EPA/630/P-02/002F 

December 2002 

Final Report 

A REVIEW OF THE REFERENCE DOSE AND
 
 

REFERENCE CONCENTRATION PROCESSES
 
 

Prepared for the 
 
 

Risk Assessment Forum
 
 

U.S. Environmental Protection Agency
 
 

Washington, DC
 
 

Reference Dose/Reference Concentration (RfD/RfC) Technical Panel 

Bob Benson (OPRA/Region 8) Edward Ohanian (OST/OW)
 
 

Gary Foureman (NCEA/ORD) Jennifer Orme-Zavaleta (NHEERL/ORD)
 
 

Lee Hofmann (PARMS/OSWER) Deborah Rice (NCEA/ORD)
 
 

Carole Kimmel (NCEA/ORD)* Jennifer Seed (OPPT/OPPTS)
 
 

Gary Kimmel (NCEA/ORD) Hugh Tilson (NHEERL/ORD)
 
 

Susan Makris (OPP/OPPTS) Vanessa Vu (SAB Staff Office, formerly
 
 

Deirdre Murphy (OAQPS/OAR) OSCP/OPPTS and NCEA/ORD)
 
 

*Technical Panel Chair 

Technical Advisors 

Amy Mills, IRIS Director, NCEA/ORD 

Bill Wood, RAF Director, NCEA/ORD 

Risk Assessment Forum
 
 

U.S. Environmental Protection Agency
 
 

Washington, DC 20460
 
 
DISCLAIMER 

This document has been reviewed in accordance with U.S. Environmental Protection 

Agency policy and approved for publication. Mention of trade names or commercial products 

does not constitute endorsement or recommendation for use. 

ii 
CONTENTS 

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

LIST OF FIGURES .......................................................... vii
 

LIST OF ABBREVIATIONS AND ACRONYMS .................................. viii
 

PREFACE ...................................................................x 
 

EXECUTIVE SUMMARY .................................................... xiii
 

1. INTRODUCTION, PURPOSE, AND SCOPE .................................. 1-1
 
 

2.	 	REVIEW OF THE CURRENT USE OF ACUTE, SHORT-TERM, AND 
 
 

LONGER-TERM REFERENCE VALUES .................................... 2-1
 
 

2.1. REVIEW OF CURRENT LESS-THAN-LIFETIME 
 
 

REFERENCE VALUES ............................................... 2-1
 
 

2.1.1. Acute Reference Exposure (ARE) Methodology ...................... 2-1
 
 

2.1.2. Acute Exposure Guidelines (AEGL) Program ........................ 2-3
 
 

2.1.3. Office of Pesticide Programs (OPP) Procedures for Setting 
 
 

Acute and Intermediate RfDs ..................................... 2-5
 
 

2.1.4. Office of Water (OW) Health Advisories (HAs) ...................... 2-6
 
 

2.1.5. Agency for Toxic Substances and Disease Registry (ATSDR)
 
 

Minimal Risk Levels (MRLs) ..................................... 2-9
 
 

2.2. SUMMARY OF CURRENT METHODS FOR SETTING ACUTE, 
 
 

SHORT-TERM, AND LONGER-TERM REFERENCE VALUES ............. 2-9
 
 

2.3. RECOMMENDATION .............................................. 2-11
 
 

3.	 	REVIEW OF TESTING GUIDELINES WITH RESPECT TO LIFE
 
 

STAGE ASSESSMENT ................................................... 3-1
 
 

3.1. EVALUATION OF CURRENT GUIDELINE 
 
 

TESTING PROTOCOLS .............................................. 3-2
 

3.1.1.	 	Exposures and Endpoints Related to General Toxicity Testing ........... 3-4
 
 

3.1.1.1. Acute and Short-Term Toxicity Studies ..................... 3-6
 
 

3.1.1.2. Subchronic and Chronic Toxicity Studies .................... 3-9
 
 

3.1.2.	 	Exposures and Endpoints Related to Evaluation of
 
 

Reproductive Toxicity ......................................... 3-10
 
 

3.1.2.1. Overview of Tests ..................................... 3-10
 
 

3.1.2.2. Gaps in Life Stage of Assessment ......................... 3-13
 
 

3.1.2.3. Gaps in Assessment Endpoints ........................... 3-14
 
 

3.1.2.4. Gaps in Duration/Latency Assessment ..................... 3-14
 
 

3.1.3.	 	Exposures and Endpoints Related to Evaluation of Neurotoxicity........ 3-15
 
 

3.1.3.1. Overview of Tests ..................................... 3-15
 
 

iii 
CONTENTS (continued) 

3.1.3.2. Gaps in Life Stage of Assessment ......................... 3-17
 
 

3.1.3.3. Gaps in Assessment Endpoints ........................... 3-18
 
 

3.1.3.4. Gaps in Duration/Latency Assessment ..................... 3-18
 
 

3.1.4.	 	Exposures and Endpoints Related to Evaluation 
 
 

of Immunotoxicity ............................................ 3-19
 
 

3.1.4.1. Overview of Tests ..................................... 3-19
 
 

3.1.4.2. Gaps in Life Stage of Assessment ......................... 3-22
 
 

3.1.4.3. Gaps in Assessment Endpoints ........................... 3-22
 
 

3.1.4.4. Gaps in Duration/Latency Assessment ..................... 3-23
 
 

3.1.5.	 	Exposures and Endpoints Related to Evaluation of Cardiovascular Toxicity 3-23
 
 

3.1.5.1. Overview of Tests ..................................... 3-23
 
 

3.1.5.2. Gaps in Life Stage of Assessment ......................... 3-23
 
 

3.1.5.3. Gaps in Assessment Endpoints ........................... 3-25
 
 

3.2.	 	CONCLUSIONS AND RECOMMENDATIONS .......................... 3-26
 
 

3.2.1. Conclusions .................................................. 3-27
 
 

3.2.2. Recommendations ............................................. 3-29
 
 

3.3.	 	OPTIONS FOR ALTERNATIVE TESTING APPROACHES ................ 3-31
 
 

3.3.1. Alternative Acute Toxicity Testing Protocol ........................ 3-31
 
 

3.3.2. Alternative Chronic Toxicity Testing Protocols ...................... 3-33
 
 

3.3.2.1. The Expanded Chronic/Carcinogenicity Study ............... 3-33
 
 

3.3.2.2. The Unified Screening Study ............................. 3-35
 
 

4.	 	FRAMEWORK FOR SETTING ACUTE, SHORT-TERM, LONGER-TERM, 
 
 

AND CHRONIC REFERENCE VALUES ..................................... 4-1
 
 

4.1. DEFINITIONS OF EXPOSURE DURATIONS FOR USE IN
 
 

SETTING REFERENCE VALUES ...................................... 4-2
 
 

4.2. PROPOSED CHANGES IN THE REFERENCE VALUE DEFINITIONS ....... 4-4
 
 

4.3. CHARACTERIZATION OF THE EXTENT OF THE HEALTH-RELATED
 
 

DATABASE FOR SETTING REFERENCE VALUES ...................... 4-7
 
 

4.3.1. Review of Studies .............................................. 4-7
 
 

4.3.1.1. Adequacy of Studies .................................... 4-8
 
 

4.3.2.	 	Issues to be Considered in Characterizing the Database
 
 

for Risk Assessment ........................................... 4-11
 
 

4.3.2.1. The Weight-of-Evidence Approach ........................ 4-11
 
 

4.3.2.2. Use of Human and Animal Data in Risk Assessment .......... 4-12
 
 

4.3.2.3. Characterization of Effects in Potentially 
 
 

Susceptible Subpopulations .............................. 4-15
 
 

4.3.3. Characterization of the Extent of the Database ...................... 4-17
 
 

4.3.3.1. Extent of the Database .................................. 4-20
 
 

4.4. DERIVATION OF REFERENCE VALUES .............................. 4-21
 
 

iv 
CONTENTS (continued) 

4.4.1. Sample Reference Values and Selection of Endpoints to 
 
 

Use as the POD for Reference Values ............................. 4-22
 
 

4.4.2. Dose Adjustment for Duration of Exposure ......................... 4-24
 
 

4.4.2.1. Duration Adjustment Procedures for Inhalation 
 
 

Exposures to Continuous-Exposure Scenarios ............... 4-24
 
 

4.4.2.2. Duration Adjustment for Inhalation Developmental
 
 

Toxicity Studies—A Current Exception .................... 4-25
 
 

4.4.2.3. Duration Adjustment for Acute Reference Values—Discontinuous
 
 

Scenarios of 24 Hours or Less ............................ 4-26
 
 

4.4.3. Derivation of a HEC or a HED ................................... 4-29
 
 

4.4.3.1. PBPK Models and Derivation of HEDs 
 
 

and HECs: Estimating Internal Dose ....................... 4-30
 
 

4.4.3.2. Default Procedures and Derivation of HECs from the RfC
 
 

Methodology: Derivation and Application of DAFs ........... 4-32
 
 

4.4.3.3. HECs and Children—A Special Case? ..................... 4-34
 
 

4.4.3.4. Deriving a HED for Oral and Dermal Exposure—Use
 
 

of BW3/4 as a Cross-Species DAF ......................... 4-35
 
 

4.4.4. Other Issues .................................................. 4-37
 
 

4.4.5. Application of Uncertainty/Variability Factors ...................... 4-38
 
 

4.4.5.1. Recommendations for Application of UFs .................. 4-40
 
 

4.4.5.2. Interspecies UF ....................................... 4-42
 
 

4.4.5.3. Intraspecies UF ....................................... 4-42
 
 

4.4.5.4. LOAEL-to-NOAEL UF ................................. 4-44
 
 

4.4.5.5. Database UF .......................................... 4-44
 
 

4.4.5.6. Subchronic-to-Chronic-Duration UF ....................... 4-45
 
 

4.4.5.7. Modifying Factor (MF) ................................. 4-46
 
 

4.4.6.	 	Future Directions ............................................. 4-47
 
 

4.4.6.1. Chemical-Specific Adjustment Factors (CSAFs) ............. 4-47
 
 

4.4.6.2. Probabilistic Approaches ................................ 4-48
 
 

4.4.7. Summary of Key Points from the Case Studies ...................... 4-49
 
 

5. RECOMMENDATIONS ................................................... 5-1
 
 

APPENDIX A: ISSUES RAISED BY THE 10X TASK FORCE ................... A-1
 

APPENDIX B:	 	 CASE STUDIES—EVALUATING AND SELECTING HEALTH
 

ENDPOINTS FOR DERIVING REFERENCE VALUES .............B-1
 

GLOSSARY .............................................................. G-1
 

REFERENCES .............................................................R-1
 

v 
LIST OF TABLES
 
 

Table 2-1. Duration definitions used for various reference values .................. 2-10
 

Table 2-2. Uncertainty/safety factors for various reference values ................. 2-12
 

Table 3-1. Approximate age at equivalent life stages in several species .............. 3-3
 

Table 3-2. Systems/endpoints evaluated by routine toxicity guideline testing protocols . . 3-5
 

Table 4-1.	 	 Factors for evaluation of the weight of evidence regarding the likelihood 
 

of effects in humans ............................................. 4-14
 

Table 4-2.	 	 Factors for evaluating of evidence regarding identification 
 

and characterization of susceptible subpopulations ..................... 4-17
 

Table 4-3. DAFs based on BW3/4 for various species ............................ 4-36
 

Table 4-4.	 	 BMDLs, ingested dose, and RfDs for various endpoints from the
 

Faroe Islands, New Zealand, and the NRC integrative analysis ........... 4-51
 

vi 
LIST OF FIGURES
 
 

Figure 3-1. Exposures and endpoints related to general toxicity evaluations ........... 3-7
 

Figure 3-2. Exposures and endpoints related to reproductive evaluations ............. 3-11
 

Figure 3-3. Exposures and endpoints for neurotoxicity evaluations ................. 3-16
 

Figure 3-4. Exposures and endpoints for immunotoxicity evaluations ............... 3-20
 

Figure 3-5. Exposures and endpoints related to cardiovascular evaluations ........... 3-24
 

Figure 3-6. Alternative acute toxicity protocol ................................. 3-32
 

Figure 3-7. Expanded chronic/carcinogenicity study ............................. 3-34
 

Figure 3-8. Unified screening study .......................................... 3-36
 

Figure 4-1.	 	 Concentration-by-duration plot showing the effect of the exponent 
 

in the Cn x T = k on extrapolation across time. ........................ 4-27
 

Figure 4-2.	 	 Current and proposed generalized procedures for deriving HECs 
 

or HEDs from animal exposures ................................... 4-31
 

vii 
LIST OF ABBREVIATIONS AND ACRONYMS 

ACE II Angiotensin converting enzyme II
 
 

ADI Acceptable Daily Intake
 
 

AEGL Acute exposure guideline level
 
 

ARE Acute reference exposure
 
 

ATSDR Agency for Toxic Substances and Disease Registry
 
 

AUC Area under the curve
 
 

BMC Benchmark concentration
 
 

BMCL Benchmark concentration lower confidence limit
 
 

BMD Benchmark dose
 
 

BMDL Benchmark dose lower confidence level
 
 

BMR Benchmark response
 
 

CatReg Categorical Regression (software)
 
 

CFSAN Center for Food Safety and Nutrition
 
 

CNS Central nervous system
 
 

CSAF Chemical-specific adjustment factor
 
 

DAF Dosimetric adjustment factor
 
 

DNT Developmental neurotoxicity
 
 

ECE-1 Endothelin-converting enzyme-1
 
 

ELISA Enzyme-linked immunosorbent assay
 
 

FDA Food and Drug Administration
 
 

FQPA Food Quality Protection Act
 
 

FIFRA Federal Insecticide, Fungicide, and Rodenticide Act
 
 

GD Gestational day
 
 

GLP Good Laboratory Practices
 
 

HA Health Advisory
 
 

HEC Human equivalent concentration
 
 

HED Human equivalent dose
 
 

IPCS International Programme on Chemical Safety
 
 

IRIS Integrated Risk Information System
 
 

LOAEL Lowest-observed-adverse-effect-level
 
 

MF Modifying factor
 
 

MOE Margin of exposure
 
 

MRL Minimal risk level
 
 

NAAQS National Ambient Air Quality Standards
 
 

NCEA National Center for Environmental Assessment
 
 

NK Natural Killer
 
 

NOAEL No-observed-adverse-effect-level
 
 

OAR Office of Air and Radiation
 
 

OECD Organisation for Economic Cooperation and Development
 
 

OPP Office of Pesticide Programs
 
 

viii 
LIST OF ABBREVIATIONS AND ACRONYMS (continued) 

OPPTS Office of Prevention, Pesticides, and Toxic Substances
 
 

OSWER Office of Solid Waste and Emergency Response
 
 

OW Office of Water
 
 

P Parental
 
 

PAD Population adjusted dose
 
 

PBPK Physiologically-based pharmacokinetic model
 
 

PFC Plaque-forming cell
 
 

PND Postnatal day
 
 

POD Point of departure
 
 

PRA Plasma renin activity
 
 

RDDR Regional deposited dose ratio
 
 

RGDR Regional gas dose ratio
 
 

RfC Reference concentration
 
 

RfD Reference dose
 
 

SAB Science Advisory Board
 
 

SPF Specific pathogen free
 
 

SRBC Sheep Red Blood Cells
 
 

TSCA Toxic Substance Control Act
 
 

TWA Time-weighted average
 
 

UF Uncertainty factor
 
 

ix 
PREFACE 

The U.S. Environmental Protection Agency (EPA) Risk Assessment Forum was 

established to promote scientific consensus on risk assessment issues and to ensure that this 

consensus is incorporated into appropriate risk assessment guidance. To accomplish this, the 

Forum assembles experts throughout EPA in a formal process to study and report on these issues 

from an Agency-wide perspective. For major risk assessment activities, the Forum has 

established Technical Panels to conduct scientific reviews and analyses. Members are chosen to 

ensure that necessary technical expertise is available. 

The RfD/RfC Technical Panel (hereafter the Technical Panel) was established by the 

Risk Assessment Forum in early 1999 in response to a request from the Agency’s 10X Task 

Force1

 to the Science Policy Council and the Forum. In the process of developing a strategy for 

implementing the Food Quality Protection Act (FQPA) relative to protecting children’s health 

and application of the 10X safety factor, the 10X Task Force produced two draft reports, one on 

toxicology (U.S. EPA, 1999b) and one on exposure data requirements (U.S. EPA, 1999c) that 

were used by the Office of Pesticide Programs to develop a policy document for implementation 

of the FQPA safety factor (U.S. EPA, 2002b). 

The draft 10X toxicology report (U.S. EPA, 1999b) raised a number of issues that relate 

to the derivation of the oral reference dose (RfD) and inhalation reference concentration (RfC). 

Examples of these issues include the following. (1) Appropriate application of a database 

uncertainty factor (UF) or modifying factor for studies that are considered necessary but are 

absent or judged inadequate that may show children to be significantly more sensitive or 

susceptible than adults. Addressing this issue also implicates aspects of other UFs that relate to 

children’s health, including the factor for inter-individual variability in humans (e.g., response of 

the aged vs. response of the younger adult or child), and the interspecies UF (e.g., young animals 

vs. young humans). (2) How to account for degree of concern for potential toxicity to children 

in the RfD/RfC process. Degree of concern, as used in the 10X toxicology report, refers to the 

characterization of the database as to the likelihood that the agent under review would have 

effects in humans within the context of dose, route, duration, and timing of exposure. (3) The 

1

The 10X Task Force was created by the EPA Administrator to explore the adequacy of 

current testing approaches for pesticides for protecting children’s health and to recommend 

approaches for implementing the additional 10X safety factor mandated by the 1996 Food 

Quality Protection Act. 

x 
use of developmental toxicity data as the basis for reference values of chronic duration (RfDs or 2 

RfCs) and the appropriate setting of acute, short-term, and longer-term reference values, 

including the application of developmental toxicity data for these shorter-duration reference 

values. (4) The appropriateness and/or rationale for adjusting the no-observed-adverse-effect 

level (NOAEL) or the benchmark dose from developmental toxicity data with inhalation 

exposures using a concentration-times-time (C x t) adjustment, as is done for other study types. 

The Technical Panel also was asked to consider the need for additional toxicity test 

protocols related to children’s health as recommended by the 10X Task Force, when such 

protocols should be required, and how the data should be interpreted for risk assessment 

purposes. These additional protocols include (1) collection of toxicokinetic data, both in adults 

and at different developmental stages; (2) direct dosing of neonates, especially when early 

exposure is of concern; (3) perinatal carcinogenesis studies and appropriate triggers for when 

they should be required; (4) developmental immunotoxicity testing and appropriate triggers; (5) 

advanced developmental neurotoxicity testing, in particular, cognitive testing that is more similar 

to that used in humans; and (6) exposure assessments that are more compatible with the doseresponse assessment. (See Appendix A for more a detailed discussion of the issues raised by the 

10X Task Force.) 

The Science Policy Council and the Risk Assessment Forum agreed that these issues 

should be examined—with input from various program offices within the Agency and from the 

outside scientific/policy community—on a broader scale than just for pesticides. This charge 

was expanded by the Forum to include a more in-depth review of a number of issues related to 

the RfD/RfC process, in part because of several other Forum activities that were underway. 

These activities included development of Framework for the Harmonization of Cancer and 

Noncancer Risk Assessment, revision of Benchmark Dose Guidance Document, and revision of 

Guidelines for Carcinogen Risk Assessment. In addition, the RfD/RfC derivation process 

(Barnes and Dourson, 1998; U.S. EPA, 1994, 2002c) had not been evaluated in detail for a 

number of years, and several scientific issues concerning children’s health, for example, 

neurotoxicity and immunotoxicity, have become increasingly important in risk assessment. 

These various but related activities have prompted the need to re-examine the RfD/RfC process 

and to coordinate these efforts with other related activities. In particular, it was important that 

efforts continue to focus on moving toward the goal of harmonization of risk assessment 

2

The term reference value is used generically here to refer to values such as the RfD, 

RfC, acute reference exposure (ARE), Health Advisory (HA), acute exposure guideline level 

(AEGL), minimal risk level (MRL), or other similar values. 

xi 
approaches for all health endpoints. This document represents the review and deliberations of 

the RfD and RfC processes by the Risk Assessment Forum Technical Panel. 

xii
 
EXECUTIVE SUMMARY 

This document summarizes the review and deliberations of the Risk Assessment Forum’s 

RfD/RfC Technical Panel and its recommendations for improvements in oral reference 

dose/inhalation reference concentration (RfD/RfC) process as well as additional efforts that are 

needed. It discusses revisions to the framework for the derivation of reference values. The 

document is a review, not guidance, but it does make recommendations that should be 

considered in the implementation of changes in the current process and/or development of 

needed guidance. 

The Technical Panel reviewed most of the issues relating to hazard characterization for 

developing reference values and the need for developing reference values for different durations 

of exposure as well as the process of deriving reference values, but it did not go into detail on the 

quantitative aspects of the dose-response process, which is being covered in other Forum 

activities. The Technical Panel views the RfD/RfC process as one that should be continually 

evolving as new information becomes available and new scientific and risk assessment 

approaches are developed. This does not mean that current RfDs or RfCs are invalid, but these 

new scientific issues should be included in the process of re-evaluating of current reference 

values. 

This document reviews and discusses a number of issues and provides conclusions and 

recommendations that are intended to improve the RfD/RfC process. The Technical Panel has 

provided specific recommendations for the development of guidance in some cases and more 

general conclusions and recommendations in others. In the latter cases, the Technical Panel felt 

that development of specific recommendations was beyond the scope of its efforts or that 

policies needed to be further developed before specific guidance could be written to implement 

the recommendations. The document is divided into five chapters: 

Chapter 1 provides an introduction, background, purpose, and scope for the project. 

Chapter 2 reviews current approaches to developing acute, short-term, and longer-term 

reference values as well as the chronic reference values, the RfD and the RfC. This chapter 

incorporates the presentations and discussions on developing less-than-lifetime values from 

briefings to the Technical Panel and a colloquium held August 2, 2000, and includes discussions 

of the proposed Acute Reference Exposure (ARE) methodology for acute inhalation exposures, 

the Acute Exposure Guideline Level (AEGL) Program, the Office of Pesticide Programs’ 

procedures for setting acute and longer-term duration RfDs, the Office of Water’s Health 

Advisories, and the Agency for Toxic Substances and Disease Registry’s Minimal Risk Levels. 

xiii 
On the basis of its review of the various approaches to setting acute, short-term, and 

longer-term reference values, the Technical Panel concurred with the recommendation of the 

10X Task Force that such values should be set, where possible, and that they should be 

incorporated into the Integrated Risk Information System (IRIS) database. In addition, the 

Technical Panel recommended that this process be done in a consistent manner using 

standardized definitions for acute, short-term, longer-term, and chronic durations that are 

consistent with current practice. These values can then be used by various program offices, 

where applicable. A framework for deriving these additional values is presented in Chapter 4. 

Chapter 3 reviews the current Office of Prevention, Pesticides and Toxic Substances’ 

harmonized health effects testing guidelines for the purpose of determining the data available for 

setting various duration reference values. The intent of this review is not to suggest that 

additional testing be conducted for each and every chemical in order to fill in the information 

gaps identified for those organ systems evaluated. Nor is it suggested that alternative testing 

protocols that are discussed in this chapter should be conducted for every chemical or become 

part of current toxicology testing requirements or that these alternative protocols are the only 

options available. Rather, it is the goal of this document to provide a basis for the development 

of innovative alternative testing approaches and the use of such data in risk assessment, and to 

then illustrate some aspects of this concept with a few examples. In reviewing the current testing 

protocols, target organs/systems that are evaluated were reviewed as was the thoroughness of 

testing with respect to life stage assessment, endpoint assessment, route, timing and duration of 

exposure, and latency to response. These issues were all considered important in evaluating 

potentially susceptible subpopulations, including life stages. The testing guideline protocols 

were reviewed overall for these issues; in addition, four biological systems were evaluated in 

depth, two that are fairly thoroughly evaluated (the reproductive and nervous systems) and two 

that are evaluated to a more limited extent (the immune and cardiovascular systems). In each 

case, an overview of the tests for the particular system is given, as well as a more specific 

discussion of gaps in life stage of assessment, gaps in assessment endpoints, and gaps in duration 

and latency assessment. 

The Technical Panel has made a number of recommendations concerning toxicity testing, 

including development of a strategy for approaches to toxicity testing, with guidance on how and 

when to use existing and newly recommended guidelines; development of guidelines or 

guideline study protocols that will provide more systematic information on toxicokinetics and 

toxicodynamics (i.e., mechanism or mode of action), including at different life stages; 

development of protocols for acute and short-term studies that provide more comprehensive data 

xiv 
for setting reference values; modification of existing guideline study protocols to provide more 

comprehensive coverage of life stages for both exposure and outcomes; collection of more 

information from less-than-lifetime exposure to evaluate latency to effect and reversibility of 

effect; development of guidelines or guideline study protocols to assess immunotoxicity, 

carcinogenicity, and cardiovascular toxicity at different life stages; and exploration of the 

feasibility of setting dermal reference values for direct toxicity at the portal of entry, including 

sensitization. 

A primary goal of this review was to provide the basis for recommendations for the 

development of a strategy for approaches to toxicity testing and for innovative alternative testing 

approaches to provide data for risk assessment. The Technical Panel is suggesting that 

alternative strategies and guidance for testing approaches be developed that incorporate 

information on toxicokinetics and mode of action early in the process, thus allowing a more 

targeted testing approach. In addition, alternative protocols are discussed that are aimed at more 

efficient use of animals and resources in combined studies that would provide more extensive 

data on life stages, endpoints, and other factors not well characterized in current testing 

approaches. Recommendations are also made about research areas that should be encouraged to 

aid in better study design and interpretation of data for risk assessment. 

Finally, an example of an alternative testing protocol for acute exposure and evaluation 

that incorporates the types of endpoints and evaluations optimal for setting acute reference 

values is discussed. Two sample alternative protocols are presented for chronic exposures and 

options are discussed for combining studies and evaluations to include a wider array of life stage 

and endpoint assessments. 

Chapter 4 discusses a number of modifications to the existing framework for use in 

deriving reference values, both for the current chronic reference values (RfD and RfC) as well as 

for acute, short-term, and longer-term reference values. The approach to reference values 

discussed here is intended for risk assessments of any type of health effect known or assumed to 

be produced through a nonlinear and/or threshold mode of action (which may include U-shaped 

or other nonmonotonic dose-response curves as well as thresholds). Thus, the Technical Panel 

recommends moving away from the dichotomy between “cancer” and “noncancer.” The term 

“noncancer” has been removed from the reference value definition, denoting the move toward 

defining approaches for low-dose estimation or extrapolation based on mode of action. Two 

case studies that illustrate many of the concepts discussed in this chapter are presented in more 

detail in Appendix B. The Technical Panel recommends including the acute, short-term, longerterm, and chronic reference values derived on the basis of the recommendations in this report in 

xv 
IRIS after appropriate internal, external, and consensus review. Standard exposure durations are 

proposed, as is a definition for the reference value, including a designation for route and duration 

of exposure. 

The Technical Panel is aware that there will be data limitations for an individual 

chemical that may preclude development of all four reference values, and it is aware that time 

and resources need to be considered when implementing these recommendations. The IRIS 

program has begun to implement a pilot program to test whether development of the expanded 

array of reference values is practical and can be accomplished without unduly delaying the 

completion of an IRIS file. As a part of the pilot, the IRIS program will need to identify the 

methods to be used in deriving these additional values. 

The Technical Panel recommends that endpoint-specific reference values should not be 

developed, including the reference dose for developmental toxicity, RfDDT. Rather, a sample 

reference value should be calculated for each relevant and appropriate endpoint and these should 

be considered in the derivation of various duration reference values. The reference values 

should be derived to be protective of all types of effects for a given duration of exposure. 

An expanded approach to the evaluation of studies and characterization of the extent of 

the database as a whole is recommended; in particular, several factors are discussed that should 

be considered in a weight-of-evidence approach for characterizing hazard for the population as a 

whole as well as for potentially susceptible subpopulations. Those considerations for assessing 

level of concern raised by the Toxicology Working Group of the 10X Task Force have been 

incorporated into this approach. 

In the context of this framework, the Technical Panel recommends a somewhat different 

approach to characterizing the extent of the database for reference values. Instead of specifying 

particular studies, this approach emphasizes the types of data needed (both in terms of human 

and animal data) for deriving reference values, and it recommends the use of a narrative 

description of the extent of the database rather than a single confidence ranking of high, medium, 

or low. To characterize the database, the Technical Panel has developed a description of a 

“minimal” database and a “robust” database as a way of describing the range of data that can be 

used for deriving a reference value, and the Panel urges the use of a great deal of scientific 

judgement in the process of summarizing the extent of the database, including its strengths and 

limitations. 

The narrative approach is intended to emphasize the types of data available (both human 

and animal) as well as the data gaps that could improve the derivation of reference values. This 

approach should encourage the use of a wider range of information in deriving reference values, 

xvi 
taking into consideration the issues of duration, timing, and route of exposure; the types and 

extent of endpoint assessments (i.e., structure and function); the life stages evaluated; and the 

potential for latent effects and/or reversibility of effects. 

Dosimetric adjustment of values for deriving a human equivalent concentration (HEC) 

for inhalation exposure is discussed, as is the derivation of a human equivalent dose (HED) for 

oral or dermal exposure. The Technical Panel recommends that duration adjustment procedures 

to continuous exposures based on concentration times time (C x t) be used as a default procedure 

for inhalation developmental toxicity studies as for other health effects from inhalation 

exposures. In addition, further evaluation of current dosimetric adjustments for deriving HECs 

should be pursued to confirm or assess the relevance for population subgroups (particularly for 

children). 

Because of the recommendation for deriving several duration reference values, the 

Technical Panel recommends that the data for the point of departure (POD) be evaluated on the 

basis of a comparison of all relevant endpoints carried through the derivation of sample reference 

values, with selection of the limiting value(s) as the final step rather than on the basis of 

selection of a single “critical study” and “critical effect.” To aid in this evaluation, the use of an 

exposure-response array is recommended as a visual display of all relevant and appropriate 

endpoints and durations of exposure in order to determine the range of numerical values for each 

reference value. 

The Technical Panel makes a number of recommendations concerning the application of 

uncertainty factors (UFs) for reference value derivation. In particular, it is imperative that the 

IRIS documentation contain a justification for the individual factors selected for each chemical 

or assessment because rigid application of UFs could lead to an illogical set of reference values. 

Although default factors of 10 are recommended, with 3 used in place of half-power values (i.e., 

100.5) when occurring singly, the exact value of the UF chosen should depend on the quality of 

the studies available, the extent of the database, and scientific judgment. Sound scientific 

judgment should be used in the application of UFs to derive reference values that are applied to 

the value chosen for the POD derived from the available database (BMDL, NOAEL, or 

LOAEL). 

The Technical Panel recommends that if there is uncertainty in more than four areas of 

extrapolation, it is unlikely that the database is sufficient to derive a reference value. Even when 

there is uncertainty in four areas, the database should be carefully evaluated to determine 

whether the derivation of a reference value is appropriate. In addition, the Technical Panel 

recommends limiting the total UF applied to a chronic reference value for any particular 

xvii 
chemical to 3000. This maximum of 3000 applies only to the UFs and does not include the 

various adjustment factors discussed in Chapter 4. 

The intraspecies UF is applied to account for variations in susceptibility within the 

human population (interhuman variability) and the possibility (given a lack of relevant data) that 

the database available is not representative of the dose/exposure-response relationship in the 

subgroups of the human population that are most sensitive to the health hazards of the chemical 

being assessed. Because the RfD/RfC is defined to be applicable to “susceptible subgroups,” 

this UF was established to account for uncertainty in that regard. In general, the Technical Panel 

reaffirms the importance of this UF, recommending that reduction of the intraspecies UF from a 

default of 10 be considered only if data are sufficiently representative of the exposure/doseresponse data for the most susceptible subpopulation(s). At the other extreme, a 10-fold factor 

may sometimes be too small because of factors that can influence large differences in 

susceptibility, such as genetic polymorphisms. The Technical Panel urges the development of 

data to support the selection of the appropriate size of this factor, but recognizes that often there 

are insufficient data to support a factor other than the default. 

The Technical Panel urges continued research and evaluation of the similarities and 

differences between the general population and susceptible subpopulations, particularly children 

and the elderly, in their responses to particular agents. From such evaluations, the protectiveness 

of the 10-fold default factor should continue to be assessed. The Technical Panel urges the 

development of data to support the selection of the appropriate size of this factor, but it 

recognizes that often there are insufficient data to support a factor other than the default. The 

database UF is intended to account for the potential for deriving an underprotective RfD/RfC as 

a result of an incomplete characterization of the chemical’s toxicity. In addition to the 

identification of toxicity information that is lacking, review of existing data may also suggest 

that a lower reference value might result if additional data were available. Consequently, in 

deciding to apply this factor to account for deficiencies in the available data set, and in 

identifying its magnitude, the assessor should consider both the data lacking and the data 

available for particular organ systems as well as life stages. The Panel considers the purpose of 

the modifying factor (MF) to be sufficiently subsumed in the general database UF, and 

recommends that use of the MF be discontinued. 

Given that there are several UFs that can be used to deal with data deficiencies as part of 

the current reference value process, and given that these are assumed to overlap to some extent, 

the Technical Panel agrees with the 10X Task Force Toxicology Working Group that the current 

interspecies, intraspecies, and database deficiency UFs, if appropriately applied using the 

xviii 
approaches recommended in this review, will be adequate in most cases to cover concerns and 

uncertainties regarding the potential for pre- and postnatal toxicity and the completeness of the 

toxicology database. In other words, an additional uncertainty factor is not needed in the 

RfC/RfD methodology because the currently available factors are considered sufficient to 

account for uncertainties in the database from which the reference values are derived (and does 

not exclude the possibility that these UFs may be decreased or increased from the default value 

of 10). The approach to using chemical-specific data for toxicokinetic and toxicodynamic 

components of the interspecies UF is part of the current RfC methodology. The Technical Panel 

encourages the Agency to develop its own guidance for chemical-specific adjustment factors 

(CSAFs) on the basis of some of the available methodologies (e.g., the International Programme 

on Chemical Safety [IPCS]). 

Several other issues discussed by the Technical Panel were considered more appropriate 

for deliberation by other panels/committees, for example, further consideration of the use of 

BMD modeling approaches for deriving reference values; harmonization of the approaches for 

HEC and HED derivation for all types of health effects; further evaluation of approaches such as 

probabilistic analysis for characterizing variability and uncertainty in toxicity reference values; 

further evaluation of appropriate adjustment of doses for duration of exposure for acute toxicity 

data; and further evaluation of duration adjustment for short-term and longer-term reference 

values analogous to the subchronic-to-chronic duration UF for chronic reference values. 

Chapter 5 summarizes the recommendations of the Technical Panel. 

xix 
1. INTRODUCTION, PURPOSE, AND SCOPE 

The RfD/RfC Technical Panel (hereafter the Technical Panel) was established by the 

U.S. Environmental Protection Agency’s (EPA’s, or the Agency’s) Risk Assessment Forum in 

early 1999 to review the current oral reference dose (RfD) and inhalation reference concentration 

(RfC) processes, in particular with respect to how well children and other potentially susceptible 

subpopulations are protected; to consider new scientific issues that have become more important 

and of greater concern in risk assessment; and to raise issues that should be explored or 

developed further for application in the RfD/RfC process. This document summarizes the 

review and deliberations of the Technical Panel and its recommendations for improvements in 

the process as well as additional efforts that are needed. It discusses revisions to the framework 

for the derivation of RfDs and RfCs. The document is a review, not guidance, but it does make 

recommendations that should be considered in the implementation of changes in the current 

process and/or development of needed guidance. 

Many of the recommendations made in this report are consistent with the Agency’s 

commitment to harmonization of health risk assessment procedures, including the harmonization 

of approaches for noncancer and cancer endpoints, and to making efficient use of animal testing 

to achieve this goal. As noted in several places in the document, all such topics have not been 

discussed and resolved by the Agency. For instance, the differences in scaling factors used for 

cancer and noncancer derivations from oral exposure data are raised as an issue that has not been 

resolved; thus, there will likely be a need for revised or further guidance on this issue. 

Although mixtures or multiple chemical exposures are not specifically discussed in this 

review, most of the recommendations are applicable to the approach to risk assessment of 

mixtures. The Agency’s mixtures risk assessment guidelines should be consulted for issues 

specific to the evaluation of mixtures (U.S. EPA, 1986, 2000a). In addition, the Agency has 

recently issued the draft Framework for Cumulative Risk Assessment (U.S. EPA, 2002a), which 

deals with the issue of multiple stressors and their overall impacts on exposure-effect 

relationships. The risk assessment approaches discussed within this framework are likely to be 

the subject of further guidance as well. 

The Technical Panel attempted to review most of the issues relating to hazard 

characterization for developing reference values, to the need for developing reference values for 

different durations of exposure, and to the process of deriving reference values. The Technical 

Panel did not go into detail on the quantitative aspects of the dose-response process, as this is 

being covered in other Forum activities (e.g., the benchmark dose [BMD] guidance document 

1-1
 
and the quantitative dose-response aspects of the cancer guidelines revision process). The 

Technical Panel approached its review from the point of view that the RfD/RfC process has been 

and should be a continually evolving process. Thus, as new information becomes available and 

new scientific and risk assessment approaches are developed, they are incorporated into new 

RfDs and RfCs as these values are developed or as current RfDs and RfCs are reevaluated. This 

process of incorporating new science does not invalidate current RfDs or RfCs, because 

consideration of these new scientific issues is included in the reevaluation of current values; 

higher or lower values or, in some cases, no change in the current value may result. 

This report provides conclusions and recommendations that are intended to improve the 

RfD/RfC process. The audience for this review is primarily the Integrated Risk Information 

System (IRIS) program, IRIS chemical managers, and other scientists within the Agency who are 

involved in developing the RfDs and RfCs, as well as IRIS users and the program offices within 

EPA that develop RfDs and RfCs or similar values (see Chapter 2), particularly resource 

managers who may be impacted by the potential for additional workload due to several of the 

recommendations. The Technical Panel has provided specific recommendations for guidance in 

some cases and more general conclusions and recommendations in others. In the latter cases, the 

Technical Panel felt that development of specific recommendations was beyond the scope of its 

efforts or that policies needed to be further developed before specific guidance could be written 

to implement the recommendations. 

The methodology recommended in the RfD document is considered generally applicable 

to both cancer and noncancer endpoints where dose-response relationships are thought to be 

either nonlinear or consistent with a threshold. Although the emphasis in this document is on the 

calculation of RfDs and RfCs, the same processes and considerations are applicable to the 

margin of exposure (MOE), as discussed in the draft cancer risk assessment guidelines (U.S. 

EPA, 1999a). 

The Technical Panel discussed a number of issues concerning a revised framework for 

the RfD/RfC process, with particular emphasis on the extent to which children and other 

potentially susceptible subpopulations are considered. The next three chapters summarize these 

issues, and several recommendations are made. Chapter 2 reviews current approaches to 

developing acute, short-term, and longer-term reference values as well as the chronic reference 

values, the RfD and the RfC. Chapter 3 reviews the current testing guidelines with respect to life 

stage assessment and discusses the gaps in life stage assessment, endpoint assessment, and 

assessment of duration and latency. Alternative testing protocols and strategies as options for 

combining studies and evaluations are discussed. 

1-2
 
Chapter 4 provides constructive commentary on the current framework used in deriving 

reference values and on the need and possibilities for calculating reference values for different 

durations and routes of exposure. In addition, an expanded approach to evaluating studies and 

characterizing the extent of the database as a whole is presented and discussed, including 

dosimetric adjustment, the application of uncertainty factors (UFs), and derivation of sample 

reference values for each appropriate and relevant endpoint to aid in selecting the point of 

departure (POD) for deriving reference values. 

The final chapter (Chapter 5) summarizes all of the recommendations of the Technical 

Panel. Two case studies that illustrate several of the recommended changes are also included as 

Appendix B. 

1-3
 
 
2. REVIEW OF THE CURRENT USE OF ACUTE, SHORT-TERM, 

AND LONGER-TERM REFERENCE VALUES 

The Technical Panel considered the recommendation of the 10X Task Force that acute, 

short-term, and longer-term reference values as well as chronic reference values should be set for 

environmental agents (see Appendix A). It is likely that the endpoints critical for setting acute, 

short-term, and longer-term reference values may differ from those for setting chronic RfDs and 

RfCs, although studies that use acute and short-term exposure conditions from which the 

appropriate data for many types of effects could be derived are not often available. Data on 

acute and short-term health effects must often be derived from observations after the first 

exposure in a repeated-exposure testing protocol. 

Several acute and short-term values currently are set for various chemical types and 

media. For example, acute and chronic oral RfDs are set for pesticides, with some intermediate 

values set for occupational and residential pesticide exposures. Health advisories (HAs) of 

several durations have been developed for drinking water. In addition, the Office of Solid Waste 

and Emergency Response, the Office of Prevention, Pesticides, and Toxic Substances (OPPTS), 

and other program offices and regional offices use values derived through the interagency acute 

exposure guidelines (AEGL) process for emergency response planning. The National Center for 

Environmental Assessment (NCEA) is currently developing the acute reference exposure (ARE) 

methodology for acute inhalation exposures. These developments are reviewed in more detail 

below. 

2.1. REVIEW OF CURRENT LESS-THAN-LIFETIME REFERENCE VALUES 

The Technical Panel was briefed by representatives of several Agency offices on the 

methods currently used to set various less-than-lifetime reference values. Subsequently, on 

August 2, 2000, a Risk Assessment Forum colloquium was held on this topic (CDM Group, Inc., 

2000). Each of the methods was presented and discussed. In addition, a recommendation by the 

Technical Panel to begin deriving acute, short-term, and longer-term reference values as well as 

chronic values and to standardize the definitions for each duration was presented and discussed. 

Each method presented is summarized below. 

2.1.1. Acute Reference Exposure (ARE) Methodology 

The ARE methodology is being developed at the request of the EPA’s Office of Air and 

Radiation. It is intended for development of reference values for acute inhalation exposures of 

2-1
 
24 hours or less. The criteria air pollutants are not included, because they are assessed within the 

National Ambient Air Quality Standards (NAAQS) setting process.1 The ARE is defined as an 

inhalation exposure of 24 hours or less that is not likely to cause noncancer adverse effects. The 

ARE can be applied to intermittent exposures or to a continuous exposure. AREs are being 

developed in order to address the acute risk aspects of risk-related provisions of the hazardous 

air pollutant sections of the 1990 Clean Air Act Amendments. The ARE methodology is 

described in a 1998 EPA external review draft document (U.S. EPA, 1998a). The method builds 

on the procedures of the RfC methodology. 

The ARE methodology includes three approaches in order to accommodate the varying 

types of data available for acute exposure. The first two approaches, the no-observed-adverseeffect level (NOAEL) and the benchmark concentration (BMC) are familiar. The third approach, 

categorical regression (CatReg), is newer. The NOAEL approach is useful for chemicals that 

have limited available data and for which no or limited dose-response relationships have been 

established. The BMC approach is suitable for analysis of studies that establish dose-response 

relationships. The CatReg approach requires multiple studies that report not only dose and 

response, but also duration; it is most applicable for data-rich chemicals. A feature of the 

CatReg approach is that effects data are grouped into severity categories (e.g., mild or severe to 

lethal) to which sophisticated regression procedures are then applied. 

Adjustments for deriving ARE values of different durations (e.g., 15 minutes or 8 hours) 

are made differently for the CatReg approach than for the NOAEL and BMC approaches. For 

any approach, the preferred adjustment procedure is to use a pharmacokinetic model, if 

available. When the NOAEL or BMD approach is used, the default procedure is to use the 

multiple of concentration times time (C x t) (Cn

 x t = k; ten Berge et al., 1986) to extrapolate 

from short to long duration and to use the same concentration as obtained for long duration to 

extrapolate from long to short duration. When more than one duration is available, interpolation 

is performed. When the CatReg approach is used, the procedure involves reading the values 

directly from the concentration duration curve that is generated by the CatReg software. These 

approaches are explained more fully and illustrated in Chapter 4. 

A minimal data set has not been defined for the ARE. Also, extrapolation from the oral 

to the inhalation route of exposure is not addressed in the ARE approach. UFs in the ARE 

approach include a lowest-observed-adverse-effect level- (LOAEL-) to-NOAEL UF of 10 and a 

1

Criteria air pollutants are those air pollutants for which NAAQS have been established 

under the Clean Air Act; at present, the six criteria air pollutants are particulate matter, ozone, 

carbon monoxide, nitrogen oxides, sulfur dioxide, and lead. 

2-2 
default value of 10 for interspecies and for intraspecies extrapolation. No factor is assigned for 

database inadequacies and study quality. 

In 1998, the EPA Science Advisory Board (SAB) reviewed the ARE methodology 

document and made a number of comments that addressed, among other things, issues about the 

NOAEL and BMC approaches, the need for addressing protection of children, the dosimetry 

adjustment and duration extrapolation, and the CatReg approach. The SAB discussed the fact 

that the CatReg model, as currently set up, forces parallelism of the concentration-duration 

curves for the various severity categories. In addition, there were concerns about judging 

severity categories across various target organs and species, and there was discussion about the 

reliability of the confidence limits around the maximum likelihood estimate and about the 

appropriateness of the approach used to accommodate group versus individual data. This 

methodology has since (March 2001) undergone an Agency review by the Risk Assessment 

Forum. The principal comments from this review concerned reevaluation of whether CatReg 

should remain as an approach in the ARE methodology and further evaluation of the procedures 

for cross-species dosimetry adjustment. Revision of the ARE methodology is currently 

underway. In addition to revising the ARE methodology and CatReg software documents, 

NCEA-Research Triangle Park will develop a framework for adding AREs to the IRIS database. 

