# NTP-CERHR Monograph on the **Potential Human Reproductive and Developmental Effects**  **of Di-isononyl Phthalate (DINP)**

March 200 3 NIH Publication No. 03-4484 

# ����������������������������������� ��������������������� ��������������������������� ��������������������������������������������

## Table of Contents

Preface

............................................................... ............................................................... ............... i

Introduction

............................................................... ............................................................... ....... ii

NTP Brief on Di-isononyl Phthalate (DINP)

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

References

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

Appendix I. NTP-CERHR Phthalates Expert Panel
Preface

............................................................... ............................................................... I-1 Expert Panel

............................................................... ....................................................... I-2

Appendix II. Phthalates Expert Panel Report on DINP
Preface

............................................................... .............................................................. II-i Chemistry, Usage and Exposure

............................................................... ....................... II-1 General Toxicological and Biological Parameters

......................................................... II-7 Developmental Toxicity Data

............................................................... ......................... II-15 Reproductive Toxicity

............................................................... .................................... II-19 Data Summary & Integration

............................................................... ......................... II-21
References

............................................................... ...................................................... II-34
Tables

............................................................... ............................................................. II-37

Appendix III. Public Comments on the Phthalates Expert Panel Reports
AdvaMed

............................................................... ......................................................... III-1 American Chemistry Council (12-7-2000)

............................................................... ..... III-5 American Chemistry Council (12-11-2000)

............................................................... ... III-7 American Chemistry Council (4-13-2001)

............................................................... ... III-58 Discovery Medical, Inc

............................................................... ................................. III-66 Environmental Working Group (11-3-2000)

............................................................... . III-67 Environmental Working Group (12-8-2000)

............................................................... . III-69
William Faber

............................................................... ................................................ III-71 Healthy Environments & Product Safety Branch

........................................................ III-81
Health Care Without Harm

............................................................... ........................... III-83 Beverly Smith

............................................................... ................................................ III-87 Swedish Chemical Inspection Agency

............................................................... .......... III-88i The National Toxicology Program (NTP) es

tablished the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) in 1998. The CERHR is a publicly accessible resource for information about adverse repro

ductive and/or developmental health effects associated with exposure to environmental and/or occupational chemicals. The CERHR
is located at the National Institute of Envi

ronmental Health Sciences (NIEHS) of the
National Institutes of Health and Dr. Michael Shelby is the director. 1

The CERHR broadly solicits nominations of chemicals for evaluation from the public and private sectors. The CERHR follows a formal process for review and evaluation of nominated chemicals that includes multiple opportunities for public comment. Chemicals are selected for evaluation based upon several factors including the following:

**•** potential for human exposure from use
and occurrence in the environment.

**•** extent of public concern.

**•** production volume.

**•** availability of scientific evidence for reproductive and/or developmental tox

icity.

The CERHR convenes a scientific expert panel that meets in a public forum to review, discuss,
and evaluate the scientific literature on the se

lected chemical. Public comment is invited pri

or to and during the meeting. The expert panel produces a report on the chemical’s reproduc

tive and developmental toxicities and provides its opinion of the degree to which exposure
to the chemical is hazardous to humans. The panel also identifies areas of uncertainty and
where additional data are needed. The CERHR expert panels use explicit guidelines to evalu

ate the scientific literature and prepare the expert panel reports. Expert panel reports are made public and comments are solicited.

Next, the CERHR prepares the NTP-CERHR monograph. The NTP-CERHR monograph in

cludes the NTP brief on the chemical evaluated, the expert panel report, and all public com

ments. The goal of the NTP brief is to provide the public, as well as government health, regu

latory, and research agencies, with the NTP’s interpretation of the potential for the chemical to adversely affect human reproductive health
or children’s health. The NTP-CERHR mono

graph is made publicly available electronically on the CERHR web site and in hard copy or
CD-ROM from the CERHR. 

### Preface

1 Information about the CERHR is available on the web at http://cerhr.niehs.nih.gov or by contacting

the director: P.O. Box 12233, MD EC-32, NIEHS, Research Triangle Park, NC 27709 919-541-3455 [phone] 919-316-4511 [fax] shelby@niehs.nih.gov [email]

Information about the NTP is available on the web

at < http://ntp-server.niehs.nih.gov > or by contact

ing the NTP Office of Liaison and Scientific Re

view at the NIEHS: liaison@starbase.niehs.nih.gov [email] 919-541-0530 [phone]i i iii In 1999, the CERHR Core Committee, an advi

sory committee composed of representatives from NTP member agencies, recommended seven phthalates for expert panel review.

These chemicals were selected because: (a) there is the potential for human exposure from their widespread use and occur

rence within the environment, (b) they have a high production volume, (c) there is substantial scientific literature addressing the reproductive and/or developmental toxicities of these chemi

cals, and (d) they are of concern to the public.

These seven phthalates are as follows:

• di(2-ethylhexyl)phthalate (DEHP)

• di-isononyl phthalate (DINP)

• di-isodecyl phthalate (DIDP)

• di-n-butyl phthalate (DBP)

• butyl benzyl phthalate (BBP)

• di-n-octyl phthalate (DnOP)

• di-n-hexyl phthalate (DnHP)

Phthalates are a group of similar chemicals widely used to soften and increase the flex

ibility of plastic consumer products such as shower curtains, medical devices, upholstery, raincoats, and soft squeeze toys. They are not bound to the plastics and can leach into the sur

rounding environment. The scientific literature on the reproductive and developmental toxici

ties of several phthalates is extensive. In addi

tion, there is widespread public concern about the safety of phthalates.

As part of the evaluation of phthalates, the CERHR convened a panel of scientific experts (Appendix I) to review, discuss, and evaluate the scientific evidence on the potential repro

ductive and developmental toxicities of each phthalate. There were three public meetings of this panel (August 17-19 and December 15- 17, 1999 and July 12-13, 2000). The CERHR received numerous public comments on the phthalates throughout the evaluation process.

The NTP has prepared an NTP-CERHR mono

graph for each phthalate. This monograph includes the NTP brief on DINP, a list of the expert panel members (Appendix I), the expert panel’s report on DINP (Appendix II), and all public comments received on the expert panel’sreports on phthalates (Appendix III). The NTPCERHR monograph is intended to serve as a single, collective source of information on the potential for DINP to adversely affect human reproduction or development. Those interested in reading this report may include individuals, members of public interest groups, and staff of health and regulatory agencies.

The NTP brief included within this report presents the NTP’s interpretation of the poten

tial for exposure to DINP to cause adverse reproductive or developmental effects in peo

ple. It is based upon information about DINP provided in the expert panel report, the public comments, and additional scientific informa

tion available since the expert panel meetings. The NTP brief is intended to provide clear, balanced, scientifically sound information on the potential for DINP exposures to result in adverse health effects on development and reproduction. 

### Introductioni i iii While there are biological and practical rea

sons for considering developmental toxicity and reproductive toxicity as 2 separate is

sues, it is important to keep in mind that life in mammals, including humans, is a cycle. In brief, the cycle includes the production of sperm and eggs, fertilization, prenatal de

velopment of the offspring, birth, post-natal development, sexual maturity, and, again, production of sperm and eggs.

In the past, toxic effects were often stud

ied in a “life stage specific” manner. Thus, concerns for developmental toxicity were addressed by exposing pregnant mothers and looking for adverse effects in fetuses. Developmental toxicity was detected as death, structural malformations, or reduced weights of the fetuses just prior to birth. Re

productive toxicity was studied by exposing sexually mature adults to the chemical of in

terest and effects were detected as impaired capacity to reproduce. Over the years, toxi

cologists realized that exposure during one part of the life cycle could lead to adverse effects that might only be apparent at a dif

ferent part of the life cycle. For example, ex

posure of a sexually mature individual to an agent capable of inducing genetic damage in eggs or sperm might have no apparent effect on the exposed individual. However, if a genetically damaged egg or sperm from that individual is involved in fertilization, the induced genetic damage might lead to death or a genetic disorder in the offspring. In this example, chemical-induced damage is detected in the next generation. In con

trast, the reproductive system begins devel

oping well before birth and continues until sexual maturity is attained. Thus, exposure of sexually immature animals, either before or following birth, to agents or conditions that adversely affect development of the reproductive system can result in structural or functional reproductive disorders. These effects may only become apparent after the exposed individual reaches the age of pu

berty or sexual maturity.

Thus, in the case of genetic damage induced in eggs or sperm, what might be considered reproductive toxicity gives rise to develop

mental disorders. Conversely, in the case of adverse effects on development of the reproductive tract, developmental toxicity results in reproductive disorders. In both these examples it is difficult to make a clear distinction between developmental and re

productive toxicity. This issue is important in considering the phthalate evaluations because evidence of developmental toxic

ity affecting reproductive capacity in later stages of the life cycle is reported for at least 3 of the phthalates - BBP, DBP, and DEHP. 

### Developmental Toxicity versus Reproductive Toxicity1NTP Brief

What is DINP? DINP is an oily, viscous liquid with the chemi

cal formula C 26 H 42 O 4 . It is a complex substance
that contains a mixture of DINP isomers such as the one shown in Fig. 1. It is one of a group of industrially important chemicals known as phthalates. Phthalates are primarily used as plasticizers to add flexibility to plastics. DINP is used to manufacture a broad range of con

sumer products such as garden hoses, pool lin

ers, flooring tiles, tarps, and toys. It is not used in medical devices and finds only limited use in food packaging.

Recent information indicates that approximate

ly 178 million kilograms (392 million pounds)
of DINP were used in the United States in
1998.

Are People Exposed to DINP?* Yes. There are several ways that people may be exposed to DINP at home or at work. Hu

man exposure to DINP can occur during the manufacture of DINP, during the manufacture of DINP-containing products, during the use of such products, or through the presence of DINP in the environment. Food does not appear to be a significant source of exposure. While inhalation, ingestion, or skin contact may ex

pose people to DINP, consumer exposure is thought to occur primarily by ingestion and
skin contact. Public concern has been raised for exposure of infants and children through the mouthing of soft toys made of DINP-contain

ing plastics. The U.S. Consumer Product Safety Commission (CPSC) convened a Chronic Haz

ard Advisory Panel on Diisononyl Phthalate (CHAP-DINP) to determine if DINP in con

sumer products poses chronic health hazards. This panel considered hazards to children from mouthing DINP-containing toys. Their report was issued in June 2001, subsequent to release of the Phthalates Expert Panel Reports (CPSC, 2001).

Because of inadequate information on human exposure to DINP, the expert panel took the conservative position of assuming that gen

eral population exposures in the United States would be less than 3-30 μg/kg bw/day (mi

crograms per kilogram body weight per day). This is the range of exposures estimated for the more widely used phthalate, DEHP. This as

sumption was supported by exposure calcula

tions (Kohn et al., 2000; David, 2000) using the urine data from Blount et al., 2000. Calculated daily exposure estimates indicate that 95% of the study population exposed to DINP was ex

posed to less than 1.7 μg/kg bw/day, and that the maximum exposure was 22 μg/kg bw/day. The CHAP-DINP (CPSC, 2001) estimated that, as a result of mouthing DINP-contain

ing toys, children 0-18 months old could be exposed to up to 280 μg/kg bw/day. Children 19-36 months old could be exposed to up to 70 μg/kg bw/day. A 15 pound child exposed to 280 μg/kg bw/day would be exposed to ap

proximately 2000 μg DINP/day. A 30 pound 

### NTP Brief on Di-isononyl Phthalate (DINP)

O O

O O **Figure 1.** Chemical structure of the DINP isomer, Di (7-methyloctyl) phthalate

* Answers to this and subsequent questions may

be: Yes, Probably, Possibly, Probably Not, No or Unknown2NTP Brief

3NTP Brief

Clear evidence of adverse effects

Some evidence of adverse effects

Limited evidence of adverse effects

Insufficient evidence for a conclusion

Limited evidence of no adverse effects

Some evidence of no adverse effects

Clear evidence of no adverse effects Developmental Toxicity child exposed to 70 μg/kg bw/day would be exposed to approximately 1000 μg DINP/day. By comparison, a small drop of water weighs approximately 30,000 μg and a grain of table salt weighs approximately 60 μg.

Can DINP Affect Human Development or Reproduction? Possibly. Although there is no direct evidence that exposure of people to DINP adversely af

fects reproduction or development, studies in laboratory animals have shown that exposure to DINP can adversely affect development, but not reproduction, in rodents (Fig. 2).

Scientific decisions concerning health risks are generally based on what is known as the “weight-of-the-evidence.” In this case, recog

nizing the lack of human data, some evidence of developmental effects, and limited evidence of no reproductive effects in animals, the NTP judges the scientific evidence sufficient to conclude that DINP might adversely affect development of the human fetus if the levels of exposure are sufficiently high (Fig. 3).

It is important to note that the levels of DINP exposure that lead to adverse effects on devel

opment in rodents are generally far higher than those experienced by people. Summary of Supporting Evidence As detailed in the expert panel report, studies of reproductive and developmental toxicity in rats have shown that exposure of the pregnant female to relatively high doses of DINP can affect development of the kidneys and skel

etal system of the fetus and result in reduced birth weights. The reproductive toxicity stud

ies reviewed by the expert panel reported no evidence of adverse effects on the reproductive system of rats.

Subsequent to release of the expert panel re

port, a rodent study was conducted to deter

mine if DINP produced antiandrogenic-like effects in male rats following exposure to 750 mg/kg bw/day from gestation day 14 through postnatal day 3 (Gray et al., 2000). Treatment resulted in female-like areolas/nipples in some male pups, and limited evidence of effects on the structure of the male reproductive tract. No effects were observed for reproductive tract development endpoints that included testis weight, anogenital distance, age of preputial separation, hypospadias or undescended testes. This study provides some evidence that DINP, like other phthalates such as DEHP and DBP, adversely affects development of the male rat reproductive system. However, the use of a sin

gle, high dose level of DINP limits the utility of

**Figure 2.** The weight of evidence that DINP causes adverse developmental or reproductive effects in laboratory animals

Reproductive Toxicity Clear evidence of adverse effects

Some evidence of adverse effects

Limited evidence of adverse effects

Insufficient evidence for a conclusion

Limited evidence of no adverse effects

Some evidence of no adverse effects

Clear evidence of no adverse effects Developmental Toxicity

Developmental Toxicity2NTP Brief

3NTP Brief

this study in evaluating human health risks.

Are Current Exposures to D INP High Enough to Cause Concern? Probably not. More data are needed to bet

ter understand the levels to which people are exposed to DINP and how these exposures vary across the population. Although the gen

eral U.S. population presently appears to be exposed to DINP at levels that are not of imme

diate concern for causing adverse reproductive or developmental effects, data are not available to permit conclusions regarding the possibility of effects in various age groups, occupations, or socioeconomic strata. Thus, the NTP offers the following conclusions.

**The NTP concurs with the conclusions of the CERHR Phthalates Expert Panel and has min** 

**** **imal concern for DINP causing adverse effects to human reproduction or fetal development.**  This is based on recent estimated DINP ex

posures in the U.S. general population. The CHAP-DINP (CPSC, 2001) convened by the U.S. Consumer Product Safety Commission concluded, “...the risk to reproductive and de

velopmental processes in humans due to DINP exposure is extremely low or nonexistent.”

**The NTP has minimal concern for develop**

  ** mental effects in children.**

This is lower than the “low concern” expressedby the expert panel and is based on the CHAPDINP estimates of potential DINP exposures in children from mouthing DINP-containing toys. The exposure levels at which developmental effects were reported in rats (143-285 mg/kg bw/day) is about 1000 times higher than the upper end of the range estimated for children’s exposure (70-280 μg/kg bw/day). 

### Figure 3. NTP conclusions regarding the possibilities that human development or reproduction might be adversely affected by exposure to DINP

Serious concern for adverse effects

Concern for adverse effects

Some concern for adverse effects

Minimal concern for adverse effects

Negligible concern for adverse effects

Insufficient hazard and/or exposure data Developmental or reproductive effects

These conclusions are based on the information available at the time this brief was prepared. As new information on toxicity and expo

sure accumulate, it may form the basis for either lowering or raising the levels of concern expressed in the conclusions.4NTP Brief 

### References:

Blount BC, Silva MJ, Caudill SP, Needham LL, Pirkle JL, Sampson EJ, Lucier GW, Jackson RJ, Brock JW. Levels of seven urinary phthalate metabolites in a human reference population. Environmental Health Perspectives 108 : 979- 982 (2000).

CPSC. Report to the U.S. Consumer Product Safety Commission by the Chronic Hazard Advisory Panel on Diisononyl Phthalate (DINP). June, 2001. (cited June 2002). < http: //www.cpsc.gov/LIBRARY/FOIA/Foia01/os/ dinp.pdf >

David RM. Exposure to Phthalate Esters. En

vironmental Health Perspectives 108 : A440 (2000).

Gray LE, Ostby J, Furr J, Price M, Rao Veera

machaneni DN, Parks L. Perinatal exposure to the phthalates DEHP, BBP, and DINP, but not DEP, DMP, or DOTP, alters sexual differentia

tion of the male rat. Toxicological Sciences 58 : 350-365 (2000).

Kohn MC, Parham F, Masten SA, Portier CJ, Shelby MD, Brock JW, Needham LL. Human exposure estimates for phthalates. Environ

mental Health Perspectives 108 : A440-A442 (2000).I-1Appendix I 

### Appendix I. NTP-CERHR Phthalates Expert Panel Report on DINP

A 16-member panel of scientists covering dis

- ciplines such as toxicology, epidemiology, and medicine was recommended by the Core Com

- mittee and approved by the Associate Director of the National Toxicology Program. Over the course of a 16-month period, the panel criti

- cally reviewed more than 500 documents on 7 phthalates and identified key studies and issues for plenary discussions. At three public meet

- ings 1 , the expert panel discussed these studies, the adequacy of available data, and identified data needed to improve future assessments. At the final meeting, the expert panel reached con

- clusions on whether estimated exposures may result in adverse effects on human reproduction or development. Panel assessments were based
on the scientific evidence available at the time of the final meeting. The expert panel reports were made available for public comment on October 10, 2000, and the deadline for public comments was December 11, 2000 ( Federal Register 65:196 [10 Oct. 2000] p60206). The Phthalates Expert Panel Report on DINP is provided in Appendix II and the public com

- ments received on that report are in Appendix III. Input from the public and interested groups throughout the panel’s deliberations was in

- valuable in helping to assure completeness and accuracy of the reports. The Phthalates Expert Panel Reports are also available on the CERHR
website < http://cerhr.niehs.nih.gov >.

