uced 37This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-241 eosinophilic infiltration in the absence of sensiti zation and challenge was not different from non1 treated, non-sensitized mice. 2 These results suggest that formaldehyde exposure ma y aggravate eosinophilic infiltration and 3 goblet cell proliferation that accompanies allergic responses. This response is associated with an 4 increase in IL-5, an eosinophilic attractant, and a n increase in RANTES, which recruits 5 eosinophils by chemotaxis in formaldehyde-exposed a nd Der f challenged animals, although the 6 effect was not statistically significantly elevated compared with Der f challenge-induced levels 7

of IL-5 and RANTES alone. 8

9

Panel A 10

Panel B 11

control FA Derf Derf + FA ab abc ++++

+++

++

+

±

-

12 13 Figure 4-17. The effects of formaldehyde inhalatio n exposures on eosinophil 14 infiltration (Panel A) and goblet cell proliferatio n (Panel B) after Der f 15 challenge in the nasal mucosa of male ICR mice afte r sensitization and 16 challenge. 17 18 Note: a

p < 0.001 compared with control group; b p < 0.001 compared with 19 formaldehyde group; c p < 0.05 compared with Der f group. 20 Source: Redrawn from Sadakane et al. (2002). 21 control FA Derf Derf + FA ab abc ++++

+++

++

+

±

-Eosinophil scoreGoblet cell scoreThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-242 Fujimaki et al. (2004a) investigated the long-term effects of low-dose formaldehyde 1 exposure on immunologic and neurological inflammati on. Female C3H/He mice were exposed 2 to 0, 0.082, 0.393, or 1.87 ppm (0, 0.1, 0.48, or 2 .3 mg/m 3 ) formaldehyde 16 hours/day, 3 5 days/week for 12 weeks. Six mice at each exposur e level were given injections of OVA plus 4 adjuvant before the initial exposure and in weeks 3 , 6, 9, and 11 of the experiment. Five mice at 5 each formaldehyde-exposure level did not receive OV A injections. One day after the last 6 exposure, mice were weighed and blood, BAL, spleen, and thymus were collected from each 7 animal. After weighing, spleens were disaggregated and spleen cells harvested for cell culture. 8 Immunophenotype of the spleen cells was determined by flow cytometry (CD4, CD8, CD3, and 9 CD19 positive cells). Lymphocyte proliferation in response to lipopolysaccharide (LPS), 10 phytohemagglutinin A (PHA), or OVA was determined a fter 72 hours in culture. Splenocytes 11 were cultured for 48 hours in the presence of LPS, PHA, and OVA (immunized mice only), and 12 supernatants were collected for cytokine analysis ( IL-4, IL-5, and IFN- γ ). Splenocytes were 13 cultured for 24 hours in the presence or absence ofOVA to assess chemokine production (MCP14

1 and MIP1- α ). Anti-OVA IgE, IgG 1 , IgG 2, and IgG 3 were quantified in blood plasma. 15 Body and thymus weights were unchanged by formaldeh yde exposure or OVA injection 16 (Fujimaki et al., 2004a), while, in non-immunized m ice, spleen weights were reduced by 17 formaldehyde exposure from 152 mg in controls to 12 8, 118, and 121 mg in mice exposed to 18 0.08, 0.4, and 1.8 ppm formaldehyde, respectively. Spleen weights tended to increase in groups 19 exposed to 400 and 2,000 ppb formaldehyde compared with controls in OVA-immunized mice 20 (control: 117.8 mg compared with 400 ppb: 168.6 mg and control: 121.0 mg compared with 21 2,000 ppb:153.2 mg, respectively) but were not stat istically significant. 22 To gain insight on the overall pulmonary inflammato ry response of mice exposed to 23 formaldehyde in both immunized and non-immunized mi ce, the total number and differential 24 count of MPs, neutrophils, lymphocytes, and eosinop hils in BAL were counted and were found 25 to be unchanged by formaldehyde in non-immunized mi ce. By contrast, in immunized mice 26 exposed to 1.8 ppm formaldehyde, the total number o f BAL cells, MPs, and eosinophils were 27 significantly increased compared with non-immunized controls (9.65 versus 2.84, 7.22 versus 28 2.74, and 2.0 versus 0.02 ×10 4 cells, respectively). 29 To further assess the pulmonary inflammatory respon se, protein levels of inflammatory 30 cytokines were determined by ELISA in BAL fluid. L evels of IL-1 β in BAL of immunized mice 31 were decreased by formaldehyde exposure ( p < 0.05 at 1.8 ppm formaldehyde), but IL-1 β levels 32 after formaldehyde exposure were not different from controls in non-immunized mice (Fujimaki 33 et al., 2004a). All other cytokines or chemokines were either unchanged (TNF- α , IL-6, and GM34 CSF) or not detected (eotaxin, MIP-1 α , and MCP-1). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-243 Various neuropeptides, such as brain-derived neurot rophic factor (BDNF), nerve growth 1 factor (NGF), and substance P are released from vag al nerve endings and mediate a neurogenic 2 inflammatory response. Levels of BDNF, NGF, and su bstance P were assessed in BAL fluid 3 and/or in plasma. BDNF was not detected in BAL or in plasma. NGF levels in immunized mice 4 were significantly higher than in non-immunized mic e in both BAL fluid and in plasma. NGF 5 levels in immunized mice were significantly attenua ted by 0.08 and 0.4 ppm formaldehyde 6 exposure (Figure 4-18) in both BAL fluid and in pla sma. Plasma level of substance P (a 7 mediator of neurogenic inflammation) was increased by formaldehyde exposures in non8 immunized mice (Figure 4-19) in both BAL fluid and plasma. This increase appears to be dose9 dependent and reaches statistical significance at 2 ,000 ppb formaldehyde exposure in non10 immunized mice compared with non-immunized controls . Similar to NGF, levels of substance P 11 increased in OVA-immunized mice compared with non-i mmunized mice in both BAL fluid and 12 plasma. Similar to NGF, levels of substance P in O VA-immunized mice were attenuated by 13 formaldehyde exposure at 80 ppb. 14

15



   6

5

4

3

2

1

0 

### OVA FA 0 80 400 2000 (ppb) 0 80 400 2000 

### - - - - + + + +

16

17 Figure 4-18. NGF in BAL fluid from formaldehyde-ex posed female C3H/He 18 mice with and without OVA sensitization. 19 20 Note: The day after the final formaldehyde inhalati on, BAL fluid was collected 21 from formaldehyde-exposed, non-immunized and formal dehyde-exposed, OVA22 immunized mice, and the production of NGF was deter mined by ELISA. Data 23 are mean ± SEM from five to six animals. ** p < 0.01. 24 25 Source: Redrawn from Fujimaki et al. (2004a). 26 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-244 1

2

3 Figure 4-19

. Plasma substance P levels in formaldehyde-exposed f emale 4 C3H/He mice with and without OVA sensitization. 5 6 Note: The day after the final formaldehyde inhalati on, plasma samples were 7 collected from formaldehyde-exposed, non-immunized, and formaldehyde8 exposed OVA-immunized mice, and the levels of subst ance P were determined by 9 ELISA. Data are mean ± SEM from five to six animal s. * p < 0.05. FA = 10 formaldehyde. 11 12 Source: Redrawn from Fujimaki et al. (2004a). 13 14 15 Fujimaki et al. (2004a) further investigated the ef fect of low-level formaldehyde exposure 16

from both immunized and non-immunized mice on the s ystemic immune response. Spleens were 17 removed from formaldehyde-exposed mice and were cul tured in the presence of LPS or PHA 18 (for non-immunized samples) or OVA (for immunized s amples). The secretory ability of 19 immunized and non-immunized spleen cells was assess ed by measuring IFN- γ release by ELISA. 20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-245 Formaldehyde exposure (1.8 ppm) increased IFN- γ fourfold in LPS-stimulated cultured spleen 1 cells from non-immunized mice. No other cytokine o r chemokine was changed by formaldehyde 2 exposure in cultured spleen cells from non-immunize d mice. In OVA-immunized mice, 3 formaldehyde had no significant effect on cytokines from stimulated spleen cells. OVA in vitro 4 stimulation significantly increased the chemokines MIP-1 and MCP-1 for control and 5 formaldehyde-treated OVA-immunized mice. The OVA-s timulated release of MCP-1 in vitro 6 was enhanced by formaldehyde exposure in a concentr ation-dependent manner, increasing 7 threefold and fourfold at 0.40 and 1.8 ppm, respect ively. Increases in MCP-1 correlate with 8 reported increases in the associated cytokine, RANT ES, which recruits eosinophils by 9 chemotaxis (Sadakane et al., 2002). These formalde hyde-induced increases in cytokine levels 10 contribute to pulmonary inflammation. The inflamma tory response is not mediated by 11 lymphocytes, since lymphocyte subsets and in vitro cell proliferation were unchanged by OVA 12 immunization or formaldehyde treatment (Fujimaki et al., 2004a). 13 Anti-OVA (IgE and IgG 2a ) levels in plasma were unchanged by formaldehyde e xposure. 14 Anti-OVA IgG 1 was reduced in immunized mice exposed to 400 ppb f ormaldehyde compared 15 with nonexposed animals. However, this effect did not persist as dose increased. Anti-OVA 16 IgG 3 was depressed in immunized mice exposed to 0.08 an d 0.4 ppm formaldehyde (Fujimaki et 17 al., 2004a). Formaldehyde exposure did not induce an inflammatory response in lung or tracheal 18 epithelium in sections viewed by light microscopy ( Fujimaki et al., 2004a). Although there was 19 a mild infiltration of mast cells into the epitheli um of OVA-immunized mice, there were no 20 effects of formaldehyde treatment on mast cell infi ltration. 21 A recent study by Lino dos Santos Franco et al. (20 09) exposed male Wistar rats for 22 3 days, 90-minutes/day, to 1% formaldehyde (by weig ht; exact doses not reported) by inhalation. 23 Of these, one group was sensitized I.P. to OVA (10 μ g), a common allergen, immediately 24 following formaldehyde exposure, and subsequently c hallenged with OVA 2 weeks later. Other 25 rats were sensitized and challenged but were not ex posed to formaldehyde. PCA reaction as well 26 as BAL analysis and whole blood analysis were condu cted. Immunohistochemical analysis of 27 platelet endothelial cell adhesion molecule-1 (PECA M-1) expression, an inflammatory mediator, 28 in lung tissue was also measured. When formaldehyd e exposure was followed by OVA 29 sensitization and challenge, decreased lung inflamm ation was reported compared with the group 30 that was OVA-sensitized but had not been exposed to formaldehyde. Reduced lung mast cell 31 degranulation was also reported in the formaldehyde /OVA group compared with the nonexposed 32 OVA group. Total circulating leukocytes, total bon e marrow cells, and lung protein expression 33 levels of PECAM-1 were also significantly decreased in formaldehyde/OVA rats compared with 34 non-formaldehyde exposed OVA rats. The reduction i n inflammatory parameters in response to 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-246 formaldehyde may be attributed to different study d esigns, since in this study animals were 1 sensitized after exposure rather than prior to expo sure. The results suggest that formaldehyde 2 may functionally alter the activity of certain cell s, like mast cells, that may downgrade an 3 appropriate immune response to antigen and might se rve to threaten lung homeostasis. Due to 4 the unique experimental design of this study, it ca nnot be directly compared with Sadakane et al. 5 (2002) or Fujimaki et al. (2004a). In addition, th is study did not intend to measure whether 6 formaldehyde can exacerbate an asthmatic response b ut rather set out to identify whether 7 formaldehyde could affect immune homeostasis. 8 In summary, studies suggest that formaldehyde expo sure may induce a predominantly 9 neurogenic inflammatory response via release of neu ropeptide, such as NGF and substance P 10 from vagal nerve endings. Formaldehyde does not ap pear to potentiate a systemic immune 11 response. However, localized pulmonary inflammatio n can be potentiated by formaldehyde 12 exposure, as indicated by the increased presence of eosinophils and certain proinflammatory 13 cytokines (IFN- γ

). This response does not appear to be mediated by classic immunogenic 14 mechanisms since studies have failed to report elev ated levels of anti-formaldehyde-specific IgE. 15 Several studies have shown that exposure to formald ehyde can facilitate allergic sensitization in 16 previously naïve animals, and it is thought that th is effect may occur due to formaldehyde’s 17 ability to increase microvascular leakage in the na sal epithelium and by causing damage to the 18 nasal barrier (Ito et al., 1996). Sadakane et al. (2002) demonstrated that formaldehyde exposure 19 can also exacerbate allergic responses by enhancing the response to challenge allergen. Thus, 20 formaldehyde may exacerbate allergic responsiveness by aggravating the sensitization response 21 in previously naïve animals by altering the permeab ility of the mucosal barrier in nasal 22 compartments. Neurogenically derived inflammation, including stimulation of the trigeminal 23 nerve and release of braykinin, suggests that the M OA for sensitization may ultimately have its 24 roots in neurogenic inflammation rather than an imm unogenic response. In addition, using a 25 different protocol, Lino dos Santos Franco et al. ( 2009) suggest that formaldehyde exposure can 26 adversely affect lung homeostasis by reducing the a ctivity of important inflammatory mediators 27 (mast cells, circulating leukocytes, PECAM-1 expres sion) when it occurs prior to sensitization, 28 thus downgrading an appropriate immune response. 29

30

4.2.1.5.2. Dermal sensitization. Wahlberg (1993) used Hartley strain guinea pigs as test 31 animals to determine the skin irritancy of a suite of industrial chemicals, including 32 formaldehyde. Aqueous solutions of the compound in a 0.1 mL volume were applied to the 33 shaved flanks of guinea pigs and gently rubbed into the skin with a cotton-tipped applicator. 34 Sites were left open and the treatments repeated on ce daily for 10 days. A number of indices of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-247 acute skin irritation were monitored, including ery thema via visual scoring and edema and skin1 fold thickness using Harpenden calipers. Varying c oncentrations of formaldehyde (up to a 10% 2 solution) induced a dose-dependent increase in skin -fold thickness. Responses also showed 3 shorter latencies at the higher concentrations. Fo r example, erythema was first observed on 4 day 2 when 10% formaldehyde was applied, day 5 (for 3%), and day 6 (for 1%). 5 Lee et al (1984) investigated the role of different routes of exposure in formaldehyde6 induced allergic sensitization. Two sets of four m ale English smooth-haired guinea pigs received 7 topical applications of 100 μ L 37% w/v formalin distributed over two shaved, dep ilated dorsal 8 sites two times over the course of 2 days at differ ent sites. The total dose was calculated as 9

74 μ g/animal. In addition, eight animals received a si ngle topical application onto a 15 mm area 10 of the dorsal surface. The applied dose of 25 μ L formaldehyde was dissolved in saline. Two 11 other groups of guinea pigs were exposed to either 6 ppm (6 hours/day for 5 days) or 10 ppm 12 (6 hours/day for 5 days) formaldehyde by inhalation . A third group of guinea pigs was exposed 13 to 10 ppm formaldehyde for 8 hours/day for 5 consec utive days by inhalation. All animals were 14 evaluated for contact sensitivity by topical applic ation of 20 mL formaldehyde diluted with 15

saline and distributed in a 15 mm area on the backs of the shaved guinea pigs (Lee et al., 1984). 16 Sites were visually inspected for erythema at 1, 6, 24, and 48 hours following the topical 17 application, and reactions were scored. No erythem a was observed in control animals. None of 18 the guinea pigs in the 6 hours/day inhalation group s (6 and 10 ppm formaldehyde) developed 19 skin sensitivity tested on day 9 (4 days after the initial exposure regimen ended). Two of four 20 guinea pigs exposed to 10 ppm formaldehyde for 8 ho urs/day for 5 consecutive days developed 21 mild skin sensitization tested on day 31. Contact sensitivity increased in a dose-dependent 22 fashion in groups of animals that had been sensitiz ed via the dermal route. Thus, dermal 23 exposure resulted in contact sensitivity. Inhalati on exposure did not consistently produce contact 24 sensitivity. 25 Arts et al. (1997) used a local lymph node assay ( LLNA) and the induction of IgE to 26 monitor the sensitization of female Wistar rats (lo w IgE-responders) and BN rats (high IgE 27 responders). For the LLNA assay, animals were sens itized by the application of varying 28 concentrations of formaldehyde in raffinated olive oil on the dorsum of both ears on days 0, 1, 29

and 2. Control animals were treated with raffinate d olive oil alone. Animals received an I.P. 30 injection of BrdU on day 5 and were subsequently sa crificed. Ear-draining lymph nodes were 31 collected, fixed, and sectioned, and the mitotic ac tivity was monitored following successive 32 incubation of the sections in anti-BrdU, biotin-lab eled rabbit anti-mouse antibody, peroxidase33 conjugated streptavidin, and 3,3-diaminobenzidine t etrahydrochloride. For serum IgE responses, 34 150 μL of different concentrations of formaldehyde were applied to the shaved flanks of rats on 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-248 day 1, then 75 μL of the same chemical at 50% of th e initial concentration were applied to the 1 dorsum of each ear on day 7. The amount of IgE in the blood was measured using ELISA but 2 appeared to be little affected by formaldehyde trea tment in either species of rat. However, the 3 ear-draining lymph nodes of both strains of rat sho wed a comparative increase in weight in 4 response to formaldehyde, and proliferation (BrdU p ositive) of paracortical cells was observed in 5 response to increasing doses of the compound. This response was most notable in BN rats 6 treated with 10% formaldehyde. Arts et al. (1997) concluded that the irritant and sensitizing 7 properties of formaldehyde may act through non-IgE- immune mechanisms. 8 Hilton et al. (1998) used the LLNA assay in female CBA/Ca (H-2 k haplotype) mice to 9 compare the skin sensitizing potencies of formaldeh yde and glutaraldehyde. The comparison 10 was set on a quantitative basis by determining the concentration of each compound necessary to 11 induce a threefold increase in lymph node cell prol iferative activity (effective concentration 12 [EC 3 ]). While both aldehydes induced a dose-dependent proliferative response, the 13 incorporation of [ 3 H]-methylthymidine was far greater in animals expos ed to glutaraldehyde 14 versus formaldehyde (with EC 3 values of 0.002–0.006 mol/L for glutaraldehyde ver sus 0.11– 15 0.18 mol/L for formaldehyde). These data indicate the potential of both chemicals to induce skin 16 sensitization, although the potency of glutaraldehy de was far greater than that of formaldehyde. 17 Xu et al. (2002) evaluated the extent to which the expression of some cytokines may 18 change as a result of cutaneous exposure to formald ehyde in mice. Female Balb/C mice were 19 skin painted with three topical applications of 100 μL of 17.5% formaldehyde or distilled water 20 with a 1-day interval between each application. Sp leen and draining lymph nodes were 21 harvested on days 3, 5, 7, 9, or 12 after the last skin painting. In some animals, contact 22 hypersensitivity was induced by applying 2% formald ehyde to both sides of mouse ears on day 3 23 following the last skin painting. For this endpoin t, the percent increase in thickness of the ears 24 was monitored. For the cytokines, mRNA expression levels of IL-2, IL-4, IL-5, IL-10, IL-12, 25 IL-13, IL-15, IL-18, and INF- γ were determined semiquantitatively by measuring th e amount of 26 individual mRNAs following amplification with the r everse transcriptase (RT)-PCR. The 27 relative amounts of cytokine mRNAs were calculated as the ratio of cytokine mRNA to that of 28 glyceraldehyde-3-phosphate dehydrogenase, as reveal ed in specific bands on an agarose gel. 29 Cutaneous formaldehyde treatment was associated wi th the long-lasting expression of 30

IL-4 and IFN- γ

mRNAs in mouse spleen and draining lymph nodes and with IL-15 mRNA only 31 in mouse spleen. Only IL-13 mRNAs displayed a tran sient increase in expression in both spleen 32 and draining lymph nodes. Levels of IL-2, IL-12, a nd IL-15 were increased in the mouse spleen 33 but not the lymph nodes. The mouse ear swelling te st gave positive correlations with enhanced 34 expression of mRNA for IL-4 and IFN- γ (Table 4-52). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-249 1 Table 4-52. Correlation coefficients among ear swe lling responses and skin 2 mRNA levels in contact hypersensitivity to formalde hyde in mice 3 4

Correlation coefficients

Variables IL-2 IL-4 IFN- γ Ear swelling 0.50 0.74 a 0.67 a

IL-2 – 0.39 0.60 IL-4 – – 0.79 a 5

a Statistically significant ( p < 0.05). 6 7 Source: Xu et al. (2002). 8 9 10 4.2.1.5.3. Summary of sensitization studies. Several animal studies report increased airway 11 resistance and BC due to inhalation exposures to fo rmaldehyde (Nielsen et al., 1999; 12 Swiecichowski et al., 1993; Biagini et al., 1989; A mdur, 1960). Changes in pulmonary 13 resistance were observed as early as 10 minutes aft er exposure (Biagini et al., 1989), and 14 reported effect levels ranged from 0.3–13 ppm. Oth er pulmonary effects were reported in 15 conjunction with BHR, such as increased tracheal re activity and decreased pulmonary elasticity 16 (Swiecichowski et al., 1993; Amdur, 1960). Althoug h BHR is a common result of Type I 17 hypersensitivity reaction to an allergen, the obser vation of BHR alone is not sufficient to 18 demonstrate that an agent induces Type 1 hypersensi tivity. 19 BHR may be directly induced both pharmacologically and neurogenically (Joos, 2003; 20 Cain, 2001; Meggs, 1995). There is little evidence that formaldehyde itself is an allergen 21 recognized by the immune system, especially via inh alation (Lee et al., 1984). Although 22 formaldehyde exposure has been reported to alter cy tokine levels and immunoglobulins in some 23 experimental systems, these immunomodulatory effect s do not support a type 1 hypersensitivity. 24 IgE was unchanged (Fujimaki et al., 2004a; Lee et a l., 1984), and cytokine profiles were not 25 consistent with the Th-2 cytokines expected in IgE mediated hypersensitivity (Fujimaki et al., 26 2004a; Ohtsuka et al., 2003). 27 Formaldehyde-induced dermal sensitization show para llel results. The physical signs of 28 irritation and sensitization are consistently shown (e.g., rashes, edema). Some involvement of 29 the immune response has been demonstrated with posi tive LLNA assays, indicating proliferation 30 of lymphocytes in lymph nodes draining the affected area (Hilton et al., 1998; Arts et al., 1997). 31 Increased expression of Th-2 cytokines in the lymph nodes of mice given dermal applications of 32 formaldehyde does indicate an immune component to t he observed sensitization. However, the 33 response does not seem to be mediated by IgE (Arts et al., 1997; Lee et al., 1984). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-250 Ito et al. (1996) reported that a tachykinin NK 1 receptor, but not the histamine H 1 or 1 bradykinin B 2 receptors, is involved in formaldehyde-induced vas cular permeability. 2 Neuropeptides NGF and substance P were affected in BAL and stimulated splenocytes from 3 formaldehyde-exposed mice, with greater effects see n in OVA-immunized mice. Tachykinins 4 (e.g., substance P and neurokinin A) are produced b y nerve cells and can directly stimulate 5 bronchoconstriction (Van Schoor et al., 2000). Sub stance P is also a mediator of neurogenic 6 inflammation. Therefore, although formaldehyde may induce some of the symptoms of type 1 7 hypersensitivity, these symptoms are more likely ne urogenic than immunogenic in origin. 8 In contrast, formaldehyde enhances immunogenic hype rsensitivity of known allergens 9 (Sadakane et al., 2002; Riedel et al., 1996; Tarkow ski and Gorski, 1995). This potentiation 10 varied based on sensitization protocols (respirator y tract versus systemic, frequency and timing 11 of immunization, allergen, etc.) and formaldehyde e xposure regimens (concentration, continuous 12 versus intermittent exposures). Taken as a whole, the results support the finding that 13 formaldehyde exposure can aggravate a type 1 hypers ensitivity response (Table 4-53). 14

15

4.2.1.6. Neurological and Neurobehavioral Function 16

4.2.1.6.1. Inhalation exposure. There are a number of published reports examining t he effects 17 of formaldehyde exposure on nervous system structur e and function. The reports evaluating 18 behavioral effects fall into three main categories: (1) behavioral responses evaluated during or 19 immediately following formaldehyde exposures, which may include effects due to the potential 20 irritant properties of the chemical, (2) acute or s hort-term exposures followed by behavioral 21

assessments conducted 2–24 hours after termination of formaldehyde exposure, which reflect 22 sustained effects of chemical exposure independent of its irritant properties, and (3) repeated 23 exposures to formaldehyde followed by neurological assessments performed throughout the 24 treatment period or several days to weeks after ter mination of treatment. In addition to reports 25 evaluating changes in behavior, there are several r eports evaluating neuropathological effects or 26 changes in brain chemistry. 27

28This document is a draft for review purposes only a nd does not constitute Agency policy 4-251 DRAFT—DO NOT CITE OR QUOTETable 4-53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due toformaldehyde in experimental animalsSpecies No./groupTreatmentaObservations LOAEL/NOAELReferenceInhalation studiesCynomolgusmonkeys9 Methacholine-sensitive monkeysexposed to 2.5 ppmformaldehyde for 10 minutesFormaldehyde increased pulmonary resistance after 2, 5, and10 minutes.LOAEL2.5 ppmBiagini et al.(1989)Hartleyguinea pigs(male)8 0.86, 3.4, 9.4, 31.1 ppmformaldehyde for 2 hours or0.11, 0.31, 0.59, 1.05 ppm for8 hoursTotal pulmonary resistance increased after 2 hoursexposure at9.4 and 31.1 ppm. Effect was reversible and returned tobaseline within 30 minutes. Total pulmonary resistance wasincreased after 8 hours exposure at 0.3 and 1 ppm.Amount ofacetylcholine needed to achieve doubled pulmonary resistancewas decreased in animals after 2 hours exposure.NA Swiecichowskiet al. (1993)Hartleyguinea pigs(male)5–7 3.4 ppm for 8 hours No changes in ex vivo tracheal constriction or inflammation. NA Swiecichowskiet al. (1993)F344 ratsand BN rats5 16 ppm 3 hours/day, 5 days Modest changes in inflammatory cytokine expression, butrespiratory and olfactory epithelial lesions were more severe inF344 rats than in BN rats.NA Ohtsuka et al.(2003)Englishsmoothhairedguinea pigs4 6, 10 ppm, 6 hours/day, 5 days,combined with provocationchallenge (2 or 4 ppm on day 7,or days 7, 22, and 29)Inhalation challenge with 6 or 10 ppm followed by bronchialchallenge failed to increase respiratory sensitivityNA Lee et al.(1984)Perlbrightwhite,DuncanHartleyguinea pigs(female)12 0, 0.13, 0.25 ppm 8 hours/day,5 days. The animals weresensitized to OVA (3 minutesexposure to 5% OVA aerosol)Anti-OVA titer was significantly elevated over controls inanimals exposed to 0.25 ppm formaldehyde and showedthatformaldehyde may sensitize previously naïve animalsto OVA.NA Riedel et al.(1996)Balb/C mice(female)4 0, 6.63 ppm 6 hours/day for10 days or 6 hours/dayonce/week for 7 weeks. Allmice were sensitized to OVAFormaldehyde administered intranasally for 6 hours/day for10 days may facilitate sensitization to allergens since anti-OVAtiters were elevated over control animals. However, the lengthand duration of exposure appears to affect development ofsensitization.NA Tarkowski andGorski (1995)This document is a draft for review purposes only a nd does not constitute Agency policy 4-252 DRAFT—DO NOT CITE OR QUOTETable 4-53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due toformaldehyde in experimental animalsSpecies No./groupTreatmentaObservations LOAEL/NOAELReferenceWistar rats(male)5–8 0, 2, 5, 15, 45 ppm for10 minutesPulmonary insufflation or blood pressure were not altered.Vascular permeability increased in concentration-dependentmanner and could be reduced by adding a NK1 selectiveantagonist.NA Ito et al.(1996)Outbred ICRmice (male)18 0.5% formaldehyde for 15minutes, once/week for 4 weeks.Both control and exposed groupswere exposed to Der f by I.P.injection 1 day beforeformaldehyde and thenchallenged with Der f after4 weeks.More pronounced RANTES production in formaldehyde-treatedand sensitized rats than in sensitized rats that had not beenexposed to formaldehyde. Formaldehyde also potentiated IL-5production associated with sensitization.NA Sadakane et al.(2002)C3H/HeJmice(female)6 0, 0.082, 0.393, 1.87 ppm16 hours/day, 5 day/week,12 weeks. Mice also given OVAplus adjuvant before exposure,and again 3, 6, 9, 11 weeks afterexposure. Some formaldehydemice did not receive any OVASubstance P and NGF were increased dose dependentlyinformaldehyde-treated, non-immunized mice but were attenuatedin formaldehyde-treated immunized mice compared withnonexposed, immunized controls.LOAEL0.082 ppmFujimaki et al.(2004a)Wistar rats(male)NA 1% Formaldehyde by weight for90 minutes for 3 days. Onegroup was sensitized to OVAafter to formaldehyde exposureand then challenged with OVAafterwards. Others weresensitized and challenged but notexposed to formaldehyde.Total circulating leukocytes, bone marrow cells, and lungprotein PECAM expression were significantly decreased informaldehyde/OVA rats compared with OVA rats.NA Lino dosSantos Francoet al. (2009)Dermal sensitizationHartleyguinea pigs5 Skin painted once/day for10 days with 0.1 mL of 1, 3, or10% formaldehydeVarying concentrations (up to 10%) induced dose-dependentincrease in skin-fold thickness. Erythema seen earlier at higherdoses (2 days at 10% formaldehyde vs 5 days at 3% or 6 days at1%).Wahlberg etal. (1993)This document is a draft for review purposes only a nd does not constitute Agency policy 4-253 DRAFT—DO NOT CITE OR QUOTETable 4-53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due toformaldehyde in experimental animalsSpecies No./groupTreatmentaObservations LOAEL/NOAELReferenceEnglishsmoothhairedguinea pigs4 Group 1: skin painted, 100 μL37% formalin twice over 2 days,Group 2: single topicalapplication of 25 μLformaldehydeGroup 3: 10 ppm 6 hours/day for5 days by inhalationTwo of four guinea pigs from group 3 had mild skinsensitization after day 31. Contact sensitivity developed in adose-dependent manner in the dermal groups (group 1and 2).Lee et al.(1984)Wistar andBN rats(female)4 Application of formaldehyde toears on days 0, 1, 2, followed byan I.P. injection of BrdU.Ear-draining lymph nodes increased in weight in response toformaldehyde, reflected in increased number of BrdU-stainedcells, most notably in BN rats (high IgE responders) treated with10% formaldehydeArts et al.(1997)CBA/CamiceNA Compared glutaraldehyde toformaldehyde to induce a locallymph node assayGlutaraldehyde and formaldehyde induced a dose-dependentproliferative response that was greater in glutaraldehyde-treatedanimalsHilton et al.(1998)Balb/c mice(female)3–5 Skin painted with 100 μL of17.5% formaldehyde every otherday for days 3, 5, 7, 9, 12Cutaneous treatment associated with long-lasting expression ofvarious cytokines from draining lymph nodes and spleen.NA Xu et al.(2002)NA = not applicable.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-254 4.2.1.6.1.1. Behavioral response 1 Clinical signs 2 Several studies that were focused on general toxici ty or carcinogenicity of formaldehyde 3 also assessed clinical signs in exposed animals, wh ich may be related to adverse effects on the 4 nervous system. Procedural details for the assessm ents, or specific data regarding findings, were 5 not provided. Signs recorded included uncoordinate d locomotion and climbing of cage walls at 6 20 ppm formaldehyde in rats (Woutersen et al., 1987 ); restlessness at 15 ppm formaldehyde in 7 rats (Morgan et al., 1986a); dyspnea, listlessness, and hunched posture at 20 ppm and ataxia at 8 40 ppm in mice (Maronpot et al., 1986); and dyspnea in rats at 14.3 ppm formaldehyde (Kerns et 9 al., 1983). Given the lack of information regardin g procedures used for these evaluations and the 10 limited reporting of results, the utility of these data is limited. 11

12

Irritant threshold detection 13 Wood and Coleman (1995) evaluated the irritant prop erties of acute formaldehyde 14 exposure in mice. Adult male Swiss mice (eight/gro up) were initially trained to terminate a 15 60-second exposure to an irritant gas (ammonia, 1,0 00 ppm) by poking their noses into a conical 16 sensor five times to produce a 60-second facial sho wer of clean air. Each test session consisted 17 of 25 exposure trials. Following training, respons e to formaldehyde was evaluated, using the 18 same testing scenario. Each day mice had a morning exposure session to ammonia and an 19 afternoon session to formaldehyde. Formaldehyde co ncentrations tested were different each day, 20 in sequence from 0, 1, 1.8, 3, 5.6, and 10 ppm (0, 1.23, 2.21, 3.68, 6.87, and 12.3 mg/m 3 ) and 21 then stepping back again from 10 to 0 ppm. Half of the animals were tested in an ascending 22 order of formaldehyde concentrations, the other hal f in a descending order. The frequency of 23 terminating exposure, error rate, and the time laps e to termination were recorded. The 24 concentration at which 50% of the formaldehyde deli veries would be expected to be terminated 25 was estimated (AC 50 ) by simple linear regression or by analysis of cov ariance on the logit 26 transform of percentage terminated as a function of log concentration. 27 All mice were trained successfully to terminate 100 % of ammonia exposures, but varied 28 responses were observed with formaldehyde exposure. In general, time taken to terminate 29 formaldehyde exposure decreased significantly with increasing formaldehyde concentration. 30 Mice terminated more exposures to 1 ppm formaldehyd e than to air alone ( p < 0.0005), and the 31 error rate, generally below 40%, did not significan tly differ with formaldehyde concentration 32

tested. Each animal had two test sessions with eac h formaldehyde concentration (once during 33 the ascending sequence and again during the descend ing sequence); both the time to termination 34 ( p < 0.0012) and AC 50 were decreased in the second series of tests. One method of estimation by 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-255 the authors yielded an AC 50 of 3.63 ppm for the first series of tests versus 1 .88 ppm for the 1 second series. A two-way repeated measures ANOVA w ith replication and concentration as 2 within variables was highly significant ( p < 0.00005). These studies indicate mice are sensi tive 3 to the irritant properties of formaldehyde at expos ure concentrations as low as 1 ppm, and 4 animals reacted more swiftly and with greater accur acy to terminate formaldehyde exposure as 5 the concentration increased. However, a wide varie ty of responses was noted on an individual 6 animal basis. Two of the eight mice terminated 90% of the trials during 1 ppm exposures and 7 80–100% of trials at all other tested formaldehyde concentrations. One mouse terminated fewer 8 than 10% of the formaldehyde exposure trials (1–10 ppm) during the testing regimen but had a 9 92% response rate to 20 ppm formaldehyde. The rema ining five mice responded with increasing 10 termination frequency as formaldehyde concentration increased from 1 to 10 ppm, with an AC 50 11 of 2.72 ppm (3.34 mg/m 3 ). 12

13

4.2.1.6.1.2. Motor activity and habituation. Malek et al. (2003a) examined open field behavior 14 of rats after acute formaldehyde exposures. Male a nd female LEW.1K rats (15/sex/group) were 15 exposed to 0, 1, 2.5, or 5 ppm (0, 1.23, 3.08, or 6 .15 mg/m 3 ) formaldehyde for 2 hours. 16 Formaldehyde was vaporized from aqueous solutions d irectly below the exposure chamber. 17 Formaldehyde levels were checked 16 times throughou t the 2-hour exposure periods. Mean 18 formaldehyde levels of 1.01 ± 0.29 ppm, 2.51 ppm (S D is missing), and 5.0 ± 0.27 ppm were 19 achieved. Locomotor activity was assessed for 3 mi nutes in an open field 2 hours after 20 termination of formaldehyde exposure and again 24 h ours later, using an automated device to 21 count the number of squares crossed. Other behavio rs were noted, including grooming (face 22 cleaning, fur licking, and scratching), rearing, sn iffing (air and floor), wall climbing, and 23

defecation. 24 The authors reported no signs of irritation or chan ges in activity or food or water intake 25 during exposure. In general, sniffing was increase d after formaldehyde exposure and movement 26 was decreased (crossed quadrants and climbing) in b oth male and female rats ( p < 0.05). 27 Significant reductions in horizontal movements (cro ssed quadrants) were observed at all dose 28 levels and were characterized by a U-shaped dose re sponse (Figure 4-20). The lowest dose 29 tested (1 ppm) demonstrated a higher level of activ ity suppression than the two higher doses, but 30 all groups were still suppressed relative to contro ls. Although female rats displayed a greater 31 level of activity overall, a similar U-shaped dose- response pattern was also observed. 32

33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-256 1 2 Figure 4-20. Motor activity in male and female rat s 2 hours after exposure to 3 formaldehyde expressed as mean number of crossed qu adrants ± SEM. 4 Greater reductions were observed in the lowest dose group, a pattern that 5 was evident in both genders. ** = different from c ontrol, p < 0.005. 6 7 Source: Drawn from data reported by Malek et al. ( 2003a). 8 9 10 Activity in the same apparatus was reassessed 24 ho urs later. As expected, controls 11 demonstrated habituation to the test apparatus, exh ibiting only 20% of the motor activity 12 observed on day 1 (Figure 4-21). In contrast, form aldehyde-treated animals failed to 13 demonstrate the same degree of habituation. Activi ty levels for males observed on day 2 were 14 60–80% of the activity levels seen on day 1. Forma ldehyde-treated females also failed to 15 habituate and actually demonstrated increases in ac tivity on day 2 relative to day 1 at all 16 formaldehyde exposure levels. 17 Number



###  

**  **

**

**

Control 1 ppm 2.5 ppm 5.0 ppm 

### Activity as crossed quadrants

Males Females



### 0 20 40 60 80 100

FormaldehydeThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-257 Male LEW.1K Rats

0% 10% 20% 30% 40% 50% 60% 70% 80% 90%

Control 1 ppm 2.5 ppm 5.0 ppm FormaldehydeHabituation (percent ) Female LEW.1K Rats

0% 50% 100% 150% 200% 250% 300%

Control 1 ppm 2.5 ppm 5.0 ppm FormaldehydeHabituation (percent)

1 2 Figure 4-21. Habituation of motor activity was obs erved in control rats 3 during the second observation period (day 2, 24 hou rs after formaldehyde 4 exposure). 5 6 Note: Habituation is shown here as the percent decr ease in number of crossings 7 between sessions from day 1 to day 2. The degree o f habituation was reduced in 8 male rats exposed to formaldehyde (left panel) sinc e their activity was closer to 9 100% of that seen on day 1. Females (right panel) had increased activity on day 2 10 (greater than 100% of activity on day 1), which is a sensitization rather than 11 habituation. 12 13 Source: Drawn from data reported by Malek et al. ( 2003a). 14 15 16 A follow-up study by Malek et al. (2003b) further e xpanded the dose-response analysis 17 for acute formaldehyde exposure. As described abov e, male and female LEW.1K rats (10 per 18 sex per group) were exposed at 0, 0.1, 0.5, or 5 pp m (0, 0.123, 0.615, or 6.15 mg/m 3 ) 19 formaldehyde for 2 hours. Formaldehyde levels were checked nine times per hour during the 20 exposure periods, and mean values were found to be 0.13 ± 0.04, 0.48 ± 0.05, and 21 5.18 ± 0.66 ppm. Open field behavior was evaluated for each animal 2 hours after formaldehyde 22 exposure. The number of crossed quadrants for both controls and a 5 ppm group were generally 23 comparable with those observed in the first study, although female values were somewhat lower. 24 Horizontal movement was decreased by formaldehyde e xposure in a dose-dependent manner 25 with significant reductions in motor activity as lo w as 0.1 ppm in males and 0.5 ppm in females 26 (Figure 4-22). The consistency of the findings acr oss studies and between genders provides 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-258 greater confidence in the effects of low-level form aldehyde exposure on this standard test of 1 neurotoxicity. 2

3

0 10 20 30 40 50 60

0 0.1 0.5 5.0 0 0.1 0.5 5.0

formaldehyde (ppm)number of crossed quadrants

male female



### *

***

*** **

***

4 Figure 4-22. Motor activity was reduced in male an d female LEW.1K rats 5 2 hours after termination of 10-minute formaldehyde exposure. 6 7 Note: Values are means ± SDs. * = different from co ntrol, p < 0.05. ** = different 8 from controls, p < 0.01. *** = different from controls, p < 0.001. 9 10 Source: Drawn from data reported in Malek et al. ( 2003b). 11 12 13 Malek et al. (2004) also assessed the capacity of f ormaldehyde to induce persistent 14 behavioral deficits in mice. Groups of 20 male AB mice received a single 2-hour exposure to 0, 15 1.1, 2.3, or 5.2 ppm (0, 1.3, 2.8, or 6.4 mg/m 3 ) formaldehyde prior to being tested 2 and 24 hours 16 after exposure for a series of behavioral responses , including ambulation (crossed squares), 17 grooming, sniffing, rearing, wall climbing, and def ecation. Even though there were no clinical 18 signs of toxicity in any of the exposed groups, a n umber of behavioral anomalies were apparent 19 in response to formaldehyde exposure, some of which persisted for at least 24 hours, as indicated 20

in Tables 4-54 and 4-55. 21

22This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-259 Table 4-54. Fluctuation of behavioral responses wh en male AB mice inhaled 1 formaldehyde in a single 2-hour exposure: effects a fter 2 hours 2 3 Formaldehyde concentration (ppm) a

Open field parameter 0 1.1 2.3 5.2 No. of crossed inner squares 34.10 ± 7.51 25.30 ± 5 .03 b 21.20 ± 3.41 b 16.10 ± 5.37 b No. of crossed peripheral squares 56.65 ± 9.68 59.5 5 ± 9.75 49.70 ± 13.24 29.15 ± 7.47 b Total no. of crossed squares 90.75 ± 11.08 84.85 ± 9.96 71.10 ± 13.91 b 44.20 ± 7.42 b Air sniffing 19.35 ± 2.5 21.50 ± 4.26 16.35 ± 3.84 c 8.10 ± 1.77 b Floor sniffing 20.95 ± 3.72 26.50 ± 4.64 b 21.35 ± 4.77 22.80 ± 4.02 Grooming 7.95 ± 2.26 7.10 ± 3.19 7.05 ± 2.48 6.55 ± 2.06 Rearing 17.85 ± 2.56 13.90 ± 3.19 b 11.30 ± 2.30 b 9.95 ± 1.61 b Wall climbing 13.20 ± 3.09 14.55 ± 2.74 13.95 ± 2.3 1 13.95 ± 1.82 No. of excreted fecal boli 0.65 ± 0.81 0.75 ± 0.85 0.80 ± 0.77 0.90 ± 1.12 4

a Values are means ± SDs. 5

b Statistical significance of differences from contro ls ( p < 0.005). 6

c Statistical significance of differences from contro ls ( p < 0.05). 7 8 Source: Malek et al. (2004). 9 10 11 Table 4-55. Fluctuation of behavioral responses wh en male AB mice inhaled 12 formaldehyde in a single 2-hour exposure: effects a fter 24 hours 13 14 Formaldehyde concentration (ppm) a

Open field parameter 0 1.1 2.3 5.2 No. of crossed inner squares 10.40 ± 2.35 9.55 ±1.7 3 9.10 ± 1.25 9.70 ± 1.13 No. of crossed peripheral squares 42.80 ± 9.27 44.8 5 ± 14.60 44.95 ± 16.56 41.10 ± 9.08 Total no. of crossed squares 53.20 ± 8.67 54.40 ± 1 4.77 54.05 ± 15.81 50.80 ± 9.15 Air sniffing 13.65 ± 2.81 13.30 ± 3.21 12.65 ± 2.70 12.30 ± 4.14 Floor sniffing 21.55 ± 3.47 15.85 ± 3.94 b 13.25 ± 4.17 b 17.65 ± 3.13 b Grooming 8.35 ± 2.56 13.95 ± 2.21 b 10.20 ± 3.33 c 11.90 ± 3.26 b Rearing 18.30 ± 4.23 12.40 ± 2.23 b 12.25 ± 2.17 b 12.00 ± 3.32 b Wall climbing 9.25 ± 2.38 8.70 ± 1.98 8.20 ± 2.14 9 .90 ± 2.27 No. of excreted fecal boli 0.80 ± 0.83 1.20 ± 0.83 1.60 ± 0.94 c 1.20 ± 0.89 15

a Values are means ± SDs. 16

b Statistical significance of differences from contro ls ( p < 0.005). 17

c Statistical significance of differences from contro ls ( p < 0.05). 18 19 Source: Malek et al. (2004). 20 21 22 Usanmaz et al. (2002) assessed spontaneous locomoto r activity (SLMA) in Balb/c mice 23 (4–14 per group, sex unspecified) after both acute and subchronic formaldehyde exposures. 24 Prior to the acute exposure, mice were acclimated t o the exposure chamber for 4 days but 25 exposed only to clean air. On the fifth day, mice (six/group, sex unspecified) were exposed for 26 3 hours at 0, 1.8, 3.2, 4.5, 6.4, 9.7, or 14.8 ppm (0, 2.2, 3.9, 5.5, 7.9, 11.9, or 18.2 mg/m 3 ) 27 formaldehyde. Mice were removed from the exposure chamber, and SLMA behavior was 28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-260 evaluated by direct observation for 5 minutes. In addition to horizontal and vertical movement, 1 wet dog shake (WDS) behavior was noted. In a separ ate trial, Balb/c mice (six/group, sex 2 unspecified) were exposed to 8.2 ppm formaldehyde f or 1 week, 2 ppm formaldehyde for 3 2 weeks, or 3.3 ppm formaldehyde for 3 weeks (3 hou rs/day, 5 days/week) compared with 4 controls exposed only to air. SLMA behavior was ob served for 5 minutes after the last exposure. 5 Mice exposed to 8.2 ppm formaldehyde for 1 week, 3. 3 ppm formaldehyde for 2 weeks, and 6 2 ppm formaldehyde for 3 weeks lost weight over the course of the treatment ( p < 0.05). All 7 other treatment groups had weight gain similar to c ontrol mice. 8 As shown in Figure 4-23, acute 3-hour formaldehyde exposures resulted in a dose9 dependent decrease in SLMA. Decreases in horizonta l activity were significant for the three 10 highest dose groups (6.4, 9.7, and 14.8 ppm), and d ecreases in vertical activity were significant 11 for all six formaldehyde treatment groups. SLMA wa s similarly decreased following subchronic 12 exposures (data not shown here). Although the expe rimental protocol included longer exposures 13 and a slightly longer observation period (5 versus 3 minutes) than in Malek et al. (2003a, b), the 14 results are consistent, indicating decreased activi ty in formaldehyde-exposed animals several 15 hours after exposure was ended. 16

17

100

80

60

40

20

0 SLMA after acute exposures





 

 

  

Ambulatory Activity Vertical Activity Control FA 1.8 ppm FA 3.2 ppm FA 4.5 ppm FA 6.4 ppm FA 9.7 ppm FA 14.8 ppm

18

19 Figure 4-23. The effects of the acute formaldehyde (FA) exposures on the 20 ambulatory and vertical components of SLMA. 21 22 Note: FA = formaldehyde exposure concentration. ** = p < 0.01 from controls. 23 24 Source: Usanmaz et al. (2002). 25 26 27 Usanmaz et al. (2002) noted an increase in WDS, aft er the acute exposures, as a possible 28 preconvulsive effect. However, the mice were only observed for 5 minutes, and it is unclear how 29This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-261 the researchers distinguished between a WDS due to an irritating odor and a preconvulsive 1 movement. No other study has noted convulsive effe cts from formaldehyde exposure in any 2 species. A second set of trials was reported in th e same paper that sought to evaluate 3 formaldehyde effects on CNS excitability. Balb/c m ice (six/group, sex unspecified) were 4 exposed to 0, 1.8, 6.4, or 14.8 ppm (0, 2.2, 7.9, o r 18.2 mg/m 3 ) formaldehyde for 3 hours. 5 Subchronic exposures were at 2 ppm (2.5 mg/m 3 ) formaldehyde for 3 weeks or 3.3 ppm 6 (4.1 mg/m 3 ) formaldehyde for 2 weeks. Seizures were induced by I.P. injection of 7 pentilenetetrazole (PTZ), and the incidence, severi ty, and course of induced seizures were 8 recorded. The PTZ injection induced seizures in 83 , 88, and 91% of controls, with 16, 38, and 9 67% mortality in controls in the three trials. Mor tality was highly variable in treatment groups as 10 well. The authors report that PTZ-induced seizures were decreased in incidence by acute 11 formaldehyde exposure in a dose-dependent fashion w ith only 33% of mice exposed to 14.8 ppm 12 formaldehyde experiencing seizures versus 91% in co ntrol mice ( p < 0.05 at the highest dose 13 only). However, the methodology for observing and scoring seizures is unclear. Additionally, 14 there was high mortality and high variability of r esults for the three similarly treated control 15 groups. Therefore, it is difficult to assess data quality and interpret these findings. 16 Boja et al. (1985) exposed male Sprague-Dawley rat s to air or to formaldehyde at 5, 10, 17 or 20 ppm for 3 hours on 2 consecutive days. On th e second day, half the rats received the same 18 exposure as the previous day, while half the rats w ere switched (e.g., half those rats receiving air 19 the first day received formaldehyde the second day, and half those receiving formaldehyde the 20 first day received air the second day), for a total of four possible exposure combinations. During 21 the exposure period, activity levels were monitored by observation, once per minute for the first 22 hour and once every 5 minutes for the second hour. At the end of the second exposure session, 23

rats were sacrificed and brains removed for neuroch emical analysis (see Section 4.2.1.6.1.5). 24 Behavioral results were described in detail only f or control and 5 ppm groups. During 25 the first exposure session, activity levels of form aldehyde-exposed animals were significantly 26 decreased (approximately 50% of control levels). O n the second day of exposure, those animals 27 previously exposed to formaldehyde exhibited partia l recovery, those experiencing their first 28 formaldehyde exposure behaved similarly to those in itially exposed on the first day, and those 29 animals exposed to formaldehyde for a second time h ad a greater decrease in activity than during 30 the first exposure (to approximately 30% of control levels). The authors stated that a similar 31 effect was seen in animals exposed at 10 ppm but th at results at 20 ppm were not interpretable 32 (data were not presented). Overall, the decreased activity seen in this study is consistent with 33 effects seen by other authors. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-262 Senichenkova (1991) exposed pregnant female rats to 0 or 0.5 mg/m 3 (0 or 403 ppb) 1 formaldehyde on gestation days (GDs) 1–19 for 4 hou rs/day. Reproductive aspects of this study 2 will be discussed in the reproduction section; howe ver, results from behavioral assessments 3

conducted on the neonates are discussed here. The author stated that maturation of motor 4 reflexes (assessed as surface righting and pendular reflex), open field behavior, and maze 5 learning ability were assessed. Detailed descripti ons of procedures and results were not provided 6 for all assessments, but it was stated that motor r eflex development did not differ in treated and 7 control animals. Open field motor activity assessm ents in 40-day-old (juvenile) offspring 8 revealed an increase in squares visited and an incr eased frequency of rearing on the second and 9 third days of testing, indicating a lack of habitua tion in the offspring of formaldehyde-treated 10 dams; similar levels of activity by both measures w ere found on the first test day. Counts of 11

defecation and urination were increased on all 3 da ys of testing. Increased exploratory behavior, 12 described as increased impulses, was also noted in a learning task (not otherwise described), but 13 the author stated that learning rate and ability of the formaldehyde-treated group was not 14 different from controls (no data were provided). 15 Mobility and neuromuscular excitability (not otherw ise described) in offspring of female 16 white rats were also evaluated by Sheveleva (1971). Dams were exposed to 0.005 or 17 0.0005 mg/L (approximately 4,000 or 400 ppb, respec tively) formaldehyde on GDs 1–19. 18 Spontaneous mobility (over 15 minutes) and neuromus cular excitability were evaluated in 19 offspring at 1 or 2 months of age (other results fr om this study are discussed under 20 developmental toxicity, above). At 1 month, sponta neous mobility was reduced at the low dose 21 in males (52% of control levels; p < 0.01) but not at the high dose, and at both dose s in females 22 (to 64 and 56% of control levels at the mid dose an d high dose, respectively; p < 0.02). At two 23 months, there was a dose-related increase in activi ty for both sexes, statistically significant ( p < 24 0.001) in high-dose females only (391% of control l evels). 25

26

4.2.1.6.1.3. Learning and memory. The effects of repeated formaldehyde exposures on l earning 27 were investigated by Malek et al. (2003c), using a labyrinth swim maze. In this task, animals are 28 required to make a series of consecutive right or l eft turns to gain access to an escape platform 29 (Malek et al., 2003c). Adult male and female LEW.1 K rats (15/sex/group) were exposed to 0, 30 0.1, 0.5, or 5.4 ppm (0, 0.123, 0.615, or 6.64 mg/m 3 ) formaldehyde 2 hours/day for 31 10 consecutive days. Formaldehyde levels were chec ked eight times throughout the 2-hour 32 exposure periods. Mean formaldehyde levels of 0.1 ± 0.02, 0.5 ± 0.1, and 5.4 ± 0.65 ppm were 33 achieved. Body weight was measured on days 1, 5, a nd 10 of the experiment. Two days prior to 34 beginning the formaldehyde exposures, all subjects were given an acclimation trial in which they 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-263 were individually placed into the water-filled basi n at the start position and allowed to navigate 1 to the escape platform with manual assistance to le arn the correct route. Thereafter, the water 2 labyrinth test was run on each day of formaldehyde treatment, 2 hours after completion of each 3 daily exposure. Time taken to complete the test an d errors made were recorded for each rat 4 (Table 4-56). An error was defined as swimming tow ard the start position or circling in the same 5 position without moving forward toward the escape p latform. Rats were sacrificed at the end of 6 the experiment, and tissues were taken from the lun g, heart, thymus, kidney, liver, pancreas, 7 skeletal muscle, and spleen. Tissues were fixed an d prepared for histologic examination by light 8 microscope. No differences were noted in food cons umption or body weight gain for either male 9 or female rats (Malek et al., 2003c). No treatment -related differences in organ pathology were 10 reported (with the possible exception of focal micr oatelectasis (lung collapse at the microscopic 11 level) seen in two to three animals in each formald ehyde-exposed group but not control animals). 12

13 Table 4-56. Effects of formaldehyde exposure on co mpletion of the labyrinth 14 test by male and female LEW.1K rats 15 16 Swimming time (sec) Error rate (mean) Male rats Day 1 Day 6 Day 10 Day 1 Day 6 Day 10 Control 105 12.2 6.33 7.4 0.5 0.0 0.1 ppm a 100 12.9 6.07 7.7 5.0 c 3.2 c 0.5 ppm 97 16.7 c 7.60 b 7.6 4.4 c 1.8 c 5.4 ppm 105 25.7 c 10.9 c 7.7 5.0 c 2.8 c Swimming time (sec) Error rate (mean) Female rats Day 1 Day 6 Day 10 Day 1 Day 6 Day 10 Control 103 12.5 6.47 7.9 0 0.0 0.1 ppm 96 12.3 7.53 7.1 5.2 c 3.0 c 0.5 ppm 97 14.6 c 7.60 b 8.0 4.6 c 2.2 c 5.4 ppm 98 23.5 c 9.73 c 7.9 5.2 c 2.6 c 17

a Rats were exposed to formaldehyde for 2 hours/day, for 10 consecutive days. 18

b Different from control, p < 0.05. 19

c Different from control, p < 0.005. 20 21 Source: Malek et al. (2003c). 22 23 24 A clear learning curve was evident in control anima ls, with rats completing the task in 25 less time and with fewer errors over days (Table 4- 56). Although the number of errors decreased 26 with increasing experience in all groups, error rat es in formaldehyde-exposed rats at all doses 27 were consistently higher than those observed in con trols, starting on day 3 (Figure 4-24). All 28 control animals performed without errors by day 6, whereas all treated animals were still making 29 two to three errors on day 10, the final day of tes ting. Time required (latency) to complete the 30 maze was also reduced over days. Although this mea sure of performance was not as sensitive as 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-264 error rate, formaldehyde-induced deficits were stil l evident in the 0.5 and 5.4 ppm exposure 1 groups of both sexes. 2

3

4

5

Figure 4-24. Effects of formaldehyde exposure on t he error rate of female 6 LEW.1K rats performing the water labyrinth learning test

. 7 8 Source: Drawn from data reported in Malek et al. ( 2003c). 9 10 11 Impaired performance on formaldehyde-treated subjec ts cannot be attributed to 12 alterations in swimming ability, since latencies to complete the maze were identical for 0 and 13 0.1 ppm groups, yet acquisition of the task was sti ll impaired in the 0.1 ppm group based on 14 number of errors committed (see Figure 4-24). This study reports an adverse effect level of 15 0.1 ppm for increased error rate in the labyrinth w ater test, and all dose groups were equally 16 impaired across a broad range of exposures, 0.1–5.4 ppm. An independent estimate of 17 swimming speed was not included, so motor competenc y could not be directly evaluated. 18 However, comparable latency scores and error rates at the beginning of testing across all groups 19 and latency scores that track together over days su ggest that impaired swimming ability does 20 account for the observed differences in latency, wh ich are most likely reflective of the increased 21This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-265 number of errors in treated animals (errors usually increase the distance traveled and thus time 1 required for completion of the trial). 2 Pitten et al. (2000) evaluated the effects of very brief formaldehyde exposures 3 (10 minutes) but prolonged duration (90 days) on pr eviously learned performance in a land 4 version of the labyrinth maze. Adult male and fema le Wistar rats (13/group) were trained on the 5 task for 14 days, two trials/day. Animals were req uired to make a series of five left or right turns 6 from the entrance of the maze to retrieve a piece o f cheese placed in the goal box at the opposite 7 end. Animals were guided by the experimenter throu gh the maze during this acclimation phase 8 until all subjects were able to retrieve the food w ithout aid. After animals were trained (but prior 9 to formaldehyde exposure), performance was assessed once daily for 11 days, and the latency to 10 complete the maze as well as the number of errors c ommitted when traversing from the entrance 11 to the goal box was recorded. Animals were then as signed to one of three dose groups (five to 12 eight/sex/group) such that task performance was equ ivalent across groups prior to 13 commencement of formaldehyde exposures. Animals we re exposed to 0 ppm, 2.6 ppm (0.25% 14 formaldehyde solution to yield 3.06 ± 0.77 mg/m 3 ), or 4.6 ppm (0.70% formaldehyde solution to 15 yield 5.55 ± 1.27 mg/m 3 ) formaldehyde 10 minutes/day, 7 days/week for 90 d ays. Animals were 16 assessed for performance in the maze every seventh day, at least 22 hours after the exposure on 17 the previous day. At the end of the 90-day exposur e period, monitoring of maze performance 18 continued once every 10 days for an additional 30 d ays. All rats were sacrificed at the end of the 19 postexposure trials and tissue sections were prepar ed for histologic examination by light 20 microscopy, including liver, trachea, lung, kidney, heart, spleen, pancreas, testicle, and brain. 21 No treatment-related changes in food or water consu mption weight gain or in histologic samples 22 obtained at the termination of the experiment were observed. 23 Pitten et al. (2000) reported that no gender differ ences existed as a function of 24 formaldehyde treatment; therefore, data were presen ted by combining sexes. Control rats 25 showed no change in error rate but a slight decreas e in running time through the maze during the 26 course of the experiment. The formaldehyde-exposed groups began with a similar performance 27 level and error rate as controls, but their perform ance degraded over the course of formaldehyde 28 exposure. By the fourth week of exposure, increase d numbers of errors were evident in both 29 exposed groups relative to controls. This trend re ached statistical significance at the 12-week 30 time point, with a greater than twofold increase in number of errors ( p < 0.05). Formaldehyde31

treated rats also tended to have increased run time s through the maze ( p = 0.04), but no 32 difference was seen by formaldehyde concentration. By 4 weeks after termination of exposure, 33 no statistical differences among the three groups w ere evident, but the tendency for the two 34 exposed groups to make more errors and have longer latencies remained. Since Pitten et al. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-266 (2000) tested animals after the task was acquired, these results indicate deficits in the retention of 1 a previously learned task. 2 Lu et al. (2008) evaluated the effects of formaldeh yde on performance of mice in a 3 Morris water maze. Kunming mice (five males/group) were exposed to formaldehyde at 0.2, 1, 4 or 3 mg/m 3 6 hours/day for 7 days (measured concentrations: 0 .2 ± 0.01, 0.99 ± 0.04, and 5

3.03 ± 0.16 mg/m 3 ). Mice were trained to locate a hidden platform i n a large, circular tank 6 (106 cm diameter, 31 cm deep). Each animal receive d four training trials per day, beginning 7 30 minutes after the end of exposure. During train ing, latency to locate the platform was 8 recorded for each trial, with a maximum of 60 secon ds, after which the animal was guided to the 9 platform. After the last day of training, an addit ional trial was conducted with the platform 10 removed (the probe trial); time spent in each maze quadrant was measured to determine the time 11 the animal spent searching for the platform in the correct area of the maze. Performance in the 12 water maze, measured as mean escape latency across the seven training trials, was significantly 13 impaired in the 3 mg/m 3 group. No significant difference was seen at 1 mg /m 3 , although there 14 appeared to be an increased latency during the seco nd day of testing. During the probe trial, 15 control animals spent significantly more time in th e correct quadrant, but neither formaldehyde16 exposed group did so. Results of this study indica te deficits in learning and retention of the 17 Morris water maze following formaldehyde exposure, with greater effects seen in the higher dose 18

group. 19 Apfelbach and Reibenspies (1991) published a brief report of formaldehyde effects on 20 olfactory learning. Ferrets were exposed to 0.25 p pm (0.31 mg/m 3 ) formaldehyde gas 21 continuously for 6 months. A Y-shaped maze was use d to test odor detection, discrimination 22 between odors, and odor threshold. Ferrets were co nditioned to distinguish ethyl acetate 23 (0.1 vol%) from clean air. Untreated ferrets achie ved 75% success after an average of 110 trials. 24 However, formaldehyde-treated ferrets required on a verage 320 trials to reach a 75% success 25 rate. A 90% success rate was achieved by untreated ferrets after 420 trials. However, this level 26 of success was not reached in formaldehyde-treated ferrets. 27 The same researchers also tested olfactory function in formaldehyde-treated ferrets, as 28 summarized in Section 4.2.1.7 (Apfelbach et al., 19 92). A decrease in olfactory discrimination 29 and a reduction in the percentage of olfactory cell s in the olfactory epithelium were reported 30 after 3–12 months exposure to 0.25 or 0.5 ppm forma ldehyde. Decreased olfactory sensitivity in 31 rats exposed to 0.25 or 0.5 ppm formaldehyde has al so been reported by the same researchers 32 (Weiler and Apfelbach, 1992; Apfelbach and Weiler, 1991), and Weiler and Apfelbach (1992) 33 reported in an abstract that shifts in olfactory th resholds were greater when exposure was 34 initiated at PND 30 than at adult ages. Given the documented changes in olfactory thresholds, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-267 observed changes in olfactory learning would likely be confounded by the potential for 1 decreased olfactory function by formaldehyde exposu res, and definitive conclusions regarding 2 formaldehyde effects specific to learning cannot be made based on these studies. 3

4

4.2.1.6.1.4. Neurosensitization. Sorg et al. (1996) studied the potential for formal dehyde 5 exposure to induce sensitization in the CNS, possib ly through the limbic pathways in the brain. 6 The authors hypothesized that multiple chemical sen sitivity (MCS) has an onset and progression 7 similar to CNS sensitization and may, therefore, be a similar process. These experiments were 8 conducted to test this hypothesis and to determine whether formaldehyde exposure could be used 9

as a model for MCS. Behavioral sensitization can b e initiated by psychostimulants (e.g., 10 cocaine) and manifest as increased locomotor activi ty upon subsequent challenge with the 11

stimulant. 12 Sorg et al. (1996) evaluated cross-sensitization of cocaine-induced increases in activity 13 from an initial formaldehyde exposure. Female Spra gue-Dawley rats (eight to nine) were 14 exposed to 0 or 11 ppm (0 or 13.5 mg/m 3 ) formaldehyde 1 hour/day for 7 days. Locomotor 15 activity was measured (by photocell) after saline i njection (1 day postexposure) and after cocaine 16 injection (2 days postexposure). A similar protoco l was conducted on days 36 and 37 17 postexposure. Motor activity levels following sali ne injection were similar for controls and 18 formaldehyde-treated rats. However, formaldehyde e xposure initiated sensitization to cocaine as 19 evidenced by a greater increase in locomotor activi ty in mice treated with formaldehyde 20 followed by cocaine ( p < 0.05) with an average count of crossed grids grea ter than 40,000 (2 21 hours) in treated animals compared with 25,000 (2 h ours) in controls. The cross-sensitization 22 was transient, with no treatment effects on cocaine -induced activity either 29 or 37 days 23 postexposure. When examining individual data, the authors suggested that the formaldehyde24 treated groups in both cases have a cluster of high responders (HRs), suggesting some animals 25 may have been more sensitive. A second group of si milarly treated female rats was pretested for 26 locomotor activity and divided into subgroups of HR s or low responders (LRs). They were then 27 given a panel of neurobehavioral tests: anxiety (el evated plus maze, day 11); memory (passive 28 avoidance training, day 12; passive avoidance test, day 19); and nociceptive test (day 20). Trunk 29 blood corticosterone levels were determined during stress on day 35 postexposure. No 30 significant treatment differences were found in the passive avoidance test, nociception, or 31 corticosterone levels (basal or stress induced). O n the elevated plus maze, a two-way ANOVA 32 indicated no overall formaldehyde treatment effects , but the HR rats had higher open arm time 33 ratios (indicating greater anxiety) regardless of t reatment. Within the treatment groups, the 34 difference in behavior between HR and LR subgroups was only significant for the formaldehyde- 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-268 treated rats (

p < 0.05). Authors suggested that cross-sensitizatio n to cocaine-induced locomotor 1 activity was caused by enhanced dopamine transmissi on within the mesolimbic system (ventral 2 tegmental area to nucleus accumbens projection) fol lowing repeated formaldehyde exposure. A 3 critical role of the hypothalamic-pituitary-adrenal (HPA) axis has also been implicated in cross4

sensitization. 5 Sorg et al. (1998) and Sorg and Hochstatter (1999) further explored formaldehyde6 induced behavioral sensitization using the cocaine model. In contrast to the results following 7 exposure to 11 ppm (Sorg et al., 1996), rats expose d to only 1 ppm for 7 days showed no cross8 sensitization to cocaine injection. However, anima ls exposed to 1 ppm formaldehyde for 9

4 weeks exhibited increased cocaine-induced vertica l activity (with no difference in horizontal 10 activity) for 4–6 weeks after cessation of exposure . Activity levels of formaldehyde-exposed rats 11 were approximately threefold those of control rats 3–4 days postexposure and still 1.5-fold 12 control levels at 4–6 weeks postexposure ( p < 0.05). 13 Sorg et al. (2001) examined changes in corticostero ne levels in rats with and without 14 formaldehyde treatment. Basal corticosterone level s in trunk blood were established in naïve 15 male Sprague-Dawley rats taken directly from their home cage immediately prior to sacrifice. In 16 an acute trial, male rats were exposed to 0, 0.7, o r 2.4 ppm (0, 0.86, or 2.96 mg/m 3 ) 17 formaldehyde for either 20 or 60 minutes, and trunk blood was collected for corticosterone 18 analysis. Therefore, these rats were challenged wi th a new environment (the exposure chamber) 19 in the presence or absence of formaldehyde. In a s eparate trial, basal and challenged 20 corticosterone levels were measured after repeated exposure (1 hour/day, 5 days/week for 2 or 21 4 weeks). Basal corticosterone levels were measure d in trunk blood immediately after removing 22 the animal from its home cage. Challenged corticos terone levels were measured after rats were 23 placed into the exposure chamber for a final 20-min ute exposure. Body weight was measured at 24 the beginning of each week of exposure and was unch anged by formaldehyde treatment. 25

Corticosterone levels were increased over basal lev els when rats were placed in the 26 exposure chamber for 20 minutes (Figure 4-25, panel a) but returned to basal levels after 27 60 minutes in the exposure chamber (not shown). Th is response may reflect the stress of the new 28

environment and acclimatization after 60 minutes in the chamber. Corticosterone levels were the 29 same in the presence or absence of formaldehyde, in dicating no treatment effect. 30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-269 1

Panel a Panel b Panel c 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 Figure 4-25. Basal and stress-induced trunk blood corticosterone levels in 22 male LEW.1K rats after formaldehyde inhalation expo sures. 23 24 Note: Panel a: no pretreatment, corticosterone leve ls after 20-minute 25 formaldehyde exposure. Panels b and c show both ba sal and induced 26 corticosterone levels after a 2- or 4-week pretreat ment to formaldehyde 27 1 hour/day. Challenge to induce corticosterone was a 20-minute reexposure at the 28 formaldehyde level tested. 29 30 Source: Sorg et al. (2001). 31 32

Control animals exhibited an increase in basal cort icosterone after 2 weeks, which 33 returned to naïve levels after 4 weeks (Figure 4-25 , panels b and c). Formaldehyde-treated rats 34 demonstrated a comparable increase in basal cortico sterone levels at 2 weeks, but these levels 35

did not return to naïve levels at 4 weeks as seen w ith controls. Control and 0.7 ppm exposed rats 36 showed a similar response to challenge (the final 2 0-minute exposure). However, rats exposed to 37 2.4 ppm were hyperresponsive, with exaggerated cort icosterone levels during this final exposure. 38

Differences in basal corticosterone levels after fo rmaldehyde exposure and the 39 hyperresponsiveness seen in animals exposed at 2.4 ppm provide evidence of 40 formaldehyde-induced perturbations of the HPA axis. Authors suggested that elevated 41 corticosterone levels induced by repeated formaldeh yde exposures may contribute to the cross42 sensitization to cocaine-induced motor activity. 43 Formaldehyde-induced changes in the HPA axis may co ntribute to behavioral effects of 44 formaldehyde exposure reported by Sorg et al. (2004 ) and Sorg and Hochstatter (1999). The 45 authors also reported an enhanced conditioning to o dor in animals previously exposed to 46 Corticosterone levels after 2 weeks of repeated formaldehyde exposure

0 20 40 60 80 100 120 140 160 180 200

Control 0.07 2.4 Formaldehyde (ppm) Basal

ChallengeSerumcorticosterone (ng/mL)

0 20 40 60 80 100 120 140 160 180 200

Control 0.07 2.4

Formaldehyde (ppm ) Basal Challenge Corticosterone levels: with no pre-exposure to formaldehydeSerum corticosterone (ng/mL)

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Control 0.07 2.4

Formaldehyde (ppm) Corticosterone levels after 4 weeks of repeated formaldehyde exposureSerum corticosterone (ng/mL) Basal

