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www.epa.gov/iris



# TOXICOLOGICAL REVIEW

OF

BIPHENYL

(CAS No. 92

- 52 -4)



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

July 2011

NOTICE

nt is an Interagency Science Consultation draft. This information is distributed
purpose of pre-dissemination peer review under applicable information quality
t has not been formally disseminated by EPA. It does not represent and should not

to represent any Agency determination or policy. It is being circulated for review
al accuracy and science policy implications.

U.S. Environmental Protection Agency
Washington, DC This docume
solely for the
guidelines. I

be construed

of its technic

### ii DRAFT

- DO NOT CITE OR QUOTE DISCLAIMER

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

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

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

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

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

### iii DRAFT

- DO NOT CITE OR QUOTE CONTENTS

— TOXICOLOGICAL REVIEW OF BIPHENYL (CAS No. 92-52-4)

LIST OF TABLES ......................................................................................................................... vi

LIST OF ABBREVIATIONS AND ACRONYMS ........................................................................x
FOREWORD ............................................................................................................................ xii

AUTHORS, CONTRIBUTORS, AND REVIEWERS

............................................................... xiii

1. INTRODUCTION ......................................................................................................................1

2. CHEMICAL AND PHYSICAL INFORMATION ....................................................................3

3. TOXICOKINETICS ...................................................................................................................6

3.1. ABSORPTION ...................................................................................................................6

3.2. DISTRIBUTI ON .................................................................................................................7

3.3. METABOLISM ..................................................................................................................8

3.3.1. Identification of Metabolites ........................................................................................8

3.3.1.1. Results from in vivo Animal Studies ..................................................................8

3.3.1.2. Results from in vitro Studies with Animal and Human Cells or Tissues .........10
3.3.2. Metabolic Pathways ...................................................................................................11

3.3.2.1. Description of Metabolic Scheme and Enzymes Involved ...............................11
3.3.3. Regulation of Metabolism, Sites of Metabolism, and Relationships to Toxic

Effects .......................................................................................................................14
3.3.3.1. Evidence for Induction of Phase I and II Enzymes ...........................................14

3.3.3.2. Demonstrated Tissue Sites of Metabolism .......................................................16

3.3.3.3. Possible Relationships Between Metabolites and Toxic Effects ......................16

3.4. ELIMINATION ................................................................................................................17
3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS ................18

4. HAZARD IDENTIFICATION .................................................................................................19

4.1. STUDIES IN HUMANS ...................................................................................................19

4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN

ANIMALS

— ORAL AND INHALATION .....................................................................23
4.2.1. Oral Exposure ............................................................................................................24
4.2.1.1. Subchronic Toxicity ..........................................................................................24
4.2.1.2. Chronic Toxicity and Carcinogenicity ..............................................................25

4.2.2. Inhalation Studies.......................................................................................................42

4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES

— ORAL AND

INHALATION .................................................................................................................44
4.3.1. Oral Exposure ............................................................................................................44
4.3.2. Inhalation Exposure ...................................................................................................47

4.4. OTHER DURATION

- OR ENDPOINT

- SPECIFIC STUDIES .......................................47

4.4.1. Acute and Short

- term Toxicity Data ..........................................................................47
4.4.2. Kidney/Urinary Tract Endpoint Studies ....................................................................49
4.4.3. Biphenyl as a Tumor Promoter ..................................................................................53

4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE

OF ACTION .....................................................................................................................54
4.5.1. Effects on the Urinary Tract of Rats ..........................................................................54

4.5.2. Ef fects on the Liver of Mice ......................................................................................56

### iv DRAFT

- DO NOT CITE OR QUOTE 4.5.3. Estrogenic Effects ......................................................................................................56
4.5.4. Effects on Apoptosis ..................................................................................................57

4.5.5. Mitochondrial Effects ................................................................................................58
4.5.6. Genotoxicity ...............................................................................................................58

4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS .................................................68

4.6.1. Oral ............................................................................................................................74

4.6.2. Inhalation ...................................................................................................................74

4.6.3. Mode-of-Action Information .....................................................................................75

4.7. EVALUATION OF CARCINOGENICITY .....................................................................76 4.7.1. Summary of Overall Weight of Evidence ..................................................................76
4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence.................................79

4.7.3. Mode-of-Action Information .....................................................................................81

4.7.3.1. Mode-of- Action Information for Bladder Tumors in Male Rats ......................81

4.7.3.2. Mode-of- Action Information for Liver Tumors in Female Mice .....................87

4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES .................................................90
4.8.1. Possible Childhood Susceptibility .............................................................................90

4.8.2. Possible Gender Differences ......................................................................................91

4.8.3. Other ..........................................................................................................................91

5. DOSE

- RESPONSE ASSESSMENTS ......................................................................................93

5.1. ORAL REFERENCE DOSE (RfD) ..................................................................................93
5.1.1. Choice of Principal Study and Critical Effect— with Rationale and Justification .....93
5.1.2. Methods of Analysis—Including Models ..................................................................96

5.1.3. RfD Derivation— Including Application of Uncertainty Factors (UFs) ..................104

5.1.4. Previous RfD Assessment ........................................................................................105
5.2. INHALATION REFERENCE CONCENTRATION (RfC) ..........................................106
5.2.1. Choice of Principal Study and Critical Effect— with Rationale and Justification ...106

5.2.2. Previous RfC Assessment ........................................................................................107

5.3. UNCERTAINTIES IN THE RfD and RfC .....................................................................107

5.4. CANCER ASSESSMENT ..............................................................................................108
5.4.1. Choice of Study/ Data

- with Rational and Justification ....................................109

5.4.2. Dose

- Response Data ..........................................................................................109
5.4.3. Dose Adjustments and Extrapolation Method(s) ...............................................110
5.4.4. Oral Slope Factor and Inhalation Unit Risk .......................................................113

5.4.5. Uncertainties in Cancer Risk Values .......................................................................113
5.4.5.1. Oral Slope Factor ............................................................................................113

5.4.5.2. Inhalation Unit Risk ........................................................................................114

5.4.6. Previous Cancer Assessment ...................................................................................115

6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND

DOSE RESPONSE ...........................................................................................116

6.1. HUMAN HAZARD POTENTIAL .................................................................................116

6.1.1. Noncancer ................................................................................................................116

6.1.2. Cancer ......................................................................................................................117

6.2. DOSE RESPO NSE .........................................................................................................118

6.2.1. Noncancer/Oral ........................................................................................................118

6.2.2. Noncancer/Inhalation ...............................................................................................118

6.2.3. Cancer/Oral ..............................................................................................................118

6.2.4. Cancer/Inhalation .....................................................................................................119

### 7. REFERENCES .......................................................................................................................120

APPENDIX A. SU MMARY OF EXTERNAL PEER REVIEW AND PUBLIC

COMMENTS AND DISPOSITION ................................................................ A-1

APPENDIX B. BENCHMARK DOSE CALCULATIONS FOR THE REFERENCE

DOSE ................................................................................................................B-1

APPENDIX C. BENCHMARK MODELING FOR THE ORAL SLOPE FACTOR ................C-1

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### vi DRAFT

- DO NOT CITE OR QUOTE LIST OF TABLES

Table 2 -1. Physicochemical properties of biphenyl ........................................................................4

Table 3 -1. Metabolites of biphenyl identified in urine, feces, and bile of male albino rats .......... 9

Table 4

- 1. Biphenyl concentrations in the air of a Finnish paper mill producing biphenylimpregnated fruit wrapping paper ............................................................................. 20

Table 4 -2. Nerve conduction velocities of 24 persons exposed to biphenyl: comparison
with 60 unexposed males .......................................................................................... 21

Table 4 -3. Exposure data and clinical featu res for five Parkinson’s Disease patients with
occupational exposure to biphenyl ............................................................................ 23

Table 4 -4. Incidences of urinary bladder lesion s in male and female F344 rats exposed to
biphenyl in the diet for 2 years .................................................................................. 27

Table 4 -5. Incidences of ureter and kidney lesions in male and female F344 rats exposed to
biphenyl in the diet for 2 years .................................................................................. 29

Table 4 -6. Body and organ weight data for male and female rats administered biphenyl in
the diet for 2 years ..................................................................................................... 33

Table 4 -7. Survival rate, body weight, food consumption, and daily biphenyl intake in
mice fed diets containing biphenyl for 2 years .......................................................... 36

Table 4

- 8. Dose

- related changes in selected clinical chemistry values from male and

female BDF 1 

### mice exposed to biphenyl via the diet for 2 years ............................... 37

Table 4 -9. Incidences of gross and histopathological findings in male and female BDF 1 

### mice fed diets containing biphenyl for 2 years .......................................................... 38

Table 4

- 10. Incidences of selected tumor types among controls and mice administered
biphenyl orally for 18 months .................................................................................. 41

Table 4

- 11. Incidences of selected histopathologic lesions in tissues of CD -1 mice exposed
to biphenyl vapors 7 hours/day, 5 days/week for 13 weeks .................................... 44

Table 4 -12. Prenatal effects following oral administration of biphenyl to pregnant Wistar

rats on GDs 6 –15 ..................................................................................................... 45

Table 4 -13. Summary of reproductive data in albino rats exposed to dietary biphenyl .............. 47

Table 4 -14. Number of Wistar rats exposed to biphenyl and the degree of change in kidney
weight and cellular architecture ............................................................................... 51

Table 4 -15. Content of biphenyl sulphate conjugates in urine and urinary crystals from
F344 rats treated with biphenyl and potassium bicarbonate (to elevate the pH

and K + 

### concentration of the urine) ........................................................................... 55

Table 4 -16. Genotoxicity test results for biphenyl....................................................................... 59

Table 4 -17. Genotoxicity test results for biphenyl metabolites ................................................... 63

Table 4 -18. Summary of major studies evaluating effects of biphenyl after oral

administration in rats and mice ................................................................................ 69

### vii DRAFT

- DO NOT CITE OR QUOTE Table 4 -19. Summary of major studies evaluating effects of biphenyl after inhalation
exposure in rats and mice ......................................................................................... 73

Ta ble 5-1. BMD modeling datasets for incidences of nonneoplastic effects in the urinary
tract of male and female F344 rats exposed to biphenyl in the diet for 2 years ...... 97

Table 5

- 2. BMD modeling datasets for body weight, selected clinical chemistry results,
and histopathological kidney effects in male and female BDF 1 

### mice exposed
to biphenyl in the diet for 2 years ............................................................................ 98

Table 5

- 3. BMD modeling dataset for incidence of litters with fetal skeletal anomalies
from Wistar rat dams administered biphen yl by gavage on GDs 6–15 ................... 99

Table 5

- 4. Summary of BMDs/BMDLs for selected nonneoplastic effects following oral
exposure of rats and mice to biphenyl ................................................................... 102

Table 5 -5. Incidence data for tumors in the urinary bladder of male and female F344 rats
exposed to biphenyl in the diet for 2 years ............................................................ 109

Table 5

- 6. Incidence data for liver tumors in male and female BDF 1 

### mice fed diets
containing biphenyl for 2 years .............................................................................. 110

Table 5 -7. Scaling factors for determining HEDs to use for BMD modeling of fema le

BDF 1 

### mouse liver tumor incidence data from Umeda et al. (2005) ....................... 111

Table 5 -8. Incidence of liver adenomas or carcinomas (combine d) in female BDF 1 

### mice
fed diets containing biphenyl for 2 years ............................................................... 112

Table 5 -9. POD and oral slope factor derived from liver tumor incidence data from BDF 1 

### female mice exposed to biphenyl in the diet for 2 years ........................................ 113

Table B -1. BMD modeling datasets for incidenc es of nonneoplastic effects in the urinary
tract of male and female F344 rats exposed to biphenyl in the diet for 2 years .... B-1

Table B- 2. BMD modeling datasets for body weight, selected clinical chemistry results,
and histopathological kidney effects in male and female BDF 1 

### mice exposed
to biphenyl in the diet for 2 years .......................................................................... B-2

Table B

- 3. BMD modeling dataset for incidence of litters with fetal skeletal anomalies
from Wistar rat dams administered biphenyl by gavage on GDs 6–15 ................. B-3

Table B -4. Summary of BMD modeling results for incidence of renal nodular transitional
cell hyperplasia in male F344 rats exposed to biphenyl in the diet for 2 years ..... B-3

Table B -5. Summary of BMD modeling results for incidence of renal nodular transitional
cell hyperplasia in female F344 rats exposed to biphenyl in the diet for 2 years .. B-5

Table B -6. Summary of BMD modeling results for incidence of renal simple tran sitional
cell hyperplasia in male F344 rats exposed to biphenyl in the diet for 2 years ..... B-7

Table B -7. Summary of BMD modeling results for incidence of renal simple transitional
cell hyperplasia in female F344 rats exposed to biphenyl in the diet for 2 years .. B-9

Table B -8. Summary of BMD modeling results for incidence of mineralization in renal
pelvis of male F344 rats exposed to biphenyl in the diet for 2 years ................... B-11

Table B -9. Summary of BMD modeling results for incidence of mineralization in renal
pelvis of female F344 rats exposed to biphenyl in the diet for 2 years ............... B-13

### viii DRAFT

- DO NOT CITE OR QUOTE Table B -10. Summary of BMD modeling results for incidence of hemosiderin deposits in
the kidney of female F344 rats exposed to biphenyl in the diet for 2 years ........ B-15

Table B -11. Summary of BMD modeling results for incidence of papillary mineralization
in the kidney of male F344 rats exposed to biphenyl in the diet for 2 years ....... B-17

Table B -12. Summary of BMD modeling results for incidence of papillary mineralization
in the kidney of female F344 rats exposed to biphenyl in the diet for 2 years .... B-19

Table B -13. Summary of BMD modeling results for incidence of combined transitional
cell hyperplasia in the bladder of male F344 rats exposed to biphenyl in the
diet for 2 years ..................................................................................................... B-21

Table B -14. Summary of BMD modeling results for incidence of mineralization in the
kidney (inner stripe outer medulla) of male BDF 1 

### mice exposed to biphenyl in
the diet for 2 years ................................................................................................ B-23

Table B -15. Summary of BMD modeling results for incidence of mineralization in the
kidney (inner stripe outer medulla) of female BDF 1 

### mice exposed to biphenyl
in the diet for 2 years ........................................................................................... B-25

Table B

- 16. BMD m odel results for serum LDH activity in female BDF 1 

### mice exposed to
biphenyl in the diet for 2 years ............................................................................ B-27

Table B

- 17. BMD modeling results for serum AST activity in female BDF 1 

### mice exposed
to biphenyl in the diet for 2 years ........................................................................ B-28

Table B -18. BMD modeling result s for serum ALT activity in female BDF 1 

### mice exposed
to biphenyl in the diet for 2 years ........................................................................ B-31

Table B -19. BMD modeling results for serum AP activity in female BDF 1 

### mice exposed to
biphenyl in the diet for 2 years ............................................................................ B-32

Table B -20. BMD modeling results for changes in BUN levels (mg/dL) in male BDF 1 

### mice exposed to biphenyl in the diet for 2 years ................................................. B-33

Table B -21. BMD modeling results for changes in BUN levels (mg/dL) in female BDF 1 

### mice exposed to biphenyl in the diet for 2 years ................................................. B-36

Table B -22. BMD modeling results for changes in mean terminal body weight in male

BDF 1 

### mice exposed to biphenyl in the diet for 2 years ....................................... B-37

Table B

- 23. BMD modeling results for changes in mean terminal body weight in female

BDF 1 

### mice exposed to biphenyl in the diet for 2 years ....................................... B-38

Tabl e B

- 24. Summary of BMD modeling results for incidence of litters with fetal skeletal
anomalies from Wistar rat dams administered biphenyl by gavage on GDs 6

– 15.......................................................................................................................... B-40

Table C

- 1. Incidences of liver adenomas or carcinomas (combined) in female BDF 1 

### mice
fed diets containing biphenyl for 2 years ............................................................... C-1

Table C -2. Model predictions for liver tumors (adenomas or carcinomas combined) in

female BDF 1 

### mice exposed to biphenyl in the diet for 2 years ............................. C-2

### LIST OF FIGURES

3-1. Schematic presentation of the metabolic pathways of biphenyl. ...........................................13

5- 1. NOAELs and LOAELs for noncancer effects in rats and mice from repeated oral

exposure to biphenyl. .............................................................................................................94

5- 2. BMDs and BMDLs for selected noncancer effects in rats and mice from repeated oral
exposure to biphenyl. ...........................................................................................................103

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### x DRAFT

- DO NOT CITE OR QUOTE LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygienists

AIC Akaike’s Information Criterion

ALT alanine aminotransfer ase
ALP alkaline phosphatase

AP alkaline phosphatase

AST aspartate aminotransferase

BBN N-butyl-N- (4 -hydroxybutyl)nitrosamine

BMD benchmark dose

BMR benchmark response

BMDS Benchmark Dose Software

BrdU 5-bromo-2-deoxyuridine

BUN blood urea nitrogen

CA chromosomal aberration

CASRN Chemical Abstracts Service Registry Number

CHL Chinese hamster lung

CHO Chinese hamster ovary

CYP cytochrome P-450

CVSF conduction velocity of the slowest motor fibers

DF degrees of freedom

DNA deoxyribonucleic acid

EEG electroencephalography

EHEN N-ethyl-N-hydroxyethylnitrosamine

EMG electromyographic

ENMG electroneuromyography

GC gas chromatography

GC

- MS gas chromatography- mass spectrometry

GD gestation day

GOT glutamate oxaloacetate transaminase

GPT glutamate pyruvate transaminase

HED human equivalent doses

HGPRT hypoxanthine guanine phosphoribosyl transferase

HPLC high-performance liquid chromatography
i.p. intraperitoneal or intraperitoneally

IRIS Integrated Risk Information System

K o/w 

### octanol/water partition coefficient

K

m 

### Michaelis constant

LD 50 

### median lethal dose

LDH lactate dehydrogenase

LOAEL lowest -observed-adverse- effect level

MCV motor conduction velocity

NOAEL no-observed-adverse- effect level

PBPK physiologically based pharmacokinetic

PD Parkinso n’s disease

POD point of departure

PPAR peroxisome proliferator activated receptors

RfC reference concentration

### xi DRAFT

- DO NOT CITE OR QUOTE RfD reference dose

ROS reactive oxygen species

RR relative risk

SCE sister chromatid exchange

SD standard deviation

TLV threshold limit value

TMS trimethylsilyl

TWA time

- weighted average

UDS unscheduled DNA synthesis

UF uncertainty factors

UGT uridine diphosphate glucuronosyl transferase

U.S. EPA U.S. Environmental Protection Agency

### xii DRAFT

- DO NOT CITE OR QUOTE FOREWORD

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

for the hazard and dose-response assessment in IRIS pertaining to chronic exposure to biphenyl.

It is not intended to be a comprehensive treatise on the chemical or toxicological nature of

biphenyl.

The intent of Section 6, Major Conclusions in the Characterization of Hazard and Dose

Response, is to present the major conclusions reached in the derivation of the reference dose,

reference concentration and cancer assessment, where applicable, and to characterize the overall

confidence in the quantitative and qualitative aspects of hazard and dose response by addressing

the quality of data and related uncertainties. The discussion is intended to convey the limitations

of the assessment and to aid and guide the risk assessor in the ensuing steps of the risk

assessment process.

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

the reader is referred to EPA ’ s IRIS Hotline at (202) 566 -1676 (phone), (202) 566-1749 (fax), or

hotline.iris@epa.gov (email address).

### xiii DRAFT

- DO NOT CITE OR QUOTE AUTHORS, CONTRIBUTORS, AND REVI EWERS

CHEMICAL MANAGER / AUTHOR

Zheng (Jenny) Li , Ph.D, DABT

U.S. EPA, ORD/NCEA
Washington, DC

CONTRIBUTORS

Christine Yuyang Cai, MS, PCP

U.S. EPA, ORD/NCEA
Washington, DC

J. Connie Kang- Sickel , Ph.D

U.S. EPA, ORD/NCEA
Washington, DC

CONTRACTOR SUPPORT

George Holdsworth, Ph.D

Lutz W. Weber, Ph.D, DABT
Oak Ridge Institute for Science and Education
Oak Ridge, TN

David Wohlers, Ph.D
Joan Garey, Ph.D

Peter McClure, Ph.D, DABT

SRC, Inc.
Syracuse, NY

REVIEWERS

This document has been provided for review to EPA scientists.

INTERNAL EPA REVIEWERS

Maureen Gwinn, Ph.D, DABT

U.S. EPA, ORD/NCEA
Washington, DC

Margaret Pratt, Ph.D

U.S. EPA, ORD/NCEA
Washington, DC

### 1 DRAFT

- DO NOT CITE OR QUOTE 1. INTRODUCTION 1

2

3 

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



### Information System (IRIS) Summary of the hazard and dose- response assessment of biphenyl. 5



### IRIS Summaries may include oral reference dose (RfD) and inhalation reference concentration 6



### (RfC) values for chronic and other exposure durations, and a carcinogenicity assessment. 7



### The RfD and RfC, if derived, provide quantitative information for use in risk assessments 8



### for health effects known or assumed to be produced through a nonlinear (presumed threshold) 9



### mode of action. The RfD (expressed in units of mg/kg- day) is defined as an estimate (with 10



### uncertainty spanning perhaps an order of magnitude) of a daily exposure to the human 11



### population (including sensitive subgroups) that is likely to be without an appreciable risk of 12



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

### ) is 13



### analogous to the oral RfD, but provides a continuous inhalation exposure estimate. The 14



### inhalation RfC considers toxic effects for both the respiratory system (portal -of-entry) and for 15



### effects peripheral to the respiratory system (extrarespiratory or systemic effects). Reference 16



### values are generally derived for chronic exposures (up to a lifetime), but may also be derived for 17



### acute ( ≤24 hours), short- term (>24 hours up to 30 day s), and subchronic (>30 days up to 10% of 18



### lifetime) exposure durations, all of which are derived based on an assumption of continuous 19



### exposure throughout the duration specified. Unless specified otherwise, the RfD and RfC are 20



### derived for chronic exposure duration. 21



### The carcinogenicity assessment provides information on the carcinogenic hazard 22



### potential of the substance in question and quantitative estimates of risk from oral and inhalation 23



### exposure may be derived. The information includes a weight-of-evidence judgment of the 24



### likelihood that the agent is a human carcinogen and the conditions under which the carcinogenic 25



### effects may be expressed. Quantitative risk estimates may be derived from the a pplication of a 26



### low-dose extrapolation procedure. If derived, the oral slope factor is a plausible upper bound on 27



### the estimate of risk per mg/kg -day of oral exposure. Similarly, a plausible inhalation unit risk is 28



### an upper bound on the estimate of risk p er μg/m 3 

### air breathed. 29



### Development of these hazard identification and dose- response assessments for biphenyl 30



### has followed the general guidelines for risk assessment as set forth by the National Research 31



### Council (NRC, 1983). U.S. Environmental Protection Agency (U.S. EPA) Guidelines and Risk 32



### Assessment Forum Technical Panel Reports that may have been used in the development of this 33



### assessment include the following: Guidelines for the Health Risk Assessment of Chemical 34



### Mixtures (U.S. EPA, 1986a), Guidelines for Mutagenicity Risk Assessment (U.S. EPA, 1986b), 35



### Recommendations for and Documentation of Biological Values for Use in Risk Assessment (U.S. 36



### EPA, 1988), Guidelines for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), Interim 37



### Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, 38

### 2 DRAFT

- DO NOT CITE OR QUOTE 1994a), Methods for Derivation of Inhalation Reference Concentrations and Application of 1



### Inhalation Dosimetry (U.S. EPA, 1994b), Use of the Benchmark Dose Approach in Health Risk 2



### Assess ment (U.S. EPA, 1995), Guidelines for Reproductive Toxicity Risk Assessment (U.S. EPA, 3



### 1996), Guidelines for Neurotoxicity Risk Assessment (U.S. EPA, 1998), Science Policy Council 4



### Handbook : Risk Characterization (U.S. EPA, 2000a), Benchmark Dose Technical Guidance 5



### Document (U.S. EPA, 2000b), Supplementary Guidance for Conducting Health Risk Assessment 6



### of Chemical Mixtures (U.S. EPA, 2000c), A Review of the Reference Dose and Reference 7



### Concentration Processes (U.S. EPA, 2002), Guidelines for Carcinogen Risk Assessment (U.S. 8



### EPA, 2005a) , Supplemental Guidance for Assessing Susceptibility from Early- Life Exposure to 9



### Carcinogens (U.S. EPA, 2005b), Science Policy Council Handbook: Peer Review (U.S. EPA, 10



### 2006a), and A Framework for Assessing Health Risks of Envi ronmental Exposures to Children 11



### (U.S. EPA, 2006b). 12



### The literature search strategy employed for this compound was based on the Chemical 13



### Abstracts Service Registry Number (CASRN) and at least one common name. Any pertinent 14



### scientific information submitted b y the public to the IRIS Submission Desk was also considered 15



### in the development of this document. The relevant literature was reviewed through June 2011. 16

17

### 3 DRAFT

- DO NOT CITE OR QUOTE 2. CHEMICAL AND PHYSICAL INFORMATION 1

2

3 

### Pure biphenyl is a white or colorless crystalline solid that usually forms leaflets or scales; 4



### commercial preparations may be yellowish or slightly tan ( NLM , 2007). Biphenyl is said to 5



### have a pleasant odor that is variably described as pecul iar, butter -like, or resembling geraniums 6



### (NLM, 2007; IPCS, 1999). Biphenyl melts at 69°C and has a vapor pressure of 8.93 **×** 10 -3 

### mm 7



### Hg at 25°C, making it likely to enter the environment in its vaporized form (NLM, 2007). If 8



### particle -bound biphenyl is precipitated to the ground, it is likely to be reintroduced to the 9



### atmosphere by volatilization. The water solubility of biphenyl is 7.48 mg/L at 25 o 

### C. The 10



### logarithm of the octanol/water partition coefficient (K o/w 

### ) of biphenyl of 3.98 suggests a potential 11



### for bioaccumulation (NLM, 2007). B ecause it is biodegraded with an estimated half

- life of 2 12



### and 3 days in air and water, respectively (NLM, 2007), and is metabolized rapidly by humans 13



### and animal s (see Section 3), bioaccumulation does not occur (IPCS, 1999). Biphenyl is 14



### ubiquitous in the environment, with reported indoor air concentrations of 0.16–1 μg/m 3 

### and 15



### outdoor levels of approximately 0.03 μg/m 3 

### (IPCS, 1999). The physicochemical properties of 16



### biphenyl are summarized in Table 2-1. 17

18

19

### 4 DRAFT

- DO NOT CITE OR QUOTE Ta ble 2

- 1. Physicochemical properties of biphenyl

Synonyms Diphenyl, 1,1 ′

- biphenyl, 1,1 ′

- diphenyl, bibenzene, phenylbenzene,

lemonene, Carolid AL, Phenador

- X, Tetrosine LY

CASRN 92

- 52 -4

Chemical structure

Chemical formula C 12 H 10

Molecular weight 154.2

Melting point 69°C

Boiling point 256°C

Specific gravity 1.041 g/cm 3 at 20°C

Vapor pressure 8.93 **×** 10 -3 mm Hg at 25°C

L og K o/w 4.01

4.11 a

4.17 or 5.27

– 5.46 b

Water solubility 7.48 mg/L at 25°C

Henry’s law constant 3.08 **×** 10 -4 atm -m 3 /mol at 25°C

Conversion factors 1 ppm = 6.31 mg/m 3 ; 1 mg/m 3 = 0.159 ppm

a Monsanto (1979).

b Estimated by different methods: Dow (1971).

Source: NLM (2007).

1



### Biphenyl exists naturally as a component of crude oil or coal tar. I t is primarily produced 2



### by debromination/dimerization of bromobenzene, is isolated as a byproduct of the 3



### hydrodealkylation of toluene (yield approximately 1%), or is synthesized by catalytic 4



### dehydrocondensation of benzene. Biphenyl is currently not registered for use as a pesticide in 5



### the United States , but is still used in other countries as a fungistat, most commonly to preserve 6



### packaged citrus fruits or in plant disease control (NLM, 2007). The major uses of biphenyl today 7



### are as chemical synthesis intermediates (among them , the sodium salt of 2-hydroxy-biphenyl, a 8



### pesticide known as Dowicide 1), as dye carriers in polyester dyeing, and as components in heat 9



### transfer fluids (in particular Dowtherm A or Therminol **** VP -1, consisting of 26.5% biphenyl 10



### and 73.5% diphenyl oxide). Hist orically, biphenyl was the primary byproduct in the 11



### manufacture of polychlorinated biphenyls (PCBs) until PCBs were banned in the 1970’s (U.S. 12



### EPA 1978). The purity of t echnical biphenyl range s from 93–99.9%. The prevalent impurities 13



### in technical prepara tions are terphenyls, a side product from the dehydrocondensation of 14

### 5 DRAFT

- DO NOT CITE OR QUOTE benzene. Biphenyl is rated as a high-volume production chemical. Annual U.S. production in 1



### 1990 was approximately 1.6 **×** 10 4 

### metric tons (NLM, 2007). 21 

### 3. TOXICOKINETICS

2

3

4 

### 3.1. ABSORPTION

5 

### No quantitative studies on the absorption of biphenyl have been conducted in humans.

6 

### However, evidence of hepa tic toxicity produced by a probable combination of inhalation and

7 

### dermal exposures to biphenyl was identified a s the likely cause of death of a worker in a

8 

### biphenyl-impregnated fruit wrapping paper production facility and provides prima facie

9 

### qualitative evidence of absorption in a human subject (Häkkinen et al. , 1973). This worker had

10 

### 11 year s of exposure to biphenyl ; at the time of his death

, air measurements in the factory were

11 

### as high as 123 mg /m 3 

### . Evidence of hepatic and nervous system toxicity was also observed in

12 

### eight co-workers (Häkkinen et al., 1973).

6 DRAFT

- D O NOT CIT E OR QUOTE Animal studies in rats, rabbits, guinea pigs, and pigs ind icate that biphenyl is rapidly and 13



### readi ly absorbed following oral exposure, as evidenced by the detection of metabolites in urine 14



### and bile ( Meyer, 1977; Meyer and Scheline, 1976; Meyer et al. , 1976a, b). Results from a study 15



### with rats administere d radiolabeled biphenyl indicate extensive oral absorption (about 85% of 16



### administered dose) (Meyer et al., 1976a, see below), whereas re sults from studies of rabbits, 17



### guinea pigs, and pigs administered nonlabeled biphenyl indicate less extensive oral absor ption in 18



### the range of 28–49% of the administered dose (Meyer, 1977; Meyer et al., 1976b). 19



### In the most quantitative assessment of absorption using radiolabeled biphenyl, male 20



### albino rats (n = 3; body weight = 200–300 g) given an oral dose of 100 mg /kg (0.7–1.0 μ Ci) of 21



### [ 14 

### C]-biphenyl (in soy oil) excreted 75– 80% of the radioactivity in their urine within the first 22



### 24 hours, with a total average urinary excretion of 84.8% and fecal excretion of 7.3% during the 23



### 96-hour post-dosing period (Meyer et al., 1976a). Only a trace of [ 14 

### C]-CO 2 

### was detected in 24



### expired air and <1% of the radioactivity was recovered from tissues obtained at the 96-hour 25



### sacrifice of the rats. These results indicate that at least 85% of the administe red dose was 26



### absorbed in rats. 27



### Less quantitative estimates of oral absorption have been provided in analytical studies of 28



### biphenyl and metabolites in urine and feces from rabbits (Meyer, 1977), guinea pigs (Meyer, 29



### 1977), and pigs (Meyer et al., 1976b) following oral administration of single 100-mg/kg doses of 30



### unlabeled biphenyl. 31



### Male White Land r abbits and Sff:PIR guinea pigs were given biphenyl (100 mg /kg) by 32



### gavage in soy oil, and ur ine and feces were collected at 24-hour intervals , up to 96 hour s after 33



### administration (Meyer, 1977) . The phenolic metabolites of biphenyl were analyzed as 34



### trimethylsilyl (TMS) ethers by combined gas chromatography/mass spectrometry (GC/MS) 35



### (guinea pigs) or GC (rabbits). The biphenyl was hydroxylated to monohydroxylated biphenyls 36



### and minor amounts of dihydroxylated derivatives, with the main route of excretion being through 37



### the urine in both sp ecies and the major metabol ite being 4-hydroxybiphenyl. In guinea pigs 38

### 7 DRAFT

- DO NOT CITE OR QUOTE (n = 3), t he mass of i dentified metabolites in urine collected for 24 or 96 hour s accounted for 1



### 29.5 or 32.9% of the administered dose, respectively. In the first 24 hour s, biphenyl and 2



### biphenyl metabolites in feces accounted for 20.3% of the dose; most of this (14.3%) was 3



### biphenyl, presumably unabsorbed. Bile was collected for 24 hours from another group of two 4



### bile -cannulated guinea pigs dosed with 100 mg /kg biphenyl. No unchanged biphenyl was 5



### detected in th e collected bile, but conjugated mono- and dihydroxy metabolites accounted for 6



### about 3% of the administered dose. The results with guinea pigs indicate that at least 33% of the 7



### administered dose was abso rbed. In rabbits, uri nary metabolites accounted for 49.1% of the 8



### dose, with most of this (25.4% on the first day and 15.9% on the second day ) eliminated as 9



### conjugates. In the first 24 hours, biphenyl and metabolites in feces accounted for 1.6% of the 10



### dose with 1.4% being biph enyl. These results indicate that at least 49 % of the administered dose 11



### was absorbed in rabbits

. 12



### Absorption of single oral 100 mg/kg doses of biphenyl (in soy oil or propylene glycol) 13



### has also been demonstrated in male and female Danish Landrace pigs weig hing 31–35 kg (Meyer 14



### et al., 1976b). Metabolites identified in urine collected at four 24- hour intervals after dose 15



### administration included mono -, di- , and trihydroxybiphenyls , detected as TMS ethers by GC/MS 16



### after enzyme hydrolysis of the samples by β

- gl ucuronidase and sulphatase. Metabolites 17



### identified and quantified in 24-hour urine samples accounted for averages of 17.5 and 26.5% of 18



### the dose administered in soy oil to two female pigs and in propylene glycol to two male pigs

, 19



### respectively. Unchanged biphenyl was not detected in the urine samples. Metabolites in urine 20



### collected for 96 hours accounted for averages of 27.6 and 44.8% of the doses administered to 21



### female and male pigs, respectively. No phenolic metabolites of biphenyl were det ected in feces 22



### collected for 96 hours. Unchanged biphenyl was not detected in the feces collected from male 23



### pigs, but the amount of unchanged biphenyl in feces from the two female pigs accounted for 24

25 

### 18.4 and 5% of the administered dose. These results indicate that at least about 28 and 45% of

26 

### oral 100 mg/kg doses of biphenyl were absorbed in female and male pigs, respectively. It is

27 

### uncertain if the gender difference was due to vehicle differences or actual gender differences in

28 

### absorption efficiency.

29 

### No animal studies were located examining quantitative aspects of absorption of biphenyl

30 

### by the respiratory tract or skin.

31

32 

### 3 .2. DISTRIBUTION

33 

### No information was located regarding distribution of absorbed biphenyl in humans and

34 

### limited animal data are availab le . Meyer et al. (1976a ) orally administered 100 mg/kg

[ 14 35 

### C]-biphenyl to male albino rats and measured radioactivity in the lung, heart, kidney, brain,

36 

### spleen, liver, skeletal muscles, peritoneal fat, genital tract, and gastrointestinal tract at 96 hours

37 

### after dosing . M ost of the radioactivity was excreted in urine (84.8%) and feces (7.3%) over the

38 

### 96-hour period, and only 0.6% of the administered radioactivity remained in the animals at

### 8 DRAFT

- DO NOT CITE OR QUOTE 1 

### 96 hour s: 0.1% was found in peritoneal fat, 0.3% in th e gastrointestinal tract (including its

2 

### contents) 0.1% in skeletal muscles , and 0.1% in the genital tract. Levels of r adioactivity in other

3 

### examined tissues were very low. The results indicate that absorbed biphenyl is not preferentially

4 

### stored in tissue s and is rapidly excreted, principally through the urine.

5

6 

### 3.3. METABOLISM

7 

### 3.3.1. Identification of Metabolites

8 

### 3.3.1.1. Results from in vivo Animal Studies

9 

### No human studies have been identified on the in vivo metabolism of biphenyl. However,

10 

### the in vivo metabolism of biphenyl has been studied extensively in laboratory animals. These

11 

### studies have determined that in rats, rabbits, pigs, dogs, mice, and guinea pigs, biphenyl is

12 

### converted into a range of hydroxylated metabolites ( Halpaap -Wood et al., 1981 a; Meyer, 1977;

13 

### Meyer and Scheline

, 1976; Meyer et al. , 1976a, b). These metabolites have been detected in

14 

### urine both as nonconjugated comp ounds and as acidic conjugates.

15 

### The derivation of urinary metabolites and their subsequent analysis with GC has resulted

16 

### in the identification of more than 10 mono-, di-, and trihydroxybiphenyl metabolites from the

17 

### urine of rats, pigs, guinea pigs, and rabbits ( Meyer , 1977; Meyer and Scheline

, 1976; Meyer et

18 

### al. , 1976a, b). These metabolites have been found as mercapturic acid conjugates and

19 

### glucuronide conjugates (Millburn et al., 1967). Comparable metabolites have been identified

among mammalian species tested, although quantitative differences in metabo lite formation are 20



### evident among species. A major meta bolite in the rat, mouse, guinea pig, rabbit, and pig was 21



### reportedly 4-hydroxybiphenyl ( Halpaap -Wood et al. 1981a; Meyer, 1977; Meyer and Scheline, 22



### 1976 ). 4,4'-Dihydroxybiphenyl was identified as a major metabolite in the pig (Meyer et al., 23



### 1976b) and the rat ( Halpaap -Wood et al., 1981a; Meyer and Scheline, 1976), while 3,4-di24



### hydroxybiphenyl was a major urinary metabolite in two strains of mice (Halpaap -Wood et al., 25



### 1981a). Table 3- 1 reviews the metabolites that have been identified in the excreta and bile of 26



### male albino rats given single doses of 100 mg biphenyl/kg , as reported by Meyer and Scheline 27



### (1976). 28

29

### 9 DRAFT

- DO NOT CITE OR QUOTE Table 3

- 1. Metabolites of biphenyl identified in urine, feces

, and bile of male

albino rats

a Metabolite Urine Feces Bile

Day 1 Day 2 Days 3 + 4 Days 1

– 4 Day 1 Day 1

Biphenyl 0.1 0.1 ND b 0.2 ND ND

2

- Hydroxybiphenyl 0.4 0.5 0.1 1.0 0.3 0.1

3

- Hydroxybiphenyl 0.9 0.4 0.3 1.6 0.5 0.5

4

- Hydroxybiphenyl 6.8 0.7 0.2 7.7 1.0 1.5

3,4

- Dihydroxybiphenyl 0.6 0.2 ND 0.8 ND 0.1

3,4’

- Dihydroxybiphenyl 1.5 0.3 0.8 2.6 ND 0.3

4,4’

- Dihydroxybiphenyl 9.6 1.7 0.1 11.4 1.8 1.9

2,5

- Dihydroxybiphenyl Trace ND ND Trace ND ND

Methoxy

- hydroxybiphenyls 0.1 ND ND 0.1 ND 0.1

Methoxy

- dihydroxybiphenyls 0.5 0.3 0.1 0.9 ND ND

3,4,4’

- Trihydroxybiphenyl 1.8 0.9 0.5 3.2 1.1 0.7

Total 22.3 5.1 2.1 29.5 4.7 5.2

a Values are percent of administered dose.

b ND = not detected.

Source: Meyer and Scheline (1976).

1



### The hydroxylation of biphenyl to produce 2-hydroxybiphenyl is a minor pathway in rats 2



### and mice, but is more easily detected in mice than r ats (Halpaap -Wood et al., 1981a, b). 3



### Following intraperitoneal (i.p.) injection of [ 14 

### C]-labeled biphenyl (30 mg/kg), the pattern of 4



### perc entages of radioactivity detected in urinary metabolites showed a relatively greater ability to 5



### produce 2-hydroxybiphenyl in mice than rats. In Sprague

- Dawley rats, metabolites identified in 6



### order of abundance were (with percentage of total urinary radioactivity noted in parentheses): 7



### 4,4’- dihydroxybiphenyl (44.5%); 4 -hydroxybiphenyl (28.5%); 3,4,4’-trihydroxybiphenyl (8.8%); 8



### 3,4’- dihydroxybiphenyl (8 .5%); 3,4-dihydroxybiphenyl (5.1 %); 3

- hydroxybiphenyl (1.8%); and 9



### 2-hydroxybiphenyl (1.5%). In DBA/2Tex m ice, major identified metabolites were: 4-hydroxy10



### biphenyl (39.5%); 3,4 -dihydroxybiphenyl (30.3%); 4,4’-dihydroxybiphenyl (10.2%); 11



### 3,4,4’-trihydroxybiphenyl (6.2%); 3-hydroxybiphenyl (4.3%); and 2- hydroxybiphenyl (4.2%). 12



### In rats, 2,3-, 2,4-, and 2,5-dih yroxybiphenyl were detected at trace levels (<0.1%), whereas , in 13



### mice , these metabolites were detected at levels of 0.3%, 0.8%, and 0.7%, respectively (Halpaap14



### Wood et al., 1981a). 15



### No in vivo studies have been identified that directly investigate differential metabolism of 16



### biphenyl between males and females of any species. However, studies on urinary crystals and 17



### calculi formation and composition after chronic exposure to biphenyl in the diet indicate that 18



### male F344 rats are more susceptible than females to the formation of urinary bladder calculi 19

### 10 DRAFT

- DO NOT CITE OR QUOTE (O hnishi et al., 2001, 2000a, b). Urinary bladder calculi in males were predomina ntly composed 1



### of t he insoluble potassium salt of 4-hydroxybiphenyl-O-sulph ate, whereas the less freq uently 2



### occurring urinary bladder calculi in females were composed mainly of 4-hydroxybiphenyl and 3



### potassium sulphate, hydrolysis products of 4-hydroxybiphenyl-O-sulph ate (O hnishi et al., 2001, 4

5 

### 2000a, b). These observations are consistent with observations that male rats have relatively

6 

### higher urinary potassium concentrations and pH values than female rats, and with the hypothesis

7 

### that gender differences in these urinary conditions (rather than gender differences in metabolism

8 

### of biphenyl) may be responsible for the gender differences in urinary calculi formation and the

9 

### subsequent development of non-neoplastic (hyperplasia) and neoplastic (papillomas and

10 

### carcinomas) lesions in male, but not female, F344 rats (Umeda et al., 2002; Ohnishi et al., 2001,

11 

### 2000a, b).

12

13 

### 3.3.1.2. Results from in vitro Studies w ith Animal a nd Human Cells o r Tissues

14 

### The metabolism of biphenyl in vitro has been investigated using tissue s of human origin,

15 

### resulting in evidence that the human metabolism of biphenyl is qualitatively simil ar to, but may

16 

### be quantitatively different from, rat metabolism. Benford et al. (1981) measured 2- , 3 -, and

17 

### 4-hydroxylation of biphenyl in microsomes prepared from the livers of five rats (sex not

18 

### identified) and four humans (sex not identified). The reaction products, after solvent extraction

19 

### and high-performance liquid chromatography ( HPLC ) quantitation, revealed that 2-hydroxylase

20 

### in the rat was 35 times higher than in humans, while 3- and 4-hydroxylases in humans were

21 

### 1.5 and 1.2 times higher t han in rats.

22 

### The evidence from studies of human tissue samples exposed to biphenyl metabolites in

vitro suggests differential Phase II metabolism contingent upon tissue origin. Powis et al. (1988 ) 23



### have shown that p -hydroxybiphenyl is conjugated with glucuronic acid and sulphate in human 24



### liver and kidney tissue slices. In the liver, glucuronidation was the favored conjugation pathway, 25



### while sulphation was favored in the kidney . Powis et al. (1989) also compared Phase I biphenyl 26



### metabolism in human (from surgery), dog (mongrel), and rat (male F344) liver slices and 27



### primary hepatocytes. It was found that liver slices from all three species had a similar capacity 28



### to metabolize biphenyl, ~3.5 nmol biphenyl/minute per g tissue, while hepatocyte preparations 29



### fr om rats had about 4 times the metabolic capacity of dog hepatocytes and about 20 times that of 30



### human hepatocytes. Powis et al. (1989) speculated that hepatocytes from dog and human liver 31



### slices may have experienced more damage during isolation than rat hepatocytes. 32



### A study of the sulphation of biphenyl metabolites in human surgical tissue samples was 33



### conducted by Pacifici et al. (1991) . T issue samples of various types (liver, intestinal mucosa, 34



### lung, kidney, bladder, and brain) were obtained from surger ies of patients of both sexes between 35



### the ages of 49 and 76 year s of age (each patient contributed only one tissue type, so that within

36



### patient organ comparisons were not made) and prepared 12,000 and 105,000 g supernatants to 37



### study sulphation of biphenyl metabolites, specifically 2-, 3-, and 4-hydroxybiphenyl. 38

### 11 DRAFT

- DO NOT CITE OR QUOTE Sulphotransferase activity for each of these substrates was detected in all tissues studied, 1



### although marked tissue dependence was observed, with the highest activity found in the liver and 2



### the lowest in the brain. The Michaelis constant ( K

m 

### ) of sulphotransferase was dependent on the 3



### substrate , but not on tissue type, with K

m 

### varying over a 500-fold range. The highest values of 4



### K

m 

### were found with 4-hydroxybiphenyl and the lowest were found with 3-hydroxybiphenyl. 5



### Several studies of biphenyl metabolism with in vitro animal systems support the findings 6



### from the in vivo urinary metabolite investigations that: (1) a range of hydroxylated biphenyl 7



### metabolites are formed , (2) 4-hydroxybiphenyl is a major metabolite , and (3) hydroxylated 8



### biphenyl metabolites are conjugated to glucuronic acid or sul ph ate. Wiebkin et al. (1984, 1976) 9



### reported that isolated rat and hamster hepatocytes metabolized biphenyl primarily to 10



### 4-hydroxybiphenyl and also to 4,4'-hydroxybiphenyl, both of which were then conjugated. A 11



### small amount of 2-hydroxybiphenyl was produced. When 4-hydroxybiphenyl was incubated 12

13 

### with the hepatocytes, it was hydroxylated to 4,4'-dihydroxybiphenyl. Pretreat me nt of the

14 

### animals with either 5,6 -benzoflavone or phenobarbital had little effect on the conjugate

15 

### formation rate in the in vitro experiment. Bianco et al. (1979) reported that rat hepatic

16 

### microsomes metabolize biphenyl to 4 -, 2-, and 3-hydroxybiphenyl, which are conjugated to form

17 

### glucuronides and sulph ates. The 4 -hydroxybiphenyl isomer was the major metabolite. The

18 

### formation of 4-hydroxybiphenyl as a major metabolite in the hamster, mouse, and rabbit was

19 

### confirmed by Billings and McMahon (1978). 2- Hyd roxybiphenyl and 3-hydroxybiphenyl were

20 

### detected in a lower amount in a ratio of 2:1 by hamster and rabbit microsomes, and in a 1:1 ratio

21 

### by mouse microsomes. In contrast, almost all hydroxylation of biphenyl in rat microsomes g ave

22 

### rise to 4 -hydroxybiphe nyl.

23

24 

### 3.3.2. Metabolic Pathways

25 

### 3.3.2.1. Description of Metabolic Scheme a nd Enzymes Involved

26 

### Burke and Bridges (1975) suggested that biphenyl metabolism is mediated by

27 

### cytochrome P-450 (CYP) monooxygenases. Evidence of an arene oxide intermediate, which

28 

### may participate in binding to cellular macromolecules, was repor ted by Billings and McMahon

29 

### (1978). Support for CYP metabolism of biphenyl was provided by Halpaap-Wood et al.

30 

### (1981 a, b), who reported that greater amounts of hydroxybiphenyls were obtained in in vitro

31 

### assays using liver homogenates when rats were treated first with β -naphthoflavone, 3-methyl32 

### cholanthrene or Aroclor 1254, which are known CYP inducers. In C57BL/6Tex mice, CYP

33 

### induction with β -naphthoflavone led to relatively greater amounts of urinary excretion of

34 

### 2-hydroxybiphenyl, compared with uninduced mice, whereas pret reatment with β -naphtho35 

### flavone led to increases in urinary excretion of 2-, 3-, and 4-hydroxybiphenyl in Sprague- Dawley

36 

### rats and was without influence on the pattern of hydroxybiphenyl metabolites in DBA/2Tex mice

37 

### (Halpaap -Wood et al., 1981a).

### 12 DRAFT

- DO NOT CITE OR QUOTE Figure 3- 1 details combined evidence from the Halpaap -Wood et al. (1981 a, b) and 1



### Meyer and Scheline (1976) studi es on the metabolic pathways of biphenyl. While sulph ates and 2



### glucuronides are formed on all three metabolic levels illustrated, only monosulph ates and 3



### monoglucuronides are identified. Monomethyl ethers are formed from dihydroxy and trihydroxy 4



### metabolite s alone. Glucuronides at the dihydroxy and trihydroxy levels are additionally labeled 5



### with a question mark to suggest that, while these metabolites are likely, they have not been 6



### identified. 7

### 13 DRAFT

- DO NOT CITE OR QUOTE OH OH OH

OH OH OH

OH

OH OH

OH

OH

OH

OH OH

OH OH Biphenyl

4-Hydroxybiphenyl 2-Hydroxybiphenyl 3-Hydroxybiphenyl CYP

CYP

CYP ar,ar'-Dihydroxybiphenyl ar,ar-Dihydroxybiphenyl

ar,ar,ar'-Trihydroxybiphenyl

Sulphonotransferase

UGT

COMT Monohydroxysulfate -glucuronide

Dihydroxy-monosulfate -monoglycuronide (?) -monomethyl ether

Trihydroxy-monosulfate -monoglucuronide (?) -monomethyl ether +

+ +

1

2

3 

### ar = aryl group; COMT = catechol -O- methyltransferase; UGT = uridine

4 

### diphosphate glucuronosyl transferase ; q uestion marks denote tentative metabolites

5 

### (see text).

6

7 

### Sources: Halpaap -Wood et al. (1981 a, b); Meyer and Scheline (1976).

8



### Figure 3

- 1. Schematic presentation of the meta bolic pathways of biphenyl. 9

10

### 14 DRAFT

- DO NOT CITE OR QUOTE The metabolic scheme in Figure 3 -1 does not include the possible redox cycling of 1



### 2,5-dihydroxybiphenyl (also known as phenylhydroquinone), which involves CYP

- mediated 2



### cycling between phenylhydroquinone and phenylbenzoquinone leading to the generation of 3



### reactive oxygen species (ROS) (Balakrishnan et al. 2002; Kwok et al., 1999). This pathway is 4



### thought to play a role in the carcinogenic effect of 2-hydroxybiphenyl (also known as 5



### ortho -phenylphenol), a broad spectrum fungicide that , like biphenyl, induces urinary bladder 6



### tumors in chronically exposed male rats (Kwok et al., 1999). Free 2,5-dihydroxybiphenyl and its 7



### glucuronide or sulphate conjugates are readily d etected in the urine of rats exposed to 8



### 2-hydroxybiphenyl, and the formation of 2,5-dihydoxybiphenyl and phenylbenzoquinone is the 9



### principal metabolic pathway for 2-hydroxybiphenyl in the rat , especially at high exposure levels 10



### associated with urinary bladder tumor formation (Kwok et al., 1999; Morimoto et al., 1989; 11



### Nakao et al., 1983; Reitz et al., 1983; Meyer and Scheline, 1976). In contrast, the formation of 12



### 4- hydroxybiphenyl and 4,4’ -dih ydroxybiphenyl is the principal metabolic pathway for biphenyl 13



### in rats and mice , and 2,5-dihydroxybiphenyl was not detected, or only detected at trace levels, in 14



### the urine of rats exposed to 100 mg biphenyl/kg (Meyer and Scheline, 1976 ; see Table 3 -1). In 15



### mice exposed to i.p. doses of [ 14 

### C]- biphenyl (30 mg/kg), radioactivity in 2 -hydroxybiphenyl and 16



### 2,5-dihydroxybiphenyl in the urine accounted for only about 5% of the total radioactivity 17



### detected in urinary metabolites (Halpaap -Wood et al., 1981a). 18

19



### 3.3.3. Regulation of Metabolism

, Sites of Metabolism , and Relationships to Toxic E ffects 20



### 3.3.3.1. Evidence for Induction of Phase I and II E nzymes 21



### No studies of Phase I or II enzyme induction using liver microsomes of human origin 22



### were identified. However, a number of studies have been conducted in rodents to investigate the 23



### induction of Phase I enzymes that catalyze biphenyl hydroxylation. For example, Creaven and 24



### Parke (1966) reported that pretreatment of weanling Wistar rats or ICI mice with phenobarbital 25



### (an inducer of CYP3A4, 2B6, and 2C8 as reported by Parkinson and Ogilvie, 2008) or 26



### 3-methylcholanthrene (an inducer of CYP1A2 as reported by Parkinson and Ogilvie, 2008) 27



### increased NADPH -dependent activities of liver microsomes to produce 2-hydroxybiphenyl and 28



### 4-hydroxybiphenyl from biphenyl to varying degrees depending on the inducer. Haugen (1981) 29



### reported that pretreatment of male CD rats with phenobarbital or 3- methylcholanthrene increased 30



### NADPH -dependent activities of liver microsomes to produce 2-, 3-, and 4-hydroxybiphenyl from 31



### biphenyl, again to varying degrees depending on the inducer. Stuehmeier et al. (1982) reported 32



### that phenobarbital pretreatment of male C57BL/6JHan mice induced liver microsomal activities 33



### to produce 4-hydroxybiphenyl, but not 2-hydroxybiphenyl, from biphenyl, whereas 34



### 3-methylcholanthrene induced a ctivities for both 4 - and 2-hydroxylation of biphenyl. Halpaap35



### Wood et al. (1981a ) reported that pretreatment of male Sprague

- Dawley rats with 36



### β -naphthoflavone (an inducer of CYP1A2 as reported by Parkinson and Ogilvie, 2008; also 37



### known as 5,6-benzoflavone) enhanced the urinary excretion of 2-, 3-, and 4-hydroxybiphenyl, 38

### 15 DRAFT

- DO NOT CITE OR QUOTE 3,4- dihydroxybiphenyl, and 3,4,4’ -trihydroxybiphenyl following i.p. administration of 30 mg 1