1 Phthalate Expert Panel meeting dates were: August

17-19, 1999, in Alexandria, VA; December 15-17, 1999, in Research Triangle Park, NC; and July 12- 13, 2000, in Arlington, VA.I-2Appendix I Robert Kavlock, Ph.D. (chair)
EPA/ORD Research Triangle Park, NC

Kim Boekelheide, M.D., Ph.D. Brown University Providence, RI

Robert Chapin, Ph.D.
NIEHS Research Triangle Park, NC

Michael Cunningham, Ph.D.
NIEHS Research Triangle Park, NC

Elaine Faustman, Ph.D. University of Washington Seattle, WA

Paul Foster, Ph.D. Chemical Industry Institute of Toxicology Research Triangle Park, NC

Mari Golub, Ph.D.
Cal/EPA Davis, CA

Rogene Henderson, Ph.D. Inhalation Toxicology Research Institute Albuquerque, NM Irwin Hinberg, Ph.D.
Health Canada Ottawa, Ontario, Canada

Ruth Little, Sc.D.
NIEHS Research Triangle Park, NC

Jennifer Seed, Ph.D.
EPA/OPPT Washington, DC

Katherine Shea, M.D. North Carolina State University Raleigh, NC

Sonia Tabacova, M.D., Ph.D.

FDA Rockville, MD

Shelley Tyl, Ph.D. Research Triangle Institute Research Triangle Park, NC

Paige Williams, Ph.D. Harvard University Cambridge, MA

Tim Zacharewski, Ph.D. Michigan State University, East Lansing, MI 

### Appendix I. NTP-CERHR Phthalates Expert Panel **(Name and Affiliation)**

# ����������������������������������� ��������������������� ��������������������������� ��������������������������������������������Appendix II


# NTP-CERHR EXPERT PANEL REPORT

on Di-isononyl Phthalate

October 2000 NTP-CERHR-DINP-00

### TABLE OF CONTENTS

1.0 CHEMISTRY, USAGE, AND EXPOSURE

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

1.1 Chemistry

............................................................... ............................................................... ...... 1
1.2 Exposure and Usage

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

2.0 GENERAL TOXICOLOGICAL AND BIOLOGICAL PARAMETERS

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

2.1 General Toxicity

............................................................... ........................................................... 6
2.1.1 Human Data

............................................................... ....................................................... 6
2.1.2 Experimental Animal Data

............................................................... ................................. 6
2.2 Toxicokinetics

............................................................... .............................................................. 8
2.3 Genetic Toxicity

............................................................... ......................................................... 11

3.0 DEVELOPMENTAL TOXICITY DATA

............................................................... ..................................... 12

3.1 Human Data

............................................................... ............................................................... . 12
3.2 Experimental Animal Toxicity

............................................................... .................................... 12
3.2.1 Prenatal Development

............................................................... ...................................... 12
3.2.2 Postnatal Development

............................................................... ..................................... 14

4.0 REPRODUCTIVE TOXICITY

............................................................... .................................................... 20

4.1 Human Data

............................................................... ............................................................... . 20
4.2 Experimental Animal Toxicity

............................................................... .................................... 20

5.0 DATA SUMMARY & INTEGRATION

............................................................... ....................................... 25

5.1 Summary

............................................................... .............................................................. 25
5.1.1 Human Exposure

............................................................... .............................................. 25
5.1.1.1 Utility of Data to the CERHR Evaluation

........................................................... 25
5.1.2 General Biological and Toxicological Data

............................................................... ..... 26
5.1.2.1 Utility of Data to the CERHR Evaluation

........................................................... 27
5.1.3 Developmental Toxicity

............................................................... ................................... 27
5.1.3.1 Utility of Data to the CERHR Evaluation

........................................................... 31
5.1.4 Reproductive Toxicity

............................................................... ...................................... 31
5.1.4.1 Utility of Data to the CERHR Evaluation

........................................................... 36
5.2 Integrated Evaluation

............................................................... ................................................. 36
5.3 Expert Panel Conclusions

............................................................... .......................................... 37
5.4 Critical Data Needs

............................................................... .................................................... 38

6.0 REFERENCES

............................................................... ............................................................... .............. 39

7.0 TABLES

............................................................... ............................................................... ........................ 44Appendix IIAppendix II

II-i 

## PREFACE

The National Toxicology Program (NTP) and the National Institute of Environmental Health Sciences established the NTP Center for the Evaluation of Risks to Human Reproduction (CERHR) in June, 1998. The purpose of the Center is to provide timely, unbiased, scientifically sound evaluations of human and experimental evidence for adverse effects on reproduction, including development, caused by agents to which humans may be exposed.

The following seven phthalate esters were selected for the initial evaluation by the Center: butyl benzyl phthalate, di(2-ethylhexyl) phthalate, di-isodecyl phthalate, di-isononyl phthalate, di-n-butyl phthalate, di-n-hexyl phthalate, and di-n-octyl phthalate. Phthalate esters are used as plasticizers in a wide range of polyvinyl chloride-based consumer products. These chemicals were selected for the initial evaluation by the CERHR based on their high production volume, extent of human exposures, use in children’s products, published evidence of reproductive or developmental toxicity, and public

concern.

This evaluation is the result of three public Expert Panel meetings and 15 months of deliberations by a 16-member panel of experts made up of government and non-government scientists. This report has been reviewed by the CERHR Core Committee made up of representatives of NTP-par

ticipating agencies, by CERHR staff scientists, and by members of the Phthalates Expert Panel. This report is a product of the Expert Panel and is intended to (1) interpret the strength of scientific evidence that a given exposure or exposure circumstance may pose a hazard to reproduction and the health and welfare of children; (2) provide objective and scientifically thorough assessments of the scientific evidence that adverse reproductive/development health effects are associated with expo

sure to specifi c chemicals or classes of chemicals, including descriptions of an y uncertainties that would diminish confidence in assessment of risks; and (3) identify knowledge gaps to help establish research and testing priorities.

The Expert Panel Reports on phthalates will be a central part of the subsequent NTP report that will also include public comments on the Panel Reports and any relevant information that has become available since completion of the Expert Panel Reports. The NTP report will be transmitted to the appropriate Federal and State Agencies, the public, and the scientific community.

The NTP-CERHR is headquartered at NIEHS, Research Triangle Park, NC and is staffed and administered by scientists and support personnel at NIEHS and at Sciences International, Inc., Alexandria, Virginia.

Reports can be obtained from the website < http://cerhr.niehs.nih.gov / > or from:
CERHR Sciences International, Inc. 1800 Diagonal Road, Suite 500 Alexandria, VA 22314-2808 Telephone: 703-838-9440II-iiAppendix II 

### A Report of the CERHR Phthalates Expert Panel :

Name Affiliation

Robert Kavlock, PhD (Chair) National Health and Environmental Effects Research Laboratory/ USEPA, Research Triangle Park, NC Kim Boekelheide, MD, PhD Brown University, Providence, RI Robert Chapin, PhD NIEHS, Research Triangle Park, NC Michael Cunningham, PhD NIEHS, Research Triangle Park, NC Elaine Faustman, PhD University of Washington, Seattle, WA Paul Foster, PhD Chemical Industry Institute of Toxicology, RTP, NC Mari Golub, PhD California Environmental Protection Agency, Sacramento, CA Rogene Henderson, PhD Lovelace Respiratory Research Institute, Albuquerque, NM Irwin Hinberg, PhD Health Canada, Ottawa, Ontario, Canada Ruth Little, ScD NIEHS, Research Triangle Park, NC Jennifer Seed, PhD Office of Toxic Substances/USEPA, Washington, DC Katherine Shea, MD, MPH Duke University, Durham, NC Sonia Tabacova, MD, PhD Food and Drug Administration, Rockville, MD Rochelle Tyl, PhD, DABT Research Triangle Institute, Research Triangle Park, NC Paige Williams, PhD Harvard University, Boston, MA Timothy Zacharewski, PhD Michigan State University, East Lansing, MI

With the Support of CERHR Staff:

**NTP/NIEHS**

Michael Shelby, PhD Director, CERHR Christopher Portier, PhD Acting Associate Director, NTP Gloria Jahnke, DVM Technical Consultant Lynn Goldman, MD Technical Consultant

**Sciences International, Inc**

**.**

John Moore, DVM, DABT Principal Scientist Annette Iannucci, MS Toxicologist Ann Walker, MS, ELS Information Specialist and Technical EditorAppendix II

II-1 

### 1.0 CHEMISTRY, USAGE, AND EXPOSURE

**1.1 Chemistry** 

**Figure 1: Chemical Structure of a Di-isononyl Phthalate Isomer Di (7-methyloctyl) phthalate** 

O O

O O

DINP is a complex substance assigned two different CAS Registry Numbers (RN). CAS RN 68515-48-0 (designated DINP-1 in this document) is manufactured from octene that is converted to alcohol moieties consisting mainly of 3,4-, 4,6-, 3,6-, 3,5, 4,5-, and 5,6-dimethyl-heptanol-1. CAS RN 28553-12-0 (DINP-2) is produced from n-butene that is converted primarily to methyloctanols and dimethylheptanols. The 28553-12-0 CAS RN also represents DINP-3 which is produced from n-butene and isobutene that are converted to alcohols, with 60% consisting of methylethyl hex

anols. According to the American Chemistry Council (ACC; formerly CMA), DINP-3 is no longer produced (1). The ACC (2) has stated that although DINP is a complex substance, it is not variable due to the stability of the alcohol manufacturing process. The two types of DINP are considered commercially interchangeable.

DINP is an oily, viscous liquid at standard temperature and pressure.

**Table 1: Physicochemical Properties of DINP**

**Property Value** 

Chemical Formula C 26 H 42 O 4 Molecular Weight 419
Melting Point -48 o C
Boiling Point 370 o C
Specific Gravity 0.97
Solubility in Water Insoluble (<0.001 mg/L)
Log K

ow 9 (3)

**1.2 Exposure and Usage**  Humans may be exposed to DINP by the oral, dermal, and inhalation routes of exposure. Occupa

tional exposure occurs primarily through inhalation and dermal contact, while consumer exposure occurs primarily through oral and dermal routes. Exposure of children to DINP through children’s products is a public concern.II-2Appendix IIAppendix II

II-3 Occupational exposure DINP, like other phthalate esters, is manufactured within a closed system under negative pressure. However, some exposures may occur during the loading and unloading of railroad cars and trucks. Slightly higher exposures may occur during the production of PVC products because of elevated temperatures and more open processes. ACC (1) cites six studies that indicate exposures are below 1 mg/m 3 during production of phthalates and below 2 mg/m 3 during production of PVC. As discussed in Section 2.2, dermal exposure is not expected to result in significant absorption into the body.

Consumer exposure The range of products that contain DINP is quite broad. The use, categories, and amounts used of DINP in 1998 are given in Table 2.

**Table 2: Calculated 1998 US Consumption of DINP (thousands of metric tons)**

**End Use Subtotal**  **Total**

Film and Sheet 13
Stationary and Wood Veneer 6

Pool Liners 1

Other 6
Flooring 48

Tiles 23

Sheets 25

Artificial Leather 3

Coated Fabrics 21
Tarps 16
Conveyor Belts 1

Other 4
Dip Coating/Slush Molded 30

Gloves 15
Toys 6

Traffic Cones <1

Other ~9
Tubings and Profiles 7

Profiles 5

Garden Hoses 2

Wire and Cables 32

Shoes/Shoe Soles 9
Under-Body Coating 7 Sealants (carpet backing) 8

GRAND TOTAL 178 (1)II-2Appendix IIAppendix II

II-3 DINP is a general purpose plasticizer with a broad range of applications used in flexible PVC. It is widely used in the toy, construction, and general consumer product markets. It has limited use in food packaging and is not used in medical applications.

Because of physicochemical similarities between DINP and DEHP, general exposure to DINP is probably very similar to exposure to DEHP, but few monitoring data were located. Based on data for other phthalates, one could speculate that environmentally-contaminated food represents a pri

mary route for human exposure. However, the data are scant in support of this view.

DINP’s solubility in water is extremely low; levels are often below the analytical detection limit. Vapor pressure is also extremely low, so measured concentrations in air are not available. Modeling based on physicochemical properties of DINP can be compared to similar models for DEHP.

Food In 1996, dinonyl phthalate (isomer not specified) was identified but not quantified in 4 of 12 infant formulas from the UK (4). In a follow-up survey conducted by the Ministry of Agriculture, Fish

eries, and Food (MAFF) (5), DINP was not specifically targeted, but there was no evidence of its presence in 39 samples of infant formula from the UK. In a UK survey of fatty foods (e.g., dairy products, meats, fish, eggs, and oils), DINP was not detected at an analytical limit of 0.01 mg/kg (6).

Toys PVC plastics are often used in children’s products. Different phthalates are constituents of PVC; DINP is currently the predominant plasticizer (7). Other phthalates, including DEHP, have been or are also used (8, 9). US toy manufacturers began voluntary removal of DEHP from pacifiers and nipples in 1986 (10). Few studies pertaining to plasticizers in children’s products were found in the peer-reviewed literature. Additional information is available from industry groups and several gov

ernment agencies. The Expert Panel did not perform a comprehensive review of available data, but believes the information it reviewed reflects the general state of knowledge.

As reported by the Consumer Product Safety Commission (CPSC) (7), Chen measured DINP in 31 of 35 toys and found a concentration range of 15.1–54.4 % dry weight. Health Canada (11) ana

lyzed 41 children’s products made in the US, China, and Thailand for the presence of DINP and DEHP. DINP was detectable in 27 of the 41 products in concentrations that ranged from 3.9 to 44% dry weight. Criteria for the selection of products were not discussed in any of these surveys. No information on market share, length of availability on the market, or estimates of the numbers of products in circulation was noted in any study. Only Health Canada listed product number, country of origin, manufacturer/distributor, and brand. All studies listed a product description. Marin (9) analyzed 15 samples of materials used in toys in Spain. The authors noted that the PVC contained a mixture of plasticizers including DINP, DEHP, and DIDP, but reported only the DEHP content.

The estimation of actual exposure of children to phthalates contained in children’s products has
been studied. In vitro studies using various agitation and impaction approaches yield a wide range of extraction of DINP from toys. CPSC used stainless steel pistons, 11 cm 2 of each product, and simulant saliva to obtain extraction rates for their 31 DINP-positive children’s products. MigrationII-4Appendix IIAppendix II

II-5 was log normally distributed with a mean rate of 8.2 – 9.83 μg/11 cm 2 /hour and a range of 1– 48 μg/11 cm 2 /hour. Both CPSC (7) and Health Canada (11) failed to find any correlation between release rate of DINP under experimental conditions and total DINP content.

The Dutch Consensus Group reported a small study by Meuling and Rijk (12) using 20 adult vol

unteers. A control specimen without DINP and three specimens with DINP were used; specimen1 contained 38% DINP. Specimens 2 and 3 came from different parts of the same commerciallyavailable teething ring, representing different shapes for mouthing, and contained 43% DINP each.
All three were 10 cm 2 total area. All 20 volunteers were instructed to suck and bite on the control specimen for 10–15 minutes, all saliva was collected, volunteers rested 5 minutes and then they performed 4 separate sessions on the same test piece of specimen 1, resting 5 minutes between each session. This procedure was repeated with half (n=10) of the volunteers on specimen 2 and the other half (n=10) on specimen 3. DINP extraction from specimen 1 was 1.38 (0.3–8.3) μg/min, from specimen two 2.44 (0.9–8.9) μg/min, and from specimen three 1.63 (0.9–5.7) μg/min. The mean across all groups was 1.8 μg/10cm2/min (or 120 μg/11cm 2 /hour). There was no correlation between extraction and pH or protein content of the saliva. Release rates over the various 15-minute intervals seemed consistent. The increase in extraction of Specimen 2 was thought to be due to the finger-like shape resulting in different mouthing behaviors from those employed on the disk-like shape of Specimens 1 and 3.

CPSC (7) reported a similar protocol using 10 adult volunteers and 5 toys, and found a mean migration rate of 241.3 μg/11cm 2 /hour. This rate was 39.5 times higher than the average rate obtained by impaction with disks cut from the same 5 toys, but was similar to the ranges in the Dutch simulation study.

Exposure of children to DINP from PVC toys was estimated by Fiala et al. (13) in Austria. DINP
levels were measured in the saliva of 10 adult volunteers who first sucked on and then sucked and
chewed on 10–15 cm 2 pieces of teether (containing about 36% DINP) for 1 hour. In the experi

ment where the volunteers only sucked on the sample, the migration rates of DINP ranged from 297–1,452 μg/dm 2 /hour with a mean migration rate of 832±397 μg/dm 2 /hour. Using assumptions of an 8 kg body weight, 3-hour exposure time (12), and 10 cm2 mouthing area, mean and maximum exposure levels of 31.25 μg/kg bw/day and 54.4 μg/kg bw/day, respectively, were estimated. For the experiment where the adults chewed on the sample, migration rates of DINP in 9 adults ranged from 768−2152 μg/dm 2 /hour. Using the same assumptions from the first experiment, a maximum exposure level of 84.5 μg/kg bw/day was estimated.

CPSC (7), the Dutch Consensus Group (12), and Health Canada (11) ha ve attempted to calculate daily intake based upon the leaching rates described above. The Dutch Group used Monte Carlo simulation and estimates of mouthing time and the leaching rates from the in vivo study of 20 adults. Mouthing time was derived from parent observations and logging of mouthing time of 42 children aged 3−35 months (Table 3). Mouthing time was calculated for the time children were awake, but not eating, during ten 15 minute observation periods over 2 days. Logs were kept of objects mouthed; the objects were divided into those intended for mouthing and those not intended for mouthing. The Dutch calculations used total mouthing time excluding time spent mouthing pac

-II-4Appendix IIAppendix II

II-5 ifiers. Because the greatest exposure levels were determined for children within the ages of 3−12 months, the results for that age group are summarized in Table 4.

