# NTP-CERHR Monograph on the **Potential Human Reproductive and Developmental Effects**  **of Di- n**  **-Octyl Phthalate (DnOP)**

May 2003 NIH Publication No. 03-4488 

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

## Table of Contents

Preface

............................................................... ............................................................... ............... i Introduction

............................................................... ............................................................... ....... ii NTP Brief on Di- n -Octyl Phthalate (DnOP)

............................................................... ..................... 1 References

............................................................... ............................................................... .......... 3 Appendix I. NTP-CERHR Phthalates Expert Panel Preface

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

............................................................... ....................................................... I-2 Appendix II. Phthalates Expert Panel Report on DnOP Preface

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

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

......................................................... II-4 Developmental Toxicity Data

............................................................... ........................... II-8 Reproductive Toxicity

............................................................... ...................................... II-9 Data Summary & Integration

............................................................... ......................... II-11 References

............................................................... ...................................................... II-18 Tables

............................................................... ............................................................. II-21 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) established 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 selected chemical. Public comment is invited prior to and during the meeting. The expert panel produces a report on the chemical’s reproductive and developmental toxicities and provides its opinion of the degree to which exposure to the chemical is hazard

ous to humans. The panel also identifies areas of uncertainty and where additional data are needed. The CERHR expert panels use explicit guidelines to evaluate 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 monographincludes the NTP brief on the chemical evaluated, the expert panel report, and all public comments. The goal of the NTP brief is to pro

vide the public, as well as government health, regulatory, and research agencies, with the NTP’s interpretation of the potential for the chemical to adversely affect human reproduc

tive health or children’s health. The NTPCERHR monograph 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 contact

ing the director: NIEHS, P.O. Box 12233, MD EC-32, 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 devel

opmental toxicities of these chemicals, 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 DnOP, a list of the expert panel members (Appendix I), the expert panel’s report on DnOP (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 DnOP 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 monograph presents the NTP’s interpretation of the poten

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

ple. It is based upon information about DnOP 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 DnOP 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 two 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, postnatal development, sexual maturity, and, again, production of sperm and eggs. In the past, toxic effects were often studied 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 DnOP? DnOP is an oily substance manufactured by reaction of phthalic anhydride and n-octanol in the presence of a catalyst. The structure of DnOP is shown in Fig. 1. Its chemical formula is C 24 H 38 O 4 . It is one of a group of industri

ally important chemicals known as phthalates. Phthalates are used primarily as plasticizers to add flexibility to plastics. Available informa

tion indicates that there are no commercial uses of pure DnOP. However, it makes up approxi

mately 20% of the commercially important phthalate mixture known as C6–10 phthalate. This mixture is used in the manufacture of a variety of commercial products including flooring and carpet tiles, tarps, pool liners, and garden hoses. DnOP is approved by the FDA as an indirect food additive and is used in seam cements, bottle cap liners, and conveyor belts. DnOP-containing compounds are not known to be used in medical devices or toys. Based on an estimated annual production of 50 million pounds of C6–10 phthalate, 10 million pounds (20% of 50 million) of DnOP is pro

duced each year.

Are People Exposed to DnOP?* Yes. There are several ways that people may be exposed to DnOP at home or at work. Human exposure can occur during the manufactureof DnOP, during the manufacture of DnOPcontaining products, during the use of such products, or through the presence of DnOP in the environment. Environmental exposures can occur through air, water, food, or contact with DnOP-containing products. Studies have shown that DOP (isomer not specified) is detectable in a variety of food products and household dust samples but the data are not sufficient to permit a confident estimate of general population exposure. Because of in

adequate information on human exposure to DnOP, the expert panel took the conservative position of assuming that general population exposures in the United States would be 3–30 μg/kg bw/day (micrograms per kilogram body weight per day). This is the range of exposures estimated for the more widely used phthalate, DEHP. By comparison, a small drop of water weighs approximately 30,000 μg and a grain of table salt weighs approximately 60 μg. Can DnOP Affect Human Development or Reproduction? Probably not. There is no direct evidence that exposure of people to DnOP adversely affects reproduction or development. Limited studies with mice and rats show that high exposures to DnOP may cause adverse developmental effects but no evidence of reproductive effects was observed (Fig. 2). Scientific decisions concerning health risks aregenerally based on what is known as “weightof-the-evidence.” There are no exposure or effects data in humans. There is limited evi

dence of developmental effects in animals at high doses, and some evidence of no reproduc

tive effects in animals. The NTP judges the sci

entific evidence to indicate DnOP is not likely to affect human reproductive systems. The data 

### NTP Brief on Di- **n** -Octyl Phthalate (DnOP)

O O

O O **Figure 1.** Chemical structure of DnOP

* 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 are insufficient to make a judgment on possible developmental effects. (Fig. 3). Summary of Supporting Evidence As presented in the expert panel report, two de

velopmental toxicity studies—one in rats and one in mice—were available for evaluation. In the rat study, pregnant dams were treated with DnOP by intraperitoneal injection on gestation days 5, 10, and 15. Fetuses were evaluated on gestation day 20. The two dose levels used were very high, approximately 5,000 and 10,000 mg/kg bw/day. Increased malformations and reduced body weights were observed in fe

tuses at both dose levels. In the mouse study, pregnant dams were treated by gavage with approximately 10,000 mg/kg bw/day on ges

tation days 6–13 and allowed to deliver pups. DnOP treatment resulted in smaller litter sizes and reduced weight gain on postnatal days 1–3. However, birth weights and pup survival to day 3 were not affected. The reproductive toxicity of DnOP was evalu

ated in a mouse continuous breeding study. Animals were exposed through dosed feed to approximately 1,800, 3,600, or 7,500 mg/ kg bw/day. No adverse reproductive effects were observed in the treated parents or their offspring. Likewise, no reproductive effects were observed in two limited studies in male rats where oral exposure to DnOP for 4 days or 13 weeks did not affect testes weights or histology.

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

Reproductive Toxicity



### Figure 3. NTP conclusions regarding the possibilities that human development or reproduction might be adversely affected by exposure to DnOP
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 Reproductive effects Developmental effects2NTP Brief

3NTP Brief No new data on the developmental or repro

ductive effects of DnOP have become available since the expert panel report was completed. Are Current Exposures to DnOP High Enough to Cause Concern? Probably not. No studies have been conducted in humans and only a few animal studies are available. However, the animal studies evalu

ated indicate that there are no effects on re

production and that developmental effects may occur at high exposure levels. Although no firm data are available on human exposures, it appears that the U.S. general population is not exposed to DnOP levels that are of immediate concern for causing adverse reproductive or developmental effects. The NTP offers the fol

lowing conclusions. **The NTP concurs with the CERHR Phthalates Expert Panel’s conclusion that there is negli** 
**** **gible concern for effects on adult reproductive systems.**  This conclusion is based on the assumption that humans are exposed to levels of DnOP less than 30 μg/kg bw/day. Although DnOP is reported to be lethal to mouse fetuses at high doses, data are not avail

able to determine exposure levels at which no adverse effects occur. Therefore, the NTP concludes that there is insufficient information on developmental toxicity to reach a conclusion regarding the potential for DnOP to adversely affect human development. 