### biphenyl/kg body weight. In contrast, pretreatment of male C57BL/6Tex mice with 2



### β -naphthoflavone did not increase the overall urinary excretion of biphenyl metabolites 3



### following i.p. administration of 60 mg biphenyl/kg, but shifted the principal metabolite from 4



### 4-hydroxybiphenyl to 2-hydroxybiphenyl and 2,5- dihydroxybiphenyl (Halpaap -Wood et al., 5



### 1981a ). Wiebkin et al. (1984) reported that β -naphthoflavone pretreatment of male Lewis rats or 6



### male Syrian g olden hamsters induced biphenyl hydroxylation activities in freshly isolated 7



### pancreatic acinar cells or hepatocytes. From these observations and e xamination of patterns of 8



### inhibition of biphenyl hydroxylation activities by CYP inhibitors (e.g., α -naphthoflavone and 9



### 1-benzyl-imidazole) under non-induced and induced conditions (see Haugen, 1981), it is 10



### apparent that multiple CYP enzymes (e.g., CYP1A2 and CYP3A4) are likely involved in 11



### biphenyl hydroxylation. However, no studies were located that used more modern techniques 12



### (such as CYP knockout mice) to identify the principal CYP enzymes involved in the initial 13



### hydroxylation of biphenyl or the formati on of the dihydroxy- or trihydroxybiphenyl metabolites. 14



### Several animal studies were located examining the possible coordinated induction of 15



### Phase I enzymes with Phase II enzymes catalyzing the conjugation of hydroxylated biphenyl 16



### metabolites to sul ph ate orglucuronic acid. Hepatocytes from rats (strain and sex were not noted) 17



### pretreated with the CYP inducers, phenobarbital or 3- methylcholanthrene, produced glucuronide 18



### and sulphate conjugates of 4-hydroxybiphenyl when incubated with biphenyl (Wiebkin et al., 19



### 1978). Glucuronide conjugates were predominant under these “CYP -induced” conditions, 20



### whereas hepatocytes from non -induced control rats produced predominant sulph ate conjugates of 21



### 4-hydroxybiphenyl. These results suggest that induction (or possibly activation) of 22



### glucuronidation enzymes may be coordinated with the induction of CYP enzymes. In contrast, 23



### pretreatment of male Lewis rats with β -naphthoflavone (an inducer of CYP1A2) did not enhance 24



### activities of freshly isolated pancreatic acinar cells to co njugate 4-hydroxybiphenyl with sulph ate 25



### or glucuronic acid, but the influence of this pretreatment on the conjugation capacity of 26



### hepatocytes was not examined in this study (Wiebkin et al., 1984). In another study, uridine 27



### diphosphate glucuronosyl transfe rase (UGT) activities with 1

- naphthol or 3 -hydroxy28



### benzo[a]pyrene as substrates were higher in liver microsomes from male Wistar rats pretreated 29



### with Aroclor 1254 (an inducer of several CYP enzymes) or phenobarbital, respectively, 30



### compared with microsomes from control rats without pretreatment with CYP inducers (Bock et 31



### al., 1980). Although Bock et al. (1980) measured UGT activities in microsomes from several 32



### tissues from non-induced rats with 4-hydroxybiphenyl as a substrate, no comparisons between 33



### induce d and non -induced conditions were made using 4-hydroxybiphenyl as substrate. Paterson 34



### and Fry (1985) reported that hepatocytes or liver slices from male Wistar rats pretreated with 35



### β -naphthoflavone showed decreased rates of glucuronidation of 4-hydroxybip henyl, compared 36



### with hepatocytes or liver slices from rats without β -naphthoflavone pretreatment. Results from 37



### this database provide equivocal evidence that the induction of Phase I enzymes catalyzing the 38

### 16 DRAFT

- DO NOT CITE OR QUOTE hydroxylation of biphenyl may be coordinated with induction of Phase II enzymes catalyzing 1



### glucuronidation of hydroxylated biphenyl metabolites. 2

3



### 3.3.3.2. Demonstrated Tissue Sites of Me tabolism 4



### CYP enzymes catalyzing hydroxylation of biphenyl and other substrates are present in 5



### most, if not all, mammalian tissues, but the highest levels of activities are normally found in liver 6



### (Parkinson and Ogilvie, 2008). In a study of male Sprague- Dawley rat s, cytochrome P450 7



### content was 20 - to 40-fold higher in the microsomes from liver than from lung, although 8



### biphenyl-4-hydrolase activity was only 1.7- fold higher in the microsomes from liver than from 9



### lung (Matsubara et al., 1974). Wiebkin et al. (1984) observed 200- and 1,000- fold higher rates 10



### of biphenyl metabolism in 5,6-benzoflavone- pretreated hepatocytes compared to similarly 11



### treated pancreatic acinar cells from male Lewis rats and Syrian golden hamsters, respectively

. 12



### Activities for enzymes catalyzing the conjugation of hydroxybiphenyls and other 13



### hydroxylated aromatic compounds with glucuronic acid or sulph ate have been detected in a 14



### number of mammalian tissues, and, similar to CYP, the highest levels are found in the liver 15



### (Parkinson and Ogi lvie, 2008). Available data for conjugation activities with hydroxybiphenyls 16



### in various mammalian tissues are consistent with this concept. Sulphotransferase activities with 17



### 2-, 3-, or 4-hydroxybiphenyl as substrates in microsomes from several human tissues showed an 18



### approximate 100- to 500-fold range with the following order: liver > ileum > lung > colon > 19



### kidney > bladder > brain (Pacifici et al., 1991). UGT activities with 4-hydroxybiphenyl as 20



### substrate in microsomes from several male Wistar rat tiss ues showed the following order: liver > 21



### intestine > kidney > testes ≈ lung; activities were below the limit of detection in microsomes 22



### from skin and spleen (Bock et al., 1980). 23

24



### 3.3.3.3. Possible Relationships Between Metabolites and Toxic Effects 25



### I ncrea sed formation of urinary tract crystals and calculi in F344 rats chronic ally exposure 26



### to biphenyl in the diet has been well documented. This phenomenon occurs predominantly in 27



### males and can ultimately lead to non -neoplastic and neoplastic changes in the urinary bladder 28



### (Umeda et al. , 2002). Ohnishi et al. (2001, 2000a, b, 1998) have proposed mechanistic roles for 29



### the potassium salt of the 4 -hydroxybiphenyl sul ph ate conjugate, high urine potassium 30



### concentrations, and relatively high urine pH in producing urinary calculi, which are found in 31



### 86% of male F344 rats and only 16% of female rats exposed to high biphenyl concentrations in 32



### the diet (4

, 500 ppm) for 2 years (Umeda et al., 2002). Gender differences in calculi composition 33



### were also observed, with calcu li in male F344 rats being mainly composed of potassium 4- 34



### hydroxybiphenyl-O- sulph ate and calculi in female rats composed mainly of 4 -hydroxybiphenyl 35



### and potassium sulphate, presumably produced by the hydrolysis of 4-hydroxybiphenyl-O36



### sulphate in the urine (O hnishi et al., 2000a, b). As discussed earlier, these observations are 37



### consistent with the hypothesis that gender differences in urinary conditions (higher urine 38

### 17 DRAFT

- DO NOT CITE OR QUOTE potassium concentrations and pH) may be responsible for the gender differences in urinary 1



### calculi formation and the subsequent development of nonneoplastic and neoplastic lesions in 2



### male, but not female, F344 rats (Umeda et al., 2002; Ohnishi et al., 2001, 2000a, b). 3

4



### 3.4. ELIMINATION 5



### No studies were located on the route or rate of elimination of biphenyl in humans , but 6



### results from studies of orally exposed animals indicate that absorbed biphenyl is rapidly 7



### elimin ated from the body, principally as conjugated hydroxylated metabolites in the urine. 8



### The most quantitative data on the routes and rates of elimination come from a study of 9



### rats following administration of radiolabeled biphenyl (Meyer et al., 1976a). Urine collected for 10



### 24 hour s after the oral administration of 100 mg /kg [ 14 

### C]-labeled biphenyl in soy oil to male 11



### albino rats contained 75 .8% of the administered radioactivity, compared with 5.8% detected in 12



### feces collected in the same period

. Ninety

- six hour s after dose administration, < 1% of the 13



### administered radioactivity remained in tissu es, 84.8% was in collected urine, 7.3% was in fece s, 14



### and 0.1% was in collected expired air (Meyer et al., 1976a). Although chemical identity analysis 15



### of fecal radioactivity was not conducted by Meyer et al. (1976a), results from GC/MS analyses 16



### of bile collected from bile -cannulated rats given single 100 mg /kg doses of unlabeled biphenyl 17



### indicate that biliary excretion of metabolites represents a minor pathway of elimination (Meyer 18



### and Scheline, 1976) . In bile collected for 24 hours, unchanged biphenyl was not detected and 19



### conjugated metabolites a ccounted for 5.2% of the admini stered dose; in contrast, conjugated 20



### metabolites of biphenyl in 24-hour urine accounted for 22.3% of the dose (Meyer and Scheline, 21



### 1976). 22



### Supporting evidence for the importance of urinary elimination of conjugated metabolites 23



### is pro vided by the results of other studies, which analyzed biphenyl and biphenyl metabolites by 24



### GC/MS or GC in urine and feces collected from rabbits (Meyer, 1977), guinea pigs ( Meyer, 25



### 1977), and pigs (Meyer et al., 1976b) following oral administration of 100 mg/kg doses of 26



### unlabeled biphenyl. In 24 -hour urine samples, unchanged biphenyl was not detected, and total 27



### metabol ites accounted for averages of 25.4% of the administered dose in rabbits, 31.3% in 28



### guinea pigs, 17.5% in female pigs, and 26.4% in male pigs. As in rats, biliary excretion 29



### represents a minor elimination pathway in guinea pigs and rabbits ; metabolites detected in bile 30



### `collected for 24 hours from bile

- cannulated guinea pigs accounted for 3.3% of the administered 31



### dose, but for only 0.3% of the dose in bile collected for 7 hours from a rabbit given 100 mg /kg 32



### biphenyl (Meyer, 1977). Neither unchanged biphenyl nor hydroxylated biphenyl metabolites 33



### were detected in bile collected from a bile -cannulated pig for 24 hour s after administration of 34



### 100 mg/kg biphenyl (Meyer et al., 1976b). 35



### No studies were located examining quantitative aspects of elimination in animals 36



### following inhalation or dermal exposure to biphenyl. 37

38

### 18 DRAFT

- DO NOT CITE OR QUOTE 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PB P K) MODELS 1



### No studies were located on the development of PB PK models for biphenyl in animals or 2



### humans. 3

41 

### 4. HAZARD IDENTIFICATION

2

3

4 

### 4.1. STUDIES IN HUMANS

5 

### Limited human data include assessments of workers exposed to biphenyl during

6 

### production of biphenyl- impregnated fruit wrapping paper at one mill in Finland (Seppäläinen and

7 

### Häkkinen, 1975; Häkkinen et al., 1973, 1971) and another mill in Sweden (Wastensson et al.,

8 

### 2006) and a single case report of reversible hepatotoxic effects attributed to biphenyl exposure

9 

### (Carella an d Bettolo, 1994).

10

11 

### Seppäläinen and Häkkinen, 1975; Häkkinen et al., 1973, 1971

12 

### Häkkinen and colleagues assessed the health of paper mill workers exposed to biphenyl

13 

### during the production of biphenyl-impregnated paper used to wrap citrus fruits. In 1959,

14 

### workers complained about a strong odor and irritation to the throat and eyes. Air measurements

15 

### made at various locations within the facility in June of 1959 resulted in estimated average

16 

### biphenyl concentrations of 4.4–128 mg /m 3 

### (Table 4 -1). In 1969, a 32- year

- old worker at the

17 

### facility, who had worked for 11 year s in the oil room where biphenyl levels were particularly

18 

### high, became ill. Despite aggressive medical intervention, the patient grew worse and died. Key

19 

### features at autopsy included necrosis of most liver cells, severe, but unspecified changes in the

19 DRAFT

- DO NOT CITE OR QUOTE kidneys, degeneration of the heart muscles, hyperactive bone marrow, and edematous changes in 20



### the brain (Häkkinen et al., 1973, 1971). Subsequent measurements of biphenyl in the workplace 21



### air (Ja nuary 1970) resulted in estimated average concentrations ranging from 0.6 to 123 mg /m 3 22



### (Table 4 -1). Measurements taken in both 1959 and 1971 indicated that biphenyl air 23



### concentrations at multiple work areas greatly exceeded the current ACGIH (2008) threshold limit 24



### value ( TLV ) of 0.2 ppm (1.3 mg/m 3 

### ). In the location where biphenyl was mixed with paraffin oil 25



### (the oil room), biphenyl occurred both as a vapor and as a dust, suggesting the possibility of both 26



### dermal and inhalation exposures. 27

28

### 20 DRAFT

- DO NOT CITE OR QUOTE Table 4 -1. B iph enyl concentrations in the air of a Finnish paper mill
producing b iphenyl

- impregnated fruit wrapping paper

Sampling center locations Average concentrations 3 ) (mg/m

June 1959 January 1970

Paper mill hall

In front of paper reel 17.9 7.2

Behind impregnating roller 128.0 64.0

Near paper machine 7.2 1.5

Near rolling machine 4.4 0.6

Oil

- room

Near measuring container 19.5 3.5

Above measuring container (lid open) No data 123.0

Near mixing container No data 15.5

During addition of b iphenyl to mixing container No data 74.5

Source: Häkkinen et al. (1973).

1



### Thirty -one male workers at the Finnish facility were engaged in the biphenyl2



### impregnation process; two other workers (one male stock keeper and one female paper cutter ) 3



### were thought to have been exposed to biphenyl and were therefore included in the study. 4



### Common complaints among these workers included fatigue, headache, gastrointestinal 5



### discomfort, numbness and aching of the limbs, and general fatigue; laboratory tests revealed 6



### elevated se rum aspartate aminotran sferase (AST) and alanine aminotransferase (ALT) (which 7



### can indicate inflammation or damage to liver cells) in 10 of the 33 workers (Häkkinen et al., 8



### 1973). Eight of the 33 worker s were admitted to the hospital for further examinati on, including 9



### liver biopsy. The majority of the 33 workers were subjected to neurophysiological examinations, 10



### including electroencephalography (EEG) and electroneuromyography (ENMG, consisting of 11



### nerve conduction velocity and electromyographic [EMG] tests). Seppäläinen and Häkkinen 12



### (1975) published the most comprehensive results of the neurophysiological examinations. In all, 13



### 24 subjects (including the 8 hospitalized workers) underwent neurophysiological examinations. 14



### Exposure to biphenyl was terminated immediately following the initial neurophysiological 15



### examinations, and 11 and 7 of these subjects were retested 1 and 2 year s later, respectively. 16



### EEG r esults

. At initial examination, 10 of the 24 workers had abnormal EEGs, which 17



### included diffuse slow wa ve abnormalities (6 cases), lateral spike and slow wave discharges 18



### (2 case s), posterior slowing only (1 case ), and mild slow wave abnormality in the right temporal 19



### area (1 case ). Six subjects exhibited unusual distribution of alpha rhythm with alpha activ ity also 20



### prominent in the frontal areas. Four of the subjects exhibited no EEG abnormalities. In general, 21



### the EEG results observed at initial examination were qualitatively similar in the 11 subjects 22



### reexamined 1 year later. Exceptions included addition al diffuse slow wave abnormalities in the 23



### two subjects initially exhibiting only spike and wave discharges and the disappearance of the one 24

### 21 DRAFT

- DO NOT CITE OR QUOTE case of mild temporal local abnormality. There was no discernable improvement in the EEGs of 1



### the seven subjects reexamined after 2 years. 2



### ENMG r esults

. As shown in Table 4-2, the 24 biphenyl- exposed worker s exhibited no 3



### significant differences in mean maximal motor conduction velocity (MCV) relative to those of a 4



### control group consisting of 60 healthy Finnish males, but significantly ( p < 0.001) slower mean 5



### conduction velocity of the slowest motor fibers (CVSF) of the ulnar nerves. Results at the 1- year 6



### follow up of 11 of the biphenyl-exposed workers revealed no significant changes in initial 7



### conduction velocity meas ures, but at the 2-year reexamination of 7 of the 11 subjects, the MCVs 8



### of the median and deep peroneal nerves were significantly slower ( p < 0.02 and p < 0.01, 9



### respectively) compared to the initial measurements. Abnormal EMGs among the biphenyl10



### exposed workers included diminished numbers of motor units on maximal muscle contraction 11



### (10 subjects) and fibrillations in some muscles (7 subjects). Workers exhibiting abnormal EMGs 12



### typically displayed slowing of some nerve conduction velocities as well. Of tho se 11 subjects 13



### undergoing repeat ENMG examination after 1 year , 5 subjects showed an increased level of 14



### ENMG abnormality, while 4 remained unchanged and 2 had diminished abnormalities. At the 15



### end of 2 year s, three of seven subjects displayed diminished EN MG abnormalities, three of seven 16



### were unchanged, and one of seven had the abnormality increased. 17

18



### Table 4

- 2. Nerve conduction velocities of 24 persons exposed to biphenyl:
comparison with 60 unexposed males

Nerve Biphenyl group
( mean ± SD) Control group
(mean ± SD) t

- test

Median

MCV 57.7 ± 6.3 58.0 ± 3.8 Not significant

Ulnar

MCV 56.3 ± 4.6 56.6 ± 4.0 Not significant

CVSF 41.4 ± 5.2 45.5 ± 3.2 p < 0.001

Deep peroneal

MCV 50.2 ± 5.4 50.3 ± 3.5 Not significant

CVSF 37.7 ± 3.9 38.2 ± 5.6 Not significant

Posterior tibial

MCV 43.4 ± 3.9 42.4 ± 4.7 Not significant

SD = standard deviation

Source: Seppäläinen and Häkkinen (1975).

19



### Seppäläinen and Häkkinen (1975) noted that subjects often exhibited signs of dysfunction 20



### in both the peripheral nervous system, as evidenced by abnormal ENMGs, and the central 21



### nervous system, as evidenced by abnormal EEGs and abnormal distribution of alpha activity. 22



### Only five subjects ( four men and the only woman in the biphenyl-exposed group) were found to 23

### 22 DRAFT

- DO NOT CITE OR QUOTE have completely normal neurophysiological records. The authors interpreted their data to 1



### indicate that biphenyl can attack the nervous system at different levels, the sites of greatest 2



### vulnerability being the brain and peripheral nerves. Compound- related anomalies in nerve 3



### conduction, EEG, and ENMG signals, while small, were consistent with the persistence of 4



### incapacity and the incidence of subjective symptoms. 5

6



### Carella and Bettolo, 1994 7



### Carella and Bettolo (1994) published a case report of a 46- year -old female who had 8



### suffered from periodic asthenia while working over a 25- year period at a fruit -packing facility 9



### where biphenyl-impregnated paper was used. The patient presented with hepatomegaly, 10



### neutrophilic leukocytosis, and clinical chemist ry findings indicative of hepatic perturbation. For 11



### example, the activities of liver

- specific enzymes in serum were 62 mU/mL for AST, 90 mU/mL 12



### for ALT, 320 mU/mL for alkaline phosphatase (AP), and 970 IU/L for gamma glutamyl 13



### transferase . Examination of a liver biopsy taken from the subject showed a polymorphic 14



### inflammatory infiltrate with eosinophils in the portal and lobular regions. These findings are 15



### indicative of chronic hepatitis. 16



### Following cessation of work in citrus packing, the patient’s asthenia gradually 17



### disappeared and the serum enzyme abnormalities returned to normal. This permitted the 18



### speculation that, in the absence of any other obvious causes of the liver abnormality, 19



### occupational exposure to biphenyl may have been the principal etiologic al factor. It is possible 20



### that, for this patient, exposure was via all of the major exposure pathways, inhalation, oral, and 21



### dermal, with the latter route predominating. 22

23



### Wastensson et al. , 2006 24



### At a facility manufacturing biphenyl-impregnated paper in Sweden, a cluster of five cases 25



### of Parkinson’s disease (PD) among the employees was investigated. Since, according to the 26



### national average, only 0.9 cases would be expected from the 255 employees at the facility 27



### (relative risk [RR] 5.6 [95% confidence interval 1.9– 13]), it was suspected that the elevated PD 28



### at the facility may have been related to biphenyl exposure. Four of the subjects worked in the 29



### vicinity of a rewinder/dryer, while the fifth attended to another rewinder. Although no ambient 30



### biphenyl levels were available for the subjects’ work space, it was thought likely that the level of 31



### biphenyl in air would be greater than the existing TLV of 1.3 mg/m 3 

### (0.2 ppm) based on 32



### measurements at a Finnish paper mill with similar production practice s (Häkki nen et al., 1973). 33



### Two subjects may have been exposed to higher levels of biphenyl than the others when they 34



### created the paraffin oil/biphenyl mixture. 35



### In addition to comparing existing PD cases to national trends, Wastensson et al. (2006) 36



### examined the medical records of 222 former employees who had died. Nine cases of PD were 37



### found among the decedents, compared with 4.3 cases of PD expected from data on the lifetime 38

### 23 DRAFT

- DO NOT CITE OR QUOTE risk of developing PD in the general population. This comparison yielded an RR of 2.1, w ith a 1



### 95% confidence interval of 0.96–4.0. 2



### Clinical features and exposure data for the five living subjects, all of whom were 3



### diagnosed with PD by a neurologist at a local hospital, are summarized in Table 4 -3. With one 4



### exception, the patients were in comparatively good health on initial diagnosis. The exception 5



### was a 53

- year -old male who had diabetes mellitus and withdrew from the study before his 6



### neurological condition could be confirmed. Assuming that the diagnoses of PD were valid, the 7



### central issue is whether these data indicate a causal relationship between PD and exposure to 8



### biphenyl. Wastensson et al. (2006) discussed this issue in the context of other studies that have 9



### pointed to a possible cause -and- effect relationship between pesticide exposur e and PD, but were 10



### unable to draw any firm conclusions fr om their limited data

. 11

12



### Table 4 -3. Exposure data and clinical features for five Parkinson’s Disease
patients with occupational exposure to biphenyl

Case

1 2 3 4 5

Exposure data

Age 63 63 58 54 63

Workplace PM3 PM3 PM4 PM3 PM3

Years of e xposure a 12 4 9 4 2

Age at onset of exposure 19 26 17 18 21

Age at onset of symptoms 52 55 44 51 55

Clinical features

Resting tremor + + + + +

Cogwheel rigidity + + +

– +

Brady kinesia + + + +

– Positive response to levodopa b + + + + +

a Exposure to biphenyl about one

- third of each year

. b All five patients improved with levodopa, which is a medication for Parkinson’s Disease.

PM = paper mill

Source: Wastensson et al. (2006).

13

14 

### 4.2. SUBCHRONIC AND CHRONIC STUDIES AND CANCER BIOASSAYS IN

15 

### ANIMALS

— ORAL AND INHALATION

16 

### Overview

. Available oral data for biphenyl include two well-designed chronic toxicity

17 

### and carcinogenicity studies, one in F344 rats (Umeda et al., 2002) and one in BDF 1 

### mice (Umeda

18 

### e t al., 2005). Increased incidence of urinary bladder transitional cell papillomas and carcinomas,

19 

### associated with the formation of urinary bladder calculi, occurred in male, but not female,

F344 rats at the highest tested dietary concentration, 4 ,500 ppm, but were not found at lower 20

### 24 DRAFT

- DO NOT CITE OR QUOTE exposure levels of 1 ,500 or 500 ppm. Non- neoplastic kidney lesions (simple transitional cell 1



### hyperplasia in the renal pelvis and hemosiderin deposits) were found in female F344 rats at 2



### biphenyl dietary concentrations ≥ 1, 500 ppm (Umeda et al., 2002). Several other rat studies 3



### provide supporting evidence that the kidney and other urinary tract regions are critical targets for 4



### biphenyl in rats (Shiraiwa et al., 1989; Ambrose et al., 1960; Pecchiai and Saffiotti, 1957; Dow 5



### Chemical Co., 1953). In BDF 1 

### mice, increased incidence of liver tumors (hepatocellular 6



### adenomas and carcinomas) and non-neoplastic effects on the kidney (mineralization) and liver 7



### (increased activities of plasma ALT and AST) were found in females exposed to biphenyl dietary 8



### concentrations of 2,000 or 6,000 ppm (Umeda et al., 2005). In contrast, no carcinogenic 9



### responses or noncancer adverse effects were found in female ddY mice exposed to 5

, 000 ppm 10



### biphenyl in the diet for 2 years (Imai et al., 1983) or in B6C3F 1 

### and B6AKF 1 

### mice exposed to 11



### 517 ppm biphenyl in the diet for 18 months (Innes et al., 1969; NCI , 1968). 12



### No chronic inhalation toxicity studies in animals are available. In subchronic inhalation 13



### toxicity studies, respiratory tract irritation and in creased mortality following exposure to dusts of 14



### biphenyl (7 hours/day, 5 days/week for up to about 90 days) w ere reported in mice exposed to 15



### 5 mg/m 3 

### and in rats exposed to 300 mg/m 3 

### , but not in rabbits exposed to 300 mg/m 3 

### (Deichmann 16



### et al., 1947; Monsanto, 1946). Congestion or edema of the lung, kidney, and liver, accompanied 17



### by hyperplasia with inflammation of the trachea, was found in CD-1 mice exposed to biphenyl 18



### vapors at 25 or 50 ppm (158 or 315 mg/m 3 

### ) for 13 weeks (Sun Co mp an y Inc., 1977b). 19



### Detaile d study descriptions for all available subchronic and chronic toxicity and 20



### carcinogenicity studies follow. 21

22



### 4.2.1. Oral Exposure 23



### 4.2.1.1. Subchronic Toxicity 24



### Dow Chemical Co., 1953 25



### Twenty -one-day-old female Long-Evans rats (8/group) were exposed to 0, 0.01, 0.03, or 26



### 0.1% biphenyl in the diet for 90 days. Body weights were monitored 3 times/week, and the 27



### weights of the liver, kidneys, adrenals, and spleen were recorded at necropsy. Sections of heart, 28



### liver, kidney, spleen, adrenals, pancreas, ovary, uterus, stomach, small and large intestine, 29



### voluntary muscle, lung, thyroid, and pitui tary from each rat were preserved in formalin. 30



### Hematoxylin and eosin stained sections of the preserved sections from two rats of each group 31



### were examined pathologically. 32



### Based on U.S. EPA (1988) subchronic reference values for body weight and food 33



### consumption in female Long-Evans rats, doses of biphenyl estimated for the dietary levels of 34



### 0.01, 0.03, and 0.1% are estimated to have been 10, 30, and 100 mg /kg-day, respectively. There 35



### were no significant treatment -related effects on body weight, food consumption, or organ 36



### weights. Results of histopathologic examinations were unremarkable. Biphenyl-exposed groups 37



### exhibited lower average plasma blood urea nitrogen ( BUN ) levels than controls (28.2, 25.7, and 38

### 25 DRAFT

- DO NOT CITE OR QUOTE 26.3 mg percent for low- , mid -, and high-dose groups, respectively, compared to 35.3 mg percent 1



### for controls), although the statistical significance of these apparent treatment

- related differences 2



### was not reported and the biological significance is uncertain. 3

4



### Umeda et al., 2004 5



### Six

- week

- old BDF 1 

### mice (10/sex/group) were exposed to biphenyl at dietary 6



### concentrations of 0, 500, 2,000, 4,000, 8,000, 10,000, or 16,000 ppm for 13 weeks. To overcome 7



### possible problems with taste aversion, mice assigned to the 8,000 and 10,000 ppm groups were 8



### fed 4,000 ppm dietary biphenyl for the first week and 8,000 or 10,000 ppm for the remaining 9



### 12 weeks. Mice designated to receive 16,000 ppm were fed 4,000 ppm dietary biphenyl for the 10



### first week, 8,000 ppm for the second week, and 16,000 ppm for the remaining 11 we eks. 11



### Animals were checked daily for clinical signs; body weight and food consumption were recorded 12



### weekly; organ weights were noted at term; and liver sections were processed for light 13



### microscopic examination. Electron microscopy was carried out on liver tissue from one control 14



### and one 16,000 ppm female. 15



### Based on U.S. EPA (1988) subchronic default reference values for body weight and food 16



### consumption (average values for combined sexes), doses of biphenyl for the dietary 17



### concentrations of 500, 2,000, 4,000, 8,000, 10,000, and 16,000 ppm are estimated to have been 18



### 93, 374, 747, 1,495, 1,868, and 2,989 mg/kg-day, respectively. A single 16,000 ppm female 19



### mouse died during the study; all other mice survived until terminal sacrifice. Final body weights 20



### of mice of both sexes in the 8,000, 10,000, and 16,000 ppm groups were significantly lower than 21



### gender- matched controls (for males: 83.3, 84.9, and 75.1% of controls; for females: 93.7, 91.6, 22



### and 85.8% of controls, respectively). Umeda et al. (2004) noted that absolute liver weights were 23



### significantly higher in 8,000 and 16,000 ppm female mice, but did not include the extent of these 24



### increases in the study report. Light microscopic examination of liver specimens from all 25



### 16,000 ppm female mice revealed enlarge d centrilobular hepatocytes, the cytoplasm of which 26



### was filled with numerous eosinophilic fine granules. Upon electron microscopic examination, 27



### these eosinophilic granules were identified as peroxisomes, indicative of a peroxisome 28



### proliferative effect in the liver of the 16,000 ppm female mice. Evidence of histopathologic liver 29



### lesions was not found in females of the 8,000 or 10,000 ppm groups. There were no signs of 30



### treatment

- related increased liver weight or histopathologic evidence of clearly enlarged 31



### hepatocytes in any of the biphenyl- treated groups of male mice. 32

33



### 4.2.1.2. Chronic Toxicity and Carcinogenicity 34



### 4.2.1.2.1. Chronic rat s tudies 35



### Umeda et al., 2002 36



### In a chronic toxicity and carcinogeni ci ty study of F344 rats (50/sex/group), biphenyl was 37



### administered in the diet for 2 year s at concentrations of 0, 500, 1,500, or 4,500 ppm . All animals 38

### 26 DRAFT

- DO NOT CITE OR QUOTE were examined daily for clinical signs; body weights and food intake were determined once a 1



### week for the first 14 weeks and every 4 weeks thereafter. Urinalysis was performed on all 2



### surviving rats during week 105. Upon necropsy, weights of all major organs were recorded; all 3



### major organs and tissues were subjected to histopathologic examination. 4



### The study report included a plot of mean body weights during t he 2- year study, but did 5



### not include food consumption data. Estimated doses, therefore, were calculated using time6



### weighted average (TWA) body weights from the graphically-depicted data (Figure 1 of Umeda 7



### et al., 2002) and U.S. EPA (1988) chronic reference values for food consumption in F344 rats. 8



### The resulting estimated doses for the 500, 1,500, and 4,500 ppm exposure groups were 36.4, 110, 9



### and 378 mg/kg-day, respectively, for males and 42.7, 128, and 438 mg/kg-day, respectively, for 10



### females . The study authors reported significantly lower mean body weights among 4,500 ppm 11



### rats of both sexes compared to their respective controls. Mean body weights of 4,500 ppm male 12



### and female rats were lower than those of controls throughout most of the study period and were 13



### approximately 20% lower than respective controls at terminal sacrifice . There was no significant 14



### effect on mean body weights of 500 or 1,500 ppm males or females. Survival of low - and mid15



### dose male and female rats was not significantly different fro m controls. The study authors 16



### reported that 3/50 of the 4,500 ppm female rats died after 13 –26 week s of biphenyl exposure and 17



### attributed the deaths to marked mineralization of the kidneys and heart. However, they also 18



### indicated that survival of this grou p was not adversely affected thereafter. Significantly 19



### decreased survival was noted only for the group of 4,500 ppm male rats, 19/50 of which died 20



### prior to terminal sacrifice. The first death occurred around treatment week 36; this rat exhibited 21



### urinary bladder calculi. Survival data for the other groups were not provided. Evidence of 22



### hematuria was first noted in 4,500 ppm male rats around week 40 and was observed in a total of 23



### 32/50 of the 4,500 ppm males during the remainder of the treatment period; 14 of these rats 24



### appeared anemic. Hematuria and bladder tumors were primarily considered as causes of death 25



### among the 4,500 ppm males (n = 19) that died prior to terminal sacrifice. Urinalysis performe d 26



### during the final treatment week revealed significantly increased urinary pH in the 31 remaining 27



### 4,500 ppm male rats (pH of 7.97 vs. 7.66 for controls; p < 0.05); occult blood was noted in the 28



### urine of 23 of these males. Urine samples in 10/37 surviving 4,500 ppm females tested positive 29



### for occult blood. Significant increases in relative kidney weights of 4,500 ppm males and 30



### females and absolute kidney weights of 4,500 ppm males were reported

, but actual data were not 31



### presented. 32



### Gross pathologic examinat ions at premature death or terminal sacrifice revealed the 33



### presence of calculi in the bladder of 43/50 of the 4,500 ppm males and 8/50 of the 4,500 ppm 34



### females (Table 4 -4); these lesions were not seen in 500 or 1,500 ppm male or female rats. The 35



### bladder c alculi in the male rats were white, yellow, brown, gray, and black in color, ranged from 36



### 0.3 to 1.0 cm in size, and exhibited triangular, pyramidal, cuboidal, and spherical shapes. The 37



### bladder calculi in the female rats were white and yellow in color, of uniform spheroidal shape, 38

### 27 DRAFT

- DO NOT CITE OR QUOTE and similar in size to those of the male rats. Forty -one of the 4,500 ppm male rats exhibited 1



### polyp- like or papillary nodules protruding into the lumen from the bladder wall; bladder calculi 2



### were noted in 38 of these males. Four of the eight calculi -bearing 4,500 ppm female rats also 3



### exhibited thickening of the bladder wall. It was noted that 30/32 of the 4,500 ppm male rats with 4



### hematuria also exhibited kidney or urinary bladder calculi. 5

6



### Table 4 -4 . Incidences of urinary blad der lesions in male and female F344
rats exposed to biphenyl in the diet for 2 years

Males (n = 50) Females (n = 50)

Dietary concentration (ppm) 0 500 1,500 4,500 0 500 1,500 4,500

Calculated dose (mg/kg

- d) 0 36.4 110 378 0 42.7 128 438

Lesion

Transitional cell

Simple hyperplasia a 0 0 0 12 b 0 0 1 1

Nodular hyperplasia a 0 0 0 40 b 1 0 0 5 c

Papillary hyperplasia a 0 0 0 17 b 0 0 0 4

Combined 0 0 0 45 1 0 1 10 b

Papilloma 0 0 0 10 b 0 0 0 0

Carcinoma 0 0 0 24 b 0 0 0 0

Papilloma or c arcinoma
(combined) 0 0 0 31 b 0 0 0 0

Squamous cell

Metaplasia a 0 0 0 19 b 0 0 0 4

Hyperplasia a 0 0 0 13 b 0 0 0 1

Papilloma or carcinoma
(combined) 0 0 0 1 0 0 0 0

Inflammatory polyp a 0 0 0 10 b 0 0 0 0

Calculi 0 0 0 43 b 0 0 0 8 b

a The number is the sum of animals with severity grades of slight, moderate, marked, or severe.

b Significantly different from control group ( p < 0.01) according to Fisher’s exact test.

c Significantly different from control group ( p < 0.05) according to Fishe r’s exact test.

Source: Umeda et al. (2002).

7



### Histopathologic examinations at death or terminal sacrifice revealed no indications of 8



### biphenyl-induced tumors or tumor- related lesions in organs or tissues other than those associated 9



### with the urinary tract. As shown in Table 4-4, neoplastic and nonneoplastic lesions of the 10



### urinary bladder were essentially limited to the 4,500 ppm rats and predominantly the males. 11



### Only 4,500 ppm male rats exhibited papilloma (10/50) or carcinoma (24/50) of transitiona l cell 12



### epithelium

, three of which exhibited both papilloma and carcinoma. Most of the transitional cell 13



### carcinomas (20/24) projected into the lumen, and the tumor cells invaded the entire body wall. 14



### Bladder calculi were found in all 24 males with transit ional cell carcinoma and 8/10 of the males 15

### 28 DRAFT

- DO NOT CITE OR QUOTE with transitional cell papilloma. Among noncancerous responses in the bladder, simple, nodular, 1



### and papillary hyperplasias were evident in 4,500 ppm animals. These hyperplasias developed in 2



### the focal area of the bladder epithelium. Simple hyperplasia occurred less frequently than 3



### nodular and papillary hyperplasias; furthermore, simple hyperplasia was almost always 4



### accompanied by either nodular or papillary hyperplasia in the 4,500 ppm males. Ten of the 5



### 4,500 ppm males had polyps in the bladder epithelium, which were composed of spindle fibers 6



### proliferated around transitional epithelial cells accompanied by inflammatory infiltration of 7



### submucosal bladder epithelium. Squamous metaplasia was noted on the surface of the polyps, 8



### which were found at different loci than the bladder tumors. 9



### Table 4 -5 summarizes the incidences of lesions of the ureter and kidney in the male and 10



### female rats. The incidence of simple transitional cell hyperplasia in the ureter was greater in the 11



### 4,500 ppm males than the 4,500 ppm fem ales. Other responses, such as mineralization of the 12



### corticomedullary junction, were increased over controls to a greater extent in males compared to 13



### females. In the renal pelvis, simple and nodular hyperplasia was frequently observed in 14



### 4,500 ppm males and 500 and 1,500 ppm females. Responses such as papillary necrosis, infarct, 15



### and hemosiderin deposition occurred predominantly in exposed females. 16

17

### 29 DRAFT

- DO NOT CITE OR QUOTE Table 4 -5 . Incidences of ureter and kidney lesions in male and female

F344 rats exposed to biphenyl in t he diet for 2 years

Males (n = 50) Females (n = 50)

Dietary concentration (ppm) 0 500 1,500 4,500 0 500 1,500 4,500

Calculated d ose (mg/kg

- d) 0 36.4 110 378 0 42.7 128 438

Response

Ureter

Transitional cell hyperplasia

Simple hyperplasia 1 0 0 8 a 0 0 0 2

Nodular hyperplasia 0 0 0 1 0 0 0 0

Dilatation 0 0 0 14 a 0 0 0 6 b

Kidney

Renal pelvis

Transitional cell hyperplasia

Simple hyperplasia 6 8 5 19 c 3 5 12 c 25 a

Nodular hyperplasia 0 1 1 21 a 0 0 1 12 a

Squamous metaplasia 0 0 0 2 0 0 0 0

Mineralization 9 6 10 18 b 12 12 18 27 a

Desquamation 1 0 0 11 a 0 0 0 2

Calculi 0 0 0 13 a 0 0 0 3

Other

Mineralization of
corticomedullary junction 0 0 0 10 a 21 2 26 18

Mineralization of papilla 9 9 14 23 c 2 6 3 12 a

Papillary necrosis 0 0 0 7 d 0 0 0 23 a

Infarct 0 0 0 0 1 0 0 8 c

H emosiderin deposits 0 0 0 0 4 8 22 a 25 a

Chronic nephropathy 45 45 43 34 33 35 30 26

a Significantly different from control group ( p < 0.0 1 ) according to χ 2 test.

b Significantly different from control group ( p < 0.05) according to Fisher’s exact test.

c Significantly different from control group ( p < 0.0 5 ) according to χ 2 test.

d Significantly different from control group ( p < 0.01) according to Fisher’s exact test.

Source: Umeda et al. (2002).

1



### In summary, the chronic toxicity and carcinogenicity study of male and female F344 rats 2



### administered biphenyl in the diet for 2 year s (Umeda et al., 2002) provides evidence for 3



### biphenyl-induced bladder tumors in males, but not females, based on the development of 4



### transitional cell papillomas and carcinomas in the 4,500 ppm (438 mg/kg -day) males (Table 4 -4). 5



### This study identified a no -observed- adverse

- effect level ( NOAEL ) of 500 ppm (42.7 mg/kg-day) 6



### and a lowest -observed-adve rse

- effect level ( LOAEL ) of 1,500 ppm (128 mg/kg-day) for 7



### nonneoplastic kidney lesions (simple transitional cell hyperplasia in the renal pelvis and 8



### hemosiderin deposits) in female F344 rats exposed to biphenyl in the diet for 2 years. 9

### 30 DRAFT

- DO NOT CITE OR QUOTE 1

2 

### Shiraiwa et al., 1989

The chronic toxicity of b iphenyl was assessed in Wistar rats (50/sex/group) administered 3



### the chemical at 0, 0.25, or 0.5% (0, 2,500, or 5,000 ppm) in the diet for up to 75 weeks. The rats 4



### were observed daily for clinical signs. Body weight and food consumption were measured 5



### weekly. At death or scheduled sacrifice, gross pathologic examinations were performed and all 6



### organs were removed and preserved. Other than body weight and compound consumption data, 7



### the published results of this study were limited to kidney weight data and urolithiasis findings. 8



### Based on reported values for mean daily biphenyl intake (mg biphenyl/rat) and mean initial and 9



### final body weights for each study group, doses of biphenyl at the 0.25 and 0.5% dietary levels 10



### are estimate d to have been 165 and 353 mg/kg-day for males, respectively, and 178 and 11



### 370 mg/kg-day for females, respectively. Mean final body weights in both 2,500 and 5,000 ppm 12



### groups of biphenyl- exposed male and female rats were significantly lower (approximately 15 and 13



### 25% lower; p < 0.01) than their respective controls. Absolute and relative kidney weights of 14



### control and biphenyl

- exposed rats were similar , with the exception of significantly increased 15



### ( p < 0.001) mean relative kidney weight in 2,500 ppm female rats. The study authors reported 16



### the occurrence of hematuria (in both the 2,500 and 5,000 ppm groups) as early as week 16 and 17



### stated that it was more recognizable a t 60 weeks. Kidney stone formation was reported in 18



### 6/46 and 1/43 of the 2,500 ppm males and females, respectively, and in 19/47 and 20/39 of the 19



### 5,000 ppm males and females, respectively. Detection of stones in other regions of the urinary 20



### tract was esse ntially limited to the 5 ,000 ppm groups and included the ureter (2/47 males and 21



### 2/39 females) and urinary bladder (13/47 males and 6/39 females). Kidney stones were hard, 22



### black , and located from the pelvic area to the medullary region. Stones in the uret er were hard, 23



### black , and composed of protein. Stones in the urinary bladder were hard, yellowish- white, round 24



### to oval in shape, and composed of ammonium magnesium phosphate. Histologically, kidneys 25



### with stones exhibited obstructive pyelonephritis accompanied by hemorrhage, lymphocytic 26



### infiltration, tubular atrophy, cystic changes of tubules, and fibrosis. Urinary bladders with stones 27



### exhibited simple or diffuse hyperplasia and papillomatosis of the mucosa; however, neoplastic 28



### lesions were not seen. No c ontrol rats (44 males and 43 females) showed stones in the kidney, 29



### ureter, or urinary bladder. The lowest exposure level in this study, 2,500 ppm i n the diet for 30



### 75 weeks, was a LOAEL for formation of kidney stones associated with pyelonephritis in Wistar 31



### rats (dose levels of 165 and 178 mg/kg- day for males and females, respectively) . Urinary 32



### bladder stones associated with simple or diffuse hyperplasia and papillomatosis of the mucosa of 33



### the urinary bladder was observed at the highest exposure level, 5 ,000 ppm biphenyl in the diet 34



### (dose levels of 3 53 and 370 mg/kg- day for males and females, respectively)

. 35



### Shiraiwa et al. (1989) also reported the results of an initiation-promotion study in male 36



### Wistar rats (25/group) that included three groups administered a basal diet for 2 weeks followed 37



### by diets containing 0, 0.125, or 0.5% b iphenyl (0, 1 ,250, or 5,000 ppm) for 34 weeks. Three 38

### 31 DRAFT

- DO NOT CITE OR QUOTE other groups received diets containing 0.1% N-ethyl-N-hydroxyethylnitrosamine (EHEN, an 1



### initiator of kidney tumors in rats) for 2 weeks followed by diets containing 0, 0.125, or 0.5% 2



### b iphenyl (0, 1 ,250, or 5,000 ppm) for 34 weeks. Initial and final body weights were recorded. 3



### At terminal sacrifice, gross pathologic examinations were performed. The study repor t included 4



### information regarding kidney weights, but did not indicate whether weights of other organs were 5



### measured. Kidney and urinary bladder were fixed; kidneys were sectioned transversely (10

– 6



### 12 serial slices) and urinary bladders were cut into 4 –6 se rial slices. The authors used a 7



### computer-linked image analyzer to determine the incidence of kidney lesions and dysplastic foci. 8



### The presence of stones in the kidney and urinary bladder was assessed qualitatively using an 9



### infrared spectrophotometer. Bas ed on reported values for mean daily biphenyl intake (mg 10



### biphenyl/rat) and average body weight (mean initial body weight + one- half the difference 11



### between mean initial and mean final body weight) for each study group, doses of biphenyl at the 12



### 0.125 and 0.5% dietary levels are estimated to have been 59.28 and 248.3 mg/kg-day, 13



### respectively, for rats on basal diet alone for the first 2 weeks and 62.0 and 248.2 mg/kg-day, 14



### respectively, for rats receiving EHEN in the diet for the first 2 weeks. The mean final b ody 15



### weight of the rats receiving basal diet followed by diet containing 0.5% biphenyl was 16



### significantly lower ( p < 0.001) than that of controls (0.389 ± 22 vs. 0.432 ± 30 kg). It was stated 17



### that relative kidney weights were increased this group of biphenyl-exposed rats compared to the 18



### basal diet control group, but the actual data were not presented. Stones were detected only in the 19



### rats receiving 0.5% biphenyl in the diet; incidences were 4/25 (kidney), 1/25 (ureter), and 20



### 3/25 (urinary bladder) in rats th at had received that basal diet for the first 2 weeks. Similar 21



### results regarding final body weight and the detection of stones in the urinary tract were reported 22



### for the rats that had received EHEN in the diet prior to the administration of biphenyl. 23



### Inc idences of dysplastic foci and renal cell tumors were determined in the kidneys of all groups 24



### of rats. Only rats that had received EHEN during the initial 2 weeks exhibited neoplastic kidney 25



### lesions (dysplastic foci, renal cell tumors). For the EHEN + 0% biphenyl, EHEN + 0.125% 26



### biphenyl, and EHEN + 0.5% biphenyl groups, incidences of rats with dysplastic foci were 25/25, 27



### 21/25, and 25/25, respectively, and incidences of rats with renal cell tumors were 13/25, 12/25, 28



### and 7/25, respectively. Under the conditions of this study, biphenyl did not exhibit tumor 29



### promoting characteristics for the kidney tumor initiator, EHEN. 30

31



### Ambrose et al., 1960 32



### Weanling albino rats (15/sex/group) were administered biphenyl in the diet at 33



### concentrations of 0, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, or 1% for 2 years (10, 50, 100, 500, 1,000, 34



### 5,000, or 10,000 ppm). Body weights were monitored every week during the period of active 35



### growth and then at 50-day intervals. Hemoglobin was monitored every 100 days in control and 36



### high-dose rats; at 500, 600, and 700 days in rats receiving 0.5% biphenyl, and at 500 and 37



### 600 days in rats receiving 0.1% dietary biphenyl. A 98-day paired- feeding experiment was 38

### 32 DRAFT

- DO NOT CITE OR QUOTE conducted in which control rats were provided the same amount of food that rats of the 0.5 and 1



### 1.0% dietary biphenyl groups consumed to assess whether possible differences in growth would 2



### indicate a biphenyl exposure- related toxicological response or decreased palatability. At 3



### necropsy, the weights of liver, kidneys, heart, and testes were determined for all groups except 4



### those receiving 1.0% biphenyl in the diet. Stained sections of heart, lung, liver, kidney, adrenal, 5



### spleen, pancreas, stomach, intestine, bladder, thyroid, brain, pituitary, and gonads were prepared 6



### for histopathologic ex aminations. In some cases, bone marrow smears were prepared. 7



### The study report of Ambrose et al. (1960) did not include sufficient information from 8



### which daily biphenyl doses could be calculated. Biphenyl d oses are estimated at 1, 4, 8 , 42, 84, 9



### 420, and 840 mg/kg- day for the dietary levels of 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, and 1.0%, 10



### respectively , based on U.S. EPA (1988) reference values for body weight and food consumption 11



### in F344 rats (averages of values for males and females ). There is greater un certainty in the dose 12



### estimates at the two highest exposure levels because the magnitude of reported decreased food 13



### consumption in these groups was not specified in the study report. Decreased longevity was 14



### apparent in male and female rats of the 0.5 and 1.0% biphenyl exposure groups, but was not 15



### evident at lower exposure levels. Growth rates appeared similar among controls and groups 16



### exposed to biphenyl levels ≤0.1%. At the two highest exposure levels, markedly decreased 17



### growth was evident, but was attributable to decreased food consumption and indicative of 18



### decreased palatability based on results of the paired

- feeding experiment. Decreased hemoglobin 19



### levels were reported in male and female rats of the two highest exposure levels after 300

– 20



### 400 and 500–600 days, respectively, but were considered at least partially related to lower food 21



### consumption in these groups relative to controls . Selected organ weights are summarized in 22



### Table 4 -6. T here were no statistically significant treatment

- related effects on o rgan weights at 23



### dietary levels ≤0.1%, which were below those associated with decreases in food consumption, 24



### body weight, and survival (i.e., 0.5 and 1.0%). Relative liver and kidney weights of female rats 25



### of the 0.5% biphenyl exposure group were si gnificantly ( p < 0.05) increased, approximately 26



### 45 and 215% higher than those of respective controls. The only significant compound- related 27



### histopathological change occurred in the kidneys, which, in all members of the two highest 28



### exposure groups, showed irregular scarring, lymphocytic infiltration, tubular atrophy, and tubular 29



### dilation associated with cyst formation. Some evidence of hemorrhage was present, and calculi 30



### were frequently noted in the renal pelvis. Evidence of metaplasia in the epithelium o f the renal 31



### pelvis did not implicate neoplastic activity, and, taking the histopathological results as a whole, 32



### there appeared to be no clear -cut, compound-related tumor development. However , the small 33



### number of animals in each group and the decreased survival in the two highest dose groups may 34



### have impaired the ability to detect late -developing tumors. The study identified 1, 000 ppm 35



### biphenyl in the diet (84 mg/kg -day) as a NOAEL and 5 ,000 ppm (420 mg/kg -day) as the LOAEL 36



### for kidney effects including tubular atrophy and dilation associated with cyst formation and 37



### calculi formation in the renal pelvi s of albino rats of both sexes. 38

### 33 DRAFT

- DO NOT CITE OR QUOTE 1



### Table 4 -6. Body and organ weight data for male and female rats
administered biphenyl in the diet for 2 years

Percent biphenyl

in diet Days on

diets Number

of rats Mean body weight
(g) ± SE Mean relative organ w eight (g) ± SE

Liver Kidneys Heart Testes

Males

0.0 745 9 396 ± 24.6 2.89 ± 0.16 0.75 ± 0. 02 0.32 ± 0. 0 15 0.72 ± 0.03

0.001 744 8 424 ± 5.1 2.66 ± 0.06 0.70 ± 0. 03 0.28 ± 0.008 0.62 ± 0.07

0.005 747 10 383 ± 19.8 2.84 ± 0.15 0.73 ± 0. 02 0.30 ± 0.01 0.56 ± 0.06

0.01 752 11 394 ± 14.2 2.47 ± 0.07 0.72 ± 0. 0 1 0.31 ± 0.008 0.67 ± 0.07

0.05 730 13 371 ± 15.8 3.03 ± 0.12 0.74 ± 0. 02 0.31 ± 0.007 0.65 ± 0.06

0.1 746 10 366 ± 23.7 2.98 ± 0.19 0.83 ± 0. 05 0.34 ± 0.012 0.60 ± 0.08

0.5 746 2 345 3.12 1.17 0.36 0.36

Females

0.0 745 9 333 ± 9.4 3.11 ± 0.15 0.65 ± 0.01 0.33 ± 0.01 NA

0.001 744 6 369 ± 13.4 3.21 ± 0.17 0.62 ± 0.02 0.28 ± 0.07 NA

0.005 747 5 335 ± 16.6 2.81 ± 0.28 0.64 ± 0.02 0.31 ± 0.03 NA

0.01 752 11 341 ± 9.1 3.46 ± 0.74 0.62 ± 0.02 0.30 ± 0.01 NA

0.05 730 5 306 ± 12.5 3.51 ± 0.12 0.68 ± 0.02 0.31 ± 0.01 NA

0.1 746 5 327 ± 6.8 3.18 ± 0.10 0.65 ± 0.01 0.32 ± 0.01 NA

0.5 746 5 226 ± 25.8 4.52 ± 0.20 a 1.39 ± 0.14 a 0.46 ± 0.04 NA

a S ignificantly different from controls ( p < 0.05) according to two

- tailed Student’s t

- test.

