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# TOXICOLOGICAL REVIEW OF

FORMALDEHYDE

INHALATION TOXICITY

(CAS No. 50-00-0)



## In Support of Summary Information on the

Integrated Risk Inform ation System (IRIS)



# VOLUME II of IV

Hazard Characterization

March 17, 2010

NOTICE

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

solely for the purpose of pre-di ssemination peer review under a pplicable information quality

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

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

of its technical accuracy and science policy implications.

U.S. Environmental Protection Agency

Washington, DC

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

DRAFT

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- ii DISCLAIMER 1

2

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

distributed solely for the purpose of pre

- dissemination peer review under ap plicable information 4

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

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

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

8

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

DRAFT

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- iii CONTENTS

— TOXICOLOGICAL REVIEW OF FORMALDEHYDE 1

(CAS No. 50

- 00

- 0) 2

3

4

LIST OF TABLES

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

LIST OF FIGURES

................................ ................................ ................................ ....................... xx

LIST OF ABBREVIATIONS AND ACRONYMS

................................ ................................ .... xxv

FOREWORD

................................ ................................ ................................ ............................ xxxii

AUTHORS, CONTRIBUTORS, AND REVIEWERS

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

VOLUME I

1. INTRODUCTION

................................ ................................ ................................ .................. 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. Derm al Contact

................................ ................................ ............................... 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 Metabo lism (Non

- Enzymatic)

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

- 5

3.1.2.3. Exogenous Sources of Formaldehyde Production

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

- 5

3.1.2.4. FA

- GSH Conjugate as a Method of Systemic Distribution

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

- 6

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

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

- 6

3.1.2.6. Levels of Endogenous For maldehyde 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 F ormaldehyde 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 con stitute 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. EXCR ETION

................................ ................................ ................................ .............. 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 Form aldehyde 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 Mode ling 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 th e Rat and Rhesus DPX Data

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

- 51

3.6.7. Uncertainty in Prediction of Human DPX Concentrations

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

- 53

VOLUME II

4. HAZARD CHARACTERIZATION

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

- 1

4.1. HUMAN STUDIES

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

- 1

4.1.1. Noncancer Health Effects

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

- 1

4.1.1.1. Sensory Irritation (Eye, Nose, Throat Irri tation)

............................... 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 E xposure 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 con stitute 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. Summary: Carcinogenic Hazard in Humans

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

- 107

4.2. ANIMAL STUDIES

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

- 109

4.2.1. Noncancer Health Effects

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

- 110

4.2.1.1. Reflex Bradypnea

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

- 110

4.2.1.2. Respiratory Tract Pathology

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

- 120

4.2.1.3. Gastrointestinal Tract and Systemic Toxicity

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

- 201

4. 2.1.4. Immune Function

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

- 216

4.2.1.5. Hypersensitivity and Atopic Reactions

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

- 225

4.2.1.6. Neurological and Neurobehavioral Function

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

- 250

4.2.1.7. Reproductive and Developmental Toxicity

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

- 285

4.2.2. Carcinogenic Potential: Animal Bioassays

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

- 324

4.2 .2.1. Respiratory Tract

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

- 324

4.2.2.2. Gastrointestinal Tract

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

- 326

4.2.2.3. Lymphohematopoietic Cancer

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

- 328

4.2.2.4. Summary

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

- 335

4.3. GENOTOXICITY

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

- 335

4.3.1. Formaldehyde

- DNA Reactions

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

- 335

4.3.1.1. DNA

- Protein Cross

- Links (DPXs)

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

- 336

4.3.1. 2. DNA Adducts

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

- 341

4.3.1.3. DNA

- DNA Cross

- Links (DDXs)

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

- 343

4.3.1.4. Single Strand Breaks

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

- 344

4.3.1.5. Other Genetic Effects of Formaldehyde in Mammalian Cells

...... 4

- 345

4.3.2. In Vitro Clastogenicity

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

- 345

4.3.3. In Vitro Mutagenicity

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

- 347

4.3.3.1 . Mutagenicity in Bacterial Systems

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

- 347

4.3.3.2. Mutagenicity in Non

- mammalian Cell Systems

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

- 353

4.3.3.3. Mutagenicity in Mammalian Cell Systems

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

- 353

4.3.4. In Vivo Mammalian Genotoxicity

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

- 360

4.3.4.1. Genotoxicity in Laboratory Animals

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

- 360

4.3 .4.2. Genotoxicity in Humans

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

- 362

4.3.5. Summary of Genotoxicity

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

- 370

4.4. SYNTHESIS AND MAJOR EVALUATION OF NONCARCINOGENIC

EFFECTS

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

- 371

4.4.1. Sensory Irritation

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

- 376

4.4.2. Pulmonary Function

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

- 379

4.4.3. Hypersensitivity and Atopic Reactions

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

- 382

4.4.4. Upper Respiratory Tract Histopathology

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

- 383

4.4.5. Toxicogenomic and Molecular Data that May Inform MOAs

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

- 385

4.4.6. Noncancer Modes of Actions

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

- 387

4.4.7. Immunotoxicity

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

- 389This document is a draft for review purposes only and does not con stitute Agency policy.

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- vi CONTENTS (continued)

4.4.8. Effects on the Nervous Syst em

................................ ................................ .... 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 Toxici ty

................................ .................. 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 Rep roductive 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. Lymphohematopoieti c Malignancies

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

- 408

4.5.2.1. Background

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

- 408

4.5.2.2. All LHP Malignancies

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

- 410

4.5.2.3. All Leukemia

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

- 414

4.5.2.4. Subtype Analysis

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

- 418

4.5.2.5. Myeloid Leukemia

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

- 419

4.5.2.6. Solid Tumors of Lymphoid Origin

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

- 421

4.5.2.7. Supporting Evidenc e from Animal Bio

- Assays for

Formaldehyde

- Induced Lymphohematopoietic Malignancies

..... 4

- 423

4.5.3. Carcinogenic Mode(s) of Action

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

- 427

4.5.3.1. Mechanistic Data for Formaldehyde

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

- 428

4.5.3.2. Mode of Action Evaluation for Upper Respiratory Tract

Cancer (Nasopharyngeal Cancer, Sino

- Nasal)

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

- 439

4.5.3.3. Mode(s) of Action for Lymphohematpoietic Malignancies

.......... 4

- 446

4.5.4. Hazard Characterization for Formaldlehyde Carcinogenicity

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

- 453

4.6. SUSCEPTIBLE PO PULATIONS

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

- 454

4.6.1. Life Stages

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

- 454

4.6.1.1. Early Life Stages

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

- 455

4.6.1.2. Later Life Stages

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

- 459

4.6.1.3. Conclusions on Life

- Stage Susceptibility

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

- 459

4.6.2. Health/Disease Status

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

- 460

4.6.3. Nutritional Status

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

- 461

4.6.4. Gender Differences

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

- 462

4.6.5. Genetic Differences

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

- 462This document is a draft for review purposes only and does not con stitute Agency policy.

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- vii CO NTENTS (continued)

4.6.6. Co

- Exposures

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

- 464

4.6.6.1. Cumulative Risk

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

- 464

4.6.6.2. Aggregate Exposure

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

- 465

4.6.7. Uncertainties of Database

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

- 465

4.6.7.1. Uncertainties of Exposure

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

- 465

4.6.7.2. Uncertainties of Effect

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

- 466

4.6.8. Summary of P otential Susceptibility

................................ ............................ 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. Developmental and Reproductive Toxicity

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

- 25

5.1.2. Summary of Critical Effects and Candidate RfCs

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

- 33

5.1.2.1. Selection of Studies for Candidate RfC Derivation

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

- 33

5.1.2.2. Derivation of Candidate RfCs from Key Studies

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

- 40

5.1.2.3. Evaluation of the Study

- Specific Candidate RfC

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

- 66

5.1.3. Database Uncertainties in the RfC Derivation

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

- 69

5.1.4. Uncertainties in the RfC Derivation

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

- 72

5.1.5. Previous Inhalation Assessment

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

- 74

5.2. QUANTITATIVE C ANCER ASSESSMENT BAS ED ON THE NATIONAL

CANCE R INSTITUTE COHORT S TUDY

................................ .............................. 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 Cance r Mortality

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

- 79

5.2.2.3. Prediction of Lifetime Extra Risk of Nasopharyngeal Cancer

Incidence

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

- 81

5.2.2.4. Sources of Uncertainty

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

- 83

5.2.3. Lymphohematopoietic Cancer

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

- 88

5.2.3.1. Exposure

- Response Modeling of the National Cancer

Institute Cohort

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

- 88This document is a draft for review purposes only and does not con stitute Agency policy.

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- viii CONTENTS (continued)

5.2.3.2. Prediction of Lifetime Extra Risks for Hodgkin Lymphoma

and Leukemia Mortality

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

- 91

5.2.3.3. Prediction of Lifetime Extra Risks for Hodgkin Lymphoma

and Leukemia Incidence

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

- 93

5.2.3.4. Sources of Uncertainty

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

- 95

5.2.4. Conclusions on Cancer Unit Risk Estimates Based on Human Data

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

- 99

5.3. DOSE

- RESPONSE MODELING OF RISK OF SQUAMOUS 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. Na sal Tumor Incidence Data

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

- 104

5.3.1.2. Mechanistic Data

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

- 105

5.3.2. The CIIT Biologically Based Dose

- Response Modeling

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

- 106

5.3.2.1. Major Results of the CIIT Modeling Effort

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

- 111

5.3.3. This Assessment’s Conclusions from Evaluation of Dose

- Respons e

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. Comp arison 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 INHALATIO N

.. 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 (ADAF s)

.................... 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, Met abolism, and Excretion

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

- 1

6.1.3. Noncancer Health Effects in Humans and Laboratory Animals

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

- 4

6.1.3.1. Sensory Irritation

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

- 4

6.1.3.2. Respiratory Tract Pathology

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

- 5

6.1.3.3. Effects on Pulmonary Function

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

- 8

6.1.3.4. Asthmatic Responses and Increa sed 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 con stitute 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. Carcinogenicity in Human and Laboratory Animals

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

- 14

6.1.4.1. Carcinogenicity in Humans

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

- 14

6.1.4.2. Carcinogenicity in Laboratory Animals

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

- 20

6.1.4.3. Carcinogenic Mode(s) of Action

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

- 21

6.1.5. Cancer Hazard Characterization

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

- 24

6.2. DOSE

- RESPONSE CHARACTERIZ ATION

................................ ........................... 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 Deriv ation

....................... 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 D ata

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

- 36

6.2.2.4. Extrapolation Aporoaches

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

- 37

6.2.2.5. Inhalation Unit Risk Estimates for Cancer

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

- 41

6.2.2.6. Early

- Life Susceptibility

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

- 41

6.2.2.7. Uncertainties in the Quantitative Risk Estimates

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

- 42

6.2.2.8. Conclusions

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

- 45

6.3. SUMMARY AND CONCLUSIONS

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

- 45

REFERENCES

................................ ................................ ................................ ............................ R

- 1

VOLUME IV

APPENDIX A: SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC

COMMENTS AND DISPOSITIONS

................................ ............................. A

- 1

APPENDIX B: SIMULATIONS OF INTERINDIVIDUAL AND ADULT

- TO

- CHILD

VARIABILITY IN REACTIVE GAS UPTAKE IN A SMALL SAMPLE

OF PEO PLE (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 ALTERNATIV E

IMPLEMENTATIONS

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

- 1

APPENDIX F: SENSITIVITY ANALYSIS OF BBDR MODEL FOR FORMALDEHYDE

INDUCED RESPIRATORY CANCER IN HUMANS

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

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

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- x CONTENTS (continued)

APPENDIX G: EVALUATION OF THE CANCER DOSE

- RESPONSE MODELING

OF GENOMIC DATA FOR FORMALDEHYDE RISK ASSE SSMENT

..... G

- 1

APPENDIX 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 con stitute Agency policy.

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- xi LIST OF TABLES

Table 2

- 1. Physicochemical properties of formaldehyde

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

- 2

Table 2

- 2. Ambie nt air levels by land use category

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

- 6

Table 2

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

- occupational)

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

- 8

Table 3

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

...... 3

- 8

Table 3

- 2. Formaldehyde kinetics in human and rat tissue samples

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

- 21

Table 3

- 3. Allelic frequencies of ADH3 in human populations

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

- 25

Table 3

- 4. Percent distribution of airborne [ 14 C]

- formaldehyde in F344 rats

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

- 26

Table 3

- 5. Apparent formaldehyde levels in exhaled breath of individua ls 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 c oncentrations 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. Coh ort 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 ref lex bradypnea in various

strains of rats

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

- 113

Table 4

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

mice exposed to 15 ppm

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

- 116

Table 4

- 8. Exposure regimen for cross

- tolerance study

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

- 117This document is a draft for review purposes only and does not con stitute Agency policy.

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- xii LIST OF TABLES (continued)

Table 4

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

respiratory tract

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

- 127

Table 4

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

nasoturbinates

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

- 129

Table 4

- 11. Enzymatic activities in nasal respirator y epithelium of male Wistar rats

exposed to formaldehyde, ozone, or both

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

- 130

Table 4

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

to 0, 15, or 145.6 ppm formaldehyde for 6 hours

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

- 138

Table 4

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

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

formaldehyde for 6 hours

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

- 139

Table 4

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

exposed to forma ldehyde via inhalation

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

- 140

Table 4

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

formaldehyde

— short term studies

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

- 143

Table 4

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

exposed to formaldehyde

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

- 146

Table 4

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

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

formaldehyde 6 hours/day for 13 weeks

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

- 148

Table 4

- 18. Formaldehyde

- induced nonneopl astic histopathologic changes in male

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

and examined at the end of 130 weeks inclusive of exposure

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

- 149

Table 4

- 19. Formaldehyde

- induced nasal tumors in male albino SPF Wistar rats

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

examined at the end of 130 weeks inclusive of exposure

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

- 150

Table 4

- 20. Formaldehyde effects on nasal epithelium for various concentration

- by

time products in male albino Wistar rats

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

- 153

Tab le 4

- 21. Rhinitis 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 con stitute 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 anima ls; data pooled for males and

females

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

- 155

Table 4

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

formaldehyde

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

- 156

Table 4

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

cerebral cortex of male Wistar rats exposed to formaldehyde via inhalation

for 4 and 13 weeks

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

- 158

Table 4

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

- treated

male Wistar rats

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

- 158

Table 4

- 26. Total lung cytochrome P450 measurements of control a nd formaldehyde

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. Histopathologic findings and severity scores in the naso

- and

maxilloturbinates of female Sprague

- Dawley rats exposed to inhaled

formaldehyde and wood dust for 104 weeks

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

- 166

Table 4

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

Sprague

- Dawley rats exposed to inhaled formaldehyde or HCl alone and

in combination for a lifetime

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

- 170

Table 4

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

inhaled formaldehyde for 2 years

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

- 173

Table 4

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

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

bioassay

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

- 174

Table 4

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

rats exposed to in haled formaldehyde for 2 years

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

- 175

Table 4

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

to formaldehyde

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

- 183

Table 4

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

from male Wistar rats after inhalation exposures to formaldehyde

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

- 194This document is a draft for review purposes only and does not con stitute 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 proliferat ion, LI and ULLI, in male F344 rats

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

- 196

Table 4

- 37. Formaldehyde

- induced changes in cell proliferation and (ULLI) in the nasal

passages of male F344 rats exposed 6 hours/day

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

- 198

Table 4

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

regional site location of squamous cell carcinomas in formaldehyde

exposed rats for correlation to cell proliferation rates

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

- 199

Table 4

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

respiratory tract

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

- 202

Tabl e 4

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

after drinking

- water exposure to formaldehyde for 4 weeks

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

- 206

Table 4

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

after drinking

- wat er 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. Sum mary 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 formal dehyde exposure

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

- 218

Table 4

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

phagocyte system (MPS) in female B6C3F1 mice after a 3

- week, 15 ppm

formaldehyde exposure ( 6 hours/day, 5 days/week)

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

- 219

Table 4

- 47. Formaldehyde exposure regimens for determining the effects of

formaldehyde exposure on pulmonary S. aureus infection

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

- 221

Table 4

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

exposure in experimental animals

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

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- xv LIST OF TABLES (continued)

T able 4

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

routes of exposure: inhalation, dermal, and injection

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

- 232

Table 4

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

inhalation, topical applicat ion, or footpad injection

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

- 233

Table 4

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

formaldehyde

- exposed male ICR mice with and without Der f sensitization

..... 4

- 240

Table 4

- 52. Correlation coefficients among ear swelling resp onses and skin mRNA

levels in contact hypersensitivity to formaldehyde in mice

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

- 249

Table 4

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

sensitization due to formaldehyde in experimental animals

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

- 251

Table 4

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

formaldehyde in a single 2

- hour exposure: effects after 2 hours

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

- 259

Table 4

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

formaldehyde in a single 2

- hour exposure: effects after 24 hours

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

- 259

Table 4

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

male and female LEW.1K rats

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

- 263

Table 4

- 57. Summary of neurological and neurobehavioral studies in inhaled

formaldehyde in experimental animals

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

- 279

Tabl e 4

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

dams exposed via inhalation from mating through gestation

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

- 289

Table 4

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

male Wistar rats

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

- 298

Table 4

- 60. Formaldehyde effects on adult male albino Wistar rats

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

- 299

Table 4

- 61. Formaldehyde effects on testosterone levels and seminiferous tubule

diameters in Wistar rats following 91 days of exposure

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

- 300

Table 4

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

epithelial height in Wistar rats following 18 weeks of exposure

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

- 302

Table 4

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

formaldehyde

- treated mice

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

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- xvi LIST OF TABLES (continued)

Table 4

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

gestation

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

- 303

Table 4

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

abnormal sperm after oral administration of formaldehyde to male

Wistar rats

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

- 305

Ta ble 4

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

exposed intraperitoneally

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

- 306

Table 4

- 67. Incidence of sperm head abnormalities in formaldehyde

- treated rats

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

- 307

Table 4

- 68. Dominant lethal mutations after exposure o f 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. Sum mary of reported developmental effects in formaldehyde dermal

exposure studies

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

- 318

Table 4

- 72. Summary of reported reproductive effects in formaldehyde inhalation

studies

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

- 319

Table 4

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

.......... 4

- 322

Table 4

- 74. Summary of reported reproductive effects in formaldehyde intraperitoneal

studies

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

- 323

Table 4

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

lymphoma

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

- 329

Table 4

- 76. Formaldehyde

- DNA reactions (DPX for mation)

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

- 340

Table 4

- 77. Formaldehyde

- DNA reactions (DNA adduct formation)

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

- 343

Table 4

- 78. Formaldehyde

- DNA interactions (single stranded breaks)

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

- 344

Table 4

- 79. Other genetic effects of formaldehyde in mammalian cells

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

- 346

Table 4

- 80. In vitro c lastogenicity of formaldehyde

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

- 348

Table 4

- 81. Summary of mutagenicity of formaldehyde in bacterial systems

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

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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 stu dents 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 labora tory workers and

controls

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

- 370

Table 4

- 89. Summary of human cytogenetic studies

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

- 372

Table 4

- 90. Summary of cohort and case

- control studies which evaluated the

incidence of all LHP cancers in formaldehyde

- exposed populations

(ICD

- 8 Codes: 200

- 209) and all leukemias (ICD

- 8 Codes: 204

- 207)

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

- 412

Table 4

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

disease groupings within the broad category of all lymphohematopoetic

malignancies

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

- 419

Table 4

- 92. Summary of stud ies which provide mortality statistics for myeloid

leukemia sub

- types

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

- 420

Table 4

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

multiple myeloma from cohort analyses of formaldehyde exposed workers

...... 4

- 422

Table 4

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

lymphoma

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

- 424

Table 4

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

Sprague

- Dawley rats

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

- 425

Table 4

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

exposure

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

- 469This document is a draft for review purposes only and does not con stitute 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 rats

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

- 23

Table 5

- 3. Developmental and reproductive toxicity PODs including duration

adjustments

– key human study

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

- 31

Table 5

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

health endpoint category

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

- 36

Table 5

- 5. Adjustment for nonoccupational exposures to formaldehyde

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

- 64

Table 5

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

supporting studies

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

- 68

Table 5

- 7. Relative risk estimates for mortality from nasopharyngeal malignanc ies

(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 e stimates 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 cumulative 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 mortality from Hodgkin lymphoma

(ICD

- 8 code 201) and leukemia (ICD

- 8 codes 204

– 207) by level of

formaldehyde exposure for different exposure metrics

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

- 90

Table 5

- 13. Regression coefficients for Hodgkin lymphoma and leukemia mortality

fro m 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 con stitute Agency policy.

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- xix LIST OF TABLES (continued)

Table 5

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

continuous exposure to formaldehyde

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

- 91

Table 5

- 16. 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

Table 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 i nhalation unit risk estimates for Hodgkin

lymphoma incidence from formaldehyde exposure, based on Beane

Freeman et al. (2009) log

- linear trend analyses for cumulative exposure

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

- 94

Table 5

- 19. EC 005 , LEC 005 , and inhalation unit risk estimates for leukem ia incidence

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

log

- linear trend analyses for cumulative exposure

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

- 94

Table 5

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

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

- 100

Table 5

- 21. Calculation of comb ined cancer incidence unit risk estimate at 0.1 ppm

.......... 5

- 100

Table 5

- 22. Summary of tumor incidence in long

- term bioassays on F344 rats

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

- 105

Table 5

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

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

- 125

Table 5

- 24. Formaldehyde

- induced rat tumor incidences

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

- 128

Table 5

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

formaldehyde flux and DPX

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

- 134

Table 5

- 26. Summary of inhalation unit risk estimates

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

- 135

Table 5

- 27. Total cancer risk from exposure to a constant f ormaldehyde exposure

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 specifi ed extra cancer risk levels for selected formaldehyde

- related

cancers

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

- 36

Table 6

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

animal data

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

- 42This document is a draft for review purposes only and does not con stitute 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

- mediate d protein modifications

................................ ...................... 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 nas al passages of F344 rat, rhesus monkey, and human

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

- 36

Figure 3

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

simulations with water

- dye studies

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

- 37

Figure 3

- 8. Lateral view of nasal wall mass flux of inhaled formaldehy de 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 formaldehy de

exposure concentration in an adult male human

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

- 43

Figure 3

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

model compared with pressure drop measurements made in two hollow

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

in vivo

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

- 45

Figure 3

- 12. Formaldehyde

- DPX dosimetry in the 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 con stitute Agency policy.

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- xxi LIST OF FIGURES (conti nued)

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 resp iratory 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 autor adiography 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 formaldehy de

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

- 137

Figure 4

- 9. Frequency and location by cross

- section level of squamous metaplasia in

the nasal cavity of F344 rats exposed to formaldehyde via inhalation

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

- 172

Figure 4

- 10. Effect of formaldehyde exposure on cell proliferation of the respirat ory

mucosa of rats and mice

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

- 190

Figure 4

- 11. Alveolar MP Fc

- mediated phagocytosis from mice exposed to 5 ppm

formaldehyde, 10 mg/m 3 carbon black, or both

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

- 223

Figure 4

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

following formaldehyde exposure in guinea pigs after OVA sensitization and

OVA challenge

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

- 235

Figure 4

- 13. OVA

- specific IgG1 (1B) in formaldehyde

- treated sensitized guinea pigs

prior to OVA challenge

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

- 235

Figure 4

- 14. Anti

- OVA titers in female Balb/C mice expo sed 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 re ceptor antagonists 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 con stitute 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 gob let cell proliferation (Panel B) after Der f

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

challenge

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

- 241

Figure 4

- 18. NGF in BAL fluid from formaldehyde

- exposed female C3H/He mice

with and without OA sensitization

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

- 243

Figur e 4

- 19. Plasma Substance P levels in formaldehyde

- exposed female C3H/He

mice with and without OVA sensitization

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

- 244

Figure 4

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

formaldehyde expressed as mean number of crossed quadran ts ± SEM

.......... 4

- 256

Figure 4

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

second observation period (day 2, 24 hours after formaldehyde exposure)

..... 4

- 257

Figure 4

- 22. Motor activity was reduced in male and female LEW.1K rats 2 hour s

after termination of 10

- minute formaldehyde exposure

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

- 258

Figure 4

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

ambulatory and vertical components of SLMA

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

- 260

Figure 4

- 24. Effects of formaldehyde exposure on the error rate of fe male 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 f ormaldehyde

- exposed mice

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

- 274

Figure 4

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

indicated exposure groups

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

- 325

Figure 4

- 28. Leukemia incidence in Sprague

- Dawley rats exposed to formaldehyde

in drinking water for 2 year s

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

- 330

Figure 4

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

24 months

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

- 332

Figure 4

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

formaldehyde for 24 months

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

- 333

Figure 4

- 31. Cumulative incidence or tum or 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 con stitute 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 averag e intensity of formaldehyde exposure and

the risk of lymphohematopoietic malignancy

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

- 416

Figure 4

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

- 29

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

(HUV EC)

................................ ................................ ................................ ......... 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 f ollowing formaldehyde

exposure at 32, 170, 390, or 890 ppb

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

- 21

Figure 5

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

performing the water labyrinth learning test

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

- 24

Figure 5

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

exposure index category with 8

- hour time weighted average formaldehyde

exposure concentration of 219 ppb

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

- 27

Figure 5

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

relation to indoor residential formaldehyde concentrations.

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

- 41

Figure 5.5. Odds ratios for physician

- diagnosed asthma in children associated with 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 a nd sensory irritation (eye irritation)

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

- 53This document is a draft for review purposes only and does not con stitute 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 for myeloid, lymphoid, and

all leukemia

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

- 98

Figure 5

- 11. Schematic of integration of pharmacokinetic and pharmacodynamic

components in the CIIT model

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

- 109

Figure 5

- 12. Fit to the rat tumor incidence data using the model and assumption s in

Conolly et al. (2003)

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

- 112

Figure 5

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

characterized by partitioning the nasal surface by formaldehyde flux to

the tissue per ppm of exposure concentration, resulting in 20 flux bins

.......... 5

- 120

Figure 5

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

.......... 5

- 120

Figure 5

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

two BBDR models

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

- 124

Figure 5

- 16. Replot of log

- probit fit of the combi ned 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. Mul tistage Weibull model fit of tumor incidence data compared with

KM estimates of spontaneous tumor incidence

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

- 132This document is a draft for review purposes only and does not con stitute Agency policy.

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- xxv LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygienists

ADAF age

- dependent adjustment factors

A DH alcohol dehydrogenase

ADS anterior dorsal septum

AIC Akaike Information Criterion

AIE average intensity of exposure

AIHA American Industrial Hygiene Association

ALB albumin

ALDH aldehyde dehydrogenase

ALL acute lymphocytic leukemia

ALM anterior lateral meatus

ALP alkaline phosphatase

ALS amyotrophic lateral sclerosis

ALT alanine aminotransferase

AML acute myelogenous leukemia

AMM anterior medial maxilloturbinate

AMPase adenosine monophosphatase

AMS anterior medial septum

ANAE alpha

- naphth ylacetate 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 Thor acic 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)eth er

BDNF brain

- derived neurotrophic factor

BEIR biologic effects of ionizing radiation

BfR German Federal Institute for Risk Assessment

BHR bronchial hyperresponsiveness

BMC benchmark concentration

BMCL 95% lower bound on the benchmark concentration

B MCR binuclated micronucleated cell ratefluoresce

BMD benchmark dose

BMDL 95% lower bound on the benchmark doseThis document is a draft for review purposes only and does not con stitute 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 chromosomal aberrations

CalEPA California Environmental Protection Agency

CAP College of American Pathologists

CASRN Chemical Abstracts Service Registry Number

CAT catalase

CBMA cytokinesis

- blocked micronucleus assay

CBMN cytokinesis

- b locked 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 In stitute of Toxicology

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 protein s

CS conditioned stimulus

C × t concentration times time

DA Daltons

DAF dosimetric adjustment factor

DDX DNA

- DNA cross

- links

DEI daily exposure index

DEN diethylnitrosamine

Der f common dust mite allergen

DMG dimethylglycine

DMGDH dimethylglycine dehydrogenase

DNA deoxyribonucleic acid

DOPAC 3,4

- dihydroxyphenylacetic acid

DPC / DPX DNA

- protein cross

- links

EBV Epstein

- Barr virus

EC effective concentration

ED effective dose

EHC Environmental Health Committee

ELISA enzyme

- linked immunosorbent a ssayThis document is a draft for review purposes only and does not con stitute Agency policy.

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- xxvii LIST OF ABBREVIATIONS AND ACRONYMS (continued)

EPA U.S. Environmental Protection Agency

ERPG emergency response planning guideline

ET ethmoid turbinates

FALDH formaldehyde dehydrogenase

FDA U.S. Food and Drug Administration

FDR fecundability de nsity ratio

FEF forced expiratory flow

FEMA Federal Emergency Management Agency

FEV1 forced expiratory volume in 1 second

FISH fluorescent in situ hybridization

FSH follicle

- stimulating hormone

FVC forced vital capacity

GALT gut

- associated lymphoid tissue

GC

- MS gas chromatography

- mass spectrometry

GD gestation day

GI gastrointestinal

GO gene ontology

G6PDH glucose

- 6

- phosphate dehydrogenase

GPX glutathione peroxidase

GR glutathione reductase

GM

- CSF granulocyte macrophage

- colony

- stimulating factor

GSH reduced glutathione

GSNO S

- nitrosoglutathione

GST glutathione S

- transferase

HAP hazardous air pollutant

Hb hemoglobin

HCl hydrochloric acid

HCT hematocrit

HEC human equivalent concentration

5

- HIAA 5

- hydroxyindoleacetic acid

hm hydroxymethyl

H MGSH S

- hydroxymethylglutathione

HPA hypothalamic

- pituitary adrenal

HPG hypothalamo

- pituitary

- gonadal

HPLC high

- performance liquid chromatography

HPRT hypoxanthine

- guanine phosphoribosyl transferase

HR high responders

HSA human serum albumin

HSDB Haz ardous Substances Data Bank

Hsp heat shock protein

HWE healthy worker effect

I cell initiated cell

IARC International Agency for Research on CancerThis document is a draft for review purposes only and does not con stitute Agency policy.

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- xxviii LIST OF ABBREVIATIONS AND ACRONYMS (continued)

ICD International Classification of Diseases

IF inter facial

IFN interferon

Ig immunoglobulin

IL interleukin

I.P. intraperitoneal

IPCS International Programme on Chemical Safety

IRIS Integrated Risk Information System

K

m Michaels

- Menton constant

KM Kaplan

- Meier

LD 50 median lethal dose

LDH lactate de hydrogenase

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 sensit ivity

MCV mean corpuscular volume

MDA malondialdehyde

MEF maximal expiratory flow

ML myeloid leukemia

MLE maximum likelihood estimate

MMS methyl methane sulfonate

MMT medial maxilloturbinate

MN micronucleus, micronuclei

MNNG N

- methyl

- N

- nitro

- N

- nitr osoguanidine

MOA mode of action

MoDC monocyte

- derived dendritic cell

MP macrophage

MPD multistage polynomial degree

MPS mononuclear phagocyte system

MRL minimum risk levelThis document is a draft for review purposes only and does not con stitute Agency policy.

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- xxix LIST OF ABBREVIATIONS AND ACRONYMS (continued)

mRNA messenger ribonucleic a cid

MVE

- 2 Murray Valley encephalitis virus

MVK Moolgavkar, Venzon, and Knudson

N cell normal cell

NaCl sodium choride

NAD+ nicotinamide adenine dinucleotide

NADH reduced nicotinamide adenine dinucleotide

NALT nasally associated lymphoid tissue

NATA National

- Scale Air Toxics Assessment

NCEA National Center for Environmental Assessment

NCHS National Center for Health Statistics

NCI National Cancer Institute

NEG Nordic Expert Group

NER nucleotide excision repair

NGF nerve growth factor

NHL non

- Ho dgkin’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

- hydroxymethyldeoxya denosine

N 4

- hmdC N 4

- hydroxymethylcytidine

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

- m ethyl

- 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 He alth Administration

OTS Office of Toxic Substances

OVA ovalbumin

PBPK physiologically based pharmacokinetic

PC Philadelphia chromosome

PCA passive cutaneous anaphylaxis

PCMR proportionate cancer mortality ratio

PCNA proliferating cell nuclear antige n

PCR polymerase chain reaction

PCV packed cell volumeThis document is a draft for review purposes only and does not con stitute 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 plaq ue

- forming cell

PG periglomerular

PHA phytohemagglutinin

PLA2 phospholipase A2

PI phagocytic index

PLM posterior lateral meatus

PMA phorbol 12

- myristate 13

- acetate

PMR proportionate mortality ratio

PMS posterior medial septum

PND postnatal day

POD point of departure

POE portal of entry

PTZ pentilenetetrazole

PUFA polyunsaturated fatty acids

PWULLI population weighted unit length labeling index

RA reflex apnea

RANTES regulated upon activation, normal T

– cell expressed and secreted

RB reflex bra dypnea

RBC red blood cells

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 transcriptase

SAB Science Advisory Board

SCC squamous cell carcinoma

SCE sister chromatid exchange

SCG sodium cromoglycate

SD standard deviation

SDH succinate dehydrogenase; sarcosine dehydrogenase

SEER Surveillance, Epidemiology, a nd End Results

SEM standard error of the mean

SEN sensitizer

SH sulfhydryl

SHE Syrian hamster embryo

SLMA spontaneous locomotor activityThis document is a draft for review purposes only and does not con stitute Agency policy.

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- xxxi LIST OF ABBREVIATIONS AND ACRONYMS (continued)

SMR standardized mortality ratio

SNP single nucleotide polymorp hism

SOD superoxide dismutase

SOMedA N 6

- sulfomethyldeoxyadenosine

Sp1 specificity protein

SPIR standardized proportionate incidence ratio

SSAO semicarbozole

- sensitive amine oxidase

SSB single strand breaks

STEL short

- term exposure limit

TBA tumor be aring animal

TH T

- lymphocyte helper

THF tetrahydrofolate

TK toxicokinetics

TL tail length

TLV threshold limit value

TNF tumor necrosis factor

TP total protein

TRI Toxic Release Inventory

TRPV transient receptor potential vanilloid

TWA time

- weight ed average

TZCA thiazolidine

- 4

- carboxylate

UCL upper confidence limit

UDS unscheduled DNA synthesis

UF uncertainty factor

UFFI urea formaldehyde foam insulation

ULLI unit length labeling index

URT upper respiratory tract

USDA U.S. Department of Agr iculture

VC vital capacity

VOC volatile organic compound

WBC white blood cell

WDS wet dog shake

WHO World Health Organization

WHOROE World Health Organization Regional Office for EuropeThis document is a draft for review purposes only and does not con stitute Agency policy.

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- xxxii 

### 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 4. HAZARD CHARACTER I ZATION 1

2

3

4.1. HUMAN STUDIES 4

This chapter discusses epidemiologic studies of site

- specific cancers and other adverse 5

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

literature describing inhalation exposure and i ts potential carcinogenic and noncarcinogenic 7

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

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

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

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

outcomes. 12

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

- resp iratory 13

tract cancers. The first part discusses site

- specific cancers that are chiefly located in the 14

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

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

on non

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

to formaldehyde than respiratory epithelium

— mainly, lymphohematopoietic (LHP) cancer, brain 18

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

20

4.1.1. Noncancer Health Effects 21

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

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

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

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

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

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

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

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

bradypnea (RB) (also known as reflex apnea [RA]). Formaldehyde

- induced sensory irritation 30

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

Formaldehyde

- induced neuro genic sensitization and atopy may result in lifelong health effects 32

from short

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

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

sneezing, RB, and BC, and sensitization. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 2 Eye, nose, and throat irritation in response to formaldehyde inhalation exposure is well 1

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

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

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

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

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

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

sensory irritation. The endpoints for assessing irritation include self

- reporting of adverse 8

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

- blink 9

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

irritation b ut studies have documented other types of irritation, including dermal irritation 11

eczema and dermatitis. 12

13

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

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

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

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

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

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

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

making, and formaldehyde

- based resins; and (3) anatomy students who are exposed under well

21

defined conditions during academic courses where they are examining formaldehyde

- preserved 22

cadavers. 23

24

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

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

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

Lehnen (1987). In this cross

- sectional stud y of nearly 2,000 Minnesota residents living in 392 28

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

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

formaldehyde. Techn icians administered a symptom questionnaire to participating residents at 31

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

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

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

were taken from two rooms of the home, usually the bedroom and living room, and were kept 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 3 refrigerated until analysis. Respondents were not aware of the resul ts of the formaldehyde 1

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

from Ritchie and Lehnen (1987) provide a clear dose

- response relationship in the percentage of 3

residential occupants reporting eye, nose, a nd throat irritation. Specifically, eye irritation 4

responses increase from 1

– 2% in homes with formaldehyde concentrations lower than 0.1 ppm 5

to 12

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

– 6

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

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

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

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

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

- selected 11

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

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

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

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

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

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

throat. 18

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

Ritchie and Lehnen (1987) are corroborated by Hanrahan et al. (1984) who conducted a cross

20

sectional survey by using a random sample of mobile homes from mobile home parks in 21

Wisconsin. Sixty

- one teenage and adult residents participated. Health questionnaires were self

22

administered by each occupant. Respondents were blinded to the results of their home 23

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

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

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

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

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

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

statistically significant conc entration

- response relationship was reported in graphical form, 30

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

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

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

eye irritation with 12

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

(1984) showed approximately 17.5% predicted prevalence of burning eyes. T he prevalence of 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 4 burning eyes rose linearly to approximately 65% prevalence at 0.5 ppm, with some diminishment 1

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

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

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

nearly an identical distribution of formaldehyde concentrations in participating and 5

nonparticipating homes. Demographic char acteristics of some of the non

- respondents were 6

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

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

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

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

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

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

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

1991; Thun et al., 1982; Dally et al., 1981). Dally et al. (19 81) collected data in 100 “complaint 14

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

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

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

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

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

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

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

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

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

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

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

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

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

Eye irritation was positively ass ociated with formaldehyde with a clear concentration response 28

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

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

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

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

by formaldehyde exposure and chronic disease prevalence. Thun et al. (19 82) reported increased 33

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

insulated with urea

- formaldehyde foam relative to nearby homes without urea foam 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 5 formaldehyde insulation (UFFI); however, there were no measurem ents of formaldehyde 1

concentration taken in this study. 2

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

occupational exposures within mobile and non

- mobile daycare centers. They reported the mean 4

concentration in mobi le and non

- mobile day care centers were 350 (200

– 450) ppb and 65 (40

– 5

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

workers (n = 70) in the mobile units as compared with those in non

- mobile units (n = 34 ). The 7

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

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

percentages among those working in non

- mobile units. 10

11

4.1.1.1.1.2. Occupational epidemiology.

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

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

mean concentration of 0.40 mg/m 3 . Alexandersson and He denstierna (1988) reported that the 23

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

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

with 18 controls (nonexposed indi viduals working at the same factory). The frequency of eye 26

irritation was 65.8% among those exposed and 16.7% among controls. No controls reported 27

nose/throat irritation, but about 40% of those exposed did. 28 Horvath et al. (1988) compared irritation symptoms 12

between 109 workers at a particleboard manufacturing plant and 264 workers at food plants as a 13

control group. The mean 8

- hour time

- weighted average (TWA) formaldehyde concent rations 14

between these two groups were 0.69 ppm (range 0.17

– 2.93) and 0.05 ppm (range 0.03

– 0.12), 15

respectively. Eye, nose, and throat irritation were more common among the former group 16

(prevalence of symptoms during a work shift: throat sore or burning

— tes t 22.0%, controls 3.9%; 17

cough

— test 34.9%, controls 18.9%; burning of nose

— test 28.4%, controls 2.0%; stuffy nose

— 18

test 33.9%, controls 14.2%; itching of nose

— test 21.1%, controls 7.9%; eyes burning or 19

watering

— test 39.5%, controls 9.1%; eyes itching

— test 19 .3%, controls 7.1%). 20

A Swedish study conducted at a chemical plant f ound nasal and eye discomfort were 29

reported by 64 and 24%, respectively, of workers (n = 70) exposed to formaldehyde (range 0.05

– 30

0.50 mg/m 3 with a mean of 0.26 mg/m 3 ) versus 25 and 6%, respectively, of the control group 31

made up of clerks from the local go vernment (n = 36). In addition, the majority of workers 32

exposed to formaldehyde reported that their symptoms were relieved during weekends and 33

vacations (Holmström and Wilhelmsson, 1988). Another study by the same authors 34

(Wilhelmsson and Holmström, 1992 ) reported similar results. In this study irritation prevalent 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 6 among 66 workers from a formaldehyde

- producing plant was compared with that seen among 1

36 community clerks. The workers were exposed to 0.26 mg/m 3 of formaldehyde (range 0.05

– 2

0.6 mg/m 3 ). The clerks were exposed to an average of 0.09 mg/m 3 . Nasal and eye discomfort 3

were reported at rates of 53 and 24%, respectively, among the workers. Among the community 4

clerks, 3 and 6%, respectively, reported discomfort. 5

Holness and Nethercott (1989) re ported significant increases in eye irritation (42 versus 6

21%) and nose irritation (44 versus 16%) among 84 funeral service workers as compared with 7

38 controls (students and individuals from a service organization). The former group had been 8

actively emb alming for approximately 10 years and had nearly twice the pack

- years smoked as 9

the controls. The exposure concentration in both groups was 0.36 and 0.02 ppm, respectively. 10

11

4.1.1.1.1.3. Epidemiology on laboratory students

In a study of 24 formaldehyde

- exposed anatomy students (personal breathing zone 17

samples 0.73 ppm, range 0.49

– 0.93), the prevalence of eye irritation befor e the start of a cadaver 18

dissection class was 16%, while after the class, the prevalence was 59%. The increase in eye 19

irritation was most pronounced, but increases were also observed in the prevalence of irritation 20

of the nose (21%) and throat (15%) (Krie bel et al., 1993). The authors also reported a tendency 21

for this increase in intensity between the beginning and end of class to diminish over the 22

10

- week course, especially for eye irritation. However, although the intensity of the irritation 23

diminished , eye irritation was still present among the students after 10 weeks of intermittent 24

exposure. The report of increase in post

- versus pre

- class irritation symptoms in this study was 25

no greater for asthmatic students (n = 5) compared with non

- asthmatic stu dents. 26 . Several studies have monitor ed sensory 12

irritation in medical/physical therapy students exposed to formaldehyde during anatomy courses. 13

These studies have particular advantages: the student population generally has no former 14

occupational exposure, and, oftentimes, pre

- class survey da ta serve as the control, providing a 15

better basis for assessing the effects of formaldehyde exposure. 16

Takahashi et al. (2007) showed that 143 medical students reported various symptoms 27

(including eye and throat irritation) and that the percentage of students reporting symptoms 28

increased between the beginning and end of the course 2 months later. After the first day of 29

class, approximately 35% of students reported eye soreness and about 15% reported throat 30

irritation. After the course ended, these rates were close to 70% for eye soreness and slightly 31

above 40% for throat irritation. The reported average room formaldehyde concentration was 32

2.12 ppm (range 1.7

– 2.4), while the gas samplers worn on the students’ chests averaged 2.4 ppm 33

(range 1.8

– 3.8). Another study of students in an anatomy laboratory class in Japan (Takigawa et 34

al., 2005) measured formaldehyde concentrations and irritation symptoms before and after the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 7 installation of a ventilation system. This system reduced the median personal formaldehyde 1

exposure concentration from 2.7 to 0.72 ppm. Before installation of the ventilation system , the 2

students complained about exacerbation of all the sensory irritation symptoms on average. The 3

increase in 8 out of 25 symptoms was significantly reduced after installing general ventilation 4

( p < 0.05). After installation of the ventilation system, a dose

- dependent relationship with 5

formaldehyde was seen for irritated eyes but not for itchy nose. 6

Akbar

- Khanzadeh et al. (1994) detected mean personal area levels of formaldehyde at 7

1.24 ppm and a range of 0.1

– 2.94 ppm from personal air sampling device s. Almost 90% of the 8

students in this study reported eye irritation, 74% reported nose irritation, and close to 30% 9

reported throat irritation during or after exposure to formaldehyde during the laboratory period 10

after having completed at least 6 weeks of laboratory sessions with formaldehyde exposure. In 11

addition, Uba et al. (1989) demonstrated that symptoms of eye, nose, and throat irritation were 12

correlated with formaldehyde exposure among medical students by comparing students’ 13

responses on a question naire completed after a lab with formaldehyde exposure to a 14

questionnaire completed after a lab with no formaldehyde exposure. The authors compared 15

questionnaires completed prior to students’ first anatomy lab to a questionnaire completed 16

7 months later. Reports of cough were more frequent after the 7 months. These students were 17

exposed to a mean level of 1.9 ppm (range 0.1

– 5.0) while dissecting (measured using portable 18

infrared spectrophotometer), and a TWA from all laboratory activities ranged from bel ow limits 19

of detection to 0.93 (measured using personal sampling devices in the students’ breathing zones). 20

21

4.1.1.1.2. Acute studies: controlled chamber exposures. Results from controlled human studies 22

demonstrate eye, nose, and throat irritation in a ssociation with formaldehyde exposure (Lang et 23

al., 2008; Yang et al., 2001; Krakowiak et al., 1998; Kulle, 1993; Green et al., 1989, 1987; Kulle 24

et al., 1987; Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 25

1984; B ender et al., 1983; Anderson et al., 1983; Weber

- Tschopp et al., 1977; Andersen, 1979; 26

Schuck et al., 1966). A key advantage of chamber studies is the ability to monitor and closely 27

control formaldehyde concentrations during exposure. However, chamber st udies may also be 28

limited by other aspects of the study design, including small number of participants, use of 29

healthy volunteers, short exposure durations (a few minutes), and often studies were conducted 30

with only one exposure group and at relatively hig h concentrations (>1 ppm). The lack of 31

multiple exposure levels in many studies limits the understanding of exposure

- response 32

relationships. Additionally, numerous reports that demonstrate multiple symptoms of eye, nose, 33

and throat irritation at levels a t or above 1 ppm did not explore lower levels of exposure and can 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 8 only be used for primary hazard identification ( Yang et al., 2001; Green et al., 1989, 1987; 1

Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 1984 ). 2

T he National Aeronautical and Space Administration conducted experiments in closed

3

environment living, including environmental monitoring and air quality. James et al. (2002) 4

quantified air pollutants, including formaldehyde, during 30, 60, and 90

- day test s in a closed 5

chamber study of a Lunar

- Mars life support chamber. Unfortunately, the detection methods used 6

during the 30

- day test were not sensitive enough to detect formaldehyde at levels below 2 7

mg/m 3 . Thus, badge samples were obtained in the 60

- day a nd 90

- day tests and provided greater 8

detection sensitivity (to 0.02 mg/m 3 ). Measured values of formaldehyde increased over time. In 9

the 60

- day test, formaldehyde levels were well above accepted limits (data not shown). Health 10

effects data are limited si nce there were only four crew members. One crew member reported 11

eye and upper airway irritation at formaldehyde concentrations of 0.25 mg/m 3 (308 ppb) on day 12

15. It should also be noted that astronauts are exceptionally healthy individuals, and these dat a 13

should be interpreted carefully when determining expected health effects in the general 14

population. The experimenters determined that formaldehyde levels increased as temperature 15

increased. Formaldehyde was also linked to murals lining the chamber and was subsequently 16

removed before executing the 90

- day study. Between days 0 and 60, formaldehyde levels 17

remained between 0.02 and 0.04 mg/m 3 , with one sharp peak that occurred at day three to 0.07 18

mg/m 3 . Between days 60 and 90, formaldehyde concentrations increased to 0.07 to 0.09 mg/m 3

. 19

The increase was attributed to an incomplete oxidation of methanol in a catalytic bed rather than 20

in excessive off

- gassing of formaldehyde. No crew members reported any adverse effects in the 21

90

- day study. 22

A few studies have been conducted that specifically address sensitive populations 23

(asthmatics) and/or individuals during exercise, which can exacerbate asthma (further details of 24

these studies are in Section 4.1.1.3, Effects on Asthmatics). In Sauder et al. (1986), 8

- minute 25

bicycle exercise was completed multiple times during the exposure period (3 hours). However, 26

irritation symptoms were only reported after 2 hours of exposure and do not address whether 27

changes occurred during the periods of exercise. Overall, repo rts of eye, nose, and throat 28

irritation increased with exposure to formaldehyde (3 ppm) compared with reports of irritation 29

with no exposure to formaldehyde. Green et al. (1987) report that eye, nose, and throat irritation 30

symptoms were greater immediatel y after exercise during exposure to 3 ppm formaldehyde. 31

Additionally, the response levels were similar between asthmatic (n = 16) and non

- asthmatic 32

(n = 22) subjects. Similar effects of exercise on certain symptoms, such as throat irritation, were 33

report ed in 15 asthmatic subjects exposed to 2 ppm formaldehyde at rest and after exercise 34

(Witek et al., 1987). 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 9 Kulle (1993) and Kulle et al. (1987) enrolled 19 healthy volunteers and exposed them to a 1

range of formaldehyde concentrations. At 2 ppm, 53% report ed mild or moderate eye irritation 2

(32% mild, 21% moderate). At 3 ppm, 100% of subjects exposed at this level (n = 9) reported 3

irritation. The reported increase in irritation was shown to correspond with increasing 4

formaldehyde concentration in a linear fashion. Mild nose/throat irritation was present among 5

37% of those exposed to 2 ppm of formaldehyde. Odor detection was very similar to the 6

distribution seen for eye irritation. Nineteen subjects performed light to moderate exercise while 7

exposed to 2 ppm; there was no increase in report of eye irritation, but nose/throat irritation did 8

increase. The data were reanalyzed (Kulle, 1993), and thresholds for irritation were found to be 9

0.5

– 1 ppm for eye irritation and 1 ppm for nose/throat irritation. 10

Yan g et al. (2001) reported that eight individuals exposed to varying levels of 11

formaldehyde (1.65, 2.99, and 4.31 ppm) had mild to moderate eye irritation during the 5

- minute 12

exposures. The increase in irritation was detected at 30 seconds with exposure to 1.65 ppm of 13

formaldehyde. The highest severity ratings at this concentration occurred between 60 and 14

90 seconds. Frequency of eye blinking was also measured. The peak in blinking rate occurred 15

after about 1 minute of exposure and then decreased almost b ack to a normal rate after 5 minutes 16

of exposure. Higher formaldehyde concentrations were associated with increased frequency of 17

blinking compared with the 1.65 ppm exposure. 18

Other studies have examined responses across multiple exposure levels. For exam ple, 19

Weber

- Tschopp et al. (1977) used two different methods of studying irritation resulting from 20

formaldehyde exposure. For one, they exposed subjects (n = 33) to an increasing level of 21

formaldehyde (maximum exposure was 3.2 ppm). This design precluded evaluation of distinct 22

effects at different exposure levels. The researchers addressed this by examining another group 23

of subjects (n = 48) that were exposed to 0, 1, 2, 3, or 4 ppm five times for 90 seconds. Levels of 24

nasal and throat irritation for thi s discontinuous exposure were slightly higher than the irritation 25

levels reported among those with continuous exposure. However, this was reversed for eye 26

irritation; those with continuous exposure reported higher levels of irritation than those with 27

disc rete exposures. An objective measure, eye

- blinking rate, was measured for those with 28

continuous exposure and was found to have a statistically significant increase at 1.7 ppm. 29

Bender et al. (1983) conducted a study that enrolled individuals who “responde d” to 30

formaldehyde at 1.3 and 2.2 ppm and did not report irritation to the clean air control. They 31

found that, among these subjects, exposure to 1 ppm of formaldehyde (n = 27) resulted in the 32

reporting of eye irritation with a median response time of 78 s econds. Reports of irritation were 33

given as less than slightly irritating for formaldehyde concentrations of 0.3

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

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- 10 Assessment of sensory irritation for pain and discomfort often relies on self

- reporting, 1

using symptom questionnaires and severity ra tings (e.g., mild, moderate, severe). In the case of 2

formaldehyde, subjective ratings of eye irritation correlate positively with eye

- blinking 3

frequency (Lang et al., 2008). Lang et al. (2008) saw an increase in eye blinking after 4

195 minutes of exposure to formaldehyde at 0.5 ppm with four peak exposures of 1 ppm. After 5

this amount of time and formaldehyde exposure, there was also an increase in moderate eye 6

redness. Weber

- Tschopp et al. (1977) reported that, among concentrations ranging from 0.03 to 7

3 .2 ppm, eye

- blinking frequency was increased at 1.7 ppm; similarly Yang et al. (2001) reported 8

increased blinking at >1.5 ppm (the lowest concentration examined). There are studies that 9

suggest that psychological factors (e.g., anxiety) can impact the per ception of irritation

— and 10

perhaps more so at lower concentrations (Lang et al., 2008; Ihrig et al., 2006; Dalton, 2003). 11

However, when Lang et al. (2008) controlled for mood prior to exposure, subjective symptoms 12

of eye, nasal, and olfactory irritation we re significantly related to exposure (0.5 ppm) 13

Schuck et al. (1966) performed a study that also examines self

- reported eye irritation as 14

well as blinking rate. Fourteen individuals were exposed to formaldehyde concentrations 15

ranging from 0 to 1 ppm. Inc reased irritation was reported with increasing formaldehyde 16

concentration. One subject, judged to be the least sensitive, was still able to detect formaldehyde 17

levels as low as 0.01 ppm. In addition, the authors examined the blinking rate of participants

, 18

which they found was related to irritation intensity. 19

Andersen (1979) and Anderson and Molhave (1983) reported on a controlled experiment 20

in which 16 individuals were exposed to varying levels of formaldehyde for five hours and rated 21

their level of disco mfort over the exposure period. Discomfort occurred within 1 hour at 22

formaldehyde exposure levels of 1 and 2 mg/m 3 (Andersen and Molhave, 1983) After 2 hours, 23

increasing discomfort was reported among the groups exposed to 0.3 and 0.5 mg/m 3 . Subject 24

repor ted that discomfort was mainly conjunctival irritation and dryness in the nose and throat. 25

Subjects complained at all four concentrations of formaldehyde: 0.3, 0.5, 1.0, and 2.0 mg/m 3 and 26

of 16 subjects, 3, 5, 15, and 15 subjects complained at each respec tive exposure concentration 27

(Andersen and Molhave, 1983). 28

Controlled chamber studies have also been conducted on various populations of 29

previously exposed individuals to determine if formaldehyde exposure potentiates an 30

individual’s response to acute expo sures. Schachter et al. (1987) reported on 15 laboratory 31

workers “frequently exposed to formaldehyde” (no quantification of exposure is given; however, 32

the workers report being exposed for 1 to 7 days per week from a range of 1 to 21 years). Tests 33

perfor med at the start of the study found that these individuals had pulmonary function similar to 34

that seen in healthy individuals. The workers in this study reported subjective measures of eye, 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 11 nose, and throat irritation after 40 minutes of exposure to 2 ppm of formaldehyde. However, the 1

2 ppm acute exposure in this study may be sufficiently high to induce significant irritation in 2

most individuals. Krakowiak et al. (1998) reported that 10 asthmatics with occupational 3

exposure to formaldehyde (via formaldeh yde solutions or pure gaseous formaldehyde) exhibited 4

similar symptom scores to healthy controls (never exposed to formaldehyde in the workplace) 5

exposed to 0.4 ppm formaldehyde for 2 hours. The mean symptom scores and standard 6

deviation (SD), which inclu ded information on sneezing, rhinorrhea, mucosal edema, and 7

itching, were 4.6 ± 1.6 (mean ± SD) for asthmatics and 4.3 ± 1.2 for healthy subjects 8

immediately after inhalation. These dropped to 1.8 ± 1.2 and 1.2 ± 1.3, respectively, 4 hours 9

after the expos ure. It is unclear if sensitive individuals may not be represented in either of these 10

groups, as the workers were tolerating their exposures during the work shift “healthy worker” 11

effect. However, residents (n = 9) exposed to formaldehyde in their homes, who complained 12

about adverse effects from the material, but with no occupational exposure reported eye, nose, 13

and throat irritation at a similar rate as controls (individuals in homes without formaldehyde or 14

individuals in homes with formaldehyde but not reporting adverse effects [n = 9]) after a 15

90

- minute exposure to 1 ppm (Day et al., 1984). The number of individuals reporting eye 16

irritation, nasal congestion, and throat irritation were seven, three, and two among sensitive 17

individuals and eight, four, and three among controls, respectively. These individuals may be 18

considered a sensitive population since they had “previously complained of various 19

nonrespiratory effects from the UFFI in their homes” (household concentrations unknown). 20

21

4.1.1.2. Pulmon ary Function 22

Workers chronically exposed to formaldehyde have exhibited signs of reduced lung 23

function consistent with BC, inflammation, or chronic obstructive lung disease. Lung function 24

deficits have been reported both in pre

- shift versus post

- shift me asurements and as a result of 25

chronic exposures (Pourmahabadian et al., 2006; Herbert et al., 1994; Malaka and Kodama, 26

1990; Alexandersson and Hedenstierna, 1989; Alexandersson et al., 1982). Decreases in 27

spirometric values, including vital capacity (VC), forced expiratory volume (FEV), forced vital 28

capacity (FVC), and FEV/FVC have been reported. Chronic studies also report increased 29

respiratory symptoms, such as cough, increased phlegm, asthma, chest tightness, and chest colds, 30

in exposed workers (Pourma habadian et al., 2006; Herbert et al., 1994; Malaka and Kodama, 31

1990; Alexandersson and Hedenstierna, 1989; Alexandersson et al., 1982). Similar findings 32

have been reported for low

- level residential formaldehyde exposure, including decreased peak 33

expirato ry flow (PEF) rates (Krzyzanowski et al., 1990). 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 12 Worker exposures that report cross

- shift differences in spirometric values are consistent 1

with formaldehyde

- induced sensory irritation. Additionally, concordance has been reported 2

between subjective irrita nt response and measured changes in pulmonary function, further 3

supporting the possibility that cross

- shift and short

- term evidence of BC may be a reflexive 4

response to sensory irritation. Absolute values for lung function parameters are likely to vary by 5

gender, age, height, and smoking status and are best compared when normalized to the expected 6

lung function based on these variables (Schoenberg et al., 1978). Individual variation can also be 7

addressed by each subject serving as his/her control with mea surements taken before, during, and 8

after exposure. Analysis of the percent change in various parameters in this context may have 9

greater sensitivity to detect exposure

- related changes in function. 10

In addition to individual variation in baseline lung func tion, there is variation in bronchial 11

responsiveness. Reduced lung function parameters in response to methacholine challenge is a 12

standard test for BC, and this can be used to define responsive, sensitive, or susceptible 13

individuals. Since formaldehyde

- i nduced BC is measured with these lung function tests, 14

variability in bronchial responsiveness may impact interpretation of formaldehyde

- induced 15

changes. Experiments with sensitive individuals can help address this question. However, 16

results need to be no rmalized in some way to account for differences in responsiveness before 17

formaldehyde exposure. Researchers have in some cases excluded hyperresponsive individuals 18

or presented results as a proportion or percent of the unexposed value for each individual. 19

However, excluding sensitive individuals may bias results towards the null. 20

The American Thoracic Society (ATS) published an official statement on what 21

constitutes an adverse health effect of air pollution (ATS, 2000). According to the ATS 22

statement, e xposure that increases the risk of an adverse effect to the entire population can be 23

considered adverse, even though it may not increase the risk of any individual to an unacceptable 24

level. For example, a population of asthmatics could have a distribution of lung function such 25

that no individual has a level associated with significant impairment. Exposure to an air 26

pollutant could shift the distribution to lower levels that still do not bring any individual to a 27

level that is associated with clinically re levant effects. However, this would be considered to be 28

adverse because individuals within the population would have diminished reserve function and 29

therefore would be at increased risk if affected by another agent. 30

31

4.1.1.2.1. Epidemiologic literature. The potential adverse effects of formaldehyde exposure on 32

pulmonary function in humans can be examined on several time scales of interest. The 33

epidemiologic literature supports the assessment of exposures among exposed anatomy medical 34

students where all participants have well

- defined and similar duration of exposure (i.e., a 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 13 semester

- long class) (Kriebel et al., 2001, 1993; Akbar

- Khanzadeh and Mlynek, 1997; Akbar

1

Khanzadeh et al., 1994; Uba et al., 1989; Fleisher, 1987), among individuals living or worki ng in 2

buildings with formaldehyde exposure (Franklin et al., 2000; Krzyzanowski et al., 1990; Main 3

and Hogan, 1983), and among workers (industrial, manufacture, mortuary, hospital staff, etc.) 4

( Ostojic et al., 2006; Herbert et al., 1994; Khamgaonkar and Fu lare, 1991; Malaka and Kodama, 5

1990; Nunn et al., 1990; Alexandersson and Hedenstierna, 1989; Holness and Nethercott, 1989; 6

Holmström and Wilhelmsson, 1988; Horvath et al., 1988; Kilburn et al., 1985; Alexandersson et 7

al., 1982). 8

The observed effects in th e previously unexposed anatomy students provide additional 9

information on acute exposures in two naïve populations (Kriebel et al., 2001, 1993) as well as 10

special insight into the intermediate stages of possible sensitization (Kriebel et al., 1993). 11

Krieb el and colleagues (1993) examined the pre

- laboratory and post

- laboratory PEF in students 12

attending anatomy classes once per week. They found the strongest pulmonary response when 13

examining the average cross

- laboratory decrement in PEF in the first 2 weeks of the study when 14

formaldehyde concentrations collected in the breathing zones had a geometric average 15

concentration of 0.73 ppm. Overall, the students exhibited a 2% decrement in PEF, while the 16

students with any history of asthma showed a 7.3% decrement in PEF. These findings of acute 17

decreases in PEF following students’ initial anatomy sessions were corroborated by the Kriebel 18

et al. (2001) study, which used a similar study design applied to another class of anatomy 19

students. 20

The Kriebel et al. (1993) study also shows how the acute effects of formaldehyde 21

exposure were altered following several weeks of weekly episodic exposure. By the fifth week 22

of class, the pre

- and post

- laboratory measurements of PEF were no longer reflecting a clearly 23

demonstrated acute effect, but, following the seventh week of episodic exposure, both pre

- and 24

post

- laboratory PEF continued to drop steadily until the class adjourned after 10 weeks. While 25

the acute effects of formaldehyde exposure appeared to diminish after several weeks of exposure, 26

the intermediate effect across 9 weeks was a 24 L/minute drop in PEF that was statistically 27

significant ( p < 0.01 after statistical control for random person effects, asthma, interaction 28

between time and asthma, and eye as well as nose symptoms of irritation). 29

Similar studies among medical students have been performed. In one study, 34 exposed 30

and 12 control students completed pulmonary function tests before and after their work in the 31

laboratory (approximately 3 hours) (Akbar

- Khanzade h et al., 1994). The time

- weighted average 32

exposure ranged from 0.07

- 2.94 ppm. More than 94% of the subjects were exposed to >0.3 ppm 33

and 31.7% were exposed to >0.5 ppm. Comparing pre

- and post

- exposures among the exposed 34

students, on average FVC decrea sed by 1.4%, FEV 3 decreased by 1.2%, FEV 1 /FVC increased by 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 14 1.6%, and FVC 25

– 75% increased 2.5%. These average percent changes in the control group are

– 1

0.3, 1.30, 2.31, and 0.6%, respectively. The researchers also calculated correlation coefficients 2

by ex amining the relationship between lung function and formaldehyde concentration, but no 3

association was found. Akbar

- Khanzadeh and Mlynek (1997) performed another study with 50 4

exposed students and 36 controls and reported a larger increase in lung function among controls 5

when compared with cases after 1

– 3 hours of exposure (FVC 3.0 versus 0.9, FEV 1 4.1 versus 6

1.2, FEV 3 3.3 versus 0.8, forced expiratory flow during the middle of the FVC [FEF 25

– 75% ] 6.1 7

versus 0.7). These differences between cases and contro ls remained for FEV 1 , FEV 3 , and FEF 25

– 8

75% after 3 hours. 9

A study of 103 medical students was performed over a period of 7 months in which the 10

students were exposed to formaldehyde at a time weighted average of <1 ppm with peaks of 5 11

ppm during anatomy la boratory sessions (Uba et al., 1989). Twelve students were asthmatics. 12

Unlike the studies by Kriebel et al. (2001, 1993), these researchers did not find a change in 13

pulmonary function over the course of 7 months. The mean percent change for pulmonary 14

fu nction before and after the exposure did change slightly, with measures showing decreases in 15

function at the end of the laboratory session (measurements taken at the 7

- month time point: 16

FVC

– 0.79%, FEV 1

– 0.48%, FEF 25

– 75% 0.07%, FEV 1 /FVC 0.24%). 17

Finally, Fl eisher (1987) gave self

- administered questionnaires to medical students after 18

completing an anatomy laboratory session (formaldehyde exposure measures as <1 ppm) and a 19

pathology/microbiology laboratory session (no formaldehyde exposure). Over 8% of studen ts 20

reported experiencing shortness of breath during the laboratory with formaldehyde exposure, but 21

none of the students reported shortness of breath in the laboratory session with no exposure. No 22

objective measurements of formaldehyde exposure were used. 23

Three studies have been performed that examine formaldehyde exposure from the 24

buildings in which individuals live or work. One study included children 6

– 13 years of age and 25

measured the levels of formaldehyde in their homes. There was no association betw een FVC or 26

FEV and the indoor concentrations of formaldehyde, although there were signs of lower airway 27

inflammation as measured by levels of exhaled nitric oxide (NO) in children exposed to average 28

formaldehyde levels ≥0.05 ppm (Franklin et al., 2000). M unicipal employees with their children 29

(613 adults and 298 children) were randomly sampled in another study of home exposures 30

(Krzyzanowski et al., 1990). Residential exposures to formaldehyde were based on repeated 31

samples from each individual’s kitchen, living area, and bedroom. The average formaldehyde 32

concentration was 26 ppb, with a maximum sample value of 140 ppb. The majority of subjects 33

(83%) lived in homes with 2

- week average concentrations below 40 ppb. Subjects’ peak 34

expiratory flow rates (PE FRs) were determined four times daily, in the morning, at noon, in the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 15 early evening, and before bed, for 2 weeks. A statistically significant linear relationship between 1

increased formaldehyde exposure and decreased PEFR was reported in children but not adults. 2

All statistical models controlled for socioeconomic status, tobacco smoking (current active or 3

environmental tobacco smoking), and nitrogen dioxide concentrations. Among adults, there was 4

a statistically significant nonlinear relationship with de creased morning PEFR for formaldehyde 5

concentration <40 ppb. 6

Main and Hogan (1983) reported on a group of individuals (n = 21) working in two 7

mobile trailers for 34 months and exposed to levels of formaldehyde ranging from 0.12 to 8

1.6 ppm (mean age 38 ± 9 years, 76% male, 19% nonsmokers). The control population was 9

comprised of individuals who did not work in the trailers (n = 18; mean age 30 ± 6, 50% male, 10

22% nonsmokers). There were no differences between the exposure and control groups’ percent 11

pred icted FEV 1 or FVC regardless of smoking status. 12

Several studies allowed for the examination of potential chronic effects of formaldehyde 13

exposure. These included an occupational study by Malaka and Kodama (1990) that reported 14

pre

- shift pulmonary function as a percentage of expected among the formaldehyde exposed 15

compared with comparable people not exposed to formaldehyde. This study found that an 16

average 8

- hour TWA formaldehyde exposure of 1.13 ppm from area samples was associated 17

with statistically signi ficant decrements in FEV 1 , FEV 1 /FVC, and FEF 25

– 75% compared with a 18

referent population. The strongest response was for FEF 25

– 75%, which showed a 12% drop in 19

observed function compared with expected function in the unexposed, but it is unclear how to 20

inter pret the potential chronic adverse effect(s) with just the magnitude of the decrement and the 21

length of the average occupational tenure at this plywood facility (6.5 years), which was not 22

reported by exposure status. 23

A study comparing oriented strand boa rd workers (exposed to formaldehyde) with oil/gas 24

field plant workers (not exposed to formaldehyde) demonstrated a difference in pulmonary 25

function between the two groups (Herbert et al., 1994). The groups were similar in regard to 26

measured FVC and FEV 1 ( controlled for age, height, and smoking), but the workers exposed to 27

formaldehyde had lower FEV 1 /FVC. In addition, those exposed to formaldehyde showed a 28

decrease in FVC and FEV 1 after their shift, with an average pre

- and post

- shift difference of 29

47 mL ( p = 0.022) and 39 mL ( p = 0.044) for FVC and FEV 1 , respectively (however change 30

could not be compared with the controls of this study because no post

- shift measurements were 31

taken). Two other occupational studies found no association between formaldehyde and lung 32

function (Holness and Nethercott, 1989; Horvath et al., 1988). One of these studies was 33

conducted among funeral workers and an unexposed control group (Holness and Nethercott, 34

1989). There was no difference in pulmonary function of the two group s at baseline. After 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 16 exposure, there was no change in lung function for the exposed or unexposed when comparing 1

lung function tests done immediately before and after an embalming procedure (for controls the 2

repeat measures were taken approximately 2

– 3 hou rs after the first measure) (changes in 3

percentage predicted FVC and FEV 1 were 0.88 ± 2.95 and

– 0.03 ± 2.40 for exposed and 1.13 ± 4

3.98 and 1.45 ± 4.43 for unexposed). Further analysis showed no association between 5

formaldehyde levels and changes in lung function. Another study (Horvath et al., 1988) found 6

no differences in pre

- shift pulmonary function between the exposed (workers at a particleboard 7

and molded products operation, formaldehyde measured using individual monitors ranged from 8

0.17

– 2.93 ppm) a nd controls (workers from nearby food processing facilities, formaldehyde 9

measured using individual monitors ranged from 0.03

– 0.12 ppm). However, the authors did find 10

a post

- shift decline in FVC and FEV 1 among controls and FEV 1 and FEF 25

– 75% among workers 11

when using paired comparisons for each group. When assigning all controls a formaldehyde 12

exposure value of 0.05 ppm, there was a correlation detected in pre

- and post

- shift pulmonary 13

function changes and formaldehyde, though no specific details on regress ion analysis were 14

provided. 15

A study performed in India (Khamgaonkar and Fulare, 1991) examined individuals 16

working in anatomy and histopathology departments and exposed to formaldehyde (mean 17

1.00 ppm, range 0.036

– 2.27). Controls (individuals not working in laboratories with 18

formaldehyde) were exposed to an average of 0.102 ppm formaldehyde (range 0

– 0.52). Lung 19

function tests were performed on a Monday morning after days of no exposure in order to 20

examine chronic effects. The FVC and FEV 1 % of the expose d group, respectively, were 17.12 21

and 22.94% reduced compared with the control group (Khamgaonkar and Fulare, 1991); 22

however, while the pool of cases and controls were frequency

- matched on age and gender, there 23

was no mention by the investigators of normal izing the pulmonary function metrics by gender 24

and height, which would have made for more appropriate comparisons. Kilburn et al. (1985) 25

also demonstrated reduced pulmonary function (lower percent predicted FVC, FEV 1 , and 26

FEF 25

- 75% ) among workers occupati onally exposed to formaldehyde when compared with 27

individuals working at jobs without formaldehyde exposure. 28

Two occupational studies found no association between formaldehyde exposure and 29

deficits in pulmonary function (Ostojic et al., 2006; Holmström and Wilhelmsson, 1988). 30

Ostojic et al. (2006) examined nonsmoking male health service professionals working in 31

pathoanatomic laboratories with 8 hours of formaldehyde exposure per day at an unspecified 32

concentration for at least 4 years (n = 16). The contro l group was comprised of sixteen age

- and 33

stature

- matched nonsmoking male controls. There was no difference in mean FVC or FEV 1 34

between exposed and controls. The researchers also examined values for diffusing lung capacity 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 17 for carbon monoxide and membran e diffusion capacity, which were similar between the exposed 1

and control groups. However, blood volume of pulmonary capillaries was found to be higher in 2

the exposed group. Holmström and Wilhelmsson (1988) recruited individuals from a chemical 3

plant wher e formaldehyde and formaldehyde products were made (n = 70). Exposure levels 4

varied from 0.05

– 0.5 mg/m 3 . A control group was mostly comprised of clerks for the local 5

government (n = 36). No difference in FEV% was detected between the groups. Mean FVC w as 6

lower than expected among the exposed group (expected values were based on age, sex, smoking 7

habits, height, and weight). This study went further and measured changes in pulmonary 8

function for those employed more than 5 years and reported no signs of i ncreasing restrictivity 9

after 5 years. No associations were seen and there was no correlation between pulmonary 10

function and cumulative dose of formaldehyde (Holmström and Wilhelmsson, 1988). 11

There have been only two studies that have reported on the lo ngitudinal follow

- up of 12

workers exposed to formaldehyde (Nunn et al., 1990; Alexandersson and Hedenstierna, 1989). 13

The Alexandersson and Hedenstierna (1989) investigation not only examined the acute effects of 14

exposure across shift but was able to do so a mong some of the same workers that had been 15

studied 5 years earlier (Alexandersson et al., 1982). Statistically significant decreases in 16

FEV 1 /FVC and FEF 25

– 75% were noted over the intervening 5 years in nonsmokers after correction 17

for normal aging and ref erence lung function spirometry values. The decrease in FEF 25

– 75% was 18

0.212 ± 0.066 L/second (mean ± SD) for each year of exposure and was highly significant ( p < 19

0.01). For comparison with the 12% drop in the same pulmonary metric reported by Malaka and 20

Kodama (1990) over an estimated 6.5 years, the extrapolated percentage decrease in FEF 25

– 75% 21

was computed for the Alexandersson and Hedenstierna (1989) study by using the reported yearly 22

decrement applied to the pre

- shift values at the time of the initial study period. From the 23

predicted value of 4.57 L/second, a decrease of 0.168 L/second for each year of exposure 24

regardless of smoking status was calculated. For 6.5 years of exposure, this would result in a 25

24% drop in FEF 25

– 75% . Formaldehyde concentra tions were estimated at 0.42 ppm in the first 26

Alexandersson et al. (1982) study and at 0.50 ppm in the Alexandersson and Hedenstierna (1989) 27

study. The study by Nunn et al. (1990) assessed the decrease in FEV 1 with age. The researchers 28

calculated the dec rease in FEV 1 to be 42 mL/year among workers exposed to formaldehyde and 29

41 mL/year for workers who were not exposed to formaldehyde. Thus, they showed no 30

association between formaldehyde exposure and decreased FEV 1

. 31

There are a few important limitation s to consider in these occupational studies of 32

formaldehyde exposure. First, an often

- shared weakness is the absence of data on, and 33

appropriate statistical control of, potential confounding by occupational co

- exposures. Also, 34

studies that did not report pre

- shift pulmonary function as a percentage of expected function 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 18 contribute less to an assessment of potential chronic effects because, post

- hoc, it is difficult to 1

calibrate the multiple pulmonary function data for cross

- study comparison without knowled ge of 2

the age, gender, smoking status, height, year of birth, etc., that are important determinants of the 3

pulmonary function metrics of concern. 4

5

4.1.1.2.2. Acute studies: controlled chamber exposures. Pulmonary effects of acute 6

formaldehyde exposure ha ve been studied in both healthy volunteers and sensitive populations 7

under controlled conditions. Controlled chamber studies have the advantage of measured 8

controlled exposures, but other factors can limit the usefulness of the studies, especially when 9

st udy populations are small and there is high variability in the measured parameters. 10

In general, acute formaldehyde exposures (0.5

– 3 ppm) have not induced significant 11

pulmonary deficits in healthy, non

- exercising volunteers (Kulle et al., 1987; Schachter et al., 12

1986; Witek et al., 1986; Day et al., 1984; Andersen and Molhave, 1983). However, it is unclear 13

whether the data analysis in these reports had the statistical power to substantiate the small 14

deficits reported in occupational and student studies. All four reports had relatively small study 15

groups of healthy individuals (n = 19 [Kulle et al., 1987], n = 16 [Andersen and Molhave, 1983], 16

n = 15 [Schachter et al., 1986], n = 15 [Witek et al., 1986], and n = 9 [Day et al., 1984]), and in 17

some cases the group was further divided by gender. Two studies report the absolute values of 18

the lung function parameters without adjustment to individual expected function or the 19

unexposed baseline for each individual (Kulle et al., 1987; Andersen and Molhave, 1983). As 20

discussed, this decreases the power of the study to detect formaldehyde

- induced changes in 21

pulmonary function. In contrast, Witek et al. (1986) and Schachter et al. (1986) report lung 22

function as a percent of baseline (although not normalized for age gender and height). Each 23

study showed an increase in FEV 1 in formaldehyde

- exposed individuals at rest and increases in 24

maximal expiratory flow (MEF) at 50% of expired vital capacity ( MEF50%) (Witek et al., 1986; 25

Schachter et al., 1986). However, in both reports the SDs of changes in lung function parameters 26

are quite large, nearly equaling the reported value and exceeding it in several cases. The absence 27

of normalized raw data, combined with large individual variation, limit the interpretation of these 28

s tudies. A small study (Day et al., 1984) that included nine healthy individuals showed no 29

changes in FEV or FVC after 90 minutes of exposure to 1 ppm of formaldehyde. A more recent 30

study (Lang et al., 2008) of 21 healthy volunteers exposed to a range of formaldehyde 31

concentrations (0.0 to 0.15 or 0.3 ppm) reports no formaldehyde

- related pulmonary deficits. 32

However, data were not shown, and it is unclear whether the authors compared absolute or 33

relative values of lung function and what variation in lung f unction was present in the study 34

population. Additionally, the authors did not provide the criteria used to gauge deficit of lung 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 19 function. If a clinically significant deficit was defined (e.g., 20%), then more subtle changes in 1

pulmonary function, as su pported in other studies, would not have been reported. 2

Similar to these studies of healthy individuals, acute controlled studies including 3

asthmatics also report no changes in pulmonary function associated with formaldehyde exposure 4

(Ezratty et al., 200 7; Harving et al., 1990; Green et al., 1987; Sauder et al., 1987; Witek et al., 5

1987, 1986), including studies of individuals thought to have formaldehyde

- induced bronchial 6

asthma (Krakowiak et al., 1998); however, the number of asthmatic individuals inclu ded in each 7

of these studies was small. (The details of these studies have been reported elsewhere in this 8

chapter. Briefly, the number of asthmatics in the study/total number of individuals in the study 9

are as follows: Ezratty et al. [2007]

— 12/12; Harvi ng et al. [1990]

— 15/15; Green et al. [1987]

— 10

16/38; Sauder et al. [1987]

— 9/9; Witek et al. [1987]

— 15/15; Witek et al. [1986]

— 15/30; 11

Krakowiak et al. [1998]

— 10/20]. The same is true for individuals who are frequently exposed to 12

formaldehyde either at work ( n = 15) (Schachter et al., 1987) or at home (n = 18) (Day et al., 13

1984). 14

Small but statistically significant deficits in pulmonary function due to acute 15

formaldehyde exposure (2 or 3 ppm) have been reported in healthy volunteers during exercise 16

(Green et al., 1989, 1987; Sauder et al., 1986; Schachter et al., 1986). Although changes in lung 17

function parameters averaged over experimental groups were generally small, some individuals 18

exhibited clinically significant deficits, even after only 2 hours of expo sure. Deficits in FEV 1 19

and FEF 25

– 75% in the first 30 minutes of a 2

- hour exposure at 3 ppm formaldehyde were 2 and 20

7%, respectively. Changes in lung function were not statistically significant after 60 and 21

180 minutes of exposure (Sauder et al., 1986), e ven when assessed as absolute rather than 22

relative measurements. Thirteen percent (5 of 38 subjects) demonstrated formaldehyde

- induced 23

clinically significant deficits when exposed at 3 ppm during exercise (defined by Green et al. 24

(1987) as decrease in FEV 1 > 10% of control). 25

26

4.1.1.3. Asthma 27

A large number of studies have investigated the potential association between 28

formaldehyde exposure and a continuum of adverse health effects ranging from decrements in 29

pulmonary function to asthma. In general, epide miologic studies of adults have reported varied 30

results between null findings and positive findings. However, The National Research Council 31

concluded in its report on Formaldehyde that, “Formaldehyde has been shown to cause bronchial 32

asthma in humans” (NR C, 1981), citing numerous studies demonstrating the induction of asthma 33

following exposure to formaldehyde (Hendrick and Lane, 1975, 1977; Laffont and Noceto, 1961; 34

Nova and Touraine, 1957; Paliard et al., 1949; Popa et al., 1969; Sakula, 1975; Schoenberg and 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 20 Mitchell, 1975; Turiar, 1952; Vaughan, 1939). In a subsequent review article on formaldehyde 1

and the health effects that have been associated with it, Stenton and Hendrick (1994) reported on 2

formaldehyde and asthma in occupational settings and starkly describe the “...first detailed case 3

report of formaldehyde asthma confirmed by specific inhalation challenge test occurring in a 4

nursing sister on a renal dialysis unit. Her symptoms were suggestive of late asthmatic reactions 5

occurring 4 to 5 hours after heavy exposures. The occurrence of late reactions was confirmed in 6

a series of challenge tests that involved the painting of formalin onto cardboard pieces within a 7

confined space” (Stenton and Hendrick, 1994; Hendrick 1997). The results of the challeng e tests 8

are illustrated in Figure 4

- 1 . 9

10

11

12

13

14

Figure 4

- 1. Delayed asthmatic reaction following the inhalation of 15

formaldehyde after “painting” 100% formalin for 20 minutes. Challenge 2 16

was premedicated with inhaled betamethasone 200 μg. 17

Source: (Sten ton and Hendrick, 1994) 18

19

Five years later, the two nurses were re

- challenged with the nurse who had left the 20

dialysis unit having no response to the subsequent challenge while the nurse who had remained 21

working in the unit developed mild late asthmatic r esponse with peripheral blood eosinophilia 22

(Stenton and Hendrick, 1994; Hendrick et al., 1982). Stenton and Hendrick (1994) concluded 23

that these studies “provide clear evidence of formaldehyde’s ability to induce asthma” but no 24

indication of the exposure concentrations to induce it. In a follow

- up study of dialysis unit 25This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 21 staffers exposed to formaldehyde as a sterilizing agent, 8/28 people reported respiratory 1

symptoms and a prolonged increase in circadian rhythm of peak expiratory flow rate was seen in 2

one subject (Hendrick and Lane, 1983) implying an increase in airway responsiveness (Stenton 3

and Hendrick, 1994). It should be noted, however, that while there did appear to be a clear 4

response to formalin, it is not known what contribution to the response wa s attributable to 5

formaldehyde and what contribution might have been attributable to methanol. Further, while 6

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

do not offer information on the concentrations a t which adverse effects would expected in a 8

population. 9

There is at least one clinical study in humans that investigated whether exposure to a low 10

level of formaldehyde (500 **μ** g/m 3 ) would enhance inhaled allergen responses (Ezratty et al., 11

2007). Twelve subjects with intermittent asthma were exposed to either formaldehyde or 12

purified air in a double

- blind crossover study for 1 hour. Following exposure (8 hours), airway 13

respon siveness to methacholine challenge was measured. No significant effects on 14

methacholine

- induced bronchial hyperresponsiveness (BHR) were detected due to formaldehyde 15

exposure. 16

Numerous epidemiologic studies have investigated adverse effects in populations

. 17

Decreased peak expiratory flow rates (PEFR) are an important component in the diagnosis of 18

asthma and there is evidence of formaldehyde

- induced decrements in PEFR (see Section 19

4.1.1.2). However, the diagnosis of asthma is both a more serious health cond ition and 20

diagnostically more complex than decreased PEFR alone and is evaluated here as a distinct 21

endpoint. A number of epidemiologic studies have investigated the potential association 22

between formaldehyde exposure and a continuum of adverse health eff ects from pulmonary 23

function to asthma. 24

The association between formaldehyde and asthma has been studied by examining 25

occupational exposures (Fransman et al., 2003; Malaka and Kodama, 1990), school

- related 26

exposures (Zhao et al., 2008; Smedje and Norback, 2001; Norback et al., 2000) and residential 27

exposures (Matsunaga et al., 2008; Tavernier et al., 2006; Gee et al., 2005; Delfino et al., 2003; 28

Rumchev et al., 2002; Garrett et al., 1999; Palczynski et al., 1999; Norback et al., 1995; 29

Krzyzanowski et al., 1 990). The two occupational studies examined the respiratory health of 30

plywood workers (Fransman et al., 2003; Malaka and Kodama, 1990). The most recent of these 31

was conducted in New Zealand by Fransman et al. (2003). Personal samples of formaldehyde 32

exp osure were taken. The mean level of exposure was 0.08 mg/m 3 (65 ppb) and the majority of 33

samples were below the limit of detection which was reported to be 0.03 mg/m 3 (24 ppb). 34

Compared with those with low levels of formaldehyde exposure, workers with hi gh levels of 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 22 exposure were more likely to report having asthma (OR=4.3 [95% CI]: 0.7

– 27.7]). The 1

association was not seen when examining formaldehyde exposure and use of asthma medication. 2

The second study of plywood workers was completed in Indonesia. Background levels of 3

formaldehyde ranged from 0.003 to 0.07 ppm. The highest concentration of formaldehyde 4

detected in an air sample was in the particleboard unit (range 1.16 to 3.48 ppm). Asthma, which 5

was defined as “have you ever had an attack of whee zing that made you feel short of breath?”, 6

was found to be positively associated with formaldehyde exposure (Malaka and Kodama, 1990). 7

Studies of exposure to formaldehyde at schools have been performed in China (Zhao et 8

al., 2008) and in Sweden (Smedje and Norback, 2001). In the study from China (Zhao et al., 9

2008), mean levels of formaldehyde were reported to be 2.3 μg/m 3 (range 1.0

– 5.0 μg/m 3 ) 10

indoors and 5.8 μg/m 3 (range 5.0

– 7.0 μg/m 3 ) outdoors. Cumulative asthma and daytime attacks 11

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

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

levels were found to be associated with nocturnal attacks of breathlessness. In Sweden (Smedj e 14

and Norback, 2001), the levels of formaldehyde measured indoors were higher (mean 4, range 15

<5.0

– 72 μg/m 3 ). One difference between this Swedish study and the study performed in China is 16

that the Swedish study examined the incidence of asthma over a 4

- yea r period. This study did 17

not report an association between formaldehyde exposure and the incidence of asthma (OR 1.2 18

[95% CI: 0.8

– 1.7]) among the whole study population. However, when the investigators 19

stratified on history of atopy, they reported that a mong children without a history of atopy, a new 20

diagnosis of asthma was significantly more likely at higher concentrations of formaldehyde (OR 21

1.7 per 10 μg /m 3 [95% CI: 1.1

– 2.6]) and at higher total concentrations of mold (OR=4.7 per 10

- 22

fold increased in total molds [95% CI: 1.2

- 18.4] in the classroom air. The finding for adverse 23

effects of formaldehyde and mold did not appear to control for the other exposure and no 24

information on the potential correlation between the two exposures was provided. In orde r to 25

evaluate the potential for confounding of the reported formaldehyde association by the reported 26

mold association, the magnitude of effects must be compared on an appropriate scale since the 27

magnitude of an odds ratio depends on the magnitude of the ch ange in exposure level that is 28

expected to produce increased risk. Standardizing the units to the reported geometric mean 29

standard deviation, the result for formaldehyde (GSM=2.3 μg /m 3 ) is OR 1 =1.13 per GSD and the 30

result for mold is OR 2 =1.02 for a compar ison of risks at the GSM to 10*GSM and OR 3

1 OR per GSD=exp[ln(OR per μg /m 3 )/ 10 μg /m 3 * 2.3 μg /m 3 ]= exp[ln(1.7)/10*2.3]=1.13 =1.06 for 31

2 OR per GSD=exp[ln(OR per 10

- fol d increase )/ (9*GSM)*1.6 μg /m 3 ]= exp[ln(4.7)/162*1.6]=1.02

3 OR per GSD=exp[ln(OR per 10

- fold increase )/ (9*Minimum)*1.6 μg /m 3 ]= exp[ln(4.7)/45*1.6]=1.06This document is a draft for review purposes only and does not constitute Agency policy.

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- 23 a comparison of risks at the minimum value of total molds (5*10 3 /m 3 ) to 10*minimum. As it 1

appears that the magnitude of the formaldehyde effect is substantially stronger than that of the 2

mold effect (fol lowing standardization of exposure increment) it can be concluded that the 3

reported formaldehyde effect could not have been the spurious result of uncontrolled 4

confounding by mold. 5

The results of studies measuring residential exposure to formaldehyde and a sthma are 6

varied, with some demonstrating an association and others finding no relationship. A recent 7

study (Matsunaga et al., 2008) found no association between 24

- hour formaldehyde and 8

prevalence of asthma when pregnant women with an exposure ≥47 ppb we re compared to those 9

with exposure to <18 ppb. However, they did report at increased risk of atopic eczema. It 10

should be noted that this study did not assess the risk of incident asthma. A study utilizing self

11

reported asthma prevalence as an outcome als o found no association with levels of formaldehyde 12

(mean 25.9 μg/m 3 , range 2.0

– 66.8 μg/m 3 ) (Palczynski et al., 1999) although they did note that 13

the incidence of allergic diseases was highest in the highest formaldehyde exposure group but 14

that the group wa s too small for statistical evaluation. 15

A study performed by Tuthill (1984) measured formaldehyde exposure for children 16

grades K through 6 by using a combination of proxy variables. Overall, there was no 17

association, but some individual variables did show an increased risk. For example, the reported 18

risk ratio for having new construction or remodeling performed in the house in the past 4 months 19

was 2.5 (95% CI: 1.7

– 3.9). The risk ratio for having new or upholstered furniture in the house 20

(brought into th e house within the past 4 months) was 2.2 (95% CI: 1.2

– 3.9). 21

The study by Delfino et al. (2003) assessed whether ambient formaldehyde concentration 22

measured at a central monitoring site were associated with asthma symptoms. The study 23

examined 22 10

– 15 yea r olds with at least 1 year of physician

- diagnosed asthma and living in a 24

nonsmoking household. The mean levels of formaldehyde were measured to be 7.21 ppb (range 25

4.27

– 14.02 ppb). There was a positive association between asthma symptom scores (comparing 26

children who report symptoms interfering with their daily activities versus those with no 27

symptoms or symptoms not great enough to affect their daily activities) and high current levels 28

of formaldehyde (OR 1.90 [95% CI: 1.13

– 3.19]). 29

Three studies (Taverni er et al., 2006; Gee et al., 2005; Garrett et al., 1999) were 30

performed by matching children with and without asthma and comparing the levels of 31

formaldehyde in their homes. Gee et al. (2005) reported median formaldehyde levels of 0.03 32

ppm in living rooms and 0.04 ppm in bedrooms. Analyses were limited to univariate 33

comparisons of formaldehyde levels for cases of existing asthma and controls without asthma. 34

The concentrations did not differ in a statistically significant manner. The study by Gee et al. 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 24 (2005) was followed up with a more sophisticated analysis of the same children in the same 1

homes. Tavernier et al. (2006) reiterated the earlier finding by Gee et al (2005) that 2

formaldehyde was not found to be associated with existing asthma. Tavernier e t al. (2006) did 3

not report the measured levels of formaldehyde but gave the OR for the highest tertile of 4

exposure compared with the lowest tertile of exposure as 0.99 (95% CI: 0.39

– 2.50). The width 5

of this confidence interval suggests that these finding s would still be consistent with two

- fold 6

increase in risk. 7

Garrett et al. (1999) reported on the risk of allergy and asthma

- like respiratory symptoms 8

due to formaldehyde exposure in a cross

- sectional survey of households with children with (n = 9

53) or wit hout (n = 88) doctor

- diagnosed asthma. Formaldehyde exposure was characterized by 10

4 seasonal in

- home sampling events across the year for bedrooms and 4

– day passive samples 11

collected in living rooms, kitchens and outdoors. Statistically significant linear trends for 12

increased risk of having asthma were seen with increasing formaldehyde levels (p < 0.02); 13

however, the ORs for the association did not remain statistically significant after controlling for 14

parental allergy and asthma (exact ORs and 95% CIs not given). Garrett et al (1999) also 15

evaluated the prevalence and severity of allergic sensitization to 12 common allergens and 16

reported increased prevalence with increasing formaldehyde concentration in the home. The 17

respiratory symptom score was also inc reased and demonstrated a significant effect for 18

formaldehyde in a multiple regression after adjusting for multiple risk factors and interactions. 19

For the atopy and respiratory symptom endpoints, severity/incidence was increased in the 20

medium (20

– 50 μg/m 3 ) and high (>50 μg/m 3 ) exposure groups relative to the low (<20 μg/m 3 ) 21

exposure group, based on the highest of four seasonal 4

- day formaldehyde measurements in the 22

home. The associations between formaldehyde concentrations and severity of allergic 23

sensiti zation are clearly shown and further substantiated with multivariate regression controlling 24

for potential confounders. In logistic regressions, both the prevalence and severity of allergic 25

sensitization to 12 common allergens increased with increasing for maldehyde concentration in 26

the home. The crude association for atopy with an increase in formaldehyde concentration per 27

10 μg/m 3 was OR=1.34 which increased when adjusted for parental asthma and gender to and 28

odds ratio of 1.42 per 10 μg/m 3 (95% CI: 0.99

- 2.04). Passive smoking, the presence of pets, 29

indoor nitrogen dioxide concentrations, airborne fungal spores and house

- dust

- mite allergens did 30

not influence the effect estimates and were unlikely to be confounders. Additionally, a 31

calculated respiratory symptom score was increased and demonstrated a significant relationship 32

to increased formaldehyde concentration in a multiple linear regression after adjusting for 33

multiple risk factors and interactions. For each of these endpoints, severity/incidence was 34

increased in the medium (20

– 50 μg/m 3 ) and high (>50 μg/m 3 ) exposure groups relative to the 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 25 low (<20 μg/m 3 ) exposure group, based on the highest of four seasonal 4

- day formaldehyde 1

measurements in the home. 2

Residential formaldehyde exposure was associat ed with an increased risk of asthma in a 3

population

- based case

- control study of 192 children aged 6 months to 3 years (Rumchev et al., 4

2002). The study, which comprises 88 cases of children discharged from the emergency 5

department of a children’s hospita l in Perth, Australia, with a primary diagnosis of asthma and 6

104 controls, provides a positive exposure

- response relationship. Seasonal in

- home 7

formaldehyde measurements taken in the living room and subject’s bedroom were used to assess 8

exposure (8

- hour passive sampler). The odds ratios (ORs) for risk of asthma by formaldehyde 9

exposure level category were adjusted for numerous risk factors both familial and environmental 10

including, familial history of asthma, age, sex, smoking, presence of pets, and attr ibutes of the 11

home. Of these, age, allergic sensitization to common allergens, and family history of allergy 12

were independent risk factors for asthma (ORs of 1.09, 2.57, and 2.66, respectively). Categorical 13

analysis of the data indicates the ORs for asth ma were increased in the two highest formaldehyde 14

exposure groups, reaching statistical significance for household exposures > 60 μg/m 3 (48 ppb) 15

(OR of 1.39) . Analysis of the data with formaldehyde as a continuous variable provides a 16

statistically signif icant increase in the risk of asthma (3 % increase in risk per every 10 ug/m3 17

increase in formaldehyde level. All analyses controlled for other indoor air pollutants, allergen 18

levels, relative humidity, and indoor temperature as well as other risk factors

. 19

A study of 202 households (mean formaldehyde level of 26 ppb) found that among 20

children aged 6

– 15 years old and exposed to environmental tobacco smoke, the prevalence of 21

asthma was 45.5% for those with measured levels of formaldehyde in the kitchen >60 ppb. The 22

prevalence of asthma dropped to 15.1% for levels ≤40 ppb and 0% for 41

– 60 ppb. No trend in 23

asthma prevalence was seen for children who were not exposed to environmental tobacco smoke 24

(Krzyzanowski et al., 1990). 25

Finally, a study by Norback et a l. (1995) reported mean levels of formaldehyde were 29 26

μg/m 3 (range <5

– 110 μg/m 3 ) in the bedrooms of individuals experiencing nocturnal 27

breathlessness compared with formaldehyde levels of 17 μg/m 3 (<5

– 60 μg/m 3 ) among those 28

without nocturnal breathlessness. The OR for this association was 12.5 (95% CI: 2.0

– 77.9) and 29

the effect was substantially stronger in magnitude than the associations observed for toluene, 30

terpenes and volatile organic compounds which makes confounding by those co

- exposures 31

unlikely. 32

Fo rmaldehyde has clearly been shown to be a cause of bronchial asthma and several 33

epidemiologic studies have identified causal evidence of an adverse effect of exposure on 34

pulmonary function and the incidence of asthma. While there are studies that did not find 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 26 associations, many of those were limited by their study design, exposure measurement and the 1

definition of prevalent asthma as the health endpoint. 2

3

4.1.1.4. Respiratory Tract Pathology 4

Formaldehyde

- induced respiratory tract pathology includes infla mmation, rhinitis, goblet 5

cell hyperplasia, metaplastic changes, squamous cell hyperplasia, and impaired mucociliary 6

transport. Formaldehyde may bind to the trigeminal nerve and trigger the release of neurogenic 7

mediators of inflammation that result in ti ssue edema, lacrimation, mucus production and 8

leukocyte infiltration. How much inflammation, hyperplasia, and metaplastic change are due to 9

sensory irritation

- induced inflammatory responses compared with formaldehyde

- induced direct 10

cell damage cannot be d istinguished. Increased mucus flow and metaplastic changes may 11

progress in relation to the concentration and duration of exposure to protect the underlying 12

tissue. When the exposure exceeds protective and defensive mechanisms, permanent damage 13

results (S wenberg et al., 1983). Nonetheless, these changes serve as a sensitive indicator of 14

formaldehyde exposure, since they occur before gross cellular damage and focal lesions 15

(Monticello et al., 1989), and potentially suggest a point at which the concentratio n and duration 16

of exposure exceed the protective nature of local responses (increased mucus flow, goblet cell 17

hyperplasia, squamous metaplasia, etc.) (Swenberg et al., 1983). A number of human studies 18

have reported nasal lesions associated with exposure t o formaldehyde (Pazdrak et al., 1993; 19

Ballarin et al., 1992; Boysen et al., 1990; Holmström et al., 1989c; Edling et al., 1988), while 20

other studies have documented changes in mucociliary clearance and activity ( Holmström and 21

Wilhelmsson, 1988; Andersen a nd Molhave, 1983). These studies are summarized below. 22

23

4.1.1.4.1. Nasal lesions. Ballarin et al. (1992) did a case

- control study of 15 workers from a 24

plywood factory where urea

- formaldehyde glue is used. Mean levels of formaldehyde exposure 25

(8

- hour av erage) were estimated to be 0.09, 0.1, and 0.39 mg/m 3 in three regions of the facility 26

(sawmill, shearing press, and warehouse, respectively). Nasal respiratory samples were 27

obtained. Stained cells were scored for histopathology. Cytology examination re vealed 28

increased squamous metaplasia cells in 10 of 15 (67%) factory workers (with an average severity 29

score of 2.3) compared with one of 15 (6%) controls (with an average histology severity score of 30

1.6). In addition, one formaldehyde exposed worker (n = 15) exhibited mild dysplasia and had 31

the highest severity score (3.0). Authors suggest that these results may be due to chronic 32

irritation of the nasal respiratory mucosa. This small study reported only incidence of lesions 33

and did not score based on se verity of lesions. The lesion incidence was not reported in relation 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 27 to dose, so no dose

- response relationship could be determined, precluding the establishment of a 1

point of departure (POD). 2

Holmström et al. (1989) collected nasal biopsy samples from wor kers exposed to air or to 3

formaldehyde at a median concentration of 240 ppm. Nasal biopsy samples were scored on a 4

0

– 8 range with normal respiratory epithelium as 0 and carcinoma as 8. Observed histologic 5

changes included loss of cilia, goblet cell hype rplasia, and cuboidal and goblet cell metaplasia 6

replacing normal columnar epithelium. The incidence associated with each histologic change 7

was not reported and cannot be compared between formaldehyde

- exposed and control 8

individuals. Moreover, these biol ogically relevant changes were not analyzed independently in 9

the analysis. The mean scores were 1.56 (range, 0

– 4) for the control group and 2.16 (range, 0

– 4) 10

for the formaldehyde

- exposed group. Although the range of scores in the controls and 11

formaldehyd e

- exposed groups were the same (0

– 4), the difference in mean scores (1.56 versus 12

2.16) was statistically significant ( p < 0.05); scores were worse in the formaldehyde

- exposed 13

group. The authors reported no correlation between the duration of exposure and histologic 14

changes and no correlation between smoking habits and biopsy scores. The loss of cilia, goblet 15

cell hyperplasia, and cuboidal and squamous cell metaplasia replacing the columnar epithelium 16

were increased in the group exposed to formaldehyde and is a biologically relevant change. This 17

study provides a lowest

- observed

- adverse

- effect level (LOAEL) of 0.240 ppm for nasal 18

histopathology. 19

Edling et al. (1988) collected nasal biopsy samples from workers (n = 75) exposed to 20

formaldehyde at three plants (workers in two of these plants were also exposed to wood dust) 21

compared with a referent group (n = 25). Concentrations ranged from 0.1 to 1.1 mg/m 3 (TWA) 22

with peaks of 5 mg/m 3 . Nasal histology was scored from 0 to 8 by increasing severity, from 23

normal respiratory epithelium (0) to carcinoma (8). A normal respiratory epithelium was noted 24

in 3 of 75 workers. A loss of cilia and goblet cell hyperplasia (scores of 2) was reported in eight 25

workers. Mixed cuboid/squamous epithelium (metaplasia), stratified squamous epithelium, and 26

keratosis were reported in 58 of 75 workers (those with scores of 3, 4, and 5 were combined). 27

Dysplasia (score of 6) was reported in 6 of 75 formaldehyde

- exposed workers. None of the 28

workers had lesions that warranted a histolog ic score higher than 6. Histologic scores did not 29

correlate with duration of exposure but could not be confirmed due to poor reporting. Data from 30

the referent group were not included. A POD could not be determined from this study. 31

Boysen et al. (1990) c ollected nasal biopsy samples from workers exposed to air (n = 37) 32

or to formaldehyde (n = 37) and sometimes wood dust. The exposed workers were classified 33

into two exposure groups, 0.5

– 2 ppm and >2 ppm. Nasal biopsy samples were assessed by using 34

a hist opathology score range of 0

– 5, based on the pathology of pseudostratified columnar 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 28 epithelium (0) to dysplasia (5). Mean pathology scores for the control were decreased compared 1

with the formaldehyde

- exposed group (1.4 and 1.9, respectively) but did not r each statistical 2

significance. Little quantitative pathology data were provided, although qualitative histology 3

revealed a range of observed effects from deciliated epithelial cells to mixed stratified cuboidal, 4

squamous epithelium to dysplasia. None of the control samples received histologic severity 5

scores of 4 or 5, indicating that keratinizing stratified squamous epithelium and dysplasia were 6

not observed in controls. A wider variety of histopathologic lesions were reported in exposed 7

workers compare d with controls, and a greater number of exposed workers had histologic 8

changes compared with controls. Incidence data for each type of histopathology were not 9

reported, but the authors wrote that the degree of metaplastic alterations was more pronounced 10

among the exposed workers. An upper range for the high concentration group (>2 ppm) was not 11

reported, and median concentrations were not provided. 12

Pazdrak et al. (1993) exposed human subjects (six men, three women) to 0.4 ppm 13

formaldehyde in a chamber f or 2 hours. Approximately half of the subjects suffered from skin 14

hypersensitivity to formaldehyde, while the other subjects were healthy. An evaluation of nasal 15

lavage pretest and following formaldehyde exposure revealed that the hypersensitive and heal thy 16

groups had similarly elevated eosinophil counts at 0 hours after exposure (from 17

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

39 × 10 3 cells/mL to 69 × 10 3 cells/mL for hypersensitive subjects [ p < 0.05]). Similar 19

eo sinophil levels were also seen in both groups at 3 and 18 hours. Both groups had equivalent 20

increases in lavage albumin and total protein levels following exposure, but basophil counts were 21

unchanged. Based on evidence of formaldehyde

- induced inflammatio n, these data provide a 22

LOAEL of 0.4 ppm for nasal histopathology. 23

24

4.1.1.4.2. Mucociliary clearance. In addition to abnormal nasal histopathology, changes in 25

mucociliary clearance were also observed in some of these studies at similar exposure 26

concentra tions. The mucociliary apparatus is an important barrier to infection and exogenous 27

agents and, thus, is considered as a potential adverse effect. These effects may be due to direct 28

interaction of formaldehyde with the mucus itself or to SI

- induced infla mmation in the nasal 29

tissue that affects mucus production and creation of an effective mucosal barrier. 30

Andersen and Molhave (1983) reviewed five controlled human studies, one of which 31

(Andersen and Lundqvist, 1974) examined mucus flow rate in 16 individua ls acutely exposed to 32

0, 0.3, 0.5, 1, or 2 ppm formaldehyde for 4

– 5 hours in a chamber. Mucus flow rate was 33

decreased in the anterior and middle third of the ciliated mucosa at 0.3 ppm, but statistical 34

significance was not determined. This study included smokers and nonsmokers. The small 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 29 sample size, potential confounder effect from smoking, and lack of dose

- response relationship 1

preclude the establishment of a POD. 2

Holmström and Wilhelmsson (1988) demonstrated reduced mucociliary clearance and 3

nasal muc osal swelling in 70 workers exposed to a median formaldehyde concentration of 4

0.21 ppm, compared with a referent group of store clerks (n = 36) and was further averaged over 5

years of exposure. Mucosal swelling and mucociliary activity was measured in the nasal 6

turbinates. The authors also reported symptoms not only during the weekdays, but also over 7

weekends and vacation periods. Formaldehyde

- exposed subjects self

- reported significantly more 8

nasal discomfort, eye discomfort, deeper airway discomfort, and frequent headache than the 9

referent group. Groups exposed to formaldehyde had more pronounced mucosal swelling (10.7 10

nasal resistance score) compared with the reference group (6.5 nasal resistance score). This 11

difference persisted when data were normali zed for differential nasal congestion in the subjects. 12

Decreased mucociliary activity was seen in 3% of controls and 20% of formaldehyde

- exposed 13

subjects and reached statistical significance ( p < 0.05). It is not clear whether impaired 14

mucociliary cleara nce was a consequence of altered cell morphology or increased mucus 15

viscosity. These data provide a LOAEL of 0.21 ppm based on impaired mucociliary clearance. 16

Thus, mild nasal epithelial lesions observed in formaldehyde

- exposed workers have been 17

observed consistently at levels of about 0.20 ppm to about 2 ppm (Boysen et al., 1990; 18

Holmström et al., 1989; Edling et al., 1988). Of these, Holmström et al. (1989) and Edling et al. 19

(1988) do not appear to be confounded by exposure to wood dust. Nasal biopsy p athology from 20

formaldehyde

- exposed workers is consistent with irritant and reactive properties of 21

formaldehyde (Ballarin et al., 1992; Boysen et al., 1990; Holmström et al., 1989; Edling et al., 22

1988; Berke, 1987). Moreover, these findings are supported b y results from animal toxicity and 23

pharmacokinetic and anatomical airflow studies, indicating that, at concentrations less than 24

1 ppm, inhaled formaldehyde gas does not reach lower regions of the respiratory tract. Of the 25

available human studies that eval uated histopathology, Holmström and Wilhelmsson (1988) 26

appears to be the most robust and sensitive. The study was carefully designed and included a 27

large sample of formaldehyde

- exposed subjects who were considered separately from workers 28

exposed to combin ations of exposures (formaldehyde and wood dust). Study subjects had been 29

exposed to formaldehyde regularly for many years. The authors reported not only weekday 30

exposures but effects reported on weekends and on vacation. Total exposure was carefully 31

ca lculated and averaged. The data were controlled for potential confounders, such as smoking. 32

The endpoint of reduced mucociliary clearance has been substantiated by Andersen and Molhave 33

(1983) and Holmström et al. (1989). Animal studies have also reporte d formaldehyde

- induced 34

changes on the nasal mucosa and are highlighted in Section 4.2.1.2. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 30 4.1.1.5. Immunologic Effects 1

Numerous studies have examined the immunologic responses of individuals exposed to 2

formaldehyde. This section will discuss four specif ic areas related to immunotoxicity after 3

exposure to formaldehyde: increased upper respiratory tract (URT) infections, systemic immune 4

dysfunction, sensitization and atopy, and production of formaldehyde

- protein complexes. Some 5

studies report increased in cidence of URT infections after exposure to formaldehyde (Lyapina et 6

al., 2004; Krzyzanowski et al., 1990; Holness and Nethercott, 1989). This effect appears to 7

occur independently of systemic immune changes and may be due to damage to the mucosal 8

barrier , thus facilitating pathogen access. A number of studies have investigated the hypothesis 9

that formaldehyde may induce systemic immunomodulation (Ohtani et al., 2004a, b; Erdei et al., 10

2003; Thrasher et al., 1990, 1987; Pross et al., 1987 ). Some studies have also evaluated immune 11

system effects by investigating the role of reactive oxygen species (ROS) from respiratory burst 12

associated with immune cells (Lyapina et al., 2004; Gorski et al., 1992) and by assessing 13

chromosomal damage in immune cells (Orsièr e et al., 2006; Yu et al., 2005)

. In addition to the 14

effects of formaldehyde on asthmatics and the potential for formaldehyde exposure to exacerbate 15

asthmatic responses, reviewed in Section 4.1.1.2, numerous studies have investigated whether 16

formaldehyde may directly induce sensitization and atopic responses by measuring 17

immunoglobulin E (IgE) levels associated with formaldehyde exposure ( Ohmichi et al., 2006; 18

Vandenplas et al., 2004; Doi et al., 2003; Baba et al., 2000; Palczynski et al., 1999; Krakowiak et 19

al., 1998; Wantke et al., 1996a, b; Liden et al., 1993 ; Salkie, 1991; Grammer et al., 1990; 20

Kramps et al., 1989). Findings are largely negative and suggest that formaldehyde

- induced IgE 21

production is not likely. Lastly, studies have investigated the p roduction of formaldehyde

22

specific antibodies, formaldehyde

- albumin complexes, and formaldehyde

- heme complexes (Kim 23

et al., 2001; Carraro et al., 1997; Grammer et al., 1993, 1990; Dykewicz et al., 1991; Thrasher et 24

al., 1990). Heme complex formation is no t a strict immunologic endpoint but may trigger 25

antibody formation and thus it will be discussed in this section. This section will thus summarize 26

the human studies that have specifically addressed the increased incidence of URT infections, 27

immunotoxic en dpoints, atopy and sensitization, and formation of formaldehyde

- heme and 28

formaldehyde

- albumin complexes. 29

30

4.1.1.5.1. Increased URT infections. Diverse studies have investigated the possibility that 31

formaldehyde exposure leads to increased URT infections (Lyapina et al., 2004; Krzyzanowski 32

et al., 1990; Holness and Nethercott, 1989). Lyapina et al. (2004) studied 29 workers who were 33

occupationally exposed occupationally to formaldehyde for an average of 12.7 years through 34

contact with carbamide

- formaldehy de glue. The mean values of the average shift concentrations 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 31 of formaldehyde in the application of carbamide

- formaldehyde glue to be 0.71 ppm TWA with a 1

range of 0.32 to 1.57 ppm. The workers were divided into two subgroups, one (n = 12) that 2

suffered fr om either a long history (with clinical findings) of chronic mucous inflammation of 3

the URT with multiple relapses and a second group (n = 17) whose URT inflammations were 4

short, acute, and predominantly viral. Twenty

- one healthy subjects served as contro ls. A 5

statistically significant association of self

- reported chronic bronchitis and decreased resistance to 6

URT infection was reported in all the exposed workers compared with controls ( p = 0.02). Of 7

the workers, 41% had a history of chronic respiratory infection and frequent long

- lasting 8

infectious inflammatory relapses (group 1a). Another group (group 1b) consisted of 17 exposed 9

workers, 12 of whom had no history of recurrent viral infections of the URT. There was a 10

statistically significant associati on of frequency and duration of inflammatory relapses between 11

groups 1a and 1b. No dates were provided regarding when these measurements were made or 12

over what period of time they were calculated. 13

Krzyzanowski et al. (1990) measured formaldehyde levels in homes and recorded, by 14

way of a questionnaire, health histories from adult and child residents. Formaldehyde levels 15

were reported from samples taken for two 1

- week periods in various rooms of the home (kitchen, 16

living room, subject’s bedroom). The avera ge formaldehyde level was 26 ppb in 202 homes, and 17

levels were stratified into homes with exposure levels below 40 ppb, between 40 and 60 ppb, and 18

above 60 ppb. Incidences of doctor

- diagnosed chronic bronchitis were more prevalent in 19

children (under age 1 5) living in homes with higher formaldehyde (>60 ppb) readings in the 20

kitchen ( p < 0.001). This effect was more pronounced ( p < 0.001) in children simultaneously 21

exposed to environmental tobacco smoke. The prevalence of chronic cough was also increased 22

i n adults living in homes with measurable levels of formaldehyde, but data were not shown. 23

Holness and Nethercott (1989) assessed chronic bronchitis in 87 funeral workers, where the 24

average formaldehyde exposure was reported at 0.38 ± 0.19 ppm. Chronic br onchitis was 25

observed in 20 funeral workers (n = 87) exposed to formaldehyde compared with 3 cases of 26

chronic bronchitis in nonexposed referent controls (n = 38). 27

These studies suggest that exposure to formaldehyde may be associated with increased 28

incidenc e of chronic bronchitis. The mechanism for this association has not been elucidated. 29

Pathogens may gain access to the URT via a compromised mucosal barrier, as has been shown in 30

histopathology studies (Section 4.1.1.4). 31

32

4.1.1.5.2. Immune function. A n umber of studies have evaluated the ability of formaldehyde to 33

induce systemic immunotoxic effects ( Ohtani et al., 2004a, b ; Erdei et al., 2003; Thrasher et al., 34

1990, 1987; Pross et al., 1987 ). Some studies have reported altered innate immune responses 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 32 a ssociated with formaldehyde exposure (Erdei et al., 2003), while others have noted adaptive 1

immune response suppression associated with formaldehyde exposure (Thrasher et al., 1990, 2

1987) and changes associated with alterations to a predominant T

— lymphocyt e helper 2 (Th2) 3

pattern (Ohtani et al., 2004a, b). In contrast, Pross et al. (1987) did not observe formaldehyde

4

associated changes in systemic immune function. 5

Erdei et al. (2003) found that Haemophilus influenzae humoral biomarker (H.in.IgG), 6

Klebsiell a pneumoniae biomarker (K.pn.IgG), and elevated monocyte concentrations were 7

significantly associated with high formaldehyde concentrations in asthmatic children, compared 8

with nonsensitive children. Briefly, Erdei et al. (2003) compared the immune system responses 9

in 9

- to 11

- year

- old Hungarian school children whose respiratory systems were immunologically 10

compromised (chronic respiratory disease, asthma) and normal children who were exposed to 11

indoor air pollutants, including formaldehyde. In the homes of the children with the highest 12

levels of pollutants, 49.3% of formaldehyde measurements exceeded the Hungarian indoor 13

standard of 0.01 ppm, while 20% exceeded the World Health Organization’s (WHO’s) suggested 14

indoor level of 0.09 ppm. The authors exclud ed from consideration all measurements that 15

exceeded WHO’s air quality guidelines in one unidentified city to prevent a “city

- related bias,” 16

since these measurements occurred entirely in that city. The average formaldehyde 17

concentration in the 123 homes t ested was 14 ppm with a range of 0.5 to 46 ppm. H.in.IgG and 18

K.pn.IgG were significantly associated with high formaldehyde concentrations ( p < 0.013 and 19

p < 0.049, respectively) in sensitive children compared with nonsensitive children. These 20

markers wer e also correlated with high levels of nitrogen dioxide, the number of cigarettes 21

smoked, and exposure to paint, volatile organic compounds, and solvents. Additionally, indoor 22

formaldehyde exposure was significantly associated with increased monocyte conce ntrations 23

( p < 0.017) that are important to the innate immune response (inflammation) in diseased tissue. 24

The authors concluded that the elevation of immune biomarkers in sensitive children with 25

respiratory disease is likely the result of high concentrati ons of toxic indoor air pollutants, 26

including formaldehyde. 27

Thrasher et al. (1987) assessed the effects of formaldehyde exposure on cellular 28

immunity and antibody formation in eight exposed and eight unexposed individuals. The 29

exposed group consisted of t hree males and five females. Seven of the exposed individuals had 30

resided in mobile homes for periods ranging from 2 to 7 years; the eighth was a laboratory 31

worker who resided in a newly decorated, energy

- efficient apartment. Air monitoring in four of 32

th e homes revealed formaldehyde vapor concentrations ranging from 0.07 to 0.55 ppm. Venous 33

blood samples were collected from all subjects and T

- and B

- cells were counted and monitored 34

for blastogenesis. When IgG and IgE antibodies to formaldehyde were moni tored in serum, no 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 33 IgE antibodies to formaldehyde were detected in exposed or control subjects. IgG antibody titers 1

in exposed subjects ranged from 1:8 to 1:256 but essentially were undetected (1:4) in seven of 2

the controls. T

- and B

- cell numbers were si gnificantly lower ( p < 0.05) in mobile home residents 3

(48 and 12.6%, respectively) compared with those of control subjects (65.9 and 14.75%, 4

respectively). As determined by incorporation of 3 H

- thymidine into 48

- hour unaltered 5

lymphocytes, phytohemagglutin in

- stimulated T

- and B

- cell blastogenesis was significantly 6

depressed ( p < 0.01) in cells of mobile home residents compared with those of control subjects 7

(17,882 and 28,576 cpm, respectively). Thrasher et al. (1987) concluded that exposure to 8

formaldehyd e decreases the proportion of peripheral T cells. 9

In a later study, Thrasher et al. (1990) evaluated five groups of subjects with varying 10

levels and durations of formaldehyde exposure. The groups consisted of (1) asymptomatic 11

chiropractic students exposed during anatomy classes (controls with only intermittent exposure 12

to formaldehyde), (2) mobile home residents, (3) office workers, (4) patients with multiple 13

symptoms who had been removed from the source of formaldehyde for at least a year, and 14

(5) occupat ionally exposed patients. All groups were assessed for immunologic function via 15

white cell, lymphocyte, and T

- cell counts, T

- helper/suppressor ratios and B

- cell counts. When 16

compared with controls (chiropractic students), the patient groups had significa nt elevations in 17

formaldehyde antibody titers and B

- cell titers. 18

Ohtani et al. (2004a, b) reported effects of exposure to formaldehyde and diesel exhaust 19

particles on cytokine production by human monocyte

- derived dendritic cells (MoDCs) and 20

T cells in vitr o. Dendritic cells were stimulated with CD40 ligand and interferon (IFN)

- **γ** , T cells 21

with anti

- CD3/CD28 antibodies. Cytokine proteins and mRNA levels were measured in 22

supernatants by enzyme

- linked immunosorbent assay (ELISA) and polymerase chain reaction 23

(PCR), respectively. Formaldehyde and diesel exhaust particles significantly increased tumor 24

necrosis factor (TNF)

- **α** levels and suppressed interleukin (IL)

- 12p40 protein and mRNA levels 25

in MoDCs. The same treatment suppressed protein synthesis and mRNA e xpression of IFN

- **γ** 26

and IL

- 10 in T cells. The authors concluded that their findings support a role of formaldehyde 27

and diesel exhaust particles in altering the immune response to a Th2

- dominant pattern that 28

furthers allergic inflammation. Further details, such as exposure concentrations and 29

experimental protocols, are not available. 30

In contrast, Pross et al. (1987) concluded that formaldehyde does not induce altered 31

immune activity. The authors evaluated the immunologic response of asthmatic subjects 32

expo sed to UFFI off

- gas products. Subjects consisted of 23 individuals with a history of 33

asthmatic symptoms attributed to UFFI and 4 individuals with asthma unrelated to UFFI off

- gas 34

products. All subjects were exposed in an environmental chamber according t o the following 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 34 sequence: (1) room air (placebo) for 30 minutes; (2) 1 ppm formaldehyde gas for 3 hours; (3) 1

UFFI particles (4 μm diameter, 0.5 particles/mL) for 3 hours, commencing 48 hours after 2

formaldehyde gas exposure; and (4) UFFI off

- gas products fo r 3 hours, commencing 48 hours 3

after UFFI particle exposure. There was a significant increase in the percentage and absolute 4

number of eosinophils and basophils in the subjects who lived in UFFI homes but no differences 5

between exposure groups with respec t to lymphocyte subpopulations either before or after UFFI 6

exposure. However, when T8 suppressor cells were counted, values in the UFFI

- exposed group 7

pre

- exposure and postexposure, a small but statistically significant ( p < 0.01) increase in T8 cell 8

count was observed. The biological significance of this increase in T8 cell count in exposed 9

asthmatics is not known. Pross et al. (1987) concluded that short

- term exposure to formaldehyde 10

was not immunosuppressive and did not result in systemic immune reacti vity. 11

Respiratory burst from immune cells creates ROS that can incur further cellular damage. 12

Several studies have evaluated, either directly or indirectly, the potential role of ROS as potential 13

mediators of formaldehyde

- associated effects, particularly those caused by immune cells. Gorski 14

et al. (1992) measured chemiluminescence resulting from the release of free radicals from 15

granulocytes of healthy and formaldehyde

- sensitive subjects. Thirteen subjects with contact 16

dermatitis who were occupationally exposed to formaldehyde and five healthy volunteers 17

participated in the study. All underwent skin

- prick tests for common allergens as well as a 18

histamine inhalation provocation test. Subjects were exposed to 0.5 mg/m 3 (0.41 ppm) 19

formaldehyde for 2 hours, and the PEFR was measured immediately before exposure, after 60 20

and 120 minutes of exposure, and 6 and 21 hours after completion of exposure. Peripheral blood 21

granulocyte chemiluminescence was measured in the presence of luminol. Free radical 22

production was increased significantly within 30 minutes of beginning the exposure in subjects 23

with allergic dermatitis and remained elevated for 24 hours compared with baseline values. 24

Gorski et al. (1992) concluded that granulocyte chemiluminescence did not incre ase in healthy, 25

formaldehyde

- exposed patients but was diagnostic for formaldehyde

- sensitive patients. These 26

results also suggest a putative role for oxidative damage associated with formaldehyde exposure, 27

particularly in sensitized individuals. 28

Lyapina et al. (2004) also reported effects of formaldehyde exposure on neutrophil 29

respiratory burst activity (NRBA), the capacity of polymorphonuclear leukocytes to produce 30

reactive oxygen radicals in response to chemical or microbial stimuli using flow cytometry. 31

Briefly, Lyapina et al. (2004) studied 29 workers who were occupationally exposed to 32

formaldehyde for an average of 12.7 years through contact with carbamide

- formaldehyde glue 33

with a mean value of the average shift concentration of formaldehyde reported a s 0.71 ppm 34

TWA with a range of 0.32 to 1.57 ppm. The workers were divided into two subgroups, one (n = 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 35 12) that suffered from either a long history (with clinical findings) of chronic mucous 1

inflammation of the URT with multiple relapses, and a second gro up (n = 17) whose URT 2

inflammations were short, acute, and predominantly viral. Twenty

- one healthy subjects served 3

as controls. A suite of hematological tests and flow cytometric analysis for respiratory burst 4

activity were performed. Although no signif icant difference was observed in the spontaneous 5

and stimulated NRBA (median percentage of oxidizing cells) between the 29 exposed workers 6

with URT inflammation and the healthy controls (0.83 versus 1.35, respectively), a separate 7

comparison of the NRBA of 12 workers with chronic, repeating URT infections and 17 workers 8

with short, infrequent episodes of URT inflammations was significant (0.45 versus 1.00, p = 9

0.037). When the NRBA of the group with chronic URT infections (n = 12) was separately 10

compared w ith that of the healthy controls (n = 21), the results were also significant (0.45 versus 11

1.35, p = 0.012). Individuals with chronic URT infections have reduced NRBA that could be 12

due to formaldehyde exposure. Neutrophils respond to tissue damage or loca l invasion of 13

microorganisms and act to phagocytize foreign cells. If neutrophilic activity is hampered or 14

altered by formaldehyde exposure, then the ability to fight infection will be diminished, leading 15

to prolonged infection. However, no dose

- response pattern of formaldehyde exposure could be 16

determined from this study. 17

Other investigators have reported chromosomal damage in immune cells due to 18

formaldehyde (Orsière et al., 2006; Yu et al., 2005). Yu et al. (2005) evaluated chromosomal 19

damage in lymp hocytes from 151 exposed and nonexposed workers from a plywood factory 20

detected by comet assay. The authors reported that chromosomal damage was statistically 21

elevated in lymphocytes from formaldehyde

- exposed workers compared with controls. 22

However, no i nformation on exposure duration or levels was provided. Orsière et al. (2006) 23

studied DNA damage in lymphocytes from 59 hospital employees with formaldehyde exposures 24

from pathology and anatomy laboratories in five hospitals. Controls were 37 workers fro m the 25

same hospitals, matched on gender, age, and smoking habits, with no known exposure to 26

genotoxic agents. Study participations were excluded if workers had a history of radio

- or 27

chemotherapy or had used therapeutic medications that were known to be m utagenic. 28

Occupational exposure was determined through 15

- minute and 8

- hour personal air sampling 29

during a typical workday. Mean formaldehyde concentrations were 2 ppm (range: <0.1

– 30

20.4 ppm) for 15

- minute sampling and 0.1 ppm (range: <0.1

– 0.7 ppm) for 8

- hour sampling. No 31

change in DNA damage was found between the beginning and end of the workday among 32

exposed workers (3.9 ± 0.6 versus 3.6 ± 0.5 relative light units/ng DNA). However, exposed 33

workers had significant elevations in the binucleated micronucl eated cell rate (BMCR) per 34

1,000 cells compared with controls (16.9 ± 9.3 versus 11.1 ± 6.0%; p < 0.001), but BMCR did 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 36 not appear to be correlated with formaldehyde concentration. Linear regression analysis showed 1

that the effect for exposure remained aft er adjusting for gender, age, smoking, and drinking 2

habits. For 18 exposed and 18 control workers who underwent cytokinesis

- blocked 3

micronucleus assay (CBMA) combined with fluorescent in situ hybridization (FISH) with pan

4

centromeric DNA probe, results sh owed that the frequency of micronuclei (MN) containing only 5

one centromere (C1+MN) was elevated among the exposed compared with unexposed workers 6

(11.0 ± 6.2% versus 3.1 ± 2.4%; p < 0.001). The effect of exposure remained significant after 7

controlling for gender, age, smoking, and drinking habits. Results from Yu et al. (2005) and 8

Orsière et al. (2006) suggest that formaldehyde exposure may promote chromosomal damage 9

leading to micronucleated lymphocytes. 10

Compromised lymphocyte function may significantly contribute to altered immune 11

status. The mechanism underlying this effect has not been elucidated. 12

13

4.1.1.5.3. Sensitization and atopy. Numerous studies have documented formaldehyde

- induced 14

exacerbation of asthmatic responses (see Section 4.1.1.2). Th e mechanism of this effect has not 15

been clarified and has led investigators to assess the potential for formaldehyde to directly induce 16

formation of formaldehyde

- specific antibodies, leading to allergic responsiveness. One case 17

report showed systemic alle rgic reactions (e.g., anaphylaxis) to formaldehyde in a patient 18

undergoing hemodialysis (Maurice et al. [1986] referenced in Thrasher et al. [1990]). Some 19

studies have evaluated the potential association of formaldehyde

- specific IgE in already

20

sensitized individuals (Baba et al., 2000; Palczynski et al., 1999). Other studies have 21

investigated whether formaldehyde can directly induce IgE in nonsensitized individuals. Most of 22

the studies have not identified presence of formaldehyde

- specific IgE ( Ohmichi et al., 2006; 23

Krakowiak et al., 1998; Grammer et al., 1993, 1990; Kramps et al., 1989; Thrasher et al., 1987) 24

and are summarized below. A few studies (Vandenplas et al., 2004; Doi et al., 2003; Liden et 25

al., 1993) reported positive IgE against formaldehyde, associated with exposure, but the IgE 26

titers were either transient (Vandenplas et al., 2004) or were positive in a small subset of 27

previously sensitized subjects (2 of 15) (Liden et al., 1993). Doi et al. (2003) detected IgE 28

against formaldehyde in two a sthmatic children (out of 122 asthmatic children), but the response 29

severity did not correlate with exposure level. 30

Palczynski et al. (1999) evaluated whether exposure to formaldehyde might facilitate 31

specific sensitization to common allergens. The study population was comprised of residents of 32

apartments built in 1989

– 1990. Only households with children from 5

– 15 years were eligible for 33

the study. A random sample of 120 apartments was selected in which lived a total of 465 34

persons aged 5

– 65 years. Indi vidual demographic characteristics and medical histories were 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 37 determined by questionnaire. Residents were tested, using the skin

- prick method, for allergen 1

response to a variety of materials, such as household dust, pollens, and feathers. Total serum IgE 2

levels were measured, and the presence of formaldehyde

- specific IgE antibodies was determined. 3

Measured mean levels of formaldehyde were 21.05 ± 8.94 ppb. No significant relationship 4

between respiratory allergy prevalence and indoor exposure to formalde hyde was detected. 5

Significant increases in serum IgE levels were found in children exposed to both environmental 6

tobacco smoke and formaldehyde. 7

Baba et al. (2000) investigated whether production of formaldehyde

- specific IgE could be 8

detected in adult as thmatics. Formaldehyde exposure levels were not documented. 9

Formaldehyde

- IgE was detected in two asthmatic patients (n = 80), one male and one female, but 10

the titer of IgE did not parallel the severity of the asthmatic responses and could not be linked t o 11

formaldehyde exposure. Thus, formaldehyde

- specific IgE

- mediated allergy was rare in adult 12

chronic asthmatics. 13

Several studies have examined serum for formaldehyde

- specific IgE antibodies in groups 14

of formaldehyde

- exposed humans ( Ohmichi et al., 2006 ; Kr akowiak et al., 1998; Wantke et al., 15

1996a, b; Salkie, 1991 ; Grammer et al., 1990 ; Kramps et al., 1989). While formaldehyde

16

specific IgE was reported in one study (Wantke et al., 1996a), results from most other studies 17

failed to find a consistently strong association between formaldehyde

- specific IgE or IgG 18

antibodies in groups of formaldehyde

- exposed humans. 19

Wantke et al. (1996a) detected elevated levels of formaldehyde

- specific IgE in 24 of 62 20

8

- year

- old children who were students in three particleboard

- paneled classrooms in which the 21

estimated formaldehyde air concentrations were 0.075, 0.069, and 0.043 ppm. In a health 22

survey, the children reported headaches (29/62), fatigue (21/62), dry nasal mucosa (9/62), rhinitis 23

(23/62), cough (15/62), and noseble eds (14/62). The number of children with symptoms in each 24

classroom decreased with decreasing formaldehyde concentration (49, 47, and 24, respectively, 25

for the 0.075, 0.069, and 0.043 ppm classrooms). However, the investigators reported that 26

elevated lev els of specific IgE did not correlate with the number and severity of symptoms. 27

When the children were evaluated after 3 months in a new school that did not have particleboard 28

paneling and had lower ambient formaldehyde concentrations (0.029, 0.023, and 0 .026 ppm), the 29

number of children reporting symptoms decreased significantly from earlier figures, and, when 30

measured in 20 of the children, the mean serum levels of formaldehyde

- specific IgE declined 31

significantly compared with pre

- moving mean levels. 32

In contrast, a study by Krakowiak et al. (1998) measured serum IgE levels in asthmatic 33

and healthy subjects as part of a larger study to characterize the mechanism of 34

formaldehyde

- induced nasal and bronchial response in asthmatic subjects with suspected 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 38 forma ldehyde allergy. Ten subjects reported to have formaldehyde rhinitis and asthma and 1

10 healthy subjects underwent a 2

- hour inhalation challenge in an exposure chamber with 2

formaldehyde at a concentration of 0.5 mg/m 3 (0.41 ppm). Formaldehyde

- specific ser um IgE 3

antibodies were measured, and cellular, biochemical, and mediator changes were assessed in 4

nasal lavage before, immediately after, and at 4 and 24 hours after challenge. Challenges with 5

formaldehyde caused only transient symptoms of rhinitis in bot h groups. Furthermore, none of 6

the subjects thought to have occupational asthma developed clinical symptoms of bronchial 7

irritation. No specific IgE antibodies to formaldehyde were detected in persons with 8

occupational exposure to formaldehyde. No diffe rences in the nasal response to formaldehyde 9

were found between subjects reported to have occupational allergic respiratory diseases and 10

healthy subjects ( p > 0.05). The study showed that inhaled formaldehyde at a level as low as 11

0.5 mg/m 3 did not induce a specific allergic response either in the upper or in the lower part of 12

the respiratory tract. In addition, it demonstrated that there was no difference in nasal response 13

to formaldehyde between asthmatic subjects occupationally exposed to formaldehyde a nd 14

healthy subjects. 15

Similarly, formaldehyde

- specific IgE antibodies were detected in only 1 serum sample 16

(out of 86) from four groups of formaldehyde

- exposed subjects (Kramps et al., 1989). The 17

subject with detected formaldehyde

- specific IgE displayed al lergic symptoms. The groups 18

included (1) 28 subjects living or working in places with formaldehyde

- containing construction 19

materials (e.g., chipboard) and estimated formaldehyde concentrations ranging from 0.08 to 20

0.37 ppm, (2) 18 occupationally exposed s ubjects from an anatomy laboratory and in other 21

unspecified industries where air concentrations were not measured, (3) 12 hospital attendants 22

who worked with formaldehyde

- sterilized hemodialysis equipment, and (4) 28 hemodialysis 23

patients coming into conta ct with equipment sterilized with formaldehyde. Other subjective 24

symptoms, such as headache, eye irritation, and respiratory complaints, were reported by 25

24/28 subjects in the construction material group and confirm that formaldehyde is an irritant 26

(revie wed in Section 4.1.1.1). Durations of exposure or length of employment were not reported 27

for the subjects in this study. 28

Grammer et al. (1990) studied the immunologic nature of formaldehyde sensitivity in 29

37 workers who were examined by a group of physici ans in response to complaints of 30

formaldehyde

- related illness. Air sampling of formaldehyde ranged from 0.003 to 0.078 ppm, 31

but specific levels were not tied to specific workplace areas. Blood samples were collected and 32

assayed for IgE and IgG activity a gainst formaldehyde. None of the workers had IgG activity 33

against formaldehyde. No IgE antibodies were detected in the other 32 workers. The authors 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 39 concluded that there was no evidence of an immunologically mediated response to formaldehyde 1

in this gro up of workers. 2

Formaldehyde

- specific IgE was not detected in any of a group of 45 medical students 3

before or after the students attended a 4

- week anatomy dissecting course (Wantke et al., 1996b). 4

Estimates of ambient air concentrations of formaldehyde ran ged from 0.059 to 0.219 ppm (0.124 5

± 0.05 ppm; mean ± SD). However, the survey revealed frequencies of irritation symptoms that 6

were consistent with other studies (e.g., itching of the skin in 33/45 students, headache in 15/45, 7

and burning eyes in 13/45). 8

Similarly, Ohmichi et al. (2006) were unable to correlate formaldehyde exposure with 9

specific IgE production among eight students attending a gross anatomy laboratory. 10

Formaldehyde exposure was estimated to range from 0.33 to 1.47 ppm during the laborato ry 11

sessions. The sample size was small, and IgE levels varied substantially (ranging from <19 to 12

>5,000 international units/mL). Compared with IgE levels taken 90 minutes prior to the start of 13

the first session, IgE levels measured shortly after the last session and up to 23 days following 14

the last session showed no association with exposure. 15

Salkie (1991) investigated the prevalence of formaldehyde

- specific IgE in practicing 16

pathologists who complained of formaldehyde sensitivity. Exposure levels were n ot reported. 17

Serum samples were assayed for total IgE and formaldehyde

- specific IgE. Of the 46 subjects, 18

29 self

- reported atopy that was confirmed in 12 subjects by positive IgE. Moreover, 29 subjects 19

complained of formaldehyde

- specific sensitivity. Ho wever, zero subjects had formaldehyde

20

specific IgE, and there was no evidence that atopic individuals were more sensitive to 21

formaldehyde than non

- atopic individuals. The authors noted that atopic individuals may have 22

selectively reduced their exposure to formaldehyde. 23

Vandenplas et al. (2004) evaluated a case study of a 31

- year

- old male who was 24

accidentally exposed to formaldehyde for 2 hours. The exposure level was not provided. The 25

subject had smoked a pack of cigarettes a day for 13 years and was adm itted to the emergency 26

room for asthmatic symptoms. Eight days following exposure, increased levels of 27

formaldehyde

- specific IgE antibodies were detected but could not be detected in subsequent 28

assessments. 29

A clinical study by Liden et al. (1993) evaluate d IgE

- specific antibodies against 30

formaldehyde in 23 subjects who had previously tested positive for skin sensitization by skin 31

prick test. Subjects were exposed to formaldehyde by skin patch (1% formaldehyde in water). 32

Ten of the subjects were classifie d as atopic Though 15 of 23 of the sensitized subjects were 33

also sensitive to formaldehyde applied by skin patch, formaldehyde

- IgE was positive in 2 of 15 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 40 individuals who were not classified as atopic. No dose

- response relationship could be 1

determined f rom the study design of this study. 2

Doi et al. (2003) conducted a clinical study in 155 children of which 122 were 3

asthmatics. No specific exposure to formaldehyde was documented. IgE against formaldehyde 4

was determined in blood. Formaldehyde

- specific I gE was found in two asthmatic children. 5

Thus, while several studies have documented formaldehyde

- specific IgE, the occurrence is rare 6

and may be transient. Asthmatic children may be more predisposed to form formaldehyde

7

specific IgE than non

- atopic indiv iduals or adults. The formation of formaldehyde

- specific IgE 8

is quite rare. 9

10

4.1.1.5.4. F ormaldehyde

- albumin and formaldehyde

- heme complexes

. Numerous studies have 11

shown that formaldehyde can bind to blood proteins as formaldehyde

- heme and formaldehyde

12

human serum albumin (formaldehyde

- HSA) complexes (Carraro et al., 1997; Grammer et al., 13

1993, 1990; Dykewicz et al., 1991; Thrasher et al., 1990). Kim et al. (2001) failed to identify 14

IgE against formaldehyde

- HSA complexes in one case

- control subject foll owing industrial 15

occupational exposure to formaldehyde. These complexes may serve to traffic formaldehyde 16

throughout the bloodstream and throughout the body. While formaldehyde may be too small to 17

engender an immune response, these complexes may be able to trigger formaldehyde

- protein

18

specific antibodies, leading to an immune response, including sensitization. 19

Thrasher et al. (1990) evaluated five groups of subjects as follows with varying levels 20

and durations of formaldehyde exposure: asymptomatic chirop ractic students exposed during 21

anatomy classes (controls with only intermittent exposure to formaldehyde), mobile home 22

residents, office workers, patients with multiple symptoms who had been removed from the 23

source of formaldehyde for at least a year, and occupationally exposed patients. All groups were 24

assessed for production of antibodies against formaldehyde

- HSA. The level of autoantibodies 25

was significantly elevated in patients exposed long

- term to formaldehyde. From the data, 26

Thrasher et al. (1990) concluded that exposure to formaldehyde stimulates IgG antibody 27

production to formaldehyde

- HSA. 28

Grammer et al. (1990) studied the immunologic nature of formaldehyde sensitivity in 29

37 workers who were examined by a group of physicians in response to complai nts of 30

formaldehyde

- related illness. Air sampling of formaldehyde ranged from 0.003 to 0.078 ppm, 31

but specific levels were not tied to specific workplace areas. Blood samples were collected and 32

assayed for IgE and IgG activity against formaldehyde and fo rmaldehyde

- HSA. None of the 33

workers had IgG activity against formaldehyde. Five workers had IgE against both HSA alone 34

and against formaldehyde

- HSA complexes. No IgE antibodies were detected in the other 32 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 41 workers. The authors concluded that there was no evidence of an immunologically mediated 1

response to formaldehyde in this group of workers. 2

Grammer et al. (1993) described the evaluation of a worker with bronchospasm 3

symptoms caused by formaldehyde exposure. The worker was evaluated by means of ELIS A, 4

cutaneous tests, and methacholine and formaldehyde inhalation challenges. The ELISA showed 5

that the worker had positive IgE and IgG titers to formaldehyde

- HSA. The worker also had a 6

positive cutaneous test for formaldehyde

- HSA but a negative methachol ine challenge at 25 7

mg/mL and negative formaldehyde inhalation challenges at exposure concentrations of 0.3, 1, 3, 8

and 5 ppm for 20 minutes. The worker might have developed a positive response if a higher 9

concentration of formaldehyde had been used for th e challenge, but it is more probable that the 10

worker’s symptoms were not caused by immunologically mediated asthma. 11

Dykewicz et al. (1991) evaluated whether IgE or IgG antibodies to formaldehyde were 12

related to formaldehyde exposure or to respiratory sympt oms arising from such an exposure. 13

The authors studied 55 potentially exposed subjects (hospital histology technicians, internal 14

medicine residents, pathology residents, current smokers, and persons with known workplace 15

exposure to formaldehyde) and compa red them to controls with no history of formaldehyde 16

exposure. Reported workplace formaldehyde concentrations were 0.2

– 0.64 ppm for pathology 17

residents, 0.64 ppm for histology technicians, and 0.6

– 11 ppm for miscellaneous formaldehyde 18

exposure scenarios. Workplace air concentrations were not measured for the other occupations. 19

Occupational exposure to formaldehyde averaged 12.45 years for histology technicians, 20

0.38 years for medical residents, 3.21 years for pathology residents, and 18.34 years for five 21

subjects exposed to formaldehyde in miscellaneous workplaces. Blood samples were analyzed 22

for IgE and IgG reactivity with formaldehyde

- HSA complexes. Three subjects had IgE against

- 23

HSA; these three and two others had low levels of anti

- formaldehyde

- HSA IgG. The presence of 24

IgG and IgE antibodies to formaldehyde was not clearly related to formaldehyde exposure or 25

pack

- years of smoking. One subject had both IgE and IgG antibodies and also suffered from eye 26

and respiratory symptoms when exposed to formal dehyde at his workplace. However, the 27

authors concluded that they could not establish a relationship between IgE and IgG levels and 28

formaldehyde exposure. This study has several limitations. First, the volunteers (hospital 29

workers) may not be representa tive of exposed workers in the general population. One of the 30

exposure groups comprised cigarette smokers. Although the study focused on formaldehyde 31

antibodies, which would be unaffected by the other chemicals, respiratory symptoms among 32

smokers would r eflect exposures to the constituents of smoke. Dykewicz et al. (1991) concluded 33

that immunologically mediated asthma caused by formaldehyde is extremely rare and may not 34

exist at all. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 42 Carraro et al. (1997) reported development of a reliable assay to effec tively measure 1

formaldehyde

- HSA complexes in smokers, ex

- smokers, and nonsmokers. A correlation between 2

formaldehyde

- HSA antibodies and smoking status was detected. This study did not correlate 3

formaldehyde exposure and formaldehyde

- HSA antibodies. 4

Give n that formaldehyde is a sensory irritant that is particularly bothersome to 5

individuals with compromised lung function or asthma, numerous studies have assessed the 6

ability of formaldehyde to induce immunotoxic effects. Some studies have documented 7

incre ased rates of chronic bronchitis and URT infections associated with exposure to 8

formaldehyde, which suggests a possible immunomodulatory effect. However, of the numerous 9

articles that have investigated systemic immunomodulatory effects due to formaldehyde 10

(Lyapina et al., 2004; Gorski et al., 1992; Thrasher et al., 1990, 1988, 1987; Pross et al., 1987), 11

few have reported significant immune modulation related to formaldehyde exposure. Significant 12

decreases in specific adaptive immune cell populations do no t appear correlated to formaldehyde 13

exposure (Erdei et al., 2003; Gorski et al., 1992; Thrasher et al., 1990, 1987; Pross et al., 1987). 14

Thus, the tendency for increased infection rates associated with formaldehyde may not be related 15

to altered immune fun ction. Perhaps altered mucociliary clearance and disturbed mucosal barrier 16

may provide greater access for pathogens and result in greater infection rates. Moreover, 17

formaldehyde has been associated with exacerbation of asthmatic or atopic responses, 18

part icularly in sensitized individuals. However, this effect does not appear to occur by increased 19

IgE or formaldehyde

- specific IgE levels (Ohmichi et al., 2006; Palczynski et al., 1999; 20

Krakowiak et al., 1998; Wantke et al., 1996b; Salkie, 1991; Grammer et a l., 1990; Kramps et al., 21

1989). Thus, formaldehyde

- associated enhanced allergic responses does not appear to be due to 22

direct induction of sensitization and may not occur via an immunologic mechanism. Lastly, the 23

formation of formaldehyde

- heme and formal dehyde

- HSA complexes has been well documented 24

(Grammer et al., 1993, 1990; Dykewicz et al., 1991; Thrasher et al., 1990) and may serve as a 25

biomarker of exposure (Carraro et al., 1997). Moreover, these complexes may provide a means 26

by which formaldehyde t ravels throughout the bloodstream and may drive antibody formation 27

that may lead to immune activation. 28

29

4.1.1.6. Neurological/Behavioral 30

There is some suggestion of neurological impairment in humans following occupational 31

exposure to formaldehyde; the dat a are limited and the results from several studies are potentially 32

confounded by exposure to other solvents. Two studies of histology technicians with 33

occupational exposure to formaldehyde and other solvents found neurological deficits and poorer 34

performa nce on neurocognitive tests associated with formaldehyde exposure (Kilburn et al., 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 43 1987, 1985). In another study, Kilburn and Warshaw (1992) found no change from initial 1

performance, for as long as 4 years, in follow

- up evaluations of histology technician s with 2

continuing exposure to formaldehyde. In a preliminary report from a prospective study, 3

Weiskopf et al. (2009) found a strong association between duration of formaldehyde exposure 4

and death from amyotrophic lateral sclerosis (ALS). In a controlled exposure study, Bach et al. 5

(1990) found that, when workers with chronic formaldehyde exposure were challenged with an 6

acute formaldehyde exposure, they exhibited poorer performance on some neurocognitive tests 7

compared with workers without chronic exposur e undergoing the same acute challenge 8

conditions. In another controlled exposure study, Lang et al. (2008) found equivocal changes in 9

reaction time following an acute exposure. 10

11

4.1.1.6.1. Epidemiological studies. Kilburn et al. (1985) reported that a group of 76 female 12

histology technicians displayed statistically significantly greater frequencies of neurobehavioral 13

deficits (lack of concentration and loss of memory, disturbed sleep, impaired balance, variations 14

in mood, and irritability), than did a r eferent group of 56 unexposed female clerical workers. 15

The technicians had been employed from 2 to 37 years (mean 12.8 years). Analysis of 16

workplace air samples indicated the presence of several solvents, ranging from 0.2 to 1.9 ppm for 17

formaldehyde, 3.2 to 102 ppm for xylene, 2 to 19.1 ppm for chloroform, and 8.9 to 12.6 ppm for 18

toluene. Subsequently, Kilburn et al. (1987) administered a battery of 10 tests to 305 female 19

histology technicians to assess various aspects of cognitive and motor function. T he researchers 20

analyzed the results by regression analysis with age, years of smoking, and hours per day of 21

exposure to formaldehyde and other solvents as explanatory variables. Increased daily hours of 22

exposure to formaldehyde were significantly correlat ed with decreased performance in several 23

tests (including several types of memory, dexterity, and balance), whereas hours of daily 24

exposure to other solvents were only correlated with decreased performance in a single memory 25

test. In a later prospective s tudy of performance, 318

– 494 histology technicians were tested in a 26

battery of neurobehavioral tests, and testing for a subset of subjects was repeated yearly for up to 27

4 years. No statistically significant decrement in performance was found when initial test results 28

were compared with retest results to evaluate effects of continuing occupational exposure to 29

formaldehyde (or other solvents) or possible effects of aging (Kilburn and Warshaw, 1992). 30

Kilburn (1994) later reported that three anatomists and on e railroad worker, occupationally 31

exposed to airborne formaldehyde for 14

– 30 years, each showed impaired performance on 32

several neurobehavioral tests (e.g., choice reaction time, abnormal balance, digit symbol, and 33

perceptual motor speed). 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 44 Weisskopf et al. (2009) evaluated the association between chemical exposure and death 1

from ALS, using the cohort of 987,229 people from the prospective Cancer Prevention Study II 2

of the American Cancer Society. From 1989

– 2004, 1,156 deaths from ALS were identified from 3

m ortality records from the National Death Index. Exposure assessment occurred prior to follow

4

up and was based on a questionnaire; participants were asked about current exposure to 12 types 5

of chemicals and whether they had been regularly exposed in the pa st. After controlling for a 6

number of potentially confounding factors (including age, sex, smoking status, military service, 7

education, alcohol intake, occupation, vitamin use, and exposure to other chemicals), it was 8

found that exposure to formaldehyde f or a known duration was statistically significantly 9

associated with increased risk of death from ALS ( p < 0.0001) with a relative risk (RR) of 2.47 10

(95% CI: 1.58

– 3.86) based on 22 deaths. Weisskopf et al. (2009) reported that the association 11

had a strongl y significant dose

- response relationship, with increased duration of exposure 12

associated with increased RR of ALS mortality with a reported p value for continuous trend of 13

0.0004. Multivariate adjusted rate ratios were 1.5 for known formaldehyde exposures less than 14

4 years, 2.1 for 4

– 10 years, and 4.1 for >10 years. Although the authors indicated that these 15

results need independent verification, the results of this study of the nearly one million people 16

followed for 15 years is unlikely to be biased due t o its longitudinal design. 17

18

4.1.1.6.2. Controlled exposure studies. Bach et al. (1990) examined whether cognitive and 19

motor performance of humans responded acutely to formaldehyde exposure and whether 20

previous chronic exposure to formaldehyde affected th e responses observed following acute 21

exposure. Thirty

- two men with at least 5 years of occupational exposure to formaldehyde and 22

29 matched controls were exposed to formaldehyde at concentrations of 0.04, 0.21, 0.48, or 23

1.10 ppm for 5.5 hours. During the exposure period, symptoms were assessed by using a 24

standardized questionnaire, and subjects were evaluated in four tests designed to estimate several 25

aspects of cognitive function. Testing was performed once prior to exposure and twice during 26

the exposur e period. The authors noted that the typical dose

- related symptoms of respiratory 27

irritation were not seen in this study. Previously unexposed subjects reported more headaches, 28

“heavy head,” and physical tiredness than the exposed workers. In both occup ationally exposed 29

and unexposed subjects, decreased performance in an addition test was significantly correlated 30

with increasing concentration of formaldehyde. Compared with previously unexposed subjects, 31

occupationally exposed subjects showed significant ly decreased performance in three other tests, 32

although the effect was not dose related. The study did not adjust for several potential 33

confounders, including prior exposure to other chemical agents, and the age and health status of 34

the cases and controls . Authors concluded that their data indicated that acute exposure to 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 45 formaldehyde might cause acute effects on CNS functions but that more investigation was 1

needed to verify their results. 2

In a study evaluating chemosensory irritation, Lang et al. (2008) assessed possible 3

changes in reaction time during an acute (4

- hour) exposure to formaldehyde concentrations 4

between 0

– 0.5 ppm (some exposure sessions also included short concentration peaks of up to 5

1 ppm) with or without a masking agent (ethyl acetate). Twenty

- one healthy volunteers were 6

exposed once per day to each of 10 different exposure combinations in random order (for a total 7

of 10 sessions per subject). Reaction time was tested before and after each exposure session. 8

Significant increases in reac tion time were seen at 0.3 ppm formaldehyde, with or without 9

masking agent, but not at 0.5 ppm. The significance of these findings is unclear. 10

Performance of 16 healthy volunteers on addition, multiplication, and card punching 11

tasks was measured by Anders en and Molhave (1983) before and during a 5

- hour exposure to 12

formaldehyde at concentrations up to 2 mg/m 3 . The authors reported that formaldehyde 13

exposure had no effect on performance, but results were not presented. 14

15

4.1.1.6.3. Summary. The limited inf ormation currently available from human studies does not 16

permit a definitive conclusion regarding an association between formaldehyde exposure and 17

human neurotoxicity. There is, however, sufficient information to raise a serious concern for this 18

type of e ffect, and additional studies are needed. 19

20

4.1.1.7

. Developmental and Reproductive Toxicity 21

Epidemiologic studies suggest a convincing relationship between occupational exposure 22

to formaldehyde and adverse reproductive outcomes in women. Several of these studies deal 23

with spontaneous abortion following maternal occupational formaldehyde exposure (Taskinen et 24

al., 1999, 1994; John et al., 1994; Seitz and Baron, 1990; Hemminki et al., 1985, 1982; Axelsson 25

et al., 1984), but not all reported a significant as sociation between exposure and spontaneous 26

abortion. A study of fecundability found an increase in time to pregnancy among female 27

workers exposed to formaldehyde (Taskinen et al., 1999). Three studies that examined the effect 28

of occupational exposures on the incidence of congenital malformation produced mixed results 29

(Dulskiene and Gražulevičiene, 2005; Taskinen et al., 1994; Hemminki et al., 1985). A 30

population

- based, semi

- ecologic study found an association between atmospheric formaldehyde 31

exposure and low birth weight (Gražulevičiene et al., 1998). 32

33

4.1.1.7.1. Spontaneous abortion. Several epidemiologic studies report a relationship between 34

occupational exposure to formaldehyde and increases in risk of spontaneous abortion following 35

maternal occupa tional formaldehyde exposure (Taskinen et al., 1999, 1994; John et al., 1994; 36This document is a draft for review purposes only and does not constitute Agency policy.

