EAP on DEHP in Medical Devices MDB Report: An Exposure and Toxicity Assessment

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A presentation of the report entitled "DEHP in Medical Devices" was made to the Expert Advisory Panel April 26, 2001. The presentation notes which summarize the technical document are available in both official languages. Unfortunately, the full technical document is available in English only.

Medical Devices Bureau
Therapeutic Products Directorate
Health Products & Foods Branch
Health Canada

July 2001
Revised: February 2002

Prepared by: Irwin Hinberg, Ph.D, Head, Criteria & Assessment,
Research & Surveillance Division, Medical Devices Bureau
Therapeutic Products Directorate

EXECUTIVE SUMMARY

This report reviews the scientific and medical literature on the toxicity of
di(2-ethylhexyl) phthalate (DEHP), assesses the available information on human exposure to DEHP from medical procedures, and evaluates the specific concerns raised about the safety of DEHP. It identifies the medical procedures associated with the highest exposures and judges whether these exposures pose a significant risk to patients undergoing them. The report does not determine whether the health risks from DEHP exposure outweigh its benefits.

There are very few human data from which to characterize the toxicity of DEHP. Therefore, the evaluation of human risk from medical exposures (mostly intravenous procedures) must be extrapolated from studies in experimental animals (mostly studies of toxicity following oral exposures), where species differences in metabolism are important considerations. Uncertainties exist in the performance of such extrapolations. The report has identified toxicologic end points in animals and determined their relevance to humans based on exposure information (the route, duration, and amount) and other relevant factors (e.g., metabolism and toxicologic mechanism).

Several important exposures in neonates and in the fetus have not been reported in the scientific literature. These include exposures from parenteral and enteral feeding, ventilators, combinations of simultaneous exposures, and placental transfer of maternally derived DEHP/ MEHP from medical procedure. The report provides estimates of these exposures based on theoretical calculations. The qualitative risk assessment in the report does not depend on the accuracy of these estimates.

There are no data on the carcinogenicity of DEHP or its major metabolites in humans. DEHP induces liver tumors in rats and mice when administered in the diet at high doses. The International Agency for Research on Cancer (IARC) recently concluded that the mechanism by which DEHP increases the incidence of hepatocellular tumors in rats and mice (peroxisome proliferation) is not relevant to humans and declared DEHP as "not classifiable as to carcinogenicity to humans." The report accepts these conclusions.

There are no data on the reproductive and developmental toxicity of DEHP or its major metabolites in humans. The report concludes that oral exposure to DEHP can cause reproductive and developmental toxicity in rodents. Testicular toxicity is the main adverse effect observed in most rodent species. Analysis of the data obtained with rats suggests a NOAEL of 3.7 -14 mg/kg/day for testicular toxicity for oral exposure and a NOAEL of 60 mg/kg/day for testicular toxicity from intravenous exposure. The report notes that the available data are inadequate for the reliable estimation of a NOAEL based on malformations in the reproductive tract of the fetus during gestational exposure, which may be the most sensitive end point for developmental and reproductive toxicity of DEHP. Based on a review of the toxicokinetics of DEHP in rodents and primates and possible mechanisms of action of DEHP-inducing reproductive toxicity, the report concludes that the rodent data are relevant to predicting that DEHP has the potential to produce adverse reproductive effects in humans. However, in terms of risk assessment, route of exposure and dose are important parameters which need to be taken into account when extrapolating across species.

The report reviews reports of adverse effects in humans exposed to DEHP and concludes that the studies are insufficient in design and outcome to demonstrate any cause-effect relationship between human exposure to DEHP and the occurrence of toxicity.

The report concludes that significant data gaps, including insufficient dose-response data on adverse effects on the reproductive tract caused by gestational exposure and the uncertainties in extrapolating animal data to humans, make it impossible to obtain a quantitative estimate of human risks associated with DEHP exposure from medical devices.

The report identifies sub-populations (e.g, dialysis patients) who have long-term exposures to relatively high doses of DEHP and others, such as infants and the developing fetus, who are exposed to relatively high doses at the most vulnerable periods of development.

The report concludes that young children, particularly critically ill infants undergoing certain medical procedures, represent a population at increased risk for the adverse effects of DEHP because of increased exposure on a mg/kg basis relative to adults, and because of potentially increased sensitivity to some of the adverse effects of DEHP. Extracorporeal membrane oxygenation, the medical procedure that results in exposure of patients to the highest levels of DEHP to patients on a mg/kg basis, is used almost exclusively on newborn infants. Parenteral exposures can approach the NOAEL for intravenous exposure in rats.

PREFACE

In the early 1970s, a number of studies reported that
di(2-ethylhexyl) phthalate (DEHP), a plasticizer found in a wide variety of medical devices made from polyvinyl chloride, leached out of the devices during use. The studies identified DEHP and its metabolites in the blood and other tissues of patients. Numerous studies have shown that in rodents, DEHP causes cancer of the liver, developmental and reproductive toxicity, nephrotoxicity, and pulmonary toxicity. The relevance of these finding to humans continues to be debated, despite three decades of research into DEHP toxicity and human exposure.

Studies conducted by Health Canada in 1988-89, concluded that the levels of DEHP and its metabolites found in blood or blood components that were stored or processed in PVC bags or tubing did not represent a significant health risk. This finding was confirmed by a number of independent studies. In fact, several studies suggested that the low levels of phthalates that leached out blood bags could protect red blood cells during their prolonged storage.

On November 27, 1998, Health Canada issued an advisory regarding children's toys manufactured with phthalates. As a result, the media and some advocacy groups raised concerns about the safety of DEHP in medical devices. This report has been prepared in response to these concerns.

In preparing this report, the Medline and Toxline databases were searched to find the medical and scientific reports on human exposure to DEHP, particularly from medical procedures, animal and in vitro studies of the toxicity, toxicokinetics and mechanism of toxicity of DEHP, and reports of adverse effects in humans. The databases included over 1000 publications that meet these criteria. The toxicity of DEHP, particularly its carcinogenicity and reproductive and developmental toxicity in experimental animals, has been reviewed in several recent publications (T: 1-9). In particular, the Expert Panel on Phthalates of the Center for Evaluation of Risks to Human Reproduction, National Toxicology Program (NTP-CERHR), US Department of Health and Human Services, recently published a comprehensive review of the reproductive and developmental toxicity of DEHP (T: 2). Through its membership on panel, the Medical Devices Bureau actively participated in the NTP-CERHR review. Part of this report's review of DEHP exposure was originally prepared for the NTP-CERHR review. In addition, the report's review of reproductive and developmental toxicity generally reflects the position of the NTP-CERHR review, although Health Canada did conduct an independent review.

The report is not exhaustive. Instead, it identifies and critically reviews the key scientific papers dealing with different aspects of exposure and toxicity to: identify the critical toxicity endpoints; estimate the no-observed-adverse-effect-levels (NOAEL) or lowest-observed-adverse-effect-levels (LOAEL); assess the relevance of the findings in animals for patients exposed to DEHP from medical procedures; identify data gaps in current knowledge; and assesses whether exposure to DEHP from medical procedures may cause adverse health effects in humans. The report does not determine whether the health risks from DEHP exposure outweigh its benefits.

The first draft of this report was released in July, 2001 together with a request for written comments from all interested persons. The only written comments received were from the Phthalate Esters Panel of the American Chemistry Council. The revised report takes these comments into account.

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Table of Contents

EXECUTIVE SUMMARY
PREFACE

1. EXPOSURE TO DEHP IN MEDICAL DEVICES

1.1. Introduction

1.1.1. PVC Used in Medical Devices
1.1.2. Properties of DEHP
1.1.3. DEHP-Containing Medical Devices

1.2. Levels of DEHP Extracted from Medical Devices

1.2.1. DEHP in Blood and Blood Products
1.2.2. DEHP Extracted by IV Solutions
1.2.3. DEHP Extracted by Peritoneal Dialysis Solution
1.2.4. DEHP Extracted from Medical Tubing

1.2.4.1. DEHP Extraction from Cardiopulmonary Bypass Tubing
1.2.4.2. DEHP Extraction from Infusion Lines during TPN Administration
1.2.4.3. DEHP Extraction from Infusion Lines during Enteral Feeding
1.2.4.4. DEHP Extraction from Infusion Lines during Drug Delivery
1.2.4.5. DEHP Extraction from Respiratory Tubing

1.2.4.5.1. Heated Respiratory Tubing (Adults)
1.2.4.5.2. Heated Respiratory Tubing (Neonates)
1.2.4.5.3. Oxygen Supply Tubing (Adults)
1.2.4.5.4. Oxygen Supply Tubing (Neonates)
1.2.4.5.5. Tracheal or Tracheotomy Tubes (Adults)
1.2.4.5.6. Tracheal or Tracheotomy Tubes (Neonates)

1.3. Estimates of Human Exposure

1.3.1. Introduction
1.3.2. Adult Exposures

1.3.2.1. Long-Term Adult Exposures

1.3.2.1.1. Hemodialysis
1.3.2.1.2. Continuous Ambulatory Peritoneal Dialysis
1.3.2.1.3. Long-Term Transfusions
1.3.2.1.4. Long-Term Total Parenteral Nutrition

1.3.2.2. Short-Term Adult Exposures

1.3.2.2.1. Short-Term Transfusion of Blood Components
1.3.2.2.2. Extracorporeal Membrane Oxygenation
1.3.2.2.3. IV Infusion of Drugs

1.3.3. Neonatal Exposures

1.3.3.1. Replacement Blood Transfusions
1.3.3.2. Extracorporeal Membrane Oxygenation
1.3.3.3. Total Parenteral Nutrition
1.3.3.4. Respiratory Therapy

1.3.3.4.1. Ventilation using Heated Respiratory Tubing
1.3.3.4.2. Oxygen Therapy
1.3.3.4.3. Respiratory Therapy using an Endotracheal Tube

1.3.3.5. Prenatal Exposure

1.3.4. Multiple Exposures
1.3.5. MEHP 31
1.3.6. Conclusions - Human Exposure

2. DEHP TOXICITY

2.1. Introduction
2.2. Absorption, Distribution, Metabolism and Excretion of DEHP
2.3. General Toxicity

2.3.1. Acute Toxicity
2.3.2. Subchronic and Chronic Toxicity

2.4. Genotoxicity

2.4.1. Gene Mutations in Bacteria, Fungi and Drosophila

2.4.2. in vitro Studies of Mammalian Cells

2.4.3. in vivo Studies
2.4.4. Evaluation

2.5. Carcinogenicity

2.5.1. Experimental Data

2.5.2. Evaluation
2.6. Reproductive and Developmental Toxicity

2.6.1. Reproductive Toxicity

2.6.1.1. Introduction
2.6.1.2. Adverse Reproductive Effects
2.6.1.3. Species Differences in Sensitivity to Reproductive Toxicity
2.6.1.4. Age-Differences in Vulnerability to Reproductive Toxicity
2.6.1.5. Mechanism of Reproductive Toxicity
2.6.1.6. LOAEL and NOAEL for Reproductive Toxicity

2.6.1.6.1. Oral Exposure
2.6.1.6.2. Parenteral Exposure

2.6.2. Developmental Toxicity

2.7. Adverse Human Health Effects

2.7.1. Pulmonary Toxicity
2.7.2. Peritoneal Sclerosis in Patients on Peritoneal Dialysis
2.7.3. Liver Cholestasis in Infants on ECMO
2.7.4. Cardiovascular Toxicity
2.7.5. Nephrotoxicity
2.7.6. Summary

3. CONCLUSIONS

3.1. Exposure
3.2. Toxicity
3.3. Relative Risks

3.3.1. Adults
3.3.2. Critically Ill Infants and Young Children
3.3.3. Pregnant Women
3.3.4. Recommendations

APPENDIX 1
REFERENCES

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List of Tables

Table 1a: Physical Properties of DEHP
Table 1b. Best Estimate of Vapour Pressure of DEHP based on Menzel, 1997
Table 2. Typical Formulation of a PVC Blood Bag
Table 3: Manufacturer's recommended application for blood bags sold in Canada
Table 4: DEHP in Selected Blood Components Under Similar Conditions
Table 5: Blood Levels of DEHP measured Before and After Selected Medical Procedures
Table 6: Reported DEHP Exposures by Medical Procedures (Adults)
Table 7a. Daily Dose of DEHP Delivered by Hemodialysis
Table 7b. Estimated DEHP Received from Total Parenteral Nutrition
Table 8: Reported DEHP Exposures in Neonates
Table 9: MEHP Exposure from Medical Procedures
Table 10. Daily Exposure of Patients and the General Population to DEHP
Table 11. Acute Toxicity of Single Dose of DEHP
Table 12. Hepatocellular Tumours Reported in the NTP Study
Table 13a. LOAEL and NOAEL for Reproductive Toxicity of DEHP in Rodents
Table 13b. Developmental Toxicity of DEHP and its Metabolites Following Oral Exposure
Table 14. Alternatives to DEHP-Plasticized PVC

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1. EXPOSURE TO DEHP IN MEDICAL DEVICES

1.1. Introduction

1.1.1. PVC Used in Medical Devices

A wide variety of medical devices are made of polyvinyl chloride (PVC) or contain PVC components. A 1992 survey (Blass et al., 1992) found that PVC had the largest market share - 25% - of all polymers used in medical devices in Western Europe, the U.S. and Japan. PVC resins are hard, brittle compounds due to the strong attraction between hydrogen and chlorine atoms of adjacent polymer chains. PVC used in medical devices therefore contains a relatively high proportion (20-40%) of plasticizer, which provides it with desired mechanical properties. These include: flexibility, strength, suitability for use at a wide range of temperatures and a variety of sterilization processes, resistance to kinking, optical clarity, weldability, barrier capability, and bondability. Plasticized PVC can also be softened and shaped into many configurations without cracking or leaking, which is of great importance in many medical device applications.

The added plasticizer consists of a polar ester group and a linear part.It is not chemically bound to the PVC polymer but, instead, exists in the PVC matrix in a semi-solid or gel-like structure. The ester group of the plasticizer binds to the hydrogen and chlorine atoms of the PVC polymer through Van der Waals forces, while the linear part acts as a buffer between PVC polymer chains, increasing the distance between them. This weakens the binding between adjacent polymer chains, imparting much greater flexibility and other desirable physical properties to the polymer material.

A number of high-boiling-point phthalate esters and other chemicals such as adipates, citrates and trimellitates have been used to soften PVC. However,
di(2-ethylhexyl) phthalate (DEHP) is the plasticizer of choice for PVC-based medical devices because it is best able to provide medical devices with their desired mechanical properties.

Figure 1. Chemical Structure of Di(2-Ethylhexyl) Phthalate.

1.1.2. Properties of DEHP

Di(2-ethylhexyl) phthalate (DEHP) is produced by reacting 2-ethylhexanol with phthalic anhydride in the presence of acid or metal catalyst at high temperature. Figure 1 shows the chemical structure of DEHP.

DEHP is a colorless liquid with a slight odor. It is insoluble in water, miscible with mineral oil and hexane, and soluble in most organic solvents. DEHP readily dissolves in body fluids such as saliva, blood and plasma. Some of its physical properties are listed in Table 1a, while Table 1b lists the vapour pressure at different temperatures.

Table 1a: Physical Properties of DEHP

Table 1b. Best Estimate of Vapour Pressure of DEHP based on Menzel, 1997

Many values have been given for the solubility of DEHP in water, ranging from 1 to 340 g/L ( Boese, 1984; Howard, 1985; Staples, 1997; Staples, 2000; Scholz, 1997; Parkerton, 2000). According to the latest Kow (octanol water partition) values measured by HPLC (Staples, 1997), the water solubility for DEHP is 0.18-2.8 g/l.

It is now generally accepted that these lower values represent the true water solubility of phthalates and that the higher ones reflect the ability of phthalates to form relatively stable and homogeneous dispersions in water under laboratory conditions (Staples, 1997; Staples, 2000).
A wide range of values has been reported for the vapour pressure of DEHP. Values for 25 °C range from about 10-6 to 10-3 Pa (Howard et al., 1985; Montgomery and Welkom, 1990; Menzel,1997; Cousins and MacKay, 2000). Many of the reported values may have been obtained with impure DEHP. Menzel showed that interference from impurities produces inaccurately high values and measured the vapour pressure of 99.5% pure DEHP at temperatures ranging from 60 °C to 216 °C.

Several recent reviews of DEHP have extrapolated the Menzel's results to obtain estimates of vapour pressures for temperatures below 60 °C. Extrapolating from 60 °C (the lowest temperature studied by Menzel (1997) introduces considerable uncertainty into the vapour pressure estimates for
25 °C and 37 °C. Furthermore, the extrapolation methods used in the different reviews were not identical, resulting in significant differences 150% in the values reported. The estimates obtained from the best fit of Menzel's experimental data using TableCurve 2D appear to be the most reliable and are the ones reported in Table 1b.

PVC-based medical devices generally contain 20-40% DEHP by weight. For a 500 mL blood bag, this represents approximately 10 g of DEHP. In addition to the plasticizer, a number of other additives, including stabilizers, fillers, colorants, and lubricants may be added to the polymer formulation to ensure that the final product meets specific requirements. Since PVC is the most widely used plastic in medical devices, standards have been adopted for PVC formulations used in medical applications (ASTM, 1993; European Pharmacopoeia). A typical formulation is shown in Table 2.

Table 2. Typical Formulation of a PVC Blood Bag

Since the DEHP in PVC is not chemically bound to PVC, it can leach out when a PVC-containing medical device comes in contact with fluids such as blood, plasma, and drug solutions, or it can be released when the device is heated. The rate at which DEHP and other plasticizers migrate from the medical device depends on the storage conditions: temperature of the fluid in contact with the device; the amount of fluid in contact with the PVC; the contact time; the extent of shaking or flow rate of the fluid; and the lipophilicity of the fluid. Higher temperatures and shaking increase the migration rate. Since DEHP is lipophilic, it will readily be extracted by lipid-containing fluids such as whole blood, plasma, platelet concentrate, IV lipid emulsions, total parenteral nutrition solutions, and solutions containing Polysorbate 80, and other formulation aids used to solubilize some intravenous medications. DEHP is extracted more rapidly into these types of fluids than into non-lipid containing solutions, such as the saline priming solutions used for hemodialysis and extracorporeal membrane oxygenation.

In establishing the safety of PVC-based medical devices, it is essential to estimate the extent of DEHP leaching during medical use. In contrast to DEHP, tri-2 (ethylhexyl) trimellitate (TEHTM), an alternative to DEHP used in platelet bags, is very insoluble in plasma. Its higher molecular weight, higher boiling point and different molecular structure (with increased stearic hindrance) all contribute to its lower rate of extraction from medical devices.

1.1.3. DEHP-Containing Medical Devices

DEHP is the primary plasticizer used in the manufacture of a wide variety of medical devices. These include: blood and blood-component storage bags, intravenous solution containers, and administration sets, respiratory tubing, extracorporeal membrane oxygenation therapy tubing sets, continuous ambulatory peritoneal dialysis (CAPD), hemodialysis sets, autopheresis sets, nasogastric feeding tubes, catheters, enema packs, needle hubs, urology products, medical gloves, masks and oxygen tents. Other hospital equipment, including basins, bed pans, specimen collection bags, and inflatable splints, are also made with PVC; however, these devices do not come in contact with breached tissue or blood.

Medical procedures with PVC-containing devices such hemodialysis, transfusion of whole blood, platelets or plasma, extracorporeal oxygenation, cardiopulmonary bypass, and administration of intravenous fluids exposes the patient to DEHP via the intravenous route; respiratory therapy (e.g., artificial ventilation) exposes the patient to DEHP via the inhalation route; and nasogastric tubes expose the patient to DEHP by ingestion.

PVC-free or phthalate-free alternatives exist for many uses of PVC in medical devices. For example, there are currently five types of blood bags on the Canadian market. The first three are blood bags made of polyvinyl chloride plasticized with either DEHP, tri-2 (ethylhexyl) trimellitate (TEHTM) or
butyryl-tri-n-hexyl-citrate (BTHC). DEHP and TEHTM are phthalate derivatives, while BTHC is a citrate plasticizer. The other two types are made of different materials, polyolefin and ethylene vinyl acetate (EVA), both of which have no added plasticizer. The application of the different types of bags depends on the characteristics and properties of each of the plastics. The applications recommended for each type is summarized in Table 3.

Table 3: Manufacturer's recommended application for blood bags sold in Canada

 

Currently, both Canadian blood operators [Canadian Blood Services (CBS) and Héma Québec (HQ)] use blood bags and plasmapheresis bags made of PVC-DEHP, manufactured by Med-Sep and Haemonetics, respectively, and plateletpheresis bags made of PVC-BTHC, manufactured by Cobe.

1.2. Levels of DEHP Extracted from Medical Devices

1.2.1. DEHP in Blood and Blood Products

There are extensive data on the levels of DEHP extracted into blood, blood components, and blood derivatives. The mean levels of DEHP reported in blood or blood products ranges from 0 to 650 mg/mL, depending on storage conditions and blood product. Table 4 lists selected data on DEHP levels in various blood products.

Table 4: DEHP in Selected Blood Components Under Similar Conditions1

1 Expressed as the mean (range) and/or ± SD
2 Abbreviation: ACD, Acid Citrate Dextrose; CPD, Citrate Phosphate Dextrose; AHF, anti-haemophilic factor; FP, frozen plasma; NSA, normal serum albumin; PPR, plasma protein factor.

The wide variability in DEHP levels reported in Table 4 can be explained by the differences in storage temperature, duration of storage, the source of the blood product, levels of lipids in the blood and plasma, and the analytical methods used to measure DEHP.

Freezing blood, such as red blood cells or plasma components during storage, prevents the migration of DEHP (Contreras et al, 1974; Vessman and Rietz, 1974; Rock et al. 1986). When blood products are frozen shortly after collection, such as for fresh frozen plasma (less than 8 hours after collection) or RP15 (recovered plasma frozen within 15 hours of collection), minimal amounts of DEHP leach into the collected material. As expected, Table 4 shows that products manufactured from frozen plasma, which is a requirement for derivatives sold in Canada, contain little or no DEHP and only low levels of its metabolite, mono-ethylhexyl phthalate (MEHP).

As shown in Table 4, Contreras et al. (1974) reported that levels of DEHP in whole blood nearing the end of its recommended storage shelf-life (42 days at 4^oC) may exceed 600 mg/mL. However, patients requiring a transfusion generally will receive RBC, platelets, or fresh frozen plasma, rather than whole blood, which is rarely administered. Reported levels of DEHP in RBCs range from 4 to 123 mg/mL.

The table also shows that levels of DEHP exceeding 540 mg/mL have been reported in stored plasma (Dine, 1991). However, this may not be important clinically, since fresh frozen plasma is currently used instead of unfrozen stored plasma. Reported levels of DEHP in fresh frozen plasma range from 6 to 27 mg/mL.

Since DEHP is converted to mono(2-ethylhexyl) phthalate (MEHP) by plasma esterases (Albro and Thomas, 1973a; Peck et al., 1979), blood and blood products contaminated with DEHP may also contain MEHP. The source of plasma has a direct and significant impact on the levels of DEHP and MEHP.

Platelets and other blood components stored in bags made from the PVC-free or phthalate-free alternatives listed in Table 3 contain no DEHP and, depending on the alternative selected, zero levels or reduced levels of other plasticizers. The following results have been reported.

PVC-TEHTM. TEHTM is 100-fold less leachable than DEHP. Very low levels of TEHTM have been reported in platelet concentrates (0 to 0.2 mg, 5 days , 22^oC, shaking) (Simon, 1983; Gerlai et al., 1987; Chawla and Hinberg, 1992) and red blood cells (not detectable stored at 4^oC) (Carmen, 1993) stored in PVC-TEHTM bags.

PVC-BTHC. BTHC leaks into plasma at a level 60 to 70 percent less than DEHP (Simon, 1983; Snyder et al. 1992; Carmen, 1993) . Platelet concentrates stored for 7 days have been reported to contain 20 mg of BTHC per unit PVC (Simon, 1983; Carmen, 1993).

Polyolefin plastic contains no added plasticizers in polyolefin plastic. Although it contains the the anti-oxidant (BHBB), this substance was not detected in platelet concentrates after 5 days of storage at 22 ^oC and shaking (Rock et al, 1986; Labow et al, 1986). DEHP has been reported in platelet concentrates collected and stored in polyolefin bags; however, its presence was linked to a contaminated sterilization unit, not to the actual composition of the plastic (Labow et al, 1986).

EVA plastic contains no added plasticizer. There have been no reports of plasticizers detected in blood components stored in EVA bags.

The current practice in Canada is to store platelets in DEHP-free bags. Patients receiving transfusions of platelets may nevertheless be exposed to DEHP as a PVC infusion set is used to administer the platelets. (See below).

1.2.2. DEHP Extracted by IV Solutions

Since DEHP has very low solubility in water - less than 3 µg/L at 20^oC (Table 1a) - very little PVC will leach into normal saline solutions from PVC storage bags even after long periods of storage (Dine et al., 1991). However, several studies have found that if normal saline and glucose solutions in PVC bags are agitated, DEHP may from an emulsion, increasing the amount of DEHP extracted into the solutions (Smistad et al., 1989, Roksvaag et al. 1990, Holllifiel, 1959, Howard et al. 1985, Defoe et al. 1990). Values of 285 µg/L (Holllifiel, 1959), 340 µg/L (Howard et al. 1985), and 360 µg/L (Defoe et al. 1990) have been reported for water and 160 µg/L for saline (Howard et al. 1985), suggesting that DEHP has lower solubility or emulsion formation capacity in saline than in water.

A variety of drugs are administered intravenously by adding them to PVC intravenous bags. The rate at which DEHP is extracted from the bags into the drug solution depends on the hydrophobicity of the drug formulation.

Pharmaceutical solvents such as ethanol and polyethylene glycol do not affect the extraction of DEHP from PVC storage bags (Pearson and Trissel, 1993). In contrast, a variety of formulation aids, including Polysorbate 80 (Tween 80) and castor oil (Cremophor EL), dramatically increase the rate of DEHP extraction (Pearson and Trissel, 1993, Trissel, 1998,). For example, according to one report (Pearson and Trissel, 1993), 3.1 to 237 mg of DEHP leached into a litre of Polysorbate 80 solution stored in a PVC bag for one day at 24⁰C.