NA = not applicable; SE = standard error of the mean

Source : Ambrose et al. (1960)

2

3 

### Pecchiai and Saffiotti, 1957

Male al bino rats (8/group; strain not stated) were given biphenyl in the diet for up to 4



### 13 months at concentrations resulting in estimated doses of 250 or 450 mg/kg-day. Upon 5



### sacrifice, liver, kidney, spleen, heart, lung, thyroid, parathyroid, adrenal, pancrea s, testis, 6



### stomach , and intestine were processed for histopathological examination. At 2- month interim 7



### sacrifices, moderate degenerative changes in liver and kidney were observed at both dose levels. 8



### Liver effects consisted of moderate degeneration and h ypertrophy of the Kupffer cells with a 9



### generally well -preserved structure. Renal glomeruli were undamaged, but tubuli showed mild 10



### signs of degeneration. The liver and kidney effects did not appear to increase in severity in rats 11



### treated for up to 13 months. Other histopathologic effects noted in the biphenyl- treated rats 12



### included hypertrophied splenic reticular cells, small follicles with sparse colloid and 13



### desquamation of follicular epithelium in the thyroid, and hyperplastic and hyperkeratinized 14



### forest omach epithelium with occasional desquamation. Although the study report did not 15



### includ e tumor incidence data for the two dose groups, the study authors reported neoplastic 16



### lesions in the forestomach of three biphenyl- treated ra ts. Two of the rats exhibited papillomas of 17

### 34 DRAFT

- DO NOT CITE OR QUOTE the forestomach epithelium (one after 7 weeks and one after 7 months of treatment); a squamous 1



### cell carcinoma was diagnosed in the other rat after 1 year of treatment . The study authors noted 2



### two sequential resp onses to chronic biphenyl exposure: degenerative changes of nuclei and 3



### cytoplasm in the parenchyma of liver and kidney, spleen, thyroid, and adrenals within 2 months 4



### followed within 1 month or more by functional-regenerative changes that resulted in hyper plasia 5



### and nuclear hypertrophy of liver and kidney parenchyma as well as functional hyperactivity of 6



### the thyroid and parathyroid. Signs of cirrhosis were not seen, but irritation and hyperplasia were 7



### evident in the lower urinary tract. The lowest dose, 250 mg/kg-day biphenyl, was an apparent 8



### LOAEL for nonneoplastic degenerative changes in the liver, kidney, thyroid, and parathyroid of 9



### male albino rats resulting in hyperplasia of liver, kidney, and thyroid. 10

11



### Dow Chemical Co., 1953 12



### Sprague- Dawley rats (12/s ex/group) were exposed to biphenyl in the diet for 2 years at 13



### exposure levels of 0, 0.01, 0.1, or 1% (0, 100, 1,000, or 10,000 ppm). Body weights were 14



### monitored twice weekly for 3 months, then weekly. Blood samples were taken from all animals 15



### at the star t of the experiment, approximately every 3 months thereafter, and at term. Hemoglobin 16



### levels, red and white blood cell counts and differential cell counts, and BUN concentrations were 17



### recorded. At death or scheduled necropsy, organ weights were recorded for liver, lung, kidneys, 18



### heart, and spleen. Sections from heart, liver, kidney, spleen, adrenals, pancreas, gonads, 19



### stomach, small and large intestine, voluntary muscle, lung, bladder, and brain were fixed and 20



### stained for histopathologic examination. 21



### Based on U.S. EPA (1988) chronic reference values for body weight and food 22



### consumption in Sprague-Dawley rats (average values for combined sexes), doses of biphenyl for 23



### the dietary levels of 0.01, 0.1, and 1% are estimated to have been 7, 73 , and 732 mg/kg-day, 24



### respectively. It is unclear to what extent the data in the study were compromised by an outbreak 25



### of pneumonia that affected the colony during the course of the experiment. Survival was poor in 26



### control males, all of which had died by 18 months. Only two of the females receiving 0.1% 27



### biphenyl in the diet survived to the end of the 21 st 

### month, and none had survived by the end of 28



### the 23 rd 

### month. However, the authors considered the decreased survival in this group of females 29



### to have been compound- relat ed. Striking biphenyl concentration-related reductions in body 30



### weight gain were observed among the groups, although, in monitoring food efficiency, the 31



### authors indicated that the reduced growth was likely due to a lower daily consumption of food 32



### rather th an to the toxicological consequences of biphenyl. There were no clear indications of 33



### exposure- related changes in hematological parameters, but the authors reported significant 34



### ( p < 0.05) increases in average (combined sexes) relative liver and kidney weights at the highest 35



### exposure level , compared with control values (4.71 vs. 3.05 g/100 g and 1.68 vs. 1.00 g/100 g, 36



### respectively) . Histopathologic examinations revealed dilatation of the kidney tubules, an effect 37



### that appeared to be associated with seco ndary inflammation, uremia, disruption of the filtration 38

### 35 DRAFT

- DO NOT CITE OR QUOTE system, and an increase in BUN in affected animals. Since tubular dilatation was evident in 1



### controls as well as treated animals, the authors presented their data on a semiquantitative severity 2



### scale (0

– 4) in which 0 = no observed changes, 1 = tissue changes in occasional isolated areas, 3



### 2 = tissue changes in multiple areas, 3 = tissue changes in numerous areas, and 4 = extensive 4



### tissue changes involving all or almost all areas. Among the controls, l ow

- , mid -, and high-dose 5



### rats, respective i ncidences for tubular dilatation with severity scores ≥2 were 1/12, 6/1 2, 7 /1 2, 6



### and 11/12 for males and 1/12, 3/12, 4/12, and 11/12 for females. Respectively, i ncidences for 7



### tubular dilatation with severity score s ≥3 were 0/12, 1/12, 2/12, and 9/12 for males and 1/12, 8



### 2/12, 2/12, and 11/12 for females. Severity scores ≥ 3 for tubular dilatation are considered to 9



### represent adverse renal effects. Calcification and intratubular inflammation were frequently 10



### observed at the highest biphenyl exposure level. T he incidence data for renal tubular dila ta tion 11



### with a severity score ≥ 3 indicate that 100 ppm biphenyl in the diet (73 mg/kg-day) was a 12



### N OAEL and that 1,000 ppm (732 mg/kg- day) was a LOAEL for renal effects in Spra gue- Dawley 13



### rats. The small number of rats in th e exposure groups and the decreased surviva l at the highest 14



### exposure level may have impaired the ability to detect late -developing tumors in this study. 15

16



### 4.2.1.2.2. Chronic m ouse s tudies 17



### Umeda et al., 2005 18



### In a chronic toxicity and carcinogeni ci ty study of BDF 1 

### mice (50/sex/group), biphenyl 19



### was administered in the diet for 2 year s at concentrations of 0, 667, 2,000 or 6,000 ppm . All 20



### animals were observed daily for clinical signs and mortality. Body weights and food 21



### consumption were recorded weekly for the first 14 week s and every 4 weeks thereafter. 22



### Hematological and clinical chemistry parameters were measured in blood samples drawn from 23



### all 2

- year survivors just prior to terminal sacrifice. At death or t erminal sacrifice, gross 24



### pathological examinations were performed and organs were removed and weighed. Specific 25



### tissues prepared for microscopic examination were not listed in the study report, but included 26



### liver and kidney. 27



### There were no overt clinical s igns or effects on food consumption or survival among 28



### biphenyl- exposed mice of either sex compared to respective controls. However, mean terminal 29



### body weights of 2,000 and 6,000 ppm mice of both sexes were significantly less than those of 30



### respective contr ols (Table 4 -7). Based on body weight and food consumption data, the study 31



### authors estimated that the 667, 2,000, and 6,000 ppm dietary levels resulted in average daily 32



### biphenyl doses of 97, 291, and 1,050 mg /kg-day in the males and 134, 414, and 1,420 mg /kg-day 33



### in the females (Table 4 -5). 34

35

### 36 DRAFT

- DO NOT CITE OR QUOTE Table 4 -7 . Survival rate, body weight, food consumption, and daily biphenyl
intake in mice fed diets containing biphenyl for 2 years

Biphenyl in diet (ppm) Survival at term Average (± SD) body
weight at term (g) Average food
consumption (g/d ) Average dose
(mg/kg

- d)

Males

0 35/50 46.9 ± 4.9 5.6 0

667 41/50 43.1 ± 7.9 5.5 97

2,000 41/50 42.9 ± 6.0 a 5.5 291

6,000 39/50 32.4 ± 3.6 b 5.4 1

, 050

Females

0 31/50 34.0 ± 4.0 5.9 0

667 22/50 32.5 ± 3.3 5.8 134

2,000 25/50 30.5 ± 3.1 b 5.9 414

6,000 32/49 25.5 ± 3.0 b 5.9 1

, 420

a Significantly different from controls ( p < 0.05) according to Dunnett’s test.

b Significantly different from controls ( p < 0.01) according to Dunnett’s test.

Source: Umeda et al. (2005).

1



### Although there were no compound- related changes in hematological parameters, some 2



### clinical chemistry parameters showed marked changes in relation to dose, including a biphenyl 3



### dose- related increase in BUN that achieved statistical significance in 6,000 ppm males and 4



### females and 2,000 ppm males. Particularly striking were dose- related increases in activities of 5



### the plasma enzymes AP, lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase 6



### ( GOT; also referred to as AST), and glutamate pyruvate transaminase ( GPT; also referred to as 7



### ALT) in the female mice. These data are shown in Table 4 -8 and are suggestive of biphenyl8



### related hepatocellular disruption. Umeda et al. (2005) noted that females with malignant liver 9



### tumors exhibited extremely high AST, ALT, and LDH activities. Biphenyl effects on these 10



### parameters in males were less obvious, a lthough AP activity was significantly greater than 11



### controls in 6,000 ppm males (261 ± 102 vs. 178 ± 111 IU/L) (Table 4 -8). 12

13

### Table 4 -8. Dose- related changes in selected clinical chemistry values from

male and female BDF 1 

### mice exposed to biphenyl via the d iet for 2 years

Males

Biphenyl dietary
concentration (ppm) 0 667 2,000 6,000

Dose (mg/kg

- d ) 0 97 291 1,050

Endpoint (mean ± SD) n = 34 n = 39 n = 37 n = 37

AST (IU/ L ) 85 ± 92 58 ± 38 69 ± 60 88 ± 151

ALT (IU/ L ) 73 ± 113 34 ± 31 36 ± 49 43 ± 80

AP (IU/ L ) 178 ± 111 155 ± 30 169 ± 36 261 ± 102 a

LDH (IU/ L ) 321 ± 230 252 ± 126 432 ± 868 283 ± 200

BUN (mg/d L ) 20.2 ± 3.6 22.0 ± 4.0 23.2 ± 4.4 b 22.9 ± 2.7 a

Females

Biphenyl dietary
concentration (pp m) 0 667 2,000 6,000

Dose (mg/kg

- d ) 0 134 414 1,420

Endpoint (mean ± SD) n = 28 n = 20 n = 22 n = 31

AST (IU/ L ) 75 ± 27 120 ± 110 211 ± 373 a 325 ± 448 a

ALT (IU/ L ) 32 ± 18 56 ± 46 134 ± 231 a 206 ± 280 a

AP (IU/ L ) 242 ± 90 256 ± 121 428 ± 499 556 ± 228 a

LDH (IU/ L ) 268 ± 98 461 ± 452 838 ± 2,000 1,416 ± 4,161 b

BUN (mg/d L ) 14.9 ± 2.0 14.8 ± 3.4 21.0 ± 20.5 23.8 ± 11.7 a

a Significantly different from controls ( p < 0.01) according to Dunnett’s test.

b Significantly different from controls ( p < 0.05) according to Dunnett’s test.

ALT (GPT) = alanine aminotransferase (glutamic pyruvic transaminase); AP (ALP) = alkaline phosphatase;

AST (GOT) = aspartate aminotransferase (glutamic oxaloacetic transaminase)

Source: Umeda et al. (2005).

1

2 

### The only apparent exposure- related effect on organ weights was 1.3-, 1.4-, and 1.6-fold

37 DRAFT

- DO NOT CITE OR QUOTE increases in relative liver weights of 667, 2,000, and 6,000 ppm f e male mice, respectively (the 3



### data for liver weight group means and standard deviations [SDs] were not presented in Umeda et 4



### al . [2005]). Incidenc es of gross and histopathological findings are presented in Table 4 -9. Gross 5



### pathologic examinations revealed biphenyl dose- related increased incidences of liver nodules in 6



### females, but not males. The nodules were round- or oval-shaped cystic or solid ma sses 7



### approximately 3–23 mm in diameter of the largest axis. Histopathological examinations 8



### revealed that 5, 16, and 19 of the nodule-bearing 667, 2,000, and 6,000 ppm female mice also 9



### exhibited proliferative lesions of hepatocellular origin. Significantl y increased incidences of 10



### basophilic cell foci were observed in 2,000 and 6,000 ppm female mice. Although incidences of 11



### basophilic cell foci were significantly increased in 667 ppm male mice as well, a dose- related 12



### effect was not evident because incidence s of this lesion were not significantly increased in 13



### 2,000 or 6,000 ppm males compared to controls. Incidences of hepatocellular adenomas and 14

### 38 DRAFT

- DO NOT CITE OR QUOTE combined incidences of hepatocellular adenomas or carcinomas were significantly increased in 1



### the 2,000 and 6,000 ppm females and Peto’s trend tests confirmed significant positive trends for 2



### dose- related increased incidences of hepatocellular adenomas ( p < 0.05) and combined 3



### incidences of hepatocellular adenomas or carcinomas ( p < 0.01). Incidences of hepatocellular 4



### carcinomas were significantly increased in 2,000 ppm females , but not 667 or 6,000 ppm 5



### females. However, Umeda et al. (2005) noted that the incidences of hepatocellular carcinomas 6



### (5/50 or 10%) in each of the 667 and 6,000 ppm groups of females exceeded a range of historical 7



### control data (26 hepatocellular carcinomas in 1,048 female mice [2.5% incidence] ) at the 8



### laboratory where the study was conducted. No significant biphenyl exposure- related effects on 9



### liver tumor in cidences were seen in male mice. Incidences of desquamation of the urothelium in 10



### the renal pelvis were increased in 6,000 ppm male and female mice. Incidences of 11



### mineralization in the inner stripe of the outer medulla of the kidney were significantly inc reased 12



### in the 2,000 and 6,000 ppm female mice. 13

14



### Table 4 -9 . Incidences of gross and histopathological findings in male and

female BDF 1 

### mice fed diets containing biphenyl for 2 years

Parameter Dietary concentration of biphenyl (ppm)

Males Females

0 667 2,000 6,000 0 667 2,000 6,000

Average dose (mg/kg

- d )

0 97 291 1

, 050 0 134 414 1

, 420

Necropsy

Liver nodules 20/50 16/49 14/50 11/50 7/50 13/50 24/50 26/49

Histopathology

Liver

Adenoma 8/50 6/49 7/50 3/50 2/50 3/50 12/50 a 10/49 a

Carcinoma 8/50 8/49 5/50 4/50 1/50 5/50 7/50 a 5/49

Adenoma or carcinoma
(combined) 16/50 12/49 9/50 7/50 3/50 8/50 16/50 b 14/49 a

Basophilic cell foci 0/50 6/49 b 1/50 2/50 1/50 1/50 12/50 b 6/49 a

Clear cell foci 0/50 6/49 b 2/50 0/50 2/50 1/50 3/50 2/49

Eosinophilic cell foci 0/50 0/49 0/50 0/50 0/50 1/50 0/50 0/49

Kidney

Desquamation: pelvis 0/50 0/49 0/50 10/50 b 4/50 0/50 0/50 15/49 b

Mineralization inner stripe

– outer medulla 9/50 8/49 14/50 14/50 3/50 5/50 12/50 a 26/49 b

a Significantly different from controls ( p < 0.05) according to Fisher’s exact test.

b Significantly different from controls ( p < 0.01) according to Fisher’s exact test.

Source: Umeda et al. (2005).

15

16 

### In summary, the chronic toxicity and carcinogenicity study of male and female BDF 1

17 

### mice administered biphenyl in the diet for 2 year s (Umeda et al., 2005) provides evidence for

### 39 DRAFT

- DO NOT CITE OR QUOTE biphenyl-induced liver tumors in females, but not males, based on significantly increased 1



### incidences of hepatocellular adenomas and combined carcinomas or adenomas in the female 2



### mice receiving biphenyl from the diet at 414 and 1,420 mg/kg-day (Table 4 -9). This study 3



### identified a NOAEL of 13 4 mg/kg -day and a LOAEL of 414 mg/kg -day for nonneoplastic 4



### effects (mineralization in the kidney and significantly increased plasma ALT and AST activities) 5



### in female BDF 1 

### mice exposed to biphenyl in the diet for 2 year s. 6

7



### Imai et al., 1983 8



### Groups of female ddY mice were fed diets containing 0 (n = 37 mice) or 0.5% 9



### (n = 34 mice) biphenyl (5,000 ppm) in the diet for 2 years. This study also included groups 10



### exposed to dietary concentrations of 0.2% thiabendazole or a mixture of 0.25% biphenyl and 11



### 0.1% thiabendazole (results from this part of the study are not further described herein). Food 12



### consumption, body weights, and survival were assessed at intervals throughout exposure. At 13



### terminal sacrifice, several organs were weighed and prepared for microscopic histology (brain, 14



### pituitary, thymus, liver , spleen, pancreas, lung, heart, adrenal, kidney, ovaries, and uterus); in 15



### addition, the thyroid, stomach, small intestine, and large intestine were prepared for histology 16



### only. Urine samples collected from 10 control and 9 treated mice at terminal sacrifice were 17



### analyzed for protein glucose, ketones, bilirubin, urobilogen, and pH. Blood samples collected at 18



### the terminal sacrifice from 12 control and 9 treated mice were assessed for hematological 19



### endpoints, and serum samples (n = 6 for control and treated groups ) were also assessed for 20



### biochemical endpoints including GOT

, GPT , AP, cholinesterase, glucose, albumin, and total 21



### protein. Based on U.S. EPA (1988) methodology for estimating food consumption rates from 22



### body weight data and the reported average terminal body weight for the 5,000 ppm mice 23



### (0.037 kg), an oral dose of 855 mg/kg-day is estimated from the dietary exposure. Exposure to 24



### biphenyl did not influence survival, food consumption, or growth compared with controls (as 25



### shown in Figures 1, 2, and 3 in Imai et al. [1983]). No marked exposure- related effects were 26



### found on terminal organ and body weights (Tables 5 and 6 in Imai et al. [1983]) or on the 27



### urinalytic, hematologic, or serum biochemical endpoints (Tables 2, 3, and 4 in Imai et al. 28



### [1983]). Histological examination revealed no increased incidence of non- neoplastic lesions in 29



### examined tissues in the 5 ,000 ppm biphenyl group, compared with the control group (Table 7 in 30



### Imai et al. [1983]). The only tissues showing tumors at elevated incidence in the 5

, 000 ppm 31



### mice, compared with the control group, were the lung (11/34 [32.4%] vs. 9/37 [24.3%] in 32



### controls) and lymphatic tissues (lymphomas: 5/34 [14.7%] v s. 4/37 [10.8%]; leukemia: 3/34 33



### [8.8%] vs. 2/37 [5.4%]), but these increases are not statistically significant ( p > 0.05 by the 34



### Fisher ’s exact test). In summary, exposure of female ddY mice to 5,000 ppm biphenyl in the diet 35



### for 2 years was a NOAEL for non -neoplastic lesions, survival, body and organ weight changes, 36



### and changes in urinalytic, hematologic, and serum chemistry endpoints. In contrast to the 2- year 37



### bioassay with BDF 1 

### mice that found increased liver tumors in female mice exposed to dietary 38

### 40 DRAFT

- DO NOT CITE OR QUOTE concentrations ≥2 ,000 ppm (Umeda et al., 2005), no carcinogenic response occurred in female 1



### ddY mice exposed to 5, 000 ppm biphenyl in the diet (estimated dose of 855 mg/kg- day) for 2



### 2 years (Imai et al., 1983). 3

4



### Innes et al., 1969; NCI , 1968 5



### The carcinogenic potentials of 130 chemicals, including biphenyl, were assessed in a 6



### protocol that exposed groups of two strains of F 1 hybrid mice (18/sex/strain/group), produced by 7



### mating female C57BL/6 mice to either male C3H/Anf mice (F1 de signated as strain A ) or male 8



### AKR mice (F1 designated as stra in B) to individual chemicals by the oral route for 18 months. 9



### Four groups of untreated controls and a group of gelatin vehicle controls (18/sex/strain/group) 10



### were included in the study. In the case of biphenyl, the chemical was administered via gavage to 11



### mice for 3 weeks, starting at the age of 7 days at 215 mg biphenyl/kg body weight in 0.5% 12



### gela tin (the report of Innes et al. [1969] appears to have erroneously reported the gavage dose as 13



### 2.5 mg/kg). Thereafter, and for the rest of the experimental period, biphenyl was mixed with 14



### chow to a final concentration of 517 ppm. The gavage dose level and food concentration of 15



### biphenyl were selected to reflect the maximum tolerated dose identified in preliminary range16



### finding single- dose subcutaneous injection and single

- and repeated -dose oral administration 17



### studies. Initial gavage dose and dietary levels of biphenyl were not adjusted for weight gain 18



### during the 18-month study. Based on U.S. EPA (1988) chronic reference values for body weight 19



### and food consumption in strain A mice (average values for combined sexes), an average oral 20



### dose of 91 mg/kg- day is estimated from the dietary exposure. Blood smears were prepared from 21



### mice that showed splenomegaly, liver enlargement , or lymph adenopathy at necropsy. At term, 22



### mice were examined for a ny gross pathological featur es. Major organs were processed for 23



### histopathologic examination (including total chest contents, liver, spleen, kidneys with adrenals, 24



### stomach, and genital organs). Innes et al. (1969) reported i ncidences for hepatomas, pulmonary 25



### tumors, and lymphomas in control mice (Table 5 of Innes et al., 1969) and for tested chemicals 26



### that were judged to give “high tumor yield” (Table 7 of Innes et al., 1969); biphenyl was 27



### reported to be noncarcinogenic, but tumor incidence data for b iphenyl were not reported. The 28



### NCI (1968) report included tabulated incidences of hepatomas, pulmonary tumors, and 29



### lymphomas in control mice and biphenyl- treated mice, which are summarized in Table 4 -10. In 30



### summary, the results provide no evidence of a carcinogenic response to 18 months of oral 31



### exposure to biphenyl (215 mg/kg by gavage for 3 weeks, followed by dietary exposure to 32



### 517 ppm biphenyl). 33

34

### 41 DRAFT

- DO NOT CITE OR QUOTE Table 4 -10 . Incidences of selected tumor types among controls and mice
administered biphenyl orally for 18 months

Group Incidences of selected tumor types a

Hepatoma Pulmonary tumors Reticular cell sarcoma

Strain A male mice

Controls 8/79 5/79 5/79

Biphenyl

- treated 2/17 3 /17 1/17

Strain A female mice

Controls 0/87 3 /87 4/87

Biphenyl

- treated 0/18 1/18 0/18

Strain B male mice

Controls 5/90 10/90 1/90

Biphenyl

- treated 3 /17 1/17 0/17

Strain B female mice

Controls 1/82 3/82 4/82

Biphenyl

- treated 0/17 0/17 4/17

a Tumor incidences were tallied from those mice for which histopathologic examinations were performed

.

Source: NCI (1968)

.
1

2 

### 4.2.1.2.3. Chronic studies in other animal species

3 

### Monsanto, 1956

Mongrel dogs (two males and one female/group) were administered 0, 2.5, or 25 mg/kg 4



### biphenyl in corn oil by capsule 5 days/week for 1 year. Dogs were examined daily for clinical 5



### signs and weighed weekly. Blood samples were withdrawn at 3-month intervals to measure such 6



### hematological parameters as hemoglobin, hematocrit, blood cell count, sedimentation rate, 7



### icterus index, bromosulphalein retention, and, among clinical chemistry parameters, BUN. Urine 8



### samples were obtained at similar intervals to measure specific gravity, sugar, protein, bile 9



### pigments, occult blood, and microscopic sediment. Samples of urine from the high-dose dogs 10



### were collected during week 18, pooled, and analyzed for the presence of biphenyl and 11



### metabolites. At termination, gross necr opsies were performed, and sections of large and small 12



### intestine, pancreas, ovary or testis, adrenal, urinary bladder, stomach, lung, thyroid, brain, heart, 13



### spleen, and liver were prepared for histopathologic examination. Although slight fluctuations 14



### were seen in body weight during the study, the dogs generally exhibited a net weight gain. 15



### Fluctuations in hematological parameters and urine analysis were inconsistent and not 16



### considered compound-related. Gross pathological examination of the dogs showed no obviously 17



### compound- related effects. Histopathologic examinations revealed lung congestion consistent 18



### with bronchial pneumonia in one high-dose dog; histopathology was unremarkable for each of 19



### the other dogs in the study. 20

21

### 42 DRAFT

- DO NOT CITE OR QUOTE Dow Chemical Co., 1953 1



### Dow Chemi cal Co. (1953) described a biphenyl feeding experiment in which four groups 2



### of Rhesus monkeys (two males and one female/group) were exposed to 0, 0.01, 0.1, or 1% 3



### biphenyl in chow for 1 year, during which time most of the animals experienced ill health not 4



### related to biphenyl exposure. Despite this caveat, hematological parameters were normal. The 5



### authors considered an increase in relative liver weight in high -dose monkeys (4.65 g/100 g body 6



### weight vs. 3.90 g/100 g body weight in controls) to possibly be compound- related. 7

8



### 4.2.2. Inhalation Studies 9



### Deichmann et al., 1947; Monsanto, 1946 10



### In three separate experiments, albino rabbits (sex and strain not stated), Sprague- Dawley 11



### rats (sex not stated), and mice (sex and strain not stated) were repeatedly exposed to dusts 12



### composed of 50% biphenyl attached to celite for 7 hours/day, 5 day s/week. In the first 13



### experiment, 3 rabbits and 10 rats were exposed to an average concentration of 300 mg /m 3 

### on 14



### each of 64 days over a period of 94 day s. The rats exhibite d irritation of the nasal mucosa 15



### accompanied by serosang uineous discharge. Five of the rats died prior to term, and the survivors 16



### lost weight. The rabbits exhibited no exposure-related adverse signs. In the second experiment, 17



### three rabbits and six rats were exposed to an average concentration of 40 mg/m 3 

### on each of 18



### 46 days over a total period of 68 days. One rat died prior to term. The surviving rats showed 19



### signs of mucous membrane irritation, but appeared to gain weight at a normal rate. The rabbits 20



### exhibited no exposure-related adverse signs. In the third experiment, 12 mice and 4 rats were 21



### exposed to an average concentration of 5 mg /m 3 

### on each of 62 days over a total period of 22



### 92 day s. While the rats were unaffected at this concentration, all of t he mice showed signs of 23



### irritation of the upper respiratory tract and two died prior to term. Bronchopulmonary lesions 24



### (including acute emphysema, congestion, edema, bronchitis, widespread lobular pneumonia, and 25



### multiple pulmonary abscesses) were reported in rats from experiments 1 and 2 and in mice of 26



### experiment 3. Some unspecified minor liver and kidney lesions were also noted. Based on the 27



### results of these three experiments, a LOAEL of 5 mg /m 3 

### for upper respiratory tract irritation in 28



### the mice was ide ntified. 29

30



### Sun Company Inc., 1977b 31



### Groups of CD-1 mice (50/sex/group) were exposed to airborne biphenyl at vapor 32



### concentrations of 0, 25, or 50 ppm (0, 157.7, and 315.3 mg /m 3 

### , respectively) for 7 hours/day, 33



### 5 day s/week for 13 weeks. Mice were maintained and exposed to biphenyl in groups of 5 (for a 34



### total of 10 groups/sex/exposure group). All animals were checked daily for clinical signs and 35



### mortality, and body weight data were collected. Upon completion of the 13- week exposure 36



### period, surviving mice wer e placed in metabolic cages for 12 -hour collection of urine for 37



### urinalysis. Blood samples were collected for blood chemistry and hematology assessments. 38

### 43 DRAFT

- DO NOT CITE OR QUOTE Gross and histopathologic examinations were performed on all mice. Ten surviving 1



### mice/sex/group were held for a 30- day recovery period prior to terminal sacrifice. 2



### During the first few days of biphenyl exposure, some of the test material crystallized in 3



### the delivery system; analysis of biphenyl exposure levels was not performed on these days. 4



### Daily meas ured biphenyl exposure concentrations were highly variable during the first half of 5



### the 13- week exposure period, whereas subsequently measured concentrations were closer to 6



### target concentrations. For example, during the first 45 exposure sessions, measured daily 7



### biphenyl concentrations in the 50 ppm target groups ranged from as low as 5 ppm to as high as 8



### 102 ppm and subsequent measurements ranged from 48 to 55 ppm. Mean biphenyl 9



### concentrations (± 1 SD ) calculated for the entire 13 weeks of exposure were 2 5 ± 7 and 10



### 50 ± 16 ppm for the 25 and 50 ppm target groups, respectively. The authors reported the loss of 11



### 46/100 of the 25 ppm mice due to overheating and cannibalization. Since the overheating event 12



### occurred after 46 exposures, the overall study duration ran for 117 day s to ensure that 13



### replacement mice received a total of 65 exposures as called for in the protocol. The study report 14



### did not mention results of clinical observations

, and mortality data were not specifically 15



### summarized. There were no clear indications of exposure-related effects on body weights. 16



### Results of urinalysis, hematology, and clinical chemistry did not indicate any clear exposure17



### related changes that could be attributed to biphenyl toxicity. Gross and histopathological 18



### examinations revealed congest ed and hemorrhagic lungs, hyperplasia of the trachea with 19



### inflammation accompanied by a high incidence of pneumonia, and congestion and edema in liver 20



### and kidney of biphenyl-exposed mice (see Table 4 -11). The pathologist considered the 21



### congestion in the lung, liver, and kidney a likely effect of the anesthetic used for killing the mice, 22



### although control mice did not exhibit these effects at 13- week sacrifice . The hemorrhagic lungs 23



### and tracheal hyperplasia were considered effects of biphe nyl exposure. Results from the 30-day 24



### recovery groups suggest that the biphenyl exposure- related pulmonary effects were reversible. 25



### This study identified a LOAEL of 25 ppm for histopathologic lung, liver, and kidney lesions in 26



### male and female CD

- 1 mice e xposed to biphenyl by inhalation for 7 hours/day, 5 day s/week for 27



### 13 weeks. 28

### 44 DRAFT

- DO NOT CITE OR QUOTE 1



### Table 4 -11. Incidences of selected histopathologic lesions in tissues of CD -1
mice exposed to biphenyl vapors 7 hours/day, 5 days/week for 13 weeks

Effect 13

- Week exposure groups a

0 ppm 25 ppm 50 ppm

Pulmonary congestion, edema 0/80 95/98 71/71

Pneumonia 0/80 15/98 20/71

Tracheal hyperplasia 0/80 80/98 70/71

Hepatic congestion, edema 0/80 87/98 71/71

Renal congestion, edema 0/80 87/98 71/71

a The study report presented incidences of histopathologic lesions for com bined male and female mice only; n o statistical analyses were conducted.

Source: Sun Co mpany Inc. (1977b).

2

3 

### 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES

— ORAL AND INHALATION

4.3.1. Oral Exposure 4



### Khera et al., 1979 5



### Pregnant female Wistar rats (18 –20 group) were gavaged with 0, 125, 250, 500, or 6



### 1,000 mg/kg-day biphenyl in corn oil on gestation days (GDs) 6–15. Body weights of dams 7



### were recorded on GDs 1, 6 –15, and 22, at which point all dams were sacrif iced. Parameters 8



### evaluated at autopsy included the number of corpora lutea, fetal weights and viability, and early 9



### resorptions. Two

- thirds of the live fetuses/litter were examined for skeletal development and the 10



### rest were examined for the presence of vi sceral abnormalities. Five of the 20 high-dose dams 11



### died prior to sacrifice. Doses ≤500 mg /kg-day produced no clinical signs of maternal toxicity or 12



### evidence of treatment

- related effects on maternal weight gain. As shown in Table 4 -12, a 13



### significantly i ncreased number of dams without live fetuses was observed in the high-dose 14



### group, compared with controls . Mean numbers of corpora lutea and live fetuses in the high-dose 15



### dams were similar to those of controls and dams of all other dose levels. However, t he percent 16



### of dead fetuses and resorption sites was clearly higher in the high-dose group, and the numbers 17



### of anomalous fetuses and litters bearing anomalous fetuses appeared to increase with increasing 18



### dose. Khera et al. (1979) noted that the slight incr eases in the number of fetuses with anomalies, 19



### such as missing and unossified sternebrae or delayed calvarial ossification, were not statistically 20



### significant, but, as shown in Table 4-12 , the incidence of litters with any type of fetal anomalies 21



### (“anomalous litters/number examined”) was elevated ( p < 0.05 by Fisher ’s exact test) at 22



### 500 mg/kg-day, but not at lower doses, compared with control incidences. This study identified 23



### a NOAEL of 500 mg/kg-day and a LOAEL of 1,000 mg/kg-day for frank maternal toxicity 24



### (increased mortality and decreased dams with live fetuses) and lethal fetal effects. For less 25

### 45 DRAFT

- DO NOT CITE OR QUOTE severe developmentally toxic effects (increased incidence of anomalous litters), 500 mg/kg-day 1



### was a LOAEL and 250 mg/kg-da y was a NOAEL. 2

3



### Table 4 -12 . Prenatal effects following oral administration of biphenyl to
pregnant Wistar rats on GDs 6–15

Effect Dose (mg/kg

- d )

0 125 250 500 1,000

Rats without

mated live fetuses at term/number

2 /18 0/20 1 /19 2 /20 11 /20 a

Corpora lutea/pregnancy (mean ± SE) 12.6 ± 0.4 12.9 ± 0.4 13.7 ± 0.5 13.3 ± 0.4 12.5 ± 0.7

Live fetuses/pregnancy (mean ± SE) 11.3 ± 0.7 11.8 ± 0.6 11.9 ± 0.6 11.2 ± 0.5 10.7 ± 1.3

Dead or resorbed fetuses (%) 4.8 3.3 6.1 7.8 13.7 b

Fetal weight (g mean ± SE) 5.1 ± 0.1 5.3 ± 0.1 5.2 ± 0.1 5.2 ± 0.1 4.5 ± 0.3

Anomalous fetuses/number examined 17/176 22/236 22/213 35/199 c 25/107 c

Anomalous litters/number examined 8/16 11/20 13/18 15/18 c 6/9

Anomalies (number of fetuses affected)

Wavy ribs, uni

- and bilateral 3 7 9 8 5

Extra ribs, uni

- and bilateral 9 12 9 15 6

13th rib, small sized 1 1 2 1 0

Sternebrae, missing or unossified 4 3 4 16 17

Calvarium, delayed ossification 0 2 0 0 8

Miscellaneous 1 1 1 0 0

a Signi ficantly ( p < 0.05) different from control incidence according to Fisher’s exact test. Five dams died prior to

scheduled sacrifice, five other dams were not pregnant at term , and one dam had seven resorption sites and no live

fetuses

. b Derived from nine pregnant dams with li ve fetuses and one dam with seven reso rptions and no live fetuses. The
study author stated that the percentage of dead or resorbed fetuses in the 1

, 000 mg/kg dose group was not
statistically significantly different from controls.

c Significantly ( p < 0.05) different from controls according to Fisher’s exact test.

Source: Khera et al. (1979).



### Dow Chemical Co., 1953

Dow Chemical Co. (1953) reported the results of a multigenerational study in which 4

5

6



### groups of 4-month-old male and female Long Evans rats ( three males and nine females/group) 7



### were fed diets containing 0, 0.01, 0.1, or 1.0% biphenyl. Based on U.S. EPA (1988) subchronic 8



### reference values for body weight and food consumption in male and female Long Evans rats, 9



### doses of biphenyl for the dietary levels of 0.01, 0.1, and 1.0% are estimated to have been 9, 89, 10



### and 887 mg/kg- day, respectively , for the males and 10, 101, and 1,006 mg/kg-day, respectively, 11



### for the females

. Average cross -gender doses for males and females were 10, 95, and 947 mg/kg12



### day. For breeding, three females were placed together with one male. Following the breeding 13



### phase, females were separated and number of litters cast , number of days between mating and 14

### 46 DRAFT

- DO NOT CITE OR QUOTE delivery, and average number of pups/litter at delivery were recorded. F1 p ups were weigh ed 1



### and culled to seven/litter at 2 days of age and weaned at 3 weeks of age, and weights were 2



### recorded weekly for postnatal weeks 3 –6 . The F1 rats were continued on the same diets as their 3



### parents, and, at 10 weeks of age, nine F1 females and three F1 males were mated to produce an 4



### F2 generation of pups. F2 pups were selected (by the same procedure) for mating and 5



### production of an F3 generation that were sacrificed at 3 weeks of age; twelve F3 pups from each 6



### diet group were subjected to gross pathologic examinations. 7



### There were no significant differences between controls and 0.01 and 0.1% biphenyl- fed 8



### groups regarding litters cast; gestation length ; or average number or weight of pups/litter at birth 9



### or at 3 or 6 weeks of age. D ecreased fertility in the 1% biphenyl -fed group of females was 10



### observed (6/9, 7/9, and 8/9 confirmed pregnancies for the three successive generations of 1.0% 11



### biphenyl-fed groups vs. 8/9, 9/9, and 8/9 confirmed pregnancies for controls). Averaged for F1, 12



### F2, and F3 pups combined, the 1.0% biphenyl-fed group exhibited significantly ( p < 0.05) 13



### decreased number of pups/litter at birth ( 6.2 /litter vs . 8. 6/litter for controls) and lower average 14



### body weight at 3 weeks of age (36 vs. 48 g for controls) and 6 weeks of age (78 vs. 113 g for 15



### controls) . Gross pathologic evaluations of F3 weanlings revealed no signs of biphenyl treatment16



### related effects. There was no evidence of a cumulative effect over the three generations. The 17



### study authors indicated that the decreased fertility, smaller litter size, and reduced rate of growth 18



### in the 1.0% biphenyl-fed group may have been associated with unpalata bility and resultant 19



### decreased food intake. 20

21



### Ambrose et al., 1960 22



### The research report of Ambrose et al. (1960) contains a subsection in which the 23



### reproductive toxicity of biphenyl was examined in two experimental series. In the first 24



### experiment, weanling albino rats were administered 0 or 0.1% biphenyl (5 males and 25



### 10 females/group) or 0.5% biphenyl (3 males and 9 females) in the diet for 60 day s prior to 26



### mating. In the second experiment, groups of 90-day-old albino rats were administered 0 or 0.1% 27



### biphenyl ( 4 males and 8 females/group) or 0.5% biphenyl ( 3 males and 9 females) in the diet for 28



### 11 days prior to mating. Based on U.S. EPA (1988) subchronic reference values for body weight 29



### and food consumption in rats of unspecified strain (average values for combined sexes), doses of 30



### biphenyl for the dietary levels of 0.1 and 0.5% are estimated to have been 105 and 525 mg /kg31



### day, respectively. All rats were maintained on their respective diets throughout mating and until 32



### the progeny of all litters were weaned. Insufficient information is provided in the report to 33



### permit a judgment as to whether di etary exposure to biphenyl was associated with reproductive 34



### deficits. However, the authors presented tabular data for number of rats casting litters, total 35



### born, and range of litter size (Table 4-13) and concluded that the compound had no significant 36



### effe ct on reproduction. 37

38

### 47 DRAFT

- DO NOT CITE OR QUOTE Table 4 -13. Summary of reproductive data in albino rats exposed to dietary
biphe nyl

Experimental series Diet Dams with litters Total offspring Litter size (range)

First a Control 9/10 59 3

– 9

0.1% biphenyl 10/10 67 2

– 10

0.5% biphenyl 8/9 53 3–9

Second b Control 8/8 64 5

– 13

0.1% biphenyl 6/8 63 3

– 10

0.5% biphenyl 8/9 48 3–9

a Weanling rats on diets for 60 days before mating.

b 90

- Day

- old rats on diets for 11 days before mating.

Source: Ambrose et al. (1960).

1

2 

### 4.3.2. Inhalation Exposure

No studies were identified that examined the reproductive/developmental toxicity of 3



### biphenyl via the inhalation route. 4

5



### 4.4. OTHER DURATION

- OR ENDPOINT

- SPECIFIC STUDIES 6



### 4.4.1. Acute and Short- term Toxicity Data 7



### Acute oral toxicity studies of biphenyl provide median lethal dose ( LD 50 

### ) values ranging 8



### from 2 ,180 to 5,040 mg/kg for rats (Monsanto, 1976; Pecchiai and Saffiotti, 1957; Union 9



### Carbide, 1949; Deichmann et al., 1947) and an LD 50 

### value of 2 ,410 mg/kg for rab bits 10



### (Deichmann et al., 1947). Dow Chemical Co., (1939) reported 100% survival and 100% lethal 11



### doses of 1,600 and 3,000 mg /kg, respectively, in rats. Clinical signs commonly observed 12



### following single oral dosing in these studies included increased respiration, lacrimation, loss of 13



### appetite and body weight, and muscular weakness. Deaths occurred in the first few days 14



### following dosing. Typical targets of histopathologic lesions were lungs, liver, and upper 15



### gastrointestinal tract. 16



### In another acute study, P ecchiai and Saffiotti (1957) administered single gavage doses of 17



### biphenyl at 1–2.5, 3–6, 7, 9–11, or 10– 13 mg/kg to groups of rats (n = 2– 10) and observed them 18



### for up to 7 months following dosing. Histopathological changes to the liver, kidney, thyroid, 19



### p arathyroid, and gastrointestinal mucosa were reported in biphenyl- treated rats; however , the 20



### study report did not provide information regarding numbers of treatment- related deaths or 21



### incidences of lesions in the various treatment groups. Among surviving rats, signs of 22



### regeneration were evident within 1 –4 month s after treatment. By 7 month s after treatment

, most 23



### of the changes had disappeared

, but hepatocytes displayed modest vacuolization of the 24

### 48 DRAFT

- DO NOT CITE OR QUOTE cytoplasm and numerous binucleate cells in the periphery of the lobules. In renal tubuli, a 1



### moderate number of cytoplasmic granules were observed. 2

3



### Sun Company Inc., 1977a 4



### Groups of mice (10/sex of unspecified strain) were exposed to biphenyl by inhalation for 5



### 4 hour s at average analytical concentrations of 14.11 , 38.40, or 42.80 ppm (89.0, 242.2, and 6



### 270.0 mg /m 3 

### , respectively) and observed for up to 14 days following exposure. Clinical signs of 7



### hyperactivity and mild respiratory discomfort were noted during exposure, but resolved during 8



### postexposure observation. A solitary male mouse of the 42.80 ppm group died after 2 hour s of 9



### exposure, but this death was not attributed to biphenyl exposure. All other mice survived 10



### throughout the 14 -day postexposure observation period. Slight lung congestion was noted in 11



### most mice upon gross pathological examination. 12



### Sun Company Inc. (1977a) also provided details of a study in which groups of mice 13



### (10/sex of unspecified strain) were exposed to biphenyl for 7 hour s/day, 5 day s/week for 2 weeks 14



### at average analytical concentratio ns of 0, 24.8, or 54.75 ppm (0, 156.4, and 345.5 mg /m 3



### , 15



### respectively). Five animals/group were sacrificed immediately after exposure; the remaining 16



### animals were sacrificed following a 14 -day recovery period. Clinical signs were monitored 17



### daily. Gross pathologic examinations at necropsy included assessment of lungs, trachea, heart, 18



### spleen, liver, kidneys, stomach, and intestines. Histopathologic examinations included tissues 19



### from lung, trachea, kidney, spleen, and liver. The study authors reported signs of hyperactivity 20



### in some mice during the first few exposure periods. One female mouse of the 24.8 ppm 21



### exposure group died prior to the third exposure session and one control female mouse died prior 22



### the final exposure session. No abnormal clinical signs were seen during the 14-day recovery 23



### period. Gross and histopathologic examinations revealed no signs of exposure- related adverse 24



### effects. 25

26



### Deichmann et al., 1947; Monsanto, 1946 27



### Four rabbits (sex and strain unspecified) received up to 20 daily doses of 500 mg /kg 28



### “purified” biphenyl to the skin; the compound was applied as a 25% preparation in olive oil. 29



### Three rabbits received the same concentration of technical biphe nyl. The compound was left on 30



### the skin for 2 hour s and then washed off with soap and water. Some biphenyl derivatives were 31



### similarly assessed. One rabbit receiving purified biphenyl died after eight applications, and the 32



### rest of the animals survived to term. The only reported sublethal effect clearly associated with 33



### biphenyl exposure was that of weight loss, averaging 45 and 172 g for the rabbits receiving 34



### purified and technical biphenyl, respectively. 35

36

### 49 DRAFT

- DO NOT CITE OR QUOTE 4.4.2. Kidney/Urinary Tract Endpoint Studies 1



### Seve ral endpoint

- specific studies assessed biphenyl

- induced urinary tract effects in rats 2



### (Shibata et al., 1989a, b; Kluwe, 1982; Søndergaard and Blom, 1979; Booth et al., 1961) and 3



### provide support to findings of the chronic oral rat studies described in Section 4.2.1.2 (Chronic 4



### Toxicity and Carcinogenicity). Detailed descriptions of these endpoint- specific studies are 5



### presented below. 6

7



### Booth et al., 1961 8



### In a preliminary study, five adult rats (sex and strain unspecified) were administered 9



### biphenyl in the diet at 1% (w/w) for 26 days followed by a 29-day postexposure recovery period 10



### for a total study period of 55 days. Total urine volume and the volume of sulfosalicylic acid

11



### precipi table sediment were recorded from urine collected from all five rats on study days 4, 8, 12



### 18, 20, and 26 (exposure days), and study days 28, 32, 35, and 54 (recovery period). Volumes of 13



### both urine and sulfosalicylic acid- precipitable sediment increased fro m 7 and 0.56 mL, 14



### respectively, on exposure day 4 to 32 and 2.24 mL, respectively, on exposure day 20. Both 15



### values remained relatively high (approximately 27 and 2.2 mL, respectively) on exposure day 26 16



### and decreased to approximately 14 and 0.8 mL, respect ively , by the end of the recovery period. 17



### Fractionation and analysis of the precipitate suggested the presence of p -hydroxybiphenyl and its 18



### glucuronide. The study authors indicated that similar effects were noted in male and female rats 19



### receiving biphenyl at a level of 0.5% in the diet, but not a t the 0.1% dietary level. 20



### A follow-up study employed 42 rats of each sex and exposure group and biphenyl dietary 21



### levels of 0, 0.1, 0.25, or 0.5% (w/w). Biphenyl doses are estimated at 83.7, 209, and 419 mg/kg22



### day for the dietary levels of 0.1, 0.25, and 0.5%, respectively, based on U.S. EPA (1988) chronic 23



### reference values for body weight and food consumption in F344 rats (averages of values for 24



### males and females). Rats were exposed for up to 165 day s and followed for 0, 30, or 60 day s of 25



### recovery. Urine samples were collected periodically from five rats/sex/ exposure group. Interim 26



### sacrifices of five rats/sex/exposure group were performed after 30, 60, and 120 days on the diet 27



### in order to assess the prog ression of biphenyl -induced histopathological effects on the kidney. 28



### As noted in the preliminary study, the rats of the 0.5% exposure group in the follow-up study 29



### exhibited gradual increases in the urine volume and sulfosalicylic acid- precipitable sedimen t and 30



### decreased in both parameters during postexposure recovery. The study authors indicated that 31



### these effects were much less pronounced in the 0.25% exposure group and absent in the 0.1% 32



### exposure group. At the 0.5% exposure level, kidney lesions were noted in 1/5 of the males 33



### (several small cysts and dilated tubules in the medulla and inner cortex) and 2/5 of the females 34



### (mild local tubular dilation with some epithelial flattening) following 30 day s of exposure. 35



### Similar, but more extensive, kidney lesions were noted in 3/5 males and 5/5 females following 36



### 60 days of exposure. The kidney lesions were even more prominent following 120 day s of 37



### exposure. Reported histopathologic findings in the kidneys of rats from the 0.25% exposure 38

### 50 DRAFT

- DO NOT CITE OR QUOTE group were limited to a single instance of an unspecified “prominent kidney lesion” at 60 days, 1



### and one small calcul us in the pelvis of one rat and a small calcareous deposit in the renal 2



### pyramid of another rat following 120 days of exposure. Based on available information in the 3



### study report, there were no apparent assessments of urinary and histopathologic renal effects at 4



### the end of the 165-day treatment period. However, during the 60-day postexposure recovery 5



### period, rats of the 0.5% biphenyl exposure group exhibited a regression of kidney lesions and 6



### improvement in urine quality. 7

8



### Kluwe, 1982 9



### Kluwe (1982) examined changes in urine composition and kidney morphology in F344 10



### rats exposed to biphenyl. Groups of male F344 rats were administered biphenyl (in corn oil) by 11



### single gavage dosing at 0, 250, 500, or 1,000 mg /kg and observed for 15 days following 12



### treatment. Body weights were recorded , and urine was collected on days 1, 2, 3, 4, 8, and 13



### 15 following treatment for urinalysis. Interim sacrifices were performed on eight c ontrol and 14



### eight high -dose rats on posttreatment days 1, 2, 3, 8, and 15 for assessment of weight and 15



### histopathology of the kidney. The study authors presented body weight data as mean percent 16



### (n = 6) of preexposure body weight; results of urinalyses were presented as mean values (n = 6) 17



### for each group. There were no significant effects on body weight in th e low -dose group. Mean 18



### body weight gains of mid - and high- dose groups w ere consistently 6–10% lower than control 19



### values ( p < 0.05), beginning as early as day 2 following the initiation of dosing and continuing 20



### through day 15. Dose-related increases in polyuria, proteinuria, and glucosuria were observed on 21



### day 1; polyuria and glucosuria were no longer apparent by day 4 and proteinuria resolved 22



### between d ays 8 and 15. The study authors presented no data to indicate that single oral dosing 23



### caused changes in kidney weight. Histopathologic examinations of kidneys revealed renal 24



### papillary necrosis in 8/32 high- dose rats; this effect was observed as early as day 1 and persisted 25



### during the 15-day posttreatment period. 26



### Kluwe et al. (1982) conducted a similar experiment in which groups of male F344 rats 27



### received biphenyl at doses of 0, 250, or 500 mg /kg-day by gavage for 14 days. In this 28



### experiment, polyuria persisted throughout the treatment period; glucosuria was no longer 29



### apparent by day 4 and proteinuria resolved between treatment days 8 and 15. Relative kidney 30



### weight of high-dose rats was significantly increased during the second half of the treatment 31



### perio d, but the magnitude of this effect was small and considered by the study authors to be of 32



### little biological significance. There was some indication of tubular dilatation in focal areas of 33



### kidneys from the high- dose rats. 34

35



### Søndergaard and Blom, 1979 36



### Group s of male and female SPF

- Wistar rats were administered diets consisting of 37



### semisynthetic chow and biphenyl at concentrations resulting in biphenyl doses of 0, 50, 150, 300, 38

### 51 DRAFT

- DO NOT CITE OR QUOTE or 450 mg /kg

- day. Other groups were administered diets consisting of commercial ch ow and 1



### biphenyl at concentrations resulting in biphenyl doses of 0, 50, 150, 300, 500, or 1,000 mg/kg2



### day. The treatment period lasted for up to 21 day s. The numbers of male and female rats in each 3



### treatment group are specified in Table 4-14 . Urine was collected on days 4, 10, and 17 for 4



### urinalysis. At terminal sacrifice, absolute and relative kidney weights were determined and 5



### kidney tissues were prepared for light and electron microscopic assessment. Apparently, interim 6



### sacrifices (days 1, 2, 4, and 10) were performed in order to assess the activity of AP in proximal 7



### tubules. Table 4-14 presents semiquantitative study results, which include increases in urine 8



### volume/specific gravity and relative kidney weight, as well as polycystic kidney changes. No 9



### changes in AP levels were seen as a result of biphenyl exposure. The kidney effects of biphenyl 10



### appeared to be more pronounced when added to the semisynthetic diet vs. the commercial diet, 11



### with 50 mg/kg-day as a LOAEL for the onset of kidney changes. 12

13



### T able 4 -14 . Number of Wistar rats exposed to biphenyl and the degree of
change in kidney weight and cellular architecture

Exposure
(mg/kg -d) N umber of animals
(male/female) Relative kidney weight

increases Cystic change Increases of urine

vol ume /spec ific gravity

Semisynthetic diet

0 3/14

– – – /

–

50 4/3 +

–

150 0/10 + * ●/●

300 14/14 +++ ***

450 4/4 +++ ***

Commercial chow

0 10/20

– – – /

–

50 10/10

– –

150 10/10

– –

300 10/10

– –

500 a 0/10 + b

– ●/●

1,000 a 0/10 +++ b ** ●/●

a Dose for 14 days.

b Absolute organ weight.

+ = statistically significant compared with controls ( p < 0.05), as calculated by the authors (Student’s t

- test);

+++ = statistically significant compared with controls ( p < 0.001), as calculated by the authors (Student’s t- test) ;

* = less than one

- third of the area ; ** = less than two

- thirds of the area ; *** = greater than two

- thirds of the area ;

● = effect; – = no effect.

Source: Søndergaard and Blom (1979).