ChallengeThis document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-270 repeated formaldehyde. Male and female Sprague-Daw ley rats (60–80 days of age) were 1 exposed at 1 ppm (1.23 mg/m 3 ) formaldehyde 1 hour/day, 5 days/week for 4 weeks (Sorg and 2 Hochstatter, 1999). Two weeks after exposure ended , rats were trained to the conditioned fear 3 task. Rats were conditioned to a fear response by either odor only or odor associated with 4 footpad shock. Orange-oil extract was used as the odor conditioned stimulus (CS). One day 5 after conditioning, rats were reintroduced into the environment without an odor cue, and time 6 spent motionless in the freezing posture (freezing) was observed. On day 2 after conditioning, 7 rats were placed in a novel environment, and time s pent in the freezing posture was evaluated in 8 the absence and then the presence of odor. This wa s repeated on day 12 after conditioning to 9 measure the loss of the freezing response to the co nditioned odor. 10 Both treated and exposed rats showed similar respon ses on reintroduction into the 11 conditioning environment in the absence of an odor cue on day 1 (Sorg and Hochstatter, 1999). 12 As expected, rats conditioned with a footpad shock demonstrated greater time motionless than 13 odor-trained only rats, and there was no difference between control and formaldehyde-treated 14 rats. However, in the presence of odor on days 2 a nd 12, formaldehyde-exposed rats who were 15 conditioned with odor associated with foot shock sp ent significantly more time freezing than 16 odor-only trained rats ( p < 0.05); control animals on those days showed no di fference in time 17 freezing in the presence and absence of odor. The authors concluded that the formaldehyde18 treated rats had more difficulty than controls in e xtinguishing the fear response to the 19 conditioned odor, and speculated that an enhancemen t of the fear-conditioned response by 20 formaldehyde pretreatment supports the hypothesis t hat sensitization may include effects through 21 the limbic system of the brain. 22 In a second experiment, adult male and female Sprag ue-Dawley rats were exposed at 0 or 23 2 ppm (2.45 mg/m 3 ) formaldehyde 1 hour/day, 5 days/week for 4 weeks (Sorg et al., 2004). Two 24 to 3 weeks after exposure ended, rats were trained to the conditioned fear task. Rats were given 25 a foot shock either associated with an odor (paired group) or unassociated with an odor (unpaired 26 group). Orange-oil extract was used as the odor CS . After training, freezing behavior was 27 assessed (1) in the same context in the absence of odor (1 day), (2) in a new context in the 28 presence and absence of the CS (5 consecutive days) , and (3) in another novel context in the 29 presence and absence of the CS. 30 Formaldehyde-exposed male rats demonstrated increas ed conditioned fear response to an 31 odor CS (orange oil) paired with foot shock with no change in the degree of conditioning to the 32 context. For female rats, formaldehyde exposure di d not affect the percent of time spent 33 freezing, either in the conditioning context or the novel context in the absence of the conditioned 34 odor. In contrast, male rats spent an increased ti me freezing in a novel context in the presence of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-271 odor, indicating a greater conditioned fear respons e to the olfactory cue ( p < 0.05). This is in 1 agreement with the previous study where formaldehyd e effects were seen in the presence of the 2 conditioning odor but not the environment (Sorg and Hochstatter, 1999). However, in this study 3

female rats did not exhibit a similar enhancement o f fear conditioning to the olfactory CS. 4 The authors suggested that repeated exposure to low levels of formaldehyde acts as a 5 stressor in much the same way as inescapable foot s hock, with resulting sensitized responses 6 within the olfactory/limbic pathways (Sorg et al., 2004). This interpretation is consistent with 7 work described above in which augmented basal corti costerone levels following repeated 8 formaldehyde exposures were demonstrated. However, while the fear conditioning in the present 9 study and cross-sensitization to cocaine described above (Sorg and Hochstatter, 1999) occurred 10 3–4 weeks after termination of exposure to formalde hyde, the duration of corticosterone 11 elevation induced by repeated exposure to formaldeh yde has not been determined. It is possible 12 that augmentation of corticosterone levels followin g formaldehyde exposure results from direct 13 action of formaldehyde on the HPA axis. Experiment s designed to compare HPA activation 14 following standard stressors (repeated inescapable foot shock or restraint stress), stress induced 15 by other irritants (chemicals with strong irritant odors but no CNS action), and repeated 16 formaldehyde exposures are necessary to dissociate primary from secondary action of 17 formaldehyde on CNS function in this paradigm. It is also possible that enhanced conditioning 18 to an odor stimulus results from formaldehyde-induc ed increases in airway irritation, rendering 19 the conditioned odor stimulus a more salient cue, p roducing a conditioned response that is not 20 extinguished as readily as in air-exposed controls. However, damage of the nasal mucosa and 21 lesions would be expected to be minimal at 1 ppm fo rmaldehyde exposures and most likely 22 resolved 2 weeks after exposure was ended (see Sect ion 4.2.1.2). Therefore, a more salient cue 23 for fear conditioning to odor due to physical irrit ation is not likely. Alternatively, formaldehyde 24 may act to up regulate olfactory activity, producin g a stronger sense of odor during conditioning. 25

26

4.2.1.6.1. 5. Neurochemistry and neuropathology. Several studies that were focused on general 27 toxicity or carcinogenicity of formaldehyde also as sessed histopathology in exposed animals, 28 including pathological evaluation of the brain. In all cases, details of the pathological evaluation 29 were not provided. Reported results stated that no significant lesions were seen on unspecified 30 tissues (Appelman et al., 1988; Maronpot et al., 19 86; Kerns et al., 1983) or that an increase in 31 relative brain weight (data not provided) was consi dered of no toxicological significance 32 (Woutersen et al., 1987). The absence of procedura l information, or specific reported results, 33 limits the utility of this information. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-272 Boja et al. (1985) measured changes in several neu rotransmitters (norepinephrine, 1 dopamine, 5-hydroxytryptamine) and their major meta bolites (3,4-dihydroxyphenylacetic acid 2 [DOPAC] and 5-hydroxyindoleacetic acid [5-HIAA]) fo llowing one or two 3-hour exposures to 3 formaldehyde at 0, 5, 10, or 20 ppm. Animals were sacrificed immediately following the second 4 exposure, and brains were immediately removed, froz en, and sectioned. Regions of interest were 5 analyzed by high-pressure liquid chromatography wit h electrochemical detection. Authors stated 6

that neurotransmitter concentrations were measured in multiple brain regions, but results were 7 reported only for the 5 ppm exposure and only for t he hypothalamus. No change was seen in 8 concentrations of norepinephrine or 5-hydroxytrypta mine for any exposure paradigm. For those 9 animals exposed twice to formaldehyde, there was a slight (statistically significant) increase in 10 dopamine and a larger (approximately fourfold) incr ease in 5-HIAA. DOPAC was increased 11 (approximately 30%) in animals receiving formaldehy de during the second exposure only. 12 Recent work by Hayashi et al. (2004) indicates that formaldehyde exposure increases the 13 activity of periglomerular (PG) cells in the main o lfactory bulb. Tyrosine hydroxylase activity 14 was measured as a marker for activity of olfactory function. The authors surmised that 15 expression levels of this enzyme are useful markers since it has been reported that the protein is 16 up regulated after sensory stimulation and is down regulated by odor deprivation or when the 17 olfactory epithelium is removed (Cho et al., 1996; Stone et al., 1991; McLean and Shipley, 1988; 18 Baker et al., 1983). Eight-week-old female C3H/HeN mice were exposed at 0, 0.08, 0.4, or 19 2 ppm (0, 0.1, 0.49, or 2.45 mg/m 3 ) formaldehyde 16 hours/day for 1 day or 12 weeks 20 (5 days/week). Formaldehyde exposure did not affec t body weight. Mice were sacrificed 21 24 hours after exposure; the brains were removed, f ixed, and prepared for sectioning. One side 22 of the olfactory bulb was sliced into 40 μm-thick s erial frontal sections and immuno-stained for 23 tyrosine hydroxylase activity. The number of tyros ine hydroxylase-positive PG cells was 24 determined by examining digital photomicrographs of three tissue sections, averaging the counts 25 from 10–15 glomeruli per section. 26 Neither the size of the olfactory bulb (rostrocauda l, dorsoventral, and mediolateral 27 lengths) nor the total number of PG cells was chang ed by formaldehyde exposure. The number 28 of tyrosine hydroxylase-positive PG cells per glome rulus was unchanged by a single 29 formaldehyde exposure but increased after 12 weeks of repeated exposures. The increases were 30 similar among treatment groups: 5.54 ± 0.31 at 0.80 ppm, 5.18 ± 0.60 at 0.4 ppm, and 6.0 ± 0.83 31 at 2 ppm or 196, 167, and 196% of controls, respect ively. As an indicator of activity, it is not 32 unexpected that the enzyme was up regulated after r epeated exposure to an odorous compound. 33 Hayashi et al. (2004) hypothesize that the increase d tyrosine hydroxylase activity is an indication 34 of increased sensitivity and, therefore, may be a m odel for MCS. However, it is unknown if the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-273 increase in enzyme activity after repeated exposure s is transient or could result in sensitization. 1 Tyrosine hydroxylase is the first enzyme in the dop amine synthetic pathway, but the role of 2 dopamine in PG cells is not known. Further researc h would be needed to understand the 3 potential for formaldehyde to act as a sensitizing agent in this model. 4 In an abstract, Kakeyama et al. (2004) outline the results of experiments to address the 5 effects of subchronic exposure to low levels of for maldehyde on changes in neurotransmitter6 related mRNA expressions in mice forebrains. An un stated number of female C3H/He mice 7 were exposed 16 hours/day, 5 days/week to 400 ppb ( 0.49 mg/m 3 ) formaldehyde for 12 weeks. 8 The authors used RT-PCR methodologies to quantify m RNA encoding for the glutamate receptor 9 subunits GluR1 and GluR2, the dopamine receptor D1, and the serotonin receptor 5-HT1A in the 10 neocortex, hippocampus, amygdala, and hypothalamus. Raised levels of mRNA expression were 11 observed for GluR1 in the neocortex and hippocampus ; GluR1, GluR2, and the dopamine 12 receptor D1 in the amygdala; and the serotonin rece ptor 5-HT1A in the hypothalamus. Reduced 13 mRNA expression was observed for GluR2 in the hippo campus and neocortex. When other 14 mice were subjected to a radiofrequency-induced les ion of the hippocampus then exposed to 15 formaldehyde for 12 weeks as before, the altered ex pression of GluR1 and GluR2 in the 16 neocortex was abolished. However, the increment of mRNA expression of 5-HT1A in the 17 hypothalamus was further enhanced. In demonstratin g that formaldehyde affects neocortical 18 GluR1 and GluR2 mRNA expressions through a hippocam pal function, Kakeyama et al. (2004) 19 concluded that subchronic exposure to low concentra tions of formaldehyde can affect neural 20

transmission in the forebrain. 21 Fujimaki et al. (2004b) examined the effects of for maldehyde on NGF in the brain and 22 hippocampus. Ten female C3H/HeN mice/group were ex posed to 0, 80, 400, or 2,000 ppb (0, 23 0.1, 0.5, or 2.45 mg/m 3 ) formaldehyde 16 hours/day, 5 days/week for 12 wee ks. Some groups of 24 mice received the same treatment after I.P. injecti on of 10 μg of OVA and 2 mg alum prior to the 25 commencement of formaldehyde exposure. For this su bgroup, booster injections of OVA were 26 administered on days 21, 42, 63, and 77 during the formaldehyde exposure regimen. 27 Quantitative measures of NGF and BDNF in homogenate s of whole brain and hippocampus were 28 obtained by ELISA and mRNA determination. The amou nt of NGF protein in whole brains 29 remained unchanged in the non-immunized mice. Howe ver, brain NGF levels were significantly 30 increased in OVA-immunized mice exposed to 80 and 4 00 ppb (but not 2,000 ppb) 31 formaldehyde (Figure 4-26). This result was confir med by parallel increases in the 32 concentrations of hippocampal NGF mRNA that were pr oduced in immunized mice exposed to 33 formaldehyde at the same concentrations. However, there were no comparable increases in the 34 amounts of brain-derived neurotrophic factor in eit her immunized or non-immunized mice. In 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-274 discussing the mechanisms potentially associated wi th their results, Fujimaki et al. (2004b) 1 considered it likely that low-level exposure to for maldehyde could enhance NGF production 2 through the stimulation of the HPA axis together wi th immunization. 3

4

5

6 7 Figure 4-26. NGF production in the brains of forma ldehyde-exposed mice. 8 9 Note: Female C3H mice were exposed to formaldehyde 16 hours/day, 10 5 days/week for 12 weeks. NGF in homogenates of wh ole brain and 11 hippocampus were measured by ELISA. Values are mea ns ± SEM (n = 5–6). 12 * = p < 0.05 and ** = p < 0.01 versus control mice, as calculated by the a uthors. 13 14 Source: Redrawn from Fujimaki et al. (2004b). 15 16 17 The enhancement of NGF in the brains of immunized m ice exposed to formaldehyde 18 gave rise to the suggestion that NGF may promote th e survival of hippocampal neurons when 19 challenged with formaldehyde. To examine whether o r not apoptosis plays a role in this process, 20 Tsukahara et al. (2006) measured the effects of for maldehyde on apoptotic mechanisms 21 regulating the survival and death of cells and on N -methyl-D-aspartate (NMDA) receptors. 22 Female C3H/HeN mice (13/group) were exposed to 0 or 400 (393 ± 34) ppb (0 or 490.8 μg/m 3 ) 23 formaldehyde 16 hours/day, 5 days/week for 12 weeks . Seven control and formaldehyde-treated 24 mice were immunized with 10 μg OVA plus 2 mg alumin um hydroxide prior to exposure. 25 Subsequently, these mice received OVA via aerosol a s a booster during weeks 3, 6, 9, and 11. 26 Hippocampi were dissected from all animals 1 day af ter the final exposure and homogenized in 27 hypotonic buffer. The 12,000 rpm supernatants were analyzed by Western blotting for the 28 presence of the proteins BCl-2 (which inhibits apop tosis) and Bax (which opposes BCl-2 action 29 and promotes apoptosis) and the NMDA receptor subty pes 2A and 2B (NR2A and NR2B). 30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-275 Immunohistochemical analysis was also carried out f or the presence of active caspase-3, an 1 apoptosis marker. 2 The levels of NR2A and NR2B were unaffected by expo sure to formaldehyde in either 3 immunized or non-immunized mice. Likewise, the num ber of caspase-3 immunoreactive cells 4 did not change as a result of formaldehyde exposure . However, when measured amounts of 5

Bcl-2 and Bax were normalized to the amount of β -tubulin, the ratio Bcl-2/Bax was significantly 6 increased in immunized mice exposed to formaldehyde . Non-immunized mice did not show this 7 apparently compound-related response. Consistent w ith the concept that the proportions of Bcl-2 8 and Bax are critical for the regulation of cell sur vival and death, the authors interpreted their data 9 as an indication that changes to the ratio of Bcl-2 /Bax expressions might be an important 10 adaptive response to the effects of formaldehyde, s uch that the antiapoptotic changes might 11 contribute to the protection of hippocampal neurons from the pernicious effects of formaldehyde 12 exposure itself. 13 The same research group used the immunized mouse mo del to determine whether 14 formaldehyde exposure affected mRNA expression of g enes related to synaptic plasticity 15 (Ahmed et al., 2007). Ten female C3H/HeN mice were exposed to 0 or 400 ppb formaldehyde 16 16 hours/day, 5 days/week for 12 weeks. All mice w ere immunized with 10 μg OVA plus 2 mg 17 aluminum hydroxide prior to initial formaldehyde ex posure then treated in weeks 3, 6, 9, and 11 18

with aerosolized OVA as a booster. Five treated an d control animals were I.P. injected with 1 19 mg/kg MK-801, a noncompetitive NMDA receptor agonis t before the last formaldehyde 20 exposure. At term, hippocampi were dissected and f rozen at −80 o C until processing. At that 21 point, total mRNA was extracted and first strand cD NA was synthesized by using reverse 22 transcriptase. Expression levels of various protei ns/receptors, including NMDA NR2A and 23 NR2B receptor subunits, dopamine D1 and D2 receptor s, cyclic AMP responsive element24 binding proteins (CREB-1 and CREB-2), and the trans cription factors FosB and ∆ FosB were 25 determined by using the PCR. The expression level of each mRNA species was expressed 26 relative to the sample’s content of 18S rRNA. The total protein lysate was also assayed for 27 pCREB by Western blotting. 28 In the first of a sequence of histograms, Ahmed et al. (2007) demonstrated a significant 29 increase in mRNA expression of NR2A as a result of formaldehyde exposure. However, this 30

effect was abolished in animals treated with MK-801 . A similar trend in the mRNA expression 31 of NR2B in response to formaldehyde exposure did no t achieve statistical significance. MK-801 32 treatment significantly reduced receptor in mRNA ex pression in the presence of formaldehyde. 33 The authors provided data showing an increased expr ession of dopamine D1 and D2 receptor 34 mRNA response to formaldehyde, in both cases abolis hed by treatment with MK-801. The 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-276 expression of CREB-1 mRNA also conformed to the pat tern of being increased as a result of 1 formaldehyde exposure but abolished by MK-801. How ever, the expression of CREB-2 and 2

FosB/ ∆ FosB was unaffected by formaldehyde. When normaliz ed to the amount of β -tubulin, 3 there were no significant effects of formaldehyde e xposure and MK-801 treatment on the protein 4 levels of pCREB. Finally, there was no significant difference in the expression of transient 5 receptor potential vanilloid receptor (TRPV1) betwe en control and formaldehyde-exposed mice, 6 and MK-801 itself did not significantly alter the m RNA level of TRPV1. In seeking to explain 7 their results, the authors speculated that low-leve l exposure of immunized mice to formaldehyde 8 had an effect on hippocampal synaptic plasticity at the mRNA level, as evidenced by the 9 enhancement of mRNA for NR2A, the dopamine D1 and D 2 receptors, and CREB1, with up 10 regulation compensating for the sustained levels of enhanced protein expression under low-level 11 formaldehyde exposure. The interpretation of these changes in NR2A mRNA, in the context of 12 the results of Tsukahara et al. (2006), showing no change in NR2A and NR2B protein 13 expression, was not discussed. 14

15

4.2.1.6.1.6. Neurogenesis. Two papers have examined the effects of subacute ex posure to 16 formaldehyde on the overall size (volume) of discre te cellular areas of the hippocampus in 17 neonatal rats. The researchers also used an optica l fractionator counting method to derive a 18 plausible estimate of cell number. Aslan et al. (2 006) studied the effects of formaldehyde 19 exposure on the number and volume of granular cells in the hippocampal dentate gyrus. 20 Sarsilmaz et al. (2007) examined the impact of post natal formaldehyde exposure on brain 21 hemisphere volume and on the size and cell number o f pyramidal cells in the cornu ammonis 22 region of the hippocampus. The in-life phase was t he same in each study, featuring the exposure 23 of 10 neonatal male Wistar rats/group to 0, 6, and 12 ppm (0, 7.36, and 14.7 mg/m 3 ) 24 formaldehyde 6 hours/day, 5 days/week for 30 days. Five rats/group were sacrificed at that point 25 (PND 30), while the rest were maintained without fu rther treatment until PND 90. 26 For both pyramidal and granular areas, a much lower number of cells was seen on 27 PND 90 versus PND 30 ( p < 0.001). This response was evident irrespective of the amount of 28 exposure to formaldehyde and is consistent with nor mal brain development. Compound-specific 29 effects of formaldehyde on the volume and number of granular and pyramidal cells varied by 30 dose and over the two time points. There was a sma ll increase in the volume of the granular cell 31 layer of the dentate gyrus in rats sacrificed on PN D 30 ( p < 0.001) in response to increasing 32 formaldehyde concentration (Aslan et al., 2006), wi th no significant change in neuron number; 33 the increased volume (now accompanied by an increas e in neuron number) was still evident at 34 the low-exposure level on PND 90 ( p < 0.01) but not at the high dose. Brain hemispher e volume 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-277 was decreased at both concentrations on PND 30 ( p < 0.01) but was increased at both 1 concentrations (

p < 0.01, with a larger magnitude of effect at 6 ppm ) on PND 90 (Sarsilmaz et 2 al., 2007). In the hippocampal cornu ammonis regio n, the volume of the pyramidal cell layer on 3 PND 30 was increased in low-dose animals ( p < 0.001) but decreased in high dose animals ( p < 4 0.001) as compared with control values; neither gro up was significantly different from controls 5

on PND 90. There was a dose-related decrease in t otal neuron number in the cornu ammonis on 6 PND 30 (

p < 0.01 for both doses); on PND 90 the decrease in n euron number remained 7 statistically significant in both treatment groups ( p < 0.01), but there was no longer any 8 difference in the magnitude of the effect between d oses (Sarsilmaz et al., 2007). 9 In a third study from the same laboratory, Songur e t al. (2008) used the same exposure 10 paradigm to evaluate changes in oxidant and antioxi dant systems in the cerebellum of perinatally 11 exposed rats. Exposure was carried out as in Aslan et al. (2006) and Sarsilmaz et al. (2007), 12 described above. On PND 30 or 90, cerebellums from seven male rats per group were evaluated 13 for levels of malondialdehyde (MDA), NO, superoxide dismutase activity (SOD), and 14 glutathione peroxidase (GPX) activity. Dose-relate d increases in NO (approximately 20–80%), 15 MDA (100–160%), and GPX (25–60%) and dose-related d ecreases in SOD (20–30%) were seen 16 on PND 30. In general, the magnitude of change fro m control levels was maintained on PND 90, 17 with the exception of MDA levels in 6 ppm animals, which appeared to approach control levels 18 at 90 days. The authors stated that these findings indicated that formaldehyde exposure may 19 cause neurotoxicity via the production of oxidative damage in the brain. Persistence of the effect 20 to the 90-day time point (30 days after cessation o f exposure) supports the possibility that 21 formaldehyde may cause long-lasting or permanent ch anges in the brain following early life 22 exposure. These results are consistent with the ea rlier studies by Aslan et al. (2006) and 23 Sarsilmaz et al. (2007), finding permanent changes in brain structure (although in a different 24 brain region) following early life exposure. 25

26

4.2.1.6.1.7. Summary of formaldehyde effects on neurobehavioral and neuropathological 27 measures, following exposure via inhalation. As has been demonstrated in mice (Wood and 28 Coleman, 1995), it is possible that rats experience respiratory tract irritation during low-level 29 formaldehyde exposure. Perturbations in nervous sy stem function reported with formaldehyde 30 exposure include reductions in motor activity, lack of habituation, impairment in acquisition of a 31 new learning task, deficits in retention of a previ ously learned task, increases in corticosterone 32 levels, sensitization to cocaine-induced locomotor activity, and enhanced fear conditioning using 33 an olfactory CS (Table 4-57). Many of these effect s were observed at exposure levels at or 34 below 1 ppm, and some persisted days to weeks after termination of exposure. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-278 Malek et al. (2004, 2003a, b) detected behavioral c hanges in rats and mice tested 2 to 1 24 hours postexposure. The mechanism of these beha vioral changes is unknown, and available 2

data do not allow dissociation of direct effects on the nervous system and behavioral responses to 3 the irritant effects of formaldehyde (control exper iments [e.g., using a different aversive odor 4 with or without irritant properties] were not inclu ded). Given that behavioral changes were 5 observed hours to days after cessation exposure (i. e., beyond the time required for formaldehyde6 induced irritation to subside), it is unlikely that these behavioral changes were caused by 7 formaldehyde-induced irritation. Similarly, althou gh it is possible that systemic effects of 8 formaldehyde exposure might cause reduced motor act ivity during or immediately following 9 exposure, it is unlikely that these effects can acc ount for the differences in responses of male rats 10 24 hours after exposure (Malek et al., 2003a). Fur thermore, a follow-up study demonstrated 11 reduced motor activity in animals 2 hours after a 2 -hour exposure to much lower levels of 12 formaldehyde (0.1 ppm), which fall well below the l evels identified by Wood and Coleman 13 (1995) as the AC 50 for formaldehyde in mice (Malek et al., 2003b). 14 Two studies reported significant reductions in lear ning or retention following brief 15 periods of repeated exposure to low levels of forma ldehyde (Malek et al., 2003c; Pitten et al., 16 2000) (Table 4-57). Malek et al. (2003c) reported an increased number of errors in acquiring a 17 water maze task; testing took place daily 2 hours a fter termination of a 2-hour exposure. The 18 work of Pitten et al. (2000) revealed that brief ex posures over many weeks led to increases in 19 errors performing a previously learned task and tha t the magnitude of the effect increased over 20 the course of the exposure period. Testing occurre d remote from the time of exposure (22 hours 21 after the previous exposure), and the deficits appe ared to persist for several weeks after exposure 22 terminated, minimizing the possibility that these e ffects were related to irritant properties of 23 formaldehyde. Although the exposure levels were mo derately high (2.6–4.6 ppm) and continued 24 over several months, the duration of a single expos ure event was very brief (10 minutes). 25 Sorg and Hochstatter (1999) and Sorg et al. (2004, 2001) suggest that behavioral 26 sensitization associated with low-level formaldehyd e exposure was linked to alterations in HPA 27

control of corticosterone. Cross-sensitization to the locomotor activity-enhancing properties of 28 cocaine and changes in response to a conditioned fe ar paradigm were observed in animals 29 exposed several weeks earlier to repeated low-level formaldehyde. Direct activation of 30 mesolimbic dopamine pathways or activation of condi tioned fear response in the amygdala by 31 formaldehyde could underlie these behavioral effect s; these observations were also seen by study 32 authors as consistent with a formaldehyde-induced s tress response. 33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-279 Table 4-57. Summary of neurological and neurobehav ioral studies of inhaled formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/ NOAEL Reference Irritant detection threshold Male Swiss mice 8 0, 1, 1.8, 3,

5.6, or 10 ppm

60-second

exposure episode to determine irritant

response Sensitivity of mice to acute formaldehyde levels

determines the median concentration at which 50% of exposures were terminated by the subject (AC 50 ) decreased upon repeat exposure. AC 50 = 3.63 for first series, AC 50 = 1.88 ppm for second series. Time to exposure termination decreased with increasing formaldehyde concentration. Time to termination was decreased in repeat

exposures. NA b Wood and Coleman (1995)

Motor activity and habituation Male and female LEW.1K

rats 10/sex 0, 1, 2.5, or 5

ppm for 2 hours Reduced horizontal activity : Number of crossed

quadrants reduced 2 hours after exposure at all doses for males and females. Reduced habituation : Exposed rats exhibited greater activity than controls when reintroduced into the testing environment 24 hours later (males and females, all doses). LOAEL = 1 ppm 2 hours Malek et al.

(2003a)

Male and female LEW.1K

rats 10/sex 0, 0.1, 0.5, or 5

ppm for 2 hours Reduced horizontal activity : Number of crossed

quadrants reduced 2 hours after exposure at all doses for males and females. LOAEL = 0.1 ppm 2 hours in males Malek et al.

(2003b)

Male AB mice 5–7/sex 0, 1.1, 2.3, or

5.2 ppm for 2 hours Reduced horizontal activity : Number of crossed

quadrants reduced 2 hours after exposure at all doses. LOAEL = 1.1 ppm 2 hours Malek et al.

(2004)

Balb/c mice 6 0, 1.8, 3.2, 4.5,

6.4, 9.7, or 14.8 ppm for 3 hours Reduced horizontal and vertical activity : Dosedependent decreases in crossed quadrants and rearing. Significant for males at 1.8 ppm and greater ( p < 0.01). Significant for females at 6.4 ppm or greater ( p < 0.01). LOAEL = 1.8 ppm 3 hours in males Usanmaz et

al. (2002)

Balb/c mice 6 3.3 ppm for 2

weeks or 2 ppm for 3 weeks

3 hours/day, 5 days/week Reduced horizontal and vertical activity

decreases in crossed quadrants and rearing.

3.3 ppm (2 weeks) and 2 ppm (1 week) ( p < 0.01, p < 0.05). LOAEL = 2 ppm 3 weeks Usanmaz et

al. (2002)

SpragueDawley

rats 8 0, 5, 10, or 20

ppm; 3 hours/day for 1 or 2 days Reduced activity levels on both days. Decreases

seen at 5 and 10 ppm; data reported only for 5

ppm group LOAEL = 5 ppm, 3 hours Boja et al.

(1985)This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-280 Table 4-57. Summary of neurological and neurobehav ioral studies of inhaled formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/ NOAEL Reference Rats 0 or 0.5 mg/m 3

(0.4 ppm) on GDs 1–19, 4 hours/day Increased motor activity on the 2 nd and 3 rd days of testing (reduced habituation) in offspring exposed in utero. Increased number of squares entered ( p < 0.01) and frequency of rearing ( p < 0.05). LOAEL = 0.4 ppm, gestational Senichenkova

(1991)

Rats 15 0, 0.005, or

0.0005 mg/L (approximately 4 or 0.4 ppm), GDs 1–19 Changes in motor activity at one and two months

in offspring exposed in utero. Decreased spontaneous mobility at 1 month in both sexes, increased activity at 2 months in both sexes. LOAEL = 0.4 ppm, gestational Sheveleva

(1971)

Learning and memory Adult male and female LEW.1K

rats 15/sex 0, 0.1, 0.5, or

5.4 ppm 2 hours for 10 consecutive days Impairment in acquisition of a new task : Male

and female rats at all formaldehyde exposures had significantly more errors in completing a water labyrinth (

p < 0.01). Male and female rats had longer times to completion of the maze at 0.5 and 5.4 ppm ( p < 0.05, p < 0.01). LOAEL = 0.1 ppm 2 hours/ 10 days Malek et al.

(2003c)

Adult male and female Wistar

rats 5–8/

sex 0, 2.6, 4.6 ppm

10 minutes/day for 90 consecutive days Deficit in the retention of a learned task : Male

and female rats committed significantly more errors ( p < 0.05) and took more time to complete the land maze in across the course of the experiment (

p < 0.04). LOAEL = 2.6 ppm 10 minutes/ 90 days Pitten et al.

(2002)

Ferrets 0.25 ppm Impairment in acquisition of a new task : Exposed ferrets only achieved a 75% success rate in training to discriminate odors in a Y-maze versus 90% success rate in controls. Note: The results are confounded with other effects on the olfactory epithelium. The same researchers also reported a decrease in olfactory sensitivity and a reduction in percentage of olfactory cells in similarly treated animals. None established Apfelbach

and Reibenspies (1991) (abstract only)

Male juvenile and adult

rats 5/group 0.25, 0.5 ppm Decreases in olfactory thresholds, in juvenile animals but not in adults ( p < 0.002). LOAEL =

250 ppm in juveniles Weiler and

Apfelbach (1992) (abstract only)This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-281 Table 4-57. Summary of neurological and neurobehav ioral studies of inhaled formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/ NOAEL Reference Neurosensitization endpoints FemaleSpragueDawley

rats 8–9 0 or 11 ppm

1 hour/day, 7 days Increase in cocaine-induced activity : Increased

quadrants crossed after cocaine injection 1 and 2 days after exposure (

p = 0.05 and p < 0.04, respectively).

No change in corticosterone levels 28 days postexposure. No change in nociceptive or passive avoidance test or plus-maze (21, 20, and 13 days postexposure, respectively) (21 days). LOAEL = 11 ppm/ 7 days (unbounded) Sorg et al.

(1996)

Female and maleSpragueDawley

rats Various

up to 24/group 11 ppm, 1

hour/day, 7 days

1 ppm, 1 hour/day, 7 days

1 ppm, 1 hour/day, 5 days/week, 4 weeks Increase in cocaine-induced activity : Increase in

rearing after cocaine injection 1 day after exposure but not 4– 6 weeks after exposure; 11 ppm for 7 days or 1 ppm for 4 weeks.

No change in rats exposed at 1 ppm for 1 week. LOAEL = 1 ppm 4 weeks

NOAEL = 1 ppm 7 days Sorg and

Hochstatter (1999)

Female and maleSpragueDawley

rats Various

up to 24/group 1 ppm, 2

hours/day, 5 days/week, 4 weeks Increased conditioned fear response in

formaldehyde-treated, foot-shock-conditioned rats, twofold (

p < 0.05). LOAEL = 1 ppm 4 weeks Sorg and

Hochstatter (1999)

MaleSpragueDawley

rats 4–9 or

16 0, 0.7, or 2.4

ppm for 20 or 60 minutes

0, 0.7, or 2.4

ppm 1 hour/day, 5 days/week for 2 or 4 weeks No change in corticosterone in acute (20- and

60-minute) exposures.

Increase in basal corticosterone : 0.7 ppm for 2 or 4 weeks.

Hyperresponsive corticosterone response to environment : 2.4 ppm for 2 or 4 weeks. LOAEL = 0.7 ppm/ 2 weeks

NOAEL = 0.7 ppm/ 20 minutes Sorg et al.

(2001)

Female and maleSpragueDawley

rats 4–9 or

16 0 or 2 ppm

1 hour/day, 5 days/week for 4 weeks Increased conditioned fear response to an

olfactory cue in formaldehyde-treated, footshock-conditioned male rats. Measured as increased time freezing when presented with a novel environment (

p < 0.05).

No effect in female rats

. LOAEL = 2 ppm/ 4 weeks

NOAEL = 2 ppm/ 4 weeks Sorg et al.

(2004)This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-282 Table 4-57. Summary of neurological and neurobehav ioral studies of inhaled formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/ NOAEL Reference Neurochemistry and neuropathology8-weekold female C3H/ HeN mice 5 0, 0.08, 0.4, or

1 ppm

16 hours/day, 5 days/week for 1 day or 12 weeks No change in size of main olfactory bulb by

several measures. No change in numbers of PG cells. No change in tyrosine hydroxylase immunopositive PG cells after 1 day.

Increase in tyrosine hydroxylase-immunopositive PG cells after 12 weeks to 196, 167, and 196% of controls at 0.08, 0.40, and 1 ppm, respectively. LOAEL = 0.08 ppm/ 12 weeks Hayashi et al.

(2004)

Adult maleSpragueDawley

rats 8 0, 5, 10, 20

ppm 3 hours/day, 1 or 2 days No change in norepinephrine or 5-

hydroxytryptamine in hypothalamus. Increase in DOPAC in hypothalamus after one

exposure. Increase in dopamine and 5-HIAA in hypothalamus after two exposures. LOAEL = 5 ppm/ 3 hours Boja et al.

(1985)

Neurogenesis Neonatal Wistar

rats 5 0, 6, or 12

ppm, 6 hours/day, 5 days/week for 30 days Changes in volume of granular cell layer of the

dentate gyrus in the hippocampus at postnatal days 30 and 90 (

p < 0.001) LOAEL = 6 ppm/ 30 days Aslan et al.

(2006)

Neonatal Wistar

rats 5 0, 6, or 12

ppm, 6 hours/day, 5 days/week for 30 days Decreases in brain hemisphere volume at PND

30 ( p < 0.01)

Changes in volume and cell numbers in the CA region of the hippocampus on PND 30 ( p < 0.01) LOAEL = 6 ppm/ 30 days Sarsilmaz et

al. (2007)

Neonatal Wistar

rats 7 0, 6, or 12

ppm, 6 hours/day, 5 days/week for 30 days Changes in oxidant and antioxidant systems in

cerebellum on PNDs 30 and 90 ( p = 0.017– 0.001)

. Increases in MDA, NO, and GSH-Px and decreases in SOD at both time points. LOAEL = 6 ppm/ 30 days Songur et al.

(2008)

a Treatment is given as the formaldehyde concentratio n in air (ppm) with the length of exposure each day and the duration of treatment in days, as available.

b NA = not available.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-283 Limited data regarding possible neurochemical chang es in the brains of formaldehyde1 exposed, immunized mice (Ahmed et al., 2007; Fujima ki et al., 2004b; Hayashi et al., 2004; 2 Kakeyama et al., 2004) and rats (Boja et al., 1985) provided conflicting information, and the 3 implications of these data regarding possible forma ldehyde neurotoxicity are difficult to 4

determine. 5 In developmental exposure paradigms, changes in bra in structure (Sarsilmaz et al., 2007; 6 Aslan et al., 2006), brain chemistry (Songur et al. , 2008), and motor activity (Senichenkova, 7 1991; Sheveleva, 1971) were seen following neonatal or in utero exposure to formaldehyde. In 8 addition, Weiler and Apfelbach (1992) found juvenil e animals to be more sensitive to 9 formaldehyde-induced changes in olfactory threshold s when compared with adult animals. 10 These studies raise concern about possible long-las ting neurological effects of early exposure to 11 formaldehyde. It is important to note, however, th at exposure levels in these studies were higher 12 (250–6,000 ppb) than those producing the behavioral effects in adults described above. 13 Overall, available data provide substantial eviden ce of behavioral effects, including 14 motor activity changes and changes in learning and retention, following repeated exposure to 15 relatively low levels of formaldehyde. These effec ts were seen in multiple laboratories, in 16 studies conducted by different authors, and using d ifferent behavioral paradigms. These 17 conclusions are also supported by more limited data , indicating possible developmental effects 18 on the nervous system, including changes in brain s tructure and in the behavior of offspring; the 19 developmental findings are less robust since they w ere seen only in individual laboratories and 20 occurred following exposure to higher concentration s of formaldehyde. Studies evaluating 21 developmental neurotoxicity at lower doses, compara ble to those used in the adult studies, were 22 not available. None of the available data provide sufficient information to allow a determination 23 of the mechanism for these behavioral changes, alth ough it is unlikely that they are attributable 24 to the irritant properties of formaldehyde. The da ta regarding behavioral sensitization provide 25 some support for a stress-related mechanism for tho se findings, but the applicability of this 26 mechanism to the behavioral changes seen in the oth er studies, including the learning deficits, 27

has not been evaluated. 28

29

4.2.1.6.2. Oral exposure. Available data regarding neurotoxic effects of form aldehyde exposure 30 following oral exposure are very limited. Several chronic or subchronic oral toxicity studies 31 evaluated changes in brain weight or histopathology in rats or dogs following repeated oral 32 exposures to formaldehyde at doses as high as 300 m g/kg-day, administered in drinking water 33 (Til et al., 1989, 1988; Tobe et al., 1989; Johanns en et al., 1986). Although data were not 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-284 presented in the publications, all stated that no c hanges in brain weight or pathology were seen in 1 the standard evaluations performed in these studies

. 2 Two studies evaluated changes in behavior followin g exposure to formaldehyde in 3 drinking water (Venkatakrishna-Bhatt et al., 1997; Venkatakrishna-Bhatt and Panchal, 1992). 4 Venkatakrishna-Bhatt and Panchal (1992) evaluated c hanges in performance on a conditioned 5 avoidance task in adult male albino rats (five/grou p). Animals were exposed to formaldehyde in 6 drinking water (10 mg/mL) or by I.P. injection (10 mg/kg) for 60 days. Although it was stated 7 that water consumption was recorded, the data were not presented, and thus actual exposure 8

levels cannot be documented. Prior to the initiati on of exposure, rats were trained on the 9 conditioned avoidance task (climbing a wooden pole in response to a warning buzzer, thus 10 avoiding electric shock from a floor grid). Rats w ere trained to a predetermined performance 11 criterion (not described); animals not achieving th e criterion were removed from study. Training 12 and testing conditions (e.g., retest interval and d uration of sessions) were not well described. 13 Data were presented as percent response in behavior al performance (apparently separately for the 14 escape or avoidance aspects of the task) or percent age decrease in response. No control data 15 were presented, and pretreatment performance was no t described. Figures presented 16 performance at 10-day intervals, starting with day 0, with each data point stated to represent the 17 mean for five experimental sets; again, the interva l between experimental sets and the number of 18 trials per set was not specified. Although the aut hors concluded that a deficit in performance 19 was demonstrated, the data as presented were diffic ult to interpret and the conclusion could not 20 be verified based on the data as presented. 21 Venkatakrishna Bhatt and Panchal (1992) examined ch anges in performance on a 22 conditioned avoidance response, presumably using a procedure similar to the one described 23 above. Albino rats (sex not specified, five/group) were exposed to formaldehyde in drinking 24 water at 0.2 or 0.5 mg/mL for 90 days. As describe d above, rats were trained in performing the 25 task prior to the start of exposure. Venkatakrishn a-Bhatt Bhatr and Panchal (1992) stated that 26 there was a dose-related deterioration of performan ce, but no data were presented to support 27

these conclusions. 28 In summary, available data are insufficient to con duct a reliable assessment of neurotoxic 29 effects of formaldehyde following oral exposure. L imited data suggest a lack of overt 30 neuropathological changes at doses up to 300 mg/kg- day (Til et al., 1989, 1988; Tobe et al., 31 1989; Johannsen et al., 1986), but detailed informa tion regarding the types of neuropathological 32 evaluations performed in those studies is not avail able, and thus no firm conclusions can be 33 drawn regarding the potential for neuropathological effects. The two available studies evaluating 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-285 behavioral changes are not considered to provide us eful information, and thus effects on nervous 1 system function could not be evaluated. 2

3

4.2.1.6.3. Summary. Overall, there is strong evidence that formaldehyde exposure via 4 inhalation may cause adverse effects on nervous sys tem function in experimental animals at 5 relatively low levels of exposure (LOAELs as low as 100 ppb). Although human data regarding 6 neurotoxicity following formaldehyde inhalation are limited, available data provide support that 7 the types of effects seen in humans are similar to those found in animal studies. Evidence from 8 available human controlled inhalation exposure stud ies indicates that humans may be affected at 9 doses similar to those used in animal studies; howe ver, the human data are extremely limited. 10 There are insufficient data to evaluate the potent ial for neurotoxicity following oral 11 exposure to formaldehyde. Limited evaluations of b rain weight or histopathology in available 12

chronic or subchronic oral studies found no evidenc e of formaldehyde-induced changes (Til et 13 al., 1989, 1988; Tobe et al., 1989; Johannsen et al ., 1986). However, reliable studies examining 14 nervous system function or focused studies of neuro pathology following oral exposure to 15 formaldehyde are not available. 16

17

4.2.1.6.4. Other considerations. Major data gaps were found regarding the evaluation of 18 changes in nervous system structure or function fol lowing formaldehyde exposure by both the 19

inhalation or oral routes. 20 With respect to inhalation exposure, none of the a vailable human studies resulted in data 21 sufficient to conduct a reliable dose-response asse ssment for changes in nervous system function. 22

Most of the available animal inhalation studies use d short exposure durations (acute or 23 short-term), precluding a reliable evaluation of ne urotoxicity following chronic exposure. 24 Available data for neurodevelopmental exposures are also quite limited, consisting of evaluation 25 of neuropathology in only one brain region and func tional evaluations focused only on changes 26 in motor activity. 27 Major data gaps also exist regarding neurotoxicity following oral exposure, with no 28 relevant human data and extremely limited animal da ta. Available oral exposure studies were 29 insufficient to permit a reliable evaluation of the potential for neurotoxicity following oral 30 exposure to formaldehyde. 31

32

4.2.1.7. Reproductive and Developmental Toxicity 33 The potential for developmental and reproductive ef fects after formaldehyde exposure by 34 the inhalation route has generally been considered low, since formaldehyde, as a reactive gas, is 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-286 not expected to penetrate past the POE (NEG, 2003; IPCS, 2002; Collins et al., 2001). 1 Nevertheless, a number of animal studies have demon strated effects of formaldehyde on pre- and 2 postnatal development and on the reproductive syste m. For example, developmental toxicity 3

was observed in two studies that evaluated a standa rd battery of developmental endpoints 4 resulting from inhalation exposure on GDs 6–10 (Mar tin, 1990; Saillenfait et al., 1989). 5 Similarly, oral exposures resulted in developmental effects when administered during 6 comparable gestational windows (Marks et al., 1980; Hurni and Ohder, 1973). There have also 7 been reports that identified developmental effects at lower-level formaldehyde exposures that 8 were administered throughout gestation (Senichenkov a and Chebotar, 1996; Senichenkova, 9 1991; Kitaev et al., 1984; Sheveleva, 1971; Gofmekl er and Bonashevskaya, 1969; Gofmekler, 10 1968; Pushkina et al., 1968). Postnatal functional consequences of developmental exposures 11 have also been identified (Sarsilmaz et al., 2007; Aslan et al., 2006; Weiler and Apfelbach, 1992; 12 Senichenkova, 1991; Sheveleva, 1971). Additionally , a number of studies suggest that 13 formaldehyde adversely affects the male reproductiv e system after both inhalation and oral 14 exposures (Xing et al., 2007; Zhou et al., 2006; Öz en et al., 2005, 2002; Sarsilmaz et al., 1999; 15 Chowdhury et al., 1992; Cassidy et al., 1983; Gusev a, 1972). This section reviews the available 16 published studies assessing reproductive and develo pmental endpoints of formaldehyde. 17

18

4.2.1.7.1. Inhalation studies addressing developmental and rep roductive toxicity. Saillenfait et 19 al. (1989) reported a comprehensive and well-docume nted developmental study in Sprague20 Dawley rats. Pregnant rats were exposed beginning on GD 6 in order to cover critical stages of 21 development (e.g., implantation and major organogen esis). Female Sprague-Dawley rats 22 (25/group) were exposed to 0, 5, 10, 20, or 40 ppm (0, 6.15, 12.3, 24.6, or 49.2 mg/m 3 ) 23 formaldehyde 6 hours/day on GDs 6–20. The onset of pregnancy was determined by the 24 presence of sperm in a vaginal smear. Dams were ex posed to formaldehyde in a dynamic flow 25 chamber, and formaldehyde concentrations were deter mined to be 0, 5.17 ± 0.51, 9.92 ± 0.88, 26 20.04 ± 0.88, and 38.96 ± 3.70 ppm. Dams were weig hed on GDs 0, 6, and 21 and sacrificed on 27 day 21. Upon examination, uterine weights, fetal w eights, sex ratio, number of implantation and 28 resorption sites, and live and dead fetuses were re corded. Fetuses were examined for external 29 malformations and cleft palate. One-half of viable fetuses were sectioned to assess soft-tissue 30 alterations. The other half were fixed, stained wi th alizarin red S, and examined for skeletal 31

alterations. 32 Body weight gain of dams and body weight of male an d female fetuses were reduced by 33 exposure to 40 ppm formaldehyde to 49, 78, and 81% of control values, respectively ( p < 0.01) 34 (Saillenfait et al., 1989). Reduced weight gain in dams remained significantly decreased when 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-287 uterine weight was accounted for ( p < 0.01). Mean fetal weight of male pups was reduce d at 1 maternal exposures of 20 and 40 ppm formaldehyde (5 .53 and 4.42 g versus 5.61 g in controls). 2 Decreased fetal body weight in females was only see n at 40 ppm (4.27 g versus 5.24 g in 3 controls). All other pregnancy endpoints were unch anged by formaldehyde exposure (e.g., 4 uterine weight, implantation and resorption sites, live fetuses, dead fetuses, and sex ratios). No 5 major malformations were noted in fetuses. Some mi nor soft tissue and skeletal anomalies, such 6 as dilated ureter, missing sternebrae, extra fourte enth rib, and rudimentary thirteenth rib 7 (statistics not given), were reported. However, th ese effects occurred at similar frequencies in 8 control and treatment groups. The incidence of del ayed ossification of the thoracic vertebrae 9 was 8.7% in fetuses from the 40 ppm exposure group versus 1.8% in controls. However, this 10 difference was not statistically significant. Over all, from these results formaldehyde was neither 11 lethal to embryos nor teratogenic, only exhibiting fetotoxic effects at exposures of 20 ppm and 12 above. These are levels where there was a signific ant decrease in fetal body weight. 13 Martin (1990) conducted a similar study exposing pr egnant rats during similar stages of 14 development. Mated female Sprague-Dawley rats (25/ group) were exposed to 2, 5, or 10 ppm 15 (2.46, 6.15, or 12.3 mg/m 3 ) formaldehyde 6 hours/day on GDs 6–15. The study included two 16 control groups: dams placed in the exposure chamber s once a day but exposed only to clean air 17 and dams fed and housed similarly to the experiment al groups but never put into the inhalation 18 chambers. The method of formaldehyde vapor generat ion and details of the exposure chamber 19 were not described. Mean formaldehyde exposure con centrations were reported as 1.88, 4.88, 20 and 9.45 ppm (variability not given, analytical met hod not discussed). Food consumption and 21 body weight were recorded. On GD 20, rats were sac rificed, and the following pregnancy 22 parameters were recorded: live fetuses, dead fetuse s and resorptions, number of corpora lutea, 23 fetal weights, sex ratios, and preimplantation and postimplantation losses. Fetuses were 24 examined for major malformations, minor external an d visceral anomalies, and minor skeletal 25 anomalies (details not given). Weight gain and foo d consumption in dams were said to be 26 reduced at 10 ppm ( p < 0.05). Formaldehyde exposure of the dams at 5 an d 10 ppm led to an 27

increased incidence of reduced ossification of the pubic and ischial bones in fetuses on GD 20 28 ( p < 0.05). Reduced ossification correlated with lowe r fetal weights, and the author considered 29 both of these findings a result of larger litter si ze and, therefore, not related to formaldehyde 30 exposure. However, no tables presenting the data o r statistical analysis were provided. All other 31 pregnancy parameters and fetal anomalies were descr ibed as unaffected by formaldehyde 32 exposure. However, without data presented for the assessed endpoints, background rates of 33 malformations, trends in the data, and variability, it is difficult to evaluate the Martin (1990) 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-288 comparisons. However, the author’s observations of reduced fetal weight and increased 1

incidence of reduced ossification are consistent wi th the results of Saillenfait et al. (1989). 2 Kilburn and Moro (1985) studied similar endpoints b ut included formaldehyde exposure 3 during earlier gestational windows. The study repo rt, only available in abstract form and not 4 found as a subsequent published article, does not p rovide many methodological details. Female 5 rats (number and strain not reported) were exposed to 0 or 30 ppm (0 or 37.2 mg/m 3 ) 6 formaldehyde 8 hours/day during GDs 3–17, 3–12, 8–1 2, or 9–11. A second experiment 7 included pair-fed controls for dams exposed to 30 p pm formaldehyde during GDs 3–17, 3–12, 8 and 8–12. The authors reported reductions in fetal and maternal weight gain that were greater 9 than decreases in pair-fed controls. Fetal anomali es were noted after 15 days of gestational 10 exposure (e.g., altered organ size and undescended testes). Although the report indicates some 11 maternal toxicity and fetotoxic effects (for exampl e, stunted growth), lack of study details and 12 clear reporting make this report of negligible util ity in human health risk assessment. 13 There are several early studies that examined devel opmental effects of formaldehyde 14 exposure administered throughout gestation (Gofmekl er and Bonashevskaya, 1969; Gofmekler, 15 1968; Pushkina et al., 1968). It is unclear if the se reports represent the same or overlapping 16 experimental groups. They were performed in the sa me laboratory and are reported with a 17 similar level of detail. The source of formaldehyd e, method of vapor generation, exposure 18 conditions (dynamic versus static), confirmation of exposure concentrations, study design, and 19 data presentation details were not provided. Absen ce of such critical information detracts from 20 the quality of these studies as a coherent record o f experimental information, and, thus, these 21 findings can only be utilized qualitatively in the formaldehyde risk assessment. 22 In the Gofmekler (1968) study, female rats (36, str ain not specified) were continuously 23 exposed at 0, low, or high formaldehyde concentrati ons beginning 10–15 days prior to mating 24 (target concentrations of 0, 0.01, or 0.81 ppm form aldehyde [0, 0.01, or 1 mg/m 3 ]). The author 25 reported a 14–15% increase in pregnancy duration an d a decrease in litter size (data not shown). 26 However, males and females were mated 6–10 days, an d no information was provided on how 27 mating and conception were confirmed. No external malformations were attributed to 28 formaldehyde exposure. Concentration-dependent inc reases in pup body weight and decreases in 29 lung and liver weight were attributed to formaldehy de exposure. Pup weights were increased 30 from 5.6 g in controls to 6.0 and 6.3 g in groups 1 and 2, respectively ( p < 0.01 and p < 0.001). 31 Formaldehyde exposure increased pup adrenal weight in both groups and pup thymus and kidney 32 weight in group 2 only (Table 4-58). 33

34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-289 Table 4-58. Effects of formaldehyde on body and or gan weights in rat pups 1 from dams exposed via inhalation from mating throug h gestation 2 3 Relative organ weights (mg/10 g body weight)

Exposure (ppm) a Body weight

(g) Thymus Heart Lung Liver Adrenals Kidney

0 5.6 26 61.4 287.1 587.7 3.2 51.4 0.01 6.0 b 25.1 61.5 230.2 c 557.7 d 4.2 c 53.4 0.81 6.3 c 31.7 c 64.5 223.2 c 550.8 b 3.8 d 55.7 b 4

a Dams were exposed to formaldehyde continuously from 10–15 days prior to mating. Exposure concentratio ns 5 were not validated. 6

b Different from controls, p < 0.01. 7

c Different from controls, p < 0.001. 8

d Different from controls, p < 0.05. 9 10 Source: Gofmekler (1968). 11 12 In a study by Gofmekler and Bonashevskaya (1969), t he researchers evaluated organ 13 histopathology in pups from similarly treated dams. Pregnant female albino rats were 14 continuously exposed at two formaldehyde concentrat ions (groups 1 and 2, as described above). 15 Adult males were similarly exposed during mating. Offspring were examined for malformations, 16 and the organs were fixed and sectioned for histopa thologic examination, including hematoxylin 17 and eosin staining, Brachet stain for RNA, and Feul gen stain for DNA. Liver and kidney 18 sections were also stained with Schiff’s reagent (w hich reacts with aldehydes), with Sudan III for 19 lipids, and Pearl’s stain for iron. Placenta, uter us, and ovaries from the dams and testes of the 20 males were sectioned, stained, and evaluated. The authors stated that formaldehyde induced no 21 external anomalies (reported elsewhere, but no refe rence given). The authors also noted 22 involution of lymphoid tissue and changes in liver, mild hypertrophy of Kupffer cells, and 23 numerous extramedullary myelopoietic centers in pup s from dams in group 2. Pups from both 24 treatment groups showed reduced glycogen content in the myocardium and the presence of iron 25 in Kupffer cells. There was a localized increase i n positive reaction to Schiff’s reagent in the 26

basement membrane and intertubular connective tissu e of the kidneys. The authors suggested 27

that this was an indication of functional alteratio ns in the renal tubular apparatus. All other 28 tissues examined and histochemical staining indicat ed no differences due to formaldehyde 29

exposure. 30 Researchers in the same laboratory (Pushkina et al. , 1968) studied the effects of 31 formaldehyde exposure on vitamin C (ascorbic acid, an antioxidant) and nucleic acid levels in 32 dams and fetuses as general measures of toxicity. Female white rats (n = 160) were continuously 33 exposed at two formaldehyde concentrations (groups 1 and 2, as described above) from 20 days 34 prior to mating (6–10 days) and then throughout ges tation. Dams were sacrificed and ascorbic 35

acid and nucleic acid levels determined in harveste d organs (methods referenced but not 36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-290 described). No visible malformations in pups were noted. Formaldehyde exposure increased 1 fetal body weight and organ weight in both groups ( data not given). There was an average of 2 11.3 fetuses per litter for control dams versus 9.8 and 8.6 for groups 1 and 2, respectively. The 3 authors reported that formaldehyde exposure decreas ed DNA levels and increased RNA levels in 4 organs (further details not provided). Formaldehyd e exposure resulted in lower vitamin C levels 5 in the whole fetus (76 and 75% of controls) and in maternal liver specimens (82 and 88% of 6 controls) for exposure groups 1 and 2, respectively ( p < 0.05). In contrast, vitamin C was higher 7 in fetal liver (127% of controls) in group 1 ( p < 0.05). The significance of these differences is 8 unknown. The authors considered the results as gen eral measures of biochemical changes and 9

therefore toxic. 10 The reports of Gofmekler and Bonashevskaya (1969), Gofmekler (1968), and Pushkina et 11 al. (1968) lack key methodological details. As dis cussed above, exposure conditions and actual 12 formaldehyde concentrations cannot be validated. A lthough methods were not thoroughly 13 detailed, results were reported in data tables with statistics and detailed descriptions of observed 14 pathological changes. However, without validation of exposure concentrations, these findings 15 can only be considered qualitatively. 16 In another study, Sheveleva (1971) exposed female m ongrel (i.e., not a homogeneous 17 genetic strain) white rats to 0, 0.0005, or 0.005 m g/L (0, 0.5, or 5 mg/m 3 ) (0, 0.4, or 4 ppm) 18 formaldehyde on GDs 1–19 (where GD 1 was defined as the day that spermatozoa were detected 19 in vaginal smears) for 4 hours/day. In each group, 15 dams were terminated on GD 20 for 20 evaluation of ovarian corpora lutea, uterine implan tation sites, pre- and postimplantation loss, 21 number of live fetuses, fetal length and weight, an d external examination for malformations. 22 Additionally, in each group, six dams were allowed to deliver their litters. Developmental 23 landmarks were monitored (i.e., ear and eye opening , incisor eruption, emergence of hair coat), 24 and the pups were further evaluated at 1 and 2 mont hs of age for body weight, threshold of 25 neuromuscular excitability, total oxygen consumptio n in 1 hour per 100 g of weight, and 26 spontaneous mobility over 10 minutes. Maternal tox icity (recorded on GD 17) included 27 significantly (

p < 0.05) decreased leukocyte counts in both treated groups and a number of 28 additional findings at 0.005 mg/L (i.e., significan t reductions in the threshold of neuromuscular 29 excitability, rectal temperature, and blood hemoglo bin level) as well as an increase in 30 spontaneous mobility over 15 minutes. (It is noted that a significant reduction in blood 31 hemoglobin was also observed by Sanotskii et al. [1 976] in pregnant rats, following 20 days of 32 gestational inhalation exposure for 4 hours/day to formaldehyde at 6 mg/m 3 [4.83 ppm].) Fetal 33

examinations on GD 20 identified a 50–70% increase in mean preimplantation loss in both 34 formaldehyde-exposed groups. When pups were 1 mont h of age, a reduction in spontaneous 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-291 mobility was noted in both treated groups; in pups at 2 months of age, an increase in mobility 1 was observed in the 0.005 mg/L group. Also, when p ups were 2 months of age, there were 2 alterations in hemoglobin levels and leukocyte coun ts in both treated groups. Detailed 3 descriptions of some study methodologies (particula rly in regard to neurological and behavioral 4 assessments) were not provided in the published pap er. 5 Based on a review of the work by Gofmekler (1968) a nd various epidemiologic studies 6 available at the time, Kitaev et al. (1984) hypothe sized that formaldehyde may exert toxic effects 7 in the early days of gestation. To study embryotox ic effects of formaldehyde inhalation 8 exposures, mature female Wistar rats (five to nine per group) were exposed to 0.41 or 1.22 ppm 9 (0.5 or 1.5 mg/m 3 ) formaldehyde 4 hours/day, 5 days/week for 4 month s (Kitaev et al., 1984). 10 Rats were exposed in dynamic flow chambers and form aldehyde levels measured gravimetrically 11 (but not reported). Females were mated on day 120 of exposure and mating confirmed by the 12 presence of sperm in a vaginal smear. Embryos were harvested on the second or third day of 13 pregnancy (GD 2 or 3) and examined by both light an d phase contrast microscopy for changes in 14 morphology (i.e., evidence of embryonic degeneratio n). Additionally, maternal weight gain and 15 organ weights (ovaries, uterus, and adrenal glands) and blood samples (HCT, Hb, and TP) were 16 monitored as indicators of general toxicity. These parameters were unchanged by formaldehyde 17 exposure. Formaldehyde exposure at 1.22 ppm for 4 months resulted in an increased number of 18 degenerating embryos on GD 3 (14.9 versus 4.4% in c ontrols) and a smaller increase of 10.2% 19 (versus 5.1% in controls; statistical significance not assessed) on GD 2. Indications of 20 degeneration included reduced size and changes in a ppearance (granulation of the ooplasm, 21 wrinkling and degradation of nuclear material). Ho wever, it is unclear if litter effects were 22 accounted for in the statistical analyses, and it i s unknown how the affected embryos were 23 distributed between litters. For dams exposed to 0 .41 ppm formaldehyde, the number of 24 degenerated embryos was not increased on day 2 (3.8 versus 5.1% in controls) but was increased 25 on day 3 (9.1 versus 4.4% in controls; again, unkno wn if statistically significant) after maternal 26 exposure to 1.22 ppm formaldehyde. This observatio n may be coincidental since it was seen in 27

dams sacrificed on GD 2 but not in those sacrificed on GD 3. Kitaev et al. (1984) considered 28 these findings to indicate that repeated exposure t o formaldehyde over a 4-month period can 29 disturb reproductive function, resulting in adverse effects early in embryonic development. 30 To further explore the effects of inhalation exposu res to formaldehyde on reproductive 31 function, Kitaev et al. (1984) conducted a second s eries of experiments on 200 similarly treated 32 female rats. After 4 months of repeated formaldehy de exposure at 0.41 or 1.22 ppm as described 33 above, organ weights (ovaries and uterus) and blood levels of gonadotropic hormones and 34 progesterone were determined. However, the day and time of hormone measurement were not 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-292 given in the report, and normal diurnal variations in these hormones could affect the reported 1 findings if time of day was not accounted for. The length of the estrous cycle was unchanged 2 during exposure. Formaldehyde exposure modulated g onadotropin levels and relative ovarian 3 weight, suggesting low-level effects on the female rat reproductive system prior to mating 4 (Kitaev et al., 1984). Ovarian weight and blood le vels of luteinizing hormone (LH) were both 5 significantly increased after exposures at 0.41 ppm formaldehyde but remained at control levels 6 in rats exposed at 1.22 ppm. Blood levels of folli cle-stimulating hormone (FSH) were increased 7 approximately 66% from control after 1.22 ppm forma ldehyde exposure ( p < 0.05). 8 Progesterone levels were unchanged by formaldehyde treatment. Kitaev et al. (1984) suggested 9 a role of the hypothalamus-pituitary system based o n increased ovary weight, a greater number 10 of degenerated embryos, and increased LH in rats ex posed at 0.41 ppm. They postulated that 11 these effects were not seen at 1.22 ppm due to a to xic effect exhibited as embryonic 12 degeneration, thus the absence of a dose-response d id not alter the interpretation of the validity 13 of the adverse response. The study NOAEL was not d etermined, and the study LOAEL was 0.4 14 ppm (0.5 mg/m 3 ), based upon increased early embryo loss and on ma ternal outcomes (increased 15 ovarian weight and increased blood LH levels) follo wing 4 months of formaldehyde treatment. 16 For the finding of increase blood FSH levels, the e ndpoint NOAEL was 0.4 ppm (0.5 mg/m 3 ) 17 and the LOAEL was 1.2 ppm (1.5 mg/m3). 18 Senichenkova and Chebotar (1996) and Senichenkova ( 1991) examined reproductive and 19 developmental effects of daily formaldehyde exposur e on GDs 1–19 of pregnancy, including the 20 potential effect of formaldehyde exposure on develo pment early in gestation. Additionally, since 21 anemia adversely affects fetal development, Seniche nkova and Chebotar (1996) also examined 22 formaldehyde effects in iron-deficient dams to dete rmine whether co-exposure further 23 compromises fetal development. In both studies, fe male white rats were exposed to 0 or 24 0.41 ppm formaldehyde (0 or 0.5 mg/m 3 ), 4 hours/day on GDs 1–19. Formaldehyde 25 concentrations in the dynamic exposure chambers wer e measured gravimetrically to confirm the 26 exposure concentration but were not reported (metho ds not provided). It is unclear if gravimetric 27 measurements would be sensitive or accurate enough to validate these low-exposure 28 concentrations without a better understanding of th e methodology. This uncertainty in exposure 29 conditions should be considered in evaluating the r eported results. 30 Mongrel female white rats were exposed at a target concentration of 0 or 0.41 ppm (0 or 31 0.5 mg/m 3 ) formaldehyde 4 hours/day on GDs 1–19 (Senichenkov a, 1991). On GD 20, a subset 32

of the dams was sacrificed and examined for number of corpora lutea, implantation and 33 resorption sites, live/dead fetuses, and fetal weig hts. Fetuses were examined for gross pathology 34 of the internal organs and skeleton (details not gi ven). Blood pH, partial pressure of CO 2 , and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-293 partial pressure of oxygen were measured in both da ms and embryos. The remaining dams were 1 brought to term to study postnatal effects of forma ldehyde exposure. Rat pups were observed on 2 PNDs 1–25 for viability, physical development, and maturation rate of motor reflexes. Behavior 3 of juvenile offspring (PND 40) was studied in an op en field test, and maze learning was tested at 4 sexual maturity. In a follow-up report, Senichenko va and Chebotar (1996) present blood 5 chemistry data, pregnancy outcome, and developmenta l data for similarly treated dams and their 6 pups in a chemical model of iron deficiency. Intra peritoneal injections of the iron-chelating 7 agent bipyridyl were given on GDs 12–15 at the thre shold embryotoxic dose (1 mL, 25% 8 solution). On day 20, the dams were sacrificed and dams and fetuses examined as described 9 above. In addition to blood pH, partial pressure o f carbon dioxide and partial pressure of 10 oxygen, acid metabolic products (not detailed), and true bicarbonates were reported for maternal 11

and fetal blood. A review of the data from these r eports indicates there may be an overlap of the 12 study groups. Neither paper presents the entire da ta set; thus, for tranparency and brevity, the 13 following text discusses the combined findings from both studies as if they were a single study 14 Formaldehyde exposure did not affect such indicator s of pregnancy outcome as number 15 of corpora lutea, implantation and resorption sites , and live and dead fetuses, all of which were 16 unchanged (Senichenkova and Chebotar, 1996; Seniche nkova, 1991). Although fetal weight was 17 slightly increased by formaldehyde exposure, 2.35 v ersus 2.24 g in controls ( p < 0.001), neither 18 fetal length nor bone length were changed (femur an d humerus) (Senichenkova and Chebotar, 19 1996; Senichenkova, 1991). Often, increased fetal weight is the result of early physical 20 development, and other signs of development, such a s ossification, would be expected to be 21 enhanced as well. The average number of bone cente rs per limb was increased by formaldehyde 22 exposure from 2.45 and 2.66 to 2.78 and 2.91 in con trols for metacarpal and metatarsal bone 23 centers, respectively ( p < 0.05) (Senichenkova, 1991); these findings were c onsistent with 24 increased growth and weight. In contrast, Senichen kova (1991) reported a decrease in the 25 number of embryos with ossification centers in the hyoid bone (100% in controls versus 91% for 26 formaldehyde exposure, p < 0.05), consistent with the results of Saillenfai t et al. (1989) and 27 Martin (1990). However, litter size, a factor infl uencing fetal weight, was not provided, and it is 28 unclear if Senichenkova (1991) took litter size int o account in the analysis. 29 Senichenkova and Chebotar (1996) reported increased blood acidosis and decreased 30 blood alkaline reserves (bicarbonates and total CO 2 ) in formaldehyde-treated dams and their 31 embryos (

p < 0.05). However, this finding should be considere d in light of the fact that chronic 32 blood acidosis may increase bone remodeling and dec rease bone density in adults. It is unknown 33 if the reported blood acidosis could reduce ossific ation rates in developing embryos. A better 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-294 understanding of exposure conditions and the acid m etabolic products measured is needed to 1 determine the biological relevance of the reported changes in blood acid balance. 2 Iron deficiency, induced by injections of bipyridyl (an iron-chelating agent), was found to 3

be fetotoxic. Iron-deficient dams with no formalde hyde exposure had higher rates of 4 postimplantation death than controls (12.6 ± 5.5 ve rsus 4.8 ± 1.3%). Formaldehyde exposure in 5 conjunction with iron deficiency increased postimpl antation death to 23.1 ± 5.9%. Fetal weight 6 and litter size were not reported for the bipyridyl treatment groups, but bipyridyl treatment in 7 conjunction with formaldehyde resulted in a decreas ed number of metatarsal bone centers (2.21 8 ± 0.12 versus 2.72 ± 0.08 in controls; p < 0.001). This decrease was also significant when 9 compared with formaldehyde or bipyridyl alone ( p < 0.02). However, all pregnancy outcome 10 parameters were not reported for the bipyridyl trea tment. 11 Fetal anomalies were reported after formaldehyde ex posure and were increased by iron 12 deficiency. The incidence of litters with internal organ anomalies was increased from 1.4% in 13 controls to 14.2% in formaldehyde-treated dams (Sen ichenkova, 1991). Undescended testes 14 were the predominant anomaly described: 20.8% in li tters from formaldehyde-treated dams 15 versus 1.2% in controls ( p < 0.05) (Senichenkova, 1991). Similar findings wer e reported by 16 Senichenkova and Chebotar (1996). Bipyridyl treatm ent in conjunction with formaldehyde 17 exposure increased the overall incidence of fetal a nomalies (13.8 ± 2.1% in controls versus 6.6 ± 18 1.8% with iron deficiency alone) (Senichenkova and Chebotar, 1996). However, there are 19 discrepancies between the two papers in the reporti ng of the anomalies, and it is unclear whether 20 the experimental groups overlap between papers, whe re some parameters are identical (which 21 would lead to double counting of the same animal, i ncluding identical SDs) and others are 22 different. Additionally, the reporting is unclear with respect to the basis of the incidence rates 23 reported (for example, overall incidence versus inc idence within litter or incidence of litters with 24 anomalies). Unclear reporting, together with some of the uncertainties regarding exposure 25 conditions, suggests that the data may be of limite d quality to support risk assessment. 26 In the second phase of the studies, pups were deliv ered and postnatal development was 27 assessed (Senichenkova, 1991). Eruption of the upp er and lower incisors was delayed in pups 28 from formaldehyde-treated dams, occurring on PND 14 versus PND 12 in controls ( p < 0.01). 29 All other measures of physical postnatal developmen t were unchanged by formaldehyde. To 30 evaluate postnatal functional outcomes following in utero exposure to formaldehyde, an open 31 field test was conducted in juvenile rats on 3 cons ecutive days (PNDs 40–42). Juvenile rats from 32 formaldehyde-treated dams exhibited increased mobil ity (crossed squares), rearings, and 33 defecations/urinations compared with control rats o n the second and third open field tests 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-295 ( p < 0.05). There were no differences seen in the maz e-learning test assessed in mature offspring 1 of formaldehyde-treated dams (Senichenkova, 1991). 2 Additional assessments of formaldehyde exposure on neurological development are 3 described above in the section on neurological and behavioral toxicity in animal studies (Section 4 4.2.1.6). In brief, studies conducted by Sarsilmaz et al. (2007) and Aslan et al. (2006) exposed 5 10 neonatal male Wistar rats/group to 0, 6, or 12 p pm (0, 7.36, or 14.7 mg/m 3 ) formaldehyde 6 6 hours/day, 5 days/week for 30 days. At that time , five rats/group were killed and subjected to 7 neuropathological examination; the remaining rats w ere maintained until PND 90, at which time 8 they were killed and evaluated. Aslan et al. (2006 ) examined the number and volume of granular 9 cells in the hippocampal dentate gyrus, while Sarsi lmaz et al. (2007) examined the size and 10 number of the pyramidal cells in the cornu ammonis of the hippocampus. In both studies, lower 11 numbers of cells were observed in both treated grou ps at PND 90 as compared with PND 30. 12 Although the effects of treatment on the volume and number of granular and pyramidal cells 13 were somewhat inconsistent, a significant decrease in the number of neurons in the pyramidal 14 cell layer of the hippocampal cornu ammonis was obs erved at both PNDs 30 and 90 (Sarsilmaz 15 et al., 2007). 16 One other study reported effects on nervous system function following exposure to 17 formaldehyde during postnatal development. An abst ract by Weiler and Apfelbach (1992) 18 described a study in which juvenile rats (strain no t specified) were exposed to 0.25 ppm 19 (0.31 mg/m 3 ) formaldehyde from PNDs 30–160 or adult rats were exposed to 0.5 ppm 20 (0.62 mg/m 3 ) formaldehyde for 90 days. Decreased olfactory se nsitivity (i.e., increased olfactory 21 thresholds) was observed and was greater when the e xposure was initiated in the young rats, as 22 compared with the adults. 23 Evaluation of offspring following prenatal, perinat al, and/or juvenile inhalation exposures 24 to formaldehyde have also been reported by Kum et a l. (2007), Sandikci et al. (2007), and 25 Songur et al. (2005). Kum et al. (2007) exposed fe male Sprague-Dawley rats (six dams/group) 26 and their offspring to 0 or 6 ppm (0 or 7.4 mg/m 3 ) formaldehyde for 8 hours/day in separate 27 groups with exposures starting on GD 1, on postpart urition day 1, or in offspring at 4 weeks of 28 age and continuing for 6 weeks. In another group, exposures were initiated in adult rats. Mean 29 body and liver weights were significantly decreased in the offspring exposed in utero and in 30 early postnatal life, and mean liver weights were a lso significantly decreased in rats with juvenile 31 exposures. However, neither body weight nor liver weight was affected in the group with 32 exposure initiating at an adult age, suggesting a l ife-stage-related susceptibility to formaldehyde33 induced hepatic toxicity. Evaluation of biomarkers of oxidative stress revealed significantly 34 increased catalase (CAT) and MDA in the livers of o ffspring that were exposed prenatally, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-296 significantly decreased GSH levels in the livers of offspring that were exposed in early postnatal 1 life, and significantly decreased SOD levels in the livers of offspring that were exposed starting 2 at 4 weeks of age. No biomarkers were altered in t he livers of rats exposed to formaldehyde only 3

as adults. 4 A similar study design was used by Sandikci et al. (2007) to examine the effects of 0 or 5 6 ppm (0 or 7.4 mg/m 3 ) formaldehyde on bronchus associated lymphoid tiss ue (BALT) 6 following pre- and perinatal, juvenile, or adult ex posures of 6 weeks duration in Sprague-Dawley 7 rats (six/group). The presence of the lysosomal en zyme alpha-naphthylacetate esterase (ANAE) 8 served as a marker of T-lymphocytes in peripheral b lood and tissue sections. Significant 9 increases in ANAE-positive T-lymphocytes were found in BALT in all but the in utero exposed 10 groups as compared with control; this outcome is co nsistent with the postnatal development of 11 BALT in the rat. In peripheral blood, ANAE-positiv e lymphocyte ratios were significantly ( p < 12 0.001) increased as compared with controls at all l ife stages tested. 13 Songur et al. (2005) examined the effect (and rever sibility) of formaldehyde exposures 14 during the early postnatal period on zinc, copper, and iron levels and activity of SOD in the lung 15 tissue of Wistar rats. Litters (12–14/group) were exposed to 0, 6, or 12 ppm (0, 7.4, or 16 14.9 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for 30 days . Trace element and 17 biochemical analyses were conducted on PND 30 or 90 . Decreased SOD activity, decreased 18 levels of copper and iron levels, and increased zin c levels were observed in the lungs of treated 19 groups following 30 days of treatment and at 90 day s (i.e., 60 days posttreatment). Survival was 20

not affected in neonatal rats. Clinical observatio ns during treatment included evidence of 21 respiratory irritation and toxicity. Body weight a nd food and water consumption were also 22 nonsignificantly decreased as compared with control s. 23 There are several reports in the literature regardi ng formaldehyde effects after inhalation 24 exposure on the male reproductive system in animals (Galalipour et al., 2007; Zhou et al., 2006; 25 Özen et al., 2005, 2002; Sarsilmaz et al., 1999; Wo utersen et al., 1987; Maronpot et al., 1986; 26 Guseva, 1972). The earliest report examined the ef fect of simultaneous exposures to 27 formaldehyde from air and water (Guseva, 1972). Ma le rats (n = 12, strain not specified) were 28 co-exposed to formaldehyde in air and drinking wate r 4 hours/day, 5 days/week for 6 months. 29 There were three exposure levels in the experiment of different air and drinking water 30 concentrations: (1) 0.41 ppm (0.5 mg/m 3 ) formaldehyde in air and 0.1 mg/L in water; (2) 0. 20 31 ppm (0.25 mg/m 3 ) formaldehyde in air and 0.01 mg/L in water; or (3 ) 0.10 ppm (0.12 mg/m 3 ) 32 formaldehyde in air and 0.005 mg/L in water. Repro ductive function was assessed by mating 33 two females per male. The time for the onset of pr egnancy and the number of pregnancies per 34 treatment group were recorded. A subset of dams wa s sacrificed on GD 20 of pregnancy, and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-297 the number and weight of fetuses was determined. P ostnatal development of the remaining dams 1 was tracked (e.g., times of eye opening and develop ment of hair coat). Nucleic acid levels were 2 determined in the testes of formaldehyde-exposed ra ts. Gonadotropic response was assessed by 3 injecting an emulsion of pituitaries from exposed m ale rats into unexposed infantile females and 4 measuring the weight ratios of the uterus and ovari es. Formaldehyde exposure reduced nucleic 5

acid levels in testes to 88 and 92% of controls in groups 1 and 2, respectively. No other 6 formaldehyde-induced differences were found. 7 Woutersen et al. (1987) and Maronpot et al. (1986) examined tissue sections from testes 8 and ovaries of exposed animals as part of studies p rimarily addressing respiratory tract toxicity 9 (see Section 4.2.1.2.2.4 for complete study details ). Maronpot et al. (1986) exposed female and 10 male B6C3F1 mice to 0, 2, 4, 10, 20, and 40 ppm (0, 2.45, 4.91, 12.3, 24.5, and 49.1 mg/m 3 ) 11 formaldehyde 6 hours/day, 5 days/week for 13 weeks. Decreased weight gain due to 12 formaldehyde exposure was seen in both male and fem ale mice. Additionally, there was 80% 13 mortality at the highest exposure (40 ppm). The au thors reported endometrial hypoplasia and 14 lack of ovarian luteal tissue in females exposed to 40 ppm, but no compound-related changes 15 were observed in testes sectioned and viewed by lig ht microscopy. 16 In a study by Appleman et al. (1988), male Wistar rats (40/group) with undamaged or 17 damaged (via bilateral intranasal electrocoagulatio n) nasal mucosa were exposed for 13 or 18 52 weeks to 0, 0.1, 1, or 10 ppm (0, 0.124, 1.24, o r 12.4 mg/m 3 ) formaldehyde 6 hours/day, 19 5 days/week. At study termination, mean body weigh t was decreased, but relative testes weight 20 was increased in the 10 ppm group (interpreted as a non-adverse outcome that was associated 21 with the decreased body weight). No treatment-rela ted histopathologic findings were reported 22 for male reproductive organs (although it is not cl ear to what extent they were evaluated since 23 the primary focus of the study was on the nasal epi thelium). 24 Following up on earlier reports of decreased Leydig cell quality in rats administered I.P. 25 injections of formaldehyde (Chowdhury et al. [1992] , described in Section 5.2.1.8.3), Sarsilmaz 26 et al. (1999) studied the effects of formaldehyde i nhalation on Leydig cells. Adult male Wistar 27 rats (30) were exposed to 0, 10, or 20 ppm (0, 12.3 , or 24.6 mg/m 3 ) formaldehyde 8 hours/day, 28 5 days/week for 4 weeks. Animals were observed dai ly and weighed weekly. Rats were 29 sacrificed on day 29 and autopsied, and testes were weighed, fixed, and sectioned for histologic 30 examination. Signs of irritation from formaldehyde exposure were noted (frequent eye blinking, 31 excessive licking, increased frequency of nose clea ning, interrupted breathing, and sneezing). 32 Body weight gain was reduced by formaldehyde exposu re from 17.7% gain in control rats to 33 4.66 and 2.63% in rats exposed at 10 and 20 ppm, re spectively ( p < 0.001). As shown in 34 Table 4-59, relative testes weights were unaffected (reported as proportions but more likely to be 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-298 percentages), although trends and numerical differe nces were similar to those reported by Özen 1 et al. (2002). Sarsilmaz et al. (1999) found that both Leydig cell quantity and the percentage of 2 cells with normal nuclei were reduced by formaldehy de treatment. Although the dose-dependent 3 reduction in Leydig cell quantity was statistically significant at both exposure levels, the study 4

authors considered the data to be within the normal range. 5

6 Table 4-59. Formaldehyde effects on Leydig cell qu antity and nuclear 7 damage in adult male Wistar rats 8 9

Appearance of nucleus e,g

Inhalation

exposure a Relative

testes weight b,c,d Leydig cell

quantity c,e,f Normal Pyknotic Karyorectic Karyolytic Control 0.93 (0.03) 47.27 (7.8) 98 2 0 0 10 ppm 0.92 (0.06) 45.04 (7.8) h 92 2 4 2 20 ppm 0.89 (0.06) 44.36 (7.5) i 76 9 10 5 10

a Rats were exposed 8 hours/day, 5 days/week for 4 we eks. 11

b Stated to represent the ratio of the last day’s tes ticle weight to the body weight but more likely to be the percent 12 of body weight. 13

c Cells within 100 defined areas. 14

d n = 10. 15

e For each exposure group, 100 defined locations were assessed. 16

f

n = 100. 17

g Values presented as percentage of cells. 18

h Different from control ( p < 0.05), as reported by the authors. 19

i Different from control ( p < 0.01), as reported by the authors. 20 21 Source: Sarsilmaz et al. (1999). 22 23 24 It was hypothesized that decreased Leydig cell qual ity may have been the result of 25 oxidative stress induced by formaldehyde exposure. Özen et al. (2002), in the same laboratory, 26 investigated changes in testicular iron, copper, an d zinc levels as measures of oxidative stress 27 and damage. Adult male albino Wistar rats (seven/g roup) were exposed at 0, 10, or 20 ppm (0, 28 12.2, or 24.4 mg/m 3 ) formaldehyde 8 hours/day, 5 days/week for either 4 or 13 weeks. Rats 29 were observed daily and weighed once a week. Rats were sacrificed at the end of the exposure 30 period and autopsied, and the testes were removed a nd weighed. Zinc, copper, and iron levels 31 were determined in testes tissue by using atomic ab sorption spectrophotometry. Both weight 32 gain and relative testes weight were decreased in a concentration-dependent and duration33 dependent manner (Table 4-60). Both zinc and coppe r levels in rat testes were reduced in a 34 concentration- and duration-dependent manner by for maldehyde exposure. For example, zinc 35 was reduced from 277 to 107 mg/kg after a 4-week × 20 ppm exposure and from 260 to 36 95 mg/kg after a 12-week × 20 ppm exposure ( p < 0.001) (Table 4-60). Iron levels in testes were 37This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-299 increased from 30 to 39 mg/kg after a 4-week 20 ppm exposure ( p < 0.01) and from 33 to 1 58 mg/kg after 12 weeks at 20 ppm ( p < 0.05). The authors suggested that alterations in trace 2

element levels in the testes were consistent with o xidative damage and may have contributed to 3 changes in Leydig cell function. These researchers also reported alterations in trace metals in 4 lung tissue from Wistar rats exposed to formaldehyd e 8 hours/day, 5 days/week for 4 or 5 13 weeks. Iron levels were increased at 5 ppm for 13 weeks and 10 ppm for either 4 or 6 13 weeks. Zinc levels decreased for all formaldehy de exposures. In both cases, the authors 7

attributed elevated iron levels to oxidative stress . In addition to citing the role of zinc as a 8 cofactor of cytoplasmic Cu-Zn-SOD, the authors sugg ested that zinc may have been consumed 9 by increased FALDH activity. Although oxidative st ress and increased FALDH activity may be 10 relevant to the POE and therefore impact the lung, it is less clear how these changes would occur 11 in the testes. Taken together, the reports of Özen et al. (2002) and Sarsilmaz et al. (1999) 12 suggested a LOAEL of 10 ppm 8 hours/day for 4 weeks for changes in Leydig cell quantity and 13 quality, decreased testes weight, and changes in tr ace metal content (zinc, copper, and iron). 14