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- 46 Seitz and Baron, 1990; Axelsson et al., 1984). Increased RRs were in the range of 1.7 to more 1

than 3.0. However, other studies (Hemminki et al., 1985, 1982) found no associatio n between 2

occupational formaldehyde exposure and spontaneous abortion. Paternal occupational exposure 3

to formaldehyde was not related to spontaneous abortion (Lindbohm et al., 1991). 4

The earliest report of an association between spontaneous abortion and f ormaldehyde 5

exposure comes from a Swedish cohort study of female laboratory workers (Axelsson et al., 6

1984). Subjects were women born in 1935 or later and worked in a university laboratory during 7

1968

– 1979. There were 745 women who responded to a mailed questionnaire (response rate = 8

95%), 556 of whom reported on 1,180 pregnancies that resulted in 997 births. Exposure to 9

formaldehyde was estimated based on answers to the questionnaires. Formaldehyde exposure 10

was reported only in connection with 10 pregn ancies, of which 5 went to term, 3 were reported 11

as miscarriages, and 2 were terminated by induced abortion. Excluding the latter, the 12

spontaneous abortion rate among women exposed to formaldehyde in the first trimester was 3/8 13

(37.5%) compared with 14/14 8 (9.5%) in the population of laboratory workers not exposed to 14

any solvent in the first trimester. 15

While not computed by the authors, the OR can be calculated as 5.7 (95% CI: 1.2

– 26.6). 16

The exposure assessment on which this result is based was methodolog ically weak but unlikely 17

to be a source of bias. Given the exploratory nature of this study, potential confounders were not 18

controlled for, but no other co

- exposure was more strongly related to the increased risk of 19

miscarriage, so this result is not like ly to be explained by confounding. Selection bias is also an 20

unlikely explanation given the high participation rate. However, although this association of an 21

increased risk of pregnancy loss with formaldehyde exposure is statistically significant, the CI is 22

wide and chance may be a possible explanation for this finding. 23

In a 1988 Health Hazard Evaluation, the National Institute for Occupational Safety and 24

Health (NIOSH) investigated complaints of adverse reproductive outcomes at a plant where 25

work pants w ere cut and sewn with a fabric that was treated with a resin that releases 26

formaldehyde (Seitz and Baron, 1990). In a NIOSH laboratory, the fabric released 163 to 1,430 27

μg of formaldehyde/gram of cloth. TWA personal breathing space formaldehyde levels ranged 28

from trace to 0.46 ppm, while workstation values ranged from 0.32 to 0.70 ppm. The 29

investigators studied the outcomes by using a mailed questionnaire. The response rate for 30

current employees was 98%. There were 296 pregnancies among a cohort of 188 women. The 31

investigators found increased rates of spontaneo us abortion, premature birth, and congenital 32

malformations. The crude rate of spontaneous abortion was 21% among women working at the 33

plant while pregnant (4 of 19 pregnancies), 15% among women employed elsewhere while 34

pregnant (11 of 71 pregnancies), and 5% among women at home while pregnant (10 of 206 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 47 pregnancies). The investigators did not explain how workers employed elsewhere or at home 1

during pregnancy were categorized compared with current workers, nor did they calculate RRs. 2

As calculated from da ta presented in Table 5 of the monograph, the crude OR (not corrected for 3

multiple observations per woman) for those pregnant while currently working at the plant 4

compared with all others was 3.2 (95% CI: 0.8

– 12). There were also excess congenital 5

malform ations (13 versus 2%) and premature births (13 versus 4%) among the live births (both 6

based on two births each in the exposed group) from the women who were pregnant while 7

employed at the textile plant compared with women who stayed at home. After the NIO SH 8

investigation, changes were made in the plant to improve ventilation. 9

Because the report provides insufficient details of the methodology and the fact that there 10

was no personal exposure classification in this study, it is difficult to validate the fi ndings in this 11

report. The results did not take into account other potential risk factors for spontaneous abortion 12

or correct for multiple pregnancies per woman. Furthermore, the marked differences between 13

the “home” and “work” pregnancies were difficult to interpret. 14

A cohort study of effects of paternal occupational exposures in Finland by Lindbohm et 15

al. (1991) found that exposure to formaldehyde had little effect on the rate of spontaneous 16

abortions among 99,186 pregnancies listed in the national hosp ital discharge register. The 17

analysis was limited to births/spontaneous abortions in 1976 and from May 1980 to April 1982. 18

Spontaneous abortion incidence came from the hospital discharge register and data from 19

outpatient clinics. There were 808 pregnant wives among potentially formaldehyde

- exposed 20

fathers. Exposure to formaldehyde was based on employment information listed in the Finnish 21

1980 census. Compared with pregnancies among wives of unexposed spouses, the age and 22

socioeconomic level

- adjusted OR s were 1.1 for low paternal exposure to formaldehyde and 1.0 23

for moderate to high paternal exposure. Paternal occupational exposures to ethylene oxide, 24

gasoline/benzene, and rubber industry chemicals were associated with spontaneous abortion. 25

The authors hypothesized that the mode of action (MOA) for spontaneous abortion following 26

male exposure to chemicals is genetic damage to germ cells. 27

The indirect exposure assessment was a substantial limitation of this study. Some 28

confounders in a study of this typ e could not be controlled for (smoking history, previous 29

spontaneous abortions, alcohol use), and census data could not provide completely accurate 30

information, potentially masking associations between paternal formaldehyde exposure and 31

spontaneous abortio n. 32

A case

- control study by Taskinen et al. (1994) of effects of maternal occupational 33

exposure to chemicals used in laboratories in Finland indicated that exposure to formalin, which 34

is a 37% aqueous solution of formaldehyde, was related to an increased ri sk of spontaneous 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 48 abortion. The investigators studied subjects from payrolls of Finnish state

- employed laboratory 1

workers, the laboratory workers’ union, and a register of workers occupationally exposed to 2

carcinogens. These records were cross

- referenced with the hospital discharge register. The 3

investigators selected women who had a single spontaneous abortion during the period 1973

– 4

1986 and two controls who had delivered a baby without malformations. The final sample size 5

was 208 cases and 329 control s after refusals and other exclusions. The response rate was 6

82.4%. 7

Information on occupational exposure, health status, medication, contraception, and 8

pregnancy history came from mailed questionnaires. Industrial hygienists’ construction of an 9

exposure index was based on the subjects’ descriptions of their work assignments, use of 10

solvents including estimates of quantity used, and use of a fume hood. ORs were adjusted for 11

employment, smoking, alcohol consumption, parity, previous miscarriage, birth cont rol failure, 12

febrile disease during pregnancy, and other organic solvents found in laboratory work. 13

Spontaneous abortion was associated with 3

– 5 days per week of formalin exposure (OR 3.5 14

[95% CI: 1.1

– 11.2]). A contemporaneous study of formaldehyde conce ntrations in similar 15

Finnish workplaces (pathology and/or histology laboratories) reported workroom air to range 16

from 0.01 to 7 ppm with a mean of 0.45 ppm formaldehyde (Heikkilä et al., 1991 [as cited in 17

Taskinen et al., 1994]) and that the highest exposu res occurred during emptying of sample 18

containers, dish washing, and preparation of formaldehyde solution. 19

Although the results of this study indicate an increased risk between spontaneous 20

abortion and exposure to formaldehyde/formalin, the women were also exposed to several 21

chemicals concurrently, of which toluene (OR 4.7 [95% CI: 1.4

– 15.9]) and xylene (OR 3.1 [95% 22

CI: 1.3

– 7.5]) were also significantly associated with the incidence of spontaneous abortion. 23

However, the investigators reported that the wome n were more likely to be co

- exposed to 24

formalin and xylene, which would make confounding by toluene less likely, and, since xylene 25

was not as strongly associated with the outcome as was formaldehyde, it too is unlikely to fully 26

explain the reported relatio nship between formaldehyde and increased risk of spontaneous 27

abortion. While it is possible that exposure misclassification may have occurred because of the 28

indirect assessment of workplace chemical exposure, an overall conclusion is that, since the 29

expos ure assessment was conducted by industrial hygienists, it is unlikely that this form of bias 30

will have impacted the results of the study to any great extent. 31

In a U.S. study (John et al., 1994), the results of a case

- control study of cosmetologists 32

also su pported an association between spontaneous abortion and the use of formaldehyde

- based 33

disinfectants. The study population came from the 1988 North Carolina cosmetology license 34

registry. Women on this list who were 22

– 36 years of age were screened to find those who were 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 49 recently pregnant. The cases were full

- time cosmetologists who experienced a spontaneous 1

abortion before gestational week 20 during 1983

– 1988. The most recent spontaneous abortion 2

was used as the reference case. Controls were full

- time c osmetologists who delivered a live 3

infant during the same time period. 4

Information was based on mailed questionnaires. Women were not told the purpose of 5

the study in order to avoid selection and recall bias. Of 8,356 women who received the 6

screening que stionnaires, 72.5% responded. Of those, 1,696 qualified for the study and 73.6% 7

completed a more detailed questionnaire. Among them, 96 women were “absolutely sure” they 8

had a spontaneous abortion and qualified as cases. There were 1,058 live births tha t qualified as 9

controls. Exposure assessment included identification of disinfectants used as well as types of 10

chemicals used on hair, use of gloves, hours worked, number of procedures involving chemicals, 11

and use of manicure products. Presence of formal dehyde in the cosmetology profession in 12

general was confirmed in two NIOSH hazard reports (Almaguer and Klein, 1991; Almaguer and 13

Blade, 1990). ORs were adjusted for age, smoking, pregnancy characteristics, other jobs, hours 14

worked, education (cosmetology school), hours standing per week, number of chemical 15

procedures per week, hair dyes per week, bleachings per week, permanents per week, use of 16

gloves, beauty salon characteristics, and use of alcohol or formaldehyde disinfectants. 17

An elevated OR of 2.1 (9 5% CI: 1.0

– 4.3) was reported with the use of formaldehyde

18

based disinfectants adjusted for maternal characteristics and other workplace exposures. Other 19

chemical exposures were also associated with spontaneous abortion, including number of 20

chemical servic es per week, hair dyes, bleaches, and alcohol

- based disinfectants. Strengths of 21

this study include adjustment for important confounding risk factors for spontaneous abortion, 22

detailed collection of interview

- based information, a favorable response rate, a nd the fact that the 23

index population had a high likelihood of formaldehyde exposure. These data provide overall 24

support for an association between formaldehyde exposure and spontaneous abortion. 25

In a retrospective cohort study by Taskinen et al. (1999) o f female woodworkers in 26

Finland, exposure to formaldehyde was associated with delayed conception and spontaneous 27

abortion. The subjects, recruited from a woodworkers’ union and other businesses involving 28

wood processing, were linked to a national register of births. Women were included if they were 29

born between 1946 and 1975, had a live birth at age 20

– 40 years during 1985

– 1995, had worked 30

in the wood processing industry for at least 1 month, and had first employment in the wood 31

processing industry beginn ing at least 6 months before the index pregnancy. The first pregnancy 32

that fulfilled the above criteria was the index pregnancy. There were 1,094 women with these 33

criteria. Information about personal characteristics, pregnancies, and exposures were coll ected 34

from mailed questionnaires for which the response rate was 64%. After other exclusions 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 50 (primarily infertility history, unknown time to pregnancy, and contraceptive failure), the final 1

sample included 602 women. 2

Estimates of mean daily exposure to fo rmaldehyde were based on measurements taken at 3

the women’s factories of employment during the early 1990s. Where measurements were 4

unavailable, measurements from equivalent industries were used. An exposure index 5

representing a TWA exposure was establish ed for every person in the study based on the 6

concentration of workplace formaldehyde multiplied by the proportion of the workday exposed 7

to formaldehyde. The investigators categorized TWA formaldehyde exposure into categories of 8

low (mean of 18 ppb), med ium (mean of 76 ppb), and high (mean of 219 ppb) exposure. 9

Time

- to

- pregnancy data were analyzed by a discrete proportional hazards regression 10

procedure with, as the outcome, a fecundability density ratio (FDR), in which a ratio of average 11

incidence densiti es of pregnancies for exposed women was compared with that of the employed, 12

unexposed women. As explained by Taskinen et al. (1999), an FDR significantly below unity 13

suggests that conception was delayed. The age

- , employment

- , smoking

- , alcohol consumpti on

- , 14

parity

- , and menstrual irregularity

- adjusted FDR was 0.64 (95% CI: 0.43

– 0.92) for women 15

exposed to high formaldehyde levels compared with the unexposed controls, indicating that there 16

was a substantial delay in time to conception in this group of wome n. Among a subset of women 17

with high exposure who did not use gloves, the FDR was even lower (0.51 [95% CI: 0.28

– 0.92]), 18

suggesting that these results might be explained in part through dermal contact with 19

formaldehyde or might indicate an individual’s fa ilure to follow appropriate precautions, which 20

might have increased inhalation exposures in other ways. Exposure to solvents, wood dust and 21

other dusts, and phenols was not associated with decreased fecundability. 22

The investigators further conducted an an alysis of the risk of spontaneous abortion after 23

carefully including only women who had the same workplace during the year of the spontaneous 24

abortion as they had during the beginning of the time

- to

- pregnancy period. Spontaneous 25

abortion was associated wi th formaldehyde exposure in the low exposure group (OR = 2.4 [95% 26

CI: 1.2

– 4.8]), in the medium exposure group (OR = 1.8 [95% CI: 0.8

– 4.0]), and in the high 27

exposure group (OR = 3.2 [95% CI: 1.2

– 8.3]). Endometriosis was also associated with the 28

highest for maldehyde level (OR = 4.5 [95% CI: 1.0

– 20.0]). 29

This study by Taskinen et al. (1999) was a well

- designed population

- based case

- control 30

study that appears to have been well executed and appropriately analyzed. The study population 31

of Finnish women was well defined and adequately selected so as to allow for meaningful 32

comparisons of health effects between individuals with different levels of exposure to 33

formaldehyde. The participation rate was 64%, which is low enough to raise a concern about the 34

potential f or selection bias. However, the authors noted that selection bias has not influenced the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 51 results of other reproductive epidemiology studies reporting results on smoking, irregular 1

menstruation, and earlier miscarriages, which are known to lengthen the tim e to pregnancy 2

(Bolumar et al., 1996; Sallmén et al., 1995; Baird and Wilcox, 1985). Furthermore, there is no 3

evidence to support conjecture that an individual’s decision to participate in this study would be 4

differential with respect to their workplace f ormaldehyde exposures while being non

- differential 5

with respect to the other exposures of interest, including organic solvents, wood dust, and 6

phenols. Since the women who chose to participate in this study were not likely to be aware of 7

the specific hypo theses under investigation, nor could they have known the formaldehyde 8

exposures that were independently estimated by an industrial hygienist, selection bias is not a 9

likely explanation for the findings of adversity. 10

Data on pregnancy history, including sp ontaneous abortions, were collected by 11

questionnaire. Spontaneous abortion is the most common adverse outcome of pregnancy (Klein 12

et al., 1989), and retrospective self

- report of spontaneous abortion has been found to match well 13

with prospectively collecte d reproductive histories (Wilcox and Horney, 1984). Many 14

spontaneous abortions, however, are missed with self

- reporting with the magnitude likely 15

exceeding 25%, but only rarely do women self

- report false positive events (Wilcox and Horney, 16

1984). The eff ect of such an undercount is to cause a bias towards the null when the likelihood 17

of undercounting is unrelated to formaldehyde exposure. The implication is that the observed 18

association of increased risk of spontaneous abortion associated with occupation al exposure to 19

formaldehyde may be an underestimation of the true risk. 20

Two studies (Hemminki et al., 1985, 1982) specifically assessed the effects of 21

formaldehyde exposure and reported no significant increase in the risk of spontaneous abortion. 22

Hemminki and colleagues (1982) conducted a retrospective cohort study of nurses who were 23

potentially exposed to chemical sterilizing agents, including formaldehyde, ethylene oxide, and 24

glutaraldehyde. The risk of having a spontaneous abortion among the women on t he sterilizing 25

staff was compared with that among the control population of nursing auxiliaries whom the 26

supervisory nurses thought to be unexposed to the chemical sterilizing agents during the previous 27

three decades. However, no measurements of the chemi cal sterilizing agents were taken. 28

Information about exposure to chemical sterilizing agents was obtained from the supervising 29

nurses. When the women were conducting sterilizing procedures during their pregnancies, the 30

frequency of spontaneous abortion w as 15.1% compared with 4.6% for the nonexposed 31

pregnancies among the sterilizing staff. The increased frequency of spontaneous abortion 32

correlated with exposure to ethylene oxide but not with exposure to glutaraldehyde or 33

formaldehyde. The investigators reported that ethylene oxide concentrations have been 34

measured in many sterilizing units in Finnish hospitals; 8

- hour weighted mean concentrations 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 52 have ranged from 0.1 to 0.5 ppm with peak concentrations up to 250 ppm (measurements by the 1

Finnish Institute of Occupational Health) (Hemminki et al, 1982). No measurements of 2

glutaraldehyde concentrations were available. Hemminki et al. (1982) reported that exposure to 3

formaldehyde in the sterilization units may be minimal, particularly when gas chambers are used. 4

The range of formaldehyde concentrations measured in sterilizing units has been reported as 5

0.03

– 3.5 ppm. 6

It is not clear that the unexposed women who served as controls were an appropriate 7

comparison group to the sterilizing staff. The investigat ors reported that, among the sterilizing 8

staff, those women who were unexposed during pregnancy experienced a rate of spontaneous 9

abortion of 4.6% but that, among the comparison population of nursing auxiliaries who were 10

presumed to be unexposed, the rate of spontaneous abortion was 10.5%. Had the nursing 11

auxiliaries been an appropriate comparison group, it would be expected that their rate of 12

spontaneous abortion would be similar to the unexposed sterilizing staff. Given this anomaly in 13

study design and the unknown concentrations of formaldehyde exposure that were assessed as 14

positive or negative by supervisory nurses regarding occupational exposures in the previous 15

30 years, it is concluded that this report of no association between formaldehyde exposure and 16

the risk of spontaneous abortion does not temper the conclusion that formaldehyde exposure has 17

been shown to increase the risk of spontaneous abortion. 18

A second study by the same lead author (Hemminki et al., 1985) used a different study 19

design to re assess the hypothesis that chemical exposures common in the field of nursing could 20

be risk factors for spontaneous abortion. This case

- control study found no increase in the risk of 21

spontaneous abortion associated with exposure to formaldehyde. The head nurses at each 22

hospital were asked by the investigators whether each case or control had been exposed to 23

formaldehyde during a given 3

- month period corresponding to the first trimester of a study 24

participant’s pregnancy during 1973

– 1979. Formaldehyde expo sure was assessed as positive or 25

negative for either use as a sterilizing agent or use of sterilized instruments. The reported crude 26

OR for formaldehyde exposure was 0.6; no CIs were provided. From the data reported in 27

Table 2 in Hemminki et al. (1985), the unadjusted OR and its CI can be computed post hoc as 28

OR (0.70 [95% CI: 0.28

– 1.73]). The authors acknowledged that the study failed to distinguish 29

between sterilizing work and the use of sterilized instruments, where only very small exposures 30

could be expected. Given the likelihood of extreme exposure misclassification and the 31

presentation of only crude results without control of potential confounding for formaldehyde, 32

these results do not appear to be exculpatory of a true causal association between f ormaldehyde 33

exposure and the risk of spontaneous abortion. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 53 A meta

- analysis of formaldehyde exposure and spontaneous abortion was conducted by 1

Collins et al. (2001b). However, the published results should be interpreted with caution. This 2

meta

- analysis in cluded one very large null study of paternal formaldehyde exposure along with 3

seven studies of maternal exposure. The two null studies by Hemminki et al. (1985, 1982) were 4

also included without consideration of the potentially extreme exposure misclassifi cation that 5

may have attenuated any true adverse effect. Nevertheless, the overall reported meta

- analytic 6

RR for parental formaldehyde exposure based on eight maternal and paternal exposure studies 7

was 1.6 (95% CI: 0.9

– 2.7). For case

- control studies the RR was 1.8 (95% CI: 0.7

– 4.8), and for 8

cohort studies the RR was 1.7 (95% CI: 1.2

– 2.3). Collins et al. (2001b) argued that the method 9

of exposure evaluation may have influenced the observed results; they stated that several of the 10

studies whose exposures w ere based on the investigator’s judgment were likely misclassified, 11

which may have obscured the true relationship, while other studies that assessed exposure based 12

on self

- reporting could have suffered from recall bias. They report that RRs were higher fo r 13

studies based on self

- reported exposures (RR = 1.9 [95% CI: 1.3

– 2.6]) than those based on 14

objective exposure assessments (RR = 1.5 [95% CI: 0.6

– 3.7]) and suggested that this difference 15

might reflect recall bias in the exposure assessment. However, for r ecall bias to have been 16

operable in these studies, the women who provided self

- reported data on pregnancy history and 17

occupational exposure would have had to appreciate that the hypothesis of interest was the 18

specific effect of formaldehyde on the risk of spontaneous abortion. In the specific case of the 19

study by Taskinen and colleagues (1999), the investigator also looked at the effects of other 20

exposures, such as organic solvents, dust, and phenols, and did not report adverse effects. It is 21

therefore un likely that the women providing exposure data were doing so in a manner indicative 22

of recall bias. If the supposition of non

- differential misclassification error in exposure is indeed 23

correct, the observed results of the meta

- analysis would likely have be en biased towards the null. 24

Therefore, the true RR for maternal formaldehyde could be higher than Collins et al. (2001b) 25

reported and would likely be statistically significant. Had the study of paternal exposure been set 26

aside, the meta

- analysis almost s urely would have shown a statistically significant increase in the 27

risk of spontaneous abortion associated with maternal formaldehyde exposure. This single study 28

reported a null finding based on exposure assessment from census records of employment, and, 29

as the largest of the studies in the meta

- analysis, it contributed the greatest weight. 30

Lastly, Collins and coworkers (2001b) suggested that there were potential confounding 31

factors in each of the workplaces that might have produced the observed findings o f increased 32

risk of spontaneous abortion associated with formaldehyde. While each of these occupational 33

studies focused on women who were co

- exposed to formaldehyde and other chemicals, the 34

occupational groups were quite different and had different sets o f co

- exposures. The 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 54 woodworkers in the Taskinen et al. (1999) study were potentially co

- exposed to organic solvents 1

related to painting and lacquering, dusts, and phenols, none of which was shown to be an 2

independent predictor of adverse risk. The cosmet ologists studied by John et al. (1994) were 3

co

- exposed to hair dyes, bleach, alcohol

- based disinfectants, and chemicals specific to services, 4

such as fingernail sculpturing, but, in analyses that were specifically adjusted for other work 5

exposures and thei r potentially confounding effects, the investigators reported an OR of 2.1 6

(95% CI: 1.0

– 4.3) for the use of formaldehyde

- based disinfectants. The laboratory workers 7

studied by Axelsson et al. (1984) were potentially co

- exposed to a wide range of solvents, but the 8

miscarriage rate was highest among those exposed to formaldehyde, and, for a potential 9

confounder to entirely explain an observed effect of another exposure, it must be more strongly 10

associated with the adverse outcome. 11

It does not appear that the collective results of formaldehyde exposures associated with 12

increased risk of spontaneous abortion

— often in spite of exposures being crudely measured

— 13

can be explained by information bias or confounding. 14

The findings by Taskinen et al. (1999) of reduced fertility and increased risk of 15

spontaneous abortion are internally consistent and coherent with other reports of increased risk 16

of pregnancy loss associated with exposure to formaldehyde (John et al., 1994; Taskinen et al., 17

1994; Seitz and Baron, 1990; Ax elsson et al., 1984). Absent evidence of alternative explanation 18

for these findings, it is concluded that exposure to formaldehyde is associated with pregnancy 19

loss and diminished fertility. 20

21

4.1.1.7.2. Congenital malformations. Only three studies have reported on the epidemiologic 22

evidence of an association between formaldehyde exposure and the risks of births having 23

congenital malformations. In the earliest study by Hemminki et al. (1985), the investigators 24

presented an analysis of 34 congenital malfo rmations from the Finnish Register of Congenital 25

Malformations and compared them with a group of 95 controls from those used in the larger 26

study. An association was found between formaldehyde exposure and malformations based on 27

three exposed cases (OR = 1 .8). 28

The case

- control study by Taskinen et al. (1994) of effects of occupational exposure to 29

chemicals used in laboratories in Finland examined the potential effects of exposure to formalin 30

on both spontaneous abortions and congenital malformation. The in vestigators reported on a 31

study of 36 laboratory workers with a child registered in the Finnish Register of Congenital 32

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

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

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- 55 A Lithuanian study (Dulskiene an d Gražulevičiene, 2005) for which only a brief 1

summary is available in English investigated the risk of congenital heart malformations as a 2

result of exposure to 43 different agents. The number of births included in the study was not 3

given in the English abstract. Exposure to residential ambient formaldehyde concentrations of 4

>2.42 μg/m 3 (0.002 ppm) was associated with a 24% increase in the risk of congenital heart 5

malformations (OR = 1.24 [95% CI: 0.81

– 2.07]). The details of this study are unavailable i n 6

English translation, making it impossible to critically analyze details, such as co

- exposure and 7

other possible confounders. 8

9

4.1.1.7.3. Low birth weight. A case

- control study by Gražulevičiene et al. (1998) examined the 10

association of low birth weight (<2,500 grams) and air pollutants, including formaldehyde, 11

particulates, sulfur dioxide, lead, ozone, and nitrogen dioxide, measured in 12 areas in the city o f 12

Kaunas, Lithuania. This city has conducted environmental pollutant measurements since 1993, 13

and the investigators classified formaldehyde exposure based on the area of residence of the 14

study subjects. Formaldehyde levels in the 12 districts of Kaunas i n 1994 ranged from 1.36 to 15

5.28 μg/m 3 (0.0011

– 0.0043 ppm), with a citywide average of 3.14 μg/m 3 (0.0026 ppm). 16

Information on infants came from a birth registry. There were 244 cases of low birth weight and 17

4,089 normal controls born in 1994. Personal d ata came from record

- based prenatal interviews, 18

and pregnancy data came from hospital records. 19

The crude RR of low birth weight among women exposed to the highest airborne 20

formaldehyde level was 1.68 (95% CI: 1.24

– 2.27). After adjustment for age, occupati on, 21

hazardous work, education, marital status, smoking, hypertension, and other air pollutants, the 22

OR was still elevated but no longer statistically significant (OR 1.37 [95% CI: 0.90

– 2.09]). 23

Although formaldehyde exposure was the only single air polluta nt associated with low birth 24

weight, factors such as smoking, marital status, and pregnancy

- related factors had more of an 25

impact on birth weight. Total suspended particulates (OR 2.58 [95% CI: 1.34

– 4.99]) and 26

hazardous work (OR 2.62 [95% CI: 1.12

– 6.10]), which was not defined by the authors, were also 27

related to low birth weight. 28

Aside from studies of birth weight deficits from tobacco smoke and occupational 29

exposure, the literature on exposure to ambient air pollutants to support the investigators’ 30

hypot hesis is limited. The strength of the association between total suspended particulates and 31

low birth weight supports the idea that incidence of birth weight <2,500 grams may be related to 32

atmospheric pollution, although this finding may not be specific to formaldehyde. Because of 33

the large number of variables evaluated in the analysis, large fluctuations in the atmospheric 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 56 formaldehyde measurements, co

- exposure to other pollutants, and geographic variability of low 1

birth weight, it is difficult to estimat e the impact of formaldehyde alone on low birth weight. 2

3

4.1.1.7.4. Summary. Although all studies on potential developmental toxicity of formaldehyde 4

have limitations and do not uniformly report positive results, the associations between 5

spontaneous abor tion, delayed conception, or reproductive outcomes and formaldehyde exposure 6

in multiple studies cannot be dismissed, because several studies report concordant findings 7

across several populations and study methodologies. The results of most of the studies with 8

positive findings were adjusted for many potentially confounding factors that may be related to 9

spontaneous abortion and infertility, including smoking and alcohol use, pregnancy and 10

reproductive history, and other chemical exposures. 11

The association between fertility and formaldehyde (Taskinen et al., 1999) stands out 12

because of its strong quantitative statistical analysis, adequate sample size, and rigorous exposure 13

assessment. This study was designed to specifically assess the effect of formaldehy de on 14

reproductive outcomes. Furthermore, it was the only study with an exposure assessment based 15

on quantitative measurements from the subject’s workplace. Moreover, the investigators 16

conducted a multivariable survival analysis that approximates a longi tudinal life table or person

17

year analysis while simultaneously adjusting for important confounders. The findings were 18

strengthened by statistically significant associations between formaldehyde and spontaneous 19

abortion and endometriosis. The fact that t he use of gloves may reduce the reproductive effect of 20

formaldehyde supports the dose

- response relationship in this study, and the lack of an association 21

between time to pregnancy and any other workplace exposures strengthens the specificity of 22

formaldehyd e effects. The results also support associations reported between formaldehyde and 23

increased risk of spontaneous abortion because subfertility and spontaneous abortion are 24

biologically linked (subclinical pregnancy losses are increased among women with fe rtility 25

problems) (Gray and Wu, 2000; Hakim et al., 1995), and both subfertility and spontaneous 26

abortion may be related to sensitivity to environmental agents (Correa et al., 1996). 27

28

4.1.1.8

. Oral Exposure Effects on the Gastrointestinal Tract 29

No human epidemiology studies exist to determine an association between oral exposure 30

of formaldehyde and adverse health effects in the gastrointestinal (GI) tract. 31

32

4.1.1.9. Summary: Noncarcinogenic Hazard in Humans 33

Formaldehyde has clearly and consistently been shown to be a potent sensory irritant 34

with a variety of adverse health effects. Eye, nose, and throat irritation as a result of 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 57 formaldehyde exposure has been documented in a wide range of epidemiologic studies. Workers 1

chronically exposed to formaldehyd e have exhibited signs of reduced lung function consistent 2

with BC, inflammation, or chronic obstructive lung disease. A well

- conducted residential 3

epidemiology study has convincingly shown a concentration response for decreased pulmonary 4

function among c hildren with increased formaldehyde exposures. Several cross

- sectional studies 5

have described associations between increased concentrations of formaldehyde and increased 6

prevalence of asthma. However, two case

- control studies that focused on risk factors for the 7

initial physician diagnosis of asthma, which is indicative of atopic switching, have also shown 8

concentration

- dependent adverse effects associated with formaldehyde exposure. 9

Results of research on the effects of formaldehyde on tissue histology s uggest that 10

formaldehyde is also responsible for reduced mucociliary clearance and the induction of 11

histopathologic lesions in the nose. In addition, there is evidence of neurological impairment in 12

several studies of formaldehyde

- exposed histology technic ians, but confounding exposures to 13

other neurotoxic solvents and inconsistent results prevent drawing definitive conclusions 14

concerning the neurotoxicity of formaldehyde from these studies. 15

Finally, there is epidemiologic evidence that formaldehyde is ass ociated with adverse 16

reproductive outcomes. Four of six occupational studies found an increased risk of spontaneous 17

abortion among formaldehyde

- exposed women. Results of other studies suggested associations 18

among formaldehyde and congenital malformations , low birth weight, and endometriosis. The 19

strongest evidence of an association between formaldehyde and an adverse reproductive outcome 20

came from a well

- conducted study of infertility in women employed in the wood processing 21

industry. This study found a greater than threefold increased risk of spontaneous abortion, a 22

nearly 50% decrease in a measure of delayed conception indicating reduced fertility, and 23

increased time to pregnancy associated with average daily formaldehyde exposures of 0.15

– 24

1 ppm. 25

26

4.1. 2. Cancer Health Effects 27

4.1.2.1. Respiratory Tract Cancer 28

4.1.2.1.1. NPC. NPC is a very rare form of cancer. The incidence is less than 1 per 100,000 29

persons throughout most parts of the world. The most common form of NPC arises from the 30

epithelial cells lining the nasopharynx. This presentation constitutes between 75 and 100% of all 31

NPCs. There are two types, squamous cell carcinoma (SCC) and nonkeratinizing carcinoma. In 32

the U.S., the 5

- year survival rate for NPC is about 25% (Burt et al., 1992) . Certain risk factors 33

have been implicated in its etiology, including Epstein

- Barr virus (EBV), wood dust and 34This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 58 particles applied to wood in its treatment, exhaust fumes, occupational smoke, and nitrosamines. 1

The epidemiologic studies of NPC are summarize d in Table 4

- 1. 2

3

4.1.2.1.1.1. Cohort studies.

Several of these studies measured exposure to formaldehyde at 10 production facilities 11

that contributed to a cohort that has been studied by Blair et al. (1987, 1986 ) and Hauptmann et 12

al. (2004). The National Cancer Institute (NCI) conducted a mortality study of solid tumors 13

among a cohort of 25,619 workers who were employed in these 10 plants that produced or used 14

formaldehyde in the U.S. before 1966 (Blair et al., 1987, 1986). Subjects were followed to 15

January 1, 1980, accruing approximately 600,000 person

- years of follow

- up. Hauptmann et al. 16

(2004) updated the cohort to December 31, 1994 and reported a significant excess risk of NPC in 17

exposed workers based on U. S. population death rates (standardized mortality ratio [SMR] = 2.1 18

[95% CI: 1.05

– 4.21]). In addition to the SMR based on an external comparison population, RRs 19

were presented based on internal comparisons of similar workers in order to minimize potential 20

selection bias due to the well

- known healthy worker effect (HWE). For NPC, RRs increased 21

with several different exposure metrics, including average exposure intensity, cumulative 22

exposure, highest peak exposure, and duration of exposure to formaldehyde ( p values for tests 23

for trends were 0.066, 0.025, <0.001, and 0.147, respectively). These results were based on 24

primary data analyses of the health and exposure data collected by the NCI, according to their 25

research protocol and analyzed accordingly. As s uch, the reported statistical p values may be 26

appropriately interpreted as showing that these workers were at increased risk of NPC associated 27

with exposure to formaldehyde. These NCI investigations controlled for potential selection bias 28

due to the HWE a nd for several potential confounders, including calendar year, age, sex, race, 29

and pay category. There was no evidence of any differential measurement error that could have 30

produced the observation of a spurious association. Any non

- differential measurem ent error 31

would likely have led to an observed effect of formaldehyde that was less than that which would 32

otherwise have been observed in the absence of measurement error. 33 The International Agency for Research on Cancer (IARC) reported

4

on eleven cohort studies of formaldehyde

- exposed industry workers (Marsh et al., 2002, 1996, 5

1994; Andjelkovich et al., 1995; Gardner et al., 19 93; Bertazzi et al., 1989, 1986; Stayner et al., 6

1988; Blair et al., 1987, 1986; Edling et al., 1987) and results from eight cohort studies of 7

professional workers (Hall et al., 1991; Hayes et al., 1990; Stroup et al., 1986; Harrington and 8

Oakes, 1984; Lev ine et al., 1984; Walrath and Fraumeni, 1984, 1983; Friedman and Ury, 1983) 9

(IARC, 2006). 10This document is a draft for reviewpurposes only and does not constituteAgency policy4-59 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

Hauptmann et al. (2004) Retrospective cohort

mortality study of 25,619

workers employed at 10

formaldehyde plants in the

U.S. followed from either

plant start

- up or first

employment through 1994.

The 10 plants produced

formaldehyde (3 plants),

molding compounds

(3 plants), photographic

film (2 plants), plywood

(1 plant), and formaldehyde

resins (6 plants). Exposure estimates a based

on job titles, tasks, visits

to plants by study

industrial hygienists, and

monitoring data

measurements. Peak

exposure = short

- term

excursions >8

- hour TWA

formaldehyde intensity

and knowledge of job

tasks. Workers

contributed pre

- exposure person time to nonexposed

category. RRs we re from

Poisson regression

models, using a 15

- year lag to account for tumor

latency. Overall

Nonexposed SMR 1.56 (95% CI: 0.39

– 23) (2)

Exposed SMR 2.10 (95% CI: 1.05

– 21) (8)

Peak exposure (ppm)

0 RR b 1.00 (95% CI: NS) (2)

>0 to <2.0 N/A (95% CI: NS) (0)

2.0 to <4.0 N/A (95% CI: NS) (0)

4.0 or greater 1.83 (95% CI: NS) (7)

Trend p < 0.001

Average intensity of exposure (ppm)

0 RR b 1.00 (95% CI: NS) (2)

≤0.5 N/A (95% CI: NS) (0)

0.5 to <1.0 0.38 (95% CI: NS) (1)

1.0 or greater 1.67 (95% CI: NS) (6)

Trend p = 0.066

Cumulative exposure (ppm

- years)

0 RR b 2.40 (95% CI: NS) (2)

>0 to <1.5 1.00 (95% CI: NS) (3)

1.5 to <5.5 1.19 (95% CI: NS) (1)

5.5 or more 4.14 (95% CI: NS) (3)

Trend p = 0.025

Duration (years)

0 RR b 1.77 (95% CI: NS) (2)

>0 to <5 1.00 (95% CI: NS) (4)

5 to <15 0.83 (95% CI: NS) (1)

15 or more 4.18 (9 5% CI: NS) (2)

Trend p = 0.147

Marsh et al. (2002) Retrospective cohort

mortality study of 7,328

workers hired up to 1984 Worker

- specific exposure a

from job exposure matrix

based on available Cohort study

Overall

U.S. SMR 4.94 (95% CI: 1.99

– 10) (7)This document is a draft for reviewpurposes only and does not constituteAgency policy4-60 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

and followed until 1998 in

one plant from Hauptmann

et al. (2004). Mortality was

compared with death rates

in two Connecticut counties

and the U.S. sporadic sampling data

from 1965 −1987, job

descriptions, and verbal

job descript ions by plant

personnel and industrial

hygienists. Exposures

ranked on a 7

- point scale

with exposure range

assigned to each rank.

17% of jobs validated with

company monitoring data;

remaining 83% based on

professional judgment.

Assumed pre

- 1965

exposure levels same as

post

- 1965 levels. County SMR 5.00 (95% CI: 2.01

– 10) (7)

Short

- term worker (<1 year)

SMR 5.35 (95% CI: 1.46

– 14) (4)

Long

- term worker (1 or more years)

SMR 4.59 (95% CI: 0.95

– 13) (3)

Year of hire

1941 −1946 SMR (0)

1947 −1956 SMR 8.13 (95% CI: 2.98

– 18) (6)

1957 or later SMR 2.63 (95% CI: 0.07

– 15)

Cumulative exposure (ppm

- years) county

Unexposed SMR (0)

0 to <0.004 SMR 3.97 (95% CI: 0.10

– 22) (1)

0.004

– 0.219 SMR 5. 89 (95% CI: 1.22

– 17) (3)

0.22+ SMR 7.51 (95% CI: 1.55

– 22) (3)

Average exposure (ppm) county

Unexposed SMR (0)

0 to <0.03 SMR 2.41 (95% CI: 0.06

– 13) (1)

0.03

– 0.159 SMR 15.30 (95% CI: 4.16

– 39) (4)

0.16+ SMR 4.13 (95% CI : 0.50

– 15) (2)

Duration of exposure to >0.2 ppm (years)

Unexposed SMR 3.01 (95% CI: 0.36

– 11) (2)

0 to <1 SMR 4.81 (95% CI: 0.58

– 17.4) (2)

1

– 9 SMR 4.04 (95% CI: 0.10

– 22.51) (1)

10+ SMR 27.60 (95% CI: 3.34

– 100) (2)

Duratio n of exposure to ≥0.7 ppm (years)

Unexposed SMR 3.64 (95% CI: 0.99

– 9.31) (4)

<1 SMR 9.51 (95% CI: 1.15

– 34.4) (2)

1+ SMR 11.07 (95% CI: 0.28

– 61.67) (1)This document is a draft for reviewpurposes only and does not constituteAgency policy4-61 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

Marsh et al. (2002) A nested case

- control

analysis of all pharyngeal

cancer cases also conducted

with four controls randomly

selected from cohort and

matched on age, year of

birth, race, and sex.

Conditional logistic model

used for nested case

- control

analysis. Nested case

- control analysis c

Duration of exposure to >0.2 ppm (ye ars)

Unexposed OR 1.00 (8)

0 to <1 OR 1.13 (95% CI: 0.24

– 5.29) (6)

1

– 9 OR 1.38 (95% CI: 0.18

– 9.03) (3)

10+ OR 9.49 (95% CI: 0.55

– 701) (5)

Dur ation of exposure to ≥0.7 ppm (years)

Unexposed OR 1.00 (16)

<1 OR 0.52 (95% CI: 0.08

– 2.45) (4)

1+ OR 1.11 (95% CI: 0.06

– 11.3) (2)

Hayes et al. (1990) Proportionate mortality

cohort study of 4,046 U.S.

male embalmers and funeral

di rectors who died between

1975 and 1985. Exposure presumed. Overall PMR 2.16 (4)

Hansen and Olsen (1995) Proportionate incidence

study of 2,041 men with

cancer who died between

1970 and 1984, identified

from the Danish Cancer

Registry and matche d with

the Danish Supplementary

Pension Fund, whose

longest work experience

occurred at least 10 years

before the cancer diagnosis.

The SPIR measured the

proportion of cases of NPC

in formaldehyde

- associated

companies relative to the

proportion of cases o f NPC

among all employees in

Denmark. Linked companies through

tax records to the national

Danish Product Register. Overall SPIR 1.3 (95% CI: 0.03

– 3.2) (4)This document is a draft for reviewpurposes only and does not constituteAgency policy4-62 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

Olsen et al. (1984) Case

- control study of 314

cases of NPC from Danish

Cancer Regist ry linked to

the Registry during 1970

– 1982. Three controls/case

sampled with cancer of the

colon, rectum, breast, and

prostate by age, sex, and

year of diagnosis of cases. Employment histories

after 1964 from files

maintained by Danish

Cancer Registry ev aluated

by industrial hygienists. Men OR 0.7 (95% CI: 0.3

– 1.7)

Women OR 2.6 (95% CI: 0.3

– 22)

West et al. (1993) Case

- control study of 104

non

- Chinese incident NPC

cases from the Philippine

General Hospital matched

with 104 hospital and 101

community controls. Personal interview,

including job history.

Industrial hygienists

blinded to case

- control

status reviewed and rated

jobs as likely or unlikely

to be exposed. Analysis

by length of exposure,

length of exposure lagged

10 years, time since first

exposure, and age at first

exposure, based on date of

interview or death. Length of exposure (years)

<15 RR d 2.7 (95% CI: 1.1

– 6.6)

15 or more 1.2 (95% CI: 0.5

– 3.2)

Length of exposure lagged 10 years (years)

<15 R R d 1.6 (95% CI: 0.7

– 3.8)

15 or more 2.1 (95% CI: 0.7

– 6.2)

Years since first exposure

<25 RR d 1.3 (95% CI: 0.6

– 3.2)

25 or more 2.9 (95% CI: 1.1

– 7.6)

Age at first exposure (years)

<25 RR d 2.7 (95% CI: 1.1

– 6.6)

25 or older 1.2 (95% CI: 0.5

– 3.3)This document is a draft for reviewpurposes only and does not constituteAgency policy4-63 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

Roush et al. (1987) Population

- based case

control study of 173 male

cases from the Connecticut

Tumor Registry who died of

any cause from 1935

– 1975.

605 male controls randomly

selected from state death

c ertificates during same

time period. Four categories: I,

probably exposed most of

working life; II, probably

exposed most of working

life and probably exposed

20+ years before death;

III, probably exposed most

of working life and

probably to high level in

some year; IV, probably

exposed most of working

life and probably exposed

to high level 20+ years

before death. Exposure levels

I OR e 1.0 (95% CI: 0.6

– 1.7)

II 1.3 (95% CI: 0.7

– 2.4)

III 1.4 (95% CI: 0.6

– 3.1)

IV 2.3 (95% CI: 0. 9

– 6.0)

Vaughan et al. (1986a) Population

- based case

control study of 27

incidence cases of NPC

(during 1980

– 1983) from a

13

- county area (Washington

State Cancer Surveillance

System) and 552 matched

controls from random digit

dialing in same are a, for

occupational exposures. Interview

- based

information on lifetime

occupational exposure to

formaldehyde with cases,

next of kin, and controls.

Exposure from available

hygiene data, NIOSH and

other data, and NCI job

exposure linkage system.

Exposure levels based on

investigator’s judgment.

Exposure score A : weighted sum of no. years

spent per job (weight =

estimated formaldehyde

level). B : weighted sum

of no. years spent per job

with 15

- year lag (latency). Intensity

Low OR f 1.2 (95% CI: 0.5

– 3.3)

Medium/high 1.4 (95% CI: 0.4

– 4.7)

No. years exposed

1

– 9 OR f 1.2 (95% CI: 0.5

– 3.1)

10 or more 1.6 (95% CI: 0.4

– 5.8)

Exposure score A: no lag

5

– 19 OR f 0.9 (95% CI: 0.2

– 3.2)

20 or more 2.1 (95% CI: 0.6

– 7 .8)

Exposure score B: 15

- year lag

5

– 19 OR f 1.7 (95% CI: 0.5

– 5.7)

20 or more 2.1 (95% CI: 0.4

– 10)This document is a draft for reviewpurposes only and does not constituteAgency policy4-64 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

Vaughan et al. (1986b) Population

- based case

control study of 27

incidence cases on NPC

(during 1980

– 1983) from a

13

- county area (Washington

State Cancer Surveillance

System) and 552 matched

controls from random digit

dialing in same area, for

residential exposures. No direct measurements.

Interview information

from cases/controls or

next of kin: residence in

past 50 years, use of

particleboard or plywood,

and lifetime occupational

and chemical exposure

history. Years of residence in mobile home

1

– 9 OR g 2.1 (95% CI: 0.7

– 6.6)

10 or more 5.5 (95% CI: 1.6

– 19)

Years of exposure to particleboar d

1

– 9 OR g 1.4 (95% CI: 0.5

– 3.4)

10 or more 0.6 (95% CI: 0.2

– 2.3)

Exposure source

Occupation only OR g 1.7 (95% CI: 0.5

– 5.7)

Mobile home only 2.8 (95% CI: 1.0

– 7.9)

Both 6.7 (95% CI: 1.2

– 39)

Vaughan et al. (2000) Po pulation

- based case

control study of 196

incident epithelial NPC

patients identified from 5

U.S. cancer registries from

1987

– 1993 matched with

244 controls from random

digit dialing in the same

geographic regions. Interviewed for lifetime

occupational and chemical

exposure. Exposure

estimates by industrial

hygienist without

knowledge of case

controls status.

Probability of exposure:

definitely not or unlikely

(<10%); possible ( ≥10%

and <50%); probable

(>50% and <90%); and

definite ≥90%). Jobs with

potent ial exposure

assigned estimated

concentration levels based

on TWA: low (<10 ppm),

moderate ( ≥10 and

<50 ppm), and high

( ≥50 ppm). Possible, probable, or definite exposure (61 cases, 76 controls)

Ever OR h 1.6 (95% CI: 1.0

– 2.8)

Duration (years)

1

– 5 OR h 0.9 (95% CI: 0.4

– 2.1)

6

– 17 1.9 (95% CI: 0.9

– 4.4)

18 or more 2.7 (95% CI: 1.2

– 6.0)

Trend p = 0.014

Cumulative exposure (ppm

- years)

0.05

– 0.40 OR h 0.9 (95% CI: 0.4

– 2.0)

0.41

– 1.10 1.8 (95% CI: 0.8

– 4.1)

≥1.10 3.0 (95% CI: 1.3

– 6.6)

Trend p = 0.033

Probable or definite exposure (27 cases, 30 controls)

Ever OR h 2.1 (95% CI: 1.1

– 4.2)

Duration (years)

1

– 5 OR h 2.0 (95% CI: 0.8

– 5.0)

6

– 17 3.3 (95% CI: 0.9

– 12)

18 or more 1.6 (95% CI: 0.5

– 5.6)

Trend p = 0.069This document is a draft for reviewpurposes only and does not constituteAgency policy4-65 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

Cumulative exposure (ppm

- years)

0.05

– 0.40 OR h 1.9 (95% CI: 0.7

– 4.9)

0.41

– 1.10 2.6 (95% CI: 0.7

– 9.5)

≥1.10 2.2 (95% CI: 0.7

– 7.0)

Trend p = 0.13

Definite exposure (10 cases, 2 controls)

Ever OR h 13.3 (95% CI: 2.5

– 70)

Hildesheim et al. (2001) Population

- based case

control study of 375

incident cases from two

Taiwanese hospitals

between 7/15/91 and

12/31/94. 325 controls

came from a random sample

of househo lds from a

national household

registration system and

were age, sex, and area

- of

residence matched. Tumors

were histologically

confirmed. All subjects

were tested for the EBV.

Exposure metrics were

stratified by seropositivity. In

- person interviews

coll ected information on

risk factors and job history

for jobs held >1 year,

including length of time

job held, type of industry,

and tasks, tools, and

materials used on the job.

Industrial hygienist

assigned Standard

Industry Classification/

Standard Occup ational

Classification codes to

jobs, assigning each a

probability and intensity

of exposure on a 0

– 9

scale. Exposure metrics

were duration, average

intensity (intensity scale),

average probability

(probability scale),

cumulative (average

intensity), year s since 1st

exposure, and age at 1st

exposure. Analysis of Ever exposed RR i 1.4 (95% CI: 0.9

– 2.2)

Duration (years)

≤10 RR i 1.3 (95% CI: 0.69

– 2.3)

>10 RR i 1.6 (95% CI: 0.91

– 2.9)

Trend p = 0.08

EBV pos j

Ever exposed RR i 2.7 (95% CI: 1.2

– 6.2)

≤10 RR i 2.8 (95% CI: 0.8

– 9.7)

>10 2.6 (95% CI: 0.9

– 7.7)

Cumulative exposure (average intensity

- years)

<25 RR i 1.3 (95% CI: 0.7

– 2.4)

≥25 RR i 1.5 (95% CI: 0.9

– 2.7)

Trend p = 0.10

EBV pos j

<25 RR i 4.0 (95% CI: 0.9

– 17)

≥25 RR i 2.2 (95% CI: 0.8

– 5.8)

Years since 1st exposure

<20 RR i 2.3 (95% CI: 1.0

– 5.8)

≥20 RR i 1.2 (95% CI: 0.8

– 2.0)This document is a draft for reviewpurposes only and does not constituteAgency policy4-66 Table 4

- 1. Cohort and case

- control studies of formaldehyde cancer and NPC

Refer ence Study design Exposure assessment Results; statistical significance (number observed deaths)

nonkeratinizing or

undifferentiated tumors

yielded similar results as

overall a nalysis. EBV pos h

<25 RR i 2.3 (95% CI: 0.5

– 10)

≥25 RR i 2.8

Age at 1st exposure

<20 RR i 1.3 (95% CI: 0.8

– 2.0)

≥20 RR i 3.4 (95% CI: 0.9

– 12)

EBV pos j

<25 RR i 2.6 (95% CI: 1.1

– 6.5)

≥25 RR i 3.1 (95% CI: 0.4

– 24)

a Exposure estimates by Hauptmann et al. (2004) were 10 times higher than those of Marsh et al. (2002).

b Adjusted for calendar year, age, sex, race, and pay category (salaried versus wage).

c Results for cumulative and average intensity of exposure are n ot included here because condition logistic regression produces unstable estimates for this small

number of cases.

d Adjusted for years since first exposure to dust and exhaust fumes.

e Adjusted for age at death, year at death, and availability of occupation al information (Roush et al., 1987).

f Adjusted for cigarette smoking, alcohol consumption, gender, and age.

g Adjusted for ethnic origin and cigarette smoking.

h Adjusted for age, sex, race, SEER site, cigarette usage, proxy status, and education.

i Adjusted for age, sex, education, and ethnicity.

j EBV seropositives defined as positive for one of the following anti

- EBV antibodies known to be associated with NPC: viral capsid antigen IgA, EBV nuclear

antigen 1 IgA, early antigen IgA, DNA binding protein IgG, an d anti

- DNase IgG.

N/A = not applicable, NS = not significant, PMR = proportionate mortality ratio.This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 67 Following these reports of increased risk of NPC associated with formaldehyde exposure, 1

a series of post hoc analyses of similar data were undertaken by Mars h and coworkers (Marsh et 2

al., 2007a, b, 2002, 1996; Marsh and Youk, 2005). Briefly, these studies focused on the specific 3

findings from a single plant in the NCI cohort (Wallingford, Connecticut) that generated the 4

majority of the NPC cases. 5

In the most recent subsequent report, Marsh et al. (2007a) continues to argue against a 6

formaldehyde

- NPC association. Although earlier reports speculated on anecdotal evidence that 7

the statistically significant excess risk of NPC observed at the Wallingford, Connect icut, plant 8

reflected the influence of unmeasured nonoccupational risk factors associated with employment 9

outside the plant, the new report (Marsh et al., 2007a) suggests that occupational or hobby

10

related work in silversmithing may have confounded the obs erved effect of formaldehyde on the 11

increased risk of NPC. In this report, Marsh et al. (2007a) show that their subjectively assessed 12

work in silversmithing is strongly associated with NPC. While the reported ORs are indeed quite 13

high, the estimates are extremely unstable and it is not clear how many a priori hypotheses were 14

tested for statistical significance. There are no citations of an association between silversmithing 15

exposures and NPC in the medical literature. Marsh and coworkers mention that th ere was 16

concordance of silver manufacturing history in the Wallingford, Connecticut, area. If 17

silversmithing exposures are indeed independent risk factors for NPC, it would be expected that 18

the rates of NPC in the surrounding counties with historical silv er

- related exposures would be 19

elevated. However they are not increased, as evidenced by the comparability of the increased 20

rates of NPC among the plant workers compared with both the national and local county rates 21

that were very similar (Marsh et al., 20 07a). The comparable rates indicate the counties’ rates of 22

NPC were very similar to the national rates and weaken an association between silversmithing 23

and NPC. Given the many post hoc reexaminations of alternative hypotheses to explain the 24

original NCI findings, it is more likely that silversmithing is an artifactual confounder. 25

26

4.1.2.1.1.2. Professional cohort studies. Two cohort studies of professional groups, such as 27

anatomists, pathologists, embalmers, and funeral directors, examined the risk of N PC and 28

formaldehyde exposure. In general, measurements of formaldehyde concentrations were not 29

available in studies of professionals but are generally below 1 ppm (IARC, 1995; Korczynski, 30

1994; Stewart et al., 1992; Moore and Ogrodnik, 1986). Hayes et al . (1990) reported an excess 31

risk of NPC among male professional embalmers and funeral directors, based on 4 deaths with 32

1.9 expected based on age, gender, and calendar

- year

- specific proportions of deaths in the U.S. 33

population. Hansen and Olsen (1995) stu died male Danish cancer patients employed in 34

companies in which formaldehyde was used or produced. Only a slight excess risk of NPC was 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 68 found, based on 4 cases with 3.2 expected (standardized proportionate incidence ratio [SPIR] 1

1.3). Hansen and Olsen (1 995) also reported on a significantly elevated risk of sinonasal cancer. 2

3

4.1.2.1.1.3. Case

- control studies.

Three case

- control studies have been conducted since the 1995 IARC report. Armstrong 11

et al. (2000) fou nd no association between formaldehyde exposure and NPC (adjusted OR = 0.71 12

[95% CI: 0.34

– 1.43]), controlling for wood dust and industrial heat. Using data from the 13

Surveillance Epidemiology and End Results (SEER) program, Vaughan et al. (2000) found an 14

O R for ever

- exposed persons of 3.1 (95% CI: 1.0

– 9.6) among cases of epithelial NPC, 15

suggesting differences in the etiology of cancers at this site. There was a trend of increasing risk 16

of NPC with increasing duration of exposure and cumulative exposure, co ntrolling for wood dust 17

exposure. Finally, Hildesheim et al. (2001) found that exposure to formaldehyde produced 18

modest risk elevations for duration of exposure (OR = 1.6 for 10 years or less and 1.2 for over 19

10 years of exposure), for cumulative exposure (ORs were 1.3 for <25 years of exposure and 1.5 20

for 25+ years of exposure), and for years since first exposure. Among those with EBV, the OR 21

was 2.7 (95% CI: 1.2

– 6.2) for ever

- exposed persons. The risk was significantly higher among 22

exposed persons whos e work history was within the last 10 years (OR = 4.7 [95% CI: 1.1

– 20.0]) 23

and for those followed 20+ years after exposure (OR = 2.8 [95% CI: 1.1

– 7.6]). 24 Five case

- control studies (West et al., 1993; Roush et al.,

4

1987; Vaughan et al., 1986a, b; Olsen et al., 1984) reviewed by IARC in 1995 provided evidence 5

of excess risks of NPC due to formaldehyde. Most of these studies report significant and 6

nonsignificant elevations in risk of NPC in the range of 1.5

– 3.0, with some higher than 5.0. In 7

its report, IARC (1995) concluded that, taking the data as a whole, formaldehyde appears to have 8

a causal role in the induction of NPC, recognizing that the conclusion is based on small numbers 9

of cancer cases. 10

25

4.1.2.1.1.4. Summary of NPC studies. Findings from the large NCI cohort studies of NPC risk 26

due to fo rmaldehyde exposure clearly show a consistent pattern of increased risk with increased 27

exposures. Post hoc reanalyses have challenged the interpretation of these findings but have not 28

been able to dispute the reported excess in NPC mortality (Marsh et al. , 2007a, b, 2002, 1996; 29

Marsh and Youk, 2005). The major questions that have been raised by Marsh and coworkers 30

highlight the observation that the NPC findings appear to depend on the results of 1 of the 10 31

plants that made up the NCI cohort. While it is theoretically possible for coexposures at that 32

plant or among those workers to act as potential confounders or modifiers of the observed effect 33

of formaldehyde on increased risk of NPC, there is no solid evidence of such a relationship that 34

would outweigh or supersede the reported adverse effects of formaldehyde exposure. While all 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 69 of the cohort members at the Wallingford, Connecticut, plant were also exposed to particulates, 1

the NCI investigators did observe a dose

- response relationship with formaldehyde among 2

individuals with high particulate exposures, thereby strengthening the causal interpretation of the 3

formaldehyde relationship with an increased risk of NPC. The described association of a 4

potential occupational relationship with silversmithing and NPC has no basis in the medical 5

literature and is inconsistent with the supposition that this activity is common in the locality of 6

Wallingford, Connecticut, but has not been associated with increased rates of NPC in 7

surrounding New Haven County (Connectic ut). Marsh and coworkers did report significantly 8

increased rates of pharyngeal cancer (including NPCs) among workers from the Wallingford 9

plant compared with both the county and national rates. It is more plausible that the observed 10

association at the W allingford plant reflects higher formaldehyde exposures than at other plants. 11

The exposure levels at Plant 2 were even higher than at the Wallingford plant and were 12

associated with a fivefold increase in risk associated with NPC, even though this was base d on a 13

single observed case and was not significant. 14

In addition to the evidence from the NCI cohort studies, modest additional evidence is 15

found in the professional cohort studies of Hayes et al. (1990) and Hansen and Olsen (1995). 16

The rarity of the dis ease and difficulties in obtaining valid and reliable historical exposure 17

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

from the results of several case

- control studies that support an increased risk of N PC from 19

exposure to formaldehyde. The studies of Vaughan et al. (2000) and Hildesheim et al. (2001) 20

provide evidence of an association of NPC with exposure to formaldehyde. Vaughan et al. 21

(2000) found a dose

- response relationship of NPC with increasing e xposure to formaldehyde, as 22

did Hildesheim et al. (2001). These studies, in general, are easier to conduct and may provide 23

more statistical power for a specific level of risk estimate than do cohort studies. 24

25

4.1.2.1.2. Nasal and paranasal cancer 26

4.1.2.1 .2.1. Case

- control studies. Eight case

- control studies were evaluated in the 1995 IARC 27

monograph regarding the risk of nasal cavity and accessory sinuses from exposure to 28

formaldehyde (Luce et al., 1993; Roush et al., 1987; Hayes et al., 1986; Olsen and Asnaes, 1986; 29

Vaughan et al., 1986a, b; Brinton et al., 1984; Olsen et al., 1984). Of three studies that identified 30

a cell type, two reported a positive finding of sinonasal cancer (Hayes et al., 1986; Olsen and 31

Asnaes, 1986). One of the positive studies did not report any exposure to the potentially 32

confounding influence of wood dust, while the other two did report an adjustment for exposure 33

to wood dust. Of the remaining five studies where a cell type was not identified, only Roush et 34

al. (1987) and Ol sen et al. (1984) found positive results. The remaining studies (Vaughan et al., 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 70 1986a, b; Brinton et al., 1984) from the 1995 IARC monograph did not find associations between 1

exposure and sinonasal cancer. Study details of the epidemiologic studies of n asal and paranasal 2

cancer are summarized in Table 4

- 2. Vaughan et al. (1986b) matched 53 sinonasal cancer 3

patients to 552 controls. Potential residential exposure to formaldehyde was estimated by 4

utilizing residence in a mobile home with or without the p resence of UFFI or particleboard or 5

plywood as a surrogate for exposure. The authors found an OR of 1.5 for sinonasal cancer in 6

subjects reporting residence of 10 or more years in a mobile home with UFFI before diagnosis. 7

A higher OR (1.8) was reported f or less than 10 years of mobile home residence. However, 8

because actual formaldehyde levels in the subjects’ mobile homes are unknown, the exposure 9

estimates are, at best, imprecise surrogates that typically have the effect of attenuating any true 10

risk. In Vaughan et al. (1986a), the same cases and controls were examined for occupational 11

exposures to formaldehyde, but no increase in risk of nasal or paranasal cancer was reported. 12

More recently, Luce et al. (2002) pooled data from 12 case

- control studies. Combined, 13

these studies had 195 adenocarcinomas and 432 SCCs of the sinonasal passages compared with 14

3,136 controls. The authors reported a significant increase in the risk of sinonasal 15

adenocarcinoma in men (adjusted OR = 3.0 [95% CI: 1.5

– 5.7]; 91 cases ) and in women (adjusted 16

OR = 6.2 [95% CI: 2.0

– 19.7]; 5 cases) with a high probability of exposure to formaldehyde. For 17

SCCs, the ORs were more modest: OR = 1.2 in men and OR = 1.5 in women for a high 18

probability of exposure to formaldehyde. In an analys is of 11 formaldehyde

- exposed cases of 19

sinonasal adenocarcinomas who were not exposed to wood dust, there was an elevated risk in 20

men (OR = 1.9; 3 cases) and a significantly increased risk in women (OR = 11.1 [95% CI: 3.2

– 21

38.0]; 5 cases) with a high probab ility of exposure to formaldehyde. Limitations of these studies 22

were the lack of information about the actual levels or intensity of exposure to formaldehyde, 23

exposure to multiple occupational carcinogens, and the small number of cases in some 24

subgroups. In spite of those limitations, which generally obfuscate the observation of a true 25

underlying effect, these studies identified effects of formaldehyde that were statistically 26

significant predictors of sinonasal cancers. 27

28This document is a draft for reviewpurposes only and does not constituteAgency policy4-71 Table 4

- 2. Case

- control studies of formaldehyde and nasal and paranasal cancer

Reference Study design Exposure assessment Results, statistical significance (number of cases)

Olsen and Asnaes

(1986) Case

- control study of

histologically confirmed cases

of squamous cell

carcinoma /lymphoepithelioma

of the sinonasal cavities and

paranasal cancers in 215 men

and adenocarcinomas of the

sinonasal cavities and

paranasal cancers in 39 men

matched with 2,465 controls

with other cancers from the

Danish Cancer Registry,

1970

– 1982. Employmen t histories

after 1964 from files

maintained by Danish

Cancer Registry

estimated by industrial

hygienists. Squamous cell carcinoma/lymphoepithelioma

Ever vs. never

Formaldehyde only RR 2.0 (95% CI: 0.7

– 5.9)

Formaldehyde + wood

dust RR 1.6 (95% CI: 0.8

– 3.3)

10 or more years since first exposure

Formaldehyde only RR 1.4 (95% CI: 0.3

– 6.4)

Formaldehyde + wood

dust RR 1.8 (95% CI: 0.7

– 4.4)

Adenocarcinoma

Ever vs. Never

Formald ehyde only RR 7.0 (95% CI: 1.1

– 44)

Formaldehyde + wood

dust RR 40.0 (95% CI: 22

– 71)

10 or more years since first exposure

Formaldehyde only RR 9.5 (95% CI: 1.6

– 58)

Formaldehyde + wood

dust RR 44.0 (95% CI: 22

– 88)

Hayes et al. (1986) Case

- control study of 91 men

with SCC of the nasal cavity

and paranasal sinuses, from

clinical records of six medical

institutions in the Netherlands.

195 controls from living and

deceased males from

municipal residence regist ries,

from 1978

– 1981. Cases selected from

clinical records of six

institutions in the

Netherlands. 91 male

patients and 195

controls from living and

deceased males from

municipal residence

registries with little or

no exposure to wood

dust. Industrial

h ygienists evaluated job

histories according to

probability of exposure

based on job records. Industrial hygienist A

Any exposure RR 3.0 (90% CI: 1.3

– 6.4)

Moderate exposure 2.7 (90% CI: 1.0

– 7.2)

High exposure 3.1 (90% CI: 0.9

– 10.0)

Industrial hygienist B

Any exposure RR 1.9 (90% CI: 1.0

– 3.6)

Moderate exposure 1.4 (90% CI: 0.5

– 3.4)

High exposure 2.4 (90% CI: 1.1

– 5.1)This document is a draft for reviewpurposes only and does not constituteAgency policy4-72 Table 4

- 2. Case

- control studies of formaldehyde and nasal and paranasal cancer

Reference Study design Exposure assessment Results, statistical significance (number of cases)

Roush et al. (1987) Population

- based case

- control

study of 198 male cases of

sino nasal cancer from the

Connecticut Tumor Registry

who died of any cause in

1935

– 1975. Controls were

605 males dying in

Connecticut during the same

time period, randomly selected

from state death certificates. Occupations from city

directories and

evalua tion of job by

industrial hygienist who

classed exposure into I

, probably exposed to

some level most of

working life; II

, probably exposed to

some level most of

working life and

probably exposed to

some level 20+ years

before death; III

, probably exposed t o

some level most of

working life and

probably exposed to a

high level in some

years; IV Exposure levels Sinonasal cancer

, probably

exposed to some level

most of working life

and probably exposed

to a high level 20+

years before death. I OR a 0.8 (95% CI: 0.5

– 1.3)

II 1.0 (95% CI: 0.5

– 1.8)

III 1.0 (95% CI: 0.5

– 2.2)

IV 1.5 (95% CI: 0.6

– 3.9)

Luce et al. (1993) Case

- control study of men

with sinonasal cancer

(histologically confirmed), 77

with adenoc arcinoma, 59 with

squamous cell carcinomas,

and 25 tumors of other types,

matched with 409 controls

from 27 French hospitals and Industrial hygienist

estimation based on job

histories from personal

interviews. Subjects

were broken out into no

exposure, p ossible

exposure, or probable/definite Adenocarcinoma b

Possible exposure OR c 1.28 (95% CI: 0.16

– 10)

Probable/definite exposure

Average level

**≤** 2 OR c 4.15 (95% CI: 0.96

– 18)

**>** 2 5.33 (95% CI: 1.28

– 22)

Duration (yea rs)

**≤** 20 OR c 1.03 (95% CI: 0.18

– 5.77)

**>** 20 6.86 (95% CI: 1.69

– 28)This document is a draft for reviewpurposes only and does not constituteAgency policy4-73 Table 4

- 2. Case

- control studies of formaldehyde and nasal and paranasal cancer

Reference Study design Exposure assessment Results, statistical significance (number of cases)

from lists of names supplied

by patients. exposure. Those

classed as

probable/definite

exposure further

categorized into three

levels of frequency of

exposure during a

norm al workweek: 1 =

<5% of the time; 2 = 5

– 30% of the time; and 3

= >30% of the time.

Concentration was

categorized into 3

levels: low (<0.0 ppm);

medium (0.1

– 1 ppm);

and high (>1 ppm).

The exposure index =

concentration ×

frequency. Cumulative

level = sum of exposure

indices. Average level

= cumulative

level/duration and

ranged from 1 to 9.

Nearly all cases had had

wood dust exposure. Cumulative level (years)

**≤** 30 OR c 1.13 (95% CI: 0.19

– 6.95)

30

– 60 2.66 (95% CI: 0.38

– 19)

**>** 60 6.91 (95% CI: 1.69

– 28)

Age 1st exposed (years)

**≤** 15 OR c 9.99 (95% CI: 1.85

– 54)

16

– 20 4.12 (95% CI: 0.95

– 18)

**>** 20 2.74 (95% CI: 0.58

– 13)

Dat e 1st exposed (years)

After 1954 OR c 6.02 (95% CI: 1.18

– 31)

Before 1954 4.26 (95% CI: 1.06

– 17)

Other cell type carcinoma b

Possible exposure OR c 0.81 (95% CI: 0.15

– 4.36)

Probable/definite exposure

Average level

**≤** 2 OR c 1.67 (95% CI: 0.51

– 5.42)

**>** 2 3.04 (95% CI: 0.95

– 9.7)

Duration (years)

**≤** 20 OR c 2.82 (95% CI: 0.94

– 8.4)

**>** 20 1.62 (95% CI: 0.48

– 5.51)

Cumulative level (years)

**≤** 30 OR c 2.18 (95% CI: 0.65

– 7.31)

**>** 30 2.21 (95% CI: 0.73

– 6.73)

Age 1st exposed (years)

**≤** 20 OR c 2.03 (95% CI: 0.63

– 6.54)

**>** 20 2.36 (95% CI: 0.76

– 7.33)

Date 1st exposed (years)

After 1954 OR c 0.48 (95% CI: 0.05

– 4.35)

Before 1954 3.27 (95% CI: 1.15

– 9.33)

Luce et al. (2002) Pooled analysis of 195

adenocarcinomas and 432

squamous cell carcinomas of Job exposure matrix

based on interview data

developed for pooled Adenocarcinoma

High probability of exposure

Men OR d 3.0 (95% CI: 1.5

– 5.7)This document is a draft for reviewpurposes only and does not constituteAgency policy4-74 Table 4

- 2. Case

- control studies of formaldehyde and nasal and paranasal cancer

Reference Study design Exposure assessment Results, statistical significance (number of cases)

the sinus/nasal cavity matched

with 3,136 controls from 12

case

- control studies. analysis. Indus trial

hygiene data used to

develop indices of

exposure. 11

formaldehyde cases

reported no exposure to

wood dust. Women OR e 6.2 (95% CI: 2.0

– 20)

Squamou s cell carcinoma

High probability of exposure

Men OR e 1.2 (95% CI: 0.8

– 1.8)

Women OR e 1.5 (95% CI: 0.6

– 3.8)

Brinton et al. (1984) Case

- control study of 160

patients with cancer of the

nasal cavity and paranasal

sinuses from four N orth

Carolina and Virginia

hospitals matched with 290

hospital controls with other

conditions, based on

occupational exposures. Interview data on job

history. Estimation of

exposure based on

industry type. Only

two cases employed in

industry associated w ith

formaldehyde. There

were no deaths in the

high exposure category. Overall male and female RR 0.35 (95% CI: 0.1

– 1.8)

Residence in mobile home OR f 0.6 (95% CI: 0.2

– 1.7)

Years of exposure to particleboard

1 to 9 OR f 1 .8 (95% CI: 09

– 3.8)

10 or more 1.5 (95% CI: 0.7

– 3.2)

Olsen et al. (1984) Case

- control study of 488

cases of nasal cancer linked to

the Danish Cancer Registry

during 1970

– 1982. Controls

were individuals with cancer

of the colon, rectum, breast,

and prostate. Three controls

per case were selected for the

same distributions of age, sex,

and year of diagnosis as cases. Employment histories

after 1964 from files

maintained by Danish

Cancer Registry

estimated by industrial

hygienists. Men

Formaldehyde only

Ever exposed RR 2.8 (95% CI: 1.8

– 4.3) (33)

Exposure to wood dust and

formaldehyde RR 3.1 (95% CI: 1.8

– 5.3) (23)

Ever exposed RR 3.5 (95% CI: 2.2

– 5.6) (28)

1 st exposure >10 years or

more before d iagnosis RR 4.1 (95% CI: 0.2.3

– 7.3) (20)

Hansen and Olsen

(1995) Proportionate incidence study

of 2,041 men with sinonasal

cancer who died between 1970

and 1984 identified from the

Danish Cancer Registry

matched with the Danish Linked companies

through tax records to

national Danish Product

Register, where

companies must report

amount of Overall SPIR 2.3 (95% CI: 1.3

– 4.0) (13)

Low formaldehyde SPIR 0.8 (95% CI: 0.02

– 4.4) ( 1)

Formaldehyde, no wood

dust SPIR 3.0 (95% CI: 1.4

– 5.7) (9)

SPIR 5.0 (95% CI: 0.5

– 13) (2)

Unknown SPIR 1.0 (95% CI: 0.03

– 6.1) (1)This document is a draft for reviewpurposes only and does not constituteAgency policy4-75 Table 4

- 2. Case

- control studies of formaldehyde and nasal and paranasal cancer

Reference Study design Exposure assessment Results, statistical significance (number of cases)

Supplementary Pension Fund

whose longest work

experience occurred at least 10

years before the cancer

diagnosis. The measure of

risk was the SPIR, which

measured the proportion of

cases of sinonasal cancer in

formaldehyde

- associated

companies relative to the

proportion of cases of

sinonasal cancer among all

employees in Denmark. formaldehyde used per

year.

Coggon et al. (2003) Cohort mortality study of

14,014 men employed in 6

factories of the chemical

industry in Great Britain from

periods during which

formaldehyde was produced.

Cohort followed through

2000. Sinonasal cancer

mortality SMRs based on

English and Welsh age and

calendar

- year

- specific

mortality rates. Exposures as sessment

based on data

abstracted from

company records. Each

job categorized as

background, low,

moderate, high, or

unknown levels. For

analysis of sinonasal

cancer, no gradient used

because of small

number of observed

cases. Overall SMR 0.87 (95% CI: 0 .11

– 3.14) (2)

a Adjusted for age at death, year at death, and availability of occupational information.

b All had medium to high exposure to wood dust.

c Adjusted for age and exposure to glues and adhesives.

d Adjusted for age and study.

e Adjusted for age, study, and cumulative exposure to dust.

f Adjusted for cigarette smoking, alcohol consumption, gender, and age.This document is a draft for review purposes only and does not constitute Agency policy.

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- 76 4.1.2.1.2.2. Cohort studies of nasal and paranasal cancer.

Three subsequent cohort studi es reported on nasal and paranasal cancer. Hansen and 11

Olsen (1995), in a proportional incidence study, found a significantly increased risk of sinonasal 12

cavity cancer (SPIR = 2.3 [95% CI: 1.3

– 4.0]; 13 observed) in 265 Danish industries, where 13

2,041 of 91, 182 cancer patients had at least 10 years of continuous formaldehyde

- related work 14

experience before diagnosis. Coggon et al. (2003), in a cohort study of 14,014 employees in six 15

chemical factories in Great Britain, found only 2 deaths from sinonasal cance r (2.3 expected 16

based on national death rates in Great Britain). Finally, Hauptmann et al. (2004) evaluated the 17

sinonasal cancer risk in the NCI cohort and found three cases (SMR = 1.19 [95% CI: 0.38

– 3.68]) 18

among those with a 15

- year lag period. 19 IARC (1995) also reported the

1

results of several cohort studies of profess ional and industrial workers for nasal and paranasal 2

cancer (Andjelkovich et al., 1995; Gardner et al., 1993; Hall et al., 1991; Hayes et al., 1990; 3

Bertazzi et al., 1989; Edling et al., 1987; Blair et al., 1986; Stroup et al., 1986; Harrington and 4

Oakes, 1984; Levine et al., 1984; Walrath and Fraumeni, 1984, 1983; Friedman and Ury, 1983). 5

Only a few studies reported any cases of sinonasal cancer. No cases of this type of cancer were 6

reported in any of the studies of professional workers examined by the I ARC. Only 2 cases (2.2 7

expected) were reported by Blair et al. (1986), and only 1 case (1.7 expected) was reported by 8

Gardner et al. (1993). The likelihood of finding this rare tumor type in a long

- term cohort study 9

is low. 10

20

4.1.2.1. 2.3. Summary of nasal and paranasal cancers.

31 The pooled case

- control study of Luce 21

et al. (2002) provides strong evidence of an association between formaldehyde exposure and 22

increased risk of sinonasal adenocarcinoma. The cohort studies may not have had sufficient 23

statistical power to show an association, and studies that did not distinguish cancer type may 24

have aggregated a truly causal relationship with a noncausal relationship with SCC. In summary, 25

there appears to be increased risk of sinonasal canc er associated with formaldehyde exposure 26

with or without exposure to wood dust. The effect appears to be stronger when the risk is 27

stratified by cancer type with higher risks of adenocarcinoma compared with SCC. Taken 28

together with the NPC findings in th e neighboring tissue, it is concluded that there is evidence of 29

higher risks of sinonasal cancer associated with exposure to formaldehyde. 30

4.1.2.1.3

. Other respiratory tract cancers

. Of six cohort studies of buccal/pharynx cancer in 32

studies of professio nals reviewed by IARC (Hayes et al., 1990; Logue et al., 1986; Stroup et al., 33

1986; Levine et al., 1984; Walrath and Fraumeni, 1984, 1983), no evidence of a risk associated 34

with exposure to formaldehyde was reported (see Table 4

- 3). In studies of industri al worker 35

cohorts where buccal/pharynx cancer was examined (Andjelkovich et al., 1995; Stayner et al., 36This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 77 1988; Blair et al., 1986), only one (Stayner et al., 1988) reported an excess risk of death from this 1

tumor (SMR = 3.4, based on four deaths in a cohort of 6,741 white women). Three case

- control 2

studies (Merletti et al., 1991; Vaughan et al., 1986a, b) did not find an association between oral 3

cavity, oropharyngeal, and hypopharyngeal cancers and formaldehyde exposure. However, 4

Merletti et al. (1991) foun d an elevated OR of 1.8 associated with probable or definite exposure 5

to formaldehyde in a study of 86 patients matched with 373 controls. There was no risk of 6

laryngeal cancer associated with formaldehyde in a case

- control study (Wortley et al., 1992) of 7

235 patients with laryngeal cancer and 547 controls. The OR in that study was 1.0 (adjusted for 8

age, smoking, drinking, and level of education). IARC (1995) concluded that there was little 9

evidence of an increased risk of laryngeal cancer. 10

11

4.1.2.1.3.1. Cohort studies of other respiratory tract cancers.

The Marsh et al. (2002) update also derived elevated risk estimates for oropharyngeal, 20

hypopharyngeal, and pharyngeal

- unspecified cancers. The SMRs ranged from 2.11 to 2.25 21

based on U.S. rates and 1.52 to 1.89 based on county rates. When combined with NPC 22

International Classification of Death (ICD) codes 146

– 149 to increase statistical pow er, the total 23

pharyngeal cancer SMRs were significant based on U.S. death rates (SMR = 2.63, n = 22, p < 24

0.01) and county death rates (SMR = 2.23, n = 22, p < 0.01) and remained significant for both 25

short

- term (less than 1 year) and long

- term exposures. F urthermore, using the exposure 26

estimates of Marsh et al. (1996), both cumulative and average exposure to formaldehyde resulted 27

in elevated SMRs, some of which were significant for pharyngeal cancer. Coggon et al. (2003) 28

identified 14 cases of cancer of th e larynx (13.1 expected) in their cohort of formaldehyde

29

exposed factory workers. Pinkerton et al. (2004) found an excess risk of mortality from buccal 30

cavity cancer (SMR = 1.33, four deaths observed) and a deficit of the risk of pharyngeal cancer 31

(SMR = 0.64, three observed). Since the number of observed deaths was small and the risk 32

estimates were subject to much variation for both studies, no conclusions about cancer risk can 33

be drawn. 34 Hansen and Olsen (1995) 12

reported a 10% increase in the risk of cancer of the buccal cavity and pharynx (SPIR = 1.1) in 13

their proportional incidence study of Danish workers. Marsh et al. (1996) reported no excess 14

risk of buccal cavity cancer cases (SMR = 1.31) based on U.S. rates and no excess based on state 15

mortality rates (SMR = 1.0). For oropharyngeal cancer, the SMR was 1.84 (based on two cases), 16

the SMR for hypopharyngeal cancer was 1.41 (based on one case), and the SMR for laryngeal 17

cancer was 1.47 (based on six cases). The latter risks were elevated even when SMRs were 18

derived from Connecticut mortality rates. 19This document is a draft for reviewpurposes only and does not constituteAgency policy4-78 Table 4

- 3. Epidemiologic studies of formaldehyde and pharyngeal c ancer (includes nasopharyngeal cancer)

Reference Study design Exposure assessment Results, statistical significance (number observed deaths for cohort study)

Marsh et al. (2002) Retrospective cohort

mortality study of 7,328

workers hired up to 1984

an d followed until 1998 in

one plant from Blair et al.

(1986, 1987) and

Hauptmann et al. (2004).

Mortality was compared

with death rates in two

Connecticut counties and

U.S. A nested case

- control

analysis was also conducted

with 4 controls matched on

age, year of birth, race, and

sex randomly selected from

cohort. Conditional logistic

model was used for nested

case

- control analysis. Worker

- specific exposures

from job exposure matrix

were based on available

sporadic sampling data from

1965

– 1987, job descri ptions,

and verbal job descriptions

by plant personnel and

industrial hygienists.

Exposures were then ranked

on a 7

- point scale. An

exposure range was assigned

to each rank. 17% of jobs

validated with company

monitoring data, remaining

83% based on prof essional

judgment. Pre

- 1965 levels of

formaldehyde were assumed

to be the same as post

- 1965

levels. Cohort study

Overall

U.S. SMR 2.63 (95% CI: 1.65

– 3.98) (22)

County SMR 2.23 (95% CI: 1.40

– 3.38) (22)

Short

- term worker (<1 y ear)

SMR 2.35 (95% CI: 1.22

– 4.11) (12)

Long

- term worker (1 or more years)

SMR 2.10 (95% CI: 1.01

– 3.86) (10)

Cumulative exp. (ppm

- years) county

Unexposed SMR 1.24 (95% CI: 0.15

– 4.49) (2)

>0 to <0.004 SMR 3.31 (95% CI: 1.22

– 7.21) (6)

0.004

– 0.219 SMR 2.06 (95% CI: 0.83

– 4.24) (7)

0.22+ SMR 2.30 (95% CI: 0.92

– 4.73) (7)

Average Exposure (ppm) county

Unexposed SMR 1.24 (95% CI: 0.15

– 4.49) (2)

>0 to <0.03 SMR 2.02 (95% CI: 0.74

– 4.40) (6)

0.03

– 0.159 SMR 3.82 (95% CI: 1.54

– 7.88) (7)

0.16+ SMR 2.03 (95% CI: 0.82

– 4.19) (7)

Exposure to **≤** 0.2 ppm SMR 1.72 (95% CI: 0.74

– 3.39) (8)

Exposure to >0.2 ppm SMR 2.68 (95% CI: 1.46

– 4.49) (14)

Exposure to **≤** 0.7 ppm SMR 2.12 (95% CI: 1 .21

– 3.45) (16)

Nested case

- control analysis

Cumulative exp. (ppm

- years)

<0.004 OR 1.00 (8)

0.004

– 0.219 OR 0.71 (95% CI: 0.20

– 2.43) (7)

0.22+ OR 0.79 (95% CI: 0.18

– 3.20) (7)

Average exposure (ppm)

<0.03 OR 1.00 (8)

0.03

– 0.159 OR 1.71 (95% CI: 0.47

– 6.10) (7)

0.16+ OR 0.99 (95% CI: 0.27

– 3.55) (7)

Exposure to >0.2 ppm OR 1.35 (95% CI: 0.45

– 4.25) (14)

Exposure to >0.7 ppm OR 1.60 (95% CI: 0.15

– 9.77) (6)This document is a draft for reviewpurposes only and does not constituteAgency policy4-79 Table 4

- 3. Epidemiologic studies of formaldehyde and pharyngeal c ancer (includes nasopharyngeal cancer)

Reference Study design Exposure assessment Results, statistical significance (number observed deaths for cohort study)

Coggon et al. (2003) Cohort mor tality study of

14,014 chemical workers

employed in 6 British

factories. Based on data abstracted

from company records. Each

job was categorized as having

background, low, moderate,

high, or unknown levels of

formaldehyde. Overall SMR 1.55 (95% CI: 0.87

– 2.56) (15)

High exposure SMR 1.91 (95% CI: 0.70

– 4.17) (6)

Shangina et al.

(2006) Multicentered, hospital

based case

- control study in

four European countries;

men only. Cancer cases: 34

hypopharyngeal; 316

laryngeal. Controls: 728

hospita l patients with

various conditions. Exposures determined by

local industrial hygienists,

chemists, and physicians.

Coding was established and

standardized. Categories

were developed for 73

agents; frequency was

estimated as the proportion of

time a worke r was exposed.

Linear trends were examined

for duration in years,

weighted duration in hours,

and cumulative exposure. Formaldehyde Laryngeal cancer:

Ever vs. never

Highest cumulative

(>22,700 mg/m 3

- hours)

vs. lowest

Tests of trends:

Years exposed

Cumul ative exposure OR

OR

p = 0.06

p = 0.07 1.68

3.12 (95% CI: 0.85

– 3.31)

(95% CI: 1.23

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

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- 80 Hauptmann et al. (2004) combined URT cancers (cancers of the salivary gland, mouth, 1

nasopharynx, nasal cavity, and larynx). For average inte nsity of exposure (AIE), the RR was 2

1.69 for the medium exposure category (0.5 to <1.0 ppm) and 2.21 ( p < 0.05) for the high 3

exposure category (>1.0 ppm). For peak exposure, the RR was 1.24 for the medium exposure 4

category (2.0 to <4.0 ppm) and 1.65 for t he high exposure category (>4.0 ppm). For cumulative 5

exposure, the RR was 1.92 for the medium exposure category (1.5 to <5.5 ppm) but 0.86 in the 6

high exposure category (>5.5 ppm

- years). The dose trends for these analyses, while suggestive 7

for average an d peak exposures, were not statistically significant. 8

9

4.1.2.1.3.2. Case

- control studies of other respiratory cancers.

In a case

- control study, Laforest et al. (2000) examined 201 patients w ith squamous cell 18

hypopharyngeal cancer and 296 patients with squamous cell laryngeal cancer, who were matched 19

to 296 controls with cancers of other sites in 15 French hospitals. Adjusting for potential 20

confounders, the OR of hypopharyngeal cancer in pati ents with a high probability of exposure to 21

formaldehyde was 3.78 (95% CI: 1.50

– 9.49). The ORs were significantly increased with both 22

exposure durations and high cumulative level of exposure. 23 Gustavsson et al. (1998) 10

conducted a case

- control study of 545 cases of SCC of the oral cavity, oropharynx, hypopharynx, 11

larynx, and es ophagus, frequency

- matched by age and region with 641 controls. Regression 12

analyses among 545 male cases showed elevated but nonsignificant risks of SCC of the oral 13

cavity (OR = 1.28), esophagus (OR = 1.90), and larynx (RR = 1.45) associated with 14

formalde hyde exposure. However, several of the carcinoma types were statistically significantly 15

associated with exposure to welding fumes, polyaromatic hydrocarbons, asbestos, and metal 16

dust. 17

Marsh et al. (2002) conducted a nested case

- control study of t he 22 pharyngeal cancer 24

deaths in the Wallingford, Connecticut, plant cohort. Each of the pharyngeal cancer deaths was 25

matched on race, sex, age, and year of birth to four controls from the cohort. Twenty of the 26

22 cases were exposed to formaldehyde, yie lding an OR of 3.04 after adjustment for smoking 27

and year of hire. There was little or no association of pharyngeal cancer incidence in these 28

workers with either average or cumulative exposure, based on the exposure estimates in this 29

study. There was a s uggested trend of increasing OR with increasing duration of exposure for 30

any formaldehyde exposure as well as for formaldehyde exposure >0.2 ppm. The results of this 31

nested case

- control analysis are inconclusive because of its low statistical power and 32

qu estionable exposure estimates, which differed substantially from those estimated by the NCI 33

(see Section 4.1.1.1). In addition, the relatively flat dose

- response curve in the nested case

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

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- 81 control study contradicts the positive dose

- response curves reported (particularly for NPCs) in 1

the same study, based on SMRs derived from county and U.S. death rates in the cohort analysis. 2

Shangina et al. (2006) conducted a multicentered case

- control study in Europe and Russia 3

of 34 cases of hypopharyngeal cancer, 316 ca ses of laryngeal cancer, and 728 controls. With 4

regard to formaldehyde exposure, a nonsignificant positive association was found for laryngeal 5

cancer (OR = 1.68, 95% CI: 0.85

– 3.31). Trends over increasing exposure were found for 6

duration of exposure in y ears ( p = 0.06) and for cumulative exposures ( p = 0.07). The 7

investigators reported an OR of 3.12 (95% CI: 1.23

– 7.91) for the highest cumulative exposure 8

group (>22,700 mg/m 3

- hours) compared with the unexposed group. 9

10

4.1.2.1.3.3. Summary of other respir atory cancers.

17 The evidence for a compound

- specific 11

effect on the risk of buccal/pharynx, oral cavity, oropharynx, hypopharynx, and laryngeal 12

cancers as a result of exposure to formaldehyde is minimal. Only the study by Laforest et al. 13

(2000) and, to a l esser extent, that by Shangina et al. (2006) provided evidence of an association 14

between formaldehyde and these tumors. However, even the study of Laforest et al. (2000) had 15

major limitations that made the evidence of an association suggestive at best. 16

4 .1.2.1.4. Lung Cancer. None of the cohort studies of workers in specific professions indicated 18

excess risks of lung cancer. Of the professional studies reviewed, the RRs range from an 19

extremely low value (SMR) of 0.2 in Hall et al. (1991), based on nine deaths, to an RR 20

(proportional mortality ratio) of 1.1, based on 70 lung cancer deaths in Walrath and Fraumeni 21

(1983). 22

23

4.1.2.1.4.1. Industrial worker cohort studies of lung cancer.

Several indust rial cohort studies (Andjelkovich et al., 1995; Bertazzi et al., 1989, 1986; 29

Stayner et al., 1988; Edling et al., 1987) reported no significant excess risks of lung cancer from 30

exposure to formaldehyde. No consistent association between formaldehyde expos ure and lung 31

cancer was found in several reports of the NCI 10

- plant cohort originally investigated by Blair et 32

al. (1987, 1986). Hauptmann et al. (2004) gives the most recent report on this cohort, which has 33

been studied in part or in its entirety by sev eral others (Marsh et al., 1994, 1992a, b; Sterling and 34

Weinkam, 1994, 1989a, b, 1988; Robins et al., 1988; Liebling et al., 1984; Fayerweather et al., 35 Evidence of a relationship 24

between formaldehyde exposure and lung cance r is conflicting, with some studies showing 25

modest increases while others show significant deficits in risk. There is, at best, only weak 26

evidence from several studies to suggest that exposure to formaldehyde is associated with lung 27

cancer. 28This document is a draft for review purposes only and does not constitute Agency policy.

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- 82 1983; Wong, 1983; Marsh, 1982). Marsh et al. (1996) reported small but significantly increased 1

risks of respiratory cancer in males at one plant from the cohort. The SMRs were 1.22 based on 2

U.S. rates, 1.34 based on Connecticut rates, and 1.28 based on county rates. 3

Similarly, Gardner et al. (1993) and Acheson et al. (1984) found a significant but modest 4

association between lung cancer and formaldehyde exposure (SMR = 1.2 [95% CI: 1.1

– 1.4]). In 5

workers hired after 1964, the SMR was 1.1. No trends by level or duration of exposure were 6

found. Pinkerton et al. (2004) and Stayner et al. (1988) studied a coh ort of 11,030 workers in 7

three garment plants and found an SMR of 1.1 for lung cancer. Some studies have found modest 8

elevations in risk of lung cancer with formaldehyde exposure, some of which were significant. 9

Coggon et al. (2003) updated the Gardner e t al. (1993) study of industrial workers. By taking 10

data from all six factories together, results showed a statistically significant excess risk of lung 11

cancer in the high

- exposure category when compared with British national mortality rates (SMR 12

= 1.58 [ 95% CI: 1.40

– 1.78]) and to local mortality rates (SMR = 1.28 [95% CI: 1.13

– 1.44]). 13

Callas et al. (1996) reanalyzed the cumulative exposure of 279 lung cancer cases among white 14

male workers from the NCI study, which comprised 80% of the NCI cohort (Blair e t al., 1986). 15

The analysis revealed modest RRs of 1.46, 1.27, and 1.38 for lung cancer in the cumulative 16

exposure categories 0.05 to 0.5, 0.51 to 5.5, and greater than 5.5 ppm

- years, respectively. None 17

of these RRs were significant. Finally, Matanoski ( 1991) reported a significant deficit in the risk 18

of respiratory cancer (SMR = 0.56 [95% CI: 0.44

– 0.70]; 77 observed) in pathologists 19

presumably exposed to formaldehyde based on U.S. mortality rates. 20

21

4.1.2.1.4.2. Case

- control studies.

De Stefani et al. (2005) conducted a case

- control study of 338 adenocarcinomas of the 30

lung in male patients admitted to four Montevide o hospitals from 1994 to 2000. The highest 31

ORs were for smoking (6.0 [95% CI: 3.3

– 11]) and for former smokers (4.0 [95% CI: 2.1

– 7.3]). 32

In addition, three agents (i.e., asbestos, silica dust, and formaldehyde) indicated significant 33

excess risks of lung ad enocarcinoma after adjusting for smoking history. A significant exposure

34 Several case

- contr ol studies of lung cancer (Partanen et al., 22

1990; Gerin et al., 1989; Bond et al., 1986; Coggon et al., 1984; Fayerweather et al., 1983; 23

Anderson et al., 1982) showed no excess lung cancer risk associated with potential exposure to 24

formaldehyde when analyz ed by length of exposure, intensity, and potential exposure 5, 10, or 25

15 years before death or by combinations of these factors. By contrast, Coggon et al. (1984) 26

reported a statistically significant increase in risk of lung cancer among male patients wit h any 27

potential exposure to formaldehyde based on occupations listed on death certificates (SMR = 1.5 28

[95% CI: 1.2

– 1.8]). 29This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 83 duration relationship was found for formaldehyde for 21+ years of exposure (OR = 3.0 [95% CI: 1

1.6

– 5.8]; p = 0.004 for trend). 2

3

4.1.2.1.4.3. Summary of lung cancer.

13 The evidence for an association between formaldehyde

4

and lung cancer is limited. Only one study has found a statistically significant effect (Coggon et 5

al., 2003). However, there may be other explanations rather than exposure to formaldehyde for 6

this association

. Excep t for the findings of De Stefani et al. (2005), other studies of lung cancer 7

and exposure to formaldehyde have not supported this finding, including several well

- done 8

cohort studies that were specifically designed to evaluate lung cancer. Until the Coggon et al. 9

(2003) study of British formaldehyde workers is replicated or reevaluated to determine the cause 10

of the excessive lung cancer risk, evidence from that study alone is insufficient at this time to 11

support an association between lung cancer and formal dehyde exposure. 12

4.1.2.1.5. Summary of respiratory tract cancers. Recent studies of NPC continue to support an 14

association with exposure to formaldehyde even at low levels. In some studies, the association 15

between formaldehyde and NPC persisted even wh en adjusted for the effect of potential 16

confounders (Hauptmann et al., 2004). Data from some reports have suggested a dose

- response 17

relationship (Hauptmann et al., 2004; Marsh et al., 2002; Vaughan et al., 2000). 18

The risk of NPC was significantly elevated among industrial workers with cumulative 19

exposure, average exposure, and peak exposure to formaldehyde (Hauptmann et al., 2004). The 20

studies of the single Wallingford plant by Marsh et al. (2002, 1996, 1994) and Marsh and Youk 21

(2005) also revealed a dose

- response trend, although the absolute exposure level estimates were 22

much lower. The relatively flat dose

- response curve seen in the nested case

- control study by 23

Marsh et al. (2002) of all pharyngeal cancers was inconsistent with the positive dose

- respons e 24

curves reported in the same paper based on county and U.S. death rates, particularly for NPC. 25

Also of interest was the finding of a statistically significant increase in the risk of NPC in 26

formaldehyde

- exposed workers who were seropositive for EBV in th e Hildesheim et al. (2001) 27

study. 28

The pooled analysis by Luce et al. (2002) provides evidence of a relationship of sinonasal 29

cancer, particularly adenocarcinoma, with formaldehyde. However, as with some of the studies 30

of NPC, the findings are potentially confounded by concurrent exposure to wood dust. When 31

wood dust exposure was adjusted for in the analysis, the resulting risks were still positive but 32

based on small numbers and, as a result, subject to much variability. The more recent studies 33

continued to reveal small significant and nonsignificant associations among cancers of 34This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 84 buccal/pharynx, oral cavity, hypopharynx, and larynx and exposure to formaldehyde. However, 1

the estimates were always based on small numbers. 2

A recent study that reported stati stically significant lung cancer in association with a high 3

level of exposure to formaldehyde was conducted by Coggon et al. (2003). The investigators 4

suggested that unknown lifestyle factors, including smoking, could be responsible for the 5

finding. Desp ite the results of their analysis, the authors were unconvinced that formaldehyde 6

was the agent responsible for the elevation in lung cancer risk. However, De Stefani et al. (2005) 7

also reported a statistically significant risk of lung adenocarcinoma in f ormaldehyde

- exposed 8

hospital patients even when smoking was controlled for in their analyses. 9

In all studies of formaldehyde and lung cancer, smoking remains an important 10

confounder and possibly an effect modifier. Residual confounding of smoking or othe r 11

respiratory exposures (e.g., wood dust or chemical or particular exposures) must always be 12

considered. 13

14

4.1.2.2. Non

- Respiratory Tract Cancer 15

4.1.2.2.1. LHP cancers

. Cancers of the hematopoietic system include lymphosarcoma, 16

reticulosarcoma, Hodgki n’s disease, non

- Hodgkin’s disease, multiple myeloma, and all types of 17

leukemia, including lymphoid and myeloid. Virtually all of the studies of LHP cancers and 18

formaldehyde are cohort studies and are divided into two groups: professional and industrial. 19

Several of the studies of professional groups were reviewed in an IARC (1995) monograph and 20

are briefly discussed in the next section regarding their findings on cancer of the LHP system. 21

One case

- control study of non

- Hodgkin’s lymphoma is discussed at t he end of this section. 22

23

4.1.2.2.1.1. Professional cohort studies.

Harrington and Shannon (1975) conducted a cohort mortality study of 2,079 British 31

pathologists (1955

– 1973) and 12,944 British m edical laboratory technicians (1963

– 1973). When 32

compared with death rates for England and Wales, the all

- cause SMR for the pathologists was 33

0.60 versus 0.67 for the laboratory technicians. There was a significant increase in the risk of 34

lymphatic and hem atopoietic neoplasia (SMR 2.0; 8 observed with 3.3 expected; p < 0.01) 35 Several cohort studies have been undertaken by 24

professional groups (i.e., anatomists, pathologists, embalmers, and funeral directors) because 25

their careers are likely to bring them into c ontact with formaldehyde. Some studies have 26

reported an increase in the risk of myelogenous leukemia and other LHP cancers (see Table 4

- 4). 27

A few of the increased risks were statistically significant. None of the studies of professionals 28

have used perso nal exposure measurements of formaldehyde or other chemicals, making 29

specificity for any single exposure difficult to determine. 30This document is a draft for review purposes only and does not constitute Agency policy.

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- 85 among male pathologists. However, the SMR for technicians was only 0.6 (3 observed). The 1

low SMRs suggest that these professionals have a healthier profile compared with the British 2

p opulation. No actual exposure estimates are available. 3

Harrington and Oakes (1984) expanded the above study to include 2,307 male and 413 4

female pathologists and laboratory technicians. Mortality was only examined from 1973 until 5

1980; deaths that occurr ed before 1974 were not included in the update. The SMR for leukemia 6

was 0.91 in men and 9.26 (based on one case) in women. Although the earlier LHP cancer 7

deaths were not included in this analysis, the investigators say in their conclusion that their 8

pr evious suggestion of an increase in certain lymphatic neoplasia was not confirmed in the 9

present study because of small numbers. The exceptionally low SMRs suggest that this group of 10

professionals enjoyed a healthier lifestyle compared with the British po pulation as a whole. Just 11

as in the earlier studies of these professionals, no exposure estimates are available. 12

Hall et al. (1991) expanded the above study by including the newest members of the 13

Royal College of Pathologists. The cohort totaled 4,512 in dividuals, although only 3,069 males 14

and 803 females were included in the analysis. The reasons for this discrepancy were not 15

specified, although the authors mentioned that an unknown number of expected deaths for 16

Northern Irish and female Scottish pathol ogists were not calculated, 32 pathologists were lost in 17

follow

- up, and cause of death was unknown for 9 individuals. Follow

- up was extended from 18

1980 to 1986. Mortality was enumerated from 1974 to 1987, a period of time that differed from 19

both of the ea rlier studies described above. There were statistically not significant excess risks 20

for lymphatic and hematopoietic cancer (SMR 1.44; 10 observed) and leukemia (SMR 1.52; 21

4 observed) for both sexes combined, based on mortality rates in England and Wales. 22

Separately, there was 1 female death in the lymphatic and hematopoietic cancer category (0.57 23

expected). The most striking observation in this study is that, despite the low cancer mortality 24

(SMR 0.45 for all cancer; 53 observed but 118.19 expected), th ere was still an excess (but not 25

statistically significant) risk of hematopoietic cancers. This finding of an extremely low risk for 26

all cancers suggests that population death rates may not be appropriate as a referent group

— for 27

example, the SMRs for lung cancer (0.19) and nonneoplastic respiratory diseases (0.23) were 28

significantly decreased, suggesting a lower prevalence of smoking among the pathologists 29

compared with the general population of England and Wales. However, the finding of a possibly 30

increa sed risk of LHP cancers should be analyzed further by selecting a more appropriate 31

reference population (another professional group without exposure to formaldehyde) or by 32

utilizing internal comparisons. 33

Walrath and Fraumeni (1983) conducted a proportionat e mortality study of all embalmers 34

and funeral directors licensed in the state of New York between 1902 and 1980 who were known 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 86 to have died between 1925 and 1980. The investigators requested death certificates for 1,678 1

persons but received only 1,263 (7 5%). The investigators restricted their analysis to 1,132 2

males. The distribution of the causes of death was compared with the age

- , race

- , and calendar

3

year

- specific proportions of deaths for each cause among the male U.S. population. Duration of 4

expos ure was approximated by time since first license. While the methodology could not be 5

applied in all calculations because of data gaps, excess risks were found for lymphatic and 6

hematopoietic cancers, with a proportionate mortality ratio (PMR) of 1.2 (obse rved 25), and for 7

leukemia, with a PMR of 1.32 and proportionate cancer mortality ratio (PCMR) of 1.19 8

(12 observed). The PMRs were not affected when the estimates were stratified by latency 9

(<35 years or 35 years since first license) or by age at first l icense. Because the cause of death 10

could not be determined for nearly 25% of the study group, the risk estimates could be 11

underestimated. The metrics, PMR, and PCMR are not stochastic processes. An increase in one 12

cause would produce decreases in all th e other causes. 13

Using the proportionate mortality method, Walrath and Fraumeni (1984) studied 1,007 14

deceased white male embalmers, members of the California Bureau of Funeral Directing and 15

Embalming, whose deaths occurred between 1925 and 1980. The decede nts had to have been 16

licensed to practice between 1916 and 1978. For lymphatic and hematopoietic cancer, the PMR 17

was 1.22 (19 deaths observed). For leukemia alone, the PMR was 1.75 and significant 18

(12 deaths observed, p < 0.05). Among embalmers licensed for 20 years or longer, the risk of 19

leukemia increased and was also significant (PMR 2.21; 8 observed; p < 0.05). But this study, 20

like the study of New York embalmers, had the same limitations discussed above. The 21

investigators did not provide informati on on the number of embalmers for whom no cause of 22

death could be found. 23

Levine et al. (1984) conducted a cohort mortality study of 1,477 male Ontario 24

undertakers first licensed between 1928 and 1957 and followed until the end of 1977. Out of 25

359 subjects who had died, there were 8 deaths from lymphatic and hematopoietic cancers 26

compared with 6.5 expected. Additionally, there were 4 deaths from leukemia versus 27

2.5 expected. Because death rates were not available for Ontario before 1950, person

- years and 28

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

undertakers. 30

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

members of the American Association of Anatomists between 18 88 and 1969. The investigators 32

derived SMRs from the U.S. white male population and used members of the American 33

Psychiatric Association (APA) as a comparison group. Vital status was ascertained between 34

1925 and 1979. Women were excluded from analysis b ecause of the small numbers. Only 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 87 738 deaths were observed versus 1,133.9 expected, based on U.S. death rates (SMR 0.65), 1

possibly indicating a sizable HWE. However, a slight increase in the risk of lymphatic and 2

hematopoietic cancers (SMR 1.2; 18 observ ed) and the risk of leukemia (SMR 1.5; 10 observed) 3

was evident. A significant increase in the risk of brain cancer (SMR 2.7; 10 observed; p < 0.05) 4

was also reported. When the leukemia analysis was restricted to the myeloid type, the SMR 5

increased to 8. 8, based on five deaths ( p < 0.05). The analysis using the APA group was 6

restricted to deaths that occurred between 1900 and 1969. This restriction removed five 7

leukemia deaths and person

- years from the analysis because they likely died after 1969. Beca use 8

of this, there were only 3 leukemia deaths versus 3.6 expected, based on APA death rates. The 9

investigators concluded that the etiological agent had not been definitively identified, mentioning 10

that a wide range of solvents, stains, and preservatives, including formaldehyde, are used to 11

prepare biological specimens. 12

Logue et al. (1986) conducted a cohort study of male radiologists and pathologists 13

registered with the Radiation Registry of Physicians and the College of American Pathologists 14

(CAP) betwee n 1962 and 1977. Although the main focus was on determining mortality in 15

radiologists from exposure to ionizing radiation, mortality was also ascertained for pathologists 16

alone. To derive SMRs, expected deaths were the sum of the products of person

- years times 17

death rates for both cohorts during the follow

- up period in white males only. However, there 18

were no exposure measurements, and the SMRs were not adjusted for calendar time. Of 5,585 19

members of the CAP, 496 had died by December 31, 1977. Although the SMR was 0.48 for 20

pathologists for cancer of the lymphatic and hematopoietic system, for the more specific 21

category of leukemia and aleukemia the SMR was 1.06 (neither was significant). For 22

radiologists, the SMRs were 0.78 and 1.55, respectively, also not significant. Cause of death 23

could not be determined for 8% of the deaths. Although age

- adjusted rates for leukemia were 24

also calculated for each cohort, they were only used for comparison between the two separate 25

professional groups. 26

Hayes et al. (1 990) conducted a proportionate mortality study of 3,649 deceased white 27

and 397 deceased nonwhite U.S. male embalmers and funeral directors who had died between 28

1975 and 1985, using records from local licensing boards, state funeral directors’ associations in 29

32 states and the District of Columbia, the National Funeral Directors’ Association, and state 30

offices of vital statistics (n = 894). Expected deaths by cause were derived from 5

- year age

- and 31

calendar

- year

- specific proportions of deaths among appropri ate race groups from the U.S. 32

population. No measured exposure data were available. A PCMR would be derived by 33

excluding noncancer causes of death. Statistically significant excesses in hematopoietic and 34

lymphatic cancers were found in white (PMR 1.31 [ 95% CI: 1.06

– 1.59]; 100 observed) and 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 88 nonwhite (PMR 2.41 [95% CI: 1.35

– 3.97]; 15 observed) embalmers and funeral directors. The 1

combined PMR was 1.39 (95% CI: 1.15

– 1.63). The excess risk was higher for myeloid 2

leukemia ( ML) (PMR 1.61 [95% CI: 1.02

– 2.41]; 23 observed) and for other unspecified 3

leukemias (PMR 2.08 [95% CI: 1.21

– 3.34]; 17 observed) in white males. The risks were 4

elevated in nonwhite males based on only a few cases (PMR 1.33 [95% CI: 1.10

– 1.60]; 5

4 observed). 6

Matanoski (1991) conducted a stud y of 6,111 male pathologists for NIOSH. Members of 7

the cohorts were part of an earlier unpublished study. Twenty

- nine thousand psychiatrists were 8

used as a comparison group. Both samples were selected from the membership rolls of 9

professional associatio ns. A total of 3,787 pathologists died between 1940 and 1978. Women 10

were excluded from the analysis. Of the population of psychiatrists, 4,788 died by 1980. U.S. 11

age

- and calendar

- time

- specific death rates from 1925 were used to develop SMRs. Separate 12

SMRs were based on psychiatrists’ death rates. The risk of hematopoietic cancer (excluding 13

Hodgkin’s disease) was elevated (SMR 1.25; 57 observed) based on U.S. white males. For 14

leukemia, the SMR was 1.35 (31 observed). The SMR for leukemia among psych iatrists was 15

0.83 (35 observed). Compared with leukemia in psychiatrists, the SMR for pathologists was 16

1.68 (95% CI: 1.14

– 2.38). The SMR for other lymphatic cancers was 1.53 (16 observed) and for 17

LHP cancer 1.22 (64 observed). Comparing the pathologists ’ death rates to those of psychiatrists 18

could be thought to have greater validity than if death rates for the U.S. population as a whole 19

had been used, because of shared socioeconomic circumstances and access to medical care 20

between the two professional gr oups. Differences in access to health care might have been 21

greater for subjects in the earlier part of the study, because improved diagnosis and medical care 22

for LHP cancers became more broadly available later in the study period. By using SMRs based 23

on U.S. death rates, which include those who do not have adequate access to medical care, the 24

difference between expected and observed deaths would be reduced. This is less likely to occur 25

when one professional group is compared with another professional gro up, assuming 26

psychiatrists and pathologists have equal access to care. 27

28

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

studies that show associations between LHP cancer and formaldehyde. The studies by Mars h et 30

al. (1994), Blair et al. (1986), and Acheson et al. (1984) and the later update by Gardner et al. 31

(1993) provide estimates of exposure to formaldehyde. The remaining studies generally rely 32

either on duration of exposure (number of years in the job) a s a surrogate (Pinkerton et al., 2004) 33

or provide no exposure assessment. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 89 Marsh et al. (1994), in an early study of the Wallingford plant, which is also part of the 1

Hauptmann et al. (2004, 2003) and Blair et al. (1986) studies, found SMRs of 0.89 and 0.91, 2

based on U.S. and county death rates, respectively (25 observed deaths). The authors did not 3

further discuss this cancer site until after Hauptmann et al. (2003) was published. Blair et al. 4

(1986) reported on 4,396 deaths from all causes in the 10 forma ldehyde

- associated factories that 5

made up the NCI cohort of 26,561 workers employed before January 1, 1966. There was little 6

evidence of an association with LHP system cancer (SMR 0.91; 56 observed) in exposed white 7

men, who dominated the cohort. 8

Hauptman n et al. (2003) updated the cohort mortality study of Blair et al. (1986) that 9

consisted of predominantly the same (25,619) workers from 10 plants. The primary focus of this 10

analysis was cancer of the LHP system, including leukemia. The description and d emographics 11

of the current study are the same as those reported by Blair et al. (1986) and Stewart et al. 12

(1986). In the current update, follow

- up was extended through December 31, 1994. The 13

additional 15 years of follow

- up increased the number of deaths from 4,349 to 8,486. Exposures 14

were not updated for the 4% of workers still in exposed jobs in 1980, but eliminating exposure 15

estimates for these workers did not change the results since exposures received after this date 16

were considered so low as to con tribute little to the analysis by the authors. 17

Peak exposure categories were defined as nonexposed, low (0.1

– 1.9 ppm), medium (2.0

– 18

3.9 ppm), and high (4.0 ppm or greater). Average intensity categories of exposure were defined 19

as nonexposed, low (0.1

– 0.4 p pm), medium (0.5 to <0.9 ppm), and high ( ≥1.0 ppm). Cumulative 20

exposure was defined as nonexposed, low (0.1

– 1.4 ppm

- years), medium (1.5

– 5.4 ppm

- years), 21

and high ( ≥5.5 ppm

- years). Duration of exposure was defined as 0, 0.1

– 4.9 years, 5.0

– 22

14.9 years, and ≥ 15 years. The median TWA exposure level was 0.45 ppm, range 0.01

– 23

4.25 ppm. Only 2.6% of the workers had average exposure intensities of 2 ppm or higher, and 24

14.3% had peak exposures of 4 ppm or higher. A total of 3,201 workers had no exposure. The 25

medi an duration in formaldehyde

- exposed jobs was 2 years. The median TWA intensity for 26

formaldehyde exposure was 0.5 ppm among exposed workers. 27

A Poisson regression model was stratified for calendar year, age, sex, race, and pay 28

category (salary/wage). A min imum latency period of 2 years between exposure and death from 29

a potentially exposure

- related LHP cancer was assumed by the investigators to prevent the 30

inclusion of exposures not likely to contribute to the development of LHP cancer because of their 31

timin g. Other lag times were evaluated that did not improve the regression models. 32

There were 2,099 cancer deaths. Hauptmann et al. (2003) reported that mortality from all 33

causes, all cancers, and LHP malignancies were significantly lower among the unexposed (SMRs 34

0.77, 0.65, and 0.62, respectively). Among the exposed, the SMRs were 0.95, 0.90, and 0.80, 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 90 respectively, for mortality from all causes, all cancers, and LHP malignancies. These SMRs in 1

part reflect the HWE caused by using external U.S. population death rates as a referent. 2

Unexposed workers also may have differed from the exposed workers in other ways. The 3

remaining analyses used internal comparisons, avoiding the HWE. The referent group in this 4

analysis was the low exposure group rather than th e unexposed group, because nonexposed 5

workers, who are primarily managers, secretaries, and other non

- production personnel, were 6

considered likely to have different socioeconomic characteristics than workers in the production 7

areas. 8

Statistically significa nt positive associations were found for LHP malignancies and 9

leukemia, particularly myeloid, in certain higher exposure categories in comparison with 10

employees in the lowest exposure categories (Table 4

- 4). In the highest peak exposure level, the 11

RR for L HP malignancies was 1.87 (95% CI: 1.27

– 2.75; 64 observed) and for ML was 3.46 12

(95% CI: 1.27

– 9.43; 14 observed) compared with employees in the low exposure peak level. For 13

workers with high peak exposure levels, the RR for LHP malignancies was 1.71 (95% CI : 14

1.14

- 2.58; 49 observed) and 2.43 (95% CI: 0.81

– 7.25) for ML. The trend tests for slope were 15

highly statistically significant for both LHP malignancies ( p < 0.002) and ML ( p < 0.009). 16

Significant results for LHP cancers were also seen with the average in tensity exposure 17

metric. RRs were 1.63 ( p < 0.05) and 1.50 ( p < 0.05) for the medium and high categories, 18

respectively. The risk of ML was also significantly increased (RR = 2.49) in the highest 19

exposure category. In contrast, Hauptmann et al. (2003) di d not find statistically significant 20

associations of formaldehyde with LHP cancer, either by cumulative exposure or years of 21

duration. However, there were positive associations for leukemia (RR = 1.39) and ML (RR = 22

1.35) when exposure was 15 years or long er. 23

The authors concluded that formaldehyde may cause leukemia, particularly ML, in 24

humans. However, because results from other studies were inconsistent, they suggested caution 25

in drawing definite conclusions. A biological basis for the significant exce ss risk of LHP cancer 26

remains unclear. The authors pointed out several studies that indicate changes that are consistent 27

with chromosomal changes in formaldehyde

- exposed persons, such as increased frequencies of 28

(MN (He et al., 1998; Kitaeva et al., 1996; Suruda et al., 1993), sister chromatid exchanges 29

(SCEs) (Shaham et al., 2002, 1997; Yager et al., 1986), chromosomal aberrations (CAs) (He et 30

al., 1998; Bauchinger and Schmid, 1985), and DNA

- protein cross

- links (DPXs) (Shaham et al., 31

1997, 1996a) in perip heral lymphocytes of humans exposed to formaldehyde. 32

Hauptmann et al. (2003) identified 11 suspected carcinogens used at the plants: 33

antioxidants (unspecified), asbestos, carbon black, dyes and pigments, hexamethylenetetramine, 34

melamine, phenol, plasticize rs, urea, wood dust, and benzene. Some workers were employed as 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 91 chemists and laboratory technicians. The investigators did not find substantial changes in the 1

risk estimates after adjusting for exposure to these substances or for working as a chemist or 2

laboratory technician. They also eliminated the 586 benzene

- exposed persons from their 3

analysis and found similar results (benzene is a known human leukemogen). Smoking was not 4

likely to explain an increased risk of leukemia in this cohort, because no in crease was seen for 5

smoking

- related diseases, including lung cancer. The cohort consisted predominantly of males 6

(88%). Strengths include the fact that the cohort was large and there was a long period of 7

follow

- up that was 96.6% successful. Internal ana lyses eliminated the HWE. One potential 8

limitation that could lead to an underestimate of risks is the 3.4% or 866 lost to follow

- up. 9

The study by Hauptmann et al. (2003) has been criticized extensively by several experts 10

representing the formaldehyde ind ustry (Tarone and McLaughlin, 2005; Casanova et al., 2004; 11

Cole and Axten, 2004; Collins, 2004; Collins and Lineker, 2004). Most of the same criticisms 12

have been repeated in other critiques by the above

- mentioned authors and have been addressed in 13

the dis cussions concerning the details of the methodology. However, a few new issues have 14

arisen from these critiques, as follows. One issue pertains to a concern that person

- years at risk 15

of death may have been assigned wrongly to the highest “peak” category o f exposure for the 16

duration of the study period. For example, there is inconsistency in the fact that only 4% of the 17

original cohort (Blair et al., 1986) had average exposures equaling or exceeding 2 ppm yet 45% 18

of the person

- years were assigned to the pe ak exposure category. Average exposures are time

19

weighted exposures that can have brief excursions over 4 ppm and still average 2 ppm or less. 20

Only for the peak exposure surrogate were person

- year values assigned to the peak category 21

following the exposu re, because it is a test for the possibility that biological changes could have 22

been initiated from that brief high exposure that might increase the risk of cancer. If these 23

genetic changes are irrevocable, then the risk of cancer could be increased and s ubsequent person 24

× years should be assigned to that higher risk category. 25

According to Casanova et al. (2004), the assignment of peak exposures in the Hauptmann 26

et al. (2003) study was questionable because they were based on professional judgement. 27

Howeve r, there are adequate grounds for hypothesizing that the assignment of peak exposure 28

was completed before determination of vital status and cause of death. It is always possible that 29

some subjects may be subject to misclassification. Hauptmann et al. (20 03) chose this metric 30

partly because it more closely resembled the exposure that embalmers and pathologists may have 31

received from formaldehyde. This same criticism could be said about the Coggon et al. (2003) 32

study as well. 33

Hauptmann et al. (2003) have a lso been criticized because the metric “cumulative 34

exposure” was not significant and did not show a trend. No adequate explanation has been given 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 92 by Hauptmann et al. (2003) except that it is possible that this metric is not as sensitive for this 1

agent. D uration of exposure was only weakly associated with a trend of increasing risk. After 2

15 years’ duration, there appeared to be a slight increase in ML based on 10 cases (RR = 1.35 3

[95% CI: 0.56

– 3.24]). For all leukemia the risk in workers who were expose d 15 or more years 4

was somewhat higher (RR = 1.39 [95% CI: 0.78

– 2.49]) based on 22 cases. 5

Another of the criticisms from these authors discussed the lack of a biologically plausible 6

explanation for how leukemia could result from exposure to formaldehyde wh en there appears to 7

be no recognizable indication of the presence of formaldehyde in excessive quantities in the 8

blood of animals or any associated metabolites in experimental research animals. Hauptmann et 9

al. (2004b) responded that there is evidence tha t genotoxic effects can be detected in vivo in the 10

bone marrow of rats and in human peripheral lymphocytes. 11

Stayner et al. (1988) conducted a cohort study of 11,030 workers (82% female) followed 12

from 1955 or the beginning date of exposure through 1982 in t hree garment factories. Personnel 13

records from three garment manufacturing facilities, one in Pennsylvania and two in Georgia, 14

were used to assemble a cohort of workers who attained a minimum of 3 months of exposure 15

after the introduction of formaldehyde into these facilities. Formaldehyde resins were used to 16

treat fabrics, beginning in 1955 and 1959. Although formaldehyde levels were available on a 17

subset of the employees from monitoring data available from surveys completed in 1981 and 18

1984, they were not used in this analysis. Instead, the results were stratified by duration and 19

latency. SMRs were based on U.S. population mortality rates. Based on six cases, the SMRs for 20

leukemia were 2.43 and 3.81 among workers with 20 or more years since first exp osure or at 21

least 10 years of exposure, respectively. In their conclusions, the authors suggested that, 22

although the numbers of deaths from LHP cancers were small, the risks were related to duration 23

and latency. 24

Pinkerton et al. (2004) updated the Stayner et al. (1988) study by adding 16 years of 25

follow

- up. No new exposure information was added. The mean TWA exposure in 1981

– 1984 26

for the three plants was 0.15 ppm. No additional information regarding earlier industrial hygiene 27

data was available, althoug h the authors stated that the levels of exposure to formaldehyde were 28

greater in the years before 1980. Stayner et al. (1988) cited independent studies of exposure 29

levels in similar garment factories in the 1960s that seemed to indicate that the formaldeh yde 30

levels during that period ranged from 0.9 to 2.7 ppm (Blejer and Miller, 1966) in one garment 31

manufacturing area. Another report (Shipkovitz, 1966) of 10

- minute personal exposure samples 32

indicated a range from 0.3 to 2.7 ppm in eight garment plants. In another study (Ahmad and 33

Whitson, 1973), the levels ranged from 2 to 10 ppm. Goldstein (1973) calculated that 34

concentrations in the cutting rooms of garment plants dropped from 10 ppm in 1968 to less than 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 93 2 ppm in 1973 because of an improvement in the resin treating process. The authors assumed 1

that exposure ceased in 1981 and 1983. This produced an underestimate of exposure based on 2

duration of employment for about 11% of the cohort who were still actively employed after those 3

dates. Stayner et al. (1988) speculated that the risks of cancer of the buccal cavity, leukemia, and 4

other LHP neoplasia may have been due to exposure to the highest potential formaldehyde levels 5

in the industry between 1955 and 1962, because the resin used to treat permanent p ress fabrics 6

still contained a relatively large amount of formaldehyde. 7

The SMRs were derived from age

- , race

- , and calendar

- time

- adjusted U.S. mortality 8

rates. The analysis was repeated using Georgia or Pennsylvania mortality rates. In addition to 9

the p rimary analysis of the underlying cause of death, the analysis used all causes listed on the 10

death certificates to evaluate multiple cause mortality. As a referent for this, the analysis relied 11

on multiple cause death rates available since 1960 from the N ational Death Index maintained by 12

the U.S. Centers for Disease Control and Prevention (CDC). 13

Altogether, 608 cancer deaths were observed. The SMR for all cancer was 0.89 (95% CI: 14

0.82

– 0.97). The overall SMR for leukemia was 1.09 (24 deaths) and 1.44 (15 deaths) for ML. 15

After 10 years of exposure, the risk for ML was 2.19. Exposure prior to 1963 was associated 16

with a risk of 1.61. Among garment workers followed for 20 or more years from initial 17

exposure, the SMR was significantly elevated for ML (1.91; p < 0.05; 13 deaths), as was the 18

SMR for multiple cause leukemia (1.92 [95% CI: 1.08

– 3.17]; 15 deaths) in the subgroup with 19

10 or more years of exposure to formaldehyde and who were followed for 20 or more years after 20

first exposure. The multiple cause mo rtality for ML for this subgroup of workers was also 21

significant (SMR 2.55 [95% CI: 1.10

– 5.03]; 8 deaths). 22

The study by Stayner et al. (1988) has only limited power to detect excess risks of rare 23

cancers, such as NPC and nasal cancer (13 and 16%, respectiv ely). Limitations to the 24

interpretations of the findings include a lack of any monitoring data before 1981, particularly 25

during the critical time period 1955 to 1962, and lack of personal exposure estimates for any 26

members of the cohort. The possibility exists that misclassification may still be present because 27

the intensity of exposure to formaldehyde decreased as improvements were made in the resin 28

systems used to treat fabrics (e.g., a person who worked 5 years beginning in 1955 might have 29

been subject to greater exposure than a person who worked 5 years beginning in 1993). 30

However, workers from the 1950s and 1990s were both placed in the same category of having 31

worked fewer than 10 years. The median duration of exposure was 3.3 years. Work histories 32

were not updated in the follow

- up study; however, the low or background exposure levels that 33

probably existed after 1981 were not likely to contribute substantially to the risk of cancer. The 34

use of mortality data to estimate risk, when the case fatality rate was less than 100% for most 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 94 cancer sites evaluated, could potentially produce an underestimate of the actual risk. Despite 1

these limitations, this study provides additional evidence of an association between leukemia, 2

especially ML, and formaldehyde in comparison with the general population. 3

Gardner et al. (1993) reported that the risk of leukemia was not statistically significant 4

(SMR 0.9) based on 15 deaths among workers employed before 1965. Only four leukemia 5

deaths were observed after 1964 thro ugh 1989, producing an SMR of 0.9. 6

When Coggon et al. (2003) updated the above cohort study of 14,014 men first employed 7

before 1965 in six factories by adding 11 additional years of follow

- up (ending December 31, 8

2000), no increase in the risk of leukemia or related cancers of the hematopoietic system was 9

reported, either in the entire cohort (SMR 0.91; 31 observed) or in the group with the highest 10

formaldehyde exposure (>2 ppm) (SMR 0.71; 8 observed). Similar results were obtained for 11

Hodgkin’s disease, non

- Hodgkin’s lymphoma, and multiple myeloma. No other cancers of the 12

hematopoietic system were evaluated, and no additional analyses were performed to assess a 13

possible leukemia risk. However, the main finding from this study was a marked association of 14

lung cancer with formaldehyde (discussed in the lung cancer section). This study’s main focus 15

was respiratory disease, lung cancer, and stomach cancer, not LHP cancers. For cancers of the 16

LHP system, there was neither latency evaluation nor internal com parisons. The HWE is also 17

potentially a problem. 18

Andjelkovich et al. (1995) studied a cohort of 3,929 male iron foundry workers 19

potentially exposed to formaldehyde between January 1, 1960, and December 31, 1989, in which 20

127 cancer deaths had occurred dur ing the observation period. An industrial hygienist, after 21

reviewing work histories, categorized formaldehyde exposure into four levels corresponding to 22

the approximate midpoint of the ranges: none, low (0.05 ppm), medium (0.55 ppm), and high 23

(1.5 ppm) fo r exposure to formaldehyde. Boundaries of these exposure categories were not 24

given. The authors warned that the assignment of exposure levels was not perfect because 25

“subjective judgment had to be applied in many instances.” SMRs were based on U.S. male 26

mortality rates, but actual ranges were not specified. The authors also compared the exposed to 27

2,032 nonexposed workers from the same company. The population

- based SMR for 28

hematopoietic cancer in the exposed population was 0.59 (based on seven observed deaths). For 29

leukemia the SMR was 0.43, based on two deaths. There were no other analyses for leukemia or 30

LHP cancers in this study. Because of the uncertainty about workers’ true formaldehyde 31

exposure, there was no analysis by level of exposure, durat ion, or latency. There were also very 32

few LHP cancers in the cohort. Thus, these results neither support nor refute an association of 33

formaldehyde exposure with LHP cancers. The main focus of this paper was on lung cancer risk. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 95 Bertazzi et al. (1989, 19 86), in a cohort mortality study, followed 1,330 male workers 1

from 1959 through 1986 at a formaldehyde resin plant in Italy. The workers had to have been 2

employed for at least 30 days at the plant sometime between 1959 and 1980 to be included in the 3

study . Their mortality was compared with national and local rates adjusted for age and calendar 4

time period. No individual exposure estimates were available, but mean levels were estimated to 5

be between 0.2 and 3.8 mg/m 3 (0.16 and 3.1 ppm) during the period 1 974

– 1979. The authors 6

found an SMR of 2.01 (five deaths observed) for cancer of the lymphatic and hematopoietic 7

system. The study’s limitations included incomplete work histories, small numbers of deaths, 8

and a follow

- up period that may not have been suf ficient to allow for a latency period for the 9

development of LHP cancers. As before, the results neither support nor refute an association of 10

formaldehyde exposure with LHP cancers. 11

Edling et al. (1987) reported on the incidence of disease in a cohort of 521 blue collar 12

Swedish workers in plants where abrasives bound with formaldehyde resins were manufactured. 13

Formaldehyde levels ranged from 0.1 to 1.0 mg/m 3 (0.08

– 0.8 ppm). The workers in the cohort 14

were employed between 1955 and 1983, and incidence rate s were calculated from 1958 through 15

1981. There were only 24 total cancer cases (28.5 expected) of which 2 (1.0 expected) were 16

lymphomas and 2 (0.5 expected) were multiple myelomas. Expected cases were determined 17

through the Swedish National Cancer Regis ter. No other LHP cancers were observed. This 18

study lacked the power to detect any significant associations between LHP cancer and exposure 19

to formaldehyde. 20

Dell and Teta (1995) conducted a cohort mortality study of 5,932 male employees of a 21

New Jersey p lastics manufacturing, research, and development facility. The workers, who had 22

been employed during the period 1946

– 1967, were followed

- up for an average of 32 years. 23

SMRs were based on U.S. and New Jersey mortality rates. Hourly workers (n = 3,853) we re 24

analyzed separately from the 2,079 salaried employees. Although no excess risk was evident for 25

hematopoietic cancer in hourly workers (SMR 0.93; 28 observed), there was an SMR of 1.69 26

(95% CI: 1.07

– 2.53; 23 observed) among salaried workers. This assoc iation was further 27

narrowed to mainly research and development workers (eight leukemia deaths observed with 28

three expected, for an SMR of 2.67). No common exposure was found when work history 29

records were examined. The decedents were mostly associated wi th process development in two 30

research pilot plants, where chemical engineers, lab technicians, and plant operators executed 31

small

- scale product development. Although notebooks referred to benzene and toluene solvents, 32

no definite connection was made with formaldehyde or any of the solvents. No ambient air 33

measurements of formaldehyde were available. The findings cannot be assumed to be due to 34

formaldehyde exposure because of the presence of other potential leukemogens. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 96 Blair et al. (1993) conducted a st udy that evaluated the risk of non

- Hodgkin’s lymphoma 1

from exposure to formaldehyde. This was a population

- based, case

- control, interview

- based 2

study of 1,867 white males of whom 622 cases had the disease and 1,245 were controls. 3

Subjects had lived in Io wa and Minnesota between 1980 and 1983. This study was exploratory 4

and designed to find associations with any environmental exposures and non

- Hodgkin’s 5

lymphoma. Subjects or next of kin were interviewed to determine what exposures the cases and 6

controls may have received based on agricultural exposures, work histories, medical conditions, 7

and family history. Extra effort was made to collect information about occupation, industrial 8

exposures, and other selected exposures. The analysis revealed an OR of 1 .2 for exposure to 9

formaldehyde. Similar associations were found for metals and other substances in the study. 10

This study, because it did not select cases and controls from a population with possible 11

formaldehyde exposure, could not detect specific relat ionships between formaldehyde and 12

non

- Hodgkin’s lymphoma. 13

14

4.1.2.2.1.3. Summary of non

- respiratory tract cancers.

Additional support linking LHP cancer and formaldehyde comes from a study of garment 29

workers (Pinkerton et al., 2004) and studies of pathologists and other medical workers exposed 30

t o formaldehyde (Matanoski, 1991; Blair et al., 1990; Hayes et al., 1990; Stroup et al., 1986; 31

Walrath and Fraumeni, 1984, 1983; Harrington and Shannon, 1975). Hayes et al. (1990) and 32

Stroup et al. (1986) also reported significant excess risks of ML. 33 The Hauptmann et al. (2003) study 15

appears to provide the strongest evidence of an association for ML in particular. Statistically 16

significa nt positive associations were found for LHP malignancies and leukemia, particularly 17

ML, in certain higher exposure categories in comparison with employees in the lowest exposure 18

categories. In the highest peak exposure level, the RR for LHP malignancies w as 1.87 (95% CI: 19

1.27

– 2.75; 64 observed) and for ML was 3.46 (95% CI: 1.27

– 9.43; 14 observed) compared with 20

employees in the low

- exposure peak level. For workers with high

- peak exposure levels, the RR 21

for LHP malignancies was 1.71 (95% CI: 1.14

– 2.58; 49 o bserved) and 2.43 (95% CI: 0.81

– 7.25) 22

for ML. The trend tests for slope were highly statistically significant for both LHP malignancies 23

( p < 0.002) and ML ( p < 0.009). Significant results for LHP cancers were also seen with the 24

average intensity exposure metric. RRs were 1.63 ( p < 0.05) and 1.50 ( p < 0.05) for the medium 25

and high categories, respectively. The risk of ML was also significantly increased (RR = 2.49) 26

in the highest exposure category. In contrast, results showed no associations of formalde hyde 27

with LHP cancer, either by cumulative exposure or years of duration. 28

Sever al reports have challenged the association between LHP cancer and formaldehyde 34

(Casanova et al., 2004; Cole and Axten, 2004; Collins, 2004; Collins and Lineker, 2004; Coggon 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 97 et al., 2003; Casanova and Heck, 1987; Heck et al., 1985). These papers argue tha t a biological 1

explanation for the excess risk of LHP cancer or leukemia remains unclear. Absent a plausible 2

MOA by which formaldehyde could cause these cancers, many investigators have been unable to 3

accept the reported increased risks identified in the epidemiologic literature. Some researchers 4

have argued against the biological plausibility of formaldehyde

- induced lymphoreticular cancers 5

based solely on the assumption that formaldehyde as a reactive gas does not penetrate past the 6

POE. This argument i s relevant to diseases for which transformation of stem cells in the bone 7

marrow is essential. However, cancers that arise from more mature cells present outside of the 8

bone marrow compartment cannot be dismissed with this argument. Although often groupe d for 9

analysis, the lymphohemoreticular system cancers represent many distinct malignancies that may 10

arise from discrete cell types in different tissues throughout the body. For example, acute 11

lymphocytic leukemia (ALL) is believed to arise from the trans formation of a lymphoid stem cell 12

in the bone marrow, resulting in a blood

- borne leukemia of immature cells of the lymphoid cell 13

line. However, if transformation occurs in a mature lymphocyte (e.g., post

- germinal center B 14

cell), a chronic lymphocytic leuk emia (CLL) results. Although etiologically different, these 15

cases would both be lymphocytic leukemia. When considering biological plausibility of an 16

exogenous agent increasing the incidence of ALL, bone marrow toxicity would be expected. 17

However, when c onsidering the biological plausibility of CLL, bone marrow toxicity would not 18

be essential. Mutation or epigenetic changes attained in the mature cell may be passed on to 19

daughter cells during response to antigen and eventually lead to transformation. So the 20

etiologies of these two leukemias need not be similar. In contrast, a non

- Hodgkin’s lymphoma 21

results from transformation of a mature B or T cell resulting in a solid tumor. The etiology of 22

this cancer is actually similar to CLL. A recent reclassifi cation of lymphoid malignancies by the 23

WHO designates adult B

- cell leukemias and lymphomas as the same disease, with ALL as a 24

separate disease. Therefore, mortality analysis by ICD code and the standard groupings of those 25

codes does not reflect the biolog y of the cancers. 26

Considering the whole class of LHP cancers, there is a range of biological plausibility for 27

an agent whose primary action is at the POE. Acute leukemias (ALL and acute myelogenous 28

leukemia [AML]), believed to arise from transformation of stem cells in the bone marrow, are 29

less plausible, although trafficking of stem cells to different tissues would be an alternative 30

etiology for exogenous compounds acting at the POE. In contrast CLL, lymphomas, multiple 31

myelomas (from plasma B cells), an d unspecified cancers may involve an etiology in peripheral 32

tissues to include cells, cell aggregates, germinal centers, and lymph nodes. An association of 33

these cancers to an exogenous agent acting at the POE is biologically plausible. 34

35This document is a draft for reviewpurposes only and does not constituteAgency policy4-98 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

Harrington and

Shannon (1975) Cohort mortality study of 2,079

pat hologists and laboratory

technicians from the Royal

College of Pathologists and the

Pathological Society of Great

Britain from 1955

– 1973. The

comparison population came

from national mortality data. Presumed exposure to

formaldehyde tissue

fixative. Path ologists

All cause mortality SMR 0.60

LHP cancers SMR 2.0 p < 0.01 (8)

Hodgkin’s disease SMR 1.4 (1)

Leukemia SMR 0.6 (1)

Technicians

All cause mortality SMR 0.67

LHP cancers SMR 0. 5 (3)

Hodgkin’s disease SMR

– (0)

Leukemia SMR 0.5 (1)

Harrington and

Oakes (1984) Cohort mortality study of 2,720

pathologists from the Royal

College of Pathologists and the

Pathological Society of Great

Britain from 1974

– 1980. Vital

status obtained from the census, a

national health registry, and other

sources. SMRs developed from

the English, Scottish, Irish, and

Welsh populations. Presumed exposure to

formaldehyde tissue

fixative. All causes

Men SMR 0.56

Women SMR 0.99

Leukemia

Men SMR 0.91 (90% CI: 0.05

– 4.29) (1)

Women SMR 9.26 (90% CI: 0.47

– 43.9) (1)

Other LHP cancers

Men SMR 0.53 (90% CI: 0.03

– 2.54) (1)

Women SMR

– – (0)

Hall et al. (1991) Cohort mortality study of 4,512

patho logists from the Royal

College of Pathologists and the

Pathological Society of Great

Britain from 1974

– 1987. Vital

status obtained from the census, a

national health registry, and other

sources. SMRs developed from

the English and Welsh

populations. Pre sumed exposure to

formaldehyde tissue

fixative. All cause mortality

Men SMR 0.43 (176)

Women SMR 0.65 (18)

Hodgkin’s disease SMR 1.21 (95% CI: 0.03

– 6.71) (1)

All cancers SMR 1.44 (95% CI: 0.69

– 2.63) (10)

Leukemia SMR 1.52 ( 95% CI: 0.41

– 3.89) (4)

Levine et al. (1984) Cohort mortality study of 1,477 Presumed exposure to All LHP cancers SMR 1.24 (8)This document is a draft for reviewpurposes only and does not constituteAgency policy4-99 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

male Ontario undertakers first

licensed 1928

– 1957, followed

from 1950 to 1977. SMRs

developed from Ontario

mortality rates. formaldehyde tissue

fixa tive. Leukemia SMR 1.60 (4)

Stroup et al. (1986) Cohort mortality study of 2,317

white male members of the

American Association of

Anatomists from 1888 to 1969

who died 1925

– 1979. SMRs

developed using U.S. po pulation

mortality rates. Presumed exposure to

formaldehyde tissue

fixative. All cause mortality SMR 0.65 (95% CI: 0.60

– 0.70) (738)

All LHP cancers SMR 1.2 (95% CI: 0.7

– 2.0) (18)

Lymphosarcoma and

reticulosarcoma SMR 0.7 (95% CI: 0.1

– 2 .5) (2)

Hodgkin’s disease SMR

– – (0)

Leukemia SMR 1.5 (95% CI: 0.7

– 2.7) (10)

Other lymphatic SMR 2.0 (95% CI: 0.7

– 4.4) (6)

Logue et al. (1986) Cohort mortality study of 4,485

pathologists who were members

of the College of American

Patholo gists, 1962

– 1972,

followed for mortality through

1977. SMRs developed from

U.S. population mortality rates. Presumed exposure to

formaldehyde tissue

fixative. LHP cancer other than

leukemia SMR 0.48 (NR)

Leukemia SMR 1.06 (NR)

Matanoski (1991) Cohort mortality study of 6,111

male pathologists from

membership rolls of the

American Medical Association

1912

– 1950. Mortality was

followed through 1978. SMRs

developed from U.S. population

white male mortality rates. Presume d exposure to

formaldehyde tissue

fixative. All cancer SMR 0.78 (95% CI: 0.71

– 0.85) (508)

All LHP cancers SMR 1.25 (95% CI: 0.95

– 1.62) (57)

Lymphosarcoma and

reticulosarcoma SMR 1.31 (95% CI: 0.66

– 2.35) (11)

Hodgkin’s disea se SMR 0.36 (95% CI: 0.04

– 1.31) (2)

Leukemia SMR 1.35 (95% CI: 0.92

– 1.92) (31)

Other lymphatic SMR 1.54 (95% CI: 0.82

– 2.63) (13)

Hauptmann et al.

(2003) Retrospective cohort mortality

study of 25,619 workers

employed at 10 formaldehyde

plants i n the U.S. followed from

either the plant start

- up or first Exposure estimates

based on job titles,

tasks, visits to plants by

study industrial

hygienists, and All LHP cancers

Exposed SMR 0.80 (95% CI: 0.69

– 0.94) (161)

Unexposed SMR 0.62 (95 % CI: 0.39

– 1.00) (17)

Peak exposure (ppm)

employment through 1994. monitoring data through 0 RR 1.08 (95% CI: 0.60

– 1.94) (17)This document is a draft for reviewpurposes only and does not constituteAgency policy4-100 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

SMRs calculated using sex

- , age

-

, race

- , and calendar

- year

specific U.S. mortality rates.

RRs estimated using Poisson

regression stratified by calendar

year, age, sex, and race; adjusted

for pay category. 1980. Peak exposure

defined as short

- term
excursions exceeding

the 8

- hour TWA

formaldehyde intensity

and knowledge of job

tasks. Exposures to 11

other compounds were

identified. Workers

contributed pr e

exposure person

- time to

nonexposed category.

Poisson regression

models used a 2

- year lag to account for tumor

latency. 0.1

– 1.9 RR 1.00 Reference value (48)

2.0 to <4.0 RR 1.71 (95% CI: 1.14

– 2.58) (49)

4 .0 or greater RR 1.87 (95% CI: 1.27

– 2.75) (64)

Trend p = 0.002

Average exposure (ppm)

0 RR 0.91 (95% CI: 0.52

– 1.59) (17)

0.1

– 0.4 RR 1.00 Reference value (81)

0.5 to <1.0 RR 1.63 (95% CI: 1.11

– 2.37) (42)

1.0 or greater RR 1.5 0 (95% CI: 1.01

– 2.24) (38)

Trend p = 0.050

Cumulative exposure(ppm

- years)

0 RR 0.74 (95% CI: 0.42

– 1.30) (17)

0.1

– 1.4 RR 1.00 Reference value (94)

1.5 to 5.4 RR 0.79 (95% CI: 0.52

– 1.21) (29)

5.5 or greater RR 1.03 (95% CI: 0.70

– 1.52) (38)

Trend p = 0.157

Leukemia

Peak exposure (ppm)

0 RR 0.78 (95% CI: 0.25

– 2.43) (4)

0.1

– 1.9 RR 1.00 Reference value (16)

2.0 to <4.0 RR 2.04 (95% CI: 1.04

– 4.01) (20)

4.0 or greater RR 2.46 (95% CI: 1.31

– 4.62) (29)

Trend p = 0.001

Average exposure (ppm)

0 ppm RR 0.56 (95% CI: 0.19

– 1.66) (4)

0.1

– 0.4 RR 1.00 Reference value (32)

0.5 to <1.0 RR 1.52 (95% CI: 083

– 2.79) (16)

1.0 or greater RR 1.68 (95% CI: 0.91

– 3.08) (17)This document is a draft for reviewpurposes only and does not constituteAgency policy4-101 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

Trend p = 0.193

Cumulative exposure (ppm

- years)

0 RR 0.48 (95% CI: 0.16

– 1.42) (4)

0.1

– 1.4 RR 1.00 Reference value (35)

1.5

– 5.4 RR 0.90 (95% CI: 0.47

– 1.73) (13)

5.5 or greater RR 1.14 (95% CI: 0.63

– 2.07) (17)

Trend p = 0.183

Hodgkin’s disease

Peak exposure (ppm)

0 RR 0.51 (95% CI: 0.06

– 4.52) (1)

0.1

– 1.9 RR 1.00 Reference value (5)

2.0 to <4.0 RR 3.45 (95% CI: 0.98

– 12.2) (7)

4.0 or greater RR 3.35 (95% CI: 0.97

– 11.6) (8)

Trend p = 0.014

Average exposure (ppm)

0 RR 0.46 (95% CI: 0.05

– 3.93) (1)

0.1

– 0.4 RR 1.00 Reference value (7)

0.5 to <1.0 RR 4.70 (95% CI: 1.61

– 13.8) (8)

1.0 or greater RR 3.12 (95% CI: 0.91

– 10.7) (5)

Trend p = 0.022

Cumulative (ppm

- years)

0 RR 0.29 (95% CI: 0.04

– 2.34) (1)

0.1

– 1.4 RR 1.00 Reference value (12)

1.5

– 5.4 RR 1.35 (95% CI: 0.45

– 3.99) (5)

5.5 or greater RR 1.17 (95% CI: 0.31

– 4.46) (3)

Trend p = 0.037

ML

Peak exposure (ppm)

0 RR 0.67 (95% CI: 0.12

– 3.61) (2)

0.1 to 1.9 RR 1.00 Reference value (6)

2.0 to <4.0 RR 2.43 (95% CI: 0.81

– 7.25) (8)

4.0 or greater RR 3.46 (95% CI: 1.27

– 9.43) (14)This document is a draft for reviewpurposes only and does not constituteAgency policy4-102 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

Trend p = 0.003

Average exposure (ppm)

0 RR 0.41 (95% CI: 0.08

– 1.95) (2)

0.1 to 0.4 RR 1.00 Reference value (14)

0.5 to <1.0 RR 1.15 (95% CI: 0.41

– 3.23) (5)

1.0 or greater RR 2.49 (95% CI: 1.03

– 6.03) (9)

p = 0.086

Cumulative (ppm

- years)

0 RR 0.32 (95% CI: 0.07

– 1 .51) (2)

0.1

- 1.4 RR 1.00 Reference value (17)

1.5

- 5.4 RR 0.57 (95% CI: 0.19

– 1.73) (4)

5.5 or greater RR 1.02 (95% CI: 0.40

– 2.55) (7)

Trend p = 0.123

Pinkerton et al.

(2004) Cohort mortality study of 11,098

workers in 3 garment plant s exposed **≥** 3 months after

formaldehyde was introduced.

Women comprised 81.7% of the

cohort. Vital status was followed

through 1998. SMRs were

calculated by using sex

- , age

- ,

race

- , and calendar

- year

- specific

U.S. mortality rates. Multiple

cause SMRs we re derived from

all contributing causes from

death certificates. Data for 549 randomly

selected employees in 5

departments in 1981 and

1984 used to estimate

overall exposure levels.

Levels presumed to be

0.09

– 0.20 ppm. All LHP cancers SMR 0.97 (95% CI: 0 .74

– 1.26) (59)

Lymphosarcoma and

reticulosarcoma SMR 0.85 (95% CI: 0.28

– 1.99) (5)

Hodgkin's disease SMR 0.55 (95% CI: 0.07

– 1.98) (2)

Other lymphatic SMR 0.97 (95% CI: 0.64

– 1.40) (28)

Leukemia SMR 1.09 (95% CI: 0.70

– 1.62) (24)

Mortality since 1960

Lymphocytic leukemia SMR 0.60 (95% CI: 0.12

– 1.75) (3)

ML SMR 1.44 (95% CI: 0.80

– 2.37) (15)

10+ years of

exposure SMR 2.19 NS (8)

20+ years since 1st

exposure SMR 1.91 p > 0.05 (13)

Multiple cause leukemia

10+ years of

exposure and 20+

years since 1st

exposure SMR 1.92 (95% CI: 1.08

– 3.17) (15)This document is a draft for reviewpurposes only and does not constituteAgency policy4-103 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

Multiple cause ML

20+ years since 1st

exposure SMR 2.02 (95% CI: 1.13

– 3.34) (15)

10+ years of

ex posure and 20+

years since 1st

exposure SMR 2.55 (95% CI: 1.10

– 5.03) (8)

Coggon et al. (2003) Cohort mortality study of 14,014

men employed in 6 factories of

the chemical industry in Great

Britain from periods during

which formaldehyd e was produced. Cohort mortality

followed from 1941 through

2000. SMRs based on English

and Welsh age

- and calendar

year

- specific mortality rates. Exposure assessment

based on data abstracted

from company records.

Jobs categorized as

background, low,

mo derate, high, or

unknown levels. Non

- Hodgkin's lymphoma

Overall SMR 0.98 (95% CI: 0.67

– 1.39) (31)

High exposure SMR 0.89 (95% CI: 0.41

– 1.70) (9)

Leukemia

Overall SMR 0.91 (95% CI: 0.62

– 1.29) (31)

High exposure SMR 0. 71 (95% CI: 0.31

– 1.39) (8)

Multiple myeloma

Overall SMR 0.86 (95% CI: 0.48

– 1.41) (15)

High exposure SMR 1.18 (95% CI: 0.48

– 2.44) (7)

Andjelkovich et al.

(1995) Cohort mortality study of 3,929

automotive industry iron foundry

workers exposed from 1960

– 1987 and followed through 1989.

SMRs calculated using sex

- , age

-

, race

- , and calendar

- year

specific U.S. mortality rates. Exposure assessment

based on review of

work histories by an

industrial hygienist. All LHP cancers SMR 0.59 (95% CI: 0.23

– 1.21) (7)

Leukemia SMR 0.43 (95% CI: 0.05

– 1.57) (2)

Bertazzi et al. (1986) Cohort mortality study of 1,330

male workers in an Italian resin

plant. Subjects were employed

any time between 1959 and 1980

for at least 30 days. Vital s tatus
followed through 1986. SMRs

calculated using sex

- , age

- , race

- , and calendar

- year

- specific

national and local mortality rates. Exposure assessment

based on reconstruction

of work history. All LHP cancers SMR 2.01 (5)This document is a draft for reviewpurposes only and does not constituteAgency policy4-104 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

Edling et al. (1987) Cohort mortality and incidence

study of 521 Swedish workers in

an abrasive production plant with

at least 5 years of employment

between 1955 and 1983,

followed through 1983. Exposure level of

1

– 5 μg/m 3

. Lymphoma SMR 2.0 (95% CI: 0.2

– 7.2) (2)

Multiple myelo ma SMR 4.0 (95% CI: 0.5

– 14) (2)

Dell and Teta (1995) Cohort mortality study of 5,932

male employees of a New Jersey

plastics manufacturing, research

and development facility. Examination of work

histories to identify jobs

where formaldehyde

was involved. All LHP cancers

Hourly workers SMR 0.93 (28)

Salaried workers SMR 1.69 (23)

Leukemia

Hourly workers SMR 0.98 (12)

Salaried workers SMR 1.98 (11)

Walrath and

Fraumeni (1983) Proportionate mortality cohort

study of 1,132 w hite male

embalmers licensed to practice

between 1902 and 1980 in New

York who died between 1925

and 1980 identified from

registration files. Deaths were

compared with age

- , race

- , and

calendar

- year

- expected numbers

of deaths from the U.S.

population. No direct

measurements.
Presumed exposure to

formaldehyde tissue

fixative. All LHP cancers

PMR 1.15 (21)

Lymphosarcoma and reticulosarcoma

PMR 1.08 (4)

Hodgkin’s disease

PMR 1.0 (2)

Other lymphatic lymphoma

PMR 1.18 (5)

Leukemia

PMR 1.32 (10)

Walrath and

Fraumeni (1984) Proportionate mortality cohort

study of 1,007 white male

embalmers from the California

Bureau of Funeral Directing and

Embalming who died between

1925 and 1980. Deaths were

compared with age

- and

calendar

- year

- expected numbers No direct

measurements.
Presumed exposure to

formaldehyde tissue

fixative. All LHP cancers

PMR 1.22 (19)

Lymphosarcoma and reticulosarcoma

PMR 0.97 (3)

Hodgkin’s disease

PMR

– (0)

Other lymphatic lymphoma

PMR 1.33 (4)This document is a draft for reviewpurposes only and does not constituteAgency policy4-105 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

of deaths from the U.S.

population. Leukemia

PMR 1.75 p < 0.05 (12)

Licensed <20 years PMR 1.24 (4)

Licensed **≥** 20 years PMR 2.21 p < 0.05 (8)

Hayes et al. (1990) Proportionate mortality cohort

study of 3,649 deceased white

and 397 deceased nonwhite U.S.

male embalmers and funeral

directors, derived from licensing

boards and funeral director

associations in the 32 states and

the District of Columbia.

Occupation was confirmed on

death cert ificate. Deaths were

compared with age

- and

calendar

- year

- expected numbers

of deaths from the U.S.

population. No direct

measurements.
Presumed exposure to

formaldehyde tissue

fixative. All LHP cancers

PMR 1.39 (95% CI: 1.15

– 1.63) (115)

Race

White PMR 1.31 (95% CI: 1.06

– 1.59) (100)

Nonwhite PMR 2.41 (95% CI: 1.35

– 3.97) (15)

Occupation on death certificate

Embalmer PMR 1.23 (95% CI: 0.78

– 1.85) (23)

Funeral director PMR 1.56 (95% CI: 1.23

– 1.94) (78)

Other PMR 1.30 (95% CI: 0.67

– 2.28) (12)

Age at death

<60 PMR 1.35 (95% CI: 0.88

– 1.98) (26)

60

– 74 PMR 1.72 (95% CI: 1.33

– 2.19) (66)

**≥** 75 PMR 1.16 (95% CI: 0.74

– 1.74) (23)

Hodgkin’s disease

PMR 0.72 (95% CI: 0.15

– 2.10) (3)

Non

- Hodgkin’s lymphoma

PMR 1.26 (95% CI: 0.87

– 1.76) (34)

Lymphosarcoma and reticulosarcoma

PMR 1.12 (95% CI: 0.58

– 1.96) (12)

Multipl e myeloma

PMR 1.37 (95% CI: 0.84

– 2.12) (20)

Other lymphatic lymphoma

PMR 1.35 (95% CI: 0.85

– 2.01) (22)

Lymphatic leukemia

PMR 0.74 (95% CI: 0.29

– 1.53) (7)

ML

PMR 1.57 (95% CI: 1.01

– 2.34) (24)

Other leukemiaThis document is a draft for reviewpurposes only and does not constituteAgency policy4-106 Table 4

- 4. Epid emiologic studies of formaldehyde and lymphohematopoietic cancers

Reference Study design Exposure assessment Results, statistical significance (number of observed deaths for cohort

study)

PMR 2.28 (95% CI: 1.39

– 3.52) (20)

Blair et al. (1993) Population

- based case

- control

study of 622 white men with

LHP cancers. Cancers selected

from Iowa and Minnesota cancer

surveillance networks diagnosed

between 10/80 and 9/82. 1,245

matched controls for living cases

selected by random digit dialing

if younger than age 65 and from

Medicare records if 65 or older.

Study focused on agricultural

exposures. Personal interviews of

subjects or next of kin

included job histories,

agricultural exposures,

and che mical exposures.

Job titles used to create

job exposure matrix.

