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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
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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. 
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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. DISTRIBUTION .................................................................................................................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. Effects on the Liver of Mice ......................................................................................56
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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 RESPONSE .........................................................................................................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. SUMMARY 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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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 features for five Parkinson’s Disease patients with 

occupational exposure to biphenyl ............................................................................ 23

Table 4-4. Incidences of urinary bladder lesions 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 BDF1 mice exposed to biphenyl via the diet for 2 years ............................... 37

Table 4-9. Incidences of gross and histopathological findings in male and female BDF1

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
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Table 4-19. Summary of major studies evaluating effects of biphenyl after inhalation 

exposure in rats and mice ......................................................................................... 73

Table 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 BDF1 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 biphenyl 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 BDF1 mice fed diets 

containing biphenyl for 2 years .............................................................................. 110

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

BDF1 mouse liver tumor incidence data from Umeda et al. (2005) ....................... 111

Table 5-8. Incidence of liver adenomas or carcinomas (combined) in female BDF1 mice 

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

Table 5-9. POD and oral slope factor derived from liver tumor incidence data from BDF1

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

Table B-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 .... B-1

Table B-2. BMD modeling datasets for body weight, selected clinical chemistry results, 

and histopathological kidney effects in male and female BDF1 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 transitional 

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
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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 BDF1 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 BDF1 mice exposed to biphenyl 

in the diet for 2 years ........................................................................................... B-25

Table B-16. BMD model results for serum LDH activity in female BDF1 mice exposed to 

biphenyl in the diet for 2 years ............................................................................ B-27

Table B-17. BMD modeling results for serum AST activity in female BDF1 mice exposed 

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

Table B-18. BMD modeling results for serum ALT activity in female BDF1 mice exposed 

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

Table B-19. BMD modeling results for serum AP activity in female BDF1 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 BDF1

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 BDF1

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 

BDF1 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 

BDF1 mice exposed to biphenyl in the diet for 2 years ....................................... B-38

Table 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 BDF1 mice 

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

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

female BDF1 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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LIST OF ABBREVIATIONS AND ACRONYMS

ACGIH American Conference of Governmental Industrial Hygienists 

AIC Akaike’s Information Criterion

ALT alanine aminotransferase

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 

Ko/w octanol/water partition coefficient

Km Michaelis constant

LD50 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 Parkinson’s disease

POD point of departure 

PPAR peroxisome proliferator activated receptors 

RfC reference concentration
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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 
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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 information about this assessment or other questions relating to IRIS, 

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

hotline.iris@epa.gov (email address). 
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AUTHORS, CONTRIBUTORS, AND REVIEWERS

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 
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1 1. INTRODUCTION

2 

3 

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

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

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

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

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

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

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

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

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

deleterious effects during a lifetime. The inhalation RfC (expressed in units of mg/m3 13 ) is 

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

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

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

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

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

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

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

21 derived for chronic exposure duration. 

22 The carcinogenicity assessment provides information on the carcinogenic hazard 

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

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

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

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

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

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

an upper bound on the estimate of risk per μg/m3 29 air breathed.

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

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

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

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

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

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

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

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

38 Policy for Particle Size and Limit Concentration Issues in Inhalation Toxicity (U.S. EPA, 
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1 1994a), Methods for Derivation of Inhalation Reference Concentrations and Application of 

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

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

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

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

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

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

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

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

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

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

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

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

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

15 scientific information submitted by the public to the IRIS Submission Desk was also considered 

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

17 
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1 2. CHEMICAL AND PHYSICAL INFORMATION

2 

3 

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

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

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

(NLM, 2007; IPCS, 1999). Biphenyl melts at 69°C and has a vapor pressure of 8.93 × 10-3 7 mm 

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

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

atmosphere by volatilization. The water solubility of biphenyl is 7.48 mg/L at 25o 10 C. The 

11 logarithm of the octanol/water partition coefficient (Ko/w) of biphenyl of 3.98 suggests a potential 

12 for bioaccumulation (NLM, 2007). Because it is biodegraded with an estimated half-life of 2 

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

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

ubiquitous in the environment, with reported indoor air concentrations of 0.16–1 μg/m3 15 and 

outdoor levels of approximately 0.03 μg/m3 16 (IPCS, 1999). The physicochemical properties of 

17 biphenyl are summarized in Table 2-1. 

18

19
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Table 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 C12H10

Molecular weight 154.2

Melting point 69°C

Boiling point 256°C

Specific gravity 1.041 g/cm3 at 20°C

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

Log Ko/w 4.01

4.11a

4.17 or 5.27–5.46b

Water solubility 7.48 mg/L at 25°C

Henry’s law constant 3.08 × 10-4 atm-m3

/mol at 25°C

Conversion factors 1 ppm = 6.31 mg/m3

; 1 mg/m3 = 0.159 ppm

a

Monsanto (1979).

b

Estimated by different methods: Dow (1971).

Source: NLM (2007).

1 

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

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

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

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

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

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

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

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

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

11 and 73.5% diphenyl oxide). Historically, biphenyl was the primary byproduct in the 

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

13 EPA 1978). The purity of technical biphenyl ranges from 93–99.9%. The prevalent impurities 

14 in technical preparations are terphenyls, a side product from the dehydrocondensation of 
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1 benzene. Biphenyl is rated as a high-volume production chemical. Annual U.S. production in 

1990 was approximately 1.6 × 104 2 metric tons (NLM, 2007). 
1 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 hepatic toxicity produced by a probable combination of inhalation and 

7 dermal exposures to biphenyl was identified as 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 years of exposure to biphenyl; at the time of his death, air measurements in the factory were 

11 as high as 123 mg/m3. Evidence of hepatic and nervous system toxicity was also observed in 

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

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13 Animal studies in rats, rabbits, guinea pigs, and pigs indicate that biphenyl is rapidly and 

14 readily absorbed following oral exposure, as evidenced by the detection of metabolites in urine 

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

16 with rats administered radiolabeled biphenyl indicate extensive oral absorption (about 85% of 

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

18 guinea pigs, and pigs administered nonlabeled biphenyl indicate less extensive oral absorption in 

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

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

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

[

14 22 C]-biphenyl (in soy oil) excreted 75–80% of the radioactivity in their urine within the first 

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

96-hour post-dosing period (Meyer et al., 1976a). Only a trace of [14 24 C]-CO2 was detected in 

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

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

27 absorbed in rats.

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

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

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

31 unlabeled biphenyl. 

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

33 gavage in soy oil, and urine and feces were collected at 24-hour intervals, up to 96 hours after 

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

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

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

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

38 the urine in both species and the major metabolite being 4-hydroxybiphenyl. In guinea pigs 
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1 (n = 3), the mass of identified metabolites in urine collected for 24 or 96 hours accounted for 

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

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

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

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

6 detected in the collected bile, but conjugated mono- and dihydroxy metabolites accounted for 

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

8 administered dose was absorbed. In rabbits, urinary metabolites accounted for 49.1% of the 

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

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

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

12 was absorbed in rabbits. 

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

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

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

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

17 after enzyme hydrolysis of the samples by β-glucuronidase and sulphatase. Metabolites 

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

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

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

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

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

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

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

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 available. 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. Most 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 
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1 96 hours: 0.1% was found in peritoneal fat, 0.3% in the gastrointestinal tract (including its 

2 contents) 0.1% in skeletal muscles, and 0.1% in the genital tract. Levels of radioactivity in other 

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

4 stored in tissues 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., 1981a; Meyer, 1977; 

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

14 urine both as nonconjugated compounds 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 

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

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

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

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

24 1976b) and the rat (Halpaap-Wood et al., 1981a; Meyer and Scheline, 1976), while 3,4-di25 hydroxybiphenyl was a major urinary metabolite in two strains of mice (Halpaap-Wood et al., 

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

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

28 (1976). 

29
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Table 3-1. Metabolites of biphenyl identified in urine, feces, and bile of male 

albino rats 

Metabolitea

Urine Feces Bile

Day 1 Day 2 Days 3 + 4 Days 1–4 Day 1 Day 1

Biphenyl 0.1 0.1 NDb 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

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

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

Following intraperitoneal (i.p.) injection of [

14 4 C]-labeled biphenyl (30 mg/kg), the pattern of 

5 percentages of radioactivity detected in urinary metabolites showed a relatively greater ability to 

6 produce 2-hydroxybiphenyl in mice than rats. In Sprague-Dawley rats, metabolites identified in 

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

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

9 3,4’-dihydroxybiphenyl (8.5%); 3,4-dihydroxybiphenyl (5.1%); 3-hydroxybiphenyl (1.8%); and 

10 2-hydroxybiphenyl (1.5%). In DBA/2Tex mice, major identified metabolites were: 4-hydroxy11 biphenyl (39.5%); 3,4-dihydroxybiphenyl (30.3%); 4,4’-dihydroxybiphenyl (10.2%); 

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

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

14 mice, these metabolites were detected at levels of 0.3%, 0.8%, and 0.7%, respectively (Halpaap15 Wood et al., 1981a). 

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

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

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

19 male F344 rats are more susceptible than females to the formation of urinary bladder calculi 
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1 (Ohnishi et al., 2001, 2000a, b). Urinary bladder calculi in males were predominantly composed 

2 of the insoluble potassium salt of 4-hydroxybiphenyl-O-sulphate, whereas the less frequently 

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

4 potassium sulphate, hydrolysis products of 4-hydroxybiphenyl-O-sulphate (Ohnishi et al., 2001, 

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 with Animal and Human Cells or Tissues

14 The metabolism of biphenyl in vitro has been investigated using tissues of human origin, 

15 resulting in evidence that the human metabolism of biphenyl is qualitatively similar 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 than in rats.

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

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

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

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

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

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

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

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

30 from rats had about 4 times the metabolic capacity of dog hepatocytes and about 20 times that of 

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

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

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

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

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

36 the ages of 49 and 76 years of age (each patient contributed only one tissue type, so that within37 patient organ comparisons were not made) and prepared 12,000 and 105,000 g supernatants to 

38 study sulphation of biphenyl metabolites, specifically 2-, 3-, and 4-hydroxybiphenyl. 
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1 Sulphotransferase activity for each of these substrates was detected in all tissues studied, 

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

3 the lowest in the brain. The Michaelis constant (Km) of sulphotransferase was dependent on the 

4 substrate, but not on tissue type, with Km varying over a 500-fold range. The highest values of 

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

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

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

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

9 biphenyl metabolites are conjugated to glucuronic acid or sulphate. Wiebkin et al. (1984, 1976) 

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

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

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

13 with the hepatocytes, it was hydroxylated to 4,4'-dihydroxybiphenyl. Pretreatment 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 sulphates. 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-Hydroxybiphenyl 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 gave 

22 rise to 4-hydroxybiphenyl.

23

24 3.3.2. Metabolic Pathways 

25 3.3.2.1. Description of Metabolic Scheme and 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 reported by Billings and McMahon 

29 (1978). Support for CYP metabolism of biphenyl was provided by Halpaap-Wood et al. 

30 (1981a, 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 pretreatment 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). 
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1 Figure 3-1 details combined evidence from the Halpaap-Wood et al. (1981a, b) and 

2 Meyer and Scheline (1976) studies on the metabolic pathways of biphenyl. While sulphates and 

3 glucuronides are formed on all three metabolic levels illustrated, only monosulphates and 

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

5 metabolites alone. Glucuronides at the dihydroxy and trihydroxy levels are additionally labeled 

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

7 identified. 
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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; question marks denote tentative metabolites 

5 (see text).

6 

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

8 

9 Figure 3-1. Schematic presentation of the metabolic pathways of biphenyl. 

10
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1 The metabolic scheme in Figure 3-1 does not include the possible redox cycling of 

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

3 cycling between phenylhydroquinone and phenylbenzoquinone leading to the generation of 

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

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

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

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

8 glucuronide or sulphate conjugates are readily detected in the urine of rats exposed to 

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

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

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

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

13 4-hydroxybiphenyl and 4,4’-dihydroxybiphenyl is the principal metabolic pathway for biphenyl 

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

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

mice exposed to i.p. doses of [14 16 C]-biphenyl (30 mg/kg), radioactivity in 2-hydroxybiphenyl and 

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

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

19

20 3.3.3. Regulation of Metabolism, Sites of Metabolism, and Relationships to Toxic Effects 

21 3.3.3.1. Evidence for Induction of Phase I and II Enzymes

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

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

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

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

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

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

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

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

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

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

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

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

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

35 3-methylcholanthrene induced activities for both 4- and 2-hydroxylation of biphenyl. Halpaap36 Wood et al. (1981a) reported that pretreatment of male Sprague-Dawley rats with 

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

38 known as 5,6-benzoflavone) enhanced the urinary excretion of 2-, 3-, and 4-hydroxybiphenyl, 
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1 3,4-dihydroxybiphenyl, and 3,4,4’-trihydroxybiphenyl following i.p. administration of 30 mg 

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

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

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

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

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

7 male Syrian golden hamsters induced biphenyl hydroxylation activities in freshly isolated 

8 pancreatic acinar cells or hepatocytes. From these observations and examination of patterns of 

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

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

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

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

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

14 hydroxylation of biphenyl or the formation of the dihydroxy- or trihydroxybiphenyl metabolites. 

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

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

17 metabolites to sulphate or glucuronic acid. Hepatocytes from rats (strain and sex were not noted) 

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

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

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

21 whereas hepatocytes from non-induced control rats produced predominant sulphate conjugates of 

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

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

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

25 activities of freshly isolated pancreatic acinar cells to conjugate 4-hydroxybiphenyl with sulphate 

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

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

28 diphosphate glucuronosyl transferase (UGT) activities with 1-naphthol or 3-hydroxy29 benzo[a]pyrene as substrates were higher in liver microsomes from male Wistar rats pretreated 

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

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

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

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

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

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

36 β-naphthoflavone showed decreased rates of glucuronidation of 4-hydroxybiphenyl, compared 

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

38 this database provide equivocal evidence that the induction of Phase I enzymes catalyzing the 
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1 hydroxylation of biphenyl may be coordinated with induction of Phase II enzymes catalyzing 

2 glucuronidation of hydroxylated biphenyl metabolites. 

3 

4 3.3.3.2. Demonstrated Tissue Sites of Metabolism

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

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

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

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

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

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

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

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

13 Activities for enzymes catalyzing the conjugation of hydroxybiphenyls and other 

14 hydroxylated aromatic compounds with glucuronic acid or sulphate have been detected in a 

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

16 (Parkinson and Ogilvie, 2008). Available data for conjugation activities with hydroxybiphenyls 

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

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

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

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

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

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

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

24

25 3.3.3.3. Possible Relationships Between Metabolites and Toxic Effects

26 Increased formation of urinary tract crystals and calculi in F344 rats chronically exposure 

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

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

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

30 the potassium salt of the 4-hydroxybiphenyl sulphate conjugate, high urine potassium 

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

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

33 the diet (4,500 ppm) for 2 years (Umeda et al., 2002). Gender differences in calculi composition 

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

35 hydroxybiphenyl-O-sulphate and calculi in female rats composed mainly of 4-hydroxybiphenyl 

36 and potassium sulphate, presumably produced by the hydrolysis of 4-hydroxybiphenyl-O37 sulphate in the urine (Ohnishi et al., 2000a, b). As discussed earlier, these observations are 

38 consistent with the hypothesis that gender differences in urinary conditions (higher urine 
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1 potassium concentrations and pH) may be responsible for the gender differences in urinary 

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

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

4 

5 3.4. ELIMINATION

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

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

8 eliminated from the body, principally as conjugated hydroxylated metabolites in the urine. 

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

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

24 hours after the oral administration of 100 mg/kg [14 11 C]-labeled biphenyl in soy oil to male 

12 albino rats contained 75.8% of the administered radioactivity, compared with 5.8% detected in 

13 feces collected in the same period. Ninety-six hours after dose administration, <1% of the 

14 administered radioactivity remained in tissues, 84.8% was in collected urine, 7.3% was in feces,

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

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

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

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

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

20 conjugated metabolites accounted for 5.2% of the administered dose; in contrast, conjugated 

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

22 1976). 

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

24 is provided by the results of other studies, which analyzed biphenyl and biphenyl metabolites by 

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

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

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

28 metabolites accounted for averages of 25.4% of the administered dose in rabbits, 31.3% in 

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

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

31 `collected for 24 hours from bile-cannulated guinea pigs accounted for 3.3% of the administered 

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

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

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

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

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

37 following inhalation or dermal exposure to biphenyl. 

38
18 DRAFT - DO NOT CITE OR QUOTE

1 3.5. PHYSIOLOGICALLY BASED PHARMACOKINETIC (PBPK) MODELS

2 No studies were located on the development of PBPK models for biphenyl in animals or 

3 humans. 

4 
1 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 and 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/m3 (Table 4-1). In 1969, a 32-year-old worker at the 

17 facility, who had worked for 11 years 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

20 kidneys, degeneration of the heart muscles, hyperactive bone marrow, and edematous changes in 

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

air (January 1970) resulted in estimated average concentrations ranging from 0.6 to 123 mg/m3 22

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

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

value (TLV) of 0.2 ppm (1.3 mg/m3 25 ). In the location where biphenyl was mixed with paraffin oil 

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

27 dermal and inhalation exposures. 

28
20 DRAFT - DO NOT CITE OR QUOTE

Table 4-1. Biphenyl concentrations in the air of a Finnish paper mill 

producing biphenyl-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 biphenyl to mixing container No data 74.5

Source: Häkkinen et al. (1973).

1 

2 Thirty-one male workers at the Finnish facility were engaged in the biphenyl3 impregnation process; two other workers (one male stock keeper and one female paper cutter) 

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

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

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

7 elevated serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT) (which 

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

9 1973). Eight of the 33 workers were admitted to the hospital for further examination, including 

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

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

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

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

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

15 Exposure to biphenyl was terminated immediately following the initial neurophysiological 

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

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

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

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

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

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

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

23 reexamined 1 year later. Exceptions included additional diffuse slow wave abnormalities in the 

24 two subjects initially exhibiting only spike and wave discharges and the disappearance of the one 
21 DRAFT - DO NOT CITE OR QUOTE

1 case of mild temporal local abnormality. There was no discernable improvement in the EEGs of 

2 the seven subjects reexamined after 2 years.

3 ENMG results. As shown in Table 4-2, the 24 biphenyl-exposed workers exhibited no 

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

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

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

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

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

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

10 respectively) compared to the initial measurements. Abnormal EMGs among the biphenyl11 exposed workers included diminished numbers of motor units on maximal muscle contraction 

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

13 typically displayed slowing of some nerve conduction velocities as well. Of those 11 subjects 

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

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

16 end of 2 years, three of seven subjects displayed diminished ENMG abnormalities, three of seven 

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

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

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

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

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

23 Only five subjects (four men and the only woman in the biphenyl-exposed group) were found to 
22 DRAFT - DO NOT CITE OR QUOTE

1 have completely normal neurophysiological records. The authors interpreted their data to 

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

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

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

5 incapacity and the incidence of subjective symptoms. 

6 

7 Carella and Bettolo, 1994 

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

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

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

11 neutrophilic leukocytosis, and clinical chemistry findings indicative of hepatic perturbation. For 

12 example, the activities of liver-specific enzymes in serum were 62 mU/mL for AST, 90 mU/mL 

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

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

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

16 indicative of chronic hepatitis.

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

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

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

20 occupational exposure to biphenyl may have been the principal etiological factor. It is possible 

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

22 dermal, with the latter route predominating.

23

24 Wastensson et al., 2006 

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

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

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

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

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

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

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

biphenyl in air would be greater than the existing TLV of 1.3 mg/m3 32 (0.2 ppm) based on 

33 measurements at a Finnish paper mill with similar production practices (Häkkinen et al., 1973). 

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

35 created the paraffin oil/biphenyl mixture.

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

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

38 found among the decedents, compared with 4.3 cases of PD expected from data on the lifetime 
23 DRAFT - DO NOT CITE OR QUOTE

1 risk of developing PD in the general population. This comparison yielded an RR of 2.1, with a 

2 95% confidence interval of 0.96–4.0. 

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

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

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

6 was a 53-year-old male who had diabetes mellitus and withdrew from the study before his 

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

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

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

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

11 unable to draw any firm conclusions from their limited data. 

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 exposurea 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 levodopab + + + + +

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 BDF1 mice (Umeda 

18 et 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, 

20 F344 rats at the highest tested dietary concentration, 4,500 ppm, but were not found at lower 
24 DRAFT - DO NOT CITE OR QUOTE

1 exposure levels of 1,500 or 500 ppm. Non-neoplastic kidney lesions (simple transitional cell 

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

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

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

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

6 Chemical Co., 1953). In BDF1 mice, increased incidence of liver tumors (hepatocellular 

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

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

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

10 responses or noncancer adverse effects were found in female ddY mice exposed to 5,000 ppm 

11 biphenyl in the diet for 2 years (Imai et al., 1983) or in B6C3F1 and B6AKF1 mice exposed to 

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

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

14 toxicity studies, respiratory tract irritation and increased mortality following exposure to dusts of 

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

5 mg/m3 and in rats exposed to 300 mg/m3, but not in rabbits exposed to 300 mg/m3 16 (Deichmann 

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

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

vapors at 25 or 50 ppm (158 or 315 mg/m3 19 ) for 13 weeks (Sun Company Inc., 1977b). 

20 Detailed study descriptions for all available subchronic and chronic toxicity and 

21 carcinogenicity studies follow.

22

23 4.2.1. Oral Exposure 

24 4.2.1.1. Subchronic Toxicity

25 Dow Chemical Co., 1953 

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

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

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

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

30 voluntary muscle, lung, thyroid, and pituitary from each rat were preserved in formalin. 

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

32 were examined pathologically. 

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

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

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

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

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

38 exhibited lower average plasma blood urea nitrogen (BUN) levels than controls (28.2, 25.7, and 
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1 26.3 mg percent for low-, mid-, and high-dose groups, respectively, compared to 35.3 mg percent 

2 for controls), although the statistical significance of these apparent treatment-related differences 

3 was not reported and the biological significance is uncertain. 

4 

5 Umeda et al., 2004 

6 Six-week-old BDF1 mice (10/sex/group) were exposed to biphenyl at dietary 

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

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

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

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

11 first week, 8,000 ppm for the second week, and 16,000 ppm for the remaining 11 weeks. 

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

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

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

15 and one 16,000 ppm female.

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

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

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

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

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

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

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

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

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

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

26 16,000 ppm female mice revealed enlarged centrilobular hepatocytes, the cytoplasm of which 

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

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

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

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

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

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

33

34 4.2.1.2. Chronic Toxicity and Carcinogenicity

35 4.2.1.2.1. Chronic rat studies

36 Umeda et al., 2002 

37 In a chronic toxicity and carcinogenicity study of F344 rats (50/sex/group), biphenyl was 

38 administered in the diet for 2 years at concentrations of 0, 500, 1,500, or 4,500 ppm. All animals 
26 DRAFT - DO NOT CITE OR QUOTE

1 were examined daily for clinical signs; body weights and food intake were determined once a 

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

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

4 major organs and tissues were subjected to histopathologic examination. 

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

6 not include food consumption data. Estimated doses, therefore, were calculated using time7 weighted average (TWA) body weights from the graphically-depicted data (Figure 1 of Umeda 

8 et al., 2002) and U.S. EPA (1988) chronic reference values for food consumption in F344 rats. 

9 The resulting estimated doses for the 500, 1,500, and 4,500 ppm exposure groups were 36.4, 110, 

10 and 378 mg/kg-day, respectively, for males and 42.7, 128, and 438 mg/kg-day, respectively, for 

11 females. The study authors reported significantly lower mean body weights among 4,500 ppm 

12 rats of both sexes compared to their respective controls. Mean body weights of 4,500 ppm male 

13 and female rats were lower than those of controls throughout most of the study period and were 

14 approximately 20% lower than respective controls at terminal sacrifice. There was no significant 

15 effect on mean body weights of 500 or 1,500 ppm males or females. Survival of low- and mid16 dose male and female rats was not significantly different from controls. The study authors 

17 reported that 3/50 of the 4,500 ppm female rats died after 13–26 weeks of biphenyl exposure and 

18 attributed the deaths to marked mineralization of the kidneys and heart. However, they also 

19 indicated that survival of this group was not adversely affected thereafter. Significantly 

20 decreased survival was noted only for the group of 4,500 ppm male rats, 19/50 of which died 

21 prior to terminal sacrifice. The first death occurred around treatment week 36; this rat exhibited 

22 urinary bladder calculi. Survival data for the other groups were not provided. Evidence of 

23 hematuria was first noted in 4,500 ppm male rats around week 40 and was observed in a total of 

24 32/50 of the 4,500 ppm males during the remainder of the treatment period; 14 of these rats 

25 appeared anemic. Hematuria and bladder tumors were primarily considered as causes of death 

26 among the 4,500 ppm males (n = 19) that died prior to terminal sacrifice. Urinalysis performed 

27 during the final treatment week revealed significantly increased urinary pH in the 31 remaining 

28 4,500 ppm male rats (pH of 7.97 vs. 7.66 for controls; p < 0.05); occult blood was noted in the 

29 urine of 23 of these males. Urine samples in 10/37 surviving 4,500 ppm females tested positive 

30 for occult blood. Significant increases in relative kidney weights of 4,500 ppm males and 

31 females and absolute kidney weights of 4,500 ppm males were reported, but actual data were not 

32 presented.

33 Gross pathologic examinations at premature death or terminal sacrifice revealed the 

34 presence of calculi in the bladder of 43/50 of the 4,500 ppm males and 8/50 of the 4,500 ppm 

35 females (Table 4-4); these lesions were not seen in 500 or 1,500 ppm male or female rats. The 

36 bladder calculi in the male rats were white, yellow, brown, gray, and black in color, ranged from 

37 0.3 to 1.0 cm in size, and exhibited triangular, pyramidal, cuboidal, and spherical shapes. The 

38 bladder calculi in the female rats were white and yellow in color, of uniform spheroidal shape, 
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1 and similar in size to those of the male rats. Forty-one of the 4,500 ppm male rats exhibited 

2 polyp-like or papillary nodules protruding into the lumen from the bladder wall; bladder calculi 

3 were noted in 38 of these males. Four of the eight calculi-bearing 4,500 ppm female rats also 

4 exhibited thickening of the bladder wall. It was noted that 30/32 of the 4,500 ppm male rats with 

5 hematuria also exhibited kidney or urinary bladder calculi. 

6 

Table 4-4. Incidences of urinary bladder 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 hyperplasiaa 0 0 0 12b 0 0 1 1

Nodular hyperplasiaa 0 0 0 40b 1 0 0 5c

Papillary hyperplasiaa 0 0 0 17b 0 0 0 4

Combined 0 0 0 45 1 0 1 10b

Papilloma 0 0 0 10b 0 0 0 0

Carcinoma 0 0 0 24b 0 0 0 0

Papilloma or carcinoma

(combined)

0 0 0 31b 0 0 0 0

Squamous cell

Metaplasiaa 0 0 0 19b 0 0 0 4

Hyperplasiaa 0 0 0 13b 0 0 0 1

Papilloma or carcinoma

(combined)

0 0 0 1 0 0 0 0

Inflammatory polypa 0 0 0 10b 0 0 0 0

Calculi 0 0 0 43b 0 0 0 8b

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 Fisher’s exact test.

Source: Umeda et al. (2002).

7 

8 Histopathologic examinations at death or terminal sacrifice revealed no indications of 

9 biphenyl-induced tumors or tumor-related lesions in organs or tissues other than those associated 

10 with the urinary tract. As shown in Table 4-4, neoplastic and nonneoplastic lesions of the 

11 urinary bladder were essentially limited to the 4,500 ppm rats and predominantly the males. 

12 Only 4,500 ppm male rats exhibited papilloma (10/50) or carcinoma (24/50) of transitional cell 

13 epithelium, three of which exhibited both papilloma and carcinoma. Most of the transitional cell 

14 carcinomas (20/24) projected into the lumen, and the tumor cells invaded the entire body wall. 

15 Bladder calculi were found in all 24 males with transitional cell carcinoma and 8/10 of the males 
28 DRAFT - DO NOT CITE OR QUOTE

1 with transitional cell papilloma. Among noncancerous responses in the bladder, simple, nodular, 

2 and papillary hyperplasias were evident in 4,500 ppm animals. These hyperplasias developed in 

3 the focal area of the bladder epithelium. Simple hyperplasia occurred less frequently than 

4 nodular and papillary hyperplasias; furthermore, simple hyperplasia was almost always 

5 accompanied by either nodular or papillary hyperplasia in the 4,500 ppm males. Ten of the 

6 4,500 ppm males had polyps in the bladder epithelium, which were composed of spindle fibers 

7 proliferated around transitional epithelial cells accompanied by inflammatory infiltration of 

8 submucosal bladder epithelium. Squamous metaplasia was noted on the surface of the polyps, 

9 which were found at different loci than the bladder tumors. 

10 Table 4-5 summarizes the incidences of lesions of the ureter and kidney in the male and 

11 female rats. The incidence of simple transitional cell hyperplasia in the ureter was greater in the 

12 4,500 ppm males than the 4,500 ppm females. Other responses, such as mineralization of the 

13 corticomedullary junction, were increased over controls to a greater extent in males compared to 

14 females. In the renal pelvis, simple and nodular hyperplasia was frequently observed in 

15 4,500 ppm males and 500 and 1,500 ppm females. Responses such as papillary necrosis, infarct, 

16 and hemosiderin deposition occurred predominantly in exposed females. 

17
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Table 4-5. Incidences of ureter and kidney 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

Response

Ureter

Transitional cell hyperplasia

Simple hyperplasia 1 0 0 8a 0 0 0 2

Nodular hyperplasia 0 0 0 1 0 0 0 0

Dilatation 0 0 0 14a 0 0 0 6b

Kidney

Renal pelvis

Transitional cell hyperplasia

Simple hyperplasia 6 8 5 19c 3 5 12c 25a

Nodular hyperplasia 0 1 1 21a 0 0 1 12a

Squamous metaplasia 0 0 0 2 0 0 0 0

Mineralization 9 6 10 18b 12 12 18 27a

Desquamation 1 0 0 11a 0 0 0 2 

Calculi 0 0 0 13a 0 0 0 3

Other

Mineralization of 

corticomedullary junction 0 0 0 10a 21 2 26 18

Mineralization of papilla 9 9 14 23c 2 6 3 12a

Papillary necrosis 0 0 0 7d 0 0 0 23a

Infarct 0 0 0 0 1 0 0 8c

Hemosiderin deposits 0 0 0 0 4 8 22a 25a

Chronic nephropathy 45 45 43 34 33 35 30 26

a

Significantly different from control group (p < 0.01) 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 < 0.05) 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 

2 In summary, the chronic toxicity and carcinogenicity study of male and female F344 rats 

3 administered biphenyl in the diet for 2 years (Umeda et al., 2002) provides evidence for 

4 biphenyl-induced bladder tumors in males, but not females, based on the development of 

5 transitional cell papillomas and carcinomas in the 4,500 ppm (438 mg/kg-day) males (Table 4-4). 

6 This study identified a no-observed-adverse-effect level (NOAEL) of 500 ppm (42.7 mg/kg-day) 

7 and a lowest-observed-adverse-effect level (LOAEL) of 1,500 ppm (128 mg/kg-day) for 

8 nonneoplastic kidney lesions (simple transitional cell hyperplasia in the renal pelvis and 

9 hemosiderin deposits) in female F344 rats exposed to biphenyl in the diet for 2 years.
30 DRAFT - DO NOT CITE OR QUOTE

1 

2 Shiraiwa et al., 1989 

3 The chronic toxicity of biphenyl was assessed in Wistar rats (50/sex/group) administered 

4 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 

5 were observed daily for clinical signs. Body weight and food consumption were measured 

6 weekly. At death or scheduled sacrifice, gross pathologic examinations were performed and all 

7 organs were removed and preserved. Other than body weight and compound consumption data, 

8 the published results of this study were limited to kidney weight data and urolithiasis findings. 

9 Based on reported values for mean daily biphenyl intake (mg biphenyl/rat) and mean initial and 

10 final body weights for each study group, doses of biphenyl at the 0.25 and 0.5% dietary levels 

11 are estimated to have been 165 and 353 mg/kg-day for males, respectively, and 178 and 

12 370 mg/kg-day for females, respectively. Mean final body weights in both 2,500 and 5,000 ppm 

13 groups of biphenyl-exposed male and female rats were significantly lower (approximately 15 and 

14 25% lower; p < 0.01) than their respective controls. Absolute and relative kidney weights of 

15 control and biphenyl-exposed rats were similar, with the exception of significantly increased 

16 (p < 0.001) mean relative kidney weight in 2,500 ppm female rats. The study authors reported 

17 the occurrence of hematuria (in both the 2,500 and 5,000 ppm groups) as early as week 16 and 

18 stated that it was more recognizable at 60 weeks. Kidney stone formation was reported in 

19 6/46 and 1/43 of the 2,500 ppm males and females, respectively, and in 19/47 and 20/39 of the 

20 5,000 ppm males and females, respectively. Detection of stones in other regions of the urinary 

21 tract was essentially limited to the 5,000 ppm groups and included the ureter (2/47 males and 

22 2/39 females) and urinary bladder (13/47 males and 6/39 females). Kidney stones were hard, 

23 black, and located from the pelvic area to the medullary region. Stones in the ureter were hard, 

24 black, and composed of protein. Stones in the urinary bladder were hard, yellowish-white, round 

25 to oval in shape, and composed of ammonium magnesium phosphate. Histologically, kidneys 

26 with stones exhibited obstructive pyelonephritis accompanied by hemorrhage, lymphocytic 

27 infiltration, tubular atrophy, cystic changes of tubules, and fibrosis. Urinary bladders with stones 

28 exhibited simple or diffuse hyperplasia and papillomatosis of the mucosa; however, neoplastic 

29 lesions were not seen. No control rats (44 males and 43 females) showed stones in the kidney, 

30 ureter, or urinary bladder. The lowest exposure level in this study, 2,500 ppm in the diet for 

31 75 weeks, was a LOAEL for formation of kidney stones associated with pyelonephritis in Wistar 

32 rats (dose levels of 165 and 178 mg/kg-day for males and females, respectively). Urinary 

33 bladder stones associated with simple or diffuse hyperplasia and papillomatosis of the mucosa of 

34 the urinary bladder was observed at the highest exposure level, 5,000 ppm biphenyl in the diet 

35 (dose levels of 353 and 370 mg/kg-day for males and females, respectively). 