2.1.2. Acute Exposure Guidelines (AEGL) Program 

The primary purpose of the AEGL program is to develop guideline levels for once-in-alifetime short-term exposures to airborne concentrations of acutely toxic chemicals (NRC, 2000). 

AEGLs are needed for a wide variety of emergency planning, response, and prevention 

applications. AEGLs represent threshold exposure limits for the general public and are 

applicable to emergency exposure periods ranging from 10 minutes to 8 hours. Specific values 

are set for 10 minutes, 30 minutes, 1 hour, 4 hours, and 8 hours. It is believed that the 

recommended exposure levels are applicable to the general population, including infants and 

children and other individuals (e.g., asthmatics) who may be sensitive or susceptible. It is 

recognized that certain individuals who may be subject to unique or idiosyncratic responses 

could experience the effects described at concentrations below the corresponding AEGL level. 

The AEGL-1, AEGL-2, and AEGL-3 levels are distinguished by varying degrees of 

severity of toxic effects. With increasing airborne concentrations above each AEGL level there 

is a progressive increase in the likelihood of occurrence and the severity of effects described for 

each level. 

2-3
 

AEGL-1 is the airborne concentration of a substance above which it is predicted that the 

general population, including susceptible individuals, could experience notable discomfort, 

irritation, or certain asymptomatic, nonsensory effects. However, the effects would not be 

disabling and would be transient and reversible upon cessation of exposure. 

AEGL-2 is the airborne concentration of a substance above which it is predicted that the 

general population, including susceptible individuals, could experience irreversible or other 

serious, long-lasting adverse health effects or an impaired ability to escape. 

AEGL-3 is the airborne concentration of a substance above which it is predicted that the 

general population, including susceptible individuals could experience life-threatening health 

effects or death. 

Airborne concentrations below AEGL-1 represent exposure levels that could produce 

mild and progressively increasing odor, taste, and sensory irritation or certain asymptomatic, 

nonsensory effects. 

UFs are used for extrapolations. If there are no appropriate human data, an interspecies 

UF of 1, 3, or 10 is used. The factors considered when deciding on a specific value include (1) 

the species tested (type, appropriateness, and range), (2) the toxicological endpoint observed and 

the likely mechanism of action, (3) the range of response in the species tested, (4) the variability 

of response among the species tested, and (5) pharmacokinetic differences among the species 

tested. An intraspecies UF of 1, 3, or 10 is also used. The factors considered when assigning a 

specific value include (1) the toxicological endpoint observed and the likely mechanism of 

action, (2) the range of response among humans and subpopulations, and (3) pharmacokinetic 

differences among individuals. Individual factors of 3 are often used to ensure that the final 

values are not overly conservative. 

Adjustment for duration is conducted using the equation Cn

 x t = k. If data are available 

for the endpoint of concern, the value of n is derived from regression analysis. If data are not 

available for the endpoint of concern, the value of n is usually derived from lethality data by 

regression analysis and used for the other endpoints. If the study duration is greater than 1 hour, 

the 10-minute value is usually assigned equal to the 30-minute value. If no data are available to 

derive a value of n, a value of 3 is used to extrapolate to shorter durations, and a value of 1 is 

used to extrapolate to longer durations. As mentioned above, this procedure is further explained 

and illustrated in Chapter 4. 

2-4
 

2.1.3. Office of Pesticide Programs (OPP) Procedures for Setting Acute and 

Intermediate RfDs 

OPP developed methodologies for acute dietary as well as occupational and residential 

risk assessments during the process of re-registration following the 1988 revision to the Federal 

Insecticide, Fungicide, and Rodenticide Act (FIFRA). In 1998, a guidance document, 

Toxicology Endpoint Selection Process (U.S. EPA, 1998b), was presented to the FIFRA 

Scientific Advisory Panel for review and comment. This document, which provided the basis for 

procedures that are still in place, describes toxicology endpoint selection for less-than-lifetime 

dietary and occupational/residential risk assessments for pesticides. It includes guidance on 

evaluating toxicity studies that are relevant for use, selecting appropriate endpoints for hazard 

identification, the process of hazard identification, the influence of dermal absorption in hazard 

identification, the criteria for the use of the NOAEL and the LOAEL, and the use of MOEs in 

risk assessments. Since this guidance was first issued, some changes have evolved, such as the 

replacement of the acute MOE with the acute RfD and the addition of standard consideration of 

short- and intermediate-term incidental nondietary ingestion exposures for toddlers. 

Toxicology Endpoint Selection Process (U.S. EPA, 1998b) describes the types of studies 

that are most likely to provide appropriate endpoints for the various exposure durations and risk 

assessments that will be conducted for each pesticide. OPP can rely on the availability of a wide 

variety of standard guideline toxicity studies from which to select endpoints because such studies 

are required by regulation for any pesticide registration (40 CFR Part 158). Additionally, OPP 

considers other sources of toxicology data, such as studies published in the open literature, as 

appropriate. 

For the establishment of the acute RfD, OPP uses a weight-of-evidence approach in 

evaluating all the available data. Three guideline studies have been found to be particularly 

useful by OPP: the acute neurotoxicity study, the prenatal developmental toxicity study, and the 

developmental neurotoxicity (DNT) study. 

Acute effects from subchronic and chronic dietary studies are also used in the 

establishment of the acute dietary RfD. Careful scrutiny of toxicological data from early in the 

first week of treatment can sometimes identify effects that can be described as acute. However, 

for a number of reasons, this option has not often been used. These reasons include the absence 

of detailed toxicological observations other than morbidity and mortality checks in subchronic 

and chronic studies before the end of the first week of treatment (i.e., after 7 days of treatment), 

the nature of the dietary exposure (i.e., each daily exposure results from an extended period of 

nightly feeding rather than from a discrete acute dose), and the possibility that apparent adverse 

2-5
 
effects during the first week of treatment may be related to palatability issues as the animals 

adjust to treated feed. 

OPP does not calculate short- or intermediate-term references doses. However, risk 

assessments are conducted for incidental nondietary ingestion exposures to toddlers—a very 

specific population subgroup—that result from the use of a pesticide in and around the home or 

other nonoccupational sources such as schools, parks, and golf courses. The post-application 

risk assessment considers or accounts primarily for incidental ingestion of (1) the dry pesticide 

materials (granules or pellets) used to treat outdoor residential areas, (2) pesticide residues in soil 

that are ingested by toddlers who play in treated areas (e.g., yards, gardens, playgrounds) as a 

result of normal mouthing activities, and (3) pesticide residues that are transferred to the skin of 

toddlers playing in treated areas and are subsequently ingested as a result of hand-to-mouth 

transfer. These risk assessments consider short-term (1 day to 1 month) and intermediate-term 

(1–6 months) exposure durations. Risks are expressed as MOEs. The MOE approach is used 

because these exposures are considered to be nondietary in source and are based on high-end 

values or (when adequate site- or chemical-specific field data are unavailable) on assumptions. 

OPP also conducts short-term, intermediate, and long-term (longer than 6 months) dermal 

and inhalation risk assessments for occupational and residential exposures. The MOE approach 

is also used to calculate the risk for these nondietary exposure scenarios. A difficulty that OPP 

often faces when conducting these risk assessments is that dermal absorption and inhalation 

toxicity data are often not available for food-use pesticides; in that case, appropriate assumptions 

are applied, and the available oral toxicity data are converted for use in dermal and inhalation 

risk assessment. 

Toxicology Endpoint Selection Process (U.S. EPA, 1998b) does not address the use of 

UFs in acute dietary risk assessment. In practice, however, the same 10-fold inter- and 

intraspecies UFs are used in calculating the acute dietary RfD as are used for the chronic RfD. 

Other standard UFs may be used when appropriate (e.g., the LOAEL-to-NOAEL threefold 

factor). Others are not appropriate for an acute risk assessment, for example, the threefold 

subchronic-to-chronic factor. However, no standard set of “core” studies has been defined for 

acute dietary risk assessment; therefore, a database UF is not used. If appropriate endpoints and 

doses cannot be selected for acute dietary risk assessment from the studies in the database, then 

an acute RfD is not calculated. 

2.1.4. Office of Water (OW) Health Advisories (HAs) 

The OW HA program was initiated in 1978 to provide guidance on unregulated 

contaminants found in drinking water. Since then, HAs have also been developed for regulated 

2-6
 
contaminants. HAs are derived for contaminants that are known to or are likely to occur in 

drinking water and that may cause adverse, noncarcinogenic health effects (Orme and Ohanian, 

1991). The approach for developing HAs is based on recommendations from the National 

Academy of Sciences (NAS, 1977). HAs are developed for specific exposure durations (1 day, 

10 days, longer-term, and lifetime) that reflect different emergency contamination situations. 

HAs are not legally enforceable, but they do serve as technical guidance to assist in emergency 

spills or contamination situations or for determining unreasonable risks to health under sections 

1415 and 1416 of the Safe Drinking Water Act. They also are issued at the request of State or 

local governments or to fill a need for criteria, guidelines, or standards. HAs undergo scientific 

peer review and can function as a preliminary risk assessment, if necessary. 

The following assumptions are used in setting the various HAs. The 1-day HA represents 

a concentration of the contaminant in drinking water that is considered protective of adverse 

noncancer health effects in a 10 kg child. The 10 kg child serves as the protected individual for 

the less-than-lifetime HAs because a child of this size is likely to receive a greater dose on a 

mg/kg basis. This 1-day HA can serve as a guideline for each day for up to 5 consecutive days 

of exposure. The 1-day HA is usually derived from experimental studies of 7 days duration or 

less. 

The 10-day HA is considered protective of these effects in a 10 kg child for each day for 

up to 14 days of continuous exposure and may be based on experimental studies of 30-day 

duration or less. 

The longer-term HA, which is based on subchronic exposure studies covering 10% of an 

animal’s lifetime, is considered protective of an exposure period in humans of up to 7 years (i.e., 

10% of an individual’s lifetime). The longer-term HA is developed to protect both a 10 kg child 

and a 70 kg adult. 

The lifetime HA is considered protective of lifetime exposures and is usually based on 

chronic or subchronic or other more relevant experimental data. The Lifetime HA is based on 

the chronic oral RfD, adjusted for a 70 kg adult drinking 2 L water per day; the value is 

apportioned by a relative source contribution, for example, 20% of the toxicant represented by 

intake of water. 

HA levels are generally based on available, well-conducted studies that involve humans 

or animals. Data from drinking water studies are preferred; however, data from dietary or 

gavage studies can also be used. In the absence of oral data, studies by other routes of exposure, 

such as inhalation or injection, are considered. Following identification of an appropriate study 

to develop a HA, the NOAEL or the LOAEL is adjusted for water consumption by the protected 

2-7
 
individual. For a child, the assumed water consumption level is 1 L/day; for an adult, 2 L/day is 

used. 

When data are absent for setting a 1-day or a 10-day HA, OW uses scientific judgment on 

how to handle any given situation on the basis of the overall weight of evidence. In the absence 

of short-term toxicity studies, a subchronic or chronic study may be used to develop a less-thanlifetime HA. Given the pressure under which HAs need to be calculated, many assessments are 

based on whatever toxicological data are available and on scientific judgment. Although this 

may be an overly conservative approach, OW considers the error to be protective of public 

health. 

OW applies the same factors for minimum data as those outlined in the Agency’s RfD 

methodology. For example, in emergency situations, missing data are accounted for by applying 

another factor of 3 or 10. Or, for instance, where inhalation data might be applied to estimate a 

HA based on water consumption, a factor may be applied to account for differences in 

absorption. Judgments based on toxicokinetic and toxicodynamic considerations are reached 

through intensive consultation. 

Calculation of HAs is straightforward and familiar, and in most cases the NOAEL/UF 

approach is used. For each of the less-than-lifetime HA values, it is assumed that all of an 

individual’s exposure to a contaminant comes from a drinking water source. The calculation of 

the lifetime HA differs from that of the less-than-lifetime values in that a relative source 

contribution factor is included. This factor adjusts the exposure to reflect the portion that is 

likely to be contributed from drinking water. Unless actual exposure data are available, a default 

factor of 20% is used to reflect the assumed contribution to exposure from drinking water. Also, 

in cases where there is limited evidence suggesting a carcinogenic potential of a contaminant, an 

additional “policy” factor of 10 is applied in calculating the lifetime HA. 

The methodology for developing HAs was reviewed by the SAB and the FIFRA 

Scientific Advisory Panel in 1986. Each HA that is developed undergoes external peer review 

and Agency review before it is released to the public. The availability of the HAs is announced 

in the Federal Register and distributed through the Safe Drinking Water Hotline and the Water 

Docket and by the Office of Science and Technology in OW. In addition, HAs have been 

published in a collection of books and are available in English, Japanese, and Italian. 

2-8
 

2.1.5. Agency for Toxic Substances and Disease Registry (ATSDR) Minimal Risk Levels 

(MRLs) 

The ATSDR is tasked with establishing MRLs, which are defined as 

“ ... an estimate of daily human exposure to a hazardous substance that is likely 

to be without appreciable risk of adverse noncancer health effects over a specified 

route and duration of exposure.” 

MRLs are considered by ATSDR to be substance-specific estimates intended to be 

screening levels in identifying contaminants and potential health effects that may be of concern; 

they do not define clean-up or action levels. The derivation procedures for MRLs have many 

similarities and parallels to the derivation of RfDs and RfCs; MRLs are based on careful 

scientific consideration of noncancer health effects only, not on consideration of cancer effects. 

A list of various procedural specifics employed in the derivation of MRLs, including specific 

effects and the level of severity, is codified in a Federal Register notice (ATSDR, 1996). The 

definition of an MRL differs expressly from EPA’s definition of an RfD or an RfC in that both 

route and duration are included. The current routes of concern for MRL derivation are oral and 

inhalation (but not dermal). 

The EPA procedures and methodologies discussed above address the issue of duration 

through a variety of extrapolation procedures. For MRLs, however, duration is addressed by 

providing for the designation of MRLs in three different duration categories: acute = <14 days, 

intermediate = 15–364 days, and chronic = >365 days. These duration categories are absolute 

and apply to all species, regardless of relative life span. Thus, it is possible for a contaminant to 

have a total of six different MRL values: two routes by three different durations. 

The use of UFs is a parallel practice in RfD/RfC and MRL derivation. The UFs used by 

ATSDR are intraspecies 1, 3, 10; interspecies 1, 3, 10; and LOAEL/NOAEL 3, 10. The 

modifying factor (MF) can include database considerations, that is, deficiencies in the data or 

overestimates from bioaccumulative chemicals. 

2.2. SUMMARY OF CURRENT METHODS FOR SETTING ACUTE, SHORT-TERM, 

AND LONGER-TERM REFERENCE VALUES 

In summary, several methods are used by various EPA programs for setting acute, shortterm, and longer-term reference values. The definitions for each of the durations used for the 

methods reviewed are included in Table 2-1. Because there are some differences in these 

2-9
 
Table 2-1. Duration definitions used for various reference values 

Reference value duration Definition 

Acute 

ARE Inhalation single continuous exposure values for durations 

< 24 hrs (to be protective of intermittent exposures) 

AEGL 10 and 30 min; 1, 4, and 8 hrs 

OPP acute RfD Maximum 1-day dietary exposure 

OW 1-day HA 1 day (5 successive daily doses) 

ATSDR acute MRL <14 days 

Standardized definitiona 24 hrs or less 

Short-term 

ARE NA 

AEGL NA 

OPP short-term RfD 1 day–1 month 

OW 10-day HA 10 days (7–14 successive daily doses) 

ATSDR MRL NA 

Standardized definitiona >24 hrs up to 30 days 

Longer-term 

ARE NA 

AEGL NA 

OPP intermediate RfD 1–6 months 

OW longer-term HA Approximately 10% of life span in humans (90 days to 1 

year in test species) 

ATSDR intermediate MRL 15–364 days 

Standardized definitiona >30 days up to approximately 10% of the life span in 

humans (>30–90 days in typically used laboratory species) 

a

 See Chapter 4 for further discussion of these definitions. 

2-10
 
definitions, standardized definitions were discussed at the Risk Assessment Forum Colloquium 

(CDM Group, 2000), and these are shown in Table 2-1. Definitions for durations are further 

discussed in Chapter 4. 

A comparison of the UFs applied for various reference values is shown in Table 2-2. 

Although there is some variation in the UFs applied, those for animal-to-human extrapolation 

(UA), for within-human variability (UH), and for LOAEL-to-NOAEL (UL) are fairly consistent. 

Less consistent is the way in which database deficiencies (UD) are taken into consideration, 

particularly for pesticides where the Food Quality Protection Act (FQPA) safety factor is used to 

account for deficiencies in the database related to children’s health risks. 

Duration extrapolation for each of these values was also reviewed. Some type of 

duration adjustment of the NOAEL or the BMD is done for the ARE and the AEGL methods, 

and there appears to be consistency in the use of Cn

 x t for extrapolating from shorter to longer 

exposures but in using the same value (i.e., no duration adjustment) when extrapolating from 

longer to shorter exposures. Duration extrapolation is not done for the OPP RfDs, the OW HAs, 

or the ATSDR MRLs. 

2.3. RECOMMENDATION 

On the basis of its review of the various approaches to setting acute, short-term, and 

longer-term reference values, the Technical Panel concurred with the recommendation of the 

10X Task Force that acute, short-term, and longer-term reference values should be set, where 

possible, and that they be incorporated into the IRIS database. In addition, the Technical Panel 

recommended that these values be set in a consistent manner, using standardized definitions for 

acute, short-term, longer-term, and chronic durations that are consistent with current practice. 

These values can then be used by various program offices, where applicable. A scheme for 

deriving these additional values is presented in Chapter 4. 

2-11
 

Table 2-2. Uncertainty/safety factors for various reference values 

Reference value 

UFa 

UA UH UL UD FQPAb 

ARE 1, 3, 10 1, 3, 10 1, 3, 10 ND NA 

AEGL 1, 3, 10 1, 3, 10 3c NDd NA 

OPP acute and 

intermediate RfDs 

10 10 3, 10 NDe 10+ 

OW HAs 1, 3, 10 1, 3, 10 1, 3, 10 case-specific NA 

ATSDR MRLs 1, 3, 10 1, 3, 10 1, 3, 10 NDd NA 

a

 Uncertainty factors: UA = animal-to-human; UH = within-human variability; 

UL = LOAEL-to-NOAEL; UD = database deficiency.
 
 b

 Additional safety factor required under FQPA.
 
 c

 Endpoint = lethality, not really a LOAEL-to-NOAEL adjustment in this case.
 
 d

 Database deficiencies considered, and a factor may be included for intermediate RfDs if, for
 


example, there is no reproduction and fertility study.
 
 e

 Overlaps with the FQPA safety factor (see U.S. EPA, 2002b)
 


ND = not done
 


NA = not applicable
 


2-12
 

3. REVIEW OF TESTING GUIDELINES WITH RESPECT TO 

LIFE STAGE ASSESSMENT 

As a first step in determining the data necessary for setting various duration reference 

values for protecting potentially susceptible subpopulations, the Technical Panel reviewed the 

current OPPTS Series 870 health effects testing guidelines2

 to determine what information is 

gathered in these studies. The intent of this review is not to suggest that additional testing be 

conducted for each and every chemical in order to fill in the information gaps identified for those 

organ systems evaluated. Nor is it suggested that the alternative testing protocols that are 

discussed in this chapter should be conducted for every chemical or become part of current 

toxicology testing requirements or that these alternative protocols are the only options available. 

Rather, it is the goal of this document to provide a basis for the development of innovative 

alternative testing approaches and the use of such data in risk assessment and to then illustrate 

some aspects of this concept with a few examples. 

Development of a toxicology testing paradigm that is based not on rigid conformance to a 

list of required guideline screening studies but rather on the application of knowledge about the 

chemical is encouraged. Under such a paradigm, both the selection of studies that would be 

required as well as the design of the tests themselves could be influenced by other substantive 

and reliable information about the chemical. For example, the incorporation of toxicokinetic and 

mode-of-action data early in the development of the testing strategy for a chemical would 

provide particularly valuable direction for development of research protocols. 

Other input could include toxicity and dose-response data from other guideline or 

nonguideline studies, in vitro screening assays, structure-activity relationships, studies that 

examine age-related sensitivity or susceptibility to chemical exposure, and information on 

potential or actual exposure to humans. These data could be used to inform a more targeted 

approach in the design of individual studies or of an overall testing strategy and might in some 

cases result in a reduction in the number of animals used in testing or support a position that a 

traditionally required toxicology test should be waived. 

The purpose of the review of the current OPPTS guidelines was to understand which 

target organ systems are evaluated in current testing protocols and how thorough the testing 

protocols are with respect to life stage assessment; endpoint assessment; route, timing, and 

2

The guidelines are available on the OPPTS web page (http://www.epa.gov/docs/ 

OPPTS_Harmonized/870_Health_Effects_Test _Guidelines/Series/). 

3-1 
duration of exposure; reversibility; and latency to response. These issues were all considered of 

importance in evaluating potentially susceptible subpopulations, including children. The 

following sections give an overview of the current testing protocols evaluated in this way and, 

for certain organ/functional systems, provide a more in-depth analysis as to whether and how 

current protocols address these issues. The organs/functional systems that were examined in 

greater detail included the reproductive and the nervous systems, which were selected to 

represent systems that are thought to be rather well-evaluated. The immune and the 

cardiovascular systems were selected for review because the current evaluation of these systems 

is limited. It should be noted that testing guidelines were not originally designed with a focus on 

evaluations of different life stages or different durations of exposure. Therefore, a number of 

gaps in life stage assessment, endpoint assessment, timing and duration of exposure, 

reversibility, and latency to response were noted for each organ system that is reviewed in depth. 

The last section provides recommendations for alternative testing approaches that are 

designed to make more efficient use of animals and resources in combined studies that would 

provide more extensive data on life stages, endpoints, and other factors not well characterized in 

current testing approaches. 

3.1. EVALUATION OF CURRENT GUIDELINE TESTING PROTOCOLS 

The following tables and figures summarize the exposures and endpoints covered in 

current testing guidelines, what is covered for each organ system/endpoint measured, and the 

relative depth of evaluation for each system/endpoint. In addition, the life stages covered by 

exposures and outcomes are illustrated. The discussions that correspond to the figures give an 

overview of the tests that are currently available and the gaps in assessment of life stages, 

endpoints, timing and duration of exposure, and latency to response. Together, these analyses 

provide a clear picture of the testing guidelines currently available, the systems/endpoints 

measured, the life stages during which exposures and outcomes are measured, the timing and 

duration of exposures included, and the degree of detail covered for both structural and 

functional outcomes. 

In order to make comparisons among laboratory animal species and humans in terms of 

life stages covered, the approximate ages that correspond to specific events or life stages (e.g., 

birth, weaning, puberty, etc.) in different species are shown in Table 3-1, and these events/life 

stages are indicated in the figures. In a few cases, no data could be found on appropriate ages 

corresponding to particular life stages. In particular, the ages for mature adults and older adults 

often were not available, and there is some controversy about what constitutes old age in today’s 

3-2
 
Table 3-1. Approximate age at equivalent life stages in several species 

3-3
 

Rat Mouse Rabbit Beagle dog 

Life stage Age Life stage Age Life stage Age Life stage Age Li

Embryonic GD 0–16 Embryonic GD 0–15 Embryonic GD 0–19 Embryonic GD 0–30? Em

Fetala GD 16–22 

(22–23 days) 

Fetal GD 15–20 

(18–22 days) 

Fetal GD 19–32 

(30–32 days) 

Fetal GD 30–63 

(53–71 days) 

Fet

Neonateb PND 0–14 Neonate PND 0–14 Neonate PND 0–21? Neonate PND 0–21 Neo

Weaningc PND 21 Weaning PND 21 

(19–28) 

Weaning PND 42 

(42–56) 

Weaning PND 42 Infa

Tod

Young PND 22–35 Young PND 21–35 Young PND 42–? Young 1.5–5 mos Pre

Ele

sch

Puberty PND 35–60 Puberty PND 35–? Puberty 3–8 mos Puberty 5–7 mos Ad

Sexual maturity 

2.5–3 mos Breeding 

age 

1.5–2 mos Breeding 

age 

6–9 mos Breeding 

age 

12 mos You

Mature adult 

5–18 mos Mature 

adult 

Mature adult 

Mature adult 

Ma

Old adult 18 mos–2 

yrs+ 

Old adult Old adult Old adult -15 yrs Old 

a Range of gestation length in parentheses.b Some neonatal events in rodents occur in utero in humans.c Range of weaning ages in parentheses.

GD = gestation dayPND = postnatal day
population. A background paper on aging commissioned as part of this review discusses this 

issue to some extent (Versar Inc., 2001a). In animal studies, the use of dietary restriction has 

been shown to affect aging and life span to a significant extent, so the issue of what constitutes 

an older animal is also somewhat controversial. 

3.1.1. Exposures and Endpoints Related to General Toxicity Testing 

Table 3-2 provides an overview of the biological systems and other endpoints that are 

evaluated by routine toxicity test designs. The table includes all of the routine test designs that 

are available in Agency testing guidelines for evaluating toxicity and most of the test designs 

that focus on specific biological functions. The acute and subchronic studies are intended to give 

general information on the potential toxicity of an agent by screening the major organ systems, 

in particular, the liver, the kidney, and the gastrointestinal tract. This information can be used to 

determine where to look in more detail at specific organ system structure and function. The 

chronic studies, which are usually done in combination with a carcinogenicity study, evaluate 

general toxicity in all major organ systems. Several testing guidelines have been developed with 

the idea that certain systems should be evaluated frequently in more detail (e.g., the nervous 

system) or that the general toxicity studies do not provide any indication of a potential for effects 

(e.g., reproductive and developmental toxicity studies). More detailed information about 

specific aspects of guideline test designs for certain systems (e.g., life stages covered, exposure 

periods, outcomes measured, etc.) is included in the figures. 

Table 3-2 is shaded and marked to indicate the extent of the evaluation of a particular 

system/endpoint within a particular test design. indicates that the system/endpoint is a 

primary focus of the particular test design and that detailed assessment of the dose-response 

relationship of an exposure is carried out within some defined life stage and exposure period for 

major elements of the system/endpoint. indicates those systems/endpoints for which 

some histopathology or clinical measure of system function is carried out. X indicates 

those systems/endpoints that are assessed in some observational or gross manner. “0” indicates 

that the system/endpoint cannot be included, generally because of the design of the test. Blank 

cells indicate that the system/endpoint is not presently included but could be if the test design 

were altered appropriately. 

It is obvious from the table that few systems/endpoints are examined in any significant 

detail. The systems/endpoints under the acute test designs are for the most part observational in 

nature. The acute inhalation toxicity with histopathology guideline (40 CFR 799.9135) was 

developed under the Toxic Substances Control Act for characterizing the exposure-response 

3-4
 
3-5

 Table 3-2. by routine toxicity guideline testing protocolsa

Guidelineb

Systems 

Lungrespiratory Cardio- vascular Hema- tologic Musculo- skeletal Skin Eye Gastro- intest- inal Kidney- urinary Liver Immuno- logical Repro- ductive Neuro- logical Endocrino- logical Pharmaco -kinetic- metabolic M

 Acute, oral X X X X X X X X 

 Acute, inhalation XX X X X X X X X X 

 Acute, dermal X X X XX X X X X X 

 Subchronic, oral XX XX XX XX X XX XX XX XX XX XX XX XX 

 Subchronic, inhalation XX XX XX XX X XX XX XX XX XX XX XX XX 

 Subchronic, dermal XX XX XX XX XX XX XX XX XX XX XX XX XX 

 21-day, dermal XX XX XX XX XX XX XX XX XX XX XX XX XX 

 Chronic, oral XX XX XX XX X XX XX XX XX XX XX XX XX 

 Chronic, inhalation XX XX XX XX X XX XX XX XX XX XX XX XX 

 Chronic, dermal XX XX XX XX XXX XX XX XX XX XX XX XX XX 

 Prenatal developmental toxicity

X XX XX X XX X XX X XX XX 

 Reproduction and fertility effects

X X X X X X XX XXX X X 

 Neurotoxicity, acute XXX  Neurotoxicity, subchronic XXX  Neurotoxicity, acute-delayed XXX  Neurotoxicity, subchronic-delayed XXX  Neurotoxicity, chronic XXX  Developmental neurotoxicity X XXX X

 Operant behavior XXX  Peripheral nerve function XXX  Sensory evoked potential XXX  Eye irritation, primary XX 0 

 Dermal irritation, primary XX 0 

 Dermal, sensitization X X 0  Dermal, penetration X XX 

 Metabolism/pharmacokinetics XXX 

 Genetic toxicity 0 

 Immunotoxicity XXX 0 

a X e observational or gross endpoints are included; indicates level X plus histopathology or some clini

 function. ental toxicity study includes a more in-depth structural evaluation. indicates the major 

 0 indicates that this endpoint cannot be included as a major aspect in this protocol. A blank indicates that an aspect is not routinely 

b A Series 870 guideline(s) exists for conducting each of the above tests.

Systems/endpoints evaluated 

X X X

indicates that som

The prenatal developm
relationship for sensitive endpoints following acute inhalation exposure and the toxicologic 

response following acute high exposures (see further discussion in section 3.1.1.1). Acute 

toxicity information is useful in establishing reference values for short-duration exposures and 

for establishing dose-ranges for subchronic and chronic studies. The subchronic and chronic test 

designs evaluate most endpoints with somewhat greater detail than do the acute test designs. 

Although the histopathology and/or clinical measures of system function are screening in nature, 

there is greater confidence that with this level of examination the dose-response relationship will 

be more clearly defined. Nevertheless, it should be recognized that most systems/endpoints are 

evaluated at a screening level, and detailed analyses of pathology and function are generally not 

carried out. Even in those test designs that do incorporate detailed analyses, these analyses are 

limited in regard to the life stages, exposure periods, and measures that are assessed. 

Figure 3-1 shows the study designs that are used for general toxicity testing 

superimposed on a time line that indicates the life stages during which exposure occurs (hatched 

bars) and endpoints are measured (indicated in the boxes). The guideline studies shown 

represent the minimum requirement for derivation of a chronic oral RfD. Similar studies are 

required for the chronic inhalation RfC, with appropriate endpoints for inhalation exposure and 

toxicity included. In some cases, only a 90-day subchronic study is available instead of the 

chronic studies shown. Because the relative length of time between life stages varies among 

species, the placement of exposures and endpoints on the figures is not necessarily to scale. The 

following sections discuss the studies that address acute and short-term toxicity as well as 

chronic toxicity. Similar figures related to specific organ system toxicity testing are shown in 

subsequent sections. 

3.1.1.1. Acute and Short-Term Toxicity Studies
 

3.1.1.1.1. Overview of tests. The primary purpose of the guideline acute toxicity tests (870.1100
 

acute oral; 870.1200 acute dermal; and 870.1300 acute inhalation) and other short-term studies
 

(e.g., 14–28-day studies, no OPPTS guidelines available) is to identify hazards (focusing on
 

route-specific lethality) from short-term exposure studies, provide a basis for classification and
 

3-6
 
3-7

Preconception Emb/Fetal Juvenile Adulthood 

Prenatal Developmental 

Toxicity Study

Reproduction and

Fertility Effects Study

ConceptionBirth Weaning Maturity Sene

Adolescence

Puberty

P

F1

Rodent Combined Chronic/

Carcinogenicity Study

NB/Preweaning

F1 sexual 

maturation

F1 & F2 litter size, sex ratio, F1 & F2

survival, weight, gross structure, 

brain, spleen, thymus weights

Life Stages

Guideline Study Designs: Satellite g

followed 28 

Fetal survival, weight,

gross structure

Dog Chronic Study

Satellite group

followed 28 days

Clinical observations, viab

body weight, food & wa

consumption, clinical path

(every 6 months)

Opthalmolog

structure & 

Neurotoxicity evaluation

Neurotoxicity evaluation

P & F1 estrous cycles, s

measures, fertility, preg

maintenance, parturition

weight, gross structu

histology

Preconception Emb/Fetal Juvenile Adulthood 

Prenatal Developmental 

Toxicity Study

Reproduction and

Fertility Effects Study

ConceptionBirth Weaning Maturity Sene

Adolescence

Puberty

P

F1

Rodent Combined Chronic/

Carcinogenicity Study

NB/Preweaning

F1 sexual 

maturation

F1 & F2 litter size, sex ratio, F1 & F2

survival, weight, gross structure, 

brain, spleen, thymus weights

Life Stages

Guideline Study Designs: Satellite g

followed 28 

Fetal survival, weight,

gross structure

Dog Chronic Study

Satellite group

followed 28 days

Clinical observations, viab

body weight, food & wa

consumption, clinical path

(every 6 months)

Opthalmolog

structure & 

Neurotoxicity evaluation

Neurotoxicity evaluation

P & F1 estrous cycles, s

measures, fertility, preg

maintenance, parturition

weight, gross structu

histology

Emb = Embryonic 

Figure 3-1. Exposures and endpoints related to general toxicity evaluations. Endpoints shown are fo

oral exposures; endpoints specific to inhalation and dermal exposure are included for studies 

those routes of exposure.

NB = Newborn P = Parental F1 and F2 = 1st and 2nd filial generations
labeling, and enable the selection of exposure ranges for longer-term studies.3 Acute guideline 

studies are conducted in young adult animals, with a 14-day post-exposure observation period. 

Other than mortality, the endpoints include cage-side observations, body weight at the end of the 

observation period, gross pathology changes at necropsy, and histopathological examination of 

organs showing evidence of gross pathology in animals surviving 24 hours or more. Two other 

available guideline studies cover acute exposures followed by extensive assessment of a specific 

organ system. The first is the acute inhalation toxicity study with histopathology (40 CFR 

799.9135), which was developed for hazardous air pollutants. This study includes assessments 

of liver, kidney, and broncho alveolar lavage samples for several indicators of cellular damage 

(e.g., total protein, cell count, percent leukocytes) and a phagocytosis assay to determine 

macrophage activity. For the respiratory tract histopathology, detailed specifications are 

provided. 

The second expanded study that includes observations following an acute exposure is the 

acute neurotoxicity study (870.6200), which was developed for the evaluation of neurotoxic 

chemicals and includes assessments of functional behavior and motor activity at the time of peak 

effect and again at 14-days post-treatment and histopathology of the central and peripheral 

nervous systems at 14-days post-treatment. The prenatal developmental toxicity study 

(870.3700) in two species (typically rats and rabbits) and the DNT study (870.6300) can also 

provide relevant data for acute risk assessment because maternal observations are often recorded 

daily and there is a presumption that effects during development may result from a single 

exposure. 

3.1.1.1.2. Gaps in life stage of assessment. Acute/short-term testing is done only in prenatally 

exposed animals and in young adults. No direct information is available from any of these 

studies on acute or short-term exposure in postweaning young animals or aged animals. 

3

Alternative test protocols have been adopted by the Organization for Economic 

Cooperation and Development for acute toxicity testing for oral, dermal, and inhalation 

exposure, including the fixed-dose procedure, the acute toxic class method, and the up-and-down 

procedure. All are designed to minimize animal usage and provide minimal hazard and doseresponse information for classification, labeling, and dose selection. In the future, EPA plans to 

put primary reliance on the up-and-down procedure for testing of technical grade pesticides, 

although the other tests may be acceptable in some circumstances, e.g., testing of pesticidal 

products. These studies are not designed to provide information for use in less-than-lifetime risk 

assessments. 

3-8 
3.1.1.1.3. Gaps in assessment endpoints. Data on only a limited number of toxicological 

endpoints are available from guideline acute toxicity (lethality) studies except in the case of the 

acute inhalation toxicity guideline study with histopathology and the acute neurotoxicity study. 

Consequently, these studies often are not suitable for use in deriving reference values unless 

additional data, such as those from subchronic studies (e.g., hematological, clinical, histology of 

more organs), are collected. Some data from animals examined at early times might be available 

in guideline subchronic or chronic studies. These data could augment the results from guideline 

acute studies. 

3.1.1.1.4. Gaps in duration of exposure/latency to response assessment. There is no guideline 

study for short-term toxicity testing, although the prenatal developmental toxicity studies in rats 

and rabbits and the DNT study include repeated dosing of maternal animals for periods of less 

than 25 days. Because of the post-exposure observation period in acute guideline studies and in 

the DNT study, some information on latency to effect and reversibility of effect may be 

available. 

3.1.1.2. Subchronic and Chronic Toxicity Studies 

The subchronic exposure studies (870.3100, 870.3150, 870.3200, 870.3250, 870.3465) 

are used for setting chronic RfDs and RfCs when a chronic study is not available. The guideline 

studies for chronic exposures (870.4100, 870.4200, 870.4300) (1 year in rodents, although the 

typical study is a combined chronic and carcinogenicity study with a 2-year exposure) provide an 

in-depth look at a number of organ systems, and in some cases they evaluate both structure and 

function (see Figure 3-1). The chronic study in nonrodents, usually dogs, involves a 12-month 

exposure with similar endpoints assessed as in rodents. The prenatal developmental toxicity 

study (870.3700) in two species (typically rats and rabbits), the DNT study (870.6300), and the 

reproduction and fertility effects study (870.3800), typically in rats, are also considered in setting 

chronic RfDs or RfCs. 

3.1.1.2.1. Gaps in life stage of assessment. The subchronic and chronic studies are conducted 

in young adult animals, with exposure in the chronic/carcinogenicity study continuing into old 

age. No information is available from chronic studies in pre- or postnatal animals. Exposures in 

subchronic study protocols do not include pre- or postnatal development, although the 

reproduction and fertility effects study does provide data on subchronic exposures in animals 

that are exposed before birth, through prenatal and postnatal development up to mating of the F1 

3-9
 
males and females and through pregnancy (F1 young adult females). No subchronic toxicity 

evaluations are conducted in aged animals. No chronic studies are conducted in pre- or postnatal 

animals, although aged animals are exposed and evaluated as part of the chronic study protocol. 

3.1.1.2.2. Gaps in assessment endpoints. The greatest gaps appear to be the lack of routine 

testing for subchronic neurotoxicity in adults, immunotoxicity testing in adults, and more 

thorough toxicokinetics in animals at various life stages. Gaps in assessment endpoints during 

prenatal and postnatal development are discussed in the next section. Assessment endpoints for 

routine toxicity testing in old age are completely lacking, as is background information on 

endpoints related to the aging process itself. 

3.1.1.2.3. Gaps in duration/latency assessment. Chronic studies that include prenatal and 

postnatal exposure into old age are lacking. The so-called chronic study in dogs is actually a 

short-term study, as it does not cover at least 10% of the life span. Chronic studies that include a 

satellite group in which exposure is stopped after 12 months in rodents do assess latency to 

response for a brief period of time (28 days or more). 

3.1.2. Exposures and Endpoints Related to Evaluation of Reproductive Toxicity 

3.1.2.1. Overview of Tests 

The reproductive organs are examined structurally in a number of general guideline 

screening studies, including the 90-day subchronic study (OPPTS 870.3100, 870.3150, 

870.3250, 870.3465), chronic/carcinogenicity studies (OPPTS 870.4100, 870.4200, 870.4300), 

the prenatal developmental toxicity study (OPPTS 870.3700), and the reproduction and fertility 

effects study (OPPTS 870.3800), which is a two-generation reproduction study. In addition, 

extensive assessment of numerous functional aspects of the reproductive system is conducted in 

the reproduction and fertility effects study. Specific functional effects on the reproductive 

system of male animals can also be assessed in the rodent dominant lethal assay (OPPTS 

870.5450). As illustrated in Figure 3-2, these studies include a variety of both structural and 

functional assessments of the reproductive system over a wide sampling of life stages. 

In guideline subchronic and chronic/carcinogenicity studies, gross structural evaluation 

and general qualitative histopathology are conducted on reproductive organs and tissues. The 

animals in these studies are adults, but at the time of organ assessment they may be young (e.g., 

rats 45 days to 5 months of age from a subchronic study), mature (e.g., rats 5–18 months of age 

from a reproduction study), or old animals (e.g., rats 18 months to 2 years of age from a chronic 

study), depending on the protocol. 

3-10
 
3-11

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental 

Toxicity Study

Reproduction andFertility Effects Study

ConceptionBirth Weaning Maturity Sene

Developmental Neurotoxicity

Study

F1 sexual

maturation

Adolescence

Puberty

P

F1

F1 & F2 litter size, sex ratio,

survival, weight, gross structure, 

brain, spleen, thymus weights

Embryonic death 

Rodent Dominant 

Lethal Assay

Litter size, sex ratio, survival, weight,

gross structure, developmental

landmarks including sexual maturation

Litter size, sex ratio, fetal survival,

weight, gross structure

P & F1 Estrous cycle

measures, fertility, p

maintenance, part

organ weight, gross 

histology

Subchronic or Chronic 

Toxicity Study

Organ weight, histo

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental 

Toxicity Study

Reproduction andFertility Effects Study

ConceptionBirth Weaning Maturity Sene

Developmental Neurotoxicity

Study

F1 sexual

maturation

Adolescence

Puberty

P

F1

F1 & F2 litter size, sex ratio,

survival, weight, gross structure, 

brain, spleen, thymus weights

Embryonic death 

Rodent Dominant 

Lethal Assay

Litter size, sex ratio, survival, weight,

gross structure, developmental

landmarks including sexual maturation

Litter size, sex ratio, fetal survival,

weight, gross structure

P & F1 Estrous cycle

measures, fertility, p

maintenance, part

organ weight, gross 

histology

Subchronic or Chronic 

Toxicity Study

Organ weight, histo

Emb = Embryonic 

Figure 3-2. 

NB = Newborn P = Parental F1 and F2 = 1st and 2nd filial generations

Exposures and endpoints related to reproductive evaluations.
Standard guideline prenatal developmental toxicity studies are designed to evaluate the 

potential effects of the test substance on the developing fetus. Observations on the reproductive 

capacity of the maternal animals in this study generally consist only of clinical observations 

(including any abnormalities of pregnancy maintenance) and gross necropsy data (including 

uterine). Selected fetuses are examined for gross structural changes to the internal reproductive 

organs. In studies that employ methods of serial sectioning in the process of soft tissue 

examination, a limited macroscopic evaluation of the internal structure and integrity of the 

reproductive organs is performed; however, the fetal tissues are not examined microscopically. 

Additionally, there are no assessments of organ function in this study design. 

In the guideline reproduction and fertility effects study, rats are exposed to the test 

substance over the duration of two generations, beginning when the first generation animals are 

young adults of approximately 6–9 weeks of age. Daily exposure continues during all phases of 

development and reproductive function. Adult animals of both generations are killed as mature 

adults, generally prior to reaching reproductive senescence (that is, the cessation of normal 

reproductive function) or an age that would be considered geriatric in that species. Assessments 

of reproductive capability and function are conducted at least once in each generation. These 

assessments include direct evaluation of the age of sexual maturation, estrous cyclicity 

(immediately prior to mating), sperm measures (at termination), mating success, fertility and 

fecundity, implantation, pregnancy maintenance, gestation duration, parturition, and success of 

lactation (e.g., maternal nurturing and nesting behavior). 

Indirect assessments of some reproductive functions are also evaluated. These 

observations are based on evidence of normality in a structure, function, or process that is 

dependent on normal functioning of the component parts, including, for example, hormonal 

homeostasis, ejaculation, accessory gland function, placental function, milk production, pup 

nursing behavior or ability, and, to some extent, reproductive senescence (although the adult 

animals are terminated at the end of each generation, when they are only around 6 months of 

age; therefore, there are no assessments conducted in older rats). Gross structural assessments of 

the whole animal are conducted on adult and immature animals throughout the course of the 

study; gross internal (organ) structural assessments are conducted on offspring that are killed at 

litter standardization (postnatal day [PND] 4), weaning (PND 21), and termination of each 

generation (mature adults). Histopathological evaluation of the reproductive organs (gonads and 

accessory structures) is conducted only in the mature parental adult animals that are killed at the 

termination of each generation. The guideline specifies a very focused pathological examination 

of the reproductive organs in this study. 

3-12
 
The dominant lethal assay is not conducted for every chemical, but it may be conducted 

in response to a concern raised by other developmental or reproductive toxicity findings in the 

database. In this study, sexually mature adult males are treated with the test substance to 

determine whether there is an effect in the germinal tissue that does not cause dysfunction in the 

gamete but is lethal to the fertilized egg or developing embryo. Exposed males are mated with 

untreated females, and uterine contents are evaluated. Evidence of pre- and/or postimplantation 

loss is generally thought to be indicative of treatment-related chromosomal damage in germinal 

tissue. 

3.1.2.2. Gaps in Life Stage of Assessment 

Determination of gaps in the assessment of potential effects of any chemical across all 

life stages requires consideration of both the exposure period and the time of assessment. In the 

prenatal developmental toxicity study, animals are exposed from implantation through gestation. 

The reproductive organs are examined for gross structural changes, but no microscopic 

examination is conducted. There is no follow-up of the animals to determine the functional 

consequences of prenatal exposure. In the reproduction and fertility effects study, the F1 

animals are exposed from preconception throughout prenatal and postnatal development until 

after mating. The reproductive organs are examined macroscopically at weaning and adulthood. 

The maturation of the reproductive system is assessed, as is its function. Thus, the study 

provides a fairly thorough assessment of structure and function following exposure during many 

critical periods of development. In the parental generation, the animals are exposed as young 

adults, and the structure and function of the reproductive organs are assessed. 

The dominant lethal study, when conducted, assesses a single aspect of the function of 

the reproductive system for one sex, although a detailed structural assessment is not conducted. 