Industrial hygienist

estimated probability

and intensity of

exposures to large

numbers of substances. Non

- Hodgkin’s

lymphoma

(formaldehyde

exposure) OR a 1.2 (95% CI: 0.9

– 1.7)

Funeral s ervice worker OR a 2.1 (95% CI: 0.5

– 7.9) (6)

a Adjusted for age, state, smoking, family history of malignant proliferative disease, agricultural exposure to pesticides, use of dye, and direct/surrogate response

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

DRAFT

— DO NOT CITE OR QUOTE 4

- 107 4.1.2.2.2. Brain and CNS cancer

. Several studies of professional groups discussed earlier 1

investigated brain and other CNS cancers among those exposed to formaldehyde on the job. 2

Several of these studies found that exposure increased risk two to three times among exposed 3

profess ionals (Hall et al., 1991; Stroup et al., 1986; Walrath and Fraumeni, 1984), while others 4

found modest or no increase in risk (Hayes et al., 1990; Levine et al., 1984; Walrath and 5

Fraumeni, 1983). 6

None of the industrial cohort worker mortality studies of exposure to formaldehyde found 7

a clear relationship between formaldehyde exposure and risk of brain or CNS cancer (Pinkerton 8

et al., 2004; Coggon et al., 2003; Andjelkovich et al., 1995; Gardner et al., 1993; Stayner et al., 9

1988; Blair et al., 1987, 1986) . To date, no case

- control studies of brain and CNS cancer have 10

been completed. In the Hauptmann et al. (2004) study, the authors reported that no clear 11

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

13

4.1.2.2.3. Pancr eatic and other cancers. Two studies (Kernan et al., 1999; Dell and Teta, 1995) 14

have found increases in the risk of pancreatic cancer in association with possible exposure to 15

formaldehyde. Collins et al. (2001a) conducted a meta

- analysis of fourteen stud ies (Kernan et 16

al., 1999; Andjelkovich et al., 1995; Hansen and Olsen, 1995; Gardner et al., 1993; Hall et al., 17

1991; Matanoski, 1991; Hayes et al., 1990; Gerin et al., 1989; Stayner et al., 1988; Blair et al., 18

1986; Stroup et al., 1986; Levine et al., 198 4; Walrath and Fraumeni, 1984, 1983) and found a 19

small increase in risk (RR = 1.1 [95% CI: 1.0

– 1.3]). 20

Other sites that have been examined are stomach cancer (Coggon et al., 2003) (SMR = 21

1.47; p < 0.05), intraoccular melanoma (Holly et al., 1996) (OR = 2.9 [95% CI: 1.2

– 7.0]), and 22

thyroid cancer among women (Wong et al., 2006) (OR = 8.33 [95% CI: 1.16

– 60.0]; 2 cases). 23

However, without further substantiation, it is difficult to infer causation based on these isolated 24

results alone. 25

26

4.1.2.3. Summary: Carcino genic Hazard in Humans 27

The weight of the epidemiologic evidence at this time supports a link between 28

formaldehyde exposure and NPC in humans. This conclusion is based on the longitudinal cohort 29

study of Hauptmann et al. (2004) as well as the case

- control studies of NPC and formaldehyde 30

exposure completed by Hildesheim et al. (2001), Vaughan et al. (2000), and several additional 31

case

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

of the other cohort studies found little or no increased risk of NPC from exposure to 33

formaldehyde. However, Hauptmann et al. (2004) employed different exposure metrics and 34

based their analyses on conservative internal comparisons that limited the potential for the HWE 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 108 to obscure true ef fects. The case

- control studies that provide additional evidence of an 1

association between NPC and formaldehyde have more power and generally rely on better 2

diagnoses of NPC. Better ascertainment of histologic types of tumors can sometimes also be 3

obtain ed if the cases are taken from cancer registries. The NPC risk is also supported by 4

experimental evidence in animals in which formaldehyde induces nasal cancers (Section 4.2.2). 5

Since the physiology of the rat nasal passage is somewhat different from tha t of humans, it is not 6

possible to obtain a direct site

- specific correspondence between the species. However, in both 7

species, the tumors are found within the same area of the URT where maximum exposure can be 8

expected to occur. 9

Several researchers have c hallenged the conclusion of a relationship between 10

formaldehyde and NPC. Those critical of the link argue that, given the wide variability in results 11

across studies and competing explanations, conclusions about any link from the existing studies 12

are prema ture. The difficulty in attaining consensus on whether formaldehyde influences the risk 13

of NPC in humans arises from several limitations inherent in epidemiologic methods and 14

exposure assessment, as well as from the characteristics of the disease. The mo st prominent of 15

these limitations are the rarity of the cancer and imprecise estimates of exposure. Because NPC 16

is a very rare cancer with an incidence of less than 1 per 100,000, it is difficult to obtain precise 17

estimates of risk from cohort studies. A lthough case

- control studies are better suited for 18

studying rare conditions, they are limited in obtaining valid and precise exposure assessments. A 19

further problem with exposure assessment is isolating formaldehyde exposure from other 20

potential chemical or particulate exposures that may influence risk of NPC. Imprecise exposure 21

assessment and the inability to isolate formaldehyde exposure from other exposures are largely 22

the bases on which Marsh and coworkers have challenged the NCI cohort study (Marsh e t al., 23

2007a, b, 2002, 1996; Marsh and Youk, 2005). Marsh and coworkers (Marsh et al., 2007a) show 24

that subjectively assessed exposure to silversmithing is tentatively associated with NPC. Given 25

that there were no prior citations of an association betwee n silversmithing exposures and NPC in 26

the medical literature and given the many post hoc reexaminations of alternative hypotheses to 27

explain the original NCI findings, it is more likely that silversmithing is an artifactual potential 28

confounder. 29

It may be expected that, without new approaches for obtaining more accurate and precise 30

estimates of exposure, further follow

- up of current cohorts and future epidemiologic studies of 31

formaldehyde and NPC will face the same limitations and criticisms found with exis ting studies. 32

These limitations notwithstanding, the epidemiologic studies reviewed here represent what may 33

be currently discernable about a formaldehyde

- NPC link in humans by using rigorous 34

observational methods. As such, concluding any influence of for maldehyde must be made on the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 109 weight of all human and animal evidence in the face of known and expected limitations in study 1

design and exposure assessment. 2

The results of two well

- designed cohort studies found a positive association between 3

formaldehyde

- exposed professionals, such as pathologists, embalmers, and funeral directors, and 4

LHP cancer, particularly ML. The largest cohort study of formaldehyde has the most extensive 5

exposure assessment (Blair et al., 1986; Stewart et al., 1986), and the cohort was followed for a 6

median duration of 35 years (Hauptmann et al., 2003). By using cumulative exposure measures 7

not previously used and by using internal comparison groups, significant increases in the risk of 8

cancer of the LHP system, particularly ML, wer e reported. This study demonstrated that 9

formaldehyde was a risk factor for LHP cancers, independent of other risk factors, such as 10

benzene and smoking. Hauptmann et al. (2003) found statistically significant dose trends for 11

peak exposure and AIE. Pinke rton et al. (2004) also found a significant increase in the risk of 12

ML in garment workers 20 years after their initial exposure and in workers with 10 or more years 13

of exposure. Additionally, several studies of pathologists, embalmers, and other medical 14

w orkers reported greater numbers of observed deaths from leukemia than expected although 15

many studies of these groups suffer from a substantial HWE based on comparisons with external 16

death rates. Two of these studies, Hayes et al. (1990) and Stroup et al. (1986), also report a 17

significantly excess risk of ML in embalmers, funeral directors, and anatomists. 18

There is a range of biological plausibility for an agent whose primary action is at the 19

POE. Acute leukemias (ALL and AML), believed to arise from trans formation of stem cells in 20

the bone marrow, are less plausible. In contrast chronic lymphatic leukemia, lymphomas, 21

multiple myelomas (from plasma B cells), and unspecified cancers may involve an etiology in 22

peripheral tissues to include cells, cell aggreg ates, germinal centers, and lymph nodes. An 23

association of these cancers to an exogenous agent acting at the POE is biologically plausible. 24

It is the conclusion of this assessment that the weight of the epidemiologic evidence at 25

this time supports a link between formaldehyde exposure and carcinogenicity in humans. 26

27

4.2

. ANIMAL STUDIES 28

This section discusses the available laboratory animal data on the toxicity of inhalation, 29

oral, and dermal exposures to formaldehyde. A comprehensive database of laborator y animal 30

studies is available for formaldehyde, including numerous 2

- year bioassays by both the 31

inhalation and oral exposure routes and dermal application studies. Although a large portion of 32

the literature reports studies focused on toxic effects at the site of contact or POE, general 33

systemic effects as well as neurobehavioral effects, reproductive and developmental effects, 34

immunologic changes, and sensitization are well represented in the literature as well. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 110 RB is a reduction in ventilation rate, min ute volume, and other physiological parameters 1

experienced by rodents exposed to an irritant/reactive gas. Although humans and nonhuman 2

primates do not exhibit the same change in respiratory rate, these studies are included in Section 3

4.2.1.1 in order to better understand the effects on RB in interpreting rodent studies presented in 4

the balance of the chapter. Additionally, although binding to the trigeminal nerve and 5

subsequent downstream events do not result in pulmonary changes in humans, the mechanism 6

itself plays a role in understanding other adverse health effects observed in humans. 7

The available data for the three exposure routes confirm direct formaldehyde

- induced 8

toxicity in tissues present at the POE. These observations are consistent with the 9

physicochemical characteristics, reactivity, and metabolic pathways of formaldehyde as 10

discussed in Chapter 3. Indications of cell damage, cell proliferation, and inflammatory 11

responses are similar for each route of exposure, therefore effects at the POE for inhalation and 12

oral exposures are described first (Sections 4.2.1.2 and 4.2.1.3, respectively). Given the well

13

established nature of these health effects and the wealth of literature for inhalation exposures, 14

complete study summaries for respiratory t ract effects are provided. Studies are organized by 15

study duration

— acute, subchronic and chronic

— where some of the chronic bioassays were 16

designed to address carcinogenic potential. Section 4.2.2 pulls together the findings of chronic 17

studies across the routes of exposure to evaluate the carcinogenic potential of formaldehyde 18

exposure. 19

Although a majority of the oral and inhalation studies focus on health effects at the 20

POE

— respiratory tract and GI tract

— the general systemic toxicity of formaldehyde is ad dressed 21

where it was integral to the study. Therefore, body weight and organ weight changes, gross 22

pathology, organ histopathology outside of the POE, blood and urine chemistry, and other 23

biochemical measures may be included in these study summaries. An overview of general 24

systemic findings is provided in Section 4.4 for all routes of exposure. 25

Studies addressing immune function, neurobehavioral effects, sensitization, and 26

reproductive and developmental effects are addressed across routes of exposure. Th e specialized 27

nature of these studies requires discrete treatment, and inclusion of data across routes of 28

exposure allows for a synthesis of the available information. 29

30

4.2.1. Noncancer Health Effects 31

4.2.1.1. Reflex Bradypnea 32

Reflex bradypnea (RB), wh ich is believed to be a protective response, is often observed 33

in rodents exposed to reactive gases. It is primarily characterized by marked decreases in 34

activity, respiratory rate, body temperature, and metabolic rate. RB is not seen in humans and 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 111 nonhu man primates. An understanding of the RB is important to the interpretation of many of 1

the animal bioassays examining formaldehyde

- induced health effects. Of chief concern is that 2

the physiological effects of RB, described below, may interfere with appro priate interpretation of 3

adverse effects noted with formaldehyde exposure. It is important to distinguish between an 4

effect directly related to RB versus formaldehyde exposure. Additionally the effects of RB may 5

mask or alter formaldehyde

- induced health effects. Secondly, differential respiratory effects of 6

RB due to species and strain will result in differential inhaled doses at the same exposure level. 7

This needs to be considered both when comparing the results of animal studies and in 8

extrapolation t o humans. Finally, although humans do not experience RB, the mechanism of RB 9

as a reflex response to trigeminal nerve stimulation assists in understanding human health related 10

to localized and reflex responses due to trigeminal nerve stimulation. 11

Irritant gases have been shown to decrease body temperature, heart rate, and blood 12

pressure as well as alter blood chemistry in rodents (Pauluhn, 2003, 1996; Jaeger and Gearhart, 13

1982). Because of their small size, mice can rapidly lower their body temperatures a nd thus their 14

metabolic rate and ventilation rate. The hypothermia that results from RB can directly affect 15

nearly all biological processes (Gordon et al., 2008). Formaldehyde exposure can dramatically 16

lower ventilation rate and reduce body temperature i n mice by as much as 4°C, and it has been 17

posited that decreased oxygen supply is likely to have profound effects on organisms with 18

substantial oxygen demands (Jaeger and Gearhart, 1982). The effects of RB are reversible, 19

though it may take several minute s to several hours to return to pre

- exposure conditions 20

(Pauluhn, 1996; Jaeger and Gearhart, 1982). 21

The literature on sensory irritation is broad; many studies have investigated species 22

differences, dose response relationships, tolerance, and cross

- toler ance to other sensory irritants 23

(see Tables 4

- 5 and 4

- 6). This discussion focuses on the changes in respiratory rate and minute 24

volume during formaldehyde exposure. Sensory irritation is often quantified as the statistically 25

derived exposure concentratio n that results in a 50% reduction in respiratory rate (RD 50 ) in 26

rodents (ASTM, 2000; Kane et al., 1979). Kane and Alarie (1977) evaluated various aspects of 27

sensory irritation, including establishing the RD 50 , exploring the reproducibility of response, 28

in vestigating the effect of tracheal cannulation, and determining the potential for tolerance with 29

repeated exposure or pre

- exposure in male Swiss

- Webster mice, caused by formaldehyde and 30

acrolein. The RD 50 was established by exposing four mice for 10 minut es at each concentration 31

across a range representing approximately 10 to 80% reduction in respiration and calculated by 32

using least squares regression. The RD 50 and its 95% CI for formaldehyde were calculated to be 33

3.1 (2.1

– 4.7) ppm (3.8 [2.58

– 5.77] mg/m 3 ). The tracheal cannulation experiments demonstrated 34

that the effect on respiratory rate was caused by URT sensory irritation. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 112 Table 4

- 5 . Respiratory effects of formaldehyde

- induced reflex bradypnea in 1

various strains of mice 2

3

Species/strain No./

group T reatment a Respiratory effects Reference

Male Swiss

Webster mice 4 Duration: 10 minutes.

Exposure: up to 100 ppm. RD 50 a = 3.1 ppm

(95% CI: 2.1

– 4.7). Kane and Alarie

(1977)

Male Swiss

Webster mice 8 Duration: 3 hours/day for

3 days. Exposure: 0.52, 0 .44,

1.16, 1.83, 3.10, 5.35, 5.60, and

11.2 ppm. RD 50 = 3.4 ppm

(95% CI: 2.4

– 4.7). Kane and Alarie

(1977)

Male Swiss

Webster mice 4 Duration: 10 minutes (head

only). Exposure: up to 10 ppm. RD 50 = 3.2 ppm

(95% CI: 2.1

– 4.7). Steinhagen and

Barr ow (1984)

Male Swiss OF 1 mice 6 Single 5

- minute exposure to

four unspecified

concentrations. RD 50 = 5.3 ppm. De Ceaurriz et al.

(1981)

Male B6C3F1 mice 4 Duration: 10 minutes (head

only).

Exposure: Range up to 10 ppm. RD 50 = 4.9 ppm

(95% CI: 3.9

– 6. 4). Steinhagen and

Barrow (1984)

Male B6C3F1 mice 4 Duration: 10 minutes (head

only). Exposure: up to 15 ppm
Pretreatment: 2, 6, or 15 ppm

6 hours/day for 4 days. Naïve mice: RD 50 = 4.4 ppm

(95% CI: 0.9

– 5.0)

Pretreated mice: RD 50 =

4.3 ppm (95% CI: 3.4

– 5.5). Chang et al.

(1981); Barrow et

al. (1983)

Male C57BL6/F1

mice 3 Whole

- body exposure for up to

2 hours. After 1.25 hours:

Tidal volume reduced by

33%; 68% reduction in

respiratory frequency; CO 2 production reduced by

50%; %; body temperatu re dropped from 37.8 to

34.7°C. Jaeger and

Gearhart (1982)

4

a Exposure concentration that results in a 50% reduction in respiratory rate. 5This document is a draft for review purposes only and does not constitute Agency policy.

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- 113 Table 4

- 6. Respiratory effects of formaldehyde

- induced reflex bradypnea in various 1

strains of rats 2

3

Species/strain No ./

group Treatment Respiratory effects Reference

Male Crl

- CD rats 4 RD 50 = 13.8 ppm.

Male Wistar rats 4 30

- minute nose

- only exposure

to a range of formaldehyde

concentrations. RD 50 = 10.0 ppm. Cassee et al.

(1996)

Male F344 rats 4 Duration: 10 minut es (head

only). Exposure: up to

56 ppm.
Pretreatment:2, 6, or 15 ppm

6 hours/day for 4 days Naïve rats: RD 50 = 13.1 ppm

(95% CI: 10.6

– 17.5)

Pretreated rats: RD 50 = 10.8 ppm

(95% CI: 7.6

– 16.9) Chang et al.

(1981); Barrow

et al. (1983)

Male F344 rats 4 Single 10

- minute head

- only

exposure to a range of

concentrations.

Pretreatment: 15 or 28 ppm

formaldehyde or 10 ppm

chlorine. Baseline RD 50 = 31.7 ppm.

Pre

- exposure to formaldehyde

induced tolerance at 28 ppm

(RD 50 = 20.2 ppm) but not

15 ppm.

Pre

- exposure to chlorine

- induced

tolerance to formaldehyde

(RD 50 ranged from 64.5 to

115 ppm, depending on

exposure duration). Chang and

Barrow (1984)

Male F344 rats ND 10 minute exposure to acrolein

or acetaldehyde (head only).

Pre

- exposed to f ormaldehyde

at 15 ppm for 6 hours/day for

9 days. Pre

- exposure to formaldehyde

induced tolerance:

Acetaldehyde (RD 50 = 2,991 ppm

in naive versus 10,601 ppm in

preconditioned animals)

Acrolein (RD 50 = 6 ppm in naïve

versus 29.6 ppm in preconditioned

anim als). Babiuk et al.

(1985)

Male Charles

Rivers CD rats 3 Whole

- body exposure for up

to 2 hours. After 0.7 hours:

Tidal volume reduced by

22%; 20% reduction in

respiratory frequency; CO 2 production unaffected. Jaeger and

Gearhart (1982)

4

5

Across the liter ature there is fairly good agreement on RD 50 values for various strains of 6

mice (Table 4

- 5), ranging from 3.1 ppm in male Swiss

- Webster mice to 4.9 ppm in male 7

B6C3F1 mice. Rats are less sensitive, with RD 50 values ranging from 10 ppm in male Wistar 8

rats to 31.7 ppm in male F344 rats. No reported RD 50 for female rodents exposed to 9

formaldehyde exists. 10

Jaeger and Gearhart (1982) evaluated the effect of formaldehyde on respiratory rate, tidal 11

volume, minute volume, carbon dioxide (CO 2 ) production (exhaled t o air) as a reflection of total 12

metabolism, and core body temperature in male Charles River CD rats and male C57BL6/F1 13This document is a draft for review purposes only and does not constitute Agency policy.

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- 114 mice. Animals (three/concentration) were exposed to 15 ppm (18.4 mg/m 3 ) formaldehyde for up 1

to 2 hours. Mice exhibited a greater decrea se in respiratory frequency and minute volume 2

compared with the rats. CO 2 production and body temperature were also affected to a greater 3

extent in the mice (Table 4

- 5). The authors postulated that the decreased body temperature in 4

mice would likely lead to decreased biologic action of formaldehyde in the tissue. 5

6

4.2.1.1.1. Tolerance. Tolerance is defined as an increase in the concentration required to elicit 7

the same degree of RB response and was evaluated by Kane and Alarie (1977). In the first set of 8

experiments, mice (four/concentration) were exposed 3 hours/day for 4 days at the concentration 9

associated with either a 30 or 50% decrease in respiratory frequency (specific concentrations not 10

given) (Kane and Alarie, 1977). Naïve animals served as co ntrols for each day. The maximum 11

response increased with each additional day of exposure, and the diminution of response that was 12

typically exhibited after 60 minutes of exposure in naïve animals was markedly delayed. In the 13

second set of experiments, mi ce were exposed to a formaldehyde concentration at one

- tenth the 14

RD 50 (i.e., 0.3 ppm) 3 hours/day for 3 days. On the fourth day the animals underwent a similar 15

exposure protocol to identify the concentration that resulted in an RD 50 , following the above 16

p rotocol. No change in the RD 50 was demonstrated. Both of these experiments indicate no 17

change in tolerance with either type of pretreatment in Swiss

- Webster mice. 18

Chang and Barrow (1984) tested whether tolerance would develop in male F344 19

(CDF[F344]Crl/B r) rats exposed to formaldehyde. Exposure to formaldehyde at 15 ppm 20

(18.4 mg/m 3 ) for 6 hours/day, 5 days/week failed to induce tolerance. However, tolerance was 21

observed following exposure to 28 ppm (34.4 mg/m 3 ) formaldehyde for 4 days. The 22

concentratio n

- response curve in these animals was significantly different than that of naïve 23

animals, with an increase in the RD 50 estimate for this exposure duration from 31.7 to 70.2 ppm. 24

25

4.2.1.1.2. Cross

- species differences in inhaled dose. Formaldehyde

- induced RB lowers both 26

respiratory rate and tidal volume and thus reduces the inhaled dose of formaldehyde at a given 27

exposure concentration. Chang et al. (1983) and Barrow et al. (1983) evaluated the species 28

differences and the effective inhaled dose between rat s and mice, since mice seem to be more 29

sensitive to formaldehyde

- induced RB and do not exhibit tolerance as shown in F344 rats. 30

Groups (four/concentration) of male F344 rats and male B6C3F1 mice were exposed to 31

formaldehyde concentration ranges of 6.2

– 48 ppm (7.6

– 59 mg/m 3 ) or 0.78

– 14.0 ppm (0.96

– 32

17.2 mg/m 3 ), respectively, for 10 minutes. Pretreated animals used in the tolerance experiments 33

were exposed to formaldehyde at 2, 6, or 15 ppm (2.45, 7.36, or 18.4 mg/m 3 ) 6 hours/day for 34

4 days prior to determina tion of the RD 50 and concentration response across the same ranges. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 115 A concentration

- dependent decrease in respiratory rate was seen in both naïve and 1

pretreated rats during formaldehyde exposure. Tolerance (defined as a decrease in respiratory 2

rate follow ed by a subsequent return to control values) occurred after 4 minutes of exposure and 3

was more pronounced at concentrations above 4 ppm. Concentration

- response relationships 4

were very similar for naïve and pretreated rats, and the RD 50 s were similar for b oth groups 5

(naïve = 13.1 ppm [95% CI: 10.6

– 17.5]; pretreated = 10.8 ppm [95% CI: 7.6

– 16.9 ]). In 6

contrast, naïve or pretreated mice did not develop tolerance during exposures. An examination 7

of concentration

- response relationships for mice showed similar RD 50 values (naïve = 4.4 ppm 8

[95% CI: 0.9

– 5.0 ] and pretreated = 4.3 ppm [95% CI: 3.4

– 5.5]) compared with rats, although 9

the slopes of the concentration

- response regressions were statistically different (Figure 4

- 2). 10

11

12

13

Figur e 4

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

14

pretreated male B6C3F1 mice and F344 rats. 15

16

Source: Redrawn from Chang et al. (1983). 17

18

19

Exposure of naïve or pretreated rats resulted in an increased (compensatory) tidal 20

volume. Howe ver, the increase in tidal volume did not compensate entirely for the decrease in 21

ventilation rate and was only concentration dependent in pretreated rats. Comparison of tidal 22

volume from naïve and pretreated mice exposed to formaldehyde showed a slight i ncrease in 23

naïve animals but a decrease in pretreated ones. The effect of formaldehyde exposure on tidal 24 1 1 0

1 0 0

9 0

8 0

7 0

6 0

5 0

4 0

3 0

2 0

1 0

0 2 4 6 8 1 0 1 2 0 2 4 0 3 6 0 0 2 4 6 8 1 0 1 2 0 2 4 0 3 6 0 B

6 C

3 F

1 M i c e F 3 4 4 R a t s

N a ï v e ( 6 p p m )

P r e t r e a t e d ( 6 p p m )

N a ï v e ( 1 5 p p m )

P r e t r e a t e d ( 1 5 p p m )

T i m e ( m i n

. ) d u r i n g e x p o s u r eThis document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 116 volume was concentration dependent in both groups of mice. These results indicate that tidal 1

volume does not compensate entirely for the decrease in respiratory rate and that the 2

compensation is slightly greater in rats than in mice. 3

These studies (Barrow et al., 1983; Chang et al., 1983) showed that B6C3F1 mice sustain 4

RB, whereas F344 rats develop tolerance more readily both during exposure and with 5

pretreatment. Thus, these results suggest that the rat may be the more sensitive species for the 6

effects of inhaled formaldehyde due in part to the difference in sensitivity between mice and rats 7

as evidenced by an RD 50 of 4.9 versus 31.7 ppm and the abil ity of rats to develop tolerance while 8

mice appear to sustain RB. Barrow et al. (1983) used the results of these experiments to estimate 9

an inhaled dose equivalent to the exposure concentration of 15 ppm for the strains of mice and 10

rats used in the chroni c formaldehyde bioassays by Kerns et al. (1983) and Monticello and 11

Morgan (1994) described in Section 4.1.2 as follows: 12

13

Inhaled dose (μg/min

- cm 2 ) = 14

Nasal cavity surface area (cm 2 ) (5

- 1) 16 HCHO concentration (μg/L) × minute volume (L/min) 15

17

As shown i n Table 4

- 7, because mice were observed to be able to decrease their minute 18

volume by approximately 75% as compared with 45% in rats, a twofold higher inhaled dose 19

would be expected in rats versus mice. This difference may be relevant to the increased 20

inc idence of SCC in the nasal cavity seen in F344 rats when compared with B6C3F1 mice. 21

22

Table 4

- 7. Inhaled dose of formaldehyde to nasal mucosa of F344 rats and 23

B6C3F1 mice exposed to 15 ppm 24

25

Parameter F344 rats B6C3F1 mice

HCHO concentration (μg/L) 18.4 18 .4

Minute volume (L/min) 0.114 0.012

URT surface area (cm 2 ) 13.44 2.89

Inhaled dose (μg/min/cm 2 ) 0.156 0.076

26

Source: Barrow et al. (1983). 27

28

29

4.2.1.1.3. Cross

- tolerance. Cross

- tolerance of chemically

- induced reflex responses has been 30

examined in seve ral systems in order to better understand the specificity and nature of the 31

interaction of reactive chemicals (such as formaldehyde with chlorine) with the trigeminal nerve 32

involved in the RB. Development of cross

- tolerance to formaldehyde following preex posure to 33This document is a draft for review purposes only and does not constitute Agency policy.

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- 117 chlorine or to chlorine following preexposure to formaldehyde was shown to be a function of the 1

duration of the pretreatment in male F344 rats (Chang and Barrow, 1984) (Table 4

- 8). A 7

- day 2

recovery period resulted in only a slight loss of cross

- tolerance from a 4

- day pre

- exposure to 3

either chlorine or formaldehyde (data not shown). The cross

- tolerance between formaldehyde 4

and chlorine demonstrated in the Chang and Barrow (1984) study suggests that these chemicals 5

may act via a common mechanism a nd may involve the trigeminal nerve. In rats, cross

- tolerance 6

was induced after chlorine exposure but not after formaldehyde exposure, which suggests that 7

the trigeminal nerve may have different reactive sites that are differentially activated, depending 8

on the stimulus. 9

10

Table 4

- 8. Exposure regimen for cross

- tolerance study 11

12

Pre

- exposure Chlorine RD 50
FA

- pretreated Naïve

Formaldehyde 15 ppm

6 hours/day 1 day 22.6 ppm

10.9 ppm 4 days 16.8 ppm

10 days 64.5 ppm

Pre

- exposure Formaldehyde RD 50 Cl

- pretreated Naive

Chlorine 10 ppm

6 hours/day 1 day 64.5 ppm

31.7 4 days 66 ppm

10 days 115 ppm

13

Source: Chang and Barrow (1984). 14

15

16

Babiuk et al. (1985) evaluated the potential for formaldehyde pretreatment to cause cross

17

tolerance with va rious other inhaled aldehydes, including acetaldehyde and acrolein. Male F344 18

rats were pretreated with 15 ppm (18.4 mg/m 3 ) formaldehyde 6 hours/day for 9 days and 19

challenged on the 10 th day with the second aldehyde for 10 minutes at various concentration s 20

(four rats/concentration) to establish an RD 50 . Exposure to acetaldehyde and acrolein, the two 21

smallest molecules in the series of aldehydes tested, resulted in cross

- tolerance. The RD 50 and 22

its 95% CI for acetaldehyde were estimated at 2,991 (95% CI: 2,411

– 3,825) ppm in the naïve 23

rats, and this was increased by approximately 3.5

- fold to 10,601 (95% CI: 7,902

– 15,442) ppm in 24

the rats pretreated with formaldehyde. With acrolein, the RD 50 increased approximately fivefold, 25

from 6.0 (95% CI: 3.5

– 18.1) ppm t o 29.6 (95% CI: 15.6

– 93.0) ppm. Cross

- tolerance with 26

formaldehyde has only been demonstrated with acetaldehyde, acrolein, and chlorine (Babiuk et 27

al., 1985; Chang and Barrow, 1984), suggesting that it is not a generalized phenomenon. 28

Whether the phenome non of tolerance involves modulation of specific trigeminal nerve 29

receptors or whether it results from less specific chemical injury of the nasal mucosa has not 30This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 118 been determined. For example, different mechanisms lead to stimulation of the trigeminal nerve 1

and are likely to control the decrease in respiratory rate. In particular, acetaldehyde might 2

interact with sensory nerves via an amino group (Steinhagen and Barrow, 1984; Schauenstein et 3

al., 1977), whereas the receptor

- binding site for formaldehyde and acrolein is believed to be a 4

thiol group. Furthermore, different binding sites exist on the trigeminal nerve for different 5

irritants (Nielsen, 1991). Thus, Bos et al. (1992) concluded that the data on tolerance or 6

“desensitization” versus “sensitization ” (as defined strictly on the basis of the respiratory apneic 7

response) may be the result of adaptation or reversible/irreversible adverse changes. The 8

mechanisms underlying sensitization or desensitization are not well characterized. 9

10

4.2.1.1.4. Formald ehyde binding and activation of trigeminal nerve afferent activity. Kane 11

and Alarie (1978) evaluated the effect of 11 combinations of acrolein and formaldehyde on 12

respiratory rate in outbred specific

- pathogen

- free male Swiss

- Webster mice. Exposure 13

concen trations ranged from 0.12

– 8.97 ppm (0.28

– 21 mg/m 3 ) for acrolein and 0.37

– 9.73 ppm 14

(0.45

– 11.9 mg/m 3 ) for formaldehyde. The data were evaluated using a simple model of 15

competitive antagonism. Comparing the observed and predicted responses indicated no appa rent 16

differences, and paired t

- tests showed no statistical significance. The authors concluded that 17

acrolein and formaldehyde acted at the same receptor site and acted as competitive antagonists 18

when exposure occurred simultaneously. 19

Kulle and Cooper (197 5) investigated the effects of formaldehyde on trigeminal nerve 20

afferent activity in adult male Sprague

- Dawley rats. The authors isolated both the ethmoid and 21

nasopalatine branches of the trigeminal nerve and recorded afferent signaling as electrical 22

acti vity while reactive gases (formaldehyde, ozone, and amyl alcohol) were passed through the 23

nasal passages of the anesthetized animals. The authors reported that both branches of the 24

trigeminal nerve responded similarly to all three chemicals, and they ther efore conducted the 25

balance of their experiments on the nasopalatine branch of the nerve. Nerve response was 26

calculated as the difference between exposed and control activity, and the threshold for a positive 27

response was arbitrarily defined as an increas e of 0.1 spikes per second. The sensory threshold 28

was determined by extrapolation from the measured nerve response to a range of formaldehyde 29

concentrations (0.5

– 2.5 ppm) or ozone (5.0

– 29 ppm) for an exposure duration of 2 minutes. 30

Amyl alcohol exposure (0.3

– 10.0 ppm) lasted for 25 seconds. Threshold was arbitrarily defined 31

as an increase of 0.1 spikes per second. The mean thresholds were 0.25 ppm for formaldehyde, 32

5.0 ppm for ozone, and 0.30 ppm for amyl alcohol, suggesting that the trigeminal nerve is highly 33

sensitive to formaldehyde and amyl alcohol compared with ozone exposure. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 119 In a second set of experiments, Kulle and Cooper (1975) investigated the effects of 1

prolonged formaldehyde

- exposure on the odor response to amyl alcohol. Rats were pre

- expose d 2

to a series of amyl alcohol concentrations (0.3, 0.7, 1.0, 3.3, 6.7, or 10.0 ppm [1.08, 2.52, 3.6, 3

11.9, 24, or 36 mg/m 3 ]) then a 1

- hour continuous formaldehyde exposure (0, 0.5, 1.0, 1.5, or 4

2.0 ppm [0, 0.61, 1.23, 1.84, or 2.45 mg/m 3 ]). There was a pr ogressive decrease in odor 5

response to amyl alcohol with increasing stimulus of formaldehyde concentration ( p < 0.01, 6

analysis of variance [ANOVA]). The response to formaldehyde concentration was described by 7

a power function Y = 0.741 × X 1.47 , where X is the formaldehyde concentration. The effects of 8

exposure to 2.0 ppm were similar, regardless of whether it was presented immediately as a 9

separate exposure or as the final concentration of a progressively increasing series. The response 10

to amyl alcohol d id not fully recover within the 1

- hour extended recovery period. Thus, it 11

appeared that the afferent function depression was not due to receptor adaptation or insufficient 12

time for formaldehyde diffusion away from receptor sites. 13

In an attempt to elucidat e the basis of the differential effects of various types of 14

aldehydes on sensory irritation, Tsubone and Kawata (1991) recorded the afferent activity of the 15

surgically isolated ethmoidal nerve (a branch of the trigeminal nerve) during delivery of 0.32

– 16

4.7 ppm (0.39

– 5.77 mg/m 3 ) formaldehyde, 0.18

– 7.2 ppm (0.41

– 16.5 mg/m 3 ) acrolein, and 134

– 17

2,232 ppm (241

– 4,021 mg/m 3 ) acetaldehyde into the cannulated URT of male Wistar rats 18

(six/aldehyde) at a flow rate of 200 mL/minutes for 22 seconds. Only one aldehyde was used in 19

each animal and each exposure was repeated two to four times at different concentrations. The 20

activity of the nerve was recorded as the number of electrical discharges for a total period of 21

100 seconds, including pre

- inhalation (30 second), inhal ation (22 second), and post

- inhalation 22

(48 second) periods. Nitrogen was used as the control gas and as the vehicle to dilute the 23

aldehyde gases in order to not interfere with the gas chromatography used to analyze the 24

exposures. The vapor concentrations associated with a 50% increase in nerve activity over the 25

level of control gas were calculated as approximately 1.8, 1.2, and 908 ppm for formaldehyde, 26

acrolein, and acetaldehyde, respectively. These results are consistent with the findings of 27

Steinhagen and Barrow (1984) and the hypothesis that the differences in RD 50 are due to 28

differences in chemical reactivity in the tissue. 29

In summary, RB is a phenomenon observed in rodents exposed to reactive gases, 30

believed to be a protective response to the irrita nt properties of the gas. In comparative studies, 31

rats appear more sensitive to irritant gases since they have a more pronounced RB response 32

compared with mice at a given concentration of formaldehyde and because the dose required to 33

elicit a bradypneic r esponse is higher in rats than in mice. Interestingly, only rats appear to 34

develop tolerance to irritant gases, while mice sustain an RB response. When formaldehyde 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 120 exposure is studied in concert with other reactive gases like chlorine and other aldehyde s like 1

acetaldehyde and acrolein, cross

- tolerance developed. However, the mechanism underlying this 2

response is unknown. It is thought that RB may occur as a result of stimulation of the trigeminal 3

nerve. Thus, although RB appears to be a phenomenon spe cific to rodents, the mechanism by 4

which it occurs, trigeminal nerve stimulation, may be applicable to understanding MOAs in other 5

species, such as primates and humans, particularly in regard to sensitization. 6

7

4.2.1.2. Respiratory Tract Pathology 8

The da tabase for evaluating the POE toxicity in the respiratory tract of inhaled 9

formaldehyde is robust, with well

- designed studies that span a duration range of a few hours to 10

chronic 2

- year bioassays. Toxicity testing has been performed in various species, in cluding 11

mice, rats, hamsters, guinea pigs, dogs, and nonhuman primates. Although a few studies include 12

examination of tissues outside of the URT, the majority of studies focus on changes in cell 13

proliferation and cell pathology in the nasal mucosa. Both mice and rats are well

- defined animal 14

models with standard histologic sections established to evaluate various regions of the nasal 15

passages, divided into Levels 1 to 5 and illustrated in Figure 4

- 3. Pathology of the nasal mucosa 16

will be discussed with re ference to these sections, and the region examined will be stipulated 17

(e.g., nasoturbinates, maxilloturbinates, or ethmoid turbinates [ETs]). Additionally, pathology of 18

the respiratory epithelium will be distinguished from effects on the olfactory epithel ium, 19

although the nature of the lesions is similar. 20

21

4.2.1.2.1. Mucociliary clearance

. The mucociliary apparatus of the URT is the first line of 22

defense against airborne toxicants. Comprising a thick mucus layer (epiphase), hydrophase, and 23

ciliated epit helium, the mucociliary apparatus may entrain, neutralize, and remove particulates 24

and airborne chemicals from inspired air (Figure 4

- 4). The mucus serves to entrain or neutralize 25

and remove exogenous agents from the nasal epithelium (e.g., particles, rea ctive chemicals). As 26

reviewed by Kim et al. (2003), the nasal mucus contains proteins, glycoprotein, and lipids but is 27

primarily water (95%) and is propelled along by movement of the underlying cilia. Degradation 28

in the continuity or function of the muco ciliary apparatus, which provides protection to the nasal 29

epithelium, would result in higher levels of gases and particles reaching the nasal epithelium 30

itself and greater penetration of chemicals into the respiratory tract. Therefore, breakdown and 31

disru ption of mucociliary function are adverse effects, since a key bodily defense to exogenous 32

agents (including infectious agents) is damaged. 33

34

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

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- 121 1

Figure 4

- 3. Sagittal view of the rat nose (nares oriented to the left). 2

3

Note: The figure shows the normal di stribution of nasal mucosae and the section 4

levels used in contemporary histopathology (Brenneman et al., 2000; Mery et al., 5

1994). Sections 1, 2, 4, and 5 correspond to Levels I, II, III, and IV as proposed 6

by Young (1981). S = squamous, T/R = transitio nal/respiratory, O = olfactory 7

mucosa. 8

9

Source: Brenneman et al. (2000). 10

11

12This document is a draft for review purposes only and does not constitute Agency policy.

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- 122 1

Figure 4

- 4 . Main components of the nasal respiratory epithelium. 2

3

Note: OM = osmiophilic membrane; EP = epiphase; HY = hypophase; Cl = cilia; 4

MV = microvilli; CJ = cell junct ion; CC = ciliated cell; NCC = non

- ciliated cell; 5

GC = goblet cell; NE = nerve; GL = gland; BV = blood vessel; ECS = 6

extracellular space; BM = basement membrane. 7

8

Source: Morgan et al. (1986d). 9

10

Mucus flow slows upon formaldehyde exposure, despite an incr ease in the ciliary 11

beat of the underlying epithelial cells, which propel the mucus across the nasal epithelium 12

(Morgan et al., 1986a, c, d; 1983). These findings are consistent with other studies since 13

airborne pollutants and reactive gases have been sho wn to decrease mucus flow rates in 14

several animal models (Mannix et al., 1983; Iravani, 1974; Carson et al., 1966; Dalhamn, 15

1956; Cralley, 1942). In addition to slowing flow, the mucus layer has been observed 16

breaking up as it floats on the epiphase, crea ting gaps in the epiphase and revealing the 17

hydrophase below (Morgan et al., 1986c, d). Formaldehyde reacts with glycoproteins in 18

the mucus of the epiphase, creating cross

- links between these large molecules; this is 19

believed to increase the viscosity of the mucus. 20This document is a draft for review purposes only and does not constitute Agency policy.

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- 123 In their first experiments, Morgan et al. (1983) describe progressive mucostasis (slowing 1

of mucous flow) and ciliastasis (disruption of ciliary beat) with increasing days of exposure to 2

formaldehyde in male F344 rats (15 ppm; 6 hours/day for 1, 2, 4, or 9 days). Ciliastasis occurred 3

with greater frequency and across more regions of the nasoturbinate with subsequent days of 4

exposure. After 9 days, mucostasis was recorded in all but two regions evaluated. Although the 5

severity and time course o f these changes varied across regions of the nose, the process followed 6

a similar pattern: decreased flow, increased ciliary action, mucostasis, and ciliastasis. Since the 7

formaldehyde

- induced deficits in mucociliary function increased with days of exposu re, activity 8

did not fully recover between exposures (18 hours) (Morgan et al., 1983). Therefore, the 9

severity and extent of adverse effects are dependent on both the concentration of exposure and 10

duration (in this case, days of repeated exposures). 11

In su bsequent studies, Morgan et al. (1986c) examined the exposure

- response 12

relationship of formaldehyde effects on mucociliary function and functional recovery 18 hours 13

after exposure ceased. Exposure regimens similar to the above experiment included addition al 14

exposure concentrations (0.5, 2, and 6 ppm) and an additional time point of 15 days duration. 15

Exposure at 2 and 6 ppm resulted in the same progression of effects on mucus flow and ciliary 16

beat. Considering both severity and extent of effects a clear e xposure

- response relationship was 17

demonstrated. Additionally, within each exposure group, effects progressed both in severity and 18

extent by duration of exposure to formaldehyde (from 1 to 4, 9, and 15 days of exposure) 19

(Morgan et al., 1986c). 20

Flow and cil iary beat were not reduced, but rather increased, in epithelium from rats 21

exposed to 0.5 ppm formaldehyde. Mucus flow in 2 of 10 areas assessed was clearly increased 22

(275 and 200% of controls) after 4 days of exposure to 0.5 ppm formaldehyde. Two other 23

e pithelial regions showed a similar trend (150% of controls), but this change was not statistically 24

significant. Interestingly, measurements made in corresponding areas after 9 days of exposure 25

did not show an increase, and measurements in one region were reduced to 37% of control. 26

Although it is not known whether the observed increase in mucus flow rate is a subtle indication 27

of an adaptive response to a low level irritant, the increase appears to be transient. It is not 28

known if flow rate would continue to decrease below control levels for repeated exposures at 29

0.5 ppm for longer than 9 days. 30

The regions affected at 15 ppm generally included the lateral aspects of the nasoturbinate 31

and both the dorsal and medial aspects of the maxilloturbinate. In gener al there was an anterior 32

to posterior effect with increasing concentration and time. Additionally, impaired mucociliary 33

function was more extensive with greater concentration and length of exposure. Nasal lesions 34

were seen on the nasal epithelium and cor related with those areas where some inhibition of 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 124 ciliary function was measured. Areas without mucus flow but that still retained ciliary function 1

did not develop epithelial lesions. Morgan et al. (1986c) reported “coagulated mucus,” viewed 2

as a “continu ous membrane” over the epithelium after 6 hours of exposure to 15 ppm 3

formaldehyde. Minor cell damage and infiltrating neutrophils and monocytes were also seen in 4

these areas. The coagulated mucus was not seen in similarly exposed rats that were allowed 5

18 hours of recovery before sacrifice. However, ciliated cells were damaged, and there was a 6

greater presence of neutrophils and macrophages (MPs) after this recovery period. The authors 7

noted that, as the exposure continued, these areas exhibited increa sed signs of inflammation and 8

epithelial damage, eventually resulting in “severe degenerative changes.” 9

Morgan et al. (1986a) refined their study design to implement a nose

- only exposure to 10

formaldehyde in order to better examine the progression of changes in mucociliary function 11

during short

- term exposure, allowing examination of mucus flow immediately following 12

exposure. Three F344 rats/group were exposed to 15 ppm (18.4 mg/m 3 ) formaldehyde for 10, 13

20, 45, or 90 minutes or 6 hours. Two groups of rats we re exposed to 2 ppm to determine a no 14

effect level for 90 minutes or 6 hours. The extent and severity of mucostasis and ciliastasis seen 15

after a 6

- hour 15 ppm (18.4 mg/m 3 ) formaldehyde exposure and a 1

- hour recovery period were 16

similar to the earlier stud y (Morgan et al., 1986a), indicating that similar exposure conditions 17

were reached with this nose

- only apparatus. Ciliastasis and mucostasis were both less severe and 18

less extensive in a time

- dependent manner and at the earlier time points of 10, 20, 45, and 19

90 minutes. Significant recovery was seen in mucociliary function by allowing a 1

- hour 20

recovery between exposure and sacrifice. Regions of both the nasal septum and lateral wall, 21

which exhibited no mucus flow when examined immediately after a 6

- hour exposure, had 22

measurable flow after the 1

- hour recovery period. Similar recovery was seen at all durations of 23

exposure. No decreases in mucociliary function were seen after exposure for either 90 minutes 24

or 6 hours at 2 ppm formaldehyde. However, given evidence of recovery (Morgan et al., 1986a) 25

and the time taken to dissect and view the tissues ex vivo may have obscured more subtle effects. 26

To assess more immediate effects on mucociliary apparatus, Morgan et al. (1984a) have 27

examined formaldehyde effect s on the mucociliary apparatus of isolated frog palates. This 28

system allowed observation of mucociliary function during exposure. Unexposed frog palates 29

were covered by a continuous sheet of mucus of variable thickness, which was observed to flow 30

in stre ams across the palette, exhibiting a wave

- like form in some areas of the epiphase. The 31

authors reported particle movement in a lower, less viscous layer that was consistent with a less 32

viscous underlying hydrophase, similar to that described in rat mucosa . The basal mucus flow 33

rate was 0

– 4 mm/minute, with localized ciliary activity. Short periods of increased mucus flow 34

were associated with seemingly spontaneous increases in ciliary beat. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 125 Formaldehyde exposure resulted in an initial increase in ciliary beat and mucus flow rate 1

in all palates exposed at 1.37, 4.36, and 9.58 ppm formaldehyde (but not 0.23 ppm). With 2

increasing formaldehyde concentration and time of exposure, mucostasis was evident as mucus 3

became stiff and eventually rigid. Ciliary beat continued after mucostasis was reached until 4

palates were exposed to 4.36 and 9.48 ppm formaldehyde, when ciliastasis was reached. The 5

time course to peak mucus flow rate, mucostasis, and ciliastasis was concentration dependent, 6

with mucostasis reached i n less than 3 minutes at 9.48 ppm. In contrast, increased mucus flow 7

peaked at 8 minutes in palettes exposed at 1.52 ppm formaldehyde, which, though declining, 8

remained above basal levels after 25 minutes with no mucostasis or ciliastasis noted at this le vel. 9

Fló

- Neyret et al. (2001) demonstrated reduced mucociliary clearance and decreased 10

frequency of ciliary beats by using a similar isolated frog palette mucociliary apparatus. 11

However the palates were exposed by formaldehyde in the Ringer’s solution i n which the palates 12

were placed (0, 1.25, 2.5, or 5 ppm). Also, mucus was removed from the palettes and did not 13

come into direct contact with the formaldehyde. Despite these differences, formaldehyde caused 14

mucociliary clearance to decrease in a time

- an d concentration

- dependent manner; mucostasis 15

occurred after 60 minutes of exposure to 5 ppm formaldehyde (Figure 4

- 5). Ciliary beat was 16

decreased in a time

- dependent manner at 2.5 and 5 ppm exposure but increased at 1.25 ppm 17

formaldehyde (Figure 4

- 5). Re duced mucociliary clearance at 2.5 and 5 ppm was consistent with 18

the reduced ciliary beat. However, clearance decreased at 1.25 ppm formaldehyde, where there 19

was an apparent increase in ciliary beat. The authors suggest this may be a result of disrupting 20

the harmonic movement of the cilia, impairing effective mucociliary clearance. Based on study 21

results, the authors hypothesize that changes in ciliary beat, including excitation at lower 22

exposures, are likely to be a direct effect of formaldehyde on epit helial cells or other cellular 23

components of the mucosa. 24

In summary, numerous studies have identified impaired mucociliary clearance activity 25

associated with formaldehyde exposure (Table 4

- 9). Although low

- dose and short

- term 26

exposures first increase ci liary beat, impaired mucus flow, slowed ciliary beat, and eventual 27

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

These effects are both concentration and duration dependent and can be seen in as few as 29

15 minutes from exposure. Repeated inhalation exposures in rats indicate the effect does not 30

fully recovery in an 18

- hour period between exposures, contributing to greater impairment over 31

extended periods of exposure. 32

33This document is a draft for review purposes only and does not constitute Agency policy.

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- 126 1

2

3

4

5

6

7

8

9

Figure 4

- 5 . Decreased mucus clearance and ciliary beat in isolated frog palates 10

exposed to formaldehyde after 3 days in culture. 11

12

Source: Fló

- Neyret et al. (2001). 13

14

Morgan et al. (1983) suggested that the initial stimulation of ciliary activity may be a 15

defensive response to the ir ritant gas, possibly indicating some penetration of formaldehyde to 16

the underlying epithelial cells. Later effects of mucostasis may be a result of cross

- linking of 17

mucus glycoproteins by formaldehyde, creating a rigid mucus that is not able to flow even with a 18

rigorous ciliary beat. It is unknown if the eventual cessation of ciliary beat is a result of 19

compound

- related effects on ciliated epithelium as formaldehyde diffuses through the mucus or 20

an indirect effect associated with mucostasis. However, in vitro experiments by Fló

- Neyret et al. 21

(2001) indicate that formaldehyde in solution, supporting isolated frog palates without mucus, 22

resulted in the same sequence of effects, including increased ciliary beat at the lowest exposure. 23

These data suggest a r ole of formaldehyde beyond its ability to form protein cross

- links in 24

mucociliary proteins. 25

26

4.2.1.2.2. Cellular pathology . This section summarizes studies that have investigated cellular 27

pathology in the URT and in the lung. Below, full study descript ions are provided for both 28

short

- term and subchronic duration studies (including, where appropriate, how cell proliferation 29

relates to the observed formaldehyde

- induced pathology). 30

31This document is a draft for reviewpurposes only and does not constituteAgency policy4-127 Table 4

- 9 . Summary of formaldehyde effects on mucociliary function in th e upper respiratory tract

Species N a Treatment Measure of

mucociliary

function Summary of results by location Reference

Male F344

rats 10 15 ppm formaldehyde

6 hours/day for 1, 2, 4,

or 9 days Mucus flow and

ciliary beat Mucostasis in regions 2, 3, 4 , 5, and 8 for all rats after a single

dose.Mucostasis in all but two regions evaluated by day 9. Ciliastasis

followed mucostasis. Morgan et al.

(1983)

Male F344

rats 6 0, 0.5, 2, 6, or 15 ppm

formaldehyde

6 ourshours/day for 1,

4, 9, or 15 days Mucus fl ow and

ciliary beat and

histopathologic

analysis Flow or ciliary beat were increased at 0.5 ppm.

After1After 1 day, slowed or halted mucociliary flow at 15 ppm after 6

hours.

After 9 days, slowed or halted mucociliary flow decreased or completely

stopped i n all nasal regions evaluated.

Regions affected included lateral aspect of the nasoturbinate and dorsal and

medial aspects of maxilloturbinate. Morgan et al.

(1986c)

Male F344

rats 3 per

group 15 ppm formaldehyde

for 10, 20, 45, or

90 minutes or 6 hours M ucus flow and

ciliary beat Ciliastasis and mucostasis increased in a time

- and concentration

- dependent

manner, with maximal response at 6 hours.

Significant recovery was observed when a 1

- hour recovery period occurred

between exposure and sacrifice. Morgan et al.

(1986a)

Isolated frog

palates Not

stated 0.23, 1.37, 4.36, or

9.58 ppm

formaldehyde Mucus flow rates

and histopathology Ciliary beat and mucus flow increased from baseline at 1.37, 4.36, and

9.58 ppm. Over time, mucus became rigid, and ciliastasis occurred Morgan et al.

(1984a)

Isolated frog

palates 4 0, 1.25, 2.5, and 5 ppm

formaldehyde every 15

minutes for 60 minutes Mucociliary

clearance and ciliary

beat Ciliary beat decreased in a time

- dependent manner at 2.5 and 5,0 ppm but

was increased at 1 .25 ppm.

Mucostasis occurred after 60 minutes at 5 ppm. Fló

- Neyret et

al. (2001)

N = number of animals in study.This document is a draft for review purposes only and does not constitute Agency policy.

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- 128 4.2.1.2.2.1. Nasal pathology short

- term studies.

Scanning electron microscope examination of nasoturbinates showed increased mucus, 12

erythrocyte infiltration, swelling of microvillus cells, and some c ell separation in formaldehyde

13

treated rats. Nasoturbinates examined 1 day after exposure showed greater effects, including cell 14

damage, matted cilia, and blebbing of cell membranes. Damage to microvillus cells of the 15

maxilloturbinate included deformed c ilia, cell swelling and rupture, and lack of typical microvilli 16

on the cell margins. As in the nasoturbinates, damage was more marked 24 hours after exposure. 17

The epithelium of the ETs exhibited less cell damage than in the nasal and maxillary regions, 18

w ith the slight lesions noted in the upper (ET1) portion and little to no damage noted on the mid 19

and lower (ET2 and ET3) regions. Examination of transverse tissue sections revealed swollen 20

goblet cells and stretched epithelial cells that formed an epithel ial lining approximately 40% 21

taller than the lining seen in control rats. There was also a patchy loss of ciliated cells in the 22

respiratory epithelium, where columnar cells were present. 23 Inhalation of formaldehyde for a few hours has

1

been shown to result in damage of the nasa l mucosa, depending on the exposure concentration. 2

Bhalla et al. (1991) observed changes in cell morphology in male Sprague

- Dawley rat nasal 3

epithelia after a single 4

- hour exposure to 10 ppm (12.3 mg/m 3 ) formaldehyde. Three exposed 4

rats were sacrificed 1 hour and 24 hours after exposure, with two control rats at each time point. 5

Noses were fixed, decalcified, and sliced along the midsagittal plane through the nasal septum. 6

The exposed turbinates were examined by scanning electron microscopy. Transvers e sections 7

through the hard palate, at the level of the incisive papillae, were prepared for light microscopy 8

from similarly exposed rats (n = 10). The authors provided detailed descriptions of cell epithelial 9

organization in untreated rat turbinates and changes observed in formaldehyde

- treated rats, as set 10

forth below. No statistical analysis was provided. 11

Buckley et al. (1984) investigated the respiratory tract lesions ass ociated with several 24

sensory irritants. As part of this investigation, male Swiss

- Webster mice were exposed to 25

3.13 ppm (3.85 mg/m 3 ) formaldehyde 6 hours/day for 5 days. A total of nine chemicals were 26

tested in parallel. The report indicates there were 24

– 34 mice in each group, although not 27

detailed for each chemical. One

- half of the treatment group and unexposed controls were 28

sacrificed immediately after the last exposure. The remaining exposed mice were sacrificed 29

72 hours later. The head, trachea, and lungs were fixed and heads decalcified. Five sections 30

were taken of each nose at levels equivalent to standard levels 2

– 6 (Figure 4

- 3) and were 31

examined by light microscopy. Details on lung and trachea sections were not given. 32

Formaldehyde induced l esions in the respiratory epithelium of exposed mice, including 33

inflammation, exfoliation, erosion, ulceration, necrosis, and squamous metaplasia. The section 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 129 level for these effects was not given. No effects were reported in the squamous epithelium, 1

olf actory epithelium, trachea, or lungs of formaldehyde

- exposed mice. 2

Monteiro

- Riviere and Popp (1986) evaluated damage to the respiratory epithelium due to 3

acute formaldehyde exposures. Male F344 (CDF [F344]/CrlBr) rats (three to five per group) 4

were expose d at 0.5, 2.0, 6.0, or 15 ppm (0.62, 2.5, 7.4, or 18.5 mg/m 3 ) formaldehyde 5

6 hours/day for either 1, 2, or 4 days. Rats were sacrificed either immediately after exposure or 6

18 hours later (Table 4

- 10). After fixation and decalcification, blocks of tissue were collected 7

from transverse sections of the skull. The first block of tissue, 1 μm thick, was taken just 8

posterior of the incisor teeth. The second block was taken halfway between the first block and 9

the incisive papillae. The dorsal nasal conchae, lateral wall, and ventral nasal conchae were 10

microdissected, postfixed, and viewed by transmission electron microscopy (Monteiro

- Riviere 11

and Popp, 1986). 12

13

Table 4

- 10. Concentration regimens for ultrastructural evaluation of male 14

CDF rat nasoturbinates 15

16

Fo rmaldehyde a,b Duration Time of sacrifice Observations

0.5 ppm (3) 6 hours for 1 day

6 hours for 4 days 18 hours later No lesions.

Altered ciliary configuration.

2.0 ppm (3) 6 hours for 1 day

6 hours for 4 days 18 hours later No lesions.

Altered ciliary c onfiguration.

6 ppm

(5 each group) 6 hours for 1 day

6 hours for 1 day

6 hours for 2 days

6 hours for 4 days Immediately

18 hours later Focal lesions on dorsal and nasal conchae and

lateral wall.

Severity of lesions increased with exposure

duration.

1 5 ppm

(5 each group) 6 hours for 1 day

6 hours for 2 days 18 hours later Focal lesions on dorsal and nasal conchae and

lateral wall.

Severity of lesions increased with exposure

duration.

Severity of lesions increased with concentration.

17

a Number of expose d rats is shown in parentheses. 18

b Five control rats were examined for each experiment. 19

Source: Monteiro

- Riviere and Popp (1986). 20

21

No lesions were observed at either 0.5 or 2.0 ppm formaldehyde for either 1 day or 22

4 days, evaluated 18 hours after exposure. However, an unusual altered ciliary configuration, 23

including blebbing of the cell membrane, was observed in almost all formaldehyde

- treated rats, 24

whereas it was only “occasionally noted” in control rats. Focal lesions in the dorsal and ventral 25

conchae an d lateral wall were seen in rats exposed at 6 and 15 ppm for 1 day and sacrificed 26

immediately after exposure. These lesions included cytoplasmic and autophagic vacuoles, loss 27

of microvilli, and hypertrophy. Lesions increased in severity with both exposur e concentration 28This document is a draft for review purposes only and does not constitute Agency policy.

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- 130 and duration. Neutrophil infiltration and intercellular edema were seen after 1 day at 6 and 1

15 ppm. Nonkeratinized squamous metaplasia was noted after 4 days at 6 and 15 ppm in treated 2

rats. Cell death and sloughing were noted after onl y 2 days of exposure at 6 ppm formaldehyde. 3

As described above, Cassee and Feron (1994) examined the effects of intermittent 4

exposure to formaldehyde (3.5 ppm [4.3 mg/m 3 ]), ozone (0.44 ppm [0.86 mg/m 3 ]), or a 5

combination of the two on changes to the rat na sal epithelium. Exposure occurred through six 6

consecutive 12

- hour cycles in which rats were exposed for 8 hours and then not exposed for a 7

further 4 hours. Rats were weighed before the first and after the last exposure periods and 8

sacrificed immediately after the last exposure. To collect tissue for biochemical analysis, skulls 9

were split sagittally and the respiratory epithelium collected. Tissues from six rats were pooled 10

and homogenized to enable the measurement of glutathione (GSH) and the activitie s of the 11

following enzymes: glutathione S

- transferase (GST), glutathione peroxidase (GPX), glucose

- 6

- 12

phosphate dehydrogenase (G6PDH), glutathione reductase (GR), alcohol dehydrogenase (ADH), 13

and formaldehyde dehydrogenase (FALDH). The remaining heads were fixed, decalcified, and 14

sectioned (standard cross sections [Figure 4

- 3]). 15

All groups, including controls, lost weight during the course of treatment. Rats exposed 16

to formaldehyde, ozone, or both lost more weight than controls ( p < 0.05, p < 0.01, and 17

p < 0.001, respectively). Formaldehyde treatment alone increased GPX from 48.6 to 18

64.0 μmole/minute

- mg protein ( p < 0.05) (Table 4

- 11). Formaldehyde exposure, in conjunction 19

with ozone, decreased GST from 490 to 389 μmole/minute

- mg protein ( p < 0.05). No other 20

enzyme activities or tissue GSH levels were affected by formaldehyde exposure. 21

22

Table 4

- 11. Enzymatic activities in nasal respiratory epithelium of male 23

Wistar rats exposed to formaldehyde, ozone, or both 24

25

Enzyme Controls a Formaldehyde

(3.5 ppm) Oz one (0.4 ppm) Both b

ADH 2.66 (0.99) 3.53 (0.13) 3.40 (0.33) 2.42 (0.61)

GST 490 (32) 494 (24) 514 (4) 389 (28) c

GPX 48.6 (4.3) 64.0 (7.9) c 55.6 (2.0) 54.5 (0.3)

G6PDH 58.9 (7) 60.8 (4.7) 65.8 (1.0) 45.5 (6.8)

GR 275 (16) 288.2 (16) 279 (17) 236 (14)

FALDH 0.77 (0.03) 0.68 (0.04) 0.68 (0.07) 0.80 (0.08)

26

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

μ mole/minute/mg of cytosolic protein. 28

b Rats were exposed intermittently, 12

- hour cycles of 8 hours exposed and 4 hours unexposed, for 3 days. 29

c Different from control, p < 0.05. 30

31

Source: Cassee and Feron (1994). 32

33This document is a draft for review purposes only and does not constitute Agency policy.

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- 131 Formaldehyde

- exposed rats exhibited lesions in the nasal epithelium at levels 2 and 3 of 1

the nose, with effects slightly more severe in level 2. Lesions observed include necrosis, 2

hyperplasia accompanied by squamous metaplasia, and rhinitis. Exposure to formaldehyde in 3

the presence of ozone resulted in more severe squamous metaplasia (statistics not given). These 4

findings are similar to those of Monteiro

- Riviere and Popp (1986), indicating that single or 5

repeated exposures can result in cell damage and death. Cell death and increased cell 6

proliferation were seen here after 3 days of repeated exposures to 3.5 ppm formaldehyde. While 7

no increases were see n in olfactory epithelium, frank necrosis, squamous metaplasia, and 8

hyperplasia of both ciliated and nonciliated epithelium were noted at level 2 and 3. 9

Javdan and Taher (2000) exposed male and female albino Wistar rats (five/group) at 0, 2, 10

or 5 ppm (0, 2 .5, or 6.2 mg/m 3 ) formaldehyde 8 hours/day for either 3 or 30 days. Transverse 11

tissue sections at the base of the incisive teeth and the first palatine folds were examined by light 12

microscopy. Lesions reported after 3 days of exposure to 2 ppm formaldehy de included chorion 13

congestion, cell disarrangement, squamous hyperplasia, atypical mitosis, and epithelial 14

hyperplasia. Similar lesions were seen after 30 days but were more severe. Effects at 5 ppm 15

formaldehyde included goblet cell proliferation, olfac tory epithelial hyperplasia, calcified 16

regions, and an abscess on the chorion. These lesions were more severe after 30 days of 17

exposure. 18

Kamata et al. (1996a, b) conducted several high

- dose studies by inhalation in rats. 19

Specifically they exposed male F3 44 rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m 3 ) 20

formaldehyde for 6 hours ( Kamata et al., 1996a) . In a subsequent study in the same laboratory, 21

male F344 rats were exposed to either 0, 15, or 145 ppm (0, 18.5, or 178 mg/m 3 ) formaldehyde 22

nose only for 6 hours ( Kamata et al., 1996b) . Congestion was noted in the nasal cavities of 23

formaldehyde

- exposed rats and was more severe at 145.6 ppm (Kamata et al., 1996b). Rats 24

exposed to 15 ppm formaldehyde had lesions in the nasal turbinate and trachea (not d etailed) 25

(Kamata et al., 1996b). A slight hypersecretion of mucus was noted in the tracheal epithelium in 26

the absence of histopathologic changes. Rats exposed to 145.6 ppm had more dramatic lesions 27

that penetrated more deeply into the respiratory tract. Hyperkeratosis of the squamous 28

epithelium was found at level 1 of the nasal cavity. Hypersecretion, desquamation, and irregular 29

mucosal epithelium were seen in levels 2, 3, 4, and 5 of the nasal cavity, with more severe 30

changes noted in the nasal septum. Increased secretion and desquamation of mucosal cells 31

occurred in the trachea, and a slight hyperplasia of the alveolar wall was noted in rats exposed to 32

145.6 ppm formaldehyde (Kamata et al., 1996b) 33

Hester et al. (2003) carried out a transcriptional ana lysis of the nasal epithelium of male 34

F344 rats 24 hours after nasal instillation of 40 μL of 400 mM formaldehyde. Immediately after 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 132 sacrifice, cells were harvested from the nasal cavity for RNA extraction. The authors found 1

several phase I and II enzyme s, indicative of oxidative stress, to be elevated. They also reported 2

the greatest increase in inflammatory genes, such as iNOS and neuropeptides. In an effort to 3

phenotypically link any gene changes to pathology, Hester et al. (2003) noted that this exp osure 4

scenario has been demonstrated to induce regenerative hyperplasia with minimal cytotoxicity. In 5

this regard, they observed no significant change in nine genes involved in three apoptotic 6

pathways. 7

In an expansion of their earlier study, Hester et al. (2005) carried out a transcriptional 8

analysis of the nasal epithelium of male F344 rats that had been exposed to formaldehyde by 9

nasal instillation for a single exposure, 5 days of exposures, or 28 days of exposure. In addition, 10

this study also attemp ted to characterize the comparative toxicity of glutaraldehyde with 11

structurally similar formaldehyde (van Birgelen et al., 2000). Thus, four animals per group were 12

instilled with 40 μL of deionized water (control group), 40 μL of 400 mM formaldehyde, or 13

40 μL of 20 mM glutaraldehyde. Phenotypically, both aldehydes induced similar 14

histopathologic changes. 15

Both aldehydes induced similar changes in DNA repair and apoptotic pathways initially, 16

but the patterns of gene changes were different after about 5 day s of exposure. Eight genes were 17

differentially expressed between formaldehyde and glutaraldehyde that indicated different 18

pathways for DNA repair, including recombination, base excision repair, and nucleotide excision 19

repair. Within this group, replicati on protein 70 and DNA excision repair ERCC1 showed a 20

twofold induction by formaldehyde compared with glutaraldehyde. Since both of these genes 21

and their products function by recognizing and removing damaged DNA bases, Hester et al. 22

(2005) hypothesized tha t formaldehyde

- exposed cells may remove damaged bases more 23

efficiently than glutaraldehyde

- exposed cells 24

25

4.2.1.2.2.2. Lung pathology: short

- term studies.

Ionescu et al. (1978) described prog ressive damage in pulmonary tissue of adult male 30

rabbits exposed to an aerosol of 3% formaldehyde solution 3 hours/day for up to 50 days 31

(method of aerosol generation or particle size were not provided). An equivalent air 32

concentration was not reported an d cannot be derived from the information given. Animals were 33

sacrificed at several time points (3, 7, 15, 20, 30, and 50 days), and fragments of the caudal lobes 34

of both lungs were taken to examine bronchi (intrapulmonary and distal) and lung parenchyma. 35 In addition to nasal pathology, several 26

researchers specifically investigated formaldehyde

- induced effects in the trachea, bronchi, and 27

pulmonary tissues of the deep respiratory tract in a variety of species (Lino dos Santos Franco et 28

al., 2006; Kamata et al., 1996a, b; Schreibner et al., 1979; Ionescu et al., 1978). 29This document is a draft for review purposes only and does not constitute Agency policy.

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- 133 Enzymatic activity was characterized in frozen sections for β

- galactosidase, adenosine 1

triphosphatase (ATPase), adenosine monophosphatase (AMPase), lactate dehydrogenase (LDH), 2

malate dehydrogenase, succinate dehydrogenase (SDH), acid phosphatase, Tween

- 60 esterase, 3

naphthol

- AS

- D

- acetate esterase, proline oxidase, hydroxyproline epimerase, leucyl 4

aminopeptidase, and β

- glucuronidase. A portion of the lung was fixed and sectioned and viewed 5

by light microscopy to determine changes in cell populations and tissue pathology. 6

In addition, biochemical analysis revealed th at enzymatic activity of β

- galactosidase, 7

ATPase, AMPase, LDH, malate dehydrogenase, and SDH were all unchanged by formaldehyde 8

exposure across the course of treatment (Ionescu et al., 1978). The activities of several enzymes 9

were increased through the co urse of exposure, including acid phosphatase, Tween

- 60 esterase, 10

naphthol

- AS

- D

- acetate esterase, proline oxidase, and hydroxyproline epimerase. Although no 11

details were reported, the authors described the changes as progressive, with the increase in 12

proli ne oxidase and hydroxyproline epimerase seen only in the second half of the treatment 13

course. The activities of two enzymes, leucyl aminopeptidase and β

- glucuronidase, were 14

observed to decrease rapidly (time frame not provided) (Ionescu et al., 1978) 15

Histologic changes in the lung tissue were noted after only 3 days of exposure and were 16

generally progressive throughout the course of treatment. Early cha nges in the bronchial 17

epithelium included increased mucus secretion, hyperplasia, and hypertrophy of epithelial cells. 18

Lymphocyte infiltration was noted in many areas, and a limited thickening of the alveolar walls 19

was reported after 3 days of exposure. Epithelial cell lesions, thickening of the alveolar, and 20

infiltration of lymphocytes increased as exposure continued. Mucus cells increased as much as 21

40% after 40 days of treatment. After 40 days of treatment, Ionescu et al. (1978) observed 22

“destructive and fibrotic lesions” and provided a detailed description of progressive lesions. 23

Schreiber et al. (1979) also examined histologic changes in lung tissue after high 24

formaldehyde exposures. Syrian golden hamsters (34, sex not stated) were exposed to 250 p pm 25

(308 mg/m 3 ) formaldehyde 1 hour/day for 1, 2, 5, or 15 days. Five hamsters in each treatment 26

group were sacrificed 2 days after exposure was ended. Three hamsters in each group were 27

sacrificed 1, 2, or 6 weeks after exposure ended to determine if form aldehyde

- induced changes 28

regressed over time. Tracheal washing was carried out to collect cytologic samples in each 29

animal prior to sacrifice. Samples were fixed, stained, and examined by light microscopy. 30

Lungs and tracheae were removed en bloc and fix ed, and 20, 1 μm thick cross sections were 31

taken (location not detailed). The remaining respiratory tissue was sectioned at 200 μm 32

intervals. Sections were stained and viewed by light microscopy. 33

Abnormal epithelial cells were found in tracheal washings from formaldehyde

- exposed 34

hamsters. Schreibner et al. (1979) described cells with lobulated nuclei and a coarse chromatin 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 134 pattern, especially in cells showing signs of degeneration (e.g., vacuolization of nuclei and 1

cytoplasm) (Schreiber et al., 1979). C ell number and damage were not quantified, and there was 2

no discussion of the effects of exposure duration on treatment, if any, on these observations. 3

Tracheal washing was normal 2 and 6 weeks after the end of exposure, indicating that the 4

cytological ch anges were reversible (Schreiber et al., 1979). 5

Formaldehyde exposure caused multifocal lesions in the mucociliary epithelium in the 6

trachea and larger bronchi. Dysplastic and poorly differentiated squamous metaplastic foci 7

replaced ciliated epithelium (S chreiber et al., 1979). Abnormal nuclear membranes, tonofibrils 8

around the nuclei, the appearance of nucleoli, and heterochromatin condensation were distinct in 9

the formaldehyde

- treated hamsters. These changes, observed 2 days after formaldehyde 10

exposure , were reversible over time and not seen 2 and 6 weeks later. 11

Because of the similarity of form and physiology of rhesus monkey URTs to the human 12

respiratory tract, the effects of short

- term formaldehyde exposure were evaluated in both nasal 13

and lung tissu e in these monkeys by Monticello et al. (1989). Male rhesus monkeys (nine/group) 14

were exposed to 6 ppm formaldehyde (7.4 mg/m 3 ) 6 hours/day for 5 days/week for either 1 or 15

6 weeks. Control animals were exposed to the same regimen of filtered air for 1 we ek. Monkeys 16

were weighed during the course of exposure and observed for clinical signs of irritation or 17

sickness. Monkeys were intravenously injected with [ 3 H]

- thymidine 18 hours after the last 18

formaldehyde treatment to evaluate induced cell proliferatio n. Sections of the nasal passages, 19

trachea, larynx, lung carina, and duodenum were processed for histoautoradiography. Tissues 20

fixed and sectioned for examination by light microscopy included nose, adrenal, sternum (bone 21

marrow), duodenum, esophagus, eye s, gallbladder, heart, kidney, liver, lymph nodes, pancreas, 22

stomach, spleen, and tongue. The nose was cut into a series of transverse sections, 3 μm thick, 23

and sections from five levels were examined (Figure 4

- 6). Lung lobes were trimmed 24

midsagittally a nd sectioned with care to include airway bifurcations. Sections of the nasal 25

passages, trachea (cross section), larynx (cross section), lung carina (frontal section), and 26

duodenum were also processed for histoautoradiography. 27

There were no significant cha nges in body weight over the course of the experiment. 28

Oronasal breathing was noted in the first 15 minutes of formaldehyde exposure (Monticello et 29

al., 1989). Monkeys did experience eye irritation (mild lacrimation and conjunctival hyperemia) 30

during exp osure. 31

32This document is a draft for review purposes only and does not constitute Agency policy.

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- 135 1

Figure 4

- 6. Diagram of nasal passages, showing section levels chosen for 2

morphometry and autoradiography in male rhesus monkeys exposed to 3

formaldehyde. 4

5

Source: Redrawn from Monticello et al. (1989). 6

7

Formaldehyde

- related lesions were reporte d in the nasal passages, tracheas, and in the 8

larynx of treated animals (Figure 4

- 7) (Monticello et al., 1989). Nasal epithelium from treated 9

animals exhibited many of the histologic lesions described in rodent studies, including loss of 10

goblet cells, los s of cilia, epithelial hyperplasia, squamous metaplasia, and neutrophilic 11

inflammatory response in the respiratory epithelium. The lesions were more severe after 12

6 weeks of exposure and were present over a greater percentage of the epithelium compared wit h 13

the 1

- week exposure group ( p < 0.05) (Figure 4

- 7). 14

15This document is a draft for review purposes only and does not constitute Agency policy.

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- 136 

### B 2 5

2 0

1 5

1 0

5

0 C D E

L E V E L 

### B 2 5

2 0

1 5

1 0

5

0 C D E

L E V E L 

### 1 W K E X P

6 W K E X P C O N T R O L

A ) B )

1

2

Figure 4

- 7. Formaldehyde

- induced cell proliferation in male rhesus monkeys 3

exposed to formaldehyde 4

5

Note: Animals were exposed to 6 ppm formaldehyde 6 hours/day, 5 days/week 6

for 1 or 6 weeks. Bar g raph depicting mean labeling indices for the respiratory 7

epitheli um at levels B −E. A: One

- week exposure group. B: Six

- week exposure 8

group. *Statistically different from controls ( p ≤ 0.05). Statistically different 9

from 1

- week exposure group ( p ≤ 0.05). 10

11

Source: Redrawn from Monticello et al. (1989). 12

13

14

There was a d istinct anterior to posterior gradient in both 1

- week and 6

- week treatment 15

groups in which the anterior regions had a higher percentage of impacted epithelium (Monticello 16

et al., 1989). However, the longer duration exposure produced significantly more les ions in the 17

larynx and trachea compared with those observed after only 1 week of exposure ( p < 0.05). No 18

formaldehyde

- related lesions were reported for the epithelium of the maxillary sinus, a structure 19

not present in rodents. Labeling indices (LIs) from the histoautoradiograms indicated increased 20

cell proliferation in transitory, respiratory, and olfactory epithelial cells after the 6

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

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- 137 formaldehyde exposure (Figure 4

- 8) (Monticello et al., 1989). Similar trends were seen after 1

only 1 week but were s tatistically significant only in the respiratory epithelium. Although 2

increased proliferation in the trachea and carina was statistically significant after 1 week of 3

exposure, the greater increases seen after 6 weeks of exposure, compared with controls, w ere not 4

statistically significant. A small sample size (n = 3) and high variability may have contributed to 5

the lack of statistical significance. Monticello et al. (1989) noted that increased cell proliferation 6

was seen in locations with minimal histolog ic changes, indicating proliferation may be a more 7

sensitive predictor of adverse health effects of formaldehyde exposure. 8

9

6 0

5 0

4 0

3 0

2 0

1 0

0

B C D E L / T

L E V E L 6 0

5 0

4 0

3 0

2 0

1 0

0

B C D E L / T

L E

V E L B ) A ) 1 W K E X P

6 W K E X P

10

11

Figure 4

- 8. Formaldehyde

- induced lesions in male rhesus monkeys exposed 12

to formaldehyde. 13

14

Note: Animals were exposed to 6 ppm fo rmaldehyde 6 hours/day, 5 days/week 15

for 1 or 6 weeks. Bar graph showing levels B

– E of the nasal passages and the 16

larynx/trachea (L/T), depicting percent surface area with formaldehyde

- induced 17

lesions. Morphometry of level A was excluded due to the simila rity of normal 18

features of transitional epithelium to formaldehyde

- induced lesions in the 19

respiratory epithelium. A: One

- week exposure group. B: Six

- week exposure 20

group. 21

*Statistically different from controls ( p ≤ 0.05). 22

| Statistically different from 1

- week exposure group ( p ≤ 0.05). 23

Source: Redrawn from Monticello et al. (1989). 24

25

There are two reports in the literature assessing changes in pulmonary tissues after acute 26

formaldehyde exposures (Kamata et al., 1996a, b). Kamata et al. (1996a) exposed male F344 27This document is a draft for review purposes only and does not constitute Agency policy.

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- 138 rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m 3 ) formaldehyde for 6 hours. Lung lavage 1

samples were collected and the fluid analyzed for the lipids, free cholesterol, phosphatidyl 2

ethanolamine, phos phatidyl choline, sphingomyelin, and triglyceride. 3

The bronchoalveolar lavage (BAL) was analyzed for triglycerides, cholesterol, and 4

phosphatidyl choline. As in the first experiment ( Kamata et al., 1996a), triglyceride 5

concentration was reduced in the lav age of treated animals, in this case, to 16% of controls in 6

lavage in those rats exposed to 145.6 ppm formaldehyde (Table 4

- 12). Cholesterol concentration 7

was unchanged and phosphatidyl choline was increased to 220% of that of control rats as a result 8

of exposure to 145.6 ppm formaldehyde. However, BAL lipids were unchanged in 15 ppm 9

exposed rats. Triglycerides were reduced in unwashed lung tissue from formaldehyde

- treated 10

rats in a concentration

- dependent manner and free fatty acids were reduced in rats exposed to 11

145.6 ppm formaldehyde. Neither triglyceride nor sphingomyelin was detected in lung lavage 12

fluid from the high treatment group. 13

14

Table 4

- 12. Lipid analysis of lung tissue and lung lavage from male F344 15

rats exposed to 0, 15, or 145.6 ppm form aldehyde for 6 hours 16

17

Control a 15 ppm a 145 ppm a

Lung tissue

Free fatty acids (mg/g lung) 3.30 (0.7) 3.11 (1.23) 1.41 (0.63) b

Triglyceride (mg/g lung) 1.55 (0.23) 0.74 (0.14) c 0.62 (0.17) c

Cholesterol (mg/g lung) 1.72 (0.10) 1.41 (0.25) 1.16 (0.55)

Phosphatidyl ethanolamine (mg/g lung) 7.41 (1.81) 7.46 (2.28) 5.49 (1.78)

Phosphatidyl choline (mg/g lung) 11.0 (1.49) 9.65 (3.21) 7.53 (3.52)

Sphingomyelin (mg/g lung) 3.44 (0.75) 3.13 (1.28) 2.51 (0.95)

Lung lavage

Triglyceride (mg/lung) 0.31 (0. 10) 0.24 (0.09) 0.05 (0.02) c

Cholesterol (mg/lung) 0.04 (0.01) 0.04 (0.01) 0.04 (0.01)

Phosphatidyl choline (mg/lung) 0.66 (0.23) 0.84 (0.35) 1.45 (0.31) c

18

a SD given in parentheses. 19

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

c Significant differen ce from controls ( p < 0.01). 21

22

Source: Kamata et al. (1996b). 23

24

Concentration

- dependent decreases were seen in nonprotein sulfhydryl (SH) groups and 25

lipooxygenase in nasal mucosa homogenate and nonprotein SH groups in lung tissue 26

homogenate (Table 4

- 13). I ncreases in both lipooxygenase and LDH activities were found in 27

lung tissue homogenate from formaldehyde

- exposed rats. 28

29This document is a draft for review purposes only and does not constitute Agency policy.

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- 139 Table 4

- 13. Formaldehyde effects on biochemical parameters in nasal 1

mucosa and lung tissue homogenates from male F344 rats exposed to 0, 15, 2

or 145.6 ppm formaldehyde for 6 hours 3

4

Control a 15 ppm a 145 ppm a

Nasal mucosa b

Nonprotein SH groups (μM/g tissue) c 1.64 (0.50) 1.29 (0.28) 0.73 (0.21) f

Lipid peroxides (μM/g tissue) 118 (23) 71 (16) f 59 (18) f

Glucose

- 6

- dehydrogenase (U/g tis sue) d 1.96 (0.10) 1.87 (0.07) 2.07 (0.13)

Lung e

Nonprotein SH groups (μM/g tissue) c 1.83 (0.18) 1.70 (0.11) 1.29 (0.28) f

Lipid peroxides (μM/g tissue) 72 (8) 95 (15) f 93 (8) g

Glutathione reductase (U/g tissue) d 0.42 (0.25) 0.25 (0.05) 0.22 (0.05)

L actate dehydrogenase (U/g tissue) d 77.37 (9.28) 88.69 (7.66) 93.62 (4.99) f

5

a SD given in parentheses. 6

b 5 or 10% nasal mucosa homogenates. 7

c nmol malonaldehyde/g tissue. 8

d Units per gram tissue. 9

e 20% lung homogenates. 10

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

g Significant difference from controls ( p < 0.05). 12

13

Source: Kamata et al. (1996b). 14

15

16

Lino dos Santos Franco et al. (2006) studied the effects of inhaled formaldehyde on lung 17

injury and changes in airway reactivity in rats. The extent of local an d systemic inflammation 18

was assessed by changes in leukocyte counts in BAL fluid, blood, bone marrow, and spleen. 19

Changes of reactivity of isolated tracheae and intrapulmonary bronchi in response to 20

methacholine were monitored in response to formaldehyde exposure. The authors exposed male 21

Wistar rats to formaldehyde generated from a 1% solution of formalin. However, they provided 22

insufficient information for the exposure concentration to be determined. Groups of six animals 23

were exposed to formaldehyde for either 0, 30, 60, or 90 minutes on 4 consecutive days. All 24

experiments were carried out 24 hours after the final exposure. 25

The authors reported a significantly increased number of leukocytes in the BAL fluid of 26

animals exposed to formaldehyde via inha lation. The effect reached a maximum for the longer 27

exposure duration (90 minutes). Compared with controls, rats exposed to formaldehyde 28

90 minutes/day for 4 days also displayed an increase in the number of total blood leucocytes 29

(1.4 ± 0.06 × 10 4 versu s 0.8 ± 0.01 × 10 4 cells/mm 3 ). These values are means ± standard error of 30

the mean (SEM) for six animals/group. The effect appeared to reflect changes in the 31

mononuclear cell population (1.1 ± 0.02 × 10 4 versus 0.6 ± 0.003 × 10 4 cells/mm 3 ) rather than 32

pe ripheral blood neutrophils (0.2 ± 0.003 × 10 4 cells/mm 3 in test animals and controls). There 33

was also an apparently compound

- related increase in the total cell count in the spleen 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 140 (112.7 ± 4.4 × 10 6 versus 94.2 ± 5.5 × 10 6 cells). However, a change in t he number of cells 1

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

45.0 ± 4.3 × 10 6 cells). Lino dos Santos Franco et al. (2006) provided data on dose

- dependent 3

changes in methacholine

- induced contractions in isola ted tracheae and bronchi obtained from 4

formaldehyde

- exposed and control rats. Although the maximal contractile response induced by 5

methacholine in tracheae of formaldehyde

- treated rats was unchanged compared with controls, 6

contractions in isolated bronchi were significantly weaker than those observed in controls. 7

The authors examined the effect of formaldehyde inhalation on rat lung mast cells. 8

Degranulation and significant neutrophil infiltration were features of the response to 9

formaldehyde (Table 4

- 14)

. 10

11

Table 4

- 14 . Mast cell degranulation and neutrophil infiltration in the lung of 12

rats exposed to formaldehyde via inhalation 13

14

Treatment group Mast cell degranulation

(cells/mm 2 ) a Neutrophil infiltration

(cells/mm 2 ) a

Controls 0 b 0.3 ± 0.2

Formaldehyde

- e xposed 2.0 ± 0.4 c 5.2 ± 1.7 c

15

a Values are means ± SEM; n = 6. 16

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

c No statistical analysis was provided by the authors for these changes. 18

19

Source: Lino dos Santos Franco et al. (2006)

. 20

21

22

Selected pharmacological agents were used to explore the mechanism by which exposure 23

to formaldehyde might have brought about the observed lung infiltration and bronchial 24

hyporesponsiveness. Lino dos Santos Franco et al. (2006) provided data showing t hat separate 25

pretreatment of the animals with compound 48/80, sodium cromoglycate (SCG), and 26

indomethacin reduced the formaldehyde effect on neutrophil release into BAL but had no effect 27

on mononuclear cell counts. Compound 48/80 and SCG also reversed the formaldehyde

- induced 28

reduction in bronchial response to methacholine, but indomethacin had the opposite effect 29

(causing an additional decrease in bronchial responsiveness). In broad terms, these findings 30

were thought to implicate mast cells as a possible mediator of the toxicological effects of 31

formaldehyde. Histologically, a significantly increased number of degranulated mast cells were 32

evident in the pulmonary tissue of rats that were exposed to formaldehyde. 33

Lino dos Santos Franco et al. (2006) also e xamined the regulatory role of NO on 34

formaldehyde

- induced bronchial activity. Nitrites generated by cultured cells of BAL from 35

formaldehyde

- treated rats increased about threefold compared with those from controls. 36This document is a draft for review purposes only and does not constitute Agency policy.

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- 141 However, pretreatment with the NO syntha se inhibitor, N

- nitro

- L

- arginine methyl ester, 1

prevented the formaldehyde

- induced bronchial hyporesponsiveness to methacholine but had no 2

effect on pulmonary leukocyte recruitment. These data implicate the existence of distinct 3

mechanisms for the inductio n of lung inflammation versus bronchial hyporeactivity. Further 4

support for this concept came from an experiment in which rats were pretreated with capsaicin to 5

examine the involvement of sensory fibers in lung inflammation and the bronchial 6

hyporesponsiv eness induced by formaldehyde inhalation. Although the treatment did not 7

influence formaldehyde

- induced bronchial hyporesponsiveness to methacholine, the number of 8

leukocytes recovered in the BAL fluid were reduced compared with those of rats exposed to 9

f ormaldehyde alone. 10

11

4.2.1.2.2.3. Extrapulmonary effects: short

- term studies

In a subsequent study in the same laboratory (Kamata et al., 1996b), male F344 rats were 28

exposed to either 0, 1 5, or 145 ppm (0, 18.5, or 178 mg/m 3 ) formaldehyde nose only for 6 hours 29

( Kamata et al., 1996b) . Fifteen animals were treated at each level and separated into subgroups 30

of five animals each for tissue collection and the determination of other endpoints. Blood 31

samples were collected from one subgroup to determine such hematological and clinical 32

chemistry parameters as RBC count, Hb, PCV, MCV, MCHC, WBC count, and plasma levels of 33

TP, ALB, BUN, glucose, phospholipids, triglycerides, total cholesterol, LDH, alkaline 34

phosphatase (ALP), aspartate aminotransferase (AST), alanine aminotransferase (ALT), and 35 . Kamata et al. (1996a) exposed male 12

F344 rats to 0, 128.4, or 294.5 ppm (0, 158, or 362 mg/m 3 ) formaldehyde for 6 hours. In 13

addition, blood samples were monitored for hematology and clinical chemistry parameters, 14

including red blood cell (RBC) count, hemoglobin (Hb), packed cell volume (PCV), mean 15

corpuscular volume (MCV), mean corpuscular hemoglobin concentration (MCHC), white blood 16

cell (WBC) count, and plasma levels of total p rotein (TP), albumin (ALB), blood urea nitrogen 17

(BUN), glucose, phospholipids, triglycerides, total cholesterol, cholinesterase, and LDH. Male 18

rats exposed to 294.5 ppm formaldehyde had increased RBC count, Hb, hematocrit (HCT), 19

MCV, and serum glucose ( p < 0.05) compared with controls (Kamata et al., 1996a). There were 20

concentration

- related decreases in serum measures of TP, ALB, and phospholipids ( p < 0.05). 21

BUN was decreased in rats exposed to 128.4 ppm but increased in the higher treatment group 22

( p < 0.05). Phospholipid analysis of the lung surfactant indicated a decrease in the production in 23

formaldehyde

- treated animals ( p < 0.05). Total free cholesterol, phosphatidyl ethanolamine, and 24

phosphatidyl choline were reduced to 60, 55, and 38% of controls for rats treated with 294.5 ppm 25

formaldehyde ( p < 0.05). Sphingomyelin was reduced to 32% of controls in the low treatment 26

group ( p < 0.05). 27This document is a draft for review purposes only and does not constitute Agency policy.

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- 142 G6PDH. BAL was collected from five animals of each group and analyzed for phospholipids. 1

Lung homogenate from five animals in each treatment group was analy zed for nonprotein SH 2

groups, lipid peroxides, and total lipids. The 20,000 × g supernatant of the lung homogenate was 3

assayed for the activities of GR, G6PDH, and LDH. Similarly, nonprotein SH groups and lipid 4

peroxidase were measured in homogenates of excised nasal mucosa. At autopsy, organs (brain, 5

heart, lung, liver, kidney, spleen, and testis) were weighed and tracheae and nasal turbinates 6

examined. After fixation and decalcification, five sections across the nose were taken, 7

corresponding to stand ard sections 1

– 5 (Figure 4

- 3). 8

Several blood parameters were affected after these acute exposures. The WBC count was 9

slightly increased, from 4.7 × 10 3 cells/mm 3 in control rats to 5.1 × 10 3 cells/mm 3 and 6.1 × 10 3 10

cells/mm 3 at 15 and 145.6 ppm formaldehy de, respectively (Kamata et al., 1996b). Serum levels 11

of AST and LDH decreased in an apparent concentration

- dependent manner (AST 68 and 54% 12

of controls and LDH 48 and 28% of controls, respectively). Serum levels of G6PDH and ALT 13

were decreased similarly across exposure groups at 45 and 78% of controls, respectively. 14

A synopsis of respiratory pathology findings following short

- term exposure to 15

formaldehyde is presented in Table 4

- 15. 16

17

4.2.1.2.2.4. Nasal pathology : subchronic studies.

Although control mice gained weight, mice exposed to 40 ppm formaldehyde lost weight 26

during the 13

- week exposures. Expressed b y the authors as a percent of weight gain in controls, 27

the weight losses were −235% in males and −168.6% in females. Early mortality for both male 28

and female mice exposed to 40 ppm was 80%. Although gross and histochemical effects in 29

excised pieces from each organ system were evaluated, endometrial hypoplasia in mice treated 30

with 40 ppm was the only effect noted outside the respiratory system. The authors considered 31

this effect secondary to the observed respiratory tract lesions and frank toxicity at 40 ppm 32

formaldehyde. 33 In a study by Maron pot et al. (1986), female 18

and male B6C3F1 mice (10/group) were exposed at 0, 2, 4, 10, 20, or 40 ppm (0, 2.46, 4.92, 19

12.3, 24.6, or 49.2 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for 13 weeks. Clinical 20

observations were made daily, and mice were weighe d weekly. At autopsy, tissue sections from 21

each organ system (approximately 50 tissues per mouse) were fixed, stained, and examined by 22

light microscopy. Noses were fixed, decalcified, and transversely trimmed at three levels: the 23

incisor teeth, midway be tween the incisor teeth and first molar teeth, and the second molar teeth 24

(corresponding to sections 2, 3, and 4 in Figure 4

- 3). 25

34This document is a draft for reviewpurposes only and does not constituteAgency policy4-143 Table 4

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

— short

- term studies

Species/strain No./

group Treatment Respiratory effects LOAEL/NOAEL Reference

Nasal pathology

Male Sprague

Dawley rats 3 Single 4

- hour exposure to

10 ppm formaldehyde. Marked histopathologic changes to the nasoturbinates,

maxilloturbinates, ethmoidal turbinates, and goblet and

microvillus cells. LOAEL = 10 ppm. Bhalla et al.

(1991)

Male Swiss

Webster mice 24

– 34 0 or 3.13 ppm formaldehyde

6 hours/day for 5 days. Histopathologic lesions to the respiratory epithelium,

including inflammation, exfoliation, erosion,

ulceration, necrosis, and squamous metaplasia. LOAEL = 3.13 ppm. Buckley et al.

(1984)

Male F344 rats 3

– 5 0, 0.5, 2, 6, or 15 ppm

6 hours/day for 1, 2, or 4 days. Histopathologic lesions to the nasal conchae, lateral

wall, and ventral nasal conchae. NOAEL = 2 ppm for

focal lesions. Some

changes in ciliary

configuration were

evident at all exposures. Monteiro

- Riviere

and Popp (1986)

Male Wistar rats 20 0 or 3.5 ppm formaldehyde

through six consecutive 12

- hour

cycles in which rats were

exposed for 8 hours; 10 were

unexposed for 4 hours. The activity of GPX was increased in respiratory

epithelium homogenates. The nasa l respiratory

epithelium showed frank necrosis. LOAEL = 3.5 ppm. Cassee and Feron

(1994)

Male F344 rats 5 0, 6, or 15 ppm

[ 14 C]

- formaldehyde 6 hours/day

for a single day (naïve group).

A pretreated group was exposed

to 6 or 15 ppm formaldehyde

6 hours/da y for 4 days prior to

[ 14 C]

- formaldehyde exposure. Cellular necrosis to the nasal epithelium. 10.05%

cellular proliferation. LOAEL = 6 ppm. Chang et al.

(1983)

Male and female

Wistar rats 5/sex 0, 2, or 5 ppm formaldehyde

8 hours/day for 3 or 30 days. Ce ll disarrangement, squamous hyperplasia, atypical

mitosis, and epithelial hyperplasia. NOAEL = 2 ppm. Javdan and Taher

(2000)

Male F344 rats 15 0, 15, or 145.6 ppm

formaldehyde for a single 6

- hour

exposure. Histopathologic lesions in the nasal turbinates and

trachea LOAEL = 15 ppm. Kamata et al.

(1996b)

Male rhesus

monkeys 9 0 or 6 ppm formaldehyde

6 hours/day for 1 or 6 weeks.

[ 3 H]

- thymidine was injected

prior to sacrifice. Histopathologic lesions, including loss of goblet cells,

loss of cilia, epitheli al hyperplasia, squamous

metaplasia, and neutrophilic inflammation. LOAEL = 6 ppm. Monticello et al.

(1989)This document is a draft for reviewpurposes only and does not constituteAgency policy4-144 Table 4

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

— short

- term studies

Species/strain No./

group Treatment Respiratory effects LOAEL/NOAEL Reference

Tracheal and lung pathology

Syrian golden

hamsters (sex

unstated) 5 0 or 250 ppm 1 hour/day for 1,

2, 5, or 15 days. Abnormal cells in tracheal la vage, an effect that was

reversed on cessation of treatment. LOAEL = 250 ppm. Schreiber (1979)

Male rabbits

(strain unstated) ND Aerosol generated from a 3%

formaldehyde solution

3 hours/day for up to 50 days

(air concentration unknown). Necrosis of the b ronchi and lung parenchyma.

Increased activities of acid phosphatase, Tween

- 60

esterase, naphthol

- AS

- D

- acetate esterase, proline

oxidase, and hydroxyproline epimerase. Reduced

activities of leucyl aminopeptidase and β

glucuronidase. Adverse histopatholo gic changes. ND. Ionescu et al.

(1978)

Male F344 rats 6 0, 128.4, or 294.5 ppm for a

single 6

- hour exposure. Phospholipid content was reduced in lung surfactant,

for example, sphingomyelin to 43% of controls in the

low

- concentration group. LOAEL = 128.4 p pm. Kamata et al.

(1996a)

Male F344 rats 15 0, 15, or 145.6 ppm

formaldehyde for a single 6

- hour

exposure. Biochemical changes in lung homogenates. Altered

lipid content of BAL in high concentration rats. LOAEL = 15 ppm. Kamata et al.

(1996b)

Male Wista r rats 6 Aerosol generated from a 1%

formalin solution 0, 30, 60, or

90 minutes/day on 4 consecutive

days (air concentration

unknown). Increased leukocyte count in bronchoalveolar fluid.

Degranulation of mast cells and increased neutrophil

infiltration. N D. Lino dos Santos

Franco et al.

(2006)

Extrapulmonary effects

Male F344 rats 6 0, 128.4, or 294.5 ppm for a

single 6

- hour exposure. . LOAEL = 128.4 ppm. Kamata et al.

(1996a)

Male F344 rats 15 0, 15, or 145.6 ppm

formaldehyde for a single 6

- hour

exposu re. . LOAEL = 15 ppm. Kamata et al.

(1996b)

ND = not determined; LOAEL = lowest

- observed

- adverse

- effect level; NOAEL = no

- observed

- adverse

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

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- 145 While no statistical comparison was provided, respiratory tract lesions showed an 1

increased incidenc e with concentration, as well as an increased distribution throughout the 2

respiratory tract (Table 4

- 16). No lesions were seen in the nasal cavity, larynx, trachea, or lung 3

of control mice or mice treated with 2 ppm formaldehyde. Minimal squamous metapla sia in the 4

nasal cavity was noted in 1 of 10 male mice treated with 4 ppm formaldehyde, but none were 5

observed in the female mice. However, squamous metaplasia was observed in all mice in the 6

higher treatment groups (10, 20, and 40 ppm). Lesions became m ore severe and penetrated more 7

deeply into the respiratory tract as exposure concentration increased. Where lesions were present 8

in the nasal cavities of all mice exposed to 10 ppm, similar lesions were reported in the larynx 9

and trachea of some animals e xposed to 20 ppm and all animals exposed to 40 ppm 10

formaldehyde. Mice exposed to 40 ppm formaldehyde exhibited lesions as deep as the lung, 11

including squamous metaplasia, submucosal fibrosis inflammation, and epithelial hyperplasia. 12

The findings of Maronp ot et al. (1986) indicated a no

- observed

- adverse

- effect level 13

(NOAEL) of 4 ppm and a LOAEL of 10 ppm in mice, based on squamous metaplasia in the 14

nasal epithelium. Although a LOAEL of 10 ppm was observed, there was 80% mortality for 15

both sexes at 40 ppm, indicating a very narrow range between the first observed adverse health 16

effects and frank effect concentrations in mice for this 13

- week treatment. 17

In a study by Woutersen et al. (1987), male and female albino SPF Wistar rats (10/group) 18

were exposed to 0, 1, 10, or 20 ppm (0, 1.23, 12.3, or 24.6 mg/m 3 ) formaldehyde 6 hours/day, 19

5 days/week for 13 weeks. Rats were checked daily and weighed weekly. Three longitudinal 20

sections of lungs, trachea, and larynx and six standard cross sections of the nose were ta ken for 21

microscopic examination. Two rats per exposure group were similarly treated for 3 days and 22

sacrificed 18 hours later, and nasoturbinates were dissected to measure cell proliferation. 23

Woutersen et al. (1987) noted that the majority of the dose

- de pendent increases in cell 24

proliferation seen at section level 3 after 3 days of repeated 6

- hour exposures to 10 and 20 ppm 25

(12.3 and 24.6 mg/m 3 ) formaldehyde occurred in areas of the epithelium showing “clear 26

squamous metaplasia and hyperplasia.” Cell pro liferation rates in metaplastic epithelium of 27

29.5 and 33.2% were much higher than the 1.4 to 2.8% proliferation in the visibly unaffected 28

respiratory epithelium from rats exposed at 10 ppm formaldehyde. Although there was a slight 29

trend towards increased cell proliferation in the visibly unaffected epithelium of exposed animals 30

compared with unexposed controls, the majority of increased cell proliferation resulting from 31

exposure to 10 and 20 ppm formaldehyde was attributed to the metaplastic epithelium 32

(W outersen et al., 1987). 33This document is a draft for reviewpurposes only and does not constituteAgency policy4-146 Table 4

- 16. Location and incidence of respiratory tract lesions in B6C3F1 mice exposed to

formaldehyde

Location of respiratory

tract lesions Control 2 ppm 4 ppm 10 ppm 20 ppm 40 ppm

Male Female Male Female Male Female Male Fe male Male Female Male Female

Nasal cavity

Squamous metaplasia

Seropurulent inflammation – a

– –

– –

– –

– 1/10

– –

– 10/10

4/10 10/10

– 10/10

10/10 10/10

8/10 10/10

10/10 10/10

10/10

Larynx

Squamous metaplasia

– – – – – – – – 6/9 3/9 10/10 7/8

Trachea

Squamous metaplasia

Epithelial hyperplasia

Seropurulent inflammation

Submucosal fibrosis –

–

–

– –

–

–

– –

–

–

– –

–

–

– –

–

–

– –

–

–

– –

–

–

– 1/10

–

–

– 3/10

4/10

–

– 5/10

2/10

–

– 10/10

2/10

8/10

9/10 10/10

--- 5/10

5/ 10

Lung (Bronchus)

Squamous metaplasia

Inflammation

Submucosal fibrosis –

–

– –

–

– ND b

ND

ND ND

ND

ND ND

ND

ND ND

ND

ND –

–

– –

–

– –

–

– –

–

– 4/10

3/10

2/10 3/10

2/10

–

a Dash indicates no lesions recorded in that treatment group.

b ND = no data.

Source: Maronpot et al. (1986).This document is a draft for review purposes only and does not constitute Agency poli cy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 147 Statistically significant increases were seen in focal respiratory epithelial hyperplasia and 1

keratinization in both male and female rats at the highest treatment level (20 ppm) (Table 4

- 17). 2

Male rats also had statistically significant increases in observed respiratory epithelial squamous 3

metaplasia, focal olfactory epithelial thinning, and rhinitis. Both male and female rats treated 4

with 10 ppm formaldehyde showed statistically significant increases in squamou s metaplasia, 5

hyperplasia, and keratinization of the respiratory epithelium (Woutersen et al., 1987). 6

Disarrangement of the respiratory epithelium was only significantly increased in female rats, but 7

this change was observed at both the 10 and 20 ppm trea tment levels. Although some lesions 8

were observed in animals treated with 1 ppm formaldehyde, their incidences were not 9

statistically significant and the findings were equivocal. 10

Feron et al. (1988) examined recovery of formaldehyde

- induced nasal lesions after 11

subchronic exposures. Male albino SPF Wistar rats (50

– 55/group) were exposed to 0, 10, or 12

20 ppm (0, 12.3, or 24.6 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for either 4, 8, or 13

13 weeks. All groups were observed for a total of 130 weeks, includi ng treatment and recovery. 14

Rats were weighed weekly for the first 13 weeks and monthly thereafter. Rats (five/group) were 15

sacrificed immediately after the end of exposure (4, 8, or 13 weeks). The balance of the rats 16

were sacrificed after 130 weeks, incl usive of exposure time. At sacrifice, noses were fixed and 17

sectioned by using standard section levels. 18

Formaldehyde exposure (20 ppm) was associated with reduced body weight throughout 19

the exposure period (4, 8, or 13 weeks). However, body weight in thes e groups matched that of 20

controls after 8, 40, and 100 weeks, respectively. Rats exposed to 10 ppm for 8 or 12 weeks had 21

slightly decreased body weight (further details not given). 22

Nonneoplastic lesions were reported in the nasal mucosa of rats exposed to either 10 or 23

20 ppm formaldehyde and examined immediately after exposure was discontinued (4, 8, or 24

13 weeks). Lesions increased in severity with both exposure duration and concentration (details 25

of severity and incidence were not provided). Rhinitis, hyperplasia, and squamous metaplasia of 26

the respiratory epithelium were seen in rats from both dose groups, but changes in olfactory 27

epithelia were only seen in rats exposed to 20 ppm, where cell disruption, thinning of the 28

epithelium, and simple cuboidal or squamous metaplasia were also reported. Changes in the 29

dorsomedial region, at the junction of the respiratory and olfactory epithelium, were similar to 30

those seen in the olfactory epithelium of rats exposed to 20 ppm formaldehyde. A similar 31

concentrat ion

- and duration

- dependent increase in histopathologic changes in nasal epithelium 32

was observed after the full 130 weeks, which included 126, 122, or 117 weeks of recovery for 33

the three duration groups, 4, 8, and 13 weeks, respectively (Table 4

- 17). 34

35This document is a draft for review purposes only and does not constitute Agency poli cy.

DRAFT

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- 148 Tabl e 4

- 17. Formaldehyde effects (incidence and severity) on histopathologic 1

changes in the noses and larynxes of male and female albino SPF Wistar rats 2

exposed to formaldehyde 6 hours/day for 13 weeks 3

4

Concentration of formaldehyde (ppm)

0 1 10 20 0 1 10 20

Respiratory epithelium Severity Males Females

Diffuse squamous

metaplasia Slight

– a

– – – – – – 3

Moderate

– – – 5 b

– – – 4

Severe

– – – 5 b

– – – 3

Focal squamous

metaplasia Very slight

– 1

– – – – 1

– Slight

– 1 6 b

– – – 7 c

–
Moderate

– – 4

– – – 2

– Focal hyperplasia Very slight

– – 1 1

– – 2 1

Slight

– – 6 b 7 c

– 1 6 b 6 b

Moderate

– – 1

– – – – – Focal disarrangement Very slight

– – 1

– – – 2 1

Slight

– – 3

– – 1 6 b 6 b

Moderate

– – 1

– – – – – Focal keratinization Very slight

– 2 6 b 1

– – 6 b 6 b

Slight

– – 3 6 b

– – 2 4

Moderate

– – – 1

– – – – Olfactory epithelium

Focal thinning Slight

– – – 2

– – – 2

Moderate

– – – 1

– – – 2

Severe

– – – 5 b

– – – 2

Focal squamous

metaplasia Slight

– – – 4

– – – 3

Moderate

– – – 4

– – – 1

Focal keratinization Very slight

– – – 1

– – – – Slight

– – – 2

– – – – Rhinitis

– 2 5 b 10 c

– – 3 2

Larynx

Squamous metaplasia Very slight

– – – 3

– NE d NE

– Slight

– – – 1

– NE NE

–
Moderate

– – – 1

– NE NE

– Keratinizatio n Slight

– – – 2

– NE NE

– 5

a Dash indicates no lesions reported. 6

b Different from control, p < 0.05. 7

c Different from control, p < 0.01. 8

d NE = not evaluated. 9

10

Source: Woutersen et al. (1987). 11

12

13

Feron et al. (1988) did not provide a direct comparison among lesions reported at the 14

interim sacrifice and terminal sacrifice after the extended recovery period. However, similar 15

lesions were reported after the recovery period, including focal hyperplasia and stratified 16

squamous metaplasia of the respiratory epithe lium, stratified cuboidal or squamous metaplasia in 17

the dorsomedial area, and replacement of olfactory epithelium. The incidence and severity of 18This document is a draft for review purposes only and does not constitute Agency poli cy.

DRAFT

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- 149 these lesions in rats exposed to 20 ppm formaldehyde were statistically different from control 1

animals, regard less of exposure duration (Table 4

- 18). 2

3

Table 4

- 18. Formaldehyde

- induced nonneoplastic histopathologic changes in 4

male albino SPF Wistar rats exposed to 0, 10, or 20 ppm formaldehyde 5

(6 hours/day, 5 days/week) and examined at the end of 130 weeks inclu sive of 6

exposure 7

8

4 Weeks 8 Weeks 13 Weeks

Formaldehyde, ppm 0 10 20 0 10 20 0 10 20

Total noses examined 44 44 45 45 44 43 45 44 44

Respiratory epithelium focal hyperplasia

Very slight 0 0 0 0 1 3 0 5 a 2

Slight 0 3 8 b 2 2 12 b 1 6 14 b

Mode rate 0 0 1 0 1 0 0 0 4

Respiratory epithelium focal stratified squamous

metaplasia

Very slight 3 6 14 b 8 16 17 a 2 10 a 2

Slight 4 2 19 b 2 1 20 b 3 18 b 26 b

Moderate 0 2 3 0 0 2 1 5 14 b

Severe 0 0 0 0 0 0 0 0 1

Respiratory/olfactory epitheliu m stratified

cuboidal or squamous metaplasia 0 0 4 0 0 17 b 0 2 23 b

Rhinitis 7 7 18 a 4 6 22 a 8 11 23 b

Olfactory epithelium replacement by respiratory

epithelium and regeneration

Very slight 0 0 0 0 0 2 0 0 1

Slight 1 0 6 0 0 14 b 0 0 12 b

Moder ate 0 0 1 0 0 3 0 0 12 b

Severe 0 0 0 0 0 1 0 0 1

9

a Significantly different from control, p < 0.05. 10

b Significantly different from control, p < 0.01. 11

12

Source: Feron et al. (1988). 13

14

Although a slight increase in changes to the olfactory epithelium and dors omedial area 15

was seen in rats treated with 20 ppm formaldehyde for only 4 weeks, these differences were 16

significant and more severe in the 8

- and 13

- week treatment groups. Replacement of olfactory 17

epithelium by respiratory epithelium was described as slig ht after 8 weeks of exposure and slight 18

to moderate after 13 weeks of exposure in the 20 ppm treatment groups. Therefore, 19

formaldehyde

- induced lesions were not resolved after a considerable nonexposure recovery 20

period of up to 126 weeks (Feron et al., 198 8). 21

Feron et al., (1988) derived a correlation between the development of nonneoplastic 22

changes in nasal epithelium and the development of nasal tumors as a result of these subchronic 23

formaldehyde exposures. Two SCCs were reported in rats exposed to 10 pp m formaldehyde but 24This document is a draft for review purposes only and does not constitute Agency poli cy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 150 were not considered to be formaldehyde related because of their locations (nasolacrimal duct, 1

incisor tooth). Six tumors were observed in the 20 ppm, 13

- week exposure group (Table 4

- 19) 2

of which three of the tumors were SCCs similar to those observed as a result of chronic 3

formaldehyde exposure. Two polypoid adenomas also were reported in rats exposed to 20 ppm 4

formaldehyde. Feron et al. (1988) concluded that subchronic exposures to 20 ppm formaldehyde 5

could result in an increase in na sal tumors, an effect that followed observation of cellular 6

proliferation. 7

8

Table 4

- 19. Formaldehyde

- induced nasal tumors in male albino SPF Wistar 9

rats exposed to formaldehyde (6 hours/day, 5 days/week for 13 weeks) and 10

examined at the end of 130 weeks i nclusive of exposure 11

12

Tumor type 0 ppm 10 ppm 20 ppm

No. of rats exposed for 4 weeks 44 44 45

Polypoid adenoma 0 0 1 a

SCC 0 0 1

No. of rats exposed for 8 weeks 45 44 43

Polypoid adenoma 0 0 1 a

SCC 2 1 1

No. of rats exposed for 13 weeks 45 44 44

SCC 0 1 3 a

Cystic squamous cell carcinoma 0 0 1

Carcinoma in situ 0 0 1 a

Ameloblastoma 0 0 1

13

a Tumor considered to be associated with formaldehyde exposure. 14

15

Source: Feron et al. (1988). 16

17

18

A companion study from the same laboratory examined the effects o f lower concentration 19

formaldehyde exposures (Zwart et al., 1988). Male and female albino Wistar rats (50/group) 20

were exposed to 0, 0.3, 1, or 3.0 ppm (0, 0.37, 1.2, or 3.7 mg/m 3 ) formaldehyde 6 hours/day, 21

5 days/week for 13 weeks. Body weight, general c ondition, and behavior were recorded weekly. 22

No effects of formaldehyde exposure on body weight changes were noted, and growth was 23

considered comparable among different exposure groups and controls. Rats were sacrificed 24

during week 14, and noses were fix ed and sectioned (exact time after exposure ended not given). 25

Six standard cross sections were examined for each animal by light microscopy, anterior to 26

posterior. Noses were fixed and decalcified, and six standard cross sections were taken and 27

developed

. 28

No formaldehyde

- related lesions were reported in the respiratory epithelium at section 29

level 3 after 13 weeks of formaldehyde exposure (0.1 ppm, 1 ppm, or 3 ppm). Signs of 30This document is a draft for review purposes only and does not constitute Agency poli cy.

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- 151 inflammation (rhinitis, sinusitis, mononuclear cell infiltrates) were observed in 1

formaldehyde

- treated rats, but there was no concentration

- response relationship (data not 2

provided). Formaldehyde

- related pathology in the anterior part of level 2 epithelium was 3

reported in 37/50 males and 21/50 female rats exposed to 3.0 ppm for 13 wee ks. Both 4

keratinized and unkeratinized squamous metaplasia were present, and disarranged cells and 5

hyperplastic respiratory epithelium were found in the transitional zone between squamous and 6

pseudostratified epithelium at level 2. Foci of keratinized sq uamous epithelium, glandularization 7

of goblet cells, and deciliated epithelium were observed by electron microscopy in anterior 8

sections of level 2 of rats exposed to 3 ppm formaldehyde. Epithelial cells with irregularly 9

shaped and strongly indented nucle i were described at level 2 in animals exposed to 0.3 and 10

1 ppm formaldehyde and were considered to be disarranged as well at 3 ppm formaldehyde 11

exposures. 12

Although early cell proliferation at level 3 corresponded to basal cell hyperplasia at 13

3 days, neith er effect persisted for the course of the exposure. The authors speculate that this is 14

an indication of an adaptive response, perhaps through increased function of the mucociliary 15

apparatus present at level 3. In contrast, the early changes at section le vel 2 were less dramatic 16

but persisted through 13 weeks, including clear formaldehyde

- related pathology. 17

Concentration times time (C × t) issues have been investigated for histopathology as well 18

as for cellular proliferation, outlined above. Specifically, Wilmer et al. (1989, 1987) compared 19

the effects of 8

- hour continuous and 8

- hour intermittent formaldehyde exposure in two studies. 20

Fifty male albino Wistar rats (10/group) were exposed to different exposure regimens to achieve 21

similar compound

- related C × t products. A C × t product of 40 ppm

- hours (49.2 mg/m 3

- hours) 22

was attained by an 8

- hour exposure to 5 ppm (6.2 mg/m 3 ) or a 4

- hour exposure to 10 ppm (12.3 23

mg/m 3 ) (Wilmer et al., 1987). Similarly, an 80 ppm

- hours (98.4 mg/m 3

- hours) C × t product was 24

at tained from continuous 10 ppm exposure or intermittent 20 ppm (24.6 mg/m 3 ) exposure. Rats 25

were exposed to one of these regimens 8 hours/day for either 3 days (two/group) or 4 weeks 26

(eight/group). Eighteen hours after exposure ended, rats were injected wi th [ 3 H]

- thymidine and 27

sacrificed 2 hours later. Noses were fixed and decalcified, and six standard cross sections were 28

taken and developed. 29

Thinning and disarrangement of the respiratory epithelium, squamous metaplasia, basal 30

cell hyperplasia, and rhiniti s were seen in formaldehyde

- treated rats. Lesions were most severe 31

in group 4 (20 ppm intermittent). Groups 2 and 3 had similar lesions (10 ppm intermittent and 32

continuous). Rats in group 1 had mild lesions. Formaldehyde concentration was the major 33

det erminate in severity of nasal lesions. Formaldehyde effects were less severe in group 1 than 34

in group 3, even though the C × t product was the same, indicating concentration rather than 35This document is a draft for review purposes only and does not constitute Agency poli cy.

DRAFT

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- 152 duration or cumulative exposure correlates to severity. Epithelial l esions in group 3 rats were 1

similar among rats exposed to 10 ppm, regardless of duration (groups 2 and 3). 2

In a follow

- up study, Wilmer et al. (1989) assessed both cellular proliferation and 3

histologic lesions in Wistar rats exposed to formaldehyde in grou ps that differed by 4

concentration and time. Group A served as a control group (0 ppm). Group B was exposed to 5

1 ppm for 8 hours, group C to 2 ppm for 8 hours, group D to 2 ppm for 4 hours (30 minutes for 6

8 hours), and group E to 4 ppm for 4 hours (30 min utes for 8 hours). The experimental design 7

and cellular proliferation results are illustrated in Table 4

- 20. Intermittent exposures at 2 and 4 8

ppm resulted in formaldehyde

- related histopathologic lesions similar to those reported by Zwart 9

et al. (1988). Disarrangement and squamous metaplasia in respiratory epithelium were observed 10

at 4 ppm (Table 4

- 20). Disarrangement, nest

- like infolds, goblet cell hyperplasia, and rhinitis 11

were observed at 2 ppm. Rats exposed continuously for 8 hours at 2 ppm formald ehyde had 12

fewer lesions than rats intermittently exposed to 2 ppm and were not statistically different from 13

controls. Although lesions were noted in rats given the continuous 1 ppm, 8

- hour treatment, 14

their incidence was not significantly different from th e controls (Table 4

- 20). It should be noted 15

that the control rats in this study were reported to have a higher frequency of lesions than 16

controls in two previous studies from this laboratory employing the same techniques (Zwart et 17

al., 1988; Woutersen et al., 1987). For example, lesions noted in the respiratory epithelium of 25 18

control rats included 13 disarrangements, 13 basal cell hyperplasia, and 5 each of goblet cell 19

hyperplasia, nest

- like infolds, and squamous metaplasia. This is in contrast to the data of 20

Woutersen et al. (1987), who reported no lesions in the respiratory epithelium of 20 control rats 21

(male and female). Although Zwart et al. (1988) discussed inflammatory lesions in control rats, 22

no mention was made of the other scored lesions in co ntrol animals. Overall, Wilmer et al. 23

(1989) reported clear adverse effects at 2 ppm formaldehyde, resulting from intermittent 24

exposure for 8 hours/day, 5 days/week for 13 weeks. The indication of no effects at 1 ppm and 2 25

ppm continuous exposure should be considered with some caution, given the unusual incidence 26

of lesions in the control animals. 27

The results reported by Wilmer et al. (1989, 1987) indicate a greater influence of 28

concentration, rather than exposure regimen (continuous versus intermittent) on formaldehyde 29

toxicity. However, these studies were conducted as repeated 8

- hour exposure regimens over a 30

course of days or weeks. Therefore both regimens allowed for a 16

- hour recovery time before 31

the next reexposure and do not represent a true contin uous exposure. This research group has 32

speculated that defensive adaptation of the nasal mucosa may include the function of the 33

mucociliary apparatus (Feron et al., 1989). Morgan et al. (1986a) have shown formaldehyde 34

effects on mucus flow and ciliary be at in F344 rats to result from hourly exposures to 15 ppm 35This document is a draft for review purposes only and does not constitute Agency poli cy.

DRAFT

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- 153 formaldehyde. However, effects seen in repeated 8

- hour exposures may not correspond to those 1

effects resulting from shorter duration exposures to higher formaldehyde concentrations. 2

3

Table 4

- 20 . F ormaldehyde effects on nasal epithelium for various 4

concentration

- by

- time products in male albino Wistar rats 5

6

Respiratory epithelium

at crosssection level 2 Exposure regimen ( number of animals )

A (25) B (22) C (24) D (23) E (25)

0 ppm 1 ppm 2 ppm 2 pp m 4 ppm

8

- Hour

continuous 8

- Hour

continuous 8

- Hour

intermittent 8

- Hour

intermittent

Disarrangement

Focal

Diffuse 12

1 4

1 8

0 3 a

15 b 8

11 c

Necrosis

Focal

Diffuse 4

0 3

0 0

0 2

2 3

2

Basal cell hyperplasia

Focal

Diffuse 9

4 4

0 6

0 11

4 10

11

Squamous metaplasia

Focal 5 0 1 7 16 c

Keratinization 0 0 1 0 3

Nest

- like infolds

Focal

Diffuse 5

0 4

3 11

1 14 c

0 7

1

Goblet cell hyperplasia

Focal

Diffuse 0

5 1

2 1

8 2

13 b 1

10

Rhinitis 3 2 3 16 c 8

7

a p < 0.05, compared with gr oup A. 8

b p < 0.001, compared with group A. 9

c p <0.01, compared with group A. 10

11

Source: Wilmer et al. (1989). 12

13

Rusch et al. (1983a, b) performed a comparative study of formaldehyde effects on the 14

nasal epithelium in F344 rats, Syrian golden hamsters, and cyn omolgus monkeys. Groups of 15

animals were exposed at 0, 0.2, 1, or 3 ppm (0, 0.25, 1.2, or 3.7 mg/m 3 ) formaldehyde 16

22 hours/day, 7 days/week for 26 weeks. Six male monkeys, 10 male and 10 female hamsters, 17

and 20 male and 20 female rats were exposed at each exposure level. The experiment was run in 18

two trials, each with its own control group: trial 1 at 0.2 or 1 ppm and trial 2 at 3 ppm. Animals 19

were weighed weekly and physically assessed (details not given). At sacrifice, organ weights 20

were recorded for the kidney, adrenals, heart, and liver. Tissue sections of the lung (4), trachea, 21

and nasal turbinates (4) of each animal were examined by light microscopy (section locations not 22This document is a draft for review purposes only and does not constitute Agency poli cy.

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- 154 given). Additionally, sections were examined by electron microscopy for rat s in the control and 1

1 ppm treatment groups (five rats per group). 2

Body weights of both male and female rats in the 3 ppm treatment group were depressed 3

by 20% between week 2 and the end of the 26

- week exposure. Absolute liver weights were 4

decreased in th ese animals as well (26% lower in males and 12% lower in females, p < 0.05). 5

This decrease in liver weight remained significant for male rats when normalized for body 6

weight (a ratio of 2.9 in treated versus 3.16 in controls) but not for female rats. No significant 7

body weight or organ weight changes were seen in hamsters or monkeys. Increased incidences 8

of congestion (36/156), hoarseness (32/156), and nasal discharge (62/156) were observed in 9

monkeys in the 3.0 ppm treatment group versus no hoarseness o r congestion and only five 10

observations of nasal discharge in 156 observations for control monkeys. Increased nasal 11

congestion was noted in the two lower treatment groups of monkeys: 30/156 and 12

45/156 observations, respectively, versus 9/156 observations in nasal discharge in the controls. 13

The authors reported an increase in nasal discharge and lacrimation in treated hamsters but no 14

increases in symptoms in rats. However, observations of adverse symptoms in the control rats 15

were greater than 10% on some measures. 16

Rhinitis increased in rats in the 3 ppm treatment group, and the incidence in controls was 17

notable (Table 4

- 21). All groups of monkeys showed some rhinitis, and no treatment effects 18

were observed in either monkeys or hamsters. Monkeys and rats in the high treatment group 19

(3 ppm) had a greater incidence of lesions in the nasoturbinate epithelium (Table 4

- 22). Rusch 20

et al. (1983a, b) noted that most lesions were mild to moderate but were “somewhat more 21

severe” in the high treatment group. Hamste rs did not exhibit a similar increase, with few 22

lesions noted in the nasal epithelium. Overall, these studies show a clear increase in adverse 23

health effects at 3 ppm for rats and monkeys, with no adverse effects seen in hamsters at this 24

treatment level o r rats and monkeys at the lower concentrations (0.2 ppm and 1 ppm). 25

26

Table 4

- 21. Rhinitis observed in formaldehyde

- treated animals; data pooled for 27

male and female animals 28
29

F344 rats Cynomolgus monkeys Syrian golden

hamsters

Trial 1:

I, Control

II, 0.2 ppm III, 1 ppm 17/38

14/39

14/38 4/6

4/6

5/6 0/14

0/4

0/11

Trial 2:

IV, Control

V, 3 ppm 12/40

25/39 2/6

4/6 0/9

2/16

30

Source: Rusch et al. (1983a, b). 31This document is a draft for review purposes only and does not constitute Agency poli cy.

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- 155 Table 4

- 22. Epithelial lesions found in the middle region of nasoturbinates of 1

formaldeh yde

- treated and control animals; data pooled for males and 2

females 3

4

F344 rats Cynomolgus monkeys Syrian golden

hamsters

Basal cell

hyperplasia Squamous

metaplasia/

hyperplasia Squamous metaplasia/

hyperplasia Nasal epithelium

Trial 1:

I, Control

II, 0.2 ppm

III, 1 ppm 0/38

0/38

0/36 2/38

1/38

3/36 0/6

0/6

1/6 No lesions noted

Trial 2:

IV, Control

V, 3 ppm 4/39

25/37 3/39

23/37 0/6

6/6 No lesions noted

5

Source: Rusch et al. (1983a, b). 6

7

8

Andersen et al. (2008) examined the e ffect of formaldehyde exposure at several 9

concentrations and durations. This study comprised histopathology and cell proliferation data, as 10

well as genomic analyses at Level II of the nasal cavity. Toxicogenomics analysis was 11

performed only at Level II b ecause this was the region where the most severe lesions have been 12

reported in chronic bioassays (Andersen et al., 2008; Monticello et al., 1991; Kerns et al., 1983). 13

More specifically, Andersen et al. (2008) stated that the histopathologic and cell proli feration 14

effects at Levels II and III (with similar tissue structure) (Monticello et al., 1991) provided 15

phenotypic anchoring for the genetic analysis. Table 4

- 23 summarizes many of the broad 16

phenotypic findings. 17

The primary conclusions of this study w ith regard to the histopathology and cell 18

proliferation are as follows: 19

**•** The presence of inflammatory cell infiltrates in the nasal epithelial tissue of F344 rats is 20

highly variable and provides no coherent pattern with dose or duration at levels below 21

6 pp m. 22

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

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

**•** Cell proliferation (as measured by labeling indices) was significantly elevated in 25

Levels I

– III at 6 ppm on day 5 and Level I on day 15, leading to the conclusion that 26

significant changes in cell proliferation may not occur at exposures to ≤2 ppm. 27

**•** A significant decrease in cell density was observed at Level I in animals exposed to 28

6 ppm formaldehyde for 15 days, which was posited to be related to tissue remodeling in 29

response to this concentration. 30This document is a draft for reviewpurposes only and does not constituteAgency policy4-156 Table 4

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

ppm Response D1 D1R D5 D6 D6R D15

0 0.7 2 6 15 0 0.7 2 6 15 0 0.7 2 6 15 0 0.7 2 6 1 5 0 0.7 2 6 15 0 0.7 2 6 15

I 0 1 6 8

– 4 2 1 7

– 1 1 5 8

– 5 2 4 7

– 6 1 3 7

– 3 1 0 5

–
H 0 0 0 0

– 0 1 3 8

– 0 0 3 8

– 0 0 1 8

– 0 0 2 8

– 0 0 2 7

–
M 0 0 0 0

– 0 0 0 0

– 0 0 0 7

– 0 0 0 0

– 0 0 0 0

– 0 0 0 0

– P1 39±9 37±15 65±40 155±89 a 79±55 56±37 51±44 119±38 a

P2

– – – a

– – – – P3

– – – a

– – – – CD 321±30 336±64 377±141 400±61 362±61 340±57 321±37 293±53 b

G

– 0 1 42 745

– 0 0 0

– – 0 15 28

– – 0 0 9

– – – – – – – 0 0 54

D = day; R = recovery.

I = infiltrations (number out of 8 total animals); H = hyperplasia (number/8); M = metaplasia (number/8).

P1

– P3 = proliferation at levels I

– III (ULLI).

CD = cell density (cells/mm) at Level I.

G = genes signific antly altered at Level II of nasal epithelial tissue.

a Significantly elevated ULLI and LI at Level I on day 5 or significantly elevated lLI lat Level I on day 15; index a without numerical value indicates significant

increases in ULLI in all subregions of Levels II and III at day 5.

b Statistically significant difference from control ( p < 0.05).

Source: Andersen et al. (2008).This document is a draft for review purposes only and does not consti tute Agency policy.

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- 157 Based on their analysis of the microarray data, Andersen et al. (2008) concluded that no 1

genes were significantly altered by exp osure to 0.7 ppm from 1 to 15 days. Exposure to 2 ppm 2

primarily resulted in gene changes at 5 days of exposure, but not thereafter. One gene was 3

significantly increased on day 1, but the authors did not identify that gene. At 6 and 15 ppm, 4

42 and 745 ge nes were altered at day 1, respectively. After 5 days, gene changes were only 5

observed at 6 ppm (15 ppm was not examined after day 1). These findings support conclusions 6

reached by their laboratory in an earlier analysis. Thus, the primary conclusion in the Andersen 7

et al. (2008) study is that genomic changes, including those suggestive of mutagenic effects, did 8

not temporally precede or occur at lower doses than phenotypic changes in the tissue. The 9

implications of this finding will be examined later i n Section 4.5. 10

11

4.2.1.2.2.5. Lung pathology: subchronic studies.

Özen et al. (2003a) measured zinc, copper, and iron content in lung tissue from 19

formaldehyde

- exposed Wistar rats. Adult male rats were exposed to 0, 5, or 15 ppm (0, 6.2, or 20

18.5 mg/m 3 ) formaldehyde 8 hours/day, 5 days/week for either 4 or 13 weeks. Rats were 21

checked daily and weighed weekly. At sacrifice, rats were autopsied and examined for gross 22

pathological changes. Lung tissue was homogenized and ana lyzed for zinc, copper, and iron. 23 Studies have also investigated the ability for 12

formaldehyde to induce pathology in the trachea, bronchi, and lung tissue. These studies have 13

reported tracheal tissue changes, lung inflamma tion, necrosis, changes to the biochemistry of 14

BAL fluid and lung surfactant in a variety of species. Özen et al. (2003a) noted changes in zinc 15

concentration in the lung tissue following exposure for formaldehyde. Dallas et al. (1989) and 16

Dinsdale et al. (1993) observed changes in P450 enzyme activity in the lung associated with 17

formaldehyde exposure. 18

Body weight gain was depressed in all treatment groups in a concentration

- dependent 24

manner ( p < 0.001) (Table 4

- 24). Formaldehyde

- exposed rats consumed less food and water 25

than controls and showed unsteady breathing, incr eased nose cleaning, excessive licking, 26

frequent sneezing, and nasal mucosa hemorrhages. Significant decreases were seen in the zinc 27

content of lungs after either 5 or 10 ppm formaldehyde exposure (Table 4

- 25). Copper content 28

was unchanged from controls in all treatment regimens, whereas iron content was increased after 29

4 weeks of 5 ppm exposure and after 13 weeks of either 5 or 10 ppm formaldehyde exposure 30

(Özen et al., 2003a). 31

32

33This document is a draft for review purposes only and does not consti tute Agency policy.

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- 158 Table 4

- 24. Percent body weight gain and concentrations of iron, zinc, and 1

copper in cerebral cortex of male Wistar rats exposed to formaldehyde via 2

inhalation for 4 and 13 weeks 3

4

Exposure (mg/m 3 ) Weight gain (%) a Zinc (mg/kg) a Copper (mg/kg) a Iron (mg/kg) a

4

- week data

0 20.11 ± 2.87 120 ± 6.03 4.60 ± 0.42 25.07 ± 2.83

6.1 7.27 ± 1.49 e 130 ± 7.26 c 5.60 ± 0.50 b 23.00 ± 2.32

12.2 5.24 ± 1.52 e 185 ± 10.36 e 5.80 ± 0.60 d 22.14 ± 1.95 b

13

- week data

0 60.53 ± 7.84 123 ± 6.22 4.67 ± 0.38 24.92 ± 2.84

6.1 38.41 ± 2.53 e 155 ± 7.94 e 5.41 ± 0.56 c 22.00 ± 2.41

12.2 25.87 ± 1.32 e 163 ± 6.03 e 6.10 ± 0.73 e 21.00 ± 1.96 b

5

a Values are means ± SDs (n = 7). 6

7

Statistical significance of differences versus controls, as calculated by the authors: 8

b p < 0.05. c p < 0.02. d p < 0.002. e p < 0.001. 9

10

Source: Özen et al. (2003b). 11

12

13

Table 4

- 25. Zi nc, copper, and iron content of lung tissue from formaldehyde

14

treated male Wistar rats 15

16

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

0 ppm Control 20.7 (1.6) 0.39 (0.05) 12.5 (0.8)

5 ppm 4 weeks 16.1 (1.3) d 0.32 (0.07) 12.9 (1.0)

10 ppm 4 weeks 13. 8 (1.2) e 0.36 (0.04) 17.5 (1.3) e

0 ppm Control 20.0 (1.6) 0.39 (0.05) 12.7 (0.4)

5 ppm 13 weeks 15.3 (1.4) e 0.37 (0.04) 17.9 (1.1) e

10 ppm 13 weeks 13.0 (1.1) e 0.39 (0.05) 22.4 (1.4) e

17

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

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

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

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

e p < 0.001, compared with controls, as calculated by the au thors. 22

23 Source: Özen et al. (2003a). 24

25

There are two reports of lung cytochrome P450 levels after formaldehyde exposure. The 26

first report by Dallas et al. (1989) describes concentration

- and duration

- dependent changes in P450 27

levels. Male Sprague

- Dawley rats were exposed at 0, 0.5, 3.0, or 15 ppm (0, 0.62, 3.7, or 18.5 28

mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for 1 day, 4 days, 12 weeks, or 24 weeks. There 29

were six rats in each exposure group, but the experiment was run in two parts, with three rats in 30

each subgroup. Rats were sacrificed after 1 day, 4 days, 12 weeks, or 24 weeks of exposure, and 31

liver microsomes were prepared. TP and P450 content were determined on each sample. 32This document is a draft for review purposes only and does not consti tute Agency policy.

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- 159 Average P450 levels in control groups ranged from 17

– 76 pmol P450/mg p rotein. 1

However, no P450 was detected in lung from formaldehyde

- treated animals after 1 day of 2

exposure, with a method detection limit of approximately 10 pmol P450/mg protein. In contrast, 3

P450 levels were elevated significantly above controls in a conc entration

- dependent manner after 4

4 days of formaldehyde exposure (Table 4

- 26). Although P450 levels remained elevated in some 5

experimental groups after 12 and 24 weeks of exposure, results were variable and less dramatic. 6

7

Table 4

- 26. Total lung cytochro me P450 measurements of control and 8

formaldehyde

- treated male Sprague

- Dawley rats 9

10

1 Day a,b 4 Days 12 Weeks 24 Weeks

Formaldehyde Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2 Expt. 1 Expt. 2

0 ppm 17 (6) 44 (13) 39 (11) 23 (3) 29 (10) 19 (23) 76 (4 9) 18 (11)

0.5 ppm ND ND 103 (52) 137 (14) e 87 (11) d 35 (7) 172 (12) c 38 (9)

3.0 ppm ND ND 357 (10) e 278 (100) e 91 (10) d 67 (34) 92 (103) 30 (15)

15 ppm ND ND 362 (38) e 334 (4) e 130 (2) e 56 (6) 151 (9) 48 (7) c

11

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

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

c Different from control, p < 0.05. 14

d Different from control, p < 0.01. 15

e Different from control, p < 0.001, as calculated by the authors. 16

N D = not detected above the limit of detection, approximately 10 pmol/mg protein. 17

18

Source: Dallas et al. (1989). 19

20

21

A later study by Dinsdale et al. (1993) attempted to confirm the increase in P450 levels 22

reported by Dallas et al. (1989). In their first ex periment, Dinsdale et al. (1993) treated male 23

Sprague

- Dawley rats at approximately 10 ppm (12.3 mg/m 3 ) formaldehyde 6 hours/day for 24

4 days. The formaldehyde vapor was generated from formalin by a concentric jet atomizer. For 25

the second experiment, Dinsda le et al. (1993) similarly exposed rats to formaldehyde, but the gas 26

was generated by the thermal depolymerization of paraformaldehyde as was done by Dallas et al. 27

(1989). The concentration of P450 and activity of several P450 isozymes were measured in lu ng 28

microsomes (pentoxyresorufin O

- dealkylase, benzyloxyresorufin O

- dealkylase, ethoxyresorufin 29

O

- dealkylase, and 2

- aminofluorene N

- hydroxylation). ALP and γ

- glutamyl transpeptidase 30

activity were measured in BAL fluid collected from each animal. No changes were seen in BAL 31

enzyme activity or the activity of lung microsomes for the P450 substrates tested. Cytochrome 32

P450 levels were unchanged in experiment 1, where formaldehyde was generated from formalin. 33

Cytochrome P450 levels were increased in experiment 2 with formaldehyde generated from 34

paraformaldehyde (Table 4

- 27). 35

36This document is a draft for review purposes only and does not consti tute Agency policy.

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- 160 Table 4

- 27. Cytochrome P450 levels in formaldehyde

- treated rats 1

2

Group Experiment 1

(formalin) a Experiment 2

(paraformaldehyde) a

(nmol/mg protein)

Control 82 ± 30 85 ± 5

10 ppm formaldehyde 73 ± 27 125 ± 23 b

3

a Values are means ± SDs (n = 3

– 5). 4

b Different from controls, p < 0.05. 5

6

Source: Dinsdale et al. (19 93). 7

8

4.2.1.2.2.6. Extrapulmonary toxicity: subchronic studies.

Hematological parameters were unaffected by formaldehyde treatment. The lung tiss ue 22

of all species exhibited interstitial inflammation after 90 days of formaldehyde exposure 23

(detailed description not provided). Formaldehyde

- treated rats and guinea pigs also had focal 24

chronic inflammation in heart and kidney tissue sections. However, the authors were uncertain 25

whether the observed changes to heart and kidney were due to formaldehyde exposure. 26 Several studies have investigated

9

toxicity in organs other than those associated with the respiratory tract. An earlier cross

- species 10

study examined changes in lung tissue resulting from co ntinuous exposure (Coon et al., 1970). 11

Animals were exposed to 3.7 ppm (4.6 mg/m 3 ) formaldehyde for 90 days. Five species of 12

animals were studied: male and female Sprague

- Dawley and Long

- Evans derived rats (15), male 13

and female Princeton

- derived guinea p igs (15), male New Zealand albino rabbits (3), male 14

squirrel monkeys ( Saimiri sciureus ) (3), and purebred male beagle dogs (2). Blood samples were 15

taken for Hb concentration, HCT, leukocyte counts, and serum levels of BUN, AST, ALT, ALP, 16

and LDH. Section s of heart, lung, liver, kidney, and spleen were fixed and examined from each 17

species (details of method not provided). Brain, spinal cord, and adrenal tissue also were 18

examined in monkeys and dogs as well as thyroid from dogs. Liver and kidney sections were 19

stained for reduced nicotinamide adenine dinucleotide, lactate, isocitrate, and β

- hydroxybutyrate. 20

Tissue sections of the nasal mucosa were not examined in this study. 21

As mentioned above, Woutersen et al. (1987) exposed male and female albino SPF 27

Wistar rats (10/group) to 0, 1, 10, or 20 ppm (0, 1.23, 12.3, or 2 4.6 mg/m 3 ) formaldehyde 28

6 hours/day, 5 days/week for 13 weeks. Rats were checked daily and weighed weekly. During 29

week 13, blood samples were taken for Hb, PCV, RBC count, and a differential count of 30

leukocytes. Urine samples were also analyzed. At sac rifice, blood samples were analyzed for 31

ALB, creatinine, glucose, TP, BUN, and the enzyme activities (AST, ALT, and ALP). GSH and 32

protein content were determined in liver homogenates. Organs were examined and weighed: 33

adrenals, brain, heart, kidneys, liv er, lungs, ovaries, pituitary, spleen, testes, thymus, and thyroid. 34This document is a draft for review purposes only and does not consti tute Agency policy.

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- 161 No gross pathological changes were seen upon autopsy, but body weights decreased in 1

both male and female rats at the 20 ppm treatment level. Of the organs weighed, 6 of 11 had 2

significant ly increased relative rates in male rats exposed to 20 ppm formaldehyde. Relative 3

brain weight was increased in female rats at the same treatment level (Woutersen et al., 1987). 4

Clinical chemistry parameters of liver and kidney function and hematological parameters 5

were also measured after the 13

- week treatment by Woutersen et al. (1987). Compared with 6

those of controls, activities of AST, ALT, and ALP were significantly elevated in plasma from 7

the 20 ppm treated male rats (by 124, 132, and 126%, respecti vely; p < 0.05). Total plasma 8

protein was reduced to 95% of controls in the same animals. Although there was an observed 9

increase in BUN in male rats treated with 1 ppm, this was not considered a treatment effect. 10

Furthermore, no statistically significa nt differences were seen for these parameters in female rats 11

at any concentration level (Woutersen et al., 1987). 12

Sul et al. (2007) exposed Sprague

- Dawley rats to 0, 5, and 10 ppm formaldehyde for 13

6 hours/day (5 days/week) for 2 weeks and collected lung samples for tissue damage and 14

genomic analysis. According to their results, 21 genes were altered in a dose

- dependent manner 15

by microarray analysis; 2 were up regulated and 19 were down regulated in the lung tissue of 16

animals exposed to formaldehyde. Ho wever, six of the nine genes further analyzed by PCR did 17

not show dose dependency (authors did not comment). Although the authors briefly describe the 18

functions and potential implications for changes in the expression of some of the altered genes, 19

there i s no discussion of the relationship between these altered genes (i.e., there is no pathway 20

analysis). 21

In 2006, Im et al. (2006) published a proteomic analysis using the same exposure 22

protocols (possibly using the same animals as in the Sul et al. [2007] s tudy, although neither 23

study makes reference to the other). Im et al. (2006) examined DNA damage in lymphocytes 24

and liver tissues, as well as protein and lipid oxidation in plasma and liver samples. Similar to 25

changes reported in the lung (discussed else where), using two

- dimensional electrophoresis and 26

matrix

- assisted laser desorption ionization time

- of

- flight mass spectrometry, the authors also 27

reported dose

- dependent changes in the levels of 32 proteins in plasma (19 up, 13 down). None 28

of the changes i n plasma proteins correspond to the changes in lung reported by Sul et al. (2007). 29

Again, no pathway analysis was provided. Interestingly, Im and colleagues (2006) also 30

demonstrated a dose

- dependent increase in plasma IL

- 4 and dose

- dependent decrease in IFNγ, 31

perhaps indicative of Th

- 2

- mediated inflammatory response. An overview of formaldehyde 32

exposure

- related pathology in the respiratory system of laboratory animals is presented in 33

Table 4

- 28. 34This document is a draft for reviewpurposes only and does not constituteAgency policy4-162 Table 4

- 28 . Summary of respiratory tract pathology from i nhalation exposures to formaldehyde, subchronic studies

Species/strain No./

group Treatment a Respiratory effects LOAEL/NOAEL Reference

Nasal pathology

B6C3F1 mice

(male and

female) 10 0, 2, 4, 10, 20, or 40 ppm

formaldehyde 6 hours/day,

5 days/week for 13 weeks Minimal squamous metaplasia in 1 of 10 mice

(4 ppm). Squamous metaplasia observed in all mice

at 10 and 20 ppm. NOAEL = 4 ppm Maronpot et al.

(1986)

SPF Wistar Rats

(male and

female) 10 0, 1, 10, or 20 ppm

formaldehyde 6 hours/day, 5

days/week for 13 weeks Increased respiratory epithelial hyperplasia and

keratinization at 20 ppm; squamous metaplasia at

10 ppm in males and females. NOAEL = 1 ppm Woutersen et al.

(1987)

SPR Wister rats

(male) 50

– 55 0, 10, or 20 ppm formaldehyde

for 6 hours/day, 5 days/week

for 4, 8, or 13 weeks Rhinitis, hyperplasia, and squamous metaplasia in

respiratory epithelium at all doses (number of weeks

not specified).

Squamous metaplasia of olfactory epithelium at

20 ppm (number of weeks not specified) NOAEL = 1 ppm Fer on et al. (1988)

Wistar rats (male

and female) 50 0, 0.3, 1, or 3.0 ppm

formaldehyde 6 hours/day,

5 days/week for 13 weeks Keratinized and non

- keratinized squamous

metaplasia in level 2 epithelium in 37/50 male and

21/50 female rats at 3 ppm for 13 weeks. NOAEL = 1 ppm Zwart et al. (1988)

Wistar rats (male) 10 40 ppm

- hours (8 hours at

5 ppm, 4 hours at 10 ppm) or

80 ppm hours (10 ppm

continuous or 20 ppm

intermittently) Thinning and disarrangement of respiratory

epithelium, squamous metaplasia, most sever e in

20 hours intermittent exposure NOAEL = 10 ppm Wilmer et al.

(1987)

Wistar rats (male) 10 0, 8, or 16 ppm, given either

continuously or intermittently Disarrangement and squamous metaplasia at 4 ppm.

Continuous exposure yielded less severe lesions

th an intermittent exposure LOAEL = 8 ppm Wilmer et al.

(1989)

F344 rats (male

and female),

Syrian golden

hamsters (male

and female),

cynomolgus

monkeys 20 rats,

10

hamsters,

6 monkeys 0.0.2, 1, or 3 ppm

22 hours/day, 7 days/week,

26 weeks Rats: rhinitis at 3 ppm, increased incidence of nasal

lesions at 3 ppm.

Monkeys: rhinitis at all doses, increased incidence

of nasal lesions at 3 ppm.

Hamsters: no significant nasal lesions. NOAEL = 1 ppm Rusch et al.

(1983a, b)

Tracheal and lung pathology

Wistar rats (m ale) 6 0, 5, 15 ppm for 8 hours/day,

5 days/week, 4 or 13 weeks Significant decreases in zinc content in lung, copper

unchanged, iron increased in lung. LOAEL = 5 ppm Özen et al. (2003)This document is a draft for reviewpurposes only and does not constituteAgency policy4-163 Table 4

- 28 . Summary of respiratory tract pathology from i nhalation exposures to formaldehyde, subchronic studies

Species/strain No./

group Treatment a Respiratory effects LOAEL/NOAEL Reference

Sprague

- Dawley

rats (male) 6 but n = 5

in some

trials 0, 0.5, 3.0, 15 ppm 6

hours/day, 5 days/week, for 1

day, 4 days, 12 weeks, 24

weeks Increased P450 levels after 4 days at 3 ppm. NOAEL = 0.5 ppm Dallas et al.

(1989)

Sprague

- Dawley

rats (male) 5 0 or 10 ppm 6 hours/day,

4 days using both formalin and

paraformaldehyde P4 50 levels increased at 10 ppm only in groups

treated with paraformaldehyde. LOAEL = 10 ppm Dinsdale et al.

(1993)

Extrapulmonary effects

Rats and guinea

pigs 15 3.7 ppm for 90 days Focal chronic inflammation in heart and kidney

tissue. LOAEL = 3.7 ppm Co on et al. (1970)

SPF Wistar rats

(male and

female) 10 0, 1, 10, or 20 ppm

6 hours/day, 5 days/week for

13 weeks Relative brain weight increased in female rats at

20 ppm; increased AST, ALT, ALP in plasma at

20 ppm. NOAEL = 10 ppm Woutersen et al.

(1987)This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 164 4.2.1.2.3. Chronic inhalation bioassays. The respiratory pathology observed in chronic 1

bioassays is consistent with the subchronic studies. As exposure concentration and duration of 2

exposure are increased, the pathology becomes more severe and penetrat es more deeply into the 3

respiratory tract. These effects are progressive over time. Tumors are reported in several 4

bioassays, primarily SCCs. Experimental results regarding both the severity of respiratory tract 5

pathology as well as the tumor incidence vary by species strain and experimental design. As 6

discussed above rodents experience RB, and species differences in respiratory and physiological 7

depression would result in differences in absorbed dose in the respiratory tract, given the same 8

exposure co ncentration (Chang and Barrow, 1983). Additionally, differences in the nasal 9

architecture result in species

- dependent variation of formaldehyde absorption (flux) within the 10

respiratory tract (see Section 3.4). Therefore, chronic studies are discussed by species for greater 11

clarity. 12

13

4.2.1.2.3.1. Mice.

In a comprehensive study conducted by Swenberg et al. (1980) (also reported in Kerns et 25

al. [1983]) in conjunction with Chemical Industry Institute of Toxicology (CIIT) and Battelle 26

Columbus Laboratories, male and female C57BL/6 × C3H F 1 (B6C3F 1 ) mice (approximately 27

120/sex/concentration) were exposed to 0, 2.0, 5.6, or 14.3 ppm (0, 2.45, 6.87, or 17.5 mg/m 3 ) 28

formaldehyde 6 hours/day, 5 days/week for 24 months. This exposure period was followed by 29

up to 6 months of nonexposure to evaluate recovery . Interim sacrifices were conducted at 6, 12, 30

18, 24, 27, and 30 months (due to unscheduled deaths, no male mice were sacrificed at 18 or 31

27 months). Exposure generation was accomplished by sublimation of paraformaldehyde, and 32

exposures were conducted in whole

- body chambers. Detailed sectioning and examination of the 33

nasal passages were conducted at each interim sacrifice, beginning at 12 months, and for all 34

unscheduled deaths. Gross organ pathology was noted for all animals and complete 35 Early experiments by Horton et al. (1963) subjected mice (C3H, sex 14

unspecified) to extreme formaldehyde concentrations (0, 0.05, 0.1, and 0.2 mg/L or 41

– 163 ppm) 15

in an attempt to simulate lung pathology reported in humans exposed to cigarette smoke. The 16

mice were exposed 1 hour/day, 3 days a week for up to 35 weeks. The authors did not note the 17

effects of RB or provide any information on pathology of the URT. There was a clear increase 18

in histologic ch anges in the tracheobronchial epithelium by exposure, including basal

- cell 19

hyperplasia, stratification squamous cell metaplasia and atypical metaplasia. Subsequent 20

exposures to various combinations of formaldehyde and coal tar did result in squamous cell 21

tumors. The findings of Horton et al. (1963) suggest a role for formaldehyde in lung cancer 22

under some conditions. However, the exposure design and early deaths in the treatment groups 23

severely limit the usefulness of these data in human health risk asse ssment. 24This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 165 histopathologic examination was conducted on all animals in the control and high

- exposure 1

groups. There were no differences in survival in any exposure group compared with controls. 2

Generally, poor survival in all groups of male mice was attributed to fighting and infe ctions of 3

the urogenital tract associated with group housing; 78, 77, 81, and 82 unscheduled deaths were 4

recorded before 24 months in the 0, 2.0, 5.6, and 14.3 ppm treatment groups, respectively ( 5

n = 119, 120, 120, and 119 males, respectively). After th e interim sacrifices (6 and 12 months) 6

only 17

– 22 male mice survived to the 24

- month scheduled sacrifice. Female mice had much 7

greater survival with only 30, 34, 19, and 34 unscheduled deaths prior to the 24

- month sacrifice. 8

The authors did not note the effects of RB in mice, although the RD 50 for a 10

- minute exposure 9

for male B6C3F1 mice has been reported at 4.9 ppm and 4.4 ppm (Steinhagen and Barrow, 1984; 10

Chang et al., 1981). 11

The first examination of the nasal cavities was conducted at the 12

- month interim 12

sacrifice. Inflammation in the nasal turbinates was evident in mice in the 2 and 6 ppm treatment 13

groups (14/20 and 18/20, respectively), including adenitis of the nasal lacrimal duct, lacrimal 14

duct, and vomeronasal gland. Inflammation was not pre sent in mice exposed at 15 ppm, 15

although serous rhinitis was seen in 4 of 20 animals. At 18 months, mice exposed at 2 and 16

6 ppm no longer exhibited adenitis in the nasoturbinates. Epithelial dysplasia was evident in 4 of 17

20 mice at 6 ppm exposure. Mice in the high

- exposure group had significantly greater nasal 18

pathology; epithelial dysplasia and squamous metaplasia were reported in 18/19 and 17/19 19

female mice, respectively, exposed to 15 ppm. After 24 months, squamous epithelial hyperplasia 20

of the nasol acrimal duct (29/45) and atrophy of the olfactory epithelium (18/45) were also noted 21

in animals from the high

- exposure group (male and female) (Battelle Columbus Laboratories, 22

1981). Similar pathology was reported in only a small fraction of mice exposed at 2 and 6 ppm 23

(5/48 and 11/60, respectively). 24

Three months after cessation of exposure, only nine female mice were available for 25

sacrifice, but within this small sample the data suggested recovery of nasal lesions: epithelial 26

dysplasia (4/9), squamous metaplasia (2/9), atrophy of the olfactory epithelium (1/9), and 27

squamous epithelial hyperplasia of the nasolacrimal duct (1/9) (Battelle Columbus Laboratories, 28

1981). 29

Of the 17 male mice that survived to 24 months in the 14.3 ppm exposure group, 2 had 30

S CC in the nasal cavity ( p < 0.05). Of the two tumor

- bearing mice, one exhibited significant 31

epithelial pathology, including rhinitis, dysplasia, squamous metaplasia, and hyperplasia. 32

Squamous metaplasia of the nasolacrimal duct was the only related patho logy reported for the 33

second mouse. No SCCs were found in female mice, although 48 mice survived to 24 months. 34

The authors reported no other formaldehyde

- related tumors. However, comparisons were based 35This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 166 on summary tables by organ. Although lymphomas wer e analyzed by organ and site (e.g., 1

increase in salivary gland lymphoma considered separately from mandibular lymphoma), later 2

reanalysis of lymphoma in female mice, based on tumor

- bearing animals (TBAs), does indicate 3

an association with formaldehyde expo sure. 4

5

4.2.1.2.3.2. Rats.

There were no differences in mortality among the groups at any time during the study 15

period. Rats exposed to formaldehyde were reported to have exhibited yellow discoloration of 16

the fur, and many displayed eye i rritation. Formaldehyde exposure, with and without wood dust, 17

induced squamous metaplasia, keratinization, and dysplasia of the nasal epithelium (Table 4

- 29). 18 Holmström et al. (1989a) evaluated co

- exposure of inhaled formaldehyde

6

with wood dust in 16 female Sprague

- Dawley rats/group. Rats were exposed in whole

- body 7

chambers for 6 hours/day, 5 days/week for 104 weeks to formaldehyde alone at 12.4 ± 1.1 ppm 8

(15.21 ± 1.35 mg/m 3 ), wood dust alone (25 mg/m 3 ), or both wood dust (25 mg/m 3 ) and 9

formaldehyde (12.7 ± 1.0 ppm) or to room air as the control. The wood dust was generated from 10

grinding of beech. Microscopic measurements of the w ood particles indicated that 11

approximately 70% had a geometric diameter of about 10 μm, while 10

– 20% were about 5 μm or 12

less. Animals were sacrificed at 104 weeks and histopathology was performed on five transverse 13

sections of the nasal cavity (Figure 4

- 3 ) and the lungs (not otherwise specified). 14

19

Table 4

- 29. Histopathologic findings and severity scores in the naso

- and 20

maxilloturbinates of female Sprague

- Dawley rats exposed to inhaled 21

formaldehyde and wood dust for 104 weeks 22

23

Treatment Pronounced

squamous metaplasia Pronounced

squamous metaplasia

with

keratinization Pronounced

squamous metaplasia

with

presence of

dysplasia Sum of rats

wit h

pronounced

metaplasia

and/or

dysplasia CCSCC Histologic scores

at the level of

naso

- and

maxilloturbinates

(mean ± SD)

Formaldehyde

group (n = 16) 7 2 1 10 1 2.25 ± 1.73 a

Formaldehyde

wood dust group

(n = 15) 7 1 4 12 0 2.6 ± 1.88 a

Wood dust group

(n = 15) 0 0 0 0 0 1.86 ± 0.83 b

Control group

(n = 15) 0 0 0 0 0 1.07 ± 0.70

24

a p < 0.01. 25

b p < 0.05. 26

27

Source: Holmström et al. (1989a). 28This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 167 Among the five levels of the nasal cavity that were examined, Holmström et al. (1989a) 1

p resented findings for the naso

- and maxilloturbinates since formaldehyde

- induced tumors had 2

been associated with this level (Morgan et al., 1986a, b). The data also suggested an effect of 3

wood dust on formaldehyde

- induced nasal pathology, with a slightly higher histologic score and 4

greater incidence of dysplasia than formaldehyde exposure alone. One SCC (1/16) occurred in 5

the group exposed to formaldehyde only but not in the group exposed to formaldehyde and wood 6

dust. Microscopic examination of the lung s revealed that emphysema (diagnostic criteria not 7

specified) was more prevalent in both groups exposed to wood dust compared with the control 8

group ( p < 0.05). There was no significant difference in pulmonary epithelial histopathology 9

among the groups. 10

T obe et al. (1985) also evaluated F344 rats (32/group) exposed to inhaled formaldehyde 11

for 28 months. Exposures were for 6 hours/day, 5 days/week to formaldehyde concentrations of 12

0, 0.3, 2, and 14 ppm (0, 0.37, 2.45, and 17.2 mg/m 3 ). Fourteen of 32 rats (44%) in the high 13

concentration group developed nasal SCCs, compared with none in the other exposed groups and 14

the control group. Tobe et al. (1985) reported increased rhinitis, hyperplasia, and squamous 15

metaplasia of the nasal respiratory epithelium, inc luding in the low

- exposure group (0.3 ppm.) 16

However, some level of rhinitis, hyperplasia, and metaplasia were also present in controls. 17

Without a more complete report, it is unknown whether or not the pathology reported at 0.3 ppm 18

was a formaldehyde

- rela ted effect. 19

Kamata et al. (1997) evaluated the effects of inhaled formaldehyde in male F344 20

(F344/DuCrj) rats (32/group) exposed for 28 months. Formaldehyde exposure was generated by 21

metering 37% formalin (containing 10% methanol) into a sprayer in a glas s bottle and diluting 22

with room air. Concentration in the chamber was monitored twice daily by the acetyl acetone 23

method. Exposures were for 6 hours/day, 5 days/week at nominal formaldehyde concentrations 24

of 0, 0.3, 2.0, and 15 ppm (0, 0.37, 2.45, and 18 .4 mg/m 3 ). Actual levels were 0, 0.3 ± 0.07, 2.17 25

± 0.32, and 14.85 ± 2.22 ppm (mean ± SD). Rats in the 0 ppm group were given methanol to 26

inhale at the same concentration (4.2 ppm) as the 15 ppm group. A room control no

- exposure 27

group was also included in the study. All animals were observed for clinical signs once a day 28

during the study. Body weights and food consumption were recorded weekly. Five animals per 29

group, randomly selected at the end of 12, 18, and 24 months, and all surviving animals at 30

28 months were sacrificed for hematological measurements (Hb, RBCs, PCV, MCV, mean 31

corpuscular hemoglobin [MCH], MCHC, and WBCs), biochemical determinations (TP, ALB, 32

BUN, ALP, AST, ALT, glucose, albumin/globulin ratio, phospholipids, triglycerides, and to tal 33

cholesterol), and pathological examinations. Wet weights were taken on brain, heart, lungs, 34

liver, kidneys, spleen, testes, and adrenal gland of each rat. Histopathology was performed on all 35This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 168 moribund or dead animals and those at specified sacrifices on all gross lesions and the following 1

tissues: pituitary, thyroid, nasal cavity, trachea, esophagus, stomach, small and large intestines, 2

prostate gland, urinary bladder, muscle, femur, sciatic nerve, spinal cord, and mesenteric lymph 3

nodes. Histopatholo gic sections of the nose were obtained from five anatomical levels, but these 4

did not correspond to the typical levels taken in other bioassays. Most notably, section level B 5

was anterior and not posterior to the incisor teeth. The incidence data for nas al histopathology 6

were not reported with respect to section level location, with the exception that the 7

nonproliferative lesions and tumors reported were described to occur predominantly at levels B 8

and C. 9

Yellow discoloration of the coats occurred in anim als exposed at the 2 and 15 ppm levels. 10

Significant decreases in body weight and food consumption were observed in the high 11

concentration (15 ppm) group throughout the exposure period, and elevated mortality was noted 12

at 28 months (88.3 versus 31.8% in co ntrols). The first death occurred after 6 versus 18 months 13

in the control group. Other effects noted in the 15 ppm exposure group include decreased 14

triglycerides, reduced liver weight (both relative and absolute), and increased relative adrenal 15

weights. 16

Treatment

- related macroscopic and histopathologic findings were limited to the nasal 17

cavity. Squamous cell metaplasia was reported in all treatment groups: 16% (0.3 ppm), 37.5% 18

(2 ppm), and 91% (15 ppm) of exposed rats. Epithelial hyperplasia was simila rly present in 12.5, 19

22, and 91% of the animals, respectively. Since a no

- effect level could not be determined, the 20

authors reported benchmark doses (BMDs) of 0.25 and 0.24 ppm for squamous cell metaplasia 21

and epithelial hyperplasia (10% response.). Addi tional lesions only occurring in the 15 ppm 22

dose group were papillary hyperplasia (2/32), SCC (13/32), squamous cell papilloma (3/32), and 23

sarcoma (1/32). The majority of the tumors were located at levels B and C of the nasal cavity. 24

Albert et al. (1982) and Sellakumar et al. (1985) reported on a set of lifetime studies 25

performed in male Sprague

- Dawley rats to evaluate the effects of inhaled formaldehyde alone 26

and in combination with hydrochloric acid (HCl). Rats were exposed 6 hours/day, 5 days/week 27

for life. In the first experiment (Albert et al., 1982), 8

- week

- old male inbred Sprague

- Dawley 28

rats (n = 99) were exposed to a mixture of 10 ppm (12.3 mg/m 3 ) HCl and 14 ppm (17.2 mg/m 3 ) 29

formaldehyde, and there were two control groups: air

- sham and untreated ( n = 50). 30

Bis(chloromethyl)ether (BCME), a known animal carcinogen (Albert et al., 1975; Kuschner et 31

al., 1975; Figueroa et al., 1973; Laskin et al., 1971), is formed when formaldehyde and HCl are 32

mixed. BCME concentrations were estimated at about 1 ppb in the formaldehyde

- HCl mixed 33

exposures, based on levels in the mixing chamber. Complete necropsies were conducted when 34

animals died naturally or were killed when moribund. Histologic sections were taken from the 35This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 169 nasal cavity, larynx, trachea, pulmonary lobes, liver, bladder, kidney, spleen, and other organs 1

with gross pathologic alterations. 2

Exposure to the mixed gases (formaldehyde

- HCl

- BCME) induced nasal lesions, 3

including epithelial hyperplasia (71%), squamous metaplasia (64%), squamous papilloma (3%)

, 4

and SCC (25%) (Albert et al., 1982). Although a few squamous metaplasia were noted in the 5

larynx, trachea, and bronchi, these lesions were also noted in controls. Mortality in exposed rats 6

was significantly increased over controls and was approximately 30% when the first carcinoma 7

was reported (233 days). Mortality in exposed rats rose quickly to approximately 60% after the 8

first year of exposure. Therefore, the authors used a life

- table method to calculate a mortality

9

corrected cumulative incidence, reporting a corrected cumulative incidence of 77% at 720 days 10

after first exposure. 11

In the second experiment performed in this laboratory (Sellakumar et al., 1985; Albert et 12

al., 1982), Sprague

- Dawley rats were similarly exposed to HCl (10 ppm) alone, fo rmaldehyde 13

alone (15 ppm), or a combination of both. The combination exposure was generated in two 14

different ways to better understand the influence of BCME formation on study results: premixed 15

at high concentrations and gases fed separately into the inle t air supply at the target 16

concentrations. BCME concentration measured by a gas chromatography/mass spectrometry 17

method in the premixed chamber varied between 0.1 and 0.4 ppb. Cage

- side observations and 18

necropsy procedures were as described in Albert et al. (1982) with the exception of the histologic 19

preparation of the head. The head was cut transversely into four tissue blocks, and sections were 20

taken from the face of each. 21

Animals exposed to formaldehyde alone and formaldehyde

- HCl (premixed or non

22

prem ixed) showed a marked decrease in body weight after 16 weeks. After 32 weeks rats 23

exposed to the premixed formaldehyde

- HCl (with BCME) had higher mortality compared with 24

the other mixed gas exposures ( p < 0.05). Nasal pathology was similar among rats exp osed to 25

formaldehyde alone or the mixed gases (Table 4

- 30). Desquamation of respiratory epithelial 26

cells was reported in the respiratory epithelium that covers the nasomaxillary turbinates and the 27

nasal septum (approximately section levels 2 and 3). Olfa ctory epithelium in the ET frequently 28

showed an inflammatory reaction with seropurulent exudate filling the lumen. Squamous 29

metaplasia and hyperplasia were reported in the larynx and trachea in all treatment groups. 30

Tumors arose primarily from the nasomax illary turbinates and nasal septum. The SCCs 31

were predominantly moderate to well differentiated, with excessive amounts of keratin occluding 32

the lumen, killing the animals by asphyxiation. Statistical comparisons by the log rank test (Peto 33

test) showed t hat tumor incidence was increased in the premixed formaldehyde

- HCl combined 34

exposure group over formaldehyde alone or the combined formaldehyde

- HCl (not premixed). 35This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 170 There were no significant differences in the latency among groups, with the average latency 1

varying from 603 to 645 days. Rats exposed to HCl exposure alone did not develop tumors. 2

The esthesioneuroepithelioma is a unique tumor type observed with a high incidence in 3

an earlier inhalation study of rats exposed to BCME (Kuschner et al., 1975), su ggesting that the 4

higher incidence of nasal tumors observed in the premixed

- combined formaldehyde

- HCl

5

exposure group may have been due to BCME (Krimsky, 1986) since this premixed protocol was 6

the one most likely to generate BCME. Sellakumar et al. (1985) refuted this assertion, stating 7

that this singular tumor occurred in the absence of other changes in the ethmoid region or in the 8

lungs where BCME was also demonstrated to cause tumors. Furthermore, exposure was 9

approximately one

- tenth the cumulative dose in the Kuschner et al. (1975) study that was 10

associated with a single similar tumor. Sellakumar et al. (1985) attributed the higher incidence 11

in the premixed

- combination group to traces of other alkylating agents (not BCME) that could 12

have been formed. The results demonstrate that animals exposed to either a combination of 13

formaldehyde

- HCl or to formaldehyde alone develop nasal tumors, principally SCCs, at about 14

the same frequency, indicating that HCl plays little or no role in the carcinogenicity of inh aled 15

formaldehyde. 16

17

Table 4

- 30. Histopathologic changes (including tumors) in nasal cavities of 18

male Sprague

- Dawley rats exposed to inhaled formaldehyde or HCl alone 19

and in combination for a lifetime 20

21

Observation Premixed

HCl

- HCHO Non

- premixed

HCl

- HCHO HCHO HCl Air Colony

Number of animals examined 100 100 100 99 99 99

Rhinitis 74 75 74 81 72 70

Epithelial or squamous hyperplasia 54 53 57 62 51 45

Squamous metaplasia 64 68 60 9 5 6

Polyp or papilloma 13 11 10 0 0 0

SCC 45 27 38 0 0 0

Adenocarci noma 1 2 0 0 0 0

Mixed carcinoma 0 0 1 0 0 0

Fibrosarcoma 1 0 1 0 0 0

Esthesioneuroepithelioma 1 0 0 0 0 0

Larynx

Hyperplasia 11 22 21 22 2 2

Squamous metaplasia 10 15 4 0 0 0

Trachea

Hyperplasia 18 32 21 26 6 2

Squamous metaplasia 9 8 7 0 0 0

22

Source: Sellakumar et al. (1985). 23

24

25This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 171 In a companion study to the chronic mouse study described above (Kerns et al., 1983; 1

Swenberg et al., 1980), groups of F344 rats (approximately 120/sex/concentration) were exposed 2

to 0, 2.0, 5.6, or 14.3 ppm (0, 2.45, 6.87, or 17.5 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week 3

for 24 months. This exposure period was followed by up to 6 months of nonexposure to evaluate 4

recovery. Interim sacrifices were conducted at 6, 12, 18, 24, 27, and 30 months. Study 5

pa rameters and methods were as described above. 6

Formaldehyde exposure increased mortality of both male and female rats in all treatment 7

groups ( p < 0.05 for 6 and 15 ppm groups). Severe treatment

- related mortality was seen at the 8

highest exposure group begi nning at 12 months with only 30% surviving to the 24 months. 9

There were no alterations in clinical chemistry, neurofunctional, or ophthalmological 10

measurements considered to be related to formaldehyde exposure. A concentration

- dependent 11

increase in yello w discoloration of the hair coat was observed. This discoloration dissipated over 12

the 3

- month postexposure period. Rats in the highest

- concentration group were dyspneic 13

( p < 0.01) and emaciated ( p < 0.05) and had many facial swellings that on closer exam ination 14

were revealed to be carcinomas protruding through the nasal cavity. Neoplastic lesions in the 15

URT were first observed clinically at day 358 in females and day 432 in males. 16

Macroscopically, these lesions originated in the anterior portion of the nasal cavity and, in a few 17

instances, extended into the ETs. 18

Figure 4

- 9 shows the frequency of squamous metaplasia by location in the noses of rats 19

sacrificed at various time points along the 2

- year exposure period. Histopathologic lesions were 20

confined t o the nasal cavity and proximal trachea in concentration

- dependent fashion. The 21

morphologic diagnosis of squamous metaplasia was used to designate zones of altered 22

epithelium that were characterized by the presence of a well

- differentiated germinal layer 23

(stratum germinativum) and superficial layers of epithelium (stratum spinosum and stratum 24

corneum). Keratin was produced only in areas of squamous metaplasia. Epithelial dysplasia was 25

detected earlier than squamous metaplasia and was characterized by a m ucosa that had 26

undergone a transition from nonciliated simple cuboidal to one that was several cells thick and 27

squamoid with an organization and polarity of the individual cells that had changed from vertical 28

to horizontal with respect to the basement memb rane. Similar histomorphologic changes have 29

also been called basal cell hyperplasia and epidermoid metaplasia (e.g., Albert et al. [1982]). 30

Figure 4

- 9 clearly illustrates that concentration is a dominant determinant of lesion distribution. 31

At low concen trations the lesions occur only in the most anterior region (cross

- section level 1). 32

At 5.6 ppm, the squamous metaplasia in levels 1, 2, and 3 was also associated with purulent 33

rhinitis and epithelial dysplasia. At the highest concentration, the lesions progress to the more 34

distal URT, with lesions evident in level 5 and no difference in the incidence at level 1 or 2 35This document is a draft for review purposes only and doe s not constitute Agency policy.