Several studies (Venkataramanan et al, 1986, Mazur et al., 1989, Pearson and Trissel, 1993, Mazzo, et al., 1997, Trissel, 1998) have identified a variety of drug formulations that significantly increase the extraction of DEHP from the PVC container into the solution. These drugs include:

Cefoperazone (Cefobid Bulk)
Chlordiazepoxide HCL (Librium)
Ciprofloxacin (Cipro IV)
Cimetidene (Tagamet)
Cyclosporine (Sandimmune)
Etoposide (VePesid)
Fluconazole (Diflucan)
Metronidazole HCl (Flagyl IV)
Micronazole (Monistat IV)
Paclitaxel (Taxol)
Tacrolimus (Prograf)
Taxotere (Docetaxel)
Teniposide (Vumon)
Total parenteral nutrition formulations
Vitamin A

One study (Venkataramanan et al 1986) reported that the concentration of DEHP in a Cyclosporine solution in 5% dextrose stored in a PVC bag at room temperature reached 31 µg/mL after 8 hr and 104 µg/mL after 24 hr of shaking.

The highest DEHP concentrations are reached when the drugs are pre-mixed in the IV bags and the pre-mixed solution is agitated for 24 hr.

More recently, Faouzi et al. (1999) found that pre-mixing and storage of teniposide in PVC bags for 48 hours at room temperature extracted 52 mg DEHP from the bags. These investigators also found that DEHP released from the administration sets could contribute significantly to the total amount of DEHP infused into the patient. These findings have been confirmed in a study by Loff et al. (2000), which simulated the infusion of midazolam, fentanyl, and propofol in neonates. This study is discussed in greater detail in Section 1.2.4.4.

Clinicians and nurses appear to be familiar with the increased leaching of DEHP from PVC containers into lipophilic drug formulations. Pearson and Trissel (1993) have recommended that these drug formulations should be prepared in non-PVC containers and administered through non-PVC tubing. The labelling of such formulations includes a warning to that effect. For example, the directions for use for Taxol include the following warning:

"Data collected for the presence of the extractable plasticizer DEHP
[di(2-ethylhexyl) phthalate] show that levels increase with time and concentration when dilutions are prepared in PVC containers. Consequently, the use of plasticized PVC containers and administration sets is not recommended."

Several brands of multi-layer bags (polypropylene on the outside, nylon in the middle, and polyethylene on the inside) are available as an alternative to PVC bags for the storage and use in the administration of IV drugs. These bags are not manufactured with DEHP. However, Sarbach et al. (1996) recently reported phthalate levels ranging from 128 to 149 µg/mL in saline that had been stored in one brand of trilaminate bag. The observation that the phthalate came from the polyurethane adhesive suggests that the phthalate was not DEHP but phthalic acid, which may have formed by the hydrolysis of phthalate anhydride, the curing agent for the polyurethane adhesive.

Total parenteral nutrition (TPN) formulations contain amino acids, dextrose, electrolytes and lipids. TPN solutions are administered within 24-36 hours of mixing. The presence of lipids has been shown to increase extraction of DEHP from PVC bags (Mazur et al. 1989). In TPN formulations without added lipids, there was no measurable amount of DEHP. In TPN formulations with added lipids, the concentration of DEHP in the TPN solution increases with time and storage temperature. Mazur et al. (1989) found that the highest concentration of DEHP reached within this time was 3.1 µg/mL. In contrast, Allwood et al. (1986) had previously reported that the concentration of DEHP in a TPN solution containing 20% intralipid reached 40 µg/mL after 24-hr storage in a PVC bag.

According to Dickerson et al. (1997), the majority of clinicians in the US use DEHP-free containers (non-DEHP plasticized PVC or polyolefin) to mix and deliver TPN containing lipids. Polyethylene syringes are generally used for pre-mature and newborn infants. Although PVC-free infusion sets are available for this purpose, infusion lines are almost always made of PVC and are required for pump-assisted lipid administration, which is the procedure used for neonates and pediatric patients.

1.2.3. DEHP Extracted by Peritoneal Dialysis Solution

In this procedure only aqueous solutions come in contact with PVC and, therefore, relatively small amount of DEHP are extracted. Nassburger et. al (1987) found levels of DEHP in peritoneal dialysis solution ranging from 0.004 to 0.011 µg/mL, while Mettang (1996) reported levels between 0.021 to
0.130 µg/mL in the dialysis solution. Both studies reported much higher levels of MEHP, mono(2-ethylhexyl) phthalate, a major metabolite of DEHP, which is more toxic than DEHP. Assuming a total of 8 L fluid/day, these results suggests that the total amount of DEHP delivered to the patient should not exceed (0.130 mg/L) x 8,000 L/day (~ 1 mg/day). The actual amount of DEHP absorbed by the patient should be less, since some of the DEHP will remain in the perfusate that is drained from the peritoneum and discarded.

1.2.4. DEHP Extracted from Medical Tubing

PVC tubing is used in a large number of medical applications such as haemodialysis, extracorporeal membrane oxygenation, cardiopulmonary bypass procedures, IV delivery of TPN emulsions, enteral feeding, and mechanical ventilation of infants and adults. Easterling et al. (1974) reported the extraction by plasma of up to 10 mg of DEHP from PVC infusion sets over a period of 5 hr. Loff et al. (2000) recently reported that perfusion of platelet rich plasma through PVC tubing used in transfusions extracted 13.9 µg DEHP/mL, which correspond to a total of 4.9 mg for the transfusion of 1 unit (350 mL) of platelet rich plasma, while fresh frozen plasma extracted 24.9 µg DEHP/mL, corresponding to a total of 8.7 mg DEHP/unit. in vitro experiments reviewed in Huber et al. (1996), indicate that DEHP is extracted in amounts varying from 1.5 to 6.0 mg/hour from dialysis tubing (Ono et al., 1975; Lewis et al., 1978; Gerstoft et al., 1980; Sjoberg et. al., 1985a; Nassberger et al.,1987). Levels as high as 3.5 - 4 mg/L have been reported to be extracted per hour during extracorporeal membrane oxygenation (Schneider, 1989). However, Karle et al. (1997) reported much lower extraction rates during extracorporeal membrane oxygenation and no detectable levels of DEHP extracted from heparin-coated tubing. This study suggests that extracorporeal membrane oxygenation using heparin-coated tubing would expose the patient to little or no DEHP. The wide variability in reported rates of DEHP extraction from medical tubing is due to differences in composition, length and surface area of the tubing used, flow rate, temperature, and the composition of the infusate. As a result, the results obtained with one medical procedure in one medical centre generally cannot be extrapolated to other procedures carried out in other centres.

1.2.4.1. DEHP Extraction from Cardiopulmonary Bypass Tubing

Cardiopulmonary bypass is used in a number of cardiac surgical procedures, including heart valve replacement, coronary bypass graft surgery, correction of congenital heart defects, and heart transplants. Since a large amount of PVC tubing is used for the heart-lung bypass circuits, DEHP extraction from the tubing may represent a significant source of DEHP exposure. There are no published reports on rates of DEHP extraction from cardiopulmonary bypass tubing. However, Barry et al. (1989) have measured the total dose of DEHP delivered to the patient in a variety cardiac bypass procedures. The total dose reported by Barry et al. in Table 6, e.g., 15.4 - 72.9 mg/procedure for coronary artery bypass graft procedures, includes the DEHP extracted from the containers used to store the blood products transfused into the patient and the DEHP extracted from the cardiopulmonary bypass tubing.

1.2.4.2. DEHP Extraction from Infusion Lines during TPN Administration

Although DEHP-free tubing is available for gravity infusion of TNP emulsions, PVC tubing is generally required for the pump-assisted administration of TPN emulsions that contain lipids. The amount of PVC extracted from the tubing will depend on the total surface area of the tubing. Since the surface area is about the same for an adult as for an infant, the infant would receive a higher dose of DEHP/kg bw than an adult.

There are very limited data on the extraction of DEHP from PVC infusion lines during TPN procedures. Easterling et al. (1974) reported that up to 10.2 mg DEHP could be released from an infusion set in 5 hours. However, the total amount of DEHP released over the 24-hour perfusion period was not measured. More recently, Loff et al. (2000) measured the extraction of DEHP from PVC infusion lines by lipids under conditions typical for newborn intensive care units. They found that a TPN formulation containing 20% lipids extracted 10.2 mg DEHP from the infusion tubing in one day. (Total volume infused = 20 mL).

1.2.4.3. DEHP Extraction from Infusion Lines during Enteral Feeding

Many critically-ill patients, particularly patients in nursing facilities or at home, receive nutritional support by enteral feeding instead of parenteral nutrition. PVC enteral feeding tubes are often used in this procedure, although polyurethane tubing is also available for this purpose. Exposure to DEHP from enteral infusions has not been studied and published data on the rate of DEHP extraction from PVC tubing used in other medical applications cannot be used to obtain accurate exposure estimates. However an order of magnitude estimate of the contribution of the PVC enteral tubing to DEHP exposure may be obtained by combining the data of Loff et al. (2000) on the leaching of DEHP from PVC tubing exposed to a 20% lipid mixture plasma with the data obtained by Khaliq et al. (1992) on the decrease in rate of DEHP extraction with time.

The perfusion line used by Loff et al. (2000) was 225 cm long. The authors did not report the internal diameter. However, in response to a request for addition information, the authors provided us with additional information about the perfusion lines used, which suggests that the inner diameter of the parenteral infusion line used was 0.11 cm (the inner diameter of the NutrakittuqTM brand we examined. The surface area of the tubing exposed to the lipid mixture would then be 77.9 cm² (i.e., 2 x 3.1459 x 0.055 cm x 225 cm) and therefore, DEHP was extracted at an average rate of 131 µg/cm² /day during the first day. However, Khaliq et al. (1992) found that the rate of extraction averaged over fifteen days was approximately 25% that of the first day. Therefore, the average rate of release of DEHP from a typical PVC enteral feeding tube (length = 100 cm; inner diameter = 0.53 cm; inner surface area = 166 cm²) kept in place for 15 days is estimated to be

0.25 x (131 µg/cm² /day) x 166 cm² x 0.001 mg/ g = 5.4 mg/day.

This estimate is associated with considerable uncertainty in view of the absence of experimental data for enteral feeding.

1.2.4.4. DEHP Extraction from Infusion Lines during Drug Delivery

Faouzi et al. (1999) and Loff et al. (2000) recently reported that DEHP released from PVC administration sets may contribute significantly to the total amount of DEHP infused into the patient during IV infusion of drugs. In a simulated study of the infusion of propofol for low dose sedation of neonates, Loff et al. (2000) found that 6.5 mg DEHP were extracted from the PVC administration set.

1.2.4.5. DEHP Extraction from Respiratory Tubing

Some components of breathing circuits used both in hospitals and at home, notably oxygen supply and humidifier tubing, possibly some heated respiratory tubing (see below), suction catheters, and endotracheal tubes are commonly made from PVC plasticized with DEHP. However, there are no reliable published data on the rate of extraction of DEHP from these devices under clinical conditions of use. The rate will depend on the following factors.

1. The leaching of DEHP from the internal surface of respiratory breathing systems will be influenced by the flow rate through the tubing, and this will vary between individuals. For example, although the standard adult quietly breathes 500 mL (Tidal Volume) at a rate of 12 breaths per minute (Respiratory Rate) resulting in a mean flow of 6 L/min (Minute Volume = Tidal Volume x Respiratory Rate), a newborn would have a mean flow of about 1/10th of this value. Tidal Volume per body mass is nearly a constant (e.g. 7.8 mL/kg for an adult male and 7.1 mL/kg for a neonate), so there is a wide variation in the tidal volume between a 70 kg male and a 3 kg neonate. Since the respiratory rate also varies with age, the mean flow rates (Minute Volume) will vary considerably between individuals, and thus their exposure to DEHP. Furthermore, the above values relate to individuals breathing spontaneously. However, because of the need to overcome equipment deadspace, tidal volumes are higher during controlled ventilation of apatient requiring ventilatory assistance. Furthermore, physiogical conditions may require higher tidal and minute volumes (depending upon the disease state necessitating ventilatory care), so that tidal volumes of 12 mL/kg are typical during controlled ventilation (and Minute Volume settings on ventilators can exceed 10 L/min). Consequently, the mean flow rate through respiratory tubing, and thus the amount of DEHP released, can be expected to vary over a wide range between individuals and the mode of ventilation. In considering adult exposure, it is convenient to think of a mean flow rate of 5 to10 L/min, as being representative, and a minute flow rate of 10 L/min as useful for estimating worst-case scenarios.

2. A more important consideration in the leaching process would be the variation in respiratory rate associated with the normal minute volumes of 5 to 10 L/min. Since minute volume is simply the tidal volume times the respiratory rate, the same minute volume can be achieved with a respiratory rate that varies by a factor of 3 as long as the tidal volume varies inversely by the same amount. It is not unusual to see high respiratory rates
(e.g. >=30 bpm) in adults with a correspondingly shallow breathing (tidal volume).

The significance of this to the leaching process, is that the tidal volume determines the flow velocity in the respiratory tubing, and the flow velocity will influence the convective mass transfer process at the corrugated tube surface. This is a complicated fluid process that is usually not amenable to good theoretical calculations (not only is the tube corrugated but it is bent and twisted in use resulting in non-axial flow components) and would normally require empirical data. However, the process might be simplified by thinking of the two extremes in flow rates as allowing turbulent (high flow - high tidal volumes and low respiratory rates) versus more laminar (low flow - low tidal volumes and high respiratory rates) flow conditions. Under turbulent flow conditions the boundary layer is quite small so that the vapour pressure gradient between the core flow and the tube surface (with corrugations) is maximal to that under laminar flow conditions where the flow boundary layer would be much greater. Consequently, there will be a higher mass transfer of material from the tube surface (particularly in the corrugations which act as little pools) to the main channel flow under turbulent or high flow conditions than under the laminar or low flow conditions. With a respiratory rate of 3 to 1, for the same tidal volume, clinically one would expect a marked difference in the amount of leached product between these two extremes.

Finally, the ventilator inspiratory to expiratory ratio of the respiratory cycle will also influence the flow velocity in the tube (i.e. 900 mL tidal volume in 1 sec versus 1.5 sec for a mean flow of 54 L/min versus 36 L/min). Consequently, the flow conditions at the tube surface relevant to the mass transfer of leached product can vary by an order of magnitude, even though the same minute volume is maintained.

3. The diameter of adult size tubing is uniform amongst various manufacturers. However, the length of the tubing can vary from 1.5 to 1.8 meters (and down to 1.2 meters with pediatric or infant sizes), so that the surface area associated with leaching would vary by about 20 %. Apart from flow velocity in the tube, the surface area will clearly influence the total mass transfer of DEHP.

4. Heated versus unheated tubes will obviously influence the mass transfer of DEHP from the tube surface, because of the difference in vapour pressure. Estimates of vapour pressure using a temperature of 37 °C are a better approximation of the elevated temperatures in the circuit than the use of room temperature. Heated respiratory circuits are maintained at an elevated temperature to minimize the condensation of the humidified airstream from the humidifier along the tube (rain out), and the objective is to maintain the temperature at the patient end at about 34 °C to 37 °C (a thermistor at the patient end provides feedback to maintain temperatures at these levels. However, it is important to recognize that it is the bulk air flow in the tube that is being maintained at the elevated temperature (to prevent rain out). Thus, while the core flow temperature would be maintained at this elevated temperature, there will be a thermal gradient between the core flow and the room air outside the tube. The actual wall temperature will fall somewhere between the core flow at 34 °C or 37 °C and the room temperature. The flow boundary layer on the inside of the tube to that outside will determine the actual temperature of tube, which is relevant to the vapour pressure determination of DEHP at the tube surface. In principle, the boundary layer outside the tube will be governed by free convective conditions in contrast to the inside of the tube, where forced convection occurs. Consequently, there should be much greater thermal resistance on the outside of the tube than that on the inside, so that the wall temperature should be higher than the
27 °C temperature that would represent the midway point of the thermal gradient (34 + 20)/2 if the thermal resistance on the inside and outside were equal. These values need to be determined empirically, as the theoretical predictions are subject to considerable errors from the assumptions that would be made.

Using 37 °C as a worst case scenario is a reasonable assumption. However, clinically the amount of DEHP leached will be influenced by the actual wall temperature of the tube, because of the influence on vapour pressure. In the same context, the influence of respiratory rate and flow velocity on the inner boundary layer, as noted in 2 above, will also influence the inner thermal resistance and thus the temperature of the tube. Consequently, this would also increase the variation in product leached from the tube.

1.2.4.5.1. Heated Respiratory Tubing (Adults)

These coaxial tubes are used when long term ventilator support is necessary and are connected to a ventilator, which provides the pneumatic supply for positive pressure breathing. The inspiratory limb delivers fresh gas to the patient during inspiration, and the expiratory limb effectively opens up to allow flow during expiration.

Although peak flow rates created by the ventilator can exceed 60 L/min, this only occurs at the peak of the inspiratory cycle. In the inspiratory leg this flow cycle only occurs during patient inspiration, so that the flow rate drops to zero during expiration and the respiratory pause before the next breath. Consequently the mean flow rate through the tubing is simply the tidal volume times the number of breaths per minute, which is known as the minute volume. This is the flow involved for mass transfer. Flow in the expiratory leg occurs from passive expiration and results in lower peak flows but has the same mean flow through a respiratory cycle.

The worst-case scenario involves breathing air saturated with DEHP continuously for 24 hours/day. The amount of DEHP received by the patient would be given by

Daily mass transfer of DEHP (mg/day) =

Concentration of DEHP (mg/L) x Mean Flow Rate (L/min) x min/day

Using a typical value of 10 L/min for respiratory breathing circuits and assuming a vapour pressure of 4.8x10^-4 Pa at 37 °C (Table 1b) we obtain the following estimate of the maximum amount of DEHP extracted from heated respiratory tubing during mechanical ventilation of an adult.

For flow at patient end of the circuit, the fraction of DEHP, assuming fully saturated, would be 4.8 x 10^-9 (1 atm = 10⁵ Pa). Thus, for a minute flow of
10 L/min, the minute volume of DEHP is

4.8x10^-9 x 10 L/min = 48 x 10^-9 L/min

The daily volume is therefore

48 x 10^-9 L/min x 60 x 24 min/day = 6.9 x 10^-5 L/day

Since the MW = 390.57, and the standard volume at 37 °C is
25.4 L (i.e. 22.4 x (1+37/273)), then the corresponding mass is
6.9 x 10^-5 L/day x 390.57/25.4 gm/L= 1.06 x 10^-3 gm/day = 1.06 mg/day.

We would have to have supersaturation or different vapour pressures to achieve much higher values.

As the humidified air enters the beginning of the tube (from the humidifier), the partial pressure of DEHP will be zero, so that a maximum gradient will exist to allow the diffusion of DEHP across the boundary layer. As this bolus of air migrates along the tubing and collects DEHP, the partial pressure of DEHP will rise, reducing the vapour pressure gradient between the core flow and the tube surface. Consequently, the beginning of the tube should provide the primary mass transfer of DEHP to the core flow, with the patient end of the tube contributing little, if the core partial pressure approaches the vapour pressure at 37 °C. Consequently, it is difficult to imagine a circumstance in which the above value of 1.06 mg/day would not represent the maximum inspired by the patient.

The amount of DEHP extracted from anaesthesia breathing circuits would be similar, since these circuits are functionally equivalent to respiratory breathing circuits and similar minute flow volumes.

1.2.4.5.2. Heated Respiratory Tubing (Neonates)

Respiratory standards for a newborn utilize a tidal volume of 20 mL and a respiratory rate of 36 for a minute volume of 720 mL. However, flow through the respiratory circuit is typically 2 L/min with the valve closing in the expiratory limb for a sufficient time to achieve the desired tidal volume and respiratory rate, while the remaining flow is ventilated to the room. The tubes are shorter, typically about 1.2 meters, resulting in a surface area of 0.4 to 0.7 that of the adult. The internal diameter is half that of the adult tubing, or about 1 cm, so that the cross sectional flow area of the infant tube would be only 1/4 that of the adult. Consequently, the mean flow velocity would be about
4 times higher than that in the adult circuit, for the same volume flow rate. However, the peak flow rates for an infant ventilator would typically be 2 to
4 L/min (the maximum pediatric setting is usually about 20 L/min), whereas 40 to 60 L/min would be common for adults (since maximum flow rates of
120 L/min are typical with some as high as 300 L/min). Thus, in comparison with the pre-term infant study results, we would expect an adult to have volume flow rates of about 20 times that of the infant, which would imply a mean flow velocity in the adult tube of about 5 times that of the infant. Consequently, one would expect a smaller boundary layer thickness in the adult circuit in comparison with the infant circuit, which would suggest that greater quantities of DEHP should be leached from the adult circuit per unit area of surface. Given the larger surface area of the adult circuit (perimeter is 2 x infant and length is about 1.5 x infant), the surface area difference alone would suggest about 3 times as much DEHP leaches from the adult circuit as from the infant circuit. And unless the flow conditions in the infant circuit already allow for maximum diffusion of DEHP across the boundary layer (so that the thinner boundary layer in the adult circuit has no impact), one would expect the higher flow velocities in the adult circuit to add to the amount of DEHP convected away from the tube surface (particularly the corrugations).

In the worst-case scenario, the amount of DEHP extracted per day would be

(1/3) x 6.9 x 10^-5 L/day x 390.57/25.4 gm/L = 0.35 mg/day.

Roth et al. (1988) studied five pre-term infants who were ventilated using heated respiratory tubing and humidified air. They reported that concentrations of DEHP in the condensate collected from the water traps of the respirator tubing that ranged from <0.001 to 4.1 mg/L Based on these values, Roth et al. (1988) estimated that the infants could have received inhalation doses of DEHP ranging from 1 to 4.2 mg DEHP/hour These reported DEHP concentrations in the condensate appear to have been serious overestimates due to a sampling error. Furthermore, DEHP levels in the condensate cannot provide a reliable estimate of inhalation exposure since the infants were not exposed to the condensate. In particular, the results obtained by Roth et al., 1988, may not be provide a good estimate of the exposure to DEHP during mechanical ventilation of infants for the following reasons.

1. The study was conducted in 1988, when the use of unheated tubing would have been typical of that period of time. The respiratory tubing used by Roth et al. does not appear to be the type that uses heating wires to maintain temperature throughout the inspiratory limb. Rather, the humidifier gas temperature was in the 50-60 °C range, so that the temperature would have dropped along the tube length until reaching 30 °C to 32 °C at the patient end (i.e. at the endotracheal tube). This could be significant in terms of the amount of DEHP leached, as there would be temperature gradient of about a 20 °C along the length of tube. Thus, the machine end, attached to the humidifier output, would initially be in the 50-60 °C range, so that the tube surface would be initially be closer to these temperatures with a correspondingly higher vapour pressure for "evaporating" the DEHP at the tube surface. If it were possible to achieve saturation of the air bolus at these higher temperatures (at the machine end) then one would expect some condensation of the DEHP along with the water vapour along the tube length as the temperature dropped. This would be expected to influence the amount DEHP collected in the water trap, depending on location. Thus, the circuit used by Roth et al. (1988), would favor higher concentrations of leached DEHP than would be typical with the current practice of using heated tubing with an effectively lower mean wall temperature.

2. The paper notes that the water trap was collected sporadically 4 or 5 times during the 24-hour period. Since the time interval between sampling would have been about 5 or 6 hours, the impact of preferential evapouration of water from the trap during this time interval would have to be considered. Since the DEHP would have a much lower vapour pressure than water vapour, depending upon the temperature and dynamics, one could envisage selective evapouration of the water vapour from the trap such that the concentration of DEHP in solution could increase above that percentage which is condensing. Possible cycling in temperature of the humidifier might influence this process.

3. Another significant factor may arise from the fact that most of the water condensation occurring along the walls of the tubing drains under gravity towards the water trap (to avoid injury to the patient). Thus, the DEHP could be leaching into this water condensate and then draining towards the trap without actually being airborne.

Consequently, it is likely that the levels of DEHP measured in the water traps could be artificially high relative to the levels inhaled by the infants.

Further studies would obviously be needed to validate the findings of Roth et al. (1988), since this is the only study to report on DEHP levels in a mechanical ventilation system with a heated respirator tubing system and humidified air and is based on a limited number of observations. However, the type of circuit used by Roth et al. (1988), has been replaced in current practice by the use of heated tubing with a lower mean wall temperature. Furthermore, respiratory tubing used with ventilators is currently made from polyethylene. Health Canada recently confirmed this by testing the thirteen leading North American brands.

1.2.4.5.3. Oxygen Supply Tubing (Adults)

This tubing is used to deliver oxygen from a flow meter that is attached to an oxygen regulator connected to a gas cylinder, or with the flow meter directly attached to a hospital's central gas pipeline system through an outlet on the wall. This tubing would have the widest application in respiratory therapy, since any patient receiving some supplemental oxygen would have this tubing attached to a face mask or nasal prongs. The population exposure from this source significantly exceeds the cases where breathing assistance is necessary (i.e. ventilators with breathing circuits and tracheal or tracheotomy tubes). This tubing is used not only be used in the hospital but also in patients homes attached to an oxygen cylinder, an oxygen concentrator, or liquid oxygen supplies (with or without humidification), as well as in mobile applications with small gas cylinders.

The oxygen flow rates through this tubing range from 2 L/min to 15 L/min, depending upon the level of support necessary to maintain blood oxygen levels within normal range. Values of < 6 L/min are typical of the more common applications.

The worst-case scenario (exposure to DEHP saturated air at 37 °C and a flow rate of 15 L/min for the duration of the procedure), would result in a DEHP exposure of 15/10 the DEHP exposure from heated PVC respiratory tubing; i.e., 1.6 mg DEHP/day.

1.2.4.5.4. Oxygen Supply Tubing (Neonates)

This tubing is rarely used for neonates, except for resuscitation. The worst case estimate of the amount of DEHP extracted per day would be 1/3 the amount extracted during adult use; i.e., 0.5 mg/day.

1.2.4.5.5. Tracheal or Tracheotomy Tubes (Adults)

These devices provide the interface between the patient airway and the breathing circuit or oxygen supply tubing.

The flow rates through these would be the same as that for breathing circuits (i.e. at the minute volume flow rate required by the patient), and the only exception is that flow is bidirectional. Whereas the oxygen supply tubing and respiratory circuit only has gas flow in one direction, the tracheal tube will be exposed to flow during both inspiration and expiration. Consequently, although the minute flow rate is the same as for circuits, say 10 L/min, if DEHP was fully absorbed in the patients lungs, then the same minute flow would contribute to a loss during expiration. However, from the concept of patient exposure to DEHP, it is only the inspiratory flow segment that is relevant, leading to the same worst case estimate of DEHP exposure as for breathing circuits, i.e., 1.1 mg/day.

1.2.4.5.6. Tracheal or Tracheotomy Tubes (Neonates)

Latini et al. (1999) recently measured the DEHP content of neonatal endotracheal tubes before and after use and reported a loss of
0.06-0.12 mg DEHP per mg sample after use. This would represent a loss of up to 60-120 mg DEHP for a typical 1 g endotracheal tube, i.e.,
11- 22 mg DEHP per day, since the average length of the procedure was 129.5 hrs. (Additional details obtained from the authors.)