36 Shiraiwa et al. (1989) also reported the results of an initiation-promotion study in male 

37 Wistar rats (25/group) that included three groups administered a basal diet for 2 weeks followed 

38 by diets containing 0, 0.125, or 0.5% biphenyl (0, 1,250, or 5,000 ppm) for 34 weeks. Three 
31 DRAFT - DO NOT CITE OR QUOTE

1 other groups received diets containing 0.1% N-ethyl-N-hydroxyethylnitrosamine (EHEN, an 

2 initiator of kidney tumors in rats) for 2 weeks followed by diets containing 0, 0.125, or 0.5% 

3 biphenyl (0, 1,250, or 5,000 ppm) for 34 weeks. Initial and final body weights were recorded. 

4 At terminal sacrifice, gross pathologic examinations were performed. The study report included 

5 information regarding kidney weights, but did not indicate whether weights of other organs were 

6 measured. Kidney and urinary bladder were fixed; kidneys were sectioned transversely (10–

7 12 serial slices) and urinary bladders were cut into 4–6 serial slices. The authors used a 

8 computer-linked image analyzer to determine the incidence of kidney lesions and dysplastic foci. 

9 The presence of stones in the kidney and urinary bladder was assessed qualitatively using an 

10 infrared spectrophotometer. Based on reported values for mean daily biphenyl intake (mg 

11 biphenyl/rat) and average body weight (mean initial body weight + one-half the difference 

12 between mean initial and mean final body weight) for each study group, doses of biphenyl at the 

13 0.125 and 0.5% dietary levels are estimated to have been 59.28 and 248.3 mg/kg-day, 

14 respectively, for rats on basal diet alone for the first 2 weeks and 62.0 and 248.2 mg/kg-day, 

15 respectively, for rats receiving EHEN in the diet for the first 2 weeks. The mean final body 

16 weight of the rats receiving basal diet followed by diet containing 0.5% biphenyl was 

17 significantly lower (p < 0.001) than that of controls (0.389 ± 22 vs. 0.432 ± 30 kg). It was stated 

18 that relative kidney weights were increased this group of biphenyl-exposed rats compared to the 

19 basal diet control group, but the actual data were not presented. Stones were detected only in the 

20 rats receiving 0.5% biphenyl in the diet; incidences were 4/25 (kidney), 1/25 (ureter), and 

21 3/25 (urinary bladder) in rats that had received that basal diet for the first 2 weeks. Similar 

22 results regarding final body weight and the detection of stones in the urinary tract were reported 

23 for the rats that had received EHEN in the diet prior to the administration of biphenyl. 

24 Incidences of dysplastic foci and renal cell tumors were determined in the kidneys of all groups 

25 of rats. Only rats that had received EHEN during the initial 2 weeks exhibited neoplastic kidney 

26 lesions (dysplastic foci, renal cell tumors). For the EHEN + 0% biphenyl, EHEN + 0.125% 

27 biphenyl, and EHEN + 0.5% biphenyl groups, incidences of rats with dysplastic foci were 25/25, 

28 21/25, and 25/25, respectively, and incidences of rats with renal cell tumors were 13/25, 12/25, 

29 and 7/25, respectively. Under the conditions of this study, biphenyl did not exhibit tumor 

30 promoting characteristics for the kidney tumor initiator, EHEN.

31

32 Ambrose et al., 1960 

33 Weanling albino rats (15/sex/group) were administered biphenyl in the diet at 

34 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, 

35 5,000, or 10,000 ppm). Body weights were monitored every week during the period of active 

36 growth and then at 50-day intervals. Hemoglobin was monitored every 100 days in control and 

37 high-dose rats; at 500, 600, and 700 days in rats receiving 0.5% biphenyl, and at 500 and 

38 600 days in rats receiving 0.1% dietary biphenyl. A 98-day paired-feeding experiment was 
32 DRAFT - DO NOT CITE OR QUOTE

1 conducted in which control rats were provided the same amount of food that rats of the 0.5 and 

2 1.0% dietary biphenyl groups consumed to assess whether possible differences in growth would 

3 indicate a biphenyl exposure-related toxicological response or decreased palatability. At 

4 necropsy, the weights of liver, kidneys, heart, and testes were determined for all groups except 

5 those receiving 1.0% biphenyl in the diet. Stained sections of heart, lung, liver, kidney, adrenal, 

6 spleen, pancreas, stomach, intestine, bladder, thyroid, brain, pituitary, and gonads were prepared 

7 for histopathologic examinations. In some cases, bone marrow smears were prepared.

8 The study report of Ambrose et al. (1960) did not include sufficient information from 

9 which daily biphenyl doses could be calculated. Biphenyl doses are estimated at 1, 4, 8, 42, 84, 

10 420, 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%, 

11 respectively, based on U.S. EPA (1988) reference values for body weight and food consumption 

12 in F344 rats (averages of values for males and females). There is greater uncertainty in the dose 

13 estimates at the two highest exposure levels because the magnitude of reported decreased food 

14 consumption in these groups was not specified in the study report. Decreased longevity was 

15 apparent in male and female rats of the 0.5 and 1.0% biphenyl exposure groups, but was not 

16 evident at lower exposure levels. Growth rates appeared similar among controls and groups 

17 exposed to biphenyl levels ≤0.1%. At the two highest exposure levels, markedly decreased 

18 growth was evident, but was attributable to decreased food consumption and indicative of 

19 decreased palatability based on results of the paired-feeding experiment. Decreased hemoglobin 

20 levels were reported in male and female rats of the two highest exposure levels after 300–

21 400 and 500–600 days, respectively, but were considered at least partially related to lower food 

22 consumption in these groups relative to controls. Selected organ weights are summarized in 

23 Table 4-6. There were no statistically significant treatment-related effects on organ weights at 

24 dietary levels ≤0.1%, which were below those associated with decreases in food consumption, 

25 body weight, and survival (i.e., 0.5 and 1.0%). Relative liver and kidney weights of female rats 

26 of the 0.5% biphenyl exposure group were significantly (p < 0.05) increased, approximately 

27 45 and 215% higher than those of respective controls. The only significant compound-related 

28 histopathological change occurred in the kidneys, which, in all members of the two highest 

29 exposure groups, showed irregular scarring, lymphocytic infiltration, tubular atrophy, and tubular 

30 dilation associated with cyst formation. Some evidence of hemorrhage was present, and calculi 

31 were frequently noted in the renal pelvis. Evidence of metaplasia in the epithelium of the renal 

32 pelvis did not implicate neoplastic activity, and, taking the histopathological results as a whole, 

33 there appeared to be no clear-cut, compound-related tumor development. However, the small 

34 number of animals in each group and the decreased survival in the two highest dose groups may 

35 have impaired the ability to detect late-developing tumors. The study identified 1,000 ppm 

36 biphenyl in the diet (84 mg/kg-day) as a NOAEL and 5,000 ppm (420 mg/kg-day) as the LOAEL 

37 for kidney effects including tubular atrophy and dilation associated with cyst formation and 

38 calculi formation in the renal pelvis of albino rats of both sexes. 
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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 weight (g) ± SE

Liver Kidneys Heart Testes

Males

0.0 745 9 396 ± 24.6 2.89 ± 0.16 0.75 ± 0.02 0.32 ± 0.015 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 ± 0.01 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 ± 0.20a 1.39 ± 0.14a 0.46 ± 0.04 NA

a

Significantly 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 

4 Male albino rats (8/group; strain not stated) were given biphenyl in the diet for up to 

5 13 months at concentrations resulting in estimated doses of 250 or 450 mg/kg-day. Upon 

6 sacrifice, liver, kidney, spleen, heart, lung, thyroid, parathyroid, adrenal, pancreas, testis, 

7 stomach, and intestine were processed for histopathological examination. At 2-month interim 

8 sacrifices, moderate degenerative changes in liver and kidney were observed at both dose levels. 

9 Liver effects consisted of moderate degeneration and hypertrophy of the Kupffer cells with a 

10 generally well-preserved structure. Renal glomeruli were undamaged, but tubuli showed mild 

11 signs of degeneration. The liver and kidney effects did not appear to increase in severity in rats 

12 treated for up to 13 months. Other histopathologic effects noted in the biphenyl-treated rats 

13 included hypertrophied splenic reticular cells, small follicles with sparse colloid and 

14 desquamation of follicular epithelium in the thyroid, and hyperplastic and hyperkeratinized 

15 forestomach epithelium with occasional desquamation. Although the study report did not 

16 include tumor incidence data for the two dose groups, the study authors reported neoplastic 

17 lesions in the forestomach of three biphenyl-treated rats. Two of the rats exhibited papillomas of 
34 DRAFT - DO NOT CITE OR QUOTE

1 the forestomach epithelium (one after 7 weeks and one after 7 months of treatment); a squamous 

2 cell carcinoma was diagnosed in the other rat after 1 year of treatment. The study authors noted 

3 two sequential responses to chronic biphenyl exposure: degenerative changes of nuclei and 

4 cytoplasm in the parenchyma of liver and kidney, spleen, thyroid, and adrenals within 2 months 

5 followed within 1 month or more by functional-regenerative changes that resulted in hyperplasia 

6 and nuclear hypertrophy of liver and kidney parenchyma as well as functional hyperactivity of 

7 the thyroid and parathyroid. Signs of cirrhosis were not seen, but irritation and hyperplasia were 

8 evident in the lower urinary tract. The lowest dose, 250 mg/kg-day biphenyl, was an apparent 

9 LOAEL for nonneoplastic degenerative changes in the liver, kidney, thyroid, and parathyroid of 

10 male albino rats resulting in hyperplasia of liver, kidney, and thyroid. 

11

12 Dow Chemical Co., 1953 

13 Sprague-Dawley rats (12/sex/group) were exposed to biphenyl in the diet for 2 years at 

14 exposure levels of 0, 0.01, 0.1, or 1% (0, 100, 1,000, or 10,000 ppm). Body weights were 

15 monitored twice weekly for 3 months, then weekly. Blood samples were taken from all animals 

16 at the start of the experiment, approximately every 3 months thereafter, and at term. Hemoglobin 

17 levels, red and white blood cell counts and differential cell counts, and BUN concentrations were 

18 recorded. At death or scheduled necropsy, organ weights were recorded for liver, lung, kidneys, 

19 heart, and spleen. Sections from heart, liver, kidney, spleen, adrenals, pancreas, gonads, 

20 stomach, small and large intestine, voluntary muscle, lung, bladder, and brain were fixed and 

21 stained for histopathologic examination. 

22 Based on U.S. EPA (1988) chronic reference values for body weight and food 

23 consumption in Sprague-Dawley rats (average values for combined sexes), doses of biphenyl for 

24 the dietary levels of 0.01, 0.1, and 1% are estimated to have been 7, 73, and 732 mg/kg-day, 

25 respectively. It is unclear to what extent the data in the study were compromised by an outbreak 

26 of pneumonia that affected the colony during the course of the experiment. Survival was poor in 

27 control males, all of which had died by 18 months. Only two of the females receiving 0.1% 

biphenyl in the diet survived to the end of the 21st 28 month, and none had survived by the end of 

the 23rd 29 month. However, the authors considered the decreased survival in this group of females 

30 to have been compound-related. Striking biphenyl concentration-related reductions in body 

31 weight gain were observed among the groups, although, in monitoring food efficiency, the 

32 authors indicated that the reduced growth was likely due to a lower daily consumption of food 

33 rather than to the toxicological consequences of biphenyl. There were no clear indications of 

34 exposure-related changes in hematological parameters, but the authors reported significant 

35 (p < 0.05) increases in average (combined sexes) relative liver and kidney weights at the highest 

36 exposure level, compared with control values (4.71 vs. 3.05 g/100 g and 1.68 vs. 1.00 g/100 g, 

37 respectively). Histopathologic examinations revealed dilatation of the kidney tubules, an effect 

38 that appeared to be associated with secondary inflammation, uremia, disruption of the filtration 
35 DRAFT - DO NOT CITE OR QUOTE

1 system, and an increase in BUN in affected animals. Since tubular dilatation was evident in 

2 controls as well as treated animals, the authors presented their data on a semiquantitative severity 

3 scale (0–4) in which 0 = no observed changes, 1 = tissue changes in occasional isolated areas, 

4 2 = tissue changes in multiple areas, 3 = tissue changes in numerous areas, and 4 = extensive 

5 tissue changes involving all or almost all areas. Among the controls, low-, mid-, and high-dose 

6 rats, respective incidences for tubular dilatation with severity scores ≥2 were 1/12, 6/12, 7/12, 

7 and 11/12 for males and 1/12, 3/12, 4/12, and 11/12 for females. Respectively, incidences for 

8 tubular dilatation with severity scores ≥3 were 0/12, 1/12, 2/12, and 9/12 for males and 1/12, 

9 2/12, 2/12, and 11/12 for females. Severity scores ≥3 for tubular dilatation are considered to 

10 represent adverse renal effects. Calcification and intratubular inflammation were frequently 

11 observed at the highest biphenyl exposure level. The incidence data for renal tubular dilatation 

12 with a severity score ≥3 indicate that 100 ppm biphenyl in the diet (73 mg/kg-day) was a 

13 NOAEL and that 1,000 ppm (732 mg/kg-day) was a LOAEL for renal effects in Sprague-Dawley 

14 rats. The small number of rats in the exposure groups and the decreased survival at the highest 

15 exposure level may have impaired the ability to detect late-developing tumors in this study. 

16

17 4.2.1.2.2. Chronic mouse studies

18 Umeda et al., 2005 

19 In a chronic toxicity and carcinogenicity study of BDF1 mice (50/sex/group), biphenyl 

20 was administered in the diet for 2 years at concentrations of 0, 667, 2,000 or 6,000 ppm. All 

21 animals were observed daily for clinical signs and mortality. Body weights and food 

22 consumption were recorded weekly for the first 14 weeks and every 4 weeks thereafter. 

23 Hematological and clinical chemistry parameters were measured in blood samples drawn from 

24 all 2-year survivors just prior to terminal sacrifice. At death or terminal sacrifice, gross 

25 pathological examinations were performed and organs were removed and weighed. Specific 

26 tissues prepared for microscopic examination were not listed in the study report, but included 

27 liver and kidney. 

28 There were no overt clinical signs or effects on food consumption or survival among 

29 biphenyl-exposed mice of either sex compared to respective controls. However, mean terminal 

30 body weights of 2,000 and 6,000 ppm mice of both sexes were significantly less than those of 

31 respective controls (Table 4-7). Based on body weight and food consumption data, the study 

32 authors estimated that the 667, 2,000, and 6,000 ppm dietary levels resulted in average daily 

33 biphenyl doses of 97, 291, and 1,050 mg/kg-day in the males and 134, 414, and 1,420 mg/kg-day 

34 in the females (Table 4-5). 

35
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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 ± 6.0a 5.5 291

6,000 39/50 32.4 ± 3.6b 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 ± 3.1b 5.9 414

6,000 32/49 25.5 ± 3.0b 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 

2 Although there were no compound-related changes in hematological parameters, some 

3 clinical chemistry parameters showed marked changes in relation to dose, including a biphenyl 

4 dose-related increase in BUN that achieved statistical significance in 6,000 ppm males and 

5 females and 2,000 ppm males. Particularly striking were dose-related increases in activities of 

6 the plasma enzymes AP, lactate dehydrogenase (LDH), glutamate oxaloacetate transaminase 

7 (GOT; also referred to as AST), and glutamate pyruvate transaminase (GPT; also referred to as 

8 ALT) in the female mice. These data are shown in Table 4-8 and are suggestive of biphenyl9 related hepatocellular disruption. Umeda et al. (2005) noted that females with malignant liver 

10 tumors exhibited extremely high AST, ALT, and LDH activities. Biphenyl effects on these 

11 parameters in males were less obvious, although AP activity was significantly greater than 

12 controls in 6,000 ppm males (261 ± 102 vs. 178 ± 111 IU/L) (Table 4-8). 

13
Table 4-8. Dose-related changes in selected clinical chemistry values from 

male and female BDF1 mice exposed to biphenyl via the diet 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 ± 102a

LDH (IU/L) 321 ± 230 252 ± 126 432 ± 868 283 ± 200

BUN (mg/dL) 20.2 ± 3.6 22.0 ± 4.0 23.2 ± 4.4b 22.9 ± 2.7a

Females

Biphenyl dietary 

concentration (ppm) 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 ± 373a 325 ± 448a

ALT (IU/L) 32 ± 18 56 ± 46 134 ± 231a 206 ± 280a

AP (IU/L) 242 ± 90 256 ± 121 428 ± 499 556 ± 228a

LDH (IU/L) 268 ± 98 461 ± 452 838 ± 2,000 1,416 ± 4,161b

BUN (mg/dL) 14.9 ± 2.0 14.8 ± 3.4 21.0 ± 20.5 23.8 ± 11.7a

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

3 increases in relative liver weights of 667, 2,000, and 6,000 ppm female mice, respectively (the 

4 data for liver weight group means and standard deviations [SDs] were not presented in Umeda et 

5 al. [2005]). Incidences of gross and histopathological findings are presented in Table 4-9. Gross 

6 pathologic examinations revealed biphenyl dose-related increased incidences of liver nodules in 

7 females, but not males. The nodules were round- or oval-shaped cystic or solid masses 

8 approximately 3–23 mm in diameter of the largest axis. Histopathological examinations 

9 revealed that 5, 16, and 19 of the nodule-bearing 667, 2,000, and 6,000 ppm female mice also 

10 exhibited proliferative lesions of hepatocellular origin. Significantly increased incidences of 

11 basophilic cell foci were observed in 2,000 and 6,000 ppm female mice. Although incidences of 

12 basophilic cell foci were significantly increased in 667 ppm male mice as well, a dose-related 

13 effect was not evident because incidences of this lesion were not significantly increased in 

14 2,000 or 6,000 ppm males compared to controls. Incidences of hepatocellular adenomas and 
38 DRAFT - DO NOT CITE OR QUOTE

1 combined incidences of hepatocellular adenomas or carcinomas were significantly increased in 

2 the 2,000 and 6,000 ppm females and Peto’s trend tests confirmed significant positive trends for 

3 dose-related increased incidences of hepatocellular adenomas (p < 0.05) and combined 

4 incidences of hepatocellular adenomas or carcinomas (p < 0.01). Incidences of hepatocellular 

5 carcinomas were significantly increased in 2,000 ppm females, but not 667 or 6,000 ppm 

6 females. However, Umeda et al. (2005) noted that the incidences of hepatocellular carcinomas 

7 (5/50 or 10%) in each of the 667 and 6,000 ppm groups of females exceeded a range of historical 

8 control data (26 hepatocellular carcinomas in 1,048 female mice [2.5% incidence]) at the 

9 laboratory where the study was conducted. No significant biphenyl exposure-related effects on 

10 liver tumor incidences were seen in male mice. Incidences of desquamation of the urothelium in 

11 the renal pelvis were increased in 6,000 ppm male and female mice. Incidences of 

12 mineralization in the inner stripe of the outer medulla of the kidney were significantly increased 

13 in the 2,000 and 6,000 ppm female mice.

14

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

female BDF1 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 12/50a 10/49a

Carcinoma 8/50 8/49 5/50 4/50 1/50 5/50 7/50a 5/49

Adenoma or carcinoma

(combined) 16/50 12/49 9/50 7/50 3/50 8/50 16/50b 14/49a

Basophilic cell foci 0/50 6/49b 1/50 2/50 1/50 1/50 12/50b 6/49a

Clear cell foci 0/50 6/49b 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 10/50b 4/50 0/50 0/50 15/49b

Mineralization inner stripe–

outer medulla 9/50 8/49 14/50 14/50 3/50 5/50 12/50a 26/49b

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 BDF1

17 mice administered biphenyl in the diet for 2 years (Umeda et al., 2005) provides evidence for 
39 DRAFT - DO NOT CITE OR QUOTE

1 biphenyl-induced liver tumors in females, but not males, based on significantly increased 

2 incidences of hepatocellular adenomas and combined carcinomas or adenomas in the female 

3 mice receiving biphenyl from the diet at 414 and 1,420 mg/kg-day (Table 4-9). This study 

4 identified a NOAEL of 134 mg/kg-day and a LOAEL of 414 mg/kg-day for nonneoplastic 

5 effects (mineralization in the kidney and significantly increased plasma ALT and AST activities) 

6 in female BDF1 mice exposed to biphenyl in the diet for 2 years.

7 

8 Imai et al., 1983 

9 Groups of female ddY mice were fed diets containing 0 (n = 37 mice) or 0.5% 

10 (n = 34 mice) biphenyl (5,000 ppm) in the diet for 2 years. This study also included groups 

11 exposed to dietary concentrations of 0.2% thiabendazole or a mixture of 0.25% biphenyl and 

12 0.1% thiabendazole (results from this part of the study are not further described herein). Food 

13 consumption, body weights, and survival were assessed at intervals throughout exposure. At 

14 terminal sacrifice, several organs were weighed and prepared for microscopic histology (brain, 

15 pituitary, thymus, liver, spleen, pancreas, lung, heart, adrenal, kidney, ovaries, and uterus); in 

16 addition, the thyroid, stomach, small intestine, and large intestine were prepared for histology 

17 only. Urine samples collected from 10 control and 9 treated mice at terminal sacrifice were 

18 analyzed for protein glucose, ketones, bilirubin, urobilogen, and pH. Blood samples collected at 

19 the terminal sacrifice from 12 control and 9 treated mice were assessed for hematological 

20 endpoints, and serum samples (n = 6 for control and treated groups) were also assessed for 

21 biochemical endpoints including GOT, GPT, AP, cholinesterase, glucose, albumin, and total 

22 protein. Based on U.S. EPA (1988) methodology for estimating food consumption rates from 

23 body weight data and the reported average terminal body weight for the 5,000 ppm mice 

24 (0.037 kg), an oral dose of 855 mg/kg-day is estimated from the dietary exposure. Exposure to 

25 biphenyl did not influence survival, food consumption, or growth compared with controls (as 

26 shown in Figures 1, 2, and 3 in Imai et al. [1983]). No marked exposure-related effects were 

27 found on terminal organ and body weights (Tables 5 and 6 in Imai et al. [1983]) or on the 

28 urinalytic, hematologic, or serum biochemical endpoints (Tables 2, 3, and 4 in Imai et al. 

29 [1983]). Histological examination revealed no increased incidence of non-neoplastic lesions in 

30 examined tissues in the 5,000 ppm biphenyl group, compared with the control group (Table 7 in 

31 Imai et al. [1983]). The only tissues showing tumors at elevated incidence in the 5,000 ppm 

32 mice, compared with the control group, were the lung (11/34 [32.4%] vs. 9/37 [24.3%] in 

33 controls) and lymphatic tissues (lymphomas: 5/34 [14.7%] vs. 4/37 [10.8%]; leukemia: 3/34 

34 [8.8%] vs. 2/37 [5.4%]), but these increases are not statistically significant (p > 0.05 by the 

35 Fisher’s exact test). In summary, exposure of female ddY mice to 5,000 ppm biphenyl in the diet 

36 for 2 years was a NOAEL for non-neoplastic lesions, survival, body and organ weight changes, 

37 and changes in urinalytic, hematologic, and serum chemistry endpoints. In contrast to the 2-year 

38 bioassay with BDF1 mice that found increased liver tumors in female mice exposed to dietary 
40 DRAFT - DO NOT CITE OR QUOTE

1 concentrations ≥2,000 ppm (Umeda et al., 2005), no carcinogenic response occurred in female 

2 ddY mice exposed to 5,000 ppm biphenyl in the diet (estimated dose of 855 mg/kg-day) for 

3 2 years (Imai et al., 1983). 

4 

5 Innes et al., 1969; NCI, 1968 

6 The carcinogenic potentials of 130 chemicals, including biphenyl, were assessed in a 

7 protocol that exposed groups of two strains of F1 hybrid mice (18/sex/strain/group), produced by 

8 mating female C57BL/6 mice to either male C3H/Anf mice (F1 designated as strain A) or male 

9 AKR mice (F1 designated as strain B) to individual chemicals by the oral route for 18 months. 

10 Four groups of untreated controls and a group of gelatin vehicle controls (18/sex/strain/group) 

11 were included in the study. In the case of biphenyl, the chemical was administered via gavage to 

12 mice for 3 weeks, starting at the age of 7 days at 215 mg biphenyl/kg body weight in 0.5% 

13 gelatin (the report of Innes et al. [1969] appears to have erroneously reported the gavage dose as 

14 2.5 mg/kg). Thereafter, and for the rest of the experimental period, biphenyl was mixed with 

15 chow to a final concentration of 517 ppm. The gavage dose level and food concentration of 

16 biphenyl were selected to reflect the maximum tolerated dose identified in preliminary range17 finding single-dose subcutaneous injection and single- and repeated-dose oral administration 

18 studies. Initial gavage dose and dietary levels of biphenyl were not adjusted for weight gain 

19 during the 18-month study. Based on U.S. EPA (1988) chronic reference values for body weight 

20 and food consumption in strain A mice (average values for combined sexes), an average oral 

21 dose of 91 mg/kg-day is estimated from the dietary exposure. Blood smears were prepared from 

22 mice that showed splenomegaly, liver enlargement, or lymph adenopathy at necropsy. At term, 

23 mice were examined for any gross pathological features. Major organs were processed for 

24 histopathologic examination (including total chest contents, liver, spleen, kidneys with adrenals, 

25 stomach, and genital organs). Innes et al. (1969) reported incidences for hepatomas, pulmonary 

26 tumors, and lymphomas in control mice (Table 5 of Innes et al., 1969) and for tested chemicals

27 that were judged to give “high tumor yield” (Table 7 of Innes et al., 1969); biphenyl was 

28 reported to be noncarcinogenic, but tumor incidence data for biphenyl were not reported. The 

29 NCI (1968) report included tabulated incidences of hepatomas, pulmonary tumors, and 

30 lymphomas in control mice and biphenyl-treated mice, which are summarized in Table 4-10. In 

31 summary, the results provide no evidence of a carcinogenic response to 18 months of oral 

32 exposure to biphenyl (215 mg/kg by gavage for 3 weeks, followed by dietary exposure to 

33 517 ppm biphenyl). 

34
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Table 4-10. Incidences of selected tumor types among controls and mice 

administered biphenyl orally for 18 months

Group

Incidences of selected tumor typesa

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 

4 Mongrel dogs (two males and one female/group) were administered 0, 2.5, or 25 mg/kg 

5 biphenyl in corn oil by capsule 5 days/week for 1 year. Dogs were examined daily for clinical 

6 signs and weighed weekly. Blood samples were withdrawn at 3-month intervals to measure such 

7 hematological parameters as hemoglobin, hematocrit, blood cell count, sedimentation rate, 

8 icterus index, bromosulphalein retention, and, among clinical chemistry parameters, BUN. Urine 

9 samples were obtained at similar intervals to measure specific gravity, sugar, protein, bile 

10 pigments, occult blood, and microscopic sediment. Samples of urine from the high-dose dogs 

11 were collected during week 18, pooled, and analyzed for the presence of biphenyl and 

12 metabolites. At termination, gross necropsies were performed, and sections of large and small 

13 intestine, pancreas, ovary or testis, adrenal, urinary bladder, stomach, lung, thyroid, brain, heart, 

14 spleen, and liver were prepared for histopathologic examination. Although slight fluctuations 

15 were seen in body weight during the study, the dogs generally exhibited a net weight gain. 

16 Fluctuations in hematological parameters and urine analysis were inconsistent and not 

17 considered compound-related. Gross pathological examination of the dogs showed no obviously 

18 compound-related effects. Histopathologic examinations revealed lung congestion consistent 

19 with bronchial pneumonia in one high-dose dog; histopathology was unremarkable for each of 

20 the other dogs in the study. 

21
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1 Dow Chemical Co., 1953 

2 Dow Chemical Co. (1953) described a biphenyl feeding experiment in which four groups 

3 of Rhesus monkeys (two males and one female/group) were exposed to 0, 0.01, 0.1, or 1% 

4 biphenyl in chow for 1 year, during which time most of the animals experienced ill health not 

5 related to biphenyl exposure. Despite this caveat, hematological parameters were normal. The 

6 authors considered an increase in relative liver weight in high-dose monkeys (4.65 g/100 g body 

7 weight vs. 3.90 g/100 g body weight in controls) to possibly be compound-related.

8 

9 4.2.2. Inhalation Studies 

10 Deichmann et al., 1947; Monsanto, 1946 

11 In three separate experiments, albino rabbits (sex and strain not stated), Sprague-Dawley 

12 rats (sex not stated), and mice (sex and strain not stated) were repeatedly exposed to dusts 

13 composed of 50% biphenyl attached to celite for 7 hours/day, 5 days/week. In the first 

experiment, 3 rabbits and 10 rats were exposed to an average concentration of 300 mg/m3 14 on 

15 each of 64 days over a period of 94 days. The rats exhibited irritation of the nasal mucosa 

16 accompanied by serosanguineous discharge. Five of the rats died prior to term, and the survivors 

17 lost weight. The rabbits exhibited no exposure-related adverse signs. In the second experiment, 

three rabbits and six rats were exposed to an average concentration of 40 mg/m3 18 on each of 

19 46 days over a total period of 68 days. One rat died prior to term. The surviving rats showed 

20 signs of mucous membrane irritation, but appeared to gain weight at a normal rate. The rabbits 

21 exhibited no exposure-related adverse signs. In the third experiment, 12 mice and 4 rats were 

exposed to an average concentration of 5 mg/m3 22 on each of 62 days over a total period of 

23 92 days. While the rats were unaffected at this concentration, all of the mice showed signs of 

24 irritation of the upper respiratory tract and two died prior to term. Bronchopulmonary lesions 

25 (including acute emphysema, congestion, edema, bronchitis, widespread lobular pneumonia, and 

26 multiple pulmonary abscesses) were reported in rats from experiments 1 and 2 and in mice of 

27 experiment 3. Some unspecified minor liver and kidney lesions were also noted. Based on the 

results of these three experiments, a LOAEL of 5 mg/m3 28 for upper respiratory tract irritation in 

29 the mice was identified.

30

31 Sun Company Inc., 1977b 

32 Groups of CD-1 mice (50/sex/group) were exposed to airborne biphenyl at vapor 

concentrations of 0, 25, or 50 ppm (0, 157.7, and 315.3 mg/m3 33 , respectively) for 7 hours/day, 

34 5 days/week for 13 weeks. Mice were maintained and exposed to biphenyl in groups of 5 (for a 

35 total of 10 groups/sex/exposure group). All animals were checked daily for clinical signs and 

36 mortality, and body weight data were collected. Upon completion of the 13-week exposure 

37 period, surviving mice were placed in metabolic cages for 12-hour collection of urine for 

38 urinalysis. Blood samples were collected for blood chemistry and hematology assessments. 
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1 Gross and histopathologic examinations were performed on all mice. Ten surviving 

2 mice/sex/group were held for a 30-day recovery period prior to terminal sacrifice.

3 During the first few days of biphenyl exposure, some of the test material crystallized in 

4 the delivery system; analysis of biphenyl exposure levels was not performed on these days. 

5 Daily measured biphenyl exposure concentrations were highly variable during the first half of 

6 the 13-week exposure period, whereas subsequently measured concentrations were closer to 

7 target concentrations. For example, during the first 45 exposure sessions, measured daily 

8 biphenyl concentrations in the 50 ppm target groups ranged from as low as 5 ppm to as high as 

9 102 ppm and subsequent measurements ranged from 48 to 55 ppm. Mean biphenyl 

10 concentrations (± 1 SD) calculated for the entire 13 weeks of exposure were 25 ± 7 and 

11 50 ± 16 ppm for the 25 and 50 ppm target groups, respectively. The authors reported the loss of 

12 46/100 of the 25 ppm mice due to overheating and cannibalization. Since the overheating event 

13 occurred after 46 exposures, the overall study duration ran for 117 days to ensure that 

14 replacement mice received a total of 65 exposures as called for in the protocol. The study report 

15 did not mention results of clinical observations, and mortality data were not specifically 

16 summarized. There were no clear indications of exposure-related effects on body weights. 

17 Results of urinalysis, hematology, and clinical chemistry did not indicate any clear exposure18 related changes that could be attributed to biphenyl toxicity. Gross and histopathological 

19 examinations revealed congested and hemorrhagic lungs, hyperplasia of the trachea with 

20 inflammation accompanied by a high incidence of pneumonia, and congestion and edema in liver 

21 and kidney of biphenyl-exposed mice (see Table 4-11). The pathologist considered the 

22 congestion in the lung, liver, and kidney a likely effect of the anesthetic used for killing the mice, 

23 although control mice did not exhibit these effects at 13-week sacrifice. The hemorrhagic lungs 

24 and tracheal hyperplasia were considered effects of biphenyl exposure. Results from the 30-day 

25 recovery groups suggest that the biphenyl exposure-related pulmonary effects were reversible. 

26 This study identified a LOAEL of 25 ppm for histopathologic lung, liver, and kidney lesions in 

27 male and female CD-1 mice exposed to biphenyl by inhalation for 7 hours/day, 5 days/week for 

28 13 weeks.
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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 groupsa

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 combined male and female mice only; no 

statistical analyses were conducted.