DRAFT

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- 17 2 across the various sacrifice times. Statistically significant ( p < 0.05) regression of the lesion was 1

evident at most locations at the 27

- m onth sacrifice (3 months postexposure) (e.g., level 1 in the 2

2 ppm group, all levels of the 5.6 ppm group, and levels 4 and 5 of the 14.3 ppm group). 3

4

6

1 2

1 8

2 4

2 7 M o n t h s

M o n t h s

M o n t h s

M o n t h s

M o n t h s 1 0 0

8 0

6 0

4 0

2 0

1 0 0

8 0

6 0

4 0

2 0

1 0 0

8 0

6 0

4 0

2 0

L o c a t i o n o f s q u a m o u s m e t a p l a s i a i n n a s a l c a v i t y L e v e l I L e v e l I I L e v e l I I I L e v e l I V L e v e l V 

## A

B

C

5

Figure 4

- 9. Frequency and location by cross

- section level of squamous 6

metaplasia in the nasal cavity of F344 rats exposed to formaldehyde via 7

inhalation. 8

9

Note: Exposure concentrations were 2.0 ppm (A), 5.6 ppm (B), or 14.3 ppm (C). 10

Nasal cavity levels 2, 3, 4, and 5 were not evaluated at the 6

- and 12

- month 11

interim sacrifices in the 14.3 ppm exposure g roup. 12

13

Source: Redrawn from Kerns et al. (1983). 14

15This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 173 Furthermore, progression of lesions distally to the lower respiratory tract (LRT) occurred 1

only in the high concentration group. Tracheal pathology observed at 18 months included 2

multifocal areas of minim al to mild epithelial hyperplasia, epithelial dysplasia, or squamous 3

metaplasia. There were no significant tracheal lesions present in the 0, 2.0, or 5.6 ppm exposure 4

groups, and tracheal lesions were not observed during the postexposure period in the 14. 3 ppm 5

exposure group. 6

Table 4

- 31 provides the summary data of all neoplastic lesions in the nasal cavity of 7

exposed rats. The adjusted cumulative incidence rates of SCC in male and female rats from the 8

14.3 ppm exposure group at 24 months were 67 and 87 %, respectively. In this group, the 9

formation of zones of squamous metaplasia with zones of squamous epithelial hyperplasia and 10

increased keratin production appeared to precede areas of squamous papillary hyperplasia with 11

foci of cellular atypia. More ad vanced lesions included carcinoma in situ and invasive SCC of 12

the nasal turbinates. The neoplasia were extremely osteolytic and were associated with excessive 13

keratin production and mild to severe purulent rhinitis. In many animals from the high

- exposure 14

group (with or without carcinoma), the excessive accumulation of keratin and inflammatory 15

exudates within the lumen of the URT caused severe dyspnea and death. Polypoid adenomas 16

were also observed in eight rats (four/sex) from the low

- exposure group, six male rats from the 17

intermediate

- exposure group, and six rats (five males, one female) from the high

- exposure group 18

in level 1, 2, or 3. One control male rat had a similar lesion. When adjusted and unadjusted data 19

were analyzed, no significant difference s were observed in pair

- wise analyses; however, a 20

significant adjusted trend ( p < 0.05) was reported for male rats. There was no evidence of 21

progression from polypoid adenoma to SCC. 22

23

Table 4

- 31. Summary of neoplastic lesions in the nasal cavity of F344 rats 24

exposed to inhaled formaldehyde for 2 years 25

26

Formaldehyde

(ppm) Sex No. of nasal

cavities

evaluated SCC Nasal

carcinoma Undifferentiated

carcinoma or

sarcoma Carcino

sarcoma Polypoid

adenoma Osteo

chondroma

0 M 118 0 0 0 0 1 1

F 114 0 0 0 0 0 0

2.0 M 118 0 0 0 0 4 0

F 118 0 0 0 0 4 0

5.6 M 119 1 0 0 0 6 0

F 116 1 0 0 0 0 0

14.3 M 117 51 1 a 2 a 1 4 0

F 115 52 1 0 0 1 0

27

a One rat in this group also had an SCC. 28

29

Source: Kerns et al. (1983). 30

31This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 174 Morgan et al. (1986b) performed an additional an alysis of the slides and tissues 1

from the Kerns et al. (1983) study to more precisely determine the location of each tumor 2

recorded. Additional sections were cut from the existing tissue blocks if a full slide set 3

(i.e., five sections) was unavailable for each animal. For each animal, the location of 4

each tumor was recorded on diagrams of the cross section of the nose, and an attempt to 5

determine the site of origin was made based on the center of the tumor mass. The results 6

for each case were assigned an accuracy rating that was based on the degree of 7

confidence that the pathologist had in the designated site of origin. Results for SCCs are 8

shown in Table 4

- 32. 9

10

Table 4

- 32. Apparent sites of origin for the SCCs in the nasal cavity of F344 11

rats exposed t o 14.3 ppm of formaldehyde gas in the Kerns et al. (1983) 12

bioassay 13

14

Sex Accuracy

rating Number of

animals Total SCC (%) a

Area 1 b Area 2 b Area 3 b Area 4 b Unable to

determine

Male High

Low 36

25 56

56 28

20 14

8 3

0 NA

16

Female High

Low 45

15 62

47 27

33 7

13 4

0 NA

7

Totals 121 57 26 10 3 4

15

a Rounded to nearest whole number. 16

b Area 1 = lateral aspect of the nasoturbinate and adjacent lateral wall; Area 2 = midventral septum; Area 3 = dorsal 17

septum and roof of dorsal meatus; Area 4 = dorsal and l ateral aspect of the maxilloturbinate. 18

NA = not applicable. 19

20

Source: Morgan et al. (1986b). 21

22

23

In the 14.3 ppm exposure group, 98/103 rat noses had adequate numbers and quality of 24

slides for mapping the SCC distribution. Single neoplasia were present in 8 0 (40/sex), while 25

multiple neoplasia were present in 9 males (21 neoplasia) and 9 females (20 neoplasia). The 26

results were similar for cases with high or low accuracy. For example, more than half (57%) of 27

the SCCs occurred on the lateral side of the naso turbinate and adjacent lateral wall at the front of 28

the nose (levels 1 and 2); approximately 25% were located on the midventral nasal septum 29

(levels 2 and 3); and about 10% were on the dorsal septum and roof of the dorsal meatus 30

(levels 1, 2, and 3). A sm all number (3%) were found on the maxilloturbinate (levels 2 and 3), 31

which only involved the medial aspect. All other regions of the nose where SCC was found were 32

considered to be involved as a result of invasion from one or more of the above sites. Ther e 33This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 175 were two tumors in the 5.6 ppm group: one male had a single neoplasm on the ventral nasal 1

septum (level 3) while a female had an SCC from the lateral aspect of the maxilloturbinate to the 2

adjacent lateral wall (level 2). 3

On the basis of the morphology of 19 small neoplasia in this study and in additional work 4

described below (Morgan, 1997; Monticello et al., 1996), it was further concluded that the SCCs 5

arose from the epithelium lining the airway and not from the underlying glandular epithelium. 6

This m apping procedure and that of Monticello et al. (1996) described below were in good 7

concordance and showed a clear site specificity; most of the SCC arose in the anterior lateral 8

meatus (ALM) (57%), which is lined by transitional epithelium, and the midvent ral nasal septum 9

(26%), which is lined by respiratory epithelium (Morgan, 1997). 10

The CIIT performed a second bioassay on inhaled formaldehyde in 9

- week

- old male 11

F344 (CDF[F344]/CrlBr) rats (Monticello et al., 1996). The rats were exposed 6 hours/day, 5 12

days/week for 24 months to 0, 0.7, 2, 6, 10, and 15 ppm (0, 0.86, 2.45, 7.36, 12.3, and 13

18.4 mg/m 3 ) formaldehyde. Study objectives were to repeat the Kerns et al. (1983) bioassay, 14

better defining the concentration response relationship and to seek a corre lation between 15

localized data on tumor sites and concomitant cell proliferation assays. Histopathology was 16

performed on six cross

- section levels of the nasal cavity on every animal of an unscheduled 17

death and all those of the terminal sacrifice after 24 m onths. The distribution of lesions for each 18

individual animal was recorded onto epithelial maps of the nasal cavity at 30 selected levels 19

designed to permit accurate localization (Mery et al., 1994). Cell proliferation was measured in a 20

subset of animals (five per treatment group) at 3, 6, 18, and 24 months of exposure in each of the 21

nasal regions to which tumors were mapped (Table 4

- 33). 22

23

Table 4

- 33. Incidence and location of nasal squamous cell carcinoma in male 24

F344 rats exposed to inhaled formaldehyd e for 2 years 25

26

Formaldehyde

concentration

(ppm) No. of

nasal

cavities

examined Nasal location

No. of

animals

with

SCC a Anterior

lateral

meatus Posterior

lateral

meatus Anterior

mid

septum Posterior

mid

septum Anterior

dorsal

septum Anterior

medial

maxillo

turbinate Maxillary

sinus

0 90 0 0 0 0 0 0 0 0

0.7 90 0 0 0 0 0 0 0 0

2 96 0 0 0 0 0 0 0 0

6 90 1 0 0 0 0 0 0 1

10 90 12 2 0 0 0 0 0 20

15 147 17 9 8 1 3 4 0 69

27

a Total number of animals with SCCs, including those too large to allocate a nd those located in a site not listed in this 28

table. 29

Source: Monticello et al. (1996). 30This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 176 Yellow discoloration of the fur, a consistent response to formaldehyde in rats, was 1

observed in the rats exposed to 10 and 15 ppm formaldehyde. There were numerous pre mature 2

deaths in the 15 ppm exposure group, resulting in significantly decreased survival relative to 3

controls (18.8 versus 35.7%; p < 0.001). Survival was higher in the three lowest exposure 4

groups and statistically comparable to controls in the 10 ppm e xposure group (35.7 versus 5

31.3%, respectively). 6

Control animals showed no histopathologic evidence of disease in the nasal passages. 7

Buccal cavity SCC, not associated with the nasal cavity, was present in 2 of 90 control animals. 8

This was considered a n incidental finding and within the spontaneous incidence range reported 9

for this strain of rat. Buccal SCCs were observed in three animals at 15 ppm and in one animal 10

at 2 ppm. All other neoplastic responses in the respiratory tract were confined to the nose and 11

considered to have originated from the epithelium lining the nasal airways. The nasal neoplasia 12

included SCCs and polypoid (transitional) adenomas and were similar in morphologic 13

characteristics to those described in the Kerns et al. (1983) chro nic bioassay. The incidence of 14

nasal SCCs by location is summarized in Table 4

- 33, which demonstrates a clear concentration

15

response relationship. No SCCs occurred in the two lowest exposure groups or in the controls. 16

One nasal rhabdomyosarcoma and two nasal adenocarcinomas were reported in animals in the 17

highest treatment groups. 18

Regional analysis indicated that the SCCs arose in nasal regions lined with transitional or 19

respiratory epithelium and were most common in the lateral meatus and the midseptu m (Table 20

4

- 33). Within the lateral meatus and mid

- septum, there was clear evidence of a higher tumor 21

incidence rate in the anterior sample site ( p = 0.001 and 0.02, respectively). Smaller numbers of 22

SCCs were observed on the medial aspect of the maxillot urbinate and the dorsal septum and on 23

the posterior lateral wall and lining of the nasopharyngeal meatus (data not shown). No SCCs 24

were observed in the maxillary sinus, with the exception of one animal exposed to 15 ppm that 25

had a small tumor in the wall of the ostium of this sinus. Tumor rates across the seven nasal 26

epithelial sites are presented in Table 4

- 33. There was an increasing tumor response between the 27

10 and 15 ppm exposure groups in all sites, except in the ALM. The SCC rates at 10 and 15 pp m 28

were virtually identical (13.3 and 11.6%, respectively), which is probably attributable to the 29

occurrence of many large neoplasia in the lateral meatus site that were not suitable and not 30

counted in the analysis. 31

The nonlinear tumor response is mirrored by a highly nonlinear response in cell 32

proliferation measured after 3, 6, 12, and 18 months of exposure. Significant treatment

- induced 33

responses in cell proliferation indices at these time points were only observed at the two highest 34

exposure concentratio ns (10 and 15 ppm). Other treatment

- induced lesions, predominantly 35This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 177 epithelial hypertrophy, hyperplasia, squamous metaplasia, and mixed inflammatory cell 1

infiltrate, were also most severe at these two exposure concentrations. Significant distortion and 2

de struction of the nasoturbinate architecture occurred in many animals exposed to 15 ppm. 3

Nasal turbinate adhesions and olfactory degeneration (usually confined to the walls of the 4

anterior dorsal medial meatus) also occurred in animals exposed to 10 and 15 ppm. Lesions in 5

the 6 ppm exposure group were limited to focal squamous metaplasia in the anterior regions. 6

As discussed briefly above, small numbers of polypoid adenomas were also induced by 7

formaldehyde exposure and were similar in acinar

- like structur e and location to those in the 8

Kerns et al. (1983) bioassay. No polypoid adenomas occurred in the control animals or in the 9

0.7, 2, or 6 ppm exposure groups. A clear concentration response was observed in the 10 and 10

15 ppm exposure groups. Five of 90 an imals (5.6%) in the 10 ppm exposure group and 14 of 11

147 animals (9.5%) in the 15 ppm exposure group had a polypoid adenoma. Most of these 12

polypoid adenomas (79%) were located in or adjacent to the lateral meatus. The significance of 13

these tumors for risk assessment remains to be determined (Morgan, 1997). 14

Appelman et al. (1988) studied the effects of bilateral intranasal electrocoagulation 15

damage on susceptibility to inhaled formaldehyde in male SPF Wistar (Cpb: WU) rats. Rats 16

were exposed 6 hours/day, 5 days/week for 13 or 52 weeks to 0, 0.1, 1.0, or 10 ppm (0, 0.12, 17

1.23, or 12.3 mg/m 3 ) formaldehyde. These concentrations were chosen because the various 18

short

- term studies performed in the same laboratory (described in Section 4.2.1.2) showed that 19

formal dehyde was noncytotoxic to the nasal mucosa at levels of 0.3, 1.0, and 2.0 ppm, slightly 20

cytotoxic at 3 and 4 ppm, and strongly cytotoxic at 10 and 20 ppm (Zwart et al., 1988; Wilmer et 21

al., 1987; Woutersen et al., 1987). Furthermore, because nasal tumors have only been found at 22

exposure concentrations that also induced severe degenerative, hyperplastic, and metaplastic 23

changes in the nasal epithelium (Griesemer et al., 1985; Squire and Cameron, 1984), Feron et al. 24

(1984) and the investigators at the TNO

- C IVO Toxicology and Nutrition Institute postulated that 25

formaldehyde at a subcytotoxic concentration was only a very weak initiator without promoting 26

activity. Appelman et al. (1988) used an electrocoagulation method in this study to evaluate if 27

damage to the mucosa followed by compensatory cell proliferation might render the epithelium 28

vulnerable to subcytotoxic levels of formaldehyde. One

- half of the rats used in the study 29

(10/group) were damaged bilaterally and then subjected to the first 6

- hour exposur e to 30

formaldehyde approximately 20

– 26 hours after the electrocoagulation procedure. Ten 31

undamaged rats/group were also exposed at each concentration for either 13 or 52 weeks. 32

Histopathologic examination included six standard cross

- section levels in the nose; livers of all 33

rats killed at 14 weeks and of all control and 10 ppm exposed rats killed in week 53; larynges, 34

tracheas, and lungs of all rats of the control and 10 ppm exposed rats killed in week 53; and 35This document is a draft for review purposes only and doe s not constitute Agency policy.

DRAFT

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- 178 organs and tissues of control and 10 ppm expos ed rats with an undamaged nasal mucosa killed in 1

week 53. 2

Yellow discoloration of the fur occurred in all animals of the two highest exposure 3

groups. Growth retardation was observed in the animals killed with or without damaged noses 4

after 2 weeks of expo sure to 10 ppm formaldehyde. No toxicologically significant findings in 5

the body weights or organ weights of any animals in the other exposure groups were observed. 6

No relevant differences between groups were found in any of the hematological or urinary 7

parameters with the exception of frequent oliguria ( p < 0.05) in the top exposure group without 8

nasal coagulation and killed in week 53. Three

- way ANOVA revealed a significant increase in 9

TP content of the liver in rats with damaged noses as compared with rats with undamaged noses, 10

and there was a significant negative correlation between the formaldehyde exposure level and TP 11

in these same rats. Hepatic GSH was positively correlated with both nasal damage and age of 12

the animals. No treatment

- related gros s findings were observed in animals sacrificed at either 14 13

or 53 weeks except for yellow discoloration of the fur in rats exposed at the two highest 14

concentrations. No changes observed in the larynx, trachea, lungs, liver, or other tissues 15

evaluated were regarded as related to formaldehyde. 16

Few nasal lesions were noted in intact rats exposed at 0.1 or 1 ppm for either 13 or 52 17

weeks (n = 10/group). Focal squamous metaplasia was noted in a single animal exposed at 18

1 ppm for 13 weeks. Rats exposed at 10 p pm formaldehyde demonstrated clear pathology in the 19

respiratory epithelium progressing from 13 to 52 weeks, including squamous metaplasia, basal 20

cell hyperplasia, and focal rhinitis. Additionally, focal nest

- like infolds of the epithelium were 21

present in 4 of 10 rats at 52 weeks, and minor changes to the olfactory epithelium were noted 22

(thinning/disarrangement and focal basal cell hyperplasia.) 23

All rats with damaged nasal passages exhibited similar minor pathology of the respiratory 24

epithelium at 13 and 5 2 weeks (squamous metaplasia, focal basal cell hyperplasia, and focal 25

rhinitis). Formaldehyde

- related effects were note at 52 weeks, where the squamous metaplasia 26

of the respiratory epithelium was no longer noted in controls (versus 13 weeks) but was clea rly 27

present in all formaldehyde

- treatment groups, including progression from focal to diffuse lesions 28

(at 1 and 10 ppm) and keratinization (3/10 and 4/10 at 0.1 ppm and 10 ppm, respectively). The 29

formaldehyde effects on the respiratory epithelium were muc h more severe in rats with damaged 30

nasal passages, with all animals demonstrating thinning and disarrangement of the olfactory 31

epithelium and 8 of 10 rats exhibiting “loosely arranged submucosal tissue.” Squamous 32

metaplasia and focal rhinitis of the olfac tory epithelium were seen in less than half of the 33

formaldehyde

- treated rats with damage. No changes in the olfactory epithelium due only to 34

electrocoagulation were encountered. 35This document is a draft for review purposes only and doe s not constitute Agency policy.

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- 179 The most notable effects of nasal damage from electrocoagulation were the one s at the 1

highest formaldehyde exposure (10 ppm) on the olfactory epithelium. Damage to the respiratory 2

epithelium also occurred more posteriorly in rats with damaged noses. Since electrocoagulation 3

often induced damage that included partial or complete l oss of turbinates and septal perforation, 4

a likely explanation for the posterior distribution of the damage is an abnormal airflow pattern. 5

This gross damage to the nasal structure may have also disrupted normal mucous production and 6

flow. Therefore, for maldehyde

- induced pathology appearing deeper in the nasal passages, 7

including the respiratory epithelium, may be due to formaldehyde penetrating more deeply into 8

the nasal passages and resulting in greater tissue doses in these areas. 9

Woutersen et al. (198 9) conducted a lifetime study in parallel to the 1

- year study 10

described above for Appelman et al. (1988). Male Wistar rats with nasal damage induced by 11

electrocoagulation (60/group) or without nasal damage (30/group) were exposed 6 hours/day, 12

5 days/week to the same concentrations as in the previous study (0, 0.1, 1.0, and 10 ppm) for 13

28 months or for 3 months followed by a 25

- month observation period. The general condition 14

and behavior of the animals were checked daily. Body weight, organ weight, and gr oss 15

pathology were evaluated as described for Appelman et al. (1988). Histopathologic examination 16

was conducted on all animals at the standard six cross sections (see Figure 4

- 3). 17

No remarkable findings on behavior were observed except for yellowing of th e fur in 18

animals at the two highest concentrations. There were no relevant differences in mortality (data 19

not shown). Growth retardation was observed relative to controls in animals with or without 20

damaged noses exposed to 10 ppm from day 14 onward. Bod y weights were generally slightly 21

lower in formaldehyde

- exposed animals with an intact nasal mucosa and slightly higher in 22

exposed animals with damaged noses than in the corresponding controls. 23

The effects of formaldehyde exposure on the respiratory and ol factory epithelium after 24

28 months of exposure were similar to those reported for 52 weeks exposure (Appelman et al., 25

1988): rhinitis, squamous metaplasia with some keratinization of the respiratory epithelium, and 26

thinning/disarrangement and slight squamo us metaplasia of the olfactory epithelium at the 27

10 ppm exposure. Effects attenuated from the anterior to posterior sections (I

– II, III, IV, and V

– 28

VI). A low incidence of olfactory epithelium replaced by respiratory epithelium (<10%) and 29

vacuolation and atrophy of olfactory cells (<10%) was reported, this in part may be due to the 30

larger study size (30 rats per group versus 10). Squamous metaplasia in levels I

– II of the 31

respiratory epithelium at 10 ppm was the only treatment

- related pathology remaining i n rats 32

exposed for 3 months followed by a 25

- month recovery period. 33

Similarly, as reported by Appelman et al. (1988), rats with noses damaged by 34

electrocoagulation did demonstrate increased pathology of the respiratory epithelium. 35This document is a draft for review purposes only and doe s not constitute Agency policy.

DRAFT

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- 180 Formaldehyde exposure at 10 ppm exacerbated these changes, and effects were noted in more 1

posterior sections than in rats without nasal damage (levels III, IV, and V). Olfactory pathology 2

was also greater in formaldehyde

- treated rats: basal cell hyperplasia, replacement of olfac tory 3

epithelium by respiratory epithelium (10

– 20% at level III and <10% at level IV). Although the 4

incidences are low, there is some evidence that effects on the olfactory epithelium may be 5

increased at the lower formaldehyde exposures (0.1 and 1 ppm.) A nalysis of the number of 6

animals with olfactory pathology would be helpful to better understand the potential of low

- level 7

formaldehyde effects on these less frequent lesions. Interestingly, the recovery of the olfactory 8

and respiratory epithelium seen in rats with undamaged nasal cavities after a 25

- month recovery 9

period was not evident in rats with damaged noses. Formaldehyde

- exposure effects are only 10

present at the 10 ppm exposure for the respiratory epithelium (squamous metaplasia, basal cell 11

hyperpla sia), and the formaldehyde

- related effects on the olfactory epithelium 12

(thinning/disarrangement, basal cell hyperplasia, and replacement by respiratory epithelium) are 13

seen at 0.1 and 1.0 ppm as well. 14

A single SCC, 1 out of 30 rats, was found in each 28

- mo nth formaldehyde

- treatment 15

group (1/26, 1/28, and 1/26, respectively) but not in any control animals (n = 52). SCCs were 16

also noted in rats with noses damaged by electrocoagulation (1/54, 1/58, 0/56, and 15/58 for 17

control rats and the formaldehyde

- treatme nt groups, respectively). These data clearly indicate a 18

synergistic effect of high formaldehyde exposure and nasal damage on the formation of SCCs in 19

rats. One adenosquamous carcinoma and one adenocarcinoma were also reported as increasing 20

the frequency to 17/58 for all tumors. Additionally SCC was present in two rats in the 0.1 and 21

1 ppm 3

- month exposure groups with damaged noses only, although only one SCC was reported 22

in the 10 ppm 3

- month groups with and without damaged noses. Rats not surviving to 23

28 months are included in these results, as well as the histopathology reported above. Since no 24

mortality data are reported, it should be noted that the incidence of both nasal lesions and tumors 25

are not controlled for early deaths. 26

In total, 30 tumors we re examined from this study. In general, the tumors (26/30 or 87%) 27

were SCCs, and 69% (18/26) of these clearly originated from the respiratory epithelium lining 28

the septum or nasal turbinates. The eight other SCCs, derived from the epithelium lining the 29

nasolacrimal duct, were seen in connection with severe odontodystrophy and periodontitis or 30

might have originated from the skin or salivary glands. Four remaining rats bearing a nasal 31

tumor developed a small polypoid adenoma located on the nasoturbinate, an adenocarcinoma 32

originating from the olfactory epithelium, an adenosquamous carcinoma of the respiratory 33

epithelium lining the septum or turbinates, or a carcinoma in situ of epithelium in the 34

nasolacrimal duct. 35This document is a draft for review purposes only and doe s not constitute Agency policy.

DRAFT

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- 181 4.2.1.2.3.3. Hamsters

The second set of experiments by Dalbey (1982) ex amined interaction of formaldehyde 12

exposure on tumor formation from DEN administered subcutaneously. The five treatment 13

groups included: (1) controls (n = 50); (2) formaldehyde only (n = 50); (3) DEN 0.5 mg, once 14

per week for 10 weeks (n = 100); (4) forma ldehyde exposure for life with DEN injection for the 15

first 10 weeks given 48 hours after formaldehyde exposure (n = 27); and (5) DEN injection for 16

10 weeks, followed by formaldehyde exposure for life (n = 23). In all groups hamsters were 17

exposed at 30 ppm formaldehyde 5 hours/day, once a week. Histopathologic examinations were 18

conducted as above. 19 . Dalbey (1982) e xamined the effects of inhaled formaldehyde alone for a

1

lifetime or combined with diethylnitrosamine (DEN) in an initiation

- promotion study design 2

using male Syrian golden hamsters. For the first experiment, hamsters were exposed at either 0 3

or 10 ppm (0 or 12.3 mg/m 3 ) formaldehyde in whole body chambers 5 hours/day, 5 days/week 4

for a lifetime (132 controls, 88 exposed). Histopathologic evaluations were carried out on two 5

transverse sections of the nasal turbinates (otherwise not specified), longitudinal sections of 6

larynx and trachea, and all lung lobes cut along the bronchus prior to embedding. In the 7

formaldehyde

- only (10 ppm) experiment, mortality was increased relative to unexposed controls 8

( p < 0.05). No tumors and little evidence of toxicity to th e nasal epithelium were observed. 9

There was no increase in rhinitis. Epithelial hyperplasia and metaplasia were increased in 10

formaldehyde

- treated animals (5% incidence) versus none observed in controls. 11

Although weekly exposures to formaldehyde alone (30 ppm once a week) did not 20

influence mortality, treatment with DEN alone significantly ( p < 0.05) increased mor tality above 21

that of untreated controls, and mortality was further elevated ( p < 0.05) in the two groups 22

exposed to both DEN and formaldehyde compared with DEN alone. No respiratory tract tumors 23

were observed in untreated animals or those receiving only f ormaldehyde. DEN treatment alone 24

resulted in a high incidence (77%) of tumors (nasal, larynx, trachea, and lung). Formaldehyde 25

pre

- or posttreatment did not further increase the number of TBAs

- . All tumors observed were 26

classified as adenomas. Formalde hyde pretreatment nearly doubled the number of tumors per 27

animal in the trachea (but not lung or larynx) ( p < 0.05). This increase in tumors initiated by 28

DEN given 48 hours after formaldehyde exposure suggests a role of formaldehyde

- induced 29

changes in th e respiratory tract in tumor promotion (e.g., cell proliferation and inflammation). 30

31

4.2.1.2.3.4. Summary

. Chronic rodent studies of inhalation exposure to formaldehyde provide a 32

consistent picture of the agent’s toxicity

— especially on the URT

— on which m ost studies focus. 33

All three species tested

— hamsters, mice, and rats

— had some degree of hyperplastic and 34

metaplastic change in the nasal passages. The pathology defined in acute and subchronic 35This document is a draft for review purposes only and doe s not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 182 exposures is similarly described in chronic studies, where pr ogression, severity, and presence in 1

more posterior sections of the nose increase with both the concentration and duration of 2

exposure. 3

Pathology of the respiratory epithelium includes rhinitis, goblet cell hyperplasia, 4

pseudoepithelial cell hyperplasia , squamous metaplasia, and dysplasia (see Table 4

- 34). At 5

higher exposures and longer durations of exposure, similar effects are seen on the olfactory 6

epithelium, present further into the nasal passages. In addition to hyperplasia and squamous 7

metaplasia , thinning and disarrangement of the olfactory epithelium noted and, in a few cases, 8

cell damage and replacement of olfactory epithelium with respiratory epithelium appear, 9

including loss of sensory cells (Woutersen et al., 1989; Kerns et al., 1983; Battel le Columbus 10

Laboratories, 1981). 11

Clear species differences in the severity of lesions are present. Although the bioassays in 12

mice, hamsters, and rats do represent similar exposure concentrations and duration of exposure, 13

hamsters exhibit little pathology and rats (three strains tested) exhibit gross toxicity and even 14

increased mortality. Mice similarly exposed exhibit a range of effects on the respiratory 15

epithelium but not near the severity seen in rats. Many factors may contribute to these observed 16

sp ecies differences. As Chang and Barrow (1983) reported, the increased RB of mice seems to 17

be protective of POE damage in comparison to that of rats. The reduced ventilation rate and 18

minute volume of rodents in the presence of a reactive gas can reduce th e effective delivered 19

dose at the same exposure concentration (Chang and Barrow, 1983). Additionally, as illustrated 20

in the computational fluid dynamic (CFD) modeling (see Section 3.5), there are species 21

differences in nasal architecture that influence ar eas of formaldehyde absorption or flux into the 22

tissue. Localized differences in mucus flow and production as well as metabolic enzymes have 23

also been posited as having roles in differential toxicity of formaldehyde on the URT (see 24

Chapter 3). 25

Formaldehy de

- induced tumors were present in exposed rats and mice and primarily 26

involved SCCs later in life (Kamata et al., 1997; Tobe et al., 1985; Kerns et al., 1983; Swenberg 27

et al., 1980). Although exposure of male Syrian hamsters to either 10 or 30 ppm did not result in 28

formaldehyde

- induced nasal tumors, a classic initiation

- promotion assay with DEN

- induced 29

tumor formation did indicate that formaldehyde increased the tumor burden per animal, where 30

DEN induced tumors in 77% of the animals (Dalbey, 1982). This s tudy suggests a role for 31

promotion in the observed carcinogenicity of formaldehyde. Less clear are the implications of 32

the synergistic effect of formaldehyde exposures and gross damage to the respiratory epithelium 33

by electrocoagulation on tumor formation (Woutersen et al., 1989). 34

35This document is a draft for reviewpurposes only and does not constituteAgency policy4-183 Table 4

- 34. Summary of respiratory tract pathology from chronic inhalation exposures to formaldehyde

Species/strain No./

group Treatment a Respiratory effects Noncancer

LOAEL/NOAEL Reference

Chronic bioassays

C3H mice (sex

unstated) Mice

60 0, 41, 82, or 163 ppm

1 hour/day, 3 days/week for up

to 35 weeks.

Low

- and mid

- group mice then

exposed at either 122 or

244 ppm during weeks 35

– 70. Pathology: Histologic changes in the tracheobronchial

epithelium by exposure, includin g basal

- cell

hyperplasia, stratification squamous cell metaplasia, and

atypical metaplasia

.

Carcinogenicity: No SCC formation was evident in

mice exposed to formaldehyde alone. LOAEL = 41 ppm

No evidence of

carcinogenicity Horton et al. (1963)

Mal e and female

B6C3F1 mice 120/sex 0, 2, 5.6, or 14.3 ppm

6 hours/day, 5 days/week for 24

months.

The protocol featured a 6

- month

recovery period. Interim

sacrifices occurred at 6, 12, 18,

24, and 30 months. Pathology: Rhinitis; hyperplasia, dysplasia, and

squamous metaplasia of the nasal epithelium; atrophy of

the olfactory epithelium; glandular adenitis and

nasolacrimal duct hyperplasia and metaplasia.

Carcinogenicity: Nasal SCC in male mice at 24 months

(2/17). No SCC in female mice. LOAEL = 2 ppm

Evidence of

carcinogenicity Swenberg et al.

(1980); Kerns et al.

(1983); CIIT

(1982) ; Battelle

Columbus

Laboratories (1981)

Female Sprague

Dawley rats Rats

16 0 or 12.4 ppm

formaldehyde ± wood dust

6 hours/day, 5 days/week for

104 weeks. Patholog y: Squamous metaplasia and dysplasia.

Carcinogenicity: One of 16 rats exposed to

formaldehyde alone developed SCCs. LOAEL = 12.4 ppm

Support for

carcinogenicity Holmström et al.

(1989a)

Male and female

F344 rats 32/sex 0, 0.3, 2, or 14 ppm

6 hours/ day, 5 days/week for

28 months. Pathology: I ncreased rhinitis, hyperplasia, and

squamous metaplasia of the nasal respiratory epithelium

Carcinogenicity: Nasal SCCs in high concentration

rats (44%). LOAEL = 0.3 ppm

Support for

carcinogenicity Tobe et al. (1985)

Male F344 rats 32 0, 0.3, 2, or 15 ppm

6 hours/day, 5 days/week for

28 months. Pathology: Squamous cell metaplasia and epithelial

hyperplasia.

Carcinogenicity: SCC (13/32), squamous cell

papilloma (3/32), and sarcoma (1/32). LOAEL = 0.3 p pm

BMD 10 = 0.24 ppm

Evidence of

carcinogenicity Kamata et al. (1997)This document is a draft for reviewpurposes only and does not constituteAgency policy4-184 Table 4

- 34. Summary of respiratory tract pathology from chronic inhalation exposures to formaldehyde

Species/strain No./

group Treatment a Respiratory effects Noncancer

LOAEL/NOAEL Reference

Male Sprague

Dawley rats 100 0 or 15 ppm 6 hours/day,

5 days/week for life. Pathology: Squamous metaplasia, epithelial

hyperplasia, and polyps/papillomas.

Carcinogenicity: SCCs for med in the nasomaxillary

turbinates and nasal septum (25%). LOAEL = 15 ppm

Evidence of

carcinogenicity Albert et al. (1982);

Sellakumar et al.

(1985)

Male and female

F344 rats 120/sex 0, 2, 5.6, or 14.3 ppm

6 hours/day, 5 days/week for

24 months. Th e protocol

featured a 6

- month recovery

period. Interim sacrifices

occurred at 6, 12, 18, 24, and

30 months. Pathology: Lesions of the nasal cavity were the

primary effects, including squamous metaplasia and

epithelial dysplasia, hyperkeratosis, goblet ce ll

hyperplasia, and rhinitis.

Salivary gland: atrophy, squamous metaplasia, and

sialadenitis.

Carcinogenicity: SCCs were evident in the nasal

cavity of high concentration rats, plus some polypoid

adenomas. LOAEL = 2 ppm

Evidence of

carcinogenicity Swenberg et al.

(1980); Kerns et al.

(1983); CIIT (1982);

Battelle Columbus

Laboratories (1981);

Morgan et al.

(1986b)

Male F344 rats 90 and 150

controls 0, 0.7, 2, 6, 10, or 15 ppm

6 hours/day, 5 days/week for

24 months. Pathology: Olfactory degenera tion, squamous

metaplasia, epithelial hypertrophy and hyperplasia, and

mixed inflammatory cell infiltrate.

Carcinogenicity: SCCs and polypoid adenomas in the

nasal cavity LOAEL = 2 ppm

Evidence of

carcinogenicity Monticello et al.

(1996)

Male SPF Wi star

rats 10 0, 0.1, 1, or 10 ppm

6 hours/day, 5 days/week for 13

or 52 weeks.

An electrocoagulation method

was applied to damage the

noses of 1⁄2 of each study group. Pathology: Formaldehyde

- induced focal changes to the

respiratory and olfactory epithel ium, including rhinitis,

hyperplasia, and metaplasia (10 ppm).

In rats with damaged noses: squamous metaplasia of the

respiratory epithelium increased at all formaldehyde

exposures. Pathology of the olfactory epithelium

increased at the 10 ppm exposure.

Carcinogenicity: No tumors noted; 1

- year study LOAEL = 0.1 ppm

in rats with damaged

nasal passages

NOAEL = 1 ppm for

rats with intact noses Appelman et al.

(1988)This document is a draft for reviewpurposes only and does not constituteAgency policy4-185 Table 4

- 34. Summary of respiratory tract pathology from chronic inhalation exposures to formaldehyde

Species/strain No./

group Treatment a Respiratory effects Noncancer

LOAEL/NOAEL Reference

Male Wistar rats 30 (without

nasal

damage),

60 (with

nasal

damage) 0, 0.1, 1, and 10 pp m 6 hours/day, 5 days/week for 28

months or for 3 months with a

25

- month observation period.

An electrocoagulation method

was applied to damage the nasal

cavity. Pathology: Intact noses: squamous metaplasia in the

high concentration group exposed for 28 months and

degeneration of the olfactory epithelium.

Changes were more severe in animals with damaged

noses .

Carcinogenicity: SCCs developed in 15/60 rats with

damaged noses exposed at 10 ppm. In other groups, the

incidence of nasal tumors was low irrespective of the

state of nasal damage. NOAEL = 1 ppm

Evidence of

carcinogenicity Woutersen et al.

(1989)

Male Syrian

golden hamsters Hamsters

88 treated

132

controls. 0 or 10 ppm formaldehyde

5 hours/day, 5 days/week for

life. Pathology: Inc reased mortality. Epithelial hyperplasia

and metaplasia increased in formaldehyde

- treated

animals (5% incidence)

Carcinogenicity: No tumors reported. LOAEL = 10 ppm

No evidence of

carcinogenicity Dalbey (1982)

Male Syrian

golden hamsters 50 0 or 30 p pm 5 hours/day, 1 day/week for life

± injections with 0.5 mg DEN. Pathology: Increased mortality in conjunction with

DEN

— above DEN

- only treated animals. Respiratory

pathology not reported.

Carcinogenicity: Only hamsters receiving DEN

developed tumors (77%, adenomas). There was an

increase in the number of tumors per TBAs in the

trachea of animals exposed to formaldehyde 48 hours

prior to DEN (but no increase in TBAs). LOAEL = 30 ppm.

Evidence for

formaldehyde as a

promoter Dalbey (1982)This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 186 4.2.1.2.4. Summary of respiratory pathology.

Progression of lesions can be viewed a s progression from the anterior to posterior 12

sections of the nasal cavity or as a progression in severity of lesions at a particular location (e.g., 13

level or region) of the nasal passages. In both cases, progression is evident with increasing 14

exposure con centration and with increasing duration of exposure ( Kamata et al., 1997; 15

Monticello et al., 1996; Morgan et al., 1986b; Takahashi et al., 1986; Sellakumar et al., 1985; 16

Kerns et al., 1983; Albert et al., 1982 ). The data suggest that concentration and dur ation of 17

exposure do not act in a simply cumulative manner (e.g., C × t). Additionally the influence of 18

concentration, duration, and repeated exposure may be different for various effects. For 19

example, some lesions may be transient (e.g., low

- exposure ce ll proliferation), others may have a 20

threshold and vary little after that (e.g., rhinitis). Additionally, as the nasal epithelium responds 21

with both adaptive and adverse epithelial changes, the absorption of formaldehyde into the tissue 22

at that location m ay be reduced. As respiratory epithelium transitions to squamous metaplasia, 23

the effective tissue dose of formaldehyde increases posterior to these lesions. As barriers to 24

formaldehyde flux into the tissue develop (e.g., squamous metaplasia, keratinizati on), 25

formaldehyde penetrates more deeply into the nasal passages (Kimbell et al., 2006). Therefore, 26

although both concentration and duration of exposure do effect the adverse effect, the 27

relationship is difficult to define and in fact may be different for various adverse effects. 28 The progressive pathology of the nasal passages

1

from inhalation exposure to formaldehyde is well documented, especially in rodents (rats and 2

mice) (see Tables 4

- 9, 4

- 15, 4

- 28, 4

- 34). Although there are specie s differences in tissue dose 3

(Section 3.4) due to variations in nasal architecture and breathing patterns, the nature and 4

progression of the pathology is fairly well conserved across species, including nonhuman 5

primates. The observed formaldehyde

- induced pathology includes disruption of the mucociliary 6

apparatus, rhinitis ( s erous and purolent), hyperplasia (cell proliferation), metaplasia (transition of 7

cell type), dysplasia (disarrangement of cells), nest

- like infolds and invaginations of the 8

epithelium, thinning of the epithelial layer and focal to diffuse lesions, atrophy of the olfactory 9

epithelium, thickening and keratinization (usually of squamous metaplasia), tumors (adenoma, 10

sarcoma, carcinoma) (Section 4.2.2). 11

Respiratory histopathology has been commonly reported in response to exposure to 29

formaldehyde in rats and mice (Lino dos Santos Franco et al., 2006; Javden and Taher, 2000; 30

Kamata et al., 1996a, b; Cassee and Feron, 1994; Bhalla e t al., 1991; Monteiro

- Riviere and Popp, 31

1986; Buckley et al., 1984; Chang et al., 1983), rabbits (Ionescu et al., 1978), hamsters 32

(Schreibner et al., 1979), and rhesus monkeys (Monticello et al., 1989). The histopathologic 33

lesions ranged from inflammation to ulceration, necrosis, and metaplasia that occurred in nasal 34

turbinates, maxilloturbinates, and goblet and microvillus cells (Bhalla et al., 1991). These effects 35This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 187 were observed at a variety of doses (e.g., 10 ppm for 4 hours, 3.13 ppm for 6 hours for 1, 2, or 1

4 days, 6 or 15 ppm). Wilmer et al. (1989, 1987) assessed whether a dose and time

- dependent 2

interaction (C × t) is associated with histopathologic lesions. Results indicated that 3

concentration, rather than duration or cumulative exposure, correlat es best with severity of 4

lesions (Wilmer et al., 1989, 1987). 5

Histopathologic lesions and changes to biochemistry have been reported in the lung as 6

well, though these effects were observed following a high dose of formaldehyde. In addition, 7

changes in cli nical chemistry, P450 expression and activity in lung tissue, and gene expression 8

that is phenotypically anchored to the observed respiratory pathology have been reported. 9

Extrapulmonary effects have also been noted, including changes in liver chemistry, relative brain 10

weight, and focal, chronic inflammation in the heart and kidney. Most of these changes occurred 11

at exposures of 20 ppm, and those that occurred at lower formaldehyde exposures (3.7 ppm) 12

could not be strictly correlated with formaldehyde exp osure. 13

Some researchers have reported formaldehyde

- induced effects in the pulmonary region in 14

rats, mice, and rabbits. Kamata et al. (1996a) observed reduced lipid content of pulmonary 15

surfactant in rats exposed to 128.4 or 294.5 ppm formaldehyde. Kamata et al. (1996b) reported 16

biochemical changes in lung homogenates and altered lipid content of BAL at 145.6 ppm 17

formaldehyde. Lino dos Santos Franco et al. (2006) observed increased leukocytes (and 18

neutrophils) and degranulated mast cells recovered in BAL fluid (concentration of 1% formalin 19

not provided). In rabbits, Ionescu et al. (1978) observed frank necrosis of lung parenchyma after 20

aerosol inhalation of 3% formalin for 3 hours/day for 50 days (concentration of formaldehyde 21

not provided). These pulmon ary effects may be due to frank toxicity resulting from the high 22

dose of formaldehyde used in these studies. 23

Several recent toxicogenomics studies have assessed gene expression changes in nasal 24

and lung tissue in animals and in humans by using in vivo and in vitro approaches. Hester et al. 25

(2005, 2003) documented changes in gene expression associated with DNA repair and apoptosis 26

in nasal tissue from male rats after a single instillation of formaldehyde. Other gene expression 27

changes were observed in thos e genes related to xenobiotic metabolism and in cell cycle and 28

repair. These preliminary results provide an initial basis for forming a phenotypically anchored 29

set of gene expression changes associated with exposure to formaldehyde and may assist in 30

deter mining the underlying MOA, as will be discussed in Section 4.5. Sul et al. (2007) 31

investigated gene expression genes in lung tissue from formaldehyde

- exposed rats. Yang et al. 32

(2005) performed a proteomics analysis by using lung tissue extracted from for maldehyde

33

exposed rats. Two studies used human tracheal cell lines to investigate formaldehyde

- induced 34

gene expression changes in vitro (Lee et al., 2008, 2007). However, the relevance of these 35This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 188 findings to actual exposures remains unknown. In total, tox icogenomics studies hold promise, 1

but they must be interpreted with caution until results can be replicated and phenotypically 2

linked to observable changes. 3

Thus, formaldehyde

- induced respiratory pathology has been commonly described in the 4

nasal passages and includes cellular proliferation, mucociliary function, and histopathologic 5

lesions. Pulmonary effects have been documented as well but at high doses. The nasal 6

pathology may occur as a result of both concentration and duration components of exposure. 7

8

4.2.1.2.5. Cell proliferation. Formaldehyde

- induced cell proliferation has been demonstrated 9

under range of exposure conditions in vivo and in vitro as well (Chapter 3). Formaldehyde

10

induced mitogenesis may be a primary effect (as demonstrated in the in vitro work) or secondary 11

to adaptive responses and tissue remodeling (Swenberg et al., 1983). This section provides a 12

comprehensive discussion of formaldehyde effects on cell proliferation in the epithelial tissues in 13

the respiratory tract. The majori ty of the work discussed investigates cell proliferation with 14

in vivo labeling of proliferating cells, although additional methods, such as flow

- cytometry, have 15

been employed in some instances. 16

Swenberg et al. (1986) conducted a series of experiments in rodents to assess cell 17

proliferation in the nasal mucosa after formaldehyde inhalation. Radiolabeled thymidine 18

[ 3 H]

- thymidine was injected intraperitoneally (I.P.) into male F344 rats and B6C3F1 mice after 19

formaldehyde exposure to assess the extent of in vivo incorporation into proliferating cells. Two 20

hours later, animals were sacrificed and the nasal passages were fixed, embedded, and sectioned 21

to examine the nasal mucosa. Slides were exposed for 12 weeks and developed to identify cells 22

that incorporat ed the radiolabeled thymidine. The percentage of labeled cells, as indicated by the 23

presence of five or more grains over the nucleus, was determined by visual count. A total of 24

4,000 or 1,500 cells were counted per section for rats and mice, respectively

. 25

The first set of studies reported by Swenberg et al. (1986) compared the dose response of 26

rats and mice. Animals were exposed to 0, 0.5, 2, 6, or 15 ppm (0, 0.61, 2.45, 7.36, or 27

18.4 mg/m 3 ) formaldehyde 6 hours/day for 3 days. Tritiated thymidine for c ell labeling was 28

injected 2 hours after the end of exposure. No change in the percentage of labeled cells was seen 29

after 0.5 or 2 ppm formaldehyde exposure. However, the nasal passages of rats exposed at 6 and 30

15 ppm showed 10

- to 20

- fold increases over controls in LI at level 2. A similar cell 31

proliferation response was seen in mice treated with 15 ppm formaldehyde, although no increase 32

over control was seen in mice exposed to 6 ppm formaldehyde. These findings are consistent 33

with other data that indic ate rats are more sensitive to formaldehyde exposure than mice. This 34

may be due to differences in the reflex apneic response between the two species. As discussed in 35This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 189 Section 4.2.1.1, mice maintain decreases in minute volume in response to formaldehyde, w hich 1

results in a lower overall effective internal dose to the mice. 2

Comparing cell proliferation rates after 2 versus 18 hours of exposure, Swenberg et al. 3

(1986) found that the longer exposure duration gave twice the cell proliferation rates after 4

repe ated exposures. Therefore, these researchers conducted a second dose

- response study to 5

examine cell proliferation 18 hours after exposure instead of the shorter exposure duration. The 6

dose

- response study varied dose as well as duration of treatment. Rat s were exposed 6 hours/day 7

to either 0.5, 2, or 6 ppm (0.61, 2.45, or 7.36 mg/m 3 ) formaldehyde over periods of 1, 3, or 9 8

days. Formaldehyde exposure at 0.5, 2, or 6 ppm for 1 day increased cell proliferation in the 9

nasal epithelium. However, these incre ases were transient, and cell proliferation was not 10

increased after 3 or 9 days of exposure to 0.5 ppm or 2 ppm formaldehyde. Although still 11

elevated after a 3

- day exposure to 6 ppm formaldehyde, cell proliferation returned to control 12

values after 9 days of exposure to 6 ppm formaldehyde (Swenberg et al., 1986). Therefore, 13

although concentration is a major determinant of cell proliferation, duration of exposure also 14

influenced formaldehyde

- induced cell proliferation in the nasal epithelium. 15

Swenberg et al . (1986) directly tested the effects of cumulative exposure versus 16

concentration for both mice and rats. Animals were treated with one of three regimens, resulting 17

in the same C × t product: 3 ppm × 12 hours, 6 ppm × 6 hours, or 12 ppm × 3 hours, each 18

ex posure resulting in 36 ppm

- hours. The animals were exposed once a day for either 3 or 9 days. 19

Tritiated thymidine was injected 18 hours after exposure to label of proliferating cells. Tissue 20

sections from levels 1 and 2 of the nasal passages were examin ed in each case, and the 21

percentage of cells labeled was reported as the percentage of proliferating cells (Figure 4

- 10). 22

Cell proliferation at level 1 in the nasal cavity was much greater than at level 2 for all C × 23

t combinations of formaldehyde exposure in both mice and rats (Figure 4

- 10). The authors noted 24

that level 1 is more anterior and lacks significant defense from the mucociliary apparatus, which 25

may account for the observed greater sensitivity to formaldehyde. At all C × t exposure 26

products, 3 days of exposure resulted in greater cell proliferation than 9 days of exposure. This 27

was true for both species and for both examined levels of the nasal cavity. The decrease in cell 28

proliferation by day 9 is consistent with data on rats labeled 18 hours postexposure (Swenberg et 29

al., 1986). 30

31

32This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 190 1

3 0

2 0

1 0

0

M I C E R A T S M I C E R A T S 1 0

5

0

C O N T R O L 3 x 1 2 6 x 6 1 2 x 3

1 5

1 0

5

0

M O U S E R A T 3

2

1

0

M I C E R A T S ( a ) ( b )

( d ) ( c ) 2

3

Figure 4

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

respiratory mucosa of rats and mice. 5

6

Note: a and b are data following 3 days of exposure; c and d are for 9 days of 7

exposure. a and c are from level 1 (most anterior); b and d are from level 2. 8

[ 3 H]

- thymidine was administered 18 hours after the last exposure. 9

10

Source: Swenberg et al. (1986). 11

12

13

When comparing C × t exposures for a single species and location, the findings are more 14

compl ex. Cell proliferation in level 2 of the nasal passages appeared to be more dependent on 15

concentration than on duration or cumulative exposure, with the strongest response seen for 16

12 ppm formaldehyde in combination with the shortest exposure period, 3 ho urs (Figure 4

- 10). 17

This pattern was observed in both rats and mice after 3 days of exposure and in rats after 9 days 18

of exposure. No increases in cell proliferation at level 2 were seen for any C × t combination in 19

mice after 9 days. In contrast, increa ses in cell proliferation at level 1 of the nasal passages were 20

not strictly concentration dependent. After a 3

- day exposure, no clear differences were seen 21

among different C × t treatments for either mice or rats, suggesting cumulative exposure may be 22This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 191 th e important metric. Therefore, it may be concluded that cell proliferation for level 1 of the 1

nasal passages, where there is less protection of the epithelium, is influenced by concentration, 2

time, and duration of exposure. Cell proliferation at level 2 appeared to be more dependent on 3

concentration than time of exposure (Swenberg et al., 1986). 4

Cassee and Feron (1994) reported a qualitative increase in histochemical staining for 5

proliferating cell nuclear antigen (PCNA) in the respiratory epithelium of t he nasoturbinates, 6

maxilloturbinates, septum, and lateral wall at levels 2 and 3 of rat nasal passages after repeated 7

exposures to 3.5 ppm (4.29 mg/m 3 ) formaldehyde 22 hours/day for 3 days. While no increases 8

were seen in olfactory epithelium, frank necro sis, squamous metaplasia, and hyperplasia of both 9

ciliated and nonciliated epithelium were noted at these section levels. 10

Quantitative cell proliferation studies have been conducted by several researchers in the 11

same laboratory (Reuzel et al., 1990; Wilmer et al., 1989; Zwart et al., 1988; Wilmer et al., 1987; 12

Woutersen et al., 1987) (Summary Table 4

- 39). These studies build off of those of Swenberg et 13

al. (1986), who labeled proliferating cells with [ 3 H]

- thymidine in assessing cell proliferation 14

within th e nasal mucosa. The studies, all performed in male albino Wistar rats and using a 15

similar experimental design, provide the basis for comparing different exposure levels and dose 16

regimens across studies. Wilmer et al. (1987) demonstrate a concentration

- de pendent increase in 17

cell proliferation after 3 days of repeated 8

- hour exposures at 5, 10, or 20 ppm (6.13, 12.3, or 18

24.6 mg/m 3 ) formaldehyde, regardless of continuous versus interrupted exposure conditions 19

(2.83, 8.87, and 19.8 versus 0.86% proliferation in controls). Similar trends were seen when the 20

repeated continuous exposures were extended for 4 weeks, but cell proliferation was not 21

maintained at the same levels. As observed by Swenberg et al. (1986), these results suggest that 22

duration of repeated exposures may be an important determinant of cell proliferation rates. 23

Woutersen et al. (1987) reported that the majority of the dose

- dependent increases in cell 24

proliferation seen at section level 3 after 3 days of repeated 6

- hour exposures to 10 and 20 p pm 25

(12.3 and 24.6 mg/m 3 ) formaldehyde occurred in areas of the epithelium showing “clear 26

squamous metaplasia and hyperplasia.” Cell proliferation rates in metaplastic epithelium of 27

29.5 and 33.2% were much higher than the 1.4 to 2.8% proliferation in the visibly unaffected 28

respiratory epithelium from rats exposed at 10 ppm formaldehyde. Although there was a slight 29

trend towards increased cell proliferation in the visibly unaffected epithelium of exposed animals 30

compared with unexposed controls, the majori ty of increased cell proliferation resulting from 31

exposure to 10 and 20 ppm formaldehyde was attributed to the metaplastic epithelium. 32

Similarly, dose

- dependent increases in cell proliferation seen at level 3 after 3 days of 33

repeated 6

- hour exposures at 0. 3, 1, and 3 ppm (0.37, 1.23, and 3.68 mg/m 3 ) formaldehyde 34

( p < 0.001) corresponded to focal basal cell hyperplasia and loss of cilia (Woutersen et al., 1987). 35This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 192 No necrosis or focal erosion was noted at these levels of formaldehyde exposure. Cell 1

prolifera tion was not sustained at this location, and no lesions were noted after 13 weeks of 2

repeated 6

- hour exposures. The authors hypothesized that defensive mechanisms, such as the 3

mucociliary apparatus, may have provided greater protection of the mucosa at le vel 3. Swenberg 4

et al. (1986) drew a similar conclusion when evaluating extended exposures, suggesting that 5

more posterior sections had a greater adaptive ability than those anterior sections with little 6

mucociliary function. Both Woutersen et al. (1987) and Swenberg et al. (1986) reported 7

sustained cell proliferation and development of lesions in the more anterior cross section. 8

Repeated exposures to 3 ppm formaldehyde (6 hours/day) resulted in significant increases in cell 9

proliferation in the epitheli al cells at level 2, with accompanying disarrangement, focal 10

hyperplasia, and squamous metaplasia (Woutersen et al., 1987). Although no cell death was 11

observed at level 2 when viewed by light microscopy, “strongly indented and disarranged nuclei” 12

were see n by electron microscopy, which may be consistent with apoptosis (Woutersen et al., 13

1987). However, later work in the same laboratory indicated no increased cell proliferation at 14

levels 2 or 3 in male Wistar rats exposed to formaldehyde at 1 or 2 ppm (1.2 3 and 2.45 mg/m 3 ) 15

(8

- hour repeated exposures for 3 days or 13 weeks) and only minimal response in rats exposed at 16

4 ppm formaldehyde (interrupted 8

- hour exposures for 3 days or 13 weeks) (Wilmer et al., 17

1989). 18

Reuzel et al. (1990) published the only report in which formaldehyde effects on cell 19

proliferation were studied for longer daily exposure durations: 22 hours/day versus 6

– 20

8 hours/day. Male Wistar rats were exposed to formaldehyde, ozone, or the combination of the 21

two 22 hours/day for 3 consecutive da ys. The concentrations of formaldehyde were 0.3, 1.0, or 22

3.0 ppm (0.37, 1.23, or 3.68 mg/m 3 ). Rats were injected with [ 3 H]

- thymidine 2 hours rather than 23

18 hours after the last exposure. Cell proliferation was quantified by enumerating the percentage 24

of labeled cells in fixed and stained tissue sections. Cell proliferation on the nasoturbinates, 25

maxilloturbinates, lateral wall, and septum at levels 2 and 3 were quantified and reported 26

separately. Cell proliferation was increased at all locations in lev el 2 at 3 ppm formaldehyde 27

exposure ( p < 0.05) but not at 0.3 or 1 ppm exposures (Summary Table 4

- 39). Whereas 28

proliferation of cells in the nasoturbinate, maxilloturbinate, and septum was nearly undetectable 29

in control animals, 4, 5, and 3% proliferation was reported after repeated 22

- hour exposures to 3 30

ppm formaldehyde. Basal proliferation in the lateral wall was greater than in other areas, 31

approximately 1% increasing to 6% after exposure to 3 ppm formaldehyde. Although basal 32

levels of cell prolifera tion were slightly higher in all areas of level 3, formaldehyde had no 33

significant effects on cell proliferation in the level 3 areas evaluated. There was a slight trend for 34

increases at 3 ppm, but all proliferation rates were below 1%. Exposure to 3 ppm formaldehyde 35This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 193 also damaged the respiratory epithelium at levels 2 and 3, where cell disarrangement and 1

hyperplastic and metaplastic lesions were reported. 2

Roemer et al. (1993) investigated the effects of formaldehyde exposure on cell 3

proliferation in the t rachea and lung in addition to nasal mucosa. Male Sprague

- Dawley rats 4

were exposed head only to 2, 6, or 20 ppm (2.45, 7.36, or 24.5 mg/m 3 ) formaldehyde 6 hours/day 5

for either 1 or 3 days. Proliferating cells were labeled with 5

- bromodeoxyuridine (BrdU), the 6

label injected 16

– 22 hours after formaldehyde exposure ended. Free lung cells were harvested 7

by tracheal lavage, and the majority of isolated cells were MPs (>97%). Epithelial cells were 8

isolated from the nasal and tracheal mucosa by dissection, phy sical disaggregation, and enzyme 9

treatment to release epithelial cells. All cells were fixed and stained with fluorescent dyes to 10

detect BrdU and total DNA. Flow cytometry was used to determine the percentage of BrdU

11

labeled cells as a measure of cell pr oliferation. Cells undergoing unscheduled DNA synthesis 12

(e.g., DNA repair) were excluded by cell cycle analysis. 13

The proportion of BrdU

- labeled cells from the nose and trachea increased two

- to 14

threefold above control values after a single 6

- hour exposu re to formaldehyde (Table 4

- 35). The 15

lowest effective dose for increased cell proliferation was 2 ppm for nose and tracheal cell 16

proliferation ( p < 0.05). However, increased proliferation in the nasal mucosa at the lowest dose 17

was transient, returning to control levels after a 3

- day exposure. Cell proliferation remained 18

increased in the nasal mucosa after exposure to 6 or 10 ppm (7.36 or 12.3 mg/m 3 ) formaldehyde 19

for 3 days. In contrast, proliferation of tracheal cells appeared to be reduced as a result of a 20

3

- day exposure to 2 or 6 ppm formaldehyde. A similar trend was seen in free lung cells, but the 21

differences were not statistically significant. 22

The flow cytometry employed by Roemer et al. (1993) allowed for subtle changes in 23

proliferation rates to be measured with good discrimination. However, the method of cell 24

isolation did not allow examination of proliferation rates in discrete regions of the mucosa, 25

which may have attenuated the magnitude of the response. Additionally, proliferation rates 26

re present a mix of cell types that were not separated in this analysis, making the findings difficult 27

to interpret. This may be especially noteworthy in the free lung cells that were reportedly 28

primarily MPs. 29

30

31This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 194 Table 4

- 35. Cell proliferation in nasal muco sa, trachea, and free lung cells 1

isolated from male Wistar rats after inhalation exposures to formaldehyde 2

3

1 Day a Control 2 ppm 6 ppm 20 ppm

Nose 1.3 b 2.4 c 3.7 c 2.7

Trachea 1.2 3.1 c 2.1 c 2.8

Lung d 1.8 2.6 3.3 3.1

3 Days Control 2 ppm 6 ppm 20 ppm

Nos e 1.3 1.4 2.5 c 2.3 c

Trachea 1.2 0.3 c 0.6 c 2.5 c

Lung 1.8 2.2 2.4 5.1

4

a Exposures were 6 hours/day. 5

b Proliferation is measured as the percent of BrdU

- labeled cells. 6

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

d The majority of free lung cells were M Ps (97%). 8

9

Source: Roemer et al. (1993). 10

11

12

Monticello et al. (1990) investigated whether changes in cell proliferation rate correlated 13

with areas of cell injury or with areas that developed tumors due to formaldehyde exposures by 14

using a unique metric of cell proliferation. They hypothesized that treatment

- related effects on 15

cell populations could influence the apparent cell proliferation measured as LI, even though no 16

proliferative effect had occurred. For example, cell death could give an apparent incr eased 17

proliferation as a LI (% cells proliferating) by reducing the total number of cells present. This 18

would be especially true for a stratified epithelium, where the number of basal cells in active 19

proliferation may not change but cells above the basal layer might die or slough off, thereby 20

reducing the overall number of population of cells counted. The unit length labeling index 21

(ULLI) metric was developed to normalize proliferation rates against length of basal membrane 22

rather than cell population. H owever, application of a ULLI to the pseudostratified epithelium of 23

the nasal mucosa introduced additional complexities. First, undamaged mucosa has a single 24

layer of epithelial cells that have the capability for cell proliferation. Second, cells only be come 25

layered in response to cell damage as a protective measure. Therefore, the total cells present and 26

the linear cell density should be considered, as well as the number and density of proliferating 27

cells, in developing an understanding of the prolifera tive response of these tissues to toxic insult. 28

Monticello et al. (1990) directly compared the apparent effects of formaldehyde exposure 29

on cell proliferation when quantified as an LI or as a ULLI. Male F344 rats were divided into 30

groups (n = 6) and expos ed to 0, 2, 6, or 15 ppm (0, 2.45, 7.36, or 18.4 mg/m 3 ) formaldehyde 31

6 hours/day, 5 days/week for 12 weeks. Rats were administered [ 3 H]

- thymidine continuously for 32

the last 5 days of exposure by surgically implanted osmotic pumps. After sacrifice, nasal 33This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 195 p assages were fixed, and sections from standard level 3 were prepared for examination. Cell 1

proliferation was quantified at the midseptum and the lateral meatus at this level. Basement 2

membrane length, total number of cells present, and number of labeled proliferating cells were 3

recorded for each location. Each of these areas also was scored for the presence of nasal lesions. 4

The formaldehyde

- related lesions included epithelial hyperplasia, squamous metaplasia, 5

and acute inflammation. These lesions were most severe in animals exposed to 15 ppm, mild at 6

6 ppm, but absent at 2 ppm. Cell proliferation, measured either as LI or ULLI, was increased in 7

the level 3 septum and lateral meatus after 13 weeks of exposure to 15 ppm formaldehyde but 8

not to 6 or 2 ppm (Table 4

- 36). There was a slight increase in both cell number and labeled cells 9

in the lateral meatus of rats exposed to 6 ppm formaldehyde, but both measures of proliferation 10

were unchanged from controls. The increased proliferation in the lateral meat us at 15 ppm was 11

entirely due to an increased number of labeled cells. Total cells were unchanged at 15 ppm; 12

therefore, both LIs demonstrated a similar increase over control. In addition to increased labeled 13

cells in the septum at 15 ppm, total cells wer e increased from 470 to 640 ( p < 0.05). Where the 14

total cells and linear cell density were increased, the ULLI was proportionally increased over the 15

LI. These observations are consistent with the development of squamous metaplasia and 16

hyperplasia seen at 15 ppm. However, while both LI and ULLI showed an eightfold increase in 17

cell proliferation in the lateral meatus, they gave different results in the septum where cell 18

number was increased by formaldehyde treatment. LI increased 19

- fold and ULLI 25

- fold with 19

repeated exposures to 15 ppm formaldehyde. Although these data are based on only 5

– 6 20

animals/group, and only in an extended study, the results suggest that the ULLI and LI may not 21

be proportional under all conditions studied. In similar experiments the LI and ULLI provided 22

different indices of proliferation in the olfactory epithelium after methyl bromide exposure 23

(Monticello et al., 1990). Methyl bromide exposure decreased cell number/mm of basement 24

membrane in a time

- dependent manner, and the LI a nd ULLI were not proportional across these 25

changes. At day 3 there was an increase in labeled cells but a decrease in total cells; therefore, 26

the LI was increased greater than 20

- fold, where the ULLI was only increased eightfold. The 27

authors endeavored t o explain why the ULLI and LI yielded different findings. Where ULLI is a 28

more time

- efficient method of assessing cell proliferation, the authors suggested that 29

representative areas should be quantified by LI to better understand the nature of increased 30

U LLI. 31

32

33This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 196 Table 4

- 36. The effect of repeated formaldehyde inhalation exposures for 1

3 months on cell count, basal membrane length, proliferation cells, and two 2

measures of cell proliferation, LI and ULLI, in male F344 rats 3

4

Formaldehyde exposure level

(6 hou rs/day, 5 days/week for 3 months)

0 ppm 2 ppm 6 ppm 15 ppm

Lateral meatus

Total cells

BM length (mm) b

Cells/mm BM

Labeled cells

LI

ULLI 1,800 ± 100

12.7 ± 0.6

150 ± 5

130 ± 10

7.2% c

10.2 cells/mm d 1,800 ± 150

11.9 ± 0.5

1 50 ± 10

130 ± 20

7.2%

10.9 cells/mm 2,300 a ± 1700

13.4 ± 0.3

170 ± 10

210 ± 30

9.1%

15.7 cells/mm 1,900 ± 160

11.6 ± 0.7

150 ± 5

1,400 ± 130

73.7%

120.7 cells/mm

Septum

Total cells

BM length (mm)

Cells/mm BM

Labeled cells

LI

ULLI 470 ± 20

2.9 ± 0.1

160 ± 10

20 ± 1

4.3%

6.9 cells/mm 460 ± 30

2.7 ± 0.1

170 ± 10

40 ± 10

8.7%

14.8 cells/mm 470 ± 20

2.9 ± 0.1

160 ± 3

10 ± 2

2.1%

3.45 cells/mm 640 a ± 20

2.9 ± 0.1

220 a ± 10

250 ± 50

39%

86.2 cells/mm

5

a Different from con trol, p < 0.05. 6

b BM is basal membrane length in mm. 7

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

d Calculated from group averages: ULLI = (labeled cells)/BM length 9

10

Source: Monticello et al. (1990). 11

12

13

Monticello et al. (1990) reported similar results in a contemporary abstract; although 14

treatment groups were slightly different than in the above experiments, the findings were similar. 15

Rats were exposed to 0, 0.7, 2, 6, 10, or 15 ppm (0, 0.86, 2.45, 7.36, 12.3, or 18.4 mg/m 3 ) 16

formaldehyd e 6 hours/day for 4 days, 6 weeks, or 3 months. ULLIs were determined in the 17

septum and lateral meatus (methods not detailed). It is not stated whether [ 3 H]

- thymidine 18

labeling was carried out by injection or continuous infusion. Significant increases in cell 19

proliferation were reported after repeated exposures to 6, 10, and 15 ppm for 4 days and 6 weeks. 20

After 3 months of exposure, cell proliferation was still increased in rats exposed to 10 and 21

15 ppm formaldehyde. The authors noted that, although inc reased cell proliferation was seen at 22

earlier time points, sustained increased cell proliferation was only seen at 10 and 15 ppm, which 23

they considered the clearly carcinogenic doses. 24

Monticello et al. (1991) applied the ULLI measurements in evaluating for maldehyde 25

effects on cell proliferation after short

- term and subchronic repeated exposures. Six male F344 26

rats/group were exposed to 0, 0.7, 2, 6, 10, or 15 ppm (0, 0.86, 2.45, 7.36, 12.3, or 18.4 mg/m 3 ) 27This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 197 formaldehyde 6 hours/day for 1, 4, or 9 days and fo r 6 weeks, using a 5 days/week regimen. 1

Rats were injected with [ 3 H]

- thymidine 18 hours postexposure to label proliferating cells. All 2

animals were sacrificed 2 hours later. Nasal passages were fixed, and sections from levels 2 and 3

3 were prepared for e xamination. Cell proliferation was quantified for three locations in level 2 4

(specifically, the lateral meatus, midseptum, and medial aspect of the maxilloturbinate) and for 5

two regions of level 3 (the lateral wall and midventral septum). Each of these a reas also was 6

scored for the presence of nasal lesions. 7

As discussed above, proliferating cells were visually identified by the number of grains 8

over the nucleus, 10 grains indicating a proliferating cell. Cell proliferation was quantified as the 9

number o f proliferating cells per length of basement membrane (cells/mm) and reported as a 10

ULLI. The report does not indicate the length of membrane viewed for each section as an 11

indication of how representative the counts are for each region. Lesions associated with 12

formaldehyde exposure may change the density of cells/mm of basement membrane (Monticello 13

et al., 1990). Areas of disarranged cells, erosion, metaplasia, or layering of epithelial cells may 14

exhibit different cell profiles. These processes would alt er cell density, and therefore the ULLI, 15

independent of differential proliferation rates. As such, it is not expected to be proportional to 16

cell proliferation rates across conditions that have the potential to change cell density 17

(Monticello et al., 1990)

. 18

No formaldehyde

- induced epithelial lesions or increases in the ULLI were seen in rats 19

exposed to 0.7 or 2.0 ppm formaldehyde, regardless of duration (Table 4

- 37). Formaldehyde

20

induced lesions were present in all regions of the nasal epithelium after exp osures to 10 and 21

15 ppm formaldehyde, regardless of duration (Monticello et al., 1991). Incidence and severity of 22

the lesions increased with concentration and duration of treatment and were correlated to areas 23

with increased cell proliferation. Rats expo sed to 6 ppm formaldehyde developed lesions in the 24

level 2 nasal passages, where the ULLI was clearly elevated, but not in the deeper level 3 25

passages. For example, no formaldehyde

- related lesions were seen at the lateral meatus and 26

septum of level 3 at 1 , 4, and 9 days of repeated exposure at 6 ppm, although cell proliferation 27

was increased. This transient increase in ULLI returned to near

- control levels after 6 weeks of 28

repeated exposure (Table 4

- 37). Monticello et al. (1991) suggested that cell prolif eration is a 29

more sensitive indicator of cellular response and not necessarily dependent on cellular necrosis. 30

31

32This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 198 Table 4

- 37. Formaldehyde

- induced changes in cell proliferation (ULLI) in 1

the nasal passages of male F344 rats exposed 6 hours/day 2

3

Location a E xposure concentration

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

Level 2: lateral meatus

1 day

4 days

9 days

6 weeks 2.16

1.46

1.44

0.91 1.31

1.37

1.20

0.88 2.36

1.72

1.73

1.36 16.9 b

30.5 b

23.5 b

14.4 b 11.2 b

20.9 b

28.6 b

23.9 b 12.7 b

25.8 b

24.6 b

28.7 b

Level 2: midseptum

1 day

4 days

9 days

6 weeks 1.08

1.03

1.09

0.41 1.01

0.97

0.80

0.24 1.69

0.67

0.97

0.68 3.85

10.0 b

10.9 b

2.10 17.9 b

26.1 b

19.6 b

21.4 b 16.7 b

29.1 b

29.1 b

25.9 b

Level 2: medial

maxilloturbinate

1 day

4 days

9 days

6 weeks 2.49

1.36

1.38

1.02 1.75

1.54

0.80

1.21 2.81

1.09

1.48

1.11 18.15 b

25.03 b

22.54 b

16.32 b 5.9

20.3 b

21.0 b

26.1 b 5.3

19.4 b

28.7 b

25.1 b

Level 3: lateral meatus

1 day

4 days

9 days

6 weeks 1.83

1.10

1.36

0.98 1.72

1.27

1.40

0.91 2.46

1.09

1.74

0.86 7.53 b,,c

8.77 b,

, c

7.35 b, c

2.08 14.5 b

20.0 b

30.6 b

24.2 b 16.4 b

30.8 b

40.4 b

34.8 b

Level 3: midseptum

1 day

4 days

9 days

6 weeks 3.02

2.81

1.68

2.18 1.74

3.09

1.06

1.54 2.39

1.43

1.43

2.57 4.20

9.22 b,

, c

9.50 b,

, c

2.58 24.4 b

18.7 b

28.6 b

14.0 b 19.3 b

34.4 b

32.5 b

27.5 b

4

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

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

c In dicates a location where epithelial lesions were not seen by light microscopy. 7

8

Source: Monticello et al. (1991). 9

10

11

The sustained cell proliferation at the lateral meatus and midseptum in rats exposed to 10 12

and 15 ppm formaldehyde, locations where SCCs ar e known to arise, supports a role for 13

compensatory cell proliferation in tumor development. However, Monticello et al. (1991) noted 14

that regional differences in sustained cell proliferation do not always correspond to the 15

occurrence of nasal tumors, prima rily SCCs, in formaldehyde

- exposed rats. Where sustained 16

cell proliferation has been demonstrated in the medial maxilloturbinate (MMT) at level 2 17

(Monticello et al., 1991), SCCs have not been found to originate in this area at similar exposures 18

(Monticell o et al., 1996; Woutersen et al., 1989). Monticello et al. (1991) suggested that the 19

findings of Bermudez and Allen (1984), indicating that the epithelial cells of the maxilloturbinate 20This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 199 are more resistant to the genotoxic effects of DEN, support the possib ility that differences in 1

regional tissue susceptibility may contribute to site specificity of formaldehyde

- related SCCs. 2

Monticello et al. (1996) further explored the correlation between measures of cell 3

proliferation and tumor site by modifying the ULLI to take into consideration the total number of 4

cells in a region that may be subject to increased cell proliferation. The population weighted 5

ULLI (PWULLI) is the product of the expected number of cells on a three

- dimensional surface 6

in the nasal mucosa a nd the ULLI of a cross section of that surface. For this series of 7

experiments, six male F344 rats/group were exposed to 0, 0.7, 2, 6, 10, or 15 ppm (0, 0.86, 2.45, 8

7.36, 12.3, or 18.4 mg/m 3 ) formaldehyde for up to 24 months with interim sacrifices at 3, 6, 12, 9

and 18 months. Before each interim sacrifice [ 3 H]

- thymidine was continuously injected for the 10

last 5 days of exposure through a surgically implanted pump. Nasal passages were prepared, and 11

six standard sections were taken and developed as above fo r [ 3 H]

- thymidine

- labeled cells. 12

Stained tissue sections were viewed in order to map all nasal tumors. A ULLI was determined 13

for each region (details not provided). The total cell population of each nasal region was 14

estimated from control animals sacrifi ced at 3 months (Table 4

- 38). Cell profiles were counted 15

across 0.5 mm of basement membrane length at two locations for each region (site not specified). 16

Total cells per region were estimated from these counts and the modeled surface area expected in 17

eac h region (Fluid Dynamics Analysis Package version 7.0). It is unclear if one or more rats 18

were used to quantify cell population. Cell counts and variability were not reported. 19

20

Table 4

- 38. Cell population and surface area estimates in untreated male 21

F34 4 rats and regional site location of squamous cell carcinomas in 22

formaldehyde

- exposed rats for correlation to cell proliferation rates 23

24

Nasal region Total cells

(number) a Area

(mm 2 ) b Cell density

(cell/mm 2 ) SCC incidence c

10 ppm 15 ppm

Anterior late ral meatus 976,000 59.5 16,400 12 17

Anterior midseptum 184,000 10.5 17,500 0 1

Anterior dorsal septum 128,000 3.84 33,300 0 3

Anterior medial

maxilloturbinate 104,000 7.63 13,600 0 4

Posterior lateral meatus 508,000 38.1 13,300 2 9

Po sterior midseptum 190,000 10.8 17,600 0 1

Maxillary sinus 884,000 38 23,300 0 0

Region not specified c

-- -- -- 6 25

25

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

of the re gion. 27

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

c The number of animals bearing a tumor located in the region. Animals were exposed 6 hours/day for 24 months 29

prior to sacrifice. 30

Source: Monticello et al. (1996). 31This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 200 ULLIs were quantifie d by region of the nasal passages in order to correlate with regional 1

localization of tumors. For example, the anterior midseptum included cells from the midseptum 2

from approximately standard section levels 2 to 3. An anterior to posterior pattern of 3

for maldehyde effects, especially differences in cell proliferation rates, has been well established. 4

As such, cell proliferation rates would be expected to vary across the nasal regions used in this 5

analysis. Areas considered to possibly be preneoplastic we re not quantified for this work. 6

Monticello et al. (1996) reported increased ULLIs in the ALM and the MMT of rats exposed to 7

10 or 15 ppm at all time points (3, 6, 12, and 18 months) but provided no indication of variability 8

or a statistical analysis, mak ing it difficult to determine where true differences may exist. Some 9

caution should be used in interpreting the ULLI counts assigned for each region. 10

The PWULLI was calculated by multiplying the reported ULLIs by the calculated cell 11

populations by region. SCC incidence by region had a greater correlation to the calculated 12

PWULLI than the ULLI, R 2 = 0.88 versus R 2 = 0.46, respectively. The authors noted that the 13

relative lack of correlation with the ULLI was influenced by findings at the maxilloturbinate 14

where cell proliferation was high but SCC incidence was low. Other tumor types were not 15

included in the analysis (polypoid adenomas, adenocarcinomas, and rhabdomyosarcomas). 16

Additionally, 54 of the SCC tumors could not be accurately localized and were ex cluded from 17

the analysis, resulting in exclusion of 30 and 39% of animals with SCCs in the 10 and 15 ppm 18

treatment groups, respectively. The authors cautioned that the absence of these data might have 19

skewed the regional analysis of tumor location. Altho ugh the purpose of weighting the ULLIs 20

by total population of cells available in each region is to better represent the chance of a tumor 21

arising in each region, the cancer incidence was represented by the number of animals, not the 22

number of tumors, per r egion. Based on the exclusion of location data (up to 40% of the 23

animals), lack of variability and significance reported for the ULLI for cell counts, and SCC 24

incidence considered by animal rather than by tumor, the significance of a greater correlation b y 25

PWULLI versus ULLI is of questionable value. 26

Monticello et al. (1989) also assessed formaldehyde

- induced cell proliferation and 27

regional site location of lesions in the respiratory tract of rhesus monkeys (see Section 4.2.1.2.2.2 28

for a full study descrip tion). LIs from the histoautoradiograms indicated increased cell 29

proliferation in transitory, respiratory, and olfactory epithelial cells after the 6

- week 30

formaldehyde exposure. Similar trends were seen after only 1 week but were statistically 31

significan t only in the respiratory epithelium. Although increased proliferation in the trachea and 32

carina was statistically significant after 1 week of exposure, the greater increases seen after 33

6 weeks of exposure were not statistically significant. A small samp le size (n = 3) and high 34

variability may have contributed to the lack of statistical significance. The authors noted that 35This document is a draft for review purpo ses only and does not constitute Agency policy.

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- 201 increased cell proliferation was seen in locations with minimal histologic changes, indicating 1

proliferation may be a more sensitive predictor of adverse health effects of formaldehyde 2

exposure. Table 4

- 39 provides a summary of formaldehyde

- induced cell proliferation data. 3

4

4.2.1.3. Gastrointestinal Tract and Systemic Toxicity 5

As with inhalation, the POE is thought to be the principal target tissue in response to oral 6

exposure. A concentration

- dependent pattern of toxicity longitudinally down the GI tract has 7

been observed upon oral exposure. Some evidence (Til et al., 1989, 1988) suggests that, with 8

regard to oral exposure, duration in addition to concentration is important in the development of 9

toxicity. 10

Formalin and paraformaldehyde were used to dose animals in oral toxicity studies. 11

Formalin contains 12

– 15% methanol as a preservative to inhibit the polymerization of 12

formaldehyde and subsequent precipitation as paraformaldehyde (Kiernan, 2000). The presence 13

of methanol in formalin may confound the results of a formaldehyde study. Methanol has been 14

shown to be a developmental and neurologic toxin (e.g., Degitz et al. [2004a, b]; R ogers et al. 15

[2004, 2002]; Weiss et al. [1996]; Sharpe et al. [1982]). Oral dosing with paraformaldehyde is 16

preferred because it allows for the preparation of methanol

- free formaldehyde in the laboratory 17

by dissolving paraformaldehyde in slightly basic wa ter. 18

19

4.2.1.3.1. Short

- term and subchronic studies. Til et al. (1988) evaluated the oral toxicity of 20

formaldehyde and acetaldehyde in a subacute study in Wistar (Cpb:WU; Wistar random) rats. 21

Groups of rats (10/sex/dose) were exposed to paraformaldehyd e dissolved in drinking water at 0, 22

5, 25, and 125 mg/kg

- day for 4 weeks. The control group was comprised of 20 rats of each sex. 23

To account for potential effects of decreased water consumption in treated animals, an additional 24

control group of 10 male a nd 10 female rats was given drinking water in an amount equal to the 25

amount of liquid consumed by the group given the highest dose. Examination of the GI tract was 26

performed in all dose groups and included the tongue, esophagus, and stomach. Histopatholo gy 27

for the other tissues was performed on high

- dose and control animals. 28

29This document is a draft for reviewpurposes only and does not constituteAgency policy4-202 Table 4

- 39. Summary of formaldehyde effects on cell proliferation in the upper respiratory tract

Species N a Treatment b Measure of cell

proliferation Summary of results by locatio n c Reference

Male F344

rats;

male

B6C3F1

mice NR d 0.5, 2, 6, or 15 ppm

6 hours/day for

3 days LI: percent labeled cells

on tissue sections

( 3 H

- thymidine I.P. d

2 hours postexposure) Level 2: Rats exhibited greater increased cell proliferation than mice.

No increase seen in rats or mice at 0.5 or 2.0 ppm.

No increase seen in mice at 6 ppm, but rats had 20

- fold increase in

proliferation.

10

- to 20

- fold increase seen in both rats and mice at 15 ppm. Swenberg et

al. (1986)

Male F344

rats NR 0.5, 2, or 6 p pm 6 hours/day

1, 3, or 9 days LI: percent labeled cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure) Level 2: Transient increase in cell proliferation on day 1 at 0.5 and 2.0 ppm. Increase in cell proliferation on days 1, 3, and 9 by 6 ppm

. Swenberg et

al. (1986)

Male F344

rats;

male

B6C3F1

mice NR 3 ppm for 12 hours,

6 ppm for 6 hours, or

12 ppm for 3 hours

3 or 9 days LI: percent labeled cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Level 1:

3 days: Gre ater increased proliferation in rats than mice. Increases

similar for various concentrations yielding the same C × t product.

9 days: Mice exhibited duration

- dependent increases in proliferation,

inverse to concentration for constant C × t.

Level 2

3 day s: Concentration

- dependent increase in cell proliferation.

9 days: Concentration

- dependent increase in cell proliferation in rats; no

increase in mice. Swenberg et

al. (1986)

Male F344

rats 4

– 5 15 ppm

6 hours/day

1 or 5 days LI: percent labeled cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Level 2: Increase in cell proliferation in respiratory epithelium,

nasoturbinates, maxilloturbinates, and lateral wall.

1 day: 5.51 f versus 0.43% in controls

5 days: 10.1% f Chang et al.

(1983) e

Male

BC3F1

mice 4

– 5 15 ppm

6 hours/day

1 or 5 days LI: percent labeled cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Level 2: Increase in cell proliferation in respiratory epithelium,

nasoturbinates, maxilloturbin ates, and lateral wall.

1 day: 2.14 f versus 0.27% in controls

5 day: 3.42% f Chang et al.

(1983) e

Male

albino

Wistar rats 5 d 3.5 ppm

8 hours, twice a day

for 3 days Qualitative staining for

PCNA on tissue sections Levels 2 and 3: Increase in cell prolife ration in respiratory epithelium,

nasoturbinates, maxilloturbinates, septum, and lateral wall. Cassee and

Feron (1994) eThis document is a draft for reviewpurposes only and does not constituteAgency policy4-203 Table 4

- 39. Summary of formaldehyde effects on cell proliferation in the upper respiratory tract

Species N a Treatment b Measure of cell

proliferation Summary of results by locatio n c Reference

Male

albino

Wistar rats 3 0, 5, or 10 ppm

8 hours/day

continuously for

3 days or 4 weeks, or

0, 10, or 20 ppm

8 hours/day

intermitt ent g

for 3 days or 4 weeks LI: percent labeled cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Section level not stipulated in report.

3 days: 0.86% in controls

2.83% f at 5 ppm continuous

8.87% f at 10 ppm conti nuous

9.80% f at 10 ppm interrupted

19.8% f at 20 ppm interrupted 4 weeks: 0.68% in controls

1.33% at 5 ppm continuous

8.85% h at 10 ppm continuous

3.41% f at 10 ppm interrupted

13.9% f at 20 ppm interrupted Wilmer et al.

(1987) e

Male

albino

Wistar rats 2 0, 1, 10, or 20 ppm

6 hours/day for

3 days LI: percent labeled cells

(18 hour postexposure ex

vivo 3 H

- thymidine labeled

excised mucosa) Level 3

Metaplastic epithelium: increased prolife ration

31.4% at 10 ppm, 37.6% at 20 ppm

Visibly unaffected respiratory epithelium

1.6% in controls

2.6% at 10 ppm, 2.8% at 20 ppm Woutersen et

al. (1987) e

Male

albino

Wistar rats 5 0, 1, or 2 ppm

8 hours/day

continuous ly for

3 days or 4 weeks,

or 0, 2, or 4 ppm

8 hours/day

intermittent g

for 3 days or 4 weeks LI: percent labeled cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Level 2

3 days: No change from controls

0.60% in controls

0.34% at 1 ppm continuous

0.61% at 2 ppm continuous

0.29% at 2 ppm interrupted

0.58% at 4 ppm interrupted 4 weeks: no change from controls

1.03% in controls

0.81% at 1 ppm continuou s

0.91% at 2 ppm continuous

1.16% at 2 ppm interrupted

2.86% at 4 ppm interrupted Wilmer et al.

(1989) e

Male and

female

albino

Wistar rats 5 0, 0.3, 1, 3 ppm

6 hours/day,

5 days/week

for 3 days or

13 weeks. LI: percent labe led cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Level 2: Increased cell proliferation at days 3 and 13 weeks ( p < 0.001).

Level 3: Transient dose

- dependent increase at 1 and 3 ppm;

only seen at day 3

( p < 0.001).

Note: Results pooled by sex. Data shown graphically on log

- normal scale. Zwart et al.

(1988) e

Male

Wistar rats 5 0, 0.3, 1, or 3 ppm

22 hours/day for

3 days LI: percent labeled cells

on tissue sections (2 hours

postexposure ex vivo 3 H

thymidin e

- labeled excised

mucosa) Level 2: 3 ppm increased cell proliferation in nasoturbinates,

maxilloturbinates, septum, and lateral wall ( p < 0.05).

Level 3: No significant increases in cell proliferation. Reuzel et al.

(1990)This document is a draft for reviewpurposes only and does not constituteAgency policy4-204 Table 4

- 39. Summary of formaldehyde effects on cell proliferation in the upper respiratory tract

Species N a Treatment b Measure of cell

proliferation Summary of results by locatio n c Reference

Male

Sprague

Dawley

rats 3

– 5 0, 2, 6, or 20 ppm

6 hours/day for

1 or 3 days LI: percent labeled cells

by flow cytometry

(5

- bromodeoxyuridine I.P.

18 hours postexposure for

2 hours) Respiratory and olfactory epithelial cells.

1 day: 1.3% in controls

2.4% at 2 ppm f

3.7% at 6 ppm f

2.7% at 20 ppm f

Tracheal epithelial cells

1 day: 1.2% in controls

3.1% at 2 ppm f

2.1% at 6 ppm

2.8% at 20 ppm f

Free lung cells (>97% MPs):

no significant change. 3 days:

1.4% at 2 ppm

2.5% at 6 ppm f

2.3% at 20 ppm f

3 days:

0.3% at 2 ppm f

0.6% at 6 ppm f

2.5% at 20 ppm f Roemer et al.

(1993)

Male F344

rats 6 0.7, 2, 6, 10, or

15 ppm 6 hours/day,

5 days/week for 1, 4,

or 9 days or 6 weeks ULLI (unit length LI) ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Level 3

No increases in cell proliferation at 0.7 or 2 ppm.

Level 4

ULLI increases in locations without lesions at 6 ppm.

Increases in ULLI at all locations at 10 an d 15 ppm. Monticello et

al. (1991)

Male

rhesus

monkeys 3 6 ppm

6 hours/day for

1 or 6 weeks LI: percent labeled cells

on tissue sections ( 3 H

thymidine I.P. 18 hours

postexposure for 2 hours) Nasal passages: Duration

- dependent increase in cell proliferati on at all levels

(B

– E) in transitional, respiratory, and olfactory epithelium.

Increased cell proliferation in areas with minimal lesions.

Larynx: trend for increased proliferation

Trachea: increased cell proliferation

1 week : 1.14 versus 0.55% in c ontrols

6 weeks: 3.73%

Carina of trachea: increased cell proliferation.

1 week: 1.34 versus 0.43% in controls

6 weeks: 3.60%

Respiratory bronchioles: no increase in proliferation. Monticello et

al. (1989)

a N = number of animals per treatme nt group.

b Treatment is given as the concentration of formaldehyde, duration of exposure each day, and length of the experiment in days and weeks.

c Standard section levels of the nasal passages as shown in Figure 4

- 3 are given for experiments in rats or mi ce.

d NR = not reported; I.P. = intraperitoneally.

e Study is described in full in section 4.2.1.2.2.4.

. f Different from control, p < 0.05.

g Intermittent exposures were 30 minutes per hour for 8 hours.

h Data from one animal only.This document is a draft for re view purposes only and does not constitute Agency policy.

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- 205 The rats appeared to be h ealthy throughout the study, and no effects on growth occurred 1

despite significant decreases in food and water intake that occurred at the high dose (125 mg/kg

2

day). Yellow discoloration of the fur occurred in the rats on the high dosage from week 3 3

onwar d. There were no significant changes in hematology among the exposed groups except for 4

slight (not statistically different) increases in PCVs in the water

- restricted group and in high

- dose 5

males. The high

- dose groups of the formaldehyde exposed and in th e water

- restricted controls 6

had slightly increased urine density, but again this was not statistically significant. Plasma TP 7

and ALB levels were decreased in the males of the highest dose group. No changes in organ 8

weights occurred except for relative k idney weights that were slightly increased in the females of 9

the high

- dose group. Gross pathological findings were restricted to the GI tract and revealed a 10

thickening of the limiting ridge of the forestomach in all animals exposed at the highest dose tha t 11

was accompanied by a yellowish discoloration of the mucosa. These latter changes were not 12

observed in the acetaldehyde

- exposed animals. Treatment

- related histopathologic changes were 13

seen in the GI tract only. Slight (8/20) or moderate (12/20) focal h yperkeratosis of the 14

forestomach and slight focal atrophic gastritis occurred in animals of the high

- dose groups only 15

(Table 4

- 40). One female had moderate focal papillomatous hyperplasia. No histopathologic 16

changes were observed in any animals of the lo wer

- dose groups. The study established a 17

LOAEL and NOAEL for epithelial changes in the GI tract of male and female Wistar rats 18

exposed to formaldehyde in drinking water at 125 mg/kg

- day and 25 mg/kg

- day, respectively. 19

Johannsen et al. (1986) performed a s ubchronic study by using rats and dogs exposed to 20

paraformaldehyde dissolved in drinking water. Groups of albino Sprague

- Dawley rats (15/sex) 21

were administered the equivalent of 0, 50, 100, or 150 mg/kg

- day in their drinking water for 22

91 consecutive days. Pure

- bred beagle dogs (four/sex/group) were fed a diet with added aqueous 23

formaldehyde to approximate 0, 50, 75, or 100 mg/kg

- day. Dogs were observed daily and rats at 24

frequent intervals for behavioral reactions. Body weights and food and water intake were 25

recorded on a weekly basis in both species. Hematology (HCT, Hb, total and differential 26

leukocyte counts), clinical chemistry (blood sugar, BUN, ALP, AST and ALT in dogs only), and 27

urine analyses (color, appearance, pH, specific gravity, sugar, prote in, and microscopic elements) 28

were evaluated in 10 male and 10 female rats selected from each test group and in all dogs. 29

Organ weights were recorded for the adrenals, gonads, hearts, kidneys, livers, lungs, and thyroids 30

in each species. Histopathology w as performed on a set of over 20 or 30 tissues and organs from 31

rats or dogs, respectively, in the high

- dose and control groups only. 32

33

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

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- 206 Table 4

- 40 . Summary of lesions observed in the gastrointestinal tracts of 1

Wistar rats after drinking

- water exposure to f ormaldehyde for 4 weeks 2

3

Type of lesion Formaldehyde (mg/kg

- day)

0 5 25 125

Number of male rats examined

20 10 10 10

Focal hyperkeratosis of forestomach

Very slight 3 0 0 0

Slight 1 0 0 4

Moderate 0 0 0 6

Focal gastritis

Slight 0 0 0 2

Moderate 0 0 0 1

Dilated fundic glands (single or a few) 0 0 0 0

Submucosal mononuclear cell infiltrate 0 0 0 1

Type of lesion Number of female rats examined

20 10 10 10

Focal hyperkeratosis of forestomach

Very slight 6 0 0 2

Slight 0 0 0 2

Moderate 0 0 0 6

Focal gastritis

Very slight 0 0 0 1

Slight 0 0 0 1

Moderate 0 0 0 1

Focal papillomatous hyperplasia 0 0 0 1

Polymorphonuclear leukocytic infiltration 0 0 0 1

4

Source: Til et al. (1988). 5

6

7

No deaths or abnormal reactions were observed in either species. Significant reductions 8

in weight gain were observed in dogs of both sexes at 100 mg/kg

- day, in rats of both sexes at 9

150 mg/kg

- day, and in male rats at 100 mg/kg

- day of formaldehyde. There was a dose

- related 10

decrease in liqui d consumption of both sexes in rats given formaldehyde, but there was no 11

overall difference in mean food intake or feed efficiency, so the reductions in body weight gain 12

were considered to be systemic effects. Dogs administered formaldehyde had reduced fo od 13

consumption and feed efficiency at all doses tested. No significant effects on hematology, 14

clinical chemistry, or urine analyses were observed in either species. No effects in either species 15

were reported on organ weights. The GI mucosa in both speci es was reported to appear normal 16

with no indication of irritation. This study suggests a NOAEL of 150 mg/kg

- day in Sprague

17

Dawley rats and of 100 mg/kg

- day in beagle dogs for formaldehyde in drinking water. 18

Differences in the results for the rats with th ose reported in other studies (Til et al., 1989, 1988; 19

Tobe et al., 1989) may be due to strain differences or duration of the exposure. The dog may be 20

a more sensitive species than the rat based on these results and on those of 2

- week pilot studies. 21This document is a draft for re view purposes only and does not constitute Agency policy.

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- 207 4.2.1 .3.2. Chronic bioassays: oral exposure to formaldehyde

. The same laboratory that tested 1

formaldehyde and acetaldehyde in a 4

- week study (Til et al., 1988) performed a chronic bioassay 2

with formaldehyde in drinking water. Til et al. (1989) administered p araformaldehyde dissolved 3

in drinking water to Wistar rats (Cpb: WU; Wistar random) (70/sex/dose). Interim sacrifices 4

(10/sex/dose) were performed at 12 and 18 months. Formaldehyde was administered in drinking 5

water to provide target doses of 0, 5, 25, a nd 125 mg/kg

- day. The mean formaldehyde doses 6

administered were 0, 1.2, 15, or 82 mg/kg

- day for males and 0, 1.8, 21, or 109 mg/kg

- day for 7

females. Concentrations were adjusted weekly for the first 12 weeks based on dose estimates 8

derived from body weigh t and liquid consumption data. Such adjustments were made every 9

4 weeks from weeks 12 to 52 and kept constant. Fresh solutions of the test concentrations were 10

prepared weekly and stored at 15oC. 11

Endpoints examined included daily observations for conditio n and behavior, body weight 12

at weekly intervals for the first 12 weeks and then every 4 weeks thereafter, liquid intake weekly, 13

and food intake weekly for the first 12 weeks and then every 2 weeks for the remainder of the 14

study. Samples of blood were take n for hematological and clinical chemistry analyses on weeks 15

26 and 103. Analysis of blood glucose and urine pH, density, and volume was performed on 16

samples at weeks 27, 52, 78, and 104. Pooled urine samples were also evaluated for glucose, 17

occult blood , ketones, urobilinogen, and bilirubin in samples at weeks 27 and 104. Weights of 18

all major organs were recorded at interim sacrifices and at term. Gross and histopathologic 19

examinations were carried out on all major tissues of the rats in the high

- dose and control 20

groups. The livers, lungs, stomach, and noses were examined in all rats. Additionally, the 21

adrenals, kidneys, spleens, testes, thyroids, ovaries, pituitaries, and mammary glands (for 22

females) were examined in all sacrificed animals at weeks 5 3 and 79 and at term. 23

The general health and behavior of the rats were not affected in any of the formaldehyde

24

exposed groups. Slight yellowing of the fur did occur in the animals exposed at the mid and high 25

doses from week 3 onward. The mean body weight s were decreased in the males from week 1 26

and in the females from week 24 onward. At the high dose, liquid consumption was significantly 27

decreased in both sexes, and food intake was significantly decreased in the males. There were no 28

toxicologically sign ificant effects on hematological, urinary, or clinical chemistry parameters. 29

Decreases in absolute heart, liver, and testis (males) weights were attributed to lower body 30

weights. Relative kidney weights were increased in females of the high

- dose group, a nd relative 31

brain weights were increased in both sexes of the high

- dose group. Relative testis weight was 32

increased in males. Treatment

- related changes in gross pathology were restricted to the 33

forestomach. Histopathologic examinations at the two interi m sacrifices and final sacrifice 34This document is a draft for re view purposes only and does not constitute Agency policy.

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- 208 revealed GI tract changes. Renal changes were observed in the high

- dose group at final 1

sacrifice. There was no indication of treatment

- related effects in other tissues. 2

As shown in Table 4

- 41, significant histopathology i n the GI tract was limited to the 3

forestomach and stomach of rats in the high

- dose groups. Some progression with duration of 4

exposure may have occurred by week 105 because GI lesions were observed in the lower dose 5

groups at this time point, whereas none were observed in these groups at interim sacrifices. The 6

histopathologic changes included papillary epithelial hyperplasia in the forestomach that was 7

frequently accompanied by hyperkeratosis on the limiting ridge or its vicinity. The mucosa 8

showed an irregular layer of hyperplastic basal cells, but no atypical nuclei or other subcellular 9

structures were observed. Chronic atrophic gastritis occurred to varying degrees in the stomachs 10

of all high

- dose rats. In some cases the inflammatory process involved the entire mucosa and 11

was seen to extend to the whole muscularis mucosae and met the criteria for ulceration. 12

Histologic examination also showed that the incidence and degree of renal papillary 13

necrosis was increased in animals of the high

- dose groups at the terminal sacrifice. This change 14

was located at the tip of the papilla and was characterized by patchy necrosis of interstitial cells, 15

capillaries, and loops of Henle. There was no evidence of a dose

- related response in chronic 16

nephropathy. The incid ence of chronic nephropathy was lower in the males of the high

- dose 17

group than in controls. In females, the incidence was slightly higher in the test groups than in 18

controls but only achieved statistical significance at the lowest dose. It is likely that the decrease 19

in liquid intake incurred in the high

- dose groups contributed to the increased incidence and 20

degree of renal papillary necrosis observed in the high

- dose animals because dehydration has 21

been shown to enhance its production by various analgesi cs. 22

The results of this chronic bioassay indicated that formaldehyde is cytotoxic to the 23

epithelial mucosa of the nonglandular (forestomach) and glandular stomach with a LOAEL of 82 24

and 109 mg/kg

- day and a NOAEL of 15 and 21 mg/kg

- day in males and females, respectively. 25

The findings provided no evidence of carcinogenicity in either the GI tract or systemic sites for 26

formaldehyde administered in drinking water to Wistar rats at doses as high as 82 mg/kg

- day. 27

28

29This document is a draft for re view purposes only and does not constitute Agency policy.

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- 209 Table 4

- 41. Incidence of lesions observed in t he gastrointestinal tracts of 1

Wistar rats after drinking

- water exposure to formaldehyde for 2 years 2

3

Incidence of lesions with formaldehyde dose

(mg/kg

- day) a

Males Females

0 1.2 15 82 0 1.8 21 109

Week 53

Number of rats examined b 9 10 10 10 10 10 10 9

Forestomach

Focal papillary epithelial hyperplasia 0 0 0 7 0 0 0 5

Glandular stomach

Chronic atrophic gastritis 0 0 0 10 c 0 0 0 9 c

Focal ulceration 0 0 0 3 0 0 0 1

Focal mononuclear cell infiltrate 1 0 3 0 2 0 0 0

Atypical glandular hyperplasia 0 0 0 0 0 0 0 1

Week 79

Number of rats examined 10 10 10 10 10 9 10 10

Forestomach

Focal papillary epithelial hyperplasia 2 1 1 8 1 0 1 9

Glandular stomach

Chronic atrophic gastritis 0 0 0 10 c 0 0 0 10 c

Focal ulceration 0 0 0 2 0 0 0 0

Focal squamou s metaplasia 0 0 0 1 0 0 0 0

Submucosal inflammatory cell

infiltrate 1 2 0 0 0 0 0 0

Focal mononuclear cell infiltrate 0 1 0 0 1 1 0 0

Glandular dilation 2 4 4 1 2 2 4 0

Week 105

Number of rats examined b 47 45 44 47 48 49 47 48

Forestomach

Focal pa pillary epithelial hyperplasia 1 2 1 45 c 1 0 2 45 c

Focal hyperkeratosis 2 6 4 24 c 3 5 3 33 c

Focal ulceration 1 1 1 8 0 0 2 5

Focal acanthosis 1 0 2 1 0 0 0 1

Focal basic cell hyperplasia 0 1 1 0 1 0 0 0

Diverticulum 0 0 1 0 0 0 0 0

Exophytic papillom a 0 1 0 0 1 0 0 0

Glandular stomach

Chronic atrophic gastritis 0 0 0 46 c 0 0 0 48 c

Focal ulceration 0 0 0 11 c 0 0 0 10 c

Glandular hyperplasia 0 1 0 20 c 0 0 0 13 c

Mineralization 3 2 1 0 0 0 0 0

Focal inflammatory cell infiltrate 5 3 2 0 2 3 1 0

4

a In cidence in rats that died or were killed when moribund during the experiment or were killed at 5

week 53, 79, or 105. 6

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

c The values differ significantly (Fisher’s exact test) from the control value ( p < 0.00 1). 8

9

Source: Til et al. (1989). 10

11

12This document is a draft for re view purposes only and does not constitute Agency policy.

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- 210 Tobe et al. (1989) performed a chronic toxicity study of Wistar rats (Slc:Wistar) exposed 1

to paraformaldehyde dissolved in drinking water. Groups of 20 male and 20 female rats were 2

given formaldehyde solution in their dr inking water at concentrations of 0, 0.02, 0.10, and 0.50% 3

for 24 months. Interim sacrifices of six randomly chosen rats from each group were performed 4

after 12 and 18 months. Based on the estimated average amount of water intake and body 5

weight, the act ual doses of formaldehyde in either sex were reported to be 0, 10, 50, and 6

300 mg/kg

- day. Fresh test solutions were prepared twice each week. The rats were observed 7

daily for the entire study. Body weights and water and diet intake were measured once we ekly 8

or biweekly. Hematology (RBC, WBC, and Hb) and serum clinical chemistry (TP, ALB, BUN, 9

uric acid, total cholesterol, inorganic phosphorous, ALP, AST, and ALT) were made at each 10

necropsy. Organ weights were measured for the brain, heart, lung, liver, kidney, spleen, adrenal, 11

testis or ovary, pituitary, and thyroid. These organs and the stomach, small and large intestine, 12

pancreas, uterus, lymph nodes, and all tumors were examined histopathologically. 13

The general condition of animals in the high

- dose group was poor with significantly 14

reduced body weight gain as well as intake of water and diet. An increase in mortality was also 15

observed in this group. Some clinical chemistry parameters were altered in this group. No 16

significant changes in absolute o r relative organ weights were observed. Mortality was 100% in 17

the high

- dose group by 24 months. At the 12

- month sacrifice, hyperplasia of the squamous 18

epithelium with or without hyperkeratosis was observed in the forestomach of all high

- dose 19

animals (12/ 12). Basal cell hyperplasia with growth into the submucosa was also observed in 20

most cases (10/12). Erosions and/or ulcers with submucosal inflammatory cell infiltrates were 21

observed in the glandular stomach of most rats (10/12). Regenerative changes of the glandular 22

epithelium (glandular hyperplasia) were noticed in most cases (10/12) along the limiting ridge of 23

the fundic mucosa. No lesions were observed in the glandular stomach at the 50 mg/kg

- day 24

dose, and forestomach hyperplasia was observed in onl y one of six males and in one of eight 25

females at 18 and 24 months. No lesions in either the forestomach or glandular stomach were 26

observed in rats treated at 10 mg/kg

- day. 27

This study corroborates the Til et al. (1989) study and shows that the main target s for 28

formaldehyde toxicity administered by drinking water to rats are the forestomach and glandular 29

stomach. Although the lesions observed at the 50 mg/kg

- day were minimal in this study, Tobe et 30

al. (1989) designated the NOAEL at 10 mg/kg

- day, further su pporting the NOAEL of 15 mg/kg

31

day from the Til et al. (1989) study. 32

Takahashi et al. (1986) studied the effects of formaldehyde in an initiation

- promotion 33

model of stomach carcinogenesis in male outbred Wistar rats (Shizuoka Laboratory Center, 34

Shizuoka). Rats (n = 17) were given 100 mg/L of N

- methyl

- N1

- nitro

- N

- nitrosoguanidine 35This document is a draft for re view purposes only and does not constitute Agency policy.

DRAFT

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- 211 (MNNG) in drinking water and a diet supplemented with 10% sodium chloride (NaCl) for the 1

first 8 weeks as an initiation phase. This was followed by 0.5% formalin (which contains 12

– 2

15% methanol) in drinking water for 32 weeks as the promotion phase of the protocol. A 3

comparison group (n = 10) was given stock water and diet without any supplementation for the 4

first 8 weeks followed by 0.5% formalin in drinking water for 32 weeks. A nimals were observed 5

daily and weighed once every 4 weeks. Small pieces of the stomach and other tissues in the 6

peritoneal cavity were fixed for histopathologic examination. 7

Body weight gain was reduced by exposure to MNNG with sodium chloride, and 8

formal dehyde exposure during the promotion phase exacerbated this effect. Histopathologic 9

investigations were restricted to the GI tract. Formaldehyde was shown to statistically increase 10

the incidence of lesions in the forestomach and stomach in the animals in itiated with MNNG 11

with NaCl as compared with controls receiving no initiation (Table 4

- 42). Increases in 12

papilloma in the forestomach, adenomatous hyperplasia in the fundus, and adenocarcinoma in 13

the pylorus were observed. Histopathology in the animals r eceiving formaldehyde alone during 14

weeks 9 through 32 showed an increase in forestomach papillomas but with no lesions in the 15

glandular stomach (Table 4

- 42). The adenomatous hyperplasia were defined as proliferative, 16

noninvasive mucosal lesions, and the a denocarcinomas were defined as well differentiated and 17

composed of typical glandular structures, demonstrating a tubular pattern and cellular or 18

structural atypism without metastasis. No definition of criteria for papilloma diagnosis was 19

provided. The fi ndings in this study are inconsistent with those of Til et al. (1989), who found 20

no evidence of carcinogenicity in a 2

- year bioassay at comparable concentrations (assuming 37% 21

formaldehyde in formalin results in 0.19% formaldehyde in this study). As discu ssed above, the 22

differences may be due to differences in the strains of rat or in the diagnostic criteria. The lack 23

of more than one test concentration precludes dose

- response analysis of this study and provides 24

only a stand

- alone LOAEL of 0.2% formaldehy de in drinking water. The lack of consumption 25

data precludes an estimation of dose in mg/kg

- day. 26This document is a draft for reviewpurposes only and does not constituteAgency policy4-212 Table 4

- 42. Effect of formaldehyde on gastroduodenal carcinogenesis initiated by MNNG and NaCl in male

Wistar rats exposed to formaldehyde (0.5% formalin) i n drinking water for 8 weeks

No MNNG initiation prior to 8

- week oral exposure to formaldehyde (0.5% formalin in drinking water)

Gastroduodenal

carcinoma Forestomach

papillomas Glandular stomach tumors

Fundus Pylorus Duodenum

Adenocarcinoma Adeno matous

hyperplasia Adenocarcinoma Preneoplastic

hyperplasia Adenocarcinoma

Control 0% 0% 0% 0% 0% 0% 0%

Formaldehyde 0% 80% a 0% 0% 0% 0% 0%

MNNG initiation (100 mg/L in drinking water for 8 weeks) prior to 8

- week oral exposure to formaldehyde (0.5% form alin in drinking water)

Gastroduodenal

carcinoma Forestomach

papillomas Glandular stomach tumors

Fundus Pylorus Duodenum

Adenocarcinoma Adenomatous

hyperplasia Adenocarcinoma Preneoplastic

hyperplasia Adenocarcinoma

Control 13.3% 0% 0% 0% 3.3% 2 3.3% 10.0%

Formaldehyde 29.4% 88.2% a 0% 88.2% a 23.5% b 41.2% 5.9%

a Significantly different from control animals with MNNG initiation, p < 0.01.

b Significantly different from control animals with MNNG initiation, p < 0.05.

Source: Takahashi et al. (1986 ).This document is a draft for review purposes only and does not constitute Agency policy.

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- 213 Soffritti et al. (1989) administered formaldehyde in drinking water to Sprague

- Dawley 1

rats at different ages. Formaldehyde was produced via the Formox process, which yields carbon 2

monoxide, dimethyl ether, and small quantities of CO 2 and formic acid. In one experiment 3

denoted as BT 7001, rats (50/sex/dose) that were 7 weeks old were administered 0, 10, 50, 100, 4

500, 1,000, or 1,500 mg/L for 104 weeks. As is usual for experiments carried out at the 5

Ramazzini Foundation, all animals were maintained un til natural death at which point they were 6

necropsied and examined histopathologically. In experiment BT 7005, 25

- week

- old breeder rats 7

or offspring (beginning postnatal day [PND] 12) were provided drinking water with 0 or 2,500 8

mg/L formaldehyde for 104 weeks. Fluid and food consumption were measured weekly for the 9

first 13 weeks and then every 2 weeks thereafter. Individual body weights were recorded for the 10

first 13 weeks and then every 2 weeks thereafter. Histopathology was routinely performed on al l 11

major tissues, including oral and nasal cavities, the GI tract (esophagus, stomach, and intestines 12

[4 levels]), primary and secondary lymph organs (thymus, spleen, subcutaneous lymph nodes, 13

mesenteric lymph nodes, mediastinal lymph nodes, and femur [bone marrow]), head and face 14

bones, and other organ systems (liver, kidney, bladder, reproductive, and various glands). 15

Noncancer health effects were not reported. 16

No GI neoplasia were observed in any of the control rats (experiments BT 7001 and BT 17

7005). Hi storical controls for the BT experimental colony (n = 5,259) indicate an incidence of 18

approximately 1% for benign neoplasia (papillomas and acanthomas) and an incidence of 0.19% 19

for malignant stomach neoplasia (adenomas, SCCs, and adenocarcinomas, fibrosar comas, and 20

leiomyosarcomas taken together). Therefore, the size of the control groups (18

– 50 rats/sex) 21

makes detection of background neoplasia unlikely. Similarly, one or two tumors noted in a 22

treatment group (n = 18

– 50) would represent an apparent incre ase in these relatively rare tumors. 23

Although stomach and intestinal tumors were found in rats exposed to formaldehyde in drinking 24

water, the low incidence makes it difficult to discern any dose

- response effect for individual 25

neoplasia. The authors repor t formaldehyde

- induced GI tract neoplasia to include benign tumors 26

(papillomas and acanthomas of the forestomach and adenomas) and malignant tumors 27

(adenocarcinomas and leiomyosarcomas). The majority of malignant tumors were present in the 28

duodenum, jejun um, and ileum. Benign and malignant neoplasia were consistently noted in the 29

two highest exposure groups: 1500 mg/mL formaldehyde in experiment BT 7001 and 30

2,500 mg/mL in experiment BT 7005 (Table 4

- 43). Comparison of overall GI neoplasia in 31

breeder and offspring rats of experiment BT 7005 suggests that rats exposed beginning on 32

PND 12 had a greater incidence of malignant tumors. However, it should be noted that, although 33

there may be an apparent increase in overall tumors, summing across sites and locat ions is 34

needed before a response can be seen. Even then, several data points are based on a single 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 214 observed tumor. However, nonspecific MOAs, such as mutagenicity and regenerative 1

proliferation, would be expected to act on all cell types at the POE, and summing tumor types 2

may have some utility. These results constitute a weak positive result for cancer due to oral 3

exposure to formaldehyde. 4

5

Table 4

- 43. Summary of benign and malignant gastrointestinal tract 6

neoplasia reported in male and female Sprague

- Dawley rats exposed to 7

formaldehyde in drinking water at different ages 8

9

Experiment Dose Sex Total benign

tumors Total malignant

tumors All tumors

Historical

controls Not

applicable M 1.08% 0.31% 1.4%

F 0.97% 0.41% 1.4%

BT 7001

(7 weeks old) Contr ol M

– a

– –

F

– – – 1,000 mg/mL M

– 2% b 2% b

F 2% b

– 2% b

1,500 mg/mL M 4% 6% 10%

F 6%

– 6%

BT 7005

breeders

(25 weeks

old) Control M

– – –

F

– – – 2,500 mg/mL M

– 5.6% b 5.6% b

F 5.6% b

– 5.6% b

BT 7005

offspring

(PND 12) Control M

– – –

F

– – –

2,500 mg/mL M 5.6% 8.3% 13.9%

F

– 21.6% 21.6%

10

a Dash indicates no tumors reported. An incidence was not reported. 11

b Percentage is based on the observation of a single neoplasm. 12

13

Source: Soffritti et al. (1989). 14

15

Oral exposure to formalde hyde resulted in a dose

- dependent increase in all 16

hemolymphoreticular neoplasia in both male and female rats in experiment BT 7001 (Table 17

4

- 44) (Soffritti et al., 1989). The most frequent neoplasia noted were lymphoblastic leukemias 18

and lymphomas. The au thors combined lymphoblastic leukemias and lymphosarcomas for 19

analysis and summing across sites. This analysis is appropriate since there is broad consensus 20

that “neoplasms presenting as solid tumors and those presenting with blood and marrow 21

involvement are biologically the same disease but with different clinical presentations,” as stated 22

in the recent WHO reclassification of hematological malignancies (Harris et al., 2000b). 23

Inspection of the data tables suggests that the only treatment

- related effects occurred at 1,000 24This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 215 and 1,500 mg/L. The reported incidence of lymphoid neoplasia and of all hemolymphoreticular 1

neoplasia in rats exposed to the vehicle (methyl alcohol, 15 mg/L) was similar to results reported 2

in rats exposed to 50 or 100 mg/L formaldehyd e in drinking water. Soffritti et al. (1989) 3

provided no statistical analysis of the data. Although the authors report a similar increase in 4

experiment BT 7005, the apparent 5% increase is representative of a single animal in a treatment 5

group of 18 and may not represent a true increase, the study’s usefulness for 6

hemolymphoreticular neoplasia being somewhat limited by study size. 7

8

Table 4

- 44. Incidence of hemolymphoreticular neoplasia reported in male 9

and female Sprague

- Dawley rats exposed to formaldehy de in drinking 10

water from 7 weeks old for up to 2 years (experiment BT 7001) 11

12

Treatment Sex Lymphoid

neoplasia (%) Other leukemias

(%) All leukemias and

lymphomas (%)

Control M 4

– a 4

F 1 2 3

Vehicle

control M 10

– 10

F 2 4 6

10 mg/mL M 2

– 2

F 4

– 4

50 mg/mL M 10

– 10

F 8

– 8

100 mg/mL M 8 2 10

F 4 4 8

500 mg/mL M 12 4 16

F 4 4 8

1,000 mg/mL M 12

– 12

F 10 4 14

1,500 mg/mL M 22

– 22

F 10 4 14

13

a Dash indicates no neoplasm was reported. 14

15

Source: Adapted from Soffritti et al. (1989). 16

17

18

The study of Soffritti et al. (1989) does provide qualitative corroboration of evidence 19

from other studies that observed formaldehyde toxicity in the forestomach and stomach. 20

However, the dosages required to induce such lesions in this study were higher than in other 21

studies. Soffritti et al. (1989) is the only animal study of oral exposure to formaldehyde that 22

reports an increase in lymphoblastic leukemia or lymphosarcoma. These occurred at the two 23

highest doses of 1,000 and 1,500 mg/L used in the stu dy of animals exposed from 7 weeks of age 24

(experiment BT 7001). 25

26This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 216 4.2.1.3.3. Summary of toxicity in the GI tract. Short

- term and subchronic exposures to 1

formaldehyde via drinking water for 4 weeks yielded slight to moderate histopathologic lesions 2

(foca l hyperkeratosis) at 125 mg/kg

- day in male and female Wistar rats, as well as slight focal 3

gastritis and submucosal infiltrate in one to two animals of both sexes (Til et al., 1988). No 4

histopathologic lesions were noted in albino Sprague

- Dawley rats or b eagle dogs that received 5

oral doses of formaldehyde in drinking water for 91 days (Johannsen et al., 1986). In both 6

studies, decreases in weight gain were noted in exposed animals compared with controls. 7

In a chronic drinking water study, Til et al. (1989 ) reported that formaldehyde is 8

cytotoxic to the epithelial mucosa of the nonglandular (forestomach) and glandular stomach with 9

a LOAEL of 82 and 109 mg/kg

- day and a NOAEL of 15 and 21 mg/kg

- day in males and female 10

Wistar rats, respectively. The findings provided no evidence of carcinogenicity in either the GI 11

tract or systemic sites for formaldehyde administered in drinking water to Wistar rats at doses as 12

high as 82 mg/kg

- day. The incidence and degree of renal papillary necrosis was increased in 13

animals of the high

- dose groups at the terminal sacrifice (Til et al., 1989). Findings by Tobe et 14

al. (1989) corroborate the Til et al. (1989) study and show that the main targets for formaldehyde 15

toxicity administered by drinking water to rats are the forestoma ch and glandular stomach. 16

Takahashi et al. (1986) studied the effects of formaldehyde in an initiation

- promotion model of 17

stomach carcinogenesis in male outbred Wistar rats (Shizuoka Laboratory Center, Shizuoka, 18

Japan). In contrast to Til et al. (1989), Takahashi et al. (1986) found increases in incidence of 19

papilloma in the forestomach, adenomatous hyperplasia in the fundus, and adenocarcinoma in 20

the pylorus in a 2

- year bioassay at comparable concentrations (assuming 37% formaldehyde in 21

formalin results in 0.19% formaldehyde in this study). Soffritti et al. (1989) administered 22

formaldehyde in drinking water to Sprague

- Dawley rats at different ages. Rats (50/sex/dose, age 23

7 weeks) were administered 0, 10, 50, 100, 500, 1,000, or 1,500 mg/L formaldehyde i n drinking 24

water for 104 weeks. The authors reported formaldehyde

- induced GI tract neoplasia that 25

included benign tumors (papillomas and acanthomas of the forestomach and adenomas) and 26

malignant tumors (adenocarcinomas and leiomyosarcomas), albeit at a re latively low incidence 27

after summing across sites and locations. Interestingly, oral exposure to formaldehyde resulted 28

in a dose

- dependent increase in all hemolymphoreticular neoplasia in both male and female rats 29

(Soffritti et al., 1989). The most frequ ent neoplasia noted were lymphoblastic leukemias and 30

lymphomas. 31

32

4.2.1.4. Immune Function 33

Leach et al. (1983) documented potential immunomodulatory effects of formaldehyde 34

inhalation exposure. F344 rats were exposed nose only to formaldehyde 6 hours/day

, 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 217 5 days/week for up to 30 days. The target concentrations for exposure were 0, 3, 16, 61, and 1

99 ppm formaldehyde (0, 3.7, 19.7, 75.0, and 122 mg/m 3 ). Body weight and food consumption 2

were recorded, and blood samples for standard hematology and immune a ssays were collected 3

(details not given). Immune measures referenced include in vitro lymphocyte transformation, 4

hemagglutination assays, and enumeration of B cells, WBCs, and RBCs. No effects were seen at 5

3 ppm formaldehyde. However, dose

- dependent res ponses were reported for weight loss, 6

decreased food consumption, increased WBCs, increased segmented neutrophils and nucleated 7

RBCs, and decreased ability to produce antibodies to sheep RBCs. The results of the 8

lymphocyte transformation assay were incons istent, with a 25

– 30% reduction in stimulation after 9

exposure to 99 ppm but an initial stimulation seen after 16 and 61 ppm exposures. Further 10

details were not available, making it difficult to determine if these reported immunomodulatory 11

effects may have been, in part or in full, secondary to effects on the URT. Subchronic exposures 12

at 61 and 99 ppm formaldehyde would be expected to result in frank toxic effects in mice (see 13

Section 4.2.1). However, these findings suggest possible immunomodulatory effec ts due to 14

formaldehyde exposure and require further exploration. 15

Dean et al. (1984) investigated the effects of formaldehyde exposure on a range of 16

indicators of immune function. Female B6C3F1 mice were exposed to 15 ppm formaldehyde 17

(18.4 mg/m 3 ) 6 hours /day, 5 days/week for 3 weeks. Three trials were run with a total of 255 18

formaldehyde

- treated mice. Body and organ weights were recorded at sacrifice for control and 19

formaldehyde

- exposed mice (10 per group). Measures of host susceptibility, cell

- mediate d 20

immunity MP function, and antibody reactions were conducted 2 to 6 days after the end of 21

exposure (Table 4

- 45). Lymphocyte subsets, spleen cellularity, bone marrow cellularity, and 22

progenitor cell subsets were enumerated. Host susceptibility and delaye d type hypersensitivity 23

were measured in vivo . Lymphocyte proliferation, natural killer cell activity, phagocytosis, 24

hydrogen peroxide production, and IgM plaque

- forming cells (PFCs) were measured ex vivo 25

after in vivo stimulation in some cases (Table 4

- 4 5). 26

Body weight, organ weights and cellularity, progenitor cell populations, blood cell 27

counts, and differentials were unchanged in formaldehyde

- treated mice (Dean et al., 1984). 28

Circulating blood monocytes were decreased in treated mice, which may be a r eflection of the 29

local inflammatory response expected in the nasal epithelium (Dean et al., 1984). However, 30

there was no corresponding decrease in peritoneal MPs. There was a trend, but no statistical 31

significance, for decreased spleen weight, cellularit y, and B cell precursors (87, 83, and 78% of 32

controls, respectively). The mean body weight of formaldehyde

- treated mice was 21.1 versus 33

20.9 g in control mice, and thymus and spleen weights were not normalized by body weight. 34

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

DRAFT

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- 218 Table 4

- 45 . Battery of im mune parameters and functional tests assessed in 1

female B6C3F1 mice after a 3 week, 15

- ppm formaldehyde exposure 2

(6 hours/day, 5 days/week) 3

4

Immune function Model Challenge Metric

Host susceptibility Tumor resistance PYB6 sarcoma cells Subcutaneous injec tion, followed

by skin palpation to track tumor

development

Tumor resistance 16F10 melanoma cells Lung tumor burden determined by

[ 125 I]UdR incorporation

Bacterial resistance Listeria monocytogenes Survival after challenge

Cell

- mediated

immunity Del ayed type

hypersensitivity Keyhole limpet

hemocyanin Radiometric index of delayed

hypersensitivity responses

Lymphocyte

proliferation T

- cell mitogen, PHA a

B

- cell mitogen, LPS b

( ex vivo ) Ex vivo proliferation, 3 days,

measured by [ 3 H]

- thymidine

incorpo ration

Lymphocyte subsets None Percentage of cells positive for cell

surface markers (Thy

- 1, Mac

- 1,

Lyt

- 1)

Natural killer cell

activity Yac

- 1 target cells

( 51 Cr labeled)

( ex vivo ) % cytotoxicity by 51 Cr release

MP function

(both resident and

MVE

- 1 eli cited

MP) Phagocytosis Sheep RBCs

( 51 Cr labeled)

( ex vivo ) 51 Cr incorporation as a measure of

RBCs phagocytized

Hydrogen peroxide

production Pharmacologic

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

Humoral cell

immunity Antibody PFC

responses, IgM

PFCs Sheep RBCs,

TVF

- LPS, or TNF

Ficoll Plaques formed

Progenitor cells Bone marrow

cellularity (femur) None Cell enumeration by a Coulter

counter

Granulocyte

- MP

progenitors None Cell enumeration by a Coulter

counter

B

- cell precursors None Clonogenic assay
5

a T

- cell mitogen, phytohemagglutinin (PHA

- P). 6

b B

- cell mitogen, lipopolysaccharide ( Escherichia coli ). 7

8

Source: Dean et al. (1984). 9

10

11

All indicators of natural killer cell function, cell

- mediated immunity, and humoral 12

immunity in formaldehyde

- treate d mice were unchanged from controls (Dean et al., 1984). 13

Phagocytic capacity of both resident and elicited peritoneal MPs was unchanged by 14

formaldehyde treatment. However, hydrogen peroxide production in elicited peritoneal MPs was 15

significantly increase d in formaldehyde

- treated mice, 78 versus 42 nmol/mg protein ( p < 0.05) 16

(Dean et al., 1984). 17

As shown in Table 4

- 46, several indicators of host resistance in the female B6C3F1 mice 18

were increased after formaldehyde exposure (Dean et al., 1984). Tumor mass and pulmonary 19This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 219 foci after B16F10 melanoma cell challenge were significantly reduced in formaldehyde

- treated 1

mice, indicating improved tumor immunity ( p < 0.05). However, following PYB6 sarcoma cell 2

challenge, formaldehyde

- treated mice had a 7.1% tumor inc idence versus 11.1% in controls, 3

which was not statistically different. Mortality due to Listeria monocytogenes (LM) was 4

decreased from 70 to 30% ( p < 0.05). Because resistance to LM is primarily MP dependent, the 5

authors speculated that this enhanced re sistance might be due in part to increased bactericidal 6

activity as was also suggested by increased hydrogen peroxide production ex vivo in elicited 7

peritoneal MPs from female mice (Dean et al., 1984). 8

9

Table 4

- 46. Summary of the effects of formaldehyde i nhalation on the 10

mononuclear phagocyte system (MPS) in female B6C3F1 mice after a 11

3

- week, 15 ppm formaldehyde exposure (6 hours/day, 5 days/week) 12

13

In vivo indicators of MPS Metric Formaldehyde effect

Cellularity Circulating monocytes Decreased a

CMF prog enitor cells No change a

Resident peritoneal MP No change a,b

Elicited peritoneal MP No change a,b

In vivo test of host resistance LM Increased resistance a

B16F10 tumor challenge Increased resistance a

PYB6 tumor challenge No significant increase a

Ex vivo indicators of MPS Cell type/activation Formaldehyde effect

H 2 O 2 production Resident, no PMA c None detected a,b

Resident, with PMA None detected a,b

Elicited, no PMA d None detected a,b

Elicited, with PMA Increased a,b

Phagocytosis Resident No change a

Elicited No change a

Assessment of MP

maturation

Leucine aminopeptidase content Resident Decreased b

Elicited No change b

Resident No change b

Elicited No change b

Acid phosphatase content Resident No change b

Binding of tumor cells Elicited No change b

Resident No change b

Lysing of tumor cells Elicited Increased at mid

- range target

- to

- effector

cell ratio b

14

a Dean et al. (1984). 15

b Adams et al. (1987). 16

c Phorbol 12

- myristate 13

- acetate (PMA). 17

d Peritoneal MPs were elicited with the pyran copoly mer Murray Valley encephalitis virus (MVE

- 2). 18

19

Sources: Adams et al. (1987); Dean et al. (1984). 20

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

DRAFT

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- 220 Overall, the observations of increased hydrogen peroxide production and increased host 1

resistance in peritoneal MPs distant from the POE suggest that formald ehyde has an effect on the 2

mononuclear phagocyte system (MPS). The authors postulated that this effect may be indirect, 3

due in part to the tissue inflammatory response in the URT or a direct systemic effect on the 4

MPS by formaldehyde exposure (Dean et al. , 1984). Subsequent studies by the same researchers 5

explored the possibility of systemic effects of formaldehyde exposure on MPS function and 6

maturation stage (Adams et al., 1987). Female B6C3F1 mice were exposed to 15 ppm 7

(18.4 mg/m 3 ) formaldehyde 6 hou rs/day, 5 days/week for 3 weeks, as before (Adams et al., 8

1987). Both resident and Murray Valley encephalitis virus (MVE

- 2)

- elicited peritoneal MPs 9

were examined for hydrogen peroxide production, enzymatic activity, phagocytic ability, 10

binding, and lysis of tumor cells (Adams et al., 1987). 11

Similar to the findings of Dean et al. (1984), formaldehyde treatment increased hydrogen 12

peroxide production almost twofold in MVE

- 2 elicited peritoneal MPs (Adams et al., 1987). As 13

summarized in Table 4

- 46, no treatme nt differences were seen in phagocytic ability in either 14

resident or elicited MPs (Adams et al., 1987). Resident peritoneal MPs from formaldehyde

15

treated mice were not different in their ability to bind or lyse tumor cells. Although 16

formaldehyde treatmen t did not increase the ability of elicited MPs to bind tumor cells, lysis of 17

the target cells (P815 tumor cells) was increased from 28 to 37% by formaldehyde treatment but 18

only at the midrange target

- to

- effector

- cell ratio tested in the assay ( p < 0.05) (A dams et al., 19

1987). Although this is statistically significant, the authors questioned the biological 20

significance of this result since it was not observed at all three target cell ratios tested. However, 21

an increase in tumor cell lysis in vitro would be consistent with the in vivo increased tumor 22

resistance previously reported (Dean et al., 1984). The in vitro lysis response curve suggests that 23

assay conditions may result in a maximum cytolysis near 40%. If so, any treatment effects on 24

lysis would be d ifficult to discern at higher effector cell ratios. 25

Jakab (1992) investigated the effect of formaldehyde exposure on the alveolar MPs and 26

resistance to respiratory infections. The first set of experiments assessed bactericidal activity by 27

directly quanti fying the pulmonary bacterial loading after exposure to Staphylococcus aureus

. 28

White female Swiss mice were exposed to formaldehyde after bacterial infection (regimens A 29

and C), before bacterial infection (regimen B), or before and after infection (regime n D) 30

(Table 4

- 47). 31

32This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 221 Table 4

- 47. Formaldehyde exposure regimens for determining the effects of 1

formaldehyde exposure on pulmonary S. aureus infection 2

3

Pre

- infection treatment Postinfection treatment Results

Regimen A None 4 hours

0, 1, 5, 10, or 15 pp m a 15 ppm, increased

bacterial loading

Regimen B 18 hours

0, 0.5, or 1 ppm b None No effect

Regimen C None 4 hours

0, 0.5, or 1 ppm No effect

Regimen D 18 hours

0, 0.5, or 1 ppm 4 hours

0, 0.5, or 1 ppm 1 ppm, increased

bacterial loading

4

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

b 0, 0.62, or 1.2 mg/m 3 formaldehyde. 6

7

Source: Jakab (1992). 8

9

10

For regimen A, mice were exposed to 0, 1, 5, 10, or 15 ppm (0, 1.2, 6.2, 12.3, or 11

18.5 mg/m 3 ) formaldehyde. For regimens B

– D, mice were exposed to 0, 0.5 , or 1 ppm (0, 6.2, or 12

1.2 mg/m 3 ) formaldehyde. A 30

- minute exposure to an infectious aerosol of S. aureus deposited 13

2 × 10 5 staphylococci in the lungs. Bacterial loading was determined in homogenized lung tissue 14

by culturing diluted aliquots for an esti mate of bacteria present immediately after loading and 4 15

hours later. Bacterial loading was expressed as a percentage change between control and 16

formaldehyde

- exposed animals. Mice exposed to 15 ppm formaldehyde for the 4 hours 17

following bacterial infecti on (regimen A) had approximately an 8% increase in bacteria, 18

indicating decreased host resistance ( p = 0.006) (Jakab, 1992) (Table 4

- 47). Mice receiving 19

lower concentrations of formaldehyde following bacterial infection did not have increased 20

pulmonary ba cterial loading. Pre

- infection exposure to 0.5 or 1.0 ppm did not change bacterial 21

loading 4 hours after infection (regimen B). However, combining an 18

- hour pre

- infection 22

formaldehyde exposure with a 4

- hour postinfection 1 ppm formaldehyde exposure incr eased 23

pulmonary bacterial loading by approximately 6.5% ( p < 0.05). This effect was not seen with 24

only a 0.5 ppm pre

- and posttreatment regimen. Increased bacterial loading indicates that 25

formaldehyde exposure (regimens A and D) reduced pulmonary bacteri al resistance. This is in 26

apparent contradiction to the findings of increased host resistance by Dean et al. (1984). 27

However, there are important differences between the studies. The studies by Jakab (1992) are 28

acute studies examining effects at the res piratory tract where direct effects are possible. 29

Additionally, in some cases, the exposures were concurrent with bacterial infection, and it is 30

difficult to distinguish the potential for formaldehyde effects directly on the mucociliary 31

apparatus as a bar rier to infection. 32This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 222 A second set of experiments in the same report (Jakab, 1992) examined the effects of 1

co

- exposure to formaldehyde and carbon black on pulmonary infection with S. aureus. The 2

particle size distribution of the carbon black aerosol was less than a 5 μm aerodynamic diameter 3

and, therefore, 98% respirable. Female Swiss mice were exposed nose only to formaldehyde and 4

carbon black. Experiments were run at two target concentrations: (1) 2.5 ppm (3.1 mg/m 3 ) 5

formaldehyde and 3.5 mg/m 3 carbon blac k or (2) 5 ppm (6.2 mg/m 3 ) formaldehyde and 6

10 mg/m 3 carbon black. Co

- exposure was given either for 4 hours after a 30

- minute S. aureus 7

infection or 4 hours/day for 4 days as a pretreatment prior to S. aureus infection. Bacterial 8

loading was determined 0 and 4 hours after the S. aureus infection to assess bacterial survival. 9

Formaldehyde

- carbon black co

- exposure did not alter bacterial survival either as a pretreatment 10

or posttreatment to bacterial exposure. However, this exposure regimen was not run fo r 11

formaldehyde or carbon black separately, and the 4 hours/day for 4 days pretreatment was not 12

included in the formaldehyde alone experiments (Table 4

- 47). 13

Jakab (1992) also assessed the phagocytic activity of alveolar MPs collected by lavage at 14

various ti me points after formaldehyde, carbon black, or co

- exposure. Female Swiss mice were 15

co

- exposed to 5 ppm (6.2 mg/m 3 ) formaldehyde and 10 mg/m 3 carbon black 4 hours/day for 16

4 days. Mice were sacrificed and alveolar MPs harvested 1, 3, 5, 25, and 40 days aft er exposure. 17

Mice exposed only to formaldehyde or carbon black were sacrificed 3, 10, 25, and 40 days after 18

exposure. Fc

- receptor

- mediated phagocytosis was assessed ex vivo by using sensitized sheep 19

RBCs. The phagocytic index (PI) was reported as the to tal number of RBCs in 100 MPs. 20

Neither formaldehyde nor carbon black exposure alone significantly changed the PI (Jakab, 21

1992). These findings are consistent with the first co

- exposure experiment, since no changes in 22

PI were seen immediately after exposu re. However, co

- exposure did decrease the PI of alveolar 23

MPs in a time

- dependent manner, with maximal decrease to less than 70% of controls by 25 days 24

after exposure (Figure 4

- 11). Decreases in the PI reflect changes in both the percentage of 25

phagocytic MPs and the number of RBCs phagocytized (Jakab, 1992). The PI recovered to 26

control levels by 40 days postexposure. 27

28This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 223 1

1 0 0 1 1 0

9 0

8 0

7 0

6 0

5 0

0 1 0 2 0 3 0 4 0

D a y s a f t e r l a s t e x p o s u r e H C H O

C a r b o n

C a r b o n + H C H O

2

3

Figure 4

- 11. Alveolar MP Fc

- mediated phagocytosis from mice exposed to 4

5 ppm formaldehyde, 10 mg/m 3 carbon black, or both. 5

6

Note: Ex posure was 4 hours/day for 4 days. Each value represents the mean ± 7

SEM of five determinations. 8

9

Source: Redrawn from Jakab (1992). 10

11

12

Holmström et al. (1989b) evaluated the effects of long

- term formaldehyde exposure on 13

antibody production. Female Spragu e

- Dawley rats were exposed to 12.6 ppm formaldehyde 14

(15.5 mg/m 3 ) 6 hours/day, 5 days/week for 22 months. Body weight, tumor incidence, and 15

pathology were reported elsewhere (Holmström et al., 1989b). Rats were given a subcutaneous 16

injection of pneumococc al polysaccharide antigens or tetanus toxoid 21 to 25 days prior to 17

sacrifice. The two vaccines chosen represent T

- cell

- dependent and T

- cell

- independent antigens, 18

respectively. Antibody titers (IgG and IgM) were determined prior to vaccination and at 19

sac rifice. Formaldehyde treatment had no effect on antibody titers either before or after 20

vaccination (Holmström et al., 1989b). 21

22

4.2.1.4.1. Summary of formaldehyde effects on immune function. Although there were initial 23

reports of systemic immunomodulatio n attributed to formaldehyde exposure (Leach et al., 1983), 24

formaldehyde effects on measures of humoral and cell

- mediated immunity were not confirmed 25

by Dean et al. (1984). The authors did report increased host resistance to both tumor and 26

bacterial tumor challenges after a 3

- week exposure to 15 ppm formaldehyde. An increased 27Phagocytic index (% of control)Phagocytic index (% of control)This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 224 resistance to these challenges, presented distal to the site of formaldehyde exposure 1

(administered subcutaneously or intravenously), suggests a systemic effect of formaldehyde 2

expos ure. In addition, increased host resistance and hydrogen peroxide release from peritoneal 3

MPs were reported and confirmed (Adams et al., 1987; Dean et al., 1984). Chronic 4

inflammation and tissue damage to the respiratory mucosa expected with formaldehyde exposure 5

may result in an up regulation of the MPS and therefore increase host immunity. It is unclear if 6

this response would be specific to formaldehyde or similar to enhancement of immune function 7

seen with chronic inflammation. 8

Jakab (1992) demonstrat ed decreased pulmonary resistance to bacterial infection where 9

animals were exposed to 15 ppm formaldehyde immediately after bacterial loading or when they 10

were given an 18

- hour pre

- exposure to formaldehyde followed by 1 ppm formaldehyde exposure 11

after bac terial loading. The authors speculated that formaldehyde may directly act on pulmonary 12

MPs, reducing their effectiveness. However, Jakab (1992) showed that there was no change in 13

Fc

- mediated phagocytosis of alveolar MPs immediately after formaldehyde exp osures. 14

Degradation of the protective mucus layer and possible epithelial cell damage may contribute to 15

more effective bacterial infection in the presence of formaldehyde without a direct action on MP 16

function. As mentioned above, degradation of the mucu s layer may result in a more potent 17

inoculation and therefore higher bacterial loading. 18

Although neither formaldehyde nor carbon black alone impacted Fc

- mediated 19

phagocytosis of alveolar MPs, Jakab (1992) demonstrated that there was decreased Fc

- mediated 20

p hagocytosis after formaldehyde and carbon black co

- exposure. Carbon black may have acted as 21

a carrier for formaldehyde, allowing higher levels of formaldehyde to be delivered more deeply 22

into the lungs than would be seen with formaldehyde alone. 23

Formald ehyde is known to break down the mucus layer protecting the respiratory tract, 24

allowing exposure of the underlying epithelium (Morgan et al., 1986a, c, d). Additionally, 25

formaldehyde can directly induce tissue inflammation through sensory irritation via s ubstance P 26

from the trigeminal nerve (Fujimaki et al., 2004a). These actions together could contribute to 27

some of the observed effects on immune response attributed to formaldehyde exposures. 28

Degradation of the protective mucus layer would make antigens more available to the immune 29

system. It has been shown that direct application of an antigen to the nasal associated lymph 30

tissue, bypassing the mucus layer, is a more effective delivery of antigen (Hou et al., 2002). 31

Therefore, increased availability of these antigens to the immune system may in part explain 32

observed increased antibody production seen against ovalbumin (OVA) or common dust mite 33

allergen (Der f) during formaldehyde exposure (Sadakane et al., 2002; Riedel et al., 1996; 34

Tarkowski and Gorski , 1995). Neurogenic inflammation may also contribute to more efficient 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 225 antigen processing and presentation by activation of resident MPs. These factors are consistent 1

with the observation that formaldehyde exposures do not affect antibody production to a ntigens 2

administered outside of the respiratory tract, even after chronic exposures (Holmström et al., 3

1989b). 4

This effect was initially observed several days after exposure was ended with maximal 5

suppression seen 25 days after a 4

- day formaldehyde exposur e. The delayed onset of this 6

response, however, suggests an effect beyond the POE effects observed at the time of exposure. 7

T able 4

- 48 presents a summary overview of the effects of formaldehyde on immune function in 8

laboratory animals. 9

10

4.2.1.5. H ypersen sitivity and Atopic Reactions 11

Adverse reactions in humans exposed to formaldehyde in the workplace and homes have 12

been reported, which are consistent with an allergic response or a chemical sensitivity (see 13

Section 4.1.1 for details). Rashes and skin reac tions are reported in some individuals after 14

dermal exposures, and in some cases exacerbation of asthma is reported after inhalation of 15

formaldehyde. However, the reports of human reactions do not allow a clear determination of 16

whether this sensitization is immunogenic or neurogenic in origin. Formaldehyde

- induced 17

sensitization may have both neurogenic and immunologic components. Numerous animal 18

studies have been conducted in order to understand the potential for sensitization to 19

formaldehyde. Although hypersensitivity and allergic sensitization are often considered solely 20

immunologic in origin, neurogenic mechanisms may result in bronchial hypersensitivity and 21

increased immunologic sensitization. Therefore, the animal studies regarding formaldehyde

22

ind uced sensitization are evaluated discretely in order to examine these etiologic possibilities. 23

Classically, hypersensitivity is characterized as an immune response to an antigen, 24

resulting in an inflammatory reaction that itself damages the tissues or is otherwise harmful 25

(Kuby, 1991). These reactions may be localized, as in topical dermatitis, or systematic, as in 26

anaphylactic shock from an allergen. Hypersensitivity can be mediated by a humoral immune 27

response or by a cell

- mediated immune response. Fo ur classes of hypersensitivity are generally 28

recognized that differ in their immune system components and functions. Although a single 29

agent (e.g., penicillin) may induce all four types of hypersensitivity, it is more usual for an agent 30

to primarily induc e one form of hypersensitivity. 31This document is a draft for reviewpurposes only and does not constituteAgency policy4-226 Table 4

- 48. Summary of immune function changes due to inhaled formaldehyde exposure in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

F344 rats 8 0, 3, 16, 61, 99 ppm

6 hours/day,

5 days/week for

4 weeks No effects at 3 ppm. Mixed results at higher doses that were not

consistent. NA b Leach et al.

(1983)

B6C3F1

mice

(female) 10 15 ppm 6 hours/day,

5 days/week for

3 weeks Increased H 2 O 2 production, and increased host resistance to t umor formation, but other immune parameters unchanged. LOAEL 15 ppm Dean et al.

(1984)

B6C3F1

mice

(female) Pooled

MPs from

a number

of mice 15 ppm 6 hours/day,

5 days/week for

3 weeks Increased H 2 O 2 production in MVE

- 2

- elicited peritoneal MPs. LOAEL 15 p pm Adams et al.

