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www.ep a.gov/iris



# TOXICOLOGICAL REVIEW OF

FORMALDEHYDE

INHALATION TOXICITY

(CAS No. 50-00-0)



## In Support of Summary Information on the Integrated Risk Information System (IRIS)



# VOLUME I of IV

Introduction, Background,

and Toxicokinetics

March 17, 2010

NOTICE

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

U.S. Environmental Protection Agency Washington, DCThis document is a draft for review purposes only and does not constitute Agency policy.
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- ii DISCLAIMER

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

distributed solely for the purpose of pre

- dissemination peer review under applicab le information

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

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

names or commercial products does not constitute endorse ment or recommendation for use.This document is a draft for review purposes only and does not constitute Agency policy.
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- iii CONTENTS

— TOXICOLOGICAL REVIEW OF FORMALDEHYDE (CAS No. 50

- 00

- 0)

LIST OF TABLES

................................ ................................ ................................ ......................... xi

LIST OF FIGURES

................................ ................................ ................................ ....................... xx LIST OF ABBREVIATIONS AND ACRONYMS

................................ ................................ ..... xxv FOREWORD

................................ ................................ ................................ ............................ xxxii AUTHORS, CONTRIBUTORS, AND REVIEWERS

................................ ............................ xxxiii

VOLUME I

1. INTRO DUCTION

................................ ................................ ................................ .................. 1

- 1

2. BACKGROUND

................................ ................................ ................................ .................... 2

- 1

2.1. PHYSICOCHEMICA L PROPERTIES OF FORM ALDEHYDE

................................ 2

- 1 2.2. PRODUCTION, US ES, AND SOURCES OF F ORMALDEHYDE

........................... 2

- 1

2.3. ENVIRONMENTAL LEVELS AND HUMAN EXP OSURE

................................ ..... 2

- 4

2.3.1. Inhalation

................................ ................................ ................................ .......... 2

- 5 2.3.2. Ingestion

................................ ................................ ................................ ......... 2

- 10

2.3.3. Dermal Cont act

................................ ................................ ............................... 2

- 11

3. TOXICOKINETICS

................................ ................................ ................................ ............... 3

- 1

3.1. CHEMICAL PROPE RTIES 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 (N on

- 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 Formaldehy de 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 Formalde hyde 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

- 16This document is a draft for review purposes only and does not constitute Agency policy.
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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 T OXICOKINETICS OF FOR MALDEHYDE 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 Formaldehyd e 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 a nd 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

- 56This document is a draft for review purposes only and does not constitute Agency policy.
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- v 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. Sum mary: 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. R espiratory 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. Mutag enicity 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. G enotoxicity 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

- 389This document is a draft for review purposes only and does not constitute Agency policy.
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- vi CONTENTS (continued)

4.4.8. Effects on the Nervous System

................................ ................................ .... 4

- 390

4.4.8.1. Irritant Threshold Detection

................................ .......................... 4

- 390

4.4.8.2. Behavioral Effects

................................ ................................ ......... 4

- 391 4.4.8.3. Neurochemistry, Neuropathology, and 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 CARCIN OGENICITY

............................ 4

- 402 4.5.1. Cancers of the Respiratory Tract

................................ ................................ .. 4

- 402 4.5.2. Lymphohematopoietic Maligna ncies

................................ ........................... 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 An imal 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 (Nasophar yngeal 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 PO PULATIONS

................................ ................................ ............ 4

- 454 4.6.1. Life Stages

................................ ................................ ................................ .... 4

- 454 4.6.1.1. Early Lif e Stages

................................ ................................ ........... 4

- 455 4.6.1.2. Later Life Stages

................................ ................................ ............ 4

- 459

4.6.1.3. Conclusions on Life

- Stage Susceptibility

................................ ..... 4

- 459

4.6.2. Health/Disease Status

................................ ................................ ................... 4

- 460

4.6.3. Nutritional Status

................................ ................................ .......................... 4

- 461

4.6.4. Gender Differences

................................ ................................ ....................... 4

- 462

4.6.5. Genetic Differences

................................ ................................ ...................... 4

- 462This document is a draft for review purposes only and does not constitute Agency policy.
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- vii CONTENTS (co ntinued)

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 S usceptibility

................................ ............................ 4

- 467

VOLUME III

5. QUANTITATIVE ASSESSMENT: INHALATION EXPOSURE

................................ ....... 5

- 1

5.1. INHALATION REF ERENCE CONCENTRATION (R f C)

................................ ........ 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. Develo pmental 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 Stu dy

- 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 C ANCER ASSESSMENT BAS ED ON THE NATIONAL

CANCER INSTITUT E 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

Mortalit y

................................ ................................ ......................... 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

- 88This document is a draft for review purposes only and does not constitute Agency policy.
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- viii CON TENTS (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. Con clusions on Cancer Unit Risk Estimates Based on Human Data

............. 5

- 99

5.3. DOSE

- RESPONSE MODELING OF RISK OF SQUAMOUS CEL L

CARCINOMA IN THE RES PIRATORY TRACT USING ANIMAL DATA

...... 5

- 102 5.3.1. Long

- Term Bioassays in Laboratory Animals

................................ ............. 5

- 104

5.3.1.1. Nasal Tumor I ncidence 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 FR OM THE QUANTITATIVE ASSESSMENT OF

CANCER RISK FROM FOR MALDEHYDE EXPOSURE B Y 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 HUM AN HAZARD POTENTIAL

................................ ................... 6

- 1 6.1.1. Exposure

................................ ................................ ................................ ........... 6

- 1 6.1.2. Absorption, Distribution, Metabolism, an d 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

- 12This document is a draft for review purposes only and does not constitute Agency policy.
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- ix CONTENTS (continued)

6.1.3.8. Effects on General Systemic Toxicity

................................ ............. 6

- 13 6.1.3.9. Summary

................................ ................................ .......................... 6

- 14

6.1.4. Car cinogenicity 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 CH ARACTERIZATION

................................ ........................... 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 CO NCLUSIONS

................................ ................................ ......... 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

IMPLEMENTA TIONS

................................ ................................ ..................... E

- 1

APPENDIX F: SENSITIVITY ANALYSIS OF BBDR MODEL FOR FORMALDEHYDE

INDUCED RESPIRATORY CANCER IN HUMANS

................................ .... F

- 1This document is a draft for review purposes only and does not constitute Agency policy.
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- x CONTENTS (continued)

APPENDIX G: EVALUATION OF THE CANCER DOSE

- RESPONSE MODELING

OF GENOMIC DATA FOR FORMALDEHYDE RISK ASSESSMENT

..... G

- 1

AP PENDIX H: EXPERT PANEL CONSULTATION ON QUANTITATIVE

EVALUATION OF ANIMAL TOXICOLOGY DATA FOR

ANALYZING CANCER RISK DUE TO INHALED FORMALDEHYDE

.. H

- 1This document is a draft for review purposes only and does not constitute Agency policy.
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- xi LIST OF TABLES

Table 2

- 1. Physicochemical properties of formaldehyde

................................ ........................... 2

- 2

Table 2

- 2. Ambient air level s 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 [ 14 C]

- formaldehyde in F344 rats

.......................... 3

- 26

Table 3

- 5. Apparent formaldehyde levels in exhaled breath of individuals attending a health fair, adjusted for methanol and ethanol 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 concentration s 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 bradypne a 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

- 117This document is a draft for review purposes only and does not constitute Agency policy.
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- xii LIST OF TABLES (continued)

Table 4

- 9. Summary o f 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 biochemica l 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 i nhalation

................................ ............................... 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

- 1 7. 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 histopa thologic 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 formaldehy de (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

- by

time products in male albino Wistar rats

................................ .............................. 4

- 153

Table 4

- 21. Rhi nitis observed in formaldehyde

- treated animals; data pooled for male

and female animals

................................ ................................ .............................. 4

- 154This document is a draft for review purposes only and does not constitute Agency policy.
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- xiii LIST OF TABLES (continued)

Table 4

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

- treated and control animals; data pool ed for males and

females

................................ ................................ ................................ ................. 4

- 155

Table 4

- 23. Cellular and molecular changes in 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 r ats 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 formaldehy de

treated 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. Histopat hologic 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 Spr ague

- 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 site s 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 formald ehyde 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 inhalat ion exposures to formaldehyde

.................... 4

- 194This document is a draft for review purposes only and does not constitute Agency policy.
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- xiv 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 UL LI, 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. Summa ry 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 exposur e

.......................... 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 regim ens 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

- 226This document is a draft for review purposes only and does not constitute Agency policy.
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- xv LIST OF TABLES (continued)

Table 4

- 49. St udy 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 footpa d 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

Ta ble 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. Effec ts 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. Formal dehyde 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 seminifero us 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

- 302This document is a draft for review purposes only and does not constitute Agency policy.
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- xvi LIST OF TABLES (continued)

Table 4

- 64. Body weight s 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. Effe ct 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 reporte d 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

T able 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 o f formaldehyde

................................ ............................. 4

- 348

Table 4

- 81. Summary of mutagenicity of formaldehyde in bacterial systems

...................... 4

- 350This document is a draft for review purposes only and does not constitute Agency policy.
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- xvii LIST OF TABLES (continued)

Table 4

- 82 Mutagenicity in mammalian cell systems

................................ ........................... 4

- 355

Table 4

- 83. Genotoxicity in laboratory animals

................................ ................................ .... 4

- 361

Table 4

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

................................ ................................ ................................ ....... 4

- 364

Table 4

- 85. MN and SCE formation in mortuary 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 leukemi as (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 provid e 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 bioa ssays 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

ex posure

................................ ................................ ................................ ............... 4

- 469This document is a draft for review purposes only and does not constitute Agency policy.
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- xviii 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 ra ts

................................ ................................ ............. 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. EC 0005 , LEC 0005 , and inhalation unit risk estimates for NPC mortality from formaldehyde exposure based on the Hauptmann et al. (2004) log

- linear trend analyses for cum ulative exposure

................................ ................................ 5

- 81

Table 5

- 11. EC 0005 , LEC 0005 , 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 mo rtality 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

- 91This document is a draft for review purposes only and does not constitute Agency policy.
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- xix LIST OF TABLES (continued)

Table 5

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

conti nuous exposure to formaldehyde

................................ ................................ ... 5

- 91

Table 5

- 16. EC 0005 , LEC 0005 , 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

Tab le 5

- 17. EC 005 , LEC 005 , 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. EC 0005 , LEC 0005 , and inhalation unit ri sk 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. EC 005 , LEC 005 , and inhalation unit risk estimates for leukemia incidence

fro m 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 incid ence 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 incide nces

................................ ..................... 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 expos ure level of 1 μg/m 3 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 r isk levels for selected formaldehyde

- related

cancers

................................ ................................ ................................ ................... 6

- 36

Table 6

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

animal data

................................ ................................ ................................ ............. 6

- 42This document is a draft for review purposes only and does not constitute Agency policy.
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- xx 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 modific ations

................................ ...................... 3

- 2

Figure 3

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

to 14 C and 3 H

- labeled formaldehyde (0.3, 2, 6, 10, 15 ppm)

.............................. 3

- 18

Figure 3

- 3. Formaldehyde clearance by ALDH2 (GSH

- 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 F3 44 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 conc entration 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 i n 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

- 20This document is a draft for review purposes only and does not constitute Agency policy.
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- xxi LIST OF FIGURES (continued)

Figure 4

- 2. Formaldehyde effects on minute volume in naïve and formaldehyde

pretreated 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

Figur e 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 ra ts and mice

................................ ................................ ................... 4

- 190

Figure 4

- 11. Alveolar MP Fc

- mediated phagocytosis from mice exposed to 5 ppm formaldehyde, 10 mg/m 3 carbon black, or both

................................ ............... 4

- 223

Figure 4

- 12. Compressed air in milliliters as parameter for airway obstruction following formaldehyde expo sure 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 antagonist s on formaldehyde

- induced vascular permeability in the trachea and bronchi of male Wistar rats

............................ 4

- 239This document is a draft for review purposes only and does not constitute Agency policy.
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- xxii LIST OF FIGURES (continued)

Figure 4

- 17. The effects of formaldehyde inhalation exposures on eosinophil infiltration (Panel A) and goblet cell prolifera tion (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 Su bstance 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

Fig ure 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 terminati on 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

- expose d 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

- 2 9. 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

- 334This document is a draft for review purposes only and does not constitute Agency policy.
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- xxiii 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 form aldehyde 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 formaldehy de 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 concen trations.

.............................. 5

- 41

Figure 5.5. Odds ratios for physician

- diagnosed asthma in children associated with in

home 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 irritati on (eye irritation)

......................... 5

- 53This document is a draft for review purposes only and does not constitute Agency policy.
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- xxiv 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 fo r myeloid, lymphoid, and

all leukemia

................................ ................................ ................................ ........ 5

- 98

Figure 5

- 11. Schematic of integration of pharmacokinetic and pharmacodynamic components in the CIIT model

................................ ................................ ......... 5

- 110

Figure 5

- 12. Fit to the rat tumor incidence data using the model and assumptions in Conolly et a l. (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 BMC 10 and

BMCL 10

................................ ................................ ................................ ............ 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 mo del fit of tumor incidence data compared with KM estimates of spontaneous tumor incidence

................................ ................ 5

- 132This document is a draft for review purposes only and does not constitute Agency policy.
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- xxv LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygienists

ADAF age

- dependent adjustment factors

ADH alcohol dehydr ogenase 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

AL P 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

- der ived 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 mi cronucleated cell ratefluoresce

BMD benchmark dose

BMDL 95% lower bound on the benchmark doseThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxvi LIST OF ABBREVIATIONS AND ACRONYMS (continued)

BMR benchmark response

BN Brown

- Norway

BrdU bromodeoxyuridine

BUN blood urea nitrogen

BW body weight

CA ch romosomal 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 Toxicolo gy CLL chronic lymphocytic leukemia

CML chronic myelogenous leukemia

CNS central nervous system

CO 2 carbon dioxide

COEHHA California Office of Environmental Health Hazard Assessment

CREB cyclic AMP responsive element binding proteins

CS conditioned s timulus

C × t concentration times time

DA Daltons

DAF dosimetric adjustment factor

DDX DNA

- DNA cross

- links

DEI daily exposure index

DEN diethylnitrosamine

Der f common dust mite allergen

DMG dimethylglycine

DMGDH dimethylglycine dehydrogenase

DNA deoxyribonucleic acid

DOPAC 3,4

- dihydroxyphenylacetic acid

DPC / DPX DNA

- protein cross

- links

EBV Epstein

- Barr virus

EC effective concentration

ED effective dose

EHC Environmental Health Committee

ELISA enzyme

- linked immunosorbent assayThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxvii LIST OF ABBRE VIATIONS 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 for ced 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 chr omatography

- 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 glutat hione

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

- hydroxymethyl glutathione

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 Da ta Bank Hsp heat shock protein

HWE healthy worker effect

I cell initiated cell

IARC International Agency for Research on CancerThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxviii LIST OF ABBREVIATIONS AND ACRONYMS (continued)

ICD International Classification of Diseases

IF interfacial

IFN interfero n Ig immunoglobulin

IL interleukin

I.P. intraperitoneal

IPCS International Programme on Chemical Safety

IRIS Integrated Risk Information System

K
m Michaels

- Menton constant

KM Kaplan

- Meier

LD 50 median lethal dose

LDH lactate dehydrogenase

LEC 95% lower bound on the effective concentration

LED 95% lower bound on the effective dose

LHP lymphohematopoietic

LI labeling index

LM Listeria monocytogenes

LMS linearized multistage

LLNA local lymph node assay

LOAEL lowest

- observed

- adverse

- effect level

LPS lipopolysaccharide

LR low responders

LRT lower respiratory tract

MA methylamine

MALT mucus

- associated lymph tissues

MCH mean corpuscular hemoglobin

MCHC mean corpuscular hemoglobin concentration

MCS multiple chemical sensitivity

MCV mean corpu scular 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 mod e of action

MoDC monocyte

- derived dendritic cell

MP macrophage

MPD multistage polynomial degree

MPS mononuclear phagocyte system

MRL minimum risk levelThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxix LIST OF ABBREVIATIONS AND ACRONYMS (continued)

mRNA messenger ribonucleic acid

MVE

- 2 Murray Valle y 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 Tox ics 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

N 6

- hmdA N 6

- hydroxymethyldeoxyadenosine

N 4

- hmdC N 4

- hy droxymethylcytidine

N 2

- hmdG N 2

- 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

OT S 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 chai n reaction

PCV packed cell volumeThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxx LIST OF ABBREVIATIONS AND ACRONYMS (continued)

PECAM platelet endothelial cell adhesion molecule

PEF peak expiratory flow

PEFR peak expiratory flow rates

PEL permissible exposure limit

PFC plaque

- forming cell

PG per iglomerular

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

PO E 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 c ells

RD 50 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 transc riptase

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 sta ndard error of the mean

SEN sensitizer

SH sulfhydryl

SHE Syrian hamster embryo

SLMA spontaneous locomotor activityThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxxi LIST OF ABBREVIATIONS AND ACRONYMS (continued)

SMR standardized mortality ratio

SNP single nucleotide polymorphism

SOD superoxide dis mutase
SOMedA N 6

- sulfomethyldeoxyadenosine Sp1 specificity protein

SPIR standardized proportionate incidence ratio

SSAO semicarbozole

- sensitive amine oxidase

SSB single strand breaks

STEL short

- term exposure limit

TBA tumor bearing animal

TH T

- lymph ocyte 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 thiazol idine

- 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 capac ity

VOC volatile organic compound

WBC white blood cell

WDS wet dog shake

WHO World Health Organization

WHOROE World Health Organization Regional Office for EuropeThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxxii FOREWORD

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

for the hazard and dose

- response assessment in IRIS pertaining to chronic inhalation exposure to

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

nature of formaldehyde.

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

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

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

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

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

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

the reader is referred to EPA’s IRIS Hotline at (202) 566

- 1676 (phone), (202) 566

- 1749 (fax), or

hotline.iris@epa.gov (email address).This document is a draft for review purposes only and does not constitute Agency policy.
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- xxxiii AUTHORS, CONTRIBUTORS, AND REVIEWERS

CHEMICAL MANAGERS

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

EPA

- ORD

- NCEA

Danielle DeVoney, PhD, D.A.B.T., PE 2

EPA

- ORD

- NCEA

AUTHORS

Thomas Bateson, PhD

EPA

- ORD

- NCEA

Susan Euling, PhD

EPA

- ORD

- NCEA

Jennifer Jinot, PhD

Susan Makris, PhD

EPA

- ORD

- NCEA

Kathleen Raffaele, PhD

EPA

- ORD

- NCEA

John S chaum, PhD

EPA

- ORD

- NCEA

Ravi Subramaniam, PhD

EPA

- ORD

- NCEA

Suryanarayana Vulimiri, PhD

EPA

- ORD

- NCEA

CONTRIBUTORS

Gillian Backus, PhD 3

Stanley Barone, PhD

EPA

- ORD

- NCEA

1 Chemical Manager since July 2003.

2 Chemical Manager since June 2009

3 Separated from the Agency prior to final revisions to document.This document is a draft for review purposes only and does not constitute Agency policy.
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- xxxiv AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)

David Bayliss, PhD 3

EPA

- ORD

- NCE A

Ted Berner, PhD

EPA

- ORD

- NCEA

David Bussard

EPA

- ORD

- NCEA

David Farrar, PhD

EPA

- ORD

- NCEA

John Fox, PhD

EPA

- ORD

- NCEA

EPA

- ORD

- NCEA

Karen Hogan, PhD

EPA

- ORD

- NCEA

Rosemarie Hakim, PhD 3

EPA

- ORD

- NCEA

Babashaheb Sonawane, PhD

EPA

- ORD

- NCEA

Chad Thompson, PhD 3

EPA

- ORD

- NCEA

Larry Valcovic, PhD 3

EPA

- ORD

- NCEA

John J. Vandenberg, PhD

EPA

- ORD

- NCEA

Lisa Vinikoor, PhD

EPA

- ORD

- NCEA

Paul White, PhD

EPA

- ORD

- NCEAThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxxv AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)

CONTRACTOR SUPPORT

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

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

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

Interagency Agreement (IAG) Pr oject No. 03

- 18. The ORISE individuals who contributed to

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

W. Weber.

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

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

No. 03

- 18). The primary scientists involved in the work were Kenny S. Crump and Cynthia Van

Landingham.