Source: Sun Company Inc. (1977b).

2 

3 4.3. REPRODUCTIVE/DEVELOPMENTAL STUDIES—ORAL AND INHALATION

4 4.3.1. Oral Exposure 

5 Khera et al., 1979 

6 Pregnant female Wistar rats (18–20 group) were gavaged with 0, 125, 250, 500, or 

7 1,000 mg/kg-day biphenyl in corn oil on gestation days (GDs) 6–15. Body weights of dams 

8 were recorded on GDs 1, 6–15, and 22, at which point all dams were sacrificed. Parameters 

9 evaluated at autopsy included the number of corpora lutea, fetal weights and viability, and early 

10 resorptions. Two-thirds of the live fetuses/litter were examined for skeletal development and the 

11 rest were examined for the presence of visceral abnormalities. Five of the 20 high-dose dams 

12 died prior to sacrifice. Doses ≤500 mg/kg-day produced no clinical signs of maternal toxicity or 

13 evidence of treatment-related effects on maternal weight gain. As shown in Table 4-12, a 

14 significantly increased number of dams without live fetuses was observed in the high-dose 

15 group, compared with controls. Mean numbers of corpora lutea and live fetuses in the high-dose 

16 dams were similar to those of controls and dams of all other dose levels. However, the percent 

17 of dead fetuses and resorption sites was clearly higher in the high-dose group, and the numbers 

18 of anomalous fetuses and litters bearing anomalous fetuses appeared to increase with increasing 

19 dose. Khera et al. (1979) noted that the slight increases in the number of fetuses with anomalies, 

20 such as missing and unossified sternebrae or delayed calvarial ossification, were not statistically 

21 significant, but, as shown in Table 4-12, the incidence of litters with any type of fetal anomalies 

22 (“anomalous litters/number examined”) was elevated (p < 0.05 by Fisher’s exact test) at 

23 500 mg/kg-day, but not at lower doses, compared with control incidences. This study identified 

24 a NOAEL of 500 mg/kg-day and a LOAEL of 1,000 mg/kg-day for frank maternal toxicity 

25 (increased mortality and decreased dams with live fetuses) and lethal fetal effects. For less 
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1 severe developmentally toxic effects (increased incidence of anomalous litters), 500 mg/kg-day 

2 was a LOAEL and 250 mg/kg-day was a NOAEL.

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 11/20a

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 13.7b

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 35/199c 25/107c

Anomalous litters/number examined 8/16 11/20 13/18 15/18c 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

Significantly (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 live fetuses and one dam with seven resorptions 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

7 groups of 4-month-old male and female Long Evans rats (three males and nine females/group) 

8 were fed diets containing 0, 0.01, 0.1, or 1.0% biphenyl. Based on U.S. EPA (1988) subchronic 

9 reference values for body weight and food consumption in male and female Long Evans rats, 

10 doses of biphenyl for the dietary levels of 0.01, 0.1, and 1.0% are estimated to have been 9, 89, 

11 and 887 mg/kg-day, respectively, for the males and 10, 101, and 1,006 mg/kg-day, respectively, 

12 for the females. Average cross-gender doses for males and females were 10, 95, and 947 mg/kg13 day. For breeding, three females were placed together with one male. Following the breeding 

14 phase, females were separated and number of litters cast, number of days between mating and 
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1 delivery, and average number of pups/litter at delivery were recorded. F1 pups were weighed 

2 and culled to seven/litter at 2 days of age and weaned at 3 weeks of age, and weights were 

3 recorded weekly for postnatal weeks 3–6. The F1 rats were continued on the same diets as their 

4 parents, and, at 10 weeks of age, nine F1 females and three F1 males were mated to produce an 

5 F2 generation of pups. F2 pups were selected (by the same procedure) for mating and 

6 production of an F3 generation that were sacrificed at 3 weeks of age; twelve F3 pups from each 

7 diet group were subjected to gross pathologic examinations. 

8 There were no significant differences between controls and 0.01 and 0.1% biphenyl-fed 

9 groups regarding litters cast; gestation length; or average number or weight of pups/litter at birth

10 or at 3 or 6 weeks of age. Decreased fertility in the 1% biphenyl-fed group of females was 

11 observed (6/9, 7/9, and 8/9 confirmed pregnancies for the three successive generations of 1.0% 

12 biphenyl-fed groups vs. 8/9, 9/9, and 8/9 confirmed pregnancies for controls). Averaged for F1, 

13 F2, and F3 pups combined, the 1.0% biphenyl-fed group exhibited significantly (p < 0.05) 

14 decreased number of pups/litter at birth (6.2/litter vs. 8.6/litter for controls) and lower average 

15 body weight at 3 weeks of age (36 vs. 48 g for controls) and 6 weeks of age (78 vs. 113 g for 

16 controls). Gross pathologic evaluations of F3 weanlings revealed no signs of biphenyl treatment17 related effects. There was no evidence of a cumulative effect over the three generations. The 

18 study authors indicated that the decreased fertility, smaller litter size, and reduced rate of growth 

19 in the 1.0% biphenyl-fed group may have been associated with unpalatability and resultant 

20 decreased food intake. 

21

22 Ambrose et al., 1960 

23 The research report of Ambrose et al. (1960) contains a subsection in which the 

24 reproductive toxicity of biphenyl was examined in two experimental series. In the first 

25 experiment, weanling albino rats were administered 0 or 0.1% biphenyl (5 males and 

26 10 females/group) or 0.5% biphenyl (3 males and 9 females) in the diet for 60 days prior to 

27 mating. In the second experiment, groups of 90-day-old albino rats were administered 0 or 0.1% 

28 biphenyl (4 males and 8 females/group) or 0.5% biphenyl (3 males and 9 females) in the diet for 

29 11 days prior to mating. Based on U.S. EPA (1988) subchronic reference values for body weight 

30 and food consumption in rats of unspecified strain (average values for combined sexes), doses of 

31 biphenyl for the dietary levels of 0.1 and 0.5% are estimated to have been 105 and 525 mg/kg32 day, respectively. All rats were maintained on their respective diets throughout mating and until 

33 the progeny of all litters were weaned. Insufficient information is provided in the report to 

34 permit a judgment as to whether dietary exposure to biphenyl was associated with reproductive 

35 deficits. However, the authors presented tabular data for number of rats casting litters, total 

36 born, and range of litter size (Table 4-13) and concluded that the compound had no significant 

37 effect on reproduction. 

38
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Table 4-13. Summary of reproductive data in albino rats exposed to dietary 

biphenyl

Experimental series Diet Dams with litters Total offspring Litter size (range)

Firsta Control 9/10 59 3–9

0.1% biphenyl 10/10 67 2–10

0.5% biphenyl 8/9 53 3–9 

Secondb 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

3 No studies were identified that examined the reproductive/developmental toxicity of 

4 biphenyl via the inhalation route. 

5 

6 4.4. OTHER DURATION- OR ENDPOINT-SPECIFIC STUDIES

7 4.4.1. Acute and Short-term Toxicity Data

8 Acute oral toxicity studies of biphenyl provide median lethal dose (LD50) values ranging 

9 from 2,180 to 5,040 mg/kg for rats (Monsanto, 1976; Pecchiai and Saffiotti, 1957; Union 

10 Carbide, 1949; Deichmann et al., 1947) and an LD50 value of 2,410 mg/kg for rabbits 

11 (Deichmann et al., 1947). Dow Chemical Co., (1939) reported 100% survival and 100% lethal 

12 doses of 1,600 and 3,000 mg/kg, respectively, in rats. Clinical signs commonly observed 

13 following single oral dosing in these studies included increased respiration, lacrimation, loss of 

14 appetite and body weight, and muscular weakness. Deaths occurred in the first few days 

15 following dosing. Typical targets of histopathologic lesions were lungs, liver, and upper 

16 gastrointestinal tract.

17 In another acute study, Pecchiai and Saffiotti (1957) administered single gavage doses of 

18 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 

19 for up to 7 months following dosing. Histopathological changes to the liver, kidney, thyroid, 

20 parathyroid, and gastrointestinal mucosa were reported in biphenyl-treated rats; however, the 

21 study report did not provide information regarding numbers of treatment-related deaths or 

22 incidences of lesions in the various treatment groups. Among surviving rats, signs of 

23 regeneration were evident within 1–4 months after treatment. By 7 months after treatment, most 

24 of the changes had disappeared, but hepatocytes displayed modest vacuolization of the 
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1 cytoplasm and numerous binucleate cells in the periphery of the lobules. In renal tubuli, a 

2 moderate number of cytoplasmic granules were observed.

3 

4 Sun Company Inc., 1977a 

5 Groups of mice (10/sex of unspecified strain) were exposed to biphenyl by inhalation for 

6 4 hours at average analytical concentrations of 14.11, 38.40, or 42.80 ppm (89.0, 242.2, and 

270.0 mg/m3 7 , respectively) and observed for up to 14 days following exposure. Clinical signs of 

8 hyperactivity and mild respiratory discomfort were noted during exposure, but resolved during 

9 postexposure observation. A solitary male mouse of the 42.80 ppm group died after 2 hours of 

10 exposure, but this death was not attributed to biphenyl exposure. All other mice survived 

11 throughout the 14-day postexposure observation period. Slight lung congestion was noted in 

12 most mice upon gross pathological examination. 

13 Sun Company Inc. (1977a) also provided details of a study in which groups of mice 

14 (10/sex of unspecified strain) were exposed to biphenyl for 7 hours/day, 5 days/week for 2 weeks 

at average analytical concentrations of 0, 24.8, or 54.75 ppm (0, 156.4, and 345.5 mg/m3 15 , 

16 respectively). Five animals/group were sacrificed immediately after exposure; the remaining 

17 animals were sacrificed following a 14-day recovery period. Clinical signs were monitored 

18 daily. Gross pathologic examinations at necropsy included assessment of lungs, trachea, heart, 

19 spleen, liver, kidneys, stomach, and intestines. Histopathologic examinations included tissues 

20 from lung, trachea, kidney, spleen, and liver. The study authors reported signs of hyperactivity 

21 in some mice during the first few exposure periods. One female mouse of the 24.8 ppm

22 exposure group died prior to the third exposure session and one control female mouse died prior 

23 the final exposure session. No abnormal clinical signs were seen during the 14-day recovery 

24 period. Gross and histopathologic examinations revealed no signs of exposure-related adverse 

25 effects.

26

27 Deichmann et al., 1947; Monsanto, 1946 

28 Four rabbits (sex and strain unspecified) received up to 20 daily doses of 500 mg/kg 

29 “purified” biphenyl to the skin; the compound was applied as a 25% preparation in olive oil. 

30 Three rabbits received the same concentration of technical biphenyl. The compound was left on 

31 the skin for 2 hours and then washed off with soap and water. Some biphenyl derivatives were 

32 similarly assessed. One rabbit receiving purified biphenyl died after eight applications, and the 

33 rest of the animals survived to term. The only reported sublethal effect clearly associated with 

34 biphenyl exposure was that of weight loss, averaging 45 and 172 g for the rabbits receiving 

35 purified and technical biphenyl, respectively. 

36
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1 4.4.2. Kidney/Urinary Tract Endpoint Studies

2 Several endpoint-specific studies assessed biphenyl-induced urinary tract effects in rats 

3 (Shibata et al., 1989a, b; Kluwe, 1982; Søndergaard and Blom, 1979; Booth et al., 1961) and 

4 provide support to findings of the chronic oral rat studies described in Section 4.2.1.2 (Chronic 

5 Toxicity and Carcinogenicity). Detailed descriptions of these endpoint-specific studies are 

6 presented below. 

7 

8 Booth et al., 1961 

9 In a preliminary study, five adult rats (sex and strain unspecified) were administered 

10 biphenyl in the diet at 1% (w/w) for 26 days followed by a 29-day postexposure recovery period 

11 for a total study period of 55 days. Total urine volume and the volume of sulfosalicylic acid12 precipitable sediment were recorded from urine collected from all five rats on study days 4, 8, 

13 18, 20, and 26 (exposure days), and study days 28, 32, 35, and 54 (recovery period). Volumes of 

14 both urine and sulfosalicylic acid-precipitable sediment increased from 7 and 0.56 mL, 

15 respectively, on exposure day 4 to 32 and 2.24 mL, respectively, on exposure day 20. Both 

16 values remained relatively high (approximately 27 and 2.2 mL, respectively) on exposure day 26 

17 and decreased to approximately 14 and 0.8 mL, respectively, by the end of the recovery period. 

18 Fractionation and analysis of the precipitate suggested the presence of p-hydroxybiphenyl and its 

19 glucuronide. The study authors indicated that similar effects were noted in male and female rats 

20 receiving biphenyl at a level of 0.5% in the diet, but not at the 0.1% dietary level. 

21 A follow-up study employed 42 rats of each sex and exposure group and biphenyl dietary 

22 levels of 0, 0.1, 0.25, or 0.5% (w/w). Biphenyl doses are estimated at 83.7, 209, and 419 mg/kg23 day for the dietary levels of 0.1, 0.25, and 0.5%, respectively, based on U.S. EPA (1988) chronic 

24 reference values for body weight and food consumption in F344 rats (averages of values for 

25 males and females). Rats were exposed for up to 165 days and followed for 0, 30, or 60 days of 

26 recovery. Urine samples were collected periodically from five rats/sex/exposure group. Interim 

27 sacrifices of five rats/sex/exposure group were performed after 30, 60, and 120 days on the diet 

28 in order to assess the progression of biphenyl-induced histopathological effects on the kidney. 

29 As noted in the preliminary study, the rats of the 0.5% exposure group in the follow-up study 

30 exhibited gradual increases in the urine volume and sulfosalicylic acid-precipitable sediment and 

31 decreased in both parameters during postexposure recovery. The study authors indicated that 

32 these effects were much less pronounced in the 0.25% exposure group and absent in the 0.1% 

33 exposure group. At the 0.5% exposure level, kidney lesions were noted in 1/5 of the males 

34 (several small cysts and dilated tubules in the medulla and inner cortex) and 2/5 of the females 

35 (mild local tubular dilation with some epithelial flattening) following 30 days of exposure. 

36 Similar, but more extensive, kidney lesions were noted in 3/5 males and 5/5 females following 

37 60 days of exposure. The kidney lesions were even more prominent following 120 days of 

38 exposure. Reported histopathologic findings in the kidneys of rats from the 0.25% exposure 
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1 group were limited to a single instance of an unspecified “prominent kidney lesion” at 60 days, 

2 and one small calculus in the pelvis of one rat and a small calcareous deposit in the renal 

3 pyramid of another rat following 120 days of exposure. Based on available information in the 

4 study report, there were no apparent assessments of urinary and histopathologic renal effects at 

5 the end of the 165-day treatment period. However, during the 60-day postexposure recovery 

6 period, rats of the 0.5% biphenyl exposure group exhibited a regression of kidney lesions and 

7 improvement in urine quality. 

8 

9 Kluwe, 1982

10 Kluwe (1982) examined changes in urine composition and kidney morphology in F344 

11 rats exposed to biphenyl. Groups of male F344 rats were administered biphenyl (in corn oil) by 

12 single gavage dosing at 0, 250, 500, or 1,000 mg/kg and observed for 15 days following 

13 treatment. Body weights were recorded, and urine was collected on days 1, 2, 3, 4, 8, and 

14 15 following treatment for urinalysis. Interim sacrifices were performed on eight control and 

15 eight high-dose rats on posttreatment days 1, 2, 3, 8, and 15 for assessment of weight and 

16 histopathology of the kidney. The study authors presented body weight data as mean percent 

17 (n = 6) of preexposure body weight; results of urinalyses were presented as mean values (n = 6) 

18 for each group. There were no significant effects on body weight in the low-dose group. Mean 

19 body weight gains of mid- and high-dose groups were consistently 6–10% lower than control 

20 values (p < 0.05), beginning as early as day 2 following the initiation of dosing and continuing 

21 through day 15. Dose-related increases in polyuria, proteinuria, and glucosuria were observed on 

22 day 1; polyuria and glucosuria were no longer apparent by day 4 and proteinuria resolved 

23 between days 8 and 15. The study authors presented no data to indicate that single oral dosing 

24 caused changes in kidney weight. Histopathologic examinations of kidneys revealed renal 

25 papillary necrosis in 8/32 high-dose rats; this effect was observed as early as day 1 and persisted 

26 during the 15-day posttreatment period. 

27 Kluwe et al. (1982) conducted a similar experiment in which groups of male F344 rats 

28 received biphenyl at doses of 0, 250, or 500 mg/kg-day by gavage for 14 days. In this 

29 experiment, polyuria persisted throughout the treatment period; glucosuria was no longer 

30 apparent by day 4 and proteinuria resolved between treatment days 8 and 15. Relative kidney 

31 weight of high-dose rats was significantly increased during the second half of the treatment 

32 period, but the magnitude of this effect was small and considered by the study authors to be of 

33 little biological significance. There was some indication of tubular dilatation in focal areas of 

34 kidneys from the high-dose rats.

35

36 Søndergaard and Blom, 1979 

37 Groups of male and female SPF-Wistar rats were administered diets consisting of 

38 semisynthetic chow and biphenyl at concentrations resulting in biphenyl doses of 0, 50, 150, 300, 
51 DRAFT - DO NOT CITE OR QUOTE

1 or 450 mg/kg-day. Other groups were administered diets consisting of commercial chow and 

2 biphenyl at concentrations resulting in biphenyl doses of 0, 50, 150, 300, 500, or 1,000 mg/kg3 day. The treatment period lasted for up to 21 days. The numbers of male and female rats in each 

4 treatment group are specified in Table 4-14. Urine was collected on days 4, 10, and 17 for 

5 urinalysis. At terminal sacrifice, absolute and relative kidney weights were determined and 

6 kidney tissues were prepared for light and electron microscopic assessment. Apparently, interim 

7 sacrifices (days 1, 2, 4, and 10) were performed in order to assess the activity of AP in proximal 

8 tubules. Table 4-14 presents semiquantitative study results, which include increases in urine 

9 volume/specific gravity and relative kidney weight, as well as polycystic kidney changes. No 

10 changes in AP levels were seen as a result of biphenyl exposure. The kidney effects of biphenyl 

11 appeared to be more pronounced when added to the semisynthetic diet vs. the commercial diet, 

12 with 50 mg/kg-day as a LOAEL for the onset of kidney changes. 

13

Table 4-14. Number of Wistar rats exposed to biphenyl and the degree of 

change in kidney weight and cellular architecture

Exposure

(mg/kg-d) 

Number of animals 

(male/female)

Relative kidney weight 

increases Cystic change

Increases of urine 

volume/specific 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 – –

500a 0/10 +b – ●/●

1,000a 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 

16 Male F344 rats (20/group) were exposed to 0 or 0.5% (w/w) biphenyl in the diet for 
52 DRAFT - DO NOT CITE OR QUOTE

1 24 weeks (Shibata et al., 1989a). After 4 weeks, 5 rats/group were injected with 100 mg/kg 

2 5-bromo-2-deoxyuridine (BrdU) and sacrificed 1 hour later. One kidney from each rat was 

3 processed for immune-histopathologic identification of BrdU as an index of cell proliferation, 

4 while the second kidney was processed for light and scanning electron microscopic examination. 

5 The remaining rats were sacrificed after 8, 16, and 24 weeks to monitor further development of 

6 morphological alterations in the renal papilla and pelvis. Survival was unaffected by treatment 

7 and biphenyl-treated animals showed no adverse clinical signs. The study authors reported that 

8 treatment resulted in significantly lower mean body weight compared to controls; food 

9 consumption was unaffected and water consumption was slightly higher than that of controls. 

10 There were no significant treatment-related effects on labeling indices of cell proliferation (BrdU 

11 incorporation) in renal papilla or pelvic epithelia and no histopathologic lesions of the renal 

12 papilla and pelvis were evident. Focal calcification of the renal medulla was observed in the 

13 majority of the biphenyl-treated rats. The study authors stated that urinalysis demonstrated an 

14 association between biphenyl exposure and microcalculi formation, but provided no additional 

15 information regarding urinalysis results.

16 In a similar study (Shibata et al., 1989b), a group of 10 male F344 rats received 0.5% 

17 (w/w) biphenyl in the diet for up to 8 weeks. Based on U.S. EPA (1988) subchronic reference 

18 values for body weight and food consumption in male F344 rats, the dose was estimated at 

19 500 mg/kg-day. At 4 weeks, five rats/group were processed as described by Shibata et al. 

20 (1989a) for assessment of BrdU incorporation, but in the urinary bladder rather than in the 

21 kidney. During week 4, urine samples were taken for urinalysis. At terminal sacrifice, urinary 

22 bladder tissues were processed for scanning electron microscopic examinations. There were no 

23 treatment-related deaths or adverse clinical signs. Although food and water consumption were 

24 similar to controls, biphenyl-treated rats showed a consistent reduction in average body weight 

25 (229 vs. 247 g after 4 weeks and 300 vs. 327 g after 8 weeks, for treated vs. controls, 

26 respectively [p < 0.01]). A greater than fourfold increase in the BrdU labeling index was 

27 observed in urinary bladder epithelium of the biphenyl-fed rats (mean percent labeling index of 

28 0.58 ± 0.31 compared to 0.13 ± 0.09 in controls; p < 0.05). Urinalysis revealed numerous 

29 microcalculi in the urinary sediment of the biphenyl-treated rats. This condition, designated as 

30 “severe” by the authors, was associated with histopathological lesions of the epithelium of the 

31 urinary bladder that included simple hyperplasia with moderate severity in 5/5 rats, moderate 

32 pleomorphic microvilli (5/5), moderate uniform microvilli (5/5), and the occurrence of ropey or 

33 leafy microridges (5/5), the latter condition designated as severe. Scanning electron microscope 

34 images of the luminal surface of bladder epithelial cells showed pleomorphic microvilli that 

35 varied in size and shape and the formation of microridges. 

36
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1 4.4.3. Biphenyl as a Tumor Promoter 

2 Tamano et al., 1993 

3 Male B6C3F1 mice (10–20/group) received the bladder carcinogen BBN at 0 or 0.05% in 

4 the drinking water for 4 weeks followed by 0 or 1% biphenyl in the feed for 32 weeks. The mice 

5 were observed for clinical signs and body weight and food consumption were monitored. At 

6 37-week terminal sacrifice, kidneys and urinary bladders were prepared for histopathological 

7 examination. No treatment-related clinical signs were observed. Mean body weight of the BBN 

8 + 1% biphenyl-treated mice was significantly (p < 0.01) lower than that of mice receiving BBN 

9 treatment only (32.2 ± 1.8 vs. 38.4 ± 2.6 g). Biphenyl treatment did not result in increased 

10 incidences of simple hyperplasia or papillary or nodular dysplasia in the BBN-initiated mice. 

11 Administration of 1% biphenyl in the feed to eight mice for 8 weeks did not significantly affect 

12 indices of cell proliferation (BrdU incorporation) in urinary bladder epithelium.

13

14 Shiraiwa et al., 1989 

15 In the initiation-promotion portion of a chronic toxicity study designed to assess the 

16 ability of biphenyl to promote carcinogenesis by EHEN in the kidney (see Section 4.2.1.2 for a 

17 detailed study description), male Wistar rats (25/group) received basal diet with either 0 or 0.1% 

18 dietary EHEN for 2 weeks, followed by a basal diet containing either 0, 0.125, or 0.5% biphenyl 

19 for 34 weeks (Shiraiwa et al., 1989). At terminal sacrifice, gross pathologic examinations were 

20 performed. Kidney and urinary bladder were fixed; kidneys were sectioned transversely (10–

21 12 serial slices) and urinary bladders were cut into 4–6 serial slices. The authors used a 

22 computer-linked image analyzer to determine the incidence of kidney lesions and dysplastic foci. 

23 The presence of stones in the kidney and urinary bladder was assessed qualitatively using an 

24 infrared spectrophotometer.

25 Based on reported values for mean daily biphenyl intake (mg biphenyl/rat) and average 

26 body weight (mean initial body weight + one-half the difference between mean initial and mean 

27 final body weight) for each study group, doses of biphenyl at the 0.125 and 0.5% dietary levels 

28 are estimated to have been 59.28 and 248.3 mg/kg-day, respectively, for rats on basal diet alone 

29 for the first 2 weeks and 62.0 and 248.2 mg/kg-day, respectively, for rats on basal diet and 

30 EHEN for the first 2 weeks. Stones were present in the kidney, ureter, and urinary bladder of 

31 high-dose rats irrespective of whether animals were initially exposed to the basal or 

32 EHEN-containing diet (combined incidences of 6/25 and 8/25, respectively). The incidence of 

33 rats with renal cell tumors after EHEN and subsequent biphenyl administration was lower than 

34 that of rats receiving EHEN followed by basal diet) (7/25 and 13/25, respectively). This finding 

35 indicates that biphenyl was not a promoter of renal cell tumors in male Wistar rats under the 

36 conditions of the study. 

37
54 DRAFT - DO NOT CITE OR QUOTE

1 Kurata et al., 1986 

2 Male F344 rats (25/group) were exposed to 0.05% N-butyl-N-(4-hydroxybutyl)

3 nitrosamine (BBN, a bladder carcinogen) in the drinking water for 4 weeks followed by diets 

4 containing either 0 or 0.5% biphenyl for 32 weeks. One group of five rats received biphenyl 

5 without pretreatment with BBN. The rats receiving biphenyl either with or without pretreatment 

6 with BBN gained less weight than control rats or those receiving only BBN. Incidences of 

7 urinary bladder hyperplasia, papilloma, and carcinoma were 17/18 (94%), 15/18 (83%), and 

8 11/18 (61%), respectively, in the group of rats that survived treatment of BBN followed by 

9 biphenyl, compared to 6/24 (25%), 3/24 (12%), and 0/24 (0%), respectively, in the rats receiving 

10 BBN only. These urinary bladder lesions were not seen in any of the five rats receiving biphenyl 

11 without BBN pretreatment. Urinary bladder calculi were found in 25% of the rats receiving 

12 BBN followed by biphenyl and in 12% of the rats receiving BBN only. Biphenyl was 

13 considered a urinary bladder tumor promoter in male F344 rats under the conditions of the study. 

14

15 Boutwell and Bosch, 1959 

16 Biphenyl was negative for tumor promotion in a skin-painting experiment in which the 

17 initiator was 0.3% 9,10-dimethyl-1,2-benzanthracene in benzene (Boutwell and Bosch, 1959). In 

18 the 16/20 mice that survived the topical application of 20% biphenyl for 16 weeks, none had 

19 developed papillomas or carcinomas as a result of treatment.

20

21 Ito et al., 1984 

22 Six-week-old male F344 rats (20–30/group) were exposed to BBN in drinking water at 

23 0.01 or 0.05% for 4 weeks, followed by 0.5% biphenyl in the feed for 32 weeks. Controls 

24 receiving only BBN and controls receiving only biphenyl were included. After sacrifice, urinary 

25 bladders were prepared for light microscopic assessment of neoplastic and cancerous lesions. 

26 The study authors reported that biphenyl exhibited moderate bladder cancer-promoting activity, 

27 but data to support this finding were not included in the study report. 

28

29 4.5. MECHANISTIC DATA AND OTHER STUDIES IN SUPPORT OF THE MODE OF 

30 ACTION

31 4.5.1. Effects on the Urinary Tract of Rats 

32 Urinary tract effects in male rats chronically exposed to biphenyl in the diet are 

33 associated with the formation of urinary bladder calculi. Mechanistic studies performed by 

34 Ohnishi and coworkers (Ohnishi et al., 2001, 2000a, b) were designed to identify urinary 

35 metabolites of biphenyl, to assess conditions leading to calculi formation, and to determine the 

36 composition of urinary crystals and calculi. Ohnishi et al. (2000a) identified sulphate conjugates 

37 of mono- and dihydroxy biphenyl metabolites in urine and urinary crystals from F344 rats treated 
55 DRAFT - DO NOT CITE OR QUOTE

with biphenyl and KHCO3 (to elevate the pH and K+ 1 concentration of the urine). Male F344 rats 

2 (five per group) were administered a diet containing 1.6% biphenyl and 5% potassium 

3 bicarbonate for 7 days (Ohnishi et al., 2000a). Urine was collected on days 6 and 7 and pooled. 

4 Urinary crystals (i.e., precipitates) were collected and dissolved in acetonitrile and were analyzed 

5 by HPLC to identify metabolites or by inductively coupled plasma spectroscopy to identify 

6 inorganic elements. As shown in Table 4-15, biphenyl sulphate conjugates in the urine consisted 

7 primarily of 3,4-dihydroxybiphenyl-3-O-sulphate (40.9% of the total biphenyl sulphate 

8 conjugates) and 3-hydroxybiphenyl (23.4%). No bisulphates were observed (Ohnishi et al., 

9 2000a). In contrast; about 90% of sulphate conjugates in urinary crystals were 4-hydroxy10 biphenyl-O-sulphate, and only 3.9 and 1.06% were 3,4-dihydroxybiphenyl-3-O-sulphate and 

11 3-hydroxybiphenyl, respectively. In a follow-up study, Ohnishi et al. (2000b) evaluated the 

12 composition of urinary calculi in male and female rats exposed to 4,500 ppm biphenyl in the diet 

13 for 104 weeks. Urinary calculi in chronically exposed male rats were composed mainly of 

14 4-hydroxybiphenyl-O-sulphate, whereas calculi in female rats were composed primarily of 

15 4-hydroxybiphenyl and potassium sulphate, the hydrolysis products of 4-hydroxybiphenyl16 O-sulphate (Ohnishi et al., 2000b). 

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 sulphate conjugates Urine (%) Urine crystals (%)

2-Hydroxybiphenyl-O-sulphate 3.32a 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 fraction (%) for each of the sulphate 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

20 he 

21 diet for 13 weeks, Ohnishi et al. (2001) reported hydronephrosis and blood in the urine only in 

22 those animals receiving biphenyl plus potassium bicarbonate. Feed consumption was not 

23 affected by the dietary additions, while water intake was greatly increased in all groups of 

24 animals that received biphenyl and/or salts. Neither high urinary potassium levels alone, as 
56 DRAFT - DO NOT CITE OR QUOTE

1 induced by cofeeding of potassium chloride, nor high urinary pH alone, as induced by cofeeding 

2 of sodium bicarbonate, were sufficient to cause kidney damage. It was concluded that a 

3 combination of high urinary pH and high potassium levels was necessary to cause precipitation 

4 of biphenyl sulphate. It was proposed that the crystalline precipitate caused obstruction that led 

5 to hydronephrosis or damaged the transitional epithelium in the bladder causing hyperplasia. 

6 

7 4.5.2. Effects on the Liver of Mice 

8 Based on findings of biphenyl-induced liver tumors in female BDF1 mice administered 

9 high dietary concentrations of biphenyl for 2 years (Umeda et al., 2005), a 13-week oral study 

10 was performed to assess whether peroxisome proliferation might be induced (Umeda et al., 

11 2004). Groups of male and female BDF1 mice (10/sex/group) were administered biphenyl in the 

12 diet at six different concentrations ranging from 500 to 16,000 ppm. Biphenyl concentrations 

13 ≥8,000 ppm resulted in significantly decreased final body weights of males and females. 

14 Significantly increased liver weights were noted in the 8,000 and 16,000 ppm groups of female 

15 mice. Evidence of peroxisome proliferation was restricted to the 16,000 ppm group of female 

16 mice and included light microscopy findings of clearly enlarged hepatocytes filled with 

17 eosinophilic fine granules and electron microscopy confirmation that the granules corresponded 

18 to increased numbers of peroxisomes. Light microscopy of livers from rats exposed to 

19 concentrations ≤8,000 ppm showed no indications of proliferation of peroxisomes. There were 

20 no indications of other biphenyl-induced liver effects in any of the groups of male mice. 

21

22 4.5.3. Estrogenic Effects

23 Several biphenyl derivatives display estrogenic activity. Schultz et al. (2002) used the 

24 Saccharomyces cerevisiae/LacZ reporter assay to study the estrogenic activity of 120 chemicals 

25 to identify chemical structures that impart estrogenic activity to a molecule. Chemicals without a 

26 hydroxy group, among them biphenyl, were inactive in this assay. The estrogenic activities of 

27 biphenyl metabolites in this assay were 4,4′-dihydroxybiphenyl (median effective concentration 

= (2.6 × 10-7 M) > 4-hydroxybiphenyl (1.2 × 10-6 M) > 3-hydroxybiphenyl (9.2 × 10-6 28 M) 

> 2-hydroxybiphenyl (1.8 × 10-5 29 M). Estrogenic activities of the corresponding hydroxylated di-, 

30 tri-, or tetrachlorobiphenyl metabolites were approximately two orders of magnitude higher, 

31 provided there were no chlorines and hydroxy groups on the same ring. 

32 Kitamura et al. (2003) used MCF-7 cells transfected with an estrogen receptor-luciferase 

33 reporter construct to test biphenyl and its metabolites for estrogenic activity. The starting point 

34 for this investigation was the structural similarity between hydroxylated metabolites of biphenyl 

35 and of 2,2-diphenyl propane, the 4,4′-dihydroxy metabolite of which is bisphenol A, a known 

36 endocrine disrupter. Biphenyl per se displayed no estrogenic activity in this assay. Metabolites 

37 of biphenyl formed by liver microsome preparations were identified after solvent extraction from 
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1 reaction media by HPLC-MS. The compounds were also tested in an in vitro competitive 

2 estrogen receptor binding assay. The biphenyl metabolites, 2-, 3-, 4-hydroxybiphenyl, and 

3 4,4′-dihydroxybiphenyl, all exhibited estrogenic activity when the cell culture contained 

4 microsomes from 3-methylcholanthrene-induced rat livers and to a lesser extent, phenobarbital5 induced rat livers, in the presence of NADPH. In the competitive estrogen receptor binding 

6 assay, 4,4′-dihydroxybiphenyl displayed weak binding affinity, while biphenyl and its 

7 monohydroxy metabolites did not show any activity. 4,4′-Dihydroxybiphenyl is one of two 

8 major biphenyl metabolites in rats and mice (Halpaap-Wood et al., 1981a, b; Meyer and 

9 Scheline, 1976), suggesting that high doses of biphenyl, in the form of this metabolite, might 

10 induce some minor estrogenic effect.