### References: No new publications were available.

These conclusions are based on the information available at the time this brief was prepared. As new in formation on toxicity and exposure accumulate, it may form the basis for either lowering or raising the levels of concern expressed in the conclusions.I-1Appendix I 

### Appendix I. NTP-CERHR Phthalates Expert Panel Report on DnOP 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 DnOP 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- n -Octyl Phthalate

October 2000 NTP-CERHR-DNOP-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

..................................................... 4
2.1 General Toxicity

............................................................... ........................................................... 4 2.2 Toxicokinetics

............................................................... .............................................................. 6 2.3 Genetic Toxicity

............................................................... ........................................................... 7
3.0 DEVELOPMENTAL TOXICITY DATA

............................................................... ....................................... 8
3.1 Human Data

............................................................... ............................................................... ... 8 3.2 Experimental Animal Toxicity

............................................................... ...................................... 8
4.0 REPRODUCTIVE TOXICITY

............................................................... ...................................................... 9
4.1 Human Data

............................................................... ............................................................... ... 9 4.2 Experimental Animal Toxicity

............................................................... ...................................... 9
5.0 DATA SUMMARY & INTEGRATION

............................................................... ....................................... 11
5.1 Summary

............................................................... .............................................................. 11 5.1.1 Human Exposure

............................................................... .............................................. 11 5.1.1.1 Utility of Data to the CERHR Evaluation

........................................................... 11 5.1.2 General Biological and Toxicological Data

............................................................... ..... 11 5.1.2.1 Utility of Data to the CERHR Evaluation

........................................................... 12 5.1.3 Developmental Toxicity

............................................................... ................................... 12 5.1.3.1 Utility of Data to the CERHR Evaluation

........................................................... 14 5.1.4 Reproductive Toxicity

............................................................... ...................................... 14 5.1.4.1 Utility of Data to the CERHR Evaluation

........................................................... 15 5.2 Integrated Evaluation

............................................................... ................................................. 15 5.3 Expert Panel Conclusions

............................................................... .......................................... 16 5.4 Critical Data Needs

............................................................... .................................................... 17
6.0 REFERENCES

............................................................... ............................................................... .............. 18
7.0 TABLES

............................................................... ............................................................... ........................ 21Appendix 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-participating 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 evi

dence that adverse reproductive/development health effects are associated with exposure to specific chemicals or classes of chemicals, including descriptions of any 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-n-Octyl Phthalate

Di-n-octyl phthalate (DnOP) (CAS Registry Number 117-84-0) is produced by reacting phthalic anhydride and n-octanol in the presence of an acid catalyst ( 1 ). Synonyms : 1,2-benzenedicarboxylic acid, dioctyl ester; phthalic acid, dioctyl ester; n-dioctyl phthalate; n-octyl phthalate; dioctyl o-benzenedicarboxylate; bis(n-octyl) phthalate. DnOP is a significant component (20%) of C6–10 phthalate mixtures ( 1 ). **Table 1: Physicochemical Properties of DnOP Property**  **Value** Chemical Formula C 24 H 38 O 4 Molecular Weight 390.54 Vapor Pressure 1.0 x 10 -7 mm Hg at 25 °C Melting Point -25 o C Boiling Point 390 o C Specific Gravity 0.978 Solubility in Water Essentially Insoluble (0.5 μg/L) Log K ow 8.06 (2) **1.2 Exposure and Usage**  There is sometimes confusion reporting data for DnOP because DEHP is often referred to in the literature as dioctyl phthalate (DOP). Unless otherwise stated, the information discussed in this exposure section refers specifically to DnOP to the best of CERHR’s knowledge. There are no known commercial uses for pure DnOP. However, DnOP constitutes approximately 20% of C6–10 phthalate substance. Commercial production of 50 million pounds of C6–10 

### O O

O OII-2Appendix IIAppendix II

II-3 phthalate in the United States in 1994 ( 3 ) equates to 10 million pounds of DnOP. C6–10 phthalate substance is used in PVC utilized in the manufacture of flooring and carpet tile, canvas tarps, swim

ming pool liners, notebook covers, traffic cones, toys, vinyl gloves, garden hoses, weather stripping, flea collars, and shoes ( 1 ). DnOP-containing phthalate substances are also used in PVC intended for food applications such as seam cements, bottle cap liners, and con veyor belts. Release of DnOP to the environment can occur during the production of C6–10 phthalates and during the incorporation of the phthalates into plastic resins. Because phthalates are not bound to plastics, DnOP can be released during the use or disposal of the product. Phthalates released to the environment can be deposited on or taken up by crops intended for human or livestock consump

tion, and thus, may enter the human food supply. **General Population Exposure** The general population is exposed to phthalates primarily through the oral and dermal routes. Based on data for other phthalates, the most likely source of DnOP exposure to humans is dietary intake. DnOP may be found in food as a result of environmental uptake during cultivation or as a result of migration from processing equipment or packaging materials. DnOP is approved by the FDA for use as an indirect food additive in sealants used for food packaging ( 4 ). In a survey of packaged fatty foods purchased from grocery stores in the UK, the total concentration of dioctyl phthalate (DOP, isomer not specified) excluding DEHP was 2.3 mg/kg in milk ( 5 ). A paper pub

lished in 1995 reported the detection of DnOP in two samples of vodka; concentrations of 57 ppb in a 100 proof sample and 131 ppb in an 80 proof vodka ( 6 ). DOP (isomer not specified) excluding DEHP was detected in 8 of 12 infant formulas from the UK at concentrations ranging from 0.21

− 1.42 mg/kg ( 7 ). Using manufacturer recommendations for feeding rates and by assuming that formula was the only nutritional source for infants, exposures to DOP isomers other than DEHP were estimated at <0.1–43 μ g/kg bw/day at birth and <0.1–24 μ g/ kg bw/day at 6 months of age by the UK Ministry of Agricutural Fisheries and Food (MAFF) ( 7 ). In a follow-up survey DOPs were not specifically targeted, but there was no evidence of their pres

ence in 39 samples of infant formulas examined ( 8 ). Pfordt and Bruns-Weller ( 9 ) reported the phthalate content of various household items in Germany. DnOP was detected in nutmeg at 0.02 mg/kg, but levels were below the limit of detection of 0.01 mg/kg in milk (breast and commercial), cream, nuts, and baby food. DnOP was detected in the dust from a vacuum cleaner bag at 40 mg/kg; 20 mg/kg DNOP was detected in a dust wipe sample from one of three homes tested. DNOP levels in textiles were measured at 0.01

− 0.08 mg/kg. A concen

tration of 0.01 mg/kg DNOP was detected in one of three samples of flooring textiles. There appears to be little or no use of DnOP-containing compounds in to ys. According to the American Chemistry Council (ACC, formerly CMA) ( 1 ), DnOP was only detected in some teethers that were tested for phthalate ester migration by the Danish Ministry of Environment and Energy. No other studies have reported the detection of DnOP in toys. Exposure to DnOP through air is also possible but expected to be minimal. Reported concentrations of DnOP in ambient air range from 0.06 to 0.94 ng/m 3 . The highest reported concentration resultedII-2Appendix IIAppendix II

II-3 in a calculated inhaled dose of 0.29 ng/kg bw/day for an adult ( 10 ). Reported concentrations in river water have ranged from 0.024 to 1 ppb. EPA estimates that DNOP levels in drinking water influents are less than 0.5 ppb ( 10 ). These levels are several orders of magnitude lower than levels found in food. The available data do not allow the confident estimation of DnOP exposures to the general popula

tion. However, a comparison of production volumes for DnOP-containing compounds versus those that contain DEHP suggests that human exposure to DnOP is well below the exposure estimate for DEHP of 3–30 μ g/kg bw/day ( 11 ). Exposures may be higher in children due to dietary preferences or mouthing of DnOP-containing articles. **Medical Exposure** There are no known current uses of DnOP-containing compounds in medical devices. **Occupational Exposure** Workers may be exposed to DnOP primarily through inhalation and dermal contact. Phthalates are manufactured within closed systems, but exposure to workers can occur during filtering or loading/ unloading of tank cars ( 1 ). Higher exposures to phthalates can occur during the production of flex

ible PVC because the processes are open and run at higher temperatures. The ACC ( 1 ) reviewed six publications demonstrating that phthalate levels in air are generally less than 1 mg/m 3 and 2 mg/m 3 during the production of phthalates and flexible PVC, respectively. Exposure levels were estimated by the ACC ( 1 ) using assumptions of a 10 m 3 /day inhalation rate and a 70 kg body weight. The resulting exposure estimates were 143 μ g/kg bw/workday and 286 μ g/kg bw/workday for workers employed in phthalate and flexible PVC manufacturing operations, respectively.
**The summary for Section 1 is located in Section 5.1.1.**II-4Appendix IIAppendix II