INTERNAL EPA REVIEWERS

Daniel Axelrad , PhD Office of Policy, Econ omics, and Innovation

Iris Camacho , PhD

Office of Pollution Prevention and Toxics

Christina Cinalli, PhD

Office of Pollution Prevention and Toxics

Rebecca Edelstein, PhD

Office of Pollution Prevention and Toxics

Ernest Falke, PhD

Office of Pollution P revention and Toxics

Stiven Foster, PhD Office of Solid Waste and Emergency Response

Greg Fritz

Office of Pollution Prevention and Toxics

Susan Griffin, PhD EPA Region 8This document is a draft for review purposes only and does not constitute Agency policy.
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- xxxvi AUTHORS, CONTRIBUTORS, AND REVIEWERS (continued)

Timothy Leighton, PhD

Office of Pesticide Programs

Elizabeth Margosches, PhD

Office of Pollution Prevention and Toxics

Timothy McMahon, PhD Office of Pesticde Programs

Julie Migrin

- Sturza, PhD Office of Policy, Economics, and Innovation

Greg Miller, PhD

Office of Children’s Health P rotection and Environmental Education

Deirdre Murphy, PhD

Office of Air and Radiation

Marion Olson, PhD EPA Region 2

Andrea Pfahles

- Hutchens, PhD

Office of Pollution Prevention and Toxics

Jennifer Seed, PhD

Office of Pollution Prevention and ToxicsThis document is a draft for review purposes only and does not constitute Agency policy.
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- xxxvii 

### This page intentionally left blank.This document is a draft for review purposes only and does not constitute Agency policy.
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- 1 1. INTRODUCTION 1

2 3 This document presents background information and justification for the Integrated Risk 4

Information System (IRIS) Summary of the hazard and dose

- response assessment of 5

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

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

assessment. 8

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

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

mode of action. The RfD (expressed in units of mg/kg

- day) is defined as an estimate (with 11

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

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

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m 3 ) is 14

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

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

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

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

acute ( ≤ 24 hours), short

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

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

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

derived for chronic exposure duration. 22

The carcinogenicity assessment provides information on the carcinogenic hazard 23

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

exposure may be deriv ed. The information includes a weight

- of

- evidence judgment of the 25

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

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

low

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

the estimate of risk per mg/kg

- day of oral exposure. Similarly, an inhalation unit risk is a 29

plausible upper bound on the estimate of risk per μg/m 3 air breathed. 30

Development of these hazard identification and dose

- response assessments for 31

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

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

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

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

for Mutagenicity Risk Assessment (U.S. EPA, 1986b)

, Recommendations for and Documentation 36This document is a draft for review purposes only and does not constitute Agency policy.
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- 2 of Biological Values for Use in Risk Assessment (U.S. EPA, 1988), Guidelines for 1

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

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

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

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

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

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

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

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

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

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

, 11

Supplemental Guidance for Assessing Susceptibility from Early

- Life Exposure to Carcinogens 12

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

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

2006b). 15

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

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

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

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

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

21This document is a draft for review purposes only and does not constitute Agency policy.
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- 1 2. BACKGROUND 1

2 3 This chapter provides an overview of the physical and chemical characteristics of 4

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

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

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

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

9

2.1. PHYSICOCHEMICAL PROPERTIES OF FORMALDEHYDE 10

Formaldehyde (CASRN 50

- 00

- 0) is the first of the series of aliphatic aldehydes and is a 11

gas at room temperature. Its molecular structure is depicted in Figure 2

- 1. It is noted for its 12

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

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

above 150 **°** C. 15

16

Figure 2

- 1. Chemical structure of formaldehyde. 17

18

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

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

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

- 1. 22

23

2.2. PRODUCTION, USES, AND SOURCES OF FORMALDEHYDE 24

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

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

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

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

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

2006). 30

31 

## C O

H HThis document is a draft for review purposes only and does not constitute Agency policy.
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- 2 Table 2

- 1. Physicochemical properties of formaldehyde 1
2

Name Formaldehyde

Interna tional Union for Pure and Applied Chemistry name Formaldehyde

Synonyms Formic aldehyde

Methanal Methyl aldehyde Methylene oxide

Oxomethane Oxymethylene

Chemical Abstracts Service Index name Formaldehyde

CASRN 50

- 00

- 0

Formula HCHO

Molecular weight 30.0 3

Density Gas: 1.067 (air = 1) Liquid: 0.815 g/mL at

– 20 **°** C

Vapor pressure 3,883 mm Hg at 25°C

Log K

ow – 0.75 to 0.35

Henry’s law constant 3.4 **×** 10

– 7 atm

- m 3 /mol at 25 **°** C

2.2 **×** 10

– 2 Pa

- m 3 /mol at 25°C

Conversion factors (25 o C, 760 mm Hg) 1 ppm = 1.23 mg/m 3 (v/v) 1 mg/m 3 = 0.81 ppm (v/v)

Boiling point

– 19.5 **°** C at 760 mm Hg

Melting point

– 92°C

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

- free aqueous solution; 50 **°** C closed cup for 37% aqueous solution with 15% methanol

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

Autoignition temperature 300 **°** C

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

Reactivity Reacts with alkalis, acids and oxidizers

3 Sources: American Conference of Governmental Industrial Hygien ists (ACGIH) (2002); 4 International Programme on Chemical Safety (IPCS) (2002); Agency for Toxic Substances and 5 Disease Registry (ATSDR) (1999); Gerberich and Seaman (1994); Walker (1975). 6 7 8 Formaldehyde is a chemical intermediate used in the production of plywood adhesives, 9

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

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

thermosetting, resin curing agents, explosives m ade from hexamethylenetetramine, urethanes, 12

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

polyacetal resins, and controlled

- release fertilizers made from urea formaldehyde concentrates 14This document is a draft for review purposes only and does not constitute Agency policy.
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- 3 (IPCS, 1989). Formaldehyd e is used in smaller quantities for the preservation and embalming of 1

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

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

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

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

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

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

usually contains about 37% formaldehyde and 12

– 15% methanol. Methanol is added to formalin 8

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

Paraformaldehyde has the formula (CH 2 O)

n where n is 8 to 100. It is essentially a solid form of 10

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

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

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

agricultural premises, and barbering equipment. Long

- chain polymers (e.g., Delrin plastic) are 14

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

residu es in the soil, photochemical processes in sea water, and forest fires (National Library of 30

Medicine [NLM], 2001)

. 31

Formaldehyde emitted to the ambient air primarily reacts with photochemically generated 32

hydroxyl radicals in the troposphere or undergoes dir ect photolysis (IPCS, 2002). Overall, half

33

lives for formaldehyde in air can vary considerably under different conditions. Estimates for 34

atmospheric residence time in several U.S. cities ranged from 0.3 hours under conditions typical 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 4 of a rainy winter ni ght to 250 hours under conditions typical of a clear summer night (assuming 1

no reaction with hydroperoxyl radicals). Given the generally short daytime residence times for 2

formaldehyde, there is limited potential for long

- range transport (IPCS, 2002). In cases where 3

organic precursors are transported long distances, however, secondary formation of 4

formaldehyde may occur far from the anthropogenic sources of the precursors. 5

Formaldehyde is released to water from the discharges of both treated and untreated 6

industrial wastewater from its production and from its use in the manufacture of formaldehyde

7

containing resins (ATSDR, 1999). Formaldehyde is also a possible drinking

- water disinfection 8

by

- product from the use of ozone and/or hydrogen peroxide. In water , formaldehyde is rapidly 9

hydrated to form a glycol, and the equilibrium favors the glycol. 10

11

2.3. ENVIRONMENTAL LEVELS AND HUMAN EXPOSURE 12

General population exposure to formaldehyde can occur via inhalation, ingestion and 13

dermal contact. Each of these pa thways and associated media levels are discussed below. 14

Formaldehyde exposure can also occur occupationally via three main scenarios: 15

16

**•** The production of aqueous solutions of formaldehyde (formalin) and their use in the 17

chemical industry (e.g., for the syn thesis of various resins, as a preservative in medical 18

laboratories and embalming fluids, and as a disinfectant). 19

**•** Release from formaldehyde

- based resins in which it is present as a residue and/or 20

through their hydrolysis and decomposition by heat (e.g., d uring the manufacture of 21

wood products, textiles, synthetic vitreous insulation products, and plastics). In general, 22

the use of phenol

– formaldehyde resins results in much lower emissions of formaldehyde 23

than those of urea

- based resins. 24

**•** The pyrolysis or combustion of organic matter (e.g., in engine exhaust gases or during 25

firefighting) (IARC, 2006). 26

27

Industries with the greatest potential for exposure include health services, business 28

services, printing and publishing, manufacture of chemicals and alli ed products, manufacture of 29

apparel and allied products, manufacture of paper and allied products, personal services, 30

machinery (except clerical), transport equipment, and furniture and fixtures (IARC, 1995). 31

32This document is a draft for review purposes only and does not constitute Agency policy.
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- 5 2.3.1. Inhalation 1

The most current ambient ai r monitoring data for formaldehyde come from EPA’s air 2

quality system database (EPA’s AirData Web site: http://www.epa.gov/air/data/index.html ) 3

(U.S. EPA, 2009b). These data have been collected from a wide variety of sources, including 4

state and local environmental agencies, but have not been collected from a statistically based 5

survey. The most recent data, for the year 2007, come from 188 monitors located in 33 states as 6

shown in Figure 2

- 2 (U.S. E PA, 2008). The annual means for these monitors range from 0.7

– 7

45.03 μg/m 3 (0.56

– 36.31 ppb) and have an overall average of 3.44 μg/m 3 (2.77 ppb). The annual 8

means are derived by EPA by averaging all available daily data from each monitor. Table 2

- 2 9

shows a breakout of the data by land use category based on the annual means from each monitor 10

for 2005, 2006, and 2007. The land use is established on the basis of the most prevalent land use 11

within 0.25 miles of the monitor. The mobile category (land near maj or highways or interstates 12

such that it is primarily impacted by mobile sources) has the highest mean levels, and 13

agricultural lands have the lowest. 14

15

16 Figure 2

- 2. Locations of hazardous air pollutant monitors. 17 Dasgupta et al. (2005) measured formaldehyd e levels in 5 U.S. cities during 1999

- 2002. 18 Samples were collected over approximately a one month period in the spring or summer. 19 Mean levels were 5.05 ppb in Nashville, TN; 7.96 ppb in Atlanta, GA; 4.49 ppb in 20 Houston, TX; 3.12 ppb in Philadelphia, PA; and 2.63 in Sydney, FL. 21This document is a draft for review purposes only and does not constitute Agency policy.
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- 6 Table 2

- 2. Ambient air levels by land use category 1
2

Formaldehyde Exposure by Category a

Agriculture Commercial Forest Industrial Mobile b Residential
Number of data points 17 166 19 61 16 282

Mean ± standard deviation 2.08 ± 0.98 3.26 ± 2.76 2.79 ± 2.17 6.28 ± 14.45 6.84 ± 7.28 2.75 ± 1.71

Minimum 0.34 0.20 0.40 0.14 2.02 0.17

Maximum 4.34 20.61 7.33 74.72 23.39 12.35 3

a Values are μg/m 3

. 4

b “Mobile” is ambient air in locations primarily impacted by mobile sources. 5
6

Source: A irData for 2005, 2006, and 2007 (U.S. EPA, 2009b)

. 7 8 9

Under the National

- Scale Air Toxics Assessment (NATA) program, EPA has conducted 10

an emissions inventory for a variety of hazardous air pollutants (HAPs), including formaldehyde 11

(U.S. EPA, 2006c). The NA TA uses the emissions inventory data to model nationwide air 12

concentrations/exposures (U.S. EPA, 2006c). The results of the 1999 ambient air concentration 13

modeling for formaldehyde suggest that county median air levels range from 0 to 6.94 μg/m 3 (0 14

– 5.59 ppb) with a national median of 0.56 μg/m 3 (0.45 ppb) (see Figure 2

- 3). Similar results 15

were found for the year 2002: county concentrations ranged from 0.12 to 9.17 μg/m 3 (0.097

– 16

7.38 ppb) with median of 0.78 μg/m 3 (0.63 ppb). NATA has not provided upda ted concentration 17

maps for 2002. The 1999 map shows the highest levels in the far west and northeastern regions 18

of the U.S. While these modeling results can be useful, it is important to consider their 19

limitations. Some of the geographical differences r esult from differences in methods used by 20

states supplying the data. For example, the high levels indicated for Idaho result from the large 21

amount of wood burned during forest fires and the relatively high emission factor that Idaho uses 22

(compared with ot her states) to estimate formaldehyde emissions from forest fires. A 23

comparison of modeling results from NATA to measured values at the same locations is 24

presented in U.S. EPA (2006c). For 1999, it was found that formaldehyde levels were 25

underestimated at 76% of the sites (n = 68). One possible reason why the NATA results appear 26

low compared to measurements is that the modeling has not accounted for secondary formation 27

of formaldehyde in the atmosphere. 28This document is a draft for review purposes only and does not constitute Agency policy.
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- 7 1

2 Figure 2

- 3. Modeled ambient air concentrations based on 1999 emissions. 3
4 5 In general, ambient levels of formaldehyde in outdoor air are significantly lower than 6

th ose measured in the indoor air of workplaces or residences (ATSDR, 1999; IARC, 1995). 7

Indoor sources of formaldehyde in air include volatilization from pressed wood products, 8

carpets, fabrics, insulation, permanent press clothing, latex paint, and paper b ags, along with 9

emissions from gas burners, kerosene heaters, and cigarettes (NLM, 2001). In general, the major 10

indoor air sources of formaldehyde can be described in two ways: 1) those sources that have the 11

highest emissions when the product is new with decreasing emission over time, as with the first 12

set in the examples above; and 2) those sources that are reoccurring or frequent such as the 13

second set of examples above. Gilbert et al. (2006) studied 96 homes in Quebec City, Canada 14

and found elevated le vels in homes with new wood or melamine furniture purchased within the 15

previous 12 months. A summary of indoor data is provided in Table 2

- 3. Results vary 16

depending on housing characteristics and date of study. 17

18This document is a draft for review purposes only and does not constitute Agency policy.
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- 8 1

Table 2

- 3. Studies on residential indoor air levels of formaldehyde (non

occupational)

Citation No. of Samples Target Population/House Type Mean (μg/m 3 ) Range (μg/m 3 )

Gold et al., 1993 Complaint homes

Older conventional homes <60 24

- 960

Hare et al., 1996 Newly built homes 91

Hare et al ., 1996 30 days after installing pressed wood 42

- 540

Gammage and Hawthorne, 1985 >1200

131

>500

260 Homes with UFFI

Homes without UFFI Complaint mobile homes

Newer mobile homes

Older mobile homes 60

- 144

30

- 84

120

- 1080

1032

300 12

- 4080

12

- 20 4

0

- 5040

Hawthorne et al., 1986 a,b 18

11

11

40 Conventional homes 0

- 5 yr

Conventional homes 5

- 15 yr Conventional homes >15 yr

Conventional homes overall 96

48

36

72 24

- 480

U.S.EPA, 1987 560 Noncomplaint, conventional, randomly selected Noncomplai nt, mobile homes, randomly selected 32

- 109

109

- 744 6

- 576

12

- 3480

Health Canada and

Environment Canada, 2001 151 Residential (Canadian) noncomplaint

homes 35 ?

- 148

Zhang et al., 1994 a,b 6 Residential, carpeted, non

- smoking

homes 66 42

- 89

Gilbert et al ., 2006 96 Residential (Canadian) 29.5 9.6

– 90.0

Shah and Singh,

1988 315 Residential & commercial 59 23

- 89

Stock, 1987 43 Conventional homes 84 96

- 216

Krzyzanowski et al.,

1990 202 Conventional homes 31

2 Note: 1 ppb = 1.2 μg/m 3 3

4

5 Salthammer et al. ( 2010) present a thorough review of formaldehyde sources and levels 6

found in the indoor environment. Based on an examination of international studies carried out in 7

2005 or later they conclude that the average exposure of the population to formaldehyde is 20 to 8

40 μ g/m 3 under normal living conditions. They used the diagram shown in Figure 2

- 4 to 9This document is a draft for review purposes only and does not constitute Agency policy.
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- 9 summarize data they found on the r ange of formaldehyde air concentrations (in ppb) in different 1

environments

. 2

3 Figure 2

- 4. Range of formaldehyde air concentrations (ppb) in different 4

environments. Source: Salthammer et al. (2010) 5
6 7 Data on formaldehyde levels in outdoor and indoor air were collected under Canada’s 8

National Air Pollution Surveillance program (IPCS, 2002; Health Canada and Environment 9

Canada, 2001). The e ffort included four suburban and four urban sites sampled in the period 10

1990

– 1998. A Monte Carlo analysis applied to the pooled data (n = 151) was used to estimate 11

the distribution of time

- weighted 24

- hour air exposures. This study suggested that mean le vels 12

in outdoor air were 3.3 μg/m 3 (2.7 ppb) and mean levels in indoor air were 35.9 μg/m 3 13

(29.2 ppb) (Health Canada and Environment Canada, 2001). The simulation analysis also 14

suggested that general population exposures averaged 33

– 36 μg/m 3 (27

– 30 ppb). 15

Since the early to mid 1980s, manufacturing processes and construction practices have 16

been changed to reduce levels of indoor formaldehyde emissions (ATSDR, 1999). A 2008 law 17

enacted by the California Air Resource Board (CARB. 2008, Final Regulation Order : Airborne 18

Toxic Control Measure to Reduce Formaldehyde Emissions from Composite Wood Products; 19

http://www.arb.ca.gov/regact/2007/compwood07/fro

- final.pdf) has limited the amount of 20

formaldehyde that can be released by specific composite wood products (i. e., hardwood 21 0.1 1 10 100 1000 Remote Air

Rural Air

Urban Air

Normal Indoor Air

Polluted Ind oor Air

Extreme ConditionsThis document is a draft for review purposes only and does not constitute Agency policy.
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- 10 plywood, particle board, and medium density fiberboard) sold, supplied, or manufactured for use 1

in California. For this reason the mean indoor air levels presented by Health Canada and 2

Environment Canada (2001) (based on samples collected from 1989

– 1995) may overestimate 3

current levels. In addition, the Canadian indoor air data may overestimate formaldehyde levels 4

in U.S. homes, because many residential homes in Canada use wood burning stoves more 5

frequently and have tighter construction (due to colder winters), leading to less dilution of indoor 6

emissions. The outdoor air levels, however, appear to have remained fairly constant over recent 7

years, and the median outdoor level from the Canadian study (2.8 μg /m 3 ) (2.3 ppb) is very 8

similar to the median of the U.S. monitoring data ( 2.83 μg /m 3 ) (2.3 ppb) in 1999

. 9

Even though formaldehyde levels in construction materials have declined, indoor 10

inhalation concerns still persist. For example, recent studies have measured formaldehyde levels 11

in mobile homes. ATSDR (2007) reported on air sampling in 96 unoccupied trailers provided by 12

the Federal Emergency Management Agency (FEMA) used as temporary housing for people 13

displaced by Hurricane Katrina. Formaldehyde levels in closed trailers averaged 1,250 ± 828 14

μg/m 3 (mean ± standard deviation [SD]) (1.04 ± 0.69 ppm), with a range of 12

– 4,390 μg/m 3 15

(0.01

– 3.66 ppm). The levels decreased to an average of 468 ± 324 μg/m 3 (0.39 ± 0.27 ppm), 16

with a range of 0.00

– 1,960 μg/m 3 (0.00

– 1.63 ppm) when the air condition ing was turned on. 17

Levels also decreased to an average of 108 ± 96 μg/m 3 (0.09 ± 0.08 ppm), with a range of 12

– 18

588 μg/m 3 (0.01

– 0.49 ppm) when the windows were opened. ATSDR (2007) found an 19

association between temperature and formaldehyde levels; higher t emperatures were associated 20

with higher formaldehyde levels in trailers with the windows closed. They also noted that 21

different commercial brands of trailers yielded different formaldehyde levels. 22

In December 2007 and January 2008, the Centers for Diseas e Control and Prevention 23

(CDC) measured formaldehyde levels in a stratified random sample of 519 FEMA

- supplied 24

occupied travel trailers, park models, and mobile homes (“trailers”) (CDC, 2008). At the time of 25

the study, sampled trailers were in use as temp orary shelters for Louisiana and Mississippi 26

residents displaced by hurricanes Katrina and Rita. The geometric mean level of formaldehyde 27

in sampled trailers was 95 μg/m 3 (77 ppb), and the range was 3.7

– 730 μg/m 3 (3

– 590 ppb). 28

29

2.3.2. Ingestion 30

Limited U .S. data indicate that concentrations in drinking water may range up to 31

approximately 10 μg/L in the absence of specific contributions from the formation of 32

formaldehyde by ozonation during water treatment or from leaching of formaldehyde from 33

polyacetyl p lumbing fixtures (IPCS, 2002). In the absence of other data, one

- half this 34

concentration (5 μg/L) was judged to be a reasonable estimate of the average formaldehyde in 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 11 Canadian drinking water. Concentrations approaching 100 μg/L were observed in a U.S. s tudy 1

assessing the leaching of formaldehyde from domestic polyacetal plumbing fixtures, and this 2

concentration was assumed to be representative of a reasonable worst case (IPCS, 2002). 3

Formaldehyde is a natural component of a variety of foodstuffs (IARC, 1 995; IPCS, 4

1989). However, foods may be contaminated with formaldehyde as a result of fumigation (e.g., 5

grain fumigation), cooking (as a combustion product), and release from formaldehyde resin

6

based tableware (IARC, 1995). Also, the compound has been us ed as a bacteriostatic agent in 7

some foods, such as cheese (IARC, 1995). There have been no systematic investigations of 8

levels of formaldehyde in a range of foodstuffs that could serve as a basis for estimation of 9

population exposure (Health Canada and E nvironment Canada, 2001). According to the limited 10

available data, concentrations of formaldehyde in food are highly variable. In the few studies of 11

the formaldehyde content of foods in Canada, the concentrations were within a range of 12

<0.03

– 14 mg/kg (H ealth Canada and Environment Canada, 2001). Data on formaldehyde levels 13

in food have been presented by Feron et al. (1991) and IPCS (1989) from a variety of studies, 14

yielding the following ranges of measured values: 15

16

**•** Fruits and vegetables: 3

– 60 mg/kg 17

**•** Meat and fish: 6

– 20 mg/kg 18

**•** Shellfish: 1

– 100 mg/kg 19

**•** Milk and milk products: 1

– 3.3 mg/kg 20

21

Daily intake of formaldehyde was estimated by IPCS (1989) to be in the range of 1.5

– 22

14 mg for an average adult. Similarly, Fishbein (1992) estimated that the intake of forma ldehyde 23

from food is 1

– 10 mg/day but discounted this on the belief that it is not available in free form. 24

Although the bioavailability of formaldehyde from the ingestion of food is not known, it is not 25

expected to be significant (ATSDR, 1999). Using U.S. Department of Agriculture (USDA) 26

(1979) consumption rate data for various food groups, Owen et al. (1990) calculated that annual 27

consumption of dietary formaldehyde results in an intake of about 4,000 mg or approximately 28

11 mg/day. 29

30

2.3.3. Dermal Contact 31

The general population may have dermal contact with formaldehyde

- containing 32

materials, such as some paper products, fabrics, and cosmetics. For example, nail hardeners 33

contain formalin, and cosmetics products such as hand creams and suntan lotions contai n 34

formaldehyde

- releasing agents (but not formaldehyde) as preservatives. Generally, though, 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 12 dermal contact is more of a concern in occupations that involve handling concentrated forms of 1

formaldehyde, such as those occurring in embalming and chemical prod uction. 2

3This document is a draft for review purposes only and does not constitute Agency policy.
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- 1 3. TOXICOKINETICS 1