(1987)

White

Swiss mice

(female) 18 0, 1, 5, 10, or

50 ppm for 18 hours

before and/or

4 hours after a

30

- minute exposure

to bacterial infection

( S. aureus ) Combining an 18

- hour pre

- exposure to formaldehyde with 4

- hour

postexposure to forma ldehyde increased bacterial loading at 1 ppm by

6.5%. LOAEL 1 ppm Jakab (1992)_

White

Swiss mice

(female) 18 5 ppm (2.6 mg/m 3 )

formaldehyde and

10 mg/m 3 carbon

black 4 hours/day for

4 days Phagocytic index was decreased by co

- exposure to formaldehyde and

carbon black but not by either insult alone. NA Jakab (1992)

Sprague

Dawley rats

(female) 5 12.6 ppm

6 hours/day,

5 days/week,

22 months Formaldehyde treatment had no effect on antibody titers either before

or after vaccination with pneumococcal polysacch aride antigen or

tetanus toxoid. NA Holmström et

al. (1989b)

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

DRAFT

— DO NOT CITE OR QUOTE 4

- 227 Chemical sensitivity generally implies a neurogenically induced sensitization (Meggs, 1

1995). A chemical may directly interact with sensory nerves, releasing mediators t hat trigger 2

inflammation, such as substance P (a tachykinin). Repeated exposure to the same chemical is 3

hypothesized to potentiate neurogenic inflammation (Meggs, 1995). The resulting signs of tissue 4

inflammation may be similar to immunogenic inflammatio n, but there would be no requirement 5

that the immune system recognize the chemical as an antigen for this type of response. 6

Therefore, a chemical may induce one or more clinical signs of atopic asthma without a type 1, 7

IgE

- mediated hypersensitivity respon se. One form of sensitivity, , directly affects sensory nerve 8

endings, resulting in neurogenic inflammation and is a well

- known health effect attributed to 9

formaldehyde. Neurogenic responses may result from the direct and acute interaction of the 10

chemic al with sensory nerve ending receptors of the trigeminal nerve that may lead to persistent 11

rhinitis and an asthma

- like reactive airway dysfunction syndrome that may develop after short

12

term human exposures (Brooks et al., 1985). Thus, there is evidence to suggest that neurogenic 13

inflammation may contribute to observed increases in formaldehyde

- induced airway 14

hyperresponsiveness and atopic responses. The available animal studies that have investigated 15

formaldehyde

- induced airway hyperresponsiveness and ato pic responses are summarized below. 16

17

4.2.1.5.1. Inhalation studies in experimental animals. This section summarizes animal studies 18

informing the role of formaldehyde

- induced chemical sensitization. The symptoms of 19

sensitization (atopy, airway hyperrespo nsiveness) are frequently associated with immunologic 20

markers (cytokine production, leukocyte infiltration histamine release, and antibody production) 21

but may be mediated by neurogenic sensory irritation, principally by activation of the trigeminal 22

nerve ( see Section 4.1.1.1 for a discussion of sensory irritation). The animal studies that 23

illuminate these neurogenic and immunologic responses are discussed outside of the classic 24

neurotoxicology and immunotoxicology study summary sections to allow synthesis of these data. 25

Sensitization to chemical exposure by inhalation often manifests as an allergic or 26

asthmatic response as characterized by BC or BHR. This sensitization may be a result of 27

immune involvement, as in the case of hypersensitivity, or a neurogen ic sensitization, where a 28

chemical may directly stimulate inflammation. Asthma is a specific manifestation of IgE

29

mediated hypersensitivity, characterized by BHR and airway inflammation, resulting in lower 30

airway obstruction (Fireman, 2003; Kuby, 1991). In asthma, an allergen capable of cross

- linking 31

membrane

- bound IgE on mast cells initiates immunogenic inflammation resulting in an influx of 32

eosinophils, neutrophils, and lymphocytes. Mediators of BC, including histamine, eicosanoids, 33

and bradykinin (Kub y, 1991), are released during this process. Prior exposure to the allergen can 34This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 228 increase allergen

- specific IgE, potentiating the allergic reaction; this is immunogenic 1

sensitization. 2

Biagini et al. (1989) evaluated the effect of a single pulmonary exposure of formaldehyde 3

on pulmonary mechanics, including BC. The researchers chose cynomolgus monkeys known to 4

be hyperreactive to methacholine (acetyl

- β

- methacholine chloride), which is a direct

- acting 5

stimulant of BC (Cain, 2001). Measures of pulmonary mecha nics included pulmonary flow 6

resistance; dynamic compliance; PEFR; FVC; FEV; FEF 25

– 75% , and 50% of VC; and FEFs 7

normalized for VC. Nine cynomolgus monkeys were exposed to increasing levels of 8

methacholine for 1 minute at 10

- minute intervals (0, 0.125, 0.5

, 2, and 8 mg/mL) as an aerosol 9

(0.065 mL/minute with a mean aerodynamic diameter of 1.0

– 1.5 μm). Pulmonary mechanics 10

were measured to establish each monkey’s response to methacholine. Methacholine challenge, 11

as the positive control, increased pulmonary fl ow resistance at increasing levels of methacholine 12

(0.125, 0.5, 2, and 8 mg/mL) to 196 ± 16, 285 ± 57, 317 ± 64, and 461 ± 120 % of baseline 13

levels, respectively. After a 2

- week recovery period, each methacholine

- sensitized monkey was 14

exposed to 2.5 ppm f ormaldehyde (generated from formalin, 15% methanol) for 10 minutes. 15

Measures of pulmonary function were performed at 2, 5, and 10 minutes after exposure. 16

Formaldehyde exposure increased pulmonary flow resistance from 11.3 ± 1.4 cm H 2 O 17

prior to formaldehyd e exposure to 16.1 ± 2.1, 16.9 ± 2.8, and 20.0 ± 3.4 cm H 2 O at 2, 5, and 18

10 minutes after 2.5 ppm formaldehyde exposure (with 142, 150, and 177 % change, 19

respectively). All other measures of formaldehyde

- induced pulmonary mechanics were not 20

significantly different from controls. Increased pulmonary flow resistance, a measure of 21

increased BC, was induced by formaldehyde challenge in previously sensitized mice. However, 22

the differences between methacholine challenge and formaldehyde challenge were not 23

stat istically significant. Although both formaldehyde challenge and methacholine challenge 24

increased pulmonary flow resistance, there was no correlation between individual methacholine 25

responsiveness and the magnitude of effect after formaldehyde exposure ( p > 0.1). Therefore, 26

although formaldehyde exposure stimulated BC similarly to a known direct stimulating agent, 27

formaldehyde may not work through the same site of action as methacholine. 28

Swiecichowski et al. (1993) assessed pulmonary resistance and airway reactivity due to 29

formaldehyde exposure alone and in response to increasing doses of acetylcholine chloride (a 30

direct

- acting BC agent) after formaldehyde exposure in vivo. Male Hartley guinea pigs (eight 31

per group) were exposed at 0.86, 3.4, 9.4, or 31.1 ppm (1.1, 4.2, 11.6, or 38.3 mg/m 3 ) 32

formaldehyde for 2 hours or at 0.11, 0.31, 0.59, or 1.05 ppm (0.14, 0.38, 0.73, or 1.29 mg/m 3 ) 33

formaldehyde for 8 hours. Total pulmonary resistance increased after 2 hours formaldehyde 34

exposure at 9.4 and 31.1 ppm and reached similar peak resistance at the end of the exposure 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 229 period. This effect was rapidly reversible, with values returning to baseline within 30 minutes 1

after exposure. Although 2

- hour exposures at 3 and 1 ppm did not increase pulmonary 2

resistance, 8

- h our exposures at 0.3 and 1 ppm did increase pulmonary resistance to similar levels 3

as the 2

- hour exposure at 30 ppm. The results indicate that both concentration and exposure time 4

impacted the measured increase in pulmonary resistance. However, a simple multiplicative 5

model (e.g., C × t) does not adequately represent the effects observed. It is noted that an 8

- hour 6

exposure at 1 ppm (8 ppm

- hours), reached approximately the same pulmonary resistance as 2 7

hours at 9.4 ppm (19 ppm

- hours). This may in part be due to a maximum practical increase in 8

pulmonary resistance in the animals. Conversely, there was no effect at 3 ppm for 2 hours 9

(6 ppm

- hours), although significant increase in pulmonary resistance was recorded after an 10

8

- hour exposure at 0.3 ppm (2.4 ppm

- hours). Formaldehyde does not appear to exert its effects 11

via a classic C × t paradigm. Exposure concentration, however, did seem to impact recovery 12

time. 13

In addition, specific pulmonary resistance and airway reactivity to increasing doses of 14

intra venous acetylcholine chloride, a direct respiratory stimulant, were measured immediately 15

after formaldehyde exposure for up to 60 minutes. Formaldehyde

- induced airway 16

hyperreactivity was defined as a decrease in the level of acetylcholine chloride needed to 17

produce twice the basal specific resistance (effective dose [ED] 200 ). The dose of acetylcholine 18

chloride required to double the specific pulmonary resistance (ED 200 ) and airway reactivity was 19

decreased in animals exposed for 2 hours to formaldehyde. W hen the duration was extended to 8 20

hours of formaldehyde exposure, the effective dose of formaldehyde required to elicit a doubled 21

pulmonary resistance (ED 200 ) in the presence of acetylcholine chloride was decreased to 22

1.07 ppm. Lower ED 200 s were recorded in formaldehyde

- treated animals. This indicates that 23

less acetylcholine was needed to produce BC when formaldehyde was present. Thus, 24

formaldehyde can exacerbate BHR. Additionally the formaldehyde

- induced effect increased 25

with duration of exposure, ind icating that time as well as exposure concentration are factors in 26

the magnitude of the response. Directly induced increases in airway hyperreactivity peaked 1 27

hour after exposure and persisted 6 hours after exposure. 28

In a second set of experiments, male Hartley guinea pigs were treated for 8 hours at 29

3.4 ppm (4.2 mg/m 3 ) in order to measure airway hyperreactivity ex vivo (Swiecichowski et al., 30

1993). After formaldehyde exposure, tracheae were excised and mounted in tissue baths, where 31

tracheal contractio n was measured in response to direct application of acetylcholine and then 32

carbachol. Tracheae from similarly exposed guinea pigs were fixed and sectioned for histologic 33

examination and were assessed for signs of inflammation. Formaldehyde exposure did n ot 34

increase ex vivo tracheal constriction and suggests that changes in airway reactivity were 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 230 produced due to both local humoral changes and neural reflexes. However, no changes in 1

epithelial cell morphology or influx of inflammatory cells were recorded e ven up to 4 days after 2

formaldehyde exposure ended. The authors speculated on possible MOAs for BHR, such as the 3

role of an irritant receptor or altered epithelial cell biochemistry. It may be that the window of 4

acute inflammation occurred early in the e xposure protocol and was resolved by the time of first 5

measurement, after 8 hours of exposure. The absence of inflammatory markers may argue 6

against a classic type 1 sensitivity. 7

The binding of an allergen to receptor bound IgE triggers degranulation of mast cells and 8

basophils, releasing mediators of type 1 hypersensitivity, including the histamine responsible for 9

BC. Brown Norway (BN) rats are known for their high capacity for IgE production and airway 10

hyperresponsiveness in response to allergens or ot her chemicals; they have often been used as a 11

model of allergic respiratory disease. Ohtsuka et al. (1997) compared the effects of 12

formaldehyde exposure on the nasal epithelium of F344 and BN rats. If the formaldehyde

13

induced inflammatory response in the nasal epithelium is IgE mediated, BN rats would be 14

expected to display more severe effects of formaldehyde exposure than F344 rats. Both strains 15

of age

- and sex

- matched rats were exposed to formaldehyde aerosol for 3 hours/day, 5 16

days/week for 2 weeks. The aerosol was generated from a 1% formaldehyde solution by a two

17

fluid atomizer, and formaldehyde level was maintained at 2 mg (1% sol.)/L (approximately 18

16 ppm or 20 mg/m 3 ), by adjusting the flow rate for formaldehyde solution to the atomizer. 19

During t he course of exposure, the following clinical signs were monitored: abnormal 20

respiration, stridor wheezing, nasal discharge, and sneezing. Rats were weighed weekly. Two 21

days postexposure, rats were sacrificed and tissues from the head, trachea, and lungs were fixed 22

and sectioned. Transverse sections were taken at the following palatal landmarks from three 23

animals: level 1 (lateral edge of incisor teeth), level 2 (between incisive papilla and the first 24

palatal ridge), and level 3 (on the second upper mola r). The nasal septa of the remaining two 25

animals were revealed for examination by electron microscopy. 26

Formaldehyde

- treated F344 rats showed less body weight gain over the 2

- week 27

treatment, resulting in lower body weight at week 1 and week 2 than F344 co ntrols ( p < 0.05 and 28

0.01). Body weights of formaldehyde

- treated BN rats were unchanged from BN controls. The 29

authors observed fewer clinical signs of respiratory irritation in the formaldehyde

- exposed BN 30

rats compared with formaldehyde

- exposed F344 rats , such as abnormal respiration (three versus 31

five) and nasal discharge (three versus five). Histologic analysis of lung and trachea tissues 32

revealed no distinct signs of inflammation in either strain. Formaldehyde exposure induced cell 33

damage in URT tiss ues. Epithelial cell damage was milder and impacted a smaller portion of the 34

URT in BN rats compared with F344 rats. Squamous metaplasia were present in the respiratory 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 231 epithelium (levels 1 and 2) in both strains in formaldehyde

- treated rats. However, a distinct 1

keratinized layer was noted in level 1 epithelium of F344 rats, and the extent of lesions in level 2 2

respiratory epithelium was much greater than that seen in BN rats. Additionally, the olfactory 3

epithelium (level 2) in formaldehyde

- exposed F344 rats exhibited degeneration, necrosis, and 4

desquamation not seen in BN rats. Mild squamous metaplasia was noted in level 3 of the 5

respiratory epithelium in the treated F344 rats but not the treated BN rats. No pulmonary 6

function measurements were taken, and, thus, no direct comparison in BHR or BC between BN 7

and F344 rats in response to formaldehyde can be made. It appears that BN rats are more 8

resistant to formaldehyde

- induced cell damage than are F344 rats, despite the fact that BN rats 9

are known to b e IgE responders. These results suggest that IgE responsiveness may be 10

protective of formaldehyde

- induced cell damage, or IgE may not play a role at all. The authors 11

note that their earlier research indicated the BN rats have well

- developed submucosal gl ands and 12

speculate that greater mucus flow may be partly responsible for the greater resistance of BN rats 13

to the histologic signs of formaldehyde toxicity. 14

In a subsequent study in the same laboratory, Ohtsuka et al. (2003) compared histology 15

and cytokine profiles in the nasal mucosa of formaldehyde

- treated F344 and BN rats. 16

Formaldehyde aerosol was generated as above and rats (nine per group) were exposed 17

3 hours/day for 5 days to approximately 16 ppm of formaldehyde (20 mg/m 3 ). Clinical signs 18

were reco rded daily, and monitored respiratory parameters included abnormal respiration, stridor 19

wheezing, nasal discharge, and sneezing. Tissue sections of the nose (five rats per group) were 20

prepared for light microscopy as above: transverse sections at levels 1 , 2, and 3. Th

- 1 cytokines 21

(IFN

- γ, IL

- 2) and Th2 cytokines (IL

- 4 and IL

- 5) were determined from the whole nasal mucosa 22

in four rats of each treatment group. 23

As expected, lesions and neutrophilic infiltration were more severe in F344 24

formaldehyde

- exposed rats compared with treated BN rats. In addition, lesions were observed in 25

all three levels of epithelium examined in F344 rats and impacted both respiratory and olfactory 26

epithelium. Mucosal lesions in formaldehyde

- treated BN rats impacted the respirator y 27

epithelium of levels 1 and 2 only. Changes in formaldehyde

- induced cytokine mRNA 28

expression were modest in both strains. Th

- 1

- related cytokines (IFN

- **γ** , Il

- 2) in formaldehyde

29

treated BN rats were significantly decreased compared with control BN rats. A similar, although 30

not statistically significant, decrease in Th

- 2 cytokines (IL

- 4, IL

- 5) was observed in 31

formaldehyde

- treated BN rats compared with unex posed BN rats. There were no treatment 32

differences in either Th

- 1 or Th

- 2 cytokine expression in formaldehyde

- treated F344 rats 33

compared with unexposed F344 rats. The modest changes in cytokine profile reported in 34

formaldehyde

- treated BN rats were not co nsistent with type 1 hypersensitivity since type 1 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 232 hypersensitivity reactions generally result in increased Th

- 2 cytokines. The mRNA expression 1

results were not corroborated with protein levels and may not have been captured at their peak 2

expression level s. 3

Lee et al. (1984) evaluated the potential for formaldehyde to act as a sensitizing agent 4

through different routes of exposure in guinea pigs. The inhalation studies will be highlighted 5

here. Dermal exposure and associated contact sensitivity results w ill be discussed in the dermal 6

exposure section ( 4.2.1.5.2 ). Three groups of male English smooth

- haired guinea pigs 7

(four/group) were exposed via inhalation to either 6 or 10 ppm (7.4 or 12.3 mg/m 3 ) 8

formaldehyde 6 hours/day for 5 consecutive days. Depend ing on the group, animals were then 9

subjected to bronchial provocation challenge with 2 or 4 ppm formaldehyde on day 7 or days 7, 10

22, and 29 after exposure (see Table 4

- 49 for clarification). 11

12

Table 4

- 49 . Study design for guinea pigs exposed to formaldeh yde through 13

different routes of exposure: inhalation, dermal, and injection 14

15

Formaldehyde

exposure Bronchial provocation

challenge Skin test Blood drawn

for antibody

titer

Group I

— Inhalation 6 ppm formaldehyde a

, days 1

– 5 Day 7

2 ppm formaldehyde a for 1

hour Day 9 Day 14

Group II

— Inhalation 10 ppm formaldehyde,

days 1

– 5 Day 7

2 ppm formaldehyde for 1 hour Day 9 Day 14

Group III

— Inhalation 10 ppm formaldehyde,

days 1

– 5 Days 7, 22, and 29

4 ppm formaldehyde for 4

hours Day 31 Day 14

Group IV

— Dermal 100 μL formalin, days 1

and 3 Day 22

2 ppm formaldehyde for 1 hour

4 ppm formaldehyde for 4

hours Day 7 Day 14

Group V

— Injection 37 mg formaldehyde

with Freund’s adjuvant Day 19

2 ppm formaldehyde Day 7 Day 14

16

Source: Lee et al. (1984). 17

18

19

Dermal and inject ion groups are shown for comparison. Pulmonary hypersensitivity was 20

assessed by measuring respiratory rate and tidal volume in response to exposure to 2 ppm 21

formaldehyde challenge for 1 hour, 2 days postexposure for all three groups, and additional 22

measur ements were taken 22 and 29 days postexposure for group III. Blood was drawn to 23

characterize IgE antibodies to formaldehyde in a passive cutaneous anaphylaxis (PCA) assay. 24

Respiratory rate was measured following initial formaldehyde exposure and again af ter bronchial 25

challenge with formaldehyde. 26This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 233 Respiratory rate (exhibited as a pause during expiration) was depressed by 45% following 1

exposure to 10 ppm formaldehyde during the first hour of exposure. During the first hour of 2

exposure, decreased respiratory rate was accompanied by a pause during expiration that has been 3

categorized as RB and indicated sensory irritation. The decreased respiratory rate is consistent 4

with URT sensory irritation and induction of the trigeminal (neurogenic) reflex (Lee et al., 5

1984). After the first hour of exposure, decreased respiratory rate was characterized by a pause 6

between breaths, which is similar to the breathing pattern seen in mice exposed to formaldehyde 7

via tracheal cannula (Alarie, 1981). This suggests a separate effect of formaldehyde on the LRT 8

after deep penetration of formaldehyde and suggests pulmonary irritation (Lee et al., 1984). 9

However, subsequent bronchial provocation challenge with either 2 or 4 ppm 10

formaldehyde for either 1 or 4 hours failed to elicit immediate or delayed

- onset respiratory 11

sensitization (Table 4

- 50). Respiratory rates were reported as being within ±20% of pre

12

challenge levels (data not shown) and did not reflect statistical significance (Lee et al., 1984). 13

Moreover, increased respira tory sensitivity was not observed in animals that had received an 14

emulsification of formaldehyde and Freund’s complete adjuvant by injection. Only two to four 15

animals given formaldehyde injections in the presence of Freund’s complete adjuvant developed 16

a low titer of antibodies to formaldehyde (Lee et al., 1984). 17

18

Table 4

- 50 . Sensitization response of guinea pigs exposed to formaldehyde 19

through inhalation, topical application, or footpad injection 20

21

Exposure route Pulmonary sensitization Dermal sensitiz ation Antibody production

Inhalation

6 ppm (Group I)

10 ppm (Group II)

10 ppm (Group III) 0/4

0/4

0/4 0/4

0/4

2/4 0/4

0/4

0/4

Topical 0/8 8/8 0/8

Injection 0/4 4/4 2/4

22

Source: Lee et al. (1984). 23

24

25

Thus, inhalation exposure to 6 or 10 ppm formaldehyde (8 hours/day for 5 days) followed 26

by bronchial challenge with 2 or 4 ppm formaldehyde failed to result in respiratory sensitivity 27

defined as greater than 20% change in respiratory rate. Second, for animals that received an 28

injection of formal dehyde with Freud’s adjuvant, it was not effective in inducing pulmonary 29

sensitivity. While neither inhaled formaldehyde challenge nor injected formaldehyde and 30

Freud’s adjuvant emulsion were effective in producing pulmonary sensitivity, this study relied 31

on increased respiratory rate as an indication of hyperresponsiveness and may not be an accurate 32This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 234 measure of hyperresponsiveness. Thus, overall, conclusions are uninformative due to study 1

design flaws. 2

Riedel et al. (1996) tested the effects of formaldehy de inhalation on the development of 3

sensitization to a known allergen. Female Perlbright

- white Dunkin

- Hartley guinea pigs (12 per 4

group) were exposed to 0, 0.13, or 0.25 ppm formaldehyde 8 hours/day for 5 consecutive days. 5

On day 5, the animals were sens itized to the common model allergen, OVA, in a 3

- minute, head

6

only exposure to an aerosol of a 5% OVA solution. A booster sensitization with OVA occurred 7

on day 19. A compressor nebulizer with an output rate of 0.75 mL/minute generated the aerosol. 8

Part icle size ranged from 0.5 to 5.0 μm. On day 26, bronchial provocation testing was conducted 9

with 1% OVA challenge (aerosol). Blood samples were taken and anti

- OVA IgG antibodies 10

were quantified by ELISA. Significant airway obstruction was defined as an increase in 11

compressed air in the lung that cannot be expired. Three guinea pigs were exposed to 12

formaldehyde (0.20 ppm) or clean air for 5 days. Immediately after exposure, lung and tracheal 13

tissues were fixed for histologic and morphometric evaluation. Wall thickness of bronch ial and 14

alveolar septa was measured systematically with a microscope

- digitizing

- table set. 15

Significant airway obstruction as measured by compressed air was seen in 3 of 12 16

controls, 8 of 12 0.13 ppm

- exposed, and 10 of 12 0.25 ppm

- treated animals after OVA challenge. 17

The average airway obstruction was increased after 0.25 ppm (mean = 0.35 mL, p < 0.01) but not 18

after 0.13 ppm formaldehyde exposure. However, individual response to OVA sensitization was 19

highly varied, and animals exhibiting a 10

- fold increase in obstruction (measured as compressed 20

air) were seen in both treatment groups (0.13 and 0.25 ppm). Even at the lower exposure (0.13 21

ppm), biologically significant responses were seen in individuals (Figure 4

- 12). 22

Specific anti

- OVA antibodies (IgG1 clas s) were not detected in animals prior to 23

sensitization or in control

- treated animals after sensitization . Measurable anti

- OVA antibodies 24

were elevated in 3 of 12 (at 0.13 ppm) and 6 of 12 (at 0.25 ppm) formaldehyde

- treated guinea 25

pigs after sensitization (Figure 4

- 13). The average anti

- OVA titer for the high

- dose group was 26

significantly higher than for controls ( p < 0.05). The individual responses at the 0.13 ppm 27

exposure level indicate that, although the average group OVA titer may not have reached 28

sta tistical significance, there was a measureable biological response in three individuals. These 29

results indicate that formaldehyde exposure can sensitize previously naïve (non

- sensitized) 30

animals to OVA. 31This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 235  

C o n t r o l s F A 0

. 1 3 p p m F A 0

. 2 5 p p m C A ( m l )

1

0

, 8

0

, 6

0

, 4

0

, 2

0

1

2

Figure 4

- 12. Compressed air in milliliters as pa rameter for airway 3

obstruction following formaldehyde exposure in guinea pigs after OVA 4

sensitization and OVA challenge

. 5

6

Note: CA = compressed air; FA = formaldehyde; ― = median; ** = p < 0.01. 7

8

Source: Redrawn from Riedel et al. (1996). 9

10

+++ 11

12

13

14

Figure 4

- 13. OVA

- specific IgG1 (1B) in formaldehyde

- treated sensitized 15

guinea pigs prior to OVA challenge. 16

17

Note: EU = experimental units; FA = f ormaldehyde; ― = median; ** = p < 0.01. 18

19

Source: Redrawn from Riedel et al. (1996). 20

21

22 C o n t r o l s F A 0

. 1 3 p p m F A 0

. 2 5 p p m  1

. 0 0 0

1 0 0

1 0 A n t i

- O V A

- l g G 1 [ E U ]This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 236 The only significant, treatment

- related histologic change was bronchial edema, with 1

thickening of the bronchial wall in formaldehyde

- exposed animals compared with non

- treated 2

animals subjected to OVA sensitization and subsequent OVA challenge. Bronchial walls were 3

measured as 40.9 ± 2.5 versus 28.2 ± 1.2 μm. No signs of inflammation in the bronchial mucosa 4

were seen with this edema. 5

Tarkowski and Gorski (1995) expose d female Balb/C mice to 0 or 6.63 ppm (0 or 6

2 mg/m 3 ) formaldehyde for either 6 hours/day for 10 days or 6 hours/day once a week for 7

7 weeks. All mice were sensitized intranasally to OVA for 10 days or once a week for 7 weeks. 8

IgE anti

- OVA titers were det ermined from sera collected from four mice every 8 days (1 day 9

after OVA booster) by PCA. A parallel experiment to compare the role of the route of 10

administration was conducted with I.P. rather than intranasal sensitization (1 μg OVA once every 11

7 days). 12

OVA titers increased similarly in control mice and mice exposed to formaldehyde once a 13

week (Figure 4

- 14). In contrast, mice exposed to formaldehyde 6 hours/day for 10 consecutive 14

days a t the beginning of the experiment had increased anti

- OVA beginning after the fourth OVA 15

sensitization, which continued to increase through seven doses of OVA to a peak of 70 PCA 16

units ( p < 0.01) (Figure 4

- 14). Anti

- OVA IgE titers were significantly differ ent between 17

formaldehyde

- treated and nonexposed mice. 18

19

20

Figure 4

- 14. Anti

- OVA titers in female Balb/C mice exposed to 6.63 ppm 21

formaldehyde for 10 consecutive days or once a week for 7 weeks. 22

23

Note:  **=** control mice;  **=** formaldehyde once a week × 7;  **=** formaldehyde 24

10 days. 25

Source: Redrawn from Tarkowski and Gorski (1995). 26

27This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 237 Intraperitoneal sensitization to OVA was much more effective than intranasal 1

sensitization, resulting in titers as high as 1,00 0 after 4 weeks. However, there were no 2

differences between controls and animals treated with formaldehyde via the I.P. route of 3

exposure. Thus, formaldehyde administered intranasally 6 hours/day for 10 days may facilitate 4

the sensitization to allergens. These changes were not observed when formaldehyde was 5

administered intranasally once a week for 10 weeks or via I.P. injection (Tarkowski and Gorski, 6

1995). The authors speculate that formaldehyde may increase permeability of respiratory 7

epithelium and destruction of immunologic barriers. Thus the respiratory tract may become 8

vulnerable to inhaled allergens after formaldehyde exposure (Tarkowski and Gorski, 1995). 9

Ito et al. (1996) conducted three experiments to examine the effects of acute 10

formaldehyd e exposure on bronchoconstriction and the mediators of vascular permeability. 11

Male Wistar rats (five to eight per group) were exposed to 0, 2, 5, 15, or 45 ppm (0, 2.5, 6.2, 12

18.5, or 55.4 mg/m 3 ) formaldehyde for 10 minutes. Baseline pulmonary insufflati on and blood 13

pressure were determined prior to formaldehyde exposure and monitored throughout the 14

experiment. Vascular leakage was measured by injection of Evans blue dye prior to the 15

experiment and determining extravasation 5 minutes postexposure. Brief ly, lungs were perfused 16

with 0.9% saline through an aortic cannula. The lower portion of the trachea and main bronchi 17

were removed, and the Evans blue dye remaining was determined and expressed as ng dye/g 18

tissue. A second experiment was conducted to det ermine if dye leakage continued to increase 19

after exposure. Seven rats were exposed to 15 ppm formaldehyde for 10 minutes, as above. 20

Evans blue dye was injected 5 minutes postexposure, and tissues were perfused and excised 21

15 minutes later. The final ex periment was conducted to determine the effect of certain receptor 22

agonists on the formaldehyde

- induced microvascular leakage. Ten groups of Wistar rats (four to 23

seven per group) were exposed to 15 ppm formaldehyde and injected with Evans blue dye, as 24

bef ore. However, each receptor agonist under test or saline sham was injected 4

– 5 minutes prior 25

to the 10

- minute formaldehyde exposure. Agonists tested included tachykinin NK 1 receptor 26

antagonist (CP

- 99,994) at 1, 3, or 6 mg/kg; a bradykinin B 2 receptor ant agonist (HOE 140) at 27

0.65 mg/kg; and a histamine H 1 receptor antagonist (ketotifen) at 1 mg/kg. 28

Formaldehyde exposure did not change pulmonary insufflation pressure or blood 29

pressure. Formaldehyde increased vascular permeability in a concentration

- depende nt manner in 30

both the trachea and main bronchi for the first 5 minutes after exposure, as measured by Evans 31

blue dye extravasation (Ito et al., 1996) (Figure 4

- 15). Vascular permeability was not increased 32

by formaldehyde exposure from 5 to 15 minutes post exposure (experiment 2). Administration of 33

a selective NK 1 receptor antagonist (CP

- 99,994) inhibited the formaldehyde

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

DRAFT

— DO NOT CITE OR QUOTE 4

- 238 permeability, reducing Evans dye extravasation to control levels at the 3 and 6 mg/kg doses 1

(Figure 4

- 16). 2

3

  

   2 0 0

1 0 0

0

2 5 1 5 4 5 2 5 1 5 4 5 R o o m

A i r F o r m a l d e h y d e ( p p m ) F o r m a l d e h y d e ( p p m ) T r a c h e a M a i n B r o n c h i

4

5

Figure 4

- 15. Vascular permeability in the trachea and bronchi of male 6

Wistar rats after 10 minutes of formaldehyde inhalation. 7

8

Note: Vascular permeability was tested by an increase in Evans blue dye 9

extravasation in the tissue. Solid bars: formaldehyde; open ba rs: room air, n = 7. 10

Values are the means ± SEM of five to seven animals. * p < 0.05 and † p < 0.01 11

versus room

- air

- exposed group (Williams’ test). 12

13

Source: Redrawn from Ito et al. (1996). 14

15

16This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 239 1

2 0 0

1 0 0

0

F o r m a l d e h y d e F o r m a l d e h y d e S h a m S h a m T r a c h e a M a i n B r o n c h i

 

 

 



        

2 3

Figure 4

- 16 . Effect of select receptor antagonists on forma ldehyde

- induced 4

vascular permeability in the trachea and bronchi male of Wistar rats. 5

6

Note: Vascular permeability was tested by an increase in Evans blue dye 7

extravasation. Rats were treated i.v. with 1, 3, or 6 mg/kg CP

- 99,996 (open bars), 8

0.65 mg/kg HOE 140 (hatched bars), 1 mg/kg ketotifen (solid bars), or vehicle 9

(shaded bars) before formaldehyde challenge. Sham: animals were exposed to the 10

sham gas for 15 ppm formaldehyde (10 minutes) after pretreatment with 0.9% 11

saline (0.5 mL/kg i.v.). Data are the means ± SEM of six to seven rats/group. 12

* p < 0.05 versus sham

- stimulated group (unpaired Student’s t test or Welch’s 13

test). † p < 0.05. †† p < 0.01 versus 0.9% saline

- pretreated, formaldehyde

14

exposed control group (Williams’ test). 15

16

Source: Redrawn from Ito et al. (1996). 17

18

Neither the bradykinin B 2 nor histamine H 1 receptor agonists affected formaldehyde

19

induced vascular permeability (Ito et al., 1996). Therefore, the immediate effect of 20

formaldehyde exposure on vascular permeability is mediated, at least in part, through the NK 1 21

receptor but does not seem to require the B 2 or H 1 receptors. This implies a role for tachykinins 22

in formaldehyde

- induced vascular permeability. These findings suggest a neurogenic 23

inflammatory response because the tachyki nins are released from sensory nerve endings in the 24

trachea and bronchi, whereas bradykinin is released from mast cells. 25

Sadakane et al. (2002) investigated the effects of formaldehyde exposure on airway 26

inflammation caused by Der f. Two groups of male ou tbred ICR mice (18/group) were exposed 27

to an aerosol of 0.5% formaldehyde solution produced by an ultrasonic nebulizer for 15 minutes, 28

once a week for 4 weeks. Two groups were similarly treated but exposed to saline aerosol only. 29

Details of the aerosol g eneration and resulting magnitude of exposure were not given. One 30

group each of control and formaldehyde

- exposed mice was sensitized to Der f by an injection 1 31

day prior to formaldehyde exposure (1.5 mg/animal). The same groups were challenged with 32This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 240 intr atracheal instillation of Der f (10 μg/animal) after 4 weeks. Three days after allergen 1

challenge, mice were sacrificed and blood plasma and lung tissue were collected. Blood plasma 2

was analyzed for Der f

- specific immunoglobulins (IgG1 and IgE). Lungs f rom nine mice in each 3

treatment group were homogenized, and Th1 cytokine IL

- 2, Th2 cytokines IL

- 4 and IL

- 5, 4

granulocyte macrophage

- colony

- stimulating factor (GM

- CSF), and the “chemokine regulated 5

upon activation, normal T

- cell expressed and secreted” (RAN TES) protein levels were quantified 6

in the supernatant via ELISA. Lungs from nine mice in each group were fixed, sectioned, and 7

stained to evaluate eosinophil infiltration, lymphocyte infiltration, goblet cell proliferation, and 8

localization of RANTES in the airway epithelium. 9

Der f

- specific IgG1 was present in blood plasma of sensitized mice but was unchanged 10

by formaldehyde exposure (Sadakane et al., 2002). IgE was too low to titer. IL

- 2 and GM

- CSF 11

were undetected in lung homogenate supernatant, and Il

- 4 was unchanged by sensitization or 12

formaldehyde exposure. However, RANTES was increased by both formaldehyde exposure and 13

allergen sensitization and challenge (Table 4

- 51). These increases were more pronounced but 14

less than additive for formaldehyde

- exposed, allergen

- sensitized mice. IL

- 5 was increased by 15

allergen but unaffected by formaldehyde exposure only. However, formaldehyde exposure 16

potentiated the IL

- 5 increase seen with allergen challenge. 17

18

Table 4

- 51

. Cytokine and chemokine levels in lung tissue homogenate 19

supernatants in formaldehyde

- exposed male ICR mice with and without 20

Der f sensitization 21

22

Group Formaldehyde Der f GM

- CSF IL

- 2 IL

- 4 IL

- 5 RANTES

1

- - ND a ND 68.1 ± .9 4.4 ± 0.3 200.1 ± 19.7

2 +

- ND ND 59.5 ± 4.3 4.1 ± 0.2 390.6 ± 37. 4 b

3

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

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

23

a None detected. 24

b p < 0.05 from control. 25

c p < 0.001 from control. 26

d p < 0.05 from Group 2. 27

e p < 0.001 from Group 2. 28

f p < 0.001 from Group 3. 29

30

S ource: Sadakane et al. (2002). 31

32

33

Der f sensitization and challenge increased eosinophil infiltration into the interstitium 34

around the bronchi and bronchioles as well as goblet cell proliferation in the bronchial 35

epithelium (Figure 4

- 17). Formaldehyde ex posure exacerbated the eosinophilic and goblet cell 36

responses to a challenge dose of Der f ( p < 0.05) (Sadakane et al., 2002). Formaldehyde

- induced 37This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 241 eosinophilic infiltration in the absence of sensitization and challenge was not different from non

1

treated, non

- sensitized mice. 2

These results suggest that formaldehyde exposure may aggravate eosinophilic infiltration and 3

goblet cell proliferation that accompanies allergic responses. This response is associated with an 4

increase in IL

- 5, an eosinophilic attract ant, and an increase in RANTES, which recruits 5

eosinophils by chemotaxis in formaldehyde

- exposed and Der f challenged animals, although the 6

effect was not statistically significantly elevated compared with Der f challenge

- induced levels 7

of IL

- 5 and RANTES alone. 8

9

Panel A 10

Panel B 11

c o n t r o l F A D e r f D e r f + F A a b a b c + + + +

+ + +

+ +

+

±

-

12

13

Figure 4

- 17 . The effects of formaldehyde inhalation exposures on eosinophil 14

infiltration (Panel A) and goblet cell proliferation (Panel B) after Der f 15

challenge in the nasal mucosa of male ICR mice after sensitization and 16

c hallenge. 17

18

Note: a p < 0.001 compared with control group; b p < 0.001 compared with 19

formaldehyde group; c p < 0.05 compared with Der f group. 20

Source: Redrawn from Sadakane et al. (2002). 21 c o n t r o l F A D e r f D e r f + F A a b a b c + + + +

+ + +

+ +

+

±

-Eosinophil scoreGoblet cell scoreThis document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 242 Fujimaki et al. (2004a) investigated the long

- term effects of low

- dose formaldehyde 1

exposure on immunologic and neurological inflammation. Female C3H/He mice were exposed 2

to 0, 0.082, 0.393, or 1.87 ppm (0, 0.1, 0.48, or 2.3 mg/m 3 ) formaldehyde 16 hours/day, 3

5 days/week for 12 weeks. Six mice at each exposure level were gi ven injections of OVA plus 4

adjuvant before the initial exposure and in weeks 3, 6, 9, and 11 of the experiment. Five mice at 5

each formaldehyde

- exposure level did not receive OVA injections. One day after the last 6

exposure, mice were weighed and blood, B AL, spleen, and thymus were collected from each 7

animal. After weighing, spleens were disaggregated and spleen cells harvested for cell culture. 8

Immunophenotype of the spleen cells was determined by flow cytometry (CD4, CD8, CD3, and 9

CD19 positive cells). Lymphocyte proliferation in response to lipopolysaccharide (LPS), 10

phytohemagglutinin A (PHA), or OVA was determined after 72 hours in culture. Splenocytes 11

were cultured for 48 hours in the presence of LPS, PHA, and OVA (immunized mice only), and 12

superna tants were collected for cytokine analysis (IL

- 4, IL

- 5, and IFN

- γ). Splenocytes were 13

cultured for 24 hours in the presence or absence of OVA to assess chemokine production (MCP

14

1 and MIP1

- α). Anti

- OVA IgE, IgG 1 , IgG 2, and IgG 3 were quantified in blood pl asma. 15

Body and thymus weights were unchanged by formaldehyde exposure or OVA injection 16

(Fujimaki et al., 2004a), while, in non

- immunized mice, spleen weights were reduced by 17

formaldehyde exposure from 152 mg in controls to 128, 118, and 121 mg in mice expo sed to 18

0.08, 0.4, and 1.8 ppm formaldehyde, respectively. Spleen weights tended to increase in groups 19

exposed to 400 and 2,000 ppb formaldehyde compared with controls in OVA

- immunized mice 20

(control: 117.8 mg compared with 400 ppb: 168.6 mg and control: 12 1.0 mg compared with 21

2,000 ppb:153.2 mg, respectively) but were not statistically significant. 22

To gain insight on the overall pulmonary inflammatory response of mice exposed to 23

formaldehyde in both immunized and non

- immunized mice, the total number and d ifferential 24

count of MPs, neutrophils, lymphocytes, and eosinophils in BAL were counted and were found 25

to be unchanged by formaldehyde in non

- immunized mice. By contrast, in immunized mice 26

exposed to 1.8 ppm formaldehyde, the total number of BAL cells, MP s, and eosinophils were 27

significantly increased compared with non

- immunized controls (9.65 versus 2.84, 7.22 versus 28

2.74, and 2.0 versus 0.02 ×10 4 cells, respectively). 29

To further assess the pulmonary inflammatory response, protein levels of inflammatory 30

c ytokines were determined by ELISA in BAL fluid. Levels of IL

- 1 β in BAL of immunized mice 31

were decreased by formaldehyde exposure ( p < 0.05 at 1.8 ppm formaldehyde), but IL

- 1 **β** levels 32

after formaldehyde exposure were not different from controls in non

- immunized mice (Fujimaki 33

et al., 2004a). All other cytokines or che mokines were either unchanged (TNF

- α, IL

- 6, and GM

34

CSF) or not detected (eotaxin, MIP

- 1α, and MCP

- 1). 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 243 Various neuropeptides, such as brain

- derived neurotrophic factor (BDNF), nerve growth 1

factor (NGF), and substance P are released from vagal nerve endings and mediate a neurogenic 2

inflammatory response. Levels of BDNF, NGF, and substance P were assessed in BAL fluid 3

and/or in plasma. BDNF was not detected in BAL or in plasma. NGF levels in immunized mice 4

were significantly higher than in non

- immunized mic e in both BAL fluid and in plasma. NGF 5

levels in immunized mice were significantly attenuated by 0.08 and 0.4 ppm formaldehyde 6

exposure (Figure 4

- 18) in both BAL fluid and in plasma. Plasma level of substance P (a 7

mediator of neurogenic inflammation) was increased by formaldehyde exposures in non

8

immunized mice (Figure 4

- 19) in both BAL fluid and plasma. This increase appears to be dose

9

dependent and reaches statistical significance at 2,000 ppb formaldehyde exposure in non

10

immunized mice compared with n on

- immunized controls. Similar to NGF, levels of substance P 11

increased in OVA

- immunized mice compared with non

- immunized mice in both BAL fluid and 12

plasma. Similar to NGF, levels of substance P in OVA

- immunized mice were attenuated by 13

formaldehyde exposu re at 80 ppb. 14

15

 

      6

5

4

3

2

1

0



### O V A

F

A 0 8 0 4 0 0 2 0 0 0 ( p p b ) 0 8 0 4 0 0 2 0 0 0 

### - - - - + + + +

16

17

Figure 4

- 18. NGF in BAL fluid from formaldehyde

- exposed female C3H/He 18

mice with and without OVA sensitization. 19

20

Note: The day after the final formaldehyde inhalation, BAL fluid was collected 21

from formaldehyde

- exposed, non

- immunized and formaldehyde

- exposed, OVA

22

immunized mice, and the production of NGF was determined by ELISA. Data 23

are mean ± SEM from five to six animals. ** p < 0.01. 24

25

Source: Redrawn from Fujimaki et al. (2004a). 26

27This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 244 1

2

3 Figure 4 -19

. Plasma substance P levels in formaldehyde

- exposed female 4

C3H/He mice with and without OVA sensitization. 5

6

Note: The day after the final formaldehyde inhalation, plasma samples were 7

collected from formaldehyde-exposed, non-immunized, and formaldehyde8

exposed OVA- immunized mice, and the levels of substance P were determined by 9

ELISA. Data are mean ± SEM from five to six animals. * p < 0.05. FA = 10

formaldehyde. 11

12

Source: Redrawn from Fujimaki et al. (2004a). 13

14

15

Fujimaki et al. (2004a) further investigated the effect of low-level formaldehyde exposure 16

from both immunized and non- immunized mice on the systemic immune response. Spleens were 17

removed from formaldehyde-exposed mice and were cultured in the presence of LPS or PHA 18

(for non-immunized samples) or OVA (for immunized samples). The secretory ability of 19

immunized and non- immunized spleen cells was assessed by measuring IFN

- **γ** release by ELISA. 20This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 245 Formaldehyde exposure (1.8 ppm) increased IFN

- γ fourfold in LPS

- stimulated cultured spleen 1

cells from non

- immunized mice. No other cytokine or chemokine was changed by formaldehyde 2

exposure in cultured spleen cells from non

- immunized mice. In OVA

- immunized mice, 3

formaldehyde had no significant effect on cytokines from stimulated spleen cells. OVA in vitro 4

stimulation significantly increased the chemokines MIP

- 1 and MCP

- 1 for control and 5

formaldehyde

- treated OVA

- immunized mice. The O VA

- stimulated release of MCP

- 1 in vitro 6

was enhanced by formaldehyde exposure in a concentration

- dependent manner, increasing 7

threefold and fourfold at 0.40 and 1.8 ppm, respectively. Increases in MCP

- 1 correlate with 8

reported increases in the associated cytokine, RANTES, which recruits eosinophils by 9

chemotaxis (Sadakane et al., 2002). These formaldehyde

- induced increases in cytokine levels 10

contribute to pulmonary inflammation. The inflammatory response is not mediated by 11

lymphocytes, since lymphocyte s ubsets and in vitro cell proliferation were unchanged by OVA 12

immunization or formaldehyde treatment (Fujimaki et al., 2004a). 13

Anti

- OVA (IgE and IgG 2a ) levels in plasma were unchanged by formaldehyde exposure. 14

Anti

- OVA IgG 1 was reduced in immunized mice ex posed to 400 ppb formaldehyde compared 15

with nonexposed animals. However, this effect did not persist as dose increased. Anti

- OVA 16

IgG 3 was depressed in immunized mice exposed to 0.08 and 0.4 ppm formaldehyde (Fujimaki et 17

al., 2004a). Formaldehyde exposur e did not induce an inflammatory response in lung or tracheal 18

epithelium in sections viewed by light microscopy (Fujimaki et al., 2004a). Although there was 19

a mild infiltration of mast cells into the epithelium of OVA

- immunized mice, there were no 20

effects of formaldehyde treatment on mast cell infiltration. 21

A recent study by Lino dos Santos Franco et al. (2009) exposed male Wistar rats for 22

3 days, 90

- minutes/day, to 1% formaldehyde (by weight; exact doses not reported) by inhalation. 23

Of these, one group was sensitized I.P. to OVA (10 **μ** g), a common allergen, immediately 24

following formaldehyde exposure, and subsequently challenged with OVA 2 weeks later. Other 25

rats were sensitized and challenged but were not exposed to formaldehyde. PCA reaction as well 26

as BAL analysis and whole blood analysis were conducted. Immunohistochemical analysis of 27

platelet endothelial cell adhesion molecule

- 1 (PECAM

- 1) expression, an inflammatory mediator, 28

in lung tissue was also measured. When formaldehyde exposure was followed by OVA 29

sensitization and chal lenge, decreased lung inflammation was reported compared with the group 30

that was OVA

- sensitized but had not been exposed to formaldehyde. Reduced lung mast cell 31

degranulation was also reported in the formaldehyde/OVA group compared with the nonexposed 32

OVA group. Total circulating leukocytes, total bone marrow cells, and lung protein expression 33

levels of PECAM

- 1 were also significantly decreased in formaldehyde/OVA rats compared with 34

non

- formaldehyde exposed OVA rats. The reduction in inflammatory paramet ers in response to 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 246 formaldehyde may be attributed to different study designs, since in this study animals were 1

sensitized after exposure rather than prior to exposure. The results suggest that formaldehyde 2

may functionally alter the activity of certain ce lls, like mast cells, that may downgrade an 3

appropriate immune response to antigen and might serve to threaten lung homeostasis. Due to 4

the unique experimental design of this study, it cannot be directly compared with Sadakane et al. 5

(2002) or Fujimaki et al. (2004a). In addition, this study did not intend to measure whether 6

formaldehyde can exacerbate an asthmatic response but rather set out to identify whether 7

formaldehyde could affect immune homeostasis. 8

In summary, studies suggest that formaldehyde e xposure may induce a predominantly 9

neurogenic inflammatory response via release of neuropeptide, such as NGF and substance P 10

from vagal nerve endings. Formaldehyde does not appear to potentiate a systemic immune 11

response. However, localized pulmonary inf lammation can be potentiated by formaldehyde 12

exposure, as indicated by the increased presence of eosinophils and certain proinflammatory 13

cytokines (IFN

- **γ** ). This response does not appear to be mediated by classic immunogenic 14

mechanisms since studies have failed to report elevated levels of anti

- formaldehyde

- specific IgE. 15

Several studies have shown that exposure to formaldehyde can facilitate allergic sen sitization in 16

previously naïve animals, and it is thought that this effect may occur due to formaldehyde’s 17

ability to increase microvascular leakage in the nasal epithelium and by causing damage to the 18

nasal barrier (Ito et al., 1996). Sadakane et al. (20 02) demonstrated that formaldehyde exposure 19

can also exacerbate allergic responses by enhancing the response to challenge allergen. Thus, 20

formaldehyde may exacerbate allergic responsiveness by aggravating the sensitization response 21

in previously naïve ani mals by altering the permeability of the mucosal barrier in nasal 22

compartments. Neurogenically derived inflammation, including stimulation of the trigeminal 23

nerve and release of braykinin, suggests that the MOA for sensitization may ultimately have its 24

ro ots in neurogenic inflammation rather than an immunogenic response. In addition, using a 25

different protocol, Lino dos Santos Franco et al. (2009) suggest that formaldehyde exposure can 26

adversely affect lung homeostasis by reducing the activity of importan t inflammatory mediators 27

(mast cells, circulating leukocytes, PECAM

- 1 expression) when it occurs prior to sensitization, 28

thus downgrading an appropriate immune response. 29

30

4.2.1.5.2. Dermal sensitization. Wahlberg (1993) used Hartley strain guinea pigs as test 31

animals to determine the skin irritancy of a suite of industrial chemicals, including 32

formaldehyde. Aqueous solutions of the compound in a 0.1 mL volume were applied to the 33

shaved flanks of guinea pigs and gently rubbed into the skin with a cotton

- t ipped applicator. 34

Sites were left open and the treatments repeated once daily for 10 days. A number of indices of 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 247 acute skin irritation were monitored, including erythema via visual scoring and edema and skin

1

fold thickness using Harpenden calipers. Var ying concentrations of formaldehyde (up to a 10% 2

solution) induced a dose

- dependent increase in skin

- fold thickness. Responses also showed 3

shorter latencies at the higher concentrations. For example, erythema was first observed on 4

day 2 when 10% formalde hyde was applied, day 5 (for 3%), and day 6 (for 1%). 5

Lee et al (1984) investigated the role of different routes of exposure in formaldehyde

6

induced allergic sensitization. Two sets of four male English smooth

- haired guinea pigs received 7

topical applicati ons of 100 **μ** L 37% w/v formalin distributed over two shaved, depilated dorsal 8

sites two times over the course of 2 days at different sites. The total dose was calculated as 9

74 **μ** g/animal. In addition, eight animals received a single topical application onto a 15 mm a rea 10

of the dorsal surface. The applied dose of 25 **μ** L formaldehyde was dissolved in saline. Two 11

other groups of guinea pigs were exposed to either 6 ppm (6 hours/day for 5 days) or 10 ppm 12

(6 hours/day for 5 days) formaldehyde by inhalation. A third group of guinea pigs was exposed 13

to 10 ppm formaldehyde for 8 hours/day for 5 consecutive days by inhalation. All animals were 14

evaluated for contact sensitivity by topical application of 20 mL formaldehyde diluted with 15

saline and distributed in a 15 mm area on the backs of the shaved guinea pigs (Lee et al., 1984). 16

Sites were visually inspected for erythema at 1, 6, 24, and 48 hours following the topical 17

application, and reactions were scored. No erythema was observed in control animals. None of 18

the guinea p igs in the 6 hours/day inhalation groups (6 and 10 ppm formaldehyde) developed 19

skin sensitivity tested on day 9 (4 days after the initial exposure regimen ended). Two of four 20

guinea pigs exposed to 10 ppm formaldehyde for 8 hours/day for 5 consecutive day s developed 21

mild skin sensitization tested on day 31. Contact sensitivity increased in a dose

- dependent 22

fashion in groups of animals that had been sensitized via the dermal route. Thus, dermal 23

exposure resulted in contact sensitivity. Inhalation exposur e did not consistently produce contact 24

sensitivity. 25

Arts et al. (1997) used a local lymph node assay (LLNA) and the induction of IgE to 26

monitor the sensitization of female Wistar rats (low IgE

- responders) and BN rats (high IgE 27

responders). For the LLNA a ssay, animals were sensitized by the application of varying 28

concentrations of formaldehyde in raffinated olive oil on the dorsum of both ears on days 0, 1, 29

and 2. Control animals were treated with raffinated olive oil alone. Animals received an I.P. 30

inje ction of BrdU on day 5 and were subsequently sacrificed. Ear

- draining lymph nodes were 31

collected, fixed, and sectioned, and the mitotic activity was monitored following successive 32

incubation of the sections in anti

- BrdU, biotin

- labeled rabbit anti

- mouse a ntibody, peroxidase

33

conjugated streptavidin, and 3,3

- diaminobenzidine tetrahydrochloride. For serum IgE responses, 34

150 μL of different concentrations of formaldehyde were applied to the shaved flanks of rats on 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 248 day 1, then 75 μL of the same chemical at 50 % of the initial concentration were applied to the 1

dorsum of each ear on day 7. The amount of IgE in the blood was measured using ELISA but 2

appeared to be little affected by formaldehyde treatment in either species of rat. However, the 3

ear

- draining lymph nodes of both strains of rat showed a comparative increase in weight in 4

response to formaldehyde, and proliferation (BrdU positive) of paracortical cells was observed in 5

response to increasing doses of the compound. This response was most notable in BN r ats 6

treated with 10% formaldehyde. Arts et al. (1997) concluded that the irritant and sensitizing 7

properties of formaldehyde may act through non

- IgE

- immune mechanisms. 8

Hilton et al. (1998) used the LLNA assay in female CBA/Ca (H

- 2 k haplotype) mice to 9

com pare the skin sensitizing potencies of formaldehyde and glutaraldehyde. The comparison 10

was set on a quantitative basis by determining the concentration of each compound necessary to 11

induce a threefold increase in lymph node cell proliferative activity (ef fective concentration 12

[EC 3 ]). While both aldehydes induced a dose

- dependent proliferative response, the 13

incorporation of [ 3 H]

- methylthymidine was far greater in animals exposed to glutaraldehyde 14

versus formaldehyde (with EC 3 values of 0.002

– 0.006 mol/L fo r glutaraldehyde versus 0.11

– 15

0.18 mol/L for formaldehyde). These data indicate the potential of both chemicals to induce skin 16

sensitization, although the potency of glutaraldehyde was far greater than that of formaldehyde. 17

Xu et al. (2002) evaluated the extent to which the expression of some cytokines may 18

change as a result of cutaneous exposure to formaldehyde in mice. Female Balb/C mice were 19

skin painted with three topical applications of 100 μL of 17.5% formaldehyde or distilled water 20

with a 1

- day int erval between each application. Spleen and draining lymph nodes were 21

harvested on days 3, 5, 7, 9, or 12 after the last skin painting. In some animals, contact 22

hypersensitivity was induced by applying 2% formaldehyde to both sides of mouse ears on day 3 23

following the last skin painting. For this endpoint, the percent increase in thickness of the ears 24

was monitored. For the cytokines, mRNA expression levels of IL

- 2, IL

- 4, IL

- 5, IL

- 10, IL

- 12, 25

IL

- 13, IL

- 15, IL

- 18, and INF

- **γ** were determined semiquantitative ly by measuring the amount of 26

individual mRNAs following amplification with the reverse transcriptase (RT)

- PCR. The 27

relative amounts of cytokine mRNAs were calculated as the ratio of cytokine mRNA to that of 28

glyceraldehyde

- 3

- phosphate dehydrogenase, as re vealed in specific bands on an agarose gel. 29

Cutaneous formaldehyde treatment was associated with the long

- lasting expression of 30

IL

- 4 and IFN

- **γ** mRNAs in mouse spleen and draining lymph nodes and with IL

- 15 mRNA only 31

in mouse spleen. Only IL

- 13 mRNAs displayed a transient increase in expression in both spleen 32

and draining lymph nodes. Levels of IL

- 2, IL

- 12, and IL

- 15 were increased in the mouse spleen 33

but not the lymph nodes. The mouse ear swelling test gave positive correlations with enhanced 34

expression of mRNA for IL

- 4 and IFN

- **γ** (Table 4

- 52). 35

36This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 249 1

Table 4

- 52. Correlation coefficients among ear swelling responses and skin 2

mRNA levels in contact hypersensitivity to formaldehyde in mice 3

4

Variables Correlation coefficients

IL

- 2 IL

- 4 IFN

- **γ** Ear swelling 0.50 0.74 a 0.67 a

IL

- 2

– 0.39 0.60

IL

- 4

– – 0.79 a

5

a Statistically significant ( p < 0.05). 6

7

Source: Xu et al. (2002). 8

9

10

4.2.1.5.3. Summary o f sensitization studies. Several animal studies report increased airway 11

resistance and BC due to inhalation exposures to formaldehyde (Nielsen et al., 1999; 12

Swiecichowski et al., 1993; Biagini et al., 1989; Amdur, 1960). Changes in pulmonary 13

resistance w ere observed as early as 10 minutes after exposure (Biagini et al., 1989), and 14

reported effect levels ranged from 0.3

– 13 ppm. Other pulmonary effects were reported in 15

conjunction with BHR, such as increased tracheal reactivity and decreased pulmonary elas ticity 16

(Swiecichowski et al., 1993; Amdur, 1960). Although BHR is a common result of Type I 17

hypersensitivity reaction to an allergen, the observation of BHR alone is not sufficient to 18

demonstrate that an agent induces Type 1 hypersensitivity. 19

BHR may be directly induced both pharmacologically and neurogenically (Joos, 2003; 20

Cain, 2001; Meggs, 1995). There is little evidence that formaldehyde itself is an allergen 21

recognized by the immune system, especially via inhalation (Lee et al., 1984). Although 22

for maldehyde exposure has been reported to alter cytokine levels and immunoglobulins in some 23

experimental systems, these immunomodulatory effects do not support a type 1 hypersensitivity. 24

IgE was unchanged (Fujimaki et al., 2004a; Lee et al., 1984), and cyto kine profiles were not 25

consistent with the Th

- 2 cytokines expected in IgE mediated hypersensitivity (Fujimaki et al., 26

2004a; Ohtsuka et al., 2003). 27

Formaldehyde

- induced dermal sensitization show parallel results. The physical signs of 28

irritation and sens itization are consistently shown (e.g., rashes, edema). Some involvement of 29

the immune response has been demonstrated with positive LLNA assays, indicating proliferation 30

of lymphocytes in lymph nodes draining the affected area (Hilton et al., 1998; Arts e t al., 1997). 31

Increased expression of Th

- 2 cytokines in the lymph nodes of mice given dermal applications of 32

formaldehyde does indicate an immune component to the observed sensitization. However, the 33

response does not seem to be mediated by IgE (Arts et al., 1997; Lee et al., 1984). 34This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 250 Ito et al. (1996) reported that a tachykinin NK 1 receptor, but not the histamine H 1 or 1

bradykinin B 2 receptors, is involved in formaldehyde

- induced vascular permeability. 2

Neuropeptides NGF and substance P were affected in B AL and stimulated splenocytes from 3

formaldehyde

- exposed mice, with greater effects seen in OVA

- immunized mice. Tachykinins 4

(e.g., substance P and neurokinin A) are produced by nerve cells and can directly stimulate 5

bronchoconstriction (Van Schoor et al., 2000). Substance P is also a mediator of neurogenic 6

inflammation. Therefore, although formaldehyde may induce some of the symptoms of type 1 7

hypersensitivity, these symptoms are more likely neurogenic than immunogenic in origin. 8

In contrast, formaldehyde enhances immunogenic hypersensitivity of known allergens 9

(Sadakane et al., 2002; Riedel et al., 1996; Tarkowski and Gorski, 1995). This potentiation 10

varied based on sensitization protocols (respiratory tract versus systemic, frequency and timing 11

of immun ization, allergen, etc.) and formaldehyde exposure regimens (concentration, continuous 12

versus intermittent exposures). Taken as a whole, the results support the finding that 13

formaldehyde exposure can aggravate a type 1 hypersensitivity response (Table 4

- 5 3). 14

15

4.2.1.6. Neurological and Neurobehavioral Function 16

4.2.1.6.1. Inhalation exposure. There are a number of published reports examining the effects 17

of formaldehyde exposure on nervous system structure and function. The reports e valuating 18

behavioral effects fall into three main categories: (1) behavioral responses evaluated during or 19

immediately following formaldehyde exposures, which may include effects due to the potential 20

irritant properties of the chemical, (2) acute or short

- term exposures followed by behavioral 21

assessments conducted 2

– 24 hours after termination of formaldehyde exposure, which reflect 22

sustained effects of chemical exposure independent of its irritant properties, and (3) repeated 23

exposures to formaldehyde follo wed by neurological assessments performed throughout the 24

treatment period or several days to weeks after termination of treatment. In addition to reports 25

evaluating changes in behavior, there are several reports evaluating neuropathological effects or 26

cha nges in brain chemistry. 27

28This document is a draft for reviewpurposes only and does not constituteAgency policy4-251 Table 4

- 53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due to

formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

Inhalation studies

Cy nomolgus

monkeys 9 Methacholine

- sensitive monkeys

exposed to 2.5 ppm

formaldehyde for 10 minutes Formaldehyde increased pulmonary resistance after 2, 5, and

10 minutes. LOAEL

2.5 ppm Biagini et al.

(1989)

Hartley

guinea pigs

(male) 8 0.86, 3.4, 9.4, 31.1 ppm formaldehyde for 2 hours or

0.11, 0.31, 0.59, 1.05 ppm for

8 hours Total pulmonary resistance increased after 2 hours exposure at

9.4 and 31.1 ppm. Effect was reversible and returned to

baseline within 30 minutes. Total pulmonary resistance was

incre ased after 8 hours exposure at 0.3 and 1 ppm. Amount of

acetylcholine needed to achieve doubled pulmonary resistance

was decreased in animals after 2 hours exposure. NA Swiecichowski

et al. (1993)

Hartley

guinea pigs

(male) 5

– 7 3.4 ppm for 8 hours No cha nges in ex vivo tracheal constriction or inflammation. NA Swiecichowski

et al. (1993)

F344 rats

and BN rats 5 16 ppm 3 hours/day, 5 days Modest changes in inflammatory cytokine expression, but

respiratory and olfactory epithelial lesions were more severe in

F344 rats than in BN rats. NA Ohtsuka et al.

(2003)

English

smooth

haired

guinea pigs 4 6, 10 ppm, 6 hours/day, 5 days,

combined with provocation

challenge (2 or 4 ppm on day 7,

or days 7, 22, and 29) Inhalation challenge with 6 or 10 ppm followed by b ronchial

challenge failed to increase respiratory sensitivity NA Lee et al.

(1984)

Perlbright

white,

Duncan

Hartley

guinea pigs

(female) 12 0, 0.13, 0.25 ppm 8 hours/day,

5 days. The animals were

sensitized to OVA (3 minutes

exposure to 5% OVA aerosol) A nti

- OVA titer was significantly elevated over controls in

animals exposed to 0.25 ppm formaldehyde and showed that

formaldehyde may sensitize previously naïve animals to OVA. NA Riedel et al.

(1996)

Balb/C mice

(female) 4 0, 6.63 ppm 6 hours/day for

10 da ys or 6 hours/day

once/week for 7 weeks. All

mice were sensitized to OVA Formaldehyde administered intranasally for 6 hours/day for

10 days may facilitate sensitization to allergens since anti

- OVA

titers were elevated over control animals. However, the l ength

and duration of exposure appears to affect development of

sensitization. NA Tarkowski and

Gorski (1995)This document is a draft for reviewpurposes only and does not constituteAgency policy4-252 Table 4

- 53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due to

formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

Wistar rats

(male) 5

– 8 0, 2, 5, 15, 45 ppm for

10 minutes Pulmonary insufflation or blood pressure were not altered.

Vascular permeability incre ased in concentration

- dependent

manner and could be reduced by adding a NK1 selective

antagonist. NA Ito et al.

(1996)

Outbred ICR

mice (male) 18 0.5% formaldehyde for 15

minutes, once/week for 4 weeks.

Both control and exposed groups

were exposed to Der f by I.P.

injection 1 day before

formaldehyde and then

challenged with Der f after

4 weeks. More pronounced RANTES production in formaldehyde

- treated

and sensitized rats than in sensitized rats that had not been

exposed to formaldehyde. Formaldehyde also potentiated IL

- 5

production associated with sensitization. NA Sadakane et al.

(2002)

C3H/HeJ

mice

(female) 6 0, 0.082, 0.393, 1.87 ppm

16 hours/day, 5 day/week,

12 weeks. Mice also given OVA

plus adjuvant before exposure,

and again 3, 6, 9, 11 weeks aft er exposure. Some formaldehyde

mice did not receive any OVA Substance P and NGF were increased dose dependently in

formaldehyde

- treated, non

- immunized mice but were attenuated

in formaldehyde

- treated immunized mice compared with

nonexposed, immunized cont rols. LOAEL

0.082 ppm Fujimaki et al.

(2004a)

Wistar rats

(male) NA 1% Formaldehyde by weight for

90 minutes for 3 days. One

group was sensitized to OVA

after to formaldehyde exposure

and then challenged with OVA

afterwards. Others were

sensitized and c hallenged but not

exposed to formaldehyde. Total circulating leukocytes, bone marrow cells, and lung

protein PECAM expression were significantly decreased in

formaldehyde/OVA rats compared with OVA rats. NA Lino dos

Santos Franco

et al. (2009)

Dermal sens itization

Hartley

guinea pigs 5 Skin painted once/day for

10 days with 0.1 mL of 1, 3, or

10% formaldehyde Varying concentrations (up to 10%) induced dose

- dependent

increase in skin

- fold thickness. Erythema seen earlier at higher

doses (2 days at 10% fo rmaldehyde vs 5 days at 3% or 6 days at

1%). Wahlberg et

al. (1993)This document is a draft for reviewpurposes only and does not constituteAgency policy4-253 Table 4

- 53: Summary of sensitization and atopy studies by inhalation or dermal sensitization due to

formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

English

smooth

haired

guinea pigs 4 Group 1: skin painted, 100 μL

37% formalin twice over 2 days,

Group 2: single topical

application of 25 μL

formaldehyde

Group 3: 10 ppm 6 hours/day f or 5 days by inhalation Two of four guinea pigs from group 3 had mild skin

sensitization after day 31. Contact sensitivity developed in a

dose

- dependent manner in the dermal groups (group 1 and 2). Lee et al.

(1984)

Wistar and

BN rats

(female) 4 Applica tion of formaldehyde to

ears on days 0, 1, 2, followed by

an I.P. injection of BrdU. Ear

- draining lymph nodes increased in weight in response to

formaldehyde, reflected in increased number of BrdU

- stained

cells, most notably in BN rats (high IgE responders ) treated with

10% formaldehyde Arts et al.

(1997)

CBA/Ca

mice NA Compared glutaraldehyde to

formaldehyde to induce a local

lymph node assay Glutaraldehyde and formaldehyde induced a dose

- dependent

proliferative response that was greater in glutaraldehyd e

- treated

animals Hilton et al.

(1998)

Balb/c mice

(female) 3

– 5 Skin painted with 100 μL of

17.5% formaldehyde every other

day for days 3, 5, 7, 9, 12 Cutaneous treatment associated with long

- lasting expression of

various cytokines from draining lymph no des and spleen. NA Xu et al.

(2002)

NA = not applicab le.This document is a draft for review purposes only and does not constitute Agency policy.

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- 254 4.2.1.6.1.1.

Clinical signs 2 Behavioral response 1

Several studies that were focused on general toxicity or carcinogenicity of formaldehyde 3

also assessed clinical signs in exposed animals, which may be related to adverse effects on the 4

nervous system. Procedural details for the assessments, or specific data regarding findings, were 5

not provided. Signs recorded included uncoordinated locomotion and climbing of cage walls at 6

20 ppm formaldehyde in rat s (Woutersen et al., 1987); restlessness at 15 ppm formaldehyde in 7

rats (Morgan et al., 1986a); dyspnea, listlessness, and hunched posture at 20 ppm and ataxia at 8

40 ppm in mice (Maronpot et al., 1986); and dyspnea in rats at 14.3 ppm formaldehyde (Kerns e t 9

al., 1983). Given the lack of information regarding procedures used for these evaluations and the 10

limited reporting of results, the utility of these data is limited. 11

12

Irritant threshold detection 13

Wood and Coleman (1995) evaluated the irritant properties of acute formaldehyde 14

exposure in mice. Adult male Swiss mice (eight/group) were initially trained to terminate a 15

60

- second exposure to an irritant gas (ammonia, 1,000 ppm) by poking their noses into a conical 16

sensor five times to produce a 60

- second fac ial shower of clean air. Each test session consisted 17

of 25 exposure trials. Following training, response to formaldehyde was evaluated, using the 18

same testing scenario. Each day mice had a morning exposure session to ammonia and an 19

afternoon session to formaldehyde. Formaldehyde concentrations tested were different each day, 20

in sequence from 0, 1, 1.8, 3, 5.6, and 10 ppm (0, 1.23, 2.21, 3.68, 6.87, and 12.3 mg/m 3 ) and 21

then stepping back again from 10 to 0 ppm. Half of the animals were tested in an asce nding 22

order of formaldehyde concentrations, the other half in a descending order. The frequency of 23

terminating exposure, error rate, and the time lapse to termination were recorded. The 24

concentration at which 50% of the formaldehyde deliveries would be e xpected to be terminated 25

was estimated (AC 50 ) by simple linear regression or by analysis of covariance on the logit 26

transform of percentage terminated as a function of log concentration. 27

All mice were trained successfully to terminate 100% of ammonia expos ures, but varied 28

responses were observed with formaldehyde exposure. In general, time taken to terminate 29

formaldehyde exposure decreased significantly with increasing formaldehyde concentration. 30

Mice terminated more exposures to 1 ppm formaldehyde than t o air alone ( p < 0.0005), and the 31

error rate, generally below 40%, did not significantly differ with formaldehyde concentration 32

tested. Each animal had two test sessions with each formaldehyde concentration (once during 33

the ascending sequence and again du ring the descending sequence); both the time to termination 34

( p < 0.0012) and AC 50 were decreased in the second series of tests. One method of estimation by 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 255 the authors yielded an AC 50 of 3.63 ppm for the first series of tests versus 1.88 ppm for the 1

secon d series. A two

- way repeated measures ANOVA with replication and concentration as 2

within variables was highly significant ( p < 0.00005). These studies indicate mice are sensitive 3

to the irritant properties of formaldehyde at exposure concentrations as lo w as 1 ppm, and 4

animals reacted more swiftly and with greater accuracy to terminate formaldehyde exposure as 5

the concentration increased. However, a wide variety of responses was noted on an individual 6

animal basis. Two of the eight mice terminated 90% o f the trials during 1 ppm exposures and 7

80

– 100% of trials at all other tested formaldehyde concentrations. One mouse terminated fewer 8

than 10% of the formaldehyde exposure trials (1

– 10 ppm) during the testing regimen but had a 9

92% response rate to 20 ppm formaldehyde. The remaining five mice responded with increasing 10

termination frequency as formaldehyde concentration increased from 1 to 10 ppm, with an AC 50 11

of 2.72 ppm (3.34 mg/m 3 ). 12

13

4.2.1.6.1.2. Motor activity and habituation.

The authors reported no signs of irritation or changes in activity or food or water intake 25

during exposure. In general, sniffing was increased after formaldehyde exposure a nd movement 26

was decreased (crossed quadrants and climbing) in both male and female rats ( p < 0.05). 27

Significant reductions in horizontal movements (crossed quadrants) were observed at all dose 28

levels and were characterized by a U

- shaped dose response (Fig ure 4

- 20). The lowest dose 29

tested (1 ppm) demonstrated a higher level of activity suppression than the two higher doses, but 30

all groups were still suppressed relative to controls. Although female rats displayed a greater 31

level of activity overall, a simi lar U

- shaped dose

- response pattern was also observed. 32 Malek et al. (2003a) exa mined open field behavior 14

of rats after acute formaldehyde exposures. Male and female LEW.1K rats (15/sex/group) were 15

exposed to 0, 1, 2.5, or 5 ppm (0, 1.23, 3.08, or 6.15 mg/m 3 ) formaldehyde for 2 hours. 16

Formaldehyde was vaporized from aqueous solution s directly below the exposure chamber. 17

Formaldehyde levels were checked 16 times throughout the 2

- hour exposure periods. Mean 18

formaldehyde levels of 1.01 ± 0.29 ppm, 2.51 ppm (SD is missing), and 5.0 ± 0.27 ppm were 19

achieved. Locomotor activity was asse ssed for 3 minutes in an open field 2 hours after 20

termination of formaldehyde exposure and again 24 hours later, using an automated device to 21

count the number of squares crossed. Other behaviors were noted, including grooming (face 22

cleaning, fur licking, and scratching), rearing, sniffing (air and floor), wall climbing, and 23

defecation. 24

33

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

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- 256 1

2

Figure 4

- 20. Motor activity in male and female rats 2 hours after exposure to 3

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

Greater reductions were observed in the lowest dose group, a pattern that 5

was evident in both genders. ** = different from control, p < 0.005. 6

7

Source: Drawn from data reported by Malek et al. (2003a). 8

9

10

Activity in the same apparatus was reassessed 24 hours later. As expected , controls 11

demonstrated habituation to the test apparatus, exhibiting only 20% of the motor activity 12

observed on day 1 (Figure 4

- 21). In contrast, formaldehyde

- treated animals failed to 13

demonstrate the same degree of habituation. Activity levels for male s observed on day 2 were 14

60

– 80% of the activity levels seen on day 1. Formaldehyde

- treated females also failed to 15

habituate and actually demonstrated increases in activity on day 2 relative to day 1 at all 16

formaldehyde exposure levels. 17 Number



###  

**  ** **

**

Control 1 ppm 2.5 ppm 5.0 ppm 

### Activity as crossed quadrants

Males

Females



### 0 20 40 60 80 100

Formaldeh ydeThis document is a draft for review purposes only and does not constitute Agency policy.

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- 257 Male LEW.1K Rats

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

Control 1 ppm 2.5 ppm 5.0 ppm

FormaldehydeHabituation (percent ) Female LEW.1K Rats

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

Control 1 ppm 2.5 ppm 5.0 ppm

FormaldehydeHabituation (percent)

1

2

Figure 4

- 21. Habituation of motor activity was observed in control rats 3

during the second observation period (day 2, 24 hours after formaldehyde 4

exposure). 5

6

Note: Habituation is shown here as the percent decrease in number of crossings 7

between sessions from day 1 to da y 2. The degree of habituation was reduced in 8

male rats exposed to formaldehyde (left panel) since their activity was closer to 9

100% of that seen on day 1. Females (right panel) had increased activity on day 2 10

(greater than 100% of activity on day 1), wh ich is a sensitization rather than 11

habituation. 12

13

Source: Drawn from data reported by Malek et al. (2003a). 14

15

16

A follow

- up study by Malek et al. (2003b) further expanded the dose

- response analysis 17

for acute formaldehyde exposure. As described above, male a nd female LEW.1K rats (10 per 18

sex per group) were exposed at 0, 0.1, 0.5, or 5 ppm (0, 0.123, 0.615, or 6.15 mg/m 3 ) 19

formaldehyde for 2 hours. Formaldehyde levels were checked nine times per hour during the 20

exposure periods, and mean values were found to b e 0.13 ± 0.04, 0.48 ± 0.05, and 21

5.18 ± 0.66 ppm. Open field behavior was evaluated for each animal 2 hours after formaldehyde 22

exposure. The number of crossed quadrants for both controls and a 5 ppm group were generally 23

comparable with those observed in t he first study, although female values were somewhat lower. 24

Horizontal movement was decreased by formaldehyde exposure in a dose

- dependent manner 25

with significant reductions in motor activity as low as 0.1 ppm in males and 0.5 ppm in females 26

(Figure 4

- 22) . The consistency of the findings across studies and between genders provides 27This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 258 greater confidence in the effects of low

- level formaldehyde exposure on this standard test of 1

neurotoxicity. 2

3

0 10 20 30 40 50 60

0 0.1 0.5 5.0 0 0.1 0.5 5.0

formaldehyde (ppm)number of crossed quadrants

male female



### *

***

*** **

***

4

Figure 4

- 22. Motor activity was reduced in male and female LEW.1 K rats 5

2 hours after termination of 10

- minute formaldehyde exposure. 6

7

Note: Values are means ± SDs. * = different from control, p < 0.05. ** = different 8

from controls, p < 0.01. *** = different from controls, p < 0.001. 9

10

Source: Drawn from data reported i n Malek et al. (2003b). 11

12

13

Malek et al. (2004) also assessed the capacity of formaldehyde to induce persistent 14

behavioral deficits in mice. Groups of 20 male AB mice received a single 2

- hour exposure to 0, 15

1.1, 2.3, or 5.2 ppm (0, 1.3, 2.8, or 6.4 mg/m 3 ) f ormaldehyde prior to being tested 2 and 24 hours 16

after exposure for a series of behavioral responses, including ambulation (crossed squares), 17

grooming, sniffing, rearing, wall climbing, and defecation. Even though there were no clinical 18

signs of toxicity in any of the exposed groups, a number of behavioral anomalies were apparent 19

in response to formaldehyde exposure, some of which persisted for at least 24 hours, as indicated 20

in Tables 4

- 54 and 4

- 55. 21

22

23This document is a draft for review purposes only and does not constitute Agency policy.

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- 259 Table 4

- 54. Fluctuation of behavioral responses when male AB mice inhaled 1

formaldehyde in a single 2

- hour exposure: effects after 2 hours 2

3

Open field parameter Formaldehyde concentration (ppm) a

0 1.1 2.3 5.2

No. of crossed inner squares 34.10 ± 7.51 25.30 ± 5.03 b 21.20 ± 3.41 b 16.10 ± 5.37 b

No. of cross ed peripheral squares 56.65 ± 9.68 59.55 ± 9.75 49.70 ± 13.24 29.15 ± 7.47 b

Total no. of crossed squares 90.75 ± 11.08 84.85 ± 9.96 71.10 ± 13.91 b 44.20 ± 7.42 b

Air sniffing 19.35 ± 2.5 21.50 ± 4.26 16.35 ± 3.84 c 8.10 ± 1.77 b

Floor sniffing 20.95 ± 3.72 26.50 ± 4.64 b 21.35 ± 4.77 22.80 ± 4.02

Grooming 7.95 ± 2.26 7.10 ± 3.19 7.05 ± 2.48 6.55 ± 2.06

Rearing 17.85 ± 2.56 13.90 ± 3.19 b 11.30 ± 2.30 b 9.95 ± 1.61 b

Wall climbing 13.20 ± 3.09 14.55 ± 2.74 13.95 ± 2.31 13.95 ± 1.82

No. of excreted fecal boli 0.65 ± 0.81 0.75 ± 0.85 0.80 ± 0.77 0.90 ± 1.12

4

a Values are means ± SDs. 5

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

c Statistical significance of differences from controls ( p < 0.05). 7

8

Source: Malek et al. (2004). 9

10

11

Table 4

- 55. Fluctuation of behavioral responses when male AB mice inhaled 12

formaldehyde in a single 2

- hour exposure: effects after 24 hours 13

14

Open field parameter Formaldehyde concentration (ppm) a

0 1.1 2.3 5.2

No. of crossed inner squares 10.40 ± 2.35 9.55 ±1.73 9 .10 ± 1.25 9.70 ± 1.13

No. of crossed peripheral squares 42.80 ± 9.27 44.85 ± 14.60 44.95 ± 16.56 41.10 ± 9.08

Total no. of crossed squares 53.20 ± 8.67 54.40 ± 14.77 54.05 ± 15.81 50.80 ± 9.15

Air sniffing 13.65 ± 2.81 13.30 ± 3.21 12.65 ± 2.70 12.30 ± 4.14

Floor sniffing 21.55 ± 3.47 15.85 ± 3.94 b 13.25 ± 4.17 b 17.65 ± 3.13 b

Grooming 8.35 ± 2.56 13.95 ± 2.21 b 10.20 ± 3.33 c 11.90 ± 3.26 b

Rearing 18.30 ± 4.23 12.40 ± 2.23 b 12.25 ± 2.17 b 12.00 ± 3.32 b

Wall climbing 9.25 ± 2.38 8.70 ± 1.98 8.20 ± 2.14 9.90 ± 2.27

No. of excreted fecal boli 0.80 ± 0.83 1.20 ± 0.83 1.60 ± 0.94 c 1.20 ± 0.89

15

a Values are means ± SDs. 16

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

c Statistical significance of differences from controls ( p < 0.05). 18

19

Sour ce: Malek et al. (2004). 20

21

22

Usanmaz et al. (2002) assessed spontaneous locomotor activity (SLMA) in Balb/c mice 23

(4

– 14 per group, sex unspecified) after both acute and subchronic formaldehyde exposures. 24

Prior to the acute exposure, mice were acclimated to the exposure chamber for 4 days but 25

exposed only to clean air. On the fifth day, mice (six/group, sex unspecified) were exposed for 26

3 hours at 0, 1.8, 3.2, 4.5, 6.4, 9.7, or 14.8 ppm (0, 2.2, 3.9, 5.5, 7.9, 11.9, or 18.2 mg/m 3 ) 27

formaldehyde. Mice were re moved from the exposure chamber, and SLMA behavior was 28This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 260 evaluated by direct observation for 5 minutes. In addition to horizontal and vertical movement, 1

wet dog shake (WDS) behavior was noted. In a separate trial, Balb/c mice (six/group, sex 2

unspecified) w ere exposed to 8.2 ppm formaldehyde for 1 week, 2 ppm formaldehyde for 3

2 weeks, or 3.3 ppm formaldehyde for 3 weeks (3 hours/day, 5 days/week) compared with 4

controls exposed only to air. SLMA behavior was observed for 5 minutes after the last exposure. 5

M ice exposed to 8.2 ppm formaldehyde for 1 week, 3.3 ppm formaldehyde for 2 weeks, and 6

2 ppm formaldehyde for 3 weeks lost weight over the course of the treatment ( p < 0.05). All 7

other treatment groups had weight gain similar to control mice. 8

As shown in F igure 4

- 23, acute 3

- hour formaldehyde exposures resulted in a dose

9

dependent decrease in SLMA. Decreases in horizontal activity were significant for the three 10

highest dose groups (6.4, 9.7, and 14.8 ppm), and decreases in vertical activity were significan t 11

for all six formaldehyde treatment groups. SLMA was similarly decreased following subchronic 12

exposures (data not shown here). Although the experimental protocol included longer exposures 13

and a slightly longer observation period (5 versus 3 minutes) tha n in Malek et al. (2003a, b), the 14

results are consistent, indicating decreased activity in formaldehyde

- exposed animals several 15

hours after exposure was ended. 16

17

1 0 0

8 0

6 0

4 0

2 0

0 S L M A a f t e r a c u t e e x p o s u r e s

 

 

   

   

     

A m b u l a t o r y A c t i v i t y V e r t i c a l A c t i v i t y C o n t r o l

F

A 1

. 8 p p m

F

A 3

. 2 p p m

F

A 4

. 5 p p m

F

A 6

. 4 p p m

F

A 9

. 7 p p m

F

A 1 4

. 8 p p m

18

19

Figure 4

- 23. The effects of the acute formaldehyde (FA) exposures on the 20

ambulatory and ver tical components of SLMA. 21

22

Note: FA = formaldehyde exposure concentration. ** = p < 0.01 from controls. 23

24

Source: Usanmaz et al. (2002). 25

26

27

Usanmaz et al. (2002) noted an increase in WDS, after the acute exposures, as a possible 28

preconvulsive effect. How ever, the mice were only observed for 5 minutes, and it is unclear how 29This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 26 1 the researchers distinguished between a WDS due to an irritating odor and a preconvulsive 1

movement. No other study has noted convulsive effects from formaldehyde exposure in any 2

specie s. A second set of trials was reported in the same paper that sought to evaluate 3

formaldehyde effects on CNS excitability. Balb/c mice (six/group, sex unspecified) were 4

exposed to 0, 1.8, 6.4, or 14.8 ppm (0, 2.2, 7.9, or 18.2 mg/m 3 ) formaldehyde for 3 h ours. 5

Subchronic exposures were at 2 ppm (2.5 mg/m 3 ) formaldehyde for 3 weeks or 3.3 ppm 6

(4.1 mg/m 3 ) formaldehyde for 2 weeks. Seizures were induced by I.P. injection of 7

pentilenetetrazole (PTZ), and the incidence, severity, and course of induced seizure s were 8

recorded. The PTZ injection induced seizures in 83, 88, and 91% of controls, with 16, 38, and 9

67% mortality in controls in the three trials. Mortality was highly variable in treatment groups as 10

well. The authors report that PTZ

- induced seizures w ere decreased in incidence by acute 11

formaldehyde exposure in a dose

- dependent fashion with only 33% of mice exposed to 14.8 ppm 12

formaldehyde experiencing seizures versus 91% in control mice ( p < 0.05 at the highest dose 13

only). However, the methodology for observing and scoring seizures is unclear. Additionally, 14

there was high mortality and high variability of results for the three similarly treated control 15

groups. Therefore, it is difficult to assess data quality and interpret these findings. 16

Boja et a l. (1985) exposed male Sprague

- Dawley rats to air or to formaldehyde at 5, 10, 17

or 20 ppm for 3 hours on 2 consecutive days. On the second day, half the rats received the same 18

exposure as the previous day, while half the rats were switched (e.g., half thos e rats receiving air 19

the first day received formaldehyde the second day, and half those receiving formaldehyde the 20

first day received air the second day), for a total of four possible exposure combinations. During 21

the exposure period, activity levels were monitored by observation, once per minute for the first 22

hour and once every 5 minutes for the second hour. At the end of the second exposure session, 23

rats were sacrificed and brains removed for neurochemical analysis (see Section 4.2.1.6.1.5). 24

Behaviora l results were described in detail only for control and 5 ppm groups. During 25

the first exposure session, activity levels of formaldehyde

- exposed animals were significantly 26

decreased (approximately 50% of control levels). On the second day of exposure, th ose animals 27

previously exposed to formaldehyde exhibited partial recovery, those experiencing their first 28

formaldehyde exposure behaved similarly to those initially exposed on the first day, and those 29

animals exposed to formaldehyde for a second time had a greater decrease in activity than during 30

the first exposure (to approximately 30% of control levels). The authors stated that a similar 31

effect was seen in animals exposed at 10 ppm but that results at 20 ppm were not interpretable 32

(data were not presente d). Overall, the decreased activity seen in this study is consistent with 33

effects seen by other authors. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 262 Senichenkova (1991) exposed pregnant female rats to 0 or 0.5 mg/m 3 (0 or 403 ppb) 1

formaldehyde on gestation days (GDs) 1

– 19 for 4 hours/day. Reproduc tive aspects of this study 2

will be discussed in the reproduction section; however, results from behavioral assessments 3

conducted on the neonates are discussed here. The author stated that maturation of motor 4

reflexes (assessed as surface righting and pend ular reflex), open field behavior, and maze 5

learning ability were assessed. Detailed descriptions of procedures and results were not provided 6

for all assessments, but it was stated that motor reflex development did not differ in treated and 7

control animal s. Open field motor activity assessments in 40

- day

- old (juvenile) offspring 8

revealed an increase in squares visited and an increased frequency of rearing on the second and 9

third days of testing, indicating a lack of habituation in the offspring of formald ehyde

- treated 10

dams; similar levels of activity by both measures were found on the first test day. Counts of 11

defecation and urination were increased on all 3 days of testing. Increased exploratory behavior, 12

described as increased impulses, was also noted in a learning task (not otherwise described), but 13

the author stated that learning rate and ability of the formaldehyde

- treated group was not 14

different from controls (no data were provided). 15

Mobility and neuromuscular excitability (not otherwise described) in offspring of female 16

white rats were also evaluated by Sheveleva (1971). Dams were exposed to 0.005 or 17

0.0005 mg/L (approximately 4,000 or 400 ppb, respectively) formaldehyde on GDs 1

– 19. 18

Spontaneous mobility (over 15 minutes) and neuromuscular excitab ility were evaluated in 19

offspring at 1 or 2 months of age (other results from this study are discussed under 20

developmental toxicity, above). At 1 month, spontaneous mobility was reduced at the low dose 21

in males (52% of control levels; p < 0.01) but not at the high dose, and at both doses in females 22

(to 64 and 56% of control levels at the mid dose and high dose, respectively; p < 0.02). At two 23

months, there was a dose

- related increase in activity for both sexes, statistically significant ( p < 24

0.001) in hig h

- dose females only (391% of control levels). 25

26

4.2.1.6.1.3. Learning and memory. The effects of repeated formaldehyde exposures on learning 27

were investigated by Malek et al. (2003c), using a labyrinth swim maze. In this task, animals are 28

required to mak e a series of consecutive right or left turns to gain access to an escape platform 29

(Malek et al., 2003c). Adult male and female LEW.1K rats (15/sex/group) were exposed to 0, 30

0.1, 0.5, or 5.4 ppm (0, 0.123, 0.615, or 6.64 mg/m 3 ) formaldehyde 2 hours/day fo r 31

10 consecutive days. Formaldehyde levels were checked eight times throughout the 2

- hour 32

exposure periods. Mean formaldehyde levels of 0.1 ± 0.02, 0.5 ± 0.1, and 5.4 ± 0.65 ppm were 33

achieved. Body weight was measured on days 1, 5, and 10 of the experim ent. Two days prior to 34

beginning the formaldehyde exposures, all subjects were given an acclimation trial in which they 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 263 were individually placed into the water

- filled basin at the start position and allowed to navigate 1

to the escape platform with manual a ssistance to learn the correct route. Thereafter, the water 2

labyrinth test was run on each day of formaldehyde treatment, 2 hours after completion of each 3

daily exposure. Time taken to complete the test and errors made were recorded for each rat 4

(Table 4

- 56). An error was defined as swimming toward the start position or circling in the same 5

position without moving forward toward the escape platform. Rats were sacrificed at the end of 6

the experiment, and tissues were taken from the lung, heart, thymus, k idney, liver, pancreas, 7

skeletal muscle, and spleen. Tissues were fixed and prepared for histologic examination by light 8

microscope. No differences were noted in food consumption or body weight gain for either male 9

or female rats (Malek et al., 2003c). No treatment

- related differences in organ pathology were 10

reported (with the possible exception of focal microatelectasis (lung collapse at the microscopic 11

level) seen in two to three animals in each formaldehyde

- exposed group but not control animals). 12

13

Ta ble 4

- 56. Effects of formaldehyde exposure on completion of the labyrinth 14

test by male and female LEW.1K rats 15

16

Male rats Swimming time (sec) Error rate (mean)

Day 1 Day 6 Day 10 Day 1 Day 6 Day 10

Control 105 12.2 6.33 7.4 0.5 0.0

0.1 ppm a 100 12.9 6. 07 7.7 5.0 c 3.2 c

0.5 ppm 97 16.7 c 7.60 b 7.6 4.4 c 1.8 c

5.4 ppm 105 25.7 c 10.9 c 7.7 5.0 c 2.8 c

Female rats Swimming time (sec) Error rate (mean)

Day 1 Day 6 Day 10 Day 1 Day 6 Day 10

Control 103 12.5 6.47 7.9 0 0.0

0.1 ppm 96 12.3 7.53 7.1 5.2 c 3 .0 c

0.5 ppm 97 14.6 c 7.60 b 8.0 4.6 c 2.2 c

5.4 ppm 98 23.5 c 9.73 c 7.9 5.2 c 2.6 c

17

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

b Different from control, p < 0.05. 19

c Different from control, p < 0.005. 20

21

Source: Malek et al. (2003c). 22

23

24

A clear learning curve was evident in control animals, with rats completing the task in 25

less time and with fewer errors over days (Table 4

- 56). Although the number of errors decreased 26

with increasing experience in all groups, error rates in fo rmaldehyde

- exposed rats at all doses 27

were consistently higher than those observed in controls, starting on day 3 (Figure 4

- 24). All 28

control animals performed without errors by day 6, whereas all treated animals were still making 29

two to three errors on day 10, the final day of testing. Time required (latency) to complete the 30

maze was also reduced over days. Although this measure of performance was not as sensitive as 31This document is a draft for review purposes only and does not constitute Agency policy.

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- 264 error rate, formaldehyde

- induced deficits were still evident in the 0.5 and 5.4 ppm expos ure 1

groups of both sexes. 2

3

4

5

Figure 4

- 24. Effects of formaldehyde exposure on the error rate of female 6

LEW.1K rats performing the water labyrinth learning test

. 7

8

Source: Drawn from data reported in Malek et al. (2003c). 9

10

11

Impa ired performance on formaldehyde

- treated subjects cannot be attributed to 12

alterations in swimming ability, since latencies to complete the maze were identical for 0 and 13

0.1 ppm groups, yet acquisition of the task was still impaired in the 0.1 ppm group bas ed on 14

number of errors committed (see Figure 4

- 24). This study reports an adverse effect level of 15

0.1 ppm for increased error rate in the labyrinth water test, and all dose groups were equally 16

impaired across a broad range of exposures, 0.1

– 5.4 ppm. An i ndependent estimate of 17

swimming speed was not included, so motor competency could not be directly evaluated. 18

However, comparable latency scores and error rates at the beginning of testing across all groups 19

and latency scores that track together over days suggest that impaired swimming ability does 20

account for the observed differences in latency, which are most likely reflective of the increased 21This document is a draft for review purposes only and does not constitute Agency policy.

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- 265 number of errors in treated animals (errors usually increase the distance traveled and thus time 1

required for completion of the trial). 2

Pitten et al. (2000) evaluated the effects of very brief formaldehyde exposures 3

(10 minutes) but prolonged duration (90 days) on previously learned performance in a land 4

version of the labyrinth maze. Adult male and female Wistar ra ts (13/group) were trained on the 5

task for 14 days, two trials/day. Animals were required to make a series of five left or right turns 6

from the entrance of the maze to retrieve a piece of cheese placed in the goal box at the opposite 7

end. Animals were gu ided by the experimenter through the maze during this acclimation phase 8

until all subjects were able to retrieve the food without aid. After animals were trained (but prior 9

to formaldehyde exposure), performance was assessed once daily for 11 days, and th e latency to 10

complete the maze as well as the number of errors committed when traversing from the entrance 11

to the goal box was recorded. Animals were then assigned to one of three dose groups (five to 12

eight/sex/group) such that task performance was equiva lent across groups prior to 13

commencement of formaldehyde exposures. Animals were exposed to 0 ppm, 2.6 ppm (0.25% 14

formaldehyde solution to yield 3.06 ± 0.77 mg/m 3 ), or 4.6 ppm (0.70% formaldehyde solution to 15

yield 5.55 ± 1.27 mg/m 3 ) formaldehyde 10 minut es/day, 7 days/week for 90 days. Animals were 16

assessed for performance in the maze every seventh day, at least 22 hours after the exposure on 17

the previous day. At the end of the 90

- day exposure period, monitoring of maze performance 18

continued once every 10 days for an additional 30 days. All rats were sacrificed at the end of the 19

postexposure trials and tissue sections were prepared for histologic examination by light 20

microscopy, including liver, trachea, lung, kidney, heart, spleen, pancreas, testicle, and brain. 21

No treatment

- related changes in food or water consumption weight gain or in histologic samples 22

obtained at the termination of the experiment were observed. 23

Pitten et al. (2000) reported that no gender differences existed as a function of 24

formal dehyde treatment; therefore, data were presented by combining sexes. Control rats 25

showed no change in error rate but a slight decrease in running time through the maze during the 26

course of the experiment. The formaldehyde

- exposed groups began with a simi lar performance 27

level and error rate as controls, but their performance degraded over the course of formaldehyde 28

exposure. By the fourth week of exposure, increased numbers of errors were evident in both 29

exposed groups relative to controls. This trend re ached statistical significance at the 12

- week 30

time point, with a greater than twofold increase in number of errors ( p < 0.05). Formaldehyde

31

treated rats also tended to have increased run times through the maze ( p = 0.04), but no 32

difference was seen by for maldehyde concentration. By 4 weeks after termination of exposure, 33

no statistical differences among the three groups were evident, but the tendency for the two 34

exposed groups to make more errors and have longer latencies remained. Since Pitten et al. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 266 (20 00) tested animals after the task was acquired, these results indicate deficits in the retention of 1

a previously learned task. 2

Lu et al. (2008) evaluated the effects of formaldehyde on performance of mice in a 3

Morris water maze. Kunming mice (five males/g roup) were exposed to formaldehyde at 0.2, 1, 4

or 3 mg/m 3 6 hours/day for 7 days (measured concentrations: 0.2 ± 0.01, 0.99 ± 0.04, and 5

3.03 ±

Apfelbach and Reibenspies (1991) published a brief report of formaldehyde effects on 20

olfactory learning. Ferrets were exposed to 0.25 ppm (0.31 mg/m 3 ) formalde hyde gas 21

continuously for 6 months. A Y

- shaped maze was used to test odor detection, discrimination 22

between odors, and odor threshold. Ferrets were conditioned to distinguish ethyl acetate 23

(0.1 vol%) from clean air. Untreated ferrets achieved 75% succes s after an average of 110 trials. 24

However, formaldehyde

- treated ferrets required on average 320 trials to reach a 75% success 25

rate. A 90% success rate was achieved by untreated ferrets after 420 trials. However, this level 26

of success was not reached in formaldehyde

- treated ferrets. 27 0.16 mg/ m 3 ). Mice were trained to locate a hidden platform in a large, circular tank

6

(106 cm diameter, 31 cm deep ). Each animal received four training trials per day, beginning 7

30 minutes after the end of exposure. During training, latency to locate the platform was 8

recorded for each trial, with a maximum of 60 seconds, after which the animal was guided to the 9

plat form. After the last day of training, an additional trial was conducted with the platform 10

removed (the probe trial); time spent in each maze quadrant was measured to determine the time 11

the animal spent searching for the platform in the correct area of the maze. Performance in the 12

water maze, measured as mean escape latency across the seven training trials, was significantly 13

impaired in the 3 mg/m 3 group. No significant difference was seen at 1 mg/m 3 , although there 14

appeared to be an increased latency dur ing the second day of testing. During the probe trial, 15

control animals spent significantly more time in the correct quadrant, but neither formaldehyde

16

exposed group did so. Results of this study indicate deficits in learning and retention of the 17

Morris w ater maze following formaldehyde exposure, with greater effects seen in the higher dose 18

group. 19

The same researchers also tested olfactory function in formaldehyde

- treated ferrets, as 28

summarized in Section 4.2.1.7 (Apfelbach et al., 1992). A decrease in olfactory discrimination 29

and a reduction in the percentage of olfac tory cells in the olfactory epithelium were reported 30

after 3

– 12 months exposure to 0.25 or 0.5 ppm formaldehyde. Decreased olfactory sensitivity in 31

rats exposed to 0.25 or 0.5 ppm formaldehyde has also been reported by the same researchers 32

(Weiler and Apf elbach, 1992; Apfelbach and Weiler, 1991), and Weiler and Apfelbach (1992) 33

reported in an abstract that shifts in olfactory thresholds were greater when exposure was 34

initiated at PND 30 than at adult ages. Given the documented changes in olfactory thresho lds, 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 267 observed changes in olfactory learning would likely be confounded by the potential for 1

decreased olfactory function by formaldehyde exposures, and definitive conclusions regarding 2

formaldehyde effects specific to learning cannot be made based on these studies. 3

4

4.2.1.6.1.4. Neurosensitization.

Sorg et al. (1996) evaluated cross

- sensitization of cocaine

- induced increases in activity 13

fr om an initial formaldehyde exposure. Female Sprague

- Dawley rats (eight to nine) were 14

exposed to 0 or 11 ppm (0 or 13.5 mg/m 3 ) formaldehyde 1 hour/day for 7 days. Locomotor 15

activity was measured (by photocell) after saline injection (1 day postexposure) a nd after cocaine 16

injection (2 days postexposure). A similar protocol was conducted on days 36 and 37 17

postexposure. Motor activity levels following saline injection were similar for controls and 18

formaldehyde

- treated rats. However, formaldehyde exposure i nitiated sensitization to cocaine as 19

evidenced by a greater increase in locomotor activity in mice treated with formaldehyde 20

followed by cocaine ( p < 0.05) with an average count of crossed grids greater than 40,000 (2 21

hours) in treated animals compared wit h 25,000 (2 hours) in controls. The cross

- sensitization 22

was transient, with no treatment effects on cocaine

- induced activity either 29 or 37 days 23

postexposure. When examining individual data, the authors suggested that the formaldehyde

24

treated groups in both cases have a cluster of high responders (HRs), suggesting some animals 25

may have been more sensitive. A second group of similarly treated female rats was pretested for 26

locomotor activity and divided into subgroups of HRs or low responders (LRs). They were then 27

given a panel of neurobehavioral tests: anxiety (elevated plus maze, day 11); memory (passive 28

avoidance training, day 12; passive avoidance test, day 19); and nociceptive test (day 20). Trunk 29

blood corticosterone levels were determined during s tress on day 35 postexposure. No 30

significant treatment differences were found in the passive avoidance test, nociception, or 31

corticosterone levels (basal or stress induced). On the elevated plus maze, a two

- way ANOVA 32

indicated no overall formaldehyde tre atment effects, but the HR rats had higher open arm time 33

ratios (indicating greater anxiety) regardless of treatment. Within the treatment groups, the 34

difference in behavior between HR and LR subgroups was only significant for the formaldehyde

35 Sorg et al. (1996) studied the potential for formaldehyde

5

exposure to induce sensitization in the CNS, possibly through the limbic pathways in the brain. 6

The authors hypothesized that multiple chemical sensiti vity (MCS) has an onset and progression 7

similar to CNS sensitization and may, therefore, be a similar process. These experiments were 8

conducted to test this hypothesis and to determine whether formaldehyde exposure could be used 9

as a model for MCS. Behav ioral sensitization can be initiated by psychostimulants (e.g., 10

cocaine) and manifest as increased locomotor activity upon subsequent challenge with the 11

stimulant. 12This document is a draft for review purposes only and does not constitute Agency policy.

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- 268 treated rat s ( p < 0.05). Authors suggested that cross

- sensitization to cocaine

- induced locomotor 1

activity was caused by enhanced dopamine transmission within the mesolimbic system (ventral 2

tegmental area to nucleus accumbens projection) following repeated formaldehy de exposure. A 3

critical role of the hypothalamic

- pituitary

- adrenal (HPA) axis has also been implicated in cross

4

sensitization. 5

Sorg et al. (1998) and Sorg and Hochstatter (1999) further explored formaldehyde

6

induced behavioral sensitization using the coc aine model. In contrast to the results following 7

exposure to 11 ppm (Sorg et al., 1996), rats exposed to only 1 ppm for 7 days showed no cross

8

sensitization to cocaine injection. However, animals exposed to 1 ppm formaldehyde for 9

4 weeks exhibited increa sed cocaine

- induced vertical activity (with no difference in horizontal 10

activity) for 4

– 6 weeks after cessation of exposure. Activity levels of formaldehyde

- exposed rats 11

were approximately threefold those of control rats 3

– 4 days postexposure and still 1. 5

- fold 12

control levels at 4

– 6 weeks postexposure ( p < 0.05). 13

Sorg et al. (2001) examined changes in corticosterone levels in rats with and without 14

formaldehyde treatment. Basal corticosterone levels in trunk blood were established in naïve 15

male Sprague

- D awley rats taken directly from their home cage immediately prior to sacrifice. In 16

an acute trial, male rats were exposed to 0, 0.7, or 2.4 ppm (0, 0.86, or 2.96 mg/m 3 ) 17

formaldehyde for either 20 or 60 minutes, and trunk blood was collected for corticoster one 18

analysis. Therefore, these rats were challenged with a new environment (the exposure chamber) 19

in the presence or absence of formaldehyde. In a separate trial, basal and challenged 20

corticosterone levels were measured after repeated exposure (1 hour/da y, 5 days/week for 2 or 21

4 weeks). Basal corticosterone levels were measured in trunk blood immediately after removing 22

the animal from its home cage. Challenged corticosterone levels were measured after rats were 23

placed into the exposure chamber for a fin al 20

- minute exposure. Body weight was measured at 24

the beginning of each week of exposure and was unchanged by formaldehyde treatment. 25

Corticosterone levels were increased over basal levels when rats were placed in the 26

exposure chamber for 20 minutes (Fig ure 4

- 25, panel a) but returned to basal levels after 27

60 minutes in the exposure chamber (not shown). This response may reflect the stress of the new 28

environment and acclimatization after 60 minutes in the chamber. Corticosterone levels were the 29

same in the presence or absence of formaldehyde, indicating no treatment effect. 30

31Panel a Panel b Panel c

Corticosterone levels after 2 weeks of

repeated formaldehyde exposure

0 20 40 60 80 100 120 140 160 180 200

Control 0.07 2.4

Formaldehyde (ppm) Basal

ChallengeSerum corticosterone (ng/mL)

0 20 40 60 80 100 120 140 160 180 200

Control 0.07 2.4

Formaldehyde (ppm ) Basal

Challenge Corticosterone levels: with no

pre-exposure to formaldehydeSerum corticosterone (ng/mL)

0 20 40 60 80 100 120 140 160 180 200

Control 0.07 2.4

Formaldehyde (ppm) Corticosterone levels after 4 weeks

of repeated formaldehyde exposureSerum corticosterone (ng/mL) Basal

Challenge 1

2

3

4

5

6

7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

22

23

24

25

26

27

28

29

30

31

32

33

34

35

36

37

38

39

40

41

42

43

44

45

46

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

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- 269 Fi gure 4 -25. Basal and stress- induced trunk blood corticosterone levels in

male LEW.1K rats after formaldehyde inhalation expo sures.

Note: Panel a: no pretreatment, corticosterone levels after 20 -minute

formaldehyde exposure. Panels b and c show both basal and induced

corticosterone levels after a 2

- or 4 -week pretreatment to formaldehyde

1 hour/day. Challenge to induce cortic osterone was a 20 -minute reexposure at the

formaldehyde level tested.

Source: Sorg et al. (2001).

Control animals exhibited an increase in basal corticosterone after 2 weeks, which

returned to naïve levels after 4 weeks (Figure 4 -25, panels b and c). Formaldehyde- treated rats

demonstrated a comparable increase in basal corticosterone levels at 2 weeks, but these levels

did not return to naïve levels at 4 weeks as seen with controls. Control and 0.7 ppm exposed rats

showed a similar response to challen ge (the final 20-minute exposure). However, rats exposed to

2.4 ppm were hyperresponsive, with exaggerated corticosterone levels during this final exposure.

Differences in basal corticosterone levels after formaldehyde exposure and the

hyperresponsiveness seen in animals exposed at 2.4 ppm provide evidence of

formaldehyde-induced perturbations of the HPA axis. Authors suggested that elevated

corticosterone levels induced by repeated formaldehyde exposures may contribute to the crosssensitization to coca ine-induced motor activity.

Formaldehyde-induced changes in the HPA axis may contribute to behavioral effects of

formaldehyde exposure reported by Sorg et al. (2004) and Sorg and Hochstatter (1999). The

authors also reported an enhanced conditioning to odor in animals previously exposed toThis document is a draft for review purposes only and does not constitute Agency policy.

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- 270 repeated formaldehyde. Male and female Sprague

- Dawley rats (60

– 80 days of age) were 1

exposed at 1 ppm (1.23 mg/m 3 ) formaldehyde 1 hour/day, 5 days/week for 4 weeks (Sorg and 2

Hochstatter, 1999). Two weeks after exposure ended, rats were trained to the conditioned fear 3

task. Rats were conditioned to a fear response by either odor only or odor associated with 4

footpad shock. Orange

- oil extract was used as the odor conditioned stimulus (CS). One day 5

after conditioning, rat s were reintroduced into the environment without an odor cue, and time 6

spent motionless in the freezing posture (freezing) was observed. On day 2 after conditioning, 7

rats were placed in a novel environment, and time spent in the freezing posture was evalu ated in 8

the absence and then the presence of odor. This was repeated on day 12 after conditioning to 9

measure the loss of the freezing response to the conditioned odor. 10

Both treated and exposed rats showed similar responses on reintroduction into the 11

condi tioning environment in the absence of an odor cue on day 1 (Sorg and Hochstatter, 1999). 12

As expected, rats conditioned with a footpad shock demonstrated greater time motionless than 13

odor

- trained only rats, and there was no difference between control and f ormaldehyde

- treated 14

rats. However, in the presence of odor on days 2 and 12, formaldehyde

- exposed rats who were 15

conditioned with odor associated with foot shock spent significantly more time freezing than 16

odor

- only trained rats ( p < 0.05); control animals on those days showed no difference in time 17

freezing in the presence and absence of odor. The authors concluded that the formaldehyde

18

treated rats had more difficulty than controls in extinguishing the fear response to the 19

conditioned odor, and speculated that an enhancement of the fear

- conditioned response by 20

formaldehyde pretreatment supports the hypothesis that sensitization may include effects through 21

the limbic system of the brain. 22

In a second experiment, adult male and female Sprague

- Dawley rats were exposed at 0 or 23

2 ppm (2.45 mg/m 3 ) formaldehyde 1 hour/day, 5 days/week for 4 weeks (Sorg et al., 2004). Two 24

to 3 weeks after exposure ended, rats were trained to the conditioned fear task. Rats were given 25

a foot shock either associated with an odor (pa ired group) or unassociated with an odor (unpaired 26

group). Orange

- oil extract was used as the odor CS. After training, freezing behavior was 27

assessed (1) in the same context in the absence of odor (1 day), (2) in a new context in the 28

presence and absence of the CS (5 consecutive days), and (3) in another novel context in the 29

presence and absence of the CS. 30

Formaldehyde

- exposed male rats demonstrated increased conditioned fear response to an 31

odor CS (orange oil) paired with foot shock with no change in the degree of conditioning to the 32

context. For female rats, formaldehyde exposure did not affect the percent of time spent 33

freezing, either in the conditioning context or the novel context in the absence of the conditioned 34

odor. In contrast, male rats spent an increased time freezing in a novel context in the presence of 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 271 odor, indicating a greater conditioned fear response to the olfactory cue ( p < 0.05). This is in 1

agreement with the previous study where formaldehyde effects were seen in the presence of th e 2

conditioning odor but not the environment (Sorg and Hochstatter, 1999). However, in this study 3

female rats did not exhibit a similar enhancement of fear conditioning to the olfactory CS. 4

The authors suggested that repeated exposure to low levels of form aldehyde acts as a 5

stressor in much the same way as inescapable foot shock, with resulting sensitized responses 6

within the olfactory/limbic pathways (Sorg et al., 2004). This interpretation is consistent with 7

work described above in which augmented basal corticosterone levels following repeated 8

formaldehyde exposures were demonstrated. However, while the fear conditioning in the present 9

study and cross

- sensitization to cocaine described above (Sorg and Hochstatter, 1999) occurred 10

3

– 4 weeks after terminati on of exposure to formaldehyde, the duration of corticosterone 11

elevation induced by repeated exposure to formaldehyde has not been determined. It is possible 12

that augmentation of corticosterone levels following formaldehyde exposure results from direct 13

ac tion of formaldehyde on the HPA axis. Experiments designed to compare HPA activation 14

following standard stressors (repeated inescapable foot shock or restraint stress), stress induced 15

by other irritants (chemicals with strong irritant odors but no CNS act ion), and repeated 16

formaldehyde exposures are necessary to dissociate primary from secondary action of 17

formaldehyde on CNS function in this paradigm. It is also possible that enhanced conditioning 18

to an odor stimulus results from formaldehyde

- induced incr eases in airway irritation, rendering 19

the conditioned odor stimulus a more salient cue, producing a conditioned response that is not 20

extinguished as readily as in air

- exposed controls. However, damage of the nasal mucosa and 21

lesions would be expected to b e minimal at 1 ppm formaldehyde exposures and most likely 22

resolved 2 weeks after exposure was ended (see Section 4.2.1.2). Therefore, a more salient cue 23

for fear conditioning to odor due to physical irritation is not likely. Alternatively, formaldehyde 24

m ay act to up regulate olfactory activity, producing a stronger sense of odor during conditioning. 25

26

4.2.1.6.1. 5. Neurochemistry and neuropathology. Several studies that were focused on general 27

toxicity or carcinogenicity of formaldehyde also assessed his topathology in exposed animals, 28

including pathological evaluation of the brain. In all cases, details of the pathological evaluation 29

were not provided. Reported results stated that no significant lesions were seen on unspecified 30

tissues (Appelman et al., 1988; Maronpot et al., 1986; Kerns et al., 1983) or that an increase in 31

relative brain weight (data not provided) was considered of no toxicological significance 32

(Woutersen et al., 1987). The absence of procedural information, or specific reported result s, 33

limits the utility of this information. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 272 Boja et al. (1985) measured changes in several neurotransmitters (norepinephrine, 1

dopamine, 5

- hydroxytryptamine) and their major metabolites (3,4

- dihydroxyphenylacetic acid 2

[DOPAC] and 5

- hydroxyindoleacetic acid [5

- HIAA]) following one or two 3

- hour exposures to 3

formaldehyde at 0, 5, 10, or 20 ppm. Animals were sacrificed immediately following the second 4

exposure, and brains were immediately removed, frozen, and sectioned. Regions of interest were 5

analyzed by hi gh

- pressure liquid chromatography with electrochemical detection. Authors stated 6

that neurotransmitter concentrations were measured in multiple brain regions, but results were 7

reported only for the 5 ppm exposure and only for the hypothalamus. No change was seen in 8

concentrations of norepinephrine or 5

- hydroxytryptamine for any exposure paradigm. For those 9

animals exposed twice to formaldehyde, there was a slight (statistically significant) increase in 10

dopamine and a larger (approximately fourfold) incre ase in 5

- HIAA. DOPAC was increased 11

(approximately 30%) in animals receiving formaldehyde during the second exposure only. 12

Recent work by Hayashi et al. (2004) indicates that formaldehyde exposure increases the 13

activity of periglomerular (PG) cells in the main olfactory bulb. Tyrosine hydroxylase activity 14

was measured as a marker for activity of olfactory function. The authors surmised that 15

expression levels of this enzyme are useful markers since it has been reported that the protein is 16

up regulated afte r sensory stimulation and is down regulated by odor deprivation or when the 17

olfactory epithelium is removed (Cho et al., 1996; Stone et al., 1991; McLean and Shipley, 1988; 18

Baker et al., 1983). Eight

- week

- old female C3H/HeN mice were exposed at 0, 0.08, 0 .4, or 19

2 ppm (0, 0.1, 0.49, or 2.45 mg/m 3 ) formaldehyde 16 hours/day for 1 day or 12 weeks 20

(5 days/week). Formaldehyde exposure did not affect body weight. Mice were sacrificed 21

24 hours after exposure; the brains were removed, fixed, and prepared for sec tioning. One side 22

of the olfactory bulb was sliced into 40 μm

- thick serial frontal sections and immuno

- stained for 23

tyrosine hydroxylase activity. The number of tyrosine hydroxylase

- positive PG cells was 24

determined by examining digital photomicrographs of three tissue sections, averaging the counts 25

from 10

– 15 glomeruli per section. 26

Neither the size of the olfactory bulb (rostrocaudal, dorsoventral, and mediolateral 27

lengths) nor the total number of PG cells was changed by formaldehyde exposure. The number 28

of tyrosine hydroxylase

- positive PG cells per glomerulus was unchanged by a single 29

formaldehyde exposure but increased after 12 weeks of repeated exposures. The increases were 30

similar among treatment groups: 5.54 ± 0.31 at 0.80 ppm, 5.18 ± 0.60 at 0.4 ppm , and 6.0 ± 0.83 31

at 2 ppm or 196, 167, and 196% of controls, respectively. As an indicator of activity, it is not 32

unexpected that the enzyme was up regulated after repeated exposure to an odorous compound. 33

Hayashi et al. (2004) hypothesize that the incre ased tyrosine hydroxylase activity is an indication 34

of increased sensitivity and, therefore, may be a model for MCS. However, it is unknown if the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 273 increase in enzyme activity after repeated exposures is transient or could result in sensitization. 1

Tyrosin e hydroxylase is the first enzyme in the dopamine synthetic pathway, but the role of 2

dopamine in PG cells is not known. Further research would be needed to understand the 3

potential for formaldehyde to act as a sensitizing agent in this model. 4

In an abstra ct, Kakeyama et al. (2004) outline the results of experiments to address the 5

effects of subchronic exposure to low levels of formaldehyde on changes in neurotransmitter

6

related mRNA expressions in mice forebrains. An unstated number of female C3H/He mice 7

were exposed 16 hours/day, 5 days/week to 400 ppb (0.49 mg/m 3 ) formaldehyde for 12 weeks. 8

The authors used RT

- PCR methodologies to quantify mRNA encoding for the glutamate receptor 9

subunits GluR1 and GluR2, the dopamine receptor D1, and the serotonin rece ptor 5

- HT1A in the 10

neocortex, hippocampus, amygdala, and hypothalamus. Raised levels of mRNA expression were 11

observed for GluR1 in the neocortex and hippocampus; GluR1, GluR2, and the dopamine 12

receptor D1 in the amygdala; and the serotonin receptor 5

- HT1A in the hypothalamus. Reduced 13

mRNA expression was observed for GluR2 in the hippocampus and neocortex. When other 14

mice were subjected to a radiofrequency

- induced lesion of the hippocampus then exposed to 15

formaldehyde for 12 weeks as before, the altered e xpression of GluR1 and GluR2 in the 16

neocortex was abolished. However, the increment of mRNA expression of 5

- HT1A in the 17

hypothalamus was further enhanced. In demonstrating that formaldehyde affects neocortical 18

GluR1 and GluR2 mRNA expressions through a h ippocampal function, Kakeyama et al. (2004) 19

concluded that subchronic exposure to low concentrations of formaldehyde can affect neural 20

transmission in the forebrain. 21

Fujimaki et al. (2004b) examined the effects of formaldehyde on NGF in the brain and 22

hippo campus. Ten female C3H/HeN mice/group were exposed to 0, 80, 400, or 2,000 ppb (0, 23

0.1, 0.5, or 2.45 mg/m 3 ) formaldehyde 16 hours/day, 5 days/week for 12 weeks. Some groups of 24

mice received the same treatment after I.P. injection of 10 μg of OVA and 2 mg alum prior to the 25

commencement of formaldehyde exposure. For this subgroup, booster injections of OVA were 26

administered on days 21, 42, 63, and 77 during the formaldehyde exposure regimen. 27

Quantitative measures of NGF and BDNF in homogenates of whole br ain and hippocampus were 28

obtained by ELISA and mRNA determination. The amount of NGF protein in whole brains 29

remained unchanged in the non

- immunized mice. However, brain NGF levels were significantly 30

increased in OVA

- immunized mice exposed to 80 and 400 ppb (but not 2,000 ppb) 31

formaldehyde (Figure 4

- 26). This result was confirmed by parallel increases in the 32

concentrations of hippocampal NGF mRNA that were produced in immunized mice exposed to 33

formaldehyde at the same concentrations. However, there were no comparable increases in the 34

amounts of brain

- derived neurotrophic factor in either immunized or non

- immunized mice. In 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 274 discussing the mechanisms potentially associated with their results, Fujimaki et al. (2004b) 1

considered it likely that low

- level exp osure to formaldehyde could enhance NGF production 2

through the stimulation of the HPA axis together with immunization. 3

4

5

6

7

Figure 4

- 26. NGF production in the brains of formaldehyde

- exposed mice. 8

9

Note: Female C3H mice were exp osed to formaldehyde 16 hours/day, 10

5 days/week for 12 weeks. NGF in homogenates of whole brain and 11

hippocampus were measured by ELISA. Values are means ± SEM (n = 5

– 6). 12

* = p < 0.05 and ** = p < 0.01 versus control mice, as calculated by the authors. 13

14

Source: Redrawn from Fujimaki et al. (2004b). 15

16

17

The enhancement of NGF in the brains of immunized mice exposed to formaldehyde 18

gave rise to the suggestion that NGF may promote the survival of hippocampal neurons when 19

challenged with formaldehyde. To exa mine whether or not apoptosis plays a role in this process, 20

Tsukahara et al. (2006) measured the effects of formaldehyde on apoptotic mechanisms 21

regulating the survival and death of cells and on N

- methyl

- D

- aspartate (NMDA) receptors. 22

Female C3H/HeN mice ( 13/group) were exposed to 0 or 400 (393 ± 34) ppb (0 or 490.8 μg/m 3 ) 23

formaldehyde 16 hours/day, 5 days/week for 12 weeks. Seven control and formaldehyde

- treated 24

mice were immunized with 10 μg OVA plus 2 mg aluminum hydroxide prior to exposure. 25

Subsequent ly, these mice received OVA via aerosol as a booster during weeks 3, 6, 9, and 11. 26

Hippocampi were dissected from all animals 1 day after the final exposure and homogenized in 27

hypotonic buffer. The 12,000 rpm supernatants were analyzed by Western blottin g for the 28

presence of the proteins BCl

- 2 (which inhibits apoptosis) and Bax (which opposes BCl

- 2 action 29

and promotes apoptosis) and the NMDA receptor subtypes 2A and 2B (NR2A and NR2B). 30This document is a draft for review purposes only and does not constitute Agency policy.

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- 275 Immunohistochemical analysis was also carried out for the presence of active caspase

- 3, an 1

apoptosis marker. 2

The levels of NR2A and NR2B were unaffected by exposure to formaldehyde in either 3

immunized or non

- immunized mice. Likewise, the number of caspase

- 3 immunoreactive cells 4

did not change as a result of formaldehyde ex posure. However, when measured amounts of 5

Bcl

- 2 and Bax were normalized to the amount of β

- tubulin, the ratio Bcl

- 2/Bax was significantly 6

increased in immunized mice exposed to formaldehyde. Non

- immunized mice did not show this 7

apparently compound

- related response. Consistent with the concept that the proportions of Bcl

- 2 8

and Bax are critical for the regulation of cell survival and death, the authors interpreted their data 9

as an indication that changes to the ratio of Bcl

- 2/Bax expressions might be an important 10

adaptive response to the effects of fo rmaldehyde, such that the antiapoptotic changes might 11

contribute to the protection of hippocampal neurons from the pernicious effects of formaldehyde 12

exposure itself. 13

The same research group used the immunized mouse model to determine whether 14

formaldehyde exposure affected mRNA expression of genes related to synaptic plasticity 15

(Ahmed et al., 2007). Ten female C3H/HeN mice were exposed to 0 or 400 ppb formaldehyde 16

16 hours/day, 5 days/week for 12 weeks. All mice were immunized with 10 μg OVA plus 2 mg 17

alu minum hydroxide prior to initial formaldehyde exposure then treated in weeks 3, 6, 9, and 11 18

with aerosolized OVA as a booster. Five treated and control animals were I.P. injected with 1 19

mg/kg MK

- 801, a noncompetitive NMDA receptor agonist before the last formaldehyde 20

exposure. At term, hippocampi were dissected and frozen at −80 o C until processing. At that 21

point, total mRNA was extracted and first strand cDNA was synthesized by using reverse 22

transcriptase. Expression levels of various proteins/receptor s, including NMDA NR2A and 23

NR2B receptor subunits, dopamine D1 and D2 receptors, cyclic AMP responsive element

24

binding proteins (CREB

- 1 and CREB

- 2), and the transcription factors FosB and ΔFosB were 25

determined by using the PCR. The expression level of eac h mRNA species was expressed 26

relative to the sample’s content of 18S rRNA. The total protein lysate was also assayed for 27

pCREB by Western blotting. 28

In the first of a sequence of histograms, Ahmed et al. (2007) demonstrated a significant 29

increase in mRNA e xpression of NR2A as a result of formaldehyde exposure. However, this 30

effect was abolished in animals treated with MK

- 801. A similar trend in the mRNA expression 31

of NR2B in response to formaldehyde exposure did not achieve statistical significance. MK

- 8 01 32

treatment significantly reduced receptor in mRNA expression in the presence of formaldehyde. 33

The authors provided data showing an increased expression of dopamine D1 and D2 receptor 34

mRNA response to formaldehyde, in both cases abolished by treatment wi th MK

- 801. The 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 276 expression of CREB

- 1 mRNA also conformed to the pattern of being increased as a result of 1

formaldehyde exposure but abolished by MK

- 801. However, the expression of CREB

- 2 and 2

FosB/ΔFosB was unaffected by formaldehyde. When normalized to t he amount of β

- tubulin, 3

there were no significant effects of formaldehyde exposure and MK

- 801 treatment on the protein 4

levels of pCREB. Finally, there was no significant difference in the expression of transient 5

receptor potential vanilloid receptor (TRPV 1) between control and formaldehyde

- exposed mice, 6

and MK

- 801 itself did not significantly alter the mRNA level of TRPV1. In seeking to explain 7

their results, the authors speculated that low

- level exposure of immunized mice to formaldehyde 8

had an effect on hippocampal synaptic plasticity at the mRNA level, as evidenced by the 9

enhancement of mRNA for NR2A, the dopamine D1 and D2 receptors, and CREB1, with up 10

regulation compensating for the sustained levels of enhanced protein expression under low

- level 11

forma ldehyde exposure. The interpretation of these changes in NR2A mRNA, in the context of 12

the results of Tsukahara et al. (2006), showing no change in NR2A and NR2B protein 13

expression, was not discussed. 14

15

4.2.1.6.1.6. Neurogenesis.

For both pyramidal and granular areas, a much lower number of cells was seen on 27

PND 90 versus PND 30 ( p < 0.001). This response was evident irrespective of the amount of 28

exposure to formaldehyde and is consistent with normal brain development. Compound

- specific 29

effects of formaldehyde on the volume and number of granular and pyramidal cel ls varied by 30

dose and over the two time points. There was a small increase in the volume of the granular cell 31

layer of the dentate gyrus in rats sacrificed on PND 30 ( p < 0.001) in response to increasing 32

formaldehyde concentration (Aslan et al., 2006), wi th no significant change in neuron number; 33

the increased volume (now accompanied by an increase in neuron number) was still evident at 34

the low

- exposure level on PND 90 ( p < 0.01) but not at the high dose. Brain hemisphere volume 35 Two papers have examined the effects of subacute exposure to 16

formaldehyde on the overall size (volume) of discrete cellular areas of the hippocampus in 17

neonatal rats. The researchers also used an optical fractionator counting method to derive a 18

plausible estimate of cell number. Aslan et al. (2006) studied the effects of formaldehyde 19

exposure on the number and volume of granular cells in the hippocampal dentate gyrus. 20

Sarsilmaz et al. (2007) examined the impact of postnatal formaldehyde exposure on brain 21

hemisphere volume and on the size and cell number of pyramidal cells in the cornu ammonis 22

region of the hippocampus. The in

- life phase was the same in each study, featuring the exposure 23

of 10 neonatal male Wistar rats/group to 0, 6, and 12 ppm (0, 7.36, and 14.7 mg/m 3 ) 24

formaldeh yde 6 hours/day, 5 days/week for 30 days. Five rats/group were sacrificed at that point 25

(PND 30), while the rest were maintained without further treatment until PND 90. 26This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 277 was decreased at both conc entrations on PND 30 ( p < 0.01) but was increased at both 1

concentrations ( p < 0.01, with a larger magnitude of effect at 6 ppm) on PND 90 (Sarsilmaz et 2

al., 2007). In the hippocampal cornu ammonis region, the volume of the pyramidal cell layer on 3

PND 30 w as increased in low

- dose animals ( p < 0.001) but decreased in high dose animals ( p < 4

0.001) as compared with control values; neither group was significantly different from controls 5

on PND 90. There was a dose

- related decrease in total neuron number in th e cornu ammonis on 6

PND 30 ( p < 0.01 for both doses); on PND 90 the decrease in neuron number remained 7

statistically significant in both treatment groups ( p < 0.01), but there was no longer any 8

difference in the magnitude of the effect between doses (Sarsil maz et al., 2007). 9

In a third study from the same laboratory, Songur et al. (2008) used the same exposure 10

paradigm to evaluate changes in oxidant and antioxidant systems in the cerebellum of perinatally 11

exposed rats. Exposure was carried out as in Aslan e t al. (2006) and Sarsilmaz et al. (2007), 12

described above. On PND 30 or 90, cerebellums from seven male rats per group were evaluated 13

for levels of malondialdehyde (MDA), NO, superoxide dismutase activity (SOD), and 14

glutathione peroxidase (GPX) activity. Dose

- related increases in NO (approximately 20

– 80%), 15

MDA (100

– 160%), and GPX (25

– 60%) and dose

- related decreases in SOD (20

– 30%) were seen 16

on PND 30. In general, the magnitude of change from control levels was maintained on PND 90, 17

with the exception of MDA levels in 6 ppm animals, which appeared to approach control levels 18

at 90 days. The authors stated that these findings indicated that formaldehyde exposure may 19

cause neurotoxicity via the production of oxidative damage in the brain. Persistence of the effect 20

to the 90

- day time point (30 days after cessation of exposure) supports the possibility that 21

formaldehyde may cause long

- lasting or permanent changes in the brain following early life 22

exposure. These results are consistent with the earlier studies by Aslan et al. (2006) and 23

Sarsilmaz et al. (2007), finding permanent changes in brain structure (although in a different 24

brain region) following early life exposure. 25

26

4.2.1.6.1.7. Summary of formaldehyde effects on neurobehavioral and neuropathological 27

measures, following exposure via inhalation. As has been demonstrated in mice (Wood and 28

Coleman, 1995), it is possible that rats experience respiratory tract irritation during low

- level 29

formaldehyde exposure. Perturbations in nervous system function repo rted with formaldehyde 30

exposure include reductions in motor activity, lack of habituation, impairment in acquisition of a 31

new learning task, deficits in retention of a previously learned task, increases in corticosterone 32

levels, sensitization to cocaine

- in duced locomotor activity, and enhanced fear conditioning using 33

an olfactory CS (Table 4

- 57). Many of these effects were observed at exposure levels at or 34

below 1 ppm, and some persisted days to weeks after termination of exposure. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 278 Malek et al. (2004, 2003 a, b) detected behavioral changes in rats and mice tested 2 to 1

24 hours postexposure. The mechanism of these behavioral changes is unknown, and available 2

data do not allow dissociation of direct effects on the nervous system and behavioral responses to 3

th e irritant effects of formaldehyde (control experiments [e.g. , using a different aversive odor 4

with or without irritant properties] were not included). Given that behavioral changes were 5

observed hours to days after cessation exposure (i.e., beyond the ti me required for formaldehyde

6

induced irritation to subside), it is unlikely that these behavioral changes were caused by 7

formaldehyde

- induced irritation. Similarly, although it is possible that systemic effects of 8

formaldehyde exposure might cause reduced motor activity during or immediately following 9

exposure, it is unlikely that these effects can account for the differences in responses of male rats 10

24 hours after exposure (Malek et al., 2003a). Furthermore, a follow

- up study demonstrated 11

reduced motor activity in animals 2 hours after a 2

- hour exposure to much lower levels of 12

formaldehyde (0.1 ppm), which fall well below the levels identified by Wood and Coleman 13

(1995) as the AC 50 for formaldehyde in mice (Malek et al., 2003b). 14

Two studies reported sign ificant reductions in learning or retention following brief 15

periods of repeated exposure to low levels of formaldehyde (Malek et al., 2003c; Pitten et al., 16

2000) (Table 4

- 57). Malek et al. (2003c) reported an increased number of errors in acquiring a 17

wate r maze task; testing took place daily 2 hours after termination of a 2

- hour exposure. The 18

work of Pitten et al. (2000) revealed that brief exposures over many weeks led to increases in 19

errors performing a previously learned task and that the magnitude of the effect increased over 20

the course of the exposure period. Testing occurred remote from the time of exposure (22 hours 21

after the previous exposure), and the deficits appeared to persist for several weeks after exposure 22

terminated, minimizing the possibi lity that these effects were related to irritant properties of 23

formaldehyde. Although the exposure levels were moderately high (2.6

– 4.6 ppm) and continued 24

over several months, the duration of a single exposure event was very brief (10 minutes). 25

Sorg and H ochstatter (1999) and Sorg et al. (2004, 2001) suggest that behavioral 26

sensitization associated with low

- level formaldehyde exposure was linked to alterations in HPA 27

control of corticosterone. Cross

- sensitization to the locomotor activity

- enhancing proper ties of 28

cocaine and changes in response to a conditioned fear paradigm were observed in animals 29

exposed several weeks earlier to repeated low

- level formaldehyde. Direct activation of 30

mesolimbic dopamine pathways or activation of conditioned fear response in the amygdala by 31

formaldehyde could underlie these behavioral effects; these observations were also seen by study 32

authors as consistent with a formaldehyde

- induced stress response. 33This document is a draft for review purposes only and does not constitute Agency policy.

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- 279 Table 4

- 57. Summary of neurological and neurobehavioral studies of inha led

formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

Irritant detection threshold

Male

Swiss

mice 8 0, 1, 1.8, 3,

5.6, or 10 ppm

60

- second

exposure episode to

determine

irritant

response Sensitivit y of mice to acute formaldehyde levels

determines the median concentration at which

50% of exposures were terminated by the subject

(AC 50 ) decreased upon repeat exposure. AC 50 = 3.63 for first series, AC 50 = 1.88 ppm for second

series.

Time to exposure te rmination decreased with

increasing formaldehyde concentration.

Time to termination was decreased in repeat

exposures. NA b Wood and

Coleman

(1995)

Motor activity and habituation

Male

and

female

LEW.1K

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

ppm for 2

hours Reduced horizontal activity : Number of crossed

quadrants reduced 2 hours after exposure at all

doses for males and females.

Reduced habituation : Exposed rats exhibited

greater activity than controls when reintroduced

into the testing environment 24 hours later

(ma les and females, all doses). LOAEL =

1 ppm

2 hours Malek et al.

(2003a)

Male

and

female

LEW.1K

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

ppm for 2

hours Reduced horizontal activity : Number of crossed

quadrants reduced 2 hours after exposure at all

doses for males and females. LOAEL =

0.1 ppm

2 hours

in males Malek et al.

(2003b)

Male

AB mice 5

– 7/sex 0, 1.1, 2.3, or

5.2 ppm for 2

hours Reduced horizontal activity : Number of crossed

quadrants reduced 2 hours after exposure at all

doses. LOAEL =

1.1 ppm

2 hours Malek et al.

(2004)

Balb/c

mice 6 0, 1.8, 3.2, 4.5,

6.4, 9.7, or

14.8 ppm for 3

hours Reduced horizontal and vertical activity : Dose

dependent decreases in crossed quadrants and

rearing.

Significant for males at 1.8 ppm and greater ( p <
0.01).

Significant for f emales at 6.4 ppm or greater ( p <
0.01). LOAEL =

1.8 ppm

3 hours

in males Usanmaz et

al. (2002)

Balb/c

mice 6 3.3 ppm for 2

weeks or

2 ppm for 3

weeks

3 hours/day,

5 days/week Reduced horizontal and vertical activity

decreases in crossed quadrants and re aring.

3.3 ppm (2 weeks) and 2 ppm (1 week) ( p <

0.01, p < 0.05). LOAEL =

2 ppm

3 weeks Usanmaz et

al. (2002)

Sprague

Dawley

rats 8 0, 5, 10, or 20

ppm;

3 hours/day for

1 or 2 days Reduced activity levels on both days. Decreases

seen at 5 and 10 ppm; da ta reported only for 5

ppm group LOAEL =

5 ppm,

3 hours Boja et al.

(1985)This document is a draft for review purposes only and does not constitute Agency policy.

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- 280 Table 4

- 57. Summary of neurological and neurobehavioral studies of inha led

formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

Rats 0 or 0.5 mg/m 3

(0.4 ppm) on

GDs 1

– 19, 4

hours/day Increased motor activity on the 2 nd and 3 rd days

of testing (reduced habituation) in offspring

exposed in utero. Increased number of squares

entered ( p < 0.01) and frequency of rearing ( p <
0.05). LOAEL =

0.4 ppm, gestational Senichenkova

(1991)

Rats 15 0, 0.005, or

0.0005 mg/L

(approximately

4 or 0.4 ppm),

GDs 1

– 19 Changes in motor activity at one and two months

in offsprin g exposed in utero. Decreased

spontaneous mobility at 1 month in both sexes,

increased activity at 2 months in both sexes. LOAEL =

0.4 ppm, gestational Sheveleva

(1971)

Learning and memory

Adult

male and

female

LEW.1K

rats 15/sex 0, 0.1, 0.5, or

5.4 ppm

2 hours for 10

consecutive

days Impairment in acquisition of a new task : Male

and female rats at all formaldehyde exposures

had significantly more err ors in completing a

water laby rinth ( p < 0.01).

Male and female rats had longer times to

comple tion of th e maze at 0.5 and 5.4 ppm ( p <

0.05, p < 0.01). LOAEL =

0.1 ppm

2 hours/

10 days Malek et al.

(2003c)

Adult

male and

female

Wistar

rats 5

– 8/

sex 0, 2.6, 4.6 ppm

10

minutes/day

for

90 consecutive

days Deficit in the retention of a learned task : Male

and f emale rats committed significantly more

errors ( p < 0.05) and took more time to complete

the land maze in across the course of the

experiment ( p < 0.04). LOAEL =

2.6 ppm

10 minutes/

90 days Pitten et al.

(2002)

Ferrets 0.25 ppm Impairment in acquisitio n of a new task : Exposed ferrets only achieved a 75% success

rate in training to discriminate odors in a Y

- maze

versus 90% success rate in controls.

Note: The results are confounded with other

effects on the olfactory epithelium. The same

researchers also reported a decrease in olfactory

sensitivity and a reduction in percentage of

olfactory cells in similarly treated animals. None

established Apfelbach

and

Reibenspies

(1991)

(abstract

only)

Male

juvenile

and

adult

rats 5/group 0.25, 0.5 ppm Decreases in olfactory thresholds, in juvenile

animals but not in adults ( p < 0.002). LOAEL =

250 ppm in

juveniles Weiler and

Apfelbach

(1992)

(abstract

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

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- 281 Table 4

- 57. Summary of neurological and neurobehavioral studies of inha led

formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

Neurosensitization endpoints

Female

Sprague

Dawley

rats 8

– 9 0 or 11 ppm

1 hour/day, 7

days Increase in cocai ne

- induced activity : Increased

quadrants crossed after cocaine injection 1 and 2

days after exposure ( p = 0.05 and p < 0.04,

respectively).

No change in corticosterone levels 28 days

postexposure.
No change in nociceptive or passive avoidance

test or plu s

- maze (21, 20, and 13 days

postexposure, respectively) (21 days). LOAEL =

11 ppm/

7 days

(unbounded) Sorg et al.

(1996)

Female

and male

Sprague

Dawley

rats Various

up to
24/group 11 ppm, 1

hour/day,

7 days

1 ppm, 1

hour/day,

7 days

1 ppm, 1

hour/day,

5 days/week,

4 weeks Increase in cocaine

- induced activity : Increase in

rearing after

cocaine injection 1 day after exposure but not 4

– 6 weeks after exposure; 11 ppm for 7 days or 1

ppm for 4 weeks.

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

1 ppm

4 weeks

NOAEL =

1 ppm

7 days Sorg and

Hochstatter

(1999)

Female

and male

Sprague

Dawley

rats Various

up to
24/group 1 ppm, 2

hours/day,

5 days/week,

4 weeks Increased conditioned fear response in

formaldehyde

- treated, foot

- shock

- conditioned

rats, tw ofold ( p < 0.05). LOAEL =

1 ppm

4 weeks Sorg and

Hochstatter

(1999)

Male

Sprague

Dawley

rats 4

– 9 or

16 0, 0.7, or 2.4

ppm for 20 or

60 minutes

0, 0.7, or 2.4

ppm 1 hour/day,

5 days/week

for 2 or 4

weeks No change in corticosterone in acute (20

- and

60

- mi nute) exposures.

Increase in basal corticosterone : 0.7 ppm for 2

or 4 weeks.

Hyperresponsive corticosterone response to

environment : 2.4 ppm for 2 or 4 weeks. LOAEL =

0.7 ppm/

2 weeks

NOAEL =

0.7 ppm/

20 minutes Sorg et al.

(2001)

Female

and male

Spra gue

Dawley

rats 4

– 9 or

16 0 or 2 ppm

1 hour/day,

5 days/week

for 4 weeks Increased conditioned fear response to an
olfactory cue in formaldehyde

- treated, foot

shock

- conditioned male rats. Measured as

increased time freezing when presented with a

novel env ironment ( p < 0.05).

No effect in female rats

. LOAEL =

2 ppm/

4 weeks

NOAEL =

2 ppm/

4 weeks Sorg et al.

(2004)This document is a draft for review purposes only and does not constitute Agency policy.

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- 282 Table 4

- 57. Summary of neurological and neurobehavioral studies of inha led

formaldehyde in experimental animals

Species No./

group Treatment a Observations LOAEL/

NOAEL Reference

Neurochemistry and neuropathology

8

- week

old

female

C3H/

HeN

mice 5 0, 0.08, 0.4, or

1 ppm

16 hours/day,

5 days/week

for 1 day or

12 weeks No change in size of main olfactory bulb by

several measures.

No change in number s of PG cells.

No change in ty rosine hydroxylase

immunopositive PG cells after 1 day.

Increase in tyrosine hydroxylase

- immunopositive

PG cells after 12 weeks to 196, 167, and 196%

of controls at 0.08, 0.40, and 1 ppm,

respectively. LOAEL =

0.08 ppm/

12 weeks Hayashi et al.

(2004)

Adult

male

Sprague

Dawley

rats 8 0, 5, 10, 20

ppm 3 hours/day, 1

or 2 days No change in norepinephrine or 5

- hydroxytryptamine in hypothalamus.

Incre ase in DOPAC in hypothalamus after one

exposure. Increase in dopamine and 5

- HIAA in

hypothalamus after two exposures. LOAEL =

5 ppm/

3 hours Boja et al.

(1985)

Neurogenesis

Neonatal

Wistar

rats 5 0, 6, or 12

ppm,

6 hours/day,

5 days/week

for 30 days Chan ges in volume of granul ar cell layer of the

dentate gy rus in the hippocampus at postnatal

days 30 and 90 ( p < 0.001) LOAEL =

6 ppm/

30 days Aslan et al.

(2006)

Neonatal

Wistar

rats 5 0, 6, or 12

ppm,

6 hours/day,

5 days/week

for 30 days Decreases in brain hemisphere volume at PND

30 ( p < 0.01)

Changes in volume and cell numbers in the CA

region of the hippocampus on PND 30 ( p <
0.01) LOAEL =

6 ppm/

30 days Sarsilmaz et

al. (2007)

Neonatal

Wistar

rats 7 0, 6, or 12

ppm,

6 hours/day,

5 days/week

for 30 da ys Changes in oxidant and antioxidant systems in

cerebellum on PNDs 30 and 90 ( p = 0.017

– 0.001)

. Increases in MDA, NO, and GSH

- Px

and decreases in SOD at both time points. LOAEL =

6 ppm/

30 days Songur et al.

(2008)

a Treatment is given as the formaldehy de concentration in air (ppm) with the length of exposure each day and the

duration of treatment in days, as available.

b NA = not available.This document is a draft for review purposes only and does not constitute Agency policy.

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- 283 Limited data regarding possible neurochemical changes in the brains of formaldehyde

1

exposed, immunized mice (Ahm ed et al., 2007; Fujimaki et al., 2004b; Hayashi et al., 2004; 2

Kakeyama et al., 2004) and rats (Boja et al., 1985) provided conflicting information, and the 3

implications of these data regarding possible formaldehyde neurotoxicity are difficult to 4

determine

. 5

In developmental exposure paradigms, changes in brain structure (Sarsilmaz et al., 2007; 6

Aslan et al., 2006), brain chemistry (Songur et al., 2008), and motor activity (Senichenkova, 7

1991; Sheveleva, 1971) were seen following neonatal or in utero exposur e to formaldehyde. In 8

addition, Weiler and Apfelbach (1992) found juvenile animals to be more sensitive to 9

formaldehyde

- induced changes in olfactory thresholds when compared with adult animals. 10

These studies raise concern about possible long

- lasting neur ological effects of early exposure to 11

formaldehyde. It is important to note, however, that exposure levels in these studies were higher 12

(250

– 6,000 ppb) than those producing the behavioral effects in adults described above. 13

Overall, available data provide substantial evidence of behavioral effects, including 14

motor activity changes and changes in learning and retention, following repeated exposure to 15

relatively low levels of formaldehyde. These effects were seen in multiple laboratories, in 16

studies conduct ed by different authors, and using different behavioral paradigms. These 17

conclusions are also supported by more limited data, indicating possible developmental effects 18

on the nervous system, including changes in brain structure and in the behavior of offs pring; the 19

developmental findings are less robust since they were seen only in individual laboratories and 20

occurred following exposure to higher concentrations of formaldehyde. Studies evaluating 21

developmental neurotoxicity at lower doses, comparable to t hose used in the adult studies, were 22

not available. None of the available data provide sufficient information to allow a determination 23

of the mechanism for these behavioral changes, although it is unlikely that they are attributable 24

to the irritant proper ties of formaldehyde. The data regarding behavioral sensitization provide 25

some support for a stress

- related mechanism for those findings, but the applicability of this 26

mechanism to the behavioral changes seen in the other studies, including the learning d eficits, 27

has not been evaluated. 28

29

4.2.1.6.2. Oral exposure. Available data regarding neurotoxic effects of formaldehyde exposure 30

following oral exposure are very limited. Several chronic or subchronic oral toxicity studies 31

evaluated changes in brain wei ght or histopathology in rats or dogs following repeated oral 32

exposures to formaldehyde at doses as high as 300 mg/kg

- day, administered in drinking water 33

(Til et al., 1989, 1988; Tobe et al., 1989; Johannsen et al., 1986). Although data were not 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 284 presented in the publications, all stated that no changes in brain weight or pathology were seen in 1

the standard evaluations performed in these studies. 2

Two studies evaluated changes in behavior following exposure to formaldehyde in 3

drinking water (Venkatakrishna

- Bhatt et al., 1997; Venkatakrishna

- Bhatt and Panchal, 1992). 4

Venkatakrishna

- Bhatt and Panchal (1992) evaluated changes in performance on a conditioned 5

avoidance task in adult male albino rats (five/group). Animals were exposed to formaldehyde in 6

drinking water (10 mg/mL) or by I.P. injection (10 mg/kg) for 60 days. Although it was stated 7

that water consumption was recorded, the data were not presented, and thus actual exposure 8

levels cannot be documented. Prior to the initiation of exposure, rats were t rained on the 9

conditioned avoidance task (climbing a wooden pole in response to a warning buzzer, thus 10

avoiding electric shock from a floor grid). Rats were trained to a predetermined performance 11

criterion (not described); animals not achieving the criter ion were removed from study. Training 12

and testing conditions (e.g., retest interval and duration of sessions) were not well described. 13

Data were presented as percent response in behavioral performance (apparently separately for the 14

escape or avoidance as pects of the task) or percentage decrease in response. No control data 15

were presented, and pretreatment performance was not described. Figures presented 16

performance at 10

- day intervals, starting with day 0, with each data point stated to represent the 17

me an for five experimental sets; again, the interval between experimental sets and the number of 18

trials per set was not specified. Although the authors concluded that a deficit in performance 19

was demonstrated, the data as presented were difficult to interpr et and the conclusion could not 20

be verified based on the data as presented. 21

Venkatakrishna Bhatt and Panchal (1992) examined changes in performance on a 22

conditioned avoidance response, presumably using a procedure similar to the one described 23

above. Albin o rats (sex not specified, five/group) were exposed to formaldehyde in drinking 24

water at 0.2 or 0.5 mg/mL for 90 days. As described above, rats were trained in performing the 25

task prior to the start of exposure. Venkatakrishna

- Bhatt Bhatr and Panchal (19 92) stated that 26

there was a dose

- related deterioration of performance, but no data were presented to support 27

these conclusions. 28

In summary, available data are insufficient to conduct a reliable assessment of neurotoxic 29

effects of formaldehyde following or al exposure. Limited data suggest a lack of overt 30

neuropathological changes at doses up to 300 mg/kg

- day (Til et al., 1989, 1988; Tobe et al., 31

1989; Johannsen et al., 1986), but detailed information regarding the types of neuropathological 32

evaluations per formed in those studies is not available, and thus no firm conclusions can be 33

drawn regarding the potential for neuropathological effects. The two available studies evaluating 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 285 behavioral changes are not considered to provide useful information, and thus e ffects on nervous 1

system function could not be evaluated. 2

3

4.2.1.6.3. Summary. Overall, there is strong evidence that formaldehyde exposure via 4

inhalation may cause adverse effects on nervous system function in experimental animals at 5

relatively low leve ls of exposure (LOAELs as low as 100 ppb). Although human data regarding 6

neurotoxicity following formaldehyde inhalation are limited, available data provide support that 7

the types of effects seen in humans are similar to those found in animal studies. Ev idence from 8

available human controlled inhalation exposure studies indicates that humans may be affected at 9

doses similar to those used in animal studies; however, the human data are extremely limited. 10

There are insufficient data to evaluate the potential for neurotoxicity following oral 11

exposure to formaldehyde. Limited evaluations of brain weight or histopathology in available 12

chronic or subchronic oral studies found no evidence of formaldehyde

- induced changes (Til et 13

al., 1989, 1988; Tobe et al., 1989; Johannsen et al., 1986). However, reliable studies examining 14

nervous system function or focused studies of neuropathology following oral exposure to 15

formaldehyde are not available. 16

17

4.2.1.6.4. Other considerations. Major data gaps were found regarding the evaluation of 18

changes in nervous system structure or function following formaldehyde exposure by both the 19

inhalation or oral routes. 20

With respect to inhalation exposure, none of the available human studies resulted in data 21

sufficient to conduct a reli able dose

- response assessment for changes in nervous system function. 22

Most of the available animal inhalation studies used short exposure durations (acute or 23

short

- term), precluding a reliable evaluation of neurotoxicity following chronic exposure. 24

Avail able data for neurodevelopmental exposures are also quite limited, consisting of evaluation 25

of neuropathology in only one brain region and functional evaluations focused only on changes 26

in motor activity. 27

Major data gaps also exist regarding neurotoxicity following oral exposure, with no 28

relevant human data and extremely limited animal data. Available oral exposure studies were 29

insufficient to permit a reliable evaluation of the potential for neurotoxicity following oral 30

exposure to formaldehyde. 31

32

4.2.1.7

. Reproductive and Developmental Toxicity 33

The potential for developmental and reproductive effects after formaldehyde exposure by 34

the inhalation route has generally been considered low, since formaldehyde, as a reactive gas, is 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 286 not expected to penetrate p ast the POE (NEG, 2003; IPCS, 2002; Collins et al., 2001). 1

Nevertheless, a number of animal studies have demonstrated effects of formaldehyde on pre

- and 2

postnatal development and on the reproductive system. For example, developmental toxicity 3

was observ ed in two studies that evaluated a standard battery of developmental endpoints 4

resulting from inhalation exposure on GDs 6

– 10 (Martin, 1990; Saillenfait et al., 1989). 5

Similarly, oral exposures resulted in developmental effects when administered during 6

co mparable gestational windows (Marks et al., 1980; Hurni and Ohder, 1973). There have also 7

been reports that identified developmental effects at lower

- level formaldehyde exposures that 8

were administered throughout gestation (Senichenkova and Chebotar, 1996 ; Senichenkova, 9

1991; Kitaev et al., 1984; Sheveleva, 1971; Gofmekler and Bonashevskaya, 1969; Gofmekler, 10

1968; Pushkina et al., 1968). Postnatal functional consequences of developmental exposures 11

have also been identified (Sarsilmaz et al., 2007; Aslan e t al., 2006; Weiler and Apfelbach, 1992; 12

Senichenkova, 1991; Sheveleva, 1971). Additionally, a number of studies suggest that 13

formaldehyde adversely affects the male reproductive system after both inhalation and oral 14

exposures (Xing et al., 2007; Zhou et al., 2006; Özen et al., 2005, 2002; Sarsilmaz et al., 1999; 15

Chowdhury et al., 1992; Cassidy et al., 1983; Guseva, 1972). This section reviews the available 16

published studies assessing reproductive and developmental endpoints of formaldehyde. 17

18

4.2.1.7.1. Inhalation studies addressing developmental and reproductive toxicity. Saillenfait et 19

al. (1989) reported a comprehensive and well

- documented developmental study in Sprague

20

Dawley rats. Pregnant rats were exposed beginning on GD 6 in order to cover critic al stages of 21

development (e.g., implantation and major organogenesis). Female Sprague

- Dawley rats 22

(25/group) were exposed to 0, 5, 10, 20, or 40 ppm (0, 6.15, 12.3, 24.6, or 49.2 mg/m 3 ) 23

formaldehyde 6 hours/day on GDs 6

– 20. The onset of pregnancy was det ermined by the 24

presence of sperm in a vaginal smear. Dams were exposed to formaldehyde in a dynamic flow 25

chamber, and formaldehyde concentrations were determined to be 0, 5.17 ± 0.51, 9.92 ± 0.88, 26

20.04 ± 0.88, and 38.96 ± 3.70 ppm. Dams were weighed on GDs 0, 6, and 21 and sacrificed on 27

day 21. Upon examination, uterine weights, fetal weights, sex ratio, number of implantation and 28

resorption sites, and live and dead fetuses were recorded. Fetuses were examined for external 29

malformations and cleft palat e. One

- half of viable fetuses were sectioned to assess soft

- tissue 30

alterations. The other half were fixed, stained with alizarin red S, and examined for skeletal 31

alterations. 32

Body weight gain of dams and body weight of male and female fetuses were reduce d by 33

exposure to 40 ppm formaldehyde to 49, 78, and 81% of control values, respectively ( p < 0.01) 34

( Saillenfait et al., 1989) . Reduced weight gain in dams remained significantly decreased when 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 287 uterine weight was accounted for ( p < 0.01). Mean fetal weigh t of male pups was reduced at 1

maternal exposures of 20 and 40 ppm formaldehyde (5.53 and 4.42 g versus 5.61 g in controls). 2

Decreased fetal body weight in females was only seen at 40 ppm (4.27 g versus 5.24 g in 3

controls). All other pregnancy endpoints w ere unchanged by formaldehyde exposure (e.g., 4

uterine weight, implantation and resorption sites, live fetuses, dead fetuses, and sex ratios) . No 5

major malformations were noted in fetuses. Some minor soft tissue and skeletal anomalies, such 6

as dilated ure ter, missing sternebrae, extra fourteenth rib, and rudimentary thirteenth rib 7

(statistics not given), were reported. However, these effects occurred at similar frequencies in 8

control and treatment groups. The incidence of delayed ossification of the thor acic vertebrae 9

was 8.7% in fetuses from the 40 ppm exposure group versus 1.8% in controls. However, this 10

difference was not statistically significant. Overall, from these results formaldehyde was neither 11

lethal to embryos nor teratogenic, only exhibiting fetotoxic effects at exposures of 20 ppm and 12

above. These are levels where there was a significant decrease in fetal body weight. 13

Martin (1990) conducted a similar study exposing pregnant rats during similar stages of 14

development. Mated female Sprague

- D awley rats (25/group) were exposed to 2, 5, or 10 ppm 15

(2.46, 6.15, or 12.3 mg/m 3 ) formaldehyde 6 hours/day on GDs 6

– 15. The study included two 16

control groups: dams placed in the exposure chambers once a day but exposed only to clean air 17

and dams fed and h oused similarly to the experimental groups but never put into the inhalation 18

chambers. The method of formaldehyde vapor generation and details of the exposure chamber 19

were not described. Mean formaldehyde exposure concentrations were reported as 1.88, 4. 88, 20

and 9.45 ppm (variability not given, analytical method not discussed). Food consumption and 21

body weight were recorded. On GD 20, rats were sacrificed, and the following pregnancy 22

parameters were recorded: live fetuses, dead fetuses and resorptions, n umber of corpora lutea, 23

fetal weights, sex ratios, and preimplantation and postimplantation losses. Fetuses were 24

examined for major malformations, minor external and visceral anomalies, and minor skeletal 25

anomalies (details not given). Weight gain and fo od consumption in dams were said to be 26

reduced at 10 ppm ( p < 0.05). Formaldehyde exposure of the dams at 5 and 10 ppm led to an 27

increased incidence of reduced ossification of the pubic and ischial bones in fetuses on GD 20 28

( p < 0.05). Reduced ossificati on correlated with lower fetal weights, and the author considered 29

both of these findings a result of larger litter size and, therefore, not related to formaldehyde 30

exposure. However, no tables presenting the data or statistical analysis were provided. Al l other 31

pregnancy parameters and fetal anomalies were described as unaffected by formaldehyde 32

exposure. However, without data presented for the assessed endpoints, background rates of 33

malformations, trends in the data, and variability, it is difficult to evaluate the Martin (1990) 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 288 comparisons. However, the author’s observations of reduced fetal weight and increased 1

incidence of reduced ossification are consistent with the results of Saillenfait et al. (1989). 2

Kilburn and Moro (1985) studied similar endpoi nts but included formaldehyde exposure 3

during earlier gestational windows. The study report, only available in abstract form and not 4

found as a subsequent published article, does not provide many methodological details. Female 5

rats (number and strain not reported) were exposed to 0 or 30 ppm (0 or 37.2 mg/m 3 ) 6

formaldehyde 8 hours/day during GDs 3

– 17, 3

– 12, 8

– 12, or 9

– 11. A second experiment 7

included pair

- fed controls for dams exposed to 30 ppm formaldehyde during GDs 3

– 17, 3

– 12, 8

and 8

– 12. The authors re ported reductions in fetal and maternal weight gain that were greater 9

than decreases in pair

- fed controls. Fetal anomalies were noted after 15 days of gestational 10

exposure (e.g., altered organ size and undescended testes). Although the report indicates s ome 11

maternal toxicity and fetotoxic effects (for example, stunted growth), lack of study details and 12

clear reporting make this report of negligible utility in human health risk assessment. 13

There are several early studies that examined developmental effects of formaldehyde 14

exposure administered throughout gestation (Gofmekler and Bonashevskaya, 1969; Gofmekler, 15

1968; Pushkina et al., 1968). It is unclear if these reports represent the same or overlapping 16

experimental groups. They were performed in the same laboratory and are reported with a 17

similar level of detail. The source of formaldehyde, method of vapor generation, exposure 18

conditions (dynamic versus static), confirmation of exposure concentrations, study design, and 19

data presentation details were not provided. Absence of such critical information detracts from 20

the quality of these studies as a coherent record of experimental information, and, thus, these 21

findings can only be utilized qualitatively in the formaldehyde risk assessment. 22

In the Gofmekl er (1968) study, female rats (36, strain not specified) were continuously 23

exposed at 0, low, or high formaldehyde concentrations beginning 10

– 15 days prior to mating 24

(target concentrations of 0, 0.01, or 0.81 ppm formaldehyde [0, 0.01, or 1 mg/m 3 ]). The a uthor 25

reported a 14

– 15% increase in pregnancy duration and a decrease in litter size (data not shown). 26

However, males and females were mated 6

– 10 days, and no information was provided on how 27

mating and conception were confirmed. No external malformations were attributed to 28

formaldehyde exposure. Concentration

- dependent increases in pup body weight and decreases in 29

lung and liver weight were attributed to formaldehyde exposure. Pup weights were increased 30

from 5.6 g in controls to 6.0 and 6.3 g in groups 1 and 2, respectively ( p < 0.01 and p < 0.001). 31

Formaldehyde exposure increased pup adrenal weight in both groups and pup thymus and kidney 32

weight in group 2 only (Table 4

- 58). 33

34

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

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- 289 Table 4

- 58. Effects of formaldehyde on body and organ weights in rat pups 1

f rom dams exposed via inhalation from mating through gestation 2

3

E xposure (pp m) a Body weight

( g) Relative organ weights (mg/10 g body weight)

Thymus Heart Lung Liver Adrenals Kidney

0 5.6 26 61.4 287.1 587.7 3.2 51.4

0.01 6.0 b 25.1 61.5 230.2 c 557.7 d 4. 2 c 53.4

0.81 6.3 c 31.7 c 64.5 223.2 c 550.8 b 3.8 d 55.7 b

4

a Dams were exposed to formaldehyde continuously from 10

– 15 days prior to mating. Exposure concentrations 5

were not validated. 6

b Different from controls, p < 0.01. 7

c Different from controls, p < 0.001. 8

d Different from controls, p < 0.05. 9

10

Source: Gofmekler (1968). 11

12

In a study by Gofmekler and Bonashevskaya (1969), the researchers evaluated organ 13

histopathology in pups from similarly treated dams. Pregnant female albino rats were 14

continuously exposed a t two formaldehyde concentrations (groups 1 and 2, as described above). 15

Adult males were similarly exposed during mating. Offspring were examined for malformations, 16

and the organs were fixed and sectioned for histopathologic examination, including hemato xylin 17

and eosin staining, Brachet stain for RNA, and Feulgen stain for DNA. Liver and kidney 18

sections were also stained with Schiff’s reagent (which reacts with aldehydes), with Sudan III for 19

lipids, and Pearl’s stain for iron. Placenta, uterus, and ovar ies from the dams and testes of the 20

males were sectioned, stained, and evaluated. The authors stated that formaldehyde induced no 21

external anomalies (reported elsewhere, but no reference given). The authors also noted 22

involution of lymphoid tissue and ch anges in liver, mild hypertrophy of Kupffer cells, and 23

numerous extramedullary myelopoietic centers in pups from dams in group 2. Pups from both 24

treatment groups showed reduced glycogen content in the myocardium and the presence of iron 25

in Kupffer cells. There was a localized increase in positive reaction to Schiff’s reagent in the 26

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

that this was an indication of functional alterations in the renal tubular apparatus . All other 28

tissues examined and histochemical staining indicated no differences due to formaldehyde 29

exposure. 30

Researchers in the same laboratory (Pushkina et al., 1968) studied the effects of 31

formaldehyde exposure on vitamin C (ascorbic acid, an antioxid ant) and nucleic acid levels in 32

dams and fetuses as general measures of toxicity. Female white rats (n = 160) were continuously 33

exposed at two formaldehyde concentrations (groups 1 and 2, as described above) from 20 days 34

prior to mating (6

– 10 days) and th en throughout gestation. Dams were sacrificed and ascorbic 35

acid and nucleic acid levels determined in harvested organs (methods referenced but not 36This document is a draft for review purposes only and does not constitute Agency policy.