**Table 3: Total Mouthing Time**

Age (months) Sample Size Mean in Minutes (SD) Min (Minutes) Max (Minutes)

3–6 5 36.9 ±19.1 14.5 67.0

6–12 14 44.0 ± 44.7 2.4 171.5

12–18 12 16.4 ± 18.2 0 53.2

18–36 11 9.3 ± 9.8 0 30.9

**Table 4: Toy Exposure Estimates for Children Aged 3–12 Months.**

Estimated Intake Level (μg/kg bw/day)
Agency Mean 95 th Percentile 99 th Percentile Maximum

RIVM* 6.53–14.4 20.7–39.7 39.8–77.3 70.7–204

CPSC 5.7 94.3

- - Health Canada** 44 73.9*** 173.5*** 320

* Exposure range for 3−6 month-old and 6−12 month-old children;

range includes results from 3 specimens tested.

** Calculated with mouthing times for teethers and other objects intended for mouthing.

*** Results using Monte Carlo simulations in children aged 3–6 months.

The approach taken by Health Canada used published data and 10,000 Monte Carlo simulations and total mouthing time from the Dutch observation study including mouthing of pacifiers, teethers and other objects intended for mouthing. CPSC used the same mouthing-time data, b ut limited its cal

culations to the mouthing time of objects not intended for mouthing. They performed a log transfor

mation of the time because of the extreme skewness in the sample and calculated a geometric mean mouthing time of 12.03 minutes (95% CI 6.2–23.3). Exposure estimates w ere made using a log linear model, the mean leaching rate from mechanical extraction from 31 consumer products and a 39.5 factor (to adjust for differences between in vitro and in vivo extraction rates). The differences in the analyses resulted in quite different exposure estimates, which explains the different conclu

sions and recommendations of the agencies.

The differences also highlight the uncertainties inherent in these calculations. Because extraction of DINP does not correlate with DINP content, because extraction is highly variable across both laboratory procedures and human subjects, and because the number and distribution of children’s products containing DINP is unknown, the amounts of DINP presented to a child cannot be well characterized. Furthermore, the estimates of mouthing behavior in the youngest and potentially highest risk group, 3–12 months, are based upon only 19 children. No discussion of developmental age, physical condition, ethnicity, or other socio-demographic indicators is included in the small parental observation study. These numbers are preliminary estimates at best. Standardization of lab

oratory techniques with correlation with in vivo simulations, better data on product distribution and use, and independent studies of mouthing behavior in babies and young children are needed. None-II-6Appendix IIAppendix II

II-7 the-less, existing models show this is a potentially significant exposure for young children. A study using larger numbers of children has been submitted by Juberg et al. (14), but could not be cited at the time of this review. According to the ACC (2), the CPSC and EU Joint Research Laboratory are working on standardizing laboratory techniques with in vivo simulations.

Dermal exposure to DINP from toys may also occur, but has not been studied specifically in chil

dren.

Exposure Estimate Based on the physicochemical characteristics of DINP and limited monitoring data, the Expert Panel believes it reasonable to assume that exposure to DINP in the general adult population is lower than exposure to DEHP, which is estimated at 3–30 μg/kg bw/day (15). Children may incur significantly greater nondietary exposures from mouthing toys and other articles containing DINP.

**The summary for Section 1 is located in Section 5.1.1.**II-6Appendix IIAppendix II

II-7 

### 2.0 GENERAL TOXICOLOGICAL AND BIOLOGICAL PARAMETERS

**2.1 General Toxicity** 

2.1.1 Human Data

There were no human data located for Expert Panel review.

2.1.2 Experimental Animal Data
BIBRA (16) (Table 7-1) conducted a 21-day dietary study in 6-week-old F344 rats where groups of 5 males and 5 females were fed concentrations of 0, 0.6, 1.2, or 2.5% DINP (M: 639, 1,192, or 2,195 mg/kg bw/day; F: 607, 1,193, or 2,289 mg/kg bw/day). The test material most likely con

sisted of a mixture of DINP represented by CAS numbers 68515-48-0 and 28553-12-0 (DINP-1 and DINP-2). A positive control group of 5 rats per sex was exposed to 1.2% DEHP (M: 1,084 mg/kg bw/day; F: 1,063 mg/kg bw/day). Body weight and food intake were measured twice weekly. On day 21, rats were killed and necropsied. Liver, kidney, and testes were preserved in formalin and examined histologically. Peroxisomal proliferation was assessed by measuring activities of peroxi

somal proliferation enzymes and by examining liver tissue by electron microscopy.

A significant decrease in weight gain was observed in the mid- and high-dose groups. Food intake was significantly reduced in males. Organ to body weight ratios that were significantly increased in all treatment groups included liver (M: 136, 173, and 232%, F: 131, 175, and 237% of control values) and kidney (M: 115, 122, and 124%, F: 107, 108, and 114% of control values). Histo

pathological changes were not observed in kidneys; changes in liver were limited to reduced cyto

plasmic basophilia in the mid- and high-dose group and increased cytoplasmic eosinophilia in thehigh-dose group. Palmitoyl-CoA (PCoA) oxidase activity was significantly increased in the midand high-dose groups (M: 452 and 1,035%; F: 376 and 1,104% increases, respectively, compared to controls) and an increase in peroxisome numbers was observed by electron microscopy in livers from the high-dose group. The activity of 11-hydroxylase and 12-hydroxylase was significantly increased in males of all dose groups and in females of the high-dose group. Significant changes observed in all treatment groups included increased total liver proteins and reductions in serum levels of cholesterol. Serum triglyceride levels were significantly reduced in all treated males, but increased in mid- and high-dose females. The testes to body weight ratio was significantly increased in the high-dose males (135% of control value), but absolute testes weights were not significantly affected. Testicular lesions were not observed with the exception of severe unilateral atrophy in one male of the mid-dose group. Treatment with 1,063−1,084 mg DEHP/kg bw/day resulted in similar effects including decreased weight gain, increased liver and kidney to body weight ratio, increased liver enzyme activities, and reduced serum levels of cholesterol and triglycerides. Moderate testicu

lar atrophy was noted in one male. Peroxisomal proliferation is of particular interest and an increase in peroxisome numbers was observed after treatment with DEHP. PCoA activity was significantly increased to 683 and 540% of control values for males and females, respectively. The increase in peroxisomal enzyme activity in rats treated with 1,063−1,084 mg/kg bw/day DEHP was greater than that obtained by treatment with DINP at 1,192−1193 mg/kg bw/day (452 and 376% of control values in males and females, respectively).II-8Appendix IIAppendix II

II-9 This study provides evidence that the liver is a target organ of DINP. A pattern similar to effects noted with DEHP is seen: increased liver weight and induction of hepatic peroxisome proliferation. The testes do not appear to be a target organ at these dose levels. The study provided a LOAEL of 0.6% (607[F] and 639[M] mg/kg bw/day) and no NOAEL was identified.

In a 2-year dietary study, (17) (Table 7-2) systemic effects resulting from DINP-1 exposure in adult (6 week old) Fischer 344 rats were evaluated. Groups of 110 rats per sex were fed diets containing 0, 0.03, 0.3, and 0.6% DINP-1 (males: 0, 15, 152, and 307 mg/kg bw/da y; females: 0, 18, 184, and 375 mg/kg bw/day). Body weight and food intake were measured weekly. Ten rats/sex/group were killed and necropsied at 6, 12, and 18 months; the remaining rats w ere killed and necropsied at the end of the 2-year study. Evaluation of hematology, urine, and blood chemistry effects was per

formed at 6, 12, 18, and 24 months. Histopathological evaluations were conducted on the liver and the kidney from all dose groups and in the remaining organs of the control and high-dose groups. Evidence of peroxisome proliferation was determined by microscopic examination of livers of 2 rats/sex/group at 24 months.

Significant reductions in body weight gain were observed in males from 18–24 months in the 152 mg/kg bw/day group and from 12–24 months in the 307 mg/kg bw/day group. Food intake lev

els were not reported. Survival was significantly decreased in females of the 184 and 375 mg/kg bw/day groups. Liver and kidney to body weight ratios were significantly increased throughout the study in both sexes in the mid- and high-dose groups (152–375 mg/kg bw/day). Spleen to body weight ratios were significantly increased in males and females of the high-dose group (307–375 mg/kg bw/day) at 24 months. A small but significant increase in adrenal to body weight ratio was reported for females in the 375 mg/kg bw/day group at 6–12 months, and in both sexes in the high-dose group (307–375 mg/kg bw/day) at 24 months. Adrenal weights were not listed in tables. Dose-related changes in liver included hepatocyte enlargement in high-dose males and females throughout treatment. At 24 months, dose-related liver effects included regenerative nodules and focal necrosis in males and females of the two highest dose groups, and spongiosis hepatitis in males of the high-dose group. In males of the mid- and high-dose groups, consistent increases in serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) activities were observed. However, for SGOT, statistical significance was obtained only at 6 and 12 months in the mid-dose group and at 6–18 months in the high-dose group. In males, increases in SGPT activity were statistically significant at 24 months in the mid-dose group and at 6 and 18 months in the high-dose group. An increased incidence of mononuclear cell leukemia (MNCL) was observed in both sexes of the mid- and high-dose groups. Peroxisome proliferation did not occur and there was no evidence of treatment-related lesions in testes or female reproduc

tive organs. The only significant dose-related changes in hematology were a reduction in red blood cell count and hemoglobin and hematocrit values in males of the 307 mg/kg bw/day group at 24 months. Urinalysis results were not listed in tables, but authors reported increased urine volumes in high-dose males at all time points and transient increases in potassium and glucose. A NOAEL of 17 mg/kg bw/day was selected by the authors.

A second 2-year dietary feeding study in F344 rats was reported by Moore et al. (18) (Table 7- 3). Groups of 70–85 F344 rats/sex/group (6 weeks old) were fed concentrations of 0, 500, 1,500,II-8Appendix IIAppendix II

II-9 6,000, and 12,000 ppm DINP-1 (males: 0, 29.2, 88.3, 359, or 733 mg/kg bw/da y; females: 0, 36.4, 109, 442, or 885 mg/kg bw/day). Body weight and food intake were measured weekly through weeks 16–17 and monthly thereafter. Standard hematological, clinical chemistry, and urinalysis parameters were measured every 26 weeks. Peroxisome proliferation was measured in 5 rats/sex in control and high-dose groups at weeks 1, 2, and 13, and in 3–5 rats/sex in the control and 2 highest dose groups at week 104. Five rats/sex/group were sacrificed and necropsied at weeks 1, 2, and 13. Fifteen rats/sex/group were killed and necropsied at week 79. The remaining rats were sacrificed and necropsied at week 104. Another group of 55 rats/sex was exposed through diet to 12,000 ppm (males 637 mg/kg bw/day; females 774 mg/kg bw/day) DINP for 78 weeks and sacrificed at week 104 in order to determine if recovery occurs after exposure to DINP has ended. Histopathological evaluations were conducted on major organs from rats in all dose groups.

Clinical signs of toxicity were observed in rats exposed to 359 mg/kg bw/day and higher, and included hunched posture, decreased activity, bodies that appeared pale and thin, and fewer feces. Rats exposed to 733–885 mg/kg bw/day experienced a statistically significant reduction in weight gain accompanied by a decrease in food intake. Survival was significantly reduced in the high-dose males with only 54% surviving to the end of the study. The body weight effect was shown to be partially reversible because male weight gain in the recovery group was not reduced at week 104; reduced weight gain in females was less pronounced. Survival was not significantly affected in the recovery group. The authors reported that the dose-related depression of body weight gain in the two highest doses was associated with clinical chemistry findings or histomorphologic effects in liver and kidney. A significant increase in the incidence of anemia, as observed by decreases in erythrocyte, hemoglobin, and hematocrit levels, was observed throughout the study in rats exposed to 359 mg/kg bw/day and higher, but was not observed in the recovery group. A significant increase in kidney to body weight ratio was observed in rats exposed to 359 mg/kg bw/day and higher from week 79 to 104 (M: 8.1 and 25% and F: 14.4 and 22% increases in 2 highest dose groups, respec

tively, at week 104). Liver to body weight ratios were significantly increased in both sexes exposed to 359 mg/kg bw/day and higher throughout the study (M: 35 and 61% and F: 26 and 71% increases at 2 highest doses, respectively, at week 104). Histological effects observed in kidneys of rats exposed to 359 mg/kg bw/day and higher at weeks 79 and 104 included an increased inci

dence and severity of renal papilla mineralization in males (59/85 and 57/85 at 2 highest doses). An increase in tubule cell pigmentation was also reported by the authors, but the incidence of the lesion appeared equal among control and dose groups (55−59 sex/dose). Urinalysis findings at week 104, which included significant increases in urine output and corresponding decreases in potassium, calcium, creatinine, and chloride levels in high-dose males, suggested compromised ability to con

centrate in the renal tubule epithelium. Serum urea levels were significantly increased during the second half of the study in rats exposed to 359 mg/kg bw/day and higher. Increases in urine volume and kidney lesions were observed in the recovery group exposed to 733 mg/kg bw/day and greater with severity approximately equal to that of the 359−442 mg/kg bw/day treatment group. Livers of rats exposed to 359 mg/kg bw/day and higher appeared enlarged and granular at weeks 79 and 104. Histopathological effects in the livers of the high-dose group included diffuse hepatocyte enlarge

ment (37/85 males and 52/85 females), cytoplasmic eosinophilia (43/85 males and 45/85 females), and Kupffer cell/bile canaliculi pigmentation (12/85 males and 17/85 females). These effects were first detected at weeks 2, 13, and 79, respectively. The authors also reported alterations in serum alanine aminotransferase and aspartate aminotransferase activity, but the changes did not appear toII-10Appendix IIAppendix II

II-11 be consistent or dose related. Non-neoplastic liver changes were found to be reversible in the recov

ery group. Peroxisomal enzyme activity was significantly increased at week 104 in females exposed to 442 mg/kg bw/day and in both sexes of the high-dose group throughout the study. The recovery group was not tested for peroxisomal enzyme activity. Histopathological changes in testes or female reproductive organs were not observed.

Neoplastic effects included a significant increase in liver adenomas (10/80 vs 4/80) and carcinomas (11/80 vs 1/80) in male rats of the high-dose group at week 104. At week 104, renal tubule cell car

cinoma was observed in 2 males of the high-dose group and 4 males of the recovery group. Mono

nuclear cell leukemia was found in 45–49% of rats in the 2 highest dose groups. Liver neoplasms were not observed in the recovery group, but the incidence of renal tubule cell carcinoma in males and mononuclear cell leukemia remained elevated compared to controls. The authors selected a NOAEL of 1,500 ppm (88.3−109 mg/kg bw/day) for this study.

In a 2-year dietary study in 6-week-old B6C3F1/CrlBR mice (19) (Table 7-4), groups of 70 mice/ sex/group ate diets that contained 0, 500, 1,500, 4,000, and 8,000 ppm DINP-1 (males: 0, 90.3, 276, 742, 1,560 mg/kg bw/day; females: 0, 112, 336, 910, 1,888 mg/kg bw/day). Body weights and food intake were measured weekly through week 16–17 and monthly thereafter. Standard, hemato

logical, clinical chemistry, and urinalysis parameters were measured every 26 weeks. Peroxisome proliferation was measured in five mice/sex in the control and high-dose group at the midpoint and end of the study. Fifteen mice/sex/group were sacrificed and necropsied at week 79. The remaining mice were sacrificed and necropsied at the end of the 2-year study. Histopathological evaluations were conducted on major organs from mice in all dose groups. Another group of mice was exposed to 8,000 ppm DINP in the diet for 78 weeks and sacrificed at week 105−106 in order to determine if recovery would occur after exposure to DINP ended.

Toxicological and non-neoplastic effects were observed in mice that received the 2 highest doses, 742 mg/kg bw/day and greater. A statistically significant reduction in weight gain occurred through

out the study; this reduction was not accompanied by a decrease in food intake. The effect was shown to be partially reversible because female weight gain in the recovery group was not reduced at week 104; reduced weight gain in males was less pronounced. Clinical signs of toxicity were observed and included abdominal swelling in males exposed to 742 mg/kg bw/day and greater, and hunched posture, decreased activity, and fewer feces in the high-dose males. Survival was significantly reduced in the high-dose males (1,560 mg/kg bw/day), with only 63% of males sur

viving until the end of the study. Survival was not significantly affected in the recovery group. A significant reduction in kidney to body weight ratio was observed in males of the 2 highest dose groups (13 and 25% reduction, respectively), whereas a significant increase in liver to body weight ratio occurred (7 and 24% increase, respectively). Females exposed to the highest dose (1,888 mg/kg bw/day) had a 37% increase in liver to body weight ratio from week 79 to 104. Histological examination revealed an increased incidence and severity of renal nephropathy in female mice of the high-dose group. Urinalysis findings, which included significant increases in urine output and corresponding decreases in sodium, potassium, and chloride levels in high-dose mice from week 52–104, suggested compromised ability to concentrate in the renal tub ule epithelium. The effects on renal structure and function proved to be partially reversible as they were less pronounced in mice of the recovery group by the end of the study. Histopathological liver changes were observedII-10Appendix IIAppendix II

II-11 in mice of the highest dose group and included diffuse hepatocyte enlargement (56/70 males and 65/70 females) and cytoplasmic eosinophilia (67/70 males and 68/70 females) and pigment (64/70 males and 53/70 females). Other hepatic effects included increased serum alanine aminotransferase and aspartate aminotransferase levels in the high dose males at various time points throughout the study. Non-neoplastic liver changes were found to be reversible in the recovery group. An increase in peroxisomal enzyme activity in mice exposed to 1,560−1,888 mg/kg bw/day indicated that hepa

tocyte enlargement was due to peroxisomal proliferation. Histopathological changes in testes or female reproductive organs were not observed.