2 3

3.1. CHEMICAL PROPERTIES AND REACTIVITY 4

Formaldehyde (HCHO) is the smallest aldehyde (30 g/mol) and is a gas at room 5

temperature. It is highly water soluble and reactive. In water, less than 0.1% of formaldehyde 6

exists un hydrated, with the majority reported to be in the hydrated form, methylene glycol 7

(CH 2 (OH) 2 ) (Priha et al., 1996) . Formaldehy de reacts readily with high and low molecular 8

weight biological constituents. 9

10

3.1.1. Binding of Formaldehyde to Proteins 11

Formaldehyde is a reactive molecule that is likely to react with both low molecular 12

weight cellular components (e.g., reduced glut athione[GSH]) as well as high molecular weight 13

components. Unlike deoxyribonucleic acid (DNA), which has some additional barriers to 14

exposure (i.e., nucleus), extracellular and intracellular proteins are obvious targets for interacting 15

with formaldehyde. Formaldehyde is a well

- known cross

- linking agent that is used in the 16

fixation of tissues, preparation of vaccines, and study of protein

- protein interactions (Metz et al., 17

2006). However, the exact nature of the protein modifications used for these purpos es is not yet 18

fully characterized (Metz et al., 2006, 2004). Figure 3

- 1 provides a general reaction scheme for 19

formaldehyde

- mediated modifications of amino acids. In step 1, formaldehyde reacts with 20

primary N

- terminal amines to form a labile methylol add uct. This adduct can undergo 21

dehydration (step 2) to form an imine, or Schiff base (

**−** N=CH 2 ). Metz et al. (2004) examined 22

the types of formaldehyde

- protein reactions that are likely to occur in vivo by synthesizing 23

several identical polypeptides with one varying amino acid (X) within the sequence VEL X VLL 24

(V=valine, E=glutamate, L=Leucine, X=varying amino acid). Several peptides with reactive 25

amino acids did not exhibit modifications, suggesting that the peptide sequence/structure affects 26

the ability of fo rmaldehyde to react with amino acids. Peptides that were modified indicated 27

formation of methylol adducts (Figure 3

- 1, step 1) or a mixture of methylol and imine adducts 28

(Figure 3

- 1, step 2). 29This document is a draft for review purposes only and does not constitute Agency policy.
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- 2 1
2 Figure 3

- 1. Formaldehyde

- mediated protein modifications

. 3
4 Note: Formaldehyde reacts with primary N

- terminal amines to form a methylol 5 adduct [1], which increases the molecular weight by 30 Da ( **∆** m). This labile 6 adduct can rearrange to form an amine, or Schiff base [2], that results in an 7

increase in MW of 12 Da. Schiff bases can react with certain amino acids to form 8

intra

- or intermolecular methylene bridges [3]. The two amino acids depicted in 9 step 3 may be within the same protein or possibly from two different proteins. 10
11 Source: Metz et al. (2004). 12
13 14

Mu cus is composed of water, electrolytes, polysaccharides, and about 0.5% soluble 15

proteins (Priha et al., 1996; Bogdanffy et al., 1987) . Bogdanffy et al. (1987) showed that 16

although human nasal mucus can bind 70% of 100 mM formaldehyde, irreversible binding of 17

[ 14 C]

- formaldehyde to serum albumin (the major protein in mucus) was shown to be insignificant 18

after a 1

- hour incubation. Irreversible binding (50% or more) did not occur until after about 7 19

hours of in cubation. These data suggest that the protein content of mucus may not provide a 20

significant formaldehyde irreversible sink. Nonetheless, the solubility of formaldehyde in mucus 21

along with mucus flow and ingestion likely indicate that much of the inhaled dose is removed

— 22

perhaps as much as 42% in rodents (IARC, 2005; Schlosser, 1999). 23This document is a draft for review purposes only and does not constitute Agency policy.
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- 3 In general, formaldehyde interacts with proteins. Studies carried out in cell culture media 1

containing serum and formaldehyde have shown that such mixtures are quite labile. For 2

example, during a 60

- minute incubation of formaldehyde with complete cell media (i.e., with 3

fetal calf serum) at 38 **°** C, gas chromatography

- mass spectrometry (GC

- MS) exhibited very 4

different peak profiles at different points during the incubation (Proc tor et al., 1986). In contrast, 5

GC

- MS chromatograms of cell media containing formaldehyde but no serum proteins appeared 6

relatively unchanged throughout the incubation. Compared to cell culture medium alone, 7

complete media were considered to provide a mo re suitable model for the hypothetical 8

interactions that formaldehyde could undergo in vivo (including perhaps blood). 9

10

3.1.2. Endogenous Sources of Formaldehyde 11

Endogenous formaldehyde is produced through a) normal cellular metabolism through 12

enzymatic or non

- enzymatic reactions, and also as a detoxification product of xenobiotics during 13

cellular metabolism: 14

15

3.1.2.1. Normal Cellular Metabolism (Enzymatic) 16

Formaldehyde is produced during normal metabolism of methanol, amino acids (e.g., 17

glycine, se rine, methionine), choline, dimethylglycine, and methylamine and through the folate

18

dependent endogenous one

- carbon pool etc. 19

i) One of the endogenous sources for formaldehyde production is methanol, 20

formed during normal cellular metabolism. However, th is fraction may also be derived 21

through consumption of fruits, vegetables and alcohol (Shelby et al . 2004; IPCS, 1997)

. 22

In studies conducted with healthy humans whose diet was devoid of methanol

- containing 23

or meth anol

- generating foods (such as cereals containing aspartame, a precursor of 24

methanol) and who abstained from alcohol consumption, the background blood levels of 25

methanol range from 0.25

– 4.7 mg/L (Reviewed in Shelby et al 2004 [CERHR]). 26

Methanol is metaboli zed to formaldehyde predominantly by hepatic alcohol 27

dehydrogenase

- 1 (ADH1) in primates and by ADH1 and catalase (CAT) in rodents, 28

ADH1 requiring nicotinamide adenine dinucleotide (NAD + ) as a cofactor. 29

ii) Dimethylglycine (DMG), one of the byproducts of choline metabolism 30

endogenously present in the body, is an indirect source of endogenous formaldehyde. 31

Two specific dehydrogenases, i) dimethylglycine dehydrogenase (DMGDH) which 32

converts DMG to sarcosine (methylglycine) and ii) sarcosine dehydrogenase ( SDH) 33

which converts sarcosine to glycine, have been shown to non

- covalently bind to the 34This document is a draft for review purposes only and does not constitute Agency policy.
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- 4 folate enzyme, tetrahydrofolate (THF). Further, these dehydrogenases form “active 1

formaldehyde” by removing the 1

- carbon groups from THF (Binzak et al., 2000)

. 2

iii) Another source of endogenous formaldehyde is methylamine (MA), an 3

intermediary component of the metabolism of adrenaline, sarcosine, creatine, lecithin, 4

and other dietary sources (Yu and Zuo 1996) . The enzyme semicarbozole

- sensitive 5

amine oxidase (SSAO), predominantly present in the plasma membrane of endothelial 6

smooth muscle cells and in circulating blood, converts methylamine to formaldehyde, 7

hydrogen peroxide and ammonia. The formaldehyde thus released has been shown to 8

cause endothelial injury eventually leading to atheroscloros is (Kalasz, 2003) . Yu et al. 9

(1997) have shown that adrenaline, released in the body as a response to stress, is known 10

to be deaminated by the enzyme monoamine oxidase, with further conversion of 11

methylamine to formaldehyde by SSAO (Yu et al . 1997) . Creatine is another precursor 12

for methylamine wh ich is metabolized by SSAO to form formaldehyde. It has been 13

shown that short

- term, high

- dose dietary supplementation of creatine in healthy humans 14

causes a significant increase in urinary methylamine and formaldehyde levels (P oortmans 15

et al., 2005)

. 16

iv) Endogenous formaldehyde is also a constituent of the one

- carbon pool, a 17

network of interrelated biochemical reactions that involve the transfer of one

- carbon 18

groups from one compound to another (usually the transfer of the hyd roxymethyl group 19

of serine to tetrahydrofolic acid). 20

21

Tyihak et al. (1998) have demonstrated that formaldehyde, but not the methyl radical or 22

methyl cation, is involved in the enzymatic transmethylation and demethylation reactions, and 23

suggested the pre sence of a formaldehyde cycle in cells for the production and removal of 24

formaldehyde utilizing the transfer through methionine  S

- adenosylmethionine  S

- adenosyl

25

homocysteine  homocysteine (Tyihak et al., 1998) . However, these studies did not clearly 26

show whether the formaldehyde released in this cycle is in free or bound form. 27

Formaldehyde has been shown to be produced in normal and leu kemic leukocytes from 28

N 5

- methyl

- THF by enzymatic degradation (Thorndike and Beck, 1977) . This is a two

- step 29

reaction involving 1) enzymatic conversion of the methyl

- THF to formaldehyde followed by 2) 30

nonenzymatic reaction of formaldehyde with an amine. Thorndike and Beck (1977) show ed that 31

leukocyte (granulocyte and lymphocyte) cell extracts from normal individuals and patients with 32

chronic lymphocytic leukemia (CLL) or chronic myelocytic leukemia (CML) incubated with 33

14 C

- methyl

- THF and saturating amounts of tryptamine produced free formaldehyde which is 34

detected as its corresponding carboline derivative formed with tryptamine. These results 35This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 5 demonstrate the activity of the enzyme N 5 , N 10

- methylene THF reductase which oxidizes N 5

- 1

methyltetrahydrofolate to N 5 , N 10 methylene THF. The authors noted that the enzyme levels 2

were in the order of normal granulocytes < normal lymphocytes < granulocytes from a CML 3

individual < lymphocytes from a CLL individual (Thorndike and Beck, 1977) , suggesting 4

increased activity of formaldehyde producing enzyme in leukemic cells compa red to normal 5

leukocytes. Overall, formaldehyde might be a byproduct as well as an intermediary product in 6

several of these reactions. 7

8

3.1.2.2. Normal Metabolism (Non

- Enzymatic) 9

i) Formaldehyde can also be formed non

- enzymatically by the spontaneous re action of 10

methanol with hydroxyl radicals, wherein cellular hydrogen peroxide is the precursor for 11

hydroxyl radicals generated through Fenton reaction (Cederbaum and Qureshi, 1982)

. 12

13

ii) Another mechanism of nonenzymatic production of formaldehyde is through lipid 14

peroxidat ion of polyunsaturated fatty acids (PUFA) (Shibamoto, 2006; Slater, 1984) . In this 15

mechanism, reactive oxygen species (ROS) generated during oxidative stress abstract a hydrogen 16

atom from a methylene group of polyu nsaturated fatty acids (PUFA) in cell membranes causing 17

autooxidation of lipids with the eventual production of free radicals (e.g., peroxy radical). It is 18

known that a certain level of oxidative stress and lipid peroxidation does occur in normal 19

individu als, and these cellular metabolic processes are likely to contribute to endogenous 20

formaldehyde production. 21

22

3.1.2.3. Exogenous Sources of Formaldehyde Production 23

Microsomal cytochrome P450 enzymes catalyze oxidative demethylation of N

- , O

- and 24

S

- methyl groups of xenobiotic compounds whereby formaldehyde is produced as a primary 25

product, which is subsequently incorporated into the one

- carbon pool by reacting with 26

tetrahydrofolic acid or is oxidized to formate (Dahl and Hadley, 1983; Heck et al., 1982) . Also, 27

some special peroxidases, such as peroxide

- dependent horseradish peroxidase enzymatically 28

catalyze xenobiotics to generate formaldehyde in the body. In particular, an ethyl peroxide

29

dependent horseradish perox idase has been shown to act on N,N

- dimethylaniline and produce 30

equimolar amounts of N

- methylaniline and formaldehyde (Kedderis and Hollenberg, 1983)

. 31

The tobacco

- specific nitrosamine nitrosamine 4

- (methylnitrosamino)

- 1

- (3

- pyridyl)

- butanone 32

(NNK) is another source of formaldehyde. It has been shown that formaldehyde is also 33

produced during the methyl hydroxylation of NNK by rat liver microsomes (Castonguay et al., 34

1991)

. Also recent studies have demonstrated the formation of formaldehyde

- DNA adducts in 35This document is a draft for review purposes only and does not constitute Agency policy.
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— DO NOT CITE OR QUOTE 3

- 6 NNK

- treated rats using a highly sensitive liquid chromatography

- electrospray ionization

- tandem 1

mass spectrometry with selected reaction monitoring (Wang et al., 2007) , suggesting formation 2

of formaldehyde from nitrosamines. Cigarette smoke is also a source of exogenously produced 3

methylamine which is converted to formaldehyde by SSAO (Yu, 1998)

. 4

5

3.1.2.4. FA

- GSH Conjugate as a Method of Systemic Distribution 6

Formaldehyde is primarily metabolized by alcohol dehydrogenase (ADH3) which uses 7

the formaldehyde

- glutathione hemiacetal adduct as the substrate. Sangha ni et al. (2000) have 8

shown that due to high circulating concentrations (50

- fold) of glutathione in human blood, the S

9

(hydroxymethyl)glutathione (HMGSH) adduct, the nonenzymatic product of formaldehyde with 10

glutathione is the major form of formaldehyde se en in vivo (Sanghani et al., 2000) . It is likely 11

that the reversibly bound HMGSH may be transported to different tissues through circulation, 12

but, specific experimental evidence is lacking. 13

14

3.1.2.5. Metabolic Products of FA Metabolism (e.g., Fo rmic Acid) 15

Formate is converted to carbon dioxide (CO 2 ) in rodents predominantly by a folate

16

dependent enzyme pathway (Dikalova et al., 2001) . Formate is also oxidized to CO 2 and water 17

by a minor pathway involving catalase located in rat liver peroxisomes (Waydhas et al., 1978; 18

Oshino et al., 1973) . In the folate

- dependent pathway, tetrahydrofolate (THF)

- mediated 19

oxidation of formate and the transfer of one

- carbon compounds between different derivatives of 20

THF has been described. 21

Endogenous levels of formate also will be affected by dietary intake of methanol

22

producing or methanol

- containing diets since methanol is initially converted to formaldehyde 23

and eventually metabo lized to formate. It has been shown in several studies in human subjects 24

who were restricted on consuming methanol producing diets, aspartame or alcohol, that the 25

endogenous blood concentrations of formate ranged from 3.8 to 19.1 mg/L (Shelby et al 2004 26

[CERHR]). The biological half life of formic acid is 77

- 90 min (Owen et al., 1990b) . The levels 27

of formate in the urine of unexposed individuals range from 11.7 to 18 mg/L (Boeniger, 1987)

. 28

One source of formic acid intake is through diet which ranges from 0.4 to 1.2 mg per day 29

(Boeniger 1987) . The half life for plasma formate is ~30 minutes or longer (Boeniger 1987)

. 30

31

3.1.2.6. Levels of Endogenous Formaldehyde in Animal and Human Tissues 32

Heck et al. (1982) estimated that endogenous levels of formaldehyde (free as well as 33

bound) in rats ranged from 0.05 to 0.5 μmole/g (1.5

- 15 μg/g) of wet tissue as analyzed by the 34

stable isotope dilution with GC

- MS method (Heck et al., 1982) . Although the levels of free 35This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 7 formaldehyde cannot be measured due to their high reactivity and short half life, they were 1

calculated by Heck et al. (1985) using an indirect method. They added a molar excess of GSH or 2

THF to the test tube containing formaldehyde in aqueous solution enabling complete binding. 3

When estimated, they observed that the amount of formaldehyde detected was equal to the total 4

amount added to the reaction suggesting that the formaldehyde measured contained both free and 5

bound forms. Further, they calculated the free formaldehyde concentration using the 6

di ssociation constant of the HMGSH adduct and cellular concentration of GSH. Human 7

formaldehyde dehydrogenase has been shown to have a dissociation constant of 1.5 mM for the 8

formaldehyde

- GSH hemithioacetal adduct (Uot ila and Koivusalo 1974) , while the folate enzyme 9

product N 5 ,N 10

- methylene

- THF has a dissociation constant of 30 mM (Kallen and Jencks 1966b; 10

a) . This could be evaluated using the Michaels

- Menton constant (K

m ) of f ormaldehyde 11

dehydrogenase for the GSH adduct (~4 μM at 25 o C), whereby they calculated the free 12

formaldehyde level to be around 3

- 7 μM or 1

- 2% of the total formaldehyde as measured by GC

13

MS in rat tissues (Heck 1982). 14

Cascieri and Clary (1992) estimated th e total body content of formaldehyde in human 15

body based on the following assumptions. For an individual with an average body wt of 70 kg 16

and with body fluids accounting for 70% of body weight, total formaldehyde content is 17

distributed in ~49 kg of body ma ss or 49 L of body fluids, owing to the water solubility and 18

uniform distribution of formaldehyde in body fluids. It has been shown that the average blood 19

concentration (mean ± S.E.) of formaldehyde in unexposed rats and humans was 2.24 ± 0.07 and 20

2.61 ± 0.14 μg/g of blood, respectively (Heck et al., 1985) , and in unexposed rhesus monkeys it 21

was 2.42 ± 0.09 μg/g of blood (Casanova et al., 1988) , overall giving an average of 22

approximately 2.5 ppm (2.5 mg/L) formaldehyde across the species. All these studies used 23

pentafluorophenyl hydra zine derived formaldehyde using GC

- MS analysis (Table 3

- 1). 24

Assuming these values, the body content of total formaldehyde is 122.5 mg (49 L x 2.5 mg/L) or 25

1.75 mg/kg body wt at any given time. Formaldehyde given intravenously to rhesus monkeys 26

has been sh own to have a half life of ~1.5 min in blood, wherein formaldehyde in blood was 27

measured by the dimedone method (McMartin et al., 1979) . Using this information Cascieri and 28

Clary (1992) calculated that the human body generates approxi mately 40.83 mg/min [(122.5 mg 29

/2 x 1.5] of formaldehyde. Biotransformation of formaldehyde to carbon dioxide in the liver 30

alone has been estimated at 22 mg/minute (Owen et al., 1990a)

. 31

Free formaldehyde is detected in body fluids and tissues using dimedone (Szarvas et al., 32

1986) or 2,4

- dinitrohenyhydrazine (DNPH) or pentafluoropheyl hydrazine (PFPH) derivative 33

(Heck et al., 1985) or as a fluorescent derivative (Luo et al . 2001) as trapping agent and detected 34

by analytical techniques such as thin

- layer chromatography (TLC), high

- performance liquid 35This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 8 chromat ography (HPLC) and gas

- chromatography mass spectrometry (GC

- MS). Data from 1

several studies is summarized in Table 3

- 1. Using 14 C

- labeled dimedone, a chemical which 2

condenses with free formaldehyde forming a product termed “formaldemethone” enabling 3

radiom etric detection, Szarvas et al (1986) estimated the levels of endogenous formaldehyde in 4

human blood plasma to be 0.4

- 0.6 μg/mL and in human urine to be 2.5

- 4 μg/mL (Szarvas et al . 5

1986)

. 6

7

Table 3

- 1. Endogenous formaldehyde levels in animal and human tissues 8 and body fluids 9

10

Tissue Method Detected as Formaldehyde levels Reference

Not specified Not specified Not specified 0.003

- 0.012 ppm (3

- 12 ng/g) Hileman 1984

Not specified GC

- MS with stable

is otope dilution method As PFPH

derivative 1.5

- 15 ppm (0.05

- 0.5 μmole/g) Heck et al 1982a

Blood GC

- MS with select ion monitoring As PFPH

derivative 2.24 ± 0.07 ppm (2.24 ± 0.07 μg/g) Heck et al 1985

Blood GC

- MS with select ion monitoring As PFPH

derivati ve 2.61 ± 0.14 ppm (2.61 ± 0.14 μg/g) Heck et al 1985

Plasma Reverse phase HPLC

fluorescent detection As product of ampicillin 1.65 ppm (1.65 μg/mL) Luo et al 2001

Heart perfusate HPLC As DNPH adduct 0.089

- 0.126 ppm (2.98

- 4.21 nmol/mL) Shibamoto 2006

Blood GC

- MS with select ion monitoring As PFPH

derivative 2.42 ± 0.09 ppm (2.42 ± 0.09 μg/g) Casanova et al

1988

Plasma Radiometric method As formalde

methone adduct 0.4

- 0.6 ppm (0.4

- 0.6 μg/mL) Szarvas et al 1986

Urine Radiometric method As formald e

methone adduct 2.5

- 4.0 ppm (2.5

- 4.0 μg/mL) Szarvas et al 1986
11 Values in the parenthesis, originally cited in the references, are converted to parts per million (ppm) as indicated. 12 PFPH, pentafluorophenyl hydrazone derivative; DNPH, dinitrophenyl hy drazine; GC

- MS, gas

- chromatography mass 13 spectrometry; HPLC, high performance liquid chromatography. 14
15
16 Hileman (1984) reported that the endogenous levels of metabolically derived 17

formaldehyde will be in the range of 3

- 12 ng/g of tissue (Hileman 1984) . So for an average 70 18

kg individual, the endogenous level of metabolically derived formaldehyde would be 210 μg to 19

840 μg (3

- 12 ng/g x 1000 g x 70). 20

21This document is a draft for review purposes only and does not constitute Agency policy.
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- 9 3.2. ABSORPTION 1

3.2.1. Oral 2

Oral absorption of [ 14 C]

- formaldehyde (7 mg/kg) in rats resulted in 40% elimination as 3

14 C

- carbon dioxide ( 14 CO 2 ), with 10% excretion in urine, 1% excretion in feces, and much o f the 4

remaining 49% retained within the carcass, presumably due to metabolic incorporation (IARC, 5

1995; Buss et al., 1964)

. 6

7

3.2.2. Dermal 8

Jeffcoat et al. (1983) reported on the disposition of various doses of [ 14 C]

- formaldehyde 9

dermally administered t o rats, guinea pigs, and monkeys. Very little (<1% of the applied dose) 10

of the radiolabel was found in the major organs excised during necropsy. As noted by the 11

authors, the disposition of formaldehyde when administered via the dermal route was markedly 12

different to that observed when the compound was administered intravenously or 13

intraperitoneally. In the latter cases, there was much evidence of metabolic activity, and 14

substantial portions of the load were expired as CO 2 . The difference appeared to be the result of 15

a reaction of dermally applied formaldehyde with macromolecules at or near the skin surface or 16

of its evaporation. In general, portions of the load that succeed in entering the circulation 17

probably do so bound to macromolecules or by incorpo ration of the radiolabel via the one

18

carbon pool. Likewise, Bartnik et al. (1985) who applied [ 14 C]

- formaldehyde to the shaved 19

backs of rats concluded that the overwhelming majority of the formaldehyde load remained 20

sequestered in the outer layers of skin at or near the site of application. At the end of the various 21

measurements, approximately 70% of the dose was found in the treated skin, with a marked 22

localization of the remaining radioactivity in the uppermost layers. This fraction of the load was 23

con sidered to be permanently sequestered, most likely as a result of irreversible binding to 24

macromolecular components. 25

26

3.2.3. Inhalation 27

Studies indicate that the majority of inhaled formaldehyde is absorbed in the upper 28

respiratory tract (URT) but that th e extent of the scrubbing in this region varies significantly 29

across species. In dogs, nearly 100% of nasally inhaled formaldehyde is absorbed (Egle, Jr., 30

1972) . Lower respiratory tract (LRT) studies designed to collect formaldehyde via a tube 31

inserted into the lower trachea revealed that nearly 95% of formaldehyde was absorbed during 32

the first pass through the upper respirato ry tract (Egle, Jr., 1972) , an effect observed with 33

multiple ventilation rates. The rat nasal passages also scrub nearly all of the inhaled 34

formaldehyde (on average ~97%) (Morgan et al., 1986). In computational dosimetry modeling 35This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 10 based on anatomically realistic representation of the human nasal airways from a single 1

individual, approximately 90% of inhaled formaldehyde was pre dicted to be absorbed in the nose 2

at resting inspiration. As the inspiratory rate increased, this fraction decreased to about 70% at 3

light exercise and to 58% at heavy exercise conditions (see Figure 1 in Kimbell et al. [2001b]). 4

The normal human breathi ng mode during heavy exercise is oronasal (with ~54% of airflow 5

being oral) (ICRP 66, 1994). Consequently, it is estimated that during heavy exercise breathing 6

(50 L/min) the flux of formaldehyde into tissue (or rate of mass transported per mm 2 of tissue 7

surface area) in the first six to eight generations of the tracheobronchial airways is comparable to 8

that in the nasal region (Overton et al., 2001). 9

It is important to note that the computer simulations mentioned above are based on 10

anatomical representat ions of a single individual. Significant anatomical variations occur in 11

human nasal airways. For example, the nasal volumes of 10 adult nonsmoking subjects between 12

18 and 50 years of age in a study in the U.S. varied between 15 and 60 mL (Santiago et al.

