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 www.epa.gov/iris

TOXICOLOGICAL REVIEW OF

FORMALDEHYDE

INHALATION TOXICITY

(CAS No. 50-00-0) 

 

In Support of Summary Information on the 

Integrated Risk Information System (IRIS)

VOLUME 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 review under applicable information quality 

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

be construed to represent any Agency determination or policy. It is being circulated for review 

of its technical accuracy and science policy implications.

U.S. Environmental Protection Agency 

Washington, DC 

1 
This document is a draft for review purposes only and does not constitute Agency policy.

II-ii DRAFT—DO NOT CITE OR QUOTE

1 DISCLAIMER

2

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

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

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

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

7 names or commercial products does not constitute endorsement or recommendation for use. 

8

9
This document is a draft for review purposes only and does not constitute Agency policy.

II-iii DRAFT—DO NOT CITE OR QUOTE

1 CONTENTS—TOXICOLOGICAL REVIEW OF FORMALDEHYDE

2 (CAS No. 50-00-0)

3

4

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

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

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

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

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

VOLUME I

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

3.1.2.6. Levels of Endogenous Formaldehyde in Animal and Human 

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

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

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

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

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

3.2.3.1. Formaldehyde Uptake Can be Affected by Effects at the 

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

3.2.3.2. Variability in the Nasal Dosimetry of Formaldehyde in 

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

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

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

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

3.3.2.1. Disposition of Formaldehyde: Differentiating covalent 

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

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CONTENTS (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

VOLUME II

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

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

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

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

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

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

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

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

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

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

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

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

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CONTENTS (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

4.4. SYNTHESIS AND MAJOR EVALUATION OF NONCARCINOGENIC

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

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

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

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

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

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

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

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

II-vi DRAFT—DO NOT CITE OR QUOTE

CONTENTS (continued)

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

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

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

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

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

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

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

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

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

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

4.4.9.4. Functional Development Outcomes (Developmental 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

4.5.2.7. Supporting Evidence from Animal Bio-Assays for 

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

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

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

4.5.3.2. Mode of Action Evaluation for Upper Respiratory Tract 

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

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

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

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

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

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

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

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

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

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

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

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

II-vii DRAFT—DO NOT CITE OR QUOTE

CONTENTS (continued)

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

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

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

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

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

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

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

VOLUME III

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

5.2. QUANTITATIVE CANCER ASSESSMENT BASED ON THE NATIONAL 

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

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

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

5.2.2.1. Exposure-Response Modeling of the National Cancer 

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

5.2.2.2. Prediction of Lifetime Extra Risk of Nasopharyngeal Cancer 

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

5.2.2.3. Prediction of Lifetime Extra Risk of Nasopharyngeal Cancer 

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

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

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

5.2.3.1. Exposure-Response Modeling of the National Cancer 

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

II-viii DRAFT—DO NOT CITE OR QUOTE

CONTENTS (continued)

5.2.3.2. Prediction of Lifetime Extra Risks for Hodgkin Lymphoma 

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

5.2.3.3. Prediction of Lifetime Extra Risks for Hodgkin Lymphoma 

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

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

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

5.3. DOSE-RESPONSE MODELING OF RISK OF SQUAMOUS CELL 

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

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

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

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

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

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

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

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

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

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

5.3.4.1. Benchmark Dose Derived from BBDR Rat Model and Flux 

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

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

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

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

5.4. CONCLUSIONS FROM THE QUANTITATIVE ASSESSMENT OF 

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

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

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

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

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

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

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND 

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

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

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

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

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

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

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

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

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

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

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

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

II-ix DRAFT—DO NOT CITE OR QUOTE

CONTENTS (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

VOLUME IV

APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

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

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

VARIABILITY IN REACTIVE GAS UPTAKE IN A SMALL SAMPLE 

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

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

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

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

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

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

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

APPENDIX F: SENSITIVITY ANALYSIS OF BBDR MODEL FOR FORMALDEHYDE 

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

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CONTENTS (continued)

APPENDIX G: EVALUATION OF THE CANCER DOSE-RESPONSE MODELING 

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

APPENDIX H: EXPERT PANEL CONSULTATION ON QUANTITATIVE 

EVALUATION OF ANIMAL TOXICOLOGY DATA FOR 

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

II-xi DRAFT—DO NOT CITE OR QUOTE

LIST OF TABLES

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

II-xii DRAFT—DO NOT CITE OR QUOTE

LIST OF TABLES (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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LIST OF TABLES (continued)

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

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

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

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

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

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

cerebral cortex of male Wistar rats exposed to formaldehyde via inhalation 

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

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

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

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

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

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

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

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

maxilloturbinates of female Sprague-Dawley rats exposed to inhaled 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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LIST OF TABLES (continued)

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

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

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

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

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

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

regional site location of squamous cell carcinomas in formaldehyde 

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

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

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

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

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

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

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

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

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

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

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

reported in male and female Sprague-Dawley rats exposed to 

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

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

female Sprague-Dawley rats exposed to formaldehyde in drinking water 

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

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

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

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

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

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

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

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

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

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

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LIST OF TABLES (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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LIST OF TABLES (continued)

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

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

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

abnormal sperm after oral administration of formaldehyde to male 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

II-xvii DRAFT—DO NOT CITE OR QUOTE

LIST OF TABLES (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

incidence of all LHP cancers in formaldehyde-exposed populations 

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

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

disease groupings within the broad category of all lymphohematopoetic 

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

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

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

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

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

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

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

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

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

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

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

II-xviii DRAFT—DO NOT CITE OR QUOTE

LIST OF TABLES (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

of continuous exposure to formaldehyde ..............................................................5-91
This document is a draft for review purposes only and does not constitute Agency policy.

II-xix DRAFT—DO NOT CITE OR QUOTE

LIST OF TABLES (continued)

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

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

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

mortality from formaldehyde exposure based on Beane Freeman et al. 

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

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

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

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

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

lymphoma incidence from formaldehyde exposure, based on Beane 

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

for specified extra cancer risk levels for selected formaldehyde-related 

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

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

animal data.............................................................................................................6-42
This document is a draft for review purposes only and does not constitute Agency policy.

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LIST OF FIGURES

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

model compared with pressure drop measurements made in two hollow 

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

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

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

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

“painting” 100% formalin for 20 minutes..........................................................4-20
This document is a draft for review purposes only and does not constitute Agency policy.

II-xxi DRAFT—DO NOT CITE OR QUOTE

LIST OF FIGURES (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

following formaldehyde exposure in guinea pigs after OVA sensitization and 

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

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

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

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

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

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

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

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

permeability in the trachea and bronchi of male Wistar rats............................4-239
This document is a draft for review purposes only and does not constitute Agency policy.

II-xxii DRAFT—DO NOT CITE OR QUOTE

LIST OF FIGURES (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

II-xxiii DRAFT—DO NOT CITE OR QUOTE

LIST OF FIGURES (continued)

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

exposure index category with 8-hour time weighted average formaldehyde 

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

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

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

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

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

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

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

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

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

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

II-xxiv DRAFT—DO NOT CITE OR QUOTE

LIST OF FIGURES (continued)

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

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

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

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

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

components in the CIIT model.........................................................................5-109

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

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

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

characterized by partitioning the nasal surface by formaldehyde flux to 

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

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

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

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

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

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

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

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

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

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

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

II-xxv DRAFT—DO NOT CITE OR QUOTE

LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygienists

ADAF age-dependent adjustment factors

ADH alcohol dehydrogenase

ADS anterior dorsal septum

AIC Akaike Information Criterion

AIE average intensity of exposure

AIHA American Industrial Hygiene Association

ALB albumin

ALDH aldehyde dehydrogenase

ALL acute lymphocytic leukemia

ALM anterior lateral meatus

ALP alkaline phosphatase

ALS amyotrophic lateral sclerosis

ALT alanine aminotransferase

AML acute myelogenous leukemia

AMM anterior medial maxilloturbinate

AMPase adenosine monophosphatase

AMS anterior medial septum

ANAE alpha-naphthylacetate esterase

ANOVA analysis of variance

APA American Psychiatric Association

ARB Air Resources Board

AST aspartate aminotransferase

ATCM airborne toxic control measure

ATP adenosine triphosphate

ATPase adenosine triphosphatase

ATS American Thoracic Society

ATSDR Agency for Toxic Substances and Disease Registry

AUC area under the curve

BAL bronchoalveolar lavage

BALT bronchus associated lymphoid tissue

BBDR biologically based dose response

BC bronchial construction

BCME bis(chloromethyl)ether

BDNF brain-derived neurotrophic factor

BEIR biologic effects of ionizing radiation

BfR German Federal Institute for Risk Assessment

BHR bronchial hyperresponsiveness

BMC benchmark concentration

BMCL 95% lower bound on the benchmark concentration

BMCR binuclated micronucleated cell ratefluoresce

BMD benchmark dose

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

II-xxvi DRAFT—DO NOT CITE OR QUOTE

LIST OF ABBREVIATIONS AND ACRONYMS (continued)

BMR benchmark response

BN Brown-Norway

BrdU bromodeoxyuridine

BUN blood urea nitrogen

BW body weight

CA chromosomal aberrations

CalEPA California Environmental Protection Agency

CAP College of American Pathologists

CASRN Chemical Abstracts Service Registry Number

CAT catalase

CBMA cytokinesis-blocked micronucleus assay

CBMN cytokinesis-blocked micronucleus

CDC U.S. Centers for Disease Control and Prevention

CDHS California Department of Health Services

CFD computational fluid dynamics

CGM clonal growth model

CHO Chinese hamster ovary

CI confidence interval

CIIT Chemical Industry Institute of Toxicology

CLL chronic lymphocytic leukemia

CML chronic myelogenous leukemia

CNS central nervous system

CO2 carbon dioxide

COEHHA California Office of Environmental Health Hazard Assessment

CREB cyclic AMP responsive element binding proteins

CS conditioned stimulus

C × t concentration times time

DA Daltons

DAF dosimetric adjustment factor

DDX DNA-DNA cross-links

DEI daily exposure index

DEN diethylnitrosamine

Der f common dust mite allergen

DMG dimethylglycine

DMGDH dimethylglycine dehydrogenase

DNA deoxyribonucleic acid

DOPAC 3,4-dihydroxyphenylacetic acid

DPC / DPX DNA-protein cross-links 

EBV Epstein-Barr virus

EC effective concentration

ED effective dose

EHC Environmental Health Committee

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

II-xxvii DRAFT—DO NOT CITE OR QUOTE

LIST OF ABBREVIATIONS AND ACRONYMS (continued)

EPA U.S. Environmental Protection Agency

ERPG emergency response planning guideline

ET ethmoid turbinates

FALDH formaldehyde dehydrogenase

FDA U.S. Food and Drug Administration

FDR fecundability density ratio

FEF forced expiratory flow

FEMA Federal Emergency Management Agency

FEV1 forced expiratory volume in 1 second

FISH fluorescent in situ hybridization

FSH follicle-stimulating hormone

FVC forced vital capacity

GALT gut-associated lymphoid tissue

GC-MS gas chromatography-mass spectrometry

GD gestation day

GI gastrointestinal

GO gene ontology

G6PDH glucose-6-phosphate dehydrogenase

GPX glutathione peroxidase

GR glutathione reductase

GM-CSF granulocyte macrophage-colony-stimulating factor

GSH reduced glutathione

GSNO S-nitrosoglutathione

GST glutathione S-transferase

HAP hazardous air pollutant

Hb hemoglobin

HCl hydrochloric acid

HCT hematocrit

HEC human equivalent concentration

5-HIAA 5-hydroxyindoleacetic acid

hm hydroxymethyl

HMGSH S-hydroxymethylglutathione

HPA hypothalamic-pituitary adrenal

HPG hypothalamo-pituitary-gonadal

HPLC high-performance liquid chromatography

HPRT hypoxanthine-guanine phosphoribosyl transferase

HR high responders

HSA human serum albumin

HSDB Hazardous Substances Data Bank

Hsp heat shock protein

HWE healthy worker effect

I cell initiated cell

IARC International Agency for Research on Cancer
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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

ICD International Classification of Diseases

IF interfacial

IFN interferon

Ig immunoglobulin

IL interleukin

I.P. intraperitoneal

IPCS International Programme on Chemical Safety

IRIS Integrated Risk Information System

Km Michaels-Menton constant

KM Kaplan-Meier

LD50 median lethal dose

LDH lactate dehydrogenase

LEC 95% lower bound on the effective concentration

LED 95% lower bound on the effective dose

LHP lymphohematopoietic

LI labeling index

LM Listeria monocytogenes

LMS linearized multistage

LLNA local lymph node assay

LOAEL lowest-observed-adverse-effect level

LPS lipopolysaccharide

LR low responders

LRT lower respiratory tract

MA methylamine

MALT mucus-associated lymph tissues

MCH mean corpuscular hemoglobin

MCHC mean corpuscular hemoglobin concentration

MCS multiple chemical sensitivity

MCV mean corpuscular volume

MDA malondialdehyde

MEF maximal expiratory flow

ML myeloid leukemia

MLE maximum likelihood estimate

MMS methyl methane sulfonate

MMT medial maxilloturbinate

MN micronucleus, micronuclei

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

MOA mode of action

MoDC monocyte-derived dendritic cell

MP macrophage

MPD multistage polynomial degree

MPS mononuclear phagocyte system

MRL minimum risk level
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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

mRNA messenger ribonucleic acid

MVE-2 Murray Valley encephalitis virus

MVK Moolgavkar, Venzon, and Knudson

N cell normal cell

NaCl sodium choride

NAD+ nicotinamide adenine dinucleotide

NADH reduced nicotinamide adenine dinucleotide

NALT nasally associated lymphoid tissue

NATA National-Scale Air Toxics Assessment

NCEA National Center for Environmental Assessment

NCHS National Center for Health Statistics

NCI National Cancer Institute

NEG Nordic Expert Group

NER nucleotide excision repair

NGF nerve growth factor

NHL non-Hodgkin’s lymphoma

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

Management Council of Australia and New Zealand

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

N6

-hmdA N6-hydroxymethyldeoxyadenosine

N4-hmdC N4-hydroxymethylcytidine

N2

-hmdG N2-hydroxymethyldeoxyguanosine

NICNAS National Industrial Chemicals Notification and Assessment Scheme

NIOSH National Institute for Occupational Safety and Health

NLM National Library of Medicine

NMDA N-methyl-D-aspartate

NO nitric oxide

NOAEL no-observed-adverse-effect level

NPC nasopharyngeal cancer

NRBA neutrophil respiratory burst activity

NRC National Research Council

NTP National Toxicology Program

OR odds ratio

OSHA Occupational Safety and Health Administration

OTS Office of Toxic Substances

OVA ovalbumin

PBPK physiologically based pharmacokinetic

PC Philadelphia chromosome

PCA passive cutaneous anaphylaxis

PCMR proportionate cancer mortality ratio

PCNA proliferating cell nuclear antigen

PCR polymerase chain reaction

PCV packed cell volume
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LIST OF ABBREVIATIONS AND ACRONYMS (continued)

PECAM platelet endothelial cell adhesion molecule

PEF peak expiratory flow

PEFR peak expiratory flow rates

PEL permissible exposure limit

PFC plaque-forming cell

PG periglomerular

PHA phytohemagglutinin

PLA2 phospholipase A2

PI phagocytic index

PLM posterior lateral meatus

PMA phorbol 12-myristate 13-acetate

PMR proportionate mortality ratio

PMS posterior medial septum

PND postnatal day

POD point of departure

POE portal of entry

PTZ pentilenetetrazole

PUFA polyunsaturated fatty acids

PWULLI population weighted unit length labeling index

RA reflex apnea

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

RB reflex bradypnea

RBC red blood cells

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

REL recommended exposure limit

RfC reference concentration

RfD reference dose

RGD regional gas dose

RGDR regional gas dose ratio

RR relative risk

RT reverse transcriptase

SAB Science Advisory Board

SCC squamous cell carcinoma

SCE sister chromatid exchange

SCG sodium cromoglycate

SD standard deviation

SDH succinate dehydrogenase; sarcosine dehydrogenase

SEER Surveillance, Epidemiology, and End Results

SEM standard error of the mean

SEN sensitizer

SH sulfhydryl

SHE Syrian hamster embryo

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

SMR standardized mortality ratio

SNP single nucleotide polymorphism

SOD superoxide dismutase

SOMedA N6-sulfomethyldeoxyadenosine

Sp1 specificity protein

SPIR standardized proportionate incidence ratio

SSAO semicarbozole-sensitive amine oxidase

SSB single strand breaks

STEL short-term exposure limit

TBA tumor bearing animal

TH T-lymphocyte helper

THF tetrahydrofolate

TK toxicokinetics

TL tail length

TLV threshold limit value

TNF tumor necrosis factor

TP total protein

TRI Toxic Release Inventory

TRPV transient receptor potential vanilloid

TWA time-weighted average

TZCA thiazolidine-4-carboxylate

UCL upper confidence limit

UDS unscheduled DNA synthesis

UF uncertainty factor

UFFI urea formaldehyde foam insulation

ULLI unit length labeling index

URT upper respiratory tract

USDA U.S. Department of Agriculture

VC vital capacity

VOC volatile organic compound

WBC white blood cell

WDS wet dog shake

WHO World Health Organization

WHOROE World Health Organization Regional Office for Europe
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1 4. HAZARD CHARACTERIZATION

2

3

4 4.1. HUMAN STUDIES

5 This chapter discusses epidemiologic studies of site-specific cancers and other adverse 

6 health effects that may be caused by exposure to formaldehyde. The primary focus is on the 

7 literature describing inhalation exposure and its potential carcinogenic and noncarcinogenic 

8 health risks. In addition, oral, dermal, and ocular exposures to formaldehyde are discussed.

9 The noncancer health effects section is organized by endpoint, beginning with sensory 

10 irritation and followed by pulmonary function, asthma, respiratory tract pathology, immunologic 

11 responses, neurological and behavioral responses, and, finally, developmental and reproductive 

12 outcomes.

13 The carcinogenicity section is divided into two parts, respiratory tract and non-respiratory 

14 tract cancers. The first part discusses site-specific cancers that are chiefly located in the 

15 respiratory tract where direct contact with formaldehyde occurs: nasopharyngeal cancers (NPCs), 

16 nasal and paranasal cancers, other respiratory tract cancers, and lung cancers. The second part 

17 on non-respiratory tract cancer discusses those cancers at other sites with more distant exposure 

18 to formaldehyde than respiratory epithelium—mainly, lymphohematopoietic (LHP) cancer, brain 

19 and central nervous system (CNS) cancer, pancreatic cancer, and cancer at other sites.

20

21 4.1.1. Noncancer Health Effects

22 4.1.1.1. Sensory Irritation (Eye, Nose, Throat Irritation)

23 As a reactive gas, formaldehyde is a sensory irritant. Sensory irritation of the eyes and 

24 respiratory tract by formaldehyde has been observed consistently in clinical and epidemiologic 

25 studies in residential and occupational populations. Binding to sensory nerves at the portal of 

26 entry (POE) results in direct sensory responses (e.g., detection of odor and tissue irritation) as 

27 well as reflex responses to the sensory irritation and neurogenic sensitization. Reflex responses 

28 result from CNS stimulation by the afferent sensory signals and include lacrimation, coughing, 

29 sneezing, and bronchial constriction (BC). An additional reflex seen in rodents is reflex 

30 bradypnea (RB) (also known as reflex apnea [RA]). Formaldehyde-induced sensory irritation 

31 may be evident after acute exposures as well as in chronically exposed individuals. 

32 Formaldehyde-induced neurogenic sensitization and atopy may result in lifelong health effects 

33 from short-term or transient exposures. For this discussion, sensory irritation will include both 

34 direct sensory response to formaldehyde exposure and reflex responses (lacrimation, coughing, 

35 sneezing, RB, and BC, and sensitization. 
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1 Eye, nose, and throat irritation in response to formaldehyde inhalation exposure is well 

2 documented (Doty et al., 2004). Broadly, studies examining these endpoints are either controlled 

3 chamber studies with a defined population (e.g., healthy volunteers or sensitive individuals), 

4 worker/student studies, or population (e.g., residential) studies. Chamber studies, by design, are 

5 acute studies, although some researchers have investigated repeated exposures. Occupational, 

6 student, and residential exposures are generally longer duration, although there is variability in 

7 exposure and duration among subjects. Endpoints include both local effects and reflex effects of 

8 sensory irritation. The endpoints for assessing irritation include self-reporting of adverse 

9 symptoms (e.g., pain, burning, itching) as well as objective measures of irritation (e.g., eye-blink 

10 counts, lacrimation) (Doty et al., 2004). The following review focuses on eye, nose, and throat 

11 irritation but studies have documented other types of irritation, including dermal irritation 

12 eczema and dermatitis. 

13

14 4.1.1.1.1. Epidemiologic literature. A wide variety of epidemiologic studies have assessed the 

15 potential effects of exposure to formaldehyde on endpoints, indicating sensory irritation of the 

16 eye, nose, and throat. These studies generally include three different types of exposure 

17 populations: (1) Residents and visitors exposed to formaldehyde in homes and mobile buildings, 

18 where formaldehyde is present from various sources, including building components, furniture 

19 and home furnishings, heating and cooking combustion as well as active and passive smoking; 

20 (2) various occupational exposures from industrial processes related to wood products, furniture

21 making, and formaldehyde-based resins; and (3) anatomy students who are exposed under well22 defined conditions during academic courses where they are examining formaldehyde-preserved 

23 cadavers.

24

25 4.1.1.1.1.1. Residential epidemiology. Among the residential epidemiology studies of 

26 formaldehyde effects on sensory irritation, one of the strongest studies based on study design, 

27 execution, analysis, and sample size was the observational study undertaken by Ritchie and 

28 Lehnen (1987). In this cross-sectional study of nearly 2,000 Minnesota residents living in 392 

29 mobile and 494 conventional homes, personal data and formaldehyde samples were collected 

30 from residents that had responded to an offer by the state health department to test homes for 

31 formaldehyde. Technicians administered a symptom questionnaire to participating residents at 

32 the time of formaldehyde sample collection. Residents were asked to close doors and windows 

33 of their homes for 12 hours before testing was conducted, and a standardized collection protocol 

34 was used for both sample collection and analysis. Measurements of formaldehyde exposure 

35 were taken from two rooms of the home, usually the bedroom and living room, and were kept 
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1 refrigerated until analysis. Respondents were not aware of the results of the formaldehyde 

2 analyses in their homes at the time they responded to the symptom questionnaire. The results 

3 from Ritchie and Lehnen (1987) provide a clear dose-response relationship in the percentage of 

4 residential occupants reporting eye, nose, and throat irritation. Specifically, eye irritation 

5 responses increase from 1–2% in homes with formaldehyde concentrations lower than 0.1 ppm 

6 to 12–32% in homes with formaldehyde concentrations ranging from 0.1 to 0.3 ppm with 86–

7 93% of residents reporting . These effects were found in the same concentration range for 

8 people living in either mobile (n = 851) or conventional (n = 1,156) homes. Similar percentages 

9 were found for nose/throat irritation. Reports of irritation were reported for smokers, passive 

10 smokers, and nonsmokers with higher percentages of irritation among smokers, followed by 

11 passive smokers and then nonsmokers. While the participants in this study were self-selected 

12 and not a random residential sample, a clear concentration response was observed, and, even if 

13 participants sought testing because they suspected that they were being exposed to formaldehyde 

14 in their homes, they could not know the measured concentration of formaldehyde when reporting 

15 their irritation symptoms, so recall bias cannot explain the concentration response. Neither can 

16 confounding be an alternative explanation since the authors reported that formaldehyde was the 

17 most important explanatory variable for all the sensory irritation effects of the eye, nose, and 

18 throat.

19 The results of an adverse association of sensory irritation with formaldehyde reported by 

20 Ritchie and Lehnen (1987) are corroborated by Hanrahan et al. (1984) who conducted a cross21 sectional survey by using a random sample of mobile homes from mobile home parks in 

22 Wisconsin. Sixty-one teenage and adult residents participated. Health questionnaires were self23 administered by each occupant. Respondents were blinded to the results of their home 

24 formaldehyde vapor measurements, which were sampled from two rooms in the homes following 

25 instruction to close windows, refrain from smoking, and turn off gas appliances for 30 minutes 

26 prior to air sampling. Logistic regression analyses were used to ascertain potential symptom risk 

27 ratio dependency on each respondent’s age, smoking status, gender, and formaldehyde 

28 concentration measures in the home. Formaldehyde concentrations ranged from 0.1 ppm to 

29 0.8 ppm with a geometric mean of 0.16 ppm. Across this concentration range, a clear and 

30 statistically significant concentration-response relationship was reported in graphical form, 

31 controlling for age, gender, and smoking status. At 0.1 ppm, the regression model showed less 

32 than 5% predicted prevalence of burning eyes. At 0.2 ppm, the midpoint of the exposure 

33 category in Ritchie and Lehnen (1987) that was reported to be the lowest adverse effect level for 

34 eye irritation with 12–32% reporting eye irritation, the regression model of Hanrahan et al. 

35 (1984) showed approximately 17.5% predicted prevalence of burning eyes. The prevalence of 
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1 burning eyes rose linearly to approximately 65% prevalence at 0.5 ppm, with some diminishment 

2 in the rate of rise up to approximately 80% prevalence at 0.8 ppm. While only 65 out of 208 

3 randomly selected homes volunteered to complete the health questionnaires, the investigators 

4 were able to complete home formaldehyde vapor measurements on all the homes and reported 

5 nearly an identical distribution of formaldehyde concentrations in participating and 

6 nonparticipating homes. Demographic characteristics of some of the non-respondents were 

7 available and were reported as nearly identical to those of participants. There was no indication 

8 of selection bias. Confounding is unlikely to explain such a strong concentration response. 

9 These findings of associations of sensory irritation with residential exposures to 

10 formaldehyde are further supported by studies that did not examine concentration response but 

11 nonetheless assessed the association of formaldehyde with sensory irritation. Similar findings to 

12 those of Ritchie and Lehnen (1987) and Hanrahan et al. (1984) have been reported in other 

13 residential studies of increased symptoms in association with formaldehyde exposure (Liu et al., 

14 1991; Thun et al., 1982; Dally et al., 1981). Dally et al. (1981) collected data in 100 “complaint 

15 structures” (65% mobile homes, 27% conventional homes). Of these, 60% were from home 

16 owners contacting the health department and 30% from physician referrals. Twenty percent of 

17 the buildings had concentrations below the limit of detection (0.1 ppm), 20% had levels at or 

18 above 0.81 ppm, and overall the concentrations ranged from below detection to above 3 ppm 

19 with an overall median of 0.35 ppm. The median levels were 0.47 and 0.10 ppm for mobile and 

20 conventional homes, respectively. No other contaminants were measured. Eye, nose, and throat 

21 irritation were reported in a high percentage of occupants (eye irritation 68%, burning eyes 60%, 

22 runny nose 60%, dry or sore throat 57%, cough 51%), but these were not reported as a function 

23 of dose or home type. Thus, there was no control group to which rates of irritation could be 

24 compared. However, symptoms reportedly stopped in 89% of occupants when they left the 

25 “complaint structure.” The most recent residential study was performed on over 1,000 mobile 

26 homes with 1,394 participants (Liu et al., 1991). Home formaldehyde concentration ranged from 

27 below 0.01 to 0.46 ppm. Analyses used logistic regression to control for potential confounders. 

28 Eye irritation was positively associated with formaldehyde with a clear concentration response 

29 demonstrated with cumulative exposure. During the summer and winter months, formaldehyde 

30 exposure was associated with burning eyes. In the winter months, formaldehyde exposure was 

31 associated with sore throat. There was no association of formaldehyde exposure with cough or 

32 running nose during either season. Liu et al. (1991) also report a synergistic effect on irritation 

33 by formaldehyde exposure and chronic disease prevalence. Thun et al. (1982) reported increased 

34 symptoms of itchy skin and “wheezing and difficulty breathing” in residents in 395 homes 

35 insulated with urea-formaldehyde foam relative to nearby homes without urea foam 
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1 formaldehyde insulation (UFFI); however, there were no measurements of formaldehyde 

2 concentration taken in this study.

3 While not strictly a residential epidemiology study, Olsen and Dossing (1982) studied 

4 occupational exposures within mobile and non-mobile daycare centers. They reported the mean 

5 concentration in mobile and non-mobile day care centers were 350 (200–450) ppb and 65 (40–

6 90) ppb, respectively. Adverse eye, nose, and throat irritation were significantly elevated in the 

7 workers (n = 70) in the mobile units as compared with those in non-mobile units (n = 34). The 

8 authors also state that a high percentage of workers in the mobile day cares reported that the 

9 symptoms disappeared after working hours; however, the authors did not report any such 

10 percentages among those working in non-mobile units.

11

4.1.1.1.1.2. Occupational epidemiology.

21 Similar results were reported for frequency of eye and nasal discomfort in a group of 

22 workers involved in the manufacture of formaldehyde resins. These workers were exposed to a 

mean concentration of 0.40 mg/m3 23 . Alexandersson and Hedenstierna (1988) reported that the 

24 frequency of eye, nose, and throat irritation was significantly greater in 38 workers exposed to 

25 formaldehyde and solvents in lacquers (average employment duration 7.8 years) as compared 

26 with 18 controls (nonexposed individuals working at the same factory). The frequency of eye 

27 irritation was 65.8% among those exposed and 16.7% among controls. No controls reported 

28 nose/throat irritation, but about 40% of those exposed did.

12 Horvath et al. (1988) compared irritation symptoms 

13 between 109 workers at a particleboard manufacturing plant and 264 workers at food plants as a 

14 control group. The mean 8-hour time-weighted average (TWA) formaldehyde concentrations 

15 between these two groups were 0.69 ppm (range 0.17–2.93) and 0.05 ppm (range 0.03–0.12), 

16 respectively. Eye, nose, and throat irritation were more common among the former group 

17 (prevalence of symptoms during a work shift: throat sore or burning—test 22.0%, controls 3.9%; 

18 cough—test 34.9%, controls 18.9%; burning of nose—test 28.4%, controls 2.0%; stuffy nose—

19 test 33.9%, controls 14.2%; itching of nose—test 21.1%, controls 7.9%; eyes burning or 

20 watering—test 39.5%, controls 9.1%; eyes itching—test 19.3%, controls 7.1%). 

29 A Swedish study conducted at a chemical plant found nasal and eye discomfort were 

30 reported by 64 and 24%, respectively, of workers (n = 70) exposed to formaldehyde (range 0.05–

0.50 mg/m3 with a mean of 0.26 mg/m3 31 ) versus 25 and 6%, respectively, of the control group 

32 made up of clerks from the local government (n = 36). In addition, the majority of workers 

33 exposed to formaldehyde reported that their symptoms were relieved during weekends and 

34 vacations (Holmström and Wilhelmsson, 1988). Another study by the same authors 

35 (Wilhelmsson and Holmström, 1992) reported similar results. In this study irritation prevalent 
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1 among 66 workers from a formaldehyde-producing plant was compared with that seen among 

36 community clerks. The workers were exposed to 0.26 mg/m3 2 of formaldehyde (range 0.05–

0.6 mg/m3). The clerks were exposed to an average of 0.09 mg/m3 3 . Nasal and eye discomfort 

4 were reported at rates of 53 and 24%, respectively, among the workers. Among the community 

5 clerks, 3 and 6%, respectively, reported discomfort. 

6 Holness and Nethercott (1989) reported significant increases in eye irritation (42 versus 

7 21%) and nose irritation (44 versus 16%) among 84 funeral service workers as compared with 

8 38 controls (students and individuals from a service organization). The former group had been 

9 actively embalming for approximately 10 years and had nearly twice the pack-years smoked as 

10 the controls. The exposure concentration in both groups was 0.36 and 0.02 ppm, respectively. 

11

4.1.1.1.1.3. Epidemiology on laboratory students

17 In a study of 24 formaldehyde-exposed anatomy students (personal breathing zone 

18 samples 0.73 ppm, range 0.49–0.93), the prevalence of eye irritation before the start of a cadaver 

19 dissection class was 16%, while after the class, the prevalence was 59%. The increase in eye 

20 irritation was most pronounced, but increases were also observed in the prevalence of irritation 

21 of the nose (21%) and throat (15%) (Kriebel et al., 1993). The authors also reported a tendency 

22 for this increase in intensity between the beginning and end of class to diminish over the 

23 10-week course, especially for eye irritation. However, although the intensity of the irritation 

24 diminished, eye irritation was still present among the students after 10 weeks of intermittent 

25 exposure. The report of increase in post- versus pre-class irritation symptoms in this study was 

26 no greater for asthmatic students (n = 5) compared with non-asthmatic students. 

12 . Several studies have monitored sensory 

13 irritation in medical/physical therapy students exposed to formaldehyde during anatomy courses. 

14 These studies have particular advantages: the student population generally has no former 

15 occupational exposure, and, oftentimes, pre-class survey data serve as the control, providing a 

16 better basis for assessing the effects of formaldehyde exposure.

27 Takahashi et al. (2007) showed that 143 medical students reported various symptoms 

28 (including eye and throat irritation) and that the percentage of students reporting symptoms 

29 increased between the beginning and end of the course 2 months later. After the first day of 

30 class, approximately 35% of students reported eye soreness and about 15% reported throat 

31 irritation. After the course ended, these rates were close to 70% for eye soreness and slightly 

32 above 40% for throat irritation. The reported average room formaldehyde concentration was 

33 2.12 ppm (range 1.7–2.4), while the gas samplers worn on the students’ chests averaged 2.4 ppm 

34 (range 1.8–3.8). Another study of students in an anatomy laboratory class in Japan (Takigawa et 

35 al., 2005) measured formaldehyde concentrations and irritation symptoms before and after the 
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1 installation of a ventilation system. This system reduced the median personal formaldehyde 

2 exposure concentration from 2.7 to 0.72 ppm. Before installation of the ventilation system, the 

3 students complained about exacerbation of all the sensory irritation symptoms on average. The 

4 increase in 8 out of 25 symptoms was significantly reduced after installing general ventilation 

5 (p < 0.05). After installation of the ventilation system, a dose-dependent relationship with 

6 formaldehyde was seen for irritated eyes but not for itchy nose. 

7 Akbar-Khanzadeh et al. (1994) detected mean personal area levels of formaldehyde at 

8 1.24 ppm and a range of 0.1–2.94 ppm from personal air sampling devices. Almost 90% of the 

9 students in this study reported eye irritation, 74% reported nose irritation, and close to 30% 

10 reported throat irritation during or after exposure to formaldehyde during the laboratory period 

11 after having completed at least 6 weeks of laboratory sessions with formaldehyde exposure. In 

12 addition, Uba et al. (1989) demonstrated that symptoms of eye, nose, and throat irritation were 

13 correlated with formaldehyde exposure among medical students by comparing students’ 

14 responses on a questionnaire completed after a lab with formaldehyde exposure to a 

15 questionnaire completed after a lab with no formaldehyde exposure. The authors compared 

16 questionnaires completed prior to students’ first anatomy lab to a questionnaire completed 

17 7 months later. Reports of cough were more frequent after the 7 months. These students were 

18 exposed to a mean level of 1.9 ppm (range 0.1–5.0) while dissecting (measured using portable 

19 infrared spectrophotometer), and a TWA from all laboratory activities ranged from below limits 

20 of detection to 0.93 (measured using personal sampling devices in the students’ breathing zones). 

21

22 4.1.1.1.2. Acute studies: controlled chamber exposures. Results from controlled human studies 

23 demonstrate eye, nose, and throat irritation in association with formaldehyde exposure (Lang et 

24 al., 2008; Yang et al., 2001; Krakowiak et al., 1998; Kulle, 1993; Green et al., 1989, 1987; Kulle 

25 et al., 1987; Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 

26 1984; Bender et al., 1983; Anderson et al., 1983; Weber-Tschopp et al., 1977; Andersen, 1979; 

27 Schuck et al., 1966). A key advantage of chamber studies is the ability to monitor and closely 

28 control formaldehyde concentrations during exposure. However, chamber studies may also be 

29 limited by other aspects of the study design, including small number of participants, use of 

30 healthy volunteers, short exposure durations (a few minutes), and often studies were conducted 

31 with only one exposure group and at relatively high concentrations (>1 ppm). The lack of 

32 multiple exposure levels in many studies limits the understanding of exposure-response 

33 relationships. Additionally, numerous reports that demonstrate multiple symptoms of eye, nose, 

34 and throat irritation at levels at or above 1 ppm did not explore lower levels of exposure and can 
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1 only be used for primary hazard identification (Yang et al., 2001; Green et al., 1989, 1987; 

2 Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 1984).

3 The National Aeronautical and Space Administration conducted experiments in closed4 environment living, including environmental monitoring and air quality. James et al. (2002) 

5 quantified air pollutants, including formaldehyde, during 30, 60, and 90-day tests in a closed 

6 chamber study of a Lunar-Mars life support chamber. Unfortunately, the detection methods used 

7 during the 30-day test were not sensitive enough to detect formaldehyde at levels below 2 

mg/m3 8 . Thus, badge samples were obtained in the 60-day and 90-day tests and provided greater 

detection sensitivity (to 0.02 mg/m3 9 ). Measured values of formaldehyde increased over time. In 

10 the 60-day test, formaldehyde levels were well above accepted limits (data not shown). Health 

11 effects data are limited since there were only four crew members. One crew member reported 

eye and upper airway irritation at formaldehyde concentrations of 0.25 mg/m3 12 (308 ppb) on day 

13 15. It should also be noted that astronauts are exceptionally healthy individuals, and these data 

14 should be interpreted carefully when determining expected health effects in the general 

15 population. The experimenters determined that formaldehyde levels increased as temperature 

16 increased. Formaldehyde was also linked to murals lining the chamber and was subsequently 

17 removed before executing the 90-day study. Between days 0 and 60, formaldehyde levels 

remained between 0.02 and 0.04 mg/m3 18 , with one sharp peak that occurred at day three to 0.07 

mg/m3

. Between days 60 and 90, formaldehyde concentrations increased to 0.07 to 0.09 mg/m3 19 . 

20 The increase was attributed to an incomplete oxidation of methanol in a catalytic bed rather than 

21 in excessive off-gassing of formaldehyde. No crew members reported any adverse effects in the 

22 90-day study. 

23 A few studies have been conducted that specifically address sensitive populations 

24 (asthmatics) and/or individuals during exercise, which can exacerbate asthma (further details of 

25 these studies are in Section 4.1.1.3, Effects on Asthmatics). In Sauder et al. (1986), 8-minute 

26 bicycle exercise was completed multiple times during the exposure period (3 hours). However, 

27 irritation symptoms were only reported after 2 hours of exposure and do not address whether 

28 changes occurred during the periods of exercise. Overall, reports of eye, nose, and throat 

29 irritation increased with exposure to formaldehyde (3 ppm) compared with reports of irritation 

30 with no exposure to formaldehyde. Green et al. (1987) report that eye, nose, and throat irritation 

31 symptoms were greater immediately after exercise during exposure to 3 ppm formaldehyde. 

32 Additionally, the response levels were similar between asthmatic (n = 16) and non-asthmatic 

33 (n = 22) subjects. Similar effects of exercise on certain symptoms, such as throat irritation, were 

34 reported in 15 asthmatic subjects exposed to 2 ppm formaldehyde at rest and after exercise 

35 (Witek et al., 1987).
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1 Kulle (1993) and Kulle et al. (1987) enrolled 19 healthy volunteers and exposed them to a 

2 range of formaldehyde concentrations. At 2 ppm, 53% reported mild or moderate eye irritation 

3 (32% mild, 21% moderate). At 3 ppm, 100% of subjects exposed at this level (n = 9) reported 

4 irritation. The reported increase in irritation was shown to correspond with increasing 

5 formaldehyde concentration in a linear fashion. Mild nose/throat irritation was present among 

6 37% of those exposed to 2 ppm of formaldehyde. Odor detection was very similar to the 

7 distribution seen for eye irritation. Nineteen subjects performed light to moderate exercise while 

8 exposed to 2 ppm; there was no increase in report of eye irritation, but nose/throat irritation did 

9 increase. The data were reanalyzed (Kulle, 1993), and thresholds for irritation were found to be 

10 0.5–1 ppm for eye irritation and 1 ppm for nose/throat irritation. 

11 Yang et al. (2001) reported that eight individuals exposed to varying levels of 

12 formaldehyde (1.65, 2.99, and 4.31 ppm) had mild to moderate eye irritation during the 5-minute 

13 exposures. The increase in irritation was detected at 30 seconds with exposure to 1.65 ppm of 

14 formaldehyde. The highest severity ratings at this concentration occurred between 60 and 

15 90 seconds. Frequency of eye blinking was also measured. The peak in blinking rate occurred 

16 after about 1 minute of exposure and then decreased almost back to a normal rate after 5 minutes 

17 of exposure. Higher formaldehyde concentrations were associated with increased frequency of 

18 blinking compared with the 1.65 ppm exposure.

19 Other studies have examined responses across multiple exposure levels. For example, 

20 Weber-Tschopp et al. (1977) used two different methods of studying irritation resulting from 

21 formaldehyde exposure. For one, they exposed subjects (n = 33) to an increasing level of 

22 formaldehyde (maximum exposure was 3.2 ppm). This design precluded evaluation of distinct 

23 effects at different exposure levels. The researchers addressed this by examining another group 

24 of subjects (n = 48) that were exposed to 0, 1, 2, 3, or 4 ppm five times for 90 seconds. Levels of 

25 nasal and throat irritation for this discontinuous exposure were slightly higher than the irritation 

26 levels reported among those with continuous exposure. However, this was reversed for eye 

27 irritation; those with continuous exposure reported higher levels of irritation than those with 

28 discrete exposures. An objective measure, eye-blinking rate, was measured for those with 

29 continuous exposure and was found to have a statistically significant increase at 1.7 ppm.

30 Bender et al. (1983) conducted a study that enrolled individuals who “responded” to 

31 formaldehyde at 1.3 and 2.2 ppm and did not report irritation to the clean air control. They 

32 found that, among these subjects, exposure to 1 ppm of formaldehyde (n = 27) resulted in the 

33 reporting of eye irritation with a median response time of 78 seconds. Reports of irritation were 

34 given as less than slightly irritating for formaldehyde concentrations of 0.3–0.9 ppm.
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1 Assessment of sensory irritation for pain and discomfort often relies on self-reporting, 

2 using symptom questionnaires and severity ratings (e.g., mild, moderate, severe). In the case of 

3 formaldehyde, subjective ratings of eye irritation correlate positively with eye-blinking 

4 frequency (Lang et al., 2008). Lang et al. (2008) saw an increase in eye blinking after 

5 195 minutes of exposure to formaldehyde at 0.5 ppm with four peak exposures of 1 ppm. After 

6 this amount of time and formaldehyde exposure, there was also an increase in moderate eye 

7 redness. Weber-Tschopp et al. (1977) reported that, among concentrations ranging from 0.03 to 

8 3.2 ppm, eye-blinking frequency was increased at 1.7 ppm; similarly Yang et al. (2001) reported 

9 increased blinking at >1.5 ppm (the lowest concentration examined). There are studies that 

10 suggest that psychological factors (e.g., anxiety) can impact the perception of irritation—and 

11 perhaps more so at lower concentrations (Lang et al., 2008; Ihrig et al., 2006; Dalton, 2003). 

12 However, when Lang et al. (2008) controlled for mood prior to exposure, subjective symptoms 

13 of eye, nasal, and olfactory irritation were significantly related to exposure (0.5 ppm) 

14 Schuck et al. (1966) performed a study that also examines self-reported eye irritation as 

15 well as blinking rate. Fourteen individuals were exposed to formaldehyde concentrations 

16 ranging from 0 to 1 ppm. Increased irritation was reported with increasing formaldehyde 

17 concentration. One subject, judged to be the least sensitive, was still able to detect formaldehyde 

18 levels as low as 0.01 ppm. In addition, the authors examined the blinking rate of participants, 

19 which they found was related to irritation intensity.

20 Andersen (1979) and Anderson and Molhave (1983) reported on a controlled experiment 

21 in which 16 individuals were exposed to varying levels of formaldehyde for five hours and rated 

22 their level of discomfort over the exposure period. Discomfort occurred within 1 hour at 

formaldehyde exposure levels of 1 and 2 mg/m3 23 (Andersen and Molhave, 1983) After 2 hours, 

increasing discomfort was reported among the groups exposed to 0.3 and 0.5 mg/m3 24 . Subject 

25 reported that discomfort was mainly conjunctival irritation and dryness in the nose and throat. 

Subjects complained at all four concentrations of formaldehyde: 0.3, 0.5, 1.0, and 2.0 mg/m3 26 and 

27 of 16 subjects, 3, 5, 15, and 15 subjects complained at each respective exposure concentration 

28 (Andersen and Molhave, 1983). 

29 Controlled chamber studies have also been conducted on various populations of 

30 previously exposed individuals to determine if formaldehyde exposure potentiates an 

31 individual’s response to acute exposures. Schachter et al. (1987) reported on 15 laboratory 

32 workers “frequently exposed to formaldehyde” (no quantification of exposure is given; however, 

33 the workers report being exposed for 1 to 7 days per week from a range of 1 to 21 years). Tests 

34 performed at the start of the study found that these individuals had pulmonary function similar to 

35 that seen in healthy individuals. The workers in this study reported subjective measures of eye, 
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1 nose, and throat irritation after 40 minutes of exposure to 2 ppm of formaldehyde. However, the 

2 2 ppm acute exposure in this study may be sufficiently high to induce significant irritation in 

3 most individuals. Krakowiak et al. (1998) reported that 10 asthmatics with occupational 

4 exposure to formaldehyde (via formaldehyde solutions or pure gaseous formaldehyde) exhibited 

5 similar symptom scores to healthy controls (never exposed to formaldehyde in the workplace) 

6 exposed to 0.4 ppm formaldehyde for 2 hours. The mean symptom scores and standard 

7 deviation (SD), which included information on sneezing, rhinorrhea, mucosal edema, and 

8 itching, were 4.6 ± 1.6 (mean ± SD) for asthmatics and 4.3 ± 1.2 for healthy subjects 

9 immediately after inhalation. These dropped to 1.8 ± 1.2 and 1.2 ± 1.3, respectively, 4 hours 

10 after the exposure. It is unclear if sensitive individuals may not be represented in either of these 

11 groups, as the workers were tolerating their exposures during the work shift “healthy worker” 

12 effect. However, residents (n = 9) exposed to formaldehyde in their homes, who complained 

13 about adverse effects from the material, but with no occupational exposure reported eye, nose, 

14 and throat irritation at a similar rate as controls (individuals in homes without formaldehyde or 

15 individuals in homes with formaldehyde but not reporting adverse effects [n = 9]) after a 

16 90-minute exposure to 1 ppm (Day et al., 1984). The number of individuals reporting eye 

17 irritation, nasal congestion, and throat irritation were seven, three, and two among sensitive 

18 individuals and eight, four, and three among controls, respectively. These individuals may be 

19 considered a sensitive population since they had “previously complained of various 

20 nonrespiratory effects from the UFFI in their homes” (household concentrations unknown). 

21

22 4.1.1.2. Pulmonary Function

23 Workers chronically exposed to formaldehyde have exhibited signs of reduced lung 

24 function consistent with BC, inflammation, or chronic obstructive lung disease. Lung function 

25 deficits have been reported both in pre-shift versus post-shift measurements and as a result of 

26 chronic exposures (Pourmahabadian et al., 2006; Herbert et al., 1994; Malaka and Kodama, 

27 1990; Alexandersson and Hedenstierna, 1989; Alexandersson et al., 1982). Decreases in 

28 spirometric values, including vital capacity (VC), forced expiratory volume (FEV), forced vital 

29 capacity (FVC), and FEV/FVC have been reported. Chronic studies also report increased 

30 respiratory symptoms, such as cough, increased phlegm, asthma, chest tightness, and chest colds, 

31 in exposed workers (Pourmahabadian et al., 2006; Herbert et al., 1994; Malaka and Kodama, 

32 1990; Alexandersson and Hedenstierna, 1989; Alexandersson et al., 1982). Similar findings 

33 have been reported for low-level residential formaldehyde exposure, including decreased peak 

34 expiratory flow (PEF) rates (Krzyzanowski et al., 1990). 
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1 Worker exposures that report cross-shift differences in spirometric values are consistent 

2 with formaldehyde-induced sensory irritation. Additionally, concordance has been reported 

3 between subjective irritant response and measured changes in pulmonary function, further 

4 supporting the possibility that cross-shift and short-term evidence of BC may be a reflexive 

5 response to sensory irritation. Absolute values for lung function parameters are likely to vary by

6 gender, age, height, and smoking status and are best compared when normalized to the expected 

7 lung function based on these variables (Schoenberg et al., 1978). Individual variation can also be 

8 addressed by each subject serving as his/her control with measurements taken before, during, and 

9 after exposure. Analysis of the percent change in various parameters in this context may have 

10 greater sensitivity to detect exposure-related changes in function.

11 In addition to individual variation in baseline lung function, there is variation in bronchial 

12 responsiveness. Reduced lung function parameters in response to methacholine challenge is a 

13 standard test for BC, and this can be used to define responsive, sensitive, or susceptible 

14 individuals. Since formaldehyde-induced BC is measured with these lung function tests, 

15 variability in bronchial responsiveness may impact interpretation of formaldehyde-induced 

16 changes. Experiments with sensitive individuals can help address this question. However, 

17 results need to be normalized in some way to account for differences in responsiveness before 

18 formaldehyde exposure. Researchers have in some cases excluded hyperresponsive individuals 

19 or presented results as a proportion or percent of the unexposed value for each individual. 

20 However, excluding sensitive individuals may bias results towards the null.

21 The American Thoracic Society (ATS) published an official statement on what 

22 constitutes an adverse health effect of air pollution (ATS, 2000). According to the ATS 

23 statement, exposure that increases the risk of an adverse effect to the entire population can be 

24 considered adverse, even though it may not increase the risk of any individual to an unacceptable 

25 level. For example, a population of asthmatics could have a distribution of lung function such 

26 that no individual has a level associated with significant impairment. Exposure to an air 

27 pollutant could shift the distribution to lower levels that still do not bring any individual to a 

28 level that is associated with clinically relevant effects. However, this would be considered to be 

29 adverse because individuals within the population would have diminished reserve function and 

30 therefore would be at increased risk if affected by another agent. 

31

32 4.1.1.2.1. Epidemiologic literature. The potential adverse effects of formaldehyde exposure on 

33 pulmonary function in humans can be examined on several time scales of interest. The 

34 epidemiologic literature supports the assessment of exposures among exposed anatomy medical 

35 students where all participants have well-defined and similar duration of exposure (i.e., a 
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1 semester-long class) (Kriebel et al., 2001, 1993; Akbar-Khanzadeh and Mlynek, 1997; Akbar2 Khanzadeh et al., 1994; Uba et al., 1989; Fleisher, 1987), among individuals living or working in 

3 buildings with formaldehyde exposure (Franklin et al., 2000; Krzyzanowski et al., 1990; Main 

4 and Hogan, 1983), and among workers (industrial, manufacture, mortuary, hospital staff, etc.) 

5 (Ostojic et al., 2006; Herbert et al., 1994; Khamgaonkar and Fulare, 1991; Malaka and Kodama, 

6 1990; Nunn et al., 1990; Alexandersson and Hedenstierna, 1989; Holness and Nethercott, 1989; 

7 Holmström and Wilhelmsson, 1988; Horvath et al., 1988; Kilburn et al., 1985; Alexandersson et 

8 al., 1982).

9 The observed effects in the previously unexposed anatomy students provide additional 

10 information on acute exposures in two naïve populations (Kriebel et al., 2001, 1993) as well as 

11 special insight into the intermediate stages of possible sensitization (Kriebel et al., 1993). 

12 Kriebel and colleagues (1993) examined the pre-laboratory and post-laboratory PEF in students 

13 attending anatomy classes once per week. They found the strongest pulmonary response when 

14 examining the average cross-laboratory decrement in PEF in the first 2 weeks of the study when 

15 formaldehyde concentrations collected in the breathing zones had a geometric average 

16 concentration of 0.73 ppm. Overall, the students exhibited a 2% decrement in PEF, while the 

17 students with any history of asthma showed a 7.3% decrement in PEF. These findings of acute 

18 decreases in PEF following students’ initial anatomy sessions were corroborated by the Kriebel 

19 et al. (2001) study, which used a similar study design applied to another class of anatomy 

20 students.

21 The Kriebel et al. (1993) study also shows how the acute effects of formaldehyde 

22 exposure were altered following several weeks of weekly episodic exposure. By the fifth week 

23 of class, the pre- and post-laboratory measurements of PEF were no longer reflecting a clearly 

24 demonstrated acute effect, but, following the seventh week of episodic exposure, both pre- and 

25 post-laboratory PEF continued to drop steadily until the class adjourned after 10 weeks. While 

26 the acute effects of formaldehyde exposure appeared to diminish after several weeks of exposure, 

27 the intermediate effect across 9 weeks was a 24 L/minute drop in PEF that was statistically 

28 significant (p < 0.01 after statistical control for random person effects, asthma, interaction 

29 between time and asthma, and eye as well as nose symptoms of irritation). 

30 Similar studies among medical students have been performed. In one study, 34 exposed 

31 and 12 control students completed pulmonary function tests before and after their work in the 

32 laboratory (approximately 3 hours) (Akbar-Khanzadeh et al., 1994). The time-weighted average 

33 exposure ranged from 0.07-2.94 ppm. More than 94% of the subjects were exposed to >0.3 ppm 

34 and 31.7% were exposed to >0.5 ppm. Comparing pre- and post-exposures among the exposed 

35 students, on average FVC decreased by 1.4%, FEV3 decreased by 1.2%, FEV1/FVC increased by 
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1 1.6%, and FVC25–75% increased 2.5%. These average percent changes in the control group are –

2 0.3, 1.30, 2.31, and 0.6%, respectively. The researchers also calculated correlation coefficients 

3 by examining the relationship between lung function and formaldehyde concentration, but no 

4 association was found. Akbar-Khanzadeh and Mlynek (1997) performed another study with 50 

5 exposed students and 36 controls and reported a larger increase in lung function among controls 

6 when compared with cases after 1–3 hours of exposure (FVC 3.0 versus 0.9, FEV1 4.1 versus 

7 1.2, FEV3 3.3 versus 0.8, forced expiratory flow during the middle of the FVC [FEF25–75%] 6.1 

8 versus 0.7). These differences between cases and controls remained for FEV1, FEV3, and FEF25–

9 75% after 3 hours. 

10 A study of 103 medical students was performed over a period of 7 months in which the 

11 students were exposed to formaldehyde at a time weighted average of <1 ppm with peaks of 5 

12 ppm during anatomy laboratory sessions (Uba et al., 1989). Twelve students were asthmatics. 

13 Unlike the studies by Kriebel et al. (2001, 1993), these researchers did not find a change in 

14 pulmonary function over the course of 7 months. The mean percent change for pulmonary 

15 function before and after the exposure did change slightly, with measures showing decreases in 

16 function at the end of the laboratory session (measurements taken at the 7-month time point: 

17 FVC –0.79%, FEV1 –0.48%, FEF25–75% 0.07%, FEV1/FVC 0.24%).

18 Finally, Fleisher (1987) gave self-administered questionnaires to medical students after 

19 completing an anatomy laboratory session (formaldehyde exposure measures as <1 ppm) and a 

20 pathology/microbiology laboratory session (no formaldehyde exposure). Over 8% of students 

21 reported experiencing shortness of breath during the laboratory with formaldehyde exposure, but 

22 none of the students reported shortness of breath in the laboratory session with no exposure. No 

23 objective measurements of formaldehyde exposure were used.

24 Three studies have been performed that examine formaldehyde exposure from the 

25 buildings in which individuals live or work. One study included children 6–13 years of age and 

26 measured the levels of formaldehyde in their homes. There was no association between FVC or 

27 FEV and the indoor concentrations of formaldehyde, although there were signs of lower airway 

28 inflammation as measured by levels of exhaled nitric oxide (NO) in children exposed to average 

29 formaldehyde levels ≥0.05 ppm (Franklin et al., 2000). Municipal employees with their children 

30 (613 adults and 298 children) were randomly sampled in another study of home exposures 

31 (Krzyzanowski et al., 1990). Residential exposures to formaldehyde were based on repeated 

32 samples from each individual’s kitchen, living area, and bedroom. The average formaldehyde 

33 concentration was 26 ppb, with a maximum sample value of 140 ppb. The majority of subjects 

34 (83%) lived in homes with 2-week average concentrations below 40 ppb. Subjects’ peak 

35 expiratory flow rates (PEFRs) were determined four times daily, in the morning, at noon, in the 
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1 early evening, and before bed, for 2 weeks. A statistically significant linear relationship between 

2 increased formaldehyde exposure and decreased PEFR was reported in children but not adults. 

3 All statistical models controlled for socioeconomic status, tobacco smoking (current active or 

4 environmental tobacco smoking), and nitrogen dioxide concentrations. Among adults, there was 

5 a statistically significant nonlinear relationship with decreased morning PEFR for formaldehyde 

6 concentration <40 ppb. 

7 Main and Hogan (1983) reported on a group of individuals (n = 21) working in two 

8 mobile trailers for 34 months and exposed to levels of formaldehyde ranging from 0.12 to 

9 1.6 ppm (mean age 38 ± 9 years, 76% male, 19% nonsmokers). The control population was 

10 comprised of individuals who did not work in the trailers (n = 18; mean age 30 ± 6, 50% male, 

11 22% nonsmokers). There were no differences between the exposure and control groups’ percent 

12 predicted FEV1 or FVC regardless of smoking status.

13 Several studies allowed for the examination of potential chronic effects of formaldehyde 

14 exposure. These included an occupational study by Malaka and Kodama (1990) that reported 

15 pre-shift pulmonary function as a percentage of expected among the formaldehyde exposed 

16 compared with comparable people not exposed to formaldehyde. This study found that an 

17 average 8-hour TWA formaldehyde exposure of 1.13 ppm from area samples was associated 

18 with statistically significant decrements in FEV1, FEV1/FVC, and FEF25–75% compared with a 

19 referent population. The strongest response was for FEF25–75%, which showed a 12% drop in 

20 observed function compared with expected function in the unexposed, but it is unclear how to 

21 interpret the potential chronic adverse effect(s) with just the magnitude of the decrement and the 

22 length of the average occupational tenure at this plywood facility (6.5 years), which was not 

23 reported by exposure status. 

24 A study comparing oriented strand board workers (exposed to formaldehyde) with oil/gas 

25 field plant workers (not exposed to formaldehyde) demonstrated a difference in pulmonary 

26 function between the two groups (Herbert et al., 1994). The groups were similar in regard to 

27 measured FVC and FEV1 (controlled for age, height, and smoking), but the workers exposed to 

28 formaldehyde had lower FEV1/FVC. In addition, those exposed to formaldehyde showed a 

29 decrease in FVC and FEV1 after their shift, with an average pre- and post-shift difference of 

30 47 mL (p = 0.022) and 39 mL (p = 0.044) for FVC and FEV1, respectively (however change 

31 could not be compared with the controls of this study because no post-shift measurements were 

32 taken). Two other occupational studies found no association between formaldehyde and lung 

33 function (Holness and Nethercott, 1989; Horvath et al., 1988). One of these studies was 

34 conducted among funeral workers and an unexposed control group (Holness and Nethercott, 

35 1989). There was no difference in pulmonary function of the two groups at baseline. After 
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1 exposure, there was no change in lung function for the exposed or unexposed when comparing 

2 lung function tests done immediately before and after an embalming procedure (for controls the 

3 repeat measures were taken approximately 2–3 hours after the first measure) (changes in 

4 percentage predicted FVC and FEV1 were 0.88 ± 2.95 and –0.03 ± 2.40 for exposed and 1.13 ± 

5 3.98 and 1.45 ± 4.43 for unexposed). Further analysis showed no association between 

6 formaldehyde levels and changes in lung function. Another study (Horvath et al., 1988) found 

7 no differences in pre-shift pulmonary function between the exposed (workers at a particleboard 

8 and molded products operation, formaldehyde measured using individual monitors ranged from 

9 0.17–2.93 ppm) and controls (workers from nearby food processing facilities, formaldehyde 

10 measured using individual monitors ranged from 0.03–0.12 ppm). However, the authors did find 

11 a post-shift decline in FVC and FEV1 among controls and FEV1 and FEF25–75% among workers

12 when using paired comparisons for each group. When assigning all controls a formaldehyde 

13 exposure value of 0.05 ppm, there was a correlation detected in pre-and post-shift pulmonary 

14 function changes and formaldehyde, though no specific details on regression analysis were 

15 provided. 

16 A study performed in India (Khamgaonkar and Fulare, 1991) examined individuals 

17 working in anatomy and histopathology departments and exposed to formaldehyde (mean 

18 1.00 ppm, range 0.036–2.27). Controls (individuals not working in laboratories with 

19 formaldehyde) were exposed to an average of 0.102 ppm formaldehyde (range 0–0.52). Lung 

20 function tests were performed on a Monday morning after days of no exposure in order to 

21 examine chronic effects. The FVC and FEV1% of the exposed group, respectively, were 17.12 

22 and 22.94% reduced compared with the control group (Khamgaonkar and Fulare, 1991); 

23 however, while the pool of cases and controls were frequency-matched on age and gender, there 

24 was no mention by the investigators of normalizing the pulmonary function metrics by gender 

25 and height, which would have made for more appropriate comparisons. Kilburn et al. (1985) 

26 also demonstrated reduced pulmonary function (lower percent predicted FVC, FEV1, and 

27 FEF25-75%) among workers occupationally exposed to formaldehyde when compared with 

28 individuals working at jobs without formaldehyde exposure.

29 Two occupational studies found no association between formaldehyde exposure and 

30 deficits in pulmonary function (Ostojic et al., 2006; Holmström and Wilhelmsson, 1988). 

31 Ostojic et al. (2006) examined nonsmoking male health service professionals working in 

32 pathoanatomic laboratories with 8 hours of formaldehyde exposure per day at an unspecified 

33 concentration for at least 4 years (n = 16). The control group was comprised of sixteen age- and 

34 stature-matched nonsmoking male controls. There was no difference in mean FVC or FEV1

35 between exposed and controls. The researchers also examined values for diffusing lung capacity 
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1 for carbon monoxide and membrane diffusion capacity, which were similar between the exposed 

2 and control groups. However, blood volume of pulmonary capillaries was found to be higher in 

3 the exposed group. Holmström and Wilhelmsson (1988) recruited individuals from a chemical 

4 plant where formaldehyde and formaldehyde products were made (n = 70). Exposure levels 

varied from 0.05–0.5 mg/m3 5 . A control group was mostly comprised of clerks for the local 

6 government (n = 36). No difference in FEV% was detected between the groups. Mean FVC was 

7 lower than expected among the exposed group (expected values were based on age, sex, smoking 

8 habits, height, and weight). This study went further and measured changes in pulmonary 

9 function for those employed more than 5 years and reported no signs of increasing restrictivity 

10 after 5 years. No associations were seen and there was no correlation between pulmonary 

11 function and cumulative dose of formaldehyde (Holmström and Wilhelmsson, 1988). 

12 There have been only two studies that have reported on the longitudinal follow-up of 

13 workers exposed to formaldehyde (Nunn et al., 1990; Alexandersson and Hedenstierna, 1989). 

14 The Alexandersson and Hedenstierna (1989) investigation not only examined the acute effects of 

15 exposure across shift but was able to do so among some of the same workers that had been 

16 studied 5 years earlier (Alexandersson et al., 1982). Statistically significant decreases in 

17 FEV1/FVC and FEF25–75% were noted over the intervening 5 years in nonsmokers after correction 

18 for normal aging and reference lung function spirometry values. The decrease in FEF25–75% was 

19 0.212 ± 0.066 L/second (mean ± SD) for each year of exposure and was highly significant (p < 

20 0.01). For comparison with the 12% drop in the same pulmonary metric reported by Malaka and

21 Kodama (1990) over an estimated 6.5 years, the extrapolated percentage decrease in FEF25–75% 

22 was computed for the Alexandersson and Hedenstierna (1989) study by using the reported yearly 

23 decrement applied to the pre-shift values at the time of the initial study period. From the 

24 predicted value of 4.57 L/second, a decrease of 0.168 L/second for each year of exposure 

25 regardless of smoking status was calculated. For 6.5 years of exposure, this would result in a 

26 24% drop in FEF25–75%. Formaldehyde concentrations were estimated at 0.42 ppm in the first 

27 Alexandersson et al. (1982) study and at 0.50 ppm in the Alexandersson and Hedenstierna (1989) 

28 study. The study by Nunn et al. (1990) assessed the decrease in FEV1 with age. The researchers 

29 calculated the decrease in FEV1 to be 42 mL/year among workers exposed to formaldehyde and 

30 41 mL/year for workers who were not exposed to formaldehyde. Thus, they showed no 

31 association between formaldehyde exposure and decreased FEV1. 

32 There are a few important limitations to consider in these occupational studies of 

33 formaldehyde exposure. First, an often-shared weakness is the absence of data on, and 

34 appropriate statistical control of, potential confounding by occupational co-exposures. Also, 

35 studies that did not report pre-shift pulmonary function as a percentage of expected function 
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1 contribute less to an assessment of potential chronic effects because, post-hoc, it is difficult to 

2 calibrate the multiple pulmonary function data for cross-study comparison without knowledge of 

3 the age, gender, smoking status, height, year of birth, etc., that are important determinants of the 

4 pulmonary function metrics of concern.

5

6 4.1.1.2.2. Acute studies: controlled chamber exposures. Pulmonary effects of acute 

7 formaldehyde exposure have been studied in both healthy volunteers and sensitive populations 

8 under controlled conditions. Controlled chamber studies have the advantage of measured 

9 controlled exposures, but other factors can limit the usefulness of the studies, especially when 

10 study populations are small and there is high variability in the measured parameters. 

11 In general, acute formaldehyde exposures (0.5–3 ppm) have not induced significant 

12 pulmonary deficits in healthy, non-exercising volunteers (Kulle et al., 1987; Schachter et al., 

13 1986; Witek et al., 1986; Day et al., 1984; Andersen and Molhave, 1983). However, it is unclear 

14 whether the data analysis in these reports had the statistical power to substantiate the small 

15 deficits reported in occupational and student studies. All four reports had relatively small study 

16 groups of healthy individuals (n = 19 [Kulle et al., 1987], n = 16 [Andersen and Molhave, 1983], 

17 n = 15 [Schachter et al., 1986], n = 15 [Witek et al., 1986], and n = 9 [Day et al., 1984]), and in 

18 some cases the group was further divided by gender. Two studies report the absolute values of 

19 the lung function parameters without adjustment to individual expected function or the 

20 unexposed baseline for each individual (Kulle et al., 1987; Andersen and Molhave, 1983). As 

21 discussed, this decreases the power of the study to detect formaldehyde-induced changes in 

22 pulmonary function. In contrast, Witek et al. (1986) and Schachter et al. (1986) report lung 

23 function as a percent of baseline (although not normalized for age gender and height). Each 

24 study showed an increase in FEV1 in formaldehyde-exposed individuals at rest and increases in 

25 maximal expiratory flow (MEF) at 50% of expired vital capacity (MEF50%) (Witek et al., 1986; 

26 Schachter et al., 1986). However, in both reports the SDs of changes in lung function parameters 

27 are quite large, nearly equaling the reported value and exceeding it in several cases. The absence 

28 of normalized raw data, combined with large individual variation, limit the interpretation of these 

29 studies. A small study (Day et al., 1984) that included nine healthy individuals showed no 

30 changes in FEV or FVC after 90 minutes of exposure to 1 ppm of formaldehyde. A more recent 

31 study (Lang et al., 2008) of 21 healthy volunteers exposed to a range of formaldehyde 

32 concentrations (0.0 to 0.15 or 0.3 ppm) reports no formaldehyde-related pulmonary deficits. 

33 However, data were not shown, and it is unclear whether the authors compared absolute or 

34 relative values of lung function and what variation in lung function was present in the study 

35 population. Additionally, the authors did not provide the criteria used to gauge deficit of lung 
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1 function. If a clinically significant deficit was defined (e.g., 20%), then more subtle changes in 

2 pulmonary function, as supported in other studies, would not have been reported. 

3 Similar to these studies of healthy individuals, acute controlled studies including 

4 asthmatics also report no changes in pulmonary function associated with formaldehyde exposure 

5 (Ezratty et al., 2007; Harving et al., 1990; Green et al., 1987; Sauder et al., 1987; Witek et al., 

6 1987, 1986), including studies of individuals thought to have formaldehyde-induced bronchial 

7 asthma (Krakowiak et al., 1998); however, the number of asthmatic individuals included in each 

8 of these studies was small. (The details of these studies have been reported elsewhere in this 

9 chapter. Briefly, the number of asthmatics in the study/total number of individuals in the study 

10 are as follows: Ezratty et al. [2007]—12/12; Harving et al. [1990]—15/15; Green et al. [1987]—

11 16/38; Sauder et al. [1987]—9/9; Witek et al. [1987]—15/15; Witek et al. [1986]—15/30; 

12 Krakowiak et al. [1998]—10/20]. The same is true for individuals who are frequently exposed to 

13 formaldehyde either at work (n = 15) (Schachter et al., 1987) or at home (n = 18) (Day et al., 

14 1984).

15 Small but statistically significant deficits in pulmonary function due to acute 

16 formaldehyde exposure (2 or 3 ppm) have been reported in healthy volunteers during exercise 

17 (Green et al., 1989, 1987; Sauder et al., 1986; Schachter et al., 1986). Although changes in lung 

18 function parameters averaged over experimental groups were generally small, some individuals 

19 exhibited clinically significant deficits, even after only 2 hours of exposure. Deficits in FEV1

20 and FEF25–75% in the first 30 minutes of a 2-hour exposure at 3 ppm formaldehyde were 2 and 

21 7%, respectively. Changes in lung function were not statistically significant after 60 and 

22 180 minutes of exposure (Sauder et al., 1986), even when assessed as absolute rather than 

23 relative measurements. Thirteen percent (5 of 38 subjects) demonstrated formaldehyde-induced 

24 clinically significant deficits when exposed at 3 ppm during exercise (defined by Green et al. 

25 (1987) as decrease in FEV1 > 10% of control).

26

27 4.1.1.3. Asthma

28 A large number of studies have investigated the potential association between 

29 formaldehyde exposure and a continuum of adverse health effects ranging from decrements in 

30 pulmonary function to asthma. In general, epidemiologic studies of adults have reported varied 

31 results between null findings and positive findings. However, The National Research Council 

32 concluded in its report on Formaldehyde that, “Formaldehyde has been shown to cause bronchial 

33 asthma in humans” (NRC, 1981), citing numerous studies demonstrating the induction of asthma 

34 following exposure to formaldehyde (Hendrick and Lane, 1975, 1977; Laffont and Noceto, 1961; 

35 Nova and Touraine, 1957; Paliard et al., 1949; Popa et al., 1969; Sakula, 1975; Schoenberg and 
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1 Mitchell, 1975; Turiar, 1952; Vaughan, 1939). In a subsequent review article on formaldehyde 

2 and the health effects that have been associated with it, Stenton and Hendrick (1994) reported on 

3 formaldehyde and asthma in occupational settings and starkly describe the “...first detailed case 

4 report of formaldehyde asthma confirmed by specific inhalation challenge test occurring in a 

5 nursing sister on a renal dialysis unit. Her symptoms were suggestive of late asthmatic reactions 

6 occurring 4 to 5 hours after heavy exposures. The occurrence of late reactions was confirmed in 

7 a series of challenge tests that involved the painting of formalin onto cardboard pieces within a 

8 confined space” (Stenton and Hendrick, 1994; Hendrick 1997). The results of the challenge tests 

9 are illustrated in Figure 4-1 . 

10

11

12

13

14

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

16 formaldehyde after “painting” 100% formalin for 20 minutes. Challenge 2 

17 was premedicated with inhaled betamethasone 200 μg. 

18 Source: (Stenton and Hendrick, 1994)

19

20 Five years later, the two nurses were re-challenged with the nurse who had left the 

21 dialysis unit having no response to the subsequent challenge while the nurse who had remained 

22 working in the unit developed mild late asthmatic response with peripheral blood eosinophilia 

23 (Stenton and Hendrick, 1994; Hendrick et al., 1982). Stenton and Hendrick (1994) concluded 

24 that these studies “provide clear evidence of formaldehyde’s ability to induce asthma” but no 

25 indication of the exposure concentrations to induce it. In a follow-up study of dialysis unit 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 staffers exposed to formaldehyde as a sterilizing agent, 8/28 people reported respiratory 

2 symptoms and a prolonged increase in circadian rhythm of peak expiratory flow rate was seen in 

3 one subject (Hendrick and Lane, 1983) implying an increase in airway responsiveness (Stenton 

4 and Hendrick, 1994). It should be noted, however, that while there did appear to be a clear 

5 response to formalin, it is not known what contribution to the response was attributable to 

6 formaldehyde and what contribution might have been attributable to methanol. Further, while 

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

8 do not offer information on the concentrations at which adverse effects would expected in a 

9 population.

10 There is at least one clinical study in humans that investigated whether exposure to a low 

level of formaldehyde (500 μg/m3 11 ) would enhance inhaled allergen responses (Ezratty et al., 

12 2007). Twelve subjects with intermittent asthma were exposed to either formaldehyde or 

13 purified air in a double-blind crossover study for 1 hour. Following exposure (8 hours), airway 

14 responsiveness to methacholine challenge was measured. No significant effects on 

15 methacholine-induced bronchial hyperresponsiveness (BHR) were detected due to formaldehyde 

16 exposure.

17 Numerous epidemiologic studies have investigated adverse effects in populations. 

18 Decreased peak expiratory flow rates (PEFR) are an important component in the diagnosis of 

19 asthma and there is evidence of formaldehyde-induced decrements in PEFR (see Section 

20 4.1.1.2). However, the diagnosis of asthma is both a more serious health condition and 

21 diagnostically more complex than decreased PEFR alone and is evaluated here as a distinct 

22 endpoint. A number of epidemiologic studies have investigated the potential association 

23 between formaldehyde exposure and a continuum of adverse health effects from pulmonary 

24 function to asthma.

25 The association between formaldehyde and asthma has been studied by examining 

26 occupational exposures (Fransman et al., 2003; Malaka and Kodama, 1990), school-related 

27 exposures (Zhao et al., 2008; Smedje and Norback, 2001; Norback et al., 2000) and residential 

28 exposures (Matsunaga et al., 2008; Tavernier et al., 2006; Gee et al., 2005; Delfino et al., 2003; 

29 Rumchev et al., 2002; Garrett et al., 1999; Palczynski et al., 1999; Norback et al., 1995; 

30 Krzyzanowski et al., 1990). The two occupational studies examined the respiratory health of 

31 plywood workers (Fransman et al., 2003; Malaka and Kodama, 1990). The most recent of these 

32 was conducted in New Zealand by Fransman et al. (2003). Personal samples of formaldehyde 

exposure were taken. The mean level of exposure was 0.08 mg/m3 33 (65 ppb) and the majority of 

samples were below the limit of detection which was reported to be 0.03 mg/m3 34 (24 ppb). 

35 Compared with those with low levels of formaldehyde exposure, workers with high levels of 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 exposure were more likely to report having asthma (OR=4.3 [95% CI]: 0.7–27.7]). The 

2 association was not seen when examining formaldehyde exposure and use of asthma medication. 

3 The second study of plywood workers was completed in Indonesia. Background levels of 

4 formaldehyde ranged from 0.003 to 0.07 ppm. The highest concentration of formaldehyde 

5 detected in an air sample was in the particleboard unit (range 1.16 to 3.48 ppm). Asthma, which 

6 was defined as “have you ever had an attack of wheezing that made you feel short of breath?”, 

7 was found to be positively associated with formaldehyde exposure (Malaka and Kodama, 1990).

8 Studies of exposure to formaldehyde at schools have been performed in China (Zhao et 

9 al., 2008) and in Sweden (Smedje and Norback, 2001). In the study from China (Zhao et al., 

2008), mean levels of formaldehyde were reported to be 2.3 μg/m3 (range 1.0–5.0 μg/m3 10 ) 

indoors and 5.8 μg/m3 (range 5.0–7.0 μg/m3 11 ) outdoors. Cumulative asthma and daytime attacks 

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

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

14 levels were found to be associated with nocturnal attacks of breathlessness. In Sweden (Smedje 

15 and Norback, 2001), the levels of formaldehyde measured indoors were higher (mean 4, range 

<5.0–72 μg/m3 16 ). One difference between this Swedish study and the study performed in China is 

17 that the Swedish study examined the incidence of asthma over a 4-year period. This study did 

18 not report an association between formaldehyde exposure and the incidence of asthma (OR 1.2 

19 [95% CI: 0.8–1.7]) among the whole study population. However, when the investigators 

20 stratified on history of atopy, they reported that among children without a history of atopy, a new 

21 diagnosis of asthma was significantly more likely at higher concentrations of formaldehyde (OR 

1.7 per 10 μg /m3 22 [95% CI: 1.1–2.6]) and at higher total concentrations of mold (OR=4.7 per 10-

23 fold increased in total molds [95% CI: 1.2-18.4] in the classroom air. The finding for adverse 

24 effects of formaldehyde and mold did not appear to control for the other exposure and no 

25 information on the potential correlation between the two exposures was provided. In order to 

26 evaluate the potential for confounding of the reported formaldehyde association by the reported 

27 mold association, the magnitude of effects must be compared on an appropriate scale since the 

28 magnitude of an odds ratio depends on the magnitude of the change in exposure level that is 

29 expected to produce increased risk. Standardizing the units to the reported geometric mean 

standard deviation, the result for formaldehyde (GSM=2.3 μg /m3) is OR1 30 =1.13 per GSD and the 

result for mold is OR2

=1.02 for a comparison of risks at the GSM to 10*GSM and OR3

 1 OR per GSD=exp[ln(OR per μg /m3

)/ 10 μg /m3 * 2.3 μg /m3]=exp[ln(1.7)/10*2.3]=1.13

31 =1.06 for 

2 OR per GSD=exp[ln(OR per 10-fold increase)/ (9*GSM)*1.6 μg /m3

]=exp[ln(4.7)/162*1.6]=1.02

3 OR per GSD=exp[ln(OR per 10-fold increase)/ (9*Minimum)*1.6 μg /m3

]=exp[ln(4.7)/45*1.6]=1.06
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a comparison of risks at the minimum value of total molds (5*103/m3 1 ) to 10*minimum. As it 

2 appears that the magnitude of the formaldehyde effect is substantially stronger than that of the 

3 mold effect (following standardization of exposure increment) it can be concluded that the 

4 reported formaldehyde effect could not have been the spurious result of uncontrolled 

5 confounding by mold.

6 The results of studies measuring residential exposure to formaldehyde and asthma are 

7 varied, with some demonstrating an association and others finding no relationship. A recent 

8 study (Matsunaga et al., 2008) found no association between 24-hour formaldehyde and 

9 prevalence of asthma when pregnant women with an exposure ≥47 ppb were compared to those 

10 with exposure to <18 ppb. However, they did report at increased risk of atopic eczema. It 

11 should be noted that this study did not assess the risk of incident asthma. A study utilizing self12 reported asthma prevalence as an outcome also found no association with levels of formaldehyde 

(mean 25.9 μg/m3

, range 2.0–66.8 μg/m3 13 ) (Palczynski et al., 1999) although they did note that 

14 the incidence of allergic diseases was highest in the highest formaldehyde exposure group but 

15 that the group was too small for statistical evaluation.

16 A study performed by Tuthill (1984) measured formaldehyde exposure for children 

17 grades K through 6 by using a combination of proxy variables. Overall, there was no 

18 association, but some individual variables did show an increased risk. For example, the reported 

19 risk ratio for having new construction or remodeling performed in the house in the past 4 months 

20 was 2.5 (95% CI: 1.7–3.9). The risk ratio for having new or upholstered furniture in the house 

21 (brought into the house within the past 4 months) was 2.2 (95% CI: 1.2–3.9).

22 The study by Delfino et al. (2003) assessed whether ambient formaldehyde concentration 

23 measured at a central monitoring site were associated with asthma symptoms. The study 

24 examined 22 10–15 year olds with at least 1 year of physician-diagnosed asthma and living in a 

25 nonsmoking household. The mean levels of formaldehyde were measured to be 7.21 ppb (range 

26 4.27–14.02 ppb). There was a positive association between asthma symptom scores (comparing

27 children who report symptoms interfering with their daily activities versus those with no 

28 symptoms or symptoms not great enough to affect their daily activities) and high current levels 

29 of formaldehyde (OR 1.90 [95% CI: 1.13–3.19]).

30 Three studies (Tavernier et al., 2006; Gee et al., 2005; Garrett et al., 1999) were 

31 performed by matching children with and without asthma and comparing the levels of 

32 formaldehyde in their homes. Gee et al. (2005) reported median formaldehyde levels of 0.03 

33 ppm in living rooms and 0.04 ppm in bedrooms. Analyses were limited to univariate 

34 comparisons of formaldehyde levels for cases of existing asthma and controls without asthma. 

35 The concentrations did not differ in a statistically significant manner. The study by Gee et al. 
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1 (2005) was followed up with a more sophisticated analysis of the same children in the same 

2 homes. Tavernier et al. (2006) reiterated the earlier finding by Gee et al (2005) that 

3 formaldehyde was not found to be associated with existing asthma. Tavernier et al. (2006) did 

4 not report the measured levels of formaldehyde but gave the OR for the highest tertile of 

5 exposure compared with the lowest tertile of exposure as 0.99 (95% CI: 0.39–2.50). The width 

6 of this confidence interval suggests that these findings would still be consistent with two-fold 

7 increase in risk.

8 Garrett et al. (1999) reported on the risk of allergy and asthma-like respiratory symptoms 

9 due to formaldehyde exposure in a cross-sectional survey of households with children with (n = 

10 53) or without (n = 88) doctor-diagnosed asthma. Formaldehyde exposure was characterized by 

11 4 seasonal in-home sampling events across the year for bedrooms and 4–day passive samples 

12 collected in living rooms, kitchens and outdoors. Statistically significant linear trends for 

13 increased risk of having asthma were seen with increasing formaldehyde levels (p < 0.02); 

14 however, the ORs for the association did not remain statistically significant after controlling for 

15 parental allergy and asthma (exact ORs and 95% CIs not given). Garrett et al (1999) also 

16 evaluated the prevalence and severity of allergic sensitization to 12 common allergens and 

17 reported increased prevalence with increasing formaldehyde concentration in the home. The 

18 respiratory symptom score was also increased and demonstrated a significant effect for 

19 formaldehyde in a multiple regression after adjusting for multiple risk factors and interactions. 

20 For the atopy and respiratory symptom endpoints, severity/incidence was increased in the 

medium (20–50 μg/m3

) and high (>50 μg/m3) exposure groups relative to the low (<20 μg/m3 21 ) 

22 exposure group, based on the highest of four seasonal 4-day formaldehyde measurements in the 

23 home. The associations between formaldehyde concentrations and severity of allergic 

24 sensitization are clearly shown and further substantiated with multivariate regression controlling 

25 for potential confounders. In logistic regressions, both the prevalence and severity of allergic 

26 sensitization to 12 common allergens increased with increasing formaldehyde concentration in 

27 the home. The crude association for atopy with an increase in formaldehyde concentration per 

10 μg/m3 28 was OR=1.34 which increased when adjusted for parental asthma and gender to and 

odds ratio of 1.42 per 10 μg/m3 29 (95% CI: 0.99-2.04). Passive smoking, the presence of pets, 

30 indoor nitrogen dioxide concentrations, airborne fungal spores and house-dust-mite allergens did 

31 not influence the effect estimates and were unlikely to be confounders. Additionally, a 

32 calculated respiratory symptom score was increased and demonstrated a significant relationship 

33 to increased formaldehyde concentration in a multiple linear regression after adjusting for 

34 multiple risk factors and interactions. For each of these endpoints, severity/incidence was

increased in the medium (20–50 μg/m3) and high (>50 μg/m3 35 ) exposure groups relative to the 
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low (<20 μg/m3 1 ) exposure group, based on the highest of four seasonal 4-day formaldehyde 

2 measurements in the home. 

3 Residential formaldehyde exposure was associated with an increased risk of asthma in a 

4 population-based case-control study of 192 children aged 6 months to 3 years (Rumchev et al., 

5 2002). The study, which comprises 88 cases of children discharged from the emergency 

6 department of a children’s hospital in Perth, Australia, with a primary diagnosis of asthma and 

7 104 controls, provides a positive exposure-response relationship. Seasonal in-home 

8 formaldehyde measurements taken in the living room and subject’s bedroom were used to assess 

9 exposure (8-hour passive sampler). The odds ratios (ORs) for risk of asthma by formaldehyde 

10 exposure level category were adjusted for numerous risk factors both familial and environmental 

11 including, familial history of asthma, age, sex, smoking, presence of pets, and attributes of the 

12 home. Of these, age, allergic sensitization to common allergens, and family history of allergy 

13 were independent risk factors for asthma (ORs of 1.09, 2.57, and 2.66, respectively). Categorical 

14 analysis of the data indicates the ORs for asthma were increased in the two highest formaldehyde 

exposure groups, reaching statistical significance for household exposures > 60 μg/m3 15 (48 ppb) 

16 (OR of 1.39). Analysis of the data with formaldehyde as a continuous variable provides a 

17 statistically significant increase in the risk of asthma (3 % increase in risk per every 10 ug/m3 

18 increase in formaldehyde level. All analyses controlled for other indoor air pollutants, allergen 

19 levels, relative humidity, and indoor temperature as well as other risk factors.

20 A study of 202 households (mean formaldehyde level of 26 ppb) found that among 

21 children aged 6–15 years old and exposed to environmental tobacco smoke, the prevalence of 

22 asthma was 45.5% for those with measured levels of formaldehyde in the kitchen >60 ppb. The 

23 prevalence of asthma dropped to 15.1% for levels ≤40 ppb and 0% for 41–60 ppb. No trend in 

24 asthma prevalence was seen for children who were not exposed to environmental tobacco smoke 

25 (Krzyzanowski et al., 1990).

26 Finally, a study by Norback et al. (1995) reported mean levels of formaldehyde were 29 

μg/m3 (range <5–110 μg/m3 27 ) in the bedrooms of individuals experiencing nocturnal 

breathlessness compared with formaldehyde levels of 17 μg/m3 (<5–60 μg/m3 28 ) among those 

29 without nocturnal breathlessness. The OR for this association was 12.5 (95% CI: 2.0–77.9) and 

30 the effect was substantially stronger in magnitude than the associations observed for toluene, 

31 terpenes and volatile organic compounds which makes confounding by those co-exposures 

32 unlikely.

33 Formaldehyde has clearly been shown to be a cause of bronchial asthma and several 

34 epidemiologic studies have identified causal evidence of an adverse effect of exposure on 

35 pulmonary function and the incidence of asthma. While there are studies that did not find 
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1 associations, many of those were limited by their study design, exposure measurement and the 

2 definition of prevalent asthma as the health endpoint.

3

4 4.1.1.4. Respiratory Tract Pathology

5 Formaldehyde-induced respiratory tract pathology includes inflammation, rhinitis, goblet 

6 cell hyperplasia, metaplastic changes, squamous cell hyperplasia, and impaired mucociliary 

7 transport. Formaldehyde may bind to the trigeminal nerve and trigger the release of neurogenic 

8 mediators of inflammation that result in tissue edema, lacrimation, mucus production and 

9 leukocyte infiltration. How much inflammation, hyperplasia, and metaplastic change are due to 

10 sensory irritation-induced inflammatory responses compared with formaldehyde-induced direct 

11 cell damage cannot be distinguished. Increased mucus flow and metaplastic changes may 

12 progress in relation to the concentration and duration of exposure to protect the underlying 

13 tissue. When the exposure exceeds protective and defensive mechanisms, permanent damage 

14 results (Swenberg et al., 1983). Nonetheless, these changes serve as a sensitive indicator of 

15 formaldehyde exposure, since they occur before gross cellular damage and focal lesions 

16 (Monticello et al., 1989), and potentially suggest a point at which the concentration and duration 

17 of exposure exceed the protective nature of local responses (increased mucus flow, goblet cell 

18 hyperplasia, squamous metaplasia, etc.) (Swenberg et al., 1983). A number of human studies 

19 have reported nasal lesions associated with exposure to formaldehyde (Pazdrak et al., 1993; 

20 Ballarin et al., 1992; Boysen et al., 1990; Holmström et al., 1989c; Edling et al., 1988), while 

21 other studies have documented changes in mucociliary clearance and activity ( Holmström and 

22 Wilhelmsson, 1988; Andersen and Molhave, 1983). These studies are summarized below.

23

24 4.1.1.4.1. Nasal lesions. Ballarin et al. (1992) did a case-control study of 15 workers from a 

25 plywood factory where urea-formaldehyde glue is used. Mean levels of formaldehyde exposure 

(8-hour average) were estimated to be 0.09, 0.1, and 0.39 mg/m3 26 in three regions of the facility 

27 (sawmill, shearing press, and warehouse, respectively). Nasal respiratory samples were 

28 obtained. Stained cells were scored for histopathology. Cytology examination revealed 

29 increased squamous metaplasia cells in 10 of 15 (67%) factory workers (with an average severity 

30 score of 2.3) compared with one of 15 (6%) controls (with an average histology severity score of 

31 1.6). In addition, one formaldehyde exposed worker (n = 15) exhibited mild dysplasia and had 

32 the highest severity score (3.0). Authors suggest that these results may be due to chronic 

33 irritation of the nasal respiratory mucosa. This small study reported only incidence of lesions 

34 and did not score based on severity of lesions. The lesion incidence was not reported in relation 
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1 to dose, so no dose-response relationship could be determined, precluding the establishment of a 

2 point of departure (POD).

3 Holmström et al. (1989) collected nasal biopsy samples from workers exposed to air or to 

4 formaldehyde at a median concentration of 240 ppm. Nasal biopsy samples were scored on a 

5 0–8 range with normal respiratory epithelium as 0 and carcinoma as 8. Observed histologic 

6 changes included loss of cilia, goblet cell hyperplasia, and cuboidal and goblet cell metaplasia 

7 replacing normal columnar epithelium. The incidence associated with each histologic change 

8 was not reported and cannot be compared between formaldehyde-exposed and control 

9 individuals. Moreover, these biologically relevant changes were not analyzed independently in 

10 the analysis. The mean scores were 1.56 (range, 0–4) for the control group and 2.16 (range, 0–4) 

11 for the formaldehyde-exposed group. Although the range of scores in the controls and 

12 formaldehyde-exposed groups were the same (0–4), the difference in mean scores (1.56 versus 

13 2.16) was statistically significant (p < 0.05); scores were worse in the formaldehyde-exposed 

14 group. The authors reported no correlation between the duration of exposure and histologic 

15 changes and no correlation between smoking habits and biopsy scores. The loss of cilia, goblet 

16 cell hyperplasia, and cuboidal and squamous cell metaplasia replacing the columnar epithelium 

17 were increased in the group exposed to formaldehyde and is a biologically relevant change. This 

18 study provides a lowest-observed-adverse-effect level (LOAEL) of 0.240 ppm for nasal 

19 histopathology.

20 Edling et al. (1988) collected nasal biopsy samples from workers (n = 75) exposed to 

21 formaldehyde at three plants (workers in two of these plants were also exposed to wood dust) 

compared with a referent group (n = 25). Concentrations ranged from 0.1 to 1.1 mg/m3 22 (TWA) 

with peaks of 5 mg/m3 23 . Nasal histology was scored from 0 to 8 by increasing severity, from 

24 normal respiratory epithelium (0) to carcinoma (8). A normal respiratory epithelium was noted 

25 in 3 of 75 workers. A loss of cilia and goblet cell hyperplasia (scores of 2) was reported in eight 

26 workers. Mixed cuboid/squamous epithelium (metaplasia), stratified squamous epithelium, and 

27 keratosis were reported in 58 of 75 workers (those with scores of 3, 4, and 5 were combined). 

28 Dysplasia (score of 6) was reported in 6 of 75 formaldehyde-exposed workers. None of the 

29 workers had lesions that warranted a histologic score higher than 6. Histologic scores did not 

30 correlate with duration of exposure but could not be confirmed due to poor reporting. Data from 

31 the referent group were not included. A POD could not be determined from this study.

32 Boysen et al. (1990) collected nasal biopsy samples from workers exposed to air (n = 37) 

33 or to formaldehyde (n = 37) and sometimes wood dust. The exposed workers were classified 

34 into two exposure groups, 0.5–2 ppm and >2 ppm. Nasal biopsy samples were assessed by using 

35 a histopathology score range of 0–5, based on the pathology of pseudostratified columnar 
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1 epithelium (0) to dysplasia (5). Mean pathology scores for the control were decreased compared 

2 with the formaldehyde-exposed group (1.4 and 1.9, respectively) but did not reach statistical 

3 significance. Little quantitative pathology data were provided, although qualitative histology 

4 revealed a range of observed effects from deciliated epithelial cells to mixed stratified cuboidal, 

5 squamous epithelium to dysplasia. None of the control samples received histologic severity 

6 scores of 4 or 5, indicating that keratinizing stratified squamous epithelium and dysplasia were 

7 not observed in controls. A wider variety of histopathologic lesions were reported in exposed 

8 workers compared with controls, and a greater number of exposed workers had histologic 

9 changes compared with controls. Incidence data for each type of histopathology were not 

10 reported, but the authors wrote that the degree of metaplastic alterations was more pronounced 

11 among the exposed workers. An upper range for the high concentration group (>2 ppm) was not 

12 reported, and median concentrations were not provided. 

13 Pazdrak et al. (1993) exposed human subjects (six men, three women) to 0.4 ppm 

14 formaldehyde in a chamber for 2 hours. Approximately half of the subjects suffered from skin 

15 hypersensitivity to formaldehyde, while the other subjects were healthy. An evaluation of nasal 

16 lavage pretest and following formaldehyde exposure revealed that the hypersensitive and healthy 

17 groups had similarly elevated eosinophil counts at 0 hours after exposure (from 

42 × 103 cells/mL to 72 × 103 18 cells/mL for healthy subjects [p < 0.05] and from 

39 × 103 cells/mL to 69 × 103 19 cells/mL for hypersensitive subjects [p < 0.05]). Similar 

20 eosinophil levels were also seen in both groups at 3 and 18 hours. Both groups had equivalent 

21 increases in lavage albumin and total protein levels following exposure, but basophil counts were 

22 unchanged. Based on evidence of formaldehyde-induced inflammation, these data provide a 

23 LOAEL of 0.4 ppm for nasal histopathology.

24

25 4.1.1.4.2. Mucociliary clearance. In addition to abnormal nasal histopathology, changes in 

26 mucociliary clearance were also observed in some of these studies at similar exposure 

27 concentrations. The mucociliary apparatus is an important barrier to infection and exogenous 

28 agents and, thus, is considered as a potential adverse effect. These effects may be due to direct 

29 interaction of formaldehyde with the mucus itself or to SI-induced inflammation in the nasal 

30 tissue that affects mucus production and creation of an effective mucosal barrier.

31 Andersen and Molhave (1983) reviewed five controlled human studies, one of which 

32 (Andersen and Lundqvist, 1974) examined mucus flow rate in 16 individuals acutely exposed to 

33 0, 0.3, 0.5, 1, or 2 ppm formaldehyde for 4–5 hours in a chamber. Mucus flow rate was 

34 decreased in the anterior and middle third of the ciliated mucosa at 0.3 ppm, but statistical 

35 significance was not determined. This study included smokers and nonsmokers. The small 
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1 sample size, potential confounder effect from smoking, and lack of dose-response relationship 

2 preclude the establishment of a POD.

3 Holmström and Wilhelmsson (1988) demonstrated reduced mucociliary clearance and 

4 nasal mucosal swelling in 70 workers exposed to a median formaldehyde concentration of 

5 0.21 ppm, compared with a referent group of store clerks (n = 36) and was further averaged over 

6 years of exposure. Mucosal swelling and mucociliary activity was measured in the nasal 

7 turbinates. The authors also reported symptoms not only during the weekdays, but also over 

8 weekends and vacation periods. Formaldehyde-exposed subjects self-reported significantly more 

9 nasal discomfort, eye discomfort, deeper airway discomfort, and frequent headache than the 

10 referent group. Groups exposed to formaldehyde had more pronounced mucosal swelling (10.7 

11 nasal resistance score) compared with the reference group (6.5 nasal resistance score). This 

12 difference persisted when data were normalized for differential nasal congestion in the subjects. 

13 Decreased mucociliary activity was seen in 3% of controls and 20% of formaldehyde-exposed 

14 subjects and reached statistical significance (p < 0.05). It is not clear whether impaired 

15 mucociliary clearance was a consequence of altered cell morphology or increased mucus 

16 viscosity. These data provide a LOAEL of 0.21 ppm based on impaired mucociliary clearance.

17 Thus, mild nasal epithelial lesions observed in formaldehyde-exposed workers have been 

18 observed consistently at levels of about 0.20 ppm to about 2 ppm (Boysen et al., 1990; 

19 Holmström et al., 1989; Edling et al., 1988). Of these, Holmström et al. (1989) and Edling et al. 

20 (1988) do not appear to be confounded by exposure to wood dust. Nasal biopsy pathology from 

21 formaldehyde-exposed workers is consistent with irritant and reactive properties of 

22 formaldehyde (Ballarin et al., 1992; Boysen et al., 1990; Holmström et al., 1989; Edling et al., 

23 1988; Berke, 1987). Moreover, these findings are supported by results from animal toxicity and 

24 pharmacokinetic and anatomical airflow studies, indicating that, at concentrations less than 

25 1 ppm, inhaled formaldehyde gas does not reach lower regions of the respiratory tract. Of the 

26 available human studies that evaluated histopathology, Holmström and Wilhelmsson (1988) 

27 appears to be the most robust and sensitive. The study was carefully designed and included a 

28 large sample of formaldehyde-exposed subjects who were considered separately from workers 

29 exposed to combinations of exposures (formaldehyde and wood dust). Study subjects had been 

30 exposed to formaldehyde regularly for many years. The authors reported not only weekday 

31 exposures but effects reported on weekends and on vacation. Total exposure was carefully 

32 calculated and averaged. The data were controlled for potential confounders, such as smoking. 

33 The endpoint of reduced mucociliary clearance has been substantiated by Andersen and Molhave 

34 (1983) and Holmström et al. (1989). Animal studies have also reported formaldehyde-induced 

35 changes on the nasal mucosa and are highlighted in Section 4.2.1.2.
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1 4.1.1.5. Immunologic Effects

2 Numerous studies have examined the immunologic responses of individuals exposed to 

3 formaldehyde. This section will discuss four specific areas related to immunotoxicity after 

4 exposure to formaldehyde: increased upper respiratory tract (URT) infections, systemic immune 

5 dysfunction, sensitization and atopy, and production of formaldehyde-protein complexes. Some 

6 studies report increased incidence of URT infections after exposure to formaldehyde (Lyapina et 

7 al., 2004; Krzyzanowski et al., 1990; Holness and Nethercott, 1989). This effect appears to 

8 occur independently of systemic immune changes and may be due to damage to the mucosal 

9 barrier, thus facilitating pathogen access. A number of studies have investigated the hypothesis 

10 that formaldehyde may induce systemic immunomodulation (Ohtani et al., 2004a, b; Erdei et al., 

11 2003; Thrasher et al., 1990, 1987; Pross et al., 1987). Some studies have also evaluated immune 

12 system effects by investigating the role of reactive oxygen species (ROS) from respiratory burst 

13 associated with immune cells (Lyapina et al., 2004; Gorski et al., 1992) and by assessing 

14 chromosomal damage in immune cells (Orsière et al., 2006; Yu et al., 2005). In addition to the 

15 effects of formaldehyde on asthmatics and the potential for formaldehyde exposure to exacerbate 

16 asthmatic responses, reviewed in Section 4.1.1.2, numerous studies have investigated whether 

17 formaldehyde may directly induce sensitization and atopic responses by measuring 

18 immunoglobulin E (IgE) levels associated with formaldehyde exposure (Ohmichi et al., 2006; 

19 Vandenplas et al., 2004; Doi et al., 2003; Baba et al., 2000; Palczynski et al., 1999; Krakowiak et 

20 al., 1998; Wantke et al., 1996a, b; Liden et al., 1993; Salkie, 1991; Grammer et al., 1990; 

21 Kramps et al., 1989). Findings are largely negative and suggest that formaldehyde-induced IgE 

22 production is not likely. Lastly, studies have investigated the production of formaldehyde23 specific antibodies, formaldehyde-albumin complexes, and formaldehyde-heme complexes (Kim 

24 et al., 2001; Carraro et al., 1997; Grammer et al., 1993, 1990; Dykewicz et al., 1991; Thrasher et 

25 al., 1990). Heme complex formation is not a strict immunologic endpoint but may trigger 

26 antibody formation and thus it will be discussed in this section. This section will thus summarize 

27 the human studies that have specifically addressed the increased incidence of URT infections, 

28 immunotoxic endpoints, atopy and sensitization, and formation of formaldehyde-heme and 

29 formaldehyde-albumin complexes.

30

31 4.1.1.5.1. Increased URT infections. Diverse studies have investigated the possibility that 

32 formaldehyde exposure leads to increased URT infections (Lyapina et al., 2004; Krzyzanowski 

33 et al., 1990; Holness and Nethercott, 1989). Lyapina et al. (2004) studied 29 workers who were 

34 occupationally exposed occupationally to formaldehyde for an average of 12.7 years through 

35 contact with carbamide-formaldehyde glue. The mean values of the average shift concentrations 
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1 of formaldehyde in the application of carbamide-formaldehyde glue to be 0.71 ppm TWA with a 

2 range of 0.32 to 1.57 ppm. The workers were divided into two subgroups, one (n = 12) that 

3 suffered from either a long history (with clinical findings) of chronic mucous inflammation of 

4 the URT with multiple relapses and a second group (n = 17) whose URT inflammations were 

5 short, acute, and predominantly viral. Twenty-one healthy subjects served as controls. A 

6 statistically significant association of self-reported chronic bronchitis and decreased resistance to 

7 URT infection was reported in all the exposed workers compared with controls (p = 0.02). Of 

8 the workers, 41% had a history of chronic respiratory infection and frequent long-lasting 

9 infectious inflammatory relapses (group 1a). Another group (group 1b) consisted of 17 exposed 

10 workers, 12 of whom had no history of recurrent viral infections of the URT. There was a 

11 statistically significant association of frequency and duration of inflammatory relapses between 

12 groups 1a and 1b. No dates were provided regarding when these measurements were made or 

13 over what period of time they were calculated.

14 Krzyzanowski et al. (1990) measured formaldehyde levels in homes and recorded, by 

15 way of a questionnaire, health histories from adult and child residents. Formaldehyde levels 

16 were reported from samples taken for two 1-week periods in various rooms of the home (kitchen, 

17 living room, subject’s bedroom). The average formaldehyde level was 26 ppb in 202 homes, and 

18 levels were stratified into homes with exposure levels below 40 ppb, between 40 and 60 ppb, and 

19 above 60 ppb. Incidences of doctor-diagnosed chronic bronchitis were more prevalent in 

20 children (under age 15) living in homes with higher formaldehyde (>60 ppb) readings in the 

21 kitchen (p < 0.001). This effect was more pronounced (p < 0.001) in children simultaneously 

22 exposed to environmental tobacco smoke. The prevalence of chronic cough was also increased 

23 in adults living in homes with measurable levels of formaldehyde, but data were not shown. 

24 Holness and Nethercott (1989) assessed chronic bronchitis in 87 funeral workers, where the 

25 average formaldehyde exposure was reported at 0.38 ± 0.19 ppm. Chronic bronchitis was 

26 observed in 20 funeral workers (n = 87) exposed to formaldehyde compared with 3 cases of 

27 chronic bronchitis in nonexposed referent controls (n = 38).

28 These studies suggest that exposure to formaldehyde may be associated with increased 

29 incidence of chronic bronchitis. The mechanism for this association has not been elucidated. 

30 Pathogens may gain access to the URT via a compromised mucosal barrier, as has been shown in 

31 histopathology studies (Section 4.1.1.4).

32

33 4.1.1.5.2. Immune function. A number of studies have evaluated the ability of formaldehyde to 

34 induce systemic immunotoxic effects (Ohtani et al., 2004a, b; Erdei et al., 2003; Thrasher et al., 

35 1990, 1987; Pross et al., 1987). Some studies have reported altered innate immune responses 
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1 associated with formaldehyde exposure (Erdei et al., 2003), while others have noted adaptive 

2 immune response suppression associated with formaldehyde exposure (Thrasher et al., 1990, 

3 1987) and changes associated with alterations to a predominant T—lymphocyte helper 2 (Th2) 

4 pattern (Ohtani et al., 2004a, b). In contrast, Pross et al. (1987) did not observe formaldehyde5 associated changes in systemic immune function.

6 Erdei et al. (2003) found that Haemophilus influenzae humoral biomarker (H.in.IgG), 

7 Klebsiella pneumoniae biomarker (K.pn.IgG), and elevated monocyte concentrations were 

8 significantly associated with high formaldehyde concentrations in asthmatic children, compared 

9 with nonsensitive children. Briefly, Erdei et al. (2003) compared the immune system responses 

10 in 9- to 11-year-old Hungarian school children whose respiratory systems were immunologically 

11 compromised (chronic respiratory disease, asthma) and normal children who were exposed to 

12 indoor air pollutants, including formaldehyde. In the homes of the children with the highest 

13 levels of pollutants, 49.3% of formaldehyde measurements exceeded the Hungarian indoor 

14 standard of 0.01 ppm, while 20% exceeded the World Health Organization’s (WHO’s) suggested 

15 indoor level of 0.09 ppm. The authors excluded from consideration all measurements that 

16 exceeded WHO’s air quality guidelines in one unidentified city to prevent a “city-related bias,” 

17 since these measurements occurred entirely in that city. The average formaldehyde 

18 concentration in the 123 homes tested was 14 ppm with a range of 0.5 to 46 ppm. H.in.IgG and 

19 K.pn.IgG were significantly associated with high formaldehyde concentrations (p < 0.013 and 

20 p < 0.049, respectively) in sensitive children compared with nonsensitive children. These 

21 markers were also correlated with high levels of nitrogen dioxide, the number of cigarettes 

22 smoked, and exposure to paint, volatile organic compounds, and solvents. Additionally, indoor 

23 formaldehyde exposure was significantly associated with increased monocyte concentrations 

24 (p < 0.017) that are important to the innate immune response (inflammation) in diseased tissue. 

25 The authors concluded that the elevation of immune biomarkers in sensitive children with 

26 respiratory disease is likely the result of high concentrations of toxic indoor air pollutants, 

27 including formaldehyde.

28 Thrasher et al. (1987) assessed the effects of formaldehyde exposure on cellular 

29 immunity and antibody formation in eight exposed and eight unexposed individuals. The 

30 exposed group consisted of three males and five females. Seven of the exposed individuals had 

31 resided in mobile homes for periods ranging from 2 to 7 years; the eighth was a laboratory 

32 worker who resided in a newly decorated, energy-efficient apartment. Air monitoring in four of 

33 the homes revealed formaldehyde vapor concentrations ranging from 0.07 to 0.55 ppm. Venous 

34 blood samples were collected from all subjects and T- and B-cells were counted and monitored 

35 for blastogenesis. When IgG and IgE antibodies to formaldehyde were monitored in serum, no 
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1 IgE antibodies to formaldehyde were detected in exposed or control subjects. IgG antibody titers 

2 in exposed subjects ranged from 1:8 to 1:256 but essentially were undetected (1:4) in seven of 

3 the controls. T- and B-cell numbers were significantly lower (p < 0.05) in mobile home residents 

4 (48 and 12.6%, respectively) compared with those of control subjects (65.9 and 14.75%, 

respectively). As determined by incorporation of 3 5 H-thymidine into 48-hour unaltered 

6 lymphocytes, phytohemagglutinin-stimulated T- and B-cell blastogenesis was significantly 

7 depressed (p < 0.01) in cells of mobile home residents compared with those of control subjects 

8 (17,882 and 28,576 cpm, respectively). Thrasher et al. (1987) concluded that exposure to 

9 formaldehyde decreases the proportion of peripheral T cells.

10 In a later study, Thrasher et al. (1990) evaluated five groups of subjects with varying 

11 levels and durations of formaldehyde exposure. The groups consisted of (1) asymptomatic 

12 chiropractic students exposed during anatomy classes (controls with only intermittent exposure 

13 to formaldehyde), (2) mobile home residents, (3) office workers, (4) patients with multiple 

14 symptoms who had been removed from the source of formaldehyde for at least a year, and 

15 (5) occupationally exposed patients. All groups were assessed for immunologic function via 

16 white cell, lymphocyte, and T-cell counts, T-helper/suppressor ratios and B-cell counts. When 

17 compared with controls (chiropractic students), the patient groups had significant elevations in 

18 formaldehyde antibody titers and B-cell titers.

19 Ohtani et al. (2004a, b) reported effects of exposure to formaldehyde and diesel exhaust 

20 particles on cytokine production by human monocyte-derived dendritic cells (MoDCs) and 

21 T cells in vitro. Dendritic cells were stimulated with CD40 ligand and interferon (IFN)-γ, T cells 

22 with anti-CD3/CD28 antibodies. Cytokine proteins and mRNA levels were measured in 

23 supernatants by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction 

24 (PCR), respectively. Formaldehyde and diesel exhaust particles significantly increased tumor 

25 necrosis factor (TNF)-α levels and suppressed interleukin (IL)-12p40 protein and mRNA levels 

26 in MoDCs. The same treatment suppressed protein synthesis and mRNA expression of IFN-γ

27 and IL-10 in T cells. The authors concluded that their findings support a role of formaldehyde 

28 and diesel exhaust particles in altering the immune response to a Th2-dominant pattern that 

29 furthers allergic inflammation. Further details, such as exposure concentrations and 

30 experimental protocols, are not available.

31 In contrast, Pross et al. (1987) concluded that formaldehyde does not induce altered 

32 immune activity. The authors evaluated the immunologic response of asthmatic subjects 

33 exposed to UFFI off-gas products. Subjects consisted of 23 individuals with a history of 

34 asthmatic symptoms attributed to UFFI and 4 individuals with asthma unrelated to UFFI off-gas 

35 products. All subjects were exposed in an environmental chamber according to the following 
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1 sequence: (1) room air (placebo) for 30 minutes; (2) 1 ppm formaldehyde gas for 3 hours; (3) 

2 UFFI particles (4 μm diameter, 0.5 particles/mL) for 3 hours, commencing 48 hours after 

3 formaldehyde gas exposure; and (4) UFFI off-gas products for 3 hours, commencing 48 hours 

4 after UFFI particle exposure. There was a significant increase in the percentage and absolute 

5 number of eosinophils and basophils in the subjects who lived in UFFI homes but no differences 

6 between exposure groups with respect to lymphocyte subpopulations either before or after UFFI 

7 exposure. However, when T8 suppressor cells were counted, values in the UFFI-exposed group 

8 pre-exposure and postexposure, a small but statistically significant (p < 0.01) increase in T8 cell 

9 count was observed. The biological significance of this increase in T8 cell count in exposed 

10 asthmatics is not known. Pross et al. (1987) concluded that short-term exposure to formaldehyde 

11 was not immunosuppressive and did not result in systemic immune reactivity.

12 Respiratory burst from immune cells creates ROS that can incur further cellular damage. 

13 Several studies have evaluated, either directly or indirectly, the potential role of ROS as potential 

14 mediators of formaldehyde-associated effects, particularly those caused by immune cells. Gorski 

15 et al. (1992) measured chemiluminescence resulting from the release of free radicals from 

16 granulocytes of healthy and formaldehyde-sensitive subjects. Thirteen subjects with contact 

17 dermatitis who were occupationally exposed to formaldehyde and five healthy volunteers 

18 participated in the study. All underwent skin-prick tests for common allergens as well as a 

histamine inhalation provocation test. Subjects were exposed to 0.5 mg/m3 19 (0.41 ppm) 

20 formaldehyde for 2 hours, and the PEFR was measured immediately before exposure, after 60 

21 and 120 minutes of exposure, and 6 and 21 hours after completion of exposure. Peripheral blood 

22 granulocyte chemiluminescence was measured in the presence of luminol. Free radical 

23 production was increased significantly within 30 minutes of beginning the exposure in subjects 

24 with allergic dermatitis and remained elevated for 24 hours compared with baseline values. 

25 Gorski et al. (1992) concluded that granulocyte chemiluminescence did not increase in healthy, 

26 formaldehyde-exposed patients but was diagnostic for formaldehyde-sensitive patients. These 

27 results also suggest a putative role for oxidative damage associated with formaldehyde exposure, 

28 particularly in sensitized individuals.

29 Lyapina et al. (2004) also reported effects of formaldehyde exposure on neutrophil 

30 respiratory burst activity (NRBA), the capacity of polymorphonuclear leukocytes to produce 

31 reactive oxygen radicals in response to chemical or microbial stimuli using flow cytometry. 

32 Briefly, Lyapina et al. (2004) studied 29 workers who were occupationally exposed to 

33 formaldehyde for an average of 12.7 years through contact with carbamide-formaldehyde glue 

34 with a mean value of the average shift concentration of formaldehyde reported as 0.71 ppm 

35 TWA with a range of 0.32 to 1.57 ppm. The workers were divided into two subgroups, one (n = 
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1 12) that suffered from either a long history (with clinical findings) of chronic mucous 

2 inflammation of the URT with multiple relapses, and a second group (n = 17) whose URT 

3 inflammations were short, acute, and predominantly viral. Twenty-one healthy subjects served 

4 as controls. A suite of hematological tests and flow cytometric analysis for respiratory burst 

5 activity were performed. Although no significant difference was observed in the spontaneous 

6 and stimulated NRBA (median percentage of oxidizing cells) between the 29 exposed workers 

7 with URT inflammation and the healthy controls (0.83 versus 1.35, respectively), a separate 

8 comparison of the NRBA of 12 workers with chronic, repeating URT infections and 17 workers 

9 with short, infrequent episodes of URT inflammations was significant (0.45 versus 1.00, p = 

10 0.037). When the NRBA of the group with chronic URT infections (n = 12) was separately 

11 compared with that of the healthy controls (n = 21), the results were also significant (0.45 versus 

12 1.35, p = 0.012). Individuals with chronic URT infections have reduced NRBA that could be 

13 due to formaldehyde exposure. Neutrophils respond to tissue damage or local invasion of 

14 microorganisms and act to phagocytize foreign cells. If neutrophilic activity is hampered or 

15 altered by formaldehyde exposure, then the ability to fight infection will be diminished, leading 

16 to prolonged infection. However, no dose-response pattern of formaldehyde exposure could be 

17 determined from this study.

18 Other investigators have reported chromosomal damage in immune cells due to 

19 formaldehyde (Orsière et al., 2006; Yu et al., 2005). Yu et al. (2005) evaluated chromosomal 

20 damage in lymphocytes from 151 exposed and nonexposed workers from a plywood factory 

21 detected by comet assay. The authors reported that chromosomal damage was statistically 

22 elevated in lymphocytes from formaldehyde-exposed workers compared with controls. 

23 However, no information on exposure duration or levels was provided. Orsière et al. (2006) 

24 studied DNA damage in lymphocytes from 59 hospital employees with formaldehyde exposures 

25 from pathology and anatomy laboratories in five hospitals. Controls were 37 workers from the 

26 same hospitals, matched on gender, age, and smoking habits, with no known exposure to 

27 genotoxic agents. Study participations were excluded if workers had a history of radio- or 

28 chemotherapy or had used therapeutic medications that were known to be mutagenic. 

29 Occupational exposure was determined through 15-minute and 8-hour personal air sampling 

30 during a typical workday. Mean formaldehyde concentrations were 2 ppm (range: <0.1–

31 20.4 ppm) for 15-minute sampling and 0.1 ppm (range: <0.1–0.7 ppm) for 8-hour sampling. No 

32 change in DNA damage was found between the beginning and end of the workday among 

33 exposed workers (3.9 ± 0.6 versus 3.6 ± 0.5 relative light units/ng DNA). However, exposed 

34 workers had significant elevations in the binucleated micronucleated cell rate (BMCR) per 

35 1,000 cells compared with controls (16.9 ± 9.3 versus 11.1 ± 6.0%; p < 0.001), but BMCR did 
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1 not appear to be correlated with formaldehyde concentration. Linear regression analysis showed 

2 that the effect for exposure remained after adjusting for gender, age, smoking, and drinking 

3 habits. For 18 exposed and 18 control workers who underwent cytokinesis-blocked 

4 micronucleus assay (CBMA) combined with fluorescent in situ hybridization (FISH) with pan5 centromeric DNA probe, results showed that the frequency of micronuclei (MN) containing only 

6 one centromere (C1+MN) was elevated among the exposed compared with unexposed workers 

7 (11.0 ± 6.2% versus 3.1 ± 2.4%; p < 0.001). The effect of exposure remained significant after 

8 controlling for gender, age, smoking, and drinking habits. Results from Yu et al. (2005) and 

9 Orsière et al. (2006) suggest that formaldehyde exposure may promote chromosomal damage 

10 leading to micronucleated lymphocytes.

11 Compromised lymphocyte function may significantly contribute to altered immune 

12 status. The mechanism underlying this effect has not been elucidated.

13

14 4.1.1.5.3. Sensitization and atopy. Numerous studies have documented formaldehyde-induced 

15 exacerbation of asthmatic responses (see Section 4.1.1.2). The mechanism of this effect has not 

16 been clarified and has led investigators to assess the potential for formaldehyde to directly induce 

17 formation of formaldehyde-specific antibodies, leading to allergic responsiveness. One case 

18 report showed systemic allergic reactions (e.g., anaphylaxis) to formaldehyde in a patient 

19 undergoing hemodialysis (Maurice et al. [1986] referenced in Thrasher et al. [1990]). Some 

20 studies have evaluated the potential association of formaldehyde-specific IgE in already21 sensitized individuals (Baba et al., 2000; Palczynski et al., 1999). Other studies have 

22 investigated whether formaldehyde can directly induce IgE in nonsensitized individuals. Most of 

23 the studies have not identified presence of formaldehyde-specific IgE (Ohmichi et al., 2006; 

24 Krakowiak et al., 1998; Grammer et al., 1993, 1990; Kramps et al., 1989; Thrasher et al., 1987) 

25 and are summarized below. A few studies (Vandenplas et al., 2004; Doi et al., 2003; Liden et 

26 al., 1993) reported positive IgE against formaldehyde, associated with exposure, but the IgE 

27 titers were either transient (Vandenplas et al., 2004) or were positive in a small subset of 

28 previously sensitized subjects (2 of 15) (Liden et al., 1993). Doi et al. (2003) detected IgE 

29 against formaldehyde in two asthmatic children (out of 122 asthmatic children), but the response 

30 severity did not correlate with exposure level.

31 Palczynski et al. (1999) evaluated whether exposure to formaldehyde might facilitate 

32 specific sensitization to common allergens. The study population was comprised of residents of 

33 apartments built in 1989–1990. Only households with children from 5–15 years were eligible for 

34 the study. A random sample of 120 apartments was selected in which lived a total of 465 

35 persons aged 5–65 years. Individual demographic characteristics and medical histories were 
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1 determined by questionnaire. Residents were tested, using the skin-prick method, for allergen 

2 response to a variety of materials, such as household dust, pollens, and feathers. Total serum IgE

3 levels were measured, and the presence of formaldehyde-specific IgE antibodies was determined. 

4 Measured mean levels of formaldehyde were 21.05 ± 8.94 ppb. No significant relationship 

5 between respiratory allergy prevalence and indoor exposure to formaldehyde was detected. 

6 Significant increases in serum IgE levels were found in children exposed to both environmental 

7 tobacco smoke and formaldehyde.

8 Baba et al. (2000) investigated whether production of formaldehyde-specific IgE could be 

9 detected in adult asthmatics. Formaldehyde exposure levels were not documented. 

10 Formaldehyde-IgE was detected in two asthmatic patients (n = 80), one male and one female, but 

11 the titer of IgE did not parallel the severity of the asthmatic responses and could not be linked to 

12 formaldehyde exposure. Thus, formaldehyde-specific IgE-mediated allergy was rare in adult 

13 chronic asthmatics.

14 Several studies have examined serum for formaldehyde-specific IgE antibodies in groups 

15 of formaldehyde-exposed humans (Ohmichi et al., 2006; Krakowiak et al., 1998; Wantke et al., 

16 1996a, b; Salkie, 1991; Grammer et al., 1990; Kramps et al., 1989). While formaldehyde17 specific IgE was reported in one study (Wantke et al., 1996a), results from most other studies 

18 failed to find a consistently strong association between formaldehyde-specific IgE or IgG 

19 antibodies in groups of formaldehyde-exposed humans.

20 Wantke et al. (1996a) detected elevated levels of formaldehyde-specific IgE in 24 of 62 

21 8-year-old children who were students in three particleboard-paneled classrooms in which the 

22 estimated formaldehyde air concentrations were 0.075, 0.069, and 0.043 ppm. In a health 

23 survey, the children reported headaches (29/62), fatigue (21/62), dry nasal mucosa (9/62), rhinitis 

24 (23/62), cough (15/62), and nosebleeds (14/62). The number of children with symptoms in each 

25 classroom decreased with decreasing formaldehyde concentration (49, 47, and 24, respectively, 

26 for the 0.075, 0.069, and 0.043 ppm classrooms). However, the investigators reported that 

27 elevated levels of specific IgE did not correlate with the number and severity of symptoms. 

28 When the children were evaluated after 3 months in a new school that did not have particleboard 

29 paneling and had lower ambient formaldehyde concentrations (0.029, 0.023, and 0.026 ppm), the 

30 number of children reporting symptoms decreased significantly from earlier figures, and, when 

31 measured in 20 of the children, the mean serum levels of formaldehyde-specific IgE declined 

32 significantly compared with pre-moving mean levels.

33 In contrast, a study by Krakowiak et al. (1998) measured serum IgE levels in asthmatic 

34 and healthy subjects as part of a larger study to characterize the mechanism of 

35 formaldehyde-induced nasal and bronchial response in asthmatic subjects with suspected 
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1 formaldehyde allergy. Ten subjects reported to have formaldehyde rhinitis and asthma and 

2 10 healthy subjects underwent a 2-hour inhalation challenge in an exposure chamber with 

formaldehyde at a concentration of 0.5 mg/m3 3 (0.41 ppm). Formaldehyde-specific serum IgE 

4 antibodies were measured, and cellular, biochemical, and mediator changes were assessed in 

5 nasal lavage before, immediately after, and at 4 and 24 hours after challenge. Challenges with 

6 formaldehyde caused only transient symptoms of rhinitis in both groups. Furthermore, none of 

7 the subjects thought to have occupational asthma developed clinical symptoms of bronchial 

8 irritation. No specific IgE antibodies to formaldehyde were detected in persons with 

9 occupational exposure to formaldehyde. No differences in the nasal response to formaldehyde 

10 were found between subjects reported to have occupational allergic respiratory diseases and 

11 healthy subjects (p > 0.05). The study showed that inhaled formaldehyde at a level as low as 

0.5 mg/m3 12 did not induce a specific allergic response either in the upper or in the lower part of 

13 the respiratory tract. In addition, it demonstrated that there was no difference in nasal response 

14 to formaldehyde between asthmatic subjects occupationally exposed to formaldehyde and 

15 healthy subjects.

16 Similarly, formaldehyde-specific IgE antibodies were detected in only 1 serum sample 

17 (out of 86) from four groups of formaldehyde-exposed subjects (Kramps et al., 1989). The 

18 subject with detected formaldehyde-specific IgE displayed allergic symptoms. The groups 

19 included (1) 28 subjects living or working in places with formaldehyde-containing construction 

20 materials (e.g., chipboard) and estimated formaldehyde concentrations ranging from 0.08 to 

21 0.37 ppm, (2) 18 occupationally exposed subjects from an anatomy laboratory and in other 

22 unspecified industries where air concentrations were not measured, (3) 12 hospital attendants 

23 who worked with formaldehyde-sterilized hemodialysis equipment, and (4) 28 hemodialysis 

24 patients coming into contact with equipment sterilized with formaldehyde. Other subjective 

25 symptoms, such as headache, eye irritation, and respiratory complaints, were reported by 

26 24/28 subjects in the construction material group and confirm that formaldehyde is an irritant 

27 (reviewed in Section 4.1.1.1). Durations of exposure or length of employment were not reported 

28 for the subjects in this study.

29 Grammer et al. (1990) studied the immunologic nature of formaldehyde sensitivity in 

30 37 workers who were examined by a group of physicians in response to complaints of 

31 formaldehyde-related illness. Air sampling of formaldehyde ranged from 0.003 to 0.078 ppm, 

32 but specific levels were not tied to specific workplace areas. Blood samples were collected and 

33 assayed for IgE and IgG activity against formaldehyde. None of the workers had IgG activity 

34 against formaldehyde. No IgE antibodies were detected in the other 32 workers. The authors 
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1 concluded that there was no evidence of an immunologically mediated response to formaldehyde 

2 in this group of workers.

3 Formaldehyde-specific IgE was not detected in any of a group of 45 medical students 

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

5 Estimates of ambient air concentrations of formaldehyde ranged from 0.059 to 0.219 ppm (0.124 

6 ± 0.05 ppm; mean ± SD). However, the survey revealed frequencies of irritation symptoms that 

7 were consistent with other studies (e.g., itching of the skin in 33/45 students, headache in 15/45, 

8 and burning eyes in 13/45).

9 Similarly, Ohmichi et al. (2006) were unable to correlate formaldehyde exposure with 

10 specific IgE production among eight students attending a gross anatomy laboratory. 

11 Formaldehyde exposure was estimated to range from 0.33 to 1.47 ppm during the laboratory 

12 sessions. The sample size was small, and IgE levels varied substantially (ranging from <19 to 

13 >5,000 international units/mL). Compared with IgE levels taken 90 minutes prior to the start of 

14 the first session, IgE levels measured shortly after the last session and up to 23 days following 

15 the last session showed no association with exposure.

16 Salkie (1991) investigated the prevalence of formaldehyde-specific IgE in practicing 

17 pathologists who complained of formaldehyde sensitivity. Exposure levels were not reported. 

18 Serum samples were assayed for total IgE and formaldehyde-specific IgE. Of the 46 subjects, 

19 29 self-reported atopy that was confirmed in 12 subjects by positive IgE. Moreover, 29 subjects 

20 complained of formaldehyde-specific sensitivity. However, zero subjects had formaldehyde21 specific IgE, and there was no evidence that atopic individuals were more sensitive to 

22 formaldehyde than non-atopic individuals. The authors noted that atopic individuals may have 

23 selectively reduced their exposure to formaldehyde.

24 Vandenplas et al. (2004) evaluated a case study of a 31-year-old male who was 

25 accidentally exposed to formaldehyde for 2 hours. The exposure level was not provided. The 

26 subject had smoked a pack of cigarettes a day for 13 years and was admitted to the emergency 

27 room for asthmatic symptoms. Eight days following exposure, increased levels of 

28 formaldehyde-specific IgE antibodies were detected but could not be detected in subsequent 

29 assessments.

30 A clinical study by Liden et al. (1993) evaluated IgE-specific antibodies against 

31 formaldehyde in 23 subjects who had previously tested positive for skin sensitization by skin 

32 prick test. Subjects were exposed to formaldehyde by skin patch (1% formaldehyde in water). 

33 Ten of the subjects were classified as atopic Though 15 of 23 of the sensitized subjects were 

34 also sensitive to formaldehyde applied by skin patch, formaldehyde-IgE was positive in 2 of 15 
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1 individuals who were not classified as atopic. No dose-response relationship could be 

2 determined from the study design of this study.

3 Doi et al. (2003) conducted a clinical study in 155 children of which 122 were 

4 asthmatics. No specific exposure to formaldehyde was documented. IgE against formaldehyde 

5 was determined in blood. Formaldehyde-specific IgE was found in two asthmatic children. 

6 Thus, while several studies have documented formaldehyde-specific IgE, the occurrence is rare 

7 and may be transient. Asthmatic children may be more predisposed to form formaldehyde8 specific IgE than non-atopic individuals or adults. The formation of formaldehyde-specific IgE 

9 is quite rare.

10

11 4.1.1.5.4. Formaldehyde-albumin and formaldehyde-heme complexes. Numerous studies have 

12 shown that formaldehyde can bind to blood proteins as formaldehyde-heme and formaldehyde13 human serum albumin (formaldehyde-HSA) complexes (Carraro et al., 1997; Grammer et al., 

14 1993, 1990; Dykewicz et al., 1991; Thrasher et al., 1990). Kim et al. (2001) failed to identify 

15 IgE against formaldehyde-HSA complexes in one case-control subject following industrial 

16 occupational exposure to formaldehyde. These complexes may serve to traffic formaldehyde 

17 throughout the bloodstream and throughout the body. While formaldehyde may be too small to 

18 engender an immune response, these complexes may be able to trigger formaldehyde-protein19 specific antibodies, leading to an immune response, including sensitization.

20 Thrasher et al. (1990) evaluated five groups of subjects as follows with varying levels 

21 and durations of formaldehyde exposure: asymptomatic chiropractic students exposed during 

22 anatomy classes (controls with only intermittent exposure to formaldehyde), mobile home 

23 residents, office workers, patients with multiple symptoms who had been removed from the 

24 source of formaldehyde for at least a year, and occupationally exposed patients. All groups were 

25 assessed for production of antibodies against formaldehyde-HSA. The level of autoantibodies 

26 was significantly elevated in patients exposed long-term to formaldehyde. From the data, 

27 Thrasher et al. (1990) concluded that exposure to formaldehyde stimulates IgG antibody 

28 production to formaldehyde-HSA.

29 Grammer et al. (1990) studied the immunologic nature of formaldehyde sensitivity in 

30 37 workers who were examined by a group of physicians in response to complaints of 

31 formaldehyde-related illness. Air sampling of formaldehyde ranged from 0.003 to 0.078 ppm, 

32 but specific levels were not tied to specific workplace areas. Blood samples were collected and 

33 assayed for IgE and IgG activity against formaldehyde and formaldehyde-HSA. None of the 

34 workers had IgG activity against formaldehyde. Five workers had IgE against both HSA alone 

35 and against formaldehyde-HSA complexes. No IgE antibodies were detected in the other 32 
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1 workers. The authors concluded that there was no evidence of an immunologically mediated 

2 response to formaldehyde in this group of workers.

3 Grammer et al. (1993) described the evaluation of a worker with bronchospasm 

4 symptoms caused by formaldehyde exposure. The worker was evaluated by means of ELISA, 

5 cutaneous tests, and methacholine and formaldehyde inhalation challenges. The ELISA showed 

6 that the worker had positive IgE and IgG titers to formaldehyde-HSA. The worker also had a 

7 positive cutaneous test for formaldehyde-HSA but a negative methacholine challenge at 25 

8 mg/mL and negative formaldehyde inhalation challenges at exposure concentrations of 0.3, 1, 3, 

9 and 5 ppm for 20 minutes. The worker might have developed a positive response if a higher 

10 concentration of formaldehyde had been used for the challenge, but it is more probable that the 

11 worker’s symptoms were not caused by immunologically mediated asthma.

12 Dykewicz et al. (1991) evaluated whether IgE or IgG antibodies to formaldehyde were 

13 related to formaldehyde exposure or to respiratory symptoms arising from such an exposure. 

14 The authors studied 55 potentially exposed subjects (hospital histology technicians, internal 

15 medicine residents, pathology residents, current smokers, and persons with known workplace 

16 exposure to formaldehyde) and compared them to controls with no history of formaldehyde 

17 exposure. Reported workplace formaldehyde concentrations were 0.2–0.64 ppm for pathology 

18 residents, 0.64 ppm for histology technicians, and 0.6–11 ppm for miscellaneous formaldehyde 

19 exposure scenarios. Workplace air concentrations were not measured for the other occupations. 

20 Occupational exposure to formaldehyde averaged 12.45 years for histology technicians, 

21 0.38 years for medical residents, 3.21 years for pathology residents, and 18.34 years for five

22 subjects exposed to formaldehyde in miscellaneous workplaces. Blood samples were analyzed 

23 for IgE and IgG reactivity with formaldehyde-HSA complexes. Three subjects had IgE against -

24 HSA; these three and two others had low levels of anti-formaldehyde-HSA IgG. The presence of 

25 IgG and IgE antibodies to formaldehyde was not clearly related to formaldehyde exposure or 

26 pack-years of smoking. One subject had both IgE and IgG antibodies and also suffered from eye 

27 and respiratory symptoms when exposed to formaldehyde at his workplace. However, the 

28 authors concluded that they could not establish a relationship between IgE and IgG levels and 

29 formaldehyde exposure. This study has several limitations. First, the volunteers (hospital 

30 workers) may not be representative of exposed workers in the general population. One of the 

31 exposure groups comprised cigarette smokers. Although the study focused on formaldehyde 

32 antibodies, which would be unaffected by the other chemicals, respiratory symptoms among 

33 smokers would reflect exposures to the constituents of smoke. Dykewicz et al. (1991) concluded 

34 that immunologically mediated asthma caused by formaldehyde is extremely rare and may not 

35 exist at all.
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1 Carraro et al. (1997) reported development of a reliable assay to effectively measure 

2 formaldehyde-HSA complexes in smokers, ex-smokers, and nonsmokers. A correlation between 

3 formaldehyde-HSA antibodies and smoking status was detected. This study did not correlate 

4 formaldehyde exposure and formaldehyde-HSA antibodies.

5 Given that formaldehyde is a sensory irritant that is particularly bothersome to 

6 individuals with compromised lung function or asthma, numerous studies have assessed the 

7 ability of formaldehyde to induce immunotoxic effects. Some studies have documented 

8 increased rates of chronic bronchitis and URT infections associated with exposure to 

9 formaldehyde, which suggests a possible immunomodulatory effect. However, of the numerous 

10 articles that have investigated systemic immunomodulatory effects due to formaldehyde

11 (Lyapina et al., 2004; Gorski et al., 1992; Thrasher et al., 1990, 1988, 1987; Pross et al., 1987), 

12 few have reported significant immune modulation related to formaldehyde exposure. Significant 

13 decreases in specific adaptive immune cell populations do not appear correlated to formaldehyde 

14 exposure (Erdei et al., 2003; Gorski et al., 1992; Thrasher et al., 1990, 1987; Pross et al., 1987). 

15 Thus, the tendency for increased infection rates associated with formaldehyde may not be related 

16 to altered immune function. Perhaps altered mucociliary clearance and disturbed mucosal barrier 

17 may provide greater access for pathogens and result in greater infection rates. Moreover, 

18 formaldehyde has been associated with exacerbation of asthmatic or atopic responses, 

19 particularly in sensitized individuals. However, this effect does not appear to occur by increased 

20 IgE or formaldehyde-specific IgE levels (Ohmichi et al., 2006; Palczynski et al., 1999; 

21 Krakowiak et al., 1998; Wantke et al., 1996b; Salkie, 1991; Grammer et al., 1990; Kramps et al., 

22 1989). Thus, formaldehyde-associated enhanced allergic responses does not appear to be due to 

23 direct induction of sensitization and may not occur via an immunologic mechanism. Lastly, the 

24 formation of formaldehyde-heme and formaldehyde-HSA complexes has been well documented 

25 (Grammer et al., 1993, 1990; Dykewicz et al., 1991; Thrasher et al., 1990) and may serve as a 

26 biomarker of exposure (Carraro et al., 1997). Moreover, these complexes may provide a means 

27 by which formaldehyde travels throughout the bloodstream and may drive antibody formation 

28 that may lead to immune activation.

29

30 4.1.1.6. Neurological/Behavioral

31 There is some suggestion of neurological impairment in humans following occupational 

32 exposure to formaldehyde; the data are limited and the results from several studies are potentially 

33 confounded by exposure to other solvents. Two studies of histology technicians with 

34 occupational exposure to formaldehyde and other solvents found neurological deficits and poorer 

35 performance on neurocognitive tests associated with formaldehyde exposure (Kilburn et al., 
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1 1987, 1985). In another study, Kilburn and Warshaw (1992) found no change from initial 

2 performance, for as long as 4 years, in follow-up evaluations of histology technicians with 

3 continuing exposure to formaldehyde. In a preliminary report from a prospective study, 

4 Weiskopf et al. (2009) found a strong association between duration of formaldehyde exposure 

5 and death from amyotrophic lateral sclerosis (ALS). In a controlled exposure study, Bach et al. 

6 (1990) found that, when workers with chronic formaldehyde exposure were challenged with an 

7 acute formaldehyde exposure, they exhibited poorer performance on some neurocognitive tests 

8 compared with workers without chronic exposure undergoing the same acute challenge 

9 conditions. In another controlled exposure study, Lang et al. (2008) found equivocal changes in 

10 reaction time following an acute exposure. 

11

12 4.1.1.6.1. Epidemiological studies. Kilburn et al. (1985) reported that a group of 76 female 

13 histology technicians displayed statistically significantly greater frequencies of neurobehavioral 

14 deficits (lack of concentration and loss of memory, disturbed sleep, impaired balance, variations 

15 in mood, and irritability), than did a referent group of 56 unexposed female clerical workers. 

16 The technicians had been employed from 2 to 37 years (mean 12.8 years). Analysis of 

17 workplace air samples indicated the presence of several solvents, ranging from 0.2 to 1.9 ppm for 

18 formaldehyde, 3.2 to 102 ppm for xylene, 2 to 19.1 ppm for chloroform, and 8.9 to 12.6 ppm for 

19 toluene. Subsequently, Kilburn et al. (1987) administered a battery of 10 tests to 305 female 

20 histology technicians to assess various aspects of cognitive and motor function. The researchers 

21 analyzed the results by regression analysis with age, years of smoking, and hours per day of 

22 exposure to formaldehyde and other solvents as explanatory variables. Increased daily hours of 

23 exposure to formaldehyde were significantly correlated with decreased performance in several 

24 tests (including several types of memory, dexterity, and balance), whereas hours of daily 

25 exposure to other solvents were only correlated with decreased performance in a single memory 

26 test. In a later prospective study of performance, 318–494 histology technicians were tested in a 

27 battery of neurobehavioral tests, and testing for a subset of subjects was repeated yearly for up to 

28 4 years. No statistically significant decrement in performance was found when initial test results 

29 were compared with retest results to evaluate effects of continuing occupational exposure to 

30 formaldehyde (or other solvents) or possible effects of aging (Kilburn and Warshaw, 1992). 

31 Kilburn (1994) later reported that three anatomists and one railroad worker, occupationally 

32 exposed to airborne formaldehyde for 14–30 years, each showed impaired performance on 

33 several neurobehavioral tests (e.g., choice reaction time, abnormal balance, digit symbol, and 

34 perceptual motor speed).
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1 Weisskopf et al. (2009) evaluated the association between chemical exposure and death 

2 from ALS, using the cohort of 987,229 people from the prospective Cancer Prevention Study II 

3 of the American Cancer Society. From 1989–2004, 1,156 deaths from ALS were identified from 

4 mortality records from the National Death Index. Exposure assessment occurred prior to follow5 up and was based on a questionnaire; participants were asked about current exposure to 12 types 

6 of chemicals and whether they had been regularly exposed in the past. After controlling for a 

7 number of potentially confounding factors (including age, sex, smoking status, military service, 

8 education, alcohol intake, occupation, vitamin use, and exposure to other chemicals), it was 

9 found that exposure to formaldehyde for a known duration was statistically significantly 

10 associated with increased risk of death from ALS (p < 0.0001) with a relative risk (RR) of 2.47 

11 (95% CI: 1.58–3.86) based on 22 deaths. Weisskopf et al. (2009) reported that the association 

12 had a strongly significant dose-response relationship, with increased duration of exposure 

13 associated with increased RR of ALS mortality with a reported p value for continuous trend of 

14 0.0004. Multivariate adjusted rate ratios were 1.5 for known formaldehyde exposures less than 

15 4 years, 2.1 for 4–10 years, and 4.1 for >10 years. Although the authors indicated that these 

16 results need independent verification, the results of this study of the nearly one million people 

17 followed for 15 years is unlikely to be biased due to its longitudinal design.

18

19 4.1.1.6.2. Controlled exposure studies. Bach et al. (1990) examined whether cognitive and 

20 motor performance of humans responded acutely to formaldehyde exposure and whether 

21 previous chronic exposure to formaldehyde affected the responses observed following acute 

22 exposure. Thirty-two men with at least 5 years of occupational exposure to formaldehyde and 

23 29 matched controls were exposed to formaldehyde at concentrations of 0.04, 0.21, 0.48, or 

24 1.10 ppm for 5.5 hours. During the exposure period, symptoms were assessed by using a 

25 standardized questionnaire, and subjects were evaluated in four tests designed to estimate several 

26 aspects of cognitive function. Testing was performed once prior to exposure and twice during 

27 the exposure period. The authors noted that the typical dose-related symptoms of respiratory 

28 irritation were not seen in this study. Previously unexposed subjects reported more headaches, 

29 “heavy head,” and physical tiredness than the exposed workers. In both occupationally exposed 

30 and unexposed subjects, decreased performance in an addition test was significantly correlated 

31 with increasing concentration of formaldehyde. Compared with previously unexposed subjects, 

32 occupationally exposed subjects showed significantly decreased performance in three other tests, 

33 although the effect was not dose related. The study did not adjust for several potential 

34 confounders, including prior exposure to other chemical agents, and the age and health status of 

35 the cases and controls. Authors concluded that their data indicated that acute exposure to 
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1 formaldehyde might cause acute effects on CNS functions but that more investigation was 

2 needed to verify their results.

3 In a study evaluating chemosensory irritation, Lang et al. (2008) assessed possible 

4 changes in reaction time during an acute (4-hour) exposure to formaldehyde concentrations 

5 between 0–0.5 ppm (some exposure sessions also included short concentration peaks of up to 

6 1 ppm) with or without a masking agent (ethyl acetate). Twenty-one healthy volunteers were 

7 exposed once per day to each of 10 different exposure combinations in random order (for a total 

8 of 10 sessions per subject). Reaction time was tested before and after each exposure session. 

9 Significant increases in reaction time were seen at 0.3 ppm formaldehyde, with or without 

10 masking agent, but not at 0.5 ppm. The significance of these findings is unclear.

11 Performance of 16 healthy volunteers on addition, multiplication, and card punching 

12 tasks was measured by Andersen and Molhave (1983) before and during a 5-hour exposure to 

formaldehyde at concentrations up to 2 mg/m3 13 . The authors reported that formaldehyde 

14 exposure had no effect on performance, but results were not presented.

15

16 4.1.1.6.3. Summary. The limited information currently available from human studies does not 

17 permit a definitive conclusion regarding an association between formaldehyde exposure and 

18 human neurotoxicity. There is, however, sufficient information to raise a serious concern for this 

19 type of effect, and additional studies are needed.

20

21 4.1.1.7. Developmental and Reproductive Toxicity

22 Epidemiologic studies suggest a convincing relationship between occupational exposure 

23 to formaldehyde and adverse reproductive outcomes in women. Several of these studies deal 

24 with spontaneous abortion following maternal occupational formaldehyde exposure (Taskinen et 

25 al., 1999, 1994; John et al., 1994; Seitz and Baron, 1990; Hemminki et al., 1985, 1982; Axelsson 

26 et al., 1984), but not all reported a significant association between exposure and spontaneous 

27 abortion. A study of fecundability found an increase in time to pregnancy among female 

28 workers exposed to formaldehyde (Taskinen et al., 1999). Three studies that examined the effect 

29 of occupational exposures on the incidence of congenital malformation produced mixed results 

30 (Dulskiene and Gražulevičiene, 2005; Taskinen et al., 1994; Hemminki et al., 1985). A 

31 population-based, semi-ecologic study found an association between atmospheric formaldehyde 

32 exposure and low birth weight (Gražulevičiene et al., 1998). 

33

34 4.1.1.7.1. Spontaneous abortion. Several epidemiologic studies report a relationship between 

35 occupational exposure to formaldehyde and increases in risk of spontaneous abortion following 

36 maternal occupational formaldehyde exposure (Taskinen et al., 1999, 1994; John et al., 1994; 
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1 Seitz and Baron, 1990; Axelsson et al., 1984). Increased RRs were in the range of 1.7 to more 

2 than 3.0. However, other studies (Hemminki et al., 1985, 1982) found no association between 

3 occupational formaldehyde exposure and spontaneous abortion. Paternal occupational exposure 

4 to formaldehyde was not related to spontaneous abortion (Lindbohm et al., 1991).