This study cannot be used to provide an accurate estimate of neonatal exposure to DEHP from the endotracheal tubes since an unknown proportion of the DEHP extracted by air and mucus would not reach the infants lungs. For example, some of the DEHP extracted by mucus would be removed by suctioning during the procedure. This potentially important source of exposure needs to be further studied in a clinical setting.

The worst case estimate of the amount of DEHP delivered to a neonate by respiratory air stream would be 1/3 the amount delivered to an adult; i.e.,
0.5 mg/day.

DEHP could also be extracted from the endotracheal tube into respiratory tract mucus and this may, in fact be the major source of DEHP exposure from endotracheal tubes. There are no published data on the rate of extraction of DEHP from PVC tubing by mucus. However, the worst case estimate of the amount of DEHP extracted by mucus and other body fluids from a neonatal endotracheal tube could be calculated from the surface area of the tube, which is known, and an estimate of the rate of extraction of DEHP from PVC by mucus, which is unknown.

The neonatal endotracheal tubes used by Latini had an inner diameter of
0.25 cm, an outer diameter of 0.4 cm and a length of 8 cm. Therefore, the total surface area is

2 ðrh = 8 x 2 x 3.1459 x [(0.4/2) + 0.25/2] = 17.6 cm².

Therefore, based on the data of obtained by Loff et al. (2000) for the rate of extraction of DEHP from PVC infusion lines by a 20% lipid solution, the amount of DEHP extracted by mucus from neonatal endotracheal tubes should not exceed

131 g/cm² /day) x 17.6 cm² x 0.001 mg/ g = 2.3 mg.

Some of the DEHP extracted by mucus would likely be removed by suctioning during the procedure instead of being swallowed.

Therefore, the maximum amount of DEHP extracted daily from an endotracheal tube would be equal to (DEHP delivered by respiratory air stream ) + (DEHP delivered by mucus); i.e., (0.5 +2.3) mg/day = 2.8 mg/day, which is much less than the 11 - 22 mg/day reported by Latini et al. (2000).

1.3. Estimates of Human Exposure

1.3.1. Introduction

Exposure of the general population to DEHP occurs from food, water and air, via inhalation and ingestion. Occupational exposure to DEHP occurs during the manufacture and processing of this compound. Patients are exposed to DEHP during a wide variety of medical procedures that use medical devices made of PVC. The highest acute and chronic human exposures occur in infants undergoing intensive therapeutic procedures.

The largest general population exposure to DEHP is dietary, followed by indoor air. Few studies on indoor air exist, but exposures may be measurable in µg/m³. Dietary exposures result from both bioaccumulation in certain foods as well as from leaching of DEHP during processing, packaging and storing. Food surveys show a range of DEHP content, but fatty foods including dairy, fish, meat and oils contain the most. (Meek, 1996; Huber, 1996).

The total estimated daily intake is highest in children age 0.5 - 4 and all children less than age 19 are estimated to have higher overall exposures to DEHP from all sources than adults. A 1996 survey by Health Canada (NHW, 1996) suggests that exposure to DEHP from food has decreased significantly since 1989 to about 8 µg/kg bw/day for children age 1- 4 and 2 µg/kg bw/day for adults age 20-70. However, dietary estimates will differ from country to country because DEHP is introduced through various food processing and packaging techniques which differ internationally. The range of exposure in the general population from all sources (food, air, water, plastic objects) excluding medical and occupational is estimated to be 3 - 30 µg/kg-bw/day. (CEPA, Priority Substances List assessment Report, 1994; Huber et al, 1996 Doull, 1999).

The US Centre for Disease Control (CDC) has measured monoester metabolites of seven commonly used phthalates in urine samples from a reference population of 289 adult humans (Blount, 2000). An analysis of the results using two different methodologies showed that for DEHP, the average exposure was under 1 µg/kg bw/day, the 95^th percentile exposure was under 4 µg/kg bw/day, while the maximum exposure was 46 µg/kg bw/day
(Kohn, 2000, David 2000). More recently, the CDC reported urine monoester concentrations in 1024 individuals (CDC, 2001). The data suggest a median DEHP exposures of 0.7 µg/kg bw/day, and a 90th percentile exposure of
2.1 µg/kg bw/day.

Occupational exposure to DEHP occurs during the manufacture and processing of this compound. Workers may be exposed to relatively high concentrations during the compounding of DEHP with PVC resins (ASTDR, 1993). The major route of occupational exposure is inhalation (Doull, 1999). Maximum occupational exposures should not exceed 0.7 mg/kg-bw/day, if the workplace air concentrations meet the OSHA standard.

The major route of DEHP from medical procedures is intravenous, through infusion of blood, blood products or lipid solutions, hemodialysis, or other bypass procedures. In these circumstances, patients are exposed to both DEHP and its monoester metabolite, mono(2-ethylhexyl) phthalate (MEHP). DEHP migrates from the PVC bag during storage and from the tubing used during infusion and MEHP is formed by the metabolism of DEHP by blood during storage. Exposure to DEHP may also occur by inhalation (e.g., ventilators) and by ingestion (e.g., nasogastric tubes).

DEHP exposure from medical procedures is highly variable and determined by the handling of the devices during storage and use, and the fluids that come in contact with the medical device. Long storage or time of use, increased temperature, and agitation all increase the leaching of DEHP from medical devices. Leaching is also enhanced by increased lipid content or by the lipophilic nature of liquids that contact DEHP in medical devices. Unanticipated exposure may result from infusing parenteral drugs or infusates like lipid solutions, through DEHP-containing infusion sets against manufacturer specifications. Variability in reported exposure estimates can result from the analytical techniques used to measure DEHP, the care with which contamination from analytic equipment is prevented, and the methods used to estimate the total dose received by the patient.

Exposures to DEHP from medical procedures can be short-term (e.g., a single blood transfusion), chronic (e.g., hemodialysis), or sub-chronic (e.g., ECMO). Chronic or recurrent treatments like hemodialysis in chronic renal failure patients or multiple long-term transfusions in cancer victims can result in cumulatively high exposures. Intensive procedures such as exchange transfusions in neonates can result in acutely high exposures.

The magnitudes of the exposures are highly variable and have not been extensively studied. The most reliable studies of DEHP exposure measure delivered dose using area under the curve calculations for hemodialysis (Pollack et al., 1985; Faouzi et al., 1999) and exchange transfusion (Sjoberg et al., 1985b). However, most exposures estimates reported in the literature are based on spot measurements of DEHP in the patient's blood or calculated from the published rates of DEHP leaching from the medical apparatus. Neither approach is as accurate as the AUC measurement approach. A number of important exposures, including exposures from long-term transfusion of blood and blood components, parenteral and enteral feeding, breathing circuits, or combinations of simultaneous medical procedures have not been measured in vivo. Published estimates of DEHP exposures from these procedures must therefore be based on data on the rates of extraction of DEHP from the devices used in the procedures and scenario assumptions.

Exposure calculations not based upon in vivo measurements are the least accurate, since they are subject to large errors arising from uncertainties in leaching rates and scenario assumptions. For example, using data from Rubin (1976) on DEHP in pooled platelets, twice a week platelet transfusions in a pancytopenic patient could result in total exposure over 6 months from a low estimate of 2.7 grams to a high estimate of 8.6 grams. Exposure estimates will be increased by assuming long storage time, room or body temperature storage or use, and high range concentrations or large volume procedures, and will correspondingly be lowered by assuming the opposite. The variability in measured DEHP concentrations, number of parameters that can affect leaching, and numerous variables in clinical use make precise determination of exposures impossible. However, the estimates are useful for order of magnitude comparisons of DEHP exposures from different medical procedures.

Table 5, adapted from the NTP-CERHR on DEHP (CERHR, 2000), shows published data on measured blood levels of DEHP and MEHP before and after selected medical procedures.

Table 5: Blood Levels of DEHP measured Before and After Selected Medical Procedures

 

1.3.2. Adult Exposures

Adults may be exposed acutely or chronically to DEHP. Table 6 summarizes published exposure estimates for adults based on spot measurements of DEHP in blood or delivered doses using AUC calculations. Estimates of exposures associated with other medical procedures based on rates of extraction of DEHP and scenario assumption, although much less reliable than in vivo data, need also be considered.

Table 6: Reported DEHP Exposures by Medical Procedures (Adults)

 

1 Expressed as the mean (range) and/or ± SD
2 Based on extrapolation from one hr result
3 Dose retained by patient
4 AUC = Area Under Curve; CABG = Coronary Artery Bypass Graft;
CAPD = Continuous Ambulatory Peritoneal Dialysis

It is useful to distinguish between chronic (long-term) and acute (short-term) and adult exposure.

1.3.2.1. Long-Term Adult Exposures

These include exposures from hemodialysis, continuous ambulatory peritoneal dialysis (CAPD), transfusions of blood and blood products to patients with leukemia, aplastic anemia, sickle cell anemia or clotting disorders, administration of TPN therapy to critically ill patients, and enteral nutrition of critically ill patients.

1.3.2.1.1. Hemodialysis

Hemodialysis is the procedure which results in the highest cumulative dose of DEHP because of the nature the procedure and the frequency and long-term duration of the treatment. The most reliable numbers come from studies that involve the calculation of the differences from AUC of pre- and post-infusion measurements of DEHP over the 3-5 hour hemodialysis session.

The mean DEHP doses reported per hemodialysis session ranged from 30 to 105 mg, with an average mean value of about 75 mg. The mean value of 30 mg/day is from Lewis et al. (1978), which is an older study in which the results were not corrected for incomplete extraction nor for DEHP absorbed during dialysis. Faouzi et al. (1999) recently reported an average of 75 mg DEHP (Range: 44 - 197 mg) was infused into a patient during a dialysis session. However, an average of only 16.6 mg DEHP (range: 3.6 - 59.6 mg) was retained by the patient.

As shown in Table 6, doses reported for individual patients ranged from 3.6 to 360 mg/session. The wide range of estimates may be due to patient variation (e.g., hematocrit, triglycerides and serum cholesterol), differences in dialysis protocols, and differences in the circuits themselves. All of these studies, though carefully conducted, are based upon small patient numbers, which further complicates the reliability of exposure estimates.

Assuming three dialysis sessions per week, the doses reported in the three studies using direct measurements and AUC calculations, correspond to the following daily doses for a 70 kg patient.

Table 7a. Daily Dose of DEHP Delivered by Hemodialysis

1.3.2.1.2. Continuous Ambulatory Peritoneal Dialysis

Mettang et al (1996) found that serum concentrations of DEHP measured in patients during continuous ambulatory peritoneal dialysis (CAPD) ranged from 0.032-0.21 µg/mL, with a mean value of 0.79 µg/mL. This corresponds to daily dose of 20 µg DEHP/kg bw/day.

1.3.2.1.3. Long-Term Transfusions

There are no published data on DEHP levels in patients undergoing long-term transfusion of blood and blood products. All published exposure estimates are based on the use of limited data on the levels of DEHP in stored blood and blood components (Table 4, Section 1.2.1.) together with different scenario assumptions (Jacobsen, 1977; (Rock et al., 1986; Cole et al., 1981; Rubin and Ness, 1989; Huber et al., 1996; and Doull et al., 1999) As pointed previously, these estimates are subject to considerable uncertainly. Higher exposure estimates are obtained by assuming long storage time, room or body temperature storage or use, and high range concentrations or large volume procedures. Some of the scenarios used in the earlier studies do not take into account current transfusion practices. Other exposure estimates (Doull et al., 1999) incorrectly assume a linear rate of extraction of DEHP over an extended period of time. All estimates have ignored the contribution of DEHP extracted from the infusion line, although the data of Easterling et al. (1974) and Loff et al. (2000), which was discussed in Section 3.4, suggest that this assumption may not be justified.

Jacobson (1977) estimated that the total dose of DEHP received by patients with leukemia who receive multiple transfusions whole blood, platelets and RBCs for a whole year ranged from 150 to 1500 mg, while patients with aplastic anemia would receive between 56 to 365 mg. These values correspond to a daily dose of 6 to 59 µg DEHP/kg bw/day for patients with leukemia and 2 to 14 µg DEHP/kg bw/day for patients with aplastic anemia (assuming a 70 kg patient). Doull (1999) estimated DEHP exposure from transfusion of platelets in a patient with leukemia to be 36 µg/kg bw/day, assuming a dose of 13 mg DEHP/unit of platelets and the transfusion of
70 units/year. However, these findings do not appear to be relevant to Canadian patients. As pointed out in Section 3.1, patients requiring transfusions in Canada generally receive packed red blood cells, platelets, or fresh frozen plasma, rather than whole blood, which is rarely administered. Platelets and other blood components stored in bags made from the PVC-free or phthalate-free alternatives.

It has been reported that hemophiliacs given prophylactic or maintenance treatment with clotting factors (non-recombinant) could be exposed to levels up to 800 mg/year (Rubin and Ness, 1989; Consumer Product Safety Commission, 1985), i.e., up to 30 µg/kg bw/day. However, in Canada, most haemophiliacs are no longer being treated with concentrates. Hemophilia A patients are now being treated with recombinant Factor VIII and Hemophilia B patients with recombinant Factor IX. They are therefore not exposed to plasticizers from blood collection packs.

Patients with sickle cell anemia typically receive 1 - 2 units of packed red blood cells every 2 to 4 weeks. Plonait et al. (1993) reported levels of DEHP in packed red blood cells ranging from 4 to 123 g/mL (Table 4). Assuming the highest DEHP level reported, the maximum DEHP dose received by a patient with sickle cell anemia would therefore be

0.123 mg/mL x 350 mL/unit x 2 units/14 days/70 kg bw = 0.090 mg/kg bw/day

= 90 g/kg bw/day.

1.3.2.1.4. Long-Term Total Parenteral Nutrition

There are no published data on DEHP levels in adults receiving total parenteral nutrition. Although the absence of consistent data on the rate of DEHP extraction from PVC storage bags and infusion lines make it is impossible to obtain reliable estimates of adult exposure from this procedure, the limited data available (Sections 1.2.4.1 and 1.2.4.3.) indicate that it may be an important source of DEHP exposure. Estimates of exposure based on the data of Mazur et al. (1989) and Loff et al. (2000) are shown in Table 7b.

Table 7b. Estimated DEHP Received from Total Parenteral Nutrition

1.3.2.2. Short-Term Adult Exposures

These include exposures from short-term blood transfusions, including large volume transfusions of blood and blood products in trauma patients or patients with gastrointestinal bleeding, patients undergoing surgical procedures or extracorporeal membrane oxygenation (ECMO) procedures, and IV infusion of drugs.

All published short-term exposure estimates are based on limited data on blood levels of DEHP measured in patients before and after specific medical procedures (Table 5 and 6) or DEHP levels in stored blood and blood components (Table 4) together with different scenario assumptions (Jacobsen, 1977; (Rock et al., 1986; Cole et al., 1981; Rubin and Ness, 1989; Huber et al., 1996; and Doull et al., 1999). These estimates are subject to considerable uncertainly for the reasons discussed in Section 1.3.1 and vary widely depending on factors such as storage time and temperature.

1.3.2.2.1. Short-Term Transfusion of Blood Components

Short-Term Multi-Transfusions

The highest published estimates of DEHP exposure from short-term blood transfusions assume multi-transfusions of blood or blood components that have been stored for a prolonged period of time. For example, Sjoberg et al. (1985b) estimated that an adult receiving 2.5 L (5 units) of whole blood stored for 21 days would receive a DEHP dose of 1.3 to 2.6 mg/kg body weight, while Jaeger and Rubin (1972) estimated that a gunshot victim, transfused with 63 units of whole blood could receive a DEHP dose of 8.5 mg/kg body weight. Although these estimates are consistent with reported levels of DEHP in whole blood (Table 4), they may no longer be clinically relevant. As pointed out in Section 1.3.2.1.3, patients requiring transfusions will generally receive red blood cells suspended in additive solutions, platelets (stored in non-PVC storage bags), and fresh frozen plasma, rather than whole blood, which is rarely administered. Table 4 shows that much lower DEHP levels have generally been found in these blood components.

Transfusions of Blood Products in Adults During Surgical Procedures

Barry et al. (1989) reported high daily doses of DEHP for a number of cardiac bypass procedures in adults. As shown in Table 6, the highest daily dose - 2.4 mg/kg bw/day - was reported for an artificial heart transplantation procedure (Jarvik bridge).

1.3.2.2.2. Extracorporeal Membrane Oxygenation

Butch et al. (1996) reported that adult undergoing extracorporeal membrane oxygenation (ECMO) may receive up to 46 units of blood products per day (mean = 21 units) as a combination of RBCs, platelet concentrates, fresh frozen plasma, and cryoprecipitate) during ECMO therapy. The levels of DEHP reported in Table 4 for RBCs, platelet concentrates, fresh frozen plasma, and cryoprecipitate, suggest that this could expose the patient to more than 10 mg DEHP/kg bw/day (if 46 units are transfused/day) or about
3 mg DEHP/kg bw/day (21 units are transfused/day) during the 10 to 20 days of ECMO therapy. The amount of DEHP released from the PVC tubing used in ECMO therapy has not been measured in adults. However, studies of infants on ECMO, discussed below in Section 1.3.3.2, suggest that the tubing may contribute an additional 0.3 mg DEHP/kg bw/day to the daily DEHP dose.

1.3.2.2.3. IV Infusion of Drugs

The data presented in Section 1.2.2 indicate that the infusion of saline, glucose, non-lipophilic drugs and other aqueous IV solutions would result in a daily DEHP dose of less than 5 µg/kg body weight/day.

When pre-mixed, stored at room temperature in PVC bags for 48 hours and delivered used PVC infusion sets, the infusion of lipophilic drug formulations may expose the patient to relatively large doses of DEHP - more than
0.5 mg/kg bw/day for some drugs and up to 1.5 mg/kg bw when the mixture is also agitated for 24 hours (Venkataramanan et al., 1986 Pearson and Trestle, 1993). However, as pointed out in Section 1.2.2, it is unlikely that PVC bags and tubing would be used to administer lipophilic drug solutions, since published guidelines (Pearson and Trestle, 1993) and the product labelling contraindicate their use for this purpose. The administration of lipophilic drug formulations according to the manufacturer's directions for use would result is a daily DEHP dose of less than 0.05 mg/kg bw/day.

1.3.3. Neonatal Exposures

Neonatal exposures need to be assessed separately. As explained in Section 2.6.1.4, neonates are believed to be the subpopulation most vulnerable to the potentially adverse developmental effects of DEHP.

Table 8 lists some exposure estimates for neonates based on spot measurements of DEHP in blood or delivered doses using AUC calculations. Exposures associated with other medical procedures based on rates of extraction of DEHP and scenario assumptions, although less reliable than in vivo data, need also be considered.

Table 8: Reported DEHP Exposures in Neonates

1Depending on the circuit.

There are no published direct measurements of DEHP exposures from replacement blood transfusions and hyperalimentation with protein and lipids even though these procedures are routine procedures in neonatal intensive care units. Surveys of transfusion practices in neonatal intensive care units in the US (Levy et al., 1993; Ringer et al., 1998) indicate that 80% of low birth-weight infants receive multiple transfusions. Respiratory therapy is another common procedure for pre-term infants, whose lungs are frequently not fully developed. DEHP exposures. Unfortunately, there are no reliable published data on DEHP exposures from respiratory therapy or even on the release of DEHP from PVC tubing and other components of breathing circuits under normal conditions of use.

1.3.3.1. Replacement Blood Transfusions

The data from Plonait et al. (1993), shown in Table 8, suggest that infants receive a DEHP dose of 1.2 - 22.6 mg /kg bw/day in an exchange transfusion, while the study by Sjoberg et al. (1985b) suggest a dose of
0.8 - 3.3 mg /kg bw/day.

Exposures for replacement blood transfusions, usually in volumes of 10 mL/kg per transfusion, can also be estimated using published data on DEHP levels in whole blood, based on assumptions about the age of the blood, storage temperatures, and specifics of blood products selected (e.g., whole blood vs reconstituted packed red blood cells). For example, using the data from both Sjoberg et al. (1985a, 1985b) studies on DEHP in whole blood used in exchange transfusions, a standard transfusion of 10 mL/kg of whole blood would expose a neonate to an average of 490 µg/kg/transfusion (Range:140 - 850 µg/kg bw/transfusion). On the other hand, the data of Peck et al. (1979) give an average dose of of 750 µg/kg bw/transfusion (Range: 540 - 10,000 µg/kg bw per transfusion). Table 4, which list DEHP levels in different blood components, suggests that current transfusion practices, in which packed red cell concentrates reconstituted with fresh frozen plasma are used as replacement in neonates, may result in significantly lower DEHP exposures. However, the DEHP levels recently reported by Loff et al. (2000) in different blood components stored in PVC bags, plasma-rich plasma and fresh frozen plasma (Table 4), suggest the following DEHP exposures: 72 - 304 µg/kg bw/transfusion of packed red blood cells; 342 - 614 µg/kg bw/transfusion of platelet-rich plasma; and 112 - 3,390 µg/kg bw/transfusion of fresh frozen plasma. Although, the results reported for fresh frozen plasma may not be very reliable since they show a very wide variation in DEHP levels, these results suggest that transfusions of blood components may still be expose neonates to relatively high levels of DEHP.

In addition, the contribution of DEHP extracted from the PVC infusion lines during transfusion also needs to be taken into account. As reported in Section 1.2.4.3, Loff et al. (2000) found that fresh frozen plasma (FFP) extracted 25 µg DEHP/mL during the transfusion, while platelet-rich plasma extracted 13.9 µg DEHP/mL, which represents contribution of 250 µg DEHP/kg bw/transfusion in the case of FFP and 139 µg DEHP/kg bw/transfusion for platelet-rich plasma.

1.3.3.2. Extracorporeal Membrane Oxygenation

Extracorporeal membrane oxygenation (ECMO), which is used to treat respiratory failure in both premature infants and term infants when pulmonary function and other studies suggest that mechanical ventilation will be unsuccessful or cause undue harm, gives one of the highest short-term exposures to DEHP. Two studies, using different ECMO circuits, have estimated DEHP exposure from ECMO based on measurements in a small numbers of infants. Schneider et al. (1989) reported serum levels of 26.9 µg/mL for a 3-10 day course of treatment and 33.5 µg/mL after 24 days of ECMO therapy. They estimated that a neonate could be exposed to as much as 42,000 µg/kg-bw DEHP after 3 days and 140,000 µg DEHP kg-bw after 10 days of ECMO therapy, or 14 mg DEHP/kg bw/day. More recently, Karle et al. (1997) studied three different ECMO circuits and reported much lower levels of exposure: 0 to 34.9 mg DEHP/kg bw/ treatment, depending on the circuit used. The circuit, which used heparin-coated PVC tubing, did not leach DEHP, resulting in no DEHP exposure from the ECMO circuit. For the two uncoated circuits, Karle et al. estimated exposures of 4,700 µg/kg bw and 10,000 µg/kg bw after 3 days which rose to 15,500 µg/kg bw and 34,900 µg/kg bw after 10 days of treatment, i.e., about 1.6 mg/kg bw/day for the first uncoated circuit and 3.5 mg/kg bw/day for the second uncoated circuit. These estimates were based on periodic blood sampling and the in vitro leaching rates of the three circuits. Infants with similarly severe medical conditions but treated with mechanical ventilation rather than ECMO were used as controls. There was no accumulation of DEHP in the mechanically ventilated babies. AUC calculations were not performed. The differences between the results reported by Karle et al. and those of Schneider et al. may be attributed to the smaller surface areas of the newer ECMO circuits used by Karle et al. and possibly to differences in the percentage of DEHP in the tubing used.

The in vitro results obtained with the heparin-coated circuit, suggest that detectable levels of DEHP would not be released during ECMO procedures that use heparin-coated tubing. Heparin-coated ECMO circuits are commercially available in Canada.

1.3.3.3. Total Parenteral Nutrition

Neonates who cannot breast feed or bottle feed generally receive their nutrition intravenously. There are no published in vivo data on DEHP exposure from this medical procedure. The limited data on the extraction of DEHP from PVC infusion lines during TPN procedures have been discussed in Section 1.2.4.2. The measurements recently made by Loff et al. (2000) of the extraction of DEHP from PVC infusion lines by lipids under conditions typical for newborn intensive care units suggest a daily DEHP dose of
5.1 mg/kg bw/day from this source of exposure.

1.3.3.4. Respiratory Therapy

There are no reliable published data on DEHP exposures from respiratory therapy or even on the release of DEHP from PVC tubing and other components of breathing circuits under normal conditions of use.

1.3.3.4.1. Ventilation using Heated Respiratory Tubing

Roth et al. (1988) studied five pre-term infants who were mechnically ventilated using heated respiratory tubing and humidified air and reported levels of DEHP in the condensate collected from the water traps of the respirator tubing that ranged from <0.001 to 4.1 mg/L. Based on these values, Roth et al. estimated that the infants could have received inhalation doses of DEHP ranging from <0.001 to 4.2 mg/hour; i.e., < 0.01 to 50 mg/kg bw/day. However, as explained in detail in Section 1.2.4.5.2, the reported DEHP concentrations in the condensate appear to have been seriously overestimated due to a sampling error. Furthermore, DEHP levels in the condensate cannot provide a reliable estimate of inhalation exposure since the infants were not exposed to the condensate.

The worst possible case of DEHP exposure during ventilation would occur if infants were exposed to air saturated with DEHP throughout the procedure. As detailed in Section 1.2.4.5.2 for the circuit used by Roth et al. (1988) the DEHP exposure under these conditions would not exceed 0.35 mg/day
(0.2 mg/kg bw/day for a 2-kg infant) and is likely to be much lower.

The type of circuit used by Roth et al. (1988), has been replaced in current practice by heated tubing with a lower mean wall temperature. Furthermore, respiratory tubing used with ventilators is currently made from polyethylene. Health Canada recently confirmed this by testing the thirteen leading North American brands.

1.3.3.4.2. Oxygen Therapy

Oxygen therapy, in the hospital, at home and in mobile applications, requires the use of oxygen supply tubing, which is generally made of PVC. There are no published estimates of DEHP exposures from oxygen therapy. As discussed in Section 1.2.4.5.4, in the worst possible case scenario this procedure would expose an infant to 0.5 mg DEHP/day; i.e., 0.25 mg/kg bw/day. However, the actual exposure is likely to be much lower.

1.3.3.4.3. Respiratory Therapy using an Endotracheal Tube

Respiratory therapy in infants frequently requires intubation with an endotracheal tube. Latini et al. (1999) recently reported a loss of 0.06-0.12 mg DEHP/mg neonatal endotracheal tube/procedure. Since the average length of the procedure was 129.5 hrs (details obtained from the authors.), this would represent a loss of 11-22 mg DEHP/day. However, as pointed out in Section 3.4.5.6, this study can not be used to provide an accurate estimate of neonatal exposure to DEHP from the endotracheal tubes since an unknown proportion of the DEHP extracted by air and mucus would not reach the infants lungs.