11

12 4.5.4. Effects on Apoptosis

13 Kokel and Xue (2006) tested a series of benzenoid chemicals (including mesitylene, 

14 cyclohexane, benzene, toluene, and biphenyl) for their ability to suppress apoptosis in the 

15 nematode, Caenorhabditis elegans, a model suitable for the characterization of carcinogens that 

16 act by way of apoptosis inhibition. The study included wild type and three strains of C. elegans

17 mutants; the ced-3(n2438) mutant (which carries a partial loss-of-function mutation in the ced18 3 gene), the ced-3(n2273) mutant (also partly defective in cell death), and the ced-(n2433) 

19 mutant (a strong loss-of-function ced-3 mutant). Effects on apoptosis were assessed by counting 

20 the numbers of cells that should have died during embryogenesis, but inappropriately survived. 

21 The results indicated that these chemicals did not significantly affect apoptosis in wild type 

22 C. elegans. However, inhibition of apoptosis was apparent in mutant strains ced-3(n2438) and 

23 ced-3(n2273) exposed to benzene, toluene, or biphenyl. The study authors interpreted these 

24 results as indicative of apoptosis-inhibitory activity that does not depend on mutations in a 

25 specific cell-death gene. A lack of apparent apoptosis-inhibitory activity in the strong loss-of26 function ced-3(n2433) mutant was interpreted as indicative that inhibition of apoptosis, rather 

27 than transformation of cell fates, caused the increase in extra cell observed in the other two 

28 mutant strains. All three chemicals also displayed embryotoxicity. Biphenyl and naphthalene 

29 were both shown to suppress apoptosis in C. elegans mutant strain ced-3(n2438) by causing 

30 overexpression of the CED-3 caspase. The authors proposed that benzenoid chemicals that can 

31 form quinones suppress apoptosis in C. elegans via this reactive intermediate, although this was 

32 proven only for benzene, toluene, and naphthalene. 

33 Regulation of apoptosis during embryogenesis is critical, and a recent study by Tan et al. 

34 (2011) showed that inhibition of apoptosis during this stage of development may have 

35 detrimental effects on the nervous system. No literature was identified, however, that 

36 specifically supports an association between inhibition of apoptosis by biphenyl and effects on 

37 embryogenesis. 

38
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1 4.5.5. Mitochondrial Effects 

2 Nishihara (1985) assessed the effects of biphenyl on the respiratory and energy linked 

3 activities of rat liver mitochondria that had been isolated from male Wistar rats. Biphenyl (5–

4 60 μg/mL in acetone solvent) was added to liver mitochondria and effects on rates of succinate 

5 oxidation and α-ketoglutarate/malate oxidation were assessed by measuring oxygen 

6 consumption. Solvent controls were included in the study. Biphenyl significantly inhibited state 

7 3 respiration at concentrations ≥20 μg/mL. The inhibition was greater for α-ketoglutarate/malate 

8 oxidation than for succinate oxidation. State 4 respiration was significantly stimulated by 

9 biphenyl; the effect was greater in magnitude for succinate than for α-ketoglutarate/malate 

10 oxidation. Biphenyl also altered mitochondrial membrane permeability, as evidenced by the 

instantaneous release of endogenous K+ 11 , leading to instantaneous dissipation of the 

12 mitochondrial membrane potential. Inhibition of state 3 respiration is generally considered to 

13 reflect an interference with electron transport. The study author suggested that the biphenyl14 induced stimulation of state 4 respiration may be explained by an uncoupling action on 

15 respiration. 

16

17 4.5.6. Genotoxicity

18 Biphenyl. The results of genotoxicity studies of biphenyl are summarized in Table 4-16. 

19 Reverse mutation assays using Salmonella typhimurium and Escherichia coli provide 

20 consistently negative results both with and without the addition of a mammalian metabolic 

21 activation system (rat S9 mix). Biphenyl was not genotoxic in a host-mediated deoxyribonucleic 

22 acid (DNA) repair assay of E. coli in the presence of S9 (Hellmér and Bolcsfoldi, 1992). In rec 

23 assays of Bacillus subtilis, two studies reported negative results both with and without S9 

24 (Garrett et al., 1986; Kojima and Hiraga, 1978), one study reported negative results without S9 

25 (Kawachi et al., 1980) and one study reported equivocal results with S9 (Hanada, 1977). 

26 Biphenyl was reported to induce mitotic recombination both with and without S9 in 

27 Saccharomyces cerevisiae strain D3 (Pagano et al., 1988), but not in S. cerevisiae strain Diploid 

28 D7 (Garrett et al., 1986). 

29
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Table 4-16. Genotoxicity test results for biphenyl

Strain or test Test substance 

Metabolic 

activationa

Organism system Endpoint concentrations +S9 –S9 Reference

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 1–105 μg/mLb – – 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 

104-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– 104-fold range – – Probst et al., 1981

WP uvrA–, polA– 1–105 μg/mL – – Garrett et al., 1986

B/γ 

B/γ 

WP – 2try ,

WP – 2try–hcr

Units provided 

Japanese

in – Hanada, 1977

B/γ WP – 2try–hcr ≤1,000 μg/mLb – – 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
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Table 4-16. Genotoxicity test results for biphenyl

Metabolic 

Strain or test Test substance activationa

Organism system Endpoint concentrations +S9 –S9 Reference

B. subtilis Not given Rec assay NS NT – Kawachi et al., 1980

recA– 1–105 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

1–105 μg/mL – – Garrett et al., 1986

Diploid D7 10-5

10-5

or 10-3

Ma

M + + Pagano et al., 1988

Tests with cultured mammalian cells

Hamster V79 Mutation 5–100 μg/mL

100 μg/mLb

+ – 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/mLa

– 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 1–105 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 μMc – 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 activationa

Organism system Endpoint concentrations +S9 –S9 Reference

Mouse L5178Y/TK+/– Mutation 50–500 μM

150 μMa

– Garberg et al., 1988

50–1,500 μM

500 μMa

+ 

98.7–395 μM

98.7 μMa

+d Wangenheim and 

Bolcsfoldi, 1988, 1986

5–60 μM

10 μMa

+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 = sister 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 (Purchase 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

1 and Matheson, 1985); and unscheduled DNA synthesis in human lung WI-38 lung fibroblasts 

2 (with or without S9; Garrett et al., 1986). 

3 Positive results were obtained for CA in CHL fibroblasts (Sofuni et al., 1985) and 

4 mutations in Chinese hamster V79 cells (Glatt et al., 1992) in the presence, but not absence, of 

S9. Biphenyl induced forward mutations in mouse L5178Y/TK+/- 5 lymphoma cells with and 

6 without S9 (Wangenheim and Bolcsfoldi, 1988, 1986); another study provided similar results in 

7 the presence, but not the absence, of S9 (Garberg et al., 1988). Significant increases in SCE 

8 (< twofold higher than solvent controls), CA (two- to fourfold higher than solvent controls), and 

9 micronuclei (approximately 2.5-fold higher than solvent controls) were reported in human 

10 peripheral blood lymphocytes exposed to biphenyl for 24–48 hours at concentrations ≥50 μL/mL 

11 (Rencüzoğullari et al., 2008).

12 Evaluations of the potential genotoxicity of biphenyl in vivo have been performed in rats, 

13 mice, and silkworms. Biphenyl did not induce SCE or CA in bone marrow cells of rats or 

14 mutations in silkworms, but limited information is available for these studies (Kawachi et al., 

15 1980). In a Comet assay, positive results were reported for DNA damage in stomach, blood, 

16 liver, bone marrow, kidney, bladder, lung, and brain cells of CD-1 mice administered single 

17 doses of 2,000 mg biphenyl/kg (Sasaki et al., 2002, 1997). It is unknown if the DNA damage 

18 was caused by direct reaction with biphenyl or its metabolites, or by indirect damage from 

19 cytotoxicity or ROS generated from redox cycling of hydroquinone metabolites. 

20 Biphenyl metabolites. Table 4-17 summarizes results from genotoxicity tests of several 

21 biphenyl metabolites, 2-hydroxybiphenyl (also known as o-phenylphenol), 4-hydroxybiphenyl 

22 (the principal metabolite of biphenyl), and 2,5-dihydroxybiphenyl. 2-Hydroxybiphenyl and its 

23 sodium salt have received the most research attention because they are used as fungicides and 

24 anti-bacterial agents and have been found to cause urinary bladder tumors in male F344 rats with 

25 chronic exposure to high concentrations in the diet (see Balakrishna et al., 2002; Kwok et al., 

26 1999; Smith et al., 1998 for review). 

27
63 DRAFT - DO NOT CITE OR QUOTE

Table 4-17. Genotoxicity test results for biphenyl metabolites

Metabolic 

Organism

Strain or test 

system Endpoint

Test substance 

concentrations

activationa

+S9 –S9 Reference

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 – 2try–hcr

WP – 2try

1–1,000 μg/mL

1,000 μg/mLa

+/– +/– 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/mLa

– Yoshida et al., 1978 

Rat Liver DNA DNA adducts, 

[

32P]-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. Genotoxicity test results for biphenyl metabolites

Organism

Strain or test 

system Endpoint

Test substance 

concentrations

Metabolic 

activationa

+S9 –S9 Reference

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,

[

32P]-post 

labeling 

method

10 or 20 mg applied 

to skin

+ Pathak and Roy, 1993

Rat F344/bladder 

epithelium

DNA adduct,

[

32P]-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 

[

14C]-2-hydroxybiphenyl, 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/platec 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. Genotoxicity test results for biphenyl metabolites

Metabolic 

Organism

Strain or test 

system Endpoint

Test substance 

concentrations

activationa

+S9 –S9 Reference

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

[

32P]-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, but 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 micronuclei 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-hydroxybiphenyl/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 the urinary bladder of male or 
66 DRAFT - DO NOT CITE OR QUOTE

1 female rats intravesically injected with 0.05 or 0.1% phenylbenzoquinone, but not with injections 

2 of 0.05% 2-hydroxybiphenyl or 2,5-dihydroxybiphenyl, although DNA damage was found in 

3 urinary bladders from male F344 rats fed the sodium salt of 2-hydroxybiphenyl in the diet for 

4 3 months at 1,000 or 2,000 ppm, but not at 500 or 250 ppm (Morimoto et al., 1989). Topical 

5 application of 10 or 20 mg of the sodium salt of 2-hydroxybiphenyl or 5 mg of 2,5-dihydroxy6 biphenyl to the skin of female CD-1 mice produced several DNA adducts in the skin that were 

detected by the [32 7 P]-post labeling technique (Pathak and Roy, 1993). Similar adducts were 

8 formed in vitro when DNA was incubated with 2-hydroxybiphenyl (1 mM) in the presence 

9 metabolic activation from rat skin homogenates, a CYP system, or a prostaglandin synthase 

10 system (Pathak and Roy, 1993). In contrast, Smith et al. (1998), using a similar technique to that 

11 used by Pathak and Roy (1993), were unable to detect exposure-related DNA adducts in bladder 

12 epithelial tissue from male F344 rats fed 800, 4,000, 8,000, or 12,500 ppm 2-hydroxybiphenyl in 

13 the diet for 13 weeks. In this experiment, increased bladder cell epithelium proliferation (i.e., 

14 increased BrdU incorporation) was observed at 8,000 and 12,500 ppm, dietary concentrations 

15 associated with the development of urinary bladder tumors in chronically exposed rats (Smith et 

16 al., 1998). Kwok et al. (1999) found no evidence of binding of radioactivity to DNA extracted 

from the bladder epithelium of male F344 rats given single gavage doses of [14 17 C]-labeled 

18 2-hydroxybiphenyl at 15, 50, 250, 500, or 1,000 mg/kg, but increased protein binding occurred 

19 with increasing doses of 250, 500, and 1,000 mg/kg. Kwok et al. (1999) noted that the increase 

20 in protein binding increased with increasing dose levels of 250, 500, and 1,000 mg/kg, in parallel 

21 with increasing incidence of bladder epithelial lesions (hyperplasia, papillomas, and carcinomas) 

22 in rats chronically exposed to 2-hydroxybiphenyl in the diet at 0, 269, and 531 mg/kg. 

23 Increased micronuclei (about threefold increase over controls) and increased cell 

24 proliferation (>200-fold increased incorporation of BrdU in DNA) were found in the bladder 

25 epithelium of male F344 rats exposed to 2% (2,000 ppm) 2-hydroxybiphenyl in the diet for 

26 2 weeks, without evidence for hypo- or hyperploidy as assayed by fluorescence in situ 

27 hybridization with a DNA probe for rat chromosome 4 (Balakrishnan et al., 2002). Similar 

28 exposure to 2% NaCl or 2% 2-hydroxybiphenyl + 2% NaCl , produced about two- or six-fold 

29 increases of micronuclei in the bladder epithelium, respectively, but neither treatment stimulated 

30 bladder epithelium cell proliferation to the same degree as 2% 2-hydroxybiphenyl in the diet 

31 (Balakrishan et al., 2002). 2-Hydroxybiphenyl reportedly did not induce SCE in the bone 

32 marrow of rats, but exposure parameters were not specified in the report by Kawachi et al. 

33 (1980). The mechanism of 2-hydroxybiphenyl-induced micronuclei is not understood, but, as 

34 discussed by Balakrishan et al. (2002), possible mechanisms include: (1) DNA damage from 

35 ROS from redox cycling between 2,5-dihydroxybiphenyl and phenylbenzoquinone, 

36 (2) interference of the mitotic spindle through covalent modification of proteins, (3) inhibition of 

37 enzymes regulating DNA replication, or (4) micronuclei generation as a secondary response to 

38 cytotoxicity or regenerative hyperplasia. 
67 DRAFT - DO NOT CITE OR QUOTE

1 Bacterial mutation assays of the major biphenyl metabolite, 4-hydroxybiphenyl, yielded 

2 negative results in all but one case that was accompanied by overt cytotoxicity (Narbonne et al., 

3 1987). 2,5-Dihydroxybiphenyl (i.e., phenylhydroquinone) caused in vitro damage to human 

4 DNA from plasmidpbcNI in the presence of Cu(II) (Inoue et al., 1990), DNA adducts when 

5 applied to mouse skin (Pathak and Roy, 1993), but did not cause DNA damage when injected 

6 intravesically into the urinary bladder of F344 rats at a concentration of 0.05% (Morimoto et al., 

7 1989). 

8 In summary, the overall weight of evidence for biphenyl genotoxicity from short-term 

9 tests is negative or equivocal (Table 4-16). Biphenyl did not induce mutations in a variety of 

10 bacterial test systems (in the absence or presence of exogenous metabolic activation), but in vitro 

11 assays of genotoxicity in mammalian test systems yielded a mix of negative and positive results, 

12 with positive results mostly in the presence of metabolic activation. In tests of clastogenic 

13 effects in mammalian systems, biphenyl induced SCE, CAs, and micronuclei in cultured human 

14 peripheral blood lymphocytes (Rencüzoğullari et al., 2008) and CAs in one assay of CHL 

15 fibroblasts in the presence, but not the absence, of rat liver metabolic activation (Sofuni et al., 

16 1985). However, biphenyl did not induce clastogenic effects (in the presence of metabolic 

17 activation) in other assays with Chinese hamster fibroblasts (Ishidate et al., 1984; Ishidate and 

18 Odashima, 1977) or CHO cells (Yoshida et al., 1978). In the only adequately reported in vivo 

19 genotoxicity studies with biphenyl, single oral doses of 2,000 mg/kg of biphenyl or 

20 2-hydroxybiphenyl induced DNA damage in several organs of CD-1 mice (including liver and 

21 bladder), but it is uncertain if the damage was due to a direct effect on DNA by biphenyl or its 

22 metabolites or indirectly due to cytotoxicity or ROS generated by redox cycling of a 

23 hydroquinone metabolite of 2-hydroxybiphenyl (Sasaki et al., 2002, 1997). 

24 The overall weight of evidence for 2-hydroxybiphenyl genotoxicity suggests that 

25 oxidative DNA damage from redox cycling between 2,5-dihydroxybiphenyl and phenylbenzo26 quinone is possible (Sasaki et al., 2002, 1997; Pathak and Roy, 1993; Morimoto et al., 1989), but 

27 no evidence for DNA adducts or DNA binding in urinary bladder epithelium tissue was found in 

28 rats following short-term (Kwok et al., 1999) or subchronic (Smith et al., 1998) oral exposure to 

29 2-hydroxybiphenyl at high doses associated with the formation of urinary bladder tumors. 

30 Increased micronuclei in urinary bladder epithelium were detected in rats exposed to 2% 

31 2-hydroxybiphenyl or its sodium salt in the diet for 14 days (Balakrishnan et al., 2002). The 

32 mechanism of this clastogenic effect is uncertain, but could involve micronuclei formation in 

33 secondary response to cytotoxicity or regenerative cell proliferation, DNA damage from ROS

34 generated from redox cycling of a hydroquinone metabolite, or protein modifications leading to 

35 mitotic spindle interference or inhibition of enzymes important in DNA replication.

36 4-Hydroxybiphenyl, the predominant metabolite of biphenyl, was not mutagenic in 

37 bacterial testing at noncytotoxic concentrations (Narbonne et al., 1987; Hanada, 1977). 

38 2,5-Dihydroxybiphenyl (i.e., phenylhydroquinone) caused in vitro damage to human DNA from 
68 DRAFT - DO NOT CITE OR QUOTE

1 plasmidpbcNI in the presence of Cu(II) (Inoue et al., 1990) and DNA adducts when applied to 

2 mouse skin (Pathak and Roy, 1993), but did not cause DNA damage when injected intravesically 

3 into the urinary bladder of F344 rats at a concentration of 0.05% (Morimoto et al., 1989). 

4 

5 4.6. SYNTHESIS OF MAJOR NONCANCER EFFECTS

6 Tables 4-18 and 4-19 include the major studies and the observed effects for oral and 

7 inhalation exposure to biphenyl, respectively. 
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 

(statistical significance 

not reported and 

biological significance 

is uncertain).

Dow Chemical 

Co., 1953a

Mice, BDF1

(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 of 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, SpragueDawley

(12/sex/group)

Diet 0, 7, 73, 

2 yrs

or 732 73 732 Renal effects (tubular 

dilatation, calcification, 

and intratubular 

inflammation).

Dow Chemical 

Co., 1953a
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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, BDF1

(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, 1956a

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. 1953a

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. 
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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. 1953a

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/m3), duration 3 (mg/m ) 3 (mg/m ) Effect(s) at the LOAEL References

Rabbits, albino 300 mg/m3 (7 hours/day, 5 days/wk) ND ND ND Deichmann et al., 1947

(3/dose) 64 days over 94 days period

Rats, SpragueDawley (10/dose) 

Rabbits, albino 40 mg/m3 (7 hours/day, 5 days/wk) ND ND ND 

(3/dose) 46 days over 68 days period

Rats, SpragueDawley (6/dose) 

Mice (12/dose) 5 mg/m3 (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/m3 (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 1977a

hyperplasia, and congestion and edema in the liver 

and kidney) in both sexes.

1 

a 2 Report was not published.

3 ND = not determined
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1 4.6.1. Oral 

2 Biphenyl displays a relatively low acute oral toxicity, with LD50 values in laboratory 

3 animals in the 2–3 g/kg range (see Section 4.4.1). The major symptoms of biphenyl intoxication 

4 typically associated with short-term, high-dose oral exposure of animals are labored breathing, 

5 loss of body weight, and weakness. Following medium- or long-term oral exposure, reduced 

6 body weight gain has been reported frequently (Umeda et al., 2005, 2004, 2002; Ambrose et al., 

7 1960; Dow Chemical Co., 1953) and attributed to low palatability of the feed (Ambrose et al., 

8 1960; Dow Chemical Co., 1953); however, the feed intake data of Umeda et al. (2005) in mice 

9 did not support this notion. Increased liver and kidney weights were observed frequently 

10 (Umeda et al., 2004, 2002; Søndergaard and Blom, 1979; Ambrose et al., 1960; Monsanto, 1956; 

11 Dow Chemical Co., 1953). A reduction in hemoglobin levels of rats receiving biphenyl for 

12 700 days was reported (Ambrose et al., 1960). Signs of liver damage (increased serum activities 

13 of ALT, AST, AP, and LDH) were observed in mice (Umeda et al., 2005). Pathological effects 

14 on the urinary system dominated the spectrum of symptoms in dogs (Monsanto, 1956), rats 

15 (Umeda et al., 2002; Dow Chemical Co., 1953), and mice (Umeda et al., 2005). 

16 Urinary system effects, such as increased urine volume with increased specific gravity, 

17 polycystic changes, nephritis, and precipitation of free 4-OH-biphenyl and its glucuronide in 

18 urine are commonly reported following oral exposure to biphenyl (Kluwe, 1982; Søndergaard 

19 and Blom, 1979; Monsanto, 1976; Booth et al., 1961). Calculi appeared in the urine of male rats 

20 only (Umeda et al., 2002; Ohnishi et al., 2001, 2000a, b; Shibata et al., 1989b; Ambrose et al., 

21 1960). Urothelial hyperplasia with increased indices of cell proliferation have been described in 

22 rats but not in mice and were attributed to irritation by calculi (Umeda et al., 2005, 2002; Shibata 

23 et al., 1989b). Tubular dilatation and morphological changes in papillae and pelvis, kidney 

24 stones, obstructive pyelonephritis, tubular atrophy, fibrosis, and pelvic hyperplasia were 

25 observed (Shibata et al., 1989a, b; Shiraiwa et al., 1989; Takita, 1983; Kluwe, 1982; Booth et al., 

26 1961). 

27 Increased incidences of fetuses with skeletal anomalies were reported following gavage 

28 administration of biphenyl to Wistar rats during gestation (Khera et al., 1979). A three29 generation study in rats (Dow Chemical Co., 1953) found general reproductive toxicity at high 

30 doses (about 947 mg/kg-day).

31

32 4.6.2. Inhalation 

33 In a case study of workers engaged in the production of biphenyl-impregnated paper, 

34 Häkkinen et al. (1973, 1971) observed liver damage (elevated levels of serum AST and ALT; 

35 incipient cirrhosis and fatty changes in biopsy specimens) and effects on the central and 

36 peripheral nervous systems (polyneuritic symptoms [abnormal EEGs and ENMGs], giddiness, 

37 fatigue) that were attributed to long-term exposure to high concentrations of biphenyl. In one 

38 fatal case, autopsy revealed kidney and bone marrow damage and heart muscle degeneration, as 
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1 well as brain edema (Häkkinen et al., 1973, 1971). More recently the possibility has been 

2 discussed that long-term exposure to biphenyl might contribute to the onset of PD (Wastensson 

3 et al., 2006). The workplace conditions reported for these studies (Wastensson et al., 2006; 

4 Häkkinen et al., 1973, 1971) suggested that inhalation represented the predominant route of 

5 exposure, but dermal absorption as well as oral uptake (hand to mouth) might have occurred at a 

6 significant level.

7 In mice, short-term biphenyl inhalation at concentrations as high as 54.75 ppm 

(345.5 mg/m3 8 ) appeared to cause no symptoms (Sun Company Inc., 1977a). In another study, 

9 3 rabbits, 4–6 rats, or 12 mice/group were exposed to biphenyl by inhalation for 7–13 weeks at 

concentrations ranging from 5 to 300 mg/m3 10 (Deichmann et al., 1947). No adverse effects were 

11 observed in rabbits, while rats and mice showed irritation of mucous membranes and succumbed 

12 to high concentrations. Mice were far more sensitive than rats in these experiments, additionally 

13 showing congestion and hemorrhage of the lungs (Deichmann et al., 1947). Repeated exposure 

of mice to biphenyl at vapor concentrations of 25 or 50 ppm (157.75 or 315.5 mg/m3 14 ) for 

15 13 weeks resulted in high incidences of pneumonia and tracheal hyperplasia, and high incidences 

16 of congestion and edema in the lungs, liver, and kidney (Sun Company Inc., 1977b). 

17 Reproductive or developmental studies using the inhalation route of exposure were not 

18 identified. 

19

20 4.6.3. Mode-of-Action Information

21 The studies described above have demonstrated that exposure to biphenyl may lead to a 

22 variety of noncancer health effects (i.e., weight loss, liver toxicity, urinary tract toxicity). 

23 However, there is not sufficient information to determine the mode of action for noncancer 

24 health effects following exposure to biphenyl. 

25 Weight loss or lack of weight gain has been consistently associated with oral exposure to 

26 biphenyl (Umeda et al., 2005, 2002; Ambrose et al., 1960; Dow Chemical Co., 1953). The work 

27 of Nishihara (1985) provides a possible explanation for this toxic effect. This author found that, 

28 in vitro, biphenyl can act as an uncoupler of respiration. It may be speculated that long-term, 

29 high-dose exposure to biphenyl uncouples mitochondrial respiration to a certain extent, resulting 

30 in a futile cycle that diverts the use of nutrients from building body mass into maintaining 

31 necessary energy stores. It is not clear at what level of in vivo exposure this effect might become 

32 operative. 

33 Several of the oral animal studies (Umeda et al., 2005; Sun Company Inc., 1977b; 

34 Pecchiai and Saffiotti, 1957; Dow Chemical Co., 1953; Deichmann et al., 1947) and the 

35 epidemiological study by Häkkinen et al. (1973) provide evidence that the liver is a target for 

36 biphenyl toxicity by any route of exposure. This evidence consists of changes in blood 

37 parameters that are indicative of liver toxicity; however, in animal studies, liver histopathology 

38 does not support or explain this finding. Evidence for damage to the nervous system, as 
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1 suggested by Häkkinen et al. (1973) and Seppäläinen and Häkkinen (1975), has not been 

2 reproduced in animal studies. The limited evidence for an estrogenic activity of 

3 4,4′-dihydroxybiphenyl (Kitamura et al., 2003; Schultz et al., 2002) is insufficient to assign a 

4 clear endocrine-disrupting effect to this important metabolite of biphenyl.

5 Damage to the urinary tract has been observed consistently in animals but not in humans. 

6 The work of Ohnishi et al. (2001, 2000a, b) provides tenable evidence that, in the rat, this is due 

7 to the precipitation in the urinary tract of crystals consisting mostly of 4-hydroxybiphenyl. 

8 These crystals irritate the epithelia of ureters and bladder, leading to chronic inflammation and 

9 possibly cancer as well as obstruction of the urinary tract with subsequent hydronephrosis. The 

10 work of Ohnishi et al. (2001, 2000b) has made it clear that, at least in their animal model, two 

11 conditions are required for this event to occur: (1) the pH in the urine of the animals needs to be 

12 higher than normal and (2) elevated potassium levels need to accompany the elevated pH 

13 because it is the potassium salt of 4-hydroxybiphenyl sulphate that has the lowest solubility in 

14 high-pH urine. No damage to the urinary tract was observed in rabbits exposed via inhalation to 

15 biphenyl for up to 13 weeks (Deichmann et al., 1947). Although this mode of action is likely to 

16 explain the effects of biphenyl in the urinary tract of rats, it is unclear whether or not it has any 

17 bearing on humans that are likely exposed by inhalation. 

18 Gombar et al. (1991) developed structure activity relationship computer models for four 

19 types of chemical compounds (carboaromatic, heteroaromatic, alicyclic, acyclic) to estimate the 

20 teratogenic potential of 171 compounds (for which teratogenic data exist in >900 publications) in 

21 an overall procedure (dosage, maternal toxicity, and affected organ systems were not factored 

22 into these preliminary models). The models considered species, route of administration, and 

23 duration and timing of exposure. Experimental endpoints entered into the model were number of 

24 dams; maternal toxicity; teratogenic endpoints; numbers of viable implants, resorptions, and 

25 abnormal fetuses; and dead/live fetus ratio. Fetal deaths per se, runting, delayed ossification, and 

26 minor skeletal abnormalities such as extra or missing ribs were not rated as teratogenic effects. 

27 The computerized modeling uses a coding system that represents only “heavy” atoms (i.e., no 

28 hydrogens). The models included molecule fragments and their electronic descriptors to 

29 represent functional groups, molecular shape descriptors, and connectivity descriptors. The 

30 results of the calculations were presented as 24 different structural descriptor values. After 

31 eliminating two types of results (outliers and “statistically influential”), the models returned a 

32 96% correct classification of the teratogenic potential of chemicals. Biphenyl and 

33 2-hydroxybiphenyl were negative in this computerized evaluation.

34

35 4.7. EVALUATION OF CARCINOGENICITY

36 4.7.1. Summary of Overall Weight of Evidence

37 Under EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a), the 

38 database for biphenyl provides “suggestive evidence of carcinogenic potential.” This cancer 
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1 weight-of-evidence descriptor is based on urinary bladder tumors (transitional cell papillomas 

2 and carcinomas) in male F344 rats (Umeda et al., 2002) and liver tumors (hepatocellular 

3 adenomas and carcinomas) in female BDF1 mice (Umeda et al., 2005) exposed to biphenyl in the 

4 diet for 104 weeks. Earlier chronic toxicity and carcinogenicity assessments in orally exposed 

5 animals found no clear evidence of biphenyl-induced carcinogenicity in rats (Shiraiwa et al., 

6 1989; Ambrose et al., 1960; Pecchiai and Saffiotti, 1957; Dow Chemical Co., 1953), mice (Imai 

7 et al., 1983; Innes et al., 1969; NCI, 1968), dogs (Monsanto, 1956), or Rhesus monkeys (Dow 

8 Chemical Co., 1953). The earlier studies had limitations including small numbers of animals in 

9 exposure groups and shorter-than-lifetime durations of exposure due to design or decreased 

10 survival unrelated to tumor development, with the exception of a mouse study that found no 

11 evidence of carcinogenic responses in female ddY mice exposed to 5,000 ppm biphenyl in the 

12 diet for 2 years (Imai et al., 1983). 

13 Considerable evidence suggests that the development of urinary bladder tumors in male 

14 rats exposed to biphenyl depends on the sustained occurrence of urinary bladder calculi 

15 composed of precipitated 4-hydroxybiphenyl-O-sulphate, based on: (1) close correlation 

16 between urinary bladder calculi formation and development of urinary bladder tumors in male 

17 rats exposed to biphenyl, (2) dose-response and temporal concordance between biphenyl-induced 

18 urinary calculi formation, regenerative hyperplasia, and urinary bladder tumor development, 

19 (3) an overall negative or equivocal weight of evidence for the genotoxicity of biphenyl and 

20 metabolites, and (4) the wide body of evidence that other nongenotoxic or weakly genotoxic 

21 chemicals produce urinary bladder tumors in rodents at high exposure levels by a mode of action 

22 involving calculi formation, followed by ulceration or inflammation and regenerative cell 

23 proliferation (IARC, 1999b). Mode-of-action information is sufficient to conclude that these 

24 tumors are high-dose phenomena; without the development of calculi, urinary bladder tumors are 

25 not expected. The proposed mode of action is expected to be relevant to humans at exposure 

26 levels sufficient to cause urinary bladder calculi in humans, because calculi in humans have been 

27 associated with urinary bladder irritation, regeneration, and cancer (IARC, 1999b; Cohen, 1998, 

28 1995) and the metabolism of biphenyl to sulphate conjugates of hydroxylated biphenyl 

29 metabolites has been demonstrated in human tissues.

30 For liver tumors, a proposed mode of action (Umeda et al., 2004) includes activation of 

31 peroxisome proliferator activated receptors (PPARs) by biphenyl or its metabolites in liver cells 

32 or direct or indirect (through ROS) reactions with DNA in liver cells to produce mutations 

33 leading to tumor initiation. However, available data are insufficient to establish a mode of 

34 action for liver tumors in female mice (See Section 4.7.3.2.2.1 for more information). In the 

35 absence of information to indicate otherwise, the development of liver tumors in female mice 

36 with chronic exposure to biphenyl is assumed to be relevant to humans. EPA acknowledges that 

37 some mouse strains (e.g., B6C3F1) are relatively susceptible to liver tumors and the background 

38 incidence of this tumor can be high. For these reasons, use of mouse liver tumor data in risk 
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1 assessment has been a subject of controversy (King-Herbert and Thayer, 2006). The BDF1

2 mouse used in the Umeda et al. (2005) bioassay is a cross between female C57BL/6 and male 

3 DBA/2 mice (Charles River Laboratories International, Inc., 1999), both of which are considered 

4 to be relatively resistant to liver tumor induction (Maronpot, 2009). In the Umeda et al. (2005) 

5 bioassay, the incidences of tumors in male and female concurrent control mice were 32 and 6%, 

6 respectively. The relatively low background incidence of liver tumors in female control mice 

7 from Umeda et al. (205) minimizes the possible confounding of compound-related liver tumors

8 in this sex.

9 The descriptor of “suggestive evidence of carcinogenic potential” is appropriate when the 

10 weight of evidence is suggestive of carcinogenicity, i.e., a concern for potential carcinogenic 

11 effects in humans is raised, but the data are judged not sufficient for a stronger conclusion (U.S. 

12 EPA, 2005a). As discussed in Section 4.2.1.2, biphenyl exposure produced an increased 

13 incidence of urinary bladder tumors in male F344 rats (Umeda et al., 2002) and liver tumors in 

14 female BDF1 mice (Umeda et al., 2005). Such data could be considered consistent with the 

15 descriptor of “likely to be carcinogenic to humans.” As stated in the Guidelines for Carcinogen 

16 Risk Assessment (U.S. EPA, 2005a), a “likely” descriptor may include “an agent that has tested 

17 positive in animal experiments in more than one species, sex, strain, site, or exposure route, with 

18 or without evidence of carcinogenicity in humans.” Biphenyl did induce tumors in two species 

19 (rat and mouse) and at two sites (liver and urinary bladder); however, tumor findings across the 

20 biphenyl database and the interpretation of some of these findings indicate some uncertainties 

21 regarding the potential human carcinogenicity of biphenyl. 