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- 290 described). No visible malformations in pups were noted. Formaldehyde exposure increased 1

fetal body weight and organ weight in both groups (data not given). There was an average of 2

11.3 fetuses per litter for control dams versus 9.8 and 8.6 for groups 1 and 2, respectively. The 3

authors reported that formaldehyde exposure decreased DNA levels and increased RN A levels in 4

organs (further details not provided). Formaldehyde exposure resulted in lower vitamin C levels 5

in the whole fetus (76 and 75% of controls) and in maternal liver specimens (82 and 88% of 6

controls) for exposure groups 1 and 2, respectively ( p < 0.05). In contrast, vitamin C was higher 7

in fetal liver (127% of controls) in group 1 ( p < 0.05). The significance of these differences is 8

unknown. The authors considered the results as general measures of biochemical changes and 9

therefore toxic. 10

The r eports of Gofmekler and Bonashevskaya (1969), Gofmekler (1968), and Pushkina et 11

al. (1968) lack key methodological details. As discussed above, exposure conditions and actual 12

formaldehyde concentrations cannot be validated. Although methods were not thor oughly 13

detailed, results were reported in data tables with statistics and detailed descriptions of observed 14

pathological changes. However, without validation of exposure concentrations, these findings 15

can only be considered qualitatively. 16

In another study , Sheveleva (1971) exposed female mongrel (i.e., not a homogeneous 17

genetic strain) white rats to 0, 0.0005, or 0.005 mg/L (0, 0.5, or 5 mg/m 3 ) (0, 0.4, or 4 ppm) 18

formaldehyde on GDs 1

– 19 (where GD 1 was defined as the day that spermatozoa were detected 19

in vaginal smears) for 4 hours/day. In each group, 15 dams were terminated on GD 20 for 20

evaluation of ovarian corpora lutea, uterine implantation sites, pre

- and postimplantation loss, 21

number of live fetuses, fetal length and weight, and external examination for malformations. 22

Additionally, in each group, six dams were allowed to deliver their litters. Developmental 23

landmarks were monitored (i.e., ear and eye opening, incisor eruption, emergence of hair coat), 24

and the pups were further evaluated at 1 and 2 months of age for body weight, threshold of 25

neuromuscular excitability, total oxygen consumption in 1 hour per 100 g of weight, and 26

spontaneous mobility over 10 minutes. Maternal toxicity (recorded on GD 17) included 27

significantly ( p < 0.05) decreased leu kocyte counts in both treated groups and a number of 28

additional findings at 0.005 mg/L (i.e., significant reductions in the threshold of neuromuscular 29

excitability, rectal temperature, and blood hemoglobin level) as well as an increase in 30

spontaneous mobil ity over 15 minutes. (It is noted that a significant reduction in blood 31

hemoglobin was also observed by Sanotskii et al. [1976] in pregnant rats, following 20 days of 32

gestational inhalation exposure for 4 hours/day to formaldehyde at 6 mg/m 3 [4.83 ppm].) Fetal 33

examinations on GD 20 identified a 50

– 70% increase in mean preimplantation loss in both 34

formaldehyde

- exposed groups. When pups were 1 month of age, a reduction in spontaneous 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 291 mobility was noted in both treated groups; in pups at 2 months of age, an increase in mobility 1

was observed in the 0.005 mg/L group. Also, when pups were 2 months of age, there were 2

alterations in hemoglobin levels and leukocyte counts in both treated groups. Detailed 3

descriptions of some study methodologies (particularly in regard to neurological and behavioral 4

assessments) were not provided in the published paper. 5

Based on a review of the work by Gofmekler (1968) and various epidemiologic studies 6

available at the time, Kitaev et al. (1984) hypothesized that formaldehyde may exert toxic effects 7

in the early days of gestation. To study embryotoxic effects of formaldehyde inhalation 8

exposures, mature female Wistar rats (five to nine per group) were exposed to 0.41 or 1.22 ppm 9

(0.5 or 1.5 mg/m 3 ) formaldehyde 4 hours/day, 5 days/ week for 4 months (Kitaev et al., 1984). 10

Rats were exposed in dynamic flow chambers and formaldehyde levels measured gravimetrically 11

(but not reported). Females were mated on day 120 of exposure and mating confirmed by the 12

presence of sperm in a vaginal smear. Embryos were harvested on the second or third day of 13

pregnancy (GD 2 or 3) and examined by both light and phase contrast microscopy for changes in 14

morphology (i.e., evidence of embryonic degeneration). Additionally, maternal weight gain and 15

organ weights (ovaries, uterus, and adrenal glands) and blood samples (HCT, Hb, and TP) were 16

monitored as indicators of general toxicity. These parameters were unchanged by formaldehyde 17

exposure. Formaldehyde exposure at 1.22 ppm for 4 months resulted in an in creased number of 18

degenerating embryos on GD 3 (14.9 versus 4.4% in controls) and a smaller increase of 10.2% 19

(versus 5.1% in controls; statistical significance not assessed) on GD 2. Indications of 20

degeneration included reduced size and changes in appear ance (granulation of the ooplasm, 21

wrinkling and degradation of nuclear material). However, it is unclear if litter effects were 22

accounted for in the statistical analyses, and it is unknown how the affected embryos were 23

distributed between litters. For da ms exposed to 0.41 ppm formaldehyde, the number of 24

degenerated embryos was not increased on day 2 (3.8 versus 5.1% in controls) but was increased 25

on day 3 (9.1 versus 4.4% in controls; again, unknown if statistically significant) after maternal 26

exposure to 1.22 ppm formaldehyde. This observation may be coincidental since it was seen in 27

dams sacrificed on GD 2 but not in those sacrificed on GD 3. Kitaev et al. (1984) considered 28

these findings to indicate that repeated exposure to formaldehyde over a 4

- mont h period can 29

disturb reproductive function, resulting in adverse effects early in embryonic development. 30

To further explore the effects of inhalation exposures to formaldehyde on reproductive 31

function, Kitaev et al. (1984) conducted a second series of expe riments on 200 similarly treated 32

female rats. After 4 months of repeated formaldehyde exposure at 0.41 or 1.22 ppm as described 33

above, organ weights (ovaries and uterus) and blood levels of gonadotropic hormones and 34

progesterone were determined. However, the day and time of hormone measurement were not 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 292 given in the report, and normal diurnal variations in these hormones could affect the reported 1

findings if time of day was not accounted for. The length of the estrous cycle was unchanged 2

during exposure. Formaldehyde exposure modulated gonadotropin levels and relative ovarian 3

weight, suggesting low

- level effects on the female rat reproductive system prior to mating 4

(Kitaev et al., 1984). Ovarian weight and blood levels of luteinizing hormone (LH) were bo th 5

significantly increased after exposures at 0.41 ppm formaldehyde but remained at control levels 6

in rats exposed at 1.22 ppm. Blood levels of follicle

- stimulating hormone (FSH) were increased 7

approximately 66% from control after 1.22 ppm formaldehyde ex posure ( p < 0.05). 8

Progesterone levels were unchanged by formaldehyde treatment. Kitaev et al. (1984) suggested 9

a role of the hypothalamus

- pituitary system based on increased ovary weight, a greater number 10

of degenerated embryos, and increased LH in rats exposed at 0.41 ppm. They postulated that 11

these effects were not seen at 1.22 ppm due to a toxic effect exhibited as embryonic 12

degeneration, thus the absence of a dose

- response did not alter the interpretation of the validity 13

of the adverse response. Th e study NOAEL was not determined, and the study LOAEL was 0.4 14

ppm (0.5 mg/m 3 ), based upon increased early embryo loss and on maternal outcomes (increased 15

ovarian weight and increased blood LH levels) following 4 months of formaldehyde treatment. 16

For the f inding of increase blood FSH levels, the endpoint NOAEL was 0.4 ppm (0.5 mg/m 3 ) 17

and the LOAEL was 1.2 ppm (1.5 mg/m3). 18

Senichenkova and Chebotar (1996) and Senichenkova (1991) examined reproductive and 19

developmental effects of daily formaldehyde exposure o n GDs 1

– 19 of pregnancy, including the 20

potential effect of formaldehyde exposure on development early in gestation. Additionally, since 21

anemia adversely affects fetal development, Senichenkova and Chebotar (1996) also examined 22

formaldehyde effects in iron

- deficient dams to determine whether co

- exposure further 23

compromises fetal development. In both studies, female white rats were exposed to 0 or 24

0.41 ppm formaldehyde (0 or 0.5 mg/m 3 ), 4 hours/day on GDs 1

– 19. Formaldehyde 25

concentrations in the dynamic ex posure chambers were measured gravimetrically to confirm the 26

exposure concentration but were not reported (methods not provided). It is unclear if gravimetric 27

measurements would be sensitive or accurate enough to validate these low

- exposure 28

concentrations without a better understanding of the methodology. This uncertainty in exposure 29

conditions should be considered in evaluating the reported results. 30

Mongrel female white rats were exposed at a target concentration of 0 or 0.41 ppm (0 or 31

0.5 mg/m 3 ) formald ehyde 4 hours/day on GDs 1

– 19 (Senichenkova, 1991). On GD 20, a subset 32

of the dams was sacrificed and examined for number of corpora lutea, implantation and 33

resorption sites, live/dead fetuses, and fetal weights. Fetuses were examined for gross pathology 34

of the internal organs and skeleton (details not given). Blood pH, partial pressure of CO 2 , and 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 293 partial pressure of oxygen were measured in both dams and embryos. The remaining dams were 1

brought to term to study postnatal effects of formaldehyde exposur e. Rat pups were observed on 2

PNDs 1

– 25 for viability, physical development, and maturation rate of motor reflexes. Behavior 3

of juvenile offspring (PND 40) was studied in an open field test, and maze learning was tested at 4

sexual maturity. In a follow

- up report, Senichenkova and Chebotar (1996) present blood 5

chemistry data, pregnancy outcome, and developmental data for similarly treated dams and their 6

pups in a chemical model of iron deficiency. Intraperitoneal injections of the iron

- chelating 7

agent bipy ridyl were given on GDs 12

– 15 at the threshold embryotoxic dose (1 mL, 25% 8

solution). On day 20, the dams were sacrificed and dams and fetuses examined as described 9

above. In addition to blood pH, partial pressure of carbon dioxide and partial pressure o f 10

oxygen, acid metabolic products (not detailed), and true bicarbonates were reported for maternal 11

and fetal blood. A review of the data from these reports indicates there may be an overlap of the 12

study groups. Neither paper presents the entire data set; thus, for tranparency and brevity, the 13

following text discusses the combined findings from both studies as if they were a single study 14

Formaldehyde exposure did not affect such indicators of pregnancy outcome as number 15

of corpora lutea, implantation and r esorption sites, and live and dead fetuses, all of which were 16

unchanged (Senichenkova and Chebotar, 1996; Senichenkova, 1991). Although fetal weight was 17

slightly increased by formaldehyde exposure, 2.35 versus 2.24 g in controls ( p < 0.001), neither 18

fetal length nor bone length were changed (femur and humerus) (Senichenkova and Chebotar, 19

1996; Senichenkova, 1991). Often, increased fetal weight is the result of early physical 20

development, and other signs of development, such as ossification, would be expec ted to be 21

enhanced as well. The average number of bone centers per limb was increased by formaldehyde 22

exposure from 2.45 and 2.66 to 2.78 and 2.91 in controls for metacarpal and metatarsal bone 23

centers, respectively ( p < 0.05) (Senichenkova, 1991); these findings were consistent with 24

increased growth and weight. In contrast, Senichenkova (1991) reported a decrease in the 25

number of embryos with ossification centers in the hyoid bone (100% in controls versus 91% for 26

formaldehyde exposure, p < 0.05), consist ent with the results of Saillenfait et al. (1989) and 27

Martin (1990). However, litter size, a factor influencing fetal weight, was not provided, and it is 28

unclear if Senichenkova (1991) took litter size into account in the analysis. 29

Senichenkova and Chebo tar (1996) reported increased blood acidosis and decreased 30

blood alkaline reserves (bicarbonates and total CO 2 ) in formaldehyde

- treated dams and their 31

embryos ( p < 0.05). However, this finding should be considered in light of the fact that chronic 32

blood a cidosis may increase bone remodeling and decrease bone density in adults. It is unknown 33

if the reported blood acidosis could reduce ossification rates in developing embryos. A better 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 294 understanding of exposure conditions and the acid metabolic products me asured is needed to 1

determine the biological relevance of the reported changes in blood acid balance. 2

Iron deficiency, induced by injections of bipyridyl (an iron

- chelating agent), was found to 3

be fetotoxic. Iron

- deficient dams with no formaldehyde exposu re had higher rates of 4

postimplantation death than controls (12.6 ± 5.5 versus 4.8 ± 1.3%). Formaldehyde exposure in 5

conjunction with iron deficiency increased postimplantation death to 23.1 ± 5.9%. Fetal weight 6

and litter size were not reported for the bipyridyl treatment groups, but bipyridyl treatment in 7

conjunction with formaldehyde resulted in a decreased number of metatarsal bone centers (2.21 8

± 0.12 versus 2.72 ± 0.08 in controls; p < 0.001). This decrease was also significant when 9

compared with f ormaldehyde or bipyridyl alone ( p < 0.02). However, all pregnancy outcome 10

parameters were not reported for the bipyridyl treatment. 11

Fetal anomalies were reported after formaldehyde exposure and were increased by iron 12

deficiency. The incidence of litters with internal organ anomalies was increased from 1.4% in 13

controls to 14.2% in formaldehyde

- treated dams (Senichenkova, 1991). Undescended testes 14

were the predominant anomaly described: 20.8% in litters from formaldehyde

- treated dams 15

versus 1.2% in control s ( p < 0.05) (Senichenkova, 1991). Similar findings were reported by 16

Senichenkova and Chebotar (1996). Bipyridyl treatment in conjunction with formaldehyde 17

exposure increased the overall incidence of fetal anomalies (13.8 ± 2.1% in controls versus 6.6 ± 18

1.8% with iron deficiency alone) (Senichenkova and Chebotar, 1996). However, there are 19

discrepancies between the two papers in the reporting of the anomalies, and it is unclear whether 20

the experimental groups overlap between papers, where some parameters are identical (which 21

would lead to double counting of the same animal, including identical SDs) and others are 22

different. Additionally, the reporting is unclear with respect to the basis of the incidence rates 23

reported (for example, overall incidence vers us incidence within litter or incidence of litters with 24

anomalies). Unclear reporting, together with some of the uncertainties regarding exposure 25

conditions, suggests that the data may be of limited quality to support risk assessment. 26

In the second phase of the studies, pups were delivered and postnatal development was 27

assessed (Senichenkova, 1991). Eruption of the upper and lower incisors was delayed in pups 28

from formaldehyde

- treated dams, occurring on PND 14 versus PND 12 in controls ( p < 0.01). 29

All ot her measures of physical postnatal development were unchanged by formaldehyde. To 30

evaluate postnatal functional outcomes following in utero exposure to formaldehyde, an open 31

field test was conducted in juvenile rats on 3 consecutive days (PNDs 40

– 42). Ju venile rats from 32

formaldehyde

- treated dams exhibited increased mobility (crossed squares), rearings, and 33

defecations/urinations compared with control rats on the second and third open field tests 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 295 ( p < 0.05). There were no differences seen in the maze

- lear ning test assessed in mature offspring 1

of formaldehyde

- treated dams (Senichenkova, 1991). 2

Additional assessments of formaldehyde exposure on neurological development are 3

described above in the section on neurological and behavioral toxicity in animal studi es (Section 4

4.2.1.6). In brief, studies conducted by Sarsilmaz et al. (2007) and Aslan et al. (2006) exposed 5

10 neonatal male Wistar rats/group to 0, 6, or 12 ppm (0, 7.36, or 14.7 mg/m 3 ) formaldehyde 6

6 hours/day, 5 days/week for 30 days. At that time, f ive rats/group were killed and subjected to 7

neuropathological examination; the remaining rats were maintained until PND 90, at which time 8

they were killed and evaluated. Aslan et al. (2006) examined the number and volume of granular 9

cells in the hippocamp al dentate gyrus, while Sarsilmaz et al. (2007) examined the size and 10

number of the pyramidal cells in the cornu ammonis of the hippocampus. In both studies, lower 11

numbers of cells were observed in both treated groups at PND 90 as compared with PND 30. 12

A lthough the effects of treatment on the volume and number of granular and pyramidal cells 13

were somewhat inconsistent, a significant decrease in the number of neurons in the pyramidal 14

cell layer of the hippocampal cornu ammonis was observed at both PNDs 30 and 90 (Sarsilmaz 15

et al., 2007). 16

One other study reported effects on nervous system function following exposure to 17

formaldehyde during postnatal development. An abstract by Weiler and Apfelbach (1992) 18

described a study in which juvenile rats (strain not s pecified) were exposed to 0.25 ppm 19

(0.31 mg/m 3 ) formaldehyde from PNDs 30

– 160 or adult rats were exposed to 0.5 ppm 20

(0.62 mg/m 3 ) formaldehyde for 90 days

. Decreased olfactory sensitivity (i.e., increased olfactory 21

thresholds) was observed and was greater when the exposure was initiated in the young rats, as 22

compared with the adults. 23

Evaluation of offspring following prenatal, perinatal, and/or juvenile inhalation exposures 24

to formaldehyde have also been reported by Kum et al. (2007), Sandikci et al. (2007) , and 25

Songur et al. (2005). Kum et al. (2007) exposed female Sprague

- Dawley rats (six dams/group) 26

and their offspring to 0 or 6 ppm (0 or 7.4 mg/m 3 ) formaldehyde for 8 hours/day in separate 27

groups with exposures starting on GD 1, on postparturition day 1, or in offspring at 4 weeks of 28

age and continuing for 6 weeks. In another group, exposures were initiated in adult rats. Mean 29

body and liver weights were significantly decreased in the offspring exposed in utero and in 30

early postnatal life, and mean live r weights were also significantly decreased in rats with juvenile 31

exposures. However, neither body weight nor liver weight was affected in the group with 32

exposure initiating at an adult age, suggesting a life

- stage

- related susceptibility to formaldehyde

33

i nduced hepatic toxicity. Evaluation of biomarkers of oxidative stress revealed significantly 34

increased catalase (CAT) and MDA in the livers of offspring that were exposed prenatally, 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 296 significantly decreased GSH levels in the livers of offspring that were exposed in early postnatal 1

life, and significantly decreased SOD levels in the livers of offspring that were exposed starting 2

at 4 weeks of age. No biomarkers were altered in the livers of rats exposed to formaldehyde only 3

as adults. 4

A similar study desig n was used by Sandikci et al. (2007) to examine the effects of 0 or 5

6 ppm (0 or 7.4 mg/m 3 ) formaldehyde on bronchus associated lymphoid tissue (BALT) 6

following pre

- and perinatal, juvenile, or adult exposures of 6 weeks duration in Sprague

- Dawley 7

rats (six /group). The presence of the lysosomal enzyme alpha

- naphthylacetate esterase (ANAE) 8

served as a marker of T

- lymphocytes in peripheral blood and tissue sections. Significant 9

increases in ANAE

- positive T

- lymphocytes were found in BALT in all but the in ute ro exposed 10

groups as compared with control; this outcome is consistent with the postnatal development of 11

BALT in the rat. In peripheral blood, ANAE

- positive lymphocyte ratios were significantly ( p < 12

0.001) increased as compared with controls at all life s tages tested. 13

Songur et al. (2005) examined the effect (and reversibility) of formaldehyde exposures 14

during the early postnatal period on zinc, copper, and iron levels and activity of SOD in the lung 15

tissue of Wistar rats. Litters (12

– 14/group) were expos ed to 0, 6, or 12 ppm (0, 7.4, or 16

14.9 mg/m 3 ) formaldehyde 6 hours/day, 5 days/week for 30 days. Trace element and 17

biochemical analyses were conducted on PND 30 or 90. Decreased SOD activity, decreased 18

levels of copper and iron levels, and increased zinc levels were observed in the lungs of treated 19

groups following 30 days of treatment and at 90 days (i.e., 60 days posttreatment). Survival was 20

not affected in neonatal rats. Clinical observations during treatment included evidence of 21

respiratory irritati on and toxicity. Body weight and food and water consumption were also 22

nonsignificantly decreased as compared with controls. 23

There are several reports in the literature regarding formaldehyde effects after inhalation 24

exposure on the male reproductive syste m in animals (Galalipour et al., 2007; Zhou et al., 2006; 25

Özen et al., 2005, 2002; Sarsilmaz et al., 1999; Woutersen et al., 1987; Maronpot et al., 1986; 26

Guseva, 1972). The earliest report examined the effect of simultaneous exposures to 27

formaldehyde from air and water (Guseva, 1972). Male rats (n = 12, strain not specified) were 28

co

- exposed to formaldehyde in air and drinking water 4 hours/day, 5 days/week for 6 months. 29

There were three exposure levels in the experiment of different air and drinking wate r 30

concentrations: (1) 0.41 ppm (0.5 mg/m 3 ) formaldehyde in air and 0.1 mg/L in water; (2) 0.20 31

ppm (0.25 mg/m 3 ) formaldehyde in air and 0.01 mg/L in water; or (3) 0.10 ppm (0.12 mg/m 3 ) 32

formaldehyde in air and 0.005 mg/L in water. Reproductive function was assessed by mating 33

two females per male. The time for the onset of pregnancy and the number of pregnancies per 34

treatment group were recorded. A subset of dams was sacrificed on GD 20 of pregnancy, and 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 297 the number and weight of fetuses was determined. Po stnatal development of the remaining dams 1

was tracked (e.g., times of eye opening and development of hair coat). Nucleic acid levels were 2

determined in the testes of formaldehyde

- exposed rats. Gonadotropic response was assessed by 3

injecting an emulsion o f pituitaries from exposed male rats into unexposed infantile females and 4

measuring the weight ratios of the uterus and ovaries. Formaldehyde exposure reduced nucleic 5

acid levels in testes to 88 and 92% of controls in groups 1 and 2, respectively. No oth er 6

formaldehyde

- induced differences were found. 7

Woutersen et al. (1987) and Maronpot et al. (1986) examined tissue sections from testes 8

and ovaries of exposed animals as part of studies primarily addressing respiratory tract toxicity 9

(see Section 4.2.1.2.2 .4 for complete study details). Maronpot et al. (1986) exposed female and 10

male B6C3F1 mice to 0, 2, 4, 10, 20, and 40 ppm (0, 2.45, 4.91, 12.3, 24.5, and 49.1 mg/m 3 ) 11

formaldehyde 6 hours/day, 5 days/week for 13 weeks. Decreased weight gain due to 12

formald ehyde exposure was seen in both male and female mice. Additionally, there was 80% 13

mortality at the highest exposure (40 ppm). The authors reported endometrial hypoplasia and 14

lack of ovarian luteal tissue in females exposed to 40 ppm, but no compound

- rela ted changes 15

were observed in testes sectioned and viewed by light microscopy. 16

In a study by Appleman et al. (1988), male Wistar rats (40/group) with undamaged or 17

damaged (via bilateral intranasal electrocoagulation) nasal mucosa were exposed for 13 or 18

52 weeks to 0, 0.1, 1, or 10 ppm (0, 0.124, 1.24, or 12.4 mg/m 3 ) formaldehyde 6 hours/day, 19

5 days/week. At study termination, mean body weight was decreased, but relative testes weight 20

was increased in the 10 ppm group (interpreted as a non

- adverse outcome t hat was associated 21

with the decreased body weight). No treatment

- related histopathologic findings were reported 22

for male reproductive organs (although it is not clear to what extent they were evaluated since 23

the primary focus of the study was on the nasal epithelium). 24

Following up on earlier reports of decreased Leydig cell quality in rats administered I.P. 25

injections of formaldehyde (Chowdhury et al. [1992], described in Section 5.2.1.8.3), Sarsilmaz 26

et al. (1999) studied the effects of formaldehyde inhal ation on Leydig cells. Adult male Wistar 27

rats (30) were exposed to 0, 10, or 20 ppm (0, 12.3, or 24.6 mg/m 3 ) formaldehyde 8 hours/day, 28

5 days/week for 4 weeks. Animals were observed daily and weighed weekly. Rats were 29

sacrificed on day 29 and autopsied, and testes were weighed, fixed, and sectioned for histologic 30

examination. Signs of irritation from formaldehyde exposure were noted (frequent eye blinking, 31

excessive licking, increased frequency of nose cleaning, interrupted breathing, and sneezing). 32

Bo dy weight gain was reduced by formaldehyde exposure from 17.7% gain in control rats to 33

4.66 and 2.63% in rats exposed at 10 and 20 ppm, respectively ( p < 0.001). As shown in 34

Table 4

- 59, relative testes weights were unaffected (reported as proportions but more likely to be 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 298 percentages), although trends and numerical differences were similar to those reported by Özen 1

et al. (2002). Sarsilmaz et al. (1999) found that both Leydig cell quantity and the percentage of 2

cells with normal nuclei were reduced by for maldehyde treatment. Although the dose

- dependent 3

reduction in Leydig cell quantity was statistically significant at both exposure levels, the study 4

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

6

Table 4

- 59. Formaldehyde effects on Leydig cel l quantity and nuclear 7

damage in adult male Wistar rats 8

9

Inhalation

exposure a Relative

testes

weight b,c,d Leydig cell

quantity c,e,f Appearance of nucleus e,g

Normal Pyknotic Karyorectic Karyolytic

Control 0.93 (0.03) 47.27 (7.8) 98 2 0 0

10 ppm 0.92 ( 0.06) 45.04 (7.8) h 92 2 4 2

20 ppm 0.89 (0.06) 44.36 (7.5) i 76 9 10 5

10

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

b Stated to represent the ratio of the last day’s testicle weight to the body weight but more likely to be the percent 12

of body weight. 13

c Cells within 100 defined areas. 14

d n = 10. 15

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

f n = 100. 17

g Values presented as percentage of cells. 18

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

i Different from control ( p < 0.01), as reported by the authors. 20

21

Source: Sarsilmaz et al. (1999). 22

23

24

It was hypothesized that decreased Leydig cell quality may have been the result of 25

oxidative stress induced by formaldehyde exposure. Özen et al. (2002), in the same laboratory, 26

inv estigated changes in testicular iron, copper, and zinc levels as measures of oxidative stress 27

and damage. Adult male albino Wistar rats (seven/group) were exposed at 0, 10, or 20 ppm (0, 28

12.2, or 24.4 mg/m 3 ) formaldehyde 8 hours/day, 5 days/week for eithe r 4 or 13 weeks. Rats 29

were observed daily and weighed once a week. Rats were sacrificed at the end of the exposure 30

period and autopsied, and the testes were removed and weighed. Zinc, copper, and iron levels 31

were determined in testes tissue by using ato mic absorption spectrophotometry. Both weight 32

gain and relative testes weight were decreased in a concentration

- dependent and duration

33

dependent manner (Table 4

- 60). Both zinc and copper levels in rat testes were reduced in a 34

concentration

- and duration

- dependent manner by formaldehyde exposure. For example, zinc 35

was reduced from 277 to 107 mg/kg after a 4

- week × 20 ppm exposure and from 260 to 36

95 mg/kg after a 12

- week × 20 ppm exposure ( p < 0.001) (Table 4

- 60). Iron levels in testes were 37This document is a draft for review purposes only and does not constitute Agency policy.

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- 299 increased from 30 to 39 mg/kg after a 4

- week 20 ppm exposure ( p < 0.01) and from 33 to 1

58 mg/kg after 12 weeks at 20 ppm ( p < 0.05). The authors suggested that alterations in trace 2

element levels in the testes were consistent with oxidative damage and may have contribu ted to 3

changes in Leydig cell function. These researchers also reported alterations in trace metals in 4

lung tissue from Wistar rats exposed to formaldehyde 8 hours/day, 5 days/week for 4 or 5

13 weeks. Iron levels were increased at 5 ppm for 13 weeks and 1 0 ppm for either 4 or 6

13 weeks. Zinc levels decreased for all formaldehyde exposures. In both cases, the authors 7

attributed elevated iron levels to oxidative stress. In addition to citing the role of zinc as a 8

cofactor of cytoplasmic Cu

- Zn

- SOD, the auth ors suggested that zinc may have been consumed 9

by increased FALDH activity. Although oxidative stress and increased FALDH activity may be 10

relevant to the POE and therefore impact the lung, it is less clear how these changes would occur 11

in the testes. Tak en together, the reports of Özen et al. (2002) and Sarsilmaz et al. (1999) 12

suggested a LOAEL of 10 ppm 8 hours/day for 4 weeks for changes in Leydig cell quantity and 13

quality, decreased testes weight, and changes in trace metal content (zinc, copper, and i ron). 14

15

Table 4

- 60. Formaldehyde effects on adult male albino Wistar rats 16

17

Inhalation

exposure a Weight gain

(%) Testes weight

(%) Zinc

(mg/kg) Copper

(mg/kg) Iron

(mg/kg)

4 Weeks

Control 19.1 (2.7) 0.94 (0.03) 277 (16) 6.4 (0.42) 30 (2.7)

10 ppm 5.8 (2.4) b 0.92 (0.02) c 132 (8.9) b 4.2 (0.33) b 35 (2.8) d

20 ppm 2.4 (0.6) b 0.91 (0.01) c 107 (6.9) b 3.3 (0.27) b 39 (3.1) d

13 Weeks

Control 55.9 (2.3) 0.91 (0.01) 269 (15) 6.0 (0.34) 33 (2.6)

10 ppm 34.7 (3.5) b 0.84 (0.03) b 112 (8.1) b 3.6 (0.30) b 52 (3.5) b

20 ppm 20.8 (1.4) b 0.82 (0.03) b 95 (6.4) b 1.9 (0.17) b 58 (3.0) b

18

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

seven animals.. 20

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

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

d Different from control, p < 0.01, as calculated by the authors. 23

24 Source: Özen et al. (2002). 25

26

27 In another study that assessed testicular toxicity (Özen et al., 2005), male Wistar rat s 28

(18/group) were exposed by inhalation to 0, 5, or 10 ppm (0, 6.2, or 12.4 mg/m 3 ) formaldehyde 29

8 hours/day, 5 days/week for 91 days. In

- life observations in exposed rats included clinical signs 30

of respiratory irritation and decreased mean food and water consumption. At study termination, 31

serum testosterone levels and mean seminiferous tubule diameters were significantly decreased 32

from control in a dose

- responsive manner (Table 4

- 61). Immunohistochemical staining of testis 33This document is a draft for review purposes only and does not constitute Agency policy.

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- 300 tissues showed increased locali zation of heat shock protein (Hsp) 70 in the cytoplasm of 1

spermatogonia, spermatocytes, and spermatids of treated animals compared with controls (not 2

shown here). 3

4

Table 4

- 61. Formaldehyde effects on testosterone levels and 5

seminiferous tubule diameters i n Wistar rats following 91 days of 6

exposure 7

8

Inhalation

exposure a Testosterone levels

(ng/dL) Seminiferous tubule

diameters

(μm)

n = 6 n = 100

Control 406.54 ± 16.82 259.22 ± 16.18

10 ppm 244.01 ± 23.86 b 236.17 ± 13.09 c

20 ppm 141.30 ± 08.56 b 233.24 ± 10.13 c

9

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

SEMs. 11

b Different from control, p < 0.0001, by one

- way ANOVA, as calculated by the authors. 12

c Different from control, p < 0.001, by one

- way ANOVA, as calculated b y the authors. 13

14 Source: Özen et al. (2005). 15

16

17

Zhou et al. (2006) investigated the effect of formaldehyde on the testes and the protective 18

effect of vitamin E against oxidative damage by formaldehyde in adult male rats. In this study, 19

adult male Sprague

- Dawley rats (10/group) were treated for 2 weeks in the following groups: 20

(1) control rats were administered physiological saline by oral gavage, (2) rats were administered 21

physiological saline by gavage and exposed to 10 mg/m 3 (8.05 ppm) formaldehyde by in halation 22

for 12 hours/day, and (3) rats were administered daily gavage doses of 30 mg/kg vitamin E and 23

exposed to 10 mg/m 3 (8.05 ppm) formaldehyde by inhalation for 12 hours/day. Formaldehyde 24

treatment resulted in significantly decreased ( p < 0.05) mean t estis weight. Histopathologic 25

findings in treated rats included atrophy of seminiferous tubules, decreased spermatogenic cells, 26

and seminiferous cells that were “disintegrated” and shed into the lumina, which was 27

azoospermic. Interstitial tissue was edem atous with vascular dilatation and hyperemia. In the 28

formaldehyde

- treated group, epididymal sperm count and percentage of motile sperm were 29

significantly decreased, and the percentage of abnormal sperm was increased ( p < 0.05), as 30

compared with control. Evaluation of biochemical markers in testes tissue showed the activities 31

of testicular SOD, GPX, and GSH were decreased; MDA levels were significantly increased as 32

compared with control. All observed effects of formaldehyde treatment (i.e., decreased test es 33

weight, biochemical alterations, histopathologic effects, and sperm count, motility, and 34

morphology findings) were attenuated by administration of 30 mg/kg

- day vitamin E. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 301 In a study by Golalipour et al. (2007), testicular changes of increasing severity with 1

increasing duration were reported. A total of 28 Wistar rats, aged 6

- 7 weeks old, were divided 2

into four groups including three FA treatment groups (4 hours of exposure/day, 4 days/week for 3

18 weeks; 2 hours of exposure/day, 4 days/week for 18 weeks; 4 hours of exposure/day, 2 4

days/week for 18 weeks) and one untreated control. The three FA

- treated groups were exposed 5

via inhalation to formaldehyde. The mean concentration of FA vapor, based on three 6

measurements during the study (stated as the beginn ing, during, and end of the study period), 7

was reported as 1.5 ppm. At the end of the study period, the rats were sacrificed by ether 8

anesthesia and subsequent cervical dislocation. The left testis was dissected from each rat and a 9

specimen was taken fro m each testis. Tissues were fixed, embedded, sectioned (at 4 μm), and 10

stained with hematoxylin and eosin. Using a light microscope, a histopathological examination 11

was performed on the testes tissues, including morphometric evaluation of the diameter and 12

height of 20 seminiferous tubules/testis. Golalipour et al. (2007) reported an FA exposure 13

frequency (or duration)

- dependent increase in testicular germ cells and seminiferous tubule 14

defects. In the most frequent duration treatment group, a severe decrease in germ cells in >85% 15

of the seminiferous tubules a nd arrested spermatogenesis were observed. In the mid

- level 16

frequency of duration treatment group, a decrease in the number of germ cells and an increased 17

thickness of the basement membrane of 75% of the tubules was observed. In the lowest level 18

duration treatment group, a disruption in the Sertoli and germinal cell arrangement, and 19

increased spacing between germ cells was observed. Further, the seminiferous tubule diameter 20

(STD) and seminiferous epithelial height (SEH) was most decreased among the treat ment groups 21

(exhibiting the greatest decrease in the group with the greatest hours and days of exposure) 22

compared to the control (Table 4

- 62). The results of this study are consistent with the findings of 23

other studies of male reproductive system outcomes with inhalation FA exposure (e.g., Özen et 24

al., 2005 and Zhou et al., 2006). 25

Xing et al. (2007) also studied the effects of formaldehyde on sperm development and 26

reproductive capacity in adult male mice. In this study, male mice (12/group, strain not 27

sp ecified) were exposed to 0, 21, 42, or 84 mg/m 3 (0, 16.9, 33.8, or 67.6 ppm) formaldehyde via 28

inhalation for 13 weeks at 2 hours/day, 6 days/week. The males were mated to untreated females 29

in a dominant lethal protocol, and sperm morphology was assessed a t study termination. The 30

percent abnormal sperm was increased significantly ( p < 0.05) in all treated groups, as was the 31

rate of resorptions ( p < 0.01) (Table 4

- 63). The mean number of live fetuses/litter was decreased 32

in all treated groups, with statist ical significance achieved at 84 mg/m 3 . Although this study did 33

not assess the number of corpora lutea per dam, thereby precluding the calculation of 34This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4-302 preimplantation loss, it is nevertheless indicative of formaldehyde-induced sperm morphology

changes and dominant lethal effects in male mice.

Table 4

- 62. Effects of formaldehyde exposure on seminiferous tubule

diameter and epithelial height in Wistar rats following 18 weeks of

exposure 1

2

3

4

5

6

7

Seminiferous tubule Seminiferous tubule

Inhalation diameters height

a exposure (μm) (μm)

n = 7 n = 7

Control 252.12 ± 4.82 82.77 ± 2.00

1.5 ppm, 4 h/d, 4 d/w 204.55 ± 3.29 b 65.26 ± 1.43 b

1.5 ppm, 2 h/d, 4 h/w 232.45 ± 2.42 b 69.46 ± 1.78 b

1.5 ppm, 2 h/d, 2 d/w 238.94 ± 4.37 b 72.80 ± 2.03 b

8

a Values are means ± SEMs.

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

Source: Golalipour et al. (2007).

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

formaldehyde

- treated mice 9

10

11

12

13

14

15

16

17

Dose

3 ) (mg/m Sperm abnormalities Reproductive capacity

Total abnormal

sperm heads Aberration

(%) rate

Mean live fetuses/litter Resorption

(%) rate

0 391 3.53 ± 0.98 11.00 ± 1.01 2.273

21 568 5.48 ± 1.45 10.67 ± 1.16 9.380 b

42 849 6.15 ± 1.36 9.63 ± 2.83 10.390 b

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

18

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

b Significantly different from controls ( p < 0.01), as calculated by the authors.

Source: Xing et al. (2007).

4.2.1.7.2. Oral exposure studies addressing deve lopmental and reproductive toxicity. No

contemporary testing guideline studies, such as a prenatal developmental toxicity study or twogeneration reproductive toxicity study, have been performed by the oral route for formaldehyde.

However, a number of studies have evaluated developmental toxicity and reproductive

parameters in rats, mice, and dogs.

Hurni and Ohder (1973) tested the developmental toxicity of formaldehyde administered

as a 40% w/v solution containing 11–14% w/w methanol in 9–10 pregnant beagle dogs that 19

20

21

22

23

24

25

26

27

28

29

30

31This document is a draft for review purposes only and does not constitute Agency policy.

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- 303 received the compound in their diet on GDs 4

– 56. Commercial grade formaldehyde (as a 40% 1

solution) was sprayed on the pellets prior to feeding. Each animal was allotted a diet of 300 g of 2

chow (reduced to 200 g 1 week prior to term) that wa s promptly consumed (within 5

– 3

10 minutes) before the formaldehyde volatilized appreciably. The concentrations of 4

formaldehyde in the chow were 0, 125, or 375 ppm, equivalent to doses of 0, 3.1, or 5

9.4 mg/kg

- day, respectively. Dams were allowed to deliver normally and weight gain, gestation 6

length, number of litters, litter size, number of live pups, number of pups surviving through 7

weaning, and pup weights weekly for the first 8 weeks were monitored as indices of the potential 8

reproductive/developmental t oxicity of formaldehyde. There were no formaldehyde

- related 9

effects in any of the parameters other than progressive pup weights, which were lower by group 10

in litters of dams exposed to formaldehyde (Table 4

- 64). A developmental impact of 11

formaldehyde was evident in this strain of dog under the conditions of the experiment. At birth, 12

mean pup body weights were 4 and 8.4% less than control for the low

- and high

- dose groups, 13

respectively; at 8 weeks of age, the pup weight decrements were 8.3% for the low do se and 14

12.5% for the high dose, as compared with control, and established a LOAEL of 125 ppm. The 15

contribution of methanol, which is a developmental toxin (Deglitz et al., 2004; Rogers et al., 16

2004) to these outcomes is not known. No internal or skeletal malformations were observed in 17

any of the 264 live

- born and 20 still

- born pups. 18

19

Table 4

- 64. Body weights of pups born to beagles exposed to 20

formaldehyde during gestation 21

22

Time (weeks) Formaldehyde concentration in chow (ppm)

0 125 375

Average body w eight (g)

0 321 308 294

1 547 526 467

2 818 755 706

3 1,078 987 944

4 1,264 1,247 1,166

5 1,601 1,512 1,429

6 2,020 1,816 1,741

7 2,449 2,263 2,145

8 2,957 2,712 2,587

23

Source: Hurni and Ohder (1973). 24

25

26

Marks et al. (1980) conducted a developmen tal toxicity study of formaldehyde in CD

- 1 27

mice in which 29

– 35 pregnant animals were gavaged on GDs 6

– 15 with aqueous formaldehyde 28

(containing 10

– 15% methanol) at 74, 148, and 185 mg/kg

- day. Seventy

- six controls were 29

gavaged with water alone. All dams we re sacrificed on GD 18, and the numbers of implantation 30This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 304 sites in each uterine horn were counted. The high dose of formaldehyde was toxic to the dams, 1

as indicated by the deaths of 22 of 34 mice before GD 18. Thus, the dose of 148 mg/kg

- day was 2

a NOAEL fo r maternal toxicity in this study. However, it is unclear to what extent an estimated 3

concurrent dose of up to 75 mg/kg

- day methanol may have contributed to this toxic response. 4

To assess the developmental toxicity of formaldehyde, live fetuses were weig hed individually, 5

sexed, and examined for external, visceral, and skeletal malformations. Fetuses of surviving 6

high

- dose dams and of those of other groups did not show an increased incidence of 7

malformations. Therefore, Marks et al. (1980) concluded that formaldehyde did not induce fetal 8

abnormalities and that the 185 mg/kg

- day dose level was a NOAEL for the developmental 9

toxicity of formaldehyde, nor were fetotoxic effects of methanol apparent under the study 10

experimental conditions. 11

Seidenberg and Becke r (1987) and Seidenberg et al. (1986) included formaldehyde 12

(purity not indicated) in a survey of the behavior of potential toxicants in a developmental 13

toxicity screening assay (Chernoff and Kavlock, 1982). The protocol featured the administration 14

of a b orderline to xic dose to 26

– 30 pregnant ICR/SIM mice on GDs 8 −12. Dams were allowed 15

to deliver, and the neonates were examined, counted, and weighed on PNDs 1 and 3. The 16

selected formaldehyde dose of 540 mg/kg

- day was fatal for 11/30 dams, but the average weight 17

gain among surviving dams was little changed compared with controls (3.9 ± 2.3 versus 18

4.0 ± 1.0 g). Similarly, there were no changes in perinatal responses in the neonates of exposed 19

dams compared with controls. For example, the average values for number of neonates/litter, 20

percent survival, and fetal weights on PNDs 1 and 3 were closely similar to those of controls. 21

Evidence of toxicity to the male reproductive system has been observed following oral 22

administration of formaldehyde in a 40% w/v solution conta ining 11

– 14% w/w methanol. 23

Cassidy et al. (1983) administered single oral doses of 100 or 200 mg/kg to five male Wistar 24

rats/group. Testes from these animals and 20 controls were excised and examined for 25

spermatogenic abnormalities 11 days after dosing. A significant (19%) increase in testicular 26

sperm head counts was observed in rats exposed to 200 mg/kg

- day formaldehyde as compared 27

with controls (Table 4

- 65). The percentage of abnormal sperm heads was also significantly 28

increased (5%) in the 200 mg/kg

- day dose group compared with controls. These data suggest 29

that formaldehyde can induce morphologic abnormalities in the germ cells of male experimental 30

animals at dose levels that did not significantly affect testis weights. The contribution of 31

methanol to these outcomes is unknown. 32

33

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

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- 305 Table 4

- 65. Testicular weights, sperm head counts, and percentage incidence 1

of abnormal sperm after oral administration of formaldehyde to male Wistar 2

rats 3

4

Dose (mg/kg) Mean testes weight (g) Mean sperm heads ×

10 6 /g testi s Abnormal sperm heads (%)

0 3.30 175 4.76

100 3.27 166 5.22

200 3.16 209 a 9.77 a

5

a Significantly different from controls ( p < 0.001), as calculated by the authors. 6

7

Source: Cassidy et al. (1983). 8

9

10

Postmortem evaluation of the reproductive organs was conducted in a number of oral 11

studies that ranged between 4 weeks and 2 years in duration (Til et al., 1989, 1988; Tobe et al., 12

1989; Johanssen et al., 1986). Johannsen et al. (1986) administered 0, 50, 100, or 150 mg/kg

- day 13

formaldehyde in the drinking w ater to Sprague

- Dawley rats (15/sex/group) for 91 days and 0, 50, 14

75, or 100 mg/kg

- day formaldehyde in basal diet to beagle dogs (4/sex/group) for 91 days; the 15

study reported no treatment

- related effects on absolute or relative gonad weights or 16

histopathol ogy for either species. In a 4

- week drinking water study conducted by Til et al. 17

(1988), formaldehyde was administered to Wistar rats (10/sex/treated group) at nominal levels of 18

0, 25, and 125 mg/kg

- day; gonad organ weights and histopathology were not aff ected by 19

treatment. Tobe et al. (1989) conducted a chronic (24

- month) study in Wistar rats 20

(20/sex/group), with drinking water concentrations of 0, 0.02, 0.1, or 0.5%. According to the 21

study report, gonad weights were measured and histopathology was cond ucted, but no treatment

22

related findings were noted. In a chronic (105

- week) study (Til et al., 1989) in Wistar rats 23

(70/sex/group), formaldehyde was administered in the drinking water at mean actual levels of 0, 24

1.2, 15, or 82 mg/kg

- day to males and 0, 1 .8, 21, or 109 mg/kg

- day to females; serial sacrifices 25

were conducted at 53, 79, and 105 weeks of study. At study termination (105 weeks), mean 26

testes weights were 30% increased ( p < 0.01) in high

- dose males as compared with controls, and 27

histopathology e valuation revealed Leydig cell tumors in treated males (incidences of 0/50, 3/50, 28

3/50, and 2/50 for the control through high

- dose groups, respectively; historical control tumor 29

incidence data were not provided). The study authors did not judge these find ings to be 30

treatment related. By design, none of the subacute to chronic studies included measures of 31

reproductive function (e.g., estrous cyclicity, sperm measures, or reproductive performance). 32

With the exception of Til et al. (1989), detailed mean org an weight and histopathology incidence 33

data were not provided in the published reports, and Til et al. (1988) only included tumor (not 34

non

- tumor) data. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 306 4.2.1.7.3. Intraperitoneal studies addressing developmental and reproductive toxicity. Other 1

studies i n which formaldehyde was administered by I.P. injection have confirmed the potential 2

effects on the male reproductive system. 3

Chowdhury et al. (1992) administered I.P. injections of 0, 5, 10, or 15 mg/kg

- day 4

formaldehyde to Charles foster adult male rats ( 10/group) for 30 days. On study day 31, blood 5

was collected for serum testosterone measurements and the rats were sacrificed. The testes were 6

removed, weighed, fixed in Bouin’s solution, and processed for histopathology. The study 7

authors reported adver se findings in all treated groups, including significant ( p < 0.01) mean 8

body weight gains, serum testosterone levels, and testes weights. Histopathologic evaluation 9

revealed normal spermatogenic processes and Leydig cells in control animals. However, in 10

treated rats, gradual cellular degeneration in seminiferous tubules and in Leydig cells was 11

observed. Marked nuclear damage was noted in the 10 and 15 mg/kg

- day groups, with 12

significantly ( p < 0.001) decreased Leydig cell populations and nuclear diameter s observed in all 13

treated groups. Additionally, a decrease in 3β

- Δ 5

- hydroxy steroid dehydrogenase was noted in 14

the Leydig cell region of treated rat testes. 15

In a 30

- day study performed by Majumder and Kumar (1995), 10 mg/kg

- day 16

formaldehyde was administe red I.P. to eight male Wistar rats. All animals were sacrificed at 17

term, and the testes, prostate, seminal vesicles, and epididymides were excised and weighed. 18

With the use of methodologies that were not described in the report other than by reference to the 19

Laboratory Manual for the Examination of Human Semen and Sperm

- Cervical Mucus 20

Interaction (WHO, 1987), sperm counts, motility, and viability were compared with those of 10 21

controls (injected I.P. with water alone). As shown in Table 4

- 66, striking re ductions in sperm 22

count and motility were noted in formaldehyde

- treated rats compared with controls. Sperm 23

viability was also significantly reduced by formaldehyde treatment, though to a lesser overall 24

extent than sperm count and motility. 25

26

Table 4

- 66. E ffect of formaldehyde on spermatogenic parameters in male 27

Wistar rats exposed intraperitoneally 28

29

Parameters Control (n = 10) Treated (n = 8)

Sperm count (10 6 /mL) 46.30 ± 5.01 20.40 ± 2.01 a

Sperm viability (%) 87.10 ± 0.83 72.60 ± 2.32 a

Sperm motility (% ) 75.00 ± 10.90 22.00 ± 6.40 a

30

a Significantly different from controls ( p < 0.0001), as calculated by the authors. 31

32

Source: Majumder and Kumar (1995). 33

34

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

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- 307 Majumder and Kumar (1995) also carried out an in vitro experiment in which sperm from 1

normal rats were incubated with different concentrations of aqueous formaldehyde at 2

concentrations ranging from 125 pg/mL to 2.5 μg/mL. Viability of control sperm remained close 3

to 80% for a period of 1 hour, whereas the presence of formaldehyde dose

- dependently reduced 4

viability. Thus, only 50% spermatozoa were viable for 30 minutes in the presence of 5 ng/mL 5

formaldehyde compared with 6 minutes in the presence of 500 ng/mL. Sperm motility also was 6

sensitive to the presence of formaldehyde. Less than 10% of sperm was motile for 10 minutes in 7

the presence of 125 pg/mL formaldehyde. The authors of the study considered their data to be 8

good evidence that functional parameters of spermatozoa, such as viability and motility, can be 9

adversely affected by exposure to formald ehyde. Moreover, they suggested that the cumulative 10

effects of I.P. administration of formaldehyde on the male rat reproductive system raise an alert 11

that formaldehyde might impair the reproductive health of males who are occupationally exposed 12

to the compound. 13

Odeigah (1997) conducted two short

- term in vivo assays to examine sperm head 14

abnormalities and dominant lethal mutations. In the sperm assessment, five daily I.P. injections 15

of 0, 0.125, 0.25, or 0.5 mg/kg formaldehyde were administered to male alb ino rats (six/group; 16

strain not specified). The rats were killed 3 weeks after the last injection, and epididymal sperm 17

counts and abnormalities were assessed. A dose

- related decrease in sperm count was observed, 18

and significantly increased incidences of sperm head abnormalities were found at all treatment 19

levels (Table 4

- 67). 20

21

Table 4

- 67. Incidence of sperm head abnormalities in formaldehyde

22

treated rats 23

24

Dose (mg/kg) Total abnormal

sperm heads Frequency (%) ± SEM

0 90 1.50 ± 0.11

0.125 184 3.09 ± 0. 16 a

0.25 436 7.27 ± 0.30 b

0.5 514 8.57 ± 0.33 b

25

a Significantly different from controls ( p < 0.05), as calculated by the authors. 26

b Significantly different from controls ( p < 0.001), as calculated by the authors. 27

28

Source: Odeigah (1997). 29

30

31

In the domi nant lethal assay (Odeigah, 1997), five daily I.P. injections of 0, 0.125, 0.25, 32

or 0.5 mg/kg formaldehyde were administered to male rats (5 control rats, 12/treated group). 33

Subsequently, each male was mated with two untreated virgin females per week for 34

3 consecutive weeks. The females were killed 13 days after the midpoint of the mating period 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 308 and evaluated for live and dead uterine implants. In general, the number of live embryos was 1

decreased with treatment, and the number of dead implants was increa sed (Table 4

- 68). 2

Additionally, there was a reduction in fertile matings in females mated 1

– 7 days after the males 3

had been treated. This study did not assess the number of corpora lutea and therefore precluded 4

the determination of preimplantation loss. Nevertheless, it is indicative of dominant lethal 5

effects on the male germ cells. 6

7

Table 4

- 68. Dominant lethal mutations after exposure of male rats to 8

formaldehyde 9

10

Dose

(mg/kg) Time of

mating

(days) Fertile

matings a (%) Implants per

female a

(mean ± SE) Live embryos

per female

(mean ± SE) Dead implants

per female

(mean ± SE) Dominant

lethal

mutation

index b

0 0

– 21 96.67 (29) 7.86 ± 0.2 (29) 7.43 ± 0.3 0.43 ± 0.8 0

0.125 1

– 7 75.0 (18) 7.18 ± 0.3 (18) 5.95 ± 0.2 1.23 ± 0.5 19.92

8

– 14 79.17 (19) 7.38 ± 0 .5 (19) 6.30 ± 0.5 1.08 ± 0.3 15.21

15

– 21 91.67 (22) 7.68 ± 0.2 (22) 6.89 ± 0.3 0.79 ± 0.5 7.27

0.25 1

– 7 33.33 (8) 5.75 ± 0.3 (8) 2.05 ± 0.3 3.70 ± 0.4 72.41

8

– 14 50.0 (12) 6.60 ± 0.2 (12) 3.91 ± 0.2 2.69 ± 0.2 47.38

15

– 21 87.5 (21) 7.25 ± 0.4 (21) 6.63 ± 0.3 0.62 ± 0.5 10.77

0.5 1

– 7 25.0 (6) 5.05 ± 0.03 (6) 1.10 ± 0.5 3.95 ± 0.22 85.20

8

– 14 29.17 (7) 5.27 ± 0.01 (7) 1.50 ± 0.6 3.77 ± 0.28 79.81

15

– 21 83.33 (20) 7.08 ± 0.04 (20) 5.79 ± 0.4 1.29 ± 0.17 22.07

11

a Number of females with implants pre sented in parentheses. 12

b Dominant lethal mutation index: 13

Index = 1

– (Live implants experiment group per female)

(Live implants of control group per female) 15 × 100 14

16

Source: Odeigah (1997). 17

18

19

4.2.1.7.4. Dermal exposure studies addressi ng developmental and reproductive toxicity. In a 20

study designed to assess the embryotoxic effects of dermal exposure to formaldehyde, Overman 21

(1986) applied 0.5 mL of a 37% formaldehyde solution directly to the dorsal skin of female 22

Syrian golden hamster s (four

– six/group) on GDs 8, 9, 10, or 11 for 2 hours. To prevent 23

grooming during the treatment period, the animals were anesthetized with Nembutal. At the end 24

of the 2

- hour treatment period, the application site was washed thoroughly to remove any 25

remai ning formaldehyde. The dams were terminated on GD 15 (i.e., one day prior to expected 26

delivery, since the typical gestation period for the Syrian golden hamster is 16

– 18 days). The 27

fetuses were removed and fixed in either Bouin’s solution or 95% ethanol for visceral or skeletal 28

evaluation, respectively. The uteri were examined for implantation sites. Fixed fetuses were 29This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 309 weighed, measured (crown

- rump), and examined for external abnormalities; fetuses that had 1

been placed in Bouin’s fixative were evaluated for visceral anomalies by using a free

- hand 2

sectioning technique, and those that were placed in ethanol were macerated, stained, and cleared 3

for skeletal examination. In this study, the dams exhibited signs of dermal irritation and 4

irritability, but the author reported no treatment

- related effects on maternal body weight gain. 5

The percent of resorption sites was increased (although not significantly) in treated litters as 6

compared with control (0, 4.2, 8.1, 4.6, and 3.2% resorbed implantation sites for c ontrol and 7

GDs 8, 9, 10, and 11 treatment groups, respectively). No treatment

- related effects on fetal 8

weight, length, or visceral or skeletal development were observed. 9

10

4.2.1.7.5. Summary of reproductive and developmental effects. Formaldehyde exposur es up to 11

40 ppm 6 hours/day from GDs 6

– 15 or 6

– 20 did not result in external or internal malformations 12

(Martin, 1990; Saillenfait et al., 1989). Martin (1990) reported delayed skeletal ossification and 13

dose

- dependent decreases in fetal body weight at 5 pp m. Formaldehyde exposure at 40 ppm to 14

pregnant female Sprague

- Dawley rats reduced fetal body weights in male and female progeny 15

and in male pups of dams exposed to 20 ppm formaldehyde (Saillenfait et al., 1989). Based on 16

these studies (Table 4

- 69), the L OAEL for developmental effects in rats is 5 ppm, with a 17

NOAEL of 2 ppm for decreased fetal weight and delayed skeletal ossification, based on 18

inhalation exposures during GDs 6

– 20. 19

Developmental studies during earlier gestational windows of inhalation expos ure to 20

formaldehyde have reported additional adverse health effects, including delayed ossification, 21

changes in relative organ weight, undescended testes, biochemical changes (e.g., ascorbic acid 22

and nucleic acids), and blood acidosis ( Senichenkova and Che botar, 1996; Senichenkova, 1991; 23

Kilburn and Moro, 1985; Gofmekler and Bonashevskaya, 1969; Gofmekler, 1968; Pushkina et 24

al., 1968) . Kitaev et al. (1984) hypothesized that formaldehyde may affect reproductive function 25

by stimulating the hypothalamo

- pituit ary

- gonadal (HPG) axis based on their observations of 26

increased ovary weight, increased number of ovulating cells, and changes in blood levels of 27

gonadotropins (LH and FSH). Evidence of preimplantation loss, which may be related to HPG 28

disruption, was obs erved in this study and by Sheveleva (1971). Additional studies are needed to 29

better understand developmental effects of formaldehyde exposure during early gestational 30

windows. 31

The prenatal developmental toxicity of oral and dermal exposures to formaldehy de has 32

not been thoroughly studied. Reductions in postnatal growth in beagle pups was observed by 33

Hurni and Ohder (1973) following in utero exposure to 125 ppm maternal dietary formaldehyde 34

during GDs 4

– 56 in beagle dogs. However, gavage dosing during ge station of mice to overtly 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 310 maternally toxic doses (Seidenberg and Becker, 1987; Marks et al., 1980) (Table 4

- 70) and 1

dermal application during gestation to hamsters at a dose that caused dermal irritation and 2

irritability (Overman, 1986) did not result in any observed fetal toxicity (Table 4

- 71). 3

Few studies identified effects on maternal toxicity or female reproductive capacity. As 4

summarized in Table 4

- 69, exposure of rat dams at 10

– 40 ppm formaldehyde during pregnancy 5

has been shown to result in signi ficantly decreased weight gain (Martin, 1990; Saillenfait et al., 6

1989; Kilburn and Moro, 1985). Maronpot et al. (1986) reported endometrial hypoplasia with a 7

lack of ovarian luteal tissue in female rats exposed at 40 ppm but not at 20 ppm. Changes in LH 8

and FSH levels were reported in dams exposed to 0.41 ppm formaldehyde by Kitaev et al. 9

(1984), establishing an unbounded LOAEL for maternal toxicity. 10

Studies designed to assess male reproductive system endpoints in rats following repeated 11

inhalation expos ures to formaldehyde have shown concentration

- dependent decreases in Leydig 12

cell number and quality, effects on seminiferous tubules, decreases in testes weight, alterations in 13

sperm measures, decreased testosterone levels, alterations in trace metals in t he testes, and/or 14

dominant lethal effects (Zhou et al., 2006; Ö zen et al., 2005, 2002; Zhou et al., 2006Sarsilmaz et 15

al., 1999) (Table 4

- 72). Based on available studies, the LOAEL for changes in the male 16

reproductive system in rats following 5 days/week o f inhalation exposures is 5 ppm for 3 months 17

of daily exposures and 10 ppm for 4 weeks of daily exposures; these dose levels are unbounded. 18

Abnormal sperm were also noted in mice at an inhalation dose of 16.9 ppm 2 hours/day, 6 19

days/week for 13 weeks (Xin g et al., 2007), but, in contrast, Maronpot et al. (1986) reported no 20

histologic abnormalities in male mice after formaldehyde exposures at 40 ppm 6 hours/day, 5 21

days/week for 13 weeks. Varied results among studies may be due to species differences or 22

dif ferences in methods. Although several oral subchronic and chronic studies with formaldehyde 23

did not identify effects on the testes (Tobe et al., 1989; Til et al., 1988; Johanssen et al., 1986), 24

Cassidy et al. (1983) observed spermatogenic abnormalities af ter a single oral dose of 200 mg/kg 25

to rats, and a chronic drinking water study in rats (Til et al., 1989) reported low incidences of 26

Leydig cell tumors in all treated groups, compared with none in control (Table 4

- 73). 27

Additionally, studies utilizing I.P . injection of formaldehyde in rats have demonstrated testes and 28

sperm anomalies (Majumder and Kumar, 1995; Chowdhury et al., 1992) and dominant lethal 29

effects (Odeigah, 1997) (Table 4

- 74). 30This document is a draft for reviewpurposes only and does not constituteAgency policy4-311 Table 4

- 69. Summary of reported developmental effects in formald ehyde inhalation exposure studies

Species,

strain, sex n/Group Dose; time of

treatment a Reported study findings b LOAEL/NOAEL c

Reference Maternal Offspring Maternal Offspring

Rat, strain

NR, female 12 0, 0.01, or 0.81 ppm

(reported as 0.012,

and 1 mg/m 3 ) d ; continuous dosing

10

– 15 days prior to

mating and during

gestation At 0.01 and 0.81 ppm:

↑ pregnancy duration

(dose

- dependent data

not shown) e Fetuses:

At 0.01 and 0.81 ppm:

↓ fetuses/dam (dose dep., data

not shown) e

↑ body wt (dose dep., sta t. sig.)

↓ lung and liver wt (dose dep.,

stat. sig.)

↑ adrenal wt (dose dep., stat.

sig.)

At 0.81 ppm: ↑ thymus and

kidney wt (stat. sig.) L: 0.01 ppm

N: ND L: 0.01 ppm

N: ND Gofmekler

(1968)

Rat,

“albino”

strain NR,

female 12 0, 0.01, or 0.81 pp m (reported as 0.012,

and 1 mg/m 3 ) d ; continuous dosing

10

– 15 days prior to

mating and during

gestation NE Age of assessment NR.

At 0.81 ppm:

histologic effects in liver (e.g., ↑

extramedullary hematopoietic

centers), kidney (e.g., ↑

polymorphism of renal epithelial

cell nuclei)

and thymus NE e N: 0.01 ppm

L: 0.08 ppm Gofmekler and

Bonashevskaya

(1969) f,h

Rat, strain

NR, male 4 Inhalation and

drinking water

co

- exposure: 0;

0.10 ppm plus

0.005 mg/L water;

0.20 ppm plus

0.01 mg/L water; or

0.41 ppm plus

0.1 mg/L water; all

treatments

4 hours/day,

5 days/week for

6 months No effects No effects ND g ND Guseva (1972)This document is a draft for reviewpurposes only and does not constituteAgency policy4-312 Table 4

- 69. Summary of reported developmental effects in formald ehyde inhalation exposure studies

Species,

strain, sex n/Group Dose; time of

treatment a Reported study findings b LOAEL/NOAEL c

Reference Maternal Offspring Maternal Offspring

Rat, strain

NR, female NR Expt 1:

0 or 30 ppm d

Expt 2: 0, pair

- fed

control (15, 10, or

5 days), or 30 ppm;

8 hours/day for

15 days (GDs 3

– 17),

10 days (GDs 3

– 12),

5 days (GDs 8

– 12),

or 3 days (GDs 9

– 11) At 30 ppm

50% mortality (10 and

15 day exp)

↓ wt gain (duration

dep.; 3, 5, 10, and 15

day exp.)

↓ wt of liver, kidney,

spleen and thymus

↑ wt of lung and

adrenal e Fetuses:

At 30 ppm

↓ fetal wt and growth (duration

dep., 10 and 15 day exp.)

↑ dev. defects (undescended

testes, large hearts, small

thymuses, small lungs) e N: ND

L: 30 ppm e N: ND

L: 30 ppm e Kilburn and

Moro (1985) f Ab

Rat, Wistar,

female Embryo

dev expt:

5

– 9/grou p (42 adult

animals);

maternal

effects:

NR (200

adult

females

total) i 0, 0.4, or 1.2 ppm

(converted from

reported 0, 0.5 or

1.5 mg/m 3 );

4 hours/day,

5 days/week for

4 months; exposed

females mated to

unexposed males on

120 th day exp. At 0.4 ppm:

↑ wt of o varies (stat.

sig e )

↑ LH level (stat. sig. e )

At 1.2 ppm:

↑ FSH level in blood

(stat. sig.; nonsig. at

0.4 ppm e ) At 0.4 ppm:

↑ no. of embryos and 2

blastomere stage embryos (stat.

sig. in 2 nd day preg.)

At 1.2 ppm:

↑ no. degenerating embryos

(stat. sig. in 3 rd day preg.) L: 0.4 ppm

N: ND L: 0.4 ppm

N: ND Kitaev et al.

(1984) g

Rat,

Sprague

Dawley,

female and

offspring of

both sexes 25 0 (air control

group), 0 (room

control group), 2, 5,

or 10 ppm;

6 hours/day

GDs 6

– 15.

Exposed females

mated to unexp osed

males At 10 ppm:

↓ food consumption

(stat. sig.)

↓ wt gain (stat. sig.) At 5 and 10 ppm:

Fetuses:

↑ incidence of reduced

ossification of pubic and ischial

bones (stat. sig. compared with

air control group)

↓ fetal wts (nonsig.)

↑ litter size (nonsig.) L: 10 ppm

N: 5 pp m L: 5 ppm

N: 2 ppm Martin (1990) e,fThis document is a draft for reviewpurposes only and does not constituteAgency policy4-313 Table 4

- 69. Summary of reported developmental effects in formald ehyde inhalation exposure studies

Species,

strain, sex n/Group Dose; time of

treatment a Reported study findings b LOAEL/NOAEL c

Reference Maternal Offspring Maternal Offspring

Rat, “white”

strain NR,

female and

offspring of

both sexes 5

– 12 (NR

for

formalde

hyde only) 0, 0.01, or 0.81 ppm

(reported as 0.012

and 1 mg/m 3 ) d ;

continuous

10

– 15 days prior to

mating through

gestation At 0.01 and 0.81:

↓ vit. C level in liver

(stat. sig.)

↓ vit. C level in

placenta

(nonsig.) Fetuses:

At 0.01 and 0.81 ppm:

↓ fetuses/female e

↑ body wt and organ wt (data not

shown e )

↓ vit. C level in whole fetus (stat.

sig.)

At 0.01 ppm:

↓ vit. C level in fetal liver (sta t.
sig.) L: 0.01 ppm

N: ND L: 0.01

N: ND Pushkina et al.

(1968) f

Rat,

Sprague

Dawley,

female and

offspring of

both sexes 25 0 (air control), 5, 10,

20, 40 ppm; 6

hours/day,

GDs 6

– 20.

Exposed females

mated to unexposed

males. GD 21 dams:

At 5 ppm:

↑ absolute body wt gain

(5 ppm only)

At 40 ppm:

↓ body wt gain GDs 6

– 21 (stat. sig.)

↓ absolute body wt gain

(stat. sig., dose

dependent trend 20 and

40 ppm) GD 21 fetuses:

At 20 and 40 ppm:

↓ fetal body wt, male (stat. sig.)

At 40 ppm:

Delayed ossificat ion of thoracic

vertebrae (stat. sig., trend

20 ppm)

↑ unossified sternebrae (nonsig.

at 40 ppm)

↓ fetal body wt, female (stat.

sig.) L: 40 ppm

N: 20 ppm L: 20 ppm

N: 10 ppm Saillenfait et al.

(1989)This document is a draft for reviewpurposes only and does not constituteAgency policy4-314 Table 4

- 69. Summary of reported developmental effects in formald ehyde inhalation exposure studies

Species,

strain, sex n/Group Dose; time of

treatment a Reported study findings b LOAEL/NOAEL c

Reference Maternal Offspring Maternal Offspring

Rat,

mongrel

white,

female and

offspring of

both se xes NR i 0 or 0.41 ppm

(reported as 0.5

mg/m 3 )

formaldehyde

(also a 3rd group of

gasoline exposure,

not described in this

table); 4 hours/day

GDs 1

– 19 Dams GD 20:

↓ corpora lutea

(nonsig.), embryos dead

before implantation (not

stat. sig.), and

implante d embryos

(nonsig.)

↑ blood pCO 2 (stat.

sig.) Fetuses (GD 20):

Stat. sig. findings include

↑ fetal wt

↑ litters w/internal organ

anomalies

↓ fetuses w/ossification centers

in hyoid bone

↑ metacarpal bone centers

↑ metatarsal bone centers

↑ developmenta l defects

↑ blood pCO 2 and pO 2

Pups: ↓ pup wt

Dev. delays (data not shown) L: 0.41 ppm

N: ND L: 0.41 ppm

N: ND Senichenkova

(1991)

Mouse,

mongrel,

female and

offspring of

both sexes NR (254

dams) i 0 + ethyl alcohol;

0.41 ppm

formaldehyde;

0 .41 ppm formaldehyde +

bipyridyl;

4 hours/day

GDs 1

– 19.

Induced maternal

iron deficiency

anemia by I.P.

bipyridyl injections

on GDs 12

– 15;

controls injected

w/25% ethyl

alcohol. Dams GD 20:

formaldehyde alone:

↑ blood pCO 2 (stat.

sig.)

formaldehyde +

bipy ridyl:

↓ blood acid metabolic

products (stat. sig.)

↓ blood true

bicarbonates and CO 2 conc. (stat. sig.) Fetuses (GD 20):

Formaldehyde alone:

↑ cryptorchidism

Formaldehyde + bipyridyl:

↑ birth defects (stat. sig.)

↓ dev. delay (stat. sig.)

↓ blood aci d

- base measures of

embryos (stat. sig.) L: 0.41 ppm

N: ND L: 0.41 ppm

N: ND Senichenkova

and Chebotar

(1996)This document is a draft for reviewpurposes only and does not constituteAgency policy4-315 Table 4

- 69. Summary of reported developmental effects in formald ehyde inhalation exposure studies

Species,

strain, sex n/Group Dose; time of

treatment a Reported study findings b LOAEL/NOAEL c

Reference Maternal Offspring Maternal Offspring

Rat,

mongrel,

white,

female and

offspring of

both sexes 15/group

terminated

GD 20,

6/group

littered 0, 0.0005, or

0.005 mg/L (0, 0.4,

or 4 ppm) , GDs 1

– 19, 4 hours/day At 0.4 ppm: ↓ leukocyte

counts

At 4 ppm: ↓ leukocyte

counts; reduced

threshold of

neuromuscular

excitability, ↓ rectal

temperature, ↓ blood

hemoglobin; ↑

spontaneous mobility At 0.4 ppm: ↑ preimplantation

loss; at 1 mo. of age, ↓

spontaneous mobility; at 2 mo.

of age, ↓ hemoglobin levels and

leukocyte counts

At 4 ppm: ↑ preimplantation

loss; at 1 and 2 mo. of age, ↓

spontaneous mobility; at 2 mo.

of age, ↓ hemoglobin levels and

leukocyte counts L: 0.4 ppm

N: ND L: 0.4 ppm

N: ND Sheveleva

(1971)

Rat,

S prague

Dawley,

female and

offspring of

both sexes 6 dams 0 or 6 ppm

8 hours/day,

6 weeks, starting at

GD 1, PND 1,

4 weeks of age, or

adult age NE In offspring exposed in utero and

during early postnatal life: ↓

mean BW and liver weight; ↑

markers of oxida tive stress

In offspring exposed as

juveniles: ↓ mean liver weight; ↑

markers of oxidative stress

In offspring exposed only as

adults: no effect NE L: 6 ppm

N: ND Kum et al.

(2007)

Rat,

Sprague

Dawley,

female and

offspring of

both sexes 6 dams 0 or 6 ppm

8 hours/day;

6 weeks, starting at

GD 1, PND 1,

4 weeks of age, or

adult age NE In offspring exposed in early

postnatal life, as juveniles, or as

adults, ↑ ANAE

- positive T

lymphocytes in BALT

In all exposure initiation groups,

↑ ANAE

- positive lympho cyte

ratios NE L: 6 ppm

N: ND Sandikci et al.

(2007)This document is a draft for reviewpurposes only and does not constituteAgency policy4-316 Table 4

- 69. Summary of reported developmental effects in formald ehyde inhalation exposure studies

Species,

strain, sex n/Group Dose; time of

treatment a Reported study findings b LOAEL/NOAEL c

Reference Maternal Offspring Maternal Offspring

Rat, Wistar,

female and

offspring of

both sexes 12

– 14

dams 0, 6, or 12 ppm,

6 hours/day;

5 days/week,

30 days NE At 6 and 12 ppm, at postnatal

days 30 and 90: respiratory

irritation and toxicity; decr . BW,

FC, WC; ↓ SOD activity, ↓

levels of copper and iron levels

in lungs, ↑ zinc levels in lungs NE L: 6 ppm

N: ND Songur et al.

(2005)

ND: not determined; NE: not evaluated; NR: not reported; Ab: abstract only; wt: weight; stat. sig.: statistically significant ; p: pressure; l: length;

To convert concentrations in air (at 25°C) from mg/m 3 to ppm: 1 ppm = 1.23 mg/m 3 ; 1 mg/m 3 = 0.813 ppm

.

a Treatment is given as the formaldehyde concentration in air (ppm) with the length of exposure each day and the duration of tr eatm ent in days, as available.

b Studies with negative findings are included.

c L: LOAEL; N: NOAEL.

d Exposure concentrations not validated; details of formaldehyde vapor generation not reported; exposure during gestation not w ell characterized in study report.

e No statistics provided.

f Lack of study details.

g See Table 4

- 72 for reproductive effects.

h Gofmekler and Bonashevskaya (1969) seem to report on different findings from the same study (i.e., same animals) as Gofmekler (1968).

i Number/group not clear from study report.This document is a draft for reviewpurposes only and does not constituteAgency policy4-317 Table 4

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

Species,

strain, sex n/

Group Dose; time of

treatment Reported developmental effects a LOAEL/NOAEL b

Reference Maternal Offspring Maternal O ffspring

Dog,

beagle,

female and

pups 9

– 10 0, 125, or

375 ppm, dietary,

GDs 4

– 56 No effects At 125 and 375 ppm:

↓ birth wt and wt gain

through postnatal week 8 L: ND

N: 375 ppm L: 125 ppm

N: ND Hurni and

Ohder (1973)

Mouse,

CD

- 1,

female 29

– 35

total 0 , 74, 148, or

185 mg/kg

- day,

GDs 6

– 15

(aqueous

formaldehyde

solution

contained 10

– 15% methanol) At 185 mg/kg

- day:

Mortality No effects at GD 18 L: 185 mg/kg

- day

N: 148 mg/kg

- day L: ND

N: 185 mg/kg

- day Marks et al.

(1980)

Mouse,

ICR/SIM,

female 26

– 30

tot al 0 or 540 mg/kg

day, GDs 8

– 12 At 540 mg/kg

- day:

Mortality No effects in pups on PND 1

and 3 L: 540 mg/kg

- day

N: ND L: ND

N: 540 mg/kg

- day Seidenberg and

Becker (1987)

ND: not determined.

a Studies with negative findings are included.

b L: LOAEL; N: NOA EL.This document is a draft for reviewpurposes only and does not constituteAgency policy4-318 Table 4

- 71. Summary of reported developmental effects in formaldehyde dermal exposure studies

Species,

strain, sex n/

Group Dose; time of

treatment a Reported developmental effects LOAEL/NOAEL a

Reference Maternal Offspring Maternal Offsprin g

Hamster,

Syrian

golden ,

female 4

– 6 0 or 37%; 0.5 mL

applied to dorsal skin

(hair clipped) for 2 hours

then washed; GDs 8, 9,

10, or 11 Signs of dermal irritation

and irritability At all GDs o f treatment, ↑

percent resorptions (not sig.) L: 37%

N: ND L: 37%

N: ND Overman (1986)

ND, not determined

a L: LOAEL; N: NOAEL.This document is a draft for reviewpurposes only and does not constituteAgency policy4-319 Table 4

- 72. Summary of reported reproductive effects in formaldehyde inhalation studies

Species,

strain, sex n/

Group Dose; time of treatment a Reported reproductive effects b LOAEL/

NOAEL c Reference

Rat, strain

NR, male 4 Inhalation plus drinking water

co

- exposure: 0; 0.10 ppm plus

0.005 mg/L water; 0.20 ppm plus 0.01 mg/L water; or 0.41

ppm plus 0.1 mg/L water; all

trea tments; 4 hours/day,

5 days/week for 6 months.

Exposed males mated to

unexposed females At 0.20 ppm + 0.01 mg/L water and 0.41 ppm + 0.1 mg/ L water:

↓ nucleic acid in testes (dose dep.; data not shown; stat. sig.) L: 0.20 ppm

+ 0.01 mg/L

water

N: ND Guseva

(1972) d,e

Rat,

Wistar,

female NR

(200

female) 0, 0.4, or 1.2 ppm ;

4 hours/day, 5 days/week for

4 months.

Exposed females mated to

unex posed males on 120 th day

of exposure. At 0.4 ppm:

↑ wt of ovaries (stat. sig. e )

↑ LH level in blood (stat. sig. at 0.4 ppm f )

At 1.2 ppm:

↑ FSH level in blood (stat. sig., dose dep. trend f ) L: 0.4 ppm h

N: ND h Kitaev et al.

(1984) e

Mouse,

B6C3F1,

male a nd

female 10 0, 2, 4, 10, 20, or 40 ppm;

6 hours/day, 5 days/week

for 13 weeks Males and females:

At 20 ppm: ↓ wt gain

At 40 ppm: ↑ mortality (13 weeks exp.); ↓ wt loss

Females:

At 40 ppm: ↑ Uterine endometrial and ovarian hypoplasia L: 20 ppm

N: ND Maronpot et

al. (1986)

Rat, albino

Wistar,

male 7 6 groups: 0, 10, or 20 ppm;

8 hours/day, 5 days/week for

4 weeks (subacute) or

13 weeks (subchronic) At 10 and 20 ppm (both durations):

↓ wt gain (stat. sig., dose dep.)

↓ relative testes wt (stat. sig., dose and conc. dep.)

↓ zinc and copper in testes (stat. sig., dose and conc. dep.)

↑ iron in testes (stat. sig., dose and conc. dep.)

No effect on testes wt. L: 10 ppm

N: ND Özen et al.

(2002) f

Rat,

Wistar,

male 18 0, 5, or 10 ppm; 8 hours/day,

5 day s/week, 91 days At 5 and 10 ppm:

clinical signs of respiratory irritation, ↓ BW, FC, WC; ↓serum

testosterone; ↓ mean seminiferous tubule diameters; ↑ localization

of heat shock protein 70 in cytoplasm of spermatogonia,

spermatocytes, and spermatids L: 5 pp m

N: ND Özen et al.

(2005)This document is a draft for reviewpurposes only and does not constituteAgency policy4-320 Table 4

- 72. Summary of reported reproductive effects in formaldehyde inhalation studies

Species,

strain, sex n/

Group Dose; time of treatment a Reported reproductive effects b LOAEL/

NOAEL c Reference

Rat, albino

Wistar,

male 10 0, 10, or 20 ppm

8 hours/day, 5 days/week for

4 weeks Dose NR:

Irritation: standing hair, interrupted breathing, ↑ eye blinking,

licking, nose cleaning, and sneezing.

At 10 and 20 ppm:

↓ Body wt gai n (dose dep.; stat. sig.)

↓ Leydig cell quantity (stat. sig.)

↑ Nuclear damage of Leydig cells (dose dep.; stat. sig.) L: 10 ppm

N: ND Sarsilmaz et

al. (1999)

Mouse,

strain not

specified,

male 12 0, 21, 42, or 84 mg/m 3 (0,

16.9, 33.8, or 67.6 ppm);

2 hours/day, 6 days/week;

13 weeks. Exposed males

mated to unexposed females. In all treated groups: ↑ percentage abnormal sperm, ↑ resorption

rate, and ↓ live fetuses L: 16.9 ppm

N: ND Xing et al.

(2007)

Rat,

Wistar,

male 7 0 or 1.5 ppm, 18 weeks

FA ex posures: (1) 4 hours/day, 4 days/week

(2) 2 hours/day; 4 days/week

(3) 2 hours/day, 4 days/week In all treated groups: sig. ↓ seminiferous tubular diameter and

epithelial height. Other effects in exposure groups:

(1) sig. ↓ germ cells; arrested spermat ogenesis

(2) ↓ cells, increased thickness in basal membrane

(3) ↑ spaces between germ cells; disrupted association between

Sertoli and germinal cells L: 1.5 ppm

N: ND Golalipour et

al. (2007)

Rat,

Sprague

Dawley,

male 10 (1) 0 (gavage saline);

(2) 10 m g/m 3 (8.05 ppm),

12 hours/day, 2 weeks; or

(3) 10 mg/m 3 (8.05 ppm),

12 hours/day, 2 weeks, plus

30 mg/kg

- day vitamin E orally At 10 mg/m 3 : sig. ↓ testis weight, atrophy of seminiferous tubules,

↓ spermatogenic cells, disintegrated and sloughed seminifero us cells; edematous interstitial tissue with vascular dilatation and

hyperemia; ↓ epididymal sperm count and percentage motile

sperm, ↑ percentage abnormal sperm; ↓ SOD, GSH

- Px, GSH and

↑ MDA in testes; vitamin E attenuated all effects L: 8.05 ppm

N: ND Zhou et al.

(2006)

Rat,

Wistar,

male 40 0, 0.1, 1, or 10 ppm;

6 hours/day, 5 days/week, 13

or 52 weeks No effects: testis weight; histopathologic findings g L: ND

N: 10 ppm Appleman et

al. (1986)

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

b Studies with negative findings are included.

c L: LOAEL; N: NOAEL.

d Guseva (1972) was a drinking water and inhalation study.

e Developmental effects shown in Table 4

- 69.

f Statistics not provided in study report.This document is a draft for reviewpurposes only and does not constituteAgency policy4-321 g Focus of study was not the reproductive system; only reproductive system findings are addressed in the table; NOAEL and LOAEL in table are based only on

reproductive system findings.

h For increased FSH, the LOAEL was 1.2 ppm (1.5 mg/m 3 ) and the NOAEL was 0.4 ppm (0.5 mg/m 3 ).

ND: not determined; NR: not reported.

To convert concentrations in air (at 25°C) from mg/m 3 to ppm: 1 ppm = 1.23 mg/m 3 ; 1 mg/m 3 = 0.813 ppm

.This document is a draft for reviewpurposes only and does not constituteAgency policy4-322 Table 4

- 73. Summary of reported reproductive e ffects in formaldehyde oral studies

Species,

strain, sex n/

Group Dose; time of treatment Reported reproductive effects a LOAEL/

NOAEL b Reference

Rat,

Wistar,

male 5 (20

control) 0, 100, or 200 mg/kg, single

gavage dose At 200 mg/kg:

↑ (19%) testicular s perm head counts (stat. sig.)

↑ (5%) abnormal sperm head (stat. sig.) L: 200 mg/kg

N: 100 mg/kg Cassidy et al.

(1983)

Rat,

Sprague

Dawley,

both sexes 15 0, 50, 100, or 150 mg/kg

- day,

drinking water; 91 days No effects: absolute or relative gonad weights;

histopathologic findings of reproductive organs c L: ND

N: 150 mg/kg

- day Johanssen et

al. (1986)

Dog,

beagle,

both sexes 4 0, 50, 75, or 100 mg/kg

- day,

dietary; 91 days No effects: absolute or relative gonad weights;

histopathologic findings of reproduct ive organs c L: ND

N: 100 mg/kg

- day

Rat,

Wistar,

both sexes 10 0, 25, or 120 mg/kg

- day,

drinking water, 4 weeks No effects: gonad weights; histopathologic findings of

reproductive organs c L: ND

N: 120 mg/kg

- day Til et al.

(1988)

Rat,

Wistar,

both sexes 70 0, 1.2, 15, or 82 mg/kg

- day

(males), 0, 1.8, 21, or

109 mg/kg

- day (females), d

drinking water; 105 weeks In all treated groups:

Leydig cell tumors observed at 105 weeks of study c

At 82 mg/kg

- day:

↑ mean testes weights L: 1.2 mg/kg

- day

N: ND Til et al.

(1989)

Rat,

Wistar,

both sexes 20 0, 0.02, 0.1, or 0.5% in

drinking water; 24 months No effects: gonad weights; histopathologic findings of

reproductive organs c L: ND

N: 0.5% Tobe et al.

(1989)

ND, not determined.

a Studies with negative findings are i ncluded.

b L: LOAEL; N: NOAEL.

c Focus of study was not the reproductive system; only reproductive system findings are addressed in the table; NOAEL and LOAEL in table are based only on

reproductive system findings.

d Actual concentrations.This document is a draft for reviewpurposes only and does not constituteAgency policy4-323 Table 4

- 74. Sum mary of reported reproductive effects in formaldehyde intraperitoneal studies

Species,

strain, sex n/

Group Dose; time of treatment a Reported reproductive effects a LOAEL/

NOAEL b Reference

Rat,

Charles

foster,

male 10 0 or 5 mg/kg

- day; 30 days ↓ body wei ght gain

↓ Leydig cell population and cell nuclear diameter

↓ serum T levels

↓ testes weights

cellular degeneration of seminiferous tubules L: 5 mg/kg

- day

N: ND Chowdhury et

al. (1992)

Rat,

Wistar,

male 8 0 or 10 mg/kg

- day; 30 days ↓ sperm count, motilit y and sperm viability L: 10 mg/kg

- day

N: ND Majumder and

Kumar (1995)

Rat,

“albino”

strain NR,

male 6 0, 0.125, 0.250, or

0.60 mg/kg

- day; 5 days At all treatment levels:

↓ sperm count and ↑ sperm head abnormalities (3 weeks

after the last injection) L: 0 .125 mg/kg

- day

N: ND Odeigah

(1997)

Rat,

“albino”

strain NR,

male 12 0, 0.125, 0.250, or 0.60

mg/kg

- day; 5 days. Exposed

males mated to unexposed

females. At all treatment levels (at GD 13):

Delayed time to mating

↓ mean no. implants and live embryos

↑ dead implants and dominant lethal index following

mating to untreated females L: 0.125 mg/kg

- day

N: ND

a Studies with negative findings are included.

b L: LOAEL; N: NOAEL.

ND: not determined; NR: not reported; T: testosterone.This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 324 4.2.2. Carcinogenic Pot ential: Animal Bioassays 1

Chronic animal studies (inhalation and oral exposures) chronicle tumor incidence in a 2

variety of rodent models. Study descriptions are provided above in detail (Section 4.2.1, 3

Table 4

- 34). The study results are evaluated here for both routes of exposure in context of how 4

they inform the carcinogenic potential for the three major affected systems: respiratory tract, GI 5

tract, and LHP system. Experimental design and implementation must be carefully considered 6

when interpreting stud y results. For example, some key factors involved in evaluating cancer 7

bioassays include study size, organs/tissues examined, and study length (especially for late

- in

8

life tumors). 9

10

4.2.2.1. Respiratory Tract 11

In the respiratory tract, only nasal tumors a re considered formaldehyde induced in rodent 12

studies. The majority of studies were conducted using rats (F344, Wistar, or Sprague

- Dawley), 13

and all studies of 18 months or greater in mice and rats show evidence of formaldehyde

- induced 14

nasal carcinogenicity . The nasal tumors are primarily SCCs, although papillomas, polypoid 15

adenoma, adenocarcinoma, fibrosarcoma, and esthesioneuroepithelioma have been reported 16

(Kamata et al., 1997; Monticello et al., 1996; Morgan et al., 1986a, b; Takahashi et al., 1986; 17

Sel lakumar et al., 1985; Kerns et al., 1983; Albert et al., 1982). Although hyperplasia, dysplasia, 18

and squamous metaplasia of the respiratory epithelium have been observed beyond the nasal 19

cavity, other respiratory tract tumors have not been significantly i ncreased by formaldehyde 20

exposure alone. 21

Increased tumor incidence and decreased latency are correlated with increasing 22

formaldehyde exposure concentration. Reviewing data from the only lifelong inhalation study 23

with multiple exposure groups, SCC is first noted at 8 and 9 months for high exposed (15 ppm) 24

female and male F344 rats autopsied as “early deaths” prior to the 12 month sacrifice, with an 25

incidence of 43% over the course of the study (unadjusted for mortality) (Kerns et al., 1983). In 26

contrast on ly two SCCs were found in male and female rats sacrificed after 24 months of 27

exposure (incidence of SCC 2.5% at 24 months) (Kerns et al., 1983). In a follow

- up study by 28

Monticello et al. (1996), the incidence of SCC in rats exposed at 15 ppm was 47% with the first 29

tumor noted at 12 months. The incidence of SCC in male rats exposed at 10 ppm was 22% with 30

the first SCC noted at 18 months. Moreover, of 90 rats exposed at 6 ppm for 20 months only one 31

SCC was noted. No SCCs were detected in rats exposed at 0 .7 or 2 ppm formaldehyde. These 32

incidence rates are not mortality adjusted and include animals from each scheduled sacrifice (3, 33

6, 12, and 18 months). In a lifelong study of male Sprague

- Dawley rats exposed at 10 ppm 34

formaldehyde, the cumulative nasal t umor incidence was calculated as a function of time of 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 325 exposure (Figure 4

- 27) (Sellakumar et al., 1985). After 2 years of exposure, the probability of 1

nasal carcinoma was greater than 60%. 2

3

4

5

Figure 4

- 27. Mortality corrected cumulative incidences of nas al carcinomas 6

in the indicated exposure groups. 7

8

Source: Sellakumar et al. (1985). 9

10

There is some evidence that less

- than

- lifetime exposure to formaldehyde can induce nasal 11

tumors over an extended observation period. Two studies, both in male Wistar rats , report nasal 12

tumors in response to less

- than

- lifetime exposures (Woutersen et al., 1989; Feron et al., 1988). 13

A 13

- week exposure at 20 ppm resulted in four nasal tumors (three SCCs), a cystic SCC of the 14

nasolacrimal duct, and an epithelial tumor on the mandible, for a total of six tumors observed 15

over 30 months of observation (Feron et al., 1988). No tumors were noted in 13

- week controls. 16

A limited number of formaldehyde

- related tumors were noted due to 4 or 8 weeks of exposure 17

followed by 30 months of observation. Although the tumor incidence of these less

- than

- lifetime 18

exposures is low, this is consistent with the 2

- year bioassays in Wistar rats. Wistar rats are more 19

resilient to formaldehyde

- induced nasal toxicity than F344 or SD rats (Section 4.2. 1), and only 1 20

of 26 (4%) Wistar rats exposed at 10 ppm for 28 months developed SCC (Woutersen et al., 21

1989) versus 22% in F344 rats (Monticello et al., 1996). 22

Woutersen et al. (1989) also examined the effect of severe nasal damage from 23

electrocoagulatio n on formaldehyde

- induced SCC in Wistar rats. Nasal tumors were noted in 24

formaldehyde

- exposed rats without damaged noses (exposed for only 3 months and observed for 25This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 326 25 months). However, the low incidence of tumors in each treatment group (1/26, 2/60, 2/6 0, 1

1/58) indicates these data should be considered suggestive even though no SCCs were noted in 2

control rats with or without damaged noses (n = 83). The studies by Woutersen et al. (1989) did 3

demonstrate a synergistic effect of nasal damage from electroco agulation and 10 ppm 4

formaldehyde exposure (3 months), where 15/58 rats had SCC versus 1/26 with undamaged 5

noses. The study was originally designed to examine the effect of formaldehyde on the damaged 6

tissue on cancer promotion. However, it is unclear if the synergistic effect of formaldehyde 7

exposure on damaged nasal tissue is an effect of formaldehyde on the damaged cells and joint 8

effects of a mutagen with regenerative proliferation from the nasal damage. It is also possible 9

the damaged nasal passages may alter airflow in the nasal passages, resulting in significantly 10

different flux of formaldehyde into the tissue. 11

There is a single inhalation study (Dalbey, 1982) that investigates the role of promotion 12

in formaldehyde

- induced cancer. Although hamster s exhibit little to no effects of formaldehyde 13

on the nasal mucosa or other respiratory tract tissues (Rusch et al., 1983a, b; Dalbey, 1982), 14

DEN

- induced respiratory adenomas were increased with formaldehyde exposure (10 ppm) 15

48 hours prior to DEN injectio n (but not by formaldehyde alone or formaldehyde exposure after 16

DEN injection). The number of tracheal tumors per TBA was doubled by formaldehyde 17

exposure. The study authors note that adenomas should be considered independent tumors and 18

that the increase in tracheal tumors is of biological significance even given the incidence of 19

TBAs (77%, DEN alone), was not further increased by formaldehyde exposure. It is of 20

particular interest that a promotion study in hamsters is positive, since so little nasal pat hology 21

occurs with formaldehyde exposure. The absence of significant hyperplasia and tissue damage 22

in these animals suggests that formaldehyde may induce subtle changes in the respiratory tract 23

mucosa that permit formaldehyde to act as a tumor promoter. 24

25

4.2.2.2. Gastrointestinal Tract 26

As with the respiratory tract, the proximal portion of the GI tract exhibits formaldehyde

27

induced lesions in the forestomach and glandular stomach (Soffritti et al., 1989; Til et al., 1989; 28

Tobe et al., 1989; Takahashi et a l., 1986). However, data are mixed regarding the carcinogenic 29

potential of formaldehyde in the GI tract from oral exposures. 30

Two independent 2

- year cancer bioassays in Wistar rats exposed to formaldehyde in 31

drinking water were both negative; they reported no tumors found at the 24

- month sacrifice (Til 32

et al., 1989; Tobe et al., 1989). Til et al. (1989) exposed rats to a range of formaldehyde doses 33

(0, 1.2, 15, or 82 mg/kg

- day) and evaluated 44

– 49 animals per sex per dose group at 24 months 34

of exposure. N o formaldehyde

- related tumors were found. A smaller study by Tobe et al. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 327 (1989) failed to note any tumors after a 2

- year exposure at 0, 10, 50 or 300 mg/kg

- day (eight rats 1

per sex per treatment group). 2

In contrast, two lifelong studies in male and female Sprague

- Dawley rats provide support 3

for formaldehyde

- induced GI tract tumors (Soffritti et al., 1989). Both stomach and intestinal 4

tumors are rare; low background rates are expected in this colony of Sprague

- Dawley rats. 5

These studies demonstrate an incr ease in tumors (although rare) correlated with exposure to 6

formaldehyde and significantly increased susceptibility to early

- lifetime exposure. The authors 7

provide a detailed report of the background rates of various stomach and intestinal neoplasia for 8

ma le (n = 2,677) and female (n = 2,582) rats (Soffritti et al., 1989). From this background pool, 9

the total incidence of benign and malignant tumors in the stomach and intestine combined is only 10

1.4% (combining all sites and locations). The majority of tum ors are located in the stomach (1% 11

benign, 0.2% malignant). Usually, a very large study population is needed to detect increases in 12

rare tumor types. In this study, the study size of each treatment group was relatively small. 13

Thus, only a few TBAs are r esponsible for the observed increases. Additionally, a clear dose

14

response relationship is not evident (perhaps due to the low incidence) despite the fact that the 15

greatest tumor incidence was observed in the highest treatment group. As presented above, 16

apparent increases in both stomach and intestinal neoplasia are noted in formaldehyde

- treated 17

rats (ranging from 1 to 6% by type). When summed across the GI tract, tumor incidence in the 18

highest treatment group was 8% versus 1.4% in historical controls. Despite the limitations of 19

group size and lack of dose response, the findings do support the carcinogenic potential for 20

formaldehyde administered orally. Moreover, these findings are not inconsistent from Tobe et 21

al. (1989) and Til et al. (1989) because t he study design is significantly different. 22

The second study reported by Soffritti et al. (1989) demonstrates early lifetime 23

susceptibility with GI tumor incidence of 21.6% in females (n = 37) and 13.9% in males (n = 36) 24

after exposure to formaldehyde. Sp rague

- Dawley rats were exposed to formaldehyde in drinking 25

water for 2 years (0 or 2,500 mg/L). Exposures began on GD 12 in the offspring. The most 26

common tumor detected was intestinal leiomyosarcoma (13.5% in female offspring) with a 27

background rate of 0.04% in female rats in the colony. 28

Soffritti et al. (1989) stands alone in supporting formaldehyde

- induced GI tumors. These 29

findings are largely attributed to a unique study design that included lifelong observation, 30

neonatal exposure, examination of i ndividual tumor types as well as combined rare tumor types 31

for analysis, and summation of tumors across locations. The study design results in a more 32

sensitive assay for rare tumors. Thus, Soffritti et al. (1989) utilized a more appropriate design 33

and an alysis for detecting rare tumors and should not be compared with the results by Tobe et al. 34

(1989) and Til et al. (1989). 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 328 There is evidence that formaldehyde may act as a tumor promoter by the oral route as 1

well as the inhalation route (discussed above). Takahashi et al. (1986) reported an increase in 2

MNNG

- initiated GI cancers with formaldehyde exposure (29.4 versus 13.3% TBA in controls); 3

the greatest difference in tumor

- containing versus non

- tumorigenic mice was associated with 4

adenocarcinoma in the gla ndular stomach (23.5 versus 3.3% in controls). Additionally, 5

forestomach papillomas and preneoplastic hyperplasia in the glandular stomach were increased 6

with formaldehyde exposure alone. 7

The data indicate carcinogenic potential from formaldehyde ingestio n in drinking water. 8

Formaldehyde may act in part as a tumor

- promoting agent and shows clear increased 9

susceptibility from early lifetime exposures. 10

11

4.2.2.3. Lymphohematopoietic Cancer 12

The majority of chronic animal bioassays do not report either leukem ia or lymphoma, but 13

many of these studies did not have adequate study length or study design to detect these 14

malignancies. Many studies focused the histopathology on the nasal passages and respiratory 15

tract (Monticello et al., 1996; Holmström et al., 1989 a; Woutersen et al., 1989; Appleman et al., 16

1988; Dalbey, 1982; Horten et al., 1963). Kamata et al. (1997) did examine additional organs, 17

but there were only five animals at each sacrifice. Similarly, the oral study by Takahashi et al. 18

(1989) focused on the stomach and intestines. Tobe et al. (1989) only included 20 Wistar rats 19

per group with interim sacrifices. Therefore, few studies can inform the carcinogenic potential 20

of formaldehyde on the LHP system. Table 4

- 75 lists the chronic bioassays that ha ve the 21

potential to detect LHP malignancies. 22

Soffritti et al. (1989) first published an observation of formaldehyde

- induced leukemia in 23

animal studies. These study results have been criticized for their combination of lymphatic 24

leukemia and lymphoma. How ever, this classification is consistent with the current WHO 25

classification of lymphoid malignancies in humans where adult B

- and T

- cell leukemias and 26

lymphomas are considered the same disease (Harris, 2000a). Although there may be a slight 27

vehicle effect , a dose response is still readily apparent among the formaldehyde

- treated groups 28

(Figure 4

- 28). In contrast, the 2

- year bioassay in Wistar male in female rats (Til et al., 1989) was 29

clearly negative for leukemia and lymphoma with only four TBAs in all tr eatment groups 30

sacrificed at 24 months. Moreover, the drinking water levels were similar at the highest dose of 31

both Til et al. (1989) and Soffritti et al. (1989). However, the two study designs differ in length, 32

which may have influenced results since l eukemia is a late

- in

- life malignancy in rodents. Two

33

year survival in the Soffritti et al. (1989) study varied between 50 and 60%. These animals were 34

available to develop leukemia after the 2

- year window of the Til et al. (1989) study. Any 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 329 potential rol e of strain differences is unknown. Overall the results of Soffritti et al. (1989) are 1

strong since they indicate an exposure

- response relationship in a lifelong study appropriate for 2

late

- in

- life malignancies. Unlike the GI tract tumors, increased LHP m alignancies were not 3

associated with early

- life exposure to formaldehyde in drinking water. 4

5

Table 4

- 75. Summary of chronic bioassays which address rodent leukemia 6

and lymphoma 7

8

Study Histopathology Endpoint Results Comments

Drinking water exposure

Soff ritti et

al. (1986) Complete histopathology Male and female Sprague

- Dawley rats

Lymphocytic

leukemia and

lymphosarcoma Increased, showing

a dose

- response Lifelong study

High exposure of

1,500 mg/L in water

Til et al.

(1989) Complete histopathology

in control and high

- dose

group (15 ppm) Male and female Wistar Rats

Lymphoma,

leukemia No increase

(three lymphomas

and one leukemia

found in 200

animals at the 2

- year sacrifice) 2

- year bioassay

High exposure of

approximately 1,900 mg/L

(82 mg/kg for males an d

109 mg/kg for females)

Inhalation exposures

Sellakumar

et al. (1985);

Albert et al.

(1982) Necropsy focused on

respiratory tract: also liver,

spleen, kidney, and testes

and organs demonstrating

gross pathology Male rats, Sprague

- Dawley

Lymphoma No i ncrease Lifelong study, high

mortality at 24 months

(>80%)

Battelle,

Columbus

Laboratories

(1981) Complete histopathology

in controls and high

- dose

group (15 ppm) Male rats, F344

Leukemia, all No increase Extended study, high

mortality

Complete histopathology

in controls and high

- dose

group (15 ppm) Female rats, F 344

Leukemia, all Increase in

mortality

- adjusted

incidence;

p = 0.0056 a Extended study, high

mortality. Apparent

elevation in 2 and 6 ppm

treatment groups as well;

statistical comparison t o controls is problematic.

All organs in controls and

high

- dose group (15 ppm) Female mice, C57BL/6xC3HF1

Lymphoma, all 26% increase in

15 ppm group, 16%

in controls;

p = 0.0617 Extended study.

All mice included in

statistics conducted by

BattelleColu mbus

Laboratories.

9

a Original statistical analysis provided by Battelle Columbus Laboratories. Significance set at p < 0.0167. Analysis 10

of adjusted data where time to lesion and survivorship were considered (Cox [1972] and Tarone [1975]). 11

12This document is a draft for review purposes only and does not constitute Agency policy.

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- 330 Male and Female Sprague-Dawley Rats Exposed to

Formaldehyde in Drinking Water

0 5 10 15 20 25

0 10 50 100 500 1000 1500 Vehicle

Exposure (mg/l formaldehyde)All Leukemia (%) All

Female

Male

1

2

Figure 4

- 2 8. Leukemia incidence in Sprague

- Dawley rats exposed to 3

formaldehyde in drinking water for 2 years. 4

5

Note: Animals were observed until natural death. The vehicle control contained 6

the level of methanol in drinking water for the high

- dose group (1,500 mg/ L). 7

8

Source: Soffritti et al. (1989). 9

10

11

Sellakumar et al. (1985) conducted a lifelong inhalation study in male Sprague

- Dawley 12

rats exposed at 10 ppm formaldehyde. Organs outside of the respiratory tract were routinely 13

examined (liver, kidneys, and teste s), including any organ exhibiting gross pathology, so there 14

was some ability to detect leukemia and lymphoma. However, spleen, thymus, and lymph nodes 15

were not routinely examined, limiting detection of smaller lesions. Although Sellakumar et al. 16

(1985) was a lifelong study, there was a high mortality rate at 2 years (>80% from the figure), 17

again limiting the power of this study to detect late

- in

- life malignancies. Nonetheless, this study 18

did not indicate formaldehyde

- induced lymphoma or leukemia. 19

The l argest and most comprehensive study of carcinogenic health effects from 20

formaldehyde inhalation exposures is the study conducted at the Battelle Columbus Laboratory 21

(1981) and reported by Kerns et al. (1980) and Swenberg et al. (1983). Although the summar y 22

reports of this study do not discuss leukemia or lymphoma rates, mouse lymphoma and rat 23

leukemia were selected by the study pathologist and biostatistician for analysis (Battelle 24

Columbus Laboratories, 1981). Statistical analysis performed by Battelle, which accounted for 25This document is a draft for review purposes only and does not constitute Agency policy.

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- 331 time to lesion and survivorship rates, indicated a statistically significant increase in female rat 1

leukemia ( p = 0.0003) and near significant increase in female mouse lymphoma ( p = 0.06). No 2

trend analysis could be conducted since onl y gross pathology was conducted on mid

- dose mice 3

and rats (2 and 6 ppm). EPA has further analyzed these data to better understand the significance 4

of these findings. 5

As noted in the study description (Section 4.2.1.2.3), both male and female rats at the 6

h ighest exposure (15 ppm) exhibited significant early deaths due to nasal lesions (see Figure 7

4

- 29). No female rats in the highest exposure group remained after the 24

- month sacrifice 8

(which included only 14 animals). Nine male rats were examined after 24 months. Since the 9

first leukemia in rats was noted at 21 months, the early deaths prior to that time reduced the 10

number of animals in which the leukemia could have been observed. Unadjusted data do not 11

show an increase in female rat leukemia, but, when data are adjusted to account for the early 12

deaths, dramatically different results are apparent. In the unadjusted data, leukemia incidence is 13

expressed as the number of cases over total animals examined (including early deaths and 14

interim sacrifices). By using this methodology, there is a lower incidence of leukemia in high 15

exposed animals, and a slightly higher incidence of leukemia in the mid

- exposed groups 16

compared with controls (Figure 4

- 30, panel A). However, when the leukemia incidence rates are 17

ca lculated only for female rats that survived to at least 21 months (the first case noted), 18

formaldehyde

- induced increases in leukemia are evident in all treatment groups relative to 19

controls (Figure 4

- 30, panel B). These results are consistent with the ori ginal statistical analysis 20

conducted by the researchers at the Battelle Columbus Laboratories (1981). Although elevated 21

in all treatment groups, no exposure

- response relationship is evident. The lack of a dose

22

response relationship may in part be due to the fact that no animals in the high

- exposure group 23

survived past 24 months. Additionally, the 6 ppm (mid

- exposure) group had significant early 24

deaths between 21 to 24 months compared with the 2 ppm and control groups. A similar 25

reanalysis of data from m ale rats did not reveal any relationship between formaldehyde treatment 26

and leukemia incidence. 27

Male and female mice exposed to formaldehyde for 24 months did not experience the 28

same rate of formaldehyde

- related mortality (Kerns et al., 1983). However, si gnificant early 29

deaths were observed in male mice due to infighting. Therefore, the data for male mice may not 30

inform incidence of late

- in

- life tumor, such as lymphoma. As discussed above in the full study 31

description, full histopathology, including sple en, liver, thymus, and lymph nodes, was only done 32

on the control and high

- exposure group (15 ppm) mice. When comparing unadjusted incidence 33

lymphoma

- bearing mice, there is a clear elevation lymphoma in formaldehyde

- exposed female 34

mice (28%) over controls (22%) (Figure 4

- 31). 35

36This document is a draft for review purposes only and does not constitute Agency policy.

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- 332 18 mos 18 to 21 21 to 24 > 24 0 ppm 2 ppm

6 ppm

15 ppm

0 5 10 15 20 25

1

2

Figure 4

- 29. Unscheduled deaths in female F344 rats exposed to 3

formaldehyde for 24 months. 4

5

Source: Battelle Columbus Laboratories (1981). 6

7

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

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- 333 1

Panel A: Unadjusted Data Panel B: Adjusted Data 2

3

0% 5% 10% 15% 20% 25% 30%

18 mo 21 Mo 24 mo 27 mo 30 Mo

0 ppm 2 ppm 6 ppm 15 ppm 0% 5% 10% 15% 20% 25% 30%

21 mos 24 mos 27 mos 30 mos

0 ppm 2 ppm 6 ppm 15 ppm 4

5

Figure 4

- 30. Cumulative leuk emia incidence in female F344 rats exposed to 6

formaldehyde for 24 months. 7

8

Note: Panel A shows the unadjusted data where incidence rates include all 9

scheduled sacrifices and early deaths up to the time point shown. Panel B shows 10

incidence of leukemia only in rats who survived at least 21 months. 11

12

Source: Battelle Columbus Laboratories (1981). 13

14

15

16This document is a draft for review purposes only and does not constitute Agency policy.

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- 334 0% 1% 9% 18%

1% 9% 22% 28%

0% 5% 10% 15% 20% 25% 30%

12 Mo 18 Mo 24 Mo 30 Mo Control

15 ppm

1

2

Figure 4

- 31. Cumulative incidence of tumor bearing animals for lymphoma 3

in female mice exposed to formaldehyde for 24 months. 4

5

Note: Mice from the 6

- month int erim sacrifice are not included since only nasal 6

passages were examined ( p < 0.05). 7

8

Source: Battelle Columbus Laboratories (1981). 9

10

11

Although results are somewhat mixed between studies, there is evidence in the animal 12

bioassays for formaldehyde

- induced L HP malignancies. Differences in study design may 13

account in part for mixed results. The lifelong drinking water study by Soffritti et al. (1989) may 14

have allowed for malignancies to develop late in life, whereas the drinking water study by Til et 15

al. (19 89) sacrificed all animals at 24 months. Even though the exposure levels were similar, the 16

studies are not directly comparable. Similarly it is hard to directly compare results from the two 17

major inhalation studies in rats. Although Sellakumar et al. (1 985) is a lifelong study, the 18

mortality for rats was greater than 80% at 2 years. Additionally, the pathology examination was 19

much less rigorous than in the Battelle Columbus Laboratories (1981) study, perhaps missing 20

smaller lesions. Therefore, the incr ease in formaldehyde

- induced leukemia seen in female F344 21

rats late

- in

- life (Battelle Columbus Laboratories [1981]) may reflect a more sensitive study 22

design. Finally, strain differences may account for different susceptibility as well. The two 23

positive rat studies, by different routes of exposure, along with a positive result for 24This document is a draft for review purposes only and does not constitute Agency policy.

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- 335 formaldehyde

- induced mouse lymphoma, make a substantive case for the potential of 1

formaldehyde

- induced LHP malignancies. 2

3

4.2.2.4. Summary 4

Formaldehyde is toxic at the POE. Si milar lesions, including increased cell proliferation, 5

DPX, and focal lesions, are noted in the GI tract or URT, depending on route of exposure. 6

Similarly, formaldehyde

- induced tumors are noted at the POE for both routes of exposure. 7

Additionally, data e xist for both routes of exposure to indicate that formaldehyde may act in part 8

as a tumor promoter. 9

When evaluating the studies with adequate study design to assess LHP malignancies, 10

results are mixed by strain, sex, route of exposure, and length of study. The three positive 11

studies (Soffritti et al., 1986; Battelle Columbus Laboratories, 1981) had the best histopathologic 12

examinations and greater sensitivity for detection of late

- in

- life tumors. Based on these results, 13

sufficient evidence is available in animal studies to support formaldehyde

- induced LHP 14

malignancies. 15

16

4.3. GENOTOXICITY 17

Formaldehyde has been extensively studied for its mutagenic and genotoxic activity in a 18

variety of assay systems. The first reported mutagenic activity of formaldehyde w as when 19

Rapoport (1946) described the induction of sex

- linked recessive lethals in drosophila larvae fed 20

on a medium containing formalin. A variety of genotoxic and mutagenic effects have been 21

subsequently demonstrated in both in vitro and in vivo test sy stems, including the formation of 22

DPXs, point mutations, DNA strand breaks, increased MNs, and CAs (Auerbach et al 1977; Ma 23

and Harris, 1988; Conaway et al 1996; IARC 1995; 2006). 24

In this section, reactions of formaldehyde with cellular macromolecules, s uch as DNA 25

and proteins, and formaldehyde

- induced clastogenicity are described. In addition, the evidence 26

for formaldehyde

- induced mutations is considered in the context of the current EPA cancer 27

guidelines (U.S. EPA, 2005a) . Particular emphasis is given to the genotoxic effects of 28

formaldehyde in humans, described in Section 4.3.4.2

. 29

30

4.3.1. Formaldehyde

- DNA Reactions 31

Formaldehyde is a reactive chemical and interacts with DNA in several ways, forming 32

D PXs, DNA adducts, and DNA

- DNA cross

- links (DDXs) (Fennell, 1994; Casanova et al., 1989; 33

Heck and Casanova, 1987; Casanova

- Schmitz et al., 1984a, b; Casanova

- Schmitz and Heck, 34

1983; Ohba et al., 1979; Dönecke, 1978; B rutlag et al., 1969) . Formaldehyde also may facilitate 35

the formation of adducts between other chemicals (endogenous or xenobiotic) and DNA 36This document is a draft for review purposes only and does not constitute Agency policy.

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- 336 (Fennell, 1994; Koppel et al., 1991; Casanova et al., 1989; Heck and Casanova, 1987; Lam et al., 1

1985; Casanova

- Sch mitz et al., 1984a; Casanova

- Schmitz and Heck, 1983; Ohba et al., 1979; 2

Dönecke, 1978; Brutlag et al., 1969). 3

The high reactivity of formaldehyde results in little specificity in reaction sites, indicating 4

that a range of adducts and cross

- links might be expected. However, the spectrum of 5

formaldehyde

- DNA reaction products is difficult to quantify in vivo as many of these are labile 6

and difficult to measure directly (Fennell, 1994; Casanova et al., 1989) . Additio nally, 7

formaldehyde is metabolically incorporated into nucleic acids, and therefore DNA and RNA 8

assays incorporating radiolabeled formaldehyde need careful interpretation to distinguish 9

between covalently bound and metabolically incorporated formaldehyde (Casanova et al., 1989; 10

Heck and Casanova, 1987; Casanova

- Schmitz et al., 1984a, b; Casanova

- Schmitz and Heck, 11

1983) . Hence, reports of formaldehyde

- DNA reactivity in cell

- free system results may not 12

provide a usefu l measure of exposure (Fennell, 1994) . Besides, the question of biological 13

relevance must also be considered. On the other hand, methods used to extract and measure 14

DNA

- formaldehyde reaction products after in vivo exposures should be evaluated to ensure that 15

formaldehyde reaction products are neither created nor removed during sample preparation 16

(Fennell, 1994; Casanova et al., 1989)

. 17

18

4.3.1.1. DNA

- Protein Cross

- Links (DPXs ) 19

Evidence from numerous experimental models, ranging from cell

- free systems to single 20

cells and in vivo animal and human exposures, suggests that formaldehyde reacts readily with 21

DNA forming DPXs (Reviewed in Conaway et al 1996; IARC 2006). As shown in T able 4

- 76, 22

cross

- links between histones and DNA have been demonstrated in isolated chromatin samples on 23

exposure to formaldehyde from earlier studies (Ohba et al., 1979; Dönecke, 1978; Brutlag et al., 24

1969). Several in vitro studies demonstrated induction of DPX by formaldehyde exposure in 25

bacteria (Wilkins and McLeod 1976), yeast (Magana

- Schwencke and Ekert, 1978) and 26

mammalian cells including animal cells (Chinese hamster ovary cells, Chinese hamster V79 lung 27

epithelial cells, mouse leukemia L1210 cells , mouse hepatocytes, rat Yoshida lymphsarcoma 28

cells, rat C18 tracheal epithelial cells, rat hepatocytes, rat nasal, tracheal epitheial cells and aortic 29

endothelial cells) and human cells (lung and bronchial epithelial cells, fibrobasts, white blood 30

cells, peripheral blood lymphocytes, Epstein

- Barr Virus

- Burkitt’s lymphoma cells, Jurkat E6

- 1 31

cells, HeLa cells, lymphoblastoid cells, gastric mucosal cells and whole blood) as summarized in 32

Table 4

- 76. 33

Ross and Shipley (1980) showed that formaldehyde induces S SBs and DPXs; SSBs are 34

formed at concentrations >200 μM and a reduction of radiation

- induced breaks (indirect measure 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 337 of DPXs) at 50 μM. The authors used a [ 14 C]

- thymidine

- incorporated mouse L1210 cell line to 1

monitor formaldehyde

- induced DNA strand break s and DPXs. They exposed cells to varying 2

concentrations of formaldehyde for 2.5 hours. An alkaline

- elution technique in the presence or 3

absence of proteinase K was used to measure strand breaks. In order to detect DPXs, some cells 4

were exposed to 300 R of X

- rays immediately after formaldehyde treatment. Formaldehyde

5

induced DPXs were repaired 24 hours after the compound was removed from the culture (Ross 6

and Shipley, 1980)

. 7

Casanova

- Schmitz and Heck (1983) have shown that homogenates of rat nasal mucosa 8

incubated with formaldehyde in vitro followed by extraction w ith a strong aqueous

- immiscible 9

organic solvent demonstrated increased DPX formation in DNA obtained after enzymatic 10

proteolysis from the aqueous

- organic interface, termed as “interfacial DNA”. In the same study, 11

they have shown that DNA isolated from the nasal, but not olfactory, mucosa of rats exposed to 12

formaldehyde (2, 6, 15, and 30 ppm 6 hours/day for 2 days) via inhalation showed significant 13

increase in DPXs in the interfacial DNA ≥6 ppm, which was shown to be linear in the exposure 14

range of 2

– 30 ppm (2.45

– 36.8 mg/m 3 ). However, DNA in the aqueous phase did not show DPX 15

formation. Thus, the cross

- linked DNA that could be extracted from the interface after 16

proteolysis was considered to be supporting evidence of chemically induced DPX formation. 17

The in ability of this study to detect DPXs at lower levels of formaldehyde exposure is likely be 18

due to the protective mechanism of GSH, which catalyzes the conversion of formaldehyde to 19

formate. 20

So, in a later study, Casanova and Heck (1987) reported that GSH depletion caused an 21

increase in DPX formation in the interfacial DNA in the nasal mucosa of F344 rats when a dual

22

isotope ( 3 H/ 14 C) method was used. The dual isotope method helps in making the distinction 23

between metabolic incorporation and covalent bindin g of formldehyde. Oxidatoin by removal of 24

one hydrogen atom is required for metabolic incorporation of formaldehyde into cellular 25

macromolecules, but not in the formation of DNA adducts or DNA

- protein crosslinks. Thus, the 26

ratio of 3 H/ 14 C of DNA containin g DPX will be higher than the macromoleucules where 27

formaldehyde is metabolically incorporated. However, the authors further demonstrated that, 28

when the double isotope method was used, the 3 HCHO is oxidized significantly more slowly 29

than H 14 CHO under thes e conditions, resulting in an overestimate of the concentration of cross

30

links due to an isotopic effect on the oxidation of 3 HCHO catalyzed by formaldehyde 31

dehydrogenase (FDH). Besides, this method leaves residual formaldehyde that is likely to form 32

DNA adducts by reacting with deoxyribonucleosides in the DNA hydrolysates (Heck and 33

Casanova, 1987)

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

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- 338 To overcome this, Casanova et al. (1989) used an improved method, which is based not 1

on the analysis of residual formaldehyde bound to deoxyribonucleosides in DNA hydrolysates 2

but on the determination of the total 14 C

- formaldehyde bound to DNA. This study showed that 3

formaldehyde was exclusively bound to interfacial DNA, indicating the formation of DPXs. 4

Hydrolysis of DPXs in different samples quantitatively released formaldehyde. Besides, DPX 5

formation was detectable at all concentrations of exposure to formaldehyde (0.3

– 10 ppm for 6

6 hours). Overall, these studies clearly show that formaldehyde induces DPXs in nasal epithelial 7

cells of rodents. However, there are no published rodent studies that assess DPXs beyond the 8

nasal passages of the URT. 9

Formaldehyde

- induced DPXs were also found in the nasal mucosa and extra

- nasal tissues 10

of rhesus monkeys exposed to 0, 0.71, 2, or 6 ppm (0, 0.86, 2.45, or 7.36 mg/m 3 ) formaldehyde 11

6 hours/day for 3 days (Casanova et al., 1991) . These data were used as a basis for cross

- species 12

prediction of formaldehyde

- induced DPXs in humans. The presence of DPXs in rhesus monkeys 13

confirms formaldehyde‘s DNA reactivity as a general effect. Additionally, DPXs were detected 14

in the larynx/trachea/carina (pooled sample) and in intrapulmonary airways of monkeys exposed 15

to 2 or 6 ppm formaldehyde. These data demonstrate d irect effects of formaldehyde on DNA in 16

tissues that correspond to observed tumor sites in humans (nasal and nasopharynx). 17

Bermudez and Delehanty (1986) observed the formation of DPXs, scheduled (S) and 18

unscheduled DNA synthesis (UDS), and synthesis of RN A when cultured F344 rat nasal 19

epithelial cells from the naso

- and maxillary turbinates were incubated with formaldehyde. 20

Unscheduled and scheduled DNA synthesis was stimulated (0.05

– 0.1 mM) and then inhibited 21

(0.1

– 1 mM), depending on the formaldehyde con centration. Experiments by Cosma et al. (1988) 22

and Cosma and Marchok (1988) showed the induction of DPXs and DNA SSBs in cultured C18 23

rat tracheal epithelial cells exposed to 200 **μ** M formaldehyde for 90 minutes (Cosm a et al., 1988; 24

Cosma and Marchok, 1988)

. 25

Several human cells (epithelial cells, fibroblasts, buccal cells) or cell lines 26

(lymphoblastoid cells) exposed to formaldehyde have been shown to form DPX (Craft et al 27

1987; Costa et al 1997; Emri et al 2004; Li e t al 2004). 28

Craft et al. (1987) detected DPXs by alkaline elution in TK6 human lymphoblastoid cells 29

immediately after a 2

- hour exposure (zero time) to 0, 15, 50, 75, 100, 150, 300, and 600 μM 30

formaldehyde with a significant nonlinear increase in DPXs above 50 μM conce ntration, which 31

correlated with the onset of cytotoxicity, but DPXs were completely removed in cultures held for 32

24 hours before processing. In the zero

- time sample, significant increases in DPXs were first 33

observed at 50 μM and increased linearly up to 1 50 μM. In cells held for 24 hours, there was no 34

detectable increase in DPXs. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 339 However, Costa et al. (1997) detected DPXs with paraformaldehyde (which dissociates to 1

release formaldehyde) at doses that were cytotoxic (>0.003%) but could not discriminate 2

bet ween the DPX

- inducing and cytotoxic effects of this chemical in EBV, human Burkitt’s 3

lymphoma cells (Costa et al., 1997). Grafström et al. (1983) reported that the number of DPXs 4

induced by 100 **μ** M formaldehyde in vitro in human epithelial cells and fibrob lasts of bronchial 5

origin was similar and that the frequency of these cross

- links was proportional to the 6

concentration of formaldehyde. Besides the bronchial epithelial cells and fibroblasts, the authors 7

also noted that formaldehyde exposure resulted in DPXs and DNA SSBs in skin fibroblasts and 8

DNA excision repair

- deficient skin fibroblasts. However, formaldehyde was only moderately 9

cytotoxic to normal bronchial epithelial cells and fibroblasts at concentrations that induced 10

substantial DNA damage. Repa ir of the formaldehyde

- induced DNA SSBs and DPXs appeared 11

to be inhibited by the continued presence of formaldehyde in the culture medium (Grafström et 12

al., 1984). 13

Emri et al. (2004) detected a significant increase in DPX formation in primary human 14

skin fi broblasts and keratinocytes at 8 hours of exposure in vitro to formaldehyde at 25 **μ** M with 15

an approximately linear increase up to 100 **μ** M. These cells were exposed to 0, 12.5, 25, 50, and 16

100 **μ** M formaldehyde for 8 hours and then exposed to 250 **μ** M methyl met hane sulfonate 17

(MMS) for 2.5 hours. The induction of DPX formation was measured by the ability of 18

formaldehyde to reduce DNA migration in the comet assay induced by MMS in this study (Emri 19

et al., 2004)

. 20

Li et al. (2004) measured DNA damage in primary human buccal cells by using the 21

comet assay. The appearance of SSBs, suggesting compound

- induced fragmentation of DNA, 22

occurred at formaldehyde concentrations of 5 and 7.5 **μ** M. At higher concentrations, the 23

response slope decreased, indicating DPXs or DDXs (Li et al., 2004) . The same laboratory 24

reported similar findings in primary human peripheral blood lymphocytes and HeLa cells (Liu et 25

al., 2006). Peak response for SSBs was seen at 10 **μ** M in both cells, with higher concentrations 26

resulting in cross

- link formation. SSBs in HeLa cells induced by 10 **μ** M formaldehyde were 27

repaired by 60 minutes after cells were washed to remove formaldehyde. 28

Schmid and Speit (2007) tested formaldehyde for its ability to induce DPXs in blood 29

cultures. They used an indirect m ethod to monitor DPX formation in which the extent of DNA 30

migration in the comet assay in response to γ radiation was compared in formaldehyde

- treated 31

cultures versus controls. A concentration of 25 μM was required for DPX formation, and repair 32

of these l esions was rapid, with DPXs induced by concentrations of formaldehyde up to 100 μM 33

and completely removed after 8 hours. 34

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

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- 340 Table 4

- 76. Formaldedyde

- DNA reactions (DPX formation) 1

2

Species/Strain Cell/Strain Result References

DNA Interaction

DP X formation in vitro

In vitro Nucleohistone + Brutlag et al 1969

In vitro Nucleohistone + Doenecke 1978

In vitro Nucleohistone + Ohba et al 1979

Bacteria + Wilkins and McLeod 1976

Yeast Saccharomyces cerevisiae + Magana

- Schwencke and Ekert 1978

Hams ter/Chinese Ovary cells + Marinari et al 1984

Hamster/Chinese Ovary cells + Zhitkovich and Costa 1992

Hamster/Chinese Ovary cells + Olin et al 1996

Hamster/Chinese Ovary cells + Garcia et al 2009

Hamster/Chinese V79 lung epithelial cells + Swenberg et al 1983

Hamster/Chinese V79 lung epithelial cells + Merk and Speit 1998

Hamster/Chinese V79 lung epithelial cells + Merk and Speit 1999

Hamster/Chinese V79 lung epithelial cells + Speit et al 2007

Mouse Leukemia L1210 cells + Ross and Shipley, 1980

Mo use Leukemia L1210 cells + Ross et al 1981

Mouse Hepatocytes + Casanova and Heck 1997

Mouse Hepatocytes + Casanova et al 1997

Rat Yoshida lymphosarcoma cells + O'Connor and Fox 1987

Rat C18 tracheal epithelial cell line + Cosma and Marchok 1988

Rat/F3 44 Hepatocytes + Casanova and Heck 1997

Rat/F344 Nasal mucosa + Casanova

- Schmitz and Heck 1983

Rat/F344 Nasal epithelium + Bermudez et al., 1986

Rat/F344 Primary tracheal epithelial

cells + Cosma et al., 1988

Rats/Wistar Aorta endothelial cells + Lin e t al 2005

Human Lung/bronchial epithelial cells + Fornace et al 1982

Human Bronchial cell + Grafstrom et al., 1983

Human Bronchial/ Skin fibroblast + Grafstrom et al., 1984

Human Lung/bronchial epithelial cells + Grafstrom et al 1984

Human Lung/bronch ial epithelial cells + Saladino et al 1985

Human Lung/bronchial epithelial cells + Grafstrom et al 1986

Human Foreskin fibroblasts + Snyder and Van Houten 1986

Human Lung/bronchial epithelial cells + Grafstrom 1990

Human Bronchial/Skin fibroblasts + Ol in et al 1996

Human White blood cell + Shaham et al., 1996

Human EBV

- Burkitt's lymphoma cells + A Costa et al., 1997

Human Gastric mucosa cells + Blasiak et al 2000

Human Peripheral lymphocyte + Quievryn and Zhitkovich 2000

Human Fibroblast cells + Spe it et al 2000

Human Lymphocyte + Andersson et al., 2003This document is a draft for review purposes only and does not constitute Agency policy.

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- 341 Human Primary skin fibroblasts and

keratinocytes + Emri et al 2004

Human Buccal cells + Li et al 2004

Human Jurkat E6

- 1 cells + Saito et al 2005

Human Peripheral lymphocyte + Liu et al 2006

Huma n HeLa cells + Liu et al 2006

Human Whole blood + Schmid and Speit 2007

Human Lung/bronchial epithelial cells + Speit et al 2008

Human Lymphoblastoid/TK6 + Craft et al., 1987

Rat/F344 DPX formation in vivo

Nasal mucosa + Casanova

- Schmitz and Hec k 1983

Rat/F344 Nasal mucosa + Casanova

- Schmitz et al 1984b

Rat/F344 Nasal mucosa + Lam et al 1985

Rat/F344 Nasal mucosa + Heck et al 1986

Rat/F344 Nasal mucosa + Heck Hd and Casanova 1987

Rat/F344 Tracheal implants + Cosma et al., 1988

Rat/F344 Nasa l mucosa + Casanova et al 1989

Rat/F344 Nasal mucosa + Casanova et al 1994

Rhesus monkeys Nasal, larynx, trachea, and

carina + Casanova et al 1991

Human White blood cell + Shaham et al., 1996

Human Peripheral lymphocyte + Shaham et al., 1997

Human Per ipheral lymphocyte + Shaham et al., 2003

1

‘+’ indicates a positive test result 2

‘

- ’ indicates a negative test result 3

A indicates that DNA

- protein cross

- links formed at cytotoxic concentrations 4

5

6

4.3.1.2. DNA Adducts 7

In addition to the formation of DPX, there is evidence that formaldehyde forms 8

hydroxymethyl (hm) DNA adducts in vitro in a variety of cell

- free systems (Zhong and Que Hee, 9

2004a; Cheng et al., 2003; Kennedy et al., 1996; Fennell, 1994; Beland et al., 1984) and nasal 10

epithelial cells (Zh ong and Que Hee 2004b). In cell

- free systems, formaldehyde directly reacts 11

with DNA forming hmDNA adducts (Cheng et al., 2003; Kennedy et al., 1996; Fennell, 1994; 12

McGhee and von Hippel, 1977a, b, 1975a, b). 13

Beland et al. (1984) first reported hmDNA addu cts in Chinese hamster ovary (CHO) cells 14

incubated with 1 mM of radiolabeled formaldehyde. After a 2

- hour incubation, small amounts of 15

N 6

- hmdA were detected with concomitant metabolic incorporation of formaldehyde. Various 16

forms of hmDNA adducts, includi ng N 6

- hydroxymethyldeoxyadenosine (N 6

- hmdA), 17

N 4

- hydroxymethyldeoxycytidine (N 4

- hmdC), and N 2

- hydroxymethyldeoxyguanosine (N 2

- 18

hmdG), were detected by high performance liquid chromatography (HPLC) following in vitro 19

reaction between formaldehyde and calf thy mus DNA or individual deoxynucleotides. 20This document is a draft for review purposes only and does not constitute Agency policy.

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- 342 32 P

- postlabeling studies allowed for much greater analytical sensitivity but did not 1

confirm the level of N 6

- sulfomethyldeoxyadenosine found by HPLC. However, either estimate 2

of adduct formation is much less than the estimate of DPX formation (120 pmol/mg DNA) in 3

similarly treated rat nuclei (Heck and Casanova, 1987)

. 4

Casanova et al. (1989) demonstrated that detection of hmDNA adduct formation was 5

sensitive to the methodology used, particu larly the buffer used for sample preparation. 6

Specifically, Tris buffer can prevent hmDNA adduct formation due to the abundance of 7

formaldehyde

- reactive primary amine sites in the buffer. In contrast, the tertiary amine sites that 8

predominate in Bis

- Tris buffer do not react with formaldehyde. 9

Zhong and Que Hee (2004a) observed hmDNA adducts (N 6

- hmdA, N 2

- hmdG, and 10

N 4

- hmdC) in placental DNA exposed to 100 ppm formaldehyde in vitro for 20 hours at 37°C 11

followed by hydrolysis of formaldehyde

- reacted DNA usi ng bis

- Tris buffer. However, 12

deoxythymidine did not form hydroxymethyl derivatives in this study (Zhong and Que Hee, 13

2004a) . On the other hand, the same investigators were able to detect N 6

- hmdA and N 2

- hmdG 14

adducts in human nasal epithelial cells cultured in the presence of 0, 10, 25, 50, 100, 2 50, 400, or 15

500 μg/mL formaldehyde and using Tris buffer during hydrolysis of adducted DNA. The 16

toxicity threshold for <90% viability appeared to be between 100 and 250 μg/mL initial 17

formaldehyde culture concentration, and even at 500 **μ** g/mL concentration it was not toxic, with 18

a viability was 70% in this study (Zhong and Que Hee, 2004b)

. 19

The only report of formaldehyde

- induced hmDNA adducts in vivo is a recent study 20

(Wang et al., 2007), showing an indirect evidence of formation of formaldehyde

- induced N 6

- 21

hmdA in hepatic and pulmonary DNA from rats exposed to N

- nitrosodimethylamine and 22

4

- (methylnitrosamino)

- 1

- (3

- pyridyl)

- 1

- butanone. 23

Since the formaldehyde adducts are labile, Fennel (1994) developed a method by 24

derivatizing them with sodium bisulfite to their sulfomethyl form, whereby he detected 25

N 6

- sulfomethyldeoxyadenosine (SOMedA) and N 2

- sulfomethyldeoxyguanosine by using HPLC. 26

However, the levels of SOMedA in DNA isolated following incubation of radiolabeled 27

formaldehyde with isolated rat hepatic nuclei were similar to thos e in control nuclei. And in 28

human TK6 lymphoblastoid cells treated with formaldehyde, detection of SOMedA adducts was 29

precluded by additional radioactive sports. These observations suggest that N 6

- 30

sulfomethyldeoxyadenosine adducts are formed at very low levels in formaldehyde

- incubated rat 31

nuclei and that measurement of hydroxymethyldeoxyadenosine would not provide a useful 32

measure of formaldehyde exposure (Fennell, 1994)

. 33

34

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

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- 343 Table 4

- 77. Formaldedyde

- DNA reactions (DNA adduct formation) 1

2

Species/Strain Cell/Strain Result References

DNA Interaction

DNA Adduct Formation in vitro

Cell

- free system Deoxyribonucleosides + Cheng et al 2008

Cell

- free system Guanosine + Kennedy et al 1996

Cell

- free system Gu anosine + Cheng et al 2003

Placental DNA In vitro + Zhong and Que Hee 2004a

Calf thymus In vitro + Beland et al 1984

Calf thymus DNA In vitro + Von Hippel and Wang 1971

Cell

- free system In vitro + McGhee and von Hippel 1975a

Cell

- free system In vitro + McGhee and von Hippel 1975b

Cell

- free system In vitro + McGhee and von Hippel 1977a

Cell

- free system In vitro + McGhee and von Hippel 1977b

Cell

- free system In vitro + Fennell 1994

Cell

- free system In vitro + Cheng et al 2003

Rat Nuclei + Fennell 19 94

Rats Nasal epithelial cells + Casanova et al 1989

Hamster CHO cells + Beland et al 1984

Rat Nuclei + Heck Hd and Casanova 1987

Human Nasal epithelial cells + Zhong and Que Hee 2004b

DNA adduct formation in vivo

Drosophila Larvae + Alderson, 1985

Rats Indirect evidence + Wang et al 2007

3

‘+’ indicates a positive test result 4

‘

- ’ indicates a negative test result 5

6

7

4.3.1.3. DNA

- DNA Cross

- Links (DDXs) 8

Formaldehyde, besides forming DPXs and DNA adducts, has also been shown to form 9

DDX in vitro . Li et al. (2004) showed that formaldehyde induces DNA strand breaks at low

10

exposure concentration and DDXs and DPXs at higher concentrations in buccal cells. The 11

authors also showed that formaldehyde induces DDXs in human peripheral blood lymphocytes 12

e xposed in vitro when the concentration was more than 25 **μ** M. However, the formation of 13

DDXs has not been demonstrated in vivo, and the relevance of these modifications in 14

formaldehyde

- induced genotoxicity is not known at the moment. 15

Overall, formaldehyde f orms predominantly DPXs that are detected in cell

- free systems 16

and single cells in vitro and in animal and human tissues in vivo. In rodents, DPXs are formed in 17

nasal epithelia but not in extra

- nasal passages, which are completely removed within a day aft er 18

formation. The DPXs are detected in nasal and extra

- nasal tissues of monkeys, suggestive of 19This document is a draft for review purposes only and does not constitute Agency policy.

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- 344 direct effects of formaldehyde in tissues that correspond to observed tumor sites (nasal and 1

nasopharynx) in humans. Besides, this is used as a basis for cross

- species comparison with 2

humans. Formaldehyde

- DNA adducts are labile, constituting a minor fraction of the DNA

3

reaction products. DPXs but not DNA adducts appear to play an important role in the 4

genotoxicity of formaldehyde. 5

6

4.3.1.4. Single Strand Break s 7

Formaldehyde has been shown to induce DNA single strand breaks in a number of mammalian 8

cell systems in vitro as well as in vivo as shown in Table 4

- 78. Additionally, there is some 9

evidence that DNA single strand breaks (SSBs) may be induced directly by formaldehyde 10

reactivity (Grafström et al., 1984). 11

12

Table 4

- 78. Formaldehyde

- DNA interactions (single strand breaks) 13

14

Species/Strain Cell/Strain Result References

DNA single strand breaks (in vitro)

Hamster/Chinese V79 lung epithelial cells

- Speit et al 2007a

Mouse Leukemia L1210 cells (+) Ross and Shipley, 1980

Mouse Leukemia L1210 cells

- Ross et al 1981

Rat Hepatocytes + Demkowicz

- Dobrzanski and

Castonguay 1992

Rat Yoshida lymphosarcoma cells + O'Connor and Fox 1987

Rat/F344 trachea Ep ithelial cell/ Primary culture + Cosma et al., 1988

Human Bronchial cell/Skin fibroblast + Grafstrom et al., 1984

Human Peripheral blood lymphocytes + Liu et al 2006

Human HeLa cells + Liu et al 2006

Human Skin fibroblast + Snyder and Van Houten, 1986

Human Lung/bronchial epithelial cells + Saladino et al 1985

Human Lung/bronchial epithelial cells + Grafstrom et al 1984

Human Lung/bronchial epithelial cells + Grafstrom 1990

Human Lung/bronchial epithelial cells + Fornace et al 1982

Human Lung/bronc hial epithelial cells + Vock et al 1999

Human Skin keratinocytes/fibroblasts

- Emri et al 2004

DNA single strand breaks (in vivo)

Mouse Liver (maternal) + Wang and Liu 2006

Mouse Liver (fetal) + Wang and Liu 2006

Rats/Spraque

- Dawley Lung cells + Sul e t al 2007

Rat Lymphocytes + Im et al 2006

Rat Lymphocytes + Im et al 2006

15

‘no’ indicates test was not done in vivo; ‘+’ indicates a positive test result; ‘yes’ indicates test was done in vivo 16

;

- indicates a negative test result; (+) indicates a wea k positive test result. 17This document is a draft for review purposes only and does not constitute Agency policy.

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- 345 4.3.1.5. Other Genetic Effects of Formaldehyde in Mammalian Cells 1

Formaldehyde induces several other genetic and related effects in mammalian cells which 2

are evaluated by in vitro assays such as unscheduled DNA synthesis (UDS), DNA repair 3

inhibition and cell transformation as summarized in Table 4

- 79. 4

UDS, which represents DNA repair activity has been reported in nasal epithelial cells of 5

F344 rats (Bermudez and Allen 1984; Bermudez and Delahanty 1986), rat hepatocytes (Williams 6

e t al 1989) and Syrian hamster embryo cells (Hamaguchi and Tsutsui 2000). UDS was observed 7

in HeLa cells (Martin et al 1978), but not in human bronchial epithelial cells (Doolittle et al 8

1985) upon formaldehyde exposure. These studies suggest that followin g formaldehyde

- induced 9

DNA damage was followed by DNA repair. 10

Studies involving human bronchial epithelial cells and skin fibroblasts or keratinocytes 11

(Grafstrom et al 1984; Emri et al 2004), DNA repair proficient or

– deficient cell lines (e.g. 12

xeroderma pigmentosum) or cell lines hypersensitive to DNA

- DNA crosslinks (e.g. Fanconi’s 13

anemia) (Speit et al 2000) it has been shown that formaldehyde causes DNA repair inhibition at a 14

concentration range of 0.125 mM to 10 mM). Emri et al (2004) have shown that D NA repair 15

was inhibited in human keratinocytes and fibroblasts after irradiation with UVB and UVC, but 16

not UVA follwed by treatment with low concentrations of formaldehyde (10 **μ** M). They 17

observed that DNA SSB induced by UVB or UVC irradiation alone were repaired within 3

- 6 18

hours of exposure, while cells with UV irradiation followed by formaldehyde exposure still had 19

the strand breaks at the same timepoints suggesting that formald ehyde is likely to contribute to 20

UV

- induced carcinogenesis. 21

22

4.3.2. In Vitro Clastogenicity 23

Clastogenic effects, including increased MNs, CAs, and SCEs are also reported in a range 24

of in vitro study systems as shown in Table 4

- 80. 25

Miyachi and Tsutsui (2005) measured the induction of sister chromatid exchanges 26

(SCEs) in Syrian hamster embryo (SHE) cells. Cells were exposed to 0, 3.3, 10, and 33 **μ** M 27

formaldehyde for 24 hours. SCE levels after 3.3 **μ** M formaldehyde were not different from 28

controls, but sig nificant increases were observed at both 10 and 33 **μ** M. Toxicity as measured by 29

reduced cloning efficiency was seen only at 33 **μ** M (Miyachi and Tsutsui, 2005) . The same 30

laboratory used SHE cells to measure the induc tion of CAs (Hikiba et al., 2005) . Cells were 31

exposed to 0, 33, 66, and 99 **μ** M formaldehyde for 24 hours prior to staining for analysis and the 32

percentages of aberrant metaphases were 0, 6, 6, and 71, respectively. The aberrations were 33

predominantly chromosome gaps and chromosomal breaks and exchanges. The relative colony

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

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- 346 forming efficiency remained high (at least 85%) for the concentrations of formaldehyde used in 1

the experiment (Hikiba et al., 2005)

. 2

3

Table 4

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

5

Species/Strain Cell/Strain Result References

Unschedule DNA synthesis (UDS)

Rat/F344 Nasal epithelial cells + Bermudez and Allen 1984

Rat/F3 44 Nasal epithelial cells + Bermudez and Delehanty 1986

Rat Hepatocytes + Williams et al 1989

Hamster/Syrian Embryo cells + Hamaguchi and Tsutsui 2000

Human HeLa cells + Martin et al 1978

Human Bronchial epithelial cells

- Doolittle et al 1985

D NA repair inhibition

Human Bronchial epithelial cells/skin

fibroblasts + Grafstrom et al 1984

Human Normal fibroblasts (MRC5CV),

XPA cell line, & FA cell line + Speit et al 2000

Human Skin fibroblasts/keratinocytes + Emri et al 2004

6

XPA, xeroderma pig mentosum, complementation group A (deficient in NER pathway) 7

FA, Fanconi's anemia (cell line has genetic defect leading to hypersensitivity to DNA

- DNA cross links; NER, 8

nucleotide excision repair 9

10

11

Schmid and Speit (2007) observed that SCEs were in duced in lymphocytes of whole 12

blood cultures at a formaldehyde concentration of 200 μM, an effect apparently associated with 13

cytotoxicity. This was indicated by a concomitant reduction in the proliferative index. These 14

authors also observed the formation of MNs in their cultures. This effect was statistically 15

significant at a formaldehyde concentration of 300 μM and above. However, MN formation was 16

confined to those cultures in which formaldehyde treatment commenced 44 hours after the start 17

of the cultur e. This prompted the conclusion that the level of DPX formation would need to be 18

high for MN formation and that the cells must be exposed after the first mitosis. In examining 19

MN formation more closely, Schmid and Speit (2007) used the FISH technique, employing a 20

“biotin

- labeled pan

- centromeric chromosome paint specific for all human centromeres.” 21

Indicative that formaldehyde was inducing a clastogenic (rather than aneugenic) effect, 81% of 22

MNs in binucleated cells were centromere

- negative. 23This document is a draft for review purposes only and does not constitute Agency policy.

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- 347 In summary, f ormaldehyde forms MNs, SCEs, and CAs in isolated animal and human 1

cells following in vitro exposure (Table 4

- 80). 2

3

4.3.3. In Vitro Mutagenicity 4

Mutations may occur during repair of formaldehyde

- induced DNA damage (DPXs, DNA 5

adducts, SSBs, or clastogenic effects) or as a result of replication errors during mitogenesis. The 6

in vitro evidence for formaldehyde

- induced mutations is strengthened by examining the 7

correlation between these genotoxic and clastogenic events and induction of mutations. 8

Therefore st udies are presented with respect to relevance to one or more of the following lines of 9

evidence for mutagenicity recommended for consideration in the EPA guidance (U.S. EPA, 10

2005a) : (1) that the chemical is DNA reactive and/or has the ability to bind to DNA, (2) that the 11

chemical generates positive results in in vitro mutagenic test systems (specifically gene 12

mutations and CAs), and (3) that the chemical induces indications of genetic damage in in vivo 13

tes ts (specifically gene mutations and CAs). Numerous studies have demonstrated 14

formaldehyde

- induced DNA mutations under a variety of experimental conditions (reviewed in 15

IARC 1995, 2006; Ma and Harris 1988; Auerbach et al. 1977; Conaway et al 1996; NTP 2009 ). 16

17

4.3.3.1. Mutagenicity in Bacterial Systems 18

A number of research reports describe the mutagenicity of formaldehyde in bacterial test 19

systems using reverse and forward mutation assays as well as specific strains detecting deletions, 20

insertions and po int mutations. Among the bacterial strains, Salmonella typhimurium TA102 and 21

the Escherichia coli strains containing an AT base pair at the primary reversion site are often 22

used to detect oxidative compounds, cross

- linking agents and hydrazines. In an ea rly National 23

Toxicology Program (NTP) collaborative study with three laboratories, formaldehyde 24

consistently tested positive for mutagenicity in Salmonella typhimurium strain TA100 in the 25

presence of a rat or hamster liver S9 activating system (Haworth et al., 1983) . Formaldehyde 26

was mutagenic w ith and without metabolic activation in a number of other studies using in 27

reverse mutation assays with S. typhimurium strains TA98, TA100, TA102, TA104, TA2638, 28

and TA2638a and E. coli strains WP2 (pkM101), WP2 uvr A (pkM101), and hrs/r30R (Ryden et 29

al., 2 000; Dillon et al., 1998; Watanabe et al., 1996; Le Curieux et al., 1993; O’Donovan and 30

Mee, 1993; Zielenska and Guttenplan, 1988; Schmid et al., 1986; Connor et al., 1983, 1985; 31

Orstavik and Hongslo, 1985; Takahashi et al., 1985; Fiddler et al., 1984; Fre i et al., 1984; 32

Donovan et al., 1983), while other studies (Muller et al., 1993; Jung et al., 1992; Wilcox et al., 33

1990; Marnett et al., 1985) show both positive and negative results. These results are 34

summarized in Table 4

- 81 and some of the studies are described in greater detail. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 348 Table 4

- 80. In vitro clastogenicity of formaldehyde

. 1

2

Species Cell/Tissue origin Without

activation With

activation References

Cytogenetic Assays

Chromosomal aberratoins (CA)

Hamster/Chinese Ovary cells (+) (+ ) Galloway et al., 1985

Hamster/Chinese Ovary cells

- ND Dresp and Bauchinger, 1988

Hamster/Chinese Ovary cells + ND Natarajan et al 1983

Mouse Lymphoma cells + ND Speit and Merk 2002

Hamster/Syrian Embryo cells + ND Hikiba et al 2005

Hamster/Syrian E mbryo cells + ND Hagiwara et al 2006

Hamster/Chinese Ovary cells + ND Garcia et al 2009

Hamster/Chinese Lung fibroblasts + ND Ishidate Jr et al 1981

Human Lymphocytes + + Schmid et al 1986

Human Lymphocytes + ND Miretskaya and Shvartsman 1982

Human Ly mphocytes + ND Dresp and Bauchinger, 1988

Human Fibroblasts + ND Levy et al 1983

Micronucleus (MN)

Hamster/Chinese V79 lung epthelial cells + ND Speit et al 2007b

Hamster/Chinese V79 lung epthelial cells + ND Merk and Speit 1998

Human Whole blood cult ures + ND Schmid and Speit 2007

Human Human MRC5CV (normal)

and XP(Repair

- deficient) and

FA (repair

- deficient) cell lines + a ND Speit et al 2000

Sister Chromatid Exchange (SCE)

Hamster/Chinese Ovary cells (+) (+) Galloway et al., 1985

Hamster/Chinese O vary cells + ND Natarajan et al 1983

Hamster/Chinese Ovary cells + ND Garcia et al 2009

Hamster/Chinese Ovary cells + ND Obe and Beek 1979

Hamster/Syrian Embryo cells + ND Miyachi and Tsutsui 2005

Hamster/Chinese V79 lung epithelial cells + (+) Basler et al. 1985

Hamster/Chinese V79 lung epithelial cells + ND Speit et al 2007b

Hamster/Chinese V79 lung epithelial cells + ND Merk and Speit 1998, 1999

Hamster/Chinese V79 lung epithelial cells + ND Neuss and Speit 2008

Human A549 lung epithelial cells + ND Neuss and Speit 2008

Human A549 + V79 (co

- cultivated) + c ND Neuss and Speit 2008

Human A549 + V79 (co

- cultivated)

- d ND Neuss and Speit 2008

Human Lymphocytes + b ND Garry et al., 1981

Human Lymphocytes + ND Krieger and Garry 1983

Human Lymphocyt es + ND Schmid et al 1986

Human Lymphocytes + ND Obe and Beek 1979

Human Whole blood cultures + ND Schmid and Speit 2007This document is a draft for review purposes only and does not constitute Agency policy.

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- 349 Premature chromosome Condensation (PCC)

Hamster/Chinese Ovary cells + ND Dresp and Bauchinger, 1988

1

‘+’ indicates a posi tive test result 2

‘ND’ indicates test was not done 3

- indicates a negative test result 4

(+) indicates a weak positive test result 5

a MN frequency increased in repair

- deficient cell lines compared to normal cell lines 6

b indicates SCE with significant loss of cell viability 7

c A549 cells exposed for 1 h with formaldehyde then co

- cultivated with V79 cells 8

d A549 cells exposed for 1 h with formaldehyde, cells washed and then co

- cultivated with V79 cells 9

XP, xeroderma pigmentosum; FA = Fanconi's ane mia. 10

11

12This document is a draft for review purposes only and does not constitute Agency policy.