, 13

2001), and disease states can result in considerable further variation (Singh et al., 1998). 14

Species differences in kinetic factors have been argued to be the key determinants of 15

species

- specific lesion distributions for formaldehyde and other reactive inhaled gases. Airway 16

geometry is an important determinant of inhaled

- formaldehyde dosimetry in the respiratory tract 17

and its differences across species. These issues will be discussed in a later section on dosimetry 18

modeling. 19

20

3.2.3.1. Formaldehyde U ptake Can Be Affected by Effects at the Portal of Entry 21

Certain formaldehyde

- related effects have the potential to modulate its uptake and 22

clearance. The mucociliary apparatus of the upper respiratory tract is the first line of defense 23

against airborne to xins. Comprising a thick mucus layer (epiphase), hydrophase, and a ciliated 24

epithelium, the mucociliary apparatus may entrain, neutralize, and remove particulates and 25

airborne chemicals from inspired air. As reviewed by Wolfe (1986), airborne pollutants and 26

reactive gases have been shown to decrease mucus flow rates in several animal models (Mannix 27

et al., 1983; Iravani, 1974; Carson et al., 1966; Dalhamn, 1956; Cralley, 1942). Degradation in 28

the continuity or function of this mucociliary apparatus could result in a lower clearance of 29

inhaled pollutants at the portal of entry. 30

Morgan et al. (1983) first reported defects in mucociliary function in F344 rats exposed 31

to 15 ppm formaldehyde 6 hours/day for 1

– 9 days. Mucostasis occurred in several regions i n all 32

rats after a single 15 ppm exposure. Ciliastasis occurred with greater frequency and across more 33

regions of the nasoturbinate in subsequent days of exposure. The authors observed that 34

mucostasis preceded ciliastasis in most cases, and vigorous cili ary activity was noted in areas 35This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 11 without mucus flow . Morgan et al. (1984a) also studied formaldehyde effects on the mucociliary 1

apparatus of isolated frog palates in vitro. Mucostasis was evident as mucus became stiff and 2

eventually rigid with increasing formaldehyde concentration and time of exposur e. Ciliary beat 3

continued even after mucostasis, but ciliastasis ultimately occurred when exposure reached 4 and 4

9 ppm. 5

When a rodent is exposed to an irritant, its inhaled dose and pattern of deposition can be 6

profoundly affected by reflex bradypnea, a p rotective reflex seen in rodents but not in humans. 7

Reflex bradypnea can occur when the trigeminal nerve is exposed to a sufficient concentration of 8

an irritant, such as formaldehyde. It is manifest as markedly decreased activity or prostration, 9

reduced metabolism, hypothermia (as much as 5oC), significantly reduced respiratory rate and 10

minute volume, and altered blood and brain chemistry. Because of their small size, rodents are 11

able to rapidly lower their metabolism and body temperature and therefore t heir oxygen demand. 12

The consequence is that their inhaled dose of an irritating chemical is dramatically lowered. 13

Reflex bradypnea is quantified as the RD 50 , which is the concentration of a chemical that results 14

in a 50% decrease in respiratory rate. It can take as much as two hours for rodents to fully 15

recover from the effects of reflex bradypnea. The clinical manifestations of reflex bradypnea can 16

easily be misconstrued as toxicity. None of the studies described in this assessment took into 17

account th e fact that reflex bradypnea may have confounded the results. Reflex bradypnea is 18

discussed in depth in section 4.2.1.1. 19

Sensory irritation studies suggest that formaldehyde activates the trigeminal nerve by 20

activating nociceptors through the modification of receptor amino acids, possibly including thiol 21

groups. Cassee et al. (1996) measured sensory irritation to formaldehyde, acetaldehyde, and 22

acrolein in male Wistar rats, following a 30

- minute nose

- only exposure. Formaldehyde and 23

acrolein elicited simi lar responses, whereas acetaldehyde was far less irritating. The authors 24

suggested that the differences in sensitivity to the aldehydes might be explained by differences in 25

physicochemical properties and by regional differences in activities of detoxifyin g enzymes for 26

each chemical. In addition, it has been suggested that acetaldehyde might interact with sensory 27

nerves via an amino group (S teinhagen and Barrow, 1984) , whereas the receptor

- binding site for 28

formaldehyde and acrolein is believed to be a thiol group. Differential binding sites for sensory 29

irritants in the trigeminal nerve have been reported (Nielsen, 1991)

. 30

Sensory irritation effects are discussed in depth in Chapter 4 but are noted here because 31

stimulation of the trigeminal nerve by formaldehyde can result in sig nificantly lower pulmonary 32

ventilation, and formaldehyde exposure in rodents at concentrations that approach the RD 50

. 33

Barrow et al. (1983) have estimated the “inhaled dose” equivalent to an exposure concentration 34

of 15 ppm in mice and rats used in the ch ronic formaldehyde bioassays by Kerns et al. (1983) 35This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 12 and Monticello and Morgan (1994). Their results indicate that, because mice are observed to 1

decrease their minute volume by approximately 75% as compared to 45% in rats, a twofold 2

greater inhaled dose wo uld be expected in rats versus mice. This difference may be relevant to 3

the increased incidence of squamous cell carcinoma of the nasal cavity in F344 rats as compared 4

to B6C3F1 mice. Chang et al. (1983) estimated a reduction of 25% in the minute volume of 5

F344 rats. Yokley et al. (2008) have recently published a model that accounts for physiological 6

changes in ventilation rate induced by sensory irritation in rats. Thus, the “standard” minute 7

volumes used for rats and mice need to be adjusted downward when calculating dosimetric 8

adjustment factors for extrapolation of adverse effects to humans (Thompson et al., 2008). This 9

question is further discussed in the section on modeling the dosimetry. 10

Another effect that modulates dosimetry is the dynamic tis sue remodeling of nasal 11

airways that occurs as a consequence of exposure to reactive gases. For example, formaldehyde 12

dosimetry is influenced by the occurrence of squamous metaplasia, an adaptive tissue conversion 13

to squamous that occurs in nasal epitheli um exposed to toxic levels of formaldehyde. The 14

metaplasia has been observed to occur in rats at exposure concentrations of 3 ppm and higher 15

(Kimbell et al., 1997). Squamous epithelium is known to absorb considerably less formaldehyde 16

than other epitheli al types (Kimbell et al., 1997). Overall, the highest flux levels of 17

formaldehyde in the simulations of the rat nose in Kimbell et al. (2001a) are estimated in the 18

region just posterior to the nasal vestibule. A consequence of squamous metaplasia would b e to 19

“push” the higher levels of formaldehyde flux toward the more distal regions of the nose 20

(Kimbell et al., 1997). Subramaniam et al. (2008) discussed this issue further in the context of 21

uncertainties in the modeling of formaldehyde dosimetry. 22

23

3.2.3. 2. Variability in the Nasal Dosimetry of Formaldehyde in Adults and Children 24

Garcia et al. (2009) used computational fluid dynamics to study human variability in the 25

nasal dosimetry of reactive, water

- soluble gases in 5 adults and 2 children, aged 7 and 8 years 26

old. They considered two model categories of gases, corresponding to maximal and moderate 27

absorption at the nasal lining. We focus here only on the “maximum uptake” simulations in 28

Garcia et al. (2009). In this case, the gas was considered so highly reactive and soluble that it 29

was reasonable to assume an infinitely fast reaction of the absorbed gas with compounds in the 30

airway lining. Although such a gas could be reasonably considered to represent formaldehyde, 31

these results cannot be fully utilized to inform quantitative estimates of formaldehyde dosimetry 32

(and it does not appear to have been the intent of the authors either). This is because the same 33

boundary condition corresponding to maximal uptake was applied on the vestibular lining of the 34

nose as well as on the transitional and transitional epithelial lining on the rest of the nose. This is 35This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 13 not appropriate for formaldehyde as the lining on the nasal vestibule is made of keratinized 1

epithelium which is considerably less absorbing than the rest of the nose (Kimbell et al. 2001). 2

The Garcia et al. (2009) study and the results of their analyses have been further 3

described and evaluated in Appendix 3

- 1. Overall uptake efficiency, average flux (rate of gas 4

absorbed per unit surface area of the nasal l ining) and maximum flux levels over the entire nasal 5

lining did not vary substantially between adults (1.6

- fold difference in average flux and much 6

less in maximum flux), and the mean values of these quantities were comparable between adults 7

and children. These results are in agreement with conclusions reached by Ginsberg et al. (2005) 8

that overall extrathoracic absorption of highly and moderately reactive and soluble gases 9

(corresponding to category 1 and 2 reactive gases as per the scheme in USEPA [1994]) is similar 10

in adults and children. On the other hand, Figure 6A of the paper (reproduced as Figure A in 11

Appendix 3

- 1), shows significant interhuman variability in flux values at specific points on the 12

nasal walls. The local flux of formaldehyde varies amo ng individuals by a factor of 3 to 5 at 13

various distances along the septal axis of the nose. 14

15

3.3. DISTRIBUTION 16

3.3.1. Levels in Blood 17

Inhalation studies in several species indicate that exposure to formaldehyde does not 18

result in elevated levels in blo od. These studies were carried out over a wide range of exposure 19

concentrations and durations. Rats exposed to 14 ppm formaldehyde for 2 hours exhibited no 20

increase in blood formaldehyde levels [2.25 ± 0.07 μg/(g blood) in treated animals compared 21

with 2 .24 ± 0.07 μg/(g blood) in control animals] when measured by GC

- MS using a stable 22

isotope dilution technique (Heck et al., 1985, 1982) . Similarly, mean formaldehyde blood levels 23

in humans (n = 6) expos ed to 1.9 ppm formaldehyde for 40 minutes in a walk

- in chamber (2.77 ± 24

0.28 μg/g blood) were not statistically different from measurements in the same population 25

before exposure (mean of 2.61 ± 0.14 μg/g) (Heck and Casanova

- Schmitz, 1984). The 26

variability in the levels was large. At the individual level, the data showed both increase and 27

decrease in blood levels relative to pre

- exposure levels, which was attributed by the authors as 28

plausibly due to temporal variations in baseline levels in humans, partic ularly since the 29

experiment did not control food intake prior to exposure. Studies in rhesus monkeys have 30

revealed endogenous formaldehyde levels (2.4 μg/g blood) comparable to humans and that levels 31

were also unaltered following exposure to 6 ppm formald ehyde via inhalation 6 hours/day for 32

4 weeks, measurements being taken at both 7 minutes and 45 hours post final exposure 33

(Casanova et al., 1988)

. 34This document is a draft for review purposes only and does not constitute Agency policy.
DRAFT

— DO NOT CITE OR QUOTE 3

- 14 It is important to keep in mind that the GC

- MS method is not capable of detecting 1

irreversibly bound formaldehyde; for example, formaldehyde levels detected by this method, 2

even in the anterior nasal mucos a of rats exposed to 6 ppm of formaldehyde, were not elevated 3

over control levels. Furthermore, the GC

- MS method does not differentiate between free and 4

reversibly bound adducts of formaldehyde (Heck et al., 1982). Thus, measured levels represent 5

total f ormaldehyde concentration that includes free formaldehyde as well as reversibly bound 6

adducts. Based on the known Michaelis

- Menten constant, Km, for formaldehyde dehydrogenase 7

with respect to the GSH adduct formation, Heck et al. (1982) estimated under ce rtain 8

assumptions that free formaldehyde comprised only about 1

– 2% of the total formaldehyde 9

measured by their method. Furthermore, as shown by Metz et al. (2006, 2004), formaldehyde 10

reactions with primary amino and thiol groups can, in a second step, rea ct with many other 11

amino acids to form stable methylene bridges. Presumably, such reactions would not be 12

detectable by using the methods employed by Heck et al. (1982). 4

Results from an earlier experiment using radiolabeled formaldehyde in rats are consistent 17

with the conclusion based on the GC

- MS measurements of no appreciable increase in blood 18

levels of formaldehyde. Following a 6

- hour exposure of F344 rats to 15 ppm of [ 14 C]

- 19

formaldehyde (Heck et al., 1983), the concentrations of 14 C in the nasal mucosa wer e 28

- fold 20

higher than those in the blood. The observed half

- life of the terminal phase of the radioactivity 21

was long (55 hours); on the other hand, it is known that the half

- life of free formaldehyde in the 22

rat blood is very short. Therefore, the authors concluded that the radioactivity was likely due to 23

modification of macromolecules or metabolic incorporation rather than slow metabolic clearance 24

of formaldehyde. The terminal decline of the radioactivity in the packed cell fraction of the 25

blood was much slower and observed to be consistent with incorporation into erythrocytes. 26 Thus, the limited 13

interpretation of GC

- MS measurements of blood levels suggests that formaldehyde does not 14

appreciably reach the blood, is rapidly metabolized or interacts with macromolecules when it 15

escapes metabolism, or is otherwise undetected. 16

In the same paper, Heck et al. (1983) report on the similarity in the pharmacokinetics of 27

radiolabeled formaldehyde and radiolabeled formate in the rat blood, supporting their hyp othesis 28

that oxidation of formaldehyde to formate and subsequent incorporation of this compound 29

through one

- carbon metabolism were major factors in the disposition of formaldehyde. Studies 30

by Gottschling et al. (1984) have also established that the main product of metabolic clearance of 31

4 Additionally, note that, although Heck et al. (1982) demonstrated that formaldeh yde concentration can be accurately measured from glutathione and tetrahydrofolate adducts, similar experiments were not performed by using protein samples or cellular extracts (i.e., in the presence of various amino acids). In addition, standard curves f or predicting formaldehyde concentration in tissues were generated in aqueous solutions rather than biological samples.This document is a draft for review purposes only and does not constitute Agency policy.
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- 15 formaldehyde is formate, which is either further metabolized to CO 2 and water, incorporated into 1

the one

- carbon pool, and/or eliminated in the urine as a sodium salt at about 13 mg/L urine. 2

3

3.3.2. Levels in Various Tiss ues 4

The radiolabeling studies indicated high levels of 14 C in the rat nasal mucosa (equivalent 5

concentrations of 14 C

- formaldehyde in the nasal mucosa of rats naïvely exposed to 15 ppm 6

14 C

- formaldehyde were 2,148 ± 255 nmol/g compared with 76 ± 11 nmol/g in plasma). In 7

contrast, the GC

- MS studies did not detect elevated formaldehyde in this region. This is not to 8

be interpreted as a discrepancy, because the radiolabeling study did not distinguish among 9

radiolabeled species and thus the measured radioactivi ty could potentially be free or bound 10

formaldehyde, formate, or any [ 14 C] metabolically incorporated into macromolecules. 11

In concurrent studies, Casanova

- Schmitz et al. (1984) resolved the question as to whether 12

the higher [ 14 C] levels in the nasal mucosa were a consequence of GSH depletion and a 13

subsequent reduction in GSH

- dependent clearance of formaldehyde. An important result in 14

these studies was that there was no significant difference in labeling in either the nasal mucosa or 15

in plasma between naïve F344 rats and those pre

- exposed to unlabeled 15 ppm formaldehyde 16

6 hours/day for the 9 previous days. These findings indicated little or no apparent effect on the 17

disposition of formaldehyde following short

- term exposure to relatively high levels of 18

forma ldehyde. In contrast, Farooqui et al. (1986) reported decreases in GSH in several tissues 19

3 hours after a sublethal I.P. injection of formaldehyde but not after 6 and 9 hours. Taken 20

together, these data suggest that formaldehyde exposure does not result in long

- term alterations 21

in cellular GSH levels and that repeated inhalation exposure does not alter the dosimetry to the 22

bloodstream or formaldehyde body burden. 23

Heck et al. (1983) determined the 14 C concentrations in different tissues in the F344 rat 24

bod y by exposing rats in a head

- only chamber to various concentrations (5

– 24 ppm) of 25

radiolabeled formaldehyde for 6 hours. (Concentrations of 14 C in internal organs and tissues 26

relative to that in plasma did not appear to vary much as exposure concentrations increased; 27

therefore only averages over the concentration range were reported.) Except for the esophagus, 28

levels in the heart, spleen, lung, intestines, liver, and kidney were 1

– 3 times higher relative to 29

that in plasma. Labeling in the esophagus was hi gh (fivefold relative to plasma). The authors 30

attributed this relatively higher dose to mucociliary action in the nose and trachea. The data also 31

indicate that the brain, testes, and erythrocytes appear to have about threefold lower 14 C levels 32

than plas ma. Pre

- exposure to formaldehyde (for 9 days) did not alter the measured radioactivity 33

in the nasal mucosa or plasma. Thus, it was concluded that the single exposure findings may 34

also be qualitatively extended to chronic exposures. 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 16 The total radiolabel m easured in the bone marrow (femur) of F344

- rats exposed for 6 1

hours to 0.3

– 15 ppm of radiolabeled formaldehyde in the Casanova et al. (1984) experiment was 2

high (generally within a factor of 0.5 of the total labeling in the nasal respiratory mucosa). 3

Nearl y half of the 14 C was contained in the DNA in this tissue presumably on account of the high 4

rate of cell turnover in the bone marrow, indicating that the carbon derived from 14 C

5

formaldehyde was utilized for DNA synthesis (Casanova

- Schmitz et al., 1984). 6

Chang et al. (1983) described visceral labeling (via autoradiography) in rats, following 7

exposure to 15 ppm [ 14 C]

- formaldehyde 6 hours/day for 4 days. The authors attributed this 8

labeling to mucociliary clearance and grooming

- related ingestion of formalde hyde. 9

In summary, following exposure to radiolabeled formaldehyde, the radioactivity was very 10

high in the nasal mucosa but was also extensively distributed to various tissues. In particular, 11

levels in the bone marrow were high. On the other hand, formald ehyde levels in the blood 12

measured by GC

- MS were not significantly elevated. Thus, the authors considered it unlikely 13

that the elevated 14 C in various tissues was due to free formaldehyde. Instead, these levels were 14

thought to arise from either rapid met abolic incorporation or formation of covalent adducts or 15

incorporation via carboxylation reactions of the 14 CO 2 formed during metabolism. 16

The data presented thus far in this section illustrate that measuring the distribution of the 17

absorbed formaldehyde b ased on 14 C

- radiolabeling and GC

- MS studies alone is problematic 18

because it is difficult to resolve (through these studies) whether it is free, reversibly bound, 19

irreversibly bound, formate, one

- carbon pool, etc. This is of significance with regard to 20

und erstanding the availability of the absorbed formaldehyde. More indirect methods had to be 21

developed to further examine the disposition of formaldehyde; however, as discussed below, the 22

interpretation of these approaches may also not be straightforward. 23

24

3 .3.2.1. Disposition of Formaldehyde: Differentiating Covalent Binding and Metabolic 25

Incorporation 26

The motivation in presenting this section is twofold, as follows: 27

1. As concluded above, subsequent studies were necessary to ascertain whether measured 28

radio labeling in different experiments was due to formaldehyde adducts or incorporation 29

of [ 14 C] one

- carbon units of formaldehyde into macromolecules via the one

- carbon pool. 30

2. DNA protein cross

- links (DPXs) formed by formaldehyde (covalently bound in this case) 31

have been regarded as a surrogate dose metric for the intracellular concentration of 32

formaldehyde (Hernandez et al., 1994; Casanova et al., 1991, 1989). This is particularly 33

relevant because of the nonlinear dose response for DPX formation due to saturat ion of 34This document is a draft for review purposes only and does not constitute Agency policy.
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- 17 enzymatic defenses at high concentrations (Casanova et al., 1991, 1989). Thus, the 1

ability to measure DPX is an important development. 2

3

An important question is whether the formaldehyde disposed in the form of DPX is 4

detected in remote tissues. A set of elegant but complex experiments involving dual isotope 5

labeling ( 14 C and 3 H) was carried out to this end by the Heck and Casanova

- Schmitz and their 6

coworkers. Casanova

- Schmitz et al. (1984) and Casanova

- Schmitz and Heck (1983) used dual 7

isotope lab eling of formaldehyde as a way to partially distinguish between formaldehyde adducts 8

formation and metabolic incorporation. In separate experiments, F344 rats were exposed to 3 H