22 Both the liver tumors and urinary bladder tumors induced by dietary exposure to biphenyl 

23 each occurred in only one sex and only one species. Liver tumors were induced in female BDF1

24 mice only, and urinary bladder tumors occurred in male F344 rats only. The incidence of liver 

25 adenomas and carcinomas (separate and combined) in Umeda et al. (2002) was increased over 

26 control in all groups of exposed female mice; however, the liver tumor incidence plateaued at the 

27 mid- and high-dose groups (incidence of adenoma and carcinoma combined in the control and 

28 low-, mid-, and high-dose groups were 3/48, 8/50, 16/49, and 14/48, respectively). Further, 

29 female ddY mice exposed to 5000 ppm biphenyl in the diet for 2 years showed no increased 

30 incidence of liver tumors (Imai et al., 1983). Urinary bladder tumors in F344 male rats induced 

31 by dietary biphenyl exposure appear to be a high-dose phenomenon closely related to the 

32 formation of calculi. A mode of action analysis (see Section 4.7.3.1) supports the conclusion 

33 that exposures that do not lead to urinary bladder calculi will not produce tumors. While the 

34 proposed mode of action for urinary bladder tumors in male rats is considered relevant to 

35 humans, there is evidence that humans are likely to be less susceptible to these tumors than rats. 

36 As discussed in Section 4.7.3.1.4.2, the rodent horizontal quadruped stature is expected to 

37 promote calculi residency time in the bladder without causing obstruction, whereas the anatomy 

38 of the urinary tract in humans and their upright bipedal stature result in more ready excretion of 
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1 calculi in the urine or therapeutic removal of calculi that form obstructions (Cohen and Ellwein, 

2 1992; Matanowki, 1981). Overall, the mode of action analysis suggests that biphenyl is not 

3 likely to induce urinary bladder tumors in humans at environmental exposure levels. In light of 

4 the above considerations related to biphenyl-induced female mouse liver tumors and male rat 

5 bladder tumors, EPA concluded that the currently available information is most consistent with a 

6 determination that the database for biphenyl provides “suggestive evidence of carcinogenic 

7 potential.” 

8 U.S. EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) indicate that 

9 for tumors occurring at a site other than the initial point of contact, the cancer descriptor may 

10 apply to all routes of exposure that have not been adequately tested at sufficient doses. An 

11 exception occurs when there is convincing toxicokinetic data that absorption does not occur by 

12 other routes. Information available on the carcinogenic effects of biphenyl demonstrates that 

13 tumors occur in tissues remote from the site of absorption following chronic oral exposure 

14 (urinary bladder in male rats and liver in female mice). No information on the carcinogenic 

15 effects of biphenyl via the inhalation or dermal routes in humans and animals is available. 

16 Quantitative data demonstrating rapid and extensive absorption of biphenyl are restricted to the 

17 oral route of exposure; a case report of hepatic toxicity produced by a probable combination of 

18 inhalation and dermal exposures in a worker in a biphenyl-impregnated fruit wrapping paper 

19 production facility provides qualitative evidence of absorption by these routes (Häkkinen et al., 

20 1973). Therefore, based on the observance of systemic tumors following oral exposure and 

21 assumed absorption by all routes of exposure, it is assumed that an internal dose will be achieved 

22 regardless of the route of exposure. Therefore, EPA considers the biphenyl database to provide 

23 “suggestive evidence of carcinogenic potential” by all routes of exposure. 

24

25 4.7.2. Synthesis of Human, Animal, and Other Supporting Evidence

26 Available human studies were not designed to evaluate associations between exposure to 

27 biphenyl and occurrence of cancer (see Section 4.1). 

28 As discussed in Section 4.2, carcinogenicity studies in animals are limited to the oral 

29 exposure route. In well-designed cancer bioassays of F344 rats (Umeda et al., 2002) and BDF1

30 mice (Umeda et al., 2005), dietary exposure to biphenyl resulted in the occurrence of urinary 

31 bladder tumors in male rats and significantly increased incidences in liver tumors in female mice.

32 Earlier chronic toxicity and carcinogenicity assessments found no clear evidence of 

33 biphenyl-induced carcinogenicity in orally exposed rats, mice, dogs, or Rhesus monkeys. 

34 However, these studies were generally limited in design, with the exception of a study reporting 

35 no evidence of carcinogenic responses in female ddY mice (n = 34 mice vs. 37 control mice) 

36 exposed to 5,000 ppm biphenyl in the diet for 2 years (Imai et al., 1983). In a study of Wistar 

37 rats, sufficient numbers of animals (50/sex/group) were exposed to biphenyl in the diet at 

38 concentrations up to 5,000 ppm, but only for 75 weeks (Shiraiwa et al., 1989). Some of the male 
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1 rats exhibited urinary bladder calculi and simple or diffuse hyperplasia and papillomatosis of the 

2 urinary bladder mucosa in the absence of neoplastic lesions, but the study may have been 

3 terminated prior to eventual urinary bladder tumor development. Ambrose et al. (1960) exposed 

4 albino rats (15/sex/exposure level) to biphenyl in the diet at concentrations up to 10,000 ppm for 

5 2 years (10, 50, 100, 500, 1,000, 5,000, or 10,000 ppm); however, decreased survival in rats 

6 exposed to 5,000 or 10,000 ppm, presumably from decreased food consumption, and the 

7 relatively small numbers of animals in each exposure group may have impaired the ability to 

8 detect late-developing tumors. In another study, groups of Sprague-Dawley rats (12/sex/group) 

9 received biphenyl in the diet at concentrations up to 10,000 ppm for up to 2 years (Dow 

10 Chemical Co., 1953). However, this study suffered from a pneumonia outbreak, particularly 

11 among control males, and the relatively small numbers of animals and the decreased survival 

12 may have impaired the ability to detect late-developing tumors. A study of male albino rats 

13 included small numbers of rats (8/group) and a short (13 months) exposure period (Pecchiai and 

14 Saffiotti, 1957). A study of B6C3F1 or B6AkF1 mice exposed to biphenyl in the diet for only 

15 18 months (Innes et al., 1969; NCI, 1968) included relatively small numbers of mice 

16 (18/sex/group) and only one exposure level (517 ppm) that was similar to the concentration 

17 (667 ppm) without carcinogenic effect in the Umeda et al. (2005) 24-month BDF1 mouse 

18 bioassay. The dog study included two males and one female, a high dose of 25 mg/kg-day, and 

19 an exposure period of only 1 year (Monsanto, 1956). Rhesus monkeys (two males and one 

20 female) were exposed to biphenyl in the diet at a concentration of 10,000 ppm, but for only 

21 1 year (Dow Chemical Co., 1953). 

22 The overall weight of evidence for biphenyl genotoxicity from short-term tests is 

23 negative or equivocal. Biphenyl did not induce mutations in a variety of bacterial test systems, 

24 but both negative and positive results were obtained in mammalian in vitro test systems (see 

25 section 4.5.6. for references). Single oral doses of 2,000 mg biphenyl/kg induced DNA damage 

26 (detected by the Comet assay) in several organs of CD-1 mice (including the liver and bladder), 

27 but it is uncertain if the damage was due to a direct effect on DNA or was an indirect effect due 

28 to cytotoxicity or ROS generated by redox cycling of phenylhydroquinone, a major urinary 

29 metabolite of 2-hydroxybiphenyl and a minor metabolite of biphenyl in rats. 

30 The overall weight of evidence for 2-hydroxybiphenyl genotoxicity suggests that 

31 oxidative DNA damage from ROS from redox cycling between 2,5-dihydroxybiphenyl and 

32 phenylbenzoquinone is possible. DNA damage was detected in liver and bladder of CD-1 mice 

33 exposed to 2,000 mg/kg of 2-hydroxybiphenyl (Sasaki et al., 2002, 1997) and in the urinary 

34 bladder of male F344 rats fed the sodium salt of 2-hydroxybiphenyl at 1 or 2% in the diet for 3–

5 months (Morimoto et al., 1989). DNA adducts were detected by [32 35 P]-post labeling in skin of 

36 CD-1 mice after topical application of the sodium salt of 2-hydroxybiphenyl or phenylhydro37 quinone (Pathak and Roy, 1993), and increased micronuclei were detected in urinary bladder 

38 epithelium of male F344 rats exposed to 2,000 ppm 2-hydroxybiphenyl or 2,000 ppm NaCl plus 
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1 2,000 ppm 2-hydroxybiphenyl in the diet for 2 weeks (Balakrishnan et al., 2002). However, 

2 increased binding of radioactivity to DNA was not detected in DNA extracted from urinary 

3 bladder epithelium of male F344 rats exposed to single gavage doses of 2-hydroxybiphenyl as 

4 high as 1,000 mg/kg (Kwok et al., 1999), and DNA adducts were not detected in urinary bladder 

5 epithelium of male F344 rats exposed for 13 weeks to biphenyl dietary concentrations as high as 

6 12,500 ppm (Smith et al., 1998). The mechanism by which 2-hydroxybiphenyl may induce 

7 micronuclei in the urinary bladder epithelium is uncertain, but could involve micronuclei 

8 generation as a secondary response to cytotoxicity or regenerative cell proliferation, DNA 

9 damage from ROS from redox cycling of 2,5-dihydroxybiphenyl, or protein modifications 

10 leading to mitotic spindle interference or inhibition of enzymes important in DNA replication 

11 (Balakrishnan et al., 2002). The hydroxylation of biphenyl to produce 2-hydroxybiphenyl is a 

12 minor pathway in rats and mice (Halpaap-Wood et al., 1981a, b; Meyer and Scheline, 1976). 

13 2-Hydroxybiphenyl and 2,5-dihydroxybiphenyl collectively accounted for less than 2% of 

14 metabolites in urine of rats administered single oral doses of 100 mg biphenyl/kg (Meyer and 

15 Scheline, 1976) or single i.p. doses of 30 mg biphenyl/kg (Halpaap-Wood et al., 1981a). In mice 

16 given i.p. doses of 30 mg biphenyl/kg, these metabolites accounted for less than 5% of urinary 

17 metabolites (Halpaap-Wood et al., 1981a). 

18 

19 4.7.3. Mode-of-Action Information

20 4.7.3.1. Mode-of-Action Information for Bladder Tumors in Male Rats

21 4.7.3.1.1. Hypothesized mode of action. The best-supported hypothesis proposes a mode of 

22 action whereby the formation of urinary bladder calculi (from the precipitation of 4-

23 hydroxybiphenyl-O-sulphate) is a key event in the development of urinary bladder tumors in 

24 male rats fed high levels of biphenyl in the diet for 2 years. According to this hypothesis, the 

25 calculi (occurring in association with increased urinary pH and potassium, and predominantly 

26 composed of 4-hydroxybiphenyl-O-sulphate) cause irritation to transitional epithelial cells of the 

27 urinary bladder leading to sustained cell proliferation, which promotes the development of 

28 initiated cells in the urinary bladder with progression to papillomas and carcinomas.

29

30 4.7.3.1.2. Experimental support for the hypothesized mode of action

31 4.7.3.1.2.1. Strength, consistency, and specificity of association, including support for the 

32 hypothesized mode of action in male rats. The formation of urinary bladder calculi, 

33 predominantly composed of potassium 4-hydroxybiphenyl-O-sulphate, is strongly, consistently, 

34 and specifically associated with the formation of urinary bladder tumors in male rats chronically 

35 exposed to high dietary concentrations of biphenyl. Several findings support this association. 

36 Urinary bladder calculi were formed at a high prevalence (43/50; 86%) in a group of male rats 

37 exposed to biphenyl in the diet at a concentration of 4,500 ppm, but were absent in male rats 

38 receiving diets containing 0, 500, or 1,500 ppm biphenyl (Umeda et al., 2002). These 
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1 observations were consistent with the detection of urinary bladder transitional cell papilloma 

2 (10/50; 20%), carcinoma (24/50; 48%), and papilloma or carcinoma (31/50; 62%) in the 

3 4,500 ppm group of male rats and total absence of urinary bladder papilloma or carcinoma in the 

4 control, 500, or 1,500 ppm groups of male rats. Bladder calculi were found in all 24 of the male 

5 rats with urinary bladder transitional cell carcinoma and in 8/10 of the male rats with transitional 

6 cell papilloma.

7 The association between urinary bladder calculus formation and development of urinary 

8 bladder tumors is both gender and species specific. Urinary bladder calculi, of similar size to 

9 those observed in males, were observed at much lower incidence (8/50; 16%) in the 4,500 ppm 

10 female rats, but they were of more uniform color (white and yellow versus white, yellow, brown, 

11 gray, and black in males) and shape (spheroidal vs. triangular, pyramidal, cubical, and spheroidal 

12 in males) and primarily composed of 4-hydroxybiphenyl and potassium bisulphate (which are 

13 hydrolysis products of potassium 4-hydroxybiphenyl-O-sulphate) (Umeda et al., 2002; Ohnishi 

14 et al., 2000b). No urinary bladder calculi were found in the 500 and 1,500 ppm groups of female 

15 rats. Transitional cell hyperplasia was found in 10/50 4,500-ppm female rats, but no urinary 

16 bladder transitional cell papillomas or carcinomas were seen in any of the biphenyl-exposed 

17 groups of female rats. Furthermore, there was no evidence of biphenyl-induced urinary bladder 

18 calculi or bladder tumors in male or female BDF1 mice receiving dietary biphenyl at 

19 concentrations as high as 6,000 ppm for 2 years (Umeda et al., 2005). 

20 Urinary bladder calculi in male rats were associated with significantly increased urinary 

21 pH (average pH of 7.97 in the 4,500 ppm group at the final week of exposure compared to 

22 7.66 in controls) (Umeda et al., 2002) and were composed primarily of potassium 

23 4-hydroxybiphenyl-O-sulphate (Ohnishi et al., 2000b). The urine pH of female rats exposed to 

24 4,500 ppm for 104 weeks (pH = 7.26) was not elevated compared with controls (pH = 7.29) 

25 (Umeda et al., 2002), and urinary calculi of a different composition than male rats (i.e., 

26 4-hydroxybiphenyl and potassium bisulphate, compared with potassium 4-hydroxybiphenyl27 O-sulphate in males) were found in only 8/50 4,500-ppm females (Ohnishi et al., 2000b). From 

28 these observations, it appears that the formation of the calculi results from the precipitation of the 

29 potassium salt of the sulphate conjugate of 4-hydroxybiphenyl under the elevated pH conditions 

30 of the male rat urine. The mechanism responsible for increased urinary pH is unknown, although 

31 Ohnishi et al. (2001, 2000a, b) proposed that gender differences in urinary conditions, such as 

32 pH and potassium concentrations, and sulphatase activities in kidneys, may be responsible for 

33 the gender differences in urinary calculi composition and formation and the subsequent 

34 development of urinary bladder tumors in male, but not female, F344 rats.

35 Relatively strong, consistent, and specific associations between calculi formation and 

36 transitional cell hyperplasia and between transitional cell hyperplasia and the development of 

37 transitional cell tumors in the urinary bladder have been shown in male F344 rats chronically 

38 exposed to high concentrations of biphenyl in the diet. Urinary bladder transitional cell 
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1 hyperplasia (simple, nodular, papillary) occurred in 45/50 (90%) male rats receiving biphenyl in 

2 the diet for 2 years at the same dietary concentration (4,500 ppm) at which high prevalences of 

3 both urinary bladder calculi formation (43/50; 86%) and transitional cell tumors (31/50 62%) 

4 were observed (Umeda et al., 2002). Forty-two of the 45 male rats with urinary bladder 

5 transitional cell hyperplasia also exhibited urinary bladder calculi. In another study, evidence of 

6 biphenyl-induced calculi formation (microcalculi in the urine) and increased indices of urinary 

7 bladder transitional cell proliferation (greater than fourfold increase in BrdU incorporation) in 

8 male F344 rats has been reported following as little as 4–8 weeks of dietary exposure to 

9 5,000 ppm biphenyl (Shibata et al., 1989b). 

10 The most convincing evidence that degenerative changes in the urinary bladder 

11 epithelium lead to tumor formation is the site-concordance of associations between calculi 

12 formation in the urinary bladder, transitional cell proliferation, transitional cell hyperplasia, and 

13 transitional cell tumors (Umeda et al., 2002). In addition, the strong associations between 

14 urinary tract calculi formation, ulcerations or inflammation, and subsequent hyperplasia 

15 combined with repeated, high-level exposure to other chemicals that cause urinary bladder 

16 tumors in rodents, including melamine, uracil, and the sodium salt of 2-hydroxybiphenyl (IARC 

17 1999a, b, c; Cohen, 1998; 1995) provide further evidence that degenerative changes are involved 

18 in the etiology of rodent urinary bladder tumors. It is not unusual to see extensive proliferation 

19 or hyperplasia in bladder epithelium in response to urinary calculi from other rodent bladder 

20 tumorigens without an associated ulceration or intense inflammatory response. In male rats 

21 exposed to 4,500 ppm biphenyl, increasing numbers of rats with clinical hematuria were 

observed beginning at about the 40th 22 week of exposure, and histologic examinations at study 

23 termination revealed focal hyperplasia in 45/50 rats, providing some evidence of calculi-induced 

24 bladder epithelial damage followed by cell proliferation (Umeda et al., 2002). Over the course of 

25 the study, 94% of male rats with hematuria had bladder or kidney calculi, but hematuria was not 

26 found in any biphenyl-exposed females. In addition, with 8 weeks, but not 4 weeks, of exposure 

27 to 5,000 ppm biphenyl in the diet, moderate urinary bladder epithelial hyperplasia and 

28 microcalculi in urine were observed in 5/5 male F344 rats, but no descriptions of degenerative 

29 changes were provided; these observations are consistent with a rapid repair response to 

30 epithelial damage from biphenyl-induced urinary tract calculi (Shibata et al., 1989b). 

31 The ability of repeated biphenyl exposure to promote previously initiated urinary bladder 

32 cells to bladder tumors is supported by results of a bladder tumor initiation-promotion study 

33 (Kurata et al., 1986). Incidences of urinary bladder hyperplasia, papilloma, and carcinoma were 

34 significantly increased in male F344 rats initiated with dietary BBN for 4 weeks followed by 

35 5,000 ppm biphenyl in the diet for 32 weeks, compared with rats receiving BBN only for 

36 4 weeks. For example, 94 and 83% of rats treated with BBN followed by biphenyl developed 

37 urinary bladder hyperplasia and papillomas, respectively, compared with 25 and 12% of rats 

38 exposed to BBN alone. 
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1 The hypothesis that the mode of action involves the development of urinary bladder 

2 tumors in biphenyl-exposed male rats is further supported by an overall negative or equivocal 

3 weight of evidence for the genotoxicity of biphenyl. As discussed earlier, there are consistently 

4 negative results for biphenyl in bacterial mutation assays and inconsistent positive results for 

5 biphenyl in in vitro mammalian assays mostly in the presence of metabolic activation. There is 

6 evidence that 2,5-dihydroxybiphenyl (i.e., phenylhydroquinone), the principal urinary metabolite 

7 in rats exposed to high doses of 2-hydroxybiphenyl, can undergo redox cycling to produce ROS

8 that may damage DNA and lead to tumor-initiating mutations; however, 2-hydroxybiphenyl is a 

9 minor urinary metabolite of biphenyl in rats and 2,5-dihydroxybiphenyl was not detected in urine 

10 of rats exposed to oral doses of 100 mg biphenyl/kg (Meyer and Scheline, 1976). 

11

4.7.3.1.2.2. Dose-response concordance

21

12 . Dose-response relationships for urinary bladder 

13 calculi formation, transitional cell hyperplasia, and transitional cell tumor development show 

14 concordance in the 2-year oral study of rats (Umeda et al., 2002). In male rats, urinary calculi, 

15 nonneoplastic lesions (epithelial hyperplasia), and neoplastic lesions (papillomas and 

16 carcinomas) of the urinary bladder were observed only at the highest exposure level 

17 (4,500 ppm); no urinary bladder calculi, transitional cell hyperplasia, or transitional cell tumors 

18 were found in control, 500, or 1,500 ppm male rats. Furthermore, urinary bladder calculi were 

19 found in 43/45 high-dose male rats, in all 24 male rats with transitional cell carcinoma, and in 

20 8/10 of the male rats with transitional cell papilloma.

4.7.3.1.2.3. Temporal relationship

34

22 . Results from the 2-year oral study in rats (Umeda et al., 

23 2002) provide some evidence of a progression from urinary bladder calculi formation to the 

24 development of bladder tumors. Urinary bladder calculi were observed in the first 4,500 ppm 

25 male rat that died (week 36), evidence of blood in the urine was observed in 4,500 ppm male rats 

26 by week 40, and incidences of bladder calculi and bloody urine that paralleled increases in 

27 mortality and tumor formation were observed throughout the remainder of the study. In addition, 

28 results of a short-term oral study demonstrate that microcalculi can be detected in the urine of 

29 male rats after as little as 4 weeks of dietary exposure to 5,000 ppm biphenyl and that 

30 hyperplasia of urinary bladder epithelium can be detected at least by week 8 (Shibata et al., 

31 1989b). Presumably, the development of biphenyl-induced urinary bladder tumors requires a 

32 longer exposure period to urinary calculi of sufficient size, shape, and composition to induce 

33 urinary bladder epithelial damage and a sustained proliferative response. 

35 4.7.3.1.2.4. Biological plausibility and coherence. The proposed mode of action is consistent 

36 with the current understanding of cancer biology and is supported by the wide body of evidence 

37 that other chemicals with primarily nongenotoxic profiles produce urinary bladder tumors in 

38 rodents at high exposure levels by a mode of action involving calculi formation, ulceration or 
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1 inflammation, and regenerative cell proliferation ( IARC, 1999a, b, c; Cohen, 1998, 1995). 

2 Additional information could strengthen the plausibility and coherence of the proposed mode of 

3 action to explain the occurrence of biphenyl-induced urinary bladder tumors in male rats. These 

4 additional data include results from investigations of earlier time points in the proposed temporal 

5 progression from calculi formation to epithelial damage, regenerative cell proliferation, and 

6 tumor development and further investigations into the factors underlying gender-specific 

7 differences in precipitation of 4-hydroxybiphenyl-O-sulphate to form bladder calculi in rats.

8 

9 4.7.3.1.3. Other possible modes of action for bladder tumors in male rats. Although the 

10 weight of evidence from short-term standard genotoxicity tests with biphenyl and 

11 4-hydroxybiphenyl is predominantly negative, evidence is available that suggests that oral 

12 exposure to high doses of 2-hydroxybiphenyl is associated with the development of urinary 

13 bladder tumors in male rats. The induction of genotoxic effects in the urinary bladder epithelium 

14 leading to tumor initiation is proposed to occur via redox cycling between 2,5-

15 dihydroxybiphenyl and phenylbenzoquinone (Balakrishnan et al., 2002; Kwok et al.,1999; 

16 Pathak and Roy, 1993; Morimoto et al., 1989). However, the strong, consistent, and specific 

17 association between the occurrence of urinary bladder calculi composed of 4-hydroxybiphenyl18 O-sulphate and development of urinary bladder tumors in male but not female rats, the evidence 

19 that 2-hydroxybiphenyl is a minor urinary metabolite of biphenyl and, finally, that 2,5-

20 dihydroxybiphenyl was not detected in the urine of biphenyl-exposed rats, demonstrate that the 

21 support for a genotoxic mode of action involving key mutational events from biphenyl or its 

22 metabolites in the urinary bladder leading to initiation of tumor cells is not compelling. 

23 Additional support for a proposed genotoxic mode of action would come from studies showing 

24 formation of 2,5-dihydroxylbiphenyl and phenylbenzoquinone in the urinary bladder epithelium 

25 of rats exposed to low doses of biphenyl. 

26

27 4.7.3.1.4. Conclusions about the hypothesized mode of action for bladder tumors in male rats.

4.7.3.1.4.1. Support for the hypothesized mode of action in rats

33 To summarize, chronic exposure of male rats to a high dietary concentration of biphenyl 

34 (4,500 ppm) caused increased urinary pH and high prevalence of urinary bladder calculi (from 

35 the precipitation of 4-hydroxybiphenyl-O-sulphate in the urine), transitional cell hyperplasia, and 

36 transitional cell tumors. Incidences of male rats with calculi and those with bladder tumors were 

37 strongly correlated, and chronic exposure of male rats to lower dietary concentrations of 

38 biphenyl (500 and 1,500 ppm) did not increase urinary pH and did not cause calculi formation, 

28 . There is strong evidence that 

29 urinary bladder tumors in male rats chronically exposed to biphenyl in the diet is a high-dose 

30 phenomenon involving sustained occurrence of calculi in the urinary bladder leading to 

31 transitional cell damage, sustained regenerative cell proliferation, and eventual promotion of 

32 spontaneously initiated tumor cells in the urinary bladder epithelium. 
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1 transitional cell hyperplasia, or bladder tumor development. There were relatively strong 

2 associations between incidences of rats with calculi and those with transitional cell hyperplasia 

3 and between incidences of rats with transitional cell hyperplasia and bladder tumors. In contrast, 

4 high concentrations of biphenyl in the diet of female rats had no effect on urinary pH, caused a 

5 much lower prevalence of urinary bladder calculi of a different composition, and resulted in no 

6 urinary bladder tumors. The urinary bladder calculi in the male rats were mainly composed of 

7 the conjugated biphenyl metabolite, potassium 4-hydroxybiphenyl-O-sulphate, whereas those of 

8 the female rats were predominantly composed of 4-hydroxybiphenyl and potassium bisulphate 

9 (which are hydrolysis products of potassium 4-hydroxybiphenyl-O-sulphate). There was no 

10 evidence of urinary bladder calculi formation or tumor development in male and female mice 

11 exposed to similar dietary concentrations of biphenyl. Results of a tumor initiation-promotion 

12 study in male rats support the proposal that biphenyl-induced sustained cell proliferation 

13 promotes initiated tumor cells in the urinary bladder. Finally, results of genotoxicity tests with 

14 biphenyl are predominantly negative or equivocal at best. The preponderance of evidence 

15 supports a mode of action for biphenyl in male rats only involving urinary tract calculi 

16 formation, urinary epithelium damage, sustained regenerative cell proliferation and hyperplasia, 

17 and subsequent bladder tumor formation. There is evidence that 2,5-dihydroxybiphenyl can 

18 undergo redox cycling to produce ROS that may damage DNA leading to tumor-initiating 

19 mutations, but it was not detected in urine of rats exposed to oral doses of 100 mg biphenyl/kg 

20 and its metabolic precursor, 2-hydroxybiphenyl, is a minor urinary metabolite of biphenyl in rats 

21 (Meyer and Scheline, 1976). 

22

23 4.7.3.1.4.2. Relevance of the hypothesized mode of action to humans. Although there are no 

24 studies in humans examining possible associations of biphenyl exposure with urinary bladder 

25 calculi formation or cancer, urinary bladder calculi have been reported in humans following 

26 exposure to other chemicals (IARC, 1999b; Cohen 1998, 1995). Urinary bladder calculi are, in 

27 general, expected to be irritating and lead to reparative cell proliferation regardless of 

28 composition or species; however, based on the anatomy of the urinary tract in humans and their 

29 upright, bipedal stature, calculi are either quickly excreted in urine or cause obstruction leading 

30 to pain and subsequent therapeutic removal of the calculi (Cohen, 1998, 1995). In contrast, the 

31 rodent horizontal quadruped stature is expected to promote calculi residency time in the bladder 

32 without causing obstruction (Cohen, 1998, 1995). In white populations, 95% of bladder tumors 

33 are transitional cell carcinomas such as those found in male rats exposed to high concentrations 

34 of biphenyl. IARC (1999b) noted that several case-control studies of urinary bladder cancer in 

35 white human populations found relative risks for an association between a history of urinary tract 

36 stones and bladder carcinomas ranging from about 1.0 to 2.5, suggesting a causative link. Thus, 

37 the proposed mode of action is expected to be relevant to humans at exposure levels sufficient to 

38 cause urinary bladder calculi in humans, because: (1) calculi resulting from human exposure to 
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1 other substances have been associated with urinary bladder irritation, regeneration, and cancer 

2 (IARC, 1999b; Cohen 1998, 1995) and (2) sulphate conjugation of hydroxylated biphenyl 

3 metabolites has been demonstrated in human tissues (as briefly reviewed in Section 3.3). 

4 The underlying physiological factors determining the precipitation of 4-hydroxybiphenyl5 O-sulphate in urine to form calculi in male rats, but not female rats, exposed to high dietary 

6 biphenyl concentrations are unknown. Given this lack of understanding for rats and the absence 

7 of specific human data on biphenyl-induced calculi or urinary stones, there is uncertainty in 

8 extrapolation of the dose-response relationship for biphenyl-induced calculi formation in male 

9 rats to humans. 

10

11 4.7.3.1.4.3. Populations or lifestages particularly susceptible to the hypothesized mode of 

action

21

12 . IARC (1999b) noted that increased risks for bladder carcinoma in humans have been 

13 associated with cigarette smoking, exposure to infectious agents, such as Shistosoma 

14 haematobium, causing urinary tract inflammation, and a history for urinary tract infections in 

15 general. As such, people with these types of exposure or history may be particularly susceptible 

16 to the formation of urinary calculi and urinary bladder cancer, but evidence supporting this 

17 inference is lacking. In addition, there are conditions (bladder diverticuli, neurogenic bladder, 

18 and staghorn renal pelvic calculi) that can increase the residency time of calculi in humans; thus, 

19 individuals with these conditions may also be particularly susceptible to biphenyl-induced 

20 bladder tumors under the hypothesized mode of action. 

22 4.7.3.2. Mode-of-Action Information for Liver Tumors in Female Mice

23 Evidence that chronic oral exposure to biphenyl can cause liver tumors comes from the 2-

24 year BDF1 mouse bioassay by Umeda et al. (2005). Exposure to 2,000 or 6,000 ppm biphenyl in 

25 the diet, but not to 667 ppm, produced increased incidences of hepatocellular adenomas or 

26 carcinomas in female mice, but no carcinogenic response in male BDF1 mice. Earlier studies 

27 found no carcinogenic response in B6C3F1 or B6AkF1 mice exposed to 517 ppm biphenyl in the 

28 diet for 18 months (Innes et al., 1969; NCI, 1968) or in ddY female mice exposed to 5,000 ppm 

29 biphenyl in the diet for 2 years (Imai et al., 1983). The only investigations into the mode of 

30 action for biphenyl-induced liver tumors in mice involve examinations of indicators of 

31 peroxisome proliferation following biphenyl exposure (Umeda et al., 2004; Sunouchi et al., 

32 1999). Thus, a mode of action involving PPARs is proposed and an evaluation of the supporting 

33 data follows. 

34

35 4.7.3.2.1. Hypothesized mode of action for liver tumors in female mice. Proliferation of 

36 peroxisomes is regulated by a class of ligand-activated transcription factors known as PPARs. 

37 PPARα regulates induction of the peroxisome proliferation response in rodents and is thought to 

38 mediate at least some of the responses for hepatocarcinogens, including initiation of cellular 
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1 events leading to transformation. Peroxisome proliferators (PPARα agonists) are a structurally 

2 diverse group of non- or weakly mutagenic chemicals that induce a suite of responses including 

3 the induction of tumors in rats and mice (Klaunig et al., 2003). 

4 Klaunig et al. (2003) have proposed a mode of action for PPARα agonists involving the 

5 following key events. PPARα agonists activate PPARα to transcribe genes involved in 

6 peroxisome proliferation, cell cycling/apoptosis, and lipid metabolism. The changes in gene 

7 expression lead to changes in cell proliferation and apoptosis, and to peroxisome proliferation. 

8 Suppression of apoptosis coupled with increased cell proliferation allows transformed cells to 

9 persist and proliferate, resulting in preneoplastic hepatic foci and ultimately promotion of tumor 

10 growth via selective clonal expansion. Peroxisome proliferation may lead to oxidative stress, 

11 which potentially contributes to the proposed mode of action by causing indirect DNA damage 

12 and/or by causing cytotoxicity leading to reparative cell proliferation. PPARα agonists also 

13 inhibit gap junction intercellular communication and stimulate non-parenchymal hepatic Kupffer 

14 cells; these events are also thought to stimulate cell proliferation. Increases in the size and 

15 number of peroxisomes and induction of peroxisome-related gene expression (e.g., palmitoyl16 CoA oxidase and acyl-CoA oxidase) are regarded as indicators that the PPARα agonism mode of 

17 action is operative.

18

19 4.7.3.2.2. Experimental support for the hypothesized mode of action for liver tumors in female 

20 mice. 