5 The earliest report of an association between spontaneous abortion and formaldehyde 

6 exposure comes from a Swedish cohort study of female laboratory workers (Axelsson et al., 

7 1984). Subjects were women born in 1935 or later and worked in a university laboratory during 

8 1968–1979. There were 745 women who responded to a mailed questionnaire (response rate = 

9 95%), 556 of whom reported on 1,180 pregnancies that resulted in 997 births. Exposure to 

10 formaldehyde was estimated based on answers to the questionnaires. Formaldehyde exposure 

11 was reported only in connection with 10 pregnancies, of which 5 went to term, 3 were reported 

12 as miscarriages, and 2 were terminated by induced abortion. Excluding the latter, the 

13 spontaneous abortion rate among women exposed to formaldehyde in the first trimester was 3/8 

14 (37.5%) compared with 14/148 (9.5%) in the population of laboratory workers not exposed to 

15 any solvent in the first trimester.

16 While not computed by the authors, the OR can be calculated as 5.7 (95% CI: 1.2–26.6). 

17 The exposure assessment on which this result is based was methodologically weak but unlikely 

18 to be a source of bias. Given the exploratory nature of this study, potential confounders were not 

19 controlled for, but no other co-exposure was more strongly related to the increased risk of 

20 miscarriage, so this result is not likely to be explained by confounding. Selection bias is also an 

21 unlikely explanation given the high participation rate. However, although this association of an 

22 increased risk of pregnancy loss with formaldehyde exposure is statistically significant, the CI is 

23 wide and chance may be a possible explanation for this finding.

24 In a 1988 Health Hazard Evaluation, the National Institute for Occupational Safety and 

25 Health (NIOSH) investigated complaints of adverse reproductive outcomes at a plant where 

26 work pants were cut and sewn with a fabric that was treated with a resin that releases 

27 formaldehyde (Seitz and Baron, 1990). In a NIOSH laboratory, the fabric released 163 to 1,430 

28 μg of formaldehyde/gram of cloth. TWA personal breathing space formaldehyde levels ranged 

29 from trace to 0.46 ppm, while workstation values ranged from 0.32 to 0.70 ppm. The 

30 investigators studied the outcomes by using a mailed questionnaire. The response rate for 

31 current employees was 98%. There were 296 pregnancies among a cohort of 188 women. The 

32 investigators found increased rates of spontaneous abortion, premature birth, and congenital 

33 malformations. The crude rate of spontaneous abortion was 21% among women working at the 

34 plant while pregnant (4 of 19 pregnancies), 15% among women employed elsewhere while 

35 pregnant (11 of 71 pregnancies), and 5% among women at home while pregnant (10 of 206 
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1 pregnancies). The investigators did not explain how workers employed elsewhere or at home 

2 during pregnancy were categorized compared with current workers, nor did they calculate RRs. 

3 As calculated from data presented in Table 5 of the monograph, the crude OR (not corrected for 

4 multiple observations per woman) for those pregnant while currently working at the plant 

5 compared with all others was 3.2 (95% CI: 0.8–12). There were also excess congenital 

6 malformations (13 versus 2%) and premature births (13 versus 4%) among the live births (both 

7 based on two births each in the exposed group) from the women who were pregnant while 

8 employed at the textile plant compared with women who stayed at home. After the NIOSH 

9 investigation, changes were made in the plant to improve ventilation. 

10 Because the report provides insufficient details of the methodology and the fact that there 

11 was no personal exposure classification in this study, it is difficult to validate the findings in this 

12 report. The results did not take into account other potential risk factors for spontaneous abortion 

13 or correct for multiple pregnancies per woman. Furthermore, the marked differences between 

14 the “home” and “work” pregnancies were difficult to interpret.

15 A cohort study of effects of paternal occupational exposures in Finland by Lindbohm et 

16 al. (1991) found that exposure to formaldehyde had little effect on the rate of spontaneous 

17 abortions among 99,186 pregnancies listed in the national hospital discharge register. The 

18 analysis was limited to births/spontaneous abortions in 1976 and from May 1980 to April 1982. 

19 Spontaneous abortion incidence came from the hospital discharge register and data from 

20 outpatient clinics. There were 808 pregnant wives among potentially formaldehyde-exposed 

21 fathers. Exposure to formaldehyde was based on employment information listed in the Finnish 

22 1980 census. Compared with pregnancies among wives of unexposed spouses, the age and 

23 socioeconomic level-adjusted ORs were 1.1 for low paternal exposure to formaldehyde and 1.0 

24 for moderate to high paternal exposure. Paternal occupational exposures to ethylene oxide, 

25 gasoline/benzene, and rubber industry chemicals were associated with spontaneous abortion. 

26 The authors hypothesized that the mode of action (MOA) for spontaneous abortion following 

27 male exposure to chemicals is genetic damage to germ cells.

28 The indirect exposure assessment was a substantial limitation of this study. Some 

29 confounders in a study of this type could not be controlled for (smoking history, previous 

30 spontaneous abortions, alcohol use), and census data could not provide completely accurate 

31 information, potentially masking associations between paternal formaldehyde exposure and 

32 spontaneous abortion.

33 A case-control study by Taskinen et al. (1994) of effects of maternal occupational 

34 exposure to chemicals used in laboratories in Finland indicated that exposure to formalin, which 

35 is a 37% aqueous solution of formaldehyde, was related to an increased risk of spontaneous 
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1 abortion. The investigators studied subjects from payrolls of Finnish state-employed laboratory 

2 workers, the laboratory workers’ union, and a register of workers occupationally exposed to 

3 carcinogens. These records were cross-referenced with the hospital discharge register. The 

4 investigators selected women who had a single spontaneous abortion during the period 1973–

5 1986 and two controls who had delivered a baby without malformations. The final sample size 

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

7 82.4%.

8 Information on occupational exposure, health status, medication, contraception, and 

9 pregnancy history came from mailed questionnaires. Industrial hygienists’ construction of an 

10 exposure index was based on the subjects’ descriptions of their work assignments, use of 

11 solvents including estimates of quantity used, and use of a fume hood. ORs were adjusted for 

12 employment, smoking, alcohol consumption, parity, previous miscarriage, birth control failure, 

13 febrile disease during pregnancy, and other organic solvents found in laboratory work. 

14 Spontaneous abortion was associated with 3–5 days per week of formalin exposure (OR 3.5 

15 [95% CI: 1.1–11.2]). A contemporaneous study of formaldehyde concentrations in similar 

16 Finnish workplaces (pathology and/or histology laboratories) reported workroom air to range 

17 from 0.01 to 7 ppm with a mean of 0.45 ppm formaldehyde (Heikkilä et al., 1991 [as cited in 

18 Taskinen et al., 1994]) and that the highest exposures occurred during emptying of sample 

19 containers, dish washing, and preparation of formaldehyde solution.

20 Although the results of this study indicate an increased risk between spontaneous 

21 abortion and exposure to formaldehyde/formalin, the women were also exposed to several 

22 chemicals concurrently, of which toluene (OR 4.7 [95% CI: 1.4–15.9]) and xylene (OR 3.1 [95% 

23 CI: 1.3–7.5]) were also significantly associated with the incidence of spontaneous abortion. 

24 However, the investigators reported that the women were more likely to be co-exposed to 

25 formalin and xylene, which would make confounding by toluene less likely, and, since xylene 

26 was not as strongly associated with the outcome as was formaldehyde, it too is unlikely to fully 

27 explain the reported relationship between formaldehyde and increased risk of spontaneous 

28 abortion. While it is possible that exposure misclassification may have occurred because of the 

29 indirect assessment of workplace chemical exposure, an overall conclusion is that, since the 

30 exposure assessment was conducted by industrial hygienists, it is unlikely that this form of bias 

31 will have impacted the results of the study to any great extent.

32 In a U.S. study (John et al., 1994), the results of a case-control study of cosmetologists 

33 also supported an association between spontaneous abortion and the use of formaldehyde-based 

34 disinfectants. The study population came from the 1988 North Carolina cosmetology license 

35 registry. Women on this list who were 22–36 years of age were screened to find those who were 
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1 recently pregnant. The cases were full-time cosmetologists who experienced a spontaneous 

2 abortion before gestational week 20 during 1983–1988. The most recent spontaneous abortion 

3 was used as the reference case. Controls were full-time cosmetologists who delivered a live 

4 infant during the same time period.

5 Information was based on mailed questionnaires. Women were not told the purpose of 

6 the study in order to avoid selection and recall bias. Of 8,356 women who received the 

7 screening questionnaires, 72.5% responded. Of those, 1,696 qualified for the study and 73.6% 

8 completed a more detailed questionnaire. Among them, 96 women were “absolutely sure” they 

9 had a spontaneous abortion and qualified as cases. There were 1,058 live births that qualified as 

10 controls. Exposure assessment included identification of disinfectants used as well as types of 

11 chemicals used on hair, use of gloves, hours worked, number of procedures involving chemicals, 

12 and use of manicure products. Presence of formaldehyde in the cosmetology profession in 

13 general was confirmed in two NIOSH hazard reports (Almaguer and Klein, 1991; Almaguer and 

14 Blade, 1990). ORs were adjusted for age, smoking, pregnancy characteristics, other jobs, hours 

15 worked, education (cosmetology school), hours standing per week, number of chemical 

16 procedures per week, hair dyes per week, bleachings per week, permanents per week, use of 

17 gloves, beauty salon characteristics, and use of alcohol or formaldehyde disinfectants.

18 An elevated OR of 2.1 (95% CI: 1.0–4.3) was reported with the use of formaldehyde19 based disinfectants adjusted for maternal characteristics and other workplace exposures. Other 

20 chemical exposures were also associated with spontaneous abortion, including number of 

21 chemical services per week, hair dyes, bleaches, and alcohol-based disinfectants. Strengths of 

22 this study include adjustment for important confounding risk factors for spontaneous abortion, 

23 detailed collection of interview-based information, a favorable response rate, and the fact that the 

24 index population had a high likelihood of formaldehyde exposure. These data provide overall 

25 support for an association between formaldehyde exposure and spontaneous abortion.

26 In a retrospective cohort study by Taskinen et al. (1999) of female woodworkers in 

27 Finland, exposure to formaldehyde was associated with delayed conception and spontaneous 

28 abortion. The subjects, recruited from a woodworkers’ union and other businesses involving 

29 wood processing, were linked to a national register of births. Women were included if they were 

30 born between 1946 and 1975, had a live birth at age 20–40 years during 1985–1995, had worked 

31 in the wood processing industry for at least 1 month, and had first employment in the wood 

32 processing industry beginning at least 6 months before the index pregnancy. The first pregnancy 

33 that fulfilled the above criteria was the index pregnancy. There were 1,094 women with these 

34 criteria. Information about personal characteristics, pregnancies, and exposures were collected 

35 from mailed questionnaires for which the response rate was 64%. After other exclusions 
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1 (primarily infertility history, unknown time to pregnancy, and contraceptive failure), the final 

2 sample included 602 women.

3 Estimates of mean daily exposure to formaldehyde were based on measurements taken at 

4 the women’s factories of employment during the early 1990s. Where measurements were 

5 unavailable, measurements from equivalent industries were used. An exposure index 

6 representing a TWA exposure was established for every person in the study based on the 

7 concentration of workplace formaldehyde multiplied by the proportion of the workday exposed 

8 to formaldehyde. The investigators categorized TWA formaldehyde exposure into categories of 

9 low (mean of 18 ppb), medium (mean of 76 ppb), and high (mean of 219 ppb) exposure.

10 Time-to-pregnancy data were analyzed by a discrete proportional hazards regression 

11 procedure with, as the outcome, a fecundability density ratio (FDR), in which a ratio of average 

12 incidence densities of pregnancies for exposed women was compared with that of the employed, 

13 unexposed women. As explained by Taskinen et al. (1999), an FDR significantly below unity 

14 suggests that conception was delayed. The age-, employment-, smoking-, alcohol consumption-, 

15 parity-, and menstrual irregularity-adjusted FDR was 0.64 (95% CI: 0.43–0.92) for women 

16 exposed to high formaldehyde levels compared with the unexposed controls, indicating that there 

17 was a substantial delay in time to conception in this group of women. Among a subset of women 

18 with high exposure who did not use gloves, the FDR was even lower (0.51 [95% CI: 0.28–0.92]), 

19 suggesting that these results might be explained in part through dermal contact with 

20 formaldehyde or might indicate an individual’s failure to follow appropriate precautions, which 

21 might have increased inhalation exposures in other ways. Exposure to solvents, wood dust and 

22 other dusts, and phenols was not associated with decreased fecundability.

23 The investigators further conducted an analysis of the risk of spontaneous abortion after 

24 carefully including only women who had the same workplace during the year of the spontaneous 

25 abortion as they had during the beginning of the time-to-pregnancy period. Spontaneous 

26 abortion was associated with formaldehyde exposure in the low exposure group (OR = 2.4 [95% 

27 CI: 1.2–4.8]), in the medium exposure group (OR = 1.8 [95% CI: 0.8–4.0]), and in the high 

28 exposure group (OR = 3.2 [95% CI: 1.2–8.3]). Endometriosis was also associated with the 

29 highest formaldehyde level (OR = 4.5 [95% CI: 1.0–20.0]).

30 This study by Taskinen et al. (1999) was a well-designed population-based case-control 

31 study that appears to have been well executed and appropriately analyzed. The study population 

32 of Finnish women was well defined and adequately selected so as to allow for meaningful 

33 comparisons of health effects between individuals with different levels of exposure to 

34 formaldehyde. The participation rate was 64%, which is low enough to raise a concern about the 

35 potential for selection bias. However, the authors noted that selection bias has not influenced the 
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1 results of other reproductive epidemiology studies reporting results on smoking, irregular 

2 menstruation, and earlier miscarriages, which are known to lengthen the time to pregnancy 

3 (Bolumar et al., 1996; Sallmén et al., 1995; Baird and Wilcox, 1985). Furthermore, there is no 

4 evidence to support conjecture that an individual’s decision to participate in this study would be 

5 differential with respect to their workplace formaldehyde exposures while being non-differential 

6 with respect to the other exposures of interest, including organic solvents, wood dust, and 

7 phenols. Since the women who chose to participate in this study were not likely to be aware of 

8 the specific hypotheses under investigation, nor could they have known the formaldehyde 

9 exposures that were independently estimated by an industrial hygienist, selection bias is not a 

10 likely explanation for the findings of adversity.

11 Data on pregnancy history, including spontaneous abortions, were collected by 

12 questionnaire. Spontaneous abortion is the most common adverse outcome of pregnancy (Klein 

13 et al., 1989), and retrospective self-report of spontaneous abortion has been found to match well 

14 with prospectively collected reproductive histories (Wilcox and Horney, 1984). Many 

15 spontaneous abortions, however, are missed with self-reporting with the magnitude likely 

16 exceeding 25%, but only rarely do women self-report false positive events (Wilcox and Horney, 

17 1984). The effect of such an undercount is to cause a bias towards the null when the likelihood 

18 of undercounting is unrelated to formaldehyde exposure. The implication is that the observed 

19 association of increased risk of spontaneous abortion associated with occupational exposure to 

20 formaldehyde may be an underestimation of the true risk.

21 Two studies (Hemminki et al., 1985, 1982) specifically assessed the effects of 

22 formaldehyde exposure and reported no significant increase in the risk of spontaneous abortion. 

23 Hemminki and colleagues (1982) conducted a retrospective cohort study of nurses who were 

24 potentially exposed to chemical sterilizing agents, including formaldehyde, ethylene oxide, and 

25 glutaraldehyde. The risk of having a spontaneous abortion among the women on the sterilizing 

26 staff was compared with that among the control population of nursing auxiliaries whom the 

27 supervisory nurses thought to be unexposed to the chemical sterilizing agents during the previous 

28 three decades. However, no measurements of the chemical sterilizing agents were taken. 

29 Information about exposure to chemical sterilizing agents was obtained from the supervising 

30 nurses. When the women were conducting sterilizing procedures during their pregnancies, the 

31 frequency of spontaneous abortion was 15.1% compared with 4.6% for the nonexposed 

32 pregnancies among the sterilizing staff. The increased frequency of spontaneous abortion 

33 correlated with exposure to ethylene oxide but not with exposure to glutaraldehyde or 

34 formaldehyde. The investigators reported that ethylene oxide concentrations have been 

35 measured in many sterilizing units in Finnish hospitals; 8-hour weighted mean concentrations 
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1 have ranged from 0.1 to 0.5 ppm with peak concentrations up to 250 ppm (measurements by the 

2 Finnish Institute of Occupational Health) (Hemminki et al, 1982). No measurements of 

3 glutaraldehyde concentrations were available. Hemminki et al. (1982) reported that exposure to 

4 formaldehyde in the sterilization units may be minimal, particularly when gas chambers are used. 

5 The range of formaldehyde concentrations measured in sterilizing units has been reported as 

6 0.03–3.5 ppm.

7 It is not clear that the unexposed women who served as controls were an appropriate 

8 comparison group to the sterilizing staff. The investigators reported that, among the sterilizing 

9 staff, those women who were unexposed during pregnancy experienced a rate of spontaneous 

10 abortion of 4.6% but that, among the comparison population of nursing auxiliaries who were 

11 presumed to be unexposed, the rate of spontaneous abortion was 10.5%. Had the nursing 

12 auxiliaries been an appropriate comparison group, it would be expected that their rate of 

13 spontaneous abortion would be similar to the unexposed sterilizing staff. Given this anomaly in 

14 study design and the unknown concentrations of formaldehyde exposure that were assessed as 

15 positive or negative by supervisory nurses regarding occupational exposures in the previous 

16 30 years, it is concluded that this report of no association between formaldehyde exposure and 

17 the risk of spontaneous abortion does not temper the conclusion that formaldehyde exposure has 

18 been shown to increase the risk of spontaneous abortion.

19 A second study by the same lead author (Hemminki et al., 1985) used a different study 

20 design to reassess the hypothesis that chemical exposures common in the field of nursing could 

21 be risk factors for spontaneous abortion. This case-control study found no increase in the risk of 

22 spontaneous abortion associated with exposure to formaldehyde. The head nurses at each 

23 hospital were asked by the investigators whether each case or control had been exposed to 

24 formaldehyde during a given 3-month period corresponding to the first trimester of a study 

25 participant’s pregnancy during 1973–1979. Formaldehyde exposure was assessed as positive or 

26 negative for either use as a sterilizing agent or use of sterilized instruments. The reported crude 

27 OR for formaldehyde exposure was 0.6; no CIs were provided. From the data reported in 

28 Table 2 in Hemminki et al. (1985), the unadjusted OR and its CI can be computed post hoc as 

29 OR (0.70 [95% CI: 0.28–1.73]). The authors acknowledged that the study failed to distinguish 

30 between sterilizing work and the use of sterilized instruments, where only very small exposures 

31 could be expected. Given the likelihood of extreme exposure misclassification and the 

32 presentation of only crude results without control of potential confounding for formaldehyde, 

33 these results do not appear to be exculpatory of a true causal association between formaldehyde 

34 exposure and the risk of spontaneous abortion.
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1 A meta-analysis of formaldehyde exposure and spontaneous abortion was conducted by 

2 Collins et al. (2001b). However, the published results should be interpreted with caution. This 

3 meta-analysis included one very large null study of paternal formaldehyde exposure along with 

4 seven studies of maternal exposure. The two null studies by Hemminki et al. (1985, 1982) were 

5 also included without consideration of the potentially extreme exposure misclassification that 

6 may have attenuated any true adverse effect. Nevertheless, the overall reported meta-analytic 

7 RR for parental formaldehyde exposure based on eight maternal and paternal exposure studies 

8 was 1.6 (95% CI: 0.9–2.7). For case-control studies the RR was 1.8 (95% CI: 0.7–4.8), and for 

9 cohort studies the RR was 1.7 (95% CI: 1.2–2.3). Collins et al. (2001b) argued that the method 

10 of exposure evaluation may have influenced the observed results; they stated that several of the 

11 studies whose exposures were based on the investigator’s judgment were likely misclassified, 

12 which may have obscured the true relationship, while other studies that assessed exposure based 

13 on self-reporting could have suffered from recall bias. They report that RRs were higher for 

14 studies based on self-reported exposures (RR = 1.9 [95% CI: 1.3–2.6]) than those based on 

15 objective exposure assessments (RR = 1.5 [95% CI: 0.6–3.7]) and suggested that this difference 

16 might reflect recall bias in the exposure assessment. However, for recall bias to have been 

17 operable in these studies, the women who provided self-reported data on pregnancy history and 

18 occupational exposure would have had to appreciate that the hypothesis of interest was the 

19 specific effect of formaldehyde on the risk of spontaneous abortion. In the specific case of the 

20 study by Taskinen and colleagues (1999), the investigator also looked at the effects of other 

21 exposures, such as organic solvents, dust, and phenols, and did not report adverse effects. It is 

22 therefore unlikely that the women providing exposure data were doing so in a manner indicative 

23 of recall bias. If the supposition of non-differential misclassification error in exposure is indeed 

24 correct, the observed results of the meta-analysis would likely have been biased towards the null. 

25 Therefore, the true RR for maternal formaldehyde could be higher than Collins et al. (2001b) 

26 reported and would likely be statistically significant. Had the study of paternal exposure been set 

27 aside, the meta-analysis almost surely would have shown a statistically significant increase in the 

28 risk of spontaneous abortion associated with maternal formaldehyde exposure. This single study 

29 reported a null finding based on exposure assessment from census records of employment, and, 

30 as the largest of the studies in the meta-analysis, it contributed the greatest weight.

31 Lastly, Collins and coworkers (2001b) suggested that there were potential confounding 

32 factors in each of the workplaces that might have produced the observed findings of increased 

33 risk of spontaneous abortion associated with formaldehyde. While each of these occupational 

34 studies focused on women who were co-exposed to formaldehyde and other chemicals, the 

35 occupational groups were quite different and had different sets of co-exposures. The 
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1 woodworkers in the Taskinen et al. (1999) study were potentially co-exposed to organic solvents 

2 related to painting and lacquering, dusts, and phenols, none of which was shown to be an 

3 independent predictor of adverse risk. The cosmetologists studied by John et al. (1994) were 

4 co-exposed to hair dyes, bleach, alcohol-based disinfectants, and chemicals specific to services, 

5 such as fingernail sculpturing, but, in analyses that were specifically adjusted for other work 

6 exposures and their potentially confounding effects, the investigators reported an OR of 2.1 

7 (95% CI: 1.0–4.3) for the use of formaldehyde-based disinfectants. The laboratory workers 

8 studied by Axelsson et al. (1984) were potentially co-exposed to a wide range of solvents, but the 

9 miscarriage rate was highest among those exposed to formaldehyde, and, for a potential 

10 confounder to entirely explain an observed effect of another exposure, it must be more strongly 

11 associated with the adverse outcome.

12 It does not appear that the collective results of formaldehyde exposures associated with 

13 increased risk of spontaneous abortion—often in spite of exposures being crudely measured—

14 can be explained by information bias or confounding. 

15 The findings by Taskinen et al. (1999) of reduced fertility and increased risk of 

16 spontaneous abortion are internally consistent and coherent with other reports of increased risk 

17 of pregnancy loss associated with exposure to formaldehyde (John et al., 1994; Taskinen et al., 

18 1994; Seitz and Baron, 1990; Axelsson et al., 1984). Absent evidence of alternative explanation 

19 for these findings, it is concluded that exposure to formaldehyde is associated with pregnancy 

20 loss and diminished fertility.

21

22 4.1.1.7.2. Congenital malformations. Only three studies have reported on the epidemiologic 

23 evidence of an association between formaldehyde exposure and the risks of births having 

24 congenital malformations. In the earliest study by Hemminki et al. (1985), the investigators 

25 presented an analysis of 34 congenital malformations from the Finnish Register of Congenital 

26 Malformations and compared them with a group of 95 controls from those used in the larger 

27 study. An association was found between formaldehyde exposure and malformations based on 

28 three exposed cases (OR = 1.8).

29 The case-control study by Taskinen et al. (1994) of effects of occupational exposure to 

30 chemicals used in laboratories in Finland examined the potential effects of exposure to formalin 

31 on both spontaneous abortions and congenital malformation. The investigators reported on a 

32 study of 36 laboratory workers with a child registered in the Finnish Register of Congenital 

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

34 malformations.
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1 A Lithuanian study (Dulskiene and Gražulevičiene, 2005) for which only a brief 

2 summary is available in English investigated the risk of congenital heart malformations as a 

3 result of exposure to 43 different agents. The number of births included in the study was not 

4 given in the English abstract. Exposure to residential ambient formaldehyde concentrations of 

>2.42 μg/m3 5 (0.002 ppm) was associated with a 24% increase in the risk of congenital heart 

6 malformations (OR = 1.24 [95% CI: 0.81–2.07]). The details of this study are unavailable in 

7 English translation, making it impossible to critically analyze details, such as co-exposure and 

8 other possible confounders.

9

10 4.1.1.7.3. Low birth weight. A case-control study by Gražulevičiene et al. (1998) examined the 

11 association of low birth weight (<2,500 grams) and air pollutants, including formaldehyde, 

12 particulates, sulfur dioxide, lead, ozone, and nitrogen dioxide, measured in 12 areas in the city of 

13 Kaunas, Lithuania. This city has conducted environmental pollutant measurements since 1993, 

14 and the investigators classified formaldehyde exposure based on the area of residence of the 

15 study subjects. Formaldehyde levels in the 12 districts of Kaunas in 1994 ranged from 1.36 to 

5.28 μg/m3 (0.0011–0.0043 ppm), with a citywide average of 3.14 μg/m3 16 (0.0026 ppm). 

17 Information on infants came from a birth registry. There were 244 cases of low birth weight and 

18 4,089 normal controls born in 1994. Personal data came from record-based prenatal interviews, 

19 and pregnancy data came from hospital records.

20 The crude RR of low birth weight among women exposed to the highest airborne 

21 formaldehyde level was 1.68 (95% CI: 1.24–2.27). After adjustment for age, occupation, 

22 hazardous work, education, marital status, smoking, hypertension, and other air pollutants, the 

23 OR was still elevated but no longer statistically significant (OR 1.37 [95% CI: 0.90–2.09]). 

24 Although formaldehyde exposure was the only single air pollutant associated with low birth 

25 weight, factors such as smoking, marital status, and pregnancy-related factors had more of an 

26 impact on birth weight. Total suspended particulates (OR 2.58 [95% CI: 1.34–4.99]) and 

27 hazardous work (OR 2.62 [95% CI: 1.12–6.10]), which was not defined by the authors, were also 

28 related to low birth weight.

29 Aside from studies of birth weight deficits from tobacco smoke and occupational 

30 exposure, the literature on exposure to ambient air pollutants to support the investigators’ 

31 hypothesis is limited. The strength of the association between total suspended particulates and 

32 low birth weight supports the idea that incidence of birth weight <2,500 grams may be related to 

33 atmospheric pollution, although this finding may not be specific to formaldehyde. Because of 

34 the large number of variables evaluated in the analysis, large fluctuations in the atmospheric 
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1 formaldehyde measurements, co-exposure to other pollutants, and geographic variability of low 

2 birth weight, it is difficult to estimate the impact of formaldehyde alone on low birth weight.

3

4 4.1.1.7.4. Summary. Although all studies on potential developmental toxicity of formaldehyde 

5 have limitations and do not uniformly report positive results, the associations between 

6 spontaneous abortion, delayed conception, or reproductive outcomes and formaldehyde exposure 

7 in multiple studies cannot be dismissed, because several studies report concordant findings 

8 across several populations and study methodologies. The results of most of the studies with 

9 positive findings were adjusted for many potentially confounding factors that may be related to 

10 spontaneous abortion and infertility, including smoking and alcohol use, pregnancy and 

11 reproductive history, and other chemical exposures.

12 The association between fertility and formaldehyde (Taskinen et al., 1999) stands out 

13 because of its strong quantitative statistical analysis, adequate sample size, and rigorous exposure 

14 assessment. This study was designed to specifically assess the effect of formaldehyde on 

15 reproductive outcomes. Furthermore, it was the only study with an exposure assessment based 

16 on quantitative measurements from the subject’s workplace. Moreover, the investigators 

17 conducted a multivariable survival analysis that approximates a longitudinal life table or person18 year analysis while simultaneously adjusting for important confounders. The findings were 

19 strengthened by statistically significant associations between formaldehyde and spontaneous 

20 abortion and endometriosis. The fact that the use of gloves may reduce the reproductive effect of 

21 formaldehyde supports the dose-response relationship in this study, and the lack of an association 

22 between time to pregnancy and any other workplace exposures strengthens the specificity of 

23 formaldehyde effects. The results also support associations reported between formaldehyde and 

24 increased risk of spontaneous abortion because subfertility and spontaneous abortion are 

25 biologically linked (subclinical pregnancy losses are increased among women with fertility 

26 problems) (Gray and Wu, 2000; Hakim et al., 1995), and both subfertility and spontaneous 

27 abortion may be related to sensitivity to environmental agents (Correa et al., 1996).

28

29 4.1.1.8. Oral Exposure Effects on the Gastrointestinal Tract

30 No human epidemiology studies exist to determine an association between oral exposure 

31 of formaldehyde and adverse health effects in the gastrointestinal (GI) tract.

32

33 4.1.1.9. Summary: Noncarcinogenic Hazard in Humans

34 Formaldehyde has clearly and consistently been shown to be a potent sensory irritant 

35 with a variety of adverse health effects. Eye, nose, and throat irritation as a result of 
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1 formaldehyde exposure has been documented in a wide range of epidemiologic studies. Workers 

2 chronically exposed to formaldehyde have exhibited signs of reduced lung function consistent 

3 with BC, inflammation, or chronic obstructive lung disease. A well-conducted residential 

4 epidemiology study has convincingly shown a concentration response for decreased pulmonary 

5 function among children with increased formaldehyde exposures. Several cross-sectional studies 

6 have described associations between increased concentrations of formaldehyde and increased 

7 prevalence of asthma. However, two case-control studies that focused on risk factors for the 

8 initial physician diagnosis of asthma, which is indicative of atopic switching, have also shown 

9 concentration-dependent adverse effects associated with formaldehyde exposure.

10 Results of research on the effects of formaldehyde on tissue histology suggest that 

11 formaldehyde is also responsible for reduced mucociliary clearance and the induction of 

12 histopathologic lesions in the nose. In addition, there is evidence of neurological impairment in 

13 several studies of formaldehyde-exposed histology technicians, but confounding exposures to 

14 other neurotoxic solvents and inconsistent results prevent drawing definitive conclusions 

15 concerning the neurotoxicity of formaldehyde from these studies. 

16 Finally, there is epidemiologic evidence that formaldehyde is associated with adverse 

17 reproductive outcomes. Four of six occupational studies found an increased risk of spontaneous 

18 abortion among formaldehyde-exposed women. Results of other studies suggested associations 

19 among formaldehyde and congenital malformations, low birth weight, and endometriosis. The 

20 strongest evidence of an association between formaldehyde and an adverse reproductive outcome 

21 came from a well-conducted study of infertility in women employed in the wood processing 

22 industry. This study found a greater than threefold increased risk of spontaneous abortion, a 

23 nearly 50% decrease in a measure of delayed conception indicating reduced fertility, and 

24 increased time to pregnancy associated with average daily formaldehyde exposures of 0.15–

25 1 ppm.

26

27 4.1.2. Cancer Health Effects

28 4.1.2.1. Respiratory Tract Cancer

29 4.1.2.1.1. NPC. NPC is a very rare form of cancer. The incidence is less than 1 per 100,000 

30 persons throughout most parts of the world. The most common form of NPC arises from the 

31 epithelial cells lining the nasopharynx. This presentation constitutes between 75 and 100% of all 

32 NPCs. There are two types, squamous cell carcinoma (SCC) and nonkeratinizing carcinoma. In 

33 the U.S., the 5-year survival rate for NPC is about 25% (Burt et al., 1992). Certain risk factors 

34 have been implicated in its etiology, including Epstein-Barr virus (EBV), wood dust and 
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1 particles applied to wood in its treatment, exhaust fumes, occupational smoke, and nitrosamines. 

2 The epidemiologic studies of NPC are summarized in Table 4-1.

3

4.1.2.1.1.1. Cohort studies.

11 Several of these studies measured exposure to formaldehyde at 10 production facilities 

12 that contributed to a cohort that has been studied by Blair et al. (1987, 1986) and Hauptmann et 

13 al. (2004). The National Cancer Institute (NCI) conducted a mortality study of solid tumors 

14 among a cohort of 25,619 workers who were employed in these 10 plants that produced or used 

15 formaldehyde in the U.S. before 1966 (Blair et al., 1987, 1986). Subjects were followed to 

16 January 1, 1980, accruing approximately 600,000 person-years of follow-up. Hauptmann et al. 

17 (2004) updated the cohort to December 31, 1994 and reported a significant excess risk of NPC in 

18 exposed workers based on U.S. population death rates (standardized mortality ratio [SMR] = 2.1 

19 [95% CI: 1.05–4.21]). In addition to the SMR based on an external comparison population, RRs 

20 were presented based on internal comparisons of similar workers in order to minimize potential

21 selection bias due to the well-known healthy worker effect (HWE). For NPC, RRs increased 

22 with several different exposure metrics, including average exposure intensity, cumulative 

23 exposure, highest peak exposure, and duration of exposure to formaldehyde (p values for tests 

24 for trends were 0.066, 0.025, <0.001, and 0.147, respectively). These results were based on 

25 primary data analyses of the health and exposure data collected by the NCI, according to their 

26 research protocol and analyzed accordingly. As such, the reported statistical p values may be 

27 appropriately interpreted as showing that these workers were at increased risk of NPC associated 

28 with exposure to formaldehyde. These NCI investigations controlled for potential selection bias 

29 due to the HWE and for several potential confounders, including calendar year, age, sex, race, 

30 and pay category. There was no evidence of any differential measurement error that could have 

31 produced the observation of a spurious association. Any non-differential measurement error 

32 would likely have led to an observed effect of formaldehyde that was less than that which would 

33 otherwise have been observed in the absence of measurement error. 

4 The International Agency for Research on Cancer (IARC) reported 

5 on eleven cohort studies of formaldehyde-exposed industry workers (Marsh et al., 2002, 1996, 

6 1994; Andjelkovich et al., 1995; Gardner et al., 1993; Bertazzi et al., 1989, 1986; Stayner et al., 

7 1988; Blair et al., 1987, 1986; Edling et al., 1987) and results from eight cohort studies of 

8 professional workers (Hall et al., 1991; Hayes et al., 1990; Stroup et al., 1986; Harrington and 

9 Oakes, 1984; Levine et al., 1984; Walrath and Fraumeni, 1984, 1983; Friedman and Ury, 1983) 

10 (IARC, 2006). 
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

Hauptmann et al. (2004) Retrospective cohort 

mortality study of 25,619 

workers employed at 10 

formaldehyde plants in the 

U.S. followed from either 

plant start-up or first 

employment through 1994. 

The 10 plants produced 

formaldehyde (3 plants), 

molding compounds 

(3 plants), photographic 

film (2 plants), plywood 

(1 plant), and formaldehyde 

resins (6 plants).

Exposure estimatesa based 

on job titles, tasks, visits

to plants by study 

industrial hygienists, and 

monitoring data 

measurements. Peak 

exposure = short-term 

excursions >8-hour TWA 

formaldehyde intensity 

and knowledge of job 

tasks. Workers 

contributed pre-exposure 

person time to nonexposed 

category. RRs were from 

Poisson regression 

models, using a 15-year 

lag to account for tumor 

latency. 

Overall

Nonexposed SMR 1.56 (95% CI: 0.39–23) (2)

Exposed SMR 2.10 (95% CI: 1.05–21) (8)

Peak exposure (ppm) 

0 RRb 1.00 (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 < 0.001

Average intensity of exposure (ppm)

0 RRb 1.00 (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 greater 1.67 (95% CI: NS) (6)

Trend p = 0.066

Cumulative exposure (ppm-years)

0 RRb 2.40 (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 = 0.025

Duration (years)

0 RRb 1.77 (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)

Trend p = 0.147

Marsh et al. (2002) Retrospective cohort 

mortality study of 7,328 

workers hired up to 1984 

Worker-specific exposurea

from job exposure matrix 

based on available 

Cohort study

Overall

U.S. SMR 4.94 (95% CI: 1.99–10) (7)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

and followed until 1998 in 

one plant from Hauptmann 

et al. (2004). Mortality was 

compared with death rates 

in two Connecticut counties 

and the U.S. 

sporadic sampling data 

from 1965−1987, job 

descriptions, and verbal 

job descriptions by plant 

personnel and industrial 

hygienists. Exposures 

ranked on a 7-point scale 

with exposure range 

assigned to each rank. 

17% of jobs validated with 

company monitoring data; 

remaining 83% based on 

professional judgment. 

Assumed pre-1965 

exposure levels same as 

post-1965 levels.

County SMR 5.00 (95% CI: 2.01–10) (7)

Short-term worker (<1 year)

SMR 5.35 (95% CI: 1.46–14) (4)

Long-term worker (1 or more years)

SMR 4.59 (95% CI: 0.95–13) (3)

Year of hire

1941−1946 SMR (0)

1947−1956 SMR 8.13 (95% CI: 2.98–18) (6)

1957 or later SMR 2.63 (95% CI: 0.07–15)

Cumulative exposure (ppm-years) county

Unexposed SMR (0)

0 to <0.004 SMR 3.97 (95% CI: 0.10–22) (1)

0.004–0.219 SMR 5.89 (95% CI: 1.22–17) (3)

0.22+ SMR 7.51 (95% CI: 1.55–22) (3)

Average exposure (ppm) county

Unexposed SMR (0)

0 to <0.03 SMR 2.41 (95% CI: 0.06–13) (1)

0.03–0.159 SMR 15.30 (95% CI: 4.16–39) (4)

0.16+ SMR 4.13 (95% CI: 0.50–15) (2)

Duration of exposure to >0.2 ppm (years)

Unexposed SMR 3.01 (95% CI: 0.36–11) (2)

0 to <1 SMR 4.81 (95% CI: 0.58–17.4) (2)

1–9 SMR 4.04 (95% CI: 0.10–22.51) (1)

10+ SMR 27.60 (95% CI: 3.34–100) (2)

Duration of exposure to ≥0.7 ppm (years) 

Unexposed SMR 3.64 (95% CI: 0.99–9.31) (4)

<1 SMR 9.51 (95% CI: 1.15–34.4) (2)

1+ SMR 11.07 (95% CI: 0.28–61.67) (1)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

Marsh et al. (2002) A nested case-control 

analysis of all pharyngeal 

cancer cases also conducted 

with four controls randomly 

selected from cohort and 

matched on age, year of 

birth, race, and sex. 

Conditional logistic model 

used for nested case-control 

analysis. 

Nested case-control analysisc

Duration 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)

1+ OR 1.11 (95% CI: 0.06–11.3) (2)

Hayes et al. (1990) Proportionate mortality 

cohort study of 4,046 U.S. 

male embalmers and funeral 

directors who died between 

1975 and 1985.

Exposure presumed. Overall PMR 2.16 (4)

Hansen and Olsen (1995) Proportionate incidence 

study of 2,041 men with 

cancer who died between 

1970 and 1984, identified 

from the Danish Cancer 

Registry and matched with 

the Danish Supplementary 

Pension Fund, whose 

longest work experience 

occurred at least 10 years 

before the cancer diagnosis. 

The SPIR measured the 

proportion of cases of NPC 

in formaldehyde-associated 

companies relative to the 

proportion of cases of NPC 

among all employees in 

Denmark.

Linked companies through 

tax records to the national 

Danish Product Register.

Overall SPIR 1.3 (95% CI: 0.03–3.2) (4)
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purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

Olsen et al. (1984) Case-control study of 314 

cases of NPC from Danish 

Cancer Registry linked to 

the Registry during 1970–

1982. Three controls/case 

sampled with cancer of the 

colon, rectum, breast, and 

prostate by age, sex, and 

year of diagnosis of cases. 

Employment histories 

after 1964 from files 

maintained by Danish 

Cancer Registry evaluated 

by industrial hygienists. 

Men OR 0.7 (95% CI: 0.3–1.7)

Women OR 2.6 (95% CI: 0.3–22)

West et al. (1993) Case-control study of 104 

non-Chinese incident NPC 

cases from the Philippine 

General Hospital matched 

with 104 hospital and 101 

community controls.

Personal interview, 

including job history. 

Industrial hygienists 

blinded to case-control 

status reviewed and rated 

jobs as likely or unlikely 

to be exposed. Analysis 

by length of exposure, 

length of exposure lagged 

10 years, time since first 

exposure, and age at first 

exposure, based on date of 

interview or death.

Length of exposure (years)

<15 RRd 2.7 (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 RRd 1.6 (95% CI: 0.7–3.8)

15 or more 2.1 (95% CI: 0.7–6.2)

Years since first exposure

<25 RRd 1.3 (95% CI: 0.6–3.2)

25 or more 2.9 (95% CI: 1.1–7.6)

Age at first exposure (years)

<25 RRd 2.7 (95% CI: 1.1–6.6)

25 or older 1.2 (95% CI: 0.5–3.3)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

Roush et al. (1987) Population-based casecontrol study of 173 male 

cases from the Connecticut 

Tumor Registry who died of 

any cause from 1935–1975. 

605 male controls randomly 

selected from state death 

certificates during same 

time period.

Four categories: I,

probably exposed most of 

working life; II, probably 

exposed most of working 

life and probably exposed 

20+ years before death; 

III, probably exposed most 

of working life and 

probably to high level in 

some year; IV, probably 

exposed most of working 

life and probably exposed 

to high level 20+ years 

before death.

Exposure levels

I ORe 1.0 (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)

Vaughan et al. (1986a) Population-based casecontrol study of 27 

incidence cases of NPC 

(during 1980–1983) from a 

13-county area (Washington 

State Cancer Surveillance 

System) and 552 matched 

controls from random digit 

dialing in same area, for 

occupational exposures.

Interview-based 

information on lifetime 

occupational exposure to 

formaldehyde with cases, 

next of kin, and controls. 

Exposure from available 

hygiene data, NIOSH and 

other data, and NCI job 

exposure linkage system. 

Exposure levels based on 

investigator’s judgment. 

Exposure score A: 

weighted sum of no. years 

spent per job (weight = 

estimated formaldehyde 

level). B: weighted sum 

of no. years spent per job 

with 15-year lag (latency).

Intensity

Low ORf 1.2 (95% CI: 0.5–3.3)

Medium/high 1.4 (95% CI: 0.4–4.7)

No. years exposed 

1–9 ORf 1.2 (95% CI: 0.5–3.1)

10 or more 1.6 (95% CI: 0.4–5.8)

Exposure score A: no lag

5–19 ORf 0.9 (95% CI: 0.2–3.2)

20 or more 2.1 (95% CI: 0.6–7.8)

Exposure score B: 15-year lag

5–19 ORf 1.7 (95% CI: 0.5–5.7)

20 or more 2.1 (95% CI: 0.4–10)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

Vaughan et al. (1986b) Population-based casecontrol study of 27 

incidence cases on NPC 

(during 1980–1983) from a 

13-county area (Washington 

State Cancer Surveillance 

System) and 552 matched 

controls from random digit 

dialing in same area, for 

residential exposures.

No direct measurements. 

Interview information 

from cases/controls or 

next of kin: residence in

past 50 years, use of 

particleboard or plywood, 

and lifetime occupational 

and chemical exposure 

history.

Years of residence in mobile home

1–9 ORg 2.1 (95% CI: 0.7–6.6)

10 or more 5.5 (95% CI: 1.6–19)

Years of exposure to particleboard

1–9 ORg 1.4 (95% CI: 0.5–3.4)

10 or more 0.6 (95% CI: 0.2–2.3)

Exposure source

Occupation only ORg 1.7 (95% CI: 0.5–5.7)

Mobile home only 2.8 (95% CI: 1.0–7.9)

Both 6.7 (95% CI: 1.2–39)

Vaughan et al. (2000) Population-based casecontrol study of 196 

incident epithelial NPC 

patients identified from 5 

U.S. cancer registries from 

1987–1993 matched with 

244 controls from random 

digit dialing in the same 

geographic regions.

Interviewed for lifetime 

occupational and chemical 

exposure. Exposure 

estimates by industrial 

hygienist without 

knowledge of casecontrols status. 

Probability of exposure: 

definitely not or unlikely 

(<10%); possible (≥10% 

and <50%); probable 

(>50% and <90%); and 

definite ≥90%). Jobs with 

potential exposure 

assigned estimated 

concentration levels based 

on TWA: low (<10 ppm), 

moderate (≥10 and 

<50 ppm), and high 

(≥50 ppm).

Possible, probable, or definite exposure (61 cases, 76 controls)

Ever ORh 1.6 (95% CI: 1.0–2.8)

Duration (years)

1–5 ORh 0.9 (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 = 0.014

Cumulative exposure (ppm-years)

0.05–0.40 ORh 0.9 (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 = 0.033

Probable or definite exposure (27 cases, 30 controls)

Ever ORh 2.1 (95% CI: 1.1–4.2)

Duration (years)

1–5 ORh 2.0 (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)

Trend p = 0.069
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-65

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

Cumulative exposure (ppm-years)

0.05–0.40 ORh 1.9 (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 = 0.13

Definite exposure (10 cases, 2 controls)

Ever ORh 13.3 (95% CI: 2.5–70)

Hildesheim et al. (2001) Population-based casecontrol study of 375 

incident cases from two 

Taiwanese hospitals 

between 7/15/91 and 

12/31/94. 325 controls 

came from a random sample 

of households from a 

national household 

registration system and 

were age, sex, and area-ofresidence matched. Tumors 

were histologically 

confirmed. All subjects 

were tested for the EBV. 

Exposure metrics were 

stratified by seropositivity.

In-person interviews 

collected information on 

risk factors and job history 

for jobs held >1 year, 

including length of time 

job held, type of industry, 

and tasks, tools, and 

materials used on the job. 

Industrial hygienist 

assigned Standard 

Industry Classification/ 

Standard Occupational 

Classification codes to 

jobs, assigning each a 

probability and intensity 

of exposure on a 0–9 

scale. Exposure metrics 

were duration, average 

intensity (intensity scale), 

average probability 

(probability scale), 

cumulative (average 

intensity), years since 1st 

exposure, and age at 1st 

exposure. Analysis of 

Ever exposed RRi 1.4 (95% CI: 0.9–2.2)

Duration (years)

≤10 RRi 1.3 (95% CI: 0.69–2.3)

>10 RRi 1.6 (95% CI: 0.91–2.9)

Trend p = 0.08

EBV posj

Ever exposed RRi 2.7 (95% CI: 1.2–6.2)

≤10 RRi 2.8 (95% CI: 0.8–9.7)

>10 2.6 (95% CI: 0.9–7.7)

Cumulative exposure (average intensity-years)

<25 RRi 1.3 (95% CI: 0.7–2.4)

≥25 RRi 1.5 (95% CI: 0.9–2.7)

Trend p = 0.10

EBV posj

<25 RRi 4.0 (95% CI: 0.9–17)

≥25 RRi 2.2 (95% CI: 0.8–5.8)

Years since 1st exposure

<20 RRi 2.3 (95% CI: 1.0–5.8)

≥20 RRi 1.2 (95% CI: 0.8–2.0)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-66

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

Reference Study design Exposure assessment Results; statistical significance (number observed deaths)

nonkeratinizing or 

undifferentiated tumors 

yielded similar results as 

overall analysis.

EBV posh

<25 RRi 2.3 (95% CI: 0.5–10)

≥25 RRi 2.8

Age at 1st exposure 

<20 RRi 1.3 (95% CI: 0.8–2.0)

≥20 RRi 3.4 (95% CI: 0.9–12)

EBV posj

<25 RRi 2.6 (95% CI: 1.1–6.5)

≥25 RRi 3.1 (95% CI: 0.4–24)

a

Exposure estimates by Hauptmann et al. (2004) were 10 times higher than those of Marsh et al. (2002). 

b

Adjusted for calendar year, age, sex, race, and pay category (salaried versus wage).

c

Results for cumulative and average intensity of exposure are not included here because condition logistic regression produces unstable estimates for this small 

number of cases.

d

Adjusted for years since first exposure to dust and exhaust fumes.

e

Adjusted for age at death, year at death, and availability of occupational information (Roush et al., 1987). f

Adjusted for cigarette smoking, alcohol consumption, gender, and age.

g

Adjusted for ethnic origin and cigarette smoking.

h

Adjusted for age, sex, race, SEER site, cigarette usage, proxy status, and education.

i

Adjusted for age, sex, education, and ethnicity.

j

EBV seropositives defined as positive for one of the following anti-EBV antibodies known to be associated with NPC: viral capsid antigen IgA, EBV nuclear 

antigen 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 and does not constitute Agency policy.

4-67 DRAFT—DO NOT CITE OR QUOTE

1 Following these reports of increased risk of NPC associated with formaldehyde exposure, 

2 a series of post hoc analyses of similar data were undertaken by Marsh and coworkers (Marsh et 

3 al., 2007a, b, 2002, 1996; Marsh and Youk, 2005). Briefly, these studies focused on the specific 

4 findings from a single plant in the NCI cohort (Wallingford, Connecticut) that generated the 

5 majority of the NPC cases. 

6 In the most recent subsequent report, Marsh et al. (2007a) continues to argue against a 

7 formaldehyde-NPC association. Although earlier reports speculated on anecdotal evidence that 

8 the statistically significant excess risk of NPC observed at the Wallingford, Connecticut, plant 

9 reflected the influence of unmeasured nonoccupational risk factors associated with employment 

10 outside the plant, the new report (Marsh et al., 2007a) suggests that occupational or hobby11 related work in silversmithing may have confounded the observed effect of formaldehyde on the 

12 increased risk of NPC. In this report, Marsh et al. (2007a) show that their subjectively assessed 

13 work in silversmithing is strongly associated with NPC. While the reported ORs are indeed quite 

14 high, the estimates are extremely unstable and it is not clear how many a priori hypotheses were 

15 tested for statistical significance. There are no citations of an association between silversmithing 

16 exposures and NPC in the medical literature. Marsh and coworkers mention that there was 

17 concordance of silver manufacturing history in the Wallingford, Connecticut, area. If 

18 silversmithing exposures are indeed independent risk factors for NPC, it would be expected that 

19 the rates of NPC in the surrounding counties with historical silver-related exposures would be 

20 elevated. However they are not increased, as evidenced by the comparability of the increased 

21 rates of NPC among the plant workers compared with both the national and local county rates 

22 that were very similar (Marsh et al., 2007a). The comparable rates indicate the counties’ rates of 

23 NPC were very similar to the national rates and weaken an association between silversmithing 

24 and NPC. Given the many post hoc reexaminations of alternative hypotheses to explain the 

25 original NCI findings, it is more likely that silversmithing is an artifactual confounder.

26

27 4.1.2.1.1.2. Professional cohort studies. Two cohort studies of professional groups, such as 

28 anatomists, pathologists, embalmers, and funeral directors, examined the risk of NPC and 

29 formaldehyde exposure. In general, measurements of formaldehyde concentrations were not 

30 available in studies of professionals but are generally below 1 ppm (IARC, 1995; Korczynski, 

31 1994; Stewart et al., 1992; Moore and Ogrodnik, 1986). Hayes et al. (1990) reported an excess 

32 risk of NPC among male professional embalmers and funeral directors, based on 4 deaths with 

33 1.9 expected based on age, gender, and calendar-year-specific proportions of deaths in the U.S. 

34 population. Hansen and Olsen (1995) studied male Danish cancer patients employed in 

35 companies in which formaldehyde was used or produced. Only a slight excess risk of NPC was 
This document is a draft for review purposes only and does not constitute Agency policy.

4-68 DRAFT—DO NOT CITE OR QUOTE

1 found, based on 4 cases with 3.2 expected (standardized proportionate incidence ratio [SPIR] 

2 1.3). Hansen and Olsen (1995) also reported on a significantly elevated risk of sinonasal cancer.

3

4.1.2.1.1.3. Case-control studies.

11 Three case-control studies have been conducted since the 1995 IARC report. Armstrong 

12 et al. (2000) found no association between formaldehyde exposure and NPC (adjusted OR = 0.71 

13 [95% CI: 0.34–1.43]), controlling for wood dust and industrial heat. Using data from the 

14 Surveillance Epidemiology and End Results (SEER) program, Vaughan et al. (2000) found an 

15 OR for ever-exposed persons of 3.1 (95% CI: 1.0–9.6) among cases of epithelial NPC, 

16 suggesting differences in the etiology of cancers at this site. There was a trend of increasing risk 

17 of NPC with increasing duration of exposure and cumulative exposure, controlling for wood dust 

18 exposure. Finally, Hildesheim et al. (2001) found that exposure to formaldehyde produced 

19 modest risk elevations for duration of exposure (OR = 1.6 for 10 years or less and 1.2 for over 

20 10 years of exposure), for cumulative exposure (ORs were 1.3 for <25 years of exposure and 1.5 

21 for 25+ years of exposure), and for years since first exposure. Among those with EBV, the OR 

22 was 2.7 (95% CI: 1.2–6.2) for ever-exposed persons. The risk was significantly higher among 

23 exposed persons whose work history was within the last 10 years (OR = 4.7 [95% CI: 1.1–20.0]) 

24 and for those followed 20+ years after exposure (OR = 2.8 [95% CI: 1.1–7.6]).

4 Five case-control studies (West et al., 1993; Roush et al., 

5 1987; Vaughan et al., 1986a, b; Olsen et al., 1984) reviewed by IARC in 1995 provided evidence 

6 of excess risks of NPC due to formaldehyde. Most of these studies report significant and 

7 nonsignificant elevations in risk of NPC in the range of 1.5–3.0, with some higher than 5.0. In 

8 its report, IARC (1995) concluded that, taking the data as a whole, formaldehyde appears to have 

9 a causal role in the induction of NPC, recognizing that the conclusion is based on small numbers 

10 of cancer cases. 

25

26 4.1.2.1.1.4. Summary of NPC studies. Findings from the large NCI cohort studies of NPC risk 

27 due to formaldehyde exposure clearly show a consistent pattern of increased risk with increased 

28 exposures. Post hoc reanalyses have challenged the interpretation of these findings but have not 

29 been able to dispute the reported excess in NPC mortality (Marsh et al., 2007a, b, 2002, 1996; 

30 Marsh and Youk, 2005). The major questions that have been raised by Marsh and coworkers 

31 highlight the observation that the NPC findings appear to depend on the results of 1 of the 10 

32 plants that made up the NCI cohort. While it is theoretically possible for coexposures at that 

33 plant or among those workers to act as potential confounders or modifiers of the observed effect 

34 of formaldehyde on increased risk of NPC, there is no solid evidence of such a relationship that 

35 would outweigh or supersede the reported adverse effects of formaldehyde exposure. While all 
This document is a draft for review purposes only and does not constitute Agency policy.

4-69 DRAFT—DO NOT CITE OR QUOTE

1 of the cohort members at the Wallingford, Connecticut, plant were also exposed to particulates, 

2 the NCI investigators did observe a dose-response relationship with formaldehyde among 

3 individuals with high particulate exposures, thereby strengthening the causal interpretation of the 

4 formaldehyde relationship with an increased risk of NPC. The described association of a 

5 potential occupational relationship with silversmithing and NPC has no basis in the medical 

6 literature and is inconsistent with the supposition that this activity is common in the locality of 

7 Wallingford, Connecticut, but has not been associated with increased rates of NPC in 

8 surrounding New Haven County (Connecticut). Marsh and coworkers did report significantly 

9 increased rates of pharyngeal cancer (including NPCs) among workers from the Wallingford 

10 plant compared with both the county and national rates. It is more plausible that the observed 

11 association at the Wallingford plant reflects higher formaldehyde exposures than at other plants. 

12 The exposure levels at Plant 2 were even higher than at the Wallingford plant and were 

13 associated with a fivefold increase in risk associated with NPC, even though this was based on a 

14 single observed case and was not significant. 

15 In addition to the evidence from the NCI cohort studies, modest additional evidence is 

16 found in the professional cohort studies of Hayes et al. (1990) and Hansen and Olsen (1995). 

17 The rarity of the disease and difficulties in obtaining valid and reliable historical exposure 

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

19 from the results of several case-control studies that support an increased risk of NPC from 

20 exposure to formaldehyde. The studies of Vaughan et al. (2000) and Hildesheim et al. (2001) 

21 provide evidence of an association of NPC with exposure to formaldehyde. Vaughan et al. 

22 (2000) found a dose-response relationship of NPC with increasing exposure to formaldehyde, as 

23 did Hildesheim et al. (2001). These studies, in general, are easier to conduct and may provide 

24 more statistical power for a specific level of risk estimate than do cohort studies.

25

26 4.1.2.1.2. Nasal and paranasal cancer

27 4.1.2.1.2.1. Case-control studies. Eight case-control studies were evaluated in the 1995 IARC 

28 monograph regarding the risk of nasal cavity and accessory sinuses from exposure to 

29 formaldehyde (Luce et al., 1993; Roush et al., 1987; Hayes et al., 1986; Olsen and Asnaes, 1986; 

30 Vaughan et al., 1986a, b; Brinton et al., 1984; Olsen et al., 1984). Of three studies that identified 

31 a cell type, two reported a positive finding of sinonasal cancer (Hayes et al., 1986; Olsen and 

32 Asnaes, 1986). One of the positive studies did not report any exposure to the potentially 

33 confounding influence of wood dust, while the other two did report an adjustment for exposure 

34 to wood dust. Of the remaining five studies where a cell type was not identified, only Roush et 

35 al. (1987) and Olsen et al. (1984) found positive results. The remaining studies (Vaughan et al., 
This document is a draft for review purposes only and does not constitute Agency policy.

4-70 DRAFT—DO NOT CITE OR QUOTE

1 1986a, b; Brinton et al., 1984) from the 1995 IARC monograph did not find associations between 

2 exposure and sinonasal cancer. Study details of the epidemiologic studies of nasal and paranasal 

3 cancer are summarized in Table 4-2. Vaughan et al. (1986b) matched 53 sinonasal cancer 

4 patients to 552 controls. Potential residential exposure to formaldehyde was estimated by 

5 utilizing residence in a mobile home with or without the presence of UFFI or particleboard or 

6 plywood as a surrogate for exposure. The authors found an OR of 1.5 for sinonasal cancer in 

7 subjects reporting residence of 10 or more years in a mobile home with UFFI before diagnosis. 

8 A higher OR (1.8) was reported for less than 10 years of mobile home residence. However, 

9 because actual formaldehyde levels in the subjects’ mobile homes are unknown, the exposure 

10 estimates are, at best, imprecise surrogates that typically have the effect of attenuating any true 

11 risk. In Vaughan et al. (1986a), the same cases and controls were examined for occupational 

12 exposures to formaldehyde, but no increase in risk of nasal or paranasal cancer was reported.

13 More recently, Luce et al. (2002) pooled data from 12 case-control studies. Combined, 

14 these studies had 195 adenocarcinomas and 432 SCCs of the sinonasal passages compared with 

15 3,136 controls. The authors reported a significant increase in the risk of sinonasal 

16 adenocarcinoma in men (adjusted OR = 3.0 [95% CI: 1.5–5.7]; 91 cases) and in women (adjusted 

17 OR = 6.2 [95% CI: 2.0–19.7]; 5 cases) with a high probability of exposure to formaldehyde. For 

18 SCCs, the ORs were more modest: OR = 1.2 in men and OR = 1.5 in women for a high 

19 probability of exposure to formaldehyde. In an analysis of 11 formaldehyde-exposed cases of 

20 sinonasal adenocarcinomas who were not exposed to wood dust, there was an elevated risk in 

21 men (OR = 1.9; 3 cases) and a significantly increased risk in women (OR = 11.1 [95% CI: 3.2–

22 38.0]; 5 cases) with a high probability of exposure to formaldehyde. Limitations of these studies 

23 were the lack of information about the actual levels or intensity of exposure to formaldehyde, 

24 exposure to multiple occupational carcinogens, and the small number of cases in some 

25 subgroups. In spite of those limitations, which generally obfuscate the observation of a true 

26 underlying effect, these studies identified effects of formaldehyde that were statistically 

27 significant predictors of sinonasal cancers.

28
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-71

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

Reference Study design Exposure assessment Results, statistical significance (number of cases)

Olsen and Asnaes 

(1986)

 

 

Case-control study of 

histologically confirmed cases 

of squamous cell 

carcinoma/lymphoepithelioma 

of the sinonasal cavities and 

paranasal cancers in 215 men 

and adenocarcinomas of the 

sinonasal cavities and 

paranasal cancers in 39 men 

matched with 2,465 controls 

with other cancers from the 

Danish Cancer Registry, 

1970–1982.

Employment histories 

after 1964 from files 

maintained by Danish 

Cancer Registry 

estimated by industrial 

hygienists.

Squamous cell carcinoma/lymphoepithelioma

Ever vs. never

Formaldehyde only RR 2.0 (95% CI: 0.7–5.9)

Formaldehyde + wood 

dust RR 1.6 (95% CI: 0.8–3.3)

10 or more years since first exposure 

Formaldehyde only RR 1.4 (95% CI: 0.3–6.4)

Formaldehyde + wood 

dust RR 1.8 (95% CI: 0.7–4.4)

Adenocarcinoma

Ever vs. Never

Formaldehyde only RR 7.0 (95% CI: 1.1–44)

Formaldehyde + wood 

dust RR 40.0 (95% CI: 22–71)

10 or more years since first exposure 

Formaldehyde only RR 9.5 (95% CI: 1.6–58)

Formaldehyde + wood 

dust RR 44.0 (95% CI: 22–88)

Hayes et al. (1986) Case-control study of 91 men 

with SCC of the nasal cavity 

and paranasal sinuses, from 

clinical records of six medical 

institutions in the Netherlands. 

195 controls from living and 

deceased males from 

municipal residence registries, 

from 1978–1981.

Cases selected from 

clinical records of six 

institutions in the 

Netherlands. 91 male 

patients and 195 

controls from living and 

deceased males from 

municipal residence 

registries with little or 

no exposure to wood 

dust. Industrial 

hygienists evaluated job 

histories according to 

probability of exposure 

based on job records.

Industrial hygienist A

Any 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 B

Any 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)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-72

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

Reference Study design Exposure assessment Results, statistical significance (number of cases)

Roush et al. (1987) Population-based case-control 

study of 198 male cases of 

sinonasal cancer from the 

Connecticut Tumor Registry 

who died of any cause in 

1935–1975. Controls were 

605 males dying in 

Connecticut during the same 

time period, randomly selected 

from state death certificates. 

Occupations from city 

directories and 

evaluation of job by 

industrial hygienist who 

classed exposure into I, 

probably exposed to 

some level most of 

working life; II, 

probably exposed to 

some level most of 

working life and 

probably exposed to 

some level 20+ years 

before death; III, 

probably exposed to 

some level most of 

working life and 

probably exposed to a 

high level in some 

years; IV

Exposure levels Sinonasal cancer

, probably 

exposed to some level 

most of working life 

and probably exposed 

to a high level 20+ 

years before death.

I ORa 0.8 (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)

Luce et al. (1993) Case-control study of men 

with sinonasal cancer 

(histologically confirmed), 77 

with adenocarcinoma, 59 with 

squamous cell carcinomas, 

and 25 tumors of other types, 

matched with 409 controls 

from 27 French hospitals and 

Industrial hygienist 

estimation based on job 

histories from personal 

interviews. Subjects 

were broken out into no 

exposure, possible 

exposure, or 

probable/definite 

Adenocarcinomab 

Possible exposure ORc 1.28 (95% CI: 0.16–10)

Probable/definite exposure

Average level

≤2 ORc 4.15 (95% CI: 0.96–18)

>2 5.33 (95% CI: 1.28–22)

Duration (years)

≤20 ORc 1.03 (95% CI: 0.18–5.77)

>20 6.86 (95% CI: 1.69–28)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-73

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

Reference Study design Exposure assessment Results, statistical significance (number of cases)

from lists of names supplied 

by patients.

exposure. Those 

classed as 

probable/definite 

exposure further 

categorized into three 

levels of frequency of 

exposure during a 

normal workweek: 1 = 

<5% of the time; 2 = 5–

30% of the time; and 3 

= >30% of the time. 

Concentration was 

categorized into 3 

levels: low (<0.0 ppm); 

medium (0.1–1 ppm); 

and high (>1 ppm). 

The exposure index = 

concentration × 

frequency. Cumulative 

level = sum of exposure 

indices. Average level 

= cumulative 

level/duration and 

ranged from 1 to 9. 

Nearly all cases had had 

wood dust exposure.

Cumulative level (years)

≤30 ORc 1.13 (95% CI: 0.19–6.95)

30–60 2.66 (95% CI: 0.38–19)

>60 6.91 (95% CI: 1.69–28)

Age 1st exposed (years)

≤15 ORc 9.99 (95% CI: 1.85–54)

16–20 4.12 (95% CI: 0.95–18)

>20 2.74 (95% CI: 0.58–13)

Date 1st exposed (years)

After 1954 ORc 6.02 (95% CI: 1.18–31)

Before 1954 4.26 (95% CI: 1.06–17)

Other cell type carcinomab 

Possible exposure ORc 0.81 (95% CI: 0.15–4.36)

Probable/definite exposure

Average level

≤2 ORc 1.67 (95% CI: 0.51–5.42)

>2 3.04 (95% CI: 0.95–9.7)

Duration (years)

≤20 ORc 2.82 (95% CI: 0.94–8.4)

>20 1.62 (95% CI: 0.48–5.51)

Cumulative level (years)

≤30 ORc 2.18 (95% CI: 0.65–7.31)

>30 2.21 (95% CI: 0.73–6.73)

Age 1st exposed (years)

≤20 ORc 2.03 (95% CI: 0.63–6.54)

>20 2.36 (95% CI: 0.76–7.33)

Date 1st exposed (years)

After 1954 ORc 0.48 (95% CI: 0.05–4.35)

Before 1954 3.27 (95% CI: 1.15–9.33)

Luce et al. (2002) Pooled analysis of 195 

adenocarcinomas and 432 

squamous cell carcinomas of 

Job exposure matrix 

based on interview data 

developed for pooled 

Adenocarcinoma

High probability of exposure

Men ORd 3.0 (95% CI: 1.5–5.7)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-74

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

Reference Study design Exposure assessment Results, statistical significance (number of cases)

the sinus/nasal cavity matched 

with 3,136 controls from 12 

case-control studies.

analysis. Industrial 

hygiene data used to 

develop indices of 

exposure. 11 

formaldehyde cases 

reported no exposure to 

wood dust.

Women ORe 6.2 (95% CI: 2.0–20)

Squamous cell carcinoma

High probability of exposure

Men ORe 1.2 (95% CI: 0.8–1.8)

Women ORe 1.5 (95% CI: 0.6–3.8)

Brinton et al. (1984) Case-control study of 160 

patients with cancer of the 

nasal cavity and paranasal 

sinuses from four North 

Carolina and Virginia 

hospitals matched with 290 

hospital controls with other 

conditions, based on 

occupational exposures.

Interview data on job 

history. Estimation of 

exposure based on 

industry type. Only 

two cases employed in 

industry associated with 

formaldehyde. There 

were no deaths in the 

high exposure category.

Overall male and female RR 0.35 (95% CI: 0.1–1.8)

Residence in mobile home ORf 0.6 (95% CI: 0.2–1.7)

Years of exposure to particleboard

1 to 9 ORf 1.8 (95% CI: 09–3.8)

10 or more 1.5 (95% CI: 0.7–3.2)

Olsen et al. (1984) Case-control study of 488 

cases of nasal cancer linked to 

the Danish Cancer Registry 

during 1970–1982. Controls 

were individuals with cancer 

of the colon, rectum, breast, 

and prostate. Three controls 

per case were selected for the 

same distributions of age, sex, 

and year of diagnosis as cases.

Employment histories 

after 1964 from files 

maintained by Danish 

Cancer Registry 

estimated by industrial 

hygienists.

Men

Formaldehyde only

Ever exposed RR 2.8 (95% CI: 1.8–4.3) (33)

Exposure to wood dust and 

formaldehyde RR 3.1 (95% CI: 1.8–5.3) (23)

Ever exposed RR 3.5 (95% CI: 2.2–5.6) (28)

1st exposure >10 years or 

more before diagnosis RR 4.1 (95% CI: 0.2.3–7.3) (20)

Hansen and Olsen 

(1995)

Proportionate incidence study 

of 2,041 men with sinonasal 

cancer who died between 1970 

and 1984 identified from the 

Danish Cancer Registry 

matched with the Danish 

Linked companies 

through tax records to 

national Danish Product 

Register, where 

companies must report 

amount of 

Overall SPIR 2.3 (95% CI: 1.3–4.0) (13)

Low formaldehyde SPIR 0.8 (95% CI: 0.02–4.4) (1)

Formaldehyde, no wood 

dust

SPIR 3.0 (95% CI: 1.4–5.7) (9)

SPIR 5.0 (95% CI: 0.5–13) (2)

Unknown SPIR 1.0 (95% CI: 0.03–6.1) (1)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-75

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

Reference Study design Exposure assessment Results, statistical significance (number of cases)

Supplementary Pension Fund 

whose longest work 

experience occurred at least 10 

years before the cancer 

diagnosis. The measure of 

risk was the SPIR, which 

measured the proportion of 

cases of sinonasal cancer in 

formaldehyde-associated 

companies relative to the 

proportion of cases of 

sinonasal cancer among all 

employees in Denmark. 

formaldehyde used per 

year.

Coggon et al. (2003) Cohort mortality study of 

14,014 men employed in 6 

factories of the chemical 

industry in Great Britain from 

periods during which 

formaldehyde was produced. 

Cohort followed through 

2000. Sinonasal cancer 

mortality SMRs based on 

English and Welsh age and 

calendar-year-specific 

mortality rates.

Exposures assessment 

based on data 

abstracted from 

company records. Each 

job categorized as 

background, low, 

moderate, high, or 

unknown levels. For 

analysis of sinonasal 

cancer, no gradient used 

because of small 

number of observed 

cases.

Overall SMR 0.87 (95% CI: 0.11–3.14) (2)

a

Adjusted for age at death, year at death, and availability of occupational information.

b

All had medium to high exposure to wood dust. 

c

Adjusted for age and exposure to glues and adhesives. 

d

Adjusted for age and study.

e

Adjusted for age, study, and cumulative exposure to dust. f

Adjusted for cigarette smoking, alcohol consumption, gender, and age.
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4.1.2.1.2.2. Cohort studies of nasal and paranasal cancer.

11 Three subsequent cohort studies reported on nasal and paranasal cancer. Hansen and 

12 Olsen (1995), in a proportional incidence study, found a significantly increased risk of sinonasal 

13 cavity cancer (SPIR = 2.3 [95% CI: 1.3–4.0]; 13 observed) in 265 Danish industries, where 

14 2,041 of 91,182 cancer patients had at least 10 years of continuous formaldehyde-related work 

15 experience before diagnosis. Coggon et al. (2003), in a cohort study of 14,014 employees in six 

16 chemical factories in Great Britain, found only 2 deaths from sinonasal cancer (2.3 expected 

17 based on national death rates in Great Britain). Finally, Hauptmann et al. (2004) evaluated the 

18 sinonasal cancer risk in the NCI cohort and found three cases (SMR = 1.19 [95% CI: 0.38–3.68]) 

19 among those with a 15-year lag period.

1 IARC (1995) also reported the 

2 results of several cohort studies of professional and industrial workers for nasal and paranasal 

3 cancer (Andjelkovich et al., 1995; Gardner et al., 1993; Hall et al., 1991; Hayes et al., 1990; 

4 Bertazzi et al., 1989; Edling et al., 1987; Blair et al., 1986; Stroup et al., 1986; Harrington and 

5 Oakes, 1984; Levine et al., 1984; Walrath and Fraumeni, 1984, 1983; Friedman and Ury, 1983). 

6 Only a few studies reported any cases of sinonasal cancer. No cases of this type of cancer were 

7 reported in any of the studies of professional workers examined by the IARC. Only 2 cases (2.2 

8 expected) were reported by Blair et al. (1986), and only 1 case (1.7 expected) was reported by 

9 Gardner et al. (1993). The likelihood of finding this rare tumor type in a long-term cohort study 

10 is low. 

20

4.1.2.1.2.3. Summary of nasal and paranasal cancers.

31

21 The pooled case-control study of Luce 

22 et al. (2002) provides strong evidence of an association between formaldehyde exposure and 

23 increased risk of sinonasal adenocarcinoma. The cohort studies may not have had sufficient 

24 statistical power to show an association, and studies that did not distinguish cancer type may 

25 have aggregated a truly causal relationship with a noncausal relationship with SCC. In summary, 

26 there appears to be increased risk of sinonasal cancer associated with formaldehyde exposure 

27 with or without exposure to wood dust. The effect appears to be stronger when the risk is 

28 stratified by cancer type with higher risks of adenocarcinoma compared with SCC. Taken 

29 together with the NPC findings in the neighboring tissue, it is concluded that there is evidence of 

30 higher risks of sinonasal cancer associated with exposure to formaldehyde.

32 4.1.2.1.3. Other respiratory tract cancers. Of six cohort studies of buccal/pharynx cancer in 

33 studies of professionals reviewed by IARC (Hayes et al., 1990; Logue et al., 1986; Stroup et al., 

34 1986; Levine et al., 1984; Walrath and Fraumeni, 1984, 1983), no evidence of a risk associated 

35 with exposure to formaldehyde was reported (see Table 4-3). In studies of industrial worker 

36 cohorts where buccal/pharynx cancer was examined (Andjelkovich et al., 1995; Stayner et al., 
This document is a draft for review purposes only and does not constitute Agency policy.

4-77 DRAFT—DO NOT CITE OR QUOTE

1 1988; Blair et al., 1986), only one (Stayner et al., 1988) reported an excess risk of death from this 

2 tumor (SMR = 3.4, based on four deaths in a cohort of 6,741 white women). Three case-control 

3 studies (Merletti et al., 1991; Vaughan et al., 1986a, b) did not find an association between oral 

4 cavity, oropharyngeal, and hypopharyngeal cancers and formaldehyde exposure. However, 

5 Merletti et al. (1991) found an elevated OR of 1.8 associated with probable or definite exposure 

6 to formaldehyde in a study of 86 patients matched with 373 controls. There was no risk of 

7 laryngeal cancer associated with formaldehyde in a case-control study (Wortley et al., 1992) of

8 235 patients with laryngeal cancer and 547 controls. The OR in that study was 1.0 (adjusted for 

9 age, smoking, drinking, and level of education). IARC (1995) concluded that there was little 

10 evidence of an increased risk of laryngeal cancer.

11

4.1.2.1.3.1. Cohort studies of other respiratory tract cancers.

20 The Marsh et al. (2002) update also derived elevated risk estimates for oropharyngeal, 

21 hypopharyngeal, and pharyngeal-unspecified cancers. The SMRs ranged from 2.11 to 2.25 

22 based on U.S. rates and 1.52 to 1.89 based on county rates. When combined with NPC 

23 International Classification of Death (ICD) codes 146–149 to increase statistical power, the total 

24 pharyngeal cancer SMRs were significant based on U.S. death rates (SMR = 2.63, n = 22, p < 

25 0.01) and county death rates (SMR = 2.23, n = 22, p < 0.01) and remained significant for both 

26 short-term (less than 1 year) and long-term exposures. Furthermore, using the exposure 

27 estimates of Marsh et al. (1996), both cumulative and average exposure to formaldehyde resulted 

28 in elevated SMRs, some of which were significant for pharyngeal cancer. Coggon et al. (2003) 

29 identified 14 cases of cancer of the larynx (13.1 expected) in their cohort of formaldehyde30 exposed factory workers. Pinkerton et al. (2004) found an excess risk of mortality from buccal 

31 cavity cancer (SMR = 1.33, four deaths observed) and a deficit of the risk of pharyngeal cancer 

32 (SMR = 0.64, three observed). Since the number of observed deaths was small and the risk 

33 estimates were subject to much variation for both studies, no conclusions about cancer risk can 

34 be drawn.

12 Hansen and Olsen (1995) 

13 reported a 10% increase in the risk of cancer of the buccal cavity and pharynx (SPIR = 1.1) in 

14 their proportional incidence study of Danish workers. Marsh et al. (1996) reported no excess 

15 risk of buccal cavity cancer cases (SMR = 1.31) based on U.S. rates and no excess based on state 

16 mortality rates (SMR = 1.0). For oropharyngeal cancer, the SMR was 1.84 (based on two cases), 

17 the SMR for hypopharyngeal cancer was 1.41 (based on one case), and the SMR for laryngeal 

18 cancer was 1.47 (based on six cases). The latter risks were elevated even when SMRs were 

19 derived from Connecticut mortality rates.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-78

Table 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)

Marsh et al. (2002) Retrospective cohort 

mortality study of 7,328 

workers hired up to 1984 

and followed until 1998 in 

one plant from Blair et al. 

(1986, 1987) and 

Hauptmann et al. (2004). 

Mortality was compared 

with death rates in two 

Connecticut counties and 

U.S. A nested case-control 

analysis was also conducted 

with 4 controls matched on 

age, year of birth, race, and 

sex randomly selected from 

cohort. Conditional logistic 

model was used for nested 

case-control analysis. 

Worker-specific exposures 

from job exposure matrix 

were based on available 

sporadic sampling data from 

1965–1987, job descriptions, 

and verbal job descriptions 

by plant personnel and 

industrial hygienists. 

Exposures were then ranked 

on a 7-point scale. An 

exposure range was assigned 

to each rank. 17% of jobs 

validated with company 

monitoring data, remaining 

83% based on professional 

judgment. Pre-1965 levels of 

formaldehyde were assumed 

to be the same as post-1965 

levels.

Cohort study

Overall

U.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) county

Unexposed 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) county

Unexposed 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 ppm SMR 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 ppm SMR 2.12 (95% CI: 1.21–3.45) (16)

Nested case-control analysis

Cumulative 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)

Exposure to >0.7 ppm OR 1.60 (95% CI: 0.15–9.77) (6)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-79

Table 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)

Coggon et al. (2003) Cohort mortality study of 

14,014 chemical workers 

employed in 6 British 

factories. 

Based on data abstracted 

from company records. Each 

job was categorized as having 

background, low, moderate, 

high, or unknown levels of 

formaldehyde.

Overall SMR 1.55 (95% CI: 0.87–2.56) (15)

High exposure SMR 1.91 (95% CI: 0.70–4.17) (6)

Shangina et al. 

(2006)

Multicentered, hospitalbased case-control study in 

four European countries; 

men only. Cancer cases: 34 

hypopharyngeal; 316 

laryngeal. Controls: 728 

hospital patients with 

various conditions.

Exposures determined by 

local industrial hygienists, 

chemists, and physicians. 

Coding was established and 

standardized. Categories 

were developed for 73 

agents; frequency was 

estimated as the proportion of 

time a worker was exposed. 

Linear trends were examined 

for duration in years, 

weighted duration in hours, 

and cumulative exposure.

Formaldehyde

Laryngeal cancer:

Ever vs. never

Highest cumulative 

(>22,700 mg/m3

-hours) 

vs. lowest

Tests of trends:

Years exposed

Cumulative exposure

OR

OR

p = 0.06

p = 0.07

1.68

3.12

(95% CI: 0.85–3.31)

(95% CI: 1.23–7.91)
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1 Hauptmann et al. (2004) combined URT cancers (cancers of the salivary gland, mouth, 

2 nasopharynx, nasal cavity, and larynx). For average intensity of exposure (AIE), the RR was 

3 1.69 for the medium exposure category (0.5 to <1.0 ppm) and 2.21 (p < 0.05) for the high 

4 exposure category (>1.0 ppm). For peak exposure, the RR was 1.24 for the medium exposure 

5 category (2.0 to <4.0 ppm) and 1.65 for the high exposure category (>4.0 ppm). For cumulative 

6 exposure, the RR was 1.92 for the medium exposure category (1.5 to <5.5 ppm) but 0.86 in the 

7 high exposure category (>5.5 ppm-years). The dose trends for these analyses, while suggestive 

8 for average and peak exposures, were not statistically significant.

9

4.1.2.1.3.2. Case-control studies of other respiratory cancers.

18 In a case-control study, Laforest et al. (2000) examined 201 patients with squamous cell 

19 hypopharyngeal cancer and 296 patients with squamous cell laryngeal cancer, who were matched 

20 to 296 controls with cancers of other sites in 15 French hospitals. Adjusting for potential 

21 confounders, the OR of hypopharyngeal cancer in patients with a high probability of exposure to 

22 formaldehyde was 3.78 (95% CI: 1.50–9.49). The ORs were significantly increased with both 

23 exposure durations and high cumulative level of exposure. 

10 Gustavsson et al. (1998) 

11 conducted a case-control study of 545 cases of SCC of the oral cavity, oropharynx, hypopharynx, 

12 larynx, and esophagus, frequency-matched by age and region with 641 controls. Regression 

13 analyses among 545 male cases showed elevated but nonsignificant risks of SCC of the oral 

14 cavity (OR = 1.28), esophagus (OR = 1.90), and larynx (RR = 1.45) associated with 

15 formaldehyde exposure. However, several of the carcinoma types were statistically significantly 

16 associated with exposure to welding fumes, polyaromatic hydrocarbons, asbestos, and metal 

17 dust.

24 Marsh et al. (2002) conducted a nested case-control study of the 22 pharyngeal cancer 

25 deaths in the Wallingford, Connecticut, plant cohort. Each of the pharyngeal cancer deaths was 

26 matched on race, sex, age, and year of birth to four controls from the cohort. Twenty of the 

27 22 cases were exposed to formaldehyde, yielding an OR of 3.04 after adjustment for smoking 

28 and year of hire. There was little or no association of pharyngeal cancer incidence in these 

29 workers with either average or cumulative exposure, based on the exposure estimates in this 

30 study. There was a suggested trend of increasing OR with increasing duration of exposure for 

31 any formaldehyde exposure as well as for formaldehyde exposure >0.2 ppm. The results of this 

32 nested case-control analysis are inconclusive because of its low statistical power and 

33 questionable exposure estimates, which differed substantially from those estimated by the NCI 

34 (see Section 4.1.1.1). In addition, the relatively flat dose-response curve in the nested case-
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1 control study contradicts the positive dose-response curves reported (particularly for NPCs) in 

2 the same study, based on SMRs derived from county and U.S. death rates in the cohort analysis.

3 Shangina et al. (2006) conducted a multicentered case-control study in Europe and Russia 

4 of 34 cases of hypopharyngeal cancer, 316 cases of laryngeal cancer, and 728 controls. With 

5 regard to formaldehyde exposure, a nonsignificant positive association was found for laryngeal 

6 cancer (OR = 1.68, 95% CI: 0.85–3.31). Trends over increasing exposure were found for 

7 duration of exposure in years (p = 0.06) and for cumulative exposures (p = 0.07). The 

8 investigators reported an OR of 3.12 (95% CI: 1.23–7.91) for the highest cumulative exposure 

group (>22,700 mg/m3 9 -hours) compared with the unexposed group.

10

4.1.2.1.3.3. Summary of other respiratory cancers.

17

11 The evidence for a compound-specific 

12 effect on the risk of buccal/pharynx, oral cavity, oropharynx, hypopharynx, and laryngeal 

13 cancers as a result of exposure to formaldehyde is minimal. Only the study by Laforest et al. 

14 (2000) and, to a lesser extent, that by Shangina et al. (2006) provided evidence of an association 

15 between formaldehyde and these tumors. However, even the study of Laforest et al. (2000) had 

16 major limitations that made the evidence of an association suggestive at best.

18 4.1.2.1.4. Lung Cancer. None of the cohort studies of workers in specific professions indicated 

19 excess risks of lung cancer. Of the professional studies reviewed, the RRs range from an 

20 extremely low value (SMR) of 0.2 in Hall et al. (1991), based on nine deaths, to an RR 

21 (proportional mortality ratio) of 1.1, based on 70 lung cancer deaths in Walrath and Fraumeni 

22 (1983).

23

4.1.2.1.4.1. Industrial worker cohort studies of lung cancer.

29 Several industrial cohort studies (Andjelkovich et al., 1995; Bertazzi et al., 1989, 1986; 

30 Stayner et al., 1988; Edling et al., 1987) reported no significant excess risks of lung cancer from 

31 exposure to formaldehyde. No consistent association between formaldehyde exposure and lung 

32 cancer was found in several reports of the NCI 10-plant cohort originally investigated by Blair et 

33 al. (1987, 1986). Hauptmann et al. (2004) gives the most recent report on this cohort, which has 

34 been studied in part or in its entirety by several others (Marsh et al., 1994, 1992a, b; Sterling and 

35 Weinkam, 1994, 1989a, b, 1988; Robins et al., 1988; Liebling et al., 1984; Fayerweather et al., 

24 Evidence of a relationship 

25 between formaldehyde exposure and lung cancer is conflicting, with some studies showing 

26 modest increases while others show significant deficits in risk. There is, at best, only weak 

27 evidence from several studies to suggest that exposure to formaldehyde is associated with lung 

28 cancer.
This document is a draft for review purposes only and does not constitute Agency policy.

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1 1983; Wong, 1983; Marsh, 1982). Marsh et al. (1996) reported small but significantly increased 

2 risks of respiratory cancer in males at one plant from the cohort. The SMRs were 1.22 based on 

3 U.S. rates, 1.34 based on Connecticut rates, and 1.28 based on county rates. 

4 Similarly, Gardner et al. (1993) and Acheson et al. (1984) found a significant but modest 

5 association between lung cancer and formaldehyde exposure (SMR = 1.2 [95% CI: 1.1–1.4]). In 

6 workers hired after 1964, the SMR was 1.1. No trends by level or duration of exposure were 

7 found. Pinkerton et al. (2004) and Stayner et al. (1988) studied a cohort of 11,030 workers in 

8 three garment plants and found an SMR of 1.1 for lung cancer. Some studies have found modest 

9 elevations in risk of lung cancer with formaldehyde exposure, some of which were significant. 

10 Coggon et al. (2003) updated the Gardner et al. (1993) study of industrial workers. By taking 

11 data from all six factories together, results showed a statistically significant excess risk of lung 

12 cancer in the high-exposure category when compared with British national mortality rates (SMR 

13 = 1.58 [95% CI: 1.40–1.78]) and to local mortality rates (SMR = 1.28 [95% CI: 1.13–1.44]). 

14 Callas et al. (1996) reanalyzed the cumulative exposure of 279 lung cancer cases among white 

15 male workers from the NCI study, which comprised 80% of the NCI cohort (Blair et al., 1986). 

16 The analysis revealed modest RRs of 1.46, 1.27, and 1.38 for lung cancer in the cumulative 

17 exposure categories 0.05 to 0.5, 0.51 to 5.5, and greater than 5.5 ppm-years, respectively. None 

18 of these RRs were significant. Finally, Matanoski (1991) reported a significant deficit in the risk 

19 of respiratory cancer (SMR = 0.56 [95% CI: 0.44–0.70]; 77 observed) in pathologists 

20 presumably exposed to formaldehyde based on U.S. mortality rates. 

21

4.1.2.1.4.2. Case-control studies.

30 De Stefani et al. (2005) conducted a case-control study of 338 adenocarcinomas of the 

31 lung in male patients admitted to four Montevideo hospitals from 1994 to 2000. The highest 

32 ORs were for smoking (6.0 [95% CI: 3.3–11]) and for former smokers (4.0 [95% CI: 2.1–7.3]). 

33 In addition, three agents (i.e., asbestos, silica dust, and formaldehyde) indicated significant 

34 excess risks of lung adenocarcinoma after adjusting for smoking history. A significant exposure22 Several case-control studies of lung cancer (Partanen et al., 

23 1990; Gerin et al., 1989; Bond et al., 1986; Coggon et al., 1984; Fayerweather et al., 1983; 

24 Anderson et al., 1982) showed no excess lung cancer risk associated with potential exposure to 

25 formaldehyde when analyzed by length of exposure, intensity, and potential exposure 5, 10, or 

26 15 years before death or by combinations of these factors. By contrast, Coggon et al. (1984) 

27 reported a statistically significant increase in risk of lung cancer among male patients with any 

28 potential exposure to formaldehyde based on occupations listed on death certificates (SMR = 1.5 

29 [95% CI: 1.2–1.8]).
This document is a draft for review purposes only and does not constitute Agency policy.

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1 duration relationship was found for formaldehyde for 21+ years of exposure (OR = 3.0 [95% CI: 

2 1.6–5.8]; p = 0.004 for trend).

3

4.1.2.1.4.3. Summary of lung cancer.

13

4 The evidence for an association between formaldehyde 

5 and lung cancer is limited. Only one study has found a statistically significant effect (Coggon et 

6 al., 2003). However, there may be other explanations rather than exposure to formaldehyde for 

7 this association. Except for the findings of De Stefani et al. (2005), other studies of lung cancer 

8 and exposure to formaldehyde have not supported this finding, including several well-done 

9 cohort studies that were specifically designed to evaluate lung cancer. Until the Coggon et al. 

10 (2003) study of British formaldehyde workers is replicated or reevaluated to determine the cause 

11 of the excessive lung cancer risk, evidence from that study alone is insufficient at this time to 

12 support an association between lung cancer and formaldehyde exposure.

14 4.1.2.1.5. Summary of respiratory tract cancers. Recent studies of NPC continue to support an 

15 association with exposure to formaldehyde even at low levels. In some studies, the association 

16 between formaldehyde and NPC persisted even when adjusted for the effect of potential 

17 confounders (Hauptmann et al., 2004). Data from some reports have suggested a dose-response 

18 relationship (Hauptmann et al., 2004; Marsh et al., 2002; Vaughan et al., 2000).

19 The risk of NPC was significantly elevated among industrial workers with cumulative 

20 exposure, average exposure, and peak exposure to formaldehyde (Hauptmann et al., 2004). The 

21 studies of the single Wallingford plant by Marsh et al. (2002, 1996, 1994) and Marsh and Youk 

22 (2005) also revealed a dose-response trend, although the absolute exposure level estimates were 

23 much lower. The relatively flat dose-response curve seen in the nested case-control study by 

24 Marsh et al. (2002) of all pharyngeal cancers was inconsistent with the positive dose-response 

25 curves reported in the same paper based on county and U.S. death rates, particularly for NPC. 

26 Also of interest was the finding of a statistically significant increase in the risk of NPC in 

27 formaldehyde-exposed workers who were seropositive for EBV in the Hildesheim et al. (2001) 

28 study. 

29 The pooled analysis by Luce et al. (2002) provides evidence of a relationship of sinonasal 

30 cancer, particularly adenocarcinoma, with formaldehyde. However, as with some of the studies 

31 of NPC, the findings are potentially confounded by concurrent exposure to wood dust. When 

32 wood dust exposure was adjusted for in the analysis, the resulting risks were still positive but 

33 based on small numbers and, as a result, subject to much variability. The more recent studies 

34 continued to reveal small significant and nonsignificant associations among cancers of 
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1 buccal/pharynx, oral cavity, hypopharynx, and larynx and exposure to formaldehyde. However, 

2 the estimates were always based on small numbers. 

3 A recent study that reported statistically significant lung cancer in association with a high 

4 level of exposure to formaldehyde was conducted by Coggon et al. (2003). The investigators 

5 suggested that unknown lifestyle factors, including smoking, could be responsible for the 

6 finding. Despite the results of their analysis, the authors were unconvinced that formaldehyde 

7 was the agent responsible for the elevation in lung cancer risk. However, De Stefani et al. (2005) 

8 also reported a statistically significant risk of lung adenocarcinoma in formaldehyde-exposed 

9 hospital patients even when smoking was controlled for in their analyses. 

10 In all studies of formaldehyde and lung cancer, smoking remains an important 

11 confounder and possibly an effect modifier. Residual confounding of smoking or other 

12 respiratory exposures (e.g., wood dust or chemical or particular exposures) must always be 

13 considered. 

14

15 4.1.2.2. Non-Respiratory Tract Cancer

16 4.1.2.2.1. LHP cancers. Cancers of the hematopoietic system include lymphosarcoma, 

17 reticulosarcoma, Hodgkin’s disease, non-Hodgkin’s disease, multiple myeloma, and all types of 

18 leukemia, including lymphoid and myeloid. Virtually all of the studies of LHP cancers and 

19 formaldehyde are cohort studies and are divided into two groups: professional and industrial. 

20 Several of the studies of professional groups were reviewed in an IARC (1995) monograph and 

21 are briefly discussed in the next section regarding their findings on cancer of the LHP system. 

22 One case-control study of non-Hodgkin’s lymphoma is discussed at the end of this section.

23

4.1.2.2.1.1. Professional cohort studies. 

31 Harrington and Shannon (1975) conducted a cohort mortality study of 2,079 British 

32 pathologists (1955–1973) and 12,944 British medical laboratory technicians (1963–1973). When 

33 compared with death rates for England and Wales, the all-cause SMR for the pathologists was 

34 0.60 versus 0.67 for the laboratory technicians. There was a significant increase in the risk of 

35 lymphatic and hematopoietic neoplasia (SMR 2.0; 8 observed with 3.3 expected; p < 0.01) 

24 Several cohort studies have been undertaken by 

25 professional groups (i.e., anatomists, pathologists, embalmers, and funeral directors) because 

26 their careers are likely to bring them into contact with formaldehyde. Some studies have 

27 reported an increase in the risk of myelogenous leukemia and other LHP cancers (see Table 4-4). 

28 A few of the increased risks were statistically significant. None of the studies of professionals 

29 have used personal exposure measurements of formaldehyde or other chemicals, making 

30 specificity for any single exposure difficult to determine.
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1 among male pathologists. However, the SMR for technicians was only 0.6 (3 observed). The 

2 low SMRs suggest that these professionals have a healthier profile compared with the British 

3 population. No actual exposure estimates are available.

4 Harrington and Oakes (1984) expanded the above study to include 2,307 male and 413 

5 female pathologists and laboratory technicians. Mortality was only examined from 1973 until 

6 1980; deaths that occurred before 1974 were not included in the update. The SMR for leukemia 

7 was 0.91 in men and 9.26 (based on one case) in women. Although the earlier LHP cancer 

8 deaths were not included in this analysis, the investigators say in their conclusion that their 

9 previous suggestion of an increase in certain lymphatic neoplasia was not confirmed in the 

10 present study because of small numbers. The exceptionally low SMRs suggest that this group of 

11 professionals enjoyed a healthier lifestyle compared with the British population as a whole. Just 

12 as in the earlier studies of these professionals, no exposure estimates are available.

13 Hall et al. (1991) expanded the above study by including the newest members of the 

14 Royal College of Pathologists. The cohort totaled 4,512 individuals, although only 3,069 males 

15 and 803 females were included in the analysis. The reasons for this discrepancy were not 

16 specified, although the authors mentioned that an unknown number of expected deaths for 

17 Northern Irish and female Scottish pathologists were not calculated, 32 pathologists were lost in 

18 follow-up, and cause of death was unknown for 9 individuals. Follow-up was extended from 

19 1980 to 1986. Mortality was enumerated from 1974 to 1987, a period of time that differed from 

20 both of the earlier studies described above. There were statistically not significant excess risks 

21 for lymphatic and hematopoietic cancer (SMR 1.44; 10 observed) and leukemia (SMR 1.52; 

22 4 observed) for both sexes combined, based on mortality rates in England and Wales. 

23 Separately, there was 1 female death in the lymphatic and hematopoietic cancer category (0.57 

24 expected). The most striking observation in this study is that, despite the low cancer mortality 

25 (SMR 0.45 for all cancer; 53 observed but 118.19 expected), there was still an excess (but not 

26 statistically significant) risk of hematopoietic cancers. This finding of an extremely low risk for 

27 all cancers suggests that population death rates may not be appropriate as a referent group—for 

28 example, the SMRs for lung cancer (0.19) and nonneoplastic respiratory diseases (0.23) were 

29 significantly decreased, suggesting a lower prevalence of smoking among the pathologists 

30 compared with the general population of England and Wales. However, the finding of a possibly 

31 increased risk of LHP cancers should be analyzed further by selecting a more appropriate 

32 reference population (another professional group without exposure to formaldehyde) or by 

33 utilizing internal comparisons.

34 Walrath and Fraumeni (1983) conducted a proportionate mortality study of all embalmers 

35 and funeral directors licensed in the state of New York between 1902 and 1980 who were known 
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1 to have died between 1925 and 1980. The investigators requested death certificates for 1,678 

2 persons but received only 1,263 (75%). The investigators restricted their analysis to 1,132 

3 males. The distribution of the causes of death was compared with the age-, race-, and calendar4 year-specific proportions of deaths for each cause among the male U.S. population. Duration of 

5 exposure was approximated by time since first license. While the methodology could not be 

6 applied in all calculations because of data gaps, excess risks were found for lymphatic and 

7 hematopoietic cancers, with a proportionate mortality ratio (PMR) of 1.2 (observed 25), and for 

8 leukemia, with a PMR of 1.32 and proportionate cancer mortality ratio (PCMR) of 1.19 

9 (12 observed). The PMRs were not affected when the estimates were stratified by latency 

10 (<35 years or 35 years since first license) or by age at first license. Because the cause of death 

11 could not be determined for nearly 25% of the study group, the risk estimates could be 

12 underestimated. The metrics, PMR, and PCMR are not stochastic processes. An increase in one 

13 cause would produce decreases in all the other causes.

14 Using the proportionate mortality method, Walrath and Fraumeni (1984) studied 1,007 

15 deceased white male embalmers, members of the California Bureau of Funeral Directing and 

16 Embalming, whose deaths occurred between 1925 and 1980. The decedents had to have been 

17 licensed to practice between 1916 and 1978. For lymphatic and hematopoietic cancer, the PMR 

18 was 1.22 (19 deaths observed). For leukemia alone, the PMR was 1.75 and significant 

19 (12 deaths observed, p < 0.05). Among embalmers licensed for 20 years or longer, the risk of 

20 leukemia increased and was also significant (PMR 2.21; 8 observed; p < 0.05). But this study, 

21 like the study of New York embalmers, had the same limitations discussed above. The 

22 investigators did not provide information on the number of embalmers for whom no cause of 

23 death could be found.

24 Levine et al. (1984) conducted a cohort mortality study of 1,477 male Ontario 

25 undertakers first licensed between 1928 and 1957 and followed until the end of 1977. Out of 

26 359 subjects who had died, there were 8 deaths from lymphatic and hematopoietic cancers 

27 compared with 6.5 expected. Additionally, there were 4 deaths from leukemia versus 

28 2.5 expected. Because death rates were not available for Ontario before 1950, person-years and 

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

30 undertakers.

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

32 members of the American Association of Anatomists between 1888 and 1969. The investigators 

33 derived SMRs from the U.S. white male population and used members of the American 

34 Psychiatric Association (APA) as a comparison group. Vital status was ascertained between 

35 1925 and 1979. Women were excluded from analysis because of the small numbers. Only 
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1 738 deaths were observed versus 1,133.9 expected, based on U.S. death rates (SMR 0.65), 

2 possibly indicating a sizable HWE. However, a slight increase in the risk of lymphatic and 

3 hematopoietic cancers (SMR 1.2; 18 observed) and the risk of leukemia (SMR 1.5; 10 observed) 

4 was evident. A significant increase in the risk of brain cancer (SMR 2.7; 10 observed; p < 0.05) 

5 was also reported. When the leukemia analysis was restricted to the myeloid type, the SMR 

6 increased to 8.8, based on five deaths (p < 0.05). The analysis using the APA group was 

7 restricted to deaths that occurred between 1900 and 1969. This restriction removed five 

8 leukemia deaths and person-years from the analysis because they likely died after 1969. Because 

9 of this, there were only 3 leukemia deaths versus 3.6 expected, based on APA death rates. The 

10 investigators concluded that the etiological agent had not been definitively identified, mentioning 

11 that a wide range of solvents, stains, and preservatives, including formaldehyde, are used to 

12 prepare biological specimens.

13 Logue et al. (1986) conducted a cohort study of male radiologists and pathologists 

14 registered with the Radiation Registry of Physicians and the College of American Pathologists 

15 (CAP) between 1962 and 1977. Although the main focus was on determining mortality in 

16 radiologists from exposure to ionizing radiation, mortality was also ascertained for pathologists 

17 alone. To derive SMRs, expected deaths were the sum of the products of person-years times 

18 death rates for both cohorts during the follow-up period in white males only. However, there 

19 were no exposure measurements, and the SMRs were not adjusted for calendar time. Of 5,585 

20 members of the CAP, 496 had died by December 31, 1977. Although the SMR was 0.48 for 

21 pathologists for cancer of the lymphatic and hematopoietic system, for the more specific 

22 category of leukemia and aleukemia the SMR was 1.06 (neither was significant). For 

23 radiologists, the SMRs were 0.78 and 1.55, respectively, also not significant. Cause of death 

24 could not be determined for 8% of the deaths. Although age-adjusted rates for leukemia were 

25 also calculated for each cohort, they were only used for comparison between the two separate 

26 professional groups.

27 Hayes et al. (1990) conducted a proportionate mortality study of 3,649 deceased white 

28 and 397 deceased nonwhite U.S. male embalmers and funeral directors who had died between 

29 1975 and 1985, using records from local licensing boards, state funeral directors’ associations in 

30 32 states and the District of Columbia, the National Funeral Directors’ Association, and state 

31 offices of vital statistics (n = 894). Expected deaths by cause were derived from 5-year age- and 

32 calendar-year-specific proportions of deaths among appropriate race groups from the U.S. 

33 population. No measured exposure data were available. A PCMR would be derived by 

34 excluding noncancer causes of death. Statistically significant excesses in hematopoietic and 

35 lymphatic cancers were found in white (PMR 1.31 [95% CI: 1.06–1.59]; 100 observed) and 
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1 nonwhite (PMR 2.41 [95% CI: 1.35–3.97]; 15 observed) embalmers and funeral directors. The 

2 combined PMR was 1.39 (95% CI: 1.15–1.63). The excess risk was higher for myeloid 

3 leukemia (ML) (PMR 1.61 [95% CI: 1.02–2.41]; 23 observed) and for other unspecified 

4 leukemias (PMR 2.08 [95% CI: 1.21–3.34]; 17 observed) in white males. The risks were 

5 elevated in nonwhite males based on only a few cases (PMR 1.33 [95% CI: 1.10–1.60]; 

6 4 observed).

7 Matanoski (1991) conducted a study of 6,111 male pathologists for NIOSH. Members of 

8 the cohorts were part of an earlier unpublished study. Twenty-nine thousand psychiatrists were 

9 used as a comparison group. Both samples were selected from the membership rolls of 

10 professional associations. A total of 3,787 pathologists died between 1940 and 1978. Women 

11 were excluded from the analysis. Of the population of psychiatrists, 4,788 died by 1980. U.S. 

12 age- and calendar-time-specific death rates from 1925 were used to develop SMRs. Separate

13 SMRs were based on psychiatrists’ death rates. The risk of hematopoietic cancer (excluding 

14 Hodgkin’s disease) was elevated (SMR 1.25; 57 observed) based on U.S. white males. For 

15 leukemia, the SMR was 1.35 (31 observed). The SMR for leukemia among psychiatrists was 

16 0.83 (35 observed). Compared with leukemia in psychiatrists, the SMR for pathologists was 

17 1.68 (95% CI: 1.14–2.38). The SMR for other lymphatic cancers was 1.53 (16 observed) and for 

18 LHP cancer 1.22 (64 observed). Comparing the pathologists’ death rates to those of psychiatrists 

19 could be thought to have greater validity than if death rates for the U.S. population as a whole 

20 had been used, because of shared socioeconomic circumstances and access to medical care 

21 between the two professional groups. Differences in access to health care might have been 

22 greater for subjects in the earlier part of the study, because improved diagnosis and medical care 

23 for LHP cancers became more broadly available later in the study period. By using SMRs based 

24 on U.S. death rates, which include those who do not have adequate access to medical care, the 

25 difference between expected and observed deaths would be reduced. This is less likely to occur 

26 when one professional group is compared with another professional group, assuming 

27 psychiatrists and pathologists have equal access to care.

28

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

30 studies that show associations between LHP cancer and formaldehyde. The studies by Marsh et 

31 al. (1994), Blair et al. (1986), and Acheson et al. (1984) and the later update by Gardner et al. 

32 (1993) provide estimates of exposure to formaldehyde. The remaining studies generally rely 

33 either on duration of exposure (number of years in the job) as a surrogate (Pinkerton et al., 2004) 

34 or provide no exposure assessment.
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1 Marsh et al. (1994), in an early study of the Wallingford plant, which is also part of the 

2 Hauptmann et al. (2004, 2003) and Blair et al. (1986) studies, found SMRs of 0.89 and 0.91,

3 based on U.S. and county death rates, respectively (25 observed deaths). The authors did not 

4 further discuss this cancer site until after Hauptmann et al. (2003) was published. Blair et al. 

5 (1986) reported on 4,396 deaths from all causes in the 10 formaldehyde-associated factories that 

6 made up the NCI cohort of 26,561 workers employed before January 1, 1966. There was little 

7 evidence of an association with LHP system cancer (SMR 0.91; 56 observed) in exposed white 

8 men, who dominated the cohort.

9 Hauptmann et al. (2003) updated the cohort mortality study of Blair et al. (1986) that 

10 consisted of predominantly the same (25,619) workers from 10 plants. The primary focus of this 

11 analysis was cancer of the LHP system, including leukemia. The description and demographics 

12 of the current study are the same as those reported by Blair et al. (1986) and Stewart et al. 

13 (1986). In the current update, follow-up was extended through December 31, 1994. The 

14 additional 15 years of follow-up increased the number of deaths from 4,349 to 8,486. Exposures 

15 were not updated for the 4% of workers still in exposed jobs in 1980, but eliminating exposure 

16 estimates for these workers did not change the results since exposures received after this date 

17 were considered so low as to contribute little to the analysis by the authors.

18 Peak exposure categories were defined as nonexposed, low (0.1–1.9 ppm), medium (2.0–

19 3.9 ppm), and high (4.0 ppm or greater). Average intensity categories of exposure were defined 

20 as nonexposed, low (0.1–0.4 ppm), medium (0.5 to <0.9 ppm), and high (≥1.0 ppm). Cumulative 

21 exposure was defined as nonexposed, low (0.1–1.4 ppm-years), medium (1.5–5.4 ppm-years), 

22 and high (≥5.5 ppm-years). Duration of exposure was defined as 0, 0.1–4.9 years, 5.0–

23 14.9 years, and ≥15 years. The median TWA exposure level was 0.45 ppm, range 0.01–

24 4.25 ppm. Only 2.6% of the workers had average exposure intensities of 2 ppm or higher, and 

25 14.3% had peak exposures of 4 ppm or higher. A total of 3,201 workers had no exposure. The 

26 median duration in formaldehyde-exposed jobs was 2 years. The median TWA intensity for 

27 formaldehyde exposure was 0.5 ppm among exposed workers.

28 A Poisson regression model was stratified for calendar year, age, sex, race, and pay 

29 category (salary/wage). A minimum latency period of 2 years between exposure and death from 

30 a potentially exposure-related LHP cancer was assumed by the investigators to prevent the 

31 inclusion of exposures not likely to contribute to the development of LHP cancer because of their 

32 timing. Other lag times were evaluated that did not improve the regression models.

33 There were 2,099 cancer deaths. Hauptmann et al. (2003) reported that mortality from all 

34 causes, all cancers, and LHP malignancies were significantly lower among the unexposed (SMRs 

35 0.77, 0.65, and 0.62, respectively). Among the exposed, the SMRs were 0.95, 0.90, and 0.80, 
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1 respectively, for mortality from all causes, all cancers, and LHP malignancies. These SMRs in 

2 part reflect the HWE caused by using external U.S. population death rates as a referent. 

3 Unexposed workers also may have differed from the exposed workers in other ways. The 

4 remaining analyses used internal comparisons, avoiding the HWE. The referent group in this 

5 analysis was the low exposure group rather than the unexposed group, because nonexposed 

6 workers, who are primarily managers, secretaries, and other non-production personnel, were 

7 considered likely to have different socioeconomic characteristics than workers in the production 

8 areas.

9 Statistically significant positive associations were found for LHP malignancies and 

10 leukemia, particularly myeloid, in certain higher exposure categories in comparison with 

11 employees in the lowest exposure categories (Table 4-4). In the highest peak exposure level, the 

12 RR for LHP malignancies was 1.87 (95% CI: 1.27–2.75; 64 observed) and for ML was 3.46 

13 (95% CI: 1.27–9.43; 14 observed) compared with employees in the low exposure peak level. For 

14 workers with high peak exposure levels, the RR for LHP malignancies was 1.71 (95% CI: 

15 1.14-2.58; 49 observed) and 2.43 (95% CI: 0.81–7.25) for ML. The trend tests for slope were 

16 highly statistically significant for both LHP malignancies (p < 0.002) and ML (p < 0.009).

17 Significant results for LHP cancers were also seen with the average intensity exposure 

18 metric. RRs were 1.63 (p < 0.05) and 1.50 (p < 0.05) for the medium and high categories, 

19 respectively. The risk of ML was also significantly increased (RR = 2.49) in the highest 

20 exposure category. In contrast, Hauptmann et al. (2003) did not find statistically significant 

21 associations of formaldehyde with LHP cancer, either by cumulative exposure or years of 

22 duration. However, there were positive associations for leukemia (RR = 1.39) and ML (RR = 

23 1.35) when exposure was 15 years or longer.

24 The authors concluded that formaldehyde may cause leukemia, particularly ML, in 

25 humans. However, because results from other studies were inconsistent, they suggested caution 

26 in drawing definite conclusions. A biological basis for the significant excess risk of LHP cancer 

27 remains unclear. The authors pointed out several studies that indicate changes that are consistent 

28 with chromosomal changes in formaldehyde-exposed persons, such as increased frequencies of 

29 (MN (He et al., 1998; Kitaeva et al., 1996; Suruda et al., 1993), sister chromatid exchanges 

30 (SCEs) (Shaham et al., 2002, 1997; Yager et al., 1986), chromosomal aberrations (CAs) (He et 

31 al., 1998; Bauchinger and Schmid, 1985), and DNA-protein cross-links (DPXs) (Shaham et al., 

32 1997, 1996a) in peripheral lymphocytes of humans exposed to formaldehyde.

33 Hauptmann et al. (2003) identified 11 suspected carcinogens used at the plants: 

34 antioxidants (unspecified), asbestos, carbon black, dyes and pigments, hexamethylenetetramine, 

35 melamine, phenol, plasticizers, urea, wood dust, and benzene. Some workers were employed as 
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1 chemists and laboratory technicians. The investigators did not find substantial changes in the 

2 risk estimates after adjusting for exposure to these substances or for working as a chemist or 

3 laboratory technician. They also eliminated the 586 benzene-exposed persons from their 

4 analysis and found similar results (benzene is a known human leukemogen). Smoking was not 

5 likely to explain an increased risk of leukemia in this cohort, because no increase was seen for 

6 smoking-related diseases, including lung cancer. The cohort consisted predominantly of males 

7 (88%). Strengths include the fact that the cohort was large and there was a long period of 

8 follow-up that was 96.6% successful. Internal analyses eliminated the HWE. One potential 

9 limitation that could lead to an underestimate of risks is the 3.4% or 866 lost to follow-up.

10 The study by Hauptmann et al. (2003) has been criticized extensively by several experts 

11 representing the formaldehyde industry (Tarone and McLaughlin, 2005; Casanova et al., 2004; 

12 Cole and Axten, 2004; Collins, 2004; Collins and Lineker, 2004). Most of the same criticisms 

13 have been repeated in other critiques by the above-mentioned authors and have been addressed in 

14 the discussions concerning the details of the methodology. However, a few new issues have 

15 arisen from these critiques, as follows. One issue pertains to a concern that person-years at risk 

16 of death may have been assigned wrongly to the highest “peak” category of exposure for the 

17 duration of the study period. For example, there is inconsistency in the fact that only 4% of the 

18 original cohort (Blair et al., 1986) had average exposures equaling or exceeding 2 ppm yet 45% 

19 of the person-years were assigned to the peak exposure category. Average exposures are time20 weighted exposures that can have brief excursions over 4 ppm and still average 2 ppm or less. 

21 Only for the peak exposure surrogate were person-year values assigned to the peak category 

22 following the exposure, because it is a test for the possibility that biological changes could have 

23 been initiated from that brief high exposure that might increase the risk of cancer. If these 

24 genetic changes are irrevocable, then the risk of cancer could be increased and subsequent person 

25 × years should be assigned to that higher risk category.

26 According to Casanova et al. (2004), the assignment of peak exposures in the Hauptmann 

27 et al. (2003) study was questionable because they were based on professional judgement. 

28 However, there are adequate grounds for hypothesizing that the assignment of peak exposure 

29 was completed before determination of vital status and cause of death. It is always possible that 

30 some subjects may be subject to misclassification. Hauptmann et al. (2003) chose this metric 

31 partly because it more closely resembled the exposure that embalmers and pathologists may have 

32 received from formaldehyde. This same criticism could be said about the Coggon et al. (2003) 

33 study as well.

34 Hauptmann et al. (2003) have also been criticized because the metric “cumulative 

35 exposure” was not significant and did not show a trend. No adequate explanation has been given 
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1 by Hauptmann et al. (2003) except that it is possible that this metric is not as sensitive for this 

2 agent. Duration of exposure was only weakly associated with a trend of increasing risk. After 

3 15 years’ duration, there appeared to be a slight increase in ML based on 10 cases (RR = 1.35 

4 [95% CI: 0.56–3.24]). For all leukemia the risk in workers who were exposed 15 or more years 

5 was somewhat higher (RR = 1.39 [95% CI: 0.78–2.49]) based on 22 cases.

6 Another of the criticisms from these authors discussed the lack of a biologically plausible 

7 explanation for how leukemia could result from exposure to formaldehyde when there appears to 

8 be no recognizable indication of the presence of formaldehyde in excessive quantities in the 

9 blood of animals or any associated metabolites in experimental research animals. Hauptmann et 

10 al. (2004b) responded that there is evidence that genotoxic effects can be detected in vivo in the 

11 bone marrow of rats and in human peripheral lymphocytes.

12 Stayner et al. (1988) conducted a cohort study of 11,030 workers (82% female) followed 

13 from 1955 or the beginning date of exposure through 1982 in three garment factories. Personnel 

14 records from three garment manufacturing facilities, one in Pennsylvania and two in Georgia, 

15 were used to assemble a cohort of workers who attained a minimum of 3 months of exposure 

16 after the introduction of formaldehyde into these facilities. Formaldehyde resins were used to 

17 treat fabrics, beginning in 1955 and 1959. Although formaldehyde levels were available on a 

18 subset of the employees from monitoring data available from surveys completed in 1981 and 

19 1984, they were not used in this analysis. Instead, the results were stratified by duration and 

20 latency. SMRs were based on U.S. population mortality rates. Based on six cases, the SMRs for 

21 leukemia were 2.43 and 3.81 among workers with 20 or more years since first exposure or at 

22 least 10 years of exposure, respectively. In their conclusions, the authors suggested that, 

23 although the numbers of deaths from LHP cancers were small, the risks were related to duration 

24 and latency.

25 Pinkerton et al. (2004) updated the Stayner et al. (1988) study by adding 16 years of 

26 follow-up. No new exposure information was added. The mean TWA exposure in 1981–1984 

27 for the three plants was 0.15 ppm. No additional information regarding earlier industrial hygiene 

28 data was available, although the authors stated that the levels of exposure to formaldehyde were 

29 greater in the years before 1980. Stayner et al. (1988) cited independent studies of exposure 

30 levels in similar garment factories in the 1960s that seemed to indicate that the formaldehyde 

31 levels during that period ranged from 0.9 to 2.7 ppm (Blejer and Miller, 1966) in one garment 

32 manufacturing area. Another report (Shipkovitz, 1966) of 10-minute personal exposure samples 

33 indicated a range from 0.3 to 2.7 ppm in eight garment plants. In another study (Ahmad and 

34 Whitson, 1973), the levels ranged from 2 to 10 ppm. Goldstein (1973) calculated that 

35 concentrations in the cutting rooms of garment plants dropped from 10 ppm in 1968 to less than 
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1 2 ppm in 1973 because of an improvement in the resin treating process. The authors assumed 

2 that exposure ceased in 1981 and 1983. This produced an underestimate of exposure based on 

3 duration of employment for about 11% of the cohort who were still actively employed after those 

4 dates. Stayner et al. (1988) speculated that the risks of cancer of the buccal cavity, leukemia, and 

5 other LHP neoplasia may have been due to exposure to the highest potential formaldehyde levels 

6 in the industry between 1955 and 1962, because the resin used to treat permanent press fabrics 

7 still contained a relatively large amount of formaldehyde.

8 The SMRs were derived from age-, race-, and calendar-time-adjusted U.S. mortality 

9 rates. The analysis was repeated using Georgia or Pennsylvania mortality rates. In addition to 

10 the primary analysis of the underlying cause of death, the analysis used all causes listed on the 

11 death certificates to evaluate multiple cause mortality. As a referent for this, the analysis relied 

12 on multiple cause death rates available since 1960 from the National Death Index maintained by 

13 the U.S. Centers for Disease Control and Prevention (CDC).

14 Altogether, 608 cancer deaths were observed. The SMR for all cancer was 0.89 (95% CI: 

15 0.82–0.97). The overall SMR for leukemia was 1.09 (24 deaths) and 1.44 (15 deaths) for ML. 

16 After 10 years of exposure, the risk for ML was 2.19. Exposure prior to 1963 was associated 

17 with a risk of 1.61. Among garment workers followed for 20 or more years from initial 

18 exposure, the SMR was significantly elevated for ML (1.91; p < 0.05; 13 deaths), as was the 

19 SMR for multiple cause leukemia (1.92 [95% CI: 1.08–3.17]; 15 deaths) in the subgroup with 

20 10 or more years of exposure to formaldehyde and who were followed for 20 or more years after 

21 first exposure. The multiple cause mortality for ML for this subgroup of workers was also 

22 significant (SMR 2.55 [95% CI: 1.10–5.03]; 8 deaths).

23 The study by Stayner et al. (1988) has only limited power to detect excess risks of rare 

24 cancers, such as NPC and nasal cancer (13 and 16%, respectively). Limitations to the 

25 interpretations of the findings include a lack of any monitoring data before 1981, particularly 

26 during the critical time period 1955 to 1962, and lack of personal exposure estimates for any 

27 members of the cohort. The possibility exists that misclassification may still be present because 

28 the intensity of exposure to formaldehyde decreased as improvements were made in the resin 

29 systems used to treat fabrics (e.g., a person who worked 5 years beginning in 1955 might have 

30 been subject to greater exposure than a person who worked 5 years beginning in 1993). 

31 However, workers from the 1950s and 1990s were both placed in the same category of having 

32 worked fewer than 10 years. The median duration of exposure was 3.3 years. Work histories

33 were not updated in the follow-up study; however, the low or background exposure levels that 

34 probably existed after 1981 were not likely to contribute substantially to the risk of cancer. The 

35 use of mortality data to estimate risk, when the case fatality rate was less than 100% for most 
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1 cancer sites evaluated, could potentially produce an underestimate of the actual risk. Despite 

2 these limitations, this study provides additional evidence of an association between leukemia, 

3 especially ML, and formaldehyde in comparison with the general population.

4 Gardner et al. (1993) reported that the risk of leukemia was not statistically significant 

5 (SMR 0.9) based on 15 deaths among workers employed before 1965. Only four leukemia 

6 deaths were observed after 1964 through 1989, producing an SMR of 0.9.

7 When Coggon et al. (2003) updated the above cohort study of 14,014 men first employed 

8 before 1965 in six factories by adding 11 additional years of follow-up (ending December 31, 

9 2000), no increase in the risk of leukemia or related cancers of the hematopoietic system was 

10 reported, either in the entire cohort (SMR 0.91; 31 observed) or in the group with the highest 

11 formaldehyde exposure (>2 ppm) (SMR 0.71; 8 observed). Similar results were obtained for 

12 Hodgkin’s disease, non-Hodgkin’s lymphoma, and multiple myeloma. No other cancers of the 

13 hematopoietic system were evaluated, and no additional analyses were performed to assess a 

14 possible leukemia risk. However, the main finding from this study was a marked association of

15 lung cancer with formaldehyde (discussed in the lung cancer section). This study’s main focus 

16 was respiratory disease, lung cancer, and stomach cancer, not LHP cancers. For cancers of the 

17 LHP system, there was neither latency evaluation nor internal comparisons. The HWE is also 

18 potentially a problem.

19 Andjelkovich et al. (1995) studied a cohort of 3,929 male iron foundry workers 

20 potentially exposed to formaldehyde between January 1, 1960, and December 31, 1989, in which 

21 127 cancer deaths had occurred during the observation period. An industrial hygienist, after 

22 reviewing work histories, categorized formaldehyde exposure into four levels corresponding to 

23 the approximate midpoint of the ranges: none, low (0.05 ppm), medium (0.55 ppm), and high 

24 (1.5 ppm) for exposure to formaldehyde. Boundaries of these exposure categories were not 

25 given. The authors warned that the assignment of exposure levels was not perfect because 

26 “subjective judgment had to be applied in many instances.” SMRs were based on U.S. male

27 mortality rates, but actual ranges were not specified. The authors also compared the exposed to 

28 2,032 nonexposed workers from the same company. The population-based SMR for 

29 hematopoietic cancer in the exposed population was 0.59 (based on seven observed deaths). For 

30 leukemia the SMR was 0.43, based on two deaths. There were no other analyses for leukemia or 

31 LHP cancers in this study. Because of the uncertainty about workers’ true formaldehyde 

32 exposure, there was no analysis by level of exposure, duration, or latency. There were also very 

33 few LHP cancers in the cohort. Thus, these results neither support nor refute an association of 

34 formaldehyde exposure with LHP cancers. The main focus of this paper was on lung cancer risk.
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1 Bertazzi et al. (1989, 1986), in a cohort mortality study, followed 1,330 male workers 

2 from 1959 through 1986 at a formaldehyde resin plant in Italy. The workers had to have been 

3 employed for at least 30 days at the plant sometime between 1959 and 1980 to be included in the 

4 study. Their mortality was compared with national and local rates adjusted for age and calendar 

5 time period. No individual exposure estimates were available, but mean levels were estimated to 

be between 0.2 and 3.8 mg/m3 6 (0.16 and 3.1 ppm) during the period 1974–1979. The authors 

7 found an SMR of 2.01 (five deaths observed) for cancer of the lymphatic and hematopoietic 

8 system. The study’s limitations included incomplete work histories, small numbers of deaths, 

9 and a follow-up period that may not have been sufficient to allow for a latency period for the 

10 development of LHP cancers. As before, the results neither support nor refute an association of 

11 formaldehyde exposure with LHP cancers.

12 Edling et al. (1987) reported on the incidence of disease in a cohort of 521 blue collar 

13 Swedish workers in plants where abrasives bound with formaldehyde resins were manufactured. 

Formaldehyde levels ranged from 0.1 to 1.0 mg/m3 14 (0.08–0.8 ppm). The workers in the cohort 

15 were employed between 1955 and 1983, and incidence rates were calculated from 1958 through 

16 1981. There were only 24 total cancer cases (28.5 expected) of which 2 (1.0 expected) were 

17 lymphomas and 2 (0.5 expected) were multiple myelomas. Expected cases were determined 

18 through the Swedish National Cancer Register. No other LHP cancers were observed. This 

19 study lacked the power to detect any significant associations between LHP cancer and exposure 

20 to formaldehyde.

21 Dell and Teta (1995) conducted a cohort mortality study of 5,932 male employees of a 

22 New Jersey plastics manufacturing, research, and development facility. The workers, who had 

23 been employed during the period 1946–1967, were followed-up for an average of 32 years. 

24 SMRs were based on U.S. and New Jersey mortality rates. Hourly workers (n = 3,853) were 

25 analyzed separately from the 2,079 salaried employees. Although no excess risk was evident for 

26 hematopoietic cancer in hourly workers (SMR 0.93; 28 observed), there was an SMR of 1.69 

27 (95% CI: 1.07–2.53; 23 observed) among salaried workers. This association was further 

28 narrowed to mainly research and development workers (eight leukemia deaths observed with 

29 three expected, for an SMR of 2.67). No common exposure was found when work history 

30 records were examined. The decedents were mostly associated with process development in two 

31 research pilot plants, where chemical engineers, lab technicians, and plant operators executed 

32 small-scale product development. Although notebooks referred to benzene and toluene solvents, 

33 no definite connection was made with formaldehyde or any of the solvents. No ambient air 

34 measurements of formaldehyde were available. The findings cannot be assumed to be due to 

35 formaldehyde exposure because of the presence of other potential leukemogens.
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1 Blair et al. (1993) conducted a study that evaluated the risk of non-Hodgkin’s lymphoma 

2 from exposure to formaldehyde. This was a population-based, case-control, interview-based 

3 study of 1,867 white males of whom 622 cases had the disease and 1,245 were controls. 

4 Subjects had lived in Iowa and Minnesota between 1980 and 1983. This study was exploratory 

5 and designed to find associations with any environmental exposures and non-Hodgkin’s 

6 lymphoma. Subjects or next of kin were interviewed to determine what exposures the cases and 

7 controls may have received based on agricultural exposures, work histories, medical conditions, 

8 and family history. Extra effort was made to collect information about occupation, industrial 

9 exposures, and other selected exposures. The analysis revealed an OR of 1.2 for exposure to 

10 formaldehyde. Similar associations were found for metals and other substances in the study. 

11 This study, because it did not select cases and controls from a population with possible 

12 formaldehyde exposure, could not detect specific relationships between formaldehyde and 

13 non-Hodgkin’s lymphoma.

14

4.1.2.2.1.3. Summary of non-respiratory tract cancers.

29 Additional support linking LHP cancer and formaldehyde comes from a study of garment 

30 workers (Pinkerton et al., 2004) and studies of pathologists and other medical workers exposed 

31 to formaldehyde (Matanoski, 1991; Blair et al., 1990; Hayes et al., 1990; Stroup et al., 1986; 

32 Walrath and Fraumeni, 1984, 1983; Harrington and Shannon, 1975). Hayes et al. (1990) and 

33 Stroup et al. (1986) also reported significant excess risks of ML.

15 The Hauptmann et al. (2003) study 

16 appears to provide the strongest evidence of an association for ML in particular. Statistically 

17 significant positive associations were found for LHP malignancies and leukemia, particularly 

18 ML, in certain higher exposure categories in comparison with employees in the lowest exposure 

19 categories. In the highest peak exposure level, the RR for LHP malignancies was 1.87 (95% CI: 

20 1.27–2.75; 64 observed) and for ML was 3.46 (95% CI: 1.27–9.43; 14 observed) compared with 

21 employees in the low-exposure peak level. For workers with high-peak exposure levels, the RR 

22 for LHP malignancies was 1.71 (95% CI: 1.14–2.58; 49 observed) and 2.43 (95% CI: 0.81–7.25) 

23 for ML. The trend tests for slope were highly statistically significant for both LHP malignancies 

24 (p < 0.002) and ML (p < 0.009). Significant results for LHP cancers were also seen with the 

25 average intensity exposure metric. RRs were 1.63 (p < 0.05) and 1.50 (p < 0.05) for the medium 

26 and high categories, respectively. The risk of ML was also significantly increased (RR = 2.49) 

27 in the highest exposure category. In contrast, results showed no associations of formaldehyde 

28 with LHP cancer, either by cumulative exposure or years of duration. 

34 Several reports have challenged the association between LHP cancer and formaldehyde 

35 (Casanova et al., 2004; Cole and Axten, 2004; Collins, 2004; Collins and Lineker, 2004; Coggon 
This document is a draft for review purposes only and does not constitute Agency policy.

4-97 DRAFT—DO NOT CITE OR QUOTE

1 et al., 2003; Casanova and Heck, 1987; Heck et al., 1985). These papers argue that a biological 

2 explanation for the excess risk of LHP cancer or leukemia remains unclear. Absent a plausible 

3 MOA by which formaldehyde could cause these cancers, many investigators have been unable to 

4 accept the reported increased risks identified in the epidemiologic literature. Some researchers 

5 have argued against the biological plausibility of formaldehyde-induced lymphoreticular cancers 

6 based solely on the assumption that formaldehyde as a reactive gas does not penetrate past the 

7 POE. This argument is relevant to diseases for which transformation of stem cells in the bone 

8 marrow is essential. However, cancers that arise from more mature cells present outside of the 

9 bone marrow compartment cannot be dismissed with this argument. Although often grouped for 

10 analysis, the lymphohemoreticular system cancers represent many distinct malignancies that may 

11 arise from discrete cell types in different tissues throughout the body. For example, acute 

12 lymphocytic leukemia (ALL) is believed to arise from the transformation of a lymphoid stem cell 

13 in the bone marrow, resulting in a blood-borne leukemia of immature cells of the lymphoid cell 

14 line. However, if transformation occurs in a mature lymphocyte (e.g., post-germinal center B 

15 cell), a chronic lymphocytic leukemia (CLL) results. Although etiologically different, these 

16 cases would both be lymphocytic leukemia. When considering biological plausibility of an 

17 exogenous agent increasing the incidence of ALL, bone marrow toxicity would be expected. 

18 However, when considering the biological plausibility of CLL, bone marrow toxicity would not 

19 be essential. Mutation or epigenetic changes attained in the mature cell may be passed on to 

20 daughter cells during response to antigen and eventually lead to transformation. So the 

21 etiologies of these two leukemias need not be similar. In contrast, a non-Hodgkin’s lymphoma 

22 results from transformation of a mature B or T cell resulting in a solid tumor. The etiology of 

23 this cancer is actually similar to CLL. A recent reclassification of lymphoid malignancies by the 

24 WHO designates adult B-cell leukemias and lymphomas as the same disease, with ALL as a 

25 separate disease. Therefore, mortality analysis by ICD code and the standard groupings of those 

26 codes does not reflect the biology of the cancers.

27 Considering the whole class of LHP cancers, there is a range of biological plausibility for 

28 an agent whose primary action is at the POE. Acute leukemias (ALL and acute myelogenous 

29 leukemia [AML]), believed to arise from transformation of stem cells in the bone marrow, are 

30 less plausible, although trafficking of stem cells to different tissues would be an alternative 

31 etiology for exogenous compounds acting at the POE. In contrast CLL, lymphomas, multiple 

32 myelomas (from plasma B cells), and unspecified cancers may involve an etiology in peripheral 

33 tissues to include cells, cell aggregates, germinal centers, and lymph nodes. An association of 

34 these cancers to an exogenous agent acting at the POE is biologically plausible.

35
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-98

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

Harrington and 

Shannon (1975)

Cohort mortality study of 2,079 

pathologists and laboratory 

technicians from the Royal 

College of Pathologists and the 

Pathological Society of Great 

Britain from 1955–1973. The 

comparison population came 

from national mortality data. 

Presumed exposure to 

formaldehyde tissue 

fixative.

Pathologists

All cause mortality SMR 0.60

LHP cancers SMR 2.0 p < 0.01 (8)

Hodgkin’s disease SMR 1.4 (1)

Leukemia SMR 0.6 (1)

Technicians

All cause mortality SMR 0.67

LHP cancers SMR 0.5 (3)

Hodgkin’s disease SMR – (0)

Leukemia SMR 0.5 (1)

Harrington and 

Oakes (1984)

Cohort mortality study of 2,720 

pathologists from the Royal 

College of Pathologists and the 

Pathological Society of Great 

Britain from 1974–1980. Vital 

status obtained from the census, a 

national health registry, and other 

sources. SMRs developed from 

the English, Scottish, Irish, and 

Welsh populations. 

Presumed exposure to 

formaldehyde tissue 

fixative.

All causes

Men SMR 0.56

Women SMR 0.99

Leukemia

Men SMR 0.91 (90% CI: 0.05–4.29) (1)

Women SMR 9.26 (90% CI: 0.47–43.9) (1)

Other LHP cancers

Men SMR 0.53 (90% CI: 0.03–2.54) (1)

Women SMR – – (0)

Hall et al. (1991) Cohort mortality study of 4,512 

pathologists from the Royal 

College of Pathologists and the 

Pathological Society of Great 

Britain from 1974–1987. Vital 

status obtained from the census, a 

national health registry, and other 

sources. SMRs developed from 

the English and Welsh 

populations. 

Presumed exposure to 

formaldehyde tissue 

fixative.

All cause mortality

Men SMR 0.43 (176)

Women SMR 0.65 (18)

Hodgkin’s disease SMR 1.21 (95% CI: 0.03–6.71) (1)

All cancers SMR 1.44 (95% CI: 0.69–2.63) (10)

Leukemia SMR 1.52 (95% CI: 0.41–3.89) (4)

Levine et al. (1984) Cohort mortality study of 1,477 Presumed exposure to All LHP cancers SMR 1.24 (8)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-99

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

male Ontario undertakers first 

licensed 1928–1957, followed 

from 1950 to 1977. SMRs 

developed from Ontario 

mortality rates.

formaldehyde tissue 

fixative.

Leukemia SMR 1.60 (4)

Stroup et al. (1986) Cohort mortality study of 2,317 

white male members of the 

American Association of 

Anatomists from 1888 to 1969 

who died 1925–1979. SMRs 

developed using U.S. population 

mortality rates. 

Presumed exposure to 

formaldehyde tissue 

fixative.

All cause mortality SMR 0.65 (95% CI: 0.60–0.70) (738)

All LHP cancers SMR 1.2 (95% CI: 0.7–2.0) (18)

Lymphosarcoma and 

reticulosarcoma SMR 0.7 (95% CI: 0.1–2.5) (2)

Hodgkin’s disease SMR – – (0)

Leukemia SMR 1.5 (95% CI: 0.7–2.7) (10)

Other lymphatic SMR 2.0 (95% CI: 0.7–4.4) (6)

Logue et al. (1986) Cohort mortality study of 4,485 

pathologists who were members 

of the College of American 

Pathologists, 1962–1972, 

followed for mortality through 

1977. SMRs developed from 

U.S. population mortality rates. 

Presumed exposure to 

formaldehyde tissue 

fixative.

LHP cancer other than 

leukemia

SMR 0.48 (NR)

Leukemia SMR 1.06 (NR)

Matanoski (1991) Cohort mortality study of 6,111 

male pathologists from 

membership rolls of the 

American Medical Association 

1912–1950. Mortality was 

followed through 1978. SMRs 

developed from U.S. population 

white male mortality rates.

Presumed exposure to 

formaldehyde tissue 

fixative.

All cancer SMR 0.78 (95% CI: 0.71–0.85) (508)

All LHP cancers SMR 1.25 (95% CI: 0.95–1.62) (57)

Lymphosarcoma and 

reticulosarcoma

SMR 1.31 (95% CI: 0.66–2.35) (11)

Hodgkin’s disease SMR 0.36 (95% CI: 0.04–1.31) (2)

Leukemia SMR 1.35 (95% CI: 0.92–1.92) (31)

Other lymphatic SMR 1.54 (95% CI: 0.82–2.63) (13)

Hauptmann et al. 

(2003)

Retrospective cohort mortality 

study of 25,619 workers 

employed at 10 formaldehyde 

plants in the U.S. followed from 

either the plant start-up or first 

Exposure estimates 

based on job titles, 

tasks, visits to plants by 

study industrial 

hygienists, and 

All LHP cancers

Exposed SMR 0.80 (95% CI: 0.69–0.94) (161)

Unexposed SMR 0.62 (95% CI: 0.39–1.00) (17)

Peak exposure (ppm)

employment through 1994. monitoring data through 0 RR 1.08 (95% CI: 0.60–1.94) (17)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-100

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

SMRs calculated using sex-, age-

, race-, and calendar-yearspecific U.S. mortality rates. 

RRs estimated using Poisson 

regression stratified by calendar 

year, age, sex, and race; adjusted 

for pay category.

1980. Peak exposure 

defined as short-term 

excursions exceeding 

the 8-hour TWA 

formaldehyde intensity 

and knowledge of job 

tasks. Exposures to 11 

other compounds were 

identified. Workers 

contributed preexposure person-time to 

nonexposed category. 

Poisson regression 

models used a 2-year 

lag to account for tumor 

latency.

0.1–1.9 RR 1.00 Reference value (48)

2.0 to <4.0 RR 1.71 (95% CI: 1.14–2.58) (49)

4.0 or greater RR 1.87 (95% CI: 1.27–2.75) (64)

Trend p = 0.002

Average exposure (ppm)

0 RR 0.91 (95% CI: 0.52–1.59) (17)

0.1–0.4 RR 1.00 Reference value (81)

0.5 to <1.0 RR 1.63 (95% CI: 1.11–2.37) (42)

1.0 or greater RR 1.50 (95% CI: 1.01–2.24) (38)

Trend p = 0.050

Cumulative exposure(ppm-years)

0 RR 0.74 (95% CI: 0.42–1.30) (17)

0.1–1.4 RR 1.00 Reference value (94)

1.5 to 5.4 RR 0.79 (95% CI: 0.52–1.21) (29)

5.5 or greater RR 1.03 (95% CI: 0.70–1.52) (38)

Trend p = 0.157

Leukemia

Peak exposure (ppm)

0 RR 0.78 (95% CI: 0.25–2.43) (4)

0.1–1.9 RR 1.00 Reference value (16)

2.0 to <4.0 RR 2.04 (95% CI: 1.04–4.01) (20)

4.0 or greater RR 2.46 (95% CI: 1.31–4.62) (29)

Trend p = 0.001

Average exposure (ppm)

0 ppm RR 0.56 (95% CI: 0.19–1.66) (4)

0.1–0.4 RR 1.00 Reference value (32)

0.5 to <1.0 RR 1.52 (95% CI: 083–2.79) (16)

1.0 or greater RR 1.68 (95% CI: 0.91–3.08) (17)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-101

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

Trend p = 0.193

Cumulative exposure (ppm-years)

0 RR 0.48 (95% CI: 0.16–1.42) (4)

0.1–1.4 RR 1.00 Reference value (35)

1.5–5.4 RR 0.90 (95% CI: 0.47–1.73) (13)

5.5 or greater RR 1.14 (95% CI: 0.63–2.07) (17)

Trend p = 0.183

Hodgkin’s disease

Peak exposure (ppm)

0 RR 0.51 (95% CI: 0.06–4.52) (1)

0.1–1.9 RR 1.00 Reference value (5)

2.0 to <4.0 RR 3.45 (95% CI: 0.98–12.2) (7)

4.0 or greater RR 3.35 (95% CI: 0.97–11.6) (8)

Trend p = 0.014

Average exposure (ppm)

0 RR 0.46 (95% CI: 0.05–3.93) (1)

0.1–0.4 RR 1.00 Reference value (7)

0.5 to <1.0 RR 4.70 (95% CI: 1.61–13.8) (8)

1.0 or greater RR 3.12 (95% CI: 0.91–10.7) (5)

Trend p = 0.022

Cumulative (ppm-years)

0 RR 0.29 (95% CI: 0.04–2.34) (1)

0.1–1.4 RR 1.00 Reference value (12)

1.5–5.4 RR 1.35 (95% CI: 0.45–3.99) (5)

5.5 or greater RR 1.17 (95% CI: 0.31–4.46) (3)

Trend p = 0.037

ML 

Peak exposure (ppm)

0 RR 0.67 (95% CI: 0.12–3.61) (2)

0.1 to 1.9 RR 1.00 Reference value (6)

2.0 to <4.0 RR 2.43 (95% CI: 0.81–7.25) (8)

4.0 or greater RR 3.46 (95% CI: 1.27–9.43) (14)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

Trend p = 0.003

Average exposure (ppm)

0 RR 0.41 (95% CI: 0.08–1.95) (2)

0.1 to 0.4 RR 1.00 Reference value (14)

0.5 to <1.0 RR 1.15 (95% CI: 0.41–3.23) (5)

1.0 or greater RR 2.49 (95% CI: 1.03–6.03) (9)

p = 0.086

Cumulative (ppm-years)

0 RR 0.32 (95% CI: 0.07–1.51) (2)

0.1-1.4 RR 1.00 Reference value (17)

1.5-5.4 RR 0.57 (95% CI: 0.19–1.73) (4)

5.5 or greater RR 1.02 (95% CI: 0.40–2.55) (7)

Trend p = 0.123

Pinkerton et al. 

(2004)

Cohort mortality study of 11,098 

workers in 3 garment plants 

exposed ≥3 months after 

formaldehyde was introduced. 

Women comprised 81.7% of the 

cohort. Vital status was followed 

through 1998. SMRs were 

calculated by using sex-, age-, 

race-, and calendar-year-specific 

U.S. mortality rates. Multiple 

cause SMRs were derived from 

all contributing causes from 

death certificates.

Data for 549 randomly 

selected employees in 5 

departments in 1981 and 

1984 used to estimate 

overall exposure levels. 

Levels presumed to be 

0.09–0.20 ppm.

All LHP cancers SMR 0.97 (95% CI: 0.74–1.26) (59)

Lymphosarcoma and 

reticulosarcoma

SMR 0.85 (95% CI: 0.28–1.99) (5)

Hodgkin's disease SMR 0.55 (95% CI: 0.07–1.98) (2)

Other lymphatic SMR 0.97 (95% CI: 0.64–1.40) (28)

Leukemia SMR 1.09 (95% CI: 0.70–1.62) (24)

Mortality since 1960

Lymphocytic leukemia SMR 0.60 (95% CI: 0.12–1.75) (3)

ML SMR 1.44 (95% CI: 0.80–2.37) (15)

10+ years of

exposure

SMR 2.19 NS (8)

20+ years since 1st 

exposure 

SMR 1.91 p > 0.05 (13)

Multiple cause leukemia

10+ years of 

exposure and 20+ 

years since 1st 

exposure

SMR 1.92 (95% CI: 1.08–3.17) (15)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

Multiple cause ML

20+ years since 1st 

exposure 

SMR 2.02 (95% CI: 1.13–3.34) (15)

10+ years of 

exposure and 20+ 

years since 1st 

exposure

SMR 2.55 (95% CI: 1.10–5.03) (8)

Coggon et al. (2003) Cohort mortality study of 14,014 

men employed in 6 factories of 

the chemical industry in Great 

Britain from periods during 

which formaldehyde was 

produced. Cohort mortality 

followed from 1941 through 

2000. SMRs based on English 

and Welsh age- and calendaryear-specific mortality rates.

Exposure assessment 

based on data abstracted 

from company records. 

Jobs categorized as 

background, low, 

moderate, high, or 

unknown levels.

Non-Hodgkin's lymphoma 

Overall SMR 0.98 (95% CI: 0.67–1.39) (31)

High exposure SMR 0.89 (95% CI: 0.41–1.70) (9)

Leukemia 

Overall SMR 0.91 (95% CI: 0.62–1.29) (31)

High exposure SMR 0.71 (95% CI: 0.31–1.39) (8)

Multiple myeloma 

Overall SMR 0.86 (95% CI: 0.48–1.41) (15)

High exposure SMR 1.18 (95% CI: 0.48–2.44) (7)

Andjelkovich et al. 

(1995)

Cohort mortality study of 3,929 

automotive industry iron foundry 

workers exposed from 1960–

1987 and followed through 1989. 

SMRs calculated using sex-, age-

, race-, and calendar-yearspecific U.S. mortality rates. 

Exposure assessment 

based on review of 

work histories by an 

industrial hygienist.

All LHP cancers SMR 0.59 (95% CI: 0.23–1.21) (7)

Leukemia SMR 0.43 (95% CI: 0.05–1.57) (2)

Bertazzi et al. (1986) Cohort mortality study of 1,330 

male workers in an Italian resin 

plant. Subjects were employed 

any time between 1959 and 1980 

for at least 30 days. Vital status 

followed through 1986. SMRs 

calculated using sex-, age-, race-, 

and calendar-year-specific 

national and local mortality rates. 

Exposure assessment 

based on reconstruction 

of work history.

All LHP cancers SMR 2.01 (5)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

Edling et al. (1987) Cohort mortality and incidence 

study of 521 Swedish workers in 

an abrasive production plant with 

at least 5 years of employment 

between 1955 and 1983, 

followed through 1983. 

Exposure level of 

1–5 μg/m3.

Lymphoma SMR 2.0 (95% CI: 0.2–7.2) (2)

Multiple myeloma SMR 4.0 (95% CI: 0.5–14) (2)

Dell and Teta (1995) Cohort mortality study of 5,932 

male employees of a New Jersey 

plastics manufacturing, research 

and development facility.

Examination of work 

histories to identify jobs 

where formaldehyde 

was involved.