14

15 

### Shibata et al., 1989a, b

Male F344 rats (20/group) were exposed to 0 or 0.5% (w/w) biphenyl in the diet for 16

### 52 DRAFT

- DO NOT CITE OR QUOTE 24 week s (Shibata et al., 1989a). After 4 week s, 5 rats/group were injected with 100 mg /kg 1



### 5-bromo-2- deoxyuridine (BrdU) and sacrificed 1 hour later. One kidney from each rat was 2



### processed for immune-histopathologic identification of BrdU as an index of cell proliferation, 3



### while the second kidney was processed for light and scanning electron microscopic examination. 4



### The remaining rats were sacrificed after 8, 16, and 24 we ek s to monitor further development of 5



### morphological alterations in the renal papilla and pelvis. Survival was unaffected by treatment 6



### and biphenyl- treated animals showed no adverse clinical signs. The study authors reported that 7



### treatment resulted in sig nificantly lower mean body weight compared to controls; food 8



### consumption was unaffected and water consumption was slightly higher than that of controls. 9



### There were no significant treatment

- related effects on labeling indices of cell proliferation (BrdU 10



### in corporation) in renal papilla or pelvic epithelia and no histopathologic lesions of the renal 11



### papilla and pelvis were evident. Focal calcification of the renal medulla was observed in the 12



### majority of the biphenyl-treated rats. The study authors stated th at urinalysis demonstrated an 13



### association between biphenyl exposure and microcalculi formation, but provided no additional 14



### information regarding urinalysis results. 15



### In a similar study (Shibata et al., 1989b), a group of 10 male F344 rats received 0.5% 16



### (w/w ) biphenyl in the diet for up to 8 week s. Based on U.S. EPA (1988) subchronic reference 17



### values for body weight and food consumption in male F344 rats, the dose was estimated at 18



### 500 mg/kg-day. At 4 week s, five rats/group were processed as described by Shi bata et al. 19



### (1989a) for assessment of BrdU incorporation, but in the urinary bladder rather than in the 20



### kidney. During week 4, urine samples were taken for urinalysis. At terminal sacrifice, urinary 21



### bladder tissues were processed for scanning electron mi croscopic examinations. There were no 22



### treatment -related deaths or adverse clinical signs. Although food and water consumption were 23



### similar to controls, biphenyl -treated rats showed a consistent reduction in average body weight 24



### (229 vs. 247 g after 4 week s and 300 vs. 327 g after 8 weeks, for treated vs. controls, 25



### respectively [ p < 0.01]). A greater than fourfold increase in the BrdU labeling index was 26



### observed in urinary bladder epithelium of the biphenyl- fed rats (mean percent labeling index of 27



### 0.58 ± 0.31 compared to 0.13 ± 0.09 in controls; p < 0.05). Urinalysis revealed numerous 28



### microcalculi in the urinary sediment of the biphenyl- treated rats. This condition, designated as 29



### “severe” by the authors, was associated with histopathological lesions of th e epithelium of the 30



### urinary bladder that included simple hyperplasia with moderate severity in 5/5 rats, moderate 31



### pleomorphic microvilli (5/5), moderate uniform microvilli (5/5), and the occurrence of ropey or 32



### leafy microridges (5/5), the latter condition designated as severe. Scanning electron microscope 33



### images of the luminal surface of bladder epithelial cells showed pleomorphic microvilli that 34



### varied in size and shape and the formation of microridges. 35

36

### 53 DRAFT

- DO NOT CITE OR QUOTE 4.4.3. Biphenyl as a Tumor Promoter 1



### Tamano et al., 1993 2



### Male B6C3F 1 

### mice (10 –20/group) received the bladder carcinogen BBN at 0 or 0.05% in 3



### the drinking water for 4 weeks followed by 0 or 1% biphenyl in the feed for 32 weeks. The mice 4



### were observed for clinical signs and body weight and food consumption were monitored. At 5



### 37-week terminal sacrifice, kidneys and urinary bladders were prepared for histopathological 6



### examination. No treatment-related clinical signs were observed. Mean body weight of the BBN 7



### + 1% biphenyl- treated mice was significantly ( p < 0.01) lower than that of mice receiving BBN 8



### treatment only (32.2 ± 1.8 vs. 38.4 ± 2.6 g). Biphenyl treatment did not result in increased 9



### incidences of simple hyperplasia or papillary or nodular dysplasia in the BBN

- initiated mice. 10



### Administration of 1% b iphenyl in the feed to eight mice for 8 weeks did not significantly affect 11



### indices of cell proliferation (BrdU incorporation) in urinary bladder epithelium. 12

13



### Shiraiwa et al., 1989 14



### In the initiation -promotion portion of a chronic toxicity study designed to assess the 15



### ability of biphenyl to promote carcinogenesis by EHEN in the kidney (see Section 4.2.1.2 for a 16



### detailed study description), male Wistar rats (25/group) received basal diet with either 0 or 0.1% 17



### dietary EHEN for 2 week s, followed by a basal diet containing either 0, 0.125, or 0.5% biphenyl 18



### for 34 weeks (Shiraiwa et al., 1989). At terminal sacrifice, gross pathologic examinations were 19



### performed. Kidney and urinary bladder were fixed; kidneys were sectioned transversely (10– 20



### 12 serial slices) and urinary bladders were cut into 4

– 6 serial slices. The authors used a 21



### computer-linked image analyzer to determine the incidence of kidney lesions and dysplastic foci. 22



### The presence of stones in the kidney and urinary bladder was assessed qualitatively using an 23



### infrared spectrophotometer. 24



### Based on reported values for mean daily biphenyl intake (mg biphenyl/rat) and average 25



### body weight (mean initial body weight + one- half the difference between mean initial and mean 26



### final body weight) for each study group, doses of biphenyl at the 0.125 and 0.5% dietary levels 27



### are estimated to have been 59.28 and 248.3 mg /kg-day, respectively, for rats on basal diet alone 28



### for the first 2 week s and 62.0 and 248.2 mg /kg-day, respectively, for rats on basal diet and 29



### EHEN for the f irst 2 week s. Stones were present in the kidney, ureter, and urinary bladder of 30



### high- dose rats irrespective of whether animals were initially exposed to the basal or 31



### EHEN -containing diet (combined incidences of 6/25 and 8/25, respectively). The incidence of 32



### rats with renal cell tumors after EHEN and subsequent biphenyl administration was lower than 33



### that of rats receiving EHEN followed by basal diet) (7/25 and 13/25, respectively). This finding 34



### indicates that biphenyl was not a promoter of renal cell tumors in male Wistar rats under the 35



### conditions of the study. 36

37

### 54 DRAFT

- DO NOT CITE OR QUOTE Kurata et al., 1986 1



### Male F344 rats (25/group) were exposed to 0.05% N-butyl-N- (4 -hydroxybutyl) 2



### nitrosamine (BBN, a bladder carcinogen) in the drinking water for 4 week s followed by diets 3



### containing either 0 or 0.5% biphenyl for 32 week s. One group of five rats received biphenyl 4



### without pretreatment with BBN. The rats receiving biphenyl either with or without pretreatment 5



### with BBN gained less weight than control rats or those receiving only BBN. Incidences of 6



### urinary bladder hyperplasia, papilloma, and carcinoma were 17/18 (94%), 15/18 (83%), and 7



### 11/18 (61%), respectively, in the group of rats that survived treatment of BBN followed by 8



### biphenyl, compared to 6/24 (25%), 3/24 (12%), and 0/24 (0%), respectively, in the rats receiving 9



### BBN only. These urinary bladder lesions were not seen in any of the five rats receiving biphenyl 10



### without BBN pretreatment. Urinary bladder calculi were found in 25% of the rats receiving 11



### BBN followed by biphenyl and in 12% of the rats receiving BBN only. Biphenyl was 12



### considered a urinary bladder tumor promoter in male F344 rats under the conditions of the study. 13

14



### Boutwell and Bosch, 1959 15



### Biphenyl was negative for tumor promotion in a skin-painting experiment in which the 16



### initiator was 0.3% 9,10-dimethyl-1,2- benzanthracene in b enzene (Boutwell and Bosch, 1959). In 17



### the 16/20 mice that survived the topical application of 20% biphenyl for 16 weeks, none had 18



### developed papillomas or carcinomas as a result of treatment. 19

20



### Ito et al., 1984 21



### Six

- week -old male F344 rats (20–30/group) were exposed to BBN in drinking water at 22



### 0.01 or 0.05% for 4 week s, followed by 0.5% biphenyl in the feed for 32 week s. Controls 23



### receiving only BBN and controls receiving only biphenyl were included. After sacrifice, urinary 24



### bladders were prepared for light microscopic assessment of neoplastic and cancerous lesions. 25



### The study authors reported that biphenyl exhibited moderate bladder cancer- promoting activity, 26



### but data to support this finding were not included in the study report. 27

28



### 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF 29



### ACTION 30



### 4.5.1. Effects on the Urinary Tract of Rats 31



### Urinary tract effects in male rats chronically exposed to biphenyl in the diet are 32



### associated with the formation of urinary bladder calculi. Mechanistic studies pe rformed by 33



### Ohnishi and coworkers (Ohnishi et al., 2001, 2000a, b) were designed to identify urinary 34



### metabolites of biphenyl, to assess conditions leading to calculi formation, and to determine the 35



### composition of urinary crystals and calculi. Ohnishi et al. (2000a) identified sulphate conjugates 36



### of mono - and dihydroxy biphenyl metabolites in urine and urinary crystals from F344 rats treated 37

### 55 DRAFT

- DO NOT CITE OR QUOTE with biphenyl and KHCO 3 

### (to elevate the pH and K + 

### concentration of the urine). Male F344 rats 1



### (five per group) were a dministered a diet containing 1.6% biphenyl and 5% potassium 2



### bicarbonate for 7 days (Ohnishi et al., 2000a). Urine was collected on days 6 and 7 and pooled. 3



### Urinary crystals (i.e., precipitates) were collected and dissolved in acetonitrile and were analy zed 4



### by HPLC to identify metabolites or by inductively coupled plasma spectroscopy to identify 5



### inorganic elements. As shown in Table 4-15, biphenyl sulphate conjugates in the urine consisted 6



### primarily of 3,4-dihydroxybiphenyl-3-O-sulphate (40.9% of the total biphenyl sulphate 7



### conjugates) and 3

- hydroxybiphenyl (23.4%). No bisulphates were observed (Ohnishi et al., 8



### 2000a). In contrast; about 90% of sulphate conjugates in urinary crystals were 4-hydroxy9



### biphenyl-O-sulph ate , and only 3.9 and 1.06% were 3,4-dihydroxybiphenyl-3-O- sulphate and 10



### 3-hydroxybiphenyl, respectively. In a follow -up study, Ohnishi et al. (2000b) evaluated the 11



### composition of urinary calculi in male and female rats exposed to 4,500 ppm biphenyl in the diet 12



### for 104 weeks. Urinary calculi in chronically exposed mal e rats were composed mainly of 13



### 4-hydroxybiphenyl-O- sulphate, whereas calculi in female rats were composed primarily of 14



### 4-hydroxybiphenyl and potassium sulphate, the hydrolysis products of 4-hydroxybiphenyl15



### O- sulphate ( Ohnishi et al., 2000b). 16

17



### Table 4- 15. Content of biphenyl sulphate conjugates in urine and urinary
crystals from F344 rats treated with biphenyl and potassium bicarbonate (to
elevate the pH and K + 

### concentration of the urine)

Biphenyl sulph ate conjugates Urine (%) Urine crystals (%)

2

- Hydroxybiphenyl

- O

- sulphate 3.32 a 0.06

3

- Hydroxybiphenyl

- O

- sulphate 23.37 1.06

4

- Hydroxybiphenyl

- O

- sulphate 11.94 89.45

4,4’

- Dihydroxybiphenyl

- O

- sulphate 7.17 3.11

2,5

- Dihydroxybiphenyl

- O

- sulphate 5.62 0.02

3,4

- Dihydroxybiphenyl

- 3

- O

- sulphate 40.88 3.90

3,4

- Dihydroxybiphenyl

- 4

- O

- sulphate 2.27 2.28

2,3

- Dihydroxybiphenyl

- 3

- O

- sulphate 5.43 0.12

a The component f raction (%) for each of the sulph ate conjugates was estimated from the ratio of the liquid
chromatography tandem mass spectrometry peak area of the sulfate to the total area.

Source : Ohnishi et al. (2000a)

.



### Using the same experimental protocol as that described in Ohnishi et al. (2000a), but

adding potassium bicarbonate (5%), potassium chloride (5%)

, or sodium bicarbonate ( 8 %) to t 18

19



### he 20



### diet for 13 weeks , Ohnishi et al. (2001) reported hydronephrosis and blood in the urine only in 21



### those animals receiving biphenyl plus potassium bicarbonate. Feed consumption was not 22



### affected by the dietary additions, while water intake was greatly increased in all groups of 23



### animals that received biphenyl and/or salts. Neither high urinary potassium levels alone, as 24

### 56 DRAFT

- DO NOT CITE OR QUOTE induced by cofeeding of potassium chloride, nor high urinary pH alone, as induced by cofeeding 1



### of sodium bicarbonate, were sufficient to cause kidney damage. It was concluded that a 2



### combination of high urinary pH and high potassium levels was necessary to cause precipitation 3



### of biphenyl sul ph ate . It was proposed that the crystalline precipitate caused obstruction that led 4



### to hydronephrosis or damaged the transitional epithelium in the bladder causing hyperplasia. 5

6



### 4.5.2. Effects on the Liver of Mice 7



### Based on findings of biphenyl-induced liver tumo rs in female BDF 1 

### mice administered 8



### high dietary concentrations of biphenyl for 2 year s (Umeda et al., 2005), a 13-week oral study 9



### was performed to assess whether peroxisome proliferation might be induced (Umeda et al., 10



### 2004). Groups of male and female BDF 1 

### mice (10/sex/group) were administered biphenyl in the 11



### diet at six different concentrations ranging from 500 to 16,000 ppm. Biphenyl concentrations 12



### ≥8,000 ppm resulted in significantly decreased final body weights of males and females. 13



### Significantly increased liver weights were noted in the 8,000 and 16,000 ppm groups of female 14



### mice. Evidence of peroxisome proliferation was restricted to the 16,000 ppm group of female 15



### mice and included light microscopy findings of clearly enlarged hepatocytes filled wi th 16



### eosinophilic fine granules and electron microscopy confirmation that the granules corresponded 17



### to increased numbers of peroxisomes. Light microscopy of livers from rats exposed to 18



### concentrations ≤8,000 ppm showed no indications of proliferation of pero xisomes. There were 19



### no indications of other biphenyl- induced liver effects in any of the groups of male mice. 20

21



### 4.5.3. Estrogenic E ffects 22



### Several biphenyl derivatives display estrogenic activity. Schultz et al. (2002) used the 23



### Saccharomyces cerevisiae/LacZ reporter assay to study the estrogenic activity of 120 chemicals 24



### to identify chemical structures that impart estrogenic activity to a molecule. Chemicals without a 25



### hydroxy group, among them biphenyl, were inactive in this assay. The estrogenic activit ies of 26



### biphenyl metabolites in this assay were 4,4 ′ -dihydroxybiphenyl (median effective concentration 27



### = (2.6 **×** 10 -7 

### M) > 4 -hydroxybiphenyl (1.2 **×** 10 -6 

### M) > 3 -hydroxybiphenyl (9.2 **×** 10 -6 

### M) 28



### > 2- hydroxybiphenyl (1.8 **×** 10 -5 

### M). Estrogenic activities of the corresponding hydroxylated di-, 29



### tri -, or tetrachlorobiphenyl metabolites were approximately two orders of magnitude higher, 30



### provided there were no chlorines and hydroxy groups on the same ring. 31



### Kitamura et al. (2003) used MCF

- 7 cells transfected with an estrogen receptor

- luciferase 32



### reporter construct to test biphenyl and its metabolites for estrogenic activity. The starting point 33



### for this investigation was the structural similarity between hydroxylated metabolites of biph enyl 34



### and of 2,2-diphenyl propane, the 4,4 ′ -dihydroxy metabolite of which is bisphenol A, a known 35



### endocrine disrupter. Biphenyl per se displayed no estrogenic activity in this assay. Metabolites 36



### of biphenyl formed by liver microsome preparations were iden tified after solvent extraction from 37

### 57 DRAFT

- DO NOT CITE OR QUOTE reaction media by HPLC-MS. The compounds were also tested in an in vitro competitive 1



### estrogen receptor binding assay. The biphenyl metabolites, 2-, 3-, 4-hydroxybiphenyl, and 2



### 4,4 ′ -dihydroxybiphenyl, all exhibited estr ogenic activity when the cell culture contained 3



### microsomes from 3 -methylcholanthrene-induced rat livers and to a lesser extent, phenobarbital4



### induced rat livers, in the presence of NADPH. In the competitive estrogen receptor binding 5



### assay, 4,4 ′ -dihydroxybiphenyl displayed weak binding affinity, while biphenyl and its 6



### monohydroxy metabolites did not show any activity. 4,4 ′ -Dihydroxybiphenyl is one of two 7



### major biphenyl metabolites in rats and mice (Halpaap -Wood et al., 1981 a, b ; Meyer and 8



### Scheline, 1976), suggesting that high doses of biphenyl, in the form of this metabolite, might 9



### induce some minor estrogenic effect. 10

11



### 4.5.4. Effects on A poptosis 12



### Kokel and Xue (2006) tested a series of benzenoid chemicals (including mesitylene, 13



### cyclohexane, benzene, toluene, and biphenyl) for their ability to suppress apoptosis in the 14



### nematode, Caenorhabditis elegans , a model suitable for the characterization of carcinogens that 15



### act by way of apoptosis inhibition. The study included wild type and three strains of C. elegans 16



### mutants; the ced

- 3(n2438) mutant (which carries a partial loss -of-function mutation in the ced17



### 3 gene), the ced -3(n2273) mutant (also partly defective in cell death), and the ced-(n2433) 18



### mutant (a strong loss-of-function ced-3 mutant). Effects on apopto sis were assessed by counting 19



### the numbers of cells that should have died during embryogenesis, but inappropriately survived. 20



### The results indicated that these chemicals did not significantly affect apoptosis in wild type 21



### C. elegans . However, inhibition of apoptosis was apparent in mutant strains ced -3(n2438) and 22



### ced -3(n2273) exposed to benzene, toluene, or biphenyl. The study authors interpreted these 23



### r esults as indicative of apoptosis- inhibitory activity that does not depend on mutations in a 24



### specific ce ll

- death gene. A lack of apparent apoptosis- inhibitory activity in the strong loss -of25



### function ced-3(n2433) mutant was interpreted as indicative that inhibition of apoptosis, rather 26



### than transformation of cell fates, caused the increase in extra cell obse rved in the other two 27



### mutant strain s. All three chemicals also displayed embryotoxicity. Biphenyl and naphthalene 28



### were both shown to suppress apoptosis in C. elegans mutant strain ced -3(n2438) by causing 29



### overexpression of the CED- 3 caspase. The authors proposed that benzenoid chemicals that can 30



### form quinones suppress apoptosis in C. elegans via this reactive intermediate, although this was 31



### proven only for benzene, toluene, and naphthalene. 32



### Regulation of apoptosis during embryogenesis is critical , and a recent study by Tan et al. 33



### (2011) show ed that inhibition of apoptosis during this stage of development may have 34



### detrimental effects on the nervous system. N o literature was identified , however, that 35



### specifically supports an association between inhibition of apoptosis by biphenyl and effects on 36



### embryogenesis. 37

38

### 58 DRAFT

- DO NOT CITE OR QUOTE 4.5.5. Mitochondrial Effects 1



### Nishihara (1985) assessed the effects of biphenyl on the respiratory and energy linked 2



### activities o f rat liver m itochondria that had been isolated from male Wistar rats. Biphenyl (5– 3



### 60 μ g/mL in acetone solven t ) was added to liver mitochondria and effects on rates of succinate 4



### oxidation and α

- ketoglutarate/malate oxidation were assessed by measuring oxygen 5



### consumption. Solvent controls were included in the study. Biphenyl significantly inhibited state 6



### 3 respiration at concentrations ≥20 μ g/mL . The inhibition was greater for α

- ketoglutarate/malate 7



### oxidation than for succinate oxidation. State 4 respiration was significantly stimulated by 8



### biphenyl; the effect was greater in magnitude for succinate than for α

- ketoglutarate/malate 9



### oxidation. Biphenyl also altered mitochondrial membrane permeability, as evidenced by the 10



### instantaneous release of endogenous K + 

### , leading to instantaneous dissipation of the 11



### mitochondrial membrane potential. Inhibition of state 3 respiration is generally considered to 12



### reflect an interference with electron transport. The study author suggested that the biphenyl13



### induced stimulation of state 4 respiration may be explained by an uncoupling action on 14



### respiration. 15

16



### 4.5.6 . Genotoxicity 17



### Biphenyl. The results of genotoxicity studies of biphenyl are summarized in Table 4-16. 18



### Reverse mutation assays using Salmonella typhimurium and Escherichia coli provide 19



### consistently negative results both with and without the addition of a mammalian metabolic 20



### activation system (rat S9 mix). Biphenyl was not genotoxic in a host- mediated deoxyribonucleic 21



### acid ( DNA ) repair assay of E. coli in the presence of S9 (Hellmér and Bolcsfoldi, 1992). In rec 22



### assays of Bacillus subtilis , two studies reported negative results both with and without S9 23



### (Garrett et al., 1986; Kojima and Hiraga, 1978), one study reported negative results without S9 24



### (Kawachi et al., 1980) and one study reported equivocal results with S9 (Hanada, 1977). 25



### Biphenyl was reported to induce mitotic recombination both with and without S9 in 26



### Saccharomyces cerevisiae strain D3 (Pagano et al., 1988), but not in S. cerevisiae strain Diploid 27



### D7 (Garrett et al., 1986). 28

29

### 59 DRAFT

- DO NOT CITE OR QUOTE Table 4 -16 . Genotoxicity test results for biphenyl

Strain or test Test substance Metabolic

activation a

Reference Organism system Endpoint concentrations +S9

– S9

Bacterial and prokaryotic assays

S. typhimurium TA98, 100 Mutation NS

– NT Bos et al., 1988

TA98,

1538 100, 1535, NS

– NT Purchase et al., 1978

TA98, 100 NS

– – Kawachi et al., 1980

TA97, 98, 100 1

– 100 μg/plate

– – Brams et al., 1987

TA98, 1535 5– 1,000 b μg/plate

– NT Narbonne et al., 1987

TA98, 100,

YG1041 b 5

– 250 μg/plate

– – Chung

2002 and Adris, 2003,

TA98, 100,

1535,1537, 1538,

C3076, D3052, G46 0.1

– 1,000 μg/mL

– – Cline

1977 and McMahon,

TA98, 100, 1537 1– 10 5 μg/mL b

– – Garrett

Waters et

et al.,

al., 1986;

1982

TA98, 100 25

– 800 μg/plate

– – Glatt et al., 1992

TA1535, 1536,

1537

- 1, 1538 -1 Units provided
Japanese in

– Hanada, 1977

TA98, 100 1

– 1,000 μg/plate

– – Kojima

1978 and Hiraga,

TA98,

1537 100, 1535, 1

– 100 μg/plate

– – Haworth et al., 1983

TA98, 100 0.15

– 2 μg/plate

– – Houk et al., 1989

TA98, 100,

1537, 2637 1535, Up to 5 mg/plate

– NT Ishidate et al., 1984

TA98, 100,

1535, 1537,

2636 1532,

1538, 0.1

– 500 b μg/plate

– – Pagano
1983) et al., 1988;

C3076, D3052,

G46, TA98, 1000,

1535, 1537, 1538 10 4

- fold range

– – Probst et al., 1981

TA98, 100,

1537, 1538, 1535,

1978 77 μg/plate

– – Westinghouse, 1977

E. coli Chromotest Mutation 2.4

– 154 μg/mL

– – Brams et al., 1987

WP2, WP2 uvrA

– 1

– 1,000 μg/mL

– – Cline

1977 and McMahon,

WP2, WP2 uvrA

– 10 4

- fold range

– – Probst et al., 1981

WP uvrA

–

, polA

– 1

– 10 5 μg/mL

– – Garrett et al., 1986

B/γ B/γ – WP 2 try

,

– WP 2 try – hcr Units provided
Japanese in

– Hanada, 1977

B/γ

– WP 2 try – hcr ≤1,000 μg/mL b

– – Kojima

1978 and Hiraga,

K

- 12 uvrB/recA +

K

- 12 uvrB/recA

– Host

- mediated

DNA repair Up to 161 mM

– NT Hellmér and

Bolcsfoldi, 1992

### 60 DRAFT

- DO NOT CITE OR QUOTE Table 4 -16 . Genotoxicity test results for biphenyl

Metabolic

Strain or test Test substance activation a

Reference Organism system Endpoint concentrations +S9

– S9

B. subtilis Not given Rec assay NS NT

– Kawachi et al., 1980

recA

– 1

– 10 5 μg/mL

– – Garrett et al., 1986

+ ) H17 (rec

– ) M45 (rec Units provided
Japanese in +/

– +/

– Hanada, 1977

+ ) H17 (rec

– ) M45 (rec 1 or 10 mg

– – Kojima

1978 and Hiraga,

S. cerevisiae D3 Mitotic

recombination 1

– 10 5 μg/mL

– – Garrett et al., 1986

Diploid D7 10

- 5

10 -5 or 10

- 3

M a M + + Pagano et al., 1988

Tests with cultured mammalian cells

Hamster V79 Mutation 5

– 100 μg/mL
100 μg/mL b + – Glatt et al. (1992)

DON SCE 0.1

– 1 mM

– Abe and Sasaki, 1977

CHL NS NT

– Kawachi et al., 1980

CA NS NT

– Kawachi et al., 1980

Up to 25 μg/mL

– NT Ishidate et al., 1984

Up to 60 μg/mL

– NT Ishidate and

1977 Odashima,

75

– 125 μg/mL +

– Sofuni et al., 1985

DON 0.1

– 1 mM

– Abe and Sasaki, 1977

Kidney Cell

transformation 0.025

– 250 μg/mL

– NT Purchase et al., 1978

V79 ≤100 μg/mL +

– Glatt et al., 1992

CHO CA 3.1

– 200 μg/mL
100 μg/mL a – Yoshida et al., 1978

Human Peripheral blood
lymphocytes SCE 10

– 70 μL/mL NT +/

– Rencüzoğullari

2008 et al.,

CA 10

– 70 μL/mL NT +

Micronuclei 10

– 70 μL/mL NT +

Diploid lung

fibroblast Cell

transformation 0.025

– 250 μg/mL

– NT Purchase et al., 1978

Liver

- derived cells 0.025

– 250 μg/mL

– NT Purchase et al., 1978

HSBP diploid

fibroblast lung DNA repair 100 μM

_ Snyder

1985 and Matheson,

WI

- 38 lung

fibroblasts UDS 1– 10 5 μg/mL

– – Garrett

Waters et

et al.,

al., 1986;

1982

Rat Primary hepatocyte 0.01

– 100 μM

– Hsia et al., 1983a, b

100 μM

– Probst et al., 1981

Excision
repair 0.01

– 1,000 μM

– Brouns et al., 1979

DNA repair 100 μM c

– Williams et al., 1989

Immortalized liver
epithelial cells HGPRT

mutation 100 μM

– Williams, 1980

### 61 DRAFT

- DO NOT CITE OR QUOTE Table 4 -16 . Genotoxicity test results for biphenyl

a Metabolic

Strain or test Test substance activation a

Reference Organism system Endpoint concentrations +S9

– S9

Mouse L5178Y/TK +/

– Mutation 50

– 500 μM
150 μM a – Garberg et al., 1988

50

– 1,500 μM
500 μM a +

98.7

– 395 μM
98.7 μM a + d Wangenheim and

Bolcsfoldi, 1988, 1986

5

– 60 μM
10 μM a + d

In vivo tests

Rat Bone marrow SCE NS

– Kawachi et al., 1980

CA NS

–

Mouse CD

- 1/stomach,
colon, liver, kidney,
bladder, lung, brain,

bone marrow DNA damage,
Comet assay 10

– 2,000 mg/kg + Sasaki et al., 2002

Mouse CD

- 1/stomach,
liver, kidney,
bladder, lung, brain,

bone marrow DNA damage,
Comet assay 2,000 mg/kg + Sasaki et al., 1997

Silkworm Mutation NS

– Kawachi et al., 1980

Lowest concentration resulting in cytotoxicity.

b Lowest concentration resulting in precipitation.

c Highest concentration not causing cytotoxicity

. d Positive result only at cytotoxic concentrations.

CA = chromosomal aberrations; CHL = Chinese hamster lung; CHO = Chinese hamster ovary;

HGPRT = hypoxanthine guanine phosphoribosyl transferase; NS = not specified; NT = not tested; +/

– = weakly
positive or equivocal result; empty cell = no information available; SCE = si ster chromatid exchanges;

UDS = unscheduled DNA synthesis

1

2 

### Assays of biphenyl-exposed cultured mammalian cells provide mixed results. In the

3 

### absence of exogenous metabolic activation, biphenyl produced negative results for sister

4 

### chromatid exchanges (SCE) and/or chromosomal aberrations (CA) in the DON Chinese hamster

5 

### cell line (Abe and Sasaki, 1977) or Chinese hamster lung (CHL) fibroblasts (Sofuni et al., 1985;

6 

### Kawachi et al. , 1980); cell transformations in Chinese hamster kidney cells (Pur chase et al.,

7 

### 1978) and human diploid lung fibroblasts (Purchase et al., 1978); unscheduled DNA synthesis,

8 

### excision repair, and DNA repair in rat hepatocytes (Brouns et al., 1979); and hypoxanthine

9 

### guanine phosphoribosyl transferase (HGPRT) mutation in rat immortalized liver epithelial cells

10 

### (Williams, 1980). In the presence of S9 mix, biphenyl produced negative results for CAs in

11 

### CHL fibroblasts (Ishidate et al., 1984; Ishidate and Odashima, 1977) or Chinese hamster ovary

12 

### (CHO ) cells (Yoshida et al., 1978 ); DNA repair in human HSBP diploid lung fibroblasts (Snyder

### 62 DRAFT

- DO NOT CITE OR QUOTE and Matheson, 1985); and unscheduled DNA synthesis in human lung WI-38 lung fibroblasts 1



### (with or without S9; Garrett et al., 1986). 2



### Positive results were obtained for CA in CHL fibroblasts (Sofuni et al., 1985) and 3



### mutations in Chinese hamster V79 cells (Glatt et al., 1992) in the presence, but not absence, of 4



### S9. Biphenyl induced forward mutations in mouse L5178Y/TK +/- 

### lymphoma cells with and 5



### without S9 (Wangenheim and Bolcsfoldi, 1988, 19 86); another study provided similar results in 6



### the presence, but not the absence, of S9 (Garberg et al., 1988). Significant increases in SCE 7



### (< twofold higher than solvent controls), CA (two- to fourfold higher than solvent controls), and 8



### micronuclei (app roximately 2.5- fold higher than solvent controls) were reported in human 9



### peripheral blood lymphocytes exposed to biphenyl for 24–48 hours at concentrations ≥50 μL/mL 10



### (Rencüzoğullari et al., 2008). 11



### Evaluations of the potential genotoxicity of biphenyl in vivo have been performed in rats, 12



### mice, and silkworms. Biphenyl did not induce SCE or CA in bone marrow cells of rats or 13



### mutations in silkworms, but limited information is available for these studies (Kawachi et al., 14



### 1980). In a Comet assay, positive results were reported for DNA damage in stomach, blood, 15



### liver, bone marrow, kidney, bladder, lung, and brain cells of CD- 1 mice administered single 16



### doses of 2,000 mg biphenyl/kg (Sasaki et al., 2002, 1997). It is unknown if the DNA damage 17



### was caused by direct reaction with biphenyl or its metabolites, or by indirect damage from 18



### cytotoxicity or ROS generated from redox cycling of hydroquinone metabolites. 19



### Biphenyl metabolites. Table 4 -17 summarizes results from genotoxicity tests of several 20



### biphenyl metabolites, 2-hydroxybiphenyl (also known as o -phenylphenol), 4-hydroxybiphenyl 21



### (the principal metabolite of biphenyl), and 2,5-dihydroxybiphenyl. 2-Hydroxybiphenyl and its 22



### sodium salt have received the most research attention because they are used as fungicides an d 23



### anti-bacterial agents and have been found to cause urinary bladder tumors in male F344 rats with 24



### chronic exposure to high concentrations in the diet (see Balakrishna et al., 2002; Kwok et al., 25



### 1999; Smith et al., 1998 for review). 26

27

### 63 DRAFT

- DO NOT CITE OR QUOTE Table 4 -17. Genotox icity test results for biphenyl metabolites

Metabolic

Organism Strain or test

system Endpoint Test substance

concentrations activation a

Reference +S9

– S9

2- Hydroxybiphenyl in vitro tests

S. typhimurium TA98, TA100 Mutation NS

– – Kawachi et al., 1980

TA98,

1537 100, 1535, 3.3

– 250 μg/plate

– – Haworth et al., 1983

TA98, 100 1

– 1,000 μg/plate

– – Kojima and Hiraga, 1978

TA97a, 102 1

– 100 μg/plate

– – Fujita et al., 1985

TA98, 100,

1537, 2637 1535, Up to 0.5 mg/plate

– NT Ishidate et al., 1984

TA98, 100 NS +/

– +/

– Nishioka

1978 and Ogasawara,

TA1535,

1538 -1

TA1536 1537

- 1, Units provided
Japanese in +/

–

+ Hanada, 1977

E. coli B/γ B/γ – WP 2 try – hcr

– WP 2 try 1

– 1,000 μg/mL
1,000 μg/mL a +/

– +/

– Kojima and Hiraga, 1978

WP2 lacking

catalase and Streptomycin

resistance 0– 10 μM NT + Tani et al., 2007

superoxide

dismutase mutation

WP2, WP2 uvrA

–

, CM571, WP100 DNA repair NS + + Nishioka

1978 and Ogasawara,

B. subtilis Not given Rec assay 10

– 10,000 mg/plate

– – Kawachi et al., 1980

+ ) H17 (rec

– ) M45 (rec Units provided
Japanese in + + Kojima and Hiraga,

Hanada, 1977 1978;

Hamster CHL CA NS NT

– Kawachi et al., 1980

Up to 0.05 mg/mL

– NT Ishidate et al., 1984

CHO 3.1

– 200 μg/mL
94 μg/mL a – Yoshida et al., 1978

Rat Liver DNA DNA adducts,

[ 32 P]

- post labeling

method 1 mM, in presence

of rat skin
homogenate, CYP,

or prostaglandin
synthase activation

systems + b Pathak and Roy, 1993

2- Hydroxybiphenyl in vivo tests

Rat Bone marrow SCE NS

– Kawachi et al., 1980

F344/bladder
epithelium Micronuclei

Hyperdiploidy/
hypodiploidy 2,000 ppm

14 days in diet, +

– Balakrishnan et al., 2002

Cell
proliferation +

### 64 DRAFT

- DO NOT CITE OR QUOTE Table 4 -17. Genotox icity test results for biphenyl metabolite s

Organism Strain or test

system Endpoint Test substance

concentrations Metabolic

activation a

Reference +S9

– S9

Rat F344/bladder
epithelium DNA damage,

alkaline elution

assay 1,000 or 2,000 ppm,

sodium salt in diet

for 3 months; no
damage at 250 or
500 ppm + Morimoto et al., 1989

Mouse CD

- 1/stomach,

colon, liver,
kidney, bladder,
lung DNA damage,
Comet assay 10

– 2,000 mg/kg + Sasaki et al., 2002

Mouse CD

- 1/brain,

marrow bone DNA damage,
Comet assay 10

– 2,000 mg/kg

– Sasaki et al., 2002

Mouse CD

- 1/stomach,
liver, kidney,
bladder, lung DNA damage,
Comet assay 2,000 mg/kg + Sasaki et al., 1997

Mouse CD

- 1/brain,

marrow bone DNA damage,
Comet assay 2,000 mg/kg

– Sasaki et al., 1997

Mouse CD

- 1/skin DNA adduct,

[ 32 P]

- post labeling

method 10 or 20 mg applied

to skin + Pathak and Roy, 1993

Rat F344/bladder
epithelium DNA adduct,

[ 32 P]

- post labeling

method

Cell
proliferation 800

– 12,500 ppm

diet in

–

+ Smith et al., 1998

Rat F344/bladder
epithelium DNA binding 15

– 1,000 mg/kg by
gavage, labeled with

[ 14 C] -2- hydroxy

biphenyl, uniformly
labeled in phenol
ring – Kwok et al., 1999

Silkworm Mutation NS

– Kawachi et al., 1980

4- Hydroxybiphenyl in vitro tests

S. typhimurium TA98

TA1535 Mutation 5

– 1,000 μg/plate

c 1,000 μg/plate +

– NT

NT Narbonne et al., 1987

TA1535, 1536,

1537

- 1, 1538

- 1 Units provided
Japanese in

– Hanada, 1977

B. subtilis + ) H17 (rec

– ) M45 (rec Rec assay Units provided
Japanese in

– – Hanada, 1977

2,5

- Dihydroxybiphenyl in vitro or in vivo tests

Human DNA fragments
from plasmid
pbcNI DNA damage,
Comet assay 0.1 mM + d Inoue et al., 1990

### 65 DRAFT

- DO NOT CITE OR QUOTE Table 4 -17. Genotox icity test results for biphenyl metabolites

Metabolic

Organism Strain or test

system Endpoint Test substance

concentrations activation a

Reference +S9

– S9

Rat F344/bladder
epithelium DNA damage,

alkaline elution

assay 0.05% injected
intravesically into

bladder wall e

– Morimoto et al., 1989

Mouse CD

- 1/skin DNA adduct, 10 or 20 mg applied + Pathak and Roy, 1993

[ 32 P]

- post to skin
labeling

method

a Lowest concentration resulting in cytotoxicity.

b Metabolic activation system derived from rat skin homogenate.

c Lowest concentration resulting in precipitation.

d Positive response only in the presence of Cu(II)

e Injection with 0.05% or 0.1% phenylbenzoquinone, a metabolite of 2,5

- dihydroxybiphenyl, produced DNA
damage at concentrations of 0.05 or 0.1%, but not at 0.005 or 0.0005%.

NS = not specified; NT = not tested; +/

– = weakly positive or equivocal result; empty cell = no information

available

1

2 

### In bacterial mutagenicity tests or in vitro mammalian tests of 2

- hydroxybiphenyl, results

3 

### were mostly negative or equivocal, b ut other tests with bacterial systems suggest that oxidative

4 

### DNA damage following metabolism of 2-hydroxybiphenyl to 2,5-dihydroxybiphenyl is possible

5 

### (see Table 4 -17 for references). 2 -Hydroxybiphenyl induced DNA repair in E. coli strains both

6 

### with and without S9 (Nishioka and Ogasawara, 1978). Tani et al. (2007) provided evidence that

7 

### redox cycling of a semiquinone/quinone pair causes oxidative DNA damage following exposure

8 

### of a mutant E. coli strain (WP2, lacking catalase and superoxide dismutase) to 2-hydroxy9 

### biphenyl : 2-hydroxybiphenyl induced streptomycin resistance mutations in the mutant, but not

10 

### in the wild type. Exposure of B. subtilis to 2-hydroxybiphenyl both with and without S9 in the

11 

### rec assay yielded positive (Kojima and Hiraga, 1978; Hanada, 1977) and negative (Kawachi et

12 

### al., 1980) results. 2-Hydroxybiphenyl did not induce CAs in CHL fibroblasts without S9 in one

13 

### study (Kawachi et al., 1980), or with S9 in other studies of CHL fibroblasts (Ishidate et al., 1984)

14 

### and CHO cells (Yoshida et al., 1978).

15 

### Results from in vivo mammalian genotoxicity test systems provide limited evidence for

16 

### possible genotoxic actions (DNA damage and mic ronuclei formation) from 2 -hydroxybiphenyl

17 

### through its metabolites , 2,5- dihydroxybiphenyl and phenylbenzoquinone (Table 4

- 17).

18 

### DNA damage was detected by the Comet assay in the urinary bladder of CD

- 1 mice

19 

### administered single oral doses of 2,000 mg 2 -hyd ro xybiphenyl/kg, but it is unknown if the

20 

### damage was due to cytotoxicity, direct reaction of DNA with 2- hydroxybiphenyl or its

21 

### metabolites, or possible oxidative DNA damage from redox cycling of 2,5-dihydroxybiphenyl

22 

### (Sasaki et al., 2002, 1997). DNA damage was also detected in th e urinary bladder of male or

### 66 DRAFT

- DO NOT CITE OR QUOTE female rats intravesically injected with 0.05 or 0.1% phenylbenzoquinone, but not with injections 1



### of 0.05% 2-hydroxybiphenyl or 2,5-dihydroxybiphenyl, although DNA damage was found in 2



### urinary bladders fro m male F344 rats fed the sodium salt of 2- hydroxybiphenyl in the diet for 3



### 3 months at 1,000 or 2,000 ppm, but not at 500 or 250 ppm (Morimoto et al., 1989). Topical 4



### application of 10 or 20 mg of the sodium salt of 2-hydroxybiphenyl or 5 mg of 2,5-dihydroxy5



### biphenyl to the skin of female CD- 1 mice produced several DNA adducts in the skin that were 6



### detected by the [ 32 

### P] -post labeling technique (Pathak and Roy, 1993). Similar adducts were 7



### formed in vitro when DNA was incubated with 2-hydroxybiphenyl (1 mM) i n the presence 8



### metabolic activation from rat skin homogenates, a CYP system, or a prostaglandin synthase 9



### system (Pathak and Roy, 1993). In contrast, Smith et al. (1998), using a similar technique to that 10



### used by Pathak and Roy (1993), were unable to detect exposure-related DNA adducts in bladder 11



### epithelial tissue from male F344 rats fed 800, 4,000, 8,000, or 12,500 ppm 2- hydroxybiphenyl in 12



### the diet for 13 weeks. In this experiment, increased bladder cell epithelium proliferation (i.e., 13



### increased Brd U inco rporation) was observed at 8,000 and 12,500 ppm, dietary concentrations 14



### associated with the development of urinary bladder tumors in chronically exposed rats (Smith et 15



### al., 1998). Kwok et al. (1999) found no evidence of binding of radioactivity to DNA ext racted 16



### from the bladder epithelium of male F344 rats given single gavage doses of [ 14 

### C]-labeled 17



### 2-hydroxybiphenyl at 15, 50, 250, 500, or 1,000 mg/kg, but increased protein binding occurred 18



### with increasing doses of 250, 500, and 1,000 mg/kg. Kwok et al. (1999) noted that the increase 19



### in protein binding increased with increasing dose levels of 250, 500, and 1,000 mg/kg, in parallel 20



### with increasing incidence of bladder epithelial lesions (hyperplasia, papillomas, and carcinomas) 21



### in rats chronically exposed to 2-hydroxybiphenyl in the diet at 0, 269, and 531 mg/kg. 22



### Increased micronuclei (about three fold increase over controls) and increased cell 23



### proliferation (>200-fold increased incorporation of BrdU in DNA) were found in the bladder 24



### epithelium of male F344 rats exposed to 2% (2,000 ppm) 2-hydroxybiphenyl in the diet for 25



### 2 weeks, without evidence for hypo- or hyperploidy as assayed by fluorescence in situ 26



### hybridization with a DNA probe for rat chromosome 4 (Balakrishnan et al., 2002). Similar 27



### exposure to 2% NaCl or 2% 2 -hydroxybiphenyl + 2% NaCl , produced about two - or six -fold 28



### increases of micronuclei in the bladder epithelium, respectively, but neither treatment stimulated 29



### bladder epithelium cell proliferation to the same degree as 2% 2

- hydroxybiphenyl in the diet 30



### (Balakrishan et al., 2002). 2-Hydroxybiphenyl reportedly did not induce SCE in the bone 31



### marrow of rats, but exposure parameters were not specified in the report by Kawachi et al. 32



### (1980). The mechanism of 2-hydroxybiphenyl- induced micronuclei is not understood, but, as 33



### discussed by Balakrishan et al. (2002), possible mechanisms include: ( 1) DNA damage from 34



### ROS from redox cycling between 2,5-dihydroxybiphenyl and phenylbenzoquinone, 35



### (2) interference of the mitotic spindle through covalent modification of proteins, ( 3) inhibition of 36



### enzymes regulating DNA replication, or (4) micronuclei generation as a secondary response to 37



### cytotoxicity or regenerative hyperplasia. 38

### 67 DRAFT

- DO NOT CITE OR QUOTE Bacterial mutation assays of the major biphenyl metabolite, 4-hydroxybiphenyl, yielded 1



### negative results in all but one case that was accompanied by overt cytotoxic ity (Narbonne et al., 2



### 1987). 2,5- Dihydroxybiphenyl (i.e., phenylhydroquinone) caused in vitro damage to human 3



### DNA from plasmidpbcNI in the presence of Cu(II) (Inoue et al., 1990), DNA adducts when 4



### applied to mouse skin (Pathak and Roy, 1993), but did not cause DNA damage when injected 5



### intravesically into the urinary bladder of F344 rats at a concentration of 0.05% (Morimoto et al., 6



### 1989). 7



### In summary, the overall weight of evidence for biphenyl genotoxicity from short- term 8



### tests is negative or equivocal (Table 4 -16). Biphenyl did not induce mutations in a variety of 9



### bacterial test systems (in the absence or presence of exogenous metabolic activation), but in vitro 10



### assays of genot oxicity in mammalian test systems yielded a mix of negative and positive results, 11



### with positive results mostly in the presence of metabolic activation. In tests of clastogenic 12



### effects in mammalian systems, biphenyl induced SCE, CAs, and micronuclei in cultured human 13



### peripheral blood lymphocytes (Rencüzoğullari et al., 2008) and CAs in one assay of CHL 14



### fibroblasts in the presence, but not the absence, of rat liver metabolic activation (Sofuni et al., 15



### 1985). However, biphenyl did not induce clastogenic effe cts (in the presence of metabolic 16



### activation) in other assays with Chinese hamster fibroblasts (Ishidate et al., 1984; Ishidate and 17



### Odashima, 1977) or CHO cells (Yoshida et al., 1978). In the only adequately reported in vivo 18



### genotoxicity studies with biphenyl, single oral doses of 2,000 mg/kg of biphenyl or 19



### 2-hydroxybiphenyl induced DNA damage in several organs of CD-1 mice (including liver and 20



### bladder), but it is uncertain if the damage was due to a direct effect on DNA by biphenyl or its 21



### metabo lites or indirectly due to cytotoxicity or ROS generated by redox cycling of a 22



### hydroquinone metabolite of 2-hydroxybiphenyl (Sasaki et al., 2002, 1997). 23



### The overall weight of evidence for 2-hydroxybiphenyl genotoxicity suggests that 24



### oxidative D NA damage fr om redox cycling between 2,5-dihydroxybiphenyl and phenylbenzo25



### quinone is possible (Sasaki et al., 2002, 1997; Pathak and Roy, 1993; Morimoto et al., 1989), but 26



### no evidence for DNA adducts or DNA binding in urinary bladder epithelium tissue was found in 27



### ra ts following short-term (Kwok et al., 1999) or subchronic (Smith et al., 1998) oral exposure to 28



### 2-hydroxybiphenyl at high doses associated with the formation of urinary bladder tumors. 29



### Increased micronuclei in urinary bladder epithelium were detected in r ats exposed to 2% 30



### 2-hydroxybiphenyl or its sodium salt in the diet for 14 days (Balakrishnan et al., 2002). The 31



### mechanism of this clastogenic effect is uncertain, but could involve micronuclei formation in 32



### secondary response to cytotoxicity or regenerativ e cell proliferation, DNA damage from ROS 33



### generated from redox cycling of a hydroquinone metabolite, or protein modifications leading to 34



### mitotic spindle interference or inhibition of enzymes important in DNA replication. 35



### 4- Hydroxybiphenyl, the predominant metabolite of biphenyl, was not mutagenic in 36



### bacterial testing at noncytotoxic concentrations (Narbonne et al., 1987; Hanada, 1977). 37



### 2,5-Dihydroxybiphenyl (i.e., phenylhydroquinone) caused in vitro damage to human DNA from 38

### 68 DRAFT

- DO NOT CITE OR QUOTE plasmidpbcNI in the presence of Cu(II) (In oue et al., 1990) and DNA adducts when applied to 1



### mouse skin (Pathak and Roy, 1993), but did not cause DNA damage when injected intravesically 2



### into the urinary bladder of F344 rats at a concentration of 0.05% (Morimoto et al., 1989). 3

4



### 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS 5



### Table s 4- 18 and 4 -19 include the major studies and the observed effects for oral and 6



### inhalation exposure to biphenyl, respectively. 7

### 69 DRAFT

- DO NOT CITE OR QUOTE Table 4

- 18. Summary of major studies evaluating effects of biphenyl after oral administration in rats and mice

Species, strain Exposure route Dose (mg/kg

- d),

duration NOAEL
(mg/kg

- d) LOAEL
(mg/kg

- d) Effect(s) at the

LOAEL Comments Reference

Subchronic studies

Rat, Long

- Evans (female, 8/group) Diet 0, 10, 30,

90 d or 100 ND ND Lower average plasma

BUN levels in all
exposed groups
(statisti c al significance
not reported and
biological significance
is uncertain). Dow Chemical

Co., 1953 a

Mice, BDF 1 (10/sex/group) Diet 0, 93, 347, 747,

1495, 1868, or 2989 M: 747

F: 1868 M: 1495 M: Decreased body
weight

. To overcome possible
problems with taste

aversion, animals in the Umeda et

2004 al.,

13 wks F: 2989 F: Decreased body
weight >10% and
histopathological
changes within the liver
(enlarged centrilobular
hepatocytes with
numerous eosinophilic
fine granules in the
cytoplasm). 3 highest dose groups

received lower doses

for the first 1

- 2 wks of
exposure followed by

the final dose for the
remaining time.

Chronic studies

Rats, F344 Diet M: 0, 36.4, 110, or M: 110 M: 378 M: Bladder tumors and Umeda et al.,
(50/sex/group) 378 transitional cell
hyperplasia. 2002

F: 0, 42.7, 128, or

438 F: 42.7 F: 128 F: Nonneoplastic
kidney lesions (simple

transitional cell

2 yrs hyperplasia in the renal
pelvis and hemosiderin
deposits).

### 70 DRAFT

- DO NOT CITE OR QUOTE Table 4

- 18. Summary of major studies evaluating effects of biphenyl after oral administration in rats and mice

Species, strain Exposure route Dose (mg/kg

- d),

duration NOAEL
(mg/kg

- d) LOAEL
(mg/kg

- d) Effect(s) at the

LOAEL Comments Reference

Rats, Wistar
(50/sex/group) Diet M: 0, 165, or 353

F: 0, 178, or 370 M: ND

F: ND M: 165

F: 178 Formation of kidney

stones associated with
pyelonephritis in both Shiraiwa et

1989 al.,

sexes.
75 wks

Rats, Wistar
(male, 25/group) Diet Control groups:
basal diet for 2 wks
followed by
exposure at 0, 59.28,

or 248.3 for 34 wks

Exposure groups: diet containing 0.1%

EHEN for 2 wks Control:

59.28

Exposure:

62 Control:

248.3

Exposure:

248.2 Formation o f kidney

stones associated with
pyelonephritis in both

sexes. Biphenyl did not

exhibit tumor
promoting

characteristics for the
kidney tumor initiator,

EHEN, under the
conditions of this study.

followed by 0, 62,

or 248.2 for 34 wks

Rats, albino
(weanling,
15/sex/group) Diet 0, 1, 4, 8, 42,

420, and 840

2 yrs 84, 84 420 Kidney effects
including tubular
atrophy and dilation
associated with cyst

formation and calculi Ambrose et

1960 al.,

formation in the renal
pelvis of both sexes.

Rats, albino
(male, 8/group) Diet 0, 250,

13 mo or 450 ND 250 Nonneoplastic
degenerative changes
in the liver, kidney,
thyroid, and
parathyroid resulting in
hyperplasia of liver,
kidney, and thyroid. Pecchiai

Saffiotti, and

1957

Rats, Sprague

Dawley
(12/sex/group) Diet 0, 7, 73,

2 yrs or 732 73 732 Renal effects (tubular

dilatation, calcification,

and intratubular

inflam m ation). Dow Chemical

Co., 1953 a

### 71 DRAFT

- DO NOT CITE OR QUOTE Table 4

- 18. Summary of major studies evaluating effects of biphenyl after oral administration in rats and mice

Species, strain Exposure route Dose (mg/kg

- d),

duration NOAEL
(mg/kg

- d) LOAEL
(mg/kg

- d) Effect(s) at the

LOAEL Comments Reference

Mice, BDF 1 (50/sex/group) Diet M: 0, 97, 291,

1050 or M: 97 M: 291 M: Decreased body
weight

. Umeda et

2005 al.,

F: 0, 134, 414,

1420

2 yrs or F: 134 F: 414 F: Nonneoplastic
effects (mineralization
in the kidney and
significantly increased
plasma ALT and AST
activities) in female

mice.

Mice, ddY
(female, 34

- 37/group) Diet 0

2 or 855

yrs 855 ND No adverse effects

observed at the highest

dose tested. Imai et al., 1983

Mice, hybrid
(2 strains,
18/sex/strain/group) Gavage (215
mg/kg body weight
in 0.5% gelatin) for

the first 3 wks,
followed by dietary
exposure for the
remaining time 0 or 91

18 mo 91 ND No evidence
carcinogenic of a

response. Two strains of F1
hybrid mice were
produced by mating

female C57BL/6 mice

with either male

C3H/Anf mice or male

AKR mice. Innes

NCI, et al.,

1968 1969

Dogs, Mongrel Capsule in corn oil 0, 2.5 or 25 ND ND ND Monsanto, 1956 a

5 d/wk for 1 yr
Monkey, Rhesus
(2 M/dose, 1F/dose) Diet 0, 0.01,
for 1 yr 0.1, or 1% ND ND ND Dow Chemical

Co. 1953 a

Reproductive and developmental studies

Rats, Wistar

(18

- 20/dose),

pregnant Gavage in corn oil 0, 125, 250, 500 or

1,000 on GDs 6

- 15. Dam: 500 Dam:

1000 Dam: maternal toxicity
(increased mortality),

increased in dead
fetuses and resorption. Khera et al., 1979

Offspring:

250 Offspring:

500 Offspring: missing and

unossified sternebrae,
delayed calvarial

ossification.

### 72 DRAFT

- DO NOT CITE OR QUOTE Table 4

- 18. Summary of major studies evaluating effects of biphenyl after oral administration in rats and mice

1 Species, strain Exposure route Dose (mg/kg

- d),

duration NOAEL
(mg/kg

- d) LOAEL
(mg/kg

- d) Effect(s) at the

LOAEL Comments Reference

Rats, Long Evans
(9 F/dose; 3 M/dose) Diet M: 9, 89, or 887

F: 10, 101, or 1006

continuous breeding M: ND

F: 101 M: ND

F: 1006 M: ND

F: decreased fertility,

litter size, reduced fetal
growth rate. The effects seen in the
high dose group may

be associated with
unpalatability and

resultant decreased

food intake. Dow Chemical

Co. 1953 a

Rats, Albino (F/M) Diet 0, 105, or 525 ND ND ND Ambrose et

1960 al.,

continuous breeding

2 a Report was not peer reviewed.

3 F = female; M = male; ND = not determined

### 73 DRAFT

- DO NOT CITE OR QUOTE Table 4

- 19. Summary of major studies evaluating effects of biphenyl after inhalation exposure in rats and mice

NOAEL LOAEL
Species, strain Dose (mg/m 3 ), duration 3 ) (mg/m 3 ) (mg/m Effect(s) at the LOAEL References

Rabbits, albino 300 mg/m 3 (7 hours/day, 5 days/wk) ND ND ND Deichmann et al., 1947
(3/dose) 64 days over 94 days period

Rats, Sprague

Dawley (10/dose)

Rabbits, albino 40 mg/m 3 (7 hours/day, 5 days/wk) ND ND ND
(3/dose) 46 days over 68 days period

Rats, Sprague

Dawley (6/dose)

Mice (12/dose) 5 mg/m 3 (7 hours/day, 5 days/wk) ND 5 Mice: upper respiratory tract irritation (acute
62 days over 92 days period emphysema, congestion, edema, bronchitis, lobular
Rats, Sprague

- pneumonia, and multiple pulmonary abscesses)
Dawley (4/dose)

Mice, CDI 0, 157.7, or 315.3 mg/m 3 (7 hours/day, ND 157.7 Histopathologic lung, liver and kidney lesions Sun Company Inc.,
(50/sex/dose) 5 days/week), 13 weeks (congested and hemorrhagic lungs, tracheal 1977 a
hyperplasia, and congestion and edema in the liver
and kidney) in both sexes.