A worst case estimate of DEHP exposure, which takes into account the DEHP delivered to a neonate by both the respiratory air stream and respiratory tract mucus, is 2.8 mg/day, or about 1.4 mg/kg bw/day for a 2-kg neonate. Section 1.2.4.5.6 provides details of this calculation. A neonate intubated with an endotracheal tube is generally exposed to an additional dose of DEHP from other components of the respiratory circuit. For example, if oxygen therapy is administered, a worst case exposure estimate would be (2.8 + 0.5) mg/day, i.e., 3.4 mg/day or about 1.7 mg/kg bw/day for a 2-g neonate.

Although neonatal exposures will be lower than the worst case estimates, these estimates indicate that respiratory therapy may be an important source of exposure that needs to be studied in different clinical settings.

1.3.3.5. Prenatal Exposure

Prenatal exposure through medical procedures also occurs but has not been studied. Pregnancy is possible for women on chronic hemodialysis, and DEHP is known to cross the placenta. These patients give birth and some breast-feed their children. Maternal transfusions or intrauterine fetal blood transfusions and in utero fetal surgery are additional, though uncommon, reasons for exposure.

There are no published data on DEHP levels in breast milk from mothers undergoing hemodialysis. However, rough estimates of exposure of infants to DEHP from milk of nursing mothers on dialysis may be obtained from the DEHP concentrations reported in the milk of women who have not undergone medical treatments that would expose them to high levels of DEHP. A value for the human milk to human plasma partition coefficient, is needed to obtain this estimate. Published values are not available. However, the partition coefficient can be estimated by comparing reported plasma concentrations of DEHP in normal subjects (around 0.1 µg/mL) and concentrations reported in breast milk by Pfordt et al. (1999).

Pfordt et al. (1999) reported 10 to 110 µg DEHP/kg milk in five breast-feeding women (mean: 34 µg/kg, median: 43 µg/kg). Since the plasma concentration of DEHP is around 100 µg/L (0.1 µg/mL), this suggests a milk to plasma partition coefficient of 0.43. Although higher exposure levels have been reported in the past, according to Faouzi et al. (1999), plasma levels of DEHP in patients on hemodialyis reach 3000 µg/L after a 4-hour dialysis session.

Based on these results, a nursing infant, whose daily intake is 150 mL milk/kg bw/day, would receive 3000 µg/L x (milk/plasma partition coefficient) x (0.150 L/kg bw/day) DEHP from a mother on hemodialysis, i.e.,
193 µg DEHP/kg bw/day. Since plasma levels of DEHP as high as 8000 µg/L have been reported in patients undergoing dialysis, a nursing infant could receive up to 514 µg DEHP/kg bw/day from a mother on hemodialysis.

1.3.4. Multiple Exposures

All published studies of DEHP exposure from medical devices consider only single exposure sources. For many patients, particularly critically ill neonates, examining single sources of exposure (e.g., ECMO or ventilation) may significantly under-estimate the total exposure. For example, neonates who require ECMO, also require multiple replacement blood transfusions (Luban, 1995; Rosenberg et al., 1994; Bjerke et al., 1992; Green et al., 1990; Minifee et al., 1990), parenteral feeding (Pettignano et al., 1998; Piena et al., 1998) , medications, and IV fluids (Loff et al., 2000). Many of these other medical procedures may substantially increase DEHP exposure. The total DEHP exposure from combinations of simultaneous exposures may vary dramatically from medical centre to centre, depending on the treatment protocols used (Rosenberg et al., 1994; Bjerke et al., 1992).

1.3.5. MEHP

Patients who undergo medical procedures may also be exposed to the monoester metabolite of DEHP, mono(2-ethylhexyl) phthalate (MEHP), which is believed to be the active metabolite responsible for many of the reported adverse reproductive and developmental effects of DEHP in rodents. Exposure results from in vitro conversion of extracted DEHP during storage of blood products and from in vivo conversion of circulating DEHP to MEHP. DEHP is converted to MEHP in vivo via enzymatic conversion by plasma lipases (Albro and Thomas, 1973b; Peck et al, 1979).

in vitro conversion is enhanced by increased storage time and temperature (Cole, 1981). Storage at 4°C significantly inhibits conversion to MEHP and storage at -30°C prevents it entirely (Rock, 1978; Cole, 1981). Processes conducted at physiological temperatures will therefore increase exposure to MEHP. Heparin is reported to induce lipase activity, while DEHP itself induces extrahepatic lipase activity (Rovano et al., 1984) . Thus, exposure to heparinized blood or medical devices may increase the rate of conversion of DEHP to MEHP.

in vivo conversion of DEHP to MEHP depends on the route of exposure. Orally administered DEHP is converted more rapidly to MEHP than intravenously administered DEHP. After oral administration, DEHP is rapidly absorbed from the gut of rodents, mostly in the form of the monoester because of the rapid hydrolysis of the DEHP by gut lipases (Eriksson, 1986; Gaunt, 1982; Pollack, 1985; Sjoberg, 1985; Albro, 1982). In primates, including humans and marmosets, a smaller proportion of DEHP is hydrolyzed and absorbed as the monoester, because of less lipase activity in primate intestine (Rhodes, 1982; ICI, 1982; Rhodes, 1983; Shell, 1982; Schmid, 1985; Rhodes, 1984). Neonates have much lower levels of pancreatic lipase than adults (Lee, 1983). However, they can convert orally administered DEHP to MEHP via lingual, gastric (Lee, 1993; Armand,1996), hepatic (Terada and Nakanuma, 1995), and plasma lipoprotein lipases (Rovamo et al., 1984, 1988), and lipase in breast milk ( Hamosh, 1996).

Most medical exposures occur intravenously. Conversion of DEHP administered intravenously to MEHP, which is catalyzed by plasma and hepatic lipases, is much slower than that observed when equivalent doses are administered orally (Huber, 1996). Rovamo et al. (1984, 1988) found that levels of plasma and hepatic lipase enzymes in infants may be greater than levels found in adults and pre-term infants may convert DEHP to MEHP more rapidly than at-term infants, since they have higher lipoprotein lipase levels. In addition, heparin and DEHP itself both induces plasma lipase activity (Mocchiutti and Bernal, 1997) . This suggests that exposure to heparinized blood or medical devices or repeated exposure to medical procedures, which commonly occurs in pre-term neonates, may increase the rate of conversion of DEHP to MEHP.

MEHP has been measured in stored blood, blood products and peritoneal dialysate (Peck, 1979; Sjoberg, 1985a; Sjoberg, 1985b; Rock, 1978; Labow, 1986; Cole, 1981). However, insufficient data are available to calculate in vitro conversion rates, making it difficult to interpret the results. Few data have been published on MEHP exposure from medical procedures. These are shown in Table 9.

Table 9: MEHP Exposure from Medical Procedures

MEHP always accompanies DEHP which is almost always present in much larger concentrations. Total MEHP exposure is therefore the sum of exposure from MEHP formed in vitro and exposure from MEHP formed by in vivo hydrolysis of DEHP. There are no reliable data on the rate of in vivo conversion of circulating DEHP to MEHP and estimates of the total MEHP exposure from different medical procedures are unavailable.

1.3.6. Conclusions - Human Exposure

Medical exposures to DEHP and MEHP are highly variable and have only been partially studied. Most single-source exposure scenarios can be generally be estimated within one order of magnitude, but additional variability is possible depending on assumptions made. The best studies of single-source exposure measure delivered dose using AUC calculations for dialysis (Pollack, 1985, Faouzi, 1999), and exchange transfusion (Sjoberg, 1985a, 1985b).

Table 10 summarizes the exposure data. It shows estimates of the daily dose of DEHP received by the general population and by patients undergoing a variety of medical procedures. These exposures were calculated from the data tables in the individual published reports. The daily dose received from hemodialysis was calculated by assuming 156 sessions per year in a 70 kg adult. Replacement transfusion was calculated for the average, highest, and lowest DEHP concentrations reported in the blood bags used for the exchange transfusions in the Sjoberg studies (1985a; 1985b). Most other published exposure estimates are based on spot measurements of DEHP in blood in a few individuals, or calculated from leaching rates from DEHP tubing, assuming linear extraction.

Table 10. Daily Exposure of Patients and the General Population to DEHP

1For 70 kg adult, 156 sessions/yr
2Calculated from reported area under curve (AUC) dose to patients.
3 Based on concentration of DEHP measured in serum
4 Based on estimates of concentration of DEHP in stored blood and blood components
5 Based on estimated rates of DEHP extraction from PVC storage bags and infusion lines
6 Calculated from reported DEHP concentration in blood bags;
7 Based on in vitro measurements
8Worst-case estimate
9 Based on additional information received from authors. This represents total amount of DEHP lost from the endotracheal tube, not the exposure.

The data in the table lead to the following important conclusions about neonatal and adult exposures.

Exposures in Infants

1. Infants undergoing routine replacement blood transfusions may be exposed to doses of DEHP 1-2 orders of magnitude above general population exposures. Infants undergoing intensive therapies may be exposed to levels up to 3 orders of magnitude above general exposures.

2. Double volume exchange transfusion is the short-term procedure reported to give the highest acute exposure - up to 23 mg/kg bw/day.

3. Extracorporeal membrane oxygenation (ECMO) is the sub-acute medical treatment which gives one of the highest daily DEHP exposures per kg body weight and the highest daily exposure over a prolonged period of time - up to 14 mg/kg/day during the 3 to 30-day treatment period.

4. Other medical procedures that result in very high exposures relative to the general population exposure are cardiac bypass procedures, total parenteral nutrition therapy, infusion of lipophilic drugs using PVC bags and tubing (which is contraindicated in the directions for use), and possibly, respiratory therapy.

Exposures in Adults

1. Blood transfusions to the trauma patient is the short-term procedure which gives the highest acute exposure in adults - up to 8.5 mg/kg bw/day.

2. Other short-term medical procedures that give relatively high DEHP exposures are blood transfusions during ECMO, cardiopulmonary bypass procedures, and infusion of lipophilic drugs using PVC bags and tubing (contrary to the directions for use).

3. Long-term hemodialysis is the chronic procedure that exposes the patient to the highest total dose of DEHP and the highest chronic daily dose. Chronic DEHP exposures in adults undergoing hemodialysis can be 1-2 orders of magnitude above the general population exposure.

The exposure estimates in Table 10 do not include estimates of concomitant MEHP exposure, since reliable estimates are generally not available. Estimates of medical exposures from simultaneous procedures in the same patient (e.g., ventilation, nasogastric tubes, transfusion, and parenteral feeding) are also not included, since these exposures have not been quantified. Medical exposures, particularly those for critically- ill infants, may result in DEHP doses considerably above those documented for single medical procedures.

2. DEHP TOXICITY

2.1. Introduction

The toxicity of DEHP has been reviewed in several recent publications (T: 1-9). The present toxicity assessment critically reviews the animal and in vitro toxicity studies and reports of adverse effects in humans, and assesses whether exposure to DEHP from medical procedures may cause adverse health effects in humans. Key scientific papers were reviewed to:

• identify the critical toxicity endpoints;
• identify the no-observed-adverse-effect-levels (NOAEL) or lowest-observed-adverse-effect-levels (LOAEL);
• assess the relevance of the findings in animals for patients exposed to DEHP from medical procedures; and
• identify data gaps in current knowledge.

2.2. Absorption, Distribution, Metabolism and Excretion of DEHP

The absorption, distribution, metabolism and excretion of DEHP have been thoroughly reviewed by Huber et al. (T: 3) and, more recently, by the NTP-CERHR Expert Panel Report (T: 2). Although there is very little data on human metabolism, it is generally believed that human metabolism of DEHP is similar to that of primates (T:2). This section provides a summary the information on DEHP metabolism relevant to human risk assessment.

DEHP is rapidly metabolized after oral exposure. In the first step, DEHP is hydrolyzed by lipases in the gut and absorbed as the monoester,
mono(2-ethylhexyl) phthalate (MEHP), and the corresponding alcohol,
2-ethanolhexanol (2-EH) (T: 196, 214-216). Rhodes et al. (26) found that marmosets excreted radioactivity amounting to 2% of a 2000 mg/kg bw dose of DEHP in the urine versus 50% in rats, while fecal excretion was 90% in marmosets and 20% in rats (T: 26). This shows that at high doses of DEHP, intestinal absorption and/or hydrolysis of DEHP occurs more rapidly in rodents than in primates, including humans, and a smaller proportion of DEHP is hydrolyzed and absorbed as the monoester, probably because of lower lipase activity in primate intestine (T: 26, 217-219). However, when 100 mg/kg DEHP was given to rats and cynomolgus monkeys, 28% of the total radioactivity was excreted in the urinary of monkeys vs 33% in rats and 49% was excreted in the feces of the monkeys versus 51% in rats (T: 189). These results suggest that there were no significant differences between rats and primates in rates of DEHP absorption and/or hydrolysis at lower and more medically relevant doses.

Intestinal absorption of DEHP/MEHP and/or conversion of DEHP to MEHP occurs more readily in young rodents than in adults (T: 43, 196). The DEHP absorbed by the intestines may also be converted to MEHP by plasma or hepatic lipases. Thus, for the same DEHP exposure, young rodents would receive a higher systemic and testes dose than mature rodents. These findings suggest that developing rodents may be susceptible to toxicity from DEHP at lower levels of exposure.

Although human neonates have much lower levels of pancreatic lipase than adults (T: 220), they can convert orally administered DEHP to MEHP via lingual, gastric (T: 220 - 222), hepatic (T: 223-224), and plasma lipoprotein lipases (T: 226-228), and lipase in breast milk (T: 225). Rovano et al. (T: 226) found that some of these enzymes may be present at higher levels in infants than in adults. Lee et al. (1993) found that gastric lipase activity increased to a peak at 30-32 weeks gestational age, suggesting that older pre-term neonates may be more efficient in converting DEHP to MEHP than at term infants. DiPalma et al. (1991) found there was no significant differences between levels of lipase in the gastric body of children aged 5-19 months and those in adults. Thus, neonates and older children may be able to convert DEHP to MEHP via gastric, hepatic, and plasma lipases as efficiently as adults.

Inhaled DEHP is absorbed as the parent compound from the lung
(T:2, 5, 96); dermal absorption of DEHP is slow (T: 229-232).

Once absorbed into the blood stream, DEHP and its metabolites are widely distributed throughout the body, apparently without accumulation in tissues (T: 5, 96). The liver, kidneys, lung, pancreas, and plasma contain lipases which convert DEHP to MEHP and EH. DEHP is hydrolyzed to the monoester and the alcohol by these lipases after parenteral DEHP exposure. However, hydrolysis occurs more rapidly in the gut, because of higher lipase activity. Pollack et al. (T: 216) reported that MEHP levels after parenteral DEHP exposure were ~1-5% of those seen after oral DEHP exposure. However, the same authors also found that the time averaged circulating concentrations of MEHP during dialysis were similar to those of DEHP (T:234). Peck and Albro et al. (T: 235) found that MEHP made up 7% of the administered DEHP dose excreted in the urine of two leukemia patients who received platelets stored in PVC bags.

Rovamo et al. (T: 226-228) found that levels of plasma and hepatic lipase enzymes in infants may be greater than levels found in adults, and pre-term infants may convert DEHP to MEHP more rapidly than at-term infants, since they have higher lipoprotein lipase levels. In addition, heparin and DEHP itself both induce plasma lipase activity (T: 21). Exposure to heparinized blood or medical devices or repeated exposure to medical procedures, which commonly occurs in pre-term neonates, may therefore increase the rate of conversion of DEHP to MEHP.

Rats primarily metabolize DEHP by oxidation to diacids, while primates, including humans, predominately form MEHP and many other secondary metabolites. Adult humans and other primates excrete the monoester as a glucuronide, while rodents metabolize it further. The toxicological significance of this species difference is unclear. Oral doses of DEHP and the metabolites are rapidly excreted via urine and feces (T: 236-237). The parent compound, DEHP, has not been detected in the urine of any species studied, but is detected in feces in amounts inversely related to the degree of absorption. No significant retention in organs and tissues was observed in any species studied, with less than 1% dose retained a few days after administration.
(T: 2).

DEHP and MEHP have been detected in the milk of lactating female rats
(T: 239, 240) and there is one report that DEHP and MEHP can cross the placenta (T:238).

Children do not have mature glucuronidation pathways until they are 3 months old. This important clearance mechanism is therefore not fully available to neonates and young infants (T: 85). The ability to metabolize DEHP may also be compromised in individuals with kidney disease and other health problems ( T: 241-244).

Karle et al. (20) reported higher levels of DEHP in plasma in infants in the first few days of ECMO therapy but did not determine if DEHP became more efficiently metabolized during the treatment or if it was redistributed to various tissues.

2.3. General Toxicity

2.3.1. Acute Toxicity

The acute toxicity of a single dose of DEHP has been evaluated in a number of species of rodents using oral, dermal, inhalation and intravenous routes of administration (T: 5, 10-12). LD-50 values derived from these studies are shown in Table 11.

Table 11. Acute Toxicity of Single Dose of DEHP

These data show that in all the specimens tested, DEHP had a very low degree of acute toxicity when given as a single dose. DEHP delivered intravenously had a higher acute toxicity than orally administered DEHP (T:80).

2.3.2. Subchronic and Chronic Toxicity

The repeated-dose toxicity of DEHP has been evaluated in a number of animal species, both over a short-term (few weeks) and a life-time (2 years)
(T: 5, 13-29). Most of these studies used rodents and the oral route of administration. The studies show that rodents are the most sensitive species, followed by hamsters, guinea pigs, and primates. The studies have been recently reviewed in the recent NTP-CERHR Expert Panel Report (T: 2). This section summarizes the major findings of the key papers.

DEHP produces a variety of subchronic and chronic toxic effects in developing and adult animals. The main target organs of DEHP toxicity are the liver
(T: 1-9, 13-14, 16-18, 30-34), kidneys (T: 1-9, 35-38) reproductive tract (testis, ovaries, secondary sex organs) (T: 1-9, 29, 39-46) lungs (T: 47-50), and heart (T: 51-52 ). As discussed below, testicular toxicity is the most sensitive toxicity end point reported to date. Developmental toxicity has also been reported in a large number of rodent studies in which DEHP was administered in diet or by gavage (T: 2, 40, 41, 53-71).

The toxic effects on the liver, which are generally the first sign of DEHP toxicity in rodents, include liver enlargement due to hepatocyte hypertrophy and hyperplasia, a marked increase in the size and number of peroxisomes and an increase in the activity of enzymes associated with this process, and necrosis and reduction in liver functions (T: 2, 3, 14, 16, 72-76). These effects are similar to those observed following exposure to all peroxisome proliferators (T: 2, 3, 5, 77, 78), which, in addition to phthalate ester compounds, which are a structurally divers group of chemicals that includes some hypolipidemic drugs (e.g., clofibrate), phenoxy herbicides, and solvents. Long-term exposure to peroxisome proliferators is associated with liver tumours in rats and mice. The current thinking regarding the molecular mechanisms of peroxisome proliferator-induced hepatocarcinogenesis is discussed in the section on carcinogenesis.

A study of the subchronic oral toxicity of DEHP in the rat by Poon et al.
(T: 29) illustrates the type of adverse effects that are seen in target organs in most rodent studies. In this key study, groups of male and female young adult Sprague-Dawley rats (10 rats/group) were exposed to 0, 5, 50, 500 and 5000 ppm DEHP in the diet for 13 weeks. According to the authors, these dietary concentrations corresponded to average DEHP doses of 0, 0.4, 3.7, 38, and 375 mg/kg bw/day, respectively, for the male rats and 0, 0.4, 4.2, 42, and
419 mg/kg bw/day for the female rats. The following parameters were assessed at the end of the study: hematology, clinical chemistry, organ weights, and histopathology. There were no clinical signs of toxicity in the treated groups and no significant differences in body weights and food consumption between treated and control groups.

This study provides evidence that the liver and testes are target organs of DEHP in Sprague-Dawley rats. The high dose of DEHP administered produced an enlargement of the liver and peroxisome proliferation, a slight enlargement of kidneys, and mild histological changes in the thyroid. Testicular toxicity was also observed. Effects included Sertoli cell vacuolation at lower doses and bilateral, multifocal, or complete atrophy of the seminiferous tubules with complete loss of spermatogenesis and cytoplasmic vacuolation of the Sertoli cells lining the tubules, at higher doses.

David et al.(T: 13) recently observed similar testicular effects in a chronic study of Fischer-344 rats. The 6-week-old rats (50-80 males/group) were fed diets containing 0, 100, 500, 2,500, or 12,500 ppm DEHP (0, 5.8, 29, 147, and 789 mg/kg bw/day for males) for 104 weeks. Testes weight (absolute and relative) was reduced in rats of the high-dose group. Aspermatogenesis was observed in 10/10 rats of the 789 mg/kg bw/day group at study week 78. The effect was not observed in rats treated with 147 mg/kg bw/day or in the control group. At study week 105, the incidence of aspermatogenesis was significantly increased in rats exposed to 29 mg/kg bw/day and higher doses. The percentage of rats affected from the control to high-dose group was 58, 64, 78, 74, and 97%, respectively. The NTP-CRHRH Expert Panel on Phthalates noted that the study utilized suboptimal testis fixation, which may obscure an early vacuolar lesion, which DEHP produces.

These two key studies are discussed in greater detail in Section 2.6.1 (Reproductive Toxicity).

Developing animals appear to be more susceptible to the adverse effects of DEHP than older animals. Dostal et al. (T: 79) compared the hepatic peroxisome proliferation and hypolipidimic effects of DEHP in neonatal and adult rats. Five oral daily doses of 0, 10, 100, 1000 and 2000 mg/kg were given to rats beginning at 6, 14, 16, 21, 42 and 86 days of age. Suckling rats, 1-3 weeks old, suffered severe growth retardation at doses of 1,000 mg/kg and death at 2,000 mg/kg. Interestingly, deaths occurred at doses of 1,000 mg/kg in 14-day old rats but not in 16-day old rats. These results suggested that neonatal and suckling rats were more sensitive to the lethal and growth retardation effects of DEHP than adult rats. In contrast, the extent of peroxisome proliferation in the liver was similar in all ages.

Several oral exposure studies have found that developing animals show signs of testicular toxicity at much lower doses of DEHP than adults. The adverse effects were earlier on-set and less reversible (T: 40, 81, 82). These findings are supported by several recent oral exposure studies (T: 44, 83-84) showing that prenatal exposure of rats to DEHP (T: 44) and a related phthalate,
di-n-butyl phthalate (T: 44, 83-84), causes significant adverse effects in the reproductive system at doses that produce no toxic effects in adults. However, an IV exposure study by Sjoberg et al. (T: 76) did not find that 25-day-old rats had a greater susceptibility to DEHP toxicity than 40-day-old rats.

An in vitro study of cultured germ cells and Sertoli cells by Gray and Beamand (42) found that a significantly larger number of germ cell detached from Sertoli cells in cell cultures from younger animals than in cell cultures from older animals incubated in the same concentration of MEHP. These findings suggest that the developing testis may be more susceptible to damage from DEHP exposure than the mature testis.

Gray and Gangolli (T: 43) reported greater intestinal absorption of DEHP/MEHP in developing rodents than in adults, suggesting that developing rodents may be susceptible to DEHP toxicity at lower levels of exposure. Humans rely on the glucuronidation of MEHP to eliminate DEHP from the body. Infants do not have a mature glucuronidation pathway until they are 3 months old (T: 85). Since this important DEHP clearance mechanism is not fully available to neonates and young infants, they may eliminate DEHP more slowly than adults and, consequently, may be more vulnerable to DEHP toxicity.

There are very few published reports on the effects of DEHP administered intravenously (IV) to animals. (T: 30, 76, 86). In the only well-designed and controlled IV study, Sjoberg et al. (T: 76) studied the effects of IV administration of DEHP on the hepatic histology, serum enzymes and testicular histology in 40-day-old Sprague Dawley rats. The time-weighted average doses of DEHP were 2.5, 25, and 250 mg/kg bw/day. The results showed a dose-related decrease in body weight gain, an increase in relative liver weight at the middle and highest doses but no change in the clinical chemistry parameters. Liver and kidney histology appeared unchanged except for an increase in hepatic peroxisomes of 32, 26, and 41% in the 2.5, 25, and 250 mg/kg dose groups, respectively.

There was no change in the relative weight of the reproductive organs and no abnormalities in testis structure were observed under the light microscope. However, electron microscopy revealed that in 3/5 animals exposed to the highest dose there were slight enlargements of the smooth endoplasmic reticulum in Sertoli cells, which is the earliest hallmark of the DEHP testis lesion. There were also slight structural changes in early spermatocytes.

This key study shows that IV exposure to DEHP produces testicular toxicity in rats. However, it is likely that the 40-day-old rats were not at the most sensitive developmental age, since several studies have shown than younger developing rats appear to be more vulnerable to testicular toxicity than older animals (T: 40, 42-44, 81-84). Furthermore, the effect on testicular development, which may be a more sensitive end point, was not studied. This study is analyzed is discussed in more detail in Section 2.6.1.6 (LOAEL and NOAEL for Reproductive Toxicity).

Jacobson et al. (T: 30, 31) studied hepatic effects in 6-month-old rhesus monkeys receiving transfusions of plasma from DEHP-plasticized bags over a 6-month or 1 year period. The average total exposures to DEHP for the three groups of monkeys transfused weekly for one year with platelet-rich plasma stored at 22°C were: Group 1 (transfused weekly for 1 year with platelet-rich plasma stored at 20°C): 27 mg/kg bw; Group 2 (transfused weekly for 1 year with platelet-rich plasma stored at 40°C ): 8 mg/kg bw; and Group 3 (transfused biweekly for 6 months with platelet poor plasma stored at 22°C): 32 mg/kg bw. Three of the seven monkeys exposed to DEHP showed some impairment of hepatic perfusion and altered BSP clearance, which persisted up to 14 months after the exposure. Abnormal histological findings, consisting of vacuolized cells, necrosis, increased numbers of Kupffer cells or inflammatory cell infiltration, were observed at one year in six of the seven monkeys exposed to DEHP and for up to 14 months after treatment. The control monkeys showed no evidence of abnormal liver function or histology.

The hepatic effects reported by Jacobsen et al. (T: 30, 31) appear to be minor and presumably reversible alterations of liver function. In addition, several factors confound the interpretation of the results: 1) the inconsistent responses in the two groups that received the largest (and similar) DEHP exposure; 2) pooled plasma was re-transfused into the monkeys, which raises concerns about the possibility of an allergic reaction to foreign proteins; and 3) the authors reported that there was a tuberculosis outbreak in the monkey colony, which may have contributed to the hepatic effects.