15 Table 4-60. Formaldehyde effects on adult male alb ino Wistar rats 16 17

Inhalation

exposure a Weight gain

(%) Testes weight

(%) Zinc

(mg/kg) Copper

(mg/kg) Iron

(mg/kg) 4 Weeks Control 19.1 (2.7) 0.94 (0.03) 277 (16) 6.4 (0.42) 30 (2.7) 10 ppm 5.8 (2.4) b 0.92 (0.02) c 132 (8.9) b 4.2 (0.33) b 35 (2.8) d 20 ppm 2.4 (0.6) b 0.91 (0.01) c 107 (6.9) b 3.3 (0.27) b 39 (3.1) d

13 Weeks Control 55.9 (2.3) 0.91 (0.01) 269 (15) 6.0 (0.34) 33 (2.6) 10 ppm 34.7 (3.5) b 0.84 (0.03) b 112 (8.1) b 3.6 (0.30) b 52 (3.5) b 20 ppm 20.8 (1.4) b 0.82 (0.03) b 95 (6.4) b 1.9 (0.17) b 58 (3.0) b 18

a Formaldehyde exposure was 8 hours/day, 5 days/week for either 4 or 13 weeks. Values are means ± SDs o f 19 seven animals.. 20

b Different from control, p < 0.001, as calculated by the authors. 21

c Different from control, p < 0.05, as calculated by the authors. 22

d Different from control, p < 0.01, as calculated by the authors. 23 24 Source: Özen et al. (2002). 25 26 27 In another study that assessed testicular toxicity (Özen et al., 2005), male Wistar rats 28 (18/group) were exposed by inhalation to 0, 5, or 1 0 ppm (0, 6.2, or 12.4 mg/m 3 ) formaldehyde 29 8 hours/day, 5 days/week for 91 days. In-life obse rvations in exposed rats included clinical signs 30 of respiratory irritation and decreased mean food a nd water consumption. At study termination, 31

serum testosterone levels and mean seminiferous tub ule diameters were significantly decreased 32 from control in a dose-responsive manner (Table 4-6 1). Immunohistochemical staining of testis 33This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-300 tissues showed increased localization of heat shock protein (Hsp) 70 in the cytoplasm of 1 spermatogonia, spermatocytes, and spermatids of tre ated animals compared with controls (not 2 shown here). 3

4 Table 4-61. Formaldehyde effects on testosterone l evels and 5 seminiferous tubule diameters in Wistar rats follow ing 91 days of 6

exposure 7 8

Inhalation

exposure a Testosterone levels (ng/dL) Seminiferous tubule diameters ( μ m) n = 6 n = 100 Control 406.54 ± 16.82 259.22 ± 16.18 10 ppm 244.01 ± 23.86 b 236.17 ± 13.09 c 20 ppm 141.30 ± 08.56 b 233.24 ± 10.13 c 9

a Formaldehyde exposure was 8 hours/day, 5 days/week for 91 weeks. Values are means ± 10 SEMs. 11

b Different from control, p < 0.0001, by one-way ANOVA, as calculated by the au thors. 12

c Different from control, p < 0.001, by one-way ANOVA, as calculated by the aut hors. 13 14 Source: Özen et al. (2005). 15 16 17 Zhou et al. (2006) investigated the effect of form aldehyde on the testes and the protective 18 effect of vitamin E against oxidative damage by for maldehyde in adult male rats. In this study, 19 adult male Sprague-Dawley rats (10/group) were trea ted for 2 weeks in the following groups: 20 (1) control rats were administered physiological sa line by oral gavage, (2) rats were administered 21 physiological saline by gavage and exposed to 10 mg /m 3 (8.05 ppm) formaldehyde by inhalation 22 for 12 hours/day, and (3) rats were administered da ily gavage doses of 30 mg/kg vitamin E and 23 exposed to 10 mg/m 3 (8.05 ppm) formaldehyde by inhalation for 12 hours /day. Formaldehyde 24 treatment resulted in significantly decreased ( p < 0.05) mean testis weight. Histopathologic 25 findings in treated rats included atrophy of semini ferous tubules, decreased spermatogenic cells, 26 and seminiferous cells that were “disintegrated” an d shed into the lumina, which was 27 azoospermic. Interstitial tissue was edematous wit h vascular dilatation and hyperemia. In the 28 formaldehyde-treated group, epididymal sperm count and percentage of motile sperm were 29 significantly decreased, and the percentage of abno rmal sperm was increased ( p < 0.05), as 30 compared with control. Evaluation of biochemical m arkers in testes tissue showed the activities 31 of testicular SOD, GPX, and GSH were decreased; MDA levels were significantly increased as 32 compared with control. All observed effects of for maldehyde treatment (i.e., decreased testes 33 weight, biochemical alterations, histopathologic ef fects, and sperm count, motility, and 34 morphology findings) were attenuated by administrat ion of 30 mg/kg-day vitamin E. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-301 In a study by Golalipour et al. (2007), testicular changes of increasing severity with 1 increasing duration were reported. A total of 28 W istar rats, aged 6-7 weeks old, were divided 2 into four groups including three FA treatment group s (4 hours of exposure/day, 4 days/week for 3 18 weeks; 2 hours of exposure/day, 4 days/week for 18 weeks; 4 hours of exposure/day, 2 4 days/week for 18 weeks) and one untreated control. The three FA-treated groups were exposed 5 via inhalation to formaldehyde. The mean concentra tion of FA vapor, based on three 6 measurements during the study (stated as the beginn ing, during, and end of the study period), 7 was reported as 1.5 ppm. At the end of the study p eriod, the rats were sacrificed by ether 8 anesthesia and subsequent cervical dislocation. Th e left testis was dissected from each rat and a 9 specimen was taken from each testis. Tissues were fixed, embedded, sectioned (at 4 μ m), and 10 stained with hematoxylin and eosin. Using a light microscope, a histopathological examination 11 was performed on the testes tissues, including morp hometric evaluation of the diameter and 12 height of 20 seminiferous tubules/testis. Golalipo ur et al. (2007) reported an FA exposure 13 frequency (or duration)-dependent increase in testi cular germ cells and seminiferous tubule 14 defects. In the most frequent duration treatment g roup, a severe decrease in germ cells in >85% 15 of the seminiferous tubules and arrested spermatoge nesis were observed. In the mid-level 16 frequency of duration treatment group, a decrease i n the number of germ cells and an increased 17

thickness of the basement membrane of 75% of the tu bules was observed. In the lowest level 18 duration treatment group, a disruption in the Serto li and germinal cell arrangement, and 19 increased spacing between germ cells was observed. Further, the seminiferous tubule diameter 20 (STD) and seminiferous epithelial height (SEH) was most decreased among the treatment groups 21 (exhibiting the greatest decrease in the group with the greatest hours and days of exposure) 22 compared to the control (Table 4-62). The results of this study are consistent with the findings of 23 other studies of male reproductive system outcomes with inhalation FA exposure (e.g., Özen et 24 al., 2005 and Zhou et al., 2006). 25 Xing et al. (2007) also studied the effects of form aldehyde on sperm development and 26 reproductive capacity in adult male mice. In this study, male mice (12/group, strain not 27 specified) were exposed to 0, 21, 42, or 84 mg/m 3 (0, 16.9, 33.8, or 67.6 ppm) formaldehyde via 28 inhalation for 13 weeks at 2 hours/day, 6 days/week . The males were mated to untreated females 29 in a dominant lethal protocol, and sperm morphology was assessed at study termination. The 30 percent abnormal sperm was increased significantly ( p < 0.05) in all treated groups, as was the 31 rate of resorptions ( p < 0.01) (Table 4-63). The mean number of live fet uses/litter was decreased 32 in all treated groups, with statistical significanc e achieved at 84 mg/m 3 . Although this study did 33 not assess the number of corpora lutea per dam, the reby precluding the calculation of 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-302 preimplantation loss, it is nevertheless indicative of formaldehyde-induced sperm morphology 1 changes and dominant lethal effects in male mice. 2

3 Table 4-62. Effects of formaldehyde exposure on se miniferous tubule 4 diameter and epithelial height in Wistar rats follo wing 18 weeks of 5

exposure 6 7

Inhalation

exposure a Seminiferous tubule diameters ( μ m) Seminiferous tubule

height ( μ m) n = 7 n = 7 Control 252.12 ± 4.82 82.77 ± 2.00 1.5 ppm, 4 h/d, 4 d/w 204.55 ± 3.29 b 65.26 ± 1.43 b 1.5 ppm, 2 h/d, 4 h/w 232.45 ± 2.42 b 69.46 ± 1.78 b 1.5 ppm, 2 h/d, 2 d/w 238.94 ± 4.37 b 72.80 ± 2.03 b 8

a Values are means ± SEMs. 9

b Different from control, p < 0.05, as calculated by the authors. 10 11 Source: Golalipour et al. (2007). 12 13

14 Table 4-63. Incidence of sperm abnormalities and d ominant lethal effects in 15 formaldehyde-treated mice 16 17 Sperm abnormalities Reproductive capacity

Dose (mg/m 3 ) Total abnormal

sperm heads Aberration rate (%) Mean live fetuses/litter Resorption rate (%) 0 391 3.53 ± 0.98 11.00 ± 1.01 2.273 21 568 5.48 ± 1.45 10.67 ± 1.16 9.380 b

42 849 6.15 ± 1.36 9.63 ± 2.83 10.390 b

84 974 9.24 ± 2.13 a 9.04 ± 2.98 a 12.440 b 18

a Significantly different from controls ( p < 0.05), as calculated by the authors. 19

b Significantly different from controls ( p < 0.01), as calculated by the authors. 20 21 Source: Xing et al. (2007). 22 23 24 4.2.1.7.2. Oral exposure studies addressing developmental and reproductive toxicity. No 25 contemporary testing guideline studies, such as a p renatal developmental toxicity study or two26 generation reproductive toxicity study, have been p erformed by the oral route for formaldehyde. 27 However, a number of studies have evaluated develop mental toxicity and reproductive 28 parameters in rats, mice, and dogs. 29 Hurni and Ohder (1973) tested the developmental tox icity of formaldehyde administered 30 as a 40% w/v solution containing 11–14% w/w methano l in 9–10 pregnant beagle dogs that 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-303 received the compound in their diet on GDs 4–56. C ommercial grade formaldehyde (as a 40% 1 solution) was sprayed on the pellets prior to feedi ng. Each animal was allotted a diet of 300 g of 2 chow (reduced to 200 g 1 week prior to term) that w as promptly consumed (within 5– 3 10 minutes) before the formaldehyde volatilized app reciably. The concentrations of 4 formaldehyde in the chow were 0, 125, or 375 ppm, e quivalent to doses of 0, 3.1, or 5 9.4 mg/kg-day, respectively. Dams were allowed to deliver normally and weight gain, gestation 6 length, number of litters, litter size, number of l ive pups, number of pups surviving through 7 weaning, and pup weights weekly for the first 8 wee ks were monitored as indices of the potential 8 reproductive/developmental toxicity of formaldehyde . There were no formaldehyde-related 9 effects in any of the parameters other than progres sive pup weights, which were lower by group 10 in litters of dams exposed to formaldehyde (Table 4 -64). A developmental impact of 11 formaldehyde was evident in this strain of dog unde r the conditions of the experiment. At birth, 12 mean pup body weights were 4 and 8.4% less than con trol for the low- and high-dose groups, 13 respectively; at 8 weeks of age, the pup weight dec rements were 8.3% for the low dose and 14 12.5% for the high dose, as compared with control, and established a LOAEL of 125 ppm. The 15 contribution of methanol, which is a developmental toxin (Deglitz et al., 2004; Rogers et al., 16 2004) to these outcomes is not known. No internal or skeletal malformations were observed in 17 any of the 264 live-born and 20 still-born pups. 18

19 Table 4-64. Body weights of pups born to beagles e xposed to 20 formaldehyde during gestation 21 22 Formaldehyde concentration in chow (ppm) 0 125 375 Time (weeks) Average body weight (g) 0 321 308 294 1 547 526 467 2 818 755 706 3 1,078 987 944 4 1,264 1,247 1,166 5 1,601 1,512 1,429 6 2,020 1,816 1,741 7 2,449 2,263 2,145 8 2,957 2,712 2,587 23 Source: Hurni and Ohder (1973). 24 25 26 Marks et al. (1980) conducted a developmental toxic ity study of formaldehyde in CD-1 27 mice in which 29–35 pregnant animals were gavaged o n GDs 6–15 with aqueous formaldehyde 28 (containing 10–15% methanol) at 74, 148, and 185 mg /kg-day. Seventy-six controls were 29 gavaged with water alone. All dams were sacrificed on GD 18, and the numbers of implantation 30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-304 sites in each uterine horn were counted. The high dose of formaldehyde was toxic to the dams, 1 as indicated by the deaths of 22 of 34 mice before GD 18. Thus, the dose of 148 mg/kg-day was 2 a NOAEL for maternal toxicity in this study. Howev er, it is unclear to what extent an estimated 3 concurrent dose of up to 75 mg/kg-day methanol may have contributed to this toxic response. 4 To assess the developmental toxicity of formaldehyd e, live fetuses were weighed individually, 5 sexed, and examined for external, visceral, and ske letal malformations. Fetuses of surviving 6 high-dose dams and of those of other groups did not show an increased incidence of 7 malformations. Therefore, Marks et al. (1980) conc luded that formaldehyde did not induce fetal 8 abnormalities and that the 185 mg/kg-day dose level was a NOAEL for the developmental 9 toxicity of formaldehyde, nor were fetotoxic effect s of methanol apparent under the study 10 experimental conditions. 11 Seidenberg and Becker (1987) and Seidenberg et al. (1986) included formaldehyde 12 (purity not indicated) in a survey of the behavior of potential toxicants in a developmental 13 toxicity screening assay (Chernoff and Kavlock, 198 2). The protocol featured the administration 14 of a borderline toxic dose to 26–30 pregnant ICR/SI M mice on GDs 8−12. Dams were allowed 15 to deliver, and the neonates were examined, counted , and weighed on PNDs 1 and 3. The 16 selected formaldehyde dose of 540 mg/kg-day was fat al for 11/30 dams, but the average weight 17 gain among surviving dams was little changed compar ed with controls (3.9 ± 2.3 versus 18 4.0 ± 1.0 g). Similarly, there were no changes in perinatal responses in the neonates of exposed 19 dams compared with controls. For example, the aver age values for number of neonates/litter, 20 percent survival, and fetal weights on PNDs 1 and 3 were closely similar to those of controls. 21 Evidence of toxicity to the male reproductive syste m has been observed following oral 22 administration of formaldehyde in a 40% w/v solutio n containing 11–14% w/w methanol. 23 Cassidy et al. (1983) administered single oral dose s of 100 or 200 mg/kg to five male Wistar 24 rats/group. Testes from these animals and 20 contr ols were excised and examined for 25 spermatogenic abnormalities 11 days after dosing. A significant (19%) increase in testicular 26 sperm head counts was observed in rats exposed to 2 00 mg/kg-day formaldehyde as compared 27 with controls (Table 4-65). The percentage of abno rmal sperm heads was also significantly 28 increased (5%) in the 200 mg/kg-day dose group comp ared with controls. These data suggest 29 that formaldehyde can induce morphologic abnormalit ies in the germ cells of male experimental 30 animals at dose levels that did not significantly a ffect testis weights. The contribution of 31

methanol to these outcomes is unknown. 32

33 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-305 Table 4-65. Testicular weights, sperm head counts, and percentage incidence 1 of abnormal sperm after oral administration of form aldehyde to male Wistar 2 rats 3 4

Dose (mg/kg) Mean testes weight (g) Mean sperm heads × 10 6 /g testis Abnormal sperm heads (%) 0 3.30 175 4.76 100 3.27 166 5.22 200 3.16 209 a 9.77 a 5

a Significantly different from controls ( p < 0.001), as calculated by the authors. 6 7 Source: Cassidy et al. (1983). 8 9 10 Postmortem evaluation of the reproductive organs wa s conducted in a number of oral 11 studies that ranged between 4 weeks and 2 years in duration (Til et al., 1989, 1988; Tobe et al., 12 1989; Johanssen et al., 1986). Johannsen et al. (1 986) administered 0, 50, 100, or 150 mg/kg-day 13 formaldehyde in the drinking water to Sprague-Dawle y rats (15/sex/group) for 91 days and 0, 50, 14 75, or 100 mg/kg-day formaldehyde in basal diet to beagle dogs (4/sex/group) for 91 days; the 15 study reported no treatment-related effects on abso lute or relative gonad weights or 16 histopathology for either species. In a 4-week dri nking water study conducted by Til et al. 17 (1988), formaldehyde was administered to Wistar rat s (10/sex/treated group) at nominal levels of 18 0, 25, and 125 mg/kg-day; gonad organ weights and h istopathology were not affected by 19 treatment. Tobe et al. (1989) conducted a chronic (24-month) study in Wistar rats 20 (20/sex/group), with drinking water concentrations of 0, 0.02, 0.1, or 0.5%. According to the 21 study report, gonad weights were measured and histo pathology was conducted, but no treatment22 related findings were noted. In a chronic (105-wee k) study (Til et al., 1989) in Wistar rats 23 (70/sex/group), formaldehyde was administered in th e drinking water at mean actual levels of 0, 24 1.2, 15, or 82 mg/kg-day to males and 0, 1.8, 21, o r 109 mg/kg-day to females; serial sacrifices 25 were conducted at 53, 79, and 105 weeks of study. At study termination (105 weeks), mean 26 testes weights were 30% increased ( p < 0.01) in high-dose males as compared with contro ls, and 27 histopathology evaluation revealed Leydig cell tumo rs in treated males (incidences of 0/50, 3/50, 28 3/50, and 2/50 for the control through high-dose gr oups, respectively; historical control tumor 29 incidence data were not provided). The study autho rs did not judge these findings to be 30 treatment related. By design, none of the subacute to chronic studies included measures of 31 reproductive function (e.g., estrous cyclicity, spe rm measures, or reproductive performance). 32 With the exception of Til et al. (1989), detailed m ean organ weight and histopathology incidence 33 data were not provided in the published reports, an d Til et al. (1988) only included tumor (not 34 non-tumor) data. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-306 4.2.1.7.3. Intraperitoneal studies addressing developmental and reproductive toxicity. Other 1 studies in which formaldehyde was administered by I .P. injection have confirmed the potential 2 effects on the male reproductive system. 3 Chowdhury et al. (1992) administered I.P. injection s of 0, 5, 10, or 15 mg/kg-day 4 formaldehyde to Charles foster adult male rats (10/ group) for 30 days. On study day 31, blood 5

was collected for serum testosterone measurements a nd the rats were sacrificed. The testes were 6 removed, weighed, fixed in Bouin’s solution, and pr ocessed for histopathology. The study 7 authors reported adverse findings in all treated gr oups, including significant ( p < 0.01) mean 8 body weight gains, serum testosterone levels, and t estes weights. Histopathologic evaluation 9 revealed normal spermatogenic processes and Leydig cells in control animals. However, in 10 treated rats, gradual cellular degeneration in semi niferous tubules and in Leydig cells was 11 observed. Marked nuclear damage was noted in the 1 0 and 15 mg/kg-day groups, with 12 significantly (

p < 0.001) decreased Leydig cell populations and nuc lear diameters observed in all 13 treated groups. Additionally, a decrease in 3 β

- ∆ 5 -hydroxy steroid dehydrogenase was noted in 14 the Leydig cell region of treated rat testes. 15 In a 30-day study performed by Majumder and Kumar ( 1995), 10 mg/kg-day 16 formaldehyde was administered I.P. to eight male Wi star rats. All animals were sacrificed at 17 term, and the testes, prostate, seminal vesicles, a nd epididymides were excised and weighed. 18 With the use of methodologies that were not describ ed in the report other than by reference to the 19 Laboratory Manual for the Examination of Human Seme n and Sperm-Cervical Mucus 20

Interaction (WHO, 1987), sperm counts, motility, and viability were compared with those of 10 21 controls (injected I.P. with water alone). As show n in Table 4-66, striking reductions in sperm 22 count and motility were noted in formaldehyde-treat ed rats compared with controls. Sperm 23 viability was also significantly reduced by formald ehyde treatment, though to a lesser overall 24 extent than sperm count and motility. 25

26

Table 4-66. Effect of formaldehyde on spermatogeni c parameters in male 27 Wistar rats exposed intraperitoneally 28 29 Parameters Control (n = 10) Treated (n = 8)

Sperm count (10 6 /mL) 46.30 ± 5.01 20.40 ± 2.01 a Sperm viability (%) 87.10 ± 0.83 72.60 ± 2.32 a Sperm motility (%) 75.00 ± 10.90 22.00 ± 6.40 a 30

a Significantly different from controls ( p < 0.0001), as calculated by the authors. 31 32 Source: Majumder and Kumar (1995). 33 34 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-307 Majumder and Kumar (1995) also carried out an in vi tro experiment in which sperm from 1

normal rats were incubated with different concentra tions of aqueous formaldehyde at 2 concentrations ranging from 125 pg/mL to 2.5 μg/mL. Viability of control sperm remained close 3 to 80% for a period of 1 hour, whereas the presence of formaldehyde dose-dependently reduced 4 viability. Thus, only 50% spermatozoa were viable for 30 minutes in the presence of 5 ng/mL 5 formaldehyde compared with 6 minutes in the presenc e of 500 ng/mL. Sperm motility also was 6 sensitive to the presence of formaldehyde. Less th an 10% of sperm was motile for 10 minutes in 7 the presence of 125 pg/mL formaldehyde. The author s of the study considered their data to be 8 good evidence that functional parameters of spermat ozoa, such as viability and motility, can be 9 adversely affected by exposure to formaldehyde. Mo reover, they suggested that the cumulative 10 effects of I.P. administration of formaldehyde on t he male rat reproductive system raise an alert 11 that formaldehyde might impair the reproductive hea lth of males who are occupationally exposed 12 to the compound. 13 Odeigah (1997) conducted two short-term in vivo ass ays to examine sperm head 14

abnormalities and dominant lethal mutations. In th e sperm assessment, five daily I.P. injections 15 of 0, 0.125, 0.25, or 0.5 mg/kg formaldehyde were a dministered to male albino rats (six/group; 16 strain not specified). The rats were killed 3 week s after the last injection, and epididymal sperm 17

counts and abnormalities were assessed. A dose-rel ated decrease in sperm count was observed, 18 and significantly increased incidences of sperm hea d abnormalities were found at all treatment 19 levels (Table 4-67). 20

21 Table 4-67. Incidence of sperm head abnormalities in formaldehyde22 treated rats 23 24

Dose (mg/kg) Total abnormal sperm heads Frequency (%) ± SEM 0 90 1.50 ± 0.11 0.125 184 3.09 ± 0.16 a 0.25 436 7.27 ± 0.30 b

0.5 514 8.57 ± 0.33 b 25

a Significantly different from controls ( p < 0.05), as calculated by the authors. 26

b Significantly different from controls ( p < 0.001), as calculated by the authors. 27 28 Source: Odeigah (1997). 29 30 31 In the dominant lethal assay (Odeigah, 1997), five daily I.P. injections of 0, 0.125, 0.25, 32 or 0.5 mg/kg formaldehyde were administered to male rats (5 control rats, 12/treated group). 33 Subsequently, each male was mated with two untreate d virgin females per week for 34

3 consecutive weeks. The females were killed 13 da ys after the midpoint of the mating period 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-308 and evaluated for live and dead uterine implants. In general, the number of live embryos was 1 decreased with treatment, and the number of dead im plants was increased (Table 4-68). 2 Additionally, there was a reduction in fertile mati ngs in females mated 1–7 days after the males 3 had been treated. This study did not assess the nu mber of corpora lutea and therefore precluded 4 the determination of preimplantation loss. Neverth eless, it is indicative of dominant lethal 5 effects on the male germ cells. 6

7 Table 4-68. Dominant lethal mutations after exposu re of male rats to 8 formaldehyde 9 10

Dose (mg/kg) Time of

mating (days) Fertile

matings a (%) Implants per

female a (mean ± SE) Live embryos

per female (mean ± SE) Dead implants

per female (mean ± SE) Dominant

lethal mutation index b 0 0–21 96.67 (29) 7.86 ± 0.2 (29) 7.43 ± 0.3 0.43 ± 0.8 0 0.125 1–7 75.0 (18) 7.18 ± 0.3 (18) 5.95 ± 0.2 1.23 ± 0.5 19.92 8–14 79.17 (19) 7.38 ± 0.5 (19) 6.30 ± 0.5 1.08 ± 0.3 15.21 15–21 91.67 (22) 7.68 ± 0.2 (22) 6.89 ± 0.3 0.79 ± 0.5 7.27 0.25 1–7 33.33 (8) 5.75 ± 0.3 (8) 2.05 ± 0.3 3.70 ± 0.4 72.41 8–14 50.0 (12) 6.60 ± 0.2 (12) 3.91 ± 0.2 2.69 ± 0 .2 47.38 15–21 87.5 (21) 7.25 ± 0.4 (21) 6.63 ± 0.3 0.62 ± 0.5 10.77 0.5 1–7 25.0 (6) 5.05 ± 0.03 (6) 1.10 ± 0.5 3.95 ± 0.22 85.20 8–14 29.17 (7) 5.27 ± 0.01 (7) 1.50 ± 0.6 3.77 ± 0 .28 79.81 15–21 83.33 (20) 7.08 ± 0.04 (20) 5.79 ± 0.4 1.29 ± 0.17 22.07 11

a Number of females with implants presented in parent heses. 12

b Dominant lethal mutation index: 13 Index = 1 – (Live implants experiment group per fem ale) × 100 14 (Live implants of control grou p per female) 15 16 Source: Odeigah (1997). 17 18 19 4.2.1.7.4. Dermal exposure studies addressing developmental an d reproductive toxicity. In a 20 study designed to assess the embryotoxic effects of dermal exposure to formaldehyde, Overman 21 (1986) applied 0.5 mL of a 37% formaldehyde solutio n directly to the dorsal skin of female 22 Syrian golden hamsters (four–six/group) on GDs 8, 9 , 10, or 11 for 2 hours. To prevent 23 grooming during the treatment period, the animals w ere anesthetized with Nembutal. At the end 24 of the 2-hour treatment period, the application sit e was washed thoroughly to remove any 25 remaining formaldehyde. The dams were terminated o n GD 15 (i.e., one day prior to expected 26 delivery, since the typical gestation period for th e Syrian golden hamster is 16–18 days). The 27

fetuses were removed and fixed in either Bouin’s so lution or 95% ethanol for visceral or skeletal 28 evaluation, respectively. The uteri were examined for implantation sites. Fixed fetuses were 29This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-309 weighed, measured (crown-rump), and examined for ex ternal abnormalities; fetuses that had 1 been placed in Bouin’s fixative were evaluated for visceral anomalies by using a free-hand 2 sectioning technique, and those that were placed in ethanol were macerated, stained, and cleared 3 for skeletal examination. In this study, the dams exhibited signs of dermal irritation and 4 irritability, but the author reported no treatment- related effects on maternal body weight gain. 5 The percent of resorption sites was increased (alth ough not significantly) in treated litters as 6 compared with control (0, 4.2, 8.1, 4.6, and 3.2% r esorbed implantation sites for control and 7 GDs 8, 9, 10, and 11 treatment groups, respectively ). No treatment-related effects on fetal 8 weight, length, or visceral or skeletal development were observed. 9

10

4.2.1.7.5. Summary of reproductive and developmental effects. Formaldehyde exposures up to 11 40 ppm 6 hours/day from GDs 6–15 or 6–20 did not re sult in external or internal malformations 12 (Martin, 1990; Saillenfait et al., 1989). Martin ( 1990) reported delayed skeletal ossification and 13 dose-dependent decreases in fetal body weight at 5 ppm. Formaldehyde exposure at 40 ppm to 14 pregnant female Sprague-Dawley rats reduced fetal b ody weights in male and female progeny 15 and in male pups of dams exposed to 20 ppm formalde hyde (Saillenfait et al., 1989). Based on 16 these studies (Table 4-69), the LOAEL for developme ntal effects in rats is 5 ppm, with a 17 NOAEL of 2 ppm for decreased fetal weight and delay ed skeletal ossification, based on 18 inhalation exposures during GDs 6–20. 19 Developmental studies during earlier gestational wi ndows of inhalation exposure to 20 formaldehyde have reported additional adverse healt h effects, including delayed ossification, 21 changes in relative organ weight, undescended teste s, biochemical changes (e.g., ascorbic acid 22 and nucleic acids), and blood acidosis (Senichenkov a and Chebotar, 1996; Senichenkova, 1991; 23 Kilburn and Moro, 1985; Gofmekler and Bonashevskaya , 1969; Gofmekler, 1968; Pushkina et 24 al., 1968). Kitaev et al. (1984) hypothesized that formaldehyde may affect reproductive function 25 by stimulating the hypothalamo-pituitary-gonadal (H PG) axis based on their observations of 26 increased ovary weight, increased number of ovulati ng cells, and changes in blood levels of 27 gonadotropins (LH and FSH). Evidence of preimplant ation loss, which may be related to HPG 28 disruption, was observed in this study and by Sheve leva (1971). Additional studies are needed to 29 better understand developmental effects of formalde hyde exposure during early gestational 30

windows. 31 The prenatal developmental toxicity of oral and der mal exposures to formaldehyde has 32 not been thoroughly studied. Reductions in postnat al growth in beagle pups was observed by 33 Hurni and Ohder (1973) following in utero exposure to 125 ppm maternal dietary formaldehyde 34 during GDs 4–56 in beagle dogs. However, gavage do sing during gestation of mice to overtly 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-310 maternally toxic doses (Seidenberg and Becker, 1987 ; Marks et al., 1980) (Table 4-70) and 1 dermal application during gestation to hamsters at a dose that caused dermal irritation and 2 irritability (Overman, 1986) did not result in any observed fetal toxicity (Table 4-71). 3 Few studies identified effects on maternal toxicity or female reproductive capacity. As 4 summarized in Table 4-69, exposure of rat dams at 1 0–40 ppm formaldehyde during pregnancy 5 has been shown to result in significantly decreased weight gain (Martin, 1990; Saillenfait et al., 6 1989; Kilburn and Moro, 1985). Maronpot et al. (19 86) reported endometrial hypoplasia with a 7 lack of ovarian luteal tissue in female rats expose d at 40 ppm but not at 20 ppm. Changes in LH 8 and FSH levels were reported in dams exposed to 0.4 1 ppm formaldehyde by Kitaev et al. 9 (1984), establishing an unbounded LOAEL for materna l toxicity. 10 Studies designed to assess male reproductive system endpoints in rats following repeated 11 inhalation exposures to formaldehyde have shown con centration-dependent decreases in Leydig 12 cell number and quality, effects on seminiferous tu bules, decreases in testes weight, alterations in 13 sperm measures, decreased testosterone levels, alte rations in trace metals in the testes, and/or 14 dominant lethal effects (Zhou et al., 2006; Özen et al., 2005, 2002; Zhou et al., 2006Sarsilmaz et 15 al., 1999) (Table 4-72). Based on available studie s, the LOAEL for changes in the male 16 reproductive system in rats following 5 days/week o f inhalation exposures is 5 ppm for 3 months 17 of daily exposures and 10 ppm for 4 weeks of daily exposures; these dose levels are unbounded. 18 Abnormal sperm were also noted in mice at an inhala tion dose of 16.9 ppm 2 hours/day, 6 19 days/week for 13 weeks (Xing et al., 2007), but, in contrast, Maronpot et al. (1986) reported no 20 histologic abnormalities in male mice after formald ehyde exposures at 40 ppm 6 hours/day, 5 21 days/week for 13 weeks. Varied results among studi es may be due to species differences or 22 differences in methods. Although several oral subc hronic and chronic studies with formaldehyde 23 did not identify effects on the testes (Tobe et al. , 1989; Til et al., 1988; Johanssen et al., 1986), 24 Cassidy et al. (1983) observed spermatogenic abnorm alities after a single oral dose of 200 mg/kg 25 to rats, and a chronic drinking water study in rats (Til et al., 1989) reported low incidences of 26 Leydig cell tumors in all treated groups, compared with none in control (Table 4-73). 27 Additionally, studies utilizing I.P. injection of f ormaldehyde in rats have demonstrated testes and 28 sperm anomalies (Majumder and Kumar, 1995; Chowdhur y et al., 1992) and dominant lethal 29 effects (Odeigah, 1997) (Table 4-74). 30This document is a draft for review purposes only a nd does not constitute Agency policy 4-311 DRAFT—DO NOT CITE OR QUOTETable 4-69. Summary of reported developmental effects in formaldehyde inhalation exposure studiesReported study findingsbLOAEL/NOAELcSpecies,strain, sex n/GroupDose; time oftreatmentaMaternal Offspring Maternal OffspringReferenceRat, strainNR, female12 0, 0.01, or 0.81 ppm(reported as 0.012,and 1 mg/m3)d;continuous dosing10–15 days prior tomating and duringgestationAt 0.01 and 0.81 ppm:↑pregnancy duration(dose-dependent datanot shown)eFetuses:At 0.01 and 0.81 ppm:↓fetuses/dam (dose dep., datanot shown)e↑body wt (dose dep., stat. sig.)↓lung and liver wt (dose dep.,stat. sig.)↑adrenal wt (dose dep., stat.sig.)At 0.81 ppm:↑thymus andkidney wt (stat. sig.)L: 0.01 ppmN: NDL: 0.01 ppmN: NDGofmekler(1968)Rat,“albino”strain NR,female12 0, 0.01, or 0.81 ppm(reported as 0.012,and 1 mg/m3)d;continuous dosing10–15 days prior tomating and duringgestationNE Age of assessment NR.At 0.81 ppm:histologic effects in liver (e.g.,↑extramedullary hematopoieticcenters), kidney (e.g.,↑polymorphism of renal epithelialcell nuclei)and thymusNEeN: 0.01 ppmL: 0.08 ppmGofmekler andBonashevskaya(1969)f,hRat, strainNR, male4 Inhalation anddrinking waterco-exposure: 0;0.10 ppm plus0.005 mg/L water;0.20 ppm plus0.01 mg/L water; or0.41 ppm plus0.1 mg/L water; alltreatments4 hours/day,5 days/week for6 monthsNo effects No effects NDgND Guseva (1972)This document is a draft for review purposes only a nd does not constitute Agency policy 4-312 DRAFT—DO NOT CITE OR QUOTETable 4-69. Summary of reported developmental effects in formaldehyde inhalation exposure studiesReported study findingsbLOAEL/NOAELcSpecies,strain, sex n/GroupDose; time oftreatmentaMaternal Offspring Maternal OffspringReferenceRat, strainNR, femaleNR Expt 1:0 or 30 ppmdExpt 2: 0, pair-fedcontrol (15, 10, or5 days), or 30 ppm;8 hours/day for15 days (GDs 3–17),10 days (GDs 3–12),5 days (GDs 8–12),or 3 days (GDs 9–11)At 30 ppm50% mortality (10 and15 day exp)↓wt gain (durationdep.; 3, 5, 10, and 15day exp.)↓wt of liver, kidney,spleen and thymus↑wt of lung andadrenaleFetuses:At 30 ppm↓fetal wt and growth (durationdep., 10 and 15 day exp.)↑dev. defects (undescendedtestes, large hearts, smallthymuses, small lungs)eN: NDL: 30 ppmeN: NDL: 30 ppmeKilburn andMoro (1985)fAbRat, Wistar,femaleEmbryodev expt:5–9/group(42 adultanimals);maternaleffects:NR (200adultfemalestotal)i0, 0.4, or 1.2 ppm(converted fromreported 0, 0.5 or1.5 mg/m3);4 hours/day,5 days/week for4 months; exposedfemales mated tounexposed males on120thday exp.At 0.4 ppm:↑wt of ovaries (stat.sige)↑LH level (stat. sig.e)At 1.2 ppm:↑FSH level in blood(stat. sig.; nonsig. at0.4 ppme)At 0.4 ppm:↑no. of embryos and 2blastomere stage embryos (stat.sig. in 2ndday preg.)At 1.2 ppm:↑no. degenerating embryos(stat. sig. in 3rdday preg.)L: 0.4 ppmN: NDL: 0.4 ppmN: NDKitaev et al.(1984)gRat,SpragueDawley,female andoffspring ofboth sexes25 0 (air controlgroup), 0 (roomcontrol group), 2, 5,or 10 ppm;6 hours/dayGDs 6–15.Exposed femalesmated to unexposedmalesAt 10 ppm:↓food consumption(stat. sig.)↓wt gain (stat. sig.)At 5 and 10 ppm:Fetuses:↑incidence of reducedossification of pubic and ischialbones (stat. sig. compared withair control group)↓fetal wts (nonsig.)↑litter size (nonsig.)L: 10 ppmN: 5 ppmL: 5 ppmN: 2 ppmMartin (1990)e,fThis document is a draft for review purposes only a nd does not constitute Agency policy 4-313 DRAFT—DO NOT CITE OR QUOTETable 4-69. Summary of reported developmental effects in formaldehyde inhalation exposure studiesReported study findingsbLOAEL/NOAELcSpecies,strain, sex n/GroupDose; time oftreatmentaMaternal Offspring Maternal OffspringReferenceRat, “white”strain NR,female andoffspring ofboth sexes5–12 (NRforformaldehyde only)0, 0.01, or 0.81 ppm(reported as 0.012and 1 mg/m3)d;continuous10–15 days prior tomating throughgestationAt 0.01 and 0.81:↓vit. C level in liver(stat. sig.)↓vit. C level inplacenta(nonsig.)Fetuses:At 0.01 and 0.81 ppm:↓fetuses/femalee↑body wt and organ wt (data notshowne)↓vit. C level in whole fetus (stat.sig.)At 0.01 ppm:↓vit. C level in fetal liver (stat.sig.)L: 0.01 ppmN: NDL: 0.01N: NDPushkina et al.(1968)fRat,SpragueDawley,female andoffspring ofboth sexes25 0 (air control), 5, 10,20, 40 ppm; 6hours/day,GDs 6–20.Exposed femalesmated to unexposedmales.GD 21 dams:At 5 ppm:↑absolute body wt gain(5 ppm only)At 40 ppm:↓body wt gain GDs 6–21 (stat. sig.)↓absolute body wt gain(stat. sig., dosedependent trend 20 and40 ppm)GD 21 fetuses:At 20 and 40 ppm:↓fetal body wt, male (stat. sig.)At 40 ppm:Delayed ossification of thoracicvertebrae (stat. sig., trend20 ppm)↑unossified sternebrae (nonsig.at 40 ppm)↓fetal body wt, female (stat.sig.)L: 40 ppmN: 20 ppmL: 20 ppmN: 10 ppmSaillenfait et al.(1989)This document is a draft for review purposes only a nd does not constitute Agency policy 4-314 DRAFT—DO NOT CITE OR QUOTETable 4-69. Summary of reported developmental effects in formaldehyde inhalation exposure studiesReported study findingsbLOAEL/NOAELcSpecies,strain, sex n/GroupDose; time oftreatmentaMaternal Offspring Maternal OffspringReferenceRat,mongrelwhite,female andoffspring ofboth sexesNRi0 or 0.41 ppm(reported as 0.5mg/m3)formaldehyde(also a 3rd group ofgasoline exposure,not described in thistable); 4 hours/dayGDs 1–19Dams GD 20:↓corpora lutea(nonsig.), embryos deadbefore implantation (notstat. sig.), andimplanted embryos(nonsig.)↑blood pCO2(stat.sig.)Fetuses (GD 20):Stat. sig. findings include↑fetal wt↑litters w/internal organanomalies↓fetuses w/ossification centersin hyoid bone↑metacarpal bone centers↑metatarsal bone centers↑developmental defects↑blood pCO2and pO2Pups:↓pup wtDev. delays (data not shown)L: 0.41 ppmN: NDL: 0.41 ppmN: NDSenichenkova(1991)Mouse,mongrel,female andoffspring ofboth sexesNR (254dams)i0 + ethyl alcohol;0.41 ppmformaldehyde;0.41 ppmformaldehyde +bipyridyl;4 hours/dayGDs 1–19.Induced maternaliron deficiencyanemia by I.P.bipyridyl injectionson GDs 12–15;controls injectedw/25% ethylalcohol.Dams GD 20:formaldehyde alone:↑blood pCO2(stat.sig.)formaldehyde +bipyridyl:↓blood acid metabolicproducts (stat. sig.)↓blood truebicarbonates and CO2conc. (stat. sig.)Fetuses (GD 20):Formaldehyde alone:↑cryptorchidismFormaldehyde + bipyridyl:↑birth defects (stat. sig.)↓dev. delay (stat. sig.)↓blood acid-base measures ofembryos (stat. sig.)L: 0.41 ppmN: NDL: 0.41 ppmN: NDSenichenkovaand Chebotar(1996)This document is a draft for review purposes only a nd does not constitute Agency policy 4-315 DRAFT—DO NOT CITE OR QUOTETable 4-69. Summary of reported developmental effects in formaldehyde inhalation exposure studiesReported study findingsbLOAEL/NOAELcSpecies,strain, sex n/GroupDose; time oftreatmentaMaternal Offspring Maternal OffspringReferenceRat,mongrel,white,female andoffspring ofboth sexes15/groupterminatedGD 20,6/grouplittered0, 0.0005, or0.005 mg/L (0, 0.4,or 4 ppm), GDs 1–19, 4 hours/dayAt 0.4 ppm:↓leukocytecountsAt 4 ppm:↓leukocytecounts; reducedthreshold ofneuromuscularexcitability,↓rectaltemperature,↓bloodhemoglobin;↑spontaneous mobilityAt 0.4 ppm:↑preimplantationloss; at 1 mo. of age,↓spontaneous mobility; at 2 mo.of age,↓hemoglobin levels andleukocyte countsAt 4 ppm:↑preimplantationloss; at 1 and 2 mo. of age,↓spontaneous mobility; at 2 mo.of age,↓hemoglobin levels andleukocyte countsL: 0.4 ppmN: NDL: 0.4 ppmN: NDSheveleva(1971)Rat,SpragueDawley,female andoffspring ofboth sexes6 dams 0 or 6 ppm8 hours/day,6 weeks, starting atGD 1, PND 1,4 weeks of age, oradult ageNE In offspring exposed in utero andduring early postnatal life:↓mean BW and liver weight;↑markers of oxidative stressIn offspring exposed asjuveniles:↓mean liver weight;↑markers of oxidative stressIn offspring exposed only asadults: no effectNE L: 6 ppmN: NDKum et al.(2007)Rat,SpragueDawley,female andoffspring ofboth sexes6 dams 0 or 6 ppm8 hours/day;6 weeks, starting atGD 1, PND 1,4 weeks of age, oradult ageNE In offspring exposed in earlypostnatal life, as juveniles, or asadults,↑ANAE-positive Tlymphocytes in BALTIn all exposure initiation groups,↑ANAE-positive lymphocyteratiosNE L: 6 ppmN: NDSandikci et al.(2007)This document is a draft for review purposes only a nd does not constitute Agency policy 4-316 DRAFT—DO NOT CITE OR QUOTETable 4-69. Summary of reported developmental effects in formaldehyde inhalation exposure studiesReported study findingsbLOAEL/NOAELcSpecies,strain, sex n/GroupDose; time oftreatmentaMaternal Offspring Maternal OffspringReferenceRat, Wistar,female andoffspring ofboth sexes12–14dams0, 6, or 12 ppm,6 hours/day;5 days/week,30 daysNE At 6 and 12 ppm, at postnataldays 30 and 90: respiratoryirritation and toxicity; decr. BW,FC, WC;↓SOD activity,↓levels of copper and iron levelsin lungs,↑zinc levels in lungsNE L: 6 ppmN: NDSongur et al.(2005)ND: not determined; NE: not evaluated; NR: not reported; Ab: abstract only; wt: weight; stat. sig.: statistically significant; p: pressure; l: length;To convert concentrations in air (at 25°C) from mg/m3to ppm: 1 ppm = 1.23 mg/m3; 1 mg/m3= 0.813 ppm.aTreatment is given as the formaldehyde concentration in air (ppm) with the length of exposure each dayand the duration of treatment in days, as available.bStudies with negative findings are included.cL: LOAEL; N: NOAEL.dExposure concentrations not validated; details of formaldehyde vapor generation not reported; exposureduring gestation not well characterized in study report.eNo statistics provided.fLack of study details.gSee Table 4-72 for reproductive effects.hGofmekler and Bonashevskaya (1969) seem to report on different findings from the same study (i.e., same animals) as Gofmekler (1968).iNumber/group not clear from study report.This document is a draft for review purposes only a nd does not constitute Agency policy 4-317 DRAFT—DO NOT CITE OR QUOTETable 4-70. Summary of reported developmental effects in formaldehyde oral exposure studiesReported developmental effectsaLOAEL/NOAELbSpecies,strain, sexn/GroupDose; time oftreatmentMaternal Offspring Maternal OffspringReferenceDog,beagle,female andpups9–10 0, 125, or375 ppm, dietary,GDs 4–56No effects At 125 and 375 ppm:↓birth wt and wt gainthrough postnatal week 8L: NDN: 375 ppmL: 125 ppmN: NDHurni andOhder (1973)Mouse,CD-1,female29–35total0, 74, 148, or185 mg/kg-day,GDs 6–15(aqueousformaldehydesolutioncontained 10–15% methanol)At 185 mg/kg-day:MortalityNo effects at GD 18 L: 185 mg/kg-dayN: 148 mg/kg-dayL: NDN: 185 mg/kg-dayMarks et al.(1980)Mouse,ICR/SIM,female26–30total0 or 540 mg/kgday, GDs 8–12At 540 mg/kg-day:MortalityNo effects in pups on PND 1and 3L: 540 mg/kg-dayN: NDL: NDN: 540 mg/kg-daySeidenberg andBecker (1987)ND: not determined.aStudies with negative findings are included.bL: LOAEL; N: NOAEL.This document is a draft for review purposes only a nd does not constitute Agency policy 4-318 DRAFT—DO NOT CITE OR QUOTETable 4-71. Summary of reported developmental effects in formaldehyde dermal exposure studiesReported developmental effects LOAEL/NOAELaSpecies,strain, sexn/GroupDose; time oftreatmentaMaternal Offspring Maternal OffspringReferenceHamster,Syriangolden ,female4–6 0 or 37%; 0.5 mLapplied to dorsal skin(hair clipped) for 2 hoursthen washed; GDs 8, 9,10, or 11Signs of dermal irritationand irritabilityAt all GDs of treatment,↑percent resorptions (not sig.)L: 37%N: NDL: 37%N: NDOverman (1986)ND, not determinedaL: LOAEL; N: NOAEL.This document is a draft for review purposes only a nd does not constitute Agency policy 4-319 DRAFT—DO NOT CITE OR QUOTETable 4-72. Summary of reported reproductive effects in formaldehyde inhalation studiesSpecies,strain, sexn/GroupDose; time of treatmentaReported reproductive effectsbLOAEL/NOAELcReferenceRat, strainNR, male4 Inhalation plus drinking waterco-exposure: 0; 0.10 ppm plus0.005 mg/L water; 0.20 ppmplus 0.01 mg/L water; or 0.41ppm plus 0.1 mg/L water; alltreatments; 4 hours/day,5 days/week for 6 months.Exposed males mated tounexposed femalesAt 0.20 ppm + 0.01 mg/L water and 0.41 ppm + 0.1 mg/L water:↓nucleic acid in testes (dose dep.; data not shown;stat. sig.)L: 0.20 ppm+ 0.01 mg/LwaterN: NDGuseva(1972)d,eRat,Wistar,femaleNR(200female)0, 0.4, or 1.2 ppm ;4 hours/day, 5 days/week for4 months.Exposed females mated tounexposed males on 120thdayof exposure.At 0.4 ppm:↑wt of ovaries (stat. sig.e)↑LH level in blood (stat. sig. at 0.4 ppmf)At 1.2 ppm:↑FSH level in blood (stat. sig., dose dep. trendf)L: 0.4 ppmhN: NDhKitaev et al.(1984)eMouse,B6C3F1,male andfemale10 0, 2, 4, 10, 20, or 40 ppm;6 hours/day, 5 days/weekfor 13 weeksMales and females:At 20 ppm:↓wt gainAt 40 ppm:↑mortality (13 weeks exp.);↓wt lossFemales:At 40 ppm:↑Uterine endometrial and ovarian hypoplasiaL: 20 ppmN: NDMaronpot etal. (1986)Rat, albinoWistar,male7 6 groups: 0, 10, or 20 ppm;8 hours/day, 5 days/week for4 weeks (subacute) or13 weeks (subchronic)At 10 and 20 ppm (both durations):↓wt gain (stat. sig., dose dep.)↓relative testes wt (stat. sig., dose and conc. dep.)↓zinc and copper in testes (stat. sig., dose and conc. dep.)↑iron in testes (stat. sig., dose and conc. dep.)No effect on testes wt.L: 10 ppmN: NDÖzen et al.(2002)fRat,Wistar,male18 0, 5, or 10 ppm; 8 hours/day,5 days/week, 91 daysAt 5 and 10 ppm:clinical signs of respiratory irritation,↓BW, FC, WC;↓serumtestosterone;↓mean seminiferous tubule diameters;↑localizationof heat shock protein 70 in cytoplasm of spermatogonia,spermatocytes, and spermatidsL: 5 ppmN: NDÖzen et al.(2005)This document is a draft for review purposes only a nd does not constitute Agency policy 4-320 DRAFT—DO NOT CITE OR QUOTETable 4-72. Summary of reported reproductive effects in formaldehyde inhalation studiesSpecies,strain, sexn/GroupDose; time of treatmentaReported reproductive effectsbLOAEL/NOAELcReferenceRat, albinoWistar,male10 0, 10, or 20 ppm8 hours/day, 5 days/week for4 weeksDose NR:Irritation: standing hair, interrupted breathing,↑eye blinking,licking, nose cleaning, and sneezing.At 10 and 20 ppm:↓Body wt gain (dose dep.; stat. sig.)↓Leydig cell quantity (stat. sig.)↑Nuclear damage of Leydig cells (dose dep.; stat. sig.)L: 10 ppmN: NDSarsilmaz etal. (1999)Mouse,strain notspecified,male12 0, 21, 42, or 84 mg/m3(0,16.9, 33.8, or 67.6 ppm);2 hours/day, 6 days/week;13 weeks. Exposed malesmated to unexposed females.In all treated groups:↑percentage abnormal sperm,↑resorptionrate, and↓live fetusesL: 16.9 ppmN: NDXing et al.(2007)Rat,Wistar,male7 0 or 1.5 ppm, 18 weeksFA exposures:(1) 4 hours/day, 4 days/week(2) 2 hours/day; 4 days/week(3) 2 hours/day, 4 days/weekIn all treated groups: sig.↓seminiferous tubular diameter andepithelial height. Other effects in exposure groups:(1) sig.↓germ cells; arrested spermatogenesis(2)↓cells, increased thickness in basal membrane(3)↑spaces between germ cells; disrupted association betweenSertoli and germinal cellsL: 1.5 ppmN: NDGolalipour etal. (2007)Rat,SpragueDawley,male10 (1) 0 (gavage saline);(2) 10 mg/m3(8.05 ppm),12 hours/day, 2 weeks; or(3) 10 mg/m3(8.05 ppm),12 hours/day, 2 weeks, plus30 mg/kg-day vitamin E orallyAt 10 mg/m3: sig.↓testis weight, atrophy of seminiferous tubules,↓spermatogenic cells, disintegrated and sloughed seminiferouscells; edematous interstitial tissue with vasculardilatation andhyperemia;↓epididymal sperm count and percentage motilesperm,↑percentage abnormal sperm;↓SOD, GSH-Px, GSH and↑MDA in testes; vitamin E attenuated all effectsL: 8.05 ppmN: NDZhou et al.(2006)Rat,Wistar,male40 0, 0.1, 1, or 10 ppm;6 hours/day, 5 days/week, 13or 52 weeksNo effects: testis weight; histopathologic findingsgL: NDN: 10 ppmAppleman etal. (1986)aTreatment is given as the formaldehyde concentration in air (ppm) with the length of exposure each dayand the duration of treatment in days.bStudies with negative findings are included.cL: LOAEL; N: NOAEL.dGuseva (1972) was a drinking water and inhalation study.eDevelopmental effects shown in Table 4-69.fStatistics not provided in study report.This document is a draft for review purposes only a nd does not constitute Agency policy 4-321 DRAFT—DO NOT CITE OR QUOTEgFocus of study was not the reproductive system; only reproductive system findings are addressed in thetable; NOAEL and LOAEL in table are based only onreproductive system findings.hFor increased FSH, the LOAEL was 1.2 ppm (1.5 mg/m3) and the NOAEL was 0.4 ppm (0.5 mg/m3).ND: not determined; NR: not reported.To convert concentrations in air (at 25°C) from mg/m3to ppm: 1 ppm = 1.23 mg/m3; 1 mg/m3= 0.813 ppm.This document is a draft for review purposes only a nd does not constitute Agency policy 4-322 DRAFT—DO NOT CITE OR QUOTETable 4-73. Summary of reported reproductive effects in formaldehyde oral studiesSpecies,strain, sexn/GroupDose; time of treatment Reported reproductive effectsaLOAEL/NOAELbReferenceRat,Wistar,male5 (20control)0, 100, or 200 mg/kg, singlegavage doseAt 200 mg/kg:↑(19%) testicular sperm head counts (stat. sig.)↑(5%) abnormal sperm head (stat. sig.)L: 200 mg/kgN: 100 mg/kgCassidy et al.(1983)Rat,SpragueDawley,both sexes15 0, 50, 100, or 150 mg/kg-day,drinking water; 91 daysNo effects: absolute or relative gonad weights;histopathologic findings of reproductive organscL: NDN: 150 mg/kg-dayDog,beagle,both sexes4 0, 50, 75, or 100 mg/kg-day,dietary; 91 daysNo effects: absolute or relative gonad weights;histopathologic findings of reproductive organscL: NDN: 100 mg/kg-dayJohanssen etal. (1986)Rat,Wistar,both sexes10 0, 25, or 120 mg/kg-day,drinking water, 4 weeksNo effects: gonad weights; histopathologic findingsofreproductive organscL: NDN: 120 mg/kg-dayTil et al.(1988)Rat,Wistar,both sexes70 0, 1.2, 15, or 82 mg/kg-day(males), 0, 1.8, 21, or109 mg/kg-day (females),ddrinking water; 105 weeksIn all treated groups:Leydig cell tumors observed at 105 weeks of studycAt 82 mg/kg-day:↑mean testes weightsL: 1.2 mg/kg-dayN: NDTil et al.(1989)Rat,Wistar,both sexes20 0, 0.02, 0.1, or 0.5% indrinking water; 24 monthsNo effects: gonad weights; histopathologic findingsofreproductive organscL: NDN: 0.5%Tobe et al.(1989)ND, not determined.aStudies with negative findings are included.bL: LOAEL; N: NOAEL.cFocus of study was not the reproductive system; only reproductive system findings are addressed in thetable; NOAEL and LOAEL in table are based only onreproductive system findings.dActual concentrations.This document is a draft for review purposes only a nd does not constitute Agency policy 4-323 DRAFT—DO NOT CITE OR QUOTETable 4-74. Summary of reported reproductive effects in formaldehyde intraperitoneal studiesSpecies,strain, sexn/Group Dose; time of treatmentaReported reproductive effectsaLOAEL/NOAELbReferenceRat,Charlesfoster,male10 0 or 5 mg/kg-day; 30 days↓body weight gain↓Leydig cell population and cell nuclear diameter↓serum T levels↓testes weightscellular degeneration of seminiferous tubulesL: 5 mg/kg-dayN: NDChowdhury etal. (1992)Rat,Wistar,male8 0 or 10 mg/kg-day; 30 days↓sperm count, motility and sperm viability L: 10 mg/kg-dayN: NDMajumder andKumar (1995)Rat,“albino”strain NR,male6 0, 0.125, 0.250, or0.60 mg/kg-day; 5 daysAt all treatment levels:↓sperm count and↑sperm head abnormalities (3 weeksafter the last injection)L: 0.125 mg/kg-dayN: NDRat,“albino”strain NR,male12 0, 0.125, 0.250, or 0.60mg/kg-day; 5 days. Exposedmales mated to unexposedfemales.At all treatment levels (at GD 13):Delayed time to mating↓mean no. implants and live embryos↑dead implants and dominant lethal index followingmating to untreated femalesL: 0.125 mg/kg-dayN: NDOdeigah(1997)aStudies with negative findings are included.bL: LOAEL; N: NOAEL.ND: not determined; NR: not reported; T: testosterone.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-324 4.2.2. Carcinogenic Potential: Animal Bioassays 1 Chronic animal studies (inhalation and oral exposur es) chronicle tumor incidence in a 2 variety of rodent models. Study descriptions are p rovided above in detail (Section 4.2.1, 3 Table 4-34). The study results are evaluated here for both routes of exposure in context of how 4 they inform the carcinogenic potential for the thre e major affected systems: respiratory tract, GI 5 tract, and LHP system. Experimental design and imp lementation must be carefully considered 6 when interpreting study results. For example, some key factors involved in evaluating cancer 7 bioassays include study size, organs/tissues examin ed, and study length (especially for late-in8 life tumors). 9

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4.2.2.1. Respiratory Tract 11 In the respiratory tract, only nasal tumors are con sidered formaldehyde induced in rodent 12 studies. The majority of studies were conducted us ing rats (F344, Wistar, or Sprague-Dawley), 13 and all studies of 18 months or greater in mice and rats show evidence of formaldehyde-induced 14 nasal carcinogenicity. The nasal tumors are primar ily SCCs, although papillomas, polypoid 15 adenoma, adenocarcinoma, fibrosarcoma, and esthesio neuroepithelioma have been reported 16 (Kamata et al., 1997; Monticello et al., 1996; Morg an et al., 1986a, b; Takahashi et al., 1986; 17 Sellakumar et al., 1985; Kerns et al., 1983; Albert et al., 1982). Although hyperplasia, dysplasia, 18 and squamous metaplasia of the respiratory epitheli um have been observed beyond the nasal 19 cavity, other respiratory tract tumors have not bee n significantly increased by formaldehyde 20 exposure alone. 21 Increased tumor incidence and decreased latency are correlated with increasing 22 formaldehyde exposure concentration. Reviewing dat a from the only lifelong inhalation study 23 with multiple exposure groups, SCC is first noted a t 8 and 9 months for high exposed (15 ppm) 24 female and male F344 rats autopsied as “early death s” prior to the 12 month sacrifice, with an 25 incidence of 43% over the course of the study (unad justed for mortality) (Kerns et al., 1983). In 26 contrast only two SCCs were found in male and femal e rats sacrificed after 24 months of 27 exposure (incidence of SCC 2.5% at 24 months) (Kern s et al., 1983). In a follow-up study by 28 Monticello et al. (1996), the incidence of SCC in r ats exposed at 15 ppm was 47% with the first 29

tumor noted at 12 months. The incidence of SCC in male rats exposed at 10 ppm was 22% with 30 the first SCC noted at 18 months. Moreover, of 90 rats exposed at 6 ppm for 20 months only one 31 SCC was noted. No SCCs were detected in rats expos ed at 0.7 or 2 ppm formaldehyde. These 32 incidence rates are not mortality adjusted and incl ude animals from each scheduled sacrifice (3, 33 6, 12, and 18 months). In a lifelong study of male Sprague-Dawley rats exposed at 10 ppm 34 formaldehyde, the cumulative nasal tumor incidence was calculated as a function of time of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-325 exposure (Figure 4-27) (Sellakumar et al., 1985). After 2 years of exposure, the probability of 1 nasal carcinoma was greater than 60%. 2

3

4 5 Figure 4-27. Mortality corrected cumulative incide nces of nasal carcinomas 6 in the indicated exposure groups. 7 8 Source: Sellakumar et al. (1985). 9 10 There is some evidence that less-than-lifetime expo sure to formaldehyde can induce nasal 11 tumors over an extended observation period. Two st udies, both in male Wistar rats, report nasal 12 tumors in response to less-than-lifetime exposures (Woutersen et al., 1989; Feron et al., 1988). 13 A 13-week exposure at 20 ppm resulted in four nasal tumors (three SCCs), a cystic SCC of the 14 nasolacrimal duct, and an epithelial tumor on the m andible, for a total of six tumors observed 15 over 30 months of observation (Feron et al., 1988). No tumors were noted in 13-week controls. 16 A limited number of formaldehyde-related tumors wer e noted due to 4 or 8 weeks of exposure 17 followed by 30 months of observation. Although the tumor incidence of these less-than-lifetime 18 exposures is low, this is consistent with the 2-yea r bioassays in Wistar rats. Wistar rats are more 19 resilient to formaldehyde-induced nasal toxicity th an F344 or SD rats (Section 4.2.1), and only 1 20 of 26 (4%) Wistar rats exposed at 10 ppm for 28 mon ths developed SCC (Woutersen et al., 21 1989) versus 22% in F344 rats (Monticello et al., 1 996). 22 Woutersen et al. (1989) also examined the effect of severe nasal damage from 23 electrocoagulation on formaldehyde-induced SCC in W istar rats. Nasal tumors were noted in 24 formaldehyde-exposed rats without damaged noses (ex posed for only 3 months and observed for 25This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-326 25 months). However, the low incidence of tumors i n each treatment group (1/26, 2/60, 2/60, 1 1/58) indicates these data should be considered sug gestive even though no SCCs were noted in 2 control rats with or without damaged noses (n = 83) . The studies by Woutersen et al. (1989) did 3 demonstrate a synergistic effect of nasal damage fr om electrocoagulation and 10 ppm 4 formaldehyde exposure (3 months), where 15/58 rats had SCC versus 1/26 with undamaged 5 noses. The study was originally designed to examin e the effect of formaldehyde on the damaged 6 tissue on cancer promotion. However, it is unclear if the synergistic effect of formaldehyde 7 exposure on damaged nasal tissue is an effect of fo rmaldehyde on the damaged cells and joint 8 effects of a mutagen with regenerative proliferatio n from the nasal damage. It is also possible 9 the damaged nasal passages may alter airflow in the nasal passages, resulting in significantly 10 different flux of formaldehyde into the tissue. 11 There is a single inhalation study (Dalbey, 1982) t hat investigates the role of promotion 12 in formaldehyde-induced cancer. Although hamsters exhibit little to no effects of formaldehyde 13 on the nasal mucosa or other respiratory tract tiss ues (Rusch et al., 1983a, b; Dalbey, 1982), 14 DEN-induced respiratory adenomas were increased wit h formaldehyde exposure (10 ppm) 15 48 hours prior to DEN injection (but not by formald ehyde alone or formaldehyde exposure after 16 DEN injection). The number of tracheal tumors per TBA was doubled by formaldehyde 17 exposure. The study authors note that adenomas sho uld be considered independent tumors and 18 that the increase in tracheal tumors is of biologic al significance even given the incidence of 19 TBAs (77%, DEN alone), was not further increased by formaldehyde exposure. It is of 20 particular interest that a promotion study in hamst ers is positive, since so little nasal pathology 21 occurs with formaldehyde exposure. The absence of significant hyperplasia and tissue damage 22 in these animals suggests that formaldehyde may ind uce subtle changes in the respiratory tract 23 mucosa that permit formaldehyde to act as a tumor p romoter. 24

25

4.2.2.2. Gastrointestinal Tract 26 As with the respiratory tract, the proximal portion of the GI tract exhibits formaldehyde27 induced lesions in the forestomach and glandular st omach (Soffritti et al., 1989; Til et al., 1989; 28 Tobe et al., 1989; Takahashi et al., 1986). Howeve r, data are mixed regarding the carcinogenic 29 potential of formaldehyde in the GI tract from oral exposures. 30 Two independent 2-year cancer bioassays in Wistar r ats exposed to formaldehyde in 31 drinking water were both negative; they reported no tumors found at the 24-month sacrifice (Til 32 et al., 1989; Tobe et al., 1989). Til et al. (1989 ) exposed rats to a range of formaldehyde doses 33 (0, 1.2, 15, or 82 mg/kg-day) and evaluated 44–49 a nimals per sex per dose group at 24 months 34 of exposure. No formaldehyde-related tumors were f ound. A smaller study by Tobe et al. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-327 (1989) failed to note any tumors after a 2-year exp osure at 0, 10, 50 or 300 mg/kg-day (eight rats 1 per sex per treatment group). 2 In contrast, two lifelong studies in male and femal e Sprague-Dawley rats provide support 3 for formaldehyde-induced GI tract tumors (Soffritti et al., 1989). Both stomach and intestinal 4 tumors are rare; low background rates are expected in this colony of Sprague-Dawley rats. 5 These studies demonstrate an increase in tumors (al though rare) correlated with exposure to 6 formaldehyde and significantly increased susceptibi lity to early-lifetime exposure. The authors 7 provide a detailed report of the background rates o f various stomach and intestinal neoplasia for 8 male (n = 2,677) and female (n = 2,582) rats (Soffr itti et al., 1989). From this background pool, 9 the total incidence of benign and malignant tumors in the stomach and intestine combined is only 10 1.4% (combining all sites and locations). The majo rity of tumors are located in the stomach (1% 11 benign, 0.2% malignant). Usually, a very large stu dy population is needed to detect increases in 12 rare tumor types. In this study, the study size of each treatment group was relatively small. 13 Thus, only a few TBAs are responsible for the obser ved increases. Additionally, a clear dose14 response relationship is not evident (perhaps due t o the low incidence) despite the fact that the 15 greatest tumor incidence was observed in the highes t treatment group. As presented above, 16 apparent increases in both stomach and intestinal n eoplasia are noted in formaldehyde-treated 17 rats (ranging from 1 to 6% by type). When summed a cross the GI tract, tumor incidence in the 18 highest treatment group was 8% versus 1.4% in histo rical controls. Despite the limitations of 19 group size and lack of dose response, the findings do support the carcinogenic potential for 20 formaldehyde administered orally. Moreover, these findings are not inconsistent from Tobe et 21 al. (1989) and Til et al. (1989) because the study design is significantly different. 22 The second study reported by Soffritti et al. (1989 ) demonstrates early lifetime 23 susceptibility with GI tumor incidence of 21.6% in females (n = 37) and 13.9% in males (n = 36) 24 after exposure to formaldehyde. Sprague-Dawley rat s were exposed to formaldehyde in drinking 25 water for 2 years (0 or 2,500 mg/L). Exposures beg an on GD 12 in the offspring. The most 26 common tumor detected was intestinal leiomyosarcoma (13.5% in female offspring) with a 27 background rate of 0.04% in female rats in the colo ny. 28 Soffritti et al. (1989) stands alone in supporting formaldehyde-induced GI tumors. These 29 findings are largely attributed to a unique study d esign that included lifelong observation, 30 neonatal exposure, examination of individual tumor types as well as combined rare tumor types 31 for analysis, and summation of tumors across locati ons. The study design results in a more 32 sensitive assay for rare tumors. Thus, Soffritti e t al. (1989) utilized a more appropriate design 33 and analysis for detecting rare tumors and should n ot be compared with the results by Tobe et al. 34 (1989) and Til et al. (1989). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-328 There is evidence that formaldehyde may act as a tu mor promoter by the oral route as 1 well as the inhalation route (discussed above). Ta kahashi et al. (1986) reported an increase in 2 MNNG-initiated GI cancers with formaldehyde exposur e (29.4 versus 13.3% TBA in controls); 3 the greatest difference in tumor-containing versus non-tumorigenic mice was associated with 4 adenocarcinoma in the glandular stomach (23.5 versu s 3.3% in controls). Additionally, 5 forestomach papillomas and preneoplastic hyperplasi a in the glandular stomach were increased 6 with formaldehyde exposure alone. 7 The data indicate carcinogenic potential from forma ldehyde ingestion in drinking water. 8 Formaldehyde may act in part as a tumor-promoting a gent and shows clear increased 9 susceptibility from early lifetime exposures. 10

11

4.2.2.3. Lymphohematopoietic Cancer 12 The majority of chronic animal bioassays do not rep ort either leukemia or lymphoma, but 13 many of these studies did not have adequate study l ength or study design to detect these 14 malignancies. Many studies focused the histopathol ogy on the nasal passages and respiratory 15 tract (Monticello et al., 1996; Holmström et al., 1 989a; Woutersen et al., 1989; Appleman et al., 16 1988; Dalbey, 1982; Horten et al., 1963). Kamata e t al. (1997) did examine additional organs, 17 but there were only five animals at each sacrifice. Similarly, the oral study by Takahashi et al. 18 (1989) focused on the stomach and intestines. Tobe et al. (1989) only included 20 Wistar rats 19 per group with interim sacrifices. Therefore, few studies can inform the carcinogenic potential 20 of formaldehyde on the LHP system. Table 4-75 list s the chronic bioassays that have the 21 potential to detect LHP malignancies. 22 Soffritti et al. (1989) first published an observat ion of formaldehyde-induced leukemia in 23 animal studies. These study results have been crit icized for their combination of lymphatic 24 leukemia and lymphoma. However, this classificatio n is consistent with the current WHO 25 classification of lymphoid malignancies in humans w here adult B- and T-cell leukemias and 26 lymphomas are considered the same disease (Harris, 2000a). Although there may be a slight 27 vehicle effect, a dose response is still readily ap parent among the formaldehyde-treated groups 28 (Figure 4-28). In contrast, the 2-year bioassay in Wistar male in female rats (Til et al., 1989) was 29 clearly negative for leukemia and lymphoma with onl y four TBAs in all treatment groups 30 sacrificed at 24 months. Moreover, the drinking wa ter levels were similar at the highest dose of 31 both Til et al. (1989) and Soffritti et al. (1989). However, the two study designs differ in length, 32 which may have influenced results since leukemia is a late-in-life malignancy in rodents. Two33 year survival in the Soffritti et al. (1989) study varied between 50 and 60%. These animals were 34 available to develop leukemia after the 2-year wind ow of the Til et al. (1989) study. Any 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-329 potential role of strain differences is unknown. O verall the results of Soffritti et al. (1989) are 1 strong since they indicate an exposure-response rel ationship in a lifelong study appropriate for 2 late-in-life malignancies. Unlike the GI tract tum ors, increased LHP malignancies were not 3 associated with early-life exposure to formaldehyde in drinking water. 4

5 Table 4-75. Summary of chronic bioassays which add ress rodent leukemia 6 and lymphoma 7 8 Study Histopathology Endpoint Results Comments Drinking water exposure Male and female Sprague-Dawley rats Soffritti et al. (1986) Complete histopathology Lymphocytic leukemia and lymphosarcoma Increased, showing

a dose-response Lifelong study

High exposure of 1,500 mg/L in water Male and female Wistar Rats Til et al. (1989) Complete histopathology

in control and high-dose group (15 ppm) Lymphoma, leukemia No increase

(three lymphomas and one leukemia found in 200 animals at the 2-year sacrifice) 2-year bioassay

High exposure of approximately 1,900 mg/L (82 mg/kg for males and 109 mg/kg for females)

Inhalation exposures Male rats, Sprague-Dawley Sellakumar et al. (1985); Albert et al. (1982) Necropsy focused on

respiratory tract: also liver, spleen, kidney, and testes and organs demonstrating gross pathology Lymphoma No increase Lifelong study, high mortality at 24 months (>80%)

Male rats, F344 Complete histopathology in controls and high-dose group (15 ppm) Leukemia, all No increase Extended study, high mortality

Female rats, F344 Complete histopathology in controls and high-dose group (15 ppm) Leukemia, all Increase in mortality-adjusted incidence;

p = 0.0056 a Extended study, high

mortality. Apparent elevation in 2 and 6 ppm treatment groups as well; statistical comparison to controls is problematic. Female mice, C57BL/6xC3HF1 Battelle, Columbus Laboratories (1981)

All organs in controls and

high-dose group (15 ppm) Lymphoma, all 26% increase in 15 ppm group, 16% in controls;

p = 0.0617 Extended study.