Neoplastic effects included increased incidences of hepatic adenomas and carcinomas combined in females exposed to 336 mg/kg bw/day (10/60 versus 3/70), and adenomas (15/60 and 13/60 versus 10/70) and carcinomas (17/60 and 20/60 versus 10/70) in males exposed to 742 and 1,560 mg/kg bw/day, respectively, and in females exposed to 1,888 mg/kg bw/day (18 adenomas and 18 carcinomas/70 versus 2 adenomas and 1 carinoma/70). The occurrence of hepatic neoplasms was lower in the recovery group compared to the high dose mice exposed for the duration of the study with an incidence of 37−39% versus 50−56%. Based on hepatic neoplasms, the authors selected a NOAEL of 500 ppm (112 mg/kg bw/day) for females and 1,500 ppm (276 mg/kg bw/day) for
males.

Hall et al. (20) (Table 7-5) exposed sixteen 25-month-old marmosets (2/sex/group) by gavage for 13 weeks with DINP (CAS number not provided) in 1% methylcellulose and 0.5% Tween at con

centrations of 0, 100, 500, or 2,500 mg/kg bw/day. Clofibrate was administered as a positive control at 500 mg/kg bw/day. Analysis was conducted for hematology (weeks 0, 6, and 13), blood chemis

try (weeks 0, 4, and 13), estradiol and testosterone levels (week 12), and urine composition (weeks 0, 5, and 12). The main organs (including but not limited to liver, testes, and epididymides) were weighed and examined histologically (testes and epididymides were preserved in Bouin’s). Peroxi

somal proliferation was determined by measuring cyanide-insensitive PCoA oxidase activity.

Clinical signs observed in the marmosets included ungroomed coats and localized reddening of the skin around the anus and legs which was likely caused by excretion of test substance in feces. One male exposed to 2,500 mg/kg bw/day experienced a 13% weight loss and had reduced activity and a hunched posture. Weight loss or decreased weight gain was observed in 2 males and 1 female exposed to 2,500 mg/kg bw/day. Peroxisome proliferation was not evident as indicated by a lack of dose-related increases in PCoA oxidase activity. There were no DINP-treatment related changes in estradiol or testosterone levels, hematology, blood chemistry, organ weights, urine composition, or microscopic findings. The authors identified a NOAEL of 500 mg/kg bw/day.

Administration of the positive control, clofibrate, did result in an approximate 100% increase in PCoA oxidase activity. Other effects in positive control animals included an increase in 11-hydrox

ylase activity in males, reduced weight gain, anemia, and a slight increase in relative and absolute kidney weight.

Pugh et al. (21) gavaged 2-year-old (prepubertal) cynomolgus monkeys (4/group) with 0 or 500 mg/kg bw/day DINP-1 in methylcellulose for 14 days (Web Table 9). According to Short et al. (22),II-12Appendix IIAppendix II

II-13 500 mg/kg bw/day is the maximum dose that can be absorbed by the monkeys. On day 15, the ani

mals were sacrificed and the tissues were removed, weighed, and fixed in formalin for histopatho

logical evaluation. Hematology, serum chemistry, and urine analysis were conducted. Peroxisomal proliferation was examined by measuring peroxisomal beta oxidation activity and replicative DNA synthesis. Gap junctional intercellular communication was determined in liver. There were no clini

cal signs or changes in body weight gain. A significant increase in blood neutrophil numbers and decrease in lymphocyte count were the only effects reported. There were no testicular or hepatic lesions and no effects on any of the systemic parameters examined.

Mode of Action The renal neoplasia in male rats appears to be due to alpha-2-micro globulin nephropathy which is a mechanism not considered relevant to humans (23). However, an increased rate of nephropathy wasseen in female mice exposed to 1,888 mg/kg bw/day which would not be consistent with the alpha2-microglobulin mechanism. The Moore (18) study demonstrated liver tumors in rats only in the highest-dose males. Peroxisome proliferation in rats was observed at the highest dose in males and females, and the second highest dose in females but not males. No liver tumors were observed in either sex at the second highest dose level. In addition, no liver tumors were noted in the recovery groups. These results are consistent with a peroxisome proliferation mode of action for hepatic tumor induction. Unfortunately, peroxisome proliferation was assayed in mice only at the highest dose, and liver tumors were also observed at lower doses.

**2.2 Toxicokinetics** 

**Phthalate Moiety**

Absorption

Rodents: Dermal Dermal absorption of 14 C-DINP was studied in male Fischer 344 rats (24) in both conditioned (pretreatment with non-labeled DINP) and non-conditioned skin. Following exposure, the dosed area was occluded. Under all conditions, the amount absorbed after 7 days ranged from 2 to 4% of the dose. Approximately 93−99% of the administered radioactivity was recovered at the site of appli

cation. Radioactivity in feces and gut of the exposed rats suggested some excretion via the biliary

route. In in vitro studies comparing absorption of DEHP through human and rat skin (25), absorp

tion through human skin was slower than through rat skin. Therefore, the dermal absorption rate of DINP is also expected to be slower through human versus rat skin. Studies conducted by Deisinger et al. (26) have demonstrated that dermal absorption of DEHP from a plasticized film is slower than dermal absorption of neat DEHP. It is reasonable to assume that these results apply to DINP.

Rodents: Oral Oral absorption of 14 C-DINP (dose=2,500 mg/kg) was studied (27) in conditioned (pre-treatment with non-labeled DINP) and non-conditioned male albino rats. The rats were administered 0.5 mL of radiolabeled DINP by gavage and the dose was estimated at approximately 2,500 mg/kg bw by the Expert Panel based on the density of DINP and reported rat body weights. Within 72 hours, 85% of the administered dose was excreted in the feces, most within the first 24 hours. The rest ofII-12Appendix IIAppendix II

II-13 the dose was excreted in urine (average of 12%) or remained in the tissues (trace amounts). Thus, the oral absorption was approximately 12%. In studies at Midwest Research Institute (28), male and female Fischer 344 rats were dosed orally either in a single or in 5 daily doses of 50, 150, or 500 mg/kg. At least 49% of the single low dose was absorbed. Absorption was decreased at the high single dose and at all doses following repeated exposures.

Biotransformation
Most of the 14 C collected in the urine of rats following a single oral dose of 14 C-DINP was in the form of phthalic acid or side-chain oxidation products of the monoester (MINP) (28). The relative amount of phthalic acid in the urine decreased at the high dose. The monoester itself, as well as the diester, was present in only trace amounts. In feces, 8 and 41% of the radioactivity was associ

ated with the diester following administration of a low (50 mg/kg) or a high (500 mg/kg) oral dose
of 14 C-DINP. This indicates saturation of metabolism at the high dose. The remainder of the fecal radioactivity was associated with the monoester or its side-chain oxidation products. Major metabo

lites in the liver were the monoester and its side-chain oxidation products. The same metabolites and phthalic acid were in testes. Fat contained the monoester and its oxidation products. Repeated exposures revealed similar metabolites in the tissues. In summary, in the rat, DINP was de-esterfied to the monoester, which was further metabolized by side-chain oxidation of the ester group or by hydrolysis to phthalic acid. Formation of oxidation products appeared to increase following the high dose or repeated dosing, while the hydrolysis to phthalic acid decreased (28).

Distribution In albino rats receiving 0.5 mL of 14 C-DINP (approximately 2,500 mg/kg bw as estimated by the Expert Panel) after 5 days of dosing with the same amount of unlabeled DINP (27), no tissue stud

ied had over 0.001% per gram of the administered dose after 3 days. The liver contained the most radioactivity on a total tissue basis. In male and female Fischer 344 rats receiving single or repeated
oral doses of 14 C-DINP (28), radioactivity also cleared from the tissues rapidly, but analysis of tis

sues soon (within 1 hour) after the exposure indicated that the highest levels were in liver (4.7% of administered dose), kidneys (0.31%), and blood (1.62 %). Fat and testes contained small amounts of metabolites. No bioaccumulation occurred over 72 hours postdosing.

Excretion The major routes of excretion for orally administered DINP in rats were urine and feces, with about equal amounts excreted by either route at low doses, but more excreted in feces at high doses (28). Repeated dosing caused no accumulation of DINP or its metabolites in b lood or tissue, but resulted in increased formation and elimination of the monoester side-chain oxidation products (28).

Side Chain-associated Toxicokinetics A major metabolite of DINP, the monoester, MINP, is further oxidized in the side chain.



### 2.3 **Genetic Toxicity**

DINP was tested in the Ames assay, Chinese hamster ovary (CHO) cells for chromosomal aberra

tions, the mouse lymphoma forward mutation assay (L5178Y TK -/- cell line), the primary rat hepa

-II-14Appendix IIAppendix II

II-15 tocyte unscheduled DNA synthesis assay, and in an in vitro transformation assay using clone 1–13 of Balb/c-3T3 A31 mouse cells. Where appropriate, exogenous metabolic activation systems were used. Many of the assays were conducted according to GLP standards (29). Based on the results of these studies, DINP is not considered mutagenic in bacterial mutation assa ys and mammalian gene assays and is not clastogenic in one cytogenetic assay in vitro with CHO cells and in one in vivo assay with bone marrow cells of Fischer rats. This suggests that DINP is not genotoxic in vivo or in

vitro (29)

Cell transformation studies give various results. The experimental conditions in the assays were not quite identical and the results are not inconsistent. Such positi ve results are in accord with those of well known peroxisome proliferators (29). DINP tested negative in the L5178Y mouse lymphoma mutation assay and the Balb/3T3 cell transformation assay (30). The data from the mutation and cell transformation assay were reviewed by OECD.

**The summary for Section 2, including general toxicity, toxicokinetics, and genetic toxicity is**
**located in Section 5.1.2.**II-14Appendix IIAppendix II

II-15 

### 3.0 DEVELOPMENTAL TOXICITY DATA

**3.1 Human Data** 

There were no human data located for Expert Panel review.

**3.2 Experimental Animal Data** 

Two rat studies evaluating prenatal developmental toxicity of DINP by gavage were reviewed, as were the developmental toxicity aspects of a two-generation study in rats. Prenatal developmental toxicity of isononyl alcohol, a primary metabolite, was also evaluated.

Using Sprague Dawley rats, Waterman et al. (31) (Table 7-7) evaluated DINP-1 (CAS No. 68515- 48-0) and diisodecyl phthalate (DIDP) in 24 rats/group gavage treated with 0, 100, 500, and 1,000 mg/kg bw/day on gd 6

**−** 15. For both compounds, maternal toxicity was observed at 1,000 mg/kg bw/day expressed as reduced weight gain and food consumption. Fetal results were presented in terms of affected litters and fetuses. Skeletal variations were observed at the 500 and 1,000 mg/kg bw/day treatment levels. A dose-related increase in litters with lumbar ribs (25.0, 20.2, 54.2, and 78.3%) was observed, which was statistically significant at the high dose. A dose-related increase in the percent of fetuses with rudimentary lumbar ribs was observed (3.7, 5.4, 18.6, and 34.5%) with statistical signifi cance attained in the mid- and high-dose groups. The percent of fetuses with supernumerary cervical ribs was statistically increased only in the high dose group (1.6, 1.6, 1.0, and 5.7%), but the 2.5-fold increase in litter incidence in the high-dose group was not statistically significant (12.5, 12.0, 8.3, and 30.4%). There was a dose-related increase in the percentage of litters with dilated renal pelves (0, 12.0, 16.7, and 26.1%) that attained statistical significance at the highest dose. The percentage of fetuses with dilated renal pelves was significantly increased at all treatment levels (0, 3.7, 4.0, and 4.5% at 0, 100, 500, and 1,000 mg/kg bw/day, respectively). The interpretation of results by Waterman et al. (31) included the maternal and developmental LOAEL of 1,000 mg/kg bw/day with a NOAEL of 500 mg/kg bw/day, with a conclusion that DINP “is not teratogenic or a selective developmental toxicant.” The Expert Panel agreed with the authors selection of a maternal NOAEL; however, the Panel concluded that fetal data indicated that developmental toxicity was present at 500 mg/kg bw/day. The Expert Panel communicated to the study sponsor that there were improved and more current approaches to the analysis of fetal incidence data. The sponsor reanalyzed the fetal incidence data of interest using the GEE approach (32). This is a pup-level analysis within a model that uses the generalized estimating equation approach to account for the litter effect, i.e., the correlation between outcomes measured on pups within the same litter. The dose groups were tested pairwise versus controls; this gave similar results to a trend test based on a dose-response model fi t with all dose levels up to that of interest included. These reanalysis data (33), presented below, are consistent with the Expert Panel’s interpretation of the vertebral data.II-16Appendix IIAppendix II

II-17 **Table 5: Mean Percent of Pups in Litter with Effect of Interest (significance level)** 

Dose Group (mg/kg bw/day DINP)

**0 100**  **500 1,000** 

Skeletal Variations 16.4 15.0 (0.91) 28.3* (0.05) 43.4** (0.001)

Visceral Variations 0.5 3.3 (0.08) 3.7 (0.09) 5.8* (0.04)

Rudimentary Lumbar Ribs 3.5 4.7 (0.18) 18.1** (0.001) 34.2** (0.001)

Supernumerary Cervical Ribs 1.6 1.5 (0.81) 1.0 (0.64) 5.5* (0.05) *p ≤ 0.05, **p ≤ 0.01

The GEE methodology could not be used to test the dilated renal pelves data because of the zero incidence in the control. Two solutions were tried. First, the zero incidence in the control was altered by changing one pup to “affected.” Second, an alternative statistical analysis that considers litter effects was used (34). The results of these statistical tests show reasonable agreement and are shown below.

**Table 6: Mean Percent of Pups in Litter with Dilated Renal Pelves (significance level using two methods)** 

Dose Group (mg/kg bw/day DINP)

**0 100**  **500 1,000** 

Renal Pelves 0.0 3.3 3.7 5.3
Sig. using added control event Sig. using nested analysis (0.06) (0.18) (0.10) (0.14) (0.05)* (0.04)*

These results diminish the Expert Panel’s initial concern that developmental toxicity effects based on dilated renal pelves may extend to lower doses. The Panel now concludes that the 100 mg/kg bw/day dose is a NOAEL.

Using the model-fi tting approach, the sponsor also calculated benchmark doses (BMDs) at the 5 and 10% excess risk level, based on a multiplicative (or ‘extra’) excess risk function, for the rudi

mentary lumbar rib variant. At the 5% excess risk level, the BMD05 (and 95% lower confidence interval, estimated by a bootstrap approach) was 193 mg/kg bw/day (162 mg/kg bw/day). Bench

mark doses were not calculated for other variants.

Hellwig et al. (35) (Table 7-6) evaluated the comparative developmental toxicity of a number of phthalates including three separate DINP materials. The material with CAS RN 68515-48-0 was identified as DINP-1, and two materials with CAS RN 28553-12-0, but from different production lines, were identified as DINP-2 and DINP-3. See Section 1.1 for description of chemical differenc es. Each DINP was administered by gavage in olive oil at 0, 40, 200, and 1,000 mg/kg bw/day to 8−10 sperm-positive Wistar females/group on gd 6–15. The dams were killed on gd 20 and implantationII-16Appendix IIAppendix II

II-17 sites were examined. Fetuses were weighed and examined for external malformations; half of the
fetuses were examined for skeletal malformations and the other half for visceral malformations.

For DINP-1, maternal toxicity at the high dose consisted of reduced food consumption and increased relative liver (~6%; not statistically significant) and kidney (~13%) weights. There were no treatment-related effects on the number of live fetuses/dam or fetal weight. Developmental tox

icity was evident at the highest dose by a statistically significant increase in percent fetuses/litter with variations (35.3, 41.5, 29.5, and 58.4% in the 0, 40, 200, and 1,000 mg/kg bw/day groups, respectively). These variations consisted of rudimentary cervical and/or accessory 14th rib(s). A modest increase in dilated renal pelves in the high dose group was also noted by the Expert Panel (8.9% of fetuses in 78% of control litters versus 16.8% of fetuses in 90% of treated litters). There were no maternal or developmental effects at 40 or 200 mg/kg bw/day.

For DINP-2, there was no statistically significant, dose-related evidence of maternal toxicity. However, a non-significant increase in relative liver (~5%) and kidney (~7%) weight did occur. The authors stated that developmental toxicity effects were limited to an increased fetal incidence of accessory 14th lumbar ribs at the high dose. The Expert Panel also noted a modest increase in dilated renal pelves in the high-dose group (8.9% of fetuses in 78% of control litters versus 10.6% of fetuses in 80% of treated litters).

For DINP-3, maternal toxicity was present at the high dose, expressed as reduced mean body weight gain and reduced food consumption during some portions of the treatment period. Relative liver weights (~11%) were also increased at the high dose and a non-significant increase in rela

tive kidney weights (~9%) was observed. Developmental toxicity was evidenced by a statistically significant increase in percent fetuses/litter with variations at the highest dose (35.3, 29.6, 39.5, and 60.7% in the 0, 40, 200, and 1,000 mg/kg bw/day groups, respectively). Specific types of develop

mental toxicity observed in the high-dose group at increased incidences included skeletal retarda

tion (unossified or incompletely ossified sternebrae) and skeletal variations (rudimentary cervical and/or accessory 14th rib[s]). The authors assumed that low incidences of soft tissue variations (hydroureter), visceral malformations affecting the urogenital tract (agenesis of kidneys and ure

ters), and skeletal malformations affecting the long bones (shortened and bent humerus and femur) observed at the high dose were treatment-related. The Expert Panel also observed that dilated renal pelves were slightly increased in the high dose group (8.9% of fetuses in 78% of control litters versus 16.7% of fetuses in 100% of treated litters). A maternal and developmental NOAEL of 200 and LOAEL of 1,000 mg/kg bw/day, respectively, were identified by the Expert Panel and were in concurrence with effect levels identified by authors.