21 4.7.3.2.2.1. Strength, consistency, specificity of association, including support for the 

hypothesized mode of action in mice

34 The available data do not demonstrate strong, consistent, or specific associations between 

35 key events in the proposed mode of action and the development of liver tumors in female mice 

36 exposed to biphenyl. Klaunig et al. (2003) proposed that an adequate data set to support a 

37 PPARα agonism mode of action should meet the following demonstration criteria, most of which 

22 . There is limited support for a possible association 

23 between biphenyl-induced proliferation of peroxisomes and liver tumors, because the following 

24 findings were reported in female BDF1 mice (which developed liver tumors following dietary 

25 exposure to 2,000 or 6,000 ppm) but not in male BDF1 mice (which did not develop liver tumors 

26 following exposure to concentrations as high as 6,000 ppm biphenyl). Dietary exposure of 

27 female BDF1 mice to 16,000 ppm biphenyl for 13 weeks induced hepatocellular peroxisomes as 

28 evidenced by light microscopy detection of enlarged hepatocytes filled with eosinophilic fine 

29 granules and electron microscopy confirmation that the granules corresponded to increased 

30 numbers of peroxisomes (Umeda et al., 2004). Significantly increased activities were measured 

31 for potassium cyanide-insensitive palmitoyl CoA oxidation in liver homogenate (up to 1.9-fold) 

32 and lauric acid 12-hydroxylation in liver microsomes (up to 3.8-fold) from female BDF1 mice 

33 given oral doses up to 5.2 mmol/kg-day (800 mg/kg-day) for 3 days (Sunouchi et al., 1999).
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1 as noted in parentheses have not been investigated for biphenyl or its metabolites: (1) activation 

2 of PPARα (no data), (2) expression of peroxisomal genes including PPARα-mediated expression 

3 of cell cycle, growth, and apoptosis, and nonperoxisomal lipid gene expression (no data), 

4 (3) peroxisomal proliferation (limited data for biphenyl in mice as summarized in previous 

5 paragraph) and perturbation of cell proliferation and apoptosis (no data for mouse liver), 

6 (4) inhibition of gap junction intercellular communication (no data), (5) hepatocyte oxidative 

7 stress (no data), (6) Kupffer cell-mediated events (no data), and (7) selective clonal expansion 

8 (no data). 

9 

4.7.3.2.2.2. Dose-response concordance

17

10 . The available data do not show concordance between 

11 the dose-response relationships for liver tumors in female BDF1 mice exposed for 2 years to 

12 biphenyl in the diet (liver tumors at 2,000 or 6,000 ppm, but not 667 ppm; Umeda et al., 2005) 

13 and liver peroxisome proliferation, the only key event in the proposed mode of action that has 

14 been investigated. Umeda (2004) reported that, compared with controls, increased liver 

15 peroxisomes were detected in female BDF1 mice exposed to 16,000 ppm biphenyl in the diet for 

16 13 weeks, but not in mice exposed to 500, 2,000, 4,000, 8,000, or 10,000 ppm. 

4.7.3.2.2.3. Temporal relationship

24

18 . Indicators of liver peroxisome proliferation were elevated 

19 in female mice, but not male mice, with oral exposure durations of 3 days following exposure to 

20 800 mg/kg-day (increased activities of potassium cyanide-insensitive palmitoyl CoA oxidation 

21 and lauric acid 12-hydroxylation; Sunouchi et al. 1999) and 13 weeks following exposure to 

22 16,000 ppm in the diet, but not at lower dietary concentrations (increased numbers of liver 

23 peroxisomes; Umeda et al. 2004). 

4.7.3.2.2.4. Biological plausibility and coherence

28

25 . The data are inadequate to evaluate the 

26 biological plausibility and coherence of the proposed mode of action as it relates to liver tumors 

27 in female mice exposed to biphenyl. 

29 4.7.3.2.3. Other possible modes of action for liver tumors in mice. As discussed in 

30 Section 4.5.5, the overall weight of evidence from short-term standard genotoxicity tests with 

31 biphenyl and 4-hydroxybiphenyl is predominantly negative. A genotoxic mode of action for 

32 biphenyl-induced liver tumors in mice could be proposed based on the large metabolic capacity 

33 of the mouse liver to convert biphenyl to hydroxylated metabolites and evidence that metabolites 

34 of 2-hydroxybiphenyl (2,5-dihydroxybiphenyl and 2,5’-benzoquinone) can produce DNA 

35 damage (Tani et al., 2007; Balakrishnan et al., 2002; Sasaki et al. 2002, 1997; Pathak and Roy, 

36 1993; Morimoto et al., 1989). However, hydroxylation of biphenyl to produce 2-hydroxy37 biphenyl appears to be a minor metabolic pathway in mice administered single intraperitoneal 

38 doses of 30 mg biphenyl/kg (Halpaap-Wood et al., 1981a), and the available data are inadequate 
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1 to establish that this genotoxic mode of action operates in the biphenyl induction of liver tumors 

2 in mice. There have been no in vitro or in vivo investigations of biphenyl-induced DNA adducts 

3 or ROS generation in mouse liver cells or of possible gender differences in the production of 

4 biphenyl-induced DNA adducts or other genotoxic events. Current mode-of-action information 

5 is inadequate to provide plausible explanations for why female BDF1 mice exposed to high 

6 dietary concentrations of biphenyl develop liver tumors, but male BDF1 mice exposed to 

7 6,000 ppm and female ddY mice exposed to 5,000 ppm do not (Umeda et al., 2005; Imai et al., 

8 1983). 

9 

10 4.7.3.2.4. Conclusions about the hypothesized mode of action for liver tumors in mice. 

11 A PPARα agonism mode of action for liver tumors in female mice exposed to 2,000 or 4,000 

12 ppm biphenyl in the diet for 2 years is not adequately supported by the experimental data. This 

13 is based on the lack of concordance between dose-response relationships for biphenyl-induced 

14 liver tumors and proliferation of hepatocellular peroxisomes in female mice. Evidence for 

15 increased hepatocellular peroxisomes in female mice was only found with 13-week exposure to 

16 16,000 ppm biphenyl and not at several concentrations ≤10,000 ppm (Umeda et al., 2004). 

17 Furthermore, a series key events demonstrating PPARα agonism mode of action have not been 

18 identified. 

19 Available data are inadequate to support alternative modes of action that propose direct 

20 or indirect genotoxic events from reactive biphenyl metabolites or ROS, respectively, as key 

21 events. Results from standard short-term genotoxicity tests are mostly negative or equivocal for 

22 biphenyl and 4-hydroxybiphenyl. Although there is some evidence for DNA damage from ROS 

23 generated from redox cycling between 2,5-dihydroxybiphenyl and phenylbenzoquinone, there 

24 are no investigations into the metabolic formation of 2-hydroxybiphenyl, 2,5-dihydroxybiphenyl, 

25 and phenylbenzoquinone in livers of biphenyl-exposed mice exposed to a range of biphenyl 

26 doses, no in vitro or in vivo investigations of biphenyl-induced DNA adducts or ROS generation 

27 in mouse liver cells, and no investigations of possible gender differences in capability to produce 

28 biphenyl-induced DNA adducts or other genotoxic events. 

29

30 4.8. SUSCEPTIBLE POPULATIONS AND LIFE STAGES

31 4.8.1. Possible Childhood Susceptibility

32 No specific information was identified that would point specifically towards an early 

33 childhood susceptibility for biphenyl toxicity. However, the developmental profiles of 

34 superoxide dismutase and catalase in humans that were reported by McElroy et al. (1992) 

35 indicate that the activities of both enzymes may be comparatively low before and at birth, 

36 placing humans in the perinatal period at an increased risk of adverse effects elicited by quinoid 

37 metabolites of biphenyl. Specifically, Buonocore et al. (2001) drew attention to the fact that the 
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1 human brain has relatively low superoxide dismutase activity at birth. Given the limited data on 

2 age-specific ROS scavenging enzymes, any suggestions of childhood susceptibility to biphenyl is 

3 speculative. 

4 Studies in animals provide evidence that biphenyl metabolism is mediated by CYP1A2 

5 and CYP3A4 (Haugen, 1981). Phase II enzymes, such as sulphotransferases (SULTs) and 

6 uridine diphosphate glucuronosyl transferases (UGTs), may be involved in conjugation activities 

7 with hydroxybiphenyls in mammalian tissues (Pacifici et al., 1991; Bock et al., 1980). CYP1A2 

8 expression is negligible in the early neonatal period, but is significantly increased to 50% of 

9 adult levels by one year of age (Sonnier and Cresteil, 1998). In general, SULTs and UGTs, 

10 depending on the isoforms, also exhibit differential expression during human development 

11 (Duanmu et al., 2006; Strassburg et al., 2002). To the extent that metabolism increases or 

12 reduces the toxicity of biphenyl, changes in the expression of Phase I and II enzymes during 

13 development can influence susceptibility to biphenyl toxicity. Specific isoforms of cytochrome 

14 P450s and Phase II enzymes have not been identified as the principal catalyzers involved in 

15 biphenyl metabolism and the effect of differences in enzyme expression on childhood 

16 susceptibility to biphenyl has not been established. 

17

18 4.8.2. Possible Gender Differences

19 Benford and Bridges (1983) evaluated the sex- and tissue-specific induction of biphenyl 

20 2-, 3-, and 4-hydroxylase activities in microsomal preparations or primary hepatocyte cultures 

21 from male and female Wistar rats. No differences in biphenyl hydroxylase activities were 

22 observed between the sexes. However, there were some sex differences in the way tissues 

23 responded to the action of enzyme inducers. For example, the CYP1A inducer α-naphthoflavone 

24 strongly induced 2-hydroxylase in male liver but had no effect on female liver. Betamethasone 

25 induced 2-hydroxylase activity in female liver but inhibited it in male liver. The available 

26 limited human data do not suggest that gender differences exist in the response to biphenyl 

27 exposure. However, available animal data suggest gender-related differences in susceptibility to 

28 tumors (i.e., bladder tumors in male but not female F344 rats and increased incidences of liver 

29 tumors in female but not male BDF1 mice administered biphenyl in the diet for a lifetime).

30

31 4.8.3. Other 

32 The limited information on the specifics of biphenyl metabolism and toxic effects in 

33 humans does not allow a meaningful assessment of populations that might be highly susceptible 

34 to the adverse effects of biphenyl. For example, there is as yet no clear attribution of CYP 

35 isozymes to the various biphenyl hydroxylases and no information on which sulphotransferases

36 and glucuronidases conjugate hydroxylated biphenyl metabolites. It is known that many CYP 

37 isozymes, as well as glucuronidases, exist in polymorphic forms with catalytic activities that 
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1 differ from the wild type. In addition, such enzyme polymorphisms display specific distributions 

2 across populations and ethnicities that might put some at increased risk and others at decreased 

3 risk of adversity from biphenyl exposure. This lack of information represents a data gap.
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1 5. DOSE-RESPONSE ASSESSMENTS

2 

3 

4 5.1. ORAL REFERENCE DOSE (RfD)

5 5.1.1. Choice of Principal Study and Critical Effect—with Rationale and Justification

6 No information was located regarding possible associations between oral exposure to 

7 biphenyl and health outcomes in humans. 

8 As discussed in Section 4.6.1, the major and most sensitive targets of toxicity following 

9 oral exposure to biphenyl are the liver, urinary system, body weight, and developing organism 

10 (see Figure 5-1). In the rat, chronic oral studies identified the kidney and urinary bladder as 

11 critical noncancer targets (see Figure 5-1 for LOAELs and NOAELs found in these studies). 

12 Kidney effects observed include: renal pelvis transitional cell hyperplasia and hemosiderin 

13 deposits in female F344 rats at doses ≥128 mg/kg-day (Umeda et al., 2002); kidney stone 

14 formation and obstructive pyelonephritis with tubular atrophy, tubular cysts, and fibrosis in male 

15 and female Wistar rats at 165 and 370 mg/kg-day, respectively (Shiraiwa et al., 1989); renal 

16 lymphocytic infiltration, tubular atrophy, and tubular cysts in male and female albino rats at 

17 doses ≥420 mg/kg-day (Ambrose et al., 1960); mild renal tubular degeneration in male albino 

18 rats at 250 or 450 mg/kg-day (Pecchiai and Saffioti, 1957; not plotted in Figure 5-1 because 

19 quantitative data were not included in the study report); and renal tubular dilatation in male and 

20 female Sprague-Dawley rats at 732 mg/kg-day (Dow Chemical Co., 1953). An increased 

21 incidence of urinary bladder hyperplasia associated with calculi or “stones” was observed in 

22 male and female F344 rats at 378 and 438 mg/kg-day, but not at 110 and 128 mg/kg-day, 

23 respectively (Umeda et al., 2002). Elevated incidences of the same lesion were observed in male 

24 and female Wistar rats at 353 and 370 mg/kg-day, respectively (Shiraiwa et al., 1989). In 

25 contrast, urinary bladder hyperplasia and calculi were not observed in male or female albino rats 

26 at doses as high as 840 mg/kg-day (Ambrose et al., 1960) or in male or female Sprague-Dawley 

27 rats exposed to doses as high as 732 mg/kg-day (Dow Chemical Co., 1953). 
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1 

2 

3 Figure 5-1. NOAELs and LOAELs for noncancer effects in rats and mice from repeated oral exposure to biphenyl.

0

200

400

600

800

1000

1200

1400

1600

NOAEL LOAEL

Drop-lines span highest to lowest doses tested

Female 

BDF1

mouse

(1)

Female

ddY 

mouse

(2)

Male

BDF1

mouse

(1)

Female

ddY 

mouse

(2)

Female

BDF1

mouse

(1)

Female

ddY 

mouse

(2)

Female

F344 rat

(3)

Male

Wistar rat

(4)

Male,

female

albino rat

(5)

Male,

female

SpragueDawley rat 

(6)

Male

F344 rat

(3)

Male,

female

albino rat

(5)

Male

Wistar rat

(4)

Male,

female

SpragueDawley rat 

(6)

Wistar rat

(7)

Livera Body weightb Kidneyc Urinary bladderd Developmentale

a

Increased plasma liver enzymes in BDF1 mice.

b

Decreased body weight (>10% lower than controls) in BDF1 mice. c

Increased incidences of kidney lesions including: mineralization in outer medulla in BDF1 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 et 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
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1 

2 In mice, chronic oral toxicity studies identified the liver, kidney, and body weight as 

3 critical noncancer targets (see Figure 5-1 for NOAELs and LOAELs for these effects). In BDF1

4 mice, significantly (p < 0.05) increased plasma levels of enzymes indicative of liver damage 

5 were observed at dose levels of 1,050 mg/kg-day in males and ≥414 mg/kg-day in females 

6 (Umeda et al., 2005), but no exposure-related changes in liver enzymes were observed in female 

7 ddY mice at 885 mg/kg-day (Imai et al., 1983). Significantly increased incidence of 

8 mineralization of the renal outer medulla was observed in female BDF1 mice at 414 and 

9 1,420 mg/kg-day (Umeda et al, 2005), but exposure-related histological changes in the kidney 

10 were not found in female ddY mice at 885 mg/kg-day (Imai et al., 1983). Following the same 

11 pattern of apparent strain difference in susceptibility to biphenyl toxicity, body weights were 

12 decreased by >10% in male BDF1 mice at ≥291 mg/kg-day and females at ≥414 mg/kg-day 

13 (Umeda et al., 2005), but body weights in female ddY mice exposed to 885 mg/kg-day were 

14 similar to control values (Imai et al., 1983). Shorter duration oral exposure (13 weeks) of mice 

15 to biphenyl at higher dietary concentrations (estimated doses ≥1,500 mg/kg-day) has also been 

16 shown to affect body and/or liver weights in mice (Umeda et al., 2004). 

17 In the only available oral developmental toxicity study (Khera et al., 1979), frank 

18 maternal toxicity (increased mortality [5/20 vs. 0/18 in controls] and decreased number of dams 

19 with live fetuses [9/20 vs. 16/18 in controls]) occurred at the highest dose (1,000 mg/kg-day). 

20 Significantly increased incidences of fetuses with skeletal anomalies were noted at doses 

21 ≥500 mg/kg-day. The NOAEL and LOAEL of 250 and 500 mg/kg-day for delayed skeletal 

22 development are noted in Figure 5-1.

23 The 2-year dietary studies in F344 rats (Umeda et al., 2002) and BDF1 mice (Umeda et 

24 al., 2005) and the developmental study in Wistar rats (Khera et al., 1979) were selected as 

25 candidate principal studies for deriving the RfD because they provide the best available data 

26 (adequate number of dose groups and dose spacing, sufficient group sizes, comprehensive 

27 endpoint assessment and quantitation of results) to describe dose-response relationships for the 

28 critical effects in rats and mice associated with chronic or gestational oral exposure to biphenyl.

29 In the 2-year dietary study of male and female F344 rats, biphenyl was administered in 

30 the diet at 0, 500, 1,500, or 4,500 ppm (respective estimated doses were 36.4, 110, and 

31 378 mg/kg-day for males and 42.7, 128, and 438 mg/kg-day for females) (Umeda et al., 2002). 

32 At the highest dose, noncancer effects included significantly increased incidence of rats with 

33 transitional cell hyperplasia in the renal pelvis, renal mineralization and hemosiderin deposits, 

34 and urinary bladder transitional cell hyperplasia. Noncancer effects at the mid-dose level were 

35 restricted to significantly increased incidences of females with renal transitional cell hyperplasia 

36 and hemosiderin deposits. There were no significant biphenyl-related effects in low-dose males 

37 or females.
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1 In the 2-year dietary study of male and female BDF1 mice, biphenyl was administered in 

2 the diet at 0, 667, 2,000, or 6,000 ppm (respective estimated doses were 0, 97, 291, and 

3 1,050 mg/kg-day for males, and 0, 134, 414, and 1,420 mg/kg-day for females) (Umeda et al., 

4 2005). At the two highest dose levels, noncancer effects included increased incidence of mice 

5 with renal mineralization, increased levels of BUN, increased levels of serum enzymes indicative 

6 of liver damage, and decreased terminal body weights. No exposure-related effects were 

7 observed at the lowest exposure level.

8 In the oral developmental toxicity study, pregnant Wistar rats were exposed by gavage to 

9 0, 125, 250, 500, or 1,000 mg biphenyl/kg-day on GDs 6–15 (Khera et al., 1979). Significantly 

10 increased numbers of fetuses with skeletal anomalies (wavy ribs, extra ribs, small 13th rib, 

11 missing or unossified sternebrae, delayed ossification of the calvarium) were noted at doses 

12 ≥500 mg/kg-day, and the number of litters exhibiting any of these anomalies was significantly 

13 higher at the 500 mg/kg-day dose level relative to controls.

14 Candidate critical effects from the chronic study in F344 rats (Umeda et al., 2002) were: 

15 (1) nodular or simple transitional cell hyperplasia in the renal pelvis of males and females, 

16 (2) mineralization in the renal pelvis or renal papillary mineralization in males and females, 

17 (3) renal hemosiderin deposits in females, and (4) transitional cell hyperplasia in the urinary 

18 bladder of males. Candidate critical effects from the chronic study in BDF1 mice (Umeda et al., 

19 2005) were: (1) decreased body weight in males and females, (2) mineralization of the renal 

20 inner stripe-outer medulla in males and females, (3) BUN in males and females, and (4) serum 

21 liver enzyme activities (AST [GOT], ALT [GPT], AP [ALP], and LDH) in females. The 

22 candidate critical effect from the rat oral developmental toxicity study (Khera et al., 1979) was 

23 litters with fetal skeletal anomalies from Wistar rat dams exposed during gestation. 

24

25 5.1.2. Methods of Analysis—Including Models

26 Dichotomous datasets modeled include selected nonneoplastic lesions in the urinary 

27 system of male and female F344 rats (Table 5-1) exposed to biphenyl in the diet for 2 years

28 (Umeda et al., 2002), mineralization in the kidney of male and female BDF1 mice (Table 5-2) 

29 exposed to biphenyl in the diet for 2 years (Umeda et al., 2005), and litters with skeletal 

30 anomalies from Wistar rat dams (Table 5-3) administered biphenyl by gavage on GDs 6–15 

31 (Khera et al., 1979). 

32
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Table 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 

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 21c 0 0 1 12c

Simple transitional cell hyperplasia 6 8 5 19d 3 5 12d 25c

Mineralization 9 6 10 18e 12 12 18 27d

Other kidney effects

Hemosiderin depositf 0 0 0 0 4 8 22c 25c

Papillary mineralization 9 9 14 23d 2 6 3 12c

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 (0.030 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 selected for quantitative analysis. g

Female data for incidences of combined transitional cell hyperplasia not selected for quantitative analysis. 

Source: Umeda et al. (2002).

.

1 
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Table 5-2. BMD modeling datasets for body weight, selected clinical 

chemistry results, and histopathological kidney effects in male and female 

BDF1 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 ± 4.4a 22.9 ± 2.7b

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 ± 6.0a 32.4 ± 3.6b

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 12c 26d

Clinical chemistry parameter n = 28 n = 20 n = 22 n = 31

AST (IU/L) 75 ± 27 120 ± 110 211 ± 373b 325 ± 448b

ALT (IU/L) 32 ± 18 56 ± 46 134 ± 231b 206 ± 280b

AP (IU/L) 242 ± 90 256 ± 121 428 ± 499 556 ± 228b

LDH (IU/L) 268 ± 98 461 ± 452 838 ± 2,000 1,416 ± 4,161a

BUN (mg/dL) 14.9 ± 2.0 14.8 ± 3.4 21.0 ± 20.5 23.8 ± 11.7b

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 ± 3.1b 25.5 ± 3.0b

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 
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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 anomaliesa/litters 8/16 11/20 13/18 15b/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 

2 All available core dichotomous models in the EPA Benchmark Dose Software (BMDS) 

3 (version 2.1.2) were fit to the incidence data for each dataset. The multistage model was run for 

4 all polynomial degrees up to n-1 (where n is the number of dose groups including control). 

5 Adequate model fit was judged by three criteria: goodness-of-fit p-value (p ≥ 0.1), visual 

6 inspection of the dose-response curve, and a value of <2 for the largest scaled residual for any 

7 data point in the dataset (including the control). Among all of the models providing adequate fit 

8 to the data, the lowest BMDL was selected as the potential point of departure (POD) when the 

9 difference between the BMDLs estimated from these models was more than threefold; otherwise, 

10 the BMDL from the model with the lowest Akaike’s Information Criterion (AIC) was chosen as 

11 the candidate POD. In accordance with U.S. EPA (2000b) guidance, BMDs and BMDLs 

12 associated with an extra risk of 10% were calculated for all models. In the absence of 

13 information to identify the biologically significant level of response for an endpoint, a 

14 (benchmark response) BMR of 10% extra risk is typically chosen as a response level for 

15 dichotomous data and is recommended for the BMR when using dichotomous models to 

16 facilitate a consistent basis of comparison across assessments and endpoints. 

17 A BMR of 10% extra risk was selected to derive the POD for development effects from 

18 the Khera et al. (1979) study because the endpoints were characterized as affected litters. A 

19 BMR of 5% extra risk has typically been used for quantal data in reproductive and 

20 developmental studies when data are available to characterize individual pups within litters (U.S. 

21 EPA, 2000b). Since this level of reporting was not available, nested models could not be used. 

22 Thus, a BMR of 10% extra risk among affected litters was employed in order to better 

23 approximate a 5% extra risk in affected offspring and to recognize the litter as the experimental 

24 unit. BMDs and BMDLs associated with extra risk of 5% for all endpoints were also calculated 

25 for comparison. 

26 When core models failed to provide adequate fit to the data, optimizations of the models 

27 (model restriction adjustments, specification of initial parameters, and use of alternative models) 
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1 were attempted in an effort to achieve adequate fit. If these optimizations failed to achieve better 

2 fit, the highest dose was dropped and the entire modeling procedure was repeated. If an adequate 

3 fit could not be achieved after dropping the highest dose, then the dataset was determined to be 

4 unsuitable for BMD modeling.

5 For continuous data, all core continuous models available in the EPA BMDS 

6 (version 2.1.2) were first applied to the data while assuming constant variance. If the data were 

7 consistent with the assumption of constant variance (p ≥ 0.1), then the fit of all the continuous 

8 models to the mean were evaluated while assuming constant variance. In the absence of 

9 information to indicate the biologically significant level of response, BMDs and BMDLs were 

10 calculated based on a benchmark response (BMR) representing a change of 1 SD from the 

11 control. BMDs and BMDLs for decreased body weight were also calculated for a BMR of 10% 

12 decrease from the control (i.e., 10% relative deviation [RD]) because a 10% decrease in body 

13 weight is generally considered to represent a minimally biologically significant effect. For

14 serum enzyme activities (AST, ALT, AP, LDH), BMDs and BMDLs were also calculated for a 

15 BMR of 100% increase from the control (i.e., twofold or 1 RD; BMD1RD and BMDL1RD). 

16 Several expert organizations, particularly those concerned with early signs of drug-induced 

17 hepatotoxicity, have identified an increase in liver enzymes (AST, ALT, AP) compared with 

18 concurrent controls of two- to fivefold as an indicator of concern for hepatic injury (EMEA, 

19 2006; Boone et al., 2005). Because LDH, like liver enzymes, is one of the more specific 

20 indicators of hepatocellular damage in most animal species and generally parallels changes in 

21 liver enzymes in toxicity studies where liver injury occurs, a similar twofold increase in LDH is 

22 considered indicative of liver injury in experimental animals. A similar approach was taken for 

23 BUN. 

24 Adequate model fit was judged by three criteria: goodness-of-fit p-value (p ≥ 0.1), visual 

25 inspection of the dose-response curve, and a value of <2 for the largest scaled residual for any 

26 data point in the data set (including the control). Among all of the models providing adequate fit 

27 to the data, the lowest BMDL was selected as the potential POD when the BMDLs estimated 

28 from these models varied by more than threefold; otherwise, the BMDL from the model with the 

29 lowest AIC was chosen as the candidate POD. When the test for constant variance was negative, 

30 all models were run again while applying the power model integrated into the BMDS to account 

31 for nonhomogeneous variance. When the nonhomogeneous variance model provided an 

32 adequate fit (p ≥ 0.1) to the variance data, the models were evaluated using the nonhomogeneous 

33 variance model. Model fit and POD selection proceeded as described earlier. When both tests 

34 for variance (constant and nonhomogeneous) provided inadequate fit to the variance data, model 

35 restriction adjustments were attempted in an effort to achieve adequate fit. If these 

36 manipulations failed to achieve better fit, the highest dose was dropped and the entire modeling 

37 procedure was repeated. If an adequate fit could not be achieved after dropping the highest dose, 

38 then the dataset was determined to be unsuitable for BMD modeling. 
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1 Summary modeling results are presented in Table 5-4 and Figure 5-2; more detailed 

2 modeling results are presented in Appendix B (Tables B-4 through B-24 and respective model 

3 output files). The BMDs and BMDLs shown in Table 5-4 and Figure 5-2 are those from the 

4 best-fitting models for each endpoint. For datasets to which no model could be fit, NOAELs and 

5 LOAELs were considered for the candidate POD. 

6 
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Table 5-4. Summary of BMDs/BMDLs for selected nonneoplastic effects 

following 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 BMD10 BMDL10 BMD10 BMDL10

Renal pelvis

 Transitional 

 hyperplasia

cell nodular Multistage 

3-degree

193 127 Multistage 

2-degree

274 212

 Transitional 

 hyperplasia

cell simple Gamma 314 113 Gamma 71 52

 Mineralization Log-probit 208 138 Multistage 

1-degree

88 56

Kidney - other

Hemosiderin deposit Not selectedb – – Dichotomous-Hill 45 23

Papillary mineralization Multistage 

1-degree

92 58 Logistic 292 219

Bladder BMD10 BMDL10 BMD10 BMDL10

Transitional cell hyperplasia Gamma 205 147 Not selectedb – –

BDF1 mice (Umeda et al., 2005); biphenyl in the diet for 2 yrs

Kidney BMD10 BMDL10 BMD10 BMDL10

Mineralization Log-logistic 721 276 Log-logistic 233 122

Clinical chemistry BMD1RD BMDL1RD BMD1RD BMDL1RD

AST Not selectedb – – Power 190a 122a

ALT Not selectedb – – No adequate fitc – –

LDH Not selectedb – – No adequate fitc – –

AP Not selectedb – – No adequate fitc – –

BMD1SD BMDL1SD BMD1SD BMDL1SD

BUN Linear 415a 267a No adequate fitc – – 

Body weight BMD0.1RD BMDL0.1RD BMD0.1RD BMDL0.1RD

Terminal body weight No adequate fitc – – Linear 583 511

Wistar rats (Khera et al., 1979); biphenyl by gavage to dams on GDs 6–15 BMD10 BMDL10

Litters with fetal skeletal anomalies Log-logistic 57 20

a

Adequate fit obtained only after excluding results from the highest dose group. b

“Not selected” indicates that the data set was not selected for dose-response analysis because either a treatmentrelated effect was not observed or because the response observed in the other sex in the same study was more 

robust. 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 benchmark 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)
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1 

2 

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

4 

0

50

100

150

200

250

300

350

400

450

mg/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 AST TC

hyperplasia Mineralization BUN Nodular hyperplasia Simple hyperplasia Renal pelvis mineralization

Hemosiderin Papillary mineralization 

Body 

weight

Liver Bladder Kidney Developmental

TC = transitional cell

(1) = Umeda et al. (2005); (2) = Umeda et al. (2002); (3) = Khera et al. (1979)
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1 Examination of the BMD and BMDL values in Table 5-4 and Figure 5-2 reveals 

2 BMD/BMDL pairs for four kidney effects and for the developmental effect that are clustered 

3 below BMD/BMDL pairs for the other effects. The BMDL values in this cluster range from 

4 20 to 58 mg/kg-day and identify the following as the most sensitive nonneoplastic effects 

5 associated with repeated oral exposure to biphenyl in animals: (1) renal transitional cell 

6 hyperplasia (simple) in female F344 rats (52 mg/kg-day), (2) renal mineralization in female F344 

7 rats (56 mg/kg-day), (3) renal hemosiderin deposition in female F344 rats (23 mg/kg-day), 

8 (4) renal papillary mineralization in male F344 rats (58 mg/kg-day), and (5) increased litters with 

9 fetal skeletal anomalies in Wistar rats (20 mg/kg-day). 

10 NOAEL values for endpoints with datasets for which adequate model fits could not be 

11 obtained using BMDS were higher than the BMDL values for these five kidney and 

12 developmental endpoints. These include selected clinical chemistry parameters in female BDF1

13 mice (NOAELs for LDH, AP, and BUN: 414 mg/kg-day; NOAEL for ALT: 134 mg/kg-day) and 

14 terminal body weight in male BDF1 mice (NOAEL: 97 mg/kg-day). 

15 The increased fetal skeletal anomalies in Wistar rats was selected as the critical effect for 

16 deriving an oral RfD because it was considered to be an adverse effect and resulted in the most 

17 sensitive POD (BMDL10 of 20 mg/kg-day) observed compared with other PODs for biphenyl18 induced kidney effects. 

19

20 5.1.3. RfD Derivation—Including Application of Uncertainty Factors (UFs)

21 The RfD for biphenyl was derived by dividing the POD of 20 mg/kg-day (i.e., the 

22 BMDL10 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 animals (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 applied to account for intraspecies variability in susceptibility to 

31 biphenyl, as quantitative information for evaluating toxicokinetic and toxicodynamic 

32 differences among 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 
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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 for database deficiencies was applied. The 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 other clusters of Parkinson’s disease have been reported in 

20 biphenyl 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 effects on the nervous system following biphenyl exposure. Accordingly, 

23 these epidemiologic studies do 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

27 The RfD for biphenyl was calculated as follows:

28 RfD = BMDL10 ÷ UF

29 = 20 mg/kg-day ÷ 100 

30 = 0.2 mg/kg-day 

31

32 5.1.4. Previous RfD Assessment 

33 The previous IRIS assessment for biphenyl (U.S. EPA, 1989) derived an oral RfD of 

34 0.05 mg/kg-day based on kidney damage in albino rats administered biphenyl for 2 years at 

35 dietary levels ≥0.5% (Ambrose et al., 1960). U.S. EPA considered the dietary level of 0.1% (50 

36 mg/kg-day using a food factor of 0.05/day) to represent a NOAEL due to the following: (1) 

37 uncertainty in the significance of effects observed at lower doses as compared to the more certain 

38 adverse effect level of 0.5% in the diet and (2) supporting findings of 0.1% biphenyl as a 

39 NOAEL in an unpublished report of a subchronic rat feeding study and a three-generation rat 

40 reproduction study performed by Stanford Research Institute (Dow Chemical Co., 1953). The 

41 NOAEL of 50 mg/kg-day was divided by a total UF of 1,000 (10 for extrapolation from animals 

42 to humans, 10 for protection of sensitive human subpopulations, and a modifying factor of 10 to 

43 account for intraspecies variability demonstrated in the threshold suggested by the data in the 

44 chronic animal study). 

45
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1 5.2. INHALATION REFERENCE CONCENTRATION (RfC)

2 5.2.1. Choice of Principal Study and Critical Effect—with Rationale and Justification

3 Human data are limited to assessments of possible associations between occupational 

4 exposure to biphenyl and health outcomes where inhalation is presumed to have been the major 

5 exposure route. Clinical signs and abnormal electrophysiological test results among workers 

6 exposed to biphenyl during the production of biphenyl-impregnated fruit wrapping paper provide 

7 evidence of biphenyl-induced neurological effects (Seppäläinen and Häkkinen, 1975; Häkkinen 

8 et al., 1973, 1971). Case reports include an account of periodic loss of strength and eventual 

9 signs of chronic hepatitis in a woman during a 25-year period of employment at a fruit-packing 

10 facility where biphenyl-impregnated paper was used (Carella and Bettolo, 1994) and a cluster of 

11 five cases of Parkinson’s Disease (0.9 cases expected) at a facility manufacturing biphenyl12 impregnated paper (Wastensson et al., 2006). None of these studies provided air monitoring data 

13 adequate to characterize workplace exposures to biphenyl. Therefore, data from the available 

14 human studies could not be used for dose-response analysis and derivation of an RfC. 