1

a Report was not published. 2

ND = not determined 3

### 74 DRAFT

- DO NOT CITE OR QUO TE 4.6.1. Oral 1



### Biphenyl displays a relatively low acute oral toxicity, with LD 50 

### values in laboratory 2



### animals in the 2 –3 g/kg range (see Section 4.4.1). The major symptoms of biphenyl intoxication 3



### typically associated with short -term, high-dose oral exposure of animals are labored breathing, 4



### loss of body weight, and weakness. Following medium- or long -term oral exposure, reduced 5



### body weight gain has been reported frequently (Umeda et al., 2005, 2004, 2002; Ambrose et al., 6



### 1960; Dow Chemical Co., 1953) and attributed to low palatability of the feed (Ambrose et al., 7



### 1960; Dow Chemical Co., 1953); however, the feed intake data of Umeda et al. (2005) in mice 8



### did not support this notion. Increased liver and kidney weights were observed frequently 9



### (Umeda et al., 2004, 2002; Søndergaard and Blom, 1979; Ambrose et al., 1960; Monsanto, 1956; 10



### Dow Chemical Co., 1953). A reduction in hemoglobin levels of rats receiving biphenyl for 11



### 700 day s was reported (Ambrose et al., 1960). Signs of liver damage (increased serum activities 12



### of ALT, AST, AP, and LDH) were observed in mice (Umeda et al., 2005). Pathological effects 13



### on the urinary system dominated the spectrum of symptoms in dogs (Monsanto, 1956), rats 14



### (Umeda et al., 2002; Dow Chemical Co., 1953), and mice (Umeda et al., 2005). 15



### Urinary system effects, such as increased urine volume with increased specific gravity, 16



### polycystic changes, nephritis, and precipitatio n of free 4

- OH -biphenyl and its glucuronide in 17



### urine are commonly reported following oral exposure to biphenyl (Kluwe, 1982; Søndergaard 18



### and Blom, 1979; Monsanto, 1976; Booth et al., 1961). Calculi appeared in the urine of male rats 19



### only (Umeda et al., 2002; Ohnishi et al., 2001, 2000 a, b; Shibata et al., 1989b; Ambrose et al., 20



### 1960). Urothelial hyperplasia with increased indices of cell proliferation have been described in 21



### rats but not in mice and were attributed to irritation by calculi (Umeda et al., 2 005, 2002; Shibata 22



### et al., 1989b). Tubular dilatation and morphological changes in papillae and pelvis, kidney 23



### stones, obstructive pyelonephritis, tubular atrophy, fibrosis, and pelvic hyperplasia were 24



### observed (Shibata et al., 1989a, b; Shiraiwa et al., 1989; Takita, 1983; Kluwe, 1982; Booth et al., 25



### 1961). 26



### Increased incidences of fetuses with skeletal anomalies were reported fo llowing gavage 27



### administration of biphenyl to Wistar rats during gestation (Khera et al., 1979). A three

28



### generation study in rats (Dow Chemical Co., 1953) found general reproductive toxicity at high 29



### doses ( about 947 mg/kg-day ). 30

31



### 4.6.2. Inhalation 32



### In a case study of workers engaged in the production of biphenyl-impregnated paper, 33



### Häkkinen et al. (1973, 1971) observed liver damage (el evated levels of serum AST and ALT; 34



### incipient cirrhosis and fatty changes in biopsy specimens) and effects on the central and 35



### peripheral nervous systems (polyneuritic symptoms [abnormal EEGs and ENMGs], giddiness, 36



### fatigue) that were attributed to long

- term exposure to high concentrations of biphenyl. In one 37



### fatal case, autopsy revealed kidney and bone marrow damage and heart muscle degeneration, as 38

### 75 DRAFT

- DO NOT CITE OR QUO TE well as brain edema (Häkkinen et al., 1973, 1971). More recently the possibility has been 1



### discussed that lon g-term exposure to biphenyl might contribute to the onset of PD (Wastensson 2



### et al., 2006). The workplace conditions reported for these studies (Wastensson et al., 2006; 3



### Häkkinen et al., 1973, 1971) suggested that inhalation represented the predominant rou te of 4



### exposure, but dermal absorption as well as oral uptake (hand to mouth) might have occurred at a 5



### significant level. 6



### In mice, short-term biphenyl inhalation at concentrations as high as 5 4.75 ppm 7



### (345.5 mg/m 3 

### ) appeared to cause no symptoms (Sun Company Inc., 1977a). In another study, 8



### 3 rabbits, 4–6 rats, or 12 mice/group were exposed to biphenyl by inhalation for 7–13 weeks at 9



### concentrations ranging from 5 to 300 mg /m 3 

### (Deichmann et al., 1947). No adverse effects were 10



### observed in rabbits, while rats and mice showed irritation of mucous membranes and succumbed 11



### to high concentrations. Mice were far more sensitive than rats in these experiments, additionally 12



### showing congestion and hemorrhage of the lungs (Deichmann et al., 1947). Repeated exposure 13



### of mice to biphenyl at vapor concentrations of 25 or 50 ppm (157.75 or 315.5 mg/m 3 

### ) for 14



### 13 weeks resulted in high incidences of pneumonia and tracheal hyperplasia, and high inc idences 15



### of congestion and ede ma in the lungs , liver, and kidney ( Sun Co mpany Inc., 1977b). 16



### Reproductive or developmental studies using the inhalation route of exposure were not 17



### identified. 18

19



### 4.6.3. Mode-of- Action Information 20



### The studies described above ha ve demonstrated that exposure to biphenyl may lead to a 21



### variety of noncancer health effects (i.e., weight loss, liver toxicity, urinary tract toxicity). 22



### However, there is not sufficient information to determine the mode of action for noncancer 23



### health effe cts following exposure to biphenyl. 24



### Weight loss or lack of weight gain has been consistently associated with oral exposure to 25



### biphenyl (Umeda et al., 2005, 2002; Ambrose et al., 1960; Dow Chemical Co., 1953). The work 26



### of Nishihara (1985) provides a possible explanation for this toxic effect. This author found that, 27



### in vitro, biphenyl can act as an uncoupler of respiration. It may be speculated that long- term, 28



### high-dose exposure to biphenyl uncouples mitochondrial respiration to a certain extent, resulti ng 29



### in a futile cycle that diverts the use of nutrients from building body mass into maintaining 30



### necessary energy stores. It is not clear at what level of in vivo exposure this effect might become 31



### operative. 32



### Several of the oral animal studies (Umeda et al. , 2005; Sun Company Inc., 1977b; 33



### Pecchiai and Saffiotti, 1957; Dow Chemical Co., 1953; Deichmann et al., 1947) and the 34



### epidemiological study by Häkkinen et al. (1973) provide evidence that the liver is a target for 35



### biphenyl toxicity by any route of exposure. This evidence consists of changes in blood 36



### parameters that are indicative of liver toxicity; however, in animal studies, liver histopathology 37



### does not support or explain this finding. Evidence for damage to the nervous system, as 38

### 76 DRAFT

- DO NOT CITE OR QUO TE suggested by Häkkinen et al. (1973) and Seppäläinen and Häkkinen (1975), has not been 1



### reproduced in animal studies. The limited evidence for an estrogenic activity of 2



### 4,4 ′ -dihydroxybiphenyl (Kitamura et al., 2003; Schultz et al., 2002) is insufficient to assign a 3



### clear endocrine- disrupting effect to this important metabolite of biphenyl. 4



### Damage to the urinary tract has been observed consistently in animals but not in humans. 5



### The work of Ohnishi et al. (2001, 2000 a, b) provides tenable evidence that, in the rat, this is due 6



### to the precipitation in the urinary tract of crystals consisting mostly of 4 -hydroxybiphenyl. 7



### These crystals irritate the epithelia of ureters and bladder, leading to chronic inflammation and 8



### possibly cancer as well as obstruction of the urinary tract with subsequent hydronephrosis. The 9



### work of Ohnishi et al. (2001, 2000 b) has made it clear that, at least in their animal model, two 10



### conditions are required for this event to occur: (1) the pH in the urine of the animals needs to be 11



### higher than normal and (2) elevated potassium levels need to accompany the elevated pH 12



### because it is the potassium salt of 4 -hydroxybiphenyl sulph ate that has the lowest solubility in 13



### high-pH urine. No damage to the urinary tract was observed in rabbits exposed via inhalation to 14



### biphenyl for up to 13 week s (Deichmann et al., 1947). Although this mode of action is likely to 15



### explain the effects of biphenyl in the urinary tract of rats, it is unclear whether or not it has any 16



### bearing on humans that are likely exposed by inhalation. 17



### Gombar et al. (1991) developed structure activity relationship computer models for four 18



### types of chemical compounds (carboaromatic, heteroaromatic, alicyclic, acyclic) to estimate the 19



### teratogenic potential of 171 compounds (for which teratogenic data exist in >900 publications) in 20



### an overall procedure (dosage, maternal toxicity, and affected organ systems were not factored 21



### into these preliminary models). The models considered species, route of administration, and 22



### duration and timing of exposure. Experimental endpoints entered into the model were number of 23



### dams; maternal toxicity; teratogenic endpoints; numbers of viable implants, resorptions, and 24



### abnormal fetuses; and dead/live fetus ratio. Fetal deaths per se, runting, delayed ossification, and 25



### mino r skeletal abnormalities such as extra or missing ribs were not rated as teratogenic effects. 26



### The computerized modeling uses a coding system that represents only “heavy” atoms (i.e., no 27



### hydrogens). The models included molecule fragments and their electronic descriptors to 28



### represent functional groups, molecular shape descriptors, and connectivity descriptors. The 29



### results of the calculations were presented as 24 different structural descriptor values. After 30



### eliminating two types of results (outliers and “ statistically influential”), the models returned a 31



### 96% correct classification of the teratogenic potential of chemicals. Biphenyl and 32



### 2-hydroxybiphenyl were negative in this computerized evaluation. 33

34



### 4.7. EVALUATION OF CARCINOGENICITY 35



### 4.7.1. Summary of Overall Weight of Evidence 36



### U nder EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the 37



### database for biphenyl provides “ suggestive evidence of carcinogenic potential. ” This cancer 38

### 77 DRAFT

- DO NOT CITE OR QUO TE weight-of-evidence descriptor is bas ed on urinary bladder tumors (transitional cell papillomas 1



### and carcinomas) in male F34 4 rats (Umed a et al., 2002) and liver tumors (hepatocellular 2



### adenomas and carcinomas) in femal e BDF 1 

### mice (Umeda et al., 2005) exposed to biphenyl in the 3



### diet for 104 weeks. Earlier chronic t oxicity and carcinogenicity assessments in orally exposed 4



### animals found no clear evidence of biphenyl-induced carcinogenicity in rats (Shiraiwa et al., 5



### 1989; Ambrose et al., 1960; Pecchiai and Saffiotti, 1957; Dow Chemical Co., 1953), mice (Imai 6



### et al., 19 83; Innes et al., 1969; NCI , 1968), dogs (Monsanto, 1956), or Rhesus monk eys (Dow 7



### Chemical Co., 1953). The earlier studies had limitations including small numbers of animals in 8



### exposure groups and shorter- than -lifetime durations of exposure due to design or decreased 9



### survival unrelated to tumor development, with the exception of a mouse study that found no 10



### evidence of carcinogenic responses in female ddY mice exposed to 5,000 ppm biphenyl in the 11



### diet fo r 2 years (Imai et al., 1983). 12



### Considerable evidence s uggests that the development of urinary bladder tumors in male 13



### rats exposed to biphenyl depends on the sustained occurrence of urinary bladder calculi 14



### composed of precipitated 4-hydroxybiphenyl-O-sulphate, based on: (1) close correlation 15



### between urinary bladder calculi formation and development of urinary bladder tumors in male 16



### rats exposed to biphenyl, (2) dose-response and temporal concordance between biphenyl-induced 17



### urinary calculi formation, regenerative hyperplasia, and urinary bladder tumor development, 18



### (3) an overall negative or equivocal weight of evidence for the genotoxicity of biphenyl and 19



### metabolites, and (4) the wide body of evidence that other nongenotoxic or weakly genotoxic 20



### chemicals produce urinary bladder tumors in rodents at high exposure levels by a mode of action 21



### involving calculi formation, followed by ulceration or inflammation and regenerative cell 22



### proliferation (IARC, 1999b). Mode-of- action information is sufficient to conclude that these 23



### tumors are high-dose phenomena; without the development of calculi, urinary bladder tumors are 24



### not expected. The proposed mode of action is expected to be relevant to humans at exposure 25



### levels sufficient to cause urinary bladder calculi in humans, because calculi in humans have been 26



### associated wit h urinary bladder irrita tion, regeneration, and cancer (IARC, 1999b; Cohen, 1998, 27



### 1995) and the metabolism of biphenyl to sulphate conjugates of hydroxylated biphenyl 28



### metabolites has been demonstrated in human tissues. 29



### For liver tumors, a p roposed mode of action (Umeda et al., 2004) includes activation of 30



### peroxisome proliferator activated receptors (PPARs) by biphenyl or its metabolites in liver cells 31



### or direct or indirect (through ROS ) reactions with DNA in liver cells to produce mutations 32



### leading to tumor initiation. However, available data are insufficient to establish a mode of 33



### action for liver tumors in female mice (See Section 4.7.3.2.2.1 for more information)

. In the 34



### absence of information to indicate otherwise, the development of liver tumors in female mice 35



### with chronic exposure to biphenyl is assumed to be relevant to humans. EPA acknowledges that 36



### some mouse strains (e.g., B6C3F 1 

### ) are relatively susceptible to liver tumors and the background 37



### incidence of this tumor can be high. For these reason s, use of mouse liver tumor data in risk 38

### 78 DRAFT

- DO NOT CITE OR QUO TE assessment has been a subject of controversy (King- Herbert and Thayer, 2006). The BDF 1 1



### mouse used in the Umeda et al. (2005) bioassay is a cross between female C57BL/6 and male 2



### DBA/2 mice ( Charles River Laboratories International, Inc. , 1999), both of which are considered 3



### to be relatively resistant to liver tumor induction (Maronpot , 2009). In the Umeda et al. (2005) 4



### bioassay, the incidence s of tumors in male and female concurrent control mice were 32 and 6%, 5



### respectively. The relatively low background incidence of liver tumors in female control mice 6



### from Umeda et al. (205) minimiz es the possible confounding of compound- related liver tumors 7



### in this sex. 8



### The descriptor of “suggestive evidence of carcinogenic potential” is appropriate when the 9



### weight of evidence is suggestive of carcinogeni city, i.e., a concern for potential carcinogenic 10



### effects in humans is raised, but the data are judged not sufficient for a stronger conclusion (U.S. 11



### EPA, 2005a). As discussed in Section 4.2.1.2, biphenyl exposure produced an increased 12



### incidence of urinary bladder tumors in male F344 rats (Umeda et al., 2002) and liver tumors in 13



### female BDF 1 

### mice (Umeda et al., 2005). Such data could be considered consistent with the 14



### descriptor of “likely to be carcinogenic to humans.” As stated in the Guidelines for Carcinogen 15



### Risk Assessment (U.S. EPA, 2005a), a “likely” descriptor may include “an agent that has tested 16



### positive in animal experiments in more than one species, sex , strain, site, or exposure route, with 17



### or without evidence of carcinogenicity in humans.” Biphenyl did induce tumors in two species 18



### (rat and mouse) and at two sites (liver and urinary bladder); however, tumor findings across the 19



### biphenyl database and the interpretation of some of these findings indicate some uncertainties 20



### regarding the potential human carcinogenicity of biphenyl. 21



### Both the liver tumors and urinary bladder tumors induced by dietary exposure to biphenyl 22



### each occurred in only one sex and on ly one species. Liver tumors were induced in female BDF 1 23



### mice only, and urinary bladder tumors occurred in male F344 rats only. The incidence of liver 24



### adenomas and carcinomas (separate and combined) in Umeda et al. (2002) was increased over 25



### control in al l groups of exposed female mice; however, the liver tumor incidence plateaued at the 26



### mid - and high-dose groups (incidence of adenoma and carcinoma combined in the control and 27



### low- , mid -, and high-dose groups were 3/48, 8/50, 16/49, and 14/48, respectively). Further, 28



### female ddY mice exposed to 5000 ppm biphenyl in the diet for 2 years showed no increased 29



### incidence of liver tumors (Imai et al., 1983). Urinary bladder tumors in F344 male rats induced 30



### by dietary biphenyl exposure appear to be a high-dose phen omen on closely rela ted to the 31



### formation of calculi. A mode of action analysis (see Section 4.7.3.1) supports the conclusion 32



### that exposures that do not lead to urinary bladder calculi will not prod uce tumors. While the 33



### proposed mode of action for urinary bladder tumors in male rats is considered relevant to 34



### humans, there is evidence that humans are likely to be less susceptible to these tumors than rats. 35



### As discussed in Section 4.7.3.1.4.2, the rodent horizontal quadruped stature is expected to 36



### promote calculi residency time in the bladder without causing obstruction

, whereas the anatomy 37



### of the urinary tract in humans and their upright bipedal stature result in more ready excretion of 38

### 79 DRAFT

- DO NOT CITE OR QUO TE calculi in the urine or therapeutic removal of calculi that form obstruc tions (Cohen and Ellwein, 1



### 1992; Matanowki, 1981). Overall, the mode of action analysis suggests that biphenyl is not 2



### likely to induce urinary bladder tumor s in humans at environmental exposure levels. In light of 3



### the above considerations related to biphenyl- induced female mouse liver tumors and male rat 4



### bladder tumors, EPA concluded that the currently available information is most consistent with a 5



### determination that the database for biphenyl provides “suggestive evidence of carcinogenic 6



### potential.” 7



### U.S. EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) indicate that 8



### for tumors occurring at a site other than the initial point of contact, the cancer descriptor may 9



### apply to all routes of exposure that have not been adequately tested at sufficient doses. An 10



### exception occurs when there is convincing toxicokinetic data that absorption does not occur by 11



### other routes. Information available on the carc inogenic effects of biphenyl demonstrates that 12



### tumors occur in tissues remote from the site of absorption following chronic oral exposure 13



### (urinary bladder in male r ats and liver in female mice ). No i nformation on the carcinogenic 14



### effects of biphenyl via the inhalation or dermal routes in humans and animals is available

. 15



### Quantitative d ata demon strating rapid and extensive absorption of biphenyl are restricted to the 16



### oral route of exposure; a case report of hepatic toxicity produced by a probable combination of 17



### inhalation and dermal exposures in a worker in a biphenyl-impregnated fruit wrapping p aper 18



### production facility provides qualitative evidence of absorption by these routes (Häkkinen et al., 19



### 1973). Therefore, based on the observance of systemic tumors following oral exposure and 20



### assumed absorption by all routes of exposure, it is assumed tha t an internal dose will be achieved 21



### regardless of the route of exposure. Therefore, EPA considers the biphenyl database to provide 22



### “suggestive evidence of carcinogenic potential” by all routes of exposure. 23

24



### 4.7.2. Synthesis of Human, Animal, and Other Su pporting Evidence 25



### Available human studies were not designed to evaluate associations between exposure to 26



### biphenyl and occurrence of cancer (see Section 4.1). 27



### As discussed in Section 4.2, carcinogenicity studies in animals are limited to the oral 28



### exposure route. In well-designed cancer bioassays of F344 rats (Umeda et al., 2002) and BDF 1 29



### mice (Umeda et al., 2005), dietary exposure to biphenyl resulted in the occurrence of urinary 30



### bladder tumors in male rats and significantly increased incidences in liver tumors in female mice. 31



### Earlier chronic toxicity and carcinogenicity assessments found no clear evidence of 32



### biphenyl-induced carcinogenicity in orally exposed rats, mice, dogs, or Rhesus monkeys. 33



### However, these studies were generally limited in design, with the exception of a study reporting 34



### no evidence of carcinogenic responses in female ddY mice (n = 34 mice vs . 37 control mice) 35



### exposed to 5,000 ppm biphenyl in the diet for 2 years (Imai et al., 1983). In a study of Wistar 36



### rats, sufficient numbers of animals (50/sex/group) were exposed to biphenyl in the diet at 37



### concentrations up to 5,000 ppm, but only for 75 weeks (Shiraiwa et al., 1989). Some of the male 38

### 80 DRAFT

- DO NOT CITE OR QUO TE rats exhibited urinary bladder calculi and simple or diffuse hyperplasia and papillomatosis of the 1



### urinary bladder mucosa in the absence of neoplastic lesions, but the study may have been 2



### terminated prior to eventual urinary bladder tumor development. Ambrose et al. (1960) exposed 3



### albino rats (15/sex/exposure level) to biphenyl in the diet at concentrations up to 10,000 ppm for 4



### 2 years (10, 50, 100, 500, 1,000, 5,000, or 10,000 ppm); however, decreased survival in rats 5



### exposed to 5,000 or 10,000 ppm, presumably from decreased food consumption, and the 6



### relatively small numbers of animals in each exposure g roup may have impaired the ability to 7



### detect late -developing tumors. In another study, groups of Sprague- Dawley rats (12/sex/group) 8



### received biphenyl in the diet at concentrations up to 10,000 ppm for up to 2 years (Dow 9



### Chemical Co. , 1953). However, this study suffered from a pneumonia outbreak, particularly 10



### among control males, and the relatively small numbers of animals and the decreased survival 11



### may have impaired the ability to detect late -developing tumors. A study of male albino rats 12



### included small numbers of rats (8/group) and a short (13 months) exposure period (Pecchiai and 13



### Saffiotti, 1957). A study of B6C3F 1 

### or B6AkF 1 

### mice exposed to biphenyl in the diet for only 14



### 18 months (Innes et al., 1969; NCI , 1968) included relatively small numbers of mice 15



### (18/sex/group) and only one exposure level (517 ppm) that was similar to the concentration 16



### (667 ppm) without carcinogenic effect in the Umeda et al. (2005) 24-month BDF 1 

### mouse 17



### bioassay. The dog study included two males and one female, a high dose of 25 mg/kg-day, and 18



### an exposure period of only 1 year (Monsanto, 1956). Rhesus monkeys ( two males and one 19



### female) were exposed to biphenyl in the diet at a concentration of 10,000 ppm, but for only 20



### 1 year (Dow Chemical Co., 1953). 21



### Th e overall weight of evidence for biphenyl genotoxicity from short- term tests is 22



### negative or equivocal. Biphenyl did not induce mutations in a variety of bacterial test systems, 23



### but both negative and positive results were obtained in mammalian in vitro tes t systems (see 24



### section 4.5.6. for references). Single oral doses of 2,000 mg biphenyl/kg induced DNA damage 25



### (detected by the Comet assay) in several organs of CD -1 mice (including the liver and bladder), 26



### but it is uncertain if the damage was due to a dire ct effect on DNA or was an indirect effect due 27



### to cytotoxicity or ROS generated by redox cycling of phenylhydroquinone, a major urinary 28



### metabolite of 2-hydroxybiphenyl and a minor m etabolite of biphenyl in rats

. 29



### The overall weight of evidence for 2-hydroxybiphenyl genotoxicity suggests that 30



### oxidative DNA damage from ROS from redo x cycling between 2,5-dihydroxybiphenyl and 31



### phenylbenzoquinone is possible. DNA damage was detected in liver and bladder of CD

- 1 mice 32



### exposed to 2,000 mg/kg of 2-hydroxybiphenyl (S asaki et al., 2002, 1997) and in the urinary 33



### bladder of male F344 rats fed the sodium salt of 2-hydroxybiphenyl at 1 or 2% in the diet for 3

– 34



### 5 months (Morimoto et al., 1989). DNA adducts were detected by [ 32 

### P]-post labeling in skin of 35



### CD

- 1 mice after topical application of the sodium salt of 2-hydroxybiphenyl or phenylhydro36



### quinone (Pathak and Roy, 1993), and increased micronuclei were detected in urinary bladder 37



### epithelium of male F344 rats exposed to 2,000 ppm 2-hydroxybiphenyl or 2, 000 ppm NaCl plus 38

### 81 DRAFT

- DO NOT CITE OR QUO TE 2,000 ppm 2-hydroxybiphenyl in the diet for 2 weeks (Balakrishnan et al., 2002). However, 1



### increased binding of radioactivity to DNA was not detected in DNA extracted from urinary 2



### bladder epithelium of male F344 rats exposed to single gavage doses of 2-hydroxybiphenyl as 3



### high as 1,000 mg/kg (Kwok et al., 1999), and DNA adducts were not detected in urinary bladder 4



### epithelium of male F344 rats exposed for 13 weeks to biphenyl dietary concentrations as high as 5



### 12,500 ppm (Smith et al., 1998). The mechanism b y which 2-hydroxybiphenyl may induce 6



### micronuclei in the urinary bladder epithelium is uncertain , but could involve micronuclei 7



### generation as a secondary response to cytotoxicity or regenerative cell proliferation, DNA 8



### damage from ROS from red ox cycling of 2,5-dihydroxybiphenyl, or protein modifications 9



### leading to mitotic spindle interference or inhibition of enzymes important in DNA replication 10



### (Balakrishnan et al., 2002). The hydroxylation of biphenyl to produce 2-hydroxybiphenyl is a 11



### minor pathway in rat s and mice (Halpaap -Wood et al., 1981a, b ; Meyer and Scheline, 1976). 12



### 2-Hydroxybiphenyl and 2,5-dihydroxybiphenyl collectively accounted for less than 2% of 13



### metabolites in urine of rats administered single oral doses of 100 mg biphenyl/kg (Meyer and 14



### Schel ine, 1976) or single i.p. doses of 30 mg biphenyl/kg (Halpaap -Wood et al., 1981a). In mice 15



### given i.p. doses of 30 mg biphenyl/kg, these metabolites accounted for less than 5% of u rinary 16



### metabolites (Halpaap -Wood et al., 1981a). 17

18



### 4.7.3. Mode-of- Action Information 19



### 4.7.3.1. Mode-of-Action Information for Bladder Tumors in Male Rats 20



### 4.7.3.1.1. Hypothesized mode of action. The best -supported hypothesis proposes a mode of 21



### action whereby the formation of urinary bladder calculi (from the precipitation of 4

- 22



### hydroxybiphenyl-O-sulphate) is a key event in the development of urinary bladder tumors in 23



### male rats fed high levels of biphenyl in the diet for 2 years. According to this hypothesis, the 24



### calculi (occurring in associa tion with increased urinary pH and potassium, and predominantly 25



### composed of 4-hydroxybiphenyl-O- sulphate) cause irritation to transitional epithelial cells of the 26



### urinary bladder leading to sustained cell proliferation, which promotes the development of 27



### initiated cells in the urinary bladder with progression to papillomas and carcinomas. 28

29



### 4.7.3.1.2. Experimental support for the hypothesized mode of action 30



### 4.7.3.1.2.1. Strength, consistency, and specificity of association, including support for the 31



### hypothesized mode of action in male rats

. The formation of urinary bladder calculi, 32



### predominantly composed of potassium 4-hydroxybiphenyl-O- sulphate, is strongly, consistently, 33



### and specifically associated with the formation of urinary bladder tumors in male rats chronically 34



### exposed to high dietary concentrations of biphenyl. Several findings support this association. 35



### Urinary bladder calculi were formed at a high prevalence (43/50; 86%) in a group of male rats 36



### exposed to biphenyl in the diet at a concentration of 4,500 ppm, but were absent in ma le rats 37



### receiving diets containing 0, 500, or 1,500 ppm biphenyl (Umeda et al., 2002). These 38

### 82 DRAFT

- DO NOT CITE OR QUO TE observations were consistent with the detection of urinary bladder transitional cell papilloma 1



### (10/50; 20%), carcinoma (24/50; 48%), and papilloma or carcinoma (3 1/50; 62%) in the 2



### 4,500 ppm group of male rats and total absence of urinary bladder papilloma or carcinoma in the 3



### control, 500, or 1,500 ppm groups of male rats. Bladder calculi were found in all 24 of the male 4



### rats with urinary bladder transitional cell carcinoma and in 8/10 of the male rats with transitional 5



### cell papilloma. 6



### The association between urinary bladder calculus formation and development of urinary 7



### bladder tumors is both gender and species specific. Urinary bladder calculi, of similar size to 8



### those observed in males, were observed at much lower incidence (8/50; 16%) in the 4,500 ppm 9



### female rats, but they were of more uniform color (white and yellow versus white, yellow, brown, 10



### gray, and black in males) and shape (spheroidal vs. triangular, pyramidal, cubical, and spheroidal 11



### in males) and primarily composed of 4-hydroxybiphenyl and potassium bisulphate (which are 12



### hydrolysis products of potassium 4-hydroxybiphenyl-O-sulphate) (Umeda et al., 2002; Ohnishi 13



### et al., 2000b). No urinary bladder calculi were found in the 500 and 1,500 ppm groups of female 14



### rats. Transitional cell hyperplasia was found in 10/50 4,500- ppm f emale rats, but no urinary 15



### bladder transitional cell papillomas or carcinomas were seen in any of the biphenyl -exposed 16



### groups of female rats. Furthermore, there was no evidence of biphenyl-induced urinary bladder 17



### calculi or bladder tumors in male or femal e BDF 1 

### mice receiving dietary biphenyl at 18



### concentrations as high as 6,000 ppm for 2 years (Umeda et al., 2005). 19



### Urinary bladder calculi in male rats were associated with significantly increased urinary 20



### pH (average pH of 7.97 in the 4,500 ppm group at the final week of exposure compared to 21



### 7.66 in controls) (Umeda et al., 2002) and were composed primarily of potassium 22



### 4-hydroxybiphenyl-O-sulphate (Ohnishi et al., 2000b). The urine pH of female rats exposed to 23



### 4,500 ppm for 104 weeks (pH = 7.26) was not elevated compared with controls (pH = 7.29) 24



### (Umeda et al., 2002), and urinary calculi of a different composition than male rats (i.e., 25



### 4-hydroxybiphenyl and potassium bisulphate, compared with potassium 4-hydroxybiphenyl26



### O- sulphate in males) were found in only 8/50 4,500-ppm females (Ohnishi et al., 2000b). From 27



### these observations, it appears that the formation of the calculi result s from the precipitation of the 28



### potassium salt of the sulphate conjugate of 4 -hydroxybiphenyl under the elevated pH conditions 29



### of the male rat urine. The mechanism responsible for increased urinary pH is unknown, although 30



### Ohnishi et al. (2001, 2000a, b) proposed that gender differences in urinary conditions, such as 31



### pH and potassium concentrations, and sulphatase activities in kidneys, may be responsible for 32



### the gender differences in urinary calculi composition and formation and the subsequent 33



### development of urinary bladder tumors in male, but not female, F344 rats. 34



### Relatively strong, consistent, and specific associations between cal culi formation and 35



### transitional cell hyperplasia and between transitional cell hyperplasia and the development of 36



### transitional cell tumors in the urinary bladder have been shown in male F344 rats chronically 37



### exposed to high concentrations of biphenyl in the diet. Urinary bladder transitional cell 38

### 83 DRAFT

- DO NOT CITE OR QUO TE hyperplasia (simple, nodular, papillary) occurred in 45/50 (90%) male rats receiving biphenyl in 1



### the diet for 2 years at the same dietary concentra tion (4,500 ppm) at which high prevalences of 2



### both urinary bladder calculi formation (43/50; 86%) and transitional cell tumors (31/50 62%) 3



### were observed (Umeda et al., 2002). Forty-two of the 45 male rats with urinary bladder 4



### transitional cell hyperplasia also exhibited urinary bladder calculi. In another study, evidence of 5



### biphenyl-induced calculi formation (microcalculi in the urine) and increased indices of urinary 6



### bladder transitional cell proliferation (greater than fourfold increase in BrdU incorporation) in 7



### male F344 rats has been repor ted following as little as 4 –8 weeks of dietary exposure to 8



### 5,000 ppm biphenyl (Shibata et al., 1989b). 9



### The most convincing evidence that degenerative changes in the urinary bladder 10



### epithelium lead to tumor formation is the site

- concordance of associations between calculi 11



### formation in the urinary bladder, transitional cell proliferation, transitional cell hyperplasia , and 12



### transitional cell tumors (Umeda et al., 2002) . In addition, the strong associations between 13



### urina ry tract calculi formation, ulcerations or inflammation, and subsequent hyperplasia 14



### combined with repeated, high- level exposure to other chemicals that cause urinary bladder 15



### tumors in rodents, including melamine, uracil , and the sodium salt of 2-hydroxybiphenyl (IARC 16



### 1999a, b, c; Cohen, 1998; 1995) provide further evidence that degenerative changes are involved 17



### in the etiology of rodent urinary bladder tumors. I t is not unusual to see extensive proliferation 18



### or hyperplasia in bladder epithelium in response to urinary calculi from other rodent bladder 19



### tumorigens without an associated ulceration or intense inflammatory response. In male rats 20



### exposed to 4,500 ppm biphenyl, increasing numbers of rats with clinical hematuria were 21



### observed beginning at about the 40 th 

### week of exposure, and histologic examinations at study 22



### termination revealed focal hyperplasia in 45/50 rats, providing some evidence of calculi -induced 23



### bladder epithelial damage followed by cell proliferation (Umeda et al., 2002). Over the course of 24



### the study, 94% of male rats with hematuria had bladder or kidney calculi, but hematuria was not 25



### found in any biphenyl-exposed females. In addition, with 8 weeks, but not 4 weeks, of exposure 26



### to 5,000 ppm biphenyl in the diet, moderate urinary bladder epi thelial hyperplasia and 27



### microcalculi in urine were observed in 5/5 male F344 rats, but no descriptions of degenerative 28



### changes were provided; these observations are consistent with a rapid repair response to 29



### epithelial damage from biphenyl-induced urinary tract calculi (Shibata et al., 1989b). 30



### The ability of repeated biphenyl exposure to promote previously initiated urinary bladder 31



### cells to bladder tumors is supported by results of a bladder tumor initiation-promotion study 32



### (Kurata et al., 1986). Incidence s of urinary bladder hyperplasia, papilloma, and carcinoma were 33



### significantly increased in male F344 rats initiated with dietary BBN for 4 weeks followed by 34



### 5,000 ppm biphenyl in the diet for 32 weeks, compared with rats receiving BBN only for 35



### 4 weeks. Fo r example, 94 and 83% of rats treated with BBN followed by biphenyl developed 36



### urinary bladder hyperplasia and papillomas, respectively, compared with 25 and 12% of rats 37



### exposed to BBN alone. 38

### 84 DRAFT

- DO NOT CITE OR QUO TE The hypothes is that the mode of action involves the development of urinary bladder 1



### tumors in biphenyl- exposed male rats is further supported by an overall negative or equivocal 2



### weight of evidence for the genotoxicity of biphenyl. As discussed earlier, there are consistently 3



### negative results for biphen y l in bacterial mutation assays and inconsistent positive results for 4



### biphenyl in in vitro mammalian assays mostly in the presence of metabolic activation. There is 5



### evidence that 2,5-dihydroxybiphenyl (i.e., phenylhydroquinone), the principal urinary metabolite 6



### in rats exposed to high doses of 2 -hydroxybiphenyl, can undergo redox cycling to produce ROS 7



### that may damage DNA and lead to tumor

- initiating mutations ; however, 2- hydroxybiphenyl is a 8



### minor urinary metabolite of biphenyl in rats and 2,5-dihydroxybiphenyl was not detected in urine 9



### of rats exposed to oral doses of 100 mg biphenyl/kg (Meyer and Scheline, 1976). 10

11



### 4.7.3.1.2.2. Dose

- response concordance

21 

### . Dose -response relationships for urinary bladder 12



### calculi formation, transitional cell hyperplasia , and transitional cell tumor development show 13



### concordance in the 2- year oral study of rats (Umeda et al., 2002). In male rats, urinary calculi, 14



### nonneoplastic lesions (epithelial hyperplasia), and neoplastic lesions (papillomas and 15



### carcinomas) of the urinary bladder were observed only at the highe st exposure level 16



### (4,500 ppm); no urinary bladder calculi, transitional cell hyperplasia

, or transitional cell tumors 17



### were found in control, 500, or 1,500 ppm male rats. Furthermore, urinary bladder calculi were 18



### found in 43/45 high- dose male rats

, in all 24 male rats with transitional cell carcinoma, and in 19



### 8/10 of the male rats with transiti onal cell papilloma. 20



### 4.7.3.1.2.3. Temporal relationship

34 

### . Results from the 2 -year oral study in rats (Umeda et al., 22



### 2002) provide some evidence of a progression from urinary bladder calculi formation to the 23



### development of bladder tumors. Urinary bladder calculi were observed in the first 4,500 ppm 24



### male rat that died (week 36), evidence of blood in the urine was observed in 4,500 ppm male rats 25



### by week 40, and incidences of bladder calculi and bloody urine that paralleled increases in 26



### mortality and tumor formation were observed throughout the remainder of the study. In addition, 27



### results of a short- term oral study demonstrate that microcalculi can be detected in the urine of 28



### male rats after as little as 4 weeks of dietary exposure to 5,000 ppm biphenyl and that 29



### hyperplasia of urinary bladder epithelium can be detected at least by week 8 (Shibata et al., 30



### 1989b). Presumably, the development of biphenyl- induced urinary bladder tumors requires a 31



### longer exposure period to urinary calculi of sufficient size, shape, and composition to induce 32



### urinary bladder epithelial damage and a sustained proliferative response. 33



### 4.7.3.1.2.4. Biological plausibility and coherence

. The proposed mode of action is consistent 35



### with the current understanding of cancer biology and is supported by the wide body of evidence 36



### that other chemicals with primarily non genotoxic profiles produce urinary bladder tumors in 37



### rodents at high exposure levels by a mode of action involving calculi formation, ulceration or 38

### 85 DRAFT

- DO NOT CITE OR QUO TE inflammation, and regenerative cell proliferation ( IARC, 1999 a, b, c; Cohen, 1998, 1995). 1



### Additional information could strengthen the plausibility and coherence of the proposed mode of 2



### action to explain the occurrence of biphenyl-induced urinary bladder tumors in male rats. These 3



### additional data include results from investigations of earlier time points in the proposed temporal 4



### progression from calculi formation to epithelial damage, regenerative cell proliferation, and 5



### tumor development and further investigations into the factors underlying gender- specific 6



### differences in precipitation of 4-hydroxybiphenyl-O- sulphate to form bladder calculi in rats. 7

8



### 4.7.3.1.3. Other possible modes of action for bladder tumors in male r ats

. Although the 9



### weight of evidence from short- term standard genotoxicity tests with biphenyl and 10



### 4-hydroxybiphenyl is predominantly negative, evidence is available that suggests that oral 11



### exposure to high doses of 2 -hydroxybiphenyl is associated with the development of urinary 12



### bladder tumors in male rats

. The indu c tion of genotoxic effects in the urinary bladder epithelium 13



### lead ing to tumor initiation is proposed to occur via redox cycling between 2,5- 14



### dihydroxybiphenyl and phenylbenzoquinone (Balakrishnan et al., 2002; Kwok et al.,1999; 15



### Pathak and Roy, 1993; Morimoto et al., 1989). However, the strong, consistent, and specific 16



### association between the occurrence of urinary bladder calculi composed of 4-hydroxybiphenyl17



### O-sulphate and development of urinary bladder tumors in male but not female rats , the evidenc e 18



### that 2 -hydroxybiphenyl is a minor urinary metabolite of biphenyl and , finally, that 2,5- 19



### dihydroxybiphenyl was not detected in the urine of biphenyl-exposed rats, demonstrate that the 20



### support for a genotoxic mode of action involving key mutational events from biphenyl or its 21



### metabolites in the urinary bladder leading to initiation of tumor cells is not compelling. 22



### Additional support for a proposed genotoxic mode of action would come from studies showing 23



### formation of 2,5- dihydroxylbiphenyl and phenylbenzoq uinone in the urinary bladder epithelium 24



### of rats exposed to low doses of biphenyl. 25

26



### 4.7.3.1.4. Conclusions about the hypothesized mode of action for bladder tumors in male rat s. 27



### 4.7.3.1.4.1. Support for the hypothesized mode of action in rats

To summarize, chronic exposure of male rats to a high dietary concentration of biphenyl 33



### (4,500 ppm) caused increased urinary pH and high preval ence of urinary bladder calculi (from 34



### the precipitation of 4-hydroxybiphenyl-O-sulphate in the urine), transitional cell hyperplasia, and 35



### transitional cell tumors. Incidences of male rats with calculi and those with bladder tumors were 36



### strongly correlated , and chronic exposure of male rats to lower dietary concentrations of 37



### biphenyl (500 and 1,500 ppm) did not increase urinary pH and did not cause calculi formation, 38 

### . There is strong evidence that 28



### urinary bladder tumors in male rats chronically exposed to biphenyl in the diet is a high-dose 29



### phenomenon involving sustained occurrence of calculi in the urinary bladder leading to 30



### transitional cell damage, sustained regenerative cell proliferation, and eventual promotion of 31



### spontaneously initiated tumor cells in the urinary bladder epithelium. 32

### 86 DRAFT

- DO NOT CITE OR QUO TE transitional cell hyperplasia, or bladder tumor development. There were relatively strong 1



### associations between incidences of rats with calculi and those with transitional cell hyperplasia 2



### and between incidences of rats with transitional cell hyperplasia and bladder tumors. In contrast, 3



### high concentrations of biphenyl in the diet of female rats had no effect on urinary pH, caused a 4



### much lower prevalence of urinary bladder calculi of a different composition, and resulted in no 5



### urinary bladder tumors. The urinary bladder calculi in the male rats were mainly composed of 6



### the conjugated biphenyl met abolite, potassium 4 -hydroxybiphenyl-O- sul ph ate, whereas those of 7



### the female rats were predominantly composed of 4 -hydroxybiphenyl and potassium bisulph ate 8



### (which are hydrolysis products of potassium 4-hydroxybiphenyl-O-sulph ate). There was no 9



### evidence of urinary bladder calcul i formation or tumor development in male and female mice 10



### exposed to similar dietary concentrations of biphenyl. Results of a tumor initiation-promotion 11



### study in male rats support the proposal that biphenyl- induced sustained cell pro liferation 12



### promotes initiated tumor cells in the urinary bladder. Finally, results of genotoxicity tests with 13



### biphenyl are predominantly negative or equivocal at best. The preponderance of evidence 14



### supports a mode of action for biphenyl in male rats only involving urinary tract calculi 15



### formation, urinary epithelium damage, sustained regenerative cell proliferation and hyperplasia, 16



### and subsequent bladder tumor formation. There is evidence that 2,5-dihydroxybiphenyl can 17



### undergo redox cycling to produce ROS that may damage DNA leading to tumor- initiating 18



### mutations, but it was not detected in urine of rats exposed to oral doses of 100 mg biphenyl/kg 19



### and its metabolic precursor, 2 -hydroxybiphenyl, is a minor urinary metabolite of biphenyl in rats 20



### (Meyer and S cheline, 1976). 21

22



### 4.7.3.1.4.2. Relevance of the hypothesized mode of action to humans

. Although there are no 23



### studies in humans examining possible associations of biphenyl exposure with urinary bladder 24



### calculi formation or cancer, urinary bladder calculi h ave been reported in humans following 25



### exposure to other chemicals (IA RC, 1999b; Cohen 1998, 1995). Urinary bladder calculi are , in 26



### general, expected to be irritating and lead to reparative cell proliferation regardless of 27



### composition or species ; however, based on the anatomy of the urinary tract in humans and their 28



### upright, bipedal stature, calculi are either quickly excreted in urine or cause obstruction leading 29



### to pain and subsequent therapeutic removal of the calculi (Cohen, 1998, 1995). In contrast, the 30



### rodent horizontal quadruped stature is expected to promote calculi residency time in the bladder 31



### without causing obstruction (Cohen, 1998, 1995). In white populations, 95% of bladder tumors 32



### are transitional cell carcinomas such as those found in male r ats exposed to high concentrations 33



### of biphenyl

. IARC (1999b) noted that several case-control studies of urinary bladder cancer in 34



### white human populations found relative risks for an association between a history of urinary tract 35



### stones and bladder carcino mas ranging from about 1.0 to 2.5, suggesting a causative link . Thus, 36



### the proposed mode of action is expected to be relevant to humans at exposure levels sufficient to 37



### cause urinary bladder calculi in humans, because : (1) calculi resulting from human exp osure to 38

### 87 DRAFT

- DO NOT CITE OR QUO TE other substances have been associated with urinary bladder irritation, regeneration, and cancer 1



### (IARC, 1999b; Cohen 1998, 1995) and (2) sulphate conjugation of hydroxylated biphenyl 2



### metabolites has been demonstrated in human tissues (as briefly re viewed in Section 3.3). 3



### The underlying physiological factors determining the precipitation of 4-hydroxybiphenyl4



### O-sulphate in urine to form calculi in male rats, but not female rats, exposed to high dietary 5



### biphenyl concentrations are unknown. Given this lack of understanding for rats and the absence 6



### of specific human data on biphenyl-induced calculi or urinary stones, there is uncertainty in 7



### extrapolation of the dose-response relationship for biphenyl-induced calculi formation in male 8



### rats to humans. 9

10



### 4.7.3.1.4.3. Populations or lifestages particularly susceptible to the hypothesized mode of 11



### action

21 

### . IARC (1999b) noted that increased risks for bladder carcinoma in humans have been 12



### associated with cigarette smoking, exposure to infectious agents, such as Shistosoma 13



### haematobium

, causing urinary tract inflammation, and a history for urinary tract infections in 14



### general. As such, people with these types of exposure or history may be particularly susceptible 15



### to the formation of urinary calculi and urinary bladder cancer, but evidence supporting this 16



### inference is lacking. In addition, there are conditions (bladder diverticuli, neurogenic bladder, 17



### and staghorn renal pelvic calculi) that can increase the residency time of calculi in humans; thus, 18



### individuals with these conditions may also be particularly susceptible to biphenyl-induced 19



### bladder tumors under the hypothesized mode of action. 20



### 4.7.3.2. Mode-of- Action Information for Liver Tumors in Female Mice 22



### Evidence that chronic oral exposure to biphenyl can cause liver tumors comes from the 2

- 23



### year BDF 1 

### mouse bioassay by Umeda et al. (2005). Exposure to 2,000 or 6

, 000 ppm biphenyl in 24



### the diet, but not to 667 ppm, produced increased incidences of hepatocellular adenomas or 25



### carcinomas in female mice , but no carcinog enic response in male BDF 1 

### mice. Earlier studies 26



### found no carcinogenic response in B6C3F 1 

### or B6AkF 1 

### mice exposed to 517 ppm biphenyl in the 27



### diet for 18 months (Innes et al., 1969; NCI , 1968) or in ddY female mice exposed to 5,000 ppm 28



### biphenyl in the diet for 2 years (Imai et al., 1983). The only investigations into the mode of 29



### action for biphenyl- induced liver tumors in mice involve examinations of indicators of 30



### peroxisome proliferation following biphenyl exposure (Umeda et al., 2004; Sunouchi et al., 31



### 1999). Thus, a mode of action involving PPARs is proposed and an evaluation of the supporting 32



### data follows. 33

34



### 4.7.3.2.1. Hypothesized mode of action for liver tumors in female mi ce

. Proliferation of 35



### peroxisomes is regulated by a class of ligand -activated transcription factors known as PPARs. 36



### PPARα regulates induction of the peroxisome proliferation response in rodents and is thought to 37



### mediate at least some of the responses for hepat ocarcinogens, including initiation of cellular 38

### 88 DRAFT

- DO NOT CITE OR QUO TE events leading to transformation. Peroxisome proliferators (PPARα agonists) are a structurally 1



### diverse group of non- or weakly mutagenic chemicals that induce a suite of responses including 2



### the induction of tumors in rats and mice (Klaunig et al., 2003). 3



### Klaunig et al. (2003) have proposed a mode of action for PPARα agonists involving the 4



### following key events. PPARα agonists activate PPARα to transcribe genes involved in 5



### peroxisome proliferation, cell cycling /apoptosis, and lipid metabolism. The changes in gene 6



### expression lead to changes in cell proliferation and apoptosis, and to peroxisome proliferation. 7



### Suppression of apoptosis coupled with increased cell proliferation allows transformed cells to 8



### persist and proliferate, resulting in preneoplastic hepatic foci and ultimately promotion of tumor 9



### growth via selective clonal expansion. Peroxisome proliferation may lead to oxidative stress, 10



### which potentially contributes to the proposed mode of action by causing indirect DNA damage 11



### and/or by causing cytotoxicity leading to reparative cell proliferation. PPARα agonists also 12



### inhibit gap junction int er cellular communication and stimulate non

- parenchymal hepatic Kupffer 13



### cells; these events are also thought to stimulate cell proliferation. Increases in the size and 14



### number of peroxisomes and induction of peroxisome-related gene expression (e.g., palmitoyl15



### CoA oxidase and acyl- CoA oxidase) are regarded as indicators that the PPARα agonism mode of 16



### action is operative. 17

18



### 4.7.3.2.2. Experimental support for the hypothesized mode of action for liver tumors in female 19



### mice

. 20



### 4.7.3.2.2.1. Strength, consistency, specificity of association, including support for the 21



### hypothesized mode of action in mice

The available data do not demonstrate strong, consistent

, or specific associations between 34



### key events in the proposed mode of action and the development of liver tumors in female mice 35



### exposed to biphenyl. Klaunig et al. (2003) proposed that an adequate data set to support a 36



### PPARα agonism mode of action should meet the following demonstration criteria, most of which 37 

### . There is limited support for a possible association 22



### between biphenyl-induced proliferation of peroxisomes and liver tumors, because the following 23



### findings were reported in female BDF 1 

### mice (which developed liver tumors following dietary 24



### exposure to 2,000 or 6,000 ppm) but not in male BDF 1 

### mice (which did not develop liver tumors 25



### following exposure to concentrations as high as 6,000 ppm biphenyl). Dietary exposure of 26



### female BDF 1 

### mice to 16,000 ppm biphenyl for 13 weeks induced hepatocellular peroxisomes as 27



### evidenced by light microscopy detection of enlarged hepatocytes filled with eosinophilic fine 28



### granules and electron microscopy confirmation that the granules corresponded to increased 29



### numbers of peroxisomes (Umeda et al., 2004). Significantly increased activities were measured 30



### for potassium cyanide- insensitive palmitoyl CoA oxidation in liver homogenate (up to 1.9-fold) 31



### and lauric acid 12-hydroxylation in liver microsomes (up to 3.8- fold) from female BDF 1 

### mice 32



### given oral doses up to 5.2 mmol/kg-day (800 mg/kg-day) for 3 days (Sun ouchi et al., 1999). 33

### 89 DRAFT

- DO NOT CITE OR QUO TE as noted in parentheses have not been investigated for biphenyl or its metabolites: ( 1) activation 1



### of PPARα (no data) , (2) expression of peroxisomal genes including PPARα

- mediated expression 2



### of cell cycle, growth, and apoptosis, and nonperoxisomal lipid gene expression (no data), 3



### (3) peroxisomal proliferation (limited data for biphenyl in mice as summarized in previous 4



### paragraph) and perturbation of cell proliferation and apoptosis (no data for mouse liver), 5



### (4) inhibition of gap junction intercellular communication (no data), ( 5) hepatocyte oxidative 6



### stress (no data) , ( 6) Kupffer cell

- mediated events (no data)

, and (7) selective clonal expansion 7



### (no data). 8

9



### 4.7.3.2.2.2. Dose

- response concordance

17 

### . The available data do not show concordance between 10



### the dose-response relationships for liver tumors in female BDF 1 

### mice exposed for 2 years to 11



### biphenyl in the diet (liver tumors at 2,000 or 6,000 ppm, but not 667 ppm; Umeda et al., 2005) 12



### and liver peroxisome proliferation, the only key event in the proposed mode of action that has 13



### been investigated. Umeda (2004) reported that, compared with controls, increased liver 14



### peroxisome s were detected in female BDF 1 

### mice exposed to 16,000 ppm biphenyl in the diet for 15



### 13 weeks, but not in mice exposed to 500, 2,000, 4,000, 8,000, or 10,000 ppm. 16



### 4.7.3.2.2.3. Temporal relationship

24 

### . Indicators of liver peroxisome proliferation were elevat ed 18



### in female mice, but not male mice, with oral exposure durations of 3 days following exposure to 19



### 800 mg/kg

- day (increased activities of potassium cyanide -insensitive palmitoyl CoA oxidation 20



### and lauric acid 12-hydroxylation; Sunouchi et al. 1999) and 13 weeks following exposure to 21



### 16,000 ppm in the diet, but not at lower dietary concentrations (increased numbers of liver 22



### peroxisomes; Umeda et al. 2004). 23



### 4.7.3.2.2.4. Biological plausibility and coherence

28 

### . The data are inadequate to evaluate the 25



### biological plausibility and coherence of the proposed mode of action as it relates to liver tumors 26



### in female mice exposed to biphenyl. 27



### 4.7.3.2.3. Other possible modes of action for liver tumors in mice

. As discussed in 29



### Section 4.5.5, the overall weight of evidence from short-term standard genotoxicity tests w ith 30



### biphenyl and 4-hydroxybiphenyl is predominantly negative. A genotoxic mode of action for 31



### biphenyl-induced liver tumors in mice could be proposed based on the large metabolic capacity 32



### of the mouse liver to convert biphenyl to hydroxylated metabolites and evidence that metabolites 33



### of 2 -hydroxy biphenyl (2,5 -dihydroxybiphenyl and 2,5’-benzoquinone) can produce DNA 34



### damage (Tani et al., 2007; Balakrishnan et al., 2002; Sasaki et al. 2002, 1997; Patha k and Roy, 35



### 1993; Morimoto et al., 1989 ). However, hydroxylation of biphenyl to produce 2-hydroxy36



### biphenyl appears to be a minor metabolic pathway in mice administered single intraperitoneal 37



### doses of 30 mg biphenyl/kg (Halpaap-Wood et al., 1981a), and the available data are inadequate 38

### 90 DRAFT

- DO NOT CITE OR QUO TE to establish that this genotoxic mode of action operates in the biphenyl induction of liver tumors 1



### in mice. There have been no in vitro or in vivo investigations of biphenyl- induced DNA adducts 2



### or ROS generation in mouse live r cells or of possible gender differences in the produc tion of 3



### biphenyl-induced DNA adducts or other genotoxic events. Current mode-of-action information 4



### is inadequate to provide plausible explanations for why female BDF 1 

### mice exposed to high 5



### dietary concentrations of biphenyl develop liver tumors, but male BDF 1 

### mice exposed to 6



### 6,000 ppm and female ddY mice exposed to 5,000 ppm do not (Umeda et al., 2005; Imai et al., 7



### 1983). 8

9



### 4.7.3.2.4. Conclusions about the hypothesized mode of action for liver tumor s in mice

. 10



### A PPARα agonism mode of action for liver tumors in female mice exposed to 2,000 or 4,000 11



### ppm biphenyl in the diet for 2 years is not adequately supported by the experimental data. This 12



### is based on the lack of concordance between dose- response r elationships for biphenyl -induced 13



### liver tumors and proliferation of hepatocellular peroxisomes in female mice. Evidence for 14



### increased hepatocellular peroxisomes in female mice was only found with 13-week exposure to 15



### 16,000 ppm biphenyl and not at several concentrations ≤ 10,000 ppm (Umeda et al., 2004). 16



### Furthermore, a series key events demonstrating PPARα agonism mode of action have not been 17



### identified. 18



### Available data are inadequate to support alternative modes of action that propose direct 19



### or indirect geno toxic events from reactive biphenyl metabolites or ROS, respectively, as key 20



### events. Results from standard short- term genotoxicity tests are mostly negative or equivocal for 21



### biphenyl and 4 -hydroxybiphenyl. Although there is some evidence for DNA damage f rom ROS 22



### generated from redox cycling between 2,5-dihydroxybiphenyl and phenylbenzoquinone, there 23



### are no investigations into the metabolic formation of 2-hydroxybiphenyl, 2,5-dihydroxybiphenyl, 24



### and phenylbenzoquinone in livers of biphenyl-exposed mice exposed to a range of biphenyl 25



### doses, no in vitro or in vivo investigations of biphenyl-induced DNA adducts or ROS generation 26



### in mouse liver cells , and no investigations of possible gender differences in capability to produce 27



### biphenyl-induced DNA adducts or other genotoxic events. 28

29



### 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES 30



### 4.8.1. Possible Childhood Susceptibility 31



### No specific information was identified that would point specifically towards an early 32



### childhood susceptibility for biphenyl toxicity. However, th e developmental profiles of 33



### superoxide dismutase and catalase in humans that were reported by McElroy et al. (1992) 34



### indicate that the activities of both enzymes may be comparatively low before and at birth, 35



### placing humans in the perinatal period at an incr eased risk of adverse effects elicited by quinoid 36



### metabolites of biphenyl. Specifically, Buonocore et al. (2001) drew attention to the fact that the 37

### 91 DRAFT

- DO NOT CITE OR QUO TE human brain has relatively low superoxide dismutase activity at birth. G iven the limited data on 1



### age- spec ific ROS scavenging enzymes, any suggestions of childhood susceptibility to biphenyl is 2



### speculative. 3



### Studies in animals provide evidence that biphenyl metabolism is mediated by CYP1A2 4



### and CYP3A4 (Haugen, 1981). Phase II enzymes, such as sulphotransferas es (SULTs) and 5



### uridine diphosphate glucuronosyl transferases (UGTs), may be involved in conjugation activities 6



### with hydroxybiphenyls in mammalian tissues (Pacifici et al., 1991; Bock et al., 1980). CYP1A2 7



### expression is negligible in the early neonatal period, but is significantly increased to 50% of 8



### adult levels by one year of age (Sonnier and Cresteil , 1998). In general, SULTs and UGTs, 9



### depending on the isoforms, also exhibit dif ferential express ion during human development 10



### (Duanmu et al., 2006; Strassburg et al., 2002). To the extent that metabolism increases or 11



### reduces the toxicity of biphenyl, changes in the expression of Phase I and II enzymes during 12



### development can influence susceptibility to biphenyl toxicity. S pecific isoforms of cytochrome 13



### P450s and Phase II enzymes have not been identified as the principal catalyzers involved in 14



### biphenyl metabolism and th e effect of differences in enzyme expression on childhood 15



### susceptibility to biphenyl has not been established. 16

17



### 4.8.2. Po ssible Gender Differences 18



### Benford and Bridges (1983) evaluated the sex- and tissue -specific induction of biphenyl 19



### 2-, 3-, and 4- hydroxylase activities in microsomal preparations or primary hepatocyte cultures 20



### from male and female Wistar rats. No differenc es in biphenyl hydroxylase activities were 21



### observed between the sexes. However, there were some sex differences in the way tissues 22



### responded to the action of enzyme inducers. For example, the CYP1A inducer **α** -naphthoflavone 23



### strongly induced 2 -hydroxylase in male liver but had no effect on female liver. Betamethasone 24



### induced 2- hydroxylase activity in female liver but inhibited it in male liver. The available 25



### limited human data do not suggest that gender differences exist in the response to biphenyl 26



### exposu re. However, available animal data suggest gender

- related differences in susceptibility to 27



### tumors (i.e., bladder tumors in male but not female F344 rats and increased incidences of liver 28



### tumors in female but not male BDF 1 

### mice administered biphenyl in the diet for a lifetime). 29

30



### 4.8.3. Other 31



### The limited information on the specifics of biphenyl metabolism and toxic effects in 32



### humans does not allow a meaningful assessment of populations that might be highly susceptible 33



### to the adverse effects of biphenyl. For example, there is as yet no clear at tribution of CYP 34



### isozymes to the various biphenyl hydroxylases and no information on which sulpho transferases 35



### and glucuronidases conjugate hydroxylated biphenyl metabolites. It is known that many CYP 36



### isozymes, as well as glucuronidases, exist in polymorphic forms with catalytic activities that 37

### 92 DRAFT

- DO NOT CITE OR QUO TE differ from the wild type. In addition, such enzyme polymorphisms di splay specific distributions 1



### across populations and ethnicities that might put some at increased risk and others at decreased 2



### risk of adversity from biphenyl exposure. This lack of information represents a data gap. 3

### 93 DRAFT

- DO NOT CITE OR QUO TE 5. DOSE- RESPONSE ASSESSMENTS 1

2

3

4 

### 5.1. ORAL REFERENCE DOSE (RfD)

5 

### 5.1.1. Choice of Principal Study and Critical Effect— with Rationale and Justification

No information was located regarding possible associations between oral exposure to 6



### biphenyl and health outcomes in humans. 7



### As discussed in Section 4.6.1, the major and most sensitive targets of toxicity following 8



### oral exposure to biphenyl are the liver, urinary system, body weight, and developing organism 9



### (see Figure 5

- 1). In the rat, chronic oral studies identif ied the kidney and urinary bladder as 10



### critical noncancer targets (see Figure 5 -1 for LOAELs and NOAELs found in these studies). 11



### Kidney effects observed include: renal pelvis transitional cell hyperplasia and hemosiderin 12



### deposi ts in female F344 rats at doses ≥128 mg/kg -day (Umeda et al., 2002); kidney stone 13



### formation and obstructive pyelonephritis with tubular atrophy, tubular cysts, and fibrosis in male 14



### and female Wistar rats at 165 and 370 mg/kg- day, respectively (Shiraiwa et al., 1989); renal 15



### lymphocytic infiltration, tubular atrophy, and tubular cysts in male and female albino rats at 16



### doses ≥420 mg/kg-day (Ambrose et al., 1960); mild renal tubular degeneration in male albino 17



### rats at 250 or 450 mg/kg-day (Pecchiai and Saffioti, 1957; not plotted in Figure 5-1 because 18



### quantitative data were not included in the study report); and renal tubular dilatation in male and 19



### female Sprague-Dawley rats at 732 mg/kg-day (Dow Chemical Co., 1953). An increased 20



### incidence of u rinary bladder hy perplasia associated with calculi or “stones” was observed in 21



### male and female F344 rats at 378 and 438 mg/kg-day, but not at 110 and 128 mg/kg-day, 22



### respectively (Umeda et al., 2002). Elevated incidences of the same lesion were observed in male 23



### and female Wistar rats at 353 and 370 mg/kg-day, respectively (Shiraiwa et al., 1989). In 24



### contrast, urinary bladder hyperplasia and calculi were not observed in male or female albino rats 25



### at doses as high as 840 mg/kg-day (Ambrose et al., 1960) or in male or female Sprague- Dawley 26



### rats exposed to doses as high as 732 mg/kg-day (Dow Chemical Co., 1953). 27

### 94 DRAFT

- DO NOT CITE OR QUOTE 1

2 

### Figure 5

- 1. NOAELs and LOAELs for noncancer effects in rats and mice from repeated oral exposure to biphenyl. 3 0 200 400 600 800 1000 1200 1400 1600

NOAEL LOAEL Drop

- lines span highest to lowest doses tested

Female

BDF 1

mouse

( 1 ) Female

ddY

mouse

(2) M ale

BDF 1

mouse

(1) Female

ddY

mouse

(2) Female

BDF 1

mouse

(1) Female

ddY

mouse

(2) Female

F344 rat (3) Male

Wistar rat (4) Male,

female

albino rat (5) Male,

female Sprague

Dawley rat (6) Male

F344 rat (3) Male,

female

albino rat (5) Male

Wistar rat (4) Male,

female Sprague

Dawley rat (6) Wistar rat (7)

Liver a Body weight b Kidney c Urinary bladder d Develop

mental e

a Increased plasma liver enzymes in BDF 1 mice.

b Decreased body weight (>10% lower than controls) in BDF 1 mice.

c Increased incidences of kidney lesions including: mineralization in outer medulla in BDF 1 mice; renal pelvis transitional cell hyperplasia and hemosiderin deposits in F344 rats; kidney stone formation in Wistar rats; renal tubular atrophy in albino rats; renal tubular dilatation in Sprague

- Dawley rats.

d Increased incidences of urinary bladder calculi or stones and hyperplasia in F344 rats and Wistar rats.

e Increased number of litters with fetal skeletal anomalies in Wistar rats.