Greener et al. (T: 86) studied the toxicity of DEHP administered intravenously in a 4% bovine serum albumin/0.9% sodium chloride solution to 2-4 day-old rats (12/group; strain not specified) for 18 days at doses of 0, 31, 92, or 165 mg/kg bw/day. Blood analytes were measured and the brain, heart, lungs, liver, spleen, kidneys, eyes, and digestive tract were examined histologically. The reproductive organs were not weighed or examined histologically.

At the highest dose, significant effects included reduced body weight gain, a slight increase in liver weight and liver to body weight ratio, and a small elevation of serum glutamic oxaloacetic transaminase (SGOT) activity, suggesting some liver function impairment. However, there were no conclusive histopathological changes in the liver or other organs examined. Based these results, the NOAEL for liver function impairment would be 92 mg/kg bw/day.

The transcripts of the FDA Workshop on Plasticizers, held in October 1999 (T: 245) refer to a number of early studies that found acute and subacute toxic effects following parenteral administration of DEHP in animals. In an unpublished study done under contract to NIH, Rutter et al. administered varying doses of DEHP intravenously to 14 dogs for a four-week period over six days a week. The lowest dose, 21 mg/kg bw/day, produced increased lung and livers weights. Since histopathology was not performed, the results of increased organ weights have limited usefulness in evaluating toxicity. In a follow up study, the intravenous administration of DEHP solubilized from PVC blood bags produced no adverse effects. However, the dose, which was not stated, was much lower. These findings are consistent with the earlier observation by Rubin et al. and Miripol et. al (T: 246) that DEHP solubilized with serum, Tween, ethanol, or DMSO may show greater pulmonary toxicity than the same dose of DEHP extracted by plasma from a PVC bag.

In another experiment reported in the transcripts, rats were kept hypotensive and hypovolemic for 30 min and then re-transfused to mimic exchange transfusion. The DEHP was prepared by sonication in plasma and then added back to packed cells. Pulmonary toxicity was observed at a DEHP dose of
8 mg/kg and the LD-50 was about 200 mg/kg.

In the absence of additional details, these preliminary IV administration experiments cannot be critically reviewed. The results can only be considered as suggestive of possible adverse effects.

As part of a long-term study of the carcinogenicity of DEHP in rodents, the National Toxicology Program (NTP) also evaluated the non-neoplastic effects (T: 87). For 103 weeks, groups of 50 male and female Fischer 344 rats were fed a diet containing 6,000 and 12,000 ppm DEHP while groups of male and female B6C3F1 mice were fed a diet containing 3,000 and 6,000 ppm of DEHP. The body weight gains and food consumption were reduced in the treated rats but not in the mice. Peroxisome proliferation was not evaluated in this study but evidence of testicular effects including atrophy, aspermia, loss of germinal epithelium, and degeneration of seminiferous tubules was observed in the high dose male rats and mice.

Preliminary experiments were also performed, using subchronic (13 weeks) administration, to establish the maximum tolerated dose for the carcinogenicity study. In these experiments, rats and mice, 10 per group, were administered DEHP in the diet at the following doses: rats: 0, 1,600, 3,100, 6,300, 12,500 and 25,000 ppm; mice: 0, 800, 1,600, 3,100, 6,300 and 12,500 ppm. In rats, the body weight gain was significantly reduced at the high dose, and there was testicular atrophy at the 12,500 ppm dose. The NOAEL was estimated to be 6,300 ppm, which corresponded to about
320 mg/kg bw/day. In mice, the high dose was lethal and the 3,100 ppm and 6,300 ppm doses decreased the body weights, but no other effects were noted. The LOAEL was estimated to be 800 ppm, corresponding to about
100 mg/kg bw/day.

To study the effects of chronic administration of DEHP on the liver of male rats, Ganning et al. (T: 88) administered DEHP to rats in the diet at 0, 0.02, 0.2 and 2.0% for up to 102 weeks. Low doses produced a moderate increase in the activity of certain liver enzymes, which continued throughout the treatment period. These authors also reported that liver biopsies from patients undergoing hemodialysis demonstrated an increased number of peroxisomes based on a qualitative comparison of electron micrographs of liver tissue.

This is the only publication which suggests that peroxisome proliferation occurs in humans following exposure to DEHP or to any other chemical known to induce peroxisome proliferation in rodents. However, these results would need to be verified by additional studies they are based on biopsies from only a few patients and a visual comparison of electron micrographs of the liver without quantitative estimation of the number of peroxisomes or statistical analysis of data. These findings are also inconsistent with the observation that chronic treatment of humans with relatively large doses of fibrate drugs does not induce peroxisome proliferation (T: 3, 5, 150, 151).

In another study by the same authors (T: 89), male rats were exposed to 0, 0.02, 0.2 and 2.0% DEHP in the diet for 102 weeks. Only the high dose caused a reduction in body weight (about 20% ) and a large increase in activity of enzymes associated with peroxisome proliferation. At lower doses, the enzyme activities increased slowly but continuously and by the end of the two-year treatment period the activities were similar to those found with the high dose. One year after treatment, all enzyme activities had returned to normal values. No liver tumours were detected. However, the prolonged exposure to DEHP inhibited spermatogenesis and caused general tubular atrophy. The absence of liver tumours is inconsistent with the results of the NTP studies and other studies, all of which found in liver tumour after prolonged treatment with comparable DEHP doses. Since this study was not designed to evaluate carcinogenic effects, the negative findings are to be considered unreliable. However, the results, confirm the effect of DEHP on the testis and on liver enzymes associated with peroxisome proliferation and suggest that enzyme activities may revert to normal levels one year after termination of DEHP exposure.

A number of studies have also identified the kidney as target organ for DEHP toxicity (T:13, 14, 18, 35-38) in rodents. Effects reported include significant changes in kidney weights and chronic progressive nephropathy.

In a study designed to mimic DEHP human exposure during hemodialysis, Crocker et al. (T: 23) dosed rats (strain not identified) orally with 2.2 mg DEHP/kg bw/day three times a week for one year. After a one-year treatment, none of the animals had generalized polycystic kidney of the kind found in humans. However, the kidneys had multiple areas of focal cystic alteration, which appeared to be related to an inflammatory response. Creatinine clearance was reduced after one year of treatment. Since creatinine clearance is always reduced in older rats, this finding may not be related to DEHP exposure.

The results of this paper do not provide much support for the suggestion that acquired cystic kidney disease, which often occurs in long-term hemodialysis patients (T: 92, 93), may be caused by DEHP exposure in humans undergoing dialysis. The cystic changes seen in rat kidney may be part of the normal aging process. In humans, the appearance of renal cysts may also be a late manifestation of renal disease processes that could not be seen previously. Furthermore, in humans undergoing dialysis the presence of renal impairment and exposure to drugs and chemical other than DEHP that are released from hemodialysis equipment or the dialysis solution may influence the development of cystic disease.

Ward et al. (T: 18) found that mice fed high doses of DEHP (12,000 ppm) developed a number of adverse kidney effects after four weeks, including focal tubular degeneration, atrophy, and regenerative tubular hyperplasia, which progressed to severe renal cystic tubules after 8 to 16 weeks of exposure. Arcadi et al. (T: 40) found a number of histopathological changes in the kidneys of offspring exposed orally during gestation and postnatally to DEHP during suckling at 0 - 4 weeks, including shrunken renal glomeruli, dilation of renal tubuli and light fibrosis.

However in other studies (T: 13, 14, 94) long-term or exposures to high doses of DEHP failed to produce cystic or other major lesions in the kidney of both rats and mice.

Summary of Data on Chronic and Subchronic Studies

There are several well-designed and carefully controlled studies of the toxicity of DEHP following subchronic and chronic administration. Most are rodent studies, primarily by the oral route. Organs identified as critical targets for toxicity are the liver, kidney and testis. Young animals appear to be more susceptible to the adverse effects of DEHP than older animals.

In the liver of rodents, the effects can be characterized as hepatomegaly due to hyperplasia and peroxisome proliferation in the hepatocytes, and an increase in activities of enzymes that are associated with peroxisomes. There are marked species differences in these liver changes. Rodents are sensitive whereas hamsters, guinea pigs, and monkeys are relatively insensitive at the doses that produce liver changes in rodents.

In the kidney, DEHP exposure produces increased kidney weights, increased mineralisation of the renal papilla, cell pigmentation, and chronic progressive nephropathy.

In the testis, the toxic lesions observed are atrophy, high incidence of Sertoli cell vacuolation, and germinal epithelium degeneration. These effects are discussed in more detail in Section 2.6.1 (Reproductive Toxicity).

Comparative toxicity studies have indicated that adult cynomolgus monkeys and marmosets, are non-responsive to doses at which DEHP induces lesions in the target organs of rodents (T:184).

There is very limited information on the effects of DEHP in humans. The study by Ganning et al. (T: 88) is the only one to suggests that peroxisome proliferation may occur in humans due to exposure to DEHP (T: 88). However, this suggestion is based entirely on biopsies of a few patients and the visual examination of electron micrographs of liver tissue. The number of peroxisomes present was not measured.

Based on adverse effects sometimes seen in rodents in rodents after long-term oral administration of DEHP (T: 18, 35, 40, 95), it has been suggested that exposure to DEHP during dialysis may cause cystic kidney disease in long-term dialysis patients. (T: 95). However, this is not supported by other studies in which (T: 13, 14, 94) long-term or exposures to high doses of DEHP failed to produce cystic or other major lesions in the kidney of both rats and mice.

2.4. Genotoxicity

The genotoxicity of DEHP and its major metabolites have been thoroughly evaluated over the past two decades, using a variety of in vitro and in vivo assays (T: 70, 96 - 132). Much of the in vitro data was obtained in a multicentred study conducted by the International Program on Chemical safety (T: 132). The findings have been summarized in a number of recent reports (T: 1- 3, 5, 70, 132). This section focuses on the most significant studies and findings.

2.4.1. Gene Mutations in Bacteria, Fungi and Drosophila

The mutagenicity of DEHP, its metabolites, and related phthalate esters have been investigated in a number of bacterial test systems (e.g., Salmonella typhimurium and E. coli), fungi (Aspergillus, Saccharomyces cerevisiae and Schizosaccharomyces pompe) and Drosophila using the Ames test with and without metabolic activation (T: 118, 123, 131-132). The results indicated that DEHP and its metabolites were not mutagenic.

2.4.2. in vitro Studies of Mammalian Cells

A wide variety of mammalian cells cell cultures were used to test the genotoxicity of DEHP and its metabolites. The cells were tested for evidence of strand breaks, in vitro DNA repair, sister chromatid exchange, chromosomal aberrations, selective DNA amplification, polyploidy, cell transformation, aneuploidy, and metabolic cooperation. (T: 5, 132).

The results were predominantly negative, although some rare and generally inconsistent positive results have been reported (T: 132). The most conclusive positive results were obtained in studies of cell transformation) in Syrian hamster embryo cells (SHE). Cell transformation is not an established genotoxic event, since it could also be related to cell proliferation or other non-genotoxic mechanisms, thus, the positive result in this assay may not be indicative of DEHP genotoxicity.

in vitro chromosomal effects, mainly chromosomal aberrations and sister chromatid exchanges, have been evaluated in a variety of cell lines including CHO cells, CHL cells, human lymphocytes and Syrian hamster embryo cells (SHE). Most of the results of these cytogenetic assays showed no increases in chromosomal effects caused by exposure to DEHP and its metabolites. However, three independent laboratories reported that both DEHP and MEHP induced chromosomal aberrations following exposure of SHE cells to noncytotoxic concentrations of these substances. Low threshold levels were used in some studies for a positive classification (T:5). All recent reviews of this issue (T: 1- 3, 5, 132), have concluded that DEHP does not cause cell mutations in mammalian cells, taking into account the overwhelming preponderance of negative findings, the inconsistency of some of the positive results, the possibility of other explanations for the positive results (such as the detergent effect of MEHP), and concerns about the low threshold levels used in some studies for a positive classification.

2.4.3. In Vivo Studies

The genotoxicity of DEHP has been evaluated in a number of rodent studies. Gunz et al. (T: 100) evaluated the effect of DEHP on gene mutations in transgenic mice (C57BL/6f LacI locus). They found no increase in mutation frequency of the LacI gene in the DNA isolated from the liver of the treated animals. Another study failed to detect DNA damage or DNA adducts formation in hepatocytes of rats exposed to DEHP (T:133). The chromosome aberrations and enhanced transformation in hamster embryonic cells observed by Tomita et al. (T:123) may not be an indication of genotoxicity. Instead, they may be related to chromosome instability, which was also observed in the controls. (T: 5). Autian (T: 141) and Singh et al. (T: 142) found genotoxic effects in mice after intraperitoneal or intravenous injection of DEHP. The doses ranged from one-third to two-thirds of the LD50 values in the study by Singh et al. However, these findings are inconsistent with those of other studies, which found that even higher doses of DEHP and its metabolites given orally did not induce positive results (T: 132). Barber et al. (T: 112) found an increase in micronuclei in female mice after repeated administration of MEHP. In contrast, Astill et al. (T:108) found increases in percent micronucleated cells in response to both single and repeat doses of 5 g/kg bw /day. Huber et al. (T:5) have hypothesized that the positive findings reported by Singh et al. (T: 142) and Barber et al. (T:112) may have been caused by the detergent effect of MEHP, resulting in damage to the spindle system of the chromosomes.

Albro et al. (T: 134) reported in vivo DNA binding of DEHP in rat liver. However, several investigators have failed to confirm this finding (T: 135, 136). Von Daniken et al. (T: 136) attributed their preliminary positive results to cellular uptake of intermediary metabolites of nucleotides and classified DEHP as not binding to DNA.

Several studies have measured 8-hydroxydeoxyguanosine levels in the livers of rats exposed to high levels of DEHP in the diet (T: 137-139), based on the hypothesis that the most likely mechanism for induction of genotoxicity by peroxisome proliferators is the formation of oxygen free radicals. Increased levels of 8-hydroxydeoxyguanosine in livers would, therefore, be an indication of resulting DNA damage. Takagi et al. obtained weakly positive results with DEHP. Clofibrate and ciprofibrate, more powerful peroxisome proliferators, both gave positive results but nafenopin, another strong peroxisome proliferator, did not (T: 138, 139). In contrast, Cattley and Glover (T: 137 ) reported negative results with DEHP but positive results for clofibric acid. They also found that clofibric acid stimulated the production of 8-hydroxydeoxyguanosine in mitochondrial DNA but not in nuclear DNA, which is the DNA relevant to genotoxic carcinogenicity. Taken together, these finding do not provide support for a genotoxic effect of DEHP through oxidative stress.

There is only one study on genotoxicity in humans occupationally exposed to DEHP (T: 140). Exposure levels ranged from 0.0006 to 0.01 ppm (0.01-0.16 mg/m³). The study found no evidence of an increased frequency of chromosome aberrations in blood lymphocytes. However, the study is considered to be inadequate for the evaluation of genotoxicity in DEHP in humans because of the low exposure levels and the small number of workers tested. (T: 5)

2.4.4. Evaluation

Most of studies carried out to date, have found no evidence for the genotoxicity of DEHP or its metabolites. Huber et al. (T: 5) have pointed out that the few tests that showed positive results had one or more of the following limitations. The results were (1) weak and not positive according to all criteria; (2) not reproducible; (3) obtained using an unvalidated or insufficiently validated test (e.g., based on end points that may not be indicative or genotoxicity); (4) classified by the authors themselves as "preliminary" or "at present not sufficiently confirmed"; (5) strongly dependent on experimental conditions (e.g., restricted to particular doses); (6) not found in the liver, which is the target organ for carcinogenesis in rodents; or (7) similar to findings observed with established non-carcinogens.

Taken together, the weight of evidence indicates that DEHP and its metabolites (MEHP and 2-ethylhexanol) are not genotoxic or mutagenic. All recent reviews of this topic (T: 1- 3, 5, 70, 132) have reached this conclusion. It supports the theory, discussed in the following section, that DEHP exerts its tumorigenic action in rodents via a nongenotoxic action.

2.5. Carcinogenicity

2.5.1. Experimental Data

The potential carcinogenicity of DEHP has been evaluated in a number of studies of rodents chronically exposed to DEHP in the diet. Among these, only two studies are considered to be adequate carcinogenicity bioassays. Both were performed with adequate numbers of animals and used an acceptable protocol.

In the earlier study, conducted by the National Toxicology Program (NTP)
(T: 87), groups of 50 male and female Fischer 344 rats were fed a diet containing 0, 6,000, or 12,000 ppm DEHP and groups of male and female B6C3F1 mice were fed a diet containing 0, 6,000, or 12,000 ppm DEHP for 103 weeks. This corresponded to a daily DEHP intake of 0, 322, and 674 mg/kg bw/day for male rats; 0, 394, and 774 mg/kg bw/day for female rats; 0, 672 and 1,325 mg/kg bw/day for male mice, and 0, 799 and 1,821 mg/kg bw/day for female mice. Treatment did not affect food consumption or survival rates in either species. Mean body weight gains were reduced in male rats at 6,000 and 12,000 ppm, female rats at 12,000 ppm, and female mice at 3,000 and 6,000 ppm. The results, summarized in Table 12, showed a significant dose related trend in the incidence of tumours, as well as significant differences in the pairwise comparison between the high dose and control groups in both sexes and species tested.

Table 12. Hepatocellular Tumours Reported in the NTP Study

Hepatocellular carcinomas were significantly increased in female rats at 12,000 ppm (774 mg/kg bw/day), in male mice at 6,000 ppm, (1,325 mg/kg bw/day) and female mice at 3,000 (799 mg/kg bw/day) and 6,000 ppm.
(1,821 mg/kg bw/day). The combined incidence of animals with hepatocellular carcinomas or neoplastic nodules was significantly increased in male rats at 12,000 ppm and female rats at 6,000 ppm, while the combined incidence of animals with hepatocellular carcinomas or adenomas was significantly increased in male mice at 3,000 ppm.

The NTP study did not evaluate peroxisome proliferation but did observe other significant toxic effects, notably, chronic inflammation of the kidney and severe degeneration of the seminiferous tubules, testicular atrophy in the high dose male rats and mice. The testicular effects are discussed further in the section of reproductive toxicity.

The recent 2-year study by David et al. (T: 13, 14, 16), which tested the same strains of rats and mice that had been used in the NTP study has confirmed the carcinogenicity of DEHP in rodents. The DEHP doses administered in the diet to the rats were 0, 100, 500, 2500, or 12,500 ppm, corresponding to 0, 5.8, 29, 147 and 789.0 mg/kg/day for males, and 0, 7.3, 36, 182 and 939 mg/kg/day for females. In the mouse study, the DEHP doses administered in the diet were 0, 100, 500, 1,500, or 6,000 ppm, corresponding to 0, 19, 99, 292 and 1,266 mg/kg/day for the males and 0, 24, 117, 354 and 1,458 mg/kg/day for the females. Additional groups of rats and mice were treated at the high dose for 78 weeks and then with control diet for an additional 26 weeks (the recovery groups). All relevant parameters for a chronic carcinogenicity study were evaluated. In addition, hepatic cell and peroxisome proliferation were evaluated at weeks 1, 2, 13, and 79, and at end of the study.

A significantly higher incidence of hepatocellular tumors was observed for the 2500-ppm and 12,500-ppm group of rats, and for the 500-, 1500-, and 6000-ppm groups of mice, doses at which peroxisome proliferation was significantly increased. For mice, the NOAEL for both tumors and peroxisome proliferation was 100 ppm, corresponding to 19 - 24 mg/kg bw/day. For rats, a dose of
500 ppm, corresponding to 29-36 mg/kg/day, was considered to be the NOAEL both for tumors and peroxisome proliferation. The recovery group had a significantly lower incidence of tumors than the groups fed DEHP continuously for 104 weeks.

These results indicate that high levels of peroxisome proliferation and hepatomegaly are associated with DEHP hepatocarcinogenesis in rodent liver, and that the tumorigenic process may be arrested by cessation of DEHP treatment, suggesting that extended treatment with DEHP acts to promote tumor growth. The no adverse effect values are relevant to human risk assessment only if DEHP is assumed to have a threshold for tumour induction.

Long-term studies in Syrian hamsters by Schmezer et al. [T:104] revealed no evidence of tumor induction, which suggests that the carcinogenicity of DEHP may be species-specific.

One small study of workers in a di(2-ethylhexyl) phthalate production plant found no excess of cancer mortality. However, this study did not have adequate power to detect a potential excess risk. (T: 140).

2.5.2. Evaluation

The NTP study (T: 87) and the more recent studies by David et al. (T: 13, 14, 16) provide clear and adequate evidence that DEHP is hepatocarcinogenic in mice and rats following long-term oral exposure. The results indicate that the NOAEL for both tumors and peroxisome proliferation is 19 - 24 mg/kg bw/day for mice and 29-36 mg/kg/day for rats.

The relevance of these findings to humans, which depends on the mechanism by which DEHP exerts its effect on the development of liver tumours in rodents, has been debated by regulatory agencies over the last decade (T: 1, 6, 7, 96, 43). In 1993, the U.S. Agency for Toxic Substances and Disease Registry concluded on the basis of animal data that DEHP "may be reasonably anticipated to be a carcinogen"(T:1). On the other hand, based on a review of information on the mechanism of action of DEHP in hepatocarcinogenesis in rodents, the International Programme on Chemical Safety (IPCS) (T: 7) as well as the European Union (T: 143) have removed DEHP from lists of human carcinogens. The International Agency for Research on Cancer (IARC) originally classified DEHP as "a possible human carcinogen", on the basis of evidence from animal studies and the lack of adequate data in humans (T: 149). However, IARC recently reclassified DEHP as "not classifiable as to its carcinogenicity in humans" (Group 3) based on a consideration of additional mechanistic studies (T:6). IARC's conclusion is that the mechanism by which DEHP increases the incidence of hepatocellular tumours in rats and mice is not relevant to humans.

The following discussion summarizes the evidence supporting this conclusion within the framework of the multistage model of carcinogenesis (T: 5). This model subdivides tumor induction into three steps: initiation, promotion, and progression. Initiation involves the irreversible and inheritable DNA damage in the target organ and is therefore generally associated with genotoxicity. Initiation can occur by a single very short-term exposure to a tumor initiator, theoretically even by exposure to just one molecule. Initiated (pre-neoplastic cells) can also occur spontaneously, especially in the course of aging. In the next step - promotion - exposure to a tumor promoter causes the pre-neoplastic cells to multiply preferentially to form promoted foci (cell clusters). The tumor promoter induces these changes either by stimulating cell replication or interfering with apoptosis (controlled cell death). Tumor promoters generally also cause functional changes in cells, such as changes in microsomal metabolism or peroxisome enzymes. Since many of the promotional effects are reversible, long exposures to the tumour promoter may be necessary for the carcinogenesis process to continue. Therefore, the risk assessment needs to take into account the length of exposure to the tumor promoter. In the last step - progression - the promoted foci can be transformed into malignant tumor.

A number of studies have investigated the ability of DEHP to act as a tumor-initiator. In these studies, rodents were exposed to high doses of DEHP for a short period of time and then exposed to strong tumor promoters, using well-established protocols for tumor promotion (T: 144 - 147). Even excessive doses of DEHP (50 g/kg bw/day by gavage) and treatments for periods of up to 12 weeks did not produce any liver foci. Similar negative results were obtained with strong substances known to be strong peroxisome proliferators. These studies indicate that DEHP is not a tumor initiator - a conclusion which is consistent with the conclusion in Section 2.4 (Genotoxicity) that DEHP is not genotoxic.

Several studies have assessed the ability of DEHP to act as a tumor promoter by first exposing rodents to a well-established genotoxic tumor initiator and then giving the rodents high doses of DEHP over a long period of time and measuring the liver tumors that results (T: 5, 146, 147). These studies show that DEHP is a tumor promoter in rodents.

The effects of DEHP on rodent livers - liver enlargement, a marked increase in the size and number of peroxisomes and an increase in the activity of enzymes associated with this process, reduction in liver functions, and a dose-related increase in the formation of liver tumours - are similar to those observed following exposure to all peroxisome proliferators (T: 2, 3, 5, 77, 78). In addition to phthalate ester compounds, this group of chemicals includes some hypolipidemic drugs (e.g., clofibrate), phenoxy herbicides, and solvents. The current scientific consensus is that DEHP and its metabolites induces liver toxicity and a dose-related increase in liver tumours the same way as other peroxisome proliferators - by activating the peroxisome proliferator-activated receptor-a (PPARa). The mechanism is discussed below. Although human tissues contain some PPARa (1% to 10% of the levels found in rat and mouse liver), it appears that human PPARa is not responsive to activation at doses that activate rodent receptors. This is the most likely reason why liver toxicity, peroxisome proliferation and tumor formation are not observed in humans following exposure to peroxisome proliferators (T: 3, 5, 150, 151).

It was noted in Section 2.3.2 that Ganning et al. (T: 88) reported that liver biopsies from patients undergoing hemodialysis demonstrated an increased number of peroxisomes based on a qualitative comparison of electron micrographs of liver tissue. This is the only publication which suggests that peroxisome proliferation occurs in humans following exposure to DEHP or to any other chemical known to induce peroxisome proliferation in rodents. However, these results are based on biopsies from only a few patients and a visual comparison of electron micrographs of the liver without quantitative estimation of the number of peroxisomes or statistical analysis of data. The results are also inconsistent with the observation that chronic treatment of humans with fibrate drugs at relatively large doses does not induce peroxisome proliferation (T: 3, 5, 150, 151). The authors themselves pointed out that renal insufficiency and uremia, may have been responsible for the persoxisome proliferation observed in the patients. In addition, the prevalence of viral hepatitis, which is reported to increase the number and volume of peroxisomes (T: 151b), is much higher in patients on hemodialysis.

The species-specificity of peroxisome proliferation is well-established. Rats and mice are highly sensitive to the short-term and long-term effects in liver, Syrian hamsters are less responsive, while other species, including guinea pigs, dogs, and humans are non-responsive (T:3, 5). These differences suggest that humans may not be susceptible to the hepatocarcinogenic effects of DEHP. This conclusion is consistent with the absence of any evidence of peroxisome proliferation or tumour induction in the liver of humans who have been chronically treated with the hyperlipidemic drugs, clofibrate and gemfibrozil (T:3, 5, 150-153). These peroxisome proliferators are much stronger inducers of liver tumours in rodents than DEHP. (T: 3, 5, 91). Furthermore, a number of recent in vitro studies have demonstrated that liver cells of primates, including humans, are not susceptible to peroxisome proliferation expression (T: 3, 5, 150).

For these reasons, drug regulatory agencies have concluded that drugs which induce peroxisome proliferation and a dose-related increase in liver tumours in rodents such as the fibric acid derivatives (e.g, clofibrate, fenofibrate) are non-carcinogens in humans. This conclusion is further supported by recent research into the mechanism of peroxisome proliferator-induced hepatocarcinogenesis.