All LHP cancers

Hourly workers SMR 0.93 (28)

Salaried workers SMR 1.69 (23)

Leukemia

Hourly workers SMR 0.98 (12)

Salaried workers SMR 1.98 (11)

Walrath and 

Fraumeni (1983)

Proportionate mortality cohort 

study of 1,132 white male 

embalmers licensed to practice 

between 1902 and 1980 in New 

York who died between 1925 

and 1980 identified from 

registration files. Deaths were 

compared with age-, race-, and 

calendar-year-expected numbers 

of deaths from the U.S. 

population. 

No direct 

measurements. 

Presumed exposure to 

formaldehyde tissue 

fixative.

All LHP cancers

PMR 1.15 (21)

Lymphosarcoma and reticulosarcoma

PMR 1.08 (4)

Hodgkin’s disease

PMR 1.0 (2)

Other lymphatic lymphoma

PMR 1.18 (5)

Leukemia

PMR 1.32 (10)

Walrath and 

Fraumeni (1984)

Proportionate mortality cohort 

study of 1,007 white male 

embalmers from the California 

Bureau of Funeral Directing and 

Embalming who died between 

1925 and 1980. Deaths were 

compared with age- and 

calendar-year-expected numbers 

No direct 

measurements. 

Presumed exposure to 

formaldehyde tissue 

fixative.

All LHP cancers

PMR 1.22 (19)

Lymphosarcoma and reticulosarcoma

PMR 0.97 (3)

Hodgkin’s disease

PMR – (0)

Other lymphatic lymphoma

PMR 1.33 (4)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-105

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

of deaths from the U.S. 

population.

Leukemia

PMR 1.75 p < 0.05 (12)

Licensed <20 years PMR 1.24 (4)

Licensed ≥20 years PMR 2.21 p < 0.05 (8)

Hayes et al. (1990) Proportionate mortality cohort 

study of 3,649 deceased white 

and 397 deceased nonwhite U.S. 

male embalmers and funeral 

directors, derived from licensing 

boards and funeral director 

associations in the 32 states and 

the District of Columbia. 

Occupation was confirmed on 

death certificate. Deaths were 

compared with age- and 

calendar-year-expected numbers 

of deaths from the U.S. 

population.

No direct 

measurements. 

Presumed exposure to 

formaldehyde tissue 

fixative.

All LHP cancers

PMR 1.39 (95% CI: 1.15–1.63) (115)

Race

White PMR 1.31 (95% CI: 1.06–1.59) (100)

Nonwhite PMR 2.41 (95% CI: 1.35–3.97) (15)

Occupation on death certificate

Embalmer PMR 1.23 (95% CI: 0.78–1.85) (23)

Funeral director PMR 1.56 (95% CI: 1.23–1.94) (78)

Other PMR 1.30 (95% CI: 0.67–2.28) (12)

Age at death

<60 PMR 1.35 (95% CI: 0.88–1.98) (26)

60–74 PMR 1.72 (95% CI: 1.33–2.19) (66)

≥75 PMR 1.16 (95% CI: 0.74–1.74) (23)

Hodgkin’s disease

PMR 0.72 (95% CI: 0.15–2.10) (3)

Non-Hodgkin’s lymphoma

PMR 1.26 (95% CI: 0.87–1.76) (34)

Lymphosarcoma and reticulosarcoma

PMR 1.12 (95% CI: 0.58–1.96) (12)

Multiple myeloma

PMR 1.37 (95% CI: 0.84–2.12) (20)

Other lymphatic lymphoma

PMR 1.35 (95% CI: 0.85–2.01) (22)

Lymphatic leukemia

PMR 0.74 (95% CI: 0.29–1.53) (7)

ML

PMR 1.57 (95% CI: 1.01–2.34) (24)

Other leukemia
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-106

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

Reference Study design Exposure assessment

Results, statistical significance (number of observed deaths for cohort 

study)

PMR 2.28 (95% CI: 1.39–3.52) (20)

Blair et al. (1993) Population-based case-control 

study of 622 white men with 

LHP cancers. Cancers selected 

from Iowa and Minnesota cancer 

surveillance networks diagnosed 

between 10/80 and 9/82. 1,245 

matched controls for living cases 

selected by random digit dialing 

if younger than age 65 and from 

Medicare records if 65 or older. 

Study focused on agricultural 

exposures. 

Personal interviews of 

subjects or next of kin 

included job histories, 

agricultural exposures, 

and chemical exposures. 

Job titles used to create 

job exposure matrix. 

Industrial hygienist 

estimated probability 

and intensity of 

exposures to large 

numbers of substances. 

Non-Hodgkin’s 

lymphoma 

(formaldehyde 

exposure)

ORa 1.2 (95% CI: 0.9–1.7)

Funeral service worker ORa 2.1 (95% CI: 0.5–7.9) (6)

a

Adjusted for age, state, smoking, family history of malignant proliferative disease, agricultural exposure to pesticides, use of dye, and direct/surrogate response 

to interview.
This document is a draft for review purposes only and does not constitute Agency policy.

4-107 DRAFT—DO NOT CITE OR QUOTE

1 4.1.2.2.2. Brain and CNS cancer. Several studies of professional groups discussed earlier 

2 investigated brain and other CNS cancers among those exposed to formaldehyde on the job. 

3 Several of these studies found that exposure increased risk two to three times among exposed 

4 professionals (Hall et al., 1991; Stroup et al., 1986; Walrath and Fraumeni, 1984), while others 

5 found modest or no increase in risk (Hayes et al., 1990; Levine et al., 1984; Walrath and 

6 Fraumeni, 1983). 

7 None of the industrial cohort worker mortality studies of exposure to formaldehyde found 

8 a clear relationship between formaldehyde exposure and risk of brain or CNS cancer (Pinkerton 

9 et al., 2004; Coggon et al., 2003; Andjelkovich et al., 1995; Gardner et al., 1993; Stayner et al., 

10 1988; Blair et al., 1987, 1986). To date, no case-control studies of brain and CNS cancer have 

11 been completed. In the Hauptmann et al. (2004) study, the authors reported that no clear 

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

13

14 4.1.2.2.3. Pancreatic and other cancers. Two studies (Kernan et al., 1999; Dell and Teta, 1995) 

15 have found increases in the risk of pancreatic cancer in association with possible exposure to 

16 formaldehyde. Collins et al. (2001a) conducted a meta-analysis of fourteen studies (Kernan et 

17 al., 1999; Andjelkovich et al., 1995; Hansen and Olsen, 1995; Gardner et al., 1993; Hall et al., 

18 1991; Matanoski, 1991; Hayes et al., 1990; Gerin et al., 1989; Stayner et al., 1988; Blair et al., 

19 1986; Stroup et al., 1986; Levine et al., 1984; Walrath and Fraumeni, 1984, 1983) and found a 

20 small increase in risk (RR = 1.1 [95% CI: 1.0–1.3]).

21 Other sites that have been examined are stomach cancer (Coggon et al., 2003) (SMR = 

22 1.47; p < 0.05), intraoccular melanoma (Holly et al., 1996) (OR = 2.9 [95% CI: 1.2–7.0]), and 

23 thyroid cancer among women (Wong et al., 2006) (OR = 8.33 [95% CI: 1.16–60.0]; 2 cases). 

24 However, without further substantiation, it is difficult to infer causation based on these isolated 

25 results alone.

26

27 4.1.2.3. Summary: Carcinogenic Hazard in Humans

28 The weight of the epidemiologic evidence at this time supports a link between 

29 formaldehyde exposure and NPC in humans. This conclusion is based on the longitudinal cohort 

30 study of Hauptmann et al. (2004) as well as the case-control studies of NPC and formaldehyde 

31 exposure completed by Hildesheim et al. (2001), Vaughan et al. (2000), and several additional 

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

33 of the other cohort studies found little or no increased risk of NPC from exposure to 

34 formaldehyde. However, Hauptmann et al. (2004) employed different exposure metrics and 

35 based their analyses on conservative internal comparisons that limited the potential for the HWE 
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1 to obscure true effects. The case-control studies that provide additional evidence of an 

2 association between NPC and formaldehyde have more power and generally rely on better 

3 diagnoses of NPC. Better ascertainment of histologic types of tumors can sometimes also be 

4 obtained if the cases are taken from cancer registries. The NPC risk is also supported by 

5 experimental evidence in animals in which formaldehyde induces nasal cancers (Section 4.2.2). 

6 Since the physiology of the rat nasal passage is somewhat different from that of humans, it is not 

7 possible to obtain a direct site-specific correspondence between the species. However, in both 

8 species, the tumors are found within the same area of the URT where maximum exposure can be 

9 expected to occur.

10 Several researchers have challenged the conclusion of a relationship between 

11 formaldehyde and NPC. Those critical of the link argue that, given the wide variability in results 

12 across studies and competing explanations, conclusions about any link from the existing studies 

13 are premature. The difficulty in attaining consensus on whether formaldehyde influences the risk 

14 of NPC in humans arises from several limitations inherent in epidemiologic methods and 

15 exposure assessment, as well as from the characteristics of the disease. The most prominent of 

16 these limitations are the rarity of the cancer and imprecise estimates of exposure. Because NPC 

17 is a very rare cancer with an incidence of less than 1 per 100,000, it is difficult to obtain precise 

18 estimates of risk from cohort studies. Although case-control studies are better suited for 

19 studying rare conditions, they are limited in obtaining valid and precise exposure assessments. A 

20 further problem with exposure assessment is isolating formaldehyde exposure from other 

21 potential chemical or particulate exposures that may influence risk of NPC. Imprecise exposure 

22 assessment and the inability to isolate formaldehyde exposure from other exposures are largely 

23 the bases on which Marsh and coworkers have challenged the NCI cohort study (Marsh et al., 

24 2007a, b, 2002, 1996; Marsh and Youk, 2005). Marsh and coworkers (Marsh et al., 2007a) show 

25 that subjectively assessed exposure to silversmithing is tentatively associated with NPC. Given 

26 that there were no prior citations of an association between silversmithing exposures and NPC in 

27 the medical literature and given the many post hoc reexaminations of alternative hypotheses to 

28 explain the original NCI findings, it is more likely that silversmithing is an artifactual potential 

29 confounder.

30 It may be expected that, without new approaches for obtaining more accurate and precise 

31 estimates of exposure, further follow-up of current cohorts and future epidemiologic studies of 

32 formaldehyde and NPC will face the same limitations and criticisms found with existing studies. 

33 These limitations notwithstanding, the epidemiologic studies reviewed here represent what may 

34 be currently discernable about a formaldehyde-NPC link in humans by using rigorous 

35 observational methods. As such, concluding any influence of formaldehyde must be made on the 
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1 weight of all human and animal evidence in the face of known and expected limitations in study 

2 design and exposure assessment.

3 The results of two well-designed cohort studies found a positive association between 

4 formaldehyde-exposed professionals, such as pathologists, embalmers, and funeral directors, and 

5 LHP cancer, particularly ML. The largest cohort study of formaldehyde has the most extensive 

6 exposure assessment (Blair et al., 1986; Stewart et al., 1986), and the cohort was followed for a 

7 median duration of 35 years (Hauptmann et al., 2003). By using cumulative exposure measures 

8 not previously used and by using internal comparison groups, significant increases in the risk of 

9 cancer of the LHP system, particularly ML, were reported. This study demonstrated that 

10 formaldehyde was a risk factor for LHP cancers, independent of other risk factors, such as 

11 benzene and smoking. Hauptmann et al. (2003) found statistically significant dose trends for 

12 peak exposure and AIE. Pinkerton et al. (2004) also found a significant increase in the risk of 

13 ML in garment workers 20 years after their initial exposure and in workers with 10 or more years 

14 of exposure. Additionally, several studies of pathologists, embalmers, and other medical 

15 workers reported greater numbers of observed deaths from leukemia than expected although 

16 many studies of these groups suffer from a substantial HWE based on comparisons with external 

17 death rates. Two of these studies, Hayes et al. (1990) and Stroup et al. (1986), also report a 

18 significantly excess risk of ML in embalmers, funeral directors, and anatomists.

19 There is a range of biological plausibility for an agent whose primary action is at the 

20 POE. Acute leukemias (ALL and AML), believed to arise from transformation of stem cells in 

21 the bone marrow, are less plausible. In contrast chronic lymphatic leukemia, lymphomas, 

22 multiple myelomas (from plasma B cells), and unspecified cancers may involve an etiology in 

23 peripheral tissues to include cells, cell aggregates, germinal centers, and lymph nodes. An 

24 association of these cancers to an exogenous agent acting at the POE is biologically plausible.

25 It is the conclusion of this assessment that the weight of the epidemiologic evidence at 

26 this time supports a link between formaldehyde exposure and carcinogenicity in humans.

27

28 4.2. ANIMAL STUDIES

29 This section discusses the available laboratory animal data on the toxicity of inhalation, 

30 oral, and dermal exposures to formaldehyde. A comprehensive database of laboratory animal 

31 studies is available for formaldehyde, including numerous 2-year bioassays by both the 

32 inhalation and oral exposure routes and dermal application studies. Although a large portion of 

33 the literature reports studies focused on toxic effects at the site of contact or POE, general 

34 systemic effects as well as neurobehavioral effects, reproductive and developmental effects, 

35 immunologic changes, and sensitization are well represented in the literature as well. 
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1 RB is a reduction in ventilation rate, minute volume, and other physiological parameters 

2 experienced by rodents exposed to an irritant/reactive gas. Although humans and nonhuman 

3 primates do not exhibit the same change in respiratory rate, these studies are included in Section 

4 4.2.1.1 in order to better understand the effects on RB in interpreting rodent studies presented in 

5 the balance of the chapter. Additionally, although binding to the trigeminal nerve and 

6 subsequent downstream events do not result in pulmonary changes in humans, the mechanism

7 itself plays a role in understanding other adverse health effects observed in humans.

8 The available data for the three exposure routes confirm direct formaldehyde-induced 

9 toxicity in tissues present at the POE. These observations are consistent with the 

10 physicochemical characteristics, reactivity, and metabolic pathways of formaldehyde as 

11 discussed in Chapter 3. Indications of cell damage, cell proliferation, and inflammatory 

12 responses are similar for each route of exposure, therefore effects at the POE for inhalation and 

13 oral exposures are described first (Sections 4.2.1.2 and 4.2.1.3, respectively). Given the well14 established nature of these health effects and the wealth of literature for inhalation exposures, 

15 complete study summaries for respiratory tract effects are provided. Studies are organized by 

16 study duration—acute, subchronic and chronic—where some of the chronic bioassays were 

17 designed to address carcinogenic potential. Section 4.2.2 pulls together the findings of chronic 

18 studies across the routes of exposure to evaluate the carcinogenic potential of formaldehyde 

19 exposure.

20 Although a majority of the oral and inhalation studies focus on health effects at the 

21 POE—respiratory tract and GI tract—the general systemic toxicity of formaldehyde is addressed 

22 where it was integral to the study. Therefore, body weight and organ weight changes, gross 

23 pathology, organ histopathology outside of the POE, blood and urine chemistry, and other 

24 biochemical measures may be included in these study summaries. An overview of general 

25 systemic findings is provided in Section 4.4 for all routes of exposure.

26 Studies addressing immune function, neurobehavioral effects, sensitization, and 

27 reproductive and developmental effects are addressed across routes of exposure. The specialized 

28 nature of these studies requires discrete treatment, and inclusion of data across routes of 

29 exposure allows for a synthesis of the available information. 

30

31 4.2.1. Noncancer Health Effects

32 4.2.1.1. Reflex Bradypnea

33 Reflex bradypnea (RB), which is believed to be a protective response, is often observed 

34 in rodents exposed to reactive gases. It is primarily characterized by marked decreases in 

35 activity, respiratory rate, body temperature, and metabolic rate. RB is not seen in humans and 
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1 nonhuman primates. An understanding of the RB is important to the interpretation of many of 

2 the animal bioassays examining formaldehyde-induced health effects. Of chief concern is that 

3 the physiological effects of RB, described below, may interfere with appropriate interpretation of 

4 adverse effects noted with formaldehyde exposure. It is important to distinguish between an 

5 effect directly related to RB versus formaldehyde exposure. Additionally the effects of RB may 

6 mask or alter formaldehyde-induced health effects. Secondly, differential respiratory effects of 

7 RB due to species and strain will result in differential inhaled doses at the same exposure level. 

8 This needs to be considered both when comparing the results of animal studies and in 

9 extrapolation to humans. Finally, although humans do not experience RB, the mechanism of RB 

10 as a reflex response to trigeminal nerve stimulation assists in understanding human health related 

11 to localized and reflex responses due to trigeminal nerve stimulation.

12 Irritant gases have been shown to decrease body temperature, heart rate, and blood 

13 pressure as well as alter blood chemistry in rodents (Pauluhn, 2003, 1996; Jaeger and Gearhart, 

14 1982). Because of their small size, mice can rapidly lower their body temperatures and thus their 

15 metabolic rate and ventilation rate. The hypothermia that results from RB can directly affect 

16 nearly all biological processes (Gordon et al., 2008). Formaldehyde exposure can dramatically 

17 lower ventilation rate and reduce body temperature in mice by as much as 4°C, and it has been 

18 posited that decreased oxygen supply is likely to have profound effects on organisms with 

19 substantial oxygen demands (Jaeger and Gearhart, 1982). The effects of RB are reversible, 

20 though it may take several minutes to several hours to return to pre-exposure conditions 

21 (Pauluhn, 1996; Jaeger and Gearhart, 1982). 

22 The literature on sensory irritation is broad; many studies have investigated species 

23 differences, dose response relationships, tolerance, and cross-tolerance to other sensory irritants 

24 (see Tables 4-5 and 4-6). This discussion focuses on the changes in respiratory rate and minute 

25 volume during formaldehyde exposure. Sensory irritation is often quantified as the statistically 

26 derived exposure concentration that results in a 50% reduction in respiratory rate (RD50) in 

27 rodents (ASTM, 2000; Kane et al., 1979). Kane and Alarie (1977) evaluated various aspects of 

28 sensory irritation, including establishing the RD50, exploring the reproducibility of response, 

29 investigating the effect of tracheal cannulation, and determining the potential for tolerance with 

30 repeated exposure or pre-exposure in male Swiss-Webster mice, caused by formaldehyde and 

31 acrolein. The RD50 was established by exposing four mice for 10 minutes at each concentration 

32 across a range representing approximately 10 to 80% reduction in respiration and calculated by 

33 using least squares regression. The RD50 and its 95% CI for formaldehyde were calculated to be 

3.1 (2.1–4.7) ppm (3.8 [2.58–5.77] mg/m3 34 ). The tracheal cannulation experiments demonstrated 

35 that the effect on respiratory rate was caused by URT sensory irritation.
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1 Table 4-5. Respiratory effects of formaldehyde-induced reflex bradypnea in 

2 various strains of mice

3

Species/strain

No./

group Treatmenta Respiratory effects Reference

Male SwissWebster mice

4 Duration: 10 minutes.

Exposure: up to 100 ppm.

RD50a = 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. 

RD50 = 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.

RD50 = 3.2 ppm

 (95% CI: 2.1–4.7).

Steinhagen and 

Barrow (1984)

Male Swiss OF1 

mice

6 Single 5-minute exposure to 

four unspecified 

concentrations.

RD50 = 5.3 ppm. De Ceaurriz et al. 

(1981)

Male B6C3F1 mice 4 Duration: 10 minutes (head 

only).

Exposure: Range up to 10 ppm.

RD50 = 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: RD50 = 4.4 ppm

 (95% CI: 0.9–5.0) 

Pretreated mice: RD50 = 

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; CO2

production reduced by 

50%; %; body temperature 

dropped from 37.8 to 

34.7°C.

Jaeger and 

Gearhart (1982)

4 a 5 Exposure concentration that results in a 50% reduction in respiratory rate.
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1 Table 4-6. Respiratory effects of formaldehyde-induced reflex bradypnea in various 

2 strains of rats

3

Species/strain

No./

group Treatment Respiratory effects Reference

Male Crl-CD rats 4 RD50 = 13.8 ppm. 

Male Wistar rats 4 30-minute nose-only exposure 

to a range of formaldehyde 

concentrations.

RD50 = 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: RD50 = 13.1 ppm

 (95% CI: 10.6–17.5) 

Pretreated rats: RD50 = 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 RD50 = 31.7 ppm. 

Pre-exposure to formaldehydeinduced tolerance at 28 ppm 

(RD50 = 20.2 ppm) but not 

15 ppm.

Pre-exposure to chlorine-induced 

tolerance to formaldehyde 

(RD50 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 (RD50 = 2,991 ppm 

in naive versus 10,601 ppm in 

preconditioned animals) 

Acrolein (RD50 = 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; CO2

production unaffected.

Jaeger and 

Gearhart (1982)

4

5

6 Across the literature there is fairly good agreement on RD50 values for various strains of 

7 mice (Table 4-5), ranging from 3.1 ppm in male Swiss-Webster mice to 4.9 ppm in male 

8 B6C3F1 mice. Rats are less sensitive, with RD50 values ranging from 10 ppm in male Wistar 

9 rats to 31.7 ppm in male F344 rats. No reported RD50 for female rodents exposed to 

10 formaldehyde exists.

11 Jaeger and Gearhart (1982) evaluated the effect of formaldehyde on respiratory rate, tidal 

12 volume, minute volume, carbon dioxide (CO2) production (exhaled to air) as a reflection of total 

13 metabolism, and core body temperature in male Charles River CD rats and male C57BL6/F1 
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mice. Animals (three/concentration) were exposed to 15 ppm (18.4 mg/m3 1 ) formaldehyde for up 

2 to 2 hours. Mice exhibited a greater decrease in respiratory frequency and minute volume 

3 compared with the rats. CO2 production and body temperature were also affected to a greater 

4 extent in the mice (Table 4-5). The authors postulated that the decreased body temperature in 

5 mice would likely lead to decreased biologic action of formaldehyde in the tissue.

6

7 4.2.1.1.1. Tolerance. Tolerance is defined as an increase in the concentration required to elicit 

8 the same degree of RB response and was evaluated by Kane and Alarie (1977). In the first set of 

9 experiments, mice (four/concentration) were exposed 3 hours/day for 4 days at the concentration 

10 associated with either a 30 or 50% decrease in respiratory frequency (specific concentrations not 

11 given) (Kane and Alarie, 1977). Naïve animals served as controls for each day. The maximum 

12 response increased with each additional day of exposure, and the diminution of response that was 

13 typically exhibited after 60 minutes of exposure in naïve animals was markedly delayed. In the 

14 second set of experiments, mice were exposed to a formaldehyde concentration at one-tenth the 

15 RD50 (i.e., 0.3 ppm) 3 hours/day for 3 days. On the fourth day the animals underwent a similar 

16 exposure protocol to identify the concentration that resulted in an RD50, following the above 

17 protocol. No change in the RD50 was demonstrated. Both of these experiments indicate no 

18 change in tolerance with either type of pretreatment in Swiss-Webster mice.

19 Chang and Barrow (1984) tested whether tolerance would develop in male F344 

20 (CDF[F344]Crl/Br) rats exposed to formaldehyde. Exposure to formaldehyde at 15 ppm 

(18.4 mg/m3 21 ) for 6 hours/day, 5 days/week failed to induce tolerance. However, tolerance was 

observed following exposure to 28 ppm (34.4 mg/m3 22 ) formaldehyde for 4 days. The 

23 concentration-response curve in these animals was significantly different than that of naïve 

24 animals, with an increase in the RD50 estimate for this exposure duration from 31.7 to 70.2 ppm.

25

26 4.2.1.1.2. Cross-species differences in inhaled dose. Formaldehyde-induced RB lowers both 

27 respiratory rate and tidal volume and thus reduces the inhaled dose of formaldehyde at a given 

28 exposure concentration. Chang et al. (1983) and Barrow et al. (1983) evaluated the species 

29 differences and the effective inhaled dose between rats and mice, since mice seem to be more 

30 sensitive to formaldehyde-induced RB and do not exhibit tolerance as shown in F344 rats. 

31 Groups (four/concentration) of male F344 rats and male B6C3F1 mice were exposed to 

formaldehyde concentration ranges of 6.2–48 ppm (7.6–59 mg/m3 32 ) or 0.78–14.0 ppm (0.96–

17.2 mg/m3 33 ), respectively, for 10 minutes. Pretreated animals used in the tolerance experiments 

were exposed to formaldehyde at 2, 6, or 15 ppm (2.45, 7.36, or 18.4 mg/m3 34 ) 6 hours/day for 

35 4 days prior to determination of the RD50 and concentration response across the same ranges.
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1 A concentration-dependent decrease in respiratory rate was seen in both naïve and 

2 pretreated rats during formaldehyde exposure. Tolerance (defined as a decrease in respiratory 

3 rate followed by a subsequent return to control values) occurred after 4 minutes of exposure and 

4 was more pronounced at concentrations above 4 ppm. Concentration-response relationships 

5 were very similar for naïve and pretreated rats, and the RD50s were similar for both groups 

6 (naïve = 13.1 ppm [95% CI: 10.6–17.5]; pretreated = 10.8 ppm [95% CI: 7.6–16.9 ]). In 

7 contrast, naïve or pretreated mice did not develop tolerance during exposures. An examination 

8 of concentration-response relationships for mice showed similar RD50 values (naïve = 4.4 ppm 

9 [95% CI: 0.9–5.0 ] and pretreated = 4.3 ppm [95% CI: 3.4–5.5]) compared with rats, although 

10 the slopes of the concentration-response regressions were statistically different (Figure 4-2). 

11

12

13

14 Figure 4-2. Formaldehyde effects on minute volume in naïve and formaldehyde15 pretreated male B6C3F1 mice and F344 rats.

16

17 Source: Redrawn from Chang et al. (1983).

18

19

20 Exposure of naïve or pretreated rats resulted in an increased (compensatory) tidal 

21 volume. However, the increase in tidal volume did not compensate entirely for the decrease in 

22 ventilation rate and was only concentration dependent in pretreated rats. Comparison of tidal 

23 volume from naïve and pretreated mice exposed to formaldehyde showed a slight increase in 

24 naïve animals but a decrease in pretreated ones. The effect of formaldehyde exposure on tidal 

110

100

90

80

70

60

50

40

30

20

10

0 2 4 6 8 10 120 240 360 0 2 4 6 8 10 120 240 360

B6C3F1 Mice F 344 Rats

Naïve (6 ppm)

Pretreated (6 ppm)

Naïve (15 ppm)

Pretreated (15 ppm)

Time (min.) during exposure
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1 volume was concentration dependent in both groups of mice. These results indicate that tidal 

2 volume does not compensate entirely for the decrease in respiratory rate and that the 

3 compensation is slightly greater in rats than in mice.

4 These studies (Barrow et al., 1983; Chang et al., 1983) showed that B6C3F1 mice sustain 

5 RB, whereas F344 rats develop tolerance more readily both during exposure and with 

6 pretreatment. Thus, these results suggest that the rat may be the more sensitive species for the 

7 effects of inhaled formaldehyde due in part to the difference in sensitivity between mice and rats 

8 as evidenced by an RD50 of 4.9 versus 31.7 ppm and the ability of rats to develop tolerance while 

9 mice appear to sustain RB. Barrow et al. (1983) used the results of these experiments to estimate 

10 an inhaled dose equivalent to the exposure concentration of 15 ppm for the strains of mice and 

11 rats used in the chronic formaldehyde bioassays by Kerns et al. (1983) and Monticello and 

12 Morgan (1994) described in Section 4.1.2 as follows:

13

Inhaled dose (μg/min-cm

2 14 ) = 

Nasal cavity surface area (cm2 16 ) (5-1)

15 HCHO concentration (μg/L) × minute volume (L/min)

17

18 As shown in Table 4-7, because mice were observed to be able to decrease their minute 

19 volume by approximately 75% as compared with 45% in rats, a twofold higher inhaled dose 

20 would be expected in rats versus mice. This difference may be relevant to the increased 

21 incidence of SCC in the nasal cavity seen in F344 rats when compared with B6C3F1 mice.

22

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

24 B6C3F1 mice exposed to 15 ppm

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 (cm2) 13.44 2.89

Inhaled dose (μg/min/cm2) 0.156 0.076

26

27 Source: Barrow et al. (1983).

28

29

30 4.2.1.1.3. Cross-tolerance. Cross-tolerance of chemically-induced reflex responses has been 

31 examined in several systems in order to better understand the specificity and nature of the 

32 interaction of reactive chemicals (such as formaldehyde with chlorine) with the trigeminal nerve 

33 involved in the RB. Development of cross-tolerance to formaldehyde following preexposure to 
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1 chlorine or to chlorine following preexposure to formaldehyde was shown to be a function of the 

2 duration of the pretreatment in male F344 rats (Chang and Barrow, 1984) (Table 4-8). A 7-day 

3 recovery period resulted in only a slight loss of cross-tolerance from a 4-day pre-exposure to 

4 either chlorine or formaldehyde (data not shown). The cross-tolerance between formaldehyde 

5 and chlorine demonstrated in the Chang and Barrow (1984) study suggests that these chemicals 

6 may act via a common mechanism and may involve the trigeminal nerve. In rats, cross-tolerance 

7 was induced after chlorine exposure but not after formaldehyde exposure, which suggests that 

8 the trigeminal nerve may have different reactive sites that are differentially activated, depending 

9 on the stimulus.

10

11 Table 4-8. Exposure regimen for cross-tolerance study

12

Pre-exposure

Chlorine RD50

FA-pretreated Naïve

Formaldehyde 15 ppm 

6 hours/day 

1 day 22.6 ppm

4 days 16.8 ppm 10.9 ppm

10 days 64.5 ppm

Pre-exposure Formaldehyde RD50

Cl-pretreated Naive

Chlorine 10 ppm

6 hours/day 

1 day 64.5 ppm

4 days 66 ppm 31.7

10 days 115 ppm

13

14 Source: Chang and Barrow (1984).

15

16

17 Babiuk et al. (1985) evaluated the potential for formaldehyde pretreatment to cause cross18 tolerance with various other inhaled aldehydes, including acetaldehyde and acrolein. Male F344 

rats were pretreated with 15 ppm (18.4 mg/m3 19 ) formaldehyde 6 hours/day for 9 days and 

challenged on the 10th 20 day with the second aldehyde for 10 minutes at various concentrations 

21 (four rats/concentration) to establish an RD50. Exposure to acetaldehyde and acrolein, the two 

22 smallest molecules in the series of aldehydes tested, resulted in cross-tolerance. The RD50 and 

23 its 95% CI for acetaldehyde were estimated at 2,991 (95% CI: 2,411–3,825) ppm in the naïve 

24 rats, and this was increased by approximately 3.5-fold to 10,601 (95% CI: 7,902–15,442) ppm in 

25 the rats pretreated with formaldehyde. With acrolein, the RD50 increased approximately fivefold, 

26 from 6.0 (95% CI: 3.5–18.1) ppm to 29.6 (95% CI: 15.6–93.0) ppm. Cross-tolerance with 

27 formaldehyde has only been demonstrated with acetaldehyde, acrolein, and chlorine (Babiuk et 

28 al., 1985; Chang and Barrow, 1984), suggesting that it is not a generalized phenomenon. 

29 Whether the phenomenon of tolerance involves modulation of specific trigeminal nerve 

30 receptors or whether it results from less specific chemical injury of the nasal mucosa has not 
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1 been determined. For example, different mechanisms lead to stimulation of the trigeminal nerve

2 and are likely to control the decrease in respiratory rate. In particular, acetaldehyde might 

3 interact with sensory nerves via an amino group (Steinhagen and Barrow, 1984; Schauenstein et 

4 al., 1977), whereas the receptor-binding site for formaldehyde and acrolein is believed to be a 

5 thiol group. Furthermore, different binding sites exist on the trigeminal nerve for different 

6 irritants (Nielsen, 1991). Thus, Bos et al. (1992) concluded that the data on tolerance or 

7 “desensitization” versus “sensitization” (as defined strictly on the basis of the respiratory apneic 

8 response) may be the result of adaptation or reversible/irreversible adverse changes. The 

9 mechanisms underlying sensitization or desensitization are not well characterized.

10

11 4.2.1.1.4. Formaldehyde binding and activation of trigeminal nerve afferent activity. Kane 

12 and Alarie (1978) evaluated the effect of 11 combinations of acrolein and formaldehyde on 

13 respiratory rate in outbred specific-pathogen-free male Swiss-Webster mice. Exposure 

concentrations ranged from 0.12–8.97 ppm (0.28–21 mg/m3 14 ) for acrolein and 0.37–9.73 ppm 

(0.45–11.9 mg/m3 15 ) for formaldehyde. The data were evaluated using a simple model of 

16 competitive antagonism. Comparing the observed and predicted responses indicated no apparent 

17 differences, and paired t-tests showed no statistical significance. The authors concluded that 

18 acrolein and formaldehyde acted at the same receptor site and acted as competitive antagonists 

19 when exposure occurred simultaneously.

20 Kulle and Cooper (1975) investigated the effects of formaldehyde on trigeminal nerve 

21 afferent activity in adult male Sprague-Dawley rats. The authors isolated both the ethmoid and 

22 nasopalatine branches of the trigeminal nerve and recorded afferent signaling as electrical 

23 activity while reactive gases (formaldehyde, ozone, and amyl alcohol) were passed through the 

24 nasal passages of the anesthetized animals. The authors reported that both branches of the 

25 trigeminal nerve responded similarly to all three chemicals, and they therefore conducted the 

26 balance of their experiments on the nasopalatine branch of the nerve. Nerve response was 

27 calculated as the difference between exposed and control activity, and the threshold for a positive 

28 response was arbitrarily defined as an increase of 0.1 spikes per second. The sensory threshold 

29 was determined by extrapolation from the measured nerve response to a range of formaldehyde 

30 concentrations (0.5–2.5 ppm) or ozone (5.0–29 ppm) for an exposure duration of 2 minutes. 

31 Amyl alcohol exposure (0.3–10.0 ppm) lasted for 25 seconds. Threshold was arbitrarily defined 

32 as an increase of 0.1 spikes per second. The mean thresholds were 0.25 ppm for formaldehyde, 

33 5.0 ppm for ozone, and 0.30 ppm for amyl alcohol, suggesting that the trigeminal nerve is highly 

34 sensitive to formaldehyde and amyl alcohol compared with ozone exposure.
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1 In a second set of experiments, Kulle and Cooper (1975) investigated the effects of 

2 prolonged formaldehyde-exposure on the odor response to amyl alcohol. Rats were pre-exposed 

3 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, 

11.9, 24, or 36 mg/m3 4 ]) then a 1-hour continuous formaldehyde exposure (0, 0.5, 1.0, 1.5, or 

2.0 ppm [0, 0.61, 1.23, 1.84, or 2.45 mg/m3 5 ]). There was a progressive decrease in odor 

6 response to amyl alcohol with increasing stimulus of formaldehyde concentration (p < 0.01, 

7 analysis of variance [ANOVA]). The response to formaldehyde concentration was described by 

a power function Y = 0.741 × X1.47 8 , where X is the formaldehyde concentration. The effects of 

9 exposure to 2.0 ppm were similar, regardless of whether it was presented immediately as a 

10 separate exposure or as the final concentration of a progressively increasing series. The response 

11 to amyl alcohol did not fully recover within the 1-hour extended recovery period. Thus, it 

12 appeared that the afferent function depression was not due to receptor adaptation or insufficient 

13 time for formaldehyde diffusion away from receptor sites.

14 In an attempt to elucidate the basis of the differential effects of various types of 

15 aldehydes on sensory irritation, Tsubone and Kawata (1991) recorded the afferent activity of the

16 surgically isolated ethmoidal nerve (a branch of the trigeminal nerve) during delivery of 0.32–

4.7 ppm (0.39–5.77 mg/m3

) formaldehyde, 0.18–7.2 ppm (0.41–16.5 mg/m3 17 ) acrolein, and 134–

2,232 ppm (241–4,021 mg/m3 18 ) acetaldehyde into the cannulated URT of male Wistar rats 

19 (six/aldehyde) at a flow rate of 200 mL/minutes for 22 seconds. Only one aldehyde was used in 

20 each animal and each exposure was repeated two to four times at different concentrations. The 

21 activity of the nerve was recorded as the number of electrical discharges for a total period of 

22 100 seconds, including pre-inhalation (30 second), inhalation (22 second), and post-inhalation 

23 (48 second) periods. Nitrogen was used as the control gas and as the vehicle to dilute the 

24 aldehyde gases in order to not interfere with the gas chromatography used to analyze the 

25 exposures. The vapor concentrations associated with a 50% increase in nerve activity over the 

26 level of control gas were calculated as approximately 1.8, 1.2, and 908 ppm for formaldehyde, 

27 acrolein, and acetaldehyde, respectively. These results are consistent with the findings of 

28 Steinhagen and Barrow (1984) and the hypothesis that the differences in RD50 are due to 

29 differences in chemical reactivity in the tissue.

30 In summary, RB is a phenomenon observed in rodents exposed to reactive gases, 

31 believed to be a protective response to the irritant properties of the gas. In comparative studies, 

32 rats appear more sensitive to irritant gases since they have a more pronounced RB response 

33 compared with mice at a given concentration of formaldehyde and because the dose required to 

34 elicit a bradypneic response is higher in rats than in mice. Interestingly, only rats appear to 

35 develop tolerance to irritant gases, while mice sustain an RB response. When formaldehyde 
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1 exposure is studied in concert with other reactive gases like chlorine and other aldehydes like 

2 acetaldehyde and acrolein, cross-tolerance developed. However, the mechanism underlying this 

3 response is unknown. It is thought that RB may occur as a result of stimulation of the trigeminal 

4 nerve. Thus, although RB appears to be a phenomenon specific to rodents, the mechanism by 

5 which it occurs, trigeminal nerve stimulation, may be applicable to understanding MOAs in other 

6 species, such as primates and humans, particularly in regard to sensitization.

7

8 4.2.1.2. Respiratory Tract Pathology

9 The database for evaluating the POE toxicity in the respiratory tract of inhaled 

10 formaldehyde is robust, with well-designed studies that span a duration range of a few hours to 

11 chronic 2-year bioassays. Toxicity testing has been performed in various species, including 

12 mice, rats, hamsters, guinea pigs, dogs, and nonhuman primates. Although a few studies include 

13 examination of tissues outside of the URT, the majority of studies focus on changes in cell 

14 proliferation and cell pathology in the nasal mucosa. Both mice and rats are well-defined animal 

15 models with standard histologic sections established to evaluate various regions of the nasal 

16 passages, divided into Levels 1 to 5 and illustrated in Figure 4-3. Pathology of the nasal mucosa 

17 will be discussed with reference to these sections, and the region examined will be stipulated 

18 (e.g., nasoturbinates, maxilloturbinates, or ethmoid turbinates [ETs]). Additionally, pathology of 

19 the respiratory epithelium will be distinguished from effects on the olfactory epithelium, 

20 although the nature of the lesions is similar.

21

22 4.2.1.2.1. Mucociliary clearance. The mucociliary apparatus of the URT is the first line of 

23 defense against airborne toxicants. Comprising a thick mucus layer (epiphase), hydrophase, and 

24 ciliated epithelium, the mucociliary apparatus may entrain, neutralize, and remove particulates 

25 and airborne chemicals from inspired air (Figure 4-4). The mucus serves to entrain or neutralize 

26 and remove exogenous agents from the nasal epithelium (e.g., particles, reactive chemicals). As 

27 reviewed by Kim et al. (2003), the nasal mucus contains proteins, glycoprotein, and lipids but is 

28 primarily water (95%) and is propelled along by movement of the underlying cilia. Degradation 

29 in the continuity or function of the mucociliary apparatus, which provides protection to the nasal 

30 epithelium, would result in higher levels of gases and particles reaching the nasal epithelium 

31 itself and greater penetration of chemicals into the respiratory tract. Therefore, breakdown and 

32 disruption of mucociliary function are adverse effects, since a key bodily defense to exogenous 

33 agents (including infectious agents) is damaged. 

34

35
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1

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

3

4 Note: The figure shows the normal distribution of nasal mucosae and the section 

5 levels used in contemporary histopathology (Brenneman et al., 2000; Mery et al., 

6 1994). Sections 1, 2, 4, and 5 correspond to Levels I, II, III, and IV as proposed 

7 by Young (1981). S = squamous, T/R = transitional/respiratory, O = olfactory 

8 mucosa.

9

10 Source: Brenneman et al. (2000). 

11

12
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1

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

3

4 Note: OM = osmiophilic membrane; EP = epiphase; HY = hypophase; Cl = cilia; 

5 MV = microvilli; CJ = cell junction; CC = ciliated cell; NCC = non-ciliated cell; 

6 GC = goblet cell; NE = nerve; GL = gland; BV = blood vessel; ECS = 

7 extracellular space; BM = basement membrane.

8

9 Source: Morgan et al. (1986d).

10

11 Mucus flow slows upon formaldehyde exposure, despite an increase in the ciliary 

12 beat of the underlying epithelial cells, which propel the mucus across the nasal epithelium 

13 (Morgan et al., 1986a, c, d; 1983). These findings are consistent with other studies since 

14 airborne pollutants and reactive gases have been shown to decrease mucus flow rates in 

15 several animal models (Mannix et al., 1983; Iravani, 1974; Carson et al., 1966; Dalhamn, 

16 1956; Cralley, 1942). In addition to slowing flow, the mucus layer has been observed 

17 breaking up as it floats on the epiphase, creating gaps in the epiphase and revealing the 

18 hydrophase below (Morgan et al., 1986c, d). Formaldehyde reacts with glycoproteins in 

19 the mucus of the epiphase, creating cross-links between these large molecules; this is 

20 believed to increase the viscosity of the mucus.
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1 In their first experiments, Morgan et al. (1983) describe progressive mucostasis (slowing 

2 of mucous flow) and ciliastasis (disruption of ciliary beat) with increasing days of exposure to 

3 formaldehyde in male F344 rats (15 ppm; 6 hours/day for 1, 2, 4, or 9 days). Ciliastasis occurred 

4 with greater frequency and across more regions of the nasoturbinate with subsequent days of 

5 exposure. After 9 days, mucostasis was recorded in all but two regions evaluated. Although the 

6 severity and time course of these changes varied across regions of the nose, the process followed 

7 a similar pattern: decreased flow, increased ciliary action, mucostasis, and ciliastasis. Since the 

8 formaldehyde-induced deficits in mucociliary function increased with days of exposure, activity 

9 did not fully recover between exposures (18 hours) (Morgan et al., 1983). Therefore, the 

10 severity and extent of adverse effects are dependent on both the concentration of exposure and 

11 duration (in this case, days of repeated exposures).

12 In subsequent studies, Morgan et al. (1986c) examined the exposure-response 

13 relationship of formaldehyde effects on mucociliary function and functional recovery 18 hours 

14 after exposure ceased. Exposure regimens similar to the above experiment included additional 

15 exposure concentrations (0.5, 2, and 6 ppm) and an additional time point of 15 days duration. 

16 Exposure at 2 and 6 ppm resulted in the same progression of effects on mucus flow and ciliary 

17 beat. Considering both severity and extent of effects a clear exposure-response relationship was 

18 demonstrated. Additionally, within each exposure group, effects progressed both in severity and 

19 extent by duration of exposure to formaldehyde (from 1 to 4, 9, and 15 days of exposure) 

20 (Morgan et al., 1986c).

21 Flow and ciliary beat were not reduced, but rather increased, in epithelium from rats 

22 exposed to 0.5 ppm formaldehyde. Mucus flow in 2 of 10 areas assessed was clearly increased 

23 (275 and 200% of controls) after 4 days of exposure to 0.5 ppm formaldehyde. Two other 

24 epithelial regions showed a similar trend (150% of controls), but this change was not statistically 

25 significant. Interestingly, measurements made in corresponding areas after 9 days of exposure 

26 did not show an increase, and measurements in one region were reduced to 37% of control. 

27 Although it is not known whether the observed increase in mucus flow rate is a subtle indication 

28 of an adaptive response to a low level irritant, the increase appears to be transient. It is not 

29 known if flow rate would continue to decrease below control levels for repeated exposures at 

30 0.5 ppm for longer than 9 days.

31 The regions affected at 15 ppm generally included the lateral aspects of the nasoturbinate 

32 and both the dorsal and medial aspects of the maxilloturbinate. In general there was an anterior 

33 to posterior effect with increasing concentration and time. Additionally, impaired mucociliary 

34 function was more extensive with greater concentration and length of exposure. Nasal lesions 

35 were seen on the nasal epithelium and correlated with those areas where some inhibition of 
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1 ciliary function was measured. Areas without mucus flow but that still retained ciliary function 

2 did not develop epithelial lesions. Morgan et al. (1986c) reported “coagulated mucus,” viewed 

3 as a “continuous membrane” over the epithelium after 6 hours of exposure to 15 ppm 

4 formaldehyde. Minor cell damage and infiltrating neutrophils and monocytes were also seen in 

5 these areas. The coagulated mucus was not seen in similarly exposed rats that were allowed 

6 18 hours of recovery before sacrifice. However, ciliated cells were damaged, and there was a 

7 greater presence of neutrophils and macrophages (MPs) after this recovery period. The authors 

8 noted that, as the exposure continued, these areas exhibited increased signs of inflammation and 

9 epithelial damage, eventually resulting in “severe degenerative changes.”

10 Morgan et al. (1986a) refined their study design to implement a nose-only exposure to 

11 formaldehyde in order to better examine the progression of changes in mucociliary function 

12 during short-term exposure, allowing examination of mucus flow immediately following 

exposure. Three F344 rats/group were exposed to 15 ppm (18.4 mg/m3 13 ) formaldehyde for 10, 

14 20, 45, or 90 minutes or 6 hours. Two groups of rats were exposed to 2 ppm to determine a no 

15 effect level for 90 minutes or 6 hours. The extent and severity of mucostasis and ciliastasis seen 

after a 6-hour 15 ppm (18.4 mg/m3 16 ) formaldehyde exposure and a 1-hour recovery period were 

17 similar to the earlier study (Morgan et al., 1986a), indicating that similar exposure conditions 

18 were reached with this nose-only apparatus. Ciliastasis and mucostasis were both less severe and 

19 less extensive in a time-dependent manner and at the earlier time points of 10, 20, 45, and 

20 90 minutes. Significant recovery was seen in mucociliary function by allowing a 1-hour 

21 recovery between exposure and sacrifice. Regions of both the nasal septum and lateral wall, 

22 which exhibited no mucus flow when examined immediately after a 6-hour exposure, had 

23 measurable flow after the 1-hour recovery period. Similar recovery was seen at all durations of 

24 exposure. No decreases in mucociliary function were seen after exposure for either 90 minutes 

25 or 6 hours at 2 ppm formaldehyde. However, given evidence of recovery (Morgan et al., 1986a) 

26 and the time taken to dissect and view the tissues ex vivo may have obscured more subtle effects.

27 To assess more immediate effects on mucociliary apparatus, Morgan et al. (1984a) have 

28 examined formaldehyde effects on the mucociliary apparatus of isolated frog palates. This 

29 system allowed observation of mucociliary function during exposure. Unexposed frog palates 

30 were covered by a continuous sheet of mucus of variable thickness, which was observed to flow 

31 in streams across the palette, exhibiting a wave-like form in some areas of the epiphase. The 

32 authors reported particle movement in a lower, less viscous layer that was consistent with a less 

33 viscous underlying hydrophase, similar to that described in rat mucosa. The basal mucus flow 

34 rate was 0–4 mm/minute, with localized ciliary activity. Short periods of increased mucus flow 

35 were associated with seemingly spontaneous increases in ciliary beat. 
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1 Formaldehyde exposure resulted in an initial increase in ciliary beat and mucus flow rate 

2 in all palates exposed at 1.37, 4.36, and 9.58 ppm formaldehyde (but not 0.23 ppm). With 

3 increasing formaldehyde concentration and time of exposure, mucostasis was evident as mucus 

4 became stiff and eventually rigid. Ciliary beat continued after mucostasis was reached until 

5 palates were exposed to 4.36 and 9.48 ppm formaldehyde, when ciliastasis was reached. The 

6 time course to peak mucus flow rate, mucostasis, and ciliastasis was concentration dependent, 

7 with mucostasis reached in less than 3 minutes at 9.48 ppm. In contrast, increased mucus flow 

8 peaked at 8 minutes in palettes exposed at 1.52 ppm formaldehyde, which, though declining, 

9 remained above basal levels after 25 minutes with no mucostasis or ciliastasis noted at this level. 

10 Fló-Neyret et al. (2001) demonstrated reduced mucociliary clearance and decreased 

11 frequency of ciliary beats by using a similar isolated frog palette mucociliary apparatus. 

12 However the palates were exposed by formaldehyde in the Ringer’s solution in which the palates 

13 were placed (0, 1.25, 2.5, or 5 ppm). Also, mucus was removed from the palettes and did not 

14 come into direct contact with the formaldehyde. Despite these differences, formaldehyde caused 

15 mucociliary clearance to decrease in a time- and concentration-dependent manner; mucostasis 

16 occurred after 60 minutes of exposure to 5 ppm formaldehyde (Figure 4-5). Ciliary beat was 

17 decreased in a time-dependent manner at 2.5 and 5 ppm exposure but increased at 1.25 ppm 

18 formaldehyde (Figure 4-5). Reduced mucociliary clearance at 2.5 and 5 ppm was consistent with 

19 the reduced ciliary beat. However, clearance decreased at 1.25 ppm formaldehyde, where there 

20 was an apparent increase in ciliary beat. The authors suggest this may be a result of disrupting

21 the harmonic movement of the cilia, impairing effective mucociliary clearance. Based on study 

22 results, the authors hypothesize that changes in ciliary beat, including excitation at lower 

23 exposures, are likely to be a direct effect of formaldehyde on epithelial cells or other cellular 

24 components of the mucosa. 

25 In summary, numerous studies have identified impaired mucociliary clearance activity 

26 associated with formaldehyde exposure (Table 4-9). Although low-dose and short-term 

27 exposures first increase ciliary beat, impaired mucus flow, slowed ciliary beat, and eventual 

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

29 These effects are both concentration and duration dependent and can be seen in as few as 

30 15 minutes from exposure. Repeated inhalation exposures in rats indicate the effect does not 

31 fully recovery in an 18-hour period between exposures, contributing to greater impairment over 

32 extended periods of exposure.

33
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1

2

3

4

5

6

7

8

9

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

11 exposed to formaldehyde after 3 days in culture.

12

13 Source: Fló-Neyret et al. (2001).

14

15 Morgan et al. (1983) suggested that the initial stimulation of ciliary activity may be a 

16 defensive response to the irritant gas, possibly indicating some penetration of formaldehyde to 

17 the underlying epithelial cells. Later effects of mucostasis may be a result of cross-linking of 

18 mucus glycoproteins by formaldehyde, creating a rigid mucus that is not able to flow even with a 

19 rigorous ciliary beat. It is unknown if the eventual cessation of ciliary beat is a result of 

20 compound-related effects on ciliated epithelium as formaldehyde diffuses through the mucus or 

21 an indirect effect associated with mucostasis. However, in vitro experiments by Fló-Neyret et al. 

22 (2001) indicate that formaldehyde in solution, supporting isolated frog palates without mucus, 

23 resulted in the same sequence of effects, including increased ciliary beat at the lowest exposure. 

24 These data suggest a role of formaldehyde beyond its ability to form protein cross-links in 

25 mucociliary proteins.

26

27 4.2.1.2.2. Cellular pathology . This section summarizes studies that have investigated cellular 

28 pathology in the URT and in the lung. Below, full study descriptions are provided for both 

29 short-term and subchronic duration studies (including, where appropriate, how cell proliferation 

30 relates to the observed formaldehyde-induced pathology).

31
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purposes only and does not constitute 

Agency policy

4-127

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

Species Na Treatment

Measure of 

mucociliary 

function Summary of results by location Reference

Male F344 

rats

10 15 ppm formaldehyde 

6 hours/day for 1, 2, 4, 

or 9 days

Mucus flow and 

ciliary beat

Mucostasis in regions 2, 3, 4, 5, and 8 for all rats after a single 

dose.Mucostasis in all but two regions evaluated by day 9. Ciliastasis 

followed mucostasis.

Morgan et al. 

(1983)

Male F344 

rats

6 0, 0.5, 2, 6, or 15 ppm 

formaldehyde 

6 ourshours/day for 1, 

4, 9, or 15 days

Mucus flow and 

ciliary beat and 

histopathologic 

analysis

Flow or ciliary beat were increased at 0.5 ppm.

After1After 1 day, slowed or halted mucociliary flow at 15 ppm after 6 

hours.

After 9 days, slowed or halted mucociliary flow decreased or completely 

stopped in all nasal regions evaluated.

Regions affected included lateral aspect of the nasoturbinate and dorsal and 

medial aspects of maxilloturbinate.

Morgan et al. 

(1986c)

Male F344 

rats

3 per 

group

15 ppm formaldehyde 

for 10, 20, 45, or 

90 minutes or 6 hours

Mucus flow and 

ciliary beat

Ciliastasis and mucostasis increased in a time- and concentration-dependent 

manner, with maximal response at 6 hours.

Significant recovery was observed when a 1-hour recovery period occurred 

between exposure and sacrifice.

Morgan et al. 

(1986a)

Isolated frog 

palates

Not 

stated

0.23, 1.37, 4.36, or 

9.58 ppm 

formaldehyde

Mucus flow rates 

and histopathology

Ciliary beat and mucus flow increased from baseline at 1.37, 4.36, and 

9.58 ppm.

Over time, mucus became rigid, and ciliastasis occurred

Morgan et al. 

(1984a)

Isolated frog 

palates

4 0, 1.25, 2.5, and 5 ppm 

formaldehyde every 15 

minutes for 60 minutes

Mucociliary 

clearance and ciliary 

beat

Ciliary beat decreased in a time-dependent manner at 2.5 and 5,0 ppm but 

was increased at 1.25 ppm.

Mucostasis occurred after 60 minutes at 5 ppm.

Fló-Neyret et 

al. (2001)

N = number of animals in study.
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4.2.1.2.2.1. Nasal pathology short-term studies.

12 Scanning electron microscope examination of nasoturbinates showed increased mucus, 

13 erythrocyte infiltration, swelling of microvillus cells, and some cell separation in formaldehyde14 treated rats. Nasoturbinates examined 1 day after exposure showed greater effects, including cell 

15 damage, matted cilia, and blebbing of cell membranes. Damage to microvillus cells of the 

16 maxilloturbinate included deformed cilia, cell swelling and rupture, and lack of typical microvilli 

17 on the cell margins. As in the nasoturbinates, damage was more marked 24 hours after exposure. 

18 The epithelium of the ETs exhibited less cell damage than in the nasal and maxillary regions, 

19 with the slight lesions noted in the upper (ET1) portion and little to no damage noted on the mid 

20 and lower (ET2 and ET3) regions. Examination of transverse tissue sections revealed swollen 

21 goblet cells and stretched epithelial cells that formed an epithelial lining approximately 40% 

22 taller than the lining seen in control rats. There was also a patchy loss of ciliated cells in the 

23 respiratory epithelium, where columnar cells were present.

1 Inhalation of formaldehyde for a few hours has 

2 been shown to result in damage of the nasal mucosa, depending on the exposure concentration. 

3 Bhalla et al. (1991) observed changes in cell morphology in male Sprague-Dawley rat nasal 

epithelia after a single 4-hour exposure to 10 ppm (12.3 mg/m3 4 ) formaldehyde. Three exposed 

5 rats were sacrificed 1 hour and 24 hours after exposure, with two control rats at each time point. 

6 Noses were fixed, decalcified, and sliced along the midsagittal plane through the nasal septum. 

7 The exposed turbinates were examined by scanning electron microscopy. Transverse sections 

8 through the hard palate, at the level of the incisive papillae, were prepared for light microscopy 

9 from similarly exposed rats (n = 10). The authors provided detailed descriptions of cell epithelial 

10 organization in untreated rat turbinates and changes observed in formaldehyde-treated rats, as set 

11 forth below. No statistical analysis was provided.

24 Buckley et al. (1984) investigated the respiratory tract lesions associated with several 

25 sensory irritants. As part of this investigation, male Swiss-Webster mice were exposed to 

3.13 ppm (3.85 mg/m3 26 ) formaldehyde 6 hours/day for 5 days. A total of nine chemicals were 

27 tested in parallel. The report indicates there were 24–34 mice in each group, although not 

28 detailed for each chemical. One-half of the treatment group and unexposed controls were 

29 sacrificed immediately after the last exposure. The remaining exposed mice were sacrificed 

30 72 hours later. The head, trachea, and lungs were fixed and heads decalcified. Five sections 

31 were taken of each nose at levels equivalent to standard levels 2–6 (Figure 4-3) and were 

32 examined by light microscopy. Details on lung and trachea sections were not given. 

33 Formaldehyde induced lesions in the respiratory epithelium of exposed mice, including 

34 inflammation, exfoliation, erosion, ulceration, necrosis, and squamous metaplasia. The section 
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1 level for these effects was not given. No effects were reported in the squamous epithelium, 

2 olfactory epithelium, trachea, or lungs of formaldehyde-exposed mice.

3 Monteiro-Riviere and Popp (1986) evaluated damage to the respiratory epithelium due to 

4 acute formaldehyde exposures. Male F344 (CDF [F344]/CrlBr) rats (three to five per group) 

were exposed at 0.5, 2.0, 6.0, or 15 ppm (0.62, 2.5, 7.4, or 18.5 mg/m3 5 ) formaldehyde 

6 6 hours/day for either 1, 2, or 4 days. Rats were sacrificed either immediately after exposure or 

7 18 hours later (Table 4-10). After fixation and decalcification, blocks of tissue were collected 

8 from transverse sections of the skull. The first block of tissue, 1 μm thick, was taken just 

9 posterior of the incisor teeth. The second block was taken halfway between the first block and 

10 the incisive papillae. The dorsal nasal conchae, lateral wall, and ventral nasal conchae were 

11 microdissected, postfixed, and viewed by transmission electron microscopy (Monteiro-Riviere 

12 and Popp, 1986).

13

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

15 CDF rat nasoturbinates

16

Formaldehydea,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 conchae and 

lateral wall.

Severity of lesions increased with exposure 

duration.

Severity of lesions increased with concentration.

17 a 18 Number of exposed rats is shown in parentheses. b 19 Five control rats were examined for each experiment.

20 Source: Monteiro-Riviere and Popp (1986).

21

22 No lesions were observed at either 0.5 or 2.0 ppm formaldehyde for either 1 day or 

23 4 days, evaluated 18 hours after exposure. However, an unusual altered ciliary configuration, 

24 including blebbing of the cell membrane, was observed in almost all formaldehyde-treated rats, 

25 whereas it was only “occasionally noted” in control rats. Focal lesions in the dorsal and ventral 

26 conchae and lateral wall were seen in rats exposed at 6 and 15 ppm for 1 day and sacrificed 

27 immediately after exposure. These lesions included cytoplasmic and autophagic vacuoles, loss 

28 of microvilli, and hypertrophy. Lesions increased in severity with both exposure concentration 
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1 and duration. Neutrophil infiltration and intercellular edema were seen after 1 day at 6 and 

2 15 ppm. Nonkeratinized squamous metaplasia was noted after 4 days at 6 and 15 ppm in treated 

3 rats. Cell death and sloughing were noted after only 2 days of exposure at 6 ppm formaldehyde.

4 As described above, Cassee and Feron (1994) examined the effects of intermittent 

exposure to formaldehyde (3.5 ppm [4.3 mg/m3]), ozone (0.44 ppm [0.86 mg/m3 5 ]), or a 

6 combination of the two on changes to the rat nasal epithelium. Exposure occurred through six 

7 consecutive 12-hour cycles in which rats were exposed for 8 hours and then not exposed for a 

8 further 4 hours. Rats were weighed before the first and after the last exposure periods and 

9 sacrificed immediately after the last exposure. To collect tissue for biochemical analysis, skulls 

10 were split sagittally and the respiratory epithelium collected. Tissues from six rats were pooled 

11 and homogenized to enable the measurement of glutathione (GSH) and the activities of the 

12 following enzymes: glutathione S-transferase (GST), glutathione peroxidase (GPX), glucose-6-

13 phosphate dehydrogenase (G6PDH), glutathione reductase (GR), alcohol dehydrogenase (ADH), 

14 and formaldehyde dehydrogenase (FALDH). The remaining heads were fixed, decalcified, and 

15 sectioned (standard cross sections [Figure 4-3]). 

16 All groups, including controls, lost weight during the course of treatment. Rats exposed 

17 to formaldehyde, ozone, or both lost more weight than controls (p < 0.05, p < 0.01, and 

18 p < 0.001, respectively). Formaldehyde treatment alone increased GPX from 48.6 to 

19 64.0 μmole/minute-mg protein (p < 0.05) (Table 4-11). Formaldehyde exposure, in conjunction 

20 with ozone, decreased GST from 490 to 389 μmole/minute-mg protein (p < 0.05). No other 

21 enzyme activities or tissue GSH levels were affected by formaldehyde exposure.

22

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

24 Wistar rats exposed to formaldehyde, ozone, or both

25

Enzyme Controlsa

Formaldehyde 

(3.5 ppm) Ozone (0.4 ppm) Bothb

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 (0.08)

26 a 27 Values shown are the means and SDs of three measurements of a pooled sample. Units are 

28 μmole/minute/mg of cytosolic protein. b 29 Rats were exposed intermittently, 12-hour cycles of 8 hours exposed and 4 hours unexposed, for 3 days. c 30 Different from control, p < 0.05.

31

32 Source: Cassee and Feron (1994).

33
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1 Formaldehyde-exposed rats exhibited lesions in the nasal epithelium at levels 2 and 3 of 

2 the nose, with effects slightly more severe in level 2. Lesions observed include necrosis, 

3 hyperplasia accompanied by squamous metaplasia, and rhinitis. Exposure to formaldehyde in 

4 the presence of ozone resulted in more severe squamous metaplasia (statistics not given). These 

5 findings are similar to those of Monteiro-Riviere and Popp (1986), indicating that single or 

6 repeated exposures can result in cell damage and death. Cell death and increased cell 

7 proliferation were seen here after 3 days of repeated exposures to 3.5 ppm formaldehyde. While 

8 no increases were seen in olfactory epithelium, frank necrosis, squamous metaplasia, and 

9 hyperplasia of both ciliated and nonciliated epithelium were noted at level 2 and 3.

10 Javdan and Taher (2000) exposed male and female albino Wistar rats (five/group) at 0, 2, 

or 5 ppm (0, 2.5, or 6.2 mg/m3 11 ) formaldehyde 8 hours/day for either 3 or 30 days. Transverse 

12 tissue sections at the base of the incisive teeth and the first palatine folds were examined by light 

13 microscopy. Lesions reported after 3 days of exposure to 2 ppm formaldehyde included chorion 

14 congestion, cell disarrangement, squamous hyperplasia, atypical mitosis, and epithelial 

15 hyperplasia. Similar lesions were seen after 30 days but were more severe. Effects at 5 ppm 

16 formaldehyde included goblet cell proliferation, olfactory epithelial hyperplasia, calcified 

17 regions, and an abscess on the chorion. These lesions were more severe after 30 days of 

18 exposure.

19 Kamata et al. (1996a, b) conducted several high-dose studies by inhalation in rats. 

Specifically they exposed male F344 rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m3 20 ) 

21 formaldehyde for 6 hours (Kamata et al., 1996a). In a subsequent study in the same laboratory, 

male F344 rats were exposed to either 0, 15, or 145 ppm (0, 18.5, or 178 mg/m3 22 ) formaldehyde 

23 nose only for 6 hours (Kamata et al., 1996b). Congestion was noted in the nasal cavities of 

24 formaldehyde-exposed rats and was more severe at 145.6 ppm (Kamata et al., 1996b). Rats 

25 exposed to 15 ppm formaldehyde had lesions in the nasal turbinate and trachea (not detailed) 

26 (Kamata et al., 1996b). A slight hypersecretion of mucus was noted in the tracheal epithelium in 

27 the absence of histopathologic changes. Rats exposed to 145.6 ppm had more dramatic lesions 

28 that penetrated more deeply into the respiratory tract. Hyperkeratosis of the squamous 

29 epithelium was found at level 1 of the nasal cavity. Hypersecretion, desquamation, and irregular 

30 mucosal epithelium were seen in levels 2, 3, 4, and 5 of the nasal cavity, with more severe 

31 changes noted in the nasal septum. Increased secretion and desquamation of mucosal cells 

32 occurred in the trachea, and a slight hyperplasia of the alveolar wall was noted in rats exposed to 

33 145.6 ppm formaldehyde (Kamata et al., 1996b)

34 Hester et al. (2003) carried out a transcriptional analysis of the nasal epithelium of male 

35 F344 rats 24 hours after nasal instillation of 40 μL of 400 mM formaldehyde. Immediately after 
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1 sacrifice, cells were harvested from the nasal cavity for RNA extraction. The authors found 

2 several phase I and II enzymes, indicative of oxidative stress, to be elevated. They also reported 

3 the greatest increase in inflammatory genes, such as iNOS and neuropeptides. In an effort to 

4 phenotypically link any gene changes to pathology, Hester et al. (2003) noted that this exposure 

5 scenario has been demonstrated to induce regenerative hyperplasia with minimal cytotoxicity. In 

6 this regard, they observed no significant change in nine genes involved in three apoptotic 

7 pathways. 

8 In an expansion of their earlier study, Hester et al. (2005) carried out a transcriptional 

9 analysis of the nasal epithelium of male F344 rats that had been exposed to formaldehyde by 

10 nasal instillation for a single exposure, 5 days of exposures, or 28 days of exposure. In addition, 

11 this study also attempted to characterize the comparative toxicity of glutaraldehyde with 

12 structurally similar formaldehyde (van Birgelen et al., 2000). Thus, four animals per group were 

13 instilled with 40 μL of deionized water (control group), 40 μL of 400 mM formaldehyde, or 

14 40 μL of 20 mM glutaraldehyde. Phenotypically, both aldehydes induced similar 

15 histopathologic changes.

16 Both aldehydes induced similar changes in DNA repair and apoptotic pathways initially, 

17 but the patterns of gene changes were different after about 5 days of exposure. Eight genes were 

18 differentially expressed between formaldehyde and glutaraldehyde that indicated different 

19 pathways for DNA repair, including recombination, base excision repair, and nucleotide excision 

20 repair. Within this group, replication protein 70 and DNA excision repair ERCC1 showed a 

21 twofold induction by formaldehyde compared with glutaraldehyde. Since both of these genes 

22 and their products function by recognizing and removing damaged DNA bases, Hester et al. 

23 (2005) hypothesized that formaldehyde-exposed cells may remove damaged bases more 

24 efficiently than glutaraldehyde-exposed cells

25

4.2.1.2.2.2. Lung pathology: short-term studies.

30 Ionescu et al. (1978) described progressive damage in pulmonary tissue of adult male 

31 rabbits exposed to an aerosol of 3% formaldehyde solution 3 hours/day for up to 50 days 

32 (method of aerosol generation or particle size were not provided). An equivalent air 

33 concentration was not reported and cannot be derived from the information given. Animals were 

34 sacrificed at several time points (3, 7, 15, 20, 30, and 50 days), and fragments of the caudal lobes 

35 of both lungs were taken to examine bronchi (intrapulmonary and distal) and lung parenchyma. 

26 In addition to nasal pathology, several 

27 researchers specifically investigated formaldehyde-induced effects in the trachea, bronchi, and 

28 pulmonary tissues of the deep respiratory tract in a variety of species (Lino dos Santos Franco et 

29 al., 2006; Kamata et al., 1996a, b; Schreibner et al., 1979; Ionescu et al., 1978).
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1 Enzymatic activity was characterized in frozen sections for β-galactosidase, adenosine 

2 triphosphatase (ATPase), adenosine monophosphatase (AMPase), lactate dehydrogenase (LDH), 

3 malate dehydrogenase, succinate dehydrogenase (SDH), acid phosphatase, Tween-60 esterase, 

4 naphthol-AS-D-acetate esterase, proline oxidase, hydroxyproline epimerase, leucyl 

5 aminopeptidase, and β-glucuronidase. A portion of the lung was fixed and sectioned and viewed 

6 by light microscopy to determine changes in cell populations and tissue pathology.

7 In addition, biochemical analysis revealed that enzymatic activity of β-galactosidase, 

8 ATPase, AMPase, LDH, malate dehydrogenase, and SDH were all unchanged by formaldehyde 

9 exposure across the course of treatment (Ionescu et al., 1978). The activities of several enzymes 

10 were increased through the course of exposure, including acid phosphatase, Tween-60 esterase, 

11 naphthol-AS-D-acetate esterase, proline oxidase, and hydroxyproline epimerase. Although no 

12 details were reported, the authors described the changes as progressive, with the increase in 

13 proline oxidase and hydroxyproline epimerase seen only in the second half of the treatment 

14 course. The activities of two enzymes, leucyl aminopeptidase and β-glucuronidase, were 

15 observed to decrease rapidly (time frame not provided) (Ionescu et al., 1978)

16 Histologic changes in the lung tissue were noted after only 3 days of exposure and were 

17 generally progressive throughout the course of treatment. Early changes in the bronchial 

18 epithelium included increased mucus secretion, hyperplasia, and hypertrophy of epithelial cells. 

19 Lymphocyte infiltration was noted in many areas, and a limited thickening of the alveolar walls 

20 was reported after 3 days of exposure. Epithelial cell lesions, thickening of the alveolar, and 

21 infiltration of lymphocytes increased as exposure continued. Mucus cells increased as much as 

22 40% after 40 days of treatment. After 40 days of treatment, Ionescu et al. (1978) observed 

23 “destructive and fibrotic lesions” and provided a detailed description of progressive lesions.

24 Schreiber et al. (1979) also examined histologic changes in lung tissue after high 

25 formaldehyde exposures. Syrian golden hamsters (34, sex not stated) were exposed to 250 ppm 

(308 mg/m3 26 ) formaldehyde 1 hour/day for 1, 2, 5, or 15 days. Five hamsters in each treatment 

27 group were sacrificed 2 days after exposure was ended. Three hamsters in each group were 

28 sacrificed 1, 2, or 6 weeks after exposure ended to determine if formaldehyde-induced changes 

29 regressed over time. Tracheal washing was carried out to collect cytologic samples in each 

30 animal prior to sacrifice. Samples were fixed, stained, and examined by light microscopy. 

31 Lungs and tracheae were removed en bloc and fixed, and 20, 1 μm thick cross sections were 

32 taken (location not detailed). The remaining respiratory tissue was sectioned at 200 μm 

33 intervals. Sections were stained and viewed by light microscopy.

34 Abnormal epithelial cells were found in tracheal washings from formaldehyde-exposed 

35 hamsters. Schreibner et al. (1979) described cells with lobulated nuclei and a coarse chromatin 
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1 pattern, especially in cells showing signs of degeneration (e.g., vacuolization of nuclei and 

2 cytoplasm) (Schreiber et al., 1979). Cell number and damage were not quantified, and there was 

3 no discussion of the effects of exposure duration on treatment, if any, on these observations. 

4 Tracheal washing was normal 2 and 6 weeks after the end of exposure, indicating that the 

5 cytological changes were reversible (Schreiber et al., 1979).

6 Formaldehyde exposure caused multifocal lesions in the mucociliary epithelium in the 

7 trachea and larger bronchi. Dysplastic and poorly differentiated squamous metaplastic foci 

8 replaced ciliated epithelium (Schreiber et al., 1979). Abnormal nuclear membranes, tonofibrils 

9 around the nuclei, the appearance of nucleoli, and heterochromatin condensation were distinct in 

10 the formaldehyde-treated hamsters. These changes, observed 2 days after formaldehyde 

11 exposure, were reversible over time and not seen 2 and 6 weeks later.

12 Because of the similarity of form and physiology of rhesus monkey URTs to the human 

13 respiratory tract, the effects of short-term formaldehyde exposure were evaluated in both nasal 

14 and lung tissue in these monkeys by Monticello et al. (1989). Male rhesus monkeys (nine/group) 

were exposed to 6 ppm formaldehyde (7.4 mg/m3 15 ) 6 hours/day for 5 days/week for either 1 or 

16 6 weeks. Control animals were exposed to the same regimen of filtered air for 1 week. Monkeys 

17 were weighed during the course of exposure and observed for clinical signs of irritation or 

sickness. Monkeys were intravenously injected with [3 18 H]-thymidine 18 hours after the last 

19 formaldehyde treatment to evaluate induced cell proliferation. Sections of the nasal passages, 

20 trachea, larynx, lung carina, and duodenum were processed for histoautoradiography. Tissues 

21 fixed and sectioned for examination by light microscopy included nose, adrenal, sternum (bone 

22 marrow), duodenum, esophagus, eyes, gallbladder, heart, kidney, liver, lymph nodes, pancreas, 

23 stomach, spleen, and tongue. The nose was cut into a series of transverse sections, 3 μm thick, 

24 and sections from five levels were examined (Figure 4-6). Lung lobes were trimmed 

25 midsagittally and sectioned with care to include airway bifurcations. Sections of the nasal 

26 passages, trachea (cross section), larynx (cross section), lung carina (frontal section), and 

27 duodenum were also processed for histoautoradiography.

28 There were no significant changes in body weight over the course of the experiment. 

29 Oronasal breathing was noted in the first 15 minutes of formaldehyde exposure (Monticello et 

30 al., 1989). Monkeys did experience eye irritation (mild lacrimation and conjunctival hyperemia) 

31 during exposure.

32
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1

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

3 morphometry and autoradiography in male rhesus monkeys exposed to 

4 formaldehyde.

5

6 Source: Redrawn from Monticello et al. (1989). 

7

8 Formaldehyde-related lesions were reported in the nasal passages, tracheas, and in the 

9 larynx of treated animals (Figure 4-7) (Monticello et al., 1989). Nasal epithelium from treated 

10 animals exhibited many of the histologic lesions described in rodent studies, including loss of 

11 goblet cells, loss of cilia, epithelial hyperplasia, squamous metaplasia, and neutrophilic 

12 inflammatory response in the respiratory epithelium. The lesions were more severe after 

13 6 weeks of exposure and were present over a greater percentage of the epithelium compared with 

14 the 1-week exposure group (p < 0.05) (Figure 4-7).

15
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B

25

20

15

10

5

0 C D E

LEVEL

B

25

20

15

10

5

0 C D E

LEVEL

1 WK EXP

6 WK EXP

CONTROL

A) B)

1

2

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

4 exposed to formaldehyde

5

6 Note: Animals were exposed to 6 ppm formaldehyde 6 hours/day, 5 days/week 

7 for 1 or 6 weeks. Bar graph depicting mean labeling indices for the respiratory 

8 epithelium at levels B−E. A: One-week exposure group. B: Six-week exposure 

9 group. *Statistically different from controls (p ≤ 0.05). Statistically different 

10 from 1-week exposure group (p ≤ 0.05).

11

12 Source: Redrawn from Monticello et al. (1989).

13

14

15 There was a distinct anterior to posterior gradient in both 1-week and 6-week treatment 

16 groups in which the anterior regions had a higher percentage of impacted epithelium (Monticello 

17 et al., 1989). However, the longer duration exposure produced significantly more lesions in the 

18 larynx and trachea compared with those observed after only 1 week of exposure (p < 0.05). No 

19 formaldehyde-related lesions were reported for the epithelium of the maxillary sinus, a structure 

20 not present in rodents. Labeling indices (LIs) from the histoautoradiograms indicated increased 

21 cell proliferation in transitory, respiratory, and olfactory epithelial cells after the 6-week 
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1 formaldehyde exposure (Figure 4-8) (Monticello et al., 1989). Similar trends were seen after 

2 only 1 week but were statistically significant only in the respiratory epithelium. Although 

3 increased proliferation in the trachea and carina was statistically significant after 1 week of 

4 exposure, the greater increases seen after 6 weeks of exposure, compared with controls, were not 

5 statistically significant. A small sample size (n = 3) and high variability may have contributed to 

6 the lack of statistical significance. Monticello et al. (1989) noted that increased cell proliferation 

7 was seen in locations with minimal histologic changes, indicating proliferation may be a more 

8 sensitive predictor of adverse health effects of formaldehyde exposure.

9

60

50

40

30

20

10

0 B C D E L/T

LEVEL

60

50

40

30

20

10

0 B C D E L/T

LEVEL

A) B) 1 WK EXP

6 WK EXP

10

11

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

13 to formaldehyde.

14

15 Note: Animals were exposed to 6 ppm formaldehyde 6 hours/day, 5 days/week 

16 for 1 or 6 weeks. Bar graph showing levels B–E of the nasal passages and the 

17 larynx/trachea (L/T), depicting percent surface area with formaldehyde-induced 

18 lesions. Morphometry of level A was excluded due to the similarity of normal 

19 features of transitional epithelium to formaldehyde-induced lesions in the 

20 respiratory epithelium. A: One-week exposure group. B: Six-week exposure 

21 group. 

22 *Statistically different from controls (p ≤ 0.05). 

23 | Statistically different from 1-week exposure group (p ≤ 0.05).

24 Source: Redrawn from Monticello et al. (1989).

25

26 There are two reports in the literature assessing changes in pulmonary tissues after acute 

27 formaldehyde exposures (Kamata et al., 1996a, b). Kamata et al. (1996a) exposed male F344 
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rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m3 1 ) formaldehyde for 6 hours. Lung lavage 

2 samples were collected and the fluid analyzed for the lipids, free cholesterol, phosphatidyl 

3 ethanolamine, phosphatidyl choline, sphingomyelin, and triglyceride.

4 The bronchoalveolar lavage (BAL) was analyzed for triglycerides, cholesterol, and 

5 phosphatidyl choline. As in the first experiment (Kamata et al., 1996a), triglyceride 

6 concentration was reduced in the lavage of treated animals, in this case, to 16% of controls in 

7 lavage in those rats exposed to 145.6 ppm formaldehyde (Table 4-12). Cholesterol concentration 

8 was unchanged and phosphatidyl choline was increased to 220% of that of control rats as a result 

9 of exposure to 145.6 ppm formaldehyde. However, BAL lipids were unchanged in 15 ppm 

10 exposed rats. Triglycerides were reduced in unwashed lung tissue from formaldehyde-treated 

11 rats in a concentration-dependent manner and free fatty acids were reduced in rats exposed to 

12 145.6 ppm formaldehyde. Neither triglyceride nor sphingomyelin was detected in lung lavage 

13 fluid from the high treatment group.

14

15 Table 4-12. Lipid analysis of lung tissue and lung lavage from male F344 

16 rats exposed to 0, 15, or 145.6 ppm formaldehyde for 6 hours

17

Controla 15 ppma 145 ppma

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) 1.16 (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 19 SD given in parentheses.

b 20 Significant difference from controls (p < 0.05). c 21 Significant difference from controls (p < 0.01).

22

23 Source: Kamata et al. (1996b).

24

25 Concentration-dependent decreases were seen in nonprotein sulfhydryl (SH) groups and 

26 lipooxygenase in nasal mucosa homogenate and nonprotein SH groups in lung tissue 

27 homogenate (Table 4-13). Increases in both lipooxygenase and LDH activities were found in 

28 lung tissue homogenate from formaldehyde-exposed rats. 

29
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1 Table 4-13. Formaldehyde effects on biochemical parameters in nasal 

2 mucosa and lung tissue homogenates from male F344 rats exposed to 0, 15, 

3 or 145.6 ppm formaldehyde for 6 hours

4

Controla 15 ppma 145 ppma

Nasal mucosab

Nonprotein SH groups (μM/g tissue)c 1.64 (0.50) 1.29 (0.28) 0.73 (0.21)f

Lipid peroxides (μM/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)

Lunge

Nonprotein SH groups (μM/g tissue)c 1.83 (0.18) 1.70 (0.11) 1.29 (0.28)f

Lipid peroxides (μM/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 6 SD given in parentheses.

b 7 5 or 10% nasal mucosa homogenates.

c 8 nmol malonaldehyde/g tissue.

d 9 Units per gram tissue.

e 10 20% lung homogenates.

f 11 Significant difference from controls (p < 0.01). g 12 Significant difference from controls (p < 0.05).

13

14 Source: Kamata et al. (1996b).

15

16

17 Lino dos Santos Franco et al. (2006) studied the effects of inhaled formaldehyde on lung 

18 injury and changes in airway reactivity in rats. The extent of local and systemic inflammation 

19 was assessed by changes in leukocyte counts in BAL fluid, blood, bone marrow, and spleen. 

20 Changes of reactivity of isolated tracheae and intrapulmonary bronchi in response to 

21 methacholine were monitored in response to formaldehyde exposure. The authors exposed male 

22 Wistar rats to formaldehyde generated from a 1% solution of formalin. However, they provided 

23 insufficient information for the exposure concentration to be determined. Groups of six animals 

24 were exposed to formaldehyde for either 0, 30, 60, or 90 minutes on 4 consecutive days. All 

25 experiments were carried out 24 hours after the final exposure.

26 The authors reported a significantly increased number of leukocytes in the BAL fluid of 

27 animals exposed to formaldehyde via inhalation. The effect reached a maximum for the longer 

28 exposure duration (90 minutes). Compared with controls, rats exposed to formaldehyde 

29 90 minutes/day for 4 days also displayed an increase in the number of total blood leucocytes 

(1.4 ± 0.06 × 104

versus 0.8 ± 0.01 × 104 cells/mm3 30 ). These values are means ± standard error of 

31 the mean (SEM) for six animals/group. The effect appeared to reflect changes in the 

mononuclear cell population (1.1 ± 0.02 × 104 versus 0.6 ± 0.003 × 104 cells/mm3 32 ) rather than 

peripheral blood neutrophils (0.2 ± 0.003 × 104 cells/mm3 33 in test animals and controls). There 

34 was also an apparently compound-related increase in the total cell count in the spleen 
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(112.7 ± 4.4 × 106 versus 94.2 ± 5.5 × 106 1 cells). However, a change in the number of cells 

eluted from bone marrow did not reach statistical significance (54.6 ± 1.3 × 106 2 versus 

45.0 ± 4.3 × 106 3 cells). Lino dos Santos Franco et al. (2006) provided data on dose-dependent 

4 changes in methacholine-induced contractions in isolated tracheae and bronchi obtained from 

5 formaldehyde-exposed and control rats. Although the maximal contractile response induced by 

6 methacholine in tracheae of formaldehyde-treated rats was unchanged compared with controls, 

7 contractions in isolated bronchi were significantly weaker than those observed in controls.

8 The authors examined the effect of formaldehyde inhalation on rat lung mast cells. 

9 Degranulation and significant neutrophil infiltration were features of the response to 

10 formaldehyde (Table 4-14).

11

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

13 rats exposed to formaldehyde via inhalation

14

Treatment group

Mast cell degranulation

(cells/mm2

)

a

Neutrophil infiltration

(cells/mm2)

a

Controls 0b 0.3 ± 0.2

Formaldehyde-exposed 2.0 ± 0.4c 5.2 ± 1.7c

15 a 16 Values are means ± SEM; n = 6.

b

4.2 ± 0.6 cells/mm2 17 intact mast cells were found in the lungs of controls. c 18 No statistical analysis was provided by the authors for these changes.

19

20 Source: Lino dos Santos Franco et al. (2006).

21

22

23 Selected pharmacological agents were used to explore the mechanism by which exposure 

24 to formaldehyde might have brought about the observed lung infiltration and bronchial 

25 hyporesponsiveness. Lino dos Santos Franco et al. (2006) provided data showing that separate 

26 pretreatment of the animals with compound 48/80, sodium cromoglycate (SCG), and 

27 indomethacin reduced the formaldehyde effect on neutrophil release into BAL but had no effect 

28 on mononuclear cell counts. Compound 48/80 and SCG also reversed the formaldehyde-induced 

29 reduction in bronchial response to methacholine, but indomethacin had the opposite effect 

30 (causing an additional decrease in bronchial responsiveness). In broad terms, these findings 

31 were thought to implicate mast cells as a possible mediator of the toxicological effects of 

32 formaldehyde. Histologically, a significantly increased number of degranulated mast cells were 

33 evident in the pulmonary tissue of rats that were exposed to formaldehyde.

34 Lino dos Santos Franco et al. (2006) also examined the regulatory role of NO on 

35 formaldehyde-induced bronchial activity. Nitrites generated by cultured cells of BAL from 

36 formaldehyde-treated rats increased about threefold compared with those from controls. 
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1 However, pretreatment with the NO synthase inhibitor, N-nitro-L-arginine methyl ester, 

2 prevented the formaldehyde-induced bronchial hyporesponsiveness to methacholine but had no 

3 effect on pulmonary leukocyte recruitment. These data implicate the existence of distinct 

4 mechanisms for the induction of lung inflammation versus bronchial hyporeactivity. Further 

5 support for this concept came from an experiment in which rats were pretreated with capsaicin to 

6 examine the involvement of sensory fibers in lung inflammation and the bronchial 

7 hyporesponsiveness induced by formaldehyde inhalation. Although the treatment did not 

8 influence formaldehyde-induced bronchial hyporesponsiveness to methacholine, the number of 

9 leukocytes recovered in the BAL fluid were reduced compared with those of rats exposed to 

10 formaldehyde alone.

11

4.2.1.2.2.3. Extrapulmonary effects: short-term studies

28 In a subsequent study in the same laboratory (Kamata et al., 1996b), male F344 rats were 

exposed to either 0, 15, or 145 ppm (0, 18.5, or 178 mg/m3 29 ) formaldehyde nose only for 6 hours 

30 (Kamata et al., 1996b). Fifteen animals were treated at each level and separated into subgroups 

31 of five animals each for tissue collection and the determination of other endpoints. Blood 

32 samples were collected from one subgroup to determine such hematological and clinical 

33 chemistry parameters as RBC count, Hb, PCV, MCV, MCHC, WBC count, and plasma levels of 

34 TP, ALB, BUN, glucose, phospholipids, triglycerides, total cholesterol, LDH, alkaline 

35 phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and 

12 . Kamata et al. (1996a) exposed male 

F344 rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m3 13 ) formaldehyde for 6 hours. In 

14 addition, blood samples were monitored for hematology and clinical chemistry parameters, 

15 including red blood cell (RBC) count, hemoglobin (Hb), packed cell volume (PCV), mean 

16 corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), white blood 

17 cell (WBC) count, and plasma levels of total protein (TP), albumin (ALB), blood urea nitrogen 

18 (BUN), glucose, phospholipids, triglycerides, total cholesterol, cholinesterase, and LDH. Male 

19 rats exposed to 294.5 ppm formaldehyde had increased RBC count, Hb, hematocrit (HCT), 

20 MCV, and serum glucose (p < 0.05) compared with controls (Kamata et al., 1996a). There were 

21 concentration-related decreases in serum measures of TP, ALB, and phospholipids (p < 0.05). 

22 BUN was decreased in rats exposed to 128.4 ppm but increased in the higher treatment group 

23 (p < 0.05). Phospholipid analysis of the lung surfactant indicated a decrease in the production in 

24 formaldehyde-treated animals (p < 0.05). Total free cholesterol, phosphatidyl ethanolamine, and 

25 phosphatidyl choline were reduced to 60, 55, and 38% of controls for rats treated with 294.5 ppm 

26 formaldehyde (p < 0.05). Sphingomyelin was reduced to 32% of controls in the low treatment 

27 group (p < 0.05). 
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1 G6PDH. BAL was collected from five animals of each group and analyzed for phospholipids. 

2 Lung homogenate from five animals in each treatment group was analyzed for nonprotein SH 

3 groups, lipid peroxides, and total lipids. The 20,000 × g supernatant of the lung homogenate was 

4 assayed for the activities of GR, G6PDH, and LDH. Similarly, nonprotein SH groups and lipid 

5 peroxidase were measured in homogenates of excised nasal mucosa. At autopsy, organs (brain, 

6 heart, lung, liver, kidney, spleen, and testis) were weighed and tracheae and nasal turbinates 

7 examined. After fixation and decalcification, five sections across the nose were taken, 

8 corresponding to standard sections 1–5 (Figure 4-3).

9 Several blood parameters were affected after these acute exposures. The WBC count was 

slightly increased, from 4.7 × 103 cells/mm3 in control rats to 5.1 × 103 cells/mm3 and 6.1 × 103 10

cells/mm3 11 at 15 and 145.6 ppm formaldehyde, respectively (Kamata et al., 1996b). Serum levels 

12 of AST and LDH decreased in an apparent concentration-dependent manner (AST 68 and 54% 

13 of controls and LDH 48 and 28% of controls, respectively). Serum levels of G6PDH and ALT 

14 were decreased similarly across exposure groups at 45 and 78% of controls, respectively.

15 A synopsis of respiratory pathology findings following short-term exposure to 

16 formaldehyde is presented in Table 4-15.

17

4.2.1.2.2.4. Nasal pathology: subchronic studies.

26 Although control mice gained weight, mice exposed to 40 ppm formaldehyde lost weight 

27 during the 13-week exposures. Expressed by the authors as a percent of weight gain in controls, 

28 the weight losses were −235% in males and −168.6% in females. Early mortality for both male 

29 and female mice exposed to 40 ppm was 80%. Although gross and histochemical effects in 

30 excised pieces from each organ system were evaluated, endometrial hypoplasia in mice treated 

31 with 40 ppm was the only effect noted outside the respiratory system. The authors considered 

32 this effect secondary to the observed respiratory tract lesions and frank toxicity at 40 ppm 

33 formaldehyde.

18 In a study by Maronpot et al. (1986), female 

19 and male B6C3F1 mice (10/group) were exposed at 0, 2, 4, 10, 20, or 40 ppm (0, 2.46, 4.92, 

12.3, 24.6, or 49.2 mg/m3 20 ) formaldehyde 6 hours/day, 5 days/week for 13 weeks. Clinical 

21 observations were made daily, and mice were weighed weekly. At autopsy, tissue sections from 

22 each organ system (approximately 50 tissues per mouse) were fixed, stained, and examined by 

23 light microscopy. Noses were fixed, decalcified, and transversely trimmed at three levels: the 

24 incisor teeth, midway between the incisor teeth and first molar teeth, and the second molar teeth 

25 (corresponding to sections 2, 3, and 4 in Figure 4-3).

34
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-143

Table 4-15. Summary of respiratory tract pathology from inhalation exposures to formaldehyde—short-term studies

Species/strain

No./

group Treatment Respiratory effects LOAEL/NOAEL Reference

Nasal pathology

Male SpragueDawley rats3 Single 4-hour exposure to 

10 ppm formaldehyde.

Marked histopathologic changes to the nasoturbinates, 

maxilloturbinates, ethmoidal turbinates, and goblet and 

microvillus cells.

LOAEL = 10 ppm. Bhalla et al. 

(1991)

Male SwissWebster mice24–34 0 or 3.13 ppm formaldehyde 

6 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 ppm 

6 hours/day for 1, 2, or 4 days.

Histopathologic lesions to the nasal conchae, lateral 

wall, and ventral nasal conchae.

NOAEL = 2 ppm for 

focal lesions. Some 

changes in ciliary 

configuration were 

evident at all exposures.

Monteiro-Riviere

and Popp (1986)

Male Wistar rats 20 0 or 3.5 ppm formaldehyde 

through six consecutive 12-hour 

cycles in which rats were 

exposed for 8 hours; 10 were 

unexposed for 4 hours.

The activity of GPX was increased in respiratory 

epithelium homogenates. The nasal respiratory 

epithelium showed frank necrosis.

LOAEL = 3.5 ppm. Cassee and Feron 

(1994)

Male F344 rats 5 0, 6, or 15 ppm 

[

14C]-formaldehyde 6 hours/day 

for a single day (naïve group). 

A pretreated group was exposed 

to 6 or 15 ppm formaldehyde 

6 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 female 

Wistar rats

5/sex 0, 2, or 5 ppm formaldehyde 

8 hours/day for 3 or 30 days.

Cell disarrangement, squamous hyperplasia, atypical 

mitosis, and epithelial hyperplasia.

NOAEL = 2 ppm. Javdan and Taher 

(2000)

Male F344 rats 15 0, 15, or 145.6 ppm 

formaldehyde for a single 6-hour 

exposure.

Histopathologic lesions in the nasal turbinates and 

trachea

LOAEL = 15 ppm. Kamata et al. 

(1996b)

Male rhesus 

monkeys

9 0 or 6 ppm formaldehyde 

6 hours/day for 1 or 6 weeks. 

[

3

H]-thymidine was injected 

prior to sacrifice.

Histopathologic lesions, including loss of goblet cells, 

loss of cilia, epithelial hyperplasia, squamous 

metaplasia, and neutrophilic inflammation.

LOAEL = 6 ppm. Monticello et al. 

(1989)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-144

Table 4-15. Summary of respiratory tract pathology from inhalation exposures to formaldehyde—short-term studies

Species/strain

No./

group Treatment Respiratory effects LOAEL/NOAEL Reference

Tracheal and lung pathology

Syrian golden 

hamsters (sex 

unstated)

5 0 or 250 ppm 1 hour/day for 1, 

2, 5, or 15 days.

Abnormal cells in tracheal lavage, an effect that was 

reversed on cessation of treatment.

LOAEL = 250 ppm. Schreiber (1979)

Male rabbits 

(strain unstated)

ND Aerosol generated from a 3% 

formaldehyde solution 

3 hours/day for up to 50 days 

(air concentration unknown).

Necrosis of the bronchi and lung parenchyma. 

Increased activities of acid phosphatase, Tween-60 

esterase, naphthol-AS-D-acetate esterase, proline 

oxidase, and hydroxyproline epimerase. Reduced 

activities of leucyl aminopeptidase and βglucuronidase. Adverse histopathologic changes.

ND. Ionescu et al. 

(1978)

Male F344 rats 6 0, 128.4, or 294.5 ppm for a 

single 6-hour exposure.

Phospholipid content was reduced in lung surfactant, 

for example, sphingomyelin to 43% of controls in the 

low-concentration group.

LOAEL = 128.4 ppm. Kamata et al. 

(1996a)

Male F344 rats 15 0, 15, or 145.6 ppm 

formaldehyde for a single 6-hour 

exposure.

Biochemical changes in lung homogenates. Altered 

lipid 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, or 

90 minutes/day on 4 consecutive 

days (air concentration 

unknown).

Increased leukocyte count in bronchoalveolar fluid. 

Degranulation of mast cells and increased neutrophil 

infiltration.

ND. Lino dos Santos 

Franco et al. 

(2006)

Extrapulmonary effects

Male F344 rats 6 0, 128.4, or 294.5 ppm for a 

single 6-hour exposure.

. LOAEL = 128.4 ppm. Kamata et al. 

(1996a)

Male F344 rats 15 0, 15, or 145.6 ppm 

formaldehyde for a single 6-hour 

exposure.

. LOAEL = 15 ppm. Kamata et al. 

(1996b)

ND = not determined; LOAEL = lowest-observed-adverse-effect level; NOAEL = no-observed-adverse-effect level.
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1 While no statistical comparison was provided, respiratory tract lesions showed an 

2 increased incidence with concentration, as well as an increased distribution throughout the 

3 respiratory tract (Table 4-16). No lesions were seen in the nasal cavity, larynx, trachea, or lung 

4 of control mice or mice treated with 2 ppm formaldehyde. Minimal squamous metaplasia in the 

5 nasal cavity was noted in 1 of 10 male mice treated with 4 ppm formaldehyde, but none were 

6 observed in the female mice. However, squamous metaplasia was observed in all mice in the 

7 higher treatment groups (10, 20, and 40 ppm). Lesions became more severe and penetrated more 

8 deeply into the respiratory tract as exposure concentration increased. Where lesions were present 

9 in the nasal cavities of all mice exposed to 10 ppm, similar lesions were reported in the larynx 

10 and trachea of some animals exposed to 20 ppm and all animals exposed to 40 ppm 

11 formaldehyde. Mice exposed to 40 ppm formaldehyde exhibited lesions as deep as the lung, 

12 including squamous metaplasia, submucosal fibrosis inflammation, and epithelial hyperplasia.

13 The findings of Maronpot et al. (1986) indicated a no-observed-adverse-effect level 

14 (NOAEL) of 4 ppm and a LOAEL of 10 ppm in mice, based on squamous metaplasia in the 

15 nasal epithelium. Although a LOAEL of 10 ppm was observed, there was 80% mortality for 

16 both sexes at 40 ppm, indicating a very narrow range between the first observed adverse health 

17 effects and frank effect concentrations in mice for this 13-week treatment.

18 In a study by Woutersen et al. (1987), male and female albino SPF Wistar rats (10/group) 

were exposed to 0, 1, 10, or 20 ppm (0, 1.23, 12.3, or 24.6 mg/m3 19 ) formaldehyde 6 hours/day, 

20 5 days/week for 13 weeks. Rats were checked daily and weighed weekly. Three longitudinal 

21 sections of lungs, trachea, and larynx and six standard cross sections of the nose were taken for 

22 microscopic examination. Two rats per exposure group were similarly treated for 3 days and 

23 sacrificed 18 hours later, and nasoturbinates were dissected to measure cell proliferation. 

24 Woutersen et al. (1987) noted that the majority of the dose-dependent increases in cell 

25 proliferation seen at section level 3 after 3 days of repeated 6-hour exposures to 10 and 20 ppm 

(12.3 and 24.6 mg/m3 26 ) formaldehyde occurred in areas of the epithelium showing “clear 

27 squamous metaplasia and hyperplasia.” Cell proliferation rates in metaplastic epithelium of 

28 29.5 and 33.2% were much higher than the 1.4 to 2.8% proliferation in the visibly unaffected 

29 respiratory epithelium from rats exposed at 10 ppm formaldehyde. Although there was a slight 

30 trend towards increased cell proliferation in the visibly unaffected epithelium of exposed animals 

31 compared with unexposed controls, the majority of increased cell proliferation resulting from 

32 exposure to 10 and 20 ppm formaldehyde was attributed to the metaplastic epithelium 

33 (Woutersen et al., 1987).
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-146

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

formaldehyde

Location of respiratory 

tract lesions

Control 2 ppm 4 ppm 10 ppm 20 ppm 40 ppm

Male Female Male Female Male Female Male Female Male Female Male Female

Nasal cavity

Squamous metaplasia

Seropurulent inflammation

–a

–

–

–

–

–

–

–

1/10

–

–

–

10/10

4/10

10/10

–

10/10

10/10

10/10

8/10

10/10

10/10

10/10

10/10

Larynx

Squamous metaplasia – – – – – – – – 6/9 3/9 10/10 7/8

Trachea

Squamous metaplasia

Epithelial hyperplasia

Seropurulent inflammation

Submucosal fibrosis

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

–

1/10

–

–

–

3/10

4/10

–

–

5/10

2/10

–

–

10/10

2/10

8/10

9/10

10/10

---

5/10

5/10

Lung (Bronchus)

Squamous metaplasia

Inflammation

Submucosal fibrosis

–

–

–

–

–

–

 NDb

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

ND

–

–

–

–

–

–

–

–

–

–

–

–

4/10

3/10

2/10

3/10

2/10

–

a

Dash indicates no lesions recorded in that treatment group.

b

ND = no data.

Source: Maronpot et al. (1986).
This document is a draft for review purposes only and does not constitute Agency policy.

4-147 DRAFT—DO NOT CITE OR QUOTE

1 Statistically significant increases were seen in focal respiratory epithelial hyperplasia and 

2 keratinization in both male and female rats at the highest treatment level (20 ppm) (Table 4-17). 

3 Male rats also had statistically significant increases in observed respiratory epithelial squamous 

4 metaplasia, focal olfactory epithelial thinning, and rhinitis. Both male and female rats treated 

5 with 10 ppm formaldehyde showed statistically significant increases in squamous metaplasia, 

6 hyperplasia, and keratinization of the respiratory epithelium (Woutersen et al., 1987).

7 Disarrangement of the respiratory epithelium was only significantly increased in female rats, but 

8 this change was observed at both the 10 and 20 ppm treatment levels. Although some lesions 

9 were observed in animals treated with 1 ppm formaldehyde, their incidences were not 

10 statistically significant and the findings were equivocal.

11 Feron et al. (1988) examined recovery of formaldehyde-induced nasal lesions after 

12 subchronic exposures. Male albino SPF Wistar rats (50–55/group) were exposed to 0, 10, or 

20 ppm (0, 12.3, or 24.6 mg/m3 13 ) formaldehyde 6 hours/day, 5 days/week for either 4, 8, or 

14 13 weeks. All groups were observed for a total of 130 weeks, including treatment and recovery. 

15 Rats were weighed weekly for the first 13 weeks and monthly thereafter. Rats (five/group) were 

16 sacrificed immediately after the end of exposure (4, 8, or 13 weeks). The balance of the rats 

17 were sacrificed after 130 weeks, inclusive of exposure time. At sacrifice, noses were fixed and 

18 sectioned by using standard section levels.

19 Formaldehyde exposure (20 ppm) was associated with reduced body weight throughout 

20 the exposure period (4, 8, or 13 weeks). However, body weight in these groups matched that of 

21 controls after 8, 40, and 100 weeks, respectively. Rats exposed to 10 ppm for 8 or 12 weeks had 

22 slightly decreased body weight (further details not given).

23 Nonneoplastic lesions were reported in the nasal mucosa of rats exposed to either 10 or 

24 20 ppm formaldehyde and examined immediately after exposure was discontinued (4, 8, or 

25 13 weeks). Lesions increased in severity with both exposure duration and concentration (details 

26 of severity and incidence were not provided). Rhinitis, hyperplasia, and squamous metaplasia of 

27 the respiratory epithelium were seen in rats from both dose groups, but changes in olfactory 

28 epithelia were only seen in rats exposed to 20 ppm, where cell disruption, thinning of the 

29 epithelium, and simple cuboidal or squamous metaplasia were also reported. Changes in the 

30 dorsomedial region, at the junction of the respiratory and olfactory epithelium, were similar to 

31 those seen in the olfactory epithelium of rats exposed to 20 ppm formaldehyde. A similar 

32 concentration- and duration-dependent increase in histopathologic changes in nasal epithelium 

33 was observed after the full 130 weeks, which included 126, 122, or 117 weeks of recovery for 

34 the three duration groups, 4, 8, and 13 weeks, respectively (Table 4-17).

35
This document is a draft for review purposes only and does not constitute Agency policy.

4-148 DRAFT—DO NOT CITE OR QUOTE

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

2 changes in the noses and larynxes of male and female albino SPF Wistar rats 

3 exposed to formaldehyde 6 hours/day for 13 weeks

4

Concentration of formaldehyde (ppm)

0 1 10 20 0 1 10 20

Respiratory epithelium Severity Males Females

Diffuse squamous 

metaplasia

Slight – a – – – – – – 3

Moderate – – – 5b – – – 4

Severe – – – 5b – – – 3

Focal squamous 

metaplasia

Very slight – 1 – – – – 1 –

Slight – 1 6b – – – 7c –

Moderate – – 4 – – – 2 –

Focal hyperplasia Very slight – – 1 1 – – 2 1

Slight – – 6b 7c – 1 6b 6b

Moderate – – 1 – – – – –

Focal disarrangement Very slight – – 1 – – – 2 1

Slight – – 3 – – 1 6b 6b

Moderate – – 1 – – – – –

Focal keratinization Very slight – 2 6b 1 – – 6b 6b

Slight – – 3 6b – – 2 4

Moderate – – – 1 – – – –

Olfactory epithelium

Focal thinning Slight – – – 2 – – – 2

Moderate – – – 1 – – – 2

Severe – – – 5b – – – 2

Focal squamous 

metaplasia

Slight – – – 4 – – – 3

Moderate – – – 4 – – – 1

Focal keratinization Very slight – – – 1 – – – –

Slight – – – 2 – – – –

Rhinitis – 2 5b 10c – – 3 2

Larynx

Squamous metaplasia Very slight – – – 3 – NEd NE –

Slight – – – 1 – NE NE –

Moderate – – – 1 – NE NE –

Keratinization Slight – – – 2 – NE NE –

5 a 6 Dash indicates no lesions reported.

b 7 Different from control, p < 0.05. c 8 Different from control, p < 0.01. d 9 NE = not evaluated.

10

11 Source: Woutersen et al. (1987).

12

13

14 Feron et al. (1988) did not provide a direct comparison among lesions reported at the 

15 interim sacrifice and terminal sacrifice after the extended recovery period. However, similar 

16 lesions were reported after the recovery period, including focal hyperplasia and stratified 

17 squamous metaplasia of the respiratory epithelium, stratified cuboidal or squamous metaplasia in 

18 the dorsomedial area, and replacement of olfactory epithelium. The incidence and severity of 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 these lesions in rats exposed to 20 ppm formaldehyde were statistically different from control 

2 animals, regardless of exposure duration (Table 4-18). 

3

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

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

6 (6 hours/day, 5 days/week) and examined at the end of 130 weeks inclusive of 

7 exposure

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 5a 2

Slight 0 3 8b 2 2 12b 1 6 14b

Moderate 0 0 1 0 1 0 0 0 4

Respiratory epithelium focal stratified squamous 

metaplasia 

Very slight 3 6 14b 8 16 17a 2 10a 2

Slight 4 2 19b 2 1 20b 3 18b 26b

Moderate 0 2 3 0 0 2 1 5 14b

Severe 0 0 0 0 0 0 0 0 1

Respiratory/olfactory epithelium stratified 

cuboidal or squamous metaplasia 0 0 4 0 0 17b 0 2 23b

Rhinitis 7 7 18a 4 6 22a 8 11 23b

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 14b 0 0 12b

Moderate 0 0 1 0 0 3 0 0 12b

Severe 0 0 0 0 0 1 0 0 1

9 a 10 Significantly different from control, p < 0.05. b 11 Significantly different from control, p < 0.01.

12

13 Source: Feron et al. (1988).

14

15 Although a slight increase in changes to the olfactory epithelium and dorsomedial area 

16 was seen in rats treated with 20 ppm formaldehyde for only 4 weeks, these differences were 

17 significant and more severe in the 8- and 13-week treatment groups. Replacement of olfactory 

18 epithelium by respiratory epithelium was described as slight after 8 weeks of exposure and slight 

19 to moderate after 13 weeks of exposure in the 20 ppm treatment groups. Therefore, 

20 formaldehyde-induced lesions were not resolved after a considerable nonexposure recovery 

21 period of up to 126 weeks (Feron et al., 1988).

22 Feron et al., (1988) derived a correlation between the development of nonneoplastic 

23 changes in nasal epithelium and the development of nasal tumors as a result of these subchronic 

24 formaldehyde exposures. Two SCCs were reported in rats exposed to 10 ppm formaldehyde but 
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4-150 DRAFT—DO NOT CITE OR QUOTE

1 were not considered to be formaldehyde related because of their locations (nasolacrimal duct, 

2 incisor tooth). Six tumors were observed in the 20 ppm, 13-week exposure group (Table 4-19) 

3 of which three of the tumors were SCCs similar to those observed as a result of chronic 

4 formaldehyde exposure. Two polypoid adenomas also were reported in rats exposed to 20 ppm 

5 formaldehyde. Feron et al. (1988) concluded that subchronic exposures to 20 ppm formaldehyde 

6 could result in an increase in nasal tumors, an effect that followed observation of cellular 

7 proliferation.

8

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

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

11 examined at the end of 130 weeks inclusive of exposure

12

Tumor type 0 ppm 10 ppm 20 ppm

No. of rats exposed for 4 weeks 44 44 45

Polypoid adenoma 0 0 1a

SCC 0 0 1

No. of rats exposed for 8 weeks 45 44 43

Polypoid adenoma 0 0 1a

SCC 2 1 1

No. of rats exposed for 13 weeks 45 44 44

SCC 0 1 3a

Cystic squamous cell carcinoma 0 0 1

Carcinoma in situ 0 0 1a

Ameloblastoma 0 0 1

13 a 14 Tumor considered to be associated with formaldehyde exposure.

15

16 Source: Feron et al. (1988).

17

18

19 A companion study from the same laboratory examined the effects of lower concentration 

20 formaldehyde exposures (Zwart et al., 1988). Male and female albino Wistar rats (50/group) 

were exposed to 0, 0.3, 1, or 3.0 ppm (0, 0.37, 1.2, or 3.7 mg/m3 21 ) formaldehyde 6 hours/day, 

22 5 days/week for 13 weeks. Body weight, general condition, and behavior were recorded weekly. 

23 No effects of formaldehyde exposure on body weight changes were noted, and growth was 

24 considered comparable among different exposure groups and controls. Rats were sacrificed 

25 during week 14, and noses were fixed and sectioned (exact time after exposure ended not given). 

26 Six standard cross sections were examined for each animal by light microscopy, anterior to 

27 posterior. Noses were fixed and decalcified, and six standard cross sections were taken and 

28 developed.

29 No formaldehyde-related lesions were reported in the respiratory epithelium at section 

30 level 3 after 13 weeks of formaldehyde exposure (0.1 ppm, 1 ppm, or 3 ppm). Signs of 
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4-151 DRAFT—DO NOT CITE OR QUOTE

1 inflammation (rhinitis, sinusitis, mononuclear cell infiltrates) were observed in

2 formaldehyde-treated rats, but there was no concentration-response relationship (data not 

3 provided). Formaldehyde-related pathology in the anterior part of level 2 epithelium was 

4 reported in 37/50 males and 21/50 female rats exposed to 3.0 ppm for 13 weeks. Both 

5 keratinized and unkeratinized squamous metaplasia were present, and disarranged cells and 

6 hyperplastic respiratory epithelium were found in the transitional zone between squamous and 

7 pseudostratified epithelium at level 2. Foci of keratinized squamous epithelium, glandularization 

8 of goblet cells, and deciliated epithelium were observed by electron microscopy in anterior 

9 sections of level 2 of rats exposed to 3 ppm formaldehyde. Epithelial cells with irregularly 

10 shaped and strongly indented nuclei were described at level 2 in animals exposed to 0.3 and 

11 1 ppm formaldehyde and were considered to be disarranged as well at 3 ppm formaldehyde 

12 exposures.

13 Although early cell proliferation at level 3 corresponded to basal cell hyperplasia at 

14 3 days, neither effect persisted for the course of the exposure. The authors speculate that this is 

15 an indication of an adaptive response, perhaps through increased function of the mucociliary 

16 apparatus present at level 3. In contrast, the early changes at section level 2 were less dramatic 

17 but persisted through 13 weeks, including clear formaldehyde-related pathology.

18 Concentration times time (C × t) issues have been investigated for histopathology as well 

19 as for cellular proliferation, outlined above. Specifically, Wilmer et al. (1989, 1987) compared 

20 the effects of 8-hour continuous and 8-hour intermittent formaldehyde exposure in two studies. 

21 Fifty male albino Wistar rats (10/group) were exposed to different exposure regimens to achieve 

similar compound-related C × t products. A C × t product of 40 ppm-hours (49.2 mg/m3 22 -hours) 

was attained by an 8-hour exposure to 5 ppm (6.2 mg/m3 23 ) or a 4-hour exposure to 10 ppm (12.3 

mg/m3) (Wilmer et al., 1987). Similarly, an 80 ppm-hours (98.4 mg/m3 24 -hours) C × t product was 

attained from continuous 10 ppm exposure or intermittent 20 ppm (24.6 mg/m3 25 ) exposure. Rats 

26 were exposed to one of these regimens 8 hours/day for either 3 days (two/group) or 4 weeks 

(eight/group). Eighteen hours after exposure ended, rats were injected with [3 27 H]-thymidine and 

28 sacrificed 2 hours later. Noses were fixed and decalcified, and six standard cross sections were 

29 taken and developed.

30 Thinning and disarrangement of the respiratory epithelium, squamous metaplasia, basal 

31 cell hyperplasia, and rhinitis were seen in formaldehyde-treated rats. Lesions were most severe 

32 in group 4 (20 ppm intermittent). Groups 2 and 3 had similar lesions (10 ppm intermittent and 

33 continuous). Rats in group 1 had mild lesions. Formaldehyde concentration was the major 

34 determinate in severity of nasal lesions. Formaldehyde effects were less severe in group 1 than 

35 in group 3, even though the C × t product was the same, indicating concentration rather than 
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1 duration or cumulative exposure correlates to severity. Epithelial lesions in group 3 rats were 

2 similar among rats exposed to 10 ppm, regardless of duration (groups 2 and 3).

3 In a follow-up study, Wilmer et al. (1989) assessed both cellular proliferation and 

4 histologic lesions in Wistar rats exposed to formaldehyde in groups that differed by 

5 concentration and time. Group A served as a control group (0 ppm). Group B was exposed to 

6 1 ppm for 8 hours, group C to 2 ppm for 8 hours, group D to 2 ppm for 4 hours (30 minutes for 

7 8 hours), and group E to 4 ppm for 4 hours (30 minutes for 8 hours). The experimental design 

8 and cellular proliferation results are illustrated in Table 4-20. Intermittent exposures at 2 and 4 

9 ppm resulted in formaldehyde-related histopathologic lesions similar to those reported by Zwart 

10 et al. (1988). Disarrangement and squamous metaplasia in respiratory epithelium were observed 

11 at 4 ppm (Table 4-20). Disarrangement, nest-like infolds, goblet cell hyperplasia, and rhinitis 

12 were observed at 2 ppm. Rats exposed continuously for 8 hours at 2 ppm formaldehyde had 

13 fewer lesions than rats intermittently exposed to 2 ppm and were not statistically different from 

14 controls. Although lesions were noted in rats given the continuous 1 ppm, 8-hour treatment, 

15 their incidence was not significantly different from the controls (Table 4-20). It should be noted 

16 that the control rats in this study were reported to have a higher frequency of lesions than 

17 controls in two previous studies from this laboratory employing the same techniques (Zwart et 

18 al., 1988; Woutersen et al., 1987). For example, lesions noted in the respiratory epithelium of 25 

19 control rats included 13 disarrangements, 13 basal cell hyperplasia, and 5 each of goblet cell 

20 hyperplasia, nest-like infolds, and squamous metaplasia. This is in contrast to the data of 

21 Woutersen et al. (1987), who reported no lesions in the respiratory epithelium of 20 control rats 

22 (male and female). Although Zwart et al. (1988) discussed inflammatory lesions in control rats, 

23 no mention was made of the other scored lesions in control animals. Overall, Wilmer et al. 

24 (1989) reported clear adverse effects at 2 ppm formaldehyde, resulting from intermittent 

25 exposure for 8 hours/day, 5 days/week for 13 weeks. The indication of no effects at 1 ppm and 2 

26 ppm continuous exposure should be considered with some caution, given the unusual incidence 

27 of lesions in the control animals.

28 The results reported by Wilmer et al. (1989, 1987) indicate a greater influence of 

29 concentration, rather than exposure regimen (continuous versus intermittent) on formaldehyde 

30 toxicity. However, these studies were conducted as repeated 8-hour exposure regimens over a 

31 course of days or weeks. Therefore both regimens allowed for a 16-hour recovery time before 

32 the next reexposure and do not represent a true continuous exposure. This research group has 

33 speculated that defensive adaptation of the nasal mucosa may include the function of the 

34 mucociliary apparatus (Feron et al., 1989). Morgan et al. (1986a) have shown formaldehyde 

35 effects on mucus flow and ciliary beat in F344 rats to result from hourly exposures to 15 ppm 
This document is a draft for review purposes only and does not constitute Agency policy.

4-153 DRAFT—DO NOT CITE OR QUOTE

1 formaldehyde. However, effects seen in repeated 8-hour exposures may not correspond to those 

2 effects resulting from shorter duration exposures to higher formaldehyde concentrations.

3

4 Table 4-20. Formaldehyde effects on nasal epithelium for various 

5 concentration-by-time products in male albino Wistar rats

6

Respiratory epithelium 

at crosssection level 2

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

8-Hour

continuous

8-Hour

continuous

8-Hour

intermittent

8-Hour

intermittent

Disarrangement

Focal

Diffuse

12

1

4

1

8

0

3a

15b

8

11c

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 16c

Keratinization 0 0 1 0 3

Nest-like infolds

Focal

Diffuse

5

0

4

3

11

1

14c

0

7

1

Goblet cell hyperplasia

Focal

Diffuse

0

5

1

2

1

8

2

13b

1

10

Rhinitis 3 2 3 16c 8

7 a 8 p < 0.05, compared with group A. b 9 p < 0.001, compared with group A. c 10 p <0.01, compared with group A.

11

12 Source: Wilmer et al. (1989).

13

14 Rusch et al. (1983a, b) performed a comparative study of formaldehyde effects on the 

15 nasal epithelium in F344 rats, Syrian golden hamsters, and cynomolgus monkeys. Groups of 

animals were exposed at 0, 0.2, 1, or 3 ppm (0, 0.25, 1.2, or 3.7 mg/m3 16 ) formaldehyde 

17 22 hours/day, 7 days/week for 26 weeks. Six male monkeys, 10 male and 10 female hamsters, 

18 and 20 male and 20 female rats were exposed at each exposure level. The experiment was run in 

19 two trials, each with its own control group: trial 1 at 0.2 or 1 ppm and trial 2 at 3 ppm. Animals 

20 were weighed weekly and physically assessed (details not given). At sacrifice, organ weights 

21 were recorded for the kidney, adrenals, heart, and liver. Tissue sections of the lung (4), trachea, 

22 and nasal turbinates (4) of each animal were examined by light microscopy (section locations not 
This document is a draft for review purposes only and does not constitute Agency policy.

4-154 DRAFT—DO NOT CITE OR QUOTE

1 given). Additionally, sections were examined by electron microscopy for rats in the control and 

2 1 ppm treatment groups (five rats per group).

3 Body weights of both male and female rats in the 3 ppm treatment group were depressed 

4 by 20% between week 2 and the end of the 26-week exposure. Absolute liver weights were 

5 decreased in these animals as well (26% lower in males and 12% lower in females, p < 0.05). 

6 This decrease in liver weight remained significant for male rats when normalized for body 

7 weight (a ratio of 2.9 in treated versus 3.16 in controls) but not for female rats. No significant 

8 body weight or organ weight changes were seen in hamsters or monkeys. Increased incidences 

9 of congestion (36/156), hoarseness (32/156), and nasal discharge (62/156) were observed in 

10 monkeys in the 3.0 ppm treatment group versus no hoarseness or congestion and only five 

11 observations of nasal discharge in 156 observations for control monkeys. Increased nasal 

12 congestion was noted in the two lower treatment groups of monkeys: 30/156 and 

13 45/156 observations, respectively, versus 9/156 observations in nasal discharge in the controls. 

14 The authors reported an increase in nasal discharge and lacrimation in treated hamsters but no 

15 increases in symptoms in rats. However, observations of adverse symptoms in the control rats 

16 were greater than 10% on some measures.

17 Rhinitis increased in rats in the 3 ppm treatment group, and the incidence in controls was 

18 notable (Table 4-21). All groups of monkeys showed some rhinitis, and no treatment effects 

19 were observed in either monkeys or hamsters. Monkeys and rats in the high treatment group 

20 (3 ppm) had a greater incidence of lesions in the nasoturbinate epithelium (Table 4-22). Rusch 

21 et al. (1983a, b) noted that most lesions were mild to moderate but were “somewhat more 

22 severe” in the high treatment group. Hamsters did not exhibit a similar increase, with few 

23 lesions noted in the nasal epithelium. Overall, these studies show a clear increase in adverse 

24 health effects at 3 ppm for rats and monkeys, with no adverse effects seen in hamsters at this 

25 treatment level or rats and monkeys at the lower concentrations (0.2 ppm and 1 ppm).

26

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

28 male and female animals

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

31 Source: Rusch et al. (1983a, b).
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1 Table 4-22. Epithelial lesions found in the middle region of nasoturbinates of 

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

3 females

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

6 Source: Rusch et al. (1983a, b).

7

8

9 Andersen et al. (2008) examined the effect of formaldehyde exposure at several 

10 concentrations and durations. This study comprised histopathology and cell proliferation data, as 

11 well as genomic analyses at Level II of the nasal cavity. Toxicogenomics analysis was 

12 performed only at Level II because this was the region where the most severe lesions have been 

13 reported in chronic bioassays (Andersen et al., 2008; Monticello et al., 1991; Kerns et al., 1983). 

14 More specifically, Andersen et al. (2008) stated that the histopathologic and cell proliferation 

15 effects at Levels II and III (with similar tissue structure) (Monticello et al., 1991) provided 

16 phenotypic anchoring for the genetic analysis. Table 4-23 summarizes many of the broad 

17 phenotypic findings. 

18 The primary conclusions of this study with regard to the histopathology and cell 

19 proliferation are as follows:

20 • The presence of inflammatory cell infiltrates in the nasal epithelial tissue of F344 rats is 

21 highly variable and provides no coherent pattern with dose or duration at levels below 

22 6 ppm.

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

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

25 • Cell proliferation (as measured by labeling indices) was significantly elevated in 

26 Levels I–III at 6 ppm on day 5 and Level I on day 15, leading to the conclusion that 

27 significant changes in cell proliferation may not occur at exposures to ≤2 ppm.

28 • A significant decrease in cell density was observed at Level I in animals exposed to 

29 6 ppm formaldehyde for 15 days, which was posited to be related to tissue remodeling in 

30 response to this concentration.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-156

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

ppm

Response

D1 D1R D5 D6 D6R D15

0 0.7 2 6 15 0 0.7 2 6 15 0 0.7 2 6 15 0 0.7 2 6 15 0 0.7 2 6 15 0 0.7 2 6 15

I 0 1 6 8 – 4 2 1 7 – 1 1 5 8 – 5 2 4 7 – 6 1 3 7 – 3 1 0 5 –

H 0 0 0 0 – 0 1 3 8 – 0 0 3 8 – 0 0 1 8 – 0 0 2 8 – 0 0 2 7 –

M 0 0 0 0 – 0 0 0 0 – 0 0 0 7 – 0 0 0 0 – 0 0 0 0 – 0 0 0 0 –

P1 39±9 37±15 65±40 155±89a 79±55 56±37 51±44 119±38a

P2 – – – a – – – –

P3 – – – a – – – –

CD 321±30 336±64 377±141 400±61 362±61 340±57 321±37 293±53b

G – 0 1 42 745 – 0 0 0 – – 0 15 28 – – 0 0 9 – – – – – – – 0 0 54

D = 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. a

Significantly elevated ULLI and LI at Level I on day 5 or significantly elevated lLI lat Level I on day 15; index a without numerical value indicates significant 

increases in ULLI in all subregions of Levels II and III at day 5. b

Statistically significant difference from control (p < 0.05).

Source: Andersen et al. (2008).
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1 Based on their analysis of the microarray data, Andersen et al. (2008) concluded that no 

2 genes were significantly altered by exposure to 0.7 ppm from 1 to 15 days. Exposure to 2 ppm 

3 primarily resulted in gene changes at 5 days of exposure, but not thereafter. One gene was 

4 significantly increased on day 1, but the authors did not identify that gene. At 6 and 15 ppm, 

5 42 and 745 genes were altered at day 1, respectively. After 5 days, gene changes were only 

6 observed at 6 ppm (15 ppm was not examined after day 1). These findings support conclusions 

7 reached by their laboratory in an earlier analysis. Thus, the primary conclusion in the Andersen 

8 et al. (2008) study is that genomic changes, including those suggestive of mutagenic effects, did 

9 not temporally precede or occur at lower doses than phenotypic changes in the tissue. The 

10 implications of this finding will be examined later in Section 4.5.

11

4.2.1.2.2.5. Lung pathology: subchronic studies.

19 Özen et al. (2003a) measured zinc, copper, and iron content in lung tissue from 

20 formaldehyde-exposed Wistar rats. Adult male rats were exposed to 0, 5, or 15 ppm (0, 6.2, or 

18.5 mg/m3 21 ) formaldehyde 8 hours/day, 5 days/week for either 4 or 13 weeks. Rats were 

22 checked daily and weighed weekly. At sacrifice, rats were autopsied and examined for gross 

23 pathological changes. Lung tissue was homogenized and analyzed for zinc, copper, and iron.

12 Studies have also investigated the ability for 

13 formaldehyde to induce pathology in the trachea, bronchi, and lung tissue. These studies have 

14 reported tracheal tissue changes, lung inflammation, necrosis, changes to the biochemistry of 

15 BAL fluid and lung surfactant in a variety of species. Özen et al. (2003a) noted changes in zinc 

16 concentration in the lung tissue following exposure for formaldehyde. Dallas et al. (1989) and 

17 Dinsdale et al. (1993) observed changes in P450 enzyme activity in the lung associated with 

18 formaldehyde exposure.

24 Body weight gain was depressed in all treatment groups in a concentration-dependent 

25 manner (p < 0.001) (Table 4-24). Formaldehyde-exposed rats consumed less food and water 

26 than controls and showed unsteady breathing, increased nose cleaning, excessive licking, 

27 frequent sneezing, and nasal mucosa hemorrhages. Significant decreases were seen in the zinc 

28 content of lungs after either 5 or 10 ppm formaldehyde exposure (Table 4-25). Copper content 

29 was unchanged from controls in all treatment regimens, whereas iron content was increased after 

30 4 weeks of 5 ppm exposure and after 13 weeks of either 5 or 10 ppm formaldehyde exposure 

31 (Özen et al., 2003a).

32

33
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1 Table 4-24. Percent body weight gain and concentrations of iron, zinc, and

2 copper in cerebral cortex of male Wistar rats exposed to formaldehyde via 

3 inhalation for 4 and 13 weeks

4

Exposure (mg/m3) 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 ± 1.49e 130 ± 7.26c 5.60 ± 0.50b 23.00 ± 2.32

12.2 5.24 ± 1.52e 185 ± 10.36e 5.80 ± 0.60d 22.14 ± 1.95b

13-week data

0 60.53 ± 7.84 123 ± 6.22 4.67 ± 0.38 24.92 ± 2.84

 6.1 38.41 ± 2.53e 155 ± 7.94e 5.41 ± 0.56c 22.00 ± 2.41

12.2 25.87 ± 1.32e 163 ± 6.03e 6.10 ± 0.73e 21.00 ± 1.96b

5 a 6 Values are means ± SDs (n = 7).

7

8 Statistical significance of differences versus controls, as calculated by the authors:

b

p < 0.05. cp < 0.02. dp < 0.002. e 9 p < 0.001.

10

11 Source: Özen et al. (2003b).

12

13

14 Table 4-25. Zinc, copper, and iron content of lung tissue from formaldehyde15 treated male Wistar rats

16

Concentration Durationa Zincb,c Copperb,c Ironb,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 18 Rats were exposed 8 hours/day, 5 days/week for the number of weeks indicated. b 19 Concentrations are expressed as moles/mg of tissue, wet basis.

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

d 21 p < 0.005, compared with controls, as calculated by authors. e 22 p < 0.001, compared with controls, as calculated by the authors. 

23

24 Source: Özen et al. (2003a).

25

26 There are two reports of lung cytochrome P450 levels after formaldehyde exposure. The 

27 first report by Dallas et al. (1989) describes concentration- and duration-dependent changes in P450 

28 levels. Male Sprague-Dawley rats were exposed at 0, 0.5, 3.0, or 15 ppm (0, 0.62, 3.7, or 18.5 

mg/m3 29 ) formaldehyde 6 hours/day, 5 days/week for 1 day, 4 days, 12 weeks, or 24 weeks. There 

30 were six rats in each exposure group, but the experiment was run in two parts, with three rats in 

31 each subgroup. Rats were sacrificed after 1 day, 4 days, 12 weeks, or 24 weeks of exposure, and 

32 liver microsomes were prepared. TP and P450 content were determined on each sample.
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1 Average P450 levels in control groups ranged from 17–76 pmol P450/mg protein. 

2 However, no P450 was detected in lung from formaldehyde-treated animals after 1 day of 

3 exposure, with a method detection limit of approximately 10 pmol P450/mg protein. In contrast, 

4 P450 levels were elevated significantly above controls in a concentration-dependent manner after 

5 4 days of formaldehyde exposure (Table 4-26). Although P450 levels remained elevated in some 

6 experimental groups after 12 and 24 weeks of exposure, results were variable and less dramatic.

7

8 Table 4-26. Total lung cytochrome P450 measurements of control and 

9 formaldehyde-treated male Sprague-Dawley rats

10

1 Daya,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 (23) 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 12 Rats were exposed 6 hours/day, 5 days/week for the duration shown. b 13 Cytochrome P450 expressed as pmol P450/mg of protein. Values are means (SDs) (n = 3).

c 14 Different from control, p < 0.05. d 15 Different from control, p < 0.01. e 16 Different from control, p < 0.001, as calculated by the authors.

17 ND = not detected above the limit of detection, approximately 10 pmol/mg protein.

18

19 Source: Dallas et al. (1989).

20

21

22 A later study by Dinsdale et al. (1993) attempted to confirm the increase in P450 levels 

23 reported by Dallas et al. (1989). In their first experiment, Dinsdale et al. (1993) treated male 

Sprague-Dawley rats at approximately 10 ppm (12.3 mg/m3 24 ) formaldehyde 6 hours/day for 

25 4 days. The formaldehyde vapor was generated from formalin by a concentric jet atomizer. For 

26 the second experiment, Dinsdale et al. (1993) similarly exposed rats to formaldehyde, but the gas 

27 was generated by the thermal depolymerization of paraformaldehyde as was done by Dallas et al. 

28 (1989). The concentration of P450 and activity of several P450 isozymes were measured in lung 

29 microsomes (pentoxyresorufin O-dealkylase, benzyloxyresorufin O-dealkylase, ethoxyresorufin 

30 O-dealkylase, and 2-aminofluorene N-hydroxylation). ALP and γ-glutamyl transpeptidase 

31 activity were measured in BAL fluid collected from each animal. No changes were seen in BAL 

32 enzyme activity or the activity of lung microsomes for the P450 substrates tested. Cytochrome 

33 P450 levels were unchanged in experiment 1, where formaldehyde was generated from formalin. 

34 Cytochrome P450 levels were increased in experiment 2 with formaldehyde generated from 

35 paraformaldehyde (Table 4-27).

36
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1 Table 4-27. Cytochrome P450 levels in formaldehyde-treated rats

2

Group

Experiment 1

(formalin)a

Experiment 2

(paraformaldehyde)a

(nmol/mg protein)

Control 82 ± 30 85 ± 5

10 ppm formaldehyde 73 ± 27 125 ± 23b

3 a 4 Values are means ± SDs (n = 3–5). b 5 Different from controls, p < 0.05.

6

7 Source: Dinsdale et al. (1993).

8

4.2.1.2.2.6. Extrapulmonary toxicity: subchronic studies.

22 Hematological parameters were unaffected by formaldehyde treatment. The lung tissue 

23 of all species exhibited interstitial inflammation after 90 days of formaldehyde exposure 

24 (detailed description not provided). Formaldehyde-treated rats and guinea pigs also had focal 

25 chronic inflammation in heart and kidney tissue sections. However, the authors were uncertain 

26 whether the observed changes to heart and kidney were due to formaldehyde exposure.

9 Several studies have investigated 

10 toxicity in organs other than those associated with the respiratory tract. An earlier cross-species 

11 study examined changes in lung tissue resulting from continuous exposure (Coon et al., 1970). 

Animals were exposed to 3.7 ppm (4.6 mg/m3 12 ) formaldehyde for 90 days. Five species of 

13 animals were studied: male and female Sprague-Dawley and Long-Evans derived rats (15), male 

14 and female Princeton-derived guinea pigs (15), male New Zealand albino rabbits (3), male 

15 squirrel monkeys (Saimiri sciureus) (3), and purebred male beagle dogs (2). Blood samples were 

16 taken for Hb concentration, HCT, leukocyte counts, and serum levels of BUN, AST, ALT, ALP, 

17 and LDH. Sections of heart, lung, liver, kidney, and spleen were fixed and examined from each 

18 species (details of method not provided). Brain, spinal cord, and adrenal tissue also were 

19 examined in monkeys and dogs as well as thyroid from dogs. Liver and kidney sections were 

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

21 Tissue sections of the nasal mucosa were not examined in this study.

27 As mentioned above, Woutersen et al. (1987) exposed male and female albino SPF 

Wistar rats (10/group) to 0, 1, 10, or 20 ppm (0, 1.23, 12.3, or 24.6 mg/m3 28 ) formaldehyde 

29 6 hours/day, 5 days/week for 13 weeks. Rats were checked daily and weighed weekly. During 

30 week 13, blood samples were taken for Hb, PCV, RBC count, and a differential count of 

31 leukocytes. Urine samples were also analyzed. At sacrifice, blood samples were analyzed for 

32 ALB, creatinine, glucose, TP, BUN, and the enzyme activities (AST, ALT, and ALP). GSH and 

33 protein content were determined in liver homogenates. Organs were examined and weighed: 

34 adrenals, brain, heart, kidneys, liver, lungs, ovaries, pituitary, spleen, testes, thymus, and thyroid.
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1 No gross pathological changes were seen upon autopsy, but body weights decreased in 

2 both male and female rats at the 20 ppm treatment level. Of the organs weighed, 6 of 11 had 

3 significantly increased relative rates in male rats exposed to 20 ppm formaldehyde. Relative 

4 brain weight was increased in female rats at the same treatment level (Woutersen et al., 1987).

5 Clinical chemistry parameters of liver and kidney function and hematological parameters 

6 were also measured after the 13-week treatment by Woutersen et al. (1987). Compared with 

7 those of controls, activities of AST, ALT, and ALP were significantly elevated in plasma from 

8 the 20 ppm treated male rats (by 124, 132, and 126%, respectively; p < 0.05). Total plasma 

9 protein was reduced to 95% of controls in the same animals. Although there was an observed 

10 increase in BUN in male rats treated with 1 ppm, this was not considered a treatment effect. 

11 Furthermore, no statistically significant differences were seen for these parameters in female rats 

12 at any concentration level (Woutersen et al., 1987). 

13 Sul et al. (2007) exposed Sprague-Dawley rats to 0, 5, and 10 ppm formaldehyde for 

14 6 hours/day (5 days/week) for 2 weeks and collected lung samples for tissue damage and 

15 genomic analysis. According to their results, 21 genes were altered in a dose-dependent manner 

16 by microarray analysis; 2 were up regulated and 19 were down regulated in the lung tissue of 

17 animals exposed to formaldehyde. However, six of the nine genes further analyzed by PCR did 

18 not show dose dependency (authors did not comment). Although the authors briefly describe the 

19 functions and potential implications for changes in the expression of some of the altered genes, 

20 there is no discussion of the relationship between these altered genes (i.e., there is no pathway 

21 analysis).

22 In 2006, Im et al. (2006) published a proteomic analysis using the same exposure 

23 protocols (possibly using the same animals as in the Sul et al. [2007] study, although neither 

24 study makes reference to the other). Im et al. (2006) examined DNA damage in lymphocytes 

25 and liver tissues, as well as protein and lipid oxidation in plasma and liver samples. Similar to 

26 changes reported in the lung (discussed elsewhere), using two-dimensional electrophoresis and 

27 matrix-assisted laser desorption ionization time-of-flight mass spectrometry, the authors also 

28 reported dose-dependent changes in the levels of 32 proteins in plasma (19 up, 13 down). None 

29 of the changes in plasma proteins correspond to the changes in lung reported by Sul et al. (2007). 

30 Again, no pathway analysis was provided. Interestingly, Im and colleagues (2006) also 

31 demonstrated a dose-dependent increase in plasma IL-4 and dose-dependent decrease in IFNγ, 

32 perhaps indicative of Th-2-mediated inflammatory response. An overview of formaldehyde 

33 exposure-related pathology in the respiratory system of laboratory animals is presented in 

34 Table 4-28.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-162

Table 4-28. Summary of respiratory tract pathology from inhalation exposures to formaldehyde, subchronic studies

Species/strain

No./

group Treatmenta Respiratory effects LOAEL/NOAEL Reference

Nasal pathology

B6C3F1 mice 

(male and 

female)

10 0, 2, 4, 10, 20, or 40 ppm 

formaldehyde 6 hours/day, 

5 days/week for 13 weeks

Minimal squamous metaplasia in 1 of 10 mice 

(4 ppm). Squamous metaplasia observed in all mice 

at 10 and 20 ppm.

NOAEL = 4 ppm Maronpot et al. 

(1986)

SPF Wistar Rats 

(male and 

female)

10 0, 1, 10, or 20 ppm 

formaldehyde 6 hours/day, 5 

days/week for 13 weeks

Increased respiratory epithelial hyperplasia and 

keratinization at 20 ppm; squamous metaplasia at 

10 ppm in males and females.

NOAEL = 1 ppm Woutersen et al. 

(1987)

SPR Wister rats 

(male)

50–55 0, 10, or 20 ppm formaldehyde 

for 6 hours/day, 5 days/week 

for 4, 8, or 13 weeks

Rhinitis, hyperplasia, and squamous metaplasia in 

respiratory epithelium at all doses (number of weeks 

not specified).

Squamous metaplasia of olfactory epithelium at 

20 ppm (number of weeks not specified)

NOAEL = 1 ppm Feron et al. (1988)

Wistar rats (male 

and female)

50 0, 0.3, 1, or 3.0 ppm 

formaldehyde 6 hours/day, 

5 days/week for 13 weeks

Keratinized and non-keratinized squamous 

metaplasia in level 2 epithelium in 37/50 male and 

21/50 female rats at 3 ppm for 13 weeks.

NOAEL = 1 ppm Zwart et al. (1988)

Wistar rats (male) 10 40 ppm-hours (8 hours at 

5 ppm, 4 hours at 10 ppm) or 

80 ppm hours (10 ppm 

continuous or 20 ppm 

intermittently)

Thinning and disarrangement of respiratory 

epithelium, squamous metaplasia, most severe in 

20 hours intermittent exposure

NOAEL = 10 ppm Wilmer et al. 

(1987)

Wistar rats (male) 10 0, 8, or 16 ppm, given either 

continuously or intermittently

Disarrangement and squamous metaplasia at 4 ppm. 

Continuous exposure yielded less severe lesions 

than intermittent exposure

LOAEL = 8 ppm Wilmer et al. 

(1989)

F344 rats (male 

and female), 

Syrian golden 

hamsters (male 

and female), 

cynomolgus 

monkeys

20 rats,

10 

hamsters,

6 monkeys

0.0.2, 1, or 3 ppm 

22 hours/day, 7 days/week, 

26 weeks

Rats: rhinitis at 3 ppm, increased incidence of nasal 

lesions at 3 ppm.

Monkeys: rhinitis at all doses, increased incidence 

of nasal lesions at 3 ppm.

Hamsters: no significant nasal lesions.

NOAEL = 1 ppm Rusch et al. 

(1983a, b)

Tracheal and lung pathology

Wistar rats (male) 6 0, 5, 15 ppm for 8 hours/day, 

5 days/week, 4 or 13 weeks

Significant decreases in zinc content in lung, copper 

unchanged, iron increased in lung.

LOAEL = 5 ppm Özen et al. (2003)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-163

Table 4-28. Summary of respiratory tract pathology from inhalation exposures to formaldehyde, subchronic studies

Species/strain

No./

group Treatmenta Respiratory effects LOAEL/NOAEL Reference

Sprague-Dawley 

rats (male)

6 but n = 5 

in some 

trials

0, 0.5, 3.0, 15 ppm 6 

hours/day, 5 days/week, for 1 

day, 4 days, 12 weeks, 24 

weeks

Increased P450 levels after 4 days at 3 ppm. NOAEL = 0.5 ppm Dallas et al. 

(1989)

Sprague-Dawley 

rats (male)

5 0 or 10 ppm 6 hours/day, 

4 days using both formalin and 

paraformaldehyde

P450 levels increased at 10 ppm only in groups 

treated with paraformaldehyde.

LOAEL = 10 ppm Dinsdale et al. 

(1993)

Extrapulmonary effects

Rats and guinea 

pigs

15 3.7 ppm for 90 days Focal chronic inflammation in heart and kidney 

tissue.

LOAEL = 3.7 ppm Coon et al. (1970)

SPF Wistar rats 

(male and 

female)

10 0, 1, 10, or 20 ppm 

6 hours/day, 5 days/week for 

13 weeks

Relative brain weight increased in female rats at 

20 ppm; increased AST, ALT, ALP in plasma at 

20 ppm.

NOAEL = 10 ppm Woutersen et al. 

(1987)
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1 4.2.1.2.3. Chronic inhalation bioassays. The respiratory pathology observed in chronic 

2 bioassays is consistent with the subchronic studies. As exposure concentration and duration of 

3 exposure are increased, the pathology becomes more severe and penetrates more deeply into the 

4 respiratory tract. These effects are progressive over time. Tumors are reported in several 

5 bioassays, primarily SCCs. Experimental results regarding both the severity of respiratory tract 

6 pathology as well as the tumor incidence vary by species strain and experimental design. As 

7 discussed above rodents experience RB, and species differences in respiratory and physiological 

8 depression would result in differences in absorbed dose in the respiratory tract, given the same 

9 exposure concentration (Chang and Barrow, 1983). Additionally, differences in the nasal 

10 architecture result in species-dependent variation of formaldehyde absorption (flux) within the 

11 respiratory tract (see Section 3.4). Therefore, chronic studies are discussed by species for greater 

12 clarity. 

13

4.2.1.2.3.1. Mice. 

25 In a comprehensive study conducted by Swenberg et al. (1980) (also reported in Kerns et 

26 al. [1983]) in conjunction with Chemical Industry Institute of Toxicology (CIIT) and Battelle 

27 Columbus Laboratories, male and female C57BL/6 × C3H F1 (B6C3F1) mice (approximately 

120/sex/concentration) were exposed to 0, 2.0, 5.6, or 14.3 ppm (0, 2.45, 6.87, or 17.5 mg/m3 28 ) 

29 formaldehyde 6 hours/day, 5 days/week for 24 months. This exposure period was followed by 

30 up to 6 months of nonexposure to evaluate recovery. Interim sacrifices were conducted at 6, 12, 

31 18, 24, 27, and 30 months (due to unscheduled deaths, no male mice were sacrificed at 18 or 

32 27 months). Exposure generation was accomplished by sublimation of paraformaldehyde, and 

33 exposures were conducted in whole-body chambers. Detailed sectioning and examination of the 

34 nasal passages were conducted at each interim sacrifice, beginning at 12 months, and for all 

35 unscheduled deaths. Gross organ pathology was noted for all animals and complete 

14 Early experiments by Horton et al. (1963) subjected mice (C3H, sex 

15 unspecified) to extreme formaldehyde concentrations (0, 0.05, 0.1, and 0.2 mg/L or 41–163 ppm) 

16 in an attempt to simulate lung pathology reported in humans exposed to cigarette smoke. The 

17 mice were exposed 1 hour/day, 3 days a week for up to 35 weeks. The authors did not note the 

18 effects of RB or provide any information on pathology of the URT. There was a clear increase 

19 in histologic changes in the tracheobronchial epithelium by exposure, including basal-cell 

20 hyperplasia, stratification squamous cell metaplasia and atypical metaplasia. Subsequent 

21 exposures to various combinations of formaldehyde and coal tar did result in squamous cell 

22 tumors. The findings of Horton et al. (1963) suggest a role for formaldehyde in lung cancer 

23 under some conditions. However, the exposure design and early deaths in the treatment groups 

24 severely limit the usefulness of these data in human health risk assessment.
This document is a draft for review purposes only and does not constitute Agency policy.

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1 histopathologic examination was conducted on all animals in the control and high-exposure 

2 groups. There were no differences in survival in any exposure group compared with controls. 

3 Generally, poor survival in all groups of male mice was attributed to fighting and infections of 

4 the urogenital tract associated with group housing; 78, 77, 81, and 82 unscheduled deaths were 

5 recorded before 24 months in the 0, 2.0, 5.6, and 14.3 ppm treatment groups, respectively ( 

6 n = 119, 120, 120, and 119 males, respectively). After the interim sacrifices (6 and 12 months) 

7 only 17–22 male mice survived to the 24-month scheduled sacrifice. Female mice had much 

8 greater survival with only 30, 34, 19, and 34 unscheduled deaths prior to the 24-month sacrifice. 

9 The authors did not note the effects of RB in mice, although the RD50 for a 10-minute exposure 

10 for male B6C3F1 mice has been reported at 4.9 ppm and 4.4 ppm (Steinhagen and Barrow, 1984; 

11 Chang et al., 1981). 

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

13 sacrifice. Inflammation in the nasal turbinates was evident in mice in the 2 and 6 ppm treatment 

14 groups (14/20 and 18/20, respectively), including adenitis of the nasal lacrimal duct, lacrimal 

15 duct, and vomeronasal gland. Inflammation was not present in mice exposed at 15 ppm, 

16 although serous rhinitis was seen in 4 of 20 animals. At 18 months, mice exposed at 2 and 

17 6 ppm no longer exhibited adenitis in the nasoturbinates. Epithelial dysplasia was evident in 4 of 

18 20 mice at 6 ppm exposure. Mice in the high-exposure group had significantly greater nasal 

19 pathology; epithelial dysplasia and squamous metaplasia were reported in 18/19 and 17/19 

20 female mice, respectively, exposed to 15 ppm. After 24 months, squamous epithelial hyperplasia 

21 of the nasolacrimal duct (29/45) and atrophy of the olfactory epithelium (18/45) were also noted 

22 in animals from the high-exposure group (male and female) (Battelle Columbus Laboratories, 

23 1981). Similar pathology was reported in only a small fraction of mice exposed at 2 and 6 ppm 

24 (5/48 and 11/60, respectively). 