II-5 

### 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 **Acute Studies** The LD 50 values for mice and rats are given as 13 g/kg and 53.7 g/kg, respectively ( 1 ). Dermal LD 50 values have been determined at 75 mL/kg for guinea pigs ( 1 ). **Repeat-dose Toxicity** Four studies in rodents were available for review ( 12-15 ). Systemic effects following DnOP treatment for 3, 10, or 21 days were examined in 4-week-old Wistar rats. These effects were compared to effects produced by other groups of rats being fed diet containing 20,000 ppm of di-n-hexyl phthalate (DnHP), another straight chain phthalate, or di(2- ethylhexyl) phthalate (DEHP), a branched-chained phthalate ( 14 ). A group of 12 male rats was a fed a diet containing 20,000 ppm DnOP and a control group of 18 rats was fed the basal diet. Using actual food intake levels and rat body weights on the day of sacrifice, a DnOP dose of 1,821 mg/kg bw/day was calculated. Four treated and 6 control rats were killed and necropsied after 3, 10, or 21 days of treatment. Liver histopathology, enzyme activity, and peroxisome proliferation were exam

ined. Levels of thyroid hormones in serum and thyroid histopathology were also examined ( 12 ). DnOP treatment had no effect on testes weight or the gross appearance of testes, kidney, or pan

creas ( 14 ). However, liver weight was significantly increased at 10 and 21 days of DnOP treatment with liver histology and chemistry changes seen at all 3 assessment times. After 3 days of exposure, centrilobular necrosis and glycogen loss were observed. At 10 days, centrilobular fatty accumula

tion was seen, this effect became more pronounced with increasing treatment duration. Electron microscopy (EM) showed effects on the smooth endoplasmic reticuli (proliferation and dilation) and microvilli shortening in the bile canaliculi at 3 days. At 10 days, EM also showed lipid droplets in the hepatocytes and a small increase in lysosomes and peroxisomes at 3 and 21 days respectively. Biochemical evidence for peroxisome proliferation was seen with significant increases in cyanide insensitive palmitoyl CoA oxidase at 10 and 21 days of treatment. Total catalase activity was un

changed; however, in the particulate sub-fraction it was significantly increased at 10 and 21 days of treatment. Other liver enzymes that were changed included significant decreases in 5’-nucleotidase, succinate dehydrogenase , and glucose-6-phosphate at 21 days of treatment. There was a significant, DnOP-treatment related decrease in serum thyroxine (T4); serum triiodothyronine levels (T3) were not significantly affected and microscopic changes were suggestive of thyroid hyperactivity. These changes included increased lysosomal numbers and size, enlarged Golgi apparatus, and mitochon

drial damage. When compared to the other two co-tested phthalates, DnOP induced effects on hepatic lipid accu

-II-4Appendix IIAppendix II

II-5 mulation and peroxisomal proliferation that were similar to the effects caused by DnHP, but dissim

ilar to those caused by DEHP. DEHP caused greater increases in liver weight and greater increases in mitotic activity. Less fat accumulation was seen with DEHP treatment which, when it occurred, was seen in the midzone and periportal zones rather than centrilobular regions. Biochemical evi

dence for peroxisome proliferation (cyanide insensitive palmitoyl CoA oxidation) occurred earlier with DEHP (after 3 days of treatment) and was approximately 7-fold higher than levels observed with DnOP. DnOP values were twice those observed in the control rats. An effect level of 1,821 mg/kg bw/day was observed in this study after 3 days of treatment. Al

though DEHP induced peroxisome proliferation more strongly, rats exposed to DnOP did show evi

dence for proliferation after longer treatment. Additional liver effects suggestive of other types of liver damage were seen with DnOP. Liver metabolism and the biochemical changes associated with peroxisome proliferation were also studied by Lake et al. ( 13 ) who treated rats with 1,000 mg/kg DnOP for 14 days. DnOP produced a marginal increase in liver weight compared with DEHP. There were no increases in peroxisomal enzyme activities at 1,000 or 2,000 mg/kg. Systemic effects were studied in groups of young ( ∼ 4

− 6 weeks old) Sprague-Dawley rats ( 15 ). Groups (10/sex) were fed DnOP at dietary concentrations of 0, 5, 50, 500, or 5,000 ppm (males: 0, 0.4, 3.5, 36.8, or 350 mg/kg bw/day; females: 0, 0.4, 4.1, 40.8, or 403 mg/kg bw/day) for 13 weeks (Table 7-1). Negative controls (10/sex) were fed basal diet and positive controls (10/sex) were fed 5,000 ppm DEHP (males: 345 mg/kg bw/day; females: 411 mg/kg bw/day). Rats were observed daily, and body weights and food intake were measured weekly. At the end of the exposure period rats were killed and necropsied. Parameters evaluated included histopathology (reproductive organs preserved in Zenker’s solution), hematology, blood chemistry, liver enzyme activity, peroxisome proliferation, and DnOP levels in tissues. DnOP exposure did not affect organ or body weight at any dose concentration. No hematological effects or testicular changes were observed. At the dose of 4.1 mg/kg bw/day, female rats experi

enced significant increases in plasma phosphate level. At 36.8 (M) or 40.8 (F) mg/kg bw/day, no ef

fects were observed and this level was designated by the authors as a NOAEL. At the highest DnOP exposure tested, 350.1 (M) and 402.9 (F) mg/kg bw/day, liver and thyroid effects were observed. Authors reported dose-related hepatic effects including anisokaryosis, nuclear hyperchromicity, ve

siculation, cytoplasmic vacuolation, nuclear endothelial prominence, and accentuation of zonation. Increases in hepatic ethoxyresorufin-o-deethylase activity were also seen in this high-dose group. Thyroid effects, observed at the highest dose of DnOP tested, included decreases in follicle size and colloid density. Serum T3 or T4 analyses for thyroid function were not performed. Plasma calcium levels were significantly increased in male rats at the high dose only. The DEHP-positive control group of rats, exposed to 345 (M) and 411(F) mg/kg bw/day, had ef

fects in the liver and thyroid with respect to severity of lesions and biochemical changes that were similar to those observed in the high-dose DnOP group. However, DEHP also induced peroxisomal proliferation, seminiferous tubule atrophy, Sertoli cell vacuolation, and decreased sperm levels. Also, numerous additional biochemical changes were observed with the DEHP-exposed rats, suchII-6Appendix IIAppendix II

II-7 as increases in plasma levels of albumin and inorganic phosphate, total protein (in female rats), and hepatic aminopyrine-N-demethylase and analine hydroxylase. DEHP also produced significant hematological effects such as increased platelet counts in rats of both sexes, increased WBC, and decreased mean corpuscular volume and decreased hemoglobin in females. Poon et al. ( 15 ) also evaluated levels of DnOP and DEHP in liver and fat. The authors stated that the findings suggest that both DnOP and DEHP are rapidly metabolized and excreted and that their distribution in body tissues is determined by the lipophilicity of the compounds. The reliability of the actual levels reported in the paper has been questioned ( 16 ) by noting the failure of mass spec

trometry to confirm the chemicals detected in tissues and the absence of analytical blanks when performing the analyses.
**2.2 Toxicokinetics**  Phthalate Moiety Toxicokinetics **Absorption** Few data are available describing the toxicokinetics of DnOP. Albro and Moore ( 17 ) dosed male CD rats by gavage with 0.2 mL DnOP and collected urine for analysis of metabolites. They recovered 31% of the dose in the urine by 48 hours. The monoester and some free phthalic acid were detected, but no parent DnOP was observed. Blood levels of the monoester, mono-octylphthalate, were mea

sured in rats following administration of 2,000 mg/kg of DnOP by gavage ( 18 ). The biological halflife in the blood was 3.3 hours with an area under the curve (AUC) of 1,066 μg·h/mL. Peak blood levels were observed at 3 hours following administration. **Biotransformation** Humans : In a study comparing the relative rates of monohydrolysis of DnOP by rat, baboon, and human gut preparations, Lake et al. ( 19 ) demonstrated that these species possess similar intrinsic esterase activity. Rates observed in human intestinal preparations were similar enough to the other species to conclude that human intestinal metabolism of DnOP would be expected to result in ab

sorption of the monoester similar to what occurs in rats. Rodents : Six dialkyl phthalates, including DnOP, were found to be metabolized to their monoesters and alcohol by enzymes present in gut tissues. It is generally accepted that orally-ingested phthalate diesters are primarily hydrolyzed by esterases in the wall of the small intestine, not by intestinal flora, and absorbed almost entirely as the corresponding monoester ( 20 ).
**Distribution** Following an oral dose of 2,000 mg/kg DnOP to rats, mono-octylphthalate was found in blood and testes in 3