All mice included in statistics conducted by BattelleColumbus Laboratories. 9

a Original statistical analysis provided by Battelle Columbus Laboratories. Significance set at p < 0.0167. Analysis 10 of adjusted data where time to lesion and survivors hip were considered (Cox [1972] and Tarone [1975]). 11This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-330 Male and Female Sprague-Dawley Rats Exposed to

Formaldehyde in Drinking Water

0 5 10 15 20 25

0 10 50 100 500 1000 1500 Vehicle

Exposure (mg/l formaldehyde)All Leukemia (%) All

Female

Male

1

2 Figure 4-28. Leukemia incidence in Sprague-Dawley rats exposed to 3 formaldehyde in drinking water for 2 years. 4 5 Note: Animals were observed until natural death. T he vehicle control contained 6 the level of methanol in drinking water for the hig h-dose group (1,500 mg/L). 7 8 Source: Soffritti et al. (1989). 9 10 11 Sellakumar et al. (1985) conducted a lifelong inha lation study in male Sprague-Dawley 12 rats exposed at 10 ppm formaldehyde. Organs outsid e of the respiratory tract were routinely 13 examined (liver, kidneys, and testes), including an y organ exhibiting gross pathology, so there 14 was some ability to detect leukemia and lymphoma. However, spleen, thymus, and lymph nodes 15 were not routinely examined, limiting detection of smaller lesions. Although Sellakumar et al. 16 (1985) was a lifelong study, there was a high morta lity rate at 2 years (>80% from the figure), 17 again limiting the power of this study to detect la te-in-life malignancies. Nonetheless, this study 18 did not indicate formaldehyde-induced lymphoma or l eukemia. 19 The largest and most comprehensive study of carcin ogenic health effects from 20 formaldehyde inhalation exposures is the study cond ucted at the Battelle Columbus Laboratory 21 (1981) and reported by Kerns et al. (1980) and Swen berg et al. (1983). Although the summary 22 reports of this study do not discuss leukemia or ly mphoma rates, mouse lymphoma and rat 23 leukemia were selected by the study pathologist and biostatistician for analysis (Battelle 24 Columbus Laboratories, 1981). Statistical analysis performed by Battelle, which accounted for 25This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-331 time to lesion and survivorship rates, indicated a statistically significant increase in female rat 1 leukemia (

p = 0.0003) and near significant increase in female mouse lymphoma ( p = 0.06). No 2 trend analysis could be conducted since only gross pathology was conducted on mid-dose mice 3 and rats (2 and 6 ppm). EPA has further analyzed t hese data to better understand the significance 4 of these findings. 5 As noted in the study description (Section 4.2.1.2. 3), both male and female rats at the 6 highest exposure (15 ppm) exhibited significant ear ly deaths due to nasal lesions (see Figure 7 4-29). No female rats in the highest exposure grou p remained after the 24-month sacrifice 8 (which included only 14 animals). Nine male rats w ere examined after 24 months. Since the 9 first leukemia in rats was noted at 21 months, the early deaths prior to that time reduced the 10

number of animals in which the leukemia could have been observed. Unadjusted data do not 11 show an increase in female rat leukemia, but, when data are adjusted to account for the early 12 deaths, dramatically different results are apparent . In the unadjusted data, leukemia incidence is 13 expressed as the number of cases over total animals examined (including early deaths and 14 interim sacrifices). By using this methodology, th ere is a lower incidence of leukemia in high 15 exposed animals, and a slightly higher incidence of leukemia in the mid-exposed groups 16 compared with controls (Figure 4-30, panel A). How ever, when the leukemia incidence rates are 17 calculated only for female rats that survived to at least 21 months (the first case noted), 18 formaldehyde-induced increases in leukemia are evid ent in all treatment groups relative to 19 controls (Figure 4-30, panel B). These results are consistent with the original statistical analysis 20 conducted by the researchers at the Battelle Columb us Laboratories (1981). Although elevated 21 in all treatment groups, no exposure-response relat ionship is evident. The lack of a dose22 response relationship may in part be due to the fac t that no animals in the high-exposure group 23 survived past 24 months. Additionally, the 6 ppm ( mid-exposure) group had significant early 24 deaths between 21 to 24 months compared with the 2 ppm and control groups. A similar 25 reanalysis of data from male rats did not reveal an y relationship between formaldehyde treatment 26

and leukemia incidence. 27 Male and female mice exposed to formaldehyde for 24 months did not experience the 28 same rate of formaldehyde-related mortality (Kerns et al., 1983). However, significant early 29 deaths were observed in male mice due to infighting . Therefore, the data for male mice may not 30 inform incidence of late-in-life tumor, such as lym phoma. As discussed above in the full study 31 description, full histopathology, including spleen, liver, thymus, and lymph nodes, was only done 32 on the control and high-exposure group (15 ppm) mic e. When comparing unadjusted incidence 33 lymphoma-bearing mice, there is a clear elevation l ymphoma in formaldehyde-exposed female 34 mice (28%) over controls (22%) (Figure 4-31). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-332 18 mos 18 to 21 21 to 24 > 24 0 ppm 2 ppm 6 ppm 15 ppm

0 5 10 15 20 25

1 2 Figure 4-29. Unscheduled deaths in female F344 rat s exposed to 3 formaldehyde for 24 months. 4 5 Source: Battelle Columbus Laboratories (1981). 6 7This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-333 1 Panel A: Unadjusted Data Panel B: Adjusted Data 2 3

0% 5% 10% 15% 20% 25% 30%

18 mo 21 Mo 24 mo 27 mo 30 Mo

0 ppm 2 ppm 6 ppm 15 ppm 0% 5% 10% 15% 20% 25% 30%

21 mos 24 mos 27 mos 30 mos

0 ppm 2 ppm 6 ppm 15 ppm 4 5 Figure 4-30. Cumulative leukemia incidence in fema le F344 rats exposed to 6 formaldehyde for 24 months. 7 8 Note: Panel A shows the unadjusted data where incid ence rates include all 9 scheduled sacrifices and early deaths up to the tim e point shown. Panel B shows 10 incidence of leukemia only in rats who survived at least 21 months. 11 12 Source: Battelle Columbus Laboratories (1981). 13 14

15

16This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-334 0% 1% 9% 18%

1% 9% 22% 28%

0% 5% 10% 15% 20% 25% 30%



### 12 Mo 18 Mo 24 Mo 30 Mo

Control 15 ppm

1

2 Figure 4-31. Cumulative incidence of tumor bearing animals for lymphoma 3 in female mice exposed to formaldehyde for 24 month s. 4 5 Note: Mice from the 6-month interim sacrifice are n ot included since only nasal 6 passages were examined ( p < 0.05). 7 8 Source: Battelle Columbus Laboratories (1981). 9 10 11 Although results are somewhat mixed between studies , there is evidence in the animal 12 bioassays for formaldehyde-induced LHP malignancies . Differences in study design may 13 account in part for mixed results. The lifelong dr inking water study by Soffritti et al. (1989) may 14 have allowed for malignancies to develop late in li fe, whereas the drinking water study by Til et 15 al. (1989) sacrificed all animals at 24 months. Ev en though the exposure levels were similar, the 16 studies are not directly comparable. Similarly it is hard to directly compare results from the two 17 major inhalation studies in rats. Although Sellaku mar et al. (1985) is a lifelong study, the 18 mortality for rats was greater than 80% at 2 years. Additionally, the pathology examination was 19 much less rigorous than in the Battelle Columbus La boratories (1981) study, perhaps missing 20 smaller lesions. Therefore, the increase in formal dehyde-induced leukemia seen in female F344 21 rats late-in-life (Battelle Columbus Laboratories [ 1981]) may reflect a more sensitive study 22 design. Finally, strain differences may account fo r different susceptibility as well. The two 23 positive rat studies, by different routes of exposu re, along with a positive result for 24This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-335 formaldehyde-induced mouse lymphoma, make a substan tive case for the potential of 1 formaldehyde-induced LHP malignancies. 2

3

4.2.2.4. Summary 4 Formaldehyde is toxic at the POE. Similar lesions, including increased cell proliferation, 5 DPX, and focal lesions, are noted in the GI tract o r URT, depending on route of exposure. 6 Similarly, formaldehyde-induced tumors are noted at the POE for both routes of exposure. 7 Additionally, data exist for both routes of exposur e to indicate that formaldehyde may act in part 8

as a tumor promoter. 9 When evaluating the studies with adequate study des ign to assess LHP malignancies, 10 results are mixed by strain, sex, route of exposure , and length of study. The three positive 11 studies (Soffritti et al., 1986; Battelle Columbus Laboratories, 1981) had the best histopathologic 12 examinations and greater sensitivity for detection of late-in-life tumors. Based on these results, 13

sufficient evidence is available in animal studies to support formaldehyde-induced LHP 14 malignancies. 15

16 4.3. GENOTOXICITY 17 Formaldehyde has been extensively studied for its m utagenic and genotoxic activity in a 18 variety of assay systems. The first reported mutag enic activity of formaldehyde was when 19 Rapoport (1946) described the induction of sex-link ed recessive lethals in drosophila larvae fed 20 on a medium containing formalin. A variety of geno toxic and mutagenic effects have been 21 subsequently demonstrated in both in vitro and in v ivo test systems, including the formation of 22 DPXs, point mutations, DNA strand breaks, increased MNs, and CAs (Auerbach et al 1977; Ma 23 and Harris, 1988; Conaway et al 1996; IARC 1995; 20 06). 24 In this section, reactions of formaldehyde with cel lular macromolecules, such as DNA 25 and proteins, and formaldehyde-induced clastogenici ty are described. In addition, the evidence 26 for formaldehyde-induced mutations is considered in the context of the current EPA cancer 27 guidelines (U.S. EPA, 2005a). Particular emphasis is given to the genotoxic effects of 28 formaldehyde in humans, described in Section 4.3.4. 2. 29

30 4.3.1. Formaldehyde-DNA Reactions 31 Formaldehyde is a reactive chemical and interacts w ith DNA in several ways, forming 32 DPXs, DNA adducts, and DNA-DNA cross-links (DDXs) ( Fennell, 1994; Casanova et al., 1989; 33 Heck and Casanova, 1987; Casanova-Schmitz et al., 1984a, b; Casanova-Schmitz and Heck, 34 1983; Ohba et al., 1979; Dönecke, 1978; Brutlag et al., 1969). Formaldehyde also may facilitate 35 the formation of adducts between other chemicals (e ndogenous or xenobiotic) and DNA 36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-336 (Fennell, 1994; Koppel et al., 1991; Casanova et al ., 1989; Heck and Casanova, 1987; Lam et al., 1 1985; Casanova-Schmitz et al., 1984a; Casanova-Schm itz and Heck, 1983; Ohba et al., 1979; 2 Dönecke, 1978; Brutlag et al., 1969). 3 The high reactivity of formaldehyde results in litt le specificity in reaction sites, indicating 4 that a range of adducts and cross-links might be ex pected. However, the spectrum of 5 formaldehyde-DNA reaction products is difficult to quantify in vivo as many of these are labile 6 and difficult to measure directly (Fennell, 1994; C asanova et al., 1989). Additionally, 7 formaldehyde is metabolically incorporated into nuc leic acids, and therefore DNA and RNA 8 assays incorporating radiolabeled formaldehyde need careful interpretation to distinguish 9 between covalently bound and metabolically incorpor ated formaldehyde (Casanova et al., 1989; 10 Heck and Casanova, 1987; Casanova-Schmitz et al., 1984a, b; Casanova-Schmitz and Heck, 11 1983). Hence, reports of formaldehyde-DNA reactivi ty in cell-free system results may not 12 provide a useful measure of exposure (Fennell, 1994 ). Besides, the question of biological 13

relevance must also be considered. On the other ha nd, methods used to extract and measure 14 DNA-formaldehyde reaction products after in vivo ex posures should be evaluated to ensure that 15 formaldehyde reaction products are neither created nor removed during sample preparation 16 (Fennell, 1994; Casanova et al., 1989). 17

18 4.3.1.1. DNA-Protein Cross-Links (DPXs) 19 Evidence from numerous experimental models, ranging from cell-free systems to single 20 cells and in vivo animal and human exposures, sugge sts that formaldehyde reacts readily with 21 DNA forming DPXs (Reviewed in Conaway et al 1996; I ARC 2006). As shown in Table 4-76, 22

cross-links between histones and DNA have been demo nstrated in isolated chromatin samples on 23 exposure to formaldehyde from earlier studies (Ohba et al., 1979; Dönecke, 1978; Brutlag et al., 24 1969). Several in vitro studies demonstrated induc tion of DPX by formaldehyde exposure in 25 bacteria (Wilkins and McLeod 1976), yeast (Magana-S chwencke and Ekert, 1978) and 26 mammalian cells including animal cells (Chinese ham ster ovary cells, Chinese hamster V79 lung 27 epithelial cells, mouse leukemia L1210 cells, mous e hepatocytes, rat Yoshida lymphsarcoma 28 cells, rat C18 tracheal epithelial cells, rat hepat ocytes, rat nasal, tracheal epitheial cells and aor tic 29 endothelial cells) and human cells (lung and bronch ial epithelial cells, fibrobasts, white blood 30 cells, peripheral blood lymphocytes, Epstein-Barr V irus-Burkitt’s lymphoma cells, Jurkat E6-1 31 cells, HeLa cells, lymphoblastoid cells, gastric mu cosal cells and whole blood) as summarized in 32

Table 4-76. 33 Ross and Shipley (1980) showed that formaldehyde in duces SSBs and DPXs; SSBs are 34 formed at concentrations >200 μM and a reduction of radiation-induced breaks (indirect measure 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-337 of DPXs) at 50 μM. The authors used a [ 14 C]-thymidine-incorporated mouse L1210 cell line to 1 monitor formaldehyde-induced DNA strand breaks and DPXs. They exposed cells to varying 2 concentrations of formaldehyde for 2.5 hours. An a lkaline-elution technique in the presence or 3 absence of proteinase K was used to measure strand breaks. In order to detect DPXs, some cells 4 were exposed to 300 R of X-rays immediately after formaldehyde treatment. Formaldehyde5 induced DPXs were repaired 24 hours after the compo und was removed from the culture (Ross 6 and Shipley, 1980). 7 Casanova-Schmitz and Heck (1983) have shown that ho mogenates of rat nasal mucosa 8 incubated with formaldehyde in vitro followed by ex traction with a strong aqueous-immiscible 9 organic solvent demonstrated increased DPX formatio n in DNA obtained after enzymatic 10 proteolysis from the aqueous-organic interface, ter med as “interfacial DNA”. In the same study, 11 they have shown that DNA isolated from the nasal, b ut not olfactory, mucosa of rats exposed to 12 formaldehyde (2, 6, 15, and 30 ppm 6 hours/day for 2 days) via inhalation showed significant 13

increase in DPXs in the interfacial DNA ≥ 6 ppm, which was shown to be linear in the exposure 14 range of 2–30 ppm (2.45–36.8 mg/m 3 ). However, DNA in the aqueous phase did not show DPX 15 formation. Thus, the cross-linked DNA that could b e extracted from the interface after 16 proteolysis was considered to be supporting evidenc e of chemically induced DPX formation. 17 The inability of this study to detect DPXs at lower levels of formaldehyde exposure is likely be 18 due to the protective mechanism of GSH, which catal yzes the conversion of formaldehyde to 19

formate. 20 So, in a later study, Casanova and Heck (1987) repo rted that GSH depletion caused an 21

increase in DPX formation in the interfacial DNA in the nasal mucosa of F344 rats when a dual22 isotope ( 3 H/ 14 C) method was used. The dual isotope method helps i n making the distinction 23 between metabolic incorporation and covalent bindin g of formldehyde. Oxidatoin by removal of 24 one hydrogen atom is required for metabolic incorpo ration of formaldehyde into cellular 25 macromolecules, but not in the formation of DNA add ucts or DNA-protein crosslinks. Thus, the 26

ratio of 3 H/ 14 C of DNA containing DPX will be higher than the mac romoleucules where 27 formaldehyde is metabolically incorporated. Howeve r, the authors further demonstrated that, 28 when the double isotope method was used, the 3 HCHO is oxidized significantly more slowly 29

than H 14 CHO under these conditions, resulting in an overest imate of the concentration of cross30 links due to an isotopic effect on the oxidation of 3 HCHO catalyzed by formaldehyde 31 dehydrogenase (FDH). Besides, this method leaves r esidual formaldehyde that is likely to form 32 DNA adducts by reacting with deoxyribonucleosides i n the DNA hydrolysates (Heck and 33 Casanova, 1987). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-338 To overcome this, Casanova et al. (1989) used an im proved method, which is based not 1 on the analysis of residual formaldehyde bound to d eoxyribonucleosides in DNA hydrolysates 2

but on the determination of the total 14 C-formaldehyde bound to DNA. This study showed tha t 3 formaldehyde was exclusively bound to interfacial D NA, indicating the formation of DPXs. 4 Hydrolysis of DPXs in different samples quantitativ ely released formaldehyde. Besides, DPX 5

formation was detectable at all concentrations of e xposure to formaldehyde (0.3–10 ppm for 6 6 hours). Overall, these studies clearly show that formaldehyde induces DPXs in nasal epithelial 7 cells of rodents. However, there are no published rodent studies that assess DPXs beyond the 8 nasal passages of the URT. 9 Formaldehyde-induced DPXs were also found in the na sal mucosa and extra-nasal tissues 10 of rhesus monkeys exposed to 0, 0.71, 2, or 6 ppm ( 0, 0.86, 2.45, or 7.36 mg/m 3 ) formaldehyde 11 6 hours/day for 3 days (Casanova et al., 1991). These data were used as a basis for cross-species 12 prediction of formaldehyde-induced DPXs in humans. The presence of DPXs in rhesus monkeys 13 confirms formaldehyde‘s DNA reactivity as a general effect. Additionally, DPXs were detected 14 in the larynx/trachea/carina (pooled sample) and in intrapulmonary airways of monkeys exposed 15 to 2 or 6 ppm formaldehyde. These data demonstrate direct effects of formaldehyde on DNA in 16 tissues that correspond to observed tumor sites in humans (nasal and nasopharynx). 17 Bermudez and Delehanty (1986) observed the formatio n of DPXs, scheduled (S) and 18 unscheduled DNA synthesis (UDS), and synthesis of R NA when cultured F344 rat nasal 19 epithelial cells from the naso- and maxillary turbi nates were incubated with formaldehyde. 20 Unscheduled and scheduled DNA synthesis was stimula ted (0.05–0.1 mM) and then inhibited 21 (0.1–1 mM), depending on the formaldehyde concentra tion. Experiments by Cosma et al. (1988) 22 and Cosma and Marchok (1988) showed the induction o f DPXs and DNA SSBs in cultured C18 23 rat tracheal epithelial cells exposed to 200 μ M formaldehyde for 90 minutes (Cosma et al., 1988; 24 Cosma and Marchok, 1988). 25 Several human cells (epithelial cells, fibroblasts, buccal cells) or cell lines 26 (lymphoblastoid cells) exposed to formaldehyde have been shown to form DPX (Craft et al 27 1987; Costa et al 1997; Emri et al 2004; Li et al 2 004). 28 Craft et al. (1987) detected DPXs by alkaline eluti on in TK6 human lymphoblastoid cells 29 immediately after a 2-hour exposure (zero time) to 0, 15, 50, 75, 100, 150, 300, and 600 μ M 30 formaldehyde with a significant nonlinear increase in DPXs above 50 μ M concentration, which 31 correlated with the onset of cytotoxicity, but DPXs were completely removed in cultures held for 32 24 hours before processing. In the zero-time sampl e, significant increases in DPXs were first 33

observed at 50 μ M and increased linearly up to 150 μ M. In cells held for 24 hours, there was no 34

detectable increase in DPXs. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-339 However, Costa et al. (1997) detected DPXs with par aformaldehyde (which dissociates to 1 release formaldehyde) at doses that were cytotoxic (>0.003%) but could not discriminate 2 between the DPX-inducing and cytotoxic effects of t his chemical in EBV, human Burkitt’s 3 lymphoma cells (Costa et al., 1997). Grafström et al. (1983) reported that the number of DPXs 4 induced by 100 μ M formaldehyde in vitro in human epithelial cells a nd fibroblasts of bronchial 5 origin was similar and that the frequency of these cross-links was proportional to the 6 concentration of formaldehyde. Besides the bronchi al epithelial cells and fibroblasts, the authors 7 also noted that formaldehyde exposure resulted in D PXs and DNA SSBs in skin fibroblasts and 8 DNA excision repair-deficient skin fibroblasts. Ho wever, formaldehyde was only moderately 9 cytotoxic to normal bronchial epithelial cells and fibroblasts at concentrations that induced 10 substantial DNA damage. Repair of the formaldehyde -induced DNA SSBs and DPXs appeared 11 to be inhibited by the continued presence of formal dehyde in the culture medium (Grafström et 12 al., 1984). 13 Emri et al. (2004) detected a significant increase in DPX formation in primary human 14 skin fibroblasts and keratinocytes at 8 hours of ex posure in vitro to formaldehyde at 25 μ M with 15 an approximately linear increase up to 100 μ M. These cells were exposed to 0, 12.5, 25, 50, an d 16

100 μ M formaldehyde for 8 hours and then exposed to 250 μ M methyl methane sulfonate 17 (MMS) for 2.5 hours. The induction of DPX formatio n was measured by the ability of 18 formaldehyde to reduce DNA migration in the comet a ssay induced by MMS in this study (Emri 19 et al., 2004). 20 Li et al. (2004) measured DNA damage in primary hum an buccal cells by using the 21 comet assay. The appearance of SSBs, suggesting co mpound-induced fragmentation of DNA, 22 occurred at formaldehyde concentrations of 5 and 7. 5 μ M. At higher concentrations, the 23 response slope decreased, indicating DPXs or DDXs ( Li et al., 2004). The same laboratory 24 reported similar findings in primary human peripher al blood lymphocytes and HeLa cells (Liu et 25 al., 2006). Peak response for SSBs was seen at 10 μ M in both cells, with higher concentrations 26 resulting in cross-link formation. SSBs in HeLa ce lls induced by 10 μ M formaldehyde were 27 repaired by 60 minutes after cells were washed to r emove formaldehyde. 28 Schmid and Speit (2007) tested formaldehyde for its ability to induce DPXs in blood 29 cultures. They used an indirect method to monitor DPX formation in which the extent of DNA 30 migration in the comet assay in response to γ radiation was compared in formaldehyde-treated 31 cultures versus controls. A concentration of 25 μM was required for DPX formation, and repair 32 of these lesions was rapid, with DPXs induced by co ncentrations of formaldehyde up to 100 μM 33 and completely removed after 8 hours. 34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-340 Table 4-76. Formaldedyde-DNA reactions (DPX format ion) 1 2

Species/Strain Cell/Strain Result References

DNA Interaction DPX formation in vitro In vitro Nucleohistone + Brutlag et al 1969 In vitro Nucleohistone + Doenecke 1978 In vitro Nucleohistone + Ohba et al 1979 Bacteria + Wilkins and McLeod 1976

Yeast Saccharomyces cerevisiae + Magana-Schwencke and Ekert 1978 Hamster/Chinese Ovary cells + Marinari et al 1984 Hamster/Chinese Ovary cells + Zhitkovich and Costa 1992 Hamster/Chinese Ovary cells + Olin et al 1996 Hamster/Chinese Ovary cells + Garcia et al 2009 Hamster/Chinese V79 lung epithelial cells + Swenber g et al 1983 Hamster/Chinese V79 lung epithelial cells + Merk an d Speit 1998 Hamster/Chinese V79 lung epithelial cells + Merk an d Speit 1999 Hamster/Chinese V79 lung epithelial cells + Speit e t al 2007 Mouse Leukemia L1210 cells + Ross and Shipley, 1980 Mouse Leukemia L1210 cells + Ross et al 1981 Mouse Hepatocytes + Casanova and Heck 1997 Mouse Hepatocytes + Casanova et al 1997 Rat Yoshida lymphosarcoma cells + O'Connor and Fox 1987 Rat C18 tracheal epithelial cell line + Cosma and M archok 1988 Rat/F344 Hepatocytes + Casanova and Heck 1997 Rat/F344 Nasal mucosa + Casanova-Schmitz and Heck 1 983 Rat/F344 Nasal epithelium + Bermudez et al., 1986

Rat/F344 Primary tracheal epithelial cells + Cosma et al., 1988 Rats/Wistar Aorta endothelial cells + Lin et al 200 5 Human Lung/bronchial epithelial cells + Fornace et al 1982 Human Bronchial cell + Grafstrom et al., 1983 Human Bronchial/ Skin fibroblast + Grafstrom et al., 1984 Human Lung/bronchial epithelial cells + Grafstrom e t al 1984 Human Lung/bronchial epithelial cells + Saladino et al 1985 Human Lung/bronchial epithelial cells + Grafstrom e t al 1986 Human Foreskin fibroblasts + Snyder and Van Houten 1986 Human Lung/bronchial epithelial cells + Grafstrom 1 990 Human Bronchial/Skin fibroblasts + Olin et al 1996 Human White blood cell + Shaham et al., 1996 Human EBV-Burkitt's lymphoma cells + A Costa et al., 1997 Human Gastric mucosa cells + Blasiak et al 2000 Human Peripheral lymphocyte + Quievryn and Zhitkovich 2000 Human Fibroblast cells + Speit et al 2000 Human Lymphocyte + Andersson et al., 2003This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-341 Human Primary skin fibroblasts and keratinocytes + Emri et al 2004

Human Buccal cells + Li et al 2004 Human Jurkat E6-1 cells + Saito et al 2005 Human Peripheral lymphocyte + Liu et al 2006 Human HeLa cells + Liu et al 2006 Human Whole blood + Schmid and Speit 2007 Human Lung/bronchial epithelial cells + Speit et al 2008 Human Lymphoblastoid/TK6 + Craft et al., 1987

DPX formation in vivo Rat/F344 Nasal mucosa + Casanova-Schmitz and Heck 1 983 Rat/F344 Nasal mucosa + Casanova-Schmitz et al 1984 b Rat/F344 Nasal mucosa + Lam et al 1985 Rat/F344 Nasal mucosa + Heck et al 1986 Rat/F344 Nasal mucosa + Heck Hd and Casanova 1987 Rat/F344 Tracheal implants + Cosma et al., 1988

Rat/F344 Nasal mucosa + Casanova et al 1989 Rat/F344 Nasal mucosa + Casanova et al 1994

Rhesus monkeys Nasal, larynx, trachea, and carina + Casanova et al 1991

Human White blood cell + Shaham et al., 1996 Human Peripheral lymphocyte + Shaham et al., 1997 Human Peripheral lymphocyte + Shaham et al., 2003 1 ‘+’ indicates a positive test result 2 ‘-’ indicates a negative test result 3

A

indicates that DNA-protein cross-links formed at c ytotoxic concentrations 4

5 6 4.3.1.2. DNA Adducts 7 In addition to the formation of DPX, there is evide nce that formaldehyde forms 8 hydroxymethyl (hm) DNA adducts in vitro in a variet y of cell-free systems (Zhong and Que Hee, 9 2004a; Cheng et al., 2003; Kennedy et al., 1996; Fe nnell, 1994; Beland et al., 1984) and nasal 10 epithelial cells (Zhong and Que Hee 2004b). In cel l-free systems, formaldehyde directly reacts 11 with DNA forming hmDNA adducts (Cheng et al., 2003; Kennedy et al., 1996; Fennell, 1994; 12 McGhee and von Hippel, 1977a, b, 1975a, b). 13 Beland et al. (1984) first reported hmDNA adducts i n Chinese hamster ovary (CHO) cells 14 incubated with 1 mM of radiolabeled formaldehyde. After a 2-hour incubation, small amounts of 15

N 6 -hmdA were detected with concomitant metabolic inco rporation of formaldehyde. Various 16 forms of hmDNA adducts, including N 6 -hydroxymethyldeoxyadenosine (N 6 -hmdA), 17

N 4 -hydroxymethyldeoxycytidine (N 4 -hmdC), and N 2 -hydroxymethyldeoxyguanosine (N 2

- 18 hmdG), were detected by high performance liquid chr omatography (HPLC) following in vitro 19 reaction between formaldehyde and calf thymus DNA o r individual deoxynucleotides. 20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-342 32 P-postlabeling studies allowed for much greater ana lytical sensitivity but did not 1

confirm the level of N 6 -sulfomethyldeoxyadenosine found by HPLC. However, either estimate 2

of adduct formation is much less than the estimate of DPX formation (120 pmol/mg DNA) in 3 similarly treated rat nuclei (Heck and Casanova, 19 87). 4 Casanova et al. (1989) demonstrated that detection of hmDNA adduct formation was 5 sensitive to the methodology used, particularly the buffer used for sample preparation. 6 Specifically, Tris buffer can prevent hmDNA adduct formation due to the abundance of 7 formaldehyde-reactive primary amine sites in the bu ffer. In contrast, the tertiary amine sites that 8 predominate in Bis-Tris buffer do not react with fo rmaldehyde. 9 Zhong and Que Hee (2004a) observed hmDNA adducts (N 6 -hmdA, N 2 -hmdG, and 10

N 4 -hmdC) in placental DNA exposed to 100 ppm formalde hyde in vitro for 20 hours at 37°C 11 followed by hydrolysis of formaldehyde-reacted DNA using bis-Tris buffer. However, 12 deoxythymidine did not form hydroxymethyl derivativ es in this study (Zhong and Que Hee, 13 2004a). On the other hand, the same investigators were able to detect N 6 -hmdA and N 2 -hmdG 14 adducts in human nasal epithelial cells cultured in the presence of 0, 10, 25, 50, 100, 250, 400, or 15 500 μg/mL formaldehyde and using Tris buffer during hydrolysis of adducted DNA. The 16 toxicity threshold for <90% viability appeared to b e between 100 and 250 μg/mL initial 17 formaldehyde culture concentration, and even at 500 μ g/mL concentration it was not toxic, with 18 a viability was 70% in this study (Zhong and Que He e, 2004b). 19 The only report of formaldehyde-induced hmDNA adduc ts in vivo is a recent study 20 (Wang et al., 2007), showing an indirect evidence o f formation of formaldehyde-induced N 6

- 21 hmdA in hepatic and pulmonary DNA from rats exposed to N -nitrosodimethylamine and 22 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. 23 Since the formaldehyde adducts are labile, Fennel ( 1994) developed a method by 24 derivatizing them with sodium bisulfite to their su lfomethyl form, whereby he detected 25

N 6 -sulfomethyldeoxyadenosine (SOMedA) and N 2 -sulfomethyldeoxyguanosine by using HPLC. 26 However, the levels of SOMedA in DNA isolated follo wing incubation of radiolabeled 27 formaldehyde with isolated rat hepatic nuclei were similar to those in control nuclei. And in 28 human TK6 lymphoblastoid cells treated with formald ehyde, detection of SOMedA adducts was 29 precluded by additional radioactive sports. These observations suggest that N 6

- 30 sulfomethyldeoxyadenosine adducts are formed at ver y low levels in formaldehyde-incubated rat 31 nuclei and that measurement of hydroxymethyldeoxyad enosine would not provide a useful 32 measure of formaldehyde exposure (Fennell, 1994). 33

34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-343 Table 4-77. Formaldedyde-DNA reactions (DNA adduct formation) 1 2

Species/Strain Cell/Strain Result References

DNA Interaction DNA Adduct Formation in vitro Cell-free system Deoxyribonucleosides + Cheng et al 2008 Cell-free system Guanosine + Kennedy et al 1996 Cell-free system Guanosine + Cheng et al 2003 Placental DNA In vitro + Zhong and Que Hee 2004a Calf thymus In vitro + Beland et al 1984 Calf thymus DNA In vitro + Von Hippel and Wang 1971 Cell-free system In vitro + McGhee and von Hippel 1 975a Cell-free system In vitro + McGhee and von Hippel 1 975b Cell-free system In vitro + McGhee and von Hippel 1 977a Cell-free system In vitro + McGhee and von Hippel 1 977b Cell-free system In vitro + Fennell 1994 Cell-free system In vitro + Cheng et al 2003 Rat Nuclei + Fennell 1994 Rats Nasal epithelial cells + Casanova et al 1989 Hamster CHO cells + Beland et al 1984 Rat Nuclei + Heck Hd and Casanova 1987 Human Nasal epithelial cells + Zhong and Que Hee 20 04b DNA adduct formation in vivo Drosophila Larvae + Alderson, 1985 Rats Indirect evidence + Wang et al 2007 3 ‘+’ indicates a positive test result 4 ‘-’ indicates a negative test result 5 6 7 4.3.1.3. DNA-DNA Cross-Links (DDXs) 8 Formaldehyde, besides forming DPXs and DNA adducts , has also been shown to form 9 DDX in vitro. Li et al. (2004) showed that formald ehyde induces DNA strand breaks at low10 exposure concentration and DDXs and DPXs at higher concentrations in buccal cells. The 11 authors also showed that formaldehyde induces DDXs in human peripheral blood lymphocytes 12 exposed in vitro when the concentration was more th an 25 μ M. However, the formation of 13 DDXs has not been demonstrated in vivo, and the rel evance of these modifications in 14 formaldehyde-induced genotoxicity is not known at t he moment. 15 Overall, formaldehyde forms predominantly DPXs that are detected in cell-free systems 16 and single cells in vitro and in animal and human t issues in vivo. In rodents, DPXs are formed in 17 nasal epithelia but not in extra-nasal passages, wh ich are completely removed within a day after 18

formation. The DPXs are detected in nasal and extr a-nasal tissues of monkeys, suggestive of 19This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-344 direct effects of formaldehyde in tissues that corr espond to observed tumor sites (nasal and 1 nasopharynx) in humans. Besides, this is used as a basis for cross-species comparison with 2 humans. Formaldehyde-DNA adducts are labile, const ituting a minor fraction of the DNA3 reaction products. DPXs but not DNA adducts appear to play an important role in the 4 genotoxicity of formaldehyde. 5

6 4.3.1.4. Single Strand Breaks 7 Formaldehyde has been shown to induce DNA single st rand breaks in a number of mammalian 8 cell systems in vitro as well as in vivo as shown i n Table 4-78. Additionally, there is some 9 evidence that DNA single strand breaks (SSBs) may b e induced directly by formaldehyde 10 reactivity (Grafström et al., 1984). 11

12 Table 4-78. Formaldehyde-DNA interactions (single strand breaks) 13 14

Species/Strain Cell/Strain Result References

DNA single strand breaks (in vitro) Hamster/Chinese V79 lung epithelial cells - Speit et al 2007a Mouse Leukemia L1210 cells (+) Ross and Shipley, 19 80 Mouse Leukemia L1210 cells - Ross et al 1981

Rat Hepatocytes + Demkowicz-Dobrzanski and Castonguay 1992 Rat Yoshida lymphosarcoma cells + O'Connor and Fox 1987 Rat/F344 trachea Epithelial cell/ Primary culture + Cosma et al., 1988

Human Bronchial cell/Skin fibroblast + Grafstrom et al., 1984 Human Peripheral blood lymphocytes + Liu et al 2006 Human HeLa cells + Liu et al 2006 Human Skin fibroblast + Snyder and Van Houten, 1986 Human Lung/bronchial epithelial cells + Saladino et al 1985 Human Lung/bronchial epithelial cells + Grafstrom e t al 1984 Human Lung/bronchial epithelial cells + Grafstrom 1 990 Human Lung/bronchial epithelial cells + Fornace et al 1982 Human Lung/bronchial epithelial cells + Vock et al 1999 Human Skin keratinocytes/fibroblasts - Emri et al 2 004 DNA single strand breaks (in vivo) Mouse Liver (maternal) + Wang and Liu 2006

Mouse Liver (fetal) + Wang and Liu 2006 Rats/Spraque-Dawley Lung cells + Sul et al 2007 Rat Lymphocytes + Im et al 2006

Rat Lymphocytes + Im et al 2006 15 ‘no’ indicates test was not done in vivo; ‘+’ ind icates a positive test result; ‘yes’ indicates test was done in vivo 16 ; - indicates a negative test result; (+) indicate s a weak positive test result. 17This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-345 4.3.1.5. Other Genetic Effects of Formaldehyde in Mammalian Cells 1 Formaldehyde induces several other genetic and rela ted effects in mammalian cells which 2 are evaluated by in vitro assays such as unschedule d DNA synthesis (UDS), DNA repair 3

inhibition and cell transformation as summarized in Table 4-79. 4 UDS, which represents DNA repair activity has been reported in nasal epithelial cells of 5 F344 rats (Bermudez and Allen 1984; Bermudez and De lahanty 1986), rat hepatocytes (Williams 6 et al 1989) and Syrian hamster embryo cells (Hamagu chi and Tsutsui 2000). UDS was observed 7 in HeLa cells (Martin et al 1978), but not in human bronchial epithelial cells (Doolittle et al 8 1985) upon formaldehyde exposure. These studies sug gest that following formaldehyde-induced 9 DNA damage was followed by DNA repair. 10 Studies involving human bronchial epithelial cells and skin fibroblasts or keratinocytes 11 (Grafstrom et al 1984; Emri et al 2004), DNA repair proficient or –deficient cell lines (e.g. 12 xeroderma pigmentosum) or cell lines hypersensitive to DNA-DNA crosslinks (e.g. Fanconi’s 13 anemia) (Speit et al 2000) it has been shown that f ormaldehyde causes DNA repair inhibition at a 14 concentration range of 0.125 mM to 10 mM). Emri et al (2004) have shown that DNA repair 15 was inhibited in human keratinocytes and fibroblast s after irradiation with UVB and UVC, but 16 not UVA follwed by treatment with low concentration s of formaldehyde (10 μ M). They 17 observed that DNA SSB induced by UVB or UVC irradia tion alone were repaired within 3-6 18 hours of exposure, while cells with UV irradiation followed by formaldehyde exposure still had 19 the strand breaks at the same timepoints suggesting that formaldehyde is likely to contribute to 20 UV-induced carcinogenesis. 21

22 4.3.2. In Vitro Clastogenicity 23 Clastogenic effects, including increased MNs, CAs, and SCEs are also reported in a range 24 of in vitro study systems as shown in Table 4-80. 25 Miyachi and Tsutsui (2005) measured the induction o f sister chromatid exchanges 26 (SCEs) in Syrian hamster embryo (SHE) cells. Cells were exposed to 0, 3.3, 10, and 33 μ M 27 formaldehyde for 24 hours. SCE levels after 3.3 μ M formaldehyde were not different from 28 controls, but significant increases were observed a t both 10 and 33 μ M. Toxicity as measured by 29 reduced cloning efficiency was seen only at 33 μ M (Miyachi and Tsutsui, 2005). The same 30 laboratory used SHE cells to measure the induction of CAs (Hikiba et al., 2005). Cells were 31 exposed to 0, 33, 66, and 99 μ M formaldehyde for 24 hours prior to staining for a nalysis and the 32 percentages of aberrant metaphases were 0, 6, 6, an d 71, respectively. The aberrations were 33 predominantly chromosome gaps and chromosomal break s and exchanges. The relative colony- 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-346 forming efficiency remained high (at least 85%) for the concentrations of formaldehyde used in 1 the experiment (Hikiba et al., 2005). 2

3 Table 4-79. Other genetic effects of formaldehyde in mammalian cells 4 5

Species/Strain Cell/Strain Result References

Unschedule DNA synthesis (UDS) Rat/F344 Nasal epithelial cells + Bermudez and Alle n 1984 Rat/F344 Nasal epithelial cells + Bermudez and Dele hanty 1986 Rat Hepatocytes + Williams et al 1989 Hamster/Syrian Embryo cells + Hamaguchi and Tsutsui 2000

Human HeLa cells + Martin et al 1978 Human Bronchial epithelial cells - Doolittle et al 1985

DNA repair inhibition

Human Bronchial epithelial cells/skin

fibroblasts + Grafstrom et al 1984

Human Normal fibroblasts (MRC5CV),

XPA cell line, & FA cell line + Speit et al 2000

Human Skin fibroblasts/keratinocytes + Emri et al 2 004 6 XPA, xeroderma pigmentosum, complementation group A (deficient in NER pathway) 7 FA, Fanconi's anemia (cell line has genetic defect leading to hypersensitivity to DNA-DNA cross links; NER, 8 nucleotide excision repair 9 10 11 Schmid and Speit (2007) observed that SCEs were ind uced in lymphocytes of whole 12 blood cultures at a formaldehyde concentration of 2 00 μM, an effect apparently associated with 13 cytotoxicity. This was indicated by a concomitant reduction in the proliferative index. These 14

authors also observed the formation of MNs in their cultures. This effect was statistically 15 significant at a formaldehyde concentration of 300 μM and above. However, MN formation was 16 confined to those cultures in which formaldehyde tr eatment commenced 44 hours after the start 17 of the culture. This prompted the conclusion that the level of DPX formation would need to be 18 high for MN formation and that the cells must be ex posed after the first mitosis. In examining 19 MN formation more closely, Schmid and Speit (2007) used the FISH technique, employing a 20 “biotin-labeled pan-centromeric chromosome paint sp ecific for all human centromeres.” 21 Indicative that formaldehyde was inducing a clastog enic (rather than aneugenic) effect, 81% of 22 MNs in binucleated cells were centromere-negative. 23This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-347 In summary, formaldehyde forms MNs, SCEs, and CAs i n isolated animal and human 1 cells following in vitro exposure (Table 4-80). 2

3 4.3.3. In Vitro Mutagenicity 4 Mutations may occur during repair of formaldehyde- induced DNA damage (DPXs, DNA 5 adducts, SSBs, or clastogenic effects) or as a resu lt of replication errors during mitogenesis. The 6 in vitro evidence for formaldehyde-induced mutation s is strengthened by examining the 7 correlation between these genotoxic and clastogenic events and induction of mutations. 8 Therefore studies are presented with respect to rel evance to one or more of the following lines of 9 evidence for mutagenicity recommended for considera tion in the EPA guidance (U.S. EPA, 10 2005a): (1) that the chemical is DNA reactive and/o r has the ability to bind to DNA, (2) that the 11 chemical generates positive results in in vitro mut agenic test systems (specifically gene 12 mutations and CAs), and (3) that the chemical induc es indications of genetic damage in in vivo 13 tests (specifically gene mutations and CAs). Numer ous studies have demonstrated 14 formaldehyde-induced DNA mutations under a variety of experimental conditions (reviewed in 15 IARC 1995, 2006; Ma and Harris 1988; Auerbach et al . 1977; Conaway et al 1996; NTP 2009). 16

17 4.3.3.1. Mutagenicity in Bacterial Systems 18 A number of research reports describe the mutageni city of formaldehyde in bacterial test 19 systems using reverse and forward mutation assays a s well as specific strains detecting deletions, 20 insertions and point mutations. Among the bacteria l strains, Salmonella typhimurium TA102 and 21

the Escherichia coli strains containing an AT base pair at the primary reversion site are often 22 used to detect oxidative compounds, cross-linking a gents and hydrazines. In an early National 23 Toxicology Program (NTP) collaborative study with t hree laboratories, formaldehyde 24 consistently tested positive for mutagenicity in Salmonella typhimurium strain TA100 in the 25 presence of a rat or hamster liver S9 activating sy stem (Haworth et al., 1983). Formaldehyde 26 was mutagenic with and without metabolic activation in a number of other studies using in 27 reverse mutation assays with S. typhimurium strains TA98, TA100, TA102, TA104, TA2638, 28

and TA2638a and E. coli strains WP2 (pkM101), WP2 uvr A (pkM101), and hrs/r30R (Ryden et 29 al., 2000; Dillon et al., 1998; Watanabe et al., 19 96; Le Curieux et al., 1993; O’Donovan and 30 Mee, 1993; Zielenska and Guttenplan, 1988; Schmid e t al., 1986; Connor et al., 1983, 1985; 31 Orstavik and Hongslo, 1985; Takahashi et al., 1985; Fiddler et al., 1984; Frei et al., 1984; 32 Donovan et al., 1983), while other studies (Muller et al., 1993; Jung et al., 1992; Wilcox et al., 33 1990; Marnett et al., 1985) show both positive and negative results. These results are 34

summarized in Table 4-81 and some of the studies ar e described in greater detail. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-348 Table 4-80. In vitro clastogenicity of formaldehyd e

. 1 2

Species Cell/Tissue origin Without activation With

activation References

Cytogenetic Assays Chromosomal aberratoins (CA) Hamster/Chinese Ovary cells (+) (+) Galloway et al. , 1985 Hamster/Chinese Ovary cells - ND Dresp and Bauching er, 1988 Hamster/Chinese Ovary cells + ND Natarajan et al 19 83 Mouse Lymphoma cells + ND Speit and Merk 2002 Hamster/Syrian Embryo cells + ND Hikiba et al 2005 Hamster/Syrian Embryo cells + ND Hagiwara et al 200 6 Hamster/Chinese Ovary cells + ND Garcia et al 2009 Hamster/Chinese Lung fibroblasts + ND Ishidate Jr e t al 1981 Human Lymphocytes + + Schmid et al 1986 Human Lymphocytes + ND Miretskaya and Shvartsman 19 82 Human Lymphocytes + ND Dresp and Bauchinger, 1988 Human Fibroblasts + ND Levy et al 1983 Micronucleus (MN) Hamster/Chinese V79 lung epthelial cells + ND Speit et al 2007b Hamster/Chinese V79 lung epthelial cells + ND Merk and Speit 1998 Human Whole blood cultures + ND Schmid and Speit 20 07

Human Human MRC5CV (normal) and XP(Repair-deficient) and FA (repair-deficient) cell lines + a ND Speit et al 2000

Sister Chromatid Exchange (SCE) Hamster/Chinese Ovary cells (+) (+) Galloway et al. , 1985 Hamster/Chinese Ovary cells + ND Natarajan et al 19 83 Hamster/Chinese Ovary cells + ND Garcia et al 2009 Hamster/Chinese Ovary cells + ND Obe and Beek 1979 Hamster/Syrian Embryo cells + ND Miyachi and Tsutsu i 2005 Hamster/Chinese V79 lung epithelial cells + (+) Bas ler et al. 1985 Hamster/Chinese V79 lung epithelial cells + ND Spei t et al 2007b Hamster/Chinese V79 lung epithelial cells + ND Merk and Speit 1998, 1999 Hamster/Chinese V79 lung epithelial cells + ND Neus s and Speit 2008 Human A549 lung epithelial cells + ND Neuss and Spe it 2008 Human A549 + V79 (co-cultivated) + c ND Neuss and Speit 2008 Human A549 + V79 (co-cultivated) - d ND Neuss and Speit 2008 Human Lymphocytes + b ND Garry et al., 1981 Human Lymphocytes + ND Krieger and Garry 1983 Human Lymphocytes + ND Schmid et al 1986 Human Lymphocytes + ND Obe and Beek 1979 Human Whole blood cultures + ND Schmid and Speit 20 07This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-349 Premature chromosome Condensation (PCC) Hamster/Chinese Ovary cells + ND Dresp and Bauching er, 1988 1 ‘+’ indicates a positive test result 2 ‘ND’ indicates test was not done 3 - indicates a negative test result 4 (+) indicates a weak positive test result 5

a MN frequency increased in repair-deficient cell li nes compared to normal cell lines 6 b indicates SCE with significant loss of cell viabil ity 7

c A549 cells exposed for 1 h with formaldehyde then co-cultivated with V79 cells 8

d A549 cells exposed for 1 h with formaldehyde, cell s washed and then co-cultivated with V79 cells 9 XP, xeroderma pigmentosum; FA = Fanconi's anemia. 10 11This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-350 Table 4-81. Summary of mutagenicity of formaldehyd e in bacterial systems 1 2

Metabolic activation Species Strain +S9 -S9 References

Mutagenicity Assays Reverse Mutation S. typhimurium TA98, 100, 1535, 1537, 1538 - - De Flora, 1981 S. typhimurium TA100 ND (+) Couch et al., 1982 S. typhimurium TA100 + - Haworth et al., 1983 S. typhimurium TA1535, 1537 - - Haworth et al., 1983 S. typhimurium TA98 (+) - Haworth et al., 1983 S. typhimurium TA98, TA100 + + Connor et al., 1983* S. typhimurium UTH8414, UTH8413 - - Connor et al., 1983* S. typhimurium TA97, 98, 100 + + Donovan et al., 1983 S. typhimurium TA102 + + De Flora et al., 1984 S. typhimurium TA100 + ND Frei et al., 1984 S. typhimurium TA100 ND + Fiddler et al., 1984 S. typhimurium TA100 + (+) Connor et al., 1985 S. typhimurium TA98 (+) - Connor et al., 1985 S. typhimurium UTH8414, UTH8413 - - Connor et al., 1985 S. typhimurium TA100 (+) - Ashby et al., 1985** S. typhimurium TA97, 98, 1535, 1537, 1538 - - Ashby et al., 1985** S. typhimurium TA98, 100, 102 ND (+) Takahashi et al., 1985 E. coli WP2, WP2 uvrA ND + Takahashi et al., 1985

E. coli H/R30R, HS30RuvrA ND + Takahashi et al., 1985 E. coli NG30 recA, 016polA ND - Takahashi et al., 1985 S. typhimurium TA97, 98, 100 ND - Marnett et al., 1985 S. typhimurium TA102, 104 ND + Marnett et al., 1985 S. typhimurium TA98, 100 + + Oerstavik and Hongslo, 1985 S. typhimurium TA100 + + Schmid et al., 1986 S. typhimurium TA104 + ND Zielenska and Guttenplan, 1988 S. typhimurium TA102 ND - Wilcox et al., 1990

E. coli WP2 uvrA /(pKM101) ND + Wilcox et al., 1990

E. coli WP2 (pKM101) ND - Wilcox et al., 1990 S. typhimurium TA102 + ND Jung et al., 1992 S. typhimurium TA102 ND + Le Curieux et al., 1993 S. typhimurium TA102 + ND Muller et al., 1993 S. typhimurium TA98, 100, 102 ND + O’Donovan and Mee, 1993 S. typhimurium TA1535, 1537, 1538 ND - O’Donovan and Mee, 1993

E. coli WP2 (pKM101), ND + O’Donovan and Mee, 1993 E. coli WP2uvrA (pKM101) O’Donovan and Mee, 1993

E. coli K12 (AB1157)(WT) ND + Graves et al., 1994

E. coli K12 (AB1886)/(uvrA), K12(AB2480)/(recA/uvrA) ND - Graves et al., 1994

S. typhimurium TA102, 2638 ND + Watanabe et al., 1996

E. coli WP2 (pKM101), WP2uvrA (pKM101) ND + Watanabe et al., 1996This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-351 S. typhimurium TA1535 A - - Sarrif et al., 1997 S. typhimurium TA1537 A + + Sarrif et al., 1997 S. typhimurium TA98, 100 A + - Sarrif et al., 1997 S. typhimurium TA97 A ND + Sarrif et al., 1997 S. typhimurium TA1535, 1537 B - - Sarrif et al., 1997 S. typhimurium TA98 B + + Sarrif et al., 1997 S. typhimurium TA100 B - + Sarrif et al., 1997 S. typhimurium TA100 C + + Sarrif et al., 1997 S. typhimurium TA100, 104 B + + Dillon et al., 1998

E. coli (Lac+ reversion) WP3101P, WP3106P + Ohta et al 1999 S. typhimurium TA102, 2638 B ND + Ryden et al., 2000

Forward Mutation S. typhimurium TM677 ND (+) Couch et al., 1982 S. typhimurium TM677 + + Donovan et al., 1983 S. typhimurium TM677 + + Temcharoen and Thilly, 1983

E. coli D494 uvrB + Bosworth et al 1987

Deletion Mutation

E.coli GP120, GP120A 7-2, 33694 ND + D Crosby et al., 1988

Point Mutation E.coli GP120, GP120A 7-2, 33694 ND + Crosby et al., 1988

Insertion Mutation E.coli GP120, GP120A 7-2, 33694 ND + Crosby et al., 1988 1 ‘+’ indicates a positive test result 2 ‘ND’ indicates test was not done 3 ‘-’ indicates a negative test result 4 (+) indicates a weak positive test result 5 * indicates the use of formalin in mutagenicity ass ay 6 ** indicates the use of hexamethylmelamine (HEMLA), a formaldehyde-releasing compound, in mutagenicity assay 7

A indicates use of the Standard Plate Method 8

B indicates use of the Preincubation Plate Method 9

C indicates use of the Suspension Method 10

D indicates loss of DNA 11 12 13 Formaldehyde has been shown to be mutagenic in forw ard mutation assays using S. 14 typhimurium (Couch et al 1982; Donovan et al 1983; Temcharoen and Thilly 1983) as well as in 15

E. coli (Bosworth et al 1987). Temcharoen and Thilly (198 3) examined the toxicity and 16 mutagenicity of S. typhimurium strain TM677, using forward mutation to 8-azaguani ne 17 resistance, and have shown that formaldehyde induce d both toxicity and mutagenicity at 18 minimum concentrations of 0.17 mM (–S9) and 0.33 mM (+S9). It has also been shown that 19This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-352 formaldehyde formed as an intermediate by oxidation at the methyl group of 1 N-nitrodimethylamine, a biologically active N-nitra mine of environmental significance, is 2 mutagenic to S. typhimurium TA100 strain at low concentrations and toxic above 2 μ mol/plate 3 (Frei et al., 1984). 4 Bosworth et al (1987) developed a forward mutation assay in E. coli D494 uvr B strain 5 transformed with a multi-copy mutator plasmid pGW17 00, in which mutations are scored by an 6 increase in ampicillin-resistant colonies after exp osure of bacterial cells during the logarithmic 7 growth by the test chemicals. This assay is more se nsitive to base-pair substitutions, but less 8 sensitive to frameshift mutations compared to Salmo nella/miceosome-based assays. In this assay, 9 the authors (Bosworth et al 1987) observed positive curvilinear response to formaldehyde 10 exposure. Crosby et al (1988) used four E. coli strains GP120, GP120A, 7-2, and 33694 11 containing the xanthine guanine phosphoribosyl tran sferase ( gpt ) gene (which detects point 12 mutations, deletions and insertions) tested the mut agenicity of formaldehyde by exposing for 1 13 hour at 4 and 40 mM concentrations. They observed 4 1% large insertions, 18% large deletions 14 and 41% point mutations. However, at 40 mM dose the re were 92% point mutations, a majority 15 of them (62%) being transition mutations at a singl e AT base pair in the gpt gene. In the same 16 study they observed frameshift mutations in E. coli that was transformed with naked pSV2gpt 17 plamid DNA exposed to 3.3 or 10 mM formaldehyde. Th us, the mutation pattern appear to differ 18 depending on the concentration of formaldehyde expo sure to the bacterial strain as well as the 19

nature of DNA. 20 Formaldehyde has also been shown to induce primary DNA damage in E. coli and 21 mutagenic activity in the Ames fluctuation test in S. typhimurium TA100, TA102, or TA98 22 strains (Le Curieux et al., 1993). 23 O’Donovan and Mee (1993) observed clear mutagenicit y by the pre-incubation exposure 24

method in S. typhimurium TA98, TA100, and TA102 strains and both E. coli WP2(pKM101) and 25 WP2uvrA(pKM101) strains, while the standard plate-i ncorporation assays showed consistent 26 mutagenicity only with TA100 and WP2 uvr A(pKM101) strains and no evidence of mutagenicity 27 in TA1535, TA1537, or TA1538 strains using either m ethod of exposure in the absence of 28

metabolic activation. The S. typhimurium and E. coli strains used in this study are histidine and 29 tryptophan auxotrophs, with an AT base pair at the critical mutation site within the his G and trp E 30 genes, respectively, with an intact excision repairsystem facilitating the detection of cross31 linking agents and both strains carrying the mutato r plasmid, pKM101, which enhances error32 prone repair. These salmonella strains detect fram eshift (TA98 and TA1537) and base-pair 33 substitutions (TA100, TA102, and TA1535), while the E. coli strains detect base-pair 34 substitutions (WP2 uvr A). These findings are consistent with the suggest ion that formaldehyde 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-353 induces excision-repairable lesions in bacteria and indicate that the presence of the R-factor 1 plasmid may be required for the expression of its m utagenicity in excision repair-deficient 2 salmonella (O’Donovan and Mee, 1993). 3 Dillon et al. (1998) employed salmonella strains TA 100, TA102, and TA104 because of 4 the latter two strains being more sensitive to oxid ative mutagens. Formaldehyde was clearly 5 mutagenic between 6 and 50 μ g/plate in all three strains with and without metab olic activation 6 using Aroclor-induced S9 from male F344 rats or mal e B6C3F1 mice, except for an equivocal 7 response in TA102 with mouse S9 (Dillon et al., 1998). Using a set of six tester strains 8 (WP3101–WP3106) of E. coli , each reversible by a mutation involving a single DNA base pair 9 substitution, Ohta et al. (1999) determined that fo rmaldehyde preferentially induced GC to TA 10 transversion mutations. Ryden et al. (2000) demons trated a statistically significant increase in 11

the number of revertants in S. typhimurium TA102 (2.5-fold) and TA2638a (3-fold) strains by 12 formaldehyde at ≥ 17 μ g/plate compared with solvent controls. 13 In summary, formaldehyde induces mutations in seve ral bacterial strains containing an 14 AT base pair at the primary reversion site that are used to detect oxidative compounds and cross15 linking agents without metabolic activation by exog enous enzyme-activating systems. This 16 evidence is strengthened by examining the correlati on between genotoxic and clastogenic events 17

and mutation induction. 18

19 4.3.3.2. Mutagenicity in Non-Mammalian Cell Systems 20 Formaldehyde has been shown to be mutagenic in seve ral non-mammalian systems also. 21 It has been shown to cause gene conversion, strand breaks, crosslinks, homozygosis and related 22 damage in yeasts ( Saccharomyces cervisiae ), forward and reverse mutations in molds 23 ( Neurospora crassa ), micronuclei formation in spiderworts ( Tradescantia pallida ), DNA 24 damage and mutations in several plants, genetic cro ss-over or recombination, sex-linked 25 recessive lethal mutations, dominant lethal mutatio ns, heritable translocations and gene 26 mutations in insects ( Drosophila melanogaster ) and recessive lethal mutations in nematodes 27 ( Caenorhabditis elegans ), but failed to show micronuclei formation in newt larvae ( Pleurodeles 28

waltl ) (Reviewed in Conaway 1996; IARC 2006). 29

30 4.3.3.3. Mutagenicity in Mammalian Cell Systems 31 Several studies demonstrated the mutagenicity of fo rmaldehyde in mammalian cells. In 32 its report, the Federal Panel on Formaldehyde under lined the role of formaldehyde as an inducer 33 of gene mutations and CA in a variety of test syste ms (Report of the Federal Panel on 34 Formaldehyde, 1982). Results from several studies a re summarized in Table 4-82. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-354 Snyder and Van Houten (1986) demonstrated that form aldehyde increases the levels of 1 misincorporation of bases into synthetic polynucleo tides catalyzed by E. coli DNA polymerase I, 2 indicating that the mutagenicity of formaldehyde ma y be due to covalent alteration of DNA 3 bases. They have also shown that formaldehyde-indu ced DNA damage in human fibroblasts was 4 not susceptible to repair by the typical “long patc h” excision repair mechanism. 5 Craft et al. (1987) measured the induction of mutat ions at the thymidine kinase ( tk ) locus 6 or at the ouabain resistance ( Oua r ) locus in TK6 human lymphoblastoid cells. The tk mutations 7 can result from a variety of mutational events, inc luding base pair substitution, small and large 8 deletions, and chromosome exchange events, while mu tations to Oua r require specific base pair 9 substitutions. Single treatment of formaldehyde (0 , 15, 30, 50, 125, and 150 μ M) for 2 hours 10

resulted in a nonlinear increase in tk mutagenesis with increasing slope >125 μ M (Figure 4-32). 11 To explore a dose-response effect, cells were also exposed as follows: three treatments of 50 μ M 12

for 2 hours or five treatments of 30 μ M or 10 treatments of 15 μ M for 2 hours (treatments were 13 spaced 2–4 days apart) with multiple treatments cau sing an increase in tk mutations, although 14 their combined effect was less than a single treatm ent of equivalent C × t (150 μ M for 2 hours). 15 Lymphoblasts given four treatments of 150 μ M formaldehyde for 2 hours failed to induce 16

mutations at the Oua r locus. Dose-response increases were seen in all e xposure scenarios, with 17

30 μ M being the level of statistical significance. The re was little indication of a dose-response 18 effect until the cumulative concentration was great er than 100 μ M. Formaldehyde-induced 19 DPXs were no longer evident after 24 hours of expos ure; mutants induced in the TK6 20 lymphoblast cell line showed a similar dose-respons e curve to the DPXs measured immediately 21 after exposure ended (Craft et al., 1987). 22 The same group also studied mutations induced at th e X-linked hypoxanthine-guanine 23 phosphoribosyl transferase (HPRT) locus by eight re petitive treatments of 150 μ M formaldehyde 24 in TK6 human lymphoblast cell line by Southern blot analysis, wherein half (14/30) of induced 25 mutants contained partial or complete deletions wit h most of the partial deletions showing 26 unique deletion patterns, while only a third (5/15) of spontaneous mutants had partial or 27 complete deletions, indicating that formaldehyde ca n induce large losses of DNA in human 28 lymphoblast cells (Crosby et al., 1988). 29This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-355 Table 4-82. Mutagenicity in mammalian cell systems

. 1 2

Species/Strain Cell/Strain In Vivo

test Without activation References

Mutagenicity Assays

Dominant Lethal Mutation Rat/Albino Spermatocyte, Live implants yes + ND Odeigah, 1997 Mouse Dominant lethal yes - Epstein and Shafner 19 68 Mouse Dominant lethal yes - Epstein et al 1972 Mouse Dominant lethal yes (+) Fontignie-Houbrechts 1981 Rat Dominant lethal yes (+) Kitaeva et al 1990

Deletion Mutation Hamster/Chinese V79 cells Hprt locus) no - Merk and Speit 1998

Hamster/Chinese V79 cells Hprt locus) no - Merk and Speit 1999 Hamster/Chinese V79/HPRT no + ND Grafstrom et al., 1993 Hamster/Chinese Ovary HPRT no - + Graves et al., 19 96

Mouse Lymphoma L5178Y cells (Tk +/-

locus) no + Macerer et al 1996

Mouse Lymphoma L5178Y cells no + ND Speit and Merk 2002 Human Bronchial cell no + Grafstrom et al., 1983

Human Bronchial fibroblasts/epithelial cells

(HPRT locus) no + Grafstrom et al 1985

Human Bronchial fibroblasts/epithelial cells

(HPRT locus) no + Grafstrom 1990 Human Lymphoblast/HPRT no + a ND Crosby et al., 1988 Human Lymphoblast/tk no + Craft et al 1987 Human Peripheral lymphocytes yes + ND Shaham et al 2003

Human Lymphoblast (TK6) no + Goldmacher and Thilly 1983

Point Mutation Hamster/Chinese Ovary HPRT no + ND Graves et al., 1 996 Mouse Lymphoma cell/ TK+/- no + + Blackburn et al., 1991 Mouse Lymphoma cell/ TK+/- no + ND Wangeheim and Bo lcsfoldi, 1988 Human Lymphoblast/TK6 no + ND Liber et al., 1989

Insertion Mutation Hamster/Chinese Ovary HPRT no + ND Graves et al., 1 996

Heritable Mutation Mouse Heritable mutation yes + Liu et al 2009

DNA Repair enzyme activity Human Peripheral lymphocyte yes - Hayes et al., 19 97This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-356 Cell Transformation

Mouse C3H10T1/2 cells + b Ragan and Boreiko 1981

Mouse Embryo fibroblast/C3H/10T1/2 no [+] ND Boreiko et al., 1983

Mouse Embryo fibroblast/C3H/10T1/2 no [+] ND Frazelle et al., 1983

Hamster Kidney cell/BHK-21/cI.13 no + + Plesner and Hansen, 1983

p53 mutation and/or p53 protein expression

Rats/F344 Nasal squamous cell carcinomas yes + c Recio et al 1992

Rats/F344 Nasal tumor cell lines No + Bermudez et al 1994

Rats/F344 Nasal squamous cell carcinomas Yes + d Wolf et al 1995

Human Peripheral blood lymphocytes yes + Shaham et al 2003 1 ‘no’ indicates test was not done in vivo 2 ‘+’ indicates a positive test result 3 ‘ND’ indicates test was not done 4 ‘yes’ indicates test was done in vivo 5 - indicates a negative test result 6 (+) indicates a weak positive test result 7 [+] indicates positive test result after TPA or N-m ethyl-N-nitro-N-nitrosoguanidine promoter treatment 8

a indicates loss of DNA 9

b Positive only in the presence of 12-O-tetradecanoy lphorbol 13-acetate (TPA) 10

c p53 mutations 11

d p53 mutated protein detected by immunohistochemist ry 12This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-357 1