9

and 14 C

- formaldehyde at different exposure concentrations (0.3

– 15.0 ppm), a nd the 3 H/ 14 C 10

ratios of different phases of DNA were measured. Only the highlights of the results and 11

significant issues are presented here. The overall conclusions from these experiments were as 12

follows: 13

14

**•** Labeling in the nasal mucosa was due to both co valent binding and metabolic 15

incorporation. 16

**•** DPX was formed at 2 ppm and greater concentrations in the respiratory mucosa. 17

**•** In the bone marrow, formaldehyde did not bind covalently to bone marrow 18

macromolecules at any exposure concentration. The labeling o f bone marrow 19

macromolecules was found to be entirely due to metabolic incorporation and not due to 20

covalent binding. 21

22

Macromolecules such as DNA and protein can be isolated from tissue homogenates by 23

extraction into three phases: an organic phase consist ing of proteins, an aqueous phase consisting 24

of only double

- stranded DNA, and an interfacial phase consisting of both DNA and protein. 25

Single

- stranded (but not double

- stranded) DNA was particularly likely to form adducts. DNA 26

from this interfacial phase can be further purified and has been shown to consist of DPXs 27

(Casanova

- Schmitz and Heck, 1983) . Because both [ 14 C]

- formaldehyde and [ 3 H]

- formaldehyde 28

can become incorporated into DNA and protein me tabolically as well as by cross

- linking, the 29

3 H/ 14 C ratio in such cross

- linked material should be higher than in material that primarily 30

contains metabolically incorporated formaldehyde. Figure 3

- 2 shows the labeling of tissue from 31

the nasal respiratory m ucosa and bone marrow (distal femur) in rats exposed to [ 14 C]

- 32

formaldehyde and [ 3 H]

- formaldehyde vapor. 33

34This document is a draft for review purposes only and does not constitute Agency policy.
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- 18 1

2 Figure 3

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

- and 3 H

- labeled formaldehyde (0.3, 2, 6, 10, 4

15 p pm). 5

6 Note that the small yield of interfacial (IF) phase from bone marrow tissue precluded 7 further analysis; this is prima facie evidence for the lack of significant DPXs in this 8

tissue. 9 10

Source: Casanova

- Schmitz et al. (1984a). 11

12 13

In the nasal mucosa t he interfacial phase has a significantly higher 3 H/ 14 C ratio than the 14

material in the aqueous phase. This suggests that interfacial DNA has significantly more 3 H, a 15

phenomenon likely explained by additional [ 3 H]

- formaldehyde molecules present as DPXs prio r 16

to extraction. The amount of interfacial DNA was found to have a clear dose response. These 17

cross

- links were also judged to be due to exogenous formaldehyde. Likewise, the organic phase 18

of the nasal mucosa showed a similar increase in 3 H/ 14 C ratio at higher concentrations, a result 19

that could be attributed to various inter

- and intra

- protein adducts (Metz et al., 2004; Trezl et al., 20

2003; Skrzydlewska, 1996)

. 21

In contrast, analysis of macromolecules at the distal femur location presents a different 22

pattern (Figure 3

- 2, part B). First, the interfacial phase was not detected during extraction, 23

suggesting that there were few or no DPXs to be detected. Second, there was no increase in 24

3 H/ 14 C ratio in the organic (i.e., protein) phase as a function of dose. Therefore, it was 25

concluded that either radiolabeled formaldehyde or formate reached the distal site and was 26

subsequently incorporated into macromolecules. According to the mechan istic interpretation of 27

these studies, the quantity plotted on the ordinate in Figure 3

- 2 (the ratio of 3 H/ 14 C between the 28This document is a draft for review purposes only and does not constitute Agency policy.
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- 19 tissue and the exposure gas) should approach unity as metabolism becomes saturated and more 1

adduct formation occurs, particularly for protein. Indeed, this is what is observed (see 2

Figure 3

- 2, part A). In contrast, there is no dose effect in the femur, suggesting that the labeling 3

at all doses in that tissue may be due to metabolic incorporation and not due to the parent 4

formaldehyde. 5

(Note: These data were originally shown in the absence of an analysis of isotope effects 6

on covalent binding and metabolism. Subsequent studies determined that [ 3 H]

- formaldehyde is 7

oxidized less rapidly than [ 14 C]

- formaldehyde and unlabeled formaldehyde . This suggests that 8

the 3 H/ 14 C ratio, and therefore the amount of formaldehyde covalently bound to tissue, is likely 9

overestimated because more [ 3 H]

- formaldehyde remains unmetabolized, i.e., free to bind [Heck 10

and Casanova, 1987] . The authors hypothesized that this overestimate was relatively greater at 11

the lower concentrations.) 12

Similar results were obtained in GSH

- depleted rats (Casanova and Heck, 1987) . Again, 13

these authors observed a dose

- dependent increase in the 3 H/ 14 C ratio in the interfacial DNA and 14

organic fractions of d isrupted cells of the respiratory and olfactory mucosa and no such increases 15

in bone marrow. Interestingly, at 10 ppm exposure (only), GSH

- depleted rats exhibited a higher 16

3 H/ 14 C ratio in the organic phase than did normal rats. Casanova and Heck (1987) p osited that 17

much of the covalent binding at 6 ppm and lower was due to binding to extracellular proteins, 18

whereas the higher 3 H/ 14 C ratio in GSH

- depleted rats at 10 ppm was due to more intracellular 19

binding. 20

In their first experiment to measure DPX concen trations, Casanova

- Schmidt et al. (1984) 21

and Casanova and Heck (1987) used the dual isotope method ( 3 H/ 14 C) mentioned a bove. In this 22

experiment, DPX was observed only at formaldehyde concentrations ≥2 ppm. Subsequently, 23

Casanova et al. (1989) developed a more sensitive method using high

- performance liquid 24

chromatography (HPLC) for measuring DPX. In this method, tissue h omogenates were digested 25

with a proteolytic enzyme and extracted with a phenolic solvent. DPX was detected in the nasal 26

mucosa of rats at formaldehyde concentrations as low as 0.3 ppm. This method was also used to 27

measure DPX in the nasal region, the lary nx, trachea and carina, and major intrapulmonary 28

airways (airway diameters > 2mm) of rhesus monkeys exposed for 6 hours to 0.7, 2.0 and 6.0 29

ppm of formaldehyde. DPX was detected in the nose (including the nasopharynx) at all 30

concentrations and at 2.0 and 6 .0 ppm in the larynx, trachea, carina and other lower airways. 31

However, DPX was not detectable in the bone marrow of these monkeys at any concentration. 32

Overall, Heck and Casanova

- Schmitz and their coworkers interpreted the results of these 33

various experi ments to mean that inhaled formaldehyde could not reach distant sites in the body. 34

It may be noted in this context that Shaham et al. [1996] reported elevated DPX levels in the 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 20 white blood cells of laboratory workers exposed to formaldehyde. These data ar e further 1

reported in Chapter 4.) 2

3

3.4. METABOLISM 4

Formaldehyde is primarily metabolized by glutathione

- dependent formaldehyde 5

dehydrogenase (FALDH) and aldehyde dehydrogenases (ALDHs). Numerous studies now 6

recognize FALDH as a member of the alcohol dehy drogenase (ADH) family, specifically ADH3 7

(Thompson et al., 2009; Liu et al., 2004, 2001; Hedberg et al., 2003; Høøg et al., 2003; and the 8

references in each of these). The remainder of this report will refer to FALDH as ADH3. 9

10

3.4.1. In Vitro and In Vi vo Characterization of Formaldehyde Metabolism 11

Formaldehyde is oxidized to formate by two metabolic pathways (Figure 3

- 3). The first 12

pathway involves conversion of free formaldehyde to formate by the so

- called low

- K
m 13

(K

m = 400 μM) mitochondrial aldehyde d ehydrogenase

- 2 (ALDH2). The second pathway 14

involves a two

- enzyme system that converts glutathione

- conjugated formaldehyde 15

(S

- hydroxymethylglutathione [HMGSH]) to the intermediate S

- formylglutathione, which is 16

subsequently metabolized to formate and GSH by S

- formylglutathione hydrolase. 17

18

19

20 Figure 3

- 3. Formaldehyde clearance by ALDH2 (GSH

- independent) and 21 ADH3 (GSH

- dependent). 22

The K

m value for ALDH2 and free formaldehyde is about 400 μM (Teng et al., 23 2001), whereas the K

m value for HMGSH and ADH3 is 6.5 μM (Uotila and 24 Koivusalo, 1974a,b). The ADH

- mediated reactions are reversible in the presence 25 of excess reduced nicotinamide adenine dinucleotide (NADH). 26 Source: Adapted from Teng et al. (2001). 27

28This document is a draft for review purposes only and does not constitute Agency policy.
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- 21 Though ADH3 is rate limiting in this second pathway, the aff inity of HMGSH for ADH3 1

(K

m = 6.5 μM) is about 100

- fold higher than that of free formaldehyde for ALDH2. In addition 2

to the kinetic properties, this member of the ADH gene family (Høøg et al., 2003, 2001; Liu et 3

al., 2001; Jornvall et al., 2000; Estonius et al., 1996) appears to be ubiquitously expressed in 4

organ tissues (Molotkov et al., 20 02; Ang et al., 1996a, b) , exhibits cytoplasmic and nuclear 5

localization (Fernandez et al., 2003) , and is the most abundant ADH family member in the liver 6

and brain (Galter et al., 2003)

. 7

In vitro studies have examined the clearance of formaldehyde in several human and rat 8

tissues (Table 3

- 2). Examination of formaldehyde me tabolism in the rat nasal and olfactory 9

mucosa indicates nearly identical pharmacokinetics in the rat liver on a per mg of cell lysate 10

basis (Casanova

- Schmitz et al., 1984b) . Similar results have been obtained in the absence of 11

GSH, where other ALDH family m embers oxidize formaldehyde, albeit with significantly lower 12

affinity (i.e., higher K

m ). Hedberg et al. (2000) demonstrated that human buccal tissue lysate 13

kinetics are in close agreement with those reported for purified human liver ADH3 (Uotila and 14

Koivusalo, 1974a) . Additionally, micro

- array analysis indicates that these cells express far more 15

ADH3 and S

- formylglutathione hydrolase than ALDH1 or ALDH2 (Hedberg et al., 2001 a) . The 16

results of Ovrebo et al. (2002) are not easily compared with the other studies in Table 3

- 2 17

because these studies were in intact cell cultures. However, it is apparent that the 18

pharmacokinetic values in these human cells are comparable to intact rat liver cells. 19

20

Table 3

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

22

Source K

m (μM) V

max (nmol/mg protein × min) Reference

Purified human liver ADH3 6.5 2.77 **±** 0.12 Uotila and Koivusalo (1974a, b) Rat olfactory mucosa (+ GSH) 2.6 **±** 0.5 1.77 **±** 0.12 Casanova

- Schmitz et al. (1984b) Rat olfactory mucosa ( − GSH) 647 **±** 43 4.39 **±** 0.14 Casanova

- Schmitz et al. (1984b) Rat respiratory mucosa (+ GSH) 2.6 **±** 2.6 0.90 **±** 0.24 Casanova

- Schmitz et al. (1984b) Rat respiratory mucosa ( − GSH) 481 **±** 88 4.07 **±** 0.35 Casanova

- Schmitz et al. (1984b)
Rat liver (+ GSH) 5.0 **±** 1.9 2.0 **±** 0.3 Casanova

- Schmitz et al. (1984b) Human bronchial explants a 5,100 3.3 Ovrebo et al. (2002) Human bronchial epithelial a 1,400 6.1 Ovrebo et al. (2002) Rat hepatocyte s a 1,250 4.2 Ovrebo et al. (2002) Human buccal tissue (+ GSH) 11 **±** 2 2.9 **±** 0.6 Hedberg et al. (2000)
Human buccal tissue ( − GSH) 360 **±** 90 1.2 **±** 0.7 Hedberg et al. (2000) Human keratinocytes n.d. b 14.5 **±** 1.8 Hedberg et al. (2000)

Human fibroblasts n.d. 17.9 **±** 1.4 Hedberg et al. (2000)

a These studies were carried out in intact cells by measuring the for mation of formate. This likely explains the nearly 23

1,000

- fold increase in apparent K

m , since much of the formaldehyde was likely to be bound extracellularly. The 24 remaining studies used either purified enzyme or cell lysates (as indicated) and measured th e formation of NADH. 25

b n.d. = not determined. 26

27This document is a draft for review purposes only and does not constitute Agency policy.
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- 22 The data in Table 3

- 3 along with data indicating the ubiquity of ADH3, indicate that 1

many human tissues and cells, particularly in the respiratory tract, appear to exhibit significant 2

capacity to metabolize for maldehyde. Molecular biology techniques have demonstrated the 3

importance of ADH3 in formaldehyde clearance. For example, ADH

- knockout studies have 4

shown that the median lethal dose (LD 50 ) values for formaldehyde in wild type, ADH1 −/−

, 5

ADH3 −/− , and ADH4 −/

− mice strains were 0.200, 0.175, 0.135, and 0.190 g/kg, respectively 6

(Deltour et al., 1999) . Although the statistical significance was not reported, the data indicate 7

that deletion of ADH3 increases the sensitivity of mice to formaldehyde. 8

The pharmacokinetics of forma te are complex. Formate can undergo adenosine 9

triphosphate (ATP)

- dependent addition to tetrahydrofolate (THF), which can carry either one or 10

two one

- carbon groups. Formate can conjugate with THF to form N 10

- formyl

- THF and its 11

isomer N 5

- formyl

- THF, both o f which can be converted to N 5 ,N 10

- methenyl

- THF and 12

subsequently to other derivatives that are ultimately incorporated into DNA and proteins via 13

biosynthetic pathways (Figure 3

- 4). 14

15

16

17 Figure 3

- 4. Metabolism of formate. 18 Note: 1, formyl

- THF synthetase; 2 , formyl

- THF dehydrogenase. 19
20 Source: Adapted from Black et al. (1985). 21
22 23 Elevated levels of formate in urine have been detected following inhalation of methanol 24

or formate under certain conditions (Liesivuori and Savolainen, 1987) , although the 25

interpretation of this finding is unclear. There is also evidence that formate generates CO 2 – 26This document is a draft for review purposes only and does not constitute Agency policy.
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- 23 radicals and can be metabolized to CO 2 via catalase and via the oxidation of N 10

- formyl

- THF 1

(Dikalova et al., 2001 , and references therein). The significance of formate in formaldehyde 2

toxicity is unclear. Black et al. (1985) reported that hepatic tetrahydrofolate levels in monkeys 3

are 60% of those in rats and that primates are far less efficient in clearing formate than are rats 4

and dogs. Studies involving [ 14 C]

- formate suggest that about 80% is exhaled as 14 CO 2 , 2

– 7% is 5

excreted in the urine, and about 10% undergoes metab olic incorporation (Hanzlik et al., 2005

, 6

and references therein). Mice deficient in formyl

- THF dehydrogenase ex hibit no change in LD 50 7

(via I.P. dose) for methanol or in oxidation of high doses of formate (Cook et al., 2001) . It has 8

been suggested that rodents efficiently clear formate via folate

- depen dent pathways, 9

peroxidation by catalase, or an unknown third pathway. Conversely, primates do not appear to 10

exhibit such capacity and are more sensitive to metabolic acidosis following methanol poisoning 11

(Cook et al., 2001)

. 12

13

3.4.2. Formaldehyde Exposure and Perturbation of Metabolic Pathways 14

The enzyme ADH3 has received renewed attention in recent years because of new 15

functions that have been attributed to it. ADH3 is central to the metaboli sm of formaldehyde; 16

however, exposure to formaldehyde in turn alters the activity of ADH3 (in multiple dose

17

dependent ways), thereby leading to perturbation of critical metabolic pathways. These are 18

briefly mentioned below (refer to cited papers for detai ls). 19

1. Exposure to formaldehyde increases cell replication. These proliferating epithelial and 20

inflammatory cells are rich in both the messenger ribonucleic acid (mRNA) and protein 21

of ADH3 (Nilsson et al., 2004; Hedberg et al., 2000). Studies in the rodent lung suggest 22

that increases in ADH3 in such cells dramatically alter the biology of other important 23

ADH3 substrates that are involved in protein modification and cell signaling (Que et al., 24

2005). 25

2. ADH3 also participates in the oxidation of retinol and lon g

- chain primary alcohols, as 26

well as the reduction of S

- nitrosoglutathione (GSNO) (Staab et al., 2009; Thompson et 27

al., 2009; Hedberg et al., 2003; Høøg et al., 2003; Molotkov et al., 2002; Liu et al., 2001; 28

Jornvall et al., 2000; Jensen et al., 1998). Th e activity of ADH3 toward some of these 29

substrates has been shown to be significantly increased in the presence of formaldehyde. 30

Staab et al. (2009) showed that (in cultured cells) GSNO can accelerate ADH3

- mediated 31

formaldehyde oxidation and, likewise, th at formaldehyde increases ADH3

- mediated 32

GSNO reduction nearly 25

- fold. The following effects may be noted with regard to the 33

relevance of such perturbations. 34This document is a draft for review purposes only and does not constitute Agency policy.
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- 24 a. GSNO is an endogenous bronchodilator and reservoir of nitric oxide (NO) 1

activity (Jensen et al., 1998). Details on the ADH3

- mediated reduction of GSNO 2

are shown in Thompson and Grafstrom (2008). 3

b. ADH3 is implicated in playing a central role in regulating bronchiole tone and 4

allergen

- induced hyperresponsiveness (Gerard, 2005; Que et al., 2005). 5

c. As conc luded by California Environmental Protection Agency (CalEPA) (2008), 6

“the dysregulation of NO by formaldehyde [in this manner] helps to explain the 7

variety and variability in the toxic manifestations following formaldehyde 8

inhalation.” 9

10

3.4.3. Evidence fo r Susceptibility in Formaldehyde Metabolism 11

Teng et al. (2001) provided evidence that inhibition of ADH1, ALDH2, and ADH3 has 12

significant impact on formaldehyde toxicity. The authors speculated that deficiencies in any of 13

these enzymes would confer an inc reased susceptibility to formaldehyde toxicity (Teng et al., 14

2001) . P olymorphism in ALDH2 has been shown to have implications in human risk 15

assessment, specifically with regard to acetaldehyde metabolism (Ginsberg et al., 2002) . It is 16

worth noting, however, that Teng et al. (2001) only demonstrated the importance of ALDH2 in 17

rat hepatocytes with formaldehyde concentrations of 2.5 mM and greater. Since this 18

concentration is fivefold greater than the 0.5 mM K

m for free formaldehyde, ALDH2 19

involvement is not unexpected at such high concentrations. Teng et al. (2001) also demonstrated 20

the importance of ADH1 in driving the reverse reaction (i.e., formaldehyde to methanol) by 21

coadministration of NADH

- generators. This would have the effect of prolonging the life of 22

formaldehyde by continuous recycling. This is not surprising, given that many ADH reactions 23

are reversible. However, levels of nicotinamide adenine dinucleotide (NAD + ) are normally 24

much higher than NADH. 25

T o date, two studies have reported polymorphisms in ADH3, using the new 26

nomenclature. 5

5 Other epidemiologic studies investigating links between ADH3 and oral cancer use the older nomenclature and

thus refer to Class I ADH (i.e., ADH1) enzymes. ADH3 transcription appears to be regulated by specificity protein (Sp1), with a 27

minimal promoter located at positions −34 to +61. The reported polymorphisms in ADH3 28

involve four base

- pair substitutions in the promoter region and no polymorphisms in the coding 29

region (Hedberg et al., 2001b) . The three polymorphisms include −197/−196 (GG **→** AA), −79 30

(G **→** A), and +9 (C **→** T). The genotype frequencies ar e shown in Table 3

- 3. Of these alleles, the 31

+9 (C **→** T) polymorphism (in the putative Sp1 minimal promoter region) reduced transcriptional 32This document is a draft for review purposes only and does not constitute Agency policy.
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- 25 activity twofold in in vitro reporter gene experiments. According to Hedberg et al. (2001b) , no 1

studies have demonstrated differences in ADH3 enzyme activity in humans. More recently, 2

single nucleotide polymorphisms in ADH3 have been reported to be associated with childhood 3

risk of asthma, although the functional relevance of these polymorphisms has not been published 4

(Wu et al., 2007). 5

6

Table 3

- 3. Allelic frequencies of ADH3 in human populations 7
8

Allele frequencies (%)
Population, n AA

- 197/

- 196 GG

- 197/

- 196 A

- 79 G

- 79 T +9 C +9 Chinese, 83 22 78 100

– – 100 Spanish, 95 41 59 62 38

– 100
Swedish, 96 47 53 67 33 1.5 98.5 9 Source: Adapted from Hedberg et al. (2001b). 10 11 12 Alterations in THF pathways may also have an impact on formaldehyde toxicity. These 13

could result from polymorphisms in various enzymes or differences in folate intake and 14

absorption. Species differences in tetrahydrofolate levels (Black et al., 1985) are thought to play 15

a role in the differential responses to methanol across species. Cook et al. (2001) speculate that 16

rats have redundant pathways for formate clearance that may be absent or less efficient in 17

primates. 18

19

3.5. EXCRETION 20

The main product of metabolic clearance of formaldehyde is formate, which is further 21

metabolized to CO 2 and water, incorporated into the one

- carbon pool, or eliminated in the urine. 22

There is also some evidence that formaldehyde is present in exhaled breath; however, it is 23

unclear whether this originates from endogenous sources, or is simply a function of ambient 24

formaldehyde dissolved in fluids lining POEs. The following sections describe first experiments 25

in laboratory species and then a vailable data in humans. Broadly, these studies address two 26

important questions that may be of relevance for risk assessment. First, it may be of interest to 27

know what levels of formaldehyde are exhaled for comparison with inhaled levels, and whether 28

the re is any relationship between external exposure and exhaled levels. Second, there are recent 29

studies that have attempted to relate genetic polymorphisms and changes in gene transcription 30

level to levels of putative urinary formaldehyde biomarkers. 31

32

33This document is a draft for review purposes only and does not constitute Agency policy.
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- 26 3.5 .1. Formaldehyde Excretion in Rodents 1