− 6 hours ( 21 ).
**Excretion** Following a gavage dose of 559 mg/kg for 2 days to rats, metabolites accounting for 31% of the administered dose were found in urine. The major metabolites found in urine of rats were derived from the monoester ( 17 ).II-6Appendix IIAppendix II

II-7 Side Chain-associated Toxicokinetics n-Octanol is a metabolite of DnOP. Octanol is oxidized to the fatty acid and metabolized by the fatty acid oxidation pathway. **2.3 Genetic Toxicity**  Mixtures containing DnOP have not shown conclusive evidence of mutagenicity. Barber et al. ( 22 ) tested C6–10-phthalate, which contains approximately 20% DnOP, in the mouse lymphoma muta

tion and Balb/3T3 cell transformation assays. Negative results were obtained in the cell transforma

tion assay, but results of the mouse lymphoma mutation assay were considered equivocal due to the non-dose related increase in mutation frequency that occurred in the presence and absence of S9 metabolic activation. The ACC ( 1 ) reviewed two studies of Di(n-octyl, n-decyl) phthalate, which contains DnOP as a component. Di(n-octyl, n-decyl) phthalate was reported to be negative in the Ames test and the Chi

nese hamster ovary cell/HPRT locus assay.
**The summary for Section 2, including general toxicity, toxicokinetics, and genetic toxicity, is located in Section 5.1.2.** II-8Appendix IIAppendix II

II-9 

### 3.0 DEVELOPMENTAL TOXICITY DATA **3.1 Human Data**  There were no human data located for Expert Panel review. **3.2 Experimental Animal Toxicity**  Two studies were found, one in rats by intraperitoneal (IP) injection and one in mice by gavage. A third study examined effects of metabolite exposure in rats. Singh et al. ( 23 ) administered DnOP at 0, 5, or 10 mL/kg (equivalent to 0, 4,890, and 9,780 mg/kg based on the specific gravity of DnOP of 0.978 g/mL) by IP injection to pregnant Sprague Dawley rats (Table 7− 2). The rats, 5 per group, were dosed on gd 5, 10, and 15. The control group was un

treated or dosed with distilled water, normal saline, or cottonseed oil. Dams and fetuses were evalu

ated on gd 20. Information on maternal toxicity was not reported. Fetal body weight was reduced at both doses, and incidences of gross malformations were increased in a dose-related manner (0–2% in controls, 16% at 4,890 mg/kg, and 27% at 9,780 mg/kg). The abnormalities were predominantly missing tail, anophthalmia, twisted hind legs, and hematomas. Hardin et al. ( 24 ) evaluated DnOP in the Chernoff-Kavlock assay in CD-1 mice. The mice, 40/ group, were dosed by gavage, with 9,780 mg/kg bw/day (undiluted chemical, 10 mL/kg/day) or corn oil on gd 6–13. Dams were allowed to deliver their litters; dams and pups were terminated on pnd 3. No dams died, 39/40 had live litters, and maternal weight change was similar to controls. Litter size on pnd 0 was significantly reduced (10.2) versus the control value (11.5). Birth weight was normal as was pup survival to pnd 3. However, weight gain on pnd 1–3 was significantly re

duced (0.6 g) versus the control value (1.0 g). There was no effect on ability to produce litters, litter size, sex ratio, or pup weight or viability in F 1 and F 2 litters in a continuous breeding study in CD-1 mice. Mice were exposed to 0, 1.25, 2.5, or 5% DnOP (0, 1,800, 3,600, or 7,500 mg/kg bw/day) ( 25, 26 ). Complete details of this study are included in Section 4. Hellwig and Jackh ( 27 ) investigated the prenatal toxicity of n-octanol, a primary metabolite of DnOP, when administered by gavage to pregnant Wistar rats on days 6

− 15 of gestation. There were 6 groups studied, 8

− 10 females/group: a distilled water control, an emulsifier control, and DnOP doses of 1, 5, 7.5, and 10 mmol/kg (130, 650, 945, and 1,300 mg/kg bw/day DnOP, respectively). Dose-related symptoms of clinical intoxication of the nervous system were observed with maternal death seen in the three highest dose levels. A slight decrease in food consumption and body weight gain was also recorded at these doses. However, no effects on fetal weight, viability, or develop

mental toxicity were observed. The incidence of malformations was similar to that of controls.
**The summary for Section 3 is located in Section 5.1.3.**II-8Appendix IIAppendix II

II-9 

### 4.0 REPRODUCTIVE TOXICITY
**4.1 Human Data**  There were no human data located for Expert Panel review. **4.2 Experimental Animal Toxicity**  One rodent reproductive toxicity study was found for DnOP reported by Heindel et al. ( 26 ) (also ( 25 )) (Table 7-3). In this continuous breeding study, CD-1 (Swiss) mice, 20 pairs/dose level, 40 in controls) were fed DnOP in the diet at 0, 1.25, 2.5, or 5% (w/w). Body weights and food consumption were monitored, and these concentrations gave calculated daily DnOP consumption estimates of 1,800, 3,600, and 7,500 mg/kg bw/day. Following a week of pre-mating exposure, mice were housed as breed

ing pairs for 14 weeks. Litters born during the 14-week period were evaluated and removed so that the adult pair could continue breeding. Reproductive function was measured by determining the fertility index; litters/pair; live pups per litter; and pup sex, body weight, and gross external malformations. There were no effects on ability to produce litters, litter size, sex ratio, or pup weight or viability over five successive litters. For this protocol, when no effect on fertility was seen, the last litter from both the high dose and control group was reared, and used to evaluate fertility and toxicity of the F 1 generation. In addition to the reproductive parameters evaluated in the F 0 mice, sperm morphology, estrous cycles, and selected organ weights were evaluated in the F 1 mice. The F 0 mice were discard

ed without necropsy after weaning the last litter. The F 1 animals were mated within dose groups at sexual maturity. DnOP had no effect on indices of fertility, litter size, or pup weight or viability. The control and high-dose F 1 adults were killed and necropsied after delivery of a single litter. DnOP at 7,500 mg/kg bw/day in the diet had no effect on male body weight, but increased absolute and rela

tive liver weights and decreased relative seminal vesicles weight. Sperm indices were unchanged. In females, body weight was unchanged, while relative liver and kidney weights were increased; estrous cycle was unchanged by 7,500 mg/kg bw/day DnOP consumption. As discussed in Section 2.2, Poon et al. ( 15 ) (Table 7-1) reported a subchronic-type study of DnOP. Pubertal SD rats were exposed to DnOP in diet at doses as high as 5,000 ppm (350 mg/kg bw/day) for 13 weeks when the rats were killed and necropsied. Testes were weighed and fixed in Zenker’s solution; no sperm measures were taken. Terminal weights of whole body and testis were unaf

fected by DnOP consumption. Testis histology was normal. No reproductive effects were seen, so no LOAEL was determined. The reproductive NOAEL in this study is 5,000 ppm, ~350 mg/kg bw/day, based on lack of changes in testis weight and histology as observed by light microscopy. Confidence in the quality of the study is moderate-to-high. Confi dence that this study found the true NOAEL is moderate-to-low, because guidelines for subchronic studies do not require the examina

tion of functional reproductive effects. Foster et al. ( 28 ) gavaged 12 male Sprague-Dawley rats (70

− 90 g) with DnOP at 2,800 mg/kg bw/ day for 4 days. Control animals received the corn oil vehicle. No testicular lesions were observed in the treated animals. **Mode of Action** Following exposure to a variety of phthalate monoesters over a range of doses, germ cell detach