(1) = 

### Umeda e t al., 2005 ; (2) = Imai et al., 1983 ; (3) = Umeda et al., 2002 ; (4) = Shiraiwa et al., 1989 ; (5) = Ambrose et al., 1960 ; (6) = Dow

Chemical Co., 1953 ; (7) = Khera et al., 1979

### 95 DRAFT

- DO NOT CITE OR QUOTE 1



### In mice, chronic oral toxicity studies identif ied the liver, kidney, and body weight as 2



### critical noncancer targets (see Figure 5

- 1 for NOAELs and LOAELs for these effects). In BDF 1 3



### mice, significantly ( p < 0.05) increased plasma levels of enzymes indicative of liver damage 4



### were observed at dose levels of 1,050 mg/kg- day in males and ≥414 mg/kg

- day in females 5



### (Umeda et al., 2005), but no exposure- related changes in liver enzymes were observed in female 6



### ddY m ice at 885 mg/kg-day (Imai et al., 1983). Significantly increased incidence of 7



### mineralization of the renal outer medulla was observed in female BDF 1 

### mice at 414 and 8



### 1,420 mg/kg-day (Umeda et al, 2005), but exposure- related histological changes in the kidn ey 9



### were not found in female ddY mice at 885 mg/kg-day (Imai et al., 1983). Following the same 10



### pattern of apparent strain difference in susceptibility to biphenyl toxicity, body weights were 11



### decreased by >10% in male BDF 1 

### mice at ≥291 mg/kg

- day and females at ≥414 mg/kg -day 12



### (Umeda et al., 2005), but body weights in female ddY mice exposed to 885 mg/kg-day were 13



### similar to control values (Imai et al., 1983). Shorter duration oral exposure (13 weeks) of mice 14



### to biphenyl at higher dietary concentrations (estimated doses ≥1,500 mg/kg

- day) has also been 15



### shown to affect body and/or liver weights in mice (Umeda et al., 2004). 16



### In the only available oral developmental toxicity study (Khera et al., 1979), frank 17



### maternal toxicity ( increased mortality [5/20 vs. 0/18 in controls] and decreased number of dams 18



### with live fetuses [9/20 vs. 16/18 in controls]) occurred at the highest dose (1,000 mg/kg-day). 19



### Significantly increased incidences of fetuses with skeletal anomalies were noted at doses 20



### ≥500 mg/kg-day. The NOAEL and LOAEL of 250 and 500 mg/kg- day for delayed skeletal 21



### development are noted in Figure 5- 1. 22



### The 2-year dietary studies in F344 rats (Umeda et al., 2002) and BDF 1 

### mice (Umeda et 23



### al., 2005) and the developmental study in Wistar rats (Khera et al., 197 9) were selected as 24



### candidate principal studies for deriving the RfD because they provide the best available data 25



### (adequate number of dose groups and dose spacing, sufficient group sizes, comprehensive 26



### endpoint assessment and quantitation of results) to de scribe dose -response relationships for the 27



### critical effects in rats and mice associated with chronic or gestational oral exposure to biphenyl. 28



### In the 2- year dietary study of male and female F344 rats, biphenyl was administered in 29



### the diet at 0, 500, 1,500, or 4,500 ppm ( respective estimated doses were 36.4, 110, and 30



### 378 mg/kg-day for males and 42.7, 128, and 438 mg/kg-day for females) (Umeda et al., 2002). 31



### At the highest dose, noncancer effects included significantly increased incidence of rats with 32



### transi tional cell hyperplasia in the renal pelvis, renal mineralization and hemosiderin deposits, 33



### and urinary bladder transitional cell hyperplasia. Noncancer effects at the mid

- dose level were 34



### restricted to significantly increased incidences of females with renal transitional cell hyperplasia 35



### and hemosiderin deposits. There were no significant biphenyl- related effects in l ow- dose males 36



### or females. 37

### 96 DRAFT

- DO NOT CITE OR QUOTE In the 2- year dietary study of male and female BDF 1 

### mice, biphenyl was administered in 1



### the diet at 0, 667, 2,000, or 6,000 ppm ( respective estimated doses were 0, 97, 291, and 2



### 1,050 mg/kg-day for males, and 0, 134, 414, and 1,420 mg/kg- day for females) (Umeda et al., 3



### 2005) . At the two highest dose levels, noncancer effects included increased incidence of mice 4



### with re nal mineralization, increased levels of BUN, increased levels of serum enzymes indicative 5



### of liver damage, and decreased terminal body weights. No exposure- related effects were 6



### observed at the lowest exposure level. 7



### In the oral developmental toxicity study, pregnant Wistar rats were exposed by gavage to 8



### 0, 125, 250, 500, or 1,000 mg biphenyl/kg- day on GDs 6 –15 (Khera et al., 1979). Significantly 9



### increased numbers of fetuses with skeletal anomalies (wavy ribs, extra ribs, small 13th rib, 10



### missing or unossified sternebrae, delayed ossification of the calvarium) were noted at doses 11



### ≥500 mg/kg-day, and the number of litters exhibiting any of these anomalies was significantly 12



### higher at the 500 mg/kg- day dose level relative to controls. 13



### Candidate critical effects from the chronic study in F344 rats (Umeda et al., 2002) were : 14



### (1) nodular or simple transitional cell hyperplasia in the renal pelvis of males and females

, 15



### (2) mineralization in the renal pelvis or renal papillary mineralization in males and females

, 16



### (3 ) renal hemosiderin deposits in females

, and (4) transitional cell hyperplasia in the urinary 17



### bladder of males. Candidate critical effects from the chronic study in BDF 1 

### mice (Umeda et al., 18



### 2005) were: (1) decreased body weight in males and females, (2) mineralization of the renal 19



### inner stripe

- outer medulla in males and females , (3) BUN in male s and females , and (4) serum 20



### liver enzyme activities (AST [GOT], ALT [GPT], AP [ALP] , and LDH) in females. The 21



### candidate critical effect from the rat oral developm ental toxicity study (Khera et al., 1979) was 22



### litters with fetal skeletal anomalies from Wistar rat dams exposed during gestation

. 23

24



### 5.1.2. Methods of Analysis— Including Models 25



### Dichotomous datasets modeled include selected nonneoplastic lesions in the urin ary 26



### system of male and female F344 rats (Table 5 -1) exposed to biphenyl in the diet for 2 years 27



### (Umeda et al., 2002), mineralization in the kidney of male and female BDF 1 

### mice (Table 5 -2) 28



### exposed to biphenyl in the diet for 2 years (Umeda et al., 2005), an d litters with skeletal 29



### anomalies from Wistar rat dams (Table 5 -3) administered biphenyl by gavage on GDs 6–15 30



### (Khera et al., 1979). 31

32

### 97 DRAFT

- DO NOT CITE OR QUOTE Table 5- 1. BMD modeling datasets for incidences of nonneoplastic effects in

the urin ary tract of male and female F344 r ats exposed to biphenyl in the

diet for 2 years

Males (n = 50) Females (n = 50)

Biphenyl dietary concentration (ppm) 0 500 1,500 4,500 0 500 1,500 4,500

TWA body weight (kg) a 0.411 0.412 0.408 0.357 0.251 0.246 0.246 0.216

Calculated dose (mg/kg

- d) b 0 36.4 110 378 0 42.7 128 438

Effect

Renal pelvis

Nodular transitional cell hyperplasia 0 1 1 21 c 0 0 1 12 c

Simple transitional cell hyperplasia 6 8 5 19 d 3 5 12 d 25 c

Mineralization 9 6 10 18 e 12 12 18 27 d

Other kidney effects

Hemosiderin deposit f 0 0 0 0 4 8 22 c 25 c

Papillary mineralization 9 9 14 23 d 2 6 3 12 c

Bladder

Combined transitional cell g hyperplasia 0 0 0 45 1 0 1 10

a TWA body weight calculated using graphically

- presented body weight data in the study report of Umeda et al.

(2002).

b Calculated doses based on calculated TWA body weights and chronic reference food consumption values for

F344 rats (0.03 0 and 0.021 kg/day for males and females, respectively; taken from Table 1

- 6 of U.S. EPA

, 1988)

c Significantly different from control group ( p < 0.01) according to χ 2 test.

d Significantly different from control group ( p < 0.05) according to χ 2 test.

e Significantly different from controls ( p < 0.05) according to Fisher’s exact test.

f Male data for incidences of hemosiderin deposits not se lected for quantitative analysis

. g Female data for incidences of combined transitional cell hyperplasia not selected for quantitative analysis

.

Source: Umeda et al. (2002). .

1

### 98 DRAFT

- DO NOT CITE OR QUOTE Table 5

- 2. BMD modeling datasets for body weight, selected clinical
chemistry results, and histopathological kidney effects in male and female

BDF 1 

### mice exposed to biphenyl in the diet for 2 years

a Endpoint Biphenyl concentration in the diet (ppm)

0 667 2,000 6,000

Males

Dose (mg/kg

- d) 0 97 291 1,050

Histopathological kidney effect n = 50 n = 49 n = 50 n = 50

Mineralization inner stripe

- outer medulla 9 8 14 14

Clinical chemistry parameter n = 34 n = 39 n = 37 n = 37

BUN (mg/dL) 20.2 ± 3.6 22.0 ± 4.0 23.2 ± 4.4 a 22.9 ± 2.7 b

Body weight n = 35 n = 41 n = 41 n = 39

Mean terminal body weight (g) 46.9 ± 4.9 43.1 ± 7.9 42.9 ± 6.0 a 32.4 ± 3.6 b

Females

Dose (mg/kg

- d) 0 134 414 1,420

Histopathological kidney effect n = 50 n = 50 n = 50 n = 49

Mineralization inner stripe

- outer medulla 3 5 12 c 26 d

Clinical chemistry parameter n = 28 n = 20 n = 22 n = 31

AST (IU/L) 75 ± 27 120 ± 110 211 ± 373 b 325 ± 448 b

ALT (IU/L) 32 ± 18 56 ± 46 134 ± 231 b 206 ± 280 b

AP (IU/L) 242 ± 90 256 ± 121 428 ± 499 556 ± 228 b

LDH (IU/L) 268 ± 98 461 ± 452 838 ± 2,000 1,416 ± 4,161 a

BUN (mg/dL) 14.9 ± 2.0 14.8 ± 3.4 21.0 ± 20.5 23.8 ± 11.7 b

Body weight n = 31 n = 22 n = 25 n = 32

Mean terminal body weight (g) 34.0 ± 4.0 32.5 ± 3.3 30.5 ± 3.1 b 25.5 ± 3.0 b

Significantly different from controls ( p < 0.05) according to Dunnett’s test.

b Significantly different from controls ( p < 0.01) according to Dunnett’s test.

c Significantly different from controls ( p < 0.05) according to Fisher’s exact test.

d Significantly different from controls ( p < 0.01) according to Fisher’s exact test.

ALT (GPT) = alanine aminotransferase (glutamic pyruvic transaminase); AP (ALP) = alkaline phosphatase;

AST (GOT) = aspartate aminotransferase (glutamic oxaloacetic transaminase)

Source: Umeda et al. (2005).

1

### 99 DRAFT

- DO NOT CITE OR QUOTE Table 5

- 3. BMD modeling dataset for incidence of litters with fetal skeletal

anomalies from Wistar rat dams administered biphenyl by gavage on

GDs 6–15

Effect Dose (mg/kg

- d)

0 125 250 500 1,000

Litters with fetal skeletal anomalies a / litters 8/16 11/20 13/18 15 b /18 6/9

examined

a The study authors reported one runted fetus in the control group and one fetus with kinky tail in the 250 mg/kg

- day
dose group, which may have influenced the reported incidence data for anomalous litters/litters examined.

b Significantly different from controls ( p < 0.05) according to Fisher’s exact test conducted for this review.

Source: Khera et al. (1979).

1



### All available core dichotomous models in the EPA Benchmark Dose Software (BMDS) 2



### (version 2.1.2) were fit to the incidence data for each dataset. The multistage model was run for 3



### all polynomial degrees up to n-1 (where n is the number of dose groups including control). 4



### Adequate model fit was judged by three criteria: goodness-of- fit p -value ( p ≥ 0.1), visual 5



### inspection of the dose- response curve, and a value of <2 for the largest scaled residual for any 6



### data point in the dataset (including the control). Among all of the models providing adequate fit 7



### to the data, the lowest BMDL was selected as the potential point of departure (POD) when the 8



### difference between the BMDLs estimated from these models was more than threefold; otherwise, 9



### the BMDL from the model with the lowest Akaike’s Information Criterion (AIC) was chosen as 10



### the candidate POD. In accordance with U.S. EPA (2000b) guidance, BMDs and BMDLs 11



### associated with an extra risk of 10% were calculated for all models. In the absence of 12



### information to identify the biologically significant level of response for an endpoint, a 13



### (benchmark response) BMR of 10% extra risk is typically chosen as a response level for 14



### dichotomous data and is recommended for the BMR when using dichotomous models to 15



### facilitate a consistent basis of comparison across assessments and endpoints. 16



### A BMR of 10% extra risk was selected to derive the POD for development effects from 17



### the Khera et al. (1979) study because the endpoints were characterized as affected litters. A 18



### BMR of 5% extra risk has typically been used for quantal data in reproductive and 19



### developmental studies when data are available to characterize individual pups within litters (U.S. 20



### EPA, 2000b). Since this level of reporting was not available, nested models could not be used. 21



### Thus, a BMR of 10% extra risk among affected litters was employed in order to better 22



### approximate a 5% extra risk in affected offspring and to recognize the litter as the experimental 23



### unit. BMDs and BMDLs associated with extra risk of 5% for all endpoints were also calculated 24



### for comparison. 25



### When core models failed to provide adequate fit to the data, optimizations of the models 26



### (model restriction adjustments, specification of initial parameters, and use of alternative models) 27

### 100 DRAFT

- DO NOT CITE OR QUOTE were attempted in a n effort to achieve adequate fit. If these optimizations failed to achieve better 1



### fit, the highest dose was dropped and the entire modeling procedure was repeated. If an adequate 2



### fit could not be achieved after dropping the highest dose, then the dataset was determined to be 3



### unsuitable for BMD modeling. 4



### For continuous data, all core continuous models available in the EPA BMDS 5



### (v ersion 2.1 .2 ) were first applied to the data while assuming constant variance. If the data were 6



### consistent with the assumption of constant variance ( p ≥ 0.1), then the fit of all the continuous 7



### models to the mean were evaluated while assuming constant variance. In the absence of 8



### information to indicate the biologically significant level of response, BMDs and BMDLs were 9



### calculated based on a benchmark response ( BMR ) representing a change of 1 SD from the 10



### control. BMDs and BMDLs for decreased body weight were also calculated for a BMR of 10% 11



### decrease from the control (i.e., 10% relative deviation [RD] ) because a 10% decrease in body 12



### weight is generally considered to represent a minimally biologically significant effect. For 13



### serum enzyme activities (AST, ALT, AP, LDH), BMDs and BMDLs were also calculated for a 14



### BMR of 100% increase from the control (i.e., twofold or 1 RD; BMD 1RD 

### and BMDL 1RD 

### )

. 15



### Several expert organizations, particularly those concerned with early signs of drug-induced 16



### hepatotoxicity, have identified an increase in liver enzymes (AST, ALT, AP) compared with 17



### concurrent controls of two- to fivefold as an indicator of concern for hepatic injury (EMEA, 18



### 2006; Boone et al., 2005). Because LDH, like liver enzymes, is one of the more specific 19



### indicators of hepatocellular damage in most animal species and generally parallels changes in 20



### liver enzyme s in toxicity studies where liver injury occurs, a similar twofold increase in LDH is 21



### considered indicative of liver injury in experimental animals. A similar approach was taken for 22



### BUN. 23



### Adequate model fit was judged by three criteria : goodness -of- fit p -value ( p ≥ 0.1), visual 24



### inspection of the dose- response curve, and a value of <2 for the largest scaled residual for any 25



### data point in the data set (including the control). Among all of the models providing adequate fit 26



### to the data, the lowest BMDL was selected as the potential POD when the BMDLs estimated 27



### from these models varied by more than threefold; otherwise, the BMDL from the model with the 28



### lowest AIC was chosen as the candidate POD. When the test for constant variance was negative, 29



### all models we re run again while applying the power model integrated into the BMDS to account 30



### for nonhomogeneous variance. When the nonhomogeneous variance model provided an 31



### adequate fit ( p ≥ 0.1) to the variance data, the models were evaluated using the nonhomogen eous 32



### variance model. Model fit and POD selection proceeded as described earlier. When both tests 33



### for variance (constant and nonhomogeneous) provided inadequate fit to the variance data, model 34



### restriction adjustments were attempted in an effort to achieve adequate fit. If these 35



### manipulations failed to achieve better fit, the highest dose was dropped and the entire modeling 36



### procedure was repeated. If an adequate fit could not be achieved after dropping the highest dose, 37



### then the dataset was determined to be u nsuitable for BMD modeling. 38

### 101 DRAFT

- DO NOT CITE OR QUOTE Summary modeling results are presented in Table 5 -4 and Figure 5-2 ; more detailed 1



### modeling results are presented in Appendix B (Tables B- 4 through B -24 and respective model 2



### output files). The BMDs and BMDLs shown in Table 5-4 and Figure 5-2 are those from the 3



### best -fitting models for each endpoint. For datasets to which no model could be fit, NOAELs and 4



### LOAELs were considered for the candidate POD

. 5

6

### 102 DRAFT

- DO NOT CITE OR QUOTE Table 5 -4. Summary of BMDs/ BMDLs for selected nonneoplastic effects

foll owing oral exposure of rats and mice to biphenyl

Males Females

Best fitting

model Benchmark result (mg/kg

- d) Best fitting

model Benchmark result (mg/kg

- d)

F344 rats (Umeda et al., 2002); biphenyl in the diet for 2 yrs

Kidney BMD 10 BMDL 10 BMD 10 BMDL 10

Renal pelvis

Transitional
hyperplasia cell n odular Multistage

3

- degree 193 127 Multistage

2

- degree 274 212

Transitional
hyperplasia cell s imple Gamma 314 113 Gamma 71 52

Mineralization Log

- probit 208 138 Multistage

1

- degree 88 56

Kidney

- other

Hemosiderin deposit Not select ed b

– – Dichotomous

- Hill 45 23

Papillary mineralization Multistage

1

- degree 92 58 Logistic 292 219

Bladder BMD 10 BMDL 10 BMD 10 BMDL 10

Transitional cell hyperplasia Gamma 205 147 Not selected b

– –

BDF 1 mice (Umeda et al., 2005); biphenyl in the diet for 2 yrs

Kidney BMD 10 BMDL 10 BMD 10 BMDL 10

Mineralization Log

- logistic 721 276 Log

- logistic 233 122

Clinical chemistry BMD 1RD BMDL 1RD BMD 1RD BMDL 1RD

AST Not selected b

– – Power 190 a 122 a

AL T Not select ed b

– – No adequate fit c

– –

L DH Not select ed b

– – No adequate fit c

– –

AP Not select ed b

– – No adequate fit c

– –

BMD 1SD BMDL 1SD BMD 1SD BMDL 1SD

BUN Linear 415 a 267 a No adequate fit c

– –
Body weight BMD 0.1RD BMDL 0.1RD BMD 0.1RD BMDL 0.1RD

Terminal body weight No adequate fit c

– – Linear 583 511

Wistar rats (Khera et al., 1979); biphenyl by gavage to dams on GDs 6

– 15 BMD 10 BMDL 10

Litters with fetal skeletal anomalies Log

- logistic 57 2 0

a Adequate fit obtained only after excluding results from the highest dose group.

b “N ot selected” indicates that the data set was not selected for dose

- response analysis because either a treatment

related effect was not observed or because the response observed in the other sex in the same study was more

rob ust

.

c “No adequate fit” indicates that none of the models in BMDS provided an adequate fit to the data.

BMD = maximum likelihood estimate of the dose associated with the selected BMR; BMDL = 95% lower
confidence limit on the BMD (subscripts denote benc hmark response: i.e., 10 = dose associated with 10% extra risk;

1RD = 100% relative deviation from control mean value; 0.1RD = 10% relative deviation from control mean value;

1SD = 1 SD from control mean value)

### 103 DRAFT

- DO NOT CITE OR QUOTE 1

2



### Figure 5

- 2. BMDs and BMDLs for selected noncancer effects in rats and mice from repeated oral exposure to biphenyl. 3

4 0 50 100 150 200 250 300 350 400 450mg/kg-day BMD BMDL BMD = 583 mg/kg

- d BMD = 721 mg/kg

- d

BMDL = 511 mg/kg

- d

Female mouse (1) Female mouse (1) Male rat (2) Male mouse (1) Female mouse (1) Male mouse (1) Male rat (2) Female rat (2) Male rat (2) Female rat (2) Male rat (2) Female rat (2) Female rat (2) Male rat (2) Female rat (2) Anomalous litters (3)

Terminal A ST TC hyperplasia Mineralization BUN Nodular hyperplasia Simple hyperplasia Renal pelvis

mineralization Hemo

siderin Papillary mineralization

Body weight Liver Bladder Kidney Develop

mental

TC = transitional cell

(1) = Umeda et al. (2005); (2) = Ume da et al. (2002); (3) = Khera et al. (1979)

### 104 DRAFT

- DO NOT CITE OR QUOTE Examination of the BMD and BMDL values in Table 5-4 and Figure 5

- 2 reveals 1



### BM D/BMDL pairs for four kidney effects and for the developmental effect that are clustered 2



### below BM D/BMDL pairs for the other effects. The BMDL values in this cluster range from 3



### 20 to 58 mg/kg-day and identify the following as the most sensitive nonneoplastic effects 4



### associated with repeated oral exposure to biphenyl in animals: (1) renal transitional cell 5



### hyperplasia (simple) in female F344 rats ( 52 mg/kg-day), (2) renal mineraliz ation in fe male F344 6



### rats (5 6 mg/kg-day), (3) renal hemosiderin deposition in female F344 rats (2 3 mg/kg-day), 7



### (4) renal papillary mineralization in male F344 rats (5 8 mg/kg-day), and (5) increased litters with 8



### fetal skeletal anomalies in Wistar rats ( 20 mg/kg-day). 9



### NOAEL values for endpoints with datasets for which adequate model fits could not be 10



### obtained using BMDS were higher than the BMDL values for these five kidney and 11



### developmental endpoints. These include selected clinical chemistry parameters in female BDF 1 12



### mice (NOAELs for LDH, AP, and BUN: 414 mg/kg-day; NOAEL for ALT: 134 mg/kg- day) and 13



### terminal body weight in male BDF 1 

### mice (NOAEL: 97 mg/kg -day). 14



### The increased fetal skeletal anomalies in Wistar rats was selected as the critical effect for 15



### de riving an oral RfD because it was considered to be an adverse effect and resulted in the most 16



### sensitive POD (BMDL 10 

### of 2 0 mg/kg -day) observed compared with other PODs for biphenyl17



### induced kidney effects

. 18

19



### 5.1.3. RfD Derivation— Including Application of Unc ertainty Factors (UFs) 20

21 

### The RfD for biphenyl was derived by dividing the POD of 20 mg/kg-day (i.e., the

22 

### BMDL 10 

### based on fetal skeletal anomalies in litters from biphenyl

- treated pregnant Wistar rats)

23 

### by a total UF of 100, comprised of 10 for interindividual variability and 10 for interspecies

24 

### extrapolation , as described below.

25

26 

### • An UF of 10 was applied to account for interspecies variability in extrapolation from

27 

### laboratory animal s (rats) to humans because information is not available to quantitatively

28 

### assess toxicokinetic or toxicodynamic differences between animals and humans.

29

30 

### • An UF of 10 was appli ed to account for intraspecies variabilit y in susceptibility to

31 

### biphenyl , as quantitative information for evaluating toxicokinetic and toxicodynamic

32 

### differences a mong humans are not available.

33

34 

### • An UF of 1 was applied for use of data from a subchronic study to assess potential effects

35 

### from chronic exposure because developmental toxicity resulting from a narrow period of

36 

### exposure was used as the critical effect. The developmental period is recognized as a

37 

### susceptible life stage when exposure during a time window of development is more

38 

### relevant to the induction of developmental effects than lifetime exposure.

39

40 

### • An UF of 1 was applied for extrapolation from a LOAEL to a NOAEL because the

41 

### current approach is to address this factor as one of the considerations in selecting a BMR

### 105 DRAFT

- DO NOT CITE OR QUOTE 1 

### for BMD modeling. In this case, a BMR of 10 % increase in incidence of litters with

2 

### skeletal anomalies was selected under an assumption that it represents a minimal

3 

### biologically significant change.

4

5 

### • An UF of 1 to account f or database deficiencies was applied. T he biphenyl database

6 

### includes chronic toxicity studies in rats (Umeda et al., 2002; Shiraiwa et al., 1989;

7 

### Ambrose et al., 1960; Pecchiai and Saffioti, 1957; Dow Chemical Co., 1953) and mice

8 

### (Umeda et al., 2005; Imai et al., 1983); subchronic toxicity studies in rats (Shibata et al.,

9 

### 1989a, b; Kluwe et al., 1982; Søndergaard and Blom, 1979; Booth et al., 1961) and mice

10 

### (Umeda et al., 2004); a developmental toxicity study in rats (Khera et al., 1979); and one11 

### and three-generation reproductive toxicity studies in rats (Ambrose et al., 1960; Dow

12 

### Chemical Co., 1953). Epidemiological studies provide some evidence that biphenyl may

13 

### induce functional changes in the nervous system at concentrations in excess of

14 

### occupational exposure limits. Seppäläinen and Häkkinen (1975) reported small increases

15 

### in anomalies in nerve conduction, EEG, and ENMG signals in workers exposed to

16 

### biphenyl during the production of biphenyl-impregnated paper at concentrations that

17 

### exceeded the occupational limit by up to 100- fold , and Wastensson et al. (2006) reported

18 

### a cluster of Parkinson’s disease in a Swedish factory manufacturing biphenyl19 

### impregnated paper. No o ther clusters of Parkinson’s disease have been reported in

20 

### biphen yl exposed populations, and Wastensson et al. (2006) acknowledged that chance is

21 

### an alternative explanation for this cluster. Studies in experimental animal models have

22 

### not identified effect s on the nervous system following biphenyl exposure. Accordingly,

23 

### t hese epidemiologic studies d o not suggest that the nervous system is a sensitive target of

24 

### biphenyl toxicity and therefore the lack of nervous system- specific studies is not

25 

### considered a gap in the biphenyl toxicity database.

26 

### The RfD for biphenyl wa s calculated as follows: 27



### RfD = BMDL 10 

### ÷ UF 28



### = 20 mg/kg-day ÷ 100 29 

### = 0.2 mg/kg-day 30

31



### 5.1.4. Previous RfD Assessment 32


### The previous IRIS assessment for biphenyl ( U.S. EPA , 1989) derived an oral RfD of 33



### 0.05 mg/kg-day based on kidney damage in albino rats administered biphenyl for 2 years at 34



### dietary levels ≥ 0.5% (Ambrose et al., 1960). U.S. EPA considered the dietary level of 0.1% (50 35



### mg/kg-day using a food factor of 0.05/day) to represent a NOAEL due to the following: (1) 36



### uncertainty in the significance of effects observed at lower doses as compared to the more certain 37



### adverse effect level of 0.5% in the diet and (2) supporting findings of 0.1% biphenyl as a 38



### NOAEL in an unpublished report of a subchronic rat feeding study and a three

- generation rat 39



### reproduction study performed by Stanford Research Institute (Dow Chemical Co., 1953). The 40



### NOAEL of 50 mg/kg-day was divided by a total UF of 1,000 (10 for extrapolation from animals 41



### to humans, 10 for protection of sensitive h uman subpopulations, and a modifying factor of 10 to 42



### account for intraspecies variability demonstrated in the threshold suggested by the data in the 43



### chronic animal study). 44

45

### 106 DRAFT

- DO NOT CITE OR QUOTE 5.2. INHALATION REFERENCE CONCENTRATION (RfC) 1



### 5.2.1. Choice of Principal Study an d Critical Effect

— with Rationale and Justification 2



### Human data are limited to assessments of possible associations between occupational 3



### exposure to biphenyl and health outcomes where inhalation is presumed to have been the major 4



### exposure route. Clinical si gns and abnormal electrophysiological test results among workers 5



### exposed to biphenyl during the production of biphenyl-impregnated fruit wrapping paper provide 6



### evidence of biphenyl -induced neurological effects (Seppäläinen and Häkkinen, 1975; Häkkinen 7



### et a l., 1973, 1971). Case reports include an account of periodic loss of strength and eventual 8



### signs of chronic hepatitis in a woman during a 25-year period of employment at a fruit-packing 9



### facility where biphenyl- impregnated paper was used (Carella and Betto lo, 1994) and a cluster of 10



### five cases of Parkinson’s Disease (0.9 cases expected) at a facility manufacturing biphenyl

11



### impregnated paper (Wastensson et al., 2006). None of these studies provided air monitoring data 12



### adequate to characterize workplace expos ures to biphenyl. Therefore, data from the available 13



### human studies could not be used for dose-response analysis and derivation of an RfC. 14



### Limited information is available regarding the effects of inhaled biphenyl in laboratory 15



### animals

. In mice, repeated airborne exposure to biphenyl (7 hours/day, 5 days/week for 2 weeks) 16



### at concentrations as high as 54.75 ppm (345.5 mg/m 3 

### ) appeared to cause no symptoms (Sun 17



### Co mpany Inc., 1977a). In a series of studies that included repeated inhalation exposure of 18



### rabbits, rats, and mice to atmospheres containing biphenyl for periods of 68–94 days 19



### (Deichmann et al., 1947; Monsanto, 1946), rabbits exhibited no signs of exposure- related 20



### adverse effects at concentrations as high as 300 mg/m 3 

### . Irritation of mucous membranes w as 21



### observed in rats at concentrations of 40 and 300 mg/m 3 

### . Mice were the most sensitive to inhaled 22



### biphenyl; irritation of the upper respiratory tract was noted at a concentration of 5 mg/m 3 23



### (Deichmann et al., 1947; Monsanto, 1946), but other biphenyl concentrations were not tested in 24



### this experiment . The limitations of a single exposure level and poorly- reported study details 25



### preclude the use of this study for RfC derivation. 26



### Repeated expos ure of mice to biphenyl at vapor concentrations of 25 or 50 ppm 27



### (157.75 or 315.5 mg/m 3 

### ) for 13 weeks resulted in high incidences of pneumonia and tracheal 28



### hyperplasia, and high incidences of congestion and edema in the lungs, liver, and kidney (Sun 29



### Co mpany Inc., 1977b). The following study limitations and lack of supporting data preclude the 30



### usefulness of this study for deriving an RfC for biphenyl. Measured b iphenyl exposure 31



### concentrations varied greatly during the first half of the 13-week exposure period; for example, 32



### in the high concentration group ( target concentration of 50 ppm), the measured concentrations 33



### ranged from 5 ppm to 102 ppm during the first 45 exposure sessions. High mortality in 25 ppm 34



### male mice (40/50) after 46 exposures necessitated the use of replacement animals; these 35



### replacement animals received the same total number of exposure sessions as the surviving 36



### animals from the original 25 ppm group but exposures were not concurrent. Histopathological 37



### findings were reported only for males and females combined. Reports of lung congestion and 38

### 107 DRAFT

- DO NOT CITE OR QUOTE hemorrhagic lungs in some control mice were not confirmed histopathologically , and congestion 1



### in the lung, liver, and kidney were considered by the study pathologist a likely effect of the 2



### anesthetic used for killing the mice. The severity of reported histopathologic lesions was not 3



### specified. 4



### The 13- week inhalation mouse study of Sun Company Inc. (1977b) is the only available 5



### study that employed at least subchronic-duration exposure and included multiple biphenyl 6



### exposure levels. This study is considered inadequate for RfC derivation because : ( 1) exposure 7



### levels were highly variable during the first half of the 13-week exposure period, (2) one of the 8



### exposure groups experienced high losses (46/100) due to an overheating event and 9



### cannibalization after 46 exposures, although replacement mice were subsequently added and 10



### received a total of 65 exposures, and (3) limitations in the reporting of histopathological findings. 11



### An RfC was not derived due to the significant uncertainty associated with the inhalation 12



### database for biphenyl, and route- to -route extrapolation was not supported in the absence of a 13



### PBPK model. Although an RfC cannot be derived, it should be noted t hat the available 14



### inhalation data provides some evidence that inhalation exposure to biphenyl could induce 15



### respiratory or systemic lesions. 16

17



### 5.2. 2. Previous RfC Assessment 18



### No RfC was derived in the previous (1985) IRIS assessment. 19

20



### 5.3. UNCERTAINTIES IN THE RfD and RfC 21



### Risk assessments should include a discussion of uncertainties associated with the derived 22



### toxicity values. To derive the oral RfD, the UF approach (U.S. EPA, 2002, 1994b) was applied 23



### to a POD of 1 0 mg/kg

- day (see Section 5.1). Factors were applied to the POD to account for 24



### extrapolating from responses observed in an animal bioassay to humans or a diverse human 25



### population of varying susceptibilities. Uncertainties associated with the data set used to derive 26



### the biphenyl RfD are more fully described below. 27



### The available database was determined to be inadequate for deriving a chronic inhalation 28



### RfC for biphenyl (see Section 5.2). 29

30



### Selection of the critical effect for reference value determination

. The critical effect 31



### selected for derivation of the RfD was skeletal anomalies in fetuses from rat dams administered 32



### biphenyl by gavage during GDs 6– 15 . An increased incidence of these anomalies was reported 33



### at doses ≥500 mg/kg -day ; frank maternal toxicity , including death , was observed at the highest 34



### dose level (1,000 mg/kg-day). There is some degree of uncertainty regarding the toxicological 35



### significance of the reported skeletal anomalies (wavy or extra ribs and delayed ossification most 36



### commonly observe d) and the relevance of gavage dosing used in the developmental toxicity 37



### study to human exposures. Supporting developmental toxicity studies are not available. 38

### 108 DRAFT

- DO NOT CITE OR QUOTE Dose

- response modeling

. BMD modeling was used to estimate the POD for the biphenyl 1



### RfD. BMD modeling has advantages over a POD based on a NOAEL or LOAEL because, in 2



### part, the latter are a reflection of the particular exposure concentration or dose at which a study 3



### was conducted. A NOAEL or LOAEL lacks characterization of the dose

- response c urve, and for 4



### this reason, is less informative than a POD obtained from B MD modeling. The selected model, 5



### i.e., the log- logistic model , p rovided the best mathematical fit to the experimental data set (as 6



### determined by the lowest AIC), but do es not necessa rily have greater biological support over the 7



### various models included in BMDS. Other models in BMDS yield estimates of the POD higher 8



### than the POD derived using the log- logistic m odel (by up to 5.8 -fold). 9



### Interspecies extrapolation of dosimetry and toxico dynamics. Limited information is 10



### available regarding species -specific toxicokinetic and toxicodynamic differences in biphenyl 11



### metabolism. Results of available in vitro assays of human and rat liver preparations suggest 12



### qualitative similarities and quanti tative differences in biphenyl metabolism (Powis et al., 1989, 13



### 1988; Benford et al., 1981). Available in vivo animal data demonstrate qualitative and 14



### quantitative differences between rats and mice (Halpaap -Wood et al., 1981a; Meyer and Scheline 15



### 1976; Meyer et al., 1976a). However, in vivo human data are lacking and it is uncertain which 16



### animal species, the rat or the mouse, would be more comparable to humans. Other areas of 17



### biphenyl toxicokinetics (absorption, distribution, elimination), have received so me attention in 18



### animal studies, but comparative human data are not available. PBPK model s for biphenyl to 19



### address difference s in toxicokinetics between animal and human are lacking. An UF of 10 was 20



### used to account for animal to human extrapolation in the absence of adequate comparative 21



### animal and human toxicokinetic and toxicodynamic data for biphenyl. 22



### Sensitive human populations. Heterogeneity among humans is another uncertainty 23



### associated with extrapolating doses from animals to humans. Identification of populations that 24



### might be relatively more susceptible to the toxic effects of biphenyl is not feasible because of the 25



### limited information on biphenyl metabolism and mode of action of biphenyl toxic ity

. It is 26



### known, however, that many CYP isozymes and glucuronidase exist in polymorphic forms. Such 27



### enzyme polymorphism may put some populations at increased risk from biphenyl exposure. In 28



### th e absence of biphenyl-specific data on human variation, a factor of 10 was used to account for 29



### uncertainty associated with human variation. Human variation may be larger or small er; 30



### however, biphenyl-specific data to examine the potential magnitude of over- or under- estimation 31



### are absent

. 32

33



### 5.4. CANCER ASSESSMENT 34



### 5.4.1. Choice of Study/Data— with Rationale and Justification 35


### No information was located regarding possible associations between oral exposure to 36



### biphenyl and cancer in humans. Two animal bioassays found statistically significant 37

### 109 DRAFT

- DO NOT CITE OR QUOTE 1 

### associations between lifetime oral exposure to biphenyl and tum or development. Biphenyl was

2 

### associated with urinary bladder tumors in male, but not female, F344 rats (Umeda et al., 2002)

3 

### and liver tumors in female, but not male, BDF 1 

### mice (Umeda et al., 2005). Tumor data for these

4 

### two sites w ere selected for dose

- response analysis.

5 

### No studies were identified that examined the association between inhalation exposure to

6 

### biphenyl and cancer in humans or animals.

7

8 

### 5.4.2. Dose -R esponse Data

9 

### The dose- response data for urinary bladder tumor formation resulting from lifetime oral

10 

### exposure of male and female F344 rats (Umeda et al., 2002) are shown in Table 5 -5. The dose11 

### response data for liver tumor formation resulting from lifetime oral exposure of male and female

12 

### BDF 1 

### mice (Umeda et al., 2005) are sho wn in Table 5-6. The datasets selected for BMD

13 

### analysis include urinary bladder transitional cell papilloma or carcinoma (combined) in the male

14 

### F344 rats and liver adenoma or carcinoma (combined) in the female BDF 1 

### mice.

15



### Table 5 -5 . Incidence data for tumors in the urinary bladder of male and
female F344 rats exposed to biphenyl in the diet for 2 years

Males Females

Biphenyl dietary concentration (ppm) 0 500 1,500 4,500 0 500 1,500 4,500

Calculated dose (mg/kg

- d) a 0 36.4 110 378 0 42.7 128 438

Tumor incidence c

Transitional cell

Papilloma 0 /50 0 /50 0 /50 10 /49 b

, 0 /50 0 /50 0 /50 0 /50

Carcinoma 0 /50 0 /50 0 /50 24 /49 b

, 0 /50 0 /50 0 /50 0 /50

Papilloma or carcinoma 0 50 0 /50 0 /50 31 /49 b

, 0 /50 0 /50 0 /50 0 /50

a Calculated doses based on TWA body weights (calculated from body weight data presented graphically in Figure 1
of Umeda et al., 2002) and chronic reference food consumption values for F344 rats listed in Table 1

- 6 of U.S. EPA

(1988)

. b Significantly different from control group ( p < 0.01) according to Fisher’s exact test

.

c One high

- dose male rat was excluded from the denominator because it died prior to week 52. It is assumed that
this rat did not have a tumor and was not exposed for a sufficient time to be at risk for developing a tumor. Umeda
et al. (2002) did not specify the time of appearance of the first tumor.

Source: Umeda et al. (2002).

16

### 110 DRAFT

- DO NOT CITE OR QUOTE Table 5 -6. Incidence data for liver tumors in male and female BDF 1 

### mice
fed diets containing biphenyl for 2 years

a Dietary concentration of biphenyl (ppm)

Males Females

Biphenyl dietary
concentration (ppm) 0 667 2,000 6,000 0 667 2,000 6,000

Reported dose (mg/kg

- d) 0 97 291 1,050 0 134 414 1,420

Tumor incidence c

Adenoma 8/ 50 6/49 7/ 49 3/50 2/ 48 3/50 12/ 49 a 10/4 8 a

Carcinoma 8/ 50 8/49 5/ 49 4/50 1/ 48 5/50 7/ 49 a 5/4 8

Adenoma or carcinoma 16/ 50 12/49 9/ 49 7/50 3/ 48 8/50 16/ 49 b 14/4 8 a

Significantly different from controls ( p < 0.05) according to Fisher’s exact test as reported by Umeda et al. (2005)

. b Significantly different from controls ( p < 0.01) according to Fisher’s exact test as reported by Umeda et al. (2005)

.

c One low

- dose, one mid

- dose male, two control, one mid

- dose, and two high

- dose female mice were excluded from
denominators because they died prior to week 52. It is assumed that they did not have tumors and were not exposed

for a sufficient time to be at ri sk for developing a tumor. Umeda et al. (2005) did not specify the time of appearance

of the first tumor.

So urce: Umeda et al. (2005).

1

2 

### 5.4.3. Dose Adjustments and Extrapolation Method(s)

3 

### 5.4.3.1. Bladder Tumors in Male Rats

4 

### T here is strong evidence that the occurrence of urinary bladder tumors in male rats

5 

### chronically exposed to biphenyl in the diet is a high-dose nongenotoxic phenomenon involving

6 

### occurrence of calculi in the urinary bladder leading to transitional cell damage, sustained

7 

### regenerative cell proliferation, and eventual promotion of spontaneously initiated tumor cells in

8 

### the urinary bladder epithelium (see Section 4.7.3.1 for a detailed discussion of the hypothe tized

9 

### mode of action for urinary bladder tumors in biphenyl- exposed male rats). No increased risk of

10 

### bladder tumors is expected a s long as the exposure to biphenyl is below the dose needed to form

11 

### calculi (Cohen and Ellwein, 1992). As noted in the EPA Guidelines for Carcinogen Risk

12 

### Assessment ( U.S. EPA, 2005a), a nonlinear approach to dose- response analysis is used when

13 

### there are sufficient data to ascertain the mode of action and conclude that it is not linear at low

14 

### doses and the agent does not demonstrate mutagenic or other activity consistent with linear ity at

15 

### low doses. Therefore, consistent with the cancer guidelines, a nonlinear extrapolation approach

16 

### for biphenyl-induced urinary bladder tumors was selected

.

17 

### Based on the proposed mode of action, the available evidence indicates that doses below

18 

### the oral RfD would not result in the sequence of events that includes calculus formation,

19 

### consequent epithelial cell damage, and sustained regenerative cellular proliferation

.

20 

### Accordingly, the RfD of 0.2 mg/kg-day derived for noncancer effects of biphenyl was judged to

21 

### be protective against an increased risk of biphenyl-induced urinary bladder cancer.

22

### 111 DRAFT

- DO NOT CITE OR QUOTE 5.4.3.2. Liver Tumors in Female Mice 1



### In t he study report of the ir 2- year bioassay in BDF 1 

### mice

, Umeda et al. (2005) provided 2



### averaged food consumption and biphenyl dose estimates for each exposure group (Table 1 of 3



### Umeda et al., 2005). The study report did not include average body weights for the exposure 4



### groups. Therefore, the biphenyl concentration in the food was multiplied by the corresponding 5



### average daily food consumption value to determine the average daily biphenyl intake. Dividing 6



### this average daily biphenyl intake by the author-calculated daily dose yielded the average body 7



### weight that would have been used by the study authors to ca lculate t he average daily biphenyl 8



### dose. Scaling factors were calculated using U.S. EPA (1988) reference body weight for humans 9



### (70 kg) and the average body weight for each dose group of female mice: (average body 10



### weight/70) 0.25 

### = scaling factor . The human equivalent dose ( HED ) was calculated as: HED = 11



### scaling factor × reported dose (Table 5 -7 ). 12

13



### Table 5 -7 . Scaling factors for determining HEDs to use for BMD modeling

of female BDF 1 

### mouse liver tumor incidence data from Umeda et al. (2005)

Biphenyl dietary concentration (mg/kg food) 667 2,000 6,000

Reported dose (mg/kg

- d) 134 414 1,420

Reported average food consumption (kg/d) 0.0058 0.0059 0.0059

Average mouse body weight (kg) a 0.0289 0.0285 0.0249

b Scaling factor 0.143 0.142 0.137

HED (mg/kg

- d) c 19 59 195

a ( Biphenyl concentration in food [ mg/kg food ] × reported average food consumption [ kg/day] ) ÷ reported average
daily dose of biphenyl (mg/kg

- day) = calculated average mouse body weight (kg).

b Calculated using reference body weight for humans (70 kg ; U.S. EPA, 1988 ), and the average body weights for
each dose group: mouse

- to

- human scaling factor = (average mouse body weight/70) 0.25

.

c HED = reported dose × scaling factor.

4 1



### The EPA’s Guidelines for Carcinogen Risk Assessment ( U.S. EPA, 2005a) recommend 15



### that when the weight of evidence evaluation of all available data are insufficient to establish the 16



### mode of action for a tumor site and when scientifically plausible based on the available data, 17



### linear extrapolation is used as a default approach. Accordingly, a linear approach to low -dose 18



### extrapolation for biphenyl- induced liver tumors in female mice was selected because the mode of 19



### action for this tumor site has not been established (see Section 4.7.3.2). 20



### I ncidence data for liver adenoma or carcino ma (combined) in the female mouse used to 21



### derive the oral slope factor are presented in Table 5 -8. Tumor incidence data were adjusted to 22



### account for mortalities before 52 weeks; it was assumed that animals dying before 52 weeks 23



### wer e not exposed for sufficient time to be at risk for developing tumors (see footnote a in Table 24



### 5-8). 25

26

### 112 DRAFT

- DO NOT CITE OR QUOTE Table 5 -8 . Incidence of liver adenomas or carcinomas (combined) in female

BDF 1

Biphenyl dietary concent ration (ppm) 0 667 2,000 6,000

HED (mg/kg

- d) 0 19 59 195

Tumor incidence

Adenoma or carcinoma (combined) 3/48 a 8/50 16/49 a,b 14/48 a,c 

### mice fed diets containing biphenyl for 2 years

a Two control, one mid

- dose, and two high

- dose female mice were excluded from denominators because they died
prior to week 52. It is assumed that they did not have tumors and were not exposed for a sufficient time to be at
risk for developing a tumor. Umeda et al. (2005) did not specify the time of appearance of the first tumor.

b Significantly different from control s ( p < 0.05) according to Fisher’s exact test.

c Significantly different from controls ( p < 0.01) according to Fisher’s exact test.

Source: Umeda et al. (2005).