In a two-generation reproductive toxicity study, postnatal weight gain was examined in pups of F0 and F1 dams exposed to DINP in feed at concentrations of 0, 0.2, 0.4, and 0.8% during mating (0, 182–197, 356–397, and 696–802 mg/kg bw/day) , gestation (0, 143–146, 287–288, 555–560 mg/kg bw/day), and lactation (0, 254–285, 539–553, and 1,026–1,129 mg/kg bw/day) (2, 36). Complete details of the experiment are included under Section 4. Weight gain for the F1 pups was reducedby DINP; males of the high-dose group were affected on postnatal day (pnd) 0, pups of the midand high-dose groups were affected on pnd 7 and 14 and all dose groups were affected by pnd 21. Weight gain of the F2 young during lactation was reduced in primarily the mid- and high-doseII-18Appendix IIAppendix II

II-19 groups; females of the low-dose group were affected on pnd 7, females of the mid- and high-dose groups were affected on pnd 4, 7, 14, and 21, and males of the mid- and high-dose groups were affected on pnd 7, 14, and 21. Postnatal sexual maturation was not examined. The Expert Panel identified a developmental NOAEL of 0.2%.

Hellwig and Jackh (37) evaluated the prenatal development toxicity of two types of isononyl alco

hol in Wistar rats. The type 1 alcohol consisted of isomers with a medium degree of branching and 16% isodecanol and the type 2 alcohol consisted of isomers with a lo w degree of branching. On gd 6–15, 10 rats/group were gavaged with the alcohols in water with 0.005% Cremophor EL at con

centrations of 0, 1, 5, and 10 mmol/kg bw/day which the authors stated equated to 0, 144, 720, and 1,440 mg/kg bw/day. A supplementary study was later conducted in 10 rats/group exposed to 0 or type 1 or type 2 alcohol at 7.5 mmol/kg bw/day (~1,080 mg/kg bw/day). In the main and supple

mentary studies, two groups of 10 control rats each were administered water or vehicle. Fetuses and dams were evaluated on pnd 20.

For the type 1 isononyl alcohol, complete maternal lethality occurred in the 1,440 mg/kg bw/day group and 1 of 10 dams died at the 1,080 mg/kg bw/day dose. Clinical signs/symptoms were reported to have been observed in a dose-related manner in dams that received 720 mg/kg bw/day and the two higher doses. A significant reduction in maternal body weight gain and increased fetal resorptions were observed in the 1,080 mg/kg bw/day group. Numerical reductions in fetal body weight occurred in the 720 mg/kg bw/day group, but were not statistically significant. Malforma

tions that primarily affected the heart were significantly increased in fetuses and litters of the 1,080 mg/kg bw/day group. Skeletal variations (cervical ribs) or retardations (reduced ossification of ster

nebrae) were increased in the 1,080 mg/kg bw/day group (statistically significant) and 720 mg/kg bw/day group.

Maternal mortality was also observed in the 1,440 mg/kg bw/day group treated with type 2 isono

nyl alcohol with death occurring in 3/10 dams. Maternal signs and symptoms were observed in the three highest doses. Non-significant reductions in body weight gain and marginal increases in resorption rates were observed in dams exposed to 720 mg/kg bw/day and higher. Fetal body weights were significantly reduced in the 1,440 mg/kg bw/day group. The authors reported that malformations were not significantly increased, but also reported that there were significantincreases in fetuses with skeletal variations and retardations (reduced ossification) from the highdose group (1,440 mg/kg bw/day). It is not clear which type of variation was increased. The authors stated that the number of fetuses with malformations (primarily affecting the thoracic vertebrae) was elevated in the 1,080 mg/kg bw/day group.

Data available in abstract form (38) and a study in press (39) report that oral exposure of SD rats to DINP at 750 mg/kg bw/day on gd 14 through pnd 3 resulted in reproductive malformations in male offspring (7.7%). The data were not available to the Panel for evaluation, therefore we merely note
the existence of the abstract.

**The summary for Section 3 is located in Section 5.1.3**

.II-18Appendix IIAppendix II

II-19 

### 4.0 REPRODUCTIVE TOXICITY

**4.1 Human Data** 
There were no human data located for Expert Panel review.

**4.2 Experimental Animal Toxicity** 
The reproductive toxicity of DINP-1 (CAS 68515-48-0) was reported by Waterman et al. (36) (Table 7-8). This report describes the results of both a one-generation and a two-generation study. In the two-generation study, SD rats (30/group) were given DINP in the diet at 0.2, 0.4, or 0.8% (w/w) for 10 weeks prior to mating, and through gestation and lactation. The study sponsor esti

mated doses of 0, 165, 331, and 665 mg/kg bw/day for males during premating, 0, 182, 356, and 696 mg/kg bw/day for females during premating, 0, 146, 287, and 555 mg/kg bw/day for females during gestation, and 0, 254, 539, and 1,026 mg/kg bw/day for females during lactation (2). Body weights and food consumption were measured weekly. After 10 weeks of pre-mating exposure, males and females were paired 1:1 within dose groups, and the females were monitored for vaginal sperm for up to 3 weeks. F 0 males were treated until after the delivery of their last litter and then killed and necropsied; females were killed after weaning their litters. Litters were culled to yield four male and four female pups per litter on pnd 4. At weaning, one male and one female from each litter were selected to grow to adulthood for mating; the remaining animals were examined exter

nally, then killed and discarded without necropsy. F 1 animals were fed the same diet as their parents throughout the rest of the study. As adults, the F 1 rats were mated within dose groups for 3 weeks after confirmation of vaginal sperm. Estimated doses for the F 1 rats were 0, 189, 379, and 779 mg/kg bw/day for males during premating, 0, 197, 397, and 802 mg/kg bw/day for females during premating, 0, 143, 288, and 560 mg/kg bw/day for females during gestation, and 0, 285, 553, and 1,129 mg/kg bw/day for females during lactation (2). Dams were allowed to litter and raise young until pnd 1, at which time they and their litters were killed, the adults were necropsied, and their organs weighed and preserved. Sperm measures were not made; testes were fixed in Bouin’s.

Weight gain for the F 0 rats was unaffected by DINP consumption until pnd 14 and 21, when the high-dose dams weighed less than the controls. In the F 0 rats, absolute liver weight was increased in the females of the mid dose group, and in both males and females of the high-dose group. Absolute kidney weight was increased in the two highest dose groups for males and in all exposed female groups. Absolute reproductive organ weights (testes, epididymis, prostate, and seminal vesicles) were unchanged by DINP. At the high dose, absolute left ovary weight was reduced versus control, although the weight of the right ovary was unchanged; the reduced weight of the left ovary appears anomalous. Fertility indices for the F 0 mating were unchanged by DINP; this includes litter size, measures of mating, number of dead offspring, and sex ratio. Weight gain was reduced in both F 1 and F 2 pups and these results are discussed in detail in Section 3. Body w eights during the mat

ing of the F 1 generation were variably reduced at the high dose by ~8

**−** 10%. When the F 1 animals were mated within dose groups as adults, DINP caused no change in ability to mate or bear young, litter size, pup weight or viability, or sex ratio. At the F 1 adult necropsy, absolute liver weight was increased in the high-dose females and absolute kidney weight was increased in the high-dose males. Absolute reproductive organ weights were unchanged. Livers appeared more eosinophilic in
all treated F 0 and F 1 rats; kidneys of the mid- and high-dose males had minimal-to-mild pelvic dila

tion. Testes were microscopically equivalent to controls.II-20Appendix IIAppendix II

II-21 The NOAEL for reproduction appears to be at least 0.8%, 665–779 mg/kg bw/day for males and 696–802 mg/kg bw/day in females. There is no LOAEL for reproduction, as there were no repro

ductive toxicities observed. The weight gain inhibition at 0.2% seen by pnd 21 in F 1 pups suggests a developmental LOAEL of 143–285 mg/kg bw/day during gestation through lactation. Using bench

mark dose methodology (BMD) the authors reported that 250 mg/kg bw/day represented the 95% lower confidence limit for a 5% reduction in body weight.

In the one-generation study, groups of 30 male or female animals consumed DINP-1 in the feed at 0, 0.5,1.0, or 1.5% w/w for 10 weeks prior to mating (36). Study sponsor-estimated doses for males during the premating period were reported at 301–591, 622–1,157, and 966–1,676 mg/kg bw/day in the low- to high-dose groups respectively (2). Doses for the low to high dose females were 363– 624, 734–1,169, and 1,114–1,694 mg/kg bw/day during premating, 377–404, 741–796, and 1,087– 1,186 mg/kg bw/day during gestation, and 490–923, 1,034–1,731, and 1,274–2,246 mg/kg bw/day during lactation. The females were exposed throughout mating, gestation, and lactation until pnd 21. The males were killed immediately after the mating period. At necropsy, the liver, kidneys, and reproductive organs were removed and weighed.

In this one-generation study, body weight gain was reduced at 1 and 1.5% DINP. There were no effects on indices of mating or fertility (litter size), and a reproductive NOAEL of 1,000 mg/kg bw/ day was identified. At necropsy, absolute liver and kidney weights were increased in both sexes at all dose levels. Testes absolute weights were increased at the high dose; ovary weights were reduced by ~30% at the highest dose. Offspring viability was reduced in the high-dose group. Offspring body weight gain at pnd 21 was reduced at all dose levels, as in the two-generation study.

Mode of Action Several studies have examined the ability of selected phthalate esters to compete with labeled estradiol (E2) for binding to the estrogen receptor (ER). Sources of ER protein included rat uterine cytosol (40), rainbow trout hepatic cytosol (41), recombinant human ERs (rhER) overexpressed in SF9 insect cells using the baculovirus system (42, 43) and rainbow trout ERs expressed in yeast (44). Tritiated E2 was used in the tissue cytosol binding assays while a high affinity fluorescent E2 derivative was used in the rhER binding assays. Selected phthalate esters have been examined in
a number of in vitro gene expression assays systems. The assays have used stably transfected cells (40), transiently transfected cells (40, 41), yeast based assays (40, 44-46) and vitellogenin induc

tion in rainbow trout hepatocyte cultures (44). DINP exhibited no activity in an in vitro assay that measured binding of phthalates to estrogen receptors (40) and in an assay of estrogen-induced gene expression (46). The assays did not include the addition of esterases or lipases to metabolize

DINP to MINP. In vivo assays demonstrated that DINP does not increase uterine wet weight or vaginal epithelial cell cornification in immature or mature ovariectomized rats treated with up to 2,000 mg/kg bw for 4 days. (40). There were no studies located on anti-androgenic activity, but an abstract and study in press have reported that gestational DINP exposure demasculinizes male pups (38, 39). Thyroid and estrogen serum levels were unaffected in adult marmosets at doses as high as 2,500 mg/kg bw/day for 13 weeks (20).

**The summary for Section 4 is located in Section 5.1.4.**II-20Appendix IIAppendix II

II-21 

### 5.0 DATA SUMMARY & INTEGRATION

**5.1 Summary** 

5.1.1 Human Exposure DINP, a complex substance of branched, predominantly C-9 isomers, is a general-purpose plasticizer for flexible PVC with a broad range of applications. It is widely used in the toy, construction, and general consumer product markets. It has limited use in food packaging. It is not used for medical applications.

The very limited monitoring data for DINP in air, drinking water, and surface and ground waters have usually yielded negative results (i.e., concentrations below detection limits). In the few studies of food and infant formula, the levels of DINP have not been quantitated or have been at or below the detection limit (0.01 mg/kg) (4-6). Occupational exposures to phthalates are reported to be below 1 mg/m 3 during the production of phthalates and 2 mg/m 3 during the manufacture of plasticized PVC (1).

Toys represent a unique childhood exposure to DINP since it is a major plasticizer used in chil

dren’s toys (7, 8, 11). DINP content has been measured at 15.1–54.4% dry weight in 31 toys (7), and 3.9–44% dry weight in 27/42 toys (11). Using pneumatic piston impaction in saliva simulant, DINP migration ranged from 1.0–48.4 μg/11cm 2 /hour, but there was no correlation between DINP content and migration rate (7). In vivo extraction has been studied using adult volunteers as sur

rogates for children (7, 12). In a comparison of extraction rates in 10 adults mouthing toys ver

sus laboratory simulation, ratios varied from 22.9 to 72.6 (mean 39.5) for 5 toys (7). RIVM (12) tested two different pieces of one toy and a controlled disk using 20 adults and also found higher
extraction in vivo . Using a 2-day parent observation study of 42 children, ages 3–36 months, mean mouthing times have been generated per age category with ranges from 0 minutes/day in older chil

dren to 171.5 minutes/day in the 6–12 month age group (see Section 1). These mouthing times have been used to model DINP exposure by several groups using a variety of assumptions as indicated in Table 7. Dermal exposure may also occur, but has not been studied specifically in children.

**Table 7: Toy Exposure Estimates for Children Aged 3−12 Months**

**Estimated Intake Level (μg/kg bw/day)**
**Agency Mean**  **95 th**  **Percentile 99**  **th Percentile**  **Maximum**

RIVM* 6.53–14.4 20.7–39.7 39.8–77.3 70.7–204

CPSC 5.7 94.3

– – Health Canada** 44 73.9*** 173.5*** 320

* Exposure range for 3−6 month-old and 6−12 month-old children;

range includes results from 3 specimens tested.

** Calculated with mouthing times for teethers and other objects intended for mouthing.

*** Results using Monte Carlo simulations in children aged 3–6 months.II-22Appendix IIAppendix II

II-23 5.1.1.1 Utility of Data to the CERHR Evaluation The Expert Panel believes it is reasonable to assume, based on the physicochemical characteristics of DINP and existing, though limited monitoring data, that general population exposure to DINP (excluding children) is expected to be lower than DEHP, which is estimated at 3–30 μg/kg bw/day (15). Children may be in the upper portion of the population range because of their physiologic differences compared to adults. The Panel also believes that some small children are likely to have exposures exceeding the general population estimates due to non-dietary ingestion from mouthing toys and other objects that contain DINP. Current models of non-dietary oral exposure predict that older infants and toddlers may incur exposures up to one order of magnitude higher than the upper limit of exposure expected in the general population.

5.1.2 General Biological and Toxicological Data

**General Toxicity:** There were no human data identified. Animal data consisted of two subhuman primate studies and
four rodent studies. In a 13-week gavage study, adult marmosets treated with up to 2,500 mg DINP/ kg bw/day (CAS number not specified) experienced weight loss or decreased weight gain, but there was no biochemical evidence of peroxisome proliferation or microscopic changes in organs examined, including testes and epididymides (20). Prepubertal (2-year-old) cynomolgus monkeys that were gavaged with 500 mg/kg bw/day for 2 weeks experienced changes in white blood cell numbers, but there were no testicular lesions or hepatic effects, including peroxisome proliferation (21). A 21-day repeat-dose dietary study in adult rats focused on peroxisome-proliferating effects in liver, and a LOAEL of 607 (F) and 639 (M) mg/kg bw/day was identified; a NOAEL was not established (16). Effects included increased liver weight at all dose levels in males and in females, dose-related enzymatic evidence of peroxisome proliferation, and alterations in hepatic cytoplas

mic basophilia and eosinophilia at the high dose. With the exception of severe unilateral atrophy in one male of the mid-dose group, testicular effects were not observed in males dosed with up to 2,195 mg/kg bw/day. Moderate testicular atrophy was observed in one DEHP-positive control that received 1,084 mg/kg bw/day.

There were three chronic (2-year) dietary studies reviewed that were of similar design and included toxicopathologic evaluation at several times during the study. Two studies were conducted using 6-week-old F 344 rats (17, 18), while the third used 6-week-old B6C3F 1 mice (19). Lesions in testes or female reproductive organs were not observed in any of the 3 studies, with the highest doses tested being 885 mg/kg bw/day in rats and 1,888 mg/kg bw/day in mice. Non-neoplastic liver lesions and/or changes in liver enzyme activity occurred at doses of 152 mg/kg bw/day and greater in rats, and 1,560 (M) to 1,888 (F) mg/kg bw/day in mice. Biochemical evidence of peroxisome proliferation was noted throughout the study in both sexes of rats in the Moore (18) study that were dosed with 733 (M) and 885 (F) mg/kg bw/day. Female rats receiving 442 mg/kg also had bio

chemical evidence of peroxisome proliferation when evaluated at the end of the study. Peroxisome proliferation was noted in high-dose mice (1,560 [M]; 1,888 [F] mg/kg bw/day), but the mid- and low-dose groups were not examined. The Lington et al. (17) rat study evaluated peroxisome pro

liferation by electron microscopy and saw none in two rats per sex per dose group at the end of the study. Non-neoplastic kidney lesions and changes in urinary excretion were seen in rats exposed to 307 mg/kg bw/day and higher, and in mice dosed with 1,560 (M) and 1,888 (F) mg/kg bw/day.II-22Appendix IIAppendix II

II-23 Indications of anemia, such as reductions in red blood cell numbers and hemoglobin levels, were seen in rats exposed to 307 mg/kg bw/day and higher. Hepatic neoplasia was observed only in male rats exposed to 733 mg/kg bw/day and in mice exposed to 336 (F) and 742 (M) mg/kg bw/day and higher. Renal neoplasia was only observed in male rats of the highest dose group (733 mg/kg bw/ day). The apparent qualitative difference in liver and renal effects (i.e., tumors vs hepatotoxicity) in the rat studies may reflect differences in the range of doses tested.

There were no toxicity studies with inhalation exposure.

Mode of Action: The renal neoplasia in male rats appears to be due to alpha-2-micro globulin nephropathy which is a mechanism not considered relevant to humans (23). However, an increased rate of nephropathy, was seen in female mice exposed to 1,888 mg/kg bw/day which would not be consistent with the alpha-2-microglobulin mechanism. The Moore (18) study demonstrated liver tumors in rats only in the highest-dose males. Peroxisome proliferation in rats was observed at the highest dose in males and females, and the second highest dose in females but not males. No liver tumors were observed in either sex at the second-highest dose level. In addition, no liver tumors were noted in the recov

ery groups. These results are consistent with a peroxisome proliferation mode of action for hepatic tumor induction. Unfortunately, peroxisome proliferation was assayed in mice only at the highest dose, and liver tumors were observed at lower doses.