2 Figure 4-32. DNA-protein cross-links (DPX) and thy midine kinase ( tk ) 3 mutants in TK6 human lymphoblasts exposed to formal dehyde for 2 hours. 4 5 Note:  DPXs immediately after exposure, DPXs 24 hours after exposure,  tk 6 mutants. Relative survival was 100% at 0, 15, 30, and 50 μM, 30% at 125 μM, 7 and 20% at 150 μM. 8 9 Source: Adapted from Craft et al. (1987). 10 11 12 Liber et al. (1989) followed up the findings of Cro sby et al. (1988) by performing 13 Southern blot, Northern blot, and DNA sequence anal ysis on the 16 induced and 10 spontaneous 14 human lymphoblast mutants not showing deletions. N orthern blot analysis showed that the point 15 mutations fell into four categories: normal size an d amount of RNA, normal size but reduced 16 amounts of RNA, reduced size and amounts of RNA, an d no RNA. Sequence analysis of 17

recombinant DNAs from hprt mRNA in formaldehyde-induced mutants showed a pref erential 18 AT to CG transversion at a specific site, with othe r changes represented to a lesser degree (Liber 19 et al., 1989). 20 Even in CHO cells formaldehyde has been shown to in duce hprt mutations involving 21 mostly single-base pair transversions mostly occurr ing at AT sequences, including three AT to 22 TA at position 548 of exon 8 and two AT to CG and o ne GC to TA transversion at other sites 23This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-358 (Graves et al., 1996). In another study, formaldehyde-indu ced forward mutations to 1 trifluorothymidine resistance in mouse lymphoma L51 78Y tk ± cells both in the absence and 2 presence of rat liver S9 (higher concentrations req uired for effect with S9). Both toxicity and 3 mutagenicity were abolished when FADH was incorpora ted in the exposure medium (Blackburn 4 et al., 1991). 5 Formaldehyde-induced DPXs are removed in part throu gh spontaneous hydrolysis and in 6 part due to active repair processes (Quievryn and Z hitkovich, 2000). Inhibition of specific 7 proteosomes in XP-A cells inhibited DPX repair, the reby supporting the role of enzymatic 8 degradation (Quievryn and Zhitkovich, 2000). The h alf-life of formaldehyde-induced DPXs in 9 human cell lines was consistent with the findings o f Craft et al. (1987), ranging from 11.6 to 10 13 hours (Quievryn and Zhitkovich, 2000). In the s ame report, removal of DPXs from human 11 peripheral lymphocytes was much slower, with a half -life of 18.1 hours. This difference was 12 primarily in slower active repair of DPXs, with a t 1/2 of 66.6 hours for human lymphocytes 13 versus 23.3 hours for human cell lines (Quievryn an d Zhitkovich, 2000). 14 Since DPX repair involves proteolytic removal of pr oteins from the DNA, Speit et al. 15 (2000) hypothesized that single peptides or small p eptide chains cross-linked to the DNA are 16 critical to formaldehyde-induced mutation. However , these authors did not find significant 17 difference in the induction and repair of DPXs in n ormal and DNA repair-deficient cell lines but 18 observed increased susceptibility of the repair-def icient cell lines to formaldehyde-induced MN 19 induction. In this study, a normal human cell line (MRC5CV1), a xeroderma pigmentosum cell 20 line deficient in nucleotide excision repair (NER), and a Fanconi anemia cell line, which has a 21 genetic defect leading to hypersensitivity towards DDXs, were exposed to 125, 250, and 500 μ M 22 formaldehyde for 2 hours. The authors suggest that more than one repair pathway is involved in 23 the repair of cross-links and that the altered NER pathway has more severe consequences to 24 formation of CAs than disturbed cross-link repair ( Speit et al., 2000). 25 The correlation of early DPX formation and mutation is at first counterintuitive since the 26 cross-linking of protein to DNA inhibits DNA replic ation. Without active DNA replication, 27 formaldehyde-DNA adducts and DPXs would not induce replication error and would be unlikely 28 to result in a change in DNA sequence or mutation. Recent evidence indicates that residual 29 peptides and short polypeptides that remain cross-l inked to DNA after DPX removal may in fact 30 be the cause of DPX-associated, formaldehyde-induce d mutation (Speit et al., 2000). 31 A study by Merk and Speit (1998) indicated that for maldehyde-induced DPXs did not 32 result in direct gene mutations in the hprt locus of V79 Chinese hamster cells, leading the 33 authors to speculate that formaldehyde was not muta genic. Since, the hprt locus in the V79 34 Chinese hamster cell line is primarily sensitive to point mutations and other studies show the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-359 formation of deletion mutations by formaldehyde at the same locus in human lymphoblasts 1 (Crosby et al., 1988), Merk and Speit (1998) conclu ded that the hprt mutation assay is insensitive 2

to deletion mutations. 3 Later, using the mouse lymphoma assay, Speit and Me rk (2002) demonstrated that 4 exposure to formaldehyde for 2 hours was mutagenic in a concentration-dependent manner in the 5 L5178Y mouse lymphoma cells, which was mainly contr ibuted by a strong increase in small 6 colony mutants, suggestive of CAs (Speit and Merk, 2002). Detailed analysis of both 7 spontaneous and formaldehyde-induced lesions indica tes that recombination or deletion of DNA 8

from the tk

locus was primarily responsible for the loss of he terogeneity, thereby leading to the 9 observed mutant phenotype. Therefore, it is believ ed that formaldehyde is mutagenic in the 10 L5178Y cell mouse lymphoma system by a clastogenic mechanism rather than through point 11 mutations. This finding is consistent with that of Craft et al. (1987), who demonstrated 12 formaldehyde mutagenicity at the tk locus of TK6 cells, and also with the findings of Grafström 13 et al. (1984), who demonstrated increased SSB forma tion in formaldehyde-exposed cell lines. 14 Formaldehyde has also been shown to induce cell tra nsformation in mouse embryo 15 fibroblasts (Ragan and Boreiko 1981; Boreiko et al 1983; Frazelle et al 1983) . At low 16 concentrations of 0.017 mM formaldehyde has shown t o cause cell transformation in C3H10T1/2 17 mouse cells (Ragan and Boreiko 1981) and hamster ki dney cells in vitro (Plenser and Hansen 18 1983). 19 More recently, Shaham et al. (2003) examined the fr equency of DPXs and the incidence 20 of mutant versus wild type p53 tumor suppressor gen es in the peripheral blood lymphocytes of a 21 cohort of workers exposed to formaldehyde. The adj usted mean levels of DPXs were greater in 22 the lymphocytes of exposed subjects compared with t hose of unexposed subjects, and exposure 23 to formaldehyde increased the likelihood of their h aving a higher level of pantropic p53 24 (>150 pg/mL). The authors speculated on a possible causal relationship between DPXs and 25 mutations in p53. Recio et al (1992) demonstrated point mutations in the p53 tumor suppressor 26 gene in 45% (5 out of 11) of the primary nasal squa mous cell carcinomas (SCCs) obtained from 27 F344rats that were chronically exposed to 15 ppm fo rmaldehyde for 2 years (Recio et al ., 1992). 28 In summary, the results of in vitro experiments dem onstrate the mutagenicity of 29 formaldehyde. Mutagenicity is observed below level s of significant cytolethality in mammalian 30 cell lines. Formaldehyde is clearly a DNA-reactive genotoxicant inducing lesions (DPXs) that 31 show clastogenicity (SSBs, MNs, etc.). The experim ents by Speit and Merk (2002) explore 32 mechanistic links between DPXs, clastogenicity, and the observed locus-specific mutations in 33 the mouse lymphoma in vitro testing system. 34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-360 4.3.4. In Vivo Mammalian Genotoxicity 1

4.3.4.1. Genotoxicity in Laboratory Animals 2 As discussed above, formaldehyde is clearly reactiv e at the POE in animal studies, 3 resulting in increased DPXs in the nasal mucosa. D espite formaldehyde’s reactivity and 4 mutagenicity in isolated mammalian cells, clear evi dence of mutagenicity does not emerge from 5 animal bioassays (Table 4-83). 6 In a chromosomal analysis study (Fontignie-Houbrech ts, 1981), formaldehyde given I.P. 7 at 50 mg/kg to male Q strain mice and analyzed 8–15 days after treatment did not induce any 8 chromosomal lesions in spermatocytes. Also, in ano ther study from the same group (Fontignie9

Houbrechts et al., 1982), formaldehyde (30 mg/kg) given along with hydrogen peroxide (90 10 mg/kg) as a mixture to male Q strain mice failed to produce significant increases in 11 chromosomal lesions in the spermatogonia. 12 In a different study Natarajan et al. (1983) failed to detect significant differences in MN 13 induction in bone-marrow cells or CAs in spleen cel ls of male and female CBA mice given I.P. 14 6.25, 12.5, and 25 mg/kg formaldehyde compared with saline-treated controls. However, the 15 same study showed a positive induction of MNs and C As in vitro. The authors suggest that the 16 lack of genotoxicity in vivo may be due to the inab ility of formaldehyde to reach the target cells 17 in sufficient quantity to induce biological effects

. 18 Kligerman et al. (1984) also found no difference in the incidence of SCEs or 19 chromosome breakage in the peripheral lymphocytes o f male and female F344 rats exposed to 20 formaldehyde in air at 0.5, 6, or 15 ppm (0.61, 7.3 6, or 18.4 mg/m 3 ) 6 hours/day for 5 days. 21 However, in a different study (Migliore et al., 1989), clastogenic effects, such as increas ed MNs 22 and CAs, were reported in GI epithelial cells of ma le Sprague-Dawley rats after oral exposures to 23 200 mg/kg formaldehyde. In this study, micronuclea ted cells and nuclear anomalies were 24 increased in a time-dependent manner in the stomach , duodenum, ileum, and colon of rats, and 25 the mitotic index was unchanged for these cells com pared with controls at 16, 24, and 30 hours. 26 These clastogenic effects were seen without regener ative cell proliferation, supporting 27 formaldehyde-induced mutations as primary effects o f formaldehyde rather than secondary to 28 regenerative cell proliferation. 29 Kitaeva et al. (1990) observed cytopathological and cytogenetic effects of formaldehyde 30 chronic inhalation in 0.5 and 1.5 mg/m 3 doses in the female rat’s germ and marrow cells, w here 31 formaldehyde-induced harmful effects were seen in g erm cells at <1.5 mg/m 3 doses, while the 32 reliable clastogenic and cytogenetic effects on the marrow cells were induced even at the 33 0.5 mg/m 3 dose, suggesting differences among effects of smal l doses of formaldehyde on 34 different cell systems. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-361 Table 4-83. Genotoxicity in laboratory animals. 1 2 Species/Strain Cells/Organ/Tumor Result References Cytogenetic Assays Chromosomal aberratoins (CA) Mice/Q strain Spermatocyte - Fontignie-Houbrechts e t al., 1981 Mice/Q strain Spermatogonia - Fontignie-Houbrechts et al., 1982 Mice/CBA Polychromatic erythrocytes - Natarajan et al., 1983 Mice/CBA Spleen cells - Natarajan et al., 1983

Rats/F344 Lymphocytes - Kligerman et al 1984 Rats/Spraque-Dawley Gastric epithelial cells + Migliore et al 1989

Rats/Wistar Bone marrow + Kitaeva et al 1990 Rats/Spraque-Dawley Bone marrow - Dallas et al 1992 Rats/Spraque-Dawley Pulmonary lavage cells + Dallas et al 1992

Rats/F344 Peripheral blood cells - Speit et al 2009

Micronucleus (MN)

Mouse/NMRI Bone marrow - Gocke et al 1981

Mice/CBA Femoral polychromatic erythrocyte and spleen cell - Natarajan et al., 1983

Rats/Spraque-Dawley Gastric epithelial cells + Migliore et al 1989

Sister Chromatid Exchange (SCE) Rats/F344 Lymphocyte - Kligerman et al 1984 Rats/F344 Peripheral blood cells - Speit et al 2009 3 ‘+’ indicates a positive test result 4 - indicates a negative test result 5 6 7 Dallas et al. (1992) observed a slight increase (7. 6 and 9.2%) in CAs in pulmonary lavage 8 cells from male Sprague-Dawley rats exposed to 15 p pm (18.4 mg/m 3 ) formaldehyde in air 9 6 hours/day, 5 days/week for 1 or 8 weeks by inhala tion compared with corresponding controls 10 (3.5 and 4.8%), respectively. However, the small s tudy, limited as it was to five animals/group, 11 showed statistically significant increase at the hi ghest dose tested (15 ppm) but not at lower 12 doses (0.5 and 3 ppm). In the same study, no clast ogenic effects were seen in bone marrow, 13 which is consistent with formaldehyde acting primar ily at the site of first contact. 14 Speit et al (2009) investigated the genotoxicity of formaldehyde in peripheral blood 15 samples of Fischer-344 rats exposed to 0 to 15 ppm formaldehyde by whole-body inhalation for 16 4 weeks (6 h/day, 5 days/week). In this study, the authors found no significant increase in the 17 genotoxic assays such as comet assay with or withou t gamma-irradiation of blood samples (DNA 18 migration as determined by tail movement or intensi ty), sister chromatid exchange (SCE) assay 19This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-362 and micronucleus test (MNT) compared to controls. However, rats given 50 mg/kg 1 methylmethane sulfonate (MMS) by gavage for 4 hrs ( positive control for Comet and SCE 2 assays) or 10 mg/kg cyclophosphamide (CP) given twi ce orally (positive control for MNT) 3 induced significant increase in genotoxicity in thi s study. The lack of genotoxicity in this study 4 is not surprising since earlier studies by Casanova -Schimitz et al (1984a) have shown that 5 formaldehyde does not cause toxicity to bone marrow possibly due to the inability of this 6 chemical to reach the bone marrow. Although MMS an d CP used in this study were positive in 7 the genotoxicity assays, the data from positive con trols can not be used for validation since the 8 exposure routes of formaldehyde (inhalation) and th e positive controls (oral) were different. 9 No animal studies have examined clastogenic effects of formaldehyde in nasal or 10 respiratory epithelial cells. Therefore, it is unk nown whether similar changes would occur in 11 response to exposure to formaldehyde via inhalation . However, the negative finding in bone 12

marrow cannot be considered definitive evidence on the question of the mutagenic potential of 13 formaldehyde for cells present at the POE. With we ak positive results in pulmonary lavage cells 14 and clear clastogenicity in GI epithelial cells bel ow exposures that trigger regenerative cell 15 proliferation, the existing evidence, however incom plete, supports the concept of genotoxic 16 action of formaldehyde at the POE. 17

18

4.3.4.2

. Genotoxicity in Humans 19 The majority of the studies on the effects of forma ldehyde in exposed humans have 20 measured various cytogenetic endpoints, such as MNs , SCEs, or CAs in nasal and oral mucosal 21 cells (considered to be in direct contact with form aldehyde) as well as peripheral lymphocytes. 22 Since genotoxicity at the proximal sites (oral, nas al) can be readily linked to the reactive nature 23 of formaldehyde, these studies are discussed first, noting where researchers also collected blood 24 lymphocyte samples. A subsequent discussion is foc used on results in blood lymphocytes. 25 Finally, the few studies that measured DPXs in expo sed humans are discussed. Table 4-89 26 provides a summary of human cytogenetic studies of formaldehyde. 27

28

4.3.4.2.1. Nasal, buccal, and oral mucosal cells. Epithelial cells of the URT and oral cavity are 29 potential targets of formaldehyde’s DNA reactivity and genotoxicity. Several studies indicate 30 that formaldehyde exposure results in measurable in creases in SCEs, MN formation, and DPXs 31 in nasal, buccal, and oral mucosal cells; however, these genotoxic effects vary with the type of 32 exposure. Study quality, sample size, availability of exposure measurements, and assay 33 methodology may in part contribute to variability i n study findings. The studies fall into three 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-363 general categories: workers (industrial or professi onal), students and staff attending anatomy and 1 mortuary science courses, and subjects in a control led clinical trial. 2 Ballarin et al. (1992) observed significantly highe r frequency of micronucleated cells in a 3 formaldehyde exposed group in a plywood factory com pared with controls (0.9 ± 0.47 versus 4 0.25 ± 0.22, p < 0.01). In this study, the frequency of MNs and c ytology of respiratory nasal 5 mucosal cells was examined in 15 nonsmokers exposed to levels of formaldehyde that ranged 6 between 0.1 and 0.39 mg/m 3 (~0.32 ppm) for an average of 6.8 years. Exposed s ubjects were 7 compared with age- and sex-matched controls. 8 Ye et al. (2005) reported significant increases in MNs per thousand cells in nasal mucosal 9 cells for 18 nonsmoking workers (2.70 ± 1.50) in a formaldehyde manufacturing plant in the 10 Hugei province of China as compared with controls ( 1.25 ± 0.41). In addition, higher 11 frequencies of SCEs in peripheral lymphocytes of wo rkers were also reported (8.24 ± 0.89 versus 12

6.38 ± 0.41). In this study, the average age of workers was 29 ± 6.8 years, the average duration 13 at work was 8.5 years (range 1–15 years), and the r eported 8-hour TWA was 0.985 mg/m 3 14 (0.8 ppm). The control group consisted of 23 under graduate students with an average age of 15

19 ± 2.3 years. The 8-hour TWA in the student dormitor ies was 0.011 mg/m 3 (9 ppb). A group 16 of 16 waiters with an average exposure duration of only 12 weeks and an 8-hour TWA of 17 0.107 mg/m 3 (90 ppb) was also included in the study. The incid ence of MNs and SCEs in the 18 waiters was the same as that in controls. Overall, results from this study suggest that the 19 genotoxic potential of high-level formaldehyde expo sure may have occupational risks in long20

term exposure. 21 However, in a different study, Speit et al. (2007b) showed that formaldehyde did not 22

induce MNs in exfoliated buccal mucosa cells of hum ans exposed up to a maximum of 1 ppm 23 and a cumulative exposure of 13.5 ppm-hours over 2 weeks. In this study, volunteers exposed to 24 formaldehyde in closely controlled conditions (4 ho urs/day for 10 days) with a complex 25 exposure schedule, amounting to a cumulative total of 13.5 ppm-hours (16.6 mg/m 3 -hours), were 26 used. Samples of the buccal mucosa were taken from subjects 1 week before the start of the 27 experiment, at the start of the experiment, at the conclusion of the series of exposures, and at 7, 28 14, and 21 days after the completion of exposure. Thus, the subjects served as their own 29 controls. Two thousand cells per data point were a ssessed for the frequency of MNs on slides 30 that were coded by an independent quality assurance organization. As shown in Table 4-84, the 31 frequency of MN formation was statistically unchang ed from that in controls. The apparent 32 slight increase in subjects evaluated at the conclu sion of exposure was caused by frequencies of 33 MNs in two subjects (5.0 and 4.5 MNs per 1,000 cell s). The data as reported show a high 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-364 variability, where the SD approaches or exceeds the mean for each sample point, suggestive of 1 data with an asymmetrical distribution. 2

3 Table 4-84. MN frequencies in buccal mucosa cells of volunteers exposed to 4 formaldehyde 5 6 Sampling point Group MN/1000 cells (± SD)

Control data 1 week before exposure 1 0.95 ± 0.67 Immediately before exposure series 2 0.86 ± 0.84

Test data Immediately after exposure series 3 1.33 ± 1.45 7 days after exposure 4 0.94 ± 0.73 14 days after exposure 5 0.85 ± 0.86 21 days after exposure 6 0.44 ± 0.38 a 7

a Statistically significantly different from control values ( p < 0.05), as calculated by the authors. 8 9 Source: Speit et al. (2007b). 10 11 The best evidence of formaldehyde-induced clastogen ic changes in peripheral 12 lymphocytes is found in studies of anatomy class an d mortuary class students. Since genetic 13 damage accumulates with age, the studies in younger adults, where cells are analyzed before and 14 after exposure, may have greater sensitivity and fe wer confounding factors. 15 Suruda et al. (1993) showed a 12-fold increase in t he MN frequency of epithelial cells 16 from the buccal area of the mouth in mortuary scien ce students exposed to embalming fluids 17 containing formaldehyde following an 85-day exposur e period (Table 4-85). Overall, students 18 were exposed to 0.33 ppm (0.4 mg/m 3 ) formaldehyde as an 8-hour TWA on days when 19 embalming was performed (an average of 6.9 embalmin gs). Blood, oral, and nasal samples were 20 collected pre- and postexposure. As shown in Table 4-85, nasal epithelial MNs increased by 21 22% (frequency of micronucleated lymphocytes increa sed by 28%). By contrast, SCE frequency 22 decreased by 7.5% after formaldehyde exposure. 23

24 Table 4-85. MN and SCE formation in mortuary scien ce students exposed to 25 formaldehyde for 85 days 26 27

Sampling point Buccal mucosa (MN/1,000) Nasal epithelium

(MN/1,000) Blood

(MN/1,000) Blood

(SCEs/cell) Before course 0.046 ± 0.17 0.41 ± 0.52 4.95 ± 1.72 7.72 ± 1.26 After course 0.60 ± 1.27 a 0.50 ± 0.67 6.36 ± 2.03 a 7.14 ± 0.89 28

a Statistically significant ( p < 0.05), as calculated by the authors. 29 Source: Suruda et al. (1993). 30This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-365 Another group (Titenko-Holland et al., 1996) also reported a significant increase in MN 1 frequency of buccal, but not nasal, epithelial cell s from mortuary students exposed to embalming 2 fluid. In this study, 28 out of 35 students were s ampled before and after a 90-day embalming 3 class. The mean formaldehyde exposure for the subj ects providing data on buccal cell MNs was 4 14.8 ± 7.2 ppm-hours (18.2 ± 8.8 mg/m 3 -hours) for the entire 90-day period and 16.5 ± 5.8 ppm5 hours (20.3 ± 7.1 mg/m 3 -hours) for students providing data on nasal cell M Ns. Cells were 6 recorded as having either whole chromosomes with ce ntromeres (MN + ) or acentric fragments 7 and no centromeres (MN

– ). Cells with multiple nuclei were present only in samples taken after 8 exposure to embalming fluid. There was a ninefold increase in the MN frequency in buccal cells 9 ( p < 0.5) and only a twofold increase ( p > 0.05) in nasal cells. In addition, there was a twofold 10

increase in the MN + frequency in buccal cells (Table 4-86). The autho rs suggested that 11 chromosomal breakage appears to be the primary mech anism of MN formation. 12

13 Table 4-86. Incidence of MN formation in mortuary students exposed to 14 formaldehyde for 90 days 15 16 Buccal cells (n = 19) Nasal epithelial cells (n = 1 3)

Sampling point Total MN MN + MN – Total MN MN + MN – Pre-exposure 0.6 ± 0.5 0.4 ± 0.4 0.1 ± 0.2 2.0 ± 1. 3 1.2 ± 1.3 0.5 ± 0.5 Postexposure 2.0 ± 2.0 a 1.1 ± 1.3 0.9 ± 1.1 a 2.5 ± 1.3 1.0 ± 0.8 1.0 ± 0.6 a

p value 0.007 0.08 0.005 0.20 0.31 0.03 17

a Statistically significant at the level shown, as ca lculated by the authors. 18 19 Source: Titenko-Holland et al. (1996). 20 21 22 Ying et al. (1997), however, observed higher freque ncies of MNs in the nasal exfoliative 23 cells (3.85 ± 1.48 versus 1.20 ± 0.676, paired t-te st, p < 0.001) and oral exfoliative cells (0.857 ± 24 0.558 versus 0.568 ± 0.317, p < 0.001) after formaldehyde exposure, although the re was no 25 significant increase in the frequency of lymphocyte MNs ( p > 0.05) in students exposed to 26 formaldehyde in anatomy classes (three classes per week for 3 hours over an 8-week duration). 27 In this study, blood samples and nasal swabs were c ollected before and after the study. The 28 TWA concentration of formaldehyde in anatomy labora tories and student dormitories was 0.508 29 ± 0.299 mg/m 3 and 0.012 ± 0.0025 mg/m 3 , respectively, suggesting that nasal mucosa cells 30 exposed through respiration are the primary target of formaldehyde-induced genotoxicity. 31 In a different study (Ying et al., 1999), however, the same group showed that exposure to 32 formaldehyde affected the composition of lymphocyte subsets (B cells, total T cells, T helper33 inducer cells, T cytotoxic-suppressor cells), but n o significant difference was reported between 34 lymphocyte proliferation rate and SCEs at the given levels and durations of formaldehyde 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-366 exposure. This study involved 23 nonsmoking studen ts exposed to 0.508 ± 0.299 mg/m 3 1 formaldehyde for a period of 8 weeks (3 hours , 3 t imes per week). 2 Burgaz et al. (2002) reported significantly ( p < 0.05) higher mean MN frequencies in 3

buccal mucosal cells from shoe workers as well as a natomy and laboratory workers (0.62 ± 4 0.45% and 0.71 ± 0.56%, respectively) compared with unexposed controls (0.33 ± 0.30%). In 5 this study, the measured air concentrations of form aldehyde in the breathing zone of the anatomy 6 and pathology laboratory workers were between 2 and 4 ppm (2.5 and 5 mg/m 3 ). MN count per 7 3,000 cells was measured in buccal smears from shoe workers and from anatomy and pathology 8 staff, and eighteen male university staff were used as controls. 9 In a critical review, Speit and Schmid (2006) exami ned data from studies that have 10 reported the formation of MNs in nasal or buccal ce lls of persons either environmentally or 11 occupationally exposed to formaldehyde. The author s identified a number of issues relating to 12 study design, exposure regimen, and confounding fac tors, including MN levels in nasal and 13 buccal cells well below established background leve ls, reports limited by the number of cells 14 observed, variation in standard techniques, and non -concordance between buccal and nasal 15 findings. However, the authors concluded that, des pite these limitations, the weight of evidence 16 supports the finding that formaldehyde may be genot oxic in human cells in direct contact with 17 formaldehyde. 18

19

4.3.4.2.2. Peripheral blood lymphocytes. Mature lymphocytes are present at the POE as 20 intraepithelial lymphocytes and within germinal cen ters in the mucosa. Because more 21 lymphocytes may be available in the nasal mucosa th an the oral mucosa, mouth versus nose 22 breathing may contribute to variability in findings . Since some of the lymphocytes traffic around 23 the body, it is reasonable to find clastogenic effe cts in these relatively long-lived cells reflected 24 in peripheral blood lymphocytes. Thus, lymphocytes proliferating in response to antigen would 25 be more vulnerable to DNA reactivity of formaldehyd e and to the clastogenic effects in general. 26 A cytogenetic evaluation by Fleig et al. (1982) of 15 employees exposed for an average 27 of 28 years in a formaldehyde manufacturing plant r evealed no statistically significant increase 28 in the frequency of CAs in peripheral blood lymphoc ytes compared with a matched control 29 group. Likewise, in a different study (Thomson et al., 1984), no compound-related differences 30 were evident in the frequency of CAs and MNs in lym phocytes from six pathology workers and 31 five unexposed controls. 32 Bauchinger and Schmid (1985) observed an increased incidence of CAs (dicentric and 33 ring chromosomes) in the peripheral lymphocytes of 20 male paper mill workers and supervisors 34 exposed to formaldehyde (average exposure of 14.5 y ears) compared with unexposed workers. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-367 When workers and supervisors were analyzed separate ly, significant increases were only seen for 1 supervisors. The average length of exposure for su pervisors (n = 11) and workers (n = 9) was 2 18.9 years and 7.2 years, respectively. Informatio n regarding formaldehyde concentrations for 3 the two groups was not provided. However, the inci dence of SCEs among workers was actually 4 slightly lower than among the 20 controls. In cont rast, the frequency of SCEs in peripheral 5 lymphocytes of 18 nonsmoking formaldehyde workers w as increased over controls (8.24 ± 0.89 6

versus 6.38 ± 0.41) (Ye et al., 2005) (described in Section 4.3. 4.2.1). 7 Vargová et al. (1992) observed that the percentage of aberrant cells and number of breaks 8 per cell in the peripheral blood lymphocytes of for maldehyde-exposed workers was 3.08 and 9 0.045 versus 3.6 and 0.080 in controls in a pressed board factory, respectively, suggesting both 10 groups to be at an increased risk. However, normal unexposed population had only 1–2% 11

aberrant cells. The authors also noted that the mi totic index was significantly decreased in 12 exposed workers compared with controls. 13 Kitaeva et al. (1996) evaluated the genotoxic effec ts of formaldehyde among 15 14 industrially exposed workers and 8 academic laborat ory instructors and observed an increase in 15 the frequencies of CAs and MNs in the lymphocytes o f exposed subjects compared with 16 6 unexposed controls. 17 Shaham et al. (1996, 1997) found significantly high er levels of DPXs and SCEs in 18 peripheral blood lymphocytes of workers occupationa lly exposed to formaldehyde (physicians 19 and technicians) compared with unexposed control wo rkers. The authors also observed a linear 20 relationship between years of exposure to formaldeh yde and levels of DPXs and SCEs. 21 Formaldehyde-induced genotoxicity has also been rep orted in peripheral blood 22 lymphocytes of anatomy class students and mortuary workers. Vasudeva and Anand (1996) did 23 not observe significant differences in the incidenc es of CAs between the formaldehyde exposed 24 students and the matched, unexposed controls. In t his study, peripheral blood lymphocytes from 25 30 medical students exposed to formaldehyde in a gr oss anatomy laboratory for 15 months with 26 average exposures of less than 1 ppm (1.23 mg/m 3 ) formaldehyde were used. 27

He et al. (1998) used the cytokinesis-blocked MN (CBMN) assay to detect the frequency 28 of micronucleated peripheral lymphocytes in 13 stud ents exposed to formaldehyde during a 29 12-week (10 hours/week) anatomy class. Sampling of breathing zone air showed a mean 30 concentration of 2.37 ppm (3.17 mg/m 3 ). Ten students from the same school, without expo sure 31 to formaldehyde, were used as controls. CAs and SC Es were observed in both groups, and there 32 were significant increases ( p < 0.01) in the frequencies of micronucleated cells and CAs in the 33 formaldehyde-exposed group compared with the contro l group. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-368 In a study involving 97 plasticware workers (34 mal es and 63 females) exposed to 0.5 to 1 0.9 mg/m3 formaldehyde, 4.4 to 6.2 mg/m3 styrene an d 0.5 to 0.75 mg/m3 phenol for 2 months 2 to 25 years, Lazutka et al (1999) observed signifi cantly higher CAs than controls (non-exposed 3 donors matched by age and similar smoking habits as the exposed workers). Although workers 4 with short and long exposures showed significant in creases in the frequency of CAs, the 5 cytogenetic damage did not increase with exposure d uration. 6 Sari-Minodier et al. (2001), using the CBMN assay i n anatomy/pathology laboratory 7 workers, reported higher frequency of micronucleate d peripheral blood lymphocytes than in 8

matched controls. 9 Shaham et al. (2002) observed a mean number of 0.27 SCEs per chromosome in the 10 peripheral lymphocytes of an exposed cohort compare d with 0.19 in controls ( p < 0.01). This 11 study involved 90 individuals employed in 14 hospit al pathology laboratories and 52 unexposed 12

controls. 13 Yu et al. (2005) reported dose-dependent increase i n MNs and comet assay parameters 14 (olive tail moment and comet tail length) in periph eral lymphocytes in 151 workers from two 15 plywood factories compared with 112 unexposed contr ols. The TWA exposure level in the 16 working environment was 0.1–7.88 mg/m 3 (0.08–6.42 ppm) formaldehyde compared with a 17 background level of <0.01 mg/m 3 (<0.008 ppm) formaldehyde applicable to controls. In the 18 comet assay, the authors observed olive tail moment s averaging 0.93 (0.78–1.1), 3.03 (2.49– 19 3.67), and 3.95 (3.53–4.43) for control, low-, and high-exposure individuals, respectively. For 20 the same subjects, comet tail lengths were 6.78 (6. 05–7.6), 11.25 (10.12–12.5), and 12.59 (11.8– 21 13.43), respectively. In the CBMN assay, MNs/100 c ells were 0.27 ± 0.13, 0.41 ± 0.25, and 22 0.65 ± 0.36, respectively, for control, low-, and h igh-exposure individuals. 23 In a population of 18 workers exposed to formaldehy de at a plant in China, with a mean 24 employment of 8.5 years (range 1 to 15 years), Ye e t al (2005) examined nasal and lymphocytes 25 for cytogenetic effects. This study also included a second group of 16 waiters who worked in a 26 newly fitted ball room for 12 weeks with a low leve l exposure to formaldehyde from building 27 material, tobacco smoke and furniture and a group o f 23 college students as a control group. The 28 background indoor air conentraton of 0.009 ppm form aldehyde was reported in students’ dorms. 29 Significantly increased frequencies of MNs in the nasal mucosal cells and SCEs in peripheral 30 blood lymphocytes were reported for the workers, bu t not the waiters in this study. 31 Orsière et al. (2006) reported no apparent effect o n the DNA damage in peripheral blood 32 lymphocytes as assessed by a chemiluminescence micr oplate assay in pathology and anatomy 33 laboratory workers (n = 59) before and after a 1-da y exposure to formaldehyde. This study had 34 59 exposed workers and 37 controls. However, with the CBMN assay, the authors reported 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-369 statistically significant differences in the freque ncy of binucleated micronucleated cells 1 (1.69 ± 0.93 versus 1.11 ± 0.6%) in exposed versus control subjects. Discrimination between 2 clastogenic and aneugenic events by using FISH with a pan-centromeric DNA probe resulted in a 3 higher rate of binucleated micronucleated cells (1. 91 ± 1.01 versus 1.19 ± 0.56% in controls) and 4 showed that the frequency of centromeric nuclei was higher in the exposed group than in 5 controls, though not significantly. Among the cent romeric MNs, the frequency of MNs with 6 only one centromere (C1+MN) was significantly great er in pathologists/anatomists than in 7 controls (1.1 ± 0.62 versus 0.31 ± 0.24%, p < 0.001). The authors interpreted their data on 8 monocentromeric nuclei in anatomists/pathologists a s an indication that formaldehyde exposure 9 might be associated with aneugenic (rather than cla stogenic) events. 10 Based on pooled analysis of two reports (Iarmarcova i et al., 2006a, b) (Table 4-87), MN 11 frequency ratios in the peripheral lymphocytes of c ancer patients, welders, and 12 anatomists/pathologists were significantly increase d compared with the corresponding controls. 13 The data were taken from three biomonitoring studie s by using CBMN/FISH. The incidence of 14 MNs was scored and then evaluated further for the p resence of centromere-negative MNs 15 (C−MNs), centromere-positive MNs (C+MNs), and, for the latter case, those containing a single 16 centromere (C1+MNs) and those containing two or mor e centromeres (Cx+MNs). Applying 17 their findings to considerations of the aneugenic m echanism of action of formaldehyde, the 18 authors hypothesized that the use of centromeric si gnals enables the identification of endpoints 19 representing impaired chromosomal migration (with C 1+MN formation) or centrosome 20 amplification (with Cx+MN formation). 21

22 Table 4-87. Multivariate repression models linking genomic 23 instability/occupational exposures to selected endp oints from the MN assay 24 25 Study populations Number MN a C−MN C+MN C1+MN Cx+MN Cancer patients versus controls 10/10 1.85 (1.18−2.87) 2.05

(1.07−3.94) 1.81

(1.02−3.21) 1.68

(0.80−3.53) 1.28

(0.63–2.59) Welders versus controls 27/30 1.37 (1.09−1.72) 1.39

(0.99−1.95) 1.37

(1.03−1.83) 1.10

(0.80−1.53) 1.31

(0.99−1.74) Pathologists/anatomists versus controls 18/18 1.28 (0.86−1.90) 0.79

(0.46−1.36) 1.65

(1.05−2.59) 3.29

(2.04−5.30) 0.68

(0.38−1.20) 26

a Bolded values indicate statistical significance ( p < 0.05). 27 28 Source: Iarmarcovai et al. (2006b). 29 30 31 Recently, Costa et al. (2008) observed a significan t increase in the genotoxicity of 32 formaldehyde-exposed pathological anatomy laborator y workers (n = 30) compared with 33 controls (n = 30) in cytogenetic assays. In this s tudy, the authors evaluated the level of exposure 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-370 to formaldehyde near the breathing zone of workers, and TWA of exposure was calculated for 1 each subject, giving a mean level of exposure to be 0.44 ± 0.08 ppm (range: 0.04–1.58 ppm). As 2 compared with control subjects, peripheral blood ly mphocyte cultures of formaldehyde exposed 3 workers showed significant increases in MN frequenc y (5.47 ± 0.76 versus 3.27 ± 0.69; p = 4 0.003), SCEs (6.13 ± 0.29 versus 4.49 ± 0.16; p < 0.05), and comet assay as determined by tail 5 length (TL) (60.00 ± 2.31 versus 41.85 ± 1.97; p < 0.05). In addition, Costa et al. (2008) 6 observed a positive correlation between formaldehyd e exposure levels and MN frequency ( r = 7 0.384; p = 0.001) and TL ( r = 0.333; p = 0.005) (Table 4-88). However, polymorphic genes of 8 xenobiotic metabolizing and DNA repair enzymes did not show any significant effect on the 9 genotoxic endpoints. This is the lowest level of e xposure to formaldehyde in the studies 10 observed so far, wherein a clear indication of geno toxic effects of formaldehyde was 11

demonstrated. 12

13 Table 4-88. Genotoxicity measures in pathological anatomy laboratory 14 workers and controls 15 16

MN assay SCEs Comet assay Mean MN ± SEM (range) Mean SCE ± SEM (range) Mean TL ( μ M) ± SEM (range) Controls (n = 30) 3.27 ± 0.69 (0–17) 4.49 ± 0.16 (3.10–3.06) 41.85 ± 1.97 (28.85–66.52) Exposed (n = 30) 5.47 ± 0.76 (1–17) 6.13 ± 0.29 (3.64–8.80) 60.00 ± 2.31 (33.76–99.09)

p value 0.003 <0.05 <0.05 17 Source: Costa et al. (2008). 18 19 4.3.5. Summary of Genotoxicity 20 Formaldehyde’s genotoxicity has been demonstrated i n a variety of in vitro and in vivo 21 test systems measuring a variety of genetic endpoin ts. Formaldehyde forms predominantly 22 DPXs that are detected in cell-free systems and sin gle cells in vitro. DPXs are formed in nasal 23 epithelia but not in extra-nasal passages of rodent s, which are completely removed within a day 24

after formation. In vivo data in human and mammali an cells demonstrate that formaldehyde is 25 genotoxic at the site of first contact, including c ells of the mouth or the nose. DPXs are also 26 detected in nasal and extra-nasal tissues of monkey s, suggestive of direct effects of formaldehyde 27 in tissues that correspond to observed tumor sites (nasal and nasopharynx) in humans. In 28 addition, this is used as a basis for cross-species comparison with humans. Formaldehyde-DNA 29

adducts are labile and constitute a minor fraction of the DNA-reaction products and are less 30 likely to play an important role in the genotoxicit y of formaldehyde. 31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-371 Formaldehyde clastogenicity has been demonstrated b y the induction of SCEs, SSBs, 1 MNs, and CAs in cultured mammalian cells. Formalde hyde induces mutations in salmonella and 2

escherichia bacterial strains that contain an AT ba se pair at the primary reversion site that is used 3 to detect oxidative compounds and cross-linking age nts without metabolic activation by 4 exogenous enzyme-activating systems. Formaldehyde induces mutations in cultured mammalian 5 cells at levels that do not cause significant toxic ity. Despite formaldehyde’s reactivity and 6 mutagenicity in isolated mammalian cells, clear evi dence of mutagenicity does not emerge from 7 animal bioassays. 8 Formaldehyde exposure causes differential induction of MNs in human nasal epithelial 9 and buccal epithelial cells, which is significant i n industrial exposure workers and students 10 working in anatomy or mortuary science, respectivel y. However, recent data and data from 11 larger studies support a finding of increased MNs i n blood lymphocytes, although the issue 12 remains controversial because of issues relating to study design, exposure regimen, and 13 confounding factors, including MN levels in nasal a nd buccal cells well below established 14 background levels, reports limited by the number of cells observed, variation in standard 15 techniques, and non-concordance between buccal and nasal findings (Speit and Schmid, 2006). 16 Several clastogenic effects, such as induction of M Ns, SCEs, and CAs, were seen in human 17 peripheral blood lymphocytes; however, the data are not very clear. Formaldehyde exposure also 18 caused p53 mutations in rat nasal carcinomas with t he expression of mutant p53 protein. 19 Overall, induction of DPXs as a predominant lesion in vitro and in vivo, clastogenicity, 20 and mutagenicity with locus-specific mutations in n onhuman and human cells supports the 21 concept of genotoxic action of formaldehyde at the POE. 22 A summary of the genotoxicity of formaldehyde in hu mans is presented in Table 4-89. 23

24 4.4. SYNTHESIS AND MAJOR EVALUATION OF NONCARCINOG ENIC EFFECTS 25 The adverse health effects due to formaldehyde expo sure have been extensively studied 26

in humans and in animal models. Studies of human e xposure include occupational exposures, 27 environmental exposures, and clinical studies of in tentionally exposed subjects (Section 4.1). 28 Occupational exposures are primarily due to inhalat ion and dermal contact. Animal studies are 29 available for a variety of routes of exposure, incl uding inhalation, oral, dermal, and intravenous 30 and I.P. injections (Section 4.2). Additionally, a s discussed in Chapter 3, in vitro studies address 31 biological activity and the metabolic fate of forma ldehyde. 32This document is a draft for review purposes only a nd does not constitute Agency policy 4-372 DRAFT—DO NOT CITE OR QUOTETable 4-89. Summary of human cytogenetic studesExposure time (years)Formaldehydeconcentration (ppm)CytogeneticobservationsStudy populationNRange Mean Range Mean (TWA)CAsSCEsMNsReferenceAnalyses of nasal and/or buccal cellsPlywood workers 15Age and sex matched controls 152–19 6.8 0.32–0.83(1) + nasal Ballarin et al. (1992)+ buccalMale mortuary science students 22– nasal– buccalFemale mortuary science students 7Buccal and nasal swabstaken before and afterfirst 9 weeks ofembalming course0.1–4.3 1.4– nasalSuruda et al. (1993)28+buccalbMortuary science studentsaBuccal and nasal swabstaken before and afterfirst 9 weeks ofembalming course0.1–4.3 1.4– nasalTitenko-Holland et al. (1996)Female anatomy faculty 8 NA 23.6 NA NA + buccalMale anatomy faculty 5 25.6 – buccalControls (Females) 7Kitaeva et al. (1996)25 0.06–1.06(0.508) + buccalAnatomy studentsBuccal and nasal swabstaken before and after8-week anatomy course+ nasalYing et al. (1997)Anatomy/ pathology staff 28 1–13 4.70 2–4 NA +buccalControls (University staff) 18Burgaz et al. (2002)Workers at a formaldehyde plant 18 1–15 8.5 0.8+ nasalControls 23Ye et al. (2005)Volunteers 2110 days13.5 ppm-hours – buccal Speit et al. (2007b)Analyses of peripheral lymphocytesManufacturing workers 15 23–35 28 <5 1971 –Age and sex matched controls 15<1 laterFleig et al. (1982)Pathology workers 6 4–11 0.9–5.8 –Controls 5Thomson et al. (1984)This document is a draft for review purposes only a nd does not constitute Agency policy 4-373 DRAFT—DO NOT CITE OR QUOTETable 4-89. Summary of human cytogenetic studesExposure time (years)Formaldehydeconcentration (ppm)CytogeneticobservationsStudy populationNRange Mean Range Mean (TWA)CAsSCEsMNsReferenceAnatomy studentsc8 1.08–1.99d1.2 +10-week class0.08–0.6e0.3Yager et al. (1986)Papermakers 20 2–30 14.4 <3 NA +f–Controls 20Bauchinger and Schmid (1985)Wood workers 25 <5 to <16 0.45–8.6 NA –Controls 19Vargová et al. (1992)Male embalming students 22– +Female embalming students 7Blood sampled beforeand after first 9 weeksof embalming course0.15–4.3 1.4– –Suruda et al. (1993)Manufacturing workers 15 10 Up to 4 NA + +Anatomy faculty 8 17 NA NDControls 6Kitaeva et al. (1996)Medial students 30 <1 NA –Controls 30Sampled near end of15-month term-Vasudeva and Anand (1996)Anatomy students 13 12-week class2.37g+ + +He at al. (1998)Controls (students) 10Physicians 6 2-24 10 3.1-2.8 1.6 +Technicians 7 2-25 15 +Controls (age matched/unexposed) 20Shaham et al 1997Anatomy students 23-25Blood samples takenbefore and after 8-weekanatomy course0.06–1.06(0.508)-– – Ying et al. (1999, 1997)Female anatomy/pathology lab workers10 1–16 8.9 1.2–15.1 NA +Sari-Minodier et al. (2001)Controls (Women) 27Hospital pathology workersh90 1–39 15.4 0.04–0.7i0.4 +jControls 520.72-5.6 2.24 +Shaham et al. (2002)This document is a draft for review purposes only a nd does not constitute Agency policy 4-374 DRAFT—DO NOT CITE OR QUOTETable 4-89. Summary of human cytogenetic studesExposure time (years)Formaldehydeconcentration (ppm)CytogeneticobservationsStudy populationNRange Mean Range Mean (TWA)CAsSCEsMNsReferenceWorkers at a formaldehyde plant 18 1–15 8.5 0.8+ –Controls 23Ye et al. (2005)Workers at two plywood factories 151 ND 0.08–6.42 +Controls 112Yu et al. (2005)Pathology or anatomy workers 59 ND <0.1–20.4k2k+Controls 37Orsière et al. (2006)Pathologists 18 ND 0.4–7.0k2.3k+Controls 18Iarmarcovai et al. (2006a, b)Pathological anatomy lab workers 30 0.5–27 11 0.04–1.580.44 + +Controls (21 females and 9 males) 30Costa et al. (2008)Plasticware workers 97+Controls (non-exposed donors) 902 mo to 25yrs0.5-0.9mg/m3Lazutka et al 1999Wood workers 40 +Controls 22 NR NRChebotarev et al 1986School children (1984) 20 0.26 +School children (1985) 16 0.11 +School children (1986) 18 0.03Controls (1984) 17 0Preschool controls (1984) 24 0Preschool children (1984) 13 0.17-0.3Neri et al 2006Phenolformaldehyde resin workers 310.41 +Controls 740.33-30 yr0Suskov and Sazonova 1982aSame population in Suruda et al. (1993) but different slides used. Nineteen complete slide sets for buccal analysis and 13 complete slide sets for nasalepithelialcell analysis.bNot dose related; both low- and high-exposure groups had same SCE increase.cEach student sampled before and after 10-week anatomy class.dBreathing zone samples.This document is a draft for review purposes only a nd does not constitute Agency policy 4-375 DRAFT—DO NOT CITE OR QUOTEeRoom air samples.fIncrease only in 11 supervisors. See text for details.gAverage breathing zone during dissection procedure.hExposed and controls from 14 hospitals.iLow- and high-exposure groups established but numbers not provided.jNot dose related; both low and high groups had sameSCE increase.kDescribed as “mean concentrations for sampling times of 15 minutes.”CAs = chromosomal aberrations; SCEs = sister chromatid exchanges; MNs = micronuclei; TWA = time-weighted average; ND = not determined; NA = notapplicable.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-376 Taken together, the human and animal studies suppor t numerous health effects, not only 1 at the POE as expected for a reactive gas but also on pulmonary function, neurobehavioral 2 function, reproduction, development, immunomodulati on, and sensitization (atopy, asthma). The 3 discussion below provides a description of the adve rse effects seen in each area, summarizing the 4

data for both human and animal studies. MOA data a re discussed where information regarding 5 formaldehyde’s biological activity may be linked to the observed adverse health effects. 6

7 4.4.1. Sensory Irritation 8 Sensory irritation of the eyes, nose, and throat is reported in humans upon direct contact 9 with formaldehyde gas during inhalation exposures ( Holmström and Wilhelmsson, 1988; 10 Ritchie and Lehman, 1987) and includes irritation r esulting from acute exposures (Lang et al., 11 2008; Yang et al., 2001; Krakowiak et al., 1998; Ku lle, 1993; Green et al., 1989, 1987; Kulle et 12 al., 1987; Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 13 1984; Bender et al., 1983; Weber-Tschopp et al., 1 977). Controlled exposures in inhalation 14 chambers confirm the specificity of these responses to formaldehyde exposure and allow for 15 assessment of these symptoms through both subjectiv e and objective measures (Kulle, 1993; 16 Holness and Nethercott, 1989; Green et al., 1987; K ulle et al., 1987; Sauder et al., 1986; Weber17 Tschopp et al., 1977). Eye irritation may be repor ted as itching, burning, and general discomfort. 18 Tearing, redness of the eyes, and increased blink f requency are observed and may be quantified 19 in exposure under controlled conditions (Lang et al ., 2008; Yang et al., 2001; Andersen and 20 Molhave, 1983; Weber-Tschopp et al., 1977; Schuck e t al., 1966). Eye irritation appears to be 21 the most sensitive endpoint in most individuals and may be observed after short exposures 22 (195 minutes at 0.5 ppm: Lang et al. [2008]; 30 sec onds at 1.65 ppm: Yang et al. [2001]). 23 Itching, burning, and discomfort of the nose, which may be accompanied by increased 24 mucous production (runny nose), are reported by ind ividuals exposed via inhalation (Krakowiak 25 et al., 1998; Kulle, 1993; Green et al., 1987; Kull e et al., 1987; Weber-Tschopp et al., 1977). 26 Throat irritation may also be described subjectivel y as itching and burning and is often 27 accompanied by a cough (Krakowiak et al., 1998). S ymptoms of eye and mucous membrane 28 irritation are also reported in numerous rodent stu dies and support the health effects reported in 29 humans (see Section 4.1.1.1). Although dermal cont act may result in dermatitis and an apparent 30 hypersensitivity reaction, symptoms do not present upon contact as sensory irritation. There are 31 no human or animal data that assess sensory irritat ion from oral exposures. 32 The time to onset of sensory irritation symptoms an d severity of the sensory irritation are 33

a function of both the air concentration and durati on of exposure. Additionally, nose and throat 34 irritation becomes more prominent at higher exposur es and longer duration of exposure (Kulle, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-377 1993; Kulle et al., 1987). Controlled human labora tory exposures (Yang et al., 2001; Kulle, 1 1993; Kulle et al., 1987; Cain et al., 1986; Anders en and Molhave, 1983) provide more direct 2 exposure-response evidence for sensory irritation. These studies are limited to healthy 3 nonsmoking individuals. Two studies (Cain et al., 1986; Andersen and Molhave, 1983) 4 document discomfort and irritation of the eye in re sponse to acute exposures as low as 0.25 ppm. 5 Dose-response relationships are reported in a numbe r of different ways: as an incidence of the 6 reported symptom among subjects, as a score for sev erity of the symptom, or in some cases as a 7 subjective measure, such as blink frequency for eye irritation. 8 Symptoms of sensory irritation, including eye irrit ation (burning watering, increased 9 blinking), nasal irritation (rhinitis, itching/burn ing), throat/respiratory tract irritation (wheezing

, 10 coughing, phlegm production), have been reported in numerous worker cohorts. Occupational 11 exposure environments include hospital and medical settings, students, and industrial workers 12 (Takahashi et al., 2007; Takigawa et al., 2005; Kra kowiak et al., 1998; Akbar-Khanzadeh et al., 13 1994; Uba et al., 1989; Horvath et al., 1988; Schac hter et al., 1987). Formaldehyde levels often 14 vary in a work environment and peak as well as aver age exposures may be used to report 15 occupational exposures. Although sensitive individ uals often remove themselves from an 16 irritating workplace (the HWE), eye, nose, and thro at symptoms are still reported in this 17 environment. Among workers in a plant where formal dehyde resins were used, those exposed to 18 an average of 210 ppb formaldehyde reported increas ed symptoms above those in the control 19 population (Holmström and Wilhelmsson, 1988). 20 These effects have been noted in students, particul arly medical students, who are exposed 21 to formaldehyde in cadaver labs. In a study of 24 formaldehyde-exposed anatomy students 22 (personal breathing zone samples 0.73 ppm, range 0. 49–0.93) (Kriebel et al., 1993), eye, nose, 23 and throat irritation was present when comparing ra tes of irritation from the end or middle of 24

class to before the start of class. Takahashi et a l. (2007) showed that 143 medical students 25 reported various symptoms (including eye and throat irritation) and that the percentage of 26 students reporting symptoms increased between the b eginning (measured after the first day of 27 class) and the end of the course (2 months later). After the first day of class, approximately 35% 28 of students reported eye soreness and about 15% rep orted throat irritation. 29 Sensory irritation has also been noted in occupatio nal settings. Horvath et al. (1988) 30 compared irritation symptoms between 109 workers at a particleboard manufacturing plant and 31 264 workers at food plants. Eye, nose, and throat irritation were more common among the group 32 in a particleboard manufacturing facility, exposed to a mean concentration of 0.40 mg/m 3

. 33 Similarly, Alexandersson and Hedenstierna (1988) re ported that the frequency of eye, nose, and 34 throat irritation was significantly greater (65.8%) in 38 workers exposed to formaldehyde and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-378 solvents in lacquers as compared with 18 nonexposed individuals working at the same factory 1 (16.7%). Holmström and Wilhelmsson (1988) conducte d a study at a chemical plant and 2 reported nasal and eye discomfort in 64 and 24%, re spectively, of workers (n = 70) exposed to 3 formaldehyde (range 0.05–0.50 mg/m 3 with a mean of 0.26 mg/m 3 ) versus 25 and 6%, 4 respectively, in nonexposed desk clerks (n = 36). Holness and Nethercott (1989) reported 5 significant increases in eye irritation (42 versus 21%) and nose irritation (44 versus 16%) among 6 84 funeral service workers (active embalmers, >10-y ear experience) as compared with 7 38 students and individuals from a service organiza tion. The exposure concentration in both 8 groups was 0.36 and 0.02 ppm, respectively. 9 Reports of similar symptoms are correlated to indoo r residential exposures, providing 10 exposure-response relationships for the general pop ulation in low-level chronic exposure 11 scenarios. Ritchie and Lehnen (1987) surveyed resi dents in 2,000 homes classified as having 12 formaldehyde concentration <0.1 ppm, 0.1–0.3 ppm, a nd >0.3 ppm. A LOAEL of 200 ppb was 13

established from the results of Ritchie and Lehnen (1987). Liu et al. (1991) report irritant effects 14 associated with formaldehyde exposure in mobile hom es, where formaldehyde concentrations 15 ranged from the 0.01 ppm detection limit to 0.46 pp m. Eye irritation (60%), nose/throat 16 irritation (10–20%), or headache (<10%) were report ed in residents. 17

18

MOA 19 The mucosae of the URT, oral cavity pharynx, and up per airways are complex tissues, 20 where epithelial and goblet cells predominate. In addition, the nasal mucosa is highly enervated. 21 The main nerves include the trigeminal nerve and ol factory sensory cells (olfactory epithelium, 22 the vomeronasal organ, and the organ of Masera) (Fe ron et al., 2001). A possible MOA for 23 sensory irritation includes formaldehyde-induced st imulation of the trigeminal nerve (though 24 whether formaldehyde acts as a direct agonist is un known). Trigeminal nerve stimulation in the 25 nasal passages transmits signals to the CNS, which then sends efferent signals back to the nasal 26 tissues, causing sensory irritation, and possibly s ystemically via vagal nerve stimulation, 27 resulting in more systemic effects. 28 Animal studies are potentially useful models for un derstanding mechanisms of toxicity, 29 especially where sufficient human data do not exist . While experimental animal studies provide 30 a model of secondary effects, rodents also demonstr ate RB, an effect not seen in humans. Thus, 31 species that exhibit bradypnea (like mice and rats) may not be appropriate for assessing 32 respiratory endpoints. The mechanism underlying RB includes formaldehyde binding to the 33 sensory nerve endings of the trigeminal nerve, wher e signals travel to the CNS. The vagus nerve 34 transmits the efferent signal to produce smooth mus cle contraction. The animals become 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-379 inactive, their core temperatures decrease by sever al degrees C, and their respiratory rates and 1 minute volumes decrease. However, this is not to s ay that trigeminal nerve stimulation is not an 2 appropriate potential mechanism of action in other species or in humans. Since trigeminal nerve 3 stimulation has been independently confirmed in spe cies without RB, this mechanism may be a 4 viable explanation for the observed effects. 5

6 4.4.2. Pulmonary Function 7 Workers chronically exposed to formaldehyde have ex hibited signs of reduced lung 8 function, such as BC, inflammation, and chronic obs tructive lung disease. Lung function deficits 9 have been reported in pre- versus post-shift measur ements and as a result of chronic exposures 10 (Pourmahabadian et al., 2006; Herbert et al., 1994; Malaka and Kodama, 1990; Alexandersson 11 and Hedenstierna, 1989; Alexandersson et al., 1982) . Decreases in spirometric values, including 12 VC, FEV, FVC, and FEV/FVC, have been reported. Chr onic studies (Pourmahabadian et al., 13 2006; Herbert et al., 1994; Malaka and Kodama, 1990 ; Alexandersson and Hedenstierna, 1989; 14 Alexandersson et al., 1982) also report increased r espiratory symptoms, including cough, 15 increased phlegm, asthma, chest tightness, and ches t colds, in exposed workers. 16 Students have also shown decrements in lung functio n that are associated with exposure 17 to formaldehyde in laboratories. Kriebel and colle agues (1993) observed a 2% decrement in PEF 18 in healthy students attending anatomy classes once per week and a 7.3% decrement in PEF in 19 students with histories of asthma. The strongest p ulmonary response was observed when 20 examining the average cross-laboratory decrement in PEF in the first 2 weeks of the study 21 (formaldehyde geometric average concentration of 0. 73 ppm). These findings were corroborated 22 by Kriebel et al. (2001) in which a similar study d esign was applied to another class of anatomy 23

students. 24 Similarly, Akbar-Khanzadeh et al. (1994) compared p re- and postexposure pulmonary 25 function among students before and after working 3 hours in a laboratory (n = 34). On average, 26 FVC decreased by 1.4%, FEV 3 decreased by 1.2%, FEV 1 /FVC increased by 1.6%, and FVC -25– 27

75% increased 2.5%. These average percent changes in the control group are –0.3%, 1.30%, 28 2.31%, and 0.6% but were not statistically signific ant. In a follow-up study, Akbar-Khanzadeh 29 and Mlynek (1997) recorded FEV values in 50 exposed students and 36 controls and reported a 30 larger increase in lung function among controls whe n compared with cases after 1–3 hours of 31 exposure that persisted after 3 hours after exposur e termination. In a similar study, Fleisher 32 (1987) reported that approximately 8% of students r eported experiencing shortness of breath 33 during the laboratory with formaldehyde exposure, b ut none of the students reported shortness of 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-380 breath in the laboratory session with no exposure. However, no objective measurements of 1 formaldehyde exposure were used. 2 Unlike the study by Kriebel et al. (1993), Uba et a l. (1989) did not find a change in 3 pulmonary function over the course of the 7 months in a study of 96 anatomy laboratory 4 students. These negative findings may be attribute d to differential cross-shift exposures and to 5 significant differences in FVC on exposed days. 6 Deficits in pulmonary function have been reported i n occupational or residential exposure 7 studies (Khamgaonkar and Fulare, 1991; Krzyzanowski et al., 1990; Malaka and Kodama, 1990; 8 Alexandersson and Hedenstierna, 1989; Kilburn et al ., 1985; Alexandersson et al., 1982). 9 Krzyzanowski et al. (1990) documented a significant ly decreased PEFR in children 10 (298 children) who resided in homes with an average formaldehyde concentration of 26 ppb 11 (maximum sample value of 140 ppb). Among adults, t here was a statistically significant 12 nonlinear relationship with decreased morning PEFR for formaldehyde concentration <40 ppb 13 (Krzyzanowski et al., 1990). Similarly, Malaka and Kodama (1990) reported that an average 14 8-hour TWA formaldehyde exposure of 1.13 ppm from a rea samples was associated with 15 statistically significant decrements in FEV 1 , FEV 1 /FVC, and FEF 25–75% compared with a referent 16 population. Alexandersson and Hedenstierna (1989) investigated not only the acute effects of 17 exposure across shift but also measured effects of exposure among some of the same workers 18 that had been studied 5 years earlier (Alexandersso n et al., 1982). Statistically significant 19 decreases (

p < 0.01) in FEV 1 /FVC and FEF 25–75% were noted over the intervening five years in 20 nonsmokers after correcting for aging. Similar dec rements have been documented in laboratory 21 workers in India (Khamgaonkar and Fulare, 1991) and in factory workers (Kilburn et al., 1985). 22 Alexandersson et al. (1982) reported only slight de ficits in lung function 1 day following 23 occupational formaldehyde exposure in a carpentry s hop in Sweden, where the measured 24 formaldehyde level was 0.36 ppm (0.47 mg/m 3 ). In this case, subjects were compared with 25 20 nonexposed workers. 26

Other studies have found no association between for maldehyde and lung function 27 (Ostojic et al., 2006; Holness and Nethercott, 1989 ; Holmström and Wilhelmsson, 1988; Horvath 28 et al., 1988). Ostojic et al. (2006) used an inter esting measurement, “diffusing lung capacity” 29

instead of decrements in FEV1 or similar measuremen ts. Similarly, Nunn et al. (1990) assessed 30

the decrease in FEV 1 with age and showed no association between formald ehyde exposure and 31

decreased FEV 1 . Franklin et al. (2000) did not report an associa tion between FVC or FEV and 32 the indoor concentrations of formaldehyde in childr en (ages 6–13), although there were signs of 33 lower airway inflammation as measured by levels of exhaled NO (Franklin et al., 2000). 34 Similarly, Main and Hogan (1983) did not observe di fferences between FEV 1 or FVC at the end 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-381 of the 34 months between mobile home trailer worker s compared with controls who did not work 1 in trailers. The average exposure was reported as ranging from 0.12 to 1.6 ppm. 2 Occupational studies share certain limitations, inc luding the potential for confounding by 3 occupational co-exposures. Also, studies that did not report pre-shift pulmonary function as a 4 percentage of expected function are less useful to assess potential chronic effects because, 5 post hoc, it is difficult to calibrate for cross-st udy comparison due to lack of data on important 6 pulmonary function determinants, such as age, gende r, smoking status, height, and year of birth. 7 Controlled human studies and studies in nonhuman pr imates also document changes in 8 formaldehyde-induced pulmonary dysfunction. Acute exposures of healthy non-asthmatic 9 volunteers resulted in transient decreases in pulmo nary function (e.g., decreased FEV 1 , FVC 1 , 10

FEV 3 , specific airway conductance) (Green et al., 1987; Sauder et al., 1986). Green et al. (1987) 11 noted differential responsiveness in formaldehyde-e xposed subjects; some were responders while 12 others were nonresponders. This differential respo nse suggests susceptibility in certain subjects 13 (Green et al., 1987). 14 Several animal studies document increased airway re sistance and BC following 15 inhalation exposure to formaldehyde (Nielson et al. , 1999; Swiecichowski et al., 1993; Biagini et 16 al., 1989; Amdur et al., 1960). A study using cyno molgus monkeys (Biagini et al., 1989) 17

demonstrated that methacholine-induced BC can be si milarly induced by acute formaldehyde 18 exposure (10 minutes at 2.5 ppm). Thus, formaldehy de exposure simulated BC observed after 19 methacholine challenge, but these effects may not o ccur by a similar MOA. Similar results were 20 reported in guinea pigs (Swiecichowski et al., 1993 ; Amdur et al., 1960), rats (Ohtsuka et al., 21 1997), and mice (Nielson et al., 1999). 22 Deficits in pulmonary function have been documented in occupational as well as 23

controlled chamber human studies and have been corr oborated in animal studies exposed to 24 formaldehyde. However, some of these deficits are slight or transient. Some studies did not 25 identify a statistically significant decrease in pu lmonary function, and others did not observe any 26 change at all. Pulmonary function alterations appe ar to be specifically tied to exposure regimen 27 and may be reversible but remain, nevertheless, an important symptom often associated with 28 exposure to formaldehyde. 29

30

MOA 31 Formaldehyde-induced inflammation of the airways ma y contribute to observed 32 decreases in measures of pulmonary function. Even short-term inflammatory reactions could 33

reduce the effective diameter of the conductive air ways, resulting in lower respiratory volumes in 34 a number of functional tests. Formaldehyde-induced trigeminal nerve stimulation contributes to 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-382 airway inflammation, which in turn would reduce air way function. Chronic exposures may 1

result in increased sensitization or chronic inflam matory responses, which could contribute to the 2 effects seen in the worker and residential populati ons. 3 Formaldehyde-induced pulmonary function deficits ma y also be in part a result of smooth 4 muscle contraction in repose to trigeminal nerve st imulation. Trigeminal nerve stimulation 5 transmits signals to the CNS. The resulting effere nt signal from the vagal nerve produces 6 smooth muscle contraction and may result in decreas ed pulmonary function. Efferent signaling 7

has also resulted in release of substance P and oth er neuromodulatory compounds, which may 8 contribute to BC and sensitization of pulmonary res ponses (asthma, atopy). 9

10 4.4.3. Hypersensitivity and Atopic Reactions 11 Sensitization to inhalational chemical exposure may manifest as an allergic or asthmatic 12 response that is characterized by BC or BHR. This sensitization may be a result of immune 13 involvement, as in the case of hypersensitivity, or a neurogenic sensitization, where a chemical 14 may directly stimulate inflammation. Asthma is a s pecific manifestation of IgE-mediated 15 hypersensitivity, characterized by BHR and airway i nflammation, resulting in lower airway 16 obstruction (Fireman, 2003; Kuby, 1991). 17 A variety of hypersensitivity reactions have been r eported following exposure to 18 formaldehyde. Rashes and skin reactions have been reported in some individuals after dermal 19 exposures to formaldehyde. Increased expression of Th-2 cytokines in the lymph nodes of mice 20 given dermal applications of formaldehyde does indi cate the involvement of an immune 21 component to the observed sensitization (Dearman et al., 2005; Hilton et al., 1998; Arts et al., 22 1997). However, the response does not appear to be IgE mediated (Arts et al., 1997; Lee et al., 23 1984). Gorski et al. (1992) observed an increase i n formaldehyde-mediated neutrophil burst in 24 dermatitis patients exposed in a controlled chamber study and suggests a putative role of 25 oxidative stress and reactive oxygen species (ROS). 26 Inhalation exposure has been associated with increa sed asthmatic responses in asthmatics 27 in occupational settings. While few available case reports of bronchial asthma suggest direct 28 respiratory tract sensitization to formaldehyde gas (Lemiere et al., 1995; Burge et al., 1985; 29 Hendrick et al., 1982; Hendrick and Lane, 1977, 197 5), a greater body of human data provides 30 evidence of an association between formaldehyde exp osure and exacerbation of asthmatic 31 responses in compromised individuals (Kriebel et al ., 1993) and particularly in children 32 (Rumchev et al., 2002; Garrett et al., 1999; Krzyza nowski et al., 1990). Increased asthma 33 incidence reported after inhalation exposure to for maldehyde led to a NOAEL of 30 ppb 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-383 (Rumchev et al., 2002). An increased frequency of respiratory symptoms associated with 1 asthmatic responses and formaldehyde exposure led t o a LOAEL of 30 ppb (Garrett et al., 1999). 2 Exacerbation of response after formaldehyde exposur e has been demonstrated in animal 3 studies as well. Sadakane et al. (2002) demonstrat ed that formaldehyde exposure exacerbated 4 sensitization and challenge with Der f and suggeste d that formaldehyde exposure may aggravate 5 eosinophilic infiltration and goblet cell prolifera tion that accompanies allergic responses. 6 Several animal studies report increased airway resi stance and BC due to inhalation exposures to 7 formaldehyde (Nielsen et al., 1999; Swiecichowski e t al., 1993; Biagini et al., 1989; Amdur, 8 1960). Changes in pulmonary resistance were observ ed as early as 10 minutes after exposure 9 (Biagini et al., 1989), and reported effect levels ranged from 0.3 to 13 ppm. BHR is commonly 10 associated with allergic Type I hypersensitivity re actions but is not sufficient to demonstrate that 11 an agent induces Type 1 hypersensitivity. 12

13

MOA 14 The MOA underlying this response has not been eluci dated. Formaldehyde-induced IgE 15 production has been reported in some studies (Vande nplas et al., 2004; Wantke et al., 1996a). 16 Other studies suggest that this effect does not app ear to be immunogenic in nature (Fujimaki et 17 al., 2004; Lee et al., 1984). Although formaldehyd e exposure has been reported to alter cytokine 18 levels and immunoglobulins in some experimental sys tems (Fujimaki et al., 2004a; Ohtsuka et 19 al., 2003), these immunomodulatory effects do not s upport immunogenically mediated type 1 20 hypersensitivity. 21 These decrements may be mediated via neurogenic pot entiation (Sadakane et al., 2002; 22 Riedel et al., 1996; Tarkowski and Gorski, 1995). Tarkowski and Gorski (1995) suggest that 23 formaldehyde may increase permeability of respirato ry epithelium and destruction of 24 immunologic barriers. Tachykinin NK1 receptor and various neuropeptides (NGF and substance 25 P) have been implicated in formaldehyde-induced sen sitization and lend weight of evidence to a 26 neurogenic MOA (Van Schoor et al., 2000; Ito et al. 1996). 27

28 4.4.4. Upper Respiratory Tract Histopathology 29 Several studies in occupational workers have report ed increased squamous cell 30 metaplasia and reduced mucociliary clearance in nas al and buccal swabs from humans 31 occupationally exposed to formaldehyde (Holmström e t al., 1989; Holmström and Wilhelmsson, 32 1988). Evidence of genotoxic effects include incre ased MNs and CAs in nasal and buccal 33 epithelial cells from both workers and students exp osed to formaldehyde (Ying et al., 1997; 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-384 Titenko-Holland et al., 1996; Suruda et al., 1993) and suggest a potential association between 1 genotoxicity and altered histopathology. 2 Numerous animal experimental studies in multiple st rains of rats, mice, hamsters, rabbits, 3 and monkeys describe formaldehyde-induced URT patho logy (Fló-Neyret et al., 2001; Roemer 4 et al., 1993; Reuzel et al., 1990; Monticello et al ., 1989; Zwart et al., 1988; Wilmer et al., 1987; 5 Morgan et al., 1986b, 1983; Swenberg et al., 1986; Buckley et al., 1984). Effects are first 6 observed in the anterior respiratory mucosa and pro gress through the nasal passages with 7 increasing exposure concentration and time. The fi rst observed effect includes damage to the 8 mucociliary apparatus of the nasal passages in resp onse to formaldehyde. Studies conducted 9

both in vivo and in vitro demonstrate that formalde hyde disrupts mucus flow and ciliary beat that 10 are dependent on concentration and duration of expo sure. Mucociliary apparatus deficits have 11 been recorded even after 18 hours of recovery follo wing formaldehyde exposure. The 12 breakdown of the mucociliary apparatus may allow fo r increased infection and allow the 13 underlying epithelium to come into contact with exo genous chemicals. 14 Formaldehyde is highly reactive and may impact all cells in the nasal mucosa, including 15 epithelial cells (ciliated, columnar, and cuboidal) , goblet cells, sensory neurons, and 16 intraepithelial lymphocytes. The histologic change s of these processes have been described in all 17 laboratory animals examined and progress from the a nterior nares to the posterior regions of the 18 nasal passages, including the ETs and olfactory epi thelium if the concentration and duration of 19 exposure are great enough. 20 Humans and nonhuman primates have significantly les s complex nasal passages than 21 rodents. Formaldehyde has lower peak flux in human nasal tissues compared with rodents, 22 which are obligate nose breathers, but will penetra te more deeply into the human respiratory tract 23 than in rodents, since humans lack the autonomic RA response. Additionally, humans may 24 switch to mouth breathing in the presence of an irr itant gas, thus bypassing the sensitive nasal 25 passages and increasing the tissue dose in the mout h and throat. These differences have been 26 demonstrated by using nonhuman primates where, at c omparable concentrations, tissue 27 pathology and increased cell proliferation progress ed further into the respiratory tract than in 28 rodents (Monticello et al., 1989). Nonhuman primat es share common structural respiratory 29 components and patterns of breathing and do not hav e a reflex autonomic apnea response. 30 Despite the anatomical and physiological difference s in breathing patterns and different 31 exposure parameters between humans and rodents, sim ilar toxic effects are reported in tissues at 32 the POE in humans and laboratory animals. Several occupational studies have reported 33 increased squamous cell metaplasia in nasal and buc cal samples in response to formaldehyde 34 exposure (Ballarin et al., 1992; Boysen et al., 199 0; Holmström et al., 1989), paralleling the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-385 histologic effects seen in experimental animal stud ies. A few human epidemiology studies 1 suggest increased NPCs (see Section 4.5) as well as oral/buccal tumors in response to 2 formaldehyde exposure (Shangina et al., 2006; Lafor est et al., 2000). 3 The observed formaldehyde-induced URT toxicity is r elated to its high reactivity and 4 solubility. Moreover, additional interspecies diff erences in the surface area and configuration of 5 the nasal passages and upper airways will influence the areas of high formaldehyde flux in POE 6

tissues. 7

8

MOA 9 Formaldehyde-induced damage to the mucociliary appa ratus of the nasal passages may 10 occur because formaldehyde may disrupt mucus flow a nd ciliary beat that is dependent on 11 concentration and duration of exposure. Formaldehy de reacts with the mucosal glycoproteins 12 and thus may contribute directly to the breakdown o f the mucus layer. As formaldehyde reaches 13 the cells of the pseudostratified epithelium in the nasal passages, it exerts a range of effects from 14 direct damage to cell membrane, intracellular prote ins, and DNA to alterations in GSH pools and 15 increased ROS. Adaptive effects include increased mucus flow and goblet cell proliferation as 16 well as the transition of respiratory epithelium to more insensitive cuboidal cells. With 17 continued exposure at sufficient concentration, squ amous metaplasia develops, creating a 18 protective layer of keratinized cells. Gradually, this damage exceeds the cell’s ability to 19 compensate for and repair damage; chronic nasal les ions develop, and the cells die both through 20 general necrosis as well as programmed cell death, depending on the severity of the cellular 21 damage (Monticello et al., 1989; Swenberg et al., 1 983). 22 Genotoxic effects have been reported in nasal and b uccal lesions taken from both workers 23 and students exposed to formaldehyde (Ying et al., 1997; Titenko-Holland et al., 1996; Suruda et 24 al., 1993). MN formation occurs in the more sensit ive pseudostratified epithelium of the nasal 25 passages, nasopharynx, and upper airways, since the re is only one layer of epithelial cells that are 26 constantly regenerating. However, the genotoxicity observed in buccal cells is more difficult to 27 explain, since buccal basal cells are usually cover ed by protective keratinized cell layers. Cuts, 28 sores, or other buccal lesions would increase basal epithelial cells’ vulnerability to direct 29 exposure to formaldehyde. 30