15 Limited information is available regarding the effects of inhaled biphenyl in laboratory

16 animals. In mice, repeated airborne exposure to biphenyl (7 hours/day, 5 days/week for 2 weeks) 

at concentrations as high as 54.75 ppm (345.5 mg/m3 17 ) appeared to cause no symptoms (Sun 

18 Company Inc., 1977a). In a series of studies that included repeated inhalation exposure of 

19 rabbits, rats, and mice to atmospheres containing biphenyl for periods of 68–94 days 

20 (Deichmann et al., 1947; Monsanto, 1946), rabbits exhibited no signs of exposure-related 

adverse effects at concentrations as high as 300 mg/m3 21 . Irritation of mucous membranes was

observed in rats at concentrations of 40 and 300 mg/m3 22 . Mice were the most sensitive to inhaled 

biphenyl; irritation of the upper respiratory tract was noted at a concentration of 5 mg/m3 23

24 (Deichmann et al., 1947; Monsanto, 1946), but other biphenyl concentrations were not tested in 

25 this experiment. The limitations of a single exposure level and poorly-reported study details 

26 preclude the use of this study for RfC derivation. 

27 Repeated exposure of mice to biphenyl at vapor concentrations of 25 or 50 ppm 

(157.75 or 315.5 mg/m3 28 ) for 13 weeks resulted in high incidences of pneumonia and tracheal 

29 hyperplasia, and high incidences of congestion and edema in the lungs, liver, and kidney (Sun 

30 Company Inc., 1977b). The following study limitations and lack of supporting data preclude the 

31 usefulness of this study for deriving an RfC for biphenyl. Measured biphenyl exposure 

32 concentrations varied greatly during the first half of the 13-week exposure period; for example, 

33 in the high concentration group (target concentration of 50 ppm), the measured concentrations 

34 ranged from 5 ppm to 102 ppm during the first 45 exposure sessions. High mortality in 25 ppm 

35 male mice (40/50) after 46 exposures necessitated the use of replacement animals; these 

36 replacement animals received the same total number of exposure sessions as the surviving 

37 animals from the original 25 ppm group but exposures were not concurrent. Histopathological 

38 findings were reported only for males and females combined. Reports of lung congestion and 
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1 hemorrhagic lungs in some control mice were not confirmed histopathologically, and congestion 

2 in the lung, liver, and kidney were considered by the study pathologist a likely effect of the 

3 anesthetic used for killing the mice. The severity of reported histopathologic lesions was not 

4 specified. 

5 The 13-week inhalation mouse study of Sun Company Inc. (1977b) is the only available 

6 study that employed at least subchronic-duration exposure and included multiple biphenyl 

7 exposure levels. This study is considered inadequate for RfC derivation because: (1) exposure 

8 levels were highly variable during the first half of the 13-week exposure period, (2) one of the 

9 exposure groups experienced high losses (46/100) due to an overheating event and 

10 cannibalization after 46 exposures, although replacement mice were subsequently added and 

11 received a total of 65 exposures, and (3) limitations in the reporting of histopathological findings. 

12 An RfC was not derived due to the significant uncertainty associated with the inhalation 

13 database for biphenyl, and route-to-route extrapolation was not supported in the absence of a 

14 PBPK model. Although an RfC cannot be derived, it should be noted that the available 

15 inhalation data provides some evidence that inhalation exposure to biphenyl could induce 

16 respiratory or systemic lesions. 

17

18 5.2.2. Previous RfC Assessment

19 No RfC was derived in the previous (1985) IRIS assessment. 

20

21 5.3. UNCERTAINTIES IN THE RfD and RfC

22 Risk assessments should include a discussion of uncertainties associated with the derived 

23 toxicity values. To derive the oral RfD, the UF approach (U.S. EPA, 2002, 1994b) was applied 

24 to a POD of 10 mg/kg-day (see Section 5.1). Factors were applied to the POD to account for 

25 extrapolating from responses observed in an animal bioassay to humans or a diverse human 

26 population of varying susceptibilities. Uncertainties associated with the data set used to derive 

27 the biphenyl RfD are more fully described below. 

28 The available database was determined to be inadequate for deriving a chronic inhalation 

29 RfC for biphenyl (see Section 5.2). 

30

31 Selection of the critical effect for reference value determination. The critical effect 

32 selected for derivation of the RfD was skeletal anomalies in fetuses from rat dams administered 

33 biphenyl by gavage during GDs 6–15. An increased incidence of these anomalies was reported

34 at doses ≥500 mg/kg-day; frank maternal toxicity, including death, was observed at the highest 

35 dose level (1,000 mg/kg-day). There is some degree of uncertainty regarding the toxicological 

36 significance of the reported skeletal anomalies (wavy or extra ribs and delayed ossification most 

37 commonly observed) and the relevance of gavage dosing used in the developmental toxicity 

38 study to human exposures. Supporting developmental toxicity studies are not available. 
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1 Dose-response modeling. BMD modeling was used to estimate the POD for the biphenyl 

2 RfD. BMD modeling has advantages over a POD based on a NOAEL or LOAEL because, in 

3 part, the latter are a reflection of the particular exposure concentration or dose at which a study 

4 was conducted. A NOAEL or LOAEL lacks characterization of the dose-response curve, and for 

5 this reason, is less informative than a POD obtained from BMD modeling. The selected model, 

6 i.e., the log-logistic model, provided the best mathematical fit to the experimental data set (as 

7 determined by the lowest AIC), but does not necessarily have greater biological support over the 

8 various models included in BMDS. Other models in BMDS yield estimates of the POD higher 

9 than the POD derived using the log-logistic model (by up to 5.8-fold). 

10 Interspecies extrapolation of dosimetry and toxicodynamics. Limited information is 

11 available regarding species-specific toxicokinetic and toxicodynamic differences in biphenyl 

12 metabolism. Results of available in vitro assays of human and rat liver preparations suggest 

13 qualitative similarities and quantitative differences in biphenyl metabolism (Powis et al., 1989, 

14 1988; Benford et al., 1981). Available in vivo animal data demonstrate qualitative and 

15 quantitative differences between rats and mice (Halpaap-Wood et al., 1981a; Meyer and Scheline 

16 1976; Meyer et al., 1976a). However, in vivo human data are lacking and it is uncertain which 

17 animal species, the rat or the mouse, would be more comparable to humans. Other areas of 

18 biphenyl toxicokinetics (absorption, distribution, elimination), have received some attention in 

19 animal studies, but comparative human data are not available. PBPK models for biphenyl to 

20 address differences in toxicokinetics between animal and human are lacking. An UF of 10 was 

21 used to account for animal to human extrapolation in the absence of adequate comparative 

22 animal and human toxicokinetic and toxicodynamic data for biphenyl. 

23 Sensitive human populations. Heterogeneity among humans is another uncertainty 

24 associated with extrapolating doses from animals to humans. Identification of populations that 

25 might be relatively more susceptible to the toxic effects of biphenyl is not feasible because of the 

26 limited information on biphenyl metabolism and mode of action of biphenyl toxicity. It is 

27 known, however, that many CYP isozymes and glucuronidase exist in polymorphic forms. Such 

28 enzyme polymorphism may put some populations at increased risk from biphenyl exposure. In 

29 the absence of biphenyl-specific data on human variation, a factor of 10 was used to account for 

30 uncertainty associated with human variation. Human variation may be larger or smaller; 

31 however, biphenyl-specific data to examine the potential magnitude of over- or under-estimation 

32 are absent. 

33

34 5.4. CANCER ASSESSMENT

35 5.4.1. Choice of Study/Data—with Rationale and Justification

36 No information was located regarding possible associations between oral exposure to 

37 biphenyl and cancer in humans. Two animal bioassays found statistically significant 
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1 associations between lifetime oral exposure to biphenyl and tumor 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, BDF1 mice (Umeda et al., 2005). Tumor data for these 

4 two sites were 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-Response 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 BDF1 mice (Umeda et al., 2005) are shown 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 BDF1 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 incidencec

Transitional cell

Papilloma 0/50 0/50 0/50 10/49b, 0/50 0/50 0/50 0/50

Carcinoma 0/50 0/50 0/50 24/49b, 0/50 0/50 0/50 0/50

Papilloma or carcinoma 050 0/50 0/50 31/49b, 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
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Table 5-6. Incidence data for liver tumors in male and female BDF1 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 incidencec

Adenoma 8/50 6/49 7/49 3/50 2/48 3/50 12/49a 10/48a

Carcinoma 8/50 8/49 5/49 4/50 1/48 5/50 7/49a 5/48

Adenoma or carcinoma 16/50 12/49 9/49 7/50 3/48 8/50 16/49b 14/48a

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 risk for developing a tumor. Umeda et al. (2005) did not specify the time of appearance 

of the first tumor. 

Source: 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 There 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 hypothetized

9 mode of action for urinary bladder tumors in biphenyl-exposed male rats). No increased risk of 

10 bladder tumors is expected as 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 linearity 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
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1 5.4.3.2. Liver Tumors in Female Mice

2 In the study report of their 2-year bioassay in BDF1 mice, Umeda et al. (2005) provided 

3 averaged food consumption and biphenyl dose estimates for each exposure group (Table 1 of 

4 Umeda et al., 2005). The study report did not include average body weights for the exposure 

5 groups. Therefore, the biphenyl concentration in the food was multiplied by the corresponding 

6 average daily food consumption value to determine the average daily biphenyl intake. Dividing 

7 this average daily biphenyl intake by the author-calculated daily dose yielded the average body 

8 weight that would have been used by the study authors to calculate the average daily biphenyl 

9 dose. Scaling factors were calculated using U.S. EPA (1988) reference body weight for humans 

10 (70 kg) and the average body weight for each dose group of female mice: (average body 

weight/70)0.25 11 = scaling factor. The human equivalent dose (HED) was calculated as: HED = 

12 scaling factor × reported dose (Table 5-7).

13

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

of female BDF1 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.

14

15 The EPA’s Guidelines for Carcinogen Risk Assessment (U.S. EPA, 2005a) recommend 

16 that when the weight of evidence evaluation of all available data are insufficient to establish the 

17 mode of action for a tumor site and when scientifically plausible based on the available data, 

18 linear extrapolation is used as a default approach. Accordingly, a linear approach to low-dose 

19 extrapolation for biphenyl-induced liver tumors in female mice was selected because the mode of 

20 action for this tumor site has not been established (see Section 4.7.3.2). 

21 Incidence data for liver adenoma or carcinoma (combined) in the female mouse used to 

22 derive the oral slope factor are presented in Table 5-8. Tumor incidence data were adjusted to 

23 account for mortalities before 52 weeks; it was assumed that animals dying before 52 weeks 

24 were not exposed for sufficient time to be at risk for developing tumors (see footnote a in Table 

25 5-8). 

26 
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Table 5-8. Incidence of liver adenomas or carcinomas (combined) in female 

BDF1

Biphenyl dietary concentration (ppm) 0 667 2,000 6,000

HED (mg/kg-d) 0 19 59 195

Tumor incidence

Adenoma or carcinoma (combined) 3/48a 8/50 16/49a,b 14/48a,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 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).

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 

4 used by EPA in the vast majority of quantitative cancer assessments because it is thought to 

5 reflect the multistage carcinogenic process and it fits a broad array of dose-response patterns. 

6 The multistage-cancer model was run for all polynomial degrees up to n-1 (where n is the 

7 number of dose groups including control). An extra risk of 10% tumor incidence was selected as 

8 the benchmark response. Adequate model fit was judged by three criteria: goodness-of-fit p9 value (p ≥ 0.05), visual inspection of the dose-response curve, and a value of <2 for the largest 

10 scaled residual for any data-point in the dataset (including the control). If an adequate fit to the 

11 data was not achieved using the protocol above, the other dichotomous models were fit to the 

12 data. If none of the models achieved an adequate fit for the full dataset, the highest dose was 

13 dropped and the entire modeling procedure was repeated. 

14 When liver tumor incidence data for all dose groups were modeled, none of the models in 

15 BMDS, including the multistage model, provided an adequate fit of the data (see Appendix C, 

16 Table C-2). The animals in the highest dose group, while exhibiting a statistically significantly 

17 increased incidence in liver tumors compared with controls, did not show a monotonic increase 

18 in tumor response compared with the responses at the lower doses. To better estimate responses 

19 in the low dose region, the high dose group was excluded as a means of improving the fit of the 

20 model in the region of interest. When the high-dose group was dropped, the multistage model 

21 provided an adequate fit to the data (see Appendix C, Table C-2). The BMDHED10 and 

22 BMDLHED10 using this latter dataset were 18.7 and 12.2 mg/kg-day, respectively. See Appendix 

23 C for more information. 

24 
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1 5.4.4. Oral Slope Factor and Inhalation Unit Risk

2 A low-dose linear extrapolation approach results in calculation of an oral slope factor that 

3 describes the cancer risk per unit dose of the chemical at low doses. The oral slope factor was 

4 calculated by dividing the risk (i.e., BMR of 10% extra risk) at the POD by the corresponding 

5 BMDL (0.1/BMDLHED10). Using linear extrapolation from the BMDLHED10, the human 

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 7 ) was derived for liver tumors in female BDF1 mice (Table 5-9). 

8 

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

data from BDF1 female mice exposed to biphenyl in the diet for 2 years

BMDHED10 BMDLHED10 Slope factora (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/BMDL10HED; 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 POD for this 

11 dataset), because above the POD, the fitted dose-response model better characterizes what is 

12 known about the carcinogenicity of biphenyl (i.e., the slope factor may not approximate the 

13 observed dose-response relationship adequately at exposure exceeding 12.2 mg/kg-day). 

14 An inhalation unit risk for biphenyl was not derived in this assessment. The potential 

15 carcinogenicity of inhaled biphenyl has not been evaluated in human or animal studies, and 

16 route-to-route extrapolation was not possible in the absence of a PBPK model.

17

18 5.4.5. Uncertainties in Cancer Risk Values

19 5.4.5.1. Oral Slope Factor

20 A number of uncertainties underlie the cancer unit risk for biphenyl. Table 5-10 

21 summarizes the impact on the assessment of issues such as the use of models and extrapolation 

22 approaches (particularly those underlying the Guidelines for Carcinogen Risk Assessment (U.S. 

23 EPA, 2005a), the effect of reasonable alternatives, the decision concerning the preferred 

24 approach, and its justification. 

25 The uncertainties presented in Table 5-10 have a varied impact on risk estimates. Some 

26 suggest risks could be higher than was estimated, while others would decrease risk estimates or 

27 have an impact of an uncertain direction. Several uncertainties are quantitatively characterized

28 for the significantly increased rodent tumors. These include the statistical uncertainty in the 

29 multistage modeling estimate. Due to limitations in the data, particularly regarding the MOA 

30 and relative human sensitivity and variability, the quantitative impact of other uncertainties of 

31 potentially equal or greater impact has not been explored. As a result, an integrated quantitative 

32 analysis that considers all of these factors was not undertaken.
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Table 5-10. Summary of uncertainties in the biphenyl cancer slope factor 

Consideration/ Impact on slope 

approach 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. 

Cross-species Alternatives (i.e. Administered 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/kg3/4 ↓ slope factor -day (default supported by analysis of the allometric 

approach) variation of key physiological parameters 

across mammalian species. No PBPK model is

available to derive internal doses.

Extrapolation No impact on the Nonlinear Available MOA data for urinary bladder 

procedure for rat slope factor because extrapolation. 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 

tumors 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 tumors.

Extrapolation Departure from EPA’s Multistage model to Available MOA data do not inform selection 

procedure 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 EPA’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 Model options explored 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 BMDL (default Limited size of bioassay results in sampling 

uncertainty at POD if BMD10 used rather approach for variability; lower bound is 95% confidence 

than BMDL10 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 

sensitive response, including whether children are more 

subpopulations sensitive. 

BMDL10 = 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. 
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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 

5 in mice and rats. Neither an oral slope factor nor inhalation unit risk was derived in the previous 

6 cancer assessment.

7 
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1 6. MAJOR CONCLUSIONS IN THE CHARACTERIZATION OF HAZARD AND DOSE 

2 RESPONSE

3 

4 

5 6.1. HUMAN HAZARD POTENTIAL

6 6.1.1. Noncancer 

7 Toxicokinetic studies of animals indicate that orally administered biphenyl is rapidly and 

8 readily absorbed, distributed widely to tissues following absorption, and rapidly eliminated from 

9 the body, principally as conjugated hydroxylated metabolites in the urine (Meyer, 1977; Meyer 

10 and Scheline, 1976; Meyer et al., 1976a, b). Data for absorption, distribution, and elimination 

11 are not available for inhaled or dermally applied biphenyl. Metabolism to a range of 

12 hydroxylated metabolites has been demonstrated in in vitro systems with rat and human cells and 

13 tissues. Human metabolism of biphenyl appears to be qualitatively similar to metabolism in the 

14 rat, although some reports of quantitative differences are available (Powis et al., 1989, 1988; 

15 Benford et al., 1981). 

16 Available human health hazard data consist of limited assessments of workers exposed to 

17 biphenyl during the production or use of biphenyl-impregnated fruit wrapping paper in which 

18 signs of hepatic and nervous system toxicity were observed.

19 Chronic oral studies in rats and mice identify the liver and urinary system as principal 

20 targets of biphenyl toxicity, the rat kidney being the most sensitive. Results of a developmental 

21 toxicity study in rats indicate that skeletal development is a sensitive indicator of biphenyl 

22 toxicity. In chronically exposed rats, non-neoplastic kidney lesions (simple transitional cell 

23 hyperplasia in the renal pelvis and hemosiderin deposits) were found in females at ≥1,500 ppm 

24 biphenyl in the diet (128 mg/kg-day), and urinary bladder tumors, associated with urinary 

25 bladder calculi and transitional cell hyperplasia, were found in males, but not females, at the 

26 highest tested concentration, 4,500 ppm (378 mg/kg-day) (Umeda et al., 2002). Several other rat 

27 studies provide supporting evidence that the kidney and other urinary tract regions are sensitive 

28 targets for biphenyl in rats (Shiraiwa et al., 1989; Ambrose et al., 1960; Pecchiai and Saffiotti, 

29 1957; Dow Chemical Co., 1953). In chronically exposed BDF1 mice, increased incidence of 

30 nonneoplastic effects on the kidney (mineralization) and liver (increased activities of plasma 

31 ALT and AST) were found in females exposed to ≥2,000 ppm biphenyl in the diet (414 mg/kg32 day) (Umeda et al., 2005). In contrast, no exposure-related nonneoplastic or neoplastic effects 

33 on the liver or kidney were found in female ddY mice exposed to 5,000 ppm biphenyl in the diet 

34 for 2 years (Imai et al., 1983) or in B6C3F1 and B6AKF1 mice exposed to 517 ppm biphenyl in 

35 the diet for 18 months (Innes et al., 1969; NCI, 1968). In the only available developmental 

36 toxicity study for biphenyl, increased incidences of litters with fetuses showing skeletal 

37 anomalies were reported following exposure of pregnant rats to gavage doses ≥500 mg/kg-day 

38 on GDs 6–15 (Khera et al., 1979). 
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1 Biphenyl effects on reproductive function in rats have been reported at a higher exposure 

2 level than the lowest exposure levels associated with urinary tract, liver, or developmental 

3 toxicity. No exposure-related effect on the number of dams with litters was found following 

4 exposure of male and female albino rats to up to 5,000 ppm biphenyl in the diet (525 mg/kg-day) 

5 for 11 or 60 days prior to mating (Ambrose et al., 1960). In a three-generation rat study, 

6 decreased fertility, decreased number of pups/litter, and decreased pup body weight were 

7 observed at 10,000 ppm biphenyl in the diet; (947 mg/kg-day), but not at ≤1,000 ppm (Dow 

8 Chemical Co., 1953). 

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

10 toxicity studies, respiratory tract irritation and increased mortality following exposure to dusts of 

11 biphenyl (7 hours/day, 5 days/week for up to about 90 days) were reported in mice exposed to 

5 mg/m3 and in rats exposed to 300 mg/m3, but not in rabbits exposed to 300 mg/m3 12 (Deichmann 

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

14 by hyperplasia with inflammation of the trachea, was reported in CD-1 mice exposed to biphenyl 

vapors at 25 or 50 ppm (158 or 315 mg/m3 15 ) for 13 weeks (Sun Company Inc., 1977b). 

16

17 6.1.2. Cancer 

18 No assessments are available regarding possible associations between exposure to 

19 biphenyl and increased risk of cancer in humans. 

20 In a 2-year study of F344 rats administered biphenyl in the diet, significantly increased 

21 incidences of urinary bladder tumors in males were observed at the highest dose level 

22 (378 mg/kg-day). There is strong evidence that the occurrence of urinary bladder tumors in the 

23 male rats is a high-dose nongenotoxic phenomenon involving occurrence of calculi in the urinary 

24 bladder leading to transitional cell damage, sustained regenerative cell proliferation, and eventual 

25 promotion of spontaneously initiated tumor cells in the urinary bladder epithelium. Urinary 

26 bladder calculi in the high-dose (438 mg/kg-day) female rats were observed at much lower 

27 incidence and were different in physical appearance and chemical composition; furthermore, 

28 there were no urinary bladder tumors in any of the biphenyl-exposed female rats.

29 In a 2-year study of BDF1 mice administered biphenyl in the diet, the incidence of liver 

30 tumors in female mice was significantly increased at doses ≥414 mg/kg-day, but not in males at 

31 doses up to and including 1,050 mg/kg-day. Available data are insufficient to establish a mode 

32 of action for liver tumors in female mice. 

33 Under EPA Guidelines for Carcinogenic Risk Assessment (U.S. EPA, 2005a), the 

34 database for biphenyl provides “suggestive evidence of carcinogenic potential” based on 

35 evidence of female mouse liver tumors and male rat bladder tumors. 

36
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1 6.2. DOSE RESPONSE

2 6.2.1. Noncancer/Oral 

3 The RfD of 0.2 mg/kg-day was based on an increased incidence of litters with fetal 

4 skeletal anomalies from Wistar rat dams administered biphenyl by gavage on GDs 6–15 (Khera 

5 et al., 1979). The BMDL10 of 20 mg/kg-day was selected as the POD. To derive the RfD, the 

6 POD was divided by a total UF of 100 (10 for animal-to-human extrapolation and 10 for human 

7 interindividual variability in susceptibility). The interspecies uncertainty factor was applied to 

8 account for the lack of quantitative information to assess toxicokinetic and toxicodynamic 

9 differences between animals and humans. The intraspecies uncertainty factor was applied to 

10 account for the lack of information regarding the range of responses to biphenyl in the human 

11 population. 

12 The overall confidence in the RfD assessment is medium to high. Confidence in the 

13 principal study (Khera et al., 1979) is medium to high. The design, conduct and reporting of this 

14 developmental toxicity study in Wistar rats were adequate; however, only litter average data 

15 were available that did not permit a nested analysis based on individual fetal data. Confidence in 

16 the database is high. The database is robust in that it includes chronic-duration oral exposure 

17 studies in several rat and mouse strains, a developmental toxicity study in Wistar rats, and one18 and three-generation reproductive toxicity studies in rats. 

19

20 6.2.2. Noncancer/Inhalation 

21 No inhalation RfC was derived due to the lack of studies of biphenyl toxicity following 

22 chronic exposure and studies involving subchronic exposure that were inadequate for RfC 

23 derivation. Repeated exposure of mice to biphenyl vapors for 13 weeks resulted in high 

24 incidences of pneumonia and tracheal hyperplasia, and high incidences of congestion and edema 

25 in the lungs, liver, and kidney (Sun Company Inc., 1977b); however, study limitations and lack 

26 of supporting data preclude the use of this study for deriving an RfC for biphenyl. Study 

27 limitations include highly variable biphenyl exposure concentrations during the first half of the 

28 study, high mortality after 46 exposures in one group of biphenyl-exposed mice due to an 

29 overheating event and cannibalization that necessitated the use of replacement animals, and 

30 limitations in the reporting of histopathological findings. 

31

32 6.2.3. Cancer/Oral 

33 The oral slope factor of 0.008 per mg/kg-day is based on the tumor response in the liver 

34 of female BDF1 mice exposed to biphenyl in the diet for 2 years (Umeda et al., 2005). The slope 

35 factor was derived by linear extrapolation from a human equivalent BMDL10 of 12.2 mg/kg-day 

36 for liver adenomas or carcinomas. 
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1 A nonlinear extrapolation approach for biphenyl-induced urinary bladder tumors in male 

2 rats was used because evidence show that the occurrence of urinary bladder tumors is a high3 dose nongenotoxic phenomenon involving occurrence of calculi in the urinary bladder leading to 

4 transitional cell damage, sustained regenerative cell proliferation, and eventual promotion of 

5 spontaneously initiated tumor cells in the urinary bladder epithelium. As long as the dose is 

6 below that which is needed to form calculi, no increased risk of bladder tumors is expected. 

7 Therefore, the RfD of 0.2 mg/kg-day derived for noncancer effects of biphenyl was judged to be 

8 protective against increased risk of biphenyl-induced urinary bladder cancer. 

9 

10 6.2.4. Cancer/Inhalation 

11 No human or animal data on the potential carcinogenicity of inhaled biphenyl are 

12 available. Therefore, a quantitative cancer assessment for biphenyl by the inhalation pathway 

13 was not performed.
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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

1 APPENDIX A. SUMMARY OF EXTERNAL PEER REVIEW AND PUBLIC 

2 COMMENTS AND DISPOSITION

3 

4 

5 [Page intentionally left blank]
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 BDF1 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, tallied from evaluation of fetuses from Wistar rat dams 

10 administered biphenyl by gavage on GDs 6–15 (Khera et al., 1979) is shown in Table B-3. 

11

Table B-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 

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 21c 0 0 1 12c

Simple transitional cell hyperplasia 6 8 5 19d 3 5 12d 25c

Mineralization 9 6 10 18e 12 12 18 27d

Other kidney effects

Hemosiderin depositf 0 0 0 0 4 8 22c 25c

Papillary mineralization 9 9 14 23d 2 6 3 12c

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 from Umeda et al. (2002). b

Calculated doses based on TWA body weights and chronic reference food consumption values for F344 rats

(0.030 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 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 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
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Table B-2. BMD modeling datasets for body weight, selected clinical 

chemistry results, and histopathological kidney effects in male and female 

BDF1 mice exposed to biphenyl in the diet for 2 years

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 ± 4.4a 22.9 ± 2.7b

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 ± 6.0a 32.4 ± 3.6b

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 12c 26d

Clinical chemistry parameter n = 28 n = 20 n = 22 n = 31

AST (IU/L) 75 ± 27 120 ± 110 211 ± 373b 325 ± 448b

ALT (IU/L) 32 ± 18 56 ± 46 134 ± 231b 206 ± 280b

AP (IU/L) 242 ± 90 256 ± 121 428 ± 499 556 ± 228b

LDH (IU/L) 268 ± 98 461 ± 452 838 ± 2,000 1,416 ± 4,161a

BUN (mg/dL) 14.9 ± 2.0 14.8 ± 3.4 21.0 ± 20.5 23.8 ± 11.7b

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 ± 3.1b 25.5 ± 3.0b

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
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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 

Effect

Dose (mg/kg-d)

0 125 250 500 1,000

Litters with fetal skeletal anomaliesa/litters 8/16 11/20 13/18 15/18b 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 

2 Goodness of fit statistics and benchmark results for each of the modeled biphenyl3 induced nonneoplastic effects from the chronically-exposed rats (Umeda et al., 2002) and mice 

4 (Umeda et al., 2005) and the gestationally-exposed rats (Khera et al., 1979) are summarized in 

5 Tables B-4 through B-22. Each table of modeled results for a particular effect is followed by the 

6 information from the output file of the best-fitting model for that effect.

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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

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-Probitb 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

Weibullb 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 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).

8 
B-4 DRAFT - DO NOT CITE OR QUOTE

10 Wed Jan 12 10:38:57 2011

 0

 0.1

 0.2

 0.3

 0.4

 0.5

 0.6

 0 50 100 150 200 250 300 350

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

10:38 01/12 2011

BMDL BMD

 

Multistage

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Multistage Model. (Version: 3.2; Date: 05/26/2010) 

6 Input Data File: 

7 C:\USEPA\IRIS\biphenyl\rat\renalnodularhyper\male\mst_nodhypMrev_MS_3.(d) 

8 Gnuplot Plotting File: 

9 C:\USEPA\IRIS\biphenyl\rat\renalnodularhyper\male\mst_nodhypMrev_MS_3.plt

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = background + (1-background)*[1-EXP(-

15 beta1*dose^1-beta2*dose^2-beta3*dose^3)]

16 The parameter betas are restricted to be positive

17 Dependent variable = incidence

18 Independent variable = dose

19 Total number of observations = 4

20 Total number of records with missing values = 0

21 Total number of parameters in model = 4

22 Total number of specified parameters = 0

23 Degree of polynomial = 3

24 Maximum number of iterations = 250

25 Relative Function Convergence has been set to: 1e-008

26 Parameter Convergence has been set to: 1e-008

27

28 Default Initial Parameter Values 

29 Background = 0.00721859

30 Beta(1) = 3.68302e-005

31 Beta(2) = 0

32 Beta(3) = 9.69211e-009

33

34 Asymptotic Correlation Matrix of Parameter Estimates

35 ( *** The model parameter(s) -Background -Beta(2) have been estimated at a boundary point, or 

36 have been specified by the user, and do not appear in the correlation matrix )

37 Beta(1) Beta(3)

38 Beta(1) 1 -0.95

39 Beta(3) -0.95 1

40

41 Parameter Estimates

42 95.0% Wald Confidence Interval

43 Variable Estimate Std. Err. Lower Conf. Limit Upper Conf. Limit

44 Background 0 * * * 

45 Beta(1) 0.000234424 * * *

46 Beta(2) 0 * * *

47 Beta(3) 8.31393e-009 * * * 

48 * - Indicates that this value is not calculated.

49

50 Analysis of Deviance Table
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

1 Chi^2 = 1.08 d.f. = 2 P-value = 0.5832

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 together, (126.946, 248.35 ) is a 90% two-sided confidence interval for the BMD

Table 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 M 2 odel

Goodness of fit Benchmark result (mg/kg-d)

Largest a p-value residual AIC BMD5 BMDL5 BMD10 BMDL10

b Gamma

Logistic

b Log-Logistic

Log-Probitb

(2-degree) Multistage c,d

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

Weibullb 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 associated with 5% extra risk)

Source: Umeda et al. (2002).

27

28
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 0

 0.05

 0.1

 0.15

 0.2

 0.25

 0.3

 0.35

 0.4

 0 50 100 150 200 250 300 350 400 450

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

11:48 01/13 2011

BMDL BMD

 

Multistage

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Multistage Model. (Version: 3.2; Date: 05/26/2010) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalnodularhyper/female/mst_nodhypFrev_MS_2.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalnodularhyper/female/mst_nodhypFrev_MS_2.plt

10 Thu Jan 13 11:48:49 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = background + (1-background)*[1-EXP(-

15 beta1*dose^1-beta2*dose^2)]

16 The parameter betas are restricted to be positive

17 Dependent variable = incidence

18 Independent variable = dose

19 Total number of observations = 4

20 Total number of records with missing values = 0

21 Total number of parameters in model = 3

22 Total number of specified parameters = 0

23 Degree of polynomial = 2

24 Maximum number of iterations = 250

25 Relative Function Convergence has been set to: 1e-008

26 Parameter Convergence has been set to: 1e-008

27

28 Default Initial Parameter Values 

29 Background = 0

30 Beta(1) = 0

31 

32

33 Asymptotic Correlation Matrix of Parameter Estimates

34 ( *** The model parameter(s) -Background -Beta(1) 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(2)

37 Beta(2) 1

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 * * *

45 * - Indicates that this value is not calculated.

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

1 Fitted model -32.5947 1 0.277585 3 0.9642

2 Reduced model -48.1018 1 31.2917 3 <.0001

3 

4 AIC: 67.1895

5 

 

 

 

 Goodness of Fit 

7 Scaled

8 

9 

 -

Dose 

---------

Est._Prob. 

--------------

Expected Observed 

---------------------------

 Size 

--------

Residual

-------------

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

15 Chi^2 = 0.15 d.f. = 3 P-value = 0.9853

16

17 Benchmark Dose Computation

18 Specified effect = 0.1

19 Risk Type = Extra risk 

20 Confidence level = 0.95

21 BMD = 274.422

22 BMDL = 211.518

23 BMDU = 351.444

24 Taken together, (211.518, 351.444) is a 90% two-sided confidence interval for the BMD

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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

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-Probitb 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

Weibullb 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 associated 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-Hit

Fraction 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)*CumGamma[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 

 

Dose 

----------

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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

b Gamma , Weibullb

, 

Multistage (1 d -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-Probitb 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% extra 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 50 100 150 200 250 300 350 400 450

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

14:01 01/13 2011

BMDL BMD

 

Multistage

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Multistage Model. (Version: 3.2; Date: 05/26/2010) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalsimplehyper/female/mst_simplehypFrev_MS_1.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalsimplehyper/female/mst_simplehypFrev_MS_1.plt

10 Thu Jan 13 14:01:13 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = background + (1-background)*[1-EXP(-

15 beta1*dose^1)]

16 The parameter betas are restricted to be positive

17 Dependent variable = incidence

18 Independent variable = dose

19 Total number of observations = 4

20 Total number of records with missing values = 0

21 Total number of parameters in model = 2

22 Total number of specified parameters = 0

23 Degree of polynomial = 1

24 Maximum number of iterations = 250

25 Relative Function Convergence has been set to: 1e-008

26 Parameter Convergence has been set to: 1e-008

27

28 Default Initial Parameter Values 

29 Background = 0.0607741

30 Beta(1) = 0.00145231

31

32 Asymptotic Correlation Matrix of Parameter Estimates

33 Background Beta(1)

34 Background 1 -0.61

35 Beta(1) -0.61 1

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 calculated.