25 Three months after cessation of exposure, only nine female mice were available for 

26 sacrifice, but within this small sample the data suggested recovery of nasal lesions: epithelial 

27 dysplasia (4/9), squamous metaplasia (2/9), atrophy of the olfactory epithelium (1/9), and 

28 squamous epithelial hyperplasia of the nasolacrimal duct (1/9) (Battelle Columbus Laboratories, 

29 1981).

30 Of the 17 male mice that survived to 24 months in the 14.3 ppm exposure group, 2 had 

31 SCC in the nasal cavity (p < 0.05). Of the two tumor-bearing mice, one exhibited significant 

32 epithelial pathology, including rhinitis, dysplasia, squamous metaplasia, and hyperplasia. 

33 Squamous metaplasia of the nasolacrimal duct was the only related pathology reported for the 

34 second mouse. No SCCs were found in female mice, although 48 mice survived to 24 months. 

35 The authors reported no other formaldehyde-related tumors. However, comparisons were based 
This document is a draft for review purposes only and does not constitute Agency policy.

4-166 DRAFT—DO NOT CITE OR QUOTE

1 on summary tables by organ. Although lymphomas were analyzed by organ and site (e.g., 

2 increase in salivary gland lymphoma considered separately from mandibular lymphoma), later 

3 reanalysis of lymphoma in female mice, based on tumor-bearing animals (TBAs), does indicate 

4 an association with formaldehyde exposure. 

5

4.2.1.2.3.2. Rats. 

15 There were no differences in mortality among the groups at any time during the study 

16 period. Rats exposed to formaldehyde were reported to have exhibited yellow discoloration of 

17 the fur, and many displayed eye irritation. Formaldehyde exposure, with and without wood dust, 

18 induced squamous metaplasia, keratinization, and dysplasia of the nasal epithelium (Table 4-29).

6 Holmström et al. (1989a) evaluated co-exposure of inhaled formaldehyde 

7 with wood dust in 16 female Sprague-Dawley rats/group. Rats were exposed in whole-body 

8 chambers for 6 hours/day, 5 days/week for 104 weeks to formaldehyde alone at 12.4 ± 1.1 ppm 

(15.21 ± 1.35 mg/m3), wood dust alone (25 mg/m3), or both wood dust (25 mg/m3 9 ) and 

10 formaldehyde (12.7 ± 1.0 ppm) or to room air as the control. The wood dust was generated from 

11 grinding of beech. Microscopic measurements of the wood particles indicated that 

12 approximately 70% had a geometric diameter of about 10 μm, while 10–20% were about 5 μm or 

13 less. Animals were sacrificed at 104 weeks and histopathology was performed on five transverse 

14 sections of the nasal cavity (Figure 4-3) and the lungs (not otherwise specified). 

19 

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

21 maxilloturbinates of female Sprague-Dawley rats exposed to inhaled 

22 formaldehyde and wood dust for 104 weeks

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 ± 1.73a

Formaldehydewood dust group

(n = 15) 7 1 4 12 0 2.6 ± 1.88a

Wood dust group

(n = 15) 0 0 0 0 0 1.86 ± 0.83b

Control group

(n = 15) 0 0 0 0 0 1.07 ± 0.70

24 a 25 p < 0.01. b 26 p < 0.05.

27

28 Source: Holmström et al. (1989a).
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1 Among the five levels of the nasal cavity that were examined, Holmström et al. (1989a) 

2 presented findings for the naso- and maxilloturbinates since formaldehyde-induced tumors had 

3 been associated with this level (Morgan et al., 1986a, b). The data also suggested an effect of 

4 wood dust on formaldehyde-induced nasal pathology, with a slightly higher histologic score and 

5 greater incidence of dysplasia than formaldehyde exposure alone. One SCC (1/16) occurred in 

6 the group exposed to formaldehyde only but not in the group exposed to formaldehyde and wood 

7 dust. Microscopic examination of the lungs revealed that emphysema (diagnostic criteria not 

8 specified) was more prevalent in both groups exposed to wood dust compared with the control 

9 group (p < 0.05). There was no significant difference in pulmonary epithelial histopathology 

10 among the groups.

11 Tobe et al. (1985) also evaluated F344 rats (32/group) exposed to inhaled formaldehyde 

12 for 28 months. Exposures were for 6 hours/day, 5 days/week to formaldehyde concentrations of 

0, 0.3, 2, and 14 ppm (0, 0.37, 2.45, and 17.2 mg/m3 13 ). Fourteen of 32 rats (44%) in the high 

14 concentration group developed nasal SCCs, compared with none in the other exposed groups and 

15 the control group. Tobe et al. (1985) reported increased rhinitis, hyperplasia, and squamous 

16 metaplasia of the nasal respiratory epithelium, including in the low-exposure group (0.3 ppm.) 

17 However, some level of rhinitis, hyperplasia, and metaplasia were also present in controls. 

18 Without a more complete report, it is unknown whether or not the pathology reported at 0.3 ppm 

19 was a formaldehyde-related effect.

20 Kamata et al. (1997) evaluated the effects of inhaled formaldehyde in male F344 

21 (F344/DuCrj) rats (32/group) exposed for 28 months. Formaldehyde exposure was generated by 

22 metering 37% formalin (containing 10% methanol) into a sprayer in a glass bottle and diluting 

23 with room air. Concentration in the chamber was monitored twice daily by the acetyl acetone 

24 method. Exposures were for 6 hours/day, 5 days/week at nominal formaldehyde concentrations 

of 0, 0.3, 2.0, and 15 ppm (0, 0.37, 2.45, and 18.4 mg/m3 25 ). Actual levels were 0, 0.3 ± 0.07, 2.17 

26 ± 0.32, and 14.85 ± 2.22 ppm (mean ± SD). Rats in the 0 ppm group were given methanol to 

27 inhale at the same concentration (4.2 ppm) as the 15 ppm group. A room control no-exposure 

28 group was also included in the study. All animals were observed for clinical signs once a day 

29 during the study. Body weights and food consumption were recorded weekly. Five animals per 

30 group, randomly selected at the end of 12, 18, and 24 months, and all surviving animals at 

31 28 months were sacrificed for hematological measurements (Hb, RBCs, PCV, MCV, mean 

32 corpuscular hemoglobin [MCH], MCHC, and WBCs), biochemical determinations (TP, ALB, 

33 BUN, ALP, AST, ALT, glucose, albumin/globulin ratio, phospholipids, triglycerides, and total 

34 cholesterol), and pathological examinations. Wet weights were taken on brain, heart, lungs, 

35 liver, kidneys, spleen, testes, and adrenal gland of each rat. Histopathology was performed on all 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 moribund or dead animals and those at specified sacrifices on all gross lesions and the following 

2 tissues: pituitary, thyroid, nasal cavity, trachea, esophagus, stomach, small and large intestines, 

3 prostate gland, urinary bladder, muscle, femur, sciatic nerve, spinal cord, and mesenteric lymph 

4 nodes. Histopathologic sections of the nose were obtained from five anatomical levels, but these 

5 did not correspond to the typical levels taken in other bioassays. Most notably, section level B 

6 was anterior and not posterior to the incisor teeth. The incidence data for nasal histopathology 

7 were not reported with respect to section level location, with the exception that the 

8 nonproliferative lesions and tumors reported were described to occur predominantly at levels B 

9 and C.

10 Yellow discoloration of the coats occurred in animals exposed at the 2 and 15 ppm levels. 

11 Significant decreases in body weight and food consumption were observed in the high 

12 concentration (15 ppm) group throughout the exposure period, and elevated mortality was noted 

13 at 28 months (88.3 versus 31.8% in controls). The first death occurred after 6 versus 18 months 

14 in the control group. Other effects noted in the 15 ppm exposure group include decreased 

15 triglycerides, reduced liver weight (both relative and absolute), and increased relative adrenal 

16 weights. 

17 Treatment-related macroscopic and histopathologic findings were limited to the nasal 

18 cavity. Squamous cell metaplasia was reported in all treatment groups: 16% (0.3 ppm), 37.5% 

19 (2 ppm), and 91% (15 ppm) of exposed rats. Epithelial hyperplasia was similarly present in 12.5, 

20 22, and 91% of the animals, respectively. Since a no-effect level could not be determined, the 

21 authors reported benchmark doses (BMDs) of 0.25 and 0.24 ppm for squamous cell metaplasia 

22 and epithelial hyperplasia (10% response.). Additional lesions only occurring in the 15 ppm 

23 dose group were papillary hyperplasia (2/32), SCC (13/32), squamous cell papilloma (3/32), and 

24 sarcoma (1/32). The majority of the tumors were located at levels B and C of the nasal cavity.

25 Albert et al. (1982) and Sellakumar et al. (1985) reported on a set of lifetime studies 

26 performed in male Sprague-Dawley rats to evaluate the effects of inhaled formaldehyde alone 

27 and in combination with hydrochloric acid (HCl). Rats were exposed 6 hours/day, 5 days/week 

28 for life. In the first experiment (Albert et al., 1982), 8-week-old male inbred Sprague-Dawley 

rats (n = 99) were exposed to a mixture of 10 ppm (12.3 mg/m3

) HCl and 14 ppm (17.2 mg/m3 29 ) 

30 formaldehyde, and there were two control groups: air-sham and untreated (n = 50). 

31 Bis(chloromethyl)ether (BCME), a known animal carcinogen (Albert et al., 1975; Kuschner et 

32 al., 1975; Figueroa et al., 1973; Laskin et al., 1971), is formed when formaldehyde and HCl are 

33 mixed. BCME concentrations were estimated at about 1 ppb in the formaldehyde-HCl mixed 

34 exposures, based on levels in the mixing chamber. Complete necropsies were conducted when 

35 animals died naturally or were killed when moribund. Histologic sections were taken from the 
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1 nasal cavity, larynx, trachea, pulmonary lobes, liver, bladder, kidney, spleen, and other organs 

2 with gross pathologic alterations.

3 Exposure to the mixed gases (formaldehyde-HCl-BCME) induced nasal lesions, 

4 including epithelial hyperplasia (71%), squamous metaplasia (64%), squamous papilloma (3%), 

5 and SCC (25%) (Albert et al., 1982). Although a few squamous metaplasia were noted in the 

6 larynx, trachea, and bronchi, these lesions were also noted in controls. Mortality in exposed rats 

7 was significantly increased over controls and was approximately 30% when the first carcinoma 

8 was reported (233 days). Mortality in exposed rats rose quickly to approximately 60% after the 

9 first year of exposure. Therefore, the authors used a life-table method to calculate a mortality10 corrected cumulative incidence, reporting a corrected cumulative incidence of 77% at 720 days 

11 after first exposure. 

12 In the second experiment performed in this laboratory (Sellakumar et al., 1985; Albert et 

13 al., 1982), Sprague-Dawley rats were similarly exposed to HCl (10 ppm) alone, formaldehyde 

14 alone (15 ppm), or a combination of both. The combination exposure was generated in two 

15 different ways to better understand the influence of BCME formation on study results: premixed 

16 at high concentrations and gases fed separately into the inlet air supply at the target 

17 concentrations. BCME concentration measured by a gas chromatography/mass spectrometry 

18 method in the premixed chamber varied between 0.1 and 0.4 ppb. Cage-side observations and 

19 necropsy procedures were as described in Albert et al. (1982) with the exception of the histologic 

20 preparation of the head. The head was cut transversely into four tissue blocks, and sections were 

21 taken from the face of each.

22 Animals exposed to formaldehyde alone and formaldehyde-HCl (premixed or non23 premixed) showed a marked decrease in body weight after 16 weeks. After 32 weeks rats 

24 exposed to the premixed formaldehyde-HCl (with BCME) had higher mortality compared with 

25 the other mixed gas exposures (p < 0.05). Nasal pathology was similar among rats exposed to 

26 formaldehyde alone or the mixed gases (Table 4-30). Desquamation of respiratory epithelial 

27 cells was reported in the respiratory epithelium that covers the nasomaxillary turbinates and the 

28 nasal septum (approximately section levels 2 and 3). Olfactory epithelium in the ET frequently 

29 showed an inflammatory reaction with seropurulent exudate filling the lumen. Squamous 

30 metaplasia and hyperplasia were reported in the larynx and trachea in all treatment groups.

31 Tumors arose primarily from the nasomaxillary turbinates and nasal septum. The SCCs 

32 were predominantly moderate to well differentiated, with excessive amounts of keratin occluding 

33 the lumen, killing the animals by asphyxiation. Statistical comparisons by the log rank test (Peto 

34 test) showed that tumor incidence was increased in the premixed formaldehyde-HCl combined 

35 exposure group over formaldehyde alone or the combined formaldehyde-HCl (not premixed). 
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1 There were no significant differences in the latency among groups, with the average latency

2 varying from 603 to 645 days. Rats exposed to HCl exposure alone did not develop tumors.

3 The esthesioneuroepithelioma is a unique tumor type observed with a high incidence in 

4 an earlier inhalation study of rats exposed to BCME (Kuschner et al., 1975), suggesting that the 

5 higher incidence of nasal tumors observed in the premixed-combined formaldehyde-HCl6 exposure group may have been due to BCME (Krimsky, 1986) since this premixed protocol was 

7 the one most likely to generate BCME. Sellakumar et al. (1985) refuted this assertion, stating 

8 that this singular tumor occurred in the absence of other changes in the ethmoid region or in the 

9 lungs where BCME was also demonstrated to cause tumors. Furthermore, exposure was 

10 approximately one-tenth the cumulative dose in the Kuschner et al. (1975) study that was 

11 associated with a single similar tumor. Sellakumar et al. (1985) attributed the higher incidence 

12 in the premixed-combination group to traces of other alkylating agents (not BCME) that could 

13 have been formed. The results demonstrate that animals exposed to either a combination of 

14 formaldehyde-HCl or to formaldehyde alone develop nasal tumors, principally SCCs, at about 

15 the same frequency, indicating that HCl plays little or no role in the carcinogenicity of inhaled 

16 formaldehyde.

17

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

19 male Sprague-Dawley rats exposed to inhaled formaldehyde or HCl alone 

20 and in combination for a lifetime

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 45

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

23 Source: Sellakumar et al. (1985).

24

25
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1 In a companion study to the chronic mouse study described above (Kerns et al., 1983; 

2 Swenberg et al., 1980), groups of F344 rats (approximately 120/sex/concentration) were exposed 

to 0, 2.0, 5.6, or 14.3 ppm (0, 2.45, 6.87, or 17.5 mg/m3 3 ) formaldehyde 6 hours/day, 5 days/week 

4 for 24 months. This exposure period was followed by up to 6 months of nonexposure to evaluate 

5 recovery. Interim sacrifices were conducted at 6, 12, 18, 24, 27, and 30 months. Study 

6 parameters and methods were as described above.

7 Formaldehyde exposure increased mortality of both male and female rats in all treatment 

8 groups (p < 0.05 for 6 and 15 ppm groups). Severe treatment-related mortality was seen at the 

9 highest exposure group beginning at 12 months with only 30% surviving to the 24 months. 

10 There were no alterations in clinical chemistry, neurofunctional, or ophthalmological 

11 measurements considered to be related to formaldehyde exposure. A concentration-dependent 

12 increase in yellow discoloration of the hair coat was observed. This discoloration dissipated over 

13 the 3-month postexposure period. Rats in the highest-concentration group were dyspneic 

14 (p < 0.01) and emaciated (p < 0.05) and had many facial swellings that on closer examination 

15 were revealed to be carcinomas protruding through the nasal cavity. Neoplastic lesions in the 

16 URT were first observed clinically at day 358 in females and day 432 in males. 

17 Macroscopically, these lesions originated in the anterior portion of the nasal cavity and, in a few 

18 instances, extended into the ETs.

19 Figure 4-9 shows the frequency of squamous metaplasia by location in the noses of rats 

20 sacrificed at various time points along the 2-year exposure period. Histopathologic lesions were 

21 confined to the nasal cavity and proximal trachea in concentration-dependent fashion. The 

22 morphologic diagnosis of squamous metaplasia was used to designate zones of altered 

23 epithelium that were characterized by the presence of a well-differentiated germinal layer 

24 (stratum germinativum) and superficial layers of epithelium (stratum spinosum and stratum 

25 corneum). Keratin was produced only in areas of squamous metaplasia. Epithelial dysplasia was 

26 detected earlier than squamous metaplasia and was characterized by a mucosa that had 

27 undergone a transition from nonciliated simple cuboidal to one that was several cells thick and 

28 squamoid with an organization and polarity of the individual cells that had changed from vertical 

29 to horizontal with respect to the basement membrane. Similar histomorphologic changes have 

30 also been called basal cell hyperplasia and epidermoid metaplasia (e.g., Albert et al. [1982]). 

31 Figure 4-9 clearly illustrates that concentration is a dominant determinant of lesion distribution. 

32 At low concentrations the lesions occur only in the most anterior region (cross-section level 1). 

33 At 5.6 ppm, the squamous metaplasia in levels 1, 2, and 3 was also associated with purulent 

34 rhinitis and epithelial dysplasia. At the highest concentration, the lesions progress to the more 

35 distal URT, with lesions evident in level 5 and no difference in the incidence at level 1 or 2 
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4-172 DRAFT—DO NOT CITE OR QUOTE

1 across the various sacrifice times. Statistically significant (p < 0.05) regression of the lesion was 

2 evident at most locations at the 27-month sacrifice (3 months postexposure) (e.g., level 1 in the 

3 2 ppm group, all levels of the 5.6 ppm group, and levels 4 and 5 of the 14.3 ppm group). 

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

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

7 metaplasia in the nasal cavity of F344 rats exposed to formaldehyde via 

8 inhalation.

9

10 Note: Exposure concentrations were 2.0 ppm (A), 5.6 ppm (B), or 14.3 ppm (C). 

11 Nasal cavity levels 2, 3, 4, and 5 were not evaluated at the 6- and 12-month 

12 interim sacrifices in the 14.3 ppm exposure group.

13

14 Source: Redrawn from Kerns et al. (1983).

15
This document is a draft for review purposes only and does not constitute Agency policy.

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1 Furthermore, progression of lesions distally to the lower respiratory tract (LRT) occurred 

2 only in the high concentration group. Tracheal pathology observed at 18 months included 

3 multifocal areas of minimal to mild epithelial hyperplasia, epithelial dysplasia, or squamous 

4 metaplasia. There were no significant tracheal lesions present in the 0, 2.0, or 5.6 ppm exposure 

5 groups, and tracheal lesions were not observed during the postexposure period in the 14.3 ppm 

6 exposure group. 

7 Table 4-31 provides the summary data of all neoplastic lesions in the nasal cavity of 

8 exposed rats. The adjusted cumulative incidence rates of SCC in male and female rats from the 

9 14.3 ppm exposure group at 24 months were 67 and 87%, respectively. In this group, the 

10 formation of zones of squamous metaplasia with zones of squamous epithelial hyperplasia and 

11 increased keratin production appeared to precede areas of squamous papillary hyperplasia with 

12 foci of cellular atypia. More advanced lesions included carcinoma in situ and invasive SCC of 

13 the nasal turbinates. The neoplasia were extremely osteolytic and were associated with excessive 

14 keratin production and mild to severe purulent rhinitis. In many animals from the high-exposure

15 group (with or without carcinoma), the excessive accumulation of keratin and inflammatory 

16 exudates within the lumen of the URT caused severe dyspnea and death. Polypoid adenomas 

17 were also observed in eight rats (four/sex) from the low-exposure group, six male rats from the 

18 intermediate-exposure group, and six rats (five males, one female) from the high-exposure group 

19 in level 1, 2, or 3. One control male rat had a similar lesion. When adjusted and unadjusted data 

20 were analyzed, no significant differences were observed in pair-wise analyses; however, a 

21 significant adjusted trend (p < 0.05) was reported for male rats. There was no evidence of 

22 progression from polypoid adenoma to SCC.

23

24 Table 4-31. Summary of neoplastic lesions in the nasal cavity of F344 rats 

25 exposed to inhaled formaldehyde for 2 years

26

Formaldehyde 

(ppm) Sex

No. of nasal

cavities

evaluated SCC

Nasal

carcinoma

Undifferentiated

carcinoma or

sarcoma

CarcinosarcomaPolypoid 

adenoma

Osteochondroma

0 M 118 0 0 0 0 1 1

F 114 0 0 0 0 0 0

2.0 M 118 0 0 0 0 4 0

F 118 0 0 0 0 4 0

5.6 M 119 1 0 0 0 6 0

F 116 1 0 0 0 0 0

14.3 M 117 51 1a 2a 1 4 0

F 115 52 1 0 0 1 0

27 a 28 One rat in this group also had an SCC.

29

30 Source: Kerns et al. (1983).

31
This document is a draft for review purposes only and does not constitute Agency policy.

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1 Morgan et al. (1986b) performed an additional analysis of the slides and tissues 

2 from the Kerns et al. (1983) study to more precisely determine the location of each tumor 

3 recorded. Additional sections were cut from the existing tissue blocks if a full slide set 

4 (i.e., five sections) was unavailable for each animal. For each animal, the location of 

5 each tumor was recorded on diagrams of the cross section of the nose, and an attempt to 

6 determine the site of origin was made based on the center of the tumor mass. The results 

7 for each case were assigned an accuracy rating that was based on the degree of 

8 confidence that the pathologist had in the designated site of origin. Results for SCCs are 

9 shown in Table 4-32.

10

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

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

13 bioassay

14

Sex

Accuracy

rating

Number of

animals 

Total SCC (%)a

Area 1b Area 2b Area 3b Area 4b

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 16 Rounded to nearest whole number.

b 17 Area 1 = lateral aspect of the nasoturbinate and adjacent lateral wall; Area 2 = midventral septum; Area 3 = dorsal 

18 septum and roof of dorsal meatus; Area 4 = dorsal and lateral aspect of the maxilloturbinate.

19 NA = not applicable.

20

21 Source: Morgan et al. (1986b).

22

23

24 In the 14.3 ppm exposure group, 98/103 rat noses had adequate numbers and quality of 

25 slides for mapping the SCC distribution. Single neoplasia were present in 80 (40/sex), while 

26 multiple neoplasia were present in 9 males (21 neoplasia) and 9 females (20 neoplasia). The 

27 results were similar for cases with high or low accuracy. For example, more than half (57%) of 

28 the SCCs occurred on the lateral side of the nasoturbinate and adjacent lateral wall at the front of 

29 the nose (levels 1 and 2); approximately 25% were located on the midventral nasal septum 

30 (levels 2 and 3); and about 10% were on the dorsal septum and roof of the dorsal meatus 

31 (levels 1, 2, and 3). A small number (3%) were found on the maxilloturbinate (levels 2 and 3), 

32 which only involved the medial aspect. All other regions of the nose where SCC was found were 

33 considered to be involved as a result of invasion from one or more of the above sites. There 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 were two tumors in the 5.6 ppm group: one male had a single neoplasm on the ventral nasal 

2 septum (level 3) while a female had an SCC from the lateral aspect of the maxilloturbinate to the 

3 adjacent lateral wall (level 2). 

4 On the basis of the morphology of 19 small neoplasia in this study and in additional work 

5 described below (Morgan, 1997; Monticello et al., 1996), it was further concluded that the SCCs 

6 arose from the epithelium lining the airway and not from the underlying glandular epithelium. 

7 This mapping procedure and that of Monticello et al. (1996) described below were in good 

8 concordance and showed a clear site specificity; most of the SCC arose in the anterior lateral 

9 meatus (ALM) (57%), which is lined by transitional epithelium, and the midventral nasal septum 

10 (26%), which is lined by respiratory epithelium (Morgan, 1997). 

11 The CIIT performed a second bioassay on inhaled formaldehyde in 9-week-old male 

12 F344 (CDF[F344]/CrlBr) rats (Monticello et al., 1996). The rats were exposed 6 hours/day, 5 

13 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 

18.4 mg/m3 14 ) formaldehyde. Study objectives were to repeat the Kerns et al. (1983) bioassay, 

15 better defining the concentration response relationship and to seek a correlation between 

16 localized data on tumor sites and concomitant cell proliferation assays. Histopathology was 

17 performed on six cross-section levels of the nasal cavity on every animal of an unscheduled 

18 death and all those of the terminal sacrifice after 24 months. The distribution of lesions for each 

19 individual animal was recorded onto epithelial maps of the nasal cavity at 30 selected levels 

20 designed to permit accurate localization (Mery et al., 1994). Cell proliferation was measured in a 

21 subset of animals (five per treatment group) at 3, 6, 18, and 24 months of exposure in each of the 

22 nasal regions to which tumors were mapped (Table 4-33).

23

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

25 F344 rats exposed to inhaled formaldehyde for 2 years

26

Formaldehyde

concentration

(ppm)

No. of 

nasal

cavities

examined

Nasal location

No. of

animals

with 

SCCa

Anterior

lateral

meatus

Posterior

lateral

meatus

Anterior

midseptum

Posterior

midseptum

Anterior

dorsal

septum

Anterior

medial

maxilloturbinateMaxillary

sinus

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 28 Total number of animals with SCCs, including those too large to allocate and those located in a site not listed in this 

29 table.

30 Source: Monticello et al. (1996).
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1 Yellow discoloration of the fur, a consistent response to formaldehyde in rats, was 

2 observed in the rats exposed to 10 and 15 ppm formaldehyde. There were numerous premature 

3 deaths in the 15 ppm exposure group, resulting in significantly decreased survival relative to 

4 controls (18.8 versus 35.7%; p < 0.001). Survival was higher in the three lowest exposure 

5 groups and statistically comparable to controls in the 10 ppm exposure group (35.7 versus 

6 31.3%, respectively). 

7 Control animals showed no histopathologic evidence of disease in the nasal passages. 

8 Buccal cavity SCC, not associated with the nasal cavity, was present in 2 of 90 control animals. 

9 This was considered an incidental finding and within the spontaneous incidence range reported 

10 for this strain of rat. Buccal SCCs were observed in three animals at 15 ppm and in one animal 

11 at 2 ppm. All other neoplastic responses in the respiratory tract were confined to the nose and 

12 considered to have originated from the epithelium lining the nasal airways. The nasal neoplasia 

13 included SCCs and polypoid (transitional) adenomas and were similar in morphologic 

14 characteristics to those described in the Kerns et al. (1983) chronic bioassay. The incidence of 

15 nasal SCCs by location is summarized in Table 4-33, which demonstrates a clear concentration16 response relationship. No SCCs occurred in the two lowest exposure groups or in the controls. 

17 One nasal rhabdomyosarcoma and two nasal adenocarcinomas were reported in animals in the 

18 highest treatment groups. 

19 Regional analysis indicated that the SCCs arose in nasal regions lined with transitional or 

20 respiratory epithelium and were most common in the lateral meatus and the midseptum (Table 

21 4-33). Within the lateral meatus and mid-septum, there was clear evidence of a higher tumor 

22 incidence rate in the anterior sample site (p = 0.001 and 0.02, respectively). Smaller numbers of 

23 SCCs were observed on the medial aspect of the maxilloturbinate and the dorsal septum and on 

24 the posterior lateral wall and lining of the nasopharyngeal meatus (data not shown). No SCCs 

25 were observed in the maxillary sinus, with the exception of one animal exposed to 15 ppm that 

26 had a small tumor in the wall of the ostium of this sinus. Tumor rates across the seven nasal 

27 epithelial sites are presented in Table 4-33. There was an increasing tumor response between the 

28 10 and 15 ppm exposure groups in all sites, except in the ALM. The SCC rates at 10 and 15 ppm 

29 were virtually identical (13.3 and 11.6%, respectively), which is probably attributable to the 

30 occurrence of many large neoplasia in the lateral meatus site that were not suitable and not 

31 counted in the analysis.

32 The nonlinear tumor response is mirrored by a highly nonlinear response in cell 

33 proliferation measured after 3, 6, 12, and 18 months of exposure. Significant treatment-induced 

34 responses in cell proliferation indices at these time points were only observed at the two highest 

35 exposure concentrations (10 and 15 ppm). Other treatment-induced lesions, predominantly 
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1 epithelial hypertrophy, hyperplasia, squamous metaplasia, and mixed inflammatory cell 

2 infiltrate, were also most severe at these two exposure concentrations. Significant distortion and 

3 destruction of the nasoturbinate architecture occurred in many animals exposed to 15 ppm. 

4 Nasal turbinate adhesions and olfactory degeneration (usually confined to the walls of the 

5 anterior dorsal medial meatus) also occurred in animals exposed to 10 and 15 ppm. Lesions in 

6 the 6 ppm exposure group were limited to focal squamous metaplasia in the anterior regions.

7 As discussed briefly above, small numbers of polypoid adenomas were also induced by 

8 formaldehyde exposure and were similar in acinar-like structure and location to those in the 

9 Kerns et al. (1983) bioassay. No polypoid adenomas occurred in the control animals or in the 

10 0.7, 2, or 6 ppm exposure groups. A clear concentration response was observed in the 10 and 

11 15 ppm exposure groups. Five of 90 animals (5.6%) in the 10 ppm exposure group and 14 of 

12 147 animals (9.5%) in the 15 ppm exposure group had a polypoid adenoma. Most of these 

13 polypoid adenomas (79%) were located in or adjacent to the lateral meatus. The significance of 

14 these tumors for risk assessment remains to be determined (Morgan, 1997).

15 Appelman et al. (1988) studied the effects of bilateral intranasal electrocoagulation 

16 damage on susceptibility to inhaled formaldehyde in male SPF Wistar (Cpb: WU) rats. Rats 

17 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, 

1.23, or 12.3 mg/m3 18 ) formaldehyde. These concentrations were chosen because the various 

19 short-term studies performed in the same laboratory (described in Section 4.2.1.2) showed that 

20 formaldehyde was noncytotoxic to the nasal mucosa at levels of 0.3, 1.0, and 2.0 ppm, slightly 

21 cytotoxic at 3 and 4 ppm, and strongly cytotoxic at 10 and 20 ppm (Zwart et al., 1988; Wilmer et 

22 al., 1987; Woutersen et al., 1987). Furthermore, because nasal tumors have only been found at 

23 exposure concentrations that also induced severe degenerative, hyperplastic, and metaplastic 

24 changes in the nasal epithelium (Griesemer et al., 1985; Squire and Cameron, 1984), Feron et al. 

25 (1984) and the investigators at the TNO-CIVO Toxicology and Nutrition Institute postulated that 

26 formaldehyde at a subcytotoxic concentration was only a very weak initiator without promoting 

27 activity. Appelman et al. (1988) used an electrocoagulation method in this study to evaluate if 

28 damage to the mucosa followed by compensatory cell proliferation might render the epithelium 

29 vulnerable to subcytotoxic levels of formaldehyde. One-half of the rats used in the study 

30 (10/group) were damaged bilaterally and then subjected to the first 6-hour exposure to 

31 formaldehyde approximately 20–26 hours after the electrocoagulation procedure. Ten 

32 undamaged rats/group were also exposed at each concentration for either 13 or 52 weeks. 

33 Histopathologic examination included six standard cross-section levels in the nose; livers of all 

34 rats killed at 14 weeks and of all control and 10 ppm exposed rats killed in week 53; larynges, 

35 tracheas, and lungs of all rats of the control and 10 ppm exposed rats killed in week 53; and 
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1 organs and tissues of control and 10 ppm exposed rats with an undamaged nasal mucosa killed in 

2 week 53.

3 Yellow discoloration of the fur occurred in all animals of the two highest exposure 

4 groups. Growth retardation was observed in the animals killed with or without damaged noses 

5 after 2 weeks of exposure to 10 ppm formaldehyde. No toxicologically significant findings in 

6 the body weights or organ weights of any animals in the other exposure groups were observed. 

7 No relevant differences between groups were found in any of the hematological or urinary 

8 parameters with the exception of frequent oliguria (p < 0.05) in the top exposure group without 

9 nasal coagulation and killed in week 53. Three-way ANOVA revealed a significant increase in 

10 TP content of the liver in rats with damaged noses as compared with rats with undamaged noses, 

11 and there was a significant negative correlation between the formaldehyde exposure level and TP 

12 in these same rats. Hepatic GSH was positively correlated with both nasal damage and age of 

13 the animals. No treatment-related gross findings were observed in animals sacrificed at either 14 

14 or 53 weeks except for yellow discoloration of the fur in rats exposed at the two highest 

15 concentrations. No changes observed in the larynx, trachea, lungs, liver, or other tissues 

16 evaluated were regarded as related to formaldehyde.

17 Few nasal lesions were noted in intact rats exposed at 0.1 or 1 ppm for either 13 or 52 

18 weeks (n = 10/group). Focal squamous metaplasia was noted in a single animal exposed at 

19 1 ppm for 13 weeks. Rats exposed at 10 ppm formaldehyde demonstrated clear pathology in the 

20 respiratory epithelium progressing from 13 to 52 weeks, including squamous metaplasia, basal 

21 cell hyperplasia, and focal rhinitis. Additionally, focal nest-like infolds of the epithelium were 

22 present in 4 of 10 rats at 52 weeks, and minor changes to the olfactory epithelium were noted 

23 (thinning/disarrangement and focal basal cell hyperplasia.) 

24 All rats with damaged nasal passages exhibited similar minor pathology of the respiratory 

25 epithelium at 13 and 52 weeks (squamous metaplasia, focal basal cell hyperplasia, and focal 

26 rhinitis). Formaldehyde-related effects were note at 52 weeks, where the squamous metaplasia 

27 of the respiratory epithelium was no longer noted in controls (versus 13 weeks) but was clearly 

28 present in all formaldehyde-treatment groups, including progression from focal to diffuse lesions 

29 (at 1 and 10 ppm) and keratinization (3/10 and 4/10 at 0.1 ppm and 10 ppm, respectively). The 

30 formaldehyde effects on the respiratory epithelium were much more severe in rats with damaged 

31 nasal passages, with all animals demonstrating thinning and disarrangement of the olfactory 

32 epithelium and 8 of 10 rats exhibiting “loosely arranged submucosal tissue.” Squamous 

33 metaplasia and focal rhinitis of the olfactory epithelium were seen in less than half of the 

34 formaldehyde-treated rats with damage. No changes in the olfactory epithelium due only to 

35 electrocoagulation were encountered.
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1 The most notable effects of nasal damage from electrocoagulation were the ones at the 

2 highest formaldehyde exposure (10 ppm) on the olfactory epithelium. Damage to the respiratory 

3 epithelium also occurred more posteriorly in rats with damaged noses. Since electrocoagulation 

4 often induced damage that included partial or complete loss of turbinates and septal perforation, 

5 a likely explanation for the posterior distribution of the damage is an abnormal airflow pattern. 

6 This gross damage to the nasal structure may have also disrupted normal mucous production and 

7 flow. Therefore, formaldehyde-induced pathology appearing deeper in the nasal passages, 

8 including the respiratory epithelium, may be due to formaldehyde penetrating more deeply into 

9 the nasal passages and resulting in greater tissue doses in these areas.

10 Woutersen et al. (1989) conducted a lifetime study in parallel to the 1-year study 

11 described above for Appelman et al. (1988). Male Wistar rats with nasal damage induced by 

12 electrocoagulation (60/group) or without nasal damage (30/group) were exposed 6 hours/day, 

13 5 days/week to the same concentrations as in the previous study (0, 0.1, 1.0, and 10 ppm) for 

14 28 months or for 3 months followed by a 25-month observation period. The general condition 

15 and behavior of the animals were checked daily. Body weight, organ weight, and gross 

16 pathology were evaluated as described for Appelman et al. (1988). Histopathologic examination 

17 was conducted on all animals at the standard six cross sections (see Figure 4-3).

18 No remarkable findings on behavior were observed except for yellowing of the fur in 

19 animals at the two highest concentrations. There were no relevant differences in mortality (data 

20 not shown). Growth retardation was observed relative to controls in animals with or without 

21 damaged noses exposed to 10 ppm from day 14 onward. Body weights were generally slightly 

22 lower in formaldehyde-exposed animals with an intact nasal mucosa and slightly higher in 

23 exposed animals with damaged noses than in the corresponding controls.

24 The effects of formaldehyde exposure on the respiratory and olfactory epithelium after 

25 28 months of exposure were similar to those reported for 52 weeks exposure (Appelman et al., 

26 1988): rhinitis, squamous metaplasia with some keratinization of the respiratory epithelium, and 

27 thinning/disarrangement and slight squamous metaplasia of the olfactory epithelium at the 

28 10 ppm exposure. Effects attenuated from the anterior to posterior sections (I–II, III, IV, and V–

29 VI). A low incidence of olfactory epithelium replaced by respiratory epithelium (<10%) and 

30 vacuolation and atrophy of olfactory cells (<10%) was reported, this in part may be due to the 

31 larger study size (30 rats per group versus 10). Squamous metaplasia in levels I–II of the 

32 respiratory epithelium at 10 ppm was the only treatment-related pathology remaining in rats 

33 exposed for 3 months followed by a 25-month recovery period.

34 Similarly, as reported by Appelman et al. (1988), rats with noses damaged by 

35 electrocoagulation did demonstrate increased pathology of the respiratory epithelium. 
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1 Formaldehyde exposure at 10 ppm exacerbated these changes, and effects were noted in more 

2 posterior sections than in rats without nasal damage (levels III, IV, and V). Olfactory pathology 

3 was also greater in formaldehyde-treated rats: basal cell hyperplasia, replacement of olfactory 

4 epithelium by respiratory epithelium (10–20% at level III and <10% at level IV). Although the 

5 incidences are low, there is some evidence that effects on the olfactory epithelium may be 

6 increased at the lower formaldehyde exposures (0.1 and 1 ppm.) Analysis of the number of 

7 animals with olfactory pathology would be helpful to better understand the potential of low-level 

8 formaldehyde effects on these less frequent lesions. Interestingly, the recovery of the olfactory 

9 and respiratory epithelium seen in rats with undamaged nasal cavities after a 25-month recovery 

10 period was not evident in rats with damaged noses. Formaldehyde-exposure effects are only 

11 present at the 10 ppm exposure for the respiratory epithelium (squamous metaplasia, basal cell 

12 hyperplasia), and the formaldehyde-related effects on the olfactory epithelium 

13 (thinning/disarrangement, basal cell hyperplasia, and replacement by respiratory epithelium) are 

14 seen at 0.1 and 1.0 ppm as well.

15 A single SCC, 1 out of 30 rats, was found in each 28-month formaldehyde-treatment 

16 group (1/26, 1/28, and 1/26, respectively) but not in any control animals (n = 52). SCCs were 

17 also noted in rats with noses damaged by electrocoagulation (1/54, 1/58, 0/56, and 15/58 for 

18 control rats and the formaldehyde-treatment groups, respectively). These data clearly indicate a 

19 synergistic effect of high formaldehyde exposure and nasal damage on the formation of SCCs in 

20 rats. One adenosquamous carcinoma and one adenocarcinoma were also reported as increasing 

21 the frequency to 17/58 for all tumors. Additionally SCC was present in two rats in the 0.1 and 

22 1 ppm 3-month exposure groups with damaged noses only, although only one SCC was reported 

23 in the 10 ppm 3-month groups with and without damaged noses. Rats not surviving to 

24 28 months are included in these results, as well as the histopathology reported above. Since no 

25 mortality data are reported, it should be noted that the incidence of both nasal lesions and tumors 

26 are not controlled for early deaths.

27 In total, 30 tumors were examined from this study. In general, the tumors (26/30 or 87%) 

28 were SCCs, and 69% (18/26) of these clearly originated from the respiratory epithelium lining 

29 the septum or nasal turbinates. The eight other SCCs, derived from the epithelium lining the 

30 nasolacrimal duct, were seen in connection with severe odontodystrophy and periodontitis or 

31 might have originated from the skin or salivary glands. Four remaining rats bearing a nasal 

32 tumor developed a small polypoid adenoma located on the nasoturbinate, an adenocarcinoma 

33 originating from the olfactory epithelium, an adenosquamous carcinoma of the respiratory 

34 epithelium lining the septum or turbinates, or a carcinoma in situ of epithelium in the 

35 nasolacrimal duct. 
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4.2.1.2.3.3. Hamsters

12 The second set of experiments by Dalbey (1982) examined interaction of formaldehyde 

13 exposure on tumor formation from DEN administered subcutaneously. The five treatment 

14 groups included: (1) controls (n = 50); (2) formaldehyde only (n = 50); (3) DEN 0.5 mg, once 

15 per week for 10 weeks (n = 100); (4) formaldehyde exposure for life with DEN injection for the 

16 first 10 weeks given 48 hours after formaldehyde exposure (n = 27); and (5) DEN injection for 

17 10 weeks, followed by formaldehyde exposure for life (n = 23). In all groups hamsters were 

18 exposed at 30 ppm formaldehyde 5 hours/day, once a week. Histopathologic examinations were 

19 conducted as above.

1 . Dalbey (1982) examined the effects of inhaled formaldehyde alone for a 

2 lifetime or combined with diethylnitrosamine (DEN) in an initiation-promotion study design 

3 using male Syrian golden hamsters. For the first experiment, hamsters were exposed at either 0 

or 10 ppm (0 or 12.3 mg/m3 4 ) formaldehyde in whole body chambers 5 hours/day, 5 days/week 

5 for a lifetime (132 controls, 88 exposed). Histopathologic evaluations were carried out on two 

6 transverse sections of the nasal turbinates (otherwise not specified), longitudinal sections of 

7 larynx and trachea, and all lung lobes cut along the bronchus prior to embedding. In the 

8 formaldehyde-only (10 ppm) experiment, mortality was increased relative to unexposed controls 

9 (p < 0.05). No tumors and little evidence of toxicity to the nasal epithelium were observed. 

10 There was no increase in rhinitis. Epithelial hyperplasia and metaplasia were increased in 

11 formaldehyde-treated animals (5% incidence) versus none observed in controls. 

20 Although weekly exposures to formaldehyde alone (30 ppm once a week) did not 

21 influence mortality, treatment with DEN alone significantly (p < 0.05) increased mortality above 

22 that of untreated controls, and mortality was further elevated (p < 0.05) in the two groups 

23 exposed to both DEN and formaldehyde compared with DEN alone. No respiratory tract tumors 

24 were observed in untreated animals or those receiving only formaldehyde. DEN treatment alone 

25 resulted in a high incidence (77%) of tumors (nasal, larynx, trachea, and lung). Formaldehyde 

26 pre- or posttreatment did not further increase the number of TBAs-. All tumors observed were 

27 classified as adenomas. Formaldehyde pretreatment nearly doubled the number of tumors per 

28 animal in the trachea (but not lung or larynx) (p < 0.05). This increase in tumors initiated by 

29 DEN given 48 hours after formaldehyde exposure suggests a role of formaldehyde- induced 

30 changes in the respiratory tract in tumor promotion (e.g., cell proliferation and inflammation).

31

32 4.2.1.2.3.4. Summary. Chronic rodent studies of inhalation exposure to formaldehyde provide a 

33 consistent picture of the agent’s toxicity—especially on the URT—on which most studies focus. 

34 All three species tested—hamsters, mice, and rats—had some degree of hyperplastic and 

35 metaplastic change in the nasal passages. The pathology defined in acute and subchronic 
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1 exposures is similarly described in chronic studies, where progression, severity, and presence in 

2 more posterior sections of the nose increase with both the concentration and duration of 

3 exposure. 

4 Pathology of the respiratory epithelium includes rhinitis, goblet cell hyperplasia, 

5 pseudoepithelial cell hyperplasia, squamous metaplasia, and dysplasia (see Table 4-34). At 

6 higher exposures and longer durations of exposure, similar effects are seen on the olfactory 

7 epithelium, present further into the nasal passages. In addition to hyperplasia and squamous 

8 metaplasia, thinning and disarrangement of the olfactory epithelium noted and, in a few cases, 

9 cell damage and replacement of olfactory epithelium with respiratory epithelium appear, 

10 including loss of sensory cells (Woutersen et al., 1989; Kerns et al., 1983; Battelle Columbus 

11 Laboratories, 1981).

12 Clear species differences in the severity of lesions are present. Although the bioassays in 

13 mice, hamsters, and rats do represent similar exposure concentrations and duration of exposure, 

14 hamsters exhibit little pathology and rats (three strains tested) exhibit gross toxicity and even 

15 increased mortality. Mice similarly exposed exhibit a range of effects on the respiratory 

16 epithelium but not near the severity seen in rats. Many factors may contribute to these observed 

17 species differences. As Chang and Barrow (1983) reported, the increased RB of mice seems to 

18 be protective of POE damage in comparison to that of rats. The reduced ventilation rate and 

19 minute volume of rodents in the presence of a reactive gas can reduce the effective delivered 

20 dose at the same exposure concentration (Chang and Barrow, 1983). Additionally, as illustrated 

21 in the computational fluid dynamic (CFD) modeling (see Section 3.5), there are species 

22 differences in nasal architecture that influence areas of formaldehyde absorption or flux into the 

23 tissue. Localized differences in mucus flow and production as well as metabolic enzymes have 

24 also been posited as having roles in differential toxicity of formaldehyde on the URT (see 

25 Chapter 3).

26 Formaldehyde-induced tumors were present in exposed rats and mice and primarily 

27 involved SCCs later in life (Kamata et al., 1997; Tobe et al., 1985; Kerns et al., 1983; Swenberg 

28 et al., 1980). Although exposure of male Syrian hamsters to either 10 or 30 ppm did not result in 

29 formaldehyde-induced nasal tumors, a classic initiation-promotion assay with DEN-induced 

30 tumor formation did indicate that formaldehyde increased the tumor burden per animal, where 

31 DEN induced tumors in 77% of the animals (Dalbey, 1982). This study suggests a role for 

32 promotion in the observed carcinogenicity of formaldehyde. Less clear are the implications of 

33 the synergistic effect of formaldehyde exposures and gross damage to the respiratory epithelium 

34 by electrocoagulation on tumor formation (Woutersen et al., 1989).

35
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-183

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

Species/strain

No./

group Treatmenta Respiratory effects

Noncancer

LOAEL/NOAEL Reference

Chronic bioassays

C3H mice (sex 

unstated)

Mice

60 0, 41, 82, or 163 ppm

1 hour/day, 3 days/week for up 

to 35 weeks. 

Low- and mid-group mice then 

exposed at either 122 or 

244 ppm during weeks 35–70.

Pathology: Histologic changes in the tracheobronchial 

epithelium by exposure, including basal-cell 

hyperplasia, stratification squamous cell metaplasia, and 

atypical metaplasia. 

Carcinogenicity: No SCC formation was evident in 

mice exposed to formaldehyde alone.

LOAEL = 41 ppm

No evidence of 

carcinogenicity

Horton et al. (1963)

Male and female 

B6C3F1 mice

120/sex 0, 2, 5.6, or 14.3 ppm 

6 hours/day, 5 days/week for 24 

months. 

The protocol featured a 6-month 

recovery period. Interim 

sacrifices occurred at 6, 12, 18, 

24, and 30 months.

Pathology: Rhinitis; hyperplasia, dysplasia, and 

squamous metaplasia of the nasal epithelium; atrophy of 

the olfactory epithelium; glandular adenitis and 

nasolacrimal duct hyperplasia and metaplasia.

Carcinogenicity: Nasal SCC in male mice at 24 months 

(2/17). No SCC in female mice.

LOAEL = 2 ppm 

Evidence of 

carcinogenicity

Swenberg et al. 

(1980); Kerns et al. 

(1983); CIIT 

(1982) ; Battelle 

Columbus 

Laboratories (1981)

Female SpragueDawley rats

Rats

16 0 or 12.4 ppm 

formaldehyde ± wood dust 

6 hours/day, 5 days/week for 

104 weeks.

Pathology: Squamous metaplasia and dysplasia. 

Carcinogenicity: One of 16 rats exposed to 

formaldehyde alone developed SCCs.

LOAEL = 12.4 ppm

Support for 

carcinogenicity

Holmström et al. 

(1989a)

Male and female 

F344 rats

32/sex 0, 0.3, 2, or 14 ppm 

6 hours/day, 5 days/week for 

28 months.

Pathology: Increased rhinitis, hyperplasia, and 

squamous metaplasia of the nasal respiratory epithelium

Carcinogenicity: Nasal SCCs in high concentration 

rats (44%).

LOAEL = 0.3 ppm

Support for 

carcinogenicity

Tobe et al. (1985)

Male F344 rats 32 0, 0.3, 2, or 15 ppm 

6 hours/day, 5 days/week for 

28 months.

Pathology: Squamous cell metaplasia and epithelial 

hyperplasia.

Carcinogenicity: SCC (13/32), squamous cell 

papilloma (3/32), and sarcoma (1/32). 

LOAEL = 0.3 ppm

BMD10 = 0.24 ppm

Evidence of 

carcinogenicity

Kamata et al. (1997)
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purposes only and does not constitute 

Agency policy

4-184

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

Species/strain

No./

group Treatmenta Respiratory effects

Noncancer

LOAEL/NOAEL Reference

Male SpragueDawley rats100 0 or 15 ppm 6 hours/day, 

5 days/week for life.

Pathology: Squamous metaplasia, epithelial 

hyperplasia, and polyps/papillomas. 

Carcinogenicity: SCCs formed in the nasomaxillary 

turbinates and nasal septum (25%). 

LOAEL = 15 ppm

Evidence of 

carcinogenicity

Albert et al. (1982); 

Sellakumar et al. 

(1985)

Male and female 

F344 rats

120/sex 0, 2, 5.6, or 14.3 ppm 

6 hours/day, 5 days/week for 

24 months. The protocol 

featured a 6-month recovery 

period. Interim sacrifices 

occurred at 6, 12, 18, 24, and 

30 months.

Pathology: Lesions of the nasal cavity were the 

primary effects, including squamous metaplasia and 

epithelial dysplasia, hyperkeratosis, goblet cell 

hyperplasia, and rhinitis.

Salivary gland: atrophy, squamous metaplasia, and 

sialadenitis.

Carcinogenicity: SCCs were evident in the nasal 

cavity of high concentration rats, plus some polypoid 

adenomas.

LOAEL = 2 ppm 

Evidence of 

carcinogenicity

Swenberg et al. 

(1980); Kerns et al. 

(1983); CIIT (1982); 

Battelle Columbus 

Laboratories (1981); 

Morgan et al. 

(1986b)

Male F344 rats 90 and 150 

controls

0, 0.7, 2, 6, 10, or 15 ppm 

6 hours/day, 5 days/week for 

24 months.

Pathology: Olfactory degeneration, squamous 

metaplasia, epithelial hypertrophy and hyperplasia, and 

mixed inflammatory cell infiltrate.

Carcinogenicity: SCCs and polypoid adenomas in the 

nasal cavity

LOAEL = 2 ppm 

Evidence of 

carcinogenicity

Monticello et al. 

(1996)

Male SPF Wistar 

rats

10 0, 0.1, 1, or 10 ppm 

6 hours/day, 5 days/week for 13 

or 52 weeks. 

An electrocoagulation method 

was applied to damage the 

noses of 1⁄2 of each study group.

Pathology: Formaldehyde-induced focal changes to the 

respiratory and olfactory epithelium, including rhinitis, 

hyperplasia, and metaplasia (10 ppm).

In rats with damaged noses: squamous metaplasia of the 

respiratory epithelium increased at all formaldehyde 

exposures. Pathology of the olfactory epithelium 

increased at the 10 ppm exposure.

Carcinogenicity: No tumors noted; 1-year study

LOAEL = 0.1 ppm

in rats with damaged 

nasal passages

NOAEL = 1 ppm for 

rats with intact noses

Appelman et al. 

(1988)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-185

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

Species/strain

No./

group Treatmenta Respiratory effects

Noncancer

LOAEL/NOAEL Reference

Male Wistar rats 30 (without 

nasal 

damage), 

60 (with 

nasal 

damage)

0, 0.1, 1, and 10 ppm 

6 hours/day, 5 days/week for 28 

months or for 3 months with a 

25-month observation period.

An electrocoagulation method 

was applied to damage the nasal 

cavity.

Pathology: Intact noses: squamous metaplasia in the 

high concentration group exposed for 28 months and 

degeneration of the olfactory epithelium. 

Changes were more severe in animals with damaged 

noses . 

Carcinogenicity: SCCs developed in 15/60 rats with 

damaged noses exposed at 10 ppm. In other groups, the 

incidence of nasal tumors was low irrespective of the 

state of nasal damage.

NOAEL = 1 ppm

Evidence of 

carcinogenicity

Woutersen et al. 

(1989)

Male Syrian 

golden hamsters

Hamsters

88 treated 

132 

controls.

0 or 10 ppm formaldehyde 

5 hours/day, 5 days/week for 

life.

Pathology: Increased mortality. Epithelial hyperplasia 

and metaplasia increased in formaldehyde-treated 

animals (5% incidence)

Carcinogenicity: No tumors reported.

LOAEL = 10 ppm

No evidence of 

carcinogenicity

Dalbey (1982)

Male Syrian 

golden hamsters

50 0 or 30 ppm 

5 hours/day, 1 day/week for life 

± injections with 0.5 mg DEN.

Pathology: Increased mortality in conjunction with 

DEN—above DEN-only treated animals. Respiratory 

pathology not reported.

Carcinogenicity: Only hamsters receiving DEN 

developed tumors (77%, adenomas). There was an 

increase in the number of tumors per TBAs in the 

trachea of animals exposed to formaldehyde 48 hours 

prior to DEN (but no increase in TBAs).

LOAEL = 30 ppm.

Evidence for 

formaldehyde as a 

promoter

Dalbey (1982)
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4.2.1.2.4. Summary of respiratory pathology. 

12 Progression of lesions can be viewed as progression from the anterior to posterior 

13 sections of the nasal cavity or as a progression in severity of lesions at a particular location (e.g., 

14 level or region) of the nasal passages. In both cases, progression is evident with increasing 

15 exposure concentration and with increasing duration of exposure (Kamata et al., 1997; 

16 Monticello et al., 1996; Morgan et al., 1986b; Takahashi et al., 1986; Sellakumar et al., 1985; 

17 Kerns et al., 1983; Albert et al., 1982). The data suggest that concentration and duration of 

18 exposure do not act in a simply cumulative manner (e.g., C × t). Additionally the influence of 

19 concentration, duration, and repeated exposure may be different for various effects. For 

20 example, some lesions may be transient (e.g., low-exposure cell proliferation), others may have a 

21 threshold and vary little after that (e.g., rhinitis). Additionally, as the nasal epithelium responds 

22 with both adaptive and adverse epithelial changes, the absorption of formaldehyde into the tissue 

23 at that location may be reduced. As respiratory epithelium transitions to squamous metaplasia, 

24 the effective tissue dose of formaldehyde increases posterior to these lesions. As barriers to 

25 formaldehyde flux into the tissue develop (e.g., squamous metaplasia, keratinization), 

26 formaldehyde penetrates more deeply into the nasal passages (Kimbell et al., 2006). Therefore, 

27 although both concentration and duration of exposure do effect the adverse effect, the 

28 relationship is difficult to define and in fact may be different for various adverse effects.

1 The progressive pathology of the nasal passages 

2 from inhalation exposure to formaldehyde is well documented, especially in rodents (rats and 

3 mice) (see Tables 4-9, 4-15, 4-28, 4-34). Although there are species differences in tissue dose 

4 (Section 3.4) due to variations in nasal architecture and breathing patterns, the nature and 

5 progression of the pathology is fairly well conserved across species, including nonhuman 

6 primates. The observed formaldehyde-induced pathology includes disruption of the mucociliary 

7 apparatus, rhinitis (serous and purolent), hyperplasia (cell proliferation), metaplasia (transition of 

8 cell type), dysplasia (disarrangement of cells), nest-like infolds and invaginations of the 

9 epithelium, thinning of the epithelial layer and focal to diffuse lesions, atrophy of the olfactory 

10 epithelium, thickening and keratinization (usually of squamous metaplasia), tumors (adenoma, 

11 sarcoma, carcinoma) (Section 4.2.2).

29 Respiratory histopathology has been commonly reported in response to exposure to 

30 formaldehyde in rats and mice (Lino dos Santos Franco et al., 2006; Javden and Taher, 2000; 

31 Kamata et al., 1996a, b; Cassee and Feron, 1994; Bhalla et al., 1991; Monteiro-Riviere and Popp, 

32 1986; Buckley et al., 1984; Chang et al., 1983), rabbits (Ionescu et al., 1978), hamsters 

33 (Schreibner et al., 1979), and rhesus monkeys (Monticello et al., 1989). The histopathologic 

34 lesions ranged from inflammation to ulceration, necrosis, and metaplasia that occurred in nasal 

35 turbinates, maxilloturbinates, and goblet and microvillus cells (Bhalla et al., 1991). These effects 
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1 were observed at a variety of doses (e.g., 10 ppm for 4 hours, 3.13 ppm for 6 hours for 1, 2, or 

2 4 days, 6 or 15 ppm). Wilmer et al. (1989, 1987) assessed whether a dose and time-dependent 

3 interaction (C × t) is associated with histopathologic lesions. Results indicated that 

4 concentration, rather than duration or cumulative exposure, correlates best with severity of 

5 lesions (Wilmer et al., 1989, 1987).

6 Histopathologic lesions and changes to biochemistry have been reported in the lung as 

7 well, though these effects were observed following a high dose of formaldehyde. In addition, 

8 changes in clinical chemistry, P450 expression and activity in lung tissue, and gene expression 

9 that is phenotypically anchored to the observed respiratory pathology have been reported. 

10 Extrapulmonary effects have also been noted, including changes in liver chemistry, relative brain 

11 weight, and focal, chronic inflammation in the heart and kidney. Most of these changes occurred 

12 at exposures of 20 ppm, and those that occurred at lower formaldehyde exposures (3.7 ppm) 

13 could not be strictly correlated with formaldehyde exposure.

14 Some researchers have reported formaldehyde-induced effects in the pulmonary region in 

15 rats, mice, and rabbits. Kamata et al. (1996a) observed reduced lipid content of pulmonary 

16 surfactant in rats exposed to 128.4 or 294.5 ppm formaldehyde. Kamata et al. (1996b) reported 

17 biochemical changes in lung homogenates and altered lipid content of BAL at 145.6 ppm 

18 formaldehyde. Lino dos Santos Franco et al. (2006) observed increased leukocytes (and 

19 neutrophils) and degranulated mast cells recovered in BAL fluid (concentration of 1% formalin 

20 not provided). In rabbits, Ionescu et al. (1978) observed frank necrosis of lung parenchyma after 

21 aerosol inhalation of 3% formalin for 3 hours/day for 50 days (concentration of formaldehyde 

22 not provided). These pulmonary effects may be due to frank toxicity resulting from the high 

23 dose of formaldehyde used in these studies.

24 Several recent toxicogenomics studies have assessed gene expression changes in nasal 

25 and lung tissue in animals and in humans by using in vivo and in vitro approaches. Hester et al. 

26 (2005, 2003) documented changes in gene expression associated with DNA repair and apoptosis 

27 in nasal tissue from male rats after a single instillation of formaldehyde. Other gene expression 

28 changes were observed in those genes related to xenobiotic metabolism and in cell cycle and 

29 repair. These preliminary results provide an initial basis for forming a phenotypically anchored 

30 set of gene expression changes associated with exposure to formaldehyde and may assist in 

31 determining the underlying MOA, as will be discussed in Section 4.5. Sul et al. (2007) 

32 investigated gene expression genes in lung tissue from formaldehyde-exposed rats. Yang et al. 

33 (2005) performed a proteomics analysis by using lung tissue extracted from formaldehyde34 exposed rats. Two studies used human tracheal cell lines to investigate formaldehyde-induced 

35 gene expression changes in vitro (Lee et al., 2008, 2007). However, the relevance of these 
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1 findings to actual exposures remains unknown. In total, toxicogenomics studies hold promise, 

2 but they must be interpreted with caution until results can be replicated and phenotypically 

3 linked to observable changes.

4 Thus, formaldehyde-induced respiratory pathology has been commonly described in the 

5 nasal passages and includes cellular proliferation, mucociliary function, and histopathologic 

6 lesions. Pulmonary effects have been documented as well but at high doses. The nasal 

7 pathology may occur as a result of both concentration and duration components of exposure.

8

9 4.2.1.2.5. Cell proliferation. Formaldehyde-induced cell proliferation has been demonstrated 

10 under range of exposure conditions in vivo and in vitro as well (Chapter 3). Formaldehyde11 induced mitogenesis may be a primary effect (as demonstrated in the in vitro work) or secondary 

12 to adaptive responses and tissue remodeling (Swenberg et al., 1983). This section provides a 

13 comprehensive discussion of formaldehyde effects on cell proliferation in the epithelial tissues in 

14 the respiratory tract. The majority of the work discussed investigates cell proliferation with 

15 in vivo labeling of proliferating cells, although additional methods, such as flow-cytometry, have 

16 been employed in some instances. 

17 Swenberg et al. (1986) conducted a series of experiments in rodents to assess cell 

18 proliferation in the nasal mucosa after formaldehyde inhalation. Radiolabeled thymidine 

[

3 19 H]-thymidine was injected intraperitoneally (I.P.) into male F344 rats and B6C3F1 mice after 

20 formaldehyde exposure to assess the extent of in vivo incorporation into proliferating cells. Two 

21 hours later, animals were sacrificed and the nasal passages were fixed, embedded, and sectioned 

22 to examine the nasal mucosa. Slides were exposed for 12 weeks and developed to identify cells 

23 that incorporated the radiolabeled thymidine. The percentage of labeled cells, as indicated by the 

24 presence of five or more grains over the nucleus, was determined by visual count. A total of 

25 4,000 or 1,500 cells were counted per section for rats and mice, respectively.

26 The first set of studies reported by Swenberg et al. (1986) compared the dose response of 

27 rats and mice. Animals were exposed to 0, 0.5, 2, 6, or 15 ppm (0, 0.61, 2.45, 7.36, or 

18.4 mg/m3 28 ) formaldehyde 6 hours/day for 3 days. Tritiated thymidine for cell labeling was 

29 injected 2 hours after the end of exposure. No change in the percentage of labeled cells was seen 

30 after 0.5 or 2 ppm formaldehyde exposure. However, the nasal passages of rats exposed at 6 and 

31 15 ppm showed 10- to 20-fold increases over controls in LI at level 2. A similar cell 

32 proliferation response was seen in mice treated with 15 ppm formaldehyde, although no increase 

33 over control was seen in mice exposed to 6 ppm formaldehyde. These findings are consistent 

34 with other data that indicate rats are more sensitive to formaldehyde exposure than mice. This 

35 may be due to differences in the reflex apneic response between the two species. As discussed in 
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1 Section 4.2.1.1, mice maintain decreases in minute volume in response to formaldehyde, which 

2 results in a lower overall effective internal dose to the mice. 

3 Comparing cell proliferation rates after 2 versus 18 hours of exposure, Swenberg et al. 

4 (1986) found that the longer exposure duration gave twice the cell proliferation rates after 

5 repeated exposures. Therefore, these researchers conducted a second dose-response study to 

6 examine cell proliferation 18 hours after exposure instead of the shorter exposure duration. The 

7 dose-response study varied dose as well as duration of treatment. Rats were exposed 6 hours/day 

to either 0.5, 2, or 6 ppm (0.61, 2.45, or 7.36 mg/m3 8 ) formaldehyde over periods of 1, 3, or 9 

9 days. Formaldehyde exposure at 0.5, 2, or 6 ppm for 1 day increased cell proliferation in the 

10 nasal epithelium. However, these increases were transient, and cell proliferation was not 

11 increased after 3 or 9 days of exposure to 0.5 ppm or 2 ppm formaldehyde. Although still 

12 elevated after a 3-day exposure to 6 ppm formaldehyde, cell proliferation returned to control 

13 values after 9 days of exposure to 6 ppm formaldehyde (Swenberg et al., 1986). Therefore, 

14 although concentration is a major determinant of cell proliferation, duration of exposure also 

15 influenced formaldehyde-induced cell proliferation in the nasal epithelium.

16 Swenberg et al. (1986) directly tested the effects of cumulative exposure versus 

17 concentration for both mice and rats. Animals were treated with one of three regimens, resulting 

18 in the same C × t product: 3 ppm × 12 hours, 6 ppm × 6 hours, or 12 ppm × 3 hours, each 

19 exposure resulting in 36 ppm-hours. The animals were exposed once a day for either 3 or 9 days. 

20 Tritiated thymidine was injected 18 hours after exposure to label of proliferating cells. Tissue 

21 sections from levels 1 and 2 of the nasal passages were examined in each case, and the 

22 percentage of cells labeled was reported as the percentage of proliferating cells (Figure 4-10).

23 Cell proliferation at level 1 in the nasal cavity was much greater than at level 2 for all C × 

24 t combinations of formaldehyde exposure in both mice and rats (Figure 4-10). The authors noted 

25 that level 1 is more anterior and lacks significant defense from the mucociliary apparatus, which 

26 may account for the observed greater sensitivity to formaldehyde. At all C × t exposure 

27 products, 3 days of exposure resulted in greater cell proliferation than 9 days of exposure. This 

28 was true for both species and for both examined levels of the nasal cavity. The decrease in cell 

29 proliferation by day 9 is consistent with data on rats labeled 18 hours postexposure (Swenberg et 

30 al., 1986).

31

32
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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)

2 (c) (d)

3

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

5 respiratory mucosa of rats and mice.

6

7 Note: a and b are data following 3 days of exposure; c and d are for 9 days of 

8 exposure. a and c are from level 1 (most anterior); b and d are from level 2. 

[

3 9 H]-thymidine was administered 18 hours after the last exposure.

10

11 Source: Swenberg et al. (1986).

12

13

14 When comparing C × t exposures for a single species and location, the findings are more 

15 complex. Cell proliferation in level 2 of the nasal passages appeared to be more dependent on 

16 concentration than on duration or cumulative exposure, with the strongest response seen for 

17 12 ppm formaldehyde in combination with the shortest exposure period, 3 hours (Figure 4-10). 

18 This pattern was observed in both rats and mice after 3 days of exposure and in rats after 9 days 

19 of exposure. No increases in cell proliferation at level 2 were seen for any C × t combination in 

20 mice after 9 days. In contrast, increases in cell proliferation at level 1 of the nasal passages were 

21 not strictly concentration dependent. After a 3-day exposure, no clear differences were seen 

22 among different C × t treatments for either mice or rats, suggesting cumulative exposure may be 
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1 the important metric. Therefore, it may be concluded that cell proliferation for level 1 of the 

2 nasal passages, where there is less protection of the epithelium, is influenced by concentration, 

3 time, and duration of exposure. Cell proliferation at level 2 appeared to be more dependent on 

4 concentration than time of exposure (Swenberg et al., 1986).

5 Cassee and Feron (1994) reported a qualitative increase in histochemical staining for 

6 proliferating cell nuclear antigen (PCNA) in the respiratory epithelium of the nasoturbinates, 

7 maxilloturbinates, septum, and lateral wall at levels 2 and 3 of rat nasal passages after repeated 

exposures to 3.5 ppm (4.29 mg/m3 8 ) formaldehyde 22 hours/day for 3 days. While no increases 

9 were seen in olfactory epithelium, frank necrosis, squamous metaplasia, and hyperplasia of both 

10 ciliated and nonciliated epithelium were noted at these section levels.

11 Quantitative cell proliferation studies have been conducted by several researchers in the 

12 same laboratory (Reuzel et al., 1990; Wilmer et al., 1989; Zwart et al., 1988; Wilmer et al., 1987; 

13 Woutersen et al., 1987) (Summary Table 4-39). These studies build off of those of Swenberg et 

al. (1986), who labeled proliferating cells with [3 14 H]-thymidine in assessing cell proliferation 

15 within the nasal mucosa. The studies, all performed in male albino Wistar rats and using a 

16 similar experimental design, provide the basis for comparing different exposure levels and dose 

17 regimens across studies. Wilmer et al. (1987) demonstrate a concentration-dependent increase in 

18 cell proliferation after 3 days of repeated 8-hour exposures at 5, 10, or 20 ppm (6.13, 12.3, or 

24.6 mg/m3 19 ) formaldehyde, regardless of continuous versus interrupted exposure conditions 

20 (2.83, 8.87, and 19.8 versus 0.86% proliferation in controls). Similar trends were seen when the 

21 repeated continuous exposures were extended for 4 weeks, but cell proliferation was not 

22 maintained at the same levels. As observed by Swenberg et al. (1986), these results suggest that 

23 duration of repeated exposures may be an important determinant of cell proliferation rates.

24 Woutersen et al. (1987) reported that the majority of the dose-dependent increases in cell 

25 proliferation seen at section level 3 after 3 days of repeated 6-hour exposures to 10 and 20 ppm 

(12.3 and 24.6 mg/m3 26 ) formaldehyde occurred in areas of the epithelium showing “clear 

27 squamous metaplasia and hyperplasia.” Cell proliferation rates in metaplastic epithelium of 

28 29.5 and 33.2% were much higher than the 1.4 to 2.8% proliferation in the visibly unaffected 

29 respiratory epithelium from rats exposed at 10 ppm formaldehyde. Although there was a slight 

30 trend towards increased cell proliferation in the visibly unaffected epithelium of exposed animals 

31 compared with unexposed controls, the majority of increased cell proliferation resulting from 

32 exposure to 10 and 20 ppm formaldehyde was attributed to the metaplastic epithelium.

33 Similarly, dose-dependent increases in cell proliferation seen at level 3 after 3 days of 

repeated 6-hour exposures at 0.3, 1, and 3 ppm (0.37, 1.23, and 3.68 mg/m3 34 ) formaldehyde 

35 (p < 0.001) corresponded to focal basal cell hyperplasia and loss of cilia (Woutersen et al., 1987). 
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1 No necrosis or focal erosion was noted at these levels of formaldehyde exposure. Cell 

2 proliferation was not sustained at this location, and no lesions were noted after 13 weeks of 

3 repeated 6-hour exposures. The authors hypothesized that defensive mechanisms, such as the 

4 mucociliary apparatus, may have provided greater protection of the mucosa at level 3. Swenberg 

5 et al. (1986) drew a similar conclusion when evaluating extended exposures, suggesting that 

6 more posterior sections had a greater adaptive ability than those anterior sections with little 

7 mucociliary function. Both Woutersen et al. (1987) and Swenberg et al. (1986) reported 

8 sustained cell proliferation and development of lesions in the more anterior cross section. 

9 Repeated exposures to 3 ppm formaldehyde (6 hours/day) resulted in significant increases in cell 

10 proliferation in the epithelial cells at level 2, with accompanying disarrangement, focal 

11 hyperplasia, and squamous metaplasia (Woutersen et al., 1987). Although no cell death was 

12 observed at level 2 when viewed by light microscopy, “strongly indented and disarranged nuclei” 

13 were seen by electron microscopy, which may be consistent with apoptosis (Woutersen et al., 

14 1987). However, later work in the same laboratory indicated no increased cell proliferation at 

levels 2 or 3 in male Wistar rats exposed to formaldehyde at 1 or 2 ppm (1.23 and 2.45 mg/m3 15 ) 

16 (8-hour repeated exposures for 3 days or 13 weeks) and only minimal response in rats exposed at 

17 4 ppm formaldehyde (interrupted 8-hour exposures for 3 days or 13 weeks) (Wilmer et al., 

18 1989).

19 Reuzel et al. (1990) published the only report in which formaldehyde effects on cell 

20 proliferation were studied for longer daily exposure durations: 22 hours/day versus 6–

21 8 hours/day. Male Wistar rats were exposed to formaldehyde, ozone, or the combination of the 

22 two 22 hours/day for 3 consecutive days. The concentrations of formaldehyde were 0.3, 1.0, or 

3.0 ppm (0.37, 1.23, or 3.68 mg/m3). Rats were injected with [3 23 H]-thymidine 2 hours rather than 

24 18 hours after the last exposure. Cell proliferation was quantified by enumerating the percentage 

25 of labeled cells in fixed and stained tissue sections. Cell proliferation on the nasoturbinates, 

26 maxilloturbinates, lateral wall, and septum at levels 2 and 3 were quantified and reported 

27 separately. Cell proliferation was increased at all locations in level 2 at 3 ppm formaldehyde 

28 exposure (p < 0.05) but not at 0.3 or 1 ppm exposures (Summary Table 4-39). Whereas 

29 proliferation of cells in the nasoturbinate, maxilloturbinate, and septum was nearly undetectable 

30 in control animals, 4, 5, and 3% proliferation was reported after repeated 22-hour exposures to 3 

31 ppm formaldehyde. Basal proliferation in the lateral wall was greater than in other areas, 

32 approximately 1% increasing to 6% after exposure to 3 ppm formaldehyde. Although basal 

33 levels of cell proliferation were slightly higher in all areas of level 3, formaldehyde had no 

34 significant effects on cell proliferation in the level 3 areas evaluated. There was a slight trend for 

35 increases at 3 ppm, but all proliferation rates were below 1%. Exposure to 3 ppm formaldehyde 
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1 also damaged the respiratory epithelium at levels 2 and 3, where cell disarrangement and 

2 hyperplastic and metaplastic lesions were reported.

3 Roemer et al. (1993) investigated the effects of formaldehyde exposure on cell 

4 proliferation in the trachea and lung in addition to nasal mucosa. Male Sprague-Dawley rats 

were exposed head only to 2, 6, or 20 ppm (2.45, 7.36, or 24.5 mg/m3 5 ) formaldehyde 6 hours/day 

6 for either 1 or 3 days. Proliferating cells were labeled with 5-bromodeoxyuridine (BrdU), the 

7 label injected 16–22 hours after formaldehyde exposure ended. Free lung cells were harvested 

8 by tracheal lavage, and the majority of isolated cells were MPs (>97%). Epithelial cells were 

9 isolated from the nasal and tracheal mucosa by dissection, physical disaggregation, and enzyme 

10 treatment to release epithelial cells. All cells were fixed and stained with fluorescent dyes to 

11 detect BrdU and total DNA. Flow cytometry was used to determine the percentage of BrdU12 labeled cells as a measure of cell proliferation. Cells undergoing unscheduled DNA synthesis 

13 (e.g., DNA repair) were excluded by cell cycle analysis. 

14 The proportion of BrdU-labeled cells from the nose and trachea increased two- to 

15 threefold above control values after a single 6-hour exposure to formaldehyde (Table 4-35). The 

16 lowest effective dose for increased cell proliferation was 2 ppm for nose and tracheal cell 

17 proliferation (p < 0.05). However, increased proliferation in the nasal mucosa at the lowest dose 

18 was transient, returning to control levels after a 3-day exposure. Cell proliferation remained 

increased in the nasal mucosa after exposure to 6 or 10 ppm (7.36 or 12.3 mg/m3 19 ) formaldehyde 

20 for 3 days. In contrast, proliferation of tracheal cells appeared to be reduced as a result of a 

21 3-day exposure to 2 or 6 ppm formaldehyde. A similar trend was seen in free lung cells, but the 

22 differences were not statistically significant. 

23 The flow cytometry employed by Roemer et al. (1993) allowed for subtle changes in 

24 proliferation rates to be measured with good discrimination. However, the method of cell 

25 isolation did not allow examination of proliferation rates in discrete regions of the mucosa, 

26 which may have attenuated the magnitude of the response. Additionally, proliferation rates 

27 represent a mix of cell types that were not separated in this analysis, making the findings difficult 

28 to interpret. This may be especially noteworthy in the free lung cells that were reportedly 

29 primarily MPs. 

30

31
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1 Table 4-35. Cell proliferation in nasal mucosa, trachea, and free lung cells 

2 isolated from male Wistar rats after inhalation exposures to formaldehyde

3

1 Daya Control 2 ppm 6 ppm 20 ppm

Nose 1.3b 2.4c 3.7c 2.7

Trachea 1.2 3.1c 2.1c 2.8

Lungd 1.8 2.6 3.3 3.1

3 Days Control 2 ppm 6 ppm 20 ppm

Nose 1.3 1.4 2.5c 2.3c

Trachea 1.2 0.3c 0.6c 2.5c

Lung 1.8 2.2 2.4 5.1

4 a 5 Exposures were 6 hours/day.

b 6 Proliferation is measured as the percent of BrdU-labeled cells. c 7 Statistically different from controls (p < 0.05). d 8 The majority of free lung cells were MPs (97%).

9

10 Source: Roemer et al. (1993).

11

12

13 Monticello et al. (1990) investigated whether changes in cell proliferation rate correlated 

14 with areas of cell injury or with areas that developed tumors due to formaldehyde exposures by 

15 using a unique metric of cell proliferation. They hypothesized that treatment-related effects on 

16 cell populations could influence the apparent cell proliferation measured as LI, even though no 

17 proliferative effect had occurred. For example, cell death could give an apparent increased 

18 proliferation as a LI (% cells proliferating) by reducing the total number of cells present. This 

19 would be especially true for a stratified epithelium, where the number of basal cells in active 

20 proliferation may not change but cells above the basal layer might die or slough off, thereby 

21 reducing the overall number of population of cells counted. The unit length labeling index 

22 (ULLI) metric was developed to normalize proliferation rates against length of basal membrane 

23 rather than cell population. However, application of a ULLI to the pseudostratified epithelium of 

24 the nasal mucosa introduced additional complexities. First, undamaged mucosa has a single 

25 layer of epithelial cells that have the capability for cell proliferation. Second, cells only become 

26 layered in response to cell damage as a protective measure. Therefore, the total cells present and 

27 the linear cell density should be considered, as well as the number and density of proliferating 

28 cells, in developing an understanding of the proliferative response of these tissues to toxic insult.

29 Monticello et al. (1990) directly compared the apparent effects of formaldehyde exposure 

30 on cell proliferation when quantified as an LI or as a ULLI. Male F344 rats were divided into 

groups (n = 6) and exposed to 0, 2, 6, or 15 ppm (0, 2.45, 7.36, or 18.4 mg/m3 31 ) formaldehyde 

6 hours/day, 5 days/week for 12 weeks. Rats were administered [3 32 H]-thymidine continuously for 

33 the last 5 days of exposure by surgically implanted osmotic pumps. After sacrifice, nasal 
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1 passages were fixed, and sections from standard level 3 were prepared for examination. Cell 

2 proliferation was quantified at the midseptum and the lateral meatus at this level. Basement 

3 membrane length, total number of cells present, and number of labeled proliferating cells were 

4 recorded for each location. Each of these areas also was scored for the presence of nasal lesions.

5 The formaldehyde-related lesions included epithelial hyperplasia, squamous metaplasia, 

6 and acute inflammation. These lesions were most severe in animals exposed to 15 ppm, mild at 

7 6 ppm, but absent at 2 ppm. Cell proliferation, measured either as LI or ULLI, was increased in 

8 the level 3 septum and lateral meatus after 13 weeks of exposure to 15 ppm formaldehyde but 

9 not to 6 or 2 ppm (Table 4-36). There was a slight increase in both cell number and labeled cells 

10 in the lateral meatus of rats exposed to 6 ppm formaldehyde, but both measures of proliferation 

11 were unchanged from controls. The increased proliferation in the lateral meatus at 15 ppm was 

12 entirely due to an increased number of labeled cells. Total cells were unchanged at 15 ppm; 

13 therefore, both LIs demonstrated a similar increase over control. In addition to increased labeled 

14 cells in the septum at 15 ppm, total cells were increased from 470 to 640 (p < 0.05). Where the 

15 total cells and linear cell density were increased, the ULLI was proportionally increased over the 

16 LI. These observations are consistent with the development of squamous metaplasia and 

17 hyperplasia seen at 15 ppm. However, while both LI and ULLI showed an eightfold increase in 

18 cell proliferation in the lateral meatus, they gave different results in the septum where cell 

19 number was increased by formaldehyde treatment. LI increased 19-fold and ULLI 25-fold with 

20 repeated exposures to 15 ppm formaldehyde. Although these data are based on only 5–6 

21 animals/group, and only in an extended study, the results suggest that the ULLI and LI may not 

22 be proportional under all conditions studied. In similar experiments the LI and ULLI provided 

23 different indices of proliferation in the olfactory epithelium after methyl bromide exposure 

24 (Monticello et al., 1990). Methyl bromide exposure decreased cell number/mm of basement 

25 membrane in a time-dependent manner, and the LI and ULLI were not proportional across these 

26 changes. At day 3 there was an increase in labeled cells but a decrease in total cells; therefore, 

27 the LI was increased greater than 20-fold, where the ULLI was only increased eightfold. The 

28 authors endeavored to explain why the ULLI and LI yielded different findings. Where ULLI is a 

29 more time-efficient method of assessing cell proliferation, the authors suggested that 

30 representative areas should be quantified by LI to better understand the nature of increased 

31 ULLI.

32

33
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1 Table 4-36. The effect of repeated formaldehyde inhalation exposures for 

2 3 months on cell count, basal membrane length, proliferation cells, and two 

3 measures of cell proliferation, LI and ULLI, in male F344 rats

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/mmd

1,800 ± 150

11.9 ± 0.5

150 ± 10

130 ± 20

7.2%

10.9 cells/mm

2,300a ± 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

640a ± 20

2.9 ± 0.1

220a ± 10

250 ± 50

39%

86.2 cells/mm

5 a 6 Different from control, p < 0.05. b 7 BM is basal membrane length in mm.

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

d 9 Calculated from group averages: ULLI = (labeled cells)/BM length

10

11 Source: Monticello et al. (1990).

12

13

14 Monticello et al. (1990) reported similar results in a contemporary abstract; although 

15 treatment groups were slightly different than in the above experiments, the findings were similar. 

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/m3 16 ) 

17 formaldehyde 6 hours/day for 4 days, 6 weeks, or 3 months. ULLIs were determined in the 

septum and lateral meatus (methods not detailed). It is not stated whether [3 18 H]-thymidine 

19 labeling was carried out by injection or continuous infusion. Significant increases in cell 

20 proliferation were reported after repeated exposures to 6, 10, and 15 ppm for 4 days and 6 weeks. 

21 After 3 months of exposure, cell proliferation was still increased in rats exposed to 10 and 

22 15 ppm formaldehyde. The authors noted that, although increased cell proliferation was seen at 

23 earlier time points, sustained increased cell proliferation was only seen at 10 and 15 ppm, which 

24 they considered the clearly carcinogenic doses.

25 Monticello et al. (1991) applied the ULLI measurements in evaluating formaldehyde 

26 effects on cell proliferation after short-term and subchronic repeated exposures. Six male F344 

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/m3 27 ) 
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1 formaldehyde 6 hours/day for 1, 4, or 9 days and for 6 weeks, using a 5 days/week regimen. 

Rats were injected with [3 2 H]-thymidine 18 hours postexposure to label proliferating cells. All 

3 animals were sacrificed 2 hours later. Nasal passages were fixed, and sections from levels 2 and 

4 3 were prepared for examination. Cell proliferation was quantified for three locations in level 2 

5 (specifically, the lateral meatus, midseptum, and medial aspect of the maxilloturbinate) and for 

6 two regions of level 3 (the lateral wall and midventral septum). Each of these areas also was 

7 scored for the presence of nasal lesions.

8 As discussed above, proliferating cells were visually identified by the number of grains 

9 over the nucleus, 10 grains indicating a proliferating cell. Cell proliferation was quantified as the 

10 number of proliferating cells per length of basement membrane (cells/mm) and reported as a 

11 ULLI. The report does not indicate the length of membrane viewed for each section as an 

12 indication of how representative the counts are for each region. Lesions associated with 

13 formaldehyde exposure may change the density of cells/mm of basement membrane (Monticello 

14 et al., 1990). Areas of disarranged cells, erosion, metaplasia, or layering of epithelial cells may 

15 exhibit different cell profiles. These processes would alter cell density, and therefore the ULLI, 

16 independent of differential proliferation rates. As such, it is not expected to be proportional to 

17 cell proliferation rates across conditions that have the potential to change cell density 

18 (Monticello et al., 1990).

19 No formaldehyde-induced epithelial lesions or increases in the ULLI were seen in rats 

20 exposed to 0.7 or 2.0 ppm formaldehyde, regardless of duration (Table 4-37). Formaldehyde21 induced lesions were present in all regions of the nasal epithelium after exposures to 10 and 

22 15 ppm formaldehyde, regardless of duration (Monticello et al., 1991). Incidence and severity of 

23 the lesions increased with concentration and duration of treatment and were correlated to areas 

24 with increased cell proliferation. Rats exposed to 6 ppm formaldehyde developed lesions in the 

25 level 2 nasal passages, where the ULLI was clearly elevated, but not in the deeper level 3 

26 passages. For example, no formaldehyde-related lesions were seen at the lateral meatus and 

27 septum of level 3 at 1, 4, and 9 days of repeated exposure at 6 ppm, although cell proliferation 

28 was increased. This transient increase in ULLI returned to near-control levels after 6 weeks of 

29 repeated exposure (Table 4-37). Monticello et al. (1991) suggested that cell proliferation is a 

30 more sensitive indicator of cellular response and not necessarily dependent on cellular necrosis.

31

32
This document is a draft for review purposes only and does not constitute Agency policy.

4-198 DRAFT—DO NOT CITE OR QUOTE

1 Table 4-37. Formaldehyde-induced changes in cell proliferation (ULLI) in 

2 the nasal passages of male F344 rats exposed 6 hours/day

3

Locationa

Exposure concentration

0 ppm 0.7 ppm 2 ppm 6 ppmb 10 ppmb 15 ppmb

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

16.9b

30.5b

23.5b

14.4b

11.2b

20.9b

28.6b

23.9b

12.7b

25.8b

24.6b

28.7b

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

10.0b

10.9b

 2.10

17.9b

26.1b

19.6b

21.4b

16.7b

29.1b

29.1b

25.9b

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

18.15b

25.03b

22.54b

16.32b

5.9

20.3b

21.0b

26.1b

5.3

19.4b

28.7b

25.1b

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

 7.53b,,c

 8.77b,,

c

 7.35b, c

 2.08

14.5b

20.0b

30.6b

24.2b

16.4b

30.8b

40.4b

34.8b

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 

 9.22b,,

c

 9.50b,,

c

 2.58 

24.4b

18.7b

28.6b

14.0b

19.3b

34.4b

32.5b

27.5b

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

b 6 Indicates significantly different from control, p < 0.05. c 7 Indicates a location where epithelial lesions were not seen by light microscopy.

8

9 Source: Monticello et al. (1991).

10

11

12 The sustained cell proliferation at the lateral meatus and midseptum in rats exposed to 10 

13 and 15 ppm formaldehyde, locations where SCCs are known to arise, supports a role for 

14 compensatory cell proliferation in tumor development. However, Monticello et al. (1991) noted 

15 that regional differences in sustained cell proliferation do not always correspond to the 

16 occurrence of nasal tumors, primarily SCCs, in formaldehyde-exposed rats. Where sustained 

17 cell proliferation has been demonstrated in the medial maxilloturbinate (MMT) at level 2 

18 (Monticello et al., 1991), SCCs have not been found to originate in this area at similar exposures 

19 (Monticello et al., 1996; Woutersen et al., 1989). Monticello et al. (1991) suggested that the 

20 findings of Bermudez and Allen (1984), indicating that the epithelial cells of the maxilloturbinate 
This document is a draft for review purposes only and does not constitute Agency policy.

4-199 DRAFT—DO NOT CITE OR QUOTE

1 are more resistant to the genotoxic effects of DEN, support the possibility that differences in 

2 regional tissue susceptibility may contribute to site specificity of formaldehyde-related SCCs.

3 Monticello et al. (1996) further explored the correlation between measures of cell 

4 proliferation and tumor site by modifying the ULLI to take into consideration the total number of 

5 cells in a region that may be subject to increased cell proliferation. The population weighted 

6 ULLI (PWULLI) is the product of the expected number of cells on a three-dimensional surface 

7 in the nasal mucosa and the ULLI of a cross section of that surface. For this series of 

8 experiments, six male F344 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/m3 9 ) formaldehyde for up to 24 months with interim sacrifices at 3, 6, 12, 

and 18 months. Before each interim sacrifice [3 10 H]-thymidine was continuously injected for the 

11 last 5 days of exposure through a surgically implanted pump. Nasal passages were prepared, and 

six standard sections were taken and developed as above for [3 12 H]-thymidine-labeled cells. 

13 Stained tissue sections were viewed in order to map all nasal tumors. A ULLI was determined 

14 for each region (details not provided). The total cell population of each nasal region was 

15 estimated from control animals sacrificed at 3 months (Table 4-38). Cell profiles were counted 

16 across 0.5 mm of basement membrane length at two locations for each region (site not specified). 

17 Total cells per region were estimated from these counts and the modeled surface area expected in 

18 each region (Fluid Dynamics Analysis Package version 7.0). It is unclear if one or more rats 

19 were used to quantify cell population. Cell counts and variability were not reported.

20

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

22 F344 rats and regional site location of squamous cell carcinomas in 

23 formaldehyde-exposed rats for correlation to cell proliferation rates

24

Nasal region

Total cells

(number)a

Area

(mm2)

b

Cell density

(cell/mm2)

SCC incidencec

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 specifiedc -- -- -- 6 25

25 a 26 Total cell number determined in unexposed rats as a product of representative cell counts and expected surface area 

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

c 29 The number of animals bearing a tumor located in the region. Animals were exposed 6 hours/day for 24 months 

30 prior to sacrifice.

31 Source: Monticello et al. (1996).
This document is a draft for review purposes only and does not constitute Agency policy.

4-200 DRAFT—DO NOT CITE OR QUOTE

1 ULLIs were quantified by region of the nasal passages in order to correlate with regional 

2 localization of tumors. For example, the anterior midseptum included cells from the midseptum 

3 from approximately standard section levels 2 to 3. An anterior to posterior pattern of 

4 formaldehyde effects, especially differences in cell proliferation rates, has been well established. 

5 As such, cell proliferation rates would be expected to vary across the nasal regions used in this 

6 analysis. Areas considered to possibly be preneoplastic were not quantified for this work. 

7 Monticello et al. (1996) reported increased ULLIs in the ALM and the MMT of rats exposed to 

8 10 or 15 ppm at all time points (3, 6, 12, and 18 months) but provided no indication of variability 

9 or a statistical analysis, making it difficult to determine where true differences may exist. Some 

10 caution should be used in interpreting the ULLI counts assigned for each region.

11 The PWULLI was calculated by multiplying the reported ULLIs by the calculated cell 

12 populations by region. SCC incidence by region had a greater correlation to the calculated 

PWULLI than the ULLI, R2 = 0.88 versus R2 13 = 0.46, respectively. The authors noted that the 

14 relative lack of correlation with the ULLI was influenced by findings at the maxilloturbinate 

15 where cell proliferation was high but SCC incidence was low. Other tumor types were not 

16 included in the analysis (polypoid adenomas, adenocarcinomas, and rhabdomyosarcomas). 

17 Additionally, 54 of the SCC tumors could not be accurately localized and were excluded from 

18 the analysis, resulting in exclusion of 30 and 39% of animals with SCCs in the 10 and 15 ppm 

19 treatment groups, respectively. The authors cautioned that the absence of these data might have 

20 skewed the regional analysis of tumor location. Although the purpose of weighting the ULLIs 

21 by total population of cells available in each region is to better represent the chance of a tumor 

22 arising in each region, the cancer incidence was represented by the number of animals, not the 

23 number of tumors, per region. Based on the exclusion of location data (up to 40% of the 

24 animals), lack of variability and significance reported for the ULLI for cell counts, and SCC 

25 incidence considered by animal rather than by tumor, the significance of a greater correlation by 

26 PWULLI versus ULLI is of questionable value.

27 Monticello et al. (1989) also assessed formaldehyde-induced cell proliferation and 

28 regional site location of lesions in the respiratory tract of rhesus monkeys (see Section 4.2.1.2.2.2

29 for a full study description). LIs from the histoautoradiograms indicated increased cell 

30 proliferation in transitory, respiratory, and olfactory epithelial cells after the 6-week 

31 formaldehyde exposure. Similar trends were seen after only 1 week but were statistically 

32 significant only in the respiratory epithelium. Although increased proliferation in the trachea and 

33 carina was statistically significant after 1 week of exposure, the greater increases seen after 

34 6 weeks of exposure were not statistically significant. A small sample size (n = 3) and high 

35 variability may have contributed to the lack of statistical significance. The authors noted that 
This document is a draft for review purposes only and does not constitute Agency policy.

4-201 DRAFT—DO NOT CITE OR QUOTE

1 increased cell proliferation was seen in locations with minimal histologic changes, indicating 

2 proliferation may be a more sensitive predictor of adverse health effects of formaldehyde 

3 exposure. Table 4-39 provides a summary of formaldehyde-induced cell proliferation data.

4

5 4.2.1.3. Gastrointestinal Tract and Systemic Toxicity

6 As with inhalation, the POE is thought to be the principal target tissue in response to oral 

7 exposure. A concentration-dependent pattern of toxicity longitudinally down the GI tract has 

8 been observed upon oral exposure. Some evidence (Til et al., 1989, 1988) suggests that, with 

9 regard to oral exposure, duration in addition to concentration is important in the development of 

10 toxicity.

11 Formalin and paraformaldehyde were used to dose animals in oral toxicity studies. 

12 Formalin contains 12–15% methanol as a preservative to inhibit the polymerization of 

13 formaldehyde and subsequent precipitation as paraformaldehyde (Kiernan, 2000). The presence 

14 of methanol in formalin may confound the results of a formaldehyde study. Methanol has been 

15 shown to be a developmental and neurologic toxin (e.g., Degitz et al. [2004a, b]; Rogers et al. 

16 [2004, 2002]; Weiss et al. [1996]; Sharpe et al. [1982]). Oral dosing with paraformaldehyde is 

17 preferred because it allows for the preparation of methanol-free formaldehyde in the laboratory 

18 by dissolving paraformaldehyde in slightly basic water. 

19

20 4.2.1.3.1. Short-term and subchronic studies. Til et al. (1988) evaluated the oral toxicity of 

21 formaldehyde and acetaldehyde in a subacute study in Wistar (Cpb:WU; Wistar random) rats. 

22 Groups of rats (10/sex/dose) were exposed to paraformaldehyde dissolved in drinking water at 0, 

23 5, 25, and 125 mg/kg-day for 4 weeks. The control group was comprised of 20 rats of each sex. 

24 To account for potential effects of decreased water consumption in treated animals, an additional 

25 control group of 10 male and 10 female rats was given drinking water in an amount equal to the 

26 amount of liquid consumed by the group given the highest dose. Examination of the GI tract was 

27 performed in all dose groups and included the tongue, esophagus, and stomach. Histopathology 

28 for the other tissues was performed on high-dose and control animals.

29
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-202

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

Species Na Treatmentb

Measure of cell 

proliferation Summary of results by locationc Reference

Male F344 

rats;

male 

B6C3F1 

mice

NRd

0.5, 2, 6, or 15 ppm

6 hours/day for

3 days

LI: percent labeled cells 

on tissue sections

(

3

H-thymidine I.P.d

2 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 in 

proliferation.

10- to 20-fold increase seen in both rats and mice at 15 ppm. 

Swenberg et 

al. (1986)

Male F344 

rats NR

0.5, 2, or 6 ppm

6 hours/day

1, 3, or 9 days

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure)

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 9 by 6 ppm.

Swenberg et 

al. (1986)

Male F344 

rats;

male 

B6C3F1 

mice

NR

3 ppm for 12 hours,

6 ppm for 6 hours, or 

12 ppm for 3 hours

3 or 9 days

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Level 1:

3 days: Greater increased proliferation in rats than mice. Increases 

similar for various concentrations yielding the same C × t product.

9 days: Mice exhibited duration-dependent increases in proliferation, 

inverse to concentration for constant C × t.

Level 2

3 days: Concentration-dependent increase in cell proliferation.

9 days: Concentration-dependent increase in cell proliferation in rats; no 

increase in mice.

Swenberg et 

al. (1986)

Male F344 

rats 4–5

15 ppm

6 hours/day

1 or 5 days

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Level 2: Increase in cell proliferation in respiratory epithelium,

nasoturbinates, maxilloturbinates, and lateral wall.

1 day: 5.51f versus 0.43% in controls

5 days: 10.1%f

Chang et al. 

(1983)e

Male 

BC3F1 

mice

4–5

15 ppm

6 hours/day

1 or 5 days

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Level 2: Increase in cell proliferation in respiratory epithelium,

nasoturbinates, maxilloturbinates, and lateral wall.

1 day: 2.14f versus 0.27% in controls

5 day: 3.42%f

Chang et al. 

(1983)e

Male 

albino 

Wistar rats

5d

3.5 ppm 

8 hours, twice a day

for 3 days

Qualitative staining for 

PCNA on tissue sections

Levels 2 and 3: Increase in cell proliferation in respiratory epithelium,

nasoturbinates, maxilloturbinates, septum, and lateral wall.

Cassee and 

Feron (1994)e
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-203

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

Species Na Treatmentb

Measure of cell 

proliferation Summary of results by locationc Reference

Male 

albino 

Wistar rats

3

0, 5, or 10 ppm 

8 hours/day 

continuously for

3 days or 4 weeks, or 

0, 10, or 20 ppm 

8 hours/day 

intermittentg

for 3 days or 4 weeks

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Section level not stipulated in report.

3 days: 0.86% in controls

 2.83%f at 5 ppm continuous

 8.87%f at 10 ppm continuous

 9.80%f at 10 ppm interrupted

 19.8%f at 20 ppm interrupted

 

 

4 weeks: 0.68% in controls

 1.33% at 5 ppm continuous

 8.85%h at 10 ppm continuous

 3.41%f at 10 ppm interrupted

 13.9%f at 20 ppm interrupted

Wilmer et al. 

(1987)e

Male 

albino 

Wistar rats

2

0, 1, 10, or 20 ppm

6 hours/day for

3 days

LI: percent labeled cells 

(18 hour postexposure ex 

vivo 3

H-thymidine labeled 

excised mucosa)

Level 3

 Metaplastic epithelium: increased proliferation

 31.4% at 10 ppm, 37.6% at 20 ppm

 Visibly unaffected respiratory epithelium

 1.6% in controls

 2.6% at 10 ppm, 2.8% at 20 ppm

Woutersen et 

al. (1987)e

Male 

albino 

Wistar rats

5

0, 1, or 2 ppm 

8 hours/day 

continuously for

3 days or 4 weeks,

or 0, 2, or 4 ppm 

8 hours/day 

intermittentg

for 3 days or 4 weeks

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Level 2 

3 days: No change from controls 

 0.60% in controls

 0.34% at 1 ppm continuous

 0.61% at 2 ppm continuous

 0.29% at 2 ppm interrupted

 0.58% at 4 ppm interrupted

 

4 weeks: no change from controls

 1.03% in controls

 0.81% at 1 ppm continuous

 0.91% at 2 ppm continuous

 1.16% at 2 ppm interrupted

 2.86% at 4 ppm interrupted

Wilmer et al. 

(1989)e

Male and 

female 

albino 

Wistar rats

5

0, 0.3, 1, 3 ppm

6 hours/day, 

5 days/week

for 3 days or 

13 weeks.

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Level 2: Increased cell proliferation at days 3 and 13 weeks (p < 0.001).

Level 3: Transient dose-dependent increase at 1 and 3 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)e

Male 

Wistar rats

5 0, 0.3, 1, or 3 ppm

22 hours/day for 

3 days

LI: percent labeled cells 

on tissue sections (2 hours 

postexposure ex vivo 3

Hthymidine-labeled excised 

mucosa)

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 and does not constitute 

Agency policy

4-204

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

Species Na Treatmentb

Measure of cell 

proliferation Summary of results by locationc Reference

Male 

SpragueDawley 

rats

3–5

0, 2, 6, or 20 ppm

6 hours/day for

1 or 3 days

LI: percent labeled cells 

by flow cytometry 

(5-bromodeoxyuridine I.P. 

18 hours postexposure for 

2 hours)

Respiratory and olfactory epithelial cells.

 1 day: 1.3% in controls

 2.4% at 2 ppmf

 3.7% at 6 ppmf

 2.7% at 20 ppmf

Tracheal epithelial cells

 1 day: 1.2% in controls

 3.1% at 2 ppmf

 2.1% at 6 ppm 

 2.8% at 20 ppmf

Free lung cells (>97% MPs):

 no significant change.

 

3 days: 

 1.4% at 2 ppm 

 2.5% at 6 ppmf

 2.3% at 20 ppmf

3 days: 

 0.3% at 2 ppmf

 0.6% at 6 ppmf

 2.5% at 20 ppmf

Roemer et al. 

(1993)

Male F344 

rats

6 0.7, 2, 6, 10, or 

15 ppm

6 hours/day, 

5 days/week for 1, 4, 

or 9 days or 6 weeks

ULLI (unit length LI) (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Level 3

 No increases in cell proliferation at 0.7 or 2 ppm.

Level 4

 ULLI increases in locations without lesions at 6 ppm.

 Increases in ULLI at all locations at 10 and 15 ppm.

Monticello et 

al. (1991)

Male 

rhesus 

monkeys

3

6 ppm

6 hours/day for

1 or 6 weeks

LI: percent labeled cells 

on tissue sections (3Hthymidine I.P. 18 hours 

postexposure for 2 hours)

Nasal passages: Duration-dependent increase in cell proliferation at all levels 

(B–E) in transitional, respiratory, and olfactory epithelium.

Increased cell proliferation in areas with minimal lesions.

Larynx: trend for increased proliferation

Trachea: increased cell proliferation 

 1 week : 1.14 versus 0.55% in controls

 6 weeks: 3.73%

Carina of trachea: increased cell proliferation. 

 1 week: 1.34 versus 0.43% in controls

 6 weeks: 3.60%

Respiratory bronchioles: no increase in proliferation.

Monticello et 

al. (1989)

a

N = number of animals per treatment group. b

Treatment is given as the concentration of formaldehyde, duration of exposure each day, and length of the experiment in days and weeks. c

Standard section levels of the nasal passages as shown in Figure 4-3 are given for experiments in rats or mice. d

NR = not reported; I.P. = intraperitoneally.

e

Study is described in full in section 4.2.1.2.2.4. . f

Different from control, p < 0.05. g

Intermittent exposures were 30 minutes per hour for 8 hours.

h

Data from one animal only.
This document is a draft for review purposes only and does not constitute Agency policy.

4-205 DRAFT—DO NOT CITE OR QUOTE

1 The rats appeared to be healthy throughout the study, and no effects on growth occurred 

2 despite significant decreases in food and water intake that occurred at the high dose (125 mg/kg3 day). Yellow discoloration of the fur occurred in the rats on the high dosage from week 3 

4 onward. There were no significant changes in hematology among the exposed groups except for 

5 slight (not statistically different) increases in PCVs in the water-restricted group and in high-dose 

6 males. The high-dose groups of the formaldehyde exposed and in the water-restricted controls 

7 had slightly increased urine density, but again this was not statistically significant. Plasma TP 

8 and ALB levels were decreased in the males of the highest dose group. No changes in organ 

9 weights occurred except for relative kidney weights that were slightly increased in the females of 

10 the high-dose group. Gross pathological findings were restricted to the GI tract and revealed a 

11 thickening of the limiting ridge of the forestomach in all animals exposed at the highest dose that 

12 was accompanied by a yellowish discoloration of the mucosa. These latter changes were not 

13 observed in the acetaldehyde-exposed animals. Treatment-related histopathologic changes were 

14 seen in the GI tract only. Slight (8/20) or moderate (12/20) focal hyperkeratosis of the 

15 forestomach and slight focal atrophic gastritis occurred in animals of the high-dose groups only 

16 (Table 4-40). One female had moderate focal papillomatous hyperplasia. No histopathologic 

17 changes were observed in any animals of the lower-dose groups. The study established a 

18 LOAEL and NOAEL for epithelial changes in the GI tract of male and female Wistar rats 

19 exposed to formaldehyde in drinking water at 125 mg/kg-day and 25 mg/kg-day, respectively.

20 Johannsen et al. (1986) performed a subchronic study by using rats and dogs exposed to 

21 paraformaldehyde dissolved in drinking water. Groups of albino Sprague-Dawley rats (15/sex) 

22 were administered the equivalent of 0, 50, 100, or 150 mg/kg-day in their drinking water for 

23 91 consecutive days. Pure-bred beagle dogs (four/sex/group) were fed a diet with added aqueous 

24 formaldehyde to approximate 0, 50, 75, or 100 mg/kg-day. Dogs were observed daily and rats at 

25 frequent intervals for behavioral reactions. Body weights and food and water intake were 

26 recorded on a weekly basis in both species. Hematology (HCT, Hb, total and differential 

27 leukocyte counts), clinical chemistry (blood sugar, BUN, ALP, AST and ALT in dogs only), and 

28 urine analyses (color, appearance, pH, specific gravity, sugar, protein, and microscopic elements) 

29 were evaluated in 10 male and 10 female rats selected from each test group and in all dogs. 

30 Organ weights were recorded for the adrenals, gonads, hearts, kidneys, livers, lungs, and thyroids 

31 in each species. Histopathology was performed on a set of over 20 or 30 tissues and organs from 

32 rats or dogs, respectively, in the high-dose and control groups only.

33

34
This document is a draft for review purposes only and does not constitute Agency policy.

4-206 DRAFT—DO NOT CITE OR QUOTE

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

2 Wistar rats after drinking-water exposure to formaldehyde for 4 weeks

3

Type of lesion

Formaldehyde (mg/kg-day)

0 5 25 125

Number of male rats examined

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

Type of lesion

Number of female rats examined

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

5 Source: Til et al. (1988).

6

7

8 No deaths or abnormal reactions were observed in either species. Significant reductions 

9 in weight gain were observed in dogs of both sexes at 100 mg/kg-day, in rats of both sexes at 

10 150 mg/kg-day, and in male rats at 100 mg/kg-day of formaldehyde. There was a dose-related 

11 decrease in liquid consumption of both sexes in rats given formaldehyde, but there was no 

12 overall difference in mean food intake or feed efficiency, so the reductions in body weight gain 

13 were considered to be systemic effects. Dogs administered formaldehyde had reduced food 

14 consumption and feed efficiency at all doses tested. No significant effects on hematology, 

15 clinical chemistry, or urine analyses were observed in either species. No effects in either species 

16 were reported on organ weights. The GI mucosa in both species was reported to appear normal 

17 with no indication of irritation. This study suggests a NOAEL of 150 mg/kg-day in Sprague18 Dawley rats and of 100 mg/kg-day in beagle dogs for formaldehyde in drinking water. 

19 Differences in the results for the rats with those reported in other studies (Til et al., 1989, 1988; 

20 Tobe et al., 1989) may be due to strain differences or duration of the exposure. The dog may be 

21 a more sensitive species than the rat based on these results and on those of 2-week pilot studies.
This document is a draft for review purposes only and does not constitute Agency policy.

4-207 DRAFT—DO NOT CITE OR QUOTE

1 4.2.1.3.2. Chronic bioassays: oral exposure to formaldehyde. The same laboratory that tested 

2 formaldehyde and acetaldehyde in a 4-week study (Til et al., 1988) performed a chronic bioassay 

3 with formaldehyde in drinking water. Til et al. (1989) administered paraformaldehyde dissolved 

4 in drinking water to Wistar rats (Cpb: WU; Wistar random) (70/sex/dose). Interim sacrifices 

5 (10/sex/dose) were performed at 12 and 18 months. Formaldehyde was administered in drinking 

6 water to provide target doses of 0, 5, 25, and 125 mg/kg-day. The mean formaldehyde doses 

7 administered were 0, 1.2, 15, or 82 mg/kg-day for males and 0, 1.8, 21, or 109 mg/kg-day for 

8 females. Concentrations were adjusted weekly for the first 12 weeks based on dose estimates 

9 derived from body weight and liquid consumption data. Such adjustments were made every 

10 4 weeks from weeks 12 to 52 and kept constant. Fresh solutions of the test concentrations were 

11 prepared weekly and stored at 15oC.

12 Endpoints examined included daily observations for condition and behavior, body weight 

13 at weekly intervals for the first 12 weeks and then every 4 weeks thereafter, liquid intake weekly, 

14 and food intake weekly for the first 12 weeks and then every 2 weeks for the remainder of the 

15 study. Samples of blood were taken for hematological and clinical chemistry analyses on weeks 

16 26 and 103. Analysis of blood glucose and urine pH, density, and volume was performed on 

17 samples at weeks 27, 52, 78, and 104. Pooled urine samples were also evaluated for glucose, 

18 occult blood, ketones, urobilinogen, and bilirubin in samples at weeks 27 and 104. Weights of 

19 all major organs were recorded at interim sacrifices and at term. Gross and histopathologic 

20 examinations were carried out on all major tissues of the rats in the high-dose and control 

21 groups. The livers, lungs, stomach, and noses were examined in all rats. Additionally, the 

22 adrenals, kidneys, spleens, testes, thyroids, ovaries, pituitaries, and mammary glands (for 

23 females) were examined in all sacrificed animals at weeks 53 and 79 and at term.

24 The general health and behavior of the rats were not affected in any of the formaldehyde25 exposed groups. Slight yellowing of the fur did occur in the animals exposed at the mid and high 

26 doses from week 3 onward. The mean body weights were decreased in the males from week 1 

27 and in the females from week 24 onward. At the high dose, liquid consumption was significantly 

28 decreased in both sexes, and food intake was significantly decreased in the males. There were no 

29 toxicologically significant effects on hematological, urinary, or clinical chemistry parameters. 

30 Decreases in absolute heart, liver, and testis (males) weights were attributed to lower body 

31 weights. Relative kidney weights were increased in females of the high-dose group, and relative 

32 brain weights were increased in both sexes of the high-dose group. Relative testis weight was 

33 increased in males. Treatment-related changes in gross pathology were restricted to the 

34 forestomach. Histopathologic examinations at the two interim sacrifices and final sacrifice 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 revealed GI tract changes. Renal changes were observed in the high-dose group at final 

2 sacrifice. There was no indication of treatment-related effects in other tissues.

3 As shown in Table 4-41, significant histopathology in the GI tract was limited to the 

4 forestomach and stomach of rats in the high-dose groups. Some progression with duration of 

5 exposure may have occurred by week 105 because GI lesions were observed in the lower dose 

6 groups at this time point, whereas none were observed in these groups at interim sacrifices. The 

7 histopathologic changes included papillary epithelial hyperplasia in the forestomach that was 

8 frequently accompanied by hyperkeratosis on the limiting ridge or its vicinity. The mucosa 

9 showed an irregular layer of hyperplastic basal cells, but no atypical nuclei or other subcellular 

10 structures were observed. Chronic atrophic gastritis occurred to varying degrees in the stomachs 

11 of all high-dose rats. In some cases the inflammatory process involved the entire mucosa and 

12 was seen to extend to the whole muscularis mucosae and met the criteria for ulceration.

13 Histologic examination also showed that the incidence and degree of renal papillary 

14 necrosis was increased in animals of the high-dose groups at the terminal sacrifice. This change 

15 was located at the tip of the papilla and was characterized by patchy necrosis of interstitial cells, 

16 capillaries, and loops of Henle. There was no evidence of a dose-related response in chronic 

17 nephropathy. The incidence of chronic nephropathy was lower in the males of the high-dose 

18 group than in controls. In females, the incidence was slightly higher in the test groups than in 

19 controls but only achieved statistical significance at the lowest dose. It is likely that the decrease 

20 in liquid intake incurred in the high-dose groups contributed to the increased incidence and 

21 degree of renal papillary necrosis observed in the high-dose animals because dehydration has 

22 been shown to enhance its production by various analgesics.

23 The results of this chronic bioassay indicated that formaldehyde is cytotoxic to the 

24 epithelial mucosa of the nonglandular (forestomach) and glandular stomach with a LOAEL of 82 

25 and 109 mg/kg-day and a NOAEL of 15 and 21 mg/kg-day in males and females, respectively. 

26 The findings provided no evidence of carcinogenicity in either the GI tract or systemic sites for 

27 formaldehyde administered in drinking water to Wistar rats at doses as high as 82 mg/kg-day.

28

29
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1 Table 4-41. Incidence of lesions observed in the gastrointestinal tracts of 

2 Wistar rats after drinking-water exposure to formaldehyde for 2 years

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 examinedb 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 10c 0 0 0 9c

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 10c 0 0 0 10c

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 examinedb 47 45 44 47 48 49 47 48

Forestomach

Focal papillary epithelial hyperplasia 1 2 1 45c 1 0 2 45c

Focal hyperkeratosis 2 6 4 24c 3 5 3 33c

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 46c 0 0 0 48c

Focal ulceration 0 0 0 11c 0 0 0 10c

Glandular hyperplasia 0 1 0 20c 0 0 0 13c

Mineralization 3 2 1 0 0 0 0 0

Focal inflammatory cell infiltrate 5 3 2 0 2 3 1 0

4 a 5 Incidence in rats that died or were killed when moribund during the experiment or were killed at 

6 week 53, 79, or 105.

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

c 8 The values differ significantly (Fisher’s exact test) from the control value (p < 0.001).

9

10 Source: Til et al. (1989).

11

12
This document is a draft for review purposes only and does not constitute Agency policy.

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1 Tobe et al. (1989) performed a chronic toxicity study of Wistar rats (Slc:Wistar) exposed 

2 to paraformaldehyde dissolved in drinking water. Groups of 20 male and 20 female rats were 

3 given formaldehyde solution in their drinking water at concentrations of 0, 0.02, 0.10, and 0.50% 

4 for 24 months. Interim sacrifices of six randomly chosen rats from each group were performed 

5 after 12 and 18 months. Based on the estimated average amount of water intake and body 

6 weight, the actual doses of formaldehyde in either sex were reported to be 0, 10, 50, and 

7 300 mg/kg-day. Fresh test solutions were prepared twice each week. The rats were observed 

8 daily for the entire study. Body weights and water and diet intake were measured once weekly 

9 or biweekly. Hematology (RBC, WBC, and Hb) and serum clinical chemistry (TP, ALB, BUN, 

10 uric acid, total cholesterol, inorganic phosphorous, ALP, AST, and ALT) were made at each 

11 necropsy. Organ weights were measured for the brain, heart, lung, liver, kidney, spleen, adrenal, 

12 testis or ovary, pituitary, and thyroid. These organs and the stomach, small and large intestine, 

13 pancreas, uterus, lymph nodes, and all tumors were examined histopathologically.

14 The general condition of animals in the high-dose group was poor with significantly 

15 reduced body weight gain as well as intake of water and diet. An increase in mortality was also 

16 observed in this group. Some clinical chemistry parameters were altered in this group. No 

17 significant changes in absolute or relative organ weights were observed. Mortality was 100% in 

18 the high-dose group by 24 months. At the 12-month sacrifice, hyperplasia of the squamous 

19 epithelium with or without hyperkeratosis was observed in the forestomach of all high-dose 

20 animals (12/12). Basal cell hyperplasia with growth into the submucosa was also observed in 

21 most cases (10/12). Erosions and/or ulcers with submucosal inflammatory cell infiltrates were 

22 observed in the glandular stomach of most rats (10/12). Regenerative changes of the glandular 

23 epithelium (glandular hyperplasia) were noticed in most cases (10/12) along the limiting ridge of 

24 the fundic mucosa. No lesions were observed in the glandular stomach at the 50 mg/kg-day 

25 dose, and forestomach hyperplasia was observed in only one of six males and in one of eight 

26 females at 18 and 24 months. No lesions in either the forestomach or glandular stomach were 

27 observed in rats treated at 10 mg/kg-day.

28 This study corroborates the Til et al. (1989) study and shows that the main targets for 

29 formaldehyde toxicity administered by drinking water to rats are the forestomach and glandular 

30 stomach. Although the lesions observed at the 50 mg/kg-day were minimal in this study, Tobe et 

31 al. (1989) designated the NOAEL at 10 mg/kg-day, further supporting the NOAEL of 15 mg/kg32 day from the Til et al. (1989) study.

33 Takahashi et al. (1986) studied the effects of formaldehyde in an initiation-promotion 

34 model of stomach carcinogenesis in male outbred Wistar rats (Shizuoka Laboratory Center, 

35 Shizuoka). Rats (n = 17) were given 100 mg/L of N-methyl-N1-nitro-N-nitrosoguanidine 
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1 (MNNG) in drinking water and a diet supplemented with 10% sodium chloride (NaCl) for the 

2 first 8 weeks as an initiation phase. This was followed by 0.5% formalin (which contains 12–

3 15% methanol) in drinking water for 32 weeks as the promotion phase of the protocol. A 

4 comparison group (n = 10) was given stock water and diet without any supplementation for the 

5 first 8 weeks followed by 0.5% formalin in drinking water for 32 weeks. Animals were observed 

6 daily and weighed once every 4 weeks. Small pieces of the stomach and other tissues in the 

7 peritoneal cavity were fixed for histopathologic examination.

8 Body weight gain was reduced by exposure to MNNG with sodium chloride, and 

9 formaldehyde exposure during the promotion phase exacerbated this effect. Histopathologic 

10 investigations were restricted to the GI tract. Formaldehyde was shown to statistically increase 

11 the incidence of lesions in the forestomach and stomach in the animals initiated with MNNG 

12 with NaCl as compared with controls receiving no initiation (Table 4-42). Increases in 

13 papilloma in the forestomach, adenomatous hyperplasia in the fundus, and adenocarcinoma in 

14 the pylorus were observed. Histopathology in the animals receiving formaldehyde alone during 

15 weeks 9 through 32 showed an increase in forestomach papillomas but with no lesions in the 

16 glandular stomach (Table 4-42). The adenomatous hyperplasia were defined as proliferative, 

17 noninvasive mucosal lesions, and the adenocarcinomas were defined as well differentiated and 

18 composed of typical glandular structures, demonstrating a tubular pattern and cellular or 

19 structural atypism without metastasis. No definition of criteria for papilloma diagnosis was 

20 provided. The findings in this study are inconsistent with those of Til et al. (1989), who found 

21 no evidence of carcinogenicity in a 2-year bioassay at comparable concentrations (assuming 37% 

22 formaldehyde in formalin results in 0.19% formaldehyde in this study). As discussed above, the 

23 differences may be due to differences in the strains of rat or in the diagnostic criteria. The lack 

24 of more than one test concentration precludes dose-response analysis of this study and provides 

25 only a stand-alone LOAEL of 0.2% formaldehyde in drinking water. The lack of consumption 

26 data precludes an estimation of dose in mg/kg-day.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-212

Table 4-42. Effect of formaldehyde on gastroduodenal carcinogenesis initiated by MNNG and NaCl in male 

Wistar rats exposed to formaldehyde (0.5% formalin) in drinking water for 8 weeks

No MNNG initiation prior to 8-week oral exposure to formaldehyde (0.5% formalin in drinking water)

Gastroduodenal 

carcinoma

Forestomach 

papillomas

Glandular stomach tumors

Fundus Pylorus Duodenum

Adenocarcinoma

Adenomatous 

hyperplasia Adenocarcinoma

Preneoplastic

hyperplasia Adenocarcinoma

Control 0% 0% 0% 0% 0% 0% 0%

Formaldehyde 0% 80%a 0% 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)

Gastroduodenal 

carcinoma

Forestomach 

papillomas

Glandular stomach tumors

Fundus Pylorus Duodenum

Adenocarcinoma

Adenomatous 

hyperplasia Adenocarcinoma

Preneoplastic

hyperplasia Adenocarcinoma

Control 13.3% 0% 0% 0% 3.3% 23.3% 10.0%

Formaldehyde 29.4% 88.2%a 0% 88.2%a 23.5%b 41.2% 5.9%

a

Significantly different from control animals with MNNG initiation, p < 0.01. b

Significantly different from control animals with MNNG initiation, p < 0.05.

Source: Takahashi et al. (1986).
This document is a draft for review purposes only and does not constitute Agency policy.

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1 Soffritti et al. (1989) administered formaldehyde in drinking water to Sprague-Dawley 

2 rats at different ages. Formaldehyde was produced via the Formox process, which yields carbon 

3 monoxide, dimethyl ether, and small quantities of CO2 and formic acid. In one experiment 

4 denoted as BT 7001, rats (50/sex/dose) that were 7 weeks old were administered 0, 10, 50, 100, 

5 500, 1,000, or 1,500 mg/L for 104 weeks. As is usual for experiments carried out at the 

6 Ramazzini Foundation, all animals were maintained until natural death at which point they were 

7 necropsied and examined histopathologically. In experiment BT 7005, 25-week-old breeder rats 

8 or offspring (beginning postnatal day [PND] 12) were provided drinking water with 0 or 2,500 

9 mg/L formaldehyde for 104 weeks. Fluid and food consumption were measured weekly for the 

10 first 13 weeks and then every 2 weeks thereafter. Individual body weights were recorded for the 

11 first 13 weeks and then every 2 weeks thereafter. Histopathology was routinely performed on all 

12 major tissues, including oral and nasal cavities, the GI tract (esophagus, stomach, and intestines 

13 [4 levels]), primary and secondary lymph organs (thymus, spleen, subcutaneous lymph nodes, 

14 mesenteric lymph nodes, mediastinal lymph nodes, and femur [bone marrow]), head and face 

15 bones, and other organ systems (liver, kidney, bladder, reproductive, and various glands). 

16 Noncancer health effects were not reported.

17 No GI neoplasia were observed in any of the control rats (experiments BT 7001 and BT 

18 7005). Historical controls for the BT experimental colony (n = 5,259) indicate an incidence of 

19 approximately 1% for benign neoplasia (papillomas and acanthomas) and an incidence of 0.19% 

20 for malignant stomach neoplasia (adenomas, SCCs, and adenocarcinomas, fibrosarcomas, and 

21 leiomyosarcomas taken together). Therefore, the size of the control groups (18–50 rats/sex) 

22 makes detection of background neoplasia unlikely. Similarly, one or two tumors noted in a 

23 treatment group (n = 18–50) would represent an apparent increase in these relatively rare tumors. 

24 Although stomach and intestinal tumors were found in rats exposed to formaldehyde in drinking 

25 water, the low incidence makes it difficult to discern any dose-response effect for individual 

26 neoplasia. The authors report formaldehyde-induced GI tract neoplasia to include benign tumors 

27 (papillomas and acanthomas of the forestomach and adenomas) and malignant tumors 

28 (adenocarcinomas and leiomyosarcomas). The majority of malignant tumors were present in the 

29 duodenum, jejunum, and ileum. Benign and malignant neoplasia were consistently noted in the 

30 two highest exposure groups: 1500 mg/mL formaldehyde in experiment BT 7001 and 

31 2,500 mg/mL in experiment BT 7005 (Table 4-43). Comparison of overall GI neoplasia in 

32 breeder and offspring rats of experiment BT 7005 suggests that rats exposed beginning on 

33 PND 12 had a greater incidence of malignant tumors. However, it should be noted that, although 

34 there may be an apparent increase in overall tumors, summing across sites and locations is 

35 needed before a response can be seen. Even then, several data points are based on a single 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 observed tumor. However, nonspecific MOAs, such as mutagenicity and regenerative 

2 proliferation, would be expected to act on all cell types at the POE, and summing tumor types 

3 may have some utility. These results constitute a weak positive result for cancer due to oral 

4 exposure to formaldehyde.

5

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

7 neoplasia reported in male and female Sprague-Dawley rats exposed to 

8 formaldehyde in drinking water at different ages

9

Experiment Dose Sex

Total benign 

tumors

Total malignant 

tumors All tumors

Historical 

controls

Not 

applicable

M 1.08% 0.31% 1.4%

F 0.97% 0.41% 1.4%

BT 7001

(7 weeks old)

Control M –a – –

F – – –

1,000 mg/mL M – 2%b 2%b

F 2%b – 2%b

1,500 mg/mL M 4% 6% 10%

F 6% – 6%

BT 7005

breeders

(25 weeks 

old)

Control M – – –

F – – –

2,500 mg/mL M – 5.6%b 5.6%b

F 5.6%b – 5.6%b

BT 7005

offspring

(PND 12)

Control M – – –

F – – –

2,500 mg/mL M 5.6% 8.3% 13.9%

F – 21.6% 21.6%

10

a 11 Dash indicates no tumors reported. An incidence was not reported.

b 12 Percentage is based on the observation of a single neoplasm.

13

14 Source: Soffritti et al. (1989).

15

16 Oral exposure to formaldehyde resulted in a dose-dependent increase in all 

17 hemolymphoreticular neoplasia in both male and female rats in experiment BT 7001 (Table 

18 4-44) (Soffritti et al., 1989). The most frequent neoplasia noted were lymphoblastic leukemias 

19 and lymphomas. The authors combined lymphoblastic leukemias and lymphosarcomas for 

20 analysis and summing across sites. This analysis is appropriate since there is broad consensus 

21 that “neoplasms presenting as solid tumors and those presenting with blood and marrow 

22 involvement are biologically the same disease but with different clinical presentations,” as stated 

23 in the recent WHO reclassification of hematological malignancies (Harris et al., 2000b). 

24 Inspection of the data tables suggests that the only treatment-related effects occurred at 1,000 
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1 and 1,500 mg/L. The reported incidence of lymphoid neoplasia and of all hemolymphoreticular 

2 neoplasia in rats exposed to the vehicle (methyl alcohol, 15 mg/L) was similar to results reported 

3 in rats exposed to 50 or 100 mg/L formaldehyde in drinking water. Soffritti et al. (1989) 

4 provided no statistical analysis of the data. Although the authors report a similar increase in 

5 experiment BT 7005, the apparent 5% increase is representative of a single animal in a treatment 

6 group of 18 and may not represent a true increase, the study’s usefulness for 

7 hemolymphoreticular neoplasia being somewhat limited by study size.

8

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

10 and female Sprague-Dawley rats exposed to formaldehyde in drinking 

11 water from 7 weeks old for up to 2 years (experiment BT 7001)

12

Treatment Sex

Lymphoid 

neoplasia (%)

Other leukemias 

(%)

All leukemias and 

lymphomas (%)

Control M 4 –a 4

F 1 2 3

Vehicle 

control

M 10 – 10

F 2 4 6

10 mg/mL M 2 – 2

F 4 – 4

50 mg/mL M 10 – 10

F 8 – 8

100 mg/mL M 8 2 10

F 4 4 8

500 mg/mL M 12 4 16

F 4 4 8

1,000 mg/mL M 12 – 12

F 10 4 14

1,500 mg/mL M 22 – 22

F 10 4 14

13 a 14 Dash indicates no neoplasm was reported.

15

16 Source: Adapted from Soffritti et al. (1989).

17

18

19 The study of Soffritti et al. (1989) does provide qualitative corroboration of evidence 

20 from other studies that observed formaldehyde toxicity in the forestomach and stomach. 

21 However, the dosages required to induce such lesions in this study were higher than in other 

22 studies. Soffritti et al. (1989) is the only animal study of oral exposure to formaldehyde that 

23 reports an increase in lymphoblastic leukemia or lymphosarcoma. These occurred at the two 

24 highest doses of 1,000 and 1,500 mg/L used in the study of animals exposed from 7 weeks of age 

25 (experiment BT 7001). 

26
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1 4.2.1.3.3. Summary of toxicity in the GI tract. Short-term and subchronic exposures to 

2 formaldehyde via drinking water for 4 weeks yielded slight to moderate histopathologic lesions 

3 (focal hyperkeratosis) at 125 mg/kg-day in male and female Wistar rats, as well as slight focal 

4 gastritis and submucosal infiltrate in one to two animals of both sexes (Til et al., 1988). No 

5 histopathologic lesions were noted in albino Sprague-Dawley rats or beagle dogs that received 

6 oral doses of formaldehyde in drinking water for 91 days (Johannsen et al., 1986). In both 

7 studies, decreases in weight gain were noted in exposed animals compared with controls.

8 In a chronic drinking water study, Til et al. (1989) reported that formaldehyde is 

9 cytotoxic to the epithelial mucosa of the nonglandular (forestomach) and glandular stomach with 

10 a LOAEL of 82 and 109 mg/kg-day and a NOAEL of 15 and 21 mg/kg-day in males and female 

11 Wistar rats, respectively. The findings provided no evidence of carcinogenicity in either the GI 

12 tract or systemic sites for formaldehyde administered in drinking water to Wistar rats at doses as 

13 high as 82 mg/kg-day. The incidence and degree of renal papillary necrosis was increased in 

14 animals of the high-dose groups at the terminal sacrifice (Til et al., 1989). Findings by Tobe et 

15 al. (1989) corroborate the Til et al. (1989) study and show that the main targets for formaldehyde 

16 toxicity administered by drinking water to rats are the forestomach and glandular stomach. 

17 Takahashi et al. (1986) studied the effects of formaldehyde in an initiation-promotion model of 

18 stomach carcinogenesis in male outbred Wistar rats (Shizuoka Laboratory Center, Shizuoka, 

19 Japan). In contrast to Til et al. (1989), Takahashi et al. (1986) found increases in incidence of 

20 papilloma in the forestomach, adenomatous hyperplasia in the fundus, and adenocarcinoma in 

21 the pylorus in a 2-year bioassay at comparable concentrations (assuming 37% formaldehyde in 

22 formalin results in 0.19% formaldehyde in this study). Soffritti et al. (1989) administered 

23 formaldehyde in drinking water to Sprague-Dawley rats at different ages. Rats (50/sex/dose, age 

24 7 weeks) were administered 0, 10, 50, 100, 500, 1,000, or 1,500 mg/L formaldehyde in drinking 

25 water for 104 weeks. The authors reported formaldehyde-induced GI tract neoplasia that 

26 included benign tumors (papillomas and acanthomas of the forestomach and adenomas) and 

27 malignant tumors (adenocarcinomas and leiomyosarcomas), albeit at a relatively low incidence 

28 after summing across sites and locations. Interestingly, oral exposure to formaldehyde resulted 

29 in a dose-dependent increase in all hemolymphoreticular neoplasia in both male and female rats 

30 (Soffritti et al., 1989). The most frequent neoplasia noted were lymphoblastic leukemias and 

31 lymphomas.

32

33 4.2.1.4. Immune Function

34 Leach et al. (1983) documented potential immunomodulatory effects of formaldehyde 

35 inhalation exposure. F344 rats were exposed nose only to formaldehyde 6 hours/day, 
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1 5 days/week for up to 30 days. The target concentrations for exposure were 0, 3, 16, 61, and 

99 ppm formaldehyde (0, 3.7, 19.7, 75.0, and 122 mg/m3 2 ). Body weight and food consumption 

3 were recorded, and blood samples for standard hematology and immune assays were collected 

4 (details not given). Immune measures referenced include in vitro lymphocyte transformation, 

5 hemagglutination assays, and enumeration of B cells, WBCs, and RBCs. No effects were seen at 

6 3 ppm formaldehyde. However, dose-dependent responses were reported for weight loss, 

7 decreased food consumption, increased WBCs, increased segmented neutrophils and nucleated 

8 RBCs, and decreased ability to produce antibodies to sheep RBCs. The results of the 

9 lymphocyte transformation assay were inconsistent, with a 25–30% reduction in stimulation after 

10 exposure to 99 ppm but an initial stimulation seen after 16 and 61 ppm exposures. Further 

11 details were not available, making it difficult to determine if these reported immunomodulatory 

12 effects may have been, in part or in full, secondary to effects on the URT. Subchronic exposures 

13 at 61 and 99 ppm formaldehyde would be expected to result in frank toxic effects in mice (see 

14 Section 4.2.1). However, these findings suggest possible immunomodulatory effects due to 

15 formaldehyde exposure and require further exploration. 

16 Dean et al. (1984) investigated the effects of formaldehyde exposure on a range of 

17 indicators of immune function. Female B6C3F1 mice were exposed to 15 ppm formaldehyde 

(18.4 mg/m3 18 ) 6 hours/day, 5 days/week for 3 weeks. Three trials were run with a total of 255 

19 formaldehyde-treated mice. Body and organ weights were recorded at sacrifice for control and 

20 formaldehyde-exposed mice (10 per group). Measures of host susceptibility, cell-mediated 

21 immunity MP function, and antibody reactions were conducted 2 to 6 days after the end of 

22 exposure (Table 4-45). Lymphocyte subsets, spleen cellularity, bone marrow cellularity, and 

23 progenitor cell subsets were enumerated. Host susceptibility and delayed type hypersensitivity 

24 were measured in vivo. Lymphocyte proliferation, natural killer cell activity, phagocytosis, 

25 hydrogen peroxide production, and IgM plaque-forming cells (PFCs) were measured ex vivo

26 after in vivo stimulation in some cases (Table 4-45).

27 Body weight, organ weights and cellularity, progenitor cell populations, blood cell 

28 counts, and differentials were unchanged in formaldehyde-treated mice (Dean et al., 1984). 

29 Circulating blood monocytes were decreased in treated mice, which may be a reflection of the 

30 local inflammatory response expected in the nasal epithelium (Dean et al., 1984). However, 

31 there was no corresponding decrease in peritoneal MPs. There was a trend, but no statistical 

32 significance, for decreased spleen weight, cellularity, and B cell precursors (87, 83, and 78% of 

33 controls, respectively). The mean body weight of formaldehyde-treated mice was 21.1 versus 

34 20.9 g in control mice, and thymus and spleen weights were not normalized by body weight. 

35
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1 Table 4-45. Battery of immune parameters and functional tests assessed in 

2 female B6C3F1 mice after a 3 week, 15-ppm formaldehyde exposure 

3 (6 hours/day, 5 days/week)

4

Immune function Model Challenge Metric

Host susceptibility Tumor resistance PYB6 sarcoma cells Subcutaneous injection, followed 

by skin palpation to track tumor 

development

Tumor resistance 16F10 melanoma cells Lung tumor burden determined by 

[

125I]UdR incorporation

Bacterial resistance Listeria monocytogenes Survival after challenge

Cell-mediated 

immunity

Delayed type 

hypersensitivity

Keyhole limpet 

hemocyanin

Radiometric index of delayed 

hypersensitivity responses 

Lymphocyte 

proliferation

T-cell mitogen, PHAa

B-cell mitogen, LPSb

(ex vivo)

Ex vivo proliferation, 3 days, 

measured by [3H]-thymidine 

incorporation

Lymphocyte subsets None Percentage of cells positive for cell 

surface markers (Thy-1, Mac-1,

Lyt-1)

Natural killer cell 

activity

Yac-1 target cells

(

51Cr labeled)

(ex vivo)

% cytotoxicity by 51Cr release

MP function

(both resident and 

MVE-1 elicited 

MP)

Phagocytosis Sheep RBCs

(

51Cr labeled)

(ex vivo) 

51Cr incorporation as a measure of 

RBCs phagocytized

Hydrogen peroxide 

production

Pharmacologic 

stimulation (ex vivo)

H2O2 release in culture

Humoral cell 

immunity

Antibody PFC 

responses, IgM 

PFCs

Sheep RBCs,

TVF-LPS, or TNFFicoll

Plaques formed

Progenitor cells Bone marrow 

cellularity (femur)

None Cell enumeration by a Coulter 

counter

Granulocyte-MP 

progenitors

None Cell enumeration by a Coulter 

counter

B-cell precursors None Clonogenic assay

5 a 6 T-cell mitogen, phytohemagglutinin (PHA-P). b 7 B-cell mitogen, lipopolysaccharide (Escherichia coli).

8

9 Source: Dean et al. (1984).

10

11

12 All indicators of natural killer cell function, cell-mediated immunity, and humoral 

13 immunity in formaldehyde-treated mice were unchanged from controls (Dean et al., 1984). 

14 Phagocytic capacity of both resident and elicited peritoneal MPs was unchanged by 

15 formaldehyde treatment. However, hydrogen peroxide production in elicited peritoneal MPs was 

16 significantly increased in formaldehyde-treated mice, 78 versus 42 nmol/mg protein (p < 0.05) 

17 (Dean et al., 1984).

18 As shown in Table 4-46, several indicators of host resistance in the female B6C3F1 mice 

19 were increased after formaldehyde exposure (Dean et al., 1984). Tumor mass and pulmonary 
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1 foci after B16F10 melanoma cell challenge were significantly reduced in formaldehyde-treated 

2 mice, indicating improved tumor immunity (p < 0.05). However, following PYB6 sarcoma cell 

3 challenge, formaldehyde-treated mice had a 7.1% tumor incidence versus 11.1% in controls, 

4 which was not statistically different. Mortality due to Listeria monocytogenes (LM) was 

5 decreased from 70 to 30% (p < 0.05). Because resistance to LM is primarily MP dependent, the 

6 authors speculated that this enhanced resistance might be due in part to increased bactericidal 

7 activity as was also suggested by increased hydrogen peroxide production ex vivo in elicited 

8 peritoneal MPs from female mice (Dean et al., 1984).

9

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

11 mononuclear phagocyte system (MPS) in female B6C3F1 mice after a 

12 3-week, 15 ppm formaldehyde exposure (6 hours/day, 5 days/week)

13

In vivo indicators of MPS Metric Formaldehyde effect

Cellularity Circulating monocytes Decreaseda

CMF progenitor cells No changea

Resident peritoneal MP No changea,b

Elicited peritoneal MP No changea,b

In vivo test of host resistance LM Increased resistancea

B16F10 tumor challenge Increased resistancea

PYB6 tumor challenge No significant increasea

Ex vivo indicators of MPS Cell type/activation Formaldehyde effect

H2O2 production Resident, no PMAc None detected a,b

Resident, with PMA None detected a,b

Elicited, no PMAd None detected a,b

Elicited, with PMA Increased a,b

Phagocytosis Resident No changea

Elicited No changea

Assessment of MP

maturation

Leucine aminopeptidase content

Resident Decreasedb

Elicited No changeb

Resident No changeb

Elicited No changeb

Acid phosphatase content Resident No changeb

Binding of tumor cells Elicited No changeb

Resident No changeb

Lysing of tumor cells Elicited Increased at mid-range target-to-effector 

cell ratiob

14 a 15 Dean et al. (1984).

b 16 Adams et al. (1987).

c 17 Phorbol 12-myristate 13-acetate (PMA). d 18 Peritoneal MPs were elicited with the pyran copolymer Murray Valley encephalitis virus (MVE-2).

19

20 Sources: Adams et al. (1987); Dean et al. (1984).

21
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1 Overall, the observations of increased hydrogen peroxide production and increased host 

2 resistance in peritoneal MPs distant from the POE suggest that formaldehyde has an effect on the 

3 mononuclear phagocyte system (MPS). The authors postulated that this effect may be indirect, 

4 due in part to the tissue inflammatory response in the URT or a direct systemic effect on the 

5 MPS by formaldehyde exposure (Dean et al., 1984). Subsequent studies by the same researchers 

6 explored the possibility of systemic effects of formaldehyde exposure on MPS function and 

7 maturation stage (Adams et al., 1987). Female B6C3F1 mice were exposed to 15 ppm 

(18.4 mg/m3 8 ) formaldehyde 6 hours/day, 5 days/week for 3 weeks, as before (Adams et al., 

9 1987). Both resident and Murray Valley encephalitis virus (MVE-2)-elicited peritoneal MPs 

10 were examined for hydrogen peroxide production, enzymatic activity, phagocytic ability, 

11 binding, and lysis of tumor cells (Adams et al., 1987).

12 Similar to the findings of Dean et al. (1984), formaldehyde treatment increased hydrogen 

13 peroxide production almost twofold in MVE-2 elicited peritoneal MPs (Adams et al., 1987). As 

14 summarized in Table 4-46, no treatment differences were seen in phagocytic ability in either 

15 resident or elicited MPs (Adams et al., 1987). Resident peritoneal MPs from formaldehyde16 treated mice were not different in their ability to bind or lyse tumor cells. Although 

17 formaldehyde treatment did not increase the ability of elicited MPs to bind tumor cells, lysis of 

18 the target cells (P815 tumor cells) was increased from 28 to 37% by formaldehyde treatment but 

19 only at the midrange target-to-effector-cell ratio tested in the assay (p < 0.05) (Adams et al., 

20 1987). Although this is statistically significant, the authors questioned the biological 

21 significance of this result since it was not observed at all three target cell ratios tested. However, 

22 an increase in tumor cell lysis in vitro would be consistent with the in vivo increased tumor 

23 resistance previously reported (Dean et al., 1984). The in vitro lysis response curve suggests that 

24 assay conditions may result in a maximum cytolysis near 40%. If so, any treatment effects on 

25 lysis would be difficult to discern at higher effector cell ratios. 

26 Jakab (1992) investigated the effect of formaldehyde exposure on the alveolar MPs and 

27 resistance to respiratory infections. The first set of experiments assessed bactericidal activity by 

28 directly quantifying the pulmonary bacterial loading after exposure to Staphylococcus aureus. 

29 White female Swiss mice were exposed to formaldehyde after bacterial infection (regimens A 

30 and C), before bacterial infection (regimen B), or before and after infection (regimen D) 

31 (Table 4-47). 

32
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1 Table 4-47. Formaldehyde exposure regimens for determining the effects of 

2 formaldehyde exposure on pulmonary S. aureus infection

3

Pre-infection treatment Postinfection treatment Results

Regimen A None 4 hours

0, 1, 5, 10, or 15 ppma

15 ppm, increased 

bacterial loading

Regimen B 18 hours

0, 0.5, or 1 ppmb

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/m3 5 formaldehyde. b

0, 0.62, or 1.2 mg/m3 6 formaldehyde.

7

8 Source: Jakab (1992).

9

10

11 For regimen A, mice were exposed to 0, 1, 5, 10, or 15 ppm (0, 1.2, 6.2, 12.3, or 

18.5 mg/m3 12 ) formaldehyde. For regimens B–D, mice were exposed to 0, 0.5, or 1 ppm (0, 6.2, or 

1.2 mg/m3 13 ) formaldehyde. A 30-minute exposure to an infectious aerosol of S. aureus deposited 

2 × 105 14 staphylococci in the lungs. Bacterial loading was determined in homogenized lung tissue 

15 by culturing diluted aliquots for an estimate of bacteria present immediately after loading and 4 

16 hours later. Bacterial loading was expressed as a percentage change between control and 

17 formaldehyde-exposed animals. Mice exposed to 15 ppm formaldehyde for the 4 hours 

18 following bacterial infection (regimen A) had approximately an 8% increase in bacteria, 

19 indicating decreased host resistance (p = 0.006) (Jakab, 1992) (Table 4-47). Mice receiving 

20 lower concentrations of formaldehyde following bacterial infection did not have increased 

21 pulmonary bacterial loading. Pre-infection exposure to 0.5 or 1.0 ppm did not change bacterial 

22 loading 4 hours after infection (regimen B). However, combining an 18-hour pre-infection 

23 formaldehyde exposure with a 4-hour postinfection 1 ppm formaldehyde exposure increased 

24 pulmonary bacterial loading by approximately 6.5% (p < 0.05). This effect was not seen with 

25 only a 0.5 ppm pre- and posttreatment regimen. Increased bacterial loading indicates that 

26 formaldehyde exposure (regimens A and D) reduced pulmonary bacterial resistance. This is in 

27 apparent contradiction to the findings of increased host resistance by Dean et al. (1984). 

28 However, there are important differences between the studies. The studies by Jakab (1992) are 

29 acute studies examining effects at the respiratory tract where direct effects are possible. 

30 Additionally, in some cases, the exposures were concurrent with bacterial infection, and it is 

31 difficult to distinguish the potential for formaldehyde effects directly on the mucociliary 

32 apparatus as a barrier to infection.
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1 A second set of experiments in the same report (Jakab, 1992) examined the effects of 

2 co-exposure to formaldehyde and carbon black on pulmonary infection with S. aureus. The 

3 particle size distribution of the carbon black aerosol was less than a 5 μm aerodynamic diameter 

4 and, therefore, 98% respirable. Female Swiss mice were exposed nose only to formaldehyde and 

carbon black. Experiments were run at two target concentrations: (1) 2.5 ppm (3.1 mg/m3 5 ) 

formaldehyde and 3.5 mg/m3 carbon black or (2) 5 ppm (6.2 mg/m3 6 ) formaldehyde and 

10 mg/m3 7 carbon black. Co-exposure was given either for 4 hours after a 30-minute S. aureus

8 infection or 4 hours/day for 4 days as a pretreatment prior to S. aureus infection. Bacterial 

9 loading was determined 0 and 4 hours after the S. aureus infection to assess bacterial survival. 

10 Formaldehyde-carbon black co-exposure did not alter bacterial survival either as a pretreatment 

11 or posttreatment to bacterial exposure. However, this exposure regimen was not run for 

12 formaldehyde or carbon black separately, and the 4 hours/day for 4 days pretreatment was not 

13 included in the formaldehyde alone experiments (Table 4-47).