31 4.4.5. Toxicogenomic and Molecular Data That May I nform MOAs 32 Over the past several years, studies have begun to examine the effects of formaldehyde 33 exposure on gene and protein expression. These inc lude studies on in vivo and in vitro changes 34 in the global expression of mRNA (transcriptomics) and proteins (proteomics) in the tissues and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-386 cells of humans and rodents exposed to formaldehyde . Currently (2009), nine “-omics” studies 1 from five research groups are available. These stu dies are summarized in Section 5.2 and are 2

evaluated and discussed in the context of their rel evance to informing MOAs and the dose3 response characterization briefly here. 4 In 2002, EPA released the Interim Policy on Genomics (U.S. EPA, 2002c), which 5 addresses how to use genomic data in regulatory dec ision making. Although the policy 6 encourages research in genomics, it places limits o n its use, stating that genomic data alone are 7 not sufficient as a basis for decision making. The se data thus cannot currently be utilized as the 8

critical effect in a chemical risk assessment but c an be utilized in a weight-of-evidence approach 9 on a case-by-case basis. The Science Policy Counci l developed a white paper entitled Potential 10 Implications of Genomics for Regulatory and Risk As sessment Applications at EPA (U.S. EPA, 11 2004). This report described three areas where gen omic data might be applied in risk assessment 12 at EPA: MOA analysis, susceptible population, and m ixtures assessments. The genomic data on 13 formaldehyde thus may be applied to a discussion of MOA. 14 Toxicogenomics studies have investigated the gene a nd protein expression changes 15 resulting from formaldehyde exposure in a variety o f respiratory tissues, including nasal tissue 16 (Andersen et al., 2008; Thomas et al., 2007; Hester et al., 2005, 2003), and, in lung tissue (Lee et 17 al., 2008, 2007; Sul et al., 2007; Im et al., 2006) used human tracheal cell lines to study genomic 18 changes after exposure to formaldehyde in vitro. U nfortunately, these studies are not directly 19 comparable because different gene chip technology p latforms were used in different tissues, in 20 both in vivo and in vitro study designs. In genera l, the gene and protein expression changes 21 reflect changes in apoptotic pathway genes, oxidati ve stress, and tissue remodeling. Andersen et 22 al. (2008) concluded that there was a threshold lev el where exposure to formaldehyde (6 ppm) 23 does not elicit changes in nasal epithelium of F344 rats. Overall, Andersen et al. (2008) 24 concluded that genomic changes were no more sensiti ve than tissue responses and that 25 formaldehyde, being an endogenous chemical, is well handled until some threshold is achieved 26 when toxicity rapidly ensues with genomic and histo logic changes. At about 6 ppm, this largely 27 involves tissue remodeling (and protection), but re generative hyperplasia occurs at higher doses. 28 Andersen et al. (2008) conclude that there is a thr eshold where exposure to formaldehyde does 29 not elicit changes in F344 nasal epithelial tissue over the duration examined in this study (i.e., 30 15 days). Andersen et al. (2008) argue that this i s consistent with bioassays that indicate no 31 tumor formation in rodents below 6 ppm formaldehyde

. 32 The primary conclusion in the Andersen et al. (2008 ) paper is that genomic changes, 33 including those suggestive of mutagenic effects, di d not temporally precede or occur at lower 34 doses than phenotypic changes in the tissue. The a uthors stated as follows: 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-387 “The genomic signatures related to these transition s are for cell membrane and 1 extracellular components, then inflammation and cel lular stress, and eventually 2 apoptosis. Importantly, these hierarchical models indicate that the tissue 3 responses at low dose concentrations are qualitativ ely different from those at high 4 concentrations and linear extrapolations or extrapo lations that specify similar 5 modes of action at high and low doses would be inap propriate.” 6 7

4.4.6. Noncancer Modes of Actions 8 Noncancer health effects of interest span numerous organ systems and include 9 reproductive and developmental effects, neurologica l/neurobehavioral effects, and a complex 10

interaction between inflammation and immune and adv erse pulmonary function. To date, no 11 -omics studies have examined changes in reproductiv e tissue or altered gene expression in 12 developing animals. In regard to neurological/beha vioral effects, one study (Lu et al. [2008], 13 described in Section 4.1.1.6) has reported elevatio ns in the mRNA for NMDA receptor subunits 14 in brain homogenates following exposure to 2.4 ppm. Hester et al. (2003) reported a significant 15 increase in NMDA receptor subunit transcripts, alon g with other neuropeptide genes, in nasal 16 tissue. Together, these changes may relate to form aldehyde-induced sensory irritation and, 17 perhaps, changes throughout the brain. 18 In regard to inflammatory, immune, and pulmonary ef fects, transcript and protein 19 changes in rodent tracheal tissue and lung tissue i ndicate that exposure to 3 to 38 ppm 20 formaldehyde results in genes involved in oxidative stress and cell proliferation and may 21 additionally increase airway ADH3 levels (Lee et al ., 2008; Sul et al., 2007; Yi et al., 2007; Im et 22 al., 2006; Yang et al., 2005). Together, these dat a provide evidence for adverse pulmonary 23 effects that may exacerbate or facilitate asthma. 24 In lung tissue, Yang et al. (2005) identified three proteins up regulated and one protein 25 down regulated following 15 days of exposure to abo ut 28 ppm formaldehyde. None of the 26 proteins corresponded with transcript changes repor ted by Sul et al. (2007). Interestingly, Sul et 27 al. (2007) reported that only two transcripts were significantly up regulated in the lung in 28 response to 5–10 ppm formaldehyde, while 19 were do wn regulated. In this regard, it is worth 29 considering that changes in proteins may not relate to their regulation but rather to their overall 30 percent composition in a cell (relative to other pr otein changes) before and after exposure. In 31 addition to transcript changes, Sul and colleagues (2007) reported DNA damage and lipid 32 peroxidation and noted that the observed down regul ation of GR would facilitate oxidative stress, 33 while the down regulation of phospholipase A2 (PLA2 ) might represent a mechanism for 34 mitigating lipid peroxidation. It is worth noting that an increase in either of these genes could 35 also be argued to support similar conclusions (i.e. , that GR is up regulated to increase GSH 36 levels and that PLA2 up regulation explains lipid p eroxidation); this highlights the problem with 37This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-388 interpreting these data. Nevertheless, these studi es indicate adverse effects in the rodent lung in 1 response to 5–30 ppm formaldehyde. 2 In a hypothesis-driven study by Yi et al. (2007), f ormaldehyde exposure was shown to 3 increase lung ADH3 levels. Several studies indicat e that allergic responses and hyperreactivity 4 are uncoupled and may relate to ADH3 expression and activity. Que et al. (2005) has shown 5 that, in a rodent asthma model, ADH3 knockout mice exhibit similar signs of inflammation but 6 are protected from bronchoconstriction. Lino dos S antos Franco et al. (2006) provided evidence, 7 in rodents, that formaldehyde may induce inflammato ry responses (e.g., leukocyte infiltration in 8 the lung) through neurogenic mechanisms but that br onchial tone is mediated by NO. The latter 9 effect is likely to be mediated by S-nitrosoglutath ione GSNO and thus influenced by ADH3. 10 Interestingly, the single nucleotide polymorphisms (SNPs) that Wu et al. (2007) reported as 11 linked to other polymorphisms in the promoter regio n was the one demonstrating protection 12 against asthma. Hedberg et al. (2001) reported tha t at least one SNP in the promoter region 13 reduced ADH3 expression. Together, these data indi cate that reduced ADH3 expression might 14 lower GSNO turnover and bronchial tone, thereby red ucing signs of asthma. In this regard, it is 15 conceivable that wheezing and bronchoconstriction a re the symptoms that lead to medical 16 intervention and not the inflammation per se. Thus , while ADH3 polymorphisms may not cause 17 asthma, ADH3 polymorphisms may influence hyperrespo nsivity and the likelihood of asthma 18 diagnosis. This is discussed further in Section 4. 6 on susceptible populations. Formaldehyde 19 has been shown to accelerate GSNO breakdown (Staab et al., 2008a; Yi et al., 2007); thus, 20 pulmonary responses to formaldehyde may represent a balance between mechanisms that induce 21 NO (i.e., inflammation) and those that terminate GS NO (i.e., ADH3). 22 In regard to -omics changes in blood samples, the a pparent limited distribution of 23 formaldehyde may suggest that these changes are sec ondary to effects at sites of contact but 24 could also indicate systemic distribution. As note d elsewhere in this report, bradypnea can 25 induce changes in dosimetry as well as changes in c ore body temperature and blood gases 26 (hypoxia itself induces hypothermia in rodents, and thus the reduction in minute volume and gas 27 changes may both contribute to hypothermia). These physiological responses (hypothermia and 28 hypoxia) surely induce changes in gene expression. Observed gene and protein changes in the 29 blood following formaldehyde exposure could also re late to irritation and inflammation at sites 30 of contact. In this regard, Im et al. (2006) repor ted changes in cytokines indicative of Th-2 31 responses. Altogether, the authors identified 32 p roteins altered in the plasma of rats exposed to 32 formaldehyde. Although no coherent mechanisms are apparent from these changes, the authors 33 posited that they could serve as biomarkers for for maldehyde exposure. The concordance of 34 such changes across species remains to be demonstra ted. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-389 Li et al. (2007) identified dose-response relations hips for six genes in human blood 1 samples that were putatively associated with formal dehyde exposure. Three of these genes are 2 reported to inhibit apoptosis and were posited as s upporting in vitro data by Tyihak et al. (2001); 3 however, Li and colleagues (2007) did not report an y increase in blood cell count or in Hs 680.Tr 4 cell counts in vitro (i.e., these changes were not phenotypically linked to changes in cell kinetics 5 or hematology). However, these findings are not in consistent with those of Hester et al. (2003) 6 that indicated no significant increase (or decrease ) in nine genes involved in apoptotic pathways. 7 Finally, serum and glucocorticoid-induced protein k inase 1 (SGK1) was elevated in blood 8 samples and was posited to relate to possible infla mmatory and immune responses. 9

10 4.4.7. Immunotoxicity 11

Results from studies that evaluated the immunotoxic ity of formaldehyde are mixed. For 12 example, most human studies that investigated syste mic immune effects by measuring increases 13 in formaldehyde-specific IgE are negative (Doi et a l., 2003; Kim et al., 2001; Palcynski et al., 14 1999; Krakowiak et al., 1998; Wantke et al., 1996; Grammer et al., 1990). Vandenplas et al. 15 (2004) reported a transiently positive increased fo rmaldehyde-specific IgE titer in occupationally 16 exposed workers. In contrast, Thrasher et al. (199 0, 1988, 1987) reported positive 17 formaldehyde-specific IgE titers in small (six to e ight person) case studies of exposed workers, 18 and Carraro et al. (1997) reported elevated IgE tit ers in smokers. Grammer et al. (1990) did not 19 report any differences in albumin IgE in formaldehy de-exposed workers compared with controls. 20 In a residential study, Pross et al. (1986) found t hat formaldehyde insulation in homes had no 21 effect on tested human immunologic parameters. 22 One study suggests that immune system parameters ar e perturbed by formaldehyde 23 exposure. Lyapina et al. (2004) reported decreased immune resistance in all 29 workers exposed 24 to formaldehyde. This effect was associated with d ecreased neutrophil respiratory burst activity. 25 A LOAEL of 700 ppb was established. 26

Results from animal studies are mixed as to whether formaldehyde causes 27 immunotoxicity. Leach et al. (1983) reported syste mic immunomodulation in F344 rats that was 28 attributed to formaldehyde exposure, but the formal dehyde effects on measures of humoral and 29 cell-mediated immunity were not confirmed in B6C3F1 mice (Dean et al., 1984). Jakab et al. 30 (1992) detected no differences in phagocytic abilit y of alveolar MPs from mice after 31 formaldehyde exposure. Formaldehyde-exposed rats t hat were injected with pneumococcus 32 antigen or tetanus toxoid produced antibodies in am ounts similar to nonexposed, infected control 33 animals (Holmström et al., 1989b). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-390 However, specific immune parameters appear to be af fected by formaldehyde exposure. 1 For example, increased host resistance and hydrogen peroxide release from peritoneal MPs were 2 reported and confirmed (Adams et al., 1987; Dean et al., 1984) and suggest a putative role for 3 ROS. Increased host resistance may be mediated by formaldehyde-induced chronic 4 inflammation and respiratory mucosal damage that ca uses an up regulation in MPs and therefore 5 increases host immunity. Jakab et al. (2002) repor ted reduced pulmonary bacterial resistance in 6 mice after exposure to formaldehyde, as determined by increased bacterial loading. This result 7 contrasts with Dean et al. (1984) and is attributed to differential exposure regimens and 8 experimental design. 9

10 Mode of action 11 Circulating immune cells present in the mouth and u pper airways, such as intraepithelial 12 lymphocytes, direct a local inflammatory response b ut may also contribute to systemic responses 13 through secreted cytokines and soluble factors rele ased into the bloodstream (Togias, 1999). 14 Altered host resistance and hydrogen peroxide relea se from peritoneal MPs were reported 15 and confirmed (Adams et al., 1987; Dean et al., 198 4) and suggest a putative role for ROS. 16 Indeed, increased neutrophilic ROS have been associ ated with formaldehyde-induced dermatitis 17 (Gorski et al., 1992), and, conversely, decreased n eutrophil respiratory burst activity has been 18 shown in workers with history of formaldehyde-induc ed respiratory tract inflammation (Lyapina 19 et al., 2004). Oxidative stress may occur directly as a result of formaldehyde exposure or as a 20 secondary consequence to inflammatory responses. 21

22 4.4.8. Effects on the Nervous System 23 There is considerable evidence that formaldehyde ex posure causes adverse effects on the 24 nervous system following inhalation at relatively l ow exposure levels but little or no information 25 regarding a possible mechanism of action for these effects. Data regarding adverse effects on the 26 nervous system following oral exposure are very lim ited, reflecting a data gap in this area. 27 Relevant data in animals and humans for several typ es of neurological endpoints, following 28 inhalation exposure, are summarized below. 29

30

4.4.8.1. Irritant Threshold Detection 31 Humans are exquisitely sensitive to the irritant pr operties of formaldehyde, as has been 32 discussed previously (see Section 4.1.1.1). Animal data confirm the irritant properties of this 33 compound at very low concentrations (Wood and Colem an, 1995). 34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-391 4.4.8.2. Behavioral Effects 1 Limited data in humans, as well as more robust data in animals, provide evidence of 2 behavioral changes following exposure to formaldehy de at levels as low as 0.1 ppm. Studies in 3 animals have found effects that persist for days to weeks after termination of exposure. In spite 4 of significant limitations, the available human dat a are consistent with the animal findings. 5 Several types of behavior have been evaluated in an imals following formaldehyde 6 exposures. The most consistent findings, demonstra ted by multiple laboratories and in multiple 7 species, have been changes in motor activity, habit uation, and learning/memory task 8 performance. Motor activity and habituation have b een evaluated under a variety of exposure 9 conditions, using both rats and mice. Consistent d ecreases in activity have been seen in adult 10 animals (Malek et al., 2004, 2003 a, b; Usanmaz et al., 2002). Senichenkova (1991) and 11 Sheveleva (1971) also found changes in motor activi ty in offspring following in utero exposure, 12 including decreased habituation in juvenile rats ex posed in utero. Decreased performance in 13 learning and/or memory paradigms have been seen in multiple laboratories, also in both rats and 14 mice (Lu et al., 2008; Malek et al., 2003c; Pitten et al., 2000). 15 Data from controlled human exposures are very limit ed, but studies have shown 16 decreased performance in addition tasks and reactio n time tasks following acute exposures to 17 formaldehyde (Lang et al., 2008; Bach et al., 1990) . In contrast, Andersen and Molhave (1983) 18 indicated they found no change in performance on se veral types of tasks (including addition, 19 multiplication, and card punching) following acute exposure to volunteers, but supporting data 20 were not provided. 21 Data for humans are also available from epidemiolog y studies of individuals who were 22 occupationally exposed to formaldehyde. A variety of neurobehavioral deficits, including lack 23 of concentration and loss of memory, disturbed slee p, impaired balance and dexterity, and 24 changes in mood, were identified (Kilburn et al., 1 987, 1985). However, most of the individuals 25 evaluated in these studies were also occupationally exposed to other solvents, raising questions 26 regarding possible confounding of the results due t o multiple exposures. In addition, the 27 formaldehyde exposure information provided in the s tudies is not sufficient to permit a reliable 28 dose-response assessment for the effects identified . The types of effects seen in humans in the 29 available epidemiology studies are, however, consis tent with those seen in available animal 30

studies. 31 In general (and noting the differences in exposure paradigms and types of tasks), 32 behavioral effects in animals and humans appear to occur at similar exposure levels. Animal 33 studies demonstrated LOAELs as low as 100 ppb follo wing acute or repeated exposures (Malek 34 et al., 2003b, c); human controlled exposure studie s have found effects in that same range, with 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-392 LOAELs of approximately 300 ppb following acute exp osures (Lang et al., 2008; Bach et al., 1 1990). 2

3

4.4.8.3. Neurochemistry, Neuropathology, and Mechanistic Stu dies 4 Limited data are available regarding neurochemical and neuropathological sequelae of 5 formaldehyde exposure. Studies from one laboratory (Sorg et al., 2004, 2001) have suggested 6 that behavioral sensitization to formaldehyde is li nked to alterations in HPA control of 7 corticosterone and changes in mesolimbic dopamine p athways. Neurochemical changes in 8 response to formaldehyde exposure have also been do cumented in other laboratories (Fujimaki et 9 al, 2004b; Hayashi et al., 2004). Some of these da ta appear to be conflicting, and there are no 10 definitive data supporting a specific mechanism for formaldehyde effects on the nervous system 11 at this time. Neuropathological data are also limi ted, although data from one laboratory 12 (Sarsilmaz et al., 2007; Aslan et al., 2006) sugges t a concern for changes in brain structure in 13 neonatal rats following exposure at 6 or 12 ppm. N o human data are available that address these 14 endpoints. However, a prospective cohort study of nearly one million people followed for 15 15 years reported strongly significant dose-response a ssociations between death from ALS and 16 exposure to formaldehyde of a known duration, with longer exposures associated with greater 17 risk (Weisskopf et al., 2009). This large, well-de signed prospective cohort study strongly 18 supports the causal association of neuropathologica l effects in humans following long-term 19 formaldehyde exposure. 20

21

4.4.8.4. Summary 22 Overall, there is strong evidence that formaldehyde exposure via inhalation may cause 23 adverse effects on nervous system function in exper imental animals at relatively low levels of 24 exposure (LOAELs as low as 100 ppb). Although huma n data regarding neurotoxicity following 25 formaldehyde inhalation are limited, available data provide support that the types of effects seen 26

in humans are similar to those found in animal stud ies. Evidence from available human 27 controlled inhalation exposure studies indicates th at humans may be affected at doses similar to 28 those used in animal studies; however, the human da ta are extremely limited. 29 There are insufficient data to evaluate the potenti al for neurotoxicity following oral 30 exposure to formaldehyde. Limited evaluations of b rain weight or histopathology in available 31

chronic or subchronic oral studies found no evidenc e of formaldehyde-induced changes (Til et 32 al., 1989, 1988; Tobe et al., 1989; Johannsen et al ., 1986). However, reliable studies examining 33 nervous system function or focused studies of neuro pathology following oral exposure to 34 formaldehyde are not available. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-393 4.4.8.5. Data Gaps 1 Major data gaps were found regarding the evaluation of changes in nervous system 2 structure or function following formaldehyde exposu re by both the inhalation or oral routes. 3 With respect to inhalation exposure, none of the av ailable human studies resulted in data 4 sufficient to conduct a reliable dose-response asse ssment for changes in nervous system function. 5

Most of the available animal inhalation studies use d short exposure durations (acute or short6 term), precluding a reliable evaluation of neurotox icity following chronic exposure. Available 7 data for neurodevelopmental exposures are also quit e limited, consisting of evaluation of 8 neuropathology in only one brain region and functio nal evaluations focused only on changes in 9 motor activity. 10 Major data gaps also exist regarding neurotoxicity following oral exposure, with no 11 relevant human data and extremely limited animal da ta. Available oral exposure studies were 12 insufficient to permit a reliable evaluation of the potential for neurotoxicity following oral 13 exposure to formaldehyde. 14

15 4.4.9. Reproductive and Developmental Toxicity 16

A number of studies have been identified that indic ate an effect of formaldehyde 17 exposure on reproductive and developmental outcomes . Human data are described in Section 18 4.1.1.7, and animal studies are addressed in Sectio n 4.2.1.7 of this document. 19

20

4.4.9.1. Spontaneous Abortion and Fetal Death 21 Several epidemiologic studies reported increases in risk of spontaneous abortion 22 following maternal occupational formaldehyde exposu re (Taskinen et al., 1999, 1994; John et al., 23 1994; Seitz and Baron, 1990; Axelsson et al., 1984) . While these finding have been questioned 24 (Collins et al., 2001b), upon careful examination, none of the principal biases in epidemiologic 25 studies, including information bias, selection bias , and confounding, appear to be more likely 26 causes of these reported findings than the conclusi on that they may reflect an underlying causal 27 process. While each of these occupational studies focused on women who were co-exposed to 28 formaldehyde and other chemicals, the occupational groups were quite different and had 29 different sets of co-exposures. The woodworkers in the Taskinen et al. (1999) study were 30 potentially co-exposed to organic solvents related to painting and lacquering, dusts, and phenols, 31 none of which was shown to be an independent predic tor of adverse risk. The cosmetologists 32 studied by John et al. (1994) were co-exposed to ha ir dyes, bleach, alcohol-based disinfectants, 33 and chemicals specific to services, such as fingern ail sculpturing, but, in analyses that were 34 specifically adjusted for other work exposures and their potentially confounding effects, the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-394 investigators reported an increased risk for the us e of formaldehyde-based disinfectants. The 1 laboratory workers studied by Axelsson et al. (1984 ) were potentially co-exposed to a wide range 2 of solvents, but the miscarriage rate was highest a mong those exposed to formaldehyde. For a 3 potential confounder to entirely explain an observe d effect of another exposure, it must be more 4 strongly associated with the adverse outcome. It d oes not appear that the collective results of 5 formaldehyde exposures associated with increased ri sk of spontaneous abortion—often in spite 6 of exposures being crudely measured—can be explaine d by information bias or confounding. 7 Taken together, these findings are consistent with an adverse effect of formaldehyde 8 exposure on the risk of pregnancy loss. The single study with the strongest quantitative 9 assessment of that risk is Taskinen et al. (1999), and the results presented are of sufficient quality 10 to support quantitative risk assessment by using th e LOAEL/NOAEL approach. 11 This study was a well-designed population-based cas e-control study that appears to have 12 been well executed and appropriately analyzed. The study population of Finnish women was 13 well defined and adequately selected to allow for m eaningful comparisons of health effects 14 between individuals with different levels of exposu re to formaldehyde. The participation rate 15 was 64%, which is low enough to raise a concern abo ut the potential for selection bias. 16 However, the authors noted that selection bias has not influenced the results of other 17 reproductive epidemiology studies reporting results on smoking, irregular menstruation, and 18 earlier miscarriages, which are known to lengthen t he time to pregnancy (Bolumar et al., 1996; 19 Sallmén et al., 1995; Baird and Wilcox, 1985). Fur thermore, there is no evidence to support 20 conjecture that an individual’s decision to partici pate in this study would be differential with 21 respect to their workplace formaldehyde exposures w hile being nondifferential with respect to 22 the other exposures of interest, including organic solvents, wood dust, and phenols. Since the 23 women who chose to participate in this study were n ot likely to be aware of the specific 24 hypotheses under investigation nor could they have known the formaldehyde exposures that were 25 independently estimated by an industrial hygienist, selection bias is not a likely explanation for 26 the findings of adversity. 27 Data on pregnancy history, including spontaneous ab ortions, were collected by 28 questionnaire. Spontaneous abortion is the most co mmon adverse outcome of pregnancy (Klein 29 et al., 1989), and retrospective self-report of spo ntaneous abortion has been found to match well 30 with prospectively collected reproductive histories (Wilcox and Horney, 1984). Many 31 spontaneous abortions, however, are missed with sel f-reporting, with the magnitude likely 32 exceeding 25%, but only rarely do women self-report false positive events (Wilcox and Horney, 33 1984). The effect of such an undercount is to caus e a bias towards the null when the likelihood 34 of undercounting is unrelated to formaldehyde expos ure. The implication is that the observed 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-395 association of increased risk of spontaneous aborti on associated with occupational exposure to 1 formaldehyde may be an underestimation of the true risk. 2 The findings by Taskinen et al. (1999) of reduced f ertility and increased risk of 3 spontaneous abortion are internally consistent and coherent with other reports of increased risk 4 of pregnancy loss associated with exposure to forma ldehyde (John et al., 1994; Taskinen et al., 5 1994; Seitz and Baron, 1990; Axelsson et al., 1984) . Absent evidence of alternative explanation 6 for these findings, it is concluded that exposure t o formaldehyde is associated with pregnancy 7 loss and diminished fertility. 8 In animal studies, Sheveleva (1971) noted an increa se in preimplantation loss in rats 9 exposed to 0.04 and 0.4 ppm formaldehyde by inhalat ion on GDs 1–19, and Kitaev et al. (1984) 10 observed evidence of degeneration in harvested embr yos on GD 2, following 4 months of 11 maternal inhalation exposure to 0.41 ppm formaldehy de in rats. In a second series of tests 12 reported in Kitaev et al. (1984), female rats were exposed to 0.41 and 1.22 ppm formaldehyde for 13 4 months to test the hypothesis that the embryo deg eneration could have been the result of 14 disrupted reproductive hormone levels in the dams. Ovarian weight and blood levels of LH were 15 increased at 0.41 ppm (but not at 1.22 ppm), and si gnificantly increased levels of FSH were 16 observed at 1.22 ppm. Kitaev et al. (1984) propose d that effects at the 0.41 ppm might be related 17 to disruption of the hypothalamic-pituitary axis an d that at the higher exposure level (1.22 ppm) 18 frank toxic effects to the embryo were observed. T he increased FSH levels at 1.22 ppm may also 19 be indicative of hormonal perturbations induced by formaldehyde exposure that could affect 20 pregnancy maintenance in humans. The finding of tr eatment-related increased preimplantation 21 loss in rats appears to support the evidence of spo ntaneous abortion in the epidemiologic data. In 22 addition, a dominant lethal study in rats by Odeiga h (1997) identified significant 23 postimplantation loss following pre-mating I.P. for maldehyde exposures to males, suggesting a 24 potential MOA involving germ cell toxicity. Nevert heless, a number of developmental toxicity 25 studies in rats did not report treatment-related em bryolethality following gestation exposures to 26 formaldehyde. These included inhalation studies by Martin (1990), Saillenfait et al. (1989), and 27 Kilburn and Moro (1985), a series of studies by Gof mekler and Bonashevskaya (1969), 28 Gofmekler (1968), and Pushkina et al. (1968), and s tudies by Senichenkova and Chebotar (1996) 29 and Senichenkova (1991). It is noted, however, tha t, to the extent that these studies evaluated 30 embryonic or fetal death, the observations were con ducted late in gestation and the studies may 31 not have been designed to detect the preimplantatio n losses as observed in Kitaev et al. (1984) 32 and Sheveleva (1971). Additionally, a number of th e reports for these studies did not include 33 sufficient details to engender a high degree of con fidence in the reported results. Fetal death was 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-396 also not observed in oral studies with formaldehyde in beagle dogs (Hurni and Ohder, 1973) and 1 rats (Seidenberg and Becker, 1987). 2

3

4.4.9.2. Congenital Malformations 4 The effect of occupational exposures to formaldehyd e on the incidence of congenital 5 malformations was examined by Dulskiene and Gražule vi č iene (2005), Taskinen et al. (1994), 6 and Hemminki et al. (1985). Results were mixed. 7 In animal studies, the most frequently observed str uctural anomaly noted following 8 inhalation exposures to formaldehyde during gestati on was a delay in fetal (i.e., 1 st stage) testes 9 descent (Senichenkova and Chebotar, 1996; Senichenk ova, 1991; Kilburn and Moro, 1985), 10 although similar findings were not reported by Sail lenfait et al. (1989) or Martin (1990) in what 11 appear to be well-conducted prenatal developmental toxicity studies. No study in the available 12 database specifically examined the 2 nd stage of postnatal testes descent in pups. Thus, there is no 13 evidence to determine if the observed effect repres ented a developmental delay or if it was 14 related to disruptions in male reproductive tract o ntogeny, which is dependent on normal levels 15 of fetal testicular testosterone and on the express ion of insulin-like hormone-3 (insl3) in fetal 16 Leydig cells (Klonisch et al., 2004). Senichenkova (1991) observed an increased incidence of 17 other organ anomalies following formaldehyde exposu re during gestation; however, the 18 anomalies are not characterized in the report. Alt erations on fetal organ weights and/or size were 19 noted in several studies (Kilburn and Moro, 1985; G ofmekler, 1968), but it is difficult to 20 ascertain if these findings represented agenesis, h ypoplasia, or evidence of systemic organ 21 toxicity. Histopathologic evaluation of pup tissue s following maternal gestational exposures to 22 0.01 and 0.81 ppm formaldehyde was conducted by Gof mekler and Bonashevskaya (1969), 23 revealing reduced glycogen content in the myocardiu m, the presence of iron in hepatic Kupffer 24 cells, and a positive Schiff reaction in the baseme nt membrane (indicating functional alterations 25 in the renal tubule) at both exposure levels. 26

27

4.4.9.3. Low Birth Weight and Growth Retardation 28 A population-based study (Gražulevi č iene et al., 1998) reported an association between 29 atmospheric formaldehyde exposure and low birth wei ght, with an adjusted OR of 1.37 (95% CI: 30 0.90–2.09). 31

A number of inhalation studies in rats identified r educed fetal weight as an adverse 32 outcome of in utero formaldehyde exposure and are s upportive of the association noted in 33 humans. In a study that exposed pregnant rats to f ormaldehyde during GDs 6–20, Saillenfait et 34 al. (1989) observed significantly decreased male an d female fetal rat weights (78 and 81% of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-397 control values, respectively) at 40 ppm formaldehyd e. In a study that exposed the dams from 1 GDs 6–15, Martin (1990) found decreased fetal weigh ts at exposure levels of 5 and 10 ppm. In 2 both studies, observations of reduced or delayed sk eletal ossification (i.e., the thoracic vertebrae 3 in Saillenfait et al. [1989] and the pubes and isch ia bones in Martin [1990]) were consistent with 4 the fetal weight deficits. Kilburn and Moro (1985) also reported fetal body weight decreases in 5 rats at an inhalation exposure level of 30 ppm. Co nversely, increased fetal body weight as 6 compared with controls (generally considered to be non-adverse) was noted by Gofmekler 7 (1968) and Pushkina et al. (1968) at maternal forma ldehyde exposure levels of 0.1 and 0.81 ppm 8 administered for approximately 2–3 weeks prior to m ating and then throughout gestation. 9 Increased fetal weight was also noted in rats by Se nichenkova (1991) and Senichenkova and 10 Chebotar (1996) following maternal exposures to 0.4 1 ppm formaldehyde throughout gestation. 11

Studies that assessed the effects of oral administr ation of formaldehyde on development 12 are quite limited. The only oral study identified that found a treatment-related effect on offspring 13 growth was a study using beagle dogs (Hurni and Ohd er, 1973). In this study, formaldehyde was 14 administered at doses of 3.1 or 9.4 mg/kg-day in th e feed during gestation, and pup weight 15 decrements at postnatal week 8 were 6.3 and 12% in the low- and high-dose groups, respectively. 16

17

4.4.9.4. Functional Developmental Outcomes (Developmental Ne urotoxicity) 18 Indications of effects on the developing nervous sy stem were observed in several rodent 19 studies, although no similar epidemiologic findings in children were identified. These studies 20 (Sarsilmaz et al., 2007; Aslan et al., 2006; Weiler and Apfelbach, 1992; Senichenkova, 1991; 21 Sheveleva, 1971) are described in detail in Section 4.2.1.6. In the studies by Aslan et al. (2006) 22 and Sarsilmaz et al. (2007), neonatal rats were exp osed to formaldehyde for 30 days at 6,000 or 23 12,000 ppb. Decreases in the volume of discrete ar eas of the brain were observed at both 24 exposure levels in both studies, and, additionally, decreased cell numbers were noted in a region 25 of the hippocampus in the Sarsilmaz et al. (2007) s tudy. Weiler and Apfelbach (1992) exposed 26 juvenile rats to 0.25 ppm formaldehyde for 130 days or adult rats to 0.5 ppm formaldehyde for 27 90 days. Olfactory thresholds measured in this stu dy were significantly higher in the rats that 28 had been exposed as juveniles than in those that ha d been exposed only as adults. Sheveleva 29 (1971) observed alterations in spontaneous mobility in 1- and 2-month-old pups from dams that 30 had been exposed to formaldehyde at 0.04 or 0.4 ppm throughout gestation. In the Senichenkova 31 (1991) study, maternal rats were exposed to 400 ppb formaldehyde during GDs 1–19, and 32 functional observational testing was conducted on t he juvenile offspring. Changes in open-field 33 motor activity, exploratory activity, and habituati on were observed in the offspring. 34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-398 4.4.9.5. Male Reproductive Toxicity 1 A number of laboratory animal studies have reporte d effects of formaldehyde exposure 2 on male reproductive system endpoints. These effec ts include decreased testes weight, changes 3 in Leydig cell quantity and quality, degeneration o f seminiferous tubules, decreased testosterone 4 levels, alterations in biomarkers of toxicity in th e testes, and alterations in sperm measures 5 (Galilapour et al., 2007; Xing et al., 2007; Zhou e t al., 2006; Özen et al., 2005, 2002; Sarsilmaz 6 et al., 1999; Odeigah, 1997; Majumder and Kumar, 19 95; Chowdhury et al., 1992; Til et al., 7 1989, 1988; Tobe et al., 1989; Johanssen et al., 19 86; Maronpot et al., 1986; Cassidy et al., 1983; 8 Appelman et al., 1982; Guseva, 1972). Following co ncurrent exposures to formaldehyde in air 9 and drinking water for 6 months, Guseva (1972) foun d decreases in testicular nucleic acid levels. 10 In a study conducted by Chowdhury et al. (1992), ra ts were administered I.P. injections of 11 formaldehyde for 30 days, and evidence of decreased testes weight, serum testosterone levels, 12 and Leydig cell quality was observed. Sarsilmaz et al. (1999) followed up on these findings 13 (exposing male rats to formaldehyde via inhalation at 10 and 20 ppm for 4 weeks) and found 14 decreases in Leydig cell quantity and the percentag e of cells with normal nuclei. Hypothesizing 15 that the reported decreases in Leydig cell quality may have been the result of oxidative stress and 16 damage, Özen et al. (2002) evaluated biomarkers of such changes and found that testicular zinc 17 and copper levels were decreased and iron levels we re increased following exposures of adult 18 male rats to 10 and 20 ppm formaldehyde for 4 or 13 weeks. Additionally, relative testes weight 19 was decreased in a dose- and duration-dependent man ner, although this effect had not been 20 observed by Sarsilmaz et al. (1999). Özen et al. ( 2005) noted decreased serum testosterone 21 levels, decreased seminiferous tubule diameters, an d increased levels of heat shock protein in 22 spermatogonia, spermatocytes, and spermatids of rat s following 91 days of exposure to 10 ppm 23 formaldehyde. A study by Golalipour et al. (2007) observed decreased numbers of testicular 24 germ cells, altered sprmatogenesis, and reduced sem iniferous tubular diameter and epithelial 25 height in rats following 18 weeks of formaldehyde i nhalation exposure; the severity of the 26 seminiferous tubule pathology was positively correl ated to the number of hours/week of 27 exposure. Zhou et al. (2006) found decreased testi s weight, atrophy of seminiferous tubules, 28 edematous interstitial tissue, and alteration of ep ididymal sperm count, morphology, and motility 29 in rats after 2 weeks of formaldehyde exposure at 8 ppm. Abnormal sperm were also observed in 30 mice by Xing et al. (2007) after 13-weeks of inhala tion exposure at 16.9 ppm, and Cassidy et al. 31 (1983) reported sperm abnormalities in rats followi ng a single oral dose of 200 mg/kg. 32 Additionally, Majumder and Kumar (1995) observed si gnificantly reduced sperm count, motility, 33 and viability following 30 days of I.P. injection o f 10 mg/kg-day formaldehyde to male rats. 34 Also in this study, the ability of formaldehyde to affect sperm parameters was confirmed with in 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-399 vitro testing. A study conducted by Odeigah (1997) demonstrated epididymal sperm count and 1 morphology abnormalities following five I.P. doses of ≥ 0.125 mg/kg formaldehyde and 2 additionally identified dominant lethal effects (de creased live embryos and increased dead 3 implants) following mating of treated male rats wit h untreated females. 4 Although Til et al. (1989) reported low incidences of Leydig cell tumors in 5 formaldehyde-treated rats in a chronic drinking wat er study, no alterations in testes weight or 6 histopathologic lesions of the testes were observed in subchronic inhalation studies conducted by 7 Appleman et al. (1982) or Maronpot et al. (1986) or in subchronic or chronic oral studies by 8 Johanssen et al. (1986), Til et al. (1988), or Tobe et al. (1989). 9 No epidemiologic studies have identified an associa tion between formaldehyde exposure 10 and alterations in the male reproductive system (e. g., see Ward et al. [1984]). 11

12

4.4.9.6. Female Reproductive Toxicity 13

The available database for the assessment of the ef fects of formaldehyde exposure on the 14 female reproductive system was limited. In additio n to the findings of spontaneous abortions, as 15 described above, Taskinen et al. (1999) examined fe cundability in a cohort of healthy female 16 wood-processing industry workers and found that con ception was significantly delayed in 17 women who were occupationally exposed to formaldehy de. The FDR, a ratio of average 18 incidence densities of pregnancies for exposed fema le employees compared with unexposed 19 female employees, was lower in women exposed to mea n formaldehyde levels of approximately 20 0.33 ppm (range: 0.15–1.00 ppm) compared with contr ols (adjusted FDR = 0.64 [95% CI: 21 0.28-0.92]). An FDR <1.0 is indicative of delayed conception, which is an indicator of reduced 22 fertility. The subfertility observed in this study is supportive of the association observed 23 between formaldehyde exposure and spontaneous abort ion, since subclinical pregnancy losses 24 are increased in women with compromised fertility ( Gray and Wu, 2000; Hakim et al., 1995), 25 and both spontaneous abortion and subfertility can be related to exposure to environmental 26 toxicants (Correa et al., 1996). 27 As described above, formaldehyde exposures to femal e rats resulted in decreased ovarian 28 weight and altered LH and FSH levels (Kitaev et al. , 1984). Maronpot et al. (1986) reported 29 endometrial hypoplasia and lack of ovarian luteal t issue in female mice exposed to 40 ppm 30 formaldehyde for 13 weeks. Additionally, it is not ed that, in developmental toxicity studies that 31 included repeated exposures of dams before mating a nd/or during gestation, reports of adverse 32 pregnancy outcomes were few. Gofmekler (1968) repo rted an increase in pregnancy duration 33 and decrease in litter size; however, this finding was not observed in other studies. 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-400 With the exception of spontaneous abortion and incr eased time to pregnancy, associations 1 of formaldehyde exposure with adverse female reprod uctive system outcomes were not observed 2 in the available epidemiologic data. 3

4

4.4.9.7. Mode of Action 5 A strong case cannot be made for any one MOA that e xplains one or more of the 6 reproductive and developmental outcomes observed in formaldehyde epidemiologic or 7 toxicology studies. This is due to a number of iss ues, including the following: 8 (1) inconsistencies in study findings for the toxic ology studies, which may be explained by study 9 quality issues (see detailed descriptions of studie s in Sections 4.1 and 4.2); (2) few studies that 10 allow for comparisons because no study was performe d with the same study design (e.g., stage of 11 exposure, dose, species, and strain); (3) few mecha nistic studies available to test hypothesized 12 MOAs; and (4) a bias that is pervasive in the forma ldehyde literature that outcomes observed 13 beyond the POE (the nose) are not expected from inh alation exposure, which is the route of 14 exposure for most of the developmental and reproduc tive studies. This discussion presents 15 putative MOAs that have been hypothesized by study authors and the studies that support the 16 hypothesized MOAs. The four hypothesized MOAs are not mutually exclusive. They could be 17 acting alone for certain endpoints (in which case t he others are not operable) or in concert for 18 certain endpoints. 19 The focus of this discussion is on analyzing possib le MOAs for the developmental and 20 reproductive outcomes that were noted most consiste ntly, across toxicology studies and, in some 21 cases, across human and animal studies. These outc omes include developmental delays, fetal 22 loss, and sperm quality and quantity effects. 23 An endocrine-disrupting MOA is supported by some of the reproductive and 24 developmental epidemiology and toxicology studies. For example, the decreases in fetal body 25 weight (Martin, 1990; Saillenfait et al., 1989), de layed ossifications (Senichenkova and 26 Chebotar, 1996; Senichenkova, 1991; Martin, 1990; S aillenfait et al., 1989), and delayed 27 eruption of incisors (Senichenkova, 1991) noted in rats after gestational exposure to 28 formaldehyde are consistent with developmental dela ys. In turn, developmental delays can result 29 from effects on the hypothalamic-gonadal-pituitary axis in the dam that cause hormonal level 30 changes in the pup; however, hormone levels in pups were not measured. Kilburn and Moro 31 (1985) also observed organ size changes and undesce nded testes after developmental 32 formaldehyde exposure. These outcomes can also be explained by an endocrine MOA. There 33 are three studies that directly tested for changes in hormones after formaldehyde exposure. 34 Kitaev et al. (1984) observed ovarian weight and se rum LH and FSH increases after inhaled 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-401 formaldehyde in adult female rats. Chowdhury et al . (1992) assessed serum testosterone levels 1 in adult rats after formaldehyde IP exposure and fo und significant decreased testosterone and 2 testes weights and a decrease in 3- β

, ∆ -5-hydroxy steroid dehydrogenase in Leydig cells, 3 suggesting that formaldehyde affects steroidogenesi s. Özen et al. (2002) also reported 4 significant serum testosterone level decreases as w ell as decreased mean seminiferous tubule 5 diameters. Furthermore, the steroidogenesis MOA le ading to reduced testosterone is also 6 consistent with the sperm quality and quantity decr ements observed in the studies by Özen et al. 7 (2002), Sarsilmaz et al. (1999), and Odeigah (1997) studies. 8 In human studies, delayed time to pregnancy and inc reased incidence of spontaneous 9 abortion (Taskinen et al., 1999), consistent with s ome study findings from the toxicology 10 literature, could also be explained by an endocrine MOA. Alterations in hormone levels could 11 lead to pregnancy maintenance problems. Extrapolat ing the Chowdhury et al. (1992) results of 12 the steroidogenesis MOA to females, formaldehyde ex posure could affect progesterone levels 13 required for pregnancy. However, progesterone leve ls were unchanged in the female rat in the 14 one study that assessed progesterone (Kitaev et al. , 1984). Consistent with an endocrine 15 mediated MOA, Maronpot et al. (1986) observed endom etrial hypoplasia and lack of ovarian 16 luteal tissue in females exposed to formaldehyde. 17 A second hypothesized MOA for some of the developme ntal and reproductive outcomes 18 is genotoxicity of the gametes. Such an MOA could explain pregnancy loss in humans 19 (Taskinen, et al., 1999) and preimplantation loss i n animal studies (Xing et al., 2007; Kitaev et 20 al., 1984; Sheveleva, 1971) and fetal viability (e. g., litter size decreases) after formaldehyde 21 exposure. Consistent with male gamete genotoxicity , Odeigah (1997) and Xing et al. (2007) 22 observed reduced fertile matings and live embryos, and increased dead implants in a dominant 23 lethal study. 24 Oxidative stress/damage is another MOA that is cons istent with testicular toxicity, sperm 25 effects, and reduced embryo viability. Özen et al. (2002) investigated the mechanism of 26 oxidative stress being responsible for the testes q uality effects by assessing testicular iron, 27 copper, and zinc levels. Zinc and copper levels we re reduced in the rat testes, consistent with an 28 oxidative stress MOA. Özen et al. (2002) also repo rted increased iron levels and decreased zinc 29 levels in the lung, consistent with oxidative stres s. Another study (Zhou et al., 2006) that 30 investigated the oxidative stress MOA in the testes observed significant changes in oxidative 31 stress biochemical markers (decreases in SOD, GPX, GSH, and an increase in MDA levels). 32 The authors also assessed the protective effect of coadministration with vitamin E, an 33 antioxidant, on decreased testes weight, biochemica l alterations, histopathologic effects, and on 34 sperm count, motility, and morphology. The study o f Pushkina et al. (1968) found reduced 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-402 levels of Vitamin C, another antioxidant, in the fe tus and maternal liver after formaldehyde 1

exposure. 2 The MOAs proposed are not mutually exclusive and in fact could interact with one 3 another. For example, an endocrine MOA could lead to oxidative stress, and that oxidative stress 4 could lead to genotoxicity. 5

6

4.4.9.8. Data Gaps 7 The inhalation developmental toxicity studies condu cted on formaldehyde and described 8 in Section 4.2.1.7 comprise an adequate assessment of prenatal developmental toxicity for 9 application to inhalation reference concentration d etermination. The assessments of postnatal 10 developmental toxicity and of reproductive function following inhalation of formaldehyde are 11 less complete. It is notable that, although the da tabase contains some studies that assess various 12 aspects of reproductive organ system toxicity, part icularly in males, there is no assessment of 13 multigenerational reproductive function, such as wo uld be evaluated in a two-generation 14 reproductive toxicity study, nor is there an assess ment of potential reproductive effects of 15 formaldehyde exposure in human males. 16 Adequate assessments of developmental and reproduct ive toxicity following oral 17 exposures to formaldehyde have not been conducted. 18

19

4.5. SYNTHESIS AND EVALUATION OF CARCINOGENICITY 20 4.5.1. Cancers of the Respiratory Tract 21 Epidemiologic studies of formaldehyde-exposed worke rs provide sufficient evidence of a 22 causal association between formaldehyde exposure an d upper respiratory tract (URT) cancers 23 (e.g., nasopharyngeal cancer (NPC; Section 4.1.2.1. 1), nasal and paranasal cancers (Section 24 4.1.2.1.2), and other upper respiratory tract cance rs (Section 4.1.2.1.3)). In addition, the 25 observational evidence from epidemiologic studies r eporting associations between formaldehyde 26 exposure and increased risk of NPC supports a concl usion of a causal association for this specific 27 cancer. However, epidemiologic studies of rare out comes such as NPC, which has an expected 28 incidence of 1 per 100,000 people per year in the U nited States, do not typically have great 29 statistical power to rule out the null hypothesis ( i.e. no association). However, the weight of 30

evidence of the several studies reviewed in Section 4.1.2.1.1 provide an accumulation of 31

consistent observational evidence sufficient to exc lude chance as an explanation for the 32 association. Additionally, there is insufficient e vidence of consistent confounding or other bias 33 across the studies that were considered; thus, conf ounding and bias were also ruled out as 34 explanations for the observed association. The lac k of a convincing and consistent alternative 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-403 hypothesis of causation − in spite of repeated exam inations − further supports the conclusion that 1 the association between formaldehyde exposure and i ncreased risk of NPC is causal. 2 The single strongest cohort study, Hauptmann et al. (2004), shows a statistically 3 significant exposure-response relationship between formaldehyde exposure and URT cancers. 4 Hauptmann et al. (2004) demonstrated significant ex cess risk of NPC in exposed workers based 5 on U.S. population death rates (standardized mortal ity ratio [SMR] = 2.1; 95% confidence index 6 [CI] 1.05–4.21) in a large cohort of 25,619 industr ial workers. In addition to the SMR based on 7 an external comparison population, relative risks ( RRs) were presented based on internal 8 comparisons of workers in order to minimize potenti al selection bias due to the well known 9 healthy worker effect. Statistically significant e xposure-response relationships between 10 increased formaldehyde exposure and increased risk of NPC were reported for two different 11 metrics of exposure (peak and cumulative exposure). Relative risks for NPC were also elevated 12 for increased duration of exposure to formaldehyde and for the average intensity of exposure. 13 These analyses controlled for potential confounders including calendar year, age, sex, race, and 14 pay category. While exposure measurement error is likely to be present in any epidemiologic 15 study, there was no evidence of any differential me asurement error that could have produced the 16 observation of a spurious association. Any non-dif ferential measurement error would likely have 17 attenuated the effect of formaldehyde was smaller t han that which would otherwise have been 18

observed in the absence of measurement error. 19 The case-control studies similarly also report asso ciations between formaldehyde 20 exposure and cancer mortality for NPC. Although ot her risk factors for NPC (e.g., Epstein-Barr 21 Virus) and the predominant NPC histological sub-typ e (SCC versus undifferentiated) vary 22 significantly across the world, case-control studie s consistently provide evidence of an 23 association between occupational exposure to formal dehyde and NPC (Vaughn et al., 1986a; 24 Vaughn et al., 2000; Roush et al., 1987; Hildesheim et al., 2001; West et al., 1993). In their 25 more recent study, Vaughn et al. (2000) used worker histories to estimate each individual 26 worker’s formaldehyde exposure. Workers with more t han 1.10 ppm-years of cumulative 27 exposure were found to be at significantly higher r isk of NPC, with an odds ratio (OR) of 3.0 28 (95% CI 1.3-6.6) (Vaughn et al., 2000). Two differ ent exposure metrics, duration of exposure 29 and cumulative exposure, were positively associated with increased risk of NPC, with a 30 significant test for trend ( p = 0.014 and 0.033, respectively). The OR also in creased in 31 magnitude as the probability of “Ever” having occup ational exposure increased, from OR = 1.6 32 among those whose exposure was judged to be “Possib le, probable or definite” to OR = 13.3 33 among those with “Definite” exposure ( p –trend < 0.001). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-404 NPC histological sub-type analysis indicates that t hese associations held for both SCC 1 and epithelial NPC, but not for the undifferentiati ng and nonkeritinizing NPC (Vaughn et al., 2 2000). However, formaldehyde exposure is also asso ciated with risk of NPC in Taipei, Taiwan, 3 where greater than 90% of the cases had nonkeritini zing and undifferentiated carcinomas and 4 less than 10% of the cases were diagnosed as having SCCs (Hildesheim et al., 2001). These 5 reported associations were strengthened by consider ing higher probability of exposure (RR = 6 2.6; 95% CI 1.1-6.3), greater intensity of exposure (RR = 2.1; 95% CI 1-4.2) and EBV 7 seropositive cases (RR = 2.7; 95% CI 1.2-5.9) (Hild esheim et al., 2001). Case-control studies 8 have also linked residential exposure to NPC, speci fically for years of residence in mobile homes 9 (Vaughn et al., 1986b) and the use of mosquito coil s in the Philippines (West et al., 1993). 10 Independent testing of 6 brands of East Asian mosqu ito coils evaluated the emission rates of 11 carbonyl compounds in the mosquito smoke and report ed that formaldehyde and acetaldehyde 12 had the highest emission rates. Among the three ex periments on each of the six brands, the 13 range of formaldehyde concentrations was from 0.87 μg/m 3 (1 ppb) to 25 μg/m 3 (31 ppb). 14 As a group, other URT sites of direct contact with formaldehyde upon inhalation (i.e., 15 salivary gland, mouth, nasal cavity and larynx) als o showed evidence of a trend in increasing 16 relative risks with increasing average intensity an d peak exposure in the Hauptmann et al. (2004) 17 cohort study, although these trends did not reach t he level of statistical significance. The results 18

from other cohort studies and case-control studies are mixed (between positive associations and 19 null findings) for associations between formaldehyd e exposure and specific cancers of the URT 20 (IARC, 2006). For rare cancers, extremely large co horts would be needed to have the statistical 21 power to detect an association for tumors defined b y individual sites (e.g., mouth, salivary gland, 22 hypopharnyx). Results vary in the smaller cohort s tudies, where a single case may result in an 23 elevated risk but taken together the evidence is co nsidered suggestive (Section 4.1.2). Case24

control studies have been useful to better understa nd potential associations between 25 formaldehyde exposure and rare cancers of the URT. Luce et al. (2002) evaluated pooled data 26

from 12 case-control studies and demonstrated a sta tistically significant increased risk between 27 formaldehyde exposure and sinonasal cancer. A case -control study by Gustavsson et al. (1998) 28 suggested an association between formaldehyde expos ure and oral squamous cell carcinoma 29 (SCC), esophageal, and laryngeal cancers, with odds ratios (ORs) of 1.28, 1.90, and 1.45, 30 respectively. However, the individual ORs were not statistically significant. Hypopharyngeal 31 cancer was linked with formaldehyde exposure with a n OR of 3.78 (95% CI 1.50-9.49) in 32 another case-control study (Laforest et al., 2000). While the data on site-specific cancers of the 33 URT is somewhat sparse, they are consistent with a carcinogenic hypothesis and in their large 34 cohort study, Hauptmann and colleagues (2004) concl uded that in spite of the small numbers of 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-405 deaths from cancers of the URT, the positive associ ations with average intensity and peak 1 exposure were consistent with the carcinogenicity o f formaldehyde at these sites of first contact. 2

3 Supporting animal evidence 4 Inhalation exposure: 5 Animal studies, primarily rodent bioassays, strongl y support the causal relationship 6 between of formaldehyde exposure and URT carcinogen icity. Formaldehyde-induced cancers 7 are primarily seen in the nasal passages (Kerns et al., 1983; Monticello et al., 1996; Tobe at al., 8 1985; Kamata et al, 1997; Sellakumar et al., 1985), but it should be noted that rodents, unlike 9 humans, are obligate nose breathers and have convol uted nasal turbinates. Chronic animal 10 studies (inhalation and oral exposures) report tumo r incidence in a variety of rodent models. 11 Study descriptions are provided above in detail (Se ction 4.2.1, Table 4-34). The study results are 12 evaluated here for both routes of exposure in the c ontext of how they inform the carcinogenic 13 potential at the portal of entry, specifically the URT. 14 In rodent studies of the respiratory tract, only na sal tumors are considered to be induced 15 by formaldehyde. Repeated exposures to 10-15 ppm f ormaldehyde result in gross nasal lesions 16 and high incidence of nasal tumors (See Table 4-38, Section 4.2.1). Although increased cell 17 proliferation, squamous metaplasia, dysplasia and f ocal necrotic lesions have been noted in the 18 larynx and trachea in some studies, no tumors in th ese locations have been reported in the rodent 19 studies. The majority of studies were conducted usi ng rats (F344, Wistar, or Sprague-Dawley), 20 and all studies of 18 months or greater in mice and rats show evidence of formaldehyde-induced 21 nasal carcinogenicity. The nasal tumors are primar ily SCCs, although papillomas, polypoid 22 adenomas, adenocarcinomas, fibrosarcomas, and esthe sioneuroepitheliomas have been reported 23 (Kamata et al., 1997; Monticello et al., 1996; Morg an et al., 1986a, b; Takahashi et al., 1986; 24 Sellakumar et al., 1985; Kerns et al., 1983; Albert et al., 1982). Although hyperplasia, dysplasia, 25 and squamous metaplasia of the respiratory epitheli um have been observed beyond the nasal 26 cavity, other respiratory tract tumors have not bee n reported to be significantly increased by 27 formaldehyde exposure alone. 28 Increased tumor incidence and decreased latency are correlated with increasing 29 formaldehyde exposure concentration. Reviewing dat a from the only lifelong inhalation study 30 (i.e., until "natural death") with multiple exposur e groups, nasal SCCs occurred much earlier in 31 the high-exposure animals. For example, tumors are first noted at 8 and 9 months following 32 exposure for high-exposed (15 ppm) female and male F344 rats versus tumors not arising until 33 24 months in low-exposed rats (2 ppm) (Kerns et al. , 1983). In a follow-up study by Monticello 34 et al. (1996), the incidence of SCC in rats exposed at 15 ppm was 47%, with the first tumor noted 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-406 at 12 months. The incidence of SCC in male rats ex posed at 10 ppm was 22%, with the first 1 tumors observed at 18 months after exposure. Moreo ver, of the 90 rats exposed at 6 ppm for 20 2 months, only one SCC was noted. No SCCs were detec ted in rats exposed to 0.7 or 2 ppm 3 formaldehyde. These incidence rates are not mortal ity-adjusted (see Chapter 5, section 5.3.4) 4 and include animals from each scheduled sacrifice ( 3, 6, 12, and 18 months). In a lifelong study 5 of male Sprague-Dawley rats exposed to 10 ppm forma ldehyde, the cumulative nasal tumor 6

incidence was calculated as a function of time of e xposure (Sellakumar et al., 1985). After 2 7 years of exposure, the adjusted probability of nasa l carcinoma was greater than 60%. 8 There is some evidence that less-than-lifetime expo sure to formaldehyde can induce nasal 9 tumors over an extended observation period. Two st udies, both in male Wistar rats, report nasal 10 tumors in response to less-than-lifetime exposures (Woutersen et al., 1989; Feron et al., 1988). 11 A 13-week exposure at 20 ppm followed by an observa tion period of 30 months (inclusive of 12 exposure) in Wistar rats resulted in six nasal tumo rs including three nasal SCCs, one cystic SCC 13 of the nasolacrimal duct, one carcinoma in situ and an ameloblastoma, while no tumors were 14 noted in the corresponding air-exposed controls (Fe ron et al., 1988). A limited number of 15 formaldehyde-related tumors were noted from 4 or 8 weeks of exposure followed by 30 months 16 of observation. Although the tumor incidence of th ese less-than-lifetime exposures is low, this is 17 consistent with the 2-year bioassays in Wistar rats . Wistar rats are more resilient to 18 formaldehyde-induced nasal toxicity than F344 or SD rats (Section 4.2.1), and only 1 of 26 (4%) 19 Wistar rats exposed at 10 ppm for 28 months develop ed SCC (Woutersen et al., 1989) versus 20 22% in F344 rats (Monticello et al., 1996). 21 The specificity of formaldehyde-induced tumors in t he nasal passages of rodents is 22 believed, at least in part, to be a function of tis sue dose. Computational fluid dynamics (CFD) 23 modeling used to predict formaldehyde tissue flux d uring inhalation exposures suggests that at 24 comparable concentrations, tissue flux in the nasal passages of rodents is more intense than for 25 non-human primates and humans. Modeling predicts a different pattern of formaldehyde flux 26 into URT tissues of rodents compared to humans, whe re formaldehyde penetrates more deeply 27 into the respiratory tract of primates than rodents even considering nose-only breathing for 28 primates (See Section 3.4). Humans will generally switch to mouth breathing when sensing an 29 irritating smell and during physical exertion, resu lting in direct exposures to the mouth and 30 greater tissue flux in tissues beyond the bypassed nasal passages. Therefore, species differences 31 in tissue dose may contribute to formaldehyde-induc ed tumors in humans beyond the nasal 32 passages, which are not evident in rodent bioassays

. 33

34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-407 Oral Exposure: 1 Consistent with the observed carcinogenic activity of formaldehyde on tissues at the 2 portal of entry (POE) from inhalation exposure, the re is evidence to support POE effects from 3 oral exposures as well – further strengthening the overall weight of evidence of formaldehyde’s 4 carcinogenicity. As with the respiratory tract, th e proximal portion of the gastrointestinal (GI) 5 tract exhibits formaldehyde-induced lesions specifi cally in the forestomach and glandular 6 stomach (Soffritti et al., 1989; Til et al., 1989; Tobe et al., 1989; Takahashi et al., 1986). 7 However, data are mixed regarding the carcinogenic potential of formaldehyde in the GI tract 8 from oral exposures. 9 Two independent 2-year cancer bioassays in Wistar r ats exposed to formaldehyde in 10 drinking water were both negative (Til et al., 1989 ; Tobe et al., 1989). Til et al. (1989) exposed 11 rats to a range of formaldehyde doses (0, 1.2, 15, or 82 mg/kg-day) and evaluated 44–49 animals 12 per sex per dose group at 24 months of exposure. N o formaldehyde-related tumors were found. 13 A smaller study by Tobe et al. (1989) failed to not e any tumors after a 2-year exposure at 0, 10, 14 50 or 300 mg/kg-day (eight rats per sex per treatme nt group.) 15 In contrast, two studies that included lifelong obs ervation in male and female Sprague16 Dawley rats provide support for formaldehyde-induce d GI tract tumors – one study where 17 exposure commenced at 7 weeks of age and a second s tudy conducted with breeder rats (25 18 weeks of age) and their offspring (Soffritti et al. , 1989). These studies demonstrate an increase 19 in GI tumors (although rare) correlated with exposu re to formaldehyde and significantly 20 increased susceptibility to early-lifetime exposure . The authors provide a detailed report on the 21 background rates of various stomach and intestinal neoplasia for male (n = 2,677) and female (n 22 = 2,582) rats within the colony (Soffritti et al., 1989). From this background pool, the total 23 incidence of benign and malignant tumors in the sto mach and intestine combined is only 1.4% 24 (combining all sites and locations), with the major ity of tumors located in the stomach (1% 25 benign, 0.2% malignant). In comparison to colony-s pecific background rates, apparent increases 26 in both stomach and intestinal neoplasia are noted in formaldehyde-treated rats (ranging from 1 27 to 6% by type in rats exposed beginning week 7). W hen summed across the GI tract, tumor 28 incidence in the highest treatment group was 8% ver sus 1.4% in historical controls. Elevations 29 of individual tumors or a clear dose-response relat ionship are difficult to discern for rare cancers 30 where there are only 50 animals per group. Despite the limitations of group size and lack of a 31 dose–response relationship, the findings do support the carcinogenic potential for formaldehyde 32 administered orally. 33 The second study reported by Soffritti et al. (1989 ) in Sprague-Dawley rats demonstrates 34 early lifetime susceptibility, with GI tumor incide nces of 21.6% in female (n = 37) and 13.9% in 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-408 male (n = 36) offspring after exposure to formaldeh yde beginning in utero , versus 5.6% in the 1 adult breeders. Rats were exposed to formaldehyde in drinking water for 2 years (0 or 2,500 2 mg/L). Exposures began on gestational day 12 in th e offspring. The most common tumor 3 detected was intestinal leiomyosarcoma (13.5% in fe male offspring), which has a background 4 rate of 0.04% in female rats in the colony. The in cidence of GI tumors in the adult breeders (n = 5 18 per sex) was due to one adenocarcinoma in a male rat and one pallipoma/acanthoma in a 6 female rat. Although severely limited by study siz e, their occurrence is consistent with the 7 observation of formaldehyde-induced tumors, given t he low background rates for this colony. 8 The Soffritti et al. (1989) studies stand alone in observing formaldehyde-induced GI 9 tumors. These findings are largely attributed to a unique study design that included lifelong 10 observation (i.e., until "natural death"), neonatal exposure, examination of individual tumor types 11 as well as combined rare tumor types for analysis, and summation of tumors across locations. 12 The study design results in a more sensitive assay for rare tumors as well as tumors with a long 13 latency. Thus, Soffritti et al. (1989) utilized a more appropriate design and analysis for detecting 14 rare tumors, and these findings are not diminished by the null results of Tobe et al. (1989) and 15 Til et al. (1989). 16 There is evidence that formaldehyde may act as a tu mor promoter by the oral route as 17 well as the inhalation route (discussed above). Ta kahashi et al. (1986) reported an increase in N18 Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG)-initiated GI cancers in mice with formaldehyde 19 exposure (29.4%, versus 13.3% in controls); the gre atest difference in tumor response was 20 associated with adenocarcinoma of the glandular sto mach (23.5%, versus 3.3% in controls). 21 Additionally, forestomach papillomas and preneoplas tic hyperplasia in the glandular stomach 22 were increased with formaldehyde exposure alone. 23

24 4.5.2 Lymphohematopoietic Malignancies 25

4.5.2.1. Background 26 Lymphohematopoietic (LHP) cancers include neoplasms of both lymphoid and myel oid 27 cell origins. Cancers of the immune system are des cribed as leukemia if they primarily involve 28 cells from peripheral blood and bone marrow at diag nosis and lymphomas if they constitute a 29 solid tumor (Robbins, 2004). Some forms of leukemi a which present as an immature immune 30 cell phenotype are believed to arise from lymphomye loid stem cells or progenitor cells normally 31 found in the bone marrow (e.g., acute lymphoblastic leukemia (ALL) and acute myeloid 32 leukemia (AML)) (Greaves, 2004). However, multiple myeloma, lymphomas and some 33 leukemias may arise from mature functional lymphocy tes present outside of the bone marrow 34 (Greaves, 2004; see Figure 4-33). Therefore, the u se of the general term ‘leukemia’ is not 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-409 restricted to cancers from a transformed stem cell or progenitor cell in the bone marrow but also 1 applies to cancers which arise from differentiated cells (e.g., mature lymphocytes). 2 Epidemiologic studies have reported that formaldehy de exposure is associated with both 3 leukemia and lymphomas (Chapter 4.1.2.2.1). Specif ic neoplasms reported to be associated with 4 formaldehyde exposure include myeloid leukemia, Hod gkin’s lymphoma, and multiple 5 myeloma. 6

7

20 

## H

B

B T T M Germ Line or Early Embryonic

Haemopoietic Stem cell (lymphomyeloid)

Myeloid Stem Cell Lymphoid Stem Cell

Mature lymphocyte Subsets Rare Acute Leukemia

Acute Lymphoblastic Leukemia (ALL) Acute Myeloid Leukemia (AML) Myelodysplastic syndrome Chronic Myeloid Leukemia (CML)

Childhood ALL and AML

Cutaneous T Lymphoma Adult T-cell Leukemia B Chronic Lymphocytic Leukemia non-Hodgkin’s Lymphoma B or T Prolymphocytic Leukemia Hairy Cell Leukemia Myeloma 

### Developmental origins of blood cell cancers

Source: M.F. Greaves, IARC Press, (2004) 8 Figure 4-33: Developmental origins for cancers of the lymphohematopoietic 9 system (Adapted from Greaves (2004). 10 11 12 When evaluating cancers of the LHP system, epidemio logic analysis often groups many 13 of these cancers together. In part, this may be do ne to increase the statistical strength of the 14 analysis. Additionally, since there is a potential for disease misclassification, grouping these 15 diseases is preferred by some researchers, especial ly when analyzing older mortality data. 16 Historically, misclassification may have been due t o factors such as poor histopathology and 17 diagnosis of late-stage disease, where cell line of origin may have been hard to distinguish. 18

Without the cell surface markers and molecular tool s used today to classify cells, diagnosis was 19 accomplished primarily based on histology. However , as the cancers progress, the leukemic 20 stem cells may present as less mature cells. For e xample, poor health surveillance may allow 21 chronic myeloid leukemia (CML) to remain undiagnose d until the blast crisis, often seen late in 22This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-410 the disease. This blast crisis presents as a leuke mia of relatively immature myeloid cells and 1 may have been mistaken for AML without the more rec ent techniques available for classifying 2 disease. Since cancers of the LHP system may prese nt with cell surface markers for multiple cell 3 lines, classification remains problematic in some c ases. 4 Although often grouped for analysis, the LHP cancer s represent many distinct 5 malignancies which may arise from discrete cell typ es in different tissues throughout the body 6 (Greaves, 2004). The World Health Organization (WH O) has developed a classification system 7 for both lymphoid and myeloid leukemia defined by a combination of morphology, 8 immunophenotype, genetic features and clinical feat ures (Harris et al., 2000a; Harris et al., 9 2000b). The historical nomenclature and Internatio nal Classification of Disease (ICD) codes 10 used in epidemiologic studies do not correspond to these new classification systems. For 11 example, both chronic lymphocytic leukemia (CLL) an d B-cell lymphomas arise from similar 12 cell types in the periphery, yet epidemiologic stud ies have considered them independently even 13 though they are currently considered to be the same disease in the new WHO classification 14 system − which would diminish the statistical power to detect an association. Careful re-analysis 15 of epidemiologic data addresses evidence for the cl ass as a whole (all LHP cancers) but also 16 various subclasses (e.g. myeloid versus lymphoid). 17 Therefore, the following analysis first examines th e epidemiologic evidence for all LHP 18 cancers as a class, then all leukemia, to best take advantage of the majority of publications 19 available in assessing the weight of evidence for t he carcinogenicity of formaldehyde. The 20 subsequent analysis by sub-type draws upon the avai lable evidence for specific diseases or 21 groups of diseases (e.g., myeloid leukemia, Hodgkin ’s lymphoma, non-Hodgkin’s lymphoma). 22 Although fewer data are available for the sub-type analysis, these data help clarify which cancers 23 may contribute to the consistent observation of an association between formaldehyde exposure 24 and all LHP cancers. Novel combinations of phenoty pic sub-types are presented where they are 25 etiologically relevant. The sub-type analysis fram es the subsequent mode of action (MOA) 26 analysis. 27

28

4.5.2.2. All LHP Malignancies 29 Epidemiologic studies involving formaldehyde-expose d workers provide sufficient 30 evidence of a causal association between formaldehy de exposure and all LHP malignancies 31 (Section 4.1.2.1.5). Positive associations between formaldehyde exposure and LHP cancers have 32 been reported for chemical workers (Wong et al., 19 83; Bertazzi et al., 1986), embalmers 33 (Walrath and Fraumeni, 1983, Walrath and Fraumeni, 1984; Hayes et al., 1990), anatomists and 34 pathologists (Harrington and Shannon 1975; Hall et al., 1991; Levine at al 1984; Stroup et al., 35 1986; Matanoski et al., 1989) (Table 4-90). Howeve r, clear associations (in terms of overall 36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-411 standardized mortality ratios [SMRs] or proportiona l mortality ratios [PMRs]) were not reported 1 in analyses for garment workers, iron-foundry worke rs, and a large US industrial cohort 2 (Pinkerton et al., 2004; Andjelkovich et al., 1995; Beane Freeman et al., 2009; Marsh et al., 3 1996), although associations were observed in some of these studies when exposure-response 4 relationships were considered. Several published m eta-analyses are available which more 5 formally assess the strength of association between formaldehyde exposure and mortality from 6 all LHP cancers. Pooled SMRs indicate stronger ass ociations for professional workers 7 (embalmers, anatomists and pathologists) than indus try workers (Table 4-91). Bosetti et al. 8 (2008) found similar relationships, with a pooled S MR of 1.31 (95% CI 1.16-1.47) for 9 ‘professionals’ (i.e. embalmers, anatomists and pat hologists) versus a pooled estimate of 0.85 10 (95% CI 0.74-0.96) for industrial workers. A recen t meta-analysis by Zhang et al. (2009) reports 11 a summary relative risk (RR) of 1.25 (95% CI 1.09-1 .43) for both professional and industry 12 workers for all LHP cancers (ICD 9 codes 200-209). These researchers identified 19 cohort 13 study analyses, including cohort study updates. Zh ang et al. (2009) used the reported RR from 14 the highest exposure category to increase statistic al power and reduce uncertainty regarding 15 confounding or other bias. Although study selecti on was controversial, e.g., the inclusion of 16 multiple reports from a single cohort and the use o f one cohort where only a portion of the 17 workers were formaldehyde-exposed, this meta-analys is is generally supportive of an association 18 between formaldehyde and LHP malignancies. 19 The apparent differences by industry/profession may reflect many influences, including 20 exposure potential and demographic characteristics. External analysis (use of the general 21 population for comparison) relies on the assumption that cancer incidence rates are expected to 22 be similar between the general population and the s tudy population in the absence of exposure. 23 The ‘healthy worker effect’ is well known, and ther e may be differences in the magnitude of this 24 selection bias by industry or profession. For inst ance, LHP cancer incidence and mortality have 25 many risk factors including socioeconomic status. Therefore, the consistent positive findings in 26 professional workers versus mixed results in indust rial workers could be influenced by the 27 appropriateness of the comparison to the general po pulation − that is, a differential extent of 28 selection bias. Interestingly, salaried workers, but not the hourly workers, in an Italian plastic 29 manufacturing plant had elevated SMRs for LHP cance rs (1.69 (95% CI 1.07-2.53) and 30 0.93(95% CI 0.62-1.35), respectively) (Dell and Tet a, 1995). Without knowledge of which 31 worker group is most similar to the comparison popu lation with respect to LHP cancers 32 mortality, one cannot discern if this potential eff ect of demographic variability accentuates 33 effects in professional/salaried workers or obscure s the effects in industrial/hourly-wage 34

workers. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-412 Table 4-90. Summary of cohort and case-control studies which e valuated 1 the incidence of all LHP cancers in formaldehyde-ex posed populations 2 (ICD-8 Codes: 200-209) and all leukemias (ICD-8 Cod es: 204-207). (See 3 Table 4-6 for complete study details and findings) 4 5 Study population Study details All LHP cancers Leukemia Reference SMR Analysis 1 Pathologists and technicians (n=2,079) Years of study 1955-1973 2.0 (p<0.01) {pathologists}

0.5 {technicians} 0.6

{pathologists}

0.5 {technicians} Harrington and

Shannon, 1975

Pathologists and technicians (n=2,720) 1974-1980

NR 0.91 (0.05-4.29)

men

9.26(0.47-43.9)

women Harrington and

Oakes, 1984

Pathologists (n=4,512) Years of study 1974-1987 1.44 (0.69-2.63) 1.52 (0.41-3.89) Hall et al., 1991

Ontario Undertakers (n=1,477) Mortality from 1950-1977 1.24 1.60 Levine et al.,1984

Male Anatomists (n=2,327) Mortality from 1925-1979 1.20 (0.7-2.0) 1.5 (0.7-2.7) Stroup et al., 1986

Male pathologists (n=4,485) Mortality through 1977 NR 1.06 Logue et al, 1986

Male pathologists (n=6,111) Participants from 1912-

1950 membership rolls.