1

2 

### The multistage -cancer model in the EPA BMDS (version 2.1.2), using the extra risk

3 

### option, was fit to the female mouse liver tumor incidence data. The multistage model has been

used by EPA in the vast majority of quantitative cancer assessments because it is thought to 4



### reflect the multistage carcinogenic process and it fits a broad array of dose

- response patterns. 5



### The multistage -cancer model was run for all polynomial degrees up to n-1 (where n is the 6



### number of dose groups including control). An extra ris k of 10% tumor incidence was selected as 7



### the benchmark response. Adequate model fit was judged by three criteria: goodness-of- fit p

8



### value ( p ≥ 0.05), visual inspection of the dose-response curve, and a value of < 2 for the largest 9



### scaled residual for any data -point in the dataset (including the control). If an adequate fit to the 10



### data was not achieved using the protocol above, the other dichotomous models were fit to the 11



### data. If none of the models achieved an ad equate fit for the full dataset, the highest dose was 12



### dropped and the entire modeling procedure was repeated. 13



### When liver tumor incidence data for all dose groups were modeled, none of the models in 14



### BMDS, including the multistage model, provided an adequate fit of the data (see Appendix C, 15



### Table C -2). The animals in the highest dose group, while exhibiting a statistically significantly 16



### increased incidence in liver tumors compared with controls, did not show a monotonic increase 17



### in tumor response compared wi th the respo nses at the lower doses. To better estimate responses 18



### in the low dose region, the high dose group was excluded as a means of improving the fit of the 19



### model in the region of interest. When the high-dose group was dropped, the multistage model 20



### provided an adequate fit to the data (see Appendix C, Table C-2). The BMD HED10 

### and 21



### BMDL HED10 

### using this latter dataset were 18.7 and 12.2 mg/kg-day , respectively . See Appendix 22



### C for more information. 23

24

### 113 DRAFT

- DO NOT CITE OR QUOTE 5.4.4. Oral Slope Factor and Inhalation Unit R isk 1



### A low

- dose linear extrapolation approach results in calculation of an oral slope factor that 2



### describes the cancer risk per unit dose of the chemical at low doses. The oral slope factor was 3



### calculated by dividing the risk (i.e., BMR of 10% extra risk) at t he POD by the corresponding 4



### BMDL (0.1/BMDL HED 10 

### ). Using linear extrapolation from the BMDL HED 10 

### , the human 5



### equivalent oral slope factor of 8.2 × 10 -3 

### (mg/kg-d) -1 

### (round to one significant figure, 8 × 10 -3 6



### (mg/kg-d) -1 

### ) w as derived for liver tumors in female BDF 1 

### mice ( Table 5 -9). 7

8



### Table 5 -9 . POD and oral slope factor derived from liver tumor incidence

data from BDF 1 

### female mice exposed to biphenyl in the diet for 2 years

BMD HED10 BMDL HED10 Slope factor a (risk per
Species/tissue site (mg/kg

- d) (mg/kg

- d) [mg/kg

- d])

Female mouse liver tumors 18. 7 12. 2 8.2 × 10

- 3

a Human equivalent slope factor = 0.1/BMDL 10HED ; see Appendix C for details of modeling results.

9

10 

### This slope factor should not be used with exposures >12.2 mg/kg-day (the P OD for this

dataset ), because above the POD , the fitted dose- response model better characterizes what is 11



### known about the carcinogenicity of biphenyl (i.e., the slope factor may not approximate the 12



### observed dose- response relationship ad equately at exposure exceeding 12.2 mg/kg- day)

. 13



### An inhalation unit risk for biphenyl was not derived in this assessment. The potential 14



### carcinogenicity of inhaled biphenyl has not been evaluated in human or animal studies

, and 15



### route- to -route extrapolation was not possible in the absence of a PBPK model. 16

17



### 5.4.5 . Uncertainties in Cancer Risk Values 18



### 5.4.5.1. Oral Slope Factor 19



### A number of uncertainties underlie the cancer u nit risk for biphenyl. Table 5 -10 20



### summarizes the impact on the assessment of issues such as the use of models and extrapolation 21



### approaches (particularly those underlying the Guidelines for Carcinogen Risk Assessment (U.S. 22



### EPA, 2005a), the effect of reasonable alternatives, the decision concerning the pre ferred 23



### approach, and its justification. 24



### The uncer tainties presented in Table 5 -10 have a varied impact on risk estimates. Some 25



### suggest risks could be higher than was estimated, while others would decrease risk estimates or 26



### have an impact of an uncertain direction. Several uncertainties are quantitatively characterized 27



### for the significantly increased rodent tumors. These include the statistical uncertainty in the 28



### multistage modeling estimate. Due to limitations in the data, particularly regarding the M OA 29



### and relative human sensitivity and variability, the quantitative impact of other uncertainties of 30



### potentially equal or greater impact has not been explored. As a result, an integrated quantitative 31



### analysis that considers all of these factors was not undertaken. 32

### 114 DRAFT

- DO NOT CITE OR QUOTE Table 5 -10. Summary of uncertainties in the biphenyl cancer slope factor

Consideration/ Impact on slope

a pproach factor Decision Justification

Selection of data No other studies or Umeda et al . (2005) The bioassay by Umeda et al. (2005) was a

set data sets could be studies were selected. well conducted experiment with sufficient

used to derive a slope dose groups (four dose groups

, including

factors control) and animal numbers (50 animals/sex)

per group.

Cr oss

- species A lternative s (i.e. A dministered dose was There are no data to support alternatives. Use scaling scaling by [body scaled to humans on of [body weight] 3/4 for cross

- species scaling is
weight] or [ body the basis of consistent with data that allow comparison of
weight] 2/3 ) could ↑ or equivalence of potencies in humans and animals, and it is
mg/kg 3/4 ↓ slope factor -d ay (default supported by analysis of the allometric
approach) variation of key physiological parameters
across mammalian species. No PBPK model is

av ailable to derive internal dose s

.

E xtrapolation No impact on the Nonlinear Available MOA data for urinary bladder

p rocedure for rat slope factor because extrapola tion

. The tumors support nonlinearity ( i.e., that bladder
urinary bladder the MOA for male rat RfD of 0. 2 mg/kg

- day tumor is a high

- dose phenomena, and is

tumor s bladder tumors does is considered to protect closely related to calculi formation in the
not support low

- dose against the risk of urinary bladder of male rats ).
linear extrapolation. urinary bladder tumor s

.

E xtrapolation Departure from EPA’s Multistage model to Available MOA data do not inform selection

p rocedure for Guidelines for determine the POD, of dose

- response model; linear approach in

mouse liver tumors Carcinogen Risk linear low

- dose absence of clear support for an alternative is

Assessment POD extrapolation from generally consistent with scientific
paradigm, if justified, POD (default deliberations supporting EP A’s Guidelines for

could ↓ or ↑ slope approach) Carcinogen Risk Assessment

. factor by an unknown

extent

Human relevance Human risk could ↑or Liver tumors in female It was assumed that humans are as sensitive as

of female mouse ↓, depending on mice are relevant to the most sensitive rodent gender/species

liver tumor data relative sensitivity human exposure tested; true correspondence is unknown.

Model uncertainty For poorly fitting liver Drop highest dose of M odel options explor ed with full liver tumor

tumors dataset

, the liver tumors datasets did not generate a p ≥ 0.05, which is

alternatives could ↓ or dataset

. one of the indications of dropping the highest
↑ slope factor dose according to the draft Benchmark Dose

Technical Guidance (U

. S

. EPA

, 2000b).

Statistical ↓ slope factor 1. 5

- fold BMD L (default Limited size of bioassay results in sampling
uncertainty at POD if BMD 10 used rather approach for variability; lower bound is 95% conf idence

than BMD L 10 calculating plausible interval on dose

. upper bound)

Human population Low

- dose risk ↑ to an Considered No data to support range of human
variability / unknown extent qualitatively variability/sensitivity in metabolism or

se nsitive response , including whether children are more
subpopulations sensitive.

BMDL 10 = 95% lower confidence limits on the doses associated with a 10% extra risk of cancer incidence

.

1 

### 5.4.5.2. Inhalation Unit Risk

2 

### The potential carcinogenicity of inhaled biphenyl has not been assessed. Therefore, a

3 

### quantitative cancer assessment for biphenyl by the inhalation pathway was not performed.

### 115 DRAFT

- DO NOT CITE OR QUOTE 1

2 

### 5.4.6 . Previous Cancer Assessment

3 

### In the previous IRIS cancer assessment ( U

4 

### D; not classifiable as to human carcinogenicity ba .S. EPA

, 1991), biphenyl was listed in Group

sed on no human data and inadequate studies

in mice and r ats. Neither an oral slope factor nor inhalation unit risk was derived in the previous 5



### cancer assessment. 6

7

### 116 DRAFT

- DO NOT CITE OR QUOTE 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE 1



### RESPONSE 2

3

4

5 

### 6.1. HUMAN HAZARD POTENTIAL

6 

### 6.1.1. Noncancer

Toxicokinetic studies of animals indicate that o rally adm inistered biphenyl is rapidly and 7



### readily absorbed, distributed widely to tissues following absorption, and rapidly eliminated from 8



### the body, principally as conjugated hydroxylated metabolites in the urine (Meyer, 1977; Meyer 9



### and Scheline, 1976; Meyer et al., 1976a, b). Data for absorption, distribution, and elimination 10



### are not available for inhaled or dermally applied biphenyl. Metabolism to a range of 11



### hydroxylated metabolites has been demonstrated in in vitro systems with rat and human cells and 12



### tissues. Human metabolism of biphenyl appears to be qualitatively similar to metabolism in the 13



### rat, although some reports of quantitative differences are available (Powis et al., 1989, 1988; 14



### Benford et al., 1981). 15



### Available human health hazard data consist of limited assessments of workers exposed to 16



### biphenyl during the production or use of biphenyl-impregnated fruit wrapping paper in which 17



### signs of hepatic and nervous system toxicity were observed. 18



### Chronic oral studies in rats and mice identify the liver and urinary system as principal 19



### targets of biphenyl toxicity, the rat kidney being the most sensitive. Results of a developmental 20



### toxicity study in rats indicate that skeletal development is a sensitive indicator of biphenyl 21



### toxicity. In chronically exposed rats, non- neoplastic kidney lesions (simple transitional cell 22



### hyperplasia in the renal pelvis and hemosiderin deposits) were found in females at ≥1,500 ppm 23



### biph enyl in the diet (128 mg/kg -day), and urinary bladder tumors, associated with urinary 24



### bladder calculi and transitional cell hyperplasia, were found in males, but not females, at the 25



### highest tested concentration, 4,500 ppm (378 mg/kg-day) (Umeda et al., 200 2). Several other rat 26



### studies provide supporting evidence that the kidney and other urinary tract regions are sensitive 27



### targets for biphenyl in rats (Shiraiwa et al., 1989; Ambrose et al., 1960; Pecchiai and Saffiotti, 28



### 1957; Dow Chemical Co., 1953). In chronically exposed BDF 1 

### mice, increased incidence of 29



### nonneoplastic effects on the kidney (mineralization) and liver (increased activities of plasma 30



### ALT and AST) were found in females exposed to ≥2,000 ppm biphenyl in the diet (414 mg/kg

31



### day) (Umeda et al., 2005). In contrast, no exposure- related nonneoplastic or neoplastic effects 32



### on the liver or kidney were found in female ddY mice exposed to 5,000 ppm biphenyl in the diet 33



### for 2 years (Imai et al., 1983) or in B6C3F 1 

### and B6AKF 1 

### mice exposed to 517 ppm bip henyl in 34



### the diet for 18 months (Innes et al., 1969; NCI, 1968). In the only available developmental 35



### toxicity study for biphenyl, increased incidences of litters with fetuses showing skeletal 36



### anomalies were reported following exposure of pregnant rats to gavage doses ≥500 mg/kg -day 37



### on GDs 6 –15 (Khera et al., 1979). 38

### 117 DRAFT

- DO NOT CITE OR QUOTE Biphenyl effects on reproductive function in rats have been reported at a higher exposure 1



### level than the lowest exposure levels associated with urinary tract , liver, or developmental 2



### toxicity. No exposure-related effect on the number of dams with litters was found following 3



### exposure of male and female albino rats to up to 5,000 ppm biphenyl in the diet ( 525 mg/kg -day) 4



### for 11 or 60 days prior to mating (Ambrose et al., 1960). In a three- generati on rat study, 5



### decreased fertility, decreased number of pups/litter, and decreased pup body weight were 6



### observed at 10,000 ppm biphenyl in the diet; (947 mg/kg-day), but not at ≤1,000 ppm (Dow 7



### Chemical Co., 1953). 8



### No chronic inhalation toxicity studies in a nimals are available. In subchronic inhalation 9



### toxicity studies, respiratory tract irritation and increased mortality following exposure to dusts of 10



### biphenyl (7 hours/day, 5 days/week for up to about 90 days) were reported in mice exposed to 11



### 5 mg/m 3 

### and in rats exposed to 300 mg/m 3 

### , but not in rabbits exposed to 300 mg/m 3 

### (Deichmann 12



### et al., 1947; Monsanto, 1946). Congestion or edema of the lung, kidney, and liver, accompanied 13



### by hyperplasia with inflammation of the trachea, was reported in CD -1 mice expos ed to biphenyl 14



### vapors at 25 or 50 ppm (158 or 315 mg/m 3 

### ) for 13 weeks (Sun Company Inc., 1977b). 15

16



### 6.1.2. Cancer 17



### No assessments are available regarding possible associations between exposure to 18



### biphenyl and increased risk of cancer in humans. 19



### In a 2- year study of F344 rats administered biphenyl in the diet, significantly increased 20



### incidence s of urinary bladder tumors in males were observed at the highest dose level 21



### (378 mg/kg-day). There is strong evidence that the occurrence of urinary bladder tumors in the 22



### male rats is a high -dose nongenotoxic phenomenon involving occurrence of calculi in the urinary 23



### bladder leading to transitional cell damage, sustained regenerative cell proliferation, and eventual 24



### promotion of spontaneously initiated tumor cells in the urinary bladder epithelium. Urinary 25



### bladder calculi in the high

- dose (438 mg/kg

- day) female rats were observed at much lower 26



### incidence and were different in physical appearance and chemical composition; furthermore, 27



### there were no urinary bladder tumors i n any of the biphenyl- exposed female rats. 28



### In a 2-year study of BDF 1 

### mice administered biphenyl in the diet, the incidence of liver 29



### tumors in female mice was significantly increased at doses ≥414 mg/kg -day, but not in males at 30



### doses up to and including 1,050 mg/kg-day. Available data are insufficient to establish a mode 31



### of action for liver tumors in female mice

. 32



### Under EPA Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005a), the 33



### database for b iphenyl provides “ suggestive evidence of carcinogenic potential ” based on 34



### evidence o f female mouse liver tumors and male rat bladder tumors

. 35

36

### 118 DRAFT

- DO NOT CITE OR QUOTE 6.2. DOSE RESPONSE 1



### 6.2.1. Noncancer/Oral 2



### The RfD of 0. 2 mg/kg-day was based on an increased incidence of litters with fetal 3



### skeletal anomalies from Wistar rat dams administered biphenyl by gavage on GDs 6 –15 (Khera 4



### et al., 1979). The BMDL 10 

### of 2 0 mg/kg -day was selected as the POD. To derive the RfD, the 5



### POD was divided by a total UF of 100 (10 for animal- to -human extrapolation and 10 for human 6



### interindividual variability in susceptibility). The interspecies uncertainty factor was applied to 7



### account for the lack of quantitative information to assess toxicokinetic and toxicodynamic 8



### differences be tween animals and humans. The intraspecies uncertainty factor was applied to 9



### account for the lack of information regarding the range of responses to biphenyl in the human 10



### population. 11



### The overall confidence in the RfD assessment is medium to high. Confid ence in the 12



### principal study (Khera et al., 1979) is medium to high . The design, conduct and reporting of this 13



### developmental toxicity study in Wistar rats were adequate; however, only litter average data 14



### were available that did not permit a nested analysis based on individual fetal data. Confidence in 15



### the database is high. The database is robust in that it includes chronic-duration oral exposure 16



### studies in several rat and mouse strains, a developmental toxicity study in Wistar rats, and one

17



### and three- gen eration reproductive toxicity studies in rats. 18

19



### 6.2.2. Noncancer/Inhalation 20



### No inhalation RfC was derived due to the lack of studies of biphenyl toxicity following 21



### chronic exposure and studies involving subchronic exposure that were inadequate for RfC 22



### derivation . Repeated exposure of mice to biphenyl vapors for 13 weeks resulted in high 23



### incidences of pneumonia and tracheal hyperplasia, and high incidences of congestion and edema 24



### in the lungs, liver, and kidney (Sun Company Inc., 1977b) ; h owever, study limitations and lack 25



### of supporting data preclude the use of this study for deriving an RfC for biphenyl. Study 26



### limitations include highly variable biphenyl exposure concentrations during the first half of the 27



### study, high mort ality after 46 exposures in one group of biphenyl- exposed mice due to an 28



### overheating event and cannibalization that necessitated the use of replacement animals, and 29



### limitations in the reporting of histopathological findings. 30

31



### 6.2.3. Cancer/Oral 32



### The oral s lope factor of 0.008 per mg/kg-day is based on the tumor response in the liver 33



### of female BDF 1 

### mice exposed to biphenyl in the diet for 2 years (Umeda et al., 2005). The slope 34



### factor was derived by linear extrapolation from a human equivalent BMDL 10 

### of 12.2 mg/kg-day 35



### for liver adenomas or carcinomas. 36

### 119 DRAFT

- DO NOT CITE OR QUOTE A nonlinear extrapolation approach for biphenyl-induced urinary bladder tumors in male 1



### rats was used because evidence show that the occurrence of urinary bladder tumors is a high2



### dose nongenotoxic phenomenon involving occurrence of calculi in the urinary bladder leading to 3



### transitional cell damage, sustained regenerative cell proliferation, and eventual promotion of 4



### spontaneously initiated tumor cells in the urinary bladder epithelium. As long as the dose is 5



### below that which is needed to form calculi, no increased risk of bladder tumors is expected. 6



### Therefore, the RfD of 0.2 mg/kg-day derived for noncancer effects of biphenyl was judged to be 7



### protective against increased risk of biphenyl- induced urinary bladder cancer. 8

9



### 6.2.4. Cancer/Inhalation 10



### No human or animal data on the potential carcinogenicity of inhaled biphenyl are 11



### available. Therefore, a quanti tative cancer assessment for biphenyl by the inhalation pathway 12



### was not performed. 13

### 120 DRAFT

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using the Ames test with cover letter. Submitted under TSCA Section 8D; EPA Document No. 878214672; NTIS

No. OTS0206616.

Wiebkin, P; Fry, JR; Jones, CA; et al. (1976) The metabolism of biphenyl by isolated viable rat hepatocytes.
Xenobiotica 6(12):725

– 743.

Wiebkin, P; Fry, JR; Jones, C; et al. (1978) Biphenyl metabolism in isolated rat hepatocytes: effect of induction and
nature of the conjugates. Biochem Pharmacol 27:1899

– 1907.

Wiebkin, P; Schaeffer, B; Longnecker, D; et al. (1984) Oxidative and conjugative metabolism of xenobiotics by
isolated rat and hamster acinar cells. Drug Metab Dispos 12(4):427

– 431.

Williams, G. (1980) DNA repai r and mutagenesis in liver cultures as indicators in chemical carcinogen screening,
in: mammalian cell transformation by chemical carcinogens. Adv Mod Environ Toxicol 1:273

– 296.

Williams, G; Mori, H; McQueen, C. (1989) Structure

- activity relationships in the rat hepatocyte DNA

- repair test for

300 chemicals. Mutat Res 221:263

– 286.

Yoshida, S; Masubuchi, M; Hiraga, K. (1978) Cytogenetic studies of antimicrobials on cultured cells. Tokyo
Toritsu Eisei Kenkyusho Kenkyo Nempo (Annu Rep Tokyo Metrop Res Lab Public Health) 29(2):86

– 88.

### A-1 DRAFT

- DO NOT CITE OR QUOTE APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 1



### COMMENTS AND DISPOSITION 2

3

4



### [Page intentio nally left blank ] 5

### B-1 DRAFT

- DO NOT CITE OR QUOTE 1 

### APPENDIX B. BENCHMARK DOSE CALCULATIONS FOR THE REFERENCE DOSE

2

3

4 

### Datasets used for modeling incidences of nonneoplastic effects in the urinary tract of

5 

### male and female F344 rats exposed to biphenyl in the diet for 2 years (Umeda et al., 2002 ) are

6 

### shown in Table B-1. Datasets used for modeling body weight data, selected clinical chemistry

7 

### results, and histopathological kidney effects in male and female BDF 1 

### mice exposed to biphenyl

8 

### in the diet for 2 years (Umeda et al ., 2005) are shown in Table B-2. The dataset for incidence of

9 

### litters with fetal skeletal anomalies , tal lied from evaluation of fetuses from Wistar rat dams

10 

### administered biphenyl by gavage on GD s 6 –15 (Khera et al., 1979) is shown in Table B-3.

11



### Table B -1. BMD modeling data sets for i ncidences of nonneoplastic effects in
the urinary tract of male and female F344 rats exposed to biphenyl in the

diet for 2 years

Males (n = 50) Females (n = 50)

Biphenyl dietary concentration (ppm) 0 500 1,500 4,500 0 500 1,500 4,500

TWA body weight (kg) a 0.411 0.412 0.408 0.357 0.251 0.246 0.246 0.216

Calculated d ose (mg/kg

- d ) b 0 36.4 110 378 0 42.7 128 438

Effect

Renal pelvis

Nodular transitional cell hyperplasia 0 1 1 21 c 0 0 1 12 c

Simple transitional cell hyperplasia 6 8 5 19 d 3 5 12 d 25 c

Mineralization 9 6 10 18 e 12 12 18 27 d

Other kidney effects

Hemosiderin deposit f 0 0 0 0 4 8 22 c 25 c

Papillary mineralization 9 9 14 23 d 2 6 3 12 c

Bladder

Combined transitional cell g hyperplasia 0 0 0 45 1 0 1 10

a T WA body weight calculated using graphically

- presented body weight data from Umeda et al. (2002).

b Calcul ated doses based on TWA body weight s and chronic reference food consumption values for F344 rats

(0.03 0 kg/day for males and 0.021 kg/day for females; taken from Table 1

- 6 of U.S. EPA

, 1988)

.

c Significantly different from contr ol group ( p < 0.01) according to χ 2 test.

d Significantly different from control group ( p < 0.05) according to χ 2 test.

e Significantly different from controls ( p < 0.05) according to Fisher’s exact test.

f Male data for incidences of hemosiderin deposits not selected for quantitative analysis

.. g Female data for incidences of combined transitional cell hyperplasia not selected for quantitative analysis

.

.

Source: Umeda et al. (2002).

12

### B-2 DRAFT

- DO NOT CITE OR QUOTE Table B -2. BMD modeling data sets for b ody weight, selected clinical
chemistry results, and histopathological kidney effects in male and female

BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Endpoint Biphenyl concentration in the diet (ppm)

0 667 2,000 6,000

Males

D ose (mg/kg

- d ) 0 97 291 1,050

Histopathological kidney effect n = 50 n = 49 n = 50 n = 50

Mineralization inner stripe

- outer medulla 9 8 14 14

Clinical chemistry parameter n = 34 n = 39 n = 37 n = 37

BUN (mg/d L ) 20.2 ± 3.6 22.0 ± 4.0 23.2 ± 4.4 a 22.9 ± 2.7 b

Body weight n = 35 n = 41 n = 41 n = 39

Mean terminal body weight (g) 46.9 ± 4.9 43.1 ± 7.9 42.9 ± 6.0 a 32.4 ± 3.6 b

Females

D ose (mg/kg

- d ) 0 134 414 1,420

Histopathological kidney effect n = 50 n = 50 n = 50 n = 49

Mineralization inner stripe

- outer medulla 3 5 12 c 26 d

Clinical chemistry parameter n = 28 n = 20 n = 22 n = 31

AST (IU/ L ) 75 ± 27 120 ± 110 211 ± 373 b 325 ± 448 b

ALT (IU/ L ) 32 ± 18 56 ± 46 134 ± 231 b 206 ± 280 b

AP (IU/ L ) 242 ± 90 256 ± 121 428 ± 499 556 ± 228 b

LDH (IU/ L ) 268 ± 98 461 ± 452 838 ± 2,000 1,416 ± 4,161 a

BUN (mg/d L ) 14.9 ± 2.0 14.8 ± 3.4 21.0 ± 20.5 23.8 ± 11.7 b

Body weight n = 31 n = 22 n = 25 n = 32

Mean terminal body weight (g) 34.0 ± 4.0 32.5 ± 3.3 30.5 ± 3.1 b 25.5 ± 3.0 b

a Significantly different from controls ( p < 0.05) according to Dunnett’s test.

b Significantly different from controls ( p < 0.01) according to Dunnett’s test.

c Significantly different from controls ( p < 0.05) according to Fisher’s exact test.

d Significantly different from controls ( p < 0.01) according to Fisher’s exact test.

ALT (GPT) = alanine aminotransferase (glutamic pyruvic transaminase); AP (ALP) = alkaline phosphatase;

AST (GOT) = aspartate aminotransferase (glutamic oxaloacetic transaminase)

Source: Umeda et al. (2005).

1

### B-3 DRAFT

- DO NOT CITE OR QUOTE Tab le B -3. BMD modeling data set for incidence of litters with fetal skeletal

anomal ies from Wistar rat dams administered biphenyl by gavage on
GD s 6–15

Effect Dose (mg/kg

- d )

0 125 250 500 1,000

Litters with fetal skeletal anomalies a / litters 8/16 11/20 13/18 15/ 18 b 6/9

examined

a The study authors reported one runted fetus in the control group and one fetus with kinky tail in the 250 mg/kg

- day
dose group, which may have influenced the reported incidence data for anomalous litters/litters examined.

b Significantly different from controls ( p < 0.05) according to Fisher’s exact test conducted for this review

.

Source: Khera et al. (1979).

1



### Goodness of fit statistics and benchmark results for each of the modeled biphenyl2



### induced nonneoplastic effects from the chronically-exposed rats (Umeda et al., 2002) and mice 3



### (Umeda et al., 2005) and the gestationally-exposed rats (Khera et al., 1979) are summarized in 4



### Tables B -4 through B-22. Each ta ble of modeled results for a particular effect is followed by the 5



### information from the output file of the best- fitting model for that effect. 6

7



### Table B -4. Summary of BMD modeling results for incidence of renal
nodular transitional cell hyperplasia in male F344 rats exposed to biphenyl

in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

b Gamma 0.31 0.73 95.02 169.71 74.44 212.00 120.62

Logistic 0.64 0.74 92.72 178.92 133.35 233.81 192.35

b Log

- Logistic 0.31 0.74 95.01 172.40 75.93 216.08 120.70

Log

- Probit b 0.31 0.71 95.03 163.38 89.50 202.25 128.71

Multistage (3

- degree) c,d 0.58 0.84 92.60 133.82 69.08 193.30 126.95

Probit 0.59 0.84 92.76 157.59 117.53 212.09 173.76

Weibull b 0.31 0.75 95.00 175.08 73.08 221.75 121.01

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

d Betas restricted to ≥0.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%

lower c onfidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose assoc iated with 5% extra risk)

Source: Umeda et al. (2002).

8

### B-4 DRAFT

- DO NOT CITE OR QUOTE Wed Jan 12 10:38:57 2011 10 0 0.1 0.2 0.3 0.4 0.5 0.6

0 50 100 150 200 250 300 350Fraction Affected

dose Multistage Model with 0.95 Confidence Level

10:38 01/12 2011 BMD BMDL Multistage

1

BMD and BMDL indicated are associated with an extra risk of 10%, an d are in units of mg/kg

- day

. 2

3

==================================================================== 4

Multistage Model. (Version: 3.2; Date: 05/26/2010) 5

Input Data File: 6

C: \ USEPA \ IRIS \ biphenyl \ rat \ renalnodularhyper \ male \ mst_nodhypMrev_MS_3.(d) 7

Gnuplot Plotting File: 8

C: \ USEPA \ IRIS \ biphenyl \ rat \ renalnodularhyper \ male \ mst_nodhypMrev_MS_3.plt 9

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- 14

beta1*dose^1

- beta2*dose^2

- beta3*dose^3)] 15

The parameter betas are restricted to be positive 16

Depe ndent variable = incidence 17

Independent variable = dose 18

Total number of observations = 4 19

Total number of records with missing values = 0 20

Total number of parameters in model = 4 21

Total number of specified parameters = 0 22

Degree of polynomial = 3 23

Maximum number of iterations = 250 24

Relative Function Convergence has been set to: 1e

- 008 25

Parameter Convergence has been set to: 1e

- 008 26
27

Default Initial Parameter Values 28

Background = 0.00721859 29

Beta(1) = 3.68302e

- 005 30

Beta(2) = 0 31

Beta(3) = 9.69211e

- 009 32
33

Asymptotic Correlation Matrix of Parameter Estimates 34

( *** The model parameter(s)

- Background

- Beta(2) have been estimated at a boundary point, or 35

have been specified by the user, and do not appear in the correlation matrix ) 36

Beta(1) Beta(3) 37

Beta(1) 1

- 0.95 38

Beta(3)

- 0.95 1 39
40

Parameter Est imates 41

95.0% Wald Confidence Interval 42

Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit 43

Background 0 * * * 44

Beta(1) 0.000234424 * * * 45

Beta(2) 0 * * * 46

Beta(3) 8.31393e

- 009 * * * 47

* - Indicates that this value is not calculated. 48
49

Analysis of Deviance Table 50

### B-5 DRAFT

- DO NOT CITE OR QUOTE 1
2 Model

Full model Log(likelihood)

- 43.8185 # Param's

4 Deviance Test d.f. P

- value

3 Fitted model

- 44.3014 2 0.965856 2 0.617 4 Reduced model

- 71.3686 1 55.1002 3 <.0001 5

6 AIC: 92.6029 7
8 Goodness of Fit 9 Scaled 0
1

- Dose

--------- Est._Prob.

-------------- Expected Observed

-------------------------- - Size

-------- Residual

------------ - 2 0.0000 0.0000 0.000 0.000 50 0.000 3 36.4000 0.0089 0.445 1.000 50 0.836 4 110.0000 0.0362 1.809 1.000 50

- 0.613 5 378.0000 0.4159 20.794 21.000 50 0.059 6
7 1
1
1
1
1
1
1

Chi^2 = 1.08 d.f. = 2 P

- value = 0.5832 1
18
19 Benchmark Dose Computation 20 Specified effect = 0.1 21 Risk Type = Extra risk 22 Confidence level = 0.95 23 BMD = 193.298 24 BMDL = 126.946 25 BMDU = 248.35 26 Taken togethe r, (126.946, 248.35 ) is a 90% two

- sided confidence interval for the BMD



### T able B -5. Summary of BMD modeling results for incidence of renal

odular transitional cell hyperplasia in female F344 rats exposed to biphenyl

n the diet for 2 years n

i

**χ** 2

M o del Goodness of fit Benchmark result (mg/kg

- d)

Largest

a p

- value residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

b Gamma

Logistic

b Log

- Logistic

Log

- Probit b

( 2

- degree) c,d Multistage

Probit 0.96

- 0.24 69.04 200.54 118.95 276.46 198.73

0.69 0.63 69.93 277.38 211.02 343.52 289.03

0.96

- 0.26 69.07 203.45 118.10 279.78 196.91

0.99

- 0.15 68.96 188.92 134.61 261.35 193.58

0.99

- 0.36 67.19 191.47 121.69 274.42 211.52

0.76 0.54 69.69 253.65 190.94 324.08 268.17

Weibull b 0.95

- 0.27 69.08 207.16 119.11 285.37 201.63

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Betas restricted to ≥0.

d Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associa ted with 5% extra risk)

Source: Umeda et al. (2002). 27

28

### B-6 DRAFT

- DO NOT CITE OR QUOTE 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

0 50 100 150 200 250 300 350 400 450Fraction Affected

dose Multistage Model with 0.95 Confidence Level

11:48 01/13 2011 BMD BMDL Multistage

1

BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Multistage Model. (Version: 3.2; Date: 05/26/2010) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalnodularhyper/female/mst_nodhypFrev_MS_2.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalnodularhyper/female/mst_nodhypFrev_MS_2.plt 9

Thu Jan 13 11:48:49 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- 14

beta1*dose^1

- beta2*dose^2)] 15

The parameter betas are restricted to be positive 16

Dependent variable = incidence 17

Independent variable = dose 18

Total number of observations = 4 19

Total number of records with missing values = 0 20

Total number of parameters in model = 3 21

Total number of specified parameters = 0 22

Degree of polynomial = 2 23

Maximum number of iterations = 250 24

Relative Function Convergence has been set to: 1e

- 008 25

Parameter Convergence has been set to: 1e

- 008 26
27

Default Initial Parameter Values 28

Background = 0 29

Beta(1) = 0 30
31
32

Asymptotic Correlation Matrix of Parameter Estimates 33

( *** The model parameter(s)

- Background

- Beta(1) have been estimated at a boundary point, or 34

have been specified by the user, and do not appear in the correlation matrix ) 35

Beta(2) 36

Beta(2) 1 37
38
39 Parameter Estimates 40 95.0% Wald Confidence Interval 41
42
43
44 Variable

Background

Beta(1) Estimate

0

0 Std. Err.

*

* Lower Conf. Limit

*

* Upper Conf. Limit

*

*

Beta(2) 1.39908e

- 006 * * *

*

- Indicates that this value is not calculated. 45
46
47
48
49 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 32.456 4 Test d.f. P

- value

### B-7 DRAFT

- DO NOT CITE OR QUOTE Fitted model

- 32.5947 1 0.277585 3 0.9642 1

Reduced model

- 48.1018 1 31.2917 3 <.0001 2
3

AIC: 67.1895 4
5

Goodness of Fit 7 Scaled 8
9

- Dose

--------- Est._Prob.

-------------- Expected Observed

-------------------------- - Size

-------- Residu al

------------ - 0 0.0000 0.0000 0.000 0.000 50 0.000 1 42.7000 0.0025 0.127 0.000 50

- 0.357 2 128.0000 0.0227 1.133 1.000 50

- 0.126 3 438.0000 0.2354 11.770 12.000 50 0.077 6

1
1
1
1
14

Chi^2 = 0.15 d.f. = 3 P

- value = 0.9853 15
16

Benchmark Dose Computation 17

Specified effect = 0.1 18

R isk Type = Extra risk 19

Confidence level = 0.95 20

BMD = 274.422 21

BMDL = 211.518 22

BMDU = 351.444 23

Taken together, (211.518, 351.444) is a 90% two

- sided confidence interval for the BMD 24

25

26



### Table B -6. Summary of BMD modeling results for incidence of renal simple
transitional cell hyperplasia in male F344 rats exposed to biphenyl in the

diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

b,c Gamma 0.66 0.71 184.41 284.70 55.27 313.76 113.22

Logistic 0.35

- 1.18 185.78 96.07 73.33 171.37 131.76

b Log

- Logistic 0.36 0.71 186.41 320.26 58.80 340.21 115.09

Log

- Probit b 0.36 0.71 186.41 284.12 100.23 312.44 144.14

Multistage (3

- degree) d 0.60 0.74 184.59 201.02 52.30 255.53 107.40

Probit 0.33

- 1.22 185.92 90.26 68.00 164.29 124.13

Weibull b 0.36 0.71 186.41 324.89 55.27 344.08 113.14

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit differed by less than threefold.

d Betas restricted to ≥0.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose as sociated with 5% extra risk)

Source: Umeda et al. (2002).

27

### B-8 DRAFT

- DO NOT CITE OR QUOTE 1 0 0.1 0.2 0.3 0.4 0.5

0 50 100 150 200 250 300 350 Gamma Multi-Hit Model with 0.95 Confidence Level

Gamma Multi-HitFraction Affected

BMDL BMD

dose
11:55 01/13 2011

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 3

4 ==================================================================== 5 Gamma Model. (Version: 2.15; Date: 10/28/2009) 6 Input Data File: 7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalsimplehyper/male/gam_rensimphypMrev_gamma.(d) 8 Gnuplot Plotting File: 9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalsimplehyper/male/gam_rensimphypMrev_gamma.plt 10 Thu Jan 13 11:55:07 2011 11 ==================================================================== 12 BMDS_Model_Run 13

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14 The form of the probability function is: P[response]= background+(1

- 15 background)*Cu mGamma[slope*dose,power], where CumGamma(.) is the cummulative Gamma distribution 16 function 17 Dependent variable = incidence 18 Independent variable = dose 19 Power parameter is restricted as power >=1 20 Total number of observations = 4 21 Total number of records with missing values = 0 22 Maximum number of iterations = 250 23 Relative Function Convergence has been set to: 1e

- 008 24 Parameter Convergence has been set to: 1e

- 008 25
26 Default Initial (and Specified) Parameter Values 27 Background = 0.134615 28 Slope = 0.00398471 29 Power = 2.55235 30
31 Asymptotic Correlation Matrix of Parameter Estimates 32 ( *** The model parameter(s)

- Power have been estimated at a boundary point, or have been 33 specified by the user, and do not appear in the correlation matrix ) 34
35
36
37 Background

Slope Background

1

- 0.27 Slope

- 0.27

1

38 Parameter Estimates 39 95.0% Wald Confidence Interval 40
41
42
43 Variable

Background

Slope Estimate

0.126666

0.0408652 Std. Err.

0.0271566

0.00241924 Lower Conf. Limit

0.0734404

0.0361236 Upper Conf. Limit

0.179892

0.0456068

Power 18 NA 44 NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus 45 has no standard error. 46
47
48
49 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 89.7871 4 Test d.f. P

- value

50 Fitted model

- 90.2033 2 0.832451 2 0.6595

### B-9 DRAFT

- DO NOT CITE OR QUOTE 1 Reduced model

- 97.2446 1 14.915 3 0.001891 2
3 AIC: 184.407 4
5 Goodness of Fit 6 Scaled 7
8 D ose

---------- Est._Prob.

-------------- Expected Observed Size

-------------------------------- Residual

--------------- - 9 0.0000 0.1267 6.333 6.000 50

- 0.142 0
1 1
1 36.4000 0.1267 6.333 8.000 50 0.709

12 110.0000 0.1267 6.333 5.000 50

- 0.567

378.0000 0.3800 19.000 19.000 50 0.000 13
14 Chi^2 = 0.84 d.f. = 2 P

- value = 0.6558 15
16 Benchmark Dose Computation 17 Specified effect = 0.1 18 Risk Type = Extra risk 19 Confidence level = 0.95 20 BMD = 313.755 21 BMDL = 113.219 22



### Table B

- 7. Summary of BMD modeling results for incidence of renal simple
transitional cell hyperplasia in female F344 rats exposed to biphenyl in the

diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

b Gamma

, Weibull b

, d Multistage (1

- degree) c

, 0.89 0.34 183.87 34.63 25.35 71.12 52.08

Logistic 0.28 1.29 186.14 83.08 66.43 145.87 119.22

b Log

- Logistic 0.71

- 0.26 185.77 37.52 18.90 71.51 39.91

Log

- Probit b 0.41 1.00 185.39 84.12 62.52 120.97 89.91

Probit 0.33 1.22 185.77 75.68 60.94 135.30 110.85

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Selected model; the gamma and Weibull models took the form of a 1

- degree polynomial multistage model and
produced identical goodness of fit statistics and BMD values; the model with the lowest AIC was selected because
BMDL values for models providing adequate fit differed by less than threefold.

d Betas restricted to ≥0.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associated with 5% e xtra risk)

Source: Umeda et al. (2002).

23

### B-10 DRAFT

- DO NOT CITE OR QUOTE 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 50 100 150 200 250 300 350 400 450Fraction Affected

dose Multistage Model with 0.95 Confidence Level

14:01 01/13 2011 BMD BMDL Multistage

1
BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Multistage Model. (Version: 3.2; Date: 05/26/2010) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalsimplehyper/female/mst_simplehypFrev_MS_1.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalsimplehyper/female/mst_simplehypFrev_MS_1.plt 9

Thu Jan 13 14:01:13 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- 14

beta1*dose^1)] 15

The parameter betas are restricted to be positive 16

Dependent variable = incidence 17

Independent variable = dose 18

Total number of observations = 4 19

Total number of records with missing values = 0 20

Total number of parameters in model = 2 21

Total number of specified parameters = 0 22

Degree of polynomial = 1 23

Maximum number of iterations = 250 24

Relative Function Convergence has been set to: 1e

- 008 25

Parameter Convergence has been set to: 1e

- 008 26
27

Default Initial Parameter Values 28

Background = 0.0607741 29

Beta(1) = 0.00145231 30
31

Asymptotic Correlation Matrix of Parameter Estimates 32

Background Beta(1) 33

Background 1

- 0.61 34

Beta(1)

- 0.61 1 35
36
37 Parameter Estimates 38 95.0% Wald Confidence Interval 39
40
41
42 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

Background 0.057038 * * *

Beta(1) 0.00148135 * * *

* - Indicates that this value is not cal culated. 43
44
45
46 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 89.8139 4 Test d.f. P

- value

47 Fitted model

- 89.9369 2 0.246113 2 0.884 2 48 Reduced model

- 106.633 1 33.6378 3 <.0001 49
50 AIC: 183.874 51

### B-11 DRAFT

- DO NOT CITE OR QUOTE 1 Goodness of Fit 2 Scaled 3
4 Dose

----------- Est._Prob.

------------ Expected

------------ Observed

----------- ----- Size

-------- Residual

------------ - 5 0.0000 0.0570 2.852 3.000 50 0.090 6 42.7000 0.1148 5.742 5.000 50

- 0.329 7 128.0000 0.2199 10.995 12.000 50 0.343 8 438.0000 0.5072 25.358 25.000 50

- 0.101 9

Chi^2 = 0.24 d.f. = 2 P

- value = 0.8850 10
11

Benchmark Dose Computation 12

Specified effect = 0.1 13

Risk Type = Extra risk 14

Confidence level = 0.95 15

BMD = 71.1248 16

BMDL = 52.0766 17

BMDU = 105.072 18

Taken together, (52.0766, 105.072) is a 90% two

- sided confidence interval for the BMD 19

20



### Table B

- 8. Summary of BMD modeling results for incidence of
mineralization in renal pelvis of male F344 rats exposed to biphenyl in the

diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b 0.35

- 0.75 206.13 130.11 42.91 201.71 88.15

Logistic 0.58

- 0.79 204.33 98.62 70.79 181.36 130.04

Log

- Logistic b 0.34

- 0.75 206.14 128.13 36.96 199.42 78.03

Log

- Probit b ,c 0.64

- 0.74 204.13 144.55 96.05 207.88 138.13

Multistage (1

- degree) d 0.51

- 0.84 204.60 70.84 41.20 145.51 84.62

Probit 0.57

- 0.80 204.35 94.16 66.44 175.86 123.70

Weibull b 0.34

- 0.75 206.15 131.37 42.84 205.20 88.00

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

d Betas restricted to ≥0.

BMD = maximum likelihood estimate of the dose associated wi th the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associated wit h 5% extra risk)

Source: Umeda et al. (2002).

21

### B-12 DRAFT

- DO NOT CITE OR QUOTE 0.1 0.2 0.3 0.4 0.5

0 50 100 150 200 250 300 350Fraction Affected

dose LogProbit Model with 0.95 Confidence Level

15:38 01/13 2011 BMDL BMD LogProbit

1
BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Probit Model. (Version: 3.2; Date: 10/28/2009) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/male/lnp_minpelvMrev_logprobit.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/male/lnp_minpelvMrev_logprobit.plt 9

Thu Jan 13 15:38:28 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = Background + (1

- Background) * 14

Cum Norm(Intercept+Slope*Log(Dose)), where CumNorm(.) is the cumulative normal distribution 15

function 16

Dependent variable = incidence 17

Independent variable = dose 18

Slope parameter is restricted as slope >= 1 19

Total number of observations = 4 20

Total number of records with missing values = 0 21

Maximum number of iterations = 250 22

Relative Function Convergence has been set to: 1e

- 008 23

Parameter Convergence has been set to: 1e

- 008 24

User has chosen the log transformed model 25
26

Default Initial (and Specified) Parameter Values 27

background = 0.18 28

intercept =

- 6.59931 29

slope = 1 30
31

Asymptotic Correlation Matrix of Parameter Estimates 32

( *** The model parameter(s)

- slope have been estimated at a boundary point, or have been 33

specified by the user, and do not appear in the correlation matrix ) 34

background intercept 35

background 1

- 0.46 36

intercept

- 0.46 1 37
38
39 Parameter Estimates 40 95.0% Wald Confidence Interval 41
42
43
44 Variable

background

intercept

slope Estimate

0.157045

- 6.61851

1 Std. Err.

0.0325697

0.281947

NA Lower Conf. Limit

0.0932095

- 7.17111 Upper Conf. Limit

0.22088

- 6.0659

NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus 45

has no standard error. 46
47
48
49
50 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 99.607 4 Test d.f. P

- value

51 Fitted model

- 100.0 63 2 0.91202 2 0.6338

### B-13 DRAFT

- DO NOT CITE OR QUOTE Reduced model

- 104.101 1 8.98864 3 0.02944 1
2

AIC: 204.126 3
4
5 Goodness of Fit 6 Scaled 7
8

- Dose

--------- Est._Prob.

-------------- Expected Observed

-------------------------- - Size

-------- Residual

------------ - 9 0.0000 0.1570 7.852 9.000 50 0.446 0 36.4000 0.1581 7.905 6.000 50

- 0.738 1 110.0000 0.1803 9.014 10.000 50 0.363 2 378.0000 0.3653 18.267 18.000 50

- 0.079 1
1
1
13

Chi^2 = 0.88 d.f. = 2 P

- value = 0.6434 14
15

Benchmark Dose Computation 16

Specified effect = 0.1 17

Risk Type = Extra risk 18

Confidence level = 0.95 19

BMD = 207.879 20

BMDL = 138.127 21
22



### Table B

- 9. Summary of BMD modeling results for incidence of
mineralization in renal pelvis of female F344 rats exposed to biphenyl in the

diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b 0.57

- 0.43 250.89 44.66 27.40 90.32 56.28

Logistic 0.76 0.59 249.10 64.48 48.11 123.84 92.31

Log

- Logistic b < 0.001 2.90 263.72 1.33 × 10 15 NA 1.58 × 10 15 NA

Log

- Probit b < 0.001 2.90 263.72 1.54 × 10 14 NA 2.21 × 10 14 NA

Multistage (1

- degree) c, d 0.85

- 0.44 248.89 42.68 27.40 87.67 56.28

Probit 0.77 0.57 249.08 62.20 46.34 120.41 89.56

Weibull b 0.56

- 0.44 250.89 43.32 27.40 88.56 56.28

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Betas restricted to ≥0.

d Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%

lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associated with 5% extra risk)

Source: Umeda et al. (2002).

23

### B-14 DRAFT

- DO NOT CITE OR QUOTE 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 50 100 150 200 250 300 350 400 450Fraction Affected

dose Multistage Model with 0.95 Confidence Level

16:24 01/13 2011 BMD BMDL Multistage

1
BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Multistage Model. (Version: 3.2; Date: 05/26/2010) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/female/mst_minpelvlFrev_MS_1.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/female/mst_minpelvlFrev_MS_1.plt 9

Thu Jan 13 16:24:18 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- 14

beta1*dose^1)] 15

The parameter betas are restricted to be positive 16

Dependent variable = incidence 17

Independent variable = dose 18

Total number of observations = 4 19

Total number of records with missing values = 0 20

Total number of parameters in model = 2 21

Total number of specified parameters = 0 22

Degree of polynomial = 1 23

Maximum number of iterations = 250 24

Relative Function Convergence has been set to: 1e

- 008 25

Parameter Convergence has been set to: 1e

- 008 26
27

Default Initial Parameter Values 28

Background = 0.230737 29

Beta(1) = 0.00118679 30
31

Asymptotic Correlation Matrix of Parameter Estimates 33

36 32

34
35 Background Background

1 Beta(1)

- 0.62

Beta(1)

- 0.62 1

37 Parameter Estimates 38 95.0% Wald Confidence Interval 39
40
41 Variable

Background Estimate

0.228898 Std. Err.

* Lower Conf. Limit

* Upper Conf. Limit

*

Beta(1) 0.0012018 * * *

* - Indicates that this value is not calculated. 43 42

44
45
46 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 122.276 4 Test d.f. P

- value

47 Fitted model

- 122.443 2 0.334544 2 0.846 48 Reduced model

- 128.859 1 13.1664 3 0.00429 49
50 AIC: 248.887 51

### B-15 DRAFT

- DO NOT CITE OR QUOTE 1 Goodne ss of Fit 2 Scaled 3
4

- Dose

--------- Est._Prob.

-------------- Expected Observed

-------------------------- - Size

-------- Residual

------------ - 5 0.0000 0.2289 11.445 12.000 50 0.187 6 42.7000 0.2675 13.374 12.000 50

- 0.439 7 128.0000 0.3388 16.942 18.000 50 0.316 8 438.0000 0.5445 27.224 27.000 50

- 0.064 9
10 Chi^2 = 0.33 d.f. = 2 P

- value = 0.8473 11

Benchmark Dose Computation 12

Specified effect = 0.1 13

Risk Type = Extra risk 14

Confidence level = 0.95 15

BMD = 87.669 16

BMDL = 56.2773 17

BMDU = 172.188 18

Taken together, (56.2773, 172.188) is a 90% two

- sided confidence 19

20



### Table B -10. Summary of BMD model ing results for incidence of
hemosiderin deposits in the kidney of female F344 rats exposed to biphenyl

in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b

, Weibull b

, Multistage (1

- degree) c 0.022 2.36 220.99 29.64 21.20 60.87 43.54

Logistic 0.002 2.92 225.98 66.06 52.04 123.37 97.71

Log

- Logistic b 0.093 1.75 218.35 19.21 12.74 40.56 26.89

Log

- Probit b 0.002 2.82 225.97 74.77 52.43 107.53 75.40

Probit 0.002 2.90 225.57 61.90 49.07 116.90 92.96

Dichotomous

- Hill d,e 0.9997 0.026 213.75 34.28 12.76 45.32 23.29

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Betas restricted to ≥0.

d Selected model; the o nly model with an adequate fit ( **χ** 2 p

- value > 0.1)

.

e v = 0.5 (specified), g = 0.16 (specified), intercept = 0.08 (initialized), slope = 1 (initialized).

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i .e., 10 = dose associated with 10%

extra risk; 5 = dose associated with 5% extra risk)

Source: Umeda et al. (2002).

21

### B-16 DRAFT

- DO NOT CITE OR QUOTE 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 100 200 300 400Fraction Affected

dose Dichotomous-Hill Model with 0.95 Confidence Level

09:14 01/14 2011 BMDL BMD Dichotomous-Hill

1

BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Dichotomous Hill Model. (Version: 1.2; Date: 12/11/2009) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/hemosiderin/female/dhl_hemosidFrev_dichotomous 7

hill.(d) 8

Gnuplot Plotting File: 9

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/hemosiderin/female/dhl_hemosidFrev_dichotomous 10

hill.plt 11

Fri Jan 14 09:14:35 2011 12

==================================================================== 13

BMDS_Model_Run 14

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15

The form of the probability function is: P[response] = v*g +(v

- v*g)/[1+EXP(

- intercept

16

slope*Log(dose))] where: 0 <= g < 1, 0 < v <= 1v is the maximum probability of response predicted 17

by the model, and v*g is the background estimate of that probability. 18

Dependent variable = incidence 19

Independent variable = dose 20

Parameter v is set to 0.5 21

Parameter g is set to 0.16 22

Slope parameter is restricted as slope >= 1 23

Total number of observations = 4 24

Tota l number of records with missing values = 0 25

Maximum number of iterations = 250 26

Relative Function Convergence has been set to: 1e

- 008 27

Parameter Convergence has been set to: 1e

- 008 28
29

User Inputs Initial Parameter Values 30

v =

- 9999 Specified 31

g =

- 9999 Specified 32

intercept = 0.08 33

slope = 1 34
35

Asymptotic Correlation Matrix of Parameter Estimates 36

( *** The model parameter(s)

- v

- g have been estimated at a boundary point, or have been 37

specified by the user, and do not appear in the correlation matrix ) 38
39
40
41
42 intercept

slope intercept

1

- 0.99 slope

- 0.99

1

43 Parameter Estimates 44 95.0% Wald Confidence Interval 45
46
47
48 Variable

intercept

slope Estimate

- 12.5334

2.95297 Std. Err.

5.83724

1.43635 Lower Conf. Limit

- 23.9742

0.137773 Upper Conf. Limit

- 1.09265

5.76817

49 Analysis of Deviance Table 50

### B-17 DRAFT

- DO NOT CITE OR QUOTE 1
2 M odel

Full model Log(likelihood)

- 104.876 # Param's

4 Deviance Test d.f. P

- value

3 Fitted model

- 104.876 2 0.000679954 2 0.9997 4 Reduced model

- 121.314 1 32.8756 3 <.0001 5

6 AIC: 213.752 7
8 Goodness of Fit 9 Scaled 10
11 Dose

---------- Est._Prob.

-------------- Expected Observed Size

-------------------------------- Residual

--------------- - 12 0.0000 0.0800 4.000 4.000 50 0.000 13 42.7000 0.1600 7.998 8.000 50 0.001 14 128.0000 0.4401 22.007 22.000 50

- 0.002 15 438.0000 0.4982 24.908 25.000 50 0.026 16
17 Chi^2 = 0.00 d.f. = 2 P

- value = 0.9997 18

Benchmark Dose Computation 19

Specified effect = 0.1 20

Risk Type = Extra risk 21

Confi dence level = 0.95 22

BMD = 45.3249 23

BMDL = 23.2881 24

25



### Table B

- 11. Summary of BMD modeling results for incidence of papillary
mineralization in the kidney of male F344 rats exposed to biphenyl in the

diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b 0.63

- 0.37 228.81 51.08 28.48 99.83 58.49

Logistic 0.81 0.51 226.99 70.07 52.70 131.45 98.95

Log

- Logistic b < 0.001 2.93 241.27 5.64 × 10 14 NA 6.68 × 10 14 NA

Log

- Probit b 0.001 2.93 239.27 5.13 × 10 13 NA 7.38 × 10 13 NA

Multistage (1

- degree) c, d 0.88

- 0.40 226.82 44.66 28.45 91.74 58.44

Probit 0.82 0.48 226.96 66.59 49.79 126.42 94.42

Weibull b 0.63

- 0.37 228.81 49.89 28.47 98.66 58.48

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Betas restricted to ≥0.

d Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associa ted with 5% extra risk)

Source: Umeda et al. (2002).

26

### B-18 DRAFT

- DO NOT CITE OR QUOTE 0.1 0.2 0.3 0.4 0.5 0.6

0 50 100 150 200 250 300 350Fraction Affected

dose Multistage Model with 0.95 Confidence Level

11:25 01/14 2011 BMD BMDL Multistage

1
BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Multistage Model. (Version: 3.2; Date: 05/26/2010) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/pappmineral/male/mst_papminMrev_MS_1.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/pappmineral/male/mst_papminMrev_MS_1.plt 9

Fri Jan 14 11:25:01 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- 14

beta1*dose^1)] 15

The parameter betas are restricted to be positive 16

Dependent variable = incidence 17

Independent variable = dose 18

Total number of observations = 4 19

Total number of records with missing values = 0 20

Tota l number of parameters in model = 2 21

Total number of specified parameters = 0 22

Degree of polynomial = 1 23

Maximum number of iterations = 250 24

Relative Function Convergence has been set to: 1e

- 008 25

Parameter Convergence has been set to: 1e

- 008 26
27

Default Initial Parameter Values 28

Background = 0.168963 29

Beta(1) = 0.00114658 30
31

Asymptotic Correlation Matrix of Parameter Estimates 32

Background Beta(1) 33

Background 1

- 0.62 34

Beta(1)

- 0.62 1 35
36

Parameter Estimates 37
38 95.0% Wald Confidence Interval 39
40
41
42 * - Variable Estimate Std. Err.