**T**

**oxicokinetics**

. There are no human data. DINP was orally administered to adult male albino rats at doses of 50, 150, or 500 mg/kg bw/day. It is metabolized by pancreatic lipases in the lumen of the gut and rapidly absorbed (49%) as the monoester and rapidly excreted via urine and feces with no accumulation in tissues (28). Dermal absorption of DINP is slow (<4% in 7 days) in rats (24). Dermal absorption of DINP through human skin is expected to be lower than rat skin based on
results of an in vitro study conducted with DEHP (47). There is evidence for excretion via the biliary route based on radioactivity in feces and GI tract of rats dosed dermally with 14 C-DINP.
There are no inhalation studies available.

**Genetic Toxicity**

. DINP tested negative in experiments of mutagenicity and clastogenicity including the Ames, Chinese hamster ovary cell, and rat bone marrow chromosomal aberration, mouse lymphoma mutation, unscheduled DNA synthesis, and Balb/c-3T3 mouse cell transformation assays (29, 30).II-24Appendix IIAppendix II

II-25 **Table 8: Summaries of NOAELs and LOAELs and Major Effects in General Toxicity Studies**

**Protocol & Study**
**DINP Tested and Doses (mg/kg bw/day)**  **NOAEL (mg/kg bw/**  **day) LOAEL (mg/kg bw/day)**  **& Effects Major Effects at**  **Higher Doses**

13-week repeat-dose gavage study in adult

marmosets 16–25 months of age, 1–2 per sex/group Doses: 0, 100, 500, 2,500 DINP type not specified (20) 500 2,500

↓ Weight gain or weight loss No peroxisomal proliferation No microscopic findings in

organs No higher doses in study

2-week repeat-dose gavage study in male prepubescent cynomolgus monkeys 2 years of age, 4/group Doses: 0, 500
DINP-1 (21) Not
determined 500

Changes in neutrophil and lymphocyte counts
No testicular lesions No liver effects including peroxisomal proliferation No higher doses in study

21-day repeat-dose dietary study in young
adult Fischer 344 rats 6 weeks of age at start of study, 5 rats per sex/group Doses: (M) 0, 639, 1,192, 2,195; (F) 0, 607, 1,193, 2,289 Mixture of different DINP types (16) None M: 639, F: 607

↑ Liver weight ↑ Peroxisomal proliferation

(M) ↑ Kidney weight ↑ Liver weight, and peroxisomal proliferation ↑ Kidney weight ↑ Testes weight
No testicular lesions

2-year repeat-dose dietary study in
Fischer 344 rats 6-week-old at beginning of study, 110 per sex/group Doses: (M) 0, 15, 152, 307; (F) 0, 18, 184, 375
DINP-1 (17) M: 15
F: 18 M: 152, F: 184

Hepatic effects ↑ Liver weight
Mononuclear cell leukemia ↑ Kidney weight Hepatotoxicity ↑ Liver weight
No testicular lesions
Mononuclear cell leukemia
Anemia ↑ Kidney weight and excretion

changes No peroxisomal proliferation
2-year repeat-dose dietary study in Fischer
344 rats 6 weeks of age at start of study, 70–85 per sex/group Doses: (M) 0, 29, 88, 359, 733; (F) 0, 36, 109, 442, 885
DINP-1 (18) M: 88
F: 109 M: 359, F: 442

Nephrotoxicity Excretion changes
Anemia ↑ Liver weight, peroxisomal

proliferation (F)
Mononuclear cell leukemia ↑ Kidney weight Hepatic & renal neoplasia at high dose (M)
Anemia Nephrotoxicity ↑ Liver weight and peroxisomal

proliferation
Mononuclear cell leukemia ↑ Kidney weight
No testicular lesions
2-year repeat-dose dietary study in
B6C3F 1 mice 6 weeks of age at beginning of study, 70/ sex/group Dose: (M) 0, 90, 276, 742 or 1,560; (F) 0, 112, 336, 910, 1,888
DINP-1

(19) M: 276
F: 112 M: 742, F: 336

Liver neoplasia ↑ Liver weight (M) ↓ Kidney weight (M) Liver neoplasia, hepatocyte staining variations, peroxisomal proliferation, and nephrotoxicity (F) at highest
doses ↑ Liver weight ↓ Kidney weight (M)
No testicular lesionsII-24Appendix IIAppendix II

II-25 5.1.2.1 Utility of Data to the CERHR Evaluation There are adequate subchronic and chronic data available in rats and mice and adequate subchronic data in primates to assess general toxicity by the oral route, including liver and kidney effects (16- 19). No effects have been noted in the male or female reproductive system, although these studies were not designed to fully assess this system.

Toxicokinetic data consist of oral and dermal studies in rodents. The data permit the Panel’s conclusion that dermal absorption is slow; oral absorption is rapid for the monoester formed by lipases in the gut. Dose-related kinetics of absorption across species is not known. DINP and its metabolites are rapidly excreted via urine and feces with no accumulation in tissues.

5.1.3 Developmental Toxicity There were no human studies located for Expert Panel review.

Two published prenatal developmental toxicity studies in rats were available for DINP (31, 35). The protocols for the two studies were similar and included dosing of dams by gavage on gd 6–15 with sacrifice and evaluation of fetuses on gd 20–21. Developmental toxicity was also noted in both a one-generation and a two-generation toxicity study. The effects on pup body weight are discussed below and summarized in Table 9; the reproductive effects are described in Section 5.1.4.

Hellwig et al. (35) performed their studies in Wistar rats (10/group) at doses of 0, 40, 200, and 1,000 mg/kg bw/day. Although sample size (n=10) was small, the aggregate of their work can logically be considered to be three separate studies of DINP. There was a degree of consistency across all studies. Effects were only observed at the highest dose. Relative kidney and liver weights were slightly increased in dams of the highest dose group (5–13%), but statistical significance was erratic. Fetal viability and body weight were unaffected in all three studies. Skeletal variations (rudimentary cervical ribs, accessory 14 th ribs) were numerically increased with each DINP with the number of affected fetuses per litter significantly higher than controls in two instances. There was a tendency to see dilated renal pelves at the highest dose; in one study agenesis of kidneys and ureters was assumed by the authors to be DINP-related. Skeletal (shortened and bent humerus and femur) malformations were also observed in the high-dose group of this study. It is clear that organ effects are associated with kidney and the skeletal system. For maternal and developmental effects, a NOAEL of 200 and a LOAEL of 1,000 mg/kg bw/day were identified by the Expert Panel for each DINP and are in concordance with effect levels identified by Hellwig et al. (35).

The prenatal toxicity study of Waterman et al. (31) was more informative than the Hellwig study from the standpoint of number of rats per test group and completeness of data reported. Waterman et al. (31) tested DINP-1 in Sprague-Dawley rats (25/group) at doses of 0, 100, 500, or 1,000 mg/kg bw/day. Maternal toxicity at the highest dose consisted of decreased food intake and weight gain. The authors presented and analyzed effects on offspring as percent affected fetuses and percent affected litters. Waterman et al. (31) interpreted their results as indicating a LOAEL for maternal and developmental toxicity at 1,000 mg/kg bw/day and a NOAEL of 500 mg/kg bw/day. The Panel concurred with the maternal NOAEL, but concluded there was developmental toxicity at the 500 mg/kg bw/day dose. As discussed in Section 3.2, the Panel advised the study sponsor that there were more recent and improved methods for the statistical analysis of fetal incidence data. TheII-26Appendix IIAppendix II

II-27 sponsor performed appropriate reanalyses that the Panel reviewed and found to be consistent with the Panel interpretation of skeletal variations. The Panel concludes there is a NOAEL in the study at 100 mg/kg bw/day. The BMD estimated a 5% excess risk level was 193 mg/kg bw/day (95% LCL=162 mg/kg bw/day) for rudimentary lumbar ribs, as provided by the study sponsor (33).

The Panel noted that developmental toxicity was observed in the prenatal rat studies by Waterman and Hellwig. In the study by Waterman, the urinary system was a target of effect as noted by a modest increase in dilated renal pelves at the 1,000 mg/kg dose. While only a mild increase in dilated renal pelves was observed in the three Hellwig et al. studies, in one instance more severe renal effects (hydroureter, agenesis) were seen. In studies by Waterman et al. (31) and Hellwig et al. (35), the skeletal system was the target for effect as observed by an increased incidence of cervical ribs and accessory 14 th (lumbar) ribs. These studies also evaluated the closely related phthalate DIDP where the same target organs were identified. An increase in cervical ribs and lumbar ribs was observed at the common dose of 1,000 mg/kg bw/day in the two studies. While effect on lumbar ribs was more pronounced, the effect on cervical ribs is of greater toxicological concern. Cervical ribs are seen infrequently in controls, and their presence may indicate a disruption of gene expression. There is evidence that cervical ribs may interfere with normal nerve function and blood
flow.

Differences in NOAELs between the Waterman et al. (31) and Hellwig et al. (35) studies, 100 and 200 mg/kg bw/day respectively, may be due to rat strain, and certainly to dose selection.

The two-generation reproductive study by Waterman et al. (36) suggests an adverse effect on weight gain in pups during the perinatal and pre-weaning period of life. Developmental landmarks of reproductive tract development, identified as a sensitive target with other phthalates, were not
examined. F 1 mean pup body weight was significantly reduced on pnd 0 in males at 0.8% DINP (555 and 1,026 mg/kg bw/day during gestation and lactation, respectively, as calculated by study sponsors). On pnd 7 and 14, mean male and female pup body weights were significantly reduced at 0.4% (287 and 539 mg/kg bw/day during gestation and lactation, respectively) and 0.8%, and by pnd 21, mean male and female body weights were reduced at all dose levels. In the F 2 generation, mean female pup body weights were significantly reduced at 0.4 and 0.8% on pnd 4, 7, 14, and 21 and at 0.2% (143 and 285 mg/kg bw/day during gestation and lactation, respectively) at pnd 7. Mean male pup body weights were significantly reduced at 0.4 and 0.8% at pnd 7, 14, and 21. The LOAEL for developmental effects was therefore identified as 0.2% (143–285 mg/kg bw/day during gestation through lactation) by the Expert Panel.

Studies with 2 isononyl alcohols, differing in degree of branching, demonstrated clinical signs and symptoms in pregnant rats at doses of 720 mg/kg bw/day and higher (37). Table and text discrep

ancies in dose values and reported effects at the higher dose levels were noted. Toxicity was more severe with type 1 isononyl alcohol, the alcohol that had a higher degree of branching. Maternal mortality was seen at the highest dose (1,440 mg/kg bw/day) with both alcohols and in the type 1 alcohol at 1,080 mg/kg bw/day. Fetal malformations and/or variations occurred at 1,440 mg/kg bw/ day and at 1,080 mg/kg bw/day. Slight effects that may be associated with treatment were observed at 720 mg/kg bw/day. A dose of 144 mg/kg bw/day was without effect for both isononyl alcohols.II-26Appendix IIAppendix II

II-27 **Table 9: Summary of NOAELs and LOAELs and Major Effects in Developmental Toxicity Studies** 

**Protocol & Study NOAEL**  ( **mg/kg bw/day** )

**[Benchmark dose]**

**ED 05**  **in mg/kg bw/day**  **LOAEL** ( **mg/kg bw/day** ) **and Effects Developmental**  **Effects Observed at Higher Dose Levels** 

**Maternal Developmental**  **Developmental**

Prenatal gavage study in
Wistar rats.

10/group/study received 0, 40, 200, or 1,000 mg/kg bw/ day on gd 6–15.

Dams and pups examined in late gestation.

DINP-1, DINP-2, and
DINP-3

(35) 200

Maternal & Developmental 1,000

↑ Kidney and liver

weights. 1,000

↑ Cervical and

lumbar ribsall.

↑ Urogenital

and skeletal
malformation
with DINP-3. N/A

Prenatal gavage study in

Sprague-Dawley rats.

25 per group received 0, 100, 500, or 1,000 mg/kg bw/day on gd 6–15.

Dams and pups examined in late gestation.

DINP-1

(31) 500 (Maternal)

100 *** (Developmental)

[MLE(95%LCL): 193 (162) for lumbar ribs] 1,000

↓ Weight gain. 500

↑ Fetuses with

vertebral
variations. ↑ Fetuses and litters
with visceral variations (mainly
dilated renal pelves).

↑ Fetuses and litters

with lumbar ribs.

↑ Fetuses with

cervical ribs.

Two generation reproductivedietary study in SpragueDawley rats.

30 per group were fed diets with 0, 0.2, 0.4, or 0.8% from 10 weeks prior to mating (0, 182–197, 356– 397, and 696–802 mg/kg bw/day) through gestation (0, 143–146, 287–288, and 555–560 mg/kg bw/day and lactation 0, 254–285, 539– 553, and 1,026–1,129 mg/kg bw/day during lactation)**.

DINP-1

(36)* None

[250 (95% LCL) for decreased pup weight gain] 143−285

↑ Mild

histological liver changes
in F 0 and F 1 . ↑ Kidney weight

in F 0 . 143−285

↓ Weight gain on

pnd 21in F 1 . ↓ Weight gain

on pnd 7 in F 2 females. ↓ Weight gain on pnd 0 (males), 7, 14,
and 21 in F 1 . ↓ Weight gain on pnd

4 (female), 7, 14,
and 21 in F 2 .

* Only maternal and developmental effects were listed in this table. Reproductive and male systemic effects are listed in Table 10.

** Range of doses for F 1 and F 2 dams.

*** NOAEL selected by Expert Panel is lower than study author’s selectionII-28Appendix IIAppendix II

II-29 5.1.3.1 Utility of Data to the CERHR Evaluation There are adequate data available in rats to determine that prenatal oral exposure to DINP-1 results in developmental toxicity. The results of the Waterman et al. (31) and the Hellwig et al. (35) studies were remarkably consistent with respect to DINP-1. In both studies, exposure to DINP-1 resulted in increases in lumbar and cervical ribs. In addition, the effective dose levels were similar. Hellwig et al. (35) identified a LOAEL of 1,000 mg/kg bw/day and a NOAEL of 200 mg/kg bw/day with a sample size of 10/group. The Panel identified an effect level of 500 mg/kg bw/day from the Waterman et al. (31) study (sample size of 25/group) and 100 mg/kg bw/day level represented a NOAEL. In addition, Hellwig et al. (35) showed some similarities among the three DINPs in that each resulted in an increase in lumbar and cervical ribs. It is clear that the urinary and skeletalsystems are target organs where developmental toxicity is observed. The data from the twogeneration dietary study are sufficient to demonstrate an effect on postnatal growth, with a LOAEL of 143–285 mg/kg bw/day and no NOAEL. The reduced growth is consistent in both studies. Neither prenatal study extended dosing into the late gestation period which has been shown to be a critical window of development for other phthalates. In addition, the study designs did not allow for assessment of postnatal sexual maturation. The issue of late gestational exposure was addressed in a two-generation reproductive toxicity study reviewed in Section 5.1.4. Confidence in the isononyl alcohol study is limited due to table and text discrepancies in dose values and reported effects at the higher dose levels. The study is adequate to ascribe maternal and developmental toxicity at these higher doses and to assume the lowest dose was without effect.

5.1.4 Reproductive Toxicity Structural and functional reproductive effects were examined in one- and two-generation feeding studies in rats that included in utero exposure during the entire duration of pregnancy (36). In the one-generation dose range finding study, rats were administered dietary levels of 0, 0.5, 1.0, or 1.5% DINP and in the two-generation study, rats were administered dietary levels of 0, 0.2, 0.4, or 0.8% DINP. In the two-generation study, reproductive parameters including mating, fertility, and testicular histology were unaffected in both generations at the highest dose (0.8%; 665–779 and 696–802 mg/kg bw/day in males and females, respectively) and this dose was identified as the reproductive NOAEL. Developmental effects were observed, including decreased pup weight gain (most marked on pnd 21). The effects on pup weight gain are discussed in greater detail under Section 5.1.3. Histologic effects included mild hepatic eosinophilia in both sexes of parental rats in all dose groups of both generations and dilated renal pelves in F 1 parental males of the mid- and high-dose groups. The results of the study are consistent with the one-generation pilot study that was previously conducted. In the one-generation study, fertility was unaffected in male and female rats exposed to dietary DINP concentrations as high as 1.5% (966–1,676 and 1,114–1,694 mg/kg bw/day in males and females, respectively). The findings of these studies indicate that male and female rat fertility and structure of reproductive organs are unaffected by exposure to DINP at a maternal dose of 555–1,129 mg/kg bw/day during gestation and lactation, respectively, and adult exposure to concentrations as high as 1,676 mg/kg bw/day in males and 1,694 mg/kg bw/day in
females.II-28Appendix IIAppendix II

II-29 **Mode of Action** DINP exhibited no activity in an in vitro assay that measured binding of phthalates to rat uterine cytosolic estrogen receptors (40) and in an assay of estrogen-induced gene expression (46). The assays did not include the addition of esterases or lipases to metabolize the DINP to MINP. In

vivo assays demonstrated that DINP does not increase uterine wet weight or vaginal epithelial cellcornification in immature or mature ovariectomized rats (40). There were no studies located on antiandrogenic activity. Thyroid and estrogen serum levels were unaffected in adult marmosets at doses as high as 2,500 mg/kg bw/day for 13 weeks (20).

**Table 10: Summary of NOAELs and LOAELs and Major Effects in Reproductive Toxicity Studies** 

**Protocol & Study NOAEL**  **(mg/kg bw/day) LOAEL (mg/kg bw/day)**  **and Effects Reproductive Effects**  **Observed at Higher**
**Dose Levels**

**Repro Systemic** 

Two-generation repro

ductive dietary study in Sprague-Dawley rats.

30 per group were fed diets with 0, 0.2, 0.4, or 0.8% (Males: 0, 165–189, 331–379, and 665–779 mg/kg bw/day, Females: 0, 182–197, 356–397, and 696–802 mg/kg bw/day**) from 10 weeks prior to mating through gestation and
lactation.

DINP-1

(36)* 665–779 (M); 696–802 (F) (Reproductive)

None (Systemic) No effects on reproductive

structure or function. M: 165–189;
F: 182–197

↑ Mild liver

effects in F 0 and F 1. ↑ Kidney weight

in F 0 females. None

* Only effects in parental rats are listed. Developmental effects are listed in Table 9.
** Doses during the premating period-Combined for F 0 and F 1 rats.