Mortality followed through 1978. 1.25 (0.95-1.62) 1.35 (0.92-1.92) Matanoski et al., 1989

Chemical industry workers, men (n=14,014) Mortality from 1941-2000 NR 0.91(0.62-1.39) Coggon et al., 2003

Chemical workers (n=2,026) 1.36 (0.5 – 2.95) Wong et al., 1983

Industrial workers (n=25,619) Mortality followed through

2004 0.94 (0.84-1.06) 1.02

(0.85-1.59) Beane-Freemen et

al., 2009

Industrial workers (n=7,328) 0.89 Marsh et al., 1996 {Subset of NCI cohort reported by Hauptmann et al., 2003} Garment workers (n=11,098) Mortality followed through

1998 0.97 (0.74-1.26) 1.09 (0.70-1.62) Pinkerton et al., 2004 Resin plant workers (n=1,330) Employed between 1959-

1980

Mortality through 1986 2.01 NR Bertazzi et al., 1986

Plastic manufacturing 1.69 (salaried workers) 1.98

(salaried workers) Dell and Teta,

1995This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-413 (n=5,932) 0.93 (hourly workers) 0.98

(hourly workers) PMR Analysis 1

Embalmers, New York (n=1,132) Licensed between 1925-

1980 1.15 1.32 Walrath and Fraumeni, 1983

Embalmers, CA (n=1,007) Licensed between 1925-

1980

1.22 1.75 (p<0.05)

1.24 (<20 years) 2.21 (P<0.05) (>20 years) Walrath and

Fraumeni, 1984

Embalmers, U.S. (n=4,046) 1.39 (1.15-1.63)

White 1.31 (1.06-1.59) Nonwhite 2.41 (1.35-3.97) 1.52 2

(0.98-2.35)

White 1.44 (p<0.05) Nonwhite 2.72 (p<0.05) Hayes et al., 1990

Case-Control Studies 1

American cancer Society Cancer Prevention Study II: (n=362,828 men) Results for men reporting

formaldehyde exposure, and occupations related to formaldehyde exposure 1.22 (0.84-1.77) (formaldehyde exposed)

3.44 (1.11-10.68) {formaldehyde exposure and occupation} 0.96 (0.54-1.71)

(formaldehyde exposed)

5.79 (1.44-23.25) {formaldehyde exposure and occupation} Stellman et al.,

1998

White men diagnosed with leukemia (Iowa and Minnesota) (n=622) Recruited in 1980-1983 NR 1.0 (0.7-1.4) Low 0.7 (0.2-2.6) High Blair et al., 1993

1

Notes: 2 1. Relative risk estimate (SMR, PMR, or OR) presen ted with 95% confidence intervals, where available. 3 2. PMR for leukemia for the total group calculated from the published data for lymphatic leukemia (20 4, myeloid leukemia 4 ( 205), and other/unspecified (206, 207). 5 6 7 The only study which has data to inform the effects of either exposure level or the 8 appropriateness of an external comparison group on the association between formaldehyde 9 exposure and all LHP cancer mortality is the Nation al Cancer Institute (NCI) cohort study of 10 industrial workers (Blair et al., 1986; Beane Freem an et al., 2009), which presents relative rates 11 based on internal comparisons for 3 different expos ure metrics. Although SMR analysis with an 12 external comparison group did not indicate increase d mortality from all LHP cancers (0.94, CI 13 0.84-1.06, for the exposed workers), internal analy sis using the low-exposed workers as the 14 comparison group demonstrates positive exposure-res ponse relationships for increased mortality 15This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-414 from all LHP malignancies cancers and peak exposure across the study periods (1965-2004) 1 (Figure 4-34A and 4-34B) (Beane Freeman et al., 200 9), with a statistically significant trend (p < 2 0.05) for every year since 1977. These results, in dicating a positive exposure-response 3 relationship among plant workers, who most likely h ave similar demographic characteristics, are 4 noteworthy given the apparent lack of association w hen SMRs for the same cohort are calculated 5 against mortality rates for the general population. The lack of an apparent association with 6 SMRs may be attributable to the healthy worker effe ct and/or some other difference between the 7 exposed workers and the general population. 8 Although the association between formaldehyde expos ure and all LHP cancer mortality 9 in industrial and professional cohorts is mixed, th e strength of the internal analysis of the NCI 10 cohort, in the absence of positive SMRs compared to the general population, suggests that SMR 11 analyses may not be the most appropriate methodolog y for assessing LHP cancer mortality. 12 Given the potential for demographic differences bet ween an industrial workforce and the general 13 population, the results of the internal analysis of the NCI industrial cohort provide a higher 14 quality analysis – and therefore should be given si gnificantly more weight than SMR analyses of 15 industrial workers that could not distinguish their findings from the null. Given the consistency 16 and strength of the positive associations for all L HP cancers cancer mortality in professional 17 cohorts (embalmers, anatomists and pathologists) ta ken together with the strong positive results 18 of the NCI cohort, human epidemiologic evidence are sufficient to conclude that there is a causal 19 association between formaldehyde exposure and morta lity from all LHP malignancies (as a 20 group). 21

22

4.5.2.3. All Leukemia 23 Like the analysis of all LHP cancers, an analysis of all leukemia combines diseases which 24 differ significantly in cell of origin and etiology , including acute and chronic forms of both 25 myeloid and lymphatic leukemia. This class also in cludes other leukemia (e.g., erythraemia) and 26 a general category of ‘other and unspecified leukem ia’ (ICD-8 207). Regardless, there is some 27 utility in evaluating the all leukemia mortality da ta because many studies provided results for this 28 grouping. Also, the diagnosis of leukemia versus s olid LHP tumors is fairly distinct thereby 29 limiting misclassification of the endpoint. 30

31This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-415 1 2 Figure 4-34A. Association between peak formaldehyd e exposure and the risk 3 of lymphohematopoietic malignancy

. 4 5 Relative risks for medium-peak (2.0 to <4.0 ppm) an d high-peak ( ≥ 4.0 ppm) 6 formaldehyde exposure categories compared with the low exposed category (>0 7 to <2.0 ppm) and P values for trend tests among the exposed person-ye ars for 8 lymphohematopoietic malignancies are shown by year of end of follow-up, 1965- 9 2004. Values plotted at 0.1 represent RR = 0 due t o no cases in the exposure 10 category values plotted at 20 represent RR = infini ty due to no cases in the 11 referent category. The small graphs above the relative risk plots represent the 12 exposure-response trend P values based on two-sided likelihood ratio tests ( 1 df) 13 of zero slope for continuous formaldehyde exposure among exposed person-years 14 only. The points represent the relative risk estimates based on the cumulative 15 number of cases and person-years accrued from the s tart of the study to that point 16 in time and for 2004 are equivalent to the relative risk estimates presented in 17 Table 2. HLP = lymphohematopoietic malignancies, N HL = non-Hodgkin 18 lymphoma, HDG = Hodgkin lymphoma, MM = multiple mye loma, LEU = 19 leukemia, LYL = lymphatic leukemia, MYL = myeloid l eukemia, RR = relative 20 risk. 21 22 Source: Beane Freeman, et al. (2009) 23This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-416 1 Figure 4-34B. Association between average intensit y of formaldehyde 2 exposure and the risk of lymphohematopoietic malign ancy. 3 4 Relative risks for medium (0.5 – 0.9 ppm) and high ( ≥ 1.0 ppm) average-intensity 5 formaldehyde exposure categories compared with the low exposed category (0.1 – 6 0.4 ppm) and P values for trend tests among the exposed person-yea rs for 7 lymphohematopoietic malignancies by year of end of follow-up, 1965 – 2004. 8 Values plotted at 0.1 represent RR •• = due to no c ases in the exposure category. 9 The small graphs above the relative risk plots represent the exposur e – response 10 trend P values based on two-sided likelihood ratio tests (1 df ) of zero slope for 11 continuous formaldehyde exposure among exposed pers on-years only. The points 12 represent the relative risk estimates based on the cumulative number of cases and 13 person-years accrued from the start of the study to that point in time and for 2004 14 are equivalent to the relative risk estimates prese nted in Table 3 . HLP = 15 lymphohematopoietic malignancies, NHL = non-Hodgkin lymphoma, HDG = 16 Hodgkin lymphoma, MM = multiple myeloma, LEU = 17 leukemia, LYL = lymphatic leukemia, MYL = myeloid l eukemia; RR = relative 18 risk. 19 20 Source: Beane Freeman, et al. (2009) 21This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-417 Although results are mixed across the studies (Tabl e 4-90), an association between 1 formaldehyde exposure and leukemia mortality is sup ported by cohort analyses of embalmers, 2 pathologists and anatomists (Hayes et al., 1990; Wa lrath and Fraumeni, 1983; Walrath and 3 Fraumeni 1984; Hall et al., 1991; Levine et al., 19 84; Stroup et al., 1986; Matanoski et al., 1989). 4 Formaldehyde exposure and formaldehyde-related occu pation are associated with leukemia 5 diagnosis in a case-control study (RR = 5.79 (95% C I 1.44-23.25), but not formaldehyde 6 exposure alone (RR = 0.96; 95% CI 0.54-1.71) (Stell man et al., 1998). 7 In contrast, SMR analyses of the industrial cohorts do not indicate a similar association 8 (Coggon et al., 2000; Beane Freeman et al., 2009, P inkerton et al, 2004). Although the SMR 9 analysis provided for the NCI cohort does not indic ate a positive association for all leukemia 10 using an external reference group (Beane Freeman et al., 2009), the SMR for exposed versus 11 unexposed workers within the cohort suggests all le ukemia is elevated 2.1-fold with this internal 12 comparison (95% CI 0.99-4.56) 4 . A positive exposure-response relationship furthe r strengthens 13 the association of formaldehyde exposure to leukemi a mortality (Beane Freeman et al., 2009). 14 Where the referent group is defined as ‘low exposed ’ individuals, leukemia is elevated in the 15 highest peak exposure category (RR = 1.42; 95% CI 0 .92-2.18) compared to both the referent 16 group and the unexposed category (RR = 0.59; 95% CI 0.25-1.36), and there is a statistically 17 significant trend across all groups ( p = 0.02). Categorical analysis for the average int ensity and 18 cumulative exposure metrics suggests greater mortal ity in the high-exposure groups versus the 19 ‘low exposed’ individuals (RR = 1.10 [95% CI 0.68-1 .78] and 1.11 [0.7-1.74]. respectively), but 20 analysis of individual results across the exposure- response range indicates cumulative exposure 21 is a better predictor ( p = 0.08 for trend across all exposed and unexposed.) 22 Several meta-analyses have been conducted for forma ldehyde exposure and leukemia 23 which indicate a positive association. Collins et al. (2004) report an overall RR for 18 available 24 studies of 1.1 (CI 1.0-1.2), suggesting an associat ion of leukemia with formaldehyde exposure. 25 This association was stronger for both pathologists /anatomists (1.4; CI 1.0-1.9) and embalmers 26 (RR = 1.6; 1.2-2.0) than for industrial workers (RR = 0.9; 0.8-1.0). Study design also impacted 27 the apparent strength of association, with stronger associations seen in case--control studies (RR 28 = 2.4; 0.9-6.5) versus cohort studies (RR = 1.0; 0. 9-1.2). Bosetti et al. (2008) reported an 29 association between formaldehyde exposure and leuke mia mortality with a pooled RR of 1.39 30 (95% CI 1.15-1.68) for 8 groups of professional wor kers. In the same analysis, the pooled RR 31 for the 4 industrial cohorts was 0.90 (0.75-1.07). Zhang et al. (2009) reported a pooled RR of 32

4

osed Non Exposed osed Non Exposed Obs

Obs SMR SMR Var

exp exp 1

1 ln +

=         

      This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-418 1.54 (95% CI 1.18-2.00) for all cohorts identified in their meta-analysis, although this pooled RR 1 should be considered with some caution, as myeloid leukemia alone was included in the analysis 2 where available (Zhang et al., 2009). 3

4

4.5.2.4. Subtype Analysis 5

Given the associations discussed above between for maldehyde exposure and both all 6 LHP cancers and all leukemia, further analysis is n eeded to examine if the observed increase in 7 all LHP cancers is primarily a reflection of increa sed leukemia, or if other types of LHP cancers 8 may be elevated as well. Although analysis of mort ality data by sub-type may provide a better 9 understanding of the specific disease associations, there are potential pitfalls as well. Chief 10 among these concerns are the potential for disease misclassification (especially in studies with 11 older mortality data) and lack of statistical power as the number of observed cases is reduced by 12 considering sub-types. Case control studies by des ign address specific diseases and are well13 suited for sub-type analysis, but often provide lit tle exposure information. The following 14 analysis will draw from the available data to exami ne which forms of LHP malignancies may be 15 associated with formaldehyde exposure. 16 There has been speculation that the association be tween formaldehyde exposure and 17

increases in all LHP cancers and all leukemia are d riven by increased myeloid leukemia (Pyatt et 18 al., 2008; Heck and Casanova 2004; Golden et al., 2 006). If this were the case, then mortality 19 from LHP cancers other than myeloid should not be e levated, once the excess mortality from 20 myeloid leukemia is accounted for. Only 2 studies provide the data to evaluate this hypothesis – 21 both conducted by the NCI (Hayes et al., 1990 and B eane Freeman et al., 2009). From the 22 published data, crude mortality statistics can be c alculated for alternative disease groupings 23 (Table 4-91). In the NCI embalmer study (Hayes et al., 1990), only myeloid leukemia was 24 statistically elevated in the subtype analysis. Fo r the NCI industrial cohort (Beane Freeman et 25 al., 2009), elevations were also seen for Hodgkin l ymphoma relative to the referent group. In 26 both cases, the association between formaldehyde ex posure and LHP malignancies remains when 27 myeloid leukemia is dropped from the analysis. Fur ther, similar associations are found when all 28 leukemia and myeloproliferative diseases are droppe d from the analysis and only solid tumors of 29 lymphoid origin are included (lymphosarcoma and ret iculosarcoma, Hodgkin lymphoma, non30 Hodgkin lymphoma and multiple myeloma). These rean alyses illustrate the need for a more 31 careful sub-type analysis to assess the potential f or associations between formaldehyde exposure 32

and various forms of LHP cancers. 33

34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-419 Table 4-91. Secondary analysis of published mortal ity statistics to explore 1 alternative disease groupings within the broad cate gory of all lymphohematopoietic 2 malignancies 3 4

ICD-8 Codes U.S. embalmers (Whole cohort) (Hayes et al., 1990)

PMR (95% CI) U.S. industry (peak exposure metric: >4 ppm vs. >0 to ≤ 2 ppm) (Beane Freeman et al., 2009)

Relative risk (95% CI) All Lymphohematopoietic Malignancies 200-209 1.39 a (1.15-1.67) b 1.37 c (1.03-1.81) c

Alternative Disease Groupings

Exclude Myeloid Leukemia 200-204,

206-209 1.35 a (0.99-1.85) b 1.31 d

(0.97-1.75) d,e

Solid tumors of lymphoid origin (Lymphosarcoma and reticulosarcoma, Hodgkin lymphoma, non-Hodgkin lymphoma and multiple myeloma) 200-203 1.24 a (0.84-1.84) b 1.33 d

(0.93-1.90) d,e

5

a

Exp

Obs PMR = 6

b ( ) Exp Obs PMR Var 1

1 log +

= 7

c See Table 2 of Beane Freeman et al. (2009) 8

d

765 .0 37.1 103 108 14 103 19 108

=      

× ≅                

×      

− −

=

− −

=

c c c

Group Comparison

Group referent Group Comparison

Group referent

Group referent Group referent Group Comparison Group Comparison

PT PT where PT PT

Time Person Deaths Time

Person Deaths RR 9

e 

### ( )

Group referent Group Comparison Deaths Deaths RR Var 1

1 log + = 10

11 12 4.5.2.5. Myeloid Leukemia 13 The associations between myeloid leukemia and form aldehyde exposure are strong and 14 consistent (Table 4-92). Of the four studies which formally assess myeloid leukemia mortality, 15 all are positive, including cohorts of both profess ional and industrial workers (Beane Freeman et 16 al., 2009; Hayes et al., 1990; Pinkerton at al., 20 03; Stroup et al., 1986). Although few cases 17 exist for further subtype analysis, the available d ata indicate either no differences in SMRs for 18 acute myeloid leukemia (AML) versus chronic myeloid leukemia (CML) (Hayes et al., 1990; 19 Pinkerton et al., 2003) or suggest CML is more prom inent (Blair et al., 2000; Stroup et al., 1986). 20

21This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-420 Table 4-92. Summary of studies which provide mortality statisti cs for 1 myeloid leukemia sub-types. 2 3 Reference

Study population Myeloid

Leukemia Acute

Myeloid Leukemia Chronic

Myeloid Leukemia SMR Analysis 1

Garment workers (n=11,098) 1.44 (0.08-2.37) 1.34 (0.61-2.54) 1.39

(0.38-3.56) Pinkerton at al.,

2003 Anatomists (n=2,317) NR NR 8.8* Stroup et al., 1986

Industrial workers (n=25,619) 0.90 (0.67-1.21)

SMR Ratio 1.38 (0.65-2.97) (exposed/unexposed) NR NR Beane Freeman et al., 2009

PMR Analysis 1

Embalmers, U.S. (N=4,046) 1.57 (1.01-2.34) 1.52 (0.85-2.52) 1.84

(0.79-3.62) Hayes et al., 1990

Case-Control Studies 1

White men diagnosed with leukemia (Iowa and Minnesota) (n=622) NR Low: 0.9 (0.5-1.6)

High: NR Low:

1.3 (0.6-3.1)

High: 2.9 (0.3-24.5) Blair et al., 1993

4 * Leukemia SMR 1.5 (0.7-2.7) {5 of 10 deaths due to myeloid}; Chronic Myeloid Leukemia (CML) SMR of 8.8 5 1. Relative risk estimate (SMR, PMR, or OR) presen ted with 95% confidence intervals, where available. 6 7 8 Walrath and Fraumeni (1983, 1984) note that AML is prominent in their analyses of New 9 York and California licensed embalmers; however, th ey do not provide PMR analyses for CML. 10 Walrath and Fraumeni (1983 and 1984) report leukemi a cell types - for both studies the majority 11 of myeloid leukemia are acute (5/6 and 4/6, respect ively, for New York State and California 12 embalmers). However, PMRs cannot be calculated for AML versus CML in this paper, as 13 comparison rates are not available from the 1920's through the 1960's - the timeframe with the 14 majority of deaths. The authors do contrast the ob served rate of AML in the cohort to the 15 background rate for AML in white men in the 1970s - but given the potential misclassification of 16 late stage CML as AML, especially historically, thi s may not be an appropriate comparison. 17 Additionally, one would expect older data to over-r epresent AML rather than CML due to 18 diagnosis in the early decades of CML in the blast crisis as AML. Therefore, although these 19 studies support an association between formaldehyde exposure and myeloid leukemia in general 20This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-421 (Walrath and Fraumeni, 1983; 1984), the reported AM L and CML subtype information does not 1 allow a satisfactory sub-type analysis for myeloid leukemia. 2

3 4.5.2.6. Solid Tumors of Lymphoid Origin 4 Multiple myeloma, Hodgkin lymphoma, non-Hodgkin ly mphoma, lymphosarcoma, 5 reticulosarcoma, and other lymphomas may all be der ived from immune cells outside of the bone 6 marrow compartment, in peripheral blood, in the gut and respiratory mucosa and immune tissues 7 at the POE (e.g., lymph nodes, mucosa-associated ly mphoid tissue (MALT), gut-associated 8 lymphoid tissue (GALT) (Greaves, 2004). The only m eta-analysis to specifically address 9 lymphoid malignancies found evidence for increased lymphoma (Hodgkin lymphoma (pooled 10 RR = 1.23; 95% CI 0.67-2.29) and multiple myeloma ( 1.31; 1.02-1.67), but not for non-Hodgkin 11 lymphoma (1.08; 0.86-1.35) (Zhang et al., 2009). A s seen in Table 4-93 below, individual study 12 results are mixed for these lymphoid cell-line mali gnancies, as they are for all LHP cancers and 13 all leukemia above. Although these tumors are from mature lymphocytes, there is still variability 14 in the etiology, natural history and risk factors f or the many sub-types which are included in 15 these categories. 16 There is evidence for an exposure response relatio nship for both Hodgkin lymphoma and 17 multiple myeloma in the NCI industrial cohort among exposed workers (Beane-Freeman et al., 18 2009). Clear exposure response relationships for H odgkin lymphoma are defined with all three 19 metrics of exposure, peak average intensity and cum ulative exposure (p=0.01, p=0.05 and 20 p=0.08 respectively for mortality through 2004). T hese associations have been evident from first 21 follow-up through the current publication, and stat istically significant for the majority of the 22 follow-up period demonstrating that this is a stron g and consistent finding in the NCI cohort 23 (Figure 4-34 A&B) (Beane-Freeman et al., 2009). Al though the overall SMR for multiple 24 myeloma does not indicate an association, trends ac ross time indicate consistent elevation of 25 multiple myeloma mortality with both peak and avera ge intensity of exposure, where the 26 statistical strength of the association with peak e xposure increases with follow-up (Figure 4-34 27 A&B). 28This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-422 Table 4-93. Summary of mortality statistics for Ho dgkin lymphoma, 1 lymphoma and multiple myeloma from cohort analyses of formaldehyde 2 exposed workers. 3 4

Study population Hodgkin Lymphoma Non-Hodgkin

Lymphoma Multiple

Myeloma Reference

SMR Analysis 6 Pathologists (n=2,079) 1.4 2.0 (p<0.05) NR 1 Harrington and Shannon, 1975 Pathologists (n=4,512) 1.21 (0.03-6.71) 1.44 (0.69-2.63) NR Hall et al., 1991

Male Anatomists (n=2,327) − 2 0.7 (.1-2.5) 3 2.0 (0.7-4.4) 4 NR Stroup et al., 1986

Male pathologists (n=6,111) 0.36 (0.04-1.31) 1.31 (0.66-2.35) 3 1.54 (0.82-2.63) 4 NR Matanoski et al., 1989 Chemical workers (n=2,026) 2.94 (0.33- 10.63) NR NR Wong et al., 1983

British Chemical plants (n=14,014) 0.36 (0.01-2.01) 0.89 (0.41-1.70) 1.18 (0.48- 2.44) Coggon et al., 2003

Swedish workers- abrasive production plant (n=911) NR 2.0 (0.2-7.2) 4.0 (0.5-14) Edling et al., 1987a

Industrial workers (n=25,619) 1.42 (0.96-2.10) 0.86 (0.70-1.05) 0.94 (0.71- 1.25) Beane Freemen et

al., 2009

PMR Analysis 6 Embalmers, New York (n=1,132) 0.87 (p<0.05) 1.08 3

1.22 4 NR Walrath and Fraumeni, 1983 Embalmers, CA (n=1,007) − 2 3.10 3

1.33 4 NR Walrath and Fraumeni, 1984 Embalmers, U.S. N=4,046 0.72 (0.15-2.10) 1.26 (0.87-1.76)

1.12 (0.58-1.96) 3 1.35 (0.84-2.01) 4 1.37 (0.84- 2.12 Hayes et al., 1990

Case-Control Studies 6 Women in Connecticut (n=601) NR 1.3 (1.0-1.7) NR Wang et al., 2009

White men, Iowa and Minnesota (n=622) NR 1.2 (0.9-1.7) NR Blair et al., 1993

ACS Cancer Prevention Study II (n=128) NR NR 1.8 (0.6-5.7) Boffetta et al., 1999

Men, ACS Cancer Prevention Study II (n=45,399) NR 0.92 (0.5-1.68) 2.88 (0.40-10.5) 5 0.74 (0.27- 2.02) Stellman et al., 1998

Danish workers (n=1,098) NR NR Heineman et al., 1992 Danish women (607) NR NR 1.6 (0.4-5.3) Pottern et a l., 1992 Notes : 5 1: NR is not reported 6 2. “—" no cases observed. 7 3. Lymphosarcoma and reticulosarcoma only 8 4. “other lymphoma" 9 5. Formaldehyde exposure in a wood-related occupati on. RR for wood-related occupation alone was not el evated 10 0.97 (0.55-1.73) 11 6. Relative risk estimate (SMR, PMR, or OR) presen ted with 95% confidence intervals, where available 12This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-423 4.5.2.7. Supporting Evidence from Animal Bio-Assays for Form aldehyde-Induced 1 Lymphohematopoietic Malignancies 2 Chronic animal studies provide supporting evidence for formaldehyde-induced leukemia 3 and lymphoma (Soffritti et al., 1989; Battelle Labo ratories, 1981). Although the majority of 4 chronic animal bioassays do not report either leuke mia or lymphoma, it should be noted that 5 many studies focused primarily on respiratory tract and did not provide routine examination of 6 other organs, limiting the detection of leukemia an d lymphoma (Horten et al., 1963; Holmstrom 7 et al., 1989; Wouterson et al., 1989; Appleman et a l., 1988; Monticello et al., 1996; Dalbey, 8 1982). Kamata et al. (1986) did examine additional organs, but there were only 5 animals at 9

each sacrifice. Similar issues are seen with some of the drinking water studies, where Takahashi 10 et al. (1986) focus on the stomach and intestines, and the study in Wistar rats by Tobe et al. 11 (1989) only included 20 animals per sex per exposur e group with interim sacrifices. Therefore, 12 few studies remain to be explored which are informa tive about the carcinogenic potential of 13 formaldehyde on the LHP system. Table 4-94 lists the studies from the chronic bioassays which 14 have the potential to detect LHP malignancies. 15 Soffritti et al. (1989) were the first to publish a finding of formaldehyde-induced 16 leukemia in an animal bio-assay. Sprague-Dawley ra ts (50 per each sex) were exposed to 17 formaldehyde in drinking water at 0, 10, 50, 100, 5 00, 1000, 1500 mg/L given ad libitum

. 18 Lymphoblastic leukemia and lymphosarcomas were the most common lesion reported, and 19 exhibited an apparent dose-dependent increase in bo th male and female rats (Table 4-95). All 20 hemolymphoreticular cancers summed together reflect ed similar trends, but the other types did 21 not show similar relationship with exposure (e.g. i mmunoblastic lymphosarcoma). A subsequent 22 publication of full study results reports all lesio ns together as lymphoma and leukemia, providing 23 less-specific information (Soffritti et al 2002.) Additionally, there is a large discrepancy in 24 reported lesions between the two study reports wher e nearly double the incidence is reported in 25 2002. Dr. Soffritti explains the increase in repor ted LHP malignancies as a result of tabulating 26 the full histopathological findings in the 2002 rep ort (personal communication, 23 July 2009). 27 Although the second report from Soffritti et al. (2 002) have been broadly criticized on both of 28 these points (summing of dissimilar lesions and dis crepancies in reporting), these errors do not 29 iupune the original report. The 1989 report does d istinguish between different sub-types 30 providing positive results for lymphoblastic leukem ia/lymphosarcoma and histological 31 examinations were consistently conducted between tr eatment groups – even if further results 32 were published at a later timepoint. Therefore, th e findings of Soffritti et al. (1989) are 33 considered as supportive of the biological plausibi lity of formaldehyde-induced 34 lymphohematopoietic malignancies. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-424 Table 4-94: Summary of chronic bioassays which add ress rodent leukemia 1 and lymphoma 2 3 

### Study Histopathology Endpoint Results Comments Drinking Water Exposure Male and Female Sprague-Dawley Rats

Sofritti et al. 1986 Complete histopathology Lymphocytic leukemia and lymphosarcoma Increased, showing a

dose-response Life-long study High exposure of 1,500 mg/l in

water Male and Female Wistar Rats Til et al, 1989 Complete histopathology

in control and high dose group (15 ppm) Lymphoma, leukemia No increase

(3 lymphomas and 1 leukemia found in 200 animals at the 2 yr sacrifice) 2-year bioassay High exposure of approximately 1900 mg/l (82mg/kg for males and 109 mg/kg for females) 

### Inhalation Exposures Male rats, Sprague-Dawley

Sellakumar et al., 1985 ; Albert et al., 1982 Necropsy focused on

respiratory tract : also liver, spleen, kidney and testes and organs demonstrating gross pathology Lymphoma No increase Life-long study – high mortali ty at 24 months (>80%)

Male rats, F344 Batelle, Columbus Laboratories, 1981 Complete histopathology

in control and high dose group (15 ppm) Leukemia, all No increase Extended study – high mo rtality

Female Rats, F344 Batelle, Columbus Laboratories, 1981 Complete histopathology

in control and high dose group (15 ppm) Leukemia, all Increased in mortality adjusted incidence. P=0.0056 1 Extended study – high mortality Apparent elevation in 2 ppm and 6 ppm treatment groups as well (fig5-xx)– statistical comparison to controls is problematic Female mice - C57BL/6xC3HF1 Batelle, Columbus Laboratories, 1981 All organs in control and

high dose group (15 ppm) Lymphoma, all 26% in FA-exposed (15ppm) 16% in control P=0.0617 Extended study

All mice included in statistics conducted by Batelle Lab. 4 1. Original statistical analysis provided by Batte lle, Columbus Laboratories. Significance set at P<0 .0167. Analysis of adjusted 5 data where time to lesion and survivorship were con sidered, Cox (1972, Tyrone (1975). 6This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-425 Table 4-95. Incidence of lymphoblastic leukemia an d lymphosarcoma orally 1 dosed in Sprague-Dawley rats 2 3 Lymphoblastic leukemia and lymphosarcoma Tumor bearing animals (%)

Male Female control 3 1 vehicle 8 2

10 0 2

50 4 6

100 8 4 500 8 4

1000 12 10 1500 22 10 4

Source: Soffritti et al. (1989) 5

6 7 The two-year bioassay by Til et al. (1989) in male and female Wistar rats indicates no 8 increase in leukemia and lymphoma, with only 4 tumo r-bearing animals in all treatment groups 9 sacrificed at 24 months. The drinking water levels were similar at the highest dose of both 10 studies. The major difference in study design is l ength, which may have influenced results, as 11 leukemia is a late-life malignancy in rodents. Two -year survival in the Soffritti et al. (1989) 12 study varied between 50-60%. These animals would b e available to develop leukemia after the 13 two-year window of the Til et al (1989) study. Any potential role of strain differences is 14 unknown. Overall, the results of Soffritti et al. (1986) are strong, showing an exposure-response 15 relationship, in a lifelong study, appropriate for late-life malignancies. Unlike the GI tract 16 tumors, early-life exposure to formaldehyde in drin king water did not increase LHP 17 malignancies (Soffritti et al., 1989). 18 The largest and most comprehensive study of health effects from formaldehyde 19 inhalation exposures is the study reported by Kerns et al. (1983) and Swenberg et al. (1980) 20 conducted at the Columbus Laboratory of Battelle Co rporation (1981). Although the summary 21 reports of this study do not discuss leukemia or ly mphoma rates, mouse lymphoma and rat 22 leukemia were selected by the study pathologist and biostatistician for analysis (Battelle 23 Laboratory, 1981). Statistical analysis performed by Battelle Laboratories which accounted for 24 time-to-lesion and survivorship rates did indicate a statistically significant increase in female rat 25 leukemia (P = 0.0003) and a nearly significant incr ease in female mouse lymphoma ( P = 0.06). 26 No trend analysis could be performed, as only gross pathology was conducted on mid-dose mice 27This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-426 and rats (2 and 6 ppm, respectively). EPA has furt her analyzed these data to better understand 1 the significance of these findings. The percentage of lymphoma--bearing female mice increased 2 from 18% in control mice to 28% in mice exposed at 15 ppm for 24 months (6 hr/day, 5 3 days/wk) (P<0.05). Female rat leukemia was similar ly elevated to 26%, 22% and 24% by 4 inhalation exposure to 15 ppm, 6 ppm and 2 ppm, res pectively, versus 16% in controls when 5 early deaths prior to the first observed leukemia a re removed from the analysis (21 months). In 6 contrast, leukemia was not elevated in formaldehyde -exposed male F344 rats within the same 7 study. 8 Differences in study design may account in part for mixed results. The lifelong study by 9 Soffritti et al. (1989 may have allowed for detecti on of malignancies developing late in life, 10 whereas the other drinking water study by Til et al . (1989) sacrificed all animals at 24 months. 11 Even though the exposure levels were similar, the s tudies are not directly comparable. Likewise, 12 it is hard to directly compare results from the two major inhalation studies in rats. Although a 13 life-long study, the mortality for rats in the Sell akumar et al. (1985) study was greater than 80% 14 at 2 years. Additionally, the pathology examinatio n was much less rigorous than in the Battelle 15 Laboratory study, perhaps missing smaller lesions. Therefore, the increase in formaldehyde16

induced leukemia seen in female F344 rats late in l ife (Battelle laboratories, 1981) may be 17 reflecting a more sensitive study design. Finally, strain differences may account for different 18 susceptibilities as well. In summary, the availabl e evidence from chronic animal studies 19 supports the biological plausibility of the formald ehyde-induced LHP malignancies observed in 20 epidemiologic studies. The two positive rat studie s, by different routes of exposure, along with a 21 positive result for formaldehyde-induced mouse lymp homa make a substantive case for 22 formaldehyde-induced LHP malignancies. 23 The epidemiologic studies provide sufficient eviden ce to conclude that there is a causal 24 association between formaldehyde exposure and lymph ohematopoietic malignancies. When data 25 are evaluated for all leukemia together, again ther e is sufficient evidence to establish a causal 26 association, with consistent positive results in in dividual studies as well as 3 independent pooled 27 analyses. Mortality from myeloid leukemia, as well as mortality attributed to “other and 28 unspecified leukemia” is consistently elevated wher e reported. In addition, strong evidence for a 29 causal relationship between formaldehyde exposure a nd Hodgkin lymphoma is provided by the 30 consistent associations seen between formaldehyde e xposure and Hodgkin lymphoma in the NCI 31 industrial cohort, with elevations observed across decades of follow-up and significant exposure32 response relationships for all three exposure metri cs examined in the most recent follow-up 33 (Beane Freeman et al., 2009). 34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-427 4.5.3 Carcinogenic Mode(s) of Action 1 The US EPA 2005 Guidelines for Carcinogen risk Asse ssment recommend a Mode of 2 Action (MOA) analysis when data are available for e valuation. The purpose of this MOA 3 analysis is to determine if sufficient data exist t o adequately inform the exposure-response 4 relationship for cancer below the range of observed data in either human or animal studies. 5 Since the majority of the data supporting the carci nogenicity of formaldehyde comes from 6 animal bio-assays and epidemiological studies of wo rkers, EPA must extrapolate from the 7 observed risk of cancer mortality/incidence in thos e studies to levels considered protective of 8 human health for lifelong environmental exposures. In this context, the US EPA cancer 9 guidelines provide a framework to review MOA inform ation for relevant data to establish an 10 MOA informing appropriate low-dose extrapolation. 11 The supporting data for the MOA evaluation of form aldehyde are complex, and presented 12 across multiple sections of a large document; there fore, this section includes a brief summary of 13 the biological actions of formaldehyde and key mech anistic data which are believed to be 14 relevant to the MOA evaluation (Section 4.5.3.1). This information is not intended as a stand15 alone description of the evidence for a particular mechanism, but is intended to highlight the 16 major supporting arguments and direct the reader to text providing more detailed discussion. 17 The summary of data discussed below combines what is known about the human cancer 18 of concern (nasopharyngeal cancer, sinonasal cancer , leukemia and other lymphohematopoietic 19 cancers) with the potential formaldehyde-specific m echanisms of action to postulate 20 carcinogenic modes of action for each cancer or gro up of cancers (Section 4.5.3.2 and 4.5.3.2). 21 The resulting evaluation provides multiple possible MOAs for formaldehyde-induced cancers 22 where some key mechanistic events may be commonly a t work in different tissues, and some key 23 events may be more relevant to a specific tissue/ca ncer type. Each of these MOAs is evaluated 24 with respect to its relevance to human cancer, and the overall weight of evidence for its 25 relevance to formaldehyde-related human cancer. 26 Overall, multiple MOAs considered relevant to huma ns are presented for each cancer 27 type. Although some MOAs may have a greater level of supporting evidence, this reflects in part 28 how well a particular mechanism or key event may ha ve been studied. For example, there are a 29 large number of studies across many testing systems , and levels of biological organization to 30 support the mutagenicity of formaldehyde. In contr ast other likely MOAs, such as viral 31 reactivation, have little direct mechanistic eviden ce, but the available evidence is supportive. 32 The MOAs considered most relevant to upper respira tory tract cancers (e.g. NPC and 33 sinonasal cancer) are: 1) direct mutagenicity; 2) i nhibition of DNA repair mechanisms; 3) 34 formaldehyde-induced cell proliferation; 4) cytotox icity-induced cell proliferation; 5) tumor 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-428 promotion activity; and 6) localized immunosuppress ion/viral reactivation (Section 4.5.3.2). The 1 majority of these MOAs would apply equally to immun e cells present at the site of first contact 2 and may also contribute to those lymphohematopoieti c cancers which arise from peripheral 3 immune cells (e.g. Hodgkins lymphoma, multiple myel oma and some forms of leukemia). 4 Additional MOAs are considered, specifically for fo rmaldehyde-induced leukemia are: 1) 5 damage of a circulating hematopoietic stem cell or progenitor cell at the site of first contact; and 6 2) bone marrow toxicity (Section 4.5.3.3). 7 In summary – no single MOA is singled out as the b est explanation for cancer resulting 8 from formaldehyde exposure. Only one MOA - cytotox icity induced cell proliferation – suggests 9 an exposure threshold below which the MOA would not be active. However this MOA is the 10 least applicable to humans and other MOAs are consi dered operative at exposures below 11 exposures associated with cytotoxicity-induced cell proliferation. Therefore, multiple MOAs are 12 considered supported by formaldehyde-specific mecha nistic information which provide 13 biological plausibility for the cancers observed in formaldehyde exposed populations. 14

15

4.5.3.1 Mechanistic Data for Formaldehyde 16

4.5.3.1.1 DNA Reactivity/Genotoxicity/Mutagenicity 17 An agent’s genotoxic potential and ability to induc e mutations is a key consideration in 18 assessing a carcinogenic MOA, as cancer results fro m a series of genetic and epigenetic 19 alterations affecting genes that control cell growt h, division and differentiation (Hanahan and 20 Weinberg, 2000; Vogelstein et al., 1988; Kinzler an d Vogelstein, 2002). The US EPA Cancer 21 guidelines suggest several lines of evidence which are key to evaluating a mutagenic MOA: 1) Is 22 the chemical under study DNA-reactive and/or has th e ability to bind to DNA; 2) Does the 23 chemical generate positive results in in vitro muta genic test systems (specifically gene mutations 24 and chromosomal aberrations); and 3) Does the chemi cal induce manifestations of genetic 25 damage in in vivo tests (specifically gene mutation s and chromosomal aberrations) and 4) Does 26 the chemical have properties and structure-activity relationships (SAR) similar to known 27 mutagens (US EPA, 2005). As reviewed in Section 4. 3 above, there is adequate evidence for 28 formaldehyde-induced genotoxicity and mutagenicity for consideration of these key events in 29 formaldehyde’s carcinogenic MOA. 30 Formaldehyde induces a variety of genotoxic and mut agenic events when tested both in 31 vitro and in vivo systems including DNA-protein cro sslinks (DPC or DPX), point mutations, 32 DNA single strand breaks (SSB) and chromosomal aber rations (CAs) (See Section 4.3). 33 Formaldehyde, as a reactive chemical, also forms DN A adducts and DNA-DNA crosslinks 34 (DDC) and may act to form adducts between other che micals and DNA (Brutlag et al., 1969; 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-429 Donecke, 1978; Ohba et al., 1979; Fennel, 1999; Cas anova-Schmitz and Heck, 1983; Casanova1 Schmitz and Heck, 1984; Heck and Casanova, 1987 and Casanova et al., 1989). The high 2 reactivity of formaldehyde results in little specif icity indicating that a range of adducts and 3 crosslinks might be expected. 4 Numerous studies have shown that formaldehyde induc es genotoxic and mutagenic 5 effects under a variety of experimental conditions (see section 4.3 for a detailed discussion, also 6 reviewed by IARC 2006; Ma and Harris 1988 and Auerb ach et al, 1977). As discussed, 7 formaldehyde is known to directly react with DNA fo rming DPC and DNA adducts. Mutations 8 may occur during repair of formaldehyde-induced DNA damage, or as a result of replication 9 errors during mitogenesis. Additionally, there is some evidence that DNA single strand breaks 10 (SSB) may be induced directly by formaldehyde react ivity (Grafstrom et al, 1984). Clastogenic 11 effects including increased micronuclei (MN), chrom osomal aberrations (CAs) and sister 12 chromatid exchanges (SCEs) are also reported in a r ange of in vitro study systems. 13 Formaldehyde caused a concentration-dependent icrea se in calstogenicity (e.g. MN) in 14

human cell lines deficient in either DNA nucleotide excision repair (NER) or DDC repair 15 systems even though there is no change seen in DPC induction or removal between these cell 16 lines (Speit et al., 2000). These data suggest tha t alteration of DNA repair, not DPC removal, 17 contributes to formaldehyde-induced clastogenicity. Since DPC repair involves proteolytic 18 removal of proteins from the DNA, the authors hypot hesize that single peptides or small peptide 19

chains cross-linked to the DNA as in the case of DP C are critical to formaldehyde-induced 20

mutations. 21 Formaldehyde-induced MN and CAs are associated to c oncentration-dependent 22 mutagenic effects in L5178Y mouse lymphoma cells (S peit and Merk, 2002). Detailed analysis 23 of both spontaneous and formaldehyde-induced lesion s indicate that recombination or deletion of 24 DNA from the thymidine kinase ( tk ) locus was primarily responsible for the loss of h eterogeneity 25 leading to the observed mutant phenotype. Therefor e, it is believed that formaldehyde is 26 mutagenic by a clastogenic mechanism, rather than t hrough point mutations in the L5178Y 27 mouse lymphoma cell system. This finding is consis tent with Craft et al. (1987) who 28 demonstrated formaldehyde-induced mutagenicity in t he tk locus of TK6 human lymphoblastoid 29 cells, while Graftsrom et al, (1984) demonstrated i ncreased SSBs in formaldehyde-exposed 30 human cell lines. The elegant series of experiments by Speit and Merk provide the possible links 31 between DPC, clastogenicity and locus-specific muta tions firmly demonstrating formaldehyde32 induced mutations in the in vitro mouse lymphoma te sting system. 33 Formaldehyde is genotoxic at the portal of entry (P OE) in animal studies, resulting in 34

increased DPC formation in the nasal mucosa as disc ussed above. However, there are no animal 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-430 studies which directly examine the mutagenicity in nasal or respiratory epithelial cells in the 1 early stages of exposure. It is likely that the mu tations are seen in advanced stage of the tissue 2 around the transformation stage with formaldehyde e xposure. With weak positive results in 3 pulmonary lavage cells (Dallas et al., 1992) and cl astogenicity demonstrated in gastro-intestinal 4 epithelial cells of rats (Migliore et al., 1989) , below exposure levels which trigger regenerative 5 cell proliferation, the existing evidence, although thin, supports clastogenic effects of 6 formaldehyde. 7 Clastogenic effects are consistently reported in hu mans exposed to formaldehyde in the 8 industrial workplace or during anatomy or mortuary classes (See section 4.3 for a full 9 discussion). Increased micronuclei have been repor ted in nasal epithelial cells from industry 10 workers (Ballarin et al., 1992; Ye et al., 2005), b uccal epithelial cells from anatomy and 11 mortuary science students and/or staff (Kitaeva et al., 1996; Titenko-Holland et al., 1996; Burgaz 12 et al., 2001; 2002 compared to corresponding contro ls). Comparisons of micronuclei in nasal 13 and buccal cells of anatomy students before and aft er classes where they are exposed to 14 formaldehyde indicate an increase in clastogenicity (Ying et al., 1997). An examination of 15 exfoliated buccal and nasal cells in mortuary stude nts indicates greater increases in centromere16 negative micronuclei, suggesting the effects are du e to chromosome breakage or clastogenicity 17 rather than aneuploidy (Titenko-Holland et al., 199 6). Micronuclei were also increased in a 18 dose-dependent manner in buccal cells as well as pe ripheral blood lymphocytes (PBLs) in 19 mortuary students during the course of an embalming class; however, SCEs were reduced in 20 post-exposure samples (Suruda et al., 1993). Bucca l, oral and nasal cells present at the portal of 21 entry may be directly exposed to formaldehyde and t hus reports of clastogenic effects are 22 consistent with direct interaction of formaldehyde at the POE. There is some supporting 23 evidence for the mutagenicity of formaldehyde in hu man populations. Shaham et al. (2003) 24 reported a increase in mutant p 53 protein in the PBLs of individuals with mean for maldehyde 25 exposure duration of 16 years. Additionally there was is a significant association between 26

mutant p 53 protein and DPC in this study suggesting a relat ionship between the formaldehyde’s 27 genotoxic effects. More recently, Zhang et al., (20 10) have reported aneuploidy in circulating 28 hematopietic stem cells in formaldehyde exposed wor kers with increases in both monosomy7 29 and trisomy 8. 30 In summary, there are several lines of evidence sup porting mutagenic effects of 31 formaldehyde exposure: 32 1) Formaldehyde directly interacts with DNA generat ing DPC, 33 2) DPC in tissues at the POE exhibit a dose-respons e relationship to formaldehyde 34

exposure, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-431 3) Formaldehyde-induced DPC are associated with for maldehyde-induced MN and CAs, 1 4) Mutations induced by formaldehyde due to small d eletions and rearrangements in 2 DNA in various experimental systems are consistent with formaldehyde’s observed 3 clastogenic effects (MN and CAs), 4 5) Formaldehyde-induced mutations and clastogenic e ffects occur at levels below where 5 significant cytotoxicity is detected, and 6 6) Formaldehyde exposure has been correlated to sim ilar increased MN and CAs in 7 human buccal and oral cells corresponding to sites where formaldehyde-induced tumors 8

arise. 9

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4.5.3.1.2 Inhibition of DNA repair 11 Studies indicate that formaldehyde exposure may inh ibit DNA repair mechanisms 12 directly (See Section 4.3.1.5). Graftsrom (1985) f irst documented formaldehyde effects on DNA 13 repair mechanisms, reporting that formaldehyde trea tment of human bronchial fibroblasts in vitro 14 inhibited repair of O6-methyl-guuanine adducts indu ced by N-methyl-Nitrosurea (NMU). 15 Inhibition of DNA repair in human keratinocytes and fibroblasts cultured at 10 μM 16 formaldehyde affected repair of DNA single strand b reaks from ultraviolet light but was specific 17 to UVB and UVC, not impacting repair of single stra nd breaks from UVA (Emri et al., 2004). 18 To determine if formaldehyde may have similar effec ts in exposed humans, Hayes et al., 19 (1997) assessed the activity O6-alkylguanine-DNA al kyltranferase (AGT) an enzyme critical in 20 repairing DNA damage induced by alkylating agents i n formaldehyde-exposed mortuary students 21 previously shown to have increased micronuclei in b oth buccal cells and peripheral lymphocytes 22 (Suruda et al., 1993). AGT activity was lower in m ortuary students with prior embalming 23 exposures versus students with no prior exposure (p =0.08). Seventeen of 23 students had lower 24 AGT activity after the 9 week course (p<0.05) with a larger proportion of naïve students 25 demonstrating decreased activity (7 of 8) versus pr eviously exposed students (10 of 15). 26 Although detailed exposure measurements were taken for each student, the changes in AGT 27 activity were not correlated to cumulative exposure (ppm-hrs). 28

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4.5.3.1.3 Protein to protein cross-links 30 Formaldehyde is a reactive molecule that is likely to interact with both low molecular 31 weight cellular components (e.g., reduced glutathio ne [GSH]) as well as high molecular weight 32 cellular components. Unlike nuclear DNA, which has additional membrane barriers to exposure 33 (i.e., nucleus), extracellular and intracellular pr oteins, are obvious primary targets for interacting 34 with formaldehyde. Formaldehyde is a well-known cr oss-linking agent that is used in the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-432 fixation of tissues, inactivation of toxins and vir uses (e.g. preparation of vaccines), and study of 1 protein-protein interactions (Metz et al., 2006). Using several identical synthetic polypeptides 2 differing on one amino acid, Metz et al (2004) have shown that formaldehyde initially reacts 3 with the primary amino and thiol groups of amino ac ids forming unstable methylol adducts, 4 which later are partially dehydrated forming labile Schiff bases that are capable of forming 5 crosslinks with other amino acid residues, such as arginine, asparagine, glutamine, histidine, 6 tryptophan, and tyrosine through methylene bridges, but not between two primary amino groups. 7 The same group (Metz et al 2006) has also shown tha t formaldehyde forms seven intramolecular 8 crosslinks in proteins with defined structure, such as insulin, involving arginine, tyrosine and 9 lysine and the N-terminus of insulin was converted to a imidazolidinone adducts similar to that 10 observed with the synthetic peptide (Metz et al 200 4). (Figure 3-1 provides a general reaction 11 scheme for formaldehyde-mediated modifications of a mino acids.) 12

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4.5.3.1.4 Break-down of the mucociliary apparatus 14 The mucociliary apparatus of the upper respiratory tract is the first line of defense against 15 airborne toxicants. Comprised of a thick mucus laye r (epiphase), hydrophase and ciliated 16 epithelium, the mucociliary apparatus may entrain, neutralize and remove particulates and 17 airborne chemicals from inspired air (Figure 4-4). Formaldehyde reacts with the components of 18 the mucous layer (proteins, glycoprotein, and lipid s), crosslinking proteins. Formaldehyde 19 exposure induces slowing of the mucous flow, stiffi ng and breaking up of the mucous layer and 20 eventual mucostasis where gaps have been observed e xposing the underlying hydrophase and 21 epithelium. Although ciliary beat first increases in response to formaldehyde exposure, perhaps 22 to compensate for reduced flow of the epiphase, cil iastasis ensues with both higher levels of 23 exposure, and increased duration of exposure. Alte red ciliary has been noted in as little as 15 24 minutes of exposure (1.25 ppm) with functional defi cits in the mucociliary apparatus at 30 25 minutes. Altered ciliary beat has been reported at the lowest concentration tested (0.5ppm) for a 26 single 6 hour exposure. Severity of effects increa se with both duration and level of exposure 27 (see section 4.2.1.2.1). 28

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4.5.3.1.5 Induced cell proliferation 30 There are several reports apparently demonstrating formaldehyde-induced proliferation in 31 cells below cytotoxic levels of exposure. This phe nomenon has been reported from studies 32 involving both in vitro and in vivo exposures. Tyihak et al. (2001) demonstrated sign ificantly 33 increased cell proliferation in both HT-29 human co lon carcinoma and human umbilical vein 34 endothelial cell (HUVEC) lines treated with 0.1mM (the lowest dose) formaldehyde compared 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-433 to untreated controls (P<0.0001). This effect was quantified as both an increase in cell number 1 over time (Figure 4-35), and an increase in the per centage of cells undergoing mitosis at each 2 time point. The authors also report a significant (P<0.01) inhibition of apoptosis in 3 formaldehyde-treated cells as compared to untreated cells (data not shown here). In a novel 4 system using xenotransplanted human tracheobronchia l epithelial cells, formaldehyde was shown 5 to induce increased cell proliferation at doses bel ow those required for a “massive toxic effect” 6 (Ura et al., 1989). 7

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21 Figure 4-35. Effect of various doses of formaldehy de on cell number in (A) 22 HT-29 human colon carcinoma cells and in (B) human umbilical vein 23 epithelial cells (HUVEC). 24 25 Values are average of three samples + SD; * P < 0.01 and ** P < 0.0001 26 compared to corresponding controls. 27 28 Source: Tyihak et al 2001. 29 30 31 Some animal studies have demonstrated increased ce ll proliferation after formaldehyde 32 exposures by both inhalation and ingestion (See sec tion 4.2.1). However, whether sustained 33 increases in cell proliferation over baseline rates are observed upon exposure to sub-cytotoxic 34 doses of formaldehyde remains unclear. Several of the inhalation studies demonstrate increased 35 cell proliferation in the nasal epithelium at forma ldehyde exposures levels that were sub36 cytotoxic—i.e. in the absence of significant cell d eath. Acute formaldehyde exposures (1 to 3 37 days) induced increased cell proliferation at discr ete locations in the nasal mucosa, where cell 38 0

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*This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-434 proliferation was measured as a labeling index (per centage of cells pulse-labeled with tritiated1 thymidine. Reuzel et al. (1990) reported increased cell proliferation in the nasal passages 2 including the nasoturbinates, maxilloturbinates sep tum and lateral wall in male Wistar rats 3 exposed at 3 ppm, but not at 0.3 or 1 ppm, formalde hyde for 22 hours/day for 3 days. Zwart et 4 al. (1988) reported increased cell proliferation af ter exposure to 1 or 3 ppm formaldehyde, 6 5 hours/day for 3 days or 13 weeks in male and female albino Wistar rats. These increases were 6

transient at level 3 but sustained at level 2 of th e nose and were not correlated with cytotoxicity. 7 In contrast, Wilmer et al. (1989), from the same gr oup of investigators and using a similar 8 exposure regimen, reported no increase in cell prol iferation after repeated 8–hour exposures at 1 9 or 2 ppm formaldehyde for 3 days or 4 weeks. Swenb erg et al. (1986) demonstrated a transient 10 increase in cell proliferation after a single 8–hou r exposure to 0.5 or 2 ppm formaldehyde in 11 male F344 rats but no increases after 3 days or rep eated 8–hour exposures. The authors suggest 12 that adaptive responses of the nasal mucosa contrib ute to the transient nature of formaldehyde13 induced cell proliferation. After a series of acut e studies at various formaldehyde concentrations, 14 Swenberg and coworkers concluded that, in addition to cell proliferation being concentration-, 15 dose- and time-dependent, the response varies by sp ecies and by location of exposure in the nose 16 (Swenberg et al., 1983, Swenberg et al, 1986). 17 Other methods of quantifying cell proliferation in the nasal mucosa have demonstrated 18 formaldehyde-induced cell proliferation at similar low exposure concentrations. Roemer et al. 19 (1992) measured cell proliferation by flow-cytometr y in epithelial cells harvested from the nose 20 and trachea of male Sprauge-Dawley rats exposed to 2 ppm formaldehyde for 6 hours/day for 1 21 or 3 days and found increased cell proliferation af ter the 1-day exposure. These increases were 22 transient and were not evident after 3 days of expo sure. Cassee and Feron (1994) identified 23 proliferating cells by staining for the presence of proliferating cell nuclear antigen (PCNA). 24 Formaldehyde exposure at 3.6 ppm for 6 consecutive periods of 12 hours (8-hour exposures 25 followed by 4-hour-periods of non-exposure) over th ree days, qualitatively increased the 26 expression of PCNA in respiratory epithelium at lev els 2 and 3 of the nose in albino male Wistar 27 rats (Cassee and Feron, 1994). Hyperplasia, squamo us metaplasia and frank necrosis were also 28 reported for these tissues. 29 Monticello et al. (1990, 1991, 1996) conducted in vivo cell proliferation studies in which 30 they exposed F344 rats for short durations (1, 4, 9 and 42 days) as well as much longer durations 31 (13, 26, 52 and 78 weeks) to exposure concentration s of 0, 0.7, 2.0, 6.0, 10.0 and 15.0 ppm. 32 These data are unique in that they also included lo w exposure concentrations. The authors 33 reported statistically significant increases in cel l proliferation only at 6.0 ppm and higher 34 exposure concentrations in the short duration study and only at 10.0 ppm and higher 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-435 concentrations in the longer duration study. These data have undergone considerable statistical 1 analysis in several papers as well as in this docum ent. Conolly et al. (2002, 2003) and Gaylor and 2 Conolly (2004) interpreted these data, when combine d, as indicating a non-monotonic behavior 3 at low dose. In other words, formaldehyde was judge d to result in a reduction in cell proliferation 4 at low dose in comparison to baseline rates, with i ncreased proliferation effect kicking in only at 5 exposures that were cytotoxic. However, as shown in Appendix 5-3 and in Subramaniam et al. 6 (2008), Crump et al. (2008), analysis of the indivi dual animal data shows considerable 7 uncertainty and variability, both quantitative and qualitative, in the interpretation of these cell 8 proliferation data. (For example, even the control data vary over an order of magnitude in some 9 cases. See Figures 5-22 and 5-23 in Appendix 5-3.) These analyses (which were based on the 10 replicate animal data used in the above studies) co nsidered regional formaldehyde dose to the 11 tissue (flux), nasal site and duration of exposure, as well as the number of cells at a given site. 12 The overall conclusion in Section 5.3.3 (and detail ed in Appendix 5-3) is that the cell 13 proliferation dose-response at low dose could be re asonably described by both monotonic (with 14 and without a threshold) and non-monotonic curves. 15 Only one study, by Monticello et al. (1989), quanti fied cell proliferation in primates after 16 formaldehyde exposure; this study, reported an 18- fold increase in cell proliferation in the nasal 17 epithelium (respiratory and transitional), larynx, trachea and carina of male Rhesus monkeys 18 exposed to 6 ppm formaldehyde compared to controls (See section 4.2.1 for detailed study 19 description). The authors also noted that increase d cell proliferation was seen in locations with 20 minimal histological changes, indicating proliferat ion may be a more sensitive predictor of 21 adverse health effects of formaldehyde exposure. 22

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4.5.3.1.6 Cytolethality and resulting regenerative cell proli feration 24 The toxic and cytolethal effects of formaldehyde ex posure at the POE are well 25 documented after both inhalation and oral exposures (See Section 4.2.1). The nature and 26 progression of tissue injury has been best document ed in rodent inhalation assays. Early effects 27 on the nasal mucosa include altered ciliary beat an d mucus flow, hyperplasia and metaplasia of 28 nasal epithelium (Morgan et al., 1986a; Morgan et a l., 1986b; Monteiro-Riviere and Popp, 1986; 29 Maronpot et al 1986; Rusch et al., 1983 and Montice llo et al., 1986). These first changes may be 30 considered adaptive responses. Squamous epithelium may thicken and transitional epithelium 31 may change to squamous epithelium as evidenced by s quamous hyperplasia, squamous 32 metaplasia and thickening of the epithelium in thes e anterior portions of the nose. Tissue 33 damage may be transient at lower formaldehyde expos ures as these changes serve to protect 34 tissue from formaldehyde’s reactivity. However, hi gher formaldehyde concentrations can 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-436 overwhelm these adaptive responses and result in gr oss tissue damage. Frank necrosis and focal 1 erosions have been reported in time- and concentrat ion-dependent manner in rodent bioassays. 2 Both adaptive changes and cytolethality are associa ted with cell proliferation. However, 3 where adaptive changes are successful, e.g. prevent continued toxic insult to the tissue, cell 4 proliferation is transient. Exposure regimens wher e the adaptive changes are not adequate to 5 protect the tissue, would result in continued cytox icity and cell death. Sustained damage to the 6 epithelium would result in sustained cell prolifera tion to compensate for cell death. A series of 7 rodent bioassays present convincing evidence that c hronic inhalation exposures 6 hours a day, 5 8 days a week at 6, 10 and 15 ppm formaldehyde do res ult in sustained damage to the nasal 9 epithelium, sustained cell proliferation and tumor development (Kerns et al., 1983, Morgan et al., 10 1986; Monticello et al., 1990; Monticello et al., 1 991 and Monticello et al., 1996). Work by 11

Monticello and coworkers demonstrate that chronic r epeated inhalation exposures at 6, 10 or 15 12 ppm formaldehyde result in sustained cell prolifera tion at the lateral meatus, mid-septum and 13 maxilloturbinates of rat nasal passages (Monticello et al., 1991 and Monticello et al., 1996). 14

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4.5.3.1.7 Evidence for promotion 16 There is some evidence, although mixed, that formal dehyde may promote tumor 17 development by other carcinogens, and known initiat ing agents by various routes of exposure. 18 Formaldehyde exposure in drinking water (0.5% forma lin) increased glandular stomach 19

adenocarcinomas in male Wistar rats after initiatio n with 100 mg/L, N-methyl-N’- 20 nitrosoguanidine (MNNG), compared to MNNG-only-trea ted rats (Takahashi et al., 1986). In 21 white non-inbred rats, inhalation exposures (3, 30 or 300 ug/m 3 formaldehyde 7hr/day, 5 22 days/week for 1 year) increased tumor multiplicity per animal and decreased latency of 23 benzo[a]pyrene induced tumors in white non-inbred r ats (Yanysheva et al., 1998.) Similarly, 24 formaldehyde skin application decreased tumor laten cy, in 7,12-dimethylbenz(a)anthacene 25 (DMBA) initiated hairless Oslo mice (Iversen, 1986) . Although formaldehyde exposure also 26 increased the tumor multiplicity in Syrian golden h amsters where diethylnitrosamine (DEN) 27 (0.25mg I.P.) was the tumor initiator, positive res ults were only reported for the exposure 28 regimen where hamsters were exposed to formaldehyde via inhalation 48 hours prior to DEN 29 injection, and then one a week thereafter for life. However, formaldehyde did not increase the 30 number of tumors per tumor bearing animals when onl y administered beginning one week after 31 all DEN injections. In contrast, bladder cancer wa s not enhanced by intravesical instillation of 32 0.5ml of 0.3% formalin, one week after instillation of N-methyl-N-nitrosourea (MNU) in male 33 Fisher rats (Homma et al., 1986). 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-437 The observed promotion activity of formaldehyde has been tested in several systems, by 1 different routes of exposure. By several routes of exposure, formaldehyde enhanced tumor 2 development at a site where formaldehyde did not in duce tumors alone, without the initiating 3 agent (Takahashi et al., 1986, Yanysheva et al., 19 98 and Iversen, 1986). Promotion activity in 4 these studies was evidenced by increased in tumor b earing animals (oral route), increase in 5 tumors per animal (inhalation routes) and decreased tumor latency compared to those animals 6 only exposed to the initiating agent (inhalation ro ute) (Takahashi et al., 1986, Yanysheva et al., 7 1998 and Iversen, 1986). Although these experiment s do not indicate how formaldehyde acts as 8 a promoter in these systems, it is possible formald ehyde-induced mutation, increased cell 9 proliferation or other toxic action could enhance t umor development from another agent. 10

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4.5.3.1.8 Localized Immunosuppression 12 Formaldehyde exposure has induced localized immune suppression in experimental 13 animals (Dean et al., 1984) and in exposed workers (Lyapina et al., 2004). Repeated inhalation 14 exposures in rodents depopulated the URT and pulmon ary tissues of resident macrophages, 15 resulting in a transient decrease in POE host defen ses (Admas et al., 1987). After cessation of 16 exposure, the mononuclear phagocyte (MP) population s were replenished and there was a 17 subsequent increase in host defense representing bo th increased MP numbers and increased 18 bacteriocidal activity of the MPs. These data sugg est that peak exposures of formaldehyde may 19 present localized immunosuppression for components of the mononuclear phagocyte system 20 (MPS) in tissues at the site of first contact. 21 A number of studies have evaluated the ability of formaldehyde to induce systemic 22 immunotoxic effects in humans (Ohtani et al., 2004a , b; Erdei et al., 2003; Thrasher et al., 1990, 23 1987; Pross et al., 1987). Some studies have repor ted altered innate immune responses 24 associated with formaldehyde exposure (Erdei et al. , 2003), while others have noted adaptive 25 immune response suppression associated with formald ehyde exposure (Thrasher et al., 1990, 26 1987) and changes associated with alterations to a predominant T—lymphocyte helper 2 (Th2) 27 pattern (Ohtani et al., 2004a, b). In contrast, Pr oss et al. (1987) did not observe formaldehyde28 associated changes in systemic immune function. 29 Numerous studies have reported increased respirator y tract infections in formaldehyde 30 exposed individuals both in occupational and reside ntial environments (Lyapina et al., 2004; 31 Krzyzanowski et al., 1990; Holness and Nethercott, 1989). Incidences of physician-diagnosed 32 chronic bronchitis were more prevalent in children (under age 15) living in homes with higher 33 formaldehyde (>60 ppb) readings in the kitchen ( p < 0.001) but this effect was more pronounced 34 ( p < 0.001) in children simultaneously exposed to env ironmental tobacco smoke (Kryzanowski 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-438 et al., 1990). The prevalence of chronic cough was also increased in adults living in homes with 1 measurable levels of formaldehyde, but data were no t shown. Holness and Nethercott (1989) 2 assessed chronic bronchitis in 87 funeral workers, where the average formaldehyde exposure was 3 reported at 0.38 ± 0.19 ppm. Chronic bronchitis wa s observed in 20 funeral workers (n = 87) 4 exposed to formaldehyde compared with 3 cases of ch ronic bronchitis in nonexposed referent 5 controls (n = 38). A statistically significant ass ociation of self-reported chronic bronchitis and 6 decreased resistance to URT infection was reported in formaldehyde exposed workers compared 7 with controls (

p = 0.02) (Lyapina et al., 2004). Of the workers, 4 1% had a history of chronic 8 respiratory infection and frequent long-lasting inf ectious inflammatory relapses (group 1a). 9 Another group (group 1b) consisted of 17 exposed wo rkers, 12 of whom had no history of 10

recurrent viral infections of the URT. There was a statistically significant association of 11 frequency and duration of inflammatory relapses bet ween groups 1a and 1b. 12 Lyapina et al. (2004) also reported effects of form aldehyde exposure on neutrophil 13 respiratory burst activity (NRBA), the capacity of polymorphonuclear leukocytes to produce 14 reactive oxygen radicals in response to chemical or microbial stimuli using flow cytometry. A 15 suite of hematological tests and flow cytometric an alysis for respiratory burst activity were 16 performed. Although no significant difference was observed in the spontaneous and stimulated 17 NRBA (median percentage of oxidizing cells) between the 29 exposed workers with URT 18 inflammation and the healthy controls (0.83 versus 1.35, respectively), a separate comparison of 19 the NRBA of 12 workers with chronic, repeating URT infections and 17 workers with short, 20 infrequent episodes of URT inflammations was signif icant (0.45 versus 1.00, p = 0.037). When 21 the NRBA of the group with chronic URT infections ( n = 12) was separately compared with that 22 of the healthy controls (n = 21), the results were also significant (0.45 versus 1.35, p = 0.012). 23

Individuals with chronic URT infections have reduce d NRBA that could be due to formaldehyde 24 exposure. Neutrophils respond to tissue damage or local invasion of microorganisms and act to 25 phagocytize foreign cells. If neutrophilic activit y is hampered or altered by formaldehyde 26 exposure, then the ability to fight infection will be diminished, leading to prolonged infection. 27 However, no dose-response pattern of formaldehyde e xposure could be determined from this 28 study. 29

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4.5.3.1.9 Potential for systemic transport of formaldehyde 31 In aqueous solution formaldehyde exists in equilib rium with it’s hydrated form 32 methanediol (CH 2 OH 2 ) (K d = 5.5x10- 4 ). The equilibrium favors methanediol at physiolog ical 33 temperature and pH (>99.9%) and is readily reversib le. In biological systems, as free 34 formaldehyde is removed from aqueous solution throu gh binding with serum proteins and 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-439 cellular components, the equilibrium is reestablish ed by dehydration of methanediol to free 1 formaldehyde. The reversible nature of this hydrat ion reaction describes how a pool of free 2 formaldehyde may be sustained in biological systems

. 3 There is strong and consistent evidence in biologi cal testing systems in vitro that treating 4 cells with formaldehyde in an aqueous media results in significant cytoxicity, cell proliferation, 5 clastogenic effects and clear evidence of mutationa l events (Section 4.3). Similarly, animal 6 bioassays where formaldehyde is administered in dri nking water report portal of entry toxicity 7 including hyperplasia, increased cell proliferation , focal lesions and tumors (Section 4.2.1). It 8 should be noted that URT tissues are covered by an aqueous mucous layer, through which 9 formaldehyde must pass to react the cellular compon ents of the URT. It has been postulated that 10 formaldehyde transports through this mucous layer a nd the underlying tissues as methanediol 11 (Georgieva et al., 2003). 12 The dynamic equilibrium between the hydrated and u nhydrated forms of formaldehyde in 13 biological systems is well understood. Since the h ydration reaction favors methanediol, it is 14 expected that exogenous formaldehyde which reaches the blood will primarily exist as 15 methanediol and is subject to physiological elimina tion. As free, unhydrated formaldehyde 16 continues to react with serum proteins and cellular components, the blood levels of methanediol 17 are expected to reduce as it is dehydrated to maint ain equilibrium. Although some attempts to 18 measure significant changes in free formaldehyde le vels in blood after inhalation exposure have 19 not been successful, the half-life in blood has bee n measured after i.v. injection at approximately 20 2 minutes (McMartin et al., 1979). Additionally, the detection of antibodies to formaldehyde21 hemoglobin adducts and formaldehyde-albumin adducts in exposures workers, smokers and 22 laboratory animals exposed via inhalation provides direct evidence that formaldehyde is able to 23 react with serum albumin and hemoglobin in biologic al systems (Thrasher et al., 1990, Grammer 24 et al., 1990; Grammer et al., 1993; Dykewicz et al. , 1991; Varro et al., 1997 and Li et al., 2007). 25 These data support the hypothesis that exogenous fo rmaldehyde may reach and transport through 26 the blood. If so, formaldehyde (or methanediol) ma y reach sites distal to the portal of entry. 27

28

4.5.3.2 Mode of Action Evaluation for Upper Respiratory Tra ct Cancer (Nasopharyngeal 29 Cancer, Sino-nasal) 30 From the above discussion, it can be seen that nume rous mechanisms of action for 31 formaldehyde-induced cancer can be reasonably suppo rted based on various known biological 32 actions of formaldehyde (e.g., mutation, cell proli feration, cytotoxicity, and regenerative cell 33 proliferation). Additionally, alternative actions, such as immunosuppression or viral 34 reactivation, are possible, although less data exis t to evaluate these MOAs. Rather than a single 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-440 MOA, it is plausible that a combination of these fa ctors contribute to cancer incidence in an 1 exposed population. Considering multiple factors m ay help to better understand the biological 2

and mechanistic basis for the increases in cancer i ncidence observed in exposed human 3 populations. Unlike animal bioassays, human epidem iological studies may reflect not only the 4 effects of the agent of concern but also numerous o ther risk factors (e.g., viral status, diet, 5 smoking, etc.). Additionally, human studies may be impacted by biological human variability 6 across individuals, cancer biology (sub-types), wid e variability in exposure regimens in human 7 populations, etc. Therefore, if the purposes of e xploring the carcinogenic MOA of an agent are 8 to better understand the relevance of a given carci nogen to human populations and to inform the 9 exposure-response analysis, then discussions of MOA s which recognize the interaction of an 10 agent with human variability and various risk facto rs is an appropriate analysis. 11

12 a) Direct mutagenicity of formaldehyde in cells at the site of first contact: Mutations, 13 the permanent heritable changes in the genome of th e cell, are a primary mechanism for 14 the activation of oncogenes or the inactivation of tumor suppressor genes. Mutagenicity 15 is the most widely recognized determinant of chemic al-induced carcinogenicity, and it is 16

difficult to set aside the relevance of direct form aldehyde-induced mutations from its 17 demonstrated carcinogenicity. Formaldehyde-induced mutation in mucosal cells of the 18 URT, throat and buccal cavity may serve to initiate cells, or provide subsequent mutageic 19 events to already initiated cells. Since the mucos al cells have proliferative capacity, and 20 cell proliferation is a normal tissue function, mut ations may be fixed and passed to 21 daughter cells due to baseline cell proliferation o f the tissue. 22

Relevance to humans: This MOA is relevant to humans. The well-documente d DNA 23 reactivity (e.g. DPC and DNA adducts) and clastogen icity of formaldehyde in the URT of 24 laboratory animals is a direct effect of formaldehy de on tissues of first contact. As this is 25 a direct acting agent - no distribution or metaboli sm is required for the genotoxic action – 26 there is little expected species variability. As d iscussed in chapter 3, there are species 27 differences in flux of formaldehyde into the respir atory mucosal tissues, but this 28 introduces species differences in dosimetry – not m echanism. Finally, the clastogenic 29 effects in nasal and buccal epithelial cells in for maldehyde- exposed workers confirms 30 the direct genotoxic effects of formaldehyde at the first site of contact in humans. 31