14 Jakab (1992) also assessed the phagocytic activity of alveolar MPs collected by lavage at 

15 various time points after formaldehyde, carbon black, or co-exposure. Female Swiss mice were 

co-exposed to 5 ppm (6.2 mg/m3

) formaldehyde and 10 mg/m3 16 carbon black 4 hours/day for 

17 4 days. Mice were sacrificed and alveolar MPs harvested 1, 3, 5, 25, and 40 days after exposure. 

18 Mice exposed only to formaldehyde or carbon black were sacrificed 3, 10, 25, and 40 days after 

19 exposure. Fc-receptor-mediated phagocytosis was assessed ex vivo by using sensitized sheep 

20 RBCs. The phagocytic index (PI) was reported as the total number of RBCs in 100 MPs. 

21 Neither formaldehyde nor carbon black exposure alone significantly changed the PI (Jakab, 

22 1992). These findings are consistent with the first co-exposure experiment, since no changes in 

23 PI were seen immediately after exposure. However, co-exposure did decrease the PI of alveolar 

24 MPs in a time-dependent manner, with maximal decrease to less than 70% of controls by 25 days 

25 after exposure (Figure 4-11). Decreases in the PI reflect changes in both the percentage of 

26 phagocytic MPs and the number of RBCs phagocytized (Jakab, 1992). The PI recovered to 

27 control levels by 40 days postexposure.

28
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1

100

110

90

80

70

60

50

0 10 20 30 40

Days after last exposure

HCHO

Carbon

Carbon + HCHO

2

3

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

5 ppm formaldehyde, 10 mg/m3 5 carbon black, or both.

6

7 Note: Exposure was 4 hours/day for 4 days. Each value represents the mean ± 

8 SEM of five determinations.

9

10 Source: Redrawn from Jakab (1992).

11

12

13 Holmström et al. (1989b) evaluated the effects of long-term formaldehyde exposure on 

14 antibody production. Female Sprague-Dawley rats were exposed to 12.6 ppm formaldehyde 

(15.5 mg/m3 15 ) 6 hours/day, 5 days/week for 22 months. Body weight, tumor incidence, and 

16 pathology were reported elsewhere (Holmström et al., 1989b). Rats were given a subcutaneous 

17 injection of pneumococcal polysaccharide antigens or tetanus toxoid 21 to 25 days prior to 

18 sacrifice. The two vaccines chosen represent T-cell-dependent and T-cell-independent antigens, 

19 respectively. Antibody titers (IgG and IgM) were determined prior to vaccination and at 

20 sacrifice. Formaldehyde treatment had no effect on antibody titers either before or after 

21 vaccination (Holmström et al., 1989b).

22

23 4.2.1.4.1. Summary of formaldehyde effects on immune function. Although there were initial 

24 reports of systemic immunomodulation attributed to formaldehyde exposure (Leach et al., 1983), 

25 formaldehyde effects on measures of humoral and cell-mediated immunity were not confirmed 

26 by Dean et al. (1984). The authors did report increased host resistance to both tumor and 

27 bacterial tumor challenges after a 3-week exposure to 15 ppm formaldehyde. An increased Phagocytic index (% of control) Phagocytic index (% of control)
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1 resistance to these challenges, presented distal to the site of formaldehyde exposure 

2 (administered subcutaneously or intravenously), suggests a systemic effect of formaldehyde 

3 exposure. In addition, increased host resistance and hydrogen peroxide release from peritoneal 

4 MPs were reported and confirmed (Adams et al., 1987; Dean et al., 1984). Chronic 

5 inflammation and tissue damage to the respiratory mucosa expected with formaldehyde exposure 

6 may result in an up regulation of the MPS and therefore increase host immunity. It is unclear if 

7 this response would be specific to formaldehyde or similar to enhancement of immune function 

8 seen with chronic inflammation.

9 Jakab (1992) demonstrated decreased pulmonary resistance to bacterial infection where 

10 animals were exposed to 15 ppm formaldehyde immediately after bacterial loading or when they 

11 were given an 18-hour pre-exposure to formaldehyde followed by 1 ppm formaldehyde exposure 

12 after bacterial loading. The authors speculated that formaldehyde may directly act on pulmonary 

13 MPs, reducing their effectiveness. However, Jakab (1992) showed that there was no change in 

14 Fc-mediated phagocytosis of alveolar MPs immediately after formaldehyde exposures. 

15 Degradation of the protective mucus layer and possible epithelial cell damage may contribute to 

16 more effective bacterial infection in the presence of formaldehyde without a direct action on MP 

17 function. As mentioned above, degradation of the mucus layer may result in a more potent 

18 inoculation and therefore higher bacterial loading.

19 Although neither formaldehyde nor carbon black alone impacted Fc-mediated 

20 phagocytosis of alveolar MPs, Jakab (1992) demonstrated that there was decreased Fc-mediated 

21 phagocytosis after formaldehyde and carbon black co-exposure. Carbon black may have acted as 

22 a carrier for formaldehyde, allowing higher levels of formaldehyde to be delivered more deeply 

23 into the lungs than would be seen with formaldehyde alone. 

24 Formaldehyde is known to break down the mucus layer protecting the respiratory tract, 

25 allowing exposure of the underlying epithelium (Morgan et al., 1986a, c, d). Additionally, 

26 formaldehyde can directly induce tissue inflammation through sensory irritation via substance P 

27 from the trigeminal nerve (Fujimaki et al., 2004a). These actions together could contribute to 

28 some of the observed effects on immune response attributed to formaldehyde exposures. 

29 Degradation of the protective mucus layer would make antigens more available to the immune 

30 system. It has been shown that direct application of an antigen to the nasal associated lymph 

31 tissue, bypassing the mucus layer, is a more effective delivery of antigen (Hou et al., 2002). 

32 Therefore, increased availability of these antigens to the immune system may in part explain 

33 observed increased antibody production seen against ovalbumin (OVA) or common dust mite 

34 allergen (Der f) during formaldehyde exposure (Sadakane et al., 2002; Riedel et al., 1996; 

35 Tarkowski and Gorski, 1995). Neurogenic inflammation may also contribute to more efficient 
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1 antigen processing and presentation by activation of resident MPs. These factors are consistent 

2 with the observation that formaldehyde exposures do not affect antibody production to antigens 

3 administered outside of the respiratory tract, even after chronic exposures (Holmström et al., 

4 1989b).

5 This effect was initially observed several days after exposure was ended with maximal 

6 suppression seen 25 days after a 4-day formaldehyde exposure. The delayed onset of this 

7 response, however, suggests an effect beyond the POE effects observed at the time of exposure.

8 Table 4-48 presents a summary overview of the effects of formaldehyde on immune function in 

9 laboratory animals.

10

11 4.2.1.5. Hypersensitivity and Atopic Reactions

12 Adverse reactions in humans exposed to formaldehyde in the workplace and homes have 

13 been reported, which are consistent with an allergic response or a chemical sensitivity (see 

14 Section 4.1.1 for details). Rashes and skin reactions are reported in some individuals after 

15 dermal exposures, and in some cases exacerbation of asthma is reported after inhalation of 

16 formaldehyde. However, the reports of human reactions do not allow a clear determination of 

17 whether this sensitization is immunogenic or neurogenic in origin. Formaldehyde-induced 

18 sensitization may have both neurogenic and immunologic components. Numerous animal 

19 studies have been conducted in order to understand the potential for sensitization to 

20 formaldehyde. Although hypersensitivity and allergic sensitization are often considered solely 

21 immunologic in origin, neurogenic mechanisms may result in bronchial hypersensitivity and 

22 increased immunologic sensitization. Therefore, the animal studies regarding formaldehyde23 induced sensitization are evaluated discretely in order to examine these etiologic possibilities. 

24 Classically, hypersensitivity is characterized as an immune response to an antigen, 

25 resulting in an inflammatory reaction that itself damages the tissues or is otherwise harmful 

26 (Kuby, 1991). These reactions may be localized, as in topical dermatitis, or systematic, as in 

27 anaphylactic shock from an allergen. Hypersensitivity can be mediated by a humoral immune 

28 response or by a cell-mediated immune response. Four classes of hypersensitivity are generally 

29 recognized that differ in their immune system components and functions. Although a single 

30 agent (e.g., penicillin) may induce all four types of hypersensitivity, it is more usual for an agent 

31 to primarily induce one form of hypersensitivity.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-226

Table 4-48. Summary of immune function changes due to inhaled formaldehyde exposure in experimental animals

Species

No./

group Treatmenta Observations

LOAEL/

NOAEL Reference

F344 rats 8 0, 3, 16, 61, 99 ppm 

6 hours/day, 

5 days/week for 

4 weeks

No effects at 3 ppm. Mixed results at higher doses that were not 

consistent.

NAb Leach et al. 

(1983)

B6C3F1 

mice 

(female)

10 15 ppm 6 hours/day, 

5 days/week for 

3 weeks

Increased H2O2 production, and increased host resistance to tumor 

formation, but other immune parameters unchanged.

LOAEL 15 ppm Dean et al. 

(1984)

B6C3F1 

mice 

(female)

Pooled 

MPs from 

a number 

of mice

15 ppm 6 hours/day, 

5 days/week for 

3 weeks

Increased H2O2 production in MVE-2-elicited peritoneal MPs. LOAEL 15 ppm Adams et al. 

(1987)

White 

Swiss mice 

(female)

18 0, 1, 5, 10, or 

50 ppm for 18 hours 

before and/or 

4 hours after a 

30-minute exposure 

to bacterial infection 

(S. aureus)

Combining an 18-hour pre-exposure to formaldehyde with 4-hour 

postexposure to formaldehyde increased bacterial loading at 1 ppm by 

6.5%.

LOAEL 1 ppm Jakab (1992)_

White 

Swiss mice 

(female)

18 5 ppm (2.6 mg/m3) 

formaldehyde and 

10 mg/m3 carbon 

black 4 hours/day for 

4 days

Phagocytic index was decreased by co-exposure to formaldehyde and 

carbon black but not by either insult alone.

NA Jakab (1992)

SpragueDawley rats 

(female)

5 12.6 ppm 

6 hours/day, 

5 days/week, 

22 months

Formaldehyde treatment had no effect on antibody titers either before 

or after vaccination with pneumococcal polysaccharide antigen or 

tetanus toxoid.

NA Holmström et 

al. (1989b)

NA = not applicable.
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1 Chemical sensitivity generally implies a neurogenically induced sensitization (Meggs, 

2 1995). A chemical may directly interact with sensory nerves, releasing mediators that trigger 

3 inflammation, such as substance P (a tachykinin). Repeated exposure to the same chemical is 

4 hypothesized to potentiate neurogenic inflammation (Meggs, 1995). The resulting signs of tissue 

5 inflammation may be similar to immunogenic inflammation, but there would be no requirement 

6 that the immune system recognize the chemical as an antigen for this type of response. 

7 Therefore, a chemical may induce one or more clinical signs of atopic asthma without a type 1, 

8 IgE-mediated hypersensitivity response. One form of sensitivity, , directly affects sensory nerve 

9 endings, resulting in neurogenic inflammation and is a well-known health effect attributed to 

10 formaldehyde. Neurogenic responses may result from the direct and acute interaction of the 

11 chemical with sensory nerve ending receptors of the trigeminal nerve that may lead to persistent 

12 rhinitis and an asthma-like reactive airway dysfunction syndrome that may develop after short13 term human exposures (Brooks et al., 1985). Thus, there is evidence to suggest that neurogenic 

14 inflammation may contribute to observed increases in formaldehyde-induced airway 

15 hyperresponsiveness and atopic responses. The available animal studies that have investigated 

16 formaldehyde-induced airway hyperresponsiveness and atopic responses are summarized below.

17

18 4.2.1.5.1. Inhalation studies in experimental animals. This section summarizes animal studies 

19 informing the role of formaldehyde-induced chemical sensitization. The symptoms of 

20 sensitization (atopy, airway hyperresponsiveness) are frequently associated with immunologic 

21 markers (cytokine production, leukocyte infiltration histamine release, and antibody production) 

22 but may be mediated by neurogenic sensory irritation, principally by activation of the trigeminal 

23 nerve (see Section 4.1.1.1 for a discussion of sensory irritation). The animal studies that 

24 illuminate these neurogenic and immunologic responses are discussed outside of the classic 

25 neurotoxicology and immunotoxicology study summary sections to allow synthesis of these data.

26 Sensitization to chemical exposure by inhalation often manifests as an allergic or 

27 asthmatic response as characterized by BC or BHR. This sensitization may be a result of 

28 immune involvement, as in the case of hypersensitivity, or a neurogenic sensitization, where a 

29 chemical may directly stimulate inflammation. Asthma is a specific manifestation of IgE30 mediated hypersensitivity, characterized by BHR and airway inflammation, resulting in lower 

31 airway obstruction (Fireman, 2003; Kuby, 1991). In asthma, an allergen capable of cross-linking 

32 membrane-bound IgE on mast cells initiates immunogenic inflammation resulting in an influx of 

33 eosinophils, neutrophils, and lymphocytes. Mediators of BC, including histamine, eicosanoids, 

34 and bradykinin (Kuby, 1991), are released during this process. Prior exposure to the allergen can 
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1 increase allergen-specific IgE, potentiating the allergic reaction; this is immunogenic 

2 sensitization.

3 Biagini et al. (1989) evaluated the effect of a single pulmonary exposure of formaldehyde 

4 on pulmonary mechanics, including BC. The researchers chose cynomolgus monkeys known to 

5 be hyperreactive to methacholine (acetyl-β-methacholine chloride), which is a direct-acting 

6 stimulant of BC (Cain, 2001). Measures of pulmonary mechanics included pulmonary flow 

7 resistance; dynamic compliance; PEFR; FVC; FEV; FEF25–75%, and 50% of VC; and FEFs 

8 normalized for VC. Nine cynomolgus monkeys were exposed to increasing levels of 

9 methacholine for 1 minute at 10-minute intervals (0, 0.125, 0.5, 2, and 8 mg/mL) as an aerosol 

10 (0.065 mL/minute with a mean aerodynamic diameter of 1.0–1.5 μm). Pulmonary mechanics 

11 were measured to establish each monkey’s response to methacholine. Methacholine challenge, 

12 as the positive control, increased pulmonary flow resistance at increasing levels of methacholine 

13 (0.125, 0.5, 2, and 8 mg/mL) to 196 ± 16, 285 ± 57, 317 ± 64, and 461 ± 120 % of baseline 

14 levels, respectively. After a 2-week recovery period, each methacholine-sensitized monkey was 

15 exposed to 2.5 ppm formaldehyde (generated from formalin, 15% methanol) for 10 minutes. 

16 Measures of pulmonary function were performed at 2, 5, and 10 minutes after exposure.

17 Formaldehyde exposure increased pulmonary flow resistance from 11.3 ± 1.4 cm H2O 

18 prior to formaldehyde exposure to 16.1 ± 2.1, 16.9 ± 2.8, and 20.0 ± 3.4 cm H2O at 2, 5, and 

19 10 minutes after 2.5 ppm formaldehyde exposure (with 142, 150, and 177 % change, 

20 respectively). All other measures of formaldehyde-induced pulmonary mechanics were not 

21 significantly different from controls. Increased pulmonary flow resistance, a measure of 

22 increased BC, was induced by formaldehyde challenge in previously sensitized mice. However, 

23 the differences between methacholine challenge and formaldehyde challenge were not 

24 statistically significant. Although both formaldehyde challenge and methacholine challenge 

25 increased pulmonary flow resistance, there was no correlation between individual methacholine 

26 responsiveness and the magnitude of effect after formaldehyde exposure (p > 0.1). Therefore, 

27 although formaldehyde exposure stimulated BC similarly to a known direct stimulating agent, 

28 formaldehyde may not work through the same site of action as methacholine. 

29 Swiecichowski et al. (1993) assessed pulmonary resistance and airway reactivity due to 

30 formaldehyde exposure alone and in response to increasing doses of acetylcholine chloride (a 

31 direct-acting BC agent) after formaldehyde exposure in vivo. Male Hartley guinea pigs (eight 

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/m3 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/m3 33 ) 

34 formaldehyde for 8 hours. Total pulmonary resistance increased after 2 hours formaldehyde 

35 exposure at 9.4 and 31.1 ppm and reached similar peak resistance at the end of the exposure 
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1 period. This effect was rapidly reversible, with values returning to baseline within 30 minutes 

2 after exposure. Although 2-hour exposures at 3 and 1 ppm did not increase pulmonary 

3 resistance, 8-hour exposures at 0.3 and 1 ppm did increase pulmonary resistance to similar levels 

4 as the 2-hour exposure at 30 ppm. The results indicate that both concentration and exposure time 

5 impacted the measured increase in pulmonary resistance. However, a simple multiplicative 

6 model (e.g., C × t) does not adequately represent the effects observed. It is noted that an 8-hour 

7 exposure at 1 ppm (8 ppm-hours), reached approximately the same pulmonary resistance as 2 

8 hours at 9.4 ppm (19 ppm-hours). This may in part be due to a maximum practical increase in 

9 pulmonary resistance in the animals. Conversely, there was no effect at 3 ppm for 2 hours 

10 (6 ppm-hours), although significant increase in pulmonary resistance was recorded after an 

11 8-hour exposure at 0.3 ppm (2.4 ppm-hours). Formaldehyde does not appear to exert its effects 

12 via a classic C × t paradigm. Exposure concentration, however, did seem to impact recovery 

13 time.

14 In addition, specific pulmonary resistance and airway reactivity to increasing doses of 

15 intravenous acetylcholine chloride, a direct respiratory stimulant, were measured immediately 

16 after formaldehyde exposure for up to 60 minutes. Formaldehyde-induced airway 

17 hyperreactivity was defined as a decrease in the level of acetylcholine chloride needed to 

18 produce twice the basal specific resistance (effective dose [ED]200). The dose of acetylcholine 

19 chloride required to double the specific pulmonary resistance (ED200) and airway reactivity was 

20 decreased in animals exposed for 2 hours to formaldehyde. When the duration was extended to 8 

21 hours of formaldehyde exposure, the effective dose of formaldehyde required to elicit a doubled 

22 pulmonary resistance (ED200) in the presence of acetylcholine chloride was decreased to 

23 1.07 ppm. Lower ED200s were recorded in formaldehyde-treated animals. This indicates that 

24 less acetylcholine was needed to produce BC when formaldehyde was present. Thus, 

25 formaldehyde can exacerbate BHR. Additionally the formaldehyde-induced effect increased 

26 with duration of exposure, indicating that time as well as exposure concentration are factors in 

27 the magnitude of the response. Directly induced increases in airway hyperreactivity peaked 1 

28 hour after exposure and persisted 6 hours after exposure. 

29 In a second set of experiments, male Hartley guinea pigs were treated for 8 hours at 

3.4 ppm (4.2 mg/m3 30 ) in order to measure airway hyperreactivity ex vivo (Swiecichowski et al., 

31 1993). After formaldehyde exposure, tracheae were excised and mounted in tissue baths, where 

32 tracheal contraction was measured in response to direct application of acetylcholine and then 

33 carbachol. Tracheae from similarly exposed guinea pigs were fixed and sectioned for histologic 

34 examination and were assessed for signs of inflammation. Formaldehyde exposure did not 

35 increase ex vivo tracheal constriction and suggests that changes in airway reactivity were 
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1 produced due to both local humoral changes and neural reflexes. However, no changes in 

2 epithelial cell morphology or influx of inflammatory cells were recorded even up to 4 days after 

3 formaldehyde exposure ended. The authors speculated on possible MOAs for BHR, such as the 

4 role of an irritant receptor or altered epithelial cell biochemistry. It may be that the window of 

5 acute inflammation occurred early in the exposure protocol and was resolved by the time of first 

6 measurement, after 8 hours of exposure. The absence of inflammatory markers may argue 

7 against a classic type 1 sensitivity. 

8 The binding of an allergen to receptor bound IgE triggers degranulation of mast cells and 

9 basophils, releasing mediators of type 1 hypersensitivity, including the histamine responsible for 

10 BC. Brown Norway (BN) rats are known for their high capacity for IgE production and airway 

11 hyperresponsiveness in response to allergens or other chemicals; they have often been used as a 

12 model of allergic respiratory disease. Ohtsuka et al. (1997) compared the effects of 

13 formaldehyde exposure on the nasal epithelium of F344 and BN rats. If the formaldehyde14 induced inflammatory response in the nasal epithelium is IgE mediated, BN rats would be 

15 expected to display more severe effects of formaldehyde exposure than F344 rats. Both strains 

16 of age- and sex-matched rats were exposed to formaldehyde aerosol for 3 hours/day, 5 

17 days/week for 2 weeks. The aerosol was generated from a 1% formaldehyde solution by a two18 fluid atomizer, and formaldehyde level was maintained at 2 mg (1% sol.)/L (approximately 

16 ppm or 20 mg/m3 19 ), by adjusting the flow rate for formaldehyde solution to the atomizer. 

20 During the course of exposure, the following clinical signs were monitored: abnormal 

21 respiration, stridor wheezing, nasal discharge, and sneezing. Rats were weighed weekly. Two 

22 days postexposure, rats were sacrificed and tissues from the head, trachea, and lungs were fixed 

23 and sectioned. Transverse sections were taken at the following palatal landmarks from three 

24 animals: level 1 (lateral edge of incisor teeth), level 2 (between incisive papilla and the first 

25 palatal ridge), and level 3 (on the second upper molar). The nasal septa of the remaining two 

26 animals were revealed for examination by electron microscopy. 

27 Formaldehyde-treated F344 rats showed less body weight gain over the 2-week 

28 treatment, resulting in lower body weight at week 1 and week 2 than F344 controls (p < 0.05 and 

29 0.01). Body weights of formaldehyde-treated BN rats were unchanged from BN controls. The 

30 authors observed fewer clinical signs of respiratory irritation in the formaldehyde-exposed BN 

31 rats compared with formaldehyde-exposed F344 rats, such as abnormal respiration (three versus 

32 five) and nasal discharge (three versus five). Histologic analysis of lung and trachea tissues 

33 revealed no distinct signs of inflammation in either strain. Formaldehyde exposure induced cell 

34 damage in URT tissues. Epithelial cell damage was milder and impacted a smaller portion of the 

35 URT in BN rats compared with F344 rats. Squamous metaplasia were present in the respiratory 
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1 epithelium (levels 1 and 2) in both strains in formaldehyde-treated rats. However, a distinct 

2 keratinized layer was noted in level 1 epithelium of F344 rats, and the extent of lesions in level 2 

3 respiratory epithelium was much greater than that seen in BN rats. Additionally, the olfactory 

4 epithelium (level 2) in formaldehyde-exposed F344 rats exhibited degeneration, necrosis, and 

5 desquamation not seen in BN rats. Mild squamous metaplasia was noted in level 3 of the 

6 respiratory epithelium in the treated F344 rats but not the treated BN rats. No pulmonary 

7 function measurements were taken, and, thus, no direct comparison in BHR or BC between BN 

8 and F344 rats in response to formaldehyde can be made. It appears that BN rats are more 

9 resistant to formaldehyde-induced cell damage than are F344 rats, despite the fact that BN rats 

10 are known to be IgE responders. These results suggest that IgE responsiveness may be 

11 protective of formaldehyde-induced cell damage, or IgE may not play a role at all. The authors 

12 note that their earlier research indicated the BN rats have well-developed submucosal glands and 

13 speculate that greater mucus flow may be partly responsible for the greater resistance of BN rats 

14 to the histologic signs of formaldehyde toxicity.

15 In a subsequent study in the same laboratory, Ohtsuka et al. (2003) compared histology 

16 and cytokine profiles in the nasal mucosa of formaldehyde-treated F344 and BN rats. 

17 Formaldehyde aerosol was generated as above and rats (nine per group) were exposed 

3 hours/day for 5 days to approximately 16 ppm of formaldehyde (20 mg/m3 18 ). Clinical signs 

19 were recorded daily, and monitored respiratory parameters included abnormal respiration, stridor 

20 wheezing, nasal discharge, and sneezing. Tissue sections of the nose (five rats per group) were 

21 prepared for light microscopy as above: transverse sections at levels 1, 2, and 3. Th-1 cytokines 

22 (IFN-γ, IL-2) and Th2 cytokines (IL-4 and IL-5) were determined from the whole nasal mucosa 

23 in four rats of each treatment group. 

24 As expected, lesions and neutrophilic infiltration were more severe in F344 

25 formaldehyde-exposed rats compared with treated BN rats. In addition, lesions were observed in 

26 all three levels of epithelium examined in F344 rats and impacted both respiratory and olfactory 

27 epithelium. Mucosal lesions in formaldehyde-treated BN rats impacted the respiratory 

28 epithelium of levels 1 and 2 only. Changes in formaldehyde-induced cytokine mRNA 

29 expression were modest in both strains. Th-1-related cytokines (IFN-γ, Il-2) in formaldehyde30 treated BN rats were significantly decreased compared with control BN rats. A similar, although 

31 not statistically significant, decrease in Th-2 cytokines (IL-4, IL-5) was observed in 

32 formaldehyde-treated BN rats compared with unexposed BN rats. There were no treatment 

33 differences in either Th-1 or Th-2 cytokine expression in formaldehyde-treated F344 rats 

34 compared with unexposed F344 rats. The modest changes in cytokine profile reported in 

35 formaldehyde-treated BN rats were not consistent with type 1 hypersensitivity since type 1 
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1 hypersensitivity reactions generally result in increased Th-2 cytokines. The mRNA expression 

2 results were not corroborated with protein levels and may not have been captured at their peak 

3 expression levels.

4 Lee et al. (1984) evaluated the potential for formaldehyde to act as a sensitizing agent 

5 through different routes of exposure in guinea pigs. The inhalation studies will be highlighted 

6 here. Dermal exposure and associated contact sensitivity results will be discussed in the dermal 

7 exposure section (4.2.1.5.2). Three groups of male English smooth-haired guinea pigs 

(four/group) were exposed via inhalation to either 6 or 10 ppm (7.4 or 12.3 mg/m3 8 ) 

9 formaldehyde 6 hours/day for 5 consecutive days. Depending on the group, animals were then 

10 subjected to bronchial provocation challenge with 2 or 4 ppm formaldehyde on day 7 or days 7, 

11 22, and 29 after exposure (see Table 4-49 for clarification). 

12

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

14 different routes of exposure: inhalation, dermal, and injection 

15

Formaldehyde 

exposure

Bronchial provocation 

challenge Skin test

Blood drawn 

for antibody 

titer

Group I—Inhalation 6 ppm formaldehydea, 

days 1–5

Day 7

2 ppm formaldehydea 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 μL 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

17 Source: Lee et al. (1984).

18

19

20 Dermal and injection groups are shown for comparison. Pulmonary hypersensitivity was 

21 assessed by measuring respiratory rate and tidal volume in response to exposure to 2 ppm 

22 formaldehyde challenge for 1 hour, 2 days postexposure for all three groups, and additional 

23 measurements were taken 22 and 29 days postexposure for group III. Blood was drawn to 

24 characterize IgE antibodies to formaldehyde in a passive cutaneous anaphylaxis (PCA) assay. 

25 Respiratory rate was measured following initial formaldehyde exposure and again after bronchial 

26 challenge with formaldehyde.
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1 Respiratory rate (exhibited as a pause during expiration) was depressed by 45% following 

2 exposure to 10 ppm formaldehyde during the first hour of exposure. During the first hour of 

3 exposure, decreased respiratory rate was accompanied by a pause during expiration that has been 

4 categorized as RB and indicated sensory irritation. The decreased respiratory rate is consistent 

5 with URT sensory irritation and induction of the trigeminal (neurogenic) reflex (Lee et al., 

6 1984). After the first hour of exposure, decreased respiratory rate was characterized by a pause 

7 between breaths, which is similar to the breathing pattern seen in mice exposed to formaldehyde 

8 via tracheal cannula (Alarie, 1981). This suggests a separate effect of formaldehyde on the LRT 

9 after deep penetration of formaldehyde and suggests pulmonary irritation (Lee et al., 1984).

10 However, subsequent bronchial provocation challenge with either 2 or 4 ppm 

11 formaldehyde for either 1 or 4 hours failed to elicit immediate or delayed-onset respiratory 

12 sensitization (Table 4-50). Respiratory rates were reported as being within ±20% of pre13 challenge levels (data not shown) and did not reflect statistical significance (Lee et al., 1984). 

14 Moreover, increased respiratory sensitivity was not observed in animals that had received an 

15 emulsification of formaldehyde and Freund’s complete adjuvant by injection. Only two to four 

16 animals given formaldehyde injections in the presence of Freund’s complete adjuvant developed 

17 a low titer of antibodies to formaldehyde (Lee et al., 1984). 

18

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

20 through inhalation, topical application, or footpad injection 

21

Exposure route Pulmonary sensitization Dermal sensitization 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

23 Source: Lee et al. (1984).

24

25

26 Thus, inhalation exposure to 6 or 10 ppm formaldehyde (8 hours/day for 5 days) followed 

27 by bronchial challenge with 2 or 4 ppm formaldehyde failed to result in respiratory sensitivity 

28 defined as greater than 20% change in respiratory rate. Second, for animals that received an 

29 injection of formaldehyde with Freud’s adjuvant, it was not effective in inducing pulmonary 

30 sensitivity. While neither inhaled formaldehyde challenge nor injected formaldehyde and 

31 Freud’s adjuvant emulsion were effective in producing pulmonary sensitivity, this study relied

32 on increased respiratory rate as an indication of hyperresponsiveness and may not be an accurate 
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1 measure of hyperresponsiveness. Thus, overall, conclusions are uninformative due to study 

2 design flaws.

3 Riedel et al. (1996) tested the effects of formaldehyde inhalation on the development of 

4 sensitization to a known allergen. Female Perlbright-white Dunkin-Hartley guinea pigs (12 per 

5 group) were exposed to 0, 0.13, or 0.25 ppm formaldehyde 8 hours/day for 5 consecutive days. 

6 On day 5, the animals were sensitized to the common model allergen, OVA, in a 3-minute, head7 only exposure to an aerosol of a 5% OVA solution. A booster sensitization with OVA occurred 

8 on day 19. A compressor nebulizer with an output rate of 0.75 mL/minute generated the aerosol. 

9 Particle size ranged from 0.5 to 5.0 μm. On day 26, bronchial provocation testing was conducted 

10 with 1% OVA challenge (aerosol). Blood samples were taken and anti-OVA IgG antibodies 

11 were quantified by ELISA. Significant airway obstruction was defined as an increase in 

12 compressed air in the lung that cannot be expired. Three guinea pigs were exposed to 

13 formaldehyde (0.20 ppm) or clean air for 5 days. Immediately after exposure, lung and tracheal 

14 tissues were fixed for histologic and morphometric evaluation. Wall thickness of bronchial and 

15 alveolar septa was measured systematically with a microscope-digitizing-table set.

16 Significant airway obstruction as measured by compressed air was seen in 3 of 12 

17 controls, 8 of 12 0.13 ppm-exposed, and 10 of 12 0.25 ppm-treated animals after OVA challenge. 

18 The average airway obstruction was increased after 0.25 ppm (mean = 0.35 mL, p < 0.01) but not 

19 after 0.13 ppm formaldehyde exposure. However, individual response to OVA sensitization was 

20 highly varied, and animals exhibiting a 10-fold increase in obstruction (measured as compressed 

21 air) were seen in both treatment groups (0.13 and 0.25 ppm). Even at the lower exposure (0.13 

22 ppm), biologically significant responses were seen in individuals (Figure 4-12). 

23 Specific anti-OVA antibodies (IgG1 class) were not detected in animals prior to 

24 sensitization or in control-treated animals after sensitization . Measurable anti-OVA antibodies 

25 were elevated in 3 of 12 (at 0.13 ppm) and 6 of 12 (at 0.25 ppm) formaldehyde-treated guinea 

26 pigs after sensitization (Figure 4-13). The average anti-OVA titer for the high-dose group was 

27 significantly higher than for controls (p < 0.05). The individual responses at the 0.13 ppm 

28 exposure level indicate that, although the average group OVA titer may not have reached 

29 statistical significance, there was a measureable biological response in three individuals. These 

30 results indicate that formaldehyde exposure can sensitize previously naïve (non-sensitized) 

31 animals to OVA.
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

Controls FA 0.13 ppm FA 0.25 ppm

CA (ml)

1

0,8

0,6

0,4

0,2

0

1

2

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

4 obstruction following formaldehyde exposure in guinea pigs after OVA 

5 sensitization and OVA challenge.

6

7 Note: CA = compressed air; FA = formaldehyde; ― = median; ** = p < 0.01. 

8

9 Source: Redrawn from Riedel et al. (1996).

10

11 +++

12

13

14

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

16 guinea pigs prior to OVA challenge.

17

18 Note: EU = experimental units; FA = formaldehyde; ― = median; ** = p < 0.01. 

19

20 Source: Redrawn from Riedel et al. (1996).

21

22

Controls FA 0.13 ppm FA 0.25 ppm

 1.000

100

10

Anti-OVA-lgG1 [EU]
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1 The only significant, treatment-related histologic change was bronchial edema, with 

2 thickening of the bronchial wall in formaldehyde-exposed animals compared with non-treated 

3 animals subjected to OVA sensitization and subsequent OVA challenge. Bronchial walls were 

4 measured as 40.9 ± 2.5 versus 28.2 ± 1.2 μm. No signs of inflammation in the bronchial mucosa 

5 were seen with this edema. 

6 Tarkowski and Gorski (1995) exposed female Balb/C mice to 0 or 6.63 ppm (0 or 

2 mg/m3 7 ) formaldehyde for either 6 hours/day for 10 days or 6 hours/day once a week for 

8 7 weeks. All mice were sensitized intranasally to OVA for 10 days or once a week for 7 weeks. 

9 IgE anti-OVA titers were determined from sera collected from four mice every 8 days (1 day 

10 after OVA booster) by PCA. A parallel experiment to compare the role of the route of 

11 administration was conducted with I.P. rather than intranasal sensitization (1 μg OVA once every 

12 7 days). 

13 OVA titers increased similarly in control mice and mice exposed to formaldehyde once a 

14 week (Figure 4-14). In contrast, mice exposed to formaldehyde 6 hours/day for 10 consecutive 

15 days at the beginning of the experiment had increased anti-OVA beginning after the fourth OVA 

16 sensitization, which continued to increase through seven doses of OVA to a peak of 70 PCA 

17 units (p < 0.01) (Figure 4-14). Anti-OVA IgE titers were significantly different between 

18 formaldehyde-treated and nonexposed mice. 

19

20

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

22 formaldehyde for 10 consecutive days or once a week for 7 weeks.

23

24 Note:  = control mice;  = formaldehyde once a week × 7;  = formaldehyde 

25 10 days.

26 Source: Redrawn from Tarkowski and Gorski (1995).

27
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1 Intraperitoneal sensitization to OVA was much more effective than intranasal 

2 sensitization, resulting in titers as high as 1,000 after 4 weeks. However, there were no 

3 differences between controls and animals treated with formaldehyde via the I.P. route of 

4 exposure. Thus, formaldehyde administered intranasally 6 hours/day for 10 days may facilitate 

5 the sensitization to allergens. These changes were not observed when formaldehyde was 

6 administered intranasally once a week for 10 weeks or via I.P. injection (Tarkowski and Gorski, 

7 1995). The authors speculate that formaldehyde may increase permeability of respiratory 

8 epithelium and destruction of immunologic barriers. Thus the respiratory tract may become 

9 vulnerable to inhaled allergens after formaldehyde exposure (Tarkowski and Gorski, 1995). 

10 Ito et al. (1996) conducted three experiments to examine the effects of acute 

11 formaldehyde exposure on bronchoconstriction and the mediators of vascular permeability. 

12 Male Wistar rats (five to eight per group) were exposed to 0, 2, 5, 15, or 45 ppm (0, 2.5, 6.2, 

18.5, or 55.4 mg/m3 13 ) formaldehyde for 10 minutes. Baseline pulmonary insufflation and blood 

14 pressure were determined prior to formaldehyde exposure and monitored throughout the 

15 experiment. Vascular leakage was measured by injection of Evans blue dye prior to the 

16 experiment and determining extravasation 5 minutes postexposure. Briefly, lungs were perfused 

17 with 0.9% saline through an aortic cannula. The lower portion of the trachea and main bronchi 

18 were removed, and the Evans blue dye remaining was determined and expressed as ng dye/g 

19 tissue. A second experiment was conducted to determine if dye leakage continued to increase 

20 after exposure. Seven rats were exposed to 15 ppm formaldehyde for 10 minutes, as above. 

21 Evans blue dye was injected 5 minutes postexposure, and tissues were perfused and excised 

22 15 minutes later. The final experiment was conducted to determine the effect of certain receptor 

23 agonists on the formaldehyde-induced microvascular leakage. Ten groups of Wistar rats (four to 

24 seven per group) were exposed to 15 ppm formaldehyde and injected with Evans blue dye, as 

25 before. However, each receptor agonist under test or saline sham was injected 4–5 minutes prior 

26 to the 10-minute formaldehyde exposure. Agonists tested included tachykinin NK1 receptor 

27 antagonist (CP-99,994) at 1, 3, or 6 mg/kg; a bradykinin B2 receptor antagonist (HOE 140) at 

28 0.65 mg/kg; and a histamine H1 receptor antagonist (ketotifen) at 1 mg/kg.

29 Formaldehyde exposure did not change pulmonary insufflation pressure or blood 

30 pressure. Formaldehyde increased vascular permeability in a concentration-dependent manner in 

31 both the trachea and main bronchi for the first 5 minutes after exposure, as measured by Evans 

32 blue dye extravasation (Ito et al., 1996) (Figure 4-15). Vascular permeability was not increased 

33 by formaldehyde exposure from 5 to 15 minutes postexposure (experiment 2). Administration of 

34 a selective NK1 receptor antagonist (CP-99,994) inhibited the formaldehyde-induced vascular 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 permeability, reducing Evans dye extravasation to control levels at the 3 and 6 mg/kg doses 

2 (Figure 4-16). 

3











 200

100

0

Room 2 5 15 45 2 5 15 45

Air Formaldehyde (ppm)

Formaldehyde (ppm)

Trachea Main Bronchi

4

5

6 Figure 4-15. Vascular permeability in the trachea and bronchi of male 

7 Wistar rats after 10 minutes of formaldehyde inhalation.

8

9 Note: Vascular permeability was tested by an increase in Evans blue dye 

10 extravasation in the tissue. Solid bars: formaldehyde; open bars: room air, n = 7. 

Values are the means ± SEM of five to seven animals. *p < 0.05 and † 11 p < 0.01 

12 versus room-air-exposed group (Williams’ test).

13

14 Source: Redrawn from Ito et al. (1996).

15

16
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1

200

100

0

Sham Formaldehyde Sham Formaldehyde

Trachea Main Bronchi

 











   



2

3

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

5 vascular permeability in the trachea and bronchi male of Wistar rats. 

6

7 Note: Vascular permeability was tested by an increase in Evans blue dye 

8 extravasation. Rats were treated i.v. with 1, 3, or 6 mg/kg CP-99,996 (open bars), 

9 0.65 mg/kg HOE 140 (hatched bars), 1 mg/kg ketotifen (solid bars), or vehicle 

10 (shaded bars) before formaldehyde challenge. Sham: animals were exposed to the 

11 sham gas for 15 ppm formaldehyde (10 minutes) after pretreatment with 0.9% 

12 saline (0.5 mL/kg i.v.). Data are the means ± SEM of six to seven rats/group. 

13 *p < 0.05 versus sham-stimulated group (unpaired Student’s t test or Welch’s 

14 test). †p < 0.05. ††p < 0.01 versus 0.9% saline-pretreated, formaldehyde15 exposed control group (Williams’ test).

16

17 Source: Redrawn from Ito et al. (1996).

18

19 Neither the bradykinin B2 nor histamine H1 receptor agonists affected formaldehyde20 induced vascular permeability (Ito et al., 1996). Therefore, the immediate effect of 

21 formaldehyde exposure on vascular permeability is mediated, at least in part, through the NK1

22 receptor but does not seem to require the B2 or H1 receptors. This implies a role for tachykinins 

23 in formaldehyde-induced vascular permeability. These findings suggest a neurogenic 

24 inflammatory response because the tachykinins are released from sensory nerve endings in the 

25 trachea and bronchi, whereas bradykinin is released from mast cells.

26 Sadakane et al. (2002) investigated the effects of formaldehyde exposure on airway 

27 inflammation caused by Der f. Two groups of male outbred ICR mice (18/group) were exposed 

28 to an aerosol of 0.5% formaldehyde solution produced by an ultrasonic nebulizer for 15 minutes, 

29 once a week for 4 weeks. Two groups were similarly treated but exposed to saline aerosol only. 

30 Details of the aerosol generation and resulting magnitude of exposure were not given. One 

31 group each of control and formaldehyde-exposed mice was sensitized to Der f by an injection 1 

32 day prior to formaldehyde exposure (1.5 mg/animal). The same groups were challenged with 
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1 intratracheal instillation of Der f (10 μg/animal) after 4 weeks. Three days after allergen 

2 challenge, mice were sacrificed and blood plasma and lung tissue were collected. Blood plasma 

3 was analyzed for Der f-specific immunoglobulins (IgG1 and IgE). Lungs from nine mice in each 

4 treatment group were homogenized, and Th1 cytokine IL-2, Th2 cytokines IL-4 and IL-5, 

5 granulocyte macrophage-colony-stimulating factor (GM-CSF), and the “chemokine regulated 

6 upon activation, normal T-cell expressed and secreted” (RANTES) protein levels were quantified 

7 in the supernatant via ELISA. Lungs from nine mice in each group were fixed, sectioned, and 

8 stained to evaluate eosinophil infiltration, lymphocyte infiltration, goblet cell proliferation, and 

9 localization of RANTES in the airway epithelium. 

10 Der f-specific IgG1 was present in blood plasma of sensitized mice but was unchanged 

11 by formaldehyde exposure (Sadakane et al., 2002). IgE was too low to titer. IL-2 and GM-CSF 

12 were undetected in lung homogenate supernatant, and Il-4 was unchanged by sensitization or 

13 formaldehyde exposure. However, RANTES was increased by both formaldehyde exposure and 

14 allergen sensitization and challenge (Table 4-51). These increases were more pronounced but 

15 less than additive for formaldehyde-exposed, allergen-sensitized mice. IL-5 was increased by 

16 allergen but unaffected by formaldehyde exposure only. However, formaldehyde exposure 

17 potentiated the IL-5 increase seen with allergen challenge.

18

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

20 supernatants in formaldehyde-exposed male ICR mice with and without 

21 Der f sensitization

22

Group Formaldehyde Der f GM-CSF IL-2 IL-4 IL-5 RANTES

1 - - NDa 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 ± 37.4b

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

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

23 a 24 None detected.

b 25 p < 0.05 from control. c 26 p < 0.001 from control. d 27 p < 0.05 from Group 2. e 28 p < 0.001 from Group 2. f 29 p < 0.001 from Group 3.

30

31 Source: Sadakane et al. (2002).

32

33

34 Der f sensitization and challenge increased eosinophil infiltration into the interstitium 

35 around the bronchi and bronchioles as well as goblet cell proliferation in the bronchial 

36 epithelium (Figure 4-17). Formaldehyde exposure exacerbated the eosinophilic and goblet cell 

37 responses to a challenge dose of Der f (p < 0.05) (Sadakane et al., 2002). Formaldehyde-induced 
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1 eosinophilic infiltration in the absence of sensitization and challenge was not different from non2 treated, non-sensitized mice.

3 These results suggest that formaldehyde exposure may aggravate eosinophilic infiltration and 

4 goblet cell proliferation that accompanies allergic responses. This response is associated with an 

5 increase in IL-5, an eosinophilic attractant, and an increase in RANTES, which recruits 

6 eosinophils by chemotaxis in formaldehyde-exposed and Der f challenged animals, although the 

7 effect was not statistically significantly elevated compared with Der f challenge-induced levels 

8 of IL-5 and RANTES alone.

9

10 Panel A

11 Panel B

control FA Derf Derf + FA

ab

abc

++++

+++

++

+

±

-

12

13

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

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

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

17 challenge.

18

Note: a

p < 0.001 compared with control group; b 19 p < 0.001 compared with 

formaldehyde group; c 20 p < 0.05 compared with Der f group.

21 Source: Redrawn from Sadakane et al. (2002).

control FA Derf Derf + FA

ab abc

++++

+++

++

+

±

-

Eosinophil score Goblet cell score
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1 Fujimaki et al. (2004a) investigated the long-term effects of low-dose formaldehyde 

2 exposure on immunologic and neurological inflammation. Female C3H/He mice were exposed 

to 0, 0.082, 0.393, or 1.87 ppm (0, 0.1, 0.48, or 2.3 mg/m3 3 ) formaldehyde 16 hours/day, 

4 5 days/week for 12 weeks. Six mice at each exposure level were given injections of OVA plus 

5 adjuvant before the initial exposure and in weeks 3, 6, 9, and 11 of the experiment. Five mice at 

6 each formaldehyde-exposure level did not receive OVA injections. One day after the last 

7 exposure, mice were weighed and blood, BAL, spleen, and thymus were collected from each 

8 animal. After weighing, spleens were disaggregated and spleen cells harvested for cell culture. 

9 Immunophenotype of the spleen cells was determined by flow cytometry (CD4, CD8, CD3, and 

10 CD19 positive cells). Lymphocyte proliferation in response to lipopolysaccharide (LPS), 

11 phytohemagglutinin A (PHA), or OVA was determined after 72 hours in culture. Splenocytes 

12 were cultured for 48 hours in the presence of LPS, PHA, and OVA (immunized mice only), and 

13 supernatants were collected for cytokine analysis (IL-4, IL-5, and IFN-γ). Splenocytes were 

14 cultured for 24 hours in the presence or absence of OVA to assess chemokine production (MCP15 1 and MIP1-α). Anti-OVA IgE, IgG1, IgG2, and IgG3 were quantified in blood plasma.

16 Body and thymus weights were unchanged by formaldehyde exposure or OVA injection 

17 (Fujimaki et al., 2004a), while, in non-immunized mice, spleen weights were reduced by 

18 formaldehyde exposure from 152 mg in controls to 128, 118, and 121 mg in mice exposed to 

19 0.08, 0.4, and 1.8 ppm formaldehyde, respectively. Spleen weights tended to increase in groups 

20 exposed to 400 and 2,000 ppb formaldehyde compared with controls in OVA-immunized mice 

21 (control: 117.8 mg compared with 400 ppb: 168.6 mg and control: 121.0 mg compared with 

22 2,000 ppb:153.2 mg, respectively) but were not statistically significant. 

23 To gain insight on the overall pulmonary inflammatory response of mice exposed to 

24 formaldehyde in both immunized and non-immunized mice, the total number and differential 

25 count of MPs, neutrophils, lymphocytes, and eosinophils in BAL were counted and were found 

26 to be unchanged by formaldehyde in non-immunized mice. By contrast, in immunized mice 

27 exposed to 1.8 ppm formaldehyde, the total number of BAL cells, MPs, and eosinophils were 

28 significantly increased compared with non-immunized controls (9.65 versus 2.84, 7.22 versus 

2.74, and 2.0 versus 0.02 ×104 29 cells, respectively).

30 To further assess the pulmonary inflammatory response, protein levels of inflammatory 

31 cytokines were determined by ELISA in BAL fluid. Levels of IL-1β in BAL of immunized mice 

32 were decreased by formaldehyde exposure (p < 0.05 at 1.8 ppm formaldehyde), but IL-1β levels 

33 after formaldehyde exposure were not different from controls in non-immunized mice (Fujimaki 

34 et al., 2004a). All other cytokines or chemokines were either unchanged (TNF-α, IL-6, and GM35 CSF) or not detected (eotaxin, MIP-1α, and MCP-1).
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1 Various neuropeptides, such as brain-derived neurotrophic factor (BDNF), nerve growth 

2 factor (NGF), and substance P are released from vagal nerve endings and mediate a neurogenic 

3 inflammatory response. Levels of BDNF, NGF, and substance P were assessed in BAL fluid 

4 and/or in plasma. BDNF was not detected in BAL or in plasma. NGF levels in immunized mice 

5 were significantly higher than in non-immunized mice in both BAL fluid and in plasma. NGF 

6 levels in immunized mice were significantly attenuated by 0.08 and 0.4 ppm formaldehyde 

7 exposure (Figure 4-18) in both BAL fluid and in plasma. Plasma level of substance P (a 

8 mediator of neurogenic inflammation) was increased by formaldehyde exposures in non9 immunized mice (Figure 4-19) in both BAL fluid and plasma. This increase appears to be dose10 dependent and reaches statistical significance at 2,000 ppb formaldehyde exposure in non11 immunized mice compared with non-immunized controls. Similar to NGF, levels of substance P 

12 increased in OVA-immunized mice compared with non-immunized mice in both BAL fluid and 

13 plasma. Similar to NGF, levels of substance P in OVA-immunized mice were attenuated by 

14 formaldehyde exposure at 80 ppb. 

15



 



6

5

4

3

2

1

0

OVA

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

- - - - + + + +

16

17

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

19 mice with and without OVA sensitization.

20

21 Note: The day after the final formaldehyde inhalation, BAL fluid was collected 

22 from formaldehyde-exposed, non-immunized and formaldehyde-exposed, OVA23 immunized mice, and the production of NGF was determined by ELISA. Data 

24 are mean ± SEM from five to six animals. **p < 0.01.

25

26 Source: Redrawn from Fujimaki et al. (2004a).

27
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4-244 DRAFT—DO NOT CITE OR QUOTE

1 

2

3 

4 Figure 4-19. Plasma substance P levels in formaldehyde-exposed female 

5 C3H/He mice with and without OVA sensitization.

6 

7 Note: The day after the final formaldehyde inhalation, plasma samples were 

8 collected from formaldehyde-exposed, non-immunized, and formaldehyde9 exposed OVA-immunized mice, and the levels of substance P were determined by 

10 ELISA. Data are mean ± SEM from five to six animals. *p < 0.05. FA = 

11 formaldehyde. 

12 

13 Source: Redrawn from Fujimaki et al. (2004a). 

14 

15 

16 Fujimaki et al. (2004a) further investigated the effect of low-level formaldehyde exposure 

17 from both immunized and non-immunized mice on the systemic immune response. Spleens were 

18 removed from formaldehyde-exposed mice and were cultured in the presence of LPS or PHA 

19 (for non-immunized samples) or OVA (for immunized samples). The secretory ability of 

20 immunized and non-immunized spleen cells was assessed by measuring IFN-γ release by ELISA. 
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1 Formaldehyde exposure (1.8 ppm) increased IFN-γ fourfold in LPS-stimulated cultured spleen 

2 cells from non-immunized mice. No other cytokine or chemokine was changed by formaldehyde 

3 exposure in cultured spleen cells from non-immunized mice. In OVA-immunized mice, 

4 formaldehyde had no significant effect on cytokines from stimulated spleen cells. OVA in vitro

5 stimulation significantly increased the chemokines MIP-1 and MCP-1 for control and 

6 formaldehyde-treated OVA-immunized mice. The OVA-stimulated release of MCP-1 in vitro 

7 was enhanced by formaldehyde exposure in a concentration-dependent manner, increasing 

8 threefold and fourfold at 0.40 and 1.8 ppm, respectively. Increases in MCP-1 correlate with 

9 reported increases in the associated cytokine, RANTES, which recruits eosinophils by 

10 chemotaxis (Sadakane et al., 2002). These formaldehyde-induced increases in cytokine levels 

11 contribute to pulmonary inflammation. The inflammatory response is not mediated by 

12 lymphocytes, since lymphocyte subsets and in vitro cell proliferation were unchanged by OVA 

13 immunization or formaldehyde treatment (Fujimaki et al., 2004a).

14 Anti-OVA (IgE and IgG2a) levels in plasma were unchanged by formaldehyde exposure. 

15 Anti-OVA IgG1 was reduced in immunized mice exposed to 400 ppb formaldehyde compared 

16 with nonexposed animals. However, this effect did not persist as dose increased. Anti-OVA 

17 IgG3 was depressed in immunized mice exposed to 0.08 and 0.4 ppm formaldehyde (Fujimaki et 

18 al., 2004a). Formaldehyde exposure did not induce an inflammatory response in lung or tracheal 

19 epithelium in sections viewed by light microscopy (Fujimaki et al., 2004a). Although there was 

20 a mild infiltration of mast cells into the epithelium of OVA-immunized mice, there were no 

21 effects of formaldehyde treatment on mast cell infiltration. 

22 A recent study by Lino dos Santos Franco et al. (2009) exposed male Wistar rats for 

23 3 days, 90-minutes/day, to 1% formaldehyde (by weight; exact doses not reported) by inhalation. 

24 Of these, one group was sensitized I.P. to OVA (10 μg), a common allergen, immediately 

25 following formaldehyde exposure, and subsequently challenged with OVA 2 weeks later. Other 

26 rats were sensitized and challenged but were not exposed to formaldehyde. PCA reaction as well 

27 as BAL analysis and whole blood analysis were conducted. Immunohistochemical analysis of 

28 platelet endothelial cell adhesion molecule-1 (PECAM-1) expression, an inflammatory mediator, 

29 in lung tissue was also measured. When formaldehyde exposure was followed by OVA 

30 sensitization and challenge, decreased lung inflammation was reported compared with the group 

31 that was OVA-sensitized but had not been exposed to formaldehyde. Reduced lung mast cell 

32 degranulation was also reported in the formaldehyde/OVA group compared with the nonexposed 

33 OVA group. Total circulating leukocytes, total bone marrow cells, and lung protein expression 

34 levels of PECAM-1 were also significantly decreased in formaldehyde/OVA rats compared with 

35 non-formaldehyde exposed OVA rats. The reduction in inflammatory parameters in response to 
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1 formaldehyde may be attributed to different study designs, since in this study animals were 

2 sensitized after exposure rather than prior to exposure. The results suggest that formaldehyde 

3 may functionally alter the activity of certain cells, like mast cells, that may downgrade an 

4 appropriate immune response to antigen and might serve to threaten lung homeostasis. Due to 

5 the unique experimental design of this study, it cannot be directly compared with Sadakane et al. 

6 (2002) or Fujimaki et al. (2004a). In addition, this study did not intend to measure whether 

7 formaldehyde can exacerbate an asthmatic response but rather set out to identify whether 

8 formaldehyde could affect immune homeostasis.

9 In summary, studies suggest that formaldehyde exposure may induce a predominantly 

10 neurogenic inflammatory response via release of neuropeptide, such as NGF and substance P 

11 from vagal nerve endings. Formaldehyde does not appear to potentiate a systemic immune 

12 response. However, localized pulmonary inflammation can be potentiated by formaldehyde 

13 exposure, as indicated by the increased presence of eosinophils and certain proinflammatory 

14 cytokines (IFN-γ). This response does not appear to be mediated by classic immunogenic 

15 mechanisms since studies have failed to report elevated levels of anti-formaldehyde-specific IgE. 

16 Several studies have shown that exposure to formaldehyde can facilitate allergic sensitization in 

17 previously naïve animals, and it is thought that this effect may occur due to formaldehyde’s 

18 ability to increase microvascular leakage in the nasal epithelium and by causing damage to the 

19 nasal barrier (Ito et al., 1996). Sadakane et al. (2002) demonstrated that formaldehyde exposure 

20 can also exacerbate allergic responses by enhancing the response to challenge allergen. Thus, 

21 formaldehyde may exacerbate allergic responsiveness by aggravating the sensitization response 

22 in previously naïve animals by altering the permeability of the mucosal barrier in nasal 

23 compartments. Neurogenically derived inflammation, including stimulation of the trigeminal 

24 nerve and release of braykinin, suggests that the MOA for sensitization may ultimately have its 

25 roots in neurogenic inflammation rather than an immunogenic response. In addition, using a 

26 different protocol, Lino dos Santos Franco et al. (2009) suggest that formaldehyde exposure can 

27 adversely affect lung homeostasis by reducing the activity of important inflammatory mediators 

28 (mast cells, circulating leukocytes, PECAM-1 expression) when it occurs prior to sensitization, 

29 thus downgrading an appropriate immune response.

30

31 4.2.1.5.2. Dermal sensitization. Wahlberg (1993) used Hartley strain guinea pigs as test 

32 animals to determine the skin irritancy of a suite of industrial chemicals, including 

33 formaldehyde. Aqueous solutions of the compound in a 0.1 mL volume were applied to the 

34 shaved flanks of guinea pigs and gently rubbed into the skin with a cotton-tipped applicator. 

35 Sites were left open and the treatments repeated once daily for 10 days. A number of indices of 
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1 acute skin irritation were monitored, including erythema via visual scoring and edema and skin2 fold thickness using Harpenden calipers. Varying concentrations of formaldehyde (up to a 10% 

3 solution) induced a dose-dependent increase in skin-fold thickness. Responses also showed 

4 shorter latencies at the higher concentrations. For example, erythema was first observed on 

5 day 2 when 10% formaldehyde was applied, day 5 (for 3%), and day 6 (for 1%).

6 Lee et al (1984) investigated the role of different routes of exposure in formaldehyde7 induced allergic sensitization. Two sets of four male English smooth-haired guinea pigs received 

8 topical applications of 100 μL 37% w/v formalin distributed over two shaved, depilated dorsal 

9 sites two times over the course of 2 days at different sites. The total dose was calculated as 

10 74 μg/animal. In addition, eight animals received a single topical application onto a 15 mm area 

11 of the dorsal surface. The applied dose of 25 μL formaldehyde was dissolved in saline. Two 

12 other groups of guinea pigs were exposed to either 6 ppm (6 hours/day for 5 days) or 10 ppm 

13 (6 hours/day for 5 days) formaldehyde by inhalation. A third group of guinea pigs was exposed 

14 to 10 ppm formaldehyde for 8 hours/day for 5 consecutive days by inhalation. All animals were 

15 evaluated for contact sensitivity by topical application of 20 mL formaldehyde diluted with 

16 saline and distributed in a 15 mm area on the backs of the shaved guinea pigs (Lee et al., 1984). 

17 Sites were visually inspected for erythema at 1, 6, 24, and 48 hours following the topical 

18 application, and reactions were scored. No erythema was observed in control animals. None of 

19 the guinea pigs in the 6 hours/day inhalation groups (6 and 10 ppm formaldehyde) developed 

20 skin sensitivity tested on day 9 (4 days after the initial exposure regimen ended). Two of four 

21 guinea pigs exposed to 10 ppm formaldehyde for 8 hours/day for 5 consecutive days developed 

22 mild skin sensitization tested on day 31. Contact sensitivity increased in a dose-dependent 

23 fashion in groups of animals that had been sensitized via the dermal route. Thus, dermal 

24 exposure resulted in contact sensitivity. Inhalation exposure did not consistently produce contact 

25 sensitivity.

26 Arts et al. (1997) used a local lymph node assay (LLNA) and the induction of IgE to 

27 monitor the sensitization of female Wistar rats (low IgE-responders) and BN rats (high IgE 

28 responders). For the LLNA assay, animals were sensitized by the application of varying 

29 concentrations of formaldehyde in raffinated olive oil on the dorsum of both ears on days 0, 1, 

30 and 2. Control animals were treated with raffinated olive oil alone. Animals received an I.P. 

31 injection of BrdU on day 5 and were subsequently sacrificed. Ear-draining lymph nodes were 

32 collected, fixed, and sectioned, and the mitotic activity was monitored following successive 

33 incubation of the sections in anti-BrdU, biotin-labeled rabbit anti-mouse antibody, peroxidase34 conjugated streptavidin, and 3,3-diaminobenzidine tetrahydrochloride. For serum IgE responses, 

35 150 μL of different concentrations of formaldehyde were applied to the shaved flanks of rats on 
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1 day 1, then 75 μL of the same chemical at 50% of the initial concentration were applied to the 

2 dorsum of each ear on day 7. The amount of IgE in the blood was measured using ELISA but 

3 appeared to be little affected by formaldehyde treatment in either species of rat. However, the 

4 ear-draining lymph nodes of both strains of rat showed a comparative increase in weight in 

5 response to formaldehyde, and proliferation (BrdU positive) of paracortical cells was observed in 

6 response to increasing doses of the compound. This response was most notable in BN rats 

7 treated with 10% formaldehyde. Arts et al. (1997) concluded that the irritant and sensitizing 

8 properties of formaldehyde may act through non-IgE-immune mechanisms.

Hilton et al. (1998) used the LLNA assay in female CBA/Ca (H-2k 9 haplotype) mice to 

10 compare the skin sensitizing potencies of formaldehyde and glutaraldehyde. The comparison 

11 was set on a quantitative basis by determining the concentration of each compound necessary to 

12 induce a threefold increase in lymph node cell proliferative activity (effective concentration 

13 [EC3]). While both aldehydes induced a dose-dependent proliferative response, the 

incorporation of [3 14 H]-methylthymidine was far greater in animals exposed to glutaraldehyde 

15 versus formaldehyde (with EC3 values of 0.002–0.006 mol/L for glutaraldehyde versus 0.11–

16 0.18 mol/L for formaldehyde). These data indicate the potential of both chemicals to induce skin 

17 sensitization, although the potency of glutaraldehyde was far greater than that of formaldehyde.

18 Xu et al. (2002) evaluated the extent to which the expression of some cytokines may 

19 change as a result of cutaneous exposure to formaldehyde in mice. Female Balb/C mice were 

20 skin painted with three topical applications of 100 μL of 17.5% formaldehyde or distilled water 

21 with a 1-day interval between each application. Spleen and draining lymph nodes were 

22 harvested on days 3, 5, 7, 9, or 12 after the last skin painting. In some animals, contact 

23 hypersensitivity was induced by applying 2% formaldehyde to both sides of mouse ears on day 3 

24 following the last skin painting. For this endpoint, the percent increase in thickness of the ears 

25 was monitored. For the cytokines, mRNA expression levels of IL-2, IL-4, IL-5, IL-10, IL-12, 

26 IL-13, IL-15, IL-18, and INF-γ were determined semiquantitatively by measuring the amount of 

27 individual mRNAs following amplification with the reverse transcriptase (RT)-PCR. The 

28 relative amounts of cytokine mRNAs were calculated as the ratio of cytokine mRNA to that of 

29 glyceraldehyde-3-phosphate dehydrogenase, as revealed in specific bands on an agarose gel.

30 Cutaneous formaldehyde treatment was associated with the long-lasting expression of 

31 IL-4 and IFN-γ mRNAs in mouse spleen and draining lymph nodes and with IL-15 mRNA only 

32 in mouse spleen. Only IL-13 mRNAs displayed a transient increase in expression in both spleen 

33 and draining lymph nodes. Levels of IL-2, IL-12, and IL-15 were increased in the mouse spleen 

34 but not the lymph nodes. The mouse ear swelling test gave positive correlations with enhanced 

35 expression of mRNA for IL-4 and IFN-γ (Table 4-52).

36
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1

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

3 mRNA levels in contact hypersensitivity to formaldehyde in mice

4

Variables

Correlation coefficients

IL-2 IL-4 IFN-γ

Ear swelling 0.50 0.74a 0.67a

IL-2 – 0.39 0.60

IL-4 – – 0.79a

5 a 6 Statistically significant (p < 0.05).

7

8 Source: Xu et al. (2002).

9

10

11 4.2.1.5.3. Summary of sensitization studies. Several animal studies report increased airway 

12 resistance and BC due to inhalation exposures to formaldehyde (Nielsen et al., 1999; 

13 Swiecichowski et al., 1993; Biagini et al., 1989; Amdur, 1960). Changes in pulmonary 

14 resistance were observed as early as 10 minutes after exposure (Biagini et al., 1989), and 

15 reported effect levels ranged from 0.3–13 ppm. Other pulmonary effects were reported in 

16 conjunction with BHR, such as increased tracheal reactivity and decreased pulmonary elasticity 

17 (Swiecichowski et al., 1993; Amdur, 1960). Although BHR is a common result of Type I 

18 hypersensitivity reaction to an allergen, the observation of BHR alone is not sufficient to 

19 demonstrate that an agent induces Type 1 hypersensitivity. 

20 BHR may be directly induced both pharmacologically and neurogenically (Joos, 2003; 

21 Cain, 2001; Meggs, 1995). There is little evidence that formaldehyde itself is an allergen 

22 recognized by the immune system, especially via inhalation (Lee et al., 1984). Although 

23 formaldehyde exposure has been reported to alter cytokine levels and immunoglobulins in some 

24 experimental systems, these immunomodulatory effects do not support a type 1 hypersensitivity. 

25 IgE was unchanged (Fujimaki et al., 2004a; Lee et al., 1984), and cytokine profiles were not 

26 consistent with the Th-2 cytokines expected in IgE mediated hypersensitivity (Fujimaki et al., 

27 2004a; Ohtsuka et al., 2003). 

28 Formaldehyde-induced dermal sensitization show parallel results. The physical signs of 

29 irritation and sensitization are consistently shown (e.g., rashes, edema). Some involvement of 

30 the immune response has been demonstrated with positive LLNA assays, indicating proliferation 

31 of lymphocytes in lymph nodes draining the affected area (Hilton et al., 1998; Arts et al., 1997). 

32 Increased expression of Th-2 cytokines in the lymph nodes of mice given dermal applications of 

33 formaldehyde does indicate an immune component to the observed sensitization. However, the 

34 response does not seem to be mediated by IgE (Arts et al., 1997; Lee et al., 1984). 
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1 Ito et al. (1996) reported that a tachykinin NK1 receptor, but not the histamine H1 or 

2 bradykinin B2 receptors, is involved in formaldehyde-induced vascular permeability. 

3 Neuropeptides NGF and substance P were affected in BAL and stimulated splenocytes from 

4 formaldehyde-exposed mice, with greater effects seen in OVA-immunized mice. Tachykinins 

5 (e.g., substance P and neurokinin A) are produced by nerve cells and can directly stimulate 

6 bronchoconstriction (Van Schoor et al., 2000). Substance P is also a mediator of neurogenic 

7 inflammation. Therefore, although formaldehyde may induce some of the symptoms of type 1 

8 hypersensitivity, these symptoms are more likely neurogenic than immunogenic in origin.

9 In contrast, formaldehyde enhances immunogenic hypersensitivity of known allergens 

10 (Sadakane et al., 2002; Riedel et al., 1996; Tarkowski and Gorski, 1995). This potentiation 

11 varied based on sensitization protocols (respiratory tract versus systemic, frequency and timing 

12 of immunization, allergen, etc.) and formaldehyde exposure regimens (concentration, continuous 

13 versus intermittent exposures). Taken as a whole, the results support the finding that 

14 formaldehyde exposure can aggravate a type 1 hypersensitivity response (Table 4-53).

15

16 4.2.1.6. Neurological and Neurobehavioral Function

17 4.2.1.6.1. Inhalation exposure. There are a number of published reports examining the effects 

18 of formaldehyde exposure on nervous system structure and function. The reports evaluating 

19 behavioral effects fall into three main categories: (1) behavioral responses evaluated during or 

20 immediately following formaldehyde exposures, which may include effects due to the potential 

21 irritant properties of the chemical, (2) acute or short-term exposures followed by behavioral 

22 assessments conducted 2–24 hours after termination of formaldehyde exposure, which reflect 

23 sustained effects of chemical exposure independent of its irritant properties, and (3) repeated 

24 exposures to formaldehyde followed by neurological assessments performed throughout the 

25 treatment period or several days to weeks after termination of treatment. In addition to reports 

26 evaluating changes in behavior, there are several reports evaluating neuropathological effects or 

27 changes in brain chemistry.

28
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Table 4-53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due to 

formaldehyde in experimental animals

Species No./

group

Treatmenta Observations LOAEL/

NOAEL

Reference

Inhalation studies

Cynomolgus 

monkeys

9 Methacholine-sensitive monkeys 

exposed to 2.5 ppm 

formaldehyde for 10 minutes

Formaldehyde increased pulmonary resistance after 2, 5, and 

10 minutes.

LOAEL 

2.5 ppm

Biagini et al. 

(1989)

Hartley 

guinea pigs 

(male)

8 0.86, 3.4, 9.4, 31.1 ppm 

formaldehyde for 2 hours or 

0.11, 0.31, 0.59, 1.05 ppm for 

8 hours

Total pulmonary resistance increased after 2 hours exposure at 

9.4 and 31.1 ppm. Effect was reversible and returned to 

baseline within 30 minutes. Total pulmonary resistance was 

increased after 8 hours exposure at 0.3 and 1 ppm. Amount of 

acetylcholine needed to achieve doubled pulmonary resistance 

was decreased in animals after 2 hours exposure.

NA Swiecichowski 

et al. (1993)

Hartley 

guinea pigs 

(male)

5–7 3.4 ppm for 8 hours No changes in ex vivo tracheal constriction or inflammation. NA Swiecichowski 

et al. (1993)

F344 rats 

and BN rats

5 16 ppm 3 hours/day, 5 days Modest changes in inflammatory cytokine expression, but 

respiratory and olfactory epithelial lesions were more severe in 

F344 rats than in BN rats.

NA Ohtsuka et al. 

(2003)

English 

smoothhaired 

guinea pigs

4 6, 10 ppm, 6 hours/day, 5 days, 

combined with provocation 

challenge (2 or 4 ppm on day 7, 

or days 7, 22, and 29)

Inhalation challenge with 6 or 10 ppm followed by bronchial 

challenge failed to increase respiratory sensitivity

NA Lee et al. 

(1984)

Perlbrightwhite, 

DuncanHartley 

guinea pigs 

(female)

12 0, 0.13, 0.25 ppm 8 hours/day, 

5 days. The animals were 

sensitized to OVA (3 minutes 

exposure to 5% OVA aerosol)

Anti-OVA titer was significantly elevated over controls in 

animals exposed to 0.25 ppm formaldehyde and showed that 

formaldehyde may sensitize previously naïve animals to OVA.

NA Riedel et al. 

(1996)

Balb/C mice 

(female)

4 0, 6.63 ppm 6 hours/day for 

10 days or 6 hours/day 

once/week for 7 weeks. All 

mice were sensitized to OVA

Formaldehyde administered intranasally for 6 hours/day for 

10 days may facilitate sensitization to allergens since anti-OVA 

titers were elevated over control animals. However, the length 

and duration of exposure appears to affect development of 

sensitization.

NA Tarkowski and 

Gorski (1995)
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Table 4-53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due to 

formaldehyde in experimental animals

Species No./

group

Treatmenta Observations LOAEL/

NOAEL

Reference

Wistar rats 

(male)

5–8 0, 2, 5, 15, 45 ppm for 

10 minutes

Pulmonary insufflation or blood pressure were not altered. 

Vascular permeability increased in concentration-dependent 

manner and could be reduced by adding a NK1 selective 

antagonist.

NA Ito et al. 

(1996)

Outbred ICR 

mice (male)

18 0.5% formaldehyde for 15 

minutes, once/week for 4 weeks. 

Both control and exposed groups 

were exposed to Der f by I.P. 

injection 1 day before 

formaldehyde and then 

challenged with Der f after 

4 weeks.

More pronounced RANTES production in formaldehyde-treated 

and sensitized rats than in sensitized rats that had not been 

exposed to formaldehyde. Formaldehyde also potentiated IL-5 

production associated with sensitization.

NA Sadakane et al. 

(2002)

C3H/HeJ 

mice 

(female)

6 0, 0.082, 0.393, 1.87 ppm 

16 hours/day, 5 day/week, 

12 weeks. Mice also given OVA 

plus adjuvant before exposure, 

and again 3, 6, 9, 11 weeks after 

exposure. Some formaldehyde 

mice did not receive any OVA

Substance P and NGF were increased dose dependently in 

formaldehyde-treated, non-immunized mice but were attenuated 

in formaldehyde-treated immunized mice compared with 

nonexposed, immunized controls.

LOAEL 

0.082 ppm

Fujimaki et al. 

(2004a)

Wistar rats 

(male)

NA 1% Formaldehyde by weight for 

90 minutes for 3 days. One 

group was sensitized to OVA 

after to formaldehyde exposure 

and then challenged with OVA 

afterwards. Others were 

sensitized and challenged but not 

exposed to formaldehyde.

Total circulating leukocytes, bone marrow cells, and lung 

protein PECAM expression were significantly decreased in 

formaldehyde/OVA rats compared with OVA rats.

NA Lino dos 

Santos Franco 

et al. (2009)

Dermal sensitization

Hartley 

guinea pigs

5 Skin painted once/day for 

10 days with 0.1 mL of 1, 3, or 

10% formaldehyde

Varying concentrations (up to 10%) induced dose-dependent 

increase in skin-fold thickness. Erythema seen earlier at higher 

doses (2 days at 10% formaldehyde vs 5 days at 3% or 6 days at 

1%).

Wahlberg et 

al. (1993)
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purposes only and does not constitute 

Agency policy

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Table 4-53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due to 

formaldehyde in experimental animals

Species No./

group

Treatmenta Observations LOAEL/

NOAEL

Reference

English 

smoothhaired 

guinea pigs

4 Group 1: skin painted, 100 μL 

37% formalin twice over 2 days, 

Group 2: single topical 

application of 25 μL 

formaldehyde

Group 3: 10 ppm 6 hours/day for 

5 days by inhalation

Two of four guinea pigs from group 3 had mild skin 

sensitization after day 31. Contact sensitivity developed in a 

dose-dependent manner in the dermal groups (group 1 and 2).

Lee et al. 

(1984)

Wistar and 

BN rats 

(female)

4 Application of formaldehyde to 

ears on days 0, 1, 2, followed by 

an I.P. injection of BrdU.

Ear-draining lymph nodes increased in weight in response to 

formaldehyde, reflected in increased number of BrdU-stained 

cells, most notably in BN rats (high IgE responders) treated with 

10% formaldehyde

Arts et al. 

(1997)

CBA/Ca 

mice

NA Compared glutaraldehyde to 

formaldehyde to induce a local 

lymph node assay

Glutaraldehyde and formaldehyde induced a dose-dependent 

proliferative response that was greater in glutaraldehyde-treated 

animals

Hilton et al. 

(1998)

Balb/c mice 

(female)

3–5 Skin painted with 100 μL of 

17.5% formaldehyde every other 

day for days 3, 5, 7, 9, 12

Cutaneous treatment associated with long-lasting expression of 

various cytokines from draining lymph nodes and spleen.

NA Xu et al. 

(2002)

NA = not applicable.
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4.2.1.6.1.1. 

2 Clinical signs

1 Behavioral response

3 Several studies that were focused on general toxicity or carcinogenicity of formaldehyde 

4 also assessed clinical signs in exposed animals, which may be related to adverse effects on the 

5 nervous system. Procedural details for the assessments, or specific data regarding findings, were 

6 not provided. Signs recorded included uncoordinated locomotion and climbing of cage walls at 

7 20 ppm formaldehyde in rats (Woutersen et al., 1987); restlessness at 15 ppm formaldehyde in 

8 rats (Morgan et al., 1986a); dyspnea, listlessness, and hunched posture at 20 ppm and ataxia at 

9 40 ppm in mice (Maronpot et al., 1986); and dyspnea in rats at 14.3 ppm formaldehyde (Kerns et 

10 al., 1983). Given the lack of information regarding procedures used for these evaluations and the 

11 limited reporting of results, the utility of these data is limited.

12

13 Irritant threshold detection

14 Wood and Coleman (1995) evaluated the irritant properties of acute formaldehyde 

15 exposure in mice. Adult male Swiss mice (eight/group) were initially trained to terminate a 

16 60-second exposure to an irritant gas (ammonia, 1,000 ppm) by poking their noses into a conical 

17 sensor five times to produce a 60-second facial shower of clean air. Each test session consisted 

18 of 25 exposure trials. Following training, response to formaldehyde was evaluated, using the 

19 same testing scenario. Each day mice had a morning exposure session to ammonia and an 

20 afternoon session to formaldehyde. Formaldehyde concentrations tested were different each day, 

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/m3 21 ) and 

22 then stepping back again from 10 to 0 ppm. Half of the animals were tested in an ascending 

23 order of formaldehyde concentrations, the other half in a descending order. The frequency of 

24 terminating exposure, error rate, and the time lapse to termination were recorded. The 

25 concentration at which 50% of the formaldehyde deliveries would be expected to be terminated 

26 was estimated (AC50) by simple linear regression or by analysis of covariance on the logit 

27 transform of percentage terminated as a function of log concentration.

28 All mice were trained successfully to terminate 100% of ammonia exposures, but varied 

29 responses were observed with formaldehyde exposure. In general, time taken to terminate 

30 formaldehyde exposure decreased significantly with increasing formaldehyde concentration. 

31 Mice terminated more exposures to 1 ppm formaldehyde than to air alone (p < 0.0005), and the 

32 error rate, generally below 40%, did not significantly differ with formaldehyde concentration 

33 tested. Each animal had two test sessions with each formaldehyde concentration (once during 

34 the ascending sequence and again during the descending sequence); both the time to termination 

35 (p < 0.0012) and AC50 were decreased in the second series of tests. One method of estimation by 
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1 the authors yielded an AC50 of 3.63 ppm for the first series of tests versus 1.88 ppm for the 

2 second series. A two-way repeated measures ANOVA with replication and concentration as 

3 within variables was highly significant (p < 0.00005). These studies indicate mice are sensitive 

4 to the irritant properties of formaldehyde at exposure concentrations as low as 1 ppm, and 

5 animals reacted more swiftly and with greater accuracy to terminate formaldehyde exposure as 

6 the concentration increased. However, a wide variety of responses was noted on an individual 

7 animal basis. Two of the eight mice terminated 90% of the trials during 1 ppm exposures and 

8 80–100% of trials at all other tested formaldehyde concentrations. One mouse terminated fewer 

9 than 10% of the formaldehyde exposure trials (1–10 ppm) during the testing regimen but had a 

10 92% response rate to 20 ppm formaldehyde. The remaining five mice responded with increasing 

11 termination frequency as formaldehyde concentration increased from 1 to 10 ppm, with an AC50

of 2.72 ppm (3.34 mg/m3 12 ).

13

4.2.1.6.1.2. Motor activity and habituation.

25 The authors reported no signs of irritation or changes in activity or food or water intake 

26 during exposure. In general, sniffing was increased after formaldehyde exposure and movement 

27 was decreased (crossed quadrants and climbing) in both male and female rats (p < 0.05). 

28 Significant reductions in horizontal movements (crossed quadrants) were observed at all dose 

29 levels and were characterized by a U-shaped dose response (Figure 4-20). The lowest dose 

30 tested (1 ppm) demonstrated a higher level of activity suppression than the two higher doses, but 

31 all groups were still suppressed relative to controls. Although female rats displayed a greater 

32 level of activity overall, a similar U-shaped dose-response pattern was also observed.

14 Malek et al. (2003a) examined open field behavior 

15 of rats after acute formaldehyde exposures. Male and female LEW.1K rats (15/sex/group) were 

exposed to 0, 1, 2.5, or 5 ppm (0, 1.23, 3.08, or 6.15 mg/m3 16 ) formaldehyde for 2 hours. 

17 Formaldehyde was vaporized from aqueous solutions directly below the exposure chamber. 

18 Formaldehyde levels were checked 16 times throughout the 2-hour exposure periods. Mean 

19 formaldehyde levels of 1.01 ± 0.29 ppm, 2.51 ppm (SD is missing), and 5.0 ± 0.27 ppm were 

20 achieved. Locomotor activity was assessed for 3 minutes in an open field 2 hours after 

21 termination of formaldehyde exposure and again 24 hours later, using an automated device to 

22 count the number of squares crossed. Other behaviors were noted, including grooming (face 

23 cleaning, fur licking, and scratching), rearing, sniffing (air and floor), wall climbing, and 

24 defecation.

33

34
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1

2

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

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

5 Greater reductions were observed in the lowest dose group, a pattern that 

6 was evident in both genders. ** = different from control, p < 0.005.

7

8 Source: Drawn from data reported by Malek et al. (2003a).

9

10

11 Activity in the same apparatus was reassessed 24 hours later. As expected, controls 

12 demonstrated habituation to the test apparatus, exhibiting only 20% of the motor activity 

13 observed on day 1 (Figure 4-21). In contrast, formaldehyde-treated animals failed to 

14 demonstrate the same degree of habituation. Activity levels for males observed on day 2 were 

15 60–80% of the activity levels seen on day 1. Formaldehyde-treated females also failed to 

16 habituate and actually demonstrated increases in activity on day 2 relative to day 1 at all 

17 formaldehyde exposure levels.

 Number

**

**

**

 

** 

Control 1 ppm 2.5 ppm 5.0 ppm

Activity as crossed quadrants

Males

Females

0

20

40

60

80

100

Formaldehyde
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Male LEW.1K Rats

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

Control 1 ppm 2.5 ppm 5.0 ppm

Formaldehyde

Habituation (percent )

Female LEW.1K Rats

0%

50%

100%

150%

200%

250%

300%

Control 1 ppm 2.5 ppm 5.0 ppm

Formaldehyde

Habituation (percent)

1

2

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

4 during the second observation period (day 2, 24 hours after formaldehyde 

5 exposure).

6

7 Note: Habituation is shown here as the percent decrease in number of crossings 

8 between sessions from day 1 to day 2. The degree of habituation was reduced in 

9 male rats exposed to formaldehyde (left panel) since their activity was closer to 

10 100% of that seen on day 1. Females (right panel) had increased activity on day 2 

11 (greater than 100% of activity on day 1), which is a sensitization rather than 

12 habituation.

13

14 Source: Drawn from data reported by Malek et al. (2003a).

15

16

17 A follow-up study by Malek et al. (2003b) further expanded the dose-response analysis 

18 for acute formaldehyde exposure. As described above, male and female LEW.1K rats (10 per 

sex per group) were exposed at 0, 0.1, 0.5, or 5 ppm (0, 0.123, 0.615, or 6.15 mg/m3 19 ) 

20 formaldehyde for 2 hours. Formaldehyde levels were checked nine times per hour during the 

21 exposure periods, and mean values were found to be 0.13 ± 0.04, 0.48 ± 0.05, and 

22 5.18 ± 0.66 ppm. Open field behavior was evaluated for each animal 2 hours after formaldehyde 

23 exposure. The number of crossed quadrants for both controls and a 5 ppm group were generally 

24 comparable with those observed in the first study, although female values were somewhat lower. 

25 Horizontal movement was decreased by formaldehyde exposure in a dose-dependent manner 

26 with significant reductions in motor activity as low as 0.1 ppm in males and 0.5 ppm in females 

27 (Figure 4-22). The consistency of the findings across studies and between genders provides 
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1 greater confidence in the effects of low-level formaldehyde exposure on this standard test of 

2 neurotoxicity.

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

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

6 2 hours after termination of 10-minute formaldehyde exposure.

7

8 Note: Values are means ± SDs. * = different from control, p < 0.05. ** = different 

9 from controls, p < 0.01. *** = different from controls, p < 0.001.

10

11 Source: Drawn from data reported in Malek et al. (2003b).

12

13

14 Malek et al. (2004) also assessed the capacity of formaldehyde to induce persistent 

15 behavioral deficits in mice. Groups of 20 male AB mice received a single 2-hour exposure to 0, 

1.1, 2.3, or 5.2 ppm (0, 1.3, 2.8, or 6.4 mg/m3 16 ) formaldehyde prior to being tested 2 and 24 hours 

17 after exposure for a series of behavioral responses, including ambulation (crossed squares), 

18 grooming, sniffing, rearing, wall climbing, and defecation. Even though there were no clinical 

19 signs of toxicity in any of the exposed groups, a number of behavioral anomalies were apparent 

20 in response to formaldehyde exposure, some of which persisted for at least 24 hours, as indicated 

21 in Tables 4-54 and 4-55.

22

23
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1 Table 4-54. Fluctuation of behavioral responses when male AB mice inhaled 

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

3

Open field parameter

Formaldehyde concentration (ppm)a

0 1.1 2.3 5.2

No. of crossed inner squares 34.10 ± 7.51 25.30 ± 5.03b 21.20 ± 3.41b 16.10 ± 5.37b

No. of crossed peripheral squares 56.65 ± 9.68 59.55 ± 9.75 49.70 ± 13.24 29.15 ± 7.47b

Total no. of crossed squares 90.75 ± 11.08 84.85 ± 9.96 71.10 ± 13.91b 44.20 ± 7.42b

Air sniffing 19.35 ± 2.5 21.50 ± 4.26 16.35 ± 3.84c 8.10 ± 1.77b

Floor sniffing 20.95 ± 3.72 26.50 ± 4.64b 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 ± 3.19b 11.30 ± 2.30b 9.95 ± 1.61b

Wall climbing 13.20 ± 3.09 14.55 ± 2.74 13.95 ± 2.31 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 5 Values are means ± SDs.

b 6 Statistical significance of differences from controls (p < 0.005). c 7 Statistical significance of differences from controls (p < 0.05).

8

9 Source: Malek et al. (2004).

10

11

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

13 formaldehyde in a single 2-hour exposure: effects after 24 hours

14

Open field parameter

Formaldehyde concentration (ppm)a

0 1.1 2.3 5.2

No. of crossed inner squares 10.40 ± 2.35 9.55 ±1.73 9.10 ± 1.25 9.70 ± 1.13

No. of crossed peripheral squares 42.80 ± 9.27 44.85 ± 14.60 44.95 ± 16.56 41.10 ± 9.08

Total no. of crossed squares 53.20 ± 8.67 54.40 ± 14.77 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 ± 3.94b 13.25 ± 4.17b 17.65 ± 3.13b

Grooming 8.35 ± 2.56 13.95 ± 2.21b 10.20 ± 3.33c 11.90 ± 3.26b

Rearing 18.30 ± 4.23 12.40 ± 2.23b 12.25 ± 2.17b 12.00 ± 3.32b

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 ± 0.94c 1.20 ± 0.89

15 a 16 Values are means ± SDs.

b 17 Statistical significance of differences from controls (p < 0.005). c 18 Statistical significance of differences from controls (p < 0.05).

19

20 Source: Malek et al. (2004).

21

22

23 Usanmaz et al. (2002) assessed spontaneous locomotor activity (SLMA) in Balb/c mice 

24 (4–14 per group, sex unspecified) after both acute and subchronic formaldehyde exposures. 

25 Prior to the acute exposure, mice were acclimated to the exposure chamber for 4 days but 

26 exposed only to clean air. On the fifth day, mice (six/group, sex unspecified) were exposed for 

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/m3 27 ) 

28 formaldehyde. Mice were removed from the exposure chamber, and SLMA behavior was 
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1 evaluated by direct observation for 5 minutes. In addition to horizontal and vertical movement, 

2 wet dog shake (WDS) behavior was noted. In a separate trial, Balb/c mice (six/group, sex 

3 unspecified) were exposed to 8.2 ppm formaldehyde for 1 week, 2 ppm formaldehyde for 

4 2 weeks, or 3.3 ppm formaldehyde for 3 weeks (3 hours/day, 5 days/week) compared with 

5 controls exposed only to air. SLMA behavior was observed for 5 minutes after the last exposure. 

6 Mice exposed to 8.2 ppm formaldehyde for 1 week, 3.3 ppm formaldehyde for 2 weeks, and 

7 2 ppm formaldehyde for 3 weeks lost weight over the course of the treatment (p < 0.05). All 

8 other treatment groups had weight gain similar to control mice.

9 As shown in Figure 4-23, acute 3-hour formaldehyde exposures resulted in a dose10 dependent decrease in SLMA. Decreases in horizontal activity were significant for the three 

11 highest dose groups (6.4, 9.7, and 14.8 ppm), and decreases in vertical activity were significant 

12 for all six formaldehyde treatment groups. SLMA was similarly decreased following subchronic 

13 exposures (data not shown here). Although the experimental protocol included longer exposures 

14 and a slightly longer observation period (5 versus 3 minutes) than in Malek et al. (2003a, b), the 

15 results are consistent, indicating decreased activity in formaldehyde-exposed animals several 

16 hours after exposure was ended.

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

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

21 ambulatory and vertical components of SLMA.

22

23 Note: FA = formaldehyde exposure concentration. ** = p < 0.01 from controls.

24

25 Source: Usanmaz et al. (2002).

26

27

28 Usanmaz et al. (2002) noted an increase in WDS, after the acute exposures, as a possible 

29 preconvulsive effect. However, the mice were only observed for 5 minutes, and it is unclear how 
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1 the researchers distinguished between a WDS due to an irritating odor and a preconvulsive 

2 movement. No other study has noted convulsive effects from formaldehyde exposure in any 

3 species. A second set of trials was reported in the same paper that sought to evaluate 

4 formaldehyde effects on CNS excitability. Balb/c mice (six/group, sex unspecified) were 

exposed to 0, 1.8, 6.4, or 14.8 ppm (0, 2.2, 7.9, or 18.2 mg/m3 5 ) formaldehyde for 3 hours. 

Subchronic exposures were at 2 ppm (2.5 mg/m3 6 ) formaldehyde for 3 weeks or 3.3 ppm 

(4.1 mg/m3 7 ) formaldehyde for 2 weeks. Seizures were induced by I.P. injection of 

8 pentilenetetrazole (PTZ), and the incidence, severity, and course of induced seizures were 

9 recorded. The PTZ injection induced seizures in 83, 88, and 91% of controls, with 16, 38, and 

10 67% mortality in controls in the three trials. Mortality was highly variable in treatment groups as 

11 well. The authors report that PTZ-induced seizures were decreased in incidence by acute 

12 formaldehyde exposure in a dose-dependent fashion with only 33% of mice exposed to 14.8 ppm 

13 formaldehyde experiencing seizures versus 91% in control mice (p < 0.05 at the highest dose 

14 only). However, the methodology for observing and scoring seizures is unclear. Additionally, 

15 there was high mortality and high variability of results for the three similarly treated control 

16 groups. Therefore, it is difficult to assess data quality and interpret these findings.

17 Boja et al. (1985) exposed male Sprague-Dawley rats to air or to formaldehyde at 5, 10, 

18 or 20 ppm for 3 hours on 2 consecutive days. On the second day, half the rats received the same 

19 exposure as the previous day, while half the rats were switched (e.g., half those rats receiving air 

20 the first day received formaldehyde the second day, and half those receiving formaldehyde the 

21 first day received air the second day), for a total of four possible exposure combinations. During 

22 the exposure period, activity levels were monitored by observation, once per minute for the first 

23 hour and once every 5 minutes for the second hour. At the end of the second exposure session, 

24 rats were sacrificed and brains removed for neurochemical analysis (see Section 4.2.1.6.1.5).

25 Behavioral results were described in detail only for control and 5 ppm groups. During 

26 the first exposure session, activity levels of formaldehyde-exposed animals were significantly 

27 decreased (approximately 50% of control levels). On the second day of exposure, those animals 

28 previously exposed to formaldehyde exhibited partial recovery, those experiencing their first 

29 formaldehyde exposure behaved similarly to those initially exposed on the first day, and those 

30 animals exposed to formaldehyde for a second time had a greater decrease in activity than during 

31 the first exposure (to approximately 30% of control levels). The authors stated that a similar 

32 effect was seen in animals exposed at 10 ppm but that results at 20 ppm were not interpretable 

33 (data were not presented). Overall, the decreased activity seen in this study is consistent with 

34 effects seen by other authors.
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Senichenkova (1991) exposed pregnant female rats to 0 or 0.5 mg/m3 1 (0 or 403 ppb) 

2 formaldehyde on gestation days (GDs) 1–19 for 4 hours/day. Reproductive aspects of this study 

3 will be discussed in the reproduction section; however, results from behavioral assessments 

4 conducted on the neonates are discussed here. The author stated that maturation of motor 

5 reflexes (assessed as surface righting and pendular reflex), open field behavior, and maze 

6 learning ability were assessed. Detailed descriptions of procedures and results were not provided 

7 for all assessments, but it was stated that motor reflex development did not differ in treated and 

8 control animals. Open field motor activity assessments in 40-day-old (juvenile) offspring 

9 revealed an increase in squares visited and an increased frequency of rearing on the second and 

10 third days of testing, indicating a lack of habituation in the offspring of formaldehyde-treated 

11 dams; similar levels of activity by both measures were found on the first test day. Counts of 

12 defecation and urination were increased on all 3 days of testing. Increased exploratory behavior, 

13 described as increased impulses, was also noted in a learning task (not otherwise described), but 

14 the author stated that learning rate and ability of the formaldehyde-treated group was not 

15 different from controls (no data were provided).

16 Mobility and neuromuscular excitability (not otherwise described) in offspring of female 

17 white rats were also evaluated by Sheveleva (1971). Dams were exposed to 0.005 or 

18 0.0005 mg/L (approximately 4,000 or 400 ppb, respectively) formaldehyde on GDs 1–19. 

19 Spontaneous mobility (over 15 minutes) and neuromuscular excitability were evaluated in 

20 offspring at 1 or 2 months of age (other results from this study are discussed under 

21 developmental toxicity, above). At 1 month, spontaneous mobility was reduced at the low dose 

22 in males (52% of control levels; p < 0.01) but not at the high dose, and at both doses in females 

23 (to 64 and 56% of control levels at the mid dose and high dose, respectively; p < 0.02). At two 

24 months, there was a dose-related increase in activity for both sexes, statistically significant (p < 

25 0.001) in high-dose females only (391% of control levels).

26

27 4.2.1.6.1.3. Learning and memory. The effects of repeated formaldehyde exposures on learning 

28 were investigated by Malek et al. (2003c), using a labyrinth swim maze. In this task, animals are 

29 required to make a series of consecutive right or left turns to gain access to an escape platform 

30 (Malek et al., 2003c). Adult male and female LEW.1K rats (15/sex/group) were exposed to 0, 

0.1, 0.5, or 5.4 ppm (0, 0.123, 0.615, or 6.64 mg/m3 31 ) formaldehyde 2 hours/day for 

32 10 consecutive days. Formaldehyde levels were checked eight times throughout the 2-hour 

33 exposure periods. Mean formaldehyde levels of 0.1 ± 0.02, 0.5 ± 0.1, and 5.4 ± 0.65 ppm were 

34 achieved. Body weight was measured on days 1, 5, and 10 of the experiment. Two days prior to 

35 beginning the formaldehyde exposures, all subjects were given an acclimation trial in which they 
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1 were individually placed into the water-filled basin at the start position and allowed to navigate 

2 to the escape platform with manual assistance to learn the correct route. Thereafter, the water 

3 labyrinth test was run on each day of formaldehyde treatment, 2 hours after completion of each 

4 daily exposure. Time taken to complete the test and errors made were recorded for each rat 

5 (Table 4-56). An error was defined as swimming toward the start position or circling in the same 

6 position without moving forward toward the escape platform. Rats were sacrificed at the end of 

7 the experiment, and tissues were taken from the lung, heart, thymus, kidney, liver, pancreas, 

8 skeletal muscle, and spleen. Tissues were fixed and prepared for histologic examination by light 

9 microscope. No differences were noted in food consumption or body weight gain for either male 

10 or female rats (Malek et al., 2003c). No treatment-related differences in organ pathology were 

11 reported (with the possible exception of focal microatelectasis (lung collapse at the microscopic 

12 level) seen in two to three animals in each formaldehyde-exposed group but not control animals). 

13

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

15 test by male and female LEW.1K rats

16

Male rats Swimming time (sec) Error rate (mean)

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 ppma 100 12.9 6.07 7.7 5.0 c 3.2c

0.5 ppm 97 16.7 c 7.60b 7.6 4.4 c 1.8c

5.4 ppm 105 25.7 c 10.9c 7.7 5.0 c 2.8c

Female rats Swimming time (sec) Error rate (mean)

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 3.0c

0.5 ppm 97 14.6 c 7.60b 8.0 4.6 c 2.2c

5.4 ppm 98 23.5 c 9.73c 7.9 5.2 c 2.6c

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

b 19 Different from control, p < 0.05. c 20 Different from control, p < 0.005.

21

22 Source: Malek et al. (2003c).

23

24

25 A clear learning curve was evident in control animals, with rats completing the task in 

26 less time and with fewer errors over days (Table 4-56). Although the number of errors decreased 

27 with increasing experience in all groups, error rates in formaldehyde-exposed rats at all doses 

28 were consistently higher than those observed in controls, starting on day 3 (Figure 4-24). All 

29 control animals performed without errors by day 6, whereas all treated animals were still making 

30 two to three errors on day 10, the final day of testing. Time required (latency) to complete the 

31 maze was also reduced over days. Although this measure of performance was not as sensitive as 
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1 error rate, formaldehyde-induced deficits were still evident in the 0.5 and 5.4 ppm exposure 

2 groups of both sexes.

3

4

5

6 Figure 4-24. Effects of formaldehyde exposure on the error rate of female 

7 LEW.1K rats performing the water labyrinth learning test.

8

9 Source: Drawn from data reported in Malek et al. (2003c).

10

11

12 Impaired performance on formaldehyde-treated subjects cannot be attributed to 

13 alterations in swimming ability, since latencies to complete the maze were identical for 0 and 

14 0.1 ppm groups, yet acquisition of the task was still impaired in the 0.1 ppm group based on 

15 number of errors committed (see Figure 4-24). This study reports an adverse effect level of 

16 0.1 ppm for increased error rate in the labyrinth water test, and all dose groups were equally 

17 impaired across a broad range of exposures, 0.1–5.4 ppm. An independent estimate of 

18 swimming speed was not included, so motor competency could not be directly evaluated. 

19 However, comparable latency scores and error rates at the beginning of testing across all groups 

20 and latency scores that track together over days suggest that impaired swimming ability does 

21 account for the observed differences in latency, which are most likely reflective of the increased 
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1 number of errors in treated animals (errors usually increase the distance traveled and thus time 

2 required for completion of the trial).

3 Pitten et al. (2000) evaluated the effects of very brief formaldehyde exposures 

4 (10 minutes) but prolonged duration (90 days) on previously learned performance in a land 

5 version of the labyrinth maze. Adult male and female Wistar rats (13/group) were trained on the 

6 task for 14 days, two trials/day. Animals were required to make a series of five left or right turns 

7 from the entrance of the maze to retrieve a piece of cheese placed in the goal box at the opposite 

8 end. Animals were guided by the experimenter through the maze during this acclimation phase 

9 until all subjects were able to retrieve the food without aid. After animals were trained (but prior 

10 to formaldehyde exposure), performance was assessed once daily for 11 days, and the latency to 

11 complete the maze as well as the number of errors committed when traversing from the entrance 

12 to the goal box was recorded. Animals were then assigned to one of three dose groups (five to 

13 eight/sex/group) such that task performance was equivalent across groups prior to 

14 commencement of formaldehyde exposures. Animals were exposed to 0 ppm, 2.6 ppm (0.25% 

formaldehyde solution to yield 3.06 ± 0.77 mg/m3 15 ), or 4.6 ppm (0.70% formaldehyde solution to 

yield 5.55 ± 1.27 mg/m3 16 ) formaldehyde 10 minutes/day, 7 days/week for 90 days. Animals were 

17 assessed for performance in the maze every seventh day, at least 22 hours after the exposure on 

18 the previous day. At the end of the 90-day exposure period, monitoring of maze performance 

19 continued once every 10 days for an additional 30 days. All rats were sacrificed at the end of the 

20 postexposure trials and tissue sections were prepared for histologic examination by light 

21 microscopy, including liver, trachea, lung, kidney, heart, spleen, pancreas, testicle, and brain. 

22 No treatment-related changes in food or water consumption weight gain or in histologic samples 

23 obtained at the termination of the experiment were observed.

24 Pitten et al. (2000) reported that no gender differences existed as a function of 

25 formaldehyde treatment; therefore, data were presented by combining sexes. Control rats 

26 showed no change in error rate but a slight decrease in running time through the maze during the 

27 course of the experiment. The formaldehyde-exposed groups began with a similar performance 

28 level and error rate as controls, but their performance degraded over the course of formaldehyde 

29 exposure. By the fourth week of exposure, increased numbers of errors were evident in both 

30 exposed groups relative to controls. This trend reached statistical significance at the 12-week 

31 time point, with a greater than twofold increase in number of errors (p < 0.05). Formaldehyde32 treated rats also tended to have increased run times through the maze (p = 0.04), but no 

33 difference was seen by formaldehyde concentration. By 4 weeks after termination of exposure, 

34 no statistical differences among the three groups were evident, but the tendency for the two 

35 exposed groups to make more errors and have longer latencies remained. Since Pitten et al. 
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1 (2000) tested animals after the task was acquired, these results indicate deficits in the retention of 

2 a previously learned task.

3 Lu et al. (2008) evaluated the effects of formaldehyde on performance of mice in a 

4 Morris water maze. Kunming mice (five males/group) were exposed to formaldehyde at 0.2, 1, 

or 3 mg/m3 5 6 hours/day for 7 days (measured concentrations: 0.2 ± 0.01, 0.99 ± 0.04, and 

3.03 ±

20 Apfelbach and Reibenspies (1991) published a brief report of formaldehyde effects on 

olfactory learning. Ferrets were exposed to 0.25 ppm (0.31 mg/m3 21 ) formaldehyde gas 

22 continuously for 6 months. A Y-shaped maze was used to test odor detection, discrimination 

23 between odors, and odor threshold. Ferrets were conditioned to distinguish ethyl acetate 

24 (0.1 vol%) from clean air. Untreated ferrets achieved 75% success after an average of 110 trials. 

25 However, formaldehyde-treated ferrets required on average 320 trials to reach a 75% success 

26 rate. A 90% success rate was achieved by untreated ferrets after 420 trials. However, this level 

27 of success was not reached in formaldehyde-treated ferrets.

0.16 mg/m3 6 ). Mice were trained to locate a hidden platform in a large, circular tank 

7 (106 cm diameter, 31 cm deep). Each animal received four training trials per day, beginning 

8 30 minutes after the end of exposure. During training, latency to locate the platform was 

9 recorded for each trial, with a maximum of 60 seconds, after which the animal was guided to the 

10 platform. After the last day of training, an additional trial was conducted with the platform 

11 removed (the probe trial); time spent in each maze quadrant was measured to determine the time 

12 the animal spent searching for the platform in the correct area of the maze. Performance in the 

13 water maze, measured as mean escape latency across the seven training trials, was significantly 

impaired in the 3 mg/m3 group. No significant difference was seen at 1 mg/m3 14 , although there 

15 appeared to be an increased latency during the second day of testing. During the probe trial, 

16 control animals spent significantly more time in the correct quadrant, but neither formaldehyde17 exposed group did so. Results of this study indicate deficits in learning and retention of the 

18 Morris water maze following formaldehyde exposure, with greater effects seen in the higher dose 

19 group. 

28 The same researchers also tested olfactory function in formaldehyde-treated ferrets, as 

29 summarized in Section 4.2.1.7 (Apfelbach et al., 1992). A decrease in olfactory discrimination 

30 and a reduction in the percentage of olfactory cells in the olfactory epithelium were reported 

31 after 3–12 months exposure to 0.25 or 0.5 ppm formaldehyde. Decreased olfactory sensitivity in 

32 rats exposed to 0.25 or 0.5 ppm formaldehyde has also been reported by the same researchers 

33 (Weiler and Apfelbach, 1992; Apfelbach and Weiler, 1991), and Weiler and Apfelbach (1992) 

34 reported in an abstract that shifts in olfactory thresholds were greater when exposure was 

35 initiated at PND 30 than at adult ages. Given the documented changes in olfactory thresholds, 
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1 observed changes in olfactory learning would likely be confounded by the potential for 

2 decreased olfactory function by formaldehyde exposures, and definitive conclusions regarding 

3 formaldehyde effects specific to learning cannot be made based on these studies.

4

4.2.1.6.1.4. Neurosensitization.

13 Sorg et al. (1996) evaluated cross-sensitization of cocaine-induced increases in activity 

14 from an initial formaldehyde exposure. Female Sprague-Dawley rats (eight to nine) were 

exposed to 0 or 11 ppm (0 or 13.5 mg/m3 15 ) formaldehyde 1 hour/day for 7 days. Locomotor 

16 activity was measured (by photocell) after saline injection (1 day postexposure) and after cocaine 

17 injection (2 days postexposure). A similar protocol was conducted on days 36 and 37 

18 postexposure. Motor activity levels following saline injection were similar for controls and 

19 formaldehyde-treated rats. However, formaldehyde exposure initiated sensitization to cocaine as 

20 evidenced by a greater increase in locomotor activity in mice treated with formaldehyde 

21 followed by cocaine (p < 0.05) with an average count of crossed grids greater than 40,000 (2 

22 hours) in treated animals compared with 25,000 (2 hours) in controls. The cross-sensitization 

23 was transient, with no treatment effects on cocaine-induced activity either 29 or 37 days 

24 postexposure. When examining individual data, the authors suggested that the formaldehyde25 treated groups in both cases have a cluster of high responders (HRs), suggesting some animals 

26 may have been more sensitive. A second group of similarly treated female rats was pretested for 

27 locomotor activity and divided into subgroups of HRs or low responders (LRs). They were then 

28 given a panel of neurobehavioral tests: anxiety (elevated plus maze, day 11); memory (passive 

29 avoidance training, day 12; passive avoidance test, day 19); and nociceptive test (day 20). Trunk 

30 blood corticosterone levels were determined during stress on day 35 postexposure. No 

31 significant treatment differences were found in the passive avoidance test, nociception, or 

32 corticosterone levels (basal or stress induced). On the elevated plus maze, a two-way ANOVA 

33 indicated no overall formaldehyde treatment effects, but the HR rats had higher open arm time 

34 ratios (indicating greater anxiety) regardless of treatment. Within the treatment groups, the 

35 difference in behavior between HR and LR subgroups was only significant for the formaldehyde5 Sorg et al. (1996) studied the potential for formaldehyde 

6 exposure to induce sensitization in the CNS, possibly through the limbic pathways in the brain. 

7 The authors hypothesized that multiple chemical sensitivity (MCS) has an onset and progression 

8 similar to CNS sensitization and may, therefore, be a similar process. These experiments were 

9 conducted to test this hypothesis and to determine whether formaldehyde exposure could be used 

10 as a model for MCS. Behavioral sensitization can be initiated by psychostimulants (e.g., 

11 cocaine) and manifest as increased locomotor activity upon subsequent challenge with the 

12 stimulant.
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1 treated rats (p < 0.05). Authors suggested that cross-sensitization to cocaine-induced locomotor 

2 activity was caused by enhanced dopamine transmission within the mesolimbic system (ventral 

3 tegmental area to nucleus accumbens projection) following repeated formaldehyde exposure. A 

4 critical role of the hypothalamic-pituitary-adrenal (HPA) axis has also been implicated in cross5 sensitization. 

6 Sorg et al. (1998) and Sorg and Hochstatter (1999) further explored formaldehyde7 induced behavioral sensitization using the cocaine model. In contrast to the results following 

8 exposure to 11 ppm (Sorg et al., 1996), rats exposed to only 1 ppm for 7 days showed no cross9 sensitization to cocaine injection. However, animals exposed to 1 ppm formaldehyde for 

10 4 weeks exhibited increased cocaine-induced vertical activity (with no difference in horizontal 

11 activity) for 4–6 weeks after cessation of exposure. Activity levels of formaldehyde-exposed rats 

12 were approximately threefold those of control rats 3–4 days postexposure and still 1.5-fold 

13 control levels at 4–6 weeks postexposure (p < 0.05). 

14 Sorg et al. (2001) examined changes in corticosterone levels in rats with and without 

15 formaldehyde treatment. Basal corticosterone levels in trunk blood were established in naïve 

16 male Sprague-Dawley rats taken directly from their home cage immediately prior to sacrifice. In 

an acute trial, male rats were exposed to 0, 0.7, or 2.4 ppm (0, 0.86, or 2.96 mg/m3 17 ) 

18 formaldehyde for either 20 or 60 minutes, and trunk blood was collected for corticosterone 

19 analysis. Therefore, these rats were challenged with a new environment (the exposure chamber) 

20 in the presence or absence of formaldehyde. In a separate trial, basal and challenged 

21 corticosterone levels were measured after repeated exposure (1 hour/day, 5 days/week for 2 or 

22 4 weeks). Basal corticosterone levels were measured in trunk blood immediately after removing 

23 the animal from its home cage. Challenged corticosterone levels were measured after rats were 

24 placed into the exposure chamber for a final 20-minute exposure. Body weight was measured at 

25 the beginning of each week of exposure and was unchanged by formaldehyde treatment.

26 Corticosterone levels were increased over basal levels when rats were placed in the 

27 exposure chamber for 20 minutes (Figure 4-25, panel a) but returned to basal levels after 

28 60 minutes in the exposure chamber (not shown). This response may reflect the stress of the new 

29 environment and acclimatization after 60 minutes in the chamber. Corticosterone levels were the 

30 same in the presence or absence of formaldehyde, indicating no treatment effect.

31
Panel a Panel b Panel c

 

Corticosterone levels after 2 weeks of 

repeated formaldehyde exposure

0

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Control 0.07 2.4

Formaldehyde (ppm)

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Challenge

Serum corticosterone (ng/mL)

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Control 0.07 2.4

Formaldehyde (ppm)

Basal

Challenge

Corticosterone levels: with no 

pre-exposure to formaldehyde

Serum corticosterone (ng/mL)

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Control 0.07 2.4

Formaldehyde (ppm)

Corticosterone levels after 4 weeks 

of repeated formaldehyde exposure

Serum corticosterone (ng/mL)

Basal

Challenge

1 

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

male LEW.1K rats after formaldehyde inhalation exposures.

Note: Panel a: no pretreatment, corticosterone levels after 20-minute 

formaldehyde exposure. Panels b and c show both basal and induced 

corticosterone levels after a 2- or 4-week pretreatment to formaldehyde 

1 hour/day. Challenge to induce corticosterone was a 20-minute reexposure at the 

formaldehyde level tested. 

Source: Sorg et al. (2001). 

Control animals exhibited an increase in basal corticosterone after 2 weeks, which 

returned to naïve levels after 4 weeks (Figure 4-25, panels b and c). Formaldehyde-treated rats 

demonstrated a comparable increase in basal corticosterone levels at 2 weeks, but these levels 

did not return to naïve levels at 4 weeks as seen with controls. Control and 0.7 ppm exposed rats 

showed a similar response to challenge (the final 20-minute exposure). However, rats exposed to 

2.4 ppm were hyperresponsive, with exaggerated corticosterone levels during this final exposure. 

Differences in basal corticosterone levels after formaldehyde exposure and the 

hyperresponsiveness seen in animals exposed at 2.4 ppm provide evidence of 

formaldehyde-induced perturbations of the HPA axis. Authors suggested that elevated 

corticosterone levels induced by repeated formaldehyde exposures may contribute to the crosssensitization to cocaine-induced motor activity. 

Formaldehyde-induced changes in the HPA axis may contribute to behavioral effects of 

formaldehyde exposure reported by Sorg et al. (2004) and Sorg and Hochstatter (1999). The 

authors also reported an enhanced conditioning to odor in animals previously exposed to 
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1 repeated formaldehyde. Male and female Sprague-Dawley rats (60–80 days of age) were 

exposed at 1 ppm (1.23 mg/m3 2 ) formaldehyde 1 hour/day, 5 days/week for 4 weeks (Sorg and 

3 Hochstatter, 1999). Two weeks after exposure ended, rats were trained to the conditioned fear 

4 task. Rats were conditioned to a fear response by either odor only or odor associated with 

5 footpad shock. Orange-oil extract was used as the odor conditioned stimulus (CS). One day 

6 after conditioning, rats were reintroduced into the environment without an odor cue, and time 

7 spent motionless in the freezing posture (freezing) was observed. On day 2 after conditioning, 

8 rats were placed in a novel environment, and time spent in the freezing posture was evaluated in 

9 the absence and then the presence of odor. This was repeated on day 12 after conditioning to 

10 measure the loss of the freezing response to the conditioned odor.

11 Both treated and exposed rats showed similar responses on reintroduction into the 

12 conditioning environment in the absence of an odor cue on day 1 (Sorg and Hochstatter, 1999). 

13 As expected, rats conditioned with a footpad shock demonstrated greater time motionless than 

14 odor-trained only rats, and there was no difference between control and formaldehyde-treated 

15 rats. However, in the presence of odor on days 2 and 12, formaldehyde-exposed rats who were 

16 conditioned with odor associated with foot shock spent significantly more time freezing than 

17 odor-only trained rats (p < 0.05); control animals on those days showed no difference in time 

18 freezing in the presence and absence of odor. The authors concluded that the formaldehyde19 treated rats had more difficulty than controls in extinguishing the fear response to the 

20 conditioned odor, and speculated that an enhancement of the fear-conditioned response by 

21 formaldehyde pretreatment supports the hypothesis that sensitization may include effects through 

22 the limbic system of the brain.

23 In a second experiment, adult male and female Sprague-Dawley rats were exposed at 0 or 

2 ppm (2.45 mg/m3 24 ) formaldehyde 1 hour/day, 5 days/week for 4 weeks (Sorg et al., 2004). Two 

25 to 3 weeks after exposure ended, rats were trained to the conditioned fear task. Rats were given 

26 a foot shock either associated with an odor (paired group) or unassociated with an odor (unpaired 

27 group). Orange-oil extract was used as the odor CS. After training, freezing behavior was 

28 assessed (1) in the same context in the absence of odor (1 day), (2) in a new context in the 

29 presence and absence of the CS (5 consecutive days), and (3) in another novel context in the 

30 presence and absence of the CS.

31 Formaldehyde-exposed male rats demonstrated increased conditioned fear response to an 

32 odor CS (orange oil) paired with foot shock with no change in the degree of conditioning to the 

33 context. For female rats, formaldehyde exposure did not affect the percent of time spent 

34 freezing, either in the conditioning context or the novel context in the absence of the conditioned 

35 odor. In contrast, male rats spent an increased time freezing in a novel context in the presence of 
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1 odor, indicating a greater conditioned fear response to the olfactory cue (p < 0.05). This is in 

2 agreement with the previous study where formaldehyde effects were seen in the presence of the 

3 conditioning odor but not the environment (Sorg and Hochstatter, 1999). However, in this study 

4 female rats did not exhibit a similar enhancement of fear conditioning to the olfactory CS.

5 The authors suggested that repeated exposure to low levels of formaldehyde acts as a 

6 stressor in much the same way as inescapable foot shock, with resulting sensitized responses 

7 within the olfactory/limbic pathways (Sorg et al., 2004). This interpretation is consistent with 

8 work described above in which augmented basal corticosterone levels following repeated 

9 formaldehyde exposures were demonstrated. However, while the fear conditioning in the present 

10 study and cross-sensitization to cocaine described above (Sorg and Hochstatter, 1999) occurred 

11 3–4 weeks after termination of exposure to formaldehyde, the duration of corticosterone 

12 elevation induced by repeated exposure to formaldehyde has not been determined. It is possible 

13 that augmentation of corticosterone levels following formaldehyde exposure results from direct 

14 action of formaldehyde on the HPA axis. Experiments designed to compare HPA activation 

15 following standard stressors (repeated inescapable foot shock or restraint stress), stress induced 

16 by other irritants (chemicals with strong irritant odors but no CNS action), and repeated 

17 formaldehyde exposures are necessary to dissociate primary from secondary action of 

18 formaldehyde on CNS function in this paradigm. It is also possible that enhanced conditioning 

19 to an odor stimulus results from formaldehyde-induced increases in airway irritation, rendering 

20 the conditioned odor stimulus a more salient cue, producing a conditioned response that is not 

21 extinguished as readily as in air-exposed controls. However, damage of the nasal mucosa and 

22 lesions would be expected to be minimal at 1 ppm formaldehyde exposures and most likely 

23 resolved 2 weeks after exposure was ended (see Section 4.2.1.2). Therefore, a more salient cue 

24 for fear conditioning to odor due to physical irritation is not likely. Alternatively, formaldehyde 

25 may act to up regulate olfactory activity, producing a stronger sense of odor during conditioning.

26

27 4.2.1.6.1. 5. Neurochemistry and neuropathology. Several studies that were focused on general 

28 toxicity or carcinogenicity of formaldehyde also assessed histopathology in exposed animals, 

29 including pathological evaluation of the brain. In all cases, details of the pathological evaluation 

30 were not provided. Reported results stated that no significant lesions were seen on unspecified 

31 tissues (Appelman et al., 1988; Maronpot et al., 1986; Kerns et al., 1983) or that an increase in 

32 relative brain weight (data not provided) was considered of no toxicological significance 

33 (Woutersen et al., 1987). The absence of procedural information, or specific reported results, 

34 limits the utility of this information.
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1 Boja et al. (1985) measured changes in several neurotransmitters (norepinephrine, 

2 dopamine, 5-hydroxytryptamine) and their major metabolites (3,4-dihydroxyphenylacetic acid 

3 [DOPAC] and 5-hydroxyindoleacetic acid [5-HIAA]) following one or two 3-hour exposures to 

4 formaldehyde at 0, 5, 10, or 20 ppm. Animals were sacrificed immediately following the second 

5 exposure, and brains were immediately removed, frozen, and sectioned. Regions of interest were 

6 analyzed by high-pressure liquid chromatography with electrochemical detection. Authors stated 

7 that neurotransmitter concentrations were measured in multiple brain regions, but results were 

8 reported only for the 5 ppm exposure and only for the hypothalamus. No change was seen in 

9 concentrations of norepinephrine or 5-hydroxytryptamine for any exposure paradigm. For those 

10 animals exposed twice to formaldehyde, there was a slight (statistically significant) increase in 

11 dopamine and a larger (approximately fourfold) increase in 5-HIAA. DOPAC was increased 

12 (approximately 30%) in animals receiving formaldehyde during the second exposure only.

13 Recent work by Hayashi et al. (2004) indicates that formaldehyde exposure increases the 

14 activity of periglomerular (PG) cells in the main olfactory bulb. Tyrosine hydroxylase activity 

15 was measured as a marker for activity of olfactory function. The authors surmised that 

16 expression levels of this enzyme are useful markers since it has been reported that the protein is 

17 up regulated after sensory stimulation and is down regulated by odor deprivation or when the 

18 olfactory epithelium is removed (Cho et al., 1996; Stone et al., 1991; McLean and Shipley, 1988; 

19 Baker et al., 1983). Eight-week-old female C3H/HeN mice were exposed at 0, 0.08, 0.4, or 

2 ppm (0, 0.1, 0.49, or 2.45 mg/m3 20 ) formaldehyde 16 hours/day for 1 day or 12 weeks 

21 (5 days/week). Formaldehyde exposure did not affect body weight. Mice were sacrificed 

22 24 hours after exposure; the brains were removed, fixed, and prepared for sectioning. One side 

23 of the olfactory bulb was sliced into 40 μm-thick serial frontal sections and immuno-stained for 

24 tyrosine hydroxylase activity. The number of tyrosine hydroxylase-positive PG cells was 

25 determined by examining digital photomicrographs of three tissue sections, averaging the counts 

26 from 10–15 glomeruli per section.

27 Neither the size of the olfactory bulb (rostrocaudal, dorsoventral, and mediolateral 

28 lengths) nor the total number of PG cells was changed by formaldehyde exposure. The number 

29 of tyrosine hydroxylase-positive PG cells per glomerulus was unchanged by a single 

30 formaldehyde exposure but increased after 12 weeks of repeated exposures. The increases were 

31 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 

32 at 2 ppm or 196, 167, and 196% of controls, respectively. As an indicator of activity, it is not 

33 unexpected that the enzyme was up regulated after repeated exposure to an odorous compound. 

34 Hayashi et al. (2004) hypothesize that the increased tyrosine hydroxylase activity is an indication 

35 of increased sensitivity and, therefore, may be a model for MCS. However, it is unknown if the 
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1 increase in enzyme activity after repeated exposures is transient or could result in sensitization. 

2 Tyrosine hydroxylase is the first enzyme in the dopamine synthetic pathway, but the role of 

3 dopamine in PG cells is not known. Further research would be needed to understand the 

4 potential for formaldehyde to act as a sensitizing agent in this model.

5 In an abstract, Kakeyama et al. (2004) outline the results of experiments to address the 

6 effects of subchronic exposure to low levels of formaldehyde on changes in neurotransmitter7 related mRNA expressions in mice forebrains. An unstated number of female C3H/He mice 

were exposed 16 hours/day, 5 days/week to 400 ppb (0.49 mg/m3 8 ) formaldehyde for 12 weeks. 

9 The authors used RT-PCR methodologies to quantify mRNA encoding for the glutamate receptor 

10 subunits GluR1 and GluR2, the dopamine receptor D1, and the serotonin receptor 5-HT1A in the 

11 neocortex, hippocampus, amygdala, and hypothalamus. Raised levels of mRNA expression were 

12 observed for GluR1 in the neocortex and hippocampus; GluR1, GluR2, and the dopamine 

13 receptor D1 in the amygdala; and the serotonin receptor 5-HT1A in the hypothalamus. Reduced 

14 mRNA expression was observed for GluR2 in the hippocampus and neocortex. When other 

15 mice were subjected to a radiofrequency-induced lesion of the hippocampus then exposed to 

16 formaldehyde for 12 weeks as before, the altered expression of GluR1 and GluR2 in the 

17 neocortex was abolished. However, the increment of mRNA expression of 5-HT1A in the 

18 hypothalamus was further enhanced. In demonstrating that formaldehyde affects neocortical 

19 GluR1 and GluR2 mRNA expressions through a hippocampal function, Kakeyama et al. (2004) 

20 concluded that subchronic exposure to low concentrations of formaldehyde can affect neural 

21 transmission in the forebrain.

22 Fujimaki et al. (2004b) examined the effects of formaldehyde on NGF in the brain and 

23 hippocampus. Ten female C3H/HeN mice/group were exposed to 0, 80, 400, or 2,000 ppb (0, 

0.1, 0.5, or 2.45 mg/m3 24 ) formaldehyde 16 hours/day, 5 days/week for 12 weeks. Some groups of 

25 mice received the same treatment after I.P. injection of 10 μg of OVA and 2 mg alum prior to the 

26 commencement of formaldehyde exposure. For this subgroup, booster injections of OVA were 

27 administered on days 21, 42, 63, and 77 during the formaldehyde exposure regimen. 

28 Quantitative measures of NGF and BDNF in homogenates of whole brain and hippocampus were 

29 obtained by ELISA and mRNA determination. The amount of NGF protein in whole brains 

30 remained unchanged in the non-immunized mice. However, brain NGF levels were significantly 

31 increased in OVA-immunized mice exposed to 80 and 400 ppb (but not 2,000 ppb) 

32 formaldehyde (Figure 4-26). This result was confirmed by parallel increases in the 

33 concentrations of hippocampal NGF mRNA that were produced in immunized mice exposed to 

34 formaldehyde at the same concentrations. However, there were no comparable increases in the 

35 amounts of brain-derived neurotrophic factor in either immunized or non-immunized mice. In 
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1 discussing the mechanisms potentially associated with their results, Fujimaki et al. (2004b) 

2 considered it likely that low-level exposure to formaldehyde could enhance NGF production 

3 through the stimulation of the HPA axis together with immunization.

4

5

6

7

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

9

10 Note: Female C3H mice were exposed to formaldehyde 16 hours/day, 

11 5 days/week for 12 weeks. NGF in homogenates of whole brain and 

12 hippocampus were measured by ELISA. Values are means ± SEM (n = 5–6). 

13 * = p < 0.05 and ** = p < 0.01 versus control mice, as calculated by the authors.

14

15 Source: Redrawn from Fujimaki et al. (2004b).

16

17

18 The enhancement of NGF in the brains of immunized mice exposed to formaldehyde 

19 gave rise to the suggestion that NGF may promote the survival of hippocampal neurons when 

20 challenged with formaldehyde. To examine whether or not apoptosis plays a role in this process, 

21 Tsukahara et al. (2006) measured the effects of formaldehyde on apoptotic mechanisms 

22 regulating the survival and death of cells and on N-methyl-D-aspartate (NMDA) receptors. 

Female C3H/HeN mice (13/group) were exposed to 0 or 400 (393 ± 34) ppb (0 or 490.8 μg/m3 23 ) 

24 formaldehyde 16 hours/day, 5 days/week for 12 weeks. Seven control and formaldehyde-treated 

25 mice were immunized with 10 μg OVA plus 2 mg aluminum hydroxide prior to exposure. 

26 Subsequently, these mice received OVA via aerosol as a booster during weeks 3, 6, 9, and 11. 

27 Hippocampi were dissected from all animals 1 day after the final exposure and homogenized in 

28 hypotonic buffer. The 12,000 rpm supernatants were analyzed by Western blotting for the 

29 presence of the proteins BCl-2 (which inhibits apoptosis) and Bax (which opposes BCl-2 action 

30 and promotes apoptosis) and the NMDA receptor subtypes 2A and 2B (NR2A and NR2B). 
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1 Immunohistochemical analysis was also carried out for the presence of active caspase-3, an 

2 apoptosis marker.

3 The levels of NR2A and NR2B were unaffected by exposure to formaldehyde in either 

4 immunized or non-immunized mice. Likewise, the number of caspase-3 immunoreactive cells 

5 did not change as a result of formaldehyde exposure. However, when measured amounts of 

6 Bcl-2 and Bax were normalized to the amount of β-tubulin, the ratio Bcl-2/Bax was significantly 

7 increased in immunized mice exposed to formaldehyde. Non-immunized mice did not show this 

8 apparently compound-related response. Consistent with the concept that the proportions of Bcl-2 

9 and Bax are critical for the regulation of cell survival and death, the authors interpreted their data 

10 as an indication that changes to the ratio of Bcl-2/Bax expressions might be an important 

11 adaptive response to the effects of formaldehyde, such that the antiapoptotic changes might 

12 contribute to the protection of hippocampal neurons from the pernicious effects of formaldehyde 

13 exposure itself.

14 The same research group used the immunized mouse model to determine whether 

15 formaldehyde exposure affected mRNA expression of genes related to synaptic plasticity 

16 (Ahmed et al., 2007). Ten female C3H/HeN mice were exposed to 0 or 400 ppb formaldehyde 

17 16 hours/day, 5 days/week for 12 weeks. All mice were immunized with 10 μg OVA plus 2 mg 

18 aluminum hydroxide prior to initial formaldehyde exposure then treated in weeks 3, 6, 9, and 11 

19 with aerosolized OVA as a booster. Five treated and control animals were I.P. injected with 1 

20 mg/kg MK-801, a noncompetitive NMDA receptor agonist before the last formaldehyde 

exposure. At term, hippocampi were dissected and frozen at −80o 21 C until processing. At that 

22 point, total mRNA was extracted and first strand cDNA was synthesized by using reverse 

23 transcriptase. Expression levels of various proteins/receptors, including NMDA NR2A and 

24 NR2B receptor subunits, dopamine D1 and D2 receptors, cyclic AMP responsive element25 binding proteins (CREB-1 and CREB-2), and the transcription factors FosB and ΔFosB were 

26 determined by using the PCR. The expression level of each mRNA species was expressed 

27 relative to the sample’s content of 18S rRNA. The total protein lysate was also assayed for 

28 pCREB by Western blotting.

29 In the first of a sequence of histograms, Ahmed et al. (2007) demonstrated a significant 

30 increase in mRNA expression of NR2A as a result of formaldehyde exposure. However, this 

31 effect was abolished in animals treated with MK-801. A similar trend in the mRNA expression 

32 of NR2B in response to formaldehyde exposure did not achieve statistical significance. MK-801 

33 treatment significantly reduced receptor in mRNA expression in the presence of formaldehyde. 

34 The authors provided data showing an increased expression of dopamine D1 and D2 receptor 

35 mRNA response to formaldehyde, in both cases abolished by treatment with MK-801. The 
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1 expression of CREB-1 mRNA also conformed to the pattern of being increased as a result of 

2 formaldehyde exposure but abolished by MK-801. However, the expression of CREB-2 and 

3 FosB/ΔFosB was unaffected by formaldehyde. When normalized to the amount of β-tubulin, 

4 there were no significant effects of formaldehyde exposure and MK-801 treatment on the protein 

5 levels of pCREB. Finally, there was no significant difference in the expression of transient 

6 receptor potential vanilloid receptor (TRPV1) between control and formaldehyde-exposed mice, 

7 and MK-801 itself did not significantly alter the mRNA level of TRPV1. In seeking to explain 

8 their results, the authors speculated that low-level exposure of immunized mice to formaldehyde 

9 had an effect on hippocampal synaptic plasticity at the mRNA level, as evidenced by the 

10 enhancement of mRNA for NR2A, the dopamine D1 and D2 receptors, and CREB1, with up 

11 regulation compensating for the sustained levels of enhanced protein expression under low-level 

12 formaldehyde exposure. The interpretation of these changes in NR2A mRNA, in the context of 

13 the results of Tsukahara et al. (2006), showing no change in NR2A and NR2B protein 

14 expression, was not discussed.

15

4.2.1.6.1.6. Neurogenesis.

27 For both pyramidal and granular areas, a much lower number of cells was seen on 

28 PND 90 versus PND 30 (p < 0.001). This response was evident irrespective of the amount of 

29 exposure to formaldehyde and is consistent with normal brain development. Compound-specific 

30 effects of formaldehyde on the volume and number of granular and pyramidal cells varied by 

31 dose and over the two time points. There was a small increase in the volume of the granular cell 

32 layer of the dentate gyrus in rats sacrificed on PND 30 (p < 0.001) in response to increasing 

33 formaldehyde concentration (Aslan et al., 2006), with no significant change in neuron number; 

34 the increased volume (now accompanied by an increase in neuron number) was still evident at 

35 the low-exposure level on PND 90 (p < 0.01) but not at the high dose. Brain hemisphere volume 

16 Two papers have examined the effects of subacute exposure to 

17 formaldehyde on the overall size (volume) of discrete cellular areas of the hippocampus in 

18 neonatal rats. The researchers also used an optical fractionator counting method to derive a 

19 plausible estimate of cell number. Aslan et al. (2006) studied the effects of formaldehyde 

20 exposure on the number and volume of granular cells in the hippocampal dentate gyrus. 

21 Sarsilmaz et al. (2007) examined the impact of postnatal formaldehyde exposure on brain 

22 hemisphere volume and on the size and cell number of pyramidal cells in the cornu ammonis 

23 region of the hippocampus. The in-life phase was the same in each study, featuring the exposure 

of 10 neonatal male Wistar rats/group to 0, 6, and 12 ppm (0, 7.36, and 14.7 mg/m3 24 ) 

25 formaldehyde 6 hours/day, 5 days/week for 30 days. Five rats/group were sacrificed at that point 

26 (PND 30), while the rest were maintained without further treatment until PND 90.
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1 was decreased at both concentrations on PND 30 (p < 0.01) but was increased at both 

2 concentrations (p < 0.01, with a larger magnitude of effect at 6 ppm) on PND 90 (Sarsilmaz et 

3 al., 2007). In the hippocampal cornu ammonis region, the volume of the pyramidal cell layer on 

4 PND 30 was increased in low-dose animals (p < 0.001) but decreased in high dose animals (p < 

5 0.001) as compared with control values; neither group was significantly different from controls 

6 on PND 90. There was a dose-related decrease in total neuron number in the cornu ammonis on 

7 PND 30 (p < 0.01 for both doses); on PND 90 the decrease in neuron number remained 

8 statistically significant in both treatment groups (p < 0.01), but there was no longer any 

9 difference in the magnitude of the effect between doses (Sarsilmaz et al., 2007).

10 In a third study from the same laboratory, Songur et al. (2008) used the same exposure 

11 paradigm to evaluate changes in oxidant and antioxidant systems in the cerebellum of perinatally 

12 exposed rats. Exposure was carried out as in Aslan et al. (2006) and Sarsilmaz et al. (2007), 

13 described above. On PND 30 or 90, cerebellums from seven male rats per group were evaluated 

14 for levels of malondialdehyde (MDA), NO, superoxide dismutase activity (SOD), and 

15 glutathione peroxidase (GPX) activity. Dose-related increases in NO (approximately 20–80%), 

16 MDA (100–160%), and GPX (25–60%) and dose-related decreases in SOD (20–30%) were seen 

17 on PND 30. In general, the magnitude of change from control levels was maintained on PND 90, 

18 with the exception of MDA levels in 6 ppm animals, which appeared to approach control levels 

19 at 90 days. The authors stated that these findings indicated that formaldehyde exposure may 

20 cause neurotoxicity via the production of oxidative damage in the brain. Persistence of the effect 

21 to the 90-day time point (30 days after cessation of exposure) supports the possibility that 

22 formaldehyde may cause long-lasting or permanent changes in the brain following early life 

23 exposure. These results are consistent with the earlier studies by Aslan et al. (2006) and 

24 Sarsilmaz et al. (2007), finding permanent changes in brain structure (although in a different 

25 brain region) following early life exposure.

26

27 4.2.1.6.1.7. Summary of formaldehyde effects on neurobehavioral and neuropathological 

28 measures, following exposure via inhalation. As has been demonstrated in mice (Wood and 

29 Coleman, 1995), it is possible that rats experience respiratory tract irritation during low-level 

30 formaldehyde exposure. Perturbations in nervous system function reported with formaldehyde 

31 exposure include reductions in motor activity, lack of habituation, impairment in acquisition of a 

32 new learning task, deficits in retention of a previously learned task, increases in corticosterone 

33 levels, sensitization to cocaine-induced locomotor activity, and enhanced fear conditioning using 

34 an olfactory CS (Table 4-57). Many of these effects were observed at exposure levels at or 

35 below 1 ppm, and some persisted days to weeks after termination of exposure.
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1 Malek et al. (2004, 2003a, b) detected behavioral changes in rats and mice tested 2 to 

2 24 hours postexposure. The mechanism of these behavioral changes is unknown, and available 

3 data do not allow dissociation of direct effects on the nervous system and behavioral responses to 

4 the irritant effects of formaldehyde (control experiments [e.g., using a different aversive odor 

5 with or without irritant properties] were not included). Given that behavioral changes were 

6 observed hours to days after cessation exposure (i.e., beyond the time required for formaldehyde7 induced irritation to subside), it is unlikely that these behavioral changes were caused by 

8 formaldehyde-induced irritation. Similarly, although it is possible that systemic effects of 

9 formaldehyde exposure might cause reduced motor activity during or immediately following 

10 exposure, it is unlikely that these effects can account for the differences in responses of male rats 

11 24 hours after exposure (Malek et al., 2003a). Furthermore, a follow-up study demonstrated 

12 reduced motor activity in animals 2 hours after a 2-hour exposure to much lower levels of 

13 formaldehyde (0.1 ppm), which fall well below the levels identified by Wood and Coleman 

14 (1995) as the AC50 for formaldehyde in mice (Malek et al., 2003b).

15 Two studies reported significant reductions in learning or retention following brief 

16 periods of repeated exposure to low levels of formaldehyde (Malek et al., 2003c; Pitten et al., 

17 2000) (Table 4-57). Malek et al. (2003c) reported an increased number of errors in acquiring a 

18 water maze task; testing took place daily 2 hours after termination of a 2-hour exposure. The 

19 work of Pitten et al. (2000) revealed that brief exposures over many weeks led to increases in 

20 errors performing a previously learned task and that the magnitude of the effect increased over 

21 the course of the exposure period. Testing occurred remote from the time of exposure (22 hours 

22 after the previous exposure), and the deficits appeared to persist for several weeks after exposure 

23 terminated, minimizing the possibility that these effects were related to irritant properties of 

24 formaldehyde. Although the exposure levels were moderately high (2.6–4.6 ppm) and continued 

25 over several months, the duration of a single exposure event was very brief (10 minutes).

26 Sorg and Hochstatter (1999) and Sorg et al. (2004, 2001) suggest that behavioral 

27 sensitization associated with low-level formaldehyde exposure was linked to alterations in HPA 

28 control of corticosterone. Cross-sensitization to the locomotor activity-enhancing properties of 

29 cocaine and changes in response to a conditioned fear paradigm were observed in animals 

30 exposed several weeks earlier to repeated low-level formaldehyde. Direct activation of 

31 mesolimbic dopamine pathways or activation of conditioned fear response in the amygdala by 

32 formaldehyde could underlie these behavioral effects; these observations were also seen by study 

33 authors as consistent with a formaldehyde-induced stress response.
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Table 4-57. Summary of neurological and neurobehavioral studies of inhaled 

formaldehyde in experimental animals

Species

No./

group Treatmenta 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 

(AC50) decreased upon repeat exposure. AC50 = 

3.63 for first series, AC50 = 1.88 ppm for second 

series.

Time to exposure termination decreased with 

increasing formaldehyde concentration.

Time to termination was decreased in repeat 

exposures. 

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

formaldehyde in experimental animals

Species

No./

group Treatmenta Observations

LOAEL/

NOAEL Reference

Rats 0 or 0.5 mg/m3

(0.4 ppm) on 

GDs 1–19, 4 

hours/day

Increased motor activity on the 2nd and 3rd 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)
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Table 4-57. Summary of neurological and neurobehavioral studies of inhaled 

formaldehyde in experimental animals

Species

No./

group Treatmenta Observations

LOAEL/

NOAEL Reference

Neurosensitization endpoints

Female 

SpragueDawley 

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 male 

SpragueDawley 

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 male 

SpragueDawley 

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)

Male 

SpragueDawley 

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 male 

SpragueDawley 

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

formaldehyde in experimental animals

Species

No./

group Treatmenta Observations

LOAEL/

NOAEL Reference

Neurochemistry and neuropathology

8-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 

male 

SpragueDawley 

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 concentration in air (ppm) with the length of exposure each day and the 

duration of treatment in days, as available.

b

NA = not available.
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1 Limited data regarding possible neurochemical changes in the brains of formaldehyde2 exposed, immunized mice (Ahmed et al., 2007; Fujimaki et al., 2004b; Hayashi et al., 2004; 

3 Kakeyama et al., 2004) and rats (Boja et al., 1985) provided conflicting information, and the 

4 implications of these data regarding possible formaldehyde neurotoxicity are difficult to 

5 determine.

6 In developmental exposure paradigms, changes in brain structure (Sarsilmaz et al., 2007; 

7 Aslan et al., 2006), brain chemistry (Songur et al., 2008), and motor activity (Senichenkova, 

8 1991; Sheveleva, 1971) were seen following neonatal or in utero exposure to formaldehyde. In 

9 addition, Weiler and Apfelbach (1992) found juvenile animals to be more sensitive to 

10 formaldehyde-induced changes in olfactory thresholds when compared with adult animals. 

11 These studies raise concern about possible long-lasting neurological effects of early exposure to 

12 formaldehyde. It is important to note, however, that exposure levels in these studies were higher 

13 (250–6,000 ppb) than those producing the behavioral effects in adults described above.

14 Overall, available data provide substantial evidence of behavioral effects, including 

15 motor activity changes and changes in learning and retention, following repeated exposure to 

16 relatively low levels of formaldehyde. These effects were seen in multiple laboratories, in 

17 studies conducted by different authors, and using different behavioral paradigms. These 

18 conclusions are also supported by more limited data, indicating possible developmental effects 

19 on the nervous system, including changes in brain structure and in the behavior of offspring; the 

20 developmental findings are less robust since they were seen only in individual laboratories and 

21 occurred following exposure to higher concentrations of formaldehyde. Studies evaluating 

22 developmental neurotoxicity at lower doses, comparable to those used in the adult studies, were 

23 not available. None of the available data provide sufficient information to allow a determination 

24 of the mechanism for these behavioral changes, although it is unlikely that they are attributable 

25 to the irritant properties of formaldehyde. The data regarding behavioral sensitization provide 

26 some support for a stress-related mechanism for those findings, but the applicability of this 

27 mechanism to the behavioral changes seen in the other studies, including the learning deficits, 

28 has not been evaluated.

29

30 4.2.1.6.2. Oral exposure. Available data regarding neurotoxic effects of formaldehyde exposure 

31 following oral exposure are very limited. Several chronic or subchronic oral toxicity studies 

32 evaluated changes in brain weight or histopathology in rats or dogs following repeated oral 

33 exposures to formaldehyde at doses as high as 300 mg/kg-day, administered in drinking water 

34 (Til et al., 1989, 1988; Tobe et al., 1989; Johannsen et al., 1986). Although data were not 
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1 presented in the publications, all stated that no changes in brain weight or pathology were seen in 

2 the standard evaluations performed in these studies.

3 Two studies evaluated changes in behavior following exposure to formaldehyde in 

4 drinking water (Venkatakrishna-Bhatt et al., 1997; Venkatakrishna-Bhatt and Panchal, 1992). 

5 Venkatakrishna-Bhatt and Panchal (1992) evaluated changes in performance on a conditioned 

6 avoidance task in adult male albino rats (five/group). Animals were exposed to formaldehyde in 

7 drinking water (10 mg/mL) or by I.P. injection (10 mg/kg) for 60 days. Although it was stated 

8 that water consumption was recorded, the data were not presented, and thus actual exposure 

9 levels cannot be documented. Prior to the initiation of exposure, rats were trained on the 

10 conditioned avoidance task (climbing a wooden pole in response to a warning buzzer, thus 

11 avoiding electric shock from a floor grid). Rats were trained to a predetermined performance 

12 criterion (not described); animals not achieving the criterion were removed from study. Training 

13 and testing conditions (e.g., retest interval and duration of sessions) were not well described. 

14 Data were presented as percent response in behavioral performance (apparently separately for the 

15 escape or avoidance aspects of the task) or percentage decrease in response. No control data 

16 were presented, and pretreatment performance was not described. Figures presented 

17 performance at 10-day intervals, starting with day 0, with each data point stated to represent the 

18 mean for five experimental sets; again, the interval between experimental sets and the number of 

19 trials per set was not specified. Although the authors concluded that a deficit in performance 

20 was demonstrated, the data as presented were difficult to interpret and the conclusion could not 

21 be verified based on the data as presented.

22 Venkatakrishna Bhatt and Panchal (1992) examined changes in performance on a 

23 conditioned avoidance response, presumably using a procedure similar to the one described 

24 above. Albino rats (sex not specified, five/group) were exposed to formaldehyde in drinking 

25 water at 0.2 or 0.5 mg/mL for 90 days. As described above, rats were trained in performing the 

26 task prior to the start of exposure. Venkatakrishna-Bhatt Bhatr and Panchal (1992) stated that 

27 there was a dose-related deterioration of performance, but no data were presented to support 

28 these conclusions.

29 In summary, available data are insufficient to conduct a reliable assessment of neurotoxic 

30 effects of formaldehyde following oral exposure. Limited data suggest a lack of overt 

31 neuropathological changes at doses up to 300 mg/kg-day (Til et al., 1989, 1988; Tobe et al., 

32 1989; Johannsen et al., 1986), but detailed information regarding the types of neuropathological 

33 evaluations performed in those studies is not available, and thus no firm conclusions can be 

34 drawn regarding the potential for neuropathological effects. The two available studies evaluating 
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1 behavioral changes are not considered to provide useful information, and thus effects on nervous 

2 system function could not be evaluated.

3

4 4.2.1.6.3. Summary. Overall, there is strong evidence that formaldehyde exposure via 

5 inhalation may cause adverse effects on nervous system function in experimental animals at 

6 relatively low levels of exposure (LOAELs as low as 100 ppb). Although human data regarding 

7 neurotoxicity following formaldehyde inhalation are limited, available data provide support that 

8 the types of effects seen in humans are similar to those found in animal studies. Evidence from 

9 available human controlled inhalation exposure studies indicates that humans may be affected at 

10 doses similar to those used in animal studies; however, the human data are extremely limited.

11 There are insufficient data to evaluate the potential for neurotoxicity following oral 

12 exposure to formaldehyde. Limited evaluations of brain weight or histopathology in available 

13 chronic or subchronic oral studies found no evidence of formaldehyde-induced changes (Til et 

14 al., 1989, 1988; Tobe et al., 1989; Johannsen et al., 1986). However, reliable studies examining 

15 nervous system function or focused studies of neuropathology following oral exposure to 

16 formaldehyde are not available.

17

18 4.2.1.6.4. Other considerations. Major data gaps were found regarding the evaluation of 

19 changes in nervous system structure or function following formaldehyde exposure by both the 

20 inhalation or oral routes.

21 With respect to inhalation exposure, none of the available human studies resulted in data 

22 sufficient to conduct a reliable dose-response assessment for changes in nervous system function. 

23 Most of the available animal inhalation studies used short exposure durations (acute or 

24 short-term), precluding a reliable evaluation of neurotoxicity following chronic exposure. 

25 Available data for neurodevelopmental exposures are also quite limited, consisting of evaluation 

26 of neuropathology in only one brain region and functional evaluations focused only on changes 

27 in motor activity.

28 Major data gaps also exist regarding neurotoxicity following oral exposure, with no 

29 relevant human data and extremely limited animal data. Available oral exposure studies were 

30 insufficient to permit a reliable evaluation of the potential for neurotoxicity following oral 

31 exposure to formaldehyde.

32

33 4.2.1.7. Reproductive and Developmental Toxicity

34 The potential for developmental and reproductive effects after formaldehyde exposure by 

35 the inhalation route has generally been considered low, since formaldehyde, as a reactive gas, is 
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1 not expected to penetrate past the POE (NEG, 2003; IPCS, 2002; Collins et al., 2001). 