5.1.4.1 Utility of Data to the CERHR Evaluation The data are sufficient to indicate that DINP exposures are not associated with detectable effects on reproductive function. The studies did demonstrate consistent effects on the liver (weight and histology) and kidney (weight). Given the constraints of the study design, the data demonstrate no likely reproductive toxicity at doses up to 779(M)–802(F) mg/kg bw/day in the two-generation study or at 1,676(M)–1,694(F) mg/kg bw/day in the one-generation study. However, the studies did not assess endpoints of reproductive development shown to be sensitive with other phthalates.II-30Appendix IIAppendix II

II-31 **5.2 Integrated Evaluation** 

DINP is a complex substance of branched, predominantly C-9 isomers. There are no human data from which to assess the health effects associated with DINP exposure; studies of DINP toxicity are limited to laboratory animals. In the absence of human data and barring evidence to the contrary, it is assumed that the effects observed in laboratory animals are relevant to humans.

Based upon the physicochemical similarities between DINP and DEHP and on limited DINP moni

toring data, general population exposures to DINP are expected to be lower than those to DEHP
which are estimated at 3

**−** 30 μg/kg bw/day. It is reasonable to presume that humans would be exposed primarily through the oral route. Although data are scant, the ingestion of DINP through food does not appear to be common. Children may be exposed to higher levels of DINP (up to 10– 100 fold higher) than adults because infants and small children mouth toys and other articles that contain DINP that can migrate into saliva and be swallowed. DINP is not used in medical devices, therefore intravenous exposure does not occur.

Dermal absorption of DINP is slow in rats. DINP administered orally to rats is metabolized by gut lipases to the monoester, which is rapidly absorbed. DINP and its metabolites are rapidly excreted in urine and feces, with no indication of accumulation in tissues with repeated (5 **×** ) daily doses. At low doses, approximately equal amounts of the DINP-derived material are excreted in urine and feces, with the urinary metabolites consisting of the monoester and its oxidation products, while the feces contain those metabolites plus the diester. There are no toxicokinetic studies in humans, but

in vitro studies comparing the dermal uptake of other phthalates in human and rat skin suggest that dermal uptake of DINP in humans would be negligible.

Oral exposure to DINP has been shown to cause liver and kidney toxicity in adult rats and mice, but not in marmosets. The liver effects are generally consistent with those associated with peroxi

some proliferation. Liver tumors have been noted in adult male rats exposed to 733 mg/kg bw/day, in female mice exposed to 336 mg/kg bw/day, and in male mice exposed to 742 mg/kg bw/day. Kidney tumors were noted in male rats, but these tumors are associated with a mechanism that is not believed relevant to humans (alpha-2-microglobulin). However, the increased incidence of nephropathy seen in female mice exposed to 1,888 mg/kg bw/day is not consistent with the alpha2-microglobulin mechanism.

The developmental studies available include examination of effects of prenatal exposure on prenataldevelopment, as well as a limited assessment of postnatal developmental effects in one- and twogeneration reproductive studies. The prenatal studies provide consistent results and are sufficient to establish that oral exposure to DINP causes fetal skeletal variations (lumbar and cervical ribs) and in some cases, urinary tract effects (hydroureter). The Panel was confident that 500 mg/kg bw/day was an effect level, and 100 mg/kg bw/day was a NOAEL. For one study, sponsors estimated a BMD for a 5% excess risk level of 193 mg/kg bw/day with a 95% lower confidence limit of 162 mg/kg bw/day. For the second developmental toxicity study, the Expert Panel identified a devel

opmental NOAEL of 200 mg/kg bw/day. In addition, the results of the one- and two-generation dietary reproductive toxicity studies demonstrated a consistent reduction in mean pup body weights during lactation at doses as low as 143–285 mg/kg bw/day (doses during gestation and lactation)II-30Appendix IIAppendix II

II-31 and a NOAEL could not be identified. This effect level is similar to that obtained in the more robust prenatal study, although the effects are different and a similar mode of action is not assumed.

There is evidence that isononyl alcohol, a primary metabolite of DINP, is a developmental and maternal toxicant at high (~1,000 mg/kg) oral doses in rats. These doses appear to be greater than the doses of DINP that are associated with developmental toxicity, suggesting that effects at lower doses are probably associated with the monoester. The Panel does acknowledge there are no data to permit a judgment about an interactive effect between the alcohol and monoester metabolite.

Reproductive performance and histological effects on gonads and accessory sex organs were assessed in one- and two-generation dietary studies. Parental doses of up to 0.8% in feed (665–779 [M] and 696–802[F] mg/kg bw/day) did not affect fertility or sex organ histology in either the F 0 or F 1 male or female pups. A 13-week gavage study in adult marmosets resulted in no evidence of microscopic testicular changes at doses that did adversely affect body weight gain (2,500 mg/ kg bw/day). Testicular lesions were not observed in prepubertal cynomolgus monkeys that were gavaged for 2 weeks with 500 mg/kg bw/day, reportedly the maximum dose that can be absorbed by the monkeys. Chronic 2-year studies in rats and mice gave no gross or histologic evidence of effects on testes or ovaries at doses that did cause liver and kidney effects and other clinical signs of tox

icity. Thus, the data are sufficient to conclude that neither the reproductive organs nor fertility are affected by extended oral exposure to DINP. However, the Panel noted that some endpoints which are sensitive to other phthalates (i.e., preputial separation and nipple retention) were not evaluated in the two-generation study. The Panel is aware that additional data on reproductive tract develop

ment are being developed, but as yet only abstracts are available for review. The Panel also notes that the target organs in studies with adult rats, liver and kidney, are also target organs in develop

mental and multigeneration studies. This increases the Panel’s confidence that these effects are real, and that different organ system susceptibilities between adults and young are unlikely.

**5.3 Expert Panel Conclusions** 

DINP is used in toys, construction, and general consumer products. Although data are scant, expo

sure through food appears to be lower than for DEHP. Therefore, the Expert Panel believes that adult exposure to DINP will not exceed levels of 3–30 μg/kg bw/day, the estimates derived for DEHP. Exposures to DINP are likely to be below this level, but the Panel cannot quantitate how far below. Occupational exposures could occur through inhalation and dermal contact. Limited stud

ies of occupational exposures suggest that inhalation exposure is below 1 mg/m 3 during production of DINP and below 2 mg/m 3 during production of PVC. Although estimates of dermal exposure are not available, the Expert Panel is confident that dermal exposure would not result in significant absorption into the body. Children could be exposed to DINP by eating contaminated food. How

ever, DINP has not been detected in a limited survey of infant formula. By analogy, the Expert Panel believes that children’s exposure to DINP via food will not exceed those levels estimated for DEHP. Additional exposure to children may occur due to mouthing of toys and other objects that contain DINP. Current models of non-dietary oral exposure predict that older infants and toddlers may incur exposures up to one order of magnitude higher than the upper limit of exposure expected
for adults.II-32Appendix IIAppendix II

II-33 The toxicology database is sufficient to determine that oral maternal exposure to DINP can result in developmental toxicity to the conceptus. In rats, two prenatal developmental studies have shown effects on the developing skeletal system and kidney following oral exposure to DINP. The NOAELs for these studies were 100–200 mg/kg bw/day. In addition, developmental toxicity was noted in an oral two-generation reproductive toxicity study in rats. In this study, effects on pup growth were noted. These effects may be due to prenatal and/or lactational exposures to DINP. The LOAEL for the study was 143–285 mg/kg bw/day and a NOAEL was not identified. Based on the results of the toxicology studies, oral exposure to pregnant women and oral exposure to children should be examined. The Expert Panel has minimal concern for unborn children due to ambient maternal exposure to DINP. Based on estimates of exposure to DINP in toys and other objects that children may mouth, the Expert Panel has low concern for potential health effects in children. The Expert Panel cannot judge the potential health effects in unborn children following maternal occupational exposures due to the lack of toxicology data following inhalation exposures and the lack of occupational exposure information.

The oral prenatal developmental toxicity studies and the oral two-generation reproductive toxicity study have shown no effects on the reproductive system in rats. The NOAEL for reproductive toxicity is 665–779 (M) and 696–802 (F) mg/kg bw/day. The Expert Panel noted that some endpoints of reproductive development that have been shown to be sensitive with other phthalates were not assessed in the two-generation study of DINP, and therefore the Panel has only moderate confidence in the NOAEL. The Expert Panel has minimal concern about DINP resulting in reproductive toxicity in humans.

**5.4 Critical Data Needs** 

Critical data needs are discussed under two categories: experimental studies and human exposures.

**Experimental studies.** Since some relevant endpoints (i.e., nipple retention) were lacking in many of the studies reviewed, uncertainties would be reduced if this additional information were gathered. The Expert Panel rec

ommends a sequential approach for future studies that would focus on obtaining the most critical information first; subsequent studies would be dependent upon the results of the initial study. The Panel further recognized that data gathering should be an iterative process and that recommenda

tions may change as initial tiers of data are gathered. The Expert Panel recommends that the follow

ing sequential steps be considered.

1) Conduct a perinatal developmental study in orally exposed rats that addresses landmarks of sexual maturation such as nipple retention, anogenital distance, age at testes descent, age at prepuce separation, and structure of the developing reproductive system in pubertal or adult animals exposed through development. Although a two-generation reproductive toxicity study has evaluated some relevant endpoints, the recommended study would provide greater certainty about the lack of such effects with DINP. If DINP does affect these endpoints and the effective dose levels are of possible human health concern, then the Expert Panel recommends that the following study (2) be conducted.II-32Appendix IIAppendix II

II-33 2) Conduct a perinatal developmental study with oral exposure in a non-rodent species. There are species differences in the developmental toxicity associated with other phthalates. The develop

mental effects of DINP have only been examined in the rat. Therefore, there is some uncertainty whether other species would exhibit similar responses and whether the rat is an appropriate model for assessing potential human risk.

**Human exposure.** Human exposure to DINP has not been well studied; there are no reports of levels in biological materials (blood, urine, etc.), and the environmental data consist primarily of estimates.

Patterns of use, expected environmental levels, and vulnerability of exposed population groups should dictate decisions about measuring DINP in environmental media. For example, determining DINP exposures in young children is of highest priority, based on the use patterns and vulnerability described above. Workers producing PVC products are a second priority.

Collection of biological samples de novo should be accompanied by environmental measurements to provide information on exposure sources. Existing biological samples should be utilized where available if they can provide useful information about exposure.

Although information about exposure of young children is a critical data need, manufacturers of children’s toys should be polled to determine if their products will continue to contain DINP in the future. If so, an estimate of the DINP content should be made by the manufacturer and confirmed by independent studies. Salivary extraction of DINP and better estimates of mouthing behavior, especially within the potentially highest risk group of 3–12 month-old children, using data from more children, should be carried out.II-34Appendix IIAppendix II

II-35 

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8. Rastogi SC. Gas chromatographic analysis of phthalate esters in plastic toys. Chromatographia 47: 724-726(1998).

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11. Canada H. Updated. Risk assessment on diisononyl phthalate in vinyl children’s products. Ottawa, Ontario: Consumer Products Division, Product Safety Bureau, Environmental Health Directorate, Health Protection Branch, 1998.

12. RIVM, Milieu RVVE. Phthalate release from soft PVC baby toys RIVM report 613320 002: National Institute of Public Health and the Environment, 1998.

13. Fiala F, Steiner I, Kubesch K. Migration of di-(2-ethylhexyl)phthalate (DEHP) and diisononyl phthalate (DINP) from PVC articles. (2000).

14. Juberg DR, Thompson KM, Alfano K, Coughlin RJ. An observational study of object mouthing behavior by young children. Rochester, New York: International Center for Toxicology and Medicine, Submitted to journal.

15. Doull J, Cattley R, Elcombe C, Lake B, Swenberg J, Wilkinson C, Williams G. Expert panel report on DEHP.: U.S. Environmental Protection Agency, 1998.

16. BIBRA BIBRA-. A 21-day feeding study of di-isononyl phthalate. Report No. 0495/6/85.: Chemical Manufacturers Association, 1985.

17. Lington AW, Bird MG, Plutnick RT, Stubblefield WA, Scala RA. Chronic toxicity a carcinogenic evaluation of diisononyl phthalate in rats. Fundam Appl Toxicol 36:79-89(1997).

18. Moore MRCL. Oncogenicity study in rats with di(isononyl)phthalate including ancillary hepatocellular proliferation and biochemical analyses. Covance 2598-104 Volume 1 of 5. Vienna, VA: Aristech Chemical Corporation, 1998.II-34Appendix IIAppendix II

II-35 19. Moore MRCL. Oncogenicity study in mice with di(isononyl)phthalate including ancillary hepatocellular proliferation and biochemical analyses. Covance 2598-105 Volume 1 of 6. Vienna, VA: Aristech Chemical Corporation Performing Laboratory, 1998.

20. Hall M, Matthews A, Webley L, Harling R. Effects of dii-isononyl phthalate (DINP) on peroxisomal markers in the marmoset - DINP is not a peroxisome proliferator. The Journal of Toxicological Sciences 24:237-244(1999).

21. Pugh G, Isenberg JS, Kamendulis LM, Ackley DC, Clare LJ, Brown R, Lington AW, Smith JH, Klaunig JE. Effects of di-isononyl phthalate, di-2-ethylhexyl phthalate, and clofibrate in cynomolgus monkeys. Toxicol Sci 56:181-188(2000).

22. Short RD, Robinson EC, Lington AW, Chin EC. Metabolic and peroxisome proliferation studies with di (2-ethylhexyl) phthalate in rats and monkeys. Toxicol Ind Health 3:185-194(1987).

23. Caldwell DJ. Review of mononuclear cell leukemia (MNCL) in F-344 rat bioassays and its significance to human cancer risk: A case study using alkyl phthalates. Accepted for publication in Regulatory Toxicology and Pharmacology(1999).

24. Midwest Research Institute M. Dermal disposition of 14 C-diisononyl phthalate in rats 35320. Kansas City, MI: Exxon Corporation, Medical Department, Research and Environmental Health, P.O. Box 235, East Millstone, NJ, 1983.

25. Scott RC, Dugard PH, Ramsey JD, Rhodes C. In vitro absorption of some o-phthalate diesters through human and rat skin. Environ Health Perspect 74:223-227(1987).

26. Deisinger PJ, Perry LG, Guest D. In vivo percutaneous absorption of DEHP from DEHP- plasticized polyvinyl chloride film in male Fischer 344 rats. Food Chem Toxicol 36:521-527(1998).

27. Hazelton HL-L. Metabolism study of 14 C phthalate ester in rats - Final Report 32563. Vienna, VA: Esso Research and Engineering Company, 1972.

28. Midwest Research Institute M. Single and repeated oral dose pharmacokinetics of 14 C-labeled diisononyl phthalate with cover letter. MRI project No. 7282-B. Kansas City, MI: Exxon Corporation, Medical Department, Research and Environmental Health, P.O. Box 235, East Millstone, NJ, 1983.

29. OECD. Risk assessment - 1,2-Benzenedicarboxylic acid, di-C8-10-branched alkyl esters C9-rich and di-”isononyl”phthalate CAS No.: 68515-48-0 and CAS No.: 28553-12-0: EINECS-No.: 271-090-9 and EINECS-No.: 249-079-5, 1998.

30. Barber E, Cifone M, Rundell J, Przygoda R, Astill B, Moran E, Mulholland A, Robinson E, Schneider B. Results of the L5178Y mouse lymphoma assay and the Balb/3t3 cell invitro transformation assay for eight phthalate esters. J Appl Toxicol 20:69-80(2000).

31. Waterman SJ, Ambroso JL, Keller LH, Trimmer GW, Nikiforov AI, Harris SB. Developmental toxicity of di-isodecyl and di-isononyl phthalates in rats. Reprod Toxicol 13:1-6(1999).

32. Ryan L. The use of generalized estimating equations for risk assessment in developmental toxicity. Risk Analysis 12:439-447(1992).

33. McKee R. Personal communication to Jack Moore, 2000.

34. Chen JJ. Dose-response modeling of growth for develomental toxicity. Environmetrics 7:135- 144(1996).

35. Hellwig J, Freudenberger H, Jackh R. Differential prenatal toxicity of branched phthalate esters in rats. Food Chem Toxicol 35:501-512(1997).

36. Waterman SJ, Keller LH, Trimmer GW, Freeman JJ, Nikiforov AI, Harris SB, Nicolich MJ, McKeeII-36Appendix IIAppendix II

II-37 RH. Two-generation reproduction study in rats given di-isononyl phthalate in the diet. Reprod Toxicol 14:21-36(2000).

37. Hellwig J, Jackh R. Differential prenatal toxicity of one straight-chain and five branched-chain primary alcohols in rats. Food Chem Toxicol 35:489-500(1997).

38. Ostby J, Price M, Furr J, Lambright C, Hotchkiss A, Parks l, Gray Jr LE. Perinatal exposure to the phthalates DEHP, BBP, DINP, but not DEP, DMP or DOTP permanently alters androgen-dependent tissue development in Sprague-Dawley rats. Triangle Consortium or Reproductive Biology Jan 29, 2000(2000).

39. Gray LE, Ostby J, Furr J, Price M, Veeramachaneni DNR, Parks L. Perinatal exposure to the phthalates in DEHP, BBP and DINP but not DEP, DMP or DOTP alters sexual differentiation of the male rat. Toxicol. Sci. (in press)(2000).

40. Zacharewski TR, Meek MD, Clemons JH, Wu ZF, Fielden MR, Matthews JB. Examination of the

in vitro and in vivo estrogenic activities of eight commercial phthalate esters. Toxicol Sci 46:282- 293(1998).

41. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. A variety of environmentally persistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ Health Perspect 103:582-587(1995).

42. Bolger R, Wiese TE, Ervin K, Nestich S, Checovich W. Rapid screening of environmental chemicals for estrogen receptor binding capacity. Environ Health Perspect 106:551-7(1998).