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- 350 Table 4

- 81. Summary of mutagenicity of formaldehyde in bacterial systems 1

2

Species Strain Metabolic

activation References

+S9

- S9

Mutagenicity Assays

Reverse Mutation

S. typhimurium TA98, 100, 1535, 1537, 1538

- - De Flora, 1981

S. typh imurium TA100 ND (+) Couch et al., 1982

S. typhimurium TA100 +

- Haworth et al., 1983

S. typhimurium TA1535, 1537

- - Haworth et al., 1983

S. typhimurium TA98 (+)

- Haworth et al., 1983

S. typhimurium TA98, TA100 + + Connor et al., 1983*

S. typhimuriu m UTH8414, UTH8413

- - Connor et al., 1983*

S. typhimurium TA97, 98, 100 + + Donovan et al., 1983

S. typhimurium TA102 + + De Flora et al., 1984

S. typhimurium TA100 + ND Frei et al., 1984

S. typhimurium TA100 ND + Fiddler et al., 1984

S. typhimurium TA100 + (+) Connor et al., 1985

S. typhimurium TA98 (+)

- Connor et al., 1985

S. typhimurium UTH8414, UTH8413

- - Connor et al., 1985

S. typhimurium TA100 (+)

- Ashby et al., 1985**

S. typhimurium TA97, 98, 1535, 1537, 1538

- - Ashby et al., 1985**

S. typhimurium TA98, 100, 102 ND (+) Takahashi et al., 1985

E. coli WP2, WP2 uvrA ND + Takahashi et al., 1985

E. coli H/R30R, HS30RuvrA ND + Takahashi et al., 1985

E. coli NG30 recA, 016polA ND

- Takahashi et al., 1985

S. typhimurium TA97, 98, 100 ND

- Ma rnett et al., 1985

S. typhimurium TA102, 104 ND + Marnett et al., 1985

S. typhimurium TA98, 100 + + Oerstavik and Hongslo, 1985

S. typhimurium TA100 + + Schmid et al., 1986

S. typhimurium TA104 + ND Zielenska and Guttenplan, 1988

S. typhimurium TA102 ND

- Wilcox et al., 1990

E. coli WP2 uvrA /(pKM101) ND + Wilcox et al., 1990

E. coli WP2 (pKM101) ND

- Wilcox et al., 1990

S. typhimurium TA102 + ND Jung et al., 1992

S. typhimurium TA102 ND + Le Curieux et al., 1993

S. typhimurium TA102 + ND Muller e t al., 1993

S. typhimurium TA98, 100, 102 ND + O’Donovan and Mee, 1993

S. typhimurium TA1535, 1537, 1538 ND

- O’Donovan and Mee, 1993

E. coli WP2 (pKM101), ND + O’Donovan and Mee, 1993

E. coli WP2uvrA (pKM101) O’Donovan and Mee, 1993

E. coli K12 (AB 1157)(WT) ND + Graves et al., 1994

E. coli K12 (AB1886)/(uvrA),

K12(AB2480)/(recA/uvrA) ND

- Graves et al., 1994

S. typhimurium TA102, 2638 ND + Watanabe et al., 1996

E. coli WP2 (pKM101), WP2uvrA

(pKM101) ND + Watanabe et al., 1996This document is a draft for review purposes only and does not constitute Agency policy.

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- 351 S. typhimurium TA15 35 A

- - Sarrif et al., 1997

S. typhimurium TA1537 A + + Sarrif et al., 1997

S. typhimurium TA98, 100 A +

- Sarrif et al., 1997

S. typhimurium TA97 A ND + Sarrif et al., 1997

S. typhimurium TA1535, 1537 B

- - Sarrif et al., 1997

S. typhimurium TA98 B + + Sa rrif et al., 1997

S. typhimurium TA100 B

- + Sarrif et al., 1997

S. typhimurium TA100 C + + Sarrif et al., 1997

S. typhimurium TA100, 104 B + + Dillon et al., 1998

E. coli (Lac+ reversion) WP3101P, WP3106P + Ohta et al 1999

S. typhimurium TA102, 2638 B N D + Ryden et al., 2000

Forward Mutation

S. typhimurium TM677 ND (+) Couch et al., 1982

S. typhimurium TM677 + + Donovan et al., 1983

S. typhimurium TM677 + + Temcharoen and Thilly, 1983

E. coli D494 uvrB + Bosworth et al 1987

Deletion Mut ation

E.coli GP120, GP120A 7

- 2, 33694 ND + D Crosby et al., 1988

Point Mutation

E.coli GP120, GP120A 7

- 2, 33694 ND + Crosby et al., 1988

Insertion Mutation

E.coli GP120, GP120A 7

- 2, 33694 ND + Crosby et al., 1988

1

‘+’ indicates a positive test result 2

‘ND’ indicates test was not done 3

‘

- ’ indicates a negative test result 4

(+) indicates a weak positive test result 5

* indicates the use of formalin in mutagenicity assay 6

** indicates the use of hexamethylmelamine (HEMLA), a f ormaldehyde

- releasing compound, in mutagenicity assay 7

A indicates use of the Standard Plate Method 8

B indicates use of the Preincubation Plate Method 9

C indicates use of the Suspension Method 10

D indicates loss of DNA 11

12

13

Formaldehyde has been s hown to be mutagenic in forward mutation assays using S. 14

typhimurium (Couch et al 1982; Donovan et al 1983; Temcharoen and Thilly 1983) as well as in 15

E. coli (Bosworth et al 1987). Temcharoen and Thilly (1983) examined the toxicity and 16

mutagenicity of S. typhimurium strain TM677, using forward mutation to 8

- azaguanine 17

resistance, and have shown that formaldehyde induced both toxicity and mutagenicity at 18

minimum concentrations of 0.17 mM (

– S9) and 0.33 mM (+S9). It has also been shown that 19This document is a draft for review purposes only and does not constitute Agency policy.

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- 352 formaldehyde f ormed as an intermediate by oxidation at the methyl group of 1

N

- nitrodimethylamine, a biologically active N

- nitramine of environmental significance, is 2

mutagenic to S. typhimurium TA100 strain at low concentrations and toxic above 2 **μ** mol/plate 3

(Frei et al., 1984)

. 4

Bosworth et al (1987) developed a forward mutation assay in E. coli D494 uvr B strain 5

transformed with a multi

- copy mutator plasmid pGW1700, in which mutations are scored by an 6

increase in ampicillin

- resistant colonies after exposure of bacterial cells during the logarithmic 7

growth by the test chemicals. This assay is more sensitive to base

- pair substitutions, but less 8

sensitive to frameshift mutations compared to Salmonella/miceosome

- based assays. In this assay, 9

the authors (Bosworth et al 1987) observed positive curvilinear response to formaldehyde 10

exposure. Crosby et al (1988) used four E. coli strains GP120, GP120A, 7

- 2, and 33694 11

containing the xanthine guanine phosphoribosyl transferase ( gpt ) gene (which detects point 12

mutations, deletions and insertions) tested the mutagenicity of formaldehyde by exposing for 1 13

hour at 4 and 40 mM concentrations. They observed 41% large insertions, 18% large deletions 14

and 41% point mutations. However, at 40 mM dose there were 92% point mutations, a majority 15

of them (62%) being transition mutations at a single AT base pair in the gpt gene. In the same 16

study they observed frameshift mutations in E. coli that was transformed with naked pSV2gpt 17

plamid DNA exposed to 3.3 or 10 mM formaldehyde. Thus, the mutation pattern appear to differ 18

depending on the concentration of formaldehyde exposure to the bacterial strain as well as the 19

nature of DNA. 20

Formaldehyde has also been shown to induce primary DNA damage in E. coli and 21

mutagenic activity in the Ames fl uctuation test in S. typhimurium TA100, TA102, or TA98 22

strains (Le Curieux et al., 1993)

. 23

O’D onovan and Mee (1993) observed clear mutagenicity by the pre

- incubation exposure 24

method in S. typhimurium TA98, TA100, and TA102 strains and both E. coli WP2(pKM101) and 25

WP2uvrA(pKM101) strains, while the standard plate

- incorporation assays showed consiste nt 26

mutagenicity only with TA100 and WP2 uvr A(pKM101) strains and no evidence of mutagenicity 27

in TA1535, TA1537, or TA1538 strains using either method of exposure in the absence of 28

metabolic activation. The S. typhimurium and E. coli strains used in this st udy are histidine and 29

tryptophan auxotrophs, with an AT base pair at the critical mutation site within the his G and trp E 30

genes, respectively, with an intact excision repair system facilitating the detection of cross

31

linking agents and both strains carrying the mutator plasmid, pKM101, which enhances error

32

prone repair. These salmonella strains detect frameshift (TA98 and TA1537) and base

- pair 33

substitutions (TA100, TA102, and TA1535), while the E. coli strains detect base

- pair 34

substitutions (WP2 uvr A). Thes e findings are consistent with the suggestion that formaldehyde 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 353 induces excision

- repairable lesions in bacteria and indicate that the presence of the R

- factor 1

plasmid may be required for the expression of its mutagenicity in excision repair

- deficient 2

salmo nella (O’Donovan and Mee, 1993)

. 3

Dillon et al. (1998) employed salmonella strains TA 100, TA102, and TA104 because of 4

the latter two strains being more sensitive to oxidative mutagens. Formaldehyde was clearly 5

mutagenic between 6 and 50 **μ** g/plate in all three strains with and without metabolic activation 6

using Aroclor

- induced S9 from male F344 rats or male B6C3F1 mice, except for an equivocal 7

response in TA102 with mouse S9 (Dillon et al ., 1998) . Using a set of six tester strains 8

(WP3101

– WP3106) of E. coli , each reversible by a mutation involving a single DNA base pair 9

substitution, Ohta et al. (1999) determined that formaldehyde preferentially induced GC to TA 10

transversion mutations. Ryden et al. (2000) demonstrated a statistically significant increase in 11

the number of revertants in S. typhimurium TA102 (2.5

- fold) and TA2638a (3

- fold) strains by 12

formaldehyde at ≥17 **μ** g/plate compared with solvent controls. 13

In summary, formaldehyde indu ces mutations in several bacterial strains containing an 14

AT base pair at the primary reversion site that are used to detect oxidative compounds and cross

15

linking agents without metabolic activation by exogenous enzyme

- activating systems. This 16

evidence is strengthened by examining the correlation between genotoxic and clastogenic events 17

and mutation induction. 18

19

4.3.3.2. Mutagenicity in Non

- Mammalian Cell Systems 20

Formaldehyde has been shown to be mutagenic in several non

- mammalian systems also. 21

It has been shown to cause gene conversion, strand breaks, crosslinks, homozygosis and related 22

damage in yeasts ( Saccharomyces cervisiae ), forward and reverse mutations in molds 23

( Neurospora crassa ), micronuclei formation in spiderworts ( Tradescantia pallida ), DNA 24

dama ge and mutations in several plants, genetic cross

- over or recombination, sex

- linked 25

recessive lethal mutations, dominant lethal mutations, heritable translocations and gene 26

mutations in insects ( Drosophila melanogaster ) and recessive lethal mutations in ne matodes 27

( Caenorhabditis elegans ), but failed to show micronuclei formation in newt larvae ( Pleurodeles 28

waltl ) (Reviewed in Conaway 1996; IARC 2006). 29

30

4.3.3.3. Mutagenicity in Mammalian Cell Systems 31

Several studies demonstrated the mutagenicity of formald ehyde in mammalian cells. In 32

its report, the Federal Panel on Formaldehyde underlined the role of formaldehyde as an inducer 33

of gene mutations and CA in a variety of test systems (Report of the Federal Panel on 34

Formaldehyde, 1982) . Results from several studies are summarized in Table 4

- 82. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 354 Snyder and Van Houten (1986) demonstrated that formaldehyde increases the levels of 1

misincorporation of bases into synthetic polyn ucleotides catalyzed by E. coli DNA polymerase I, 2

indicating that the mutagenicity of formaldehyde may be due to covalent alteration of DNA 3

bases. They have also shown that formaldehyde

- induced DNA damage in human fibroblasts was 4

not susceptible to repair by the typical “long patch” excision repair mechanism. 5

Craft et al. (1987) measured the induction of mutations at the thymidine kinase ( tk ) locus 6

or at the ouabain resistance ( Oua r ) locus in TK6 human lymphoblastoid cells. The tk mutations 7

can result fro m a variety of mutational events, including base pair substitution, small and large 8

deletions, and chromosome exchange events, while mutations to Oua r require specific base pair 9

substitutions. Single treatment of formaldehyde (0, 15, 30, 50, 125, and 150 μM) for 2 hours 10

resulted in a nonlinear increase in tk mutagenesis with increasing slope >125 μM (Figure 4

- 32). 11

To explore a dose

- response effect, cells were also exposed as follows: three treatments of 50 μM 12

for 2 hours or five treatments of 30 μM or 10 treatments of 15 μM for 2 hours (treatments were 13

spaced 2

– 4 days apart) with multiple treatments causing an increase in tk mutations, although 14

their combined effect was less than a single treatment of equivalent C × t (150 μM for 2 hours). 15

Lymphoblasts gi ven four treatments of 150 μM formaldehyde for 2 hours failed to induce 16

mutations at the Oua r locus. Dose

- response increases were seen in all exposure scenarios, with 17

30 μM being the level of statistical significance. There was little indication of a dos e

- response 18

effect until the cumulative concentration was greater than 100 μM. Formaldehyde

- induced 19

DPXs were no longer evident after 24 hours of exposure; mutants induced in the TK6 20

lymphoblast cell line showed a similar dose

- response curve to the DPXs me asured immediately 21

after exposure ended (Craft et al., 1987)

. 22

The same group also studied mutations induced at the X

- linked hypoxanthine

- guanine 23

phosphoribosyl transferase (HPRT) locus by eight repetitive treatments of 150 **μ** M formaldehyde 24

in TK6 human ly mphoblast cell line by Southern blot analysis, wherein half (14/30) of induced 25

mutants contained partial or complete deletions with most of the partial deletions showing 26

unique deletion patterns, while only a third (5/15) of spontaneous mutants had partial or 27

complete deletions, indicating that formaldehyde can induce large losses of DNA in human 28

lymphoblast cells (Crosby et al., 1988)

. 29

30This document is a draft for review purposes only and does not constitute Agency policy.

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- 355 Table 4

- 82. Mutagenicity in mammalian cell systems. 1

2

Species/Strain Cell/Strain In Vivo

test Without

activation References

Mutagenicity Assays

Dominant Let hal Mutation

Rat/Albino Spermatocyte, Live implants yes + ND Odeigah, 1997

Mouse Dominant lethal yes

- Epstein and Shafner 1968

Mouse Dominant lethal yes

- Epstein et al 1972

Mouse Dominant lethal yes (+) Fontignie

- Houbrechts 1981

Rat Dominant lethal yes (+) Kitaeva et al 1990

Deletion Mutation

Hamster/Chinese V79 cells Hprt locus) no

- Merk and Speit 1998

Hamster/Chinese V79 cells Hprt locus) no

- Merk and Speit 1999

Hamster/Chinese V79/HPRT no + ND Grafstrom et al., 1993

Ha mster/Chinese Ovary HPRT no

- + Graves et al., 1996

Mouse Lymphoma L5178Y cells (Tk +/

-

locus) no + Macerer et al 1996

Mouse Lymphoma L5178Y cells no + ND Speit and Merk 2002

Human Bronchial cell no + Grafstrom et al., 1983

Human Bronchial fibroblast s/epithelial cells

(HPRT locus) no + Grafstrom et al 1985

Human Bronchial fibroblasts/epithelial cells

(HPRT locus) no + Grafstrom 1990

Human Lymphoblast/HPRT no + a ND Crosby et al., 1988

Human Lymphoblast/tk no + Craft et al 1987

Human Peripheral l ymphocytes yes + ND Shaham et al 2003

Human Lymphoblast (TK6) no + Goldmacher and Thilly 1983

Hamster/Chinese Point Mutation

Ovary HPRT no + ND Graves et al., 1996

Mouse Lymphoma cell/ TK+/

- no + + Blackburn et al., 1991

Mouse Lymphoma cell/ T K+/

- no + ND Wangeheim and Bolcsfoldi, 1988

Human Lymphoblast/TK6 no + ND Liber et al., 1989

Insertion Mutation

Hamster/Chinese Ovary HPRT no + ND Graves et al., 1996

Heritable Mutation

Mouse Heritable mutation yes + Liu et al 2009

DNA Repair enzyme activity

Human Peripheral lymphocyte yes

- Hayes et al., 1997This document is a draft for review purposes only and does not constitute Agency policy.

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- 356 Cell Transformation

Mouse C3H10T1/2 cells + b Ragan and Boreiko 1981

Mouse Embryo fibroblast/C3H/10T1/2 no [+] ND Boreiko et al., 1983

Mouse Embryo fibroblast/C3H/10T1/2 no [+] ND Frazelle et al., 1983

Hamster Kidney cell/BHK

- 21/cI.13 no + + Plesner and Hansen, 1983

p53 mutation and/or p53 protein expression

Rats/F344 Nasal squamous cell carcinomas yes + c Recio et al 1992

Rats/ F344 Nasal tumor cell lines No + Bermudez et al 1994

Rats/F344 Nasal squamous cell carcinomas Yes + d Wolf et al 1995

Human Peripheral blood lymphocytes yes + Shaham et al 2003

1

‘no’ indicates test was not done in vivo 2

‘+’ indicates a positive test result 3

‘ND’ indicates test was not done 4

‘yes’ indicates test was done in vivo 5

- indicates a negative test result 6

(+) indicates a weak positive test result 7

[+] indicates positive test result after TPA or N

- methyl

- N

- nitro

- N

- n itrosoguanidine promoter treatment 8

a indicates loss of DNA 9

b Positive only in the presence of 12

- O

- tetradecanoylphorbol 13

- acetate (TPA) 10

c p53 mutations 11

d p53 mutated protein detected by immunohistochemistry 12

13This document is a draft for review purposes only and does not constitute Agency policy.

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- 357 1

2

Figure 4

- 32. DNA

- prot ein cross

- links (DPX) and thymidine kinase ( tk ) 3

mutants in TK6 human lymphoblasts exposed to formaldehyde for 2 hours. 4

5

Note:  DPXs immediately after exposure,  DPXs 24 hours after exposure,  tk 6

mutants. Relative survival was 100% at 0, 15, 30, and 50 μM, 30% at 125 μM, 7

and 20% at 150 μM. 8

9

Source: Adapted from Craft et al. (1987). 10

11

12

Liber et al. (1989) followed up the findings of Crosby et al. (1988) by performing 13

Southern blot, Northern blot, and DNA sequence analysis on the 16 in duced and 10 spontaneous 14

human lymphoblast mutants not showing deletions. Northern blot analysis showed that the point 15

mutations fell into four categories: normal size and amount of RNA, normal size but reduced 16

amounts of RNA, reduced size and amounts of RNA, and no RNA. Sequence analysis of 17

recombinant DNAs from hprt mRNA in formaldehyde

- induced mutants showed a preferential 18

AT to CG transversion at a specific site, with other changes represented to a lesser degree (Liber 19

et al., 1989)

. 20

Even in CHO cells formaldehyde has been shown to induce hprt mutatio ns involving 21

mostly single

- base pair transversions mostly occurring at AT sequences, including three AT to 22

TA at position 548 of exon 8 and two AT to CG and one GC to TA transversion at other sites 23This document is a draft for review purposes only and does not constitute Agency policy.

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- 358 (Graves et al., 1996) . In another study, formaldehyde

- induced forward mutations to 1

trifluorothymidine resistance in mouse lymphoma L5178Y tk ± cells both in the absence and 2

presence of rat liver S9 (higher concentrations required for effect with S9). Both toxicity and 3

mutagenicity were abolished when FADH was incorporated in the exposure medium (Blackburn 4

et al., 1991)

. 5

Formaldehyde

- induced DPXs are removed in part through spontaneous hydrolysis and in 6

part due to active repair processes (Quievryn and Zhitkovich, 2000) . Inhibition of specific 7

proteosomes in XP

- A cells inhibited DPX repair, thereby supporting the role of enzymatic 8

degradation (Quievryn and Zhitkovich, 2000) . The half

- life of formaldehyde

- induced DPXs in 9

human cell lines was consistent with the findings of Craft et al. (19 87) , ranging from 11.6 to 10

13 hours (Quievryn and Zhitkovich, 2000) . In the same report, removal of DPXs from human 11

peripheral lymphocytes was much slower, with a half

- life of 18.1 hours. This difference was 12

prima rily in slower active repair of DPXs, with a t 1/2 of 66.6 hours for human lymphocytes 13

versus 23.3 hours for human cell lines (Quievryn and Zhitkovich, 2000)

. 14

Since DPX repair involves proteolytic removal of protei ns from the DNA, Speit et al. 15

(2000) hypothesized that single peptides or small peptide chains cross

- linked to the DNA are 16

critical to formaldehyde

- induced mutation. However, these authors did not find significant 17

difference in the induction and repair of DPXs in normal and DNA repair

- deficient cell lines but 18

observed increased susceptibility of the repair

- deficient cell lines to formaldehyde

- induced MN 19

induction. In this study, a normal human cell line (MRC5CV1), a xeroderma pigmentosum cell 20

line deficie nt in nucleotide excision repair (NER), and a Fanconi anemia cell line, which has a 21

genetic defect leading to hypersensitivity towards DDXs, were exposed to 125, 250, and 500 **μ** M 22

formaldehyde for 2 hours. The authors suggest that more than one repair pathway is involved in 23

the repair of cross

- links and that the altered NER pathway has more severe consequences to 24

formation of CAs than disturbed cross

- link repair (Speit et al., 2000)

. 25

The correlation of early DPX formation and mutation is at first counterintuitive since the 26

cross

- linking of protein to DNA inhibits DNA rep lication. Without active DNA replication, 27

formaldehyde

- DNA adducts and DPXs would not induce replication error and would be unlikely 28

to result in a change in DNA sequence or mutation. Recent evidence indicates that residual 29

peptides and short polypeptide s that remain cross

- linked to DNA after DPX removal may in fact 30

be the cause of DPX

- associated, formaldehyde

- induced mutation (Speit et al., 2000)

. 31

A study by Merk and Speit (1998) indicated that formaldehyde

- induced DPXs did not 32

result in direct gene mutations in the hprt locus of V79 Chinese hamster cells, leading the 33

authors to speculate that formaldehyde was not mutagenic. Since, the hprt locu s in the V79 34

Chinese hamster cell line is primarily sensitive to point mutations and other studies show the 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 359 formation of deletion mutations by formaldehyde at the same locus in human lymphoblasts 1

(Crosby et al., 1988)

, Merk and Speit (1998) concluded that the hprt mutation assay is insensitive 2

to deletion mutations. 3

Later, using the mouse lymphoma assay, Speit and Merk (2002) demonstrated that 4

exposure to formaldehyde for 2 hours was mutagenic in a concentration

- dependent manner in the 5

L5178Y mouse lymphoma cells, which was mainly contributed by a strong increase in small 6

colony mutants, suggestive of CAs (Speit and Merk, 2002) . Detailed analysis of both 7

spontaneous and formaldehyde

- induced lesions indicates that recombination or deletion of DNA 8

from the tk locus was primarily responsible for the loss of heterogeneity, thereby leading to the 9

observ ed mutant phenotype. Therefore, it is believed that formaldehyde is mutagenic in the 10

L5178Y cell mouse lymphoma system by a clastogenic mechanism rather than through point 11

mutations. This finding is consistent with that of Craft et al. (1987) , who demon strated 12

formaldehyde mutagenicity at the tk locus of TK6 cells, and also with the findings of Grafström 13

et al. (1984), who demonstrated increased SSB formation in formaldehyde

- exposed cell lines. 14

Formaldehyde has also been shown to induce cell transformat ion in mouse embryo 15

fibroblasts (Ragan and Boreiko 1981; Boreiko et al 1983; Frazelle et al 1983) . At low 16

concentrations of 0.017 mM formaldehyde has shown to cause cell transformation in C3H10T1/2 17

mouse cells (Ragan and Boreiko 1981) and hamster kidney cells in vitro (Plenser and Hansen 18

1983). 19

More recently, Shaham et al. (2003) examined the frequency of DPXs and the incidence 20

of mutant versus wild type p53 tumor suppressor genes in the peripheral blood lymphocytes of a 21

cohort of workers exposed to forma ldehyde. The adjusted mean levels of DPXs were greater in 22

the lymphocytes of exposed subjects compared with those of unexposed subjects, and exposure 23

to formaldehyde increased the likelihood of their having a higher level of pantropic p53 24

(>150 pg/mL). T he authors speculated on a possible causal relationship between DPXs and 25

mutations in p53. Recio et al (1992) demonstrated point mutations in the p53 tumor suppressor 26

gene in 45% (5 out of 11) of the primary nasal squamous cell carcinomas (SCCs) obtained from 27

F344rats that were chronically exposed to 15 ppm formaldehyde for 2 years (Recio et al ., 1992)

. 28

In summary, the results of in vitro experiments demonstrate the mutagenicity of 29

formaldehyde. Mutagenicity is observed below level s of significant cytolethality in mammalian 30

cell lines. Formaldehyde is clearly a DNA

- reactive genotoxicant inducing lesions (DPXs) that 31

show clastogenicity (SSBs, MNs, etc.). The experiments by Speit and Merk (2002) explore 32

mechanistic links between DPX s, clastogenicity, and the observed locus

- specific mutations in 33

the mouse lymphoma in vitro testing system. 34

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

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- 360 4.3.4. In Vivo Mammalian Genotoxicity 1

4.3.4.1. Genotoxicity in Laboratory Animals 2

As discussed above, formaldehyde is clearly reactive at the POE in animal studies, 3

resulting in increased DPXs in the nasal mucosa. Despite formaldehyde’s reactivity and 4

mutagenicity in isolated mammalian cells, clear evidence of mutagenicity does not emerge from 5

animal bioassays (Table 4

- 83). 6

In a chromosomal analy sis study (Fontignie

- Houbrechts, 1981) , formaldehyde given I.P. 7

at 50 mg/kg to male Q strain mice and analyzed 8

– 15 days after treatment did not induce any 8

chromosomal lesions in spermatocytes. Also, in another study from the same group (Fontignie

9

Houbrechts et al., 1982) , formaldehyde (30 mg/kg) given along with hydrogen peroxide (90 10

mg/kg) as a mixture to male Q st rain mice failed to produce significant increases in 11

chromosomal lesions in the spermatogonia. 12

In a different study Natarajan et al. (1983) failed to detect significant differences in MN 13

induction in bone

- marrow cells or CAs in spleen cells of male and f emale CBA mice given I.P. 14

6.25, 12.5, and 25 mg/kg formaldehyde compared with saline

- treated controls. However, the 15

same study showed a positive induction of MNs and CAs in vitro. The authors suggest that the 16

lack of genotoxicity in vivo may be due to th e inability of formaldehyde to reach the target cells 17

in sufficient quantity to induce biological effects. 18

Kligerman et al. (1984) also found no difference in the incidence of SCEs or 19

chromosome breakage in the peripheral lymphocytes of male and female F3 44 rats exposed to 20

formaldehyde in air at 0.5, 6, or 15 ppm (0.61, 7.36, or 18.4 mg/m 3 ) 6 hours/day for 5 days. 21

However, in a different study (Migliore et al., 1989) , clastogenic effects, such as increased MNs 22

and CAs, were reported in GI epithelial cells of male Sprague

- Dawley rats after oral exposures to 23

200 mg/kg formaldehyde. In this study, micronucleated cells and nuclear anomalies were 24

increased in a time

- dependent manner in the stomach, duodenum, ileum, and colon of rats, and 25

the mitotic index was unchanged for these cells compared with controls at 16, 24, and 30 hours. 26

These clastogenic effects were seen without regenerative cell proliferation, supporting 27

formaldehyde

- induced mutations as primary effects of formaldehyde rather than secondary to 28

regenerative cell proliferation. 29

Kitaeva et al. (1990) obs erved cytopathological and cytogenetic effects of formaldehyde 30

chronic inhalation in 0.5 and 1.5 mg/m 3 doses in the female rat’s germ and marrow cells, where 31

formaldehyde

- induced harmful effects were seen in germ cells at <1.5 mg/m 3 doses, while the 32

reliab le clastogenic and cytogenetic effects on the marrow cells were induced even at the 33

0.5 mg/m 3 dose, suggesting differences among effects of small doses of formaldehyde on 34

different cell systems. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 361 Table 4

- 83. Genotoxicity in laboratory animals. 1

2

Species/Str ain Cells/Organ/Tumor Result References

Cytogenetic Assays

Mice/Q strain Chromosomal aberratoins (CA)

Spermatocyte

- Fontignie

- Houbrechts et al., 1981

Mice/Q strain Spermatogonia

- Fontignie

- Houbrechts et al., 1982

Mice/CBA Polychromatic erythrocyte s

- Natarajan et al., 1983

Mice/CBA Spleen cells

- Natarajan et al., 1983

Rats/F344 Lymphocytes

- Kligerman et al 1984

Rats/Spraque

- Dawley Gastric epithelial cells + Migliore et al 1989

Rats/Wistar Bone marrow + Kitaeva et al 1990

Rats/Spraque

- Dawley Bone marrow

- Dallas et al 1992

Rats/Spraque

- Dawley Pulmonary lavage cells + Dallas et al 1992

Rats/F344 Peripheral blood cells

- Speit et al 2009

Micronucleus (MN)

Mouse/NMRI Bone marrow

- Gocke et al 1981

Mice/CBA Femoral polychromatic

erythroc yte and spleen cell - Natarajan et al., 1983

Rats/Spraque

- Dawley Gastric epithelial cells + Migliore et al 1989

Sister Chromatid Exchange (SCE)

Rats/F344 Lymphocyte

- Kligerman et al 1984

Rats/F344 Peripheral blood cells

- Speit et al 2009

3

‘+’ indicates a positive test result 4

- indicates a negative test result 5

6

7

Dallas et al. (1992) observed a slight increase (7.6 and 9.2%) in CAs in pulmonary lavage 8

cells from male Sprague

- Dawley rats exposed to 15 ppm (18.4 mg/m 3 ) formaldehyde in air 9

6 ho urs/day, 5 days/week for 1 or 8 weeks by inhalation compared with corresponding controls 10

(3.5 and 4.8%), respectively. However, the small study, limited as it was to five animals/group, 11

showed statistically significant increase at the highest dose tested (15 ppm) but not at lower 12

doses (0.5 and 3 ppm). In the same study, no clastogenic effects were seen in bone marrow, 13

which is consistent with formaldehyde acting primarily at the site of first contact. 14

Speit et al (2009) investigated the genotoxicity of f ormaldehyde in peripheral blood 15

samples of Fischer

- 344 rats exposed to 0 to 15 ppm formaldehyde by whole

- body inhalation for 16

4 weeks (6 h/day, 5 days/week). In this study, the authors found no significant increase in the 17

genotoxic assays such as comet assa y with or without gamma

- irradiation of blood samples (DNA 18

migration as determined by tail movement or intensity), sister chromatid exchange (SCE) assay 19This document is a draft for review purposes only and does not constitute Agency policy.

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- 362 and micronucleus test (MNT) compared to controls. However, rats given 50 mg/kg 1

methylmethane sulfonate (MMS) by gavage for 4 hrs (positive control for Comet and SCE 2

assays) or 10 mg/kg cyclophosphamide (CP) given twice orally (positive control for MNT) 3

induced significant increase in genotoxicity in this study. The lack of genotoxicity in this study 4

is no t surprising since earlier studies by Casanova

- Schimitz et al (1984a) have shown that 5

formaldehyde does not cause toxicity to bone marrow possibly due to the inability of this 6

chemical to reach the bone marrow. Although MMS and CP used in this study were positive in 7

the genotoxicity assays, the data from positive controls can not be used for validation since the 8

exposure routes of formaldehyde (inhalation) and the positive controls (oral) were different. 9

No animal studies have examined clastogenic effects of formaldehyde in nasal or 10

respiratory epithelial cells. Therefore, it is unknown whether similar changes would occur in 11

response to exposure to formaldehyde via inhalation. However, the negative finding in bone 12

marrow cannot be considered definitive ev idence on the question of the mutagenic potential of 13

formaldehyde for cells present at the POE. With weak positive results in pulmonary lavage cells 14

and clear clastogenicity in GI epithelial cells below exposures that trigger regenerative cell 15

proliferati on, the existing evidence, however incomplete, supports the concept of genotoxic 16

action of formaldehyde at the POE. 17

18

4.3.4.2

. Genotoxicity in Humans 19

The majority of the studies on the effects of formaldehyde in exposed humans have 20

measured various cytogen etic endpoints, such as MNs, SCEs, or CAs in nasal and oral mucosal 21

cells (considered to be in direct contact with formaldehyde) as well as peripheral lymphocytes. 22

Since genotoxicity at the proximal sites (oral, nasal) can be readily linked to the reactiv e nature 23

of formaldehyde, these studies are discussed first, noting where researchers also collected blood 24

lymphocyte samples. A subsequent discussion is focused on results in blood lymphocytes. 25

Finally, the few studies that measured DPXs in exposed human s are discussed. Table 4

- 89 26

provides a summary of human cytogenetic studies of formaldehyde. 27

28

4.3.4.2.1. Nasal, buccal, and oral mucosal cells. Epithelial cells of the URT and oral cavity are 29

potential targets of formaldehyde’s DNA reactivity and genoto xicity. Several studies indicate 30

that formaldehyde exposure results in measurable increases in SCEs, MN formation, and DPXs 31

in nasal, buccal, and oral mucosal cells; however, these genotoxic effects vary with the type of 32

exposure. Study quality, sample s ize, availability of exposure measurements, and assay 33

methodology may in part contribute to variability in study findings. The studies fall into three 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 363 general categories: workers (industrial or professional), students and staff attending anatomy and 1

mortu ary science courses, and subjects in a controlled clinical trial. 2

Ballarin et al. (1992) observed significantly higher frequency of micronucleated cells in a 3

formaldehyde exposed group in a plywood factory compared with controls (0.9 ± 0.47 versus 4

0.25 ± 0 .22, p < 0.01). In this study, the frequency of MNs and cytology of respiratory nasal 5

mucosal cells was examined in 15 nonsmokers exposed to levels of formaldehyde that ranged 6

between 0.1 and 0.39 mg/m 3 (~0.32 ppm) for an average of 6.8 years. Exposed su bjects were 7

compared with age

- and sex

- matched controls. 8

Ye et al. (2005) reported significant increases in MNs per thousand cells in nasal mucosal 9

cells for 18 nonsmoking workers (2.70 **±** 1.50) in a formaldehyde manufacturing plant in the 10

Hugei province of China as compared with controls (1.25 **±** 0.41). In addition, higher 11

frequencies of SCEs in peripheral lymphocytes of workers were also reported (8.24 **±** 0.89 versus 12

6.38 **±** 0.41). In this study, the average age of workers was 29 **±** 6.8 years, the average duration 13

at work was 8.5 years (range 1

– 15 years), and the reported 8

- hour TWA was 0.985 mg/m 3 14

(0.8 ppm). The control group consisted of 23 undergraduate students with an average age of 15

19 **±** 2.3 years. The 8

- hour TWA in the student dormitories was 0.011 mg/m 3 (9 ppb). A group 16

of 16 waiters with an average exposure duration of only 12 weeks and an 8

- hour TWA of 17

0.107 mg/m 3 (90 ppb) was also included in the study. The incidence of MNs and SCEs in the 18

waiters was the same as that in controls. Overall, res ults from this study suggest that the 19

genotoxic potential of high

- level formaldehyde exposure may have occupational risks in long

20

term exposure. 21

However, in a different study, Speit et al. (2007b) showed that formaldehyde did not 22

induce MNs in exfoliated b uccal mucosa cells of humans exposed up to a maximum of 1 ppm 23

and a cumulative exposure of 13.5 ppm

- hours over 2 weeks. In this study, volunteers exposed to 24

formaldehyde in closely controlled conditions (4 hours/day for 10 days) with a complex 25

exposure sc hedule, amounting to a cumulative total of 13.5 ppm

- hours (16.6 mg/m 3

- hours), were 26

used. Samples of the buccal mucosa were taken from subjects 1 week before the start of the 27

experiment, at the start of the experiment, at the conclusion of the series of ex posures, and at 7, 28

14, and 21 days after the completion of exposure. Thus, the subjects served as their own 29

controls. Two thousand cells per data point were assessed for the frequency of MNs on slides 30

that were coded by an independent quality assurance o rganization. As shown in Table 4

- 84, the 31

frequency of MN formation was statistically unchanged from that in controls. The apparent 32

slight increase in subjects evaluated at the conclusion of exposure was caused by frequencies of 33

MNs in two subjects (5.0 a nd 4.5 MNs per 1,000 cells). The data as reported show a high 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 364 variability, where the SD approaches or exceeds the mean for each sample point, suggestive of 1

data with an asymmetrical distribution. 2

3

Table 4

- 84. MN frequencies in buccal mucosa cells of volu nteers exposed to 4

formaldehyde 5

6

Sampling point Group MN/1000 cells (± SD)

Control data

1 week before exposure 1 0.95 ± 0.67

Immediately before exposure series 2 0.86 ± 0.84

Test data

Immediately after exposure series 3 1.33 ± 1.45

7 days after exposu re 4 0.94 ± 0.73

14 days after exposure 5 0.85 ± 0.86

21 days after exposure 6 0.44 ± 0.38 a

7

a Statistically significantly different from control values ( p < 0.05), as calculated by the authors. 8

9

Source: Speit et al. (2007b). 10

11

The best evidence of forma ldehyde

- induced clastogenic changes in peripheral 12

lymphocytes is found in studies of anatomy class and mortuary class students. Since genetic 13

damage accumulates with age, the studies in younger adults, where cells are analyzed before and 14

after exposure, m ay have greater sensitivity and fewer confounding factors. 15

Suruda et al. (1993) showed a 12

- fold increase in the MN frequency of epithelial cells 16

from the buccal area of the mouth in mortuary science students exposed to embalming fluids 17

containing formal dehyde following an 85

- day exposure period (Table 4

- 85). Overall, students 18

were exposed to 0.33 ppm (0.4 mg/m 3 ) formaldehyde as an 8

- hour TWA on days when 19

embalming was performed (an average of 6.9 embalmings). Blood, oral, and nasal samples were 20

collect ed pre

- and postexposure. As shown in Table 4

- 85, nasal epithelial MNs increased by 21

22% (frequency of micronucleated lymphocytes increased by 28%). By contrast, SCE frequency 22

decreased by 7.5% after formaldehyde exposure. 23

24

Table 4

- 85. MN and SCE forma tion in mortuary science students exposed to 25

formaldehyde for 85 days 26

27

Sampling point Buccal mucosa

(MN/1,000) Nasal epithelium

(MN/1,000) Blood

(MN/1,000) Blood

(SCEs/cell)

Before course 0.046 ± 0.17 0.41 ± 0.52 4.95 ± 1.72 7.72 ± 1.26

After course 0.60 ± 1.27 a 0.50 ± 0.67 6.36 ± 2.03 a 7.14 ± 0.89

28

a Statistically significant ( p < 0.05), as calculated by the authors. 29

Source: Suruda et al. (1993). 30

31This document is a draft for review purposes only and does not constitute Agency policy.

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- 365 Another group (Titenko

- Holland et al., 1996) also reported a significant increase in MN 1

frequency of buccal, but not nasal, epithelial cells from mortuary students exposed to embalming 2

fluid. In this study, 28 out of 35 students were sampled before and after a 90

- day embalming 3

class. The mean formaldehyde exposure for the subjects providing data on buccal cell MNs was 4

14.8 ± 7.2 ppm

- hours (18.2 ± 8.8 mg/m 3

- hours) for the entire 90

- day period and 16.5 ± 5.8 ppm

5

hours (20.3 ± 7.1 mg/m 3

- hours) for students providing data on nasal cell MNs. Cells were 6

recorded as having either whole chromosomes with centromeres (MN + ) or acentric fragments 7

and no centromeres (MN

– ). Cells with multiple nuclei were present only in samples taken after 8

exposure to embalming fluid. There was a ninefold increase in the MN frequency in buccal cells 9

( p < 0.5) and only a twofold increase ( p > 0.05) in nasal cells. In addition, there was a twofold 10

increase in the MN + frequency in buccal cells (Table 4

- 86). The authors suggested that 11

chromosomal breakage appears to be the primary mechanism of MN formation. 12

13

Table 4

- 86. Incidence of MN formation in mortuary students exposed to 14

formaldehyde for 90 days 15

16

Sampling

point Buccal cells (n = 19) Nasal epithelial cel ls (n = 13)

Total MN MN + MN

– Total MN MN + MN

–

Pre

- exposure 0.6 ± 0.5 0.4 ± 0.4 0.1 ± 0.2 2.0 ± 1.3 1.2 ± 1.3 0.5 ± 0.5

Postexposure 2.0 ± 2.0 a 1.1 ± 1.3 0.9 ± 1.1 a 2.5 ± 1.3 1.0 ± 0.8 1.0 ± 0.6 a

p value 0.007 0.08 0.005 0.20 0.31 0.03

17

a Statistically significant at the level shown, as calculated by the authors. 18

19

Source: Titenko

- Holland et al. (1996). 20

21

22

Ying et al. (1997), however, observed higher frequencies of MNs in the nasal exfoliative 23

cells (3.85 ± 1.48 versus 1.20 ± 0.676, paired t

- test, p < 0. 001) and oral exfoliative cells (0.857 ± 24

0.558 versus 0.568 ± 0.317, p < 0.001) after formaldehyde exposure, although there was no 25

significant increase in the frequency of lymphocyte MNs ( p > 0.05) in students exposed to 26

formaldehyde in anatomy classes (th ree classes per week for 3 hours over an 8

- week duration). 27

In this study, blood samples and nasal swabs were collected before and after the study. The 28

TWA concentration of formaldehyde in anatomy laboratories and student dormitories was 0.508 29

± 0.299 mg/ m 3 and 0.012 ± 0.0025 mg/m 3 , respectively, suggesting that nasal mucosa cells 30

exposed through respiration are the primary target of formaldehyde

- induced genotoxicity. 31

In a different study (Ying et al., 1999) , however, the same group showed that exposure to 32

formaldehyde affected the composition of lymphocyte subsets (B cells, total T cells, T helper

33

inducer cel ls, T cytotoxic

- suppressor cells), but no significant difference was reported between 34

lymphocyte proliferation rate and SCEs at the given levels and durations of formaldehyde 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 366 exposure. This study involved 23 nonsmoking students exposed to 0.508 ± 0.299 mg /m 3 1

formaldehyde for a period of 8 weeks (3 hours , 3 times per week). 2

Burgaz et al. (2002) reported significantly ( p < 0.05) higher mean MN frequencies in 3

buccal mucosal cells from shoe workers as well as anatomy and laboratory workers (0.62 ± 4

0.45% and 0.71 ± 0.56%, respectively) compared with unexposed controls (0.33 ± 0.30%). In 5

this study, the measured air concentrations of formaldehyde in the breathing zone of the anatomy 6

and pathology laboratory workers were between 2 and 4 ppm (2.5 and 5 mg/m 3 ). MN count per 7

3,000 cells was measured in buccal smears from shoe workers and from anatomy and pathology 8

staff, and eighteen male university staff were used as controls. 9

In a critical review, Speit and Schmid (2006) examined data from studies that have 10

rep orted the formation of MNs in nasal or buccal cells of persons either environmentally or 11

occupationally exposed to formaldehyde. The authors identified a number of issues relating to 12

study design, exposure regimen, and confounding factors, including MN le vels in nasal and 13

buccal cells well below established background levels, reports limited by the number of cells 14

observed, variation in standard techniques, and non

- concordance between buccal and nasal 15

findings. However, the authors concluded that, despite these limitations, the weight of evidence 16

supports the finding that formaldehyde may be genotoxic in human cells in direct contact with 17

formaldehyde. 18

19

4.3.4.2.2. Peripheral blood lymphocytes. Mature lymphocytes are present at the POE as 20

intraepithelial lymphocytes and within germinal centers in the mucosa. Because more 21

lymphocytes may be available in the nasal mucosa than the oral mucosa, mouth versus nose 22

breathing may contribute to variability in findings. Since some of the lymphocytes traffic around 23

the body, it is reasonable to find clastogenic effects in these relatively long

- lived cells reflected 24

in peripheral blood lymphocytes. Thus, lymphocytes proliferating in response to antigen would 25

be more vulnerable to DNA reactivity of formaldehyde and t o the clastogenic effects in general. 26

A cytogenetic evaluation by Fleig et al. (1982) of 15 employees exposed for an average 27

of 28 years in a formaldehyde manufacturing plant revealed no statistically significant increase 28

in the frequency of CAs in periph eral blood lymphocytes compared with a matched control 29

group. Likewise, in a different study (Thomson et al., 1984) , no compound

- related differences 30

were evident in the frequency of CAs and MNs in lymphocytes from six pathology workers and 31

five unexposed controls. 32

Bauchinger and Schmid (1985) observ ed an increased incidence of CAs (dicentric and 33

ring chromosomes) in the peripheral lymphocytes of 20 male paper mill workers and supervisors 34

exposed to formaldehyde (average exposure of 14.5 years) compared with unexposed workers. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 367 When workers and superv isors were analyzed separately, significant increases were only seen for 1

supervisors. The average length of exposure for supervisors (n = 11) and workers (n = 9) was 2

18.9 years and 7.2 years, respectively. Information regarding formaldehyde concentration s for 3

the two groups was not provided. However, the incidence of SCEs among workers was actually 4

slightly lower than among the 20 controls. In contrast, the frequency of SCEs in peripheral 5

lymphocytes of 18 nonsmoking formaldehyde workers was increased o ver controls (8.24 **±** 0.89 6

versus 6.38 **±** 0.41) (Ye et al., 2005) (described in Section 4.3.4.2.1 ). 7

Vargová et al. (1992) observed that the percentage of aberrant cells and number of breaks 8

per cell in the peripheral blood lymphocytes of formaldehyde

- expose d workers was 3.08 and 9

0.045 versus 3.6 and 0.080 in controls in a pressed board factory, respectively, suggesting both 10

groups to be at an increased risk. However, normal unexposed population had only 1

– 2% 11

aberrant cells. The authors also noted that the mitotic index was significantly decreased in 12

exposed workers compared with controls. 13

Kitaeva et al. (1996) evaluated the genotoxic effects of formaldehyde among 15 14

industrially exposed workers and 8 academic laboratory instructors and observed an increas e in 15

the frequencies of CAs and MNs in the lymphocytes of exposed subjects compared with 16

6 unexposed controls. 17

Shaham et al. (1996, 1997) found significantly higher levels of DPXs and SCEs in 18

peripheral blood lymphocytes of workers occupationally exposed to formaldehyde (physicians 19

and technicians) compared with unexposed control workers. The authors also observed a linear 20

relationship between years of exposure to formaldehyde and levels of DPXs and SCEs. 21

Formaldehyde

- induced genotoxicity has also been r eported in peripheral blood 22

lymphocytes of anatomy class students and mortuary workers. Vasudeva and Anand (1996) did 23

not observe significant differences in the incidences of CAs between the formaldehyde exposed 24

students and the matched, unexposed control s. In this study, peripheral blood lymphocytes from 25

30 medical students exposed to formaldehyde in a gross anatomy laboratory for 15 months with 26

average exposures of less than 1 ppm (1.23 mg/m 3 ) formaldehyde were used. 27

He et al. (1998) used the cytokine sis

- blocked MN (CBMN) assay to detect the frequency 28

of micronucleated peripheral lymphocytes in 13 students exposed to formaldehyde during a 29

12

- week (10 hours/week) anatomy class. Sampling of breathing zone air showed a mean 30

concentration of 2.37 ppm (3.1 7 mg/m 3 ). Ten students from the same school, without exposure 31

to formaldehyde, were used as controls. CAs and SCEs were observed in both groups, and there 32

were significant increases ( p < 0.01) in the frequencies of micronucleated cells and CAs in the 33

for maldehyde

- exposed group compared with the control group. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 368 In a study involving 97 plasticware workers (34 males and 63 females) exposed to 0.5 to 1

0.9 mg/m3 formaldehyde, 4.4 to 6.2 mg/m3 styrene and 0.5 to 0.75 mg/m3 phenol for 2 months 2

to 25 years, Lazu tka et al (1999) observed significantly higher CAs than controls (non

- exposed 3

donors matched by age and similar smoking habits as the exposed workers). Although workers 4

with short and long exposures showed significant increases in the frequency of CAs, the 5

cytogenetic damage did not increase with exposure duration. 6

Sari

- Minodier et al. (2001), using the CBMN assay in anatomy/pathology laboratory 7

workers, reported higher frequency of micronucleated peripheral blood lymphocytes than in 8

matched controls. 9

Sh aham et al. (2002) observed a mean number of 0.27 SCEs per chromosome in the 10

peripheral lymphocytes of an exposed cohort compared with 0.19 in controls ( p < 0.01). This 11

study involved 90 individuals employed in 14 hospital pathology laboratories and 52 un exposed 12

controls. 13

Yu et al. (2005) reported dose

- dependent increase in MNs and comet assay parameters 14

(olive tail moment and comet tail length) in peripheral lymphocytes in 151 workers from two 15

plywood factories compared with 112 unexposed controls. The T WA exposure level in the 16

working environment was 0.1

– 7.88 mg/m 3 (0.08

– 6.42 ppm) formaldehyde compared with a 17

background level of <0.01 mg/m 3 (<0.008 ppm) formaldehyde applicable to controls. In the 18

comet assay, the authors observed olive tail moments aver aging 0.93 (0.78

– 1.1), 3.03 (2.49

– 19

3.67), and 3.95 (3.53

– 4.43) for control, low

- , and high

- exposure individuals, respectively. For 20

the same subjects, comet tail lengths were 6.78 (6.05

– 7.6), 11.25 (10.12

– 12.5), and 12.59 (11.8

– 21

13.43), respectively. In the CBMN assay, MNs/100 cells were 0.27 ± 0.13, 0.41 ± 0.25, and 22

0.65 ± 0.36, respectively, for control, low

- , and high

- exposure individuals. 23

In a population of 18 workers exposed to formaldehyde at a plant in China, with a mean 24

employment of 8.5 years (range 1 to 15 years), Ye et al (2005) examined nasal and lymphocytes 25

for cytogenetic effects. This study also included a second group of 16 waiters who worked in a 26

newly fitted ball room for 12 weeks with a low level exposure to formaldehyde from building 27

mate rial, tobacco smoke and furniture and a group of 23 college students as a control group. The 28

background indoor air conentraton of 0.009 ppm formaldehyde was reported in students’ dorms. 29

Significantly increased frequencies of MNs in the nasal mucosal cells and SCEs in peripheral 30

blood lymphocytes were reported for the workers, but not the waiters in this study. 31

Orsière et al. (2006) reported no apparent effect on the DNA damage in peripheral blood 32

lymphocytes as assessed by a chemiluminescence microplate assay in pathology and anatomy 33

laboratory workers (n = 59) before and after a 1

- day exposure to formaldehyde. This study had 34

59 exposed workers and 37 controls. However, with the CBMN assay, the authors reported 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 369 statistically significant differences in t he frequency of binucleated micronucleated cells 1

(1.69 ± 0.93 versus 1.11 ± 0.6%) in exposed versus control subjects. Discrimination between 2

clastogenic and aneugenic events by using FISH with a pan

- centromeric DNA probe resulted in a 3

higher rate of binuc leated micronucleated cells (1.91 ± 1.01 versus 1.19 ± 0.56% in controls) and 4

showed that the frequency of centromeric nuclei was higher in the exposed group than in 5

controls, though not significantly. Among the centromeric MNs, the frequency of MNs with 6

only one centromere (C1+MN) was significantly greater in pathologists/anatomists than in 7

controls (1.1 ± 0.62 versus 0.31 ± 0.24%, p < 0.001). The authors interpreted their data on 8

monocentromeric nuclei in anatomists/pathologists as an indication that fo rmaldehyde exposure 9

might be associated with aneugenic (rather than clastogenic) events. 10

Based on pooled analysis of two reports (Iarmarcovai et al., 2006a, b) (Table 4

- 87), MN 11

frequency ratios in the peripheral l ymphocytes of cancer patients, welders, and 12

anatomists/pathologists were significantly increased compared with the corresponding controls. 13

The data were taken from three biomonitoring studies by using CBMN/FISH. The incidence of 14

MNs was scored and then e valuated further for the presence of centromere

- negative MNs 15

(C −MNs), centromere

- positive MNs (C+MNs), and, for the latter case, those containing a single 16

centromere (C1+MNs) and those containing two or more centromeres (Cx+MNs). Applying 17

their findings t o considerations of the aneugenic mechanism of action of formaldehyde, the 18

authors hypothesized that the use of centromeric signals enables the identification of endpoints 19

representing impaired chromosomal migration (with C1+MN formation) or centrosome 20

amp lification (with Cx+MN formation). 21

22

Table 4

- 87. Multivariate repression models linking genomic 23

instability/occupational exposures to selected endpoints from the MN assay 24

25

Study populations Number MN a C −MN C+MN C1+MN Cx+MN

Cancer patients versus

controls 10/10 1.85

(1.18 −2.87) 2.05

(1.07 −3.94) 1.81

(1.02 −3.21) 1.68

(0.80 −3.53) 1.28

(0.63

– 2.59)

Welders versus controls 27/30 1.37

(1.09 −1.72) 1.39

(0.99 −1.95) 1.37

(1.03 −1.83) 1.10

(0.80 −1.53) 1.31

(0.99

− 1.74)

Pathologists/anatomists

versus controls 18/18 1.28

(0.86 −1.90) 0.79

(0.46 −1.36) 1.65

(1.05 −2.59) 3.29

(2.04 −5.30) 0.68

(0.38 −1.20)

26

a Bolded values indicate statistical significance ( p < 0.05). 27

28

Source: Iarmarcovai et al. (2006b). 29

30

31

Recently, Costa et al. (2008) observed a significant increase in the genotoxicity of 32

formaldehyde

- exposed pathological anatomy laboratory workers (n = 30) compared with 33

controls (n = 30) in cytogenetic assays. In this study, the authors evaluated the level of exposure 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 370 t o formaldehyde near the breathing zone of workers, and TWA of exposure was calculated for 1

each subject, giving a mean level of exposure to be 0.44 ± 0.08 ppm (range: 0.04

– 1.58 ppm). As 2

compared with control subjects, peripheral blood lymphocyte cultures o f formaldehyde exposed 3

workers showed significant increases in MN frequency (5.47 ± 0.76 versus 3.27 ± 0.69; p = 4

0.003), SCEs (6.13 ± 0.29 versus 4.49 ± 0.16; p < 0.05), and comet assay as determined by tail 5

length (TL) (60.00 ± 2.31 versus 41.85 ± 1.97; p < 0.05). In addition, Costa et al. (2008) 6

observed a positive correlation between formaldehyde exposure levels and MN frequency ( r = 7

0.384; p = 0.001) and TL ( r = 0.333; p = 0.005) (Table 4

- 88). However, polymorphic genes of 8

xenobiotic metabolizing and DNA repair enzymes did not show any significant effect on the 9

genotoxic endpoints. This is the lowest level of exposure to formaldehyde in the studies 10

observed so far, wherein a clear indication of genotoxic effects of formaldehyde was 11

demonstrated. 12

13

Tabl e 4

- 88. Genotoxicity measures in pathological anatomy laboratory 14

workers and controls 15

16

MN assay SCEs Comet assay

Mean MN ± SEM

(range) Mean SCE ± SEM

(range) Mean TL ( **μ** M) ± SEM

(range)

Controls

(n = 30) 3.27 ± 0.69

(0

– 17) 4.49 ± 0.16

(3.10

– 3.06) 41.85 ± 1.97

(28.85

– 66.52)

Exposed

(n = 30) 5.47 ± 0.76

(1

– 17) 6.13 ± 0.29

(3.64

– 8.80) 60.00 ± 2.31

(33.76

– 99.09)

p value 0.003 <0.05 <0.05

17

Source: Costa et al. (2008). 18

19

4.3.5. Summary of Genotoxicity 20

Formaldehyde’s genotoxicity has been demonstrated in a variety of in vitro and in vivo 21

test systems measuring a variety of genetic endpoints. Formaldehyde forms predominantly 22

DPXs that are detected in cell

- free systems and single cells in vitro. DPXs are formed in nasal 23

epithelia but not in extra

- nasal passages of rodents, which are completely removed within a day 24

after formation. In vivo data in human and mammalian cells demonstrate that formaldehyde is 25

genotoxic at the site of first contact, including cells of the mouth or the nose. DPXs are also 26

detected in nasal and extra

- nasal tissues of monkeys, suggestive of direct effects of formaldehyde 27

in tissues that correspond to observed tumor sites (nasal and nasopharynx) in humans. In 28

addition, this is used as a basis for cross

- species comparison with humans. Formaldehyde

- DNA 29

adducts are labile and constitute a minor fraction of the DNA

- reaction products and are less 30

likely to play an important role in the genotoxicity of formaldehyde. 31This document is a draft for review purposes only and does not constitute Agency policy.

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- 371 Formaldehyde clastogenicity has been demonstrated by the induction of SCEs, SSBs, 1

MNs, and CAs in cultured mammalian cells. Formaldehyde induces mutations in salmonella and 2

escherichia bacterial strains that contain an AT base pair at the primary reversion site that is used 3

to detect oxidative compounds and cross

- linking agents without metabolic activation by 4

exogenous enzyme

- activating systems. Formaldehyde induces mutations in cultured mammalian 5

cells at levels that do not cause signific ant toxicity. Despite formaldehyde’s reactivity and 6

mutagenicity in isolated mammalian cells, clear evidence of mutagenicity does not emerge from 7

animal bioassays. 8

Formaldehyde exposure causes differential induction of MNs in human nasal epithelial 9

and b uccal epithelial cells, which is significant in industrial exposure workers and students 10

working in anatomy or mortuary science, respectively. However, recent data and data from 11

larger studies support a finding of increased MNs in blood lymphocytes, altho ugh the issue 12

remains controversial because of issues relating to study design, exposure regimen, and 13

confounding factors, including MN levels in nasal and buccal cells well below established 14

background levels, reports limited by the number of cells observ ed, variation in standard 15

techniques, and non

- concordance between buccal and nasal findings (Speit and Schmid, 2006)

. 16

Several clastogenic effects, such as induction of MNs, SCEs, and CAs, were seen in human 17

periphe ral blood lymphocytes; however, the data are not very clear. Formaldehyde exposure also 18

caused p53 mutations in rat nasal carcinomas with the expression of mutant p53 protein. 19

Overall, induction of DPXs as a predominant lesion in vitro and in vivo, clastog enicity, 20

and mutagenicity with locus

- specific mutations in nonhuman and human cells supports the 21

concept of genotoxic action of formaldehyde at the POE. 22

A summary of the genotoxicity of formaldehyde in humans is presented in Table 4

- 89. 23

24

4.4. SYNTHESIS AN D MAJOR EVALUATION OF NONCARCINOGENIC EFFECTS 25

The adverse health effects due to formaldehyde exposure have been extensively studied 26

in humans and in animal models. Studies of human exposure include occupational exposures, 27

environmental exposures, and clin ical studies of intentionally exposed subjects (Section 4.1). 28

Occupational exposures are primarily due to inhalation and dermal contact. Animal studies are 29

available for a variety of routes of exposure, including inhalation, oral, dermal, and intravenous 30

and I.P. injections (Section 4.2). Additionally, as discussed in Chapter 3, in vitro studies address 31

biological activity and the metabolic fate of formaldehyde. 32This document is a draft for reviewpurposes only and does not constituteAgency policy4-372 Table 4

- 89. Summary of human cytogenetic studes

Study population

N Exposure time (years) F ormaldehyde

concentration (ppm) Cytogenetic

observations Reference

Range Mean Range Mean (TWA) CAs SCEs MNs

Analyses of nasal and/or buccal cells

Plywood workers 15 2

– 19 6.8 0.32

– 0.83 (1) + nasal Ballarin et al. (1992) Age and sex matched controls 15

Male mortuary science students 22 Buccal and nasal swabs

taken before and after

first 9 weeks of

embalming course 0.1

– 4.3 1.4 + buccal

Suruda et al. (1993) – nasal

Female mortuary science students 7 – buccal

– nasal

Mortuary science students a 28 Buccal and nasal swabs

taken before and after

first 9 weeks of

embalming course 0.1

– 4.3 1.4 +buccal b

Titenko

- Holland et al. (1996)

– nasal

Female anatomy faculty 8 NA 23.6 NA NA + buccal

Kitae va et al. (1996) Male anatomy faculty 5 25.6

– buccal

Controls (Females) 7

Anatomy students 25 Buccal and nasal swabs

taken before and after

8

- week anatomy course 0.06

– 1.06 (0.508) + buccal Ying et al. (1997)

+ nasal

Anatomy/ pathology staff 28 1

– 13 4.70 2

– 4 NA + buccal Burgaz et al. (2002) Controls (University staff) 18

Workers at a formaldehyde plant 18 1

– 15 8.5 0.8 + nasal Ye et al. (2005) Controls 23

Volunteers 21 10 days 13.5 ppm

- hours

– buccal Speit et al. (2007b)

Analyses of peripheral lymphocytes

Manufacturing workers 15 23

– 35 28 <5 1971

– Fleig et al. (1982) Age and sex matched controls 15 <1 later

Pathology workers 6 4

– 11 0.9

– 5.8

– Thomson et al. (1984) Controls 5This document is a draft for reviewpurposes only and does not constituteAgency policy4-373 Table 4

- 89. Summary of human cytogenetic studes

Study population

N Exposure time (years) F ormaldehyde

concentration (ppm) Cytogenetic

observations Reference

Range Mean Range Mean (TWA) CAs SCEs MNs

Anatomy students c 8 10

- week class 1.08

– 1.99 d 1.2 + Yager et al. (1986)

0.08

– 0.6 e 0.3

Papermakers 20 2

– 30 14.4 <3 NA + f

– Bauchinger and Schmid (1985)

Controls 20

Wood workers 25 <5 to <16 0.45

– 8.6 NA

– Vargová et al. (1992) Controls 19

Male embalming students 22 Blood sampled before

and after first 9 weeks

of embalming course 0.15

– 4.3 1.4

– +

Suruda et al. (1993)

Female embalming stude nts 7

– –

Manufacturing workers 15 10 Up to 4 NA + +

Kitaeva et al. (1996) Anatomy faculty 8 17 NA ND

Controls 6

Medial students 30 Sampled near end of

15

- month term <1 NA

– Vasudeva and Anand (1996) Controls 30

-

Anatomy students 13 12

- week class 2.37 g + + + He at al. (1998)

Controls (students) 10

Physicians 6 2

- 24 10 3.1

- 2.8 1.6 +

Shaham et al 1997 Technicians 7 2

- 25 15 +

Controls (age matched/unexposed) 20

Anatomy students 23

- 25 Blood samples taken

before and after 8

- week

anatomy course 0.06

– 1.06 (0.508)

- – – Ying et al. (1999, 1997)

Female anatomy/pathology lab workers 10 1

– 16 8.9 1.2

– 15.1 NA + Sari

- Minodier et al. (2001)

Controls (Women) 27

Hospital pathology workers h 90 1

– 39 15.4 0.04

– 0.7 i 0.4 + j Shaham et al. (2002)

Controls 52 0.72

- 5.6 2.24 +This document is a draft for reviewpurposes only and does not constituteAgency policy4-374 Table 4

- 89. Summary of human cytogenetic studes

Study population

N Exposure time (years) F ormaldehyde

concentration (ppm) Cytogenetic

observations Reference

Range Mean Range Mean (TWA) CAs SCEs MNs

Workers at a formaldehyde plant 18 1

– 15 8.5 0.8 +

– Ye et al. (2005) Controls 23

Workers at two plywood factories 151 ND 0.08

– 6.42 + Yu et al. (2005) Controls 112

Pathology or anatomy workers 59 ND <0.1

– 20.4 k 2 k + Orsière et al. (2006)

Controls 37

Pathologists 18 ND 0.4

– 7.0 k 2.3 k + Iarmarcovai et al. (2006a, b)

Controls 18

Pathological anatomy lab workers 30 0.5

– 27 11 0.04

– 1.58 0.44 + + Costa et al. (2008) Controls (21 females and 9 males) 30

Plasticware workers 97 2 mo to 25

yrs 0.5

- 0.9

mg/m3 + Lazutka et al 1999 Controls (non

- exposed donors) 90

Wood workers 40 + Chebotarev et al 1986 Controls 22 NR NR

School children (1984) 20 0.26 +

Neri et al 2006 School children (1985) 16 0.11 +

School c hildren (1986) 18 0.03

Controls (1984) 17 0

Preschool controls (1984) 24 0

Preschool children (1984) 13 0.17

- 0.3

Phenolformaldehyde resin workers 31

0.33

- 30 yr 0.41 +

Suskov and Sazonova 1982

Controls 74 0

a Same population in Suruda et al. (1993) but different slides used. Nineteen complete slide sets for buccal analysis and 13 c omplete slide sets for nasal epithelial

cell analysis.

b Not dose related; both low

- and high

- exposure groups had same SCE increase.

c Each student sampled before and after 10

- week anatomy class.

d Breathing zone samples.This document is a draft for reviewpurposes only and does not constituteAgency policy4-375 e Room air samples.

f Increase only in 11 supervisors. See text for details.

g Average breathing zone during dissection procedure.

h Exposed and controls from 14 hospitals.

i Low

- and high

- exposure groups established but numbers not provided.

j Not dose related; both low and high groups had same SCE increase.

k Described as “mean concentrations for sampling times of 15 minutes.”

CAs = chromosomal aberrations; SCEs = sister chromatid exchanges; MNs = micronuclei; TWA = time

- weighted average; ND = not determined; NA = not

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

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- 376 Taken together, the human and animal studies support numerous health effects, not only 1

at the POE as expected for a r eactive gas but also on pulmonary function, neurobehavioral 2

function, reproduction, development, immunomodulation, and sensitization (atopy, asthma). The 3

discussion below provides a description of the adverse effects seen in each area, summarizing the 4

dat a for both human and animal studies. MOA data are discussed where information regarding 5

formaldehyde’s biological activity may be linked to the observed adverse health effects. 6

7

4.4.1. Sensory Irritation 8

Sensory irritation of the eyes, nose, and throat i s reported in humans upon direct contact 9

with formaldehyde gas during inhalation exposures (Holmström and Wilhelmsson, 1988; 10

Ritchie and Lehman, 1987) and includes irritation resulting from acute exposures ( Lang et al., 11

2008; Yang et al., 2001; Krakowiak et al., 1998; Kulle, 1993; Green et al., 1989, 1987; Kulle et 12

al., 1987; Sauder et al., 1987, 1986; Schachter et al., 1987, 1986; Witek et al., 1987; Day et al., 13

1984; Bender et al., 1983; Weber

- Tschopp et al., 1977) . Controlled exposures in inhalation 14

c hambers confirm the specificity of these responses to formaldehyde exposure and allow for 15

assessment of these symptoms through both subjective and objective measures (Kulle, 1993; 16

Holness and Nethercott, 1989; Green et al., 1987; Kulle et al., 1987; Sauder et al., 1986; Weber

17

Tschopp et al., 1977). Eye irritation may be reported as itching, burning, and general discomfort. 18

Tearing, redness of the eyes, and increased blink frequency are observed and may be quantified 19

in exposure under controlled conditions ( Lang et al., 2008; Yang et al., 2001; Andersen and 20

Molhave, 1983; Weber

- Tschopp et al., 1977; Schuck et al., 1966). Eye irritation appears to be 21

the most sensitive endpoint in most individuals and may be observed after short exposures 22

(195 minutes at 0. 5 ppm: Lang et al. [2008]; 30 seconds at 1.65 ppm: Yang et al. [2001]). 23

Itching, burning, and discomfort of the nose, which may be accompanied by increased 24

mucous production (runny nose), are reported by individuals exposed via inhalation (Krakowiak 25

et al. , 1998; Kulle, 1993; Green et al., 1987; Kulle et al., 1987; Weber

- Tschopp et al., 1977). 26

Throat irritation may also be described subjectively as itching and burning and is often 27

accompanied by a cough (Krakowiak et al., 1998). Symptoms of eye and mucous membrane 28

irritation are also reported in numerous rodent studies and support the health effects reported in 29

humans (see Section 4.1.1.1). Although dermal contact may result in dermatitis and an apparent 30

hypersensitivity reaction, symptoms do not present upon contact as sensory irritation. There are 31

no human or animal data that assess sensory irritation from oral exposures. 32

The time to onset of sensory irritation symptoms and severity of the sensory irritation are 33

a function of both the air concentration and duration of exposure. Additionally, nose and throat 34

irritation becomes more prominent at higher exposures and longer duration of exposure (Kulle, 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 377 1993; Kulle et al., 1987). Controlled human laboratory exposures (Yang et al., 2001; Kulle, 1

1993; Kulle et al., 1987; Cain et al., 1986; Andersen and Molhave, 1983) provide more direct 2

exposure

- response evidence for sensory irritation. These studies are limited to healthy 3

nonsmoking individuals. Two studies (Cain et al., 1986; Andersen and Molhave, 1983) 4

d ocument discomfort and irritation of the eye in response to acute exposures as low as 0.25 ppm. 5

Dose

- response relationships are reported in a number of different ways: as an incidence of the 6

reported symptom among subjects, as a score for severity of the symptom, or in some cases as a 7

subjective measure, such as blink frequency for eye irritation. 8

Symptoms of sensory irritation, including eye irritation (burning watering, increased 9

blinking), nasal irritation (rhinitis, itching/burning), throat/respiratory tract irritation (wheezing, 10

coughing, phlegm production), have been reported in numerous worker cohorts. Occupational 11

exposure environments include hospital and medical settings, students, and industrial workers 12

(Takahashi et al., 2007; Takigawa et al., 2005; Krakowiak et al., 1998; Akbar

- Khanzadeh et al., 13

1994; Uba et al., 1989; Horvath et al., 1988; Schachter et al., 1987). Formaldehyde levels often 14

vary in a work environment and peak as well as average exposures may be used to report 15

occupational expo sures. Although sensitive individuals often remove themselves from an 16

irritating workplace (the HWE), eye, nose, and throat symptoms are still reported in this 17

environment. Among workers in a plant where formaldehyde resins were used, those exposed to 18

an average of 210 ppb formaldehyde reported increased symptoms above those in the control 19

population (Holmström and Wilhelmsson, 1988). 20

These effects have been noted in students, particularly medical students, who are exposed 21

to formaldehyde in cadaver labs. In a study of 24 formaldehyde

- exposed anatomy students 22

(personal breathing zone samples 0.73 ppm, range 0.49

– 0.93) (Kriebel et al., 1993), eye, nose, 23

and throat irritation was present when comparing rates of irritation from the end or middle of 24

class to before the start of class. Takahashi et al. (2007) showed that 143 medical students 25

reported various symptoms (including eye and throat irritation) and that the percentage of 26

students reporting symptoms increased between the beginning (measured after the first day of 27

class) and the end of the course (2 months later). After the first day of class, approximately 35% 28

of students reported eye soreness and about 15% reported throat irritation. 29

Sensory irritation has also been noted in occupational settings. H orvath et al. (1988) 30

compared irritation symptoms between 109 workers at a particleboard manufacturing plant and 31

264 workers at food plants. Eye, nose, and throat irritation were more common among the group 32

in a particleboard manufacturing facility, expos ed to a mean concentration of 0.40 mg/m 3

. 33

Similarly, Alexandersson and Hedenstierna (1988) reported that the frequency of eye, nose, and 34

throat irritation was significantly greater (65.8%) in 38 workers exposed to formaldehyde and 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 378 solvents in lacquers as compared with 18 nonexposed individuals working at the same factory 1

(16.7%). Holmström and Wilhelmsson (1988) conducted a study at a chemical plant and 2

reported nasal and eye discomfort in 64 and 24%, respectively, of workers (n = 70) exposed to 3

formaldeh yde (range 0.05

– 0.50 mg/m 3 with a mean of 0.26 mg/m 3 ) versus 25 and 6%, 4

respectively, in nonexposed desk clerks (n = 36). Holness and Nethercott (1989) reported 5

significant increases in eye irritation (42 versus 21%) and nose irritation (44 versus 16%) am ong 6

84 funeral service workers (active embalmers, >10

- year experience) as compared with 7

38 students and individuals from a service organization. The exposure concentration in both 8

groups was 0.36 and 0.02 ppm, respectively. 9

Reports of similar symptoms are correlated to indoor residential exposures, providing 10

exposure

- response relationships for the general population in low

- level chronic exposure 11

scenarios. Ritchie and Lehnen (1987) surveyed residents in 2,000 homes classified as having 12

formaldehyde concen tration <0.1 ppm, 0.1

– 0.3 ppm, and >0.3 ppm. A LOAEL of 200 ppb was 13

established from the results of Ritchie and Lehnen (1987). Liu et al. (1991) report irritant effects 14

associated with formaldehyde exposure in mobile homes, where formaldehyde concentrati ons 15

ranged from the 0.01 ppm detection limit to 0.46 ppm. Eye irritation (60%), nose/throat 16

irritation (10

– 20%), or headache (<10%) were reported in residents. 17

18

MOA 19

The mucosae of the URT, oral cavity pharynx, and upper airways are complex tissues, 20

where epithelial and goblet cells predominate. In addition, the nasal mucosa is highly enervated. 21

The main nerves include the trigeminal nerve and olfactory sensory cells (olfactory epithelium, 22

the vomeronasal organ, and the organ of Masera) (Feron et al., 200 1). A possible MOA for 23

sensory irritation includes formaldehyde

- induced stimulation of the trigeminal nerve (though 24

whether formaldehyde acts as a direct agonist is unknown). Trigeminal nerve stimulation in the 25

nasal passages transmits signals to the CNS , which then sends efferent signals back to the nasal 26

tissues, causing sensory irritation, and possibly systemically via vagal nerve stimulation, 27

resulting in more systemic effects. 28

Animal studies are potentially useful models for understanding mechanisms of toxicity, 29

especially where sufficient human data do not exist. While experimental animal studies provide 30

a model of secondary effects, rodents also demonstrate RB, an effect not seen in humans. Thus, 31

species that exhibit bradypnea (like mice and rats) may not be appropriate for assessing 32

respiratory endpoints. The mechanism underlying RB includes formaldehyde binding to the 33

sensory nerve endings of the trigeminal nerve, where signals travel to the CNS. The vagus nerve 34

transmits the efferent signal to produce smooth muscle contraction. The animals become 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 379 inactive, their core temperatures decrease by several degrees C, and their respiratory rates and 1

minute volumes decrease. However, this is not to say that trigeminal nerve stimulation is not an 2

appro priate potential mechanism of action in other species or in humans. Since trigeminal nerve 3

stimulation has been independently confirmed in species without RB, this mechanism may be a 4

viable explanation for the observed effects. 5

6

4.4.2. Pulmonary Function 7

Workers chronically exposed to formaldehyde have exhibited signs of reduced lung 8

function, such as BC, inflammation, and chronic obstructive lung disease. Lung function deficits 9

have been reported in pre

- versus post

- shift measurements and as a result of chronic exposures 10

(Pourmahabadian et al., 2006; Herbert et al., 1994; Malaka and Kodama, 1990; Alexandersson 11

and Hedenstierna, 1989; Alexandersson et al., 1982). Decreases in spirometric values, including 12

VC, FEV, FVC, and FEV/FVC, have been reported. C hronic studies (Pourmahabadian et al., 13

2006; Herbert et al., 1994; Malaka and Kodama, 1990; Alexandersson and Hedenstierna, 1989; 14

Alexandersson et al., 1982) also report increased respiratory symptoms, including cough, 15

increased phlegm, asthma, chest tight ness, and chest colds, in exposed workers. 16

Students have also shown decrements in lung function that are associated with exposure 17

to formaldehyde in laboratories. Kriebel and colleagues (1993) observed a 2% decrement in PEF 18

in healthy students attending anatomy classes once per week and a 7.3% decrement in PEF in 19

students with histories of asthma. The strongest pulmonary response was observed when 20

examining the average cross

- laboratory decrement in PEF in the first 2 weeks of the study 21

(formaldehyde geom etric average concentration of 0.73 ppm). These findings were corroborated 22

by Kriebel et al. (2001) in which a similar study design was applied to another class of anatomy 23

students. 24

Similarly, Akbar

- Khanzadeh et al. (1994) compared pre

- and postexposure p ulmonary 25

function among students before and after working 3 hours in a laboratory (n = 34). On average, 26

FVC decreased by 1.4%, FEV 3 decreased by 1.2%, FEV 1 /FVC increased by 1.6%, and FVC

- 25

– 27

75% increased 2.5%. These average percent changes in the control group are

– 0.3%, 1.30%, 28

2.31%, and 0.6% but were not statistically significant. In a follow

- up study, Akbar

- Khanzadeh 29

and Mlynek (1997) recorded FEV values in 50 exposed students and 36 controls and reported a 30

larger increase in lung function among contr ols when compared with cases after 1

– 3 hours of 31

exposure that persisted after 3 hours after exposure termination. In a similar study, Fleisher 32

(1987) reported that approximately 8% of students reported experiencing shortness of breath 33

during the laborator y with formaldehyde exposure, but none of the students reported shortness of 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 380 breath in the laboratory session with no exposure. However, no objective measurements of 1

formaldehyde exposure were used. 2

Unlike the study by Kriebel et al. (1993), Uba et al. (1 989) did not find a change in 3

pulmonary function over the course of the 7 months in a study of 96 anatomy laboratory 4

students. These negative findings may be attributed to differential cross

- shift exposures and to 5

significant differences in FVC on exposed days. 6

Deficits in pulmonary function have been reported in occupational or residential exposure 7

studies (Khamgaonkar and Fulare, 1991; Krzyzanowski et al., 1990; Malaka and Kodama, 1990; 8

Alexandersson and Hedenstierna, 1989; Kilburn et al., 1985; Alexande rsson et al., 1982). 9

Krzyzanowski et al. (1990) documented a significantly decreased PEFR in children 10

(298 children) who resided in homes with an average formaldehyde concentration of 26 ppb 11

(maximum sample value of 140 ppb). Among adults, there was a st atistically significant 12

nonlinear relationship with decreased morning PEFR for formaldehyde concentration <40 ppb 13

(Krzyzanowski et al., 1990). Similarly, Malaka and Kodama (1990) reported that an average 14

8

- hour TWA formaldehyde exposure of 1.13 ppm from a rea samples was associated with 15

statistically significant decrements in FEV 1 , FEV 1 /FVC, and FEF 25

– 75% compared with a referent 16

population. Alexandersson and Hedenstierna (1989) investigated not only the acute effects of 17

exposure across shift but also meas ured effects of exposure among some of the same workers 18

that had been studied 5 years earlier (Alexandersson et al., 1982). Statistically significant 19

decreases ( p < 0.01) in FEV 1 /FVC and FEF 25

– 75% were noted over the intervening five years in 20

nonsmokers a fter correcting for aging. Similar decrements have been documented in laboratory 21

workers in India (Khamgaonkar and Fulare, 1991) and in factory workers (Kilburn et al., 1985). 22

Alexandersson et al. (1982) reported only slight deficits in lung function 1 da y following 23

occupational formaldehyde exposure in a carpentry shop in Sweden, where the measured 24

formaldehyde level was 0.36 ppm (0.47 mg/m 3 ). In this case, subjects were compared with 25

20 nonexposed workers. 26

Other studies have found no association between formaldehyde and lung function 27

(Ostojic et al., 2006; Holness and Nethercott, 1989; Holmström and Wilhelmsson, 1988; Horvath 28

et al., 1988). Ostojic et al. (2006) used an interesting measurement, “diffusing lung capacity” 29

instead of decrements in FEV1 or similar measurements. Similarly, Nunn et al. (1990) assessed 30

the decrease in FEV 1 with age and showed no association between formaldehyde exposure and 31

decreased FEV 1 . Franklin et al. (2000) did not report an association between FVC or FEV and 32

the indoor concentrations of formaldehyde in children (ages 6

– 13), although there were signs of 33

lower airway inflammation as measured by levels of exhaled NO (Franklin et al., 2000). 34

Similarly, Main and Hogan (1983) did not observe differences between FEV 1 or FVC at the end 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 381 of the 34 months between mobile home trailer workers compared with controls who did not work 1

in trailers. The average exposure was reported as ranging from 0.12 to 1.6 ppm. 2

Occupational studies share certain limitations, including the potential f or confounding by 3

occupational co

- exposures. Also, studies that did not report pre

- shift pulmonary function as a 4

percentage of expected function are less useful to assess potential chronic effects because, 5

post hoc, it is difficult to calibrate for cross

- study comparison due to lack of data on important 6

pulmonary function determinants, such as age, gender, smoking status, height, and year of birth. 7

Controlled human studies and studies in nonhuman primates also document changes in 8

formaldehyde

- induced pulmo nary dysfunction. Acute exposures of healthy non

- asthmatic 9

volunteers resulted in transient decreases in pulmonary function (e.g., decreased FEV 1 , FVC 1 , 10

FEV 3 , specific airway conductance) (Green et al., 1987; Sauder et al., 1986). Green et al. (1987) 11

not ed differential responsiveness in formaldehyde

- exposed subjects; some were responders while 12

others were nonresponders. This differential response suggests susceptibility in certain subjects 13

(Green et al., 1987). 14

Several animal studies document increased a irway resistance and BC following 15

inhalation exposure to formaldehyde (Nielson et al., 1999; Swiecichowski et al., 1993; Biagini et 16

al., 1989; Amdur et al., 1960). A study using cynomolgus monkeys (Biagini et al., 1989) 17

demonstrated that methacholine

- indu ced BC can be similarly induced by acute formaldehyde 18

exposure (10 minutes at 2.5 ppm). Thus, formaldehyde exposure simulated BC observed after 19

methacholine challenge, but these effects may not occur by a similar MOA. Similar results were 20

reported in gui nea pigs (Swiecichowski et al., 1993; Amdur et al., 1960), rats (Ohtsuka et al., 21

1997), and mice (Nielson et al., 1999). 22

Deficits in pulmonary function have been documented in occupational as well as 23

controlled chamber human studies and have been corrobora ted in animal studies exposed to 24

formaldehyde. However, some of these deficits are slight or transient. Some studies did not 25

identify a statistically significant decrease in pulmonary function, and others did not observe any 26

change at all. Pulmonary fun ction alterations appear to be specifically tied to exposure regimen 27

and may be reversible but remain, nevertheless, an important symptom often associated with 28

exposure to formaldehyde. 29

30

MOA 31

Formaldehyde

- induced inflammation of the airways may contribute t o observed 32

decreases in measures of pulmonary function. Even short

- term inflammatory reactions could 33

reduce the effective diameter of the conductive airways, resulting in lower respiratory volumes in 34

a number of functional tests. Formaldehyde

- induced tri geminal nerve stimulation contributes to 35This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

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- 382 airway inflammation, which in turn would reduce airway function. Chronic exposures may 1

result in increased sensitization or chronic inflammatory responses, which could contribute to the 2

effects seen in the worker a nd residential populations. 3

Formaldehyde

- induced pulmonary function deficits may also be in part a result of smooth 4

muscle contraction in repose to trigeminal nerve stimulation. Trigeminal nerve stimulation 5

transmits signals to the CNS. The resulting eff erent signal from the vagal nerve produces 6

smooth muscle contraction and may result in decreased pulmonary function. Efferent signaling 7

has also resulted in release of substance P and other neuromodulatory compounds, which may 8

contribute to BC and sensiti zation of pulmonary responses (asthma, atopy). 9

10

4.4.3. Hypersensitivity and Atopic Reactions 11

Sensitization to inhalational chemical exposure may manifest as an allergic or asthmatic 12

response that is characterized by BC or BHR. This sensitization may be a result of immune 13

involvement, as in the case of hypersensitivity, or a neurogenic sensitization, where a chemical 14

may directly stimulate inflammation. Asthma is a specific manifestation of IgE

- mediated 15

hypersensitivity, characterized by BHR and airway in flammation, resulting in lower airway 16

obstruction (Fireman, 2003; Kuby, 1991). 17

A variety of hypersensitivity reactions have been reported following exposure to 18

formaldehyde. Rashes and skin reactions have been reported in some individuals after dermal 19

exp osures to formaldehyde. Increased expression of T h

- 2 cytokines in the lymph nodes of mice 20

given dermal applications of formaldehyde does indicate the involvement of an immune 21

component to the observed sensitization (Dearman et al., 2005; Hilton et al., 19 98; Arts et al., 22

1997). However, the response does not appear to be IgE mediated (Arts et al., 1997; Lee et al., 23

1984). Gorski et al. (1992) observed an increase in formaldehyde

- mediated neutrophil burst in 24

dermatitis patients exposed in a controlled cha mber study and suggests a putative role of 25

oxidative stress and reactive oxygen species (ROS). 26

Inhalation exposure has been associated with increased asthmatic responses in asthmatics 27

in occupational settings. While few available case reports of bronchial asthma suggest direct 28

respiratory tract sensitization to formaldehyde gas (Lemiere et al., 1995; Burge et al., 1985; 29

Hendrick et al., 1982; Hendrick and Lane, 1977, 1975), a greater body of human data provides 30

evidence of an association between formaldehy de exposure and exacerbation of asthmatic 31

responses in compromised individuals (Kriebel et al., 1993) and particularly in children 32

(Rumchev et al., 2002; Garrett et al., 1999; Krzyzanowski et al., 1990). Increased asthma 33

incidence reported after inhalatio n exposure to formaldehyde led to a NOAEL of 30 ppb 34This document is a draft for review purposes only and does not constitute Agency policy.

DRAFT

— DO NOT CITE OR QUOTE 4

- 383 (Rumchev et al., 2002). An increased frequency of respiratory symptoms associated with 1

asthmatic responses and formaldehyde exposure led to a LOAEL of 30 ppb (Garrett et al., 1999). 2

Exacerbation of respo nse after formaldehyde exposure has been demonstrated in animal 3

studies as well. Sadakane et al. (2002) demonstrated that formaldehyde exposure exacerbated 4

sensitization and challenge with Der f and suggested that formaldehyde exposure may aggravate 5

eosin ophilic infiltration and goblet cell proliferation that accompanies allergic responses. 6

Several animal studies report increased airway resistance and BC due to inhalation exposures to 7

formaldehyde (Nielsen et al., 1999; Swiecichowski et al., 1993; Biagini et al., 1989; Amdur, 8

1960). Changes in pulmonary resistance were observed as early as 10 minutes after exposure 9

(Biagini et al., 1989), and reported effect levels ranged from 0.3 to 13 ppm. BHR is commonly 10

associated with allergic Type I hypersensitivit y reactions but is not sufficient to demonstrate that 11

an agent induces Type 1 hypersensitivity. 12

13

MOA 14

The MOA underlying this response has not been elucidated. Formaldehyde

- induced IgE 15

production has been reported in some studies (Vandenplas et al., 2004; Wantke et al., 1996a). 16

Other studies suggest that this effect does not appear to be immunogenic in nature (Fujimaki et 17

al., 2004; Lee et al., 1984). Although formaldehyde exposure has been reported to alter cytokine 18

levels and immunoglobulins in some exp erimental systems (Fujimaki et al., 2004a; Ohtsuka et 19

al., 2003), these immunomodulatory effects do not support immunogenically mediated type 1 20

hypersensitivity. 21

These decrements may be mediated via neurogenic potentiation (Sadakane et al., 2002; 22

Riedel et al., 1996; Tarkowski and Gorski, 1995). Tarkowski and Gorski (1995) suggest that 23

formaldehyde may increase permeability of respiratory epithelium and destruction of 24

immunologic barriers. Tachykinin NK1 receptor and various neuropeptides (NGF and substan ce 25

P) have been implicated in formaldehyde

- induced sensitization and lend weight of evidence to a 26

neurogenic MOA (Van Schoor et al., 2000; Ito et al. 1996). 27

28

4.4.4. Upper Respiratory Tract Histopathology 29

Several studies in occupational workers have report ed increased squamous cell 30

metaplasia and reduced mucociliary clearance in nasal and buccal swabs from humans 31

occupationally exposed to formaldehyde (Holmström et al., 1989; Holmström and Wilhelmsson, 32

1988). Evidence of genotoxic effects include increased MNs and CAs in nasal and buccal 33

epithelial cells from both workers and students exposed to formaldehyde ( Ying et al., 1997; 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 384 Titenko

- Holland et al., 1996; Suruda et al., 1993 ) and suggest a potential association between 1

genotoxicity and altered histopathol ogy. 2

Numerous animal experimental studies in multiple strains of rats, mice, hamsters, rabbits, 3

and monkeys describe formaldehyde

- induced URT pathology (Fló

- Neyret et al., 2001; Roemer 4

et al., 1993; Reuzel et al., 1990; Monticello et al., 1989; Zwart et al ., 1988; Wilmer et al., 1987; 5

Morgan et al., 1986b, 1983; Swenberg et al., 1986; Buckley et al., 1984). Effects are first 6

observed in the anterior respiratory mucosa and progress through the nasal passages with 7

increasing exposure concentration and time. The first observed effect includes damage to the 8

mucociliary apparatus of the nasal passages in response to formaldehyde. Studies conducted 9

both in vivo and in vitro demonstrate that formaldehyde disrupts mucus flow and ciliary beat that 10

are dependent on concentration and duration of exposure. Mucociliary apparatus deficits have 11

been recorded even after 18 hours of recovery following formaldehyde exposure. The 12

breakdown of the mucociliary apparatus may allow for increased infection and allow the 13

underly ing epithelium to come into contact with exogenous chemicals. 14

Formaldehyde is highly reactive and may impact all cells in the nasal mucosa, including 15

epithelial cells (ciliated, columnar, and cuboidal), goblet cells, sensory neurons, and 16

intraepithelial ly mphocytes. The histologic changes of these processes have been described in all 17

laboratory animals examined and progress from the anterior nares to the posterior regions of the 18

nasal passages, including the ETs and olfactory epithelium if the concentratio n and duration of 19

exposure are great enough. 20

Humans and nonhuman primates have significantly less complex nasal passages than 21

rodents. Formaldehyde has lower peak flux in human nasal tissues compared with rodents, 22

which are obligate nose breathers, but wi ll penetrate more deeply into the human respiratory tract 23

than in rodents, since humans lack the autonomic RA response. Additionally, humans may 24

switch to mouth breathing in the presence of an irritant gas, thus bypassing the sensitive nasal 25

passages and increasing the tissue dose in the mouth and throat. These differences have been 26

demonstrated by using nonhuman primates where, at comparable concentrations, tissue 27

pathology and increased cell proliferation progressed further into the respiratory tract th an in 28

rodents (Monticello et al., 1989). Nonhuman primates share common structural respiratory 29

components and patterns of breathing and do not have a reflex autonomic apnea response. 30

Despite the anatomical and physiological differences in breathing patter ns and different 31

exposure parameters between humans and rodents, similar toxic effects are reported in tissues at 32

the POE in humans and laboratory animals. Several occupational studies have reported 33

increased squamous cell metaplasia in nasal and buccal s amples in response to formaldehyde 34

exposure (Ballarin et al., 1992; Boysen et al., 1990; Holmström et al., 1989), paralleling the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 385 histologic effects seen in experimental animal studies. A few human epidemiology studies 1

suggest increased NPCs (see Section 4.5) as well as oral/buccal tumors in response to 2

formaldehyde exposure (Shangina et al., 2006; Laforest et al., 2000). 3

The observed formaldehyde

- induced URT toxicity is related to its high reactivity and 4

solubility. Moreover, additional interspecies diff erences in the surface area and configuration of 5

the nasal passages and upper airways will influence the areas of high formaldehyde flux in POE 6

tissues. 7

8

MOA 9

Formaldehyde

- induced damage to the mucociliary apparatus of the nasal passages may 10

occur because f ormaldehyde may disrupt mucus flow and ciliary beat that is dependent on 11

concentration and duration of exposure. Formaldehyde reacts with the mucosal glycoproteins 12

and thus may contribute directly to the breakdown of the mucus layer. As formaldehyde reac hes 13

the cells of the pseudostratified epithelium in the nasal passages, it exerts a range of effects from 14

direct damage to cell membrane, intracellular proteins, and DNA to alterations in GSH pools and 15

increased ROS. Adaptive effects include increased muc us flow and goblet cell proliferation as 16

well as the transition of respiratory epithelium to more insensitive cuboidal cells. With 17

continued exposure at sufficient concentration, squamous metaplasia develops, creating a 18

protective layer of keratinized cel ls. Gradually, this damage exceeds the cell’s ability to 19

compensate for and repair damage; chronic nasal lesions develop, and the cells die both through 20

general necrosis as well as programmed cell death, depending on the severity of the cellular 21

damage (M onticello et al., 1989; Swenberg et al., 1983). 22

Genotoxic effects have been reported in nasal and buccal lesions taken from both workers 23

and students exposed to formaldehyde ( Ying et al., 1997; Titenko

- Holland et al., 1996; Suruda et 24

al., 1993). MN format ion occurs in the more sensitive pseudostratified epithelium of the nasal 25

passages, nasopharynx, and upper airways, since there is only one layer of epithelial cells that are 26

constantly regenerating. However, the genotoxicity observed in buccal cells is m ore difficult to 27

explain, since buccal basal cells are usually covered by protective keratinized cell layers. Cuts, 28

sores, or other buccal lesions would increase basal epithelial cells’ vulnerability to direct 29

exposure to formaldehyde. 30

31

4.4.5. Toxicogeno mic and Molecular Data That May Inform MOAs 32

Over the past several years, studies have begun to examine the effects of formaldehyde 33

exposure on gene and protein expression. These include studies on in vivo and in vitro changes 34

in the global expression of m RNA (transcriptomics) and proteins (proteomics) in the tissues and 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 386 cells of humans and rodents exposed to formaldehyde. Currently (2009), nine “

- omics” studies 1

from five research groups are available. These studies are summarized in Section 5.2 and are 2

e valuated and discussed in the context of their relevance to informing MOAs and the dose

3

response characterization briefly here. 4

In 2002, EPA released the Interim Policy on Genomics (U.S. EPA, 2002c), which 5

addresses how to use genomic data in regulatory de cision making. Although the policy 6

encourages research in genomics, it places limits on its use, stating that genomic data alone are 7

not sufficient as a basis for decision making. These data thus cannot currently be utilized as the 8

critical effect in a c hemical risk assessment but can be utilized in a weight

- of

- evidence approach 9

on a case

- by

- case basis. The Science Policy Council developed a white paper entitled Potential 10

Implications of Genomics for Regulatory and Risk Assessment Applications at EPA (U. S. EPA, 11

2004). This report described three areas where genomic data might be applied in risk assessment 12

at EPA: MOA analysis, susceptible population, and mixtures assessments. The genomic data on 13

formaldehyde thus may be applied to a discussion of MOA. 14

T oxicogenomics studies have investigated the gene and protein expression changes 15

resulting from formaldehyde exposure in a variety of respiratory tissues, including nasal tissue 16

(Andersen et al., 2008; Thomas et al., 2007; Hester et al., 2005, 2003), and, i n lung tissue (Lee et 17

al., 2008, 2007; Sul et al., 2007; Im et al., 2006) used human tracheal cell lines to study genomic 18

changes after exposure to formaldehyde in vitro. Unfortunately, these studies are not directly 19

comparable because different gene chip technology platforms were used in different tissues, in 20

both in vivo and in vitro study designs. In general, the gene and protein expression changes 21

reflect changes in apoptotic pathway genes, oxidative stress, and tissue remodeling. Andersen et 22

al. (20 08) concluded that there was a threshold level where exposure to formaldehyde (6 ppm) 23

does not elicit changes in nasal epithelium of F344 rats. Overall, Andersen et al. (2008) 24

concluded that genomic changes were no more sensitive than tissue responses and that 25

formaldehyde, being an endogenous chemical, is well handled until some threshold is achieved 26

when toxicity rapidly ensues with genomic and histologic changes. At about 6 ppm, this largely 27

involves tissue remodeling (and protection), but regenerative hyperplasia occurs at higher doses. 28

Andersen et al. (2008) conclude that there is a threshold where exposure to formaldehyde does 29

not elicit changes in F344 nasal epithelial tissue over the duration examined in this study (i.e., 30

15 days). Andersen et al . (2008) argue that this is consistent with bioassays that indicate no 31

tumor formation in rodents below 6 ppm formaldehyde. 32

The primary conclusion in the Andersen et al. (2008) paper is that genomic changes, 33

including those suggestive of mutagenic effects, did not temporally precede or occur at lower 34

doses than phenotypic changes in the tissue. The authors stated as follows: 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 387 “The genomic signatures related to these transitions are for cell membrane and 1

extracellular components, then inflammation and cellul ar stress, and eventually 2

apoptosis. Importantly, these hierarchical models indicate that the tissue 3

responses at low dose concentrations are qualitatively different from those at high 4

concentrations and linear extrapolations or extrapolations that specif y similar 5

modes of action at high and low doses would be inappropriate.” 6

7

4.4.6. Noncancer Modes of Actions 8

Noncancer health effects of interest span numerous organ systems and include 9

reproductive and developmental effects, neurological/neurobehavioral e ffects, and a complex 10

interaction between inflammation and immune and adverse pulmonary function. To date, no 11

- omics studies have examined changes in reproductive tissue or altered gene expression in 12

developing animals. In regard to neurological/behavior al effects, one study (Lu et al. [2008], 13

described in Section 4.1.1.6) has reported elevations in the mRNA for NMDA receptor subunits 14

in brain homogenates following exposure to 2.4 ppm. Hester et al. (2003) reported a significant 15

increase in NMDA receptor subunit transcripts, along with other neuropeptide genes, in nasal 16

tissue. Together, these changes may relate to formaldehyde

- induced sensory irritation and, 17

perhaps, changes throughout the brain. 18

In regard to inflammatory, immune, and pulmonary effects, transcript and protein 19

changes in rodent tracheal tissue and lung tissue indicate that exposure to 3 to 38 ppm 20

formaldehyde results in genes involved in oxidative stress and cell proliferation and may 21

additionally increase airway ADH3 levels (Lee et al., 2008; Sul et al., 2007; Yi et al., 2007; Im et 22

al., 2006; Yang et al., 2005). Together, these data provide evidence for adverse pulmonary 23

effects that may exacerbate or facilitate asthma. 24

In lung tissue, Yang et al. (2005) identified three proteins up reg ulated and one protein 25

down regulated following 15 days of exposure to about 28 ppm formaldehyde. None of the 26

proteins corresponded with transcript changes reported by Sul et al. (2007). Interestingly, Sul et 27

al. (2007) reported that only two transcripts were significantly up regulated in the lung in 28

response to 5

– 10 ppm formaldehyde, while 19 were down regulated. In this regard, it is worth 29

considering that changes in proteins may not relate to their regulation but rather to their overall 30

percent compos ition in a cell (relative to other protein changes) before and after exposure. In 31

addition to transcript changes, Sul and colleagues (2007) reported DNA damage and lipid 32

peroxidation and noted that the observed down regulation of GR would facilitate oxida tive stress, 33

while the down regulation of phospholipase A2 (PLA2) might represent a mechanism for 34

mitigating lipid peroxidation. It is worth noting that an increase in either of these genes could 35

also be argued to support similar conclusions (i.e., that G R is up regulated to increase GSH 36

levels and that PLA2 up regulation explains lipid peroxidation); this highlights the problem with 37This document is a draft for review purposes only and does not constitute Agency policy.

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- 388 interpreting these data. Nevertheless, these studies indicate adverse effects in the rodent lung in 1

response to 5

– 30 ppm fo rmaldehyde. 2

In a hypothesis

- driven study by Yi et al. (2007), formaldehyde exposure was shown to 3

increase lung ADH3 levels. Several studies indicate that allergic responses and hyperreactivity 4

are uncoupled and may relate to ADH3 expression and activity . Que et al. (2005) has shown 5

that, in a rodent asthma model, ADH3 knockout mice exhibit similar signs of inflammation but 6

are protected from bronchoconstriction. Lino dos Santos Franco et al. (2006) provided evidence, 7

in rodents, that formaldehyde may i nduce inflammatory responses (e.g., leukocyte infiltration in 8

the lung) through neurogenic mechanisms but that bronchial tone is mediated by NO. The latter 9

effect is likely to be mediated by S

- nitrosoglutathione GSNO and thus influenced by ADH3. 10

Interest ingly, the single nucleotide polymorphisms (SNPs) that Wu et al. (2007) reported as 11

linked to other polymorphisms in the promoter region was the one demonstrating protection 12

against asthma. Hedberg et al. (2001) reported that at least one SNP in the promo ter region 13

reduced ADH3 expression. Together, these data indicate that reduced ADH3 expression might 14

lower GSNO turnover and bronchial tone, thereby reducing signs of asthma. In this regard, it is 15

conceivable that wheezing and bronchoconstriction are the symptoms that lead to medical 16

intervention and not the inflammation per se. Thus, while ADH3 polymorphisms may not cause 17

asthma, ADH3 polymorphisms may influence hyperresponsivity and the likelihood of asthma 18

diagnosis. This is discussed further in Sect ion 4.6 on susceptible populations. Formaldehyde 19

has been shown to accelerate GSNO breakdown (Staab et al., 2008a; Yi et al., 2007); thus, 20

pulmonary responses to formaldehyde may represent a balance between mechanisms that induce 21

NO (i.e., inflammation) a nd those that terminate GSNO (i.e., ADH3). 22

In regard to

- omics changes in blood samples, the apparent limited distribution of 23

formaldehyde may suggest that these changes are secondary to effects at sites of contact but 24

could also indicate systemic distri bution. As noted elsewhere in this report, bradypnea can 25

induce changes in dosimetry as well as changes in core body temperature and blood gases 26

(hypoxia itself induces hypothermia in rodents, and thus the reduction in minute volume and gas 27

changes may bo th contribute to hypothermia). These physiological responses (hypothermia and 28

hypoxia) surely induce changes in gene expression. Observed gene and protein changes in the 29

blood following formaldehyde exposure could also relate to irritation and inflammati on at sites 30

of contact. In this regard, Im et al. (2006) reported changes in cytokines indicative of Th

- 2 31

responses. Altogether, the authors identified 32 proteins altered in the plasma of rats exposed to 32

formaldehyde. Although no coherent mechanisms ar e apparent from these changes, the authors 33

posited that they could serve as biomarkers for formaldehyde exposure. The concordance of 34

such changes across species remains to be demonstrated. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 389 Li et al. (2007) identified dose

- response relationships for six ge nes in human blood 1

samples that were putatively associated with formaldehyde exposure. Three of these genes are 2

reported to inhibit apoptosis and were posited as supporting in vitro data by Tyihak et al. (2001); 3

however, Li and colleagues (2007) did not r eport any increase in blood cell count or in Hs 680.Tr 4

cell counts in vitro (i.e., these changes were not phenotypically linked to changes in cell kinetics 5

or hematology). However, these findings are not inconsistent with those of Hester et al. (2003) 6

tha t indicated no significant increase (or decrease) in nine genes involved in apoptotic pathways. 7

Finally, serum and glucocorticoid

- induced protein kinase 1 (SGK1) was elevated in blood 8

samples and was posited to relate to possible inflammatory and immune r esponses. 9

10

4.4.7. Immunotoxicity 11

Results from studies that evaluated the immunotoxicity of formaldehyde are mixed. For 12

example, most human studies that investigated systemic immune effects by measuring increases 13

in formaldehyde

- specific IgE are negative (Doi et al., 2003; Kim et al., 2001; Palcynski et al., 14

1999; Krakowiak et al., 1998; Wantke et al., 1996; Grammer et al., 1990). Vandenplas et al. 15

(2004) reported a transiently positive increased formaldehyde

- specific IgE titer in occupationally 16

exposed w orkers. In contrast, Thrasher et al. (1990, 1988, 1987) reported positive 17

formaldehyde

- specific IgE titers in small (six to eight person) case studies of exposed workers, 18

and Carraro et al. (1997) reported elevated IgE titers in smokers. Grammer et al. ( 1990) did not 19

report any differences in albumin IgE in formaldehyde

- exposed workers compared with controls. 20

In a residential study, Pross et al. (1986) found that formaldehyde insulation in homes had no 21

effect on tested human immunologic parameters. 22

One s tudy suggests that immune system parameters are perturbed by formaldehyde 23

exposure. Lyapina et al. (2004) reported decreased immune resistance in all 29 workers exposed 24

to formaldehyde. This effect was associated with decreased neutrophil respiratory bur st activity. 25

A LOAEL of 700 ppb was established. 26

Results from animal studies are mixed as to whether formaldehyde causes 27

immunotoxicity. Leach et al. (1983) reported systemic immunomodulation in F344 rats that was 28

attributed to formaldehyde exposure, but the formaldehyde effects on measures of humoral and 29

cell

- mediated immunity were not confirmed in B6C3F1 mice (Dean et al., 1984). Jakab et al. 30

(1992) detected no differences in phagocytic ability of alveolar MPs from mice after 31

formaldehyde exposure. Fo rmaldehyde

- exposed rats that were injected with pneumococcus 32

antigen or tetanus toxoid produced antibodies in amounts similar to nonexposed, infected control 33

animals (Holmström et al., 1989b). 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 390 However, specific immune parameters appear to be affected by fo rmaldehyde exposure. 1

For example, increased host resistance and hydrogen peroxide release from peritoneal MPs were 2

reported and confirmed (Adams et al., 1987; Dean et al., 1984) and suggest a putative role for 3

ROS. Increased host resistance may be mediat ed by formaldehyde

- induced chronic 4

inflammation and respiratory mucosal damage that causes an up regulation in MPs and therefore 5

increases host immunity. Jakab et al. (2002) reported reduced pulmonary bacterial resistance in 6

mice after exposure to formald ehyde, as determined by increased bacterial loading. This result 7

contrasts with Dean et al. (1984) and is attributed to differential exposure regimens and 8

experimental design. 9

10

Mode of action 11

Circulating immune cells present in the mouth and upper airways , such as intraepithelial 12

lymphocytes, direct a local inflammatory response but may also contribute to systemic responses 13

through secreted cytokines and soluble factors released into the bloodstream (Togias, 1999). 14

Altered host resistance and hydrogen pero xide release from peritoneal MPs were reported 15

and confirmed (Adams et al., 1987; Dean et al., 1984) and suggest a putative role for ROS. 16

Indeed, increased neutrophilic ROS have been associated with formaldehyde

- induced dermatitis 17

(Gorski et al., 1992), a nd, conversely, decreased neutrophil respiratory burst activity has been 18

shown in workers with history of formaldehyde

- induced respiratory tract inflammation (Lyapina 19

et al., 2004). Oxidative stress may occur directly as a result of formaldehyde exposure or as a 20

secondary consequence to inflammatory responses. 21

22

4.4.8. Effects on the Nervous System 23

There is considerable evidence that formaldehyde exposure causes adverse effects on the 24

nervous system following inhalation at relatively low exposure levels bu t little or no information 25

regarding a possible mechanism of action for these effects. Data regarding adverse effects on the 26

nervous system following oral exposure are very limited, reflecting a data gap in this area. 27

Relevant data in animals and humans for several types of neurological endpoints, following 28

inhalation exposure, are summarized below. 29

30

4.4.8.1. Irritant Threshold Detection 31

Humans are exquisitely sensitive to the irritant properties of formaldehyde, as has been 32

discussed previously (see S ection 4.1.1.1). Animal data confirm the irritant properties of this 33

compound at very low concentrations (Wood and Coleman, 1995). 34

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

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- 391 4.4.8.2. Behavioral Effects 1

Limited data in humans, as well as more robust data in animals, provide evidence of 2

behavioral changes following exposure to formaldehyde at levels as low as 0.1 ppm. Studies in 3

animals have found effects that persist for days to weeks after termination of exposure. In spite 4

of significant limitations, the available human data are consistent with the animal findings. 5

Several types of behavior have been evaluated in animals following formaldehyde 6

exposures. The most consistent findings, demonstrated by multiple laboratories and in multiple 7

species, have been changes in motor activity, habituation, and learning/memory task 8

performance. Motor activity and habituation have been evaluated under a variety of exposure 9

conditions, using both rats and mice. Consistent decreases in activity have been seen in adult 10

animals (Malek et al., 2004, 2003 a, b; U sanmaz et al., 2002). Senichenkova (1991) and 11

Sheveleva (1971) also found changes in motor activity in offspring following in utero exposure, 12

including decreased habituation in juvenile rats exposed in utero. Decreased performance in 13

learning and/or memo ry paradigms have been seen in multiple laboratories, also in both rats and 14

mice (Lu et al., 2008; Malek et al., 2003c; Pitten et al., 2000). 15

Data from controlled human exposures are very limited, but studies have shown 16

decreased performance in addition ta sks and reaction time tasks following acute exposures to 17

formaldehyde (Lang et al., 2008; Bach et al., 1990). In contrast, Andersen and Molhave (1983) 18

indicated they found no change in performance on several types of tasks (including addition, 19

multiplicat ion, and card punching) following acute exposure to volunteers, but supporting data 20

were not provided. 21

Data for humans are also available from epidemiology studies of individuals who were 22

occupationally exposed to formaldehyde. A variety of neurobehaviora l deficits, including lack 23

of concentration and loss of memory, disturbed sleep, impaired balance and dexterity, and 24

changes in mood, were identified (Kilburn et al., 1987, 1985). However, most of the individuals 25

evaluated in these studies were also occup ationally exposed to other solvents, raising questions 26

regarding possible confounding of the results due to multiple exposures. In addition, the 27

formaldehyde exposure information provided in the studies is not sufficient to permit a reliable 28

dose

- response assessment for the effects identified. The types of effects seen in humans in the 29

available epidemiology studies are, however, consistent with those seen in available animal 30

studies. 31

In general (and noting the differences in exposure paradigms and types of tasks), 32

behavioral effects in animals and humans appear to occur at similar exposure levels. Animal 33

studies demonstrated LOAELs as low as 100 ppb following acute or repeated exposures (Malek 34

et al., 2003b, c); human controlled exposure studies have fou nd effects in that same range, with 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 392 LOAELs of approximately 300 ppb following acute exposures (Lang et al., 2008; Bach et al., 1

1990). 2

3

4.4.8.3. Neurochemistry, Neuropathology, and Mechanistic Studies 4

Limited data are available regarding neurochemical and neuropathological sequelae of 5

formaldehyde exposure. Studies from one laboratory (Sorg et al., 2004, 2001) have suggested 6

that behavioral sensitization to formaldehyde is linked to alterations in HPA control of 7

corticosterone and changes in mesolimbic dop amine pathways. Neurochemical changes in 8

response to formaldehyde exposure have also been documented in other laboratories (Fujimaki et 9

al, 2004b; Hayashi et al., 2004). Some of these data appear to be conflicting, and there are no 10

definitive data suppor ting a specific mechanism for formaldehyde effects on the nervous system 11

at this time. Neuropathological data are also limited, although data from one laboratory 12

(Sarsilmaz et al., 2007; Aslan et al., 2006) suggest a concern for changes in brain structure in 13

neonatal rats following exposure at 6 or 12 ppm. No human data are available that address these 14

endpoints. However, a prospective cohort study of nearly one million people followed for 15 15

years reported strongly significant dose

- response associations between death from ALS and 16

exposure to formaldehyde of a known duration, with longer exposures associated with greater 17

risk (Weisskopf et al., 2009). This large, well

- designed prospective cohort study strongly 18

supports the causal association of neuropath ological effects in humans following long

- term 19

formaldehyde exposure. 20

21

4.4.8.4. Summary 22

Overall, there is strong evidence that formaldehyde exposure via inhalation may cause 23

adverse effects on nervous system function in experimental animals at relatively low levels of 24

exposure (LOAELs as low as 100 ppb). Although human data regarding neurotoxicity following 25

formaldehyde inhalation are limited, available data provide support that the types of effects seen 26

in humans are similar to those found in animal stud ies. Evidence from available human 27

controlled inhalation exposure studies indicates that humans may be affected at doses similar to 28

those used in animal studies; however, the human data are extremely limited. 29

There are insufficient data to evaluate the po tential for neurotoxicity following oral 30

exposure to formaldehyde. Limited evaluations of brain weight or histopathology in available 31

chronic or subchronic oral studies found no evidence of formaldehyde

- induced changes (Til et 32

al., 1989, 1988; Tobe et al. , 1989; Johannsen et al., 1986). However, reliable studies examining 33

nervous system function or focused studies of neuropathology following oral exposure to 34

formaldehyde are not available. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 393 4.4.8.5. Data Gaps 1

Major data gaps were found regarding the evalu ation of changes in nervous system 2

structure or function following formaldehyde exposure by both the inhalation or oral routes. 3

With respect to inhalation exposure, none of the available human studies resulted in data 4

sufficient to conduct a reliable dose

- response assessment for changes in nervous system function. 5

Most of the available animal inhalation studies used short exposure durations (acute or short

6

term), precluding a reliable evaluation of neurotoxicity following chronic exposure. Available 7

data for neurodevelopmental exposures are also quite limited, consisting of evaluation of 8

neuropathology in only one brain region and functional evaluations focused only on changes in 9

motor activity. 10

Major data gaps also exist regarding neurotoxicity following oral exposure, with no 11

relevant human data and extremely limited animal data. Available oral exposure studies were 12

insufficient to permit a reliable evaluation of the potential for neurotoxicity following oral 13

exposure to formaldehyde. 14

15

4.4.9. Reproducti ve and Developmental Toxicity 16

A number of studies have been identified that indicate an effect of formaldehyde 17

exposure on reproductive and developmental outcomes. Human data are described in Section 18

4.1.1.7, and animal studies are addressed in Section 4. 2.1.7 of this document. 19

20

4.4.9.1. Spontaneous Abortion and Fetal Death 21

Several epidemiologic studies reported increases in risk of spontaneous abortion 22

following maternal occupational formaldehyde exposure (Taskinen et al., 1999, 1994; John et al., 23

1994; Seitz and Baron, 1990; Axelsson et al., 1984). While these finding have been questioned 24

(Collins et al., 2001b), upon careful examination, none of the principal biases in epidemiologic 25

studies, including information bias, selection bias, and confounding, appear to be more likely 26

causes of these reported findings than the conclusion that they may reflect an underlying causal 27

process. While each of these occupational studies focused on women who were co

- exposed to 28

formaldehyde and other chemicals, the occup ational groups were quite different and had 29

different sets of co

- exposures. The woodworkers in the Taskinen et al. (1999) study were 30

potentially co

- exposed to organic solvents related to painting and lacquering, dusts, and phenols, 31

none of which was shown to be an independent predictor of adverse risk. The cosmetologists 32

studied by John et al. (1994) were co

- exposed to hair dyes, bleach, alcohol

- based disinfectants, 33

and chemicals specific to services, such as fingernail sculpturing, but, in analyses that were 34

specifically adjusted for other work exposures and their potentially confounding effects, the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 394 investigators reported an increased risk for the use of formaldehyde

- based disinfectants. The 1

laboratory workers studied by Axelsson et al. (1984) were pote ntially co

- exposed to a wide range 2

of solvents, but the miscarriage rate was highest among those exposed to formaldehyde. For a 3

potential confounder to entirely explain an observed effect of another exposure, it must be more 4

strongly associated with the a dverse outcome. It does not appear that the collective results of 5

formaldehyde exposures associated with increased risk of spontaneous abortion

— often in spite 6

of exposures being crudely measured

— can be explained by information bias or confounding. 7

Taken t ogether, these findings are consistent with an adverse effect of formaldehyde 8

exposure on the risk of pregnancy loss. The single study with the strongest quantitative 9

assessment of that risk is Taskinen et al. (1999), and the results presented are of suff icient quality 10

to support quantitative risk assessment by using the LOAEL/NOAEL approach. 11

This study was a well

- designed population

- based case

- control study that appears to have 12

been well executed and appropriately analyzed. The study population of Finnis h women was 13

well defined and adequately selected to allow for meaningful comparisons of health effects 14

between individuals with different levels of exposure to formaldehyde. The participation rate 15

was 64%, which is low enough to raise a concern about the potential for selection bias. 16

However, the authors noted that selection bias has not influenced the results of other 17

reproductive epidemiology studies reporting results on smoking, irregular menstruation, and 18

earlier miscarriages, which are known to lengt hen the time to pregnancy (Bolumar et al., 1996; 19

Sallmén et al., 1995; Baird and Wilcox, 1985). Furthermore, there is no evidence to support 20

conjecture that an individual’s decision to participate in this study would be differential with 21

respect to their workplace formaldehyde exposures while being nondifferential with respect to 22

the other exposures of interest, including organic solvents, wood dust, and phenols. Since the 23

women who chose to participate in this study were not likely to be aware of the spe cific 24

hypotheses under investigation nor could they have known the formaldehyde exposures that were 25

independently estimated by an industrial hygienist, selection bias is not a likely explanation for 26

the findings of adversity. 27

Data on pregnancy history, inc luding spontaneous abortions, were collected by 28

questionnaire. Spontaneous abortion is the most common adverse outcome of pregnancy (Klein 29

et al., 1989), and retrospective self

- report of spontaneous abortion has been found to match well 30

with prospectively collected reproductive histories (Wilcox and Horney, 1984). Many 31

spontaneous abortions, however, are missed with self

- reporting, with the magnitude likely 32

exceeding 25%, but only rarely do women self

- report false positive events (Wilcox and Horney, 33

1984) . The effect of such an undercount is to cause a bias towards the null when the likelihood 34

of undercounting is unrelated to formaldehyde exposure. The implication is that the observed 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 395 association of increased risk of spontaneous abortion associated with occupational exposure to 1

formaldehyde may be an underestimation of the true risk. 2

The findings by Taskinen et al. (1999) of reduced fertility and increased risk of 3

spontaneous abortion are internally consistent and coherent with other reports of increased risk 4

of pregnancy loss associated with exposure to formaldehyde (John et al., 1994; Taskinen et al., 5

1994; Seitz and Baron, 1990; Axelsson et al., 1984). Absent evidence of alternative explanation 6

for these findings, it is concluded that exposure to forma ldehyde is associated with pregnancy 7

loss and diminished fertility. 8

In animal studies, Sheveleva (1971) noted an increase in preimplantation loss in rats 9

exposed to 0.04 and 0.4 ppm formaldehyde by inhalation on GDs 1

– 19, and Kitaev et al. (1984) 10

observed evidence of degeneration in harvested embryos on GD 2, following 4 months of 11

maternal inhalation exposure to 0.41 ppm formaldehyde in rats. In a second series of tests 12

reported in Kitaev et al. (1984), female rats were exposed to 0.41 and 1.22 ppm formald ehyde for 13

4 months to test the hypothesis that the embryo degeneration could have been the result of 14

disrupted reproductive hormone levels in the dams. Ovarian weight and blood levels of LH were 15

increased at 0.41 ppm (but not at 1.22 ppm), and significant ly increased levels of FSH were 16

observed at 1.22 ppm. Kitaev et al. (1984) proposed that effects at the 0.41 ppm might be related 17

to disruption of the hypothalamic

- pituitary axis and that at the higher exposure level (1.22 ppm) 18

frank toxic effects to the embryo were observed. The increased FSH levels at 1.22 ppm may also 19

be indicative of hormonal perturbations induced by formaldehyde exposure that could affect 20

pregnancy maintenance in humans. The finding of treatment

- related increased preimplantation 21

los s in rats appears to support the evidence of spontaneous abortion in the epidemiologic data. In 22

addition, a dominant lethal study in rats by Odeigah (1997) identified significant 23

postimplantation loss following pre

- mating I.P. formaldehyde exposures to ma les, suggesting a 24

potential MOA involving germ cell toxicity. Nevertheless, a number of developmental toxicity 25

studies in rats did not report treatment

- related embryolethality following gestation exposures to 26

formaldehyde. These included inhalation studi es by Martin (1990), Saillenfait et al. (1989), and 27

Kilburn and Moro (1985), a series of studies by Gofmekler and Bonashevskaya (1969), 28

Gofmekler (1968), and Pushkina et al. (1968), and studies by Senichenkova and Chebotar (1996) 29

and Senichenkova (1991). It is noted, however, that, to the extent that these studies evaluated 30

embryonic or fetal death, the observations were conducted late in gestation and the studies may 31

not have been designed to detect the preimplantation losses as observed in Kitaev et al. (1984) 32

and Sheveleva (1971). Additionally, a number of the reports for these studies did not include 33

sufficient details to engender a high degree of confidence in the reported results. Fetal death was 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 396 also not observed in oral studies with formaldehyde i n beagle dogs (Hurni and Ohder, 1973) and 1

rats (Seidenberg and Becker, 1987). 2

3

4.4.9.2. Congenital Malformations 4

The effect of occupational exposures to formaldehyde on the incidence of congenital 5

malformations was examined by D ulskiene and Gražulevičiene (2005), Taskinen et al. (1994), 6

and Hemminki et al. (1985). Results were mixed. 7

In animal studies, the most frequently observed structural anomaly noted following 8

inhalation exposures to formaldehyde during gestation was a dela y in fetal (i.e., 1 st stage) testes 9

descent (Senichenkova and Chebotar, 1996; Senichenkova, 1991; Kilburn and Moro, 1985), 10

although similar findings were not reported by Saillenfait et al. (1989) or Martin (1990) in what 11

appear to be well

- conducted prenata l developmental toxicity studies. No study in the available 12

database specifically examined the 2 nd stage of postnatal testes descent in pups. Thus, there is no 13

evidence to determine if the observed effect represented a developmental delay or if it was 14

re lated to disruptions in male reproductive tract ontogeny, which is dependent on normal levels 15

of fetal testicular testosterone and on the expression of insulin

- like hormone

- 3 (insl3) in fetal 16

Leydig cells (Klonisch et al., 2004). Senichenkova (1991) obser ved an increased incidence of 17

other organ anomalies following formaldehyde exposure during gestation; however, the 18

anomalies are not characterized in the report. Alterations on fetal organ weights and/or size were 19

noted in several studies (Kilburn and Mor o, 1985; Gofmekler, 1968), but it is difficult to 20

ascertain if these findings represented agenesis, hypoplasia, or evidence of systemic organ 21

toxicity. Histopathologic evaluation of pup tissues following maternal gestational exposures to 22

0.01 and 0.81 ppm formaldehyde was conducted by Gofmekler and Bonashevskaya (1969), 23

revealing reduced glycogen content in the myocardium, the presence of iron in hepatic Kupffer 24

cells, and a positive Schiff reaction in the basement membrane (indicating functional alteratio ns 25

in the renal tubule) at both exposure levels. 26

27

4.4.9.3. Low Birth Weight and Growth Retardation 28

A population

- based study (Gražulevičiene et al., 1998) reported an association between 29

atmospheric formaldehyde exposure and low birth weight, with an adjus ted OR of 1.37 (95% CI: 30

0.90

– 2.09). 31

A number of inhalation studies in rats identified reduced fetal weight as an adverse 32

outcome of in utero formaldehyde exposure and are supportive of the association noted in 33

humans. In a study that exposed pregnant rats to formaldehyde during GDs 6

– 20, Saillenfait et 34

al. (1989) observed significantly decreased male and female fetal rat weights (78 and 81% of 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 397 control values, respectively) at 40 ppm formaldehyde. In a study that exposed the dams from 1

GDs 6

– 15, Martin (199 0) found decreased fetal weights at exposure levels of 5 and 10 ppm. In 2

both studies, observations of reduced or delayed skeletal ossification (i.e., the thoracic vertebrae 3

in Saillenfait et al. [1989] and the pubes and ischia bones in Martin [1990]) were consistent with 4

the fetal weight deficits. Kilburn and Moro (1985) also reported fetal body weight decreases in 5

rats at an inhalation exposure level of 30 ppm. Conversely, increased fetal body weight as 6

compared with controls (generally considered to be non

- adverse) was noted by Gofmekler 7

(1968) and Pushkina et al. (1968) at maternal formaldehyde exposure levels of 0.1 and 0.81 ppm 8

administered for approximately 2

– 3 weeks prior to mating and then throughout gestation. 9

Increased fetal weight was also not ed in rats by Senichenkova (1991) and Senichenkova and 10

Chebotar (1996) following maternal exposures to 0.41 ppm formaldehyde throughout gestation. 11

Studies that assessed the effects of oral administration of formaldehyde on development 12

are quite limited. T he only oral study identified that found a treatment

- related effect on offspring 13

growth was a study using beagle dogs (Hurni and Ohder, 1973). In this study, formaldehyde was 14

administered at doses of 3.1 or 9.4 mg/kg

- day in the feed during gestation, and pup weight 15

decrements at postnatal week 8 were 6.3 and 12% in the low

- and high

- dose groups, respectively. 16

17

4.4.9.4. Functional Developmental Outcomes (Developmental Neurotoxicity) 18

Indications of effects on the developing nervous system were observed in s everal rodent 19

studies, although no similar epidemiologic findings in children were identified. These studies 20

(Sarsilmaz et al., 2007; Aslan et al., 2006; Weiler and Apfelbach, 1992; Senichenkova, 1991; 21

Sheveleva, 1971) are described in detail in Section 4 .2.1.6. In the studies by Aslan et al. (2006) 22

and Sarsilmaz et al. (2007), neonatal rats were exposed to formaldehyde for 30 days at 6,000 or 23

12,000 ppb. Decreases in the volume of discrete areas of the brain were observed at both 24

exposure levels in both studies, and, additionally, decreased cell numbers were noted in a region 25

of the hippocampus in the Sarsilmaz et al. (2007) study. Weiler and Apfelbach (1992) exposed 26

juvenile rats to 0.25 ppm formaldehyde for 130 days or adult rats to 0.5 ppm formaldehy de for 27

90 days. Olfactory thresholds measured in this study were significantly higher in the rats that 28

had been exposed as juveniles than in those that had been exposed only as adults. Sheveleva 29

(1971) observed alterations in spontaneous mobility in 1

- a nd 2

- month

- old pups from dams that 30

had been exposed to formaldehyde at 0.04 or 0.4 ppm throughout gestation. In the Senichenkova 31

(1991) study, maternal rats were exposed to 400 ppb formaldehyde during GDs 1

– 19, and 32

functional observational testing was con ducted on the juvenile offspring. Changes in open

- field 33

motor activity, exploratory activity, and habituation were observed in the offspring. 34

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

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- 398 4.4.9.5. Male Reproductive Toxicity 1

A number of laboratory animal studies have reported effects of formaldehyd e exposure 2

on male reproductive system endpoints. These effects include decreased testes weight, changes 3

in Leydig cell quantity and quality, degeneration of seminiferous tubules, decreased testosterone 4

levels, alterations in biomarkers of toxicity in the testes, and alterations in sperm measures 5

(Galilapour et al., 2007; Xing et al., 2007; Zhou et al., 2006; Özen et al., 2005, 2002; Sarsilmaz 6

et al., 1999; Odeigah, 1997; Majumder and Kumar, 1995; Chowdhury et al., 1992; Til et al., 7

1989, 1988; Tobe et al. , 1989; Johanssen et al., 1986; Maronpot et al., 1986; Cassidy et al., 1983; 8

Appelman et al., 1982; Guseva, 1972). Following concurrent exposures to formaldehyde in air 9

and drinking water for 6 months, Guseva (1972) found decreases in testicular nucleic a cid levels. 10

In a study conducted by Chowdhury et al. (1992), rats were administered I.P. injections of 11

formaldehyde for 30 days, and evidence of decreased testes weight, serum testosterone levels, 12

and Leydig cell quality was observed. Sarsilmaz et al. (1 999) followed up on these findings 13

(exposing male rats to formaldehyde via inhalation at 10 and 20 ppm for 4 weeks) and found 14

decreases in Leydig cell quantity and the percentage of cells with normal nuclei. Hypothesizing 15

that the reported decreases in Le ydig cell quality may have been the result of oxidative stress and 16

damage, Özen et al. (2002) evaluated biomarkers of such changes and found that testicular zinc 17

and copper levels were decreased and iron levels were increased following exposures of adult 18

m ale rats to 10 and 20 ppm formaldehyde for 4 or 13 weeks. Additionally, relative testes weight 19

was decreased in a dose

- and duration

- dependent manner, although this effect had not been 20

observed by Sarsilmaz et al. (1999). Özen et al. (2005) noted decreas ed serum testosterone 21

levels, decreased seminiferous tubule diameters, and increased levels of heat shock protein in 22

spermatogonia, spermatocytes, and spermatids of rats following 91 days of exposure to 10 ppm 23

formaldehyde. A study by Golalipour et al. (2 007) observed decreased numbers of testicular 24

germ cells, altered sprmatogenesis, and reduced seminiferous tubular diameter and epithelial 25

height in rats following 18 weeks of formaldehyde inhalation exposure; the severity of the 26

seminiferous tubule pathol ogy was positively correlated to the number of hours/week of 27

exposure. Zhou et al. (2006) found decreased testis weight, atrophy of seminiferous tubules, 28

edematous interstitial tissue, and alteration of epididymal sperm count, morphology, and motility 29

in rats after 2 weeks of formaldehyde exposure at 8 ppm. Abnormal sperm were also observed in 30

mice by Xing et al. (2007) after 13

- weeks of inhalation exposure at 16.9 ppm, and Cassidy et al. 31

(1983) reported sperm abnormalities in rats following a single oral dose of 200 mg/kg. 32

Additionally, Majumder and Kumar (1995) observed significantly reduced sperm count, motility, 33

and viability following 30 days of I.P. injection of 10 mg/kg

- day formaldehyde to male rats. 34

Also in this study, the ability of formaldehyde to affect sperm parameters was confirmed with in 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 399 vitro testing. A study conducted by Odeigah (1997) demonstrated epididymal sperm count and 1

morphology abnormalities following five I.P. doses of ≥0.125 mg/kg formaldehyde and 2

additionally identified domin ant lethal effects (decreased live embryos and increased dead 3

implants) following mating of treated male rats with untreated females. 4

Although Til et al. (1989) reported low incidences of Leydig cell tumors in 5

formaldehyde

- treated rats in a chronic drinkin g water study, no alterations in testes weight or 6

histopathologic lesions of the testes were observed in subchronic inhalation studies conducted by 7

Appleman et al. (1982) or Maronpot et al. (1986) or in subchronic or chronic oral studies by 8

Johanssen et al . (1986), Til et al. (1988), or Tobe et al. (1989). 9

No epidemiologic studies have identified an association between formaldehyde exposure 10

and alterations in the male reproductive system (e.g., see Ward et al. [1984]). 11

12

4.4.9.6. Female Reproductive Toxicit y 13

The available database for the assessment of the effects of formaldehyde exposure on the 14

female reproductive system was limited. In addition to the findings of spontaneous abortions, as 15

described above, Taskinen et al. (1999) examined fecundability in a cohort of healthy female 16

wood

- processing industry workers and found that conception was significantly delayed in 17

women who were occupationally exposed to formaldehyde. The FDR, a ratio of average 18

incidence densities of pregnancies for exposed female empl oyees compared with unexposed 19

female employees, was lower in women exposed to mean formaldehyde levels of approximately 20

0.33 ppm (range: 0.15

– 1.00 ppm) compared with controls (adjusted FDR = 0.64 [95% CI: 21

0.28

- 0.92]). An FDR <1.0 is indicative of delayed conception, which is an indicator of reduced 22

fertility. The subfertility observed in this study is supportive of the association observed 23

between formaldehyde exposure and spontaneous abortion, since subclinical pregnancy losses 24

are increased in women wit h compromised fertility (Gray and Wu, 2000; Hakim et al., 1995), 25

and both spontaneous abortion and subfertility can be related to exposure to environmental 26

toxicants (Correa et al., 1996). 27

As described above, formaldehyde exposures to female rats resulted in decreased ovarian 28

weight and altered LH and FSH levels (Kitaev et al., 1984). Maronpot et al. (1986) reported 29

endometrial hypoplasia and lack of ovarian luteal tissue in female mice exposed to 40 ppm 30

formaldehyde for 13 weeks. Additionally, it is note d that, in developmental toxicity studies that 31

included repeated exposures of dams before mating and/or during gestation, reports of adverse 32

pregnancy outcomes were few. Gofmekler (1968) reported an increase in pregnancy duration 33

and decrease in litter si ze; however, this finding was not observed in other studies. 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 400 With the exception of spontaneous abortion and increased time to pregnancy, associations 1

of formaldehyde exposure with adverse female reproductive system outcomes were not observed 2

in the availab le epidemiologic data. 3

4

4.4.9.7. Mode of Action 5

A strong case cannot be made for any one MOA that explains one or more of the 6

reproductive and developmental outcomes observed in formaldehyde epidemiologic or 7

toxicology studies. This is due to a number of issues, including the following: 8

(1) inconsistencies in study findings for the toxicology studies, which may be explained by study 9

quality issues (see detailed descriptions of studies in Sections 4.1 and 4.2); (2) few studies that 10

allow for comparisons be cause no study was performed with the same study design (e.g., stage of 11

exposure, dose, species, and strain); (3) few mechanistic studies available to test hypothesized 12

MOAs; and (4) a bias that is pervasive in the formaldehyde literature that outcomes obs erved 13

beyond the POE (the nose) are not expected from inhalation exposure, which is the route of 14

exposure for most of the developmental and reproductive studies. This discussion presents 15

putative MOAs that have been hypothesized by study authors and the s tudies that support the 16

hypothesized MOAs. The four hypothesized MOAs are not mutually exclusive. They could be 17

acting alone for certain endpoints (in which case the others are not operable) or in concert for 18

certain endpoints. 19

The focus of this discus sion is on analyzing possible MOAs for the developmental and 20

reproductive outcomes that were noted most consistently, across toxicology studies and, in some 21

cases, across human and animal studies. These outcomes include developmental delays, fetal 22

loss, a nd sperm quality and quantity effects. 23

An endocrine

- disrupting MOA is supported by some of the reproductive and 24

developmental epidemiology and toxicology studies. For example, the decreases in fetal body 25

weight (Martin, 1990; Saillenfait et al., 1989), de layed ossifications (Senichenkova and 26

Chebotar, 1996; Senichenkova, 1991; Martin, 1990; Saillenfait et al., 1989), and delayed 27

eruption of incisors (Senichenkova, 1991) noted in rats after gestational exposure to 28

formaldehyde are consistent with developmen tal delays. In turn, developmental delays can result 29

from effects on the hypothalamic

- gonadal

- pituitary axis in the dam that cause hormonal level 30

changes in the pup; however, hormone levels in pups were not measured. Kilburn and Moro 31

(1985) also observed organ size changes and undescended testes after developmental 32

formaldehyde exposure. These outcomes can also be explained by an endocrine MOA. There 33

are three studies that directly tested for changes in hormones after formaldehyde exposure. 34

Kitaev et a l. (1984) observed ovarian weight and serum LH and FSH increases after inhaled 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 401 formaldehyde in adult female rats. Chowdhury et al. (1992) assessed serum testosterone levels 1

in adult rats after formaldehyde IP exposure and found significant decreased testo sterone and 2

testes weights and a decrease in 3

- β, **∆**

- 5

- hydroxy steroid dehydrogenase in Leydig cells, 3

suggesting that formaldehyde affects steroidogenesis. Özen et al. (2002) also reported 4

significant serum testosterone level decreases as well as decreased mean seminiferous tubule 5

diameters. Furthermore, the steroidogenesis MOA leading to reduced testosterone is also 6

consistent with the sperm quality and quantity decrements observed in the studies by Özen et al. 7

(2002), Sarsilmaz et al. (1999), and Odeigah (1997) studies. 8

In human studies, delayed time to pregnancy and increased incidence of spontaneous 9

abortion (Taskinen et al., 1999), consistent with some study findings from the toxicology 10

literature, could also be explained by an endocrine MOA. Alterat ions in hormone levels could 11

lead to pregnancy maintenance problems. Extrapolating the Chowdhury et al. (1992) results of 12

the steroidogenesis MOA to females, formaldehyde exposure could affect progesterone levels 13

required for pregnancy. However, progeste rone levels were unchanged in the female rat in the 14

one study that assessed progesterone (Kitaev et al., 1984). Consistent with an endocrine 15

mediated MOA, Maronpot et al. (1986) observed endometrial hypoplasia and lack of ovarian 16

luteal tissue in females exposed to formaldehyde. 17

A second hypothesized MOA for some of the developmental and reproductive outcomes 18

is genotoxicity of the gametes. Such an MOA could explain pregnancy loss in humans 19

(Taskinen, et al., 1999) and preimplantation loss in animal studi es (Xing et al., 2007; Kitaev et 20

al., 1984; Sheveleva, 1971) and fetal viability (e.g., litter size decreases) after formaldehyde 21

exposure. Consistent with male gamete genotoxicity, Odeigah (1997) and Xing et al. (2007) 22

observed reduced fertile matings an d live embryos, and increased dead implants in a dominant 23

lethal study. 24

Oxidative stress/damage is another MOA that is consistent with testicular toxicity, sperm 25

effects, and reduced embryo viability. Özen et al. (2002) investigated the mechanism of 26

oxida tive stress being responsible for the testes quality effects by assessing testicular iron, 27

copper, and zinc levels. Zinc and copper levels were reduced in the rat testes, consistent with an 28

oxidative stress MOA. Özen et al. (2002) also reported increased iron levels and decreased zinc 29

levels in the lung, consistent with oxidative stress. Another study (Zhou et al., 2006) that 30

investigated the oxidative stress MOA in the testes observed significant changes in oxidative 31

stress biochemical markers (decrease s in SOD, GPX, GSH, and an increase in MDA levels). 32

The authors also assessed the protective effect of coadministration with vitamin E, an 33

antioxidant, on decreased testes weight, biochemical alterations, histopathologic effects, and on 34

sperm count, motil ity, and morphology. The study of Pushkina et al. (1968) found reduced 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 402 levels of Vitamin C, another antioxidant, in the fetus and maternal liver after formaldehyde 1

exposure. 2

The MOAs proposed are not mutually exclusive and in fact could interact with one 3

another. For example, an endocrine MOA could lead to oxidative stress, and that oxidative stress 4

could lead to genotoxicity. 5

6

4.4.9.8. Data Gaps 7

The inhalation developmental toxicity studies conducted on formaldehyde and described 8

in Section 4.2.1.7 comp rise an adequate assessment of prenatal developmental toxicity for 9

application to inhalation reference concentration determination. The assessments of postnatal 10

developmental toxicity and of reproductive function following inhalation of formaldehyde are 11

l ess complete. It is notable that, although the database contains some studies that assess various 12

aspects of reproductive organ system toxicity, particularly in males, there is no assessment of 13

multigenerational reproductive function, such as would be eva luated in a two

- generation 14

reproductive toxicity study, nor is there an assessment of potential reproductive effects of 15

formaldehyde exposure in human males. 16

Adequate assessments of developmental and reproductive toxicity following oral 17

exposures to formal dehyde have not been conducted. 18

19

4.5. SYNTHESIS AND EVALUATION OF CARCINOGENICITY 20

4.5.1. Cancers of the Respiratory Tract 21

Epidemiologic studies of formaldehyde

- exposed workers provide sufficient evidence of a 22

causal association between formaldehyde exposure and upper respiratory tract (URT) cancers 23

(e.g., nasopharyngeal cancer (NPC; Section 4.1.2.1.1), nasal and paranasal cancers (Section 24

4.1.2.1.2), and other upper respiratory tract cancers (Section 4.1.2.1.3)). In addition, the 25

observational evidence from epi demiologic studies reporting associations between formaldehyde 26

exposure and increased risk of NPC supports a conclusion of a causal association for this specific 27

cancer. However, epidemiologic studies of rare outcomes such as NPC, which has an expected 28

in cidence of 1 per 100,000 people per year in the United States, do not typically have great 29

statistical power to rule out the null hypothesis (i.e. no association). However, the weight of 30

evidence of the several studies reviewed in Section 4.1.2.1.1 provid e an accumulation of 31

consistent observational evidence sufficient to exclude chance as an explanation for the 32

association. Additionally, there is insufficient evidence of consistent confounding or other bias 33

across the studies that were considered; thus, confounding and bias were also ruled out as 34

explanations for the observed association. The lack of a convincing and consistent alternative 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 403 hypothesis of causation − in spite of repeated examinations − further supports the conclusion that 1

the association b etween formaldehyde exposure and increased risk of NPC is causal. 2

The single strongest cohort study, Hauptmann et al. (2004), shows a statistically 3

significant exposure

- response relationship between formaldehyde exposure and URT cancers. 4

Hauptmann et al. (2004) demonstrated significant excess risk of NPC in exposed workers based 5

on U.S. population death rates (standardized mortality ratio [SMR] = 2.1; 95% confidence index 6

[CI] 1.05

– 4.21) in a large cohort of 25,619 industrial workers. In addition to the S MR based on 7

an external comparison population, relative risks (RRs) were presented based on internal 8

comparisons of workers in order to minimize potential selection bias due to the well known 9

healthy worker effect. Statistically significant exposure

- respo nse relationships between 10

increased formaldehyde exposure and increased risk of NPC were reported for two different 11

metrics of exposure (peak and cumulative exposure). Relative risks for NPC were also elevated 12

for increased duration of exposure to formald ehyde and for the average intensity of exposure. 13

These analyses controlled for potential confounders including calendar year, age, sex, race, and 14

pay category. While exposure measurement error is likely to be present in any epidemiologic 15

study, there was no evidence of any differential measurement error that could have produced the 16

observation of a spurious association. Any non

- differential measurement error would likely have 17

attenuated the effect of formaldehyde was smaller than that which would otherwi se have been 18

observed in the absence of measurement error. 19

The case

- control studies similarly also report associations between formaldehyde 20

exposure and cancer mortality for NPC. Although other risk factors for NPC (e.g., Epstein

- Barr 21

Virus) and the predo minant NPC histological sub

- type (SCC versus undifferentiated) vary 22

significantly across the world, case

- control studies consistently provide evidence of an 23

association between occupational exposure to formaldehyde and NPC (Vaughn et al., 1986a; 24

Vaughn et al., 2000; Roush et al., 1987; Hildesheim et al., 2001; West et al., 1993). In their 25

more recent study, Vaughn et al. (2000) used worker histories to estimate each individual 26

worker’s formaldehyde exposure. Workers with more than 1.10 ppm

- years of cumulat ive 27

exposure were found to be at significantly higher risk of NPC, with an odds ratio (OR) of 3.0 28

(95% CI 1.3

- 6.6) (Vaughn et al., 2000). Two different exposure metrics, duration of exposure 29

and cumulative exposure, were positively associated with increas ed risk of NPC, with a 30

significant test for trend ( p = 0.014 and 0.033, respectively). The OR also increased in 31

magnitude as the probability of “Ever” having occupational exposure increased, from OR = 1.6 32

among those whose exposure was judged to be “Poss ible, probable or definite” to OR = 13.3 33

among those with “Definite” exposure ( p

– trend < 0.001). 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 404 NPC histological sub

- type analysis indicates that these associations held for both SCC 1

and epithelial NPC, but not for the undifferentiating and nonkeritinizin g NPC (Vaughn et al., 2

2000). However, formaldehyde exposure is also associated with risk of NPC in Taipei, Taiwan, 3

where greater than 90% of the cases had nonkeritinizing and undifferentiated carcinomas and 4

less than 10% of the cases were diagnosed as hav ing SCCs (Hildesheim et al., 2001). These 5

reported associations were strengthened by considering higher probability of exposure (RR = 6

2.6; 95% CI 1.1

- 6.3), greater intensity of exposure (RR = 2.1; 95% CI 1

- 4.2) and EBV 7

seropositive cases (RR = 2.7; 95% CI 1.2

- 5.9) (Hildesheim et al., 2001). Case

- control studies 8

have also linked residential exposure to NPC, specifically for years of residence in mobile homes 9

(Vaughn et al., 1986b) and the use of mosquito coils in the Philippines (West et al., 1993). 10

Indep endent testing of 6 brands of East Asian mosquito coils evaluated the emission rates of 11

carbonyl compounds in the mosquito smoke and reported that formaldehyde and acetaldehyde 12

had the highest emission rates. Among the three experiments on each of the six brands, the 13

range of formaldehyde concentrations was from 0.87 μg/m 3 (1 ppb) to 25 μg/m 3 (31 ppb). 14

As a group, other URT sites of direct contact with formaldehyde upon inhalation (i.e., 15

salivary gland, mouth, nasal cavity and larynx) also showed evidence of a trend in increasing 16

relative risks with increasing average intensity and peak exposure in the Hauptmann et al. (2004) 17

cohort study, although these trends did not reach the level of statistical significance. The results 18

from other cohort studies and case

- control studies are mixed (between positive associations and 19

null findings) for associations between formaldehyde exposure and specific cancers of the URT 20

(IARC, 2006). For rare cancers, extremely large cohorts would be needed to have the statistica l 21

power to detect an association for tumors defined by individual sites (e.g., mouth, salivary gland, 22

hypopharnyx). Results vary in the smaller cohort studies, where a single case may result in an 23

elevated risk but taken together the evidence is considere d suggestive (Section 4.1.2). Case

24

control studies have been useful to better understand potential associations between 25

formaldehyde exposure and rare cancers of the URT. Luce et al. (2002) evaluated pooled data 26

from 12 case

- control studies and demonstra ted a statistically significant increased risk between 27

formaldehyde exposure and sinonasal cancer. A case

- control study by Gustavsson et al. (1998) 28

suggested an association between formaldehyde exposure and oral squamous cell carcinoma 29

(SCC), esophageal, and laryngeal cancers, with odds ratios (ORs) of 1.28, 1.90, and 1.45, 30

respectively. However, the individual ORs were not statistically significant. Hypopharyngeal 31

cancer was linked with formaldehyde exposure with an OR of 3.78 (95% CI 1.50

- 9.49) in 32

anot her case

- control study (Laforest et al., 2000). While the data on site

- specific cancers of the 33

URT is somewhat sparse, they are consistent with a carcinogenic hypothesis and in their large 34

cohort study, Hauptmann and colleagues (2004) concluded that in sp ite of the small numbers of 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 405 deaths from cancers of the URT, the positive associations with average intensity and peak 1

exposure were consistent with the carcinogenicity of formaldehyde at these sites of first contact. 2

3

Supporting animal evidence 4

Inhalatio n exposure: 5

Animal studies, primarily rodent bioassays, strongly support the causal relationship 6

between of formaldehyde exposure and URT carcinogenicity. Formaldehyde

- induced cancers 7

are primarily seen in the nasal passages (Kerns et al., 1983; Monticell o et al., 1996; Tobe at al., 8

1985; Kamata et al, 1997; Sellakumar et al., 1985), but it should be noted that rodents, unlike 9

humans, are obligate nose breathers and have convoluted nasal turbinates. Chronic animal 10

studies (inhalation and oral exposures) re port tumor incidence in a variety of rodent models. 11

Study descriptions are provided above in detail (Section 4.2.1, Table 4

- 34). The study results are 12

evaluated here for both routes of exposure in the context of how they inform the carcinogenic 13

potential at the portal of entry, specifically the URT. 14

In rodent studies of the respiratory tract, only nasal tumors are considered to be induced 15

by formaldehyde. Repeated exposures to 10

- 15 ppm formaldehyde result in gross nasal lesions 16

and high incidence of na sal tumors (See Table 4

- 38, Section 4.2.1). Although increased cell 17

proliferation, squamous metaplasia, dysplasia and focal necrotic lesions have been noted in the 18

larynx and trachea in some studies, no tumors in these locations have been reported in the rodent 19

studies. The majority of studies were conducted using rats (F344, Wistar, or Sprague

- Dawley), 20

and all studies of 18 months or greater in mice and rats show evidence of formaldehyde

- induced 21

nasal carcinogenicity. The nasal tumors are primarily SCCs, although papillomas, polypoid 22

adenomas, adenocarcinomas, fibrosarcomas, and esthesioneuroepitheliomas have been reported 23

(Kamata et al., 1997; Monticello et al., 1996; Morgan et al., 1986a, b; Takahashi et al., 1986; 24

Sellakumar et al., 1985; Kerns et al., 1983; Albert et al., 1982). Although hyperplasia, dysplasia, 25

and squamous metaplasia of the respiratory epithelium have been observed beyond the nasal 26

cavity, other respiratory tract tumors have not been reported to be significantly increased by 27

formalde hyde exposure alone. 28

Increased tumor incidence and decreased latency are correlated with increasing 29

formaldehyde exposure concentration. Reviewing data from the only lifelong inhalation study 30

(i.e., until "natural death") with multiple exposure groups, na sal SCCs occurred much earlier in 31

the high

- exposure animals. For example, tumors are first noted at 8 and 9 months following 32

exposure for high

- exposed (15 ppm) female and male F344 rats versus tumors not arising until 33

24 months in low

- exposed rats (2 ppm) (Kerns et al., 1983). In a follow

- up study by Monticello 34

et al. (1996), the incidence of SCC in rats exposed at 15 ppm was 47%, with the first tumor noted 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 406 at 12 months. The incidence of SCC in male rats exposed at 10 ppm was 22%, with the first 1

tumors o bserved at 18 months after exposure. Moreover, of the 90 rats exposed at 6 ppm for 20 2

months, only one SCC was noted. No SCCs were detected in rats exposed to 0.7 or 2 ppm 3

formaldehyde. These incidence rates are not mortality

- adjusted (see Chapter 5, se ction 5.3.4) 4

and include animals from each scheduled sacrifice (3, 6, 12, and 18 months). In a lifelong study 5

of male Sprague

- Dawley rats exposed to 10 ppm formaldehyde, the cumulative nasal tumor 6

incidence was calculated as a function of time of exposure (Sellakumar et al., 1985). After 2 7

years of exposure, the adjusted probability of nasal carcinoma was greater than 60%. 8

There is some evidence that less

- than

- lifetime exposure to formaldehyde can induce nasal 9

tumors over an extended observation period. Two studies, both in male Wistar rats, report nasal 10

tumors in response to less

- than

- lifetime exposures (Woutersen et al., 1989; Feron et al., 1988). 11

A 13

- week exposure at 20 ppm followed by an observation period of 30 months (inclusive of 12

exposure) in Wis tar rats resulted in six nasal tumors including three nasal SCCs, one cystic SCC 13

of the nasolacrimal duct, one carcinoma in situ and an ameloblastoma, while no tumors were 14

noted in the corresponding air

- exposed controls (Feron et al., 1988). A limited num ber of 15

formaldehyde

- related tumors were noted from 4 or 8 weeks of exposure followed by 30 months 16

of observation. Although the tumor incidence of these less

- than

- lifetime exposures is low, this is 17

consistent with the 2

- year bioassays in Wistar rats. Wist ar rats are more resilient to 18

formaldehyde

- induced nasal toxicity than F344 or SD rats (Section 4.2.1), and only 1 of 26 (4%) 19

Wistar rats exposed at 10 ppm for 28 months developed SCC (Woutersen et al., 1989) versus 20

22% in F344 rats (Monticello et al., 199 6). 21

The specificity of formaldehyde

- induced tumors in the nasal passages of rodents is 22

believed, at least in part, to be a function of tissue dose. Computational fluid dynamics (CFD) 23

modeling used to predict formaldehyde tissue flux during inhalation ex posures suggests that at 24

comparable concentrations, tissue flux in the nasal passages of rodents is more intense than for 25

non

- human primates and humans. Modeling predicts a different pattern of formaldehyde flux 26

into URT tissues of rodents compared to hum ans, where formaldehyde penetrates more deeply 27

into the respiratory tract of primates than rodents even considering nose

- only breathing for 28

primates (See Section 3.4). Humans will generally switch to mouth breathing when sensing an 29

irritating smell and du ring physical exertion, resulting in direct exposures to the mouth and 30

greater tissue flux in tissues beyond the bypassed nasal passages. Therefore, species differences 31

in tissue dose may contribute to formaldehyde

- induced tumors in humans beyond the nasa l 32

passages, which are not evident in rodent bioassays. 33

34

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

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- 407 Oral Exposure: 1

Consistent with the observed carcinogenic activity of formaldehyde on tissues at the 2

portal of entry (POE) from inhalation exposure, there is evidence to support POE effects from 3

oral exposures as well

– further strengthening the overall weight of evidence of formaldehyde’s 4

carcinogenicity. As with the respiratory tract, the proximal portion of the gastrointestinal (GI) 5

tract exhibits formaldehyde

- induced lesions specifically in the fo restomach and glandular 6

stomach (Soffritti et al., 1989; Til et al., 1989; Tobe et al., 1989; Takahashi et al., 1986). 7

However, data are mixed regarding the carcinogenic potential of formaldehyde in the GI tract 8

from oral exposures. 9

Two independent 2

- year cancer bioassays in Wistar rats exposed to formaldehyde in 10

drinking water were both negative (Til et al., 1989; Tobe et al., 1989). Til et al. (1989) exposed 11

rats to a range of formaldehyde doses (0, 1.2, 15, or 82 mg/kg

- day) and evaluated 44

– 49 animals 12

per sex per dose group at 24 months of exposure. No formaldehyde

- related tumors were found. 13

A smaller study by Tobe et al. (1989) failed to note any tumors after a 2

- year exposure at 0, 10, 14

50 or 300 mg/kg

- day (eight rats per sex per treatment group.) 15

I n contrast, two studies that included lifelong observation in male and female Sprague

16

Dawley rats provide support for formaldehyde

- induced GI tract tumors

– one study where 17

exposure commenced at 7 weeks of age and a second study conducted with breeder rats (25 18

weeks of age) and their offspring (Soffritti et al., 1989). These studies demonstrate an increase 19

in GI tumors (although rare) correlated with exposure to formaldehyde and significantly 20

increased susceptibility to early

- lifetime exposure. The author s provide a detailed report on the 21

background rates of various stomach and intestinal neoplasia for male (n = 2,677) and female (n 22

= 2,582) rats within the colony (Soffritti et al., 1989). From this background pool, the total 23

incidence of benign and malig nant tumors in the stomach and intestine combined is only 1.4% 24

(combining all sites and locations), with the majority of tumors located in the stomach (1% 25

benign, 0.2% malignant). In comparison to colony

- specific background rates, apparent increases 26

in bo th stomach and intestinal neoplasia are noted in formaldehyde

- treated rats (ranging from 1 27

to 6% by type in rats exposed beginning week 7). When summed across the GI tract, tumor 28

incidence in the highest treatment group was 8% versus 1.4% in historical co ntrols. Elevations 29

of individual tumors or a clear dose

- response relationship are difficult to discern for rare cancers 30

where there are only 50 animals per group. Despite the limitations of group size and lack of a 31

dose

– response relationship, the finding s do support the carcinogenic potential for formaldehyde 32

administered orally. 33

The second study reported by Soffritti et al. (1989) in Sprague

- Dawley rats demonstrates 34

early lifetime susceptibility, with GI tumor incidences of 21.6% in female (n = 37) and 13.9% in 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 408 male (n = 36) offspring after exposure to formaldehyde beginning in utero , versus 5.6% in the 1

adult breeders. Rats were exposed to formaldehyde in drinking water for 2 years (0 or 2,500 2

mg/L). Exposures began on gestational day 12 in the offspr ing. The most common tumor 3

detected was intestinal leiomyosarcoma (13.5% in female offspring), which has a background 4

rate of 0.04% in female rats in the colony. The incidence of GI tumors in the adult breeders (n = 5

18 per sex) was due to one adenocarcin oma in a male rat and one pallipoma/acanthoma in a 6

female rat. Although severely limited by study size, their occurrence is consistent with the 7

observation of formaldehyde

- induced tumors, given the low background rates for this colony. 8

The Soffritti et al . (1989) studies stand alone in observing formaldehyde

- induced GI 9

tumors. These findings are largely attributed to a unique study design that included lifelong 10

observation (i.e., until "natural death"), neonatal exposure, examination of individual tumor t ypes 11

as well as combined rare tumor types for analysis, and summation of tumors across locations. 12

The study design results in a more sensitive assay for rare tumors as well as tumors with a long 13

latency. Thus, Soffritti et al. (1989) utilized a more appr opriate design and analysis for detecting 14

rare tumors, and these findings are not diminished by the null results of Tobe et al. (1989) and 15

Til et al. (1989). 16

There is evidence that formaldehyde may act as a tumor promoter by the oral route as 17

well as the inhalation route (discussed above). Takahashi et al. (1986) reported an increase in N

18

Methyl

- N'

- Nitro

- N

- Nitrosoguanidine (MNNG)

- initiated GI cancers in mice with formaldehyde 19

exposure (29.4%, versus 13.3% in controls); the greatest difference in tumor res ponse was 20

associated with adenocarcinoma of the glandular stomach (23.5%, versus 3.3% in controls). 21

Additionally, forestomach papillomas and preneoplastic hyperplasia in the glandular stomach 22

were increased with formaldehyde exposure alone. 23

24

4.5.2 Lymphoh ematopoietic Malignancies 25

4.5.2.1. Background 26

Lymphohematopoietic (LHP) cancers include neoplasms of both lymphoid and myeloid 27

cell origins. Cancers of the immune system are described as leukemia if they primarily involve 28

cells from peripheral blood and bone marrow at diagnosis and lymphomas if they constitute a 29

solid tumor (Robbins, 2004). Some forms of leukemia which present as an immature immune 30

cell phenotype are believed to arise from lymphomyeloid stem cells or progenitor cells normally 31

found in th e bone marrow (e.g., acute lymphoblastic leukemia (ALL) and acute myeloid 32

leukemia (AML)) (Greaves, 2004). However, multiple myeloma, lymphomas and some 33

leukemias may arise from mature functional lymphocytes present outside of the bone marrow 34

(Greaves, 20 04; see Figure 4

- 33). Therefore, the use of the general term ‘leukemia’ is not 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 4 09 restricted to cancers from a transformed stem cell or progenitor cell in the bone marrow but also 1

applies to cancers which arise from differentiated cells (e.g., mature lymphoc ytes). 2

Epidemiologic studies have reported that formaldehyde exposure is associated with both 3

leukemia and lymphomas (Chapter 4.1.2.2.1). Specific neoplasms reported to be associated with 4

formaldehyde exposure include myeloid leukemia, Hodgkin’s lymphoma , and multiple 5

myeloma. 6

7

20 

## H

B

B T T M Germ Line or

Early Embryonic

Haemopoietic

Stem cell

( lymphomyeloid )

Myeloid

Stem Cell Lymphoid

Stem Cell

Mature

lymphocyte

Subsets Rare Acute Leukemia

Acute Lymphoblastic Leukemia (ALL)

Acute Myeloid Leukemia (AML)

Myelodysplastic syndrome Chronic Myeloid Leukemia (CML)

Childhood ALL and AML

Cutaneous T Lymphoma

Adult T

- cell Leukemia

B Chronic Lymphocytic Leukemia

non

- Hodgkin ’ s Lymphoma

B or T Prolymphocytic Leukemia

Hairy Cell Leukemia

Myeloma 

### Developmental origins of blood cell cancers

Source: M.F. Greaves, IARC Press, (2004)

8

Figure 4

- 33: Developmental origins for cancers of the lymphohematopoietic 9

system (Adapted from Greaves (2004). 10

11

12

When evaluating cancers of the LHP system, epidemiologic analysis often groups many 13

of these cancers together. In part, this may be done to increase the statistical strength of the 14

analysis. Additionally, since there is a potential for disease misclassification, grouping these 15

diseases is preferred by some researchers, especially when analyzing older mortality data. 16

Historically, misclassification may have been due to factors such as poor histopathology and 17

diagnosis of late

- stage disease, where cell line of origin may have been hard to distinguish. 18

Without the cell surface markers and molecular tools used today to classify cells, diagnosis was 19

accomplished primarily based on histology. However, as the cancers progress, the leukemic 20

stem cells may present as less mature cells. For example, poor health surveillance may allow 21

chronic myeloid leukemia (CML) to remain undiagnosed until the blast crisis, often seen late in 22This document is a draft for review purposes only and does not constitute Agency policy.