-II-10Appendix IIAppendix II

II-11 ment was examined in in vitro co-cultures of Sertoli-germ cells isolated from pubertal rats. Results indicate that the n-octyl monoester is ~100-fold less potent than the 2-ethylhexyl monoester in producing this effect ( 29 ). These co-culture in vitro studies suggest that DnOP produces a similar effect to other phthalates in this model system, albeit at concentrations two orders of magnitude higher. There are no in vivo data to suggest effects on either germ cells or Sertoli cells due to DnOP exposure. 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 ( 30 ), rainbow trout hepatic cytosol ( 31 ), recombinant human ERs (rhER) overexpressed in SF9 insect cells using the baculovirus system ( 32, 33 ) and rainbow trout ERs expressed in yeast ( 34 ). Triated E2 was used in the tissue cytosol binding assays while a high affinity fluorescent E2 derivative was used in the rhER binding assays. The estrogenic activity of DnOP has been exam

ined using a battery of short-term in vitro and in vivo assays. DnOP did not compete with tritiated estradiol for binding to the rat uterine cytosolic estrogen receptor ( 30 ). Selected phthalate esters have been examined in a number of in vitro gene expression assays sys

tems. The assays have used stably transfected cells ( 30 ), transiently transfected cells ( 30, 31 ), yeast based assays ( 30, 34-36 ) and vitellogenin induction in rainbow trout hepatocyte cultures ( 34 ). DnOP did not induce any activity in in vitro gene expression assays systems and did not induce re

porter gene activity in transiently transfected MCF-7 cells ( 30 ). DnOP, in contrast to the positive control estradiol, did not significantly induce a vaginal cornifica

tion response at any of the concentrations tested (20, 200, and 2,000 mg/kg) over the course of a 5-day experiment using immature and adult ovariectomized Sprague Dawley rats ( 30 ). The effects of subcutaneous injection of 10 –4 mol of DBP, BBP, and DnOP on uterine vascular permeability following a 4-hour incubation were examined in mature ovariectomized Swiss albino mice ( 37 ). Al

though no significant effect on uterine vascular permeability was reported, it is unclear whether the authors tested DnOP or DEHP. The publication states that “dioctyl phthalate” was purchased from Aldrich chemical company.

**The summary for Section 4 is located in Section 5.1.4.**II-10Appendix IIAppendix II

II-11 

### 5.0 DATA SUMMARY & INTEGRATION **5.1 Summary**  5.1.1 Human Exposure There are no known commercial uses for pure DnOP. However, DnOP constitutes approximately 20% of the commercial mixture C6–10-phthalate. This commercial mixture has a variety of home and consumer product uses including as a plasticizer for PVC used in fl ooring and carpet tile, can

vas tarps, swimming pool liners, notebook covers, traffic cones, toys, vinyl gloves, garden hoses, weather stripping, flea collars, and shoes. Uses of PVC containing DnOP with possible food appli

cations include seam cements, bottle cap liners, and conveyor belts. Dietary : In a survey of infant formulas from the UK, the level of dioctyl phthalates (DOP) other than DEHP ranged from 0.21–1.42 mg/kg ( 7 ). In a subsequent survey in 1998, DOP isomers were not targeted, but there was no evidence that they were present in any of 39 samples of infant for

mula tested ( 8 ). There was a published report in 1995 that documented the detection of DnOP in two samples of vodka at concentrations of 57 and 131 ppb ( 6 ). In a German survey ( 9 ), DNOP was detected in nutmeg at 0.02 mg/kg but DNOP levels were below the detection limit of 0.01 mg/kg in milk (breast and commercial), cream, nuts, and baby food. Dioctyl phthalate is approved for use as an indirect food additive in sealants used for food packaging ( 4 ). Exposure Estimates: Based on levels of DOP isomers (excluding DEHP) detected in baby for

mula, infant exposures to DOP isomers other than DEHP were estimated at <0.1–43 μ g/kg bw/ day at birth and <0.1

− 24 μ g/kg bw/day at 6 months of age by MAFF ( 7 ). However, there was no evidence that DOP isomers were present in infant formulas in a survey conducted 2 years later by MAFF ( 8 ). Based on production volumes of DnOP-containing compounds versus those containing DEHP, ex

posure to DnOP in the general population is likely lower than exposure to DEHP, which was esti

mated at 3

− 30 μ g/kg bw/day ( 11 ). Exposures may be higher in children due to dietary preferences and mouthing of DnOP-containing articles. Variability in food exposure estimates is possible due to the inherent variability of food eaten by individuals based on age, sex, ethnicity, time of sam

pling, and geographical locations. In occupational settings, exposure is thought to be highest in workers of flexible PVC manufacturing facilities. Based on general levels of phthalates reported, the ACC ( 1 ) estimated an occupational exposure level of 286 μ g/kg bw/workday in this activity. 5.1.1.1 Utility of Data to the CERHR Evaluation There is very limited information on exposure and exposure pathways to DnOP in humans. DnOP is not known to be produced directly for commercial use, but is a component (20%) of commercial 6

− 10 phthalate substances. 6

− 10 Phthalates are used in a variety of consumer products. 5.1.2 General Biological and Toxicological Data Data presented in this section are derived from experimental animal and laboratory studies. Hu

man data were not found.II-12Appendix IIAppendix II

II-13 **General toxicity** A 3-week dietary study ( 12, 14 ) and a 90-day dietary study ( 15 ) in rats have been conducted. Liver effects were noted when rats were fed 1,821 mg/kg bw/day for 3, 10, or 21 days, or 350 mg/kg bw/day for 90 days. Thyroid effects also were noted in rats fed 350 mg/kg/d for 90 days and 1,821 mg/kg bw/day for 21 days. No effects were observed in the testes in either study. The sub-chronic dietary NOAEL in rats is 36 (M)

− 40 (F) mg/kg bw/day.
**Toxicokinetics** DnOP is metabolized and rapidly absorbed in the gut as the monoester and primarily excreted in the urine of rats ( 17, 18, 20 ). The major metabolites found in urine of rats were derived from the monoester ( 17 ). **Genetic toxicity** DnOP has not been tested for genetic toxicity. Mixtures containing DnOP have not shown conclu

sive evidence of mutagenicity. Barber et al. ( 22 ) tested C6–10-phthalate in the mouse lymphoma mutation and Balb/3T3 cell transformation assays. 6–10-Phthalate mixture was considered equivo

cal in the mouse lymphoma mutation assay due to a non-dose related increase in mutations in the presence and absence of metabolic activation, but tested negative in the Balb/3T3 cell transforma

tion assay. According to two studies reviewed by ACC ( 1 ), di(n-octyl, n-decyl) phthalate, which contains DnOP as a component, has been reported to be negative in the Ames test and the Chinese hamster ovary/HPRT locus assay. 5.1.2.1 Utility of Data the CERHR Evaluation The data is adequate for the examination of systemic effects and identification of the liver and thy

roid as target organs. The dataset consisted of one useful study that examined systemic effects in groups of rats exposed for 90 days to multiple doses of DnOP by the oral route, a route relevant to human exposure. Levels of DnOP in the diet were verified. The evaluation included a histological examination of various organs, including reproductive organs that were fixed in Zenker’s solution. One concern is that male rats were at the pubertal stage at the start of the study and were therefore past the age of maximum sensitivity to phthalate-induced testicular damage. However, mice were exposed during prenatal development (the most sensitive period for testicular toxicity) in a continu

ous breeding study described in the reproductive toxicity section. There is adequate general toxicokinetic data for DnOP, consisting of absorption, distribution, me

tabolism, and excretion in rodents. While studies of toxicokinetics in humans have not been located, the DnOP toxicokinetic data in rodents is consistent with the large body of data on phthalates that includes data on rodents and primates. It is reasonable to assume that the DnOP rodent data are rel

evant to humans. 5.1.3 Developmental Toxicity There are no data on the developmental toxicity of DnOP in humans. Two studies, where massive doses of DnOP were administered (4,890 and 9,780 mg/kg bw/day) to rats or mice by gavage or IP injection, suggest a potential for adverse prenatal effect or effect during the perinatal period ex

pressed as death, growth retardation, and/or malformations ( 23, 24 ). However, litter size and pup weight and mortality were unaffected in a continuous breeding study where mice were exposed to dietary concentrations up to 7,500 mg/kg bw/day ( 25, 26 ). A primary metabolite, n-octanol, gave noII-12Appendix IIAppendix II