Heck et al. (1983) determined the relative contributions of various excretion pathways in 2

F344 rats following inhalation exposure to formaldehyde . Table 3

- 4 indicates that the relative 3

excretion pathways were independent o f exposure concentration (at least between 0.63 and 4

15 ppm). Nearly 40% of inhaled [ 14 C]

- formaldehyde appeared to be eliminated via expiration, 5

probably as CO 2 (it should be recalled that nearly 100% of inhaled formaldehyde is absorbed). 6

Within 70 hours of a 6

- hour exposure to formaldehyde, about 17 and 5% were eliminated in the 7

urine and feces, respectively. Nearly 40% of inhaled [ 14 C]

- formaldehyde remained in the 8

carcass, presumably due to metabolic incorporation. 9

10

Table 3

- 4. Percent distribution of airborne [ 14 C]

- formaldehyde in F344 rats 11
12

Source Concentration of formaldehyde (ppm)

0.63 13.1 Distribution (%) a
Expired air 39.4 ± 1.5 41.9 ± 0.8

Urine 17.6 ± 1.2 17.3 ± 0.6

Feces 4.2 ± 1.5 5.3 ± 1.3

Tissues and carcass 38.9 ± 1.2 35.2 ± 0.5 13

a Va lues are means ± standard deviations (n = 4). 14 15
Source: Heck et al. (1983). 16

17

18 Mashford and Jones (1982) examined elimination pathways of formaldehyde in rats 19

exposed by I.P. injection. Urine and exhaled gases were collected from rats exposed to 4 or 20

40 mg /kg [ 14 C]

- formaldehyde. At 48 hours postinjection, 82 and 78% of the radiolabel were 21

exhaled as 14 CO 2 , whereas exhaled [ 14 C]

- formaldehyde was not detected. Mashford and Jones 22

(1982) also further identified the urinary metabolites. Five hours after injec tion of the higher 23

dose, formate was determined to comprise 80% of the urinary metabolites. The authors were 24

unable to detect cysteine derivatives observed in other studies (see below) in the urine of these 25

rats prior to or after formaldehyde exposure. T he authors stated that if formaldehyde were to be 26

excreted in urine containing cysteine, then thiazolidine

- 4

- carboxylate (TZCA) would likely be 27

produced. They speculated that species differences in urinary compounds may produce 28

formaldehyde conjugates (or artifacts). 29

Hemminki (1982) reacted formaldehyde and acetaldehyde with cysteine, 30

N

- acetylcysteine, and GSH and found that formaldehyde reacted most rapidly with cysteine to 31

form TZCA. Similarly, acetaldehyde reacted preferentially with cysteine, albeit slower than 32This document is a draft for review purposes only and does not constitute Agency policy.
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- 27 formaldehyde, to form a thiazolidine derivative. However, when each aldehyde was 1

administered I.P. (10% formaldehyde, 50% acetaldehyde), thioether concentrations (nmol/mol 2

creatinine) significantly increased in the 24 and 48 hour urine of acet aldehyde

- treated rats but 3

not formaldehyde

- treated rats. These data suggest that formaldehyde is not appreciably excreted 4

in urine and thus cysteine conjugates are not likely to represent formaldehyde exposure. 5

Most recently, Shin et al. (2007) attempted to show that formaldehyde inhalation 6

increased urinary TZCA levels in Sprague

- Dawley rats. Treated rats were exposed to 3.1 and 7

38.1 ppm formaldehyde for 6 hours/day for 2 weeks, and urine was collected for 3 days. The 8

TZCA level in four control rats wa s 0.07 ± 0.02 mg/L, whereas levels in the 3 and 38 ppm 9

groups were 0.18 ± 0.045 and 1.01 ± 0.36, respectively. Notably, the concentrations in the four 10

highest exposed animals (0.71, 0.70, 1.20, and 1.43 ppm) exhibited a nearly twofold range. 11

However, thes e comparisons are confounded if the exposures have any influence on urine 12

production and urine cysteine levels. The study does not provide any data that might allow one 13

to examine this issue. 14

15

3.5.2. Formaldehyde Excretion in Exhaled Human Breath 16

Several human and animal studies have attempted to measure the concentration of 17

formaldehyde in exhaled breath. None of the human studies were designed to distinguish 18

between exogenous (room air) and endogenous (systemic) formaldehyde in exhaled breath. In 19

orde r to discern whether endogenous formaldehyde is excreted into the lungs, test subjects must 20

breathe formaldehyde

- free air. Because subjects were breathing room air, which contained 9

- 10 21

ppb formaldehyde in two studies and unspecified concentrations in two other studies, it is 22

impossible to ascertain whether there was any endogenous formaldehyde in their exhaled breath. 23

One study demonstrates that the formaldehyde concentration is lower in exhaled breath than in 24

inhaled breath (Cáp et al., 2008). Also, non e of the human studies investigated whether there is 25

any relationship between exhaled formaldehyde levels and food intake, life stage, smoking, or 26

health status. This assessment identifies a critical research need for further studies on the 27

measurement of exhaled formaldehyde. 28

Proton transfer reaction mass spectrometry (PTR

- MS) has been applied to measure trace 29

compounds in exhaled breath including volatile organics and specifically formaldehyde. The 30

basic method of PTR

- MS is based on the transfer of prot ons from H 3 0 + to gases in exhaled 31

breath and the in

- line monitoring of products where gases are tentatively identified by the mass 32

to charge ratio ( m/z ) where a m/z of 31 is consistent with protonated formaldehyde (Hansel et al., 33

1995; Lindinger et al., 19 98). It is important to note that reaction products from methanol and 34

ethanol may also produce fragments with an m/z ratio of 31 (Kusch et al., 2008). Up to 1% of 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 28 the mass of ethanol and methanol in exhaled breath may be detected with an m/z ratio of 31 and 1

thus be identified as formaldehyde, so accurate quantitation of formaldehyde should adjust for 2 this contribution (Špan ĕl and Smith, 2008). Selected ion flow tube mass spectrometry (SIFT

3

MS) is an application of PTR

- MS developed for real

- time analysis of trace gases in breath 4 (Smith and Špan ĕl, 2005; Španĕl and Smith, 2007). 5

Moser et al. (2005) measured levels of 179 volatile organic compounds (VOCs) in the 6

exhaled breath of 344 individuals. This study was not designed to ascertain whether exhaled 7

form aldehyde is of endogenous origin, but rather to demonstrate that proton transfer reaction

8

mass spectrometry can be used as a new method for rapid screening of large collectives for risk 9

factors (e.g., smoking behavior), potential disease biomarkers, and am bient air characterization. 10

The study was conducted at a health fair. The test subjects had a mean age of 61.6 years; 63% 11

were males and 14% were smokers. Samples of room air were collected and evaluated in 12

parallel with exhaled breath samples. The aut hors note that formaldehyde was detected in room 13

air, but did not report the levels; rather they stated that the background concentrations were 14

negligible. Of the 179 volatile organic compounds measured, data were reported for 14, 15

including formaldehyde a nd formic acid. The formaldehyde levels in exhaled breath spanned 16

from 1.2 to 72.7 ppb with a median of 4.3 ppb and 75 th percentile of 6.3 ppb. No explanation 17

was provided for this wide range in values, and there was no distinction of the data by sex, ag e, 18

health, or smoking status. 19

Because the test subjects were breathing ambient indoor air which contained an 20

unspecified concentration of formaldehyde, it is impossible to ascertain whether exhaled 21

formaldehyde was from an endogenous or exogenous source, or both. Moser et al. also note that 22

significant differences in exhaled breath composition could be found between smokers and non

23

smokers for 32 of the 179 chemicals measured, but the 32 chemicals were not named and no 24

substantiating data were provided. Formaldehyde may have been among these 32 chemicals 25

since it is a major component of cigarette smoke (NLM, 2001). 26

The report by Moser et al. does not provide the limit of detection for any of the 27

compounds measured or details of the analytical method. The minimum formaldehyde level 28

reported in exhaled breath was 1.23 ppb. The method employed by Moser et al. does not adjust 29

the detection of apparent formaldehyde ( m/z ) by accounting for the contribution of methanol and 30

ethanol. The levels of methanol an d ethanol in exhaled breath were reported, however. Based 31

on 1% of the mass of these chemicals contributing to the m/z = 31 in the PTR

- MS method 32 (Špan ĕl and Smith, 2008), Table 3

- 5 demonstrates that the highest formaldehyde levels reported 33

may have been a n artifact of high methanol and ethanol in exhaled breath. Because the in

- room 34

formaldehyde concentrations were not reported, it is unknown how much of this formaldehyde 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 29 represents formaldehyde levels in inhaled air. Additionally, since these samples wer e simple 1

exhaled breath samples and not SIFT

- MS samples, it is impossible to distinguish between air 2

which reached the pulmonary region versus air which only entered the upper airways. 3

4

Table 3

- 5. Apparent formaldehyde levels in exhaled breath of individu als 5 attending a health fair, adjusted for methanol and ethanol levels which 6 contribute to the detection of the protonated species with a mass to charge 7 ratio of 31 reported as formaldehyde ( m/z = 31) 8

9

Chemical Minimum 25 th
percentile Median 75 th

percent ile 97.5 th
percentile Maximum Methanol ( m/z = 31) 13.367 106.227 161.179 243.185 643.614 1536.499

1% of methanol predicted as m/z = 31 0.13 1.06 1.61 2.43 6.44 15.36

Ethanol 11.583 23.1 34.664 64.24 549.24 9779.768 1% of ethanol predicted

as m/z = 31 0.12 0.23 0.35 0.64 5.49 97.80

Mass of m/z = 31

attributable to methanol

and ethanol 0.25 1.29 1.96 3.07 11.93 113.16

Mass of m/z = 31 reported as formaldehyde 1.23 3.1 4.26 6.33 39.8 72.7

10
Source: Moser et al. (2005). 11

12

13 Turner et al. (2008) compared l evels of volatile compounds in exhaled breath to levels in 14

emissions from the skin in five males (3 in their mid 20s, and the others 42 and 49 years old). 15

The subjects fasted overnight, and measurements were taken before and after ingesting 75 g of 16

glucos e. The source of the inhaled air was laboratory air which contained an unreported 17

concentration of formaldehyde. Formaldehyde was not detected in the exhaled breath of any 18

subjects. The limit of detection was 5 ppb or better. 19

Wang et al. (2008) measur ed the concentrations of formaldehyde and 9 other chemicals 20

in the exhaled breath of three healthy male laboratory workers. The limit of quantification for 21

formaldehyde was not reported. A series of measurements were taken in the nose and mouth, 22

and also in the oral cavity during breath holding. Table 3

- 6 presents the median formaldehyde 23

levels and geometric standard deviations for the three subjects. The authors noted that 24

formaldehyde in exhaled breath was at a level somewhat lower than the ambient ai r 25

concentration, but they could not be certain of its origin. 26

27

28This document is a draft for review purposes only and does not constitute Agency policy.
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- 30 Table 3

- 6. Measurements of exhaled formaldehyde concentrations in the 1 mouth and nose, and in the oral cavity after breath holding in three healthy 2 male laboratory workers 3

4

Subject Formalde hyde (median ppb / σg)

A Mouth 5 / 2.3

Nose 7 / 2.1 Oral cavity 5 / 2.3

B Mouth 7 / 2.3

Nose 5 / 2.1 Oral cavity 6 / 1.9

C Mouth 4 / 2.5

Nose 6 / 1.9 Oral cavity 6 / 1.9 Laboratory air 9.6 ±1.5
5 Source: Wang et al. (2008). 6

7

8 Cáp et al. (2 008) evaluated relationships between volatile organic compounds measured 9

in exhaled breath and exhaled breath condensate. Exhaled breath condensate consists of 10

aerosolized particles of airway lining fluid evolved from the airway wall by turbulent airflow 11

that serve as seeds for substantial water vapor condensation, which then serves to trap water 12

soluble volatile gases. This study also attempted to ascertain whether the source of each 13

compound was endogenous or exogenous. According to the published artic le and electronic 14 communication with Dr. Patrik Špan ĕl, a co

- author for this study, the limit of quantification was 15

3 ppb or better. Measurements of formaldehyde in the direct exhaled breath of 34 subjects (25 to 16

62 years; 11 males; 2 smokers) varied from 0 to 12 ppb with a mean of 2 ppb and a median of 1 17

ppb. Measurements of formaldehyde in exhaled breath condensate ranged from 0 to 12 ppb with 18

a mean of 2 ppb and a median of 0 ppb. Two smokers exhaled formaldehyde concentrations of 0 19

and 3 ppb. The sc atter plot in Figure 3

- 5 shows that most subjects exhaled formaldehyde 20

concentrations of 0

- 3 ppb (x axis), which was considerably lower than the ambient air 21

concentration of 9.6 ±1.5 ppb. The authors concluded that the exhaled concentrations were 22

influenc ed by exogenous levels in room air, which were not controlled. Dr. Špan ĕl offered two 23

plausible explanations for why exhaled formaldehyde levels are lower than inhaled levels. He 24

noted that some subjects were tested within several minutes of entering the laboratory from 25

outdoors, so they may not have had time to acclimate to the higher indoor air concentration. 26

Another explanation is that a substantial portion of inhaled formaldehyde, which is highly 27This document is a draft for review purposes only and does not constitute Agency policy.
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- 31 reactive, was retained in the respiratory tract and th us not exhaled; that is, 9.6 ppb of 1

formaldehyde was inhaled but only a mean of 2 ppb was exhaled. 2

In this and other human studies, there was no adjustment for an artifact in the analytical 3

method that makes it impossible to distinguish between formaldeh yde and reaction products for 4

1% of exhaled methanol and ethanol because they have the same mass to charge ratio ( m/z = 31). 5

In fact, the concentration of methanol and ethanol that is misidentified as formaldehyde exceeds 6

the reported concentrations of exh aled formaldehyde. Thus, it is highly likely that the actual 7

exhaled formaldehyde concentration in Cáp et al. (2008) was significantly lower than 2 ppb, and 8

that there was little or no endogenous formaldehyde in the exhaled breath. This would be 9

consiste nt with an animal study in which Mashford and Jones (1982) detected no exhaled 10

formaldehyde in rats injected I.P. with 40 mg/kg [ 14 C]

- formaldehyde. Over 48 hours, 78% of the 11

radioactivity was exhaled as 14 CO 2 and 11% was excreted in the urine as formate, N

12

(hydroxymethyl)urea, N,N’

- bis

- (hydroxymethyl)urea, and possibly polymethylene urea. In 13

summary, there are insufficient data at this time to confidently establish a concentration of 14

formaldehyde in exhaled breath that can be attributed to endogenous sour ces. 15

16

17 Figure 3

- 5. Scatter plot of formaldehyde concentrations measured in ppb in 18 direct breath exhalations (x axis) and exhaled breath condensate headspace 19 (y axis)

. 20

21 Source: Cáp et al. (2008). 22

23

24 3.5.3. Formaldehyde Excretion in Human Urine 25

Gottsc hling et al. (1984) examined urinary formic acid in 35 veterinary students. 26

Personal monitoring badges were worn and returned after class, and urine samples were taken 27This document is a draft for review purposes only and does not constitute Agency policy.
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- 32 prior to class and within 2 hours after the class. Mean exposure levels were about 100 ppb. 1

Baseline averages of urinary formic acid (as a sodium salt) were 12.47 mg/L and ranged from 2

2.43 to 28.38 mg/L among subjects. Postexposure formate levels were slightly elevated but were 3

not statistically significant. Moreover, formate levels decr eased in several individuals relative to 4

pre

- exposure levels. The authors concluded that variability in urinary formate may mask any 5

changes and that monitoring formate within 2 hours of exposure is not informative. It is worth 6

noting, however, that inte rpretation of this finding is confounded due to the fact that diet was not 7

controlled and because no markers for urinary normalization were employed (Boeniger, 1987)

. 8

Boeniger (1987) reviewed previously published data on formate in urine (some of which 9

were in G erman). In one occupational study, workers were exposed to an average formaldehyde 10

exposure of 1.28 mg/m 3 over a 6

- hour work shift. This implies an average intake of 6 mg; 6

In the previously described study by Shin et al. (2007), human urine samples were shown 17

to contain TZCA, although variability was not reported. A subsequent study re ported that urine 18

TZCA levels were higher in individuals living in newer apartments (0.18 ± 0.121 mg/g 19

creatinine) as compared to older apartments (0.097 ± 0.040 mg/g creatinine) (Li et al., 2007) 11

Boeniger reported a range of 2.5 to 13 mg. However, the original study reported that post

- shift 12

formate levels were 152 mg/L, whereas the levels were only 24 mg/L 6 days later (no exposure). 13

Considering that only a small percentage of inhaled formaldehyde would be excreted in urine, it 14

is unclear how (or whether) formaldehyde exposur e, with the highest total dose of 13 mg, could 15

be responsible for the observed increase. 16

7

26 . 20

The authors cited this as evidence that TZCA is a urina ry marker for formaldehyde exposure, 21

even though TZCA levels were not correlated to measured (or estimated) formaldehyde 22

exposures. The individuals also differed significantly in age (21.5 vs. 28.6, p = 0.053) and 23

differed in smoking percentage (10 vs. 27 %). Clearly these two studies do not establish a 24

relationship between human formaldehyde exposure and urine TZCA levels. 25

3.6. MODELING THE TOXICOKINETICS OF FORMALDEHYDE AND DPX 27

3.6.1. Motivation 28

Airway geometry is expected to be an important determin ant of inhaled formaldehyde 29

dosimetry in the respiratory tract and its differences across species. The uptake of formaldehyde 30

in the upper respiratory tract is highly nonhomogenous and spatially localized and exhibits 31

strong species differences. Species differences in kinetic factors have been argued to be the key 32

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

7 This study is described in greater detail in Chapter 5.This document is a draft for review purposes only and does not constitute Agency policy.
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- 33 determinants of species

- specific lesion distributions for formaldehyde and other reactive inhaled 1

gases. In the first subsection here, the advantage gained in the quantitative risk assessment by 2

modeling these differences in the upper respiratory tract is explained. Such a model was 3

constructed by using computational fluid dynamic (CFD) methods for modeling airflow and 4

regional formaldehyde uptake in the rat, rhesus monkey, and human nose by s cientists at the 5

Chemical Industries Institute of Toxicology (CIIT) Centers for Health Research (presently the 6

Hamner Institutes of Health Sciences). While frank effects were seen only in the upper 7

respiratory tract in rodents, mild lesions were also pres ent in the major bronchiolar region of the 8

rhesus monkey. Therefore, with regard to extrapolation of cancer risk from animal bioassays to 9

humans, the upper and lower human respiratory tract should both be considered potentially

The fluid dynamics modeling in the respiratory tract comp rises two steps (Kimbell et al. 19

2001): airflow through the lumen and formaldehyde uptake by the lining of the respiratory tract. 20

Flow streamlines in the CFD simulations agreed reasonably well with experimentally observed 21

patterns in casts of the rat, monk ey, and human nasal passages as well as with measurements of 22

velocity taken in hollow molds of the human nose. Pressure drop as a function of volumetric 23

flow also compared well with measurements made in rats. Unlike the airflow simulations, no 24

validation of the regional formaldehyde uptake simulations (that is, the spatial distribution of 25

uptake) was possible. (It was possible to compare simulations of overall uptake with 26

experimentally observed values.) In this assessment, several indirect qualitative an d quantitative 27

lines of evidence were relied on to provide general confidence in the formaldehyde regional 28

uptake profile in the F344 rat nasal passages. In addition to the agreement mentioned earlier 29

with respect to airflow profiles, this evidence includ es general agreement between measured and 30

predicted levels of formaldehyde DPXs (in the “high

- tumor” regions) when simulations of 31

airflow and regional formaldehyde uptake were used as input to a physiologically based 32

pharmacokinetic (PBPK) model for DPX ki netics (Cohen

- Hubal et al. 1997). Such indicators 33

are not available for the simulation of uptake patterns in the human. With regard to modeling the 34

lower respiratory tract, calculations in Overton et al. (2001) are based on an idealized 35 at 10

risk of developing forma ldehyde

- induced squamous cell carcinoma. Therefore, formaldehyde 11

dose to the entire human respiratory tract human respiratory tract was modeled in order to 12

develop a dose

- response relationship that considered the entire respiratory tract. Accordingly, in 13

the second subsection results of modeling airflow and regional formaldehyde uptake in the 14

human lower respiratory tract by Overton et al. (2001) are provided. Unsteady effects were 15

argued to be insignificant at resting breathing rates, and therefore stea dy

- state inspiratory flow 16

was assumed. Since these models have been described in various reports and publications, the 17

technical details are consigned to appendices or the literature is referenced. 18This document is a draft for review purposes only and does not constitute Agency policy.
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- 34 representation of airways in the human lung and of air flow through them (referred to in the 1

literature as “single

- path” models). Overton et al. (2001) did not attempt to validate their 2

simulations. However, the following observation may be noted in support of their model. In the 3

case of the deposition of coarse and fine particulate matter, single

- path models have traditionally 4

provided a reasonably accurate representation of the average deposition in a given generation of 5

the lung airways (i.e. airways at a given depth in the lung) for a normal human population. 6