Peroxisome proliferation is a transcription-mediated process involving activation by the peroxisome proliferator of a nuclear receptor in the rodent liver called the peroxisome proliferator-activated receptor-a (PPARa). Several mechanisms have been proposed to explain how this can lead to the formation of hepatocellular tumors in rodents (T: 3, 5, 91, 150-155). The general consensus is that PPAR a-induced mitogenesis and cell proliferation are the major mechanisms responsible for peroxisome proliferator-induced hepatocarcinogenesis in rodents (T: 3). Oxidative stress is unlikely to play a major role in peroxisome proliferator-mediated hepatocarcinogenesis, but probably plays a contributory role (T: 151, 152 ), possibly by triggering the release of TNFa by Kupffer cells, which in acts as a potent mitogen in hepatocytes (T:3, 156).

PPARa belongs to a family of nuclear hormone receptors found in various species of mammals and consists of at least three different subtypes, PPARa, PPARg and PPARd, which are encoded by different genes. These receptors regulate gene transcription by binding to specific response elements at the site of regulated genes. Consistent with their distinct physiological roles, each receptor has been shown to be bound and activated by different ligands. In the case of PPARa, a number of fatty acids serve as natural ligands, while fibrate drugs and other peroxisome proliferators as exogenous ligands. Synthetic thiazolidinediones, insulin sensitizer drugs (e.g. troglitazone) and natural prostaglandins serve as ligands for PPARg.

In rodents, PPARa regulates transcription of several genes involved in peroxisome proliferation and enzymes of fatty acid oxidation. It is also involved in modulating cholesterol levels both in humans and rodents by regulating genes of cholesterol metabolism. Chemicals which induce peroxisome proliferation in rodents, including certain hypolipidimic drugs such as clofibrate, have been shown to bind specifically to and activate PPARa. PPARa is also involved in regulating cell replication and controlled cell death (apoptosis).

The best evidence that PPARa plays a critical role in the induction of peroxisome proliferation and liver tumours in rodents comes from studies of PARa-null mice (T: 152, 155, 157-160). These mice cannot express messenger RNA and the protein for PPARa, but are otherwise identical to normal (wild-type) mice. Exposure of PARa-null mice to clofibrate and other more potent peroxisome proliferators failed to induce any responses typically observed with peroxisome proliferation, such as enzyme induction, liver enlargement or induction of PPARa target genes. Furthermore, exposure to Wy-14,643, a potent peroxisome proliferator, induced liver tumours in wild-type mice but not in PPARa-null mice (T: 152, 155, 159).

Ward et al. (T: 160) exposed PPARa null-mice (mice without the PPARa receptor) and wild-type (normal) mice to DEHP at 12,00 ppm in diet for 24 weeks. The PPARa-null mice showed no signs of liver toxicity (no clinical or hispopathological changes or changes in gene expression), while the wild-type mice developed the type of liver toxicity typically seen following exposure to high doses of DEHP. The wild-type mice also exhibited kidney and testicular toxicity that was consistent with previous findings. The PPARa-null mice showed kidney and testicular toxicity (cystic kidney lesions and testicular atrophy) but the toxicity was much less severe than that exhibited by the wild-type mice and only became evident after all the wild-type mice had died.

These results unequivocally demonstrate that liver toxicity is solely due to PPARa activation, while renal and testicular toxicity occur at least partially independently of the PPARa receptor. In addition, Peters et al. (T: 161) have shown that the fetotoxicity and teratogenicity of DEHP does not depend on PPARa receptor activation. Therefore, the observation of these toxic effects in different animals species may be directly relevant to humans risk assessment.

The other subtypes of PPAR (PPAR b, PPARd, and PPARg) may play a role in mediating the non-hepatic toxicity of DEHP. A recent in vitro study by Maloney and Waxman (T:162) provides some evidence in support of this hypothesis. DEHP and its major metabolite, MEHP, were tested for their ability to activate PPARs. The test system consisted of mammalian (COS-1) cells in culture that had been transfected with plasmids expressing mouse and human PPARa and PPARg as well as a reporter plasmid carrying luciferase with PPRE (PP response element). The transfected cells were exposed to different concentrations of test chemicals for 24 h, and their luciferase activities were measured to compare receptor activation. DEHP failed to activate either PPARa or g subtypes of either mouse or human species. On the other hand, MEHP activated both mouse and human PPARa. In addition MEHP also activated PPARg of both species. The human PPARa was found to be much less responsive than its mouse counterpart, which is consistent with previous studies (T: 3).

The activation of PPARg by MEHP in PPARa knockout mice observed by Maloney and Waxman is consistent with the hypothesis that the activation of this receptor may mediate the extraheptic toxicity observed in animals. These toxic effects may therefore be relevant to humans since PPARg is highly expressed in a wide variety of human tissues, including the testis (T: 3). However, there is currently no direct evidence linking these toxic effects to PPARg activation. Appendix 1, summarizes the information currently available on PPARg and concludes that a connection between PPARg activation and carcinogenesis is unlikely. However, a link with other toxic effects, such as those observed in the kidney and testis of mice cannot be ruled out.

Human tissues do contain some PPARa - about 1% to 10% of the levels found in rat and mouse liver. However, many of these PPAR-binding sites are occupied by competing proteins (T: 157) and may therefore not be responsive to peroxisome proliferators (T: 3).

Taken together, these studies indicate that PPARa activation plays an essential role in the induction of peroxisome proliferation and liver tumours in rodents. MEHP, a major metabolite of DEHP, activates PPARa in rodent and human livers. However, peroxisome proliferation and development of liver tumours have not been observed in humans exposed to peroxisome proliferators. The very low levels of PPARa found in human liver and the observation that most of the PPARa sites are occupied by competing proteins likely results in insufficient activation of PPARa. This makes carcinogenesis mediated by the PPARa activation pathway highly unlikely in humans.

These considerations suggest that 1) there is a threshold dose for hepatocarcinogenesis below which no tumour induction would occur in rodents; and 2) hepatocarcinogenicity data from rodents are not relevant for human risk assessment.

A hypothetical cancer risk for humans from exposure to DEHP would be expected to occur only at doses that induce peroxisome proliferation. However, there is no evidence that DEHP induces peroxisome proliferation in humans at exposures that result from medical procedures. The hepatocarcinogenicity data from rodents therefore are not considered to be relevant to human risk assessment.

2.6. Reproductive and Developmental Toxicity

The Expert Panel on Phthalates of the Center for Evaluation of Risks to Human Reproduction, National Toxicology Program (NTP-CERHR), US Department of Health and Human Services, recently published a comprehensive review of the reproductive and developmental toxicity of DEHP (T: 2). Through its membership on panel, the Medical Devices Bureau actively participated in the NTP-CERHR review. The Bureau has also conducted an independent review of the key scientific papers on this topic. An integrated assessment is presented in this section.

2.6.1. Reproductive Toxicity

2.6.1.1. Introduction

There are no data on the reproductive toxicity of DEHP or its major metabolites in humans. However, the numerous studies in experimental animals provide convincing evidence that DEHP is a reproductive toxicant in rodents. These studies have provided a consistent picture of the toxic effects on the reproductive system that are produced by exposure of rodents to DEHP during development in utero and the nursing period, after weaning, and during the adult phase. A number of well-designed and carefully controlled studies, several of which are discussed in Section 2.3 (General Toxicity), provide sufficient data to estimate LOAELs and NOAELs for the reproductive toxicity of DEHP administered orally to rodents. The few data on inhalation are not adequate for the characterization of reproductive toxicity, nor for the estimation of a NOAEL. However, there are very few well-designed and carefully controlled studies that can be used to estimate a NOAEL for the reproductive toxicity of DEHP administered intravenously to rats.

A number of studies have investigated the mechanism of reproductive toxicity, the reversibility of the toxic effects, differences in vulnerability of animals of different ages and different species. These important factors need to be taken into account in evaluating the relevance of the animal data to the assessment of risk to human.

2.6.1.2. Adverse Reproductive Effects

A wide variety of dose-dependent adverse reproductive effects have been reported in rodents. In males, these are primarily damage to the testis and, at higher doses, lower sperm counts and reduced fertility. Depending on the dose, the adverse testicular effects may include atrophy of the seminiferous tubules and the testis, and cytoplasmic vacuolation of the Sertoli cells lining the tubules, a reduced sperm count, and reduced fertility or infertility (T: 2). Testicular toxicity, which was first reported many years ago (T: 42, 171, 175), is the adverse reproductive effect that is occurs at the lowest DEHP exposure levels. At high-dose exposures, adult female rats show decreased hormone production, suppressed and delayed ovulation, ovarian dysfunction (T: 46, 176), infertility (T: 41). Lamb et al. (T: 41) also reported significant decreases in ovarian weights. However, Laskey and Berman (T: 176) reported no changes in ovarian weights or morphology.

A number of in vitro and in vivo studies have identified the Sertoli cells in the testes as primary target for the testicular toxicity of DEHP and other phthalates (T: 2, 24, 39, 45, 177-182). Sertoli cells are the supportive cells in the seminiferous epithelium of the testes. They orchestrate spermatogenesis by providing structural and nutritional support to germ cells. Sertoli cell proliferation stops at puberty and each Sertoli cell can only support the development of a fixed number of germ cells in an adult (T: 185, 186). The number of Sertoli cells is a good indication of testicular size and sperm count in the adult (T: 185, 187). A decrease in the number of Sertoli cells will therefore result in a decrease in fertility. Neonatal, pubertal, and adult exposure to DEHP all cause significant changes in the morphology and function of the Sertoli cells. However, the specific adverse effect varies with age. In very young animals - during the time of Sertoli cell division - phthalate exposure inhibits cell division (T: 177). In older animals, Sertoli cell vacuolization occurs, followed by sloughing of germ cells. (T: 2). It is not known if formation of cell vacuoles and inhibition of cell division are mechanistically linked. The identity of the active toxicant, the molecular target, the possible molecular mechanism of testicular toxicity, and the relevance of this information to human risk assessment are discussed in Section 2.6.1.5.

2.6.1.3. Species Differences in Sensitivity to Reproductive Toxicity

There are species differences in sensitivity to the testicular effects of DEHP. Rats, mice, and guinea pigs are the most sensitive (T: 2, 171), hamsters are much less sensitive (T: 74), while young adult marmosets and young adult cynomolgus monkeys exposed to high doses of DEHP show no signs of testicular toxicity. (T: 170, 183). Testicular toxicity has also been reported in ferrets (T: 75).

The study by Kurata et al. (T: 170) is the most thorough of the few studies of DEHP toxicity in primates. Kurata et al. administered groups of 4 male and 4 female marmosets doses of 0, 100, 500, or 2,500 mg/kg bw/day DEHP in corn oil by gavage for 13 weeks. The marmosets were 12-15 months old at the start of the study, indicating that they had already reached puberty at the beginning of exposure. There were no treatment-related decreases in testis weight, testosterone and estradiol levels. The concentration of testicular zinc, which decreases after toxic DEHP exposure in rats and indicates major germ cell loss, was unchanged. An examination of the testis, fixed in neutral buffered formalin, by light and electron microscopy revealed no signs of testicular toxicity even at the highest dose. Hall et al. (T: 183) recently reported similar results in adult marmosets exposed to DINP.

These results are very different from the major testicular damage produced by equivalent doses in adult mice, rats, guinea pigs, and ferrets. The absence of testicular effects, can be explained, at least in part, by the poor absorption and metabolism of the high dose of DEHP administered to the marmosets, which is discussed in Section 2.2. Rhodes et al. (T: 188) found that marmosets dosed with 2,000 mg/kg bw/day of ¹⁴C-labeled DEHP for 14 days excreted 2% of the dose in the urine in 24 hours vs 50% urinary excretion in rats, while fecal excretion was 90% in the marmosets vs 20% in rats. Thus, at high doses of DEHP, the rate of DEHP absorption and/or hydrolysis is slower in the primate than in the rat. In sharp contrast, when 100 mg/kg bw/day of ¹⁴C-labeled DEHP was administered to rats and cynomolgus monkeys, 28% was excreted in the urine of the monkeys vs 33% in the rats and 49% in the feces vs 51% in rats (T: 189). These results suggest that there may be no significant differences between rats and primates in rates of DEHP absorption and/or hydrolysis at lower (and more medically relevant) doses.

In light of these results, the NTP Expert Panel on Phthalates pointed out
(T: 2) that it is not clear whether the failure by Kurata et al. to detect testicular atrophy is due to the relative insensitivity of marmosets to the adverse testicular toxicity of DEHP or to poor absorption of the DEHP administered. The Expert Panel also noted that the study was restricted to adult marmosets. The results therefore provide no information about the reproductive toxicity of DEHP following exposure during the more vulnerable phases of life - the developing fetus, the neonate and the animal before puberty - when the testis is still developing. Section 2.6.1.4 discusses age-differences in sensitivity to reproductive toxicity.

In a recent study, Pugh et al. (T: 184) found no evidence of testicular lesions in young adult cynomolgus monkeys gavage dosed with 500 mg/kg bw/day DEHP suspended in methylcellulose for 14 days. Although a relatively small number of animals were studied and only for a relatively short period of time, the maximum likely dose that would be absorbed was administered, and appropriate target organs were studied. The results show that young adult cynomolgus monkeys are not as sensitive to the effects of DEHP as juvenile rats. Since a gavage dose of 500 mg/kg bw/day DEHP would not have caused adverse testicular changes in an adult rat, the results provide no evidence that young adult cynomolgus monkeys are less susceptible to the testicular toxicity of DEHP than adult rats. The Expert Panel therefore concluded that results are of limited use is assessing the risk of DEHP to human reproduction.

2.6.1.4. Age-Differences in Vulnerability to Reproductive Toxicity

Younger (developing) animals show adverse testicular effects at much lower doses than older ones, particularly for oral exposures, and the effects appear to be earlier-onset and less reversible (T: 40, 81-84). The lowest LOAEL estimate for reproductive toxicity reported to date is based on studies of animals exposed orally during gestation and postnatally to DEHP during weaning. These findings are also supported by several recent oral exposure studies (T: 44, 83-84) showing that prenatal exposure of rats to DEHP (T: 44) and a related phthalate, di-n-butyl phthalate (T: 44, 83-84), causes significant adverse effects in the postnatal development reproductive system at doses that produce no toxic effects in adults. These findings raise concerns about prenatal exposure to DEHP leading to postnatal toxicity. Although there are very few data on reproductive effects following DEHP exposure during the late gestational and neonatal periods, it is generally believed that these periods represent a time of potentially high sensitivity to DEHP-induced disruption of the reproductive system (T: 2).

An in vitro study of cultured germ cells and Sertoli cells by Gray and Beamand (T:42) found that a significantly larger number of germ cell detached from Sertoli cells in cell cultures from younger animals than in cell cultures from older animals when incubated in the same concentration of MEHP, suggesting that the developing testis may be more susceptible to damage from DEHP exposure than the mature testis. Lloyd and Foster (178) found that initiation of spermatogenesis depended on the interaction of FSH with the Sertoli cell in young rats, but maintenance of spermatogenesis in adults did not. MEHP interfered with FSH interaction at the receptor level, which would explain the observed increased sensitivity to testicular toxicity in young animals. (Refer to Section 2.6.1.5 for a further discussion of the molecular mechanism of reproductive toxicity).

Gray and Gangolli (T: 43) reported that intestinal absorption of DEHP/MEHP is greater in developing rodents than in adults, suggesting that developing rodents may be susceptible to DEHP toxicity at lower levels of exposure. Similarly, Sjoberg et al. (T: 196) found that gavage treatment with DEHP resulted in greater absorption of MEHP, and hence, a greater systemic dose to young versus mature rats. However, an IV exposure study by Sjoberg et al. (T: 76) did not find that 25-day-old rats had a greater susceptibility to DEHP toxicity than 40-day-old rats.

Pharmacokinetics alone cannnot explain the increased sensitivity of developing rodents (T: 236). This suggests that rodents show an age-related change in tissue sensitivity to the reproductive toxicity of DEHP and humans may as well.

Humans rely on the glucuronidation of MEHP to eliminate DEHP from the body. Infants do not have mature glucuronidation pathways until they are 3 months old. Since this important DEHP clearance mechanism is not fully available to neonates and young infants (T: 85), they may eliminate DEHP more slowly than adults. This is supported by the observation by Sjoberg et al. (T :268) that the elimination of DEHP in pre-term infants occurs more slowly than its formation and the finding by Plonait et al. (T: 269) of significant amounts of DEHP in a pre-term baby four days after exchange transfusion. These observations suggest that infants may be more vulnerable to DEHP toxicity.

2.6.1.5. Mechanism of Reproductive Toxicity

There is convincing morphological, functional, and biochemical evidence that the Sertoli cell is a cellular target for neonatal, pubertal and adult exposure to DEHP (T: 2, 24, 39, 45, 177-182). In fact, all phthalates that cause testicular toxicity produce characteristic alterations in Sertoli cell ultrastructure and function (T: 2, 177, 181, 182). In an in vitro study using co-cultures of neonatal Sertoli cell and gonocytes (precursors of spermatogonia) isolated from 2-day-old rats, Li et al. (T: 45) found that MEHP at concentrations as low as 0.1 M (0.028 g/mL) caused the detachment of the Sertoli cells from the gonocytes and also inhibited Sertoli cell proliferation, while 10 M DEHP (the highest concentration studied) did not produce these effects. These results show that MEHP, not DEHP, is the active toxicant, at least for these adverse effects.

Some Sertoli cell functions are mediated by FSH interaction with membrane bound receptors (T :2). In a study using Sertoli cell cultures, Lloyd and Foster (178) found that MEHP interfered with FSH interaction with the FSH receptor. They also observed that initiation of spermatogenesis was dependent on FSH interaction with the Sertoli cell in young rats, but was not necessary for maintenance of spermatogenesis in adults. These findings would explain the increased sensitivity to testicular toxicity in young animals.

In the rat, physiological apoptosis occurs continuously to limit the size of the germ cell population to numbers that can be adequately supported. This form of germ cell death is exaggerated after testicular insults such as toxicant treatment, radiation, and heat exposure. Studies by Richburg et al. (T: 177, 190-195) suggest that the disruption of the Sertoli cell-germ cell contacts, following exposure to MEHP lead to a dramatic increase in apoptosis in germ cells, resulting in cell destruction and loss of tissue function.

The cellular target and active toxicant for reproductive toxicity following gestational exposure to DEHP are currently unknown.

A study of PPAR-a knockout mice (mice without the PPARa receptor) showed that these animals develop testicular toxicity following exposure to DEHP, although the toxicity was delayed and less severe than that seen in the wild type mice (T: 160). These results indicate that, unlike carcinogenicity, testicular toxicity occurs independent of the PPARa receptor and peroxisome proliferation. This conclusion is further supported by the observation that the guinea pig, a species which is unaffected by the peroxisome-proliferating effects of DEHP, exhibits testicular toxicity following exposure to DEHP (T:2, 171). Therefore, differences between species in sensitivity to peroxisome proliferators are not believed to be relevant to the assessment of the risk of reproductive toxicity in human.

Maloney and Waxman recently reported that MEHP, but not DEHP, 2-EH or 2EHA, activated the PPARg receptor in PPARa knockout mice that were susceptible to testicular toxicity (T: 162). This is consistent with the hypothesis that the activation of the PPARg receptor plays a role in the reproductive toxicity of phthalates. The reproductive toxicity seen in animals following exposure to DEHP would be relevant to humans, since PPARg is highly expressed in a wide variety of human tissues, including the testis and the ovary. (T: 3). However, there is currently no direct evidence linking these toxic effects to PPARg activation.

Grey et al. (T: 206) have recently suggests that DEHP induces reproductive tract abnormalities in males fetuses (T: 40, 44, 84, 205-209) by reducing testosterone to female levels during the critical stage of reproductive tract differentiation. More than one mechanism may be required to explain the diverse toxic effects produced by DEHP on the reproductive system. However, the mechanisms proposed to date support the conclusion that the reproductive toxicity seen in animals following exposure to DEHP is relevant to humans.

2.6.1.6. LOAEL and NOAEL for Reproductive Toxicity

A number of studies have been identified as key studies for the quantitative assessment of the reproductive toxicity of DEHP. Almost all of these are oral administration studies in rodents. Data on non-oral administration (IV, inhalation) are extremely limited and are inadequate for complete characterization of reproductive toxicity or the identification of NOAELs or LOAELs by non-oral routes.

2.6.1.6.1. Oral Exposure

1. Lamb et al. (41) assessed the effect of oral exposure to DEHP on fertility and reproduction in D-1 Swiss mice, 11 weeks old at the start of exposure, using a continuous breeding protocol. There were 20 breeding pairs in each treated dose group, and 40 pairs in the control group. Pairs of male and female mice, cohabiting together as breeding pairs, were exposed to doses of 0, 14, 141, and 425 mg/kg bw/day in the diet for 14 weeks. Endpoints were clinical signs, parental body weight and food consumption, fertility (numbers of pairs producing a litter/total number of breeding pairs), number of litters/pair, number of live pups/litter, proportion of pups born alive, sex ratio, pup body weights within 24 hours of birth, and water consumption. At 425 mg/kg bw/day, no breeding pairs delivered a litter. At 141 mg/kg bw/day, fertility was significantly reduced: there were fewer litters, fewer pups/litter, and fewer pups born alive. Only the control and high-dose groups were necropsied. The high-dose males showed signs of major testicular toxicity and had decreased prostate weight, reduced sperm testosterone, and elevated LH and FSH. These results suggest a LOAEL of ~141 mg/kg bw/day, and a NOAEL of ~ 14 mg/kg bw/day, based on the reductions in litter size and in proportions of pairs having litters. Since the low dose groups were not assessed at necropsy, the LOAEL and NOAEL estimates may not be accurate.

2. Schilling et al. (T: 71a) exposed groups of 10 male and female Wistar rats to DEHP in the feed, corresponding to doses of ~110, 339, and 1,060 mg/kg bw/day. The rats were mated within dose groups after 10 weeks of treatment; the offspring pups were reared and mated as adults. Except for the irregular reduced pup survival, all the adverse reproductive effects - reduced gonad weights, reduced fertility, and male reproductive developmental abnormalities - were observed only at the highest dose, 1,060 mg/kg bw/day, leading to a LOAEL for reproductive effects of ~1,060 mg/kg bw/day and a NOAEL of ~339 mg/kg bw/day. The numbers of animals in the study were relatively small, and female reproductive function was not fully assessed. It should be noted that this study was designed to select a dose for a subsequent study - rather than provide an accurate estimate of a NOAEL or LOAEL for reproductive toxicity.

The subsequent study by Schilling et al. (T: 71b) has been completed. On December 18, 2001, BASF, the sponsor of the study, provided Health Canada a copy of the full study report together with photographs of the pathology slides. The experimental protocol is the same as the one used in the previous study (T: 71a), except that there were 25 male and 25 female Wistar rats in each group. The three DEHP doses administered in the diet were 113, 340 and 1088 mg/kg bw/day. DEHP exposure was continuous from at least 70 days premating of the first parental generation to sacrifice (i.e., from conception through gestation and birth to sacrifice for the two offspring generations). The study evaluated a number of reproductive and developmental toxicity parameters, including food consumption, body weight, fertility, viability, birth weight, organ weights, pathology, estrous cyclicity, sperm parameters, anogenital distance, preputial separation, vaginal opening, areolae, and ovarian examination.

At 1088 mg/kg bw/day, the rats showed reduced body weight gain, increased liver weight, reduced fertility, adverse effects on sperm and testes, and reduced survival and growth of offspring. At 340 mg/kg bw/day, the rats had reduced survival rates and growth of offspring, and effects on indicators of reproductive development but not at 113 mg/kg bw/day. Based on these results, the authors reported that the NOAEL for developmental toxicity (survival, growth and development of offspring) was 113 mg/kg bw/day. As part of the overall evaluation of reproductive toxicity, testes were taken from parental males, fixed in Bouin's solution, and examined under light microscopy (T: 71c). The testes from males from the first offspring generation, who were exposed to DEHP continuously from conception to the time of necropsy, showed "mild" seminiferous tubular atrophy at 340 mg/kg bw/day but not at 113 mg/kg bw/day. However, there was no evidence of Sertoli cell vacuolation in otherwise normal tubules. Thus, the NOAEL for pathological changes in the testes was 113 mg/kg bw/day.

These results are consistent with those of Poon et al. (T: 29), who reported minimal to mild tubular atrophy in Sprague-Dawley rats at approximately
370 mg/kg bw/day, but no such effects at 37 mg/kg/day. However, unlike the Poon study, there was no evidence of Sertoli cell vacuolation in otherwise normal tubules in the Schilling study. The susceptibility of Sertoli cells to vacuolization in response to DEHP appears to depend on the species of rat studied (T: 280 -281).

One limitation of the study is that the testes were examined only by light microscopy. In many previous studies of the reproductive toxicity of DEHP, the testes were studied by both light microscopy and electron microscopy, which is a more sensitive technique than light microscopy. In some of the studies, adverse effects were observed only by electron microscopy. The intravenous toxicity study by Sjoberg et al. (T: 76), which is discussed below, illustrate this point. The authors found no abnormalities in testis structure at the light microscope level. However electron microscopy showed that 3/5 of the animals exposed to the high dose of DEHP had slight enlargements of the smooth endoplasmic reticulum in Sertoli cells, the earliest hallmark of the DEHP-induced testis lesion and that there were also slight structural changes in early spermatocytes.

This recent study by Schilling et al. (T: 71b) has not yet been published in a peer-reviewed journal; nor has it been evaluated by other regulatory agencies.

3. Arcadi et al. (T: 40) studied Long-Evans rats during pre- and postnatal development. Adult rats were mated for five days and the pregnant rats, 12/group, were exposed to DEHP in the drinking water at doses estimated by the authors to correspond to 0, 3-3.5, and 30-35 mg/kg bw/day) starting from first day of gestation to postnatal day (pnd) 21. The pups were not exposed after weaning. 7 pups/dam were culled 2 days after delivery, and weighed each day for 8 days and twice a week for the next 4 weeks. At 3, 4, 5, 6, and 8 weeks of age, eight pups from each group were sacrificed and the testes, liver, and kidneys were weighed and assessed histologically. Blood samples were collected from 8 dams of each group and analyzed for DEHP Behavioral testing was performed in female pups on pnd 30. At the earlier ages evaluated, both exposed groups showed significant seminiferous epithelial disorganization and delayed development. By 56 days of age, the low-dose group showed some continued seminiferous tubular disorganization, but the effect appears to be more characterized as a delay in development, while spermatogenesis in the high-dose animals was clearly disrupted. Beam walking took longer in the high-dose females at pnd 30. Based on the authors' estimate of daily consumption of phthalate, these results suggest a LOAEL of 3 mg/kg bw/day for testicular toxicity in young rats following exposure during gestation and weaning. There was no NOAEL, since both treatment groups showed adverse effects.