II-13 sign of developmental toxicity at doses up to 1,300 mg/kg bw/day in rats ( 27 ). This dose caused se

vere intoxication and some deaths in the dams and prompts speculation whether the DnOP rat study that administered 4,890 mg/kg bw/day ( 23 ) led to severe maternal intoxication as well. The authors were silent on maternal effects. The limited study designs do not provide a basis for comparing con

sistency of response in the two species, nor do they allow meaningful assessment of dose-response relationships and determination of either LOAELs or NOAELs with any degree of confidence. The available studies do suggest a developmental toxicity response with gavage or IP administration with very high doses. **Table 2: Summaries of NOAELs and LOAELs and Major Effects in Developmental Toxicity Studies**

**Protocol and Study NOAEL**  **(mg/kg bw/ day)**  **LOAEL (mg/kg bw/day) and Effects Developmental Ef** 
**** **fects Observed at Higher Dose**  **Levels Maternal**  **Developmental** Prenatal developmental toxicity study with IPexposure in SpragueDawley rats. 5 dams/group received 0, 4,890, or 9,780 mg/ kg bw on gd 5, 10, and 15. Fetuses were evalu

ated on gd 20. ( 23 ) Maternal: Not reported. Developmental:

None. Not reported. 4,890 ↑ Fetuses with

external malformations. ↓ Fetal weight. ↑ Fetuses with external malformations. ↓ Fetal weight.

Prenatal gavage toxic

ity screening assay in CD-1 mice. 40 dams/group received 0 or 9,780 mg/kg bw/ day on gd 6

− 13. Dams and pups evalu

ated on pnd 3 for litter size, survival, and body weight changes only. ( 24 ) Maternal: Not reported. Developmental:

None. Not reported. 9,780 ↓ Litter size on

pnd 0. ↓ Pup weight gain

on pnd 1

− 3. No higher doses.

Continuous breeding study in CD-1 mice. 20 dams/group received DnOP in feed at 0, 1,800, 3,600, or 7,500 mg/kg bw/day throughout mating and gestation. (25, 26 ) Maternal: * Developmental:

7,500 * No effects on live pups/litter or pup weight.

* Only developmental effects reported in this table. See Table 3 in Section in 5.1.4 for a description of effects in parental rats. ↑ =Statistically Significant Increase ↓ =Statistically Significant DecreaseII-14Appendix IIAppendix II

II-15 5.1.3.1 Utility of Data to the CERHR Evaluation The data set is inadequate for an evaluation of developmental toxicity. In one study, small numbers of rats (n=5/group) were exposed by intraperitoneal injection, a route that is not relevant to human exposure, and there was no information on maternal toxicity. In a screening study of mice, only a single dose was administered and there was no internal examination of offspring or dams. 5.1.4 Reproductive Toxicity There were no data located on the reproductive toxicity of DnOP in humans. The continuous breed

ing design with DnOP in mice was negative at what can only be considered massive dietary doses up to 7,500 mg/kg bw/day ( 26 ). This was not a true multigeneration study because effective evalu

ation of the second generation was not performed. This lack of effect is loosely corroborated by the dietary study in rats ( 15 ) which found no histologic effects on reproductive organs after sub-chronic exposure to concentrations as large as 350 or 403 mg/kg bw/day for males and females, respec

tively. In addition, testicular lesions were not observed in male Sprague-Dawley rats gavaged with DnOP at 2,800 mg/kg bw/day for 4 days ( 28 ). Since there are no adverse reproductive effects in any study, no LOAEL can be estimated. The reproductive toxicity NOAEL in mice is 7,500 mg/kg bw/day and in rats is 350 mg/kg bw/day. The data are sufficient to conclude that DnOP causes no detectable reproductive toxicity in adult mice at doses up to ~7,500 mg/kg bw/day. The data also find no reproductive toxicity at doses up to 403 mg/kg bw/day in a subchronic dietary study in adult rats or 2,800 mg/kg bw/day in a 4-day ga

vage study in young rats, but there are no data on functional measures of reproduction. The data are insufficient to conclude that DnOP does not cause reproductive toxicity in developing rats or mice. It can be reasonably speculated, based upon both in vivo and in vitro studies, that DnOP is certainly less potent in producing male reproductive effects than the shorter-chain phthalate congeners. Following exposure to a variety of phthalate monoesters over a range of doses, germ cell detachment was examined in in vitro co-cultures of Sertoli-germ cells isolated from pubertal rats. Results indi

cate that the n-octyl monoester is ~100-fold less potent than the 2-ethylhexyl monoester in producing this effect ( 29 ). These co-culture in vitro studies suggest that DnOP produces a similar effect to other phthalates in this model system, albeit at concentrations two orders of magnitude greater. There are no in vivo data to suggest effects on either germ cells or Sertoli cells due to DnOP exposure. DnOP did not exhibit estrogenic activity in a variety of in vitro assays ( 30 ). It did not induce a sig

nificant in vivo response in ovariectomized rats ( 30 ). The results suggest that adverse effects as a result of exposure to DnOP would not be due to estrogenic activities of this phthalate.II-14Appendix IIAppendix II

II-15 **Table 3: Summaries of NOAELs, LOAELs, and Major Effects in Reproductive Toxicity Studies**

**Protocol & Study NOAEL**  **(mg/kg bw/ day)**  **LOAEL (mg/kg bw/day) and Effects**  **Effects Observed at**  **Higher Dose Levels**  **Reproductive Systemic**  **Reproductive** Continuous breeding study with mating of high dose and control F 1 offspring. 20 pairs of CD-1 mice were fed diets with DnOP (0, 1,800, 3,600, 7,500 mg/kg bw/day) for 7 days prior to mating and during a con

tinuous 98-day mating period.* (25, 26 ) Reproductive: 7,500 Systemic:

Not identified due to limited examination of lower dose groups. None
No effect on fertility, mating, sperm or estrous cycles in F 0 or high dose F 1 rats. 7,500 ↑ Liver and

kidney weight in F 1 rats No higher doses in study

* Developmental effects are reported in Table 2 in Section 5.1.3. ↑ =Statistically Significant Increase 5.1.4.1 Utility of Data to the CERHR Evaluation Data are sufficient to indicate that oral DnOP exposures are not associated with detectable effects on reproduction at doses of up to 7,500 mg/kg bw/day in mice. Adequate numbers of mice (20 pairs/group) were exposed to multiple doses of DnOP for a sufficient duration. Feed was analyzed for DnOP levels. Reproductive function and sperm quality were assessed in the F 1 mice exposed during prenatal development; thus, mice exposed during the most sensitive age were evaluated. A concern with this study is that several postnatal maturation effects (found to be the most sensitive indicators of toxicity for other phthalates) were not evaluated. Other concerns included no reporting of histopathological effects, examination of only the F 1 mice from the high-dose group, and a lack of necropsies at the lower dose levels. **5.2 Integrated Evaluation**  There are no human data from which to judge the health effects of DnOP. Based on experimen

tal literature, including toxicity studies in rats and mice with DnOP and other structurally-related phthalates, there is a reasonable basis for assuming relevance of these data for judging potential hazard to humans. There are no data indicating that DnOP is currently used in medical devices. Exposure to DnOP results from its presence as a 20% constituent of a commercial mixture of C6

− 10 phthalates. Hu

mans would gain contact from household and consumer products. Absorption through skin from such contacts are expected to be low. Absorption into the body would result from dietary sources. Presence in food might reflect migration from food packaging and a legacy of fate and transport of phthalates into the environment. Like other phthalates, DnOP is readily absorbed from the intestinal tract as a monoester, and is rapidly metabolized and excreted. The experimental animal data are insufficient to permit a firm judgment about DnOP’s potential toII-16Appendix IIAppendix II