There were mass balance errors in the CFD calculations (Kimbell et al. 2001). Mass 7

balance errors associated with formaldehyde uptake into tissue ranged from less than 14% for the 8

rat, monkey, and human at restin g minute volume to approximately 27% at the highest 9

inspiratory flow rates of 31.8 and 37 L/minute in the human. 10

If DPX formation and cell proliferation are driven by formaldehyde flux, then these 11

quantities may also be expected to exhibit site and specie s differences, thus arguing for linking 12

these quantities to the modeled formaldehyde flux (Conolly et al. 2000). 13

The third subsection evaluates PBPK models for DPX kinetics in the F344 rat and rhesus 14

monkey, using CFD simulations of formaldehyde flux to the nasal lining as input (Klein et al., 15

2009; Subramaniam et al., 2007; Conolly et al., 2000), and discusses their uncertainties. This 16

subsection further discusses issues related to the scaling of the animal models to the human. 17

This assessment uses in ternal dose metrics computed by using the models described in 18

this section so as to derive more accurate human equivalent concentrations from the animal 19

bioassays than would be obtained by averaging over the respiratory surface area. The strengths 20

and unc ertainties associated with the data and the models and their relevance to the hypothesized 21

mode of action are discussed in some length. 22

23

3.6.2. Species Differences in Anatomy: Consequences for Gas Transport and Risk 24

As discussed earlier, formaldehyde is h ighly reactive and water soluble (categorized as a 25

category 1 gas), thus its absorption in the mucus layer and tissue lining of the upper respiratory 26

tract is known to be significant. The regional inhaled dose of formaldehyde to the respiratory 27

tract of a given species depends on the amount of formaldehyde delivered by inhaled air, the 28

absorption characteristics of the nasal lining, and reactions in the tissue. The amount delivered 29

by inhaled air is a function of the major airflow patterns, air

- phase diff usion, and absorption at 30

the airway

- epithelial tissue interface. The dose of formaldehyde to the epithelial tissue, which is 31

different from the amount delivered, depends on the amount absorbed at the airway

- tissue 32

interface, water solubility, mucus

- to

- tis sue phase diffusion, and chemical reactions, such as 33

hydrolysis, protein binding, and metabolism. It has been argued strongly that species differences 34

in these kinetic factors are determinants of species

- specific lesion distributions for formaldehyde 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 35 and other inhaled gases (Moulin et al., 2002; Bogdanffy et al., 1999; Ibanes et al., 1996; 1

Monticello et al., 1996; Monticello and Morgan, 1994; Morgan et al., 1991). 2

Because of the convoluted nature of the airways in the upper respiratory tract, the 3

absorptio n of such gases in the upper respiratory tract is highly nonhomogeneous. There are 4

large differences across species in the anatomy of the upper respiratory tract (Figure 3

- 6) and in 5

airflow patterns (Figure 3

- 7). Therefore, as shown in the simulations in Figure 3

- 8, it may be 6

expected that the uptake patterns and thus risk due to inhaled formaldehyde will also show 7

strong species dependence. Morgan et al. (1991) concluded that airflow

- driven dosimetry plays 8

a critical role in determining the site specifi city of various formaldehyde

- induced responses, 9

including tumors, in the nose of the F344 rat. The convoluted geometry of the airway passages 10

in the upper respiratory tract, as seen from the cross sections of the nose in Figure 3

- 6, renders an 11

idealized r epresentation of fluid flow and uptake profiles almost impossible. For these reasons, 12

Kimbell et al. (1993), Kepler et al. (1998), and Subramaniam et al. (1998) developed 13

anatomically realistic finite

- element representations of the noses of humans, F344 r ats, and 14

rhesus monkeys. These representations were subsequently used in physical and computational 15

models (Figure 3

- 6). This assessment utilizes dosimetry derived from these representations. 16

An accurate calculation of species differences in formaldehyde dosimetry in the upper 17

respiratory tract is important to the extrapolation problem for another reason. The upper 18

respiratory tract in rats is an extremely efficient scrubber of reactive gases (97% uptake) 19

(Morgan et al., 1986), thereby protecting the low er respiratory tract from gaseous penetration. 20

On the other hand, there is considerably more fractional penetration of formaldehyde into the 21

lower respiratory tract of the rhesus monkey than in the rat (see Figure 3

- 8). Therefore, an 22

accurate determinati on of scrubbing in the upper respiratory tract is important to delineate 23

species differences in the level of risk to the lower respiratory tract. Thus, in the case of the 24

rhesus monkey, the model by Kepler et al. (1998) included the trachea. Because the human 25

model also had to address the potential for oronasal breathing, an idealized single

- path model of 26

the lower respiratory tract was attached to a model of the upper respiratory tract (Overton et al., 27

2001). It is important to note that the models ment ioned above represent nasal passages 28

reconstructed from a single individual from each species (Kimbell et al., 2001a, b; Conolly et al., 29

2000; CIIT, 1999; Subramaniam et al., 1998). This is discussed later in the context of 30

intraspecies variability. 31

The h ighly localized nature of uptake patterns shown in Figure 3

- 8 means that averaging 32

uptake over the entire nasal surface area would dilute the regional dose over areas where 33

response was observed and that an extrapolation based on such averaging would clear ly not be 34

accurate. 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 36 Figure 3

- 6. Reconstructed nasal passages of F344 rat, rhesus monkey, and 1

human. 2

3

Note: Nostril is to the right, and the nasopharynx is to the left. Right side shows 4

the finite element mesh. Left

- hand side shows tracings of airwa ys obtained from 5 cross sections of fixed heads (F344 rat and rhesus monkey) and magnetic 6 resonance image sectional scans (humans). Aligned cross sections were 7

connected to form a three

- dimensional reconstruction and finite

- element 8 computational mesh. So urce: Adapted from Kimbell et al. (2001a). Additional 9 images provided courtesy of Dr. J.S. Kimbell, CIIT Hamner Institutes. 10This document is a draft for review purposes only and does not constitute Agency policy.
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- 37 1 2 Figure 3

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

of CFD simulatio ns with water

- dye studies. 4 Note: Panels A and B show the simulated airflow pattern versus water

- dye 5 streams observed experimentally in casts of the nasal passages of rats and 6 monkeys, respectively. Panel C shows the simulated inspiration airflow pattern, 7 and the histogram depicts the simulated axial velocities (white bars) vs. 8 experimental measurements made in hollow molds of the human nasal passages. 9 Dye stream plots were compiled for the rat and monkey over the physiological 10 range of inspiration flow ra tes. Modeled flow rates in humans were 15 L/minute. 11 Source: Adapted from Kimbell et al. (2001a). 12

13This document is a draft for review purposes only and does not constitute Agency policy.
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- 38 1

F344 Rat

Rhesus Monkey

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

2000

1500

1000

500

0

2

Figure 3

- 8. Lateral view of nasal wall mass flux of inhaled formaldehyde 3 simulated in the F344 rat, rhesus monkey, and human. 4

5

Note: Nostrils are to the right. Simulations were exercised in each species at 6 steady

- state inspiration flow rates of 0.576 L/minute in the rat, 4.8 L/minute in the 7 monkey, and 15 L/minute in the human. Flux was contoured over the range from 8

0

– 2,000 pmol/(mm 2

- hour

- ppm) in each s pecies. 9 10 Source: Kimbell et al. (2001a). 11
12 13

Another factor to consider in the extrapolation is that monkeys and humans are oronasal 14

breathers while rats are obligate nose

- only breathers. Thus, for humans and monkeys, oronasal 15

or oral breathing implies a s ignificantly higher uptake in the lower respiratory tract. It is known 16

that a significant fraction of the human population breathes normally through the mouth. 17

Finally, activity profiles are also determinants of extraction efficiency (see Figure 3

- 9) and of 18

breathing route (Niinimaa et al., 1981). Given the fact that formaldehyde

- induced lesions were 19This document is a draft for review purposes only and does not constitute Agency policy.
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- 39 observed as far down the respiratory tract as the first bifurcation of the lungs in exposed 1

monkeys, the entire human respiratory tract should be considered when extrapolating data from 2

rats. 3

4

5

6 Figure 3

- 9. CFD simulations of formaldehyde flux to human nasal lining at 7 different inspiratory flow rates. 8

9 Note: Right lateral view. Uptake is shown for the non

- squamous portion of the 10 epithelium. The front port ion of the nose (vestibule) is lined with keratinized 11 squamous epithelium and is expected to absorb relatively much less 12 formaldehyde. 13
14 Source: Kimbell et al. (2001b). 15
16
17This document is a draft for review purposes only and does not constitute Agency policy.
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- 40 3.6.3. Modeling Formaldehyde Uptake in Nasal Passages 1

CIIT scientists chose the F344 strain of the rat since it was assumed to be anatomically 2

representative of its species and because it is widely used experimentally, most notably in 3

bioassays sponsored by the National Toxicology Program and at CIIT. The approximate 4

locations of squamou s, mucus

- coated, and nonmucus epithelial cells were mapped onto the 5

reconstructed nasal geometry of the CFD models. Taken together, these regions of nonmucus 6

and mucus

- coated cells comprise the entire surface area of the nasal passages (see original 7

paper s and CIIT [1999] for further details on reconstruction and morphometry). Types of nasal 8

epithelium overlaid onto the geometry of the models were assumed to be similar in 9

characteristics across all three species (rat, monkey, and human) except for thickne ss, surface 10

area, and location. Species

- specific mucosal thickness, surface area, and location were estimated 11

from the literature, from the documentation of the CFD models, or by direct measurements 12

(Conolly et al., 2000; CIIT, 1999). The nasal passages of all three species were assumed to have 13

a continuous mucus coating over all surfaces except specific areas in the nasal vestibule. As 14

discussed at the beginning of this chapter, formaldehyde hydrolyzes in water and reacts readily 15

with a number of compon ents of nasal mucus. Absorption rates of inhaled formaldehyde by the 16

nasal lining were therefore assumed to depend on where the epithelial lining is coated by mucus 17

and where it is not. 18

To calculate an airflow rate that would be comparable among species, the amount of 19

inspired air (tidal volume, V T ) was divided by the estimated time involved in inhalation (half the 20

time a breath takes, or (1/2)(1/[breathing frequency, f]). Thus, an inspiratory flow rate was 21

calculated to be 2V T f, or twice the minute volum e. Predicted flux values represent an average of 22

one nasal cycle. Minute volumes were allometrically scaled to 0.288 L/minute for a 315 g rat 23

from data given by Mauderly (1986). Simulations were therefore carried out at 0.576 L/minute 24

for the rat. 25

The f luid dynamics modeling in the respiratory tract comprises two steps: modeling the 26

airflow through the lumen (solution of Navier

- Stokes equations) and modeling formaldehyde 27

uptake by the respiratory tract lining (solution of convective

- diffusion equations f or a given 28

airflow field). Details of these simulations, including boundary conditions for air flow and mass 29

transfer, are provided in Kimbell et al. (2001a, b, 1993) and Subramaniam et al. (1998). 30

Formaldehyde absorption at the airway

- to

- epithelial tiss ue interface was assumed to be 31

proportional to the air

- phase formaldehyde concentration adjacent to the nasal lining layer in 32

monkeys and humans (see the original paper [Kimbell et al., 2001a] for a more detailed 33

elaboration of the calculations for these c oefficients). 34This document is a draft for review purposes only and does not constitute Agency policy.
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- 41 Because formaldehyde is highly water soluble and reactive, Kimbell at al. (2001a) 1

assumed that absorption occurred only during inspiration. Thus, for each breath, flux into nasal 2

passage walls (rate of mass transport in the direction perpend icular to the nasal wall per mm 2 of 3

the wall surface) was assumed to be zero during exhalation, with no backpressure to uptake built 4

up in the tissues. Overton et al. (2001) estimated the error due to this assumption to be small, 5

roughly an underestimate of 3% in comparison to cyclic breathing. Also, this assumption is the 6

same as that used in default methods for reference concentration determination and has been 7

used in other PBPK model applications to describe nasal uptake (Andersen and Jarabek, 2001). 8

9

3.6.3.1. Flux Bins 10

A novel contribution of the CIIT biologically motivated dose

- response model is that cell 11

division rates and DPX concentrations are driven by the local concentration of formaldehyde. 12

These were determined by partitioning the nasal sur face by flux, resulting in 20 “flux bins.” 13

Each bin was comprised of elements (not necessarily contiguous) of the nasal surface that 14

receive a particular interval of formaldehyde flux per ppm of exposure concentration (Kimbell et 15

al., 2001a). The spatial coordinates of elements comprising a particular flux bin were fixed for 16

all exposure concentrations, with formaldehyde flux in a bin scaling linearly with exposure 17

concentration (ppm). Thus, formaldehyde flux was expressed as pmol/(mm 2

- hour

- ppm). 18

19

3.6.3. 2. Flux Estimates 20

Formaldehyde flux was estimated for the rat, monkey, and human over the entire nasal 21

surface and over the portion of the nasal surface that was lined by nonsquamous epithelium. 22

Formaldehyde flux was also estimated for the rat and monkey over the areas where cell 23

proliferation measurements were made (Monticello et al., 1991, 1989) and over the anterior 24

portion of the human nasal passages that is lined by nonsquamous epithelium. Figure 3

- 8 shows 25

the mass flux of inhaled formaldehyde to th e lateral wall of nasal passages in the F344 rat, rhesus 26

monkey, and human (Kimbell et al., 2001a). 27

Maximum flux estimates for the entire upper respiratory tract were located in the mucus

28

coated squamous epithelium on the dorsal aspect of the dorsal medial meatus near the boundary 29

between nonmucus and mucus

- coated squamous epithelium in the rat, at the anterior or rostral 30

margin of the middle turbinate in the monkey, and in the nonsquamous epithelium on the 31

proximal portion of the mid

- septum near the bounda ry between squamous and nonsquamous 32

epithelium in the human (see Kimbell et al. [2001b] for tabulations of comparative estimates of 33

formaldehyde flux across the species). 34This document is a draft for review purposes only and does not constitute Agency policy.
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- 42 The rat

- to

- monkey ratio of the highest site

- specific fluxes in the two species was 0. 98. In 1

the rat, the incidence of formaldehyde

- induced squamous cell carcinomas in chronically exposed 2

animals was high in the anterior lateral meatus (Monticello et al., 1996). Flux predicted per ppm 3

in this site and flux predicted near the anterior or p roximal aspect of the inferior turbinate and 4

adjacent lateral walls and septum in the human were similar, with a rat

- to

- human ratio of 0.84. 5

6

3.6.3.3. Mass Balance Errors 7

O verall uptake of formaldehyde was calculated as 100% x (mass entering nostril − mass 8

exiting outlet)/(mass entering nostril). Mass balance errors for air, 100% x (mass of air entering 9

nostril − mass exiting outlet)/(mass entering nostril), and inhaled for maldehyde, 100% x (mass 10

entering nostril − mass absorbed by airway walls − mass exiting outlet)/(mass entering nostril), 11

were calculated. Mass balance errors associated with simulated formaldehyde uptake from air 12

into tissue ranged from less than 14% for the rat, monkey, and human at 7.4 and 15 L/minute to 13

approximately 27% at the highest inspiratory flow rates of 31.8 and 37 L/minute (Kimbell et al., 14

2001b). Kimbell et al. (2001b) corrected the simulation results for these errors by evenly 15

distributing t he lost mass over the entire nasal surface. 16

17

3.6.4. Modeling Formaldehyde Uptake in the Lower Respiratory Tract 18

Lesions were observed in the lower respiratory tract of rhesus monkeys exposed to 6 ppm 19

formaldehyde. Therefore it is appropriate to conside r the human lower respiratory tract as 20

potentially at risk for formaldehyde

- induced cancer. Accordingly, fluid flow and formaldehyde 21

uptake in the lower respiratory tract were also modeled for the human in the CIIT approach by 22

using dosimetry estimates for the human lower respiratory tract. 23

The single

- path idealization of the human lung anatomy captures the geometrical 24

characteristics of the airways for a given lung depth, and of airflow through these airways, in an 25

average, homogeneous sense. For particul ates, this has provided a reasonable representation of 26

the average deposition in a given generation of the lung airways for a normal human population. 27

The one

- dimensional model by Weibel (1963) is generally considered adequate unless the fluid 28

dynamics at airway bifurcations need to be explicitly modeled. However, such an idealization of 29

the lung geometry has been successfully used in various models for the dosimetry of ozone and 30

particulate and fibrous matter. Most likely, the lung geometries of the susc eptible population, 31

such as those with chronic obstructive pulmonary disease, would depart significantly from the 32

geometry described in Weibel (1963). Unlike the accurate representation of the nasal anatomy 33

used in the CFD modeling, the lung geometry is i dealized in the CIIT approach as a typical path 34

Weibel geometry. This captures the lung structure in an average, homogeneous sense for a given 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 43 lung depth. The single-path model used to calculate formaldehyde uptake in the human 1

respiratory tract (Overton et al., 2001; CIIT, 1999) applied a one-dimensional equation of mass 2

transport to each generation of an adult human symmetric, bifurcating Weibel- type respiratory 3

tract anatomical model, augmented by an upper respiratory tract. The detailed CFD modeling of 4

the upper respiratory tract was made consistent with the upper respiratory tract in the single -path 5

model by requiring that the one- dimensional version of the nasal passages have the same 6

inspiratory air -flow rate and uptake during inspiration as the CFD simulations for four daily 7

human activity levels. The reader is referred to Overton et al. (2001) for further details of the 8

simulations. Results most relevant to this assessment are shown in Figure 3 -10. 9

10 Figure 3 -10. Single- path model simulations o f surface flux per ppm of 11 formaldehyde exposure concentration in an adult male human. 12 13 Source: Overton et al. (2001). 14
15 16 The primary predictions of the model, as shown in Figure 3-10, were that more than 95% 17

of the inhaled formaldehyde would be retained and formaldehyde flux in the lower respiratory 18

tract would increase for several lung airway generations from that in the posterior

- most segment 19

of the nose and then decrease rapidly, resulting in almost zero flux to the alveolar sacs. 20This document is a draft for review purposes only and does not constitute Agency policy.
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- 44 Overton et al. (2001) modeled uptake at higher inspiratory rates, including those at 1

50 L/minute of minute volume (well beyond levels where the oronasal switch occurs in the 2

normal nasal breathing population). At these rates Figure 3

- 8 indicates that formaldehyde flux in 3

the m outh cavity is comparable (but a bit less) to that occurring in the nasal passages. Overton et 4

al. (2001) did not model uptake in the oral cavity at minute volumes less than 50 L/minute. This 5

would be of interest because mouth breathers form a large segm ent of the population. 6

Furthermore, at concentrations of formaldehyde where either odor or sensory irritation becomes 7

a significant factor, humans are likely to switch to mouth breathing even at resting inspiration. 8

At a minute volume of 50 L/minute, Ove rton et al. (2001) assumed, citing Niinimaa et al. (1981), 9

that 0.55 of the inspired fraction is through the mouth. Therefore, based on the results in 10

Figure 3

- 8, it is not unreasonable to assume that for mouth breathing conditions at resting or 11

light exe rcise inspiratory rates, average flux across the human mouth lining would be 12

comparable to the average flux across the nasal lining computed in Kimbell et al. (2001a, b). 13

14

3.6.5. Uncertainties in Formaldehyde Dosimetry Modeling 15

3.6.5.1. Verification of Predicted Flow Profiles 16

The simulated streamlines of steady

- state inspiration airflow predicted by the CFD model 17

agreed reasonably well with experimentally observed patterns of water

- dye streams made in 18

casts of the nasal passages for the rat and monkey a s shown in panels A and B in Figure 3

- 7. 19

The airflow velocity predicted by CFD model simulations of the human also agreed well with 20

measurements taken in hollow molds of the human nasal passages (panel C, Figure 3

- 8) (Kepler 21

et al., 1998; Subramaniam et a l., 1998; Kimbell et al., 1997, 1993). However, the accuracy and 22

relevance of these comparisons are limited. The profiles were verified by video analysis of dye 23

streak lines in the molds of rats and rhesus monkeys, although this method is reasonable for only 24

the major airflow streams. 25

Plots of pressure drop vs. volumetric airflow rate predicted by the CFD simulations 26

compared well with measurements made in rats in vivo (Gerde et al., 1991) and in acrylic casts 27

of the rat nasal airways (Cheng et al., 1990) as shown in Figure 3

- 11. This latter comparison 28

remains qualitative due to differences among the simulation and experiments as to where the 29

outlet pressure was measured and because no tubing attachments or other experimental apparatus 30

were included in th e simulation geometry. The simulated pressure drop values were somewhat 31

lower, possibly due to these differences. 32

Inspiratory airflow was assumed to be constant in time (steady state). Subramaniam et al. 33

(1998) considered this to be a reasonable assumpti on during resting breathing conditions based 34

on a value of 0.02 obtained for the Strouhal number. Unsteady effects are insignificant when 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 45 this number is much less than one. However, this assumption may not be reasonable for light 1

and heavy exercise breat hing scenarios. 2

3

4 Figure 3

- 11. Pressure drop vs. volumetric airflow rate predicted by the CIIT 5 CFD model compared with pressure drop measurements made in two hollow 6 molds (C1 and C2) of the rat nasal passage (Cheng et al., 1990) or in rats 7

in vivo (Gerde et al., 1991). 8
9 Source: Kimbell et al. (1997). 10
11 12

3.6.5.2. Level of Confidence in Formaldehyde Uptake Simulations 13

Unlike the airflow simulations, it was not possible to evaluate the formaldehyde uptake 14

calculations directly. Since the mass transfer boun dary conditions were set by fitting overall 15

uptake to the average experimental data for various exposure concentrations, it was not possible 16

to independently verify even the overall uptake values with empirical data. This assessment has 17

relied on several indirect qualitative and quantitative lines of evidence listed below to provide 18

general confidence in the uptake profile for the F344 rat nasal passages, as modeled in CIIT 19

(1999), when gross averages are considered over certain regions of the nasal lining

. 20

In an earlier simulation, where the nasal walls were set to be infinitely absorbing of 21

formaldehyde, uptake of inhaled formaldehyde in the upper respiratory tract was predicted to be 22