2 Nevertheless, a number of animal studies have demonstrated effects of formaldehyde on pre- and 

3 postnatal development and on the reproductive system. For example, developmental toxicity 

4 was observed in two studies that evaluated a standard battery of developmental endpoints 

5 resulting from inhalation exposure on GDs 6–10 (Martin, 1990; Saillenfait et al., 1989). 

6 Similarly, oral exposures resulted in developmental effects when administered during 

7 comparable gestational windows (Marks et al., 1980; Hurni and Ohder, 1973). There have also 

8 been reports that identified developmental effects at lower-level formaldehyde exposures that 

9 were administered throughout gestation (Senichenkova and Chebotar, 1996; Senichenkova, 

10 1991; Kitaev et al., 1984; Sheveleva, 1971; Gofmekler and Bonashevskaya, 1969; Gofmekler, 

11 1968; Pushkina et al., 1968). Postnatal functional consequences of developmental exposures 

12 have also been identified (Sarsilmaz et al., 2007; Aslan et al., 2006; Weiler and Apfelbach, 1992; 

13 Senichenkova, 1991; Sheveleva, 1971). Additionally, a number of studies suggest that 

14 formaldehyde adversely affects the male reproductive system after both inhalation and oral 

15 exposures (Xing et al., 2007; Zhou et al., 2006; Özen et al., 2005, 2002; Sarsilmaz et al., 1999; 

16 Chowdhury et al., 1992; Cassidy et al., 1983; Guseva, 1972). This section reviews the available 

17 published studies assessing reproductive and developmental endpoints of formaldehyde.

18

19 4.2.1.7.1. Inhalation studies addressing developmental and reproductive toxicity. Saillenfait et 

20 al. (1989) reported a comprehensive and well-documented developmental study in Sprague21 Dawley rats. Pregnant rats were exposed beginning on GD 6 in order to cover critical stages of 

22 development (e.g., implantation and major organogenesis). Female Sprague-Dawley rats 

(25/group) were exposed to 0, 5, 10, 20, or 40 ppm (0, 6.15, 12.3, 24.6, or 49.2 mg/m3 23 ) 

24 formaldehyde 6 hours/day on GDs 6–20. The onset of pregnancy was determined by the 

25 presence of sperm in a vaginal smear. Dams were exposed to formaldehyde in a dynamic flow 

26 chamber, and formaldehyde concentrations were determined to be 0, 5.17 ± 0.51, 9.92 ± 0.88, 

27 20.04 ± 0.88, and 38.96 ± 3.70 ppm. Dams were weighed on GDs 0, 6, and 21 and sacrificed on 

28 day 21. Upon examination, uterine weights, fetal weights, sex ratio, number of implantation and 

29 resorption sites, and live and dead fetuses were recorded. Fetuses were examined for external 

30 malformations and cleft palate. One-half of viable fetuses were sectioned to assess soft-tissue 

31 alterations. The other half were fixed, stained with alizarin red S, and examined for skeletal 

32 alterations.

33 Body weight gain of dams and body weight of male and female fetuses were reduced by 

34 exposure to 40 ppm formaldehyde to 49, 78, and 81% of control values, respectively (p < 0.01) 

35 (Saillenfait et al., 1989). Reduced weight gain in dams remained significantly decreased when 
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1 uterine weight was accounted for (p < 0.01). Mean fetal weight of male pups was reduced at 

2 maternal exposures of 20 and 40 ppm formaldehyde (5.53 and 4.42 g versus 5.61 g in controls). 

3 Decreased fetal body weight in females was only seen at 40 ppm (4.27 g versus 5.24 g in 

4 controls). All other pregnancy endpoints were unchanged by formaldehyde exposure (e.g., 

5 uterine weight, implantation and resorption sites, live fetuses, dead fetuses, and sex ratios). No 

6 major malformations were noted in fetuses. Some minor soft tissue and skeletal anomalies, such 

7 as dilated ureter, missing sternebrae, extra fourteenth rib, and rudimentary thirteenth rib 

8 (statistics not given), were reported. However, these effects occurred at similar frequencies in 

9 control and treatment groups. The incidence of delayed ossification of the thoracic vertebrae 

10 was 8.7% in fetuses from the 40 ppm exposure group versus 1.8% in controls. However, this 

11 difference was not statistically significant. Overall, from these results formaldehyde was neither 

12 lethal to embryos nor teratogenic, only exhibiting fetotoxic effects at exposures of 20 ppm and 

13 above. These are levels where there was a significant decrease in fetal body weight.

14 Martin (1990) conducted a similar study exposing pregnant rats during similar stages of 

15 development. Mated female Sprague-Dawley rats (25/group) were exposed to 2, 5, or 10 ppm 

(2.46, 6.15, or 12.3 mg/m3 16 ) formaldehyde 6 hours/day on GDs 6–15. The study included two 

17 control groups: dams placed in the exposure chambers once a day but exposed only to clean air 

18 and dams fed and housed similarly to the experimental groups but never put into the inhalation 

19 chambers. The method of formaldehyde vapor generation and details of the exposure chamber 

20 were not described. Mean formaldehyde exposure concentrations were reported as 1.88, 4.88, 

21 and 9.45 ppm (variability not given, analytical method not discussed). Food consumption and 

22 body weight were recorded. On GD 20, rats were sacrificed, and the following pregnancy 

23 parameters were recorded: live fetuses, dead fetuses and resorptions, number of corpora lutea, 

24 fetal weights, sex ratios, and preimplantation and postimplantation losses. Fetuses were 

25 examined for major malformations, minor external and visceral anomalies, and minor skeletal 

26 anomalies (details not given). Weight gain and food consumption in dams were said to be 

27 reduced at 10 ppm (p < 0.05). Formaldehyde exposure of the dams at 5 and 10 ppm led to an 

28 increased incidence of reduced ossification of the pubic and ischial bones in fetuses on GD 20 

29 (p < 0.05). Reduced ossification correlated with lower fetal weights, and the author considered 

30 both of these findings a result of larger litter size and, therefore, not related to formaldehyde 

31 exposure. However, no tables presenting the data or statistical analysis were provided. All other 

32 pregnancy parameters and fetal anomalies were described as unaffected by formaldehyde 

33 exposure. However, without data presented for the assessed endpoints, background rates of 

34 malformations, trends in the data, and variability, it is difficult to evaluate the Martin (1990) 
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1 comparisons. However, the author’s observations of reduced fetal weight and increased 

2 incidence of reduced ossification are consistent with the results of Saillenfait et al. (1989).

3 Kilburn and Moro (1985) studied similar endpoints but included formaldehyde exposure 

4 during earlier gestational windows. The study report, only available in abstract form and not 

5 found as a subsequent published article, does not provide many methodological details. Female 

rats (number and strain not reported) were exposed to 0 or 30 ppm (0 or 37.2 mg/m3 6 ) 

7 formaldehyde 8 hours/day during GDs 3–17, 3–12, 8–12, or 9–11. A second experiment 

8 included pair-fed controls for dams exposed to 30 ppm formaldehyde during GDs 3–17, 3–12, 

9 and 8–12. The authors reported reductions in fetal and maternal weight gain that were greater 

10 than decreases in pair-fed controls. Fetal anomalies were noted after 15 days of gestational 

11 exposure (e.g., altered organ size and undescended testes). Although the report indicates some 

12 maternal toxicity and fetotoxic effects (for example, stunted growth), lack of study details and 

13 clear reporting make this report of negligible utility in human health risk assessment.

14 There are several early studies that examined developmental effects of formaldehyde 

15 exposure administered throughout gestation (Gofmekler and Bonashevskaya, 1969; Gofmekler, 

16 1968; Pushkina et al., 1968). It is unclear if these reports represent the same or overlapping 

17 experimental groups. They were performed in the same laboratory and are reported with a 

18 similar level of detail. The source of formaldehyde, method of vapor generation, exposure 

19 conditions (dynamic versus static), confirmation of exposure concentrations, study design, and 

20 data presentation details were not provided. Absence of such critical information detracts from 

21 the quality of these studies as a coherent record of experimental information, and, thus, these 

22 findings can only be utilized qualitatively in the formaldehyde risk assessment. 

23 In the Gofmekler (1968) study, female rats (36, strain not specified) were continuously 

24 exposed at 0, low, or high formaldehyde concentrations beginning 10–15 days prior to mating 

(target concentrations of 0, 0.01, or 0.81 ppm formaldehyde [0, 0.01, or 1 mg/m3 25 ]). The author 

26 reported a 14–15% increase in pregnancy duration and a decrease in litter size (data not shown). 

27 However, males and females were mated 6–10 days, and no information was provided on how 

28 mating and conception were confirmed. No external malformations were attributed to 

29 formaldehyde exposure. Concentration-dependent increases in pup body weight and decreases in 

30 lung and liver weight were attributed to formaldehyde exposure. Pup weights were increased 

31 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). 

32 Formaldehyde exposure increased pup adrenal weight in both groups and pup thymus and kidney 

33 weight in group 2 only (Table 4-58).

34

35
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1 Table 4-58. Effects of formaldehyde on body and organ weights in rat pups 

2 from dams exposed via inhalation from mating through gestation

3

Exposure

(ppm)a

Body weight

(g)

Relative organ weights (mg/10 g body weight)

Thymus Heart Lung Liver Adrenals Kidney

0 5.6 26 61.4 287.1 587.7 3.2 51.4

0.01 6.0b 25.1 61.5 230.2c 557.7d 4.2c 53.4

0.81 6.3c 31.7c 64.5 223.2c 550.8b 3.8d 55.7b

4 a 5 Dams were exposed to formaldehyde continuously from 10–15 days prior to mating. Exposure concentrations 

6 were not validated.

b 7 Different from controls, p < 0.01. c 8 Different from controls, p < 0.001. d 9 Different from controls, p < 0.05.

10

11 Source: Gofmekler (1968).

12

13 In a study by Gofmekler and Bonashevskaya (1969), the researchers evaluated organ 

14 histopathology in pups from similarly treated dams. Pregnant female albino rats were 

15 continuously exposed at two formaldehyde concentrations (groups 1 and 2, as described above). 

16 Adult males were similarly exposed during mating. Offspring were examined for malformations, 

17 and the organs were fixed and sectioned for histopathologic examination, including hematoxylin 

18 and eosin staining, Brachet stain for RNA, and Feulgen stain for DNA. Liver and kidney 

19 sections were also stained with Schiff’s reagent (which reacts with aldehydes), with Sudan III for 

20 lipids, and Pearl’s stain for iron. Placenta, uterus, and ovaries from the dams and testes of the 

21 males were sectioned, stained, and evaluated. The authors stated that formaldehyde induced no 

22 external anomalies (reported elsewhere, but no reference given). The authors also noted 

23 involution of lymphoid tissue and changes in liver, mild hypertrophy of Kupffer cells, and 

24 numerous extramedullary myelopoietic centers in pups from dams in group 2. Pups from both 

25 treatment groups showed reduced glycogen content in the myocardium and the presence of iron 

26 in Kupffer cells. There was a localized increase in positive reaction to Schiff’s reagent in the 

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

28 that this was an indication of functional alterations in the renal tubular apparatus. All other 

29 tissues examined and histochemical staining indicated no differences due to formaldehyde 

30 exposure.

31 Researchers in the same laboratory (Pushkina et al., 1968) studied the effects of 

32 formaldehyde exposure on vitamin C (ascorbic acid, an antioxidant) and nucleic acid levels in 

33 dams and fetuses as general measures of toxicity. Female white rats (n = 160) were continuously 

34 exposed at two formaldehyde concentrations (groups 1 and 2, as described above) from 20 days 

35 prior to mating (6–10 days) and then throughout gestation. Dams were sacrificed and ascorbic 

36 acid and nucleic acid levels determined in harvested organs (methods referenced but not 
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1 described). No visible malformations in pups were noted. Formaldehyde exposure increased 

2 fetal body weight and organ weight in both groups (data not given). There was an average of 

3 11.3 fetuses per litter for control dams versus 9.8 and 8.6 for groups 1 and 2, respectively. The 

4 authors reported that formaldehyde exposure decreased DNA levels and increased RNA levels in 

5 organs (further details not provided). Formaldehyde exposure resulted in lower vitamin C levels 

6 in the whole fetus (76 and 75% of controls) and in maternal liver specimens (82 and 88% of 

7 controls) for exposure groups 1 and 2, respectively (p < 0.05). In contrast, vitamin C was higher 

8 in fetal liver (127% of controls) in group 1 (p < 0.05). The significance of these differences is 

9 unknown. The authors considered the results as general measures of biochemical changes and 

10 therefore toxic.

11 The reports of Gofmekler and Bonashevskaya (1969), Gofmekler (1968), and Pushkina et 

12 al. (1968) lack key methodological details. As discussed above, exposure conditions and actual 

13 formaldehyde concentrations cannot be validated. Although methods were not thoroughly 

14 detailed, results were reported in data tables with statistics and detailed descriptions of observed 

15 pathological changes. However, without validation of exposure concentrations, these findings 

16 can only be considered qualitatively.

17 In another study, Sheveleva (1971) exposed female mongrel (i.e., not a homogeneous 

genetic strain) white rats to 0, 0.0005, or 0.005 mg/L (0, 0.5, or 5 mg/m3 18 ) (0, 0.4, or 4 ppm) 

19 formaldehyde on GDs 1–19 (where GD 1 was defined as the day that spermatozoa were detected 

20 in vaginal smears) for 4 hours/day. In each group, 15 dams were terminated on GD 20 for 

21 evaluation of ovarian corpora lutea, uterine implantation sites, pre- and postimplantation loss, 

22 number of live fetuses, fetal length and weight, and external examination for malformations. 

23 Additionally, in each group, six dams were allowed to deliver their litters. Developmental 

24 landmarks were monitored (i.e., ear and eye opening, incisor eruption, emergence of hair coat), 

25 and the pups were further evaluated at 1 and 2 months of age for body weight, threshold of 

26 neuromuscular excitability, total oxygen consumption in 1 hour per 100 g of weight, and 

27 spontaneous mobility over 10 minutes. Maternal toxicity (recorded on GD 17) included 

28 significantly (p < 0.05) decreased leukocyte counts in both treated groups and a number of 

29 additional findings at 0.005 mg/L (i.e., significant reductions in the threshold of neuromuscular 

30 excitability, rectal temperature, and blood hemoglobin level) as well as an increase in 

31 spontaneous mobility over 15 minutes. (It is noted that a significant reduction in blood 

32 hemoglobin was also observed by Sanotskii et al. [1976] in pregnant rats, following 20 days of 

gestational inhalation exposure for 4 hours/day to formaldehyde at 6 mg/m3 33 [4.83 ppm].) Fetal 

34 examinations on GD 20 identified a 50–70% increase in mean preimplantation loss in both 

35 formaldehyde-exposed groups. When pups were 1 month of age, a reduction in spontaneous 
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1 mobility was noted in both treated groups; in pups at 2 months of age, an increase in mobility 

2 was observed in the 0.005 mg/L group. Also, when pups were 2 months of age, there were 

3 alterations in hemoglobin levels and leukocyte counts in both treated groups. Detailed 

4 descriptions of some study methodologies (particularly in regard to neurological and behavioral 

5 assessments) were not provided in the published paper.

6 Based on a review of the work by Gofmekler (1968) and various epidemiologic studies 

7 available at the time, Kitaev et al. (1984) hypothesized that formaldehyde may exert toxic effects 

8 in the early days of gestation. To study embryotoxic effects of formaldehyde inhalation 

9 exposures, mature female Wistar rats (five to nine per group) were exposed to 0.41 or 1.22 ppm 

(0.5 or 1.5 mg/m3 10 ) formaldehyde 4 hours/day, 5 days/week for 4 months (Kitaev et al., 1984). 

11 Rats were exposed in dynamic flow chambers and formaldehyde levels measured gravimetrically 

12 (but not reported). Females were mated on day 120 of exposure and mating confirmed by the 

13 presence of sperm in a vaginal smear. Embryos were harvested on the second or third day of 

14 pregnancy (GD 2 or 3) and examined by both light and phase contrast microscopy for changes in 

15 morphology (i.e., evidence of embryonic degeneration). Additionally, maternal weight gain and 

16 organ weights (ovaries, uterus, and adrenal glands) and blood samples (HCT, Hb, and TP) were 

17 monitored as indicators of general toxicity. These parameters were unchanged by formaldehyde 

18 exposure. Formaldehyde exposure at 1.22 ppm for 4 months resulted in an increased number of 

19 degenerating embryos on GD 3 (14.9 versus 4.4% in controls) and a smaller increase of 10.2% 

20 (versus 5.1% in controls; statistical significance not assessed) on GD 2. Indications of 

21 degeneration included reduced size and changes in appearance (granulation of the ooplasm, 

22 wrinkling and degradation of nuclear material). However, it is unclear if litter effects were 

23 accounted for in the statistical analyses, and it is unknown how the affected embryos were 

24 distributed between litters. For dams exposed to 0.41 ppm formaldehyde, the number of 

25 degenerated embryos was not increased on day 2 (3.8 versus 5.1% in controls) but was increased 

26 on day 3 (9.1 versus 4.4% in controls; again, unknown if statistically significant) after maternal 

27 exposure to 1.22 ppm formaldehyde. This observation may be coincidental since it was seen in 

28 dams sacrificed on GD 2 but not in those sacrificed on GD 3. Kitaev et al. (1984) considered 

29 these findings to indicate that repeated exposure to formaldehyde over a 4-month period can 

30 disturb reproductive function, resulting in adverse effects early in embryonic development.

31 To further explore the effects of inhalation exposures to formaldehyde on reproductive 

32 function, Kitaev et al. (1984) conducted a second series of experiments on 200 similarly treated 

33 female rats. After 4 months of repeated formaldehyde exposure at 0.41 or 1.22 ppm as described 

34 above, organ weights (ovaries and uterus) and blood levels of gonadotropic hormones and 

35 progesterone were determined. However, the day and time of hormone measurement were not 
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1 given in the report, and normal diurnal variations in these hormones could affect the reported 

2 findings if time of day was not accounted for. The length of the estrous cycle was unchanged 

3 during exposure. Formaldehyde exposure modulated gonadotropin levels and relative ovarian 

4 weight, suggesting low-level effects on the female rat reproductive system prior to mating 

5 (Kitaev et al., 1984). Ovarian weight and blood levels of luteinizing hormone (LH) were both 

6 significantly increased after exposures at 0.41 ppm formaldehyde but remained at control levels 

7 in rats exposed at 1.22 ppm. Blood levels of follicle-stimulating hormone (FSH) were increased 

8 approximately 66% from control after 1.22 ppm formaldehyde exposure (p < 0.05). 

9 Progesterone levels were unchanged by formaldehyde treatment. Kitaev et al. (1984) suggested 

10 a role of the hypothalamus-pituitary system based on increased ovary weight, a greater number 

11 of degenerated embryos, and increased LH in rats exposed at 0.41 ppm. They postulated that 

12 these effects were not seen at 1.22 ppm due to a toxic effect exhibited as embryonic 

13 degeneration, thus the absence of a dose-response did not alter the interpretation of the validity 

14 of the adverse response. The study NOAEL was not determined, and the study LOAEL was 0.4 

ppm (0.5 mg/m3 15 ), based upon increased early embryo loss and on maternal outcomes (increased 

16 ovarian weight and increased blood LH levels) following 4 months of formaldehyde treatment. 

For the finding of increase blood FSH levels, the endpoint NOAEL was 0.4 ppm (0.5 mg/m3 17 ) 

18 and the LOAEL was 1.2 ppm (1.5 mg/m3).

19 Senichenkova and Chebotar (1996) and Senichenkova (1991) examined reproductive and 

20 developmental effects of daily formaldehyde exposure on GDs 1–19 of pregnancy, including the 

21 potential effect of formaldehyde exposure on development early in gestation. Additionally, since 

22 anemia adversely affects fetal development, Senichenkova and Chebotar (1996) also examined 

23 formaldehyde effects in iron-deficient dams to determine whether co-exposure further 

24 compromises fetal development. In both studies, female white rats were exposed to 0 or 

0.41 ppm formaldehyde (0 or 0.5 mg/m3 25 ), 4 hours/day on GDs 1–19. Formaldehyde 

26 concentrations in the dynamic exposure chambers were measured gravimetrically to confirm the 

27 exposure concentration but were not reported (methods not provided). It is unclear if gravimetric 

28 measurements would be sensitive or accurate enough to validate these low-exposure 

29 concentrations without a better understanding of the methodology. This uncertainty in exposure 

30 conditions should be considered in evaluating the reported results.

31 Mongrel female white rats were exposed at a target concentration of 0 or 0.41 ppm (0 or 

0.5 mg/m3 32 ) formaldehyde 4 hours/day on GDs 1–19 (Senichenkova, 1991). On GD 20, a subset 

33 of the dams was sacrificed and examined for number of corpora lutea, implantation and 

34 resorption sites, live/dead fetuses, and fetal weights. Fetuses were examined for gross pathology

35 of the internal organs and skeleton (details not given). Blood pH, partial pressure of CO2, and 
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1 partial pressure of oxygen were measured in both dams and embryos. The remaining dams were 

2 brought to term to study postnatal effects of formaldehyde exposure. Rat pups were observed on 

3 PNDs 1–25 for viability, physical development, and maturation rate of motor reflexes. Behavior 

4 of juvenile offspring (PND 40) was studied in an open field test, and maze learning was tested at 

5 sexual maturity. In a follow-up report, Senichenkova and Chebotar (1996) present blood 

6 chemistry data, pregnancy outcome, and developmental data for similarly treated dams and their 

7 pups in a chemical model of iron deficiency. Intraperitoneal injections of the iron-chelating 

8 agent bipyridyl were given on GDs 12–15 at the threshold embryotoxic dose (1 mL, 25% 

9 solution). On day 20, the dams were sacrificed and dams and fetuses examined as described 

10 above. In addition to blood pH, partial pressure of carbon dioxide and partial pressure of 

11 oxygen, acid metabolic products (not detailed), and true bicarbonates were reported for maternal 

12 and fetal blood. A review of the data from these reports indicates there may be an overlap of the 

13 study groups. Neither paper presents the entire data set; thus, for tranparency and brevity, the 

14 following text discusses the combined findings from both studies as if they were a single study

15 Formaldehyde exposure did not affect such indicators of pregnancy outcome as number 

16 of corpora lutea, implantation and resorption sites, and live and dead fetuses, all of which were 

17 unchanged (Senichenkova and Chebotar, 1996; Senichenkova, 1991). Although fetal weight was 

18 slightly increased by formaldehyde exposure, 2.35 versus 2.24 g in controls (p < 0.001), neither 

19 fetal length nor bone length were changed (femur and humerus) (Senichenkova and Chebotar, 

20 1996; Senichenkova, 1991). Often, increased fetal weight is the result of early physical 

21 development, and other signs of development, such as ossification, would be expected to be 

22 enhanced as well. The average number of bone centers per limb was increased by formaldehyde 

23 exposure from 2.45 and 2.66 to 2.78 and 2.91 in controls for metacarpal and metatarsal bone 

24 centers, respectively (p < 0.05) (Senichenkova, 1991); these findings were consistent with 

25 increased growth and weight. In contrast, Senichenkova (1991) reported a decrease in the 

26 number of embryos with ossification centers in the hyoid bone (100% in controls versus 91% for 

27 formaldehyde exposure, p < 0.05), consistent with the results of Saillenfait et al. (1989) and 

28 Martin (1990). However, litter size, a factor influencing fetal weight, was not provided, and it is 

29 unclear if Senichenkova (1991) took litter size into account in the analysis. 

30 Senichenkova and Chebotar (1996) reported increased blood acidosis and decreased 

31 blood alkaline reserves (bicarbonates and total CO2) in formaldehyde-treated dams and their 

32 embryos (p < 0.05). However, this finding should be considered in light of the fact that chronic 

33 blood acidosis may increase bone remodeling and decrease bone density in adults. It is unknown 

34 if the reported blood acidosis could reduce ossification rates in developing embryos. A better 
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1 understanding of exposure conditions and the acid metabolic products measured is needed to 

2 determine the biological relevance of the reported changes in blood acid balance.

3 Iron deficiency, induced by injections of bipyridyl (an iron-chelating agent), was found to 

4 be fetotoxic. Iron-deficient dams with no formaldehyde exposure had higher rates of 

5 postimplantation death than controls (12.6 ± 5.5 versus 4.8 ± 1.3%). Formaldehyde exposure in 

6 conjunction with iron deficiency increased postimplantation death to 23.1 ± 5.9%. Fetal weight 

7 and litter size were not reported for the bipyridyl treatment groups, but bipyridyl treatment in 

8 conjunction with formaldehyde resulted in a decreased number of metatarsal bone centers (2.21 

9 ± 0.12 versus 2.72 ± 0.08 in controls; p < 0.001). This decrease was also significant when 

10 compared with formaldehyde or bipyridyl alone (p < 0.02). However, all pregnancy outcome 

11 parameters were not reported for the bipyridyl treatment.

12 Fetal anomalies were reported after formaldehyde exposure and were increased by iron 

13 deficiency. The incidence of litters with internal organ anomalies was increased from 1.4% in 

14 controls to 14.2% in formaldehyde-treated dams (Senichenkova, 1991). Undescended testes 

15 were the predominant anomaly described: 20.8% in litters from formaldehyde-treated dams 

16 versus 1.2% in controls (p < 0.05) (Senichenkova, 1991). Similar findings were reported by 

17 Senichenkova and Chebotar (1996). Bipyridyl treatment in conjunction with formaldehyde 

18 exposure increased the overall incidence of fetal anomalies (13.8 ± 2.1% in controls versus 6.6 ± 

19 1.8% with iron deficiency alone) (Senichenkova and Chebotar, 1996). However, there are 

20 discrepancies between the two papers in the reporting of the anomalies, and it is unclear whether 

21 the experimental groups overlap between papers, where some parameters are identical (which 

22 would lead to double counting of the same animal, including identical SDs) and others are 

23 different. Additionally, the reporting is unclear with respect to the basis of the incidence rates 

24 reported (for example, overall incidence versus incidence within litter or incidence of litters with 

25 anomalies). Unclear reporting, together with some of the uncertainties regarding exposure 

26 conditions, suggests that the data may be of limited quality to support risk assessment.

27 In the second phase of the studies, pups were delivered and postnatal development was 

28 assessed (Senichenkova, 1991). Eruption of the upper and lower incisors was delayed in pups 

29 from formaldehyde-treated dams, occurring on PND 14 versus PND 12 in controls (p < 0.01). 

30 All other measures of physical postnatal development were unchanged by formaldehyde. To 

31 evaluate postnatal functional outcomes following in utero exposure to formaldehyde, an open 

32 field test was conducted in juvenile rats on 3 consecutive days (PNDs 40–42). Juvenile rats from 

33 formaldehyde-treated dams exhibited increased mobility (crossed squares), rearings, and 

34 defecations/urinations compared with control rats on the second and third open field tests 
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1 (p < 0.05). There were no differences seen in the maze-learning test assessed in mature offspring 

2 of formaldehyde-treated dams (Senichenkova, 1991).

3 Additional assessments of formaldehyde exposure on neurological development are 

4 described above in the section on neurological and behavioral toxicity in animal studies (Section 

5 4.2.1.6). In brief, studies conducted by Sarsilmaz et al. (2007) and Aslan et al. (2006) exposed 

10 neonatal male Wistar rats/group to 0, 6, or 12 ppm (0, 7.36, or 14.7 mg/m3 6 ) formaldehyde 

7 6 hours/day, 5 days/week for 30 days. At that time, five rats/group were killed and subjected to 

8 neuropathological examination; the remaining rats were maintained until PND 90, at which time 

9 they were killed and evaluated. Aslan et al. (2006) examined the number and volume of granular 

10 cells in the hippocampal dentate gyrus, while Sarsilmaz et al. (2007) examined the size and 

11 number of the pyramidal cells in the cornu ammonis of the hippocampus. In both studies, lower 

12 numbers of cells were observed in both treated groups at PND 90 as compared with PND 30. 

13 Although the effects of treatment on the volume and number of granular and pyramidal cells 

14 were somewhat inconsistent, a significant decrease in the number of neurons in the pyramidal 

15 cell layer of the hippocampal cornu ammonis was observed at both PNDs 30 and 90 (Sarsilmaz 

16 et al., 2007).

17 One other study reported effects on nervous system function following exposure to 

18 formaldehyde during postnatal development. An abstract by Weiler and Apfelbach (1992) 

19 described a study in which juvenile rats (strain not specified) were exposed to 0.25 ppm 

(0.31 mg/m3 20 ) formaldehyde from PNDs 30–160 or adult rats were exposed to 0.5 ppm 

(0.62 mg/m3 21 ) formaldehyde for 90 days. Decreased olfactory sensitivity (i.e., increased olfactory 

22 thresholds) was observed and was greater when the exposure was initiated in the young rats, as 

23 compared with the adults.

24 Evaluation of offspring following prenatal, perinatal, and/or juvenile inhalation exposures 

25 to formaldehyde have also been reported by Kum et al. (2007), Sandikci et al. (2007), and 

26 Songur et al. (2005). Kum et al. (2007) exposed female Sprague-Dawley rats (six dams/group) 

and their offspring to 0 or 6 ppm (0 or 7.4 mg/m3 27 ) formaldehyde for 8 hours/day in separate 

28 groups with exposures starting on GD 1, on postparturition day 1, or in offspring at 4 weeks of 

29 age and continuing for 6 weeks. In another group, exposures were initiated in adult rats. Mean 

30 body and liver weights were significantly decreased in the offspring exposed in utero and in 

31 early postnatal life, and mean liver weights were also significantly decreased in rats with juvenile 

32 exposures. However, neither body weight nor liver weight was affected in the group with 

33 exposure initiating at an adult age, suggesting a life-stage-related susceptibility to formaldehyde34 induced hepatic toxicity. Evaluation of biomarkers of oxidative stress revealed significantly 

35 increased catalase (CAT) and MDA in the livers of offspring that were exposed prenatally, 
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1 significantly decreased GSH levels in the livers of offspring that were exposed in early postnatal 

2 life, and significantly decreased SOD levels in the livers of offspring that were exposed starting 

3 at 4 weeks of age. No biomarkers were altered in the livers of rats exposed to formaldehyde only 

4 as adults.

5 A similar study design was used by Sandikci et al. (2007) to examine the effects of 0 or 

6 ppm (0 or 7.4 mg/m3 6 ) formaldehyde on bronchus associated lymphoid tissue (BALT) 

7 following pre- and perinatal, juvenile, or adult exposures of 6 weeks duration in Sprague-Dawley 

8 rats (six/group). The presence of the lysosomal enzyme alpha-naphthylacetate esterase (ANAE) 

9 served as a marker of T-lymphocytes in peripheral blood and tissue sections. Significant 

10 increases in ANAE-positive T-lymphocytes were found in BALT in all but the in utero exposed 

11 groups as compared with control; this outcome is consistent with the postnatal development of 

12 BALT in the rat. In peripheral blood, ANAE-positive lymphocyte ratios were significantly (p < 

13 0.001) increased as compared with controls at all life stages tested.

14 Songur et al. (2005) examined the effect (and reversibility) of formaldehyde exposures 

15 during the early postnatal period on zinc, copper, and iron levels and activity of SOD in the lung 

16 tissue of Wistar rats. Litters (12–14/group) were exposed to 0, 6, or 12 ppm (0, 7.4, or 

14.9 mg/m3 17 ) formaldehyde 6 hours/day, 5 days/week for 30 days. Trace element and 

18 biochemical analyses were conducted on PND 30 or 90. Decreased SOD activity, decreased 

19 levels of copper and iron levels, and increased zinc levels were observed in the lungs of treated 

20 groups following 30 days of treatment and at 90 days (i.e., 60 days posttreatment). Survival was 

21 not affected in neonatal rats. Clinical observations during treatment included evidence of 

22 respiratory irritation and toxicity. Body weight and food and water consumption were also 

23 nonsignificantly decreased as compared with controls.

24 There are several reports in the literature regarding formaldehyde effects after inhalation 

25 exposure on the male reproductive system in animals (Galalipour et al., 2007; Zhou et al., 2006; 

26 Özen et al., 2005, 2002; Sarsilmaz et al., 1999; Woutersen et al., 1987; Maronpot et al., 1986; 

27 Guseva, 1972). The earliest report examined the effect of simultaneous exposures to 

28 formaldehyde from air and water (Guseva, 1972). Male rats (n = 12, strain not specified) were 

29 co-exposed to formaldehyde in air and drinking water 4 hours/day, 5 days/week for 6 months. 

30 There were three exposure levels in the experiment of different air and drinking water 

concentrations: (1) 0.41 ppm (0.5 mg/m3 31 ) formaldehyde in air and 0.1 mg/L in water; (2) 0.20 

ppm (0.25 mg/m3) formaldehyde in air and 0.01 mg/L in water; or (3) 0.10 ppm (0.12 mg/m3 32 ) 

33 formaldehyde in air and 0.005 mg/L in water. Reproductive function was assessed by mating 

34 two females per male. The time for the onset of pregnancy and the number of pregnancies per 

35 treatment group were recorded. A subset of dams was sacrificed on GD 20 of pregnancy, and 
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1 the number and weight of fetuses was determined. Postnatal development of the remaining dams 

2 was tracked (e.g., times of eye opening and development of hair coat). Nucleic acid levels were 

3 determined in the testes of formaldehyde-exposed rats. Gonadotropic response was assessed by 

4 injecting an emulsion of pituitaries from exposed male rats into unexposed infantile females and 

5 measuring the weight ratios of the uterus and ovaries. Formaldehyde exposure reduced nucleic 

6 acid levels in testes to 88 and 92% of controls in groups 1 and 2, respectively. No other 

7 formaldehyde-induced differences were found.

8 Woutersen et al. (1987) and Maronpot et al. (1986) examined tissue sections from testes 

9 and ovaries of exposed animals as part of studies primarily addressing respiratory tract toxicity 

10 (see Section 4.2.1.2.2.4 for complete study details). Maronpot et al. (1986) exposed female and 

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/m3 11 ) 

12 formaldehyde 6 hours/day, 5 days/week for 13 weeks. Decreased weight gain due to 

13 formaldehyde exposure was seen in both male and female mice. Additionally, there was 80% 

14 mortality at the highest exposure (40 ppm). The authors reported endometrial hypoplasia and 

15 lack of ovarian luteal tissue in females exposed to 40 ppm, but no compound-related changes 

16 were observed in testes sectioned and viewed by light microscopy.

17 In a study by Appleman et al. (1988), male Wistar rats (40/group) with undamaged or 

18 damaged (via bilateral intranasal electrocoagulation) nasal mucosa were exposed for 13 or 

52 weeks to 0, 0.1, 1, or 10 ppm (0, 0.124, 1.24, or 12.4 mg/m3 19 ) formaldehyde 6 hours/day, 

20 5 days/week. At study termination, mean body weight was decreased, but relative testes weight 

21 was increased in the 10 ppm group (interpreted as a non-adverse outcome that was associated 

22 with the decreased body weight). No treatment-related histopathologic findings were reported 

23 for male reproductive organs (although it is not clear to what extent they were evaluated since 

24 the primary focus of the study was on the nasal epithelium).

25 Following up on earlier reports of decreased Leydig cell quality in rats administered I.P. 

26 injections of formaldehyde (Chowdhury et al. [1992], described in Section 5.2.1.8.3), Sarsilmaz 

27 et al. (1999) studied the effects of formaldehyde inhalation on Leydig cells. Adult male Wistar 

rats (30) were exposed to 0, 10, or 20 ppm (0, 12.3, or 24.6 mg/m3 28 ) formaldehyde 8 hours/day, 

29 5 days/week for 4 weeks. Animals were observed daily and weighed weekly. Rats were 

30 sacrificed on day 29 and autopsied, and testes were weighed, fixed, and sectioned for histologic 

31 examination. Signs of irritation from formaldehyde exposure were noted (frequent eye blinking, 

32 excessive licking, increased frequency of nose cleaning, interrupted breathing, and sneezing). 

33 Body weight gain was reduced by formaldehyde exposure from 17.7% gain in control rats to 

34 4.66 and 2.63% in rats exposed at 10 and 20 ppm, respectively (p < 0.001). As shown in 

35 Table 4-59, relative testes weights were unaffected (reported as proportions but more likely to be 
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1 percentages), although trends and numerical differences were similar to those reported by Özen 

2 et al. (2002). Sarsilmaz et al. (1999) found that both Leydig cell quantity and the percentage of 

3 cells with normal nuclei were reduced by formaldehyde treatment. Although the dose-dependent 

4 reduction in Leydig cell quantity was statistically significant at both exposure levels, the study 

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

6

7 Table 4-59. Formaldehyde effects on Leydig cell quantity and nuclear 

8 damage in adult male Wistar rats

9

Inhalation 

exposurea

Relative 

testes 

weightb,c,d

Leydig cell 

quantityc,e,f

Appearance of nucleuse,g

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 11 Rats were exposed 8 hours/day, 5 days/week for 4 weeks.

b 12 Stated to represent the ratio of the last day’s testicle weight to the body weight but more likely to be the percent 

13 of body weight. c 14 Cells within 100 defined areas.

d 15 n = 10.

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

f 17 n = 100.

g 18 Values presented as percentage of cells.

h 19 Different from control (p < 0.05), as reported by the authors. i 20 Different from control (p < 0.01), as reported by the authors.

21

22 Source: Sarsilmaz et al. (1999).

23

24

25 It was hypothesized that decreased Leydig cell quality may have been the result of 

26 oxidative stress induced by formaldehyde exposure. Özen et al. (2002), in the same laboratory, 

27 investigated changes in testicular iron, copper, and zinc levels as measures of oxidative stress 

28 and damage. Adult male albino Wistar rats (seven/group) were exposed at 0, 10, or 20 ppm (0, 

12.2, or 24.4 mg/m3 29 ) formaldehyde 8 hours/day, 5 days/week for either 4 or 13 weeks. Rats 

30 were observed daily and weighed once a week. Rats were sacrificed at the end of the exposure 

31 period and autopsied, and the testes were removed and weighed. Zinc, copper, and iron levels 

32 were determined in testes tissue by using atomic absorption spectrophotometry. Both weight 

33 gain and relative testes weight were decreased in a concentration-dependent and duration34 dependent manner (Table 4-60). Both zinc and copper levels in rat testes were reduced in a 

35 concentration- and duration-dependent manner by formaldehyde exposure. For example, zinc 

36 was reduced from 277 to 107 mg/kg after a 4-week × 20 ppm exposure and from 260 to 

37 95 mg/kg after a 12-week × 20 ppm exposure (p < 0.001) (Table 4-60). Iron levels in testes were 
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1 increased from 30 to 39 mg/kg after a 4-week 20 ppm exposure (p < 0.01) and from 33 to 

2 58 mg/kg after 12 weeks at 20 ppm (p < 0.05). The authors suggested that alterations in trace 

3 element levels in the testes were consistent with oxidative damage and may have contributed to 

4 changes in Leydig cell function. These researchers also reported alterations in trace metals in 

5 lung tissue from Wistar rats exposed to formaldehyde 8 hours/day, 5 days/week for 4 or 

6 13 weeks. Iron levels were increased at 5 ppm for 13 weeks and 10 ppm for either 4 or 

7 13 weeks. Zinc levels decreased for all formaldehyde exposures. In both cases, the authors 

8 attributed elevated iron levels to oxidative stress. In addition to citing the role of zinc as a 

9 cofactor of cytoplasmic Cu-Zn-SOD, the authors suggested that zinc may have been consumed 

10 by increased FALDH activity. Although oxidative stress and increased FALDH activity may be 

11 relevant to the POE and therefore impact the lung, it is less clear how these changes would occur 

12 in the testes. Taken together, the reports of Özen et al. (2002) and Sarsilmaz et al. (1999) 

13 suggested a LOAEL of 10 ppm 8 hours/day for 4 weeks for changes in Leydig cell quantity and 

14 quality, decreased testes weight, and changes in trace metal content (zinc, copper, and iron).

15

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

17

Inhalation

exposurea

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 19 Formaldehyde exposure was 8 hours/day, 5 days/week for either 4 or 13 weeks. Values are means ± SDs of 

20 seven animals..

b 21 Different from control, p < 0.001, as calculated by the authors. c 22 Different from control, p < 0.05, as calculated by the authors. d 23 Different from control, p < 0.01, as calculated by the authors.

24

25 Source: Özen et al. (2002).

26

27

28 In another study that assessed testicular toxicity (Özen et al., 2005), male Wistar rats 

(18/group) were exposed by inhalation to 0, 5, or 10 ppm (0, 6.2, or 12.4 mg/m3 29 ) formaldehyde 

30 8 hours/day, 5 days/week for 91 days. In-life observations in exposed rats included clinical signs 

31 of respiratory irritation and decreased mean food and water consumption. At study termination, 

32 serum testosterone levels and mean seminiferous tubule diameters were significantly decreased 

33 from control in a dose-responsive manner (Table 4-61). Immunohistochemical staining of testis 
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1 tissues showed increased localization of heat shock protein (Hsp) 70 in the cytoplasm of 

2 spermatogonia, spermatocytes, and spermatids of treated animals compared with controls (not 

3 shown here).

4

5 Table 4-61. Formaldehyde effects on testosterone levels and 

6 seminiferous tubule diameters in Wistar rats following 91 days of 

7 exposure

8

Inhalation

exposurea

Testosterone levels 

(ng/dL)

Seminiferous tubule 

diameters 

(μm)

n = 6 n = 100

Control 406.54 ± 16.82 259.22 ± 16.18

10 ppm 244.01 ± 23.86b 236.17 ± 13.09c

20 ppm 141.30 ± 08.56b 233.24 ± 10.13c

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

11 SEMs.

b 12 Different from control, p < 0.0001, by one-way ANOVA, as calculated by the authors. c 13 Different from control, p < 0.001, by one-way ANOVA, as calculated by the authors.

14

15 Source: Özen et al. (2005).

16

17

18 Zhou et al. (2006) investigated the effect of formaldehyde on the testes and the protective 

19 effect of vitamin E against oxidative damage by formaldehyde in adult male rats. In this study, 

20 adult male Sprague-Dawley rats (10/group) were treated for 2 weeks in the following groups: 

21 (1) control rats were administered physiological saline by oral gavage, (2) rats were administered 

physiological saline by gavage and exposed to 10 mg/m3 22 (8.05 ppm) formaldehyde by inhalation 

23 for 12 hours/day, and (3) rats were administered daily gavage doses of 30 mg/kg vitamin E and 

exposed to 10 mg/m3 24 (8.05 ppm) formaldehyde by inhalation for 12 hours/day. Formaldehyde 

25 treatment resulted in significantly decreased (p < 0.05) mean testis weight. Histopathologic 

26 findings in treated rats included atrophy of seminiferous tubules, decreased spermatogenic cells, 

27 and seminiferous cells that were “disintegrated” and shed into the lumina, which was 

28 azoospermic. Interstitial tissue was edematous with vascular dilatation and hyperemia. In the 

29 formaldehyde-treated group, epididymal sperm count and percentage of motile sperm were 

30 significantly decreased, and the percentage of abnormal sperm was increased (p < 0.05), as 

31 compared with control. Evaluation of biochemical markers in testes tissue showed the activities 

32 of testicular SOD, GPX, and GSH were decreased; MDA levels were significantly increased as 

33 compared with control. All observed effects of formaldehyde treatment (i.e., decreased testes 

34 weight, biochemical alterations, histopathologic effects, and sperm count, motility, and 

35 morphology findings) were attenuated by administration of 30 mg/kg-day vitamin E.
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1 In a study by Golalipour et al. (2007), testicular changes of increasing severity with 

2 increasing duration were reported. A total of 28 Wistar rats, aged 6-7 weeks old, were divided 

3 into four groups including three FA treatment groups (4 hours of exposure/day, 4 days/week for 

4 18 weeks; 2 hours of exposure/day, 4 days/week for 18 weeks; 4 hours of exposure/day, 2 

5 days/week for 18 weeks) and one untreated control. The three FA-treated groups were exposed 

6 via inhalation to formaldehyde. The mean concentration of FA vapor, based on three 

7 measurements during the study (stated as the beginning, during, and end of the study period), 

8 was reported as 1.5 ppm. At the end of the study period, the rats were sacrificed by ether 

9 anesthesia and subsequent cervical dislocation. The left testis was dissected from each rat and a 

10 specimen was taken from each testis. Tissues were fixed, embedded, sectioned (at 4 μm), and 

11 stained with hematoxylin and eosin. Using a light microscope, a histopathological examination 

12 was performed on the testes tissues, including morphometric evaluation of the diameter and 

13 height of 20 seminiferous tubules/testis. Golalipour et al. (2007) reported an FA exposure 

14 frequency (or duration)-dependent increase in testicular germ cells and seminiferous tubule 

15 defects. In the most frequent duration treatment group, a severe decrease in germ cells in >85% 

16 of the seminiferous tubules and arrested spermatogenesis were observed. In the mid-level 

17 frequency of duration treatment group, a decrease in the number of germ cells and an increased 

18 thickness of the basement membrane of 75% of the tubules was observed. In the lowest level 

19 duration treatment group, a disruption in the Sertoli and germinal cell arrangement, and 

20 increased spacing between germ cells was observed. Further, the seminiferous tubule diameter 

21 (STD) and seminiferous epithelial height (SEH) was most decreased among the treatment groups 

22 (exhibiting the greatest decrease in the group with the greatest hours and days of exposure) 

23 compared to the control (Table 4-62). The results of this study are consistent with the findings of 

24 other studies of male reproductive system outcomes with inhalation FA exposure (e.g., Özen et 

25 al., 2005 and Zhou et al., 2006). 

26 Xing et al. (2007) also studied the effects of formaldehyde on sperm development and 

27 reproductive capacity in adult male mice. In this study, male mice (12/group, strain not 

specified) were exposed to 0, 21, 42, or 84 mg/m3 28 (0, 16.9, 33.8, or 67.6 ppm) formaldehyde via 

29 inhalation for 13 weeks at 2 hours/day, 6 days/week. The males were mated to untreated females 

30 in a dominant lethal protocol, and sperm morphology was assessed at study termination. The 

31 percent abnormal sperm was increased significantly (p < 0.05) in all treated groups, as was the 

32 rate of resorptions (p < 0.01) (Table 4-63). The mean number of live fetuses/litter was decreased 

in all treated groups, with statistical significance achieved at 84 mg/m3 33 . Although this study did 

34 not assess the number of corpora lutea per dam, thereby precluding the calculation of 
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preimplantation loss, it is nevertheless indicative of formaldehyde-induced sperm morphology 

changes and dominant lethal effects in male mice.

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

diameter and epithelial height in Wistar rats following 18 weeks of 

exposure

1 

2 

3 

4 

5 

6 

7 

Seminiferous tubule Seminiferous tubule 

Inhalation diameters height a exposure (μm) (μm)

n = 7 n = 7

Control 252.12 ± 4.82 82.77 ± 2.00

1.5 ppm, 4 h/d, 4 d/w 204.55 ± 3.29b 65.26 ± 1.43b

1.5 ppm, 2 h/d, 4 h/w 232.45 ± 2.42b 69.46 ± 1.78b

1.5 ppm, 2 h/d, 2 d/w 238.94 ± 4.37b 72.80 ± 2.03b

8 a Values are means ± SEMs.

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

Source: Golalipour et al. (2007).

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

formaldehyde-treated mice

9 

10 

11 

12 

13 

14 

15 

16 

17 

Dose 3 (mg/m )

Sperm abnormalities Reproductive capacity

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 ± 2.13a 9.04 ± 2.98 a 12.440 b

18 a

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

Significantly different from controls (p < 0.01), as calculated by the authors.

Source: Xing et al. (2007).

4.2.1.7.2. Oral exposure studies addressing developmental and reproductive toxicity. No 

contemporary testing guideline studies, such as a prenatal developmental toxicity study or twogeneration reproductive toxicity study, have been performed by the oral route for formaldehyde. 

However, a number of studies have evaluated developmental toxicity and reproductive 

parameters in rats, mice, and dogs.

Hurni and Ohder (1973) tested the developmental toxicity of formaldehyde administered 

as a 40% w/v solution containing 11–14% w/w methanol in 9–10 pregnant beagle dogs that 

19 

20 

21 

22 

23 

24 

25 

26 

27 

28 

29 

30 

31 
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1 received the compound in their diet on GDs 4–56. Commercial grade formaldehyde (as a 40% 

2 solution) was sprayed on the pellets prior to feeding. Each animal was allotted a diet of 300 g of 

3 chow (reduced to 200 g 1 week prior to term) that was promptly consumed (within 5–

4 10 minutes) before the formaldehyde volatilized appreciably. The concentrations of 

5 formaldehyde in the chow were 0, 125, or 375 ppm, equivalent to doses of 0, 3.1, or 

6 9.4 mg/kg-day, respectively. Dams were allowed to deliver normally and weight gain, gestation 

7 length, number of litters, litter size, number of live pups, number of pups surviving through 

8 weaning, and pup weights weekly for the first 8 weeks were monitored as indices of the potential 

9 reproductive/developmental toxicity of formaldehyde. There were no formaldehyde-related 

10 effects in any of the parameters other than progressive pup weights, which were lower by group 

11 in litters of dams exposed to formaldehyde (Table 4-64). A developmental impact of 

12 formaldehyde was evident in this strain of dog under the conditions of the experiment. At birth, 

13 mean pup body weights were 4 and 8.4% less than control for the low- and high-dose groups, 

14 respectively; at 8 weeks of age, the pup weight decrements were 8.3% for the low dose and 

15 12.5% for the high dose, as compared with control, and established a LOAEL of 125 ppm. The 

16 contribution of methanol, which is a developmental toxin (Deglitz et al., 2004; Rogers et al., 

17 2004) to these outcomes is not known. No internal or skeletal malformations were observed in 

18 any of the 264 live-born and 20 still-born pups.

19

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

21 formaldehyde during gestation

22

Time (weeks)

Formaldehyde concentration in chow (ppm)

0 125 375

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

24 Source: Hurni and Ohder (1973).

25

26

27 Marks et al. (1980) conducted a developmental toxicity study of formaldehyde in CD-1 

28 mice in which 29–35 pregnant animals were gavaged on GDs 6–15 with aqueous formaldehyde 

29 (containing 10–15% methanol) at 74, 148, and 185 mg/kg-day. Seventy-six controls were 

30 gavaged with water alone. All dams were sacrificed on GD 18, and the numbers of implantation 
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1 sites in each uterine horn were counted. The high dose of formaldehyde was toxic to the dams, 

2 as indicated by the deaths of 22 of 34 mice before GD 18. Thus, the dose of 148 mg/kg-day was 

3 a NOAEL for maternal toxicity in this study. However, it is unclear to what extent an estimated 

4 concurrent dose of up to 75 mg/kg-day methanol may have contributed to this toxic response. 

5 To assess the developmental toxicity of formaldehyde, live fetuses were weighed individually, 

6 sexed, and examined for external, visceral, and skeletal malformations. Fetuses of surviving 

7 high-dose dams and of those of other groups did not show an increased incidence of 

8 malformations. Therefore, Marks et al. (1980) concluded that formaldehyde did not induce fetal 

9 abnormalities and that the 185 mg/kg-day dose level was a NOAEL for the developmental 

10 toxicity of formaldehyde, nor were fetotoxic effects of methanol apparent under the study 

11 experimental conditions.

12 Seidenberg and Becker (1987) and Seidenberg et al. (1986) included formaldehyde 

13 (purity not indicated) in a survey of the behavior of potential toxicants in a developmental 

14 toxicity screening assay (Chernoff and Kavlock, 1982). The protocol featured the administration 

15 of a borderline toxic dose to 26–30 pregnant ICR/SIM mice on GDs 8−12. Dams were allowed 

16 to deliver, and the neonates were examined, counted, and weighed on PNDs 1 and 3. The 

17 selected formaldehyde dose of 540 mg/kg-day was fatal for 11/30 dams, but the average weight 

18 gain among surviving dams was little changed compared with controls (3.9 ± 2.3 versus 

19 4.0 ± 1.0 g). Similarly, there were no changes in perinatal responses in the neonates of exposed 

20 dams compared with controls. For example, the average values for number of neonates/litter, 

21 percent survival, and fetal weights on PNDs 1 and 3 were closely similar to those of controls.

22 Evidence of toxicity to the male reproductive system has been observed following oral 

23 administration of formaldehyde in a 40% w/v solution containing 11–14% w/w methanol. 

24 Cassidy et al. (1983) administered single oral doses of 100 or 200 mg/kg to five male Wistar 

25 rats/group. Testes from these animals and 20 controls were excised and examined for 

26 spermatogenic abnormalities 11 days after dosing. A significant (19%) increase in testicular 

27 sperm head counts was observed in rats exposed to 200 mg/kg-day formaldehyde as compared 

28 with controls (Table 4-65). The percentage of abnormal sperm heads was also significantly 

29 increased (5%) in the 200 mg/kg-day dose group compared with controls. These data suggest 

30 that formaldehyde can induce morphologic abnormalities in the germ cells of male experimental 

31 animals at dose levels that did not significantly affect testis weights. The contribution of 

32 methanol to these outcomes is unknown.

33

34
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1 Table 4-65. Testicular weights, sperm head counts, and percentage incidence 

2 of abnormal sperm after oral administration of formaldehyde to male Wistar 

3 rats

4

Dose (mg/kg) Mean testes weight (g)

Mean sperm heads × 

106/g testis Abnormal sperm heads (%)

0 3.30 175 4.76

100 3.27 166 5.22

200 3.16 209a 9.77a

5 a 6 Significantly different from controls (p < 0.001), as calculated by the authors.

7

8 Source: Cassidy et al. (1983).

9

10

11 Postmortem evaluation of the reproductive organs was conducted in a number of oral 

12 studies that ranged between 4 weeks and 2 years in duration (Til et al., 1989, 1988; Tobe et al., 

13 1989; Johanssen et al., 1986). Johannsen et al. (1986) administered 0, 50, 100, or 150 mg/kg-day 

14 formaldehyde in the drinking water to Sprague-Dawley rats (15/sex/group) for 91 days and 0, 50, 

15 75, or 100 mg/kg-day formaldehyde in basal diet to beagle dogs (4/sex/group) for 91 days; the 

16 study reported no treatment-related effects on absolute or relative gonad weights or 

17 histopathology for either species. In a 4-week drinking water study conducted by Til et al. 

18 (1988), formaldehyde was administered to Wistar rats (10/sex/treated group) at nominal levels of 

19 0, 25, and 125 mg/kg-day; gonad organ weights and histopathology were not affected by 

20 treatment. Tobe et al. (1989) conducted a chronic (24-month) study in Wistar rats 

21 (20/sex/group), with drinking water concentrations of 0, 0.02, 0.1, or 0.5%. According to the 

22 study report, gonad weights were measured and histopathology was conducted, but no treatment23 related findings were noted. In a chronic (105-week) study (Til et al., 1989) in Wistar rats 

24 (70/sex/group), formaldehyde was administered in the drinking water at mean actual levels of 0, 

25 1.2, 15, or 82 mg/kg-day to males and 0, 1.8, 21, or 109 mg/kg-day to females; serial sacrifices 

26 were conducted at 53, 79, and 105 weeks of study. At study termination (105 weeks), mean 

27 testes weights were 30% increased (p < 0.01) in high-dose males as compared with controls, and 

28 histopathology evaluation revealed Leydig cell tumors in treated males (incidences of 0/50, 3/50, 

29 3/50, and 2/50 for the control through high-dose groups, respectively; historical control tumor 

30 incidence data were not provided). The study authors did not judge these findings to be 

31 treatment related. By design, none of the subacute to chronic studies included measures of 

32 reproductive function (e.g., estrous cyclicity, sperm measures, or reproductive performance). 

33 With the exception of Til et al. (1989), detailed mean organ weight and histopathology incidence 

34 data were not provided in the published reports, and Til et al. (1988) only included tumor (not 

35 non-tumor) data.
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1 4.2.1.7.3. Intraperitoneal studies addressing developmental and reproductive toxicity. Other 

2 studies in which formaldehyde was administered by I.P. injection have confirmed the potential 

3 effects on the male reproductive system.

4 Chowdhury et al. (1992) administered I.P. injections of 0, 5, 10, or 15 mg/kg-day 

5 formaldehyde to Charles foster adult male rats (10/group) for 30 days. On study day 31, blood 

6 was collected for serum testosterone measurements and the rats were sacrificed. The testes were 

7 removed, weighed, fixed in Bouin’s solution, and processed for histopathology. The study 

8 authors reported adverse findings in all treated groups, including significant (p < 0.01) mean 

9 body weight gains, serum testosterone levels, and testes weights. Histopathologic evaluation 

10 revealed normal spermatogenic processes and Leydig cells in control animals. However, in

11 treated rats, gradual cellular degeneration in seminiferous tubules and in Leydig cells was 

12 observed. Marked nuclear damage was noted in the 10 and 15 mg/kg-day groups, with 

13 significantly (p < 0.001) decreased Leydig cell populations and nuclear diameters observed in all 

treated groups. Additionally, a decrease in 3β-Δ5 14 -hydroxy steroid dehydrogenase was noted in 

15 the Leydig cell region of treated rat testes. 

16 In a 30-day study performed by Majumder and Kumar (1995), 10 mg/kg-day 

17 formaldehyde was administered I.P. to eight male Wistar rats. All animals were sacrificed at 

18 term, and the testes, prostate, seminal vesicles, and epididymides were excised and weighed. 

19 With the use of methodologies that were not described in the report other than by reference to the 

20 Laboratory Manual for the Examination of Human Semen and Sperm-Cervical Mucus 

21 Interaction (WHO, 1987), sperm counts, motility, and viability were compared with those of 10 

22 controls (injected I.P. with water alone). As shown in Table 4-66, striking reductions in sperm 

23 count and motility were noted in formaldehyde-treated rats compared with controls. Sperm 

24 viability was also significantly reduced by formaldehyde treatment, though to a lesser overall 

25 extent than sperm count and motility.

26

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

28 Wistar rats exposed intraperitoneally

29

Parameters Control (n = 10) Treated (n = 8)

Sperm count (106/mL) 46.30 ± 5.01 20.40 ± 2.01a

Sperm viability (%) 87.10 ± 0.83 72.60 ± 2.32a

Sperm motility (%) 75.00 ± 10.90 22.00 ± 6.40a

30 a 31 Significantly different from controls (p < 0.0001), as calculated by the authors.

32

33 Source: Majumder and Kumar (1995).

34

35
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1 Majumder and Kumar (1995) also carried out an in vitro experiment in which sperm from 

2 normal rats were incubated with different concentrations of aqueous formaldehyde at 

3 concentrations ranging from 125 pg/mL to 2.5 μg/mL. Viability of control sperm remained close 

4 to 80% for a period of 1 hour, whereas the presence of formaldehyde dose-dependently reduced 

5 viability. Thus, only 50% spermatozoa were viable for 30 minutes in the presence of 5 ng/mL 

6 formaldehyde compared with 6 minutes in the presence of 500 ng/mL. Sperm motility also was 

7 sensitive to the presence of formaldehyde. Less than 10% of sperm was motile for 10 minutes in 

8 the presence of 125 pg/mL formaldehyde. The authors of the study considered their data to be 

9 good evidence that functional parameters of spermatozoa, such as viability and motility, can be 

10 adversely affected by exposure to formaldehyde. Moreover, they suggested that the cumulative 

11 effects of I.P. administration of formaldehyde on the male rat reproductive system raise an alert 

12 that formaldehyde might impair the reproductive health of males who are occupationally exposed 

13 to the compound.

14 Odeigah (1997) conducted two short-term in vivo assays to examine sperm head 

15 abnormalities and dominant lethal mutations. In the sperm assessment, five daily I.P. injections 

16 of 0, 0.125, 0.25, or 0.5 mg/kg formaldehyde were administered to male albino rats (six/group; 

17 strain not specified). The rats were killed 3 weeks after the last injection, and epididymal sperm 

18 counts and abnormalities were assessed. A dose-related decrease in sperm count was observed, 

19 and significantly increased incidences of sperm head abnormalities were found at all treatment 

20 levels (Table 4-67).

21

22 Table 4-67. Incidence of sperm head abnormalities in formaldehyde23 treated rats

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 26 Significantly different from controls (p < 0.05), as calculated by the authors. b 27 Significantly different from controls (p < 0.001), as calculated by the authors.

28

29 Source: Odeigah (1997).

30

31

32 In the dominant lethal assay (Odeigah, 1997), five daily I.P. injections of 0, 0.125, 0.25, 

33 or 0.5 mg/kg formaldehyde were administered to male rats (5 control rats, 12/treated group). 

34 Subsequently, each male was mated with two untreated virgin females per week for 

35 3 consecutive weeks. The females were killed 13 days after the midpoint of the mating period 
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1 and evaluated for live and dead uterine implants. In general, the number of live embryos was 

2 decreased with treatment, and the number of dead implants was increased (Table 4-68). 

3 Additionally, there was a reduction in fertile matings in females mated 1–7 days after the males 

4 had been treated. This study did not assess the number of corpora lutea and therefore precluded 

5 the determination of preimplantation loss. Nevertheless, it is indicative of dominant lethal 

6 effects on the male germ cells.

7

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

9 formaldehyde

10

Dose 

(mg/kg)

Time of 

mating 

(days)

Fertile 

matingsa (%)

Implants per 

femalea

(mean ± SE)

Live embryos 

per female

(mean ± SE)

Dead implants 

per female

(mean ± SE)

Dominant 

lethal 

mutation 

indexb

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 12 Number of females with implants presented in parentheses. b 13 Dominant lethal mutation index:

Index = 1 – (Live implants experiment group per female)

15 (Live implants of control group per female)

14 × 100

16

17 Source: Odeigah (1997).

18

19

20 4.2.1.7.4. Dermal exposure studies addressing developmental and reproductive toxicity. In a 

21 study designed to assess the embryotoxic effects of dermal exposure to formaldehyde, Overman 

22 (1986) applied 0.5 mL of a 37% formaldehyde solution directly to the dorsal skin of female 

23 Syrian golden hamsters (four–six/group) on GDs 8, 9, 10, or 11 for 2 hours. To prevent 

24 grooming during the treatment period, the animals were anesthetized with Nembutal. At the end 

25 of the 2-hour treatment period, the application site was washed thoroughly to remove any 

26 remaining formaldehyde. The dams were terminated on GD 15 (i.e., one day prior to expected 

27 delivery, since the typical gestation period for the Syrian golden hamster is 16–18 days). The 

28 fetuses were removed and fixed in either Bouin’s solution or 95% ethanol for visceral or skeletal 

29 evaluation, respectively. The uteri were examined for implantation sites. Fixed fetuses were 
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1 weighed, measured (crown-rump), and examined for external abnormalities; fetuses that had 

2 been placed in Bouin’s fixative were evaluated for visceral anomalies by using a free-hand 

3 sectioning technique, and those that were placed in ethanol were macerated, stained, and cleared 

4 for skeletal examination. In this study, the dams exhibited signs of dermal irritation and 

5 irritability, but the author reported no treatment-related effects on maternal body weight gain. 

6 The percent of resorption sites was increased (although not significantly) in treated litters as 

7 compared with control (0, 4.2, 8.1, 4.6, and 3.2% resorbed implantation sites for control and 

8 GDs 8, 9, 10, and 11 treatment groups, respectively). No treatment-related effects on fetal 

9 weight, length, or visceral or skeletal development were observed.

10

11 4.2.1.7.5. Summary of reproductive and developmental effects. Formaldehyde exposures up to 

12 40 ppm 6 hours/day from GDs 6–15 or 6–20 did not result in external or internal malformations 

13 (Martin, 1990; Saillenfait et al., 1989). Martin (1990) reported delayed skeletal ossification and 

14 dose-dependent decreases in fetal body weight at 5 ppm. Formaldehyde exposure at 40 ppm to 

15 pregnant female Sprague-Dawley rats reduced fetal body weights in male and female progeny 

16 and in male pups of dams exposed to 20 ppm formaldehyde (Saillenfait et al., 1989). Based on 

17 these studies (Table 4-69), the LOAEL for developmental effects in rats is 5 ppm, with a 

18 NOAEL of 2 ppm for decreased fetal weight and delayed skeletal ossification, based on 

19 inhalation exposures during GDs 6–20.

20 Developmental studies during earlier gestational windows of inhalation exposure to 

21 formaldehyde have reported additional adverse health effects, including delayed ossification, 

22 changes in relative organ weight, undescended testes, biochemical changes (e.g., ascorbic acid 

23 and nucleic acids), and blood acidosis (Senichenkova and Chebotar, 1996; Senichenkova, 1991; 

24 Kilburn and Moro, 1985; Gofmekler and Bonashevskaya, 1969; Gofmekler, 1968; Pushkina et 

25 al., 1968). Kitaev et al. (1984) hypothesized that formaldehyde may affect reproductive function 

26 by stimulating the hypothalamo-pituitary-gonadal (HPG) axis based on their observations of 

27 increased ovary weight, increased number of ovulating cells, and changes in blood levels of 

28 gonadotropins (LH and FSH). Evidence of preimplantation loss, which may be related to HPG 

29 disruption, was observed in this study and by Sheveleva (1971). Additional studies are needed to 

30 better understand developmental effects of formaldehyde exposure during early gestational 

31 windows.

32 The prenatal developmental toxicity of oral and dermal exposures to formaldehyde has 

33 not been thoroughly studied. Reductions in postnatal growth in beagle pups was observed by 

34 Hurni and Ohder (1973) following in utero exposure to 125 ppm maternal dietary formaldehyde 

35 during GDs 4–56 in beagle dogs. However, gavage dosing during gestation of mice to overtly 
This document is a draft for review purposes only and does not constitute Agency policy.

4-310 DRAFT—DO NOT CITE OR QUOTE

1 maternally toxic doses (Seidenberg and Becker, 1987; Marks et al., 1980) (Table 4-70) and 

2 dermal application during gestation to hamsters at a dose that caused dermal irritation and 

3 irritability (Overman, 1986) did not result in any observed fetal toxicity (Table 4-71). 

4 Few studies identified effects on maternal toxicity or female reproductive capacity. As 

5 summarized in Table 4-69, exposure of rat dams at 10–40 ppm formaldehyde during pregnancy 

6 has been shown to result in significantly decreased weight gain (Martin, 1990; Saillenfait et al., 

7 1989; Kilburn and Moro, 1985). Maronpot et al. (1986) reported endometrial hypoplasia with a 

8 lack of ovarian luteal tissue in female rats exposed at 40 ppm but not at 20 ppm. Changes in LH

9 and FSH levels were reported in dams exposed to 0.41 ppm formaldehyde by Kitaev et al. 

10 (1984), establishing an unbounded LOAEL for maternal toxicity.

11 Studies designed to assess male reproductive system endpoints in rats following repeated 

12 inhalation exposures to formaldehyde have shown concentration-dependent decreases in Leydig 

13 cell number and quality, effects on seminiferous tubules, decreases in testes weight, alterations in 

14 sperm measures, decreased testosterone levels, alterations in trace metals in the testes, and/or 

15 dominant lethal effects (Zhou et al., 2006; Özen et al., 2005, 2002; Zhou et al., 2006Sarsilmaz et 

16 al., 1999) (Table 4-72). Based on available studies, the LOAEL for changes in the male 

17 reproductive system in rats following 5 days/week of inhalation exposures is 5 ppm for 3 months 

18 of daily exposures and 10 ppm for 4 weeks of daily exposures; these dose levels are unbounded. 

19 Abnormal sperm were also noted in mice at an inhalation dose of 16.9 ppm 2 hours/day, 6 

20 days/week for 13 weeks (Xing et al., 2007), but, in contrast, Maronpot et al. (1986) reported no 

21 histologic abnormalities in male mice after formaldehyde exposures at 40 ppm 6 hours/day, 5 

22 days/week for 13 weeks. Varied results among studies may be due to species differences or 

23 differences in methods. Although several oral subchronic and chronic studies with formaldehyde 

24 did not identify effects on the testes (Tobe et al., 1989; Til et al., 1988; Johanssen et al., 1986), 

25 Cassidy et al. (1983) observed spermatogenic abnormalities after a single oral dose of 200 mg/kg 

26 to rats, and a chronic drinking water study in rats (Til et al., 1989) reported low incidences of 

27 Leydig cell tumors in all treated groups, compared with none in control (Table 4-73). 

28 Additionally, studies utilizing I.P. injection of formaldehyde in rats have demonstrated testes and 

29 sperm anomalies (Majumder and Kumar, 1995; Chowdhury et al., 1992) and dominant lethal 

30 effects (Odeigah, 1997) (Table 4-74).
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-311

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

Species, 

strain, sex n/Group

Dose; time of 

treatmenta

Reported study findingsb LOAEL/NOAELc

Maternal Offspring Maternal Offspring Reference

Rat, strain 

NR, female

12 0, 0.01, or 0.81 ppm 

(reported as 0.012, 

and 1 mg/m3

)

d

; 

continuous dosing 

10–15 days prior to 

mating and during 

gestation

At 0.01 and 0.81 ppm:

↑ pregnancy duration 

(dose-dependent data 

not shown)e

Fetuses: 

At 0.01 and 0.81 ppm:

↓ fetuses/dam (dose dep., data 

not 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 and 

kidney wt (stat. sig.) 

L: 0.01 ppm

N: ND

L: 0.01 ppm

N: ND

Gofmekler 

(1968)

Rat, 

“albino” 

strain NR, 

female

12 0, 0.01, or 0.81 ppm 

(reported as 0.012, 

and 1 mg/m3

)

d

; 

continuous dosing 

10–15 days prior to 

mating and during 

gestation

NE Age of assessment NR.

At 0.81 ppm:

histologic effects in liver (e.g., ↑ 

extramedullary hematopoietic 

centers), kidney (e.g., ↑ 

polymorphism of renal epithelial 

cell nuclei)

and thymus 

NEe N: 0.01 ppm

L: 0.08 ppm 

Gofmekler and 

Bonashevskaya 

(1969)f,h

Rat, strain 

NR, male

4 Inhalation and 

drinking water 

co-exposure: 0; 

0.10 ppm plus 

0.005 mg/L water; 

0.20 ppm plus 

0.01 mg/L water; or 

0.41 ppm plus 

0.1 mg/L water; all 

treatments 

4 hours/day, 

5 days/week for

6 months

No effects No effects ND g ND Guseva (1972)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-312

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

Species, 

strain, sex n/Group

Dose; time of 

treatmenta

Reported study findingsb LOAEL/NOAELc

Maternal Offspring Maternal Offspring Reference

Rat, strain 

NR, female

NR Expt 1: 

0 or 30 ppmd

Expt 2: 0, pair-fed 

control (15, 10, or 

5 days), or 30 ppm; 

8 hours/day for 

15 days (GDs 3–17), 

10 days (GDs 3–12), 

5 days (GDs 8–12), 

or 3 days (GDs 9–

11)

At 30 ppm 

50% mortality (10 and 

15 day exp)

↓ wt gain (duration 

dep.; 3, 5, 10, and 15 

day exp.)

↓ wt of liver, kidney, 

spleen and thymus

↑ wt of lung and 

adrenale

Fetuses:

At 30 ppm 

↓ fetal wt and growth (duration 

dep., 10 and 15 day exp.)

↑ dev. defects (undescended 

testes, large hearts, small 

thymuses, small lungs)e

N: ND

L: 30 ppme

N: ND

L: 30 ppme

Kilburn and 

Moro (1985)f Ab

Rat, Wistar, 

female

Embryo 

dev expt: 

5–9/group 

(42 adult 

animals);

maternal 

effects: 

NR (200 

adult 

females 

total) i

0, 0.4, or 1.2 ppm 

(converted from 

reported 0, 0.5 or 

1.5 mg/m3

); 

4 hours/day, 

5 days/week for

4 months; exposed 

females mated to 

unexposed males on 

120th day 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. at 

0.4 ppme

)

At 0.4 ppm:

↑ no. of embryos and 2 

blastomere stage embryos (stat. 

sig. in 2nd day preg.)

At 1.2 ppm:

↑ no. degenerating embryos 

(stat. sig. in 3rd day preg.)

L: 0.4 ppm

N: ND

L: 0.4 ppm

N: ND

Kitaev et al. 

(1984)g

Rat, 

SpragueDawley, 

female and 

offspring of 

both sexes

25 0 (air control 

group), 0 (room 

control group), 2, 5, 

or 10 ppm; 

6 hours/day

GDs 6–15.

Exposed females 

mated to unexposed 

males

At 10 ppm:

↓ food consumption 

(stat. sig.)

↓ wt gain (stat. sig.)

At 5 and 10 ppm:

Fetuses:

↑ incidence of reduced 

ossification of pubic and ischial 

bones (stat. sig. compared with 

air control group)

↓ fetal wts (nonsig.)

↑ litter size (nonsig.)

L: 10 ppm

N: 5 ppm

L: 5 ppm

N: 2 ppm

Martin (1990)e,f
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-313

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

Species, 

strain, sex n/Group

Dose; time of 

treatmenta

Reported study findingsb LOAEL/NOAELc

Maternal Offspring Maternal Offspring Reference

Rat, “white” 

strain NR, 

female and 

offspring of 

both sexes

5–12 (NR 

for 

formaldehyde only)

0, 0.01, or 0.81 ppm 

(reported as 0.012 

and 1 mg/m3

)

d

; 

continuous

10–15 days prior to 

mating through 

gestation

At 0.01 and 0.81:

↓ vit. C level in liver

(stat. sig.)

↓ vit. C level in 

placenta

(nonsig.)

Fetuses: 

At 0.01 and 0.81 ppm:

↓ fetuses/femalee

↑ body wt and organ wt (data not 

showne

)

↓ vit. C level in whole fetus (stat. 

sig.)

At 0.01 ppm:

↓ vit. C level in fetal liver (stat. 

sig.)

L: 0.01 ppm

N: ND 

L: 0.01

N: ND

Pushkina et al. 

(1968)f

Rat, 

SpragueDawley, 

female and 

offspring of 

both sexes

25 0 (air control), 5, 10, 

20, 40 ppm; 6 

hours/day,

GDs 6–20.

Exposed females 

mated to unexposed 

males.

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 and 

40 ppm)

GD 21 fetuses:

At 20 and 40 ppm:

↓ fetal body wt, male (stat. sig.)

At 40 ppm:

Delayed ossification of thoracic 

vertebrae (stat. sig., trend 

20 ppm)

↑ unossified sternebrae (nonsig. 

at 40 ppm)

↓ fetal body wt, female (stat. 

sig.)

L: 40 ppm

N: 20 ppm

L: 20 ppm

N: 10 ppm

Saillenfait et al. 

(1989)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-314

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

Species, 

strain, sex n/Group

Dose; time of 

treatmenta

Reported study findingsb LOAEL/NOAELc

Maternal Offspring Maternal Offspring Reference

Rat, 

mongrel 

white, 

female and 

offspring of 

both sexes

NRi 0 or 0.41 ppm 

(reported as 0.5 

mg/m3

) 

formaldehyde

(also a 3rd group of 

gasoline exposure, 

not described in this 

table); 4 hours/day

GDs 1–19 

Dams GD 20: 

↓ corpora lutea 

(nonsig.), embryos dead 

before implantation (not 

stat. sig.), and 

implanted embryos 

(nonsig.)

↑ blood pCO2 (stat. 

sig.)

Fetuses (GD 20):

Stat. sig. findings include

↑ fetal wt

↑ litters w/internal organ 

anomalies

↓ fetuses w/ossification centers 

in hyoid bone 

↑ metacarpal bone centers 

↑ metatarsal bone centers

↑ developmental defects

↑ blood pCO2 and pO2

Pups: ↓ pup wt 

Dev. delays (data not shown) 

L: 0.41 ppm

N: ND

L: 0.41 ppm

N: ND

Senichenkova 

(1991)

Mouse, 

mongrel, 

female and 

offspring of 

both sexes

NR (254 

dams) i

0 + ethyl alcohol; 

0.41 ppm 

formaldehyde; 

0.41 ppm 

formaldehyde + 

bipyridyl;

4 hours/day

GDs 1–19. 

Induced maternal 

iron deficiency 

anemia by I.P. 

bipyridyl injections 

on GDs 12–15; 

controls injected 

w/25% ethyl 

alcohol.

Dams GD 20:

formaldehyde alone:

↑ blood pCO2 (stat. 

sig.)

formaldehyde + 

bipyridyl:

↓ blood acid metabolic 

products (stat. sig.)

↓ blood true 

bicarbonates and CO2

conc. (stat. sig.)

Fetuses (GD 20):

Formaldehyde alone:

↑ cryptorchidism 

Formaldehyde + bipyridyl:

↑ birth defects (stat. sig.) 

↓ dev. delay (stat. sig.)

↓ blood acid-base measures of 

embryos (stat. sig.)

L: 0.41 ppm

N: ND

L: 0.41 ppm

N: ND

Senichenkova 

and Chebotar 

(1996)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-315

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

Species, 

strain, sex n/Group

Dose; time of 

treatmenta

Reported study findingsb LOAEL/NOAELc

Maternal Offspring Maternal Offspring Reference

Rat, 

mongrel, 

white, 

female and 

offspring of 

both sexes

15/group 

terminated 

GD 20, 

6/group 

littered

0, 0.0005, or 

0.005 mg/L (0, 0.4, 

or 4 ppm), GDs 1–

19, 4 hours/day

At 0.4 ppm: ↓ leukocyte 

counts

At 4 ppm: ↓ leukocyte 

counts; reduced 

threshold of 

neuromuscular 

excitability, ↓ rectal 

temperature, ↓ blood 

hemoglobin; ↑ 

spontaneous mobility

At 0.4 ppm: ↑ preimplantation 

loss; at 1 mo. of age, ↓ 

spontaneous mobility; at 2 mo. 

of age, ↓ hemoglobin levels and 

leukocyte counts

At 4 ppm: ↑ preimplantation 

loss; at 1 and 2 mo. of age, ↓ 

spontaneous mobility; at 2 mo. 

of age, ↓ hemoglobin levels and 

leukocyte counts

L: 0.4 ppm

N: ND

L: 0.4 ppm

N: ND

Sheveleva 

(1971)

Rat, 

SpragueDawley, 

female and 

offspring of 

both sexes

6 dams 0 or 6 ppm 

8 hours/day, 

6 weeks, starting at 

GD 1, PND 1, 

4 weeks of age, or 

adult age

NE In offspring exposed in utero and 

during early postnatal life: ↓

mean BW and liver weight; ↑ 

markers of oxidative stress

In offspring exposed as 

juveniles: ↓mean liver weight; ↑ 

markers of oxidative stress

In offspring exposed only as 

adults: no effect

NE L: 6 ppm

N: ND

Kum et al. 

(2007)

Rat, 

SpragueDawley, 

female and 

offspring of 

both sexes 

6 dams 0 or 6 ppm 

8 hours/day; 

6 weeks, starting at 

GD 1, PND 1, 

4 weeks of age, or 

adult age

NE In offspring exposed in early 

postnatal life, as juveniles, or as 

adults, ↑ ANAE-positive Tlymphocytes in BALT

In all exposure initiation groups, 

↑ ANAE-positive lymphocyte 

ratios

NE L: 6 ppm

N: ND

Sandikci et al. 

(2007)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-316

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

Species, 

strain, sex n/Group

Dose; time of 

treatmenta

Reported study findingsb LOAEL/NOAELc

Maternal Offspring Maternal Offspring Reference

Rat, Wistar, 

female and 

offspring of 

both sexes

12–14 

dams 

0, 6, or 12 ppm, 

6 hours/day; 

5 days/week, 

30 days

NE At 6 and 12 ppm, at postnatal 

days 30 and 90: respiratory 

irritation and toxicity; decr. BW, 

FC, WC; ↓ SOD activity, ↓

levels of copper and iron levels 

in lungs, ↑ zinc levels in lungs

NE L: 6 ppm

N: ND

Songur 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/m3 to ppm: 1 ppm = 1.23 mg/m3

; 1 mg/m3 = 0.813 ppm.

a

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

Studies with negative findings are included.

c

L: LOAEL; N: NOAEL. 

d

Exposure concentrations not validated; details of formaldehyde vapor generation not reported; exposure during gestation not well characterized in study report. e

No statistics provided.

f

Lack of study details.

g

See Table 4-72 for reproductive effects. h

Gofmekler and Bonashevskaya (1969) seem to report on different findings from the same study (i.e., same animals) as Gofmekler (1968). i

Number/group not clear from study report.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-317

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

Species, 

strain, sex

n/

Group

Dose; time of 

treatment

Reported developmental effects a LOAEL/NOAELb

Maternal Offspring Maternal Offspring Reference

Dog, 

beagle, 

female and 

pups

9–10 0, 125, or 

375 ppm, dietary, 

GDs 4–56

No effects At 125 and 375 ppm: 

↓ birth wt and wt gain 

through postnatal week 8

L: ND

N: 375 ppm

L: 125 ppm

N: ND

Hurni and 

Ohder (1973)

Mouse, 

CD-1, 

female

29–35 

total

0, 74, 148, or 

185 mg/kg-day, 

GDs 6–15

(aqueous 

formaldehyde 

solution 

contained 10–

15% methanol)

At 185 mg/kg-day: 

Mortality

No effects at GD 18 L: 185 mg/kg-day

N: 148 mg/kg-day

L: ND

N: 185 mg/kg-day

Marks et al. 

(1980)

Mouse, 

ICR/SIM, 

female

26–30 

total

0 or 540 mg/kgday, GDs 8–12At 540 mg/kg-day: 

Mortality

No effects in pups on PND 1 

and 3

L: 540 mg/kg-day

N: ND

L: ND

N: 540 mg/kg-day

Seidenberg and 

Becker (1987)

ND: not determined.

a

Studies with negative findings are included.

b

L: LOAEL; N: NOAEL. 
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-318

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

Species, 

strain, sex

n/

Group

Dose; time of 

treatmenta

Reported developmental effects LOAEL/NOAELa

Maternal Offspring Maternal Offspring Reference

Hamster, 

Syrian 

golden , 

female

4–6 0 or 37%; 0.5 mL 

applied to dorsal skin 

(hair clipped) for 2 hours 

then washed; GDs 8, 9, 

10, or 11

Signs of dermal irritation 

and irritability

At all GDs of treatment, ↑ 

percent resorptions (not sig.)

L: 37%

N: ND

L: 37%

N: ND

Overman (1986)

ND, not determined

a

L: LOAEL; N: NOAEL. 
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-319

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

Species, 

strain, sex

n/

Group Dose; time of treatmenta Reported reproductive effectsb

LOAEL/

NOAELc Reference

Rat, strain 

NR, male

4 Inhalation plus drinking water 

co-exposure: 0; 0.10 ppm plus 

0.005 mg/L water; 0.20 ppm 

plus 0.01 mg/L water; or 0.41 

ppm plus 0.1 mg/L water; all 

treatments; 4 hours/day, 

5 days/week for 6 months.

Exposed males mated to 

unexposed females

At 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/L 

water

N: ND

Guseva 

(1972)d,e

Rat, 

Wistar, 

female

NR 

(200 

female)

0, 0.4, or 1.2 ppm ; 

4 hours/day, 5 days/week for 

4 months.

Exposed females mated to 

unexposed males on 120th day 

of 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 ppm h

N: ND h

Kitaev et al. 

(1984)e

Mouse, 

B6C3F1, 

male and 

female

10 0, 2, 4, 10, 20, or 40 ppm; 

6 hours/day, 5 days/week

for 13 weeks 

Males and females:

At 20 ppm:↓ wt gain 

At 40 ppm: ↑ mortality (13 weeks exp.); ↓ wt loss

Females: 

At 40 ppm: ↑ Uterine endometrial and ovarian hypoplasia

L: 20 ppm

N: ND

Maronpot et 

al. (1986)

Rat, albino 

Wistar,

male

7 6 groups: 0, 10, or 20 ppm;

8 hours/day, 5 days/week for

4 weeks (subacute) or 

13 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 ppm

N: ND

Özen et al. 

(2002)f

Rat, 

Wistar, 

male

18 0, 5, or 10 ppm; 8 hours/day, 

5 days/week, 91 days

At 5 and 10 ppm:

clinical signs of respiratory irritation, ↓ BW, FC, WC; ↓serum 

testosterone; ↓ mean seminiferous tubule diameters; ↑ localization 

of heat shock protein 70 in cytoplasm of spermatogonia, 

spermatocytes, and spermatids

L: 5 ppm

N: ND

Özen et al. 

(2005)
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-320

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

Species, 

strain, sex

n/

Group Dose; time of treatmenta Reported reproductive effectsb

LOAEL/

NOAELc Reference

Rat, albino 

Wistar, 

male

10 0, 10, or 20 ppm

8 hours/day, 5 days/week for 

4 weeks 

Dose 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 ppm

N: ND

Sarsilmaz et 

al. (1999)

Mouse, 

strain not 

specified, 

male

12 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 males 

mated to unexposed females.

In all treated groups: ↑ percentage abnormal sperm, ↑ resorption 

rate, and ↓ live fetuses 

L: 16.9 ppm

N: ND 

Xing et al. 

(2007)

Rat, 

Wistar, 

male

7 0 or 1.5 ppm, 18 weeks

FA exposures:

(1) 4 hours/day, 4 days/week

(2) 2 hours/day; 4 days/week

(3) 2 hours/day, 4 days/week

In all treated groups: sig. ↓ seminiferous tubular diameter and 

epithelial 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 between 

Sertoli and germinal cells

L: 1.5 ppm

N: ND

Golalipour et 

al. (2007)

Rat, 

SpragueDawley, 

male

10 (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, plus 

30 mg/kg-day vitamin E orally 

At 10 mg/m3: sig. ↓ testis weight, atrophy of seminiferous tubules, 

↓ spermatogenic cells, disintegrated and sloughed seminiferous 

cells; edematous interstitial tissue with vascular dilatation and 

hyperemia; ↓ epididymal sperm count and percentage motile 

sperm, ↑ percentage abnormal sperm; ↓ SOD, GSH-Px, GSH and 

↑ MDA in testes; vitamin E attenuated all effects

L: 8.05 ppm

N: ND

Zhou et al. 

(2006)

Rat, 

Wistar, 

male

40 0, 0.1, 1, or 10 ppm; 

6 hours/day, 5 days/week, 13 

or 52 weeks

No effects: testis weight; histopathologic findingsg L: ND

N: 10 ppm

Appleman et 

al. (1986)

a

Treatment is given as the formaldehyde concentration in air (ppm) with the length of exposure each day and the duration of treatment in days. b

Studies with negative findings are included.

c

L: LOAEL; N: NOAEL.

d

Guseva (1972) was a drinking water and inhalation study.

e

Developmental effects shown in Table 4-69. f

Statistics not provided in study report.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-321

g

Focus of study was not the reproductive system; only reproductive system findings are addressed in the table; NOAEL and LOAEL in table are based only on 

reproductive system findings.

h For 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/m3 to ppm: 1 ppm = 1.23 mg/m3

; 1 mg/m3 = 0.813 ppm.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

4-322

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

Species, 

strain, sex

n/

Group Dose; time of treatment Reported reproductive effectsa

LOAEL/

NOAELb Reference

Rat, 

Wistar, 

male

5 (20 

control)

0, 100, or 200 mg/kg, single 

gavage dose

At 200 mg/kg:

↑ (19%) testicular sperm head counts (stat. sig.)

↑ (5%) abnormal sperm head (stat. sig.)

L: 200 mg/kg

N: 100 mg/kg

Cassidy et al. 

(1983)

Rat, 

SpragueDawley, 

both sexes

15 0, 50, 100, or 150 mg/kg-day, 

drinking water; 91 days

No effects: absolute or relative gonad weights;

histopathologic findings of reproductive organsc

L: ND

N: 150 mg/kg-day

Johanssen et 

al. (1986)

Dog, 

beagle, 

both sexes

4 0, 50, 75, or 100 mg/kg-day, 

dietary; 91 days

No effects: absolute or relative gonad weights; 

histopathologic findings of reproductive organsc

L: ND

N: 100 mg/kg-day

Rat, 

Wistar, 

both sexes

10 0, 25, or 120 mg/kg-day, 

drinking water, 4 weeks

No effects: gonad weights; histopathologic findings of 

reproductive organsc

L: ND

N: 120 mg/kg-day

Til et al. 

(1988)

Rat, 

Wistar, 

both sexes

70 0, 1.2, 15, or 82 mg/kg-day 

(males), 0, 1.8, 21, or 

109 mg/kg-day (females),d

drinking water; 105 weeks

In all treated groups:

Leydig cell tumors observed at 105 weeks of studyc

At 82 mg/kg-day:

↑ mean testes weights

L: 1.2 mg/kg-day

N: ND

Til et al.

(1989)

Rat, 

Wistar, 

both sexes

20 0, 0.02, 0.1, or 0.5% in 

drinking water; 24 months

No effects: gonad weights; histopathologic findings of 

reproductive organsc

L: ND

N: 0.5%

Tobe et al. 

(1989)

ND, not determined.

a

Studies with negative findings are included. b

L: LOAEL; N: NOAEL.

c

Focus of study was not the reproductive system; only reproductive system findings are addressed in the table; NOAEL and LOAEL in table are based only on 

reproductive system findings.

d

Actual concentrations.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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

Species, 

strain, sex

n/

Group Dose; time of treatmenta Reported reproductive effectsa

LOAEL/

NOAELb Reference

Rat, 

Charles 

foster, 

male

10 0 or 5 mg/kg-day; 30 days ↓ body weight gain

↓ Leydig cell population and cell nuclear diameter

↓ serum T levels

↓ testes weights

cellular degeneration of seminiferous tubules

L: 5 mg/kg-day

N: ND

Chowdhury et 

al. (1992)

Rat, 

Wistar, 

male

8 0 or 10 mg/kg-day; 30 days ↓ sperm count, motility and sperm viability L: 10 mg/kg-day

N: ND

Majumder and 

Kumar (1995)

Rat, 

“albino” 

strain NR, 

male

6 0, 0.125, 0.250, or 

0.60 mg/kg-day; 5 days

At all treatment levels:

↓ sperm count and ↑ sperm head abnormalities (3 weeks 

after the last injection)

L: 0.125 mg/kg-day

N: ND

Odeigah 

(1997)

Rat, 

“albino” 

strain NR, 

male

12 0, 0.125, 0.250, or 0.60 

mg/kg-day; 5 days. Exposed 

males mated to unexposed 

females.

At all treatment levels (at GD 13):

Delayed time to mating

↓ mean no. implants and live embryos

↑ dead implants and dominant lethal index following 

mating to untreated females

L: 0.125 mg/kg-day

N: ND

a

Studies with negative findings are included.

b

L: LOAEL; N: NOAEL.

ND: not determined; NR: not reported; T: testosterone.
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1 4.2.2. Carcinogenic Potential: Animal Bioassays

2 Chronic animal studies (inhalation and oral exposures) chronicle tumor incidence in a 

3 variety of rodent models. Study descriptions are provided above in detail (Section 4.2.1, 

4 Table 4-34). The study results are evaluated here for both routes of exposure in context of how 

5 they inform the carcinogenic potential for the three major affected systems: respiratory tract, GI 

6 tract, and LHP system. Experimental design and implementation must be carefully considered 

7 when interpreting study results. For example, some key factors involved in evaluating cancer 

8 bioassays include study size, organs/tissues examined, and study length (especially for late-in9 life tumors).

10

11 4.2.2.1. Respiratory Tract

12 In the respiratory tract, only nasal tumors are considered formaldehyde induced in rodent 

13 studies. The majority of studies were conducted using rats (F344, Wistar, or Sprague-Dawley), 

14 and all studies of 18 months or greater in mice and rats show evidence of formaldehyde-induced 

15 nasal carcinogenicity. The nasal tumors are primarily SCCs, although papillomas, polypoid 

16 adenoma, adenocarcinoma, fibrosarcoma, and esthesioneuroepithelioma have been reported 

17 (Kamata et al., 1997; Monticello et al., 1996; Morgan et al., 1986a, b; Takahashi et al., 1986; 

18 Sellakumar et al., 1985; Kerns et al., 1983; Albert et al., 1982). Although hyperplasia, dysplasia, 

19 and squamous metaplasia of the respiratory epithelium have been observed beyond the nasal 

20 cavity, other respiratory tract tumors have not been significantly increased by formaldehyde 

21 exposure alone.

22 Increased tumor incidence and decreased latency are correlated with increasing 

23 formaldehyde exposure concentration. Reviewing data from the only lifelong inhalation study 

24 with multiple exposure groups, SCC is first noted at 8 and 9 months for high exposed (15 ppm) 

25 female and male F344 rats autopsied as “early deaths” prior to the 12 month sacrifice, with an 

26 incidence of 43% over the course of the study (unadjusted for mortality) (Kerns et al., 1983). In 

27 contrast only two SCCs were found in male and female rats sacrificed after 24 months of 

28 exposure (incidence of SCC 2.5% at 24 months) (Kerns et al., 1983). In a follow-up study by 

29 Monticello et al. (1996), the incidence of SCC in rats exposed at 15 ppm was 47% with the first 

30 tumor noted at 12 months. The incidence of SCC in male rats exposed at 10 ppm was 22% with 

31 the first SCC noted at 18 months. Moreover, of 90 rats exposed at 6 ppm for 20 months only one 

32 SCC was noted. No SCCs were detected in rats exposed at 0.7 or 2 ppm formaldehyde. These 

33 incidence rates are not mortality adjusted and include animals from each scheduled sacrifice (3, 

34 6, 12, and 18 months). In a lifelong study of male Sprague-Dawley rats exposed at 10 ppm 

35 formaldehyde, the cumulative nasal tumor incidence was calculated as a function of time of 
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1 exposure (Figure 4-27) (Sellakumar et al., 1985). After 2 years of exposure, the probability of 

2 nasal carcinoma was greater than 60%.

3

4

5

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

7 in the indicated exposure groups.

8

9 Source: Sellakumar et al. (1985).

10

11 There is some evidence that less-than-lifetime exposure to formaldehyde can induce nasal 

12 tumors over an extended observation period. Two studies, both in male Wistar rats, report nasal 

13 tumors in response to less-than-lifetime exposures (Woutersen et al., 1989; Feron et al., 1988). 

14 A 13-week exposure at 20 ppm resulted in four nasal tumors (three SCCs), a cystic SCC of the 

15 nasolacrimal duct, and an epithelial tumor on the mandible, for a total of six tumors observed 

16 over 30 months of observation (Feron et al., 1988). No tumors were noted in 13-week controls. 

17 A limited number of formaldehyde-related tumors were noted due to 4 or 8 weeks of exposure 

18 followed by 30 months of observation. Although the tumor incidence of these less-than-lifetime 

19 exposures is low, this is consistent with the 2-year bioassays in Wistar rats. Wistar rats are more 

20 resilient to formaldehyde-induced nasal toxicity than F344 or SD rats (Section 4.2.1), and only 1 

21 of 26 (4%) Wistar rats exposed at 10 ppm for 28 months developed SCC (Woutersen et al., 

22 1989) versus 22% in F344 rats (Monticello et al., 1996). 

23 Woutersen et al. (1989) also examined the effect of severe nasal damage from 

24 electrocoagulation on formaldehyde-induced SCC in Wistar rats. Nasal tumors were noted in 

25 formaldehyde-exposed rats without damaged noses (exposed for only 3 months and observed for 
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1 25 months). However, the low incidence of tumors in each treatment group (1/26, 2/60, 2/60, 

2 1/58) indicates these data should be considered suggestive even though no SCCs were noted in 

3 control rats with or without damaged noses (n = 83). The studies by Woutersen et al. (1989) did 

4 demonstrate a synergistic effect of nasal damage from electrocoagulation and 10 ppm 

5 formaldehyde exposure (3 months), where 15/58 rats had SCC versus 1/26 with undamaged 

6 noses. The study was originally designed to examine the effect of formaldehyde on the damaged 

7 tissue on cancer promotion. However, it is unclear if the synergistic effect of formaldehyde 

8 exposure on damaged nasal tissue is an effect of formaldehyde on the damaged cells and joint 

9 effects of a mutagen with regenerative proliferation from the nasal damage. It is also possible 

10 the damaged nasal passages may alter airflow in the nasal passages, resulting in significantly 

11 different flux of formaldehyde into the tissue.

12 There is a single inhalation study (Dalbey, 1982) that investigates the role of promotion 

13 in formaldehyde-induced cancer. Although hamsters exhibit little to no effects of formaldehyde 

14 on the nasal mucosa or other respiratory tract tissues (Rusch et al., 1983a, b; Dalbey, 1982), 

15 DEN-induced respiratory adenomas were increased with formaldehyde exposure (10 ppm) 

16 48 hours prior to DEN injection (but not by formaldehyde alone or formaldehyde exposure after 

17 DEN injection). The number of tracheal tumors per TBA was doubled by formaldehyde 

18 exposure. The study authors note that adenomas should be considered independent tumors and 

19 that the increase in tracheal tumors is of biological significance even given the incidence of 

20 TBAs (77%, DEN alone), was not further increased by formaldehyde exposure. It is of 

21 particular interest that a promotion study in hamsters is positive, since so little nasal pathology 

22 occurs with formaldehyde exposure. The absence of significant hyperplasia and tissue damage 

23 in these animals suggests that formaldehyde may induce subtle changes in the respiratory tract 

24 mucosa that permit formaldehyde to act as a tumor promoter.

25

26 4.2.2.2. Gastrointestinal Tract

27 As with the respiratory tract, the proximal portion of the GI tract exhibits formaldehyde28 induced lesions in the forestomach and glandular stomach (Soffritti et al., 1989; Til et al., 1989; 

29 Tobe et al., 1989; Takahashi et al., 1986). However, data are mixed regarding the carcinogenic 

30 potential of formaldehyde in the GI tract from oral exposures.

31 Two independent 2-year cancer bioassays in Wistar rats exposed to formaldehyde in 

32 drinking water were both negative; they reported no tumors found at the 24-month sacrifice (Til 

33 et al., 1989; Tobe et al., 1989). Til et al. (1989) exposed rats to a range of formaldehyde doses 

34 (0, 1.2, 15, or 82 mg/kg-day) and evaluated 44–49 animals per sex per dose group at 24 months 

35 of exposure. No formaldehyde-related tumors were found. A smaller study by Tobe et al. 
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1 (1989) failed to note any tumors after a 2-year exposure at 0, 10, 50 or 300 mg/kg-day (eight rats 

2 per sex per treatment group).

3 In contrast, two lifelong studies in male and female Sprague-Dawley rats provide support 

4 for formaldehyde-induced GI tract tumors (Soffritti et al., 1989). Both stomach and intestinal 

5 tumors are rare; low background rates are expected in this colony of Sprague-Dawley rats. 

6 These studies demonstrate an increase in tumors (although rare) correlated with exposure to 

7 formaldehyde and significantly increased susceptibility to early-lifetime exposure. The authors 

8 provide a detailed report of the background rates of various stomach and intestinal neoplasia for 

9 male (n = 2,677) and female (n = 2,582) rats (Soffritti et al., 1989). From this background pool, 

10 the total incidence of benign and malignant tumors in the stomach and intestine combined is only 

11 1.4% (combining all sites and locations). The majority of tumors are located in the stomach (1% 

12 benign, 0.2% malignant). Usually, a very large study population is needed to detect increases in 

13 rare tumor types. In this study, the study size of each treatment group was relatively small. 

14 Thus, only a few TBAs are responsible for the observed increases. Additionally, a clear dose15 response relationship is not evident (perhaps due to the low incidence) despite the fact that the 

16 greatest tumor incidence was observed in the highest treatment group. As presented above, 

17 apparent increases in both stomach and intestinal neoplasia are noted in formaldehyde-treated 

18 rats (ranging from 1 to 6% by type). When summed across the GI tract, tumor incidence in the 

19 highest treatment group was 8% versus 1.4% in historical controls. Despite the limitations of 

20 group size and lack of dose response, the findings do support the carcinogenic potential for 

21 formaldehyde administered orally. Moreover, these findings are not inconsistent from Tobe et 

22 al. (1989) and Til et al. (1989) because the study design is significantly different.

23 The second study reported by Soffritti et al. (1989) demonstrates early lifetime 

24 susceptibility with GI tumor incidence of 21.6% in females (n = 37) and 13.9% in males (n = 36) 

25 after exposure to formaldehyde. Sprague-Dawley rats were exposed to formaldehyde in drinking 

26 water for 2 years (0 or 2,500 mg/L). Exposures began on GD 12 in the offspring. The most 

27 common tumor detected was intestinal leiomyosarcoma (13.5% in female offspring) with a 

28 background rate of 0.04% in female rats in the colony. 

29 Soffritti et al. (1989) stands alone in supporting formaldehyde-induced GI tumors. These 

30 findings are largely attributed to a unique study design that included lifelong observation, 

31 neonatal exposure, examination of individual tumor types as well as combined rare tumor types 

32 for analysis, and summation of tumors across locations. The study design results in a more 

33 sensitive assay for rare tumors. Thus, Soffritti et al. (1989) utilized a more appropriate design 

34 and analysis for detecting rare tumors and should not be compared with the results by Tobe et al. 

35 (1989) and Til et al. (1989). 
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1 There is evidence that formaldehyde may act as a tumor promoter by the oral route as 

2 well as the inhalation route (discussed above). Takahashi et al. (1986) reported an increase in 

3 MNNG-initiated GI cancers with formaldehyde exposure (29.4 versus 13.3% TBA in controls); 

4 the greatest difference in tumor-containing versus non-tumorigenic mice was associated with 

5 adenocarcinoma in the glandular stomach (23.5 versus 3.3% in controls). Additionally, 

6 forestomach papillomas and preneoplastic hyperplasia in the glandular stomach were increased 

7 with formaldehyde exposure alone.

8 The data indicate carcinogenic potential from formaldehyde ingestion in drinking water. 

9 Formaldehyde may act in part as a tumor-promoting agent and shows clear increased 

10 susceptibility from early lifetime exposures.

11

12 4.2.2.3. Lymphohematopoietic Cancer

13 The majority of chronic animal bioassays do not report either leukemia or lymphoma, but 

14 many of these studies did not have adequate study length or study design to detect these 

15 malignancies. Many studies focused the histopathology on the nasal passages and respiratory 

16 tract (Monticello et al., 1996; Holmström et al., 1989a; Woutersen et al., 1989; Appleman et al., 

17 1988; Dalbey, 1982; Horten et al., 1963). Kamata et al. (1997) did examine additional organs, 

18 but there were only five animals at each sacrifice. Similarly, the oral study by Takahashi et al. 

19 (1989) focused on the stomach and intestines. Tobe et al. (1989) only included 20 Wistar rats 

20 per group with interim sacrifices. Therefore, few studies can inform the carcinogenic potential 

21 of formaldehyde on the LHP system. Table 4-75 lists the chronic bioassays that have the 

22 potential to detect LHP malignancies.

23 Soffritti et al. (1989) first published an observation of formaldehyde-induced leukemia in 

24 animal studies. These study results have been criticized for their combination of lymphatic 

25 leukemia and lymphoma. However, this classification is consistent with the current WHO 

26 classification of lymphoid malignancies in humans where adult B- and T-cell leukemias and 

27 lymphomas are considered the same disease (Harris, 2000a). Although there may be a slight 

28 vehicle effect, a dose response is still readily apparent among the formaldehyde-treated groups 

29 (Figure 4-28). In contrast, the 2-year bioassay in Wistar male in female rats (Til et al., 1989) was 

30 clearly negative for leukemia and lymphoma with only four TBAs in all treatment groups 

31 sacrificed at 24 months. Moreover, the drinking water levels were similar at the highest dose of 

32 both Til et al. (1989) and Soffritti et al. (1989). However, the two study designs differ in length, 

33 which may have influenced results since leukemia is a late-in-life malignancy in rodents. Two34 year survival in the Soffritti et al. (1989) study varied between 50 and 60%. These animals were 

35 available to develop leukemia after the 2-year window of the Til et al. (1989) study. Any 
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1 potential role of strain differences is unknown. Overall the results of Soffritti et al. (1989) are 

2 strong since they indicate an exposure-response relationship in a lifelong study appropriate for 

3 late-in-life malignancies. Unlike the GI tract tumors, increased LHP malignancies were not 

4 associated with early-life exposure to formaldehyde in drinking water.

5

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

7 and lymphoma

8

Study Histopathology Endpoint Results Comments

Drinking water exposure

Soffritti et 

al. (1986) Complete histopathology

Male and female Sprague-Dawley rats

Lymphocytic 

leukemia and 

lymphosarcoma

Increased, showing 

a dose-response

Lifelong study

High exposure of 

1,500 mg/L in water

Til et al. 

(1989) Complete histopathology 

in control and high-dose 

group (15 ppm)

Male and female Wistar Rats

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

Sellakumar 

et al. (1985);

Albert et al. 

(1982)

Necropsy focused on 

respiratory tract: also liver, 

spleen, kidney, and testes 

and organs demonstrating 

gross pathology

Male rats, Sprague-Dawley

Lymphoma No increase Lifelong study, high 

mortality at 24 months 

(>80%)

Battelle, 

Columbus 

Laboratories 

(1981)

Complete histopathology 

in controls and high-dose 

group (15 ppm)

Male rats, F344

Leukemia, all No increase Extended study, high 

mortality

Complete histopathology 

in controls and high-dose 

group (15 ppm)

Female rats, F344

Leukemia, all Increase in 

mortality-adjusted 

incidence;

p = 0.0056a

Extended study, high 

mortality. Apparent 

elevation in 2 and 6 ppm 

treatment groups as well; 

statistical comparison to 

controls is problematic.

All organs in controls and 

high-dose group (15 ppm)

Female mice, C57BL/6xC3HF1

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 10 Original statistical analysis provided by Battelle Columbus Laboratories. Significance set at p < 0.0167. Analysis 

11 of adjusted data where time to lesion and survivorship were considered (Cox [1972] and Tarone [1975]).

12
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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

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

4 formaldehyde in drinking water for 2 years.

5

6 Note: Animals were observed until natural death. The vehicle control contained 

7 the level of methanol in drinking water for the high-dose group (1,500 mg/L).

8

9 Source: Soffritti et al. (1989).

10

11

12 Sellakumar et al. (1985) conducted a lifelong inhalation study in male Sprague-Dawley 

13 rats exposed at 10 ppm formaldehyde. Organs outside of the respiratory tract were routinely 

14 examined (liver, kidneys, and testes), including any organ exhibiting gross pathology, so there 

15 was some ability to detect leukemia and lymphoma. However, spleen, thymus, and lymph nodes 

16 were not routinely examined, limiting detection of smaller lesions. Although Sellakumar et al. 

17 (1985) was a lifelong study, there was a high mortality rate at 2 years (>80% from the figure), 

18 again limiting the power of this study to detect late-in-life malignancies. Nonetheless, this study 

19 did not indicate formaldehyde-induced lymphoma or leukemia.

20 The largest and most comprehensive study of carcinogenic health effects from 

21 formaldehyde inhalation exposures is the study conducted at the Battelle Columbus Laboratory 

22 (1981) and reported by Kerns et al. (1980) and Swenberg et al. (1983). Although the summary 

23 reports of this study do not discuss leukemia or lymphoma rates, mouse lymphoma and rat 

24 leukemia were selected by the study pathologist and biostatistician for analysis (Battelle 

25 Columbus Laboratories, 1981). Statistical analysis performed by Battelle, which accounted for 
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1 time to lesion and survivorship rates, indicated a statistically significant increase in female rat 

2 leukemia (p = 0.0003) and near significant increase in female mouse lymphoma (p = 0.06). No 

3 trend analysis could be conducted since only gross pathology was conducted on mid-dose mice 

4 and rats (2 and 6 ppm). EPA has further analyzed these data to better understand the significance 

5 of these findings.

6 As noted in the study description (Section 4.2.1.2.3), both male and female rats at the 

7 highest exposure (15 ppm) exhibited significant early deaths due to nasal lesions (see Figure 

8 4-29). No female rats in the highest exposure group remained after the 24-month sacrifice 

9 (which included only 14 animals). Nine male rats were examined after 24 months. Since the 

10 first leukemia in rats was noted at 21 months, the early deaths prior to that time reduced the 

11 number of animals in which the leukemia could have been observed. Unadjusted data do not 

12 show an increase in female rat leukemia, but, when data are adjusted to account for the early 

13 deaths, dramatically different results are apparent. In the unadjusted data, leukemia incidence is 

14 expressed as the number of cases over total animals examined (including early deaths and 

15 interim sacrifices). By using this methodology, there is a lower incidence of leukemia in high 

16 exposed animals, and a slightly higher incidence of leukemia in the mid-exposed groups 

17 compared with controls (Figure 4-30, panel A). However, when the leukemia incidence rates are 

18 calculated only for female rats that survived to at least 21 months (the first case noted), 

19 formaldehyde-induced increases in leukemia are evident in all treatment groups relative to 

20 controls (Figure 4-30, panel B). These results are consistent with the original statistical analysis 

21 conducted by the researchers at the Battelle Columbus Laboratories (1981). Although elevated 

22 in all treatment groups, no exposure-response relationship is evident. The lack of a dose23 response relationship may in part be due to the fact that no animals in the high-exposure group 

24 survived past 24 months. Additionally, the 6 ppm (mid-exposure) group had significant early 

25 deaths between 21 to 24 months compared with the 2 ppm and control groups. A similar 

26 reanalysis of data from male rats did not reveal any relationship between formaldehyde treatment 

27 and leukemia incidence.

28 Male and female mice exposed to formaldehyde for 24 months did not experience the 

29 same rate of formaldehyde-related mortality (Kerns et al., 1983). However, significant early 

30 deaths were observed in male mice due to infighting. Therefore, the data for male mice may not 

31 inform incidence of late-in-life tumor, such as lymphoma. As discussed above in the full study 

32 description, full histopathology, including spleen, liver, thymus, and lymph nodes, was only done 

33 on the control and high-exposure group (15 ppm) mice. When comparing unadjusted incidence 

34 lymphoma-bearing mice, there is a clear elevation lymphoma in formaldehyde-exposed female 

35 mice (28%) over controls (22%) (Figure 4-31).

36
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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

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

4 formaldehyde for 24 months.

5

6 Source: Battelle Columbus Laboratories (1981).

7

8
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1

2 Panel A: Unadjusted Data Panel B: Adjusted Data

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

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

7 formaldehyde for 24 months.

8

9 Note: Panel A shows the unadjusted data where incidence rates include all 

10 scheduled sacrifices and early deaths up to the time point shown. Panel B shows 

11 incidence of leukemia only in rats who survived at least 21 months.

12

13 Source: Battelle Columbus Laboratories (1981).

14

15

16
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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

3 Figure 4-31. Cumulative incidence of tumor bearing animals for lymphoma 

4 in female mice exposed to formaldehyde for 24 months.

5

6 Note: Mice from the 6-month interim sacrifice are not included since only nasal 

7 passages were examined (p < 0.05).

8

9 Source: Battelle Columbus Laboratories (1981).

10

11

12 Although results are somewhat mixed between studies, there is evidence in the animal 

13 bioassays for formaldehyde-induced LHP malignancies. Differences in study design may 

14 account in part for mixed results. The lifelong drinking water study by Soffritti et al. (1989) may 

15 have allowed for malignancies to develop late in life, whereas the drinking water study by Til et 

16 al. (1989) sacrificed all animals at 24 months. Even though the exposure levels were similar, the 

17 studies are not directly comparable. Similarly it is hard to directly compare results from the two 

18 major inhalation studies in rats. Although Sellakumar et al. (1985) is a lifelong study, the 

19 mortality for rats was greater than 80% at 2 years. Additionally, the pathology examination was 

20 much less rigorous than in the Battelle Columbus Laboratories (1981) study, perhaps missing 

21 smaller lesions. Therefore, the increase in formaldehyde-induced leukemia seen in female F344 

22 rats late-in-life (Battelle Columbus Laboratories [1981]) may reflect a more sensitive study 

23 design. Finally, strain differences may account for different susceptibility as well. The two 

24 positive rat studies, by different routes of exposure, along with a positive result for 
This document is a draft for review purposes only and does not constitute Agency policy.

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1 formaldehyde-induced mouse lymphoma, make a substantive case for the potential of 

2 formaldehyde-induced LHP malignancies.

3

4 4.2.2.4. Summary

5 Formaldehyde is toxic at the POE. Similar lesions, including increased cell proliferation, 

6 DPX, and focal lesions, are noted in the GI tract or URT, depending on route of exposure. 

7 Similarly, formaldehyde-induced tumors are noted at the POE for both routes of exposure. 

8 Additionally, data exist for both routes of exposure to indicate that formaldehyde may act in part 

9 as a tumor promoter.

10 When evaluating the studies with adequate study design to assess LHP malignancies, 

11 results are mixed by strain, sex, route of exposure, and length of study. The three positive 

12 studies (Soffritti et al., 1986; Battelle Columbus Laboratories, 1981) had the best histopathologic 

13 examinations and greater sensitivity for detection of late-in-life tumors. Based on these results, 

14 sufficient evidence is available in animal studies to support formaldehyde-induced LHP 

15 malignancies.

16

17 4.3. GENOTOXICITY

18 Formaldehyde has been extensively studied for its mutagenic and genotoxic activity in a 

19 variety of assay systems. The first reported mutagenic activity of formaldehyde was when 

20 Rapoport (1946) described the induction of sex-linked recessive lethals in drosophila larvae fed 

21 on a medium containing formalin. A variety of genotoxic and mutagenic effects have been 

22 subsequently demonstrated in both in vitro and in vivo test systems, including the formation of 

23 DPXs, point mutations, DNA strand breaks, increased MNs, and CAs (Auerbach et al 1977; Ma 

24 and Harris, 1988; Conaway et al 1996; IARC 1995; 2006). 

25 In this section, reactions of formaldehyde with cellular macromolecules, such as DNA 

26 and proteins, and formaldehyde-induced clastogenicity are described. In addition, the evidence 

27 for formaldehyde-induced mutations is considered in the context of the current EPA cancer 

28 guidelines (U.S. EPA, 2005a). Particular emphasis is given to the genotoxic effects of 

29 formaldehyde in humans, described in Section 4.3.4.2.

30

31 4.3.1. Formaldehyde-DNA Reactions

32 Formaldehyde is a reactive chemical and interacts with DNA in several ways, forming 

33 DPXs, DNA adducts, and DNA-DNA cross-links (DDXs) (Fennell, 1994; Casanova et al., 1989; 

34 Heck and Casanova, 1987; Casanova-Schmitz et al., 1984a, b; Casanova-Schmitz and Heck, 

35 1983; Ohba et al., 1979; Dönecke, 1978; Brutlag et al., 1969). Formaldehyde also may facilitate 

36 the formation of adducts between other chemicals (endogenous or xenobiotic) and DNA 
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1 (Fennell, 1994; Koppel et al., 1991; Casanova et al., 1989; Heck and Casanova, 1987; Lam et al., 

2 1985; Casanova-Schmitz et al., 1984a; Casanova-Schmitz and Heck, 1983; Ohba et al., 1979; 

3 Dönecke, 1978; Brutlag et al., 1969). 

4 The high reactivity of formaldehyde results in little specificity in reaction sites, indicating 

5 that a range of adducts and cross-links might be expected. However, the spectrum of 

6 formaldehyde-DNA reaction products is difficult to quantify in vivo as many of these are labile 

7 and difficult to measure directly (Fennell, 1994; Casanova et al., 1989). Additionally, 

8 formaldehyde is metabolically incorporated into nucleic acids, and therefore DNA and RNA 

9 assays incorporating radiolabeled formaldehyde need careful interpretation to distinguish 

10 between covalently bound and metabolically incorporated formaldehyde (Casanova et al., 1989; 

11 Heck and Casanova, 1987; Casanova-Schmitz et al., 1984a, b; Casanova-Schmitz and Heck, 

12 1983). Hence, reports of formaldehyde-DNA reactivity in cell-free system results may not 

13 provide a useful measure of exposure (Fennell, 1994). Besides, the question of biological 

14 relevance must also be considered. On the other hand, methods used to extract and measure 

15 DNA-formaldehyde reaction products after in vivo exposures should be evaluated to ensure that 

16 formaldehyde reaction products are neither created nor removed during sample preparation 

17 (Fennell, 1994; Casanova et al., 1989).

18

19 4.3.1.1. DNA-Protein Cross-Links (DPXs)

20 Evidence from numerous experimental models, ranging from cell-free systems to single 

21 cells and in vivo animal and human exposures, suggests that formaldehyde reacts readily with 

22 DNA forming DPXs (Reviewed in Conaway et al 1996; IARC 2006). As shown in Table 4-76, 

23 cross-links between histones and DNA have been demonstrated in isolated chromatin samples on 

24 exposure to formaldehyde from earlier studies (Ohba et al., 1979; Dönecke, 1978; Brutlag et al., 

25 1969). Several in vitro studies demonstrated induction of DPX by formaldehyde exposure in 

26 bacteria (Wilkins and McLeod 1976), yeast (Magana-Schwencke and Ekert, 1978) and 

27 mammalian cells including animal cells (Chinese hamster ovary cells, Chinese hamster V79 lung 

28 epithelial cells, mouse leukemia L1210 cells, mouse hepatocytes, rat Yoshida lymphsarcoma 

29 cells, rat C18 tracheal epithelial cells, rat hepatocytes, rat nasal, tracheal epitheial cells and aortic 

30 endothelial cells) and human cells (lung and bronchial epithelial cells, fibrobasts, white blood 

31 cells, peripheral blood lymphocytes, Epstein-Barr Virus-Burkitt’s lymphoma cells, Jurkat E6-1 

32 cells, HeLa cells, lymphoblastoid cells, gastric mucosal cells and whole blood) as summarized in 

33 Table 4-76. 

34 Ross and Shipley (1980) showed that formaldehyde induces SSBs and DPXs; SSBs are 

35 formed at concentrations >200 μM and a reduction of radiation-induced breaks (indirect measure 
This document is a draft for review purposes only and does not constitute Agency policy.

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of DPXs) at 50 μM. The authors used a [14 1 C]-thymidine-incorporated mouse L1210 cell line to 

2 monitor formaldehyde-induced DNA strand breaks and DPXs. They exposed cells to varying 

3 concentrations of formaldehyde for 2.5 hours. An alkaline-elution technique in the presence or 

4 absence of proteinase K was used to measure strand breaks. In order to detect DPXs, some cells 

5 were exposed to 300 R of X-rays immediately after formaldehyde treatment. Formaldehyde6 induced DPXs were repaired 24 hours after the compound was removed from the culture (Ross 

7 and Shipley, 1980).

8 Casanova-Schmitz and Heck (1983) have shown that homogenates of rat nasal mucosa 

9 incubated with formaldehyde in vitro followed by extraction with a strong aqueous-immiscible 

10 organic solvent demonstrated increased DPX formation in DNA obtained after enzymatic 

11 proteolysis from the aqueous-organic interface, termed as “interfacial DNA”. In the same study, 

12 they have shown that DNA isolated from the nasal, but not olfactory, mucosa of rats exposed to 

13 formaldehyde (2, 6, 15, and 30 ppm 6 hours/day for 2 days) via inhalation showed significant 

14 increase in DPXs in the interfacial DNA ≥6 ppm, which was shown to be linear in the exposure 

range of 2–30 ppm (2.45–36.8 mg/m3 15 ). However, DNA in the aqueous phase did not show DPX 

16 formation. Thus, the cross-linked DNA that could be extracted from the interface after 

17 proteolysis was considered to be supporting evidence of chemically induced DPX formation. 

18 The inability of this study to detect DPXs at lower levels of formaldehyde exposure is likely be 

19 due to the protective mechanism of GSH, which catalyzes the conversion of formaldehyde to 

20 formate. 

21 So, in a later study, Casanova and Heck (1987) reported that GSH depletion caused an 

22 increase in DPX formation in the interfacial DNA in the nasal mucosa of F344 rats when a dualisotope (3

H/14 23 C) method was used. The dual isotope method helps in making the distinction 

24 between metabolic incorporation and covalent binding of formldehyde. Oxidatoin by removal of 

25 one hydrogen atom is required for metabolic incorporation of formaldehyde into cellular 

26 macromolecules, but not in the formation of DNA adducts or DNA-protein crosslinks. Thus, the 

ratio of 3

H/14 27 C of DNA containing DPX will be higher than the macromoleucules where 

28 formaldehyde is metabolically incorporated. However, the authors further demonstrated that, 

when the double isotope method was used, the 3 29 HCHO is oxidized significantly more slowly 

than H14 30 CHO under these conditions, resulting in an overestimate of the concentration of crosslinks due to an isotopic effect on the oxidation of 3 31 HCHO catalyzed by formaldehyde 

32 dehydrogenase (FDH). Besides, this method leaves residual formaldehyde that is likely to form 

33 DNA adducts by reacting with deoxyribonucleosides in the DNA hydrolysates (Heck and 

34 Casanova, 1987).
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1 To overcome this, Casanova et al. (1989) used an improved method, which is based not 

2 on the analysis of residual formaldehyde bound to deoxyribonucleosides in DNA hydrolysates 

but on the determination of the total 14 3 C-formaldehyde bound to DNA. This study showed that 

4 formaldehyde was exclusively bound to interfacial DNA, indicating the formation of DPXs. 

5 Hydrolysis of DPXs in different samples quantitatively released formaldehyde. Besides, DPX 

6 formation was detectable at all concentrations of exposure to formaldehyde (0.3–10 ppm for 

7 6 hours). Overall, these studies clearly show that formaldehyde induces DPXs in nasal epithelial 

8 cells of rodents. However, there are no published rodent studies that assess DPXs beyond the 

9 nasal passages of the URT. 

10 Formaldehyde-induced DPXs were also found in the nasal mucosa and extra-nasal tissues 

of rhesus monkeys exposed to 0, 0.71, 2, or 6 ppm (0, 0.86, 2.45, or 7.36 mg/m3 11 ) formaldehyde 

12 6 hours/day for 3 days (Casanova et al., 1991). These data were used as a basis for cross-species 

13 prediction of formaldehyde-induced DPXs in humans. The presence of DPXs in rhesus monkeys 

14 confirms formaldehyde‘s DNA reactivity as a general effect. Additionally, DPXs were detected 

15 in the larynx/trachea/carina (pooled sample) and in intrapulmonary airways of monkeys exposed 

16 to 2 or 6 ppm formaldehyde. These data demonstrate direct effects of formaldehyde on DNA in 

17 tissues that correspond to observed tumor sites in humans (nasal and nasopharynx). 

18 Bermudez and Delehanty (1986) observed the formation of DPXs, scheduled (S) and 

19 unscheduled DNA synthesis (UDS), and synthesis of RNA when cultured F344 rat nasal 

20 epithelial cells from the naso- and maxillary turbinates were incubated with formaldehyde. 

21 Unscheduled and scheduled DNA synthesis was stimulated (0.05–0.1 mM) and then inhibited 

22 (0.1–1 mM), depending on the formaldehyde concentration. Experiments by Cosma et al. (1988) 

23 and Cosma and Marchok (1988) showed the induction of DPXs and DNA SSBs in cultured C18 

24 rat tracheal epithelial cells exposed to 200 μM formaldehyde for 90 minutes (Cosma et al., 1988; 

25 Cosma and Marchok, 1988).

26 Several human cells (epithelial cells, fibroblasts, buccal cells) or cell lines 

27 (lymphoblastoid cells) exposed to formaldehyde have been shown to form DPX (Craft et al 

28 1987; Costa et al 1997; Emri et al 2004; Li et al 2004). 

29 Craft et al. (1987) detected DPXs by alkaline elution in TK6 human lymphoblastoid cells 

30 immediately after a 2-hour exposure (zero time) to 0, 15, 50, 75, 100, 150, 300, and 600 μM 

31 formaldehyde with a significant nonlinear increase in DPXs above 50 μM concentration, which 

32 correlated with the onset of cytotoxicity, but DPXs were completely removed in cultures held for 

33 24 hours before processing. In the zero-time sample, significant increases in DPXs were first 

34 observed at 50 μM and increased linearly up to 150 μM. In cells held for 24 hours, there was no 

35 detectable increase in DPXs.
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1 However, Costa et al. (1997) detected DPXs with paraformaldehyde (which dissociates to 

2 release formaldehyde) at doses that were cytotoxic (>0.003%) but could not discriminate 

3 between the DPX-inducing and cytotoxic effects of this chemical in EBV, human Burkitt’s 

4 lymphoma cells (Costa et al., 1997). Grafström et al. (1983) reported that the number of DPXs 

5 induced by 100 μM formaldehyde in vitro in human epithelial cells and fibroblasts of bronchial 

6 origin was similar and that the frequency of these cross-links was proportional to the 

7 concentration of formaldehyde. Besides the bronchial epithelial cells and fibroblasts, the authors 

8 also noted that formaldehyde exposure resulted in DPXs and DNA SSBs in skin fibroblasts and 

9 DNA excision repair-deficient skin fibroblasts. However, formaldehyde was only moderately 

10 cytotoxic to normal bronchial epithelial cells and fibroblasts at concentrations that induced 

11 substantial DNA damage. Repair of the formaldehyde-induced DNA SSBs and DPXs appeared 

12 to be inhibited by the continued presence of formaldehyde in the culture medium (Grafström et 

13 al., 1984).

14 Emri et al. (2004) detected a significant increase in DPX formation in primary human 

15 skin fibroblasts and keratinocytes at 8 hours of exposure in vitro to formaldehyde at 25 μM with 

16 an approximately linear increase up to 100 μM. These cells were exposed to 0, 12.5, 25, 50, and 

17 100 μM formaldehyde for 8 hours and then exposed to 250 μM methyl methane sulfonate 

18 (MMS) for 2.5 hours. The induction of DPX formation was measured by the ability of 

19 formaldehyde to reduce DNA migration in the comet assay induced by MMS in this study (Emri

20 et al., 2004). 

21 Li et al. (2004) measured DNA damage in primary human buccal cells by using the 

22 comet assay. The appearance of SSBs, suggesting compound-induced fragmentation of DNA, 

23 occurred at formaldehyde concentrations of 5 and 7.5 μM. At higher concentrations, the 

24 response slope decreased, indicating DPXs or DDXs (Li et al., 2004). The same laboratory 

25 reported similar findings in primary human peripheral blood lymphocytes and HeLa cells (Liu et 

26 al., 2006). Peak response for SSBs was seen at 10 μM in both cells, with higher concentrations 

27 resulting in cross-link formation. SSBs in HeLa cells induced by 10 μM formaldehyde were 

28 repaired by 60 minutes after cells were washed to remove formaldehyde.

29 Schmid and Speit (2007) tested formaldehyde for its ability to induce DPXs in blood 

30 cultures. They used an indirect method to monitor DPX formation in which the extent of DNA 

31 migration in the comet assay in response to γ radiation was compared in formaldehyde-treated 

32 cultures versus controls. A concentration of 25 μM was required for DPX formation, and repair 

33 of these lesions was rapid, with DPXs induced by concentrations of formaldehyde up to 100 μM 

34 and completely removed after 8 hours.

35
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1 Table 4-76. Formaldedyde-DNA reactions (DPX formation)

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 + Swenberg et al 1983

Hamster/Chinese V79 lung epithelial cells + Merk and Speit 1998

Hamster/Chinese V79 lung epithelial cells + Merk and Speit 1999

Hamster/Chinese V79 lung epithelial cells + Speit et 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 Marchok 1988

Rat/F344 Hepatocytes + Casanova and Heck 1997

Rat/F344 Nasal mucosa + Casanova-Schmitz and Heck 1983

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 2005

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 et al 1984

Human Lung/bronchial epithelial cells + Saladino et al 1985

Human Lung/bronchial epithelial cells + Grafstrom et al 1986

Human Foreskin fibroblasts + Snyder and Van Houten 1986

Human Lung/bronchial epithelial cells + Grafstrom 1990

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., 2003
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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

Rat/F344

DPX formation in vivo

Nasal mucosa + Casanova-Schmitz and Heck 1983

Rat/F344 Nasal mucosa + Casanova-Schmitz et al 1984b

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

2 ‘+’ indicates a positive test result

3 ‘-’ indicates a negative test result

A 4 indicates that DNA-protein cross-links formed at cytotoxic concentrations

5

6

7 4.3.1.2. DNA Adducts

8 In addition to the formation of DPX, there is evidence that formaldehyde forms 

9 hydroxymethyl (hm) DNA adducts in vitro in a variety of cell-free systems (Zhong and Que Hee, 

10 2004a; Cheng et al., 2003; Kennedy et al., 1996; Fennell, 1994; Beland et al., 1984) and nasal 

11 epithelial cells (Zhong and Que Hee 2004b). In cell-free systems, formaldehyde directly reacts 

12 with DNA forming hmDNA adducts (Cheng et al., 2003; Kennedy et al., 1996; Fennell, 1994; 

13 McGhee and von Hippel, 1977a, b, 1975a, b). 

14 Beland et al. (1984) first reported hmDNA adducts in Chinese hamster ovary (CHO) cells 

15 incubated with 1 mM of radiolabeled formaldehyde. After a 2-hour incubation, small amounts of 

N6 16 -hmdA were detected with concomitant metabolic incorporation of formaldehyde. Various 

forms of hmDNA adducts, including N6

-hydroxymethyldeoxyadenosine (N6 17 -hmdA), 

N4-hydroxymethyldeoxycytidine (N4-hmdC), and N2-hydroxymethyldeoxyguanosine (N2 18 -

19 hmdG), were detected by high performance liquid chromatography (HPLC) following in vitro 

20 reaction between formaldehyde and calf thymus DNA or individual deoxynucleotides. 
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32 1 P-postlabeling studies allowed for much greater analytical sensitivity but did not 

confirm the level of N6 2 -sulfomethyldeoxyadenosine found by HPLC. However, either estimate 

3 of adduct formation is much less than the estimate of DPX formation (120 pmol/mg DNA) in 

4 similarly treated rat nuclei (Heck and Casanova, 1987). 

5 Casanova et al. (1989) demonstrated that detection of hmDNA adduct formation was 

6 sensitive to the methodology used, particularly the buffer used for sample preparation. 

7 Specifically, Tris buffer can prevent hmDNA adduct formation due to the abundance of 

8 formaldehyde-reactive primary amine sites in the buffer. In contrast, the tertiary amine sites that 

9 predominate in Bis-Tris buffer do not react with formaldehyde. 

Zhong and Que Hee (2004a) observed hmDNA adducts (N6-hmdA, N2 10 -hmdG, and 

N4 11 -hmdC) in placental DNA exposed to 100 ppm formaldehyde in vitro for 20 hours at 37°C 

12 followed by hydrolysis of formaldehyde-reacted DNA using bis-Tris buffer. However, 

13 deoxythymidine did not form hydroxymethyl derivatives in this study (Zhong and Que Hee, 

2004a). On the other hand, the same investigators were able to detect N6

-hmdA and N2 14 -hmdG 

15 adducts in human nasal epithelial cells cultured in the presence of 0, 10, 25, 50, 100, 250, 400, or 

16 500 μg/mL formaldehyde and using Tris buffer during hydrolysis of adducted DNA. The 

17 toxicity threshold for <90% viability appeared to be between 100 and 250 μg/mL initial 

18 formaldehyde culture concentration, and even at 500 μg/mL concentration it was not toxic, with 

19 a viability was 70% in this study (Zhong and Que Hee, 2004b). 

20 The only report of formaldehyde-induced hmDNA adducts in vivo is a recent study 

(Wang et al., 2007), showing an indirect evidence of formation of formaldehyde-induced N6 21 -

22 hmdA in hepatic and pulmonary DNA from rats exposed to N-nitrosodimethylamine and 

23 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone.

24 Since the formaldehyde adducts are labile, Fennel (1994) developed a method by 

25 derivatizing them with sodium bisulfite to their sulfomethyl form, whereby he detected 

N6

-sulfomethyldeoxyadenosine (SOMedA) and N2 26 -sulfomethyldeoxyguanosine by using HPLC. 

27 However, the levels of SOMedA in DNA isolated following incubation of radiolabeled 

28 formaldehyde with isolated rat hepatic nuclei were similar to those in control nuclei. And in 

29 human TK6 lymphoblastoid cells treated with formaldehyde, detection of SOMedA adducts was 

precluded by additional radioactive sports. These observations suggest that N6 30 -

31 sulfomethyldeoxyadenosine adducts are formed at very low levels in formaldehyde-incubated rat 

32 nuclei and that measurement of hydroxymethyldeoxyadenosine would not provide a useful 

33 measure of formaldehyde exposure (Fennell, 1994). 

34

35
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1 Table 4-77. Formaldedyde-DNA reactions (DNA adduct formation)

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 1975a

Cell-free system In vitro + McGhee and von Hippel 1975b

Cell-free system In vitro + McGhee and von Hippel 1977a

Cell-free system In vitro + McGhee and von Hippel 1977b

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 2004b

DNA adduct formation in vivo

Drosophila Larvae + Alderson, 1985

Rats Indirect evidence + Wang et al 2007

3

4 ‘+’ indicates a positive test result

5 ‘-’ indicates a negative test result

6

7

8 4.3.1.3. DNA-DNA Cross-Links (DDXs)

9 Formaldehyde, besides forming DPXs and DNA adducts, has also been shown to form 

10 DDX in vitro. Li et al. (2004) showed that formaldehyde induces DNA strand breaks at low11 exposure concentration and DDXs and DPXs at higher concentrations in buccal cells. The 

12 authors also showed that formaldehyde induces DDXs in human peripheral blood lymphocytes 

13 exposed in vitro when the concentration was more than 25 μM. However, the formation of 

14 DDXs has not been demonstrated in vivo, and the relevance of these modifications in 

15 formaldehyde-induced genotoxicity is not known at the moment.

16 Overall, formaldehyde forms predominantly DPXs that are detected in cell-free systems 

17 and single cells in vitro and in animal and human tissues in vivo. In rodents, DPXs are formed in 

18 nasal epithelia but not in extra-nasal passages, which are completely removed within a day after 

19 formation. The DPXs are detected in nasal and extra-nasal tissues of monkeys, suggestive of 
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1 direct effects of formaldehyde in tissues that correspond to observed tumor sites (nasal and 

2 nasopharynx) in humans. Besides, this is used as a basis for cross-species comparison with 

3 humans. Formaldehyde-DNA adducts are labile, constituting a minor fraction of the DNA4 reaction products. DPXs but not DNA adducts appear to play an important role in the 

5 genotoxicity of formaldehyde.

6

7 4.3.1.4. Single Strand Breaks

8 Formaldehyde has been shown to induce DNA single strand breaks in a number of mammalian 

9 cell systems in vitro as well as in vivo as shown in Table 4-78. Additionally, there is some 

10 evidence that DNA single strand breaks (SSBs) may be induced directly by formaldehyde 

11 reactivity (Grafström et al., 1984).

12

13 Table 4-78. Formaldehyde-DNA interactions (single strand breaks)

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, 1980

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 et al 1984

Human Lung/bronchial epithelial cells + Grafstrom 1990

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 2004

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

16 ‘no’ indicates test was not done in vivo; ‘+’ indicates a positive test result; ‘yes’ indicates test was done in vivo 

17 ; - indicates a negative test result; (+) indicates a weak positive test result.
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1 4.3.1.5. Other Genetic Effects of Formaldehyde in Mammalian Cells

2 Formaldehyde induces several other genetic and related effects in mammalian cells which 

3 are evaluated by in vitro assays such as unscheduled DNA synthesis (UDS), DNA repair 

4 inhibition and cell transformation as summarized in Table 4-79. 

5 UDS, which represents DNA repair activity has been reported in nasal epithelial cells of 

6 F344 rats (Bermudez and Allen 1984; Bermudez and Delahanty 1986), rat hepatocytes (Williams 

7 et al 1989) and Syrian hamster embryo cells (Hamaguchi and Tsutsui 2000). UDS was observed 

8 in HeLa cells (Martin et al 1978), but not in human bronchial epithelial cells (Doolittle et al 

9 1985) upon formaldehyde exposure. These studies suggest that following formaldehyde-induced 

10 DNA damage was followed by DNA repair. 

11 Studies involving human bronchial epithelial cells and skin fibroblasts or keratinocytes 

12 (Grafstrom et al 1984; Emri et al 2004), DNA repair proficient or –deficient cell lines (e.g. 

13 xeroderma pigmentosum) or cell lines hypersensitive to DNA-DNA crosslinks (e.g. Fanconi’s 

14 anemia) (Speit et al 2000) it has been shown that formaldehyde causes DNA repair inhibition at a 

15 concentration range of 0.125 mM to 10 mM). Emri et al (2004) have shown that DNA repair 

16 was inhibited in human keratinocytes and fibroblasts after irradiation with UVB and UVC, but 

17 not UVA follwed by treatment with low concentrations of formaldehyde (10 μM). They 

18 observed that DNA SSB induced by UVB or UVC irradiation alone were repaired within 3-6 

19 hours of exposure, while cells with UV irradiation followed by formaldehyde exposure still had 

20 the strand breaks at the same timepoints suggesting that formaldehyde is likely to contribute to 

21 UV-induced carcinogenesis. 

22

23 4.3.2. In Vitro Clastogenicity 

24 Clastogenic effects, including increased MNs, CAs, and SCEs are also reported in a range 

25 of in vitro study systems as shown in Table 4-80. 

26 Miyachi and Tsutsui (2005) measured the induction of sister chromatid exchanges 

27 (SCEs) in Syrian hamster embryo (SHE) cells. Cells were exposed to 0, 3.3, 10, and 33 μM 

28 formaldehyde for 24 hours. SCE levels after 3.3 μM formaldehyde were not different from 

29 controls, but significant increases were observed at both 10 and 33 μM. Toxicity as measured by 

30 reduced cloning efficiency was seen only at 33 μM (Miyachi and Tsutsui, 2005). The same 

31 laboratory used SHE cells to measure the induction of CAs (Hikiba et al., 2005). Cells were 

32 exposed to 0, 33, 66, and 99 μM formaldehyde for 24 hours prior to staining for analysis and the 

33 percentages of aberrant metaphases were 0, 6, 6, and 71, respectively. The aberrations were 

34 predominantly chromosome gaps and chromosomal breaks and exchanges. The relative colony-
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1 forming efficiency remained high (at least 85%) for the concentrations of formaldehyde used in 

2 the experiment (Hikiba et al., 2005).

3

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

5

Species/Strain Cell/Strain Result References 

Unschedule DNA synthesis (UDS)

Rat/F344 Nasal epithelial cells + Bermudez and Allen 1984

Rat/F344 Nasal epithelial cells + Bermudez and Delehanty 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 2004

6

7 XPA, xeroderma pigmentosum, complementation group A (deficient in NER pathway)

8 FA, Fanconi's anemia (cell line has genetic defect leading to hypersensitivity to DNA-DNA cross links; NER, 

9 nucleotide excision repair 

10

11

12 Schmid and Speit (2007) observed that SCEs were induced in lymphocytes of whole 

13 blood cultures at a formaldehyde concentration of 200 μM, an effect apparently associated with 

14 cytotoxicity. This was indicated by a concomitant reduction in the proliferative index. These 

15 authors also observed the formation of MNs in their cultures. This effect was statistically 

16 significant at a formaldehyde concentration of 300 μM and above. However, MN formation was 

17 confined to those cultures in which formaldehyde treatment commenced 44 hours after the start 

18 of the culture. This prompted the conclusion that the level of DPX formation would need to be 

19 high for MN formation and that the cells must be exposed after the first mitosis. In examining 

20 MN formation more closely, Schmid and Speit (2007) used the FISH technique, employing a 

21 “biotin-labeled pan-centromeric chromosome paint specific for all human centromeres.” 

22 Indicative that formaldehyde was inducing a clastogenic (rather than aneugenic) effect, 81% of 

23 MNs in binucleated cells were centromere-negative.
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1 In summary, formaldehyde forms MNs, SCEs, and CAs in isolated animal and human 

2 cells following in vitro exposure (Table 4-80).

3

4 4.3.3. In Vitro Mutagenicity

5 Mutations may occur during repair of formaldehyde-induced DNA damage (DPXs, DNA 

6 adducts, SSBs, or clastogenic effects) or as a result of replication errors during mitogenesis. The 

7 in vitro evidence for formaldehyde-induced mutations is strengthened by examining the 

8 correlation between these genotoxic and clastogenic events and induction of mutations. 

9 Therefore studies are presented with respect to relevance to one or more of the following lines of 

10 evidence for mutagenicity recommended for consideration in the EPA guidance (U.S. EPA, 

11 2005a): (1) that the chemical is DNA reactive and/or has the ability to bind to DNA, (2) that the 

12 chemical generates positive results in in vitro mutagenic test systems (specifically gene 

13 mutations and CAs), and (3) that the chemical induces indications of genetic damage in in vivo 

14 tests (specifically gene mutations and CAs). Numerous studies have demonstrated 

15 formaldehyde-induced DNA mutations under a variety of experimental conditions (reviewed in 

16 IARC 1995, 2006; Ma and Harris 1988; Auerbach et al. 1977; Conaway et al 1996; NTP 2009). 

17

18 4.3.3.1. Mutagenicity in Bacterial Systems

19 A number of research reports describe the mutagenicity of formaldehyde in bacterial test 

20 systems using reverse and forward mutation assays as well as specific strains detecting deletions, 

21 insertions and point mutations. Among the bacterial strains, Salmonella typhimurium TA102 and 

22 the Escherichia coli strains containing an AT base pair at the primary reversion site are often 

23 used to detect oxidative compounds, cross-linking agents and hydrazines. In an early National 

24 Toxicology Program (NTP) collaborative study with three laboratories, formaldehyde 

25 consistently tested positive for mutagenicity in Salmonella typhimurium strain TA100 in the 

26 presence of a rat or hamster liver S9 activating system (Haworth et al., 1983). Formaldehyde 

27 was mutagenic with and without metabolic activation in a number of other studies using in 

28 reverse mutation assays with S. typhimurium strains TA98, TA100, TA102, TA104, TA2638, 

29 and TA2638a and E. coli strains WP2 (pkM101), WP2 uvrA (pkM101), and hrs/r30R (Ryden et 

30 al., 2000; Dillon et al., 1998; Watanabe et al., 1996; Le Curieux et al., 1993; O’Donovan and 

31 Mee, 1993; Zielenska and Guttenplan, 1988; Schmid et al., 1986; Connor et al., 1983, 1985; 

32 Orstavik and Hongslo, 1985; Takahashi et al., 1985; Fiddler et al., 1984; Frei et al., 1984; 

33 Donovan et al., 1983), while other studies (Muller et al., 1993; Jung et al., 1992; Wilcox et al., 

34 1990; Marnett et al., 1985) show both positive and negative results. These results are 

35 summarized in Table 4-81 and some of the studies are described in greater detail.
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1 Table 4-80. In vitro clastogenicity of formaldehyde.