II-17 pose a developmental toxicity hazard to humans. Studies that suggest potential developmental ef

fects were of inadequate design for confident interpretation and effects were observed only at very high doses. A study of n-octanol, a primary metabolite of DnOP, reported severe maternal intoxi

cation without any effect on growth, viability, or development. It was noted that adequate data are available on DnOP to indicate adverse effects on liver at doses lower than doses that suggest devel

opmental toxicity. There are data to indicate that DnOP does not demonstrate estrogenic properties. There are experimental data on the reproductive toxicity of DnOP. The data indicate no effects in adult mice fed high doses (7,500 mg/kg bw/day). The data in rats, while negative at dietary doses up to 350 mg/kg bw/day in adults and gavage doses up to 2,800 mg/kg bw/day in young rats, did not assess a sufficient array of reproductive measures to be considered a complete evaluation. The data, while indicating a lack of effect, are insufficient to conclude with complete confidence that exposure by the oral route poses no hazard to adult reproduction. While there was a continuous breeding study that assessed the effects of exposure to DnOP during development on subsequent reproductive function later, the protocol did not completely assess two generations. **5.3 Expert Panel Conclusions**  There are no known commercial uses for pure DnOP. However, DnOP constitutes approximately 20% of the commercial mixture C6–10 phthalate. This commercial mixture has a variety of home and consumer product uses. DnOP is approved for use as an indirect food additive in sealants used for food packaging ( 4 ). There is very limited information on exposure and exposure pathways to DnOP in humans. To allow for an integrated evaluation of exposure and toxicity information, the Expert Panel made a conservative estimate (i.e., an overestimation) of general population expo

sure to DnOP that would be at or lower than DEHP exposures of 3–30 μg/kg bw/day (see DEHP review). A similar approach for studying occupational exposures in chemical manufacturing was done using measured DEHP levels to estimate that chemical worker exposure would be less than 286 μg/kg bw/workday. Two studies where massive doses of DnOP were administered intraperitoneally to rats or by gavage to mice suggest a potential for adverse prenatal effects or effects during the perinatal period expressed as death, growth retardation, and/or malformations ( 23, 24 ). However, litter size and pup weight and mortality were unaffected in a continuous breeding study where mice were orally exposed to concentrations up to 7,500 mg/kg bw/day ( 25, 26 ). A primary metabolite, n-octanol, gave no sign of developmental toxicity at doses up to 1,300 mg/kg bw/day in rats ( 27 ). Higher doses resulted in maternal lethality. The dataset is inadequate for an evaluation of developmental toxicity for the following reasons. The limited study designs do not provide a basis for compar

ing consistency of response in the two species, nor do they allow meaningful assessment of doseresponse relationships and confident determination of either LOAELs or NOAELs. The available studies suggest a developmental toxicity response with gavage or IP administration at very high doses. The developmental toxicity response suggests minimal concern from exposure during preg

nancy or the perinatal period. There is a single dietary exposure multigeneration study on DnOP in mice which was negative at exposures of 7,500 mg/kg bw/day ( 26 ). This lack of effect is loosely corroborated by the dietary study in rats ( 15 ) which found no histologic effects on reproductive organs after sub-chronic expo

-II-16Appendix IIAppendix II

II-17 sure to concentrations as large as 350 or 403 mg/kg bw/day for males and females, respectively. In addition, no testicular lesions were observed in young male rats that were gavage dosed with 2,800 mg/kg bw/day for 4 days. Since there are no adverse reproductive effects in either study, no LOAEL can be estimated. The reproductive toxicity NOAEL in mice is 7,500 mg/kg bw/day and in rats is 350 mg/kg bw/day. Data are sufficient to indicate that oral DnOP exposures are not associated with detectable effects on reproduction at doses of up to 7,500 mg/kg bw/day in mice. Therefore, the Panel has only neg

ligible concern for effects on the adult reproductive system. Taken together, the Expert Panel has determined that although all of the databases are limited or inadequate, the existing data do not sug

gest that DnOP is a potent developmental or reproductive toxicant in rodents. **5.4 Critical Data Needs**  The critical data needs are to determine if, and at what levels, humans are exposed to DnOP. DnOP is a significant constituent (20%) of a C6

− 10 phthalate product that has major commercial produc

tion and use. The public would be best served if data needs for the evaluation of risks to human reproduction focus on the commercial mixture that contains DnOP, rather than pure di-n-octyl phthalate. Information on the use of DnOP as a food additive would be useful in determining if there are critical data needs for pure DnOP.II-18Appendix IIAppendix II

II-19 

### 6.0 REFERENCES
1. CMA. Comments of the Chemical Manufacturers Association phthalate esters panel in re

sponse to request for public input on seven phthalate esters. FR Doc. 99-9484. Washington, DC: Chemical Manufacturers Association, 1999. 2. Staples CA, Peterson DR, Parkerton TF, Adams WJ. The environmental fate of phthalate es

ters: A literature review. Chemosphere 35:667-749 (1997). 3. ACC. Meeting with Aristech Chemical Company, 1999. 4. FDA. Code of Federal Regulations. 21 (1999). 5. MAFF. Phthalates in food. Joint food safety and standards group food surveillance informa

tion sheet, vol 1999:MAFF - UK, 1996; 9p. 6. HSDB. Hazardous Substances Data Bank. Bethesda:National Institutes of Health, 1999. 7. MAFF. Phthalates in infant formulae. Joint food safety and standards group food surveil

lance information sheet, vol 1999:MAFF - UK, 1996; 7p. 8. MAFF. Phthalates in infant formulae. Food surveillance information sheet 168: Joint Food Safety and Standards Group, 1998. 9. Pfordt J, Bruns-Weller E. Die phthalsaureester als eine gruppe von umwelt-chemikalien mit endokrienem potential: Niedersachsisches Ministerium fur Ernahrung, Landwirtschaft und Forsten, 1999. 10. ATSDR AfTSaDR-. Di-n-octylphthalate. Toxicological Profile. Prepared by: Sciences In

ternational, Inc. Under Subcontract to: Research Triangle Institute.: U.S. Department of Health and Human Services, Public Health Service., 1997. 11. 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. 12. Hinton RH, Mitchell FE, Mann A, Chescoe D, Price SC, Nunn A, Grasso P, Bridges JW. Ef

fects of phthalic acid esters on the liver and thyroid. Environ Health Perspect 70:195-210 (1986). 13. Lake BG, Gray TJB, Gangolli SD. Hepatic effects of phthalate esters and related com

pounds--in vivo and in vitro correlations. Environ Health Perspect 67:283-90 (1986). 14. Mann AH, Price SC, Mitchell FE, Grasso P, Hinton RH, Bridges JW. Comparison of the short-term effects of di(2-ethylhexyl) phthalate, and di (n-octyl) phthalate in rats. Toxicol Appl Pharmacol 77:116-132 (1985). 15. Poon R, Lecavalier P, Mueller R, Valli VE, Procter BG, Chu I. Subchronic oral toxicity of di-n-octyl phthalate and di (2-ethylhexyl) phthalate in the rat. Food Chem Toxicol 35:225-II-18Appendix IIAppendix II