Although this study provides additional evidence for testicular effects after gestational exposure, the NTP Expert Panel on Phthalates did not use it to identify NOAELs because of concerns about the actual amount of DEHP administered to the animals (T: 2). The DEHP was given as suspension in drinking water and there was no verification of the dose. The very low solubility of DEHP in water and the possible instability of the suspension made it essential to verify the dose.

4. In a study by Agarwal et al. (39), DEHP was administered in the diet for 60 days to mature male rats at doses corresponding to 0, 18, 69, 284 and
1,156 mg/kg/day. After the 60-day treatment, the males were mated with untreated females. At the highest dose, the weight of the testes, epididymis and prostate were reduced and there was severe testicular degeneration, a reduction in epididymal sperm density and motility, and the reproductive capacity of male rats was also reduced.

A dose-dependent reduction in total body, testes, epididymis and prostate weights was noted at the two highest doses. At the high dose, the reproductive capacity of male rats was also reduced as shown by the reduced litter size. This effect was directly correlated to the testicular degenerative effects: reduced sperm count and sperm mobility, and appearance of abnormal sperms. No significant reproductive toxicity was detected at the lower doses. These results suggest a LOAEL of 1,156 mg/kg bw/day and a NOAEL of 284 mg/kg bw/day reproductive effects. In commenting on this study, the NTP Expert Panel on Phthalates noted that the true LOAEL and NOAEL for reproductive effects may not have been identified because mature rats were studied and the most sensitive endpoints were not examined (T: 2).

5. Poon et al. (T: 29) exposed groups of young adult male and female Sprague-Dawley rats to 0, 5, 50, 500 and 5000 ppm DEHP in the diet for 13 weeks. According to the authors, these dietary concentrations corresponded to average DEHP doses of 0, 0.4, 3.7, 38, and 375 mg/kg bw/day, respectively, for the male rats and 0, 0.4, 4.2, 42, and 419 mg/kg bw/day for the female rats. At the end of the study, the hematology, clinical chemistry, weights and the histopathology of the organs was assessed.

In the testes, Sertoli cell vacuolation, characterized by the authors as "mild," was seen in 7/10 males in the 500 ppm (38 mg/kg bw/day) group, and 9/10 males in the 5,000 ppm (375 mg/kg bw/day) group showed bilateral, multifocal, or complete atrophy of the seminiferous tubules with complete loss of spermatogenesis and cytoplasmic vacuolation of the Sertoli cells lining the tubules. The LOAEL, based on the testicular effects (Sertoli cell vacuolation), was judged Poon et al. to be 38 mg/kg bw/day. This would make the NOAEL 3.7 mg/kg bw/day, which is the lowest published NOAEL for repeated-dose reproductive toxicity studies in young adult rodents.

The NTP Expert Panel on Phthalates noted that the study was thorough in design and execution but did not incorporate measures of reproductive function. There was no gestational exposure and, therefore, no evaluation of what is likely to be the most sensitive endpoint.

6. David et al. (T: 13) reported testicular effects in Fischer-344 rats in a chronic study. The 6-week-old rats (50-80 males/group) were fed diets containing 0, 100, 500, 2,500, or 12,500 ppm DEHP (0, 5.8, 29, 147, and
789 mg/kg bw/day for males) for 104 weeks. Testes weight (absolute and relative) was reduced in rats of the high-dose group. Aspermatogenesis was observed in 10/10 rats of the 789 mg/kg bw/day group at study week 78 but not in rats treated with 147 mg/kg bw/day or in the control group. At study week 105, the incidence of aspermatogenesis was significantly increased in rats exposed to 29 mg/kg bw/day and higher. The percentage of rats affected from the control to high-dose group was 58, 64, 78, 74, and 97%, respectively. The NTP-CRHRH Expert Panel on Phthalates commented on the high quality of the study but noted that it utilized suboptimal testis fixation, which may have obscured any early vacuolar lesions produced by DEHP.

David et al. (T: 13) selected a NOAEL for testicular toxicity (aspermatogenesis) of 29 mg/kg bw/day. However, the NTP-CERHR Expert Panel on Phthalates concluded that the findings indicate a NOAEL for testis effects of 5.8 mg/kg bw/day, because of the clear dose-response increase in the proportion of each group showing aspermatogenesis (T: 2). However, this NOAEL may not be reliable because of the high frequency of aspermia in the controls and the concern expressed by the authors that an increased incidence of aspermia may be a normal occurrence in the aging rat. It is therefore uncertain whether the aspermia observed at 29 mg/kg bw/day was dose-related, age-related, or a combination of both.

7. The study by Kurata et al. ( T: 170) of young adult male marmosets orally exposed (by gavage in by gavage in corn oil) for 13 weeks to 100, 500, or 2,500 mg/kg bw/day DEHP has been discussed in Section 2.6.1.3. Since no reproductive effects were detected, a LOAEL cannot be assigned. The reproductive NOAEL is 2,500 mg/kg bw/day. The results are consistent with the study by Pugh et al. (T: 184) who of found no evidence of testicular lesions in young adult cynomolgus monkeys gavage dosed with 500 mg/kg bw/day DEHP (See Section 2.6.1.3. for details.) However, as pointed out by the NTP Expert Panel on Phthalates, these studies are of no use in determining the reproductive toxicity of DEHP in animals before puberty, when the testis is still developing.

2.6.1.6.2. Parenteral Exposure

There have been very few studies of reproductive toxicity following parenteral exposure to DEHP (T: 26, 76, 173, 174, 196). The existing parenteral exposure data are generally consistent with the conclusion from the oral exposure studies that the reproductive system is a target for DEHP toxicity. However, they are of limited value for setting a NOAEL or LOAEL for reproductive toxicity because of a variety of serious design deficiencies. For example, in the studies be Agarwal et al. (173, 174), the animals had fluid-filled pouches observed to contain some DEHP, which raises concerns about incomplete absorption of the DEHP dose; the numbers of animals was relatively small; the exposures were short; the histological examination was suboptimal; and, since the animals were adults, the most sensitive endpoints were not studied.

Sjoberg et al. (T: 76) studied the effects of IV administration of DEHP on testicular histology in 40-day-old Sprague Dawley rats. The time weighted average doses of DEHP were 2.5, 25, and 250 mg/kg bw/day. There was no change in the relative weight of the reproductive organs and no abnormalities in testis structure were observed under the light microscope. However, electron microscopy revealed that in 3/5 animals exposed to the highest dose there were slight enlargements of the smooth endoplasmic reticulum in Sertoli cells, which is the earliest hallmark of the DEHP testis lesion. There were also slight structural changes in early spermatocytes. The results suggest that for reproductive changes in the 40-day-old rats, the LOAEL was
250 mg/kg bw/day and the NOAEL was 25 mg/kg bw/day.

This study used appropriate controls, appropriate histology, sensitive measures, and multiple dose levels. However, the effects reported - Sertoli cell vacuoles and germ cell structural changes - were not functional effects. Furthermore, the rats were no longer at the most sensitive developmental age. Younger rats are believed to be more vulnerable to testicular toxicity. Furthermore, the effects on testicular development, which may be a more sensitive end point, were not studied.

The Advanced Medical Technology Association (AdvaMed) recently provided Health Canada with a study on the reproductive toxicity of DEHP administered intravenously to neonatal rats for 21 days (T: 280). In the study, 2- to 5-day old male Sprague-Dawley rats, received DEHP by daily intravenous infusion at doses of 0, 60, 300, or 600 mg/kg bw/day for 21 consecutive days. DEHP dosing formulations were prepared daily by emulsifying the required amount of DEHP in Intralipid and administered via a tail vein. At the end of the 21-day treatment period (the "Primary Period"), 6 or 7 animals in each group were euthanized and necropsied. The remaining animals (9 in each group) were held without further treatment until they reached an age of about 90 days (the "Recovery Phase") and then were necropsied and examined. The following tests were performed.

At the end of the 21-day exposure period, testicular atrophy and hepatomegaly were observed in the rats exposed to DEHP doses of 300 mg/kg bw/day and 600 mg/kg bw/day. Histopathological examination of the testes of animals exposed to 300 mg/kg/day and 600 mg/kg bw DEHP showed a decrease in the diameter of the seminiferous tubules and a mild depletion of germinal epithelial cells, with focal to multifocal distribution within the testes. In addition, both the mean absolute testis weight and the testis weight adjusted relative to the body weight were decreased at exposures of 300 and 600 mg/kg bw DEHP/day at the end of both the primary treatment period and the recovery period. The NOAEL was 60 mg/kg/day. Although testicular atrophy persisted at the end of the recovery period, histopathological changes were not seen by examination under light microscopy in the recovery group previously exposed to a DEHP dose of 300 or 600 mg/kg/day for 21 days.

The functional assessment of male reproductive capacity (sperm count, sperm motility and sperm morphology) in DEHP-exposed rats conducted at the end of the recovery period showed no effect on any of these parameters.

Although recovery was seen in sperm counts, morphology and motility, testicular atrophy persisted at the end of the recovery period in rats exposed to DEHP either orally or intravenously. Even at the end of the recovery period, both the mean absolute testis weight and the testis weight adjusted relative to the body weight were decreased for the rats exposed to 300 and 600 mg/kg bw DEHP/day. This shows that the adverse testicular effects of DEHP were not completely reversed. These findings are consistent with previous studies which reported only partial reversibility of the adverse testicular effects following exposure to DEHP (T: 39, 283, 284).

The AdvaMed study is a well-designed study and has the following strengths:

• The use of 3-5 day old rats, animals at a sensitive stage in life, and the intravenous route of exposure is directly relevent to the concern about intravenous exposure of neonates to DEHP
  
  
• The doses selected, two of which certainly exceed the levels encountered during medical exposure, allow for the determination of a NOAEL.
  
  
• The solubization of DEHP in Intralipid reduces concerns about the influence of the solvent on the observed adverse effects.
  
  
• The inclusion of a recovery period provides some information on the reversibility of some of the observed adverse effects.
  
  
• The study was conducted according to the U.S. FDA Regulations for Good Laboratory Practice, and the Institute Laboratory Animal Resources Commission on Life Sciences, National Research Council: Guide for the Care and Use of Laboratory Animals.
  
  
• The tests conducted were appropriate and analytically sound.
  
  
• Except for the cases noted in the "Protocol Deviations" section of the report, the doses of DEHP adminstered to the animals were known accurately.

The study included the following limitations which need to be considered in interpreting the results:

• The study was conducted by Baxter Healthcare Corporation and commissioned by AdvaMed. There was no independent assessment of the histopathology. Only the assessment of reproductive capacity (sperm motility, total sperm count and sperm morphology) was contracted out to scientists in another company.
  
  
• A relatively small number of animals were studied at each intravenous dose (6-7 rats), which decreases the confidence in the results.
  
  
• The study did not include the electron microscopic examination of the testes. In some previous studies of of the reproductive toxicity of DEHP, adverse testicular effects were observed only by electron microscopy. For example, in the intravenous toxicity study by Sjoberg et al. (T: 76), the authors found no abnormalities in the structure of the testis at the light microscope level. However electron microscopy showed that 3/5 of the animals exposed to the high dose of DEHP had slight enlargements of the smooth endoplasmic reticulum in Sertoli cells, the earliest hallmark of the DEHP testis lesion and that there were also slight structural changes in early spermatocytes.
  
  
• The administration of high doses of DEHP to neonatal rats as a bolus via a tail vein is a novel technique. It is very different from exposure of neonates to DEHP from medical procedures, such as the parenteral feeding, in which DEHP leaches continuously from the PVC tubing for the duration of the procedure. The AdvaMed report notes that there were occasions where some of the administered dose leaked out of the injection site. Measuring DEHP blood levels would have increased the confidence in the results.
  
  
• The study exposed 3-5 day old rats to DEHP for 21 days. Thus, DEHP exposure included what is believed to be a vulnerable stage in life. However, the animals were exposed only postnatally, while humans may be exposed both prenatally and postnatally. Furthermore, some recent studies indicate that the first three weeks after birth may not be the only period during which rats are most vulnerable to DEHP toxicity. For example, a recent study by Akingbemi et al. (T: 281) indicates that exposure of 21 day old male rats to 200 mg/kg bw/day DEHP for two weeks reduced Leydig cell testosterone to 50% of the level in controls.
  
  
• Although some functional indices of male reproductive capacity recovered in DEHP-exposed rodents, fertility was not assessed by mating the animals. The conclusion that "functional impairment of reproductive capacity did not occur" is not justified.

Despite these limitations, the AdvaMed study provides the most reliable available estimate of the NOAEL for the reproductive toxicity of DEHP following intravenous exposure. It is therefore the most relevant study for assessing the potential effects of intravenous exposure to DEHP from medical procedures.

Based on the above discussion, the studies by Lamb (T: 41) and Poon (T: 29) are the most suitable for deriving LOAEL and NOAEL values for reproductive toxicity in rats following oral exposure to DEHP. The study by Sjoberg et al. (T: 76), although inadequate for the complete characterization of reproductive toxicity following parenteral exposure, provides a rough estimate of the LOAEL and NOAEL. The AdvaMed study (T: 282) provides a more reliable estimate. Table 13a lists these values.

Table 13a. LOAEL and NOAEL for Reproductive Toxicity of DEHP in Rodents

The NTP-CERHR Expert Panel on Phthalates concluded that existing data support a NOAEL between 3.7-14 mg/kg bw/day for oral exposure in rats. However, the two NOAELs within this range (Table 13a) are based on different end points: 3.7 mg/kg bw/day for Sertoli cell vacuolation and 14 mg/kg bw/day for reduced fertility and live births. It is likely that Sertoli cell vacuolation would be the earliest of the end points detected. The studies by Lamb et al. (T:41) did not look for this effect.

Phthalates are believed to be more potent reproductive toxicants at lower doses when exposure occurs during gestation (T: 43, 44, 83-84, 178). However, the existing data do not allow a reliable estimation of an NOAEL based on gestational exposure. The unpublished two-generation reproductive toxicity study of DEHP adminstered orally to Wistar rats by Schilling et al.
(T: 71b, 71c), which was reviewed in Section 2.6.1.6.1, provides a NOAEL (oral exposure) for the gestational toxicity of DEHP in Wistar rats. However, this study has not yet been published in a peer-reviewed journal; nor has it been evaluated by other regulatory agencies. Furthermore, the results may not apply to Sprague-Dawley rats, the species most frequently used to assess DEHP toxicity.

This report will therefore use 3.7 mg/kg bw/day, the value obtained by Poon et al. (T: 29), as the NOAEL for reproductive toxicity in rats orally exposed to DEHP and 60 mg/kg bw/day, the value obtained in the AdvaMed study
(T: 282), as the NOAEL for reproductive toxicity in rats exposed intravenously to DEHP. However, it will also take into account the belief that a NOAEL based on gestational exposure may be lower than this value. As a result, these NOAELs will not be used to quantitatively assess the risk posed by exposure of patients to DEHP.

2.6.2. Developmental Toxicity

There are no data on the developmental toxicity of DEHP or its major metabolites in humans. However, the numerous studies in experimental animals, largely studies of oral exposure in rodents during pregnancy, provide convincing evidence that DEHP and its metabolites (MEHP, 2-EH, 2-EHA, and phthalic acid) are developmental toxicants in rodents (T: 1-9, T: 2, 40, 41, 53-71, 197-201). These studies have consistently reported that exposure to DEHP produces malformations as well as intrauterine death and delays in development. At lower doses, developmental effects include increased prenatal mortality and fetal resorption, skeletal and limb malformations, and developmental delays. At higher doses, fetotoxic and teratogenic effects seen included: a decrease in kidney and testes weights; cardiovascular malformations (including defects in the heart and the aortic arch); eye defects; hydronephrosis; missing limb bones, finger abnormalities; malformed limbs; open neural tubes; and intrauterine death. There was also an increase in spontaneous abortions. In addition, several recent studies have reported that exposure to DEHP and certain other phthalates during gestation causes alteration in the reproductive tract of the fetus. (T: 40, 44, 84, 205-209) These include: hypospadias and vaginal pouch formation, alterations in androgen-dependent processes (i.e., testis descent, retained nipples), and malformations of the ventral prostate, seminal vesicle, levator ani plus bulbocavernosus muscles, gubernacular cord, and the epididymis (T: 205). These DEHP- induced alterations to the male reproductive tract during gestation have been discussed in Section 2.6.1 and are therefore not discussed further in this section. As pointed out in that section, these effects are believed to be the most sensitive end points for establishing NOELs for the developmental and reproductive toxicity of DEHP.

Table 13b presents a brief summary of the key studies for establishing LOAELs and NOAELs for the developmental toxicity of DEHP and its major metabolites.

Table 13b. Developmental Toxicity of DEHP and its Metabolites Following Oral Exposure

Abbreviations used: 2-EH: 2- Ethylhexanol; 2-EHA: 2-Ethylhexanoic acid; gd: gestation day.

The data in the table suggest a NOAEL of ~ 44 mg/kg bw/day for the developmental toxicity (skeletal, visceral, and external malformations) of DEHP administered orally to CD-1 mice, the most sensitive species. However, it is generally believed that the lowest dose at which developmental toxicity occurs has not yet been established, since the limited data on DEHP- induced alterations to the male reproductive tract during gestation are inadequate for determining LOAELs and NOAELs.

There are insufficient data on the developmental toxicity of DEHP administered parenterally or intraperitoneally to identify LOAELs and NOAELs for these exposure routes. In the only published IV exposure study (T: 67), no fetal toxicity was observed following the intravenous administration of DEHP to pregnant rabbits. However the doses, 1 - 5 mg/kg bw/day were lower than those used in the oral exposure studies. The lowest dose reported to produce fetal toxicity following IP administration was 1,970 mg/kg bw/day (T: 68). No fetal toxicity in pregnant rats in an inhalation study at the maximum dose of 84 mg/kg bw/day (T: 66).

In the case of orally administered MEHP, resorption rather than malformation is the most sensitive endpoint with a LOAEL of 35 mg/kg bw/day. The data also show that both 2-EH and 2-EHA administered orally are developmental toxicants in rodents with NOAELs of ~ 130 mg/kg bw/day (decreases in fetal weight) and ~ 100 mg/kg bw/day, respectively.

The mechanism of DEHP-induced developmental toxicity has not been studied as extensively that of DEHP-induced reproductive toxicity. Peters et al. observed DEHP-induced developmental toxicity (fetotoxicity and teratogenicity) in PPARa knockout mice (T: 159, 160). These effects therefore occur independently of peroxisome proliferation.

The results obtained with the PPARa knockout mice do no rule out a role for PPAR activation in DEHP-induced developmental toxicity, since rat embryos express three isotypes of PPAR (alpha, beta or delta, or gamma (T: 210, 211). Maloney and Waxman recently reported that MEHP, but not DEHP,
2-EH or 2EHA, activated the PPARg receptor in PPARa knockout mice.
(T: 162), while valproic acid, a structural isomer of 2-EHA, has been shown to activate the PPARd receptor (212). As discussed in Sections 2.5.2 and 2.6.1.5, PPARg is highly expressed in a wide variety of human tissues, including the testis (T: 2, 3). Thus, the developmental toxicity reported in rodents may be directly relevant to humans.

Studies by Peters et al. (T: 57) and Bui et. al (T: 213) provide some evidence in support of the hypothesis that the mechanism of action for DEHP-induced developmental toxicity is the induction of zinc deficiency secondary to an acute phase response. Although zinc deficiency is known to be a risk factor for developmental toxicity and an acute phase response to DEHP has been reported (T: 57), the evidence is far from conclusive.

Grey et al. (T: 206) have recently suggests that DEHP induces reproductive tract abnormalities in males fetuses (T: 40, 44, 84, 205-209) by reducing testosterone to female levels during the critical stage of reproductive tract differentiation.

As discussed in Section 2.6.1.5, more than one mechanism may be required to explain the diverse toxic developmental effects produced by DEHP. However, the mechanisms proposed to date support the conclusion that the reproductive toxicity seen in animals following exposure to DEHP is relevant to humans.

2.7. Adverse Human Health Effects

There have been no controlled epidemiological studies of toxicity in humans exposed to DEHP and there no data on the reproductive and developmental toxicity of DEHP or its major metabolites in humans. However, there are a few reports in the medical literature of a variety of adverse effects seen in patients exposed to DEHP during medical procedures. These include:

• pulmonary toxicity;
• peritoneal sclerosis in patients on peritoneal dialysis;
• liver cholestasis in infants on ECMO;
• cardiovascular toxicity; and
• nephrotoxicity.

In reviewing these reports, it is important to note that although infants undergoing intensive medical therapy are exposed to relatively high doses of DEHP, there is very little direct (clinical) evidence that DEHP exposure causes significant adverse effects in this vulnerable subpopulation.

2.7.1. Pulmonary Toxicity

Intravenous administration of DEHP to rats and dogs has been reported to produce a range of adverse on the lungs, including respiratory distress, increased lung weight, inflammation, infiltration of polymorphonuclear cells, tracheal bleeding, and pulmonary edema (T: 5, T: 12). Although some investigators have attributed these effects to the surfactant used to solubilize DEHP, Rubin and Chang reported adverse pulmonary effects in rats at doses as low as 8 to 13 mg/kg body weight following administration of DEHP solubilized in plasma without a surfactant (T: 245). Similarly, Rutter (T: 245) reported pulmonary effects in dogs (thickening of the alveolar membrane and infiltration of polymorphonuclear cells and increased lung weight) following the intravenous administration of undiluted DEHP at a dose of 100 mg/kg for six weeks. In contrast, in a study of the effects of IV administration of DEHP on 40-day-old Sprague Dawley rats, Sjoberg et al. (T:76) detected no pulmonary damage at intravenous doses as high as 250 mg/kg bw/day.

Inhalation of DEHP may produce adverse effects in the lungs similar to those reported for intravenous exposure. Klimisch et al. (T: 249) reported an increase in lung and liver weights of rats after inhalation exposure to 1 mg/L,
6 hours/day, 5 days/week for 28 days, equivalent to a DEHP dose of 230 mg/kg bw/day. These changes were accompanied by histological lung changes including a thickening of alveolar sputums and foam cell proliferation. However, Merkle et al. (T: 66) found no adverse effects in pregnant rats or their offspring exposed to 300 mg/m3 for 6 hr/day (equivalent to a dose of 80 mg/kg bw/day) on gestational days 6-15.

There have been several case reports of similar pulmonary effects in pre-term infants exposed to various medical procedures associated with DEHP exposure, such as ECMO treatment and blood transfusion.

Lung opacity increases immediately after initiation of ECMO in adults and infants and has been found to correspond to decreased pulmonary function, and severe opacity correlates strongly with mortality (T: 247, 248). Karle et al. observed that that infants on ECMO had worse chest radiographic scores than those not undergoing the procedure and suggested that DEHP may play a role in lung injury in patients on ECMO (T: 20). However, they found no correlation between the chest radiographic score and the concentration of DEHP in the serum. Furthermore, they found no evidence of pulmonary toxicity in infant undergoing ECMO, other than the chest radiographic scores.

The widely accepted explanation for the adverse effects observed in the lungs of patients undergoing ECMO is that they are caused by pressure changes across the alveolus (T: 247). High ventilator pressures (high peak inspiratory and positive end-expiratory pressures) are required to maintain oxygenation in patients before the start of ECMO. Since adequate oxygenation can be achieved at lower ventilator pressures once ECMO is started, the ventilator pressure settings are reduced to reduce the risk of barotrauma. The reduction in the alveolar-capillary pressure gradient will force fluid from the capillary into the alveolus. The reduced pressure gradient can also cause collapse of the alveoli.

Roth et al. (T: 244) reported lung disorders in three pre-term infants ventilated with heated PVC respiratory tubes for 4 weeks. According to Roth et al., this treatment exposed the infants to very high doses of DEHP. However, as discussed in Section 1.3.3.4.1, estimates of exposures were based on DEHP levels in the condensate, which cannot provide a reliable estimate of inhalation exposure, since the infants were not exposed to the condensate. Furthermore, the type of circuit used by Roth et al. has since been replaced by heated tubing with a lower mean wall temperature. In addition, commercial respiratory tubing intended for use with ventilators is currently made from polyethylene rather than PVC.

The lung disorders observed by Roth et al. (T: 244) in the three pre-term infants resembled hyaline membrane disease. In two of infants, clinical signs of the disease were observed between the 2nd and 4th weeks of treatment. These infants showed normal development in the 20-month follow up period. The third infant died 14 days after birth. The autopsy revealed atelectasis and little alveolar differentiation in the lung but no signs of hyaline membrane. Roth et al. hypothesized that DEHP was responsible for the lung disorders, possibly by interfering with the formation of alveolar surfactant. However, Karle et al. (T: 20) reported that surfactant therapy did not influence the chest X-ray scores.

As discussed in Section 1.3.3.4.3, Latini et al. (T: 9, 250) observed a loss of DEHP from endotracheal tubes used to ventilate very low birth weight infants, suggesting significant infant exposure. Based on evidence of pulmonary toxicity in animals and the lung disorders in pre-term infants reported by Roth et al. (T: 244), they hypothesized that DEHP could play a role in causing bronchopulmonary dysplasia. They also pointed out that previous in vitro studies indicated that DEHP may enhance the release of lysosomal enzymes from alveolar macrophages during phagocytosis (T: 251) and inhibit alveolar macrophage killing (T: 252). Alveolar macrophages produce and release reactive oxygen species and a wide variety of proteases that can cause lung damage and emphysema (T: 251). However, in the absence of a randomized controlled study comparing infants ventilated with and without PVC-containing tubing, there is no clinical evidence to establish a causal relationship between bronchopulmonary dysplasia and exposure to DEHP.

Transfusion-related acute lung injury, a serious complication associated with the transfusion of plasma-containing blood products, is characterized by acute respiratory distress, pulmonary edema and hypoxemia (T: 253 - 255). The preponderance of evidence suggests that transfused complement-activating antibodies (either granulocyte or HLA-specific) act as mediators, which result in granulocyte aggregation, activation, and microvascular pulmonary injury (T: 253). Silliman et al. (T: 254, 255) observed that plasma from blood stored for extended periods of time produced this type of lung injury, while plasma separated the day the blood was donated did not. Sillman et al. also found that extraction of the lipids from plasma eliminated the pulmonary toxicity from plasma stored for extended periods of time, suggesting that a substance in the plasma was responsible for the adverse effects. Since DEHP, which is lipophilic, is associated with the plasma fraction of the blood, these results are consistent with its involvement in transfusion-related acute lung injury. However, there is no clinical proof that it does play a role. Since a number of substances associated with the lipid fraction increase with storage time, one of these may play a role in the pathogenesis of lung injury.