In the subchronic and chronic studies, the animals are exposed beginning as young adults, and 

the structure—but not the function—of the reproductive organs is assessed. Therefore, the major 

gaps include (1) the lack of functional assessment (particularly the age of onset of reproductive 

senescence) in older adult animals following adult-only exposures, and (2) the lack of structural 

and functional assessments in older adult animals following developmental exposures. 

The onset of reproductive senescence can be marked by findings such as altered 

hormonal homeostasis, disruption of estrous cyclicity, diminished sperm measures (number, 

motility, or morphology), or gonadal atrophy. Studies in rodents have demonstrated the adverse 

effects of a number of agents (e.g., ionizing radiation, chemotherapeutic agents, polycyclic 

aromatic hydrocarbons, and agents that form epoxides, such as 1, 3-butadiene and 4-

vinylcyclohexene) on reproductive senescence (reviewed by Hoyer and Sipes, 1996). 

3-13
 
In humans, premature reproductive senescence has been associated with cigarette 

smoking (Jick et al., 1977). In addition to potentially diminishing fertility in individuals who are 

only slightly past prime reproductive age, early reproductive senescence can adversely affect the 

general health of the aged human. For example, hormonal alterations that are associated with 

early senescence have been linked to abnormalities of cardiovascular function, osteoporosis, and 

even a predisposition to early mortality. 

3.1.2.3. Gaps in Assessment Endpoints 

As described above, there are identifiable gaps in the endpoints that are used to assess 

reproductive toxicity in guideline studies. Currently, there is no assessment of functional 

endpoints in older animals following adult exposures, and there are no structural or functional 

endpoints assessed in older animals following developmental exposures, including reproductive 

senescence. In addition, concerns have recently been raised about the ability to detect rare 

malformations of the reproductive organs and abnormalities in the maturation of the reproductive 

system in the two-generation reproductive toxicity study. This concern relates particularly to 

endocrine-active chemicals. In the current guideline, three pups/sex/litter are examined 

macroscopically at weaning. 

Questions have been raised about whether these weanlings should be retained until day 

45 (females) or day 60 (males) to ensure that any later-appearing gross or functional changes are 

detected. This issue is currently being examined within the endocrine validation/standardization 

program. 

3.1.2.4. Gaps in Duration/Latency Assessment 

There are no studies that include acute or chronic exposures that can be used to assess the 

development of the reproductive system. As indicated above, it has been suggested that animals 

be retained until older ages in the two-generation study in order to assess later-appearing 

structural or functional changes in reproductive organs. In addition, there is no consideration of 

latent responses for reproductive toxicity, such as early onset of reproductive senescence, as a 

result of an exposure earlier in life in any of the studies that can be used to evaluate reproductive 

toxicity, except for a few endpoints in the DNT study. 

3-14
 
3.1.3. Exposures and Endpoints Related to Evaluation of Neurotoxicity 

3.1.3.1. Overview of Tests 

Observation of the animals for signs of overt toxicity and routine gross pathological 

assessment of the nervous system is required under OPPTS acute, subchronic, and chronic study 

protocols (870.100–870.400 series). In rat studies, age at initiation of testing is to be 8–12 

weeks under acute and subchronic testing protocols. In acute studies, cage-side observation and 

gross neuropathology are the only endpoints required under 870.100 (oral, dermal, or inhalation 

exposure). Motor activity, grip strength, and sensory reactivity and neuropathology are 

measured in the rodent oral study, the dermal 21–28- and 90-day subchronic studies, and the 90-

day inhalation study. In rodent subchronic studies, specific assessment for neurotoxicity is 

performed at or near the end of the study, although observations of the animals, including those 

for detection of overt neurotoxicity, are made routinely throughout the study. No specific 

functional tests for neurotoxicity are required for nonrodent subchronic studies, although 

observation and neuropathology are required. 

Chronic toxicity studies (oral, dermal, inhalation) are to be performed in two species (one 

rodent) over a 12-month period, regardless of the life span of the species. Exposure in rodents is 

to begin no later than 8 weeks of age. Motor activity, grip strength, and sensory reactivity are to 

be assessed at or near the end of the study, but no earlier than the 11th month. Clinical 

observation is performed weekly throughout the study and would presumably detect gross 

neurological abnormality. In current practice, the chronic study is often combined with the 

carcinogenicity test, in which dosing extends for 24 months in rats and 18 months in mice 

(OPPTS 870.4300). Motor activity would be performed at 11–12 months only, as in the chronic 

study, and not again until near the end of exposure. 

The neurotoxicity screening battery (870.6200) is designed to be included in acute, 

subchronic, or chronic toxicity studies (Figure 3-3). The endpoints examined extend those 

required in the 870.100 series, although there is no guidance as to when these extended batteries 

would be required. The functional observation battery includes a ranking system for general 

reactivity, activity, and gait abnormalities, as well as forelimb and hindlimb grip strength, 

landing foot splay, sensorimotor reactivity to sensory stimuli, and pain reception. Motor activity 

and a more detailed neuropathological observation are also required in this battery. For acute 

studies, assessments are made before initiation of dosing, at the estimated peak of activity within 

8 hours of dosing, and at 7 and 14 days post-dosing. For subchronic studies, assessments are 

performed pre-exposure and at 7, 8, and 13 weeks of exposure. For chronic studies, assessment 

is at pre-exposure and every 3 months post-exposure. There is no specific guidance regarding 

3-15
 
3-16

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental 

Toxicity Study

ConceptionBirth Weaning Maturity Senes

Developmental Neurotoxicity

Study

Adolescence

Puberty

Gross structure of the CNS

Motor activity

Auditory startle,

learning & memory

Neuropathology

Delayed Neurotoxicity of

Organophosphorous 

Pesticides (adult hen)

acute28-day

Neuropathy target esterase

(NTE), neuropathology, gross 

observation, motor function, 

acetylcholinesterase (AChE)

Schedule-controlled 

operant behavior,

peripheral nerve function,

sensory evoked

potentials

May b

Neurotoxicity

ScreeningBattery

acute

subchronic

(90 days)

chronic(12 months)

Chronic/carcinogenicity(24 months)

Neuropathology

Motor activity, F

strength, landing 

sensorimotor 

perce

? ? ?

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental 

Toxicity Study

ConceptionBirth Weaning Maturity Senes

Developmental Neurotoxicity

Study

Adolescence

Puberty

Gross structure of the CNS

Motor activity

Auditory startle,

learning & memory

Neuropathology

Delayed Neurotoxicity of

Organophosphorous 

Pesticides (adult hen)

acute28-day

Neuropathy target esterase

(NTE), neuropathology, gross 

observation, motor function, 

acetylcholinesterase (AChE)

Schedule-controlled 

operant behavior,

peripheral nerve function,

sensory evoked

potentials

May b

Neurotoxicity

ScreeningBattery

acute

subchronic

(90 days)

chronic(12 months)

Chronic/carcinogenicity(24 months)

Neuropathology

Motor activity, F

strength, landing 

sensorimotor 

perce

? ? ?

Emb = Embryonic 

Figure 3-3. 

NB = Newborn FOB = Functional observation battery

Exposures and endpoints for neurotoxicity evaluations.
the assessment schedule for the combined chronic/carcinogenicity study, but presumably the 

schedule required for the chronic study would be maintained. 

The DNT study protocol (870.6300) currently requires dosing of the dams from 

gestational day (GD) 6 through PND 10, although the requirement may soon be extended to 

PND 21 (i.e., until weaning). Motor activity is measured at PNDs 13, 17, 21, and 60. Auditory 

startle is measured around weaning and at PND 60, as is a test of learning and memory, which 

may be the same test or different tests at the two time points. Cage-side observation of both 

dams and pups is required, and neuropathology in the pups is required at PND 11 and at the 

termination of the study (usually PND 60). The prenatal developmental toxicity study 

(870.3700) requires dosing of the dams on GDs 6–20 in rats and 6–29 in rabbits. Gross 

structural evaluation of the nervous system is evaluated as part of the fetal examinations 

conducted in this study. 

3.1.3.2. Gaps in Life Stage of Assessment 

One of the most significant gaps revealed by Figure 3-3 is the lack of exposure or 

assessment under any protocol during old age. For example, following acute exposure, 

assessment is for 14 days in juvenile or young adult animals. The chronic exposure protocol 

extends exposure into adulthood and the combined chronic/carcinogenicity protocol extends 

exposure up to approximately the aged period in the rat, but neurotoxicology assessments are not 

performed in aged animals. Thus, none of the protocols assess potential effects of chemicals on 

aging as a function of exposure during development. This may be important, because studies in 

animals have shown that developmental exposure to agents that cause neurotoxicity, such as 

trimethyl tin, can accelerate the onset of cognitive deficits measured later in life. Other studies 

with methyl mercury have documented early-onset sensory dysfunction in monkeys exposed 

during development. Furthermore, current testing protocols do not provide information collected 

at different life stages—that is, comparison of effects of exposure during infancy, adulthood, or 

old age. This is important, because life stage-dependent differences in pharmacokinetic, and 

possibly toxicodynamic, parameters could result in quantitatively or qualitatively different 

effects at different life stages. 

Under the DNT protocol, there currently is no requirement to perform kinetic studies to 

ascertain either in utero or postnatal exposure. There is no mechanism to guarantee exposure 

postnatally (i.e., direct dosing of pups) because the compound may not be excreted into breast 

milk or it may be excreted only at very low concentrations. This is of particular importance, 

because the early postnatal period in the rodent is equivalent to a prenatal life stage in humans. 

3-17
 
There is no long-term follow-up assessment to detect delayed neurotoxic effects, a situation that 

is arguably more worrisome for developmental exposure than for exposure later in life. 

3.1.3.3. Gaps in Assessment Endpoints 

The nervous system is one of the most fully assessed organ systems in the EPA/OPPTS 

870 guidelines. Nonetheless, most of the endpoint assessments are designed to be screening 

procedures rather than sensitive assessments of nervous system function. In addition, the 

assessments required are different in the neurotoxicity screening battery than in the DNT study. 

The adult neurotoxicity screening battery does not require assessment of learning and memory or 

auditory startle. The lack of assessment of cognitive function in the neurotoxicity screening 

battery constitutes a significant omission that should be addressed. 

It may also be pointed out that even in the developmental protocol, the tests that are used 

to assess learning and memory may be very simple, potentially revealing only relatively gross 

deficits. In addition, although potentially more sensitive cognitive, sensory, and motor tests are 

available (Figure 3-3), there is no guidance as to what would trigger a requirement for these 

assessments. Except for the protocol for delayed neurotoxicity for organophosphorous pesticides 

in the hen, there is no assessment of neurochemical endpoints. Additionally, the required 

neuropathological assessments may also be considered screening. 

Minimal morphometric analysis, consisting of the thickness of “representative” layers in 

the neocortex, hippocampus, and cerebellum, is required in the DNT study. No morphometric 

analyses are required in the adult neurotoxicity testing protocols. Although more sophisticated 

tests would presumably not be performed on all agents, more sophisticated measures could be 

triggered by results from screening tests. It also may be advisable to require more sensitive tests 

in instances of particular concern, for example, adding more extensive morphometric analysis to 

the DNT protocol. 

In summary, although the nervous system is one of the most thoroughly assessed systems 

in the 870 test guideline studies, it must be kept well in mind when interpreting the results that 

these are screening tests. Positive findings must be viewed as indicative of relatively overt 

toxicity, not so-called subtle effects. 

3.1.3.4. Gaps in Duration/Latency Assessment 

One of the principles in the neurotoxicity risk assessment guidelines (U.S. EPA, 1998c) 

is that neurotoxicity could occur after one or a few exposures, such as in the case of an 

organophosphate insecticide that produces a delayed neuropathy, or only after a series of 

repeated exposures, as in the case of acrylamide. For DNT, it is assumed that a single exposure 

3-18
 
to a chemical during a critical period of development could result in an adverse effect on the 

developing nervous system. There are, however, few data that compare the effects of a single 

exposure to a chemical with the effects of the same chemical given multiple times during 

development. 

3.1.4. Exposures and Endpoints Related to Evaluation of Immunotoxicity 

3.1.4.1. Overview of Tests 

Examination of the macro- and/or microscopic structural anatomy of immune system 

organs and tissues is performed in a number of general guideline screening studies, including the 

acute inhalation toxicity with histopathology guideline (40 CFR 799.9135), the 90-day 

subchronic study (OPPTS 870.3100, 870.3150, 870.3250, 870.3465), the chronic/carcinogenicity 

studies (OPPTS 870.4100, 870.4200, 870.4300), the prenatal developmental toxicity study 

(OPPTS 870.3700), and the two-generation reproduction study (OPPTS 870.3800). In addition, 

functional assessments of the immune system are evaluated in the skin sensitization study 

(OPPTS 870.2600) and the immunotoxicity testing guideline (OPPTS 870.7800) (see Figure 3-

4). 

In the guideline immunotoxicity study, young adult rats (6–8 weeks of age) are exposed 

to the test substance for 28 days, at which time they are terminated. The spleen and thymus are 

examined macroscopically, and organ weights are recorded; a histopathological evaluation is not 

performed. Assessments of immune system function include an evaluation of the response to the 

T cell-dependent antigen, sheep red blood cells (SRBC). The SRBC antigen response assays can 

be conducted either by an antibody plaque-forming cell (PFC) assay or an immunoglobulin 

quantification by enzyme-linked immunosorbent assay (ELISA). In addition, an assessment of 

natural killer (NK) cell activity and/or enumeration of splenic or peripheral blood total B cells, 

total T cells, and T cell subpopulations may be required on a case-by-case basis. 

The skin sensitization study has been generally conducted in guinea pigs as a Guinea Pig 

Maximization Test (GPMT) or a Buehler test. In a recent review by the FIFRA Science 

Advisory Panel (U.S. EPA, 2001a), it was recommended that in the future, skin sensitization 

methods should preferentially include the local lymph node assay (LLNA), which uses young 

adult mice. The skin sensitization test involves an initial intradermal (GPMT) and/or epidermal 

(Buehler, LLNA) exposure of the test animal to a substance, followed by a challenge exposure 

approximately 1 week later. In the guinea pig tests, sensitization is determined by examining the 

reaction to the challenge exposure and comparing this reaction with that of the initial induction 

3-19
3-20

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental Toxicity Study

Reproduction andFertility Effects Study

ConceptionBirth Weaning Maturity Sene

Adolescence

Puberty

P

F1

Organ weights, gross 

structure, histology

Gross thymus/spleen structure

Chronic/carcinogenicity Study

Subchronic Study

Spleen/thymus 

weights

Clinical pathology (differential WBC,

serum immunoglobulin every 6 months

Reaction to initial induction or c

Immunotoxicity Study

Skin Sensitization Study

Gross structure & weights of t

response to T cell-dependent 

(PFC or ELISA). Case-by-case - 

total B cells, T cells and T cell 

Gross structure, thymus/sp

spleen/thymus/lymph node (2 

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental Toxicity Study

Reproduction andFertility Effects Study

ConceptionBirth Weaning Maturity Sene

Adolescence

Puberty

P

F1

Organ weights, gross 

structure, histology

Gross thymus/spleen structure

Chronic/carcinogenicity Study

Subchronic Study

Spleen/thymus 

weights

Clinical pathology (differential WBC,

serum immunoglobulin every 6 months

Reaction to initial induction or c

Immunotoxicity Study

Skin Sensitization Study

Gross structure & weights of t

response to T cell-dependent 

(PFC or ELISA). Case-by-case - 

total B cells, T cells and T cell 

Gross structure, thymus/sp

spleen/thymus/lymph node (2 

Emb = Embryonic F1= 1st filial generation 

PFC = Placque-forming cells e-linked immunosorbent assay 

WBC = White blood cells 

Figure 3-4. 

NB = Newborn P = Parental SRBC = Sheep red blo

ELISA = Enzym NK = Natural killer 

Exposures and endpoints for immunotoxicity evaluations.
exposure. In the LLNA, proliferation of lymphocytes is measured (as a function of in vivo 

radioisotope incorporation into cellular DNA) in draining lymph nodes proximal to the 

application site. Hence, although the GPMT and Buehler tests result in a qualitative assessment 

of hypersensitivity, the LLNA provides a quantitative dose-response evaluation. 

Histopathological evaluation of the skin is not required with any of these methods, but it may be 

conducted. No other immune system endpoints or organs are evaluated in this study. 

In guideline subchronic and chronic/carcinogenicity studies, an evaluation of 

macroscopic structure and general qualitative histopathology are conducted on only a few 

immune system tissues. In studies that include young adult animals (e.g., rats 45 days to 5 

months of age from a subchronic study), the spleen, thymus, and lymph nodes from two 

locations (one near to and the other distant from the site of administration) are examined; the 

spleen and thymus are weighed. In chronic and carcinogenicity study guidelines, there is no 

requirement that the thymus be examined and/or weighed. For rodents (e.g., rats or mice 18 

months to 2 years of age), it is reasonable to assume that the thymus would have undergone 

normal age-related atrophy by study termination. However, the thymus might be present at early 

interim sacrifices of rodents (e.g., at 6 months or 12 months of study) during a long-term study, 

and it would certainly be present at study termination in a canine chronic study (at which point 

the dogs are young adults of only approximately 1.5 years of age). 

Differential white cell counts in the circulating blood are examined at study termination 

in the subchronic study and at approximately 6-month intervals in long-term studies. Serum 

immunoglobulin levels may be measured at the same intervals. Perturbations may indicate 

increased immune system response to some unspecified initiator, but this information does not 

address the adequacy of immune system function. In the same manner, histopathological 

evaluation of other organ systems in the subchronic and chronic/carcinogenicity studies may 

identify cellular alterations that are nonspecific indicators of an effect on immune response, for 

example, the presence of increased numbers of macrophages in lung tissue or an increased 

incidence of inflammatory dermal lesions. 

In the reproduction and fertility effects study in rats, a macroscopic evaluation of all 

organ systems is conducted in a sample of offspring at weaning and in the mature adult parental 

animals at the termination of each generation. Additionally, the spleen and thymus are weighed 

for those pups that are necropsied at weaning; these measurements are intended to provide 

information on the need for further evaluation of the immunotoxic potential of a chemical to the 

immature animal. 

In the prenatal developmental toxicity study, an evaluation of the macroscopic structure 

of the thymus and spleen is conducted in at least half of the fetuses from each litter. 

3-21
 
3.1.4.2. Gaps in Life Stage of Assessment 

In the available guideline studies, assessments of organs with immune system function 

are conducted in fetuses following prenatal exposure, in weanling animals following pre- and 

postnatal exposure, and in young and/or mature adult animals at a variety of time points. With 

prenatal exposures and evaluation at early life stages, these assessments consist entirely of the 

evaluation of macroscopic changes, with no microscopic examination. Toxicokinetic data that 

characterize the exposure in the young (i.e., exposure of the fetus to the chemical or its 

metabolites via the placenta or of the neonate via breast milk) are not routinely required and are 

seldom available. 

Some detailed structural assessment (histopathology) is conducted in mature or older 

adult animals. Indirect assessment of immune system function is conducted in adult animals of 

various ages via the evaluation of peripheral blood cells and chemistry. Direct functional 

assessments of the immune system are conducted only in young adult animals; generally this age 

group is selected for assessment because of the anticipated robustness of the immune response. 

There is no guideline that examines potential perturbation of immune system function 

following early pre- and/or postnatal exposure (often referred to as a developmental 

immunotoxicity study). Comparisons of immune effects following exposure at various life 

stages (i.e., during in utero or postnatal development, adulthood, or old age), including data that 

analyze whether these effects are more severe in one age group or whether the effects are 

persistent, are not required. To achieve even a minimal assessment of immune system structure 

and function, a broad variety of studies would need to be conducted and assessed; yet there could 

still be relatively low confidence in the ability of the results of these combined studies to predict 

the outcome of age-specific insults to the immune system. 

3.1.4.3. Gaps in Assessment Endpoints 

There are identifiable gaps in the endpoints that are used to assess immunotoxicity in 

guideline studies. For example, for fetuses, immature animals, and old animals (rodents), 

assessments are composed entirely of the evaluation of macroscopic structural changes, with no 

histopathological or functional evaluations. In mature adult animals, thorough macroscopic and 

microscopic structural assessments, as well as routine hematological testing (e.g., blood cell 

counts), are performed; however, those assessments are generally very limited in young animals, 

and guideline requirements do not consider species differences. The only assessments of 

functional integrity of the immune system are provided by the guideline sensitization study and 

the 28-day immunotoxicity study. These studies are conducted only in young adult animals, and 

3-22
 
they include only a few examples of potential immune system response (e.g., hypersensitivity, 

humoral immunity, or nonspecific cell-mediated immunity). In very young and very old 

animals, there is no direct assessment of immunological function. No assessment of autoimmune 

effects is conducted in any of the current guideline protocols. 

3.1.4.4. Gaps in Duration/Latency Assessment 

Latent effects on immune function that result from early lifetime exposure are not 

assessed; these can include effects in aged animals that result from in utero, neonatal, or youngadult exposure. Exacerbation of effects in relation to aging and response to subsequent 

immunological challenge are not routinely or systematically assessed to any extent. The twogeneration reproduction study offers an opportunity to evaluate immunotoxic response in 

adulthood that resulted from prenatal or early postnatal exposure. In the chronic toxicity studies 

in rodents, aged animals are available for evaluation. However, in both cases there is little focus 

on the evaluation of the immune system. Only indirect evidence of perturbation of the immune 

system may be observed through macroscopic and microscopic evaluation of various organs; 

corollary functional assessment is not performed. Response to an immunological challenge is 

examined only in the hypersensitization study, and even when the results from this study are 

positive, no further specific assessment of the immune system is pursued. 

3.1.5. Exposures and Endpoints Related to Evaluation of Cardiovascular Toxicity 

3.1.5.1. Overview of Tests 

Gross observation of the heart and major vessels augmented by conditional standard 

pathology is mentioned in most applicable OPPTS Series 870 health effect guidelines (Figure 3-

5). 

3.1.5.2. Gaps in Life Stage of Assessment 

The period from birth to maturity is essentially without toxicological monitoring of 

cardiovascular endpoints for both repeated chronic and single acute-exposure regimes. 

3-23
 
3-24

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental 

Toxicity Study

ConceptionBirth Weaning Maturity Sene

Adolescence

Puberty

Gross structure

Combined Chronic Toxicity/

Carcinogenicity Test

Subchronic Toxicity Test

Acute Toxicity Test

Gross 

pathology,

no

sectioning 

instructions

stipulated

Life Stages Preconception Emb/Fetal

NB/Preweaning Juvenile Adulthood 

Guideline Study Designs:

Prenatal Developmental 

Toxicity Study

ConceptionBirth Weaning Maturity Sene

Adolescence

Puberty

Gross structure

Combined Chronic Toxicity/

Carcinogenicity Test

Subchronic Toxicity Test

Acute Toxicity Test

Gross 

pathology,

no

sectioning 

instructions

stipulated

Emb = Embryonic 

Figure 3-5. 

NB = Newborn

Exposures and endpoints related to cardiovascular evaluations.
3.1.5.3. Gaps in Assessment Endpoints 

Gross observation only of the heart is provided for in most OPPTS guidelines. 

Functional clinical or histopathological cardiac examination is not currently part of any testing 

guideline. Even gross pathology could be improved and brought into line with current 

cardiovascular evaluation by separating, weighing, and constructing right and left ventricle-tobody weight ratios to give an evaluation of cardiac hypertrophy. Also, guidelines regarding 

sectioning procedures for the heart, either number or plane, could be provided. 

No simple cardiac functional evaluation is currently available, including even systolic or 

diastolic blood pressures. It should be noted that telemetric in-dwelling echocardiograms 

(ultrasound examinations of the heart) can be used to detect occlusions and atherosclerosis and to 

detect alterations in cardiac output. Combination echocardiograms and electrocardiogram 

analysis can detect cardiac wave forms as well as heart rate variability in high- and lowfrequency power ranges (i.e., beat-to-beat changes in heart rate ascribed to varying control by the 

autonomic nervous system). Heart rate variability may be critical in explaining toxicity, as was 

shown in recent work associating exposures to fine particulate matter with decreases in heart rate 

variability in elderly humans (Creason et al., 2001). Both echocardiograms and 

electrocardiograms can be done on rats down to 100 g, well within the size range of juvenile and 

adolescent rats. 

Chemicals can produce degenerative and/or inflammatory changes in the peripheral 

blood vessels as a consequence of an excessive pharmacologic effect or by an interaction with a 

vascular structural or functional macromolecule. As a result of sustained arterial 

vasoconstriction, peripheral arterial lesions consisting of intimal proliferation and medial 

degenerative changes could result in gangrene. Also, chemicals can induce or enhance atheroma 

formation, which is characterized by endothelial damage with increased permeability, monocyte 

adhesion, and endothelial proliferation. 

Selected representative techniques to study the peripheral vascular system consist of flow 

measurement techniques (Smith et al., 1994), such as electromagnetic flowmetry, pulsed Doppler 

flowmetry, transit time flowmetry, laser Doppler fluxmetry, and laser scanner methods. These 

techniques allow investigation of blood flow in vessels as large as the aorta and as small as the 

capillary, determination of the level of perfusion in tissues, and calculation of the derived 

hemodynamic variable of resistance. 

The two major noninvasive techniques for determining microvascular velocity are the 

flying spot technique and the dual-slit technique. External ultrasound may be used to examine 

internal vascular dimensions. A noninvasive assessment of arterial flow in rodents and monkeys 

can be performed using Doppler spectrum analysis (duplex ultrasound technology) (Leopold et 

3-25
 
al., 1997). This test detects arterial compromise in extremities, functional severity, and the 

hemodynamic significance of vascular lesions. In most cases, the locations in the arteries 

involved can be designated. Information regarding the extent and effectiveness of collateral 

circulation can also be gained. This testing is a valuable tool for monitoring early flow 

compromise secondary to chronic reoccurrence of anastomotic or distal disease. 

Several blood/plasma tests for clinical assessment are in active use in cardiovascular 

research. In general, these are tests that may be used to document a cardiovascular accident 

(within 48–96 hours). Their utility for risk assessment has yet to be evaluated. Specific 

enzymes currently being used by the research community for these purposes include LDH-I, 

creatinine kinase-II, and troponin. Other enzymes useful as prognostic indicators of risk of a 

cardiovascular accident include angiotensin converting enzyme II, plasma renin activity, 

endothelin-converting enzyme-1, and catecholamines (epinephrine and norepinephrine). 

3.2. CONCLUSIONS AND RECOMMENDATIONS 

A review of current testing guidelines was conducted to determine the types of data 

available for setting reference values. The approach used was to evaluate testing guidelines from 

the point of view of (1) life stages covered, (2) endpoints assessed generally and for specific 

organ systems, (3) timing and duration of exposure, and (4) evaluation of reversibility and 

latency to response. 

The relevance of these issues to the health evaluation of children and other potentially 

susceptible subpopulations should be apparent from the gaps identified in each of the above 

sections regarding life stage assessment, endpoints assessed, timing and duration of exposures 

included in guideline studies, reversibility, and latency to response. Although a number of areas 

of toxicity testing have been discussed, this review should not be considered exhaustive, and 

other health effects may be as or more important for particular chemicals than those reviewed in 

detail here. 

Issues of particular concern for children’s health that have not been discussed in great 

detail here are effects related to asthma and other respiratory tract toxicity. For both children 

and the elderly, renal and liver function can be a major factor in the disposition, metabolism, and 

excretion of chemicals and, therefore, their toxicity. Thus, the evaluation of toxicity and the 

interpretation of data in terms of its completeness will always require scientific judgment about 

whether or not adequate data have been collected on effects of importance at the appropriate life 

stages and timing and duration of exposure, for example, for a given agent. 

Effects seen at the termination of a chronic study may be due to cumulative damage from 

a continued repeated chemical insult, but they could also be a latent response from an earlier 

3-26
 
single or short-term multiple exposure. Thus, latent effects might be revealed in chronic studies, 

but it would not be clear whether they were the result of acute/short-term exposure or the chronic 

exposure. Specific information on the latency of a response would follow only from a clearer 

understanding of the mechanism of the effect and from actual “stop exposure” protocols (e.g., 

the satellite studies depicted in Figure 3-1) or from shorter-term exposures with follow-up over a 

much longer period of time. It thus follows that any chemical database that does not have 

exposure-response studies of lifetime duration or any specific exposure-latency protocols would 

not cover the possibility of latent effects. 

Effects that persist throughout a designated post-exposure period may be considered 

irreversible; those that do not are reversible. For chronic lifetime exposures, designation of an 

effect as irreversible or reversible is academic, as exposure is presumed to be lifetime (i.e., there 

is no post-exposure period). For shorter-term values (e.g., acute, short-term) where an 

appreciable period of time post-exposure is anticipated, designation of an effect as reversible or 

irreversible becomes more relevant. Derivation of a reference value based on shorter-term 

exposure guideline protocols would have to fully consider the aspect of reversibility in 

interpretation of the data. It is important to understand the difference between an endpoint that is 

truly reversible and one that is related to or is a precursor of other adverse effects. For example, 

low birth weight may be “reversible” through catch-up growth postnatally, but it also may be 

related to developmental delays or other health outcomes that result from prenatal growth 

reduction/retardation. 

3.2.1. Conclusions 

From this review, the Technical Panel reached the following major conclusions: 

1. There are a number of gaps in life stages covered in current guideline testing 

protocols, particularly in terms of the exposure periods included. In particular, there is 

minimal evaluation of aged animals, especially after exposures that include early 

development. 

2. There are a number of gaps in the evaluation of endpoints included for certain 

systems; for example, the evaluations of the cardiovascular and immune systems in 

various guideline studies were reviewed as examples of systems that are minimally 

covered. Other systems, for example, the reproductive and nervous systems, are 

evaluated in more detail, but even in these systems there are gaps that need to be 

3-27
 
considered; notably, functional evaluations are not always included or integrated with 

structural evaluations of particular systems. 

3. Acute and short-term exposure studies are either not available or include only gross 

effects, so that the data needed to derive acute and short-term reference values are often 

not available. 

4. Latency to response and reversibility are only rarely evaluated directly. These types 

of effects could have a major impact on hazard characterization, especially in designing 

acute and short-term test guideline protocols and ultimately on the risk management 

options that can be used for intervention or prevention. 

5. Although not more specifically discussed, it is clear that there is a lack of information 

on toxicokinetics. The available data are generally limited to studies that are conducted 

in young adult animals, but there are no guideline protocols for toxicokinetic evaluations 

during development or in older age related to exposures and outcomes. 

6. The underlying assumption that the internal dose of the active form of an agent to the 

target site is the relevant measure of dose clearly underscores toxicokinetics as an 

essential tool that must be used in both hazard identification and dose-response 

evaluations. This should not only continue to be a central and critical area of exploration, 

it should be an area of direct application to assessment activities to address various 

issues, including but not limited to (a) design of studies, (b) delivery to the fetus/neonate, 

(c) dose scaling, (d) toxicokinetic and toxicodynamic considerations, and (e) route 

extrapolation. 

A white paper on pharmacokinetics commissioned by the Technical Panel (Versar Inc., 

2001b) is meant to serve as a technical resource for the application of toxicokinetics to 

these and other issues addressed throughout this document. Another white paper on 

aging (Versar Inc., 2001a) also addresses issues of changing pharmacokinetics during 

this life stage. 

7. Portal-of-entry effects (i.e., respiratory, gastrointestinal, dermal) are acknowledged as 

being important in the effects of chemicals, and they may preclude systemic toxicity as 

being sentinel. Chronic oral RfDs and inhalation RfCs have been developed for a 

3-28
 
number of agents, but rarely have dermal RfDs been derived. In some cases, oral RfDs 

and oral cancer potency factors have been used to assess systemic toxicity from dermal 

exposures. However, the dermal route of exposure can result in different patterns of 

distribution, metabolism, and excretion than those that occur from the oral route. Dermal 

contact with a chemical may also result in direct dermal toxicity, such as allergic contact 

dermatitis, urticaria reactions, chemical irritation, and skin cancer. 

The dose-response relationship for the portal-of-entry effects in skin is likely to be 

independent of any associated systemic toxicity exhibited by a particular chemical. 

Therefore, there is a long-term need for the development of dermal RfDs that consider 

both the systemic toxicity effects and the portal-of-entry effects of individual chemicals. 

In addition, there is a need for data on the dermal uptake of chemicals from soil, water, 

and air, including information about specific chemical forms and bioavailability from 

different soil types that contribute to variations in uptake. Different exposure duration 

RfDs, such as acute chemical injury to the skin, need to be developed. 

3.2.2. Recommendations 

On the basis of the review of the guideline toxicity studies, the Technical Panel makes 

the following recommendations (in no particular order) regarding the development of testing 

procedures and guidance for their use. In identifying the need for development of specific 

protocols, the Technical Panel is not recommending that these tests be used for every chemical 

or in all circumstances, as pointed out at the beginning of the chapter. 

•	 Develop a strategy for alternative approaches to toxicity testing, with guidance on 

how and when to use existing and newly recommended guidelines. Information on all 

aspects of a chemical should be considered in the strategy for testing, including 

chemical-physical characteristics, intended use, and toxicokinetic and toxicodynamic 

(mode of action) data, to allow a more efficient and targeted testing approach. In 

addition, the strategy should consider life stages in evaluating exposures and 

outcomes, as well as other sensitive subpopulations. 

•	 Develop guidelines or guideline study protocols that will provide more systematic 

information on toxicokinetics and toxicodynamics (i.e., mechanism or mode of 

action), including at different life stages. Such studies could provide information that 

would be relevant to susceptible subpopulations, including life stages (i.e., inform the 

3-29
 
selection of the intraspecies UF). Such studies also could provide information on 

species differences (i.e., inform the selection of the interspecies UF). Finally, such 

studies can provide information to conduct route-to-route extrapolations and reduce 

the number of route-specific tests required to derive a reference value. 

•	 Develop protocols for acute and short-term studies that provide more comprehensive 

data for setting reference values (see Section 3.3). The existing protocols for acute 

studies (except for the acute inhalation protocol with histopathologic evaluation) 

generally collect data only on what could be called frank effects, which may not be 

protective of more subtle effects. 

•	 Modify existing guideline study protocols to provide more comprehensive coverage of 

life stages for both exposure and outcomes (see Section 3.3). Existing guideline 

studies do not include, for example, the evaluation of toxic effects that may occur in 

old age from prenatal or early postnatal exposure (including carcinogenesis) or 

premature aging from exposure earlier in life. 

•	 Collect more information from less-than-lifetime exposure to evaluate latency to 

effect and to evaluate reversibility of effect. Existing guideline studies, with the 

exception of the acute tests and some developmental toxicity studies, expose animals 

up to the time of testing. Some form of “stop exposure” studies would provide useful 

information that could increase or decrease the level of concern for an observed toxic 

event. 

•	 Develop guidelines or guideline study protocols to assess immunotoxicity, 

carcinogenicity, and cardiovascular toxicity at different life stages. Immunotoxicity 

and cardiovascular toxicity are presently looked at only in a cursory manner. 

Carcinogenicity is currently evaluated only after chronic exposure to adult animals. 

There is a need to integrate functional measurements into evaluations of these and 

other systems. 

•	 Explore the feasibility of setting dermal reference values for direct toxicity at the 

portal of entry, including sensitization. Reference values have been derived for 

lesions in the gastrointestinal and respiratory tracts from direct exposure. The lack of 

procedures for dealing with similar effects on the skin is a glaring omission. 

3-30
 
3.3. OPTIONS FOR ALTERNATIVE TESTING APPROACHES 

The Technical Panel explored alternative testing protocols for acute toxicity testing as 

well as alternative protocols for subchronic/chronic toxicity testing. These are offered here as 

alternatives that may be used, depending on the agent being tested or the type of reference values 

needed. 

3.3.1. Alternative Acute Toxicity Testing Protocol 

The current EPA test guidelines for acute toxicity focus on the determination of an LD50 

in adult test species. A gross necropsy is conducted on the animals, and histologic evaluation of 

target organs may or may not be conducted. Therefore, very limited information is obtained 

from the current protocol that would be useful for determining an acute reference value. 

However, a number of alternative study designs are available that would provide information for 

consideration in establishing the acute reference value (Gad and Chengelis, 1998). 

One basic study design is shown in Figure 3-6. In this protocol, a control group and a 

minimum of three dose groups with 10 animals/sex/group are used. The animals are dosed once 

on day 1 and followed for 2 weeks. Clinical signs of toxicity are recorded daily, food 

consumption and body weights are recorded on days 1–4, 8, and 14. There is an interim sacrifice 

of 5 animals/sex/group at 3 days after dosing and a final sacrifice of the remaining animals at 2 

weeks after dosing. At both sacrifices, hematological and clinical chemistry analyses are 

conducted, as is a urinalysis. The animals are necropsied, organ weights are recorded, and the 

organs are examined histologically. 

Because the purpose of this study design is to provide hazard and dose-response 

information rather than determination of an LD50, the dose levels should be chosen accordingly. 

This study would initially be conducted on adult animals. As information is obtained from other 

toxicology and/or toxicokinetic studies, it may be necessary to conduct the study with animals at 

different life stages and to include other endpoints. 

3-31
 
Acclimatization 

Study day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Acclimatization 

Study day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Acclimatization 

Study day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Acclimatization 

Study day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Acclimatization 

Study day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

Acclimatization 

Body weight, 

food consumption 

Body weight, Body we

food consumption food consu

Study day 1 2 3 4 5 6 7 8 9 10 11 12 13 14 

3-32
 Dosing 

Gross necropsy, 

organ weight, 

histopathology, 

hematology, 

clinical chemistry 

Gro

org

hist

he

clinic

Figure 3-6. Alternative acute toxicity protocol. 
3.3.2. Alternative Chronic Toxicity Testing Protocols 

As stated, a review of currently available EPA guideline toxicology studies (OPPTS 870 

Series) demonstrates that there is no single protocol that addresses continuous exposure through 

all life stages of any test species. To address this issue, two possible alternative study designs 

were considered: the “expanded chronic/carcinogenicity study” and the “unified screening 

study.” These are described in some detail below and are illustrated in accompanying figures. 

The intent of this discussion is to demonstrate the advantages (and disadvantages) of exploring 

nontraditional testing paradigms; however, such discussion does not constitute a 

recommendation for implementation. For many chemicals, the existence of adequate (by 

Agency standards) stand-alone studies would preclude the need for further testing, with or 

without expanded or combined protocols such as those described below. In any case, any 

proposal to use alternative study designs in a regulatory setting should be thoroughly discussed 

by Agency and registrant scientists prior to study initiation. 

3.3.2.1. The Expanded Chronic/Carcinogenicity Study 

An example of a study design that would incorporate lifetime (in utero through old age) 

exposure is the expanded chronic/carcinogenicity study (shown in Figure 3-7), which could 

serve as a replacement for a standard guideline chronic/carcinogenicity study in rats. In this 

expanded study, female rats are assigned to treatment groups, mated, and treated with test 

substance throughout gestation and lactation. When pups are weaned on PND 21, they are 

assigned individual animal numbers and maintained within their established treatment group. 

Prenatal and early postnatal exposure to the test substance in this study is similar to that required 

for the in utero carcinogenicity study that is used to evaluate food additive chemicals for 

regulation by the Food and Drug Administration’s Center for Food Safety and Nutrition. 

The difference here is that the study duration is extended to a period of 3 years (vs. a 

typical chronic duration of 2 years for rats), with interim sacrifices scheduled at yearly intervals. 

The total number of animals used in this expanded study is greater than for a standard guideline 

chronic/carcinogenicity study because of the additional interim sacrifice; for each annual 

segment, the sacrifice of 25 rats/sex/group is required. To reduce this number, the study could 

be conducted with fewer animals per segment (e.g., 20/sex/group), or only two sacrifices could 

be scheduled (e.g., at 1.5 and 3 years). Of course, such actions will either reduce the power of 

the evaluation for tumor data or will eliminate examination of an important life phase. 

Parameters typical of a guideline chronic/carcinogenicity study are examined in this 

expanded study (e.g., mortality, clinical observations, body weight, food consumption, clinical 

3-33
 
Mate 

Gestation Lactation 

Wean and 

assign F1 pups 

Sacrifice P animals 

1 yr 2 yr 

Interim sacrifice 1 Interim sacrifice 2 

Initiate 

treatment 

Quarantine 

Immunotoxicity screen 

Clinical pathology 

Histopathology Neuropathology 

Immunotoxicity screen Clinical pathology Histopathology 

Neuropathology 

Neurobehavior Neurobehavior 

Clinical pathology 

Clinical pathology 

Clinical patholo

Immunotoxicity screen 

at 

estatio actatio In at trea en 

uarantin 

ti

e

i m

t 

t 

n n 

G L Neurobehavior 1 yr Neurobehavior 2 yr 

Clinical Clinical Clinical 

Wean and pathology Interim sacrifice 1 pathology Interim sacrifice 2 patholo

assign F1 pups 

Sacrifice P animals 

Immunotoxicity screen Immunotoxicity screen 

Immunotoxicity screen Clinical pathology Clinical pathology

Histopathology HistopathologyNeuropathology Neuropathology 

Figure 3-7. Expanded chronic/carcinogenicity study. 

Q M

e 

3-34
 e 
chemistry and hematology, ophthalmology, gross pathology, and histopathology). In addition, 

neurological and immunological evaluations are performed in the adult animals at multiple 

intervals into old age, which, along with the fact that the animals are exposed to the chemical 

during all life stages, contributes to the superiority of this study design. 

Although the temporal linear nature of this study protocol makes it less complicated to 

conduct in the laboratory, this attribute also results in the inability to easily assess some other 

important endpoints, such as prenatal development, reproduction and endocrine function, and 

DNT. Additionally, by 3 years of age, when this study would be terminated, survival in 

laboratory rats may be compromised; therefore, it may be necessary to consider using feed 

restriction to maximize the number of animals available for in vivo and post mortem assessment 

of aged animals. In addition, housing from birth in specific-pathogen-free facilities may be 

necessary to maintain sufficient viable animals for such an extended period of time (see the 

background white paper on aging, Versar Inc., 2001a). 

3.3.2.2. The Unified Screening Study 

An alternative study design, the unified screening study, is illustrated in Figure 3-8. This 

study is composed of at least four segments: a two-generation reproduction and fertility study, an 

expanded chronic/carcinogenicity study, a developmental toxicity study, and a DNT study. Each 

of these is currently conducted as a separate study. An optional continuous-breeding study 

segment could be added to the design. When conducted in the rat, the unified screening study 

assesses all life stages of the animals and provides a means to evaluate prenatal developmental 

toxicity, DNT, reproduction, and endocrine function, all within animals that are derived from the 

same gene pool and are evaluated within two generations of the progenitor rodents that are 

initially placed on study. 

The unified screening study begins as a typical two-generation reproduction and fertility 

study, with 10 weeks of treatment, mating, gestation, and lactation phases conducted according 

to OPPTS 870.3800. The F1 weanlings are selected for either the second generation of the 

reproduction and fertility study or the expanded chronic/carcinogenicity study. (As a point of 

clarification, at any point that animals are selected and/or assigned to a different study segment, 

it is assumed that the treatment group remains constant for each animal.) The parental (P) 

animals from the first generation are not immediately terminated; rather, they are transferred to a 

prenatal developmental study segment. After a short rest, they are mated. The P males can be 

terminated at any time point; the P females are continued through to caesarian section on 

approximately GD 20. The resulting F1b fetuses are processed and examined for external, soft 

tissue, and skeletal 

3-35
 
Chronic/Carcinogenicity Study Segment 

1 yr 2 yr 

Mate F1 M & F - continuous cohabitation 

Optional Continuous Breeding Study Segment 

Chronic/Carcinogenicity Study Segment 

1 yr 2 yr 

Prenatal Developmental 

Toxicity Study Segment 

Reproduction and Fertility 

Study Segment 

Initiate treatment 

10 weeks 

10 weeks 

Mate 

Mate 

Mate 

Gestation Lactation 

Gestation Lactation Wean & 

Assign F2 

C-section 

Histopathology

F1b fetal evaluations 

PND 60 

Term sac F

Histopathology F1 Neuropathology of select F2 

(P Generation) 

(F1 Generation) 

P

Quar. 

Neurobehavioral assessments 

Interim sac 1 Interim sac 2 

Developmental Neurotoxicity 

Study Segment 

Wean & 

Assign F1a 

(F2 Generation) 

(F1 Generation) 

F1 

Gestation 

Gestation Gestation Gestation Gestation Gestation Lactation 

Remove F2 pups Remove F2 pups Remove F2 pups Remove F2 pups 

Cross-over mating of F1 

control and treated paren

Prenatal Developmental 

Toxicity Study Segment 

Reproduction and Fertility 

Study Segment 

Initiate treatment 

10 weeks 

10 weeks 

Mate 

Mate 

Mate 

Gestation Lactation 

Gestation Lactation Wean & 

A ssign F2 

C-section 

Histopathology

F1b fetal evaluations 

PND 60 

Term sac F

H istopathol ogy F1 Neuropath ology of select F2 

(P Generation) 

(F1 Generation) 

P

Quar. 

Neurobehavioral assessments 

Interim sac 1 Interim sac 2 

Developmental Neurotoxicity 

Study Segment 

Wean & 

Assign F1a 

(F2 Generation) 

(F1 Generation) 

F1 

Gestation 

Gestation Gestation Gestation Gestation Gestation Lactation 

Remove F2 pups Remove F2 pups Remove F2 pups Remove F2 pups 

Cr oss-over mating of F1 

control and tr eated paren

3-36
 

Mate F1 M & F - continuous cohabitation 

Optional Continuous Breeding Study Segment 

Quar. = Quarantine P = Parental F1 and F2 = 1st and 2nd filial generations 

Figure 3-8. Unified screening study. Study lines are not drawn to scale. 
abnormalities, as is typical in an OPPTS 870.3700 study. At necropsy, however, the Pgeneration animals receive an extended postmortem examination, according to the procedures 

for the two-generation reproduction and fertility study, that includes sperm measures for the 

males and extensive histopathology of the reproductive and other organ systems for both sexes. 