32 b) Decrease in DNA repair function within cells at the site of first contact: A decrease 33 in DNA repair capacity in these tissues by formalde hyde may increase total mutations 34 over time due to either endogenous or exogenous sou rces of mutation. Although there 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-441 are only a few studies which have explored the pote ntial for formaldehyde to reduce 1 DNA repair capacity, the evidence is positive, both in vitro testing systems, and in one 2 study of occupationally exposed humans (Grafstrom 1 985; Hayes et al., 1997). 3

Relevance to humans: This MOA is considered relevant to humans. The g eneral 4 population is exposed to various carcinogens, many with mutagenic potential, at sites of 5 first contact including; air pollution, tobacco pro ducts, nitrosamines and viruses. 6 Additionally, there are endogenous sources of DNA d amage and mutagenicity in humans 7 (e.g. lipid peroxidation, oxidative stress). The d emonstration of reduced DNA repair 8 activity (O6-alkylguanine-DNA alkyltransferase acti vity) in formaldehyde-exposed 9 mortuary students suggests this toxic action of for maldehyde is possible in humans. 10

11 c) Formaldehyde-induced cell proliferation: Formaldehyde-induced cell proliferation in 12

the oral and respiratory mucosa may be considered a key event in conjunction with the 13 genotoxic effects, and induced mutational events ob served with formaldehyde exposure. 14

This MOA is intended to describe events which occur below exposure levels which 15

induce cell death and mucosal lesions. Therefore t his MOA is comprised of two key 16

events: 17

a. Formaldehyde-induced genotoxicity or mutation 18

b. Formaldehyde-induced cell proliferation 19

20 DNA replication during cell proliferation may serve to translate DNA damage or a 21 formaldehyde-related DNA lesion into a permanent ch ange in the sequence of nucleic 22 acids during replication of the DNA– e.g. ‘fix’ a m utation from DNA damage. 23 Additionally formaldehyde-induced cell proliferatio n may provide an opportunity for 24 initiated cells to proliferate, increasing the pote ntial for additional mutation events and 25 transformation. The increased cell proliferation o bserved in the mucosal tissues in direct 26 contact with formaldehyde during inhalation exposur es may serve to amplify the risk of 27

cell transformation from mutation alone. Researche rs have noted that increased cell 28 proliferation may be transient in some locations as adaptive responses compensate 29 (Swenberg 1983). However, evidence in both monkeys and rodents indicate that 30 increased cell proliferation in repeated exposures across time do result in sustained cell 31 proliferation. Data in Rhesus monkeys indicates in creased cell proliferation is observed 32 beyond the nasal cavities to the larynx, trachea an d carnia (first tracheal 33 branching)(Monticello et al., 1989). Additionally, the authors note that cell proliferation 34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-442 is a more sensitive indicator of effects on the epi thelium, observed even when minimal 1 histological changes were present. 2

Human Relevance: Both formaldehyde-induced mutation and cell prolife ration are 3 direct effects on the oral and nasal mucosa, well d ocumented in rodent models with 4 supporting evidence in human epidemiological studie s. Therefore both key events are 5 relevant to humans. As noted above, there are spec ies differences in localized flux of 6 formaldehyde into the tissues of the oral and respi ratory tract based on structural 7 differences in the airways, as well as breathing pa tterns. Although these differences may 8 effects the dosimetry of the formaldehyde absorptio n into the tissues, this only influences 9 the magnitude of response at any given location. D ata from exposed Rhesus monkeys 10 which documents formaldehyde-induced cell prolifera tion in tissues beyond the nasal 11 cavity, and tissues with minimal histological chang es supports a role for cell-proliferation 12 in the observed cancers in humans, which occur beyo nd the nasal cavities, and in tissues 13 without formaldehyde-related focal lesions. 14

15 d) Cytotoxicity-induced cell proliferation (CICP): Cell death followed by compensatory 16 cell proliferation is a reasonable MOA for agent-in duced cancer. It should be noted that 17 the exposure conditions which result in CICP in rod ents is known to result in significant 18 DNA reactivity and genotoxicity. Therefore, formal dehyde-induced mutations cannot be 19 excluded from this MOA. The animal bioassays suppo rt the carcinogenic potential of 20 formaldehyde in this context (Kerns et al., 1983; S elkemur et al, 1983 and Monticello et 21 al 1986). The majority of squamous cell carcinomas (SCCs) seen in formaldehyde22 exposed rats have been localized to the lateral mea tus and mid-septum in the nasal 23 passages (Morgan et al., 1986; Monticello et al., 1 996), while polyploid adenomas have 24 predominantly been reported at the maxilloturbinate s (Morgan et al., 1986; Monticello et 25 al., 1996). Morgan et al. (1986) speculated that t he maxilloturbinate was less susceptible 26 to SCC due to metabolic differences. However, Mont icello et al. (1996) later suggested 27 that the smaller population of cells available at t he maxilloturbinate accounted for fewer 28 SCCs observed at that site. Regardless, for those locations where SCCs do arise in rats 29 chronically exposed to formaldehyde, a clear tempor al relationship can be demonstrated 30 for dose regimens capable of producing sustained ep ithelial damage and sustained cell 31 proliferation to eventual tumor development. Conve rsely, tumors are not observed in 32 these rodent models at those sites in the nasal pas sages without sustained cell 33 proliferation. 34

35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-443 Relevance to humans: Human exposure to formaldehyde would most likely involve 1 chronic exposures to indoor levels of formaldehyde, and episodic exposures in the 2 environment or from an occupational exposure (See r eview in Chapter 2). An exposure 3 scenario parallel to that used in chronic rodent bi oassays is unlikely (e.g. 2-15 ppm 6-8 4 hours/day, 5 days/week for 10-30 months). Exposur e conditions are difficult to assess 5 especially in retrospective studies. However, only the most extreme industrial work 6 conditions would result in human exposures similar to those that produce sustained 7 compensatory cell proliferation in animal studies ( i.e. 6-15 ppm 6 hrs/day, 5 days per 8 week). Gross tissue lesions as reported in rodents from repeated chronic exposures at 6 9 and 10 ppm formaldehyde have not been reported from workplace exposure, and only 10 minor histopathological changes have been noted (Bo ysen et al., 1990 and Holmstrom 11 and Wilhelmsson et al., 1989). It is possible that workers were episodically exposed to 12 formaldehyde levels which resulted in cell death an d focal or gross lesions requiring cell 13 proliferation for tissue remodeling or repair. How ever, it is unexpected that these 14

conditions would be relevant to human environmental exposures. Therefore, although 15 regenerative cell proliferation is retained as a re asonable MOA for formaldehyde 16 carcinogenicity in experimental animals, it is uncl ear whether it is relevant to the 17 extrapolation of health risks to formaldehyde expos ures in the general environment. 18

19 e) Promotion: Several animal studies indicate that formaldehyde e xposure may promote 20 tumor formation due to other carcinogenic or initia ting agents. There are positive data 21 by several routes of exposure (oral, dermal and inh alation) and promotion has been 22 reported as an increase in tumor bearing animals, a n increase in tumors multiplicity or a 23 decrease in tumor latency with formaldehyde exposur e in conjunction with the initiating 24 agent compared to tumors from the initiating agent along, or formaldehyde alone. The 25 specific key events which may explain this promotio n effect are unknown but may 26 include several of the mechanisms discussed as pote ntial MOAs for formaldehyde: 27 mutagenicity, mitogenesis, co-carcinogenicity, immu nosuppression. Promotion is 28 considered here as a separate MOA, since these acti vities are noted for experimental 29 conditions and tumor sites where formaldehyde did n ot induce tumors in the absence of 30 the initiating agent. 31

Relevance to humans: Although the human epidemiologic literature doesn’t address 32 issues of tumor promotion, the nature of the cancer s of concern indicate that chemical 33 promotion may be relevant to cancer incidence for t hese sites. Many of the risk factors 34

for NPC and other mouth and oral and URT cancers in clude direct mutagens (e.g. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-444 smoking, dietary nitrosamines) where a promoting ag ent would be expected to increase 1

cancer incidence with these other risk factors. Ad ditionally, the well known viral risk 2 factors for cancers of the mouth and URT also sugge st a role for promoting agents to 3 human cancer incidence. Although only tangential e vidence, this does suggest that the 4 promoting activity of a chemical agent, would be re levant to the agent’s carcinogenicity 5 at these sites. Therefore, the potential for forma ldehyde to act as a promoter with other 6 initiators – is considered relevant to formaldehyde ’s carcinogenic MOA. 7

8 f) Increased URT infections / viral reactivation: Inhalation exposure to formaldehyde 9 has been shown to decrease the defenses of the body against infection through two 10 mechanisms: 1) damage to the protective mucous barr ier and function of the mucociliary 11 apparatus; and 2) localized immunosuppression. The se effects have been demonstrated 12 in both exposed humans and controlled animal experi ments. Additionally, increased 13 respiratory tract infections are associated with fo rmaldehyde exposure in several 14 populations. Common viral agents (e.g. Epstein bar r virus) are known risk factors for 15 NPC, sinonasal cancers and other URT cancers. Alth ough direct evidence does support 16 increased URT infections due to formaldehyde exposu re, and URT infections are 17 considered risk factors for URT cancers, direct evi dence for formaldehyde-related 18 infections leading to cancer is lacking. There is however one epidemiological study 19 which finds the association between formaldehyde an d NPC is strengthened in Epstein 20 barr virus sero-positive cases versus sero-negative cases. These data suggest a possible 21 role for formaldehyde in infection, viral reactivat ion, or co-carcinogenicity with a viral 22

agent. 23

24

Relevance to humans: The potential role of increased URT infections and 25 immunosuppression at the portal of entry is conside red to relevant to humans. Data in 26 humans are available to support both key events in this MOA. Additionally, 27 epidemiological studies are conducted in human popu lations where individuals may be 28 exposed to various viral agents across the study pe riod. Therefore, toxic actions by 29 formaldehyde which may increase URT infections, or viral-reactivation at the site of first 30 contact, could be acting in conjunction with viral agents to contribute, in part, to observed 31 associations between formaldehyde exposure and incr eased URT cancer. 32

33 Summary and integration of key events: 34 Each of the hypothesized MOAs discussed above to be tter understand the carcinogenic 35 potential of formaldehyde is supported by formaldeh yde-specific evidence, either in animal 36This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-445 studies, human studies or both. For those key even ts studied in animal models such as cell 1 proliferation, genotoxicity, degradation of the muc ociliary apparatus and CICP, supporting 2 evidence is available in more than one species, mul tiple strains (e.g. rats) and has been reported 3 by multiple researchers. Therefore the overall dat abase supporting these key events in laboratory 4 studies, and their corresponding MOAs is fairly lar ge. In contrast, some key events relevant to 5 humans, but less studied in animal models may have a small supporting database (e.g. increased 6 respiratory tract infections). These alternative M OAs are retained as potentially relevant to the 7 carcinogenic action of formaldehyde as the intent o f this discussion is to identify modes of action 8 will may contribute to the observation of increased upper respiratory tract cancers in exposed 9 human populations. It is noted that additional stu dy is needed to better understand the range of 10 effects formaldehyde may have at sites of first con tact in humans. 11 The MOAs which include genotoxicity, mutation, decr eased DNA repair, increased cell 12 proliferation and CICP are interrelated. Condition s which provide both a source of cell 13 proliferation and increased mutation would be expec ted to increase neoplastic transformation. 14 Formaldehyde acts on the target tissue, the respira tory epithelium, to induce each of these events. 15 However, these key events operate across different exposure ranges and present different 16 exposure response relationships. For example, form aldehyde-induced mutations would be 17 expected across the exposure range, where any incre mental increase in genotoxicity and 18 formaldehyde-related mutation would contribute to b ackground levels, with the potential to 19 increase cancer risk incrementally. In contrast, f ocal and gross lesions to the respiratory mucosa 20 due to cytolethality are not observed unless exposu re concentrations are sufficient to provide 21 localized tissue doses (flux) required to result in cell death and related compensatory cell 22 proliferation. Since tissue dose (flux) is depende nt on not only exposure concentration but also 23 duration of exposure and location in the respirator y tract (Section 3.4), and varies by species, 24 correlation of exposure concentrations to tissue re sponses directly are complex. Exposure 25 response relationships for the key events (cell pro liferation, genotoxicity, degradation of the 26 mucociliary apparatus and CICP) are reported by exp osure concentration, not tissue flux, which 27 would be a more biologically relevant measure. 28 Although the tissue dose-response relationships for formaldehyde induced mutation, 29 mitogenesis and cytolethality are different, the ef fects at the tissue level cannot be easily 30 disaggregated. At any given exposure concentration , target cells in the respiratory tract will 31 experience different effective tissue concentration s of formaldehyde. Measurement of cell 32 proliferation, DNA protein crosslinks or genotoxici ty may require examining a population of 33 cells which would have been subject to different fl ux rates of formaldehyde (See chapter 3). 34 Similarly, when evaluating the tumor dose response, cells within the target tissue will represent a 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-446 range of target tissue formaldehyde concentrations. Therefore, an integrated MOA scheme is 1 hypothesized where key events may influence the obs erved tumor response differentially across 2 the exposure response range (Figure 4-36). This sc hematic illustrates the potential for 3 genotoxicity and formaldehyde-induced mutation to o ccur where tissue dose (flux of 4 formaldehyde into the tissue is minimal). Where t issue dose is increased, formaldehyde-induced 5 cell proliferation is observed in addition to genot oxicity. As tissue dose increases and 6 formaldehyde effects on the respiratory mucosa are more severe, gross pathology including focal 7 and gross lesions due to cell death are noted. The refore, several of the MOAs presented above 8 may be operative and relevant to human exposures at exposure levels resulting in minimal tissue 9 flux – a) direct formaldehyde genitoxicity and resu lting mutation, b) inhibition of DNA repair 10 and c) formaldehyde induced cell proliferation in c onjunction with mutation. CICP, which 11 involves localized and gross tissue lesions would b e operative at higher exposure levels. There is 12 little data to inform the dose range over which the remaining hypothesized MOAs may operate 13 (promotion and increased respiratory tract infectio ns/viral action). 14

15

Mutagenesis

Basal Cell Proliferation Tumor Response

Exposure Concentration

Adaptive Cell Proliferation Induced mitogenesis

Regenerative Proliferation

A B C Integrated Dose Response Curve for Formaldehyde

16 Figure 4-36: Integrated MOA scheme for respiratory tract tumors 17

18 4.5.3.3 Mode(s) of Action for Lymphohematopoietic M alignancies 19

4.5.3.3.1 MOA evaluation for Leukemia 20 Leukemia may arise from stem cells and progenitor c ells in the bone marrow (e.g. acute 21 and chronic myeloid leukemia) or from mature lympho cytes (e.g. chronic lymphatic leukemia, 22This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-447 hairy cell leukemia) (Figure 4-33, Section 4.5.2). Although there is a consistent association 1 between formaldehyde exposure and forms of leukemia when considered as group of diseases 2 (Table 4-91, Section 4.5.2), the strongest and most consistent associations are seen specifically 3 with myeloid leukemia. Little evidence supports an association between formaldehyde exposure 4 and other specific leukemia subtypes, although two studies support a strong association between 5 formaldehyde and “other leukemia and unspecified le ukemia (ICD-9 code 207). Therefore, this 6 MOA evaluation will focus on mechanisms which may i mpact all forms of leukemia (e.g. bone 7 marrow toxicity) or those specific to myeloid leuke mia. The mechanistic data supporting the key 8 events in this analysis are presented in section 4. 5.3.1. 9

10 a) Direct effects of formaldehyde on a circulating ste m cell or progenitor cell present at 11 the portal of entry: Hematopoietic stem cells do circulate throughout the body and can 12 be harvested from peripheral blood. Formaldehyde e xhibits a range of toxic effects at the 13 site of first contact including genotoxic effects b elieved to be mediated by direct DNA 14 reactivity (Section 4.3). Formaldehyde is known to directly react with blood components 15 in formaldehyde exposed humans and animals resultin g in both hemoglobin and albumin 16 adducts (Thrasher et al., 1990, Grammer et al., 199 0; Grammer et al., 1993; Dykewicz et 17 al., 1991; Varro et al., 1997 and Li et al., 2007). Therefore, it has been hypothesized that 18 formaldehyde could react with DNA in circulating he matopoietic stem cells (Zhang et al., 19 2009) resulting in heritable mutations which may co ntribute to leukemia incidence. 20 Recently Zhang et al. (2010) have tested the hypoth esis that exogenous formaldehyde 21 may damage circulating stem cells. Clastogenic eff ects were found in circulating 22 hematopoietic stem cells cultured from formaldehyde exposed workers. The reported 23 aneuploidy was demonstrated as significant increase s in both monosomy 7 and trisomy 8. 24 These specific chromosomal changes are consistent w ith those reported for agent-induced 25 myeloid leukemia (Zhang et al., 2010). 26

Relevance to Humans: This hypothesized MOA is considered relevant to hum ans. 27 Supporting evidence is found in humans for formalde hyde direct reactivity with blood 28 proteins (e.g. albumin and hemoglobin) as well as c lastogenic effects in circulating 29 hematopoietic stem cells in formaldehyde exposed wo rkers. 30

31 b) Bone marrow toxicity: Direct bone marrow toxicity is the most studied l eukemogenic 32 action for an endogenous agent (e.g. benzene, ioniz ing radiation). It is believed that an 33 agent which exerts its toxicity on the bone marrow, resulting in translocations and 34 heritable mutations in hematopoietic stem cells may cause leukemia. It has been 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-448 hypothesized that formaldehyde may transport to the bone marrow in its hydrated form 1 (methandiol) and react with cellular proteins, and DNA casuing direct effects on 2 components of the bone marrow. Pancytopenia (a red uction in blood borne cells formed 3 in the bone marrow) is a symptom of direct bone mar row toxicity and is observed with 4 other leukemogenic agents (e.g. benzene, ionizing r adiation). A recent review of 8 5 published studies of formaldehyde exposed workers i n China by Tang et al. (2009) 6 indicates 7 of the studies provide evidence of redu ced white blood cell counts, platelet 7 levels and hemoglobin levels associated with formal dehyde exposure. A study of 8 occupationally exposed nurses provided a correlatio n between decreased white blood cell 9 counts and formaldehyde exposure (Kuo et al., 1997) . A recent study by Zhang et al. 10 (2010) provides the best evidence for bone marrow t oxicity, where they report not only a 11 reduction in white blood cell counts, but reduction s in cell counts of all the blood cells, as 12 well as increased mean cell volume. Although these reductions did not meet the clinical 13 definition of pancytopenia (when averaged across th e study population), reduction of all 14

blood borne cells formed in the bone marrow is cons istent with the bone marrow toxicity 15 associated with pancytopenia seen with other leukem ogens (Zhang et al., 2010). 16

Relevance to Humas: This hypothesized MOA is considered relevant to hum ans. 17 Supporting evidence is found in humans for bone mar row toxicity in formaldehyde 18 exposed workers. 19

20

4.5.3.3.2 MOA evaluation for Lymphomas (e.g. Hodgkin lymphoma , Multiple myeloma) 21 The general MOA for formaldehyde is based on direct chemical reactivity and toxic 22 effects at the portal of entry (POE). Formaldehyde is directly and indirectly genotoxic, and 23 reacts with cellular proteins and DNA in cells whic h it comes into contact. Additionally, 24 immunosuppression, viral reactivation and promotion effects are relevant to lymphoma and 25 related malignancies. Therefore, the key events fo r the adult cell lymphoid cancers would 26 include these actions. Lymphoid tumors (e.g. lymph ocytic leukemia, B-cell lymphoma, mantle 27 cell lymphoma [a rare form on non-Hodgkin lymphoma] and myeloma) may arise from cells 28 present at the portal of entry (POE) (Figure 4-33). The location and function of mature 29 lymphocytes contribute to their vulnerability to tr ansformation by agents at the POE. Therefore, 30 a brief summary of the immuno-biology of these cell s is provided in order to provide context for 31

the MOA evaluation: 32

33

Location: Lymphocytes are present in the oral and respiratory tract epithelium, as well 34 as in cell aggregates and tertiary immune structure s (e.g. germinal centers) in the mucosal 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-449 tissues (Zuercher and Cebra, 2002; Zuercher et al., 2002; Wu et al 1997 and Kupper et 1 al., 1990). These mucosa-associated lymphoid tissu es (MALT) provide the opportunity 2 for formaldehyde to directly interact with componen ts of the immune system present at 3 the POE (Wu et al., 1997, Claeys et al. 1996, Park et al., 2003 and Fujimura 2000). 4 Intraepithelial lymphocytes are present in the pseu dostratified epithelium of the 5 nasopharyngeal passages and there are aggregates of immune cells and germinal cells 6 present in these tissues. Crypts containing mature lymphocytes exist at the surface of the 7 nasal epithelium (Fujimura 2000). Microfold cells or M-cells form the crypts, where the 8 lymphocytes are covered by a thin membrane (Figure 4-37). Functionally, these 9 lymphocytes identify and process foreign antigens a t the POE (Fujimura 2000). 10 Therefore the mature lymphocytes within these crypt s, exposed to exogenous agents, are 11 involved in active immune responses to foreign anti gens. 12

13 Clonal Expansion: Mature lymphocytes (both B and T-cells) clonally ex pand their 14 populations in response to an exogenous antigen whe n a humoral immune response is 15 stimulated. Therefore cell proliferation is a norm al function of these mature lymphocytes 16 and occurs every time there is an infection. Cell proliferation of mature B and T-cells, 17 responsive to a particular antigen, occurs in activ e germinal centers (including those 18 within the respiratory tract). Cells may be expose d to exogenous agents during the 19 immune response, or cells responding in the germina l center may have previously been in 20 the epithelium or M-cell crypt. 21

22 Somatic Hypermutation: Normal immune function includes the process of so matic 23 hypermutation where B-cells undergo DNA rearrangeme nt of the variable region genes to 24 produce novel antibodies specific to a given antige n. This process is key to adaptive 25 immunity and demonstrated by the basic principles o f immuno-biology which underlie 26 vaccination theory. Gene sequencing of adult B-cel l lymphomas and leukemias indicate 27 that the chromosomal regions involved in somatic hy permutation correspond to known 28 oncogenes in these cancers. The vulnerability of t hese processes is evidenced by the 29 observation that approximately 90-95% of adult lymp homas and leukemias are of B-cell 30 origin (Gordon et al., 2003). Formaldehyde-induce d protein-protein crosslinking could 31 disrupt cell processes including somatic hypermutat ion and cell mitosis, resulting in 32 agent-induced translations similar to those found i n spontaneous B-cell malignancies. 33

34This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-450 17 1 Figure 4-37. Location of intra-epithelial lymphocy tes along side epithelial cells in 2 the human adenoid. (Source Fujimura et al., 2000) 3 4 a) Direct or indirect formaldehyde-induced mutation in cells at the site of first contact: 5 Immune cells including intraepithelial lymphocytes, and cells in mucosal associated 6 lymph tissue (MALT) are collocated with the epithel ial cells from which URT cancers 7 arise (Figure 4-37). Therefore the direct and indi rect mutagenic potential for 8 formaldehyde is equally applicable to components of the immune system present in these 9 tissues. Mutations, the permanent heritable change s in the genome of the cell, are a 10 primary mechanism for the activation of oncogenes o r the inactivation of tumor 11 suppressor genes. Mutagenicity is the most widely recognized determinant of chemical12 induced carcinogenicity, and it is difficult to set aside the relevance of direct 13 formaldehyde-induced mutations from its demonstrate d carcinogenicity. Formaldehyde14 induced mutation in immune cells present at the sit e of first contact, may initiate cells or 15 or provide subsequent mutagenic events to already i nitiated cells. The competence of our 16 immune system relies on the proliferation of periph eral lymphocytes in response to 17 immune challenge (infection). Additionally, herita ble changes to the variable gene 18 regions in B-cells generated during somatic hyper-m utation are essential to adaptive 19 immunity (e.g. immunization) demonstrating that per manent heritable changes in the 20 DNA of peripheral B-cells are passed to daughter ce lls and retained in the body for 21 decades. Any agent-induced mutations would be simi larly propagated and retained with 22 the potential to contribute to the transformation o f mature lymphocytes. 23This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-451 Relevance to humans: This MOA is relevant to humans. The well-documente d DNA 1 reactivity (e.g. DPC and DNA adducts) and clastogen icity of formaldehyde at the POE in 2 laboratory animals is a direct effect of formaldehy de on tissues of first contact, and these 3

mechanisms are considered relevant to humans. As w ith epithelial cells, clastogenic 4 effects in peripheral lymphocytes are documented in formaldehyde-exposed students and 5 workers, confirming the genotoxic effects of formal dehyde in immune cells, from which 6 lymphomas and related diseases may arise (Section 4 .5.2Figure 4-33). 7

8 b) Formaldehyde-induced protein-protein crosslinks may disrupt somatic9 hypermutation: Although not as well studied as DNA-protein crossli nks, formaldehyde 10 also formes crosslinks between amino acids on prote ins (Section 4.5.3.1.3 for details). 11 Specific oncogenes for malignancies which arise fro m mature B-cells are linked to errors 12 in the process of somatic hyper-mutation (Greaves e t al. 2004). If formaldehyde creates 13 protein crosslinks in competent B-cells which effec t the process of DNA rearrangement, 14 formaldehyde may generate translocations and relate d oncogenes similar to those 15 observed in observed in spontaneous B-cell malignan cies. 16

Relevance to humans: This hypothesis has not been tested in either exp osed human or 17 animal test systems. However, the link between som atic-hypermnutation and B-cell 18 oncogenes is well established and perturbation of t his process by an exogenous agent is a 19 reasonable extension of the current understanding o f the etiology of B-cell malignancies. 20

21 c) Increased URT infections / viral reactivation: Inhalation exposure to formaldehyde 22 has been shown to decrease the defenses of the body against infection through two 23 mechanisms: 1) damage to the protective mucous barr ier and function of the mucociliary 24 apparatus; and 2) localized immunosuppression (Sect ion 4.5.3.1). These effects have 25 been demonstrated in both exposed humans and contro lled animal experiments. 26 Additionally, increased respiratory tract infection s are associated with formaldehyde 27 exposure in several populations. Common viral agen ts (e.g. Epstein barr virus) are 28 known risk factors for malignancies which arise fro m mature lymphocytes. Thus, 29

increased URT infections or viral reactivation due to formaldehyde exposure may 30

influence the incidence of these cancers. 31

Relevance to humans: The potential role of increased URT infections and 32 immunosuppression at the portal of entry is conside red to be relevant to humans. Data in 33 humans are available to support both key events in this MOA. Additionally, co-exposure 34 to infectious agents (including viruses) would be e xpected in participants in an 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-452 epidemiological study, suggesting an MOA which acte d in conjunction with infectious 1 agents may be relevant to agent-induced cancer. Th erefore, toxic actions by 2 formaldehyde which may increase URT infections, or viral-reactivation at the site of first 3 contact, could be acting in conjunction with viral agents to contribute, in part, to observed 4 associations between formaldehyde exposure and incr eased lymphoma and related 5

diseases. 6

7

4.5.3.3.3 Summary and evaluation of hypothesized MOA(s) for Lymphohematopoietic 8 Malignancies 9

The well-documented direct toxic action of formalde hyde on cells at the site of first 10 contact is a general effect based on the reactivity of formaldehyde with cellular components (e.g. 11 proteins and DNA) (Section 4.5.3.1). As a general effect, it is reasonable that these toxic effects 12

would be relevant to all cells which come into cont act with formaldehyde. The current debate 13 regarding the biological plausibility of formaldehy de-induced lymphohematopoeitic 14 malignancies centers around a perspective that the diseases within this general grouping are 15 systemic cancers arising only out of bone marrow to xicity (Heck et al., 2006, Pyatt et al., 2008) 16 and that it is implausible for formaldehyde to indu ce bone marrow toxicity. The above MOA 17 evaluation expands the current debate by considerin g the impact of POE toxicity on elements of 18 the immune system and cancers might arise from thes e cells (Section 4.5.3.3.2) and by 19 presenting data which support the observation that formaldehyde is associated with bone marrow 20 toxicity and damage to circulating stem cells in ex posed humans (Section 4.5.3.3.1). 21 As significant increases in free formaldehyde in pe ripheral blood from exogenous 22 exposure has not been detected (Heck et al., 1987), it has been hypothesized that formaldehyde 23 does not transport and therefore cannot exert toxic effects outside of the tissues at the site of firs t 24 contact (Heck et al., 2006, Pyatt et al., 2008). I n contrast to this hypothesis, effects are seen in 25 formaldehyde-exposed humans which indicate systemic effects on the hematopoietic system 26 including reduced white blood cell counts, clastoge nic effects in peripheral blood lymphocytes 27 and aneuploidy in circulating stem cells (Tang et a l., 2008, Zhang et al., 2010 and Section 28 4.5.3.1). These observed effects in humans are con sistent with agent-induced bone marrow 29 toxicity and are observed with other well-studied e xogenous leukemogens (e.g. benzene and 30 ionizing radiation.) It is unknown if formaldehyde is distributed systemically to exert its effects 31 directly on cells in the bone marrow or if damage t o circulating stem cells or progenitor cells 32

would be sufficient to result in the observed effec ts in humans (Zhang et al., 2010). Additional 33 research is needed to better determine the potentia l for systemic transport of formaldehyde 34 considering both detection of its hydrated form (me thylene glycol) as well as formaldehyde 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-453 protein adducts (e.g. FA-GSH and FA-albumin). Simi larly the results of Zhang et al. (2010) 1 need to be extended (analysis for additional chromo somal aberrations) and repeated. Although 2

further evidence is needed to better understand the hypothesized mechanisms for formaldehyde3 induced effects on hematopoietic stem cells, the ob served hematotoxic effects in humans cannot 4 be set aside. Therefore, however unlikely, the cur rent data support the biological plausibility of 5 formaldehyde effects on the hematopoietic system. 6

7 4.5.4. Hazard Characterization FOR Formaldehyde Ca rcinogenicity 8 Formaldehyde is Carcinogenic to Humans by the Inhalation Route of Exposure 9 Based on the results from a large and well-followed longitudinal cohort study of 10

industrial workers and several case-control studies , the epidemiologic evidence is sufficient to 11 characterize the association between formaldehyde e xposure and nasopharyngeal cancer (NPC) 12 as causal in humans (Hauptmann et al., 2004; Hildes heim et al., 2001; Vaughan et al., 2000). As 13 a group, upper respiratory tracts sites of direct c ontact with formaldehyde upon inhalation (i.e., 14 salivary gland, mouth, nasopharynx, nasal cavity an d larynx) also showed sufficient evidence of 15

a causal association. Case-control studies have dem onstrated associations between formaldehyde 16 exposure and rare cancers of the URT. Luce et al. (2002) evaluated pooled data from 12 case17 control studies and demonstrated a statistically si gnificant increased risk between formaldehyde 18 exposure and sinonasal cancer. Hypopharyngeal canc er was linked with formaldehyde exposure 19 with an OR of 3.78 (95% CI 1.50-9.49) in another ca se-control study (Laforest et al., 2000). 20 Hauptmann and colleagues (2004) concluded that in s pite of the small numbers of deaths from 21 cancers of the URT, the positive associations with average intensity and peak exposure were 22 consistent with the carcinogenicity of formaldehyde at these sites of first contact. The finding 23 that formaldehyde inhalation causes nasal squamous cell carcinoma in animals (Section 4.2.1.2) 24 further supports the determination of a causal asso ciation of formaldehyde exposure and 25 increased risk of upper respiratory tract cancer in humans. Both humans and animals developed 26 tumors within the upper respiratory tract, the POE site expected to receive direct exposure to 27 formaldehyde. 28 Overall, there is a consistent association between formaldehyde exposure and various 29 forms of lymphohematopoietic (LHP) cancers, with al l leukemias, myeloid leukemia 30 specifically, Hodgkin lymphoma and multiple myeloma demonstrating the greatest strength and 31 consistency of results. Where exposure-response da ta exist, exposure-response trends have been 32 seen for all LHP malignancies, all leukemia, myeloi d leukemia and Hodgkin lymphoma 33 (Pinkerton et al., 2004; Beane Freeman et al., 2009 ). Taken together, the data demonstrate a 34 consistent association, across various worker popul ations, with the expected temporal association 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-454 to exposure and defined exposure–response relations hips in two different worker cohorts. The 1 strongest associations tend to be with myeloid leuk emia and Hodgkin lymphoma. The criterion 2 of reasonable biological plausibility is easily met for the majority of the diseases which 3 contribute to an observation of all LHP cancers, sp ecifically the cancers derived from mature 4 lymphocytes. The potential for formaldehyde-induce d LHP cancer is further supported by the 5 results of animal bioassays, where formaldehyde-ind uced leukemia and lymphoma had been 6 demonstrated in 3 independent studies in two specie s (rats and mice) and both sexes. 7

8

4.6. SUSCEPTIBLE POPULATIONS 9 “Susceptible subpopulations” is used here to refer to factors, such as life stage, genetics, 10 health status, etc., that may predispose individual s to greater response to an exposure. This 11 greater response could be achieved either through d ifferences in exposure to the chemical or 12 differences in underlying toxicokinetic (TK) and to xicodynamic (TD) differences between the 13 susceptible and other populations. For example, li fe stages may include the developing 14 individual before and after birth up to maturity (e .g., preconception, embryo, fetus, young child, 15 adolescent), adults, or aging individuals. Another susceptibility factor is genetics. Specifically, 16 susceptible subpopulations may also include people with specific genetic polymorphisms that 17 render them more vulnerable to a specific agent or people with specific diseases or pre-existing 18 conditions (e.g., asthmatics). The term may also re fer to gender differences, lifestyle choices, or 19 nutritional state (USEPA, 2002, Section 4.3.2.3). 20 A discussion of a comprehensive list of all possibl e susceptibility factors affecting 21 exposure and response to formaldehyde, or any chemi cal, is not possible. Therefore, the 22 discussion of susceptibility factors focuses on 1) factors hypothesized to be of importance to 23 formaldehyde; and 2) factors for which there are av ailable formaldehyde data. A partial list of 24 these factors includes gender, genetic polymorphism s, preexisting disease status, nutritional 25 status, diet, and previous or concurrent exposures to other chemicals. Qualitatively, the presence 26 of multiple susceptibility factors will increase th e variability that is seen in a population response 27 to formaldehyde toxicity. 28

29 4.6.1. Life Stages 30 Individuals at different life stages are physiologi cally, anatomically, and biochemically 31 different. Examples include physiological changes that occur through the lifespan (Selevan et 32 al., 2000). They may also have distinctive exposur e pathways (i.e., transplacental, breast milk 33 ingestion), and exhibit differences in behavior (U. S. EPA, 2006b; NRC, 1993). Early life stages 34 (i.e., during development, prior to mature adulthoo d) and the later life stages (i.e., aging) differ 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-455 greatly from mature adulthood in body composition, organ function, and many other 1 physiological parameters that can impact the TK and /or TD of chemicals and their metabolites 2 (ILSI, 2003). This section presents and evaluates the pertinent published literature available to 3 assess whether and how individuals of differing lif e stages may respond differently to 4 formaldehyde. 5

6

4.6.1.1. Early Life Stages 7 4.6.1.1.1. Factors influencing exposure and dosime try 8 For all life stages, the primary exposure routes fo r formaldehyde include inhalation and, 9 in some cases, ingestion (see Chapter 5). Some exp osure scenarios may be child specific. For 10 example, to the extent that the presence of baby fu rniture produced with formaldehyde in a 11 child’s house contributes to greater concentrations in a child’s room, exposures for very young 12 children in those circumstances may be increased (E nvironment California, 2008). As with all 13 chemicals, placental transfer and breast milk inges tion are exposure pathways that are unique to 14 early life stages. Studies assessing early life st age exposure pathways to formaldehyde have not 15 been performed. Presumably, unmetabolized formalde hyde reacts too quickly to be effectively 16 transported from mother to fetus by placental trans fer; in addition, formaldehyde is not lipophilic 17 and is therefore unlikely to accumulate in breast m ilk. However, the relevant dose metric for 18 formaldehyde-related effects may vary depending on the specific target of concern (e.g., direct 19 toxicity at the portal of entry versus systemic eff ects); insufficient information is currently 20

available to determine whether individuals in diffe rent life stages are at higher risk for exposure 21 to specific target tissues. 22 There are some calculations however which shed ligh t on lifestage differences in the 23 inhaled tissue dose at the portal of entry. Using r espiratory tract surface areas and ventilation 24 rates reported in the literature and the scheme in USEPA (1994), Ginsberg et al. (2005) 25 calculated that overall extrathoracic absorption of highly reactive and soluble gases is similar in 26 adults and children. These results are in agreement with that of Garcia et al. (2009) who used 27 computational fluid dynamics to study differences i n the nasal dosimetry of reactive, water28 soluble gases between 5 adults and 2 children, aged 7 and 8 years old. Overall uptake efficiency, 29 average flux (rate of gas absorbed per unit surface area of the nasal lining) and maximum flux 30 levels over the entire nasal lining did not vary su bstantially between adults and children (1.6-fold 31 difference in average flux and much less in maximum flux). On the other hand, the local flux of 32 formaldehyde varies between the two children by a f actor of 2 to 4 at various distances along the 33 septal axis of the nose. The results in Garcia et a l. (2009) have been further described and 34 evaluated in Appendix 3-1. Under normal resting bre athing conditions, it is expected that very 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-456 little formaldehyde is delivered to the lung. Howev er, under higher activity as well as mouth 1 breathing scenarios, both of which appear likely to happen more regularly in children, 2 formaldehyde dose to the lung will be substantial. 5 3 The toxicokinetic characteristics of formaldehyde are described in Chapter 3, with 4 absorption and distribution studies discussed in Se ctions 3.2 and 3.3. Studies to assess 5 differential absorption or distribution of formalde hyde in early life stages have not been 6 performed and represent a significant data gap. Th e metabolism of formaldehyde is described in 7 section 3.4. Expression of the enzymes that metabo lize formaldehyde (ALDH2 and FALDH, 8 and specifically ADH3) is known to be developmental ly regulated and thus may alter the TK of 9 formaldehyde in early life stages. ADH3 is ubiqui tously expressed and is present in the human 10 fetus, neonate, and 1- to 10-year-old children (Hin es and McCarver, 2002; Estonius et al., 1996). 11 During early development in rodents, when neurulati on first begins and forms collections of 12 somites along the neural tube, ADH3 activities are significantly lower (at 8–10 and 11–13 somite 13 stages) and suggest a decreased ability to detoxify formaldehyde in the early embryo (Harris et 14 al., 2003). ADH mRNA expression levels appear to b e age related, with decreased expression of 15 ADH common in premature neonates and infants up to 5 months old. Thereafter, ADH 16 expression increases and is dependent on body weigh t (Ginsberg et al., 2004). Benedetti et al. 17 (2007) reported that decreased ADH expression persi sted until age 2 to 5 years. Westerlund et 18 al. (2005) tracked the ontogeny of ADH3 specificall y and reported that ADH3 expression was 19 ubiquitous in mouse and rat embryos and was the onl y ADH enzyme to be consistently localized 20 to brain tissue, suggesting a housekeeping function . Thus, neonates and very young children 21 may have a decreased ability to metabolize formalde hyde due to differential expression of ADH3 22 in development compared that of with adults; howeve r, activity levels of this enzyme and 23 alternate pathways specific to children are not ava ilable in the literature. 24

25 4.6.1.1.2. Life-stage exposure and adverse health outcomes 26 In general, exposure to toxic agents during early d evelopment (i.e, pre-conception, 27 prenatal stages, or postnatal development) may affe ct organ development and may also lead to 28 increased disease susceptibility later in life. Fo llowing early life stage exposure to 29 formaldehyde, a number of adverse health outcomes h ave been observed, including alterations in 30 the respiratory, reproductive, and neurological sys tems. For example, the developing respiratory 31 tract may be more vulnerable to insult compared wit h an adult respiratory tract, and thus, 32 increase the severity of response. The potential f or reproductive and developmental toxicity of 33

5 For example, in the case of ozone concentrations o f 0.1 ppm, a moderately reactive gas, Ginsberg et a l. (2008) predict a 5-fold variation in the dose to the deep lung between quiet and heavy breathing conditions f or an 8-year old child.This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-457 formaldehyde is discussed in detail in Sections 4.1 .1.7 (human studies) and 4.2.1.7 (animal 1 studies), while effects on the nervous system are d iscussed in Sections 4.1.1.6 and 4.2.1.6 2 (human and animal studies, respectively). The spec ific case of formaldehyde exposure and 3 pulmonary effects is discussed in detail in Section s 4.1.1.1 to 4.1.1.4 and 4.2.1.1 to 4.2.1.4. A 4 brief summary of identified effects of formaldehyde that may indicate susceptibility during 5 particular life stages is provided below. 6

7

4.6.1.1.2.1. Pre-conception. 8 Exposure prior to conception may damage reproductiv e organs and/or germ cells that 9 could affect reproduction and/or damage the genetic makeup of the offspring. Effects on 10 reproduction are discussed in Sections 4.1.1.7 and 4.2.1.8. In summary, an epidemiological 11 study (Taskinen et al., 1999) reported significantl y delayed conception among female workers 12 exposed to formaldehyde at average daily ambient fo rmaldehyde levels; these effects could be 13 consistent with adverse effects on either pre-conce ptional and/or gestational exposure. One 14 animal study (Maronpot et al., 1986) reported endom etrial hypoplasia and lack of ovarian luteal 15 tissue in female mice exposed for 13 weeks to 40 pp m formaldehyde via inhalation, suggesting 16 the potential for treatment-related alterations to the female reproductive system. Since the 17 exposure was to the adult, the findings suggest tha t preconceptional FA exposure caused female 18 reproductive system effects that in turn could affe ct pregnancy. 19 In the rodent study of Kitaev et al. (1984), a thre e-fold increase in embryo degeneration on 20 gestational days 2–3 was observed after FA exposure to the dams during premating. Since the 21 exposure was to the adult in these three studies, t he findings suggest that preconceptional FA 22 exposure caused female reproductive system effects and/or affected the gametes. 23

24

4.6.1.1.2.2. Prenatal. 25 A population-based study (Gražulevi č iene et al., 1998) found an association between 26 atmospheric formaldehyde exposure and low birth wei ght, yielding an adjusted OR of 1.37 (95% 27 CI: 0.90–2.09). Three studies (Dulskiene and Gražu levi č iene, 2005; Taskinen et al., 1994; 28 Hemminki et al., 1985) that examined the effect of occupational exposures on the incidence of 29 congenital malformation produced mixed results. 30 Results from Taskinen et al. (1999) support associa tions between formaldehyde exposure, 31 subfertility, and spontaneous abortion. Subfertili ty and spontaneous abortion are biologically 32 linked (subclinical pregnancy losses are increased among women with fertility problems) (Gray 33 and Wu, 2000; Hakim et al., 1995), and both subfert ility and spontaneous abortion may be 34 related to sensitivity to environmental agents (Cor rea et al., 1996). 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-458 Two experimental animal studies (Martin, 1990; Sail lenfait et al., 1989) evaluated a 1 standard battery of developmental endpoints followi ng inhalation exposure on GDs 6–10, but 2 effects were minimal. Similarly, Chernoff and Kavl ock (1982), Marks et al. (1980), and Hurni 3 and Ohder (1973) reported minimal reproductive or d evelopmental effects in rodents in studies 4 in which dams were exposed orally during early gest ation. When formaldehyde was 5 administered via inhalation throughout gestation in female rats, some developmental effects, 6 including increased pup weight and decreases in lun g and liver weight in newborns, were 7 reported at 0.01 and 0.4 ppm (Senichenkova and Cheb otar, 1996; Senichenkova, 1991; Kitaev et 8 al., 1984; Gofmekler and Bonashevskaya, 1969; Gofme kler, 1968; Pushkina et al., 1968). Two 9 studies also reported changes in motor activity in offspring of dams exposed via inhalation to 0.4 10 ppm formaldehyde during gestation (Senichenkova, 19 91; Sheveleva, 1971). 11

12

4.6.1.1.2.3. Postnatal. 13 Following early life stage exposure to formaldehyde , a number of adverse postnatal 14 outcomes are possible, including effects on the dev eloping and adult respiratory, reproductive, 15 and neurological systems. The potential for increa sed risk of childhood cancer is also discussed 16

below. 17

18

4.6.1.1.2.3.1. Respiratory toxicity. 19 Formaldehyde is known to induce changes in pulmonar y function and cause pulmonary 20 irritation in human studies (Rumchev et al., 2002; Garrett et al., 1999; Krzyzanowski et al., 1990; 21 Holmström et al., 1989; Holmström and Wilhelmsson, 1988; Ritchie and Lehnen, 1987) and 22 animal studies (Ohtsuka et al., 2003, 1997; Riedel et al., 1996; Swiecichowski et al., 1993; Lee et 23 al., 1984). Exposure to formaldehyde in early life can cause damage to the lungs and 24 permanently influence airway function, resulting in increased vulnerability to toxicants later in 25 life. Thus, young children may demonstrate increas ed susceptibility to formaldehyde-related 26 health effects. Krzyzanowski et al. (1990) reporte d an association between physician-diagnosed 27

asthma and chronic bronchitis in children who lived in homes with formaldehyde levels that 28 were higher than 60 ppb, after controlling for soci oeconomic status and ethnicity. Rumchev et 29 al. (2002) reported a statistically significant inc reased risk of asthma with increased residential 30 concentrations of formaldehyde. Garrett et al. (19 99) found an increased association between 31 bedroom concentration of formaldehyde and increased risk of atopy in children. These studies 32 suggest that formaldehyde exposure may exacerbate r esponses in sensitive airways, particularly 33 in children. Exacerbation of response has also bee n noted in asthmatic adults and will be 34

discussed below. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-459 Another child-specific concern is that respiratory irritation may have greater impact on 1 lung function in children due to their smaller lung size; this is true even if the lung development 2 is normal. Irritation is commonly accompanied by i nflammation, which can have a greater 3 impact on children’s airways because they are narro wer than adult airways. Thus, less 4 inflammation is required for significant airway obs truction in children than in adults. 5

6

4.6.1.1.2.3.2. Developmental neurotoxicity. 7 In neonatal exposure paradigms, changes in brain st ructure (Sarsilmaz et al., 2007; Aslan 8 et al., 2006), and brain chemistry (Songur et al., 2008) were seen in young rats following 9 inhalation exposures (6000 or 12000 ppb, 5 days per week from postnatal day 0-30). In addition, 10 Weiler and Apfelbach (1992) found juvenile animals to be more sensitive to formaldehyde11 induced changes in olfactory thresholds when compar ed with adult animals (shifts in olfactory 12 thresholds were greater when exposure [at 250 ppb] was initiated at PND 30 than at adult ages). 13 These studies are consistent with the hypothesis th at early life exposure to FA can lead to long14 lasting neurological effects. Exposure levels in t hese studies (250–6,000 ppb) were in the same 15 range as those producing the behavioral effects in adults (as low as 100 ppb), but provide limited 16 information regarding relative sensitivity as no NO AELs were identified, and (with the exception 17 of Weiler and Apfelbach), similar parameters were n ot measured in adult animals using the same 18 exposure paradigms. 19

20

4.6.1.2. Later Life Stages 21 In general, older adults may be at risk for increas ed susceptibility to exposure to 22 environmental chemicals by virtue of their slower m etabolism and increased incidence of altered 23 health status (Benedetti et al., 2007; Ginsberg et al., 2005; U.S. EPA, 2005a). Additionally, 24 adverse effects of earlier exposure to some toxican ts may be observed in older adults as a result 25 of latency in expression of the effect (Olsen et al ., 1997; Sweeney et al., 1986). No studies have 26 examined the differential effects of formaldehyde e xposure for elderly adults (>65 years old) as 27 compared to other age groups. 28

29

4.6.1.3. Conclusions on Life-Stage Susceptibility 30 In summary, timing both of the exposure and of the assessment of health outcomes may 31 be important for understanding the relative risk of adverse effects from formaldehyde exposure 32 during different life stages. There are known deve lopmental differences in kinetics across life 33 stages, including differences in enzymes involved i n formaldehyde metabolism, but the 34 contribution of these differences to formaldehyde-r elated health effects is unknown. Similarly, 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-460 information regarding life-stage differences in res piratory physiology raises possible concern 1 regarding increased exposure to children, but studi es for formaldehyde are not available. 2 Available data do support an increased risk for adv erse effects on lung function in children. The 3 overall body of evidence shows some support, althou gh minimal, for susceptibility in 4 reproductive or developmental endpoints associated with exposure to formaldehyde. Some 5 studies observed altered development of the nervous system following formaldehyde exposure 6 during early life. Older adults may be at risk for increased susceptibility to formaldehyde 7

because of slower metabolism and clearance rates. Elderly adults have an increased probability 8 of having both altered health status and altered me tabolism, which could impact their ability to 9 process and recover from an adverse effect. The av ailable data are consistent with some life10 stage susceptibility differences for FA at the leve l of TD or TK differences, the results are 11

nonetheless inconclusive due to the number of data gaps. 12

13

4.6.2. Health/Disease Status 14 The factor for which we have the greatest evidence is pre-existing disease, and 15 specifically asthma. Numerous studies have assesse d the potential for increased susceptibility to 16 formaldehyde in asthmatics. Formaldehyde does not induce airway hyperreactivity directly 17 (Sheppard et al., 1984) and has not been shown to i ncrease airway hyperreactivity in either 18 asthmatics or non-asthmatics (Pazdrak et al., 1993; Harving et al., 1991; Kulle et al., 1987). 19 Significantly decreased forced expiratory volume (F EV 1 ) measurements were reported among 20 asthmatics in two studies (Casset et al., 2006; Gre en et al., 1987), while others did not find any 21 significant change in FEV 1 following formaldehyde exposure (Ezratty et al., 2 007; Frigas et al., 22 1984). 23 A few available case reports of bronchial asthma do suggest direct respiratory tract 24 sensitization to formaldehyde gas (Lemiere et al., 1995; Burge et al., 1985; Hendrick et al., 1982; 25 Hendrick and Lane, 1977, 1975). All cases displaye d marked changes in FEV 1 or pulmonary 26 airflow rate in response to acute challenges with f ormaldehyde gas at exposure levels <3 ppm. 27 Formaldehyde-induced IgE production has been report ed in some studies (Vandenplas et al., 28 2004; Wantke et al., 1996a). 29 There is a large quantity of human data providing e vidence of an association between 30 formaldehyde exposure and increased incidence of as thma or exacerbation of asthmatic 31 responses in compromised individuals. For example, Krzyzanowski et al. (1990) reported an 32 association between physician-diagnosed asthma and chronic bronchitis in children who lived in 33 homes with formaldehyde levels that were higher tha n 60 ppb, after controlling for 34 socioeconomic status and ethnicity. Rumchev et al. (2002) reported a statistically significant 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-461 increased risk of asthma with increased residential concentrations of formaldehyde. Garrett et al. 1 (1999) found an increased association between bedro om concentration of formaldehyde and risk 2 of atopy in children. These studies suggest that f ormaldehyde exposure may exacerbate 3 responses in sensitive airways, particularly in chi ldren. Exacerbation of response has also been 4 noted in adults. Kriebel et al. (1993) reported a greater decrease in peak expiratory flow (PEF) 5 in asthmatic medical students (7.3% decrement) comp ared with non-asthmatic medical students 6 (2% decrement) after 2 weeks exposure to formaldehy de (average concentration 0.73 ppm) in an 7 anatomy lab. This effect does not appear to be imm unogenic in nature (Fujimaki et al., 2004a; 8 Lee et al., 1984). 9 Several animal studies document increased airway re sistance and bronchial constriction 10 following inhalation exposure to formaldehyde (Niel son et al., 1999; Swiecichowski et al., 1993; 11 Biagini et al., 1989; Amdur et al., 1960). Sadakan e et al. (2002) demonstrated that formaldehyde 12 exposure exacerbated sensitization and challenge wi th a common dust mite allergen (Der f) as 13 measured by increased eosinophil infiltration into the interstitium around the bronchi and 14 bronchioles as well as goblet cell proliferation in the bronchial epithelium; they suggested that 15 formaldehyde exposure may aggravate eosinophilic in filtration and goblet cell proliferation that 16 accompanies allergic responses. The MOA underlying this response is unknown. These 17 decrements may occur indirectly in response to form aldehyde and may be mediated via 18 neurogenic potentiation (Sadakane et al., 2002; Rie del et al., 1996; Tarkowski and Gorski, 1995). 19 In particular, Tarkowski and Gorski (1995) suggest that formaldehyde may increase permeability 20 of respiratory epithelium and destruction of immuno logic barriers. Thus, the respiratory tract 21 may become vulnerable to inhaled allergens after fo rmaldehyde exposure (Tarkowski and 22 Gorski, 1995). 23 In summary, the data indicate that formaldehyde exp osure can aggravate a type I 24 hypersensitivity response and that this hypersensit ivity may in turn increase the susceptibility to 25 FA exposure in these individuals. Formaldehyde exp osure may predetermine an asthmatic 26 phenotype or may induce new incidences of asthma vi a indirect mechanisms, though definitive 27 evidence and a proposed mechanism remain to be dete rmined. Individuals that exhibit 28 chemically induced sensitivity and are exposed acut ely or chronically to formaldehyde in 29 residential and occupational settings might exhibit adverse responses at lower concentrations of 30 formaldehyde than the average healthy person. 31

32

4.6.3. Nutritional Status 33 Limited available data indicate that certain types of malnutrition may increase 34 susceptibility to formaldehyde exposure. Senichenk ova and Chebotar (1996) reported increased 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-462 fetal anomalies in fetuses from iron-deficient preg nant mice after formaldehyde exposure 1 compared with anemic mice that had not been exposed to formaldehyde. Forced iron reduction 2 (induced by addition of bipyridyl treatment in preg nant mice) in utero increased the overall 3 incidence of fetal anomalies when paired with forma ldehyde exposure (Senichenkova and 4 Chebotar, 1996). The findings are difficult to eva luate due to poor reporting and have not been 5 substantiated by other laboratories. 6

7

4.6.4. Gender Differences 8 Males and females can differ greatly in body compos ition, organ function, and many 9 other physiological parameters that may influence t he toxicokinetics of chemicals and their 10 metabolites in the body (Gochfeld, 2007; Gandhi et al., 2004). 11 The human epidemiology data set does not support an y specific gender susceptibilities 12 for noncancer effects due to formaldehyde exposure. In general, data suggest that nonpregnant 13 women, on a per kg body weight basis, may have slig htly lower air intake than men, which 14 would suggest that women may be less susceptible to inhaled pollutants like formaldehyde than 15 men, but this has not been investigated in the avai lable formaldehyde literature. 16 A few isolated reports have investigated potential gender differences in development of 17 nasal pharyngeal carcinomas following exposure to f ormaldehyde. One case-control study 18 identified a higher OR for sinonasal adenocarcinoma s in women (OR = 6.2 [95% CI: 2.2–19.7]) 19 compared with the OR observed in men (OR = 3.0 [95% CI: 1.5–5.7]) following exposure to 20 formaldehyde (Luce et al., 2002). However, the ove rall body of evidence remains scant. 21 There are a few reports concerning differential for maldehyde-induced effects on the male 22 and female reproductive systems. Özen et al. (2002 ), Sarsilmaz et al. (1999), and Woutersen et 23 al. (1987) reported reduced Leydig cell numbers in adult male rats exposed by inhalation. In 24 female mice, inhalation exposure to formaldehyde re sulted in endometrial hypoplasia and lack of 25 ovarian luteal tissue (Maronpot et al., 1986). The clinical significance of these effects in humans 26 is unknown, and due to limited data it is unclear w hether the female or male reproductive system 27 is more susceptible to perturbation by formaldehyde

. 28

29

4.6.5. Genetic Differences 30 There are some data for polymorphisms in humans tha t affect formaldehyde TK. As 31 discussed in Section 3.4, the primary metabolizing enzymes of formaldehyde are ALDH2 and 32 ADH3, with the latter enzyme considered more releva nt to low exposures. Polymorphisms in 33 ALDH2 have been shown to have implications in human risk assessment, specifically in regard 34 to acetaldehyde metabolism (Ginsberg et al., 2002). Teng et al. (2001) demonstrated the 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-463 importance of ALDH2 for formaldehyde metabolism in rat hepatocytes at fairly high 1 formaldehyde concentrations (2.5 mM and greater). Cheng et al. (2008) investigated the 2 relationship between occupational exposure to forma ldehyde and genetic polymorphisms of 3 ALDH2 and CYP2E1. There was a positive relationshi p between the concentration of formic 4 acid in the urine and ALDH2 genotypes ( χ 2 = 9.241, p < 0.05). Urinary formic acid 5 concentration may be affected by formaldehyde expos ure concentration and ALDH2 genotype 6 (Cheng et al., 2008) for individuals that have high exposure levels. Thus, although ALDH2 may 7 not be involved in formaldehyde metabolism if expos ure levels are low, polymorphisms of this 8 enzyme may lead to differences in response at highe r exposure levels. 9 Wu et al. (2007) looked for and identified two SNPs in ADH3 among a population of 10 Mexican asthmatic children 4 to 17 years of age. C arrying one or two copies of the minor allele 11 for one SNP resulted in a decreased RR of asthma (R R = 0.66–0.77). For the second SNP, 12 homozygotes for the minor allele had an RR of 1.6 f or asthma. The functional characteristics of 13 these SNPs are unknown. Studies evaluating whether any of the polymorphisms affect the 14 expression, regulation, stability, or activity of t he enzyme in vivo are lacking; therefore, the 15 relative susceptibility of individuals with differe nt polymorphisms cannot be characterized at this 16

time. 17 One study (Hedberg et al., 2001) identified three p olymorphisms in human ADH3 18 involving four base-pair substitutions in the promo ter region of which one (C → T) showed 19 reduced activity (~50–70% of control). Hedberg et a l. (2001) reported differences in allele 20 frequencies among Chinese, Spanish, and Swedish gro ups, consisting of Asian-Caucasian 21 differences and ethnic subgroups among Caucasians. Their results suggest that a small 22 percentage of Caucasians may have decreased ADH3 ex pression and thus, be more susceptible to 23 formaldehyde exposure. Additional studies to valid ate these findings have not been performed. 24 The relative activity level of these enzymes may a lso impact the metabolism of 25 formaldehyde. In pharmacokinetic studies, deletion of ADH3 increased the sensitivity of mice to 26 formaldehyde (Deltour et al., 1999) and was deleter ious to yeast (Achkor et al., 2003). These 27 results suggest that deficiencies in ADH3 may confe r an increased susceptibility to formaldehyde 28 toxicity (Teng et al., 2001). The importance of pr operly functioning enzymes also suggests that 29 genetic differences in ADH3 or ALDH2 may affect the response to formaldehyde exposure. 30 However, comparable human data are not available. 31 Race/ethnicity may be a surrogate for genetic diffe rences but racial or ethnic groups may 32 also reflect socioeconomic, and/or cultural factors that are distinct from genetics. Possible ethnic 33 differences may be related to genetic polymorphisms of enzymes ALDH2 and ADH3, relevant 34 for formaldehyde metabolism. ALDH2 variants, prese nt primarily in East Asians, are known to 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-464 have protective effects against alcoholism but were not found in the people of Indo-Trinidadian 1 descent (Moore et al., 2007) or in American Indians or Alaska natives (Ehlers, 2007). However, 2

there is no direct evidence to associate these vari ants to differential susceptibility to 3 formaldehyde exposure, nor is there direct evidence to associate these ethnic groups specifically 4 with differential susceptibility to formaldehyde. Further, no studies have specifically assessed 5 ethnic variability in responses to formaldehyde. 6 There are complex pathways through which genetic po lymorphisms in ADH3 can 7 potentially affect differential susceptibility to f ormaldehyde. Firstly, ADH3 is central to the 8 metabolism of formaldehyde. However, ADH3 itself ma y indirectly contribute to the adverse 9 effects of formaldehyde on pulmonary physiology (Th ompson et al., 2009; Staab et al., 2008a, b; 10 Thompson and Grafström, 2008). Exposure to formalde hyde is itself thought to alter the activity 11 of ADH3 resulting in the perturbation of critical m etabolic pathways. ADH3 participates in the 12 oxidation of retinol and long-chain primary alcohol s, as well as the reduction of S13 nitrosoglutathione (GSNO). The activity of ADH3 tow ard some of these substrates has been 14 shown to be significantly increased in the presence of formaldehyde. ADH3 has recently also 15 been shown to contribute to NO signaling through it s dual role in metabolizing GSNO, an 16 endogenous bronchodilator and reservoir of NO (Staa b et al., 2008a; Hess et al., 2005; Jensen et 17 al. 1998). Through its regulatory function on GSNO, ADH3 may thus play a central role in 18 regulating bronchial tone allergen-induced hyperres ponsiveness (Gerard, 2005; Que et al., 2005). 19 As concluded by California Environmental Protection Agency (CalEPA) (2008), “the 20 dysregulation of NO by formaldehyde [in this manner ] helps to explain the variety and 21 variability in the toxic manifestations following f ormaldehyde inhalation.” 22

23 4.6.6. Co-Exposures 24

4.6.6.1. Cumulative Risk 25 When considering health risks, it is important to c onsider the impact of co-exposures to 26 other agents that may interact with the chemical un der evaluation. Co-exposure to other 27 pollutants, particularly those that produce some of the same metabolites and similar health 28 effects as formaldehyde, is likely to occur in both occupational and nonoccupational settings. 29 Due to effects on metabolic enzymes (inducing and/o r inhibition) as well as direct effects 30 on organ system function, co-exposures may alter th e way in which formaldehyde is metabolized 31 and cleared from the body. Inhibition or induction of the enzymes responsible for metabolism of 32 chemicals may alter susceptibility to toxicity (Las h and Parker, 2001; IARC, 1995; U.S. EPA, 33 1985a). Smokers may be at increased risk for effec ts of formaldehyde exposure, because 34 formaldehyde is one of the components of cigarette smoke and is likely to heighten the point-of- 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-465 entry effect when combined with occupational or res idential exposures to inhaled formaldehyde. 1 However, no evidence is available to evaluate the p otential aggregate effects. 2

3 4.6.6.2. Aggregate Exposure 4 In addition, multiple routes of exposure to a singl e agent may increase the cumulative 5 risk by increasing the overall body burden of the c hemical. A human aggregate exposure model 6 developed by McKone and Daniels (1991) incorporated likely exposures from air, water, and soil 7 media through inhalation, ingestion, and dermal con tact. The authors hypothesized that the 8 aggregate exposure could be age-dependent but did n ot present any data for persons of differing 9 life stages. The role of multiple exposures on dif ferent genders, genetic susceptibility, or altered 10 health and nutrition status has not been investigat ed. The available database regarding the 11 potential for multiple routes of exposure (or aggre gate exposure) formaldehyde is limited. 12 Guseva (1972) specifically assessed the reproductiv e and developmental effects caused 13 by co-exposure to formaldehyde via both inhalation (0.25 mg/m 3 ) and ingestion (0.01 mg/L) 14 routes in male rats. The authors reported reduced nucleic acid levels in testes to 88 and 92% of 15 controls, which suggests a possible toxic gonadotro pic effect. The ability of male rats (receiving 16 combined exposure to formaldehyde at a low concentr ation level for a long period of time) to 17 reproduce was preserved since all the cohabited fem ales were impregnated. The number and 18 weight of the fetuses and newborn rat pups in the e xperimental co-exposure groups did not differ 19 substantially from those figures observed in the co ntrol group. No developmental defects or 20 anomalies were observed in the offspring for up to 1 month postnatally. Thus, at low exposures, 21 the reproductive effects due to combined ingestion and inhalation exposure are unknown. 22

23

4.6.7. Uncertainties of Database 24 There is a need to better characterize the implicat ions of formaldehyde exposures to 25 susceptible populations. A number of areas where t he database is currently insufficient are 26

identified below. 27

28

4.6.7.1. Uncertainties of Exposure 29 Although information exists on early life exposure to formaldehyde, a number of 30 uncertainties regarding children’s susceptibility r emain. First, inhalation is believed to be of 31 most concern for formaldehyde, since formaldehyde v apors are released from insulation or from 32 ambient sources of formaldehyde, including secondar y production from other pollutants involved 33 in photo-oxidant reactions. Any additional pathway s of exposure for children have not been 34 characterized. Since formaldehyde is nearly ubiqui tous in the environment, it is difficult to 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-466 quantify the total exposure. Second, children have different respiratory, metabolic, and activity 1 rates compared with healthy adults, potentially inf luencing ADME and target tissue exposure to 2 formaldehyde. However, studies to identify the spe cific changes in absorption of formaldehyde 3 and its metabolites across developmental stages and across organs have not been performed. In 4 addition, exposure prenatally may be altered based on whether formaldehyde or its metabolites 5 pass through the placenta, but placental transfer d ata are not available. Third, no quantitative 6 models have been developed to characterize these di fferences for formaldehyde. Formaldehyde7 specific PBPK models and their validation will aid in understanding the uncertainties associated 8 with formaldehyde exposure in children. 9 Given the large proportion of time that most indivi duals in the U.S. spend indoors, 10 exposure scenarios where indoor concentrations to f ormaldehyde are high (e.g., in homes or in 11 trailers; see section 2.3.1) may play a significant role and may be of particular concern to the 12 elderly or health-impaired individuals who spend re latively more time at home. Further 13 evaluation of the effects of co-exposures and pathw ays of exposure and aggregate risk is needed. 14 An estimate of the multiple exposure pathways is ne eded to know where along the dose-response 15 curve to place an incremental exposure to formaldeh yde. 16

17

4.6.7.2. Uncertainties of Effect 18 Studies specifically designed to evaluate effects a fter early and later life stage exposure 19 are needed in order to more fully characterize pote ntial life-stage-related differences in 20 formaldehyde toxicity, including the defining of cr itical windows during development. For 21 example, life-stage-specific neurotoxic and pulmona ry effects, particularly in the developing 22 fetus, need further evaluation. The preconceptiona l period may be a critical window for FA 23 exposure and reproductive and developmental effects , based on rodent studies of reproductive, 24 embryonic and gamete effects. Data specific to the carcinogenic effects of formaldehyde 25 exposure during early life stages do not exist. Th e reduction in fertility seen in some studies 26 (Gray and Wu, 2000; Taskinen et al., 1999; Hakim et al., 1995) is not adequately described and a 27 well-established MOA has not been identified, but s ome have been hypothesized including 28 altered sperm quality (Özen et al., 2002; Sarsilmaz et al., 1999; Woutersen et al., 1987). Further, 29 spontaneous abortion/fetal loss occurring early in gestation, prior to maternal knowledge of the 30 pregnancy, can lead to misclassification of the eff ect as infertility (see Sections 4.1.1.7 and 31 4.2.1.7). 32 More research is needed to clarify the role of gene tic polymorphisms in formaldehyde 33 metabolism. Similarly, data gaps pertaining to gen der differences remain. A potential impact of 34 nutritional status and iron deficiency on formaldeh yde toxicity needs further investigation. 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-467 A fair body of evidence suggests that asthmatics ar e more susceptible to formaldehyde exposure 1 than the general population, however the mechanism of action for this increased susceptibility is 2

unknown. 3 In the studies discussed above, there are a number of examples of studies that assessed 4 multiple susceptibility factors that are worth noti ng. For example, the Krzyzanowski et al. 5 (1990) study reported asthma and chronic bronchitis cases for two interacting potential 6 susceptible groups, in children and those with high exposure (due to living in homes with 7 formaldehyde levels that were higher than 60 ppb). Similarly, the Garrett et al. (1999) study 8 assessed the same two interacting potential suscept ible groups. 9 The study of Senichenkova and Chebotar (1996) asses sed developmental effects in 10

mouse fetuses after in utero iron-deficiency and FA exposure. Thus, the study findings must be 11 considered in light of possible interactions betwee n life stage exposure differences and 12

nutritional status differences. 13

Studies to understand the nature of the interaction s between the various susceptibility 14 factors for FA have not been performed. 15

16 4.6.8. Summary of Potential Susceptibility 17 There is some evidence to demonstrate susceptibilit y for various populations exposed to 18 formaldehyde. Available data are summarized in Tab le 4-96 where FA susceptibility factors are 19 presented by those with data for increased FA susce ptibility and those with data for differences 20 but with an unknown impact on FA susceptibility. 21 Exposure to FA during early developmental and later life stages may be of concern. 22 However, human exposure to the developing fetus is unknown since it is not known whether 23 formaldehyde or one of its metabolites crosses the placenta. However, there is very limited life24 stage-specific information regarding the TK of form aldehyde. Life-stage-specific TK has not 25 been characterized, and, thus, no PBPK models exist to effectively evaluate the risk to early life 26 stages. Children may be more susceptible to noncan cer health effects as a result of inhalation 27 exposure to formaldehyde due to increased respirato ry rates. There are no studies to evaluate 28 whether formaldehyde exposure in early life (e.g., pregnancy) is associated with an increased 29

risk of childhood cancer. 30 The weight of evidence supports a plausible associa tion between formaldehyde exposure 31 and aggravated asthmatic responses in humans and th is association is corroborated by limited 32 evidence from animal studies. Formaldehyde does no t appear to directly induce airway 33 hyperreactivity but may sensitize airways to subseq uent exposures. One issue in interpreting the 34 available studies that assessed the relationship be tween asthma and FA could not distinguish 35This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-468 between the cases of asthma that were due to earlie r FA exposure vs. those without a direct link 1 to FA exposure. 2 No direct link exists between formaldehyde exposure and differential susceptibility in 3 different ethnic groups, although genetic polymorph isms in the enzymes involved with 4 formaldehyde metabolism, ADH3 and ALDH2, provide so me support for differential 5 susceptibility to alcoholism in a number of ethnic groups. The evidence for differential gender 6 responses to formaldehyde exposure is equivocal. C o-exposures may result in altered 7 metabolism and clearance, but there is no evidence that co-exposures are a critical part of 8 formaldehyde-mediated differential susceptibility. 9 Thus, given the available data, increased susceptib ility to adverse effects of 10 formaldehyde is most strongly supported for three p opulations: 1) Preconception and perinatal 11 exposure based on reproductive and developmental ef fects; 2) children, whose exposure may be 12 higher by virtue of their increased activity level and respiratory rate; and 3) asthmatics who may 13 exhibit exacerbation of response to formaldehyde. 14This document is a draft for review purposes only a nd does not constitute Agency policy. DRAFT—DO NOT CITE OR QUOTE 4-469 Table 4-96. Available evidence for susceptibility factors of concern for 1 formaldehyde exposure 2 3

Factor Evidence that factors increase susceptibility to FA Evidence that factors show differences but unknown impact on susceptibility Life Stage

 Preconception

 Prenatal

 Postnatal Developmental effects reported suggesting that critical windows of exposure may be relevant:

 Reproductive outcomes (Taskinen et al., 1999; Maronpot et al., 1986)

 Embryo effects (Kitaev et al., 1984)

 Structural- and functional developmental outcomes (Martin, 1990; Saillenfait et al., 1989; Sheveleva, 1971; Seninchenkova, 1991)

 Lung function outcome (Krzyzanowski et al., 1990; Rumchev et al., 2002; Garrett et al., 1999)

 Developmental neurotoxicity (Weiler and Apfelbach, 1992)  Possible life stage level differences in some enzymes involved in FA metabolism (Harris et al., 2003; Ginsberg et al., 2004; Westerlund et al., 2005; Benedetti et al., 2007)

 Mixed reports of associations between prenatal exposure and developmental outcomes in human studies (positive association: Gražulevi č iene et al., 1998)

 Possible life stage level differences in some enzymes involved in FA metabolism (e.g., ↓ ADH expression over first 5 months; Ginsberg et al., 2004)

 Developmental neurotoxicity (Sarsilmaz et al., 2007; Aslan et al., 2006; Songur et al., 2008) Disease Status  Bronchial asthma (Lemiere et al., 1995; Burge et al., 1985; Hendrick et al., 1982; Hendrick and Lane, 1977, 1975)

 Increased airway resistance and bronchial constriction (Nielson et al., 1999; Swiecichowski et al., 1993; Biagini et al., 1989; Amdur et al., 1960)  Mixed results for forced expiratory volume (FEV1) measures affected by FA exposure in asthmatics (Casset et al., 2006; Green et al., 1987; Ezratty et al., 2007; Frigas et al., 1984)

Nutritional Status/Diet  Iron-deficiency in utero (Senichenkova and Chebotar, 1996). Genetics

 Polymorphisms  For high FA exposure: Urinary formic acid levels affected by ALDH2 genotype (Cheng et al., 2008)

 In mice, ADH3 increased sensitivity to FA (Achkor et al., 2003)  Differences among ADH3 alleles and asthma outcome (Wu et al., 2007)

 Differences among ethnic groups in ADH3 alleles (Hedberg et al., 2001)

Gender  Gender differences in incidence of nasopharyngeal carcinoma following FA exposure (Luce et al., 2002) 4 5 

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