A wide range of pulmonary complications has been reported in patients undergoing hemodialysis (T: 256). Knudson et al. (T: 257) observed adult respiratory distress-like syndrome during hemodialysis in twelve patients treated with chronic maintenance hemodialysis. Although these symptoms were similar to those sometimes observed after transfusion of plasma-containing blood products (T: 253 - 255), there was no evidence that the DEHP released during the procedure was responsible. Instead, an analysis of the data suggested that activation of complement by the dialysis membrane, leukopenia, and release of granulocyte elastase played a role in the pathogenesis of the pulmonary effects.

Several publications have suggested that DEHP could play a role in the pathogenesis of asthma (T: 47, 48, 258) Doelman et al. (T: 47, 48) reported that MEHP produced a dose-dependent decrease in methacholine-induced contraction of rat tracheal muscle. Based on the structural similarity of DEHP and MEHP to prostaglandins and thromboxanes, substances known to cause bronchospasm in the lungs, Oie et al. (T: 258) suggested that DEHP may cause asthma by stimulating production of prostaglandins and thromboxanes in the lung. However, there is no evidence that DEHP or MEHP can produce bronchoconstriction in humans or animals. Furthermore, there have been no reports of bronchoconstriction in the patients in studies of procedures associated with high DEHP exposures.

2.7.2. Peritoneal Sclerosis in Patients on Peritoneal Dialysis

Peritoneal sclerosis is a serious complication of peritoneal dialysis (T: 259-262). Its etiology is unknown but the evidence suggests that both peritonitis and exposure to dialysate contribute. Factors such as osmolarity, pH and lactate content of the dialysis solution, and dialysate degradation products generated during heat sterilization, and recurrent episodes of peritonitis may all contribute to the pathogenesis of peritoneal sclerosis (T: 259, 260, 262). Based on in vitro studies showing that DEHP increases the secretion of the cytokine, interleukin-1 (IL-1) from mononuclear human cells, Calo et al
(T: 264 ) and Fracassi et al. (T: 265 ) have suggested that DEHP may play a role in the pathogenesis of peritoneal sclerosis. Cytokines such as interleukin-1 (IL-1) are known to stimulate fibroblast production and collagen biosynthesis, both of which are necessary for the development of fibrosis of the peritoneal membrane. The observation by Stabellini (T: 262) that the introduction of DEHP into an air pouch in the subcutaneous tissue of rats produced hyperplastic alterations in the fibroblasts provides some support for the hypothesis that DEHP may plays a role in the etiology of peritoneal sclerosis. However, it does not prove that DEHP does play a role, since other substances present in the dialysate may also be capable of releasing cytokines.

2.7.3. Liver Cholestasis in Infants on ECMO

Shneider et al. (T: 266, 267) reported cholestasis in infants undergoing ECMO treatment. In one study (T: 267), the degree of cholestasis correlated with DEHP levels in the infants. While cholestasis was reported, no other signs of liver toxicity (e.g. increased serum transaminase levels) were observed. In contrast, infants on ECMO in the study by Karle et al. (T: 20) and those receiving exchange transfusions in study by Plonait et al. (T: 268) did not develop cholestatis. However, the development of cholestasis may depend on both the level and duration of exposure. Plonait et al. reported the highest mean plasma concentration of DEHP but very short exposure times (< 1 day), while the mean plasma concentration of DEHP and the duration of exposure reported by Shneider et al. were higher than those reported by Karle et al.

Shneider et al. postulated that DEHP exposure during ECMO may cause cholestasis but noted that other factors, particularly hemolysis, which is significant during ECMO, are likely to play an important role. Infants do not have mature glucuronidation pathways until they are 3 months old (T: 85). Since this important clearance mechanism is not fully available to neonates and young infants, they may not be able to eliminate the large bilirubin load produced by significant hemolysis. Sjoberg et al. reported competitive inhibition between MEHP and bilirubin for glucuronidation in the guinea pig
(T: 270). Thus, exposure to high concentrations of both DEHP/MEHP and bilirubin may decrease the rate of elimination of both substances and prolong the time of exposure to them. This would explain why exposure to high levels of DEHP may increase the risk of cholestasis. As Latini et al. Have pointed out (T: 9), this may also explain the high levels of MEHP found in hyperbilirubinemic infants undergoing exchange transfusions.

2.7.4. Cardiovascular Toxicity

Rock et al. (T: 52) reported large decreases in heart rate and blood pressure and incidents of cardiac arrest in rats infused with MEHP. The NOAEL and LOAEL for decreased heart rate - the most sensitive effect seen in this study - were 28.5 and 57 mg/kg, respectively. In a follow up study, Barry et al. (T: 271, 272) reported that in vitro exposure of human myocardial tissue to MEHP - but not to DEHP - produced arrhythmias, and a concentration-dependent reversible negative ionotropic effect (reduced force of cardiac contraction). The concentration of MEHP that gave a 50% reduction in strength of isolated muscle model was only 20 to 30-fold higher than the circulating levels measured in patients during heart surgery. Barry et al. therefore concluded that high concentrations of MEHP have the potential to cause cardiac toxicity in humans. However there is no evidence that MEHP produce effects of any clinical significance in patients. The alternative explanations for the hypotension (e.g., a decrease in plasma osmolality, volume depletion, impairment of sympathetic autonomic response, and the use of acetate in the dialysate) and the reduced cardiac output seen in hemodialysis and ECMO patients (See Section 2.7.1) seem to be more plausible.

2.7.5. Nephrotoxicity

Acquired cystic kidney disease (ACKD) is associated with serious complications, the most serious of which is the potential malignant transformation of the cysts and the development of renal cell neoplasms, ranging from adenoma to metastatic carcinoma. (T: 92, 93, 273-75). A review of the literature indicates an up to a 50-fold increased risk of renal cell carcinoma in ACKD compared to the general population (T: 275). The disease often occurs or worsens in patients who are on long-term dialysis for end-stage renal disease. The prevalence of ACKD is directly related to the duration of dialysis and the risk of cancer is directly related to the presence of cysts. (T:273- 274). It has been suggested that exposure to DEHP during dialysis may play a role in the etiology of the disease, since renal cysts are one of the adverse effects sometimes seen in rodents after long-term oral administration of DEHP (T: 18, 35, 40, 95). These studies have been discussed in Section 2.3.2, where it was pointed out that in other studies
(T: 13, 14, 94) long-term or exposures to high doses of DEHP failed to produce cystic or other major lesions in the kidney of both rats and mice. Furthermore, it is generally accepted that ACKD develops as a consequence of sustained uremia and can first manifest even before dialysis is initiated while the patient is still in chronic renal failure (T: 273). Since patients on dialysis appear to have a longer life expectancy, more are expected to develop ACKD.

Although the etiology of ACKD in dialysis patients is still being investigated, a number of mechanisms have been proposed. Growth factor-induced compensatory growth of tubular epithelium initiated by the changes of end-stage kidney disease, and probably perpetuated by activation of proto-oncogenes, seems to be an significant factor. Exposure to chemical contaminants other than DEHP, which are released from hemodialysis equipment or dialysate, may also play a role. Contributing factors in the development of cystic kidney disease, such as the role of drugs given to these patients, and the influence of other disease states have not been determined. It known that other kidney illnesses such as glomerulonephritis induce cystic kidney disease (T: 273-275).

Thus, the inconclusive data from animal studies and the observation that ACKD often occurs or worsens in patients who are on long-term dialysis are insufficient to establish a role for DEHP in the pathogenesis of ACKD or renal cancer.

2.7.6. Summary

There are very few human reports of toxicity in humans following exposure to DEHP. Although several plausible mechanisms have been proposed for involvement of DEHP in the pathogenesis of the adverse effects reported in patients undergoing ECMO, exchange transfusions, cardiopulmonary bypass and hemodialysis, a review of the few available reports of adverse effects in humans indicates that they are insufficient in design and outcome to demonstrate any cause-effect relationship between human exposure to DEHP and toxicity in humans. On the other hand, the absence of evidence does not rule out a possible role for DEHP.

3. CONCLUSIONS

3.1. Exposure

Exposure to DEHP occurs routinely during a variety of medical procedures that involve the use of medical devices made from polyvinyl chloride. Most medical exposures are intravenous involving the infusion of blood and blood products (which also result in exposure to MEHP), but inhalation during respiratory therapy and ingestion (e.g., from nasogastric tubes) also occurs.

The reliability of DEHP exposure estimates for medical procedures is limited by the small number of patients used in the studies, patient-to-patient variations, differences in treatment protocols, and differences in medical equipment used. Several important exposures in neonates and in the fetus and have not been reported in the scientific literature, including exposures from parenteral and enteral feeding, ventilators, IV fluids, or combinations of simultaneous exposures, and placental transfer of maternally-derived DEHP/MEHP from medical procedure. In fact, all published studies of DEHP exposure from medical devices consider only single exposure sources. For many patients, particularly critically ill neonates, examining single sources of exposure (e.g., ECMO or ventilation) may significantly under-estimate the total exposure. Furthermore, the total DEHP exposure from combinations of simultaneous exposures may vary dramatically from one medical centre to another, depending on the treatment protocols used.

The report has attempted identify the most reliable exposure estimate reported for each procedure (Table 10 and the associated discussion). However, since all exposure studies are based on a very small number of patients, the table also lists the range of exposures reported in all studies. Exposures encountered in some clinical settings may fall outside the ranges shown in the table.

The data indicate that in adults, transfusion of blood, cardiopulmonary bypass, and infusion of lipophilic drugs using PVC bags and tubing (contrary to the directions for use) result in very high short-term exposures relative to the general population exposure. Blood transfusions to the trauma patient is the procedure which gives the highest acute exposure in adults - up to
8.5 mg/kg bw/day. Long-term hemodialysis exposes adults to the highest chronic daily dose - 1-2 orders of magnitude above the general population exposure.

In the infants, important sources of short-term or subacute medical exposures are volume exchange transfusions, extracorporeal membrane oxygenation (ECMO), cardiac bypass procedures, total parenteral nutrition therapy, infusion of lipophilic drugs using PVC bags and tubing, and possibly, respiratory therapy. Double volume exchange transfusion is the short-term procedure which is reported to give the highest acute exposure - up to 23 mg/kg bw/day, while extracorporeal membrane oxygenation (ECMO) is the sub-acute medical treatment which gives one of the highest daily DEHP exposures per kg body weight and the highest daily exposure over a prolonged period of time - up to 14 mg/kg/day during the 3 to 30-day treatment period.

3.2. Toxicity

DEHP produces a variety of subchronic and chronic toxic effects in developing and adult animals. The main target organs are the liver, kidneys, reproductive tract, lungs, and heart.

There are no data on the carcinogenicity of DEHP or its major metabolites in humans. However, DEHP induces liver tumors in rodents when administered in the diet at high doses. A review of the available data indicates that activation of the peroxisome proliferator-activated receptor-a (PPARa) plays an essential role in the induction of peroxisome proliferation and liver tumours in rodents. MEHP, a major metabolite of DEHP, activates PPARa in both rodent and human livers. However, peroxisome proliferation and development of liver tumours have not been observed in humans exposed to peroxisome proliferators. The very low levels of PPARa found in human liver and the fact that most of the PPARa sites are occupied by competing proteins likely results in insufficient activation of PPARa. This makes carcinogenesis mediated by the PPARa activation pathway highly unlikely in humans and suggests that liver cancer data from rodents are not relevant for human risk assessment. The International Agency for Research on Cancer (IARC) recently reached the same conclusion and declared DEHP to be "not classifiable as to carcinogenicity to humans."

There are no data on the reproductive and developmental toxicity of DEHP or its major metabolites in humans. However, there are sufficient data to concludes that oral exposure to DEHP can cause reproductive and developmental toxicity in rodents. Testicular toxicity is the main lesion observed in most rodent species. Analysis of the data obtained with rats suggests a NOAEL of 3.7 - 14 mg/kg bw/day for testicular toxicity (3.7 mg/kg bw/day for Sertoli cell vacuolation) following oral exposure and a NOAEL of 60 mg/kg/day for testicular toxicity following intravenous exposure.

Testicular toxicity (vacuolation of Sertoli cells) appears to be the most sensitive toxicity end point reported to date. Phthalates are believed to be more potent reproductive toxicants at lower doses when exposure occurs during late gestation. However there are very few data on reproductive effects following DEHP exposure during the late gestational periods, making it impossible to use these effects as end points in a quantitative risk assessment.

There are very few human data from which to characterize the toxicity of DEHP. There have been a few reports in the medical literature of a variety of adverse effects seen in patients exposed to DEHP during medical procedures. These include:

• pulmonary toxicity;
• peritoneal sclerosis in patients on peritoneal dialysis;
• liver cholestasis in infants on ECMO;
• cardiovascular toxicity; and
• nephrotoxicity.

A review of these reports indicates that they are insufficient in design and outcome to demonstrate any cause-effect relationship between human exposure to DEHP and the occurrence of toxicity.

In the absence of experimental data on the toxicity of DEHP in humans, the evaluation of human risk from medical exposures (mostly intravenous) must be extrapolated from studies in experimental animals (mostly studies of toxicity following oral exposures), where species differences in metabolism are important considerations. Uncertainties exist in the performance of such extrapolations.

A review of the toxicokinetics of DEHP in rodents and primates and possible mechanisms of action of DEHP-inducing reproductive toxicity lead to the conclusion that the rodent data are relevant to predicting that DEHP has the potential to produce adverse reproductive effects in humans. However, in terms of risk assessment, route of exposure and dose are important parameters that need to be taken into account when extrapolating across species.

There are significant data gaps that prevent a quantitative assessment of the risk to humans associated with exposure to DEHP from medical devices. The NTP-CERHR Expert Panel on Phthalates (T: 2) has identified the following critical information needs.

• Data from animal studies using parenterally administered DEHP with the focus on developmental and reproductive effects. The AdvaMed study (T: 282) fills this information gap;
• Data on reproductive and developmental toxicity following exposure to DEHP in utero, particularly during late gestation, which may be the most sensitive window for phthalate reproductive toxicity. The most critical data need is: dose/response data for male and female reproductive tract malformations. A NOAEL for perinatal exposure cannot be obtained from existing data;
• Data on the reproductive and developmental toxicity of additional species, particularly primates. An insufficient number of non-rodent species have been used in developmental toxicity studies to establish a reliable relationship between inter-species differences in pharmacokinetics and developmental toxicity to reliably extrapolate to human toxicity;
• Data on the pharmacokinetics and metabolism in patients undergoing exposure to DEHP from medical treatments; and
• An improved physiologically-based pharmacokinetic (PBPK) model that would extend the existing model to more species and take into account possible exposure of the fetus.

3.3. Relative Risks

In the absence of this critical information and the existence of uncertainties about combinations of simultaneous exposures, makes it impossible to provide a quantitative risk assessment. However, based on the available data on the exposure, toxicity, toxicokinetics and mechanism of toxicity of DEHP data, the following general conclusions can be made about relative human risks associated with DEHP exposures.

3.3.1. Adults

There is very little concern that exposure to DEHP from medical procedures will cause reproductive toxicity in human adults. This conclusion is based on the following considerations.

1. Adult marmosets (primates) exposed to an extremely high oral dose of DEHP (2,500 mg/kg bw/day) showed no signs of reproductive toxicity. The same dose of DEHP produces severe testicular toxicity in juvenile rodents.

2. Adult rodents are 10 - 100 times less sensitive to the reproductive toxicity of DEHP than juvenile rodents.

3. Conversion of DEHP to MEHP, the active toxicant, involves lipases. There is less conversion of DEHP to MEHP by the parenteral route of exposure than by oral exposure, although the extent of reduction is unknown.

3.3.2. Critically Ill Infants and Young Children

There is serious concern than DEHP exposure from medical procedures may adversely affect male reproductive tract development of critically ill infants undergoing intensive medical therapy. Medical procedures of greatest concern include volume exchange transfusions, extracorporeal membrane oxygenation (ECMO), cardiac bypass procedures, total parenteral nutrition therapy, infusion of lipophilic drugs using PVC bags and tubing, and possibly, respiratory therapy. However, the benefits of medical procedures may outweigh these risks. This conclusion is based on the following considerations.

1. Parenteral exposure of infants to DEHP from medical procedures can, in some cases, approach the NOAEL for DEHP administered intravenously to rats.

2. Parenteral exposure involving infusion of blood or blood products also exposes the infant to MEHP, the active toxicant.

3. Infants may have higher levels of plasma and hepatic lipases than adults, suggesting that they may convert DEHP to MEHP more rapidly than adults.

4. Heparin and DEHP itself may induce plasma lipase activity. Thus, exposure to heparinized blood or repeated exposure to medical procedures may increase the rate of conversion of DEHP to MEHP.

5. Infants do not have mature glucuronidation pathways until they are three months old. This may result in prolonged exposure to DEHP and its metabolites.

6. The reproductive system of infants is developing and the developing testes are more susceptible to damage from DEHP exposure.

7. Young Sertoli cells are more susceptible to damage from MEHP than older cells.

8. It is not know if primate Sertoli cells are more or less sensitive than those in primates to the effects of DEHP.

9. There is less conversion of DEHP to MEHP by the parenteral route of exposure than by oral exposure (although the extent of reduction is unknown). This factor reduces the concern.

10. The following considerations are relevant to oral exposure (e.g., from nasogastric tubes):

• At high doses of DEHP, intestinal absorption and/or hydrolysis of DEHP occurs more rapidly in rodents than in primates and a smaller proportion of DEHP is hydrolyzed and absorbed as the monoester in primate intestine. However, there may be no significant difference between rats and primates in rates of DEHP absorption and/or hydrolysis at medically relevant doses;
• Although human neonates have much lower levels of pancreatic lipase than adults, they may be able to convert DEHP to MEHP as efficiently as adults via gastric, hepatic, and plasma lipases; and
• Intestinal absorption of DEHP/MEHP and/or conversion of DEHP to MEHP occurs more readily in young rodents than in adults. Thus, for the same DEHP exposure, young rodents would receive a higher systemic and testes dose than mature rodents. Children more than three months old have mature glucuronidation pathways. This reduces the concern to some extent. However, the other considerations apply. In particular, it should be noted that the reproductive system, including the Sertoli cells, is still developing and more susceptible to damage from DEHP exposure.

3.3.3. Pregnant Women

There is inadequate information to assess this risk. However, the following considerations suggest that there is a concern that pregnant women undergoing certain medical procedures may adversely affect the development of their offspring.

1. The fetus is at the most vulnerable stage of life.

2. Exposure to DEHP during gestation causes serious malformations in the male reproductive tract of the fetus. Even short-term exposure are effective in producing irreversible effects. The available data are inadequate for the reliable estimation of a NOAEL based on this adverse effect, which may be the most sensitive end point for developmental and reproductive toxicity of DEHP.

3. The active toxicant, MEHP, passes across the placenta and into breast milk.

4. Short-term parental exposure of pregnant women to DEHP from certain medical procedures may approach, if not exceed the NOAEL for reproductive and developmental toxicity in rodents.

5. There is less conversion of DEHP to MEHP by the parenteral route of exposure than by oral exposure, although the extent of reduction is unknown. This factor reduces the concern.

3.3.4. Recommendations

Based on these findings, it is recommended that Health Canada develop a policy statement on the use of DEHP in medical devices to provide health care users with guidelines on managing the risks. The policy should take into account both the risks and benefits associated with the use of DEHP. The use of DEHP as a plasticizer for blood storage bags has a clear advantage in that it prolongs the storage time of whole blood, perhaps by stabilizing the erythrocyte membrane. In addition, DEHP-containing PVC has many physical properties that make it highly suitable for fabricating medical bags and tubing. It is easily shaped, flexible, strong, optically clear, suitable for use at a wide range of temperatures, and easily sterilized. Current alternatives to DEHP-plasticized PVC used in medical devices (Table 14) consist of PVC softened with alternative plasticizers (citrates, trimellitates, and adipates) or alternate plastics (metallocene polyolefins, acetates, polyurethanes, and silicone). Some of these alternatives lack a number of the benefits of PVC plasticized with DEHP, while other are much more expensive. There is also inadequate safety data for a number of the alternatives. They may expose patients to hazards not present with devices made with DEHP. These factors must be taken into account is reviewing the acceptability of DEHP-containing medical devices for specific medical treatments.

Table 14. Alternatives to DEHP-Plasticized PVC

1. Alternative Plasticizers for PVC

Trioctyltrimellitate (TOTM)
Tri-2(ethylhexyl) trimellitate (TEHTM)
H-640, a polymeric plasticizer (Hatco Corp.; Fords, NJ)
Acetyltri-n-butyl citrate (Citroflex A-4, Morflex Inc)
Acetyltri-n-hexyl citrate (Citroflex A-6)
N-butyryl-tri-n-hexyl citrate (BTHC) (Citroflex B-6)
Polymeric adipate-type plasticizers (BP Amoco Chemicals; Naperville, IL)

2. Alternative Plastics

• Metallocene Polyolefins Polyethylene Polyethylene laminatePolyethylene/polypropylene copolymer Polyethylene coated (for tubing)
  • Polypropylene
  • Blends
  • Laminates
    
    
• Acetates
• Ethylene vinyl acetate
• Polytetrafluoroethylene
• Polyuerethanes
• Silicones

The policy statement will need to assess the following risk management options.

Phasing Out the Use of DEHP in Medical Devices

Do the findings of the report justify this option? Is this option practical? In addition to an assessment of the potential adverse health effects caused by exposure to DEHP in medical devices, careful consideration also needs to be given to the availability of safe and effective alternatives in sufficient volume and at a reasonable cost. The impact that banning DEHP is likely to have on the health care delivery system in the short-term and the long term, including the significantly higher cost of alternatives for a number of common medical procedures and the need to develop new protocols for other procedures, also has to be taken into account. The long-term costs cannot be accurately estimated since market forces are likely to dramatically lower the costs of alternative products.

Restricting the Use DEHP

Do the available data justify restricting the use of DEHP to certain procedures or certain patients?

Health Canada has taken this approach in its position statement on the safety of dental amalgam, which recommended restricting the use of dental amalgam in young children, pregnant women and other groups considered to be more vulnerable than the general population. The alternatives to dental amalgam were much more than expensive.

Labelling

Can the risks of DEHP exposure from medical procedures be managed through the use of warning labels? What information would be needed to ensure that health care providers are fully informed of the risks and the availability of alternative products? Should there be special warning labels on specific medical devices indicating that they are not recommended for use in medical procedures such that are associated with high DEHP exposures? For example, should uncoated DEHP tubing have a warning that it unsuitable for use in ECMO procedures? How effective would warnings in the labelling be?

Informed Consent

Can the risks of DEHP exposure from medical procedures be managed through informed consent? What measures would be needed to ensure that all patients are fully informed of the risks and the availability of alternative products. What information should be provided? Should special advice be given to patients who are most vulnerable or to all patients undergoing procedure associated with the higher exposures?

Technological Solutions

Are there ways of reducing the release of DEHP from medical devices? Coating DEHP-containing tubing with other inert polymers (e.g., polyethylene) or other materials may prevent the release of DEHP while retaining the desirable properties of the tubing. Polyethylene-coated DEHP tubing is commercially available. Heparin-coated tubing is commercially available for
ECMO circuits. The report discusses a study suggesting that extracorporeal membrane oxygenation using heparin-coated tubing would expose the patient to little or no DEHP. The use of ECMO sets, which consist of heparin-coated PVC tubing and non-PVC filters, appears to be the current standard practice in the U.S. because of the perceived clinical benefits (T: 277-279). Hemorrhagic disorders caused by systemic heparinization are frequent during ECMO. There is some evidence that the use of heparin-coated tubing for ECMO improves the safety of this procedure by reducing the thrombogenicity of the extracorporeal circuit and the need for systemic heparinization. Reduced postoperative blood loss and the need for fewer transfusions appear to be the main benefits of heparin-coated circuits.

APPENDIX 1

In the past few years, the physiological role of PPARg has been extensively investigated. It is now well-established that PPARg is abundantly expressed in adipose tissues of all mammals, and functions as a central regulator of adipocyte differentiation (T: 163). While its critical function has been identified as regulating systemic glucose and lipid homeostasis, other roles of PPARg may include cell-cycle regulation, differentiation of monocytes, and regulation of their functions. Since PPARg appears to have many critical cell functions, there has been a great deal of interest in identifying natural and synthetic ligands that bind and activate this receptor in order to modulate its activity. Natural ligands that bind and activate PPARg include a variety of fatty acids and eicosanoids. In addition, a group of synthetic compounds, the thiazolidinediones (TZDs), can also function as PPARg ligands. TZDs, such as troglitazone, were originally developed for the treatment of type-2 diabetes, because of their ability to enhance the activity of insulin in skeletal muscles. In addition, these ligands have a marked effect on the release of signalling molecules from the adipose tissue. Thus, PPARg appears to play a major role in the regulation of energy storage and metabolism, and cell differentiation.

It has also been demonstrated that PPARg activation results in cessation of cell growth. For instance, treatment of different cell lines with TZD induces an arrest in cell-cycle during logarithmic growth-phase (T: 164). Because of its regulatory activity in cell replication, there has been some interest to find out whether or not PPARg might be a useful target for treatment of tumours. In fact, synthetic PPARg ligands have been shown to inhibit growth of several tumour cell lines in culture (T: 165, 166).

Furthermore, there is also some evidence of inhibition of tumour cells from in vivo experiments. Suh et al. (T: 276) recently reported that a new ligand (a tyrosine analogue) of PPARg inhibited the development of breast cancer in an experimental rat model. In this study, mammary tumours were induced in rats by exposing them to nitrosomethylurea, a known carcinogen. The incidence of tumours decreased significantly when the exposed animals were also treated with the tyrosine analogue ligand of PPARg. This study, in conjunction with the in vitro experiments, provides some evidence that PPARg activation by certain ligands may have beneficial health effects. Thus, not all consequences of ligand binding or activation of this receptor should be viewed as undesirable effects.

However, in sharp contrast to the above positive findings, there are other studies which suggest that PPARg activation may have an opposite effect; i.e., it may promote tumour growth. Activation of PPARg was found to promote the development of colonic tumours in mice.

Another area of interest for extensive investigation is the role of PPARg in monocyte/macrophage function. It has been demonstrated that this receptor is expressed in significant levels in these cells, and production of inflammatory cytokines is blocked by known PPARg ligands. Thus, PPARg may function in the suppression of the inflammatory response elaborated by monocytes and macrophages. This function may also be considered as a beneficial effect of PPARg activation.

In summary, PPARg is a well characterized receptor, which appears to have a critical role as a metabolic regulator of glucose and lipid homeostasis. Nevertheless, our present knowledge, related to its involvement in regulation of cell cycle, apoptosis, and tumour development is insufficient and the data are too inconsistent to provide any conclusive evidence for its role in these processes. Thus, the physiological role of PPARg in these processes remains to be clarified. While a connection between PPARg activation and carcinogenesis is unlikely, a link with other toxic effects, such as those observed in the kidney and testis of mice cannot be ruled out.

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