The expanded chronic/carcinogenicity study segment, using F1 animals, would continue 

as described above concurrently with all other segments of the unified screening study but 

continuing well past the time that the others have been terminated. The other F1 pups that are 

selected as second-generation parental animals in the reproduction and fertility study segment 

are treated for 10 weeks and then undergo the standard reproductive functional assessments, as 

specified in the OPPTS 870.3800 guideline. Because a number of F2 pups from this generation 

will continue on into the DNT study segment, some additional observations are required during 

the lactation segment of the second generation. Specifically, F2 pups are selected and assigned 

for neurobehavioral assessments on PND 4 (at the time of litter standardization). Preweaning 

observations include weekly age-appropriate clinical/functional behavioral observations 

conducted outside of the home cage and motor activity assessments on PNDs 13 and 17. 

Additional assessments of physical, reflex, and sensory development may also be conducted 

during this period. 

At the time of weaning of the F2 pups on PND 21, those preselected for neurobehavioral 

assessment continue into the DNT segment and other weanlings are sacrificed for postmortem 

evaluations that address the considerations of both the reproduction protocol (including organ 

weight data) and the DNT protocol (requiring in situ perfusion fixation of tissues and 

neuropathology, including morphometric analysis). The DNT-segment F2 animals are evaluated 

as per OPPTS 870.6300, which includes multiple assessments of clinical and functional 

observations, motor activity, auditory startle habituation, and learning and memory. 

They are maintained until termination (with postmortem evaluations, including neuropathology 

following perfusion fixation) at approximately PND 60. 

Also at the time of weaning of the F2 pups, a decision could be made to either sacrifice 

the F1 parental animals immediately (with the usual sperm measures and postmortem 

evaluations) or to maintain them through a continuous-breeding reproduction study segment, 

sequentially mating the F1 adults for the production of five litters (the pups from these litters are 

terminated in early lactation). This continuous-breeding study segment, which would extend the 

reproduction study for about 100 additional days, uses a standardized assessment protocol that 

has been well characterized in the peer-reviewed literature (Lamb, 1985; Lamb et al., 1985; 

Morrissey et al., 1989) but does not have a corresponding OPPTS guideline. 

3-37
 
As previously stated, in this unified study protocol, the animals are both exposed and 

assessed during all life stages, and the evaluation of both structural and functional endpoints for 

multiple organ systems are maximized in the overall design, for example, by the inclusion of 

immunotoxicity and neurotoxicity endpoints. There is one notable exception to this statement in 

that reproductive senescence is not standardly examined. Nevertheless, if the two-generation 

reproduction study segment identifies problems with fertility or cyclicity, this could be pursued 

more rigorously by the addition of testing during the second or third year of the expanded 

chronic/carcinogenicity study, for example, evaluating cyclicity in aged female rats and/or 

evaluating ovarian follicular counts and atrophy at sacrifice. 

Another benefit of using the unified screening study design is that it results in the 

purchase and use of many fewer naive animals for study initiation and it increases the efficient 

utilization of animals, particularly of the F2 offspring from the reproduction study. 

Although there are obvious benefits in using a unified screening study, there are also a 

number of concerns or potential problems involved with its conduct. Although it is assumed that 

treatment levels and route of administration will remain constant across all study segments, this 

approach to dose-setting and route selection may not always be optimal for every phase. 

Generally, a temporal nonlinear design of this nature is more difficult to manage in the 

laboratory. The strain of rat generally used in toxicity studies is the Sprague-Dawley, whereas 

the Fischer 344 rat is often used in the standard chronic/carcinogenicity study. Fischer 344 rats 

have not typically been used in reproductive and developmental toxicity studies. The use of 

either strain for the unified study could compromise the use of historical data for comparison, for 

example, for the chronic/carcinogenicity study if the Sprague-Dawley is used and for the 

reproductive and developmental toxicity studies if the F344 is used. 

As study complexity increases, so does the opportunity for error. In some cases, a 

serious technical error in one study segment could compromise subsequent study segments and 

result in an extensive waste of animals and resources. As with the expanded 

chronic/carcinogenicity study discussed above (section 3.3.2.1.), survival during the 

chronic/carcinogenicity study segment in this design may need to be enhanced via feed 

restriction. Also, if the test substance interferes with reproduction or results in increased 

mortality, the number of offspring that are available for assignment to subsequent study 

segments (e.g., the selection of F2 animals for the DNT phase) may be critically reduced. An 

additional but similar problem could arise when selecting F1 animals for the expanded 

chronic/carcinogenicity study segment at the same time as for the second generation of the 

reproduction and fertility study segment, because a large number of offspring needs to be 

available all at one time. Additionally, the offspring that are assigned to the 

3-38
 
chronic/carcinogenicity segment should be genetically diverse within each dose group and 

should originate from as many litters as possible (i.e., not be siblings). 

A number of possible solutions that could be used alone or in combination to increase the 

number of F1 pups available for selection in other study phases include the following: 

1. Reducing the number of animals needed for the expanded chronic/carcinogenicity 

segment by examining fewer animals at each serial sacrifice or by abandoning the final 

year of evaluation, as described above. 

2. Reducing the number of animals assigned to the second generation of the reproduction 

and fertility study segment; however, this could compromise the number of F1 offspring 

that would be available for the DNT study segment. 

3. Standardizing litters to 10 rather than 8 pups per sex and assuming that no litter has 

less than 10 pups and that no pups die during lactation. Because some small litters and 

neonatal pup deaths almost always occur, even in controls, it is wiser to design the study 

more conservatively in order to avoid discovering that there are not enough F1 pups to 

assign to the later segment(s). 

4. Assigning additional females to the two-generation reproduction and fertility study 

segment in order to produce extra F1 pups for selection. Although even a modest 

increase in the number in each group would increase the probability of producing a 

sufficient number of F1 pups, a larger number of litters would generally be required in 

order to ensure genetic diversity among the weanlings that are assigned to the 

chronic/carcinogenicity study segment. This could be accomplished by placing 

additional P-generation females or breeding pairs on study, perhaps combined with 2:1 

mating procedures, or by mating the males with the reproduction and fertility studysegment females first and then with an extra set of females. One adverse consequence of 

placing additional females on study so that their litters can be used for selection of 

genetically diverse offspring for the chronic/carcinogenicity study segment is that this 

method results in a larger number of excess F1 weanling pups that would not be used for 

evaluations in this protocol. However, these pups could be used for other evaluations, 

such as immunotoxicity, specialized neurotoxicity tests, or adult onset disease or diseases 

of aging. 

3-39
 
Some of the above options appear to be more advantageous and preferable than others; 

however, no recommendation is proffered because the list is presented only to illustrate some of 

the many possibilities that could be used in a customized study design. It should be noted that 

simply combining the reproduction and fertility study and the DNT study when a two-generation 

reproduction and fertility study has not already been conducted greatly reduces the total number 

of animals that would be required to conduct the two studies individually. No additional animals 

are required over the reproduction and fertility study alone, and there is greater efficiency in the 

use of the F2 offspring when the DNT study is conducted in that group. 

3-40
 
4. FRAMEWORK FOR SETTING ACUTE, SHORT-TERM, 

LONGER-TERM, AND CHRONIC REFERENCE VALUES 

As noted in Chapter 2, the Technical Panel is recommending that EPA begin deriving 

acute, short-term, and longer-term reference values in addition to chronic reference values. The 

approach to reference values discussed here is intended for use in risk assessments for health 

effects known or assumed to be produced through a nonlinear and/or threshold mode of action. 

Although there has been a dichotomy between cancer and noncancer risk assessment in terms of 

the underlying assumption about the linearity or nonlinearity of the dose-response curve, there is 

a move toward harmonization among approaches for all health effects (Butterworth and 

Bogdanffy, 1999; Bogdanffy et al., 2001). This includes recognition of the possibility that some 

carcinogenic agents may work through nonlinear mechanisms (U.S. EPA, 1999a), whereas some 

agents that produce effects other than cancer may work through linear mechanisms (see 

discussion in U.S. EPA, 1998d). Thus, the decision to use a linear extrapolation approach or a 

reference value approach should take into consideration the underlying mode of action and 

presumed dose-response relationship. 

The approach described here is the default approach to be used when the assumption is a 

nonlinear and/or threshold mode of action, except for cases where other methods have been 

developed (e.g., in support of the NAAQS). This approach can and should be improved upon or 

replaced when more specific data on toxicokinetics and mode of action are available to allow the 

development of a chemical-specific or a biologically based dose-response model for prediction 

of risks to humans and to susceptible individuals within the population. The acute, short-term, 

longer-term, and chronic reference values derived on the basis of the recommendations in this 

report should be included in IRIS after appropriate internal, external, and consensus review. 

These values would then be available for use by program offices, where appropriate. 

In this chapter, we discuss the definitions of the exposure durations and the proposed 

changes in the definition of the corresponding reference values. In addition, several issues are 

discussed regarding the adequacy of studies and characterization of the extent of the database 

with regard to sufficiency of data for deriving reference values. The derivation of reference 

values also is discussed with regard to dosimetric adjustment and application of UFs. A number 

of recommendations are made with regard to this process. In particular, the Technical Panel 

recommends incorporating the concept of life stage and expanding the endpoints evaluated as 

well as consideration of duration and timing of exposure and latency to response in 

characterizing the extent of the database used for setting reference values. The Technical Panel 

4-1
strongly encourages the use of a narrative description of the database, including strengths and 

limitations, rather than a single confidence statement for support of a reference value. 

The adjustments required for derivation of the human equivalent dose (HED) for oral and 

dermal exposure and the human equivalent concentration (HEC) for inhalation exposure are 

described and discussed. This is followed by recommendations about the evaluation and 

comparison of data for the POD, based on an analysis of each potentially limiting endpoint 

carried through the reference value derivation process, followed by selection of the appropriate 

health-protective reference value. 

Finally, the Technical Panel emphasizes that considerable use of scientific judgment is 

advisable and necessary in practically all phases of the process, especially in the application of 

UFs. This review and its recommendations build on the principles in the Agency’s handbook on 

risk characterization (U.S. EPA, 2000b), which calls for transparency, clarity, consistency, and 

reasonableness in the risk assessment process. 

4.1. DEFINITIONS OF EXPOSURE DURATIONS FOR USE IN SETTING 

REFERENCE VALUES 

The Technical Panel proposes the following definitions of exposure duration as a first 

step in the development of consistent approaches for the Agency. These definitions are based on 

exposure durations for humans; analogous exposure durations for rodents are indicated for the 

longer-term and chronic durations. The definitions are not intended to be rigid specifications, 

but simply general descriptions of the relevant exposure time period. Their application is meant 

to be flexible, so that, for example, if a 4-month animal study is available, it may be used as the 

basis for both a longer-term and a chronic reference value. 

The definitions were developed on the basis of the review of values currently set by 

various program offices (see Chapter 2), and they have been standardized to be compatible with 

those definitions currently used by various program offices within the Agency. The definitions 

for various durations, as follow, were discussed at an EPA Risk Assessment Forum Colloquium 

(CDM Group Inc, 2000). 

Acute: Exposure by the oral, dermal, or inhalation route for 24 hours or less. 

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Short-term: Repeated exposure4

 by the oral, dermal, or inhalation route for more than 

24 hours, up to 30 days. 

Longer-term: Repeated exposure by the oral, dermal, or inhalation route for more than 

30 days, up to approximately 10% of the life span in humans5

 (more than 30 days up to 

approximately 90 days in typically used laboratory animal species6). 

Chronic: Repeated exposure by the oral, dermal, or inhalation route for more than 

approximately 10% of the life span in humans (more than approximately 90 days to 2 years in 

typically used laboratory animal species). 

The Technical Panel believes there is an advantage in having a central source of 

consensus reference values of various exposure durations available to risk assessors throughout 

EPA. EPA Program Offices could use the values for risk assessments in which the known or 

assumed exposure duration approximated the exposure duration in the appropriate reference 

value definition. The Panel recognizes that Program Offices may make further adjustments to 

the reference values depending on circumstances that are unique in their assessments. 

The Technical Panel recommends that the principles of sound science be used when the 

expanded array of reference values are developed. The Panel cautions that the exposureresponse relationships for all durations of exposure and issues of latency need to be carefully 

considered to ensure that there are no obvious conflicts in the series of recommended reference 

values for any specific chemical. This analysis can become complex in a case where the 

toxicological endpoint may differ for the different durations of exposure. 

The Technical Panel is aware that there will be data limitations for an individual 

chemical that may preclude development of all four reference values. For example, currently, a 

chronic RfD or RfC would not ordinarily be considered for inclusion in the IRIS database unless 

4

A repeated exposure may be either continuous, periodic, or intermittent. A continuous 

exposure is a daily exposure for the total duration of interest. A periodic exposure is one 

occurring at regular intervals, e.g., inhalation exposure 6 hrs/day, 5 days/wk or oral exposure 5 

days/wk. An intermittent exposure is one in which there is no effect of one exposure on the 

effect of the next; this definition implies sufficient time for the chemical and its metabolites to 

clear the biological system before the subsequent exposure, that is, noncumulative 

pharmacokinetics. A periodic exposure may or may not be intermittent. 

5

The lifespan value used depends on the situation under consideration. For example, an 

average of 70 years has been the typical default used for chronic exposures, but the average life 

span based on U.S. census data is 75.5 years (U.S. EPA, 1997a). 

6

Typically used laboratory animal species refers to rats, mice, and rabbits, for example. 

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a subchronic or chronic study were available. Similarly, where data of the type needed for 

deriving acute, short-term, or longer-term reference values are not available, theses values would 

not ordinarily be considered for inclusion in the IRIS database. In situations where an acute, 

short-term, or longer-term reference value is needed but appropriate data fitting the definition for 

duration are not available, then the Program Office may wish to consider several options. One 

option would be to not develop a reference value for that particular duration of exposure. 

Another option would be to use the reference value for the next longer duration of exposure as a 

conservative estimate of a reference value that would be protective for a short-term exposure 

duration. For example, the Office of Water (see Chapter 2) will use a longer-term health 

advisory for a child as a conservative estimate for a 10-day exposure in the absence of data to 

derive a 10-day health advisory. Other program-specific options might also be considered. 

The Technical Panel is aware that time and resources need to be considered when 

implementing its recommendations. The IRIS program has begun to implement a pilot program 

to test whether development of the expanded array of reference values is practical and can be 

accomplished without unduly delaying the completion of an IRIS file. As a part of the pilot, the 

IRIS program will need to identify the methods to be used in deriving these additional values. 

4.2. PROPOSED CHANGES IN THE REFERENCE VALUE DEFINITIONS 

In the process of considering definitions for different duration reference values, the 

Technical Panel discussed several issues 

that have been raised about the current 

definitions of the chronic RfD and RfC 

(Box 4-1). The following items describe 

the issues and the recommended changes. 

1. The parenthetical statement in 

the current RfD and RfC 

definitions—“with uncertainty 

spanning perhaps an order of 

magnitude”—has been variously 

used by risk assessors and risk 

managers to mean that the estimate 

is at the upper end, the lower end, 

or the middle of the range of an 

Box 4-1. Current definitions for the chronic oral RfD 

and inhalation RfC 

RfD: an estimate (with uncertainty spanning perhaps an 

order of magnitude) of a daily oral exposure to the human 

population (including sensitive subgroups) that is likely to 

be without appreciable risk of deleterious effects during a 

lifetime. It can be derived from a NOAEL, LOAEL, or 

BMD, with UFs generally applied to reflect limitations of 

the data used. Generally used in EPA’s noncancer health 

assessments. 

RfC: an estimate (with uncertainty spanning perhaps an 

order of magnitude) of a continuous inhalation exposure to 

the human population (including sensitive subgroups) that 

is likely to be without appreciable risk of deleterious effects 

during a lifetime. It can be derived from a NOAEL, 

LOAEL, or BMD, with UFs generally applied to reflect 

limitations of the data used. Generally used in EPA’s 

noncancer health assessments. 

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order of magnitude. This statement has been removed from the proposed revision of the 

definition for reference value (Box 4-2), and it is recommended that issues of 

uncertainty/variability be discussed qualitatively as part of the weight of evidence and 

characterization of the database. Attempts to adapt such a qualitative derivation process 

to formal quantitative procedures for prediction of accuracy presents major difficulties. 

A particularly obvious difficulty is that the same definition and phrase were applied to 

different reference values that may have varied markedly in their underlying data and, 

thus, their potential for accuracy. For example, the same “order-of-magnitude” range 

applied equally to a robust reference value with known exposures plus observable and 

quantifiable dose-response data derived from a segment of the human population and to a 

marginal reference value based only on animal data with minimal supporting 

information. 

Box 4-2. Proposed revisions in the reference value definitions 

Reference Value: an estimate of an exposure, designated by durationa and route, to the human population (including 

susceptible subgroupsb

) that is likely to be without an appreciable risk of adverse health effects over a lifetime. It 

is derived from a BMDL, a NOAEL, a LOAEL, or another suitable point of departure, with uncertainty/variability 

factorsc

 applied to reflect limitations of the data used. 

________________________________________ 

a

 The generalized durations are similar to those given in Section 4.1. for acute (#24 hours), short-term (up to 30 

days), longer-term (up to 10% of average lifespan), and chronic (up to a lifetime), all considered to be 

continuous exposures throughout the duration specified. 

b

 Susceptible subgroups may refer to life stages, e.g., children or the elderly, or to other segments of the 

population, e.g., asthmatics or the immune-compromised, but they are likely to be somewhat chemical specific 

and they may not be consistently defined in all cases. See below (Section 4.3.2.3) for further discussion. 

c

 See discussion later in this chapter (Section 4.4.5) on application of uncertainty/variability factors. 

The Technical Panel notes a lack of support from the external reviewers of this 

document for any such prediction and recommends that the database characterizations for 

reference values be approached in a comprehensive way, as discussed in Section 4.3 to 

ensure that they are authoritative and as complete as possible in order to yield qualitative 

information about the range that could be predicted around the individual estimates rather 

than attempting quantitative evaluations of accuracy and ranges. 

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2. The term “deleterious” is considered ambiguous by some, so it has been replaced with 

the term “adverse,” because the latter is more commonly understood in the context of 

data evaluation and selection of endpoints for setting reference values. 

3. In the spirit of harmonization of risk assessment approaches for human health effects, 

it has been recommended that health effects no longer be categorized as “cancer” or 

“noncancer” for the purposes of hazard characterization and dose-response analysis (U.S. 

EPA, 1997b, 1998d; Bogdanffy et al., 2001). As indicated earlier, the approach to 

reference values discussed here is intended for risk assessments for any type of health 

effect known or assumed to be produced through a nonlinear and/or threshold mode of 

action (which may include U-shaped or other nonmonotonic dose-response curves as well 

as thresholds). In light of this recommendation, the term “noncancer” has been removed 

from the definition, denoting the move toward defining approaches for low-dose 

estimation or extrapolation based on mode of action. It is recommended that this issue be 

considered further in the deliberations by the Risk Assessment Forum’s Technical Panel 

on a framework for harmonization of approaches for human health risk assessment. 

To fulfill the need for consistency in the designation of various duration reference values, 

the Panel recommends that the terminology for reference values be standardized. Rather than 

continuing to use RfD and RfC only to denote chronic oral and inhalation reference values, 

respectively, standardized terminology should be developed that denotes both duration and route 

of exposure. Although Technical Panel members did not come to agreement on the best way to 

do this (and we welcome alternative suggestions), the terminology shown below is offered as an 

example of the way in which consistent labels could be developed and used. Either new 

standard terminology, (e.g., reference value) could be used, or RfD and RfC could continue to be 

used, but they would always need to be accompanied by the qualifying duration of exposure and, 

in the case of the RfD, by the route of exposure. Thus, the following alternatives for 

terminology are offered: 

Acute (Oral, Dermal) Reference Value or Dose, Acute (Inhalation) Reference 

Value or Concentration: RfVAO, RfVAD, RfVAI; RfDAO, RfDAD, RfCAI or RfCA 

Short-term (Oral, Dermal) Reference Value or Dose; Short-term (Inhalation) Reference 

Value or Concentration: RfVSO, RfVSD, RfVSI; RfDSO, RfDSD, RfCSI or RfCS 

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Longer-term (Oral, Dermal) Reference Value or Dose; Longer-term (Inhalation) 

Reference Value or Concentration: RfVLO, RfVLD, RfVLI; RfDLO, RfDLD, RfCLI or RfCL 

Chronic (Oral, Dermal) Reference Value or Dose; Chronic (Inhalation) Reference Value 

or Concentration: RfVCO, RfVCD, RfVCI; RfDCO, RfDCD, RfCCI or RfCC 

The Panel recommends that endpoint- or life stage-specific reference values such as the 

RfDDT (reference dose for developmental toxicity), which were originally proposed in Guidelines 

for Developmental Toxicity Risk Assessment (U.S. EPA, 1991), not be derived. Rather, a sample 

reference value should be calculated for each relevant and appropriate endpoint and these should 

then be considered in the derivation of various duration reference values. Reference values 

should be derived to be protective of all types of effects for a given duration of exposure and are 

intended to protect the population as a whole, including potentially susceptible subgroups. Thus, 

the RfDDT concept of a critical window of exposure for some health effects is addressed in the 

adoption of the less-than-chronic reference values. This recommendation does not preclude, 

however, using specific common endpoints in the assessment of cumulative risk for mixtures or 

chemicals that have a common mode of action or for risk management purposes. 

4.3. CHARACTERIZATION OF THE EXTENT OF THE HEALTH-RELATED 

DATABASE FOR SETTING REFERENCE VALUES 

A necessary first step in hazard characterization is the critical evaluation of all pertinent 

and relevant human and animal data that are available in the open literature as well as data 

submitted to the Agency in response to various regulatory standards, data call-ins, or other 

requirements and agreements. 

4.3.1. Review of Studies 

Data will be available from a wide variety of sources, including studies conducted 

according to EPA guidelines, studies conducted by industry using Organization for Economic 

Cooperation and Development or other protocols, experimental studies conducted by academic 

researchers, epidemiology studies, case reports or series, and controlled clinical studies in 

4-7
volunteers.7 These studies will be of widely differing quality; EPA must evaluate each study to 

determine whether it is of acceptable quality. 

4.3.1.1. Adequacy of Studies 

The following list of questions could be helpful in the process of evaluating data from 

animal and human studies. 

All types of studies: 

• What was the purpose of the study and is there a clearly delineated hypothesis? 

C Is there sufficient description of the protocol, statistical analyses, and results to make 

an evaluation? 

• Were the appropriate endpoints assessed in the study?8 Were the techniques used for 

the assessment scientifically sound? 

C Were appropriate statistical techniques applied for each endpoint? Was the power of 

the study adequate to detect effects? 

C Did the study establish dose-response relationships? Was a BMD lower confidence 

level (BMDL), LOAEL or NOAEL established? 

• Is the shape of the dose-response curve consistent with the known toxicokinetics of 

the test compound? 

7

Currently, OPP is reviewing its policy concerning use of human data from studies in 

which there is intentional pesticide exposure, and it has asked the National Academy of Sciences 

for input on the acceptability of such studies and ethical criteria for their use under the Protection 

of Human Subjects Rule (the “Common Rule”) (EPA, 2001c). 

8

A chemical may cause a variety of toxic effects depending on the amount, duration, 

timing, and pattern of exposure (i.e., continuous, periodic, or intermittent). These effects may 

range from severe—such as death—to more subtle biochemical, physiological, or pathological 

changes in one or more organ systems. In addition, the effects will vary depending on their 

latency following exposure and when the observations are made. Primary attention is given in 

risk assessment to those effects in the lower exposure range and/or the effects most biologically 

appropriate for a human health risk assessment. 

4-8 
• Do effects fit with what is known about mode of action? 

C Is the dose-response curve for precursor events consistent with the dose-response 

curve for clinical effects? 

C Are the results of the study biologically plausible? 

C What uncertainties exist? Do the results of the study indicate the need for follow-up 

studies to reduce uncertainties? 

C Are the study conclusions supported by the data? 

Human studies: 

• What were the data sources for exposure, health status, and risk factors (e.g., 

questionnaires, biological measurements, exposure/work history record reviews, or 

exposure/disease registries) and what were their strengths and limitations? 

• What methods were used to control, measure, or reduce various forms of error (e.g., 

misclassification or interviewer bias, confounding factors and potential effect 

modifiers) and their potential impact on the findings? What is the validity (accuracy) 

and reliability (reproducibility) of the methods used to determine exposure and 

outcome? What were the response rates? 

• What major demographic and other personal factors were examined (e.g., age, sex, 

ethnic group, socioeconomic status, smoking status, and occupational exposure)? 

What other climate or life stage factors were important for the endpoints and 

exposures assessed? 

• Were the findings examined for biologic plausibility, internal and external 

consistency of the findings, and the influence of limitations of the design, data 

sources, and analytic methods? 

4-9
Animal studies: 

C Was the study sufficiently documented (e.g., conducted in accordance with good 

laboratory practices)? 

C Were appropriate analytical techniques used to measure the stability, homogeneity, 

and actual level of the test substance in the study (in the water, feed, air, etc.)? 

• Was an appropriate animal species used?9 Was an appropriate number of animals 

used? Were sex and age considered? 

C Were the dose levels appropriate? What was the basis for choosing the dose levels? 

C Was an appropriate method used to assign the animals to dose groups? 

• Was an appropriate route and matrix of exposure employed?10 

C Was the duration of exposure adequate for the particular study design? 

• Were possible alterations in metabolism considered at the higher exposure levels? 

9

The laboratory animals used most often are the rat, mouse, rabbit, guinea pig, hamster, 

dog, or monkey. When reviewing these studies, the risk assessor makes judgments about the 

ability of the study to predict the potential for toxicity in humans and tries to select data from the 

species that is most relevant to humans using the most defensible biological rationale. When 

available, comparative toxicokinetics can be used to support this decision. Absent a clearly 

most-relevant species, the most sensitive mammalian species is used, that is, the species that 

shows toxicity at the lowest exposure level. 

10The most appropriate route of exposure is the route for which an evaluation is to be 

made. The toxicity of the chemical may differ with route of exposure because of differences in 

mechanism of action or toxicokinetics (absorption, distribution, metabolism, and excretion). 

Development of data to establish dosimetry for the purpose of route-to-route extrapolation is 

encouraged; however, route-to-route extrapolation is inappropriate when based exclusively upon 

default assumptions regarding exposure and toxicokinetics. Even within the same route of 

exposure, responses may differ due to alterations in toxicokinetics, for example, dietary or water 

exposure versus oral gavage. 

4-10 
Professional judgment is required to decide, on the basis of a thorough review of all 

available data and studies, whether any observed effect is adverse and how the results fit with 

what is known about the underlying mode of action. These judgments require the input of 

experts trained in toxicology, statistics, and epidemiology and, often, of specialists in the 

structure and function of the target organ systems. Both the biological and the statistical 

significance of the effects are considered when making these judgments. Biological significance 

is the determination that the observed effect (a biochemical change, a functional impairment, or a 

pathological lesion) is likely to impair the performance or reduce the ability of an individual to 

function or to respond to additional challenge from the agent. Biological significance is also 

attributed to effects that are consistent with steps in a known mode of action. Statistical 

significance quantifies the likelihood that the observed effect is not due to chance alone. 

Precedence is given to biological significance, and a statistically significant change that lacks 

biological significance is not considered an adverse response. 

For many discrete or quantal endpoints (e.g., birth defects, tumors, or some discrete 

pathological changes), this judgment is more straightforward because criteria have been 

established for deciding what type and incidence of effects are to be considered to be adverse, 

and an increase above the background rate can be judged using statistical tools. In the case of 

continuous measures (e.g., body weight, enzyme changes, physiological measures), this tends to 

be more difficult, because the amount of change to be considered adverse has not been defined 

by toxicologists or health scientists. Consequently, the endpoint is often decided in the context 

of the endpoint itself, the study, and the relationship of changes in that endpoint to other effects 

of the agent. 

Decisions about the amount of change to consider adverse must always be made using 

professional judgment and must be viewed in light of all the data available on the endpoint of 

concern. All toxicological data on a chemical must be reviewed before deciding whether an 

effect is biologically significant and adverse. Using a default cutoff value to define adversity for 

continuous measures may result in an inappropriate interpretation of data and less than optimum 

evaluation of a chemical’s effects. 

4.3.2. Issues to be Considered in Characterizing the Database for Risk Assessment 

4.3.2.1. The Weight-of-Evidence Approach 

A weight-of-evidence approach such as that provided in EPA’s RfC Methodology (U.S. 

EPA, 1994) or in EPA’s proposed guidelines for carcinogen risk assessment (U.S. EPA, 1999a) 

should be used in assessing the database for an agent. This approach requires a critical 

4-11
evaluation of the entire body of available data for consistency and biological plausibility. 

Potentially relevant studies should be judged for quality and studies of high quality given much 

more weight than those of lower quality. When both epidemiological and experimental data are 

available, similarity of effects between humans and animals is given more weight. If the 

mechanism or mode of action is well characterized, this information is used in the interpretation 

of observed effects in either human or animal studies. Weight of evidence is not to be 

interpreted as simply tallying the number of positive and negative studies, nor does it imply an 

averaging of the doses or exposures identified in individual studies that may be suitable as PODs 

for risk assessment. The study or studies used for the POD are identified by an informed and 

expert evaluation of all the available evidence. 

4.3.2.2. Use of Human and Animal Data in Risk Assessment 

Adequate human data are the most relevant for assessing risks to humans. When 

sufficient human data are available to describe the exposure-response relationship for an adverse 

outcome(s) that is judged to be the most sensitive effect(s), reference values should be based on 

human data. Much more data on a wide range of endpoints typically are required to establish 

confidence that there are no effects of exposure. If sufficient human data are not available to 

provide the basis for reference values, data from animal studies must be employed. It is 

advantageous if some human data are available to compare with effects observed in animals, 

even if the human data are not adequate for quantitative analysis. Availability of data on effects 

in humans at least allows qualitative comparison with effects observed in animals for 

determining whether toxicity occurs in the same organ systems and whether the nature of the 

effects is similar or different. If no human data are available, reliance must be exclusively on 

animal data. In that case, attention should be paid to whether data are available in more than one 

species and, if so, whether the same or similar effects occur in different species and possible 

sources of any observed differences. 

One of the major default assumptions in EPA’s risk assessment guidelines is that animal 

data are relevant for humans (e.g., U.S. EPA, 1991, 1996, 1998c). Such defaults are intended to 

be used in the absence of experimental data that can provide direct information on the relevance 

of animal data. 

Several types of information should be considered when determining the relevance or 

nonrelevance of effects observed in animal models for humans. This information is used in a 

variety of ways, from determining the role of metabolism in toxicity (Is the parent chemical or a 

metabolite responsible for toxicity?), to assessing whether homologous activity would be 

4-12
expected across species (Do humans share the sensitivity of the animal model, or is the response 

due to some species-specific idiosyncratic reaction?), to determining whether or not a threshold 

is likely to exist for the response (Are repair mechanisms capable of maintaining a homeostatic 

process?). All of this information must be weighed in light of the known heterogeneity of the 

human population versus the relatively inbred status of laboratory animals used in toxicity 

testing studies and housed under carefully controlled environmental conditions. 

Table 4-1 presents several factors to consider when evaluating the weight of evidence 

about the likelihood of the occurrence of effects in humans that is based on animal data (in 

conjunction with human data, if available). The table is not necessarily intended to delineate all 

factors that may need to be considered, but rather to provide a framework for evaluation and 

interpretation. It is important to evaluate the database in a holistic manner, determining 

strengths and weaknesses that are relevant to the overall assessment. Each chemical and 

database presents a unique set of issues that must be evaluated critically and thoughtfully. 

The dose-response nature of the data is an important characteristic of the database or 

individual study. When data are dose related, that is, when the incidence and/or intensity of 

response changes in an orderly manner as a function of dose, the effect should be considered to 

be of greater importance than when there is no apparent association between exposure and 

toxicity. Note, however, that the dose-response relationship need not be monotonic. U-shaped 

(or inverted U-shaped) dose-response functions are not uncommon in toxicology. For example, 

a chemical may induce an enzyme at low doses and inhibit it at high doses. Similarly, many 

solvent-like chemicals (including alcohol) produce increased motor activity at lower doses and 

depressed activity at high doses. 

Similarly, comparative toxicokinetic/metabolism data that suggest qualitative and 

quantitative comparability to that in humans would support the relevancy of animal data. 

Evidence suggesting a difference in toxicokinetics/metabolism would require additional 

exploration regarding whether the difference(s) results in a major qualitative or quantitative 

difference in internal dose in humans. 

The similarity of effects between species is also an important aspect in characterizing 

the database. Similar effects in more than one species indicate that the effect provides increased 

weight of evidence for the risk assessment process, even if such data are not available in humans. 

In contrast, response data that show inconsistency of effects among studies and/or species that 

cannot be explained by differences in toxicokinetics/metabolism or timing and/or magnitude of 

exposure, may suggest that less emphasis be placed on the effect. “Similarity” does not 

necessarily require identical effects between species. For example, changes in motor activity in 

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Table 4-1. Factors for evaluation of the weight of evidence regarding the 

likelihood of effects in humans 

Factor Increased weight Decreased weight 

Dose-response 

relationship 

Orderly change in effect as a function 

of exposure (need not be monotonic) 

Toxicokinetics/ 

metabolism 

Qualitative and quantitative 

comparability between humans and 

animals 

Similarity of effects Similar effects in more than one 

animal species or in animals and 

humans 

Mode of action Demonstration of homologous mode 

of action in animal model and humans 

Temporal relationship Consistent temporal relationship 

between exposure and effect 

No identified relationship between exposure 

and magnitude of effect 

Qualitative and quantitative differences 

between humans and animals 

Inconsistency of effects among studies 

and/or species that cannot be explained by 

differences in timing and/or magnitude of 

exposure or toxicokinetics/metabolism 

Evidence suggesting that the mode of action 

is species specific and irrelevant to humans 

Lack of temporality between exposure and 

effect 

animals evaluated in the neurotoxicity screening test and cognitive effects in humans would 

generally be considered similar, because both are indicative of changes in nervous system 

function. 

Mode of action information is also important in understanding whether a particular 

effect may be important for humans. For example, a transient reduction in anogenital distance in 

the postnatal animal following perinatal exposure to an anti-androgen has increased weight if the 

chemical is also known to act as an anti-androgen in humans. Likewise, the interpretation of 

increased skeletal variants observed following exposure to many chemicals would be enhanced 

by data indicating that the mechanistic pathways for these agents and the overall biological 

significance defined were also a possibility in humans. Mode of action data are also important in 

determining whether various chemicals work by common modes or mechanisms of action, which 

would then be considered in a cumulative risk assessment. 

Another criterion that is important in evaluating data is the temporal relationship 

between exposure and effect. The exposure should precede the effect at an interval that is 

consistent with what is known about the toxicokinetics and mode of action of the agent. It may 

be the case, however, that higher doses produce a shorter latency to effect than do lower doses. 

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4.3.2.3. Characterization of Effects in Potentially Susceptible Subpopulations 

A dose-response analysis for potentially susceptible subpopulations should be done as 

part of the overall dose-response analysis for health effects in general. “Susceptible” in this 

context means a differential (greater) response at the same internal dose in a particular segment 

of the population due to intrinsic (possibly unknown) factors. “Susceptible subpopulations” is 

used here to refer both to life stages and to other factors that may predispose individuals to 

greater response to an exposure. Life stages may include the developing individual before and 

after birth up to maturity (e.g., embryo, fetus, young child, adolescent), adults, or aging 

individuals. Other susceptible subpopulations may include people with specific genetic 

polymorphisms that render them more vulnerable to a specific agent or people with specific 

diseases or pre-existing conditions (e.g., asthmatics). The term may also refer to gender 

differences, lifestyle choices, or nutritional state. 

It is important to recognize that little basis currently exists for a priori identification of 

susceptible subpopulations for many chemicals. Without other data to raise suspicions, only the 

evaluation of effects in various segments of the population such as those mentioned above can 

identify susceptible subpopulations for a particular chemical and a particular set of exposure 

conditions. In some situations, differential exposure rather than differential susceptibility per se 

may be the critical issue (e.g., hand-to-mouth activity in toddlers). Economic differences may 

also result in differential exposure and susceptibility. 

A great deal of attention has been given in recent years to the issue of children as a 

susceptible subpopulation. Several approaches have been proposed for characterizing the 

database concerning the potential pre- and postnatal toxicity of a particular chemical and 

providing some guidance as to the weight of evidence or degree of concern for children’s health. 

However, each approach has been developed for a slightly different purpose and, as such, is 

generally complementary to, but not the same as, the other approaches. 

EPA’s developmental toxicity (U.S. EPA, 1991) and reproductive toxicity (U.S. EPA, 

1996) risk assessment guidelines describe an approach that characterizes the database as 

sufficient or insufficient to judge whether a chemical does or does not pose a hazard within the 

context of dose, route, duration, and timing of exposure. The International Programme on 

Chemical Safety (IPCS) (IPCS, 1995) proposed an approach based on the quality of information 

gathered in developmental and reproductive toxicity studies and the types of data that were not 

available from these studies. EPA’s draft 10X toxicology report (U.S. EPA, 1999b) further 

extended the recommendations for characterizing risks to children’s health within the context of 

the FQPA by discussing issues that would increase or decrease the level of concern. 

4-15
The present report endorses and extends the recommendations of the 10X Toxicology 

Working Group’s report by incorporating the issues dealing with level of concern into a 

framework for evaluating the evidence regarding the identification and characterization of 

susceptible subpopulations (see below). A workshop was held recently to discuss aspects of a 

framework for children’s health risk assessment and to emphasize a broader perspective on the 

issues that should be considered in hazard characterization, dose-response assessment, exposure 

assessment, and risk characterization for children as a susceptible subpopulation (ILSI RSI, 

2001). 

In contrast with the attention paid to children and asthmatics as potentially susceptible 

subpopulations in recent years, little attention has been focused on risk assessment for other 

potentially susceptible subgroups. As outlined in Chapter 3, there currently are no requirements 

in EPA animal study protocols for exposure during old age or for outcome evaluations near the 

end of the life span following earlier life stage exposures. Similarly, healthy animals that are 

more genetically homogeneous than humans are used in standard toxicity testing protocols, and 

information on pre-existing conditions or genetic polymorphisms is largely unavailable from 

animal studies. 

Human studies also usually employ healthy nonelderly individuals, although some 

studies in more susceptible populations have been conducted, such as studies of the effects of air 

pollutants in asthmatics. Individuals who have identified risk factors that are not the focus of a 

study are usually excluded from the study sample. It is important to consider such characteristics 

of the database if human data are used as the basis for the risk assessment. 

As can be seen in Table 4-2, several issues must be considered in assessing the potential 

for some subpopulations, including different life stages, to have greater susceptibility than others 

to a chemical. These include the timing (life stage)-response relationship, indicating greater 

susceptibility to exposure at some life stages than at others; whether effects are of a different 

type in identifiable subgroups of the population; and the dose-response relationship, that is, 

whether effects are observed at different levels of exposure in different subpopulations. 

Another important consideration is whether effects are observed at the same dose but 

with a shorter latency in different subpopulations. Additionally, differences among groups in 

terms of the seriousness and reversibility of effects must be considered. For example, an agent 

may produce relatively mild and reversible neurological effects in adults but produce permanent 

behavioral impairment following in utero exposure. It is also important to keep in mind that 

effects that may initially appear to be reversible may re-appear later or be predictive of later 

adverse outcomes. This is probably best exemplified by certain outcomes following a 

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Table 4-2. Factors for evaluating evidence regarding identification and 

characterization of susceptible subpopulationsa 

Factor Increased weight Decreased weight 

Timing (life stage) -

response relationship 

Effects occur at greater 

magnitude at one or more life 

stage(s) 

No difference in effects at 

different life stage(s) 

Type of effect Different types of effects in 

specific subpopulations 

Same effect(s) across all 

potential subpopulations 

Dose-response 

relationship 

Effect occurs at lower exposures 

in one or more subpopulation(s) 

No evidence for differential 

dose-response across different 

subpopulations 

Latency of effect Latency to observed effect 

different in specific 

subpopulations 

No difference between 

subpopulations in latency to 

effect 

Seriousness/ 

reversibility of effects 

Effects different in seriousness or 

degree of reversibility in specific 

subpopulations and/or differences 

in later consequence of an 

initially reversible effect 

No differences between 

subpopulations in seriousness 

and/or reversibility of effects, or 

in later consequences of an 

initially reversible effect 

a

 Subpopulations may be defined by gender, individuals at different life stages (fetus, child, adult, elderly), 

differences in genetic polymorphisms, and/or pre-existing diseases or conditions that may result in differential 

sensitivity to adverse effects from exposure to a specific toxic agent. 

developmental exposure; for example, an initial depression in birth weight or weight gain or 

subtle developmental retardation may be indicators of more serious abnormalities later in life. 

4.3.3. Characterization of the Extent of the Database 

The derivation of an RfD or an RfC is a multifaceted process that involves the 

coordination of data gathering and evaluation, analysis and judgment in varying proportions, and 

integration of all the information available. A vital part of the chronic RfD and RfC derivation 

process that relies heavily on judgment, for example, is the current approach to characterizing 

the database. For example, the minimum dataset for low-confidence and high-confidence RfDs 

and RfCs has been specifically defined as follows (U.S. EPA, 1994, 2002c): minimum dataset 

for a low confidence chronic RfD or RfC is a single subchronic study. The minimum dataset for 

4-17
a high confidence chronic RfD or RfC is a chronic study in two species, a single two-generation 

reproductive toxicity study, and a developmental toxicity study in two species by the appropriate 

route of exposure. 

The Technical Panel is recommending a somewhat different approach. Instead of 

specifying particular studies, this approach emphasizes the types of data needed (in terms of both 

human and animal data) for deriving reference values and recommends the use of a narrative 

description of the extent of the database rather than a single confidence statement. The 

Technical Panel believes that this approach encourages the use of a wider range of information 

in deriving reference values that take into consideration the issues of duration and route of 

exposure, the timing of exposures, the types and extent of endpoint assessment (i.e., structural 

and function), the susceptible subpopulations evaluated, and the potential for latent effects and/or 

reversibility of effects. In addition, this approach encourages the identification of data that 

would be needed or useful for improving the risk assessment for a particular chemical or group 

of chemicals. 

To characterize the database, the Technical Panel has developed a description of a 

“minimal” database and a “robust” database as a way of describing the range of data that can be 

used for deriving a reference value (Box 4-3). A great deal of scientific judgment is necessary 

when evaluating the extent of the database for a particular chemical. Defining the extent of the 

database requires an overall evaluation and judgment as to where in the minimal–robust 

continuum the available database should be characterized. The Technical Panel purposely did 

not define additional categories between minimal and robust (moderate), and the Panel has 

serious concerns about developing such categories because of the tendency to try to characterize 

a database with single word descriptors. Instead, we strongly support a narrative description of 

the extent of the database, with emphasis on the strengths and limitations of the data. It should 

also be noted that a database that is less than minimal should not be used to derive a reference 

value. 

Rather than presenting separate “minimal” and “robust” database descriptions for each 

type of reference value that might be derived, the descriptions in Box 4-3 are intended to apply 

generally across the various reference value types (e.g., acute, short-term, longer-term , or 

chronic durations for oral, dermal, or inhalation routes of exposure). Additionally, it is expected 

that the different types of reference values for a particular chemical will be developed within the 

same assessment. In this manner, the entire database for a chemical may be relied upon in the 

development of each of the different values (e.g., important and relevant insights may be gleaned 

4-18
from toxicity studies for exposure 

durations other than those directly 

corresponding to the type of 

reference value being developed). 

A minimal database as 

defined above can be used to set 

reference values, but the limitations 

of such a database should be clearly 

recognized and discussed in the 

narrative description. For example, 

a minimal database may provide 

data on only one duration or route of 

exposure or it may be specific to 

only one endpoint or organ system. 

Thus, the uncertainties related to 

such a database will be great and 

should be reflected in the size of the 

UFs applied for reference value 

derivation (see further discussion 

below). 

On the other hand, a robust 

Box 4-3. Description of minimal and robust databases 

Minimal Database: no human data available, route-specific 

toxicity data are limited to dose-response data applicable to the 

duration in question with assessment of endpoints other than 

mortality. A study showing only effect levels for mortality or other 

extremely severe toxicity would not be sufficient to set a reference 

value. 

Robust Database: includes extensive human and/or animal 

toxicology data that cover route-specific information on many 

health endpoints, durations of exposure, timing of exposure, life 

stages and susceptible subpopulations. In the absence of complete 

human data, mechanistic and other data show the relevance of the 

animal data for predicting human response. Specifically, the doseresponse data for the reference value in question includes endpointspecific data (e.g., developmental toxicity, neurotoxicity) coupled 

with toxicokinetic information as needed for route-to-route 

extrapolation. The toxicity studies include the evaluation of a 

variety of endpoints (e.g., hematological, clinical, histology of 

target organs) and endpoints specific to any known hazard 

characterization. The database for a reference value of less-thanchronic duration has also addressed the issue of reversibility of 

effects and latency to response, taking into consideration the 

possibility that less-than-chronic exposure may lead to effects at 

some period of time after exposure. Biological and chemical 

characteristics of the exposure and outcomes, as well as known 

limits on reserve capacities and repair of damage, formthe basis for 

determining the appropriate length of follow-up. 

database would address issues of potential toxicity in humans and animals and include data on 

several durations and routes of exposure as well as a thorough assessment of a variety of health 

endpoints. It would also include sufficient data on toxicokinetics and mode action to provide 

extensive information for extrapolation of effects to humans, including potentially susceptible 

subpopulations. A complete database on a single health endpoint that does not contain 

information on other endpoints of possible relevancy would not necessarily constitute a robust 

database, nor would a database that provides complete information on one route and/or duration 

of exposure be considered robust. 