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 Bauchinger, 1988

Hamster/Chinese Ovary cells + ND Natarajan et al 1983

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 2006

Hamster/Chinese Ovary cells + ND Garcia et al 2009

Hamster/Chinese Lung fibroblasts + ND Ishidate Jr et al 1981

Human Lymphocytes + + Schmid et al 1986

Human Lymphocytes + ND Miretskaya and Shvartsman 1982

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 2007

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 1983

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 Tsutsui 2005

Hamster/Chinese V79 lung epithelial cells + (+) Basler et al. 1985

Hamster/Chinese V79 lung epithelial cells + ND Speit et al 2007b

Hamster/Chinese V79 lung epithelial cells + ND Merk and Speit 1998, 1999

Hamster/Chinese V79 lung epithelial cells + ND Neuss and Speit 2008

Human A549 lung epithelial cells + ND Neuss and Speit 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 2007
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Premature chromosome Condensation (PCC)

Hamster/Chinese Ovary cells + ND Dresp and Bauchinger, 1988

1

2 ‘+’ indicates a positive test result

3 ‘ND’ indicates test was not done

4 - indicates a negative test result

5 (+) indicates a weak positive test result

a 6 MN frequency increased in repair-deficient cell lines compared to normal cell lines

b 7 indicates SCE with significant loss of cell viability c 8 A549 cells exposed for 1 h with formaldehyde then co-cultivated with V79 cells

d 9 A549 cells exposed for 1 h with formaldehyde, cells washed and then co-cultivated with V79 cells

10 XP, xeroderma pigmentosum; FA = Fanconi's anemia.

11

12
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1 Table 4-81. Summary of mutagenicity of formaldehyde in bacterial systems

2

Species Strain

Metabolic 

activation References

+S9 -S9

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 NG30recA, 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., 1996
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S. typhimurium TA1535A - - Sarrif et al., 1997

S. typhimurium TA1537A + + Sarrif et al., 1997

S. typhimurium TA98, 100A + - Sarrif et al., 1997

S. typhimurium TA97A ND + Sarrif et al., 1997

S. typhimurium TA1535, 1537B - - Sarrif et al., 1997

S. typhimurium TA98B + + Sarrif et al., 1997

S. typhimurium TA100B - + Sarrif et al., 1997

S. typhimurium TA100C + + Sarrif et al., 1997

S. typhimurium TA100, 104B + + Dillon et al., 1998

E. coli (Lac+ reversion) WP3101P, WP3106P + Ohta et al 1999

S. typhimurium TA102, 2638B 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 D494uvrB + 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

2 ‘+’ indicates a positive test result

3 ‘ND’ indicates test was not done

4 ‘-’ indicates a negative test result

5 (+) indicates a weak positive test result

6 * indicates the use of formalin in mutagenicity assay

7 ** indicates the use of hexamethylmelamine (HEMLA), a formaldehyde-releasing compound, in mutagenicity assay A 8 indicates use of the Standard Plate Method B 9 indicates use of the Preincubation Plate Method

C 10 indicates use of the Suspension Method

D 11 indicates loss of DNA

12

13

14 Formaldehyde has been shown to be mutagenic in forward mutation assays using S. 

15 typhimurium (Couch et al 1982; Donovan et al 1983; Temcharoen and Thilly 1983) as well as in 

16 E. coli (Bosworth et al 1987). Temcharoen and Thilly (1983) examined the toxicity and 

17 mutagenicity of S. typhimurium strain TM677, using forward mutation to 8-azaguanine 

18 resistance, and have shown that formaldehyde induced both toxicity and mutagenicity at 

19 minimum concentrations of 0.17 mM (–S9) and 0.33 mM (+S9). It has also been shown that 
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1 formaldehyde formed as an intermediate by oxidation at the methyl group of 

2 N-nitrodimethylamine, a biologically active N-nitramine of environmental significance, is 

3 mutagenic to S. typhimurium TA100 strain at low concentrations and toxic above 2 μmol/plate 

4 (Frei et al., 1984).

5 Bosworth et al (1987) developed a forward mutation assay in E. coli D494 uvrB strain 

6 transformed with a multi-copy mutator plasmid pGW1700, in which mutations are scored by an 

7 increase in ampicillin-resistant colonies after exposure of bacterial cells during the logarithmic 

8 growth by the test chemicals. This assay is more sensitive to base-pair substitutions, but less 

9 sensitive to frameshift mutations compared to Salmonella/miceosome-based assays. In this assay, 

10 the authors (Bosworth et al 1987) observed positive curvilinear response to formaldehyde 

11 exposure. Crosby et al (1988) used four E. coli strains GP120, GP120A, 7-2, and 33694 

12 containing the xanthine guanine phosphoribosyl transferase (gpt) gene (which detects point 

13 mutations, deletions and insertions) tested the mutagenicity of formaldehyde by exposing for 1 

14 hour at 4 and 40 mM concentrations. They observed 41% large insertions, 18% large deletions 

15 and 41% point mutations. However, at 40 mM dose there were 92% point mutations, a majority 

16 of them (62%) being transition mutations at a single AT base pair in the gpt gene. In the same 

17 study they observed frameshift mutations in E. coli that was transformed with naked pSV2gpt 

18 plamid DNA exposed to 3.3 or 10 mM formaldehyde. Thus, the mutation pattern appear to differ 

19 depending on the concentration of formaldehyde exposure to the bacterial strain as well as the 

20 nature of DNA. 

21 Formaldehyde has also been shown to induce primary DNA damage in E. coli and 

22 mutagenic activity in the Ames fluctuation test in S. typhimurium TA100, TA102, or TA98 

23 strains (Le Curieux et al., 1993).

24 O’Donovan and Mee (1993) observed clear mutagenicity by the pre-incubation exposure 

25 method in S. typhimurium TA98, TA100, and TA102 strains and both E. coli WP2(pKM101) and 

26 WP2uvrA(pKM101) strains, while the standard plate-incorporation assays showed consistent 

27 mutagenicity only with TA100 and WP2uvrA(pKM101) strains and no evidence of mutagenicity 

28 in TA1535, TA1537, or TA1538 strains using either method of exposure in the absence of 

29 metabolic activation. The S. typhimurium and E. coli strains used in this study are histidine and 

30 tryptophan auxotrophs, with an AT base pair at the critical mutation site within the hisG and trpE 

31 genes, respectively, with an intact excision repair system facilitating the detection of cross32 linking agents and both strains carrying the mutator plasmid, pKM101, which enhances error33 prone repair. These salmonella strains detect frameshift (TA98 and TA1537) and base-pair 

34 substitutions (TA100, TA102, and TA1535), while the E. coli strains detect base-pair 

35 substitutions (WP2uvrA). These findings are consistent with the suggestion that formaldehyde 
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1 induces excision-repairable lesions in bacteria and indicate that the presence of the R-factor 

2 plasmid may be required for the expression of its mutagenicity in excision repair-deficient 

3 salmonella (O’Donovan and Mee, 1993).

4 Dillon et al. (1998) employed salmonella strains TA100, TA102, and TA104 because of 

5 the latter two strains being more sensitive to oxidative mutagens. Formaldehyde was clearly 

6 mutagenic between 6 and 50 μg/plate in all three strains with and without metabolic activation 

7 using Aroclor-induced S9 from male F344 rats or male B6C3F1 mice, except for an equivocal 

8 response in TA102 with mouse S9 (Dillon et al., 1998). Using a set of six tester strains 

9 (WP3101–WP3106) of E. coli, each reversible by a mutation involving a single DNA base pair 

10 substitution, Ohta et al. (1999) determined that formaldehyde preferentially induced GC to TA 

11 transversion mutations. Ryden et al. (2000) demonstrated a statistically significant increase in 

12 the number of revertants in S. typhimurium TA102 (2.5-fold) and TA2638a (3-fold) strains by 

13 formaldehyde at ≥17 μg/plate compared with solvent controls.

14 In summary, formaldehyde induces mutations in several bacterial strains containing an 

15 AT base pair at the primary reversion site that are used to detect oxidative compounds and cross16 linking agents without metabolic activation by exogenous enzyme-activating systems. This 

17 evidence is strengthened by examining the correlation between genotoxic and clastogenic events 

18 and mutation induction.

19

20 4.3.3.2. Mutagenicity in Non-Mammalian Cell Systems

21 Formaldehyde has been shown to be mutagenic in several non-mammalian systems also. 

22 It has been shown to cause gene conversion, strand breaks, crosslinks, homozygosis and related 

23 damage in yeasts (Saccharomyces cervisiae), forward and reverse mutations in molds 

24 (Neurospora crassa), micronuclei formation in spiderworts (Tradescantia pallida), DNA 

25 damage and mutations in several plants, genetic cross-over or recombination, sex-linked 

26 recessive lethal mutations, dominant lethal mutations, heritable translocations and gene 

27 mutations in insects (Drosophila melanogaster) and recessive lethal mutations in nematodes 

28 (Caenorhabditis elegans), but failed to show micronuclei formation in newt larvae (Pleurodeles 

29 waltl) (Reviewed in Conaway 1996; IARC 2006).

30

31 4.3.3.3. Mutagenicity in Mammalian Cell Systems 

32 Several studies demonstrated the mutagenicity of formaldehyde in mammalian cells. In 

33 its report, the Federal Panel on Formaldehyde underlined the role of formaldehyde as an inducer 

34 of gene mutations and CA in a variety of test systems (Report of the Federal Panel on 

35 Formaldehyde, 1982). Results from several studies are summarized in Table 4-82.
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1 Snyder and Van Houten (1986) demonstrated that formaldehyde increases the levels of 

2 misincorporation of bases into synthetic polynucleotides catalyzed by E. coli DNA polymerase I, 

3 indicating that the mutagenicity of formaldehyde may be due to covalent alteration of DNA 

4 bases. They have also shown that formaldehyde-induced DNA damage in human fibroblasts was 

5 not susceptible to repair by the typical “long patch” excision repair mechanism.

6 Craft et al. (1987) measured the induction of mutations at the thymidine kinase (tk) locus 

or at the ouabain resistance (Ouar 7 ) locus in TK6 human lymphoblastoid cells. The tk mutations 

8 can result from a variety of mutational events, including base pair substitution, small and large 

deletions, and chromosome exchange events, while mutations to Ouar 9 require specific base pair 

10 substitutions. Single treatment of formaldehyde (0, 15, 30, 50, 125, and 150 μM) for 2 hours 

11 resulted in a nonlinear increase in tk mutagenesis with increasing slope >125 μM (Figure 4-32). 

12 To explore a dose-response effect, cells were also exposed as follows: three treatments of 50 μM 

13 for 2 hours or five treatments of 30 μM or 10 treatments of 15 μM for 2 hours (treatments were 

14 spaced 2–4 days apart) with multiple treatments causing an increase in tk mutations, although 

15 their combined effect was less than a single treatment of equivalent C × t (150 μM for 2 hours). 

16 Lymphoblasts given four treatments of 150 μM formaldehyde for 2 hours failed to induce 

mutations at the Ouar 17 locus. Dose-response increases were seen in all exposure scenarios, with 

18 30 μM being the level of statistical significance. There was little indication of a dose-response 

19 effect until the cumulative concentration was greater than 100 μM. Formaldehyde-induced 

20 DPXs were no longer evident after 24 hours of exposure; mutants induced in the TK6 

21 lymphoblast cell line showed a similar dose-response curve to the DPXs measured immediately 

22 after exposure ended (Craft et al., 1987). 

23 The same group also studied mutations induced at the X-linked hypoxanthine-guanine 

24 phosphoribosyl transferase (HPRT) locus by eight repetitive treatments of 150 μM formaldehyde 

25 in TK6 human lymphoblast cell line by Southern blot analysis, wherein half (14/30) of induced 

26 mutants contained partial or complete deletions with most of the partial deletions showing 

27 unique deletion patterns, while only a third (5/15) of spontaneous mutants had partial or 

28 complete deletions, indicating that formaldehyde can induce large losses of DNA in human 

29 lymphoblast cells (Crosby et al., 1988). 

30
This document is a draft for review purposes only and does not constitute Agency policy.

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

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 1968

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., 1996

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 +aND 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

Hamster/Chinese

Point Mutation

Ovary HPRT no + ND Graves et al., 1996

Mouse Lymphoma cell/ TK+/- no + + Blackburn et al., 1991

Mouse Lymphoma cell/ TK+/- no + ND Wangeheim and Bolcsfoldi, 1988

Human Lymphoblast/TK6 no + ND Liber et al., 1989

Insertion Mutation

Hamster/Chinese Ovary HPRT no + ND Graves et al., 1996

Heritable Mutation

Mouse Heritable mutation yes + Liu et al 2009

DNA Repair enzyme activity

Human Peripheral lymphocyte yes - Hayes et al., 1997
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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

2 ‘no’ indicates test was not done in vivo

3 ‘+’ indicates a positive test result

4 ‘ND’ indicates test was not done

5 ‘yes’ indicates test was done in vivo 

6 - indicates a negative test result

7 (+) indicates a weak positive test result

8 [+] indicates positive test result after TPA or N-methyl-N-nitro-N-nitrosoguanidine promoter treatment a 9 indicates loss of DNA

b 10 Positive only in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) c 11 p53 mutations

d 12 p53 mutated protein detected by immunohistochemistry

13
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1

2

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

4 mutants in TK6 human lymphoblasts exposed to formaldehyde for 2 hours.

5

6 Note:  DPXs immediately after exposure, DPXs 24 hours after exposure,  tk

7 mutants. Relative survival was 100% at 0, 15, 30, and 50 μM, 30% at 125 μM, 

8 and 20% at 150 μM.

9

10 Source: Adapted from Craft et al. (1987).

11

12

13 Liber et al. (1989) followed up the findings of Crosby et al. (1988) by performing 

14 Southern blot, Northern blot, and DNA sequence analysis on the 16 induced and 10 spontaneous 

15 human lymphoblast mutants not showing deletions. Northern blot analysis showed that the point 

16 mutations fell into four categories: normal size and amount of RNA, normal size but reduced 

17 amounts of RNA, reduced size and amounts of RNA, and no RNA. Sequence analysis of 

18 recombinant DNAs from hprt mRNA in formaldehyde-induced mutants showed a preferential 

19 AT to CG transversion at a specific site, with other changes represented to a lesser degree (Liber

20 et al., 1989).

21 Even in CHO cells formaldehyde has been shown to induce hprt mutations involving 

22 mostly single-base pair transversions mostly occurring at AT sequences, including three AT to 

23 TA at position 548 of exon 8 and two AT to CG and one GC to TA transversion at other sites 
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1 (Graves et al., 1996). In another study, formaldehyde-induced forward mutations to 

trifluorothymidine resistance in mouse lymphoma L5178Y tk± 2 cells both in the absence and 

3 presence of rat liver S9 (higher concentrations required for effect with S9). Both toxicity and 

4 mutagenicity were abolished when FADH was incorporated in the exposure medium (Blackburn

5 et al., 1991).

6 Formaldehyde-induced DPXs are removed in part through spontaneous hydrolysis and in 

7 part due to active repair processes (Quievryn and Zhitkovich, 2000). Inhibition of specific 

8 proteosomes in XP-A cells inhibited DPX repair, thereby supporting the role of enzymatic 

9 degradation (Quievryn and Zhitkovich, 2000). The half-life of formaldehyde-induced DPXs in 

10 human cell lines was consistent with the findings of Craft et al. (1987), ranging from 11.6 to 

11 13 hours (Quievryn and Zhitkovich, 2000). In the same report, removal of DPXs from human 

12 peripheral lymphocytes was much slower, with a half-life of 18.1 hours. This difference was 

13 primarily in slower active repair of DPXs, with a t1/2 of 66.6 hours for human lymphocytes 

14 versus 23.3 hours for human cell lines (Quievryn and Zhitkovich, 2000). 

15 Since DPX repair involves proteolytic removal of proteins from the DNA, Speit et al. 

16 (2000) hypothesized that single peptides or small peptide chains cross-linked to the DNA are 

17 critical to formaldehyde-induced mutation. However, these authors did not find significant 

18 difference in the induction and repair of DPXs in normal and DNA repair-deficient cell lines but 

19 observed increased susceptibility of the repair-deficient cell lines to formaldehyde-induced MN 

20 induction. In this study, a normal human cell line (MRC5CV1), a xeroderma pigmentosum cell 

21 line deficient in nucleotide excision repair (NER), and a Fanconi anemia cell line, which has a 

22 genetic defect leading to hypersensitivity towards DDXs, were exposed to 125, 250, and 500 μM 

23 formaldehyde for 2 hours. The authors suggest that more than one repair pathway is involved in 

24 the repair of cross-links and that the altered NER pathway has more severe consequences to 

25 formation of CAs than disturbed cross-link repair (Speit et al., 2000). 

26 The correlation of early DPX formation and mutation is at first counterintuitive since the 

27 cross-linking of protein to DNA inhibits DNA replication. Without active DNA replication, 

28 formaldehyde-DNA adducts and DPXs would not induce replication error and would be unlikely 

29 to result in a change in DNA sequence or mutation. Recent evidence indicates that residual 

30 peptides and short polypeptides that remain cross-linked to DNA after DPX removal may in fact 

31 be the cause of DPX-associated, formaldehyde-induced mutation (Speit et al., 2000). 

32 A study by Merk and Speit (1998) indicated that formaldehyde-induced DPXs did not 

33 result in direct gene mutations in the hprt locus of V79 Chinese hamster cells, leading the 

34 authors to speculate that formaldehyde was not mutagenic. Since, the hprt locus in the V79 

35 Chinese hamster cell line is primarily sensitive to point mutations and other studies show the 
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1 formation of deletion mutations by formaldehyde at the same locus in human lymphoblasts 

2 (Crosby et al., 1988), Merk and Speit (1998) concluded that the hprt mutation assay is insensitive 

3 to deletion mutations. 

4 Later, using the mouse lymphoma assay, Speit and Merk (2002) demonstrated that 

5 exposure to formaldehyde for 2 hours was mutagenic in a concentration-dependent manner in the 

6 L5178Y mouse lymphoma cells, which was mainly contributed by a strong increase in small 

7 colony mutants, suggestive of CAs (Speit and Merk, 2002). Detailed analysis of both 

8 spontaneous and formaldehyde-induced lesions indicates that recombination or deletion of DNA 

9 from the tk locus was primarily responsible for the loss of heterogeneity, thereby leading to the 

10 observed mutant phenotype. Therefore, it is believed that formaldehyde is mutagenic in the 

11 L5178Y cell mouse lymphoma system by a clastogenic mechanism rather than through point 

12 mutations. This finding is consistent with that of Craft et al. (1987), who demonstrated 

13 formaldehyde mutagenicity at the tk locus of TK6 cells, and also with the findings of Grafström 

14 et al. (1984), who demonstrated increased SSB formation in formaldehyde-exposed cell lines. 

15 Formaldehyde has also been shown to induce cell transformation in mouse embryo 

16 fibroblasts (Ragan and Boreiko 1981; Boreiko et al 1983; Frazelle et al 1983) . At low 

17 concentrations of 0.017 mM formaldehyde has shown to cause cell transformation in C3H10T1/2 

18 mouse cells (Ragan and Boreiko 1981) and hamster kidney cells in vitro (Plenser and Hansen 

19 1983).

20 More recently, Shaham et al. (2003) examined the frequency of DPXs and the incidence 

21 of mutant versus wild type p53 tumor suppressor genes in the peripheral blood lymphocytes of a 

22 cohort of workers exposed to formaldehyde. The adjusted mean levels of DPXs were greater in 

23 the lymphocytes of exposed subjects compared with those of unexposed subjects, and exposure 

24 to formaldehyde increased the likelihood of their having a higher level of pantropic p53 

25 (>150 pg/mL). The authors speculated on a possible causal relationship between DPXs and 

26 mutations in p53. Recio et al (1992) demonstrated point mutations in the p53 tumor suppressor 

27 gene in 45% (5 out of 11) of the primary nasal squamous cell carcinomas (SCCs) obtained from 

28 F344rats that were chronically exposed to 15 ppm formaldehyde for 2 years (Recio et al ., 1992). 

29 In summary, the results of in vitro experiments demonstrate the mutagenicity of 

30 formaldehyde. Mutagenicity is observed below levels of significant cytolethality in mammalian 

31 cell lines. Formaldehyde is clearly a DNA-reactive genotoxicant inducing lesions (DPXs) that 

32 show clastogenicity (SSBs, MNs, etc.). The experiments by Speit and Merk (2002) explore 

33 mechanistic links between DPXs, clastogenicity, and the observed locus-specific mutations in 

34 the mouse lymphoma in vitro testing system.

35
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1 4.3.4. In Vivo Mammalian Genotoxicity

2 4.3.4.1. Genotoxicity in Laboratory Animals

3 As discussed above, formaldehyde is clearly reactive at the POE in animal studies, 

4 resulting in increased DPXs in the nasal mucosa. Despite formaldehyde’s reactivity and 

5 mutagenicity in isolated mammalian cells, clear evidence of mutagenicity does not emerge from 

6 animal bioassays (Table 4-83). 

7 In a chromosomal analysis study (Fontignie-Houbrechts, 1981), formaldehyde given I.P. 

8 at 50 mg/kg to male Q strain mice and analyzed 8–15 days after treatment did not induce any 

9 chromosomal lesions in spermatocytes. Also, in another study from the same group (Fontignie10 Houbrechts et al., 1982), formaldehyde (30 mg/kg) given along with hydrogen peroxide (90 

11 mg/kg) as a mixture to male Q strain mice failed to produce significant increases in 

12 chromosomal lesions in the spermatogonia. 

13 In a different study Natarajan et al. (1983) failed to detect significant differences in MN 

14 induction in bone-marrow cells or CAs in spleen cells of male and female CBA mice given I.P. 

15 6.25, 12.5, and 25 mg/kg formaldehyde compared with saline-treated controls. However, the 

16 same study showed a positive induction of MNs and CAs in vitro. The authors suggest that the 

17 lack of genotoxicity in vivo may be due to the inability of formaldehyde to reach the target cells 

18 in sufficient quantity to induce biological effects. 

19 Kligerman et al. (1984) also found no difference in the incidence of SCEs or 

20 chromosome breakage in the peripheral lymphocytes of male and female F344 rats exposed to 

formaldehyde in air at 0.5, 6, or 15 ppm (0.61, 7.36, or 18.4 mg/m3 21 ) 6 hours/day for 5 days. 

22 However, in a different study (Migliore et al., 1989), clastogenic effects, such as increased MNs 

23 and CAs, were reported in GI epithelial cells of male Sprague-Dawley rats after oral exposures to 

24 200 mg/kg formaldehyde. In this study, micronucleated cells and nuclear anomalies were 

25 increased in a time-dependent manner in the stomach, duodenum, ileum, and colon of rats, and 

26 the mitotic index was unchanged for these cells compared with controls at 16, 24, and 30 hours. 

27 These clastogenic effects were seen without regenerative cell proliferation, supporting 

28 formaldehyde-induced mutations as primary effects of formaldehyde rather than secondary to 

29 regenerative cell proliferation.

30 Kitaeva et al. (1990) observed cytopathological and cytogenetic effects of formaldehyde 

chronic inhalation in 0.5 and 1.5 mg/m3 31 doses in the female rat’s germ and marrow cells, where 

formaldehyde-induced harmful effects were seen in germ cells at <1.5 mg/m3 32 doses, while the 

33 reliable clastogenic and cytogenetic effects on the marrow cells were induced even at the 

0.5 mg/m3 34 dose, suggesting differences among effects of small doses of formaldehyde on 

35 different cell systems.
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1 Table 4-83. Genotoxicity in laboratory animals.

2

Species/Strain Cells/Organ/Tumor Result References 

Cytogenetic Assays

Mice/Q strain

Chromosomal aberratoins (CA)

Spermatocyte - Fontignie-Houbrechts et 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

4 ‘+’ indicates a positive test result

5 - indicates a negative test result

6

7

8 Dallas et al. (1992) observed a slight increase (7.6 and 9.2%) in CAs in pulmonary lavage 

cells from male Sprague-Dawley rats exposed to 15 ppm (18.4 mg/m3 9 ) formaldehyde in air 

10 6 hours/day, 5 days/week for 1 or 8 weeks by inhalation compared with corresponding controls 

11 (3.5 and 4.8%), respectively. However, the small study, limited as it was to five animals/group, 

12 showed statistically significant increase at the highest dose tested (15 ppm) but not at lower 

13 doses (0.5 and 3 ppm). In the same study, no clastogenic effects were seen in bone marrow, 

14 which is consistent with formaldehyde acting primarily at the site of first contact.

15 Speit et al (2009) investigated the genotoxicity of formaldehyde in peripheral blood 

16 samples of Fischer-344 rats exposed to 0 to 15 ppm formaldehyde by whole-body inhalation for 

17 4 weeks (6 h/day, 5 days/week). In this study, the authors found no significant increase in the 

18 genotoxic assays such as comet assay with or without gamma-irradiation of blood samples (DNA 

19 migration as determined by tail movement or intensity), sister chromatid exchange (SCE) assay 
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1 and micronucleus test (MNT) compared to controls. However, rats given 50 mg/kg 

2 methylmethane sulfonate (MMS) by gavage for 4 hrs (positive control for Comet and SCE 

3 assays) or 10 mg/kg cyclophosphamide (CP) given twice orally (positive control for MNT) 

4 induced significant increase in genotoxicity in this study. The lack of genotoxicity in this study 

5 is not surprising since earlier studies by Casanova-Schimitz et al (1984a) have shown that 

6 formaldehyde does not cause toxicity to bone marrow possibly due to the inability of this 

7 chemical to reach the bone marrow. Although MMS and CP used in this study were positive in 

8 the genotoxicity assays, the data from positive controls can not be used for validation since the 

9 exposure routes of formaldehyde (inhalation) and the positive controls (oral) were different.

10 No animal studies have examined clastogenic effects of formaldehyde in nasal or 

11 respiratory epithelial cells. Therefore, it is unknown whether similar changes would occur in 

12 response to exposure to formaldehyde via inhalation. However, the negative finding in bone 

13 marrow cannot be considered definitive evidence on the question of the mutagenic potential of 

14 formaldehyde for cells present at the POE. With weak positive results in pulmonary lavage cells 

15 and clear clastogenicity in GI epithelial cells below exposures that trigger regenerative cell 

16 proliferation, the existing evidence, however incomplete, supports the concept of genotoxic 

17 action of formaldehyde at the POE.

18

19 4.3.4.2. Genotoxicity in Humans

20 The majority of the studies on the effects of formaldehyde in exposed humans have 

21 measured various cytogenetic endpoints, such as MNs, SCEs, or CAs in nasal and oral mucosal 

22 cells (considered to be in direct contact with formaldehyde) as well as peripheral lymphocytes. 

23 Since genotoxicity at the proximal sites (oral, nasal) can be readily linked to the reactive nature 

24 of formaldehyde, these studies are discussed first, noting where researchers also collected blood 

25 lymphocyte samples. A subsequent discussion is focused on results in blood lymphocytes.

26 Finally, the few studies that measured DPXs in exposed humans are discussed. Table 4-89 

27 provides a summary of human cytogenetic studies of formaldehyde.

28

29 4.3.4.2.1. Nasal, buccal, and oral mucosal cells. Epithelial cells of the URT and oral cavity are 

30 potential targets of formaldehyde’s DNA reactivity and genotoxicity. Several studies indicate 

31 that formaldehyde exposure results in measurable increases in SCEs, MN formation, and DPXs 

32 in nasal, buccal, and oral mucosal cells; however, these genotoxic effects vary with the type of 

33 exposure. Study quality, sample size, availability of exposure measurements, and assay 

34 methodology may in part contribute to variability in study findings. The studies fall into three 
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1 general categories: workers (industrial or professional), students and staff attending anatomy and 

2 mortuary science courses, and subjects in a controlled clinical trial.

3 Ballarin et al. (1992) observed significantly higher frequency of micronucleated cells in a 

4 formaldehyde exposed group in a plywood factory compared with controls (0.9 ± 0.47 versus 

5 0.25 ± 0.22, p < 0.01). In this study, the frequency of MNs and cytology of respiratory nasal 

6 mucosal cells was examined in 15 nonsmokers exposed to levels of formaldehyde that ranged 

between 0.1 and 0.39 mg/m3 7 (~0.32 ppm) for an average of 6.8 years. Exposed subjects were 

8 compared with age- and sex-matched controls. 

9 Ye et al. (2005) reported significant increases in MNs per thousand cells in nasal mucosal 

10 cells for 18 nonsmoking workers (2.70 ± 1.50) in a formaldehyde manufacturing plant in the 

11 Hugei province of China as compared with controls (1.25 ± 0.41). In addition, higher 

12 frequencies of SCEs in peripheral lymphocytes of workers were also reported (8.24 ± 0.89 versus 

13 6.38 ± 0.41). In this study, the average age of workers was 29 ± 6.8 years, the average duration 

at work was 8.5 years (range 1–15 years), and the reported 8-hour TWA was 0.985 mg/m3 14

15 (0.8 ppm). The control group consisted of 23 undergraduate students with an average age of 

19 ± 2.3 years. The 8-hour TWA in the student dormitories was 0.011 mg/m3 16 (9 ppb). A group 

17 of 16 waiters with an average exposure duration of only 12 weeks and an 8-hour TWA of 

0.107 mg/m3 18 (90 ppb) was also included in the study. The incidence of MNs and SCEs in the 

19 waiters was the same as that in controls. Overall, results from this study suggest that the 

20 genotoxic potential of high-level formaldehyde exposure may have occupational risks in long21 term exposure.

22 However, in a different study, Speit et al. (2007b) showed that formaldehyde did not 

23 induce MNs in exfoliated buccal mucosa cells of humans exposed up to a maximum of 1 ppm 

24 and a cumulative exposure of 13.5 ppm-hours over 2 weeks. In this study, volunteers exposed to 

25 formaldehyde in closely controlled conditions (4 hours/day for 10 days) with a complex 

exposure schedule, amounting to a cumulative total of 13.5 ppm-hours (16.6 mg/m3 26 -hours), were 

27 used. Samples of the buccal mucosa were taken from subjects 1 week before the start of the 

28 experiment, at the start of the experiment, at the conclusion of the series of exposures, and at 7, 

29 14, and 21 days after the completion of exposure. Thus, the subjects served as their own 

30 controls. Two thousand cells per data point were assessed for the frequency of MNs on slides 

31 that were coded by an independent quality assurance organization. As shown in Table 4-84, the 

32 frequency of MN formation was statistically unchanged from that in controls. The apparent 

33 slight increase in subjects evaluated at the conclusion of exposure was caused by frequencies of 

34 MNs in two subjects (5.0 and 4.5 MNs per 1,000 cells). The data as reported show a high 
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1 variability, where the SD approaches or exceeds the mean for each sample point, suggestive of 

2 data with an asymmetrical distribution.

3

4 Table 4-84. MN frequencies in buccal mucosa cells of volunteers exposed to 

5 formaldehyde

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 ± 0.38a

7 a 8 Statistically significantly different from control values (p < 0.05), as calculated by the authors.

9

10 Source: Speit et al. (2007b).

11

12 The best evidence of formaldehyde-induced clastogenic changes in peripheral 

13 lymphocytes is found in studies of anatomy class and mortuary class students. Since genetic 

14 damage accumulates with age, the studies in younger adults, where cells are analyzed before and 

15 after exposure, may have greater sensitivity and fewer confounding factors. 

16 Suruda et al. (1993) showed a 12-fold increase in the MN frequency of epithelial cells 

17 from the buccal area of the mouth in mortuary science students exposed to embalming fluids 

18 containing formaldehyde following an 85-day exposure period (Table 4-85). Overall, students 

were exposed to 0.33 ppm (0.4 mg/m3 19 ) formaldehyde as an 8-hour TWA on days when 

20 embalming was performed (an average of 6.9 embalmings). Blood, oral, and nasal samples were 

21 collected pre- and postexposure. As shown in Table 4-85, nasal epithelial MNs increased by 

22 22% (frequency of micronucleated lymphocytes increased by 28%). By contrast, SCE frequency 

23 decreased by 7.5% after formaldehyde exposure. 

24

25 Table 4-85. MN and SCE formation in mortuary science students exposed to 

26 formaldehyde for 85 days

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 ± 1.27a 0.50 ± 0.67 6.36 ± 2.03a 7.14 ± 0.89

28 a 29 Statistically significant (p < 0.05), as calculated by the authors.

30 Source: Suruda et al. (1993).

31
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1 Another group (Titenko-Holland et al., 1996) also reported a significant increase in MN 

2 frequency of buccal, but not nasal, epithelial cells from mortuary students exposed to embalming 

3 fluid. In this study, 28 out of 35 students were sampled before and after a 90-day embalming 

4 class. The mean formaldehyde exposure for the subjects providing data on buccal cell MNs was 

14.8 ± 7.2 ppm-hours (18.2 ± 8.8 mg/m3 5 -hours) for the entire 90-day period and 16.5 ± 5.8 ppmhours (20.3 ± 7.1 mg/m3 6 -hours) for students providing data on nasal cell MNs. Cells were 

recorded as having either whole chromosomes with centromeres (MN+ 7 ) or acentric fragments 

and no centromeres (MN– 8 ). Cells with multiple nuclei were present only in samples taken after 

9 exposure to embalming fluid. There was a ninefold increase in the MN frequency in buccal cells 

10 (p < 0.5) and only a twofold increase (p > 0.05) in nasal cells. In addition, there was a twofold 

increase in the MN+ 11 frequency in buccal cells (Table 4-86). The authors suggested that 

12 chromosomal breakage appears to be the primary mechanism of MN formation.

13

14 Table 4-86. Incidence of MN formation in mortuary students exposed to 

15 formaldehyde for 90 days

16

Sampling 

point

Buccal cells (n = 19) Nasal epithelial cells (n = 13)

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 ± 2.0a 1.1 ± 1.3 0.9 ± 1.1a 2.5 ± 1.3 1.0 ± 0.8 1.0 ± 0.6a

p value 0.007 0.08 0.005 0.20 0.31 0.03

17 a 18 Statistically significant at the level shown, as calculated by the authors.

19

20 Source: Titenko-Holland et al. (1996).

21

22

23 Ying et al. (1997), however, observed higher frequencies of MNs in the nasal exfoliative 

24 cells (3.85 ± 1.48 versus 1.20 ± 0.676, paired t-test, p < 0.001) and oral exfoliative cells (0.857 ± 

25 0.558 versus 0.568 ± 0.317, p < 0.001) after formaldehyde exposure, although there was no 

26 significant increase in the frequency of lymphocyte MNs (p > 0.05) in students exposed to 

27 formaldehyde in anatomy classes (three classes per week for 3 hours over an 8-week duration). 

28 In this study, blood samples and nasal swabs were collected before and after the study. The 

29 TWA concentration of formaldehyde in anatomy laboratories and student dormitories was 0.508 

± 0.299 mg/m3 and 0.012 ± 0.0025 mg/m3 30 , respectively, suggesting that nasal mucosa cells 

31 exposed through respiration are the primary target of formaldehyde-induced genotoxicity. 

32 In a different study (Ying et al., 1999), however, the same group showed that exposure to 

33 formaldehyde affected the composition of lymphocyte subsets (B cells, total T cells, T helper34 inducer cells, T cytotoxic-suppressor cells), but no significant difference was reported between 

35 lymphocyte proliferation rate and SCEs at the given levels and durations of formaldehyde 
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exposure. This study involved 23 nonsmoking students exposed to 0.508 ± 0.299 mg/m3 1

2 formaldehyde for a period of 8 weeks (3 hours , 3 times per week). 

3 Burgaz et al. (2002) reported significantly (p < 0.05) higher mean MN frequencies in 

4 buccal mucosal cells from shoe workers as well as anatomy and laboratory workers (0.62 ± 

5 0.45% and 0.71 ± 0.56%, respectively) compared with unexposed controls (0.33 ± 0.30%). In 

6 this study, the measured air concentrations of formaldehyde in the breathing zone of the anatomy 

and pathology laboratory workers were between 2 and 4 ppm (2.5 and 5 mg/m3 7 ). MN count per 

8 3,000 cells was measured in buccal smears from shoe workers and from anatomy and pathology 

9 staff, and eighteen male university staff were used as controls. 

10 In a critical review, Speit and Schmid (2006) examined data from studies that have 

11 reported the formation of MNs in nasal or buccal cells of persons either environmentally or 

12 occupationally exposed to formaldehyde. The authors identified a number of issues relating to 

13 study design, exposure regimen, and confounding factors, including MN levels in nasal and 

14 buccal cells well below established background levels, reports limited by the number of cells 

15 observed, variation in standard techniques, and non-concordance between buccal and nasal 

16 findings. However, the authors concluded that, despite these limitations, the weight of evidence 

17 supports the finding that formaldehyde may be genotoxic in human cells in direct contact with 

18 formaldehyde.

19

20 4.3.4.2.2. Peripheral blood lymphocytes. Mature lymphocytes are present at the POE as 

21 intraepithelial lymphocytes and within germinal centers in the mucosa. Because more 

22 lymphocytes may be available in the nasal mucosa than the oral mucosa, mouth versus nose 

23 breathing may contribute to variability in findings. Since some of the lymphocytes traffic around

24 the body, it is reasonable to find clastogenic effects in these relatively long-lived cells reflected 

25 in peripheral blood lymphocytes. Thus, lymphocytes proliferating in response to antigen would 

26 be more vulnerable to DNA reactivity of formaldehyde and to the clastogenic effects in general. 

27 A cytogenetic evaluation by Fleig et al. (1982) of 15 employees exposed for an average 

28 of 28 years in a formaldehyde manufacturing plant revealed no statistically significant increase 

29 in the frequency of CAs in peripheral blood lymphocytes compared with a matched control 

30 group. Likewise, in a different study (Thomson et al., 1984), no compound-related differences 

31 were evident in the frequency of CAs and MNs in lymphocytes from six pathology workers and 

32 five unexposed controls. 

33 Bauchinger and Schmid (1985) observed an increased incidence of CAs (dicentric and 

34 ring chromosomes) in the peripheral lymphocytes of 20 male paper mill workers and supervisors 

35 exposed to formaldehyde (average exposure of 14.5 years) compared with unexposed workers. 
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1 When workers and supervisors were analyzed separately, significant increases were only seen for 

2 supervisors. The average length of exposure for supervisors (n = 11) and workers (n = 9) was 

3 18.9 years and 7.2 years, respectively. Information regarding formaldehyde concentrations for 

4 the two groups was not provided. However, the incidence of SCEs among workers was actually 

5 slightly lower than among the 20 controls. In contrast, the frequency of SCEs in peripheral 

6 lymphocytes of 18 nonsmoking formaldehyde workers was increased over controls (8.24 ± 0.89 

7 versus 6.38 ± 0.41) (Ye et al., 2005) (described in Section 4.3.4.2.1). 

8 Vargová et al. (1992) observed that the percentage of aberrant cells and number of breaks 

9 per cell in the peripheral blood lymphocytes of formaldehyde-exposed workers was 3.08 and 

10 0.045 versus 3.6 and 0.080 in controls in a pressed board factory, respectively, suggesting both 

11 groups to be at an increased risk. However, normal unexposed population had only 1–2% 

12 aberrant cells. The authors also noted that the mitotic index was significantly decreased in 

13 exposed workers compared with controls. 

14 Kitaeva et al. (1996) evaluated the genotoxic effects of formaldehyde among 15 

15 industrially exposed workers and 8 academic laboratory instructors and observed an increase in 

16 the frequencies of CAs and MNs in the lymphocytes of exposed subjects compared with 

17 6 unexposed controls. 

18 Shaham et al. (1996, 1997) found significantly higher levels of DPXs and SCEs in 

19 peripheral blood lymphocytes of workers occupationally exposed to formaldehyde (physicians 

20 and technicians) compared with unexposed control workers. The authors also observed a linear 

21 relationship between years of exposure to formaldehyde and levels of DPXs and SCEs.

22 Formaldehyde-induced genotoxicity has also been reported in peripheral blood 

23 lymphocytes of anatomy class students and mortuary workers. Vasudeva and Anand (1996) did 

24 not observe significant differences in the incidences of CAs between the formaldehyde exposed 

25 students and the matched, unexposed controls. In this study, peripheral blood lymphocytes from 

26 30 medical students exposed to formaldehyde in a gross anatomy laboratory for 15 months with 

average exposures of less than 1 ppm (1.23 mg/m3 27 ) formaldehyde were used. 

28 He et al. (1998) used the cytokinesis-blocked MN (CBMN) assay to detect the frequency 

29 of micronucleated peripheral lymphocytes in 13 students exposed to formaldehyde during a 

30 12-week (10 hours/week) anatomy class. Sampling of breathing zone air showed a mean 

concentration of 2.37 ppm (3.17 mg/m3 31 ). Ten students from the same school, without exposure 

32 to formaldehyde, were used as controls. CAs and SCEs were observed in both groups, and there 

33 were significant increases (p < 0.01) in the frequencies of micronucleated cells and CAs in the 

34 formaldehyde-exposed group compared with the control group. 
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1 In a study involving 97 plasticware workers (34 males and 63 females) exposed to 0.5 to 

2 0.9 mg/m3 formaldehyde, 4.4 to 6.2 mg/m3 styrene and 0.5 to 0.75 mg/m3 phenol for 2 months 

3 to 25 years, Lazutka et al (1999) observed significantly higher CAs than controls (non-exposed 

4 donors matched by age and similar smoking habits as the exposed workers). Although workers 

5 with short and long exposures showed significant increases in the frequency of CAs, the

6 cytogenetic damage did not increase with exposure duration. 

7 Sari-Minodier et al. (2001), using the CBMN assay in anatomy/pathology laboratory 

8 workers, reported higher frequency of micronucleated peripheral blood lymphocytes than in 

9 matched controls. 

10 Shaham et al. (2002) observed a mean number of 0.27 SCEs per chromosome in the 

11 peripheral lymphocytes of an exposed cohort compared with 0.19 in controls (p < 0.01). This 

12 study involved 90 individuals employed in 14 hospital pathology laboratories and 52 unexposed 

13 controls.

14 Yu et al. (2005) reported dose-dependent increase in MNs and comet assay parameters 

15 (olive tail moment and comet tail length) in peripheral lymphocytes in 151 workers from two 

16 plywood factories compared with 112 unexposed controls. The TWA exposure level in the 

working environment was 0.1–7.88 mg/m3 17 (0.08–6.42 ppm) formaldehyde compared with a 

background level of <0.01 mg/m3 18 (<0.008 ppm) formaldehyde applicable to controls. In the 

19 comet assay, the authors observed olive tail moments averaging 0.93 (0.78–1.1), 3.03 (2.49–

20 3.67), and 3.95 (3.53–4.43) for control, low-, and high-exposure individuals, respectively. For 

21 the same subjects, comet tail lengths were 6.78 (6.05–7.6), 11.25 (10.12–12.5), and 12.59 (11.8–

22 13.43), respectively. In the CBMN assay, MNs/100 cells were 0.27 ± 0.13, 0.41 ± 0.25, and 

23 0.65 ± 0.36, respectively, for control, low-, and high-exposure individuals.

24 In a population of 18 workers exposed to formaldehyde at a plant in China, with a mean 

25 employment of 8.5 years (range 1 to 15 years), Ye et al (2005) examined nasal and lymphocytes 

26 for cytogenetic effects. This study also included a second group of 16 waiters who worked in a 

27 newly fitted ball room for 12 weeks with a low level exposure to formaldehyde from building 

28 material, tobacco smoke and furniture and a group of 23 college students as a control group. The 

29 background indoor air conentraton of 0.009 ppm formaldehyde was reported in students’ dorms. 

30 Significantly increased frequencies of MNs in the nasal mucosal cells and SCEs in peripheral 

31 blood lymphocytes were reported for the workers, but not the waiters in this study. 

32 Orsière et al. (2006) reported no apparent effect on the DNA damage in peripheral blood 

33 lymphocytes as assessed by a chemiluminescence microplate assay in pathology and anatomy 

34 laboratory workers (n = 59) before and after a 1-day exposure to formaldehyde. This study had 

35 59 exposed workers and 37 controls. However, with the CBMN assay, the authors reported 
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1 statistically significant differences in the frequency of binucleated micronucleated cells 

2 (1.69 ± 0.93 versus 1.11 ± 0.6%) in exposed versus control subjects. Discrimination between 

3 clastogenic and aneugenic events by using FISH with a pan-centromeric DNA probe resulted in a 

4 higher rate of binucleated micronucleated cells (1.91 ± 1.01 versus 1.19 ± 0.56% in controls) and 

5 showed that the frequency of centromeric nuclei was higher in the exposed group than in 

6 controls, though not significantly. Among the centromeric MNs, the frequency of MNs with 

7 only one centromere (C1+MN) was significantly greater in pathologists/anatomists than in 

8 controls (1.1 ± 0.62 versus 0.31 ± 0.24%, p < 0.001). The authors interpreted their data on 

9 monocentromeric nuclei in anatomists/pathologists as an indication that formaldehyde exposure 

10 might be associated with aneugenic (rather than clastogenic) events. 

11 Based on pooled analysis of two reports (Iarmarcovai et al., 2006a, b) (Table 4-87), MN 

12 frequency ratios in the peripheral lymphocytes of cancer patients, welders, and 

13 anatomists/pathologists were significantly increased compared with the corresponding controls. 

14 The data were taken from three biomonitoring studies by using CBMN/FISH. The incidence of 

15 MNs was scored and then evaluated further for the presence of centromere-negative MNs 

16 (C−MNs), centromere-positive MNs (C+MNs), and, for the latter case, those containing a single 

17 centromere (C1+MNs) and those containing two or more centromeres (Cx+MNs). Applying 

18 their findings to considerations of the aneugenic mechanism of action of formaldehyde, the 

19 authors hypothesized that the use of centromeric signals enables the identification of endpoints 

20 representing impaired chromosomal migration (with C1+MN formation) or centrosome 

21 amplification (with Cx+MN formation).

22

23 Table 4-87. Multivariate repression models linking genomic 

24 instability/occupational exposures to selected endpoints from the MN assay

25

Study populations Number MNa 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 27 Bolded values indicate statistical significance (p < 0.05).

28

29 Source: Iarmarcovai et al. (2006b).

30

31

32 Recently, Costa et al. (2008) observed a significant increase in the genotoxicity of 

33 formaldehyde-exposed pathological anatomy laboratory workers (n = 30) compared with 

34 controls (n = 30) in cytogenetic assays. In this study, the authors evaluated the level of exposure 
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1 to formaldehyde near the breathing zone of workers, and TWA of exposure was calculated for 

2 each subject, giving a mean level of exposure to be 0.44 ± 0.08 ppm (range: 0.04–1.58 ppm). As 

3 compared with control subjects, peripheral blood lymphocyte cultures of formaldehyde exposed 

4 workers showed significant increases in MN frequency (5.47 ± 0.76 versus 3.27 ± 0.69; p = 

5 0.003), SCEs (6.13 ± 0.29 versus 4.49 ± 0.16; p < 0.05), and comet assay as determined by tail 

6 length (TL) (60.00 ± 2.31 versus 41.85 ± 1.97; p < 0.05). In addition, Costa et al. (2008) 

7 observed a positive correlation between formaldehyde exposure levels and MN frequency (r = 

8 0.384; p = 0.001) and TL (r = 0.333; p = 0.005) (Table 4-88). However, polymorphic genes of 

9 xenobiotic metabolizing and DNA repair enzymes did not show any significant effect on the 

10 genotoxic endpoints. This is the lowest level of exposure to formaldehyde in the studies 

11 observed so far, wherein a clear indication of genotoxic effects of formaldehyde was 

12 demonstrated.

13

14 Table 4-88. Genotoxicity measures in pathological anatomy laboratory 

15 workers and controls

16

MN assay SCEs Comet assay

Mean MN ± SEM

(range)

Mean SCE ± SEM

(range)

Mean TL (μM) ± 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

18 Source: Costa et al. (2008).

19

20 4.3.5. Summary of Genotoxicity

21 Formaldehyde’s genotoxicity has been demonstrated in a variety of in vitro and in vivo 

22 test systems measuring a variety of genetic endpoints. Formaldehyde forms predominantly 

23 DPXs that are detected in cell-free systems and single cells in vitro. DPXs are formed in nasal 

24 epithelia but not in extra-nasal passages of rodents, which are completely removed within a day 

25 after formation. In vivo data in human and mammalian cells demonstrate that formaldehyde is 

26 genotoxic at the site of first contact, including cells of the mouth or the nose. DPXs are also 

27 detected in nasal and extra-nasal tissues of monkeys, suggestive of direct effects of formaldehyde 

28 in tissues that correspond to observed tumor sites (nasal and nasopharynx) in humans. In 

29 addition, this is used as a basis for cross-species comparison with humans. Formaldehyde-DNA 

30 adducts are labile and constitute a minor fraction of the DNA-reaction products and are less 

31 likely to play an important role in the genotoxicity of formaldehyde. 
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1 Formaldehyde clastogenicity has been demonstrated by the induction of SCEs, SSBs, 

2 MNs, and CAs in cultured mammalian cells. Formaldehyde induces mutations in salmonella and 

3 escherichia bacterial strains that contain an AT base pair at the primary reversion site that is used 

4 to detect oxidative compounds and cross-linking agents without metabolic activation by 

5 exogenous enzyme-activating systems. Formaldehyde induces mutations in cultured mammalian 

6 cells at levels that do not cause significant toxicity. Despite formaldehyde’s reactivity and 

7 mutagenicity in isolated mammalian cells, clear evidence of mutagenicity does not emerge from 

8 animal bioassays. 

9 Formaldehyde exposure causes differential induction of MNs in human nasal epithelial 

10 and buccal epithelial cells, which is significant in industrial exposure workers and students 

11 working in anatomy or mortuary science, respectively. However, recent data and data from 

12 larger studies support a finding of increased MNs in blood lymphocytes, although the issue 

13 remains controversial because of issues relating to study design, exposure regimen, and 

14 confounding factors, including MN levels in nasal and buccal cells well below established 

15 background levels, reports limited by the number of cells observed, variation in standard 

16 techniques, and non-concordance between buccal and nasal findings (Speit and Schmid, 2006). 

17 Several clastogenic effects, such as induction of MNs, SCEs, and CAs, were seen in human 

18 peripheral blood lymphocytes; however, the data are not very clear. Formaldehyde exposure also 

19 caused p53 mutations in rat nasal carcinomas with the expression of mutant p53 protein.

20 Overall, induction of DPXs as a predominant lesion in vitro and in vivo, clastogenicity, 

21 and mutagenicity with locus-specific mutations in nonhuman and human cells supports the 

22 concept of genotoxic action of formaldehyde at the POE.

23 A summary of the genotoxicity of formaldehyde in humans is presented in Table 4-89.

24

25 4.4. SYNTHESIS AND MAJOR EVALUATION OF NONCARCINOGENIC EFFECTS

26 The adverse health effects due to formaldehyde exposure have been extensively studied 

27 in humans and in animal models. Studies of human exposure include occupational exposures, 

28 environmental exposures, and clinical studies of intentionally exposed subjects (Section 4.1). 

29 Occupational exposures are primarily due to inhalation and dermal contact. Animal studies are 

30 available for a variety of routes of exposure, including inhalation, oral, dermal, and intravenous

31 and I.P. injections (Section 4.2). Additionally, as discussed in Chapter 3, in vitro studies address 

32 biological activity and the metabolic fate of formaldehyde.
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Table 4-89. Summary of human cytogenetic studes

Study population

N

Exposure time (years) Formaldehyde 

concentration (ppm)

Cytogenetic 

observations Reference

Range Mean Range Mean (TWA) CAs SCEs MNs

Analyses of nasal and/or buccal cells

Plywood workers 15 2–19 6.8 0.32–0.83 (1) + nasal Ballarin et al. (1992) Age and sex matched controls 15

Male mortuary science students 22 Buccal and nasal swabs 

taken before and after 

first 9 weeks of 

embalming course

0.1–4.3 1.4 + buccal

Suruda et al. (1993) – nasal

Female mortuary science students 7 – buccal

– nasal 

Mortuary science studentsa 28

Buccal and nasal swabs 

taken before and after 

first 9 weeks of 

embalming course

0.1–4.3 1.4

+buccalb

Titenko-Holland et al. (1996)

– nasal

Female anatomy faculty 8 NA 23.6 NA NA + buccal

Male anatomy faculty 5 25.6 – buccal Kitaeva et al. (1996)

Controls (Females) 7

Anatomy students 25 Buccal and nasal swabs taken before and after 

8-week anatomy course

0.06–1.06 (0.508) + buccal Ying et al. (1997)

+ nasal

Anatomy/ pathology staff 28 1–13 4.70 2–4 NA + buccal Burgaz et al. (2002) Controls (University staff) 18

Workers at a formaldehyde plant 18 1–15 8.5 0.8 + nasal Ye et al. (2005) Controls 23

Volunteers 21 10 days 13.5 ppm-hours – buccal Speit et al. (2007b)

Analyses of peripheral lymphocytes

Manufacturing workers 15 23–35 28 <5 1971 – Fleig et al. (1982) Age and sex matched controls 15 <1 later

Pathology workers 6 4–11 0.9–5.8 – Thomson et al. (1984) Controls 5
This document is a draft for review 

purposes only and does not constitute 

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Table 4-89. Summary of human cytogenetic studes

Study population

N

Exposure time (years) Formaldehyde 

concentration (ppm)

Cytogenetic 

observations Reference

Range Mean Range Mean (TWA) CAs SCEs MNs

Anatomy studentsc 8 10-week class 1.08–1.99d 1.2 + Yager et al. (1986)

0.08–0.6e 0.3

Papermakers 20 2–30 14.4 <3 NA +f – Bauchinger and Schmid (1985) Controls 20

Wood workers 25 <5 to <16 0.45–8.6 NA – Vargová et al. (1992) Controls 19

Male embalming students 22 Blood sampled before 

and after first 9 weeks 

of embalming course

0.15–4.3 1.4 – +

Suruda et al. (1993)

Female embalming students 7 – –

Manufacturing workers 15 10 Up to 4 NA + +

Anatomy faculty 8 17 NA ND Kitaeva et al. (1996)

Controls 6

Medial students 30 Sampled near end of 

15-month term

<1 NA – Vasudeva and Anand (1996) Controls 30 -

Anatomy students 13 12-week class 2.37g + + + He at al. (1998)

Controls (students) 10

Physicians 6 2-24 10 3.1-2.8 1.6 +

Technicians 7 2-25 15 + Shaham et al 1997

Controls (age matched/unexposed) 20

Anatomy students 23-25

Blood samples taken 

before and after 8-week 

anatomy course

0.06–1.06 (0.508)

-

– – Ying et al. (1999, 1997)

Female anatomy/pathology lab workers 10 1–16 8.9 1.2–15.1 NA + Sari-Minodier et al. (2001)

Controls (Women) 27

Hospital pathology workersh 90 1–39 15.4 0.04–0.7i 0.4 +j

Shaham et al. (2002)

Controls 52 0.72-5.6 2.24 +
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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Table 4-89. Summary of human cytogenetic studes

Study population

N

Exposure time (years) Formaldehyde 

concentration (ppm)

Cytogenetic 

observations Reference

Range Mean Range Mean (TWA) CAs SCEs MNs

Workers at a formaldehyde plant 18 1–15 8.5 0.8 + – Ye et al. (2005) Controls 23

Workers at two plywood factories 151 ND 0.08–6.42 + Yu et al. (2005) Controls 112

Pathology or anatomy workers 59 ND <0.1–20.4k 2k + Orsière et al. (2006)

Controls 37

Pathologists 18 ND 0.4–7.0k 2.3k + Iarmarcovai et al. (2006a, b)

Controls 18

Pathological anatomy lab workers 30 0.5–27 11 0.04–1.58 0.44 + + Costa et al. (2008) Controls (21 females and 9 males) 30

Plasticware workers 97 2 mo to 25 

yrs

0.5-0.9 

mg/m3

+ Lazutka et al 1999 Controls (non-exposed donors) 90

Wood workers 40 + Chebotarev et al 1986

Controls 22 NR NR

School children (1984) 20 0.26 +

Neri et al 2006

School children (1985) 16 0.11 +

School children (1986) 18 0.03

Controls (1984) 17 0

Preschool controls (1984) 24 0

Preschool children (1984) 13 0.17-0.3

Phenolformaldehyde resin workers 31

0.33-30 yr

0.41 +

Suskov and Sazonova 1982

Controls 74 0

a

Same population in Suruda et al. (1993) but different slides used. Nineteen complete slide sets for buccal analysis and 13 complete slide sets for nasal epithelial 

cell analysis.

b

Not dose related; both low- and high-exposure groups had same SCE increase. c

Each student sampled before and after 10-week anatomy class. d

Breathing zone samples.
This document is a draft for review 

purposes only and does not constitute 

Agency policy

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e

Room air samples.

f

Increase only in 11 supervisors. See text for details.

g

Average breathing zone during dissection procedure.

h

Exposed and controls from 14 hospitals. i

Low- and high-exposure groups established but numbers not provided.

j

Not dose related; both low and high groups had same SCE increase.

k

Described 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 = not 

applicable.
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1 Taken together, the human and animal studies support numerous health effects, not only 

2 at the POE as expected for a reactive gas but also on pulmonary function, neurobehavioral 

3 function, reproduction, development, immunomodulation, and sensitization (atopy, asthma). The 

4 discussion below provides a description of the adverse effects seen in each area, summarizing the 

5 data for both human and animal studies. MOA data are discussed where information regarding 

6 formaldehyde’s biological activity may be linked to the observed adverse health effects.

7

8 4.4.1. Sensory Irritation

9 Sensory irritation of the eyes, nose, and throat is reported in humans upon direct contact 

10 with formaldehyde gas during inhalation exposures (Holmström and Wilhelmsson, 1988; 

11 Ritchie and Lehman, 1987) and includes irritation resulting from acute exposures (Lang et al., 

12 2008; Yang et al., 2001; Krakowiak et al., 1998; Kulle, 1993; Green et al., 1989, 1987; Kulle et 

13 al., 1987; Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 

14 1984; Bender et al., 1983; Weber-Tschopp et al., 1977). Controlled exposures in inhalation 

15 chambers confirm the specificity of these responses to formaldehyde exposure and allow for 

16 assessment of these symptoms through both subjective and objective measures (Kulle, 1993; 

17 Holness and Nethercott, 1989; Green et al., 1987; Kulle et al., 1987; Sauder et al., 1986; Weber18 Tschopp et al., 1977). Eye irritation may be reported as itching, burning, and general discomfort. 

19 Tearing, redness of the eyes, and increased blink frequency are observed and may be quantified 

20 in exposure under controlled conditions (Lang et al., 2008; Yang et al., 2001; Andersen and 

21 Molhave, 1983; Weber-Tschopp et al., 1977; Schuck et al., 1966). Eye irritation appears to be 

22 the most sensitive endpoint in most individuals and may be observed after short exposures 

23 (195 minutes at 0.5 ppm: Lang et al. [2008]; 30 seconds at 1.65 ppm: Yang et al. [2001]).

24 Itching, burning, and discomfort of the nose, which may be accompanied by increased 

25 mucous production (runny nose), are reported by individuals exposed via inhalation (Krakowiak 

26 et al., 1998; Kulle, 1993; Green et al., 1987; Kulle et al., 1987; Weber-Tschopp et al., 1977). 

27 Throat irritation may also be described subjectively as itching and burning and is often 

28 accompanied by a cough (Krakowiak et al., 1998). Symptoms of eye and mucous membrane 

29 irritation are also reported in numerous rodent studies and support the health effects reported in 

30 humans (see Section 4.1.1.1). Although dermal contact may result in dermatitis and an apparent 

31 hypersensitivity reaction, symptoms do not present upon contact as sensory irritation. There are 

32 no human or animal data that assess sensory irritation from oral exposures.

33 The time to onset of sensory irritation symptoms and severity of the sensory irritation are 

34 a function of both the air concentration and duration of exposure. Additionally, nose and throat 

35 irritation becomes more prominent at higher exposures and longer duration of exposure (Kulle, 
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1 1993; Kulle et al., 1987). Controlled human laboratory exposures (Yang et al., 2001; Kulle, 

2 1993; Kulle et al., 1987; Cain et al., 1986; Andersen and Molhave, 1983) provide more direct 

3 exposure-response evidence for sensory irritation. These studies are limited to healthy 

4 nonsmoking individuals. Two studies (Cain et al., 1986; Andersen and Molhave, 1983) 

5 document discomfort and irritation of the eye in response to acute exposures as low as 0.25 ppm. 

6 Dose-response relationships are reported in a number of different ways: as an incidence of the 

7 reported symptom among subjects, as a score for severity of the symptom, or in some cases as a 

8 subjective measure, such as blink frequency for eye irritation.

9 Symptoms of sensory irritation, including eye irritation (burning watering, increased 

10 blinking), nasal irritation (rhinitis, itching/burning), throat/respiratory tract irritation (wheezing, 

11 coughing, phlegm production), have been reported in numerous worker cohorts. Occupational 

12 exposure environments include hospital and medical settings, students, and industrial workers 

13 (Takahashi et al., 2007; Takigawa et al., 2005; Krakowiak et al., 1998; Akbar-Khanzadeh et al., 

14 1994; Uba et al., 1989; Horvath et al., 1988; Schachter et al., 1987). Formaldehyde levels often 

15 vary in a work environment and peak as well as average exposures may be used to report 

16 occupational exposures. Although sensitive individuals often remove themselves from an 

17 irritating workplace (the HWE), eye, nose, and throat symptoms are still reported in this 

18 environment. Among workers in a plant where formaldehyde resins were used, those exposed to 

19 an average of 210 ppb formaldehyde reported increased symptoms above those in the control 

20 population (Holmström and Wilhelmsson, 1988).

21 These effects have been noted in students, particularly medical students, who are exposed 

22 to formaldehyde in cadaver labs. In a study of 24 formaldehyde-exposed anatomy students 

23 (personal breathing zone samples 0.73 ppm, range 0.49–0.93) (Kriebel et al., 1993), eye, nose, 

24 and throat irritation was present when comparing rates of irritation from the end or middle of 

25 class to before the start of class. Takahashi et al. (2007) showed that 143 medical students 

26 reported various symptoms (including eye and throat irritation) and that the percentage of 

27 students reporting symptoms increased between the beginning (measured after the first day of 

28 class) and the end of the course (2 months later). After the first day of class, approximately 35% 

29 of students reported eye soreness and about 15% reported throat irritation.

30 Sensory irritation has also been noted in occupational settings. Horvath et al. (1988) 

31 compared irritation symptoms between 109 workers at a particleboard manufacturing plant and 

32 264 workers at food plants. Eye, nose, and throat irritation were more common among the group 

in a particleboard manufacturing facility, exposed to a mean concentration of 0.40 mg/m3 33 . 

34 Similarly, Alexandersson and Hedenstierna (1988) reported that the frequency of eye, nose, and 

35 throat irritation was significantly greater (65.8%) in 38 workers exposed to formaldehyde and 
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1 solvents in lacquers as compared with 18 nonexposed individuals working at the same factory 

2 (16.7%). Holmström and Wilhelmsson (1988) conducted a study at a chemical plant and 

3 reported nasal and eye discomfort in 64 and 24%, respectively, of workers (n = 70) exposed to 

formaldehyde (range 0.05–0.50 mg/m3 with a mean of 0.26 mg/m3 4 ) versus 25 and 6%, 

5 respectively, in nonexposed desk clerks (n = 36). Holness and Nethercott (1989) reported 

6 significant increases in eye irritation (42 versus 21%) and nose irritation (44 versus 16%) among 

7 84 funeral service workers (active embalmers, >10-year experience) as compared with 

8 38 students and individuals from a service organization. The exposure concentration in both 

9 groups was 0.36 and 0.02 ppm, respectively.

10 Reports of similar symptoms are correlated to indoor residential exposures, providing 

11 exposure-response relationships for the general population in low-level chronic exposure 

12 scenarios. Ritchie and Lehnen (1987) surveyed residents in 2,000 homes classified as having 

13 formaldehyde concentration <0.1 ppm, 0.1–0.3 ppm, and >0.3 ppm. A LOAEL of 200 ppb was 

14 established from the results of Ritchie and Lehnen (1987). Liu et al. (1991) report irritant effects 

15 associated with formaldehyde exposure in mobile homes, where formaldehyde concentrations 

16 ranged from the 0.01 ppm detection limit to 0.46 ppm. Eye irritation (60%), nose/throat 

17 irritation (10–20%), or headache (<10%) were reported in residents.

18

19 MOA

20 The mucosae of the URT, oral cavity pharynx, and upper airways are complex tissues, 

21 where epithelial and goblet cells predominate. In addition, the nasal mucosa is highly enervated. 

22 The main nerves include the trigeminal nerve and olfactory sensory cells (olfactory epithelium, 

23 the vomeronasal organ, and the organ of Masera) (Feron et al., 2001). A possible MOA for 

24 sensory irritation includes formaldehyde-induced stimulation of the trigeminal nerve (though 

25 whether formaldehyde acts as a direct agonist is unknown). Trigeminal nerve stimulation in the 

26 nasal passages transmits signals to the CNS, which then sends efferent signals back to the nasal 

27 tissues, causing sensory irritation, and possibly systemically via vagal nerve stimulation, 

28 resulting in more systemic effects.

29 Animal studies are potentially useful models for understanding mechanisms of toxicity, 

30 especially where sufficient human data do not exist. While experimental animal studies provide 

31 a model of secondary effects, rodents also demonstrate RB, an effect not seen in humans. Thus, 

32 species that exhibit bradypnea (like mice and rats) may not be appropriate for assessing 

33 respiratory endpoints. The mechanism underlying RB includes formaldehyde binding to the 

34 sensory nerve endings of the trigeminal nerve, where signals travel to the CNS. The vagus nerve 

35 transmits the efferent signal to produce smooth muscle contraction. The animals become 
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1 inactive, their core temperatures decrease by several degrees C, and their respiratory rates and 

2 minute volumes decrease. However, this is not to say that trigeminal nerve stimulation is not an 

3 appropriate potential mechanism of action in other species or in humans. Since trigeminal nerve 

4 stimulation has been independently confirmed in species without RB, this mechanism may be a 

5 viable explanation for the observed effects.

6

7 4.4.2. Pulmonary Function

8 Workers chronically exposed to formaldehyde have exhibited signs of reduced lung 

9 function, such as BC, inflammation, and chronic obstructive lung disease. Lung function deficits 

10 have been reported in pre- versus post-shift measurements and as a result of chronic exposures 

11 (Pourmahabadian et al., 2006; Herbert et al., 1994; Malaka and Kodama, 1990; Alexandersson 

12 and Hedenstierna, 1989; Alexandersson et al., 1982). Decreases in spirometric values, including 

13 VC, FEV, FVC, and FEV/FVC, have been reported. Chronic studies (Pourmahabadian et al., 

14 2006; Herbert et al., 1994; Malaka and Kodama, 1990; Alexandersson and Hedenstierna, 1989; 

15 Alexandersson et al., 1982) also report increased respiratory symptoms, including cough, 

16 increased phlegm, asthma, chest tightness, and chest colds, in exposed workers. 

17 Students have also shown decrements in lung function that are associated with exposure 

18 to formaldehyde in laboratories. Kriebel and colleagues (1993) observed a 2% decrement in PEF 

19 in healthy students attending anatomy classes once per week and a 7.3% decrement in PEF in 

20 students with histories of asthma. The strongest pulmonary response was observed when 

21 examining the average cross-laboratory decrement in PEF in the first 2 weeks of the study 

22 (formaldehyde geometric average concentration of 0.73 ppm). These findings were corroborated 

23 by Kriebel et al. (2001) in which a similar study design was applied to another class of anatomy 

24 students.

25 Similarly, Akbar-Khanzadeh et al. (1994) compared pre- and postexposure pulmonary 

26 function among students before and after working 3 hours in a laboratory (n = 34). On average, 

27 FVC decreased by 1.4%, FEV3 decreased by 1.2%, FEV1/FVC increased by 1.6%, and FVC-25–

28 75% increased 2.5%. These average percent changes in the control group are –0.3%, 1.30%, 

29 2.31%, and 0.6% but were not statistically significant. In a follow-up study, Akbar-Khanzadeh 

30 and Mlynek (1997) recorded FEV values in 50 exposed students and 36 controls and reported a 

31 larger increase in lung function among controls when compared with cases after 1–3 hours of 

32 exposure that persisted after 3 hours after exposure termination. In a similar study, Fleisher 

33 (1987) reported that approximately 8% of students reported experiencing shortness of breath 

34 during the laboratory with formaldehyde exposure, but none of the students reported shortness of 
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1 breath in the laboratory session with no exposure. However, no objective measurements of 

2 formaldehyde exposure were used.

3 Unlike the study by Kriebel et al. (1993), Uba et al. (1989) did not find a change in 

4 pulmonary function over the course of the 7 months in a study of 96 anatomy laboratory 

5 students. These negative findings may be attributed to differential cross-shift exposures and to 

6 significant differences in FVC on exposed days.

7 Deficits in pulmonary function have been reported in occupational or residential exposure 

8 studies (Khamgaonkar and Fulare, 1991; Krzyzanowski et al., 1990; Malaka and Kodama, 1990; 

9 Alexandersson and Hedenstierna, 1989; Kilburn et al., 1985; Alexandersson et al., 1982). 

10 Krzyzanowski et al. (1990) documented a significantly decreased PEFR in children 

11 (298 children) who resided in homes with an average formaldehyde concentration of 26 ppb 

12 (maximum sample value of 140 ppb). Among adults, there was a statistically significant 

13 nonlinear relationship with decreased morning PEFR for formaldehyde concentration <40 ppb 

14 (Krzyzanowski et al., 1990). Similarly, Malaka and Kodama (1990) reported that an average 

15 8-hour TWA formaldehyde exposure of 1.13 ppm from area samples was associated with 

16 statistically significant decrements in FEV1, FEV1/FVC, and FEF25–75% compared with a referent 

17 population. Alexandersson and Hedenstierna (1989) investigated not only the acute effects of 

18 exposure across shift but also measured effects of exposure among some of the same workers 

19 that had been studied 5 years earlier (Alexandersson et al., 1982). Statistically significant 

20 decreases (p < 0.01) in FEV1/FVC and FEF25–75% were noted over the intervening five years in 

21 nonsmokers after correcting for aging. Similar decrements have been documented in laboratory 

22 workers in India (Khamgaonkar and Fulare, 1991) and in factory workers (Kilburn et al., 1985).

23 Alexandersson et al. (1982) reported only slight deficits in lung function 1 day following 

24 occupational formaldehyde exposure in a carpentry shop in Sweden, where the measured 

formaldehyde level was 0.36 ppm (0.47 mg/m3 25 ). In this case, subjects were compared with 

26 20 nonexposed workers.

27 Other studies have found no association between formaldehyde and lung function 

28 (Ostojic et al., 2006; Holness and Nethercott, 1989; Holmström and Wilhelmsson, 1988; Horvath 

29 et al., 1988). Ostojic et al. (2006) used an interesting measurement, “diffusing lung capacity” 

30 instead of decrements in FEV1 or similar measurements. Similarly, Nunn et al. (1990) assessed 

31 the decrease in FEV1 with age and showed no association between formaldehyde exposure and 

32 decreased FEV1. Franklin et al. (2000) did not report an association between FVC or FEV and 

33 the indoor concentrations of formaldehyde in children (ages 6–13), although there were signs of 

34 lower airway inflammation as measured by levels of exhaled NO (Franklin et al., 2000). 

35 Similarly, Main and Hogan (1983) did not observe differences between FEV1 or FVC at the end 
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1 of the 34 months between mobile home trailer workers compared with controls who did not work 

2 in trailers. The average exposure was reported as ranging from 0.12 to 1.6 ppm.

3 Occupational studies share certain limitations, including the potential for confounding by 

4 occupational co-exposures. Also, studies that did not report pre-shift pulmonary function as a 

5 percentage of expected function are less useful to assess potential chronic effects because, 

6 post hoc, it is difficult to calibrate for cross-study comparison due to lack of data on important 

7 pulmonary function determinants, such as age, gender, smoking status, height, and year of birth.

8 Controlled human studies and studies in nonhuman primates also document changes in 

9 formaldehyde-induced pulmonary dysfunction. Acute exposures of healthy non-asthmatic 

10 volunteers resulted in transient decreases in pulmonary function (e.g., decreased FEV1, FVC1, 

11 FEV3, specific airway conductance) (Green et al., 1987; Sauder et al., 1986). Green et al. (1987) 

12 noted differential responsiveness in formaldehyde-exposed subjects; some were responders while 

13 others were nonresponders. This differential response suggests susceptibility in certain subjects 

14 (Green et al., 1987).

15 Several animal studies document increased airway resistance and BC following 

16 inhalation exposure to formaldehyde (Nielson et al., 1999; Swiecichowski et al., 1993; Biagini et 

17 al., 1989; Amdur et al., 1960). A study using cynomolgus monkeys (Biagini et al., 1989) 

18 demonstrated that methacholine-induced BC can be similarly induced by acute formaldehyde 

19 exposure (10 minutes at 2.5 ppm). Thus, formaldehyde exposure simulated BC observed after 

20 methacholine challenge, but these effects may not occur by a similar MOA. Similar results were 

21 reported in guinea pigs (Swiecichowski et al., 1993; Amdur et al., 1960), rats (Ohtsuka et al., 

22 1997), and mice (Nielson et al., 1999).

23 Deficits in pulmonary function have been documented in occupational as well as 

24 controlled chamber human studies and have been corroborated in animal studies exposed to 

25 formaldehyde. However, some of these deficits are slight or transient. Some studies did not 

26 identify a statistically significant decrease in pulmonary function, and others did not observe any 

27 change at all. Pulmonary function alterations appear to be specifically tied to exposure regimen 

28 and may be reversible but remain, nevertheless, an important symptom often associated with 

29 exposure to formaldehyde.

30

31 MOA

32 Formaldehyde-induced inflammation of the airways may contribute to observed 

33 decreases in measures of pulmonary function. Even short-term inflammatory reactions could 

34 reduce the effective diameter of the conductive airways, resulting in lower respiratory volumes in 

35 a number of functional tests. Formaldehyde-induced trigeminal nerve stimulation contributes to 
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1 airway inflammation, which in turn would reduce airway function. Chronic exposures may 

2 result in increased sensitization or chronic inflammatory responses, which could contribute to the 

3 effects seen in the worker and residential populations.

4 Formaldehyde-induced pulmonary function deficits may also be in part a result of smooth 

5 muscle contraction in repose to trigeminal nerve stimulation. Trigeminal nerve stimulation 

6 transmits signals to the CNS. The resulting efferent signal from the vagal nerve produces 

7 smooth muscle contraction and may result in decreased pulmonary function. Efferent signaling 

8 has also resulted in release of substance P and other neuromodulatory compounds, which may 

9 contribute to BC and sensitization of pulmonary responses (asthma, atopy).

10

11 4.4.3. Hypersensitivity and Atopic Reactions

12 Sensitization to inhalational chemical exposure may manifest as an allergic or asthmatic 

13 response that is characterized by BC or BHR. This sensitization may be a result of immune 

14 involvement, as in the case of hypersensitivity, or a neurogenic sensitization, where a chemical 

15 may directly stimulate inflammation. Asthma is a specific manifestation of IgE-mediated 

16 hypersensitivity, characterized by BHR and airway inflammation, resulting in lower airway 

17 obstruction (Fireman, 2003; Kuby, 1991).

18 A variety of hypersensitivity reactions have been reported following exposure to 

19 formaldehyde. Rashes and skin reactions have been reported in some individuals after dermal 

20 exposures to formaldehyde. Increased expression of Th-2 cytokines in the lymph nodes of mice 

21 given dermal applications of formaldehyde does indicate the involvement of an immune 

22 component to the observed sensitization (Dearman et al., 2005; Hilton et al., 1998; Arts et al., 

23 1997). However, the response does not appear to be IgE mediated (Arts et al., 1997; Lee et al., 

24 1984). Gorski et al. (1992) observed an increase in formaldehyde-mediated neutrophil burst in 

25 dermatitis patients exposed in a controlled chamber study and suggests a putative role of 

26 oxidative stress and reactive oxygen species (ROS).

27 Inhalation exposure has been associated with increased asthmatic responses in asthmatics 

28 in occupational settings. While few available case reports of bronchial asthma suggest direct 

29 respiratory tract sensitization to formaldehyde gas (Lemiere et al., 1995; Burge et al., 1985; 

30 Hendrick et al., 1982; Hendrick and Lane, 1977, 1975), a greater body of human data provides 

31 evidence of an association between formaldehyde exposure and exacerbation of asthmatic 

32 responses in compromised individuals (Kriebel et al., 1993) and particularly in children 

33 (Rumchev et al., 2002; Garrett et al., 1999; Krzyzanowski et al., 1990). Increased asthma 

34 incidence reported after inhalation exposure to formaldehyde led to a NOAEL of 30 ppb 
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1 (Rumchev et al., 2002). An increased frequency of respiratory symptoms associated with 

2 asthmatic responses and formaldehyde exposure led to a LOAEL of 30 ppb (Garrett et al., 1999).

3 Exacerbation of response after formaldehyde exposure has been demonstrated in animal 

4 studies as well. Sadakane et al. (2002) demonstrated that formaldehyde exposure exacerbated 

5 sensitization and challenge with Der f and suggested that formaldehyde exposure may aggravate 

6 eosinophilic infiltration and goblet cell proliferation that accompanies allergic responses. 

7 Several animal studies report increased airway resistance and BC due to inhalation exposures to 

8 formaldehyde (Nielsen et al., 1999; Swiecichowski et al., 1993; Biagini et al., 1989; Amdur, 

9 1960). Changes in pulmonary resistance were observed as early as 10 minutes after exposure 

10 (Biagini et al., 1989), and reported effect levels ranged from 0.3 to 13 ppm. BHR is commonly 

11 associated with allergic Type I hypersensitivity reactions but is not sufficient to demonstrate that 

12 an agent induces Type 1 hypersensitivity.

13

14 MOA

15 The MOA underlying this response has not been elucidated. Formaldehyde-induced IgE 

16 production has been reported in some studies (Vandenplas et al., 2004; Wantke et al., 1996a). 

17 Other studies suggest that this effect does not appear to be immunogenic in nature (Fujimaki et 

18 al., 2004; Lee et al., 1984). Although formaldehyde exposure has been reported to alter cytokine 

19 levels and immunoglobulins in some experimental systems (Fujimaki et al., 2004a; Ohtsuka et 

20 al., 2003), these immunomodulatory effects do not support immunogenically mediated type 1 

21 hypersensitivity.

22 These decrements may be mediated via neurogenic potentiation (Sadakane et al., 2002; 

23 Riedel et al., 1996; Tarkowski and Gorski, 1995). Tarkowski and Gorski (1995) suggest that 

24 formaldehyde may increase permeability of respiratory epithelium and destruction of 

25 immunologic barriers. Tachykinin NK1 receptor and various neuropeptides (NGF and substance 

26 P) have been implicated in formaldehyde-induced sensitization and lend weight of evidence to a 

27 neurogenic MOA (Van Schoor et al., 2000; Ito et al. 1996).

28

29 4.4.4. Upper Respiratory Tract Histopathology

30 Several studies in occupational workers have reported increased squamous cell 

31 metaplasia and reduced mucociliary clearance in nasal and buccal swabs from humans 

32 occupationally exposed to formaldehyde (Holmström et al., 1989; Holmström and Wilhelmsson, 

33 1988). Evidence of genotoxic effects include increased MNs and CAs in nasal and buccal 

34 epithelial cells from both workers and students exposed to formaldehyde (Ying et al., 1997; 
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1 Titenko-Holland et al., 1996; Suruda et al., 1993) and suggest a potential association between 

2 genotoxicity and altered histopathology.

3 Numerous animal experimental studies in multiple strains of rats, mice, hamsters, rabbits, 

4 and monkeys describe formaldehyde-induced URT pathology (Fló-Neyret et al., 2001; Roemer 

5 et al., 1993; Reuzel et al., 1990; Monticello et al., 1989; Zwart et al., 1988; Wilmer et al., 1987; 

6 Morgan et al., 1986b, 1983; Swenberg et al., 1986; Buckley et al., 1984). Effects are first 

7 observed in the anterior respiratory mucosa and progress through the nasal passages with 

8 increasing exposure concentration and time. The first observed effect includes damage to the 

9 mucociliary apparatus of the nasal passages in response to formaldehyde. Studies conducted 

10 both in vivo and in vitro demonstrate that formaldehyde disrupts mucus flow and ciliary beat that 

11 are dependent on concentration and duration of exposure. Mucociliary apparatus deficits have 

12 been recorded even after 18 hours of recovery following formaldehyde exposure. The 

13 breakdown of the mucociliary apparatus may allow for increased infection and allow the 

14 underlying epithelium to come into contact with exogenous chemicals.

15 Formaldehyde is highly reactive and may impact all cells in the nasal mucosa, including 

16 epithelial cells (ciliated, columnar, and cuboidal), goblet cells, sensory neurons, and 

17 intraepithelial lymphocytes. The histologic changes of these processes have been described in all 

18 laboratory animals examined and progress from the anterior nares to the posterior regions of the 

19 nasal passages, including the ETs and olfactory epithelium if the concentration and duration of 

20 exposure are great enough.

21 Humans and nonhuman primates have significantly less complex nasal passages than 

22 rodents. Formaldehyde has lower peak flux in human nasal tissues compared with rodents, 

23 which are obligate nose breathers, but will penetrate more deeply into the human respiratory tract 

24 than in rodents, since humans lack the autonomic RA response. Additionally, humans may 

25 switch to mouth breathing in the presence of an irritant gas, thus bypassing the sensitive nasal 

26 passages and increasing the tissue dose in the mouth and throat. These differences have been 

27 demonstrated by using nonhuman primates where, at comparable concentrations, tissue 

28 pathology and increased cell proliferation progressed further into the respiratory tract than in 

29 rodents (Monticello et al., 1989). Nonhuman primates share common structural respiratory 

30 components and patterns of breathing and do not have a reflex autonomic apnea response.

31 Despite the anatomical and physiological differences in breathing patterns and different 

32 exposure parameters between humans and rodents, similar toxic effects are reported in tissues at 

33 the POE in humans and laboratory animals. Several occupational studies have reported 

34 increased squamous cell metaplasia in nasal and buccal samples in response to formaldehyde 

35 exposure (Ballarin et al., 1992; Boysen et al., 1990; Holmström et al., 1989), paralleling the 
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1 histologic effects seen in experimental animal studies. A few human epidemiology studies 

2 suggest increased NPCs (see Section 4.5) as well as oral/buccal tumors in response to 

3 formaldehyde exposure (Shangina et al., 2006; Laforest et al., 2000).

4 The observed formaldehyde-induced URT toxicity is related to its high reactivity and 

5 solubility. Moreover, additional interspecies differences in the surface area and configuration of 

6 the nasal passages and upper airways will influence the areas of high formaldehyde flux in POE 

7 tissues.

8

9 MOA

10 Formaldehyde-induced damage to the mucociliary apparatus of the nasal passages may 

11 occur because formaldehyde may disrupt mucus flow and ciliary beat that is dependent on 

12 concentration and duration of exposure. Formaldehyde reacts with the mucosal glycoproteins 

13 and thus may contribute directly to the breakdown of the mucus layer. As formaldehyde reaches 

14 the cells of the pseudostratified epithelium in the nasal passages, it exerts a range of effects from 

15 direct damage to cell membrane, intracellular proteins, and DNA to alterations in GSH pools and 

16 increased ROS. Adaptive effects include increased mucus flow and goblet cell proliferation as 

17 well as the transition of respiratory epithelium to more insensitive cuboidal cells. With 

18 continued exposure at sufficient concentration, squamous metaplasia develops, creating a 

19 protective layer of keratinized cells. Gradually, this damage exceeds the cell’s ability to 

20 compensate for and repair damage; chronic nasal lesions develop, and the cells die both through 

21 general necrosis as well as programmed cell death, depending on the severity of the cellular 

22 damage (Monticello et al., 1989; Swenberg et al., 1983).

23 Genotoxic effects have been reported in nasal and buccal lesions taken from both workers 

24 and students exposed to formaldehyde (Ying et al., 1997; Titenko-Holland et al., 1996; Suruda et 

25 al., 1993). MN formation occurs in the more sensitive pseudostratified epithelium of the nasal 

26 passages, nasopharynx, and upper airways, since there is only one layer of epithelial cells that are 

27 constantly regenerating. However, the genotoxicity observed in buccal cells is more difficult to 

28 explain, since buccal basal cells are usually covered by protective keratinized cell layers. Cuts, 

29 sores, or other buccal lesions would increase basal epithelial cells’ vulnerability to direct 

30 exposure to formaldehyde.

31

32 4.4.5. Toxicogenomic and Molecular Data That May Inform MOAs

33 Over the past several years, studies have begun to examine the effects of formaldehyde 

34 exposure on gene and protein expression. These include studies on in vivo and in vitro changes 

35 in the global expression of mRNA (transcriptomics) and proteins (proteomics) in the tissues and 
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1 cells of humans and rodents exposed to formaldehyde. Currently (2009), nine “-omics” studies 

2 from five research groups are available. These studies are summarized in Section 5.2 and are 

3 evaluated and discussed in the context of their relevance to informing MOAs and the dose4 response characterization briefly here.

5 In 2002, EPA released the Interim Policy on Genomics (U.S. EPA, 2002c), which 

6 addresses how to use genomic data in regulatory decision making. Although the policy 

7 encourages research in genomics, it places limits on its use, stating that genomic data alone are 

8 not sufficient as a basis for decision making. These data thus cannot currently be utilized as the 

9 critical effect in a chemical risk assessment but can be utilized in a weight-of-evidence approach 

10 on a case-by-case basis. The Science Policy Council developed a white paper entitled Potential 

11 Implications of Genomics for Regulatory and Risk Assessment Applications at EPA (U.S. EPA, 

12 2004). This report described three areas where genomic data might be applied in risk assessment 

13 at EPA: MOA analysis, susceptible population, and mixtures assessments. The genomic data on 

14 formaldehyde thus may be applied to a discussion of MOA.

15 Toxicogenomics studies have investigated the gene and protein expression changes 

16 resulting from formaldehyde exposure in a variety of respiratory tissues, including nasal tissue 

17 (Andersen et al., 2008; Thomas et al., 2007; Hester et al., 2005, 2003), and, in lung tissue (Lee et 

18 al., 2008, 2007; Sul et al., 2007; Im et al., 2006) used human tracheal cell lines to study genomic 

19 changes after exposure to formaldehyde in vitro. Unfortunately, these studies are not directly 

20 comparable because different gene chip technology platforms were used in different tissues, in 

21 both in vivo and in vitro study designs. In general, the gene and protein expression changes 

22 reflect changes in apoptotic pathway genes, oxidative stress, and tissue remodeling. Andersen et 

23 al. (2008) concluded that there was a threshold level where exposure to formaldehyde (6 ppm) 

24 does not elicit changes in nasal epithelium of F344 rats. Overall, Andersen et al. (2008) 

25 concluded that genomic changes were no more sensitive than tissue responses and that 

26 formaldehyde, being an endogenous chemical, is well handled until some threshold is achieved 

27 when toxicity rapidly ensues with genomic and histologic changes. At about 6 ppm, this largely 

28 involves tissue remodeling (and protection), but regenerative hyperplasia occurs at higher doses. 

29 Andersen et al. (2008) conclude that there is a threshold where exposure to formaldehyde does 

30 not elicit changes in F344 nasal epithelial tissue over the duration examined in this study (i.e., 

31 15 days). Andersen et al. (2008) argue that this is consistent with bioassays that indicate no 

32 tumor formation in rodents below 6 ppm formaldehyde.

33 The primary conclusion in the Andersen et al. (2008) paper is that genomic changes, 

34 including those suggestive of mutagenic effects, did not temporally precede or occur at lower 

35 doses than phenotypic changes in the tissue. The authors stated as follows:
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1 “The genomic signatures related to these transitions are for cell membrane and 

2 extracellular components, then inflammation and cellular stress, and eventually 

3 apoptosis. Importantly, these hierarchical models indicate that the tissue 

4 responses at low dose concentrations are qualitatively different from those at high 

5 concentrations and linear extrapolations or extrapolations that specify similar 

6 modes of action at high and low doses would be inappropriate.”

7

8 4.4.6. Noncancer Modes of Actions

9 Noncancer health effects of interest span numerous organ systems and include 

10 reproductive and developmental effects, neurological/neurobehavioral effects, and a complex 

11 interaction between inflammation and immune and adverse pulmonary function. To date, no 

12 -omics studies have examined changes in reproductive tissue or altered gene expression in 

13 developing animals. In regard to neurological/behavioral effects, one study (Lu et al. [2008], 

14 described in Section 4.1.1.6) has reported elevations in the mRNA for NMDA receptor subunits 

15 in brain homogenates following exposure to 2.4 ppm. Hester et al. (2003) reported a significant 

16 increase in NMDA receptor subunit transcripts, along with other neuropeptide genes, in nasal 

17 tissue. Together, these changes may relate to formaldehyde-induced sensory irritation and, 

18 perhaps, changes throughout the brain.

19 In regard to inflammatory, immune, and pulmonary effects, transcript and protein 

20 changes in rodent tracheal tissue and lung tissue indicate that exposure to 3 to 38 ppm 

21 formaldehyde results in genes involved in oxidative stress and cell proliferation and may 

22 additionally increase airway ADH3 levels (Lee et al., 2008; Sul et al., 2007; Yi et al., 2007; Im et 

23 al., 2006; Yang et al., 2005). Together, these data provide evidence for adverse pulmonary 

24 effects that may exacerbate or facilitate asthma.

25 In lung tissue, Yang et al. (2005) identified three proteins up regulated and one protein 

26 down regulated following 15 days of exposure to about 28 ppm formaldehyde. None of the 

27 proteins corresponded with transcript changes reported by Sul et al. (2007). Interestingly, Sul et 

28 al. (2007) reported that only two transcripts were significantly up regulated in the lung in 

29 response to 5–10 ppm formaldehyde, while 19 were down regulated. In this regard, it is worth 

30 considering that changes in proteins may not relate to their regulation but rather to their overall 

31 percent composition in a cell (relative to other protein changes) before and after exposure. In 

32 addition to transcript changes, Sul and colleagues (2007) reported DNA damage and lipid 

33 peroxidation and noted that the observed down regulation of GR would facilitate oxidative stress, 

34 while the down regulation of phospholipase A2 (PLA2) might represent a mechanism for 

35 mitigating lipid peroxidation. It is worth noting that an increase in either of these genes could 

36 also be argued to support similar conclusions (i.e., that GR is up regulated to increase GSH 

37 levels and that PLA2 up regulation explains lipid peroxidation); this highlights the problem with 
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1 interpreting these data. Nevertheless, these studies indicate adverse effects in the rodent lung in 

2 response to 5–30 ppm formaldehyde. 

3 In a hypothesis-driven study by Yi et al. (2007), formaldehyde exposure was shown to 

4 increase lung ADH3 levels. Several studies indicate that allergic responses and hyperreactivity 

5 are uncoupled and may relate to ADH3 expression and activity. Que et al. (2005) has shown 

6 that, in a rodent asthma model, ADH3 knockout mice exhibit similar signs of inflammation but 

7 are protected from bronchoconstriction. Lino dos Santos Franco et al. (2006) provided evidence, 

8 in rodents, that formaldehyde may induce inflammatory responses (e.g., leukocyte infiltration in 

9 the lung) through neurogenic mechanisms but that bronchial tone is mediated by NO. The latter 

10 effect is likely to be mediated by S-nitrosoglutathione GSNO and thus influenced by ADH3. 

11 Interestingly, the single nucleotide polymorphisms (SNPs) that Wu et al. (2007) reported as 

12 linked to other polymorphisms in the promoter region was the one demonstrating protection 

13 against asthma. Hedberg et al. (2001) reported that at least one SNP in the promoter region 

14 reduced ADH3 expression. Together, these data indicate that reduced ADH3 expression might 

15 lower GSNO turnover and bronchial tone, thereby reducing signs of asthma. In this regard, it is 

16 conceivable that wheezing and bronchoconstriction are the symptoms that lead to medical 

17 intervention and not the inflammation per se. Thus, while ADH3 polymorphisms may not cause 

18 asthma, ADH3 polymorphisms may influence hyperresponsivity and the likelihood of asthma 

19 diagnosis. This is discussed further in Section 4.6 on susceptible populations. Formaldehyde 

20 has been shown to accelerate GSNO breakdown (Staab et al., 2008a; Yi et al., 2007); thus, 

21 pulmonary responses to formaldehyde may represent a balance between mechanisms that induce 

22 NO (i.e., inflammation) and those that terminate GSNO (i.e., ADH3). 

23 In regard to -omics changes in blood samples, the apparent limited distribution of 

24 formaldehyde may suggest that these changes are secondary to effects at sites of contact but 

25 could also indicate systemic distribution. As noted elsewhere in this report, bradypnea can 

26 induce changes in dosimetry as well as changes in core body temperature and blood gases 

27 (hypoxia itself induces hypothermia in rodents, and thus the reduction in minute volume and gas 

28 changes may both contribute to hypothermia). These physiological responses (hypothermia and 

29 hypoxia) surely induce changes in gene expression. Observed gene and protein changes in the 

30 blood following formaldehyde exposure could also relate to irritation and inflammation at sites 

31 of contact. In this regard, Im et al. (2006) reported changes in cytokines indicative of Th-2 

32 responses. Altogether, the authors identified 32 proteins altered in the plasma of rats exposed to 

33 formaldehyde. Although no coherent mechanisms are apparent from these changes, the authors 

34 posited that they could serve as biomarkers for formaldehyde exposure. The concordance of 

35 such changes across species remains to be demonstrated.
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1 Li et al. (2007) identified dose-response relationships for six genes in human blood 

2 samples that were putatively associated with formaldehyde exposure. Three of these genes are 

3 reported to inhibit apoptosis and were posited as supporting in vitro data by Tyihak et al. (2001); 

4 however, Li and colleagues (2007) did not report any increase in blood cell count or in Hs 680.Tr 

5 cell counts in vitro (i.e., these changes were not phenotypically linked to changes in cell kinetics 

6 or hematology). However, these findings are not inconsistent with those of Hester et al. (2003) 

7 that indicated no significant increase (or decrease) in nine genes involved in apoptotic pathways. 

8 Finally, serum and glucocorticoid-induced protein kinase 1 (SGK1) was elevated in blood 

9 samples and was posited to relate to possible inflammatory and immune responses.

10

11 4.4.7. Immunotoxicity

12 Results from studies that evaluated the immunotoxicity of formaldehyde are mixed. For 

13 example, most human studies that investigated systemic immune effects by measuring increases 

14 in formaldehyde-specific IgE are negative (Doi et al., 2003; Kim et al., 2001; Palcynski et al., 

15 1999; Krakowiak et al., 1998; Wantke et al., 1996; Grammer et al., 1990). Vandenplas et al. 

16 (2004) reported a transiently positive increased formaldehyde-specific IgE titer in occupationally 

17 exposed workers. In contrast, Thrasher et al. (1990, 1988, 1987) reported positive 

18 formaldehyde-specific IgE titers in small (six to eight person) case studies of exposed workers, 

19 and Carraro et al. (1997) reported elevated IgE titers in smokers. Grammer et al. (1990) did not 

20 report any differences in albumin IgE in formaldehyde-exposed workers compared with controls. 

21 In a residential study, Pross et al. (1986) found that formaldehyde insulation in homes had no 

22 effect on tested human immunologic parameters.

23 One study suggests that immune system parameters are perturbed by formaldehyde 

24 exposure. Lyapina et al. (2004) reported decreased immune resistance in all 29 workers exposed 

25 to formaldehyde. This effect was associated with decreased neutrophil respiratory burst activity. 

26 A LOAEL of 700 ppb was established.

27 Results from animal studies are mixed as to whether formaldehyde causes 

28 immunotoxicity. Leach et al. (1983) reported systemic immunomodulation in F344 rats that was 

29 attributed to formaldehyde exposure, but the formaldehyde effects on measures of humoral and 

30 cell-mediated immunity were not confirmed in B6C3F1 mice (Dean et al., 1984). Jakab et al. 

31 (1992) detected no differences in phagocytic ability of alveolar MPs from mice after 

32 formaldehyde exposure. Formaldehyde-exposed rats that were injected with pneumococcus 

33 antigen or tetanus toxoid produced antibodies in amounts similar to nonexposed, infected control 

34 animals (Holmström et al., 1989b).
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1 However, specific immune parameters appear to be affected by formaldehyde exposure. 

2 For example, increased host resistance and hydrogen peroxide release from peritoneal MPs were 

3 reported and confirmed (Adams et al., 1987; Dean et al., 1984) and suggest a putative role for 

4 ROS. Increased host resistance may be mediated by formaldehyde-induced chronic 

5 inflammation and respiratory mucosal damage that causes an up regulation in MPs and therefore 

6 increases host immunity. Jakab et al. (2002) reported reduced pulmonary bacterial resistance in 

7 mice after exposure to formaldehyde, as determined by increased bacterial loading. This result 

8 contrasts with Dean et al. (1984) and is attributed to differential exposure regimens and 

9 experimental design.

10

11 Mode of action

12 Circulating immune cells present in the mouth and upper airways, such as intraepithelial 

13 lymphocytes, direct a local inflammatory response but may also contribute to systemic responses 

14 through secreted cytokines and soluble factors released into the bloodstream (Togias, 1999).

15 Altered host resistance and hydrogen peroxide release from peritoneal MPs were reported 

16 and confirmed (Adams et al., 1987; Dean et al., 1984) and suggest a putative role for ROS. 

17 Indeed, increased neutrophilic ROS have been associated with formaldehyde-induced dermatitis 

18 (Gorski et al., 1992), and, conversely, decreased neutrophil respiratory burst activity has been 

19 shown in workers with history of formaldehyde-induced respiratory tract inflammation (Lyapina 

20 et al., 2004). Oxidative stress may occur directly as a result of formaldehyde exposure or as a 

21 secondary consequence to inflammatory responses.

22

23 4.4.8. Effects on the Nervous System

24 There is considerable evidence that formaldehyde exposure causes adverse effects on the 

25 nervous system following inhalation at relatively low exposure levels but little or no information 

26 regarding a possible mechanism of action for these effects. Data regarding adverse effects on the 

27 nervous system following oral exposure are very limited, reflecting a data gap in this area. 

28 Relevant data in animals and humans for several types of neurological endpoints, following 

29 inhalation exposure, are summarized below. 

30

31 4.4.8.1. Irritant Threshold Detection

32 Humans are exquisitely sensitive to the irritant properties of formaldehyde, as has been 

33 discussed previously (see Section 4.1.1.1). Animal data confirm the irritant properties of this 

34 compound at very low concentrations (Wood and Coleman, 1995).

35
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1 4.4.8.2. Behavioral Effects

2 Limited data in humans, as well as more robust data in animals, provide evidence of 

3 behavioral changes following exposure to formaldehyde at levels as low as 0.1 ppm. Studies in 

4 animals have found effects that persist for days to weeks after termination of exposure. In spite 

5 of significant limitations, the available human data are consistent with the animal findings.

6 Several types of behavior have been evaluated in animals following formaldehyde 

7 exposures. The most consistent findings, demonstrated by multiple laboratories and in multiple 

8 species, have been changes in motor activity, habituation, and learning/memory task 

9 performance. Motor activity and habituation have been evaluated under a variety of exposure 

10 conditions, using both rats and mice. Consistent decreases in activity have been seen in adult 

11 animals (Malek et al., 2004, 2003 a, b; Usanmaz et al., 2002). Senichenkova (1991) and 

12 Sheveleva (1971) also found changes in motor activity in offspring following in utero exposure, 

13 including decreased habituation in juvenile rats exposed in utero. Decreased performance in 

14 learning and/or memory paradigms have been seen in multiple laboratories, also in both rats and 

15 mice (Lu et al., 2008; Malek et al., 2003c; Pitten et al., 2000).

16 Data from controlled human exposures are very limited, but studies have shown 

17 decreased performance in addition tasks and reaction time tasks following acute exposures to 

18 formaldehyde (Lang et al., 2008; Bach et al., 1990). In contrast, Andersen and Molhave (1983) 

19 indicated they found no change in performance on several types of tasks (including addition, 

20 multiplication, and card punching) following acute exposure to volunteers, but supporting data 

21 were not provided.

22 Data for humans are also available from epidemiology studies of individuals who were 

23 occupationally exposed to formaldehyde. A variety of neurobehavioral deficits, including lack 

24 of concentration and loss of memory, disturbed sleep, impaired balance and dexterity, and 

25 changes in mood, were identified (Kilburn et al., 1987, 1985). However, most of the individuals 

26 evaluated in these studies were also occupationally exposed to other solvents, raising questions 

27 regarding possible confounding of the results due to multiple exposures. In addition, the 

28 formaldehyde exposure information provided in the studies is not sufficient to permit a reliable 

29 dose-response assessment for the effects identified. The types of effects seen in humans in the 

30 available epidemiology studies are, however, consistent with those seen in available animal 

31 studies.

32 In general (and noting the differences in exposure paradigms and types of tasks), 

33 behavioral effects in animals and humans appear to occur at similar exposure levels. Animal 

34 studies demonstrated LOAELs as low as 100 ppb following acute or repeated exposures (Malek 

35 et al., 2003b, c); human controlled exposure studies have found effects in that same range, with 
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1 LOAELs of approximately 300 ppb following acute exposures (Lang et al., 2008; Bach et al., 

2 1990).

3

4 4.4.8.3. Neurochemistry, Neuropathology, and Mechanistic Studies

5 Limited data are available regarding neurochemical and neuropathological sequelae of 

6 formaldehyde exposure. Studies from one laboratory (Sorg et al., 2004, 2001) have suggested 

7 that behavioral sensitization to formaldehyde is linked to alterations in HPA control of 

8 corticosterone and changes in mesolimbic dopamine pathways. Neurochemical changes in 

9 response to formaldehyde exposure have also been documented in other laboratories (Fujimaki et 

10 al, 2004b; Hayashi et al., 2004). Some of these data appear to be conflicting, and there are no 

11 definitive data supporting a specific mechanism for formaldehyde effects on the nervous system 

12 at this time. Neuropathological data are also limited, although data from one laboratory 

13 (Sarsilmaz et al., 2007; Aslan et al., 2006) suggest a concern for changes in brain structure in 

14 neonatal rats following exposure at 6 or 12 ppm. No human data are available that address these 

15 endpoints. However, a prospective cohort study of nearly one million people followed for 15 

16 years reported strongly significant dose-response associations between death from ALS and 

17 exposure to formaldehyde of a known duration, with longer exposures associated with greater 

18 risk (Weisskopf et al., 2009). This large, well-designed prospective cohort study strongly 

19 supports the causal association of neuropathological effects in humans following long-term 

20 formaldehyde exposure.

21

22 4.4.8.4. Summary

23 Overall, there is strong evidence that formaldehyde exposure via inhalation may cause 

24 adverse effects on nervous system function in experimental animals at relatively low levels of 

25 exposure (LOAELs as low as 100 ppb). Although human data regarding neurotoxicity following 

26 formaldehyde inhalation are limited, available data provide support that the types of effects seen 

27 in humans are similar to those found in animal studies. Evidence from available human 

28 controlled inhalation exposure studies indicates that humans may be affected at doses similar to 

29 those used in animal studies; however, the human data are extremely limited.

30 There are insufficient data to evaluate the potential for neurotoxicity following oral 

31 exposure to formaldehyde. Limited evaluations of brain weight or histopathology in available 

32 chronic or subchronic oral studies found no evidence of formaldehyde-induced changes (Til et 

33 al., 1989, 1988; Tobe et al., 1989; Johannsen et al., 1986). However, reliable studies examining 

34 nervous system function or focused studies of neuropathology following oral exposure to 

35 formaldehyde are not available.
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1 4.4.8.5. Data Gaps

2 Major data gaps were found regarding the evaluation of changes in nervous system 

3 structure or function following formaldehyde exposure by both the inhalation or oral routes.

4 With respect to inhalation exposure, none of the available human studies resulted in data 

5 sufficient to conduct a reliable dose-response assessment for changes in nervous system function. 

6 Most of the available animal inhalation studies used short exposure durations (acute or short7 term), precluding a reliable evaluation of neurotoxicity following chronic exposure. Available 

8 data for neurodevelopmental exposures are also quite limited, consisting of evaluation of 

9 neuropathology in only one brain region and functional evaluations focused only on changes in 

10 motor activity.

11 Major data gaps also exist regarding neurotoxicity following oral exposure, with no 

12 relevant human data and extremely limited animal data. Available oral exposure studies were 

13 insufficient to permit a reliable evaluation of the potential for neurotoxicity following oral 

14 exposure to formaldehyde.

15

16 4.4.9. Reproductive and Developmental Toxicity

17 A number of studies have been identified that indicate an effect of formaldehyde 

18 exposure on reproductive and developmental outcomes. Human data are described in Section 

19 4.1.1.7, and animal studies are addressed in Section 4.2.1.7 of this document.

20

21 4.4.9.1. Spontaneous Abortion and Fetal Death

22 Several epidemiologic studies reported increases in risk of spontaneous abortion 

23 following maternal occupational formaldehyde exposure (Taskinen et al., 1999, 1994; John et al., 

24 1994; Seitz and Baron, 1990; Axelsson et al., 1984). While these finding have been questioned 

25 (Collins et al., 2001b), upon careful examination, none of the principal biases in epidemiologic 

26 studies, including information bias, selection bias, and confounding, appear to be more likely 

27 causes of these reported findings than the conclusion that they may reflect an underlying causal 

28 process. While each of these occupational studies focused on women who were co-exposed to 

29 formaldehyde and other chemicals, the occupational groups were quite different and had 

30 different sets of co-exposures. The woodworkers in the Taskinen et al. (1999) study were 

31 potentially co-exposed to organic solvents related to painting and lacquering, dusts, and phenols, 

32 none of which was shown to be an independent predictor of adverse risk. The cosmetologists 

33 studied by John et al. (1994) were co-exposed to hair dyes, bleach, alcohol-based disinfectants, 

34 and chemicals specific to services, such as fingernail sculpturing, but, in analyses that were 

35 specifically adjusted for other work exposures and their potentially confounding effects, the 
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1 investigators reported an increased risk for the use of formaldehyde-based disinfectants. The 

2 laboratory workers studied by Axelsson et al. (1984) were potentially co-exposed to a wide range 

3 of solvents, but the miscarriage rate was highest among those exposed to formaldehyde. For a 

4 potential confounder to entirely explain an observed effect of another exposure, it must be more 

5 strongly associated with the adverse outcome. It does not appear that the collective results of 

6 formaldehyde exposures associated with increased risk of spontaneous abortion—often in spite 

7 of exposures being crudely measured—can be explained by information bias or confounding.

8 Taken together, these findings are consistent with an adverse effect of formaldehyde 

9 exposure on the risk of pregnancy loss. The single study with the strongest quantitative 

10 assessment of that risk is Taskinen et al. (1999), and the results presented are of sufficient quality 

11 to support quantitative risk assessment by using the LOAEL/NOAEL approach.

12 This study was a well-designed population-based case-control study that appears to have 

13 been well executed and appropriately analyzed. The study population of Finnish women was 

14 well defined and adequately selected to allow for meaningful comparisons of health effects 

15 between individuals with different levels of exposure to formaldehyde. The participation rate 

16 was 64%, which is low enough to raise a concern about the potential for selection bias. 

17 However, the authors noted that selection bias has not influenced the results of other 

18 reproductive epidemiology studies reporting results on smoking, irregular menstruation, and 

19 earlier miscarriages, which are known to lengthen the time to pregnancy (Bolumar et al., 1996; 

20 Sallmén et al., 1995; Baird and Wilcox, 1985). Furthermore, there is no evidence to support 

21 conjecture that an individual’s decision to participate in this study would be differential with 

22 respect to their workplace formaldehyde exposures while being nondifferential with respect to 

23 the other exposures of interest, including organic solvents, wood dust, and phenols. Since the 

24 women who chose to participate in this study were not likely to be aware of the specific 

25 hypotheses under investigation nor could they have known the formaldehyde exposures that were 

26 independently estimated by an industrial hygienist, selection bias is not a likely explanation for 

27 the findings of adversity.

28 Data on pregnancy history, including spontaneous abortions, were collected by 

29 questionnaire. Spontaneous abortion is the most common adverse outcome of pregnancy (Klein 

30 et al., 1989), and retrospective self-report of spontaneous abortion has been found to match well 

31 with prospectively collected reproductive histories (Wilcox and Horney, 1984). Many 

32 spontaneous abortions, however, are missed with self-reporting, with the magnitude likely 

33 exceeding 25%, but only rarely do women self-report false positive events (Wilcox and Horney, 

34 1984). The effect of such an undercount is to cause a bias towards the null when the likelihood 

35 of undercounting is unrelated to formaldehyde exposure. The implication is that the observed 
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1 association of increased risk of spontaneous abortion associated with occupational exposure to 

2 formaldehyde may be an underestimation of the true risk.

3 The findings by Taskinen et al. (1999) of reduced fertility and increased risk of 

4 spontaneous abortion are internally consistent and coherent with other reports of increased risk 

5 of pregnancy loss associated with exposure to formaldehyde (John et al., 1994; Taskinen et al., 

6 1994; Seitz and Baron, 1990; Axelsson et al., 1984). Absent evidence of alternative explanation 

7 for these findings, it is concluded that exposure to formaldehyde is associated with pregnancy 

8 loss and diminished fertility.

9 In animal studies, Sheveleva (1971) noted an increase in preimplantation loss in rats 

10 exposed to 0.04 and 0.4 ppm formaldehyde by inhalation on GDs 1–19, and Kitaev et al. (1984) 

11 observed evidence of degeneration in harvested embryos on GD 2, following 4 months of 

12 maternal inhalation exposure to 0.41 ppm formaldehyde in rats. In a second series of tests 

13 reported in Kitaev et al. (1984), female rats were exposed to 0.41 and 1.22 ppm formaldehyde for 

14 4 months to test the hypothesis that the embryo degeneration could have been the result of 

15 disrupted reproductive hormone levels in the dams. Ovarian weight and blood levels of LH were 

16 increased at 0.41 ppm (but not at 1.22 ppm), and significantly increased levels of FSH were 

17 observed at 1.22 ppm. Kitaev et al. (1984) proposed that effects at the 0.41 ppm might be related 

18 to disruption of the hypothalamic-pituitary axis and that at the higher exposure level (1.22 ppm) 

19 frank toxic effects to the embryo were observed. The increased FSH levels at 1.22 ppm may also 

20 be indicative of hormonal perturbations induced by formaldehyde exposure that could affect 

21 pregnancy maintenance in humans. The finding of treatment-related increased preimplantation 

22 loss in rats appears to support the evidence of spontaneous abortion in the epidemiologic data. In 

23 addition, a dominant lethal study in rats by Odeigah (1997) identified significant 

24 postimplantation loss following pre-mating I.P. formaldehyde exposures to males, suggesting a 

25 potential MOA involving germ cell toxicity. Nevertheless, a number of developmental toxicity 

26 studies in rats did not report treatment-related embryolethality following gestation exposures to 

27 formaldehyde. These included inhalation studies by Martin (1990), Saillenfait et al. (1989), and 

28 Kilburn and Moro (1985), a series of studies by Gofmekler and Bonashevskaya (1969), 

29 Gofmekler (1968), and Pushkina et al. (1968), and studies by Senichenkova and Chebotar (1996) 

30 and Senichenkova (1991). It is noted, however, that, to the extent that these studies evaluated 

31 embryonic or fetal death, the observations were conducted late in gestation and the studies may 

32 not have been designed to detect the preimplantation losses as observed in Kitaev et al. (1984) 

33 and Sheveleva (1971). Additionally, a number of the reports for these studies did not include 

34 sufficient details to engender a high degree of confidence in the reported results. Fetal death was 
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1 also not observed in oral studies with formaldehyde in beagle dogs (Hurni and Ohder, 1973) and 

2 rats (Seidenberg and Becker, 1987).

3

4 4.4.9.2. Congenital Malformations

5 The effect of occupational exposures to formaldehyde on the incidence of congenital 

6 malformations was examined by Dulskiene and Gražulevičiene (2005), Taskinen et al. (1994), 

7 and Hemminki et al. (1985). Results were mixed.

8 In animal studies, the most frequently observed structural anomaly noted following 

inhalation exposures to formaldehyde during gestation was a delay in fetal (i.e., 1st 9 stage) testes 

10 descent (Senichenkova and Chebotar, 1996; Senichenkova, 1991; Kilburn and Moro, 1985), 

11 although similar findings were not reported by Saillenfait et al. (1989) or Martin (1990) in what 

12 appear to be well-conducted prenatal developmental toxicity studies. No study in the available 

database specifically examined the 2nd 13 stage of postnatal testes descent in pups. Thus, there is no 

14 evidence to determine if the observed effect represented a developmental delay or if it was 

15 related to disruptions in male reproductive tract ontogeny, which is dependent on normal levels 

16 of fetal testicular testosterone and on the expression of insulin-like hormone-3 (insl3) in fetal 

17 Leydig cells (Klonisch et al., 2004). Senichenkova (1991) observed an increased incidence of 

18 other organ anomalies following formaldehyde exposure during gestation; however, the 

19 anomalies are not characterized in the report. Alterations on fetal organ weights and/or size were 

20 noted in several studies (Kilburn and Moro, 1985; Gofmekler, 1968), but it is difficult to 

21 ascertain if these findings represented agenesis, hypoplasia, or evidence of systemic organ 

22 toxicity. Histopathologic evaluation of pup tissues following maternal gestational exposures to 

23 0.01 and 0.81 ppm formaldehyde was conducted by Gofmekler and Bonashevskaya (1969), 

24 revealing reduced glycogen content in the myocardium, the presence of iron in hepatic Kupffer 

25 cells, and a positive Schiff reaction in the basement membrane (indicating functional alterations 

26 in the renal tubule) at both exposure levels.

27

28 4.4.9.3. Low Birth Weight and Growth Retardation

29 A population-based study (Gražulevičiene et al., 1998) reported an association between 

30 atmospheric formaldehyde exposure and low birth weight, with an adjusted OR of 1.37 (95% CI: 

31 0.90–2.09).

32 A number of inhalation studies in rats identified reduced fetal weight as an adverse 

33 outcome of in utero formaldehyde exposure and are supportive of the association noted in 

34 humans. In a study that exposed pregnant rats to formaldehyde during GDs 6–20, Saillenfait et 

35 al. (1989) observed significantly decreased male and female fetal rat weights (78 and 81% of 
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1 control values, respectively) at 40 ppm formaldehyde. In a study that exposed the dams from 

2 GDs 6–15, Martin (1990) found decreased fetal weights at exposure levels of 5 and 10 ppm. In 

3 both studies, observations of reduced or delayed skeletal ossification (i.e., the thoracic vertebrae 

4 in Saillenfait et al. [1989] and the pubes and ischia bones in Martin [1990]) were consistent with 

5 the fetal weight deficits. Kilburn and Moro (1985) also reported fetal body weight decreases in 

6 rats at an inhalation exposure level of 30 ppm. Conversely, increased fetal body weight as 

7 compared with controls (generally considered to be non-adverse) was noted by Gofmekler 

8 (1968) and Pushkina et al. (1968) at maternal formaldehyde exposure levels of 0.1 and 0.81 ppm 

9 administered for approximately 2–3 weeks prior to mating and then throughout gestation. 

10 Increased fetal weight was also noted in rats by Senichenkova (1991) and Senichenkova and 

11 Chebotar (1996) following maternal exposures to 0.41 ppm formaldehyde throughout gestation.

12 Studies that assessed the effects of oral administration of formaldehyde on development 

13 are quite limited. The only oral study identified that found a treatment-related effect on offspring 

14 growth was a study using beagle dogs (Hurni and Ohder, 1973). In this study, formaldehyde was 

15 administered at doses of 3.1 or 9.4 mg/kg-day in the feed during gestation, and pup weight 

16 decrements at postnatal week 8 were 6.3 and 12% in the low- and high-dose groups, respectively.

17

18 4.4.9.4. Functional Developmental Outcomes (Developmental Neurotoxicity)

19 Indications of effects on the developing nervous system were observed in several rodent 

20 studies, although no similar epidemiologic findings in children were identified. These studies 

21 (Sarsilmaz et al., 2007; Aslan et al., 2006; Weiler and Apfelbach, 1992; Senichenkova, 1991; 

22 Sheveleva, 1971) are described in detail in Section 4.2.1.6. In the studies by Aslan et al. (2006) 

23 and Sarsilmaz et al. (2007), neonatal rats were exposed to formaldehyde for 30 days at 6,000 or 

24 12,000 ppb. Decreases in the volume of discrete areas of the brain were observed at both 

25 exposure levels in both studies, and, additionally, decreased cell numbers were noted in a region 

26 of the hippocampus in the Sarsilmaz et al. (2007) study. Weiler and Apfelbach (1992) exposed 

27 juvenile rats to 0.25 ppm formaldehyde for 130 days or adult rats to 0.5 ppm formaldehyde for 

28 90 days. Olfactory thresholds measured in this study were significantly higher in the rats that 

29 had been exposed as juveniles than in those that had been exposed only as adults. Sheveleva 

30 (1971) observed alterations in spontaneous mobility in 1- and 2-month-old pups from dams that 

31 had been exposed to formaldehyde at 0.04 or 0.4 ppm throughout gestation. In the Senichenkova 

32 (1991) study, maternal rats were exposed to 400 ppb formaldehyde during GDs 1–19, and 

33 functional observational testing was conducted on the juvenile offspring. Changes in open-field 

34 motor activity, exploratory activity, and habituation were observed in the offspring.

35
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1 4.4.9.5. Male Reproductive Toxicity

2 A number of laboratory animal studies have reported effects of formaldehyde exposure 

3 on male reproductive system endpoints. These effects include decreased testes weight, changes 

4 in Leydig cell quantity and quality, degeneration of seminiferous tubules, decreased testosterone 

5 levels, alterations in biomarkers of toxicity in the testes, and alterations in sperm measures 

6 (Galilapour et al., 2007; Xing et al., 2007; Zhou et al., 2006; Özen et al., 2005, 2002; Sarsilmaz 

7 et al., 1999; Odeigah, 1997; Majumder and Kumar, 1995; Chowdhury et al., 1992; Til et al., 

8 1989, 1988; Tobe et al., 1989; Johanssen et al., 1986; Maronpot et al., 1986; Cassidy et al., 1983; 

9 Appelman et al., 1982; Guseva, 1972). Following concurrent exposures to formaldehyde in air 

10 and drinking water for 6 months, Guseva (1972) found decreases in testicular nucleic acid levels. 

11 In a study conducted by Chowdhury et al. (1992), rats were administered I.P. injections of 

12 formaldehyde for 30 days, and evidence of decreased testes weight, serum testosterone levels, 

13 and Leydig cell quality was observed. Sarsilmaz et al. (1999) followed up on these findings 

14 (exposing male rats to formaldehyde via inhalation at 10 and 20 ppm for 4 weeks) and found 

15 decreases in Leydig cell quantity and the percentage of cells with normal nuclei. Hypothesizing 

16 that the reported decreases in Leydig cell quality may have been the result of oxidative stress and 

17 damage, Özen et al. (2002) evaluated biomarkers of such changes and found that testicular zinc 

18 and copper levels were decreased and iron levels were increased following exposures of adult 

19 male rats to 10 and 20 ppm formaldehyde for 4 or 13 weeks. Additionally, relative testes weight 

20 was decreased in a dose- and duration-dependent manner, although this effect had not been 

21 observed by Sarsilmaz et al. (1999). Özen et al. (2005) noted decreased serum testosterone 

22 levels, decreased seminiferous tubule diameters, and increased levels of heat shock protein in 

23 spermatogonia, spermatocytes, and spermatids of rats following 91 days of exposure to 10 ppm 

24 formaldehyde. A study by Golalipour et al. (2007) observed decreased numbers of testicular 

25 germ cells, altered sprmatogenesis, and reduced seminiferous tubular diameter and epithelial 

26 height in rats following 18 weeks of formaldehyde inhalation exposure; the severity of the 

27 seminiferous tubule pathology was positively correlated to the number of hours/week of 

28 exposure. Zhou et al. (2006) found decreased testis weight, atrophy of seminiferous tubules, 

29 edematous interstitial tissue, and alteration of epididymal sperm count, morphology, and motility 

30 in rats after 2 weeks of formaldehyde exposure at 8 ppm. Abnormal sperm were also observed in 

31 mice by Xing et al. (2007) after 13-weeks of inhalation exposure at 16.9 ppm, and Cassidy et al. 

32 (1983) reported sperm abnormalities in rats following a single oral dose of 200 mg/kg. 

33 Additionally, Majumder and Kumar (1995) observed significantly reduced sperm count, motility, 

34 and viability following 30 days of I.P. injection of 10 mg/kg-day formaldehyde to male rats. 

35 Also in this study, the ability of formaldehyde to affect sperm parameters was confirmed with in 
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1 vitro testing. A study conducted by Odeigah (1997) demonstrated epididymal sperm count and 

2 morphology abnormalities following five I.P. doses of ≥0.125 mg/kg formaldehyde and 

3 additionally identified dominant lethal effects (decreased live embryos and increased dead 

4 implants) following mating of treated male rats with untreated females.

5 Although Til et al. (1989) reported low incidences of Leydig cell tumors in 

6 formaldehyde-treated rats in a chronic drinking water study, no alterations in testes weight or 

7 histopathologic lesions of the testes were observed in subchronic inhalation studies conducted by 

8 Appleman et al. (1982) or Maronpot et al. (1986) or in subchronic or chronic oral studies by 

9 Johanssen et al. (1986), Til et al. (1988), or Tobe et al. (1989).

10 No epidemiologic studies have identified an association between formaldehyde exposure 

11 and alterations in the male reproductive system (e.g., see Ward et al. [1984]).

12

13 4.4.9.6. Female Reproductive Toxicity

14 The available database for the assessment of the effects of formaldehyde exposure on the 

15 female reproductive system was limited. In addition to the findings of spontaneous abortions, as 

16 described above, Taskinen et al. (1999) examined fecundability in a cohort of healthy female 

17 wood-processing industry workers and found that conception was significantly delayed in 

18 women who were occupationally exposed to formaldehyde. The FDR, a ratio of average 

19 incidence densities of pregnancies for exposed female employees compared with unexposed 

20 female employees, was lower in women exposed to mean formaldehyde levels of approximately 

21 0.33 ppm (range: 0.15–1.00 ppm) compared with controls (adjusted FDR = 0.64 [95% CI: 

22 0.28-0.92]). An FDR <1.0 is indicative of delayed conception, which is an indicator of reduced 

23 fertility. The subfertility observed in this study is supportive of the association observed 

24 between formaldehyde exposure and spontaneous abortion, since subclinical pregnancy losses 

25 are increased in women with compromised fertility (Gray and Wu, 2000; Hakim et al., 1995), 

26 and both spontaneous abortion and subfertility can be related to exposure to environmental 

27 toxicants (Correa et al., 1996).

28 As described above, formaldehyde exposures to female rats resulted in decreased ovarian 

29 weight and altered LH and FSH levels (Kitaev et al., 1984). Maronpot et al. (1986) reported 

30 endometrial hypoplasia and lack of ovarian luteal tissue in female mice exposed to 40 ppm 

31 formaldehyde for 13 weeks. Additionally, it is noted that, in developmental toxicity studies that 

32 included repeated exposures of dams before mating and/or during gestation, reports of adverse 

33 pregnancy outcomes were few. Gofmekler (1968) reported an increase in pregnancy duration 

34 and decrease in litter size; however, this finding was not observed in other studies.
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1 With the exception of spontaneous abortion and increased time to pregnancy, associations 

2 of formaldehyde exposure with adverse female reproductive system outcomes were not observed 

3 in the available epidemiologic data.

4

5 4.4.9.7. Mode of Action

6 A strong case cannot be made for any one MOA that explains one or more of the 

7 reproductive and developmental outcomes observed in formaldehyde epidemiologic or 

8 toxicology studies. This is due to a number of issues, including the following: 

9 (1) inconsistencies in study findings for the toxicology studies, which may be explained by study 

10 quality issues (see detailed descriptions of studies in Sections 4.1 and 4.2); (2) few studies that 

11 allow for comparisons because no study was performed with the same study design (e.g., stage of 

12 exposure, dose, species, and strain); (3) few mechanistic studies available to test hypothesized 

13 MOAs; and (4) a bias that is pervasive in the formaldehyde literature that outcomes observed 

14 beyond the POE (the nose) are not expected from inhalation exposure, which is the route of 

15 exposure for most of the developmental and reproductive studies. This discussion presents 

16 putative MOAs that have been hypothesized by study authors and the studies that support the 

17 hypothesized MOAs. The four hypothesized MOAs are not mutually exclusive. They could be 

18 acting alone for certain endpoints (in which case the others are not operable) or in concert for 

19 certain endpoints. 

20 The focus of this discussion is on analyzing possible MOAs for the developmental and 

21 reproductive outcomes that were noted most consistently, across toxicology studies and, in some 

22 cases, across human and animal studies. These outcomes include developmental delays, fetal 

23 loss, and sperm quality and quantity effects.

24 An endocrine-disrupting MOA is supported by some of the reproductive and 

25 developmental epidemiology and toxicology studies. For example, the decreases in fetal body 

26 weight (Martin, 1990; Saillenfait et al., 1989), delayed ossifications (Senichenkova and 

27 Chebotar, 1996; Senichenkova, 1991; Martin, 1990; Saillenfait et al., 1989), and delayed 

28 eruption of incisors (Senichenkova, 1991) noted in rats after gestational exposure to 

29 formaldehyde are consistent with developmental delays. In turn, developmental delays can result 

30 from effects on the hypothalamic-gonadal-pituitary axis in the dam that cause hormonal level 

31 changes in the pup; however, hormone levels in pups were not measured. Kilburn and Moro 

32 (1985) also observed organ size changes and undescended testes after developmental 

33 formaldehyde exposure. These outcomes can also be explained by an endocrine MOA. There 

34 are three studies that directly tested for changes in hormones after formaldehyde exposure. 

35 Kitaev et al. (1984) observed ovarian weight and serum LH and FSH increases after inhaled 
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1 formaldehyde in adult female rats. Chowdhury et al. (1992) assessed serum testosterone levels 

2 in adult rats after formaldehyde IP exposure and found significant decreased testosterone and 

3 testes weights and a decrease in 3-β,∆-5-hydroxy steroid dehydrogenase in Leydig cells, 

4 suggesting that formaldehyde affects steroidogenesis. Özen et al. (2002) also reported 

5 significant serum testosterone level decreases as well as decreased mean seminiferous tubule 

6 diameters. Furthermore, the steroidogenesis MOA leading to reduced testosterone is also 

7 consistent with the sperm quality and quantity decrements observed in the studies by Özen et al. 

8 (2002), Sarsilmaz et al. (1999), and Odeigah (1997) studies. 

9 In human studies, delayed time to pregnancy and increased incidence of spontaneous 

10 abortion (Taskinen et al., 1999), consistent with some study findings from the toxicology 

11 literature, could also be explained by an endocrine MOA. Alterations in hormone levels could 

12 lead to pregnancy maintenance problems. Extrapolating the Chowdhury et al. (1992) results of 

13 the steroidogenesis MOA to females, formaldehyde exposure could affect progesterone levels 

14 required for pregnancy. However, progesterone levels were unchanged in the female rat in the 

15 one study that assessed progesterone (Kitaev et al., 1984). Consistent with an endocrine 

16 mediated MOA, Maronpot et al. (1986) observed endometrial hypoplasia and lack of ovarian 

17 luteal tissue in females exposed to formaldehyde.

18 A second hypothesized MOA for some of the developmental and reproductive outcomes 

19 is genotoxicity of the gametes. Such an MOA could explain pregnancy loss in humans 

20 (Taskinen, et al., 1999) and preimplantation loss in animal studies (Xing et al., 2007; Kitaev et 

21 al., 1984; Sheveleva, 1971) and fetal viability (e.g., litter size decreases) after formaldehyde 

22 exposure. Consistent with male gamete genotoxicity, Odeigah (1997) and Xing et al. (2007) 

23 observed reduced fertile matings and live embryos, and increased dead implants in a dominant 

24 lethal study.

25 Oxidative stress/damage is another MOA that is consistent with testicular toxicity, sperm 

26 effects, and reduced embryo viability. Özen et al. (2002) investigated the mechanism of 

27 oxidative stress being responsible for the testes quality effects by assessing testicular iron, 

28 copper, and zinc levels. Zinc and copper levels were reduced in the rat testes, consistent with an 

29 oxidative stress MOA. Özen et al. (2002) also reported increased iron levels and decreased zinc 

30 levels in the lung, consistent with oxidative stress. Another study (Zhou et al., 2006) that 

31 investigated the oxidative stress MOA in the testes observed significant changes in oxidative 

32 stress biochemical markers (decreases in SOD, GPX, GSH, and an increase in MDA levels). 

33 The authors also assessed the protective effect of coadministration with vitamin E, an 

34 antioxidant, on decreased testes weight, biochemical alterations, histopathologic effects, and on 

35 sperm count, motility, and morphology. The study of Pushkina et al. (1968) found reduced 
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1 levels of Vitamin C, another antioxidant, in the fetus and maternal liver after formaldehyde 

2 exposure.

3 The MOAs proposed are not mutually exclusive and in fact could interact with one 

4 another. For example, an endocrine MOA could lead to oxidative stress, and that oxidative stress 

5 could lead to genotoxicity.

6

7 4.4.9.8. Data Gaps

8 The inhalation developmental toxicity studies conducted on formaldehyde and described 

9 in Section 4.2.1.7 comprise an adequate assessment of prenatal developmental toxicity for 

10 application to inhalation reference concentration determination. The assessments of postnatal 

11 developmental toxicity and of reproductive function following inhalation of formaldehyde are 

12 less complete. It is notable that, although the database contains some studies that assess various 

13 aspects of reproductive organ system toxicity, particularly in males, there is no assessment of 

14 multigenerational reproductive function, such as would be evaluated in a two-generation 

15 reproductive toxicity study, nor is there an assessment of potential reproductive effects of 

16 formaldehyde exposure in human males.

17 Adequate assessments of developmental and reproductive toxicity following oral 

18 exposures to formaldehyde have not been conducted.

19

20 4.5. SYNTHESIS AND EVALUATION OF CARCINOGENICITY

21 4.5.1. Cancers of the Respiratory Tract

22 Epidemiologic studies of formaldehyde-exposed workers provide sufficient evidence of a 

23 causal association between formaldehyde exposure and upper respiratory tract (URT) cancers 

24 (e.g., nasopharyngeal cancer (NPC; Section 4.1.2.1.1), nasal and paranasal cancers (Section 

25 4.1.2.1.2), and other upper respiratory tract cancers (Section 4.1.2.1.3)). In addition, the 

26 observational evidence from epidemiologic studies reporting associations between formaldehyde 

27 exposure and increased risk of NPC supports a conclusion of a causal association for this specific 

28 cancer. However, epidemiologic studies of rare outcomes such as NPC, which has an expected 

29 incidence of 1 per 100,000 people per year in the United States, do not typically have great 

30 statistical power to rule out the null hypothesis (i.e. no association). However, the weight of 

31 evidence of the several studies reviewed in Section 4.1.2.1.1 provide an accumulation of 

32 consistent observational evidence sufficient to exclude chance as an explanation for the 

33 association. Additionally, there is insufficient evidence of consistent confounding or other bias 

34 across the studies that were considered; thus, confounding and bias were also ruled out as 

35 explanations for the observed association. The lack of a convincing and consistent alternative 
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1 hypothesis of causation − in spite of repeated examinations − further supports the conclusion that 

2 the association between formaldehyde exposure and increased risk of NPC is causal.

3 The single strongest cohort study, Hauptmann et al. (2004), shows a statistically 

4 significant exposure-response relationship between formaldehyde exposure and URT cancers. 

5 Hauptmann et al. (2004) demonstrated significant excess risk of NPC in exposed workers based 

6 on U.S. population death rates (standardized mortality ratio [SMR] = 2.1; 95% confidence index 

7 [CI] 1.05–4.21) in a large cohort of 25,619 industrial workers. In addition to the SMR based on 

8 an external comparison population, relative risks (RRs) were presented based on internal 

9 comparisons of workers in order to minimize potential selection bias due to the well known 

10 healthy worker effect. Statistically significant exposure-response relationships between 

11 increased formaldehyde exposure and increased risk of NPC were reported for two different 

12 metrics of exposure (peak and cumulative exposure). Relative risks for NPC were also elevated 

13 for increased duration of exposure to formaldehyde and for the average intensity of exposure. 

14 These analyses controlled for potential confounders including calendar year, age, sex, race, and 

15 pay category. While exposure measurement error is likely to be present in any epidemiologic 

16 study, there was no evidence of any differential measurement error that could have produced the 

17 observation of a spurious association. Any non-differential measurement error would likely have 

18 attenuated the effect of formaldehyde was smaller than that which would otherwise have been 

19 observed in the absence of measurement error.

20 The case-control studies similarly also report associations between formaldehyde 

21 exposure and cancer mortality for NPC. Although other risk factors for NPC (e.g., Epstein-Barr 

22 Virus) and the predominant NPC histological sub-type (SCC versus undifferentiated) vary 

23 significantly across the world, case-control studies consistently provide evidence of an 

24 association between occupational exposure to formaldehyde and NPC (Vaughn et al., 1986a; 

25 Vaughn et al., 2000; Roush et al., 1987; Hildesheim et al., 2001; West et al., 1993). In their 

26 more recent study, Vaughn et al. (2000) used worker histories to estimate each individual 

27 worker’s formaldehyde exposure. Workers with more than 1.10 ppm-years of cumulative 

28 exposure were found to be at significantly higher risk of NPC, with an odds ratio (OR) of 3.0 

29 (95% CI 1.3-6.6) (Vaughn et al., 2000). Two different exposure metrics, duration of exposure 

30 and cumulative exposure, were positively associated with increased risk of NPC, with a 

31 significant test for trend (p = 0.014 and 0.033, respectively). The OR also increased in 

32 magnitude as the probability of “Ever” having occupational exposure increased, from OR = 1.6 

33 among those whose exposure was judged to be “Possible, probable or definite” to OR = 13.3 

34 among those with “Definite” exposure (p–trend < 0.001).
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1 NPC histological sub-type analysis indicates that these associations held for both SCC 

2 and epithelial NPC, but not for the undifferentiating and nonkeritinizing NPC (Vaughn et al., 

3 2000). However, formaldehyde exposure is also associated with risk of NPC in Taipei, Taiwan, 

4 where greater than 90% of the cases had nonkeritinizing and undifferentiated carcinomas and 

5 less than 10% of the cases were diagnosed as having SCCs (Hildesheim et al., 2001). These 

6 reported associations were strengthened by considering higher probability of exposure (RR = 

7 2.6; 95% CI 1.1-6.3), greater intensity of exposure (RR = 2.1; 95% CI 1-4.2) and EBV 

8 seropositive cases (RR = 2.7; 95% CI 1.2-5.9) (Hildesheim et al., 2001). Case-control studies 

9 have also linked residential exposure to NPC, specifically for years of residence in mobile homes 

10 (Vaughn et al., 1986b) and the use of mosquito coils in the Philippines (West et al., 1993). 

11 Independent testing of 6 brands of East Asian mosquito coils evaluated the emission rates of 

12 carbonyl compounds in the mosquito smoke and reported that formaldehyde and acetaldehyde 

13 had the highest emission rates. Among the three experiments on each of the six brands, the 

range of formaldehyde concentrations was from 0.87 μg/m3 (1 ppb) to 25 μg/m3 14 (31 ppb). 

15 As a group, other URT sites of direct contact with formaldehyde upon inhalation (i.e., 

16 salivary gland, mouth, nasal cavity and larynx) also showed evidence of a trend in increasing 

17 relative risks with increasing average intensity and peak exposure in the Hauptmann et al. (2004) 

18 cohort study, although these trends did not reach the level of statistical significance. The results 

19 from other cohort studies and case-control studies are mixed (between positive associations and 

20 null findings) for associations between formaldehyde exposure and specific cancers of the URT 

21 (IARC, 2006). For rare cancers, extremely large cohorts would be needed to have the statistical 

22 power to detect an association for tumors defined by individual sites (e.g., mouth, salivary gland, 

23 hypopharnyx). Results vary in the smaller cohort studies, where a single case may result in an 

24 elevated risk but taken together the evidence is considered suggestive (Section 4.1.2). Case25 control studies have been useful to better understand potential associations between 

26 formaldehyde exposure and rare cancers of the URT. Luce et al. (2002) evaluated pooled data 

27 from 12 case-control studies and demonstrated a statistically significant increased risk between 

28 formaldehyde exposure and sinonasal cancer. A case-control study by Gustavsson et al. (1998) 

29 suggested an association between formaldehyde exposure and oral squamous cell carcinoma 

30 (SCC), esophageal, and laryngeal cancers, with odds ratios (ORs) of 1.28, 1.90, and 1.45, 

31 respectively. However, the individual ORs were not statistically significant. Hypopharyngeal 

32 cancer was linked with formaldehyde exposure with an OR of 3.78 (95% CI 1.50-9.49) in 

33 another case-control study (Laforest et al., 2000). While the data on site-specific cancers of the 

34 URT is somewhat sparse, they are consistent with a carcinogenic hypothesis and in their large 

35 cohort study, Hauptmann and colleagues (2004) concluded that in spite of the small numbers of 
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1 deaths from cancers of the URT, the positive associations with average intensity and peak 

2 exposure were consistent with the carcinogenicity of formaldehyde at these sites of first contact. 

3

4 Supporting animal evidence

5 Inhalation exposure:

6 Animal studies, primarily rodent bioassays, strongly support the causal relationship 

7 between of formaldehyde exposure and URT carcinogenicity. Formaldehyde-induced cancers 

8 are primarily seen in the nasal passages (Kerns et al., 1983; Monticello et al., 1996; Tobe at al., 

9 1985; Kamata et al, 1997; Sellakumar et al., 1985), but it should be noted that rodents, unlike 

10 humans, are obligate nose breathers and have convoluted nasal turbinates. Chronic animal 

11 studies (inhalation and oral exposures) report tumor incidence in a variety of rodent models. 

12 Study descriptions are provided above in detail (Section 4.2.1, Table 4-34). The study results are 

13 evaluated here for both routes of exposure in the context of how they inform the carcinogenic 

14 potential at the portal of entry, specifically the URT. 

15 In rodent studies of the respiratory tract, only nasal tumors are considered to be induced 

16 by formaldehyde. Repeated exposures to 10-15 ppm formaldehyde result in gross nasal lesions 

17 and high incidence of nasal tumors (See Table 4-38, Section 4.2.1). Although increased cell 

18 proliferation, squamous metaplasia, dysplasia and focal necrotic lesions have been noted in the 

19 larynx and trachea in some studies, no tumors in these locations have been reported in the rodent 

20 studies. The majority of studies were conducted using rats (F344, Wistar, or Sprague-Dawley), 

21 and all studies of 18 months or greater in mice and rats show evidence of formaldehyde-induced 

22 nasal carcinogenicity. The nasal tumors are primarily SCCs, although papillomas, polypoid 

23 adenomas, adenocarcinomas, fibrosarcomas, and esthesioneuroepitheliomas have been reported 

24 (Kamata et al., 1997; Monticello et al., 1996; Morgan et al., 1986a, b; Takahashi et al., 1986; 

25 Sellakumar et al., 1985; Kerns et al., 1983; Albert et al., 1982). Although hyperplasia, dysplasia, 

26 and squamous metaplasia of the respiratory epithelium have been observed beyond the nasal 

27 cavity, other respiratory tract tumors have not been reported to be significantly increased by 

28 formaldehyde exposure alone.

29 Increased tumor incidence and decreased latency are correlated with increasing 

30 formaldehyde exposure concentration. Reviewing data from the only lifelong inhalation study 

31 (i.e., until "natural death") with multiple exposure groups, nasal SCCs occurred much earlier in 

32 the high-exposure animals. For example, tumors are first noted at 8 and 9 months following 

33 exposure for high-exposed (15 ppm) female and male F344 rats versus tumors not arising until 

34 24 months in low-exposed rats (2 ppm) (Kerns et al., 1983). In a follow-up study by Monticello 

35 et al. (1996), the incidence of SCC in rats exposed at 15 ppm was 47%, with the first tumor noted 
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1 at 12 months. The incidence of SCC in male rats exposed at 10 ppm was 22%, with the first 

2 tumors observed at 18 months after exposure. Moreover, of the 90 rats exposed at 6 ppm for 20 

3 months, only one SCC was noted. No SCCs were detected in rats exposed to 0.7 or 2 ppm 

4 formaldehyde. These incidence rates are not mortality-adjusted (see Chapter 5, section 5.3.4) 

5 and include animals from each scheduled sacrifice (3, 6, 12, and 18 months). In a lifelong study 

6 of male Sprague-Dawley rats exposed to 10 ppm formaldehyde, the cumulative nasal tumor 

7 incidence was calculated as a function of time of exposure (Sellakumar et al., 1985). After 2 

8 years of exposure, the adjusted probability of nasal carcinoma was greater than 60%.

9 There is some evidence that less-than-lifetime exposure to formaldehyde can induce nasal 

10 tumors over an extended observation period. Two studies, both in male Wistar rats, report nasal 

11 tumors in response to less-than-lifetime exposures (Woutersen et al., 1989; Feron et al., 1988). 

12 A 13-week exposure at 20 ppm followed by an observation period of 30 months (inclusive of 

13 exposure) in Wistar rats resulted in six nasal tumors including three nasal SCCs, one cystic SCC 

14 of the nasolacrimal duct, one carcinoma in situ and an ameloblastoma, while no tumors were 

15 noted in the corresponding air-exposed controls (Feron et al., 1988). A limited number of 

16 formaldehyde-related tumors were noted from 4 or 8 weeks of exposure followed by 30 months 

17 of observation. Although the tumor incidence of these less-than-lifetime exposures is low, this is 

18 consistent with the 2-year bioassays in Wistar rats. Wistar rats are more resilient to 

19 formaldehyde-induced nasal toxicity than F344 or SD rats (Section 4.2.1), and only 1 of 26 (4%) 

20 Wistar rats exposed at 10 ppm for 28 months developed SCC (Woutersen et al., 1989) versus 

21 22% in F344 rats (Monticello et al., 1996). 

22 The specificity of formaldehyde-induced tumors in the nasal passages of rodents is 

23 believed, at least in part, to be a function of tissue dose. Computational fluid dynamics (CFD) 

24 modeling used to predict formaldehyde tissue flux during inhalation exposures suggests that at 

25 comparable concentrations, tissue flux in the nasal passages of rodents is more intense than for 

26 non-human primates and humans. Modeling predicts a different pattern of formaldehyde flux 

27 into URT tissues of rodents compared to humans, where formaldehyde penetrates more deeply 

28 into the respiratory tract of primates than rodents even considering nose-only breathing for 

29 primates (See Section 3.4). Humans will generally switch to mouth breathing when sensing an 

30 irritating smell and during physical exertion, resulting in direct exposures to the mouth and 

31 greater tissue flux in tissues beyond the bypassed nasal passages. Therefore, species differences 

32 in tissue dose may contribute to formaldehyde-induced tumors in humans beyond the nasal 

33 passages, which are not evident in rodent bioassays.

34

35
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1 Oral Exposure:

2 Consistent with the observed carcinogenic activity of formaldehyde on tissues at the 

3 portal of entry (POE) from inhalation exposure, there is evidence to support POE effects from 

4 oral exposures as well – further strengthening the overall weight of evidence of formaldehyde’s 

5 carcinogenicity. As with the respiratory tract, the proximal portion of the gastrointestinal (GI) 

6 tract exhibits formaldehyde-induced lesions specifically in the forestomach and glandular 

7 stomach (Soffritti et al., 1989; Til et al., 1989; Tobe et al., 1989; Takahashi et al., 1986). 

8 However, data are mixed regarding the carcinogenic potential of formaldehyde in the GI tract 

9 from oral exposures.

10 Two independent 2-year cancer bioassays in Wistar rats exposed to formaldehyde in 

11 drinking water were both negative (Til et al., 1989; Tobe et al., 1989). Til et al. (1989) exposed 

12 rats to a range of formaldehyde doses (0, 1.2, 15, or 82 mg/kg-day) and evaluated 44–49 animals 

13 per sex per dose group at 24 months of exposure. No formaldehyde-related tumors were found. 

14 A smaller study by Tobe et al. (1989) failed to note any tumors after a 2-year exposure at 0, 10, 

15 50 or 300 mg/kg-day (eight rats per sex per treatment group.) 

16 In contrast, two studies that included lifelong observation in male and female Sprague17 Dawley rats provide support for formaldehyde-induced GI tract tumors – one study where 

18 exposure commenced at 7 weeks of age and a second study conducted with breeder rats (25 

19 weeks of age) and their offspring (Soffritti et al., 1989). These studies demonstrate an increase 

20 in GI tumors (although rare) correlated with exposure to formaldehyde and significantly 

21 increased susceptibility to early-lifetime exposure. The authors provide a detailed report on the 

22 background rates of various stomach and intestinal neoplasia for male (n = 2,677) and female (n 

23 = 2,582) rats within the colony (Soffritti et al., 1989). From this background pool, the total 

24 incidence of benign and malignant tumors in the stomach and intestine combined is only 1.4% 

25 (combining all sites and locations), with the majority of tumors located in the stomach (1% 

26 benign, 0.2% malignant). In comparison to colony-specific background rates, apparent increases 

27 in both stomach and intestinal neoplasia are noted in formaldehyde-treated rats (ranging from 1 

28 to 6% by type in rats exposed beginning week 7). When summed across the GI tract, tumor 

29 incidence in the highest treatment group was 8% versus 1.4% in historical controls. Elevations 

30 of individual tumors or a clear dose-response relationship are difficult to discern for rare cancers 

31 where there are only 50 animals per group. Despite the limitations of group size and lack of a 

32 dose–response relationship, the findings do support the carcinogenic potential for formaldehyde 

33 administered orally. 