II-19 239 (1997). 16. American Chemistry Council Phthalate Esters Panel. Comments on Draft CERHR Evalua

tion of Di-n-Octyl Phthalate (DnOP):ACC, 2000. 17. Albro PW, Moore B. Identification of the metabolites of simple phthalate diesters in rat urine. Journal of Chromatography 94:209-218 (1974). 18. Oishi S. Effects of phthalic acid esters on testicular mitochondrial functions in the rat. Arch Toxicol 64:143-147 (1990). 19. Lake BG, Phillips JC, Linnell JC, Gangolli SD. The invitro hydrolysis of some phthalate diesters by hepatic and intestinal preparations from various species. Toxicol Appl Pharmcaol 39:239-248 (1977). 20. Rowland IR, Cottrell RC, Phillips JC. Hydrolysis of phthalate esters by the gastro-intestinal contents of the rat. Food Cosmet Toxicol 15:17-21 (1977). 21. Oishi S, Hiraga K. Testicular atrophy induced by phthalic acid monoesters: Effects of zinc and testosterone concentrations. Toxicology 15:1997 (1990). 22. 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). 23. Singh AR, Lawrence WH, Autian J. Teratogenicity of phthalate esters in rats. J Pharm Sci 61:51-55 (1972). 24. Hardin BD, Schuler RL, Burg JR, Booth GM, Hazelden KP, MacKenzie KM, Piccirillo VJ, Smith KN. Evaluation of 60 chemicals in a preliminary developmental toxicity test. Terato

gen Carcinogen Mutagen 7:29-48 (1987). 25. Gulati DK, Chambers R, Shaver S, Sabehrwal PS, Lamb JC. Di-n-octyl phthalate reproduc

tive and fertility assessment in CD-1 mice when administered in feed. Research Triangle Park: National Toxicology Program, 1985. 26. Heindel JJ, Gulati DK, Mounce RC, Russell SR, Lamb JCI. Reproductive toxicity of three phthalic acid esters in a continuous breeding protocol. Fundam Appl Toxicol 13:508-518 (1989). 27. Hellwig J, Jackh R. Differential prenatal toxicity of one straight-chain and five branchedchain primary alcohols in rats. Food Chem Toxicol 35:489-500 (1997). 28. Foster PMD, Thomas LV, Cook MW, Gangolli SD. Study of the testicular effects and chang

es in zinc excretion produced by some n-alkyl phthalates in the rat. Toxicol Appl Pharmacol 54:392-398 (1980). 29. Gray TJ, Beamand JA. Effects of some phthalate esters and other testicular toxins on pri

-II-20Appendix II mary cultures of testicular cells. Food Chem Toxicol 22:123-131 (1984). 30. 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). 31. Jobling S, Reynolds T, White R, Parker MG, Sumpter JP. A variety of environmentally per

sistent chemicals, including some phthalate plasticizers, are weakly estrogenic. Environ Health Perspect 103:582-587 (1995). 32. Bolger R, Wiese TE, Ervin K, Nestich S, Checovich W. Rapid screening of environmen

tal chemicals for estrogen receptor binding capacity. Environ Health Perspect 106:551-7 (1998). 33. 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). 34. 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). 35. Coldham NG, Dave M, Sivapathasundaram S, McDonnell DP, Connor C, Sauer MJ. Evalua

tion of a recombinant yeast cell estrogen screening assay. Environ Health Perspect 105:734- 742 (1997). 36. Harris CA, Henttu P, Parker MG, Sumpter JP. The estrogenic activity of phthalate esters in vitro. Environ Health Perspect 1997 105:802-811 (1997). 37. Mulligan SR, Balasubramanian AV, Kalita JC. Relative potency of xenobiotic estrogens in an acute in vivo mammalian assay. Environ Health Perspect 106:23-26 (1998).Appendix II

II-21 

### 7.0 TABLES
7.1 DnOP General Toxicity, Rats

............................................................... .................................. 22 7.2 DnOP Developmental Toxicity, Rats

............................................................... ....................... 23 7.3 DnOP Reproductive Toxicity, Rats

............................................................... .......................... 24 7.4 References

............................................................... ............................................................... 25II-22Appendix IIAppendix II

II-23Table 7-1: DNOP General Toxicity, Rats**Species, Strain, and** **Source Experimental** **Regimen Animal** **Number Dose** ***Body Weight** **Organ Weight** **Histopathology Hematology** **Chemistry Other** SpragueDawley RatPoon et al.1997(1)Young male andfemale rats werefed diets containingDNOP for 13 weeks,then killed and necropsied. A positivecontrol group wasexposed to DEHP.100100.40NENENENENE103.5(M)4.1(F)NENENENE↑PO4(F)1036.8(M)40.8(F)NENENENENENOAEL10350.1(M)402.9(F)NENEMild lesionsin liver. Thyroid folliclereduction anddecreased colloid density.No peroxisomeproliferationor testicularlesions.NE↑EROD↑Ca (M)10345(M)411(F)[DEHP]NE↑Li, Ki(M)↓TeTesticular atrophy, liver andthyroid lesions,and peroxisomalproliferation.↑WBC (F),PC↓Hb (F),MCV (F)↑Alb, PO4,Ca (M),protein (F),APD, AH*Doses measured in mg/kg bw/day.NE=No Effects↑=Statistically Significant Increase↓=Statistically Significant DecreaseF=FemaleM=MaleKi=KidneyLi=LiverTe=TestesAlb=AlbuminAPD=Aminopyrine-N-Demethylase ActivityAH=Aniline HydroxylaseCa=CalciumEROD=Ethoxyresorufin-O-deethylaseHb=HemoglobinMCV=Mean Corpuscular VolumePO4=PhosphatePC=Platelet CountWBC=White Blood CellII-22Appendix IIAppendix II

II-23Table 7-2: DNOP Developmental Toxicity, Rats**Species, Strain, and Source** **Experimental Regimen Animal** **Number Dose** ***Maternal effects Fetal Effects** Sprague-DawleyRatSingh et al. 1972(2)Prenatal developmental toxicitystudyDNOP administered by intraperitoneal injection on gd 5, 10, and15. Dams killed on gd 20, corporalutea counted and implantationsites examinedFetuses weighed, examined forviability and gross external malformations. 30−50% of fetusesexamined for skeletal malformations.5054,890Not mentioned in paper↓Fetal weight.↑External malformations(16% fetuses with grossabnormalities)59,780↓Fetal weight.↑External malformations(27% fetuses with grossabnormalities)*Doses measured in mg/kg bw/day.↓=Statistically Significant Decrease↑=Statistically Significant IncreaseII-24Appendix IIAppendix II

II-25Table 7-3: DnOP Reproductive Toxicity, Mice**Species, Strain, and** **Source Experimental Regimen** **Number a** **Dose b** **Effects**CD-1 MiceHeindel et al.1989Gulati et al.1985(3, 4)Dose range finding study.Fertility assessment through continuousbreeding for 14 weeks. DNOPadministered in feed.Body weight measured at 6 time points,clinical signs, and food and waterintake recorded.Litters counted, sexed, weighed,observed for abnormalities, andremoved following birth.Final litter raised; some control andhigh-dose F1weanlings mated forfertility assessment; F1organ weightsmeasured at necropsy.0−10,000Rough hair coat in high-dose group360201,800NE183,600NE207,500No adverse effects on sperm morphology, estrouscycles, or other reproductive parameters in F1rats.No effect on fertility index, mating index, numbersof litters produced, live pups/litter, sex ratio, or pupweight.↓Percent abnormal sperm in F1rats↓Seminal vesicle to body weight ratio in F1rats↑Liver and kidney (females) to body weight ratio inF1ratsaNumber of male and female pairs; half the number of controls used for F1studybAuthor-calculated doses (in mg/kg bw/day) based on male mice.NE=No Effect↑=Statistically Significant Increase↓=Statistically Significant DecreaseII-24Appendix IIAppendix II

II-25 

### References 1. Poon R, Lecavalier P, Mueller R, Valli VE, Procter BG, Chu I. Subchronic oral toxicity of di-n-octyl phthalate and di (2-ethylhexyl) phthalate in the rat. Food Chem Toxicol 35:225- 239 (1997). 2. Singh AR, Lawrence WH, Autian J. Teratogenicity of phthalate esters in rats. J Pharm Sci 61:51-55 (1972). 3. Heindel JJ, Gulati DK, Mounce RC, Russell SR, Lamb JCI. Reproductive toxicity of three phthalic acid esters in a continuous breeding protocol. Fundam Appl Toxicol 13:508-518 (1989). 4. Gulati DK, Chambers R, Shaver S, Sabehrwal PS, Lamb JC. Di-n-octyl phthalate reproduc

tive and fertility assessment in CD-1 mice when administered in feed. Research Triangle Park: National Toxicology Program, 1985.Appendix III 

# PUBLIC COMMENTS **ON THE PHTHALATES EXPERT PANEL REPORTS**  

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