It is clear that a robust database represents a “gold standard” that will rarely, if ever, be 

available. However, a lack of robustness does not mean that the database is deficient to the 

extent that a reference value could not be derived or that large UFs would need to be applied. 

Sound scientific judgement will be required to determine which UFs are appropriate in each 

case. 

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A critical assessment of the extent and quality of the database will inform the selection of 

the endpoints to be used to derive the reference values and the appropriate UFs. A reference 

value based on a single study would likely have a high degree of uncertainty. As more 

information from additional toxicology studies, toxicokinetic studies, structure-activity 

relationships, and human data becomes available, EPA can have greater assurance that the 

appropriate species, route of exposure, and target organ system(s) are known for each duration 

reference value needed for a human health risk assessment. As this additional information 

becomes available, the use of UFs will likely decrease. The ultimate objective is to account for 

all human health endpoints resulting from exposures over all life stages from before conception 

to the elderly adult. 

The optimum assessment considers subtle effects that impact an individual’s quality of 

life as well as so-called “frank” effects (death and major disease). The evaluation should 

encompass immediate health outcomes as well delayed responses to an exposure (i.e., latent 

responses), although most current testing guidelines do not explicitly evaluate latency to 

response. 

4.3.3.1. Extent of the Database 

The following series of questions regarding the extent of the database can help guide the 

assessment process: 

C Have adequate studies been conducted to establish the target organs/endpoints? 

C Have the effects been characterized for both sexes and all life stages? 

• Are data pertaining to potentially susceptible subpopulations available? 

C Are the responses consistent across species? Are the results of the studies 

biologically plausible? 

C Is the route and matrix of exposure relevant to the specific reference value being 

derived? 

C Is the duration of exposure appropriate for the specific reference value being derived? 

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C Is the animal species and strain appropriate for extrapolation to humans? 

C To what degree may the biological endpoints be extrapolated (qualitatively and 

quantitatively) to humans? 

C Are toxicokinetic data available? Are they available for both sexes, for relevant life 

stages, for other susceptible subpopulations? 

C Is the shape of the dose-response curve consistent with the known toxicokinetics of 

the test compound? 

C Are the metabolism and toxicokinetics in the animal species similar to those of 

humans? 

C Has the dose-response curve been replicated by or is it consistent with data from 

other laboratories and other test species? 

• Have the data for all relevant endpoints been adequately modeled by the BMD or 

other appropriate quantitative analysis to determine the most sensitive endpoint(s)? 

C How well is the toxicity characterized? Do the results of all the studies indicate the 

possibility of effects on particular systems that have not yet been explored 

sufficiently or do they indicate that additional studies may reveal effects not yet 

characterized? 

4.4. DERIVATION OF REFERENCE VALUES 

After the database has been thoroughly evaluated for quality and extent, as outlined 

above, several decisions must be made and procedures applied before the final derivation of a 

reference value. This section summarizes the current procedures and points out assumptions 

made and areas for improvement and clarification. A variety of factors related to the derivation 

of reference values is discussed, including the selection of relevant endpoints for the POD for 

various duration reference values (Section 4.4.1). Adjustment of the study dose/exposure for 

duration is described in Section 4.4.2, and derivation of a HED or HEC is discussed in Section 

4.4.3. 

4-21
Other issues are discussed briefly in Section 4.4.4, such as varying levels of response at 

the BMDL, BMCL (lower confidence limit on the BMC), or NOAEL due to varying study 

designs and test sensitivity and considerations of adversity and severity (i.e., nature of the 

response) for choosing the benchmark response (BMR) level. The nature and application of 

uncertainty/variability factors and MFs are discussed and critiqued in Section 4.4.5, and future 

directions are briefly discussed in Section 4.4.6. Section 4.4.7 summarizes key points from two 

case studies that are presented in detail in Appendix B. 

4.4.1. Sample Reference Values and Selection of Endpoints to Use as the POD for 

Reference Values 

Currently, the “critical effect” is used as the basis for the POD, and various UFs are 

applied to the dose at the critical effect to derive the RfD or the RfC. The critical effect is 

defined as “the first adverse effect, or its known precursor, that occurs to the most sensitive 

species as the dose rate of an agent increases” (U.S. EPA, 2002c). The underlying assumption is 

that if the RfD or the RfC is derived to prevent the critical effect from occurring, then no other 

effects of concern will occur; in addition, this approach assumes that the relationship of various 

health effects for a particular chemical is maintained across species. 

The Technical Panel is concerned that presenting only a single critical effect and the 

critical study from which it was derived in the IRIS summary table that appears at the beginning 

of each RfD or RfC file may not provide enough information to the reader who is unfamiliar 

with risk assessment and thus could be misleading. Presenting a single endpoint as a POD for a 

systemic effect, for example, cannot capture the nature of the dose-response curve for that 

particular endpoint, nor does it convey the possibility that other more serious endpoints may 

have a dose-response character markedly different from the less serious endpoint. For example, 

an agent may have a clear progression of responses with increasing dose that is seen as one type 

of effect at the lowest exposure level (e.g., proteinuria in the case of cadmium), but at a higher 

level it produces additional effects (proteinuria PLUS GFR decrements) and at the highest level 

even more types of effects (proteinuria PLUS GFR decrements PLUS osteomalacia). Each of 

these effects could have a markedly different dose-response character. 

Focusing on a single critical effect also does not reflect the situation in which other types 

of effects may be found at similar levels of exposure or the variety of health outcomes that may 

result when an exposure significantly exceeds the RfD or the RfC. Most importantly, in light of 

the Technical Panel’s recommendations for deriving an expanded number of reference values for 

different durations and routes of exposure, the limitations of focusing only on the critical effect 

4-22
become apparent because the most sensitive endpoint may be different for different durations or 

routes of exposure. 

Layered upon this complex consideration of dose-response is the further complication 

that all of the exposure levels producing these effects are or should be adjusted to a human 

equivalent exposure at the time of their comparison. These adjustments may profoundly affect 

what is considered the most sensitive organ or system. Effects that occur at the same external 

inhaled concentration but in different organs in the same exposed animals (e.g., effects in the 

liver and the nasal cavity) may have quite different HECs, based on the current RfC 

methodology (U.S. EPA, 1994), because the underlying basis for the adjustment used for 

systemic effects is markedly different from that used for portal-of-entry effects between animals 

and humans. This adjustment procedure is discussed further below but is noted here because of 

its interrelationship with identifying what is to be considered a critical effect. 

These aspects all support the case that a more comprehensive approach to setting 

reference values requires a more extensive and systematic analysis of endpoints than has 

typically been conducted in the past. In the approach proposed here, the selection of the POD 

would be similar to the current critical effect approach (e.g., U.S. EPA, 1994) and would include 

the use of sound scientific judgment in evaluating the strength and validity of studies and the 

extent of the database, as described in Section 4.3. In this approach, however, the selection of 

the POD would be based on consideration of all relevant and appropriate endpoints carried 

through the derivation of sample reference values, with selection of the limiting value(s) 

protective of all endpoints as the final step (the same approach would be used for deriving a 

POD for low-dose modeling, as discussed in the proposed cancer risk assessment guidelines 

[U.S. EPA, 1999a]). 

For example, the dose-response curves would be modeled for several adverse endpoints 

and the corresponding BMDs and BMCs and their lower 95% confidence limits 

(BMDLs/BMCLs) calculated (U.S. EPA, 2000c) or NOAELs determined if dose-response 

modeling is not possible. Next, duration adjustment to the continuous exposure scenario would 

be performed for each endpoint, with further adjustment to the corresponding HECs using the 

RfC methodology (U.S. EPA, 1994) or adjusted BMDLs or NOAELs for oral or dermal 

exposures (see Section 4.4.3 for further discussion). These adjusted values would represent the 

POD for each relevant endpoint. Then, uncertainty/variability factors that take into account a 

variety of issues, including chemical-specific data, such as known toxicokinetic differences 

between the laboratory animal species tested and humans, and mode of action information would 

be applied to the adjusted values for each relevant endpoint. The sample reference values would 

4-23
then be compared across endpoints and organ systems to determine which are the most relevant 

for use in deriving the final reference value for each exposure duration that will be protective of 

the human population (including susceptible subgroups). 

The Technical Panel recommends the use of a more visual and graphic exposureresponse array to depict the PODs for all relevant endpoints for various routes and durations of 

exposure, somewhat like those shown in the ATSDR toxicology profiles but with appropriate 

changes for the purpose of deriving reference values. The exposure-response array of the PODs 

would facilitate the evaluation and comparison of relevant endpoints and values. (See examples 

of the proposed approach discussed in Section 4.4.7 and in two case studies in Appendix B.) 

4.4.2. Dose Adjustment for Duration of Exposure 

Available studies from which reference values are derived seldom if ever match the intent 

of the reference value regarding species or duration. For example, chronic RfD and RfC values 

are intended by definition to be for “a continuous exposure to the... human population.” Doses 

or exposures from studies in which animals are exposed for less than a lifetime or in which 

worker populations are exposed only during working hours require adjustment to continuous 

exposure in order to be concordant with the intended duration of the reference value (see 

Rozman and Doull, 2000; Rozman et al., 2001, for further discussion). This section describes 

various procedures that are currently used by the Agency to adjust a LOAEL, a NOAEL, or a 

BMDL with regard to duration. The basis for these adjustments is discussed, as is the 

applicability of these procedures to various routes of exposure. 

The Agency has invested considerable time and effort into exploring these aspects for the 

inhalation route. A major point that will become apparent in this discussion is that 

methodologies for duration adjustment via the inhalation route are currently in place as part of 

the existing methodology for the chronic RfC and as proposed for ARE derivations, whereas no 

comparable documents yet exist for the oral or dermal routes of exposure. 

4.4.2.1. Duration Adjustment Procedures for Inhalation Exposures to 

Continuous-Exposure Scenarios 

Adjustment of duration to a continuous exposure scenario is regularly applied as a default 

procedure to studies with repeated exposures but not to single-exposure inhalation toxicity 

studies in animals and humans (U.S. EPA, 1994). Operationally, this is accomplished by 

4-24
applying a Cn

 x t product11 for both the number of hours in a daily exposure period and the 

number of days per week that the exposures are performed. In an inhalation study in which 

animals are exposed to 100 mg/m3 for 6 hours, 5 days per week, the adjustment to a continuous 

exposure concentration would consider both hours per day and days per week: 

100 mg/m3 × 6/24 hrs x 5/7 days/wk = 17.9 mg/m3, 

with 17.9 mg/m3

 being the concentration adjusted for continuous exposure. Study designs that 

include exposures 7 days/wk, for example, prenatal developmental toxicity studies and DNT 

studies, do not require the 5/7 days/wk adjustment. 

Exposures from human occupational studies are most often reported as 8-hr timeweighted averages (TWAs) and are therefore also discontinuous. Adjustment of these exposures 

to derive a HEC is explained below in Section 4.4.3. 

These adjustment procedures imply that the C × t product and not C is associated with the 

endpoints observed; this may be restated as implying that the area under the curve (AUC), C × t, 

rather than the peak concentration, C, is the dosimeter associated with toxicity. Although neither 

of these dosimeters may be demonstrable experimentally to be the appropriate measure of dose, 

the Agency uses adjustment to a continuous inhalation exposure based on the C × t relationship 

as a matter of policy. 

When applied to a discontinuous inhalation exposure regimen from an experimental 

study, adjustment to a continuous exposure will always result in a lower value of C and maintain 

a measure of total exposure, that is, C × t. Thus, application of this procedure provides an 

automatic margin of protectiveness for chemicals for which C alone may be appropriate, and it 

reflects the maximum dose for agents for which total or cumulative dose is the appropriate 

measure. When considered in this way, this policy can be regarded as being protective of public 

health. However, assessors are encouraged to look for data on specific chemicals that support 

the use of C x t or that offer alternative models for adjustment of exposure duration. 

4.4.2.2. Duration Adjustment for Inhalation Developmental Toxicity Studies—A Current 

Exception 

A notable exception to duration adjustment of inhalation exposures is for inhalation 

developmental toxicity studies in which this practice historically has not been done. The current 

11Where Cn

 = C1, as described in Section 4.4.2.3. 

4-25 
guidelines for developmental toxicity risk assessment (U.S. EPA, 1991) recommend against 

duration adjustment (i.e., from a discontinuous to a continuous exposure) as a default procedure 

unless toxicokinetic data are available to indicate an accumulation with continuous exposure. 

This is contrary to the default approach used for other types of studies in which duration 

adjustment is done without a requirement for toxicokinetic information. In fact, for other types 

of studies, toxicokinetic information is often used as the basis for moving away from the default 

adjustment. 

Furthermore, although the effects of some agents that cause developmental toxicity have 

been shown to be more a function of peak concentration (Nau, 1991), the effects of other agents 

have been shown to be related to either AUC or C, depending on the timing of exposure and the 

developmental timing of the organ system affected (Terry et al., 1994). In addition, recent 

studies have shown that the developmental effects of certain agents that have a short half-life, 

such as all-trans-retinoic acid (Tzimas et al., 1997) and ethylene oxide (Weller et al.,1999), or a 

very discrete exposure period, for example, hyperthermia (Kimmel et al., 2002), are a function of 

AUC. 

On the basis of this information and the rationale used for duration adjustment for other 

health effects (i.e., that exposure adjustment based on C × t tends to be more health protective), 

the Technical Panel recommends that duration adjustment procedures to continuous exposures 

based on C × t be used as a default procedure for inhalation developmental toxicity studies as it 

is for other health effects from inhalation exposure. The Technical Panel also urges continued 

development of data, modeling, and improved procedures for dose-duration adjustments related 

to developmental toxicity. 

4.4.2.3. Duration Adjustment for Acute Reference Values—Discontinuous Scenarios of 

24 Hours or Less 

As discussed above, the magnitude of response to a toxic chemical exposure usually 

depends on both the concentration and the duration of the exposure, such that the combination of 

these components, C × t, determines the response and, by logical extension, the internal dose of a 

chemical at the target tissue. In deriving acute, short-term, or longer-term reference values, there 

may be a need to specifically adjust or present these values under alternative C × t combinations. 

For example, an acute reference value may be required for both a 1-hour duration and an 8-hour 

duration, but the available data are from a 4-hour exposure. The current guidance on this issue is 

contained in the draft methodology for development of AREs (U.S. EPA, 1998a). This section 

presents the adjustment procedures recommended in the draft ARE methodology. 

4-26
Because of the recognized limitations of the C x t model, a modification has been 

developed such that Cn

 × T = k, with n being empirically derived. 

the values of the “n” exponent are shown in Figure 4-1. the 

Figure 4-1. Concentration-by-duration plot showing the effect of the 

exponent in the Cn

 x T = k on extrapolation across time. 

Source: Adapted from ten Berge et al., 1986. 

current version of the Agency’s ARE methodology, is based on the data of ten Berge et al. 

(1986). pirically derive values of “n” that ranged from 0.8 to 

3.5 for a number of chemicals on the basis of acute lethality. 

would indicate that the relationship described by Haber’s law holds and that the response is 

related to total dose. 

The consequences of varying 

This figure, which was derived from

These investigators were able to em

A value of 1 for the exponent “n” 

4-27
Note that for any degree of downward slope with increasing duration (lines marked with 

n = 1 or n = 0.8), an extrapolation from a longer to a shorter duration (i.e., from right to left) 

would result in a higher value for C. Extrapolating from a shorter to a longer duration (i.e., from 

left to right), however, would have a different consequence in that with any degree of downward 

slope, C would always be lower for the longer duration. Several possible approaches for 

extrapolation in this situation could be envisioned. One approach would be to assume a value of 

1 for “n,” such that Cn

 × T = k and lower values of C would always result; this approach is likely 

to be the actual case, because the value of “n” for most chemicals so far examined has shown an 

appreciable downward slope (e.g., 0.8 < n < 3.5 [ten Berge et al., 1986]). 

The optimal approach for extrapolating from one dose-duration response situation to 

another is the use of a physiologically based pharmacokinetic model (PBPK) model. The 

principle of using PBPK models as the basis for describing the correlations between level and 

duration of exposure, internal dose, and biological effect has been stated clearly by Andersen et 

al. (1987). Integration of information using PBPK models requires a chemical database that is 

rich in toxicity data; therefore, this approach is not applicable to most chemicals for which 

toxicokinetic data are scarce or nonexistent. 

In the absence of such a database to support the development of a PBPK model, the 

approach recommended by the draft ARE methodology is the use of chemical-specific data on 

duration dependence from other adequate but longer-duration data, if they exist (e.g., in 

extrapolating to 28 days using 7-day data, the 90-day repeated-dose data should also be 

considered). This is considered a conservative approach, because the duration adjustment 

approach (i.e., averaging to continuous exposure), when applied to multiple exposure studies 

always results in decreased values for C (i.e., extrapolation would be from shorter to longer 

durations on the curves in Figure 4-1). 

In the absence of chemical-specific data to inform duration adjustment, the response has 

most often been related to the simple C × t product. This is also the default in the draft ARE 

methodology for adjustment to longer durations. For adjustment to shorter durations, the ARE 

methodology conservatively recommends that there be no change in concentration. 

Further investigation would increase confidence in the basic assumptions made for the 

latter two methods of duration adjustment, including the applicability of the C x t relationship 

over spans of exposure from months to years and assessing the “conservativeness” of these 

approaches in relation to public health. Further investigation of C x t relationships relative to life 

stage is also recognized as a research need. 

4-28
4.4.3. Derivation of a HEC or a HED 

Animal data often form the basis for dose-response assessment. By definition, the IRIS 

risk values are for humans, thereby making animal-to-human extrapolation requisite. The 

specific point of this extrapolation is to estimate from animal exposure information the human 

exposure scenario that would result in the same response. The simplest manner in which this 

may be done is application of an animal-to-human UF (discussed further below), typically with a 

value of 10; in application this means that humans are assumed to be more sensitive to effects 

than are animals by a factor of 10. 

Much of the RfC methodology (U.S. EPA, 1994) focused on improving the science 

underlying the animal-to-human UF, segregating it into toxicokinetic and toxicodynamic 

components and providing generalized procedures to derive dosimetric adjustment factors 

(DAF). Application of DAFs to the animal airborne exposure values yields estimates of the 

concentration that would result in the same concentration to humans, that is, the HEC. 

Application of a DAF in the calculation of a HEC is considered to address the toxicokinetic 

aspects of the animal-to-human UF (i.e., to estimate from animal exposure information the 

human exposure scenario that would result in the same dose to a given target tissue). 

Current Agency practice is to accommodate uncertainty about the remaining 

toxicodynamic component through application of a partial animal-to-human UF (100.5, which is 

typically rounded to 3). The theoretical basis for deriving DAFs used in calculating HECs, along 

with recommendations for improvement of this process, is discussed in this section. 

Exposures from human occupational studies are most often reported as 8-hr TWAs for 

exposures during work days (5 days/wk). As with discontinuous exposures of animal studies 

(e.g., 6 hrs/day, 5 days/wk), exposures from occupational studies are also adjusted to derive 

continuous HECs relevant to the human population (U.S. EPA, 1994). As described below for 

animal data, the optimal approach is to use a biologically motivated mathematical, or PBPK, 

model. An occupational exposure can be extrapolated in the same fashion as intermittent 

exposure regimens from experimental laboratory animals, using particle deposition or PBPK 

models with human exertion (work) ventilation rates and exposure durations appropriate to the 

occupational setting. 

In the event that a PBPK model or required physicochemical and physiological 

parameters are not available, the default approach for human exposure scenarios is to adjust by 

the default occupational ventilation rate and for the intermittent work week schedule. The 

ventilation rate adjustment is based on the assumed amount of air used by a worker during the 

work period, that is, half of the daily ventilatory capacity of an adult male human is assigned (10 

4-29
m3

 of 20 m3 total) to the 8-hour occupational exposure (i.e., instead of 1/3 or 8/24 hrs) (ICRP, 

1994). By basing this adjustment on a functioning physiological parameter, that is, a fractional 

ventilatory capacity based on the assumption that activity levels are higher in this setting than in 

others, such as at rest or asleep, this adjustment may be considered to have a toxicokinetic basis. 

The 8-hour TWA concentrations are multiplied by this factor, 10/20 m3

, and the product is 

considered to be an average continuous airborne concentration. 

In parallel with the animal studies, an adjustment for days per week (usually 5/7 

days/wk) is also made, if applicable. This adjusted airborne concentration is considered to be a 

HEC. This default calculation, as with those described below for extrapolation from animal data, 

was developed for the general human population. It may be appropriate to further evaluate this 

approach or to develop an alternate default approach to ensure adequate consideration of 

intrahuman variation. 

Currently, no procedures parallel to the inhalation RfC methodology exist for deriving 

either oral or dermal human equivalents from animal data. Default factors (usually of 10) are 

routinely applied to address the issue of animal-to-human extrapolation. Thus, no parallel to the 

HEC, that is, a HED, is derived nor are other adjustments applied to the animal oral or dermal 

dose. 

This section recommends that dose adjustments similar to those by which HECs are 

estimated be explored in deriving HEDs for oral and dermal exposures. This would be 

accomplished in a manner parallel to the HEC derivation, by instituting and applying a DAF to 

animal oral or dermal exposures. Specific recommendations are also presented and discussed 

concerning the basis for deriving DAFs for HED calculation. These recommendations, along 

with current procedures for estimating human equivalent values, are illustrated in Figure 4-2. 

This figure also demonstrates how calculation of the HEC through application of a DAF is 

considered to address the toxicokinetic but not the toxicodynamic component of the animal-tohuman extrapolation. Procedures outlined in this figure for deriving a HEC may be applied to 

any animal inhalation exposure, regardless of whether it is a BMDL, a NOAEL, a LOAEL, or 

another effect level. 

4.4.3.1. PBPK Models and Derivation of HEDs and HECs: Estimating Internal Dose 

The preferred option for calculating a HED or a HEC is to use a chemical-specific PBPK 

model parameterized for the species and regions (e.g., respiratory tract) involved in the toxicity, 

as shown on the left-hand side in Figure 4-2. When sufficiently parameterized, a PBPK model is 

capable of calculating internal doses to a target organ from any exposure scenario in an animal 

4-30
Oral/Dermal/Inhalation 

Exposure 

ChemicalSpecific PBPK 

Model 

HEC/HED

Inhalation Exposure Oral/Dermal 

Exposure conditions, Exposure con

e.g., mg/m3, hr/d, d/wk e.g., mg/kg-d

Adjust to 

Continuous Exposure 

24 hr/d, 7 d/wk 

Adjust 

Continuous E

(7 d/w

Application of DAF 

(for toxicokinetics) 

Application 

(for toxicok

HEC 

Current 

HED 

Proposed 

4-31

DAF = Dosimetric Adjustment Factor 

Figure 4-2. Current and proposed generalized procedures for deriving HECs or HEDs from an
and then estimating what human exposure would result in this same internal dose, that is, the 

HED or the HEC. A formal DAF is not calculated in this process; rather, the model itself serves 

as a DAF in estimating HECs or HEDs. However, constructing a PBPK model is an 

information-intensive process that requires much chemical-specific data, including route-specific 

data. Such sophisticated data and models are available usually for only a subset of chemicals 

that have extensive databases. 

It should be noted that even these sophisticated models are often parameterized on the 

basis of adult members of the species. Many of the parameters critical to PBPK model solutions 

are sensitive to life stages, such as lung function/development in humans (Pinkerton and Joad, 

2000), for which no or few data are available. Thus, these models are available but often cannot 

specifically address species differences at life stages other than mature adults (and then usually 

males). The Technical Panel encourages research and data gathering to support the construction 

of PBPK models, it endorses attempts to produce PBPK models that are sensitive to life stages, 

and it supports fully attempts to produce template models for suites of related chemicals, as has 

recently been done by Barton et al. (2000). 

4.4.3.2. Default Procedures and Derivation of HECs from the RfC Methodology: 

Derivation and Application of DAFs 

The next lower level of complexity in deriving HECs is less data intensive than the 

PBPK approach. As shown in Figure 4-2, this procedure involves the use of species-specific 

physiologic and anatomic factors relevant to the form of pollutant (e.g., particle or gas) and 

categorized with regard to elicitation of response either locally (i.e., within the respiratory tract) 

or remotely. These factors are all employed in determining the appropriate DAF. For HECs, 

DAFs are applied to the “duration-adjusted” concentration to which the animals were exposed 

(e.g., to a weekly average). The generalized DAF procedures may also employ chemicalspecific parameters, such as mass transport coefficients, when available. In lieu of such data, 

however, default procedures that yield generalized adjustments are recommended. Although 

these generalized procedures were developed from the existing scientific understanding of the 

relevant processes, they have not been comprehensively evaluated (e.g., using data from humans 

and animals). They are explained fully in the RfC methodology (U.S. EPA, 1994). 

For example, the manner in which a HEC is calculated for a reactive gas that elicits an 

effect in the extrathoracic region of the respiratory tract (i.e., the nasal tract) of a rat is by 

creating a surface area/ventilation ratio for both humans and rats and applying it to the external 

exposure concentration for rats. The current default values used for both the human and the rat 

4-32
extrathoracic surface area are single estimates from the literature and are apparently estimated 

from adult specimens. The ventilation measure for humans is set at a default value of 20 m3

, and 

the ventilation measure for rats is based on an algorithm of body weight (from U.S. EPA, 1988). 

A major assumption made in this particular adjustment is that the distribution of a gas in 

the region of interest is uniform, although it is known to be highly nonuniform (Kimbell et al., 

1993, 1997). Data are not available to address this simplified assumption directly. Use of the 

method, for example on effects in the extrathoracic region, results in a DAF of about 0.2, such 

that the resultant HECs are about 20% of the animal-duration-adjusted concentration. Although 

information is not yet available to address this assumption, indications are that resolution with 

actual data may produce DAFs that are much closer to unity, that is, that are near the animaladjusted concentration. 

In comparison to the procedure for gases that elicit respiratory effects, calculation of a 

HEC for a category 3 gas (i.e., a gas that is relatively water-insoluble and unreactive in the 

respiratory tract and for which the site of toxicity is generally remote to the site of absorption in 

the pulmonary region) is usually accomplished by creating a ratio of the blood:gas partition 

coefficient for the laboratory animal species to the human value. The ratio is used as the DAF 

and applied to the experimental exposure concentration. In lieu of data on the values for 

blood:gas partition coefficients for the chemical or when the data indicate the ratio to be >1, the 

default assumption is that the ratio of animal coefficient to human coefficient is 1, and therefore 

the DAF would be 1. However, available data on partition coefficients for a number of 

compounds indicate that the animal/human ratio is usually >1 (Gargas et al., 1989; Jepson et al., 

1994) such that the DAF would also be >1. In the context of substituting data-derived values for 

UFs, the Technical Panel recommends further investigation into using data-derived values in 

constructing the animal/human ratios—even when much greater than 1—in place of the default. 

The default dosimetric adjustment procedure for particulate substances is an empirical 

model that estimates regional deposition only, although it is recognized that with the 

development of the relevant data, clearance and the retained dose may be used as a DAF (U.S. 

EPA, 1994). The DAF for particles is more specifically termed the regional deposited dose ratio 

and is derived from a normalizing factor (surface area being the recommended factor for all three 

regions of the respiratory tract), the ratio of animal-to-human minute volumes (where the human 

default value is the traditional adult value of 13.8 L vs. the adult value for the relevant animal), 

and the ratio of animal-to-human regional fractional deposition. Physiological parameters used 

in estimating the regional deposition include body weight, minute volume, and surface area for 

the three areas of the respiratory tract. Defaults for the human values are based on adult data 

4-33
(e.g., 70 kg body weight, 13.8 L minute ventilatory volume, etc); the animal values are also 

traditionally based on adult data. To evaluate protectiveness of these default calculations for 

different life stages, it may be appropriate to perform ratio calculations using data for other life 

stages. 

As a general recommendation, the Technical Panel encourages further consideration of 

the existing animal-to-human extrapolation procedures described in current methodologies (e.g., 

the chronic RfC methodology [U.S. EPA, 1994]) and the development of procedures for 

inhalation adjustment to incorporate the most current scientific thought and data to address, as 

needed, issues of variability due to life stage and other intrinsic factors. This consideration 

would include examining the extent to which calculating a HEC (or any recommended HED) 

addresses cross-species toxicokinetics as well as identification and parallel investigation into 

issues of toxicodynamics. 

4.4.3.3. HECs and Children—A Special Case? 

Children are often characterized as constituting a potentially susceptible subgroup 

because they could be at greater risk than adults for inhaled toxic agents (including both gases 

and particulates) for reasons relating to either toxicokinetics or toxicodynamics. It is clear for 

any of a variety of reasons related to toxicokinetics that an adult and a child breathing the same 

concentration of an agent such as a reactive gas may receive different doses to the body or to the 

lungs. A generalized theoretical approach to judging whether children would receive greater 

doses than would adults when both breathe the same concentration of a reactive gas, for 

example, would be to compare the amount of gas breathed in (which would be directly 

proportional to the ventilatory volume) with the overall surface area in the respiratory tract on 

which the gas may impinge. The current Agency default assumption used in deriving HECs for 

particles and reactive gases that elicit respiratory effects is that the surface area of the total 

respiratory tract of an adult male, estimated at 54.3 m2

, is exposed to a total daily air intake of 20 

m3

, a volume for an adult male derived from a combination 24-hour activity pattern in ICRP 

(1994) of sitting awake for 8 hours, exercising lightly for 8 hours, and sleeping for 8 hours. 

It has been well established that the human respiratory system passes through several 

distinct stages of maturation and growth that involve branching morphogenesis and cellular 

differentiation during the first several years of life and into adolescence (Pinkerton and Joad, 

2000). The proportion of surface area to ventilation volume may be markedly different during 

these developmental stages. The significance of these disproportions with regard to toxicant 

exposure overall or to the sites of active cellular differentiation have yet to be elucidated. 

4-34
The Technical Panel recommends that issues involving dose to the young from inhalation 

exposures be pursued both theoretically and experimentally in order to establish the basis on 

which children should be considered as a susceptible subpopulation for inhalation exposures. It 

should also be reiterated that this is an estimate of the toxicokinetic aspect of dose only, and 

toxicodynamic differences between the lungs of young children and adults are not addressed. 

4.4.3.4. Deriving a HED for Oral and Dermal Exposure—Use of BW3/4 as a 

Cross-Species DAF 

As indicated above, the Agency currently does not provide a procedure for calculating a 

HED for oral or dermal exposure scenarios that would parallel calculation of the inhalation HEC. 

Instead, assumptions are made regarding the comparability of ingested or applied dose, based on 

a mg/kg body-weight basis, and there is no adjustment for portal-of-entry alterations to internal 

dose or on portal-of-entry versus systemic effects. The Technical Panel recognizes the work of 

an interagency workgroup to develop and propose dosimetric adjustment procedures for both 

dermal and oral routes of exposure in order to address those aspects of cross-species dosimetric 

adjustment that are missing in Figure 4-2. Some of these proposals have already appeared in 

abstract form (Jarabek, 2000; Hanna and Jarabek, 2000; Hubal et al., 2000; Rigas et al., 2000). 

Figure 4-2 demonstrates that dosimetric adjustment procedures for estimating human 

equivalents from animal values are not consistent for different exposure routes. Other 

procedures, both from within and external to the Agency, could be explored for the purposes of 

deriving a DAF and employing it to estimate a HED. For example, in the absence of more 

sophisticated physiologically based models, the Agency has endorsed scaling of doses for 

carcinogens between species according to body mass raised to the 3/4 power (BW3/4) (U.S. EPA, 

1992). This procedure presumes that equal doses in these units (i.e., in mg/kg3/4/day) when 

administered daily over a lifetime, will result in equal lifetime cancer risks across mammalian 

species. This same relationship (i.e., BW3/4) has been affirmed to apply across entire phyla, 

including plants (Gillooly et al., 2001), for general metabolic rates. 

The basis for the less-than-full-power relationship for general metabolic processes (i.e., 

< BW1

) is thought to be related to species differences in exchange surfaces and distribution 

networks that constrain concentration and flux of metabolic reactants (West et al., 1997; Enquist 

et al., 1998). Thus, when this procedure is applied to animal data, the resulting scaled human 

dose may be viewed as a valid cross-species relationship not only of cancer potency but also for 

general metabolic processes and, by extension, for other phenomena involving the fundamental 

determinants of concentration and flux, the same ones that drive basic toxicokinetics. 

4-35
This brief analysis of the BW3/4 cross-species relationship and toxicokinetic processes 

and the Agency’s endorsement of this procedure for carcinogenic agents makes this process a 

possible candidate for estimating cross-species toxicokinetic relationships in the absence of 

adequate toxicokinetic information. That is, BW3/4 factors could be applied as DAFs for deriving 

a HED. This procedure would parallel the one used for deriving the HEC. As with the HEC, 

however, this process applies only to toxicokinetic aspects of cross-species extrapolation and 

does not address toxicodynamic differences that may exist between species. As with the HEC, 

consideration of toxicodynamics is proposed to be through application of a portion of the animalto-human extrapolation (100.5, which is typically rounded to 3). Table 4-3 shows the general 

magnitude of the DAFs that would be applied to various species to obtain the HED along with 

the default UF of 3 to cover toxicodynamic differences. 

Table 4-3. DAFs based on BW3/4 for various species 

Species Weight (kg) DAFa 

7 

4 

3 

Rabbit 2.5 2 

Human 70 1 

Mouse 0.03 

Rat 0.25 

Guinea pig 0.5 

a

 Derived on the basis of BW3/4 relationship. All variables in BW3/4 relationship containing time will scale BW-1/4, 

such that animal BW-1/4

/human BW-1/4 = DAF. 

The Technical Panel encourages consideration of cross-species extrapolation procedures 

for oral and dermal reference values, including evaluation of the most current scientific thought 

and data to address, as needed, issues of variability due to life stage and other intrinsic factors. 

This consideration would include examination of the extent to which calculation of a HED 

addresses cross-species toxicokinetics and identification and parallel investigation into issues of 

toxicodynamics. 

4-36
4.4.4. Other Issues 

The Technical Panel considered several other issues related to the application of a factor 

(data-derived or default) to the BMDL, the BMCL, the NOAEL, or the LOAEL selected as the 

POD from data considered adequate for risk assessment. In particular, there was controversy 

about the application of such a factor on the basis of the level of response at the BMD, the BMC, 

the NOAEL, or the LOAEL. For example, the use of a quantitative dose-response modeling 

approach results in the calculation of a BMD or a BMC, which is based on a particular level of 

response, that is, the BMR. The BMR is usually selected to be at the low end of the observable 

range of the data, which is dependent on the power of the study to detect changes from control 

values. The limit of sensitivity for most long-term bioassays is in the range of 10%, as 

determined from both the typical number of animals used in bioassays (~50/group) and a low 

spontaneous background rate (e.g., 0.1%) for a given effect (Haseman, 1984; Haseman et al., 

1989). 

For other types of studies, however, the limit of sensitivity may be lower or higher than 

10%. For example, in an analysis of a large number of standard prenatal developmental toxicity 

studies with an average sample size of 15–20 litters, the limit of sensitivity averaged 5% for the 

proportion of pups affected per litter, whereas when the quantal endpoint (i.e., the number of 

litters affected) was analyzed in dams from the same studies, the limit of sensitivity averaged 

30% (Allen et al., 1994). For data from some human studies (e.g., high-quality, large 

epidemiology studies), the limit of sensitivity may be in the range of 1 to 5%. 

In the BMD guidance document (U.S. EPA, 2000c), the BMDL or BMCL is 

recommended for the POD in order to ensure that a majority of the population is below the 

selected BMR. However, a concern has been raised that a BMD or BMC based on a response 

rate of >10% may not be appropriate to use in deriving an exposure to the human population 

(including sensitive or susceptible subgroups) that is likely to be without appreciable risk of 

deleterious or adverse effects (from current and proposed reference value definitions [Boxes 4-1 

and 4-2]) without application of a factor to extrapolate to a lower dose/exposure level considered 

to reflect a more appropriate level of risk (e.g., <10%). 

Similarly, the NOAEL is not necessarily a no-effect level, and it depends on the study 

design, including sample size, background rate, and response variability, which can be used to 

determine the limit of detection for a particular study. Thus, a NOAEL may be equivalent to no 

response or it may actually represent a substantial response rate. Previously, there has been no 

attempt to apply a factor to the NOAEL on the basis of power calculations, sample size, or 

response variability for deriving a POD, although professional judgment is recommended in 

deciding whether the study is acceptable for use in deriving a POD. 

4-37
Adjustment for the steepness of the 

dose-response curve has been noted as 

another critical aspect of the dose-response 

character that is not currently considered in 

the choice of a response level using either a 

BMD/BMC or a NOAEL approach. 

The Technical Panel was unable to 

fully evaluate these issues or to reach 

agreement about any recommendation for 

change to the current methodology, and it 

recommends that they be considered further 

by the Agency. The Technical Panel also 

recommends that factors such as the 

response rates at the BMD or the NOAEL, 

the power of the study, and the slope of the 

dose-response curve be included in the 

description of the database, where possible, 

as part of risk characterization. 

4.4.5. Application of 

Uncertainty/Variability Factors 

Box 4-4. Variability and Uncertainty 

Variability refers to true heterogeneity or diversity. For 

example, among a population that drinks water from the same 

source and with the same contaminant concentration, the risks 

from consuming the water may vary. This may be due to 

differences in exposure (i.e., different people drinking 

different amounts of water and having different body weights, 

different exposure frequencies, and different exposure 

durations) as well as differences in response (e.g., genetic 

differences in resistance to a chemical dose). Those inherent 

differences are referred to as variability. Differences among 

individuals in a population are referred to as inter-individual 

variability, while differences for one individual over time is 

referred to as intra-individual variability. 

Uncertainty occurs because of a lack of knowledge. It is not 

the same as variability. For example, a risk assessor may be 

very certain that different people drink different amounts of 

water but may be uncertain about how much variability there 

is in water intakes within the population. Uncertainty can 

often be reduced by collecting more and better data, while 

variability is an inherent property of the population being 

evaluated. Variability can be better characterized with more 

data, but it cannot be reduced or eliminated. Efforts to clearly 

distinguish between variability and uncertainty are important 

for both risk assessment and risk characterization. 

Source: U.S. EPA, 1997b. 

Reference values are derived in a way that attempts to account for both the uncertainty 

and the variability in the data available (see Box 4-4). The existing definition of UF in the IRIS 

glossary mixes the above concepts. The present definition for UF is as follows. 

Uncertainty Factor: One of several, generally 10-fold, factors used in operationally 

deriving the RfD and RfC from experimental data. UFs are intended to account for (1) 

the variation in sensitivity among the members of the human population (i.e., interhuman 

or intraspecies variability); (2) the uncertainty in extrapolating animal data to humans 

(i.e., interspecies variability); (3) the uncertainty in extrapolating from data obtained in a 

study with less-than-lifetime exposure to lifetime exposure (i.e., extrapolating from 

subchronic to chronic exposure); (4) the uncertainty in extrapolating from a LOAEL 

rather than from a NOAEL; and (5) the uncertainty associated with extrapolation from 

animal data when the database is incomplete. 

4-38
Following the logic above, the LOAEL-to-NOAEL extrapolation, the subchronic-tochronic extrapolation, and the database deficiency factors are UFs. The variation in 

susceptibility among members of the human population is a variability factor. When a default 

factor is used for intrahuman variability, however, this factor also contains some degree of 

uncertainty, because the range of uncertainty is not really known, although it is presumed to be 

no more than 10-fold. Rather than adding a new definition of variability factor, we propose to 

modify the wording of the UF definition as follows. 

Uncertainty/Variability Factor: One of several, generally 10-fold, default factors used 

in operationally deriving the RfD and the RfC from experimental data. The factors are 

intended to account for (1) the variation in sensitivity among the members of the human 

population (i.e., inter-individual variability); (2) the uncertainty in extrapolating animal 

data to humans (i.e., interspecies uncertainty); (3) the uncertainty in extrapolating from 

data obtained in a study with less-than-lifetime exposure to lifetime exposure (i.e., 

extrapolating from subchronic to chronic exposure); (4) the uncertainty in extrapolating 

from a LOAEL rather than from a NOAEL; and (5) the uncertainty associated with 

extrapolation when the database is incomplete. 

In setting pesticide tolerances, the FQPA directs EPA to use an additional 10-fold margin 

of safety to protect infants and children, taking into account the potential for pre- and postnatal 

toxicity and the completeness of the toxicology and exposure databases. The statute authorizes 

EPA to replace this additional 10X factor with a factor of a different value (higher or lower, 

including 1) only if, on the basis of reliable data, the resulting level of exposure would be safe 

for infants and children. The Agency use of this FQPA safety factor has been discussed in 

several documents (U.S. EPA, 1999b, c, 2002b). 

The Agency has concluded that in many cases, concerns regarding pre- and postnatal 

toxicity can be addressed by calculating an RfD or an MOE using pre- or postnatal 

developmental endpoints and applying traditional UFs to account for deficiencies in the toxicity 

data (U.S. EPA, 2002b). These traditional UFs include extrapolation from the LOAEL when a 

NOAEL is not available, extrapolation from a subchronic study to a chronic-exposure scenario 

when no chronic study data are available, and application of a database UF when there are gaps 

in the data considered essential for setting a reference value, including lack of data on children. 

In addition to considering these FQPA-relevant areas of uncertainty, which are addressed 

in the development of an RfD/RfC, OPP assessments of pesticide risk to children also consider 

applying part or all of the FQPA factor in certain situations to account for areas of residual 

4-39
uncertainty that the traditional UFs do not address or for which they are believed to be 

insufficient. These areas of residual uncertainty include exposure uncertainties and high concern 

for an observed susceptibility. This risk management approach is consistent with procedures 

used in the past for managing potential risks, although the FQPA has brought a significant new 

focus on improving the process of risk assessment relative to children’s health risks from 

environmental exposures. 

In considering the robustness of the RfC/RfD methodology and its adequacy for 

assessing hazards to infants and children, the Technical Panel also recognized the overlap of 

areas covered by the FQPA factor and those addressed by the traditional UFs. For example, the 

database UF may be invoked where data are unavailable or are insufficient to explicitly consider 

the potential sensitivity of the developing organism. The Technical Panel agrees with the 10X 

Task Force draft Toxicology Working Group report (U.S. EPA, 1999b) that the current 

interspecies, intraspecies, LOAEL-to-NOAEL, subchronic-to-chronic, and database-deficiency 

UFs, if appropriately applied using the approaches recommended in this review, will be adequate 

in most cases to cover concerns and uncertainties regarding the potential for pre- and postnatal 

toxicity and the completeness of the toxicology database. In other words, an additional UF is not 

needed in the RfC/RfD methodology because the currently available factors are considered 

sufficient to account for uncertainties in the database from which the reference values are 

derived (and it does not exclude the possibility that these UFs may be decreased or increased 

from the default value of 10). 

Guidance is needed on the use of developmental toxicity data in all reference values, 

including the appropriate application of UFs, because of the assumption that a single exposure 

during development may produce an effect (U.S. EPA, 1991) and the concomitant recognition 

that multiple exposures may result in effects at lower doses in many cases or cause tolerance in 

other cases. These issues are chemical specific, and scientific judgement about when and how to 

apply UFs must include consideration of toxicokinetics/metabolism as well as the mode of action 

for each agent. 

4.4.5.1. Recommendations for Application of UFs 

The exact value of the UFs chosen should depend on the quality of the studies available, 

the extent of the database, and scientific judgment. It is imperative that the IRIS documentation 

contain a justification for the individual UFs selected for a particular agent. The default factors 

typically used cover a single order of magnitude (i.e., 101

). By convention, in the Agency, a 

value of 3 is used in place of one-half power (i.e., 100.5) when appropriate. The Technical Panel 

recommends that these half-power values be factored as whole numbers when they occur singly 

4-40
but as powers or logs when they occur in tandem. A composite UF of 3 and 10 would be 

expressed as 30 (3 ×101

), whereas a composite UF of 3 and 3 would be expressed as 10 (100.5 

×100.5 = 101). It should be noted, in addition, that rigid application of log or 1⁄2 log units for UFs 

could lead to an illogical set of reference values; therefore, the Technical Panel emphasizes that 

application of scientific judgment is critical to the overall process. 

It is imperative that the IRIS documentation contain a justification for the individual 

factors selected for each chemical or assessment and for each duration reference value. 

Although default factors of 10 are recommended, with 3 used in place of half-power values (i.e., 

100.5) when occurring singly, the exact value of the UF chosen should depend on the quality of 

the studies available, the extent of the database, and scientific judgment. Sound scientific 

judgment should be used in the application of UFs to derive reference values that are applied to 

the value chosen for the POD derived from the available database (BMDL, NOAEL, or 

LOAEL). 

The Technical Panel recognizes that there is overlap in the individual UFs and believes 

that the application of five UFs of 10 for the chronic reference value (yielding a total UF of 

100,000) is inappropriate. In fact, in cases where maximum uncertainty exists in all five areas, it 

is unlikely that the database is sufficient to derive a reference value. Uncertainty in four areas 

may also indicate that the database is insufficient to derive a reference value. In the case of the 

RfC, the maximum UF would be 3000, whereas the maximum would be 10,000 for the RfD. 