34 The second study reported by Soffritti et al. (1989) in Sprague-Dawley rats demonstrates 

35 early lifetime susceptibility, with GI tumor incidences of 21.6% in female (n = 37) and 13.9% in 
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1 male (n = 36) offspring after exposure to formaldehyde beginning in utero, versus 5.6% in the 

2 adult breeders. Rats were exposed to formaldehyde in drinking water for 2 years (0 or 2,500 

3 mg/L). Exposures began on gestational day 12 in the offspring. The most common tumor 

4 detected was intestinal leiomyosarcoma (13.5% in female offspring), which has a background 

5 rate of 0.04% in female rats in the colony. The incidence of GI tumors in the adult breeders (n = 

6 18 per sex) was due to one adenocarcinoma in a male rat and one pallipoma/acanthoma in a 

7 female rat. Although severely limited by study size, their occurrence is consistent with the 

8 observation of formaldehyde-induced tumors, given the low background rates for this colony.

9 The Soffritti et al. (1989) studies stand alone in observing formaldehyde-induced GI 

10 tumors. These findings are largely attributed to a unique study design that included lifelong 

11 observation (i.e., until "natural death"), neonatal exposure, examination of individual tumor types 

12 as well as combined rare tumor types for analysis, and summation of tumors across locations. 

13 The study design results in a more sensitive assay for rare tumors as well as tumors with a long 

14 latency. Thus, Soffritti et al. (1989) utilized a more appropriate design and analysis for detecting 

15 rare tumors, and these findings are not diminished by the null results of Tobe et al. (1989) and 

16 Til et al. (1989). 

17 There is evidence that formaldehyde may act as a tumor promoter by the oral route as 

18 well as the inhalation route (discussed above). Takahashi et al. (1986) reported an increase in N19 Methyl-N'-Nitro-N-Nitrosoguanidine (MNNG)-initiated GI cancers in mice with formaldehyde 

20 exposure (29.4%, versus 13.3% in controls); the greatest difference in tumor response was 

21 associated with adenocarcinoma of the glandular stomach (23.5%, versus 3.3% in controls). 

22 Additionally, forestomach papillomas and preneoplastic hyperplasia in the glandular stomach 

23 were increased with formaldehyde exposure alone.

24

25 4.5.2 Lymphohematopoietic Malignancies

26 4.5.2.1. Background

27 Lymphohematopoietic (LHP) cancers include neoplasms of both lymphoid and myeloid 

28 cell origins. Cancers of the immune system are described as leukemia if they primarily involve 

29 cells from peripheral blood and bone marrow at diagnosis and lymphomas if they constitute a 

30 solid tumor (Robbins, 2004). Some forms of leukemia which present as an immature immune 

31 cell phenotype are believed to arise from lymphomyeloid stem cells or progenitor cells normally 

32 found in the bone marrow (e.g., acute lymphoblastic leukemia (ALL) and acute myeloid 

33 leukemia (AML)) (Greaves, 2004). However, multiple myeloma, lymphomas and some 

34 leukemias may arise from mature functional lymphocytes present outside of the bone marrow 

35 (Greaves, 2004; see Figure 4-33). Therefore, the use of the general term ‘leukemia’ is not 
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1 restricted to cancers from a transformed stem cell or progenitor cell in the bone marrow but also 

2 applies to cancers which arise from differentiated cells (e.g., mature lymphocytes). 

3 Epidemiologic studies have reported that formaldehyde exposure is associated with both 

4 leukemia and lymphomas (Chapter 4.1.2.2.1). Specific neoplasms reported to be associated with 

5 formaldehyde exposure include myeloid leukemia, Hodgkin’s lymphoma, and multiple 

6 myeloma. 

7

20

H

B

T B

M T

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

9 Figure 4-33: Developmental origins for cancers of the lymphohematopoietic 

10 system (Adapted from Greaves (2004).

11

12

13 When evaluating cancers of the LHP system, epidemiologic analysis often groups many 

14 of these cancers together. In part, this may be done to increase the statistical strength of the 

15 analysis. Additionally, since there is a potential for disease misclassification, grouping these 

16 diseases is preferred by some researchers, especially when analyzing older mortality data. 

17 Historically, misclassification may have been due to factors such as poor histopathology and 

18 diagnosis of late-stage disease, where cell line of origin may have been hard to distinguish. 

19 Without the cell surface markers and molecular tools used today to classify cells, diagnosis was 

20 accomplished primarily based on histology. However, as the cancers progress, the leukemic 

21 stem cells may present as less mature cells. For example, poor health surveillance may allow 

22 chronic myeloid leukemia (CML) to remain undiagnosed until the blast crisis, often seen late in 
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1 the disease. This blast crisis presents as a leukemia of relatively immature myeloid cells and 

2 may have been mistaken for AML without the more recent techniques available for classifying 

3 disease. Since cancers of the LHP system may present with cell surface markers for multiple cell 

4 lines, classification remains problematic in some cases. 

5 Although often grouped for analysis, the LHP cancers represent many distinct 

6 malignancies which may arise from discrete cell types in different tissues throughout the body 

7 (Greaves, 2004). The World Health Organization (WHO) has developed a classification system 

8 for both lymphoid and myeloid leukemia defined by a combination of morphology, 

9 immunophenotype, genetic features and clinical features (Harris et al., 2000a; Harris et al., 

10 2000b). The historical nomenclature and International Classification of Disease (ICD) codes 

11 used in epidemiologic studies do not correspond to these new classification systems. For 

12 example, both chronic lymphocytic leukemia (CLL) and B-cell lymphomas arise from similar 

13 cell types in the periphery, yet epidemiologic studies have considered them independently even 

14 though they are currently considered to be the same disease in the new WHO classification 

15 system − which would diminish the statistical power to detect an association. Careful re-analysis 

16 of epidemiologic data addresses evidence for the class as a whole (all LHP cancers) but also 

17 various subclasses (e.g. myeloid versus lymphoid). 

18 Therefore, the following analysis first examines the epidemiologic evidence for all LHP 

19 cancers as a class, then all leukemia, to best take advantage of the majority of publications 

20 available in assessing the weight of evidence for the carcinogenicity of formaldehyde. The 

21 subsequent analysis by sub-type draws upon the available evidence for specific diseases or 

22 groups of diseases (e.g., myeloid leukemia, Hodgkin’s lymphoma, non-Hodgkin’s lymphoma). 

23 Although fewer data are available for the sub-type analysis, these data help clarify which cancers 

24 may contribute to the consistent observation of an association between formaldehyde exposure 

25 and all LHP cancers. Novel combinations of phenotypic sub-types are presented where they are 

26 etiologically relevant. The sub-type analysis frames the subsequent mode of action (MOA) 

27 analysis. 

28

29 4.5.2.2. All LHP Malignancies

30 Epidemiologic studies involving formaldehyde-exposed workers provide sufficient 

31 evidence of a causal association between formaldehyde exposure and all LHP malignancies 

32 (Section 4.1.2.1.5). Positive associations between formaldehyde exposure and LHP cancers have 

33 been reported for chemical workers (Wong et al., 1983; Bertazzi et al., 1986), embalmers 

34 (Walrath and Fraumeni, 1983, Walrath and Fraumeni, 1984; Hayes et al., 1990), anatomists and 

35 pathologists (Harrington and Shannon 1975; Hall et al., 1991; Levine at al 1984; Stroup et al., 

36 1986; Matanoski et al., 1989) (Table 4-90). However, clear associations (in terms of overall 
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1 standardized mortality ratios [SMRs] or proportional mortality ratios [PMRs]) were not reported 

2 in analyses for garment workers, iron-foundry workers, and a large US industrial cohort 

3 (Pinkerton et al., 2004; Andjelkovich et al., 1995; Beane Freeman et al., 2009; Marsh et al., 

4 1996), although associations were observed in some of these studies when exposure-response 

5 relationships were considered. Several published meta-analyses are available which more 

6 formally assess the strength of association between formaldehyde exposure and mortality from 

7 all LHP cancers. Pooled SMRs indicate stronger associations for professional workers 

8 (embalmers, anatomists and pathologists) than industry workers (Table 4-91). Bosetti et al. 

9 (2008) found similar relationships, with a pooled SMR of 1.31 (95% CI 1.16-1.47) for 

10 ‘professionals’ (i.e. embalmers, anatomists and pathologists) versus a pooled estimate of 0.85 

11 (95% CI 0.74-0.96) for industrial workers. A recent meta-analysis by Zhang et al. (2009) reports 

12 a summary relative risk (RR) of 1.25 (95% CI 1.09-1.43) for both professional and industry 

13 workers for all LHP cancers (ICD 9 codes 200-209). These researchers identified 19 cohort 

14 study analyses, including cohort study updates. Zhang et al. (2009) used the reported RR from 

15 the highest exposure category to increase statistical power and reduce uncertainty regarding 

16 confounding or other bias. Although study selection was controversial, e.g., the inclusion of 

17 multiple reports from a single cohort and the use of one cohort where only a portion of the 

18 workers were formaldehyde-exposed, this meta-analysis is generally supportive of an association 

19 between formaldehyde and LHP malignancies.

20 The apparent differences by industry/profession may reflect many influences, including 

21 exposure potential and demographic characteristics. External analysis (use of the general 

22 population for comparison) relies on the assumption that cancer incidence rates are expected to 

23 be similar between the general population and the study population in the absence of exposure. 

24 The ‘healthy worker effect’ is well known, and there may be differences in the magnitude of this 

25 selection bias by industry or profession. For instance, LHP cancer incidence and mortality have 

26 many risk factors including socioeconomic status. Therefore, the consistent positive findings in 

27 professional workers versus mixed results in industrial workers could be influenced by the 

28 appropriateness of the comparison to the general population − that is, a differential extent of 

29 selection bias. Interestingly, salaried workers, but not the hourly workers, in an Italian plastic 

30 manufacturing plant had elevated SMRs for LHP cancers (1.69 (95% CI 1.07-2.53) and 

31 0.93(95% CI 0.62-1.35), respectively) (Dell and Teta, 1995). Without knowledge of which 

32 worker group is most similar to the comparison population with respect to LHP cancers 

33 mortality, one cannot discern if this potential effect of demographic variability accentuates 

34 effects in professional/salaried workers or obscures the effects in industrial/hourly-wage 

35 workers. 

36
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1 Table 4-90. Summary of cohort and case-control studies which evaluated 

2 the incidence of all LHP cancers in formaldehyde-exposed populations 

3 (ICD-8 Codes: 200-209) and all leukemias (ICD-8 Codes: 204-207). (See 

4 Table 4-6 for complete study details and findings)

5

Study population Study details All LHP cancers Leukemia Reference

SMR Analysis1

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, 

1995
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(n=5,932) 0.93

(hourly workers)

0.98

(hourly workers)

PMR Analysis1

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 Studies1

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

2 Notes:

3 1. Relative risk estimate (SMR, PMR, or OR) presented with 95% confidence intervals, where available.

4 2. PMR for leukemia for the total group calculated from the published data for lymphatic leukemia (204, myeloid leukemia

(

6

5 205), and other/unspecified (206, 207).

7

8 The only study which has data to inform the effects of either exposure level or the 

9 appropriateness of an external comparison group on the association between formaldehyde 

10 exposure and all LHP cancer mortality is the National Cancer Institute (NCI) cohort study of 

11 industrial workers (Blair et al., 1986; Beane Freeman et al., 2009), which presents relative rates 

12 based on internal comparisons for 3 different exposure metrics. Although SMR analysis with an 

13 external comparison group did not indicate increased mortality from all LHP cancers (0.94, CI 

14 0.84-1.06, for the exposed workers), internal analysis using the low-exposed workers as the 

15 comparison group demonstrates positive exposure-response relationships for increased mortality 
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1 from all LHP malignancies cancers and peak exposure across the study periods (1965-2004) 

2 (Figure 4-34A and 4-34B) (Beane Freeman et al., 2009), with a statistically significant trend (p < 

3 0.05) for every year since 1977. These results, indicating a positive exposure-response 

4 relationship among plant workers, who most likely have similar demographic characteristics, are 

5 noteworthy given the apparent lack of association when SMRs for the same cohort are calculated 

6 against mortality rates for the general population. The lack of an apparent association with 

7 SMRs may be attributable to the healthy worker effect and/or some other difference between the 

8 exposed workers and the general population.

9 Although the association between formaldehyde exposure and all LHP cancer mortality 

10 in industrial and professional cohorts is mixed, the strength of the internal analysis of the NCI 

11 cohort, in the absence of positive SMRs compared to the general population, suggests that SMR 

12 analyses may not be the most appropriate methodology for assessing LHP cancer mortality. 

13 Given the potential for demographic differences between an industrial workforce and the general 

14 population, the results of the internal analysis of the NCI industrial cohort provide a higher 

15 quality analysis – and therefore should be given significantly more weight than SMR analyses of 

16 industrial workers that could not distinguish their findings from the null. Given the consistency 

17 and strength of the positive associations for all LHP cancers cancer mortality in professional 

18 cohorts (embalmers, anatomists and pathologists) taken together with the strong positive results 

19 of the NCI cohort, human epidemiologic evidence are sufficient to conclude that there is a causal 

20 association between formaldehyde exposure and mortality from all LHP malignancies (as a 

21 group). 

22

23 4.5.2.3. All Leukemia

24 Like the analysis of all LHP cancers, an analysis of all leukemia combines diseases which 

25 differ significantly in cell of origin and etiology, including acute and chronic forms of both 

26 myeloid and lymphatic leukemia. This class also includes other leukemia (e.g., erythraemia) and 

27 a general category of ‘other and unspecified leukemia’ (ICD-8 207). Regardless, there is some 

28 utility in evaluating the all leukemia mortality data because many studies provided results for this 

29 grouping. Also, the diagnosis of leukemia versus solid LHP tumors is fairly distinct thereby 

30 limiting misclassification of the endpoint. 

31
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1

2

3 Figure 4-34A. Association between peak formaldehyde exposure and the risk 

4 of lymphohematopoietic malignancy. 

5

6 Relative risks for medium-peak (2.0 to <4.0 ppm) and high-peak (≥4.0 ppm) 

7 formaldehyde exposure categories compared with the low exposed category (>0 

8 to <2.0 ppm) and P values for trend tests among the exposed person-years for 

9 lymphohematopoietic malignancies are shown by year of end of follow-up, 1965-

10 2004. Values plotted at 0.1 represent RR = 0 due to no cases in the exposure 

11 category values plotted at 20 represent RR = infinity due to no cases in the 

12 referent category. The small graphs above the relative risk plots represent the 

13 exposure-response trend P values based on two-sided likelihood ratio tests (1 df) 

14 of zero slope for continuous formaldehyde exposure among exposed person-years 

15 only. The points represent the relative risk estimates based on the cumulative 

16 number of cases and person-years accrued from the start of the study to that point 

17 in time and for 2004 are equivalent to the relative risk estimates presented in 

18 Table 2. HLP = lymphohematopoietic malignancies, NHL = non-Hodgkin 

19 lymphoma, HDG = Hodgkin lymphoma, MM = multiple myeloma, LEU = 

20 leukemia, LYL = lymphatic leukemia, MYL = myeloid leukemia, RR = relative 

21 risk. 

22

23 Source: Beane Freeman, et al. (2009)

24
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4-416 DRAFT—DO NOT CITE OR QUOTE

1

2 Figure 4-34B. Association between average intensity of formaldehyde 

3 exposure and the risk of lymphohematopoietic malignancy.

4

5 Relative risks for medium (0.5 – 0.9 ppm) and high ( ≥ 1.0 ppm) average-intensity 

6 formaldehyde exposure categories compared with the low exposed category (0.1 –

7 0.4 ppm) and P values for trend tests among the exposed person-years for 

8 lymphohematopoietic malignancies by year of end of follow-up, 1965 – 2004. 

9 Values plotted at 0.1 represent RR •• = due to no cases in the exposure category. 

10 The small graphs above the relative risk plots represent the exposure – response 

11 trend P values based on two-sided likelihood ratio tests (1 df ) of zero slope for 

12 continuous formaldehyde exposure among exposed person-years only. The points 

13 represent the relative risk estimates based on the cumulative number of cases and 

14 person-years accrued from the start of the study to that point in time and for 2004 

15 are equivalent to the relative risk estimates presented in Table 3 . HLP = 

16 lymphohematopoietic malignancies, NHL = non-Hodgkin lymphoma, HDG = 

17 Hodgkin lymphoma, MM = multiple myeloma, LEU =

18 leukemia, LYL = lymphatic leukemia, MYL = myeloid leukemia; RR = relative 

19 risk.

20

21 Source: Beane Freeman, et al. (2009)

22
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1 Although results are mixed across the studies (Table 4-90), an association between 

2 formaldehyde exposure and leukemia mortality is supported by cohort analyses of embalmers, 

3 pathologists and anatomists (Hayes et al., 1990; Walrath and Fraumeni, 1983; Walrath and 

4 Fraumeni 1984; Hall et al., 1991; Levine et al., 1984; Stroup et al., 1986; Matanoski et al., 1989). 

5 Formaldehyde exposure and formaldehyde-related occupation are associated with leukemia 

6 diagnosis in a case-control study (RR = 5.79 (95% CI 1.44-23.25), but not formaldehyde 

7 exposure alone (RR = 0.96; 95% CI 0.54-1.71) (Stellman et al., 1998).

8 In contrast, SMR analyses of the industrial cohorts do not indicate a similar association 

9 (Coggon et al., 2000; Beane Freeman et al., 2009, Pinkerton et al, 2004). Although the SMR 

10 analysis provided for the NCI cohort does not indicate a positive association for all leukemia 

11 using an external reference group (Beane Freeman et al., 2009), the SMR for exposed versus 

12 unexposed workers within the cohort suggests all leukemia is elevated 2.1-fold with this internal 

comparison (95% CI 0.99-4.56)4

23 Several meta-analyses have been conducted for formaldehyde exposure and leukemia 

24 which indicate a positive association. Collins et al. (2004) report an overall RR for 18 available 

25 studies of 1.1 (CI 1.0-1.2), suggesting an association of leukemia with formaldehyde exposure. 

26 This association was stronger for both pathologists/anatomists (1.4; CI 1.0-1.9) and embalmers 

27 (RR = 1.6; 1.2-2.0) than for industrial workers (RR = 0.9; 0.8-1.0). Study design also impacted 

28 the apparent strength of association, with stronger associations seen in case--control studies (RR 

29 = 2.4; 0.9-6.5) versus cohort studies (RR = 1.0; 0.9-1.2). Bosetti et al. (2008) reported an 

30 association between formaldehyde exposure and leukemia mortality with a pooled RR of 1.39 

31 (95% CI 1.15-1.68) for 8 groups of professional workers. In the same analysis, the pooled RR 

32 for the 4 industrial cohorts was 0.90 (0.75-1.07). Zhang et al. (2009) reported a pooled RR of 

13 . A positive exposure-response relationship further strengthens 

14 the association of formaldehyde exposure to leukemia mortality (Beane Freeman et al., 2009). 

15 Where the referent group is defined as ‘low exposed’ individuals, leukemia is elevated in the 

16 highest peak exposure category (RR = 1.42; 95% CI 0.92-2.18) compared to both the referent 

17 group and the unexposed category (RR = 0.59; 95% CI 0.25-1.36), and there is a statistically 

18 significant trend across all groups (p = 0.02). Categorical analysis for the average intensity and 

19 cumulative exposure metrics suggests greater mortality in the high-exposure groups versus the 

20 ‘low exposed’ individuals (RR = 1.10 [95% CI 0.68-1.78] and 1.11 [0.7-1.74]. respectively), but 

21 analysis of individual results across the exposure-response range indicates cumulative exposure 

22 is a better predictor (p = 0.08 for trend across all exposed and unexposed.) 

 

4

Non osed Exposed Non osed

Exposed

SMR Obs Obs

SMR

Var

exp exp

1 1 ln = + 






























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1 1.54 (95% CI 1.18-2.00) for all cohorts identified in their meta-analysis, although this pooled RR 

2 should be considered with some caution, as myeloid leukemia alone was included in the analysis 

3 where available (Zhang et al., 2009). 

4

5 4.5.2.4. Subtype Analysis

6 Given the associations discussed above between formaldehyde exposure and both all 

7 LHP cancers and all leukemia, further analysis is needed to examine if the observed increase in 

8 all LHP cancers is primarily a reflection of increased leukemia, or if other types of LHP cancers 

9 may be elevated as well. Although analysis of mortality data by sub-type may provide a better 

10 understanding of the specific disease associations, there are potential pitfalls as well. Chief 

11 among these concerns are the potential for disease misclassification (especially in studies with 

12 older mortality data) and lack of statistical power as the number of observed cases is reduced by 

13 considering sub-types. Case control studies by design address specific diseases and are well14 suited for sub-type analysis, but often provide little exposure information. The following 

15 analysis will draw from the available data to examine which forms of LHP malignancies may be 

16 associated with formaldehyde exposure.

17 There has been speculation that the association between formaldehyde exposure and 

18 increases in all LHP cancers and all leukemia are driven by increased myeloid leukemia (Pyatt et 

19 al., 2008; Heck and Casanova 2004; Golden et al., 2006). If this were the case, then mortality 

20 from LHP cancers other than myeloid should not be elevated, once the excess mortality from 

21 myeloid leukemia is accounted for. Only 2 studies provide the data to evaluate this hypothesis –

22 both conducted by the NCI (Hayes et al., 1990 and Beane Freeman et al., 2009). From the 

23 published data, crude mortality statistics can be calculated for alternative disease groupings 

24 (Table 4-91). In the NCI embalmer study (Hayes et al., 1990), only myeloid leukemia was 

25 statistically elevated in the subtype analysis. For the NCI industrial cohort (Beane Freeman et 

26 al., 2009), elevations were also seen for Hodgkin lymphoma relative to the referent group. In 

27 both cases, the association between formaldehyde exposure and LHP malignancies remains when 

28 myeloid leukemia is dropped from the analysis. Further, similar associations are found when all 

29 leukemia and myeloproliferative diseases are dropped from the analysis and only solid tumors of 

30 lymphoid origin are included (lymphosarcoma and reticulosarcoma, Hodgkin lymphoma, non31 Hodgkin lymphoma and multiple myeloma). These reanalyses illustrate the need for a more 

32 careful sub-type analysis to assess the potential for associations between formaldehyde exposure 

33 and various forms of LHP cancers.

34

35
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1 Table 4-91. Secondary analysis of published mortality statistics to explore 

2 

3 

4 

5 

13 

14 

15 

16 

17 

18 

19 

20 

21 

6 

7 

8 

9 

10 

11 

12 

a

 Exp

Obs PMR =

b

 ( ) ExpObs

Var PMR 11 log +=

c See Table 2 of Beane Freeman et al. (2009) 

d 

765.0

37.1103

108

14103

19108

=



 





× ≅

































×



 





−

− = −

− =

c c

c

Comparison Group

referent Group

Comparison Group

referent Group

referent Group referent Group

Comparison Group Comparison Group

PT

PT

where

PT

PT

Deaths Person Time

Deaths Person Time

RR

e ( ) DeathsComparison Group Deathsreferent Group

Var RR 1 1 log = +

alternative disease groupings within the broad category of all lymphohematopoietic 

malignancies 

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

4.5.2.5. Myeloid Leukemia

 The associations between myeloid leukemia and formaldehyde exposure are strong and 

consistent (Table 4-92). Of the four studies which formally assess myeloid leukemia mortality, 

all are positive, including cohorts of both professional and industrial workers (Beane Freeman et 

al., 2009; Hayes et al., 1990; Pinkerton at al., 2003; Stroup et al., 1986). Although few cases 

exist for further subtype analysis, the available data indicate either no differences in SMRs for 

acute myeloid leukemia (AML) versus chronic myeloid leukemia (CML) (Hayes et al., 1990; 

Pinkerton et al., 2003) or suggest CML is more prominent (Blair et al., 2000; Stroup et al., 1986). 
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1 Table 4-92. Summary of studies which provide mortality statistics for 

2 myeloid leukemia sub-types. 3

Study population

Reference

Myeloid 

Leukemia

Acute

Myeloid 

Leukemia

Chronic

Myeloid 

Leukemia

SMR Analysis1

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 Analysis1

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 Studies1

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

5 * Leukemia SMR 1.5 (0.7-2.7) {5 of 10 deaths due to myeloid}; Chronic Myeloid Leukemia (CML) SMR of 8.8

6 1. Relative risk estimate (SMR, PMR, or OR) presented with 95% confidence intervals, where available.

7

8

9 Walrath and Fraumeni (1983, 1984) note that AML is prominent in their analyses of New 

10 York and California licensed embalmers; however, they do not provide PMR analyses for CML. 

11 Walrath and Fraumeni (1983 and 1984) report leukemia cell types - for both studies the majority

12 of myeloid leukemia are acute (5/6 and 4/6, respectively, for New York State and California 

13 embalmers). However, PMRs cannot be calculated for AML versus CML in this paper, as 

14 comparison rates are not available from the 1920's through the 1960's - the timeframe with the 

15 majority of deaths. The authors do contrast the observed rate of AML in the cohort to the 

16 background rate for AML in white men in the 1970s - but given the potential misclassification of 

17 late stage CML as AML, especially historically, this may not be an appropriate comparison. 

18 Additionally, one would expect older data to over-represent AML rather than CML due to 

19 diagnosis in the early decades of CML in the blast crisis as AML. Therefore, although these 

20 studies support an association between formaldehyde exposure and myeloid leukemia in general 
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1 (Walrath and Fraumeni, 1983; 1984), the reported AML and CML subtype information does not 

2 allow a satisfactory sub-type analysis for myeloid leukemia. 

3

4 4.5.2.6. Solid Tumors of Lymphoid Origin

5 Multiple myeloma, Hodgkin lymphoma, non-Hodgkin lymphoma, lymphosarcoma, 

6 reticulosarcoma, and other lymphomas may all be derived from immune cells outside of the bone 

7 marrow compartment, in peripheral blood, in the gut and respiratory mucosa and immune tissues 

8 at the POE (e.g., lymph nodes, mucosa-associated lymphoid tissue (MALT), gut-associated 

9 lymphoid tissue (GALT) (Greaves, 2004). The only meta-analysis to specifically address 

10 lymphoid malignancies found evidence for increased lymphoma (Hodgkin lymphoma (pooled 

11 RR = 1.23; 95% CI 0.67-2.29) and multiple myeloma (1.31; 1.02-1.67), but not for non-Hodgkin 

12 lymphoma (1.08; 0.86-1.35) (Zhang et al., 2009). As seen in Table 4-93 below, individual study 

13 results are mixed for these lymphoid cell-line malignancies, as they are for all LHP cancers and 

14 all leukemia above. Although these tumors are from mature lymphocytes, there is still variability 

15 in the etiology, natural history and risk factors for the many sub-types which are included in 

16 these categories.

17 There is evidence for an exposure response relationship for both Hodgkin lymphoma and 

18 multiple myeloma in the NCI industrial cohort among exposed workers (Beane-Freeman et al., 

19 2009). Clear exposure response relationships for Hodgkin lymphoma are defined with all three 

20 metrics of exposure, peak average intensity and cumulative exposure (p=0.01, p=0.05 and 

21 p=0.08 respectively for mortality through 2004). These associations have been evident from first 

22 follow-up through the current publication, and statistically significant for the majority of the 

23 follow-up period demonstrating that this is a strong and consistent finding in the NCI cohort 

24 (Figure 4-34 A&B) (Beane-Freeman et al., 2009). Although the overall SMR for multiple 

25 myeloma does not indicate an association, trends across time indicate consistent elevation of 

26 multiple myeloma mortality with both peak and average intensity of exposure, where the 

27 statistical strength of the association with peak exposure increases with follow-up (Figure 4-34 

28 A&B). 

29
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1 Table 4-93. Summary of mortality statistics for Hodgkin lymphoma, 

2 lymphoma and multiple myeloma from cohort analyses of formaldehyde 

3 exposed workers. 4

Study population Hodgkin Reference

Lymphoma

Non-Hodgkin 

Lymphoma

Multiple 

Myeloma

SMR Analysis6

Pathologists 

(n=2,079) 1.4 2.0 (p<0.05) NR1 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) 32.0 (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) 31.54 (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 Analysis6

Embalmers, New York

(n=1,132) 0.87 (p<0.05) 1.0831.224 NR Walrath and Fraumeni, 1983

Embalmers, CA

(n=1,007) − 2 3.1031.334 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 Studies6

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 al., 1992

5 Notes :

6 1: NR is not reported

7 2. “—" no cases observed.

8 3. Lymphosarcoma and reticulosarcoma only

9 4. “other lymphoma"

10 5. Formaldehyde exposure in a wood-related occupation. RR for wood-related occupation alone was not elevated 

11 0.97 (0.55-1.73)

12 6. Relative risk estimate (SMR, PMR, or OR) presented with 95% confidence intervals, where available
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1 4.5.2.7. Supporting Evidence from Animal Bio-Assays for Formaldehyde-Induced 

2 Lymphohematopoietic Malignancies

3 Chronic animal studies provide supporting evidence for formaldehyde-induced leukemia 

4 and lymphoma (Soffritti et al., 1989; Battelle Laboratories, 1981). Although the majority of 

5 chronic animal bioassays do not report either leukemia or lymphoma, it should be noted that 

6 many studies focused primarily on respiratory tract and did not provide routine examination of 

7 other organs, limiting the detection of leukemia and lymphoma (Horten et al., 1963; Holmstrom 

8 et al., 1989; Wouterson et al., 1989; Appleman et al., 1988; Monticello et al., 1996; Dalbey, 

9 1982). Kamata et al. (1986) did examine additional organs, but there were only 5 animals at 

10 each sacrifice. Similar issues are seen with some of the drinking water studies, where Takahashi 

11 et al. (1986) focus on the stomach and intestines, and the study in Wistar rats by Tobe et al. 

12 (1989) only included 20 animals per sex per exposure group with interim sacrifices. Therefore, 

13 few studies remain to be explored which are informative about the carcinogenic potential of 

14 formaldehyde on the LHP system. Table 4-94 lists the studies from the chronic bioassays which 

15 have the potential to detect LHP malignancies. 

16 Soffritti et al. (1989) were the first to publish a finding of formaldehyde-induced 

17 leukemia in an animal bio-assay. Sprague-Dawley rats (50 per each sex) were exposed to 

18 formaldehyde in drinking water at 0, 10, 50, 100, 500, 1000, 1500 mg/L given ad libitum. 

19 Lymphoblastic leukemia and lymphosarcomas were the most common lesion reported, and 

20 exhibited an apparent dose-dependent increase in both male and female rats (Table 4-95). All 

21 hemolymphoreticular cancers summed together reflected similar trends, but the other types did 

22 not show similar relationship with exposure (e.g. immunoblastic lymphosarcoma). A subsequent 

23 publication of full study results reports all lesions together as lymphoma and leukemia, providing 

24 less-specific information (Soffritti et al 2002.) Additionally, there is a large discrepancy in 

25 reported lesions between the two study reports where nearly double the incidence is reported in 

26 2002. Dr. Soffritti explains the increase in reported LHP malignancies as a result of tabulating 

27 the full histopathological findings in the 2002 report (personal communication, 23 July 2009). 

28 Although the second report from Soffritti et al. (2002) have been broadly criticized on both of 

29 these points (summing of dissimilar lesions and discrepancies in reporting), these errors do not 

30 iupune the original report. The 1989 report does distinguish between different sub-types 

31 providing positive results for lymphoblastic leukemia/lymphosarcoma and histological 

32 examinations were consistently conducted between treatment groups – even if further results 

33 were published at a later timepoint. Therefore, the findings of Soffritti et al. (1989) are 

34 considered as supportive of the biological plausibility of formaldehyde-induced 

35 lymphohematopoietic malignancies.

36
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1 Table 4-94: Summary of chronic bioassays which address rodent leukemia 

2 and lymphoma

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 mortality 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 mortality

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=0.00561

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

5 1. Original statistical analysis provided by Battelle, Columbus Laboratories. Significance set at P<0.0167. Analysis of adjusted 

6 data where time to lesion and survivorship were considered, Cox (1972, Tyrone (1975).

7
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1 Table 4-95. Incidence of lymphoblastic leukemia and lymphosarcoma orally 

2 dosed in Sprague-Dawley rats

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

5 Source: Soffritti et al. (1989)

6

7

8 The two-year bioassay by Til et al. (1989) in male and female Wistar rats indicates no 

9 increase in leukemia and lymphoma, with only 4 tumor-bearing animals in all treatment groups 

10 sacrificed at 24 months. The drinking water levels were similar at the highest dose of both 

11 studies. The major difference in study design is length, which may have influenced results, as 

12 leukemia is a late-life malignancy in rodents. Two-year survival in the Soffritti et al. (1989) 

13 study varied between 50-60%. These animals would be available to develop leukemia after the 

14 two-year window of the Til et al (1989) study. Any potential role of strain differences is 

15 unknown. Overall, the results of Soffritti et al. (1986) are strong, showing an exposure-response 

16 relationship, in a lifelong study, appropriate for late-life malignancies. Unlike the GI tract 

17 tumors, early-life exposure to formaldehyde in drinking water did not increase LHP 

18 malignancies (Soffritti et al., 1989).

19 The largest and most comprehensive study of health effects from formaldehyde 

20 inhalation exposures is the study reported by Kerns et al. (1983) and Swenberg et al. (1980) 

21 conducted at the Columbus Laboratory of Battelle Corporation (1981). Although the summary 

22 reports of this study do not discuss leukemia or lymphoma rates, mouse lymphoma and rat 

23 leukemia were selected by the study pathologist and biostatistician for analysis (Battelle 

24 Laboratory, 1981). Statistical analysis performed by Battelle Laboratories which accounted for 

25 time-to-lesion and survivorship rates did indicate a statistically significant increase in female rat 

26 leukemia (P = 0.0003) and a nearly significant increase in female mouse lymphoma (P = 0.06). 

27 No trend analysis could be performed, as only gross pathology was conducted on mid-dose mice 
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1 and rats (2 and 6 ppm, respectively). EPA has further analyzed these data to better understand 

2 the significance of these findings. The percentage of lymphoma--bearing female mice increased 

3 from 18% in control mice to 28% in mice exposed at 15 ppm for 24 months (6 hr/day, 5 

4 days/wk) (P<0.05). Female rat leukemia was similarly elevated to 26%, 22% and 24% by 

5 inhalation exposure to 15 ppm, 6 ppm and 2 ppm, respectively, versus 16% in controls when 

6 early deaths prior to the first observed leukemia are removed from the analysis (21 months). In 

7 contrast, leukemia was not elevated in formaldehyde-exposed male F344 rats within the same 

8 study. 

9 Differences in study design may account in part for mixed results. The lifelong study by 

10 Soffritti et al. (1989 may have allowed for detection of malignancies developing late in life, 

11 whereas the other drinking water study by Til et al. (1989) sacrificed all animals at 24 months. 

12 Even though the exposure levels were similar, the studies are not directly comparable. Likewise, 

13 it is hard to directly compare results from the two major inhalation studies in rats. Although a 

14 life-long study, the mortality for rats in the Sellakumar et al. (1985) study was greater than 80% 

15 at 2 years. Additionally, the pathology examination was much less rigorous than in the Battelle 

16 Laboratory study, perhaps missing smaller lesions. Therefore, the increase in formaldehyde17 induced leukemia seen in female F344 rats late in life (Battelle laboratories, 1981) may be 

18 reflecting a more sensitive study design. Finally, strain differences may account for different 

19 susceptibilities as well. In summary, the available evidence from chronic animal studies 

20 supports the biological plausibility of the formaldehyde-induced LHP malignancies observed in 

21 epidemiologic studies. The two positive rat studies, by different routes of exposure, along with a 

22 positive result for formaldehyde-induced mouse lymphoma make a substantive case for 

23 formaldehyde-induced LHP malignancies.

24 The epidemiologic studies provide sufficient evidence to conclude that there is a causal 

25 association between formaldehyde exposure and lymphohematopoietic malignancies. When data 

26 are evaluated for all leukemia together, again there is sufficient evidence to establish a causal 

27 association, with consistent positive results in individual studies as well as 3 independent pooled 

28 analyses. Mortality from myeloid leukemia, as well as mortality attributed to “other and 

29 unspecified leukemia” is consistently elevated where reported. In addition, strong evidence for a 

30 causal relationship between formaldehyde exposure and Hodgkin lymphoma is provided by the 

31 consistent associations seen between formaldehyde exposure and Hodgkin lymphoma in the NCI 

32 industrial cohort, with elevations observed across decades of follow-up and significant exposure33 response relationships for all three exposure metrics examined in the most recent follow-up 

34 (Beane Freeman et al., 2009). 

35
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1 4.5.3 Carcinogenic Mode(s) of Action

2 The US EPA 2005 Guidelines for Carcinogen risk Assessment recommend a Mode of 

3 Action (MOA) analysis when data are available for evaluation. The purpose of this MOA 

4 analysis is to determine if sufficient data exist to adequately inform the exposure-response 

5 relationship for cancer below the range of observed data in either human or animal studies. 

6 Since the majority of the data supporting the carcinogenicity of formaldehyde comes from 

7 animal bio-assays and epidemiological studies of workers, EPA must extrapolate from the 

8 observed risk of cancer mortality/incidence in those studies to levels considered protective of 

9 human health for lifelong environmental exposures. In this context, the US EPA cancer 

10 guidelines provide a framework to review MOA information for relevant data to establish an 

11 MOA informing appropriate low-dose extrapolation. 

12 The supporting data for the MOA evaluation of formaldehyde are complex, and presented 

13 across multiple sections of a large document; therefore, this section includes a brief summary of 

14 the biological actions of formaldehyde and key mechanistic data which are believed to be 

15 relevant to the MOA evaluation (Section 4.5.3.1). This information is not intended as a stand16 alone description of the evidence for a particular mechanism, but is intended to highlight the 

17 major supporting arguments and direct the reader to text providing more detailed discussion.

18 The summary of data discussed below combines what is known about the human cancer 

19 of concern (nasopharyngeal cancer, sinonasal cancer, leukemia and other lymphohematopoietic 

20 cancers) with the potential formaldehyde-specific mechanisms of action to postulate 

21 carcinogenic modes of action for each cancer or group of cancers (Section 4.5.3.2 and 4.5.3.2). 

22 The resulting evaluation provides multiple possible MOAs for formaldehyde-induced cancers 

23 where some key mechanistic events may be commonly at work in different tissues, and some key 

24 events may be more relevant to a specific tissue/cancer type. Each of these MOAs is evaluated 

25 with respect to its relevance to human cancer, and the overall weight of evidence for its

26 relevance to formaldehyde-related human cancer.

27 Overall, multiple MOAs considered relevant to humans are presented for each cancer 

28 type. Although some MOAs may have a greater level of supporting evidence, this reflects in part 

29 how well a particular mechanism or key event may have been studied. For example, there are a 

30 large number of studies across many testing systems, and levels of biological organization to 

31 support the mutagenicity of formaldehyde. In contrast other likely MOAs, such as viral 

32 reactivation, have little direct mechanistic evidence, but the available evidence is supportive. 

33 The MOAs considered most relevant to upper respiratory tract cancers (e.g. NPC and 

34 sinonasal cancer) are: 1) direct mutagenicity; 2) inhibition of DNA repair mechanisms; 3) 

35 formaldehyde-induced cell proliferation; 4) cytotoxicity-induced cell proliferation; 5) tumor 
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1 promotion activity; and 6) localized immunosuppression/viral reactivation (Section 4.5.3.2). The 

2 majority of these MOAs would apply equally to immune cells present at the site of first contact 

3 and may also contribute to those lymphohematopoietic cancers which arise from peripheral 

4 immune cells (e.g. Hodgkins lymphoma, multiple myeloma and some forms of leukemia). 

5 Additional MOAs are considered, specifically for formaldehyde-induced leukemia are: 1) 

6 damage of a circulating hematopoietic stem cell or progenitor cell at the site of first contact; and 

7 2) bone marrow toxicity (Section 4.5.3.3).

8 In summary – no single MOA is singled out as the best explanation for cancer resulting 

9 from formaldehyde exposure. Only one MOA - cytotoxicity induced cell proliferation – suggests 

10 an exposure threshold below which the MOA would not be active. However this MOA is the 

11 least applicable to humans and other MOAs are considered operative at exposures below 

12 exposures associated with cytotoxicity-induced cell proliferation. Therefore, multiple MOAs are 

13 considered supported by formaldehyde-specific mechanistic information which provide 

14 biological plausibility for the cancers observed in formaldehyde exposed populations. 

15

16 4.5.3.1 Mechanistic Data for Formaldehyde

17 4.5.3.1.1 DNA Reactivity/Genotoxicity/Mutagenicity

18 An agent’s genotoxic potential and ability to induce mutations is a key consideration in 

19 assessing a carcinogenic MOA, as cancer results from a series of genetic and epigenetic 

20 alterations affecting genes that control cell growth, division and differentiation (Hanahan and 

21 Weinberg, 2000; Vogelstein et al., 1988; Kinzler and Vogelstein, 2002). The US EPA Cancer 

22 guidelines suggest several lines of evidence which are key to evaluating a mutagenic MOA: 1) Is 

23 the chemical under study DNA-reactive and/or has the ability to bind to DNA; 2) Does the 

24 chemical generate positive results in in vitro mutagenic test systems (specifically gene mutations 

25 and chromosomal aberrations); and 3) Does the chemical induce manifestations of genetic 

26 damage in in vivo tests (specifically gene mutations and chromosomal aberrations) and 4) Does 

27 the chemical have properties and structure-activity relationships (SAR) similar to known 

28 mutagens (US EPA, 2005). As reviewed in Section 4.3 above, there is adequate evidence for 

29 formaldehyde-induced genotoxicity and mutagenicity for consideration of these key events in 

30 formaldehyde’s carcinogenic MOA.

31 Formaldehyde induces a variety of genotoxic and mutagenic events when tested both in 

32 vitro and in vivo systems including DNA-protein crosslinks (DPC or DPX), point mutations, 

33 DNA single strand breaks (SSB) and chromosomal aberrations (CAs) (See Section 4.3). 

34 Formaldehyde, as a reactive chemical, also forms DNA adducts and DNA-DNA crosslinks 

35 (DDC) and may act to form adducts between other chemicals and DNA (Brutlag et al., 1969; 
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1 Donecke, 1978; Ohba et al., 1979; Fennel, 1999; Casanova-Schmitz and Heck, 1983; Casanova2 Schmitz and Heck, 1984; Heck and Casanova, 1987 and Casanova et al., 1989). The high 

3 reactivity of formaldehyde results in little specificity indicating that a range of adducts and 

4 crosslinks might be expected. 

5 Numerous studies have shown that formaldehyde induces genotoxic and mutagenic 

6 effects under a variety of experimental conditions (see section 4.3 for a detailed discussion, also 

7 reviewed by IARC 2006; Ma and Harris 1988 and Auerbach et al, 1977). As discussed, 

8 formaldehyde is known to directly react with DNA forming DPC and DNA adducts. Mutations 

9 may occur during repair of formaldehyde-induced DNA damage, or as a result of replication 

10 errors during mitogenesis. Additionally, there is some evidence that DNA single strand breaks 

11 (SSB) may be induced directly by formaldehyde reactivity (Grafstrom et al, 1984). Clastogenic 

12 effects including increased micronuclei (MN), chromosomal aberrations (CAs) and sister 

13 chromatid exchanges (SCEs) are also reported in a range of in vitro study systems. 

14 Formaldehyde caused a concentration-dependent icrease in calstogenicity (e.g. MN) in 

15 human cell lines deficient in either DNA nucleotide excision repair (NER) or DDC repair 

16 systems even though there is no change seen in DPC induction or removal between these cell 

17 lines (Speit et al., 2000). These data suggest that alteration of DNA repair, not DPC removal, 

18 contributes to formaldehyde-induced clastogenicity. Since DPC repair involves proteolytic 

19 removal of proteins from the DNA, the authors hypothesize that single peptides or small peptide 

20 chains cross-linked to the DNA as in the case of DPC are critical to formaldehyde-induced 

21 mutations. 

22 Formaldehyde-induced MN and CAs are associated to concentration-dependent 

23 mutagenic effects in L5178Y mouse lymphoma cells (Speit and Merk, 2002). Detailed analysis 

24 of both spontaneous and formaldehyde-induced lesions indicate that recombination or deletion of 

25 DNA from the thymidine kinase (tk) locus was primarily responsible for the loss of heterogeneity 

26 leading to the observed mutant phenotype. Therefore, it is believed that formaldehyde is 

27 mutagenic by a clastogenic mechanism, rather than through point mutations in the L5178Y 

28 mouse lymphoma cell system. This finding is consistent with Craft et al. (1987) who 

29 demonstrated formaldehyde-induced mutagenicity in the tk locus of TK6 human lymphoblastoid 

30 cells, while Graftsrom et al, (1984) demonstrated increased SSBs in formaldehyde-exposed 

31 human cell lines. The elegant series of experiments by Speit and Merk provide the possible links 

32 between DPC, clastogenicity and locus-specific mutations firmly demonstrating formaldehyde33 induced mutations in the in vitro mouse lymphoma testing system.

34 Formaldehyde is genotoxic at the portal of entry (POE) in animal studies, resulting in 

35 increased DPC formation in the nasal mucosa as discussed above. However, there are no animal 
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1 studies which directly examine the mutagenicity in nasal or respiratory epithelial cells in the 

2 early stages of exposure. It is likely that the mutations are seen in advanced stage of the tissue 

3 around the transformation stage with formaldehyde exposure. With weak positive results in 

4 pulmonary lavage cells (Dallas et al., 1992) and clastogenicity demonstrated in gastro-intestinal 

5 epithelial cells of rats (Migliore et al., 1989) , below exposure levels which trigger regenerative 

6 cell proliferation, the existing evidence, although thin, supports clastogenic effects of 

7 formaldehyde. 

8 Clastogenic effects are consistently reported in humans exposed to formaldehyde in the 

9 industrial workplace or during anatomy or mortuary classes (See section 4.3 for a full 

10 discussion). Increased micronuclei have been reported in nasal epithelial cells from industry 

11 workers (Ballarin et al., 1992; Ye et al., 2005), buccal epithelial cells from anatomy and 

12 mortuary science students and/or staff (Kitaeva et al., 1996; Titenko-Holland et al., 1996; Burgaz 

13 et al., 2001; 2002 compared to corresponding controls). Comparisons of micronuclei in nasal 

14 and buccal cells of anatomy students before and after classes where they are exposed to 

15 formaldehyde indicate an increase in clastogenicity (Ying et al., 1997). An examination of 

16 exfoliated buccal and nasal cells in mortuary students indicates greater increases in centromere17 negative micronuclei, suggesting the effects are due to chromosome breakage or clastogenicity 

18 rather than aneuploidy (Titenko-Holland et al., 1996). Micronuclei were also increased in a 

19 dose-dependent manner in buccal cells as well as peripheral blood lymphocytes (PBLs) in 

20 mortuary students during the course of an embalming class; however, SCEs were reduced in 

21 post-exposure samples (Suruda et al., 1993). Buccal, oral and nasal cells present at the portal of 

22 entry may be directly exposed to formaldehyde and thus reports of clastogenic effects are 

23 consistent with direct interaction of formaldehyde at the POE. There is some supporting 

24 evidence for the mutagenicity of formaldehyde in human populations. Shaham et al. (2003) 

25 reported a increase in mutant p53 protein in the PBLs of individuals with mean formaldehyde 

26 exposure duration of 16 years. Additionally there was is a significant association between 

27 mutant p53 protein and DPC in this study suggesting a relationship between the formaldehyde’s 

28 genotoxic effects. More recently, Zhang et al., (2010) have reported aneuploidy in circulating 

29 hematopietic stem cells in formaldehyde exposed workers with increases in both monosomy7 

30 and trisomy 8.

31 In summary, there are several lines of evidence supporting mutagenic effects of 

32 formaldehyde exposure:

33 1) Formaldehyde directly interacts with DNA generating DPC, 

34 2) DPC in tissues at the POE exhibit a dose-response relationship to formaldehyde 

35 exposure,
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1 3) Formaldehyde-induced DPC are associated with formaldehyde-induced MN and CAs,

2 4) Mutations induced by formaldehyde due to small deletions and rearrangements in 

3 DNA in various experimental systems are consistent with formaldehyde’s observed 

4 clastogenic effects (MN and CAs),

5 5) Formaldehyde-induced mutations and clastogenic effects occur at levels below where 

6 significant cytotoxicity is detected, and

7 6) Formaldehyde exposure has been correlated to similar increased MN and CAs in 

8 human buccal and oral cells corresponding to sites where formaldehyde-induced tumors 

9 arise.

10

11 4.5.3.1.2 Inhibition of DNA repair

12 Studies indicate that formaldehyde exposure may inhibit DNA repair mechanisms 

13 directly (See Section 4.3.1.5). Graftsrom (1985) first documented formaldehyde effects on DNA 

14 repair mechanisms, reporting that formaldehyde treatment of human bronchial fibroblasts in vitro 

15 inhibited repair of O6-methyl-guuanine adducts induced by N-methyl-Nitrosurea (NMU). 

16 Inhibition of DNA repair in human keratinocytes and fibroblasts cultured at 10 μM 

17 formaldehyde affected repair of DNA single strand breaks from ultraviolet light but was specific 

18 to UVB and UVC, not impacting repair of single strand breaks from UVA (Emri et al., 2004). 

19 To determine if formaldehyde may have similar effects in exposed humans, Hayes et al., 

20 (1997) assessed the activity O6-alkylguanine-DNA alkyltranferase (AGT) an enzyme critical in 

21 repairing DNA damage induced by alkylating agents in formaldehyde-exposed mortuary students 

22 previously shown to have increased micronuclei in both buccal cells and peripheral lymphocytes 

23 (Suruda et al., 1993). AGT activity was lower in mortuary students with prior embalming 

24 exposures versus students with no prior exposure (p=0.08). Seventeen of 23 students had lower 

25 AGT activity after the 9 week course (p<0.05) with a larger proportion of naïve students 

26 demonstrating decreased activity (7 of 8) versus previously exposed students (10 of 15). 

27 Although detailed exposure measurements were taken for each student, the changes in AGT 

28 activity were not correlated to cumulative exposure (ppm-hrs).

29

30 4.5.3.1.3 Protein to protein cross-links

31 Formaldehyde is a reactive molecule that is likely to interact with both low molecular 

32 weight cellular components (e.g., reduced glutathione [GSH]) as well as high molecular weight 

33 cellular components. Unlike nuclear DNA, which has additional membrane barriers to exposure 

34 (i.e., nucleus), extracellular and intracellular proteins, are obvious primary targets for interacting 

35 with formaldehyde. Formaldehyde is a well-known cross-linking agent that is used in the 
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1 fixation of tissues, inactivation of toxins and viruses (e.g. preparation of vaccines), and study of 

2 protein-protein interactions (Metz et al., 2006). Using several identical synthetic polypeptides 

3 differing on one amino acid, Metz et al (2004) have shown that formaldehyde initially reacts 

4 with the primary amino and thiol groups of amino acids forming unstable methylol adducts, 

5 which later are partially dehydrated forming labile Schiff bases that are capable of forming 

6 crosslinks with other amino acid residues, such as arginine, asparagine, glutamine, histidine, 

7 tryptophan, and tyrosine through methylene bridges, but not between two primary amino groups. 

8 The same group (Metz et al 2006) has also shown that formaldehyde forms seven intramolecular 

9 crosslinks in proteins with defined structure, such as insulin, involving arginine, tyrosine and 

10 lysine and the N-terminus of insulin was converted to a imidazolidinone adducts similar to that 

11 observed with the synthetic peptide (Metz et al 2004). (Figure 3-1 provides a general reaction 

12 scheme for formaldehyde-mediated modifications of amino acids.)

13

14 4.5.3.1.4 Break-down of the mucociliary apparatus

15 The mucociliary apparatus of the upper respiratory tract is the first line of defense against 

16 airborne toxicants. Comprised of a thick mucus layer (epiphase), hydrophase and ciliated 

17 epithelium, the mucociliary apparatus may entrain, neutralize and remove particulates and 

18 airborne chemicals from inspired air (Figure 4-4). Formaldehyde reacts with the components of 

19 the mucous layer (proteins, glycoprotein, and lipids), crosslinking proteins. Formaldehyde 

20 exposure induces slowing of the mucous flow, stiffing and breaking up of the mucous layer and 

21 eventual mucostasis where gaps have been observed exposing the underlying hydrophase and 

22 epithelium. Although ciliary beat first increases in response to formaldehyde exposure, perhaps 

23 to compensate for reduced flow of the epiphase, ciliastasis ensues with both higher levels of 

24 exposure, and increased duration of exposure. Altered ciliary has been noted in as little as 15 

25 minutes of exposure (1.25 ppm) with functional deficits in the mucociliary apparatus at 30 

26 minutes. Altered ciliary beat has been reported at the lowest concentration tested (0.5ppm) for a 

27 single 6 hour exposure. Severity of effects increase with both duration and level of exposure 

28 (see section 4.2.1.2.1).

29

30 4.5.3.1.5 Induced cell proliferation

31 There are several reports apparently demonstrating formaldehyde-induced proliferation in 

32 cells below cytotoxic levels of exposure. This phenomenon has been reported from studies 

33 involving both in vitro and in vivo exposures. Tyihak et al. (2001) demonstrated significantly 

34 increased cell proliferation in both HT-29 human colon carcinoma and human umbilical vein 

35 endothelial cell (HUVEC) lines treated with 0.1mM (the lowest dose) formaldehyde compared
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1 to untreated controls (P<0.0001). This effect was quantified as both an increase in cell number 

2 over time (Figure 4-35), and an increase in the percentage of cells undergoing mitosis at each 

3 time point. The authors also report a significant (P<0.01) inhibition of apoptosis in 

4 formaldehyde-treated cells as compared to untreated cells (data not shown here). In a novel 

5 system using xenotransplanted human tracheobronchial epithelial cells, formaldehyde was shown 

6 to induce increased cell proliferation at doses below those required for a “massive toxic effect” 

7 (Ura et al., 1989).

8

9

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12

13

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15

16

17

18

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22 Figure 4-35. Effect of various doses of formaldehyde on cell number in (A) 

23 HT-29 human colon carcinoma cells and in (B) human umbilical vein 

24 epithelial cells (HUVEC).

25

26 Values are average of three samples + SD; * P < 0.01 and ** P < 0.0001 

27 compared to corresponding controls.

28

29 Source: Tyihak et al 2001.

30

31

32 Some animal studies have demonstrated increased cell proliferation after formaldehyde 

33 exposures by both inhalation and ingestion (See section 4.2.1). However, whether sustained 

34 increases in cell proliferation over baseline rates are observed upon exposure to sub-cytotoxic 

35 doses of formaldehyde remains unclear. Several of the inhalation studies demonstrate increased 

36 cell proliferation in the nasal epithelium at formaldehyde exposures levels that were sub37 cytotoxic—i.e. in the absence of significant cell death. Acute formaldehyde exposures (1 to 3 

38 days) induced increased cell proliferation at discrete locations in the nasal mucosa, where cell 

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1 proliferation was measured as a labeling index (percentage of cells pulse-labeled with tritiated2 thymidine. Reuzel et al. (1990) reported increased cell proliferation in the nasal passages 

3 including the nasoturbinates, maxilloturbinates septum and lateral wall in male Wistar rats 

4 exposed at 3 ppm, but not at 0.3 or 1 ppm, formaldehyde for 22 hours/day for 3 days. Zwart et 

5 al. (1988) reported increased cell proliferation after exposure to 1 or 3 ppm formaldehyde, 6 

6 hours/day for 3 days or 13 weeks in male and female albino Wistar rats. These increases were 

7 transient at level 3 but sustained at level 2 of the nose and were not correlated with cytotoxicity. 

8 In contrast, Wilmer et al. (1989), from the same group of investigators and using a similar 

9 exposure regimen, reported no increase in cell proliferation after repeated 8–hour exposures at 1 

10 or 2 ppm formaldehyde for 3 days or 4 weeks. Swenberg et al. (1986) demonstrated a transient 

11 increase in cell proliferation after a single 8–hour exposure to 0.5 or 2 ppm formaldehyde in 

12 male F344 rats but no increases after 3 days or repeated 8–hour exposures. The authors suggest 

13 that adaptive responses of the nasal mucosa contribute to the transient nature of formaldehyde14 induced cell proliferation. After a series of acute studies at various formaldehyde concentrations, 

15 Swenberg and coworkers concluded that, in addition to cell proliferation being concentration-, 

16 dose- and time-dependent, the response varies by species and by location of exposure in the nose 

17 (Swenberg et al., 1983, Swenberg et al, 1986). 

18 Other methods of quantifying cell proliferation in the nasal mucosa have demonstrated 

19 formaldehyde-induced cell proliferation at similar low exposure concentrations. Roemer et al. 

20 (1992) measured cell proliferation by flow-cytometry in epithelial cells harvested from the nose 

21 and trachea of male Sprauge-Dawley rats exposed to 2 ppm formaldehyde for 6 hours/day for 1 

22 or 3 days and found increased cell proliferation after the 1-day exposure. These increases were 

23 transient and were not evident after 3 days of exposure. Cassee and Feron (1994) identified 

24 proliferating cells by staining for the presence of proliferating cell nuclear antigen (PCNA). 

25 Formaldehyde exposure at 3.6 ppm for 6 consecutive periods of 12 hours (8-hour exposures 

26 followed by 4-hour-periods of non-exposure) over three days, qualitatively increased the 

27 expression of PCNA in respiratory epithelium at levels 2 and 3 of the nose in albino male Wistar 

28 rats (Cassee and Feron, 1994). Hyperplasia, squamous metaplasia and frank necrosis were also 

29 reported for these tissues.

30 Monticello et al. (1990, 1991, 1996) conducted in vivo cell proliferation studies in which 

31 they exposed F344 rats for short durations (1, 4, 9 and 42 days) as well as much longer durations 

32 (13, 26, 52 and 78 weeks) to exposure concentrations of 0, 0.7, 2.0, 6.0, 10.0 and 15.0 ppm. 

33 These data are unique in that they also included low exposure concentrations. The authors 

34 reported statistically significant increases in cell proliferation only at 6.0 ppm and higher 

35 exposure concentrations in the short duration study and only at 10.0 ppm and higher 
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1 concentrations in the longer duration study. These data have undergone considerable statistical 

2 analysis in several papers as well as in this document. Conolly et al. (2002, 2003) and Gaylor and 

3 Conolly (2004) interpreted these data, when combined, as indicating a non-monotonic behavior 

4 at low dose. In other words, formaldehyde was judged to result in a reduction in cell proliferation 

5 at low dose in comparison to baseline rates, with increased proliferation effect kicking in only at 

6 exposures that were cytotoxic. However, as shown in Appendix 5-3 and in Subramaniam et al. 

7 (2008), Crump et al. (2008), analysis of the individual animal data shows considerable 

8 uncertainty and variability, both quantitative and qualitative, in the interpretation of these cell 

9 proliferation data. (For example, even the control data vary over an order of magnitude in some 

10 cases. See Figures 5-22 and 5-23 in Appendix 5-3.) These analyses (which were based on the 

11 replicate animal data used in the above studies) considered regional formaldehyde dose to the 

12 tissue (flux), nasal site and duration of exposure, as well as the number of cells at a given site.

13 The overall conclusion in Section 5.3.3 (and detailed in Appendix 5-3) is that the cell 

14 proliferation dose-response at low dose could be reasonably described by both monotonic (with 

15 and without a threshold) and non-monotonic curves. 

16 Only one study, by Monticello et al. (1989), quantified cell proliferation in primates after 

17 formaldehyde exposure; this study, reported an 18-fold increase in cell proliferation in the nasal 

18 epithelium (respiratory and transitional), larynx, trachea and carina of male Rhesus monkeys 

19 exposed to 6 ppm formaldehyde compared to controls (See section 4.2.1 for detailed study 

20 description). The authors also noted that increased cell proliferation was seen in locations with 

21 minimal histological changes, indicating proliferation may be a more sensitive predictor of 

22 adverse health effects of formaldehyde exposure. 

23

24 4.5.3.1.6 Cytolethality and resulting regenerative cell proliferation

25 The toxic and cytolethal effects of formaldehyde exposure at the POE are well 

26 documented after both inhalation and oral exposures (See Section 4.2.1). The nature and 

27 progression of tissue injury has been best documented in rodent inhalation assays. Early effects 

28 on the nasal mucosa include altered ciliary beat and mucus flow, hyperplasia and metaplasia of 

29 nasal epithelium (Morgan et al., 1986a; Morgan et al., 1986b; Monteiro-Riviere and Popp, 1986; 

30 Maronpot et al 1986; Rusch et al., 1983 and Monticello et al., 1986). These first changes may be 

31 considered adaptive responses. Squamous epithelium may thicken and transitional epithelium 

32 may change to squamous epithelium as evidenced by squamous hyperplasia, squamous 

33 metaplasia and thickening of the epithelium in these anterior portions of the nose. Tissue 

34 damage may be transient at lower formaldehyde exposures as these changes serve to protect 

35 tissue from formaldehyde’s reactivity. However, higher formaldehyde concentrations can 
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1 overwhelm these adaptive responses and result in gross tissue damage. Frank necrosis and focal 

2 erosions have been reported in time- and concentration-dependent manner in rodent bioassays. 

3 Both adaptive changes and cytolethality are associated with cell proliferation. However, 

4 where adaptive changes are successful, e.g. prevent continued toxic insult to the tissue, cell 

5 proliferation is transient. Exposure regimens where the adaptive changes are not adequate to 

6 protect the tissue, would result in continued cytoxicity and cell death. Sustained damage to the 

7 epithelium would result in sustained cell proliferation to compensate for cell death. A series of 

8 rodent bioassays present convincing evidence that chronic inhalation exposures 6 hours a day, 5 

9 days a week at 6, 10 and 15 ppm formaldehyde do result in sustained damage to the nasal 

10 epithelium, sustained cell proliferation and tumor development (Kerns et al., 1983, Morgan et al., 

11 1986; Monticello et al., 1990; Monticello et al., 1991 and Monticello et al., 1996). Work by 

12 Monticello and coworkers demonstrate that chronic repeated inhalation exposures at 6, 10 or 15 

13 ppm formaldehyde result in sustained cell proliferation at the lateral meatus, mid-septum and 

14 maxilloturbinates of rat nasal passages (Monticello et al., 1991 and Monticello et al., 1996). 

15

16 4.5.3.1.7 Evidence for promotion 

17 There is some evidence, although mixed, that formaldehyde may promote tumor 

18 development by other carcinogens, and known initiating agents by various routes of exposure. 

19 Formaldehyde exposure in drinking water (0.5% formalin) increased glandular stomach 

20 adenocarcinomas in male Wistar rats after initiation with 100 mg/L, N-methyl-N’-

21 nitrosoguanidine (MNNG), compared to MNNG-only-treated rats (Takahashi et al., 1986). In 

white non-inbred rats, inhalation exposures (3, 30 or 300 ug/m3 22 formaldehyde 7hr/day, 5 

23 days/week for 1 year) increased tumor multiplicity per animal and decreased latency of 

24 benzo[a]pyrene induced tumors in white non-inbred rats (Yanysheva et al., 1998.) Similarly, 

25 formaldehyde skin application decreased tumor latency, in 7,12-dimethylbenz(a)anthacene 

26 (DMBA) initiated hairless Oslo mice (Iversen, 1986). Although formaldehyde exposure also 

27 increased the tumor multiplicity in Syrian golden hamsters where diethylnitrosamine (DEN) 

28 (0.25mg I.P.) was the tumor initiator, positive results were only reported for the exposure 

29 regimen where hamsters were exposed to formaldehyde via inhalation 48 hours prior to DEN 

30 injection, and then one a week thereafter for life. However, formaldehyde did not increase the 

31 number of tumors per tumor bearing animals when only administered beginning one week after 

32 all DEN injections. In contrast, bladder cancer was not enhanced by intravesical instillation of 

33 0.5ml of 0.3% formalin, one week after instillation of N-methyl-N-nitrosourea (MNU) in male 

34 Fisher rats (Homma et al., 1986).
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1 The observed promotion activity of formaldehyde has been tested in several systems, by 

2 different routes of exposure. By several routes of exposure, formaldehyde enhanced tumor 

3 development at a site where formaldehyde did not induce tumors alone, without the initiating 

4 agent (Takahashi et al., 1986, Yanysheva et al., 1998 and Iversen, 1986). Promotion activity in 

5 these studies was evidenced by increased in tumor bearing animals (oral route), increase in 

6 tumors per animal (inhalation routes) and decreased tumor latency compared to those animals 

7 only exposed to the initiating agent (inhalation route) (Takahashi et al., 1986, Yanysheva et al., 

8 1998 and Iversen, 1986). Although these experiments do not indicate how formaldehyde acts as 

9 a promoter in these systems, it is possible formaldehyde-induced mutation, increased cell 

10 proliferation or other toxic action could enhance tumor development from another agent.

11

12 4.5.3.1.8 Localized Immunosuppression

13 Formaldehyde exposure has induced localized immune suppression in experimental 

14 animals (Dean et al., 1984) and in exposed workers (Lyapina et al., 2004). Repeated inhalation 

15 exposures in rodents depopulated the URT and pulmonary tissues of resident macrophages, 

16 resulting in a transient decrease in POE host defenses (Admas et al., 1987). After cessation of 

17 exposure, the mononuclear phagocyte (MP) populations were replenished and there was a 

18 subsequent increase in host defense representing both increased MP numbers and increased 

19 bacteriocidal activity of the MPs. These data suggest that peak exposures of formaldehyde may 

20 present localized immunosuppression for components of the mononuclear phagocyte system 

21 (MPS) in tissues at the site of first contact. 

22 A number of studies have evaluated the ability of formaldehyde to induce systemic 

23 immunotoxic effects in humans (Ohtani et al., 2004a, b; Erdei et al., 2003; Thrasher et al., 1990, 

24 1987; Pross et al., 1987). Some studies have reported altered innate immune responses 

25 associated with formaldehyde exposure (Erdei et al., 2003), while others have noted adaptive 

26 immune response suppression associated with formaldehyde exposure (Thrasher et al., 1990, 

27 1987) and changes associated with alterations to a predominant T—lymphocyte helper 2 (Th2) 

28 pattern (Ohtani et al., 2004a, b). In contrast, Pross et al. (1987) did not observe formaldehyde29 associated changes in systemic immune function. 

30 Numerous studies have reported increased respiratory tract infections in formaldehyde 

31 exposed individuals both in occupational and residential environments (Lyapina et al., 2004; 

32 Krzyzanowski et al., 1990; Holness and Nethercott, 1989). Incidences of physician-diagnosed 

33 chronic bronchitis were more prevalent in children (under age 15) living in homes with higher 

34 formaldehyde (>60 ppb) readings in the kitchen (p < 0.001) but this effect was more pronounced 

35 (p < 0.001) in children simultaneously exposed to environmental tobacco smoke (Kryzanowski 
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1 et al., 1990). The prevalence of chronic cough was also increased in adults living in homes with 

2 measurable levels of formaldehyde, but data were not shown. Holness and Nethercott (1989) 

3 assessed chronic bronchitis in 87 funeral workers, where the average formaldehyde exposure was 

4 reported at 0.38 ± 0.19 ppm. Chronic bronchitis was observed in 20 funeral workers (n = 87) 

5 exposed to formaldehyde compared with 3 cases of chronic bronchitis in nonexposed referent 

6 controls (n = 38). A statistically significant association of self-reported chronic bronchitis and 

7 decreased resistance to URT infection was reported in formaldehyde exposed workers compared 

8 with controls (p = 0.02) (Lyapina et al., 2004). Of the workers, 41% had a history of chronic 

9 respiratory infection and frequent long-lasting infectious inflammatory relapses (group 1a). 

10 Another group (group 1b) consisted of 17 exposed workers, 12 of whom had no history of 

11 recurrent viral infections of the URT. There was a statistically significant association of 

12 frequency and duration of inflammatory relapses between groups 1a and 1b.

13 Lyapina et al. (2004) also reported effects of formaldehyde exposure on neutrophil 

14 respiratory burst activity (NRBA), the capacity of polymorphonuclear leukocytes to produce 

15 reactive oxygen radicals in response to chemical or microbial stimuli using flow cytometry. A 

16 suite of hematological tests and flow cytometric analysis for respiratory burst activity were 

17 performed. Although no significant difference was observed in the spontaneous and stimulated 

18 NRBA (median percentage of oxidizing cells) between the 29 exposed workers with URT 

19 inflammation and the healthy controls (0.83 versus 1.35, respectively), a separate comparison of 

20 the NRBA of 12 workers with chronic, repeating URT infections and 17 workers with short, 

21 infrequent episodes of URT inflammations was significant (0.45 versus 1.00, p = 0.037). When 

22 the NRBA of the group with chronic URT infections (n = 12) was separately compared with that 

23 of the healthy controls (n = 21), the results were also significant (0.45 versus 1.35, p = 0.012). 

24 Individuals with chronic URT infections have reduced NRBA that could be due to formaldehyde 

25 exposure. Neutrophils respond to tissue damage or local invasion of microorganisms and act to 

26 phagocytize foreign cells. If neutrophilic activity is hampered or altered by formaldehyde 

27 exposure, then the ability to fight infection will be diminished, leading to prolonged infection. 

28 However, no dose-response pattern of formaldehyde exposure could be determined from this 

29 study.

30

31 4.5.3.1.9 Potential for systemic transport of formaldehyde

32 In aqueous solution formaldehyde exists in equilibrium with it’s hydrated form 

methanediol (CH2OH2) (Kd = 5.5x10-

4 33 ). The equilibrium favors methanediol at physiological 

34 temperature and pH (>99.9%) and is readily reversible. In biological systems, as free 

35 formaldehyde is removed from aqueous solution through binding with serum proteins and 
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1 cellular components, the equilibrium is reestablished by dehydration of methanediol to free 

2 formaldehyde. The reversible nature of this hydration reaction describes how a pool of free 

3 formaldehyde may be sustained in biological systems.

4 There is strong and consistent evidence in biological testing systems in vitro that treating 

5 cells with formaldehyde in an aqueous media results in significant cytoxicity, cell proliferation, 

6 clastogenic effects and clear evidence of mutational events (Section 4.3). Similarly, animal 

7 bioassays where formaldehyde is administered in drinking water report portal of entry toxicity 

8 including hyperplasia, increased cell proliferation, focal lesions and tumors (Section 4.2.1). It 

9 should be noted that URT tissues are covered by an aqueous mucous layer, through which 

10 formaldehyde must pass to react the cellular components of the URT. It has been postulated that 

11 formaldehyde transports through this mucous layer and the underlying tissues as methanediol 

12 (Georgieva et al., 2003).

13 The dynamic equilibrium between the hydrated and unhydrated forms of formaldehyde in 

14 biological systems is well understood. Since the hydration reaction favors methanediol, it is 

15 expected that exogenous formaldehyde which reaches the blood will primarily exist as 

16 methanediol and is subject to physiological elimination. As free, unhydrated formaldehyde 

17 continues to react with serum proteins and cellular components, the blood levels of methanediol 

18 are expected to reduce as it is dehydrated to maintain equilibrium. Although some attempts to 

19 measure significant changes in free formaldehyde levels in blood after inhalation exposure have 

20 not been successful, the half-life in blood has been measured after i.v. injection at approximately 

21 2 minutes (McMartin et al., 1979). Additionally, the detection of antibodies to formaldehyde22 hemoglobin adducts and formaldehyde-albumin adducts in exposures workers, smokers and 

23 laboratory animals exposed via inhalation provides direct evidence that formaldehyde is able to 

24 react with serum albumin and hemoglobin in biological systems (Thrasher et al., 1990, Grammer 

25 et al., 1990; Grammer et al., 1993; Dykewicz et al., 1991; Varro et al., 1997 and Li et al., 2007). 

26 These data support the hypothesis that exogenous formaldehyde may reach and transport through 

27 the blood. If so, formaldehyde (or methanediol) may reach sites distal to the portal of entry. 

28

29 4.5.3.2 Mode of Action Evaluation for Upper Respiratory Tract Cancer (Nasopharyngeal 

30 Cancer, Sino-nasal)

31 From the above discussion, it can be seen that numerous mechanisms of action for 

32 formaldehyde-induced cancer can be reasonably supported based on various known biological 

33 actions of formaldehyde (e.g., mutation, cell proliferation, cytotoxicity, and regenerative cell 

34 proliferation). Additionally, alternative actions, such as immunosuppression or viral 

35 reactivation, are possible, although less data exist to evaluate these MOAs. Rather than a single 
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1 MOA, it is plausible that a combination of these factors contribute to cancer incidence in an 

2 exposed population. Considering multiple factors may help to better understand the biological 

3 and mechanistic basis for the increases in cancer incidence observed in exposed human 

4 populations. Unlike animal bioassays, human epidemiological studies may reflect not only the 

5 effects of the agent of concern but also numerous other risk factors (e.g., viral status, diet, 

6 smoking, etc.). Additionally, human studies may be impacted by biological human variability 

7 across individuals, cancer biology (sub-types), wide variability in exposure regimens in human 

8 populations, etc. Therefore, if the purposes of exploring the carcinogenic MOA of an agent are 

9 to better understand the relevance of a given carcinogen to human populations and to inform the 

10 exposure-response analysis, then discussions of MOAs which recognize the interaction of an 

11 agent with human variability and various risk factors is an appropriate analysis.

12

13 a) Direct mutagenicity of formaldehyde in cells at the site of first contact: Mutations, 

14 the permanent heritable changes in the genome of the cell, are a primary mechanism for 

15 the activation of oncogenes or the inactivation of tumor suppressor genes. Mutagenicity 

16 is the most widely recognized determinant of chemical-induced carcinogenicity, and it is 

17 difficult to set aside the relevance of direct formaldehyde-induced mutations from its 

18 demonstrated carcinogenicity. Formaldehyde-induced mutation in mucosal cells of the 

19 URT, throat and buccal cavity may serve to initiate cells, or provide subsequent mutageic 

20 events to already initiated cells. Since the mucosal cells have proliferative capacity, and 

21 cell proliferation is a normal tissue function, mutations may be fixed and passed to 

22 daughter cells due to baseline cell proliferation of the tissue. 

23 Relevance to humans: This MOA is relevant to humans. The well-documented DNA 

24 reactivity (e.g. DPC and DNA adducts) and clastogenicity of formaldehyde in the URT of 

25 laboratory animals is a direct effect of formaldehyde on tissues of first contact. As this is 

26 a direct acting agent - no distribution or metabolism is required for the genotoxic action –

27 there is little expected species variability. As discussed in chapter 3, there are species 

28 differences in flux of formaldehyde into the respiratory mucosal tissues, but this 

29 introduces species differences in dosimetry – not mechanism. Finally, the clastogenic 

30 effects in nasal and buccal epithelial cells in formaldehyde- exposed workers confirms 

31 the direct genotoxic effects of formaldehyde at the first site of contact in humans.

32

33 b) Decrease in DNA repair function within cells at the site of first contact: A decrease 

34 in DNA repair capacity in these tissues by formaldehyde may increase total mutations 

35 over time due to either endogenous or exogenous sources of mutation. Although there 
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1 are only a few studies which have explored the potential for formaldehyde to reduce 

2 DNA repair capacity, the evidence is positive, both in vitro testing systems, and in one 

3 study of occupationally exposed humans (Grafstrom 1985; Hayes et al., 1997). 

4 Relevance to humans: This MOA is considered relevant to humans. The general 

5 population is exposed to various carcinogens, many with mutagenic potential, at sites of 

6 first contact including; air pollution, tobacco products, nitrosamines and viruses. 

7 Additionally, there are endogenous sources of DNA damage and mutagenicity in humans 

8 (e.g. lipid peroxidation, oxidative stress). The demonstration of reduced DNA repair 

9 activity (O6-alkylguanine-DNA alkyltransferase activity) in formaldehyde-exposed 

10 mortuary students suggests this toxic action of formaldehyde is possible in humans.

11

12 c) Formaldehyde-induced cell proliferation: Formaldehyde-induced cell proliferation in 

13 the oral and respiratory mucosa may be considered a key event in conjunction with the 

14 genotoxic effects, and induced mutational events observed with formaldehyde exposure. 

15 This MOA is intended to describe events which occur below exposure levels which 

16 induce cell death and mucosal lesions. Therefore this MOA is comprised of two key 

17 events: 

18 a. Formaldehyde-induced genotoxicity or mutation

19 b. Formaldehyde-induced cell proliferation 

20

21 DNA replication during cell proliferation may serve to translate DNA damage or a 

22 formaldehyde-related DNA lesion into a permanent change in the sequence of nucleic 

23 acids during replication of the DNA– e.g. ‘fix’ a mutation from DNA damage. 

24 Additionally formaldehyde-induced cell proliferation may provide an opportunity for 

25 initiated cells to proliferate, increasing the potential for additional mutation events and 

26 transformation. The increased cell proliferation observed in the mucosal tissues in direct 

27 contact with formaldehyde during inhalation exposures may serve to amplify the risk of 

28 cell transformation from mutation alone. Researchers have noted that increased cell 

29 proliferation may be transient in some locations as adaptive responses compensate 

30 (Swenberg 1983). However, evidence in both monkeys and rodents indicate that 

31 increased cell proliferation in repeated exposures across time do result in sustained cell 

32 proliferation. Data in Rhesus monkeys indicates increased cell proliferation is observed 

33 beyond the nasal cavities to the larynx, trachea and carnia (first tracheal 

34 branching)(Monticello et al., 1989). Additionally, the authors note that cell proliferation 
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1 is a more sensitive indicator of effects on the epithelium, observed even when minimal 

2 histological changes were present. 

3 Human Relevance: Both formaldehyde-induced mutation and cell proliferation are 

4 direct effects on the oral and nasal mucosa, well documented in rodent models with 

5 supporting evidence in human epidemiological studies. Therefore both key events are 

6 relevant to humans. As noted above, there are species differences in localized flux of 

7 formaldehyde into the tissues of the oral and respiratory tract based on structural 

8 differences in the airways, as well as breathing patterns. Although these differences may 

9 effects the dosimetry of the formaldehyde absorption into the tissues, this only influences 

10 the magnitude of response at any given location. Data from exposed Rhesus monkeys 

11 which documents formaldehyde-induced cell proliferation in tissues beyond the nasal 

12 cavity, and tissues with minimal histological changes supports a role for cell-proliferation 

13 in the observed cancers in humans, which occur beyond the nasal cavities, and in tissues 

14 without formaldehyde-related focal lesions.

15

16 d) Cytotoxicity-induced cell proliferation (CICP): Cell death followed by compensatory 

17 cell proliferation is a reasonable MOA for agent-induced cancer. It should be noted that 

18 the exposure conditions which result in CICP in rodents is known to result in significant 

19 DNA reactivity and genotoxicity. Therefore, formaldehyde-induced mutations cannot be 

20 excluded from this MOA. The animal bioassays support the carcinogenic potential of 

21 formaldehyde in this context (Kerns et al., 1983; Selkemur et al, 1983 and Monticello et 

22 al 1986). The majority of squamous cell carcinomas (SCCs) seen in formaldehyde23 exposed rats have been localized to the lateral meatus and mid-septum in the nasal 

24 passages (Morgan et al., 1986; Monticello et al., 1996), while polyploid adenomas have 

25 predominantly been reported at the maxilloturbinates (Morgan et al., 1986; Monticello et 

26 al., 1996). Morgan et al. (1986) speculated that the maxilloturbinate was less susceptible 

27 to SCC due to metabolic differences. However, Monticello et al. (1996) later suggested 

28 that the smaller population of cells available at the maxilloturbinate accounted for fewer 

29 SCCs observed at that site. Regardless, for those locations where SCCs do arise in rats 

30 chronically exposed to formaldehyde, a clear temporal relationship can be demonstrated 

31 for dose regimens capable of producing sustained epithelial damage and sustained cell 

32 proliferation to eventual tumor development. Conversely, tumors are not observed in 

33 these rodent models at those sites in the nasal passages without sustained cell 

34 proliferation. 

35
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1 Relevance to humans: Human exposure to formaldehyde would most likely involve 

2 chronic exposures to indoor levels of formaldehyde, and episodic exposures in the 

3 environment or from an occupational exposure (See review in Chapter 2). An exposure 

4 scenario parallel to that used in chronic rodent bioassays is unlikely (e.g. 2-15 ppm 6-8 

5 hours/day, 5 days/week for 10-30 months). Exposure conditions are difficult to assess 

6 especially in retrospective studies. However, only the most extreme industrial work 

7 conditions would result in human exposures similar to those that produce sustained 

8 compensatory cell proliferation in animal studies (i.e. 6-15 ppm 6 hrs/day, 5 days per 

9 week). Gross tissue lesions as reported in rodents from repeated chronic exposures at 6 

10 and 10 ppm formaldehyde have not been reported from workplace exposure, and only 

11 minor histopathological changes have been noted (Boysen et al., 1990 and Holmstrom 

12 and Wilhelmsson et al., 1989). It is possible that workers were episodically exposed to 

13 formaldehyde levels which resulted in cell death and focal or gross lesions requiring cell 

14 proliferation for tissue remodeling or repair. However, it is unexpected that these 

15 conditions would be relevant to human environmental exposures. Therefore, although 

16 regenerative cell proliferation is retained as a reasonable MOA for formaldehyde 

17 carcinogenicity in experimental animals, it is unclear whether it is relevant to the 

18 extrapolation of health risks to formaldehyde exposures in the general environment.

19

20 e) Promotion: Several animal studies indicate that formaldehyde exposure may promote 

21 tumor formation due to other carcinogenic or initiating agents. There are positive data 

22 by several routes of exposure (oral, dermal and inhalation) and promotion has been 

23 reported as an increase in tumor bearing animals, an increase in tumors multiplicity or a 

24 decrease in tumor latency with formaldehyde exposure in conjunction with the initiating 

25 agent compared to tumors from the initiating agent along, or formaldehyde alone. The 

26 specific key events which may explain this promotion effect are unknown but may 

27 include several of the mechanisms discussed as potential MOAs for formaldehyde: 

28 mutagenicity, mitogenesis, co-carcinogenicity, immunosuppression. Promotion is 

29 considered here as a separate MOA, since these activities are noted for experimental 

30 conditions and tumor sites where formaldehyde did not induce tumors in the absence of 

31 the initiating agent. 

32 Relevance to humans: Although the human epidemiologic literature doesn’t address 

33 issues of tumor promotion, the nature of the cancers of concern indicate that chemical 

34 promotion may be relevant to cancer incidence for these sites. Many of the risk factors 

35 for NPC and other mouth and oral and URT cancers include direct mutagens (e.g. 
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1 smoking, dietary nitrosamines) where a promoting agent would be expected to increase 

2 cancer incidence with these other risk factors. Additionally, the well known viral risk 

3 factors for cancers of the mouth and URT also suggest a role for promoting agents to 

4 human cancer incidence. Although only tangential evidence, this does suggest that the 

5 promoting activity of a chemical agent, would be relevant to the agent’s carcinogenicity 

6 at these sites. Therefore, the potential for formaldehyde to act as a promoter with other 

7 initiators – is considered relevant to formaldehyde’s carcinogenic MOA. 

8

9 f) Increased URT infections / viral reactivation: Inhalation exposure to formaldehyde 

10 has been shown to decrease the defenses of the body against infection through two 

11 mechanisms: 1) damage to the protective mucous barrier and function of the mucociliary 

12 apparatus; and 2) localized immunosuppression. These effects have been demonstrated 

13 in both exposed humans and controlled animal experiments. Additionally, increased 

14 respiratory tract infections are associated with formaldehyde exposure in several 

15 populations. Common viral agents (e.g. Epstein barr virus) are known risk factors for 

16 NPC, sinonasal cancers and other URT cancers. Although direct evidence does support 

17 increased URT infections due to formaldehyde exposure, and URT infections are 

18 considered risk factors for URT cancers, direct evidence for formaldehyde-related 

19 infections leading to cancer is lacking. There is however one epidemiological study 

20 which finds the association between formaldehyde and NPC is strengthened in Epstein 

21 barr virus sero-positive cases versus sero-negative cases. These data suggest a possible 

22 role for formaldehyde in infection, viral reactivation, or co-carcinogenicity with a viral 

23 agent. 

24

25 Relevance to humans: The potential role of increased URT infections and 

26 immunosuppression at the portal of entry is considered to relevant to humans. Data in 

27 humans are available to support both key events in this MOA. Additionally, 

28 epidemiological studies are conducted in human populations where individuals may be 

29 exposed to various viral agents across the study period. Therefore, toxic actions by 

30 formaldehyde which may increase URT infections, or viral-reactivation at the site of first 

31 contact, could be acting in conjunction with viral agents to contribute, in part, to observed 

32 associations between formaldehyde exposure and increased URT cancer.

33

34 Summary and integration of key events:

35 Each of the hypothesized MOAs discussed above to better understand the carcinogenic 

36 potential of formaldehyde is supported by formaldehyde-specific evidence, either in animal 
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1 studies, human studies or both. For those key events studied in animal models such as cell 

2 proliferation, genotoxicity, degradation of the mucociliary apparatus and CICP, supporting 

3 evidence is available in more than one species, multiple strains (e.g. rats) and has been reported 

4 by multiple researchers. Therefore the overall database supporting these key events in laboratory 

5 studies, and their corresponding MOAs is fairly large. In contrast, some key events relevant to 

6 humans, but less studied in animal models may have a small supporting database (e.g. increased 

7 respiratory tract infections). These alternative MOAs are retained as potentially relevant to the 

8 carcinogenic action of formaldehyde as the intent of this discussion is to identify modes of action 

9 will may contribute to the observation of increased upper respiratory tract cancers in exposed 

10 human populations. It is noted that additional study is needed to better understand the range of 

11 effects formaldehyde may have at sites of first contact in humans. 

12 The MOAs which include genotoxicity, mutation, decreased DNA repair, increased cell 

13 proliferation and CICP are interrelated. Conditions which provide both a source of cell 

14 proliferation and increased mutation would be expected to increase neoplastic transformation. 

15 Formaldehyde acts on the target tissue, the respiratory epithelium, to induce each of these events. 

16 However, these key events operate across different exposure ranges and present different 

17 exposure response relationships. For example, formaldehyde-induced mutations would be 

18 expected across the exposure range, where any incremental increase in genotoxicity and 

19 formaldehyde-related mutation would contribute to background levels, with the potential to 

20 increase cancer risk incrementally. In contrast, focal and gross lesions to the respiratory mucosa 

21 due to cytolethality are not observed unless exposure concentrations are sufficient to provide 

22 localized tissue doses (flux) required to result in cell death and related compensatory cell 

23 proliferation. Since tissue dose (flux) is dependent on not only exposure concentration but also 

24 duration of exposure and location in the respiratory tract (Section 3.4), and varies by species, 

25 correlation of exposure concentrations to tissue responses directly are complex. Exposure 

26 response relationships for the key events (cell proliferation, genotoxicity, degradation of the 

27 mucociliary apparatus and CICP) are reported by exposure concentration, not tissue flux, which 

28 would be a more biologically relevant measure.

29 Although the tissue dose-response relationships for formaldehyde induced mutation, 

30 mitogenesis and cytolethality are different, the effects at the tissue level cannot be easily 

31 disaggregated. At any given exposure concentration, target cells in the respiratory tract will 

32 experience different effective tissue concentrations of formaldehyde. Measurement of cell 

33 proliferation, DNA protein crosslinks or genotoxicity may require examining a population of 

34 cells which would have been subject to different flux rates of formaldehyde (See chapter 3). 

35 Similarly, when evaluating the tumor dose response, cells within the target tissue will represent a 
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1 range of target tissue formaldehyde concentrations. Therefore, an integrated MOA scheme is 

2 hypothesized where key events may influence the observed tumor response differentially across 

3 the exposure response range (Figure 4-36). This schematic illustrates the potential for 

4 genotoxicity and formaldehyde-induced mutation to occur where tissue dose (flux of 

5 formaldehyde into the tissue is minimal). Where tissue dose is increased, formaldehyde-induced 

6 cell proliferation is observed in addition to genotoxicity. As tissue dose increases and 

7 formaldehyde effects on the respiratory mucosa are more severe, gross pathology including focal 

8 and gross lesions due to cell death are noted. Therefore, several of the MOAs presented above 

9 may be operative and relevant to human exposures at exposure levels resulting in minimal tissue 

10 flux – a) direct formaldehyde genitoxicity and resulting mutation, b) inhibition of DNA repair 

11 and c) formaldehyde induced cell proliferation in conjunction with mutation. CICP, which 

12 involves localized and gross tissue lesions would be operative at higher exposure levels. There is 

13 little data to inform the dose range over which the remaining hypothesized MOAs may operate 

14 (promotion and increased respiratory tract infections/viral action).

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

17 Figure 4-36: Integrated MOA scheme for respiratory tract tumors 

18

19 4.5.3.3 Mode(s) of Action for Lymphohematopoietic Malignancies

20 4.5.3.3.1 MOA evaluation for Leukemia

21 Leukemia may arise from stem cells and progenitor cells in the bone marrow (e.g. acute 

22 and chronic myeloid leukemia) or from mature lymphocytes (e.g. chronic lymphatic leukemia, 
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1 hairy cell leukemia) (Figure 4-33, Section 4.5.2). Although there is a consistent association 

2 between formaldehyde exposure and forms of leukemia when considered as group of diseases 

3 (Table 4-91, Section 4.5.2), the strongest and most consistent associations are seen specifically 

4 with myeloid leukemia. Little evidence supports an association between formaldehyde exposure 

5 and other specific leukemia subtypes, although two studies support a strong association between 

6 formaldehyde and “other leukemia and unspecified leukemia (ICD-9 code 207). Therefore, this 

7 MOA evaluation will focus on mechanisms which may impact all forms of leukemia (e.g. bone 

8 marrow toxicity) or those specific to myeloid leukemia. The mechanistic data supporting the key 

9 events in this analysis are presented in section 4.5.3.1.

10

11 a) Direct effects of formaldehyde on a circulating stem cell or progenitor cell present at 

12 the portal of entry: Hematopoietic stem cells do circulate throughout the body and can 

13 be harvested from peripheral blood. Formaldehyde exhibits a range of toxic effects at the 

14 site of first contact including genotoxic effects believed to be mediated by direct DNA 

15 reactivity (Section 4.3). Formaldehyde is known to directly react with blood components 

16 in formaldehyde exposed humans and animals resulting in both hemoglobin and albumin 

17 adducts (Thrasher et al., 1990, Grammer et al., 1990; Grammer et al., 1993; Dykewicz et 

18 al., 1991; Varro et al., 1997 and Li et al., 2007). Therefore, it has been hypothesized that 

19 formaldehyde could react with DNA in circulating hematopoietic stem cells (Zhang et al., 

20 2009) resulting in heritable mutations which may contribute to leukemia incidence. 

21 Recently Zhang et al. (2010) have tested the hypothesis that exogenous formaldehyde 

22 may damage circulating stem cells. Clastogenic effects were found in circulating 

23 hematopoietic stem cells cultured from formaldehyde exposed workers. The reported 

24 aneuploidy was demonstrated as significant increases in both monosomy 7 and trisomy 8. 

25 These specific chromosomal changes are consistent with those reported for agent-induced 

26 myeloid leukemia (Zhang et al., 2010).

27 Relevance to Humans: This hypothesized MOA is considered relevant to humans. 

28 Supporting evidence is found in humans for formaldehyde direct reactivity with blood 

29 proteins (e.g. albumin and hemoglobin) as well as clastogenic effects in circulating 

30 hematopoietic stem cells in formaldehyde exposed workers.

31

32 b) Bone marrow toxicity: Direct bone marrow toxicity is the most studied leukemogenic 

33 action for an endogenous agent (e.g. benzene, ionizing radiation). It is believed that an 

34 agent which exerts its toxicity on the bone marrow, resulting in translocations and 

35 heritable mutations in hematopoietic stem cells may cause leukemia. It has been 
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1 hypothesized that formaldehyde may transport to the bone marrow in its hydrated form 

2 (methandiol) and react with cellular proteins, and DNA casuing direct effects on 

3 components of the bone marrow. Pancytopenia (a reduction in blood borne cells formed 

4 in the bone marrow) is a symptom of direct bone marrow toxicity and is observed with 

5 other leukemogenic agents (e.g. benzene, ionizing radiation). A recent review of 8 

6 published studies of formaldehyde exposed workers in China by Tang et al. (2009) 

7 indicates 7 of the studies provide evidence of reduced white blood cell counts, platelet 

8 levels and hemoglobin levels associated with formaldehyde exposure. A study of 

9 occupationally exposed nurses provided a correlation between decreased white blood cell 

10 counts and formaldehyde exposure (Kuo et al., 1997). A recent study by Zhang et al. 

11 (2010) provides the best evidence for bone marrow toxicity, where they report not only a 

12 reduction in white blood cell counts, but reductions in cell counts of all the blood cells, as 

13 well as increased mean cell volume. Although these reductions did not meet the clinical 

14 definition of pancytopenia (when averaged across the study population), reduction of all 

15 blood borne cells formed in the bone marrow is consistent with the bone marrow toxicity 

16 associated with pancytopenia seen with other leukemogens (Zhang et al., 2010). 

17 Relevance to Humas: This hypothesized MOA is considered relevant to humans. 

18 Supporting evidence is found in humans for bone marrow toxicity in formaldehyde 

19 exposed workers.

20

21 4.5.3.3.2 MOA evaluation for Lymphomas (e.g. Hodgkin lymphoma, Multiple myeloma) 

22 The general MOA for formaldehyde is based on direct chemical reactivity and toxic 

23 effects at the portal of entry (POE). Formaldehyde is directly and indirectly genotoxic, and 

24 reacts with cellular proteins and DNA in cells which it comes into contact. Additionally, 

25 immunosuppression, viral reactivation and promotion effects are relevant to lymphoma and 

26 related malignancies. Therefore, the key events for the adult cell lymphoid cancers would 

27 include these actions. Lymphoid tumors (e.g. lymphocytic leukemia, B-cell lymphoma, mantle 

28 cell lymphoma [a rare form on non-Hodgkin lymphoma] and myeloma) may arise from cells 

29 present at the portal of entry (POE) (Figure 4-33). The location and function of mature 

30 lymphocytes contribute to their vulnerability to transformation by agents at the POE. Therefore, 

31 a brief summary of the immuno-biology of these cells is provided in order to provide context for 

32 the MOA evaluation:

33

34 Location: Lymphocytes are present in the oral and respiratory tract epithelium, as well 

35 as in cell aggregates and tertiary immune structures (e.g. germinal centers) in the mucosal 
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1 tissues (Zuercher and Cebra, 2002; Zuercher et al., 2002; Wu et al 1997 and Kupper et 

2 al., 1990). These mucosa-associated lymphoid tissues (MALT) provide the opportunity 

3 for formaldehyde to directly interact with components of the immune system present at 

4 the POE (Wu et al., 1997, Claeys et al. 1996, Park et al., 2003 and Fujimura 2000). 

5 Intraepithelial lymphocytes are present in the pseudostratified epithelium of the 

6 nasopharyngeal passages and there are aggregates of immune cells and germinal cells 

7 present in these tissues. Crypts containing mature lymphocytes exist at the surface of the 

8 nasal epithelium (Fujimura 2000). Microfold cells or M-cells form the crypts, where the 

9 lymphocytes are covered by a thin membrane (Figure 4-37). Functionally, these 

10 lymphocytes identify and process foreign antigens at the POE (Fujimura 2000). 

11 Therefore the mature lymphocytes within these crypts, exposed to exogenous agents, are 

12 involved in active immune responses to foreign antigens. 

13

14 Clonal Expansion: Mature lymphocytes (both B and T-cells) clonally expand their 

15 populations in response to an exogenous antigen when a humoral immune response is 

16 stimulated. Therefore cell proliferation is a normal function of these mature lymphocytes 

17 and occurs every time there is an infection. Cell proliferation of mature B and T-cells, 

18 responsive to a particular antigen, occurs in active germinal centers (including those 

19 within the respiratory tract). Cells may be exposed to exogenous agents during the 

20 immune response, or cells responding in the germinal center may have previously been in 

21 the epithelium or M-cell crypt.

22

23 Somatic Hypermutation: Normal immune function includes the process of somatic 

24 hypermutation where B-cells undergo DNA rearrangement of the variable region genes to 

25 produce novel antibodies specific to a given antigen. This process is key to adaptive 

26 immunity and demonstrated by the basic principles of immuno-biology which underlie 

27 vaccination theory. Gene sequencing of adult B-cell lymphomas and leukemias indicate 

28 that the chromosomal regions involved in somatic hypermutation correspond to known 

29 oncogenes in these cancers. The vulnerability of these processes is evidenced by the 

30 observation that approximately 90-95% of adult lymphomas and leukemias are of B-cell 

31 origin (Gordon et al., 2003). Formaldehyde-induced protein-protein crosslinking could 

32 disrupt cell processes including somatic hypermutation and cell mitosis, resulting in 

33 agent-induced translations similar to those found in spontaneous B-cell malignancies. 

34
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1

2 Figure 4-37. Location of intra-epithelial lymphocytes along side epithelial cells in 

3 the human adenoid. (Source Fujimura et al., 2000)

4

5 a) Direct or indirect formaldehyde-induced mutation in cells at the site of first contact: 

6 Immune cells including intraepithelial lymphocytes, and cells in mucosal associated 

7 lymph tissue (MALT) are collocated with the epithelial cells from which URT cancers 

8 arise (Figure 4-37). Therefore the direct and indirect mutagenic potential for 

9 formaldehyde is equally applicable to components of the immune system present in these 

10 tissues. Mutations, the permanent heritable changes in the genome of the cell, are a 

11 primary mechanism for the activation of oncogenes or the inactivation of tumor 

12 suppressor genes. Mutagenicity is the most widely recognized determinant of chemical13 induced carcinogenicity, and it is difficult to set aside the relevance of direct 

14 formaldehyde-induced mutations from its demonstrated carcinogenicity. Formaldehyde15 induced mutation in immune cells present at the site of first contact, may initiate cells or 

16 or provide subsequent mutagenic events to already initiated cells. The competence of our 

17 immune system relies on the proliferation of peripheral lymphocytes in response to 

18 immune challenge (infection). Additionally, heritable changes to the variable gene 

19 regions in B-cells generated during somatic hyper-mutation are essential to adaptive 

20 immunity (e.g. immunization) demonstrating that permanent heritable changes in the 

21 DNA of peripheral B-cells are passed to daughter cells and retained in the body for 

22 decades. Any agent-induced mutations would be similarly propagated and retained with 

23 the potential to contribute to the transformation of mature lymphocytes.
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1 Relevance to humans: This MOA is relevant to humans. The well-documented DNA 

2 reactivity (e.g. DPC and DNA adducts) and clastogenicity of formaldehyde at the POE in 

3 laboratory animals is a direct effect of formaldehyde on tissues of first contact, and these 

4 mechanisms are considered relevant to humans. As with epithelial cells, clastogenic 

5 effects in peripheral lymphocytes are documented in formaldehyde-exposed students and 

6 workers, confirming the genotoxic effects of formaldehyde in immune cells, from which 

7 lymphomas and related diseases may arise (Section 4.5.2Figure 4-33).

8

9 b) Formaldehyde-induced protein-protein crosslinks may disrupt somatic10 hypermutation: Although not as well studied as DNA-protein crosslinks, formaldehyde 

11 also formes crosslinks between amino acids on proteins (Section 4.5.3.1.3 for details). 

12 Specific oncogenes for malignancies which arise from mature B-cells are linked to errors 

13 in the process of somatic hyper-mutation (Greaves et al. 2004). If formaldehyde creates 

14 protein crosslinks in competent B-cells which effect the process of DNA rearrangement, 

15 formaldehyde may generate translocations and related oncogenes similar to those 

16 observed in observed in spontaneous B-cell malignancies. 

17 Relevance to humans: This hypothesis has not been tested in either exposed human or 

18 animal test systems. However, the link between somatic-hypermnutation and B-cell 

19 oncogenes is well established and perturbation of this process by an exogenous agent is a 

20 reasonable extension of the current understanding of the etiology of B-cell malignancies.

21

22 c) Increased URT infections / viral reactivation: Inhalation exposure to formaldehyde 

23 has been shown to decrease the defenses of the body against infection through two 

24 mechanisms: 1) damage to the protective mucous barrier and function of the mucociliary 

25 apparatus; and 2) localized immunosuppression (Section 4.5.3.1). These effects have 

26 been demonstrated in both exposed humans and controlled animal experiments. 

27 Additionally, increased respiratory tract infections are associated with formaldehyde 

28 exposure in several populations. Common viral agents (e.g. Epstein barr virus) are 

29 known risk factors for malignancies which arise from mature lymphocytes. Thus, 

30 increased URT infections or viral reactivation due to formaldehyde exposure may 

31 influence the incidence of these cancers. 

32 Relevance to humans: The potential role of increased URT infections and 

33 immunosuppression at the portal of entry is considered to be relevant to humans. Data in 

34 humans are available to support both key events in this MOA. Additionally, co-exposure 

35 to infectious agents (including viruses) would be expected in participants in an 
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1 epidemiological study, suggesting an MOA which acted in conjunction with infectious 

2 agents may be relevant to agent-induced cancer. Therefore, toxic actions by 

3 formaldehyde which may increase URT infections, or viral-reactivation at the site of first 

4 contact, could be acting in conjunction with viral agents to contribute, in part, to observed 

5 associations between formaldehyde exposure and increased lymphoma and related 

6 diseases.

7

8 4.5.3.3.3 Summary and evaluation of hypothesized MOA(s) for Lymphohematopoietic 

9 Malignancies

10 The well-documented direct toxic action of formaldehyde on cells at the site of first 

11 contact is a general effect based on the reactivity of formaldehyde with cellular components (e.g. 

12 proteins and DNA) (Section 4.5.3.1). As a general effect, it is reasonable that these toxic effects 

13 would be relevant to all cells which come into contact with formaldehyde. The current debate 

14 regarding the biological plausibility of formaldehyde-induced lymphohematopoeitic 

15 malignancies centers around a perspective that the diseases within this general grouping are 

16 systemic cancers arising only out of bone marrow toxicity (Heck et al., 2006, Pyatt et al., 2008) 

17 and that it is implausible for formaldehyde to induce bone marrow toxicity. The above MOA 

18 evaluation expands the current debate by considering the impact of POE toxicity on elements of 

19 the immune system and cancers might arise from these cells (Section 4.5.3.3.2) and by 

20 presenting data which support the observation that formaldehyde is associated with bone marrow 

21 toxicity and damage to circulating stem cells in exposed humans (Section 4.5.3.3.1).

22 As significant increases in free formaldehyde in peripheral blood from exogenous 

23 exposure has not been detected (Heck et al., 1987), it has been hypothesized that formaldehyde 

24 does not transport and therefore cannot exert toxic effects outside of the tissues at the site of first 

25 contact (Heck et al., 2006, Pyatt et al., 2008). In contrast to this hypothesis, effects are seen in 

26 formaldehyde-exposed humans which indicate systemic effects on the hematopoietic system 

27 including reduced white blood cell counts, clastogenic effects in peripheral blood lymphocytes 

28 and aneuploidy in circulating stem cells (Tang et al., 2008, Zhang et al., 2010 and Section 

29 4.5.3.1). These observed effects in humans are consistent with agent-induced bone marrow 

30 toxicity and are observed with other well-studied exogenous leukemogens (e.g. benzene and 

31 ionizing radiation.) It is unknown if formaldehyde is distributed systemically to exert its effects 

32 directly on cells in the bone marrow or if damage to circulating stem cells or progenitor cells 

33 would be sufficient to result in the observed effects in humans (Zhang et al., 2010). Additional 

34 research is needed to better determine the potential for systemic transport of formaldehyde 

35 considering both detection of its hydrated form (methylene glycol) as well as formaldehyde 
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1 protein adducts (e.g. FA-GSH and FA-albumin). Similarly the results of Zhang et al. (2010) 

2 need to be extended (analysis for additional chromosomal aberrations) and repeated. Although 

3 further evidence is needed to better understand the hypothesized mechanisms for formaldehyde4 induced effects on hematopoietic stem cells, the observed hematotoxic effects in humans cannot 

5 be set aside. Therefore, however unlikely, the current data support the biological plausibility of 

6 formaldehyde effects on the hematopoietic system.

7

8 4.5.4. Hazard Characterization FOR Formaldehyde Carcinogenicity

9 Formaldehyde is Carcinogenic to Humans by the Inhalation Route of Exposure

10 Based on the results from a large and well-followed longitudinal cohort study of 

11 industrial workers and several case-control studies, the epidemiologic evidence is sufficient to 

12 characterize the association between formaldehyde exposure and nasopharyngeal cancer (NPC) 

13 as causal in humans (Hauptmann et al., 2004; Hildesheim et al., 2001; Vaughan et al., 2000). As 

14 a group, upper respiratory tracts sites of direct contact with formaldehyde upon inhalation (i.e., 

15 salivary gland, mouth, nasopharynx, nasal cavity and larynx) also showed sufficient evidence of 

16 a causal association. Case-control studies have demonstrated associations between formaldehyde 

17 exposure and rare cancers of the URT. Luce et al. (2002) evaluated pooled data from 12 case18 control studies and demonstrated a statistically significant increased risk between formaldehyde 

19 exposure and sinonasal cancer. Hypopharyngeal cancer was linked with formaldehyde exposure 

20 with an OR of 3.78 (95% CI 1.50-9.49) in another case-control study (Laforest et al., 2000). 

21 Hauptmann and colleagues (2004) concluded that in spite of the small numbers of deaths from 

22 cancers of the URT, the positive associations with average intensity and peak exposure were 

23 consistent with the carcinogenicity of formaldehyde at these sites of first contact. The finding 

24 that formaldehyde inhalation causes nasal squamous cell carcinoma in animals (Section 4.2.1.2) 

25 further supports the determination of a causal association of formaldehyde exposure and 

26 increased risk of upper respiratory tract cancer in humans. Both humans and animals developed 

27 tumors within the upper respiratory tract, the POE site expected to receive direct exposure to 

28 formaldehyde.

29 Overall, there is a consistent association between formaldehyde exposure and various 

30 forms of lymphohematopoietic (LHP) cancers, with all leukemias, myeloid leukemia 

31 specifically, Hodgkin lymphoma and multiple myeloma demonstrating the greatest strength and 

32 consistency of results. Where exposure-response data exist, exposure-response trends have been 

33 seen for all LHP malignancies, all leukemia, myeloid leukemia and Hodgkin lymphoma 

34 (Pinkerton et al., 2004; Beane Freeman et al., 2009). Taken together, the data demonstrate a 

35 consistent association, across various worker populations, with the expected temporal association 
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1 to exposure and defined exposure–response relationships in two different worker cohorts. The 

2 strongest associations tend to be with myeloid leukemia and Hodgkin lymphoma. The criterion 

3 of reasonable biological plausibility is easily met for the majority of the diseases which 

4 contribute to an observation of all LHP cancers, specifically the cancers derived from mature 

5 lymphocytes. The potential for formaldehyde-induced LHP cancer is further supported by the 

6 results of animal bioassays, where formaldehyde-induced leukemia and lymphoma had been 

7 demonstrated in 3 independent studies in two species (rats and mice) and both sexes.

8

9 4.6. SUSCEPTIBLE POPULATIONS

10 “Susceptible subpopulations” is used here to refer to factors, such as life stage, genetics, 

11 health status, etc., that may predispose individuals to greater response to an exposure. This 

12 greater response could be achieved either through differences in exposure to the chemical or 

13 differences in underlying toxicokinetic (TK) and toxicodynamic (TD) differences between the 

14 susceptible and other populations. For example, life stages may include the developing 

15 individual before and after birth up to maturity (e.g., preconception, embryo, fetus, young child, 

16 adolescent), adults, or aging individuals. Another susceptibility factor is genetics. Specifically, 

17 susceptible subpopulations may also include people with specific genetic polymorphisms that 

18 render them more vulnerable to a specific agent or people with specific diseases or pre-existing 

19 conditions (e.g., asthmatics). The term may also refer to gender differences, lifestyle choices, or 

20 nutritional state (USEPA, 2002, Section 4.3.2.3).

21 A discussion of a comprehensive list of all possible susceptibility factors affecting 

22 exposure and response to formaldehyde, or any chemical, is not possible. Therefore, the 

23 discussion of susceptibility factors focuses on 1) factors hypothesized to be of importance to 

24 formaldehyde; and 2) factors for which there are available formaldehyde data. A partial list of 

25 these factors includes gender, genetic polymorphisms, preexisting disease status, nutritional 

26 status, diet, and previous or concurrent exposures to other chemicals. Qualitatively, the presence 

27 of multiple susceptibility factors will increase the variability that is seen in a population response 

28 to formaldehyde toxicity. 

29

30 4.6.1. Life Stages

31 Individuals at different life stages are physiologically, anatomically, and biochemically 

32 different. Examples include physiological changes that occur through the lifespan (Selevan et 

33 al., 2000). They may also have distinctive exposure pathways (i.e., transplacental, breast milk 

34 ingestion), and exhibit differences in behavior (U.S. EPA, 2006b; NRC, 1993). Early life stages 

35 (i.e., during development, prior to mature adulthood) and the later life stages (i.e., aging) differ 
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1 greatly from mature adulthood in body composition, organ function, and many other 

2 physiological parameters that can impact the TK and/or TD of chemicals and their metabolites 

3 (ILSI, 2003). This section presents and evaluates the pertinent published literature available to 

4 assess whether and how individuals of differing life stages may respond differently to 

5 formaldehyde.

6

7 4.6.1.1. Early Life Stages

8 4.6.1.1.1. Factors influencing exposure and dosimetry

9 For all life stages, the primary exposure routes for formaldehyde include inhalation and, 

10 in some cases, ingestion (see Chapter 5). Some exposure scenarios may be child specific. For 

11 example, to the extent that the presence of baby furniture produced with formaldehyde in a 

12 child’s house contributes to greater concentrations in a child’s room, exposures for very young 

13 children in those circumstances may be increased (Environment California, 2008). As with all 

14 chemicals, placental transfer and breast milk ingestion are exposure pathways that are unique to 

15 early life stages. Studies assessing early life stage exposure pathways to formaldehyde have not 

16 been performed. Presumably, unmetabolized formaldehyde reacts too quickly to be effectively 

17 transported from mother to fetus by placental transfer; in addition, formaldehyde is not lipophilic 

18 and is therefore unlikely to accumulate in breast milk. However, the relevant dose metric for 

19 formaldehyde-related effects may vary depending on the specific target of concern (e.g., direct 

20 toxicity at the portal of entry versus systemic effects); insufficient information is currently 

21 available to determine whether individuals in different life stages are at higher risk for exposure 

22 to specific target tissues. 

23 There are some calculations however which shed light on lifestage differences in the 

24 inhaled tissue dose at the portal of entry. Using respiratory tract surface areas and ventilation 

25 rates reported in the literature and the scheme in USEPA (1994), Ginsberg et al. (2005) 

26 calculated that overall extrathoracic absorption of highly reactive and soluble gases is similar in 

27 adults and children. These results are in agreement with that of Garcia et al. (2009) who used 

28 computational fluid dynamics to study differences in the nasal dosimetry of reactive, water29 soluble gases between 5 adults and 2 children, aged 7 and 8 years old. Overall uptake efficiency, 

30 average flux (rate of gas absorbed per unit surface area of the nasal lining) and maximum flux 

31 levels over the entire nasal lining did not vary substantially between adults and children (1.6-fold 

32 difference in average flux and much less in maximum flux). On the other hand, the local flux of 

33 formaldehyde varies between the two children by a factor of 2 to 4 at various distances along the 

34 septal axis of the nose. The results in Garcia et al. (2009) have been further described and 

35 evaluated in Appendix 3-1. Under normal resting breathing conditions, it is expected that very 
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1 little formaldehyde is delivered to the lung. However, under higher activity as well as mouth 

2 breathing scenarios, both of which appear likely to happen more regularly in children, 

formaldehyde dose to the lung will be substantial.5

4 The toxicokinetic characteristics of formaldehyde are described in Chapter 3, with 

5 absorption and distribution studies discussed in Sections 3.2 and 3.3. Studies to assess 

6 differential absorption or distribution of formaldehyde in early life stages have not been 

7 performed and represent a significant data gap. The metabolism of formaldehyde is described in 

8 section 3.4. Expression of the enzymes that metabolize formaldehyde (ALDH2 and FALDH, 

9 and specifically ADH3) is known to be developmentally regulated and thus may alter the TK of 

10 formaldehyde in early life stages. ADH3 is ubiquitously expressed and is present in the human 

11 fetus, neonate, and 1- to 10-year-old children (Hines and McCarver, 2002; Estonius et al., 1996). 

12 During early development in rodents, when neurulation first begins and forms collections of 

13 somites along the neural tube, ADH3 activities are significantly lower (at 8–10 and 11–13 somite 

14 stages) and suggest a decreased ability to detoxify formaldehyde in the early embryo (Harris et 

15 al., 2003). ADH mRNA expression levels appear to be age related, with decreased expression of 

16 ADH common in premature neonates and infants up to 5 months old. Thereafter, ADH

17 expression increases and is dependent on body weight (Ginsberg et al., 2004). Benedetti et al. 

18 (2007) reported that decreased ADH expression persisted until age 2 to 5 years. Westerlund et 

19 al. (2005) tracked the ontogeny of ADH3 specifically and reported that ADH3 expression was 

20 ubiquitous in mouse and rat embryos and was the only ADH enzyme to be consistently localized 

21 to brain tissue, suggesting a housekeeping function. Thus, neonates and very young children 

22 may have a decreased ability to metabolize formaldehyde due to differential expression of ADH3 

23 in development compared that of with adults; however, activity levels of this enzyme and 

24 alternate pathways specific to children are not available in the literature. 

3

25

26 4.6.1.1.2. Life-stage exposure and adverse health outcomes 

27 In general, exposure to toxic agents during early development (i.e, pre-conception, 

28 prenatal stages, or postnatal development) may affect organ development and may also lead to 

29 increased disease susceptibility later in life. Following early life stage exposure to 

30 formaldehyde, a number of adverse health outcomes have been observed, including alterations in 

31 the respiratory, reproductive, and neurological systems. For example, the developing respiratory 

32 tract may be more vulnerable to insult compared with an adult respiratory tract, and thus, 

33 increase the severity of response. The potential for reproductive and developmental toxicity of 

 5 For example, in the case of ozone concentrations of 0.1 ppm, a moderately reactive gas, Ginsberg et al. (2008) 

predict a 5-fold variation in the dose to the deep lung between quiet and heavy breathing conditions for an 8-year 

old child. 
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1 formaldehyde is discussed in detail in Sections 4.1.1.7 (human studies) and 4.2.1.7 (animal 

2 studies), while effects on the nervous system are discussed in Sections 4.1.1.6 and 4.2.1.6 

3 (human and animal studies, respectively). The specific case of formaldehyde exposure and 

4 pulmonary effects is discussed in detail in Sections 4.1.1.1 to 4.1.1.4 and 4.2.1.1 to 4.2.1.4. A 

5 brief summary of identified effects of formaldehyde that may indicate susceptibility during 

6 particular life stages is provided below.

7

4.6.1.1.2.1. Pre-conception.

9 Exposure prior to conception may damage reproductive organs and/or germ cells that 

10 could affect reproduction and/or damage the genetic makeup of the offspring. Effects on 

11 reproduction are discussed in Sections 4.1.1.7 and 4.2.1.8. In summary, an epidemiological 

12 study (Taskinen et al., 1999) reported significantly delayed conception among female workers 

13 exposed to formaldehyde at average daily ambient formaldehyde levels; these effects could be 

14 consistent with adverse effects on either pre-conceptional and/or gestational exposure. One 

15 animal study (Maronpot et al., 1986) reported endometrial hypoplasia and lack of ovarian luteal 

16 tissue in female mice exposed for 13 weeks to 40 ppm formaldehyde via inhalation, suggesting 

17 the potential for treatment-related alterations to the female reproductive system. Since the 

18 exposure was to the adult, the findings suggest that preconceptional FA exposure caused female 

19 reproductive system effects that in turn could affect pregnancy.

8 

20 In the rodent study of Kitaev et al. (1984), a three-fold increase in embryo degeneration on 

21 gestational days 2–3 was observed after FA exposure to the dams during premating. Since the 

22 exposure was to the adult in these three studies, the findings suggest that preconceptional FA 

23 exposure caused female reproductive system effects and/or affected the gametes.

24

4.6.1.1.2.2. Prenatal.

26 A population-based study (Gražulevičiene et al., 1998) found an association between 

27 atmospheric formaldehyde exposure and low birth weight, yielding an adjusted OR of 1.37 (95% 

28 CI: 0.90–2.09). Three studies (Dulskiene and Gražulevičiene, 2005; Taskinen et al., 1994; 

29 Hemminki et al., 1985) that examined the effect of occupational exposures on the incidence of 

30 congenital malformation produced mixed results.

25 

31 Results from Taskinen et al. (1999) support associations between formaldehyde exposure, 

32 subfertility, and spontaneous abortion. Subfertility and spontaneous abortion are biologically 

33 linked (subclinical pregnancy losses are increased among women with fertility problems) (Gray 

34 and Wu, 2000; Hakim et al., 1995), and both subfertility and spontaneous abortion may be 

35 related to sensitivity to environmental agents (Correa et al., 1996).
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1 Two experimental animal studies (Martin, 1990; Saillenfait et al., 1989) evaluated a 

2 standard battery of developmental endpoints following inhalation exposure on GDs 6–10, but 

3 effects were minimal. Similarly, Chernoff and Kavlock (1982), Marks et al. (1980), and Hurni 

4 and Ohder (1973) reported minimal reproductive or developmental effects in rodents in studies 

5 in which dams were exposed orally during early gestation. When formaldehyde was 

6 administered via inhalation throughout gestation in female rats, some developmental effects, 

7 including increased pup weight and decreases in lung and liver weight in newborns, were 

8 reported at 0.01 and 0.4 ppm (Senichenkova and Chebotar, 1996; Senichenkova, 1991; Kitaev et 

9 al., 1984; Gofmekler and Bonashevskaya, 1969; Gofmekler, 1968; Pushkina et al., 1968). Two 

10 studies also reported changes in motor activity in offspring of dams exposed via inhalation to 0.4 

11 ppm formaldehyde during gestation (Senichenkova, 1991; Sheveleva, 1971).

12

4.6.1.1.2.3. Postnatal.

14 Following early life stage exposure to formaldehyde, a number of adverse postnatal 

15 outcomes are possible, including effects on the developing and adult respiratory, reproductive, 

16 and neurological systems. The potential for increased risk of childhood cancer is also discussed 

17 below.

13

18

4.6.1.1.2.3.1. Respiratory toxicity.

20 Formaldehyde is known to induce changes in pulmonary function and cause pulmonary 

21 irritation in human studies (Rumchev et al., 2002; Garrett et al., 1999; Krzyzanowski et al., 1990; 

22 Holmström et al., 1989; Holmström and Wilhelmsson, 1988; Ritchie and Lehnen, 1987) and 

23 animal studies (Ohtsuka et al., 2003, 1997; Riedel et al., 1996; Swiecichowski et al., 1993; Lee et 

24 al., 1984). Exposure to formaldehyde in early life can cause damage to the lungs and 

25 permanently influence airway function, resulting in increased vulnerability to toxicants later in 

26 life. Thus, young children may demonstrate increased susceptibility to formaldehyde-related 

27 health effects. Krzyzanowski et al. (1990) reported an association between physician-diagnosed 

28 asthma and chronic bronchitis in children who lived in homes with formaldehyde levels that 

29 were higher than 60 ppb, after controlling for socioeconomic status and ethnicity. Rumchev et 

30 al. (2002) reported a statistically significant increased risk of asthma with increased residential 

31 concentrations of formaldehyde. Garrett et al. (1999) found an increased association between 

32 bedroom concentration of formaldehyde and increased risk of atopy in children. These studies 

33 suggest that formaldehyde exposure may exacerbate responses in sensitive airways, particularly 

34 in children. Exacerbation of response has also been noted in asthmatic adults and will be 

35 discussed below.

19 
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1 Another child-specific concern is that respiratory irritation may have greater impact on 

2 lung function in children due to their smaller lung size; this is true even if the lung development 

3 is normal. Irritation is commonly accompanied by inflammation, which can have a greater 

4 impact on children’s airways because they are narrower than adult airways. Thus, less 

5 inflammation is required for significant airway obstruction in children than in adults.

6

4.6.1.1.2.3.2. Developmental neurotoxicity.

8 In neonatal exposure paradigms, changes in brain structure (Sarsilmaz et al., 2007; Aslan 

9 et al., 2006), and brain chemistry (Songur et al., 2008) were seen in young rats following 

10 inhalation exposures (6000 or 12000 ppb, 5 days per week from postnatal day 0-30). In addition, 

11 Weiler and Apfelbach (1992) found juvenile animals to be more sensitive to formaldehyde12 induced changes in olfactory thresholds when compared with adult animals (shifts in olfactory 

13 thresholds were greater when exposure [at 250 ppb] was initiated at PND 30 than at adult ages). 

14 These studies are consistent with the hypothesis that early life exposure to FA can lead to long15 lasting neurological effects. Exposure levels in these studies (250–6,000 ppb) were in the same 

16 range as those producing the behavioral effects in adults (as low as 100 ppb), but provide limited 

17 information regarding relative sensitivity as no NOAELs were identified, and (with the exception 

18 of Weiler and Apfelbach), similar parameters were not measured in adult animals using the same 

19 exposure paradigms.

7 

20

21 4.6.1.2. Later Life Stages

22 In general, older adults may be at risk for increased susceptibility to exposure to 

23 environmental chemicals by virtue of their slower metabolism and increased incidence of altered 

24 health status (Benedetti et al., 2007; Ginsberg et al., 2005; U.S. EPA, 2005a). Additionally, 

25 adverse effects of earlier exposure to some toxicants may be observed in older adults as a result 

26 of latency in expression of the effect (Olsen et al., 1997; Sweeney et al., 1986). No studies have 

27 examined the differential effects of formaldehyde exposure for elderly adults (>65 years old) as 

28 compared to other age groups. 

29

30 4.6.1.3. Conclusions on Life-Stage Susceptibility

31 In summary, timing both of the exposure and of the assessment of health outcomes may 

32 be important for understanding the relative risk of adverse effects from formaldehyde exposure 

33 during different life stages. There are known developmental differences in kinetics across life 

34 stages, including differences in enzymes involved in formaldehyde metabolism, but the 

35 contribution of these differences to formaldehyde-related health effects is unknown. Similarly, 
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1 information regarding life-stage differences in respiratory physiology raises possible concern 

2 regarding increased exposure to children, but studies for formaldehyde are not available. 

3 Available data do support an increased risk for adverse effects on lung function in children. The 

4 overall body of evidence shows some support, although minimal, for susceptibility in 

5 reproductive or developmental endpoints associated with exposure to formaldehyde. Some 

6 studies observed altered development of the nervous system following formaldehyde exposure 

7 during early life. Older adults may be at risk for increased susceptibility to formaldehyde 

8 because of slower metabolism and clearance rates. Elderly adults have an increased probability 

9 of having both altered health status and altered metabolism, which could impact their ability to 

10 process and recover from an adverse effect. The available data are consistent with some life11 stage susceptibility differences for FA at the level of TD or TK differences, the results are 

12 nonetheless inconclusive due to the number of data gaps.

13

14 4.6.2. Health/Disease Status

15 The factor for which we have the greatest evidence is pre-existing disease, and 

16 specifically asthma. Numerous studies have assessed the potential for increased susceptibility to 

17 formaldehyde in asthmatics. Formaldehyde does not induce airway hyperreactivity directly 

18 (Sheppard et al., 1984) and has not been shown to increase airway hyperreactivity in either 

19 asthmatics or non-asthmatics (Pazdrak et al., 1993; Harving et al., 1991; Kulle et al., 1987). 

20 Significantly decreased forced expiratory volume (FEV1) measurements were reported among 

21 asthmatics in two studies (Casset et al., 2006; Green et al., 1987), while others did not find any 

22 significant change in FEV1 following formaldehyde exposure (Ezratty et al., 2007; Frigas et al., 

23 1984). 

24 A few available case reports of bronchial asthma do suggest direct respiratory tract 

25 sensitization to formaldehyde gas (Lemiere et al., 1995; Burge et al., 1985; Hendrick et al., 1982; 

26 Hendrick and Lane, 1977, 1975). All cases displayed marked changes in FEV1 or pulmonary 

27 airflow rate in response to acute challenges with formaldehyde gas at exposure levels <3 ppm. 

28 Formaldehyde-induced IgE production has been reported in some studies (Vandenplas et al., 

29 2004; Wantke et al., 1996a).

30 There is a large quantity of human data providing evidence of an association between 

31 formaldehyde exposure and increased incidence of asthma or exacerbation of asthmatic 

32 responses in compromised individuals. For example, Krzyzanowski et al. (1990) reported an 

33 association between physician-diagnosed asthma and chronic bronchitis in children who lived in 

34 homes with formaldehyde levels that were higher than 60 ppb, after controlling for 

35 socioeconomic status and ethnicity. Rumchev et al. (2002) reported a statistically significant 
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1 increased risk of asthma with increased residential concentrations of formaldehyde. Garrett et al. 

2 (1999) found an increased association between bedroom concentration of formaldehyde and risk 

3 of atopy in children. These studies suggest that formaldehyde exposure may exacerbate 

4 responses in sensitive airways, particularly in children. Exacerbation of response has also been

5 noted in adults. Kriebel et al. (1993) reported a greater decrease in peak expiratory flow (PEF) 

6 in asthmatic medical students (7.3% decrement) compared with non-asthmatic medical students 

7 (2% decrement) after 2 weeks exposure to formaldehyde (average concentration 0.73 ppm) in an 

8 anatomy lab. This effect does not appear to be immunogenic in nature (Fujimaki et al., 2004a; 

9 Lee et al., 1984). 

10 Several animal studies document increased airway resistance and bronchial constriction 

11 following inhalation exposure to formaldehyde (Nielson et al., 1999; Swiecichowski et al., 1993; 

12 Biagini et al., 1989; Amdur et al., 1960). Sadakane et al. (2002) demonstrated that formaldehyde 

13 exposure exacerbated sensitization and challenge with a common dust mite allergen (Der f) as 

14 measured by increased eosinophil infiltration into the interstitium around the bronchi and 

15 bronchioles as well as goblet cell proliferation in the bronchial epithelium; they suggested that 

16 formaldehyde exposure may aggravate eosinophilic infiltration and goblet cell proliferation that 

17 accompanies allergic responses. The MOA underlying this response is unknown. These 

18 decrements may occur indirectly in response to formaldehyde and may be mediated via 

19 neurogenic potentiation (Sadakane et al., 2002; Riedel et al., 1996; Tarkowski and Gorski, 1995). 

20 In particular, Tarkowski and Gorski (1995) suggest that formaldehyde may increase permeability 

21 of respiratory epithelium and destruction of immunologic barriers. Thus, the respiratory tract 

22 may become vulnerable to inhaled allergens after formaldehyde exposure (Tarkowski and 

23 Gorski, 1995).

24 In summary, the data indicate that formaldehyde exposure can aggravate a type I 

25 hypersensitivity response and that this hypersensitivity may in turn increase the susceptibility to 

26 FA exposure in these individuals. Formaldehyde exposure may predetermine an asthmatic 

27 phenotype or may induce new incidences of asthma via indirect mechanisms, though definitive 

28 evidence and a proposed mechanism remain to be determined. Individuals that exhibit 

29 chemically induced sensitivity and are exposed acutely or chronically to formaldehyde in 

30 residential and occupational settings might exhibit adverse responses at lower concentrations of 

31 formaldehyde than the average healthy person.

32

33 4.6.3. Nutritional Status

34 Limited available data indicate that certain types of malnutrition may increase 

35 susceptibility to formaldehyde exposure. Senichenkova and Chebotar (1996) reported increased 
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1 fetal anomalies in fetuses from iron-deficient pregnant mice after formaldehyde exposure 

2 compared with anemic mice that had not been exposed to formaldehyde. Forced iron reduction 

3 (induced by addition of bipyridyl treatment in pregnant mice) in utero increased the overall 

4 incidence of fetal anomalies when paired with formaldehyde exposure (Senichenkova and 

5 Chebotar, 1996). The findings are difficult to evaluate due to poor reporting and have not been 

6 substantiated by other laboratories. 

7

8 4.6.4. Gender Differences

9 Males and females can differ greatly in body composition, organ function, and many 

10 other physiological parameters that may influence the toxicokinetics of chemicals and their 

11 metabolites in the body (Gochfeld, 2007; Gandhi et al., 2004). 

12 The human epidemiology data set does not support any specific gender susceptibilities 

13 for noncancer effects due to formaldehyde exposure. In general, data suggest that nonpregnant 

14 women, on a per kg body weight basis, may have slightly lower air intake than men, which 

15 would suggest that women may be less susceptible to inhaled pollutants like formaldehyde than 

16 men, but this has not been investigated in the available formaldehyde literature.

17 A few isolated reports have investigated potential gender differences in development of 

18 nasal pharyngeal carcinomas following exposure to formaldehyde. One case-control study 

19 identified a higher OR for sinonasal adenocarcinomas in women (OR = 6.2 [95% CI: 2.2–19.7]) 

20 compared with the OR observed in men (OR = 3.0 [95% CI: 1.5–5.7]) following exposure to 

21 formaldehyde (Luce et al., 2002). However, the overall body of evidence remains scant.

22 There are a few reports concerning differential formaldehyde-induced effects on the male 

23 and female reproductive systems. Özen et al. (2002), Sarsilmaz et al. (1999), and Woutersen et 

24 al. (1987) reported reduced Leydig cell numbers in adult male rats exposed by inhalation. In 

25 female mice, inhalation exposure to formaldehyde resulted in endometrial hypoplasia and lack of 

26 ovarian luteal tissue (Maronpot et al., 1986). The clinical significance of these effects in humans 

27 is unknown, and due to limited data it is unclear whether the female or male reproductive system 

28 is more susceptible to perturbation by formaldehyde. 

29

30 4.6.5. Genetic Differences

31 There are some data for polymorphisms in humans that affect formaldehyde TK. As 

32 discussed in Section 3.4, the primary metabolizing enzymes of formaldehyde are ALDH2 and 

33 ADH3, with the latter enzyme considered more relevant to low exposures. Polymorphisms in 

34 ALDH2 have been shown to have implications in human risk assessment, specifically in regard 

35 to acetaldehyde metabolism (Ginsberg et al., 2002). Teng et al. (2001) demonstrated the 
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1 importance of ALDH2 for formaldehyde metabolism in rat hepatocytes at fairly high 

2 formaldehyde concentrations (2.5 mM and greater). Cheng et al. (2008) investigated the 

3 relationship between occupational exposure to formaldehyde and genetic polymorphisms of 

4 ALDH2 and CYP2E1. There was a positive relationship between the concentration of formic 

acid in the urine and ALDH2 genotypes (χ2 5 = 9.241, p < 0.05). Urinary formic acid 

6 concentration may be affected by formaldehyde exposure concentration and ALDH2 genotype 

7 (Cheng et al., 2008) for individuals that have high exposure levels. Thus, although ALDH2 may 

8 not be involved in formaldehyde metabolism if exposure levels are low, polymorphisms of this 

9 enzyme may lead to differences in response at higher exposure levels. 

10 Wu et al. (2007) looked for and identified two SNPs in ADH3 among a population of 

11 Mexican asthmatic children 4 to 17 years of age. Carrying one or two copies of the minor allele 

12 for one SNP resulted in a decreased RR of asthma (RR = 0.66–0.77). For the second SNP, 

13 homozygotes for the minor allele had an RR of 1.6 for asthma. The functional characteristics of 

14 these SNPs are unknown. Studies evaluating whether any of the polymorphisms affect the 

15 expression, regulation, stability, or activity of the enzyme in vivo are lacking; therefore, the 

16 relative susceptibility of individuals with different polymorphisms cannot be characterized at this 

17 time.

18 One study (Hedberg et al., 2001) identified three polymorphisms in human ADH3 

19 involving four base-pair substitutions in the promoter region of which one (C→T) showed 

20 reduced activity (~50–70% of control). Hedberg et al. (2001) reported differences in allele 

21 frequencies among Chinese, Spanish, and Swedish groups, consisting of Asian-Caucasian 

22 differences and ethnic subgroups among Caucasians. Their results suggest that a small 

23 percentage of Caucasians may have decreased ADH3 expression and thus, be more susceptible to 

24 formaldehyde exposure. Additional studies to validate these findings have not been performed. 

25 The relative activity level of these enzymes may also impact the metabolism of 

26 formaldehyde. In pharmacokinetic studies, deletion of ADH3 increased the sensitivity of mice to 

27 formaldehyde (Deltour et al., 1999) and was deleterious to yeast (Achkor et al., 2003). These 

28 results suggest that deficiencies in ADH3 may confer an increased susceptibility to formaldehyde 

29 toxicity (Teng et al., 2001). The importance of properly functioning enzymes also suggests that 

30 genetic differences in ADH3 or ALDH2 may affect the response to formaldehyde exposure. 

31 However, comparable human data are not available. 

32 Race/ethnicity may be a surrogate for genetic differences but racial or ethnic groups may 

33 also reflect socioeconomic, and/or cultural factors that are distinct from genetics. Possible ethnic 

34 differences may be related to genetic polymorphisms of enzymes ALDH2 and ADH3, relevant 

35 for formaldehyde metabolism. ALDH2 variants, present primarily in East Asians, are known to 
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1 have protective effects against alcoholism but were not found in the people of Indo-Trinidadian 

2 descent (Moore et al., 2007) or in American Indians or Alaska natives (Ehlers, 2007). However, 

3 there is no direct evidence to associate these variants to differential susceptibility to 

4 formaldehyde exposure, nor is there direct evidence to associate these ethnic groups specifically 

5 with differential susceptibility to formaldehyde. Further, no studies have specifically assessed 

6 ethnic variability in responses to formaldehyde. 

7 There are complex pathways through which genetic polymorphisms in ADH3 can 

8 potentially affect differential susceptibility to formaldehyde. Firstly, ADH3 is central to the 

9 metabolism of formaldehyde. However, ADH3 itself may indirectly contribute to the adverse 

10 effects of formaldehyde on pulmonary physiology (Thompson et al., 2009; Staab et al., 2008a, b; 

11 Thompson and Grafström, 2008). Exposure to formaldehyde is itself thought to alter the activity 

12 of ADH3 resulting in the perturbation of critical metabolic pathways. ADH3 participates in the 

13 oxidation of retinol and long-chain primary alcohols, as well as the reduction of S14 nitrosoglutathione (GSNO). The activity of ADH3 toward some of these substrates has been 

15 shown to be significantly increased in the presence of formaldehyde. ADH3 has recently also 

16 been shown to contribute to NO signaling through its dual role in metabolizing GSNO, an 

17 endogenous bronchodilator and reservoir of NO (Staab et al., 2008a; Hess et al., 2005; Jensen et 

18 al. 1998). Through its regulatory function on GSNO, ADH3 may thus play a central role in 

19 regulating bronchial tone allergen-induced hyperresponsiveness (Gerard, 2005; Que et al., 2005). 

20 As concluded by California Environmental Protection Agency (CalEPA) (2008), “the 

21 dysregulation of NO by formaldehyde [in this manner] helps to explain the variety and 

22 variability in the toxic manifestations following formaldehyde inhalation.” 

23

24 4.6.6. Co-Exposures 

25 4.6.6.1. Cumulative Risk

26 When considering health risks, it is important to consider the impact of co-exposures to 

27 other agents that may interact with the chemical under evaluation. Co-exposure to other 

28 pollutants, particularly those that produce some of the same metabolites and similar health 

29 effects as formaldehyde, is likely to occur in both occupational and nonoccupational settings. 

30 Due to effects on metabolic enzymes (inducing and/or inhibition) as well as direct effects 

31 on organ system function, co-exposures may alter the way in which formaldehyde is metabolized 

32 and cleared from the body. Inhibition or induction of the enzymes responsible for metabolism of 

33 chemicals may alter susceptibility to toxicity (Lash and Parker, 2001; IARC, 1995; U.S. EPA, 

34 1985a). Smokers may be at increased risk for effects of formaldehyde exposure, because 

35 formaldehyde is one of the components of cigarette smoke and is likely to heighten the point-of-
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1 entry effect when combined with occupational or residential exposures to inhaled formaldehyde. 

2 However, no evidence is available to evaluate the potential aggregate effects.

3

4 4.6.6.2. Aggregate Exposure

5 In addition, multiple routes of exposure to a single agent may increase the cumulative 

6 risk by increasing the overall body burden of the chemical. A human aggregate exposure model 

7 developed by McKone and Daniels (1991) incorporated likely exposures from air, water, and soil 

8 media through inhalation, ingestion, and dermal contact. The authors hypothesized that the 

9 aggregate exposure could be age-dependent but did not present any data for persons of differing 

10 life stages. The role of multiple exposures on different genders, genetic susceptibility, or altered 

11 health and nutrition status has not been investigated. The available database regarding the 

12 potential for multiple routes of exposure (or aggregate exposure) formaldehyde is limited.

13 Guseva (1972) specifically assessed the reproductive and developmental effects caused 

by co-exposure to formaldehyde via both inhalation (0.25 mg/m3 14 ) and ingestion (0.01 mg/L) 

15 routes in male rats. The authors reported reduced nucleic acid levels in testes to 88 and 92% of 

16 controls, which suggests a possible toxic gonadotropic effect. The ability of male rats (receiving 

17 combined exposure to formaldehyde at a low concentration level for a long period of time) to 

18 reproduce was preserved since all the cohabited females were impregnated. The number and 

19 weight of the fetuses and newborn rat pups in the experimental co-exposure groups did not differ 

20 substantially from those figures observed in the control group. No developmental defects or 

21 anomalies were observed in the offspring for up to 1 month postnatally. Thus, at low exposures, 

22 the reproductive effects due to combined ingestion and inhalation exposure are unknown.

23

24 4.6.7. Uncertainties of Database 

25 There is a need to better characterize the implications of formaldehyde exposures to 

26 susceptible populations. A number of areas where the database is currently insufficient are 

27 identified below.

28

29 4.6.7.1. Uncertainties of Exposure

30 Although information exists on early life exposure to formaldehyde, a number of 

31 uncertainties regarding children’s susceptibility remain. First, inhalation is believed to be of 

32 most concern for formaldehyde, since formaldehyde vapors are released from insulation or from 

33 ambient sources of formaldehyde, including secondary production from other pollutants involved 

34 in photo-oxidant reactions. Any additional pathways of exposure for children have not been 

35 characterized. Since formaldehyde is nearly ubiquitous in the environment, it is difficult to 
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1 quantify the total exposure. Second, children have different respiratory, metabolic, and activity 

2 rates compared with healthy adults, potentially influencing ADME and target tissue exposure to 

3 formaldehyde. However, studies to identify the specific changes in absorption of formaldehyde 

4 and its metabolites across developmental stages and across organs have not been performed. In 

5 addition, exposure prenatally may be altered based on whether formaldehyde or its metabolites 

6 pass through the placenta, but placental transfer data are not available. Third, no quantitative 

7 models have been developed to characterize these differences for formaldehyde. Formaldehyde8 specific PBPK models and their validation will aid in understanding the uncertainties associated 

9 with formaldehyde exposure in children.

10 Given the large proportion of time that most individuals in the U.S. spend indoors, 

11 exposure scenarios where indoor concentrations to formaldehyde are high (e.g., in homes or in 

12 trailers; see section 2.3.1) may play a significant role and may be of particular concern to the 

13 elderly or health-impaired individuals who spend relatively more time at home. Further 

14 evaluation of the effects of co-exposures and pathways of exposure and aggregate risk is needed. 

15 An estimate of the multiple exposure pathways is needed to know where along the dose-response 

16 curve to place an incremental exposure to formaldehyde. 

17

18 4.6.7.2. Uncertainties of Effect

19 Studies specifically designed to evaluate effects after early and later life stage exposure 

20 are needed in order to more fully characterize potential life-stage-related differences in 

21 formaldehyde toxicity, including the defining of critical windows during development. For 

22 example, life-stage-specific neurotoxic and pulmonary effects, particularly in the developing 

23 fetus, need further evaluation. The preconceptional period may be a critical window for FA 

24 exposure and reproductive and developmental effects, based on rodent studies of reproductive, 

25 embryonic and gamete effects. Data specific to the carcinogenic effects of formaldehyde 

26 exposure during early life stages do not exist. The reduction in fertility seen in some studies 

27 (Gray and Wu, 2000; Taskinen et al., 1999; Hakim et al., 1995) is not adequately described and a 

28 well-established MOA has not been identified, but some have been hypothesized including 

29 altered sperm quality (Özen et al., 2002; Sarsilmaz et al., 1999; Woutersen et al., 1987). Further, 

30 spontaneous abortion/fetal loss occurring early in gestation, prior to maternal knowledge of the 

31 pregnancy, can lead to misclassification of the effect as infertility (see Sections 4.1.1.7 and 

32 4.2.1.7).

33 More research is needed to clarify the role of genetic polymorphisms in formaldehyde 

34 metabolism. Similarly, data gaps pertaining to gender differences remain. A potential impact of 

35 nutritional status and iron deficiency on formaldehyde toxicity needs further investigation. 
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1 A fair body of evidence suggests that asthmatics are more susceptible to formaldehyde exposure 

2 than the general population, however the mechanism of action for this increased susceptibility is 

3 unknown. 

4 In the studies discussed above, there are a number of examples of studies that assessed 

5 multiple susceptibility factors that are worth noting. For example, the Krzyzanowski et al. 

6 (1990) study reported asthma and chronic bronchitis cases for two interacting potential 

7 susceptible groups, in children and those with high exposure (due to living in homes with 

8 formaldehyde levels that were higher than 60 ppb). Similarly, the Garrett et al. (1999) study 

9 assessed the same two interacting potential susceptible groups.

10 The study of Senichenkova and Chebotar (1996) assessed developmental effects in 

11 mouse fetuses after in utero iron-deficiency and FA exposure. Thus, the study findings must be 

12 considered in light of possible interactions between life stage exposure differences and 

13 nutritional status differences. 

14 Studies to understand the nature of the interactions between the various susceptibility 

15 factors for FA have not been performed.

16

17 4.6.8. Summary of Potential Susceptibility

18 There is some evidence to demonstrate susceptibility for various populations exposed to 

19 formaldehyde. Available data are summarized in Table 4-96 where FA susceptibility factors are 

20 presented by those with data for increased FA susceptibility and those with data for differences 

21 but with an unknown impact on FA susceptibility.

22 Exposure to FA during early developmental and later life stages may be of concern. 

23 However, human exposure to the developing fetus is unknown since it is not known whether 

24 formaldehyde or one of its metabolites crosses the placenta. However, there is very limited life25 stage-specific information regarding the TK of formaldehyde. Life-stage-specific TK has not 

26 been characterized, and, thus, no PBPK models exist to effectively evaluate the risk to early life 

27 stages. Children may be more susceptible to noncancer health effects as a result of inhalation 

28 exposure to formaldehyde due to increased respiratory rates. There are no studies to evaluate 

29 whether formaldehyde exposure in early life (e.g., pregnancy) is associated with an increased 

30 risk of childhood cancer.

31 The weight of evidence supports a plausible association between formaldehyde exposure 

32 and aggravated asthmatic responses in humans and this association is corroborated by limited 

33 evidence from animal studies. Formaldehyde does not appear to directly induce airway 

34 hyperreactivity but may sensitize airways to subsequent exposures. One issue in interpreting the 

35 available studies that assessed the relationship between asthma and FA could not distinguish 
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1 between the cases of asthma that were due to earlier FA exposure vs. those without a direct link 

2 to FA exposure.

3 No direct link exists between formaldehyde exposure and differential susceptibility in 

4 different ethnic groups, although genetic polymorphisms in the enzymes involved with 

5 formaldehyde metabolism, ADH3 and ALDH2, provide some support for differential 

6 susceptibility to alcoholism in a number of ethnic groups. The evidence for differential gender 

7 responses to formaldehyde exposure is equivocal. Co-exposures may result in altered 

8 metabolism and clearance, but there is no evidence that co-exposures are a critical part of 

9 formaldehyde-mediated differential susceptibility. 

10 Thus, given the available data, increased susceptibility to adverse effects of 

11 formaldehyde is most strongly supported for three populations: 1) Preconception and perinatal 

12 exposure based on reproductive and developmental effects; 2) children, whose exposure may be 

13 higher by virtue of their increased activity level and respiratory rate; and 3) asthmatics who may 

14 exhibit exacerbation of response to formaldehyde. 

15
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1 Table 4-96. Available evidence for susceptibility factors of concern for 

2 formaldehyde exposure

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: Gražulevičiene 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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