43. Nakai M, Tabira Y, Asa D, Yakabe Y, Shimyozu T, Noguchi M, Takatsuki M, Shimohigashi Y. Binding characteristics of dialkyl phthalates for the estrogen receptor. Biochemical and Biophysical Research Communications 254:311-314(1999).

44. Petit F, Le Goff P, Cravedi J-P, Valotaire Y, Pakdel F. Two complementary bioassays for screening the estrogenic potency of xenobiotics: Recombinant yeast for trout estrogen receptor and troup hepatocyte cultures. Journal of Molecular Endocrinology 19:321-335(1997).

45. Coldham NG, Dave M, Sivapathasundaram S, McDonnell DP, Connor C, Sauer MJ. Evaluation of a recombinant yeast cell estrogen screening assay. Environ Health Perspect 105:734-742(1997).

46. Harris CA, Henttu P, Parker MG, Sumpter JP. The estrogenic activity of phthalate esters in vitro

. Environ Health Perspect 1997 105:802-811(1997).

47. Scott RC, Dugard PH, Ramsey JD, Rhodes C. In vitro absorption of some o-phthalate diesters through human and rat skin. Environ Health Perspect 74:223-227(1987).II-36Appendix IIAppendix II

II-37 

### 7.0 TABLES

7.1 DINP General Toxicity, Rats

............................................................... ................................... 38
7.2 DINP General Toxicity, Rats

............................................................... ................................... 39
7.3 DINP General Toxicity, Rats

............................................................... ................................... 40
7.4 DINP General Toxicity, Mice

............................................................... .................................. 41

7.5 DINP General Toxicity, Marmosets

............................................................... ........................ 42
7.6 DINP Developmental Toxicity, Rats

............................................................... ....................... 43

7.7 DINP Developmental Toxicity, Rats

............................................................... ....................... 44
7.8 DINP Developmental Toxicity, Rats

............................................................... ....................... 45
7.9 DINP Developmental Toxicity, Monkeys

............................................................... ................ 46
7.10 References

............................................................... ............................................................... 47II-38Appendix IIAppendix II

II-39Table 7-1: DINP, General Toxicity, Rats**Species, Strain, and** **Source Experimental** **Regimen Animal** **Number/ Sex** **Dose*****Body Weight** **Organ Weight** ****Liver Effects Hematology** **Other**Fischer 344RatBIBRA 1985(1)Subchronic study :21 days.Rats (∼41−43 daysold) were fed dietswith 0, 0.6, 1.2, or2.5% DINP and thensacrificed and necropsied. Peroxisome proliferation was studiedby electron microscopy examination ofliver and measurement of peroxisomalenzyme activity.505639(M)607(F)NE↑Li, Ki↑11-OH(M) and12-OH(M).↑Protein.NA↓Serum Ch,Tg(M).51,192(M)1,193(F)↓↑Li, Ki↑PCoA(M), 11-OHand 12-OH(M).↑Protein.Histologicalchanges.NA↓Serum Ch,Tg(M).↑Serum Tg(F).52,195(M)2,289(F)↓↑Li, Ki,Te↑PCoA, 11-OHand 12-OH.↑Peroxisomes.↑Protein.Histologicalchanges.NA↓Serum Ch,Tg(M).↑Serum Tg(F).No dose-relatedtesticularlesions.↑A group of positivecontrol rats wasadministered DEHPat 1.2%.51,084(M)1,063(F)↓↑Li, Ki↑PCoA, 11-OHand 12-OH.↑Peroxisomes.↑Protein.NA↓Serum Ch,Tg(M).No dose-relatedtesticularlesions.*Dose measured in mg/kg bw/day.**Organ to body weight ratio.↑=Statistically Significant Increase↓=Statistically Significant DecreaseNA=Not AnalyzedNE=No EffectsF=FemaleM=MaleTe=TestesLi=LiverKi=Kidney11-OH=11-HydroxylasePCoA=Palmitoyl-CoA Oxidas12-OH=12-HydroxylaseTg=TriglycerideCh=CholesterolII-38Appendix IIAppendix II

II-39Table 7-2: DINP, General Toxicity, Rats**Species, Strain, and** **Source Experimental** **Regimen Animal** **Number/ Sex** **Dose*****Body Weight** **Organ Weight** ****Liver Effects Hematology** **Other**Fischer 344RatLington etal. 1997(2)Chronic study :2 years.6-week-old rats werefed diets with 0, 0.03,0.3, and 0.6% DINP1 for 2 years. 10rats/sex/group weresacrificed and necropsied at 6, 12, and18 months and therest were killed andnecropsied at the endof the study. Hematology, serum chemistry,and urinalysis wereevaluated every 6months. Peroxisomeproliferation wasexamined microscopically in 2 rats/sex/group at 24 months.110011015(M)18(F)NENENENENOAEL110152(M)184(F)↓(M;18–24 mo)↑Li, Ki(6–24 mo)Hepatic lesions(24 mo)↑SGOT (M; 6–12 mo)↑SGPT (M; 24 mo)↑MNCL.NE↓Survival (F).110307(M)375(F)↓(M;12–24 mo)↑Li, Ki(6–24 mo)↑Sp (24 mo)↑Ad (24 mo)Hepatocyteenlargement (6–24mo) and lesions (24mo)↑SGOT(M; 6–18 mo)↑SGPT(M; 6 &18 mo)↑MNCL.No evidence of peroxisomal proliferation↓RBC, Hb,Hct(M; 24 mo)↓Survival (F).↑Urine volume(M; 6–24 mo)↑Urine K andglucose(M; 6–18 mo)No evidence oftesticular damage.*Dose measured in mg/kg bw/day**Organ to body weight ratio↑=Statistically Significant Increase↓=Statistically Significant DecreaseNE=No EffectsF=FemaleM=MaleMo=MonthAd=AdrenalKi=KidneyLi=LiverSp=SpleenSGOT=Serum Glutamic Oxaloacetic TransaminaseSGPT=Serum Glutamic Pyruvic TransaminaseMNCL=Mononuclear Cell LeukemiaRBC=Red Blood CellHb=HemoglobinK=PotassiumII-40Appendix IIAppendix II

II-41Table 7-3: DINP, General Toxicity, Rats**Species, Strain, and** **Source Experimental Regimen** **Animal Number/** **Sex Dose** **Body Weight** **Organ Weight** ****Liver Effects Hematology** **Other**F344 RatsMoore 1998(3)Chronic study : 2 years.6-week old rats were fed diets with 0, 500, 1,500, 6,000,or 12,000 DINP. 5 rats/sex/dose were killed on weeks 1,2, and 13; 15 rats/sex/dosewere killed at 79 weeks; and55 rats/sex/group at 104–106weeks. Clinical evaluations(hematology, serum chemistry, and urinalysis) wereconducted every 26 weeks.Peroxisome proliferationwas examined in 5 rats/sexonly in the controls and highdose group during weeks 1,2, and 13 and in 3–5 rat/sexin the control, 359–442 mg/kg bw/day group, and highdose group at week 104.8507029.2(M)36.4(F)NENENENENE7088.3(M)109(F)NENENENENOAEL85359(M)442(F)NE↑Ki(wk 79-104)↑Li(wk 1-104)↑PCoA (F, wk 104)↑ASAT, ALAT(wk 52, 78, 104)↑AnemiaKidney lesions(M, wk 79–104)↑Serum urea(wk 26–104)MNCL (wk 104)85733(M)885(F)↓(wk9–104)↑Ki(wk 79-104)↑Li(wk 1-104)↑PCoA (wk 1–104)Lesions(wk 2–104)Neoplasia (M,wk 79–104)↑ASAT, ALAT(wk 52, 78, 104)↓ASAT, ALAT(F, wk 26)↑Anemia↓Survival (M)↑Serum urea(wk 26–104)↑Urine vol with↓Cl, Ca, K,Cre (M, wk 104)Kidney lesions(wk 79–104)Kidney neoplasm(M, wk 104)MNCL (wk 104)No testicular effectsA group of 55 rats/sex wasexposed to the high dosefor 78 weeks and sacrificedat 105–106 weeks to studyrecovery effects.*55637(M)774(F)↓(F)↑Ki(F)NE↑Urine vol with↓Cre(M)MNCL (wk 104)Kidney lesions andneoplasm (M)*Only effects observed by week 104 listed**Organ to body weight ratio↑=Statistically Significant Increase↓=Statistically Significant DecreaseNE=No EffectsF=FemaleM=MaleKi=KidneyLi=Liverwk=Weekvol=VolumeALAT=Alanine aminotransferaseCa=CalciumASAT=Aspartate aminotransferaseCl=ChlorideMNCL=Mononuclear Cell LeukemiaCre=CreatininePCoA=Palmitoyl-CoA OxidaseK=PotassiumII-40Appendix IIAppendix II

II-41Table 7-4: DINP, General Toxicity, Mice**Species, Strain, and** **Source Experimental Regimen** **Animal Number/** **Sex Dose** ***Body Weight** **Organ Weight** *****Liver Effects Hematology** **Other**B6C3F1/CrlBR miceMoore 1998(4)Chronic study: 2 years.6-week-old mice were feddiets with 0, 500, 1,500,4,000, and 8,000 ppmDINP. 15 mice/dose/ sexwere evaluated and sacrificed at 79 weeks and 55mice sex/group at 105–106weeks. Clinical evaluations(hematology, serum chemistry, and urinalysis) wereconducted every 26 weeks.Peroxisome proliferationwas examined in 5 mice/sexin the highest dose groupand controls during themidpoint and end of study.7007090.3(M)112(F)NENENENENOAEL (F)70276(M)336(F)NENE↑Neoplasia (F)NENOAEL (M)70742(M)910(F)↓(wk1–104)↓Ki(M),↑Li(M)(wk 79-104)↑Neoplasia (M)NENE701,560(M)1,888(F)↓(wk1–104)↓Ki (M),↑Li(wk 79-104)↑Neoplasiaand nonneoplasticchanges↑Serum ASAT,ALAT (M)↑PCoA(wk 79–104)↓WBC(wk 26–98)↓Survival (M)↑Nephropathy(F)↑Serum protein(M, week 104)↑Urinary vol with↓Na, Cl, K(week 52–104)No effects ontesticularhistologyA group of 55 mice/sex wasexposed to the high dosefor 78 weeks and sacrificedat 105–106 weeks to studyrecovery effects.**551,377(M)1,581(F)↓(M)↓Ki(M)↑NeoplasiaNENE*Dose measured in mg/kg bw/day.**Only effects observed by week 104 listed***Organ to body weight ratio↑=Statistically Significant Increase↓=Statistically Significant DecreaseNE=No EffectsF=FemaleM=Malewk=Weekvol=VolumeKi=KidneyLi=LiverWBC=White Blood CellALAT=Alanine aminotransferaseASAT=Aspartate aminotransferasePCoA=Palmitoyl-CoA OxidaseCl=ChlorideK=PotassiumNa=SodiumII-42Appendix IIAppendix II

II-43Table 7-5: DINP, General Toxicity, Marmosets**Species, Strain, and** **Source Experimental** **Regimen Animal** **Number/ Sex** **Dose*****Body Weight** **Organ Weight** **Histopathology Hematology** **Chemistry Other** MarmosetHall et al.1999(5)Subchronic study:13 Weeks.Male and female marmosets (16–25 monthsold) were gavaged withDINP in 1% methylcellulose and 0.5% Tween.Clofibrate was usedas a positive control.Parameters evaluatedat sacrifice included estradiol and testosteroneconcentrations, biochemical evidence ofperoxisomal proliferation, and organ weightsand histopathology.1–202100NENENENENE2500NENENENENE22,500decreaseaNENENENo changein PCoAUngroomedcoat and reddening of theskin.Thin appearance, hunchedposture andreduced activityin one male.a1–2500Clofibratedecreasea↑Ki tobodyweightratioaNE↑Anemia↑PCoA↑11-OHaDose measured in mg/kg bw/day.*Efects were not statistically significant↑=Statistically Significant IncreaseNE=No Effects11-OH = Lauric acid 11-hydroxylase activityPCoA=Palmitoyl-CoA Oxidase activityII-42Appendix IIAppendix II

II-43Table 7-6: DINP Developmental Toxicity, Rats**Species, Strain, and Source** **Experimental Regimen Number** **b Dose** **d Maternal Effects** **Fetal Effects**Wistar RatHellwig et al. 1997(7)Prenatal developmentaltoxicity study.Three types of DINPamanufactured by differentprocesses were administered in oil by gavageon gd 6–15. Dams wereweighed on gd 0, 6, 10,15, and 20 and sacrificedon gd 20. Maternal uteriwere weighed, corpora lutea were counted and implantation sites examined.Fetuses were weighed andexamined for gross external malformations. Half ofthe fetuses were examinedfor visceral malformations and the other half forskeletal malformations.909–1040↑Kidney to bodyweightratio in DINP-2.↓Implantation sites/litter in DINP-2.↓Live fetuses/litter in DINP-2.8–10200NENE9–101,000↑Kidney to bodyweightratio in DINP-1.↑Liver to bodyweightratio in DINP-3.↑Fetuses/litter with cervical ribs in:DINP-1: 11 fetuses/5litters vs 0 fetusesDINP-2: 4 fetuses/4litters vs 0 fetusesDINP-3: 12 fetuses/7litters vs 0 fetuses.↑Fetuses/litter with lumbar ribs in:DINP-1: 37 fetuses/10litters vs 0 fetusesDINP-2: 10 fetuses/5litters vs 0 fetusesDINP-3: 34 fetuses/8litters vs 0 fetuses.↑Fetuses/litter with hydroureter inDINP-3: 2fetuses/8litters vs 4fetuses/3 litters.↑Fetuses/litter with malformations in:DINP-3 (7.3 vs 4.3%).c↑Dilated renal pelves in DINP-1,2,3.a(1) CAS RN: 68515-48-0; (2) CAS RN: 28553-12-0; (3) CAS RN: 28553-12-0 (2 and 3 by different manufacturing process).bNumber of litters examined per type of DINP.cSkeletal and visceral malformations (humorous, femur, kidney, and ureter); not statistically significant.dDose measured in mg/kg bw/day.↑=Statistically Significant Increase↓=Statistically Significant DecreaseNE=No EffectII-44Appendix IIAppendix II

II-45Table 7-7: DINP, Developmental Toxicity, Rats**Species, Strain, and Source** **Experimental Regimen Number** ***Dose******Maternal Effects** **Fetal Effects**Sprague-DawleyRatWaterman et al.1999(8)Prenatal developmental toxicitystudy.DINP-1 administered in oil bygavage on gd 6–15. Sacrificed ongd 21. Dams weighed on gd 0, 6,9, 12, 15, 18, and 21. Maternaluterus and ovaries were weighed,corpora lutea were counted andimplantation sites examined.Fetuses were weighed, sexed,and examined for gross externalmalformations. Half of the fetuseswere examined for visceralmalformations and the other halffor skeletal malformations.24025100NE↑% Fetuses with dilated renal pelves(3.7vs 0%).24500NOAEL↑% Fetuses with dilated renal pelves(4 vs 0%).↑% Fetuses with lumbar ribs(19vs 4%).231,000↓Weight gain(transient).↓Food intake(transient).↑% Litters with dilated renal pelves(26vs 0%).↑% Fetuses with dilated renal pelves(4.5vs 0%).↑% Litters with lumbar ribs(78vs 25%).↑% Fetuses with lumbar ribs(35vs 4%).↑% Fetuses with cervical ribs(6vs 2%).*Number of litters examined.**Dose measured in mg/kg bw/day.↑=Statistically Significant Increase↓=Statistically Significant DecreaseNE =No EffectII-44Appendix IIAppendix II

II-45Table 7-8: DINP, Reproductive Toxicity, Rats**Species, Strain, and Source** **Experimental Regimen Animal** **Number/ Sex** **Dose*****Effects**CD RatsWaterman et al.2000(9)Two generation reproductive toxicitystudy.DINP administered in feed for 10weeks prior to mating at 0, 0.2, 0.4, or0.8%. Males treated until delivery oflast litter and females through gestationto lactation. Breeding pairs housedtogether for 3 weeks; body weight andfood intake was measured weekly. Onemale and female from each litter rearedto adulthood and remaining pups wereexamined and discarded.30030165/182/146/254↑Kidney weight in F0females.↓Weight gain in F1pups.30331/356/287/539↑Liver weight in F0females.↑Kidney weight in F0males and females.↓Weight gain in F1pups.30665/696/555/1,026↓Weight gain in F0females (pnd 14, 21).↑Liver weight in F0males and females.↑Kidney weight in F0males and females.↓Left ovary weight in F0females.↓Weight gain in F1pups.No effect on weights of male reproductiveorgans, testicular histology, or litter size,mating, offspring survival, and sex ratio.One male and female F1rat/littercontinued to receive the same dosesas parental rats and were then matedwithin dose groups during adulthood.30189/197/143/285NE30379/397/288/553↓F2pup weight gain during lactation.30779/802/560/1,129↓Body weight in F1.↑Liver weight in F1females andkidney weight in F1males.↓F2pup weight gain during lactation.No effects on mating, fertility, litter size,pup weight, survival, or sex ratio, sexorgan weights or testicular histology.*Doses (in mg/kg bw/day) in males during premating/females during premating/females during gestation/females during lactation.↑=Statistically Significant Increase↓=Statistically Significant DecreaseII-46Appendix IIAppendix II

II-47Table 7-9: DINP, General Toxicity, Monkeys**Species, Strain, and** **Source Experimental** **Regimen Animal** **Number/ Sex** **Dose*****Body Weight** **Organ Weight** **Histopathology Hematology** **Chemistry Other** CynomolgusmonkeyPugh et al.2000(6)Subacute study:2 weeks.2-year-old males weregavaged with DINP in0.5% methylcellulosefor 2 weeks andthen sacrificed andnecropsied.404500NENo effectson organto bodyweightratiosNo testicular orhepatic lesions.↑Neutrophils↓LymphocytesNENo peroxisomeproliferationNo effects ongap junctionalintracellularcommunication*Doses measured in mg/kg bw/day.NE=No Effects↓=Statistically Significant Decrease↑=Statistically Significant IncreaseII-46Appendix IIAppendix II

II-47 

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