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- 410 the disease. This blast crisis presents as a leukemia of relatively immature myeloid cells and 1

may have been mistaken for AML without the more recent techniques available for classifying 2

disease. Si nce cancers of the LHP system may present with cell surface markers for multiple cell 3

lines, classification remains problematic in some cases. 4

Although often grouped for analysis, the LHP cancers represent many distinct 5

malignancies which may arise from d iscrete cell types in different tissues throughout the body 6

(Greaves, 2004). The World Health Organization (WHO) has developed a classification system 7

for both lymphoid and myeloid leukemia defined by a combination of morphology, 8

immunophenotype, genetic features and clinical features (Harris et al., 2000a; Harris et al., 9

2000b). The historical nomenclature and International Classification of Disease (ICD) codes 10

used in epidemiologic studies do not correspond to these new classification systems. For 11

example, both chronic lymphocytic leukemia (CLL) and B

- cell lymphomas arise from similar 12

cell types in the periphery, yet epidemiologic studies have considered them independently even 13

though they are currently considered to be the same disease in the new WHO c lassification 14

system − which would diminish the statistical power to detect an association. Careful re

- analysis 15

of epidemiologic data addresses evidence for the class as a whole (all LHP cancers) but also 16

various subclasses (e.g. myeloid versus lymphoid). 17

Therefore, the following analysis first examines the epidemiologic evidence for all LHP 18

cancers as a class, then all leukemia, to best take advantage of the majority of publications 19

available in assessing the weight of evidence for the carcinogenicity o f formaldehyde. The 20

subsequent analysis by sub

- type draws upon the available evidence for specific diseases or 21

groups of diseases (e.g., myeloid leukemia, Hodgkin’s lymphoma, non

- Hodgkin’s lymphoma). 22

Although fewer data are available for the sub

- type ana lysis, these data help clarify which cancers 23

may contribute to the consistent observation of an association between formaldehyde exposure 24

and all LHP cancers. Novel combinations of phenotypic sub

- types are presented where they are 25

etiologically relevant. The sub

- type analysis frames the subsequent mode of action (MOA) 26

analysis. 27

28

4.5.2.2. All LHP Malignancies 29

Epidemiologic studies involving formaldehyde

- exposed workers provide sufficient 30

evidence of a causal association between formaldehyde exposure and all LHP malignancies 31

(Section 4.1.2.1.5). Positive associations between formaldehyde exposure and LHP cancers have 32

been reported for chemical workers (Wong et al., 1983; Bertazzi et al., 1986), embalmers 33

(Walrath and Fraumeni, 1983, Walrath and Fraumeni, 1984; Hayes et al., 1990), anatomists and 34

pathologists (Harrington and Shannon 1975; Hall et al., 1991; Levine at al 1984; Stroup et al., 35

1986; Matanoski et al., 1989) (Table 4

- 90). However, clear associations (in terms of overall 36This document is a draft for review purposes only and does not constitute Agency policy.

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- 411 standardized mortality ratios [SMRs] or proportional mortality ratios [PMRs]) were not reported 1

in analyses for garment workers, iron

- foundry workers, and a large US industrial cohort 2

(Pinkerton et al., 2004; Andjelkovich et al., 1995; Beane Freeman et al., 2009; Marsh et al., 3

1 996), although associations were observed in some of these studies when exposure

- response 4

relationships were considered. Several published meta

- analyses are available which more 5

formally assess the strength of association between formaldehyde exposure and mortality from 6

all LHP cancers. Pooled SMRs indicate stronger associations for professional workers 7

(embalmers, anatomists and pathologists) than industry workers (Table 4

- 91). Bosetti et al. 8

(2008) found similar relationships, with a pooled SMR of 1.31 (95% CI 1.16

- 1.47) for 9

‘professionals’ (i.e. embalmers, anatomists and pathologists) versus a pooled estimate of 0.85 10

(95% CI 0.74

- 0.96) for industrial workers. A recent meta

- analysis by Zhang et al. (2009) reports 11

a summary relative risk (RR) of 1.25 (9 5% CI 1.09

- 1.43) for both professional and industry 12

workers for all LHP cancers (ICD 9 codes 200

- 209). These researchers identified 19 cohort 13

study analyses, including cohort study updates. Zhang et al. (2009) used the reported RR from 14

the highest exposu re category to increase statistical power and reduce uncertainty regarding 15

confounding or other bias. Although study selection was controversial, e.g., the inclusion of 16

multiple reports from a single cohort and the use of one cohort where only a portion of the 17

workers were formaldehyde

- exposed, this meta

- analysis is generally supportive of an association 18

between formaldehyde and LHP malignancies. 19

The apparent differences by industry/profession may reflect many influences, including 20

exposure potential and demographic characteristics. External analysis (use of the general 21

population for comparison) relies on the assumption that cancer incidence rates are expected to 22

be similar between the general population and the study population in the absence of exposur e. 23

The ‘healthy worker effect’ is well known, and there may be differences in the magnitude of this 24

selection bias by industry or profession. For instance, LHP cancer incidence and mortality have 25

many risk factors including socioeconomic status. Therefo re, the consistent positive findings in 26

professional workers versus mixed results in industrial workers could be influenced by the 27

appropriateness of the comparison to the general population − that is, a differential extent of 28

selection bias. Interesting ly, salaried workers, but not the hourly workers, in an Italian plastic 29

manufacturing plant had elevated SMRs for LHP cancers (1.69 (95% CI 1.07

- 2.53) and 30

0.93(95% CI 0.62

- 1.35), respectively) (Dell and Teta, 1995). Without knowledge of which 31

worker group is most similar to the comparison population with respect to LHP cancers 32

mortality, one cannot discern if this potential effect of demographic variability accentuates 33

effects in professional/salaried workers or obscures the effects in industrial/hourly

- wa ge 34

workers. 35

36This document is a draft for review purposes only and does not constitute Agency policy.

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- 412 Table 4

- 90. Summary of cohort and case

- control studies which evaluated 1

the incidence of all LHP cancers in formaldehyde

- exposed populations 2

(ICD

- 8 Codes: 200

- 209) and all leukemias (ICD

- 8 Codes: 204

- 207). (See 3

Table 4

- 6 for complete study details and findings) 4

5

Study population Study details All LHP cancers Leukemia Reference

SMR Analysis 1

Pathologists and

technicians

(n=2,079) Years of study 1955

- 1973 2.0 (p<0.01)

{pathologists}

0.5

{technicians} 0.6

{pathologists}

0.5

{technicians} Harrington and

Shannon, 1975

Pathologists and

technicians

(n=2,720) 1974

- 1980

NR 0.91 (0.05

- 4.29)

men

9.26(0.47

- 43.9)

women Harrington and

Oakes, 1984

Pathologists

(n=4,512) Years of study 1974

- 1987 1.44

(0.69

- 2.63) 1.52 (0.41

- 3.89) Hall et al., 1991

Ontario Undertakers

(n=1,477) Mortality from 1950

- 1977 1.24 1.60 Levine et al.,1984

Male Anatomists

(n=2,327) Mortality from 1925

- 1979 1.20

(0.7

- 2.0) 1.5 (0.7

- 2.7) Stroup et al., 1986

Male pathologists

(n=4,485) Mortality through 1977 NR 1.06 Logue et al, 1986

Male pathologists

(n=6,111) Participants from 1912

- 1950 membership rolls.

Mortality followed through

1978. 1.25

(0.95

- 1.62) 1.35 (0.92

- 1.92) Matanoski et al.,

1989

Chemical industry

workers, men

(n=14,014) Mortality from 1941

- 2000

NR 0.91(0.62

- 1.39) Coggon et al.,

2003

Chemical workers

(n=2,026) 1.36

(0.5

– 2.95) Wong et al., 1983

Industrial workers

(n=25,619) Mortality followed through

2004

0.94

(0.84

- 1.06) 1.02

(0.85

- 1.59) Beane

- Freemen et

al., 2009

Industrial worke rs (n=7,328)

0.89 Marsh et al., 1996

{Subset of NCI

cohort reported by

Hauptmann et al.,

2003}

Garment workers

(n=11,098) Mortality followed through

1998 0.97

(0.74

- 1.26) 1.09 (0.70

- 1.62) Pinkerton et al.,

2004

Resin plant workers

(n=1,330) Employed b etween 1959

- 1980

Mortality through 1986 2.01 NR Bertazzi et al.,

1986

Plastic

manufacturing 1.69

(salaried workers) 1.98

(salaried workers) Dell and Teta,

1995This document is a draft for review purposes only and does not constitute Agency policy.

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- 413 (n=5,932) 0.93

(hourly workers) 0.98

(hourly workers)

PMR Analysis 1

Embalmers, New

York

(n= 1,132) Licensed between 1925

- 1980 1.15 1.32 Walrath and

Fraumeni, 1983

Embalmers, CA

(n=1,007) Licensed between 1925

- 1980

1.22 1.75 (p<0.05)

1.24

(<20 years)

2.21 (P<0.05)

(>20 years) Walrath and

Fraumeni, 1984

Embalmers, U.S.

(n=4,046) 1.39

(1.15

- 1 .63)

White 1.31

(1.06

- 1.59)

Nonwhite 2.41

(1.35

- 3.97) 1.52 2

(0.98

- 2.35)

White 1.44

(p<0.05)

Nonwhite 2.72

(p<0.05) Hayes et al., 1990

Case

- Control Studies 1

American cancer

Society Cancer

Prevention Study II:

(n=362,828 men) Results for men report ing

formaldehyde exposure,

and occupations related to

formaldehyde exposure 1.22 (0.84

- 1.77)

(formaldehyde

exposed)

3.44 (1.11

- 10.68)

{formaldehyde

exposure and

occupation} 0.96 (0.54

- 1.71)

(formaldehyde

exposed)

5.79 (1.44

- 23.25)

{formaldehyde

exposure and

occupation} Stellman et al.,

1998

White men

diagnosed with

leukemia

(Iowa and

Minnesota)

(n=622) Recruited in 1980

- 1983 NR 1.0 (0.7

- 1.4) Low

0.7 (0.2

- 2.6) High Blair et al., 1993

1

Notes: 2

1. Relative risk estimate (SMR, PMR, or OR) presented with 9 5% confidence intervals, where available. 3

2. PMR for leukemia for the total group calculated from the published data for lymphatic leukemia (204, myeloid leukemia 4

(

6 205)

, and other/unspecified (206, 207). 5

7

The only study which has data to inform the effec ts of either exposure level or the 8

appropriateness of an external comparison group on the association between formaldehyde 9

exposure and all LHP cancer mortality is the National Cancer Institute (NCI) cohort study of 10

industrial workers (Blair et al., 1986; Beane Freeman et al., 2009), which presents relative rates 11

based on internal comparisons for 3 different exposure metrics. Although SMR analysis with an 12

external comparison group did not indicate increased mortality from all LHP cancers (0.94, CI 13

0.84

- 1.06 , for the exposed workers), internal analysis using the low

- exposed workers as the 14

comparison group demonstrates positive exposure

- response relationships for increased mortality 15This document is a draft for review purposes only and does not constitute Agency policy.

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- 414 from all LHP malignancies cancers and peak exposure across the study periods ( 1965

- 2004) 1

(Figure 4

- 34A and 4

- 34B) (Beane Freeman et al., 2009), with a statistically significant trend (p < 2

0.05) for every year since 1977. These results, indicating a positive exposure

- response 3

relationship among plant workers, who most likely have si milar demographic characteristics, are 4

noteworthy given the apparent lack of association when SMRs for the same cohort are calculated 5

against mortality rates for the general population. The lack of an apparent association with 6

SMRs may be attributable to the healthy worker effect and/or some other difference between the 7

exposed workers and the general population. 8

Although the association between formaldehyde exposure and all LHP cancer mortality 9

in industrial and professional cohorts is mixed, the strength of the internal analysis of the NCI 10

cohort, in the absence of positive SMRs compared to the general population, suggests that SMR 11

analyses may not be the most appropriate methodology for assessing LHP cancer mortality. 12

Given the potential for demographic differences between an industrial workforce and the general 13

population, the results of the internal analysis of the NCI industrial cohort provide a higher 14

quality analysis

– and therefore should be given significantly more weight than SMR analyses of 15

indu strial workers that could not distinguish their findings from the null. Given the consistency 16

and strength of the positive associations for all LHP cancers cancer mortality in professional 17

cohorts (embalmers, anatomists and pathologists) taken together wi th the strong positive results 18

of the NCI cohort, human epidemiologic evidence are sufficient to conclude that there is a causal 19

association between formaldehyde exposure and mortality from all LHP malignancies (as a 20

group). 21

22

4.5.2.3. All Leukemia 23

Like the analysis of all LHP cancers, an analysis of all leukemia combines diseases which 24

differ significantly in cell of origin and etiology, including acute and chronic forms of both 25

myeloid and lymphatic leukemia. This class also includes other leukemia (e .g., erythraemia) and 26

a general category of ‘other and unspecified leukemia’ (ICD

- 8 207). Regardless, there is some 27

utility in evaluating the all leukemia mortality data because many studies provided results for this 28

grouping. Also, the diagnosis of leuk emia versus solid LHP tumors is fairly distinct thereby 29

limiting misclassification of the endpoint. 30

31This document is a draft for review purposes only and does not constitute Agency policy.

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- 415 1

2

Figure 4

- 34A. Association between peak formaldehyde exposure and the risk 3

of lymphohematopoietic malignancy

. 4

5

Relative risks for medium

- peak (2.0 t o <4.0 ppm) and high

- peak ( ≥4.0 ppm) 6

formaldehyde exposure categories compared with the low exposed category (>0 7

to <2.0 ppm) and P values for trend tests among the exposed person

- years for 8

lymphohematopoietic malignancies are shown by year of end of follo w

- up, 1965

- 9

2004. Values plotted at 0.1 represent RR = 0 due to no cases in the exposure 10

category values plotted at 20 represent RR = infinity due to no cases in the 11

referent category. The small graphs above the relative risk plots represent the 12

exposure

- response trend P values based on two

- sided likelihood ratio tests (1 df) 13

of zero slope for continuous formaldehyde exposure among exposed person

- years 14

only. The points represent the relative risk estimates based on the cumulative 15

number of cases and perso n

- years accrued from the start of the study to that point 16

in time and for 2004 are equivalent to the relative risk estimates presented in 17

Table 2. HLP = lymphohematopoietic malignancies, NHL = non

- Hodgkin 18

lymphoma, HDG = Hodgkin lymphoma, MM = multiple my eloma, LEU = 19

leukemia, LYL = lymphatic leukemia, MYL = myeloid leukemia, RR = relative 20

risk. 21

22

Source: Beane Freeman, et al. (2009) 23

24This document is a draft for review purposes only and does not constitute Agency policy.

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- 416 1

Figure 4

- 34B. Association between average intensity of formaldehyde 2

exposure and the risk of lymphohematopoietic malig nancy. 3

4

Relative risks for medium (0.5

– 0.9 ppm) and high ( ≥ 1.0 ppm) average

- intensity 5

formaldehyde exposure categories compared with the low exposed category (0.1

– 6

0.4 ppm) and P values for trend tests among the exposed person

- years for 7

lymphohematop oietic malignancies by year of end of follow

- up, 1965

– 2004. 8

Values plotted at 0.1 represent RR •• = due to no cases in the exposure category. 9

The small graphs above the relative risk plots represent the exposure

– response 10

trend P values based on two

- sid ed likelihood ratio tests (1 df ) of zero slope for 11

continuous formaldehyde exposure among exposed person

- years only. The points 12

represent the relative risk estimates based on the cumulative number of cases and 13

person

- years accrued from the start of the st udy to that point in time and for 2004 14

are equivalent to the relative risk estimates presented in Table 3 . HLP = 15

lymphohematopoietic malignancies, NHL = non

- Hodgkin lymphoma, HDG = 16

Hodgkin lymphoma, MM = multiple myeloma, LEU = 17

leukemia, LYL = lymphatic l eukemia, MYL = myeloid leukemia; RR = relative 18

risk. 19

20

Source: Beane Freeman, et al. (2009) 21

22This document is a draft for review purposes only and does not constitute Agency policy.

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- 417 Although results are mixed across the studies (Table 4

- 90), an association between 1

formaldehyde exposure and leukemia mortality is supported by cohort analyses of embalmers, 2

pathologists and anatomists (Hayes et al., 1990; Walrath and Fraumeni, 1983; Walrath and 3

Fraumeni 1984; Hall et al., 1991; Levine et al., 1984; Stroup et al., 1986; Matanoski et al., 1989). 4

Formaldehyde exposure and formaldehyde

- related occupat ion are associated with leukemia 5

diagnosis in a case

- control study (RR = 5.79 (95% CI 1.44

- 23.25), but not formaldehyde 6

exposure alone (RR = 0.96; 95% CI 0.54

- 1.71) (Stellman et al., 1998). 7

In contrast, SMR analyses of the industrial cohorts do not indicat e a similar association 8

(Coggon et al., 2000; Beane Freeman et al., 2009, Pinkerton et al, 2004). Although the SMR 9

analysis provided for the NCI cohort does not indicate a positive association for all leukemia 10

using an external reference group (Beane Free man et al., 2009), the SMR for exposed versus 11

unexposed workers within the cohort suggests all leukemia is elevated 2.1

- fold with this internal 12

comparison (95% CI 0.99

- 4.56) 4

Several meta

- analyses have been conducted for formaldehyde exposure and leukemia 23

which indicate a positive association. Collins et al. (2004) report an overall RR for 18 available 24

studies of 1.1 (CI 1.0

- 1.2), suggesting an association of leukemia with formaldehyde exposure. 25

This association was stronger for both pathologists/anatomists (1.4; CI 1.0

- 1.9) and embalmers 26

(RR = 1.6; 1.2

- 2.0) than for industrial workers (RR = 0.9; 0.8

- 1.0). Study design also impacted 27

the apparent strength of association, with stronger associations seen in case

-- control studies (RR 28

= 2.4; 0.9

- 6.5) versus cohort studies (RR = 1.0; 0.9

- 1.2). Bosetti et al. (2008) reported an 29

association between formaldehyde exposure and leukemi a mortality with a pooled RR of 1.39 30

(95% CI 1.15

- 1.68) for 8 groups of professional workers. In the same analysis, the pooled RR 31

for the 4 industrial cohorts was 0.90 (0.75

- 1.07). Zhang et al. (2009) reported a pooled RR of 32 . A positive exposure

- response relationship further strengthens 13

the association o f formaldehyde exposure to leukemia mortality (Beane Freeman et al., 2009). 14

Where the referent group is defined as ‘low exposed’ individuals, leukemia is elevated in the 15

highest peak exposure category (RR = 1.42; 95% CI 0.92

- 2.18) compared to both the ref erent 16

group and the unexposed category (RR = 0.59; 95% CI 0.25

- 1.36), and there is a statistically 17

significant trend across all groups ( p = 0.02). Categorical analysis for the average intensity and 18

cumulative exposure metrics suggests greater mortality i n the high

- exposure groups versus the 19

‘low exposed’ individuals (RR = 1.10 [95% CI 0.68

- 1.78] and 1.11 [0.7

- 1.74]. respectively), but 20

analysis of individual results across the exposure

- response range indicates cumulative exposure 21

is a better predictor ( p = 0.08 for trend across all exposed and unexposed.) 22

4

osed Non Exposed osed Non

Exposed



### Obs Obs SMR

SMR
Var

exp exp 

### 1 1 ln **+ =**  ** ** 

** ** 

** ** 

** ** 

** ** 

** ** 

** ** 

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

DRAFT

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- 418 1.54 (95% CI 1.18

- 2.00) for a ll cohorts identified in their meta

- analysis, although this pooled RR 1

should be considered with some caution, as myeloid leukemia alone was included in the analysis 2

where available (Zhang et al., 2009). 3

4

4.5.2.4. Subtype Analysis 5

Given the associations discussed above between formaldehyde exposure and both all 6

LHP cancers and all leukemia, further analysis is needed to examine if the observed increase in 7

all LHP cancers is primarily a reflection of increased leukemia, or if other types of LHP cancers 8

ma y be elevated as well. Although analysis of mortality data by sub

- type may provide a better 9

understanding of the specific disease associations, there are potential pitfalls as well. Chief 10

among these concerns are the potential for disease misclassificati on (especially in studies with 11

older mortality data) and lack of statistical power as the number of observed cases is reduced by 12

considering sub

- types. Case control studies by design address specific diseases and are well

13

suited for sub

- type analysis, but often provide little exposure information. The following 14

analysis will draw from the available data to examine which forms of LHP malignancies may be 15

associated with formaldehyde exposure. 16

There has been speculation that the association between formalde hyde exposure and 17

increases in all LHP cancers and all leukemia are driven by increased myeloid leukemia (Pyatt et 18

al., 2008; Heck and Casanova 2004; Golden et al., 2006). If this were the case, then mortality 19

from LHP cancers other than myeloid should no t be elevated, once the excess mortality from 20

myeloid leukemia is accounted for. Only 2 studies provide the data to evaluate this hypothesis

– 21

both conducted by the NCI (Hayes et al., 1990 and Beane Freeman et al., 2009). From the 22

published data, crude m ortality statistics can be calculated for alternative disease groupings 23

(Table 4

- 91). In the NCI embalmer study (Hayes et al., 1990), only myeloid leukemia was 24

statistically elevated in the subtype analysis. For the NCI industrial cohort (Beane Freeman e t 25

al., 2009), elevations were also seen for Hodgkin lymphoma relative to the referent group. In 26

both cases, the association between formaldehyde exposure and LHP malignancies remains when 27

myeloid leukemia is dropped from the analysis. Further, similar ass ociations are found when all 28

leukemia and myeloproliferative diseases are dropped from the analysis and only solid tumors of 29

lymphoid origin are included ( lymphosarcoma and reticulosarcoma, Hodgkin lymphoma, non

30

Hodgkin lymphoma and multiple myeloma). The se reanalyses illustrate the need for a more 31

careful sub

- type analysis to assess the potential for associations between formaldehyde exposure 32

and various forms of LHP cancers. 33

34

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

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- 419 Table 4 -91. Secondary analysis of published mortality statistics to explore 1

2

3

4

5

13

14

15

16

17

18

19

20

21

6

7

8

9

10

11

12 a

Exp Obs PMR
**=**

b **( )**
ExpObs PMR Var 11 log **+=**

c See Table 2 of Beane Freeman et al. (2009)

d

765.0 37.1103 108 14103 19108

**= **  ** **  ** **  ****

**× ≅**  ** **  ** ** 

** **  ** **  ** **  ** ** 

** **  ** ** 

**× **  ** **  ** **  ****

**− −** 

**=**

**− −** 

**=**

c c c

Group Comparison Group referent Group Comparison Group referent

Group referent Group referent Group Comparison Group Comparison

PT

PT where PT PT

Time Person Deaths Time Person Deaths

RR

e 

### ( )

Group referent Group Comparison Deaths Deaths RR Var 1 1 log **+ =**  a lternative disease groupings within the broad category of all lymphohematopoietic

malignancies

ICD- 8 Codes U.S. embalmers

(Whole cohort)

(Hayes et al., 1990)

PMR

(95% CI) U.S. industry

(peak exposure metric: >4

ppm vs. >0 to ≤2 ppm)

(Beane Freeman et a l., 2009)

Relative risk

(95% CI)

All Lymphohematopoietic

Malignancies 200

- 209 1.39 a

(1.15

- 1.67) b 1.37 c

(1.03

- 1.81) c

Alternative Disease Groupings

Exclude Myeloid Leukemia 200

- 204,

206

- 209 1.35 a

(0.99

- 1.85) b 1.31 d

(0.97

- 1.75) d,e

Solid tumors of lymphoid

origin

(Lymphosarcoma and

reticulosarcoma, Hodgkin

lymphoma, non -Hodgkin

lymphoma and multiple myeloma) 200

- 203 1.24 a

(0.84

- 1.84) b 1.33 d

(0.93

- 1.90) d,e

4.5.2.5. Myeloid Leukemia

The associations between myeloid leukemia and formaldehyde exposure are strong and

consistent (Table 4

- 92). Of the four studies which formally assess myeloi d leukemia mortality,

all are positive, including cohorts of both professional and industrial workers (Beane Freeman et

al., 2009; Hayes et al., 1990; Pinkerton at al., 2003; Stroup et al., 1986). Although few cases

exist for further subtype analysis, th e available data indicate either no differences in SMRs for

acute myeloid leukemia (AML) versus chronic myeloid leukemia (CML) ( Hayes et al., 1990;

Pinkerton et al., 2003) or suggest CML is more prominent (Blair et al., 2000; Stroup et al., 1986).This document is a draft for review purposes only and does not constitute Agency policy.

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- 420 Table 4

- 92. Summary of studies which provide mortality statistics for 1

myeloid leukemia sub

- types. 2

3

Study population Reference

Myeloid

Leukemia Acute

Myeloid

Leukemia Chronic

Myeloid

Leukemia

SMR Analysis 1

Garment workers

(n=11,098) 1.44 (0.08

- 2.37) 1.34

(0.61

- 2.54) 1.39

(0.38

- 3.56) Pinkerton at al.,

2003

Anatomists

(n=2,317) NR NR 8.8* Stroup et al., 1986

Industrial workers

(n=25,619) 0.90 (0.67

- 1.21)

SMR Ratio

1.38 (0.65

- 2.97)

(exposed/unexposed) NR NR Beane Freeman et

al., 2009

PMR Analysis 1

E mbalmers, U.S.

(N=4,046) 1.57 (1.01

- 2.34) 1.52

(0.85

- 2.52) 1.84

(0.79

- 3.62) Hayes et al., 1990

Case

- Control Studies 1

White men diagnosed

with leukemia

(Iowa and Minnesota)

(n=622) NR Low:

0.9 (0.5

- 1.6)

High:

NR Low:

1.3 (0.6

- 3.1)

High:

2.9 (0.3

- 24.5) Blair et al., 1993

4

* Leukemia SMR 1.5 (0.7

- 2.7) {5 of 10 deaths due to myeloid}; Chronic Myeloid Leukemia (CML) SMR of 8.8 5

1. Relative risk estimate (SMR, PMR, or OR) presented with 95% confidence intervals, where available. 6

7

8

Walrath and Fraum eni (1983, 1984) note that AML is prominent in their analyses of New 9

York and California licensed embalmers; however, they do not provide PMR analyses for CML. 10

Walrath and Fraumeni (1983 and 1984) report leukemia cell types

- for both studies the majority 11

of myeloid leukemia are acute (5/6 and 4/6, respectively, for New York State and California 12

embalmers). However, PMRs cannot be calculated for AML versus CML in this paper, as 13

comparison rates are not available from the 1920's through the 1960's

- the ti meframe with the 14

majority of deaths. The authors do contrast the observed rate of AML in the cohort to the 15

background rate for AML in white men in the 1970s

- but given the potential misclassification of 16

late stage CML as AML, especially historically, thi s may not be an appropriate comparison. 17

Additionally, one would expect older data to over

- represent AML rather than CML due to 18

diagnosis in the early decades of CML in the blast crisis as AML. Therefore, although these 19

studies support an association betw een formaldehyde exposure and myeloid leukemia in general 20This document is a draft for review purposes only and does not constitute Agency policy.

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- 421 ( Walrath and Fraumeni, 1983; 1984) , the reported AML and CML subtype information does not 1

allow a satisfactory sub

- type analysis for myeloid leukemia. 2

3

4.5.2.6. Solid Tumors of Lymphoid Origin 4

M ultiple myeloma, Hodgkin lymphoma, non

- Hodgkin lymphoma, lymphosarcoma, 5

reticulosarcoma, and other lymphomas may all be derived from immune cells outside of the bone 6

marrow compartment, in peripheral blood, in the gut and respiratory mucosa and immune tiss ues 7

at the POE (e.g., lymph nodes, mucosa

- associated lymphoid tissue (MALT), gut

- associated 8

lymphoid tissue (GALT) (Greaves, 2004). The only meta

- analysis to specifically address 9

lymphoid malignancies found evidence for increased lymphoma (Hodgkin lymphom a (pooled 10

RR = 1.23; 95% CI 0.67

- 2.29) and multiple myeloma (1.31; 1.02

- 1.67), but not for non

- Hodgkin 11

lymphoma (1.08; 0.86

- 1.35) (Zhang et al., 2009). As seen in Table 4

- 93 below, individual study 12

results are mixed for these lymphoid cell

- line malignanci es, as they are for all LHP cancers and 13

all leukemia above. Although these tumors are from mature lymphocytes, there is still variability 14

in the etiology, natural history and risk factors for the many sub

- types which are included in 15

these categories. 16

The re is evidence for an exposure response relationship for both Hodgkin lymphoma and 17

multiple myeloma in the NCI industrial cohort among exposed workers (Beane

- Freeman et al., 18

2009). Clear exposure response relationships for Hodgkin lymphoma are defined wit h all three 19

metrics of exposure, peak average intensity and cumulative exposure (p=0.01, p=0.05 and 20

p=0.08 respectively for mortality through 2004). These associations have been evident from first 21

follow

- up through the current publication, and statistical ly significant for the majority of the 22

follow

- up period demonstrating that this is a strong and consistent finding in the NCI cohort 23

(Figure 4

- 34 A&B) (Beane

- Freeman et al., 2009). Although the overall SMR for multiple 24

myeloma does not indicate an associa tion, trends across time indicate consistent elevation of 25

multiple myeloma mortality with both peak and average intensity of exposure, where the 26

statistical strength of the association with peak exposure increases with follow

- up (Figure 4

- 34 27

A&B). 28

29This document is a draft for review purposes only and does not constitute Agency policy.

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- 422 Tabl e 4

- 93. Summary of mortality statistics for Hodgkin lymphoma, 1

lymphoma and multiple myeloma from cohort analyses of formaldehyde 2

exposed workers. 3

4

Study population Reference Hodgkin

Lymphoma Non

- Hodgkin

Lymphoma Multiple

Myeloma

SMR Analysis 6

Pathol ogists

(n=2,079) 1.4 2.0 (p<0.05) NR 1 Harrington and

Shannon, 1975

Pathologists

(n=4,512) 1.21 (0.03

- 6.71) 1.44 (0.69

- 2.63) NR Hall et al., 1991

Male Anatomists

(n=2,327) − 2 0.7 (.1

- 2.5) 3

2.0 (0.7

- 4.4) 4 NR Stroup et al., 1986

Male pathologists

(n=6,111) 0.36 (0.04

- 1.31) 1.31 (0.66

- 2.35) 3

1.54 (0.82

- 2.63) 4 NR Matanoski et al.,

1989

Chemical workers

(n=2,026) 2.94 (0.33

- 10.63) NR NR Wong et al., 1983

British Chemic al plants

(n=14,014) 0.36 (0.01

- 2.01) 0.89 (0.41

- 1.70) 1.18 (0.48

- 2.44) Coggon et al., 2003

Swedish workers

- abrasive

production plant (n=911) NR 2.0 (0.2

- 7.2) 4.0 (0.5

- 14) Edling et al., 1987a

Industrial workers

(n=25,619) 1.42 (0.96

- 2.10) 0.86 (0.70

- 1. 05) 0.94 (0.71

- 1.25) Beane Freemen et

al., 2009

PMR Analysis 6

Embalmers, New York

(n=1,132) 0.87 (p<0.05) 1.08 3

1.22 4 NR Walrath and

Fraumeni, 1983

Embalmers, CA

(n=1,007) − 2 3.10 3

1.33 4 NR Walrath and

Fraumeni, 1984

Embalmers, U.S.

N=4,046 0.72 (0.15

- 2.10) 1.26 (0.87

- 1.76)

1.12 (0.58

- 1.96) 3

1.35 (0.84

- 2.01) 4 1.37 (0.84

- 2.12 Hayes et al., 1990

Case

- Control Studies 6

Women in Connecticut (n=601) NR 1.3 (1.0

- 1.7) NR Wang et al., 2009

White men, Iowa and Minnesota

(n=622) NR 1.2 (0.9

- 1.7) NR Blair et al., 1993

ACS Cancer Prevention Study

II (n=128) NR NR 1.8 (0.6

- 5.7) Boffetta et al., 1999

Men, ACS Cancer Prevention

Study II (n=45,399) NR 0.92 (0.5

- 1.68)

2.88 (0.40

- 10.5) 5 0.74 (0.27

- 2.02) Stellman et al., 1998

Danish workers (n=1,098) NR NR Heineman et al.,

1992

Danish women (607) NR NR 1.6 (0.4

- 5.3) Pottern et al., 1992

Notes : 5

1: NR is not reported 6

2. “

— " no cases observed. 7

3. Lymphosarcoma and reticulosarcoma onl y 8

4. “other lymphoma" 9

5. Formaldehyde exposure in a wood

- related occupation. RR for wood

- related occupation alone was not elevated 10

0.97 (0.55

- 1.73) 11

6. Relative risk estimate (SMR, PMR, or OR) presented with 95% confidence intervals, where available 12This document is a draft for review purposes only and does not constitute Agency policy.

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- 423 4.5.2. 7. Supporting Evidence from Animal Bio

- Assays for Formaldehyde

- Induced 1

Lymphohematopoietic Malignancies 2

Chronic animal studies provide supporting evidence for formaldehyde

- induced leukemia 3

and lymphoma (Soffritti et al., 1989; Battelle Laboratories, 1981) . Although the majority of 4

chronic animal bioassays do not report either leukemia or lymphoma, it should be noted that 5

many studies focused primarily on respiratory tract and did not provide routine examination of 6

other organs, limiting the detection of l eukemia and lymphoma (Horten et al., 1963; Holmstrom 7

et al., 1989; Wouterson et al., 1989; Appleman et al., 1988; Monticello et al., 1996; Dalbey, 8

1982). Kamata et al. (1986) did examine additional organs, but there were only 5 animals at 9

each sacrifice. Similar issues are seen with some of the drinking water studies, where Takahashi 10

et al. (1986) focus on the stomach and intestines, and the study in Wistar rats by Tobe et al. 11

(1989) only included 20 animals per sex per exposure group with interim sacrifi ces. Therefore, 12

few studies remain to be explored which are informative about the carcinogenic potential of 13

formaldehyde on the LHP system. Table 4

- 94 lists the studies from the chronic bioassays which 14

have the potential to detect LHP malignancies. 15

So ffritti et al. (1989) were the first to publish a finding of formaldehyde

- induced 16

leukemia in an animal bio

- assay. Sprague

- Dawley rats (50 per each sex) were exposed to 17

formaldehyde in drinking water at 0, 10, 50, 100, 500, 1000, 1500 mg/L given ad libitu m

. 18

Lymphoblastic leukemia and lymphosarcomas were the most common lesion reported, and 19

exhibited an apparent dose

- dependent increase in both male and female rats (Table 4

- 95). All 20

hemolymphoreticular cancers summed together reflected similar trends, but the other types did 21

not show similar relationship with exposure (e.g. immunoblastic lymphosarcoma). A subsequent 22

publication of full study results reports all lesions together as lymphoma and leukemia, providing 23

less

- specific information (Soffritti et al 2002.) Additionally, there is a large discrepancy in 24

reported lesions between the two study reports where nearly double the incidence is reported in 25

2002. Dr. Soffritti explains the increase in reported LHP malignancies as a result of tabulating 26

the full histopathological findings in the 2002 report (personal communication, 23 July 2009). 27

Although the second report from Soffritti et al. (2002) have been broadly criticized on both of 28

these points (summing of dissimilar lesions and discrepancies in reporti ng), these errors do not 29

iupune the original report. The 1989 report does distinguish between different sub

- types 30

providing positive results for lymphoblastic leukemia/lymphosarcoma and histological 31

examinations were consistently conducted between treatme nt groups

– even if further results 32

were published at a later timepoint. Therefore, the findings of Soffritti et al. (1989) are 33

considered as supportive of the biological plausibility of formaldehyde

- induced 34

lymphohematopoietic malignancies. 35

36This document is a draft for review purposes only and does not constitute Agency policy.

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- 424 Table 4

- 94: Summary of chronic bioassays which address rodent leukemia 1

and lymphoma 2

3


### Study Histopathology Endpoint Results Comments

Drinking Water Exposure

Male and Female Sprague

- Dawley Rats

Sofritti et al.

1986 Complete histopathology Lymphocytic

leukemia and

lymphosarcoma Increased, showing a

dose

- response Life

- long study

High exposure of 1,500 mg/l in

water

Male and Female Wistar Rats

Til et al, 1989 Complete histopathology

in control and high dose

group (15 ppm) Lymphoma,

leukemia No increase

(3 lymphoma s and 1

leukemia found in 200

animals at the 2 yr

sacrifice) 2

- year bioassay

High exposure of approximately

1900 mg/l

(82mg/kg for males and 109

mg/kg for females) 

### Inhalation Exposures

Male rats, Sprague

- Dawley

Sellakumar et

al., 1985 ; Albert et al.,

1982 Necropsy focused on

respiratory tract : also

liver, spleen, kidney and

testes and organs

demonstrating gross

pathology Lymphoma No increase Life

- long study

– high mortality at

24 months (>80%)

Male rats, F344

Batelle,

Columbus

Laboratories,

1981 C omplete histopathology

in control and high dose

group (15 ppm) Leukemia, all No increase Extended study

– high mortality

Female Rats, F344

Batelle,

Columbus

Laboratories,

1981 Complete histopathology

in control and high dose

group (15 ppm) Leukemia, al l Increased in mortality

adjusted incidence.

P=0.0056 1 Extended study

– high mortality

Apparent elevation in 2 ppm and

6 ppm treatment groups as well

(fig5

- xx)

– statistical comparison

to controls is problematic

Female mice

- C57BL/6xC3HF1

Batelle,

Columb us Laboratories,

1981 All organs in control and

high dose group (15

ppm) Lymphoma, all 26% in FA

- exposed

(15ppm)

16% in control

P=0.0617 Extended study

All mice included in statistics

conducted by Batelle Lab. 4

1. Original statistical analysis provided by Battelle, Columbus Laboratories. Significance set at P<0.0167. Analysis of adjusted 5

data where time to lesion and survivorship were considered, Cox (1972, Tyrone (1975). 6

7This document is a draft for review purposes only and does not constitute Agency policy.

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- 425 Table 4

- 95. Incidence of lymphoblastic leukemia and lymphosarcoma orally 1

dosed i n Sprague

- Dawley rats 2

3

Lymphoblastic leukemia and

lymphosarcoma

Tumor bearing animals

(%)

Male Female

control 3 1

vehicle 8 2

10 0 2

50 4 6

100 8 4

500 8 4

1000 12 10

1500 22 10

4

Source: Soffritti et al. (1989) 5

6

7

The two

- year bioassay by Til et al. (1989) in male and female Wistar rats indicates no 8

increase in leukemia and lymphoma, with only 4 tumor

- bearing animals in all treatment groups 9

sacrificed at 24 months. The drinking water levels were similar at the highest dose of both 10

studies. T he major difference in study design is length, which may have influenced results, as 11

leukemia is a late

- life malignancy in rodents. Two

- year survival in the Soffritti et al. (1989) 12

study varied between 50

- 60%. These animals would be available to develop leukemia after the 13

two

- year window of the Til et al (1989) study. Any potential role of strain differences is 14

unknown. Overall, the results of Soffritti et al. (1986) are strong, showing an exposure

- response 15

relationship, in a lifelong study, appropriate for late

- life malignancies. Unlike the GI tract 16

tumors, early

- life exposure to formaldehyde in drinking water did not increase LHP 17

malignancies (Soffritti et al., 1989). 18

The largest and most comprehensive study of health effects from formaldehyde 19

inhala tion exposures is the study reported by Kerns et al. (1983) and Swenberg et al. (1980) 20

conducted at the Columbus Laboratory of Battelle Corporation (1981). Although the summary 21

reports of this study do not discuss leukemia or lymphoma rates, mouse lymphom a and rat 22

leukemia were selected by the study pathologist and biostatistician for analysis (Battelle 23

Laboratory, 1981). Statistical analysis performed by Battelle Laboratories which accounted for 24

time

- to

- lesion and survivorship rates did indicate a statis tically significant increase in female rat 25

leukemia (P = 0.0003) and a nearly significant increase in female mouse lymphoma ( P = 0.06). 26

No trend analysis could be performed, as only gross pathology was conducted on mid

- dose mice 27This document is a draft for review purposes only and does not constitute Agency policy.

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- 426 and rats (2 and 6 ppm, res pectively). EPA has further analyzed these data to better understand 1

the significance of these findings. The percentage of lymphoma

-- bearing female mice increased 2

from 18% in control mice to 28% in mice exposed at 15 ppm for 24 months (6 hr/day, 5 3

days/w k) (P<0.05). Female rat leukemia was similarly elevated to 26%, 22% and 24% by 4

inhalation exposure to 15 ppm, 6 ppm and 2 ppm, respectively, versus 16% in controls when 5

early deaths prior to the first observed leukemia are removed from the analysis (21 mo nths). In 6

contrast, leukemia was not elevated in formaldehyde

- exposed male F344 rats within the same 7

study. 8

Differences in study design may account in part for mixed results. The lifelong study by 9

Soffritti et al. (1989 may have allowed for detection of malignancies developing late in life, 10

whereas the other drinking water study by Til et al. (1989) sacrificed all animals at 24 months. 11

Even though the exposure levels were similar, the studies are not directly comparable. Likewise, 12

it is hard to direc tly compare results from the two major inhalation studies in rats. Although a 13

life

- long study, the mortality for rats in the Sellakumar et al. (1985) study was greater than 80% 14

at 2 years. Additionally, the pathology examination was much less rigorous tha n in the Battelle 15

Laboratory study, perhaps missing smaller lesions. Therefore, the increase in formaldehyde

16

induced leukemia seen in female F344 rats late in life (Battelle laboratories, 1981) may be 17

reflecting a more sensitive study design. Finally, st rain differences may account for different 18

susceptibilities as well. In summary, the available evidence from chronic animal studies 19

supports the biological plausibility of the formaldehyde

- induced LHP malignancies observed in 20

epidemiologic studies. The t wo positive rat studies, by different routes of exposure, along with a 21

positive result for formaldehyde

- induced mouse lymphoma make a substantive case for 22

formaldehyde

- induced LHP malignancies. 23

The epidemiologic studies provide sufficient evidence to concl ude that there is a causal 24

association between formaldehyde exposure and lymphohematopoietic malignancies. When data 25

are evaluated for all leukemia together, again there is sufficient evidence to establish a causal 26

association, with consistent positive re sults in individual studies as well as 3 independent pooled 27

analyses. Mortality from myeloid leukemia, as well as mortality attributed to “other and 28

unspecified leukemia” is consistently elevated where reported. In addition, strong evidence for a 29

causal relationship between formaldehyde exposure and Hodgkin lymphoma is provided by the 30

consistent associations seen between formaldehyde exposure and Hodgkin lymphoma in the NCI 31

industrial cohort, with elevations observed across decades of follow

- up and signif icant exposure

32

response relationships for all three exposure metrics examined in the most recent follow

- up 33

(Beane Freeman et al., 2009). 34

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

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- 427 4.5.3 Carcinogenic Mode(s) of Action 1

The US EPA 2005 Guidelines for Carcinogen risk Assessment recommend a Mode of 2

Action (MOA) analysis when data are available for evaluation. The purpose of this MOA 3

analysis is to determine if sufficient data exist to adequately inform the exposure

- response 4

relationship for cancer below the range of observed data in either human or animal studies. 5

Since the majority of the data supporting the carcinogenicity of formaldehyde comes from 6

animal bio

- assays and epidemiological studies of workers, EPA must extrapolate from the 7

observed risk of cancer mortality/incidence in those studies to level s considered protective of 8

human health for lifelong environmental exposures. In this context, the US EPA cancer 9

guidelines provide a framework to review MOA information for relevant data to establish an 10

MOA informing appropriate low

- dose extrapolation. 11

The supporting data for the MOA evaluation of formaldehyde are complex, and presented 12

across multiple sections of a large document; therefore, this section includes a brief summary of 13

the biological actions of formaldehyde and key mechanistic data which a re believed to be 14

relevant to the MOA evaluation (Section 4.5.3.1). This information is not intended as a stand

15

alone description of the evidence for a particular mechanism, but is intended to highlight the 16

major supporting arguments and direct the reader to text providing more detailed discussion. 17

The summary of data discussed below combines what is known about the human cancer 18

of concern (nasopharyngeal cancer, sinonasal cancer, leukemia and other lymphohematopoietic 19

cancers) with the potential formalde hyde

- specific mechanisms of action to postulate 20

carcinogenic modes of action for each cancer or group of cancers (Section 4.5.3.2 and 4.5.3.2). 21

The resulting evaluation provides multiple possible MOAs for formaldehyde

- induced cancers 22

where some key mechan istic events may be commonly at work in different tissues, and some key 23

events may be more relevant to a specific tissue/cancer type. Each of these MOAs is evaluated 24

with respect to its relevance to human cancer, and the overall weight of evidence for its 25

relevance to formaldehyde

- related human cancer. 26

Overall, multiple MOAs considered relevant to humans are presented for each cancer 27

type. Although some MOAs may have a greater level of supporting evidence, this reflects in part 28

how well a particular mech anism or key event may have been studied. For example, there are a 29

large number of studies across many testing systems, and levels of biological organization to 30

support the mutagenicity of formaldehyde. In contrast other likely MOAs, such as viral 31

reacti vation, have little direct mechanistic evidence, but the available evidence is supportive. 32

The MOAs considered most relevant to upper respiratory tract cancers (e.g. NPC and 33

sinonasal cancer) are: 1) direct mutagenicity; 2) inhibition of DNA repair mecha nisms; 3) 34

formaldehyde

- induced cell proliferation; 4) cytotoxicity

- induced cell proliferation; 5) tumor 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 428 promotion activity; and 6) localized immunosuppression/viral reactivation (Section 4.5.3.2). The 1

majority of these MOAs would apply equally to immune c ells present at the site of first contact 2

and may also contribute to those lymphohematopoietic cancers which arise from peripheral 3

immune cells (e.g. Hodgkins lymphoma, multiple myeloma and some forms of leukemia). 4

Additional MOAs are considered, specific ally for formaldehyde

- induced leukemia are: 1) 5

damage of a circulating hematopoietic stem cell or progenitor cell at the site of first contact; and 6

2) bone marrow toxicity (Section 4.5.3.3). 7

In summary

– no single MOA is singled out as the best explanatio n for cancer resulting 8

from formaldehyde exposure. Only one MOA

- cytotoxicity induced cell proliferation

– suggests 9

an exposure threshold below which the MOA would not be active. However this MOA is the 10

least applicable to humans and other MOAs are cons idered operative at exposures below 11

exposures associated with cytotoxicity

- induced cell proliferation. Therefore, multiple MOAs are 12

considered supported by formaldehyde

- specific mechanistic information which provide 13

biological plausibility for the cancers observed in formaldehyde exposed populations. 14

15

4.5.3.1 Mechanistic Data for Formaldehyde 16

4.5.3.1.1 DNA Reactivity/Genotoxicity/Mutagenicity 17

An agent’s genotoxic potential and ability to induce mutations is a key consideration in 18

assessing a carcinogenic MOA, as cancer res ults from a series of genetic and epigenetic 19

alterations affecting genes that control cell growth, division and differentiation (Hanahan and 20

Weinberg, 2000; Vogelstein et al., 1988; Kinzler and Vogelstein, 2002). The US EPA Cancer 21

guidelines suggest sever al lines of evidence which are key to evaluating a mutagenic MOA: 1) Is 22

the chemical under study DNA

- reactive and/or has the ability to bind to DNA; 2) Does the 23

chemical generate positive results in in vitro mutagenic test systems (specifically gene mutati ons 24

and chromosomal aberrations); and 3) Does the chemical induce manifestations of genetic 25

damage in in vivo tests (specifically gene mutations and chromosomal aberrations) and 4) Does 26

the chemical have properties and structure

- activity relationships (SAR ) similar to known 27

mutagens (US EPA, 2005). As reviewed in Section 4.3 above, there is adequate evidence for 28

formaldehyde

- induced genotoxicity and mutagenicity for consideration of these key events in 29

formaldehyde’s carcinogenic MOA. 30

Formaldehyde induces a variety of genotoxic and mutagenic events when tested both in 31

vitro and in vivo systems including DNA

- protein crosslinks (DPC or DPX), point mutations, 32

DNA single strand breaks (SSB) and chromosomal aberrations (CAs) (See Section 4.3). 33

Formaldehyde, as a reactive chemical, also forms DNA adducts and DNA

- DNA crosslinks 34

(DDC) and may act to form adducts between other chemicals and DNA (Brutlag et al., 1969; 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 429 Donecke, 1978; Ohba et al., 1979; Fennel, 1999; Casanova

- Schmitz and Heck, 1983; Casanova

1

Schmitz an d Heck, 1984; Heck and Casanova, 1987 and Casanova et al., 1989). The high 2

reactivity of formaldehyde results in little specificity indicating that a range of adducts and 3

crosslinks might be expected. 4

Numerous studies have shown that formaldehyde induce s genotoxic and mutagenic 5

effects under a variety of experimental conditions (see section 4.3 for a detailed discussion, also 6

reviewed by IARC 2006; Ma and Harris 1988 and Auerbach et al, 1977). As discussed, 7

formaldehyde is known to directly react with DNA forming DPC and DNA adducts. Mutations 8

may occur during repair of formaldehyde

- induced DNA damage, or as a result of replication 9

errors during mitogenesis. Additionally, there is some evidence that DNA single strand breaks 10

(SSB) may be induced direct ly by formaldehyde reactivity (Grafstrom et al, 1984). Clastogenic 11

effects including increased micronuclei (MN), chromosomal aberrations (CAs) and sister 12

chromatid exchanges (SCEs) are also reported in a range of in vitro study systems. 13

Formaldehyde cau sed a concentration

- dependent icrease in calstogenicity (e.g. MN) in 14

human cell lines deficient in either DNA nucleotide excision repair (NER) or DDC repair 15

systems even though there is no change seen in DPC induction or removal between these cell 16

lines (S peit et al., 2000). These data suggest that alteration of DNA repair, not DPC removal, 17

contributes to formaldehyde

- induced clastogenicity. Since DPC repair involves proteolytic 18

removal of proteins from the DNA, the authors hypothesize that single peptide s or small peptide 19

chains cross

- linked to the DNA as in the case of DPC are critical to formaldehyde

- induced 20

mutations. 21

Formaldehyde

- induced MN and CAs are associated to concentration

- dependent 22

mutagenic effects in L5178Y mouse lymphoma cells (Speit and Merk, 2002). Detailed analysis 23

of both spontaneous and formaldehyde

- induced lesions indicate that recombination or deletion of 24

DNA from the thymidine kinase ( tk ) locus was primarily responsible for the loss of heterogeneity 25

leading to the observed mutant phenotype. Therefore, it is believed that formaldehyde is 26

mutagenic by a clastogenic mechanism, rather than through point mutations in the L5178Y 27

mouse lymphoma cell system. This finding is consistent with Craft et al. (1987) who 28

demonstrated formaldehyd e

- induced mutagenicity in the tk locus of TK6 human lymphoblastoid 29

cells, while Graftsrom et al, (1984) demonstrated increased SSBs in formaldehyde

- exposed 30

human cell lines. The elegant series of experiments by Speit and Merk provide the possible links 31

bet ween DPC, clastogenicity and locus

- specific mutations firmly demonstrating formaldehyde

32

induced mutations in the in vitro mouse lymphoma testing system. 33

Formaldehyde is genotoxic at the portal of entry (POE) in animal studies, resulting in 34

increased DPC fo rmation in the nasal mucosa as discussed above. However, there are no animal 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 430 studies which directly examine the mutagenicity in nasal or respiratory epithelial cells in the 1

early stages of exposure. It is likely that the mutations are seen in advanced st age of the tissue 2

around the transformation stage with formaldehyde exposure. With weak positive results in 3

pulmonary lavage cells (Dallas et al., 1992) and clastogenicity demonstrated in gastro

- intestinal 4

epithelial cells of rats (Migliore et al., 1989) , below exposure levels which trigger regenerative 5

cell proliferation, the existing evidence, although thin, supports clastogenic effects of 6

formaldehyde. 7

Clastogenic effects are consistently reported in humans exposed to formaldehyde in the 8

industrial wo rkplace or during anatomy or mortuary classes (See section 4.3 for a full 9

discussion). Increased micronuclei have been reported in nasal epithelial cells from industry 10

workers (Ballarin et al., 1992; Ye et al., 2005), buccal epithelial cells from anatomy and 11

mortuary science students and/or staff (Kitaeva et al., 1996; Titenko

- Holland et al., 1996; Burgaz 12

et al., 2001; 2002 compared to corresponding controls). Comparisons of micronuclei in nasal 13

and buccal cells of anatomy students before and after classe s where they are exposed to 14

formaldehyde indicate an increase in clastogenicity (Ying et al., 1997). An examination of 15

exfoliated buccal and nasal cells in mortuary students indicates greater increases in centromere

16

negative micronuclei, suggesting the ef fects are due to chromosome breakage or clastogenicity 17

rather than aneuploidy (Titenko

- Holland et al., 1996). Micronuclei were also increased in a 18

dose

- dependent manner in buccal cells as well as peripheral blood lymphocytes (PBLs) in 19

mortuary students du ring the course of an embalming class; however, SCEs were reduced in 20

post

- exposure samples (Suruda et al., 1993). Buccal, oral and nasal cells present at the portal of 21

entry may be directly exposed to formaldehyde and thus reports of clastogenic effects a re 22

consistent with direct interaction of formaldehyde at the POE. There is some supporting 23

evidence for the mutagenicity of formaldehyde in human populations. Shaham et al. (2003) 24

reported a increase in mutant p 53 protein in the PBLs of individuals with mean formaldehyde 25

exposure duration of 16 years. Additionally there was is a significant association between 26

mutant p 53 protein and DPC in this study suggesting a relationship between the formaldehyde’s 27

genotoxic effects. More recently, Zhang et al., (201 0) have reported aneuploidy in circulating 28

hematopietic stem cells in formaldehyde exposed workers with increases in both monosomy7 29

and trisomy 8. 30

In summary, there are several lines of evidence supporting mutagenic effects of 31

formaldehyde exposure: 32

1) For maldehyde directly interacts with DNA generating DPC, 33

2) DPC in tissues at the POE exhibit a dose

- response relationship to formaldehyde 34

exposure, 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 431 3) Formaldehyde

- induced DPC are associated with formaldehyde

- induced MN and CAs, 1

4) Mutations induced by for maldehyde due to small deletions and rearrangements in 2

DNA in various experimental systems are consistent with formaldehyde’s observed 3

clastogenic effects (MN and CAs), 4

5) Formaldehyde

- induced mutations and clastogenic effects occur at levels below where 5

s ignificant cytotoxicity is detected, and 6

6) Formaldehyde exposure has been correlated to similar increased MN and CAs in 7

human buccal and oral cells corresponding to sites where formaldehyde

- induced tumors 8

arise. 9

10

4.5.3.1.2 Inhibition of DNA repair 11

Studies indicate that formaldehyde exposure may inhibit DNA repair mechanisms 12

directly (See Section 4.3.1.5). Graftsrom (1985) first documented formaldehyde effects on DNA 13

repair mechanisms, reporting that formaldehyde treatment of human bronchial fibroblasts in vitro 14

inh ibited repair of O6

- methyl

- guuanine adducts induced by N

- methyl

- Nitrosurea (NMU). 15

Inhibition of DNA repair in human keratinocytes and fibroblasts cultured at 10 μM 16

formaldehyde affected repair of DNA single strand breaks from ultraviolet light but was spe cific 17

to UVB and UVC, not impacting repair of single strand breaks from UVA (Emri et al., 2004). 18

To determine if formaldehyde may have similar effects in exposed humans, Hayes et al., 19

(1997) assessed the activity O6

- alkylguanine

- DNA alkyltranferase (AGT) an enzyme critical in 20

repairing DNA damage induced by alkylating agents in formaldehyde

- exposed mortuary students 21

previously shown to have increased micronuclei in both buccal cells and peripheral lymphocytes 22

(Suruda et al., 1993). AGT activity was lower in mortuary students with prior embalming 23

exposures versus students with no prior exposure (p=0.08). Seventeen of 23 students had lower 24

AGT activity after the 9 week course (p<0.05) with a larger proportion of naïve students 25

demonstrating decreased activ ity (7 of 8) versus previously exposed students (10 of 15). 26

Although detailed exposure measurements were taken for each student, the changes in AGT 27

activity were not correlated to cumulative exposure (ppm

- hrs). 28

29

4.5.3.1.3 Protein to protein cross

- links 30

Formaldehyde is a reactive molecule that is likely to interact with both low molecular 31

weight cellular components (e.g., reduced glutathione [GSH]) as well as high molecular weight 32

cellular components. Unlike nuclear DNA, which has additional membrane barriers to exp osure 33

(i.e., nucleus), extracellular and intracellular proteins, are obvious primary targets for interacting 34

with formaldehyde. Formaldehyde is a well

- known cross

- linking agent that is used in the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 432 fixation of tissues, inactivation of toxins and viruses (e .g. preparation of vaccines), and study of 1

protein

- protein interactions (Metz et al., 2006). Using several identical synthetic polypeptides 2

differing on one amino acid, Metz et al (2004) have shown that formaldehyde initially reacts 3

with the primary amino and thiol groups of amino acids forming unstable methylol adducts, 4

which later are partially dehydrated forming labile Schiff bases that are capable of forming 5

crosslinks with other amino acid residues, such as arginine, asparagine, glutamine, histidine, 6

tryptophan, and tyrosine through methylene bridges, but not between two primary amino groups. 7

The same group (Metz et al 2006) has also shown that formaldehyde forms seven intramolecular 8

crosslinks in proteins with defined structure, such as insulin, invol ving arginine, tyrosine and 9

lysine and the N

- terminus of insulin was converted to a imidazolidinone adducts similar to that 10

observed with the synthetic peptide (Metz et al 2004). ( Figure 3

- 1 provides a general reaction 11

scheme for formaldehyde

- mediated modi fications of amino acids.) 12

13

4.5.3.1.4 Break

- down of the mucociliary apparatus 14

The mucociliary apparatus of the upper respiratory tract is the first line of defense against 15

airborne toxicants. Comprised of a thick mucus layer (epiphase), hydrophase and ciliated 16

epith elium, the mucociliary apparatus may entrain, neutralize and remove particulates and 17

airborne chemicals from inspired air (Figure 4

- 4). Formaldehyde reacts with the components of 18

the mucous layer (proteins, glycoprotein, and lipids), crosslinking proteins . Formaldehyde 19

exposure induces slowing of the mucous flow, stiffing and breaking up of the mucous layer and 20

eventual mucostasis where gaps have been observed exposing the underlying hydrophase and 21

epithelium. Although ciliary beat first increases in res ponse to formaldehyde exposure, perhaps 22

to compensate for reduced flow of the epiphase, ciliastasis ensues with both higher levels of 23

exposure, and increased duration of exposure. Altered ciliary has been noted in as little as 15 24

minutes of exposure (1.25 ppm) with functional deficits in the mucociliary apparatus at 30 25

minutes. Altered ciliary beat has been reported at the lowest concentration tested (0.5ppm) for a 26

single 6 hour exposure. Severity of effects increase with both duration and level of expos ure 27

(see section 4.2.1.2.1). 28

29

4.5.3.1.5 Induced cell proliferation 30

There are several reports apparently demonstrating formaldehyde

- induced proliferation in 31

cells below cytotoxic levels of exposure. This phenomenon has been reported from studies 32

involving both in vi tro and in vivo exposures. Tyihak et al. (2001) demonstrated significantly 33

increased cell proliferation in both HT

- 29 human colon carcinoma and human umbilical vein 34

endothelial cell (HUVEC) lines treated with 0.1mM (the lowest dose) formaldehyde compared 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 433 to untreated controls (P<0.0001). This effect was quantified as both an increase in cell number 1

over time (Figure 4

- 35), and an increase in the percentage of cells undergoing mitosis at each 2

time point. The authors also report a significant (P<0.01) inh ibition of apoptosis in 3

formaldehyde

- treated cells as compared to untreated cells (data not shown here). In a novel 4

system using xenotransplanted human tracheobronchial epithelial cells, formaldehyde was shown 5

to induce increased cell proliferation at dos es below those required for a “massive toxic effect” 6

(Ura et al., 1989). 7

8

9

10

11

12

13

14

15

16

17

18

19

20

21

Figure 4

- 35. Effect of various doses of formaldehyde on cell number in (A) 22

HT

- 29 human colon carcinoma cells and in (B) human umbilical vein 23

epithelial cells (HUVEC). 24

25

Values are average of three samples + SD; * P < 0.01 and ** P < 0.0001 26

compared to corresponding controls. 27

28

Source: Tyihak et al 2001. 29

30

31

Some animal studies have demonstrated increased cell proliferation after formaldehyde 32

exposures by both inhalation and ingestion (See section 4.2.1). However, whether sustained 33

increases in cell proliferation over baseline rates are observed upon exposure to sub

- cytotoxic 34

doses of formaldehyde remains unclear. Several of the inhalation studies demonstrate increased 35

c ell proliferation in the nasal epithelium at formaldehyde exposures levels that were sub

36

cytotoxic

— i.e. in the absence of significant cell death. Acute formaldehyde exposures (1 to 3 37

days) induced increased cell proliferation at discrete locations in the nasal mucosa, where cell 38 0 2 4 6 8 10 12 14 16 18 20

Day 1 Day 2 Day 3

Post

- treatmentCell number (104) Control

0.1 mM

1 mM

10 mM A. HT

- 29 Cells

* ** *

** **

0 2 4 6 8 10 12 14 16 18 20

Day 1 Day 2 Day 3

Post

- treatmentCell number (104) Control

0.1 mM

1 mM

10 mM B. HUVEC Cells

** **

*

0 2 4 6 8 10 12 14 16 18 20

Day 1 Day 2 Day 3

Post

- treatmentCell number (104) Control

0.1 mM

1 mM

10 mM A. HT

- 29 Cells

* ** *

** **

0 2 4 6 8 10 12 14 16 18 20

Day 1 Day 2 Day 3

Post

- treatmentCell number (104) Control

0.1 mM

1 mM

10 mM Control

0.1 mM

1 mM

10 mM A. HT

- 29 Cells

* ** *

** **

0 2 4 6 8 10 12 14 16 18 20

Day 1 Day 2 Day 3

Post

- treatmentCell number (104) Control

0.1 mM

1 mM

10 mM B. HUVEC Cells

** **

*

0 2 4 6 8 10 12 14 16 18 20

Day 1 Day 2 Day 3

Post

- treatmentCell number (104) Control

0.1 mM

1 mM

10 mM Control

0.1 mM

1 mM

10 mM B. HUVEC Cells

** **

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

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- 434 proliferation was measured as a labeling index (percentage of cells pulse

- labeled with tritiated

1

thymidine. Reuzel et al. (1990) reported increased cell proliferation in the nasal passages 2

including the nasoturbinates, maxilloturb inates septum and lateral wall in male Wistar rats 3

exposed at 3 ppm, but not at 0.3 or 1 ppm, formaldehyde for 22 hours/day for 3 days. Zwart et 4

al. (1988) reported increased cell proliferation after exposure to 1 or 3 ppm formaldehyde, 6 5

hours/day for 3 days or 13 weeks in male and female albino Wistar rats. These increases were 6

transient at level 3 but sustained at level 2 of the nose and were not correlated with cytotoxicity. 7

In contrast, Wilmer et al. (1989), from the same group of investigators and using a similar 8

exposure regimen, reported no increase in cell proliferation after repeated 8

– hour exposures at 1 9

or 2 ppm formaldehyde for 3 days or 4 weeks. Swenberg et al. (1986) demonstrated a transient 10

increase in cell proliferation after a single 8

– hour exposure to 0.5 or 2 ppm formaldehyde in 11

male F344 rats but no increases after 3 days or repeated 8

– hour exposures. The authors suggest 12

that adaptive responses of the nasal mucosa contribute to the transient nature of formaldehyde

13

induced cell prolif eration. After a series of acute studies at various formaldehyde concentrations, 14

Swenberg and coworkers concluded that, in addition to cell proliferation being concentration

- , 15

dose

- and time

- dependent, the response varies by species and by location of exp osure in the nose 16

(Swenberg et al., 1983, Swenberg et al, 1986). 17

Other methods of quantifying cell proliferation in the nasal mucosa have demonstrated 18

formaldehyde

- induced cell proliferation at similar low exposure concentrations. Roemer et al. 19

(1992) m easured cell proliferation by flow

- cytometry in epithelial cells harvested from the nose 20

and trachea of male Sprauge

- Dawley rats exposed to 2 ppm formaldehyde for 6 hours/day for 1 21

or 3 days and found increased cell proliferation after the 1

- day exposure. These increases were 22

transient and were not evident after 3 days of exposure. Cassee and Feron (1994) identified 23

proliferating cells by staining for the presence of proliferating cell nuclear antigen (PCNA). 24

Formaldehyde exposure at 3.6 ppm for 6 consec utive periods of 12 hours (8

- hour exposures 25

followed by 4

- hour

- periods of non

- exposure) over three days, qualitatively increased the 26

expression of PCNA in respiratory epithelium at levels 2 and 3 of the nose in albino male Wistar 27

rats (Cassee and Feron, 19 94). Hyperplasia, squamous metaplasia and frank necrosis were also 28

reported for these tissues. 29

Monticello et al. (1990, 1991, 1996) conducted in vivo cell proliferation studies in which 30

they exposed F344 rats for short durations (1, 4, 9 and 42 days) as w ell as much longer durations 31

(13, 26, 52 and 78 weeks) to exposure concentrations of 0, 0.7, 2.0, 6.0, 10.0 and 15.0 ppm. 32

These data are unique in that they also included low exposure concentrations. The authors 33

reported statistically significant increases in cell proliferation only at 6.0 ppm and higher 34

exposure concentrations in the short duration study and only at 10.0 ppm and higher 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 435 concentrations in the longer duration study. These data have undergone considerable statistical 1

analysis in several papers as well as in this document. Conolly et al. (2002, 2003) and Gaylor and 2

Conolly (2004) interpreted these data, when combined, as indicating a non

- monotonic behavior 3

at low dose. In other words, formaldehyde was judged to result in a reduction in cell prol iferation 4

at low dose in comparison to baseline rates, with increased proliferation effect kicking in only at 5

exposures that were cytotoxic. However, as shown in Appendix 5

- 3 and in Subramaniam et al. 6

(2008), Crump et al. (2008), analysis of the individual animal data shows considerable 7

uncertainty and variability, both quantitative and qualitative, in the interpretation of these cell 8

proliferation data. (For example, even the control data vary over an order of magnitude in some 9

cases. See Figures 5

- 22 and 5

- 23 in Appendix 5

- 3.) These analyses (which were based on the 10

replicate animal data used in the above studies) considered regional formaldehyde dose to the 11

tissue (flux), nasal site and duration of exposure, as well as the number of cells at a given site. 12

The overall conclusion in Section 5.3.3 (and detailed in Appendix 5

- 3) is that the cell 13

proliferation dose

- response at low dose could be reasonably described by both monotonic (with 14

and without a threshold) and non

- monotonic curves. 15

Only one study, by Mo nticello et al. (1989), quantified cell proliferation in primates after 16

formaldehyde exposure; this study, reported an 18

- fold increase in cell proliferation in the nasal 17

epithelium (respiratory and transitional), larynx, trachea and carina of male Rhesus monkeys 18

exposed to 6 ppm formaldehyde compared to controls (See section 4.2.1 for detailed study 19

description). The authors also noted that increased cell proliferation was seen in locations with 20

minimal histological changes, indicating proliferation may be a more sensitive predictor of 21

adverse health effects of formaldehyde exposure. 22

23

4.5.3.1.6 Cytolethality and resulting regenerative cell proliferation 24

The toxic and cytolethal effects of formaldehyde exposure at the POE are well 25

documented after both inhalation and oral exposures (See Section 4.2.1). The nature and 26

progression of tissue injury has been best documented in rodent inhalation assays. Early effects 27

on the nasal mucosa include altered ciliary beat and mucus flow, hyperplasia and metaplasia of 28

nasal epithelium (Morgan et al., 1986a; Morgan et al., 1986b; Monteiro

- Riviere and Popp, 1986; 29

Maronpot et al 1986; Rusch et al., 1983 and Monticello et al., 1986). These first changes may be 30

considered adaptive responses. Squamous epithelium may thicken and t ransitional epithelium 31

may change to squamous epithelium as evidenced by squamous hyperplasia, squamous 32

metaplasia and thickening of the epithelium in these anterior portions of the nose. Tissue 33

damage may be transient at lower formaldehyde exposures as t hese changes serve to protect 34

tissue from formaldehyde’s reactivity. However, higher formaldehyde concentrations can 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 436 overwhelm these adaptive responses and result in gross tissue damage. Frank necrosis and focal 1

erosions have been reported in time

- and c oncentration

- dependent manner in rodent bioassays. 2

Both adaptive changes and cytolethality are associated with cell proliferation. However, 3

where adaptive changes are successful, e.g. prevent continued toxic insult to the tissue, cell 4

proliferation is t ransient. Exposure regimens where the adaptive changes are not adequate to 5

protect the tissue, would result in continued cytoxicity and cell death. Sustained damage to the 6

epithelium would result in sustained cell proliferation to compensate for cell dea th. A series of 7

rodent bioassays present convincing evidence that chronic inhalation exposures 6 hours a day, 5 8

days a week at 6, 10 and 15 ppm formaldehyde do result in sustained damage to the nasal 9

epithelium, sustained cell proliferation and tumor deve lopment (Kerns et al., 1983, Morgan et al., 10

1986; Monticello et al., 1990; Monticello et al., 1991 and Monticello et al., 1996). Work by 11

Monticello and coworkers demonstrate that chronic repeated inhalation exposures at 6, 10 or 15 12

ppm formaldehyde result in sustained cell proliferation at the lateral meatus, mid

- septum and 13

maxilloturbinates of rat nasal passages (Monticello et al., 1991 and Monticello et al., 1996). 14

15

4.5.3.1.7 Evidence for promotion 16

There is some evidence, although mixed, that formaldehyde may promote tumor 17

development by other carcinogens, and known initiating agents by various routes of exposure. 18

Formaldehyde exposure in drinking water (0.5% formalin) increased glandular stomach 19

adenocarcinomas in male Wistar rats after initiation with 100 mg /L, N

- methyl

- N’

- 20

nitrosoguanidine (MNNG), compared to MNNG

- only

- treated rats (Takahashi et al., 1986). In 21

white non

- inbred rats, inhalation exposures (3, 30 or 300 ug/m 3 formaldehyde 7hr/day, 5 22

days/week for 1 year) increased tumor multiplicity per animal and decreased latency of 23

benzo[a]pyrene induced tumors in white non

- inbred rats (Yanysheva et al., 1998.) Similarly, 24

formaldehyde skin application decreased tumor latency, in 7,12

- dimethylbenz(a)anthacene 25

(DMBA) initiated hairless Oslo mice (Iversen, 1986 ). Although formaldehyde exposure also 26

increased the tumor multiplicity in Syrian golden hamsters where diethylnitrosamine (DEN) 27

(0.25mg I.P.) was the tumor initiator, positive results were only reported for the exposure 28

regimen where hamsters were expose d to formaldehyde via inhalation 48 hours prior to DEN 29

injection, and then one a week thereafter for life. However, formaldehyde did not increase the 30

number of tumors per tumor bearing animals when only administered beginning one week after 31

all DEN inject ions. In contrast, bladder cancer was not enhanced by intravesical instillation of 32

0.5ml of 0.3% formalin, one week after instillation of N

- methyl

- N

- nitrosourea (MNU) in male 33

Fisher rats (Homma et al., 1986). 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 437 The observed promotion activity of formaldehyd e has been tested in several systems, by 1

different routes of exposure. By several routes of exposure, formaldehyde enhanced tumor 2

development at a site where formaldehyde did not induce tumors alone, without the initiating 3

agent (Takahashi et al., 1986, Y anysheva et al., 1998 and Iversen, 1986). Promotion activity in 4

these studies was evidenced by increased in tumor bearing animals (oral route), increase in 5

tumors per animal (inhalation routes) and decreased tumor latency compared to those animals 6

only ex posed to the initiating agent (inhalation route) (Takahashi et al., 1986, Yanysheva et al., 7

1998 and Iversen, 1986). Although these experiments do not indicate how formaldehyde acts as 8

a promoter in these systems, it is possible formaldehyde

- induced mutat ion, increased cell 9

proliferation or other toxic action could enhance tumor development from another agent. 10

11

4.5.3.1.8 Localized Immunosuppression 12

Formaldehyde exposure has induced localized immune suppression in experimental 13

animals (Dean et al., 1984) and in expos ed workers (Lyapina et al., 2004). Repeated inhalation 14

exposures in rodents depopulated the URT and pulmonary tissues of resident macrophages, 15

resulting in a transient decrease in POE host defenses (Admas et al., 1987). After cessation of 16

exposure, the m ononuclear phagocyte (MP) populations were replenished and there was a 17

subsequent increase in host defense representing both increased MP numbers and increased 18

bacteriocidal activity of the MPs. These data suggest that peak exposures of formaldehyde may 19

p resent localized immunosuppression for components of the mononuclear phagocyte system 20

(MPS) in tissues at the site of first contact. 21

A number of studies have evaluated the ability of formaldehyde to induce systemic 22

immunotoxic effects in humans (Ohtani et al., 2004a, b; Erdei et al., 2003; Thrasher et al., 1990, 23

1987 ; Pross et al., 1987 ). Some studies have reported altered innate immune responses 24

associated with formaldehyde exposure (Erdei et al., 2003), while others have noted adaptive 25

immune response suppression associated with formaldehyde exposure (Thrasher et al., 1990, 26

1987) and changes associated with alterations to a predominant T

— lymphocyte helper 2 (Th2) 27

pattern (Ohtani et al., 2004a, b). In contrast, Pross et al. (1987) did not observe forma ldehyde

28

associated changes in systemic immune function. 29

Numerous studies have reported increased respiratory tract infections in formaldehyde 30

exposed individuals both in occupational and residential environments (Lyapina et al., 2004; 31

Krzyzanowski et al. , 1990; Holness and Nethercott, 1989). Incidences of physician

- diagnosed 32

chronic bronchitis were more prevalent in children (under age 15) living in homes with higher 33

formaldehyde (>60 ppb) readings in the kitchen ( p < 0.001) but this effect was more pron ounced 34

( p < 0.001) in children simultaneously exposed to environmental tobacco smoke (Kryzanowski 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 438 et al., 1990). The prevalence of chronic cough was also increased in adults living in homes with 1

measurable levels of formaldehyde, but data were not shown. Holness and Nethercott (1989) 2

assessed chronic bronchitis in 87 funeral workers, where the average formaldehyde exposure was 3

reported at 0.38 ± 0.19 ppm. Chronic bronchitis was observed in 20 funeral workers (n = 87) 4

exposed to formaldehyde compared with 3 cases of chronic bronchitis in nonexposed referent 5

controls (n = 38). A statistically significant association of self

- reported chronic bronchitis and 6

decreased resistance to URT infection was reported in formaldehyde exposed workers compared 7

with contr ols ( p = 0.02) (Lyapina et al., 2004). Of the workers, 41% had a history of chronic 8

respiratory infection and frequent long

- lasting infectious inflammatory relapses (group 1a). 9

Another group (group 1b) consisted of 17 exposed workers, 12 of whom had no h istory of 10

recurrent viral infections of the URT. There was a statistically significant association of 11

frequency and duration of inflammatory relapses between groups 1a and 1b. 12

Lyapina et al. (2004) also reported effects of formaldehyde exposure on neutrop hil 13

respiratory burst activity (NRBA), the capacity of polymorphonuclear leukocytes to produce 14

reactive oxygen radicals in response to chemical or microbial stimuli using flow cytometry. A 15

suite of hematological tests and flow cytometric analysis for resp iratory burst activity were 16

performed. Although no significant difference was observed in the spontaneous and stimulated 17

NRBA (median percentage of oxidizing cells) between the 29 exposed workers with URT 18

inflammation and the healthy controls (0.83 versus 1.35, respectively), a separate comparison of 19

the NRBA of 12 workers with chronic, repeating URT infections and 17 workers with short, 20

infrequent episodes of URT inflammations was significant (0.45 versus 1.00, p = 0.037). When 21

the NRBA of the group with chronic URT infections (n = 12) was separately compared with that 22

of the healthy controls (n = 21), the results were also significant (0.45 versus 1.35, p = 0.012). 23

Individuals with chronic URT infections have reduced NRBA that could be due to formaldehy de 24

exposure. Neutrophils respond to tissue damage or local invasion of microorganisms and act to 25

phagocytize foreign cells. If neutrophilic activity is hampered or altered by formaldehyde 26

exposure, then the ability to fight infection will be diminished, leading to prolonged infection. 27

However, no dose

- response pattern of formaldehyde exposure could be determined from this 28

study. 29

30

4.5.3.1.9 Potential for systemic transport of formaldehyde 31

In aqueous solution formaldehyde exists in equilibrium with it’s hydrated fo rm 32

methanediol (CH 2 OH 2 ) (K d = 5.5x10

- 4 ). The equilibrium favors methanediol at physiological 33

temperature and pH (>99.9%) and is readily reversible. In biological systems, as free 34

formaldehyde is removed from aqueous solution through binding with serum pr oteins and 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 439 cellular components, the equilibrium is reestablished by dehydration of methanediol to free 1

formaldehyde. The reversible nature of this hydration reaction describes how a pool of free 2

formaldehyde may be sustained in biological systems. 3

There is strong and consistent evidence in biological testing systems in vitro that treating 4

cells with formaldehyde in an aqueous media results in significant cytoxicity, cell proliferation, 5

clastogenic effects and clear evidence of mutational events (Section 4 .3). Similarly, animal 6

bioassays where formaldehyde is administered in drinking water report portal of entry toxicity 7

including hyperplasia, increased cell proliferation, focal lesions and tumors (Section 4.2.1). It 8

should be noted that URT tissues are c overed by an aqueous mucous layer, through which 9

formaldehyde must pass to react the cellular components of the URT. It has been postulated that 10

formaldehyde transports through this mucous layer and the underlying tissues as methanediol 11

(Georgieva et al., 2003). 12

The dynamic equilibrium between the hydrated and unhydrated forms of formaldehyde in 13

biological systems is well understood. Since the hydration reaction favors methanediol, it is 14

expected that exogenous formaldehyde which reaches the blood will p rimarily exist as 15

methanediol and is subject to physiological elimination. As free, unhydrated formaldehyde 16

continues to react with serum proteins and cellular components, the blood levels of methanediol 17

are expected to reduce as it is dehydrated to maint ain equilibrium. Although some attempts to 18

measure significant changes in free formaldehyde levels in blood after inhalation exposure have 19

not been successful, the half

- life in blood has been measured after i.v. injection at approximately 20

2 minutes (McMar tin et al., 1979). Additionally, the detection of antibodies to formaldehyde

21

hemoglobin adducts and formaldehyde

- albumin adducts in exposures workers, smokers and 22

laboratory animals exposed via inhalation provides direct evidence that formaldehyde is ab le to 23

react with serum albumin and hemoglobin in biological systems (Thrasher et al., 1990, Grammer 24

et al., 1990; Grammer et al., 1993; Dykewicz et al., 1991; Varro et al., 1997 and Li et al., 2007). 25

These data support the hypothesis that exogenous formal dehyde may reach and transport through 26

the blood. If so, formaldehyde (or methanediol) may reach sites distal to the portal of entry. 27

28

4.5.3.2 Mode of Action Evaluation for Upper Respiratory Tract Cancer (Nasopharyngeal 29

Cancer, Sino

- nasal) 30

From the above discus sion, it can be seen that numerous mechanisms of action for 31

formaldehyde

- induced cancer can be reasonably supported based on various known biological 32

actions of formaldehyde (e.g., mutation, cell proliferation, cytotoxicity, and regenerative cell 33

prolifera tion). Additionally, alternative actions, such as immunosuppression or viral 34

reactivation, are possible, although less data exist to evaluate these MOAs. Rather than a single 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 440 MOA, it is plausible that a combination of these factors contribute to cancer i ncidence in an 1

exposed population. Considering multiple factors may help to better understand the biological 2

and mechanistic basis for the increases in cancer incidence observed in exposed human 3

populations. Unlike animal bioassays, human epidemiological studies may reflect not only the 4

effects of the agent of concern but also numerous other risk factors (e.g., viral status, diet, 5

smoking, etc.). Additionally, human studies may be impacted by biological human variability 6

across individuals, cancer biolog y (sub

- types), wide variability in exposure regimens in human 7

populations, etc. Therefore, if the purposes of exploring the carcinogenic MOA of an agent are 8

to better understand the relevance of a given carcinogen to human populations and to inform the 9

e xposure

- response analysis, then discussions of MOAs which recognize the interaction of an 10

agent with human variability and various risk factors is an appropriate analysis. 11

12

a) Direct mutagenicity of formaldehyde in cells at the site of first contact: Mutation s, 13

the permanent heritable changes in the genome of the cell, are a primary mechanism for 14

the activation of oncogenes or the inactivation of tumor suppressor genes. Mutagenicity 15

is the most widely recognized determinant of chemical

- induced carcinogenicity , and it is 16

difficult to set aside the relevance of direct formaldehyde

- induced mutations from its 17

demonstrated carcinogenicity. Formaldehyde

- induced mutation in mucosal cells of the 18

URT, throat and buccal cavity may serve to initiate cells, or provide su bsequent mutageic 19

events to already initiated cells. Since the mucosal cells have proliferative capacity, and 20

cell proliferation is a normal tissue function, mutations may be fixed and passed to 21

daughter cells due to baseline cell proliferation of the tis sue. 22

Relevance to humans: This MOA is relevant to humans. The well

- documented DNA 23

reactivity (e.g. DPC and DNA adducts) and clastogenicity of formaldehyde in the URT of 24

laboratory animals is a direct effect of formaldehyde on tissues of first contact. As this is 25

a direct acting agent

- no distribution or metabolism is required for the genotoxic action

– 26

there is little expected species variability. As discussed in chapter 3, there are species 27

differences in flux of formaldehyde into the respiratory mu cosal tissues, but this 28

introduces species differences in dosimetry

– not mechanism. Finally, the clastogenic 29

effects in nasal and buccal epithelial cells in formaldehyde

- exposed workers confirms 30

the direct genotoxic effects of formaldehyde at the first site of contact in humans. 31

32

b) Decrease in DNA repair function within cells at the site of first contact: A decrease 33

in DNA repair capacity in these tissues by formaldehyde may increase total mutations 34

over time due to either endogenous or exogenous sources of mutation. Although there 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 441 are only a few studies which have explored the potential for formaldehyde to reduce 1

DNA repair capacity, the evidence is positive, both in vitro testing systems, and in one 2

study of occupationally exposed humans (Grafstrom 1985 ; Hayes et al., 1997). 3

Relevance to humans: This MOA is considered relevant to humans. The general 4

population is exposed to various carcinogens, many with mutagenic potential, at sites of 5

first contact including; air pollution, tobacco products, nitrosa mines and viruses. 6

Additionally, there are endogenous sources of DNA damage and mutagenicity in humans 7

(e.g. lipid peroxidation, oxidative stress). The demonstration of reduced DNA repair 8

activity (O6

- alkylguanine

- DNA alkyltransferase activity) in formal dehyde

- exposed 9

mortuary students suggests this toxic action of formaldehyde is possible in humans. 10

11

c) Formaldehyde

- induced cell proliferation: Formaldehyde

- induced cell proliferation in 12

the oral and respiratory mucosa may be considered a key event in conjun ction with the 13

genotoxic effects, and induced mutational events observed with formaldehyde exposure. 14

This MOA is intended to describe events which occur below exposure levels which 15

induce cell death and mucosal lesions. Therefore this MOA is comprised of two key 16

events: 17

a. Formaldehyde

- induced genotoxicity or mutation 18

b. Formaldehyde

- induced cell proliferation 19

20

DNA replication during cell proliferation may serve to translate DNA damage or a 21

formaldehyde

- related DNA lesion into a permanent change in the sequen ce of nucleic 22

acids during replication of the DNA

– e.g. ‘fix’ a mutation from DNA damage. 23

Additionally formaldehyde

- induced cell proliferation may provide an opportunity for 24

initiated cells to proliferate, increasing the potential for additional mutation events and 25

transformation. The increased cell proliferation observed in the mucosal tissues in direct 26

contact with formaldehyde during inhalation exposures may serve to amplify the risk of 27

cell transformation from mutation alone. Researchers have noted that increased cell 28

proliferation may be transient in some locations as adaptive responses compensate 29

(Swenberg 1983). However, evidence in both monkeys and rodents indicate that 30

increased cell proliferation in repeated exposures across time do result in sustained cell 31

proliferation. Data in Rhesus monkeys indicates increased cell proliferation is observed 32

beyond the nasal cavities to the larynx, trachea and carnia (first tracheal 33

branching)(Monticello et al., 1989). Additionally, the authors note that c ell proliferation 34This document is a draft for review purposes only and does not constitute Agency policy.

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- 442 is a more sensitive indicator of effects on the epithelium, observed even when minimal 1

histological changes were present. 2

Human Relevance: Both formaldehyde

- induced mutation and cell proliferation are 3

direct effects on the oral and nas al mucosa, well documented in rodent models with 4

supporting evidence in human epidemiological studies. Therefore both key events are 5

relevant to humans. As noted above, there are species differences in localized flux of 6

formaldehyde into the tissues of t he oral and respiratory tract based on structural 7

differences in the airways, as well as breathing patterns. Although these differences may 8

effects the dosimetry of the formaldehyde absorption into the tissues, this only influences 9

the magnitude of respon se at any given location. Data from exposed Rhesus monkeys 10

which documents formaldehyde

- induced cell proliferation in tissues beyond the nasal 11

cavity, and tissues with minimal histological changes supports a role for cell

- proliferation 12

in the observed can cers in humans, which occur beyond the nasal cavities, and in tissues 13

without formaldehyde

- related focal lesions. 14

15

d) Cytotoxicity

- induced cell proliferation (CICP): Cell death followed by compensatory 16

cell proliferation is a reasonable MOA for agent

- induced cancer. It should be noted that 17

the exposure conditions which result in CICP in rodents is known to result in significant 18

DNA reactivity and genotoxicity. Therefore, formaldehyde

- induced mutations cannot be 19

excluded from this MOA. The animal bioassays s upport the carcinogenic potential of 20

formaldehyde in this context (Kerns et al., 1983; Selkemur et al, 1983 and Monticello et 21

al 1986). The majority of squamous cell carcinomas (SCCs) seen in formaldehyde

22

exposed rats have been localized to the lateral me atus and mid

- septum in the nasal 23

passages (Morgan et al., 1986; Monticello et al., 1996), while polyploid adenomas have 24

predominantly been reported at the maxilloturbinates (Morgan et al., 1986; Monticello et 25

al., 1996). Morgan et al. (1986) speculated th at the maxilloturbinate was less susceptible 26

to SCC due to metabolic differences. However, Monticello et al. (1996) later suggested 27

that the smaller population of cells available at the maxilloturbinate accounted for fewer 28

SCCs observed at that site. Reg ardless, for those locations where SCCs do arise in rats 29

chronically exposed to formaldehyde, a clear temporal relationship can be demonstrated 30

for dose regimens capable of producing sustained epithelial damage and sustained cell 31

proliferation to eventual tumor development. Conversely, tumors are not observed in 32

these rodent models at those sites in the nasal passages without sustained cell 33

proliferation. 34

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

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- 443 Relevance to humans: Human exposure to formaldehyde would most likely involve 1

chronic exposures t o indoor levels of formaldehyde, and episodic exposures in the 2

environment or from an occupational exposure (See review in Chapter 2). An exposure 3

scenario parallel to that used in chronic rodent bioassays is unlikely (e.g. 2

- 15 ppm 6

- 8 4

hours/day, 5 days/ week for 10

- 30 months). Exposure conditions are difficult to assess 5

especially in retrospective studies. However, only the most extreme industrial work 6

conditions would result in human exposures similar to those that produce sustained 7

compensatory cell proliferation in animal studies (i.e. 6

- 15 ppm 6 hrs/day, 5 days per 8

week). Gross tissue lesions as reported in rodents from repeated chronic exposures at 6 9

and 10 ppm formaldehyde have not been reported from workplace exposure, and only 10

minor histopathol ogical changes have been noted (Boysen et al., 1990 and Holmstrom 11

and Wilhelmsson et al., 1989). It is possible that workers were episodically exposed to 12

formaldehyde levels which resulted in cell death and focal or gross lesions requiring cell 13

proliferati on for tissue remodeling or repair. However, it is unexpected that these 14

conditions would be relevant to human environmental exposures. Therefore, although 15

regenerative cell proliferation is retained as a reasonable MOA for formaldehyde 16

carcinogenicity i n experimental animals, it is unclear whether it is relevant to the 17

extrapolation of health risks to formaldehyde exposures in the general environment. 18

19

e) Promotion: Several animal studies indicate that formaldehyde exposure may promote 20

tumor formation due to other carcinogenic or initiating agents. There are positive data 21

by several routes of exposure (oral, dermal and inhalation) and promotion has been 22

reported as an increase in tumor bearing animals, an increase in tumors multiplicity or a 23

decrease in t umor latency with formaldehyde exposure in conjunction with the initiating 24

agent compared to tumors from the initiating agent along, or formaldehyde alone. The 25

specific key events which may explain this promotion effect are unknown but may 26

include several of the mechanisms discussed as potential MOAs for formaldehyde: 27

mutagenicity, mitogenesis, co

- carcinogenicity, immunosuppression. Promotion is 28

considered here as a separate MOA, since these activities are noted for experimental 29

conditions and tumor sites where formaldehyde did not induce tumors in the absence of 30

the initiating agent. 31

Relevance to humans: Although the human epidemiologic literature doesn’t address 32

issues of tumor promotion, the nature of the cancers of concern indicate that chemical 33

prom otion may be relevant to cancer incidence for these sites. Many of the risk factors 34

for NPC and other mouth and oral and URT cancers include direct mutagens (e.g. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 444 smoking, dietary nitrosamines) where a promoting agent would be expected to increase 1

cancer incidence with these other risk factors. Additionally, the well known viral risk 2

factors for cancers of the mouth and URT also suggest a role for promoting agents to 3

human cancer incidence. Although only tangential evidence, this does suggest that the 4

pr omoting activity of a chemical agent, would be relevant to the agent’s carcinogenicity 5

at these sites. Therefore, the potential for formaldehyde to act as a promoter with other 6

initiators

– is considered relevant to formaldehyde’s carcinogenic MOA. 7

8

f) Incr eased URT infections / viral reactivation: Inhalation exposure to formaldehyde 9

has been shown to decrease the defenses of the body against infection through two 10

mechanisms: 1) damage to the protective mucous barrier and function of the mucociliary 11

apparat us; and 2) localized immunosuppression. These effects have been demonstrated 12

in both exposed humans and controlled animal experiments. Additionally, increased 13

respiratory tract infections are associated with formaldehyde exposure in several 14

populations. Common viral agents (e.g. Epstein barr virus) are known risk factors for 15

NPC, sinonasal cancers and other URT cancers. Although direct evidence does support 16

increased URT infections due to formaldehyde exposure, and URT infections are 17

considered risk fac tors for URT cancers, direct evidence for formaldehyde

- related 18

infections leading to cancer is lacking. There is however one epidemiological study 19

which finds the association between formaldehyde and NPC is strengthened in Epstein 20

barr virus sero

- positive cases versus sero

- negative cases. These data suggest a possible 21

role for formaldehyde in infection, viral reactivation, or co

- carcinogenicity with a viral 22

agent. 23

24

Relevance to humans: The potential role of increased URT infections and 25

immunosuppressio n at the portal of entry is considered to relevant to humans. Data in 26

humans are available to support both key events in this MOA. Additionally, 27

epidemiological studies are conducted in human populations where individuals may be 28

exposed to various viral agents across the study period. Therefore, toxic actions by 29

formaldehyde which may increase URT infections, or viral

- reactivation at the site of first 30

contact, could be acting in conjunction with viral agents to contribute, in part, to observed 31

associatio ns between formaldehyde exposure and increased URT cancer. 32

33

Summary and integration of key events: 34

Each of the hypothesized MOAs discussed above to better understand the carcinogenic 35

potential of formaldehyde is supported by formaldehyde

- specific evidence, either in animal 36This document is a draft for review purposes only and does not constitute Agency policy.

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- 445 studies, human studies or both. For those key events studied in animal models such as cell 1

proliferation, genotoxicity, degradation of the mucociliary apparatus and CICP, supporting 2

evidence is available in more than one species, multipl e strains (e.g. rats) and has been reported 3

by multiple researchers. Therefore the overall database supporting these key events in laboratory 4

studies, and their corresponding MOAs is fairly large. In contrast, some key events relevant to 5

humans, but less studied in animal models may have a small supporting database (e.g. increased 6

respiratory tract infections). These alternative MOAs are retained as potentially relevant to the 7

carcinogenic action of formaldehyde as the intent of this discussion is to ide ntify modes of action 8

will may contribute to the observation of increased upper respiratory tract cancers in exposed 9

human populations. It is noted that additional study is needed to better understand the range of 10

effects formaldehyde may have at sites of first contact in humans. 11

The MOAs which include genotoxicity, mutation, decreased DNA repair, increased cell 12

proliferation and CICP are interrelated. Conditions which provide both a source of cell 13

proliferation and increased mutation would be expected to increase neoplastic transformation. 14

Formaldehyde acts on the target tissue, the respiratory epithelium, to induce each of these events. 15

However, these key events operate across different exposure ranges and present different 16

exposure response relationsh ips. For example, formaldehyde

- induced mutations would be 17

expected across the exposure range, where any incremental increase in genotoxicity and 18

formaldehyde

- related mutation would contribute to background levels, with the potential to 19

increase cancer ris k incrementally. In contrast, focal and gross lesions to the respiratory mucosa 20

due to cytolethality are not observed unless exposure concentrations are sufficient to provide 21

localized tissue doses (flux) required to result in cell death and related compe nsatory cell 22

proliferation. Since tissue dose (flux) is dependent on not only exposure concentration but also 23

duration of exposure and location in the respiratory tract (Section 3.4), and varies by species, 24

correlation of exposure concentrations to tissue responses directly are complex. Exposure 25

response relationships for the key events (cell proliferation, genotoxicity, degradation of the 26

mucociliary apparatus and CICP) are reported by exposure concentration, not tissue flux, which 27

would be a more biolog ically relevant measure. 28

Although the tissue dose

- response relationships for formaldehyde induced mutation, 29

mitogenesis and cytolethality are different, the effects at the tissue level cannot be easily 30

disaggregated. At any given exposure concentration, t arget cells in the respiratory tract will 31

experience different effective tissue concentrations of formaldehyde. Measurement of cell 32

proliferation, DNA protein crosslinks or genotoxicity may require examining a population of 33

cells which would have been sub ject to different flux rates of formaldehyde (See chapter 3). 34

Similarly, when evaluating the tumor dose response, cells within the target tissue will represent a 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 446 range of target tissue formaldehyde concentrations. Therefore, an integrated MOA scheme is 1

h ypothesized where key events may influence the observed tumor response differentially across 2

the exposure response range (Figure 4

- 36). This schematic illustrates the potential for 3

genotoxicity and formaldehyde

- induced mutation to occur where tissue dose (flux of 4

formaldehyde into the tissue is minimal). Where tissue dose is increased, formaldehyde

- induced 5

cell proliferation is observed in addition to genotoxicity. As tissue dose increases and 6

formaldehyde effects on the respiratory mucosa are more seve re, gross pathology including focal 7

and gross lesions due to cell death are noted. Therefore, several of the MOAs presented above 8

may be operative and relevant to human exposures at exposure levels resulting in minimal tissue 9

flux

– a) direct formaldehyde genitoxicity and resulting mutation, b) inhibition of DNA repair 10

and c) formaldehyde induced cell proliferation in conjunction with mutation. CICP, which 11

involves localized and gross tissue lesions would be operative at higher exposure levels. There is 12

little data to inform the dose range over which the remaining hypothesized MOAs may operate 13

(promotion and increased respiratory tract infections/viral action). 14

15

Mutagenesis

Basal Cell Proliferation

Tumor

Response

Exposure Concentration

Adaptive Cell Proliferation Induced mitogenesis

Regenerative Proliferation A B C Integrated Dose Response Curve for Formaldehyde

16 Figure 4

- 36: Integrated MOA scheme for respiratory tract tumors 17

18

4.5.3.3 Mode(s) of Action for Lymphohematopoietic Malignancies 19

4.5.3.3.1 MOA evaluation for Leukemia 20

Leukemia may arise from stem cells and progenitor cells in the bone marrow (e.g. acute 21

and chronic myeloid leukemia) or from mature lymphocytes (e.g. chronic lymphatic leukemia, 22This document is a draft for review purposes only and does not constitute Agency policy.

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- 447 hairy cell l eukemia) (Figure 4

- 33, Section 4.5.2). Although there is a consistent association 1

between formaldehyde exposure and forms of leukemia when considered as group of diseases 2

(Table 4

- 91, Section 4.5.2), the strongest and most consistent associations are seen specifically 3

with myeloid leukemia. Little evidence supports an association between formaldehyde exposure 4

and other specific leukemia subtypes, although two studies support a strong association between 5

formaldehyde and “other leukemia and unspecified leu kemia (ICD

- 9 code 207). Therefore, this 6

MOA evaluation will focus on mechanisms which may impact all forms of leukemia (e.g. bone 7

marrow toxicity) or those specific to myeloid leukemia. The mechanistic data supporting the key 8

events in this analysis are p resented in section 4.5.3.1. 9

10

a) Direct effects of formaldehyde on a circulating stem cell or progenitor cell present at 11

the portal of entry: Hematopoietic stem cells do circulate throughout the body and can 12

be harvested from peripheral blood. Formaldehyde exhibits a range of toxic effects at the 13

site of first contact including genotoxic effects believed to be mediated by direct DNA 14

reactivity (Section 4.3). Formaldehyde is known to directly react with blood components 15

in formaldehyde exposed humans and ani mals resulting in both hemoglobin and albumin 16

adducts (Thrasher et al., 1990, Grammer et al., 1990; Grammer et al., 1993; Dykewicz et 17

al., 1991; Varro et al., 1997 and Li et al., 2007). Therefore, it has been hypothesized that 18

formaldehyde could react wit h DNA in circulating hematopoietic stem cells (Zhang et al., 19

2009) resulting in heritable mutations which may contribute to leukemia incidence. 20

Recently Zhang et al. (2010) have tested the hypothesis that exogenous formaldehyde 21

may damage circulating stem cells. Clastogenic effects were found in circulating 22

hematopoietic stem cells cultured from formaldehyde exposed workers. The reported 23

aneuploidy was demonstrated as significant increases in both monosomy 7 and trisomy 8. 24

These specific chromosomal cha nges are consistent with those reported for agent

- induced 25

myeloid leukemia (Zhang et al., 2010). 26

Relevance to Humans: This hypothesized MOA is considered relevant to humans. 27

Supporting evidence is found in humans for formaldehyde direct reactivity with b lood 28

proteins (e.g. albumin and hemoglobin) as well as clastogenic effects in circulating 29

hematopoietic stem cells in formaldehyde exposed workers. 30

31

b) Bone marrow toxicity: Direct bone marrow toxicity is the most studied leukemogenic 32

action for an endogenou s agent (e.g. benzene, ionizing radiation). It is believed that an 33

agent which exerts its toxicity on the bone marrow, resulting in translocations and 34

heritable mutations in hematopoietic stem cells may cause leukemia. It has been 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 448 hypothesized that forma ldehyde may transport to the bone marrow in its hydrated form 1

(methandiol) and react with cellular proteins, and DNA casuing direct effects on 2

components of the bone marrow. Pancytopenia (a reduction in blood borne cells formed 3

in the bone marrow) is a sy mptom of direct bone marrow toxicity and is observed with 4

other leukemogenic agents (e.g. benzene, ionizing radiation). A recent review of 8 5

published studies of formaldehyde exposed workers in China by Tang et al. (2009) 6

indicates 7 of the studies provid e evidence of reduced white blood cell counts, platelet 7

levels and hemoglobin levels associated with formaldehyde exposure. A study of 8

occupationally exposed nurses provided a correlation between decreased white blood cell 9

counts and formaldehyde exposure (Kuo et al., 1997). A recent study by Zhang et al. 10

(2010) provides the best evidence for bone marrow toxicity, where they report not only a 11

reduction in white blood cell counts, but reductions in cell counts of all the blood cells, as 12

well as increased mean cell volume. Although these reductions did not meet the clinical 13

definition of pancytopenia (when averaged across the study population), reduction of all 14

blood borne cells formed in the bone marrow is consistent with the bone marrow toxicity 15

associat ed with pancytopenia seen with other leukemogens (Zhang et al., 2010). 16

Relevance to Humas: This hypothesized MOA is considered relevant to humans. 17

Supporting evidence is found in humans for bone marrow toxicity in formaldehyde 18

exposed workers. 19

20

4.5.3.3.2 MOA eva luation for Lymphomas (e.g. Hodgkin lymphoma, Multiple myeloma) 21

The general MOA for formaldehyde is based on direct chemical reactivity and toxic 22

effects at the portal of entry (POE). Formaldehyde is directly and indirectly genotoxic, and 23

reacts with cel lular proteins and DNA in cells which it comes into contact. Additionally, 24

immunosuppression, viral reactivation and promotion effects are relevant to lymphoma and 25

related malignancies. Therefore, the key events for the adult cell lymphoid cancers would 26

i nclude these actions. Lymphoid tumors (e.g. lymphocytic leukemia, B

- cell lymphoma, mantle 27

cell lymphoma [a rare form on non

- Hodgkin lymphoma] and myeloma) may arise from cells 28

present at the portal of entry (POE) (Figure 4

- 33). The location and function of mature 29

lymphocytes contribute to their vulnerability to transformation by agents at the POE. Therefore, 30

a brief summary of the immuno

- biology of these cells is provided in order to provide context for 31

the MOA evaluation: 32

33

Location: Lymphocytes are pre sent in the oral and respiratory tract epithelium, as well 34

as in cell aggregates and tertiary immune structures (e.g. germinal centers) in the mucosal 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 449 tissues (Zuercher and Cebra, 2002; Zuercher et al., 2002; Wu et al 1997 and Kupper et 1

al., 1990). These mucosa

- associated lymphoid tissues (MALT) provide the opportunity 2

for formaldehyde to directly interact with components of the immune system present at 3

the POE (Wu et al., 1997, Claeys et al. 1996, Park et al., 2003 and Fujimura 2000). 4

Intraepithelial ly mphocytes are present in the pseudostratified epithelium of the 5

nasopharyngeal passages and there are aggregates of immune cells and germinal cells 6

present in these tissues. Crypts containing mature lymphocytes exist at the surface of the 7

nasal epithelium (Fujimura 2000). Microfold cells or M

- cells form the crypts, where the 8

lymphocytes are covered by a thin membrane (Figure 4

- 37). Functionally, these 9

lymphocytes identify and process foreign antigens at the POE (Fujimura 2000). 10

Therefore the mature lymp hocytes within these crypts, exposed to exogenous agents, are 11

involved in active immune responses to foreign antigens. 12

13

Clonal Expansion: Mature lymphocytes (both B and T

- cells) clonally expand their 14

populations in response to an exogenous antigen when a humoral immune response is 15

stimulated. Therefore cell proliferation is a normal function of these mature lymphocytes 16

and occurs every time there is an infection. Cell proliferation of mature B and T

- cells, 17

responsive to a particular antigen, occurs in active germinal centers (including those 18

within the respiratory tract). Cells may be exposed to exogenous agents during the 19

immune response, or cells responding in the germinal center may have previously been in 20

the epithelium or M

- cell crypt. 21

22

Somatic H ypermutation: Normal immune function includes the process of somatic 23

hypermutation where B

- cells undergo DNA rearrangement of the variable region genes to 24

produce novel antibodies specific to a given antigen. This process is key to adaptive 25

immunity and demonstrated by the basic principles of immuno

- biology which underlie 26

vaccination theory. Gene sequencing of adult B

- cell lymphomas and leukemias indicate 27

that the chromosomal regions involved in somatic hypermutation correspond to known 28

oncogenes in thes e cancers. The vulnerability of these processes is evidenced by the 29

observation that approximately 90

- 95% of adult lymphomas and leukemias are of B

- cell 30

origin (Gordon et al., 2003). Formaldehyde

- induced protein

- protein crosslinking could 31

disrupt cell p rocesses including somatic hypermutation and cell mitosis, resulting in 32

agent

- induced translations similar to those found in spontaneous B

- cell malignancies. 33

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

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- 450 1

Figure 4

- 37. Location of intra

- epithelial lymphocytes along side epithelial cells in 2

the hum an adenoid. (Source Fujimura et al., 2000) 3

4

a) Direct or indirect formaldehyde

- induced mutation in cells at the site of first contact: 5

Immune cells including intraepithelial lymphocytes, and cells in mucosal associated 6

lymph tissue (MALT) are collocated with the epithelial cells from which URT cancers 7

arise (Figure 4

- 37). Therefore the direct and indirect mutagenic potential for 8

formaldehyde is equally applicable to components of the immune system present in these 9

tissues. Mutations, the permanent heritable changes in the genome of the cell, are a 10

primary mechanism for the activation of oncogenes or the inactivation of tumor 11

suppressor genes. Mutagenicity is the most widely recognized determinant of chemical

12

induced carcinogenicity, and it is difficult to s et aside the relevance of direct 13

formaldehyde

- induced mutations from its demonstrated carcinogenicity. Formaldehyde

14

induced mutation in immune cells present at the site of first contact, may initiate cells or 15

or provide subsequent mutagenic events to alre ady initiated cells. The competence of our 16

immune system relies on the proliferation of peripheral lymphocytes in response to 17

immune challenge (infection). Additionally, heritable changes to the variable gene 18

regions in B

- cells generated during somatic h yper

- mutation are essential to adaptive 19

immunity (e.g. immunization) demonstrating that permanent heritable changes in the 20

DNA of peripheral B

- cells are passed to daughter cells and retained in the body for 21

decades. Any agent

- induced mutations would be si milarly propagated and retained with 22

the potential to contribute to the transformation of mature lymphocytes. 23This document is a draft for review purposes only and does not constitute Agency policy.

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- 451 Relevance to humans: This MOA is relevant to humans. The well

- documented DNA 1

reactivity (e.g. DPC and DNA adducts) and clastogenicity of formal dehyde at the POE in 2

laboratory animals is a direct effect of formaldehyde on tissues of first contact, and these 3

mechanisms are considered relevant to humans. As with epithelial cells, clastogenic 4

effects in peripheral lymphocytes are documented in forma ldehyde

- exposed students and 5

workers, confirming the genotoxic effects of formaldehyde in immune cells, from which 6

lymphomas and related diseases may arise (Section 4.5.2Figure 4

- 33). 7

8

b) Formaldehyde

- induced protein

- protein crosslinks may disrupt somatic

9

hyp ermutation: Although not as well studied as DNA

- protein crosslinks, formaldehyde 10

also formes crosslinks between amino acids on proteins (Section 4.5.3.1.3 for details). 11

Specific oncogenes for malignancies which arise from mature B

- cells are linked to err ors 12

in the process of somatic hyper

- mutation (Greaves et al. 2004). If formaldehyde creates 13

protein crosslinks in competent B

- cells which effect the process of DNA rearrangement, 14

formaldehyde may generate translocations and related oncogenes similar to th ose 15

observed in observed in spontaneous B

- cell malignancies. 16

Relevance to humans: This hypothesis has not been tested in either exposed human or 17

animal test systems. However, the link between somatic

- hypermnutation and B

- cell 18

oncogenes is well establis hed and perturbation of this process by an exogenous agent is a 19

reasonable extension of the current understanding of the etiology of B

- cell malignancies. 20

21

c) Increased URT infections / viral reactivation: Inhalation exposure to formaldehyde 22

has been shown to decrease the defenses of the body against infection through two 23

mechanisms: 1) damage to the protective mucous barrier and function of the mucociliary 24

apparatus; and 2) localized immunosuppression (Section 4.5.3.1). These effects have 25

been demonstrated i n both exposed humans and controlled animal experiments. 26

Additionally, increased respiratory tract infections are associated with formaldehyde 27

exposure in several populations. Common viral agents (e.g. Epstein barr virus) are 28

known risk factors for malig nancies which arise from mature lymphocytes. Thus, 29

increased URT infections or viral reactivation due to formaldehyde exposure may 30

influence the incidence of these cancers. 31

Relevance to humans: The potential role of increased URT infections and 32

immuno suppression at the portal of entry is considered to be relevant to humans. Data in 33

humans are available to support both key events in this MOA. Additionally, co

- exposure 34

to infectious agents (including viruses) would be expected in participants in an 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 452 epi demiological study, suggesting an MOA which acted in conjunction with infectious 1

agents may be relevant to agent

- induced cancer. Therefore, toxic actions by 2

formaldehyde which may increase URT infections, or viral

- reactivation at the site of first 3

contact , could be acting in conjunction with viral agents to contribute, in part, to observed 4

associations between formaldehyde exposure and increased lymphoma and related 5

diseases. 6

7

4.5.3.3.3 Summary and evaluation of hypothesized MOA(s) for Lymphohematopoietic 8

Malignanc ies 9

The well

- documented direct toxic action of formaldehyde on cells at the site of first 10

contact is a general effect based on the reactivity of formaldehyde with cellular components (e.g. 11

proteins and DNA) (Section 4.5.3.1). As a general effect, it is re asonable that these toxic effects 12

would be relevant to all cells which come into contact with formaldehyde. The current debate 13

regarding the biological plausibility of formaldehyde

- induced lymphohematopoeitic 14

malignancies centers around a perspective that the diseases within this general grouping are 15

systemic cancers arising only out of bone marrow toxicity (Heck et al., 2006, Pyatt et al., 2008) 16

and that it is implausible for formaldehyde to induce bone marrow toxicity. The above MOA 17

evaluation expands t he current debate by considering the impact of POE toxicity on elements of 18

the immune system and cancers might arise from these cells (Section 4.5.3.3.2) and by 19

presenting data which support the observation that formaldehyde is associated with bone marrow 20

toxicity and damage to circulating stem cells in exposed humans (Section 4.5.3.3.1). 21

As significant increases in free formaldehyde in peripheral blood from exogenous 22

exposure has not been detected (Heck et al., 1987), it has been hypothesized that formalde hyde 23

does not transport and therefore cannot exert toxic effects outside of the tissues at the site of first 24

contact (Heck et al., 2006, Pyatt et al., 2008). In contrast to this hypothesis, effects are seen in 25

formaldehyde

- exposed humans which indicate sy stemic effects on the hematopoietic system 26

including reduced white blood cell counts, clastogenic effects in peripheral blood lymphocytes 27

and aneuploidy in circulating stem cells (Tang et al., 2008, Zhang et al., 2010 and Section 28

4.5.3.1). These observed effects in humans are consistent with agent

- induced bone marrow 29

toxicity and are observed with other well

- studied exogenous leukemogens (e.g. benzene and 30

ionizing radiation.) It is unknown if formaldehyde is distributed systemically to exert its effects 31

d irectly on cells in the bone marrow or if damage to circulating stem cells or progenitor cells 32

would be sufficient to result in the observed effects in humans (Zhang et al., 2010). Additional 33

research is needed to better determine the potential for system ic transport of formaldehyde 34

considering both detection of its hydrated form (methylene glycol) as well as formaldehyde 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 453 protein adducts (e.g. FA

- GSH and FA

- albumin). Similarly the results of Zhang et al. (2010) 1

need to be extended (analysis for additional chromosomal aberrations) and repeated. Although 2

further evidence is needed to better understand the hypothesized mechanisms for formaldehyde

3

induced effects on hematopoietic stem cells, the observed hematotoxic effects in humans cannot 4

be set aside. The refore, however unlikely, the current data support the biological plausibility of 5

formaldehyde effects on the hematopoietic system. 6

7

4.5.4. Hazard Characterization FOR Formaldehyde Carcinogenicity 8

Formaldehyde is Carcinogenic to Humans by the Inhalation R oute of Exposure 9

Based on the results from a large and well

- followed longitudinal cohort study of 10

industrial workers and several case

- control studies, the epidemiologic evidence is sufficient to 11

characterize the association between formaldehyde exposure an d nasopharyngeal cancer (NPC) 12

as causal in humans (Hauptmann et al., 2004; Hildesheim et al., 2001; Vaughan et al., 2000). As 13

a group, upper respiratory tracts sites of direct contact with formaldehyde upon inhalation (i.e., 14

salivary gland, mouth, nasophar ynx, nasal cavity and larynx) also showed sufficient evidence of 15

a causal association. Case

- control studies have demonstrated associations between formaldehyde 16

exposure and rare cancers of the URT. Luce et al. (2002) evaluated pooled data from 12 case

17

con trol studies and demonstrated a statistically significant increased risk between formaldehyde 18

exposure and sinonasal cancer. Hypopharyngeal cancer was linked with formaldehyde exposure 19

with an OR of 3.78 (95% CI 1.50

- 9.49) in another case

- control study (L aforest et al., 2000). 20

Hauptmann and colleagues (2004) concluded that in spite of the small numbers of deaths from 21

cancers of the URT, the positive associations with average intensity and peak exposure were 22

consistent with the carcinogenicity of formaldeh yde at these sites of first contact. The finding 23

that formaldehyde inhalation causes nasal squamous cell carcinoma in animals (Section 4.2.1.2) 24

further supports the determination of a causal association of formaldehyde exposure and 25

increased risk of upper respiratory tract cancer in humans. Both humans and animals developed 26

tumors within the upper respiratory tract, the POE site expected to receive direct exposure to 27

formaldehyde. 28

Overall, there is a consistent association between formaldehyde exposure an d various 29

forms of lymphohematopoietic (LHP) cancers, with all leukemias, myeloid leukemia 30

specifically, Hodgkin lymphoma and multiple myeloma demonstrating the greatest strength and 31

consistency of results. Where exposure

- response data exist, exposure

- res ponse trends have been 32

seen for all LHP malignancies, all leukemia, myeloid leukemia and Hodgkin lymphoma 33

(Pinkerton et al., 2004; Beane Freeman et al., 2009). Taken together, the data demonstrate a 34

consistent association, across various worker population s, with the expected temporal association 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 454 to exposure and defined exposure

– response relationships in two different worker cohorts. The 1

strongest associations tend to be with myeloid leukemia and Hodgkin lymphoma. The criterion 2

of reasonable biological pl ausibility is easily met for the majority of the diseases which 3

contribute to an observation of all LHP cancers, specifically the cancers derived from mature 4

lymphocytes. The potential for formaldehyde

- induced LHP cancer is further supported by the 5

result s of animal bioassays, where formaldehyde

- induced leukemia and lymphoma had been 6

demonstrated in 3 independent studies in two species (rats and mice) and both sexes. 7

8

4.6. SUSCEPTIBLE POPULATIONS 9

“Susceptible subpopulations” is used here to refer to facto rs, such as life stage, genetics, 10

health status, etc., that may predispose individuals to greater response to an exposure. This 11

greater response could be achieved either through differences in exposure to the chemical or 12

differences in underlying toxicokin etic (TK) and toxicodynamic (TD) differences between the 13

susceptible and other populations. For example, life stages may include the developing 14

individual before and after birth up to maturity (e.g., preconception, embryo, fetus, young child, 15

adolescent), adults, or aging individuals. Another susceptibility factor is genetics. Specifically, 16

susceptible subpopulations may also include people with specific genetic polymorphisms that 17

render them more vulnerable to a specific agent or people with specific dis eases or pre

- existing 18

conditions (e.g., asthmatics). The term may also refer to gender differences, lifestyle choices, or 19

nutritional state (USEPA, 2002, Section 4.3.2.3). 20

A discussion of a comprehensive list of all possible susceptibility factors affectin g 21

exposure and response to formaldehyde, or any chemical, is not possible. Therefore, the 22

discussion of susceptibility factors focuses on 1) factors hypothesized to be of importance to 23

formaldehyde; and 2) factors for which there are available formaldehyd e data. A partial list of 24

these factors includes gender, genetic polymorphisms, preexisting disease status, nutritional 25

status, diet, and previous or concurrent exposures to other chemicals. Qualitatively, the presence 26

of multiple susceptibility factors will increase the variability that is seen in a population response 27

to formaldehyde toxicity. 28

29

4.6.1. Life Stages 30

Individuals at different life stages are physiologically, anatomically, and biochemically 31

different. Examples include physiological change s that occur through the lifespan (Selevan et 32

al., 2000). They may also have distinctive exposure pathways (i.e., transplacental, breast milk 33

ingestion), and exhibit differences in behavior (U.S. EPA, 2006b; NRC, 1993). Early life stages 34

(i.e., during de velopment, prior to mature adulthood) and the later life stages (i.e., aging) differ 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 455 greatly from mature adulthood in body composition, organ function, and many other 1

physiological parameters that can impact the TK and/or TD of chemicals and their metaboli tes 2

(ILSI, 2003). This section presents and evaluates the pertinent published literature available to 3

assess whether and how individuals of differing life stages may respond differently to 4

formaldehyde. 5

6

4.6.1.1. Early Life Stages 7

4.6.1.1.1. Factors inf luencing exposure and dosimetry 8

For all life stages, the primary exposure routes for formaldehyde include inhalation and, 9

in some cases, ingestion (see Chapter 5). S ome exposure scenarios may be child specific. For 10

example, to the extent that the presenc e of baby furniture produced with formaldehyde in a 11

child’s house contributes to greater concentrations in a child’s room, exposures for very young 12

children in those circumstances may be increased (Environment California, 2008)

. As with all 13

chemicals, pla cental transfer and breast milk ingestion are exposure pathways that are unique to 14

early life stages. Studies assessing early life stage exposure pathways to formaldehyde have not 15

been performed. Presumably, unmetabolized formaldehyde reacts too quickly to be effectively 16

transported from mother to fetus by placental transfer; in addition, formaldehyde is not lipophilic 17

and is therefore unlikely to accumulate in breast milk. However , the relevant dose metric for 18

formaldehyde

- related effects may vary depen ding on the specific target of concern (e.g., direct 19

toxicity at the portal of entry versus systemic effects); insufficient information is currently 20

available to determine whether individuals in different life stages are at higher risk for exposure 21

to spec ific target tissues. 22

There are some calculations however which shed light on lifestage differences in the 23

inhaled tissue dose at the portal of entry. Using respiratory tract surface areas and ventilation 24

rates reported in the literature and the scheme in USEPA (1994), Ginsberg et al. (2005) 25

calculated that overall extrathoracic absorption of highly reactive and soluble gases is similar in 26

adults and children. These results are in agreement with that of Garcia et al. (2009) who used 27

computational fluid dyn amics to study differences in the nasal dosimetry of reactive, water

28

soluble gases between 5 adults and 2 children, aged 7 and 8 years old. Overall uptake efficiency, 29

average flux (rate of gas absorbed per unit surface area of the nasal lining) and maximum flux 30

levels over the entire nasal lining did not vary substantially between adults and children (1.6

- fold 31

difference in average flux and much less in maximum flux). On the other hand, the local flux of 32

formaldehyde varies between the two children by a fac tor of 2 to 4 at various distances along the 33

septal axis of the nose. The results in Garcia et al. (2009) have been further described and 34

evaluated in Appendix 3

- 1. Under normal resting breathing conditions, it is expected that very 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 456 little formaldehyde is delivered to the lung. However, under higher activity as well as mouth 1

breathing scenarios, both of which appear likely to happen more regularly in children, 2

formaldehyde dose to the lung will be substantial. 5

The toxicokinetic characteristics of formald ehyde are described in Chapter 3, with 4

absorption and distribution studies discussed in Sections 3.2 and 3.3. Studies to assess 5

differential absorption or distribution of formaldehyde in early life stages have not been 6

performed and represent a significan t data gap. The metabolism of formaldehyde is described in 7

section 3.4. Expression of the enzymes that metabolize formaldehyde (ALDH2 and FALDH, 8

and specifically ADH3) is known to be developmentally regulated and thus may alter the TK of 9

formaldehyde in early life stages. ADH3 is ubiquitously expressed and is present in the human 10

fetus, neonate, and 1

- to 10

- year

- old children (Hines and McCarver, 2002; Estonius et al., 1996). 11

During early development in rodents, when neurulation first begins and forms collections of 12

somites along the neural tube, ADH3 activities are significantly lower (at 8

– 10 and 11

– 13 somite 13

stages) and suggest a decreased ability to detoxify formaldehyde in the early embryo (Harris et 14

al., 2003). ADH mRNA expression levels appear t o be age related, with decreased expression of 15

ADH common in premature neonates and infants up to 5 months old. Thereafter, ADH 16

expression increases and is dependent on body weight (Ginsberg et al., 2004). Benedetti et al. 17

(2007) reported that decreased ADH expression persisted until age 2 to 5 years. Westerlund et 18

al. (2005) tracked the ontogeny of ADH3 specifically and reported that ADH3 expression was 19

ubiquitous in mouse and rat embryos and was the only ADH enzyme to be consistently localized 20

to brain tissue, suggesting a housekeeping function. Thus, neonates and very young children 21

may have a decreased ability to metabolize formaldehyde due to differential expression of ADH3 22

in development compared that of with adults; however, activity levels of thi s enzyme and 23

alternate pathways specific to children are not available in the literature. 24

3

25

4.6.1.1.2. Life

- stage exposure and adverse health outcomes 26

In general, exposure to toxic agents during early development (i.e, pre

- conception, 27

prenatal stages, or postnatal development) may affect organ development and may also lead to 28

increased disease susceptibility later in life. Following early life stage exposure to 29

formaldehyde, a number of adverse health outcomes have been observed, including alterations in 30

the respiratory, reproductive, and neurological systems. For example, the developing respiratory 31

tract may be more vulnerable to insult compared with an adult respiratory tract, and thus, 32

increase the severity of response. The potential for reproduct ive and developmental toxicity of 33

5 For example, in the case of ozone concentrations of 0.1 ppm, a moderatel y reactive gas, Ginsberg et al. (2008)

predict a 5

- fold variation in the dose to the deep lung between quiet and heavy breathing conditions for an 8

- year

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

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- 457 formaldehyde is discussed in detail in Sections 4.1.1.7 (human studies) and 4.2.1.7 (animal 1

studies), while effects on the nervous system are discussed in Sections 4.1.1.6 and 4.2.1.6 2

(human and animal studies, respectivel y). The specific case of formaldehyde exposure and 3

pulmonary effects is discussed in detail in Sections 4.1.1.1 to 4.1.1.4 and 4.2.1.1 to 4.2.1.4. A 4

brief summary of identified effects of formaldehyde that may indicate susceptibility during 5

particular li fe stages is provided below. 6

7

4.6.1.1.2.1. Pre

- conception.

Exposure prior to conception may damage reproductive organs and/or germ cells that 9

could affect reproduction and/or damage the genetic makeup of the offspring. Effects on 10

reproduction are discu ssed in Sections 4.1.1.7 and 4.2.1.8. In summary, an epidemiological 11

study (Taskinen et al., 1999) reported significantly delayed conception among female workers 12

exposed to formaldehyde at average daily ambient formaldehyde levels; these effects could be 13

consistent with adverse effects on either pre

- conceptional and/or gestational exposure. One 14

animal study (Maronpot et al., 1986) reported endometrial hypoplasia and lack of ovarian luteal 15

tissue in female mice exposed for 13 weeks to 40 ppm formaldehyde v ia inhalation, suggesting 16

the potential for treatment

- related alterations to the female reproductive system. Since the 17

exposure was to the adult, the findings suggest that preconceptional FA exposure caused female 18

reproductive system effects that in turn could affect pregnancy. 19

8

In the rodent study of Kitaev et al. (1984), a three

- fold increase in embryo degeneration on 20

gestational days 2

– 3 was observed after FA exposure to the dams during premating. Since the 21

exposure was to the adult in these three studi es, the findings suggest that preconceptional FA 22

exposure caused female reproductive system effects and/or affected the gametes. 23

24

4.6.1.1.2.2. Prenatal.

A population

- based study (Gražulevičiene et al., 1998) found an association between 26

atmospheric form aldehyde exposure and low birth weight, yielding an adjusted OR of 1.37 (95% 27

CI: 0.90

– 2.09). Three studies (Dulskiene and Gražulevičiene, 2005; Taskinen et al., 1994; 28

Hemminki et al., 1985) that examined the effect of occupational exposures on the inciden ce of 29

congenital malformation produced mixed results. 30 25

Results from Taskinen et al. (1999) support associations between formaldehyde exposure, 31

subfertility, and spontaneous abortion. Subfertility and spontaneous abortion are biologically 32

linked (subclinica l pregnancy losses are increased among women with fertility problems) (Gray 33

and Wu, 2000; Hakim et al., 1995), and both subfertility and spontaneous abortion may be 34

related to sensitivity to environmental agents (Correa et al., 1996). 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 458 Two experimental anim al studies (Martin, 1990; Saillenfait et al., 1989) evaluated a 1

standard battery of developmental endpoints following inhalation exposure on GDs 6

– 10, but 2

effects were minimal. Similarly, Chernoff and Kavlock (1982), Marks et al. (1980), and Hurni 3

and Ohd er (1973) reported minimal reproductive or developmental effects in rodents in studies 4

in which dams were exposed orally during early gestation. When formaldehyde was 5

administered via inhalation throughout gestation in female rats, some developmental effe cts, 6

including increased pup weight and decreases in lung and liver weight in newborns, were 7

reported at 0.01 and 0.4 ppm (Senichenkova and Chebotar, 1996; Senichenkova, 1991; Kitaev et 8

al., 1984; Gofmekler and Bonashevskaya, 1969; Gofmekler, 1968; Pushkin a et al., 1968). Two 9

studies also reported changes in motor activity in offspring of dams exposed via inhalation to 0.4 10

ppm formaldehyde during gestation (Senichenkova, 1991; Sheveleva, 1971). 11

12

4.6.1.1.2.3. Postnatal.

Following early life stage exposur e to formaldehyde, a number of adverse postnatal 14

outcomes are possible, including effects on the developing and adult respiratory, reproductive, 15

and neurological systems. The potential for increased risk of childhood cancer is also discussed 16

below. 17 13

18

4.6.1 .1.2.3.1. Respiratory toxicity.

Formaldehyde is known to induce changes in pulmonary function and cause pulmonary 20

irritation in human studies (Rumchev et al., 2002; Garrett et al., 1999; Krzyzanowski et al., 1990; 21

Holmström et al., 1989; Holmström and W ilhelmsson, 1988; Ritchie and Lehnen, 1987) and 22

animal studies (Ohtsuka et al., 2003, 1997; Riedel et al., 1996; Swiecichowski et al., 1993; Lee et 23

al., 1984). Exposure to formaldehyde in early life can cause damage to the lungs and 24

permanently influence airway function, resulting in increased vulnerability to toxicants later in 25

life. Thus, young children may demonstrate increased susceptibility to formaldehyde

- related 26

health effects. Krzyzanowski et al. (1990) reported an association between physician

- d iagnosed 27

asthma and chronic bronchitis in children who lived in homes with formaldehyde levels that 28

were higher than 60 ppb, after controlling for socioeconomic status and ethnicity. Rumchev et 29

al. (2002) reported a statistically significant increased ris k of asthma with increased residential 30

concentrations of formaldehyde. Garrett et al. (1999) found an increased association between 31

bedroom concentration of formaldehyde and increased risk of atopy in children. These studies 32

suggest that formaldehyde exp osure may exacerbate responses in sensitive airways, particularly 33

in children. Exacerbation of response has also been noted in asthmatic adults and will be 34

discussed below. 35 19This document is a draft for review purposes only and does not constitute Agency policy.

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- 459 Another child

- specific concern is that respiratory irritation may have greater imp act on 1

lung function in children due to their smaller lung size; this is true even if the lung development 2

is normal. Irritation is commonly accompanied by inflammation, which can have a greater 3

impact on children’s airways because they are narrower than adult airways. Thus, less 4

inflammation is required for significant airway obstruction in children than in adults. 5

6

4.6.1.1.2.3.2. Developmental neurotoxicity.

In neonatal exposure paradigms, changes in brain structure (Sarsilmaz et al., 2007; Aslan 8

e t al., 2006), and brain chemistry (Songur et al., 2008) were seen in young rats following 9

inhalation exposures (6000 or 12000 ppb, 5 days per week from postnatal day 0

- 30). In addition, 10

Weiler and Apfelbach (1992) found juvenile animals to be more sensiti ve to formaldehyde

11

induced changes in olfactory thresholds when compared with adult animals (shifts in olfactory 12

thresholds were greater when exposure [at 250 ppb] was initiated at PND 30 than at adult ages). 13

These studies are consistent with the hypothes is that early life exposure to FA can lead to long

14

lasting neurological effects. Exposure levels in these studies (250

– 6,000 ppb) were in the same 15

range as those producing the behavioral effects in adults (as low as 100 ppb), but provide limited 16

informati on regarding relative sensitivity as no NOAELs were identified, and (with the exception 17

of Weiler and Apfelbach), similar parameters were not measured in adult animals using the same 18

exposure paradigms. 19

7

20

4.6.1.2. Later Life Stages 21

In general, older adults may be at risk for increased susceptibility to exposure to 22

environmental chemicals by virtue of their slower metabolism and increased incidence of altered 23

health status (Benedetti et al., 2007; Ginsberg et al., 2005; U.S. EPA, 2005a). Additionally, 24

adver se effects of earlier exposure to some toxicants may be observed in older adults as a result 25

of latency in expression of the effect (Olsen et al., 1997; Sweeney et al., 1986). No studies have 26

examined the differential effects of formaldehyde exposure for elderly adults (>65 years old) as 27

compared to other age groups. 28

29

4.6.1.3. Conclusions on Life

- Stage Susceptibility 30

In summary, timing both of the exposure and of the assessment of health outcomes may 31

be important for understanding the relative risk of a dverse effects from formaldehyde exposure 32

during different life stages. There are known developmental differences in kinetics across life 33

stages, including differences in enzymes involved in formaldehyde metabolism, but the 34

contribution of these differenc es to formaldehyde

- related health effects is unknown. Similarly, 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 460 information regarding life

- stage differences in respiratory physiology raises possible concern 1

regarding increased exposure to children, but studies for formaldehyde are not available. 2

Avai lable data do support an increased risk for adverse effects on lung function in children. The 3

overall body of evidence shows some support, although minimal, for susceptibility in 4

reproductive or developmental endpoints associated with exposure to formalde hyde. Some 5

studies observed altered development of the nervous system following formaldehyde exposure 6

during early life. Older adults may be at risk for increased susceptibility to formaldehyde 7

because of slower metabolism and clearance rates. Elderly a dults have an increased probability 8

of having both altered health status and altered metabolism, which could impact their ability to 9

process and recover from an adverse effect. The available data are consistent with some life

10

stage susceptibility differen ces for FA at the level of TD or TK differences, the results are 11

nonetheless inconclusive due to the number of data gaps. 12

13

4.6.2. Health/Disease Status 14

The factor for which we have the greatest evidence is pre

- existing disease, and 15

specifically asthma. N umerous studies have assessed the potential for increased susceptibility to 16

formaldehyde in asthmatics. Formaldehyde does not induce airway hyperreactivity directly 17

(Sheppard et al., 1984) and has not been shown to increase airway hyperreactivity in eithe r 18

asthmatics or non

- asthmatics (Pazdrak et al., 1993; Harving et al., 1991; Kulle et al., 1987). 19

Significantly decreased forced expiratory volume (FEV 1 ) measurements were reported among 20

asthmatics in two studies (Casset et al., 2006; Green et al., 1987), while others did not find any 21

significant change in FEV 1 following formaldehyde exposure (Ezratty et al., 2007; Frigas et al., 22

1984). 23

A few available case reports of bronchial asthma do suggest direct respiratory tract 24

sensitization to formaldehyde gas ( Lemiere et al., 1995; Burge et al., 1985; Hendrick et al., 1982; 25

Hendrick and Lane, 1977, 1975). All cases displayed marked changes in FEV 1 or pulmonary 26

airflow rate in response to acute challenges with formaldehyde gas at exposure levels <3 ppm. 27

Formald ehyde

- induced IgE production has been reported in some studies (Vandenplas et al., 28

2004; Wantke et al., 1996a). 29

There is a large quantity of human data providing evidence of an association between 30

formaldehyde exposure and increased incidence of asthma or exacerbation of asthmatic 31

responses in compromised individuals. For example, Krzyzanowski et al. (1990) reported an 32

association between physician

- diagnosed asthma and chronic bronchitis in children who lived in 33

homes with formaldehyde levels that were hig her than 60 ppb, after controlling for 34

socioeconomic status and ethnicity. Rumchev et al. (2002) reported a statistically significant 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 461 increased risk of asthma with increased residential concentrations of formaldehyde. Garrett et al. 1

(1999) found an incre ased association between bedroom concentration of formaldehyde and risk 2

of atopy in children. These studies suggest that formaldehyde exposure may exacerbate 3

responses in sensitive airways, particularly in children. Exacerbation of response has also been 4

noted in adults. Kriebel et al. (1993) reported a greater decrease in peak expiratory flow (PEF) 5

in asthmatic medical students (7.3% decrement) compared with non

- asthmatic medical students 6

(2% decrement) after 2 weeks exposure to formaldehyde (average co ncentration 0.73 ppm) in an 7

anatomy lab. This effect does not appear to be immunogenic in nature (Fujimaki et al., 2004a; 8

Lee et al., 1984). 9

Several animal studies document increased airway resistance and bronchial constriction 10

following inhalation expos ure to formaldehyde (Nielson et al., 1999; Swiecichowski et al., 1993; 11

Biagini et al., 1989; Amdur et al., 1960). Sadakane et al. (2002) demonstrated that formaldehyde 12

exposure exacerbated sensitization and challenge with a common dust mite allergen (Der f) as 13

measured by increased eosinophil infiltration into the interstitium around the bronchi and 14

bronchioles as well as goblet cell proliferation in the bronchial epithelium; they suggested that 15

formaldehyde exposure may aggravate eosinophilic infiltration and goblet cell proliferation that 16

accompanies allergic responses. The MOA underlying this response is unknown. These 17

decrements may occur indirectly in response to formaldehyde and may be mediated via 18

neurogenic potentiation (Sadakane et al., 2002; Rie del et al., 1996; Tarkowski and Gorski, 1995). 19

In particular, Tarkowski and Gorski (1995) suggest that formaldehyde may increase permeability 20

of respiratory epithelium and destruction of immunologic barriers. Thus, the respiratory tract 21

may become vulner able to inhaled allergens after formaldehyde exposure (Tarkowski and 22

Gorski, 1995). 23

In summary, the data indicate that formaldehyde exposure can aggravate a type I 24

hypersensitivity response and that this hypersensitivity may in turn increase the susceptibi lity to 25

FA exposure in these individuals. Formaldehyde exposure may predetermine an asthmatic 26

phenotype or may induce new incidences of asthma via indirect mechanisms, though definitive 27

evidence and a proposed mechanism remain to be determined. Individu als that exhibit 28

chemically induced sensitivity and are exposed acutely or chronically to formaldehyde in 29

residential and occupational settings might exhibit adverse responses at lower concentrations of 30

formaldehyde than the average healthy person. 31

32

4.6.3. Nutritional Status 33

Limited available data indicate that certain types of malnutrition may increase 34

susceptibility to formaldehyde exposure. Senichenkova and Chebotar (1996) reported increased 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 462 fetal anomalies in fetuses from iron

- deficient pregnant mice after formaldehyde exposure 1

compared with anemic mice that had not been exposed to formaldehyde. Forced iron reduction 2

(induced by addition of b ipyridyl treatment in pregnant mice) in utero increased the overall 3

incidence of fetal anomalies when paired w ith formaldehyde exposure (Senichenkova and 4

Chebotar, 1996). The findings are difficult to evaluate due to poor reporting and have not been 5

substantiated by other laboratories. 6

7

4.6.4. Gender Differences 8

Males and females can differ greatly in body comp osition, organ function, and many 9

other physiological parameters that may influence the toxicokinetics of chemicals and their 10

metabolites in the body (Gochfeld, 2007; Gandhi et al., 2004). 11

The human epidemiology data set does not support any specific gend er susceptibilities 12

for noncancer effects due to formaldehyde exposure. In general, data suggest that nonpregnant 13

women, on a per kg body weight basis, may have slightly lower air intake than men, which 14

would suggest that women may be less susceptible to inhaled pollutants like formaldehyde than 15

men, but this has not been investigated in the available formaldehyde literature. 16

A few isolated reports have investigated potential gender differences in development of 17

nasal pharyngeal carcinomas following exposu re to formaldehyde. One case

- control study 18

identified a higher OR for sinonasal adenocarcinomas in women (OR = 6.2 [95% CI: 2.2

– 19.7]) 19

compared with the OR observed in men (OR = 3.0 [95% CI: 1.5

– 5.7]) following exposure to 20

formaldehyde (Luce et al., 2002) . However, the overall body of evidence remains scant. 21

There are a few reports concerning differential formaldehyde

- induced effects on the male 22

and female reproductive systems. Özen et al. (2002), Sarsilmaz et al. (1999), and Woutersen et 23

al. (1987) repo rted reduced Leydig cell numbers in adult male rats exposed by inhalation. In 24

female mice, inhalation exposure to formaldehyde resulted in endometrial hypoplasia and lack of 25

ovarian luteal tissue (Maronpot et al., 1986). The clinical significance of thes e effects in humans 26

is unknown, and due to limited data it is unclear whether the female or male reproductive system 27

is more susceptible to perturbation by formaldehyde. 28

29

4.6.5. Genetic Differences 30

There are some data for polymorphisms in humans that af fect formaldehyde TK. As 31

discussed in Section 3.4, the primary metabolizing enzymes of formaldehyde are ALDH2 and 32

ADH3, with the latter enzyme considered more relevant to low exposures. Polymorphisms in 33

ALDH2 have been shown to have implications in human risk assessment, specifically in regard 34

to acetaldehyde metabolism (Ginsberg et al., 2002) . Teng et al. (2001) demonstrated the 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 463 importance of ALDH2 for formaldehyde metabolism in rat hepatocytes at fairly high 1

formaldehyde concentrations (2.5 mM and greater). Cheng et al. (2008) investigated the 2

relationship between occupational exposure to formaldehyde and genetic polymorphisms of 3

ALDH2 and C YP2E1. There was a positive relationship between the concentration of formic 4

acid in the urine and ALDH2 genotypes ( **χ** 2 = 9.241, p < 0.05). Urinary formic acid 5

concentration may be affected by formaldehyde exposure concentration and ALDH2 genotype 6

(Cheng et al., 2008) for individuals that have high exposure levels. Thus, although ALDH2 may 7

not be involved in formaldehyde metabolism if exposure levels are low, polymorphisms of this 8

enzyme may lead to differences in response at higher exposure levels. 9

Wu e t al. (2007) looked for and identified two SNPs in ADH3 among a population of 10

Mexican asthmatic children 4 to 17 years of age. Carrying one or two copies of the minor allele 11

for one SNP resulted in a decreased RR of asthma (RR = 0.66

– 0.77). For the secon d SNP, 12

homozygotes for the minor allele had an RR of 1.6 for asthma. The functional characteristics of 13

these SNPs are unknown. Studies evaluating whether any of the polymorphisms affect the 14

expression, regulation, stability, or activity of the enzyme in vivo are lacking; therefore, the 15

relative susceptibility of individuals with different polymorphisms cannot be characterized at this 16

time. 17

One study (Hedberg et al., 2001) identified three polymorphisms in human ADH3 18

involving four base

- pair substitutions in the promoter region of which one (C **→** T) showed 19

reduced activity (~50

– 70% of control). Hedberg et al. (2001) reported differences in allele 20

frequencies among Chinese, Spanish, and Swedish groups, consisting of Asian

- Caucasian 21

differences and ethnic subgr oups among Caucasians. Their results suggest that a small 22

percentage of Caucasians may have decreased ADH3 expression and thus, be more susceptible to 23

formaldehyde exposure. Additional studies to validate these findings have not been performed. 24

The re lative activity level of these enzymes may also impact the metabolism of 25

formaldehyde. In pharmacokinetic studies, deletion of ADH3 increased the sensitivity of mice to 26

formaldehyde (Deltour et al., 1999) and was deleterious to yeast (Achkor et al., 2003) . These 27

results suggest that deficiencies in ADH3 may confer an increased susceptibility to formaldehyde 28

toxicity (Teng et al., 2001) . The importance of properly functioning enzymes also suggests that 29

genetic differences in ADH3 or ALDH2 may affect the response to formaldehyde exposure. 30

However, comp arable human data are not available. 31

Race/ethnicity may be a surrogate for genetic differences but racial or ethnic groups may 32

also reflect socioeconomic, and/or cultural factors that are distinct from genetics. Possible ethnic 33

differences may be relate d to genetic polymorphisms of enzymes ALDH2 and ADH3, relevant 34

for formaldehyde metabolism. ALDH2 variants, present primarily in East Asians, are known to 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 464 have protective effects against alcoholism but were not found in the people of Indo

- Trinidadian 1

desc ent (Moore et al., 2007) or in American Indians or Alaska natives (Ehlers, 2007). However, 2

there is no direct evidence to associate these variants to differential susceptibility to 3

formaldehyde exposure, nor is there direct evidence to associate these eth nic groups specifically 4

with differential susceptibility to formaldehyde. Further, no studies have specifically assessed 5

ethnic variability in responses to formaldehyde. 6

There are complex pathways through which genetic polymorphisms in ADH3 can 7

potentia lly affect differential susceptibility to formaldehyde. Firstly, ADH3 is central to the 8

metabolism of formaldehyde. However, ADH3 itself may indirectly contribute to the adverse 9

effects of formaldehyde on pulmonary physiology (Thompson et al., 2009; Staab et al., 2008a, b; 10

Thompson and Grafström, 2008). Exposure to formaldehyde is itself thought to alter the activity 11

of ADH3 resulting in the perturbation of critical metabolic pathways. ADH3 participates in the 12

oxidation of retinol and long

- chain primary alc ohols, as well as the reduction of S

13

nitrosoglutathione (GSNO). The activity of ADH3 toward some of these substrates has been 14

shown to be significantly increased in the presence of formaldehyde. ADH3 has recently also 15

been shown to contribute to NO signal ing through its dual role in metabolizing GSNO, an 16

endogenous bronchodilator and reservoir of NO (Staab et al., 2008a; Hess et al., 2005; Jensen et 17

al. 1998). Through its regulatory function on GSNO, ADH3 may thus play a central role in 18

regulating bronchia l tone allergen

- induced hyperresponsiveness (Gerard, 2005; Que et al., 2005). 19

As concluded by California Environmental Protection Agency (CalEPA) (2008), “the 20

dysregulation of NO by formaldehyde [in this manner] helps to explain the variety and 21

variabilit y in the toxic manifestations following formaldehyde inhalation.” 22

23

4.6.6. Co

- Exposures 24

4.6.6.1. Cumulative Risk 25

When considering health risks, it is important to consider the impact of co

- exposures to 26

other agents that may interact with the chemical u nder evaluation. Co

- exposure to other 27

pollutants, particularly those that produce some of the same metabolites and similar health 28

effects as formaldehyde, is likely to occur in both occupational and nonoccupational settings. 29

Due to effects on metabolic enzymes (inducing and/or inhibition) as well as direct effects 30

on organ system function, co

- exposures may alter the way in which formaldehyde is metabolized 31

and cleared from the body. Inhibition or induction of the enzymes responsible for metabolism of 32

ch emicals may alter susceptibility to toxicity (Lash and Parker, 2001; IARC, 1995; U.S. EPA, 33

1985a). Smokers may be at increased risk for effects of formaldehyde exposure, because 34

formaldehyde is one of the components of cigarette smoke and is likely to hei ghten the point

- of

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

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- 465 entry effect when combined with occupational or residential exposures to inhaled formaldehyde. 1

However, no evidence is available to evaluate the potential aggregate effects. 2

3

4.6.6.2. Aggregate Exposure 4

In addition, multiple routes of exposure to a single agent may increase the cumulative 5

risk by increasing the overall body burden of the chemical. A human aggregate exposure model 6

developed by McKone and Daniels (1991) incorporated likely exposures from air, water, and soil 7

media throug h inhalation, ingestion, and dermal contact. The authors hypothesized that the 8

aggregate exposure could be age

- dependent but did not present any data for persons of differing 9

life stages. The role of multiple exposures on different genders, genetic susce ptibility, or altered 10

health and nutrition status has not been investigated. The available database regarding the 11

potential for multiple routes of exposure (or aggregate exposure) formaldehyde is limited. 12

Guseva (1972) specifically assessed the reproducti ve and developmental effects caused 13

by co

- exposure to formaldehyde via both inhalation (0.25 mg/m 3 ) and ingestion (0.01 mg/L) 14

routes in male rats. The authors reported reduced nucleic acid levels in testes to 88 and 92% of 15

controls, which suggests a possi ble toxic gonadotropic effect. The ability of male rats (receiving 16

combined exposure to formaldehyde at a low concentration level for a long period of time) to 17

reproduce was preserved since all the cohabited females were impregnated. The number and 18

weigh t of the fetuses and newborn rat pups in the experimental co

- exposure groups did not differ 19

substantially from those figures observed in the control group. No developmental defects or 20

anomalies were observed in the offspring for up to 1 month postnatally. Thus, at low exposures, 21

the reproductive effects due to combined ingestion and inhalation exposure are unknown. 22

23

4.6.7. Uncertainties of Database 24

There is a need to better characterize the implications of formaldehyde exposures to 25

susceptible populatio ns. A number of areas where the database is currently insufficient are 26

identified below. 27

28

4.6.7.1. Uncertainties of Exposure 29

Although information exists on early life exposure to formaldehyde, a number of 30

uncertainties regarding children’s susceptibilit y remain. First, inhalation is believed to be of 31

most concern for formaldehyde, since formaldehyde vapors are released from insulation or from 32

ambient sources of formaldehyde, including secondary production from other pollutants involved 33

in photo

- oxidant reactions. Any additional pathways of exposure for children have not been 34

characterized. Since formaldehyde is nearly ubiquitous in the environment, it is difficult to 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 466 quantify the total exposure. Second, children have different respiratory, metabolic, and activity 1

rates compared with healthy adults, potentially influencing ADME and target tissue exposure to 2

formaldehyde. However, studies to identify the specific changes in absorption of formaldehyde 3

and its metabolites across developmental stages and a cross organs have not been performed. In 4

addition, exposure prenatally may be altered based on whether formaldehyde or its metabolites 5

pass through the placenta, but placental transfer data are not available. Third, no quantitative 6

models have been develo ped to characterize these differences for formaldehyde. Formaldehyde

7

specific PBPK models and their validation will aid in understanding the uncertainties associated 8

with formaldehyde exposure in children. 9

Given the large proportion of time that most indi viduals in the U.S. spend indoors, 10

exposure scenarios where indoor concentrations to formaldehyde are high (e.g., in homes or in 11

trailers; see section 2.3.1) may play a significant role and may be of particular concern to the 12

elderly or health

- impaired ind ividuals who spend relatively more time at home. Further 13

evaluation of the effects of co

- exposures and pathways of exposure and aggregate risk is needed. 14

An estimate of the multiple exposure pathways is needed to know where along the dose

- response 15

curve to place an incremental exposure to formaldehyde. 16

17

4.6.7.2. Uncertainties of Effect 18

Studies specifically designed to evaluate effects after early and later life stage exposure 19

are needed in order to more fully characterize potential life

- stage

- related d ifferences in 20

formaldehyde toxicity, including the defining of critical windows during development. For 21

example, life

- stage

- specific neurotoxic and pulmonary effects, particularly in the developing 22

fetus, need further evaluation. The preconceptional peri od may be a critical window for FA 23

exposure and reproductive and developmental effects, based on rodent studies of reproductive, 24

embryonic and gamete effects. Data specific to the carcinogenic effects of formaldehyde 25

exposure during early life stages do n ot exist. The reduction in fertility seen in some studies 26

(Gray and Wu, 2000; Taskinen et al., 1999; Hakim et al., 1995) is not adequately described and a 27

well

- established MOA has not been identified, but some have been hypothesized including 28

altered sper m quality (Özen et al., 2002; Sarsilmaz et al., 1999; Woutersen et al., 1987). Further, 29

spontaneous abortion/fetal loss occurring early in gestation, prior to maternal knowledge of the 30

pregnancy, can lead to misclassification of the effect as infertility (see Sections 4.1.1.7 and 31

4.2.1.7). 32

More research is needed to clarify the role of genetic polymorphisms in formaldehyde 33

metabolism. Similarly, data gaps pertaining to gender differences remain. A potential impact of 34

nutritional status and iron deficienc y on formaldehyde toxicity needs further investigation. 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 467 A fair body of evidence suggests that asthmatics are more susceptible to formaldehyde exposure 1

than the general population, however the mechanism of action for this increased susceptibility is 2

unkno wn. 3

In the studies discussed above, there are a number of examples of studies that assessed 4

multiple susceptibility factors that are worth noting. For example, the Krzyzanowski et al. 5

(1990) study reported asthma and chronic bronchitis cases for two int eracting potential 6

susceptible groups, in children and those with high exposure (due to living in homes with 7

formaldehyde levels that were higher than 60 ppb). Similarly, the Garrett et al. (1999) study 8

assessed the same two interacting potential suscepti ble groups. 9

The study of Senichenkova and Chebotar (1996) assessed developmental effects in 10

mouse fetuses after in utero iron

- deficiency and FA exposure. Thus, the study findings must be 11

considered in light of possible interactions between life stage expo sure differences and 12

nutritional status differences. 13

Studies to understand the nature of the interactions between the various susceptibility 14

factors for FA have not been performed. 15

16

4.6.8. Summary of Potential Susceptibility 17

There is some evidence to dem onstrate susceptibility for various populations exposed to 18

formaldehyde. Available data are summarized in Table 4

- 96 where FA susceptibility factors are 19

presented by those with data for increased FA susceptibility and those with data for differences 20

but w ith an unknown impact on FA susceptibility. 21

Exposure to FA during early developmental and later life stages may be of concern. 22

However, human exposure to the developing fetus is unknown since it is not known whether 23

formaldehyde or one of its metabolites crosses the placenta. However, there is very limited life

24

stage

- specific information regarding the TK of formaldehyde. Life

- stage

- specific TK has not 25

been characterized, and, thus, no PBPK models exist to effectively evaluate the risk to early life 26

stage s. Children may be more susceptible to noncancer health effects as a result of inhalation 27

exposure to formaldehyde due to increased respiratory rates. There are no studies to evaluate 28

whether formaldehyde exposure in early life (e.g., pregnancy) is assoc iated with an increased 29

risk of childhood cancer. 30

The weight of evidence supports a plausible association between formaldehyde exposure 31

and aggravated asthmatic responses in humans and this association is corroborated by limited 32

evidence from animal studie s. Formaldehyde does not appear to directly induce airway 33

hyperreactivity but may sensitize airways to subsequent exposures. One issue in interpreting the 34

available studies that assessed the relationship between asthma and FA could not distinguish 35This document is a draft for review purposes only and does not constitute Agency policy.

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- 468 betwee n the cases of asthma that were due to earlier FA exposure vs. those without a direct link 1

to FA exposure. 2

No direct link exists between formaldehyde exposure and differential susceptibility in 3

different ethnic groups, although genetic polymorphisms in the enzymes involved with 4

formaldehyde metabolism, ADH3 and ALDH2, provide some support for differential 5

susceptibility to alcoholism in a number of ethnic groups. The evidence for differential gender 6

responses to formaldehyde exposure is equivocal. Co

- expo sures may result in altered 7

metabolism and clearance, but there is no evidence that co

- exposures are a critical part of 8

formaldehyde

- mediated differential susceptibility. 9

Thus, given the available data, increased susceptibility to adverse effects of 10

forma ldehyde is most strongly supported for three populations: 1) Preconception and perinatal 11

exposure based on reproductive and developmental effects; 2) children, whose exposure may be 12

higher by virtue of their increased activity level and respiratory rate; a nd 3) asthmatics who may 13

exhibit exacerbation of response to formaldehyde. 14

15This document is a draft for review purposes only and does not constitute Agency policy.

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- 469 Table 4

- 96. Available evidence for susceptibility factors of concern for 1

formaldehyde exposure 2

3

Factor Evidence that factors increase

susceptibility to FA Evidence that factors show differences

but unknown impact on susceptibility

Life Stage

 Preconception

 Prenatal

 Postnatal Developmental effects reported

suggesting that critical windows of

exposure may be relevant:

 Reproductive outcomes (Taskinen et

al., 1999; Maronpot et al., 1986)

 Embryo effects (Kitaev et al., 1984)

 Structural

- and functional

developmental outcomes (Martin,

1990; Saillenfait et al., 1989;

Sheveleva, 1971; Seninchenkova,

1991)

 Lung function outcome

(Krzyzanowski et al., 1990; Rumchev

et al., 2002; Garrett et al., 1999)

 Developmental neurotoxicity (Weiler

and Apfelbach, 1992)  Possible life stage level differences in

some enzymes involved in FA

metabolism (Harris et al., 2003;

Ginsberg et al., 2004; Westerlund et al.,

2005; Benedet ti et al., 2007)

 Mixed reports of associations between

prenatal exposure and developmental

outcomes in human studies (positive

association: Gražulevičiene et al., 1998)

 Possible life stage level differences in

some enzymes involved in FA

metabolism (e.g.

, ↓ADH expression over

first 5 months; Ginsberg et al., 2004)

 Developmental neurotoxicity (Sarsilmaz

et al., 2007; Aslan et al., 2006; Songur et

al., 2008)

Disease Status  Bronchial asthma (Lemiere et al.,

1995; Burge et al., 1985; Hendrick et

al., 1982; H endrick and Lane, 1977,

1975)

 Increased airway resistance and

bronchial constriction (Nielson et al.,

1999; Swiecichowski et al., 1993;

Biagini et al., 1989; Amdur et al.,

1960)  Mixed results for forced expiratory

volume (FEV1) measures affected by FA

expo sure in asthmatics (Casset et al.,

2006; Green et al., 1987; Ezratty et al.,

2007; Frigas et al., 1984)

Nutritional Status/Diet  Iron

- deficiency in utero

(Senichenkova and Chebotar, 1996).

Genetics

 Polymorphisms  For high FA exposure: Urinary formic

acid levels affected by ALDH2

genotype (Cheng et al., 2008)

 In mice, ADH3 increased sensitivity to

FA (Achkor et al., 2003)  Differences among ADH3 alleles and

asthma outcome (Wu et al., 2007)

 Differences among ethnic groups in

ADH3 alleles (Hedberg et al., 2001 )

Gender  Gender differences in incidence of

nasopharyngeal carcinoma following FA

exposure (Luce et al., 2002)

4

5



# - End of Volume II

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