Background 0.168634 *

Beta(1) 0.00114846 *

Indicates that this value is not calculated. Lower Conf.

*

* Limit Upper Conf. Limi

*

* t

43
44
45
46 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 111.284 4 Test d.f. P

- value

47 Fitted model

- 111.409 2 0.250221 2 0.8824 48 Reduced model

- 117.634 1 12.6991 3 0.005335 49
50 AIC: 226.819 51

### B-19 DRAFT

- DO NOT CITE OR QUOTE 1 Goodness of Fit 2 Scaled 3
4

- Dose

--------- Est._Prob.

-------------- Expected Observed

-------------------------- - Size

-------- Residual

------------ - 5 0.0000 0.1686 8.432 9.000 50 0.215 6 36.4000 0.2027 10.134 9.000 50

- 0.399 7 110.0000 0.2673 13.365 14.000 50 0.203 8 378.0000 0.4614 23.071 23.000 50

- 0.020 9
10 Chi^2 = 0.25 d.f. = 2 P

- value = 0.8839 11

Benchmark Dose Computation 12

Specified effect = 0.1 13

R isk Type = Extra risk 14

Confidence level = 0.95 15

BMD = 91.741 16

BMDL = 58.4361 17

BMDU = 182.915 18

Taken together, (58.4361, 182.915) is a 90% two

- sided confidence interval for the BMD 19

20



### Table B -12. Summary of BMD modeling results for incidence of papillary
mineralization in the kidney of female F344 rats exposed to biphenyl in the

diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b 0.11 1.27 139.76 360.00 68.91 397.57 141.55

Logistic c 0.23 1.37 138.04 175.24 129.91 292.33 219.17

Log

- Logistic b 0.11 1.27 139.76 388.83 61.62 413.84 130.08

Log

- Probit b 0.11 1.27 139.76 356.94 150.95 395.27 217.08

Multistage (1

- degree) d 0.21 1.28 138.38 113.15 65.01 232.43 133.53

Probit 0.23 1.36 138.08 164.88 119.64 282.98 206.34

Weibull b 0.11 1.27 139.76 391.23 68.91 415.47 141.55

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

d Betas restricted to ≥0.

BMD = maximum likelihood estimate of the dose associated wi th the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associated with 5% extra risk)

Source: Umeda et al. (2002).

21

### B-20 DRAFT

- DO NOT CITE OR QUOTE 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4

0 50 100 150 200 250 300 350 400 450Fraction Affected

dose Logistic Model with 0.95 Confidence Level

13:00 01/14 2011 BMDL BMD Logistic

1
BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Logistic Model. (Version: 2.13; Date: 10/28/2009) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/b iphenyl/2011/BMD/rat/pappmineral/female/log_papmineralFrev_logistic.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/pappmineral/female/log_papmineralFrev_logistic.plt 9

Fri Jan 14 13:00:44 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = 1/[1+EXP(

- intercept

- slope*dose)] 14

Dependent variable = incidence 15

Indep endent variable = dose 16

Slope parameter is not restricted 17

Total number of observations = 4 18

Total number of records with missing values = 0 19

Maximum number of iterations = 250 20

Relative Function Convergence has been set to: 1e

- 008 21

Parameter Convergence has been set to: 1e

- 008 22
23

Default Initial Parameter Values 24

background = 0 Specified 25

intercept =

- 2.67819 26

slope = 0.00343504 27
28

Asymptotic Correlation Matrix of Parameter Estimates 29

( *** The model parameter(s)

- background have been estimated at a boundary point, or have been 30

specified by the user, and do not appear in the correlation matrix ) 31

intercept slope 32

intercept 1

- 0.78 33

slope

- 0.78 1 34
35
36 Parameter Estimates 37 95.0% Wald Confidence Interval 38
39
40 Variable

intercept

slope Estimate

- 2.72974

0.00353956 Std. Err.

0.364791

0.00119641 Lower Conf. Limit

- 3.44472

0.00119464 Upper Conf. Limit

- 2.01477

0.00588449 41
42
43
44 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 65.6458 4 Test d.f. P

- value

45 Fitted model

- 67.0198 2 2.74796 2 0.2531 46 Reduced model

- 71.3686 1 11.4455 3 0.009545 47
48 AIC: 138.04 49
50 Goodness of Fit 51 Scaled

### B-21 DRAFT

- DO NOT CITE OR QUOTE 1
2 Dose

---------- Est._Prob.

-------------- Expected

------------ Observed

------------- Size

---------- Residual

------------ - 3 0.0000 0.0612 3.062 2.000 50

- 0.626 4 42.7000 0.0705 3.526 6.000 50 1.366 5 128. 0000 0.0931 4.654 3.000 50

- 0.805 6 438.0000 0.2352 11.758 12.000 50 0.081 7
8 Chi^2 = 2.91 d.f. = 2 P

- value = 0.2330 9

Benchmark Dose Computation 10
1 1 Specified effect = 0.1

2 Risk Type = Extra risk 3 Confidence level = 0.95 4 BMD = 292.331 5 BMDL = 219.166

6



### Table B

- 13. Summary of BMD modeling results for incidence of combined
transitional cell hyperplasia in the bladder of male F344 rats exposed to
biphenyl in the diet for 2 years 1
1
1
1

1

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

b,c Gamma 1.00

- 0.12 34.54 186.38 125.23 205.40 146.73

Logistic 1.00 0.00 36.51 314.74 151.02 323.93 182.76

b Log

- Logistic 1.00 0.00 36.51 283.35 126.46 295.47 147.96

Log

- Probit b 1.00 0.00 36.51 227.03 122.78 241.87 140.96

Multistage (3

- degree) d 0.39

- 1.63 40.12 109.67 93.51 139.41 123.14

Probit 1.00 0.00 36.51 266.72 137.23 280.54 166.54

Weibull b 1.00 0.00 36.51 300.36 131.93 313.72 160.88

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

d Betas restricted to ≥0.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associated with 5% extra risk)

Source: Umeda et al. (2002).

17

### B-22 DRAFT

- DO NOT CITE OR QUOTE 0 0.2 0.4 0.6 0.8

1

0 50 100 150 200 250 300 350Fraction Affected

dose Gamma Multi-Hit Model with 0.95 Confidence Level

14:15 01/14 2011 BMDL BMD Gamma Multi-Hit

1

B MD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Gamma Model. (Version: 2.15; Date: 10/28/2009) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/bladdercombinedhyper/male/gam_bladcomhypMrev_gamma.(d 7

) 8

Gnu plot Plotting File: 9

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/bladdercombinedhyper/male/gam_bladcomhypMrev_gamma.pl 10

t 11

Fri Jan 14 14:15:19 2011 12

==================================================================== 13

BMDS_Model_Run 14

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 15

The form of the probability function is: P[response]= background+(1

- 16

background)*CumGamma[slope*dose,power], where CumGamma(.) is the cummulative Gamma distribution 17

function 18

Dependent variable = incidenc e 19

Independent variable = dose 20

Power parameter is restricted as power >=1 21

Total number of observations = 4Total number of records with missing values = 0 22

Maximum number of iterations = 250 23

Relative Function Convergence has been set to: 1e

- 008 24

Parameter Convergence has been set to: 1e

- 008 25
26

Default Initial (and Specified) Parameter Values 27

Background = 0.0192308 28

Slope = 0.0320399 29

Power = 8.56462 30
31

A symptotic Correlation Matrix of Parameter Estimates 32

( *** The model parameter(s)

- Background

- Power have been estimated at a boundary point, or 33

have been specified by the user, and do not appear in the correlation matrix ) 34

Slope 35

Slope 1 36
37
38 Parameter Estimates

95.0% Wald Confidence Interval 40
41
42 Variable

Background

Slope Estimate

0

0.0624215 Std. Err.

NA

0.00323795 Lower Conf. Limit Upper Conf. Limi t

0.0560752 0.0687677 39

43 Power 18 NA 44 NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus 45 has no standard error. 46
47
48
49 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 16.2541 4 Test d.f. P

- value

### B-23 DRAFT

- DO NOT CITE OR QUOTE 1 Fitted model

- 16.26 87 1 0.0290112 3 0.9987 2 Reduced model

- 106.633 1 180.757 3 <.0001 3
4 AIC: 34.5373 5

6 Goodness of Fit 7 Scaled 8
9 Dose

---------- Est._Prob.

-------------- Expected Observed Size

-------------------------------- Residual

--------------- - 10 0.0000 0.0000 0.000 0.000 50 0.000 11 36. 4000 0.0000 0.000 0.000 50

- 0.000 12 110.0000 0.0003 0.014 0.000 50

- 0.120 13 378.0000 0.8996 44.981 45.000 50 0.009 14
15 Chi^2 = 0.01 d.f. = 3 P

- value = 0.9995 16

Benchmark Dose Computation 17

Specified effect = 0.1 18

Risk Type = Extra risk 19

Confidence level = 0.95 20

BMD = 205.404 21

BMDL = 146.733 22

23



### Table B

- 14. Summary of BMD modeling results for incidence of
mineralization in the kidney (inner stripe outer medulla) of male BDF 1 

### mice
exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b

, Weibull b

, Multistage (1

- degree) c 0.46 1.03 214.84 369.24 155.65 758.4 5 319.71

Logistic 0.43 1.07 214.97 454.16 238.75 856.07 446.12

Log

- Logistic b,d 0.48 1.01 214.79 341.66 130.84 721.28 276.22

Log

- Probit b 0.33 1.24 215.51 710.74 377.36 1

, 022.10 542.66

Probit 0.44 1.07 214.95 442.78 227.50 844.26 430.21

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Betas restricted to ≥0.

d Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

BMD = maximum likelihood estimate of the dose associated with the selected
benchmark response; BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark
response: i.e., 10 = dose associated with 10% extra risk; 5 = dose associated with 5% extra risk)

Source: Umeda et al. (2005).

24

### B-24 DRAFT

- DO NOT CITE OR QUOTE 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45

0 200 400 600 800 1000Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

12:57 01/17 2011 BMDL BMD Log-Logistic

1 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Logistic Model. (Version: 2.13; Date: 10/28/2009) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/male/lnl_minmedullM_loglogistic.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/male/lnl_minmedullM_loglogistic.plt 9

Mon Jan 17 12:57:13 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = background+(1

- background)/[1+EXP(

- 14

intercept

- slope*Log(dose))] 15

Dependent variable = incidence 16

Independent variable = dose 17

Slope parameter is restricted as slope >= 1 18

Total number of observations = 4 19

Total number of records with missing values = 0 20

Maximum number of iterations = 250 21

Relative Function Convergence has been set to: 1e

- 008 22

Parameter Convergence has been set to: 1e

- 008 23

User has chosen the log transformed model 24
25

Default Initial Parameter Values 26

background = 0.18 27

intercept =

- 8.98323 28

slope = 1.06986 29
30

Asymptotic Correlation Matrix of Parameter Estimates 31

( *** The model parameter(s)

- slope have been estimated at a boundary point, or have been 32

specified by the user, and do not appear in the correlation matrix ) 33
34
35
36
37 background

intercept background

1

- 0.64 inter cept

- 0.64

1

38 Parameter Estimates 39 95.0% Wald Confidence Interval 40
41
42
43
44 * - Variable Estimate Std. Err.

background 0.185925 *

intercept

- 8.77824 *

slope 1 *

Indicates that this value is not calculated. Lower Conf. Limit

*

*

* Upper Conf. Limit

*

*

*

45
46
47
48 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 104.672 4 Test d.f. P

- value

49 Fitted model

- 105.397 2 1.44976 2 0.4844 50 Reduced model

- 106.377 1 3.40987 3 0.3326 51

### B-25 DRAFT

- DO NOT CITE OR QUOTE AIC: 214.794 1
2
3 Goodness of Fit 4 Scaled 5

6

-- Dose

-------- Est._Prob.

-------------- Expected Observed

-------------------------- - Size

-------- Residual

------------- 7 0.0000 0.1859 9.296 9.000 50

- 0.108 8 97.0000 0.1979 9.698 8.000 49

- 0.609 9 291.0000 0.2209 11.043 14.000 50 1.008 10 1050.0000 0.2993 14.963 14.000 50

- 0.298 11
12 Chi^2 = 1.49 d.f. = 2 P

- value = 0.4754 13

Benchmark Dose Computation 14

Specified effect = 0.1 15

Risk Type = Extra risk 16

Confidence level = 0.95 17

BMD = 721.275 18

BMDL = 276.216 19

20



### Table B- 15. Summary of BMD modeling results for incidence of
mineralization in the kidney (inner stripe outer medulla) of female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b 0.70

- 0.27 184.21 116.20 76.96 229.86 158.09

Logistic 0.31 1.22 184.34 257.38 205.80 451.19 369.40

Log

- Logistic b,c 0.80

- 0.18 184.12 127.12 57.98 233.39 122.40

Log

- Probit b 0.53 0.80 183.33 253.31 189.78 364.28 272.92

Multistage (1

- degree) d 0.92

- 0.34 182.23 104.00 76.86 213.63 157.88

Probit 0.38 1.14 183.96 234.00 188.80 417.63 343.46

Weibull b 0.69

- 0.28 184.22 113.82 76.94 227.40 158.04

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Selected model; the model with the lowest BMDL 10 was selected because BMDL values for models providing
adequate fit differed by more than threefold.

d Betas restricted to ≥0.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associated with 5% extra risk)

Source: Umeda et al. (2005).

1 2

### B-26 DRAFT

- DO NOT CITE OR QUOTE 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7

0 200 400 600 800 1000 1200 1400 Log-Logistic Model with 0.95 Confidence Level

Log-LogisticFraction Affected

BMDL BMD

dose
13:27 01/17 2011 1

BM D and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Logistic Model. (Version: 2.13; Date: 10/28/2009) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/female/lnl_minmedullF_loglogistic.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/female/lnl_minmedullF_loglogistic.plt 9

Mon Jan 17 13:27:41 2011 10

==================================================================== 11

BMDS_Model_Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the probability function is: P[response] = background+(1

- background)/[1+EXP(

- 14

intercept

- slope*Log(dose))] 15

Dependent variable = incidence 16

Independent variable = dose 17

Slope parameter is restricted as slope >= 1 18

Total number of observations = 4 19

Total number of records with missing values = 0 20

Maximum number of iterations = 250 21

Rela tive Function Convergence has been set to: 1e

- 008 22

Parameter Convergence has been set to: 1e

- 008 23

User has chosen the log transformed model 24
25

Default Initial Parameter Values 26

background = 0.06 27

intercept =

- 9.5037 28

slope = 1.31777 29
30
31
32
33
34
35 background

intercept

slope Asymptotic Correlation Matrix of Parameter Estimates

background intercept slope

1

- 0.48 0.44

- 0.48 1

- 0.99

0.44

- 0.99 1 36
37 Parameter Estimates 38 95.0% Wald Confidence Interval 39
40
41
42
43 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

background 0.05773 * * *

intercept

- 8.90345 * * *

slope 1.22989 * * *

* - Indicates that this value is not calculated. 44
45
46
47 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 89 .0288 4 Test d.f. P

- value

48 Fitted model

- 89.0609 3 0.0641982 1 0.8 49 Reduced model

- 107.593 1 37.1286 3 <.0001 50

### B-27 DRAFT

- DO NOT CITE OR QUOTE AIC: 184.122 1
2
3 Goodness of F it 4 Scaled 5

6

- Dose

--------- Est._Prob.

-------------- Expected Observed

-------------------------- - Size

-------- Residual

------------ - 7 0.0000 0.0577 2.887 3.000 50 0.069 8 134.0000 0.1078 5.391 5.000 50

- 0.178 9 414.0000 0.2307 11.535 12.000 50 0.156 10 1420.0000 0.5344 26.187 26.000 49

- 0.053 11
12 Chi^2 = 0.06 d.f. = 1 P

- value = 0.8006 13

Benchmark Dose Computation 14

Specified effect = 0.1 15

Risk Type = Extra risk 16

Confidence level = 0.95 17

BMD = 233.39 18
19 BMDL = 122.401
20



### Tab le B

- 16. BMD model results for serum LDH activity in female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

Variance

model

p

- value a Means

model

p

- value a Largest

residual AIC BMD 1SD BMDL 1SD BMD 1RD BMDL 1RD

All doses

Constant variance

Hill b < 0 .0001 NA 0.00 1

, 687.59 CF CF 182.66 0.0000

Linear c < 0 .0001 0.38 0.34 1

, 685.52 2

, 914.91 1

, 491.53 465.81 0.0026

Polynomial (2

- degree) c < 0 .0001 0.30 0.34 1

, 686.01 2

, 882.07 1

, 450.54 465.80 0.0011

Polynomial (3

- degree) c < 0 .0001 0.93 0.31 1

, 683.73 3

, 194.19 1

, 595.47 465.86 1.1 × 10

- 8

Power d < 0 .0001 0.93 0.31 1

, 683.73 3

, 193.16 1

, 449.38 465.81 0.0036

Non constant variance

Hill 0.91 NA

- 0.22 1

, 461.52 72.34 CF 161.83 107.12

Linear b 0.91 < 0 .0001 5.08 1

, 544.20

- 9

, 999.00 720.55 53.40 19.49

Polynomial (2

- degree) b 0.91 < 0 .0001 1.86 1

, 537.72 554.86 25.81 42.35 6.96

Polynomial (3

- degree) b 0.91 < 0 .0001 5.08 1

, 544.20

- 9

, 999.00 1

, 947.93 53.40 0.88

Power d 0.91 < 0 .0001 1.33 1

, 486.07 60.83 41.31 107.91 81.24

a Values < 0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be positive.

d Restrict power ≥ 1.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 s tandard deviation from control mean value; 1RD = dose associated with a 100% relative deviation
from control mean value); CF = computation failed; NA = not applicable (degrees of freedom for the test of mean

fit a re ≤ 0, the **χ** 2 test for fit is not valid)

S ource: Umeda et al. (2005).

21

22 

### None of the models provided an adequate fit to both the variance model and the means model.

23

### Table B -17 . BMD modeling results for serum AST activity in female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Goodness of fit Benchmark result (mg/kg

- d)

Variance Means

Model model

a p

- value model

a p

- value Largest

residual AIC BMD 1SD BMDL 1SD BMD 1RD BMDL 1RD

All doses

Constant variance

Hill b < 0 .0001 NA

- 5.69 × 10 7 1

, 264.30 6

, 722.40 566.24 213.62 0.00

c Linear

, Polynomial

d (2

- degree) c , Power < 0 .0001 0.72 0.68 1

, 260.96 1

, 826.88 1

, 205.47 595.87 135.74

Non constant variance

Hill b 0.52 NA 0.82 1

, 121.84 83.86 CF 154.69 114.05

c Linear 0.52 < 0 .0001 5.04 1

, 219.20 CF 90.71 21.60 2.76

Polynomial (2

- degree) c 0.52 < 0 .0001

- 2.55 × 10 9 8.00 0.00 CF 185.08 CF

d Power 0.52 < 0 .0001

- 2.13 1

, 164.51 106.70 69.43 150.64 110.24

Highest dose dropped

Constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

c Linear , Polynomial

(2

- degree) c , Power < 0 .0001 0.99 0.01 826.48 648.56 372.37 229.54 33.18

Non constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

c Linear 0.78 < 0 .0001 3.24 × 10 8 6 0 CF 228.57 CF

Polynomial (2

- degree) c 0.78 < 0 .0001

- 2.20 × 10 9 8 0 CF 219.67 CF

d,e Power 0.78 0.28

- 0.29 709.33 72.36 44.29 190.33 121.53

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be positive.

d Restrict power ≥ 1.

e Selected model; only model providing adequate fit to modeled variance and means.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 standard deviation from control mean value; 1RD = dose associated with a 100% rela tive deviation
from control mean value); CF = computation failed; NA = not applicable (degrees of freedom for the test of mea n

fit are ≤ 0, the **χ** 2 test for fit is not valid)

Source: Umeda et al. (2005).

1



### B-28 DRAFT

- DO NOT CITE OR QUOTE

### B-29 DRAFT

- DO NOT CITE OR QUOTE 50 100 150 200 250 300 350 400

0 50 100 150 200 250 300 350 400Mean Response

dose Power Model with 0.95 Confidence Level

10:47 01/18 2011 BMD BMDL Power

1
BMD and BMDL indicated are asso ciated with a twofold increase from control, and are in units of mg/kg

- day

. 2

3

==================================================================== 4

Power Model. (Version: 2.16; Date: 10/28/2009) 5

Input Data File: C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/AST/pow_ASTFHDD_power.(d) 6

Gnuplot Plotting File: 7

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/AST/pow_ASTFHDD_power.plt 8

Tue Jan 18 10:47:11 2011 9

==================================================================== 10

BMDS Model Run 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12

The form of the response function is: Y[dose] = control + slope * dose^power 13

Dependent variable = mean 14

Independent variable = dose 15

The power is restricted to be greater than or equal to 1 16

The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho) 17

Total number of dose groups = 3 18

Total number of records with missing values = 0 19

Maximum number of iterations = 250 20

Relative Function Convergence has been set to: 1e

- 008 21

Parameter Convergence has been set to: 1e

- 008 22
23

Default Initial Parameter Values 24

lalpha = 10.765 25

rho = 0 26

control = 75 27

slope = 0.369536 28

power = 0.980467 29
30

Asymptotic Correlation Matrix of Parameter Estimates 31

( *** The model parameter(s)

- power have been estimated at a boundary point, or have been 32

specified by the user, and do not appear in the correlation matrix ) 33

35
36 lalpha

rho lalpha

1

- 1 rho

- 1

1 control

- 0.43

0.37 slope

0.85

- 0.89 37 control

- 0.43 0.37 1

- 0.17 38 slope 0.85

- 0.89

- 0.17 1 34

39
40 Parameter Estimates 41 95.0% Wald Confidence Interval 42
43
44 Variable

lalpha

rho Estimate

- 12.9059

4.54893 Std. Err.

4.06805

0.905641 Lower Conf. Limit

- 20.8791

2.7739 Upper Conf. Limit

- 4.93268

6.32395 45 control 74.0253 5.21212 63.8097 84.2409 46
47 slope

power 0.38893

1 0.113823

NA 0.165841 0.61202

NA - Indic ates that this parameter has hit a bound impli ed by some inequality constraint and thus 48

has no standard error. 49
50

Table of Data and Estimated Values of Interest 51

### B-30 DRAFT

- DO NOT CITE OR QUOTE 1 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 2

------ -- - -------- -------- ----------- ----------- ---------- 3 0 28 75 74 27 28.1 0.183 4 134 20 120 126 110 94.6

- 0.29 5 414 22 211 235 373 390

- 0.289 6

Model Descriptions for likelihoods calculated 7
Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2 8
Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2 9
Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + rho*ln(Mu(i))) 10

Model A3 uses any fixed variance parameters that were specified by the user 11
Model R: Yi = Mu + e(i) Var{e(i)} = Sigma^2 12
13
14 Likelihoods of Interest 15
16 Model

A1 Log(likelihood)

- 410.240404 # Param's

4 AIC

828.480807 17 A2

- 350.033965 6 712.067929 18 A3

- 350.072753 5 710.145506 19 fitted

- 350.666161 4 709.332321 20 R

- 412.701435 2 829.402870 21

Explanation of Tests 22

Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R) 23

Test 2: Are Variances Homogeneous? (A1 vs A2) 24

Test 3: Are variances adequately modeled? (A2 vs. A3) 25

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 26

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 27
28

Tests of Interest 30
31 Test

Test 1 - 2*log(Likelihood Ratio) Test df

125.335 4 p

- value

<.0001 32 Test 2 120.413 2 <.0001 33 Test 3 0.0775771 1 0.7806 34 Test 4 1.18681 1 0.276 29

35

The p

- value for Test 1 is less than .05. There appears to be a difference between response 36

and/or variances among the dose levels. It seems appropriate to model the data 37
38

The p

- value for Test 2 is less than .1. A non

- homogeneous variance model appears to be 39

appropriate 40
41

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here 42
43

The p

- value for Test 4 is greater than .1. The model chosen seems to adequately describe the 44

data 45
46

Benchmark Dose Com putation 47

Specified effect = 1 48

Risk Type = Relative risk 49

Confidence level = 0.95 50

BMD = 190.33 51

BMDL = 121.534 52

53

### B-31 DRAFT

- DO NOT CITE OR QUOTE Table B -18 . BMD modeling results for serum ALT activity in female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

Variance

model

p

- value a Means

model

p

- value a Largest

residual AIC BMD 1SD BMDL 1SD BMD 1RD BMDL 1RD

All doses

Constant variance

Hill b < 0 .0001 NA 9.61 × 10

- 7 1

, 167.39 3

, 911.09 436.97 160.82 0.00

Linear c , Polynomial

(2

- degree) c , Power d < 0 .0001 0.55 0.94 1

, 164.57 1

, 613.62 1

, 106.30 412.90 38.31

Non constant variance

Hill b 0.78 NA

- 0.49 1

, 013.25 116.28 CF 148.75 121.30

Linear c 0.78 < 0 .0001 1.69 × 10 10 6 0 CF 419.08 CF

Polynomial (2

- degree) c 0.78 < 0 .0001

- 1.39 × 10 11 8 0 CF 87.64 CF

Power d 0.78 < 0 .0001

- 1.88 1

, 047.49 90.73 62.72 108.55 77.76

Highest dose dropped

Constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

Linear c

, < 0 .0001 0.79

- 0.22 756.72 518.80 324.41 116.10 0.00

Polynomial (2

- degree) c < 0 .0001 NA 4.25 × 10

- 7 758.65 488.92 325.96 170.36 0.00

Power d < 0 .0001 NA

- 3.00 × 10

- 9 758.65 497.95 325.96 167.69 0.00

Non constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

Linear c 0.89 < 0 .0001

- 2. 59 × 10 9 6 0 CF 111.13 CF

Polynomial (2

- degree) c 0.89 < 0 .0001

- 5

. 85 × 10 7 8 0 CF 169.57 CF

Power d 0.89 NA 0.10 631.43 110.52 67.61 172.25 117.98

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be positive.

d Restrict power ≥1.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 standa rd deviation from control mean value; 1RD = dose associated with a 100% relative deviation
from control mean value); CF = computat ion failed; NA = not applicable

Source: Umeda et al. (2005).

1

2 

### None of the models provided an adequate fit to both the variance model and the means model.

### B-32 DRAFT

- DO NOT CITE OR QUOTE Table B -19 . BMD modeling results for serum AP activity in female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Goodness of fit Benchmark result (mg/kg

- d)

Variance Means

Model model

a p

- value model

a p

- value Largest

residual AIC BMD 1SD BMDL 1SD BMD 1RD BMDL 1RD

All doses

Constant variance

Hill b < 0 .0001 NA

- 4.74 × 10

- 8 1

, 240.81 642.90 320.63 540.57 180.68

c Linear , Polynomial

d (2

- degree) c , Power < 0 .0001 0.31 1.32 1

, 239.14 1

, 253.51 919.17 1

, 208.38 720.75

Non constant variance

Hill b 0.006 NA

- 0.93 1

, 180.07 147.47 CF 177.26 CF

c Linear 0.006 < 0 .0001 5.04 1

, 334.76

- 9

, 999.00 244.46 28.02 0.05

Polynomial (2

- degree) c 0.006 < 0 .0001

- 2

. 57 × 10 1 1 8 0 CF 390.64 CF

Polynomial (3

- degree) c 0.006 < 0 .0001 1.89 1

, 242.58 1

, 495.81 213.20 1

, 506.34 333.91

d Power 0.006 < 0 .0001 1.41 1

, 236.21 665.13 345.69 815.01 482.17

Highest dose dropped

Constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

c Linear

, < 0 .0001 0.55

- 0.51 868.21 617.91 361.78 487.67 201.11

Polynomial (2

- degree) c < 0 .0001 0.95

- 0.05 867.85 510.80 393.46 467.69 315.45

d Power < 0 .0001 NA 1.09E

- 8 869.84 499.45 372.60 464.35 213.97

Non constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

c Linear 0.77 < 0 .0001 4.52 × 10 9 6 0 CF 465.02 CF

Polynomial (2

- degree) c 0.77 NA 0.13 794.19 287.55 183.20 480.63 334.12

d Power 0.77 NA

- 0.21 794.19 285.46 179.35 482.75 333.04

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be positive.

d Restrict power ≥ 1.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 standard deviation from control mean value; 1RD = dose associated with a 100% relative deviation
from control mean value); CF = computati on failed; NA = not applicable

Source: Umeda et al. (2005).

1

2 

### None of the models provided an adequate fit to both the variance model and the means model.

3

### B-33 DRAFT

- DO NOT CITE OR QUOTE Table B -20 . BMD modeling results for changes in BUN levels (mg/dL) in

male BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

Variance

model

p

- value a Means

model

p

- value a Largest

residual AIC BMD 1SD BMDL 1SD BMD 1RD BMDL 1RD

Males

All doses

Constant variance

Hill b 0.03 NA 0.25 540.50 CF CF CF CF

Linear c ,d , Polynomial

(2

- degree) c , Power 0.03 0.01

- 2.00 545.04 2

, 254.69 1

, 288.77 12

, 777.10 7

, 154.72

Non constant variance

Hill b 0.01 NA 0.25 542.49 CF CF CF CF

Linear c 0.01 0.28

- 1.95 540.78 3

, 134.77 1

, 690.32 15

, 745.20 8

, 512.03

Polynomial (2

- degree) c 0.01 0.13

- 2.23 542.57 2

, 029.81 1

, 459.55 4

, 649.85 3

, 312.21

Polynomial (3

- degree) c 0.01 0.13

- 2.25 542.52 1

, 688.06 1

, 324.21 2

, 974.25 2

, 291.81

Power d 0.01 0.13

- 2.32 542.51 1

, 170.31 1

, 092.10 1

, 334.64 1

, 196.80

Highest dose dropped

Constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

Linear c , Polynomial
(2

- degree) c , Power d 0.49 0.32 0.77 420.23 414.78 266.77 2

, 140.93 1

, 335.54

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be positive.

d Restrict power ≥ 1.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 standard deviation from control mean value; 1RD = dose associated with a 100% relative deviation
from control mean value); CF = computati on failed; NA = not applicable

Source: Umeda et al. (2005).

1

### B-34 DRAFT

- DO NOT CITE OR QUOTE 19 20 21 22 23 24 25

0 50 100 150 200 250 300 350 400Mean Response

dose Linear Model with 0.95 Confidence Level

11:03 01/19 2011 BMD BMDL Linear

1

2

BMD and BMDL indicated are asso ciated with a 1SD change from control , and are in units of mg/kg

- day

. 3

==================================================================== 4

Polynomial Model. (Version: 2.16; Date: 05/26/2010) 5

Input Data File: 6

C:/Storage/USEPA/IRIS/bipheny l/2011/BMD/mice/BUN/male/lin_BUNMHDD_linear.(d) 7

Gnuplot Plotting File: 8

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/BUN/male/lin_BUNMHDD_linear.plt 9

Wed Jan 19 11:03:37 2011 10

==================================================================== 11

BMDS Model Run 12

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 13

The form of the response function is: Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 14

Dependent variable = mean 15

Independent variable = dose 16

rho is set to 0 17

The polynomial coefficients are restricted to be positive 18

A constant variance model is fit 19

Total number of dose groups = 3 20

Total number of records with missing values = 0 21

Maximum number of iterations = 250 22

Relative Function Convergence has been set to: 1e

- 008 23

Parameter Convergence has been set to: 1e

- 008 24
25

Default Initial Parameter Values 26

alpha = 16.1929 27

rho = 0 Specified 28

beta_0 = 20.5429 29

beta_1 = 0.00972018 30
31

Asymptotic Correlation Matrix of Parameter Estimates 32

( *** The model parameter(s)

- rho have been estimated at a boundary point, or have been 33

specified by the user, and do not appear in the correlation matrix ) 34
35
36
37
38 alpha

beta_0

beta_1 alpha

1

- 3.8e

- 008

3.2e

- 008 beta_0

- 3.8e

- 008

1

- 0.74 beta_1

3.2e

- 008

- 0.74

1 39
40 Parameter E stimates 41 95.0% Wald Confidence Interval 42
43
44
45
46 Variable

alpha

beta_0

beta_1 Estimate

15.8907

20.576

0.0096108 Std. Err.

2.14271

0.566499

0.00317579 Lower Conf. Limit

11.6911

19.4657

0.00338636 Upper Conf. Limi

20.0904

21.6863

0.0158352 t

Table of Data and Estimated Values of Interest 47

Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 48

------ --- -------- -------- ----------- ----------- ---------- 49
50

0 34 20.2 20.6 3.6 3.99

- 0.55 51

### B-35 DRAFT

- DO NOT CITE OR QUOTE 1 97 39 22 21.5 4 3.99 0.77 2 291 37 23.2 23.4 4.4 3.99

- 0.264 3

Model Descriptions for likelihoods calculated 5 4

Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2 6

Model A2: Yij = Mu(i) + e(ij) 7
Var{e(ij)} = Sigma(i)^2 8

Model A3: Yij = Mu(i) + e(ij 9 ) Var{e(ij)} = Sigma^2 10

Model A3 uses any fixed variance parameters that were specified by the user 11

Model R: Yi = Mu + e(i) Var{e(i)} = Sigma^2 13 12

14
15 Likelihoods of Interest 16
17 Model

A1 Log(likelihood)

- 206.630664 # Param's

4 AIC

421.261329 18 A2

- 205.915695 6 423.8 31391 19 A3

- 206.630664 4 421.261329 20 fitted

- 207.115525 3 420.231050 21 R

- 211.514015 2 427.028031 22

Explanation of Tests 23
24

Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R) 25

Test 2: Are Variances Homogeneous? (A1 vs A2) 26

Test 3: Are variances adequately modeled? (A2 vs. A3) 27

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 28

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 29
30

Tests of Interest 31
32
33 Test

Test 1 - 2*log(Likelihood Ratio)

11.1966 Test df

4 p

- value

0.02444 34 Test 2 1.42994 2 0.4892 35 Test 3 1.42994 2 0.4892 36 Test 4 0.969721 1 0.3247 37

The p

- value for Test 1 is less than .05. There appears to be a difference between response 38

and/or variances among the dose levels. It seems appropriate to model the data 39
40

The p

- value for Test 2 is greater than .1. A homogeneous variance model appears to be 41

appropriate here 42
43

The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here 44
45

The p

- value for Test 4 is greater than .1. The model chosen seems to adequately describe the 46

data 47
48

Benchmark Dose Computation 49

Specified effect = 1 50

Risk Type = Estimated standard deviations from the control mean 51

Confidence level = 0.95 52

BMD = 414.775 53

BMDL = 266.77 54

55

### B-36 DRAFT

- DO NOT CITE OR QUOTE Table B -21 . BMD model ing results for changes in BUN levels (mg/dL) in

female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

Variance

model

p

- value a Means

model

p

- value a Largest

residual AIC BMD 1SD BMDL 1SD BMD 1RD BMDL 1RD

All doses

Constant variance

Hill b < 0 .0001 NA

- 3.45 × 10

- 8 603.61 CF CF CF CF

Linear c , Polynomial

(2

- degree) c , Power d < 0 .0001 0.38 1.18 601.53 1

, 869.01 1

, 224.15 2

, 507.85 1

, 434.76

Non constant variance

Hill b 0.08 NA

- 1.21 493.48 141.72 CF CF CF

Linear c , Polynomial

(2

- degree) c , Power d 0.08 <0 .0001

- 1.63 590.70 519.60 216.41 1, 191.69 683.73

Highest dose dropped

Constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

Linear c

, < 0 .0001 0.50

- 0.57 417.59 744.99 403.07 921.79 410.67

Polynomial (2

- degree) c < 0 .0001 0.82

- 0.18 417.19 555.48 413.38 627.58 432.73

Power d < 0 .0001 NA

- 2.11 × 10

- 10 419.13 430.03 414.77 436.97 417.75

Non constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

Linear c 0.23 0.07

- 1.38 300.36 180.70 114.17 1

, 416.07 916.09

Polynomial (2

- degree) c 0.23 NA

- 0.93 299.05 263.22 152.60 842.06 495.16

Power d 0.23 < 0 .0001

- 0.93 297.05 256.90 151.17 925.84 490.39

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be positive.

d Restrict power ≥ 1.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 standard deviation from control mean value; 1RD = dose associated with a 100% relative deviation
from control mean value); CF = computati on failed; NA = not applicable

Source: Umeda et al. (2005).

1

2 

### None of the models provided an adequate fit to both the variance model and the means model.

3

### B-37 DRAFT

- DO NOT CITE OR QUOTE Table B -22 . BMD modeling results for changes in mean terminal body
weight in male BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

Variance

model

p

- value a Means

model

p

- value a Largest

residual AIC BMD 1SD BMDL 1SD BMD 0.1RD BMDL 0.1RD

All doses

Constant variance

Hill b < 0 .0001 0.03

- 1.68 716.95 459.61 390.85 358.30 316.09

Linear c , Power d < 0 .0001 0.10

- 1.68 714.95 460.46 391.75 359.04 316.87

Polynomial (3

- degree) c < 0 .0001 0.03

- 1.66 716.89 498.04 392.48 390.52 317.33

Non constant variance

Hill b 0.002 NA

- 1.52 704.84 600.48 CF 421.46 325.00

Linear c

, 0.002 0.59

- 1.52 701.13 541.68 460.24 357.54 326.02

Polynomial (3

- degree) c 0.002 0.44

- 1.42 702.64 643.20 467.09 450.96 328.74

Power d 0.002 0.38

- 1.51 702.84 600.89 464.26 421.53 327.62

Highest dose dropped

Constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

Linear c , Polynomial

(2

- degree) c , Power d 0.01 0.05

- 1.49 560.11 566.99 328.79 400.33 238.24

Non constant variance

Hill b Not modeled; number of dose groups less than number of model parameters

Linear c , Polynomial

(2

- degree) c , Power d 0.18 0.001

- 1.5 562.10 561.56 308.43 398.66 235.32

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be negative.

d Restrict power ≥ 1.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 standard deviation from control mean value; 0.1RD = dose associated with a 10% relative deviation
from control mean value); CF = computat i on failed; NA = not applicable

Source: Umeda et al. (2005).

1

2 

### None of the models provided an adequate fit to both the variance model and the means model.

3

### B-38 DRAFT

- DO NOT CITE OR QUOTE Table B -23 . BMD model ing results for changes in mean terminal body
weight in female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Model Goodness of fit Benchmark result (mg/kg

- d)

Variance

model

a p

- value Means

model

a p

- value Largest

residual AIC BMD 1SD BMDL 1SD BMD 0.1RD BMDL 0.1RD

All doses

Constant variance

Hill b 0.36 0.80

- 0.21 382.59 387.90 230.17 397.06 243.57

Linear c ,d , Polynomial

e (2

- degree) c , Power 0.36 0.42

- 0.93 382.26 584.12 489.94 583.33 510.85

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Restrict n > 1.

c Coefficients restricted to be negative.

d Selected model; the model with the lowest AIC was selected because BMDL values for models providing adequate
fit did not differ by more than threefold.

e Restrict power ≥1.

BMDL = 95% lower confidence limit on the BMD (subscripts denote benchmark response: i.e., 1SD = dose

associated with 1 standard deviation from control mean value; 0.1RD = dose associated with a 10% relative deviation
from control mean value); CF = computati on failed; NA = not applicable

Source: Umeda et al. (2005).

1

24 26 28 30 32 34 36

0 200 400 600 800 1000 1200 1400Mean Response

dose Linear Model with 0.95 Confidence Level

09:20 01/20 2011 BMD BMDL Linear

2

BMD and B MDL indicated are associated with a 10% decrease from control , and are in units of mg/kg

- day

. 3

4

==================================================================== 5

Polynomial Model. (Version: 2.16; Date: 05/26/2010) 6

Input Data File: 7

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/termbdwt/female/lin_termbdwtF_linear.(d) 8

Gnuplot Plotting File: 9

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/termbdwt/female/lin_termbdwtF_linear.plt 10

Thu Jan 20 09:20:01 2011 11

==================================================================== 12

BMDS Model Run 13

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 14

The form of the response function is: Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ... 15

### B-39 DRAFT

- DO NOT CITE OR QUOTE Dependent variable = mean 1

Independent variable = dose 2

rho is set to 0 3

The polynomial coefficients are restricted to be negative 4

A constant variance model is fit 5

Total number of dose groups = 4 6

Total number of records with missing values = 0 7

Maximum number of iterations = 250 8

Relative Function Convergence has been set to: 1e

- 008 9

Parameter Convergence has been set to: 1e

- 008 10
11

Default Initial Parameter Values 12

alpha = 11.4937 13

rho = 0 Specified 14

beta_0 = 33.4391 15

beta_1 =

- 0.00571961 16
17

Asymptotic Correlation Matrix of Parameter Estimates 18

( *** The model parameter(s)

- rho have been estimated at a boundary point, or have been 19

specified by the user, and do not appear in the correlation matrix ) 20

22 21

23
24 alpha

beta_0

beta_1 alpha

1

- 9.6e

- 009

9.1e

- 009 beta_0

- 9.6e

- 009

1

- 0.67 beta_1

9.1e

- 009

- 0.67

1 25
26 Parameter Estimates 27 95.0% Wald Confidence Interval 28
29
30
31 Variable

alpha

beta_0

beta_1 Estimate

11.2518

33.4983

- 0.00574262 Std. Err.

1.5172

0.432523

0.000545303 Lower Conf. Limit

8.27818

32.6505

- 0.0068114 Upper Conf. Limit

14.2255

34.346

- 0.00467385 32
33 Table of Data and Estimated Values of Interest 34 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res. 35

------ --- -------- -------- ----------- ----------- ---------- 36 0 31 34 33.5 4 3.35 0.833 37 134 22 32.5 32.7 3.3 3.35

- 0.32 38 414 25 30.5 31.1 3.1 3.35

- 0.925 39 1420 32 25.5 25.3 3 3.35 0.264 40

Model Descriptions for likelihoods calculated 41
Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2 42
Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2 43
Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2 44

Model A3 uses any fixed variance parameters that were specified by the user 45
Model R: Yi = Mu + e(i) Var{e(i)} = Sigma^2 46
47

Likelihoods of Interest 49
50 Model

A1 Log(likelihood)

- 187.261579 # Param's

5 AIC

384.523158 51 A2

- 185.643849 8 387.287698 52 A3

- 187.261579 5 384.523158 53 fitted

- 188.129218 3 382.258435 54 R

- 226.477701 2 456.955401 55 48

Explanation of Tests 56
57

Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R) 58

Test 2: Are Variances Homogeneous? (A1 vs A2) 59

Test 3: Are variances adequately modeled? (A2 vs. A3) 60

Test 4: Does the Model for the Mean Fit? (A3 vs. fitted) 61

(Note: When rho=0 the results of Test 3 and Test 2 will be the same.) 62
63

Tests of Interest 65

66 Test

Test 1 - 2*log(Likelihood Ratio)

81.6677 Test df

6 p

- value

<.0001 67 Test 2 3.23546 3 0.3567 68 Test 3 3.23546 3 0.3567 69 Test 4 1.73528 2 0.4199 64

70

The p

- value for Test 1 is less than .05. There appears to be a difference between response 71

and/or variances among the dose levels. It seems appropriate to model the data 72
73

The p

- value for Test 2 is greater than .1. A homogeneous variance model appears to be 74

appropriate here 75

### B-40 DRAFT

- DO NOT CITE OR QUOTE 1
2 The p

- value for Test 3 is greater than .1. The modeled variance appears to be appropriate here 3
4 The p

- value for Test 4 is greater than .1. The model chosen seems to adequately describe the 5 data 6
7 Benchmark Dose Computation 8 Specified effect = 0.1 9 Risk Type = Relative risk 10 Confidence level = 0.95 11 BMD = 583.327 12 BMDL = 510.848 13
14

15



### Table B -24. Summary of BMD mod eling results for incidence of litters with

fetal skeletal anomalies from Wistar rat dams administered biphenyl by
gavage on GDs 6–15

Model Goodness of fit Benchmark result (mg/kg

- d)

**χ** 2 p

- value a Largest

residual AIC BMD 5 BMDL 5 BMD 10 BMDL 10

Gamma b

, Weibull b

, Multistage (1

- degree) c 0.31

- 1.25 106.11 54.45 24.15 111.84 49.61

Logistic 0.28 1.17 106.42 73.97 36.73 149.18 73.79

Log

- Logistic b,d 0.41

- 1.32 105.33 27.03 9.59 57.06 20.24

Log

- Probit b 0.23

- 1.59 106.55 125.14 55.10 179.97 79.23

Probit 0.28 1.20 106.50 79.59 41.02 160.27 82.37

a Values <0.10 fail to meet conventional goodness

- of

- fit criteria.

b Power restricted to ≥1.

c Betas restricted to ≥0.

d Selected model; the model with the lowest BMDL was selected because BMDL values for models providing
adequate fit differed by more than threefold; this model also had the lowest AIC.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark res ponse: i.e., 10 = dose associated with 10%

extra risk; 5 = dose associated with 5% extra risk)

Source: Khera et al. (1979).

### B-41 DRAFT

- DO NOT CITE OR QUOTE 1

0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9

1

0 200 400 600 800 1000Fraction Affected

dose Log-Logistic Model with 0.95 Confidence Level

16:06 01/14 2011 BMDL BMD Log-Logistic

2 3

BMD a nd B MDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day. 4

5

6

==================================================================== 7

Logistic Model. (Version: 2.13; Date: 10/28/2009) 8

Input Data File: 9

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/develop/anomlitt/lnl_anomlitt_loglogistic.(d) 10

Gnuplot Plotting File: 11

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/develop/anomlitt/lnl_anomlitt_loglogistic.plt 12

Fri Jan 14 16:06:43 2011 13

==================================================================== 14

BMDS_Model_Run 15

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 16

The form of the probability function is: P[response] = background+(1

- background)/[1+EXP(

- 17

intercept

- slope*Log(dose))] 18

Dependent variable = incidence 19

Independent variable = dose 20

Slope parameter is restricted as slope >= 1 21

Total number of observations = 5 22

Total number of records with missing values = 0 23

Maximum number of iterations = 250 24

Relative Function Convergence has been set to: 1e

- 008 25

Parameter Convergence has been set to: 1e

- 008 26

User has chosen the log transformed mo del 27
28

Default Initial Parameter Values 29

background = 0.5 30

intercept =

- 6.54827 31

slope = 1 32
33

Asymptotic Correlation Matrix of Parameter Estimates 34

( *** The model parameter(s)

- slope have been estimated at a boundary point, or have been 35

specified by the user, and do not appear in the correlation matrix ) 36

background inter cept 37

background 1

- 0.77 38

intercept

- 0.77 1 39
40

Parameter Estimates 41
42 95.0% Wald Confidence Interval 43
44
45
46 Variable

background

intercept

slope Estimate

0.503241

- 6.24131

1 Std. Err.

*

*

* Lower Conf. Limit

*

*

* Upper Conf. Limit

*

*

*

* - Indicates that this value is not calculated. 47
48
49
50
51 Model

Full model Analysis of Deviance Table

Log(likelihood) # Param's Deviance

- 49.327 5 Test d.f. P

- value

### B-42 DRAFT

- DO NOT CITE OR QUOTE 1 Fitted model

- 50.6629 2 2.67182 3 0.445 2 Reduced model

- 52.2232 1 5.79233 4 0.2152 3
4 AIC: 105.326 5

Goodness of Fit 6
7 Scaled 8
9 Dose

---------- Est._Prob.

-------------- Expected

----------- Observed

------------- -- Size

--------- Residual

------------ - 10 0.0000 0.5032 8.052 8.000 16

- 0.026 11 125.0000 0.6005 12.010 11.000 20

- 0.461 12 250.0000 0.6659 11.986 13.000 18 0.507 13 500.0000 0.7483 13.469 15.000 18 0.831 14 1000.0000 0.8315 7.483 6.000 9

- 1.321 15

Chi^2 = 2.90 d.f. = 3 P

- value = 0.4065 16
17

Benchmark Dose Computation 18

Specified effect = 0.1 19

Risk Type = Extra risk 20

Confidence level = 0.95 21

BMD = 57.0591 22

BMDL = 20.2399 23

### C-1 DRAFT

- DO NOT CITE OR QUOTE 1

2 

### APPENDIX C. BENCHMARK MODELING FOR THE ORAL SLOPE FACTOR

3

4



### The mouse liver tumor dataset from Umeda et al. (2005) for which dose- response 5



### modeling was performed is shown in Table C-1. 6

7



### Table C -1 . Incidences of liver adenomas or carcinomas (combined) in

female BDF 1 

### mice fed diets containing biphenyl for 2 years

Biphenyl dietary concentration (ppm) 0 667 2,000 6,000

Reported dose (mg/kg

- d) 0 134 414 1,420

HED (mg/kg

- d) 0 19 59 195

Tumor incidence

Adenoma or carcinoma (combined) 3/48 a 8/50 16/49 a,b 14/48 a,c

a Two control, one mid

- dose, and two high

- dose female mice were excluded from denominators because they died
prior to week 52. It is assumed that they did not have tumors and were not exposed for a sufficient time to be at
risk for developing a tumor. Umeda et al. (2005) did not specify the time of appearance of the first tumor.

b Significantly different from controls ( p < 0.05) according to Fisher’s exact test.

c Significantly different from controls ( p < 0.01) according to Fisher’s exact test.

Source: Umeda et al. (2005).

8



### Summaries of the BMDs, BMDLs, and the derived oral slope factors for the modeled 9



### mouse data are presented in Table C -2, followed by the plot and model output file from the best10



### fitting model. The animals in the highest dose group, while exhibiting a statistically 11



### significantly increased incidence in live r tumors compared with controls, did not show a 12



### monotonic increase in tumor response compared with the responses at the lower doses. To better 13



### estimate responses in the low dose region, the high dose group was excluded as a means of 14



### improving the fit of the model in the region of interest. 15

16

### C-2 DRAFT

- DO NOT CITE OR QUOTE Table C -2 . Model predictions for liver tumors (adenomas or carcinomas

combine d) in female BDF 1 

### mice exposed to biphenyl in the diet for 2 years

Goodness of fit Benchmark result (mg/kg

- d)

Cancer

slope

factor

Model **χ** 2 p

- value a Largest

residual AIC BMD HED 10 BMDL HED 10 (risk per
mg/kg

- d)

All doses

Multistage (1

- , 2

- ,

c Gamma , Weibull c 3

- degree) b

, 0.03 2.14 197.37 64.76 37.29 0.003

Logistic 0.01 2.31 198.96 104.91 71.27 0.001

c Log

- Logistic 0.04 1.97 196.62 50.68 26.80 0.004

Log

- Probit c 0.005 2.58 201.06 128.52 74.43 0.001

Probit 0.01 2.30 198.80 100.16 67.23 0.001

Highest dose dropped

Multistage (1

- degree) b,d 0.96 0.04 132.32 18.72 12.15 0.008

Multistage (2

- degree) b 0.96 0.04 132.32 18.72 12.15 0.008

a Values <0.05 fail to meet conventional goodness

- of

- fit criteria.

b Betas restricted to ≥0.

c Power restricted to ≥1

. d Selected model

.

BMD = maximum likelihood estimate of the dose associated with the selected benchmark response; BMDL = 95%
lower confidence limit on the BMD (subscripts denote benchmark response: i.e., HED10 = human equivalent dose

associated with 10% extra risk

Source: Umeda et al. (2005).

1

0 0.1 0.2 0.3 0.4 0.5

0 10 20 30 40 50 60Fraction Affected

dose Multistage Cancer Model with 0.95 Confidence Level

09:33 02/03 2011 BMD BMDL Multistage Cancer Linear extrapolation

2

BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg

- day

. 3

4

### C-3 DRAFT

- DO NOT CITE OR QUOTE ==================================================================== 1

Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) 2

Input Data File: 3

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/livertumor/female/revised_n/msc_livtumFrev2HDD_MS_1. 4

(d) 5

Gnuplot Plotting File: 6

C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/livertumor/female/revised_n/msc_livtumFrev2HDD_MS_1. 7

plt 8

Thu Feb 03 09:33:34 2011 9

==================================================================== 10

BMDS_Model_Run 11

~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 12

The form of the probability function is: P[response] = background + (1

- background)*[1

- EXP(

- 13

beta1*dose^1)] 14

The parameter betas are restricted to be positive 15

Dependent variable = incidence 16

Independent variable = dose 17

Total number of observations = 3 18

Total number of records with missing values = 0 19

Total number of parameters in model = 2 20

Total number of specified parameters = 0 21

Degree of polynomial = 1 22

Maximum number of iterations = 250 23

Relative Function Convergence has been set to: 2.22045e

- 016 24

Parameter Convergence has been set to: 1.49012e

- 008 25

**** We are sorry but Relative Function and Parameter Convergence are currently unavailable in 26

this model. Please keep checking the web sight for model updates which will eventually 27

incorporate these convergence criterion. Default values used. **** 28
29

Default Initial Parameter Values 30

Background = 0.0638384 31

Beta(1) = 0.00559363 32
33
34
35
36
37 Background Asymptotic Correlation Matrix of Parameter Estimates

Background Beta(1)

1

- 0.7

Beta(1)

- 0.7 1 38
39 Parameter Estimates 40 95.0% Wald Confidence Interval 41
42 Variable

Background Estimate

0.0630397 Std. Err.

* Lower Conf. Limit

* Upper Conf. Limit

*

Beta(1) 0.00562948 * * * 43

* - Indicates that this value is not calculated. 44
45
46
47 Analysis of Deviance Table

48
49 Model

Full model Log(likelihood) # Param's Deviance

- 64.1585 3 Test d.f. P

- value

50 Fitted model

- 64.1595 2 0.0019921 1 0.9644 51 Reduced model

- 70.107 1 11.8969 2 0.00261 52

AIC: 132.319 53
54

Goodness of Fit 55
56 Scaled 57
58 Dose

---------- Est._Prob.

-------------- Expected

------------ Observed

------------ -- Size

--------- Residual

------------ - 59 0.0000 0.0630 3.026 3.000 48

- 0.015 60 19.0000 0.1581 7.904 8.000 50 0.037 61 59.0000 0.3278 16.064 16.000 49

- 0.019 62

Chi^2 = 0.00 d.f. = 1 P

- value = 0.9644 63
64

Benchmark Dose Computation 65

Specified effect = 0.1 66

Risk Type = Extra risk 67

Confidence level = 0.95 68

BMD = 18.7158 69

BMDL = 12.1518 70

BMDU = 36.3895 71

Taken together, (12.1518, 36.3895) is a 90% two

- sided confidence interval for the BM D 72

Multistage Cancer Slope Factor = 0.00822924 73

74