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 0.8842 

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 

10 Chi^2 = 0.24 d.f. = 2 P-value = 0.8850

11

12 Benchmark Dose Computation

13 Specified effect = 0.1

14 Risk Type = Extra risk 

15 Confidence level = 0.95

16 BMD = 71.1248

17 BMDL = 52.0766

18 BMDU = 105.072

19 Taken together, (52.0766, 105.072) is a 90% two-sided confidence interval for the BMD

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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab 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-Logisticb 0.34 -0.75 206.14 128.13 36.96 199.42 78.03

Log-Probitb,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

Weibullb 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 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-12 DRAFT - DO NOT CITE OR QUOTE

 0.1

 0.2

 0.3

 0.4

 0.5

 0 50 100 150 200 250 300 350

Fraction Affected

dose

LogProbit Model with 0.95 Confidence Level

15:38 01/13 2011

BMDL BMD

 

LogProbit

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Probit Model. (Version: 3.2; Date: 10/28/2009) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/male/lnp_minpelvMrev_logprobit.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/male/lnp_minpelvMrev_logprobit.plt

10 Thu Jan 13 15:38:28 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = Background + (1-Background) * 

15 CumNorm(Intercept+Slope*Log(Dose)), where CumNorm(.) is the cumulative normal distribution 

16 function

17 Dependent variable = incidence

18 Independent variable = dose

19 Slope parameter is restricted as slope >= 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 User has chosen the log transformed model

26

27 Default Initial (and Specified) Parameter Values 

28 background = 0.18

29 intercept = -6.59931

30 slope = 1

31

32 Asymptotic Correlation Matrix of Parameter Estimates

33 ( *** The model parameter(s) -slope have been estimated at a boundary point, or have been 

34 specified by the user, and do not appear in the correlation matrix )

35 background intercept

36 background 1 -0.46

37 intercept -0.46 1

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

45 NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus 

46 has no standard error.

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 -100.063 2 0.91202 2 0.6338
B-13 DRAFT - DO NOT CITE OR QUOTE

1 Reduced model -104.101 1 8.98864 3 0.02944

2 

3 AIC: 204.126

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

14 Chi^2 = 0.88 d.f. = 2 P-value = 0.6434

15

16 Benchmark Dose Computation

17 Specified effect = 0.1

18 Risk Type = Extra risk 

19 Confidence level = 0.95

20 BMD = 207.879

21 BMDL = 138.127

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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab 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-Logisticb <0.001 2.90 263.72 1.33 × 1015 NA 1.58 × 1015 NA

Log-Probitb <0.001 2.90 263.72 1.54 × 1014 NA 2.21 × 1014 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

Weibullb 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 50 100 150 200 250 300 350 400 450

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

16:24 01/13 2011

BMDL BMD

 

Multistage

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Multistage Model. (Version: 3.2; Date: 05/26/2010) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/female/mst_minpelvlFrev_MS_1.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/renalmineral/female/mst_minpelvlFrev_MS_1.plt

10 Thu Jan 13 16:24:18 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = background + (1-background)*[1-EXP(-

15 beta1*dose^1)]

16 The parameter betas are restricted to be positive

17 Dependent variable = incidence

18 Independent variable = dose

19 Total number of observations = 4

20 Total number of records with missing values = 0

21 Total number of parameters in model = 2

22 Total number of specified parameters = 0

23 Degree of polynomial = 1

24 Maximum number of iterations = 250

25 Relative Function Convergence has been set to: 1e-008

26 Parameter Convergence has been set to: 1e-008

27

28 Default Initial Parameter Values 

29 Background = 0.230737

30 Beta(1) = 0.00118679

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 

 

 

 

 

 Goodness 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

12 Benchmark Dose Computation

13 Specified effect = 0.1

14 Risk Type = Extra risk 

15 Confidence level = 0.95

16 BMD = 87.669

17 BMDL = 56.2773

18 BMDU = 172.188

19 Taken together, (56.2773, 172.188) is a 90% two-sided confidence

20

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 

Model

Goodness of fit Benchmark result (mg/kg-d)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab, Weibullb, 

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-Logisticb 0.093 1.75 218.35 19.21 12.74 40.56 26.89

Log-Probitb 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-Hilld,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 only model with an adequate fit (χ2 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

 0 100 200 300 400

Fraction Affected

dose

Dichotomous-Hill Model with 0.95 Confidence Level

09:14 01/14 2011

BMDL BMD

 

Dichotomous-Hill

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Dichotomous Hill Model. (Version: 1.2; Date: 12/11/2009) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/hemosiderin/female/dhl_hemosidFrev_dichotomous 

8 hill.(d) 

9 Gnuplot Plotting File: 

10 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/hemosiderin/female/dhl_hemosidFrev_dichotomous 

11 hill.plt

12 Fri Jan 14 09:14:35 2011

13 ==================================================================== 

14 BMDS_Model_Run 

15 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

16 The form of the probability function is: P[response] = v*g +(v-v*g)/[1+EXP(-intercept17 slope*Log(dose))] where: 0 <= g < 1, 0 < v <= 1v is the maximum probability of response predicted 

18 by the model, and v*g is the background estimate of that probability.

19 Dependent variable = incidence

20 Independent variable = dose

21 Parameter v is set to 0.5

22 Parameter g is set to 0.16

23 Slope parameter is restricted as slope >= 1

24 Total number of observations = 4

25 Total number of records with missing values = 0

26 Maximum number of iterations = 250

27 Relative Function Convergence has been set to: 1e-008

28 Parameter Convergence has been set to: 1e-008

29

30 User Inputs Initial Parameter Values 

31 v = -9999 Specified

32 g = -9999 Specified

33 intercept = 0.08

34 slope = 1

35

36 Asymptotic Correlation Matrix of Parameter Estimates

37 ( *** The model parameter(s) -v -g have been estimated at a boundary point, or have been 

38 specified by the user, and do not appear in the correlation matrix )

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 

 

 

 

 

 

 

 

 

 

Model 

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

19 Benchmark Dose Computation

20 Specified effect = 0.1

21 Risk Type = Extra risk 

22 Confidence level = 0.95

23 BMD = 45.3249

24 BMDL = 23.2881

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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab 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-Logisticb <0.001 2.93 241.27 5.64 × 1014 NA 6.68 × 1014 NA

Log-Probitb 0.001 2.93 239.27 5.13 × 1013 NA 7.38 × 1013 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

Weibullb 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 associated 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 50 100 150 200 250 300 350

Fraction Affected

dose

Multistage Model with 0.95 Confidence Level

11:25 01/14 2011

BMDL BMD

 

Multistage

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Multistage Model. (Version: 3.2; Date: 05/26/2010) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/pappmineral/male/mst_papminMrev_MS_1.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/pappmineral/male/mst_papminMrev_MS_1.plt

10 Fri Jan 14 11:25:01 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = background + (1-background)*[1-EXP(-

15 beta1*dose^1)]

16 The parameter betas are restricted to be positive

17 Dependent variable = incidence

18 Independent variable = dose

19 Total number of observations = 4

20 Total number of records with missing values = 0

21 Total number of parameters in model = 2

22 Total number of specified parameters = 0

23 Degree of polynomial = 1

24 Maximum number of iterations = 250

25 Relative Function Convergence has been set to: 1e-008

26 Parameter Convergence has been set to: 1e-008

27

28 Default Initial Parameter Values 

29 Background = 0.168963

30 Beta(1) = 0.00114658

31

32 Asymptotic Correlation Matrix of Parameter Estimates

33 Background Beta(1)

34 Background 1 -0.62

35 Beta(1) -0.62 1

36

37 Parameter Estimates

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

12 Benchmark Dose Computation

13 Specified effect = 0.1

14 Risk Type = Extra risk 

15 Confidence level = 0.95

16 BMD = 91.741

17 BMDL = 58.4361

18 BMDU = 182.915

19 Taken together, (58.4361, 182.915) is a 90% two-sided confidence interval for the BMD

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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab 0.11 1.27 139.76 360.00 68.91 397.57 141.55

Logisticc 0.23 1.37 138.04 175.24 129.91 292.33 219.17

Log-Logisticb 0.11 1.27 139.76 388.83 61.62 413.84 130.08

Log-Probitb 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

Weibullb 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 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-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 50 100 150 200 250 300 350 400 450

Fraction Affected

dose

Logistic Model with 0.95 Confidence Level

13:00 01/14 2011

BMDL BMD

 

Logistic

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Logistic Model. (Version: 2.13; Date: 10/28/2009) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/pappmineral/female/log_papmineralFrev_logistic.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/pappmineral/female/log_papmineralFrev_logistic.plt

10 Fri Jan 14 13:00:44 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = 1/[1+EXP(-intercept-slope*dose)]

15 Dependent variable = incidence

16 Independent variable = dose

17 Slope parameter is not restricted

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

24 Default Initial Parameter Values 

25 background = 0 Specified

26 intercept = -2.67819

27 slope = 0.00343504

28

29 Asymptotic Correlation Matrix of Parameter Estimates

30 ( *** The model parameter(s) -background have been estimated at a boundary point, or have been 

31 specified by the user, and do not appear in the correlation matrix )

32 intercept slope

33 intercept 1 -0.78

34 slope -0.78 1

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 

10 Benchmark Dose Computation

11 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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

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-Probitb 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

Weibullb 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 50 100 150 200 250 300 350

Fraction Affected

dose

Gamma Multi-Hit Model with 0.95 Confidence Level

14:15 01/14 2011

BMDL BMD

 

Gamma Multi-Hit

1 

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/bladdercombinedhyper/male/gam_bladcomhypMrev_gamma.(d

8 ) 

9 Gnuplot Plotting File: 

10 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/bladdercombinedhyper/male/gam_bladcomhypMrev_gamma.pl

11 t 

12 Fri Jan 14 14:15:19 2011

13 ==================================================================== 

14 BMDS_Model_Run 

15 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

16 The form of the probability function is: P[response]= background+(1-

17 background)*CumGamma[slope*dose,power], where CumGamma(.) is the cummulative Gamma distribution 

18 function

19 Dependent variable = incidence 

20 Independent variable = dose

21 Power parameter is restricted as power >=1

22 Total number of observations = 4Total 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

27 Default Initial (and Specified) Parameter Values 

28 Background = 0.0192308

29 Slope = 0.0320399

30 Power = 8.56462

31

32 Asymptotic Correlation Matrix of Parameter Estimates

33 ( *** The model parameter(s) -Background -Power have been estimated at a boundary point, or 

34 have been specified by the user, and do not appear in the correlation matrix )

35 Slope

36 Slope 1

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. Limit 

 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 -16.2687 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

17 Benchmark Dose Computation

18 Specified effect = 0.1

19 Risk Type = Extra risk 

20 Confidence level = 0.95

21 BMD = 205.404

22 BMDL = 146.733

23

Table B-14. Summary of BMD modeling results for incidence of 

mineralization in the kidney (inner stripe outer medulla) of male BDF1 mice 

exposed to biphenyl in the diet for 2 years

Model

Goodness of fit Benchmark result (mg/kg-d)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab, Weibullb, 

Multistage (1-degree)c

0.46 1.03 214.84 369.24 155.65 758.45 319.71

Logistic 0.43 1.07 214.97 454.16 238.75 856.07 446.12

Log-Logisticb,d 0.48 1.01 214.79 341.66 130.84 721.28 276.22

Log-Probitb 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 1000

Fraction Affected

dose

Log-Logistic Model with 0.95 Confidence Level

12:57 01/17 2011

BMDL BMD

 

Log-Logistic

1 

2 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

3 

4 ==================================================================== 

5 Logistic Model. (Version: 2.13; Date: 10/28/2009) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/male/lnl_minmedullM_loglogistic.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/male/lnl_minmedullM_loglogistic.plt

10 Mon Jan 17 12:57:13 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-

15 intercept-slope*Log(dose))]

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 Parameter Values 

27 background = 0.18

28 intercept = -8.98323

29 slope = 1.06986

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 

35

36 

37

 

 

 

 

 

 

 

background 

intercept 

background 

 1 

-0.64 

intercept

-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

1 AIC: 214.794

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

14 Benchmark Dose Computation

15 Specified effect = 0.1

16 Risk Type = Extra risk 

17 Confidence level = 0.95

18 BMD = 721.275

19 BMDL = 276.216

20

Table B-15. Summary of BMD modeling results for incidence of 

mineralization in the kidney (inner stripe outer medulla) of female BDF1

mice exposed to biphenyl in the diet for 2 years

Model

Goodness of fit Benchmark result (mg/kg-d)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab 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-Logisticb,c 0.80 -0.18 184.12 127.12 57.98 233.39 122.40

Log-Probitb 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

Weibullb 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 BMDL10 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).

21
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-Logistic

Fraction Affected

 

BMDL BMD

dose

1 13:27 01/17 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 Logistic Model. (Version: 2.13; Date: 10/28/2009) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/female/lnl_minmedullF_loglogistic.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/minmedulla/female/lnl_minmedullF_loglogistic.plt

10 Mon Jan 17 13:27:41 2011

11 ==================================================================== 

12 BMDS_Model_Run 

13 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

14 The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-

15 intercept-slope*Log(dose))]

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 Parameter Values 

27 background = 0.06

28 intercept = -9.5037

29 slope = 1.31777

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

1 AIC: 184.122

2 

3 

 

 

 

 

 Goodness of Fit 

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

14 Benchmark Dose Computation

15 Specified effect = 0.1

16 Risk Type = Extra risk 

17 Confidence level = 0.95

18 BMD = 233.39

19 BMDL = 122.401

20

Table B-16. BMD model results for serum LDH activity in female BDF1

mice exposed to biphenyl in the diet for 2 years

Model

Goodness of fit Benchmark result (mg/kg-d)

Variance 

model

p-valuea

Means 

model

p-valuea

Largest

residual AIC BMD1SD BMDL1SD BMD1RD BMDL1RD

All doses

Constant variance

Hillb <0.0001 NA 0.00 1,687.59 CF CF 182.66 0.0000

Linearc <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

Powerd <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

Linearb 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

Powerd 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 standard 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 are ≤0, the χ2 test for fit is not valid)

Source: 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 BDF1

mice exposed to biphenyl in the diet for 2 years

Goodness of fit Benchmark result (mg/kg-d)

Variance Means 

Model

model a p-valuemodel a p-valueLargest

residual AIC BMD1SD BMDL1SD BMD1RD BMDL1RD

All doses

Constant variance

Hillb <0.0001 NA -5.69 × 107 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

Hillb 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 × 109 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

Hillb 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

Hillb Not modeled; number of dose groups less than number of model parameters

c Linear 0.78 <0.0001 3.24 × 108 6 0 CF 228.57 CF

Polynomial (2-degree)c 0.78 <0.0001 -2.20 × 109 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% relative deviation 

from control mean value); CF = computation failed; NA = not applicable (degrees of freedom for the test of mean 

fit are ≤0, the χ2 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 50 100 150 200 250 300 350 400

Mean Response

dose

Power Model with 0.95 Confidence Level

10:47 01/18 2011

BMDL BMD

 

Power

1 

2 BMD and BMDL indicated are associated with a twofold increase from control, and are in units of mg/kg-day.

3 

4 ==================================================================== 

5 Power Model. (Version: 2.16; Date: 10/28/2009) 

6 Input Data File: C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/AST/pow_ASTFHDD_power.(d) 

7 Gnuplot Plotting File: 

8 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/AST/pow_ASTFHDD_power.plt

9 Tue Jan 18 10:47:11 2011

10 ==================================================================== 

11 BMDS Model Run 

12 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

13 The form of the response function is: Y[dose] = control + slope * dose^power

14 Dependent variable = mean

15 Independent variable = dose

16 The power is restricted to be greater than or equal to 1

17 The variance is to be modeled as Var(i) = exp(lalpha + log(mean(i)) * rho)

18 Total number of dose groups = 3

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

24 Default Initial Parameter Values 

25 lalpha = 10.765

26 rho = 0

27 control = 75

28 slope = 0.369536

29 power = 0.980467

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 )

 

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

48 NA - Indicates that this parameter has hit a bound implied by some inequality constraint and thus 

49 has no standard error.

50

51 Table of Data and Estimated Values of Interest
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 

7 Model Descriptions for likelihoods calculated

8 Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

9 Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

10 Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = exp(lalpha + rho*ln(Mu(i)))

11 Model A3 uses any fixed variance parameters that were specified by the user

12 Model R: Yi = Mu + e(i) Var{e(i)} = Sigma^2

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

22 Explanation of Tests 

23 Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R)

24 Test 2: Are Variances Homogeneous? (A1 vs A2)

25 Test 3: Are variances adequately modeled? (A2 vs. A3)

26 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

27 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.)

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

36 The p-value for Test 1 is less than .05. There appears to be a difference between response 

37 and/or variances among the dose levels. It seems appropriate to model the data

38

39 The p-value for Test 2 is less than .1. A non-homogeneous variance model appears to be 

40 appropriate

41

42 The p-value for Test 3 is greater than .1. The modeled variance appears to be appropriate here

43

44 The p-value for Test 4 is greater than .1. The model chosen seems to adequately describe the 

45 data

46

47 Benchmark Dose Computation

48 Specified effect = 1

49 Risk Type = Relative risk 

50 Confidence level = 0.95

51 BMD = 190.33 

52 BMDL = 121.534

53
B-31 DRAFT - DO NOT CITE OR QUOTE

Table B-18. BMD modeling results for serum ALT activity in female BDF1

mice exposed to biphenyl in the diet for 2 years

Model

Goodness of fit Benchmark result (mg/kg-d)

Variance 

model

p-valuea

Means 

model

p-valuea

Largest

residual AIC BMD1SD BMDL1SD BMD1RD BMDL1RD

All doses

Constant variance

Hillb <0.0001 NA 9.61 × 10-7 1,167.39 3,911.09 436.97 160.82 0.00

Linearc, Polynomial 

(2-degree)c, Powerd

<0.0001 0.55 0.94 1,164.57 1,613.62 1,106.30 412.90 38.31

Non constant variance

Hillb 0.78 NA -0.49 1,013.25 116.28 CF 148.75 121.30

Linearc 0.78 <0.0001 1.69 × 1010 6 0 CF 419.08 CF

Polynomial (2-degree)c 0.78 <0.0001 -1.39 × 1011 8 0 CF 87.64 CF

Powerd 0.78 <0.0001 -1.88 1,047.49 90.73 62.72 108.55 77.76

Highest dose dropped

Constant variance

Hillb Not modeled; number of dose groups less than number of model parameters

Linearc, <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

Powerd <0.0001 NA -3.00 × 10-9 758.65 497.95 325.96 167.69 0.00

Non constant variance

Hillb Not modeled; number of dose groups less than number of model parameters

Linearc 0.89 <0.0001 -2.59 × 109 6 0 CF 111.13 CF

Polynomial (2-degree)c 0.89 <0.0001 -5.85 × 107 8 0 CF 169.57 CF

Powerd 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 standard deviation from control mean value; 1RD = dose associated with a 100% relative deviation 

from control mean value); CF = computation 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 BDF1

mice exposed to biphenyl in the diet for 2 years

Goodness of fit Benchmark result (mg/kg-d)

Variance Means 

Model

model a p-valuemodel a p-valueLargest

residual AIC BMD1SD BMDL1SD BMD1RD BMDL1RD

All doses

Constant variance

Hillb <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

Hillb 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 × 1011 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

Hillb 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

Hillb Not modeled; number of dose groups less than number of model parameters

c Linear 0.77 <0.0001 4.52 × 109 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 = computation 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 BDF1 mice exposed to biphenyl in the diet for 2 years

Model

Goodness of fit Benchmark result (mg/kg-d)

Variance 

model

p-valuea

Means 

model

p-valuea

Largest

residual AIC BMD1SD BMDL1SD BMD1RD BMDL1RD

Males

All doses

Constant variance

Hillb 0.03 NA 0.25 540.50 CF CF CF CF

Linearc,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

Hillb 0.01 NA 0.25 542.49 CF CF CF CF

Linearc 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

Powerd 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

Hillb Not modeled; number of dose groups less than number of model parameters

Linearc, Polynomial 

(2-degree)c, Powerd

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 = computation 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 50 100 150 200 250 300 350 400

Mean Response

dose

Linear Model with 0.95 Confidence Level

11:03 01/19 2011

BMDL BMD

 

Linear

1 

 2 

3 BMD and BMDL indicated are associated with a 1SD change from control, and are in units of mg/kg-day. 4 ==================================================================== 

5 Polynomial Model. (Version: 2.16; Date: 05/26/2010) 

6 Input Data File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/BUN/male/lin_BUNMHDD_linear.(d) 

8 Gnuplot Plotting File: 

9 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/BUN/male/lin_BUNMHDD_linear.plt

10 Wed Jan 19 11:03:37 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 Dependent variable = mean

16 Independent variable = dose

17 rho is set to 0

18 The polynomial coefficients are restricted to be positive

19 A constant variance model is fit

20 Total number of dose groups = 3

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 Parameter Values 

27 alpha = 16.1929

28 rho = 0 Specified

29 beta_0 = 20.5429

30 beta_1 = 0.00972018

31

32 Asymptotic Correlation Matrix of Parameter Estimates

33 ( *** The model parameter(s) -rho have been estimated at a boundary point, or have been 

34 specified by the user, and do not appear in the correlation matrix )

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 Estimates

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

47 Table of Data and Estimated Values of Interest

48 Dose N Obs Mean Est Mean Obs Std Dev Est Std Dev Scaled Res.

49 ------ --- -------- -------- ----------- ----------- ----------

50

51 0 34 20.2 20.6 3.6 3.99 -0.55
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)

6 Var{e(ij)} = Sigma^2

7 Model A2: Yij = Mu(i) + e(ij)

8 Var{e(ij)} = Sigma(i)^2

9 Model A3: Yij = Mu(i) + e(ij)

10 Var{e(ij)} = Sigma^2

11 Model A3 uses any fixed variance parameters that were specified by the user

Model R: Yi = Mu + e(i)

13 Var{e(i)} = Sigma^2

12

14

15 Likelihoods of Interest

16

17 

 

Model 

A1 

 Log(likelihood) 

-206.630664 

 # Param's 

 4 

 AIC

421.261329

18 A2 -205.915695 6 423.831391

19 A3 -206.630664 4 421.261329

20 fitted -207.115525 3 420.231050

21 R -211.514015 2 427.028031

22

23 Explanation of Tests 

24

25 Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R)

26 Test 2: Are Variances Homogeneous? (A1 vs A2)

27 Test 3: Are variances adequately modeled? (A2 vs. A3)

28 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

29 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.)

30

31 Tests of Interest 

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

38 The p-value for Test 1 is less than .05. There appears to be a difference between response 

39 and/or variances among the dose levels. It seems appropriate to model the data

40

41 The p-value for Test 2 is greater than .1. A homogeneous variance model appears to be 

42 appropriate here

43

44 The p-value for Test 3 is greater than .1. The modeled variance appears to be appropriate here

45

46 The p-value for Test 4 is greater than .1. The model chosen seems to adequately describe the 

47 data

48

49 Benchmark Dose Computation

50 Specified effect = 1

51 Risk Type = Estimated standard deviations from the control mean

52 Confidence level = 0.95

53 BMD = 414.775

54 BMDL = 266.77

55
B-36 DRAFT - DO NOT CITE OR QUOTE

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

female BDF1 mice exposed to biphenyl in the diet for 2 years

Model

Goodness of fit Benchmark result (mg/kg-d)

Variance 

model

p-valuea

Means 

model

p-valuea

Largest

residual AIC BMD1SD BMDL1SD BMD1RD BMDL1RD

All doses

Constant variance

Hillb <0.0001 NA -3.45 × 10-8 603.61 CF CF CF CF

Linearc, Polynomial 

(2-degree)c, Powerd

<0.0001 0.38 1.18 601.53 1,869.01 1,224.15 2,507.85 1,434.76

Non constant variance

Hillb 0.08 NA -1.21 493.48 141.72 CF CF CF

Linearc, Polynomial 

(2-degree)c, Powerd

0.08 <0.0001 -1.63 590.70 519.60 216.41 1,191.69 683.73

Highest dose dropped

Constant variance

Hillb Not modeled; number of dose groups less than number of model parameters

Linearc, <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

Powerd <0.0001 NA -2.11 × 10-10 419.13 430.03 414.77 436.97 417.75

Non constant variance

Hillb Not modeled; number of dose groups less than number of model parameters

Linearc 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

Powerd 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 = computation 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 BDF1 mice exposed to biphenyl in the diet for 2 years

Model

Goodness of fit Benchmark result (mg/kg-d)

Variance 

model

p-valuea

Means 

model

p-valuea

Largest

residual AIC BMD1SD BMDL1SD BMD0.1RD BMDL0.1RD

All doses

Constant variance

Hillb <0.0001 0.03 -1.68 716.95 459.61 390.85 358.30 316.09

Linearc, Powerd <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

Hillb 0.002 NA -1.52 704.84 600.48 CF 421.46 325.00

Linearc, 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

Powerd 0.002 0.38 -1.51 702.84 600.89 464.26 421.53 327.62

Highest dose dropped

Constant variance

Hillb Not modeled; number of dose groups less than number of model parameters

Linearc, Polynomial 

(2-degree)c, Powerd

0.01 0.05 -1.49 560.11 566.99 328.79 400.33 238.24

Non constant variance

Hillb Not modeled; number of dose groups less than number of model parameters

Linearc, Polynomial 

(2-degree)c, Powerd

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 = computation 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 modeling results for changes in mean terminal body 

weight in female BDF1 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-valueLargest

residual AIC BMD1SD BMDL1SD BMD0.1RD BMDL0.1RD

All doses

Constant variance

Hillb 0.36 0.80 -0.21 382.59 387.90 230.17 397.06 243.57

Linearc,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 = computation failed; NA = not applicable

Source: Umeda et al. (2005).

1 

 24

 26

 28

 30

 32

 34

 36

 0 200 400 600 800 1000 1200 1400

Mean Response

dose

Linear Model with 0.95 Confidence Level

09:20 01/20 2011

BMDL BMD

 

Linear

2 

3 BMD and BMDL indicated are associated with a 10% decrease from control, and are in units of mg/kg-day. 

 4 

5 ==================================================================== 

6 Polynomial Model. (Version: 2.16; Date: 05/26/2010) 

7 Input Data File: 

8 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/termbdwt/female/lin_termbdwtF_linear.(d) 

9 Gnuplot Plotting File: 

10 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/termbdwt/female/lin_termbdwtF_linear.plt

11 Thu Jan 20 09:20:01 2011

12 ==================================================================== 

13 BMDS Model Run 

14 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

15 The form of the response function is: Y[dose] = beta_0 + beta_1*dose + beta_2*dose^2 + ...
B-39 DRAFT - DO NOT CITE OR QUOTE

1 Dependent variable = mean

2 Independent variable = dose

3 rho is set to 0

4 The polynomial coefficients are restricted to be negative

5 A constant variance model is fit

6 Total number of dose groups = 4

7 Total number of records with missing values = 0

8 Maximum number of iterations = 250

9 Relative Function Convergence has been set to: 1e-008

10 Parameter Convergence has been set to: 1e-008

11

12 Default Initial Parameter Values 

13 alpha = 11.4937

14 rho = 0 Specified

15 beta_0 = 33.4391

16 beta_1 = -0.00571961

17

18 Asymptotic Correlation Matrix of Parameter Estimates

19 ( *** The model parameter(s) -rho have been estimated at a boundary point, or have been 

20 specified by the user, and do not appear in the correlation matrix )

 

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

41 Model Descriptions for likelihoods calculated

42 Model A1: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

43 Model A2: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma(i)^2

44 Model A3: Yij = Mu(i) + e(ij) Var{e(ij)} = Sigma^2

45 Model A3 uses any fixed variance parameters that were specified by the user

46 Model R: Yi = Mu + e(i) Var{e(i)} = Sigma^2

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

56 Explanation of Tests 

57

58 Test 1: Do responses and/or variances differ among Dose levels? (A2 vs. R)

59 Test 2: Are Variances Homogeneous? (A1 vs A2)

60 Test 3: Are variances adequately modeled? (A2 vs. A3)

61 Test 4: Does the Model for the Mean Fit? (A3 vs. fitted)

62 (Note: When rho=0 the results of Test 3 and Test 2 will be the same.)

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

71 The p-value for Test 1 is less than .05. There appears to be a difference between response 

72 and/or variances among the dose levels. It seems appropriate to model the data

73

74 The p-value for Test 2 is greater than .1. A homogeneous variance model appears to be 

75 appropriate here
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 modeling 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)

χ2 p-valuea

Largest

residual AIC BMD5 BMDL5 BMD10 BMDL10

Gammab, Weibullb, 

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-Logisticb,d 0.41 -1.32 105.33 27.03 9.59 57.06 20.24

Log-Probitb 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 response: 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 1000

Fraction Affected

dose

Log-Logistic Model with 0.95 Confidence Level

16:06 01/14 2011

BMDL BMD

 

Log-Logistic

2 

3 

4 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day.

 5 

 6 

7 ==================================================================== 

8 Logistic Model. (Version: 2.13; Date: 10/28/2009) 

9 Input Data File: 

10 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/develop/anomlitt/lnl_anomlitt_loglogistic.(d) 

11 Gnuplot Plotting File: 

12 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/rat/develop/anomlitt/lnl_anomlitt_loglogistic.plt

13 Fri Jan 14 16:06:43 2011

14 ==================================================================== 

15 BMDS_Model_Run 

16 ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~

17 The form of the probability function is: P[response] = background+(1-background)/[1+EXP(-

18 intercept-slope*Log(dose))]

19 Dependent variable = incidence

20 Independent variable = dose

21 Slope parameter is restricted as slope >= 1 

22 Total number of observations = 5

23 Total number of records with missing values = 0

24 Maximum number of iterations = 250

25 Relative Function Convergence has been set to: 1e-008

26 Parameter Convergence has been set to: 1e-008

27 User has chosen the log transformed model

28

29 Default Initial Parameter Values 

30 background = 0.5

31 intercept = -6.54827

32 slope = 1

33

34 Asymptotic Correlation Matrix of Parameter Estimates

35 ( *** The model parameter(s) -slope have been estimated at a boundary point, or have been 

36 specified by the user, and do not appear in the correlation matrix )

37 background intercept

38 background 1 -0.77

39 intercept -0.77 1

40

 

 

 

41 Parameter Estimates

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

*

*

*

47 * - Indicates that this value is not calculated.

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 

6 Goodness of Fit 

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

16 Chi^2 = 2.90 d.f. = 3 P-value = 0.4065

17

18 Benchmark Dose Computation

19 Specified effect = 0.1

20 Risk Type = Extra risk 

21 Confidence level = 0.95

22 BMD = 57.0591

23 BMDL = 20.2399
C-1 DRAFT - DO NOT CITE OR QUOTE

1 

2 APPENDIX C. BENCHMARK MODELING FOR THE ORAL SLOPE FACTOR

3 

4 

5 The mouse liver tumor dataset from Umeda et al. (2005) for which dose-response 

6 modeling was performed is shown in Table C-1. 

7 

Table C-1. Incidences of liver adenomas or carcinomas (combined) in 

female BDF1 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) 3/48a 8/50 16/49a,b 14/48a,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. cSignificantly different from controls (p < 0.01) according to Fisher’s exact test.

Source: Umeda et al. (2005).

8 

9 Summaries of the BMDs, BMDLs, and the derived oral slope factors for the modeled 

10 mouse data are presented in Table C-2, followed by the plot and model output file from the best11 fitting model. The animals in the highest dose group, while exhibiting a statistically 

12 significantly increased incidence in liver tumors compared with controls, did not show a 

13 monotonic increase in tumor response compared with the responses at the lower doses. To better 

14 estimate responses in the low dose region, the high dose group was excluded as a means of 

15 improving the fit of the model in the region of interest. 

16
C-2 DRAFT - DO NOT CITE OR QUOTE

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

combined) in female BDF1 mice exposed to biphenyl in the diet for 2 years

Goodness of fit Benchmark result (mg/kg-d)

Cancer 

slope 

factor 

Model χ2 p-valuea

Largest

residual AIC BMDHED10 BMDLHED10

(risk per 

mg/kg-d)

All doses

Multistage (1-, 2-, c Gamma , Weibullc3-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-Probitc 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 60

Fraction Affected

dose

Multistage Cancer Model with 0.95 Confidence Level

09:33 02/03 2011

BMDL BMD

 

Multistage Cancer

Linear extrapolation

2 

3 BMD and BMDL indicated are associated with an extra risk of 10%, and are in units of mg/kg-day. 

 4 
C-3 DRAFT - DO NOT CITE OR QUOTE

1 ==================================================================== 

2 Multistage Cancer Model. (Version: 1.9; Date: 05/26/2010) 

3 Input Data File: 

4 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/livertumor/female/revised_n/msc_livtumFrev2HDD_MS_1.

5 (d) 

6 Gnuplot Plotting File: 

7 C:/Storage/USEPA/IRIS/biphenyl/2011/BMD/mice/livertumor/female/revised_n/msc_livtumFrev2HDD_MS_1.

8 plt

9 Thu Feb 03 09:33:34 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 = 3

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: 2.22045e-016

25 Parameter Convergence has been set to: 1.49012e-008

26 **** We are sorry but Relative Function and Parameter Convergence are currently unavailable in 

27 this model. Please keep checking the web sight for model updates which will eventually 

28 incorporate these convergence criterion. Default values used. ****

29

30 Default Initial Parameter Values 

31 Background = 0.0638384

32 Beta(1) = 0.00559363

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

*

43 Beta(1) 0.00562948 * * *

44 * - Indicates that this value is not calculated.

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

53 AIC: 132.319

54

55 Goodness of Fit 

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

63 Chi^2 = 0.00 d.f. = 1 P-value = 0.9644

64

65 Benchmark Dose Computation

66 Specified effect = 0.1

67 Risk Type = Extra risk 

68 Confidence level = 0.95

69 BMD = 18.7158

70 BMDL = 12.1518

71 BMDU = 36.3895

72 Taken together, (12.1518, 36.3895) is a 90% two-sided confidence interval for the BMD 

73 Multistage Cancer Slope Factor = 0.00822924

74