90% in the rat for simulations corresponding to the resting minute volu me in the F344 rat. This 23

estimate compared reasonably well with the range of 91

– 98% observed by Morgan et al. 24

(1986a). 25This document is a draft for review purposes only and does not constitute Agency policy.
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- 46 Morgan et al (1991) showed general qualitative correspondence between the main routes 1

of flow and lesion distribution induced by formald ehyde in the rat nose. In their initial work 2

with a CFD model that represented a highly reactive and soluble gas, Kimbell et al. (1993) 3

described similarities in computed regional mass flux patterns and lesion distribution due to 4

formaldehyde. When the r esults from this work in the coronal section immediately posterior to 5

the vestibular region were considered, simulated flux levels over regions such as the medial 6

aspect of the maxilloturbinate and the adjacent septum (where lesions were seen) were an orde r 7

of magnitude higher than over other regions, such as the nasoturbinate (where lesions were not 8

seen). 8

The results of a PBPK model by Cohen

- Hubal et al. (1997) provide a reasonable level of 10

confidence in regional uptake simulations for the F344 rat w hen gross averages over nasal sites 11

are carried out. Cohen

- Hubal et al. (1997) linked the CFD dosimetry model for formaldehyde to 12

a PBPK model for formaldehyde

- DPX concentration in the F344 rat. This PBPK model was 13

calibrated by optimizing the model to c ombined DPX data from high

- tumor incidence and low

14

tumor incidence regions of the rat nose that were obtained in separate experiments by Casanova 15

et al. (1991, 1989). These data were obtained at 0.3, 0.7, 2.0, 6.0, and 10 ppm for both regions 16

and also at 15 ppm for the high

- tumor region. Model prediction of DPX concentrations for the 17

high

- tumor region only compared well with the experimental data, including 15 ppm for which 18

the model had not been calibrated. This is shown in Figure 3

- 12. 19

9

The CFD simulati ons do not model reflex bradypnea, a protective reflex seen in rodents 20

but not in humans. As discussed at length in sections 3.2.3.1 and 4.2.1.1, it is reasonable to 21

expect a range of 25% (Chang et al., 1983) to 45% (Barrow et al., 1983) decrease in minut e 22

volume in F344 rats at the exposure concentration of 15 ppm. Explicit omission of this effect in 23

the modeling is, however, not likely to be a source of major uncertainty in the modeled results 24

for uptake of formaldehyde in the rat nose for the following reason. The CFD model for the 25

F344 rat was calibrated to fit the overall experimental result for formaldehyde uptake in the F344 26

rat at 15 ppm exposure concentration. This was carried out by adjusting the mass transfer 27

coefficient used as boundary condi tion on the absorbing portion of the nasal lining. Thus, the 28

reflex bradypnea occurring in those experimental animals is phenomenologically factored into 29

the value used for the boundary condition. Nonetheless, some error in the localized distribution 30

of uptake patterns may be expected, even if the overall uptake is reproduced correctly. 31

8 However, this 1993 CFD model differed somewhat from the subsequent model by Kimbell et al. (2001a) used i n this assessment. In the 1993 model, the limiting mass

- transfer resistance for the gas was assumed to be in the air phase; that is, the concentration of formaldehyde was set to zero at the airway lining. Furthermore, this same boundary condition was use d on the nasal vestibule as well, while, in the more recent model, the vestibule was

considered to be non

- absorbing. Unfortunately, Kimbell at al. (2001a) did not report on correspondences betweenThis document is a draft for review purposes only and does not constitute Agency policy.
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- 47 Furthermore, since the same value for the mass transfer coefficient was used in human 1

simulations (as obtained from calibration of the rat model), there is additional unc ertainty in the 2

modeled human flux estimates. This issue was not addressed by Kimbell et al. (2001a, b), 3

Conolly et al. (2004), or Schlosser et al. (2003), and we are unable to assess the extent of this 4

error more accurately. 5

6

Figure 3

- 12. Formaldehyde

- DPX dosimetry in the F344 rat. 7 Panel A: calibration of the PBPK model using data from high and low tumor 8 incidence sites. Panel B: model prediction compared against data from high 9 tumor incidence site. Dashed line in panel A shows the extrapolation outsi de the 10 range of the calibrated data. 11

Source: Cohen

- Hubal et al. (1997). 12

13

flux patterns and lesion distribution.This document is a draft for review purposes only and does not constitute Agency policy.
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- 48 3.6.6. PBPK Modeling of DNA Protein Cross

- Links (DPXs) Formed by Formaldehyde 1

3.6.6.1. PBPK Models for DPXs 2

As can be seen from the previous sections, measuring the distribution of the absorbed 3

formaldehyde and identifying its form have proven difficult. Because of the high reactivity of 4

formaldehyde, rapid metabolism of formaldehyde, and complexity of formate clearance, dose 5

surrogates (or biomarkers) of exposure have been used to characterize the extent of absorption 6

and distribution of formaldehyde. As with other soluble and reactive gases, typical PBPK 7

models that predict steady

- state blood concentrations are not useful for predicting formaldehyde 8

dosimetry at this time. As noted previously, inhalation exposure to formaldehyde has not been 9

shown to increase blood formaldehyde levels. Thus, most modeling efforts for formaldehyde 10

have focused on disposition at the site of contact. 11

As discussed earlier, the concentration of D PXs formed by formaldehyde has been 12

treated as a surrogate for the tissue dose of formaldehyde in earlier efforts by Casanova et al. 13

(1991) and in EPA’s efforts to update its health assessment of formaldehyde (Hernandez et al., 14

1994). These efforts used d ata from rats and rhesus monkeys (Casanova et al., 1991, 1989). 15

Using DPXs in this manner allowed the incorporation of both clearance and metabolism of 16

formaldehyde and the incorporation of the effect of saturation on detoxification of formaldehyde 17

at hig her doses. Calculation of the average DPX concentration from these data was seen as a 18

surrogate for the area under the curve (AUC) of the reactive formaldehyde species in the 19

epithelium. Based on these data, Casanova et al. (1991) developed a PBPK model for predicting 20

DPXs in these species and for extrapolating to the human. 21

The Casanova et al. (1991) model consists of three anatomical compartments 22

representing different parts of the upper respiratory tract of the rhesus monkey. The results 23

indicated a 10

- fold difference in DPX formation between rats and monkeys, due primarily to 24

species differences in minute volume and differing quantities of DNA in the nasal mucosa. 25

Casanova et al. (1991) then developed a monkey/rat scaling factor for these parameter s by taking 26

the ratio of nasal mucosa tissue between the two species, a determinant that was proportional to 27

the total body weight differences between the two species. Using these scaling factors in their 28

model, the authors’ predictions in monkey (based o n the rat data) were in close agreement with 29

observed DPXs in monkey, particularly at higher formaldehyde concentrations. However, the 30

model overpredicted DPX formation in the monkey at lower formaldehyde concentrations. 31

Subsequent rat

- human and monkey

- h uman scaling results predicted much lower DPX formation 32

in man. Again, the values obtained at lower concentrations may have been overpredicted, as was 33

the case for rat

- monkey extrapolation. 34This document is a draft for review purposes only and does not constitute Agency policy.
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- 49 Georgieva et al. (2003) developed a model for the uptake and disp osition of 1

formaldehyde in the rat nasal lining. This model was designed to predict the distribution of 2

formaldehyde in the nasal mucosa. The model indicated that, at 6 ppm exposure, a steady

- state 3

elevation of 15

– 20 μM formaldehyde would be achieved wit hin 30 seconds. Furthermore, this 4

same elevation was predicted when the exposure was 6 ppm formaldehyde for 60 minutes. 5

Given that human blood formaldehyde levels are predicted to be about 100 **±** 15 μM (Heck et al., 6

1985) and assuming that blood formaldehyde concentration is roughly equivalent to the 7

concentration predicted at the basement membrane of the epithelium, this model predicts roughly 8

a 15

– 20% increase in blood formaldehyde. However, it shoul d be noted that a 40

- minute 9

inhalation exposure of humans to 1.99 ppm formaldehyde did not lead to a measurable increase 10

in blood formaldehyde (Heck et al., 1985). 11

Franks (2005) published a mathematical model for predicting the disposition of 12

formaldehyde in the human nasal mucosa and blood. The calculated concentrations of 13

formaldehyde in the mucus, the epithelium, and the blood attained steady

- state profiles within a 14

few seconds of exposure. The increase of the formaldehyde concentration in the blood wa s 15

predicted to be insignificant compared with the existing pre

- exposure levels in the body: an 16

increase of 0.00044 mg/L in blood formaldehyde following exposure to 1.9 ppm formaldehyde 17

for up to 8 hours. The model described formaldehyde concentration grad ients across the mucus, 18

epithelial, and submucosal compartments in the human nose. Transport of formaldehyde was 19

governed by the following processes: diffusional (in the mucus); a combination of diffusional, 20

two first order terms representing intrinsic re activity of formaldehyde and binding to DNA, and 21

Michaelis

- Menten kinetics representing enzymatic metabolism (in the epithelial layer); a first

22

order term representing non

- enzymatic removal governed by the blood perfusion rate (in the 23

submucosal compartmen t). The model used the values for the first order reaction rate constants 24

and the Michaelis

- Menten parameters (V
max and K

m ) estimated by Conolly et al. (2000) in their 25

model for extrapolating the rat and rhesus monkey data to the human. The modeling in F ranks 26

(2005) was not calibrated or validated against experimental data, but the predictions of 27

negligible penetration of free formaldehyde to the blood are qualitatively in agreement with the 28

conclusions in Heck et al. (1985). 29

Following the efforts by Casa nova and co

- workers, Cohen

- Hubal et al. (1997), Conolly et 30

al. (2000), and Georgieva et al. (2003) developed models that linked local formaldehyde flux 31

from CFD models to DPX predictions. The focus here will be on the Conolly et al. (2000) effort 32

for the following two reasons: it explicitly incorporates regional formaldehyde dosimetry in the 33

nasal lining by using results from CFD modeling of airflow and gas uptake and it brings data 34This document is a draft for review purposes only and does not constitute Agency policy.
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- 50 across species (rat and rhesus monkey) to bear on model calibration, such a situation being 1

relatively rare in chemical health risk assessments. 2

3

3.6.6.2. A PBPK Model for DPXs in the F344 Rat and Rhesus Monkey that Uses Local Tissue 4

Dose of Formaldehyde 5

In earlier risk assessment efforts (Hernandez et al., 1994; Casanova et al ., 1991; U.S. 6

EPA, 1991b), the average DPX concentration was considered a surrogate tissue dose metric for 7

the AUC of the reactive formaldehyde species. Conolly et al. (2003) assigned a more specific 8

role for DPXs, treating local DPX concentration as a do se surrogate indicative of the 9

intercellular concentration of formaldehyde, leading to formaldehyde

- induced mutations. These 10

authors indicated that it was not known whether DPXs directly induced mutations (Conolly et 11

al., 2003; Merk and Speit, 1998). Thi s is discussed in detail in the mode

- of

- action sections in 12

this document. The Conolly et al. (2000) model for the disposition of inhaled formaldehyde gas 13

and DPX in the rat and rhesus nasal lining is relatively simple in terms of model structure 14

because i t consists of a single well

- mixed compartment for the nasal lining as follows: 15

1. Formaldehyde flux to a given region of the nasal lining is provided as input to the 16

modeling and is obtained in turn as the result of a CFD model. This flux is defined as the 17

amount of formaldehyde delivered to the nasal lining per unit time per unit area per ppm 18

of concentration in the air in a direction transverse to the airflow. It is locally defined as 19

a function of location in the nose and the inspiratory flow rate and is linear with exposure 20

concentration. 21

2. The clearance of formaldehyde from the tissue is modeled as follows: 22

a. a saturable pathway representing enzymatic metabolism of formaldehyde, which 23

is primarily by formaldehyde dehydrogenase (involving Michaelis

- Menten 24

pa rameters V
max and K

m ) 25

b. a separate first

- order pathway, which is assumed to represent the intrinsic 26

reactivity of formaldehyde with tissue constituents (rate constant k f ) 27

c. first

- order binding to DNA that leads to DPX formation (rate constant k b ) 28

3. The clearanc e or repair of this DPX is modeled as a first order process (rate constant 29

k loss ). 30

31

DPX data 32

DPX concentrations were estimated from a study by Casanova et al. (1994) in which rats 33

were exposed 6 hours/day, 5 days/week, plus 4 days for 11 weeks to filtered air (naive) or to 0.7, 34

2, 6, or 15 ppm (0.9, 2.5, 7.4, or 18 mg/m 3 ) formaldehyde (pre

- exposed). On the 5th day of the 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 51 12th week, the rats were then exposed for 3 hours to 0, 0.7, 2, 6, or 15 ppm 14 C

- labeled 1

formaldehyde (with pre

- exposed animals exposed t o the same concentration as during the 2

preceding 12 weeks and 4 days). The animals were sacrificed and DPX concentrations 3

determined at two sites in the nasal mucosa. Conolly et al. (2000) used these naive rat data to 4

develop a PBPK model that predicted the time

- course of DPX concentrations as a function of 5

formaldehyde flux at these sites. 9

7 6

3.6.6.3. Uncertainties in Modeling the Rat and Rhesus DPX Data 8

3.6.6.3.1. Half

- life of DPX repair. In the development of the PBPK model for DPXs, Conolly 9

et al. (2000) assumed a value of 6.5 × 10

– 3 minute

– 1 for k loss , the first

- order rate constant for the 10

clearance (repair) of DPXs, such that the DPXs predicted at the end of a 6

- hour exposure to 11

15 ppm were reduced to exactly the detection limit for DPXs in 18 ho urs (the period between the 12

end of 1 day’s 6

- hour exposure and the beginning of the next). This determination of rapid 13

clearance was based on an observation by Casanova et al. (1994) that the DPX concentrations 14

observed in the pre

- exposed animals were not significantly higher than those in naïve animals (in 15

which there was no significant DPX accumulation). However, in vitro data (Quievryn and 16

Zhitkovich, 2000) indicate a much slower clearance, with an average k loss of 17

9.24 × 10

– 4 minute

– 1

. 18

Subramaniam et al. (2007) examined the Casanova et al. (1994) data and argued that 19

there was a significantly decreased (~ 40%) level of DPXs in high tumor regions of pre

- exposed 20

animals vs. naive animals at 6 and 15 ppm and that the weight of the tissues dissecte d from those 21

regions increased substantially, indicating a thickening of the tissues. After testing the outcome 22

of changing the tissue thickness in the PBPK model for DPXs, it was apparent to these authors 23

that such a change alone could not account for th e dramatic reduction in DPX levels after pre

24

exposure, even with the higher value of k loss used by Conolly et al. (2000) . Therefore, in 25

addition to the gross increase i n tissue weight, these data indicated either an induction in the 26

activity of enzymes that remove formaldehyde (aldehyde and formaldehyde dehydrogenase) or 27

other changes in the biochemical properties of the highly exposed tissue that must have occurred. 28

Gi ven such a change, Subramaniam et al. (2007) concluded that the experimental results in 29

Casanova et al. (1994) were consistent with the smaller experimental value of k loss indicated by 30

the Quievryn and Zhitkovich (2000) data. In particular, they argued th at if V

max increased with 31

exposure (in a tissue region

- and dose

- specific manner), then it was possible to explain the naïve 32

9 Note that Conolly et al. (2000) stated that they used the pre

- exposed data, but this statement appears to be in error (see Subramaniam et al. [2007]).This document is a draft for review purposes only and does not constitute Agency policy.
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- 52 vs. pre

- exposed data of Casanova et al. (1994), with the value of k loss effectively measured in 1

vitro by Quievryn and Zhitkovich (2000) . Furthermore, this value was measured directly, rat her 2

than obtained by indirect interpretation of measurements made at only two time points where 3

significant changes in the tissue had occurred. Therefore, Subramaniam et al. (2007) considered 4

the use of this lower value for k loss to be more appropriate. Consequently, they re

- implemented 5

and re

- optimized the Conolly et al. (2000) model with this modification and found that the fit so 6

obtained to the acute DPX data was excellent. The re

- implemented model will be used in this 7

assessment, and more details ca n be found in Subramaniam et al. (2007). 8

It should be noted that this slower DPX repair rate was obtained in an in vitro study by 9

using human cell lines that were transformed and immortalized. However, it appears that DPX 10

repair in normal cells would be e ven slower. When non

- transformed freshly purified human 11

peripheral lymphocytes were used instead, the half

- life for DPX repair was about 50% longer 12

than in the cultured cells (Quievryn and Zhitkovich, 2000). 13

14

3.6.6.3.2. Statistical uncertainty in paramet er estimates and extrapolation

. Klein et al. (2009) 15

developed methods for deriving statistical inferences of results from PBPK models. They used 16

the Conolly et al. (2000) model for this purpose, specifically because of the sparse time

- course 17

information in the above DPX data. However, they used the value of k loss deduced from 18

Quievryn and Zhitkovich (2000) and fitted the model simultaneously to both the rat and rhesus 19

monkey data, as opposed to the sequential fitting in Conolly et al. (2000). They found that the 20

predicted DPX concentrations were extremely sensitive to V

max and tissue thickness as was also 21

concluded by Georgieva et al. (2003) and Cohen

- Hubal et al. (1997). K

m was seen to be 22

substantially different across species, a finding that was attri buted plausibly to the involvement 23

of more than one enzyme (Klein et al., 2009; Georgieva et al., 2003). Klein et al. (2009) 24

concluded that the two efforts (Conolly et al. [2000] vs. Klein et al. [2009]) resulted in 25

substantially different predictions out side the range of the observed data over which the models 26

were calibrated. 27

The differences between these models occur in spite of the fact that both methods use all the 28

available DPX data in both species and the same model structures. At the 0.1 ppm expo sure 29

concentration, in general these authors obtained three

- to fourfold higher DPX concentrations 30

averaged over a 24

- hour period after exposure. Furthermore, the standard deviations in Klein et 31

al. (2009) for V

max and K

m were an order of magnitude higher and that for k f was 35

- fold lower 32

than the corresponding standard deviations reported in Conolly et al. (2000). The relatively 33

larger standard deviation for k f resulted in this parameter becoming negative in Conolly et al. 34

(2000) at even half the standar d deviation below the maximum likelihood estimate (MLE) value. 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 53 Note that, at a negative value of k f , formaldehyde would be produced as opposed to being 1

cleared through its intrinsic reactivity. 2

Klein et al. (2009) concluded that these “remarkable differen ces outside the range of the 3

observed data suggest caution in the use of these models in a predictive sense for extrapolating to 4

human exposures.” 5

6

3.6.7. Uncertainty in Prediction of Human DPX Concentrations 7

Conolly et al. (2000) used both the rat and rh esus monkey data to predict human DPX 8

concentrations and constructed a PBPK model for the rhesus monkey along similar lines as for 9

the F344 rat. In the rhesus monkey model, they maintained the same values of k b , k loss , and k f as 10

in the rat model but optimized the values of V

max and K

m against the rhesus monkey data from 11

Casanova et al. (1994). The rat and rhesus monkey parameters were then used to construct a 12

human model (see Conolly et al. [2000] for a more detailed report o f implementing the rhesus 13

monkey model and the extrapolations to the situation in humans). 14

For the human, the model used the value of K

m obtained in the rhesus monkey model and 15

the epithelial thickness averaged over three regions of the rhesus monkey nose. The maximum 16

rate of metabolism, V

max , which was estimated independently for the rat and rhesus monkey by 17

fitting to the DPX data available for these species, was then extrapolated to the human by 18

assuming a power law scaling with body weight (BW) (i.e., V
max = a × BW b ), and the coefficient 19

“a” and exponent “b” were derived from the independently estimated values of (V

max ) RAT and 20

(V
max ) MONKEY . Table 3

- 7 gives the values of V

max and K

m in the Conolly et al. (2000) 21

extrapolation. 22

23

Table 3

- 7. Extrapolation of parameters for enzymatic metabolism to the 24

human 25 26

Parameter F344 rat Rhesus monkey Human

V

max (pmol/min

- mm 3 ) 1,008.0 91.0 15.7

K

m (pmol/mm 3 ) 70.8 6.69 6.69
27 Source: Conolly et al. (2000). 28 29 30 The extent of mechanistic data across species, as avail able in this case, is rarely seen with 31

other chemicals, and the above scale

- up procedure was an attempt to use both the rodent and 32

primate DPX data. However, allometric relationships across species are generally based on 33

regressing data from multiple spec ies and usually multiple sources of data points. Thus, the 34

empirical strength of a power law derived by using two data points (F344 rat and rhesus 35This document is a draft for review purposes only and does not constitute Agency policy.
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- 54 monkey) is extremely weak for use as an allometric relationship that can then be used to 1

extrapolate to the human. 2

The following observations indicate the high level of uncertainty in the values of the 3

parameters V
max and K

m in the Conolly et al. (2000) models for predicting DPXs. First, K

m 4

varies by an order of magnitude across the rat and monkey models but is then considered 5

invariant between the monkey and human models (Conolly et al., 2000). Second, the values in 6

Conolly et al. (2000) for V

max /K

m , the low

- dose limit of the rate of enzymatic metabolism, is 7

roughly similar between the rat and monkey but lower by a factor of six in the human. 8

Another factor that can substantially influence the above extrapolation of DPXs in the 9

human is that Conolly et al. (2000) assumed the tissue to be a well

- mixed compartment with 10

regard to formaldehyde interaction with DNA and used the amount of formaldehyde bound to 11

DNA per unit volume of tissue as the DPX dose metric. Considering formaldehyde’s highly 12

reactive nature, the concentrations of formaldehyde and DPX are likely to have a sharp gradient 13

with distance into the nas al mucosa (Georgieva et al., 2003). Given the interspecies differences 14

in tissue thickness, there is consequent uncertainty as to whether DPX per unit volume or DPX 15

per unit area of nasal lining is the more appropriate dose metric to be used in the extrap olation. 16

In particular, it may be assumed that the cells at risk for tumor formation are only those in the 17

epithelium and that measured DPX data (in monkeys and rats) are an average over the entire 18

tissue thickness. Since the epithelial DPXs in monkeys ( and presumably humans) would then be 19

more greatly “diluted” by lower levels of DPX formation that occur deeper into the tissue than in 20

rats, it could be predicted that the ratio of epithelial to measured DPXs in monkeys and humans 21

would be much higher than the ratio in rats. 22

23 

# End of Volume I 24