This is because the derivation of RfCs and RfDs have evolved somewhat differently. The RfC 

methodology (U.S. EPA, 1994) recommends dividing the interspecies UF in half, one-half (100.5) 

each for toxicokinetic and toxicodynamic considerations, and it includes a DAF to account for 

toxicokinetic differences in calculating the HEC, thus reducing the interspecies UF to 3 for 

toxicodynamic issues. RfDs, however, do not incorporate a DAF for deriving a HED, and the 

interspecies UF of 10 is typically applied. 

The Technical Panel recommends limiting the total UF applied for any particular 

chemical to no more than 3000 and avoiding the derivation of a reference value that involves 

application of the full 10-fold UF in four or more areas of extrapolation. This maximum of 3000 

applies only to the UFs discussed in the following sections and does not include the various 

adjustment factors that have been discussed previously (Sections 4.4.2. and 4.4.3.). Similar 

concerns would need to be considered for the less-than-lifetime reference values, taking into 

account those UFs that are appropriate for each duration reference value. 

4-41
4.4.5.2. Interspecies UF 

The interspecies UF is applied to account for the extrapolation of laboratory animal data 

to humans, and it generally is presumed to include both toxicokinetic and toxicodynamic aspects. 

The toxicokinetic aspects of this factor were addressed in the section on deriving HEDs and 

HECs (Section 4.4.3). This UF is intended also to account for differences in species sensitivity 

(i.e., toxicodynamics) between the laboratory animal species used for testing and humans. 

Seldom are there data available to inform toxicodynamic differences. One-half the default 10-

fold interspecies UF (i.e., 100.5) is assumed to account for such differences, but more specific 

data should be used when available (see discussion of chemical-specific adjustment factors, 

Section 4.4.6.1 below), and the flexibility for applying a factor greater than 10 should be 

recognized. Unless data support the conclusion that the test species is more or equally as 

susceptible to the pollutant as are humans, and in the absence of any other specific toxicokinetic 

or toxicodynamic data, a default factor of 3 (in conjunction with HEC derivation) or 10 is 

applied. 

4.4.5.3. Intraspecies UF 

The intraspecies UF is applied to account for variations in susceptibility within the 

human population (interhuman variability) and the possibility (given a lack of relevant data) that 

the database available is not representative of the dose/exposure-response relationship in the 

subgroups of the human population that are most sensitive to the health hazards of the chemical 

being assessed. As the reference concentration/dose is defined to be applicable to “susceptible 

subgroups,” this UF was established to account for uncertainty in that regard. In general, the 

Technical Panel reaffirms the importance of this UF, recommending that reduction of the 

intraspecies UF from a default of 10 be considered only if data are sufficiently representative of 

the exposure/dose-response data for the most susceptible subpopulation(s). 

Various authors who have evaluated the intraspecies UF using data from animal or 

human studies (as summarized by Dourson et al. [1996]) have concluded that the 10-fold default 

factor appears to be protective when starting from a median response—by inference a NOAEL 

assumed to be from an average group of humans. Renwick and Lazarus (1998) considered data 

on toxicokinetics and toxicodynamics to support the idea that the 10-fold intraspecies factor can 

be divided into two factors to account for kinetics and dynamics. When they evaluated the 

composite 10-fold factor to account for variability in both kinetics and dynamics, they concluded 

that a 10-fold factor would cover the vast majority (>99%) of the population. These evaluations, 

however, did not specifically consider children as part of the range of human variability when 

evaluating the adequacy of the intraspecies UF. 

4-42
In papers that have evaluated this factor for the general population as well as for specific 

subpopulations, including children (Renwick and Lazarus,1998; Renwick, 1998) and the elderly 

(Abdel-Mageed et al., 2001), the 10-fold intraspecies factor appears to be sufficient in most 

cases, and chemical-specific factors often indicate a requirement for less than a l0-fold factor. 

Renwick (1998) indicated that the 10-fold factor is more likely to be sufficient if developmental 

toxicity data are available on the specific agent. Calabrese (2001) reviewed the data available on 

a number of chemical classes and concluded that the young are often more susceptible than 

adults but that there is a not-infrequent occurrence of greater susceptibility in adults. The 

sometimes greater sensitivity among the elderly than among mature adults appears to be related 

primarily to reduced renal clearance (Abdel-Mageed et al., 2001; Skowronski and AbdelRahman, 2001). 

The Technical Panel urges continued research and evaluation of the similarities and 

differences between the general population and susceptible subpopulations—particularly 

children and the elderly—in their responses to specific agents. From such evaluations, the 

protectiveness of the 10-fold default factor can continue to be assessed. 

The cases on IRIS in which the intraspecies UF has been reduced from the default of 10-

fold have been documented by Dourson et al. (1996). These include 2/46 RfCs and 13/346 RfDs 

(overall frequency 3.6%). In those cases where developmental effects were the most sensitive 

endpoint (0 RfCs, 6 RfDs), reduction of the intraspecies UF from 10 to 3 was based on data 

derived either from human data showing which age groups or time periods were most susceptible 

(e.g., methyl mercury exposure to the developing fetus) or from an animal study with support 

from strong human or other data (e.g., Aroclor 1016 in utero exposure in monkeys, strontiuminduced rachitic bones in young rats). In three cases the intraspecies UF was reduced to 1, based 

on very specific data about the particular vulnerability of infants and children within certain age 

ranges to an agent (e.g., nitrate, nitrite, fluorine/soluble fluoride). However, even within these 

populations it is possible that some variability exists, based on genetics, lifestyle, or other 

factors. 

In cases where the susceptible subpopulation is quite specifically defined (e.g., through 

knowledge of the chemical’s mode of action) so that the resultant RfC is truly applicable to the 

susceptible subpopulation (although not necessarily to hypersensitive individuals), reduction of 

the intraspecies UF is warranted. Thus, the Technical Panel supports and expands the 

recommendation of the Toxicology Working Group of the 10X Task Force (U.S. EPA, 1999b) 

that reduction of the intraspecies UF from a default of 10 be considered only if data are sufficient 

to support the conclusion that the data set on which the POD is based is representative of the 

exposure/dose-response data for the susceptible subpopulation(s). Given this, whether and how 

4-43
much the intraspecies UF may be reduced must be linked to how completely the susceptible 

subpopulation has been identified and their sensitivity described (vs. assumed). At the other 

extreme, a 10-fold factor may sometimes be too small because of factors that can influence large 

differences in susceptibility, such as genetic polymorphisms. The Technical Panel urges the 

development of data to support the selection of the appropriate size of this factor, but recognizes 

that often there are insufficient data to support a factor other than the default. 

4.4.5.4. LOAEL-to-NOAEL UF 

A UF (default 10) is typically applied to the LOAEL when a NOAEL is not available. 

The size of the LOAEL-to-NOAEL UF may be altered, depending on the magnitude and nature 

of the response at the LOAEL. It is important to consider the slope of the dose-response curve in 

the range of the LOAEL in making the determination to reduce the size of the LOAEL-toNOAEL UF. Several papers have described the magnitude of the difference between the dose at 

the LOAEL and at the NOAEL. For example, Lewis et al. (1990) and Faustman et al. (1994) 

showed that the ratio of the LOAEL-to-NOAEL in many cases was approximately threefold, but 

in a few cases the difference was as much as 10-fold. 

In general, the ratio of the doses at the LOAEL and the NOAEL is likely to vary 

considerably among studies and may not be informative. This is because the lowest dose in a 

study is often selected to ensure that no statistically significant response above control is 

observed and the next higher dose is selected to ensure that some significant response is 

observed, rather than selecting doses that will give a maximum NOAEL and a minimum 

LOAEL. Data should be carefully evaluated, taking into consideration the level of response at 

the LOAEL and the NOAEL and the slope of the dose-response curve before reducing the size of 

the UF applied to the LOAEL. 

4.4.5.5. Database UF 

The database UF is intended to account for the potential for deriving an underprotective 

RfD/RfC as a result of an incomplete characterization of the chemical’s toxicity. In addition to 

identifying toxicity information that is lacking, review of existing data may also suggest that a 

lower reference value might result if additional data were available. Consequently, in deciding 

to apply this factor to account for deficiencies in the available data set and in identifying its 

magnitude, the assessor should consider both the data lacking and the data available for 

particular organ systems as well as life stages. 

In many respects, the additional 10-fold factor for infants recommended by the National 

Research Council (NRC, 1993) and by Schilter et al. (1996) and called for in the 1996 FQPA is 

4-44
similar to the database UF. If the RfD/RfC is based on animal data, a factor of 3 is often applied 

if either a prenatal toxicity study or a two-generation reproduction study is missing, or a factor of 

10 may be applied if both are missing (Dourson et al., 1996). Dourson et al. (1992) examined 

the use of the database UF by analyzing ratios of NOAELs for chronic dog, rat, and mouse 

studies and reproductive and developmental toxicity studies in rats. They concluded that 

reproductive and developmental toxicity studies provide useful information for establishing the 

lowest NOAEL, and if one or more bioassays are missing, a factor should be used to address this 

scientific uncertainty in deriving a chronic RfD. 

If data from the available toxicology studies raise suspicions of developmental toxicity 

and signal the need for developmental data on specific organ systems (e.g., detailed nervous 

system, immune system, carcinogenesis, or endocrine system), then the database factor should 

take into account whether or not these data are available and used in the assessment and their 

potential to affect the POD for the particular duration RfD or RfC under development. 

If the RfD/RfC is based on human data, a similar assessment regarding the completeness 

of the database is necessary. Information on life stages and organ systems may come from either 

animal or human studies. If data on specific life stages or organ systems are unavailable or 

limited data suggest that availability of more extensive data might decrease the POD, this should 

be taken into account in assigning a database UF. For example, depending on the database and 

what is known about the chemical, the lack of a two-generation animal reproductive toxicity 

study might be considered a deficiency even if the reference value is based on human data. In 

any case, the size of the database factor to be applied will depend on other information in the 

database and on how much impact the missing data may have on determining the toxicity of a 

chemical and, consequently, the POD. 

4.4.5.6. Subchronic-to-Chronic-Duration UF 

As indicated earlier, a duration adjustment currently in use is the application of a UF 

when only a subchronic duration study is available to develop a chronic reference value such as 

the RfC or the RfD (U.S. EPA, 1994). A default value of 10 for this UF is applied to the 

NOAEL/LOAEL or BMDL/BMCL from the subchronic study on the assumption that effects 

from a given compound in a subchronic study occur at a 10-fold higher concentration than in a 

corresponding (but absent) chronic study. This factor would be applied subsequent to the 

adjustment of the exposures from intermittent to continuous, as above. 

The specific use of a UF applied to a subchronic study in the derivation of a chronic 

reference value is reasonable. Some work has been published on this aspect of extrapolation 

(Lewis et al., 1990; Pieters et al., 1998). Guidance for replacement of the default factor of 10 by 

4-45
CSAFs may be forthcoming. It would be appropriate to incorporate such data into applicable 

assessments. In the current practice, this factor is applied when a chronic reference value is 

derived from a database in which the critical study is of subchronic duration. No chronic 

reference value is derived if neither a subchronic nor chronic study is available. The application 

of a UF to less-than-subchronic studies is not part of the current practice, but further exploration 

of this issue may be appropriate. For short-term and longer-term reference values, the 

application of a UF analogous to the subchronic-to-chronic duration UF also needs to be 

explored, as there may be situations in which data are available and applicable but they are from 

studies in which the dosing period is considerably shorter than that for the reference value being 

derived. 

4.4.5.7. Modifying Factor (MF) 

A clear definition of intended usage for an MF is lacking. The only comments located 

about the MF are in the RfC methodology (U.S. EPA, 1994), and they indicate that the MF is 

intended to account for scientific uncertainties in the study or database that are not explicitly 

treated by other UFs. It is further stated that use of the factor depends principally on 

professional judgment and assessment. Some example applications are also given, such as 

accounting for small sample size or for poor exposure characterization in the principal study. 

The definition in the IRIS glossary gives similar examples. 

The description of the database UF shows substantial similarity to that of the MF. Text 

on the database UF indicates that this factor attempts to recognize that without a comprehensive 

array of endpoints there is uncertainty as to whether all possible toxicologic endpoints at the 

various life stages are adequately addressed. Without this information, uncertainty remains as to 

whether the critical effect chosen for RfD or RfC derivation is either the most sensitive or the 

most appropriate. There are only seven cases in IRIS for which an MF has been applied: RfDs 

for chromium III, chromium VI, nitrite, 1,1-biphenyl, and manganese and RfCs for methyl ethyl 

ketone and acetonitrile. The rationale for these varies considerably but in all cases appeared to 

be for reasons that could be considered under other UFs. 

Recent developments in the IRIS process include the obligation for risk characterization 

within the assessments. A central aspect of risk characterization includes discussing confidence 

and uncertainties in the quality of data used and the “clarity, transparency, consistency and 

reasonableness” of the assessment (U.S. EPA, 2000b). Within the risk characterization, the 

assessor has a pathway provided to discuss and analyze all aspects of uncertainty about the 

database, including the adequacy or limitations of the database, directly in the assessment. 

4-46
The Panel considers the purpose of the MF to be sufficiently subsumed in the general 

database UF. The Panel also notes that the risk characterization section of assessments may be 

used to provide a full and complete characterization of all uncertainty, including any residual 

uncertainty that may not be addressed by the other UFs. In view of these factors, the Panel 

recommends that use of the MF be discontinued. 

4.4.6. Future Directions

4.4.6.1. Chemical-Specific Adjustment Factors (CSAFs)

There is growing support for the use of CSAFs in place of DAFs (see Section 4.4.3.), and 

this will provide an incentive to fill existing data gaps (Murray and Andersen, 2001; Meek, 

2001; Meek et al., 2001; Bogdanffy et al., 2001). Additional chemical-specific data permit the 

replacement of components of interspecies or inter-individual variation with data-derived values 

in the context of the traditional default framework as developed by Renwick (1993) and revised 

by IPCS (1994). The following is a brief discussion of available methodologies that promote the 

use of CSAFs in risk assessment. 

Renwick (1993) described the use of toxicokinetic and toxicodynamic data as a means of 

replacing the traditional 10-fold safety factors for human sensitivity and experimental animal-tohuman extrapolation in developing acceptable daily intakes. His data-derived approach assigns 

default values for both toxicokinetic and toxicodynamic differences within each traditional 10-

fold safety factor. Specifically, Renwick proposed dividing both the interspecies and the interindividual UFs into a factor of 2.5 for toxicodynamics and a factor of 4.0 for toxicokinetics. 

IPCS (1994) has adopted the data-derived approach initially proposed by Renwick (1993), with a 

slight modification in the UF for inter-individual variation (3.16 for toxicodynamics and 3.16 for 

toxicokinetics). IPCS has used this approach in several of its recent risk assessments (e.g., IPCS, 

1998), and EPA is proposing a similar approach for boron (U.S. EPA, 2001b). 

IPCS has developed a draft guidance document (IPCS, 2001) to assist risk assessors in 

the use of experimental data in deriving CSAFs for interspecies differences and human 

variability in dose/concentration response assessment. CSAFs have been adopted because they 

better describe the nature of the refinement to the usual default approach. 

For several years, EPA used a more qualitative approach to modify the usual 10-fold 

default values (Dourson et al., 1996). Recently, it has used a data-derived approach as one of the 

methods to derive a UF for boron (U.S. EPA, 2001b). 

EPA has not yet established guidance for the use of chemical-specific data for deriving 

UFs, but the division of UFs into toxicodynamic and toxicokinetic components is in the RfC 

methodology (U.S. EPA, 1994). EPA’s assessments of data assume a division of both 

4-47
interspecies and intraspecies UFs into toxicokinetic and toxicodynamic components that have 

assigned default values of 3.16 (100.5 ) each. The Agency will develop its own guidance for the 

use of CSAFs in risk assessment, based on some of the available methodologies (e.g., IPCS). 

The Technical Panel would like to caution the user that for many substances there are 

relatively few data available to serve as an adequate basis to replace defaults for interspecies 

differences and human variability with more informative CSAFs. Currently, relevant data for 

consideration are often restricted to the component of uncertainty related to interspecies 

differences in toxicokinetics. Although there are fewer relevant data with which to address the 

other four components namely—interspecies (animal-to-human) differences in toxicodynamics, 

intraspecies (human) variability in toxicokinetics, intraspecies (human) variability in 

toxicodynamics, and adequacy of the database—it is anticipated that availability of such 

information will be needed to apply CSAFs. Specifically, the data-derived CSAF approach for 

any single substance is necessarily determined principally by the availability of relevant data. 

The extent of data available is, in turn, often a function of the economic importance of the 

substance, and this is frequently related to the extent of potential human exposure. 

4.4.6.2. Probabilistic Approaches 

Another approach to quantifying uncertainty in RfD or RfC derivation when data are not 

sufficient to develop a chemical-specific or biologically based dose-response model is 

probabilistic analysis. When the available data are sufficient to meaningfully characterize the 

distributions of interest, a probabilistic approach would provide results as a distribution rather 

than as a single measure for the dose/concentration-response. For example, distributions could 

be used for inputs into a toxicokinetic model to derive a distribution of internal dose metrics. 

Also, the approaches described in the draft IPCS guidance document (IPCS, 2001) are amenable 

to probabilistic analysis. 

Probabilistic analysis for human health assessments generally has been confined to the 

exposure variables. In deriving human health toxicity reference values, inter-individual 

variability in toxicokinetics and toxicodynamics is usually represented with a UF because data 

are insufficient to support a more quantitative representation of these sources of inter-individual 

variability. Several studies have been published addressing the use of probabilistic data for 

health assessments (Baird et al., 1996; Maull et al., 1997; Slob and Pieters, 1998; Swartout et al., 

1998; Brand et al., 1999; Gaylor and Kodell, 2000; Evans et al., 2001). The Technical Panel 

recommends that the Agency further evaluate approaches such as probabilistic analysis for 

characterizing variability and uncertainty in toxicity reference values. 

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4.4.7. Summary of Key Points from the Case Studies 

Two case studies were developed to illustrate many of the recommendations in this 

report. The studies are for two hypothetical chemicals: Inhalate, a synthetic halogenated 

aliphatic alkene, and Luteinate, a new pesticide that acts via the neuroendocrine system. 

The available database on Inhalate was considered adequate for deriving inhalation 

reference values for all four durations of exposure (acute, short-term, longer-term, and chronic). 

Very little is known about the mode of action for Inhalate except for the tumorigenic effects in 

liver, which are thought to be produced as a result of prolonged cytotoxicity caused by oxidative 

metabolism. Thus, a nonlinear mode of action is assumed for Inhalate carcinogenesis, and a 

chronic reference value is derived that takes into account these effects along with others seen 

after chronic exposure. Acute, short-term, longer-term, and chronic reference values were 

derived for Inhalate. This case study illustrates the use of a variety of types of data from toxicity 

testing studies in deriving a set of inhalation reference values, including carcinogenic effects 

assumed to have a nonlinear dose-response. 

Luteinate belongs to a class of chemicals known to work through a neuroendocrine mode 

of action. In order to ascertain its potency and confirm a similar mode of action, a number of 

short-term studies were conducted, followed by testing in more traditional toxicology studies to 

establish its long-term effects and dose response relationships. The data were considered 

adequate to derive oral reference values for all four durations of exposure. This case study 

provides an example of the usefulness of mode-of-action information in establishing the shortand long-term effects of Luteinate on relevant target organ systems at different life stages. Such 

information enables the development of a targeted robust data set for use in establishing 

reference values for various durations of exposure. 

A detailed summary of the case studies is provided in Appendix B. Several key points 

are described here that demonstrate the use of the proposed framework outlined in this chapter. 

First, the data are reviewed and characterized on the basis of the hazard and dose-response 

information, including consideration of the weight-of-evidence factors discussed in Section 

4.3.2, above. A narrative statement is used to describe the extent of the database for each 

chemical as well as the gaps in information that would make the database more robust. 

Dosimetric adjustments were made to derive HECs in the case of Inhalate. For Luteinate, 

adjustments for oral exposure were made on a BW1

 basis and do not incorporate the BW3/4 

scaling factor or other DAF, as further work is needed on the harmonization of approaches for 

deriving of oral and dermal HEDs. 

The data are presented both in tabular form and in graphical form as an exposure 

response array to provide a visualization of the data applicable to each duration of exposure. 

4-49
Then, the reference values are derived by considering all of the relevant data for each duration 

reference value, weighing the evidence in the database, developing sample values on the basis of 

various endpoints considered for each duration, and selecting a final reference value for each 

duration on the basis of an evaluation of each of the relevant endpoints rather than on a single 

critical study and critical effect. 

The approaches illustrated by the case studies showing derivation of multiple-duration 

reference values are not without precedent. Several offices within EPA, as well as ATSDR and 

the AEGL committee, derive multiple duration values for various purposes (see review in 

Chapter 2). The derivation of sample reference values in selecting the final reference value also 

is not a new idea. For example, EPA’s assessment for methylmercury included the derivation of 

sample RfDs from prospective longitudinal studies of the effects of in utero exposure to 

methylmercury (Table 4-4) in deriving a chronic RfD (U.S. EPA, 2001d). 

Sample RfDs were derived from a number of neuropsychological endpoints from two 

studies in which an association was observed (New Zealand and the Faroe Islands) as well as an 

integrative analysis of those studies plus a study in the Seychelles Islands in which no 

association between in utero methylmercury exposure and deficits in neuropsychological 

function were reported. The sample RfDs converged on 0.1 :g/kg/day, providing strong support 

for the appropriateness of this value. This RfD is not a developmental RfD per se, and its use is 

not restricted to pregnancy or developmental periods. The RfD, derived from an overall 

evaluation of the database, is applicable to lifetime daily exposure for all populations, including 

sensitive subgroups. 

In a recently released health assessment document on 1,3-butadiene (U.S. EPA, 2002d), 

sample RfCs were derived for determining the chronic RfC. 

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Table 4-4. BMDLs, ingested dose, and RfDs for various endpoints from 

the Faroe Islands, New Zealand, and the NRC integrative analysis 

Testb 

BNT Faroes 

Whole cohort 

PCB adjustedd 

Lowest PCB tertile 

CPT Faroes 

Whole cohort 

PCB adjusted 

Lowest PCB tertile 

CVLT Faroes 

Whole cohort 

PCB adjusted 

Lowest PCB tertile 

Finger Tap Faroes 

Whole cohort 

PCB adjusted 

Lowest PCB tertile 

Geometric mean 

Whole cohort 

PCB adjusted 

Lowest PCB tertile 

Median values 

Faroes 

New Zealand 

Smoothed values 

BNT Faroes 

CPT Faroes 

CVLT Faroes 

Finger Tap Faroes 

MCCPP New Zealand 

MCMT New Zealand 

Integrative 

All endpoints 

BMDLa 

(ppb mercury cord blood) 

58 

71 

40 

46 

49 

28 

103 

78 

52 

79 

66 

24 

68 

65 

34 

48 

24 

48 

48 

60 

52 

28 

32 

32 

Ingested doseb 

(:g/kg/day) 

1.081 

1.323 

0.745 

0.857 

0.913 

0.522 

1.920 

1.454 

0.969 

1.472 

1.230 

0.447 

1.268 

1.212 

0.634 

0.895 

0.447 

0.895 

0.895 

1.118 

0.969 

0.522 

0.596 

0.596 

RfDc 

(:g/kg/day) 

0.1 

0.1 

0.1 

0.1 

0.1 

0.05 

0.2 

0.1 

0.1 

0.1 

0.1 

0.05 

0.1 

0.1 

0.1 

0.1 

0.05 

0.1 

0.1 

0.1 

0.1 

0.05 

0.1 

0.1 

a

 BMDL05s from NRC (2000), Tables 7-4, 7-5, 7-6. Hair mercury was converted to blood mercury using a 250:1 

ratio and an assumption of equivalent maternal and cord levels. b

 Calculated using a one-compartment model. c

 Calculated using an UF of 10. 

d

 There was significant co-exposure to PCBs in the Faroe Islands study, with PCB cord tissue concentrations 

available for about half of the whole cohort. Analyses were performed adjusted for PCBs (half the cohort) as 

well as unadjusted for PCBs in those individuals in the lowest PCB tertile (i.e., one-sixth of the whole cohort). 

BNT = Boston Naming Test; CPT = Continuous Performance Test; CVLT = California Verbal Learning Test; 

MCCPP = McCarthy Perceived Performance; MCMT = McCarthy Motor Test. 

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5. RECOMMENDATIONS 

A number of recommendations have been made in other parts of this report. This chapter 

summarizes those recommendations, based on the Technical Panel’s review of the RfD and RfC 

process. The Technical Panel assumes that it will be possible to implement some of the 

recommendations in the near future, given adequate resources and personnel, whereas others will 

require additional effort. In particular, testing strategies are needed that consider toxicokinetic 

and mode of action information early in the process, as well as when to implement new testing 

guidelines in the process of developing a data package on a particular chemical. OPPTS, 

together with scientists in other parts of the Agency, will consider the recommendations to 

develop additional or alternative testing guidelines as part of the Harmonized Health Effects Test 

Guidelines (870 Series). 

As part of its deliberations, the Technical Panel considered the recommendations of the 

Toxicology Working Group of the 10X Task Force (U.S. EPA, 1999b, and Appendix A). The 

Technical Panel endorses those recommendations and extends and expands them to deal with a 

broader view of life stages, timing and duration of exposure, and evaluation of endpoints, both 

structural and functional. The recommendations are presented here in the order of the chapters 

in which they appear. Further discussion of the specific recommendations can be found in the 

earlier chapters. 

Chapter 2 

The Technical Panel concurred with the recommendation of the 10X Task Force that 

reference values should be derived, where possible, for acute, short-term, and longer-term as 

well as chronic exposures for oral, dermal, and inhalation routes and that they be included in the 

IRIS database for use by EPA programs, where applicable. The definitions for duration should 

be standardized but left flexible so they can be adjusted depending on the exposure situation of 

concern. 

Chapter 3 

The Technical Panel reviewed and evaluated current testing guidelines and testing 

approaches as a follow-up to its recommendation in Chapter 2 concerning the derivation of lessthan-lifetime reference values. This review was undertaken to determine what information is 

currently gathered with regard to life stage assessment, endpoint assessment, route and duration 

of exposure, and latency to response. The intent of this review is not to suggest that additional 

5-1
testing be conducted for each and every chemical in order to fill in the information gaps 

identified for those organ systems evaluated. Nor is it suggested that the alternative testing 

protocols discussed in this chapter be conducted for every chemical or become part of current 

toxicology testing requirements or that these alternative protocols are the only options available. 

Rather, it is the goal of this document to provide a basis for the development of innovative 

alternative testing approaches and the use of such data in risk assessment. The recommendations 

include: 

• Develop a strategy for alternative approaches to toxicity testing, with guidance on 

how and when to use existing and newly recommended guidelines. 

• Develop guidelines or guideline study protocols that will provide more systematic 

information on toxicokinetics and toxicodynamics (i.e., mechanism or mode of 

action), including at different life stages. 

• Develop protocols for acute and short-term studies that provide more 

comprehensive data for setting reference values. 

• Modify existing guideline study protocols to provide more comprehensive 

coverage of life stages for both exposure and outcomes. 

• Collect more information from less-than-lifetime exposures to evaluate latency to 

effect and reversibility of effect. 

• Develop guidelines or guideline study protocols to assess immunotoxicity, 

carcinogenicity, and cardiovascular toxicity at different life stages. 

• Explore the feasibility of setting dermal reference values for direct toxicity at the 

portal of entry, including sensitization. 

Chapter 4 

The Technical Panel discussed a number of modifications to the existing framework for 

reference value derivation, both for the current chronic reference values (RfD and RfC) and for 

the acute, short-term, and longer-term reference values. In addition, two case studies that 

5-2
illustrate many of these concepts are summarized in Chapter 4 and discussed in detail in 

Appendix B. The recommendations for improvement and expansion of the existing approaches 

are aimed at taking a broader approach to the characterization of the entire database and what 

impact that will have on the dose-response assessment and risk characterization of a chemical. 

Included are recommendations for setting several less-than-lifetime reference values, broader 

characterization of the database instead of using a checklist of a minimum set of studies for 

setting a reference value, using an exposure-response array and carrying appropriate and relevant 

endpoints through the derivation of sample reference values before deciding which endpoint(s) 

to use for the POD, and deriving reference values in a way that is protective of all relevant 

endpoints rather than setting reference values on particular endpoints (e.g., the RfDDT) but using 

a process that facilitates the evaluation of risk to particular subgroups for specific program office 

needs, including cumulative risk assessment. 

The specific recommendations follow: 

1. Include the acute, short-term, longer-term, and chronic reference values derived on the 

basis of the recommendations in this report in IRIS after appropriate internal, external, 

and consensus review. 

2. Use consistent definitions for the duration of exposure in deriving acute, short-term, 

longer-term, and chronic reference values. 

3. Use the revised definition for reference values shown in Chapter 4. This definition is 

aimed at clarifying that the approach to reference values discussed here is intended for 

risk assessments for any type of health effect known or assumed to be produced through a 

nonlinear and/or threshold mode of action (which may include U-shaped or other 

nonmonotonic dose-response curves as well as thresholds). Thus, the term “noncancer” 

has been removed from the definition in the spirit of overall harmonization of risk 

assessment approaches for human health effects because it has been recommended that 

health effects no longer be categorized as “cancer” or “noncancer” for the purposes of 

hazard characterization and dose-response analysis. This change denotes the move 

toward defining approaches for low-dose estimation or extrapolation based on mode of 

action. 

5-3
The term “deleterious” has been replaced with the term “adverse,” because the latter is 

more commonly used and understood in data evaluation and selection of endpoints for 

setting reference values. The parenthetical statement in the current RfD and RfC 

definitions, “with uncertainty spanning perhaps an order of magnitude,” has been 

removed from the proposed revision of the definition for reference value, and it is 

recommended that issues of uncertainty/variability be discussed qualitatively as part of 

the weight of evidence and characterization of the database. 

4. For consistency in the designation of various duration reference values, the Panel 

recommends that the terminology for reference values be standardized; this standardized 

terminology should reflect both duration and route of exposure. Consistent terminology 

recommendations for reference values are proposed in this report, but additional 

suggestions are welcome. 

5. The Technical Panel recommends that endpoint-specific reference values per se not be 

developed, including the RfDDT, which was originally proposed in Guidelines for 

Developmental Toxicity Risk Assessment (U.S. EPA, 1991). Rather, a sample reference 

value should be calculated for each relevant and appropriate endpoint and these should 

then be considered in the derivation of various duration reference values. The reference 

values should be derived to be protective of all types of effects for a given duration of 

exposure. 

6. An expanded approach to the evaluation of studies and characterization of the extent 

of the database as a whole is recommended; in particular, several factors are discussed 

that should be considered in a weight-of-evidence approach for characterizing hazard for 

the population as a whole as well as for potentially sensitive subpopulations. Those 

considerations for assessing level of concern raised by the Toxicology Working Group of 

the 10X Task Force (U.S. EPA, 1999b) have been incorporated into this approach. 

7. A narrative approach rather than a confidence ranking of high, medium, or low should 

be used in describing the extent of the database. The extremes for the extent of the 

database (i.e., minimal or robust) are defined in Chapter 4. The narrative approach is 

intended to emphasize the types of data available (both human and animal) as well as 

research needed to fill the data gaps that could improve the derivation of reference 

5-4
values, and it should encourage the use of a wider range of information in deriving 

reference values, taking into consideration the life stages evaluated; the issues of timing, 

duration, and route of exposure; the types and extent of endpoint assessment (i.e., 

structure and function); and the potential for latent effects and/or reversibility of 

responses. 

8. Duration adjustment procedures to continuous exposures for inhalation developmental 

toxicity studies should be done in the same way as for other health endpoints. 

9. Additional consideration of the HEC and HED derivation methodology is needed to 

confirm or assess the relevance for all population subgroups (particularly children). 

10. An exposure-response array should be used as a visual display of all relevant 

endpoints and durations of exposure, as shown in the case studies. This type of array can 

be used to evaluate the range of exposure-response data for different durations of 

exposure in order to determine the range of numerical values available for each route and 

duration reference value. 

11. The POD should be selected on the basis of an evaluation of all appropriate and 

relevant endpoints carried through to sample reference value derivation, with selection of 

the limiting value(s) as the final step, rather than on a single “critical study” and “critical 

effect.” 

12. It is imperative that the IRIS documentation contain a justification for the individual 

factors selected for each chemical or assessment, because rigid application of UFs could 

lead to an illogical set of reference values. Although default factors of 10 are 

recommended, with 3 used in place of half-power values (i.e., 100.5) when occurring 

singly, the exact value of the UF chosen should depend on the quality of the studies 

available, the extent of the database, and scientific judgment. Sound scientific judgment 

should be used in the application of UFs to derive reference values that are applied to the 

value chosen for the POD derived from the available database (BMDL, NOAEL, or 

LOAEL). 

5-5
13. The Technical Panel recommends that if there is uncertainty in more than four areas 

of extrapolation, it is unlikely that the database is sufficient to derive a reference value. 

Even when there is uncertainty in four areas, the database should be carefully evaluated 

to determine whether the derivation of a reference value is appropriate. In addition, the 

Technical Panel recommends limiting the total UF applied to a chronic reference value 

for any particular chemical to 3000. This maximum of 3000 applies only to the UFs and 

does not include the various adjustment factors discussed in Chapter 4. 

14. The intraspecies UF is applied to account for variations in susceptibility within the 

human population (interhuman variability) and the possibility (given a lack of relevant 

data) that the database available is not representative of the dose/exposure-response 

relationship in the subgroups of the human population that are most sensitive to the 

health hazards of the chemical being assessed. As the reference concentration/dose is 

defined to be applicable to “susceptible subgroups,” this UF was established to account 

for uncertainty in that regard. In general, the Technical Panel reaffirms the importance of 

this UF, recommending that reduction of the intraspecies UF from a default of 10 be 

considered only if data are sufficiently representative of the exposure/dose-response data 

for the most susceptible subpopulation(s). 

At the other extreme, a 10-fold factor may sometimes be too small because of 

factors that can influence large differences in susceptibility, such as genetic 

polymorphisms. The Technical Panel urges the development of data to support the 

selection of the appropriate size of this factor, but it recognizes that often there are 

insufficient data to support a factor other than the default. 

15. The Technical Panel urges continued research and evaluation of the similarities and 

differences between the general population and sensitive subpopulations in their 

responses to particular agents, particularly children and the elderly. From such 

evaluations, the protectiveness of the 10-fold default factor can continue to be assessed. 

16. Given that several UFs can be used to deal with data deficiencies as part of the 

current reference value process, and given that these are assumed to overlap to some 

extent, the Technical Panel agrees with the 10X Task Force Toxicology Working Group 

(U.S. EPA, 1999b) that the current interspecies, intraspecies, and database deficiency 

UFs, if appropriately applied using the approaches recommended in this review, will be 

5-6
adequate in most cases to cover concerns and uncertainties about children’s health risks. 

Any residual concerns about toxicity and/or exposure can be dealt with in risk 

characterization/risk management (e.g., by retention of all or part of the FQPA safety 

factor for pesticides). 

17. The Panel considers the purpose of the MF to be sufficiently subsumed in the general 

database UF. Therefore, the Panel recommends that use of the MF be discontinued. 

18. EPA has not yet established guidance for the use of specific data to replace UFs (i.e., 

CSAFs), but the division of the interspecies UF into toxicodynamic and toxicokinetic 

components is in the RfC methodology (U.S. EPA, 1994) and may apply to the 

intraspecies UF as well. The Agency is encouraged to develop its own guidance, based 

on some of the available methodologies (e.g., IPCS). 

The following issues were discussed by the Technical Panel but were considered more 

appropriate for discussion and recommendation by other panels/committees: 

1. There have been inconsistencies in the use of BMD modeling approaches to deriving 

RfDs and RfCs currently in IRIS. The Technical Panel was unable to fully evaluate these 

issues or to reach agreement about any recommendation for change to current 

methodology and recommends that they be considered further by the Agency. The 

Technical Panel also recommends that factors such as the response rates at the BMD or 

NOAEL, the power of the study, and slope of the dose-response curve be included in the 

description of the database, where possible, as part of risk characterization. 

2. The Technical Panel recommends harmonization of the approaches for HEC and HED 

derivation for all types of health effects. Development of the appropriate adjustment 

procedure is referred to the Harmonization Framework Technical Panel. 

3. The Technical Panel recommends that the Agency further evaluate approaches such as 

probabilistic analysis for characterizing variability and uncertainty in toxicity reference 

values. 

5-7
4. The Technical Panel recommends further evaluation of appropriate adjustment of 

doses for duration of exposure. The method derived from ten Berge et al. (1986) is raised 

as a possibility for acute exposures on the basis of its recommendation in the ARE 

methodology. Duration adjustment for short-term and longer-term reference values 

analogous to the subchronic-to-chronic duration UF for chronic reference values is raised 

in the case study and should be explored further. 

5-8
APPENDIX A: ISSUES RAISED BY THE 10X TASK FORCE 

A number of issues were raised by the 10X Task Force1

 in its discussions of the 

requirements for protecting children’s health and application of an additional 10X safety factor, 

as mandated by the 1996 FQPA. The Task Force felt that these issues, which include the 

following, should be discussed on a broader Agency-wide basis as well as with the outside 

community for both pesticides and other agents. 

1. Appropriate application of the database modifying factor for additional required 

developmental and adult toxicity studies. It appears from the data available that the 

default intraspecies 10-fold uncertainty factor may be adequate in the majority of cases 

for protecting children’s health. However, when data specific to children’s health are 

missing or inadequate for a particular agent, application of the database modifying factor 

in addition to the intraspecies variability factor may be sufficient to account for the 

possibility that children may be significantly more sensitive than adults. This issue needs 

further examination. 

2. How to account for the level of concern in the RfD/RfC process. Criteria for assessing 

the level of concern for children’s health were developed by the Toxicology Working 

Group of the 10X Task Force and include factors such as (a) human data on pre- and 

postnatal toxicity; (b) pre- and postnatal toxicity in animal studies, including effects of a 

different or similar type as those in adults; (c) dose-response nature of the experimental 

animal data, including the dose-related incidence of response, relative potency of 

response, slope of the dose-response curve when the margin of exposure is small, and 

how well the NOAEL or BMD is defined; and (d) relevance of the experimental animal 

data to humans, including toxicokinetics, similarity of the biological response, and 

knowledge of the mechanism of action. For each of these areas, criteria are given for 

estimating a level of concern for children’s health as high, moderate, or low. The level of 

concern may be taken into account in the uncertainty and modifying factors applied to the 

RfD, although there is currently no formal process for doing so. 

1

See 10X Task Force documents: Toxicology Data Requirements for Assessing Risks of 

Pesticide Exposure to Children’s Health (U.S. EPA, 1999b) and Exposure Requirements for 

Assessing Risks from Pesticide Exposure to Children’s Health (U.S. EPA, 1999c). 

A-1 
3. As indicated in the toxicology document appended to the Task Force report, the 

current default recommended for using developmental toxicity data for different duration 

reference values is to apply most endpoints for all durations. This is because it is 

assumed that most endpoints of developmental toxicity can be caused by a single 

exposure. If, however, developmental effects are more sensitive than those seen after 

longer-term exposures, then even the chronic RfD/RfC should be based on such effects to 

reduce the risk of potential greater sensitivity in children. Because the standard studies 

currently conducted for developmental toxicity involve repeated exposures, data are not 

often available on which endpoints may be induced by acute, subacute, subchronic, or 

chronic dosing regimens and, therefore, on which should be used in setting various 

duration reference values. Further consideration of the appropriate application of 

developmental toxicity endpoints to various duration reference values is recommended. 

As part of this recommendation, an in-depth review of the HED document on Hazard 

Identification—Toxicology Endpoint Selection System, should be undertaken. 

4. Appropriate setting of intermediate RfDs/RfCs for pesticides and other agents. The 

focus of the RfD and the RfC has been on chronic exposure reference values. Acute 

RfDs are also set for pesticides, and intermediate reference values are set for residential 

exposures as well as for drinking water. Data on developmental toxicity will often be a 

greater factor in calculating the acute and intermediate reference values, and exposures to 

children are more often of this type as well. Consideration should be given to setting 

intermediate reference values for environmental agents. In addition, the question of 

whether or when to set RfDs/RfCs specific for children should be considered. 

5. Appropriate adjustment of the NOAEL or the BMD from inhalation exposure studies 

for extrapolation of developmental toxicity data using less-than-continuous exposure to a 

continuous-exposure scenario. Currently, NOAELs/BMDs from inhalation exposure 

studies other than those for developmental toxicity using, for example, a 6-hr/day 

exposure regimen, are adjusted to a continuous (24 hr/day) exposure for calculating 

RfDs/RfCs. The developmental toxicity risk assessment guidelines (U.S. EPA, 1991) 

recommended against making this adjustment because it was assumed that there was a 

threshold above which exposure would have to occur before an effect would result. This 

recommendation needs to be reconsidered, along with the adjustment of NOAELS/BMDs 

in general. 

A-2
Several improvements in testing approaches were also proposed for consideration in the 

10X Task Force report as a way to improve the assessment of potential risks to children. The 

Technical Panel was asked to consider the need for such tests, when they should be required, and 

interpretation of the data for risk assessment purposes. The improvements to be considered 

include 

• pharmacokinetics that include data from different developmental stages, perhaps 

done in a tiered approach as suggested in Kimmel and Francis (1990); 

• direct dosing of neonates, especially when early exposure is of concern, because 

this is the time when differences in metabolic capability are greatest; 

• perinatal carcinogenesis studies and appropriate triggers for when they should be 

required; 

• developmental immunotoxicity testing and appropriate triggers; and 

• advanced DNT testing, in particular, cognitive testing that is more similar to that 

used in humans. 

An additional issue was how to make exposure assessments compatible with the doseresponse assessment. For example, how should the appropriate durations of exposure be 

determined for toxic endpoints of concern? Should standard exposure durations be used? 

A-3
APPENDIX B: CASE STUDIES—EVALUATING AND SELECTING 

HEALTH ENDPOINTS FOR DERIVING REFERENCE VALUES 

Two case studies were developed by the Technical Panel to illustrate many of the points 

discussed in this report. The first case study is of a hypothetical volatile chemical for which 

limited data are available and little information is known about the mode of action except that 

there is support for a nonlinear mode of action for cancer. 

The second case study is of a hypothetical endocrine disruptor for which the mode of 

action is known or assumed from other chemicals in the same class. This case study is used to 

illustrate in part how the information on mode of action can inform a more focused collection of 

data as well as the interpretation of the data and its use in risk assessment. 

In both case studies, NOAELs and LOAELs rather than BMDLs or BMCLs are used to 

derive reference values, in large part because the data are fictitious and were not developed to 

the point that they could be readily modeled. However, the Technical Panel strongly encourages 

the use of dose-response modeling and calculation of BMDLs or BMCLs for selection of the 

POD to be used as the basis for deriving reference values. 

B-1
CASE STUDY 1: INHALATE 

Inhalate, a synthetic halogenated aliphatic alkene, is a nonflammable volatile liquid at 

room temperature. The chemical enters the air through its industrial and commercial use, 

primarily as a solvent. It is also found in surface and ground water and soil upon disposal. The 

most important route of human exposure is inhalation of the chemical in the ambient and indoor 

air, although there is a lower possibility of ingestion through contaminated drinking water. 

Because of its high volatility, dermal exposure to the chemical is expected to be minimal. 

This case study illustrates the use of single or multiple health endpoints for deriving 

reference values for different durations of exposure following inhalation exposure. It also 

illustrates the harmonized approach for all effects (including cancer) that are known or assumed 

to be produced through a nonlinear or threshold mode of action. For the purpose of illustration, 

results of key studies are summarized in Table B-1, including dose-response data for different 

health endpoints relevant to different durations of exposure via inhalation exposure. Although 

oral data for this chemical were available, a brief description is included here only to show the 

consistency with which effects were seen after either inhalation or oral exposure. 

SUMMARY OF HEALTH EFFECTS INFORMATION 

Absorption, Distribution, Metabolism, and Elimination 

There is very little information on the absorption and distribution of Inhalate in humans 

and laboratory animals following oral, inhalation, or dermal exposure. However, similar effects 

are seen by oral and inhalation exposures, suggesting that Inhalate or its metabolites reach their 

target sites after absorption from either exposure route. Available in vitro metabolic studies 

indicate that Inhalate is extensively metabolized in target tissues including the liver and kidney 

of rats and mice. Limited in vitro studies with human tissues show a similar pattern of 

metabolism. As discussed below, much of Inhalate-induced toxicity appears to be due to its 

metabolites. These metabolites have been detected in the urine of rats and mice following 

inhalation and oral exposure to the parent chemical. 

Postulated Mode of Action 

No information is available on mode of action except for the carcinogenicity of Inhalate. 

The carcinogenic effects of Inhalate in rodent liver are attributed to oxidative metabolismmediated cytotoxicity in the target organ. The oxidative metabolism produces highly tissuereactive metabolites that lead to tissue injury and cell death. The persistent cell proliferation 

B-2
B-3

Table B-1. Summary results of major inhalation exposure studies on Inhalate

Species Sex

Exposure duration

and frequency

Concentrations

(mg/m3)

LOAEL/

NOAEL

(mg/m3)

HEC a

(mg/m3) Resp

Human M/F 2 hrs 4, 40, 400 40/4 40/4,

2-hr

Headache, dizziness drowsiness, anesthe

Accidental exposure NA NA - Narcosis, proteinuria6 hrs/day for 7 days(clinical exposure)b

10, 20, 100, 150 20/10 4/2 Headache, dizziness

 drowsiness

Occupational(>15 yrs)

TWA of 56 20/NA 20/NA Dizziness, forgetfuln

 serum liver enzyme levels of lysozymes

F Occupational NA NA - Menstrual disorders; 

 abortion; cardiac an of workers

Rat/mouse

M/F 4 hrs 0, 13, 24, 50 13/NA 13/NA, 

4-hr

Dose-related hypera hypoactivity, narco

6 hrs/day, 5 days/wkfor 2 wks

0, 50, 100, 200 50/NA 9/NA Dose-related hypera

 hypoactivity, narco

Rat M/F 6 hrs/day, 5 days/wk

for 13 wks

0, 30 30/NAc – Changes in fatty acid 

 brains
Table B-1. Summary results of major inhalation exposure studies on Inhalate (continued)

Species Sex

Exposure duration

and frequency

Concentrations

(mg/m3

)

LOAEL/

NOAEL

(mg/m3

)

HEC a

(mg/m3) Resp

B-4

Rat(cont)

M/F 6 hrs/day, 5days/wk

for 13 wks

0, 22, 39, 88 39/22 7/4 Dose-related hypera

 hypertrophy, vacuo hepatocytes, necros toxic nephrosis of t cells

Mouse M/F 6 hrs/day, 5 days/wk

for 13 wks

0, 28, 50, 100 50/28 9/5 Dose-related hypera

 hypertrophy, vacuo hepatocytes, necros toxic nephrosis of t cells

Rat M/F 6 hrs/day, 5 days/wk

for 104 wks

0, 11, 22, 44 22/11 4/2 Clinical signs of neu

 related liver hypert of hepatocytes, nec carcinoma; cytome of tubular epithelia

Mouse M/F 6 hrs/day, 5 days/wk

for 78 wkse

0, 11, 28, 56 28/11 5/2

Rat F GDs 70–13, 

6 hrs/day

0, 10, 90 90/10 22.5/2.5 Decreased motor act

 ataxia in dams at hi

M/F Two-generation

reproductive study, 6hrs/day, 5 days/wk

0, 28, 100 100/28 18/5 Reduced litter size, r

 offspring, sedation 

a These values are approximate HECs derived in accordance with the 

 RfC methodology (U.S. EPA, 1994) and Chapter 4 (this report) for category 3 Gases using pharmacokinetic data for Inhalate.

b Data included here for comparison; data from intentional human

 exposures are not currently used by EPA in risk assessment.

c Special study for neurotoxicity, no HEC calculatd Considered to be a lifetime exposure

 HEC = human equivalent conentration NA = not available TWA = time-weighted average GD = gestation day
presumably would lead to higher probabilities of cell mutation and subsequent cancer. Liver 

tumors were produced only at dose levels that resulted in repeated or sustained cytotoxicity and 

regenerative cell proliferation. This postulated mode of action is further supported by the 

observation in specialized studies that neither the cytotoxicity nor cell proliferation occurred in 

the CYP2E1 null mouse or in the wild type treated with a P450 inhibitor at the same exposure. 

The weight of the evidence indicates that a mutagenic mode of action via DNA reactivity is not a 

significant component of Inhalate-induced liver tumors in rats and mice. There are no data 

indicating that the mode of action observed in rodents is not also likely to apply to humans. 

Nervous System 

Inhalate has been found to elicit dose-dependent clinical signs of CNS effects in adult 

humans following acute inhalation exposure and accidental ingestion. CNS symptoms have been 

reported in several studies of occupational exposure of workers to Inhalate. Dose-dependent 

clinical signs of CNS effects have been observed in adult rats and mice exposed to Inhalate by 

inhalation following different duration of exposures. There are limited data indicating that 

prenatal exposure to Inhalate adversely affects the developing nervous system in rats and mice 

(see Growth and Development below). There are no data on the ability of Inhalate to affect the 

nervous system at other life stages (e.g., during the fetal period, infancy, childhood, or old age). 

The mechanism of action for the CNS effects has not been clearly established, but it is believed 

to be related to effects of the parent compound on lipid and fatty acid composition of the 

membranes. 

Inhalation Exposure 

Several reports available in the open literature indicate dose-dependent clinical signs of 

CNS symptoms in adults exposed acutely and subacutely via inhalation to Inhalate. Males and 

females exposed acutely to high concentrations (40–400 mg/m3

 for 2 hours) showed dosedependent effects, including headache, dizziness, incoordination, drowsiness, and anesthesia. 

No effect was reported following acute exposure to 4 mg/m3

. Similar effects were observed in 

adult human volunteers at lower concentrations (10, 20, 100, 150 mg/m3

) for 6 hours per day for 

up to 7 days, with a NOAEL of 10 mg/m3

. An acute accidental exposure of a small group of 

workers to an unknown (presumably high) concentration resulted in narcosis. 

Long-term and chronic neurotoxic effects have been reported in several studies of 

occupational exposure of workers to Inhalate in different industries. Exposure data were not 

provided in these reports; however, it can be presumed that these workers were exposed to a 

B-5
daily TWA exposure of 56 mg/m3

. Subjective neurological symptoms, including dizziness and 

forgetfulness, were consistently reported across studies. No other information on possible 

neurological effects was collected in these studies. 

Concentration-dependent clinical signs of neurological effects, including hyperactivity, 

ataxia, hypoactivity, and finally loss of consciousness, have also been reported in adult rats and 

mice following acute (13, 24, 50 mg/m3

 for 4 hours) and short-term inhalation exposure (50,100, 

200 mg/m3 6 hrs/day for 2 weeks) to Inhalate at high concentration. Similar effects were 

observed in rats exposed at 39 and 88 mg/m3 and in mice at 50 and 100 mg/m3 for 13 weeks. 

The subchronic NOAELs for rats and mice were 22 and 28 mg/m3, respectively. 

Chronic exposure to Inhalate at lower concentrations resulted in less serious clinical 

signs of CNS effects in rats (22 or 44 mg/m3

) and mice (28 or 56 mg/m3). The chronic NOAELs 

for rats and mice in these studies were both 11 mg/m3

. It should be noted that neurological 

endpoints examined in these animal studies are limited to clinical signs and histopathology. In a 

special study, changes in fatty acid composition of the brain were observed in rats exposed at 30 

mg/m3 (the only tested concentration) for 90 days. 

Pregnant rats were exposed by inhalation to Inhalate at 0, 10, or 90 mg/m3

 for 6 hrs/day 

on days 7–13 of gestation. Decreased motor activity was observed in 21- or 60-day-old pups 

from dams exposed to 90 mg/m3. A NOAEL of 10 mg/m3 for developmental neurotoxicity was 

identified in this study. 

Oral Exposure 

Acute neurological effects in adult humans after ingestion of Inhalate are similar to those 

seen after inhalation. Accidental exposure to approximately 6–8 mL (or about 100 mg/kg/day) 

resulted in narcotic effects. Neural tube defects and eye anomalies were reported in studies of 

offspring of residents exposed to drinking water contaminated with Inhalate and other solvents. 

Exposure levels were not determined in this study. 

Single oral gavage administration of Inhalate to adult rats (1000 mg/kg) caused ataxia. 

Ataxia was also observed in pregnant rats treated by gavage at 900 mg/kg on gestation days 

(GDs) 6–19. No CNS effects were reported in a chronic oral gavage study in rats and mice at 50, 

100, or 300 mg/kg/day. However, neurological endpoints examined in these studies were limited 

to clinical signs and histopathology. 

In a study that investigated the effect of Inhalate on the developing nervous system, male 

mouse pups were treated by gavage at 50 or 300 mg/kg/day for 7 days (postnatal days [PNDs] 

B-6
10–17). Hyperactivity was reported in animals during adulthood at the high dose. No studies 

with exposure throughout development (prenatal and postnatal) were available. 

Liver 

Two reports provided suggestive evidence of liver effects in workers exposed to Inhalate 

in different industries. Inhalate has been shown to induce dose-dependent liver toxicity in adult 

rats and mice following subchronic and chronic exposure by inhalation and oral gavage. Liver 

tumors were also observed in chronic studies of rats and mice. Available data support the 

conclusion that liver tumors were produced only at dose levels that resulted in repeated or 

sustained cytotoxicity and regenerative cell proliferation. Inhalate carcinogenic effects in rodent 

liver are attributed to oxidative metabolism-mediated cytotoxicity in the target organ. The 

oxidative metabolism produces highly tissue-reactive metabolites that lead to tissue injury and 

cell death. The persistent cell proliferation presumably would lead to higher probabilities of cell 

mutation and subsequent cancer. 

This postulated mode of action is further supported by the observation in specialized 

studies that neither the cytotoxicity nor cell proliferation occurred in the CYP2E1 null mouse or 

in the wild type treated with a P450 inhibitor at the same exposure. The weight of the evidence 

indicates that a mutagenic mode of action via DNA reactivity is not a significant component of 

Inhalate-induced liver tumors in rats and mice. No data exist indicating that the mode of action 

observed in rodents is not also likely to apply to humans. There are no data that provide any 

insights into possible differential sensitivity across life stages. 

Inhalation Exposure 

One study reported changes in serum levels of liver enzymes in workers exposed to the 

chemical at a daily TWA exposure concentration of about 56 mg/m3 over an 8-hour work shift. 

These workers, however, did not exhibit any clinical symptoms of liver dysfunction. 

Dose-related liver effects (liver hypertrophy, vacuolization of hepatocytes, necrosis) have 

been observed in mice following subchronic exposure (13 weeks) to Inhalate at 50 or 100 mg/m3

, 

with a NOAEL of 28 mg/m3

. Dose-related liver toxicity and hepatocellular carcinomas were 

also found in mice following chronic exposure at 28 and 56 mg/m3

. The NOAEL for liver 

toxicity in mice in this chronic study was 11 mg/m3

. 

Rats showed similar liver responses but at higher exposure concentrations following 

subchronic exposure (39 or 88 mg/m3

), with a NOAEL of 22 mg/m3. Liver toxicity and 

hepatocellular carcinomas were also observed at 22 or 44 mg/m3 in a chronic study in rats. The 

B-7
NOAEL for liver effects in rats was 11 mg/m3

. It should be noted that liver effects examined in 

these subchronic and chronic studies were limited to clinical chemistry, morphology, and 

histopathology. 

Oral Exposure 

Liver effects were observed in mice and rats treated subchronically (100, 300, 500 

mg/kg/day) or chronically (50, 100, 300 mg/kg/day) with Inhalate via oral gavage and were 

similar to those seen after inhalation exposure. Mice showed more severe effects than did rats. 

Dose-related hepatocellular carcinomas were also found in treated mice in a chronic study. 

Kidney 

Available human and animal studies indicate that Inhalate also has the potential to cause 

renal toxicity in adults. The mechanisms for the development of kidney effects in humans and 

animals are not known. No data were available to evaluate the effects of Inhalate at life stages 

other than in adults (i.e., during development or old age). 

Inhalation Exposure 

Symptoms of renal dysfunction (proteinuria, hematuria) have been associated with 

accidental human exposure to anesthetic concentrations of Inhalate. Subtle or no renal effects 

were reported in workers exposed chronically. Increased urinary levels of lysozyme and betaglucuronidase suggestive of mild renal tubular damage have been observed in workers exposed 

for an average of 15 years to a daily TWA concentration of 56 mg/m3

. 

Dose-related renal toxicity (cytomegaly, toxic nephrosis of tubular epithelial cells in the 

inner renal cortex) was induced in rats (39, 88 mg/m3) and mice (50, 100 mg/m3) exposed to 

Inhalate for 13 weeks. Subchronic NOAELs for renal effects in rats and mice were 22 mg/m3 

and 28 mg/m3, respectively. Similar renal effects were observed in a chronic study in rats (22, 

44 mg/m3

) and mice (28, 56 mg/m3). Chronic NOAELs for renal effects in rats and mice were 

both 11 mg/m3

. 

Oral Exposure 

Dose-related toxic nephropathy characterized by degenerative changes in the proximal 

convoluted tubules and necrosis of the tubular epithelium were found in rats and mice treated 

with Inhalate via oral gavage for 90 days at 100, 300, or 500 mg/kg/day and for 2 years at 50, 

B-8
100, or 300 mg/kg/day. Subchronic and chronic NOAELs for renal effects in rats and mice were 

at 100 and 50 mg/kg/day, respectively. 

Growth and Development 

Available studies in humans and animals indicate that Inhalate has the potential to cause 

developmental effects by inhalation and oral ingestion. Limitations of human studies could not 

resolve whether the observed developmental effects were causally related to the chemical or 

were a result of chance or bias. However, the epidemiologic findings are supported by animal 

studies with exposure to Inhalate by inhalation and oral gavage that show that the developing 

nervous system is the most sensitive target in rats and mice. 

Inhalation Exposure 

Epidemiologic studies of women occupationally exposed to Inhalate and other related 

solvents have reported elevated risk of spontaneous abortion and cardiac anomalies in their 

offspring. Due to limitations of these studies, an exposure-response could not be established. 

No other health endpoints were investigated in these studies. 

Pregnant rats were exposed by inhalation to Inhalate at 0, 10, or 90 mg/m3

 for 6 hrs/day 

on days 7–13 of gestation. Decreased motor activity was observed in 21- and 60-day-old pups 

from dams exposed to 90 mg/m3. A NOAEL of 10 mg/m3 for developmental effects was 

identified in this study. No studies included exposure throughout gestation and lactation to 

determine effects at other developmental stages. Data from a two-generation reproduction study 

indicated a reduction in litter size and survival of offspring in rats exposure to Inhalate at 100 

mg/m3

, a concentration that also resulted in sedation and renal effects. No effects were reported 

at 28 mg/m3

. 

Oral Exposure 

Neural tube defects and eye anomalies have been reported in studies of residents exposed 

to drinking water contaminated with Inhalate and other solvents. Exposure levels were not 

determined in this study. 

An increased incidence of micro/anophthalmia were observed in the offspring of rats 

treated with Inhalate by gavage at 900 mg/kg/day on GDs 6–19. In a study that investigated the 

effect of Inhalate on the developing nervous system, male mouse pups were treated by gavage 

with Inhalate at 50 or 300 mg/kg/day for 7 days (age 10–17 days). Hyperactivity was reported in 

animals during adulthood at the high dose. No effects were found at the low dose. No studies 

B-9
included exposure throughout gestation and lactation to determine effects at other developmental 

stages. 

Reproductive System 

Available studies in humans and animals suggest that Inhalate may have the potential to 

cause reproductive effects. The underlying mechanism of action for potential reproductive 

effects is not known. 

Inhalation Exposure 

There is suggestive evidence of spontaneous abortion and menstrual disorders among 

women occupationally exposed to Inhalate. However, no definitive conclusions can be made 

because of the limitations associated with these studies. 

Reduced litter size and reduced survival of offspring were reported in rats exposed to 

Inhalate at 100 mg/m3

 in a two-generation reproduction inhalation study; this concentration also 

resulted in sedation and renal effects. No effects were identified at 28 mg/m3. The protocol 

used, however, was not the most recent one, in which reproductive development (e.g., timing of 

puberty or anogenital distance) and adult reproductive function (semen quality, estrous cyclicity) 

are evaluated, nor were organ weights measured. No effects on the reproductive system were 

noted in any other studies. 

Oral Exposure 

No information is available on the potential reproductive effects of Inhalate in animals 

via oral exposure. 

SELECTION OF HEALTH ENDPOINTS AND DERIVATION OF REFERENCE

VALUES

Narrative Description of the Extent of the Database

No information is available on possible modes of action except for liver carcinogenic 

effects in rats and mice. In this case, the mode of action is attributed to oxidative metabolismmediated cytotoxicity and persistent cell proliferation in the liver. Persistent regenerative cell 

proliferation presumably would lead to higher probabilities of cell mutation and subsequent 

cancer. No data exist to indicate that the mode of action observed in rodents is not also likely to 

apply to humans. Pharmacokinetic data indicate that Inhalate is extensively metabolized and that 

much of its toxicity is due to the metabolites. 

B-10
The database for inhalation exposure is limited but adequate for deriving reference 

values. Some human data on acute, short-term, and longer-term exposures are available, 

although the range of endpoints evaluated and the dose-response information for different 

durations of exposure are limited. The animal data include acute, short-term, longer-term, and 

chronic studies with exposures beginning in young adult animals. The acute and short-term data 

are limited to clinical signs of morbidity and mortality, whereas the longer-term and chronic 

studies include some histopathology as well. 

A developmental neurotoxicity (DNT) study was conducted in rats with prenatal 

exposure limited to GDs 7–13 (as opposed to more extensive exposure throughout a major part 

of CNS development, e.g., GD 6 to PND 11 or 20 in the standard DNT study testing protocol). 

No other studies of prenatal or postnatal developmental toxicity were done except for 

evaluations of survival and growth in a two-generation reproduction study in rats. However, the 

protocol used was one in which reproductive development (e.g., timing of puberty or anogenital 

distance) and adult reproductive function (semen quality, estrous cyclicity) were not evaluated, 

nor were organ weights measured. No studies were conducted that considered issues related to 

the toxicity of the agent in old age, either from earlier exposures or from exposures in aged 

animals. 

The database for oral exposure is much more limited than the database for inhalation 

exposure, with acute accidental ingestion data in humans at a single, high-dose level resulting in 

narcosis and chronic drinking water exposure (no dose information) associated with an increase 

in birth defects. The animal data are likewise very limited, with a single-dose acute toxicity 

study in rats in which clinical signs of morbidity and mortality were evaluated and subchronic 

(90-day) and chronic toxicity data in rats and mice indicating effects similar to those seen with 

inhalation exposure. Prenatal developmental toxicity data were available in rats following 

exposure on GDs 6–19, and an evaluation of adult neurotoxicity was conducted in mice 

following developmental exposure on PNDs 10–17. No other developmental toxicity data, and 

no information on reproductive toxicity or adult neurotoxicity were available. No studies were 

conducted that considered issues related to the toxicity of the agent in old age, either from earlier 

exposures or from exposures in aged animals. 

Exposure-Response Array 

In addition to displaying the data in tabular form (Table B-1), an exposure-response array 

can be a useful way of visually displaying the data (see Figures B-1 through B-4) to show what 

data are available for each duration of exposure. The data shown in the graphs are the human 

equivalent concentrations (HECs) based on the dosimetric adjustments discussed in Chapter 4 of 

B-11
0

5

10152025303540

2 hrs-Human 4 hrs-Rat 4 hrs-Mouse GD 7 - 13-

Rat

Twogeneratio

Rat

NOAEL LOAEL 

Timing and duration of exposure–Species

Dose (mg/m

3

)

N

e

u

r

o

t

o

x

i

c

i

t

y

Neurotoxicity

Neurotoxicity

Developmental Neurotoxicity

Repro- Neuro-toxicity

HEC HEC

05

10152025303540

2 hrs-Human 4 hrs-Rat 4 hrs-Mouse GD 7 - 13-

Rat

Twogeneratio

Rat

NOAEL LOAEL 

Timing and duration of exposure–Species

Dose (mg/m

3

)

Neurotoxicity

Neurotoxicity

Neurotoxicity

Developmental Neurotoxicity

Repro- Neuro-toxicity

HEC HEC

B-12

Figure B-1. Exposure-response array of data considered for the Inhalate acute reference value.
B-13

05

10152025

7d-Human GD 7 - 13-

Rat

2 wks-Rat 2 wks-Mouse TwogenerationRat

NOAEL LOAEL 

Timing and duration of exposure–Species

Dose (mg/m

Neurotoxicity

Developmental Neurotoxicity

Neurotoxicity

Neurotoxicity

Repro- Neuro-toxicity 3)

HEC HEC

05

10152025

7d-Human GD 7 - 13-

Rat

2 wks-Rat 2 wks-Mouse TwogenerationRat

NOAEL LOAEL 

Timing and duration of exposure–Species

Dose (mg/m

Neurotoxicity

Developmental Neurotoxicity

Neurotoxicity

Neurotoxicity

Repro- Neuro-toxicity 3)

HEC HEC

Figure B-2. Exposure-response array of data considered for the Inhalate short-term reference valu
0 

5 

10 15 20 25 

GD 7 - 13-Rat Two-generationRat 13 wks-Rat 13 wks-Mouse 

Timing and duration of exposure–Species 

Dose (mg/m

3) 

NOAEL LOAEL NOA HEC HEC EL LOAEL 

Developmental Neurotoxicity 

Repro-, Neuro-toxicity 

Neuro-, Liver, Kidney Toxicity 

Neuro-, Liver, Kidney Toxicity 

HEC HEC

2520

Dose (mg/m

3) 

1510

B-14 5

0

GD 7 - 13-Rat Two-generationRat 13 wks-Rat 13 wks-Mouse

Timing and duration of exposure–Species 

Figure B-3. Exposure-response array of data considered for the Inhalate

longer-term reference val
0 

5 

10 15 20 25 

GD 7 - 13-Rat Two-generationRat 104 wks-Rat 78 wks-Mou

Timing and duration of exposure–Species 

Dose (mg/m

3) 

NOAEL LOAEL NOAEL HEC LOAEL HEC

Neuro-, Liver, Kidney Toxicity 

Neuro-, Liver, Kidney Toxicity 

Developmental Neurotoxicity 

Repro-, Neuro-toxicity 

HEC HEC

2520

Dose (mg/m

3) 

1510

B-15

50

GD 7 - 13-Rat Two-generationRat 104 wks-Rat 78 wks-Mou

Timing and duration of exposure–Species 

Figure B-4. Exposure-response array of data considered for the Inhalate

chronic reference value.
this report, including dosimetric adjustment of the developmental toxicity data in the same 

manner as for other types of toxicity data, as recommended in U.S. EPA (1994) and Chapter 4. 

No toxicokinetic model is available for Inhalate, so adjustments to the applied 

concentrations for dose adjustments and calculation of the HEC in this case study were done 

using the default procedures discussed in U.S. EPA (1994) and Chapter 4 of this document. As 

the effects observed are systemic, with no indications of portal-of-entry effects, the specific 

default procedures are based on the vapors of Inhalate being a category 3 gas. The principal 

parameter for interspecies extrapolation of category 3 gases, the blood:gas (air) partition 

coefficient (Hb/g), is unknown for both humans and animals and assumed to be 1. Therefore, the 

dosimetric adjustment factor (DAF) (see Chapter 4, Section 4.4.3) applied to the duration 

adjusted concentrations is 1, such that the duration adjusted values then become the HECs. 

Duration adjustment was accomplished by factoring the exposure concentration (in 

mg/m3

) by 6/24 (for hours of exposure) and, where applicable, by 5/7 for the number of days per 

week exposed. As noted in Chapter 4, Section 4.4.2.1, duration adjustments were not made to 

acute (i.e., less than 24-hr) single exposures. 

Uncertainty Factors (UFs) 

An interspecies UF (A in Table B-2) of 101/2 was applied in all cases to animal studies 

because the dosimetric adjustment procedures applied to Inhalate are considered to address the 

toxicokinetics portion of this UF, leaving the rest of the UF to cover interspecies toxicodynamics 

(U.S. EPA, 1994). An intraspecies UF (H in Table B-2) of 10 was applied in all cases because 

the data did not allow the estimation of within-human variability and the most sensitive life stage 

and/or susceptible subpopulation was not clearly identified in the database. 

A LOAEL-to-NOAEL UF (L in Table B-2) was applied to the human data for 

neurotoxicity for the longer-term and chronic reference values because only a LOAEL was 

identified in the data for those two durations of exposure. A subchronic-to-chronic (duration) 

UF (S in Table B-2) was not applied, as there were data available for the appropriate duration of 

exposure in each case. For the longer-term and chronic reference values, a duration UF was not 

applied to the data from the developmental toxicity study, as this is not the usual practice when 

considering these data for longer-term and chronic exposures. However, as noted in the report 

(Chapter 4, Section 4.4.5.6), the application of a UF analogous to the subchronic-to-chronicduration UF should be explored, as there may be situations in which data are available and 

applicable but they are from studies in which the dosing period is considerably shorter than that 

for the reference value being derived. 

B-16
B-17

Table B-2. Derivation of reference values for Inhalate—inhalation exposure

Reference

value

duration

Exposure

duration

HEC

(mg/m3

) Species

Type of

effecta

Uncertainty factorsb

Reference value

(ppm)c

Total A H L S D Sample Final

Acute 2 hrs 4 Human NT 30 1 10 1 1

3

0.13

0.03

GDs 7–13 2.5 Rat DNT 100 3 10 1 1 0.03

4 hrs 13Ld Rat/

Mouse

NT 1000 3 10 10 1 0.01

13–16 wks 5 Rat RT 100 3 10 1 1 0.05

Short-term 7 days 2 Human NT 30 1 10 1 1

3

0.07

0.03 14 days 9Ld Rat/

Mouse

NT 1000 3 10 10 1 0.01

GDs 7–13 2.5 Rat DNT 100 3 10 1 1 0.03

13–16 wks 5 Rat RT 100 3 10 1 1 0.05

Longer-term >15 yrs 20Ld Human NT 300 1 10 10 1

3

0.07

0.03

13 wks 4 Rat NT 100 3 10 1 1 0.04

13 wks 5 Mouse NT 100 3 10 1 1 0.05

GDs 7–13 2.5 Rat DNT 100 3 10 1 1e 0.03

13–16 wks 5 Rat RT 100 3 10 1 1 0.05

13 wks 4 Rat LT 100 3 10 1 1 0.04

13 wks 5 Mouse LT 100 3 10 1 1 0.05

13 wks 4 Rat KT 100 3 10 1 1 0.04

13 wks 5 Mouse KT 100 3 10 1 1 0.05

Chronic >15 yrs 20Ld Human NT 300 1 10 10 1

3

0.07

0.02

104 wks 2 Rat NT 100 3 10 1 1 0.02

78 wks 2 Mouse NT 100 3 10 1 1 0.02

GDs 7–13 2.5 Rat DNT 100 3 10 1 1e 0.03

13–16 wks 5 Rat RT 100 3 10 1 1 0.05

104 wks 2 Rat LT 100 3 10 1 1 0.02

78 wks 2 Mouse LT 100 3 10 1 1 0.02

104 wks 2 Rat KT 100 3 10 1 1 0.02

78 wks 2 Mouse KT 100 3 10 1 1 0.02

 a

 NT = neurotoxicity; DNT = developmental

 neurotoxicity; RT = reproductive toxicity;

 LT = liver toxicity; KT = kidney toxicity

 b

 A = animal-to-human (interspecies);

 H = inter-individual (intraspecies); 

 L = LOAEL-to-NOAEL; S = subchronic-

 to-chronic duration; D = database deficiency

 c

 Sample = reference value based on that particular

 endpoint, species, duration; Final = reference value for

 the entire database for a particular duration of

 exposure.

 d

 L indicates that this value is the HEC based on the

 LOAEL.

 e

 A duration UF was not applied to the data from the

 developmental neurotoxicity study for either the

 longer-term or chronic reference value; however, the

 adjustment should be considered when extrapolating

 from shorter to longer durations of exposure.

 GD = gestation day
B-18

A database UF of 101/2 was applied in all cases because there were data indicating that one of

the main target organs for Inhalate was the nervous system, that there was neurotoxicity in adults

(humans and animals), and that there were some data on developmental neurotoxicity, but with

exposure limited to only a portion of the developmental period. In addition, there was no study in

which pregnancy outcomes were evaluated (i.e., fetal survival, growth, and structural development)

except for a possible association with spontaneous abortion and cardiac anomalies in occupationally

exposed workers. These data gaps were not considered likely to reduce the NOAEL by more than a

factor of 3 because there were some data on developmental exposures from a two-generation

reproductive toxicity study. There were no reports of effects on fertility or reproduction per se, except

for a possible association with menstrual disturbances in occupationally exposed workers.

Acute Inhalation Exposure

Results of available studies indicate that acute inhalation exposure to Inhalate can result in

neurotoxic effects in human adults with a LOAELHEC of 40 mg/m3

 and a NOAELHEC of 4 mg/m3 in a 2-

hour exposure. Animal studies also show that Inhalate has the potential to cause neurotoxicity in adults

with a LOAELHEC of 13 mg/m3 in a 4-hr exposure, and developmental neurotoxicity and other

reproductive effects with LOAELHECs of 22.5 mg/m3 and 18 mg/m3 and NOAELHECs of 2.5 mg/m3

and 5 mg/m3, respectively.

Default UFs of 101/2 (animal-to-human extrapolation), 10 (inter-individual differences), and

101/2 (database deficiencies: no adequate prenatal developmental toxicity studies in two species, no

adequate developmental neurotoxicity study) were applied to all the NOAELHECs to derive sample

reference values. In addition, a UF of 10 (LOAEL to NOAEL) was applied to the 4-hr rat and mouse

LOAELHECs. Human and animal studies indicate that the nervous system is vulnerable to Inhalate

exposure. Although the sample reference values for the 4-hr adult rat and mouse exposures were

lower than that based on the developmental neurotoxicity study, the values were within a similar range

and the sample reference value for developmental neurotoxicity had less overall uncertainty. 

Therefore, the resultant reference value chosen for acute inhalation exposure is 0.03 mg/m3

 (Table B2).

Short-term Inhalation Exposure

The reference value for short-term inhalation exposure is based on the human data

NOAELHECs of 2 mg/m3

 as well as the animal developmental neurotoxicity NOAEL of 2.5 mg/m3 and
B-19

reproductive toxicity NOAEL of 5 mg/m3

 (LOAELHECs of 4 mg/m3, 22.5 mg/m3, and 18 mg/m3,

respectively). In addition, the 14 day exposures of rats and mice resulted in a LOAELHEC of 9 mg/m3. 

For the human NOAELHEC of 2 mg/m3, applying a 10-fold default UF for intraspecies uncertainty and

variability and a 101/2-fold UF for database deficiencies results in a sample reference value for shortterm inhalation exposure of 0.07 mg/m3

. Sample reference values based on animal data include default

factors of 101/2 (interspecies), 10 (intraspecies) and 101/2 (database deficiencies) applied to the

NOAELHECs for developmental neurotoxicity and reproductive toxicity (2.5 and 5 mg/m3, respectively)

and result in sample reference values of 0.03 and 0.05 mg/m3

. An additional factor of 10 (LOAEL to

NOAEL) applied to the adult neurotoxicity data in rats and mice results in a sample reference value of

0.01 mg/m3

. Given the close range of values and the lower overall uncertainty in the sample reference

value for developmental neurotoxicity than that for adult neurotoxicity, the final reference value of 0.03

mg/m3

 is chosen (Table B-2).

Longer-term Inhalation Exposure

Subchronic and chronic inhalation exposure to Inhalate can result in multiple health effects. 

Available studies demonstrate neurotoxicity in adult humans. However, dose-response information is

not available, and the presumed LOAEL (20 mg/m3) for neurotoxicity in humans is somewhat higher

than the HECs for other health endpoints (developmental, reproductive, liver, and renal effects)

observed in animal studies, where the LOAELHECs range from 7 mg/m3 to 22.5 mg/m3 and the

NOAELHECs range from 2.5 mg/m3

 to 5 mg/m3. Dose-response data for these health endpoints in

animal studies can be used as the basis for deriving a longer-term inhalation reference value for Inhalate.

UFs of 101/2 (interspecies), 10 (intraspecies), and 101/2 (database deficiencies) were applied to

NOAELHECs for the various endpoints in deriving sample reference values. If an additional factor of 3

were applied to the rat developmental toxicity data to account for the marked difference in exposure

duration in the study itself (7 days of exposure: GDs 7–13) versus the duration covered by this

reference value (up to 10% of the life span), a longer-term sample reference value of 0.01 mg/m3

 would

result. Without this additional factor, the sample reference value from the developmental toxicity study

was still the lowest value (0.03 mg/m3

), although all values from the animal studies were in a similar

range (0.03–0.05).

The final reference value chosen was 0.03 mg/m3

 to be protective of the developing individual

as well as adults (Table B-2). Whether an additional factor should be applied to the developmental

toxicity data or to other data of much shorter duration should be explored further.
B-20

Chronic Inhalation Exposure

For the chronic inhalation reference value, the LOAELHECs range from 4 mg/m3

 to18 mg/m3

and the NOAELHECs range from 2 mg/m3 to 5 mg/m3. UFs of 101/2 (interspecies), 10 (intraspecies),

and 101/2 (database deficiencies) applied to the chronic exposure NOAELHECs for neurotoxicity and

liver, kidney, and reproductive toxicity data result in sample reference values of 0.02–0.05 mg/m3

(Table B-2). Applying these UFs to the NOAELHEC of 2.5 mg/m3 for developmental toxicity yields a

sample reference value of 0.03 mg/m3, which falls within the range of chronic study-based values. If,

contrary to current practice, an additional 3- or 10-fold UF for subchronic to chronic duration were

applied to the developmental NOAELHEC, the sample reference value would be 0.01 or 0.003. As

mentioned in Section 4.4.5.6, this issue may need further exploration. In this example, several

endpoints result in a sample chronic inhalation reference value of 0.02 mg/m3

, the value chosen for the

chronic inhalation reference value.

With regard to the liver effects of Inhalate, the NOAELHECs and reference values for liver

histopathology were the same as for the tumorigenic effects; thus, the reference value based on liver

toxicity should be protective of the carcinogenic effects of Inhalate.

Overall Evaluation of Reference Values

The reference values for Inhalate were similar across all durations of exposure. This is because

the same data were used as the basis for the acute, short-term, and longer-term reference value, that is,

the effects on developmental neurotoxicity. Although there were human data appropriate for

consideration for all four durations of exposure, the endpoints examined in these studies or reports were

limited and were not indicative of effects on the developing nervous system. To be protective of

developmental life stages, it was considered appropriate to base the reference values on the

developmental neurotoxic effects, for which the sample reference value was slightly lower than for other

endpoints. For the chronic reference value, a number of sample reference values, including that for the

carcinogenic effects of Inhalate, were clustered in the same range, at 0.02 mg/m3

, slightly lower than

that for developmental neurotoxicity. In this case, it was considered appropriate to use this lower

reference value to be protective of all potential effects for lifetime exposures.
CASE STUDY 2: LUTEINATE 

INTRODUCTION 

Luteinate is a new pesticide that was developed for use as an herbicide. Environmental 

fate studies have shown that it will persist in soils and will therefore likely to move into ground 

and surface water. The general population may be exposed to Luteinate through consumption of 

food and drinking water. 

Luteinate belongs to a class of pesticides for which the neuroendocrine mode of action is 

known. It was designed to be less potent than other members of the class. In order to ascertain 

its potency, a number of short-term studies were first conducted; they confirmed a similar mode 

of action and the fact that Luteinate was less potent. Following this, Luteinate was tested in 

more traditional toxicology studies to establish its long-term effects and dose-response 

relationships. 

This case study provides an example of the usefulness of mode of action information in 

establishing the short- and long-term effects of Luteinate on relevant target organ systems at 

different life stages. Such information enables the development of a targeted robust data set for 

use in establishing reference values for various durations of exposure. 

Postulated Mode of Action 

Other members of this class of pesticides have been shown to act on the hypothalamicpituitary-ovarian axis. These pesticides affect the hypothalamus, leading to a decreased 

secretion of hypothalamic norepinephrine (NE). Decreased NE levels result in decreased release 

of gonadotropin releasing hormone (GnRH) from the hypothalamus. GnRH is the hormone 

responsible for inducing the pituitary gland to release luteinizing hormone (LH). Thus, the 

decrease in GnRH leads to a suppression of the pituitary LH release. These compounds also 

decrease the neurotransmitter dopamine, which in turn leads to a decrease in pituitary prolactin. 

Decreased LH and prolactin levels have the potential to impact several organ systems at 

different life stages. In humans, there are robust pulses of the LH surge in the fetus and prior to 

birth. The LH pulsatility continues with diminishing amplitude during the early months of 

postnatal life, and then LH secretion becomes barely detectable during much of the first decade 

of postnatal life. Around the age of 10, there is the reemergence or re-awaking of LH pulses 

while sleeping. 

The natural progression from prepubertal to postpubertal status is dependent on the 

normal function of the hypothalamic-pituitary-gonadal axis. Likewise, many of the same 

hypothalamic mechanisms that control pituitary function and the pituitary hormones themselves 

B-21
(especially LH and prolactin) play a key role in pubertal development. In the adult, ovulation is 

dependent on sufficient LH levels. Therefore, this class of pesticides impacts critical 

reproductive processes, including puberty, ovarian cyclicity, pregnancy, and lactation (milk 

quality/production). Given the role of NE and dopamine in the development of the CNS, this 

class of pesticides may also affect the developing CNS; however, relevant toxicology studies 

have not yet been conducted. In addition, suppression of prolactin during the early postnatal 

period in rodents may lead to prostate inflammation in male offspring. 

This class of pesticides has also been shown to lead to mammary tumors in SpragueDawley (SD) rats but not in other strains of rats or mice. Mammary tumors result from the 

prolonged decrease in serum LH, which leads to a cessation of ovulation and eventually causes 

the ovarian follicles to continue to secret estradiol. In concert with prolactin, estrogen acts on 

the mammary gland and leads to the formation of mammary tumors. However, the induction of 

the tumors has been shown to be due to unique features of the reproductive aging process in 

Sprague-Dawley rats; because humans do not age in the same manner, this mode of action is 

unlikely to be operative in humans. 

SUMMARY OF HEALTH EFFECTS INFORMATION 

Studies with other pesticides in this chemical class have found negligible differences in 

response (with the exception of the induction of mammary tumors in SD rats) among various 

strains of rats and between rats and mice. Therefore, all studies on Luteinate were conducted on 

SD rats. 

Luteinate exposure results in a spectrum of effects that are related to decreases in serum 

LH and decreases in prolactin. The toxicology studies are summarized below in the context of 

the specific effects associated with either decreases in LH or prolactin. In addition, the effect 

levels for specific toxicological endpoints are summarized in Table B-3. It is important to note 

that as the spectrum of effects are mechanistically related to decreases in serum LH or prolactin, 

there is great similarity in the doses that affect LH and prolactin levels and those that cause the 

related effects. 

Absorption, Distribution, Metabolism, and Elimination 

Studies in adult rats have shown that Luteinate is rapidly absorbed following oral 

exposure. It is not metabolized and is eliminated in the urine. No other information is available. 

The toxicokinetic profiles of other pesticides in this chemical class have not been extensively 

examined either. 

B-22
Table B-3. Summary of endpoints and effect levels for rat studies of Luteinate 

Response Exposure period 

Dose levels 

(mg/kg/day) 

NOAEL/LOAEL 

(mg/kg/day) 

Decreased LH Pregnant females: 

GDs 1–8 50, 100, 200 100/200 

PNDs 22–41 25, 50, 100 50/100 

Adults: 

3 days 

28 days 

6 months 

50, 100, 200 

40, 80, 160 

4, 8, 16 

100/200 

40/80 

4/8 

Two-generation reproduction study: 

10 weeks - F0 

13 weeks - F1a 

5, 10, 25, 50 

5, 10, 25, 50 

5/10 

5/10 

Disruption of 

estrous 

cyclicity 

PND 22-41 25, 50, 100 50/100 

Adults: 

28 days 

6 months 

40, 80, 160 

4, 8, 16 

40/80 

4/8 

Two-generation reproduction study: 

10 weeks - F0 

13 weeks - F1a 

5, 10, 25, 50 

5, 10, 25, 50 

5/10 

5/10 

Altered 

pregnancy 

maintenance 

Pregnant females: 

GDs 1–8 

GDs 6–10 

GDs 1–20 

50, 100, 200 

100, 200, 400 

50, 100, 200 

100/200 

200/400 

100/200 

Two-generation reproduction study: 

11–13 weeks - F0 

14–16 weeks - F1a 

5, 10, 25, 50 

5, 10, 25, 50 

5/10 

5/10 

Delayed 

parturition 

Pregnant females: 

GDs 6–10 100, 200, 400 100/200 

Two-generation reproduction study: 

14 weeks - F0 

17 weeks - F1a 

5, 10, 25, 50 

5, 10, 25, 50 

5/10 

5/10 

Delayed 

vaginal opening 

PNDs 22–41 25, 50, 100 50/100 

Two-generation reproduction study: 

4–5 weeks - F1a 5, 10, 25, 50 25/50 

Delayed 

preputial 

separation 

PND 23–53 25, 50, 100 50/100 

Two-generation reproduction study: 

6–7 weeks - F1a 5, 10, 25, 50 25/50 

B-23
Table B-3. Summary of endpoints and effect levels for rat studies of Luteinate 

(continued) 

Response Exposure period 

Dose levels 

(mg/kg/day) 

NOAEL/LOAEL 

(mg/kg/day) 

Attenuation of 

prolactin 

release 

Lactating females: 

PNDs 1–4 25, 50, 100 25/50 

Adults: 

3 days 

28 days 

6 months 

50, 100, 200 

40, 80, 160 

4, 8, 16 

100/200 

40/80 

4/8 

Two-generation reproduction study: 

10 weeks - F0 

13 weeks - F1a 

5, 10, 25, 50 

5, 10, 25, 50 

5/10 

5/10 

Increased 

prostatitis in 

offspring 

PNDs 1–4 25, 50, 100 25/50 

Adults: 

28 days 

6 months 

40, 80, 160 

4, 8, 16 

>200/NAb 

>160/NAb 

Two-generation reproduction study: 

16 weeks - F0 

16 weeks - F1a 

5, 10, 25, 50 

5, 10, 25, 50 

>50/NAb 

5/10 

Reduced 

weight of 

seminal 

vesicles and 

ventral prostate 

PNDs 23–53 25, 50, 100 50/100 

Adults: 

28 days 

6 months 

40, 80, 160 

4, 8, 16 

>200/NAb 

>160/NAb 

Two-generation reproduction study: 

16 weeks - F1a 5, 10, 25, 50 5/10 

Fetus: 

Delayed 

ossification 

Reduced fetal 

weight 

GDs 1–20 

GDs 1–20 

50, 100, 200 

50, 100, 200 

100/200 

>200/NAb 

a

 F1 exposures are indicated for the duration of postnatal exposure, but it is assumed that 

3 weeks of prenatal exposure also occurred.

b

 When no effect on a particular endpoint was noted in a study, the NOAEL is indicated 

as > the highest dose, and the LOAEL as NA (not applicable). 

B-24
Decreased LH Surge and Related Effects 

Exposure to Luteinate resulted in a significant decrease in serum LH that was dependent 

on the dose and duration of exposure. In a 3-day gavage study of SD male and female rats 

exposed to levels of 0, 50, 100, and 200 mg/kg/day, there were significant decreases in serum 

LH at 200 mg/kg/day. Serum LH was significantly reduced at doses of 80 mg/kg/day and above 

in a 28-day study and at doses of 8 mg/kg/day and above in a 6-month study of SD rats. Levels 

in the latter study were similar to those observed in the F0 rats in a two-generation reproductive 

toxicity study in which significant decreases in serum LH were observed at doses of 10 

mg/kg/day and higher following 10 weeks of exposure. 

Serum LH was also measured in pregnant dams exposed to doses of 0, 50, 100, and 200 

mg/kg/day on GDs 1–8 and was significantly reduced at 200 mg/kg/day. Exposure of weanling 

SD rats on PNDs 22–41 to doses of 0, 25, 50, and 100 mg/kg/day of Luteinate resulted in a 

significant decrease in serum LH at 100 mg/kg/day. In a two-generation reproductive toxicity 

study, serum LH was measured in the F1 generation prior to mating (13 weeks postnatal 

exposure) and was significantly reduced at doses of 10 mg/kg/day and higher. 

Disruption of Estrous Cyclicity 

In the adult female, ovulation is dependent on sufficient levels of LH. Because exposure 

to Luteinate suppresses LH, it would be anticipated to also disrupt the normal estrous cycle. 

Estrous cyclicity was abnormal in SD rats exposed to 80 mg/kg/day for 28 days and 8 mg/kg/day 

for 6 months. In the two-generation reproductive toxicity study, estrous cyclicity was abnormal 

in the F0 females exposed to 10 mg/kg/day for 10 weeks and in the F1 females exposed to 10 

mg/kg/day for 13 weeks postnatally (as well as prenatally). In addition, exposure of weanlings 

to 100 mg/kg/day on PNDs 22–41 resulted in abnormal estrous cycles. 

Pregnancy 

Altered LH levels also impact the ability of the female to maintain pregnancy as well as 

the timing of parturition. Thus, in a variety of exposure scenarios, an increase in pre- and 

postimplantation loss was observed. Implantation was affected following exposure to 200 

mg/kg/day on GDs 1–8 or 1–20 and after exposure to 400 mg/kg/day on GDs 6–10. Parturition 

was delayed following exposure to 200 mg/kg/day and above on GDs 6–10. In the twogeneration reproductive toxicity study, there was an increase in pre- and postimplantation loss 

and a delay in parturition in the F0 and F1 females at doses of 10 mg/kg/day and higher. 

B-25
Sexual Maturation 

At the time of puberty, the CNS and pituitary respond to increased concentrations of 

estradiol in a positive feedback fashion culminating in the first LH surge. Thus, exposure to 

Luteinate would likely impact sexual maturation. Vaginal opening was delayed in female SD 

rats following exposure to 100 mg/kg/day on PNDs 22–41 and preputial separation was delayed 

in males exposed to 100 mg/kg/day on PNDs 23–53. In the two-generation reproductive toxicity 

study, vaginal opening was delayed in the F1 females exposed to 50 mg/kg/day and preputial 

separation was delayed in the F1 males exposed to 50 mg/kg/day. 

Decreased Prolactin and Related Effects 

Prolactin levels were significantly decreased in adult SD rats following exposure to 200 

mg/kg/day of Luteinate for 3 days, to 80 mg/kg/day for 28 days, and to 8 mg/kg/day for 6 

months. In a study in which lactating dams were exposed during PNDs 1–4, prolactin levels 

were reduced at doses of 50 mg/kg/day. In the two-generation reproductive toxicity study, 

prolactin levels were reduced in the F0 and F1 animals at doses of 10 mg/kg/day. 

Prostatitis 

As a consequence of the reduced prolactin levels, an increased incidence of prostatitis 

was observed in males following maternal exposure to 50 mg/kg/day during lactation days 1–4, 

and in the F1 males exposed to 10 mg/kg/day and higher. Prostatitis was not observed in males 

exposed during adulthood only (i.e., F0 males, males in 28-day and 6-month studies). 

Organ Weights 

In males exposed to doses of 100 mg/kg/day Luteinate during PNDs 23–53, there was a 

decrease in absolute and relative weights of the seminal vesicles and ventral prostate. This was 

also observed in the F1 males exposed to doses of 10 mg/kg/day or greater at the time of 

terminal sacrifice following mating. Histopathological examination revealed no lesions in either 

study. No organ weight changes or histopathological lesions were noted in any of the other 

studies. 

Effects Unrelated to LH or Prolactin 

A prenatal developmental toxicity study was conducted in which pregnant SD rats were 

exposed to doses of 0, 50, 100, or 200 mg/kg/day Luteinate on GDs 1–20. In addition to the 

increase in implantation loss noted above, there was an increase in delayed ossification at 200 

B-26
mg/kg/day but no effect on fetal body weight. It is unlikely that this effect is related to LH 

levels. However, the mode of action is unknown. 

SELECTION OF HEALTH ENDPOINTS AND DERIVATION OF REFERENCE

VALUES

Narrative Description of the Extent of the Database

The database for oral exposure is quite robust and is adequate for deriving reference 

values. Because the mode of action was known for other pesticides in this chemical class, it was 

possible to study known targets during relevant life stages. Luteinate was shown to interfere 

with the pituitary-hypothalamic axis, resulting in a decrease in serum LH and prolactin. The 

decrease was shown to be dependent on dose and duration of exposure. Normal LH levels are 

known to be required for ovulation, maintenance of pregnancy, timing of parturition, and sexual 

maturation. Similarly, decreases in prolactin can lead to prostatitis and effects on the male 

reproductive organs. Each of these events were examined in several short-term studies as well as 

in traditional prenatal developmental toxicity studies and a two-generation reproductive toxicity 

study. 

An acute toxicity study of Luteinate was not conducted. However, information was 

available on serum LH and prolactin levels from other short-term exposures that was informative 

for acute exposures. The database as a whole demonstrates that there is a clear dose-duration 

relationship for Luteinate on serum LH levels. Serum LH levels were reduced following a 3-day 

exposure in adult rats and in pregnant rats on GDs 1–8 at doses of 200 mg/kg/day; longer 

exposures to weanlings on PNDs 22–41 or to adults for 28 days required a dose of 100 or 80 

mg/kg/day, respectively. The effective dose of 10 mg/kg/day was still lower following 10 to 13 

weeks exposure. Therefore, it is unlikely that serum LH levels and related effects would occur at 

doses less than 200 mg/kg/day following an acute exposure, but higher doses may actually be 

necessary. 

The situation is less clear for the decrease in prolactin and related effects. Prolactin 

levels were reduced following a 3-day exposure to adult rats at 200 mg/kg/day and following 

exposure to lactating dams at 50 mg/kg/day. A 28-day exposure to adult rats resulted in 

decreased prolactin levels at 80 mg/kg/day, whereas 10 mg/kg/day was an effective dose at 10 to 

13 weeks of exposure. Thus, although there is a clear dose-duration relationship between the 4-

and 10-week periods, the relationship is less clear for durations of less than 28 days. However, it 

is unlikely that serum prolactin levels and related effects would have NOAELs less than 25 

mg/kg/day following an acute exposure. 

B-27
Knowledge about the effect of Luteinate on serum LH and prolactin levels from these 

other studies was considered in conjunction with the prenatal developmental toxicity study for 

deriving the acute reference value. It is assumed that effects resulting from developmental 

exposures (both prenatal and postnatal) may be the result of a single exposure. Therefore, the 

fetal and offspring effects resulting from exposures during gestation and postnatally to the time 

of sexual maturation were also considered for the acute reference value. Data from the twogeneration study were not considered because there were always shorter-term studies showing 

effects at higher doses. Taken as a whole, the lack of an acute toxicity study was not considered 

to be a major data gap for Luteinate. 

A chronic study of Luteinate was not conducted. However, serum LH was decreased at 

10 mg/kg/day in the F0 and F1 animals in the two-generation reproductive toxicity study and at 8 

mg/kg/day in the 6-month study. Because there is essentially no change in the effective dose 

levels following 13 weeks of exposure and 6 months of exposure, it is unlikely that a longer 

exposure period would substantially lower the effective dose. Thus, the lack of a chronic study 

was not considered a major database deficiency. Although there is no knowledge of the effects 

of continued LH and prolactin suppression on reproductive aging, this is probably a qualitative, 

rather than a quantitative data gap. It is known from chronic bioassays of other pesticides in this 

chemical class that lifetime exposure results in mammary tumors in SD rats, but this mode of 

action is unlikely to be operative in humans due to differences in the aging process. Thus, this 

was not considered to be a data gap for Luteinate. 

A developmental neurotoxicity study was not conducted. Given that Luteinate interferes 

with two neuroreceptors, norepinephrine and dopamine, the potential for developmental 

neurotoxicity exists. Thus, the lack of knowledge regarding the developing nervous system is 

considered to be a data gap. 

Exposure-Response Array 

In addition to the display in tabular form (Table B-3), the data considered useful for 

deriving each reference value were displayed in an exposure-response array. The arrays for 

acute, short-term, longer-term, and chronic exposures are shown in Figures B-5 through B-8, 

respectively. Although data on delayed ossification were considered in deriving the longer-term 

and chronic reference values, the data were not included in Figures B-7 and B-8 because the 

doses were substantially higher than those for all other endpoints, and inclusion would have 

altered the y-axis substantially. Figure B-9 is a composite of the NOAELs for each endpoint 

B-28
with each exposure duration. In addition, the NOAELs for each relevant endpoint, the 

uncertainty factors (UFs), and the sample and final reference values are shown in Table B-4. 

Uncertainty Factors 

An interspecies UF (A in Table B-4) of 10 was applied in all cases because the data were 

insufficient to characterize toxicokinetic and toxicodynamic differences between rodents and 

humans. An intraspecies UF (H in Table B-4) of 10 was applied in all cases because the data did 

not allow the estimation of human variability. A UF for LOAEL to NOAEL (L in Table B-4) 

was not applied because the NOAEL was known for each exposure duration. A subchronic-tochronic UF (S in Table B-4) was not applied because there were data available for each exposure 

duration. A database UF of 101/2 was applied in all cases because there are concerns for the 

potential developmental neurotoxicity of Luteinate and there were no available data. 

A database UF of 101/2 (rather than a UF of 10) was applied on basis of the observation 

that for other pesticides in this chemical class, the doses that result in a decrease in 

norephinephrine and dopamine are very similar to the doses that lead to decreases in serum LH. 

Therefore, it is likely that any developmental neurotoxicity effects would be observed at similar 

doses. Because the effects of Luteinate on serum LH levels have been well characterized, 

additional developmental neurotoxicity information is unlikely to dramatically affect the 

reference values. However, a UF of 101/2 was retained to reflect underlying uncertainties of this 

data gap. 

Acute Exposure 

For the derivation of the acute reference value, a standard prenatal developmental 

toxicity study provided information on the effects of Luteinate on fetal ossification in the 

absence of an effect on fetal weight. There was no information on serum LH or prolactin and 

related effects following an acute exposure. As described above in the discussion of the extent 

of the database, it is unlikely that the NOAEL for serum LH levels and related effects would be 

less than 100 mg/kg/day following an acute exposure, and it could be higher. Similarly, it is 

unlikely that the NOAEL for serum prolactin levels and related effects would be less than 25 

mg/kg/day following an acute exposure. The effects on sexual maturation occurred at a dose of 

100 mg/kg/day (NOAEL of 50 mg/kg/day) and effects on ossification occurred at a dose of 200 

mg/kg/day (NOAEL of 100 mg/kg/day). 

Thus, derivation of the acute reference value based on these endpoints would also be 

protective of effects on serum LH and related effects, but may not cover effects on prolactin and 

B-29
Timing and Duration of Exposure 

0 

20 40 60 80 

100 120 140 160 180 200 

PNDs 1 - 4 PNDs 1 - 4 PNDs 22 -

41 

PNDs 23 -

53 

GD 1 - 20 

Dose (mg/kg/day) 

NOAEL LOAEL 

cation 

NOAEL LOAEL 

Decreased Prolactin 

Prostatitis 

Delayed Ossification 

Delayed Vaginal Opening 

Delayed Preputial Separati

200

on 

180160

B-30

Dose (mg/kg/day)140

120100

80604020

0

PNDs 1 - 4 PNDs 1 - 4 PNDs 22 -

41 

PNDs 23 -

53

GD 1 - 20

Timing and Duration of Exposure

Figure B-5. Exposure-response array of data considered for the Luteinate

acute reference value.
0 

50 

100 150 200 250 300 350 400 

3 days 28 days PNDs 

22 - 41 

28 days PNDs 

22 - 41 

PNDs 

21 - 41 

4 - 5 

weeks 

(F1) 

PNDs 

23 - 53 

6 - 7 

weeks 

(F1) 

28 days 

Timing and Duration of Exposure 

Dose (mg/kg/day) 

NOAEL LOAEL 

Decrea

sed LH 

Disrupted Estrus Cyclicity 

Decreased LH 

Disrupted Estrus Cyclicity 

Pregnancy Loss 

Pregnancy Loss 

Delayed Parturition 

Delayed Vaginal Opening 

Delayed Preputial separation 

Delayed Vaginal Opening 

Delayed Preputial Separation 

Decreased Prolactin 

Decreased Prolactin 

Porstatitis 

Decreased LH 

400350

Dose (mg/kg/day)

300250200150100

50

0 B-313 days 28 days PNDs 22 - 41 28 days PNDs 22 - 41 GD 1 - 8, 

GD 

1 - 20 

GD 

6 - 10 

GD 

6 - 10 

PNDs 

21 - 41 

4 - 5 

weeks 

(F1) 

PNDs 

23 - 53 

6 - 7 

weeks 

(F1) 

PNDs 

1 - 4 

28 days 

PNDs 

1 - 4 

Timing and Duration of Exposure

Figure B-6. Exposure-response array of data considered for the Luteinate

short-term reference va
0 

10 20 30 40 50 60 70 80 90 

100 

10 - 13 weeks 

(F0, F1) 

10 - 13 weeks 

(F0, F1) 

11 - 16 weeks 

(F0, F1) 

14 - 17 weeks 

(F0, F1) 

10 - 13 weeks 

(F0, F1) 

16 weeks (F1) 

Timing and Duration of Exposure 

Dose (mg/kg/day) 

NOAEL LOAEL 

Decreased LH Disrupted Estrus C

yclicity 

Pregnancy Loss 

Delayed Parturition 

Prostatitis Decreased Prolactin 

100

90

Dose (mg/kg/day)80

7060504030

B-3220

10

0

10 - 13 weeks 

(F0, F1) 

10 - 13 weeks 

(F0, F1) 

11 - 16 weeks 

(F0, F1) 

14 - 17 weeks 

(F0, F1) 

10 - 13 weeks 

(F0, F1) 

16 weeks (F1) 

Timing and Duration of Exposure 

Figure B-7. Exposure-response array of data considered for the Luteinate

longer-term reference v
0 

10 20 30 40 50 60 70 80 90 

100 

6 months 10 - 13 weeks 

(F0, F1) 

6 months 10 - 13 weeks 

(F0, F1) 

11 - 16 weeks 

(F0, F1) 

14 - 17 weeks 

(F0, F1) 

10 - 13 week

(F0, F1) 

Timing and Duration of Exposure 

Dose (mg/kg/day) 

100

9080

NOAEL LOAEL 

Decreased LH 

Disrupted Estrus Cyclicity 

Pregnancy Loss 

Delayed Parturition 

Decreased LH 

Disrupted Estrus Cyclicity 

Decreased Prolactin 

Dose (mg/kg/day)

70605040

B-33

302010

0

6 months 10 - 13 weeks 

(F0, F1) 

6 months 10 - 13 weeks 

(F0, F1) 

11 - 16 weeks 

(F0, F1) 

14 - 17 weeks 

(F0, F1) 

10 - 13 week

(F0, F1) 

Timing and Duration of Exposure 

Figure B-8. Exposure-response array of data considered for the Luteinate

chronic reference value.
0 

50 

100 150 200 250 

0 5 10 

Dose (mg/kg/day) 

Altered pregnancy maintenance LH reduction Estrous disruption Delayed parturition 

Prolactin release attenuation Reduced seminal vesicle weight Reduced fetal ossification Delayed preputial separation Delayed vaginal opening Prostatitis ltere pregnanc aintenan eductio strou dis uptio elaye parturitio rolact releas at enuatio e u e i a vesicl eigh e u e e a ossifi ati elaye r p tia sepa ti ay v gi a o eni ros atiti

1 2 3 4 6 7 8 9 

GDs 1-8 GDs 6-1

GDs 1-2

PNDs 1

PNDs 2

PNDs 2

3D 

28D 

6mo F0-10wk 

F1-13wk 

F1- 5wk 

F1- 7wk 

F1-16wk 

250 200

Dose (mg/kg/day) 150

100

B-34 50

0 

0 1 2 3 4 5 6 7 8 9

y n n n 

c

n n g n s o n l a o r

d

a

n

l p

n 

em s P t we

R d c

d

R d c

d 

r t ec HL r e t s d d

p e

u

l 

d

f t l E Am D l e e D in

D P 

Figure B-9. NOAELs for Luteinate with timing/duration of exposure. 

10 
Table B-4. Summary of reference values for Luteinate 

Reference 

value 

duration 

Exposure 

duration 

NOAEL 

(mg/kg/day) 

Type of 

effect 

Uncertainty factorsa 

Reference value 

(mg/kg/day) 

A H L S D Total Sample Final 

Acute PNDs 1–4 25 Prolactin 10 10 1 1 

3 

300 0.08 

0.08 

PND 1–4 25 Prostatitis 10 10 1 1 300 0.08 

PNDs 

22–41 50 

Sexual 

Maturation 

10 10 1 1 300 0.17 

PNDs 

23–53 50 

Sexual 

Maturation 

10 10 1 1 300 0.17 

GDs 1–20 100 Fetal 

ossification 

10 10 1 1 300 0.33 

Short-term 3-day 

adult 

100 LH 10 10 1 1 

3 

300 0.33 

0.08 

28-day 

adult 

40 LH 10 10 1 1 300 0.13 

PNDs 

22–41 

50 LH 10 10 1 1 300 0.17 

GDs 1–8 100 LH 10 10 1 1 300 0.33 

28-day 

adult 

40 Estrus 10 10 1 1 300 0.13 

PNDs 

22–41 

50 Estrus 10 10 1 1 300 0.17 

GDs 1–8 100 Pregnancy 

Maintenance 

10 10 1 1 300 0.33 

GDs 1–20 100 Pregnancy 

Maintenance 

10 10 1 1 300 0.33 

GDs 6–10 200 Pregnancy 

Maintenance 

10 10 1 1 300 0.67 

GDs 6–10 100 Parturition 10 10 1 1 300 0.33 

PNDs 

22–41 

50 Sexual 

Maturation 

10 10 1 1 300 0.17 

PNDs 

23–53 

50 Sexual 

Maturation 

10 10 1 1 300 0.17 

B-35
Table B-4. Summary of reference values for Luteinate (continued) 

Reference 

value 

duration 

Exposure 

duration 

NOAEL 

(mg/kg/day) 

Type of 

effect 

Uncertainty factorsa 

Reference value 

(mg/kg/day) 

A H L S D Total Sample Final 

Short-term 4–7 

weeks 

25 Sexual 

Maturation 

10 10 1 1 

3 

300 

PNDs 1–4 25 Prolactin 10 10 1 1 300 

3-day 

adult 

100 Prolactin 10 10 1 1 300 

28-day 

adult 

40 Prolactin 10 10 1 1 300 

PNDs 1–4 25 Prostatitis 10 10 1 1 300 

PNDs 

23–53 

25 Organ Wt 10 10 1 1 300 

GDs 1–20 100 Fetal 

ossification 

10 10 1 1 300 

Longerterm 10–13 

weeks 

5 LH 10 0 1 

3 

300 

10–13 

weeks 

5 Estrus 10 10 1 1 300 

11–16 

weeks 

5 Pregnancy 

Maintenance 

10 10 1 1 300 

14–17 

weeks 

5 Parturition 10 10 1 1 300 

4–7 

weeks 

25 Sexual 

Maturation 

10 10 1 1 300 

10–13 

weeks 

5 Prolactin 10 10 1 1 300 

16 weeks 5 Prostatitis 10 10 1 1 300 

16 weeks 5 Organ Wt 10 10 1 1 300 

GDs 1–20 100 Fetal 

ossification 

10 10 1 1 300 

1 1 

0.08 

0.08 

0.08 

0.33 

0.13 

0.08 

0.08 

0.33 

0.02 

0.02 

0.02 

0.02 

0.02 

0.08 

0.02 

0.02 

0.02 

0.33 

B-36
Table B-4. Summary of reference values for Luteinate (continued) 

Reference 

value 

duration 

Exposure 

duration 

NOAEL 

(mg/kg/day) 

Type of 

effect 

Uncertainty factorsa 

Reference value 

(mg/kg/day) 

A H L S D Total Sample Final 

Chronic 6 months 4 LH 10 10 1 1 

3 

300 0.01 

0.01 

6 months 4 Estrus 10 10 1 1 300 0.01 

11–16 

weeks 

5 Pregnancy 

Maintenance 

10 10 1 1 300 0.02 

14–17 

weeks 

5 Parturition 10 10 1 1 300 0.02 

10–13 

weeks 

5 Prolactin 10 10 1 1 300 0.02 

16 weeks 5 Prostatitis 10 10 1 1 300 0.02 

16 weeks 5 Organ Wt 10 10 1 1 300 0.02 

GDs 1–20 100 Fetal 

ossification 

10 10 1 1 300 0.33 

a

 A = animal-to-human (interspecies); H = inter-individual (intraspecies); L = LOAEL-to-NOAEL; 

S = subchronic-to-chronic duration; D = database deficiency 

related effects. For this reason, the NOAEL of 25 mg/kg/day for prolactin and related effects 

following a 4-day developmental (neonatal) exposure was used as the basis for the acute 

reference value, with the assumption that a single exposure during a critical period of 

development would be sufficient to produce these effects. 

Although a single-exposure LOAEL, particularly for the reduction in prolactin and 

increase in prostatitis (which showed a dependence on exposure duration in adults), might be 

slightly higher than the 4-day LOAEL, lack of data on this endpoint from a single-day exposure 

leads us to rely on the 4-day value during what appears to be a particularly sensitive time in the 

early postnatal period. Effects on prolactin (and therefore dopamine) also indicate a concern for 

developmental neurotoxicity (Figure B-5). Because there is a strong relationship between the 

data gaps of the acute toxicity study and the developmental neurotoxicity study, a database UF of 

101/2 was applied. In addition, UFs of 10 for interspecies and intraspecies uncertainty/variability 

were applied. The resulting reference value was 0.08 mg/kg/day. 

B-37
Short-term Exposure 

A variety of endpoints were examined for calculating the short-term reference value. 

Information was available on serum LH from the 3-day and 28-day adult studies as well as the 

study in which pregnant dams were exposed on GDs 1–8. Information on estrous cyclicity was 

available from the 28-day study in adult rats and in the study in which female weanlings were 

exposed on PNDs 22–41. Information on pregnancy maintenance was available from studies in 

which pregnant dams were exposed on GDs 1–8, 6–10, and 1–20, and information on parturition 

was available for exposures on GDs 6–10. 

Information on sexual maturation in females was available from the study in which 

female weanlings were exposed on PNDs 22–41, and information on sexual maturation in males 

was available from the study in which weanling male rats were exposed on PNDs 23–53. In 

addition, information on sexual maturation from the two-generation reproductive toxicity study 

was also considered relevant. The latter study also provided information on the effects of 

Luteinate on the seminal vesicles and ventral prostate. Information on prolactin was available 

from the 28-day study in adults and the study in which lactating dams were exposed on PNDs 

1–4; information on prostatitis was also available from the latter study (Figure B-6). 

Interspecies and intraspecies UFs of 10 were applied to each of these endpoints (Table B4). For the entire database available for short-term exposure, a database UF of 101/2 was applied 

in the derivation of the final reference value for the lack of a developmental neurotoxicity study. 

The resulting reference values ranged from 0.08 to 0.67 mg/kg/day, and the final reference value 

was 0.08 mg/kg/day. 

Longer-term Exposure 

For derivation of the longer-term reference value, the databases available for the acute 

and shorter-term exposures were considered. In addition, the two-generation reproductive 

toxicity study provided information following longer exposures for serum LH, estrous cyclicity, 

pregnancy maintenance, parturition, prolactin, and prostatitis (Fig. B-7). Interspecies and 

intraspecies UFs of 10 were applied to each of these endpoints (Table B-4). The resulting 

sample reference values were 0.33 mg/kg/day, based on delayed ossification and 0.08 

mg/kg/day, based on sexual maturation; all the other endpoints yielded a value of 0.02 

mg/kg/day. The latter was chosen as the final value for the longer-term reference value. 

B-38
Chronic Exposure 

Information from all of the exposure scenarios described above was considered in 

deriving the chronic reference value. In addition, information on serum LH and estrous cyclicity 

was available from the 6-month study in adult rats. For endpoints where there was a clear 

exposure-dose-effect relationship, only information from studies of the longest exposure period 

was included in Table B-4 and Figure B-8. As noted above, decreases in serum LH were 

observed at 8 mg/kg/day in the 6-month study and at 10 mg/kg/day in the two-generation 

reproductive toxicity study; the NOAELs were 4 and 5 mg/kg/day, respectively. Estrous 

cyclicity was affected at these same dose levels in the two studies. The NOAEL for all other 

endpoints in the two-generation reproductive toxicity study was also 5 mg/kg/day. 

Given the similarity in effect levels in the two studies, it is unlikely that longer exposures 

would alter the effect level. For this reason, although there is no information on the effect of 

Luteinate on reproductive aging, this is considered to be a qualitative gap in hazard 

identification, but was not considered to be a database deficiency for the purposes of deriving a 

chronic reference value. UFs of 10 for interspecies and intraspecies variability and uncertainty 

were applied to each of these endpoints (Table B-4). The reference values ranged from 0.01 to 

0.02 mg/kg/day, and 0.01 mg/kg/day was chosen as the final value. 

B-39
GLOSSARY 

NOTE: The following terms are used in this document. To the extent possible, 

definitions were taken from other EPA sources, e.g., IRIS, the Children’s Health Research 

Strategy, the RfC Methodology. In some cases, the definitions have been revised from the 

originals in IRIS for the sake of clarity or to be consistent with usage in this document. Those 

terms and definitions that are changed and/or newly proposed in this document to be added to 

IRIS are shown in italics and the definition(s) they are proposed to replace are indicated in 

brackets. A number of other terms are included in the IRIS glossary that are not listed here, 

simply because they were not used in this document. 

Acute Exposure: One dose or multiple doses of short duration spanning less than or 

equal to 24 hours. [Current IRIS definition.] 

Acute Exposure: Exposure by the oral, dermal, or inhalation route for 24 hours or less. 

[Proposed definition to replace the current Acute Exposure definition on IRIS.] 

Adverse Effect: A biochemical change, functional impairment, or pathologic lesion that 

affects the performance of the whole organism or reduces an organism's ability to respond to an 

additional environmental challenge. 

Benchmark Dose (BMD) or Concentration (BMC): A statistical lower confidence 

limit on the dose that produces a predetermined change in response rate of an adverse effect 

(called the benchmark response or BMR) compared to background. [current IRIS definition] 

Benchmark Dose (BMD) or Concentration (BMC): A dose or concentration that 

produces a predetermined change in response rate of an adverse effect (called the benchmark 

response or BMR) compared to background. [Proposed definition to replace the current 

definition on IRIS.] 

BMDL or BMCL: A statistical lower confidence limit on the dose or concentration at the 

BMD or BMC, respectively. [A new definition to be added to IRIS.] 

Benchmark Response (BMR): An adverse effect used to define a benchmark dose from 

which an RfD (or RfC) can be developed. The change in response rate over background of the 

BMR is usually in the range of 5 to 10%, which is the limit of responses typically observed in 

well-conducted animal experiments. 

Bioassay: An assay for determining the potency (or concentration) of a substance that 

causes a biological change in experimental animals. 

Bioavailability: The degree to which a substance becomes available to the target tissue 

after administration or exposure. 

G-1
Biologically Based Dose Response (BBDR) model: A predictive tool used to estimate 

potential human health risks by describing and quantifying the key steps in the cellular, tissue, 

and organismal responses as a result of chemical exposure. [Current IRIS definition.] 

Biologically Based Dose Response (BBDR) Model: A predictive model that describes 

biological processes at the cellular and molecular level linking the target organ dose to the 

adverse effect. [Proposed definition to replace the current definition on IRIS.] 

Blood-to-air Partition Coefficient: A ratio of a chemical's concentration between blood 

and air when at equilibrium. 

Chronic Exposure: Multiple exposures occurring over an extended period of time or a 

significant fraction of the animal's or the individual's lifetime. [Current IRIS definition.] 

Chronic Exposure: Repeated exposure by the oral, dermal, or inhalation route for more 

than approximately 10% of the life span in humans (more than approximately 90 days to 2 years 

in typically used laboratory animal species). [Proposed definition to replace the current 

definition for Chronic Exposure on IRIS.] 

Chronic Study: A toxicity study designed to measure the (toxic) effects of chronic 

exposure to a chemical. 

Critical Effect: The first adverse effect, or its known precursor, that occurs to the most 

sensitive species as the dose rate of an agent increases. 

Critical Study: The study that contributes most significantly to the qualitative and 

quantitative assessment of risk; also called Principal Study. 

Developmental Toxicity: Adverse effects on the developing organism that may result 

from exposure prior to conception (either parent), during prenatal development, or postnatally 

until the time of sexual maturation. The major manifestations of developmental toxicity include 

death of the developing organism, structural abnormality, altered growth, and functional 

deficiency. 

Dose: The amount of a substance available for interactions with metabolic processes or 

biologically significant receptors after crossing the outer boundary of an organism. The 

potential dose is the amount ingested, inhaled, or applied to the skin. The applied dose is the 

amount presented to an absorption barrier and available for absorption (although not 

necessarily having yet crossed the outer boundary of the organism). The absorbed dose is the 

amount crossing a specific absorption barrier (e.g., the exchange boundaries of the skin, lung, 

and digestive tract) through uptake processes. Internal dose is a more general term denoting 

the amount absorbed without respect to specific absorption barriers or exchange boundaries. 

The amount of the chemical available for interaction by any particular organ or cell is termed 

G-2
the delivered or biologically effective dose for that organ or cell. [New definition proposed to 

be added to IRIS.] 

Dose-Response Assessment: A determination of the relationship between the magnitude 

of an administered, applied, or internal dose and a specific biological response. Response can be 

expressed as measured or observed incidence, percent response in groups of subjects (or 

populations), or as the probability of occurrence within a population. [Current IRIS definition.] 

Dose-Response Assessment: A determination of the relationship between the magnitude 

of an administered, applied, or internal dose and a specific biological response. Response can 

be expressed as measured or observed incidence or change in level of response, percent 

response in groups of subjects (or populations), or the probability of occurrence or change in 

level of response within a population. [Proposed definition to replace the current definition on 

IRIS.] 

Dose-Response Relationship: The relationship between a quantified exposure (dose) 

and the proportion of subjects demonstrating specific, biological changes (response). [Current 

IRIS definition.] 

Dose-Response Relationship: The relationship between a quantified exposure (dose) and 

the proportion of subjects demonstrating specific biological changes in incidence or in degree of 

change (response). [Proposed definition to replace the current definition on IRIS.] 

Endpoint: An observable or measurable biological event or chemical concentration (e.g., 

metabolite concentration in a target tissue) used as an index of an effect of a chemical exposure. 

Epidemiology: The study of disease patterns in human populations. [Current IRIS 

definition.] 

Epidemiology: The study of the distribution and determinants of health-related states or 

events in specified populations and the application of this study to the control of health 

problems. [Proposed definition to replace the current definition on IRIS.] 

Exposure: Contact made between a chemical, physical, or biological agent and the outer 

boundary of an organism. Exposure is quantified as the amount of an agent available at the 

exchange boundaries of the organism (e.g., skin, lungs, gut). 

Exposure Assessment: An identification and evaluation of the human population 

exposed to a toxic agent, describing its composition and size, as well as the type, magnitude, 

frequency, route, and duration of exposure. 

Exposure Pathway: The physical course an environmental agent takes from the source 

to the individual exposed. 

G-3
Extrapolation, Low Dose: An estimate of the response at a point below the range of the 

experimental data, generally through the use of a mathematical model. 

Hazard: A potential source of harm. 

Hazard Assessment: The process of determining whether exposure to an agent can cause 

an increase in the incidence of a particular adverse health effect (e.g., cancer, birth defect) and 

whether the adverse health effect is likely to occur in humans. 

Hazard Characterization: A description of the potential adverse health effects 

attributable to a specific environmental agent, the mechanisms by which agents exert their toxic 

effects, and the associated dose, route, duration, and timing of exposure. [New definition 

proposed to be added to IRIS] 

Human Equivalent Concentration (HEC): The human concentration (for inhalation 

exposure) of an agent that is believed to induce the same magnitude of toxic effect as the 

experimental animal species concentration. This adjustment may incorporate toxicokinetic 

information on the particular agent, if available, or use a default procedure. 

Human Equivalent Dose (HED): The human dose (for other than the inhalation routes 

of exposure) of an agent that is believed to induce the same magnitude of toxic effect as the 

experimental animal species dose. This adjustment may incorporate toxicokinetic information 

on the particular agent, if available, or use a default procedure, such as assuming that daily oral 

doses experienced for a lifetime are proportional to body weight raised to the 0.75 power. 

Incidence: The number of new cases of a disease that develop within a specified 

population over a specified period of time. 

Incidence Rate: The ratio of new cases within a population to the total population at risk 

given a specified period of time. 

Latency Period: The time between exposure to an agent and manifestation or detection 

of a health effect of interest. 

Linear Dose Response: A pattern of frequency or severity of biological response that 

varies proportionately with the amount of dose of an agent. [Current IRIS definition.] 

Linear Dose Response: A pattern of frequency or severity of biological response that 

varies directly with the amount of dose of an agent. This linear relationship holds only at low 

doses in the range of extrapolation. [Proposed definition to replace the current definition on 

IRIS.] 

Longer-term Exposure: Repeated exposure by the oral, dermal, or inhalation route for 

more than 30 days, up to approximately 10% of the life span in humans (more than 30 days up to 

G-4
approximately 90 days in typically used laboratory animal species). [Proposed new definition to 

be used relative to the Longer-term Reference Value. Similar to the current definition for 

Subchronic Exposure. Because subchronic exposure studies will continue to be used in risk 

assessment, the latter term should be retained as well but replaced with the definition for Longerterm Exposure.] 

Lowest-Observed-Adverse-Effect Level (LOAEL): The lowest exposure level at which 

there are statistically or biologically significant increases in frequency or severity of adverse 

effects between the exposed population and its appropriate control group. Also referred to as 

lowest-effect level (LEL). [Current IRIS and RfC Methodology definition.] 

Lowest-Observed-Adverse-Effect Level (LOAEL): The lowest exposure level at which 

there are biologically significant increases in frequency or severity of adverse effects between 

the exposed population and its appropriate control group. [Proposed to replace the current 

definition in IRIS and the RfC methodology, U.S. EPA, 1994] 

Margin of Exposure (MOE): The LED10 or other point of departure divided by the 

actual or projected environmental exposure of interest. 

Mechanism of Action: The complete sequence of biological events that must occur to 

produce the toxic effect. 

Mode of Action (MOA): A less-detailed description of the mechanism of action in 

which some but not all of the sequence of biological events leading to a toxic effect is known. 

Modifying Factor (MF): A factor used in derivation of a reference dose or reference 

concentration. The magnitude of the MF reflects the scientific uncertainties of the study and 

database not explicitly treated with standard uncertainty factors (e.g., the completeness of the 

overall database). A MF is greater than zero and less than or equal to 10, and the default value 

for the MF is1. [Current definition in IRIS; this report recommends that its use be discontinued.] 

No-Observed-Adverse-Effect Level (NOAEL): The highest exposure level at which 

there are no statistically or biologically significant increases in the frequency or severity of 

adverse effect between the exposed population and its appropriate control; some effects may be 

produced at this level, but they are not considered adverse, nor precursors to adverse effects. 

[Current IRIS and RfC Methodology definition.] 

No-Observed-Adverse-Effect Level (NOAEL): The highest exposure level at which there 

are no biologically significant increases in the frequency or severity of adverse effect between 

the exposed population and its appropriate control; some effects may be produced at this level, 

but they are not considered adverse or precursors to adverse effects. [Proposed to replace the 

current definition in IRIS and the RfC methodology, U.S. EPA, 1994.] 

G-5
Nonlinear Dose Response: A pattern of frequency or severity of biological response that 

does not vary proportionately with the amount of dose of an agent. When mode of action 

information indicates that responses may not follow a linear pattern below the dose range of the 

observed data, non-linear methods for determining risk at low dose may be justified. [Current 

IRIS definition.] 

Nonlinear Dose Response: A pattern of frequency or severity of biological response that 

does not vary directly with the amount of dose of an agent. When mode of action information 

indicates that responses may fall more rapidly than dose below the range of the observed data, 

nonlinear methods for determining risk at low dose may be justified. [Proposed definition to 

replace the current definition on IRIS.] 

Physiologically Based Pharmacokinetic (PBPK) Model: Physiologically based 

compartmental model used to characterize pharmacokinetic behavior of a chemical. Available 

data on blood flow rates, and metabolic and other processes which the chemical undergoes 

within each compartment are used to construct a mass-balance framework for the PBPK model. 

[Current IRIS definition.] 

Physiologically Based Pharmacokinetic (PBPK) Model: A model that estimates the dose 

to a target tissue or organ by taking into account the rate of absorption into the body, 

distribution among target organs and tissues, metabolism, and excretion. [Proposed definition 

to replace the current definition on IRIS.] 

Point of Departure: The dose-response point that marks the beginning of a low-dose 

extrapolation. This point is most often the upper bound on an observed incidence or on an 

estimated incidence from a dose-response model. [Current IRIS definition.] 

Point of Departure: The dose-response point that marks the beginning of a low-dose 

extrapolation. This point can be the lower bound on dose for an estimated incidence or a 

change in response level from a dose-response model (BMD), or a NOAEL or LOAEL for an 

observed incidence, or change in level of response. [Proposed definition to replace the current 

definition on IRIS.] 

Ppb: A unit of measure expressed as parts per billion. Equivalent to 1 x 10-9. 

Ppm: A unit of measure expressed as parts per million. Equivalent to 1 x 10-6. 

Prevalence: The proportion of disease cases that exist within a population at a specific 

point in time relative to the number of individuals within that population at the same point in 

time. 

Reference Concentration (RfC): An estimate (with uncertainty spanning perhaps an 

order of magnitude) of a continuous inhalation exposure to the human population (including 

sensitive subgroups) that is likely to be without an appreciable risk of deleterious effects during a 

G-6
lifetime. It can be derived from a NOAEL, LOAEL, or benchmark concentration, with 

uncertainty factors generally applied to reflect limitations of the data used. Generally used in 

EPA's noncancer health assessments. [Current IRIS definition.] 

Reference Dose (RfD): An estimate (with uncertainty spanning perhaps an order of 

magnitude) of a daily oral exposure to the human population (including sensitive subgroups) that 

is likely to be without an appreciable risk of deleterious effects during a lifetime. It can be 

derived from a NOAEL, LOAEL, or benchmark dose, with uncertainty factors generally applied 

to reflect limitations of the data used. Generally used in EPA's noncancer health assessments. 

[Current IRIS definition.] 

Reference Value (RfV): An estimate of an exposure for [a given duration] to the human 

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

adverse effects over a lifetime. It is derived from a BMDL, a NOAEL, a LOAEL, or another 

suitable point of departure, with uncertainty/variability factors applied to reflect limitations of 

the data used. [Durations include acute, short-term, longer-term, and chronic and are defined 

individually in this glossary. This definition is proposed to replace those for the Reference Dose 

(RfD) and Reference Concentration (RfC). A subscript would be used with the RfV to denote 

route and duration, e.g., RfVAO for the Acute Oral Reference Value.] 

Regional Deposited Dose (RDD): The deposited dose of particles calculated for a 

respiratory tract region of interest (r) as related to an observed toxicity. For respiratory effects of 

particles, the deposited dose is adjusted for ventilatory volumes and the surface area of the 

respiratory region affected (mg/min-sq. cm). For extrarespiratory effects of particles, the 

deposited dose in the total respiratory system is adjusted for ventilatory volumes and body 

weight (mg/min-kg). 

Regional Deposited Dose Ratio (RDDR): The ratio of the regional deposited dose 

calculated for a given exposure in the animal species of interest to the regional deposited dose of 

the same exposure in a human. This ratio is used to adjust the exposure-effect level for 

interspecies dosimetric differences to derive a human equivalent concentration for particles. 

Regional Gas Dose: The gas dose calculated for the region of interest as related to the 

observed effect for respiratory effects. The deposited dose is adjusted for ventilatory volumes 

and the surface area of the respiratory region affected (mg/min-sq.cm). 

Regional Gas Dose Ratio (RGDR): The ratio of the regional gas dose calculated for a 

given exposure in the animal species of interest to the regional gas dose of the same exposure in 

humans. This ratio is used to adjust the exposure effect level for interspecies dosimetric 

differences to derive a human equivalent concentration for gases with respiratory effects. 

Risk (in the context of human health): The probability of injury, disease, or death from 

exposure to a chemical agent or a mixture of chemicals. In quantitative terms, risk is expressed 

in values ranging from zero (representing the certainty that harm will not occur) to one 

G-7
(representing the certainty that harm will occur). The following are examples of how risk is 

expressed within IRIS: E-4 or 10-4 = a risk of 1/10,000; E-5 or 10-5 = 1/100,000; E-6 or 10-6 = 

1/1,000,000. Similarly, 1.3 E-3 or 1.3 x 10-3 = a risk of 1.3/1,000=1/770; 8 E-3 or 8 x 10-3 = a 

risk of 1/125 and 1.2 E-5 or 1.2 x 10-5 = a risk of 1/83,000. [Current IRIS definition.] 

Risk: The probability of adverse effects resulting from exposure to an environmental 

agent or mixture of agents. [Proposed definition to replace the current definition on IRIS.] 

Risk Characterization: The integration of information on hazard, exposure, and doseresponse to provide an estimate of the likelihood that any of the identified adverse effects will 

occur in exposed people. [New definition proposed to be added to IRIS.] 

Risk Assessment (in the context of human health): The determination of potential 

adverse health effects from exposure to chemicals, including both quantitative and qualitative 

expressions of risk. The process of risk assessment involves four major steps: hazard 

identification, dose-response assessment, exposure assessment, and risk characterization. 

[Current IRIS definition.] 

Risk Assessment: The evaluation of scientific information on the hazardous properties of 

environmental agents (hazard characterization), the dose-response relationship (dose-response 

assessment), and the extent of human exposure to those agents (exposure assessment). The 

product of the risk assessment is a statement regarding the probability that populations or 

individuals so exposed will be harmed and to what degree (risk characterization). [Proposed 

definition to replace the current definition on IRIS.] 

Short-term Exposure: Multiple or continuous exposure to an agent for a short period of 

time, usually one week. [Current IRIS definition.] 

Short-term Exposure: Repeated exposure by the oral, dermal, or inhalation route for 

more than 24 hours, up to 30 days. [Proposed definition to replace the current definition for 

Short-term Exposure on IRIS.] 

Statistical Significance: The probability that a result [sic] likely to be due to chance 

alone. By convention, a difference between two groups is usually considered statistically 

significant if chance could explain it only 5% of the time or less. Study design considerations 

may influence the a priori choice of a different statistical significance level. [Current IRIS 

definition.] 

Statistical Significance: The probability that a result is not likely to be due to chance 

alone. By convention, a difference between two groups is usually considered statistically 

significant if chance could explain it only 5% of the time or less. Study design considerations 

may influence the a priori choice of a different level of statistical significance. [Proposed 

definition to replace the current definition on IRIS.] 

G-8
Subchronic Exposure: Exposure to a substance spanning approximately 10% of the 

lifetime of an organism. [See note for Longer-term Exposure.] 

Subchronic Study: A toxicity study designed to measure effects from subchronic 

exposure to a chemical. 

Supporting Studies: Studies that contain information useful for providing insight and 

support for conclusions. 

Susceptible Subgroups: May refer to life stages, for example, children or the elderly, or 

to other segments of the population, for example, asthmatics or the immune-compromised, but 

are likely to be somewhat chemical-specific and may not be consistently defined in all cases. 

[New definition proposed to be added to IRIS.] 

Susceptibility: Increased likelihood of an adverse effect, often discussed in terms of 

relationship to a factor that can be used to describe a human subpopulation (e.g., life stage, 

demographic feature, or genetic characteristic). [New definition proposed to be added to IRIS.] 

Systemic Effects or Systemic Toxicity: Toxic effects as a result of absorption and 

distribution of a toxicant to a site distant from its entry point, at which point effects are 

produced. Not all chemicals that produce systemic effects cause the same degree of toxicity in all 

organs. [Current IRIS definition.] 

Systemic Effects or Systemic Toxicity: Toxic effects as a result of absorption and 

distribution of a toxicant to a site distant from its entry point. [Proposed definition to replace the 

current definition on IRIS.] 

Target Organ: The biological organ(s) most adversely affected by exposure to a 

chemical substance. [Current IRIS definition.] 

Target Organ: The biological organ(s) most adversely affected by exposure to a 

chemical or physical agent. [Proposed definition to replace the current definition on IRIS.] 

Threshold: The dose or exposure below which no deleterious effect is expected to occur. 

Toxicity: The degree to which a chemical substance elicits a deleterious or adverse effect 

upon the biological system of an organism exposed to the substance over a designated time 

period. [Current IRIS definition.] 

Toxicity: Deleterious or adverse biological effects elicited by a chemical, physical, or 

biological agent. [Proposed definition to replace the current definition on IRIS.] 

G-9
Toxicodynamics: The determination and quantification of the sequence of events at the 

cellular and molecular levels leading to a toxic response to an environmental agent (also called 

pharmacodynamics). [New definition proposed to be added to IRIS.] 

Toxicokinetics: The determination and quantification of the time course of absorption, 

distribution, biotransformation, and excretion of chemicals (also called pharmacokinetics). 

[New definition proposed to be added to IRIS.] 

Toxicology: The study of harmful interactions between chemicals and biological 

systems. [current IRIS definition] 

Toxicology: The study of harmful interactions between chemical, physical, or biological 

agents and biological systems. [Proposed definition to replace the current definition on IRIS.] 

Toxic Substance: A chemical substance or agent which may cause an adverse effect or 

effects to biological systems. [Current IRIS definition.] 

Toxic Substance: A chemical, physical, or biological agent that may cause an adverse 

effect or effects to biological systems. [Proposed definition to replace the current definition on 

IRIS.] 

Uncertainty: Uncertainty occurs because of a lack of knowledge. It is not the same as 

variability. For example, a risk assessor may be very certain that different people drink different 

amounts of water but may be uncertain about how much variability there is in water intakes 

within the population. Uncertainty can often be reduced by collecting more and better data, 

whereas variability is an inherent property of the population being evaluated. Variability can be 

better characterized with more data but it cannot be reduced or eliminated. Efforts to clearly 

distinguish between variability and uncertainty are important for both risk assessment and risk 

characterization. [New definition proposed to be added to IRIS.] 

Uncertainty Factor (UF): One of several, generally 10-fold, factors used in 

operationally deriving the RfD and RfC from experimental data. UFs are intended to account for 

(1) variation in sensitivity among the members of the human population, i.e., interhuman or 

intraspecies variability; (2) the uncertainty in extrapolating animal data to humans, i.e., 

interspecies variability; (3) the uncertainty in extrapolating from data obtained in a study with 

less-than-lifetime exposure to lifetime exposure, i.e., extrapolating from subchronic to chronic 

exposure; (4) the uncertainty in extrapolating from a LOAEL rather than from a NOAEL; and (5) 

the uncertainty associated with extrapolation from animal data when the data base is incomplete. 

[current IRIS definition] 

Uncertainty/Variability Factors (UFs): One of several, generally 10-fold, default factors 

used in operationally deriving the RfD and RfC from experimental data. The factors are 

intended to account for (1) variation in sensitivity among the members of the human population 

(i.e., inter-individual variability); (2) uncertainty in extrapolating animal data to humans (i.e., 

G-10
interspecies uncertainty); (3) uncertainty in extrapolating from data obtained in a study with 

less-than-lifetime exposure to lifetime exposure (i.e., extrapolating from subchronic to chronic 

exposure); (4) uncertainty in extrapolating from a LOAEL rather than from a NOAEL; and (5) 

uncertainty associated with extrapolation when the database is incomplete. [Proposed definition 

to replace the current one for Uncertainty Factor on IRIS.] 

Variability: Variability refers to true heterogeneity or diversity. For example, among a 

population that drinks water from the same source and with the same contaminant 

concentration, the risks from consuming the water may vary. This may be due to differences in 

exposure (i.e., different people drinking different amounts of water and having different body 

weights, different exposure frequencies, and different exposure durations) as well as differences 

in response (e.g., genetic differences in resistance to a chemical dose). Those inherent 

differences are referred to as variability. Differences among individuals in a population are 

referred to as inter-individual variability; differences for one individual over time is referred to 

as intra-individual variability. [New definition proposed to be added to IRIS.] 

G-11
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